D 1035
OULU 2009
U N I V E R S I T Y O F O U L U P. O. B . 7 5 0 0 F I - 9 0 0 1 4 U N I V E R S I T Y O F O U L U F I N L A N D
U N I V E R S I TAT I S
S E R I E S
SCIENTIAE RERUM NATURALIUM
Professor Mikko Siponen
HUMANIORA
University Lecturer Elise Kärkkäinen
TECHNICA
Professor Hannu Heusala
ACTA
ASPECTS OF BONE
SUGAR BIOLOGY
PECTIN NANOCOATINGS OF HARD
TISSUE IMPLANTS
MEDICA
Professor Helvi Kyngäs
SCIENTIAE RERUM SOCIALIUM
Senior Researcher Eila Estola
SCRIPTA ACADEMICA
Information officer Tiina Pistokoski
OECONOMICA
University Lecturer Seppo Eriksson
EDITOR IN CHIEF
University Lecturer Seppo Eriksson
PUBLICATIONS EDITOR
Publications Editor Kirsti Nurkkala
ISBN 978-951-42-9319-1 (Paperback)
ISBN 978-951-42-9320-7 (PDF)
ISSN 0355-3221 (Print)
ISSN 1796-2234 (Online)
U N I V E R S I T AT I S O U L U E N S I S
Hanna Kokkonen
E D I T O R S
Hanna Kokkonen
A
B
C
D
E
F
G
O U L U E N S I S
ACTA
A C TA
D 1035
FACULTY OF MEDICINE,
INSTITUTE OF BIOMEDICINE,
DEPARTMENT OF ANATOMY AND CELL BIOLOGY,
UNIVERSITY OF OULU
D
MEDICA
ACTA UNIVERSITATIS OULUENSIS
D Medica 1035
HANNA KOKKONEN
ASPECTS OF BONE SUGAR
BIOLOGY
Pectin nanocoatings of hard tissue implants
Academic dissertation to be presented with the assent of
the Faculty of Medicine of the University of Oulu for
public defence in Auditorium A101 of the Department of
Anatomy and Cell Biology (Aapistie 7 A), on 4 December
2009, at 11 a.m.
O U L U N Y L I O P I S TO, O U L U 2 0 0 9
Copyright © 2009
Acta Univ. Oul. D 1035, 2009
Supervised by
Professor Juha Tuukkanen
Reviewed by
Docent Tiina Laitala-Leinonen
Professor Risto Renkonen
ISBN 978-951-42-9319-1 (Paperback)
ISBN 978-951-42-9320-7 (PDF)
http://herkules.oulu.fi/isbn9789514293207/
ISSN 0355-3221 (Printed)
ISSN 1796-2234 (Online)
http://herkules.oulu.fi/issn03553221/
Cover design
Raimo Ahonen
OULU UNIVERSITY PRESS
OULU 2009
Kokkonen, Hanna, Aspects of bone sugar biology. Pectin nanocoatings of hard
tissue implants
Faculty of Medicine, Institute of Biomedicine, Department of Anatomy and Cell Biology,
University of Oulu, P.O.Box 5000, FI-90014 University of Oulu, Finland
Acta Univ. Oul. D 1035, 2009
Oulu, Finland
Abstract
The improvement of implant biocompatibility is constantly under investigation. Titanium is a
standard biomaterial that performs well in dental and orthopedic implantations. However,
detrimental adverse effects resulting from e.g. biomaterial properties, inflammatory responses and
surgical procedures occasionally occur. Coating the biomaterials aims at increasing the proportion
of successful operations.
Pectins, large plant cell wall polysaccharides, are innovative, modifiable, and potentially antiinflammatory candidates for biomaterial nanocoatings. In this thesis, covalently-grafted pectin
fragments (modified hairy regions, MHRs) modified either in vitro (from apple) or in vivo (from
potato) were tested.
Cell culture vessels and titanium substratum coated with the apple-MHRs, MHR-A and a
further-tailored fragment type, MHR-B, were compared with controls for their ability to support
proliferation and differentiation of osteoclasts and osteoblasts. Cells grew and differentiated on
MHR-B and on the control surfaces; MHR-A did not perform well in these assays. Geneticallyengineered potato MHRs did not support bone cell growth to the same extent as apple MHR-B,
but nonetheless the possibility to manipulate cellular proliferation with specific in vivo –
modifications of pectins was introduced.
When implanted into rat soft tissues, neither of the apple MHRs provoked severe acute
inflammatory reactions, which indicates good in vivo - tolerance of these botanical
macromolecules. These studies illustrate the biocompatibility of MHRs, and the directions
towards which they could be further tailored. In terms of clinical use, their tolerability in vivo is
especially significant.
Keywords: biocompatible materials, glycomics, osteoblasts, osteoclasts, pectins,
prostheses and implants, titanium
To co-authors James J, the Kid B, Kenguru Köö,
Nurmio T, Miljoona P, and Hasslehoff D
6
Acknowledgements
This work was carried out at the Department of Anatomy and Cell Biology,
Institute of Biomedicine, University of Oulu, during the years 2004–2009. I
express my sincere gratitude to my mentor, Professor Juha Tuukkanen, DDS,
PhD, for his enthusiastic guidance through this project. I also thank Professors
Hannu Rajaniemi, MD, PhD and Petri Lehenkari, MD, Ph.D. – the heads of the
Department of Anatomy and Cell Biology, as well as its whole personnel,
especially the cluster bomb of our bone group.
All my co-authors as well as the other PectiCoaters are thanked for sharing
their knowledge. Docent Tiina Laitala-Leinonen and Professor Risto Renkonen
are acknowledged for reviewing this thesis, and Dr. Deborah Kaska for its
language revision. Eero Oja, Marja Paloniemi, Anna-Maija Ruonala and Paula
Salmela are acknowledged for their valuable technical assistance. This work was
financially supported by the EC-project PectiCoat (NMP4-CT-2005-517036), The
National Graduate School of Musculoskeletal Disorders and Biomaterials
(TBGS), The Academy of Finland (130795), and The Finnish medical Foundation
Duodecim, and The Foundation of Orthopedics and Traumatology.
My officemates, Mari and Virpi, are acknowledged for the collectively pedant
and seriously academic attitude towards work. I am also grateful to the pope M.
Murtomaa for the spiritual atmosphere in our room. Without you I would never
have accomplished my thesis – this slowly. Annina Sipola is acknowledged for
sharing the practical consequences of it. My co-pöntöt Minna Karjalainen and
Johanna Korvala are thanked for the former, radiant study years at the Department
of Biology. Also all my other friends, especially the “old-timers” Anne, Erika,
Jukke, Markus, Mäkiset, Paakkolanvaarat, Pete, Tuomas and WSQ, deserve my
warmest compliments for providing me the feeling of never being alone. Aside,
hedonistic bubbling deserves to be mentioned, as does all the to-be-collegially provided professional relief.
Lopuksi osoitan rakkaille vanhemmilleni Orvokille ja Kaukolle suurimmat
kiitokseni kaikesta. Ilman aina saatavilla olevaa apuanne niin tämä kuin kovin
moni muukin asia olisi jäänyt tekemättä. Myös todellista elämänviisautta edustava
isoäitini, Kotimummo Annikki, ansaitsee lämpimät kiitokset. Ihana tyttäreni Hilla
puolestaan on suurin ja tärkein muistutus siitä, millä on merkitystä.
Oulu, November 2009
Hanna Kokkonen
7
Mennään pois täältä
ennen kuin muuttuu ääni kelloissa;
tihulaismyrskyt,
ukkosenjohdattimet ihokarvoissa,
nopeus, tehokkuus, kahviautomaatit,
oikeustalojen täydet salit,
connecting people
vaikka kätelläkään ei enää saa
Postilaatikot täysinä
Niin laajat kaistat,
ettei sinuun kohta yletä
Kaikki muu sähkö
aina jotenkin
lähempänä kuin
lämmin EKG:si,
padottujen ionikanavien
liioitellut maadoitukset
Annetaan hetken olla
elinten laatikoissa,
kirurginterästen
oudoissa kummuissa,
jänteiden ääriviivat
anatomian kuvastoissa
(opetellaan ne muuten)
Mennään paikkaan, jossa
ei ole kenttää –
vain vesi virtaa,
hirvet hengittävät
8
Abbreviations
α-MEM
AGE
ALP
AMI
ANOVA
Ara
AsAP
β-GP
BMP
bp
BRU
BSA
BSP
Cbfa1
cDNA
CT
D-MEM
ECM
EtOH
FA
FBGC
FBS
FCS
FESEM
FGF
FITC
FN
GAG
Gal
GalA
Glc
GlcA
HA
HE
HM
Dulbecco`s modified Eagle`s medium, alpha modification
Advanced glycation end product
Alkaline phosphatase
Aminated surface
Analysis of variance
Arabinose
L-ascorbic acid 2-phosphate
Phosphate disodium salt pentahydrate
Bone morphogenic protein
Base pair
Bone remodeling unit
Bovine serum albumin
Bone sialoprotein
Core-binding factor alpha 1 (a.k.a. Runx2)
Complementary deoxyribonucleic acid
Calcitonin
Dulbecco`s modified Eagle`s medium
Extracellular matrix
Ethanol
Focal adhesion
Foreign body giant cell
Fetal bovine serum
Fetal calf serum
Field emission scanning electron microscopy
Fibroblast growth factor
Fluorescein isothiocyanate
Fibronectin
Glycosaminoglycan
Galactose
Galacturonic acid
Glucose
Glucuronic acid
Hydroxyapatite
Hematoxylin-eosin
High-methyl pectin
9
hMSC
IL
LM
MHR
MMP
MTT
NCP
OCIL
OCN
OPG
OPN
PBS
PFA
PTH
PTR
RANK
RANKL
RG
RGD
Rha
RNA
RT
RT-PCR
sLRP
TCPS
TGF
Ti
TNF
TRACP
TRITC
WGA
WT
10
Human mesencymal stem cells
Interleukin
Low-methyl pectin
Modified hairy region
Matrix metalloproteinase
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Non-collagenous protein
Osteoclast inhibitory lectin
Osteocalcin
Osteoprotegerin
Osteopontin
Phosphate-buffered saline
Paraformaldehyde
Parathyroid hormone
Transgenic potato -derived
Receptor activator for NF-κB
Receptor activator for NF-κB ligand
Rhamnogalacturonan
Arginine-glycine-aspartic acid
Rhamnose
Ribonucleic acid
Room temperature
Reverse transcriptase polymerase chain reaction
Small leucine-rich proteoglycan
Tissue culture polystyrene
Transforming growth factor
Titanium (uncoated)
Tumor necrosis factor
Tartrate-resistant acid phosphatase
Tetramethylrhodamine isothiocyanate
Wheat germ agglutinin
Wild type
List of original publications
This thesis is based on the following articles referred to in the text by their Roman
numerals:
I
Kokkonen HE, Ilvesaro J, Morra M, Schols HA & Tuukkanen J (2007) Effect
of Modified Pectin Molecules on the Growth of Bone Cells.
Biomacromolecules 8(2): 509–15.
II Kokkonen H, Cassinelli C, Morra M, Schols HA & Tuukkanen J (2008)
Differentiation
of
Osteoblasts
on
Pectin-Coated
Titanium.
Biomacromolecules 9(9): 2369–76.
III Kokkonen H, Niiranen H, Schols HA, Morra M, Stenbäck F & Tuukkanen J
(2009) Pectin-Coated Titanium Implants are Well-Tolerated in vivo. Journal
of Biomedical Materials Research: Part A. In press.
IV Kokkonen H, Verhoef R, Kauppinen K, Muhonen V, Jørgensen B, Damager I,
Schols H, Morra M, Ulvskov P & Tuukkanen J (2009) Proliferation of
Osteoblastic Cells on In vivo Engineered Potato Pectin Fragments.
Manuscript.
