METHODS TO QUANTITATIVELY ASSESS THE PERFORMANCE
OF CONNECTIVE TISSUE PROGENITOR CELLS IN RESPONSE
TO SURFACE MODIFIED BIOMATERIALS
BY
VIVEK P RAUT
Submitted in partial fulfillment of the requirements
For the degree of Doctor of Philosophy
Dissertation Advisor: Dr. George F. Muschler
Department of Biomedical Engineering
CASE WESTERN RESERVE UNIVERSITY
August 2013
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
_____________Vivek P Raut______________
candidate for the ____ Doctor of Philosophy __degree *.
(signed)____
Dr. Horst von Recum __________
(Chair of the committee)
___________Dr. George F Muschler_________
_
_______ _Dr. Steven Eppell_______
_____
_
____
Dr. Cathleen Carlin_____ _______
(date) __March 15, 2013___
*We also certify that written approval has been obtained for any
proprietary material contained therein.
ii
To my parents and my wife Puja, for their unconditional support, encouragement, and
sacrifices
iii
TABLE OF CONTENTS
Page
TITLE PAGE .................................................................................................................
i
COMMITTEE APPROVAL SHEET ........................................................................... ii
DEDICATION ............................................................................................................... iii
TABLE OF CONTENTS ............................................................................................... iv
LIST OF TABLES ......................................................................................................... vii
LIST OF FIGURES ....................................................................................................... viii
ACKNOWLEDGEMENTS ........................................................................................... ix
ABSTRACT ................................................................................................................... xi
Chapter 1: Preface .........................................................................................................
1
Chapter 2: Introduction to the Components of the Method ...........................................
2.1. Role of Connective Tissue Progenitor Cells (CTPs) in Bone Repair .....................
2.1.1. Definition of CTPs ...............................................................................................
2.1.2. Role of CTPs in Bone Regeneration ....................................................................
2.1.3. Difference between CTPs and MSCs ..................................................................
2.1.4. The Colony Forming Units ..................................................................................
2.2. The Colony Forming Unit Assay ............................................................................
2.2.1. Limitations of Manual Observer-Dependent Assay ............................................
2.2.2. ASTM F2944-12-Automated CFU Assay ...........................................................
2.3. Surface Modification of Biomaterials .....................................................................
2.3.1. Mechanism of Cell-Surface Interaction ...............................................................
2.3.2. Mechanism of Adhesion ......................................................................................
2.3.3. Mechanism of Migration......................................................................................
2.3.4. Mechanism of Osteoblastic Differentiation .........................................................
2.3.5. Influence of Surface properties on Cell-Biomaterial Interactions .......................
2.3.5.1. Effect of Surface Morphology and Roughness on Cell Behavior .....................
2.3.5.2. Effect of Surface Energy and Chemistry on Cell Behavior ..............................
2.3.5.3. Effect of Scaffold Osmolarity and pH on Cell Behavior ..................................
2.4. Figures and Tables ..................................................................................................
2.5. Works Cited ............................................................................................................
5
6
8
9
10
11
12
12
13
14
16
17
17
18
19
19
20
21
22
25
Chapter 3: Design and Verification of Methods to Assess the Response of Connective
Tissue Progenitor Cells to Surface Modified Biomaterials ........................................... 41
3.1. Unmet Need ............................................................................................................ 41
3.2. Proposed Solution ................................................................................................... 42
3.3. Components of the Design Relevant to Bone Tissue Regeneration ....................... 44
3.3.1. Osteoconductive Materials................................................................................... 44
3.3.2. Osteoinductive Stimuli......................................................................................... 45
3.3.3. CFU assay for CTPs ............................................................................................ 46
iv
3.4. Constraints of the design ......................................................................................... 47
3.4.1. Use of Beta-tricalcium phosphate as a biomaterial that is modified with tethered
EGF ................................................................................................................................ 47
3.4.2. Use of CTPs and osteogenic conditions to screen for pro-osteogenic biological
response.......................................................................................................................... 48
3.4.3. Use of Colonyze™, a standardized automated CFU assay to assess CTP response
........................................................................................................................................ 49
3.5. Technical Specifications of the Design................................................................... 50
3.5.1. Design of biomaterial surfaces............................................................................. 50
3.5.2. Surface density and stability of bioactive ligands................................................ 51
3.5.3. Primary and secondary outcomes of the CFU assay............................................ 51
3.5.4. CFU Analysis - Cell culture conditions, inclusion/ exclusion criteria and sample
size determination .......................................................................................................... 53
3.5.5. Criteria for success ............................................................................................... 54
3.6. Design and Verification .......................................................................................... 55
3.6.1. Assessment of existing TCP scaffolds ................................................................. 55
3.6.2. Verification .......................................................................................................... 57
3.6.3. Revised Design .................................................................................................... 57
3.6.3.1. Phase I: Design 1 .............................................................................................. 58
3.6.3.2. Design 2 ............................................................................................................ 59
3.6.3.3. Design 3 ............................................................................................................ 60
3.6.3.4. Verification ....................................................................................................... 61
3.6.4. Phase II - Optimization of Design 2 .................................................................... 62
3.6.5. Verification .......................................................................................................... 63
3.6.6. Colonyze™ algorithm .......................................................................................... 64
3.6.7. Verification of the binding and stability of tethered EGF ................................... 65
3.6.8. Quantification of the surface density of tethered EGF and stability analysis ...... 65
3.6.9. Verification .......................................................................................................... 66
3.7. Discussion ............................................................................................................... 66
3.8. Tables and Figures .................................................................................................. 70
3.9. Works Cited ............................................................................................................ 83
Chapter 4: Assessment of Human Connective Tissue Progenitor Cell Response to ΒTricalcium Phosphate Surfaces with Tethered Epidermal Growth Factor .................... 89
4.1. Abstract ................................................................................................................... 89
4.2. Introduction ............................................................................................................. 90
4.3. Methods................................................................................................................... 92
4.3.1. Fabrication of TCP surfaces ................................................................................ 93
4.3.2. Synthesis and purification of TCPBP-EGF protein ............................................. 95
4.3.3. Tethering TCPBP-EGF to TCP coverslips .......................................................... 96
4.3.4. Quantification of the amount of tethered EGF and stability analysis .................. 97
4.3.5. Isolation of cells from bone marrow and trabecular space .................................. 97
4.3.6. Staining, imaging and data analysis ..................................................................... 99
4.3.7. Data validation and analysis ................................................................................ 100
v
4.3.8. Statistical analysis ................................................................................................ 101
4.4. Results and Discussion ........................................................................................... 101
4.4.1. Characterization of 2D TCP coverslips ............................................................... 101
4.4.2. Quantification of the concentration and stability of tethered EGF on TCP
coverslips ....................................................................................................................... 102
4.4.3. Quantification of the stability of tethered EGF on TCP coverslips ..................... 102
4.4.4. CTP response to tethered TCPBP-EGF ............................................................... 102
4.4.4.1. Tethered EGF enhances CFE in bone marrow as well as trabecular surface ... 102
4.4.4.2. Tethered EGF does not influence the number of cells per colony or cell density
........................................................................................................................................ 104
4.4.4.3. Tethered EGF maintains osteogenic potential of CTPs .................................... 104
4.5. Discussion ............................................................................................................... 104
4.6. Conclusion .............................................................................................................. 106
4.7. Figures and Tables .................................................................................................. 107
4.8. Works Cited ............................................................................................................ 114
Chapter 5: Conclusions .................................................................................................. 121
5.1. The Need – Quantitative Assessment of the Effect of Scaffold Biomaterials and
Biomaterial surface Modifications on Relevant Cell Populations ................................. 121
5.2. Quantitative Assessment using Colonyze™ and Analytical Principles in the ASTM
Standard Method (F2944-12) ........................................................................................ 123
5.3. Qualitative Scoring System for Surface Preparation .............................................. 124
5.4. Characterization of Surface Preparation of TCP with and without Surface
Modification using TCPBP-EGF ................................................................................... 125
5.5. Works Cited ............................................................................................................ 126
Chapter 6: Future Directions .......................................................................................... 127
6.1. Assessment of CTP response to tethered EGF under normoxia, and the cellular stress
induced by hypoxia and pro-death cytokines................................................................. 128
6.2. Understanding the mechanism of CTP response to tethered EGF under the cellular
stress induced by hypoxia and pro-death cytokines ....................................................... 130
6.3. Assessment of the effect of tethered EGF on bone regeneration in vivo................ 133
6.4. Optimization of the surface-modification of TCP scaffolds ................................... 136
6.5. Assessment of CTP response to surface modifications of other bone graft materials
........................................................................................................................................ 138
6.6. Optimization of the assessment of the two-dimensional TCP surfaces ................. 138
6.6. Concluding Remarks ............................................................................................... 141
6.7. Works Cited ............................................................................................................ 141
vi
LIST OF TABLES
Chapter 2:
Table 1: Summary of common immobilization chemistries .......................................... 22
Table 2.Summary of common methods of growth factor tethering to biomaterial
substrates ........................................................................................................................ 23
Chapter 3:
Table 1: Primary and secondary outcomes of the method .............................................
vii
71
LIST OF FIGURES
Chapter 2:
Figure 1: General scheme for a growth factor immobilization to a substrate with Watersoluble carbodiimide ...................................................................................................... 24
Figure 2: General scheme for modifying a growth factor with a photo-reactive phenyl
azide group and subsequent immobilization on a biomaterial substrate........................ 24
Figure 3: General scheme for modifying a growth factor with a photo-reactive acrylate
group and subsequent immobilization on an acrylated biomaterial substrate ............... 25
Chapter 3:
Figure 1: The engineering design method ..................................................................... 70
Figure 2: Assessment of TheriLok™ TCP scaffolds. .................................................... 71
Figure 3: Imaging approach to quantify colony-parameters in TheriLok™ scaffolds. . 72
Figure 4: Initial proof of design of 2 D TCP coverslips ................................................ 73
Figure 5: Expected thickness of the PDMS layer as a function of spin coater speed .... 74
Figure 6: Optimization of the TCP surface design, effect of particle size and PDMS
thickness on the uniformity of TCP surfaces ................................................................. 75
Figure 7: Verification of nuclei segmentation on opaque TCP surfaces using a
Colonyze™ algorithm .................................................................................................... 81
Figure 8: Quantification of the surface density of tethered TCPBP-EGF ..................... 82
Chapter 4:
Figure 1: A schematic of presentation of tethered TCPBP-EGF on TCP surfaces. ......
Figure 2: Preparation and characterization of 2D TCP surfaces....................................
Figure 3: Quantification of the surface density of tethered TCPBP-EGF. ....................
Figure 4: Tethered EGF improves Colony Forming Efficiency (CFE) at Day 9...........
Figure 5: tethered EGF does not change number of cells per colony at Day 9 .............
Figure 6: Tethered EGF does not change relative colony density at Day 9. .................
Figure 7: tethered EGF does not change % AP expression in colony cells at Day 9 ....
viii
107
108
109
110
111
112
113
ACKNOWLEDGEMENTS
I am grateful to my dissertation advisor, Dr. George Muschler, for his guidance,
encouragement and support throughout my graduate studies. I have been fortunate to
learn research, philosophy, and the art of science from such an accomplished scientist.
His passion and dedication to science motivated me to become a better engineer,
researcher and thinker. Dr. Muschler has been a source of inspiration for me and he will
always be one of the most respected individuals in my life.
I would also like to thank my guidance committee members, Dr. Steve Eppell, Dr.
Cathy Carlin, and Dr. Horst von Recum, who have provided me with a great deal of
invaluable scientific advice and guidance, both at my committee meetings as well as in
many external discussions. I would like to offer a special thanks to Dr. Linda Griffith
from MIT, for her collaboration, scientific support, mentoring, and encouragement
throughout the research. I have had the chance to learn from brilliant minds and receive
the best guidance anyone could ask for.
I am also particularly indebted to Dr. Tom Patterson, Cynthia Boehm, and Linda
Stockdale, who provided me the guidance in the design of experiments, scientific writing,
project management and personal development. Tom’s mentorship in scientific writing
and scientific critique will always be cherished very highly. The wealth of experience,
finesse in the art of experimentation, and the calm influence of Cynthia and Linda was of
invaluable help to me; I was extremely lucky to work with such talented, hard-working,
and friendly individuals.
ix
I must also thank Linda, Jaime Rivera and Dr. Luis Alvarez, from the Griffith lab
at MIT for their scientific inputs, materials for my research, friendship, amazing
collaborative work, and some very interesting and stimulating scientific discussions. I
immensely enjoyed the time I spent with them at MIT, accomplishing some very exciting
experiments and training. Dr. Amit Vasanji and Richard Rozic from ImageIQ, Inc.
always helped me with their scientific expertise in image acquisition and processing. I
value their assistance in the timely completion of my work.
I also owe a debt of gratitude to the members of the Muschler group, both present
and past, who made Cleveland Clinic a great place to work; especially Tonya Caralla,
Sandra Villarruel, Chris Heylman, Ed Kwee, Viviane Luangphakdy, Kentaro Shinohara,
and Hui Pan with whom I have had so many wonderful discussions, both scientific and
otherwise. All together, we were a great team and enjoyed working together. I look
forward to seeing them excel in their respective careers, and collaborating with some of
them in future.
I graciously thank the BME departments at Case and Cleveland Clinic- the
faculty, staff, scientists, and students for their assistance and support.
I am forever thankful for my wonderful family back home in India, and all my
friends in Cleveland. My parents and my in-laws stood by me firmly in the best and worst
times of my graduate studies. My wife Puja was the best friend and companion I could
have asked for, and opened my world to true love and support outside work. I made some
great friends in Cleveland, who have become an extended family. This strong support
system kept me sane and provided some great moments to cherish forever. Thank you all.
x
METHODS TO QUANTITATIVELY ASSESS THE PERFORMANCE OF
CONNECTIVE TISSUE PROGENITOR CELLS IN RESPONSE TO SURFACE
MODIFIED BIOMATERIALS
BY
VIVEK P RAUT
ABSTRACT
The field of orthopaedic tissue engineering includes a broad range of treatment
options that seek to regenerate or accelerate the repair of bone tissue. Formation of new
bone by the activity of connective tissue progenitor cells (CTPs) is a central feature of
each of these treatment options. CTPs are defined as the heterogeneous population of
stem and progenitor cells that are resident in native tissues and are capable of
proliferation to give rise to progeny that will differentiate to express one or more
connective tissue phenotypes. The strategy of using a cell-based therapy to achieve bone
regeneration may include: targeting CTPs, and guiding their downstream progression
towards the osteogenic pathway using a combination of scaffolds, bioactive factors, and
mechanical and biophysical methods. Recent advancements in biomaterials science
include promising technologies for modifying biomaterial surfaces with pro-survival or
pro-osteogenic bioactive ligands. Before testing the pre-clinical efficacy of these surface
modified biomaterials in animal models, it is important to test and optimize the influence
of these materials on cells in an in vitro setting.
The purpose of this dissertation is to introduce and demonstrate a new method to
assess the response of CTPs to surface-modified biomaterials using a standardized
automated colony forming unit (CFU) assay. Individual components of the method are
xi
verified for their compliance with the technical specifications. The method is validated
using β-TriCalcium Phosphate (TCP), a bone scaffold material that was modified with
Epidermal Growth Factor (EGF), a pro-survival and proliferation bioactive ligand, using
a novel TCP-binding peptide. The amount of tethered EGF on the TCP surface was
quantified, and its stability was demonstrated. Surfaces modified with tethered EGF were
shown to enhance the colony forming efficiency of CTPs obtained from human bone
marrow and the trabecular surface of bone. These data support the potential of tethered
EGF presentation on the surface of implantable TCP biomaterials as a means of
enhancing the performance of local and transplanted CTPs in a setting of bone repair or
other tissue engineering applications.
xii
CHAPTER 1: PREFACE
The purpose of this dissertation is to introduce and demonstrate a new quantitative
method to assess the response of connective tissue progenitor (CTP) cells to surfacemodified biomaterials by applying an automated colony forming unit (CFU) assay that
has been recently adopted by the American Society for Testing and Materials (ASTM)
(ASTM F2944-12) [1] (referred here as “Colonyze™”). This new method uses
engineering design principles to combine the following components: (1) a novel method
to prepare two dimensional surfaces of a bone scaffold material, with or without surface
modification using a bioactive ligand; (2) a method to verify and optimize these surfaces
with respect to their repeatability, stability, uniformity in coverage, and smoothness; and
(3) a method to assess the material and surface modification effects on CTPs using
Colonyze™.
The combined method was validated using β-TriCalcium Phosphate (TCP), a
clinically used bone scaffold material with, and without surface modification with
Epidermal Growth Factor (EGF), a pro-survival and proliferation bioactive ligand, using
a novel TCP-binding peptide that has been developed by researchers at MIT [2]. The
unique contribution of this work is the development, verification, and validation of the
method; and the results demonstrating that the colony forming efficiency of CTPs is
enhanced in response to the surface modification of TCP with EGF.
This dissertation should be of interest to the scientific community performing
basic and translational research in the fields of tissue engineering, stem cell therapies, and
biomaterial science. The work reported in this dissertation was published, in part, in
reports and publications of the Armed Forces Institute of Regenerative Medicine
1
(AFIRM) [3], and in several national and international conference proceedings. This
research may lead to accelerating rational development of new biomaterials and their
surface modifications that improve the rate and reliability of regeneration of large
traumatic bone defects. This research is relevant to both injured warriors and to those
suffering traumatic injuries in the civilian population.
Chapter 2 of this dissertation introduces the underlying components of the
method, and principles of their use. These components include the use or targeting of
CTPs in bone tissue engineering applications; assessment of CTP performance using
Colonyze™ [1]; techniques of surface modification of biomaterials; interactions between
cells and biomaterials; and their role in the efficacy of the biomaterials in orthopaedic
tissue engineering applications. The purpose of this chapter is to provide an adequate
summary of these components and it is not intended to be a comprehensive review.
Chapter 3 introduces the design of a method to quantitatively assess the response
of CTPs to surface modified biomaterials using Colonyze™. Engineering design
principles were applied to design this method. The scope of the design was defined, and
constraints were identified based on the available knowledge and the unmet need. The
technical specifications were defined, forming the basis for verification of the compliance
of individual components.
A novel method to prepare two-dimensional surfaces of biomaterials was
developed. Three designs were tested. The design most effective in meeting technical
specifications was chosen, and optimized for uniformity in coverage and smoothness. The
feasibility of using Colonyze™ to quantify the CTP response was verified. The surface
2
density of tethered EGF and the stability of the presentation of tethered EGF on TCP
surfaces were verified.
Chapter 4 integrates these components, and describes the validation of this
method using a real-world example. TCP surfaces were modified with tethered EGF
using a novel TCP-binding peptide that has been developed in Dr. Linda Griffith’s
laboratory at MIT [2]. CTPs were harvested from bone marrow and the trabecular surface
of bone. The response of CTPs to TCP surfaces modified with tethered EGF was
quantified using Colonyze™. The results discussed in this chapter not only validated the
method, they also demonstrated that tethered EGF increases CTP colony forming
efficiency. These data and methods can be used to develop a combination product
consisting of TCP scaffolds modified with tethered EGF and bone marrow derived cells,
which could be transplanted into large orthopaedic defects. This strategy may lead to new
clinical methods that significantly improve the rate and reliability of regeneration of large
traumatic bone defects.
Chapter 5 provides the conclusions and the summary of the method discussed in
Chapters 3 and 4. This summary will help researchers apply this method to other types of
synthetic biomaterials, bioactive factors, and stem cells that can be used for orthopaedic
tissue engineering and other tissue engineering applications. It also provides guidance to
the proposed future studies in Chapter 6.
Chapter 6 proposes future studies to progress the work discussed in this
dissertation. These proposed studies will advance understanding of the effects of tethered
EGF in influencing the response of CTPs. Each study is introduced with a significance,
hypothesis and rationale, followed by the experimental design. The following proposed
3
studies are discussed: quantification of CTP response to tethered EGF under normoxia,
and cellular stress induced by hypoxia and inflammatory cytokines; characterizing the
mechanism of CTP response to tEGF under the cellular stress induced by hypoxia and
inflammatory cytokines; assessment of the effect of tethered EGF on bone regeneration;
improving the surface modification of TCP scaffolds; and assessment of CTP response to
surface modifications of other bone graft materials. These contributions enable
exploration of additional scientific questions that are significant for the field of
orthopaedic tissue engineering in general, and for the efficacy of surface modified TCP
materials in bone grafting in particular. This work is intended to advance the current
standard of orthopaedic trauma care.
Works Cited:
1.
ASTM International, W.C., PA, ASTM F2944 - 12 "Standard Test Method for
Automated Colony Forming Unit (CFU) Assays—Image Acquisition and Analysis
Method for Enumerating and Characterizing Cells and Colonies in Culture", 2012,
ASTM International, West Conshohocken, PA, 2003, astm.org.
2.
Griffith, L.G., Richard T. Lee, and Luis Manuel Alvarez., Modulating cell
behavior with engineered HER-receptor ligands, in Biological Engineering
2009,
Massachusetts Institute of Technology: Cambridge MA.
3.
Irgens, T., Armed Forces Institute of Regenerative Medicine Annual Report, in
Armed Forces Institute of Regenerative Medicine Annual Report 2009, 2010 and 2011,
ARMY MEDICAL RESEARCH AND MATERIEL COMMAND FORT DETRICK MD
ARMED FORCES INST OF REGENERATIVE MEDICINE.
4
CHAPTER 2: INTRODUCTION TO THE COMPONENTS OF THE METHOD
Current
orthopaedic
tissue-engineering
strategies
recognize
the
critical
importance of osteoprogenitor cells, biomaterials, and bioactive stimuli, among other
factors, as orthopaedic graft components. Generation of new bone is assisted by
transplanting osteogenic cells along with the scaffold material, particularly when large
defects are involved [1-12]. The local and transplanted cells can be targeted, and their
performance can be modified using a scaffold matrix that facilitates the distribution of
osteogenic cells through attachment and migration. In particular, it is desired to use a
biomaterial that is osteoconductive, i.e., graft material that facilitates the attachment and
migration of cells, and thereby the distribution of a bone healing response throughout the
grafted volume [13-15]. The performance of osteogenic cells may be further influenced
by osteoinductive stimuli, mass transport and revascularization of the defect, and by
controlling
the
mechanical
environment
[13-15].
Osteogenesis
occurs
when
osteoprogenitor cells respond to osteoconductive and/or inductive stimuli by
differentiating along the osteoblastic lineage, and contributing to new bone formation.
This chapter provides an overview of the principles of the use of connective tissue
osteo-progenitor (CTP) cells in bone tissue engineering, and methods to assess CTP
performance using an automated assay. In addition, principles of surface modification of
biomaterials are introduced, and the interactions between cells and biomaterials are
discussed, including how these interactions play a critical role in the efficacy of the
biomaterials in orthopaedic tissue engineering applications. This introduction forms a
foundation for Chapters 3 and 4, which elaborate a novel method to assess CTP response
5
to surface modified biomaterials designed for bone tissue engineering applications in
vitro.
2.1.
Role of Connective Tissue Progenitor Cells (CTPs) in Bone Repair: Stem cells
and progenitor cells are present in all adult tissues and are critical to tissue health,
maintenance, and response to injury or disease throughout life. Stem cells are the source
of all new tissues arising from repair and remodeling, and are modulated by chemical and
physical signals that control their activation, proliferation, migration, differentiation, and
survival. Stem cells give rise to progenitor cells and are distinguished from progenitor
cells by their capacity for self-renewal, or self regeneration by asymmetric cell division
[2]. In contrast, progenitor cells (also called transit cells) proliferate and expand in
number. Progenitor cells have a limited capacity for self-renewal and are committed to
progress toward a more differentiated phenotype. In bone, stem cells give rise to
progenitor cells, which advance to become pre-osteoblasts and then osteoblasts.
Various stem and progenitor cells from mammalian postnatal bone marrow have
been extensively used in tissue engineering applications because of their potential to
differentiate into specialized connective tissue cells, including bone, cartilage, muscle,
fat, tendon, ligament, fibrous tissue, perivascular adventitia, as well as blood cells [12,
16-20]. This has important implications with regard to the design of tissue engineering
strategies, in that cells derived from one tissue might be useful in forming a different
tissue.
During skeletal development and repair, bone formation can occur via the
processes of endochondral or intramembranous ossification. Endochondral ossification
6
occurs primarily in long bones while in the flat bones of the skull and also the mandible,
bone formation occurs via the process of intramembranous ossification.
During
endochondral ossification, undifferentiated mesenchymal stem cells condense at the site
of future bone formation. Cells within the condensation center differentiate into
chondrocytes to produce cartilage while those at the periphery differentiate into fibroblast
like perichondrial cells to produce perichondrium. During this process, cartilage becomes
mineralized as the chondrocytes become hypertrophic, followed by vascular invasion of
the mineralized cartilage. Subsequently, chondroclasts degrade the mineralized cartilage
allowing osteoblast migration and osteoid deposition onto the cartilaginous matrix with
replacement of the cartilage precursor with mineralized bone [21, 22].
In contrast to endochondral ossification, no cartilage precursor is formed during
intramembranous ossification. Cell differentiation occurs within a membranous,
condensed plate of mesenchymal cells present in fibrous connective tissue with
differentiation of these cells directly into those of an osteoblastic lineage. These cells
continue to differentiate and proliferate into osteoblasts that deposit bone matrix resulting
in the formation of woven bone [21, 23].
The number of stem and progenitor cells in various tissues can be assayed in vitro
by harvesting cells from the tissues and growing them in tissue culture under conditions
that promote their activation, proliferation and phenotypic differentiation. The number of
stem cells and progenitors can be estimated on the basis of the number of colony-forming
units [3, 24]. The underlying assumption of this approach is that one progenitor cell leads
to the formation of one colony containing the progeny of that progenitor cell.
7
2.1.1. Definition of CTPs: CTPs denote a combined heterogeneous population of stem
cells and progenitor cells that is capable of both proliferation and differentiation into one
or more connective tissue phenotypes. The term CTP was introduced to enable
exploration of the concentration, prevalence, diversity, and spectrum of biological
potential among cells that are natively present in different tissues, and the changes in
these parameters that can be related to aging, gender, disease and responses to chemical
conditions (e.g. hypoxia) or pharmaceuticals (e.g. steroids and nicotine) [1, 2, 24, 25]. It
is important to recognize that a CTP is a tissue resident cell that is assayed in vitro and
detected by the fact that it gives rise to a colony of progeny. However, only the colony
founding cell is a CTP. The true number of CTPs in various tissues is most often
estimated with use of the Colony-Forming Unit (CFU) assays, that is, cells that give rise
to a colony of proliferating progenitor cells in vitro under conditions that are selected to
promote activation and proliferation of one or more fractions of the connective tissue
progenitor population. In these assays, each colony represents the progeny of a founding
stem or progenitor cell. Functional biological differences between the colony-founding
cells are revealed by differences in the proliferation rate, the morphology, the migration,
and the differentiation of their progeny in each colony.
The cells that make up a proliferating colony are the progeny of the founding
CTP, but they change rapidly under in vitro conditions and quickly adopt a phenotype
that is distinctly different than the tissue resident CTP that gave rise to the colony. It is
also important to recognize that even bone forming CTPs (CTPs capable of giving rise to
progeny that can differentiate into bone –which we identify by the nomenclature CTP-O)
in a given tissue are a heterogeneous population. This is evidenced by the fact that the
8
progeny of some CTP-Os divide rapidly and others divide slowly, and that some colonies
express bone markers quickly in vitro (often without osteogenic stimuli) while others
require exogenous osteogenic stimuli. Thus, it should be evident that there is no one
phenotypic marker or set of markers that defines a CTP, because CTPs include many
types of progenitors. However, it is possible to characterize the population of CTPs from
a given tissue by applying methods that assay their concentration, prevalence, and
variation in biological potential.
2.1.2. Role of CTPs in Bone Regeneration: Bone repair requires the activity of
osteoblasts, which are derived from cells of the mesenchymal lineage, such as CTP-Os.
