Novel Design of a Three-Dimensional Biomimetic Nanofiber Scaffold:
applications in ligament tissue engineering.
John Schoenbeck, Christopher Lach, Reem Daher-Nahhas.
Lawrence Technological University – BME Senior Capstone Project – Dr. Mansoor Nasir, Fall 2012
Abstract: A commonly injured ligament in young adults that leads to degenerative diseases such as
osteoarthritis is the Anterior Cruciate Ligament (ACL) of the knee. Tissue Engineering has taken
numerous steps in the past decade towards developing in-vitro grown tissue grafts for the replacement of
diseased and damaged ligaments. Previous research has shown that three-dimensionally patterned
electrospun poly(ε-caprolactone, PCL) nanofibers hold vast potential as a biodegradable scaffold material
for ligament tissue engineering. The purpose of this research is to establish and characterize a novel
braided scaffold design for use in ligament tissue engineering applications. In this study, a second-order
triple helix braid concept was designed to mimic the hierarchal structure of collagen found in native ACL
tissues. Structural properties will be determined using environmental scanning electron microscopy
(ESEM) and mercury perfusion porosimetry. Mechanical properties will be characterized via static tensile
test. Cell compatibility will be determined by analyzing statically cultured cell-seeded scaffolds via
Live/Dead and AlamarBlue assays. Protocols have been established and data collection for structure,
mechanical, and cell viability characteristics have begun and will continue through the spring. It is
anticipated that the second-order triple helix braiding structure will enhance cellular viability and
mechanical properties when compared to controls because of its biomimetic structure. The experimental
protocols developed for this research will enable future studies to rapidly develop new fiber braiding
patterns and characterize their viability as tissue engineering scaffolds.
Background: Medical science has made significant progress in the surgical repair and replacement of
damaged ligaments over the last sixty years. The current gold standard for ACL replacement is taking a
graft from the center of the patient’s patellar tendon (PT) and implanting it into the connection sites of the
removed ACL. This process involves immense harvest site morbidity and the PT lacks the mechanical
properties of the ACL; it only marginally improves the patient’s mobility for a very limited time period.
Tissue engineering is a revolutionary approach to the repair and replacement of diseased tissues using a
combination of cells, biomaterials scaffolds, and biochemical and mechanical factors.
Biomaterials scaffolds play a pivotal role in supporting seeded cell attachment, proliferation and
differentiation. Several biomimetic scaffold designs have been demonstrated that mimic certain
advantageous features of the extracellular matrix (ECM) of the native tissues, such as the material
composition, surface chemistry, structural features and mechanical properties. We have chosen
electrospun poly(ε-caprolactone) (PCL) nanofibers as our scaffold material. These fibers will be braided
into a dense bundle intended to mimic the triple-helix hierarchal structure of collagen present in native
ACL tissue. A specific feature of the mechanical properties we are interested in reproducing accurately is
the stress/strain toe region of the braided material. This is a structural feature that appears in the material’s
stress/strain curve in which the material can be elongated without a linear increase in the strain. In native
tissues, this feature is achieved by the crimped and wavy structure of native collagen fibrils. As the
ligament is loaded, the crimps initially straighten, allowing for some elongation before the collagen fibers
begin to stretch. This wavy structure can be reproduced in textiles by braiding material fibers into helix
shapes.
Research Objectives & Methods: The primary objective of this research project is to achieve a
biomimetic three-dimensional braided scaffold design that has structural and mechanical properties
similar to those of the native ACL to support fibroblast cell attachment and proliferation. We propose that
the triple helix pattern, when braided into higher ordered patterns, will enhance the mechanical properties
of single fiber controls and elicit increased biological responses when seeded with fibroblasts. We intend
to create protocols that look at all of the key properties of a tissue engineering scaffold, a standard we
have not seen in previous literature; which generally chooses only one of these three approaches:
1. Characterizee the structuraal features of the braided sscaffolds usinng video analyysis, environm
mental
scanning electron micrroscopy (ESE
EM), porosimeetry, and a cuustom-built tennsion measurrement apparaatus.
2. Determine th
he mechanicaal properties of
o the braidedd scaffolds annd compare thhem to data from
the nativee ACL tissue
3. Evaluate thee biocompatib
bility of the brraided scaffollds by analyzzing cell alignnment, metaboolic
activity, and
a growth kinetics when seeded
s
with human
h
fibrobllasts.
The
T electrospu
un fibers are provided
p
by Dr.