11
12
Contents
Abstract
Acknowledgements
7
Abbreviations
9
List of original publications
11
Contents
13
1 Introduction
15
2 Review of the literature
17
2.1 Bone ........................................................................................................ 17
2.1.1 Bone as an organ .......................................................................... 17
2.1.2 Bone as a tissue ............................................................................ 18
2.1.3 Osteoclasts .................................................................................... 19
2.1.4 Osteoblasts.................................................................................... 20
2.1.5 Intercellular and cell-ECM interactions ....................................... 22
2.2 Carbohydrates in bone biology ............................................................... 24
2.2.1 Sugar molecules in bone and joints .............................................. 24
2.2.2 Sugar molecules and osteoclasts................................................... 25
2.2.3 Sugar molecules and osteoblasts .................................................. 27
2.3 Bone implants ......................................................................................... 29
2.3.1 Biocompatibility: Biological factors ............................................ 29
2.3.2 Biocompatibility: Physicochemical factors .................................. 30
2.3.3 Host responses to implantation ..................................................... 31
2.3.4 Coating of bone implants.............................................................. 32
2.4 Pectins ..................................................................................................... 34
2.4.1 Pectins as candidates for biomaterial applications ....................... 35
2.4.2 Modification of pectins................................................................. 36
2.4.3 Immunological aspects of pectins................................................. 38
3 Aims of the present study
39
4 Materials and methods
41
4.1 Cell cultures ............................................................................................ 41
4.1.1 Cell lines ....................................................................................... 41
4.1.2 Primary cultures ........................................................................... 41
4.2 Test materials .......................................................................................... 42
4.2.1 MHR preparations ........................................................................ 42
4.2.2 MHR-coated samples ................................................................... 43
4.2.3 Bone slices.................................................................................... 44
13
4.3 Stainings .................................................................................................. 44
4.3.1 Fluorescent labels ......................................................................... 44
4.3.2 Non-fluorescent stains .................................................................. 45
4.4 Microscopic methods .............................................................................. 45
4.4.1 Light and fluorescence microscopy .............................................. 45
4.4.2 Laser scanning confocal microscopy ............................................ 45
4.4.3 Field emission scanning electron microscopy .............................. 46
4.4.4 Image analyses.............................................................................. 46
4.5 Molecular methods .................................................................................. 46
4.5.1 RT-PCR ......................................................................................... 46
4.5.2 Enzyme activity measurement ...................................................... 47
4.6 In vivo procedures ................................................................................... 47
4.6.1 Animals and implantations ........................................................... 48
4.6.2 Histological sections ..................................................................... 48
4.7 Statistical analyses .................................................................................. 48
5 Results
49
5.1 Interactions between MHRs and bone cells ............................................ 49
5.1.1 Proliferation and differentiation of osteoclasts (I) ........................ 49
5.1.2 Proliferation and differentiation of osteoblasts (I, II, IV) ............. 49
5.2 In vivo effects of MHR-coated implants (III) .......................................... 52
6 Discussion
55
6.1 Effects of pectins on bone cells ................................................................. 55
6.1.1 Osteoclastic responses .................................................................. 55
6.1.2 Osteoblastic proliferation ............................................................. 56
6.1.3 Osteoblastic differentiation ........................................................... 58
6.2 In vivo – testing of inflammatory responses ............................................ 61
6.2.1 Fibrous capsule formation ............................................................ 62
6.2.2 Consistence of the capsules .......................................................... 62
6.3 General discussion and future prospects ................................................. 64
6.3.1 Physicochemical features of MHR-coatings ................................ 64
6.3.2 Sugar aspects ................................................................................ 67
6.3.3 Immunological aspects ................................................................. 69
7 Conclusions
71
References
73
Original publications
93
14
1
Introduction
Bone and dental implants are widely used as substitutive items in various clinical
complications resulting from e.g. erosive diseases and trauma. Materials
contacting living tissues are called biomaterials, whose interactions with the
surrounding tissue should not excessively disturb the homeostatic functions of the
cells. This inertness of biomaterials is defined as biocompatibility. However,
problems in biocompatibility often occur and may lead to e.g. inflammation,
infections or even implant loosening. One approach to improving
biocompatibility is to coat biomaterials with appropriate molecules in order to
diminish any negative effects of implants and/or to enhance desired biological
processes after implantation.
Bone tissue undergoes constant remodeling, which is conducted by boneresorbing osteoclasts and bone-forming osteoblasts. It is important that the
biomaterial, which in bone and dental implantations usually is titanium, does not
interfere with the cooperation of these cell types.
The purpose of this thesis was to investigate the feasibility of using the novel
molecular candidates, pectins, as bone and dental implant nanocoatings. Pectins
are large and structurally variable polysaccharides of botanical origin. The
interactions between cells and pectin fragments were studied both in vitro and in
vivo, focusing especially on the proliferation and differentiation of bone cells on
pectin coatings as well as on the tendency of pectin fragments to provoke
inflammatory responses. These studies may both augment our basic knowledge of
sugar biology in bone cells and in particular assess the applicability of pectincoated implants for clinical purposes.
15
16
2
Review of the literature
2.1
Bone
2.1.1 Bone as an organ
The bony skeleton of higher vertebrates provides mechanical support for the
body, protects the vital organs, and enables movement together with muscles. In
addition to these main anatomical functions, bone takes part in several
physiological processes, e.g. regulation of calcium and phosphate ion balances in
the serum, and production of blood cells (hematopoiesis) in the bone marrow of
long bones.
At the macroscopic level, two structurally different textures can be
distinguished in bones. Compact bone forms a dense cortex that surrounds other
sections of bone that form the bone marrow cavity in the diaphysis area of long
bones. In the metaphysis and epiphysis areas of a long bone the cavity is largely
occupied by a network of metabolically active trabecular (or cancellous) bone
surrounding the bone marrow. Cartilaginous growth plates are located at the
border between the metaphysis and epiphysis. The outmost layer of bone is called
the periosteum, and the inner surface between the cortex and the bone marrow is
the endosteum.
Bones ossify either via an endochondral or an intramembraneous
developmental route. In endochondral ossification, a cartilaginous prototype of a
bone is subsequently replaced by mineralized matrix. This endochondral route is
typical for long bones. In contrast, flat bones are developed via intramembraneous
ossification, in which bones are formed and fused together without an
intermediate cartilage model. A special anomaly of bone formation is the socalled ectopic ossification, which signifies the development of bone in soft tissues
outside the skeleton – a phenomenon manifesting in some relatively rare
syndromes, e.g. osteoma cutis- and fibrodysplasia ossificans progressiva
(Ruggieri et al. 1995, Cortes & Gosain 2006, Lounev et al. 2009, Suda et al.
2009).
Biological processes in bones are regulated by various hormones and local
factors, e.g. parathyroid hormone (PTH), calcitonin (CT), estrogen, 1,25dihydroxyvitamin D3 (1,25(OH2)D3), tumor necrosis factor (TNF), transforming
growth factors (TGF-α /-β), interferon gamma (IFN-γ), interleukin-1 (IL-1), and
17
bone morphogenic proteins (BMPs). Besides these molecular regulators,
environmental elements including nutrition and physical exercise, as well as
genetic heritage affect the condition of the skeleton. Age is also an important
factor; the peak bone mass is achieved in late adolescence (Matkovic et al. 1994).
2.1.2 Bone as a tissue
Bone tissue can be classified as a specialized dense connective tissue. The
structural units of cortical bone are called osteons, which represent the regions of
bone remodeling. The center of an osteon is occupied by a Haversian system
consisting of a vascular route in a central canal, which is surrounded by lamellar
layers of osteocytes located in solitary niches, i.e. lacunae. The borders of osteons
are seen as cement lines, visual remnants of previous activities of bone
remodeling.
The extracellular matrix (ECM) of bone is composed of both organic and
inorganic compounds. About 22% of the bone matrix consists of proteins, the
majority being thus composed of inorganic components; 70% mineral salts and
8% water (Lane 1979). The most abundant protein, 95% of the total protein
fraction, in bone ECM is type I collagen, in addition to which bone also contains
minor amounts of collagen types III, V, and XI. Collagen fibers are decorated
with hydroxyapatite [3Ca3(PO4)2(OH)2] crystals making the bone matrix rigid and
durable. As indicated by the chemical formula, the main components of
hydroxyapatite are calcium and phosphate (DeJong 1926), which reflects the
aforementioned physiological importance of bone tissue in the regulation of ion
homeostasis. The remaining non-collagenous proteins (NCP) produced by
osteoblasts, such as fibronectin (FN), osteocalcin (OCN), osteopontin (OPN), and
bone sialoprotein (BSP) play important roles in the interactions between bone
cells and the ECM. Additionally, OPN has been shown to be located abundantly
in the cement lines separating old and new bone matrices (Mulari et al. 2004).
Bone tissue is constantly remodeled in a finely-tuned series of complex
events taking place in so-called bone remodeling units (BRU), a process that
occurs also in vitro (Marcus 1987, Väänänen 1993, Mulari et al. 2004, Andersen
et al. 2009). The alternating phases of bone resorption and formation are mediated
by two bone cell types, osteoclasts and osteoblasts, respectively. Imbalance
between the activities of these cell types may result in various diseases, such as
the well-known osteoporosis, which is linked to decreased estrogen levels and
subsequent decreased bone mass predisposing to fractures (Takano-Yamamoto &
18
Rodan 1990). Another condition related to bone cell imbalance is osteopetrosis
(Walker 1973), in which the activity of osteoclasts is hindered resulting in
excessive thickness and also to an elevated fracture susceptibility of cortical bone
(Tuukkanen et al. 2000). Thus a well-balanced and coordinated communication
between bone cell types is crucial to maintain the appropriate functionality of
bone. In addition to osteoclasts and osteoblasts, bone tissue contains osteocytes
and bone lining cells.
2.1.3 Osteoclasts
In the remodeling cycle of bone, the mineralized matrix must be dissolved prior to
the phase of new bone formation. The resorption of bone is mediated by large and
multinuclear cells, osteoclasts, first described by Albert Kölliker (Kölliker 1873).
Osteoclasts differentiate from hematopoietic stem cells and thus share the same
developmental origin with e.g. monocytes / macrophages. Osteoclasts gain their
multinuclear state via fusion of mononuclear precursor cells (Suda et al. 1992,
rana-Chavez & Bradaschia-Correa 2009). The differentiation and activity of
osteoclasts is dependent on certain, either stimulating or inhibiting systemic
factors (e.g. PTH, CT, IL-1, TGF-α and -β, and 1,25(OH)2D3) as well as on the
proximity of osteoblasts. For example, the effects of PTH and 1,25(OH)2D3 on
osteoclasts are affected by osteoblasts (Silve et al. 1982, Narbaitz et al. 1983).
Additionally, osteoblasts produce e.g. IL-1 and -6 and prostaglandin-E2 required
for controlling osteoclast activation, and receptor activator for NF-κB (nuclear
factor kappa-light-chain-enhancer of activated B cells) i.e. RANKL (also denoted
as OPGL, TRANCE or ODF). RANKL can stimulate the differentiation and
resorptive activation of osteoclasts by binding to a RANK receptor of osteoclasts
(Suda et al. 1992, Lacey et al. 1998, Matsuzaki et al. 1998, Yasuda et al. 1999).
Another osteoblast-derived component affecting osteoclasts is osteoprotegerin
(OPG), a protein that also binds to RANK thus hindering the differentiation of
osteoclasts. Additionally, bone marrow stromal cells including osteoblasts secrete
local factors, such as macrophage colony-stimulating factor (M-CSF), required
for osteoclastic maturation (Wiktor-Jedrzejczak et al. 1990).
Osteoclasts are capable of excavating both the organic and inorganic
components of bone in an acidified resorption cavitity surrounded by an
osteoclastic membrane organization called the sealing zone (Lakkakorpi et al.
1991, Lakkakorpi & Väänänen 1991). The sealing zone is an actin-rich ring-like
structure tightly attached to the bone matrix surface, onto which it confines
19
another plasma membrane domain, the endosomally and lysosomally active
ruffled border (Baron et al. 1988, Palokangas et al. 1997). Through this
membrane area the cellular vesicles containing acidifying H+ ions generated by
e.g. carbonic anhydrase II enzyme are released into the resorption lacuna (i.e.
Howship`s lacuna). Vacuolar H+-ATPases (V-ATPases) are pivotal in the activity
of osteoclasts, and the localization of the V-ATPase subunit mRNAs change
according to the phase of the resorption cycle and corresponding polarity of an
osteoclast (Laitala-Leinonen et al. 1996). Interestingly, inhibition of V-ATPases
has been shown to alleviate the processes of rheumatoid arthritis (Niikura et al.
2007). The resulting pH gradient is responsible for dissolving hydroxyapatite,
after which the organic composite – mainly composed of collagen-I fibers –
become accessible to different proteinases, such as collagenases, lysosomal
enzymes, and cathepsin-K. Chondrocyte-produced cathepsin-K has also been
shown to play a role in the pathogenesis of transgenic osteoarthritis mouse models
due to its ability to degrade cartilage as well (Morko et al. 2004). Some matrix
metalloproteinases (MMPs) and other proteinases produced by osteoblasts are
also involved in the initiation of the resorption phase thus predisposing bone
surface to resorption (Chambers & Fuller 1985, Kusano et al. 1998, RomanRoman et al. 2003).
The remnants of the resorption are endocytosed into the osteoclast, processed
by intracellular vesicular tartrate-resistant acid phosphatase (TRACP),
transcytosed towards a basolateral functional secretory domain and excreted from
the cell through this membrane region (Salo et al. 1997, Vääräniemi et al. 2004).
Phagocytes are also thought to participate in tidying up the resorbed bone site,
albeit some evidence emphasizes the role of osteoblasts in cleaning the organic
components from the resorption lacunae preceding the deposition of new bone
matrix.
2.1.4 Osteoblasts
After osteoclastic activity, bone-forming osteoblasts migrate to the resorption site
to refill the dissolved lacuna with new bone tissue by first producing an organic
matrix called osteoid, and by subsequently (about 20 days after osteoid
deposition) mineralizing it. Osteoblasts can form mineral by means of two,
probably parallel, mechanisms: nucleation (mineral crystals occupy the hole
regions of collagen-I fibrils) and matrix vesicle production (osteoblasts secrete
20
small organelles in the vicinity of calcium phosphate grains) (Anderson 1984,
Schwartz et al. 1987, Williams & Frolik 1991).