Since bone defects are, by definition, deficient in CTP-Os, their treatment cannot be
optimized without CTP-O transplantation from another source, such as bone marrow.
Bone marrow has been widely used clinically as a source of autogenous CTP-Os. CTPOs can be harvested from a bone marrow aspirate with very low morbidity and minimal
pre-processing compared to other sources of CTP-Os such as fat and muscle. The
established clinical procedures to increase CTP-O concentration and/or reduce number of
competing non-CTP-O cell population from bone marrow are relatively uncomplicated.
These procedures involve centrifugation of the marrow to remove majority of red blood
cells and plasma, and concentrate the CTPs, white blood cells, platelets and other
nucleated cells. The CTP population increases 4-6 fold using this approach [26].
Alternatively, bone scaffold materials can be used to selectively retain the CTPs on the
scaffold surface, by transforming the bone marrow through a column of scaffold material
at a low speed, which enables retention of 40-70 % CTPs on certain scaffolds [27]. Bone
9
marrow derived CTP-Os can be transplanted to the defect site in a single intra-operative
procedure, and is a more viable alternative than transplantation of culture expanded
population such as MSCs, whose therapeutic usage requires at least two operative
procedures (cell harvest and implantation), and a complex laboratory treatment that is
time intensive and expensive. The strategy of CTP-O transplantation has led to improved
bone formation compared to scaffolds without CTP-Os [7-9, 28-34].
2.1.3. Difference between CTPs and MSCs: CTPs differ from in vitro culture
expanded cells, such as bone marrow stromal cells (BMSCs), mesenchymal stem cells
(MSCs), and adult multipotential progenitor cells (MAPCs), which are more purified and
homogeneous in nature. Mesenchymal stem cells (MSC) are defined as a population of
“purified, homogeneous culture expanded cells that retain the capacity for differentiation
in to multiple mesenchymal lineages, where mesenchymal lineages include bone,
cartilage, fat, muscle, tendon and bone marrow stroma” [19, 35]. A rigorous protocol for
demonstrating multilineage potential was defined and used in the pioneering work of Dr.
Arnold Caplan [19, 35]. This work demonstrated that not all batches of culture expanded
cells would satisfy the full set of requirements to call them MSCs. Further, culture
expansion favors cells that divide quickly. Therefore, the process of producing a batch of
MSCs removes from consideration stem cells that grow slowly. Analysis of CTPs, which
by definition does not include culture expansion, allows for information to be obtained
from all of the potential founding cells, including those that grow slowly.
By definition, to be counted as a CTP, a bone marrow derived cell needs to be
capable of giving rise to progeny that can form one of the connective tissue phenotypes.
10
In contrast, to be counted as an MSC a cell must have the capacity to differentiate into
all “mesenchymal phenotypes (bone, cartilage, tendon, muscle, fat, marrow stroma).
However, the heterogeneity presented by CTPs more closely mimics the multifunctional
nature of cells within the bone marrow environment and this heterogeneity can be
viewed as a source of valuable information that can be dissected experimentally to
understand the prevalence and kinetics of various connective tissue stem-cell
populations associated with tissue engineering strategies.
2.1.4. The Colony Forming Units: Friedenstein et al. (1970) demonstrated that bone
marrow cells explanted and grown in monolayer cultures form colonies [36]. The number
of CFUs formed was a linear, increasing function of the number of plated explanted cells.
Both time lapse photography and mixed cultures of male/female cells indicated that each
colony was derived from a single progenitor cell. Additionally, colonies derived from
different sources exhibited different morphologies [37-39]. Bone marrow cultures
contained colonies with dense centers at day ten, while pleural and peritoneal cultures
contained looser and smaller colonies [40, 41].
In vivo and in vitro studies demonstrated the pluripotent nature of the CFU
progeny [42]. Passaged progeny of bone marrow derived CFUs placed in diffusion
chambers formed osteogenic tissue [41]. In vitro colony assays methods applied to
hematopoietic progenitors have been used to demonstrate the heterogeneity of the
progenitor population and the capacity for osteogenic differentiation [37, 39, 43, 44].
Individual colonies express alkaline phosphatase (an early osteogenic marker) and vary
widely in size, morphology and level of alkaline phosphatase activity.
11
2.2. The Colony Forming Unit Assay: Cells and tissues commonly used in tissue
engineering and cellular therapy are routinely assayed and analyzed to define the number,
prevalence, biological features and biological potential of the original stem cell and
progenitor population(s). The number of stem cells and progenitor cells in various tissues
can be assayed in vitro by harvesting cells from the tissues using methods that preserve
their viability and biological potential, and culturing in vitro that leads to activation and
proliferation of stem and progenitor cells into the formation of colonies. A colony is
defined as a cluster of a predetermined number of cells present within predefined
dimensional parameters. The true number of stem cells and progenitors in a population
can be estimated on the basis of the number of colony-forming units (CFUs) observed.
The prevalence of stem cells and progenitors can be estimated on the basis of the number
of observed CFUs detected, divided by the number of total cells assayed.
2.2.1. Limitations of Manual Observer-Dependent Assay: Current standards utilize
user input for defining the presence and location of colonies based on visualization of an
entire culture surface at low magnification using a microscope. The cell culture surface
sample may be viewed in transmission light mode (unstained or with a histochemical
marker) or fluorescently with a dye or antibody. For this method, colony count is the
only measurable output parameter. The manual quantification of cell and CFU cultures
based on an observer-dependent judgment is an extremely tedious and time-consuming
task, and can be significantly impacted by user bias. In addition to being highly time and
labor intensive and subjective, manual enumeration has been shown to have a significant
12
degree of intra- and inter-observer variability, with coefficients of variation (CV) ranging
from 8.1% to 40.0% and 22.7% to 80%, respectively [45]. When multiple observers are
employed, observer fatigue can be reduced, but the accuracy and reproducibility of cell
and colony identification can be significantly compromised due to significant intra- and
inter-observer variability. Standard CVs for cell viability assessment and progenitor
(colony) type enumeration range from 19.4% to 42.9% and 46.6% to 100%, respectively
[45]. In contrast, studies focusing on bacteria, bone marrow-derived stem cells and
osteogenic progenitor cells have collectively concluded that automated enumeration
provided significantly greater accuracy, precision, and/or speed for counting and sizing
cells and colonies, relative to conventional manual methodologies. Automated methods
for enumerating cells and colonies are less biased, less time consuming, less laborious,
and provide greater qualitative and quantitative data for intrinsic characteristics of cell
and colony type and morphology.
2.2.2. ASTM F2944-12-Automated CFU Assay: A fully automated, accurate, and
standardized method for characterizing the biological potential and functional potency of
the cultured cells can be achieved by utilizing an automated, microscope-based imaging
system that is able to stitch together sequentially scanned, high resolution images to form
a single mosaic image of the entire culture surface, and subsequently employing image
processing algorithms to identify cells and assign them to individual colonies. In addition
to the calculation of prevalence, extracted parameters include colony level statistics such
as cell/nuclear density and count, colony morphology (shape and size parameters),
secondary marker coverage and effective proliferation rates.
13
Colonyze™ is an integrated software suite for image acquisition and cell and
colony analysis. This method utilizes standardized protocols for image capture of cells
and colonies derived from in vitro processing of a defined population of starting cells in a
defined field of view (FOV), and standardized protocols for image processing and
analysis. The primary outcome measure is the number of colonies present in the FOV. In
addition, the characteristics and sub-classification of individual colonies and cells within
the FOV also provides useful information, based on extant morphological features,
distributional properties, or properties elicited using secondary markers (e.g., staining or
labeling methods). The procedure, imaging-set up, hardware and software requirements,
image acquisition and processing methods and available options, data acquisition and
analysis methods, and limitations are discussed in the recently published ASTM Standard
Method, (ASTM F2944-12) [46]: “Standard Test Method for Automated Colony Forming
Unit (CFU) Assays- Image Acquisition and Analysis Method for Enumerating and
Characterizing Cells and Colonies in Culture”.
2.3.
Surface Modification of Biomaterials: Recently developed biomaterials are
designed to be not only biocompatible, and/or osteoconductive, but also to promote tissue
specific behaviors that can assist regeneration of functional target tissue. These materials
can broadly be categorized as bioactive biomaterials. The surface properties of the
biomaterial are primarily responsible for dictating cell behavior in response to and
interactions with the biomaterial. Modification of biomaterial surfaces with bioactive
ligands such as cell adhesion molecules, bioactive factors that promote cell survival,
proliferation, migration or differentiation can be used to induce selective cell interaction
14
with the specific adhesion and bioactive factor receptors expressed by autologous or
exogenous cells at the defect site.
Ligands used for surface modified biomaterials include adhesion molecules such
as RGD peptides, growth factors such as EGF, PDGF, FGF and BMPs. However, growth
factors in the physiological environment can be rapidly degraded or rendered inactive
prior to reaching their target, and biomaterial systems designed to deliver soluble growth
factors to sites of tissue repair are therefore often ineffective and costly. Large amounts
of soluble growth factors are frequently needed to effect cellular outcomes, although the
delivery of such large quantities has the potential to damage cells and tissues.
Immobilization of growth factors to biomaterial substrates has emerged as a
method for improving the stability and persistence of growth factor delivered to cells.
These goals cannot be achieved via simple growth factor adsorption, which does not
allow control over delivery, retention, orientation, or desorption rate [47, 48].
Immobilization strategies can prolong growth factor availability, allow spatial control,
and reduce the amount of growth factor required, thereby potentially reducing the cost
and increasing the efficacy of various bioactive ligands.
The process of covalently or preferentially adhering these molecules to the
biomaterial using a high affinity binding is referred to as tethering. Tethering ensures that
the signaling molecule does not diffuse away from the cells rapidly, and enables control
over the concentration and 3 dimensional conformation of the molecule that preserves its
ability to interact with receptors. The presentation of growth factors in an immobilized
form also has physiological relevance, as both soluble and matrix-bound growth factors
perform distinct functions in the in vivo environment [49, 50].
15
There are several approaches to modify the biomaterial surface with tethered
growth factors. Table 1 summarizes conjugation techniques to tether growth factors to
biomaterials [51]. Figures 1-3 depict the generalized mechanisms of tethering growth
factor to a substrate [51]. The choice of tethering approach depends upon the chemistry
of the substrate and the reactive groups on the growth factors, structure, and the location
of reactive groups relative to the receptor-binding area of the growth factor structure.
Several studies have used these conjugation chemistries to tether a variety of growth
factors in bioactive form to biomaterial substrates, and are summarized in Table 2.
2.3.1. Mechanism of Cell-Surface Interaction: When a biomaterial comes into the
contact with the human body, the proteins, lipids, saccharides and other extra-cellular
molecules present in physiological fluid get adsorbed on the biomaterial surface [52-54].
The adsorption of extracellular molecule occurs through complex and poorly understood
mechanisms. Cells arrive on the implant surface at later times. Therefore, cellbiomaterial interaction is mediated via the adsorbed protein layer [54, 55]. Biomaterials
surface properties influence cell response in terms of the kinetics and thermodynamics of
the protein adsorption. The nature of the conditioning biomolecules and their orientation
on the surface will have direct consequences on the protein adsorption and thereby the
recruitment, attachment, proliferation and differentiation of cells [54-66]. Further, the
efficacy of a biomaterial depends on the ability of the biomaterial to provide
environmental cues to the recruitment and stability of multiple cell phenotypes in culture,
proliferative and/or differentiative signals, and cell migration.
16
2.3.2. Mechanism of Adhesion: The extracellular matrix of bone is composed of 90%
collagen (type I collagen 97% and type V collagen 3%) and of 10% other proteins
(osteocalcin 20%, osteonectin 20%, bone sialoproteins 12%, proteoglycans 10%,
osteopontin, fibronectin, growth factors, bone morphogenetic proteins, etc.) [67]. All
these proteins are synthesized by osteoblasts and most are involved in adhesion [67]. In
vitro, other proteins such as fibronectin or vitronectin have been shown to be involved in
osteoblast adhesion. Some of the bone proteins, including collagen and fibronectin, have
chemotactic or adhesive properties, notably because they contain an Arg–Gly–Asp
(RGD) sequence which is specific to the fixation of cell membrane receptors like
integrins [60, 68-72].
The sites of adhesion between tissue cultured cells and substrate surfaces are
called focal contacts or adhesion plaques [73-75]. Focal contacts are closed junctions
where the distance between the substrate surface and the cell membrane is between 10–
15 nm. The focal contacts include specialized microstructures anchored within the cell to
cytoskeletal microfilaments which underlie the cell membrane. The external faces of
focal contacts present specific cell receptor proteins that facilitate cell adhesion. There
are more than 20 distinct cell-matrix receptors in the integrin family alone [76, 77]. The
type of focal adhesion and its geometry can influence the shape the cell assumes,
ultimately influencing phenotypic expression.
2.3.3. Mechanism of Migration: Cell migration is facilitated by an interaction between
the cell, its substrate and its cytoskeleton. Firstly, cells develop a projection of their
leading edge to form a lamellipodium. Cells subsequently use adhesive interactions (via
17
integrins) to generate the traction and energies required for cell movement. The last step
of the migratory cycle is the release of adhesions at the lagging end followed by cell
detachment and retraction [78, 79]. In general, cells with a low motility form strong focal
adhesions while motile cells form less adhesive structures [78, 79], and an intermediate
level of attachment force induces a maximal migration rate.
2.3.4. Mechanism of Osteoblastic Differentiation: After the attachment phase,
extracellular collagenous and non-collagenous matrix osteoblastic proteins (e.g. alkaline
phosphatase, thrombospondin, osteopontin, osteocalcin, osteonectin, type I collagen and
bone sialoprotein) are synthesized and released by cells. These proteins regulate the fate
of cells that produce them (autocrine regulation) or of other cells in their immediate
environment (paracrine regulation). A subset of these factors are called as morphogens;
which are the factors that have a capability of initiating a cascade of events leading to
differentiation of a progenitor cell into a committed end line cell, like an osteoblast or a
chondrocyte. Members of the Transforming growth factor beta superfamily play a special
role in the morphogenesis of bone and cartilage. A member of the family, the bone
morphogenetic proteins (BMPs), is particularly important in inducing Osteogenesis [8082]. When BMPs are implanted in a non-osseous mesenchymal tissue, they may induce
chondrogenesis [83-87]. If this cartilage is able to fully differentiate along the
endochondral lineage, it becomes calcified and supports subsequent vascularization and
bone formation. The type of protein and their amounts depend on the cell type and nature
of the surface.
18
2.3.5. Influence of Surface properties on Cell-Biomaterial Interactions: Surfaces are
different from the corresponding bulk of the material. They contain unsaturated bonds
which lead to the formation of surface reactive layers and adsorbed contamination layers.
Preparation technique effects such as the type of material (metal, ceramics, and
polymers), type of chemicals used for preparation, type of sterilization techniques, even
though plays a key role in determining the bulk properties, the surface topography,
chemistry or surface energy predominantly govern the surface properties. As previously
discussed, cells in contact with a surface will firstly adhere and migrate. This first phase
depends on previously described adhesion proteins. Thereafter, the quality of this
adhesion and m influences their morphology, capacity for proliferation and
differentiation.
2.3.5.1. Effect of Surface Morphology and Roughness on Cell Behavior: Cells are
also sensitive to the surface roughness and the fluctuations in surface structure.
Variations in surface texture, or microtopography, can also affect the cellular response to
an implant [88]. In case of osteoblasts, a higher percentage of cells adhere to the rougher
surface [88]. In contrast, fibroblasts prefer smoother surfaces, and epithelial cells attach
only to the smoothest surfaces [88]. The overall roughness of the surface influences the
nature of the host response. In vivo, the surface characteristics of an implant, particularly
roughness, may control tissue healing and therefore subsequent implant success [62, 89,
90]. Logically, the distribution of focal contacts dominates the mode of adhesion of
osteoblasts on the different roughnesses. On smooth surfaces, focal contacts are
distributed uniformly on the entire membrane surface which was in contact with the
19
surface. On rough surfaces, focal contacts are visible only at the extremities of cell
extensions where cell membranes were in contact with the substrate [91-93].
2.3.5.2. Effect of Surface Energy and Chemistry on Cell Behavior: The hydrophilic
and hydrophobic characteristics and the net positive or negative charge on the surface are
also of great importance for cell response. Cell adhesion is generally better on
hydrophilic surfaces [94-96]. Cell migration can be altered using the degree of
hydrophilicity of the surface. It has been shown that human skin fibroblast migration
increased with increasing hydrophilicity of the surface [97, 98]. The energy at the surface
of a biomaterial is defined by its general charge density and the net polarity of the charge.
A surface with a net positive or negative charge may be hydrophilic in character, whereas
a surface with a neutral charge may be more hydrophobic. The net effect of the surface
charge is to create a local environment with a specific surface tension, surface free energy
and energy of adhesion [54, 55]. The net surface charge also plays a key role in cell
attachment and spreading. This may be conceptualized as the interfacial free energy. As
the initial events that led to protein adsorption are altered with time, a series of interfaces
is established, each with an independent interfacial free energy. Differences in chemistry
of the functional groups of a surface also influence the cell attachment and proliferation,
although the exact mechanism is not clearly established. Presentation of ions or specific
molecules (e,g, poly-ethylene glycol) that alter the hydrophobicity of the surface can
change the orientation of binding proteins, and ultimately, the binding of cells to the
material [99, 100].
20
2.3.5.3. Effect of Scaffold Osmolarity and pH on Cell Behavior: A scaffold must
provide and maintain an environment with physiological pH (7.3 to 7.4) and osmolarity
(280 to 310 mOsmol/Litre). A deviation of pH and osmolarity from physiological
conditions may lead to cell injury [101-104]. For most scaffolds, simple hydration with
normal saline solution prior to exposing them to cell can avoid cell injury. However,
some matrices do not allow an isotonic condition to be created for cell delivery. Many
bone matrix materials that are prepared with use of solutions containing high
concentrations of low-molecular weight materials to improve handling (e.g., glycerol)
and materials that dissolve rapidly in water, releasing hyper-osmolar concentrations of
local ions (e.g., calcium sulfate) present this risk. If such materials are mixed with cells,
they can induce osmotic injury, reducing or precluding cell viability.
21
22
Reactive GF group(s) needed
Amine or carboxyl
Amine
Amine
heterobifunctional crosslinker with
an amine-reactive NHS group and a
sulfhydryl-reactive maleimide
Immobilization molecule/strategy
Water-soluble carbodiimide (e.g., EDC)
Succinimidyl ester-phenyl azide (e.g., sulfoSANPAH)
Monoacrylated PEG-succinimidyl ester
Sulfosuccinimidyl-4-(Nmaleimidomethyl)cyclohexane-1carboxylate (sulfo-SMCC)
Acrylate
Double bonds or C–H bonds, or
N–H bonds, or Nucleophiles (e.g.,
amines)
Amine or carboxyl
Reactive substrate group(s)
needed
Table 1. Summary of common immobilization chemistries.
Heterobifunctional PEG
UV light
UV light or free radical
polymerization
Coupling reaction
Immobilization mechanism
2.4.
Figures and Tables:
Immobilization molecule/strategy
Water-soluble carbodiimide (e.g.,
EDC)
Succinimidyl ester-phenyl azide
Growth
Factor
VEGF
Substrate
Reference
Collagen
[105-107]
EGF
PCL
[108, 109]
BMP-2
Collagen
[110]
VEGF
SEM
[111]
EGF
Glass
[112, 113]
EGF
Polystyrene
[114-116]
RGD
VEGF
Monoacrylated PEG-succinimidyl
ester
EGF
PDGF-BB
FGF-2
TGF-beta
Sulfo-SMCC
VEGF
VEGF
PDGF-BB
BMP-2
BMP-2
PEG-based
polymers
PEG-based
polymers
PEG-based
polymers
PEG-based
polymers
PEG-based
polymers
PEG-based
polymers
Agarose
Collagen
DBM
PCL
Chitosan
[117, 118]
[119, 120]
[121-126]
[127]
[128]
[129]
[130]
[131]
[132]
[133]
[134]
Table 2. Summary of common methods of growth factor tethering to biomaterial
substrates
23
Figure 1: General scheme for a growth factor immobilization to a substrate with Watersoluble carbodiimide
Figure 2: General scheme for modifying a growth factor with a photo-reactive phenyl
azide group and subsequent immobilization on a biomaterial substrate
24
Figure 3: General scheme for modifying a growth factor with a photo-reactive acrylate
group and subsequent immobilization on an acrylated biomaterial substrate.
2.5. Works Cited:
1.
Muschler, G.F. Human Connective Tissue Stem Cells and Progenitors - A Step
Towards Clinical Practice. in Stem Cells and Orthopaedics Educational
Workshop. Annual Meeting of the Orthopaedic Research Society. 2002. Dallas,
TX.
2.
Muschler, G.F. and R.J. Midura, Connective tissue progenitors: practical
concepts for clinical applications. Clin Orthop Relat Res, 2002(395): p. 66-80.
3.
Muschler, G.F., C. Nakamoto, and L.G. Griffith, Engineering principles of
clinical cell-based tissue engineering. J Bone Joint Surg Am, 2004. 86-A(7): p.
1541-58.
25
4.
Al-Salihi, K.A., Tissue-engineered bone via seeding bone marrow stem cell
derived osteoblasts into coral: a rat model. Med J Malaysia, 2004. 59 Suppl B: p.
200-1.
5.
Arinzeh, T.L., et al., A comparative study of biphasic calcium phosphate ceramics
for human mesenchymal stem-cell-induced bone formation. Biomaterials, 2005.
26(17): p. 3631-8.
6.
Boden, S.D., et al., The use of coralline hydroxyapatite with bone marrow,
autogenous bone graft, or osteoinductive bone protein extract for posterolateral
lumbar spine fusion. Spine, 1999. 24(4): p. 320-7.
7.
Brodke, D., et al., Bone grafts prepared with selective cell retention technology
heal canine segmental defects as effectively as autograft. J Orthop Res, 2006.
24(5): p. 857-66.
8.
Bruder, S.P., et al., The effect of implants loaded with autologous mesenchymal
stem cells on the healing of canine segmental bone defects. J Bone Joint Surg Am,
1998. 80(7): p. 985-96.
9.
Cancedda, R., P. Giannoni, and M. Mastrogiacomo, A tissue engineering
approach to bone repair in large animal models and in clinical practice.
Biomaterials, 2007. 28(29): p. 4240-50.
10.
Desai, B.M., Osteobiologics. Am J Orthop, 2007. 36(4 Suppl): p. 8-11.
11.
Fortier, L.A., et al., Concentrated bone marrow aspirate improves full-thickness
cartilage repair compared with microfracture in the equine model. J Bone Joint
Surg Am. 92(10): p. 1927-37.
26
12.
Gulotta, L.V., et al., Application of bone marrow-derived mesenchymal stem cells
in a rotator cuff repair model. Am J Sports Med, 2009. 37(11): p. 2126-33.
13.
Shahgoli, S. and M.H. Levine, Introduction and overview of bone grafting. N Y
State Dent J, 2011. 77(2): p. 30-2.
14.
Schroeder, J.E. and R. Mosheiff, Tissue engineering approaches for bone repair:
concepts and evidence. Injury, 2011. 42(6): p. 609-13.
15.
Albrektsson, T. and C. Johansson, Osteoinduction, osteoconduction and
osseointegration. Eur Spine J, 2001. 10 Suppl 2: p. S96-101.
16.
Chen, G.Q. and Q. Wu, The application of polyhydroxyalkanoates as tissue
engineering materials. Biomaterials, 2005. 26(33): p. 6565-78.
17.
Hui, J.H., et al., Mesenchymal stem cells in musculoskeletal tissue engineering: a
review of recent advances in National University of Singapore. Ann Acad Med
Singapore, 2005. 34(2): p. 206-12.
18.
Bruder, S.P., D.J. Fink, and A.I. Caplan, Mesenchymal stem cells in bone
development, bone repair, and skeletal regeneration therapy. J Cell Biochem,
1994. 56(3): p. 283-94.
19.
Caplan, A.I., Mesenchymal stem cells. J Orthop Res, 1991. 9(5): p. 641-50.
20.
Caplan, A.I., Adult mesenchymal stem cells for tissue engineering versus
regenerative medicine. J Cell Physiol, 2007. 213(2): p. 341-7.
21.
Chung, U.I., et al., Distinct osteogenic mechanisms of bones of distinct origins. J
Orthop Sci, 2004. 9(4): p. 410-4.
22.
Colnot, C., Cellular and molecular interactions regulating skeletogenesis. J Cell
Biochem, 2005. 95(4): p. 688-97.
27
23.
Kronenberg, H.M., Developmental regulation of the growth plate. Nature, 2003.
423(6937): p. 332-6.
24.
Muschler, G.F., R.J. Midura, and C. Nakamoto, Practical Modeling Concepts for
Connective Tissue Stem Cell and Progenitor Compartment Kinetics. J Biomed
Biotechnol, 2003. 2003(3): p. 170-193.
25.
Bidula, J., et al., Osteogenic progenitors in bone marrow aspirates from smokers
and nonsmokers. Clin Orthop Relat Res, 2006. 442: p. 252-9.
26.
Castillo, T.N., et al., Comparison of growth factor and platelet concentration
from commercial platelet-rich plasma separation systems. Am J Sports Med,
2011. 39(2): p. 266-71.
27.
Muschler, G.F., et al., Selective retention of bone marrow-derived cells to
enhance spinal fusion. Clin Orthop Relat Res, 2005(432): p. 242-51.
28.
Hernigou, P., et al., Percutaneous autologous bone-marrow grafting for
nonunions. Influence of the number and concentration of progenitor cells. J Bone
Joint Surg Am, 2005. 87(7): p. 1430-7.
29.
Homma, Y., G. Zimmermann, and P. Hernigou, Cellular therapies for the
treatment of non-union: The past, present and future. Injury, 2013. 44 Suppl 1: p.
S46-9.
30.
Jager, M., et al., Cell therapy in bone healing disorders. Orthop Rev (Pavia),
2010. 2(2): p. e20.
31.
Hernigou, P., et al., Cell therapy of hip osteonecrosis with autologous bone
marrow grafting. Indian J Orthop, 2009. 43(1): p. 40-5.
28
32.
Hernigou, P., et al., Percutaneous implantation of autologous bone marrow
osteoprogenitor cells as treatment of bone avascular necrosis related to sickle
cell disease. Open Orthop J, 2008. 2: p. 62-5.
33.
Hernigou, P., et al., The use of percutaneous autologous bone marrow
transplantation in nonunion and avascular necrosis of bone. J Bone Joint Surg
Br, 2005. 87(7): p. 896-902.
34.
Hernigou, P. and F. Beaujean, Treatment of osteonecrosis with autologous bone
marrow grafting. Clin Orthop Relat Res, 2002(405): p. 14-23.
35.
Caplan, A.I., Review: mesenchymal stem cells: cell-based reconstructive therapy
in orthopedics. Tissue Eng, 2005. 11(7-8): p. 1198-211.
36.
Friedenstein, A.J., R.K. Chailakhjan, and K.S. Lalykina, The development of
fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen
cells. Cell Tissue Kinet, 1970. 3(4): p. 393-403.
37.
Penfornis, P. and R. Pochampally, Isolation and expansion of mesenchymal stem
cells/multipotential stromal cells from human bone marrow. Methods Mol Biol,
2011. 698: p. 11-21.
38.
Bourin, P., et al., Mesenchymal Progenitor Cells: Tissue Origin, Isolation and
Culture. Transfus Med Hemother, 2008. 35(3): p. 160-167.
39.
Owen, M. and A.J. Friedenstein, Stromal stem cells: marrow-derived osteogenic
precursors. Ciba Found Symp, 1988. 136: p. 42-60.
40.
Friedenstein, A.J., et al., Precursors for fibroblasts in different populations of
hematopoietic cells as detected by the in vitro colony assay method. Exp Hematol,
1974. 2(2): p. 83-92.
29
41.