D Joseph Coorey’s lab at thhe Universityy of Michigann. The
ESEM, fo
orce transduceers, and mech
hanical testing
g setup are proovided by thee LTU Biomeedical Engineering
program. Cell culturing
g will be cond
ducted using equipment prrovided by Deean Moore’s L
LTU Life
Sciences program.
p
Thee tension measurement guide was constrructed at a coost of $175. A
AlamarBlue annd
Live/Dead
d cell assay kits
k for next seemester will cost
c approxim
mately $1200. Funding for additional coosts
has been generously
g
prrovided by Drr. Yawen Li.
Table
T
1 lists th
he basic featurres of the braid design based on literatuure describingg the hierarchaal
structure of
o human ligaaments. Major design parameters and coorresponding testing/charaacterization
methods are
a listed in Table
T
2.
Table 1: Hierarchal
H
braid
d design using paracord for color-coded fibber identificatioon.
Scaffold Model Fiber Identified M
Model Second O
Order Triple‐Helix Strructure Expecteed Benefits Potential Limitations
Three fibers are braided into triple heelix sub
bunits. Three sub
bunits are braided into
o a triple helix. Axial fiber aalignment, Evenly distrributed and equally sizeed pores, Superior tensile strength compared tto single fiber control
Poor lateral integrrity, Reduced capacity for elongation withou
ut increasing materiaal sstrain, Potential fo
or ssmothering seedeed cells
Table 2: Braiding design parameters and testing methods
Research Study Biomimetic Structure Mechanical Properties Biocompatibility Parameter of Interest Braid Angle Native Tissue Properties N/A
ACL Scaffold Data from Literature 30‐33 degrees [29] Testing Method
Kinovea
Braiding Tension N/A
N/A
Force Transducer
Fiber patterns Circular Loom [29] ESEM Imaging
Porosity Aligned & Crimped
[26] N/A
55% to 65% [29]
Porosimeter
Pore Size N/A
150 to 250 um [29] Porosimeter
Ultimate Tensile Strength Elastic Modulus 13 to 46 MPa [27]
46 MPa [28]
182‐292 MPa [27]
150 to 200 MPa [28] Maximum Strain 30 to 44% [27]
32% [28]
Toe Region 2 to 4.8% [27]
Up to 7.5% [28]
Cell Alignment Parallel to Strain Axis [28] N/A
Parallel to Fiber Axis [28] N/A
Tensile Test using MTS Tensile Test using MTS Tensile Test using MTS Tensile Test using MTS Confocal Microscopy/ ESEM AlamarBlue & Live/Dead Assay Cell Proliferation Preliminary Results: Experimental protocols have been established (Appendix A) for both the structural
and mechanical testing methods. Dr. Li’s Tissue Engineering Lab has provided the group with
opportunities to practice live/dead assays; we have had major success in this area and developed protocols
that will be adapted for this particular application. Initial experiments have been conducted to work out
the kinks and identify additional obstacles to the design of the research project. Figure 1 is a sample of the
load cell readings taken from the initial braiding trial. Figure 2 is an example of the Kinovea key frame
analysis used to determine the braiding angles. Figure 3 is the force/elongation data collected from the
materials testing system. Figure 4 is an ESEM image of the braided fiber.
Force (lb compression)
(
)
Stress (MPa)
5
4
3
2
1
0
0
0.2
0.4
0.6
0.8
Strain (mm/mm)
Figure 2: Stress‐stra
ain response of a
a braided PCL sccaffold.
Figu
ure 4: Kinovea A
Angle Measurem
ment
1
0.055
0.044
0.033
0.022
0.011
0
‐0.011
‐0.022
‐0.033
‐0.044
‐0.055
000:00.0
02:52.8
05:45.6
Time Point (minutess:seconds)
Figure 1: Bra iding Tension Lo
oad Cell Readinggs. Figuree 3: ESEM Imagee with Pore Meaasurements
w obtained while
w
trying to
o see if the miicrotester for the ESEM fitt our needs loooks
The stresss strain data we
very prom
mising. The wavy
w
braid patttern of the fib
bers producess a toe region from 0 to 5%
% of the strainn, this
is very clo
ose to the 4.8%
% produced by
b the crimpeed, wavy struccture of collaggen in native ACL tissues.. In
addition, our
o calculated
d elastic modu
ulus of 258 MPa
M matches well to the coorresponding native tissue
property and
a exceeds data
d from prev
vious literaturre. Our braidiing tension avveraged a littlle under 0.03 lb.