Osteoblasts originate from mesenchymal stem cells, as do cells of connective,
cartilaginous, muscle, and adipose tissue. The maturation route of osteoblasts
proceeds from a preosteoblastic cell to an osteoblast to an osteocyte. Additionally
the bone-lining cells can be regarded as a specific phenotype of the osteoblast
lineage.
Preosteoblasts are the precursors of the differentiated osteoblasts, the
veritable bone-forming cells. Several markers of mature osteoblasts can be
distinguished. For example, the production of alkaline phosphatase (ALP), a
plasma membrane -anchored enzyme indicative of mineralization, is a strong
indication of the transition towards a differentiated osteoblastic phenotype (Stein
et al. 1989, Owen et al. 1990, Aubin et al. 1995). Several isoforms of soluble
bone ALP (BALP) can be purified and identified (Magnusson & Farley 2002,
Sharp et al. 2007). The expression of osteocalcin (OCN), i.e. bone gla-protein,
serves as the clearest mineralization marker, and its serum or urine concentration
may be measured in metabolic disease as an indicator of bone turnover rate (Price
et al. 1980, Ivaska et al. 2005).
Osteoblast-specific genes require two main transcription factors, core-binding
factor alpha 1, i.e. Cbfa-1 (also known as Runx2 or Osf2) and Osterix, in order to
be transcribed (Ducy et al. 1997, Beck et al. 2001). Osteoblasts are also under the
control of various regulators, e.g. insulin-like growth factors (IGFs) and BMPs
(Ripamonti & Reddi 1992), and they also are capable of auto-regulating their
activity via TGF-β production (Robey et al. 1987).
After the formation of fresh bone tissue, some of the osteoblasts (~ 10–20%)
involved in the synthesis remain enclosed in the matrix. These cells become
osteocytes, the most abundant (90–95%) bone cell type (Frost 1960). Osteocytes
are located in separate lacunae and form an intercellular network via small
cytoplasmic projections extending through tunnel-like structures, the canaliculi.
Through these channels osteocytes embedded in mineralized – and thus
impermeable for diffusion – bone tissue is able to of receive oxygen, nutrients,
and other important molecules from the blood supply of the Haversian system.
Additionally, the tips of the processes contain connexin-based gap junctions,
through which osteocytes communicate electrically and metabolically with each
other and probably with other bone cell types as well (Doty 1981, Donahue et al.
1995).
21
The role of osteocytes in bone biology still remains relatively obscure, but
they seem to be involved in sensing and reacting to mechanical loading of the
bones (Aarden et al. 1994, Reijnders et al. 2007, You et al. 2008, Turner et al.
2009). Osteocytes respond to bone micro-damage and affect subsequent
remodeling (Kurata et al. 2006, Heino et al. 2009). Recently it has been shown
that bone also adapts to mechanical loading via modulating the RANKL
expression of osteoblasts at least immediately after stretching stimuli (Kreja et al.
2008).
Some post-proliferative osteoblastic cells have an inactive appearance at the
bone surfaces. These so-called bone-lining cells are not capable of producing
bone matrix, and they mark bone areas not participating in the remodeling
processes. Once the resorption phase starts, bone-lining cells disengage from the
bone surface. The role of bone-lining cells seems to relate to the production of
osteoclast-activating factors, e.g. cytokines. Additionally, they have been shown
to participate in linking the resorptive and formative remodeling phases by
removing collagen rudiments from resorption lacunae together with MMPs
(Everts et al. 2002). These cells may also revert to active osteoblasts (Chow et al.
1998).
2.1.5 Intercellular and cell-ECM interactions
To sustain the physiological and anatomical balance of bone tissue, the
communication and interactions of different bone cell types must be coordinated
properly. The main steps in the bone remodeling cycle are the activation,
resorption, reversal, and formation phases (Frost 1973), which are illustrated in
Figure 1.
Activation is the starting point for the remodeling of a BRU, during which the
bone surface transforms active and osteoclasts are attracted to, fuse, and
differentiate at the site to be remodeled. As mentioned earlier, both osteocytes as
well as bone-lining cells are thought to participate in the activation phase
(Chambers 1985, Martin 2000). Osteoblasts also play an important role since it
has been shown that osteoclasts cannot initiate resorption at the sites of nonmineralized osteoid; thus it must become either mineralized or degraded
(proteinases) by osteoblasts before resorption can commence (Jones et al. 1986,
Chambers & Hall 1991). The resorption phase is followed by a reversal phase
characterized by the appearance of macrophages at the resorbed bone site (Raisz
1988). The reversal phase is the link between bone resorption and formation,
22
which implies that it is during this stage when the still largely unknown signals to
osteoblasts become manifested. The macrophages (Raisz 1988) as well as bonelining cells and osteoclasts may play a role in signaling the osteoblasts by
secreting certain paracrine factors. Additionally, some factors or components of
the bone matrix revealed during resorption may act as signaling molecules for
osteoblasts so that resorption is always succeeded by the deposition of new bone
tissue, i.e. the formation phase (Mundy & Roodman 1987).
mesenchymal stem cell
hematopoietic stem cell
osteoblastic stromal cell
preosteoblast
lining cells
osteoblasts
formation
osteoclast
reversal
resorption
osteocytes
activation
Fig. 1. A schematic illustration of the bone remodeling phases. Modified from
(U.S.Department of Health and Human Services 2004).
Osteoblasts are anchorage-dependent cells. Their attachment and spreading
require so-called focal adhesions (FAs), complex protein aggregates including e.g.
paxillin and vinculin, which form a link between the actomyosin cytoskeleton of a
cell and the NCP proteins of the ECM via integrin receptors (Abercrombie et al.
1971, Ruoslahti & Pierschbacher 1987, Sastry & Burridge 2000). Herewith the
bone ECM affects various cellular functions, such as survival, motility, gene
expression, and proliferation (Damsky & Werb 1992, Huhtala et al. 1995, Moursi
et al. 1997).
Integrins are heterodimeric transmembrane receptors composed of α- and βsubunits, which bind to a specific tripeptide sequence, arginine – glycine –
aspartic acid (RGD) of NCP protein ligands (Ruoslahti & Pierschbacher 1986,
Hynes 1987). Bone cell types differ in terms of their integrin composition. For
instance, osteoblasts typically express e.g. fibronectin-specific α5β1 integrin as
23
well as α2β1 integrin, which binds to fibronectin and collagen-I. Also variable ανβintegrins are involved in osteoblastic development (Lai & Cheng 2005). Integrin
binding launches cellular signaling cascades involving e.g. protein kinases
(Boudreau & Jones 1999, Miranti et al. 1999). Integrin-mediated cell adhesion is
fundamental for osteoblastic functions including proliferation, differentiation, and
mineralization, and thus plays an important role also in the biomaterial field
(Lynch et al. 1995, Moursi et al. 1997, Gronthos et al. 1997, Siebers et al. 2005).
2.2
Carbohydrates in bone biology
Carbohydrate molecules play important roles in several biological processes; they
act as recognition molecules in cell-cell- and cell-ECM communication. For
instance, cell adhesion to ECM molecules involves not only integrins but also
some sugar-containing non-integrin receptors, such as the laminin-binding
dystroglycan (Colognato et al. 2007) and various cell surface proteoglycans
(Adams & Watt 1993). Glycans also act as antigenic and differentiation
determinants. The function of glycans is to fine-tune biological processes; these
complex molecules often located at cell surfaces are very information-dense. The
interactions between glycomolecules and proteins capable of binding them, i.e.
lectins, have been under intense investigation because they may pave the way for
the development of e.g. cancer therapeutics. (Nangia-Makker et al. 2002, Flitsch
& Ulijn 2003, Shriver et al. 2004)
2.2.1 Sugar molecules in bone and joints
In bone tissue, various macromolecules containing sugar components are located
both at bone cell surfaces as well as in the ECM. For example,
glycosaminoglycans (GAGs), such as chondroitin and heparan sulfates, are
common constituents of bone ECM. GAGs are acidic polysaccharide side chains
formed of repeated uronic acid and hexosamine sugars forming larger
proteoglycan aggregates together with a core protein (e.g. perlecan or syndecan),
which thus are strongly anionic. The resulting negative charge is considered to be
crucial for the ionic calcification of the bone matrix since the linking of
hydroxyapatite crystals with collagen fibers is affected by and partly dependent
on the bone ECM ground substance rich in proteoglycans. Proteoglycans also
form major components of articular cartilages and intervertebral discs.
24
GAGs have various functions in bone and cartilage tissues. As indicated with
murine calvarial cell and organ cultures, GAGs may modulate the effect of
growth factors (e.g. FGF-2 and TGF-β1) on bone development (Manton et al.
2007a). Heparan- and chondroitin sulfates have also been identified as regulators
of BMP-mediated differentiation of osteoblasts from hMSCs (Manton et al.
2007b). Interestingly, GAGs have recently been shown to be overexpressed in
osteosarcoma, and they may thus act as potent regulators of cancer progression by
controlling the binding of OPG to RANKL (Lamoureux et al. 2009).
In cartilage, GAGs and proteoglycans in general are crucially involved in e.g.
the responses of articular cartilage after joint immobilization – induced atrophies
(Kiviranta et al. 1994, Jortikka et al. 1997, Haapala et al. 1999). Degradation and
repair of intervertebral discs as well as knee articular cartilage also involve
changes in the levels of proteoglycans, including biglycan, decorin and
syndecan-1 (Inkinen et al. 1998, Salminen-Mankonen et al. 2005). Additionally,
the proteoglycan / collagen ratio of the intervertebral disc annulus fibrosus has
been reported to decrease due to mechanical stress in dogs, which enhances the
aggregation between the remaining proteoglycans and hyaluronan (Säämänen et
al. 1993). Hyaluronan represents a free, unsulfated form of GAG. In cartilage,
hyaluronan contributes to ECM lubrication and assembly, but its functions in
bone have remained unclear. Recently, however, hyaluronan has been asserted to
be involved in reducing inflammation-related bone resorption by inhibiting the
expression of e.g. prostaglandin-E2 and MMPs in osteoblasts (Hirata et al. 2009).
MMP-1, -8- and -13 are the traditionally-recognized collagenases that function in
rheumatoid arthritis (RA), yet human trypsin-2 also appears to be involved in
collagen-II degradation in RA (Stenman et al. 2005).
2.2.2 Sugar molecules and osteoclasts
Glycobinding proteins are generally known as lectins. In order to enter into bone
tissue from the vasculature, osteoclasts have to recognize and bind to certain
glycoproteins, such as laminin, in the vessel basement membranes. The
subsequent interactions with bone ECM glycoproteins are also crucial for
osteoclastic activity. These glycomolecule-involving interactions are mediated by
e.g. galectin-3, a β-galactoside-specific lectin first identified in the plasma
membrane of chicken osteoclasts (Gorski et al. 2002). Galectin-3 is also been
reported to participate in endochondral bone formation since it functions as a
25
substrate for MMP-9, which controls chondrocyte apoptosis and osteoclast
recruitment in this process (Ortega et al. 2005).
Interestingly, some lectins of botanical origin have specific targets in bone.
For instance, Arachis hypogaea peanut agglutinin (PNA), which recognizes β-Dgalactose(1,3)N-acetyl-D-galactosamine (Gal-β-1,3-GalNac) disaccharide, and
wheat germ agglutinin (WGA), which recognizes N-acetyl(1,4)D-glucosamine
(GlcNAc) as well as terminal neuraminic acid, bind to osteoclastic cells (Illes &
Fischer 1989, Illes et al. 1992). PNA has also been reported to bind to some bone
marrow cells, endosteal mononuclear cells as well as peripheral rat monocytes,
and can be used for osteoclast identification (Väänänen et al. 1986). WGA is
commonly exploited in bone tissue analytics: The resorption pits can be stained
with WGA, which indicates the existence of the sugar epitopes recognized by this
lectin in the resorbed bone matrix (Selander et al. 1994). Furthermore, WGA has
been reported to attenuate ALP activity, whereas neuraminidase enzyme tends to
elevate it (Sharp et al. 2007). Additionally, various BALP isoforms can be
distinguished by their different sensitivity to WGA precipitation (Magnusson &
Farley 2002).
An important sugar moiety found in several cell types is a 9-carbon,
carboxylate group -containing sialic acid, a.k.a. neuraminic acid, which usually is
the most distal sugar molecule in the cell surface glycans. Sialic acids are found
in a wide variety of organisms – also in bacteria, fungi, and plants. Two types of
neuraminic acids – N-acetyl neuraminic acid (Neu5Ac) and N-glycolylneuraminic
acid (Neu5Gc) – are expressed in mammalian tissues with the exception that
humans express only the former due to a deletion mutation in the CMP-Neu5Ac
hydroxylase (CMAH) gene. Basically, in vertebrates, three types of glycan classes
can carry sialic acid moieties: glycosphingolipids, N-glycans and O-glycans.
Among other roles, sialic acids participate in cell-virus and cell-cell fusions;
for example alpha (2,3)-linked- and alpha (2,6)-linked sialic acids take part in
osteoclastogenesis (Takahata et al. 2007). Sialic acids may also influence stem
cell therapies as contaminating xenoantigens. For example, murine Neu5Gc
probably affects the efficacy and survival of transplanted human embryonic as
well as mesenchymal stem cells (Heiskanen et al. 2007). In fact, human serum
naturally contains IgG antibodies against Neu5Gc (Nguyen et al. 2005). Some
inherited diseases, such as Salla disease, also involve sialic acids; the transport of
the cleaved sialic acid residues to lysosomal degradation by a sialin transporter is
usually defective in these syndromes (Morin et al. 2004).