Friedenstein, A.J., R.K. Chailakhyan, and U.V. Gerasimov, Bone marrow
osteogenic stem cells: in vitro cultivation and transplantation in diffusion
chambers. Cell Tissue Kinet, 1987. 20(3): p. 263-72.
42.
Ashton, B.A., et al., Formation of bone and cartilage by marrow stromal cells in
diffusion chambers in vivo. Clin Orthop Relat Res, 1980(151): p. 294-307.
43.
Kuznetsov, S.A., A.J. Friedenstein, and P.G. Robey, Factors required for bone
marrow stromal fibroblast colony formation in vitro. Br J Haematol, 1997. 97(3):
p. 561-70.
44.
Luria, E.A., A.F. Panasyuk, and A.Y. Friedenstein, Fibroblast colony formation
from monolayer cultures of blood cells. Transfusion, 1971. 11(6): p. 345-9.
45.
Powell, K.A., et al., Quantitative image analysis of connective tissue progenitors.
Anal Quant Cytol Histol, 2007. 29(2): p. 112-21.
46.
ASTM International, W.C., PA, ASTM F2944 - 12 "Standard Test Method for
Automated Colony Forming Unit (CFU) Assays—Image Acquisition and Analysis
Method for Enumerating and Characterizing Cells and Colonies in Culture",
2012, ASTM International, West Conshohocken, PA, 2003, astm.org.
47.
Sharon, J.L. and D.A. Puleo, Immobilization of glycoproteins, such as VEGF, on
biodegradable substrates. Acta Biomater, 2008. 4(4): p. 1016-23.
48.
Kuhlman, W., et al., Interplay between PEO tether length and ligand spacing
governs cell spreading on RGD-modified PMMA-g-PEO comb copolymers.
Biomacromolecules, 2007. 8(10): p. 3206-13.
49.
Barrientos, S., et al., Growth factors and cytokines in wound healing. Wound
Repair Regen, 2008. 16(5): p. 585-601.
30
50.
Ferrara, N., Binding to the extracellular matrix and proteolytic processing: two
key mechanisms regulating vascular endothelial growth factor action. Mol Biol
Cell, 2010. 21(5): p. 687-90.
51.
Masters, K.S., Covalent growth factor immobilization strategies for tissue repair
and regeneration. Macromol Biosci, 2011. 11(9): p. 1149-63.
52.
Meyer, U., et al., Basic reactions of osteoblasts on structured material surfaces.
Eur Cell Mater, 2005. 9: p. 39-49.
53.
Puleo, D.A., et al., Examination of osteoblast-orthopaedic biomaterial
interactions using molecular techniques. Biomaterials, 1993. 14(2): p. 111-4.
54.
Raut, V.P., et al., Molecular dynamics simulations of peptide-surface interactions.
Langmuir, 2005. 21(4): p. 1629-39.
55.
Agashe, M., et al., Molecular simulation to characterize the adsorption behavior
of a fibrinogen gamma-chain fragment. Langmuir, 2005. 21(3): p. 1103-17.
56.
Grausova, L., et al., Enhanced growth and osteogenic differentiation of human
osteoblast-like cells on boron-doped nanocrystalline diamond thin films. PLoS
One. 6(6): p. e20943.
57.
Zhou, H., et al., Stimulated osteoblastic proliferation by mesoporous silica
xerogel with high specific surface area. J Mater Sci Mater Med. 22(3): p. 731-9.
58.
Godek, M.L., et al., Adsorbed serum albumin is permissive to macrophage
attachment to perfluorocarbon polymer surfaces in culture. J Biomed Mater Res
A, 2009. 88(2): p. 503-19.
59.
Rouahi, M., et al., Influence of hydroxyapatite microstructure on human bone cell
response. J Biomed Mater Res A, 2006. 78(2): p. 222-35.
31
60.
Yang, X.B., et al., Human osteoprogenitor growth and differentiation on synthetic
biodegradable structures after surface modification. Bone, 2001. 29(6): p. 52331.
61.
Healy, K.E., A. Rezania, and R.A. Stile, Designing biomaterials to direct
biological responses. Ann N Y Acad Sci, 1999. 875: p. 24-35.
62.
Kieswetter, K., et al., The role of implant surface characteristics in the healing of
bone. Crit Rev Oral Biol Med, 1996. 7(4): p. 329-45.
63.
Yang, Y., et al., Theoretical study of bone sialoprotein in bone biomineralization.
Cells Tissues Organs. 194(2-4): p. 182-7.
64.
Latour, R.A., Thermodynamic perspectives on the molecular mechanisms
providing protein adsorption resistance that include protein-surface interactions.
J Biomed Mater Res A, 2006. 78(4): p. 843-54.
65.
Latour, R.A., Jr. and C.J. Rini,
Theoretical analysis
of
adsorption
thermodynamics for hydrophobic peptide residues on SAM surfaces of varying
functionality. J Biomed Mater Res, 2002. 60(4): p. 564-77.
66.
Latour, R.A., Jr., et al., Determination of apparent thermodynamic parameters for
adsorption of a midchain peptidyl residue onto a glass surface. J Biomed Mater
Res, 2000. 49(1): p. 58-65.
67.
Anselme, K., Osteoblast adhesion on biomaterials. Biomaterials, 2000. 21(7): p.
667-81.
68.
Fujisawa, R. and M. Tamura, Acidic bone matrix proteins and their roles in
calcification. Front Biosci. 17: p. 1891-903.
32
69.
Qu, Z., et al., Improving bone marrow stromal cell attachment on
chitosan/hydroxyapatite scaffolds by an immobilized RGD peptide. Biomed
Mater. 5(6): p. 065001.
70.
Santiago, L.Y., et al., Peptide-surface modification of poly(caprolactone) with
laminin-derived
sequences
for
adipose-derived
stem
cell
applications.
Biomaterials, 2006. 27(15): p. 2962-9.
71.
Smith, E., et al., RGD-grafted thermoreversible polymers to facilitate attachment
of BMP-2 responsive C2C12 cells. Biomaterials, 2005. 26(35): p. 7329-38.
72.
Gronthos, S., et al., Integrin expression and function on human osteoblast-like
cells. J Bone Miner Res, 1997. 12(8): p. 1189-97.
73.
Burridge, K. and K. Fath, Focal contacts: transmembrane links between the
extracellular matrix and the cytoskeleton. Bioessays, 1989. 10(4): p. 104-8.
74.
Fath, K.R., C.J. Edgell, and K. Burridge, The distribution of distinct integrins in
focal contacts is determined by the substratum composition. J Cell Sci, 1989. 92 (
Pt 1): p. 67-75.
75.
Burridge, K., et al., Focal adhesions: transmembrane junctions between the
extracellular matrix and the cytoskeleton. Annu Rev Cell Biol, 1988. 4: p. 487525.
76.
Aplin, A.E., et al., Signal transduction and signal modulation by cell adhesion
receptors: the role of integrins, cadherins, immunoglobulin-cell adhesion
molecules, and selectins. Pharmacol Rev, 1998. 50(2): p. 197-263.
77.
Howe, A., et al., Integrin signaling and cell growth control. Curr Opin Cell Biol,
1998. 10(2): p. 220-31.
33
78.
Cox, E.A. and A. Huttenlocher, Regulation of integrin-mediated adhesion during
cell migration. Microsc Res Tech, 1998. 43(5): p. 412-9.
79.
Huttenlocher, A., R.R. Sandborg, and A.F. Horwitz, Adhesion in cell migration.
Curr Opin Cell Biol, 1995. 7(5): p. 697-706.
80.
Damien, C.J. and J.R. Parsons, Bone graft and bone graft substitutes: a review of
current technology and applications. J Appl Biomater, 1991. 2(3): p. 187-208.
81.
Miron, R.J. and Y.F. Zhang, Osteoinduction: A Review of Old Concepts with New
Standards. J Dent Res.
82.
Yun, Y.R., et al., Administration of growth factors for bone regeneration. Regen
Med. 7(3): p. 369-85.
83.
Lee, S.Y., T. Nakagawa, and A.H. Reddi, Induction of chondrogenesis and
expression of superficial zone protein (SZP)/lubricin by mesenchymal progenitors
in the infrapatellar fat pad of the knee joint treated with TGF-beta1 and BMP-7.
Biochem Biophys Res Commun, 2008. 376(1): p. 148-53.
84.
Knippenberg, M., et al., Osteogenesis versus chondrogenesis by BMP-2 and
BMP-7 in adipose stem cells. Biochem Biophys Res Commun, 2006. 342(3): p.
902-8.
85.
Sekiya, I., D.C. Colter, and D.J. Prockop, BMP-6 enhances chondrogenesis in a
subpopulation of human marrow stromal cells. Biochem Biophys Res Commun,
2001. 284(2): p. 411-8.
86.
Hanada, K., et al., BMP-2 induction and TGF-beta 1 modulation of rat periosteal
cell chondrogenesis. J Cell Biochem, 2001. 81(2): p. 284-94.
34
87.
Kuboki, Y., et al., Two distinctive BMP-carriers induce zonal chondrogenesis and
membranous ossification, respectively; geometrical factors of matrices for celldifferentiation. Connect Tissue Res, 1995. 32(1-4): p. 219-26.
88.
Boyan, B.D., et al., Role of material surfaces in regulating bone and cartilage cell
response. Biomaterials, 1996. 17(2): p. 137-46.
89.
Schwartz, Z., et al., Underlying mechanisms at the bone-surface interface during
regeneration. J Periodontal Res, 1997. 32(1 Pt 2): p. 166-71.
90.
Kieswetter, K., et al., Surface roughness modulates the local production of growth
factors and cytokines by osteoblast-like MG-63 cells. J Biomed Mater Res, 1996.
32(1): p. 55-63.
91.
Anselme, K., B. Noel, and P. Hardouin, Human osteoblast adhesion on titanium
alloy, stainless steel, glass and plastic substrates with same surface topography. J
Mater Sci Mater Med, 1999. 10(12): p. 815-9.
92.
Anselme, K., et al., In vitro control of human bone marrow stromal cells for bone
tissue engineering. Tissue Eng, 2002. 8(6): p. 941-53.
93.
Anselme, K., et al., Qualitative and quantitative study of human osteoblast
adhesion on materials with various surface roughnesses. J Biomed Mater Res,
2000. 49(2): p. 155-66.
94.
Li, X., et al., Preparation of hydrophilic/hydrophobic porous materials. J Colloid
Interface Sci, 2008. 323(1): p. 120-5.
95.
Kwon, A., et al., Physiological significance of hydrophilic and hydrophobic
textile materials during intermittent exercise in humans under the influence of
35
warm ambient temperature with and without wind. Eur J Appl Physiol Occup
Physiol, 1998. 78(6): p. 487-93.
96.
Takahashi, H. and W.J. Finger, Dentin surface reproduction with hydrophilic and
hydrophobic impression materials. Dent Mater, 1991. 7(3): p. 197-201.
97.
Ruardy, T.G., et al., Adhesion and spreading of human skin fibroblasts on
physicochemically characterized gradient surfaces. J Biomed Mater Res, 1995.
29(11): p. 1415-23.
98.
Schakenraad, J.M., et al., Thermodynamic aspects of cell spreading on solid
substrata. Cell Biophys, 1988. 13(1): p. 75-91.
99.
Roosjen, A., et al., Stability and effectiveness against bacterial adhesion of
poly(ethylene oxide) coatings in biological fluids. J Biomed Mater Res B Appl
Biomater, 2005. 73(2): p. 347-54.
100.
Saldarriaga Fernandez, I.C., et al., In vitro and in vivo comparisons of
staphylococcal biofilm formation on a cross-linked poly(ethylene glycol)-based
polymer coating. Acta Biomater. 6(3): p. 1119-24.
101.
Burg, M.B., J.D. Ferraris, and N.I. Dmitrieva, Cellular response to hyperosmotic
stresses. Physiol Rev, 2007. 87(4): p. 1441-74.
102.
Alfieri, R.R. and P.G. Petronini, Hyperosmotic stress response: comparison with
other cellular stresses. Pflugers Arch, 2007. 454(2): p. 173-85.
103.
Lang, F., et al., Osmotic shock-induced suicidal death of erythrocytes. Acta
Physiol (Oxf), 2006. 187(1-2): p. 191-8.
104.
Capuano, P. and G. Capasso, [The importance of intracellular pH in the
regulation of cell function]. G Ital Nefrol, 2003. 20(2): p. 139-50.
36
105.
Chiang, C.K., et al., Engineering surfaces for site-specific vascular differentiation
of mouse embryonic stem cells. Acta Biomater, 2010. 6(6): p. 1904-16.
106.
Chiu, L.L., et al., Defining conditions for covalent immobilization of angiogenic
growth factors onto scaffolds for tissue engineering. J Tissue Eng Regen Med,
2011. 5(1): p. 69-84.
107.
Chiu, L.L. and M. Radisic, Scaffolds with covalently immobilized VEGF and
Angiopoietin-1 for vascularization of engineered tissues. Biomaterials, 2010.
31(2): p. 226-41.
108.
Tigli, R.S., et al., Cellular Behavior on Epidermal Growth Factor (EGF)Immobilized PCL/Gelatin Nanofibrous Scaffolds. J Biomater Sci Polym Ed, 2010.
109.
Choi, J.S., K.W. Leong, and H.S. Yoo, In vivo wound healing of diabetic ulcers
using electrospun nanofibers immobilized with human epidermal growth factor
(EGF). Biomaterials, 2008. 29(5): p. 587-96.
110.
Yamachika, E., et al., Immobilized recombinant human bone morphogenetic
protein-2 enhances the phosphorylation of receptor-activated Smads. J Biomed
Mater Res A, 2009. 88(3): p. 599-607.
111.
Anderson, S.M., et al., The phosphorylation of vascular endothelial growth factor
receptor-2 (VEGFR-2) by engineered surfaces with electrostatically or covalently
immobilized VEGF. Biomaterials, 2009. 30(27): p. 4618-28.
112.
Ogiwara, K., et al., Effect of photo-immobilization of epidermal growth factor on
the cellular behaviors. Biochem Biophys Res Commun, 2006. 345(1): p. 255-9.
37
113.
Ogiwara, K., et al., Construction of a novel extracellular matrix using a new
genetically engineered epidermal growth factor fused to IgG-Fc. Biotechnol Lett,
2005. 27(20): p. 1633-7.
114.
Stefonek, T.J. and K.S. Masters, Immobilized gradients of epidermal growth
factor promote accelerated and directed keratinocyte migration. Wound Repair
Regen, 2007. 15(6): p. 847-55.
115.
Stefonek-Puccinelli, T.J. and K.S. Masters, Regulation of cell signaling and
function via changes in growth factor presentation. Conf Proc IEEE Eng Med
Biol Soc, 2009. 2009: p. 1167-71.
116.
Ito, Y., G. Chen, and Y. Imanishi, Micropatterned immobilization of epidermal
growth factor to regulate cell function. Bioconjug Chem, 1998. 9(2): p. 277-82.
117.
Au, A., et al. The use of P(MMA-r-POEM) Comb Copolymer to Present Peptides
for Enriching the Osteoblastic Progenitor Population from Bone Marrow
Aspirates. in 7th Annual Hilton Head Workshop on Tissue Engineering. 2003. Sea
Pines Plantation, Hilton Head, SC.
118.
Au, A., et al., Formation of osteogenic colonies on well-defined adhesion peptides
by freshly isolated human marrow cells. Biomaterials, 2007. 28(10): p. 1847-61.
119.
Leslie-Barbick, J.E., et al., Micron-scale spatially patterned, covalently
immobilized vascular endothelial growth factor on hydrogels accelerates
endothelial tubulogenesis and increases cellular angiogenic responses. Tissue
Eng Part A, 2011. 17(1-2): p. 221-9.
120.
Leslie-Barbick, J.E., J.J. Moon, and J.L. West, Covalently-immobilized vascular
endothelial
38
growth
factor
promotes
endothelial
cell
tubulogenesis
in
poly(ethylene glycol) diacrylate hydrogels. J Biomater Sci Polym Ed, 2009.
20(12): p. 1763-79.
121.
Gobin, A.S. and J.L. West, Effects of epidermal growth factor on fibroblast
migration through biomimetic hydrogels. Biotechnol Prog, 2003. 19(6): p. 17815.
122.
Fan, V.H., et al., Tethered epidermal growth factor provides a survival advantage
to mesenchymal stem cells. Stem Cells, 2007. 25(5): p. 1241-51.
123.
Marcantonio, N.A., et al., The influence of tethered epidermal growth factor on
connective tissue progenitor colony formation. Biomaterials, 2009. 30(27): p.
4629-38.
124.
Platt, M.O., et al., Sustained epidermal growth factor receptor levels and
activation by tethered ligand binding enhances osteogenic differentiation of multipotent marrow stromal cells. J Cell Physiol, 2009. 221(2): p. 306-17.
125.
Rodrigues, M., et al., Surface tethered epidermal growth factor protects
proliferating and differentiating multipotential stromal cells from FasL-induced
apoptosis. Stem Cells, 2013. 31(1): p. 104-16.
126.
Tamama, K., H. Kawasaki, and A. Wells, Epidermal growth factor (EGF)
treatment on multipotential stromal cells (MSCs). Possible enhancement of
therapeutic potential of MSC. J Biomed Biotechnol, 2010. 2010: p. 795385.
127.
Saik, J.E., et al., Covalently immobilized platelet-derived growth factor-BB
promotes angiogenesis in biomimetic poly(ethylene glycol) hydrogels. Acta
Biomater, 2011. 7(1): p. 133-43.
39
128.
DeLong, S.A., J.J. Moon, and J.L. West, Covalently immobilized gradients of
bFGF on hydrogel scaffolds for directed cell migration. Biomaterials, 2005.
26(16): p. 3227-34.
129.
Mann, B.K., R.H. Schmedlen, and J.L. West, Tethered-TGF-beta increases
extracellular matrix production of vascular smooth muscle cells. Biomaterials,
2001. 22(5): p. 439-44.
130.
Rahman, N., et al., The use of vascular endothelial growth factor functionalized
agarose to guide pluripotent stem cell aggregates toward blood progenitor cells.
Biomaterials, 2010. 31(32): p. 8262-70.
131.
He, Q., et al., Improved cellularization and angiogenesis using collagen scaffolds
chemically conjugated with vascular endothelial growth factor. Acta Biomater,
2011. 7(3): p. 1084-93.
132.
Chen, L., et al., Direct chemical cross-linking of platelet-derived growth factorBB
to
the
demineralized
bone
matrix
improves
cellularization
and
vascularization. Biomacromolecules, 2009. 10(12): p. 3193-8.
133.
Zhang, H., et al., Chemically-conjugated bone morphogenetic protein-2 on threedimensional polycaprolactone scaffolds stimulates osteogenic activity in bone
marrow stromal cells. Tissue Eng Part A, 2010. 16(11): p. 3441-8.
134.
Park, Y.J., et al., Immobilization of bone morphogenetic protein-2 on a
nanofibrous chitosan membrane for enhanced guided bone regeneration.
Biotechnol Appl Biochem, 2006. 43(Pt 1): p. 17-24.
40
CHAPTER 3: DESIGN AND VERIFICATION OF METHODS TO ASSESS THE
RESPONSE OF CONNECTIVE TISSUE PROGENITOR CELLS TO SURFACE
MODIFIED BIOMATERIALS
3.1.
Unmet Need: The field of tissue engineering and regenerative medicine has
generated a broad range of biomaterials that can be applied in many different ways as
treatment options that seek to regenerate or accelerate the repair of various tissues. Bone
regeneration is one of the most intensely studied approaches. Over 50 biomaterial
products are currently available for bone regeneration. Formation of new bone by the
progeny of native connective tissue progenitor cells (CTPs) is a central feature of each of
these treatment options. The strategy of using a cell-based therapy to achieve bone
regeneration inevitably includes the potential to use biomaterials to “target” CTPs and to
use their interaction with a biomaterial surface to enhance their attachment, survival,
migration, proliferation, or differentiation. Many of these same cellular responses can be
targeted using other means such as mechanical or biophysical environment or soluble
bioactive factors. However, biomaterials surfaces represent a unique opportunity due to
the potential that they offer to control the special orientation of the cells and signals in a
tissue region as well as the concentration and presentation of specific signals.
Recent advancements in biomaterials science include promising technologies for
modifying biomaterial surfaces with pro-survival or pro-osteogenic bioactive ligands.
These surface modifications use biomaterial-specific chemistry to present bioactive
ligands in a controlled, stable and biologically active form.
Before testing the pre-clinical efficacy of these surface modified biomaterials in
animal models, it is important to test the influence of these materials on cells in an in
41
vitro setting. In vitro assays using cell culture preparations can be used to: characterize
the response of specific cell types to biomaterials and possible degradation products,
confirm the bioactivity and stability of a tethered molecule, characterize the dose
responsiveness of a tethered ligand, characterize the differences between substrate
surfaces and tethering chemistries for presentation of individual tethered ligands, In vitro
assays can also be used to advance the understanding of fundamental biological outcomes
in response to biological signals. However, in order to provide the stem cell and
regenerative medicine community with the greatest possible and generalizable benefit,
the methods available for assessment should be standardized, systematic, repeatable,
quantifiable, user-independent, automated, cost effective and applicable to a vast array of
biomaterials, surface modification strategies and cell sources. A platform offering such
methods is not yet available, and represents the unmet need in both the biomedical
research and the clinical community.
3.2.
Proposed Solution: In this dissertation, I have addressed this gap in standardized
methodology and advance the field of biomedical engineering by designing and applying
a quantitative method to assess the response of CTPs (or other cell types) to a biomaterial
surface or a surface-modified biomaterial by applying the recently adopted ASTM
standard automated colony forming unit (CFU) assay (ASTM F2944-12). This new
method uses engineering design principles to combine the following components:
(1) a novel method to prepare two dimensional surfaces of a particulate of any
bone scaffold material, with or without surface modification using a bioactive
ligand;
42
(2) a method to verify and optimize these surfaces with respect to their
repeatability, stability, uniformity in coverage, and smoothness; and
(3) a method to assess the material and surface modification effects on CTPs
using a standardized method for the automated CFU assay.
Figure 1 describes the flowchart of the design process. Constraints were identified based
on the available knowledge and the unmet need. The technical specifications were
defined, forming the basis for verification of the compliance of individual components.
The combined method was validated using β-TriCalcium Phosphate (TCP), a widely
available and clinically useful bone scaffold material.
TCP surfaces were further
evaluated with and without surface modification with Epidermal Growth Factor (EGF), a
pro-survival and proliferation bioactive ligand. Technology for tethering EGF to a TCPcontaining surface using fusion protein containing both EGF and a novel TCP-binding
peptide was developed by the Griffith Lab at MIT, as described in Chapter 4.
The methods described for surface fabrication and testing are applicable to a wide
variety of cell types, biomaterials, and bioactive ligands.
Some examples include:
ceramics (e.g., β-TriCalcium Phosphates (TCPs), hydroxyapatites, nanocrystalline
calcium phosphates), biologics (e.g., demineralized bone matrix, chitosan, lyophilized
collagen)
or
synthetic
polymers
(e.g.
Poly(methyl
methacrylate)
(PMMA),
Polycaprolactone (PCL), poly(propylene fumarate) PPF) are some examples of such
biomaterials. While this presentation is focused on materials and applications in bone
tissue regeneration, these methods can apply to materials and cell types well beyond bone
regeneration, including cells and materials relevant to: skin and wound healing, nerve
43
regeneration, muscle regeneration, tendon/ligament, vascular reconstruction, as well as
hollow and solid organs.
3.3.
Components of the Design Relevant to Bone Tissue Regeneration: Osteogenic
Connective Tissue Progenitors (CTPs): The recent developments in stem cell and
progenitor cell biology has highlighted the fact that bone marrow, trabecular surface
bone, muscle, periosteum, perivascular tissue, adipose tissue and blood can each provide
a potential niche and source for a heterogeneous population tissue derived stem cells and
progenitor cells capable of contributing to the forming bone tissues.
Bone repair
requires osteogenic Connective Tissue Progenitors (CTPs), defined as a tissue resident
population of cells that are capable of proliferation and osteoblastic differentiation. No
new bone can be formed without a source of cells capable of generating progeny that
ultimately secrete osteoid, the hallmark of bone formation. Many preclinical studies have
demonstrated improved graft performance when CTPs are added, even to small graft sites
in young healthy animals, supporting the premise that the CTP population is suboptimal
in virtually all clinical settings and that optimal performance from any osteoconductive or
osteoinductive biomaterial may require augmentation with local CTPs [1-10].
3.3.1. Osteoconductive Materials: An osteoconductive surface is one that permits the
attachment, migration, proliferation and differentiation of CTPs and other contributing
cells throughout a grafted region, and thereby facilitates the distribution of bone
formation and bridging or connection to adjacent bone structures. An ideal
osteoconductive scaffold is biocompatible, reproducible, resorbable, porous, and cost
44
effective. A variety of synthetic materials are used clinically as bone grafts, including
ceramics, collagen, non-collagenous proteins, bioactive glasses, and biodegradable
polymers.
3.3.2. Osteoinductive Stimuli: Osteoindudctive stimuli are defined as soluble factors
that modify (induce a change in) cell fate in a definitive direction towards bone
differentiation. Bone Morphogenetic Proteins (BMPs) are the prototypical inductive
signaling molecule in the musculoskeletal system. However many other factors have
important osteotropic effects. Platelet Derived Growth Factor (PDGF), basic Fibroblast
Growth Factor (bFGF) and EGF are each identified as key effectors in improving the
performance of stem and progenitor cells in orthopaedic tissue engineering applications.
EGF is a potent mitogen for osteoblastic progenitors, and is both necessary and
sufficient to induce colony growth in bone marrow-derived cells [11, 12]. EGF signaling
has been shown to prevent TNF-induced apoptosis in a cell line derived from MSCs [13,
14]. The EGF receptor (EGFR) is expressed by virtually every cell type, including stem
and progenitor cells. EGFR is a receptor tyrosine kinase that activates intracellular
signaling cascades that influence cell proliferation, migration, and differentiation. EGFEGFR interaction induces EGFR dimerization and autophosphorylation which activates
to downstream intracellular pathways. Once bound to the receptor, EGF is rapidly
internalized by receptor-mediated endocytosis leading to receptor desensitization, thereby
down-regulating the effects of exogenous EGF [15].
Osteoinductive stimuli in the form of growth factors and bioactive peptides can
also be selectively concentrated and presented by either preferential adsorption or
45
tethering- i.e. covalently linking them to a surface. There are several potential advantages
in presenting growth factors as a matrix-tethered molecule for applications in tissue
engineering. Tethering ensures that the signaling molecule does not diffuse away from
the cells rapidly, and enables control over the concentration and three dimensional
conformation of the molecule that preserves its ability to interact with receptors. In case
of epidermal growth factor (EGF), tethering it to the scaffold has shown to be competent
to bind and activate the EGF receptor, but not internalized and degraded. This mode
mimics features of the physiological presentation of EGF receptor ligands that act in
juxtacrine fashion or are matrix bound.
EGF is a candidate for relatively simple chemical modifications, compared to
other growth factors. EGF is a relatively small protein with a structure that is
characterized by a single amine group at its N-terminus. EGF has been shown to be
bioactive when tethered to biomaterials, in contrast to many other factors, which have
limited applications when presented in a tethered form [16, 17]. Currently, most growth
factors (e.g., BMPs) are implanted in very high local concentrations and released by
diffusion, making it extremely difficult to control their local concentration and rate of
clearance. In case of epidermal growth factor (EGF), tethering it to the scaffold has
shown to be competent to bind and activate the EGF receptor, but not internalized and
degraded. This mode mimics features of the physiological presentation of EGF receptor
ligands that act in juxtacrine fashion or are matrix bound.