There wass one small peeriod when brraiding began
n in which tennsion was 0.001 lb, but theree was no eviddence
of any stru
uctural differences at severral images tak
ken along thee fiber axis. W
We took severral key framess
from the video
v
of our braiding
b
proceess and noticeed that our brraid angles rannged from 355 to 40 degreees
with a ten
ndency to start low and end
d high as the excess
e
fiber taails got short.. The overall wavy shape oof
each fiber mimics the wavy structure of collagen fibrils visually. Initial pore size measurements using the
ESEM ruler tool indicate that our pore diameters are right on target. With the protocols and data analysis
procedure preliminary study complete, we have determined that the majority of the experimental process
we developed in advance will help us to achieve our desired outcomes. We have also identified several
obstacles that require additional steps in our process to overcome.
Expected Outcomes & Potential Obstacles: The expected result is an optimized braided scaffold
pattern that has similar structural and biomechanical properties to the native ACL. The optimized pattern
should also show ideal biocompatibility when seeded with ligament fibroblasts. Furthermore, our
protocols for characterizing scaffolds along a broad spectrum of key properties will encourage some
standardization of the way researchers analyze their own tissue engineering attempts. We expect that our
data will achieve statistical significance in two to three of four designated one-month time blocks for our
studies. These expectations will be verified by power analysis before the first time block and checked
after data collection by unpaired t-tests. If the fourth month is not needed to address setbacks, it will be
dedicated to further explorations such as fatigue, creep, and stress-relaxation tests or comparisons using
additional braid designs.
The first obstacle we encountered in our setup is the selection of a material testing system. The
braided PCL fibers withstood far more significant elongation than was originally expected, and the 10mm
minimum size braid sample did not fail when stretched to 20mm (the upper limit of the micro-MTS clamp
for the ESEM). As a result, we will schedule data run times with Beaumont Orthopedics to use their MTS,
which has a much longer range of travel for its crosshead. General Motors was going to provide us with
porosimetry data for our structural analysis, but are experiencing some setbacks with the equipment and
may or may not be ready in time for our intended schedule. We are reaching out to other collaborators and
contacts to find a new porosimeter, and have micro-computed tomography available through Beaumont as
a fallback plan to stay on schedule. Finally, our load cell output was displaying an upward creep in the
reading from tension into compression as we braided our fibers. This issue was overcome by separation of
the individual peak, low, and average readings from the cell. The peak reading creeps upward nonlinearly from 0 to 0.09 lb compression when the cell is zeroed and left static for 15 minutes. The low
reading, however, holds its zero level and responds to applied tension with ideal sensitivity.
Future Directions & Implications: Finding an optimal three-dimensional braiding pattern for
electrospun biodegradable polymer fibers will provide an avenue for future developments in ligament
tissue scaffolds. For starters, a custom designed fiber braiding machine could be designed to produce
more consistent braided scaffolds with optimum structure, mechanical properties, and biocompatibility.
The optimized braid patterns will also provide a platform for future enhancement studies involving
mechanical stimulation with a cyclical bioreactor. Furthermore, the study will help to guide a more
rational design for ACL tissue engineering scaffolds, and outline the process for which these braids can
be constructed and tested. The project also has important implications for the regeneration of other fibrous
tissues. Examples of these tissues include tendons, the meniscus of the knee, and the annulus fibrosus of
the intervertebral disc.
Team Members & Responsibilities:
Faculty Advisor: Yawen Li Ph.D., Technical Advisor: Tristan Maerz (Beaumont Research Group)
John Schoenbeck: Research cultivation, draft writer, scaffold and measurement device design.
John’s summer research on fiber braided scaffolds provided the groundwork for the establishment
of the project objectives. His organizational and networking capabilities provide the team with the
necessary resources and time needed to make consistent progress towards the achievement of those goals.
Christopher Lach: Scaffold braiding and sample preparation, draft illustrator, cell culturing.
Chris’s summer research on electrospun nanofibers resulted in the novel design of the braided
scaffolds. His delicate attention to detail and capacity for engineering tools and software provide the team
with consistent scaffold structure properties and the ability to analyze any variations in the same.
Reem Daher-Nahhas: Experimental protocol design, data collection and analysis, draft editor.
Reem’s summer research on ESEM and mechanical testing will enable the team to maximize the
efficiency and relevancy of the data collection process. Her persistence and patience in defining
calculation techniques and minimizing user error will ensure reliable data acquisition and analysis.
Though each member of the team has clearly defined and separable responsibilities, we conduct all
physical work collectively while approaching intellectual tasks with a divide-and-conquer approach. The
group meets three times per week to share findings and progress in project completion tasks.
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