26
The functionality of a specific sialic acid is correlated with its terminal
position and specific structure in cell surface glycochains, and as a consequence,
the lectin recognition and subsequent functions of the “host cell” can be modified
by sialic acid modification or removal (Varki 1997). For example, trafficking of
mesenchymal stem cells to bone can be affected by modifying the sialylcontaining glycans on the membrane of MSCs (Sackstein et al. 2008). Sialic acids
are recognized by e.g. C-type lectins, selectins and by so-called Siglecs, which are
immunoglobulin-related proteins and form the largest category of sialic acid –
binding molecules (Varki 1997). Selectins usually require so-called a sialylLewisX -motif (SLeX) at the terminus of the glycan to be recognized (McEver
2002, Rosen 2004). This motif is made up of sialic acid residues located in the
vicinity of a α-1-3(4)-linked fucose residue. Sialyl Lewis X -epitopes have also
recently been shown to participate in glycoprofiling the differentiation stage of
bone marrow -derived mesenchymal stem cells (Heiskanen et al. 2009). In
addition, sialyl Lewis X -based glycostructures apparently play a role in
inflammatory conditions by acting as organ-specific epitopes for the L-selectins
of infiltrating leukocytes (Renkonen et al. 2002). In relation to tissue analytics, it
is noteworthy that PNA lectin recognizes the galactose moiety uncovered by
removing the terminal sialic acid residue of glycans.
2.2.3 Sugar molecules and osteoblasts
Variable effects of GAGs on bone cells have been discovered: The heparin
concentration affects the growth and mineralization of osteoblasts (Hausser &
Brenner 2004). Recently it has also been shown that heparan sulfate containing
proteoglycans can act as co-receptors for fibroblast growth factors (FGFs), a
phenomenon underlying e.g. osteoblastic syndecan expression (Song et al. 2007).
In turn, a sulfated hyaluronan has been shown to scale up the expression of ALP
and the cell adhesion molecules N-cadherin and connexin-43 in rat osteoblasts
(Nagahata et al. 2004). Biglycan – a bone ECM small leucine-rich proteoglycan
(sLRP) – is needed for the differentiation of osteoblasts (Chen et al. 2004, Chen
et al. 2005, Parisuthiman et al. 2005). Another sLRP, decorin, is also crucial for
collagen fibrillogenesis and bone-implant contact (Klinger et al. 1998, Matsuura
et al. 2005). Decorin is also probably responsible for the proper timing of
osteoblast differentiation by delaying mineralization of incompletely fibrillated
collagen matrix (Mochida et al. 2003). Heparan sulfate proteoglycans are also
capable of inhibiting osteoblastic functionality, and this restraint is overcome by
27
the heparanase production of osteoblasts (Kram et al. 2006). Interestingly,
heparan-like polymers synthesized from dextran have also been shown to enhance
tissue repairing of bone defects by probably modulating the effects of heparanbinding growth factos (e.g. FGF) to osteoblastic cells (Blanquaert et al. 1999).
In addition to proteoglycans, osteoblasts produce several glycoproteins in
bone matrix including the aforementioned FN. Sialic acids are often linked to
glycoproteins; for example, proteins characteristic of bone and dentin tissues –
OPN, BSP (which is strictly specific for mineralized tissues), bone acidic
glycoprotein-75 (BAG-75) as well as dentin-specific dentin matrix protein -1
(DMP1) and dentine sialoprotein (DSP) – are rich in sialic acids (Oldberg et al.
1986, Somerman et al. 1988, Butler 1989, Ohnishi et al. 1991, Butler et al. 1997,
Qin et al. 2001). Along with these, osteoblasts express calcium-, collagen-, and
hydroxyapatite-binding osteonectin, which is a phosphorylated, sialic acid –
containing glycoprotein accounting for the greatest proportion of osteoblastderived NCPs in bone (Termine et al. 1981). Osteonectin has also been shown to
play a role in breast cancer metastasis to bone (Campo McKnight et al. 2006).
Additionally, the presence of the terminal sialic acid and other glycosylations on
the surface of osteoblasts is crucial for the tethering and proliferation of
hematopoietic progenitor cells (Crean et al. 2004).
An additional perspective on the links between glycomolecules and bone
tissue is represented by sugar-binding sites of osteoblasts. For example, galectin-3
is expressed also in osteoblasts, in which the galectin-3 production is altered by
so-called advanced glycation end-products (AGE) thus probably affecting
osteoblast and bone tissue metabolism (Stock et al. 2003, Mercer et al. 2004).
AGEs are produced in diabetes mellitus and probably involved in the appearance
of osteopenia since accumulated AGEs in bone ECM seem to play a role in the
regulation of osteoblast functions that are dependent on the osteoblast
differentiation phase (McCarthy et al. 1999, McCarthy et al. 2001a, McCarthy et
al. 2001b). AGEs also seem to affect osteoblastic attachment via integrins by
modifying collagen-I of the bone ECM (McCarthy et al. 2004).
Osteoblasts also express a membrane-bound osteoclast inhibitory lectin
(OCIL) capable of inhibiting osteoclast formation (Zhou et al. 2001, Hu et al.
2004). OCIL-expression of osteoblasts is regulated by various factors including
e.g. parathyroid hormone -related protein (Zheng et al. 2009). Soluble
recombinant mouse and human OCILs bind several important GAGs (Gange et
al. 2004).
28
2.3
Bone implants
Implanting substitutive devices and prostheses into pathologically destroyed or
injured tissues (as a consequence of e.g. osteoarthritis, osteoporosis, or trauma) is
a relatively common and clinically important approach to compensate for the
anatomical loss of the tissue and to restore its biological function. A standard
material in bone and dental implants is titanium (Ti), a metallic element proven to
be sufficiently physiologically inert to be used in clinical implantations. Cobaltbased alloys have also performed well, whereas the formerly used stainless steel
has proven to be too stiff for orthopedic purposes. A shape-memory alloy, nickeltitanium (NiTi), has also been widely studied as a hard tissue implant material
(Kapanen et al. 2001, Muhonen et al. 2009). Occasionally, however, implanting
procedures may lead to severe adverse effects. Inefficient implant insertion,
inflammation, and / or infection may prevent healing and result even in implant
loosening. The possible complications usually are consequences of insufficient
biocompatibility between the implant and the cells producing the surrounding
tissue.
2.3.1 Biocompatibility: Biological factors
In the case of hard tissue implants, biocompatibility refers to the interactions
between a biomaterial and bone cells. Biocompatibility is characterized by the
ability of the cells contacting the biomaterial to remain alive and functional; these
functions should not be disturbed by contact with the implant. In a broader
context, biocompatibility signifies that unacceptable harm to the recipient of a
biomaterial is avoided. Biocompatibility is a transient concept since its definition
varies with the progress in biomaterial science (Williams 2008).
In orthopedic and dental implants, osseointegration is a critical factor, which
is dependent on various interactive processes between the contacting cells –
especially osteoblasts – and biomaterials. First, cells have to attach to the
biomaterial surface. Osteoblasts adhere via integrins, the plasma membrane
receptors described above. A commonly used method of detecting integrin-based
cellular attachment in vitro is the fluorescent labeling of the protein components,
e.g. paxillin and vinculin, of FAs. Another, osteoblast-specific indicator of cell
attachment onto biomaterials involves ECM protein heparin-binding sites and
heparan sulfate proteoglycans of osteoblastic plasma membrane. This
proteoglycan-based adhesion is especially mediated by the so-called KRSR
29
(lysine-arginine-serine-arginine) peptide found in the ECM protein vitronectin,
and several studies discuss the use of these components in the improvement of
orthopedic biomaterials (Dalton et al. 1995, Dee et al. 1998, Dettin et al. 2002,
Bagno et al. 2007).
Secondly, after attachment, cells should begin to proliferate. Proliferation is
the phase of vigorous division, growth, and spreading, during which the cells
usually do not express specific differentiation markers. The tendency of a
biomaterial to support proliferation can be detected by e.g. staining the
cytoskeletal actin to visualize the cellular morphology and spreading.
Thirdly, cells have to differentiate into mature osteoblasts capable of
producing the mineral required for the effective osseointegration of a bone
implant. The differentiation competency of osteoblasts can be traced by
investigating certain differentiation markers, such as the expression of
osteoblastic genes (e.g. ALP) and mineral deposition (e.g. quantifying the calcium
produced).
The biocompatibility of bone implants proven by osseointegration is mainly
dependent on the functionality of osteoblasts. In fact, it has recently been
suggested that bone formation on artificial materials does not deviate from the
natural process of bone formation. Bone-implant bonding is created by the
interdigitation of the osteoblast-produced, non-collagenous bone cement with the
sub-micron scale topography of the biomaterial rather than a phenomenon defined
solely by the bioactive chemical features of the material surface (Davies 2007).
The activation of resorbing osteoclasts as such may be undesirable in
implantations. However, in prescreening the biocompatibility of the bonecontacting biomaterials, osteoclasts serve as a feasible cell model due to their
extremely fine-tuned and sensitive growth requirements and differentiation
processes. Additionally, balanced interactions and communication between
osteoclasts and osteoblasts are crucial around the implants as well; the signals
subsequent to resorption that attract osteoblasts to the site could be beneficial also
in the osseointegration of hard tissue implants.
2.3.2 Biocompatibility: Physicochemical factors
Metal surface microtopography has been reported to strongly affect the cells so
that increased roughness of e.g. the titanium surface seems to promote osteoblast
differentiation in parallel with decreased osteoclast formation (Boyan et al. 2002,
Schneider et al. 2003, Lossdorfer et al. 2004). The porosity of a biomaterial also
30
enhances bone formation around certain biomaterials, such as a recently reported
glass fiber – reinforced composite (Mattila et al. 2009). However, the fixation
between bone and total joint arthroplasties, their subsequent permanency and
possible need for revision variably depend on e.g. the overall implant design,
texture, porosity, polishing, wear debris production, fatigue failure, micromotion
and probably elevated hydrodynamic pressure (Bauer & Schils 1999a, Bauer &
Schils 1999b).
The attachment of the cells onto a biomaterial depends largely on e.g. the
adsorption of proteins onto the implant surface, which is the first important phase
in determining the biocompatibility between tissue and foreign materials. Protein
adsorption from surrounding fluids specifically affects the behavior of the cells
that contact the proteins (Horbett & Weathersby 1981, Horbett 1981, Steinberg et
al. 1989). Especially proteins containing the RGD sequence play a definitive role
in determining the biocompatibility of the implants. Protein molecules in the
surrounding fluids as well as those secreted by cells are adsorbed onto the implant
material surface depending, for example, on the wettability – i.e. hydrophobicity /
hydrophilicity – of the surface. The pursuant effects are conditional on the
material surface properties and on the types of proteins adsorbed (Steinberg et al.
1989, Lee & Voros 2005). Many physical properties including the wettability and
electrical charge of biomaterials are linked to the type and the size of the
molecules on a surface, which should be carefully considered when coating
biomaterials. The wettability of a biomaterial may affect integrin receptor
expression of the cells: For instance, human fetal osteoblasts prefer a hydrophilic
to a hydrophobic surface in terms of αvβ3-integrin expression and cell spreading
(Lim et al. 2005). Hydrophobicity is also generally considered as a disadvantage
for biomaterial biocompatibility (Chang et al. 2005).
2.3.3 Host responses to implantation
Implantation of a biomaterial into tissue always induces a so-called foreign body
reaction at the implantation site. This reaction can be regarded as a phase in a
wound healing reaction series, which usually involves acute/chronic inflammation
and thrombosis to a varying degree. A foreign body reaction is preceded by the
formation of fibrin-replacing fibrous connective tissue (i.e. granulation tissue),
and followed by the formation of a fibrous tissue capsule (i.e. fibrosis) around
injured tissue. The response to implantations is also characterized by the arrival of
monocytes, which subsequently mature into macrophages and fuse to form so31
called foreign body giant cells (FBGC). For example OPN is reported to inhibit
the formation of FBGCs to some extent (Tsai et al. 2005). From the carbohydrate
point of view, it is worth mentioning that macrophage fusion is partly mediated by
mannose receptors located on their plasma membranes (McNally et al. 1996,
DeFife et al. 1997).
In contrast to natural (without implants) wound healing, host responses to an
implant include protein adsorption onto the implant surface. This adsorption
might promote chronic inflammation at the implantation site. Additionally,
infections caused by bacteria, often Staphylococcus aureus and skin-associated
Staphylococcus epidermidis that attach to implant surfaces are classified as
adverse outcomes of implantations leading to a risk of implant loosening. In fact,
infection frequencies in total hip and knee joint prosthetic implantations average
between 1.5–2.5% in primary and between 3.2–5.6% in revision operations, of
which S. epidermidis accounts for 19–37.5% and S. aureus for 22–23.6% of the
cases (Lentino 2003, Rohde et al. 2007). (Gristina & Costerton 1985, Anderson
1988)
Chronic inflammation around an implant may also increase the release of
wear particles from the biomaterial surface under mechanical strain over time.