3.3.3. CFU assay for CTPs: When placed in the in vitro culture system, under
appropriate conditions, CTPs adhere to the surface on which they are plated, survive, and
46
go on to proliferate to form a colony of cells that represent the progeny of the original
founding CTP. Colony forming unit (CFU) assays have been the gold standard for the
quantitative assessment of CTPs since the 1980s. Several methods have been used to
define colonies for the purposes of quantification of the number, concentration or
prevalence of colony founding cells in a starting population. Colonies are identified
based on gross size, morphology, number of cells and expression of specific markers or
enzyme activity [18-21]. However, the vast majority of these methods have been based
on subjective and qualitative assessments based on skilled observers, which have been
shown to be prone to large variation within and between observers [22]. In this work we
have chosen to adopt automated image-based methods of colony assessment consistent
with the recently adopted ASTM Standard Method, (ASTM F2944-12) [23]: “Standard
Test Method for Automated Colony Forming Unit (CFU) Assays- Image Acquisition and
Analysis Method for Enumerating and Characterizing Cells and Colonies in Culture”. An
image capture and analysis system platform developed in the Muschler Lab and named
Colonyze™ is used for this purpose. In the form that was available at the beginning of
this project, Colonyze™ was designed and partly optimized for the assessment of
colonies on glass or tissue culture plastic surfaces, or with surface modifications applied
to glass or tissue culture plastic [24-27]. These assays are not optimized for a threedimensional scaffold or the use of an optically opaque material.
3.4.
Constraints of the design:
3.4.1. Use of Beta-tricalcium phosphate as a biomaterial that is modified with
tethered EGF: Beta-tricalcium phosphate (TCP) is a clinically used bone graft material
47
and is widely used as bone void filler [28-34]. These materials have been widely used as
bone graft materials due to their biocompatibility and similarity in composition to bone
mineral [35, 36]. TriCalcium Phosphate [Ca3(PO4)2] is not a natural component of bone
but has chemical proportions of calcium and phosphate similar to bone mineral [37].
These materials provide a biocompatible osteoconductive scaffold for new bone
formation.
The continued development and use of TCP as a bone graft material is limited by
its material properties, and the disintegration of TCP in aqueous solutions at
physiological pH which do not permit stable direct covalent chemical surface
modification using covalent binding methods. As a means of overcoming the surface
constraints of TCP related to covalent binding, the surface of TCP scaffolds was modified
in this project using tethered EGF via a high-affinity, non-covalent binding novel TCP
binding peptide TCPBP [38]. We examined the effect of tethered TCPBP-EGF on the in
vitro performance of bone marrow-derived CTPs. Verification of TCPBP surfaces
required measurement of TCPBP-EGF concentration, biological activity, and stability.
The tethering concentration of TCPBP is dependent on the concentration in tethering
solution and incubation time. The specific receptor mediated biological TCPBP-EGF
could be confirmed based on the activation of ERK in passage 3 MSCs. The stability of
tethered EGF on TCP scaffolds could be characterized using the standardized BCA assay.
3.4.2. Use of CTPs and osteogenic conditions to screen for pro-osteogenic biological
response: Cell sourcing for these methods was designed to utilize human bone marrow
or discarded bone collected from patients undergoing invasive arthroscopic surgeries
48
under an IRB approved protocol. CTPs show a large patient to patient variation in both
the concentration of cells and the prevalence of colony founding CTPs among the cells
isolated. . The number of patients recruited for the study will be determined using
statistical methods employing power analyses. As the intended usage of the surface
modified TCP biomaterials being tested for use as bone void fillers that promote
osteogenic potential of local or transplanted CTPs, the CTP response was evaluated in
osteogenic culture conditions.
3.4.3. Use of Colonyze™, a standardized automated CFU assay to assess CTP
response: The traditional CFU assay determines the observed CTP prevalence manually
by counting the number of colonies on the adherent surface. However, this method is
highly user dependant and the assessment may be influenced by user bias in
determination of a colony, and inaccuracy in colony identification. The user- to- user
variability has been shown to be as high as 40%. The method developed in this
dissertation will use an automated CFU assay that uses an algorithm that is accurate,
consistent, automated and independent of user bias, and is compliant with the ASTM
Standard Method (ASTM F2944-12) [23]: “Standard Test Method for Automated Colony
Forming Unit (CFU) Assays- Image Acquisition and Analysis Method for Enumerating
and Characterizing Cells and Colonies in Culture”.
While verification and validation of this method is demonstrated using one class
of biomaterial, one class of bioactive stimulus, and one cell type, this method can easily
be generalized to any combination of biomaterials, bioactive stimuli and cells. Potential
applications of this method are discussed in Chapter 6.
49
3.5.
Technical Specifications of the Design:
3.5.1. Design of biomaterial surfaces: Biomaterials can be prepared in a variety of
shapes, dimensions and sizes. However, the following surface properties are desired:
(1) Ease of preparation and repeatability: the method of biomaterial surface
preparation must be consistent, repeatable, and relatively easy to execute.
(2) Uniformity: Surfaces must be uniform in terms of the surface properties
(coverage, smoothness, porosity, hydrophilicity, or charges where applicable),
and interaction between the cell and surface,
(3) Stability: the surface must be stable for the duration of culture, and may not
degrade or disintegrate.
(4) Detection of colonies: the form of the biomaterial (shape and dimensions)
must be such that the cells which comprise the colonies formed by the progeny of
CTPs can be detected in an accurate and consistent manner enabling the primary
outcomes of colony formation and performance to be quantified.
In order to choose the optimal surface design, a scoring system will be
implemented for the verification step. Surfaces will be scored for:
(1) Ease of
Preparation (2) Repeatability (3) Surface Uniformity (4) Stability During Culture Period
(5) and Identification and Detection of Colonies. A scoring system is defined where the
biomaterials will be scored on the scale of 0 to 5 (5 for Excellent, 4 Above Average, 3
Average, 2 Acceptable, 1 Poor, And 0 Unacceptable).
50
Four samples from each surface condition will be evaluated. In addition to the
visual qualitative assessment, brightfield microscopy, and scanning electron microscopy
will be used to confirm the surface properties, and cell culture will be used to determine
the cell response properties. The biomaterial with the highest overall score, AND no
score below 3 in any of the criteria will be advanced to the validation step.
3.5.2. Surface density and stability of bioactive ligands: Surface density of the
bioactive ligand on the test material is measured in units of number of ligands per µm2.
On theoretical grounds, the surface area of a spread cell in contact with the biomaterial
surface is ~ 500 µm2. A typical MSC expresses 50-100,000 EGF receptors (which gives
the estimated surface density as 100-200 receptors per µm2). A significant portion of
these cells can interact with tethered EGF molecule over the course of time. Therefore a
minimum surface density that is at least 100 times more than the surface density of the
receptor on the cell surface is like to be sufficient to stimulate the EGFRs on the cell
surface to a maximum level [26, 38, 39]. For the TCPBP-EGF protein (molecular weight
77 kDa), that has been tethered to a 18 mm diameter coverslip (surface area 254 x 10 6
µm2 ), a desired surface density of 20000 molecules per µm2 translates to 650 nanograms
per coverslip. Including a 40% excess, the minimum expected amount of tethered
TCPBP-EGF is set at at least 1000 nanograms per coverslip. The repeatability and
stability of surface modification will be verified for at least 5 days.
3.5.3. Primary and secondary outcomes of the CFU assay: Primary outcomes of the
automated CFU assays will be:
51
(1) Number of colonies on the biomaterial surface,
(2) Number of cells in each colony,
(3) Colony density, a ratio of the number of cells in colony and the colony area
(4) Alkaline phosphatase (AP) positive area in the cell cytoplasm.
A colony is defined as a cluster of 8 or more cells separated by less than 142 µm
(an empiric metric based providing clustering of cells into colony objects matching
determinations made by skilled observers).
Cells are identified using a means of
counting individual cells. In this project the means of cell identification is based on
staining of fixed cells using 4',6-diamidino-2-phenylindole (DAPI), a nuclear fluorescent
stain.
DAPI stained nuclei can be identified under fluroscence with 340-380 nm
excitation and >425 nm emission wavelangths. Alkaline phosphatase (AP) is a marker for
early osteogenesis. AP expression will be determined using a alkaline phosphatase Vector
Red stain kit (Vector Labs), that produces a precipitate which has a red color in the visual
spectrum and under fluorescence with 566 nm excitation and 575-670 nm emission
wavelengths.
(1) Relative Colony Forming Efficiency (CFE): CFE of the test material
will be calculated as a ratio of the mean number of colonies on the surface of a test
material divided by the mean number of colonies on the control material. CFE will be
used to determine the influence of the test material on the colony forming ability of
CTPs. Further, all the colony data will be pooled together for each test condition for all
patient samples. A median will be calculated for each colony metric. Due to the known
wide variation in CTP prevalence and performance between individual subjects, the
52
outcome corresponding to the test surface will be divided by the outcome corresponding
to the control surface.
(2) Colony Size (metric of effective proliferation): determined as the median
number of cells per colony on the test surface standardized to the median number of cells
per colony on the control surface, (3) Relative Migration: determined as the median
number of colony density on the test surface standardized to the median number of
colony density on the control surface, (4) Osteoblastic differentiation: determined as the
median number of AP positive cell area divided by cell number on the test surface
standardized to that on the control surface. Table 1 summarizes the primary and
secondary outcomes and technical specifications:
3.5.4. CFU Analysis - Cell culture conditions, inclusion/ exclusion criteria and
sample size determination: Bone marrow derived cells are cultured for 9 days in
osteogenic conditions containing 10% fetal bovine serum, 50 μM ascorbate, 10 nM
dexamethasone. The initial plating density per surface will be 500,000 for bone marrow
or marrow space-derived cells and 200,000 for cells derived from trabecular surface.
Based on previous data, the expected range of colony prevalence at these plating densities
is 4 to 50 per surface. A prevalence of more than 50 leads to a state of confluence in
which the progeny of individual colonies often overgrow into one another compromising
the ability to identify discrete colonies. A prevalence of less than 4 colonies limits the
accuracy of the assessment - as one error in positive or negative colony identification can
cause a 25% or more difference in overall assessment. Therefore, confluent samples
53
(more than 50 colonies) or samples with less than 4 colonies are excluded from the
analysis.
3.5.5. Criteria for success: Based on historical data correlating
CTP prevalence
(observed colony formation) and in in vivo biological response [25, 40] , a minimum of
50% improvement in CFE or cell number per colony, a function of effective proliferation
rate (EPR) represents an appropriate (albeit as yet unvalidated) threshold for
demonstrating a biological response to a test condition (e.g. a surface modified
biomaterial) compared to the control (material with no surface modification) that is of
potential clinical value.
Metrics of colony density (associated with migration) and osteoblastic
differentiation, are also potentially relevant from a clinical perspective. However, these
metrics, taken by themselves may or may not measure positive aspects of biological
response from a clinical perspective, and must be interpreted in the overall context of
performance, starting with CFE and effective proliferation rate (EPR). An increased level
of osteoblastic differentiation (i.e. expression of markers or activities associated with
osteoblastic differentiation, such as alkaline phosphatase activity or PTH receptors)
indicates more CTPs committed to osteogenesis (i.e. demonstrate performance as
osteogenic CTPs). However, because differentiation is often associated with a shift away
from proliferation, osteogenesis alone, and particularly premature osteogenesis may have
the adverse effect of reducing the overall number of mature osteoblasts generated and the
resultant amount of new bone tissue. Similarly a decreased level of colony density
indicates more migration of CTP-progeny, which may be biologically desirable in
54
distributing progenitors throughout the grafted volume. However, an increased colony
density, suggesting less migration of CTP- progeny, may also be biologically desirable,
since high density may be associated with both an increased EPR and increase osteogenic
activity.
Patient-to-patient variability and the known heterogeneity of CTP in cells
harvested from bone marrow and bone tissue samples must be considered in determining
sample size when making generalizable comparisons between test conditions. A power
analysis is performed to determine the necessary sample size to yield a significant
difference with α = 0.05 and power = 0.80. In general, assessment in samples from at
least 5 and up to 20 human subjects is necessary to document the generalizability of a
given finding across subjects. Within subject samples, analysis of at least 20 and up to
200 colonies (progeny samples of individual CTPs) is necessary to document differences
between test conditions (i.e. biomaterials and surface modifications).
3.6.
Design and Verification:
3.6.1. Assessment of existing TCP scaffolds: TCP TheriLok™ scaffolds (“crosses”), a
gift from Integra® Lifesciences (Plainsboro, NJ), were used as a generic substrate for
assessment of CTP-derived colony formation on a 3D surface and as a substrate for tether
in of TCPBP-EGF to a TCP surface. Crosses are fabricated using a 3D printing
methodology (3DP) followed by sintering to shape TCP granules into a 3D TCP scaffold
of defined shape. The nominal size of the TCP TheriLok™ scaffolds is 3 mm from tip to
tip of each cross and and 1.8 mm in height. The “cross” design was intended to facilitate
55
interlocking between crosses when placed into a defect site as a means of enhancing
stability. TheriLok™ scaffolds are 60% porous with a pore size of 250 µm.
Scaffolds were first scored for compliance with technical specifications (i.e.
broken or misshaped scaffolds were discarded). Scaffolds were sterilized using a steam
autoclave (20 minutes at 121°C and 15 psi) and hydrated in 1X sterile PBS under
vacuum.
Individual scaffolds were transferred to a sterile well in a 96 well
polypropylene plate. 150 uL of bone marrow aspirate containing was gently loaded into
each well.
Crosses loaded with marrow-derived cells were cultured in osteogenic
conditions for 6 days. Culture media was replaced every 72 hours. Cells adherent to each
cross were fixed with 1:1 acetone/methanol (Fisher Scientific, Silver Spring, MD) for ten
minutes at room temperature and air dried. Prior to staining, cells were hydrated with
deionized water for 10 minutes. VECTASHIELDTM (Vector Labs, Burlingame, CA)
mounting medium containing DAPI (0.75 µg/µL) was applied in the dark overnight,
followed by a rinse using deionized water. Alkaline phosphatase activity was assayed
using SK5100 Vector Red kit (Vector Labs, Burlingame, CA) according to the
manufacturer’s instructions.
The crosses were scanned using a fluorescent microscope using a 2048 x 3072
Quantix K6303E 12 bit digital camera (Roper Scientific) attached to a Leica DMXRA
motorized microscope controlled by ImagePro imaging software. 48 individual images
were collected in a total of 40 focal planes starting at the top of the scaffold at 461 x 344
pixels (1641.16 μm x 1224.64 μm), 8-bit gray level, using a 10x objective (pixel size =
3.56 µm). Each plane was separated by 50 µm. Individual images were stitched together
to form one montaged image per plane. This allowed capture and reconstruction of 3D
56
images from the top 50 µm of the 1800 µm thick scaffold. DAPI fluorescence localizing
individual cell nuclei were obtained with UV light (340-380 nm) excitation and
fluorescence was read at >425 nm. Alkalike phosphatase expression was imaged with
566 nm excitation and 575-670 nm emission wavelengths.
3.6.2. Verification: Images associated with the assessment of colony formation on
TheriLok™ scaffolds are shown in Figures 1 and 2. A movie moving through each of the
focal planes can be provided as supplemental material. Overall, evaluation of CTPderived colony formation was found to be challenged by several factors.
TCP
TheriLok™ scaffolds are brittle and subject to fracture and deformation during
processing and handling. This required hand selection of intact scaffold. The scaffolds
are optically opaque and also geometrically complex and porous. As a result, cells and
colonies were distributed into the pores of the material and along the vertical walls of the
scaffolds, making many of the cells present unobservable in the center and along the
sides of the scaffold below a depth of 50 µm. These limitations were judged to make the
overall utility of the method of colony assessment unacceptable, as outlined below.
Average
Score
Ease of
Preparation
Repeatability
Surface
Uniformity
Stability During
Culture Period
5
2.5
2.5
2
Identificati
on and
Detection of
Colonies
0
3.6.3. Revised Design: To overcome the limitations of the TCP scaffolds, methods were
explored to develop a two dimensional presentation of the TCP scaffold surface was
designed in which TCP could be presented on the surface of a substrate, reproducing the
surface texture of the TheriLok™ scaffolds, but limiting the depth of the porosity to less
57
than 30-40 µm. This exploration followed two primary phases in design, In Phase I,
three basic designs strategies were explored using TCP particles 46-106 µm diameter:
(1) Design 1 - Manual PDMS coating with addition of TCP particles before curing.
(2) Design 2 - Spin coating PDMS with addition of TCP particles before curing
(3) Design 3 - Spin coating preparation of PDMS with TCP particles followed by
mechanically pressing particles into the PDMS surface before curing of the PDMS.
In Phase II, following the selection of the design that satisfied the criteria of
success, was chosen as a preferred strategy, the design was further explored for the
optimization with respect to two primary variables: a) PDMS thickness (15 vs 35µm) and
b) TCP particle size (46-106 vs 26-45 vs less than 26 µm), as illustrated in Figure 6.
Ultimately, Design 2 using PDMS thickness of 35µm and TCP particle size of less than
or equal to 25 µm was selected as optimal, based on the stability of particles on the
surface, superior uniformity of coverage and smoothness.
The following detailed discussion as well as the images presented in Figure 6,
summarizes this process.
3.6.3.1. Phase I: Design 1: (manual preparation of PDMS coating with TCP
particles 46-106 µm diameter and removal of non-attached particles by manual
tapping): 18 mm diameter circular glass coverslips (thickness- 0.13-0.17 mm) (VWR,
Batavia, IL) were used as a base surface. Coverslips were cleaned by swirling them for
four hours in 2% ChemSolv glass cleaner solution (Mallinckrodt Baker, Phillipsburg, NJ)
at room temperature, followed by swirling three times for 5 minutes in deionized water
All swirling steps were performed at 100 rpm using a MaxQ 2000 Rotary shaker (Thermo
58
Scientific, Brookfield, WI). Coverslips were then silanized by swirling in filtered 2%
Siliclad (Gelest, Morrisville, PA) for 20 seconds, followed by swirling three times for 5
minutes in deoinized water. Silanized coverslips were cured at 100˚C for 20 minutes, left
at room temperature overnight, and then stored in a vacuum oven at room temperature.
PDMS (polydimethlysiloxane) was used to provide an adherent surface for TCP
particles. PDMS mixture was prepared using SYLGARD 184 Silicone Elastomer kit
(Dow Corning, Midland, MI) by mixing the Elastomer Base and curing agent at 10:1
ratio. A 100 ul droplet of PDMS was placed in a parafilm-lined plastic tray. A silanized
coverslip was placed on top of the droplet, and pressed gently using a separate larger
glass coverslip (2 cm x 6 cm). TCP powder (particle size < 106 µm) was provided as a
gift from Integra Lifesciences (Plainsboro, NJ).
Six PDMS-coated coverslips were
placed in a TCP powder- PDMS side down, and pressed gently for 30 seconds. Excess
unattached TCP particles were removed by manually holding the slide at a 90 degree
angle to the table top and tapping gently against a clean surface. Coverslips were cured at
room temperature overnight, washed twice with deionized water and stored till further
use.
3.6.3.2. Design 2: (Spin coated PDMS with TCP particles 46-106 µm diameter
layered on the surface, removal of non-adherent particles using sonication and air
blowing): In this design, a PDMS layer was applied on silanized glass coverslips by the
spin coating process. SYLGARD 184 Silicone Elastomer Base and the curing agent were
mixed in a 10:1 ratio at room temperature. 1 mL PDMS mixture was prepared for a batch
of 12 coverslips. The mixture was placed in a vacuum oven at room temperature for 20
59
minutes to remove air bubbles. Silanized glass coverslips were placed on a spin coater
controller head (Headway Research, Garland, TX). A 50µL PDMS mixture was
deposited on each coverslip, and was spun at 2500 rpm for 30 seconds. Each coverslip
was manually examined to ensure that there was no lint, debris or irregularities.
TCP powder (particle size < 106 µm) was sieved on the PDMS layer immediately
after spin-coating was completed. 1 gm TCP powder was poured into the sieve (VWR
Scientific), and TCP particles were gently pushed through the sieve using a spatula. TCP
particles were allowed to deposit onto the PDMS coat. TCP coated coverslips were gently
tapped to remove unattached TCP particles, and cured in a vacuum oven at 60˚C for 2
hours at atmospheric pressure. This curing step hardens the PDMS layer, thereby
providing a stable TCP coating. TCP surfaces were prepared in batches of 12. TCP
coated coverslips were sonicated upside down for 2 minutes in deionized water to remove
non-adherent TCP particles using the Aquasonic sonicator (VWR Scientific) at 50Hertz.
A gentle air stream was used to blow any remaining loose TCP from each coverslip.
Coverslips were then dried overnight and examined under the microscope to determine
coverage and artifacts. Coverslips were then dried overnight and stored at room
temperature till further use.
3.6.3.3. Design 3: (Spin coated PDMS with TCP particles 46-106 µm diameter
layered on the surface and secondarily pressed into the PDMS): In this approach,
design 2 was modified to include a step in which the TCP layer was pressed into the
PDMS layer using a clean 250 gm glass block for 10 seconds prior to curing. Pressing the
TCP layer was expected to provide a more uniform surface. The procedure developed in
60
design 2 was repeated, except the surfaces were pressed. The resulting surfaces were
scored for compliance with technical specifications.
To test these surfaces for effectiveness in detection of colony formation, surfaces
were sterilized under UV for 45 minutes each side, and then washing twice in 70%
ethanol followed by sterile 1X PBS. Coverslips were placed in a sterile 2cm x 2 cm
LabTek chamber (Nunc, Naperville, IL) and 500,000 nucleated marrow derived cells
were plated in 2mL osteogenic culture medium. Culture media was replaced after 72
hours. Scaffolds were fixed after 9 days with 1:1 acetone methanol and stained with
DAPI and AP, and colony formation was verified qualitatively under a fluorescent
microscope using appropriate filters for DAPI and AP wavelengths.
3.6.3.4. Verification: In Phase I, all three surface designs scored better than the TCP
scaffolds on repeatability, uniformity, stability during the culture time. As shown below,
Design 2 as scored higher than Designs 1 and 3 in ease of preparation, repeatability, and
surface uniformity. Based on this assessment and overall scores, Design 2 was chosen
for further verification experiments and validation.
Average
Score
Ease of
Preparation
Repeatability
Surface
Uniformity
Stability
During
Culture
Period
Identification
and Detection of
Colonies
Overall
Score
Design
1
3
3
3.5
5
4
18.5
Design
2
Design
3
4
4.5
4.5
5
4.5
22.5
3
3
4
5
4.5
19.5
61
3.6.4. Phase II - Optimization of Design 2 (with respect to PDMS thickness and
TCP particle size): The thickness of the PDMS coating depends of the viscosity of the
PDMS solution and the spin coat speed. A thin PDMS layer with larger particle size will
make the surface non-uniform and rough, and on the other side a thick PDMS layer with
smaller particle size may compromise the exposure of TCP particles on the surface. To
optimize the surface properties we used different combinations of the PDMS thicknesses
and the TCP particle sizes we used different combinations of the PDMS thicknesses and
the TCP particle sizes.
Two spin coat speeds (2000, 5000 rpm) were used to vary the thickness of the
PDMS coating. The expected coating thickness was 35 and 15 µm, respectively [41, 42] .
Sintered TCP granules, were sieved onto the PDMS coated coverslip to obtain particles
that were ≤25 µm, 26 - 45 µm, and 46-106 µm in size. To confirm that the TCP was
exposed on the surface (not covered by PDMS) and that the TCP was intimately
entrapped by the PDMS and not resting directly on the glass coverslip, a subset of TCP
coated coverslips were demineralized by rocking coated side down in 3 mL of 1M HCL
overnight and rinsed 3X for 5 min in deionized water, and left again overnight at 4C in 3
mL of 1X PBS.
A Scanning Electron Microscope (SEM) was used to obtain high magnification
images of the surfaces. SEM images were used to confirm the removal of TCP granules
from the surface and to screen for patches of exposed glass coverslip that would imply a
non-uniform thickness of PDMS.
62
3.6.5. Verification: A scoring system introduced about was used again to assess the
surface uniformity and smoothness. Surfaces were scored on the scale of 0 to 5 (5 for
Excellent, 4 Above Average, 3 Average, 2 Acceptable, 1 Poor, And 0 Unacceptable).
See Figure 6 for visual illustration of the findings for each thickness and particle size
combination.
PDMS Thickness of 15µm and Particle size: 46-106 µm: (Figure 6. Images A-C):
SEM images before and after demineralization demonstrated non-uniformity of the
PDMS surface, as evidenced by patches of exposed glass in Figure 6B. The larger
particle size lead to a rough pattern on the surface, with crevices with a depth more than
50 µm. The 15µm thickness of PDMS layer was inadequate to hold TCP particles in
many areas. Overall surface was scored as 1.
PDMS Thickness of 15µm and Particle size: 25-45 µm: (Figure 6 D-F): The
uniformity and smoothness of the TCP layer improved considerably. Further, SEM
images of demineralized surfaces verified that a PMDS thickness of 15µm provided a
stable base for 25-45 µm TCP particles. These surfaces were scored as 3.
PDMS Thickness of 15 µm and Particle size: <=25 µm: (Figure 6 G-I): Surfaces
prepared with 15µm thick PDMS also provided a stable base for TCP particles <=25 µm
in size. However, some irregularities were observed. (Figure 6G). These surfaces were
scored as 3.
63
PDMS Thickness of 35µm and Particle size: 46-106 µm: (Figure 6. Images J-L): The
increase in the thickness of PDMS To 35 µm improved the binding of TCP particles. The
PDMS thickness was adequate to hold the TCP particles. However, the roughness of the
surface was observed to be similar to figure (A). These surfaces were also scored as 3.
PDMS Thickness of 35µm and Particle size: 25-45 µm: (Figure 6 Images M-O): the
PDMS thickness was adequate to provide a binding surface for TCP particles. The
smoothness and the uniformity in coverage improved compared to previous
combinations. These surfaces were also scored as 4.
PDMS Thickness of 35µm and Particle size: < =25 µm: (Figure 6 Images P-R):
Surfaces prepared with 35 µm thick PDMS and <=25um particle size provided the most
uniform and the smoothest surface. The average score for these surfaces was 5. This
composition was used in subsequent experiments to evaluate in vitro performance with
and without TCPBP-EGF. These scored are summarized below:
PDMS Thickness (µm)
TCP Particle Size (µm)
Average Score
46-106
1
15
25-45
3
< =25
3
46-106
3
35
25-45
4
< =25
5
3.6.6. Colonyze™ algorithm: The existing Colonyze™ image processing algorithm has
been described in detail in prior publications [22, 25, 43]. As previously defined, each
imaging application (cell source and surface often produce colonies with a different
spectrum of features. As a result, the Colonyze™ method used in an individual setting
was refined to create a defined “profile” which sets specific variables used in identifying
64
cells and clustering cells into defined colony objects (e.g. thresholds for nuclear size,
staining intensity). The Colonyze™ profile refined for these applications was verified to
accurately detect at least 95% of the nuclei that were segmented against the autofluorescent TCP background, as shown in Figure 7.
3.6.7. Verification of the binding and stability of tethered EGF: Preparation of
tethered EGF modified TCP surfaces:
Eight TCP coverslips were tethered in one
parafilm-layered plastic tray. Plastic trays and parafilms were first cleaned with 70%
ethanol. Parafilms were then treated with sterile 1% BSA prepared in sterile 1X PBS
(Calbiochem) overnight at 4˚C in a sterile chamber, rinsed twice with sterile 1X PBS, and
allowed to dry. BSA coated parafilms were kept at 4˚C till further use. TCP surfaces were
sterilized under UV for 45 minutes each side, and then washed twice in 70% ethanol
followed by sterile 1X PBS. TCPBP-EGF protein was a provided by Dr. Linda Griffith’s
laboratory at MIT (Cambridge, MA). TCPBP-EGF solution was prepared at desired
concentrations on ice using 1X PBS as a diluting agent. BSA-coated parafilms were
placed inside plastic trays. 150 µL TCPBP-EGF droplets were placed on the parafilm,
and TCP coverslips was gently placed on the droplet with the TCP side interacting with
the protein solution. The entire assembly was sealed, and incubated at 4˚C for 36 hours in
a humidifying chamber.
3.6.8. Quantification of the surface density of tethered EGF and stability analysis:
TCPBP-EGF was tethered to TCP coverslips as described above. EGF-tethered
coverslips were rinsed twice with 1X PBS, placed in fresh PBS solution, and then stored
65
at 4 C for 0, 24 hours or 5 days (N=4). To measure stability, surfaces were removed and
rinsed twice with 1X PBS. The amount of EGF associated with each coverslip was
assessed by the BCA assay (Piercenet, Rockford, IL ) [44, 45].
3.6.9. Verification: At time = 0 the mass of tethered EGF per TCP coverslip was in the
range of 6000-7000 nanograms (mean= 6187, std. dev. 779) (Figure 8A), which met the
criteria for success (at least 1000 nanograms). We demonstrated the repeatability of the
tethering process by quantifying the amount of tethered EGF using two separate batches
of coverslips using the same reagents in a repeated experiment. The P value was
calculated as 0.16 using the standardized ANOVA methods for a 95% confidence
interval; it verified that the binding was repeatable. The coefficient of variation for this
comparison was 12.5 %.
The stability of the tethered EGF surface (tethering concentration = 2.7 µM) to
TCP coverslips was assessed in triplicate after storage at 0, 1, and 5 days in 1X PBS at
4oC. No significant degradation was detected over this interval. The P value was
calculated as >0.15 using the standardized ANOVA methods for a 95% confidence
interval. For the Day 0 vs. Day 1 comparison, the P value was 0.22, where for Day 0 vs
Day 5 comparison, the P value was 0.15. The overall coefficient of variation was 7.98%.
These data verify the stability of the presentation of tethered TCPBP-EGF. Tethered EGF
presentation met the defined criteria of success.
3.7. Discussion: In this chapter, we have introduced a method to assess the CTP response
to the surface modification of a biomaterial using an automated colony forming-unit
66
assay. This method overcomes several limitations of existing methods, enabling colony
detection on an opaque surface or a 3-dimensional form of scaffold material, and the
application of automated image capture and analysis to extract colony level assessment of
the CTP response to the surface modified biomaterials.
We first determined the scope and components of the design. This design is
applicable to biomaterials that are in a solid form (such as amorphous or crystalline
ceramics, hydroxyapatites, demineralized tissue powder, or micro and nanoscale solid
polymers), and can be used to prepare a stable surface (at least for the duration of the
assay -10 days). In the application demonstrated here, we used TCP, a synthetic bone
void filler, as a biomaterial, and then modified the surface of the TCP with EGF, using a
specific binding protein. We used bone marrow aspirate and trabecular surface-derived
cells as sources of connective tissue progenitors (CTPs).
The CTP response was
quantitatively assessed using an automated CFU assay system, Colonyze™ that meets the
standards in ASTM Standard Method (F2944-12) [23].
We defined the technical specifications associated with this method. These
included primary and secondary measurable outcomes of the CFU assay, and the criteria
for success. We proposed to assess CTP performance colony forming efficiency,
proliferation, migration and osteoblastic differentiation. We further established the
inclusion/ exclusion criteria for colonies, and established criteria of success to assess
whether a surface modification is sufficient to significantly influence the CTP response.
A scoring system was designed to assess biomaterial surfaces, and surface
modification. Surfaces were scored for ease of preparation, repeatability, uniformity and
consistency in coverage, stability and colony formation. Surfaces that did not meet the
67
criteria for success were revised until the criteria were met. The best performing surface
design was optimized for particle size and PDMS thickness. Surfaces with the maximum
score were advanced to analysis using Colonyze™ to assess the CTP response.
We first used existing scaffolds (TheriLok™ TCP scaffolds) to verify whether
they satisfy the technical specifications. Existing scaffolds had several limitations,
including a lack of stability during the culture period, and an inability to quantify colony
level parameters in 3-dimensional structure due to its architecture and optical opacity.
These limitations warranted a two-dimensional representation of a scaffold material. A
novel approach to surface preparation was introduced to prepare two-dimensional TCP
surfaces. Three surface designs were prepared, and scored for the compliance to the
technical specifications defined for biomaterial surfaces. The design with the highest
score (Design 2- Spin coated PDMS with TCP particles 46-106 µm diameter layered on
the surface, removal of non-adherent particles using sonication) was advanced for
optimization. This method can be used for any type of particulate biomaterial, ceramic or
polymeric to create a two-dimensional surface on a glass coverslip using a PDMS layer
as a binder to hold the particulate surface in place on the spin-coated on the glass.
Surface topography plays a key role in the cell response. Topography has effects
on cell-surface interactions, surface charges and hydrophobicity. The cell-surface
interactions and their effect on cell response have been discussed in detail in Chapter 2.
Considering the importance of surface topography on cell response, we verified means to
measure the surface uniformity, and smoothness based on the SEM images. A scoring
system was defined, and three particle sizes and two PDMS thickness layers were
verified for compliance. The surface with <=25 µm particles size and 2000 rpm rotation
68
speed provided the best score. These surfaces were advanced to the surface modification
and colony formation tests.
We used TCP surfaces with tethered EGF as a prototype of a surface modified
biomaterial. We quantified the amount of tethered EGF in separate a series of 4
experiments, and at different time points up to 5 days. We verified that the amount of
tethered EGF was sufficient to induce a ligand- receptor interaction. We verified that
EGF can be tethered to TCP surface in a repeatable, consistent manner. We also verified
that the EGF remains stable for at least 5 days in 1X PBS.
We further verified that these surfaces prepared using these methods can be used
to assess CTP colony level response using an automated CFU assay, Colonyze™
platform. The benefits of using Colonyze™ are discussed in Chapter 2. Expanding this
range of Colonyze™ analysis capabilities from translucent two dimensional surfaces to
non-translucent textured surfaces using PDMS as a binder rather than the surface itself
opens the potential for exploration of broad range of biomaterials and surface
modification methods. Application of this method required the development of an
application-specific profile in Colonyze™ to enable accurate cell segmentation and
identification on the auto-fluorescent surface background. Further, we verified at least
90% accuracy in cell segmentation. In Chapter 4, we will use this unified approach to
validate this method by quantifying the CTP response to TCP coverslips with tethered
EGF.
69
3.8.
Tables and Figures:
Figure 1: The engineering design method: Use of engineering design principles to
design a method to assess the connective tissue progenitor cell-response to surface
modified biomaterials
70
Parameter
Definition
Primary Outcomes (Directly Measured)
A cluster of 8 or more cells separated by less than 142
Colony
µm from each other
Number of cells in each colony (counted based on
Colony Size
DAPI stained nuclei)
Ratio of the number of cells and colony area
Colony density
Area of Alkaline phosphatase Area of the colony that is stained positively for AP
expression
(AP) (AAP)
Secondary Outcomes (Calculated)
Ratio of the mean observed prevalence of colony
Relative Colony Forming
forming units on the surface of a test material divided
Efficiency (CFE)
by the mean number of colonies on the control material
Median number of cells per colony on the test surface
Colony Size (metric of
standardized to the median number of cells per colony
effective proliferation)
on the control surface
Ratio of colony density on the test surface divided by
Relative Migration (metric
colony density on the control surface.
related to Colony density)
Area of the colony that is stained positively for AP
Area Fraction of Alkaline
expression divided by total number of cells in each
phosphatase (AP) (metric of
colony
differentiation)
Table 1: Primary and secondary outcomes of the method
(B)
Figure 2: Assessment of TheriLok™ TCP scaffolds. (A) Macroscopic dimensions, (B)
Disintegration of scaffolds after hydration in 1X PBS.
71
(A)
(B)
(C)
(E)
(D)
Figure 3: Imaging approach to quantify colony-parameters in TheriLok™ scaffolds.
(A) A schematic of Z-stacking method, (B-E) Segmentation of nuclei within a plane
below 100 µm. (B,C) Note the limitation of the opacity of the scaffold in identifying the
nuclei in the middle part of the scaffold (Only the cells on the edge are segmented). (D)
Composite image of the DAPI-stained nuclei on the scaffold combining all 48 planes. (E)
Composite image of the DAPI-stained nuclei that positively expressed alkaline
phosphatase.
72
Design 1
Design 2
Design 3
Manual
preparation
of Spin coated PDMS with Modified Design 3: TCP
PDMS coating with TCP TCP particles 46-106µm particles are pressed into
particles 46-106µm diameter diameter layered on the the PDMS
surface
Figure 4: Initial proof of design of 2 D TCP coverslips: Three designs, as described in
section 5.3.2 are shown here. Second row provides the design description. Third row
depicts the preparation of PDMS layer. Fourth row depicts the deposition of PDMS
particles. Fifth row provide snapshots of these surfaces. Sixth (bottom) shows the
formation of CTP-derived colonies. Cells were fixed after 9 days of culture and stained
for alkaline phosphatase (in red).
73
*
*
*
(A)
(B)
(C)
Figure 5: Expected thickness of the PDMS layer as a function of spin coater speed:
(A) The expected thickness of Slygard-184 PDMS layer is between 15-35 µm for the
speeds of 2000-5000 rpm [42]. (B) The thickness was confirmed to be 35 µm at 2000
rpm using SEM image analysis. (C) an SEM image at the scale of 100 µm to demonstrate
the uniformity in coverage of TCP coating on PDMS.
74
(A)
(B)
X100, Angle 45°
PDMS spin coat speed = 5000 rpm
PDMS Thickness = 15 µm
TCP Particle size 46-106 µm
(C)
Figure 6: Optimization of the TCP surface design, effect of particle size and PDMS
thickness on the uniformity of TCP surfaces: (A-C) Particle size- 46-106 µm, PDMS
thickness- 15 µm. Images were obtained at 100X magnification using a Scanning
Electron Microscope (SEM). (A) SEM image at the edge of a coverslip. Note the
roughness of the surface (B) SEM image of the coverslip that was demineralized with
HCl treatment. TCP particles were removed and PDMS layer was maintained. Note that
the PDMS thickness was inadequate to provide a binding surface for TCP particles of 46106 µm size. (C) SEM image at the center of the coverslip. Note the overall consistency
of the coverage.
75
(D)
X100, Angle 45°
PDMS spin coat speed = 5000 rpm
PDMS Thickness = 15 µm
TCP Particle size 26-45 µm
(E)
(F)
Figure 6: Optimization of the TCP surface design, effect of particle size and PDMS
thickness on the uniformity of TCP surfaces: (D-F) Particle size- 26-45 µm, PDMS
thickness- 15 µm. Images were obtained at 100X magnification using a Scanning
Electron Microscope (SEM). (D) SEM image at the edge of a coverslip. Smoothness of
the surface is improved compared to image (A) (E) SEM image of the coverslip that was
demineralized with HCl treatment. TCP particles were removed and PDMS layer was
maintained. Note that, unlike image (B), the PDMS thickness was sufficient to provide a
binding surface for TCP particles of 26-45 µm size. (F) SEM image at the center of the
coverslip. Note the overall consistency of the coverage.
76
(G)
X100, Angle 45°
PDMS spin coat speed = 5000 rpm
PDMS Thickness = 15 µm
TCP Particle size <=25 µm
(H)
(I)
Figure 6: Optimization of the TCP surface design, effect of particle size and PDMS
thickness on the uniformity of TCP surfaces: (G-I) Particle size- <= 25 µm, PDMS
thickness- 15 µm. Images were obtained at 100X magnification using a Scanning
Electron Microscope (SEM). (G) SEM image at the edge of a coverslip. Smoothness of
the surface was inconsistent compared to image (D) (H) SEM image of the coverslip that
was demineralized with HCl treatment. TCP particles were removed and PDMS layer
was maintained. Note that, unlike image (B), the PDMS thickness was sufficient to
provide a binding surface for TCP particles of <= 25 µm size. (I) SEM image at the
center of the coverslip. Note that the overall consistency of the coverage is improved
compared to (C) and (F)
77
(J)
X100, Angle 45°
PDMS spin coat speed = 2000 rpm
PDMS Thickness = 35 µm
TCP Particle size 46-106 µm
(K)
(L)
Figure 6: Optimization of the TCP surface design, effect of particle size and PDMS
thickness on the uniformity of TCP surfaces: (J-L) Particle size- 46-106 µm, PDMS
thickness- 35 µm. Images were obtained at 100X magnification using a Scanning
Electron Microscope (SEM). (J) SEM image at the edge of a coverslip. Note that the
roughness of the surface was similar to (A). (K) SEM image of the coverslip that was
demineralized with HCl treatment. TCP particles were removed and PDMS layer was
maintained. Note that the PDMS thickness was adequate to provide a binding surface for
TCP particles of 46-106 µm size. (L) SEM image at the center of the coverslip. Note the
overall consistency of the coverage.
78
(N)
(M)
X100, Angle 45°
PDMS spin coat speed = 2000 rpm
PDMS Thickness = 35 µm
TCP Particle size 25-45 µm
(O)
Figure 6: Optimization of the TCP surface design, effect of particle size and PDMS
thickness on the uniformity of TCP surfaces: (M-O) Particle size- 25-45 µm, PDMS
thickness- 35 µm. Images were obtained at 100X magnification using a Scanning
Electron Microscope (SEM). (M) SEM image at the edge of a coverslip. Note that the
roughness of the surface was similar to (D). (N) SEM image of the coverslip that was
demineralized with HCl treatment. TCP particles were removed and PDMS layer was
maintained. Note that the PDMS thickness was adequate to provide a binding surface for
TCP particles of 46-106 µm size. (O) SEM image at the center of the coverslip. Note the
overall consistency of the coverage.
79
(P)
X100, Angle 45°
PDMS spin coat speed 2000 rpm
PDMS Thickness = 36 µm
TCP Particle size ≤ 25 µm
(Q)
(R)
Figure 6: Optimization of the TCP surface design, effect of particle size and PDMS
thickness on the uniformity of TCP surfaces: (P-R) Particle size- <= 25 µm, PDMS
thickness- 35 µm. Images were obtained at 100X magnification using a Scanning
Electron Microscope (SEM). (P) SEM image at the edge of a coverslip. Note that the
roughness of the surface reduced compared to that in (M). (Q) SEM image of the
coverslip that was demineralized with HCl treatment. TCP particles were removed and
PDMS layer was maintained. Note that the PDMS thickness was adequate to provide a
binding surface for TCP particles of <= 25 µm size. (O) SEM image at the center of the
coverslip. Note the overall consistency of the coverage. These surfaces provided best
smoothness, consistency in coverage and TCP exposure, and hence were chosen for the
final design.
80
(A)
(B)
(C)
(D)
Figure 7: Verification of nuclei segmentation on opaque TCP surfaces using a
Colonyze™ algorithm: (A) A montaged image of a colony on the TCP surface. Cells
were stained with DAPI, and the image was obtained using a fluorescence microscope
using DAPI excitation and emission wavelengths. (B) The Colonyze™ algorithm
segments nuclei against the auto-fluorescent TCP background. (C) A colony was
identified from (B); Nuclei were colored blue. (D) Image (C) was superimposed on image
(A) to verify that at least 95% nuclei are accurately identified.
81
8000
Amount of TCPBP-EGF per
Coverslip (nanograms)
7000
6000
5000
4000
3000
2000
1000
0
Batch 1
Batch 2
Day 0
(A)
Amount of TCPBP-EGF per
Coverslip (nanograms)
8000
7000
6000
5000
4000
3000
2000
1000
0
Day 0
Day 1
Day 5
(B)
Figure 8: Quantification of the surface density of tethered TCPBP-EGF. (A) Amount
of tethered EGF is consistent in repeated experiments. Two repeated experiments (Batch
1 and Batch 2) were conducted to quantify the amount of tethered EGF on TCP
coverslips at Day 0. Coefficient of variation: 12.5%, P=0.16. The results show
consistency within each experiment, and between experiments (B) Tethered EGF
presentation is stable for at least 5 days at 4o C. Tethered EGF was quantified at three
time points (Days 0, 1 and 5). Day 0 vs Day1: P=0.22; Day 0 vs Day 5: P=0.15. The
results show the stability of tethered EGF presentation.
82
3.9.
Works Cited:
1.
Brodke, D., et al., Bone grafts prepared with selective cell retention technology
heal canine segmental defects as effectively as autograft. J Orthop Res, 2006.
24(5): p. 857-66.
2.
Bruder, S.P., et al., The effect of implants loaded with autologous mesenchymal
stem cells on the healing of canine segmental bone defects. J Bone Joint Surg Am,
1998. 80(7): p. 985-96.
3.
Cancedda, R., P. Giannoni, and M. Mastrogiacomo, A tissue engineering
approach to bone repair in large animal models and in clinical practice.
Biomaterials, 2007. 28(29): p. 4240-50.
4.
Hernigou, P., et al., Percutaneous autologous bone-marrow grafting for
nonunions. Influence of the number and concentration of progenitor cells. J Bone
Joint Surg Am, 2005. 87(7): p. 1430-7.
5.
Homma, Y., G. Zimmermann, and P. Hernigou, Cellular therapies for the
treatment of non-union: The past, present and future. Injury, 2013. 44 Suppl 1: p.
S46-9.
6.
Jager, M., et al., Cell therapy in bone healing disorders. Orthop Rev (Pavia),
2010. 2(2): p. e20.
7.
Hernigou, P., et al., Cell therapy of hip osteonecrosis with autologous bone
marrow grafting. Indian J Orthop, 2009. 43(1): p. 40-5.
8.
Hernigou, P., et al., Percutaneous implantation of autologous bone marrow
osteoprogenitor cells as treatment of bone avascular necrosis related to sickle
cell disease. Open Orthop J, 2008. 2: p. 62-5.
83
9.
Hernigou, P., et al., The use of percutaneous autologous bone marrow
transplantation in nonunion and avascular necrosis of bone. J Bone Joint Surg
Br, 2005. 87(7): p. 896-902.
10.
Hernigou, P. and F. Beaujean, Treatment of osteonecrosis with autologous bone
marrow grafting. Clin Orthop Relat Res, 2002(405): p. 14-23.
11.
Gronthos, S. and P.J. Simmons, The growth factor requirements of STRO-1positive human bone marrow stromal precursors under serum-deprived
conditions in vitro. Blood, 1995. 85(4): p. 929-40.
12.
Marcantonio, N.A., et al., The influence of tethered epidermal growth factor on
connective tissue progenitor colony formation. Biomaterials, 2009. 30(27): p.
4629-38.
13.
Gaudet, S., et al., A compendium of signals and responses triggered by prodeath
and prosurvival cytokines. Mol Cell Proteomics, 2005. 4(10): p. 1569-90.
14.
Janes, K.A., et al., The response of human epithelial cells to TNF involves an
inducible autocrine cascade. Cell, 2006. 124(6): p. 1225-39.
15.
Schlessinger, J., Common and distinct elements in cellular signaling via EGF and
FGF receptors. Science, 2004. 306(5701): p. 1506-7.
16.
Platt, M.O., et al., Sustained epidermal growth factor receptor levels and
activation by tethered ligand binding enhances osteogenic differentiation of multipotent marrow stromal cells. J Cell Physiol, 2009. 221(2): p. 306-17.
17.
Platt, M.O., et al., Multipathway kinase signatures of multipotent stromal cells are
predictive for osteogenic differentiation: tissue-specific stem cells. Stem Cells,
2009. 27(11): p. 2804-14.
84
18.
Kuznetsov, S.A., A.J. Friedenstein, and P.G. Robey, Factors required for bone
marrow stromal fibroblast colony formation in vitro. Br J Haematol, 1997. 97(3):
p. 561-70.
19.
Luria, E.A., A.F. Panasyuk, and A.Y. Friedenstein, Fibroblast colony formation
from monolayer cultures of blood cells. Transfusion, 1971. 11(6): p. 345-9.
20.
Owen, M. and A.J. Friedenstein, Stromal stem cells: marrow-derived osteogenic
precursors. Ciba Found Symp, 1988. 136: p. 42-60.
21.
Penfornis, P. and R. Pochampally, Isolation and expansion of mesenchymal stem
cells/multipotential stromal cells from human bone marrow. Methods Mol Biol,
2011. 698: p. 11-21.
22.
Powell, K.A., et al., Quantitative image analysis of connective tissue progenitors.
Anal Quant Cytol Histol, 2007. 29(2): p. 112-21.
23.
ASTM International, W.C., PA, ASTM F2944 - 12 "Standard Test Method for
Automated Colony Forming Unit (CFU) Assays—Image Acquisition and Analysis
Method for Enumerating and Characterizing Cells and Colonies in Culture",
2012, ASTM International, West Conshohocken, PA, 2003, astm.org.
24.
Kim, E.J., et al., Modulating human connective tissue progenitor cell behavior on
cellulose acetate scaffolds by surface microtextures. J Biomed Mater Res A,
2008.
25.
Caralla, T., et al., In vivo transplantation of autogenous marrow-derived cells
following rapid intraoperative magnetic separation based on hyaluronan to
augment bone regeneration. Tissue Eng Part A, 2013. 19(1-2): p. 125-34.
85
26.
Marcantonio, N.A., Boehm, C.A., Rozic, R., Au, A., Wells, A., Muschler, G.F.,
and Griffith, L.G, Tethered epidermal growth factor increases connective tissue
progneitor colony formation. Biomaterials, Submitted.
27.
Au, A., et al., Formation of osteogenic colonies on well-defined adhesion peptides
by freshly isolated human marrow cells. Biomaterials, 2007. 28(10): p. 1847-61.
28.
Erbe, E.M., et al., Potential of an ultraporous beta-tricalcium phosphate synthetic
cancellous bone void filler and bone marrow aspirate composite graft. Eur Spine
J, 2001. 10 Suppl 2: p. S141-6.
29.
Pochon, J.P., et al., [Bone replacement using beta-tricalcium phosphate--results
of experimental studies and initial clinical case examples]. Z Kinderchir, 1986.
41(3): p. 171-3.
30.
Mellonig, J.T., P. Valderrama Mdel, and D.L. Cochran, Histological and clinical
evaluation of recombinant human platelet-derived growth factor combined with
beta tricalcium phosphate for the treatment of human Class III furcation defects.
Int J Periodontics Restorative Dent, 2009. 29(2): p. 169-77.
31.
Gan, Y., et al., The clinical use of enriched bone marrow stem cells combined
with porous beta-tricalcium phosphate in posterior spinal fusion. Biomaterials,
2008. 29(29): p. 3973-82.
32.
Franch, J., et al., Beta-tricalcium phosphate as a synthetic cancellous bone graft
in veterinary orthopaedics: a retrospective study of 13 clinical cases. Vet Comp
Orthop Traumatol, 2006. 19(4): p. 196-204.
33.
Szabo, G., et al., A prospective multicenter randomized clinical trial of
autogenous bone versus beta-tricalcium phosphate graft alone for bilateral sinus
86
elevation: histologic and histomorphometric evaluation. Int J Oral Maxillofac
Implants, 2005. 20(3): p. 371-81.
34.
Muschik, M., et al., Beta-tricalcium phosphate as a bone substitute for dorsal
spinal fusion in adolescent idiopathic scoliosis: preliminary results of a
prospective clinical study. Eur Spine J, 2001. 10 Suppl 2: p. S178-84.
35.
Kay, J.F., Calcium phosphate coatings: understanding the chemistry and biology
and their effective use. Compend Suppl, 1993(15): p. S520-5; quiz S565-6.
36.
Rey, C., Calcium phosphate biomaterials and bone mineral. Differences in
composition, structures and properties. Biomaterials, 1990. 11: p. 13-5.
37.
Jarcho, M., Calcium phosphate ceramics as hard tissue prosthetics. Clin Orthop
Relat Res, 1981(157): p. 259-78.
38.
Griffith, L.G., Richard T. Lee, and Luis Manuel Alvarez., Modulating cell
behavior with engineered HER-receptor ligands, in Biological Engineering 2009,
Massachusetts Institute of Technology: Cambridge MA.
39.
Fan, V.H., et al., Tethered epidermal growth factor provides a survival advantage
to mesenchymal stem cells. Stem Cells, 2007. 25(5): p. 1241-51.
40.
Luangphakdy, V., et al., Evaluation of osteoconductive scaffolds in the canine
femoral multi-defect model. Tissue Eng Part A, 2013. 19(5-6): p. 634-48.
41.
Wu, H., B. Huang, and R.N. Zare, Construction of microfluidic chips using
polydimethylsiloxane for adhesive bonding. Lab Chip, 2005. 5(12): p. 1393-8.
42.
Balagadde, F.K., et al., Long-term monitoring of bacteria undergoing
programmed population control in a microchemostat. Science, 2005. 309(5731):
p. 137-40.
87
43.
Villarruel, S.M., et al., The effect of oxygen tension on the in vitro assay of human
osteoblastic connective tissue progenitor cells. J Orthop Res, 2008. 26(10): p.
1390-7.
44.
Chutipongtanate, S., et al., Systematic comparisons of various spectrophotometric
and colorimetric methods to measure concentrations of protein, peptide and
amino acid: detectable limits, linear dynamic ranges, interferences, practicality
and unit costs. Talanta, 2012. 98: p. 123-9.
45.
Walker, J.M., The bicinchoninic acid (BCA) assay for protein quantitation.
Methods Mol Biol, 1994. 32: p. 5-8.
88
CHAPTER 4: ASSESSMENT OF HUMAN CONNECTIVE TISSUE
PROGENITOR CELL RESPONSE TO Β-TRICALCIUM PHOSPHATE
SURFACES WITH TETHERED EPIDERMAL GROWTH FACTOR
4.1.
Abstract: Connective Tissue Progenitors (CTPs)
are
defined as
the
heterogeneous population of stem and progenitor cells that are resident in native tissues
and are capable of proliferation to give rise to progeny that will differentiate to express
one or more connective tissue phenotypes. Preclinical studies in both small and large
animal models have suggested that the concentration and prevalence of CTPs in many, if
not all bone defect sites is suboptimal. This is supported by the evidence that the
transplantation of sources of CTPs into bone defects improve bone regeneration. A
variety of methods have been used to harvest, process and transplant CTPs from bone or
bone marrow. However, the survival of transplanted cells is threatened both by profound
hypoxia and by the presence of pro-apoptotic cytokines. This work seeks to develop
practical methods to improve CTP performance using epidermal growth factor (EGF) as a
signaling molecule. EGF increases proliferation of culture expanded stem cells without
compromising their osteogenic potential, and thus has a potential to play a critical role in
osteogenesis. EGF was tethered to a β-tricalcium phosphate (TCP) scaffold via a novel
TCP binding peptide (TCPBP). TCPCP was identified using a phage display library, a
gene fusion was created to fuse EGF to TCPBP into a single TCPBP-EGF protein, which
was then expressed in E. coli, and purified. TCPBP-EGF was tethered to a TCP surface.
Tethering reduces the EGF degradation rate, improves control over its concentration and
presentation, and enables prolonged signaling. We evaluated the effect of tethered
TCPBP-EGF (tethered EGF) on CTP performance in vitro. The amount of tethered EGF
89
on the TCP surface was quantified, and its stability was demonstrated. CTPs were
obtained from trabecular surface or bone marrow samples. Tethered EGF enhanced
colony forming efficiency of CTPs in all eight patient samples (p< 0.03), with an average
increase of 62%. These data support the potential of tethered EGF presentation on the
surface of implantable TCP biomaterials as a means of enhancing the performance of
local and transplanted CTPs in a setting of bone repair or other tissue engineering
applications.
4.2.
Introduction: Each year, roughly 500,000 bone grafting procedures are
performed in the United States to treat various forms of orthopedic fixations [1-3],
including critical segmental bone defects (defined as a defect that cannot heal without
external intervention) [4]. Bone grafts and associated devices represent a $1.5 billion
industry in the United States [1-3]. Autogenous cancellous bone (ACB) is currently the
gold standard for the graft material, and is used in ~50% of these procedures [5-7].
However, the harvest of ACB is associated with significant morbidity, including surgical
scars, blood loss, pain, prolonged surgical time and rehabilitation, and infection risk [7].