This phenomenon is likely to lead in unwanted osteoclastic activity and bone
resorption around orthopedic implants. Osteoclasts are induced by the
inflammatory cytokines and other factors, e.g. interleukins (IL-1, 6, 11), TNFs,
OPG, and RANK/+, produced by the wear particle -stimulated cells (Konttinen et
al. 2005). The resulting peri-implant osteolysis is the most common reason for
aseptic implant failure, which also may be partly mediated by lymphocyte
responses to the metal ions released (Haynes et al. 2004, Hallab et al. 2005).
Additionally, wear debris from a biomaterial may carry adherent, bacteria-specific
endotoxins, e.g. lipopolysaccharide (LPS), which also have been shown in some
cases to stimulate osteoclasts and cytokine secretion (Bi et al. 2001, Gorbet &
Sefton 2005).
In order to minimize undesirable host responses and the subsequent risk of
implant loosening, the modification of biomaterials with various molecular
coatings aims to enhance the biocompatibility of implants.
2.3.4 Coating of bone implants
The clinical improvement of bone implants by various coatings has recently been
under intense investigation. ECM components including various proteins and
32
polysaccharides that contribute significantly to the regulation of tissue
homeostasis, have been investigated as potential biomaterial coatings (Morra
2006, Furth et al. 2007). Several examples of interactions between bone cells and
biomaterial coatings have been reported: For example, collagen – a naturally
occuring protein component of bone tissue – has been tested as a coating
molecule (Morra et al. 2003a). Bioactive coatings of hydroxyapatite (HA) and
other ceramics have been found to contribute positively to joint arthroplasties and
dental implants (Dumbleton & Manley 2004, Alzubaydi et al. 2008).
Pharmacologically-tuned implants have also been prepared; for example, HAcoated titanium implants have been treated with zoledronate (a bisphosphonate) to
reduce peri-implant osteolysis (Peter et al. 2005). For bone tissue engineering, a
resorbable calcium phosphate (CaP) scaffold has produced promising results
(Lickorish et al. 2007). Also nanosized HA-based composites and bone mineralorganic mimics are being developed (Huang et al. 2008, Best et al. 2008). The
effect of polysaccharides, e.g. foreign (non-human) chitosan on osteoblasts has
been studied (Bumgardner et al. 2003, Wang et al. 2008). Other examples of
glycomolecules considered for coating applications include hyaluronan (Morra
2005), heparin (Keuren et al. 2003), alginic acid (Morra & Cassinelli 2000), and
dextran hydrogels (Ferreira et al. 2004). Plant-derived starch polysaccharides
have also been tested in the context of bone scaffolds (Salgado et al. 2007).
The substratum to be coated in the case of bone implants is usually titanium,
even though other biomaterials, such as algae-derived hydroxyapatite ceramic
bone substitute, have also provided promising in vitro results (Turhani et al.
2005). Despite the general physiological inertness of Ti, several clinical cases
indicate problems relating to titanium implantations and thus the need to seek
appropriate molecular improvements of this implant material. Furthermore,
coating may augment the beneficial features of Ti, i.e. ability to support bone cell
proliferation and differentiation, as reported with e.g. collagen (Morra et al.
2003a), HA (Goto et al. 2004), FN (Schneider & Burridge 1994), and different
adhesive peptides (Dettin et al. 2009). However, the possibility to precisely
modify and tailor the bioactive coating molecules to improve their
biocompatibility would be a significant advance. Novel candidates for this
purpose are pectins.
33
2.4
Pectins
Pectins were first identified in 1825 (Braconnot 1825a, Braconnot 1825b). These
large and complex polysaccharides form a versatile molecular group of structural
components of taxonomically higher, land-habiting plant cell walls
(dicotyledons), as well as of some algae. Pectins are most abundantly found in the
primary plant cell wall and also in the middle lamella between contiguous plant
cells. Together with hemicellulose molecules, pectins comprise the major (on
average 40% of the dry weight in fruits and vegetables) elements of the plant cell
wall matrix, within which cellulose microfibrils construct a rigid lamellar network
capable of bearing osmotic pressure as well as mechanical stress. The schematic
topography of pectin molecules in a plant cell wall is outlined in Figure 2.
Pectins play several important roles in plants; they participate in e.g.
mechanical support, morphological development, fruit ripening, and
emulsification of plant tissue (Redondo-Nevado et al. 2001, Vincken et al. 2003).
Especially the emulsifying role is noteworthy since the hydrocolloidal gelforming property of pectins is widely exploited in e.g. the food industry. This
ability of pectins to gel is due to many negatively-charged sugar units (containing
-COO- groups) in the homogalacturonan region that are prone to bind Ca2+ cations
and form so-called “eggbox” structures. This calcium cross-linking of pectin
subsequently leads to profuse hydration.
Fig. 2. Plant cell wall architecture showing the topography of pectins.
34
The structural details of pectins vary both species- and tissue-specifically but
some elements are common to all pectins. Two major structural components can
be separated: so-called smooth and hairy regions. The smooth region consists of
α-1,4-linked D-galacturonic acid (Fig. 3) residues (α-D-GalA), some of which are
esterified on the carboxyl group with methanol yielding either a high-methyl
(HM) or low-methyl (LM) homogalacturonan chain. In contrast, the hairy region
(also known as the ramified region) contains two alternating sugar residues, α1,4-linked D-galacturonosyl and α-1,2-linked L-rhamnosyl moieties, forming a
rhamnogalalacturonan-I (RG-I) backbone. The RG-I backbone might carry
various kinds of chemical groups (e.g. acetyl groups linked to hydroxyl groups of
galacturonic acid residues) and side chains (e.g. rhamnose-linked arabinan and
arabinogalactan as well as partly methanol-esterified xylogalacturonan polymers).
Pectins may also contain a conserved and complex RG-II component attached to
the homogalacturonan region (Ishii & Matsunaga 2001). The complexity and
variability of different pectins follow from the diverse combinations of the many
structural blocks, chemical groups, and side chains, which provide pectins with
varying physico-chemical as well as biological properties. (Doco et al. 1997,
Willats et al. 2001, Vincken et al. 2003, Hinz et al. 2005)
2.4.1 Pectins as candidates for biomaterial applications
Pectins are novel and interesting candidate macromolecules for implant coating
applications. In general, plant-derived polysaccharides hold a great potential in
cell behavior modulation, as already indicated with various soluble botanical
sugars and mammalian cell types (Ding et al. 2003, Brunold et al. 2004, Deters et
al. 2005). Besides these, the effects of artificial sugar-grafted polystyrene
substrata have also been assessed (Kim et al. 2003).
Due to their botanical origin, the acquisition and use of pectins would not
raise ethical questions. They are readily available and inexpensive since they are
remnant by-products of e.g. juice manufacture. Additionally, it is noteworthy that
the human body cannot degrade these plant-derived molecules, which apparently
is an intriguing feature in biomaterial implantations. Finally, the prime benefit of
screening pectins as coating molecules is their vast natural diversity combined
with the possibility to specifically tailor them in vitro and in vivo.
35
2.4.2 Modification of pectins
Pectin composition depends on the plant species and tissue types: For example,
four different pectin fractions have been isolated from apple (Schols et al. 1995).
In addition to this natural variation on pectin structures, they can be further
tailored in vitro with pectinolytic enzymes – usually as commercial, microorganism -derived enzyme mixtures designed for degrading disruptive pectin gels
and to promote the release of beneficial flavonoids in juice production. After
pectin isolation, it is possible to separate smooth and the RG-I -containing hairy
regions with endopolygalacturonase and rhamnogalacturonan hydrolase.
Polygalacturonases (acting either exo- or endo enzymes) degrade
homogalacturonan without methyl esters, and rhamnogalacturonases cut the
glycosidic bond between rhamnose and galacturonic acid residues, respectively
(Mutter et al. 1996). The charge, wettability, and polarity of these molecules can
easily be controlled by modulating the content of methyl and acetyl groups as
well as the chain length of these molecules with e.g. pectin methyl and acetyl
esterases and specific hydrolases and lyases (pectin lyases can also attack methylsubstituted homogalacturonan) (Schols et al. 1994, Schols et al. 1995, Mutter et
al. 1998a, Mutter et al. 1998b, Daas et al. 2000, van Alebeek et al. 2002, van
Alebeek et al. 2003). These enzymes are strictly specific to certain substructures
of pectin meaning that their actions are not dependent on the composition of the
whole molecule (Schols et al. 1994). Methyl esterases of fungal origin tend to act
randomly, whereas the corresponding plant-derived esterases function merely in a
blockwise manner, although many factors seem to influence this mode of action
(Micheli 2001). Acetyl esters from the homogalacturonan region are removed by
pectin acetyl esterases and from the RG-I region by rhamnogalacturonan acetyl
esterases (Searle-van Leuween et al. 1992). The attack sites of pectin-specific
enzymes are shown in Figure 3.
36
Fig. 3. General structural organization of a pectin molecule and the attack sites of
pectin-specific enzymes. Modified from (Voragen et al. 1995, Morra et al. 2004).
Pectin fragments formed by enzymatic treatments are designated as modified
hairy regions (MHRs). It may be possible to exploit the differences in the
chemical, physical, and biological properties of these molecules in the coating of
medical devices. MHR fragments can indeed be considered as nanocoatings since
they have been deposited onto surfaces to form a 6–10 nm thick coating layer.
MHRs can be covalently linked onto a surface via carbodiimide condensation.
The surface to be coated is first functionalized with allylamine plasma deposition
(aminated surface, AMI), after which a covalent bond is formed between the
deposited amino groups and the carboxyl groups of the MHRs. X-ray
photoelectron spectroscopy (XPS) has been used to verify that the immobilization
of MHRs is successful and uniform. Atomic force microscopy (AFM) has in turn
revealed that the repulsive reaction of MHRs occurs over a shorter range than e.g.
carbodiimide-condensed hyaluronan (Morra et al. 2003b). This probably reflects
the fact that, unlike e.g. the merely hydrogen-bonding hyaluronan, the ionic
repulsion of MHRs could be more significant since here the neutral side chains as
well as acetyl and methyl esterifications are emphasized. (Morra et al. 2004)
Another means of tailoring pectin fragments is their in vivo modification in
planta (McCann et al. 2001). For example, the pectin composition of potato
tubers has been engineered by expressing a fungal endo-galactanase cDNA in
transgenic potato plants (Oxenboll et al. 2000).
37
Interestingly, LM pectin hydrogels cross-linked either ionically (with Ca2+
ions) or covalently (with divinylsulfone) have also been recently evaluated for
their ability to promote apatite nucleation in the presence of simulated body fluid
(SBF) – a crucial phase in bone-bonding of biomaterials (Ichibouji et al. 2008,
Ichibouji et al. 2009). In turn, pectin nanogel cross-linked with glutaraldehyde has
shown potential as drug delivery system (Chang et al. 2007).
2.4.3 Immunological aspects of pectins
Implant failures often appear as a consequence of excessive host reactions at the
implanted tissue area. Inflammation represents the innate immune system, in
which monocytes/macrophages as well as other leukocytes play important roles.
In contrast, specific immunological responses always involve the action of
lymphocytes, i.e. antibody-producing B-cells and helper- or cytotoxic T-cells,
which possess immunological “memory” and a coordinated, very exact system of
attacking antigens.
Variable and partially opposing immunological effects of pectins have been
described. For example, citrus pectins are reported to have some antiinflammatory effects on LPS-stimulated macrophages, a promising discovery
from the implantation point of view (Chen et al. 2006). The degree of
esterification of pectins also plays a role: According to Salman and colleagues
(2008), citrus pectins with a higher degree of esterification are more prone to
inhibit the production of some pro-inflammatory cytokines and to enhance
production of the anti-inflammatory cytokines of human peripheral blood
mononuclear cells (Salman et al. 2008). On the other hand, some pectins promote
certain immunological responses, such as the stimulation of lymphocytes (Sakurai
et al. 1999, Dourado et al. 2004a) as well as complement activation (Wang et al.
1994, Michaelsen et al. 2000a). Interestingly, it has also been shown that, in some
cases, a whole pectin molecule is immunologically inert, whereas its degradation
fragments are not (Wang et al. 2003).
The use of MHRs as a bone implant nanocoating is to a great extent
dependent on how it will be tolerated by human tissues. Neutrality in this respect
is the minimum requirement for clinical applications, but naturally it would be
beneficial if MHRs exhibited active anti-inflammatory features of the MHRs. The
immunological potential of MHRs may partly be determined by the acetyl and
carboxyl content indicating that it may be possibile to adapt the immunological
reactivity of the tailored MHRs.
38
3
Aims of the present study
Modified hairy regions (MHRs) of pectins are new, innovative candidate
macromolecules for the improvement of hard tissue implant biocompatibility.
These covalently attached plant-derived polysaccharides could work as a
functional coating on bone implants since the mammalian tissues are unable to
resorb them. MHRs can also be modified to assume various important surface
structures and properties. To examine the potential of pectins, their ability to
support bone cell growth as well as their probable immunological effects were
investigated.