These co-morbidities emphasize the need for effective alternatives to autografts that
could significantly improve the outcome, reduce the morbidity and/or cost of these
procedures.
Bone repair requires the activity of osteoblasts, which are derived from osteogenic
CTPs. Connective Tissue Progenitors (CTPs) are defined as the heterogeneous population
of stem and progenitor cells that are resident in native tissues and are capable of
proliferation to give rise to progeny that will differentiate to express one or more
90
connective tissue phenotypes [8-10]. Bone marrow has been widely used as a clinical
source of autogenous CTPs [11-14]. Bone marrow can be harvested from a bone marrow
aspirate with very low morbidity and minimal pre-processing compared to other sources
of CTPs such as fat and muscle [10, 15]. Several procedures have been explored to
increase CTPs concentration and/or reduce number of competing non-CTP cell
population from bone marrow [16-18]. In large bone defects, which are often associated
with local tissue trauma such as loss of muscle, periosteum and supporting vasculature,
the performance of transplanted CTPs may be significantly hindered by a harsh wound
environment, characterized by hypoxia and pro-inflammatory signaling molecules that
may apoptosis or necrosis [8, 10, 16, 19, 20].
EGF is one of many potential modulators of stem and progenitor cells in
orthopaedic tissue engineering applications, along with Bone Morphogenetic Proteins
(BMPs), Platelet Derived Growth Factor (PDGF), and basic Fibroblast Growth Factor
(bFGF). EGF is a potent mitogen for osteoblastic progenitors, and is both necessary and
sufficient to induce colony growth in bone marrow-derived cells [21, 22]. EGF signaling
prevents TNF-induced apoptosis in culture expanded marrow stromal cells [23-28].
Receptors for EGF ligand (EGFR) are expressed by virtually every cell type, including
stem and progenitor cells. EGFR is a receptor tyrosine kinase that activates intracellular
signaling cascades that influence cell proliferation, migration, and differentiation. EGFEGFR interaction induces EGFR dimerization and autophosphorylation, which activates
to downstream intracellular pathways. Once bound to the receptor, EGF is rapidly
internalized by receptor-mediated endocytosis leading to receptor desensitization, thereby
down-regulating the effects of exogenous EGF [29]. Compared to other growth factors,
91
EGF is a relatively small protein with a structure that is suitable for chemical
modification because of its singular amine group [30, 31] .
Molecular Surface Design can be used to tether growth factors such as EGF to the
surface of a biomaterial as a means of improving control over the cellular and tissue
response to the implant material. Tethering ensures that the signaling molecule does not
diffuse away from the cells rapidly, and enables control over the concentration and 3dimensional conformation of the molecule, which preserves its ability to interact with
receptors [22, 27, 32, 33]. In contrast, most current clinical therapies deliver growth
factors by diffusion release (e.g., BMPs). When released by diffusion, it is difficult to
control their local concentration and rate of clearance.
Beta-tricalcium phosphate (TCP) is clinically used osteoconductive bone void
filler [34-40]. The ability to modify the surface of TCP with growth factors could extend
its biofunctionality and improve its clinical performance. To explore this potential, novel
methods have been developed to tether bioactive EGF molecule to the surface of a TCP
coverslip using a high-affinity, non-covalent binding of a novel TCP binding peptide
(TCPBP) (Figure 1). This chapter tests the hypothesis that tethered EGF will significantly
improve the in vitro performance of CTP when cultured on a TCP surface.
4.3.
Methods: In Chapter 3, a new method was introduced to prepare reproducible
textured 2D surfaces suitable for the assessment of cellular response to a biomaterial
surface or a surface-modified biomaterial using any relevant adherent cell type. In this
Chapter, this method was validated using TCP that was modified with Epidermal Growth
92
Factor (EGF), a pro-survival and proliferation bioactive ligand, using a novel TCPbinding peptide (TCPBP-EGF).
This method several limitations of existing methods, enabling colony detection on
an opaque surface or a highly textured presentation of a scaffold material that is intended
to be used in a 3-dimensional form. This method is based on the preparation of the
biomaterial of interest in a particulate form (generally less than 25 µm in diameter, but
50-100 µm may be acceptable for some materials), and adhering these particles to the
substrate surface using polydimethlysiloxane (PDMS).
The PDMS surface is first
prepared in a uniform thickness using spin coating. A thickness of 35 µm was selected
for TCP, but may require further optimization for larger particle sizes or materials with
different wetting properties. Using this method of preparation, the biological response to
a variety of biomaterials can be compared in a setting where the effect of texture and
larger scale structural properties is controlled or limited.
4.3.1.
Fabrication of TCP surfaces: 18 mm diameter circular glass coverslips
(thickness- 0.13-0.17 mm) (VWR, Batavia, IL) were used as a base surface. Coverslips
were cleaned by swirling them for four hours in 2% ChemSolv glass cleaner solution
(Mallinckrodt Baker, Phillipsburg, NJ) at room temperature, followed by swirling three
times for 5 minutes in deionized water All swirling steps were performed at 100 rpm
using a MaxQ 2000 Rotary shaker (Thermo Scientific, Brookfield, WI). Coverslips were
then silanized by swirling in filtered 2% Siliclad (Gelest, Morrisville, PA) for 20 seconds,
followed by swirling three times for 5 minutes in deoinized water. Silanized coverslips
93
were cured at 100˚C for 20 minutes, left at room temperature overnight, and then stored
in a vacuum oven at room temperature.
A polydimethlysiloxane (PDMS) coating was used to provide a uniform adherent
surface for TCP particles (PDMS is a silicon-based organic polymer that is inert, nontoxic, non-flammable and optically clear). A PDMS layer was applied on silanized glass
coverslips by the spincoating process. SYLGARD 184 Silicone Elastomer Base and the
curing agent were mixed in a 10:1 ratio at room temperature. 1 mL PDMS mixture was
prepared for a batch of 12 coverslips. The mixture was placed in a vacuum oven at room
temperature for 20 minutes to remove air bubbles. Silanized glass coverslips were placed
on a spin coater controller head (Headway Research, Garland, TX). A 50µL PDMS
mixture was deposited on each coverslip, and was spun at 2000 rpm for 30 seconds. Each
coverslip was manually examined to ensure that there was no lint, debris or irregularities.
Thickness of the PDMS layer depends upon the speed of rotation and viscosity of
the mixture. Based on the values reported in the literature [41, 42], the thickness of the
PDMS layer is expected to be 30-40 µm. The thickness was later confirmed using
scanning electron microscope (SEM) images. Each coverslip coated with non-cured
PDMS was manually examined to ensure that there was no lint, debris or irregularities.
TCP powder (particle size < 106 um) was a gift from Integra Orthobiologics
(Plainsboro, NJ). TCP was sieved on the PDMS layer immediately after spin-coating was
completed. 1 gm TCP powder was poured into the 25 um sieve (VWR Scientific), and
TCP particles were gently pushed through the sieve using a spatula. TCP particles were
allowed to deposit onto the PDMS coat. TCP coated coverslips were gently tapped to
remove unattached TCP particles, and cured in a vacuum oven at 60˚C for 2 hours at
94
atmospheric pressure. This curing step hardens the PDMS layer, thereby providing a
stable TCP coating. TCP surfaces were prepared in batches of 12. TCP coated coverslips
were sonicated upside down for 2 minutes in deionized water to remove non-adherent
TCP particles using the Aquasonic sonicator (VWR Scientific) at 50Hertz. A gentle air
stream was used to blow any remaining loose TCP from each coverslip. Coverslips were
then dried overnight and examined under the microscope to determine coverage and
artifacts. Coverslips were then dried overnight and stored at room temperature till further
use.
4.3.2. Synthesis and purification of TCPBP-EGF protein: The TCPBP-EGF protein
was a gift from Dr. Linda Griffith’s laboratory at MIT (Cambridge, MA). A TCPbinding peptide (TCPBP), discovered through phage display, was fused to EGF (TCPBPEGF) and produced in E. coli [43]. An amylose resin was used to purify the final fusion
protein and an on-column method was used for endotoxin removal. This on column
method was modified from a previously published protocol [44]. Briefly, the columnbound protein was washed with 25 column volumes of a 1% Triton X-114 in 20mM Trisbuffer pH 7.4 and then washed with 25 column volumes of the same buffer without
Triton X-114. The purification was carried out at 4°C.
Purified TCPBP-EGF protein was tested for endotoxin using a standardized
Limulus Amebocyte Lysate (LAL) assay. Charles River Endosafe (CRE) system, a
standardized apparatus for LAL assay, was used (LAL is a lysate from horseshoe crab
(Limulus polyphemus) amoebocytes. LAL reacts with bacterial endotoxin or
lipopolysaccharide, which is a membrane component of gram negative bacteria. This
95
reaction is the basis of the LAL assay, which is used for the quantification of bacterial
endotoxins). The sensitivity of this assay kit was 0.05-5 Endotoxin units (EU)/ mL.
All reagents, including the protein sample to be tested, were warmed to room
temperature prior to use. The TCPBP-EGF protein was diluted to the desired
concentration in endotoxin-free certified water (Gibco, Billings, MT) under UV
sterilization to avoid any contamination. 25 µL of TCPBP-EGF protein sample was
loaded in each of 4 wells (n=4) of the CRE reader. The peak endotoxin reading, spike
recovery and coefficient of variation of TCPBP-EGF protein were compared with that of
the standard curve to determine the amount of endotoxin present in the protein.
The endotoxin level in TCPBP-EGF was found to be less than 0.05 EU/mL. The
endotoxin removal step reduced endotoxin levels in untreated TCPBP-EGF more than
100-fold over treated TCPBP-EGF.
4.3.3. Tethering TCPBP-EGF to TCP coverslips: A 4 cm x 6 cm plastic tray, lined
with parafilm (Sigma-Aldrich) was used as an apparatus for the tethering process. The
parafilm provides a uniform, chemically inert, hydrophobic surface for tethering. Eight
TCP coverslips were tethered in one parafilm-layered plastic tray. Plastic lids and
parafilms were first cleaned with ethanol and sterilized under UV (60-75 J/m2) for 45
minutes on each side. Parafilms were treated with sterile 1% BSA (Calbiochem)
overnight at 4˚C in a sterile chamber, rinsed twice with sterile 1X PBS, and allowed to
dry in a sterile hood. BSA-coated parafilms were kept in a sterile container at 4˚C till
further use. The BSA treatment is used to minimize the non-specific adsorption of
TCPBP-EGF on parafilms. TCP coverslips were sterilized using UV for 45 minutes on
96
each side, and subsequently with EtOH prior to tethering. TCPBP-EGF protein solution
was sterilely prepared at 2.7 µM on ice using sterile 1X PBS as a diluting agent. Sterile
BSA-coated parafilms were placed inside sterile plastic trays. 150 µL TCPBP-EGF
droplets were placed on the parafilm, and sterile TCP coverslips was gently placed on the
droplet with the TCP side interacting with the protein solution. The entire assembly was
sealed to maintain sterility, and was incubated at 4˚C for 36 hours in a humidifying
chamber. Coverslips were washed once with sterile 1X PBS immediately before cell
culture.
4.3.4. Quantification of the amount of tethered EGF and stability analysis: TCPBPEGF was tethered to TCP coverslips as described above. EGF-tethered coverslips were
rinsed twice with 1X PBS, placed in fresh PBS solution, and then stored at 4 C for 0, 24
hours or 5 days (N=4). To measure stability, surfaces were removed and rinsed twice
with 1X PBS. The amount of EGF associated with each coverslip was assessed by the
BCA assay (Piercenet, Rockford, IL) [45, 46].
4.3.5. Isolation of cells from bone marrow and trabecular space: Bone marrow
aspirates from the iliac crest, and discard bone cores from the proximal femur of human
subjects undergoing elective hip arthroplasty procedures were obtained under in an IRB
approved protocol with informed consent. Cells were harvested from either bone marrow
aspirate (BMA), or from trabecular surface (TS) of bone. Each marrow aspirate was
limited to a two ml volume from a given needle site, to limit dilution with peripheral
blood. Each sample was aspirated into one ml of normal saline containing 1000 units of
97
Na-Heparin. Ten aspirates were harvested from both the left and the right iliac crest (20
total). Each marrow sample was suspended in 20 mL α-minimal essential medium (αMEM) (Gibco, Billings, MT) containing 2 unit/mL Na-heparin to obtain a 23 mL
suspension for each aspirate.
Each heparinized bone marrow suspension was centrifuged at 400 x g for 10
minutes at room temperature. Nucleated cells were selected using a buffy coat isolation
[47, 48] (approximate volume 1.8mL) and re-suspended in α-MEM containing 0.3%
Bovine Serum Albumin (BSA) (Calbiochem). Suspension with buffy-coated cells was recentrifuged at 400 x g for 10 min. The second buffy coat was isolated and re-suspended
in α-MEM containing 0.3% BSA.
The double-buffy-coated cells were then pooled
together and nucleated cells in each aspirate were counted using a hemocytometer. This
cell suspension was used as a source of bone marrow aspirate (BMA) derived CTPs
(Cohort 1- three patients).
To harvest cells from the trabecular surface, the cancellous bone was washed in αMEM with antibiotic/antimycotic (Life Technologies, Carlsbad, CA), cut into small
pieces (1-2 mm in diameter) with an osteotome, and washed thoroughly to remove cells
from the marrow space. Cells that remained adherent to the trabecular surface were then
recovered by collagenase type I (100U/ml, Sigma, Inc.) digestion [49]. This cell
suspension was used as a source of trabecular surface (TS) derived CTPs (Cohort 2- 5
patients).
Cells from the BMA were plated at a density of 500,000 nucleated cells per
coverslip, and cells from TS were plated at a density of 200,000 cells per coverslip,
respectively (N=3). These plating densities were based on variations in the prevalence of
98
CTPs in BMA and TS samples (Data not Shown). The plating densities were chosen to
optimize colony formation with overgrowth (confluence) of colonies. Coverslips were
placed in 2.05 x 2.05 cm2 Lab-TekTM (Nunc, Logan, UT) glass chamber slides. An
osteogenic medium (α-MEM, 10% fetal bovine serum, 50 μM ascorbate, 10 nM
dexamethasone and penicillin/streptomycin) was used for all cell culture conditions, and
was changed every 3 days. Cultures were maintained at 37˚C in a humidified atmosphere
of 5% CO2 at 20% oxygen.
4.3.6. Staining, imaging and data analysis: Cells were harvested after nine days of
culture. At the time of cell harvest, the growth medium was removed by aspiration, and
culture chambers were vigorously rinsed with 1X PBS to remove non-adherent cells and
debris. The remaining cells were fixed with 1:1 acetone/methanol (Fisher Scientific,
Silver Spring, MD) for ten minutes at room temperature and air dried. Prior to staining,
cells were hydrated with deionized water for 10 minutes. VECTASHIELDTM (Vector
Labs, Burlingame, CA) mounting medium containing DAPI (0.75 µg/µL) was applied in
the dark overnight, followed by a rinse using deionized water. Alkaline phosphatase
activity was assayed using SK5100 Vector Red kit (Vector Labs, Burlingame, CA)
according to the manufacturer’s instructions. Dry coverlips were stored in the dark at
room temperature until image acquisition.
Coverslips were imaged using a 2048 x 3072 Quantix K6303E 12 bit digital
camera (Roper Scientific) attached to a Leica DMXRA motorized microscope controlled
by ImagePro imaging software. 240 individual images were collected at 461 x 344 pixels
(1641.16 μm x 1224.64 μm), 8-bit gray level, using a 10x objective (pixel size = 3.56
99
µm). Images of DAPI fluorescence localizing individual cell nuclei were obtained with
UV light (340-380 nm) excitation and fluorescence was read at >425 nm. Images of
Alkalike Phosphatase stained cells were obtained with 566 nm excitation and 575-670 nm
emission wavelengths.
Image processing and analysis were performed using a software algorithm
developed in the Muschler laboratory using algorithms written in the C/C++
programming language and Motif X-Windows TM environment [50]. Algorithms were
designed for background correction, montaging, nuclei segmentation, and quantitative
characterization of CTP-derived colonies. Briefly, individual images of one coverslip
were background-corrected by subtracting a smoothed (15 x 15 square average filter)
background image and montaged together to create one image of the entire coverslip.
Nuclear segmentation was accomplished using a gray value threshold and area
calculation. Colony clusters were identified in an automated fashion using Colonyze™.
Euclidian Distance Map was used to group nuclei falling within 142.4 μm of at least one
other nucleus into colony clusters of at least eight nuclei. Identified colonies on each slide
were reviewed using Colonyze™ to minimize any errors. Reviewed chambers were
quantified and analyzed for different colony parameters as described below.
4.3.7. Data validation and analysis: The effect of tethered EGF on CTP metrics
(number of colonies, number of cells per colony, expression of alkaline phosphatase, cell
density) was evaluated. A mean was calculated for the number of colonies for all patients
under each condition. A median was also calculated for each colony metric for each
patient sample: number of cells (nuclei) within the colony, cell density (nuclei per mm2),
100
and percentage of alkaline phosphatase positive area divided by cell number for all
colonies from each patient sample under each condition.
Due to the known wide
variation in CTP prevalence and performance between individual subjects, the outcome
of CTP response to TCP surfaces with tethered EGF were standardized by dividing the
outcome with the corresponding outcome for TCP surfaces with no tethered EGF for
each patient sample. This ratio provided a relative measure of various parameters for
each condition compared to control.
4.3.8. Statistical analysis: Statistical analysis was performed using standard ANOVA
methods. Given the historical patient-to-patient variability and heterogeneity of CTPs in
terms of colony forming efficiency (CFE) and proliferation, power analyses indicated that
a sample size of at least 8 patient samples was sufficient to yield a significant difference
with α = 0.05 and power > 0.80.
4.4.
Results and Discussion:
4.4.1. Characterization of 2D TCP coverslips: The average mass of a PDMS-coated
glass coverslip was 152 mg (ST DEV = 1.9, n = 5). The average mass of a TCP coated
coverslip was 172 mg (ST DEV = 4.3, n=5). Based on these calculations, the average
mass of TCP per coverslip was expected to be 20 mg. The presence of TCP particles,
consistency of the thickness of the TCP layer, and TCP particle size (less than 25 µm)
was later confirmed using SEM images (Figure 2B). SEM also confirmed the uniformity
of TCP surfaces within a range of 50 µm.
101
4.4.2. Quantification of the concentration and stability of tethered EGF on TCP
coverslips: The concentration and purity of TCPBP-EGF protein (molecular weight: 77
kDa, 70% purity) was determined by J. Rivera, MIT. We demonstrated the
reproducibility of the tethering process within and between batches by quantifying the
amount of tethered EGF in two separate experiments. At time = 0 the mean amount of
tethered EGF per TCP coverslip was in the range of 6000-7000 ng (Figure 3A) (P=0.16,
coefficient of variation: 12.5 %).
4.4.3. Quantification of the stability of tethered EGF on TCP coverslips:
Quantification of tethered EGF (tethering concentration = 2.7 µM) to TCP coverslips
after storage up to 5 days in 1X PBS at 4°C demonstrated that there was no statistically
significant difference in the amount of tethered EGF remaining on the surface after
different storage times (p-value>0.15, N = 3) (Figure 3B).
4.4.4. CTP response to tethered TCPBP-EGF:
4.4.4.1. Tethered EGF enhances CFE in bone marrow as well as trabecular
surface: Formation of osteogenic colonies in culture requires cell adhesion, survival,
proliferation, and expression of differentiation markers. Osteogenic colony formation is
typically assessed by plating cells in serum-containing medium onto glass or plastic
substrates so that adhesion is mediated by adsorbed serum proteins. In the CFU assay,
CTPs were plated on surfaces at the cell loading densities determined using historical
data on CTP prevalence from bone marrow aspirates and cells isolated from the
trabecular surface (Figure 4). The number of colonies (8 or more cells in a cluster)
102
formed, number of cells per colony, colony area, along with determination of
differentiation status, were counted after 9 days of culture.
Tethered EGF increased CFE on TCP surfaces by more than 62 % when
compared to TCP surfaces without tethered EGF, including both BMA- and TS-derived
cells (P<0.02) (Figure 4). Differences in the final colony number may arise from
differences in the number of CTPs adhering initially, activation of CTPs for form
colonies or differences in proliferation. These processes are both influenced by cues from
the culture substrate and cues from soluble medium components. Because the prevalence
of CTPs within a BMA sample differs from the prevalence of CTPs from the TS, we
asked if the ability of tethered EGF to affect colony forming efficiency might be
influenced by the source of CTPs. Tethered EGF improved CFE in both BMA and TS
derived CTPs, with an average increase of 57% in BMA derived cells and 72% in TS
derived cells (P<0.02) (Figure 4). The increase in CFE was consistent across all patient
samples.
These data emphasize the robustness of the observed CFE increase associated
with tethered EGF. Tethered EGF could cause an increase in CFE by increasing
adhesion, survival, proliferation, or a combination of these processes. While an initial
enhancement of CTP activation and proliferation would likely lead to an increase in
colony number, induction of proliferation at a later point would lead to an increase in the
number of cells per colony, and possibly the colony area. Therefore, we used quantitative
image analysis to further study CTP response by examining colony number and size.
103
4.4.4.2. Tethered EGF does not influence the number of cells per colony or cell
density: The median number of cells per colony remained the same in the presence of
tethered EGF, in both BMA- and TS-derived samples (Figure 5). Median colony area was
similar as well across all patients. Because an increase in colony area could be a result of
increased cell number resulting from proliferation, or increased migration, we also
assessed colony density by dividing the number of cells per colony by median colony
area. Our analysis revealed that colonies formed in the presence of tethered EGF had a
similar density to colonies without tethered EGF (Figure 6). This suggests the possibility
that an increase in CTP CFE is predominantly governed by the events of increased
adhesion and survival, and less so by proliferation or migration. It is possible that the
effect of tethered EGF could be in combination with other synergistic biological cues.
Further studies are needed to elucidate these interactions.
4.4.4.3. Tethered EGF maintains osteogenic potential of CTPs: With Colonyze™, it
is possible to examine the number of cells expressing alkaline phosphatase within a
colony by measuring the area of a colony expressing alkaline phosphatase, and
normalizing it to the number of cells within that colony. Figure 7 shows that alkaline
phosphatase expression within a colony was similar across all conditions (p>.05 for all
comparisons). These results are similar to the previously reported influence of tethered
EGF on the alkaline phospahatase expression in culture expanded MSCs as well as CTPs
[22, 43].
4.5.
Discussion: In this study, we examined the effect of tethered EGF on CTP colony
formation from cells obtained from freshly aspirated human bone marrow as well as cells
104
isolated from the surface of trabecular bone for potential tissue engineering applications.
We demonstrated that the amount of tethered EGF was adequate to induce a biological
cell response. However, we have not yet explored the possibility of a dose response.
TCPBP-EGF construct was previously found to be biologically active on passage 3
human MSCs [J Rivera and L Griffith, MIT- unpublished results]. We also demonstrated
the stability of the tethered EGF constructs for at least 5 days of incubation. We also
demonstrated that the endotoxin levels in the protein were 10-fold below commercial
standards of protein production.
This work demonstrated an increase in the CTP colony forming efficiency. This
observation could be due to an increase in the activation of CTPs in the sample. It is also
possible that an increase in the attachment or survival of CTPs could contribute towards
this effect. This data cannot distinguish between these mechanisms. Many prior studies
have demonstrated that EGF-induced signaling is a key effector of cell survival and
proliferation [25, 51-53]. Due to the inherent limitations in working with bone marrow as
well as trabecular bone, we could not perform the CFU assay using inhibitors to verify
the dependence of colony formation on EGF-induced signaling. Therefore, we cannot
definitively rule out other possible underlying mechanisms that are not induced by
EGFR-signaling. One possibility would be that increased colony formation was solely a
result of an increase in the mechanical attachment of cells resulting from a large number
of EGF-EGFR binding events. However, this is unlikely due to the fact that the density of
tethered EGF is significantly lower than the density of other cell-surface interaction
moieties. Another possibility is that the tethered EGF is promoting the selective
adsorption of serum factors that can then promote differential cell signaling. However,
105
previous results with culture expanded CTPs using a similar experimental system suggest
that our observed effects are EGFR-mediated [22, 32].
4.6.
Conclusion:
In conclusion, this study has successfully demonstrated tethering EGF to TCP
surfaces via a novel TCP binding peptide. TCPBP-EGF was tethered to a TCP surface at
6000-7000 ng per coverslip, and was stable for at least five days of incubation. Human
bone marrow and trabecular surface- derived cells demonstrated an increase in CFE on
the TCP surfaces with tethered EGF when compared to TCP surfaces without tethered
EGF, while maintaining the osteogenic potential. There was no change due to tethered
EGF in the number of cell per colony or colony density. The CTP response to tethered
EGF was seen in both bone marrow (three out of three) and trabecular surface (five out of
five) derived CTPs. In contrast, addition of soluble EGF did not improve CFE [data not
shown]. These findings support a possible value of tethered TCPBP-EGF as a modulator
of the clinically applicable biomaterials.
In future studies, the effect of tethered EGF on CTP response could be further
explored in terms of the effect of tethered EGF on long-term differentiation and
mineralization of CTPs. The dose response relationship between concentrations of
tethered EGF and CTP response could be evaluated. Further, TCP scaffolds with tethered
EGF could be tested for their efficacy in bone regeneration in an animal model, such as
the canine femoral multidefect model that has been used previously by the investigators
to assess the efficacy of bone graft materials [17, 54, 55]. The efficacy of tethered EGF
106
in augmenting CTP response can further be tested in presence of pro-inflammatory
cytokines or hypoxia, where the survival of cells could be a challenge.
The presentation strategy of tethered EGF can be further advanced to co-tether
other relevant bioactive factors when TCP-containing scaffolds are used. A similar
strategy might be implemented to identify binding peptides to other forms of
biomaterials, and tether bioactive factors to those biomaterials via a fusion of
corresponding binding peptides and growth factors.
4.7. Figures and Tables:
Figure 1: A schematic of presentation of tethered TCPBP-EGF on TCP surfaces.
The TCPBP is the binding domain that tethers the protein. The Coil domain provides
flexibility to the EGF molecule which is important for its biological activity. The coiled
domain also provides additional stability due to the coil-coil interactions between two
TCPBP-EGF proteins. The EGF domain is linked to the TCPBP at its C-terminus.
107
50uL PDMS Droplet is spin coated
Silanized Coverslip
Spin Coater
(A)
---------------------------------------------------------------------------------------------------------------------
TCP Particles
PDMS
Silanized Coverslip
(B)
(D)
(C)
Figure 2: Preparation and characterization of 2D TCP surfaces. (A, B) A schematic
of the process that involves coating a PDMS layer on a glass coverslip using a spincoater, and deposition of TCP particles on the PDMS coat, followed by a curing process
for surface hardening, (C) SEM image of a TCP surface, demonstrating the uniformity
and smoothness of the surface with 50 um range, (D) Colony formation on the TCP
coverslip. CTPs were plated on a TCP surface, fixed after 9 days of culture, and stained
for alkaline phosphatase (in red), a marker of early osteogenesis.
108
8000
Amount of TCPBP-EGF per
Coverslip (nanograms)
7000
6000
5000
4000
3000
2000
1000
0
Batch 1
Batch 2
Day 0
(A)
Amount of TCPBP-EGF per
Coverslip (nanograms)
8000
7000
6000
5000
4000
3000
2000
1000
0
Day 0
Day 1
Day 5
(B)
Figure 3: Quantification of the surface density of tethered TCPBP-EGF. (A) Amount
of tethered EGF is consistent in repeated experiments. Two repeated experiments (Batch
1 and Batch 2) were conducted to quantify the amount of tethered EGF on TCP
coverslips at Day 0. Coefficient of variation: 12.5%, P=0.16. The results show
consistency within each experiment, and between experiments (B) Tethered EGF
presentation is stable for at least 5 days at 4o C. Tethered EGF was quantified at three
time points (Days 0, 1 and 5). Day 0 vs Day1: P=0.22; Day 0 vs Day 5: P=0.15. Data was
obtained using ANOVA. The results show the stability of tethered EGF presentation.
109
Colony Forming Efficiency
Standardized to Control
2.5
Overall Average
Patient 1
Patient 2
Patient 3
Patient 4
Patient 5
Patient 6
Patient 7
Patient 8
2
1.5
1
*
0.5
0
No TCPBP-EGF (Control)
Tethered TCPBP-EGF
(A)
Colony Forming Efficiency
Standardized to Control
2.5
2
Overall
Average
Patient 1
1.5
Patient 3
*
1
0.5
0
No TCPBP-EGF (Control)
Tethered TCPBP-EGF
(B)
Colony Forming Efficiency
Standardized to Control
2.5
Overall Average
2
*
Patient 4
Patient 5
1.5
Patient 6
Patient 7
1
Patient 8
0.5
0
No TCPBP-EGF (Control)
Tethered TCPBP-EGF
(C)
Figure 4: Tethered EGF improves Colony Forming Efficiency (CFE) at Day 9. (A)
CFE of TCP surfaces with tethered EGF standardized to control (TCP coverslips with no
tethered EGF) for all 8 patients combined. (B) Tethered EGF improves CFE of CTPs
derived from bone marrow aspirates (3 patients) (C) Tethered EGF improves CFE of
CTPs derived from trabecular surface of bone (5 patients)
110
Median Number of Cells per Colony
Standardized to Control
1.2
Overall Average
1
0.8
Patient 1
Patient 2
Patient 3
0.6
Patient 4
Patient 5
0.4
Patient 6
Patient 7
0.2
Patient 8
0
Median Number of Cells per Colony
Standardized to Control
No TCPBP-EGF (Control)
Tethered TCPBP-EGF
(A)
1.2
1
0.8
Overall
Average
Patient 1
Patient 2
0.6
0.4
0.2
0
No TCPBP-EGF (Control)
Tethered TCPBP-EGF
(B)
Median Number of Cells per Colony
Standardized to Control
(2nd Cohort- 5 Patients) Median number of cells per colony after 9 days of culture
1.2
1
Overall
Average
Patient 4
Patient 5
0.8
Patient 6
0.6
0.4
Patient 7
Patient 8
0.2
0
No TCPBP-EGF (Control)
Tethered TCPBP-EGF
(C)
Figure 5: tethered EGF does not change number of cells per colony at Day 9. (A)
Number of cells per colony on TCP surfaces with tethered EGF standardized to control
(TCP coverslips with no tethered EGF) for all 8 patients combined. (B) CTPs derived
from bone marrow aspirates (3 patients) (C) CTPs derived from trabecular surface of
bone (5 patients).
111
Median Colony Density
Standardized to Control
1.4
1.2
1
Overall Average
Patient 1
Patient 2
Patient 3
0.8
0.6
Patient 4
Patient 5
Patient 6
0.4
0.2
Patient 7
Patient 8
0
Median Colony Density
Standardized to Control
No TCPBP-EGF (Control)
1.2
1
Tethered TCPBP-EGF
(A)
Overall
Average
Patient 1
Patient 2
0.8
Patient 3
0.6
0.4
0.2
0
Median Colony Density
Standardized to Control
No TCPBP-EGF (Control)
1.4
1.2
Overall
Average
Patient 4
1
Patient 5
0.8
Patient 6
0.6
Tethered TCPBP-EGF
(B)
Patient 7
Patient 8
0.4
0.2
0
No TCPBP-EGF (Control)
Tethered TCPBP-EGF
(C)
Figure 6: Tethered EGF does not change relative colony density at Day 9. (A)
Number of cells per colony on TCP surfaces with tethered EGF standardized to control
(TCP coverslips with no tethered EGF) for all 8 patients combined. (B) CTPs derived
from bone marrow aspirates (3 patients) (C) CTPs derived from trabecular surface of
bone (5 patients).
112
Median (AP+ Area/ Cell Number)
per Colony Standardized to Control
1.6
1.4
Overall
Average
Patient 1
1.2
Patient 2
1
Patient 3
0.8
Patient 4
0.6
Patient 5
0.4
0.2
0
Median (AP+ Area/ Cell Number)
per Colony Standardized to Control
No TCPBP-EGF (Control)
1.6
1.4
Overall
Average
Patient 1
1.2
Patient 2
1
Patient 3
(A)
0.8
0.6
0.4
0.2
0
No TCPBP-EGF (Control)
Median (AP+ Area/ Cell Number)
per Colony Standardized to Control
Tethered TCPBP-EGF
Tethered TCPBP-EGF
(B)
1.6
1.4
1.2
1
0.8
0.6
Overall
Average
Patient 4
Patient 5
Patient 6
Patient 7
Patient 8
0.4
0.2
0
No TCPBP-EGF (Control)
Tethered TCPBP-EGF
(C)
Figure 7: tethered EGF does not change % AP expression in colony cells at Day 9.
(A) Percentage Alkaline phosphatase (AP) expression is quantified as AP-positive area of
the colony divided by colony cell number. % AP expression on TCP surfaces with
tethered EGF standardized to control (TCP coverslips with no tethered EGF) for all 8
patients combined. (B) CTPs derived from bone marrow aspirates (3 patients) (C) CTPs
derived from trabecular surface of bone (5 patients).
113
4.8.
Works Cited:
1.
Greenwald, A.S., et al., Bone-graft substitutes: facts, fictions, and applications. J
Bone Joint Surg Am, 2001. 83-A Suppl 2 Pt 2: p. 98-103.
2.
Miyazaki, M., et al., An update on bone substitutes for spinal fusion. Eur Spine J,
2009. 18(6): p. 783-99.
3.
Giannoudis, P.V., H. Dinopoulos, and E. Tsiridis, Bone substitutes: an update.
Injury, 2005. 36 Suppl 3: p. S20-7.
4.
Schmitz, J.P. and J.O. Hollinger, The critical size defect as an experimental model
for craniomandibulofacial nonunions. Clin Orthop Relat Res, 1986(205): p. 299308.
5.
Samartzis, D., et al., Is autograft the gold standard in achieving radiographic
fusion in one-level anterior cervical discectomy and fusion with rigid anterior
plate fixation? Spine, 2005. 30(15): p. 1756-61.
6.
Zarate-Kalfopulos, B. and A. Reyes-Sanchez, [Bone grafts in orthopedic
surgery]. Cir Cir, 2006. 74(3): p. 217-22.
7.
Younger, E.M. and M.W. Chapman, Morbidity at bone graft donor sites. J Orthop
Trauma, 1989. 3(3): p. 192-5.
8.
Muschler, G.F. and R.J. Midura, Connective tissue progenitors: practical
concepts for clinical applications. Clin Orthop Relat Res, 2002(395): p. 66-80.
9.
Muschler, G.F., C. Nakamoto, and L.G. Griffith, Engineering principles of
clinical cell-based tissue engineering. J Bone Joint Surg Am, 2004. 86-A(7): p.
1541-58.
114
10.
Patterson, T.E., et al., Cellular strategies for enhancement of fracture repair. J
Bone Joint Surg Am, 2008. 90 Suppl 1: p. 111-9.
11.
Giannini, S., et al., Histological and immunohistochemical analysis of an
allogenic bone graft engineered with autologous bone marrow mononuclear cells
in the treatment of a large segmental defect of the ulna. A case report. Chir
Organi Mov, 2008. 91(3): p. 171-5.
12.
Park, I.H., I.D. Micic, and I.H. Jeon, A study of 23 unicameral bone cysts of the
calcaneus: open chip allogeneic bone graft versus percutaneous injection of bone
powder with autogenous bone marrow. Foot Ankle Int, 2008. 29(2): p. 164-70.
13.
Tilley, S., et al., Taking tissue-engineering principles into theater: augmentation
of impacted allograft with human bone marrow stromal cells. Regen Med, 2006.
1(5): p. 685-92.
14.
Ateschrang, A., et al., Fibula and tibia fusion with cancellous allograft vitalised
with autologous bone marrow: first results for infected tibial non-union. Arch
Orthop Trauma Surg, 2009. 129(1): p. 97-104.
15.
McLain, R.F., et al., Aspiration of osteoprogenitor cells for augmenting spinal
fusion: comparison of progenitor cell concentrations from the vertebral body and
iliac crest. J Bone Joint Surg Am, 2005. 87(12): p. 2655-61.
16.
Muschler, G.F., et al., Selective retention of bone marrow-derived cells to
enhance spinal fusion. Clin Orthop Relat Res, 2005(432): p. 242-51.
17.
Caralla, T., et al., In vivo transplantation of autogenous marrow-derived cells
following rapid intraoperative magnetic separation based on hyaluronan to
augment bone regeneration. Tissue Eng Part A, 2013. 19(1-2): p. 125-34.
115
18.
Castillo, T.N., et al., Comparison of growth factor and platelet concentration
from commercial platelet-rich plasma separation systems. Am J Sports Med,
2011. 39(2): p. 266-71.
19.
Villarruel, S.M., et al., The effect of oxygen tension on the in vitro assay of human
osteoblastic connective tissue progenitor cells. J Orthop Res, 2008.
20.
Muschler, G.F., R.J. Midura, and C. Nakamoto, Practical Modeling Concepts for
Connective Tissue Stem Cell and Progenitor Compartment Kinetics. J Biomed
Biotechnol, 2003. 2003(3): p. 170-193.
21.
Gronthos, S. and P.J. Simmons, The growth factor requirements of STRO-1positive human bone marrow stromal precursors under serum-deprived
conditions in vitro. Blood, 1995. 85(4): p. 929-40.
22.
Marcantonio, N.A., et al., The influence of tethered epidermal growth factor on
connective tissue progenitor colony formation. Biomaterials, 2009. 30(27): p.
4629-38.
23.
Gaudet, S., et al., A compendium of signals and responses triggered by prodeath
and prosurvival cytokines. Mol Cell Proteomics, 2005. 4(10): p. 1569-90.
24.
Janes, K.A., et al., The response of human epithelial cells to TNF involves an
inducible autocrine cascade. Cell, 2006. 124(6): p. 1225-39.
25.
Fan, V.H., et al., Tethered epidermal growth factor provides a survival advantage
to mesenchymal stem cells. Stem Cells, 2007. 25(5): p. 1241-51.
26.
Rodrigues, M., et al., Production of reactive oxygen species by multipotent
stromal cells/mesenchymal stem cells upon exposure to fas ligand. Cell
Transplant, 2012. 21(10): p. 2171-87.
116
27.
Tamama, K., et al., Epidermal growth factor as a candidate for ex vivo expansion
of bone marrow-derived mesenchymal stem cells. Stem Cells, 2006. 24(3): p. 68695.
28.
Wells, A., et al., Motility signaled from the EGF receptor and related systems.
Methods Mol Biol, 2006. 327: p. 159-77.
29.
Schlessinger, J., Common and distinct elements in cellular signaling via EGF and
FGF receptors. Science, 2004. 306(5701): p. 1506-7.
30.
Kuhlman, W.A., et al., Chain Conformations at the Surface of a Polydisperse
Amphiphilic Comb Copolymer Film. Macromolecules, 2006. 39(15): p. 51225126.
31.
Kuhlman, W., et al., Interplay between PEO tether length and ligand spacing
governs cell spreading on RGD-modified PMMA-g-PEO comb copolymers.
Biomacromolecules, 2007. 8(10): p. 3206-13.
32.
Platt, M.O., Roman, A.J., Wells, A., Lauffenburger, D.A., and Griffith, L.G ,,
Sustained epidermal growth factor levels and activation by tethered ligand
binding enhances osteogenic differentiation of multi-potent marrow stromal cells.
J. Cell Phys, 2009.
33.
Griffith, L.G., Emerging design principles in biomaterials and scaffolds for tissue
engineering. Ann N Y Acad Sci, 2002. 961: p. 83-95.
34.
Erbe, E.M., et al., Potential of an ultraporous beta-tricalcium phosphate synthetic
cancellous bone void filler and bone marrow aspirate composite graft. Eur Spine
J, 2001. 10 Suppl 2: p. S141-6.
117
35.
Pochon, J.P., et al., [Bone replacement using beta-tricalcium phosphate--results
of experimental studies and initial clinical case examples]. Z Kinderchir, 1986.
41(3): p. 171-3.
36.
Mellonig, J.T., P. Valderrama Mdel, and D.L. Cochran, Histological and clinical
evaluation of recombinant human platelet-derived growth factor combined with
beta tricalcium phosphate for the treatment of human Class III furcation defects.
Int J Periodontics Restorative Dent, 2009. 29(2): p. 169-77.
37.
Gan, Y., et al., The clinical use of enriched bone marrow stem cells combined
with porous beta-tricalcium phosphate in posterior spinal fusion. Biomaterials,
2008. 29(29): p. 3973-82.
38.
Franch, J., et al., Beta-tricalcium phosphate as a synthetic cancellous bone graft
in veterinary orthopaedics: a retrospective study of 13 clinical cases. Vet Comp
Orthop Traumatol, 2006. 19(4): p. 196-204.
39.
Szabo, G., et al., A prospective multicenter randomized clinical trial of
autogenous bone versus beta-tricalcium phosphate graft alone for bilateral sinus
elevation: histologic and histomorphometric evaluation. Int J Oral Maxillofac
Implants, 2005. 20(3): p. 371-81.
40.
Muschik, M., et al., Beta-tricalcium phosphate as a bone substitute for dorsal
spinal fusion in adolescent idiopathic scoliosis: preliminary results of a
prospective clinical study. Eur Spine J, 2001. 10 Suppl 2: p. S178-84.
41.
Balagadde, F.K., et al., Long-term monitoring of bacteria undergoing
programmed population control in a microchemostat. Science, 2005. 309(5731):
p. 137-40.
118
42.
Wu, H., B. Huang, and R.N. Zare, Construction of microfluidic chips using
polydimethylsiloxane for adhesive bonding. Lab Chip, 2005. 5(12): p. 1393-8.
43.
Griffith, L.G., Richard T. Lee, and Luis Manuel Alvarez., Modulating cell
behavior with engineered HER-receptor ligands, in Biological Engineering 2009,
Massachusetts Institute of Technology: Cambridge MA.
44.
Salek-Ardakani, S., et al., High level expression and purification of the EpsteinBarr virus encoded cytokine viral interleukin 10: efficient removal of endotoxin.
Cytokine, 2002. 17(1): p. 1-13.
45.
Chutipongtanate, S., et al., Systematic comparisons of various spectrophotometric
and colorimetric methods to measure concentrations of protein, peptide and
amino acid: detectable limits, linear dynamic ranges, interferences, practicality
and unit costs. Talanta, 2012. 98: p. 123-9.
46.
Walker, J.M., The bicinchoninic acid (BCA) assay for protein quantitation.
Methods Mol Biol, 1994. 32: p. 5-8.
47.
Gilmore, M.J. and H.G. Prentice, Standardization of the processing of human
bone marrow for allogeneic transplantation. Vox Sang, 1986. 51(3): p. 202-6.
48.
Arnaud, F., et al., Bone marrow separation, purification and cryopreservation:
application for autologous bone marrow transplant (ABMT). Int J Artif Organs,
1985. 8(4): p. 209-14.
49.
Siclari, V.A., et al., Mesenchymal progenitors residing close to the bone surface
are functionally distinct from those in the central bone marrow. Bone, 2012.
50.
Powell, K.A., et al., Quantitative image analysis of connective tissue progenitors.
Anal Quant Cytol Histol, 2007. 29(2): p. 112-21.
119
51.
Burlacu, A., et al., Factors secreted by mesenchymal stem cells and endothelial
progenitor cells have complementary effects on angiogenesis in vitro. Stem Cells
Dev, 2013. 22(4): p. 643-53.
52.
Gibson, S., et al., Epidermal growth factor protects epithelial cells against Fasinduced apoptosis. Requirement for Akt activation. J Biol Chem, 1999. 274(25):
p. 17612-8.
53.
Joyner, D.E., et al., Potential for modulation of the fas apoptotic pathway by
epidermal growth factor in sarcomas. Sarcoma, 2011. 2011: p. 847409.
54.
Luangphakdy, V., et al., Evaluation of osteoconductive scaffolds in the canine
femoral multi-defect model. Tissue Eng Part A, 2013. 19(5-6): p. 634-48.
55.
Muschler, G.F., et al., Evaluation of bone-grafting materials in a new canine
segmental spinal fusion model. J Orthop Res, 1993. 11(4): p. 514-24.
120
CHAPTER 5: CONCLUSIONS
The overall goal of this dissertation was to contribute to the development of tissue
engineering strategies that could be used in the evaluation of cell interactions with
biomaterials or modified biomaterial surfaces. The specific design was validated using βTriCalcium Phosphate (TCP), a common biomaterial used in bone regeneration, and the
assessment of the targeted modification of the surface of TCP was performed with
respect to the in vitro performance of human bone and marrow-derived connective tissue
progenitors (CTPs) and their progeny. The assessment of cellular response to
biomaterials utilizes the recently adopted standard method for automated colony forming
unit (CFU) assay (ASTM F2944-12) [1], and is enabled by a customized software
platform developed in the Muschler Lab within the Department of Biomedical
Engineering (referred to as “Colonyze™”).
In Chapter 3, a new method was introduced to prepare reproducible textured 2D
surfaces suitable for the assessment of cellular response to a biomaterial surface or a
surface-modified biomaterial using any relevant adherent cell type. In Chapter 4, the
method was validated using TCP that was modified with Epidermal Growth Factor
(EGF), a pro-survival and proliferation bioactive ligand, using a novel TCP-binding
peptide (TCPBP-EGF). This Chapter (Chapter 5) summarizes the conclusions of Chapter
3 and 4 that may be helpful to future investigators.
5.1. The Need – Quantitative Assessment of the Effect of Scaffold Biomaterials and
Biomaterial surface Modifications on Relevant Cell Populations: Three dimensional
scaffolds that are relevant to biomedical applications are valuable as potential surfaces to
121
assess cellular interactions (attachment, survival, migration, proliferation, differentiation).
However, the vast majority of three dimensional biomaterial scaffolds are optically
opaque and also geometrically complex and porous. As a result, cells and colonies which
may adhere and respond to these materials are often distributed into the deep pores of the
material and along the vertical walls or overhangs in the scaffold structures, making
many of the cells present on the scaffolds unobservable below a depth of 30-40 um.
Moroever, even if the deeper regions of a scaffold were accessible to direct measurement,
the thickness of these structures creates large variation in the local environment around
cells (due to diffusion gradients in standard media conditions).
The TCP TheriLok™ scaffolds used in the validation of this approach had these
limitations. In addition, the TCP TheriLok™ scaffolds are brittle and subject to fracture
and deformation during processing and handling.
A new method was introduced to overcome several limitations of existing
methods, enabling colony detection on an opaque surface or a highly textured
presentation of a scaffold material that is intended to be used in a 3-dimensional form.
This method is based on the preparation of the biomaterial of interest in a particulate
form (generally less than 25 µm in diameter, but 50-100 µm may be acceptable for some
materials),
and
adhering
these
Polydimethylsiloxane (PDMS).
particles
to
the
substrate
surface
using
The PDMS surface is first prepared in a uniform
thickness using spin coating. A thickness of 35 µm was selected for TCP, but may
require further optimization for larger particle sizes or materials with different wetting
properties and densities. Using this method of preparation, the biological response to a
variety of biomaterials can be compared in a setting where the effect of texture and larger
122
scale structural properties is controlled or limited. Once a physically and chemically
stable 2D particulate surface is prepared, it is then possible to secondarily modify the
chemical surface of the particular bulk material systematically to assess the biological
effect of surface modification.
While these methods were applied and validated specifically for TCP and CTPs
and their progeny, with a focus on applications in bone tissue regeneration, these methods
can readily be applied to a variety of materials and cell types which have relevance well
beyond bone regeneration, including cells and materials relevant to: skin and wound
healing,
nerve
regeneration,
muscle
regeneration,
tendon/ligament,
vascular
reconstruction, as well as hollow and solid organs. The only intrinsic requirements in this
method is that the bulk biomaterial utilized in this process be relatively chemically stable
in a aqueous (culture media) environment at physiological pH.
A scoring system was designed to qualitatively evaluate and compare biomaterial
surface preparations. Surfaces were scored for ease of preparation and repeatability,
uniformity and consistency in coverage, stability and detection of colonies. This scoring
system can be further modified according to the requirements of the surface design, or the
available skillset of the user.
5.2. Quantitative Assessment using Colonyze™ and Analytical Principles in the
ASTM Standard Method (F2944-12): The feasibility of using Colonyze™ to facilitate
automated colony forming unit assay based on the recently adopted ASTM Standard
Method (F2944-12) was demonstrated. Colonyze™ was used to identify cells on the
material surface and to group the identified cells into colony objects, each representing
123
the progeny of a colony founding connective tissue progenitor present in the initial tissue
sample. An increase in the number of colonies formed (observed prevalence of CTPs)
when the TCP surface was modified using the TCPBP-EGF peptide provided
confirmation that this surface modification resulted in an increase in colony forming
efficiency (CFE) by tissue derived CTPs. These methods also enable the detection of
relative differences in effective proliferation rate (EPR), cell density within colonies (a
metric of migration, expression of differentiation markers, as well as metrics of cell
survival.
5.3. Qualitative Scoring System for Surface Preparation: A qualitative scoring system
was used to evaluate and rank the value of various biomaterial surface preparations for
their compliance to technical specifications. The table below summarizes the results of
the verification step. The method was verified for six technical specifications. In all
cases, the criterion for success was met.
1
2
3
4
5
6
124
Technical
Specification
Repeatability
Uniformity in
coverage
Stability during
culture period
Ability to use
Colonyze for
CFU assay
Surface density
of bioactive
ligand
Stability of
surface
modification
Criteria for Success
Measured
Performance
4
Criteria
met?
Yes
4.5
Yes
5
Yes
4.5
Yes
At least 1000 ng per
coverslip
6000-7000 ng
per coverslip
Yes
At least 5 days
5 days
Yes
No score below 3, and
the best overall score
No score below 3, and
the best overall score
No score below 3, and
the best overall score
No score below 3, and
the best overall score
5.4. Characterization of Surface Preparation of TCP with and without Surface
Modification using TCPBP-EGF: In Chapter 4, the method was validated in its entirety
using β-TriCalcium Phosphate (TCP), a bone void filler material that was modified with
Epidermal Growth Factor (EGF), a pro-survival and proliferation bioactive ligand, using
a novel TCP-binding peptide. The amount of tethered EGF on the TCP surface was
quantified, and its stability was demonstrated. The colony forming efficiency was
measured as a ratio of the mean observed prevalence of colony forming units on the
surface of TCP surfaces with tethered Effective Proliferation Rate was measured as
median number of cells per colony on the TCP surface with tethered EGF standardized to
the median number of cells per colony on the TCP surface without tethered EGF.
Migration was estimated using the relative colony density, which as measured as a ratio
of colony density on TCP surface with tethered EGF divided by colony density on TCP
surface without tethered EGF. Osteoblastic differentiation was measured in terms of the
area fraction of Alkaline phosphatase (AP) which was measured as the area of the colony
that is stained positively for AP expression divided by total number of cells in each
colony.
This work has successfully demonstrated tethering EGF to TCP surfaces via a
novel TCP binding peptide. TCPBP-EGF was tethered to a TCP surface 6000-7000
molecules per µm2, and was stable for at least five days of incubation. Human bone
marrow and trabecular surface- derived cells demonstrated an increase in CFE on the
TCP surfaces with tethered EGF when compared to TCP surfaces without tethered EGF,
while maintaining the osteogenic potential. There was no change due to tethered EGF in
the number of cell per colony or colony density. The CTP response to tethered EGF was
125
seen in both bone marrow (three out of three) and trabecular surface (five out of five)
derived CTPs. In contrast, addition of soluble EGF did not improve CFE [data not
shown].
The efficacy of the TCP surfaces with tethered EGF was verified for four
technical specifications. In two cases, the criterion for success was met. The results of
validation are summarized below:
1
2
3
4
CFU assay
Colony Forming
Efficiency
Proliferation
Migration
Osteoblastic
differentiation
At least 50%
Improvement
At least 50%
improvement
Increases
Maintains
62%
Yes
No change
No
No change
No change
No
Yes
These results demonstrated that modifying bone scaffold materials with EGF may
have a value in improving the performance of CTPs in bone repair. These data support
the potential of tethered EGF presentation on the surface of implantable TCP biomaterials
as a means of enhancing the performance of local and transplanted CTPs in a setting of
bone repair or other tissue engineering applications.
5.5.
Works Cited:
1.
ASTM International, W.C., PA, ASTM F2944 - 12 "Standard Test Method for
Automated Colony Forming Unit (CFU) Assays—Image Acquisition and Analysis
Method for Enumerating and Characterizing Cells and Colonies in Culture", 2012,
ASTM International, West Conshohocken, PA, 2003, astm.org.
126
CHAPTER 6: FUTURE DIRECTIONS
The long-term goal of this research is to advance the field of orthopaedic tissue
engineering by improving the performance of osteogenic connective tissue progenitor
cells (CTPs) transplanted in a bone graft using surface modification of the graft material.
The purpose of this dissertation is to introduce and demonstrate a new method to assess
the response of CTPs to biomaterials or surface-modified biomaterials using an
automated colony forming unit (CFU) assay that has been recently adopted by the
American Society for Testing and Materials (ASTM) [1] (referred here as
“Colonyze™”). This method is particularly applicable to biomaterials that are solid,
opaque, and water insoluble or minimally degradable. Bone graft materials such as
ceramics (e.g., β-TriCalcium Phosphates (TCPs), hydroxyapatites, nanocrystalline
calcium phosphates), biologics (e.g., demineralized bone matrix, chitosan, lyophilized
collagen)
or
synthetic
polymers
(e.g.
Poly(methyl
methacrylate)
(PMMA),
Polycaprolactone (PCL), poly(propylene fumarate) PPF) are some examples of such
biomaterials.
The method described here is validated in its entirety using β-TriCalcium
Phosphate (TCP), a common bone void filler material. TCP was also modified with
Epidermal Growth Factor (EGF), a pro-survival and proliferation bioactive ligand, using
a novel TCP-binding peptide. The amount of tethered EGF on the TCP surface was
quantified, and its stability was demonstrated. TCP surfaces with tethered EGF were
shown to enhance colony forming efficiency (CFE) of CTPs obtained from trabecular
surface or bone marrow samples. These data support the potential value of tethered EGF
presentation on the surface of implantable TCP biomaterials as a means of enhancing the
127
performance of local and transplanted CTPs in a setting of bone repair or other tissue
engineering applications.
The work discussed in this dissertation could be continued in several possible
ways: to advance the understanding of the role of tethered EGF in influencing the
osteogenic progenitor cell response; to optimize the surface modification of TCP; or to
assess CTP response to other types of biomaterials and surface modifications that are
relevant to tissue engineering. In this chapter, these proposed studies are presented with
their significance, hypothesis and rationale, followed by the design of experiments. These
proposed studies include: assessment of CTP response to tethered EGF under normoxia,
and the stress induced by hypoxia and pro-death cytokines; understanding the mechanism
of CTP response to tethered EGF under hypoxia; assessment of the effect of tethered
EGF on bone regeneration in a large animal model; optimization of the surfaced
modification of TCP scaffolds; and assessment of CTP response to other bone graft
materials with or without surface modifications.
6.1.
Assessment of CTP response to tethered EGF under normoxia, and the cellular
stress induced by hypoxia and pro-death cytokines: transplantation of osteogenic
connective tissue progenitors (CTPs) into large bone defects is a clinical therapeutic
strategy that is based on the premise that the concentration and prevalence of
osteogenic cells in these defects sites is suboptimal. The performance of transplanted
cells is threatened both by the profound local hypoxia and the presence and pro-death
chemical signals that tend to drive cells to die by apoptosis or necrosis. Reduced
oxygen tension limits energy production and triggers various signaling pathways
128
regulating proliferation, apoptosis and differentiation [2-5], and therefore can
influence the performance of CTPs and other progenitor populations. The
inflammation of bone tissue elevates hypoxic stress, by increasing the density of
metabolically active cells in a wound site. Inflammation also increased the local
release and activity of multiple pro-death ligands including Fas ligand (FasL), tumor
necrosis factor (TNF), TNF-related apoptosis-inducing ligand (TRAIL), interferon γ
(IFN-γ), and interleukin 1 (IL-1) [6-9]. In many cell types, including culture
expanded MSCs, FasL induces apoptosis by activation of caspases [10-14].