The aims of the present research are summed up as follows:
1.
2.
3.
4.
To assess the interactions between both osteoclastic and osteoblastic cells and
the MHRs of apple pectin. By testing different MHRs (MHR-A and MHR-B)
we aim to reveal those properties of pectins that stimulate the attachment and
growth of bone cells.
To test the differentiation capacity of osteoblasts on MHRs grafted onto
titanium in order to gain information about bone formation and
mineralization processes on a pectin-coated bone implant material.
To investigate the possible inflammatory reactions induced by the MHRcoated titanium implants in soft tissue in vivo.
To assess the interactions between osteoblastic cells and the MHRs of wildtype and transgenic potato pectins.
39
40
4
Materials and methods
4.1
Cell cultures
The use of both primary and cell line -derived bone cell cultures (studies I, II, and
IV) are briefly explained in the following chapters.
4.1.1 Cell lines
Murine calvarial preosteoblastic MC3T3-E1 (subclone IV) cells (ATCC®, LCG
Promochem) were maintained in (I, IV) or induced to form osteoblasts (II). The
cells were cultured in α-MEM (Dulbecco`s modified Eagle`s medium, αmodification) supplemented with 10% fetal bovine serum (FBS), 1% Lglutamine, and penicillin (100 U/ml) – streptomycin (100 µg/ml) antibiotics. In
the mineralization medium, 50 µg/ml L-ascorbic acid 2-phosphate (AsAP) and
10-2 M β-glycerol phosphate disodium salt pentahydrate (β-GP) were added. Cells
were cultured in an incubator (+ 37 ºC, 5% CO2, 95% air) in T25 cell culture
bottles, released with trypsinization, and seeded at a density of 10 000 cells/cm2
on test materials (for 2 weeks in mineralization cultures).
Human mesenchymal cells (hMC) from bone marrow (Cambrex Inc.
Walkersville, Md) were cultured (by Dr. Marco Morra and colleagues, Nobil Bio
Ricerche, Portacomaro, Italy) in D-MEM containing 10% FBS, 1% L-glutamine,
and 100 U/ml penicillin + 100 µg/ml streptomycin antibiotics. Cells were
differentiated to mineralizing osteoblasts with 0.05 mM AsAP, 10 mM βGP, 0.1
µM dexamethasone, and mesenchymal cell growth supplement (MCGS)
additives. Cells were released with trypsinization and cultivated (105
cells/titanium disc) on test materials (II).
4.1.2 Primary cultures
Osteoclasts were differentiated from a bone marrow cell pool from long bones of
1–2 day old Sprague-Dawley rat pups (pit assay; I). Endosteal cells were scraped
into α-MEM (Sigma) containing 10% FBS, 1% L-glutamine, 100 U/ml penicillin
+ 100 µg / ml streptomycin, and 20 mM HEPES buffer. The cells were collected
by centrifugation, resuspended in the medium, and cultured (50 µl cell
suspension/cm2) on test materials for 2–4 days.
41
Primary osteoblasts (II) were differentiated from the bone marrow cell pool
of 12-week-old mice (male C57/BL6). Cells were cultured in α-MEM
supplemented with 10% FBS, 1% L-glutamine, 100 U/ml penicillin + 100 µg/ml
streptomycin, 50 µg/ml AsAP, 10-2 M βGP, and 10-8 M dexamethasone. After
centrifugation, the cells were resuspended in the same medium and cultured on
test materials at a density of 106 cells/cm2 for 2 weeks.
4.2
Test materials
The preparation and covalent grafting of the apple- and potato-derived pectin
fragments (MHRs) used in the studies (I-IV) are briefly explained in the
following chapters.
4.2.1 MHR preparations
Two different MHRs isolated from apple tissue (MHR-A and MHR-B) and
genetically engineered or wild type potato MHRs (MHRPTR_ARA,
MHRP_TRGAL, and MHRP_WT) were tested in this thesis. MHR-A and MHR-B
were obtained by in vitro modification with commercial enzyme preparations
[Rapidase 600 for MHR-A and Rapidase Rliq+ for MHR-B (DSM Food
Specialties)].
As reported in Table 1, MHR-A and MHR-B differ particularly in their acetyl
group content, arabinose amount, and side chain length. MHR-A contains linear,
mainly arabinan-rich side chains composed of on average > 10 arabinan units.
These chains have been digested with arabinanases from MHR-B (which contains
only 1–2 arabinoses in a side chain) resulting thus also in a lower molecular
weight (MW). However, the total sugar molar percentage is greater in MHR-B;
especially xylogalacturonan predominates in this fragment. MHR-B possesses a
lower degree of acetylation, whereas MHR-A has abundant acetyl substitutions.
The level of methylation does not differ significantly. (Morra et al. 2004)
Potato MHRs were produced by in vivo modification in transgenic / wild type
potato (Solanum tuberosum L. cv. Posmo) tubers (cultivated by Dr. Peter Ulvskov
and colleagues, Department of Genetics and Biotechnology, University of Aarhus,
Denmark). Transgenic MHRPTR_ARA reduced in arabinan and MHRPTR_GAL
reduced in galactan were obtained by expressing specific arabinase and
galactanase enzymes in the potato plants, respectively. Wild type potato
42
(MHRP_WT) was also tested. The sugar compositions of the MHRPs are
represented in more details in the study IV.
The MHR preparations were treated and analyzed by Dr. Henk Schols and
colleagues (Laboratory of Food Chemistry, Wageningen University, Wageningen,
The Netherlands).
4.2.2 MHR-coated samples
MHRs were covalently linked (Dr. Marco Morra, Nobil Bio Ricerche,
Portacomaro, Italy) to different test materials: tissue culture polystyrene (TCPS)
dishes (I, IV), objective glasses (I), titanium discs (II) or cylindrical titanium
items (III). Briefly, the test material surfaces were functionalized with amino
groups by allylamine plasma deposition (aminated surface, AMI), which is a
commonly used plasma modification method (Puleo et al. 2002). MHRs were
subsequently linked to the surfaces via covalent bonding between the amino
groups and the carboxyl groups of the MHRs (carbodiimide condensation). The
coating protocol is described in more detail by Morra and colleagues (Morra et al.
2004). Uncoated surfaces were used as controls. Test materials were sterilized
with 10 x solution (Sigma) of penicillin-streptomycin (I, II, IV) or with ethanol
(III).
Table 1. Molar percentages (mol-%) of sugar moieties of the apple-MHRs. Partly
modified from (Morra et al. 2004).
Sugar
MHR-A (mol-%)
MHR-B (mol-%)
Arabinose
50
11
Galactose
10
20
Galacturonic acid
26
37
Methyl / 100 mol of GalA
40
34
Acetyl / 100 mol of GalA
55
11
Glucose
1
3
Mannose
0
0
Rhamnose
5
11
0.19
0.3
Xylose
8
18
% (w/w) sugar
66
78
Rhamnose:GalA
43
4.2.3 Bone slices
Slices (~1.0 cm2, thickness 0.5–1.0 mm) of cortical bone of bovine femurs were
used as a cell culture control substratum. Briefly, slices were cut, sonicated (3 x
30 s), treated with 70% EtOH (prepared by Mikko Finnilä), and used as a culture
surface for bone resorption assays (I).
4.3
Stainings
For visualizing cells (studies I, II, and IV) and histological sections (study III),
various fluorescent and non-fluorescent staining methods were used.
4.3.1 Fluorescent labels
Before staining, the cells were fixed with 3% paraformaldehyde (PFA) in PBS
(supplemented with 2–4% sucrose) for 10 min at RT and rinsed with 1x PBS.
Cell nuclei were stained with a 1.25 µg/ml solution of DNA-binding Hoechst
33258 fluorochrome (Sigma Chemical Co.) for 10 min at RT (I). F-actin was
labeled with 500 ng/ml phalloidin (Sigma Chemical Co.) conjugated with FITC
(green fluorescence) or TRITC (red fluorescence) for 20 min at 37 ºC (I, II, IV).
Paxillin (I, II, IV) and vinculin (I) dots of FAs were stained with indirect
immunofluorescence labeling. The cells were permeabilized with 0.1% (v/v)
Triton-X-100 in PBS (10 min on ice) and blocked with 0.2% BSA (30 min at RT).
The permeabilized cells were then incubated with a 1:100 dilution of a
monoclonal mouse paxillin (ZYMED Laboratories) – or vinculin – (Sigma
Chemical Co.) specific primary antibody for 45 min and with a secondary
antibody (ALEXA Fluor 568 or ALEXA Fluor 488 goat anti-mouse IgG,
Molecular Probes) for 30 min on ice.
The calcium of osteoblast cultures (II) was labeled with
tetracyclinehydrochloride antibiotic (Sigma-Aldrich), which acts as a calcium
chelator able to bind to nascent, globular calcium deposits. Tetracycline (50
ng/µl) was added into the medium of cell cultures and incubated for 5 days before
fixation.
44
4.3.2 Non-fluorescent stains
Osteoclasts (I) and macrophages (III) were stained for tartrate-resistant acid
phosphatase (TRACP) using a Leukocyte Acid Phosphatase Kit (Sigma)
according to the manufacturer`s instructions. The TRACP5b isoform is expressed
by osteoclasts and TRACP5a by activated macrophages and FBGCs (Efstratiadis
& Moss 1985a, Efstratiadis & Moss 1985b, Kadoya et al. 1994).
Histological sections (III) were stained with hematoxylin-eosin (HE). Briefly,
samples were incubated in aluminum hematoxylin for 13 min, rinsed under
running water for 5 min, differentiated in 70% ethanol containing 0.5% HCl for
15–30 s, rinsed with distilled H2O and subsequently in ammonia water (3 ml NH3
in 1000 ml H2O) for 10 s. After rinsing with distilled H2O for 1–10 min, the
samples were incubated in an eosin solution (0.5% eosin in 25–50% ethanol) for
1.5 min, differentiated by quickly dipping the samples in distilled H2O 3–6 times
and by washing them in 95% ethanol that contained 2 drops of 0.1% ammonium
hydroxide.
4.4
Microscopic methods
For microscopic visualizations, the samples were mounted in glycerol-PBS,
Pertex or Shandon Immu-MountTM (Thermo) and covered with cover slips.
4.4.1 Light and fluorescence microscopy
Light microscopy was used for the investigation of TRACP-stained osteoclasts (I)
and histological sections (III). Hoechst- and phalloidin -stained cells (I) were
visualized with a conventional epifluorescence microscope (Nikon Optiphot II)
using a suitable filter for each fluorochrome. Microscopic images were recorded
with a Sony DXC 930P video camera.
4.4.2 Laser scanning confocal microscopy
Samples stained with phalloidin (I, II, IV), paxillin (I, II, IV) and vinculin (I)
antibodies or tetracycline (II) were visualized with a confocal laser scanning
microscope (LSM 510, Zeiss) using 20×, 40×, 63× and/or 100× objectives.
45
4.4.3 Field emission scanning electron microscopy
For field emission scanning electron microscopy (FESEM) visualization of cells
and sample surfaces (IV), cut circular pieces (Ø 8.0 mm) of sample surfaces were
dehydrated in ascending ethanol series (30%  50%  70%  80%  90%, 10
min in each) and finally twice in 100% (5 min). Samples were subsequently
critical point dried (BAL-TEC CPD 030) and sputtered (Agar High Resolution
Sputter Coater) with platinum (~15 nm Pt coat). Imaging was performed with
Zeiss ULTRA Plus -machinery using 3.0 kV voltage and 4000- , 8000-, or 20000fold magnifications.
4.4.4 Image analyses
Microscopic fields of paxillin and/or vinculin (I, IV) or tetracycline (II) data
obtained with the laser scanning confocal microscope were analyzed with MCID
M4 3.0 Rev. 1.1 or with MCID-M5+ (Imaging Research, Inc., Canada) digital
image analyzing system.
4.5
Molecular methods
Molecular methods were used for assessing the ALP production and activity of
osteoblastic cells (study II).
4.5.1 RT-PCR
Total RNA was isolated from osteoblasts (II) with a GenEluteTM Mammalian
Total RNA Kit (Sigma-Aldrich) according to the manufacturer`s instructions.
For RT-PCR, RNA was reverse-transcribed with SuperScriptTM III Reverse
Transcriptase (InVitrogen) to cDNA, which was subsequently PCR-amplified
using gene-specific primers (Sigma Proligo) and DyNAzymeTM EXT DNA
Polymerase (Finnzymes) according to the manufacturer`s instructions. The primer
sequences for ALP were 5`-GCCCTCTCCAAGACATATA-3` (forward) and 5`CCATGATCACGTCGATATCC-3` (reverse) demarcating a product size of 372
bp (Qu et al. 1998, Zhao et al. 2005). For β-actin, the forward primer sequence
5`-TGGACTTCGAGCAAGAGATGG-3` and the reverse primer sequence 5`ATCTCCTTCTGCATCCTGTCG-3` demarcating a 289 bp product were used
(Wu et al. 2004, Zhao et al. 2005).
46
PCR reactions for both genes started with 30 min at 48° followed by 10 min
at 95º (ALP) or at 94º (β-actin). ALP was produced in 30 cycles of 1 min at 95º, 2
min at 55°, and 1 min at 72º. β-actin synthesis consisted of 23 cycles of 1 min at
94º, 45 s at 62º, and 45 s at 72º. Both reactions were completed with 7 min at 72º.
DNA products were subjected to 1% agarose gel electrophoresis, visualized
with SYBR® Green Nucleic Acid Stain (Fluka), and compared to GeneRulerTM
100 bp DNA ladder (Fermentas). Mouse genomic DNA and pure water were used
as negative controls for the PCR reactions.
4.5.2 Enzyme activity measurement
The specific ALP activity (i.e. the ratio of the absorbance to the cell number) of
hMSCs cultured on coated titanium samples was spectrophotometrically
measured (II) 3, 7 and 10 days after cell seeding (by Dr. Marco Morra, Nobil Bio
Ricerche, Portacomaro, Italy). For cell counting, a MTT (dimethyl thiazolyl
diphenyl tetrazolium salt) assay was used. In this assay, the yellow MTT solution
is reduced by the cells` mitochondrial dehydrogenase enzyme to insoluble blue
formazan thus indicating cell viability (Alley et al. 1988). Briefly, the cells were
incubated in MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
bromide)-sodium succinate solution for 3 h at 37 °C, after which 6.25% v/v
0.1mol/l NaOH in dimethylsulfoxide was added in order to dissolve the formazan.
The wells were agitated for 5 min until the purple color was evenly distributed,
and the absorbance was evaluated at a wavelength of 560 nm.
For ALP activity measurement, the cells were lysed with lysing solution
(0.2% Triton X-100, Sigma). The lysates were then incubated with the enzyme
substrate mixture solution containing 1 mM MgCl2 x 6H2O, 1.5 mM 2-amino-2methyl-1-propanol and 20 mM p-nitrophenylphosphate for 2 h at 37 °C. The
reaction was stopped with 1 M NaOH. The absorbance of the samples was read at
a wavelength of 405 nm with a Tecan Genios microplate reader. Absorbances
were finally compared with standard curve values obtained by the measurements
of p-nitrophenol standard solution. The reaction steps of the specific ALP activity
are described in more details in the study II.
4.6
In vivo procedures
The in vivo tolerability of MHR-coated titanium implants were assessed in the
study III.
47
4.6.1 Animals and implantations
Animals (12-week-old Sprague-Dawley rats) used in the in vivo study (III) were
obtained and handled according to the procedure approved by the Ethical
Committee of the University of Oulu and the County Administrative Board of
Southern Finland.
Coated (MHR-A, MHR-B, AMI) and uncoated (Ti) titanium cylinders (length
6 mm, diameter 1.8 mm) were implanted under the fascia of the latissimus dorsi
muscle, during which the animals were under isoflurane (4.5%  2.5% for
maintaining) gas anesthesia. Buprenorphine (Temgesic; 0,05 mg / kg s.c.) was
used for analgesia before (30 min) and after (for 3 d) the operation. After 1 or 3
weeks, the rats were euthanized with CO2 suffocation and the implants together
with the surrounding tissues were removed for histological section preparation.
4.6.2 Histological sections
Soft tissues surrounding the implants were fixed in neutral formaldehyde solution
(100 ml 36–40% formalin, 900 ml H2O, 4 g NaH2PO4, 6.5 g Na2HPO4) and
dehydrated with an ascending alcohol series: 70% (16 h)  96% (16 h)  twice
in 100% (1 h)  xylene (twice for 30 min). The samples were embedded in 55–
57 ºC paraffin, and sectioned with a microtome (Microm HM 355 S). For
hematoxylin-eosin (HE) staining (described above), the sections were
deparaffinized and rehydrated in a descending alcohol series (95%  absolute
ethanol twice; 3–5 min in each) leading to double xylene incubation (3–5 min).
After another dehydration phase following the HE staining, the sections were
mounted onto glass slides for microscopy. Sections were prepared and stained by
Mrs. Anna-Maija Ruonala and Mrs. Paula Salmela.
4.7
Statistical analyses
Statistical analyses were run with SPSS14.0 software. The normality of the
datasets was determined with a Kolmogorov-Smirnov test. For normal
distributions, a parametric analysis of variance (ANOVA) and a t-test were used.
Kruskal-Wallis and Mann-Whitney tests were used for nonparametric analyses.
The statistical significance was set at a p-value ≤ 0.05. Origin 6.0 or Origin 7.5
software was used for constructing the histograms. Statistical significance is
denoted as follows: * p < 0.05; ** p < 0.01; *** p < 0.001.
48
5
Results
5.1
Interactions between MHRs and bone cells
The growth of both bone-resorbing osteoclasts and bone-forming osteoblasts on
different, covalently-linked MHR-types were studied in this thesis.
5.1.1 Proliferation and differentiation of osteoclasts (I)
The in vitro biocompatibility between MHRs and bone cells was preliminarily
screened with osteoclasts. Primary osteoclasts differentiated from the bone
marrow cell pool were cultured on MHR-grafted and on uncoated TCPS as well
as on bone slices as control surfaces. Of the pectin fragments, MHR-B was
preferred by the cells, as indicated by cellular density, morphology, and a greater
number of TRACP-positive (TRACP+) osteoclasts. The total number of TRACP+
cells on MHR-B also exceeded that on uncoated TCPS (p < 0.01) yielding values
statistically similar to those on the bone slice surface. In addition to the total cell
number, osteoclasts containing 1–2 nuclei and 3 or more nuclei (indicated as
multinuclear cells) were calculated separately. Osteoclasts with 1–2 nuclei tended
to reject MHR-A (p < 0.001), whereas they preferred MHR-B even over uncoated
TCPS (p < 0.01) or bone slices (p < 0.05). The number of multinuclear (≤ 3
nuclei) cells did not differ between MHR-A and MHR-B. On bone, the greatest
number of multinuclear TRACP+ cells as well as the proportion of multinuclear
cells/total TRACP+ cells was observed compared to all other sample types. The
multinuclear cells/total TRACP+ cells ratio did not differ between MHRs,
whereas the ratio of TRACP+ cells/the total bone marrow pool did in favor of
MHR-B. Actin ring staining supported the data. On MHR-A, actin rings were not
detected, on MHR-B approximately 1/3 of the cells contained an actin ring, and
on bone all the osteoclasts had formed an actin ring.
5.1.2 Proliferation and differentiation of osteoblasts (I, II, IV)
MC3T3-E1 cells (I, II, IV), primary osteoblasts from mice (II), and human
mesenchymal (hMC) cells from bone marrow (II) were cultured on either coated
TCPS dishes (I, IV) or titanium discs (II) to investigate the ability of apple
(MHR-A and MHR-B) and potato (MHRPTRGAL, MHRPTRARA, MHRPTRWT)
49
modified hairy region fragments to support the attachment, spreading, and/or
differentiation of the bone-forming cells.
On the apple MHR -coated TCPS samples (I), MC3T3-E1 cells attached well
on MHR-B and uncoated TCPS but weakly on MHR-A and aminated surface
AMI. The percentages of the cells containing focal adhesions indicated by
paxillin and vinculin stainings were MHR-A: 0.0% < AMI: 4.1% < TCPS: 72.2%
< MHR-B: 81.5% (the difference between TCPS and MHR-B was not
significant). The FA spot size differences were better-defined. Both the average
area (µm2) and length (µm) of FAs on TCPS (1.45 µm2; 2.11 µm) significantly
exceeded those of MHR-B (0.57 µm2; 1.28 µm).
On titanium (II), MC3T3-E1 cells formed paxillin dots on the MHR-A
coating (as well as on all the other surface types). Primary osteoblasts, however,
formed FAs on every titanium surface type except on MHR-A. In general, MHRB and pure titanium supported FA formation best.
Osteoblastic cells showed more proliferation on MHR-B than on MHR-A, as
indicated by the cellular density and morphology. On MHR-B, the confluency and
spreading of the cells strongly resembled that on uncoated TCPS or Ti, whereas
on MHR-A mainly rounded and weakly attached cells were observed. MC3T3-E1
cells on MHR-A -coated titanium were exceptionally spread, however. On
aminated TCPS, the proliferating MC3T3-E1 cells did not form cultures that were
as dense or as well-spread as the cells on MHR-B and TCPS. This was not as
clearly manifested on titanium samples.
In addition to the apple MHRs, adhesion and proliferation of MC3T3-E1 cells
on TCPS grafted with transgenic MHRPTR_ARA (ARA), MHRPTR_GAL (GAL),
and wild-type MHRP_WT (WT) potato pectins were studied in order to
preliminarily test the optional, in vivo -based modification method of pectin
fragments (IV). Three focal adhesion parameters – length (µm), proportional area
(%), and number – of MC3T3-E1 cells were quantified. Cells grew best on TCPS
and AMI controls, while only minor differences were observed between cells
cultured on the wild type (WT) or mutant (ARA, GAL) potato samples. Even
though MC3T3-E1 cells generally seemed to prefer the wild-type potato pectin
surface (compared to either of the mutant samples), the only statistically
significant deviation between the potato samples was the greatest FA dot length
on the GAL-surface (4.37 µm) compared to ARA (4.07 µm; p < 0.01) or WT
(4.17 µm; p < 0.05). On TCPS, the average FA dot length was 4.74 µm, which
represent a significance of p < 0.001 compared to all the potato samples and p <
0.05 compared to AMI. Neither the proportional area nor the number of FA dots
50
differed significantly between potato samples. Instead, the area-% and the FA
number on TCPS exceeded significantly those on all the potato sample types (p <
0.001 compared to ARA and GAL and p < 0.01 compared to WT). These results
are reported in Table 3.
Confocal and FESEM imaging of the samples revealed that osteoblasts
spread on all the surface types to some extent, but spreading was clearly better on
TCPS and AMI controls. Between the potato samples, WT generally performed
the best, and the largest cell-free areas were detected on ARA surfaces.
Visualizing the cells from a tilted angle (50º) verified flattened cell morphology
on all the sample types. Screening the sample surfaces without cells at maximum
magnification did not reveal any micro scale deformations of the sample surfaces.
Table 3. Focal adhesion parameters [mean (± SD)] of MC3T3-E1 cells grown on
different potato MHRs and control samples (IV).
FA length (µm)
MHRPTR_ARA
MHRPTR_GAL
MHRP_WT
AMI
TCPS
3.40 (0.35)
4.37 (0.12)
4.17 (0.20)
4.51 (0.24)
4.74 (0.14)
FA area (%)
1.37 (0.01)
1.21 (0.00)
1.51 0.00)
1.94 (0.01)
2.37 (0.01)
FA number
141 (40.17)
120 (39.63)
154 (46.61)
201 (44.99)
229 (50.52)
The differentiation of osteoblasts (MC3T3-E1 cells and primary osteoblasts) on
apple MHR -coated titanium samples (II) was evaluated by quantifying the
proportional areas (%) of tetracycline-stained calcium deposits. On MHR-A, no
calcium could be detected (and was thus excluded from the statistical analyses).
On all the other sample surfaces (MHR-B, AMI, Ti), statistically similar amounts
of calcium deposits were observed, the respective values being 14.0%, 12.7%,
and 14.2% for MC3T3-E1 cells, and 26.6%, 24.4%, and 27.5% for primary
osteoblasts.
The ALP expression of osteoblasts (MC3T3-E1 cells and primary osteoblasts)
grown on titanium discs (II) was screened at different time points with RT-PCR.
With MC3T3-E1 cells, ALP mRNA was not produced by day 3 (except very
dimly on Ti), whereas at day 7 and day 15 ALP is clearly expressed in all sample
types. With primary osteoblasts (whose ALP expression was generally stronger
than that of MC3T3-E1 cells), ALP was also expressed at 10 d and 15 d quite
uniformly with the exception of a fainter product band in the 15 d MHR-A
sample.
The specific ALP activity of hMCs was determined on days 3, 7, and 10 by
measuring the enzyme activity per cell number. By day 3, the proliferating cells
51
showed only very low ALP activity. At days 7 and 10, cells grown on MHR-A
exhibited significantly lower (p < 0.01) ALP activity than cells grown on other
sample types (where no statistical differences were detected). By the day 10, the
most supportive sample type for ALP activity was MHR-B, which significantly
(p < 0.01) exceeded all the other sample types.
5.2
In vivo effects of MHR-coated implants (III)
Apple MHR (MHR-A and MHR-B) -coated and aminated (AMI) / uncoated (Ti)
control titanium cylinders were implanted under the latissimus dorsi -muscle
fascia of adult rats. After 1 or 3 weeks, the average fibrous capsule thicknesses
formed around the implants (foreign body reaction) were measured from HEstained histological sections (Fig. 4 and Fig. 5) to acertain the in vivo
responsiveness of the MHR-grafted titanium.
After 1 week, the only statistically significant difference in capsule thickness
was the more extensive fibrosis around the MHR-B implant (average capsule
thickness 42.9 µm) compared to other sample types (MHR-A: 33.2 µm, AMI:
32.5 µm, Ti: 32.3 µm). However, this difference was no longer apparent by the 3week time point, as indicated by the statistically similar capsule thickness values
(MHR-A: 22.5 µm, MHR-B: 24.2 µm, AMI: 26.4 µm, Ti: 23.7 µm). These results
are reported in figure 4. The decrease in capsule thickness between the 1 week
time point and the end of the 3 week follow up period was statistically significant
only on the MHR-B and Ti implants.