However, the response of bone marrow derived to tethered EGF in the presence of
pro-death ligands such as FasL, particularly under conditions of hypoxia is as yet
poorly understood. Also, the CTP response to tethered EGF under normal
physiological oxygen tension (normoxia) is unknown. In this context, it is relevant to
test the efficacy of TCP modified with tethered EGF in improving CTP response
under these conditions.
The goal of this study is to test the influence of tethered EGF on the CFE,
proliferation, migration and osteoblastic differentiation of CTPs under conditions of
normoxia, hypoxia, and presence of FasL. Even though the mechanism of the response
can be assessed using a hypothesis based study that is described in Section 6.2, it is
relevant to first quantify the CTP response under these conditions using the method
described in this dissertation.
Many cell types, including culture expanded MSCs, respond to hyoxia or FasL
by activating canonical signal transduction pathways including, but not limited to, the
Mitogen-activated protein kinase (MAPK)- extracellular-regulated kinase (ERK)
129
pathway [5]. The MAPK-ERK signaling can be modulated through the EGF receptor
(EGFR) [15-19]. EGFR is expressed by virtually every cell type, including stem and
progenitor cells. EGFR is a receptor tyrosine kinase that activates intracellular signaling
cascades that influence cell proliferation, migration, and differentiation. The presence of
soluble EGF reduces hypoxia-induced apoptosis via EGFR-mediated MAPK activation
[20-22]. In the presence of FasL, the presence of tethered EGF improved the initial
attachment and survival of culture-expanded MSCs by sustained MAPKK-ERK signaling
[10, 11, 14]. The mechanism of tethered EGF-mediated cell response also involves
activation of MAPK-ERK pathways [12, 23]. Therefore, TCP surfaces with tethered EGF
are expected to improve the response of CTPs, compared to TCP surfaces without EGF.
All experiments will use bone marrow as a source of CTPs. The CTP response
will be evaluated at 0.1% oxygen (to mimic the hypoxic environment that develops after
closure of the surgical wound due to lack of vascular mass transport) [24], 3% oxygen (to
mimic normoxia- the physiological conditions in bone marrow) [24], and in the presence
of 100 ng/ml FasL (a dose that is sufficient to induce a 50% cell death in culture
expanded MSCs) [10]. CTP response at 20% will be used as a positive control. The
outcome measures will be colony forming efficiency CFE, proliferation, migration, and
osteoblastic differentiation at Days 6 and 9 using Colonyze™. These results will also
form a basis for the proposed study described in Section 6.2.
6.2. Understanding the mechanism of CTP response to tethered EGF under the
cellular stress induced by hypoxia and pro-death cytokines: The mechanism by
which tethered EGF mediates effects on CFE and proliferation under hypoxia or in
130
the presence of pro-death cytokines is uncertain. The objective of this proposed
follow-up study is to advance our understanding of the mechanism(s) by which
tethered EGF influences CTP performance under these conditions. The central
hypothesis of this study is that tethered EGF-dependent improvement in the in vitro
performance of CTPs is mediated, at least in part, by the activation of the Akt,
MAPK (Mitogen Activated Protein Kinase) and/or HIF-1α (Hypoxia Inducible
Factor-1α) signaling pathways, alone or in combination. This hypothesis is based on
the following rationale: in many cells types, including culture-expanded
mesenchymal stem cells, hypoxia stimulates cell death via apoptosis [25-27] and an
increase in caspase-3 activation (an intracellular shift from inactive to active caspase3) [22, 28]. Akt activation is necessary for cell survival in hypoxia [5]. Activation of
the Akt and MAPK pathways (phosphorylation of Akt and MAPK) inhibits apoptosis
by inhibiting caspase-3 activation and stabilization of HIF-1α. In addition to
survival, activation of the MAPK pathway can improve cell proliferation and
attachment, in many cell types [19, 29]. EGF is known to increase phosphorylation
of Akt and MAPK in normoxia in many cell types [19, 29]; however, its effect on
osteogenic progenitor cells under conditions of hypoxia is unknown. This study will
define the extent to which the biological effects of a biomaterial surface on
osteogenic progenitor cells under conditions of hypoxia are mediated through one or
more of these mechanisms.
This study can be advanced by first testing the hypothesis that tethered EGF
increases the phosphorylation of Akt and MAPK, increases HIF-1α stabilization and
decreases caspase-3 activation in osteogenic progenitor cells, human dermal fibroblasts
131
and endothelial cells when cultured in hypoxia. Further, the relative effect of tethered
EGF on the survival, proliferation and attachment of osteogenic progenitor cells, dermal
fibroblasts and endothelial cells under conditions of hypoxia can be quantified. This
mechanism can further be explored by testing the hypothesis that selective inhibition of
Akt, MAPK, and/or HIF-1α pathways will reduce survival, proliferation and/or
attachment of osteogenic progenitor cells, dermal fibroblasts and endothelial cells under
conditions of hypoxia.
Passage-1 cells obtained from adult human bone marrow derived connective
tissue progenitor cells (CTPs) can be used as a clinically relevant source of CTPs for this
study. Passage-1 human dermal fibroblasts and vascular endothelial cells derived from
the same patient subject can be isolated [30-32] , and used as an in-patient control to
determine the extent to which tethered EGF effects may be specific for osteogenic
progenitor cells or generalized to other clinically relevant human cell types.
The response of cells will be evaluated at 0.1% oxygen, and in presence of 100
ng/ml FasL. The cell EGF receptor (EGFR), pEGFR, Akt, pAkt, MAPK, pMAPK, HIF1α, stabilized HIF-1α (sHIF-1α), caspase-3, and cleaved caspase-3 (cCaspase-3) levels
will be measured using standard protein immumoblotting assays at early and late cell
culture periods to assess the durability of tethered EGF effects on cells. These levels will
be standardized to the levels of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a
housekeeping gene that is often stably and constitutively expressed at high levels in most
tissues and cells. The standardized pEGFR can confirm that tethered EGF-mediated
changes in downstream signaling are due to EGFR activation. Standardized levels of
132
pAkt, pMAPK, sHIF-1α, and cCaspase-3 can be used to determine the effect of tethered
EGF on the activation of these pathways.
The effect of tethered EGF on cell survival will be determined using a
standardized live-dead cell assay. The effect of tethered EGF on proliferation can be
determined in terms of the total DNA content of cells and a standardized
Bromodeoxyuridine (BrdU)-cell proliferation assay [33]. An imaging-based cell
quantification assay can be used to quantify the number of adherent cells.
Osteogenic progenitor cells can be treated with the selective inhibitors of Akt,
MAPK, HIF-1α and caspase-3 pathways respectively prior to seeding. The relative roles
of these pathways on tethered EGF-mediated cell survival, proliferation and attachment
as well as interaction between these pathways can be characterized. Cell response
evaluated at early and late time points will determine the effect of selective inhibition as a
function of time.
The characterization of the mechanism and pathways responsible for mediating
tethered EGF effects on the survival, proliferation and/or attachment and retention of
osteogenic progenitor cells on a biomaterial surface, as well as characterization of the
dose dependent effects of tethered EGF presentation, will enable rational development of
biomaterials that are modified with tethered EGF. Characterization of the effects on
dermal fibroblasts and endothelial cells will contribute to an understanding of the likely
safety profile of biomaterials presenting tethered EGF, as well as the potential application
of tethered EGF biomaterials to other tissue engineering applications.
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6.3.
Assessment of the effect of tethered EGF on bone regeneration in vivo: The
efficacy of tethered EGF in improving bone regeneration can be tested in a large
animal bone defect model. The canine femoral multidefect (CFMD) model and the
chronic caprine tibial defect (CCTD) model are relevant animal models for this
study. The rationale for choosing an animal model for the in vivo testing of
biomaterials has been discussed in [34].
The CFMD model has been used previously to assess the efficacy of bone graft
materials with transplanted bone marrow derived cells [35-37]. This model consists of
four equally sized and equally spaced cylindrical defects measuring 15 mm in depth and
10 mm in diameter created in the femur of a single dog. Bone regeneration is assessed at
four weeks. The defects are of sufficient size such that the interior of the defect is
characterized by profound hypoxia [24], a key feature of large clinical defects that is not
modeled in small animal defects. Bone formation and revascularization within the defect
occur through a process of ingrowth that has a radially oriented outside-in pattern, which
can be readily measured and characterized using microcomputed tomography (MicroCT)
and histological methods. The CFMD model system is a robust and sensitive system with
which to make objective comparisons between materials. The CFMD model allows these
comparisons to be made in a rapid and efficient manner (only 4 weeks) using a large
animal system, in a defect of clinically relevant size. By collecting four data points from
each animal, the CFMD model maximizes the value of each subject and minimizes the
number of subjects that are needed to provide sufficient power for comparisons.
Moreover, in addition to total bone formation and histology, the CFMD model provides
134
an opportunity in one model to assess the pattern, distribution, and remodeling features
associated with a material in varying biological settings.
The CFMD model will be used for initial screening and the dose response study.
TheriLok™ (Integra Orthobiologics) TCP scaffolds will be used for in vivo studies, as
their biocompatibility, and clinical efficacy as a bone graft has been tested [38-42].
TheriLok™ scaffolds with tethered TCPBP-EGF will be prepared using methods
described in Chapters 3 and 4. The selective retention method that has been described in
detail in [43] will be used for loading bone marrow-derived CTPs to the scaffolds.
Previous studies have demonstrated the efficacy of selective retention to load freshly
aspirated bone marrow-derived cells to graft materials with an increase in CTP loading
efficiency by 40-70% [37, 43]. TheriLok™ scaffolds with tethered TCPBP-EGF will be
implanted in two defect sites and TheriLok™ scaffolds without TCPBP-EGF will be
implanted in the other two defect sites. Implants will be fixed with a metal plate to
prevent scaffold movement and loss. The implant sites will be harvested four weeks post
implantation and the defect sites will be fixed and embedded till further use. Defect sites
will be isolated from femurs and will be imaged using microCT to measure the density of
newly mineralized tissue in the defect. We will also use histomorphologic methods to
further evaluate the extent of bone formation using Giemsa, Hematoxylin & Eosin
(H&E), and Goldner’s Trichrome stained 5 μm cross sections of the tissue from the
defect site. Giemsa and H&E will be used to differentially detect infectious and
inflammatory cells, while Goldner’s Trichrome will be used to identify mineralized bone
as well as calculate a percent of bone area using thresholding of microscopy images of
the mineralized bone stain. Analysis will be performed using ImagePro software for
135
reconstruction of microCT images and calculation of newly mineralized tissue volume. A
dose response study can be conducted to determine the best possible tethered EGF dose
that leads to maximum bone regeneration.
The Muschler lab, in collaboration with the University of Minnesota and the
Institute for Surgical Research, has developed the CCTD model, which creates a 5 cm
segmental bone defect, and incorporates the loss of regional soft tissues and periosteum,
local tissue scarring, and a fibrous induced membrane that forms around a PMMA spacer
placed at the site of the defect. It is intended to better represent the complexity and
severity of the actual biological environment in which clinical bone grafts currently fail.
In this model, each animal undergoes two surgical procedures, a “Pre-procedure” in
which a 5 cm tibial defect is created and preserved with the use of a
polymethylmethacrylate (PMMA) spacer, followed by the “Treatment Procedure” in
which various clinically relevant bone grafting treatment scenarios can be implemented.
During the Pre-procedure, the 5 cm defect is created and the tibia stabilized with an
intramedullary interlocking nail. The newly created defect is filled with a PMMA spacer
to induce a membrane to form at the defect site. At the treatment procedure, the PMMA
spacer is removed and the defect is filled with the test material used in the study; the
induced membrane serves to contain the test material.
The best performing tethered EGF dose from the CFMD study can be tested for
its efficacy using this model. Preparation of graft materials will be similar to the CFMD
study. Grafts will be implanted during the Treatment Procedure, 4 weeks after the Preprocedure. The tibia will be harvested for further assessment of defect healing using
136
microCT and histological analysis 12 weeks after implantation, and fixed for analysis.
The analysis methods will be same as the ones used for the CFMD model.
6.4.
Optimization of the surface-modification of TCP scaffolds: The presentation
strategy of tethered EGF can be further optimized in terms of the dose of tethered EGF,
co-presentation of other relevant bioactive factors along with EGF, and identification of
binding peptides for other forms of biomaterials.
In this dissertation, the efficacy of tethered EGF was demonstrated using only one
dose of tethered EGF, but its optimization was not assessed. In future experiments, a dose
response of tethered EGF could be assessed. The surface density of tethered EGF on the
TCP surface can be controlled using the concentration of the protein solution used for the
tethering step [44]. A dose response study can include 4 concentrations of tethered EGF
(50, 10, 0.1, 0.05-fold the concentration of tethered EGF used in this dissertation,
respectively). A tethered EGF dose that was used in Chapter 2 (incubation of scaffolds in
2.7 uM TCPBP-EGF solution) will be used as a positive control.
Optimization of TCPBP-EGF protein is an active area of research. Researchers at
MIT are modifying the TCPBP protein construct to improve the stability and binding.
These modifications include addition of repeated binding domains, and modification of
TCPBP-EGF protein to improve binding and biofunctionality. In addition, novel proteins
constructs are being fabricated to co-tether other bioactive ligands, such as Heregulin,
with EGF using a single binding domain.
The efficacy of tethered EGF can be further assessed in terms of its effect on the
long-term differentiation and mineralization of CTPs. As shown in chapter 4, there was
137
no tethered EGF-induced effect on osteoblastic differentiation of CTPs after 9 days of
culture. However, it is conceivable that tethered EGF could affect the differentiation
process at later time points. A positive tethered EGF-induced effect on long term
differentiation and mineralization would indicate that the improved colony formation of
CTPs translates to improved long term osteogenesis.
6.5. Assessment of CTP response to surface modifications of other bone graft materials:
In chapter 3, we introduced a novel method to prepare 2-dimensional surfaces of
TCP material. These surfaces were characterized for their presentation and surface
uniformity. TCP is a class of ceramics used as bone void filler. A similar approach
could be used to prepare 2 dimensional surfaces of other amorphous and crystalline
materials used in tissue engineering applications. These materials include ceramics
(e.g.
hydroxyapatites,
nanocrystalline
calcium
phosphates),
biologics
(e.g.
demineralized bone matrix, chitosan, lyophilized collagen) or synthetic polymers
(e.g. Poly(methyl methacrylate) (PMMA), Polycaprolactone (PCL), poly(propylene
fumarate) PPF). The response of CTPs to these materials can be assessed using
Colonyze™, using the method described in Chapters 3 and 4. This method could be
used for a comparative assessment of these graft materials for their effect on CTP
performance. In addition, the efficacy of novel peptide binding domains that are
specific to these materials can be tested using this method.
6.6. Optimization of the assessment of the two-dimensional TCP surfaces: A novel
method to prepare two dimensional surfaces of a particulate of any bone scaffold material
138
was introduced in Chapter 3. This method was verified using the TCP material. These
surfaces were assessed with respect to their repeatability, stability, uniformity in
coverage, and smoothness using a semi-quantitative scoring system. High resolution
Scanning Electron Miscoscope (SEM) images were used to assess the smoothness and the
uniformity in coverage. A more quantitative method could be implemented for the
assessment in future studies. Atomic Force Microscopy (AFM) is a method to measure
the surface roughness. However, the AFM method is not well suited for the TCP surfaces
as AFM can measure the surface roughness only upto 20 micron range, whereas the
roughness of TCP surfaces was around 30-50 microns. Further, the use of AFM is limited
due to the fact that the AFM uses a very fragile sensor tip, which has a risk of damage
when used with a hard ceramic material such as TCP. Replacement of the tip can be very
expensive. To circumvent these limitations, a more suitable method to measure the
surface roughness in this range could be using non-contact, optical profilometers. There
are several optical profilometers that use non-contact mode to measure surface roughness
in the low to high micrometer range. The non-contact mode uses a light beam that is
emitted from a stationary tip. The light beam is reflected from the surface whose
roughness is being measured. The reflected beam is received at the same stationary tip
where the light beam is is originated. The time between the emitted and received light
beams is measured. The roughness of the surface is estimated using the variations in the
measured time, and is displayed in microns. In addition to the sensitivity range, the noncontact profilometer also eliminates the risk of damaging the sensor tip due to the surface
material that is being tested.
139
The uniformity of coverage of the TCP material can be assessed quantitatively
using an imaging-based approach. The low resolution (20-50 X magnification) SEM
images can be analyzed using advanced image processing software such as ImageJ or
ImagePro. The thresholds can be defined for the gray-levels that represent the TCP
particles (higher gray-level) and the PDMS layer (lower gray-level), and the image can be
analyzed to filter the relative values of the corresponding gray-levels. This method will
facilitate the quantitative assessment of the uniformity in terms of % area that is occupied
by TCP relative to the total area of the surface.
The scoring system that is introduced in Section 3.6 does not differentiate
between the relative significance of the parameters that were assessed, namely ease of
preparation, repeatability, surface uniformity, stability during culture period, and
identification and detection of colonies. Consequently, the parameters are valued at the
same level of significance (or importance). For example, a score of 3 in ease of
preparation is valued same as the score of 3 in the stability during culture period, even
though the stability during culture period is far more important in the response of cells to
the surface than the ease of preparation. This limitation of the proposed scoring system
can be circumvented by assigning a multiplier for each assessment parameter. The value
of the multiplier can be assigned based on the relative significance of the parameters. In
case of the scoring system discussed in Section 3.6, ease of preparation, repeatability,
surface uniformity, stability during culture period, and identification and detection of
colonies can be assigned the multiplier values of 1, 3, 5, 5 and 5 respectively. The
individual scores for the coverslip design will be multiplied by these values.
140
Consequently, the relative significance of the parameters will be reflected in the overall
score of the design.
6.7.
Concluding Remarks: The work in this dissertation has developed and
documented a new and novel method for quantitative assessment of the effects of
biomaterials and modified biomaterial surfaces on the performance of relevant cell
populations. Specifically relevant to bone tissue engineering, these data provided clear
evidence that the modification of a TCP biomaterial surface using a TCP binding peptide
fusion protein with EGF (TCPBP-EGF) will increase colony forming efficiency by bone
marrow-derived human connective tissue progenitors. Together with the proposed future
studies, this work can provide an important contribution towards advancing the current
standard of care of orthopaedic trauma in a clinical setting.
6.8.
Works Cited:
1.
ASTM International, W.C., PA, ASTM F2944 - 12 "Standard Test Method for
Automated Colony Forming Unit (CFU) Assays—Image Acquisition and Analysis
Method for Enumerating and Characterizing Cells and Colonies in Culture",
2012, ASTM International, West Conshohocken, PA, 2003, astm.org.
2.
Villarruel, S.M., et al., The effect of oxygen tension on the in vitro assay of human
osteoblastic connective tissue progenitor cells. J Orthop Res, 2008.
141
3.
Iwase, T., et al., Comparison of angiogenic potency between mesenchymal stem
cells and mononuclear cells in a rat model of hindlimb ischemia. Cardiovasc Res,
2005. 66(3): p. 543-51.
4.
Komatsu, D.E. and M. Hadjiargyrou, Activation of the transcription factor HIF-1
and its target genes, VEGF, HO-1, iNOS, during fracture repair. Bone, 2004.
34(4): p. 680-8.
5.
Das, R., et al., The role of hypoxia in bone marrow-derived mesenchymal stem
cells: considerations for regenerative medicine approaches. Tissue Eng Part B
Rev. 16(2): p. 159-68.
6.
Grossman, W.J., et al., Human T regulatory cells can use the perforin pathway to
cause autologous target cell death. Immunity, 2004. 21(4): p. 589-601.
7.
Wiley, S.R., et al., Identification and characterization of a new member of the
TNF family that induces apoptosis. Immunity, 1995. 3(6): p. 673-82.
8.
Dinarello, C.A., Biologic basis for interleukin-1 in disease. Blood, 1996. 87(6): p.
2095-147.
9.
Boehm, U., et al., Cellular responses to interferon-gamma. Annu Rev Immunol,
1997. 15: p. 749-95.
10.
Fan, V.H., et al., Tethered epidermal growth factor provides a survival advantage
to mesenchymal stem cells. Stem Cells, 2007. 25(5): p. 1241-51.
11.
Tamama, K., et al., Epidermal growth factor as a candidate for ex vivo expansion
of bone marrow-derived mesenchymal stem cells. Stem Cells, 2006. 24(3): p. 68695.
142
12.
Platt, M.O., Roman, A.J., Wells, A., Lauffenburger, D.A., and Griffith, L.G ,,
Sustained epidermal growth factor levels and activation by tethered ligand
binding enhances osteogenic differentiation of multi-potent marrow stromal cells.
J. Cell Phys, 2009.
13.
Tamama, K., H. Kawasaki, and A. Wells, Epidermal growth factor (EGF)
treatment on multipotential stromal cells (MSCs). Possible enhancement of
therapeutic potential of MSC. J Biomed Biotechnol. 2010: p. 795385.
14.
Rodrigues, M., et al., Surface tethered epidermal growth factor protects
proliferating and differentiating multipotential stromal cells from FasL-induced
apoptosis. Stem Cells, 2013. 31(1): p. 104-16.
15.
Jorissen, R.N., et al., Epidermal growth factor receptor: mechanisms of activation
and signalling. Exp Cell Res, 2003. 284(1): p. 31-53.
16.
Wiley, H.S., Trafficking of the ErbB receptors and its influence on signaling. Exp
Cell Res, 2003. 284(1): p. 78-88.
17.
Krampera, M., et al., HB-EGF/HER-1 signaling in bone marrow mesenchymal
stem cells: inducing cell expansion and reversibly preventing multilineage
differentiation. Blood, 2005. 106(1): p. 59-66.
18.
Leahy, D.J., Structure and function of the epidermal growth factor (EGF/ErbB)
family of receptors. Adv Protein Chem, 2004. 68: p. 1-27.
19.
Schlessinger, J., Common and distinct elements in cellular signaling via EGF and
FGF receptors. Science, 2004. 306(5701): p. 1506-7.
20.
Liu, L.Z., et al., Reactive oxygen species regulate epidermal growth factorinduced vascular endothelial growth factor and hypoxia-inducible factor-1alpha
143
expression through activation of AKT and P70S6K1 in human ovarian cancer
cells. Free Radic Biol Med, 2006. 41(10): p. 1521-33.
21.
Peng, X.H., et al., Cross-talk between epidermal growth factor receptor and
hypoxia-inducible factor-1alpha signal pathways increases resistance to
apoptosis by up-regulating survivin gene expression. J Biol Chem, 2006. 281(36):
p. 25903-14.
22.
Levy, R., et al., Apoptosis in human cultured trophoblasts is enhanced by hypoxia
and diminished by epidermal growth factor. Am J Physiol Cell Physiol, 2000.
278(5): p. C982-8.
23.
Platt, M.O., et al., Multipathway kinase signatures of multipotent stromal cells are
predictive for osteogenic differentiation: tissue-specific stem cells. Stem Cells,
2009. 27(11): p. 2804-14.
24.
Muschler, G.F., et al., The design and use of animal models for translational
research in bone tissue engineering and regenerative medicine. Tissue Eng Part B
Rev, 2010. 16(1): p. 123-45.
25.
Chang, C.P., et al., Hypoxic preconditioning enhances the therapeutic potential of
the secretome from cultured human mesenchymal stem cells in experimental
traumatic brain injury. Clin Sci (Lond), 2013. 124(3): p. 165-76.
26.
Rosova, I., et al., Hypoxic preconditioning results in increased motility and
improved therapeutic potential of human mesenchymal stem cells. Stem Cells,
2008. 26(8): p. 2173-82.
144
27.
Potier, E., et al., Prolonged hypoxia concomitant with serum deprivation induces
massive human mesenchymal stem cell death. Tissue Eng, 2007. 13(6): p. 132531.
28.
Weil, B.R., et al., Mesenchymal stem cells enhance the viability and proliferation
of human fetal intestinal epithelial cells following hypoxic injury via paracrine
mechanisms. Surgery, 2009. 146(2): p. 190-7.
29.
Klein, P., et al., A structure-based model for ligand binding and dimerization of
EGF receptors. Proc Natl Acad Sci U S A, 2004. 101(4): p. 929-34.
30.
Boyce, D.E., et al., The role of lymphocytes in human dermal wound healing. Br J
Dermatol, 2000. 143(1): p. 59-65.
31.
Ghosh, M.M., et al., A simple human dermal model for assessment of in vitro
attachment efficiency of stored cultured epithelial autografts. J Burn Care
Rehabil, 1995. 16(4): p. 407-17.
32.
Supp, D.M., K. Wilson-Landy, and S.T. Boyce, Human dermal microvascular
endothelial cells form vascular analogs in cultured skin substitutes after grafting
to athymic mice. FASEB J, 2002. 16(8): p. 797-804.
33.
BRDU
Cell
Proliferation
Assay
Reagents.
Available
from:
http://www.cellsignal.com/products/6813.html.
34.
Raut, V.P., Thomas E. Patterson, Joseph C. Wenke, Jeffrey O. Hollinger, and
George F. Muschler. "Assessment of Biomaterials." An Introduction to
Biomaterials (2011): 157., Assessment of Biomaterials, in An Introduction to
Biomaterials J.O. Hollinger, Editor. 2011, CRC Press: Boca Raton, Fl. p. 157.
145
35.
Caralla, T., et al., In vivo transplantation of autogenous marrow-derived cells
following rapid intraoperative magnetic separation based on hyaluronan to
augment bone regeneration. Tissue Eng Part A, 2013. 19(1-2): p. 125-34.
36.
Luangphakdy, V., et al., Evaluation of osteoconductive scaffolds in the canine
femoral multi-defect model. Tissue Eng Part A, 2013. 19(5-6): p. 634-48.
37.
Takigami, H., et al., Bone formation following OP-1 implantation is improved by
addition of autogenous bone marrow cells in a canine femur defect model. J
Orthop Res, 2007. 25(10): p. 1333-42.
38.
Erbe, E.M., et al., Potential of an ultraporous beta-tricalcium phosphate synthetic
cancellous bone void filler and bone marrow aspirate composite graft. Eur Spine
J, 2001. 10 Suppl 2: p. S141-6.
39.
Franch, J., et al., Beta-tricalcium phosphate as a synthetic cancellous bone graft
in veterinary orthopaedics: a retrospective study of 13 clinical cases. Vet Comp
Orthop Traumatol, 2006. 19(4): p. 196-204.
40.
Gan, Y., et al., The clinical use of enriched bone marrow stem cells combined
with porous beta-tricalcium phosphate in posterior spinal fusion. Biomaterials,
2008. 29(29): p. 3973-82.
41.
Muschik, M., et al., Beta-tricalcium phosphate as a bone substitute for dorsal
spinal fusion in adolescent idiopathic scoliosis: preliminary results of a
prospective clinical study. Eur Spine J, 2001. 10 Suppl 2: p. S178-84.
42.
Pochon, J.P., et al., [Bone replacement using beta-tricalcium phosphate--results
of experimental studies and initial clinical case examples]. Z Kinderchir, 1986.
41(3): p. 171-3.
146
43.
Muschler, G.F., et al., Selective retention of bone marrow-derived cells to
enhance spinal fusion. Clin Orthop Relat Res, 2005(432): p. 242-51.
44.
Griffith, L.G., Richard T. Lee, and Luis Manuel Alvarez., Modulating cell
behavior with engineered HER-receptor ligands, in Biological Engineering 2009,
Massachusetts Institute of Technology: Cambridge MA.
147