52
Fig. 4. HE stained tissue capsules 1 week (upper panel) and 3 weeks (lower panel)
after implantation. (50 µm scale bar) (III).
53
Fig. 5. Statistical analyses of tissue capsule thicknesses (µm) formed around different
titanium sample types A) 1 week, B) 3 weeks after implantation. (III).
Pathological interpretation of the histological sections suggested that the capsule
formation around the implants was a stromal rather than an inflammatory reaction
since
the
cell
profile
of
the
capsule
mainly
consisted
of
fibroblasts/myofibroblasts. This was further verified with TRACP-staining of the
samples. Only activated macrophages and/or FBGCs show positive TRACP
staining (TRACP+). Foreign body giant cells that would represent a strong
inflammatory reaction were not detected in capsules formed around any of the
sample types. A few activated TRACP-positive mononuclear macrophages were
visualized in all sample types at both time points, but interestingly none of these
were in the actual fibrotic capsule area. Instead, they had cumulated beneath the
peri-implant area or even farther away in loose connective and adipose tissues
perivascularly.
54
6
Discussion
6.1
Effects of pectins on bone cells
Appropriate molecular coatings of hard tissue biomaterials are sought to diminish
adverse effects of the implants and to enhance osteoblast-mediated bone
formation at the implantation site. Our results indicate the short-haired apple
pectin RG-I fragment MHR-B is more biocompatible than the longer-chained
MHR-A. This general difference is the result of variable molecular weights and
compositions of the fragments, which probably generate divergent protein
adsorption profiles on the MHR-coated surfaces. The subsequent discrepancies in
cellular behavior on these surfaces were manifested by both osteoclasts and
osteoblasts.
6.1.1 Osteoclastic responses
The preliminary testing of MHRs included in vitro experiments, i.e. cell cultures
on MHR coatings. Among the bone cell types, osteoclasts are appropriate cells for
screening of the interactions between cells and MHRs. The differentiation of
osteoclasts requires specific growth conditions. The sensitivity of these delicat
cells makes them suitable for testing the biocompatibility and the degree of
tolerance of novel implant coatings.
It is generally thought that cell spreading can be considered as a characteristic
of proliferating cells, whereas rounded cells indicate either differentiation or
apoptosis (Boudreau & Jones 1999, Ma et al. 2005). When culturing a bone
marrow -derived mixed cell pool (pit assay, I) on the MHRs, the cells generally
showed a rounded morphology on MHR-A, and a spread and confluent
appearance on MHR-B. The amount of TRACP+ osteoclasts was significantly
diminished on MHR-A compared with MHR-B, TCPS or bone. MHR-A may
promote differentiation, but the cells on MHR-A might be rounded and
aggregated for other reasons, such as improper attachment and a subsequent
decrease in cell growth. However, Hoechst staining did not reveal massive
apoptosis in cells. This effect of MHR-A on cell morphology could be further
studied by observing the cultures at additional time points.
TRACP+ osteoclasts containing 1–2 nuclei and those containing 3 or more
nuclei (multinuclear) were counted separately. Interestingly, on MHR-B, a greater
55
number of 1–2 nuclear TRACP+ cells were observed in comparison to TCPS and
even bone, and the total number of osteoclasts on MHR-B slightly exceeded that
on TCPS. However, the number of multinuclear osteoclasts was higher on bone
than on any other sample type. Based on these results, MHR-B seems to be
tolerated by osteoclasts and supports their proliferation and initial differentiation,
but terminal differentiation processes are somehow hindered. Indeed, the number
of multinuclear TRACP+ cells did not differ between the two MHRs, or between
MHR-B and TCPS (a generally cell-compatible surface) comparison. Thus it
seems likely that the natural bone substrate offers some factors that allow
osteoclastic differentiation to occur properly. Defining these signals and
components could offer interesting cues for further MHR-tailoring as well.
Actin ring formation affirmed these observations. On MHR-A, actin rings
were not detected, whereas on bone, practically all the osteoclasts showed an
actin ring. On MHR-B a few (~1/3) of osteoclasts had organized a ring. Again,
MHR-B tends to allow the formation of activated – but probably not resorbing –
osteoclasts since actin ring organization can be considered as a marker of the
substratum tolerance. Additionally, it has been shown that active osteoclasts can
flourish and migrate on surfaces even without resorptive phases (Saltel et al.
2004).
The effect of MHRs on the maturation, activation, and resorption capacity of
osteoclasts could be further studied using osteoclastogenesis cultures, in which
osteoclasts progress from a stem cell level to a fully-differentiated state with
appropriate growth factors in vitro. With osteoclastogenesis assay it would also be
possible to study how, if at all, osteoclasts resorb sugar coatings. The ability of
osteoclasts to resorb non-mineralized substrates has been reported with somewhat
contradictory results. Although it has been claimed that osteoclasts resorb only
mineralized material (Chambers et al. 1984), they have also been shown to
dissolve other, even non-biological, substrata in vitro (Jones et al. 1984).
6.1.2 Osteoblastic proliferation
The compatibility between MHRs and bone-forming osteoblasts is of interest
especially with regard to the osseointegrative potential of the coating candidates.
The proliferation and differentiation of osteoblasts were examined on both apple
MHRs (MHR-A and MHR-B) as well as on transgenic (MHRPTR_ARA,
MHRPTR_GAL) and wild type (MHRP_WT) potato MHRs). Osteoblastic
proliferation was examined by screening the cells` ability to adhere and spread on
56
the surfaces, whereas differentiation was evaluated by their competence to excrete
osteoid matrix and to convert to a mineralizing phenotype.
As a proliferative indicator, the abundance and certain parameters of focal
adhesions (FAs) were determined for MC3T3-E1 osteoblastic cells (I, IV)
cultured on MHR-grafted TCPS samples. MC3T3-E1 cells are classified as
immortal (but non-cancerous) cells, which implies that they grow rather
efficiently and hence are appropriate for introductory biocompatibility screening
for anchorage-dependent cells. MC3T3-E1 cells spread and attach well onto both
MHR-B and uncoated TCPS, whereas proliferation is clearly hindered on both
MHR-A and AMI surfaces, as indicated by a diminished density and a rounded
morphology of the cells. The proportion of FA-containing cells did not differ
between MHR-B and TCPS, but interestingly the size of the FAs did. On TCPS,
FAs were larger (calculated both as FA lengths and areas) and the difference was
statistically significant. This may indicate that MHR-B allows the cells to adhere
and spread, but the FAs formed on that surface may not be as fully developed as
on the TCPS control.
Differences in MC3T3-E1 cellular behavior were also observed on the
potato-MHR samples (Table 3.). The TCPS control surface performed the best, as
expected, in terms of all the parameters tested (i.e. FA length, proportional area,
and the total number). Surprisingly, in these samples, the aminated surface
performed nearly as well as TCPS, differing significantly only with regard to the
length of the FAs. The cells also spread well on the AMI surface, unlike with
previous apple MHR cultures. In contrast to the apple MHR, none of the potato
MHRs supported cellular growth to the extent of the controls. The wild type (WT)
MHR appeared to be the best substrate, since it allowed the growth of a more
confluent and spread cell population compared to either of the transgenic samples
(ARA or GAL). However, despite the more detached appearance of the cells on
GAL, the longest FA dots were evident on this surface type. Nonetheless, neither
the proportional area nor the number of FAs on GAL exceeded the corresponding
values on ARA or WT (on which the absolute values were actually higher than on
GAL, but the differences were not statistically significant). Based on these facts it
seems that the galactose-deficient MHR type does not properly support cellular
growth, even though the few formed FAs managed to constitute lengthy dots.
ARA may thus be considered as intermediate between the GAL and WT types
with regard to osteoblastic proliferation. However, none of the potato MHRs
appears to achieve the level of the apple MHR-B, but on the other hand they do
not seem to perform as faintly as the MHR-A fragment.
57
Even though these potato MHRs did not perform appreciably well (but were
not clearly incompatible with cells either), it is important to note that also the in
vivo -produced MHRs affect bone cell behavior. Enzymes expressed in transgenic
plants for RG-I modification offer an alternative method besides the in vitro
enzyme treatments. The results also provide some preliminary information about
the significance of certain monosaccharide units of the MHR molecules. ARA
represents an arabinan-poor (~ 70% decrease) MHR-type, which has been
produced by expressing endo-α-L-1,5-arabinanase in the Golgi membranes of the
potato tuber cells (Skjøt et al. 2002). Correspondingly, lowering the degree of
galactosylation in the GAL sample was obtained by expressing endo-β-1,4galactanase resulting in ~ 30% reduction of galactose moieties (Oxenboll et al.
2000). Thus osteoblast attachment seems to react to the depletion of galactose
since on the GAL surface, the FA dots were the longest although paradoxically
they showed the lowest area and number. Thus the galactose deficiency appears to
be relative unsupportive for FA formation, but the few FAs formed managed to
form a long dot structure. On the other hand, the reduction of the arabinose
content did not cause obvious differences. Since the wild type MHR that
contained the default amounts of both arabinose and galactose performed better
than either of the transformants, it may be deduced that both monosaccharides
play some role in osteoblast adhesion. However, molecular analyses (by Dr. Peter
Ulvskov and colleagues, data not shown) have shown that unexpectedly, the
transgenic expressions of arabinanase and galactanase do not affect only the
arabinan and galactan side chains, respectively, but also other structures of the
RG-I molecule. The ARA resembles more the wild type, which might explain the
lack of any statistically significant FA deviations between them. However, in the
GAL sample, the backbone of the RG-I as well as the galactan chains are altered.
As a result, the MHR type deviates more from the WT. The WT and ARA
backbones consist of approximately the same number of Rha residues, whereas
the GAL backbone carries a Rha unit chain that is twice that long. Moreover, the
length of the GalA homogalacturonan chain is nearly double that in WT or ARA.
Thus in vivo modification of MHRs may alter not only the specific side chains,
but also the overall RG-I structure.
6.1.3 Osteoblastic differentiation
MC3T3-E1 cells, primary murine bone marrow cells, and human mesenchymal
stem cells (hMSCs) were differentiated using osteogenic media into mineralizing
58
osteoblasts on apple MHR (MHR-A and MHR-B) -coated titanium (II). Uncoated
(Ti) and aminated (AMI) titanium served as control surfaces. The ability of the
samples to support osteoblastic differentiation was screened by defining the
extent of calcium deposition as well as the ALP gene expression levels of the
cells. As expected, on pure Ti, the cells were both abundant and well-attached. As
in the proliferation cultures on TCPS, MHR-B again manifested better cell
compatibility than MHR-A. Cell confluency on MHR-A still remained the lowest.
However, MC3T3-E1 cells formed FAs on MHR-A (and AMI) surfaces probably
due to differences in the sample surface topography, i.e. higher roughness (Ra ~
0.300 nm) of titanium in comparison to TCPS (Park et al. 2001). The microrough
topography together with a relatively high wettability can produce capillary
effects, which are not characteristics of TCPS surfaces. In contrast, primary
murine osteoblasts did not form detectable FAs on MHR-A. Instead, the primary
cells did not appear to be viable and failed to organize an actin cytoskeleton on
MHR-A.
Quantitative evaluation of osteoblastic mineralization in vitro may often be
difficult, especially when using small culture areas, from which the isolation of
calcium for further analyses may be inadequate. In this thesis, the mineralization
capacity of osteoblasts growing on titanium discs was determined by quantifying
the calcium deposits that were stained in situ with tetracycline, a fluorescent
antibiotic used for labeling calcium in cell cultures among other applications
(Chentoufi et al. 1993, Wan et al. 2002, Mulari et al. 2004). Nevertheless,
tetracycline can diffuse into the cells to some extent and subsequently bind to free
intracellular calcium. This was considered in the microscopic analyses and only
the brightest, clearly extracellular calcium-containing mineral depositions were
measured.
The deposition of calcium by both MC3T3-E1 cells and primary murine
osteoblasts clearly differed on the two MHR samples. On the MHR-A surface,
practically no calcium could be detected – thus reflecting the inability of
osteoblasts to differentiate on this coating. However, on all the other sample
types, i.e. MHR-B, AMI and Ti, equivalent quantities of deposited calcium were
evident. Primary osteoblasts produced calcium more abundantly than MC3T3-E1
cells grown on the corresponding surfaces – an expected feature in primary vs.
cell line cultures. However, when testing metallic biomaterials, it should be kept
in mind that the differentiation progression may also be influenced by properties
other than the nanocoatings. For example, MC3T3-E1 cells have been shown to
commence mineralization later on titanium than on plastic (Matsuura et al. 2005).
59
The quantification of mineral depositions was further evaluated by analyzing
osteoblastic differentiation at the molecular level. The expression of ALP by
osteoblasts was studied either with enzyme activity measurements (hMSCs) or
RT-PCR (MC3T3-E1 cells and primary murine osteoblasts). As demonstrated by
DNA gel electrophoresis with MC3T3-E1 cells, ALP gene expression could not
be detected 3 days after cell seeding on any coating type (except very faintly on
Ti). This is the default situation during the proliferation phase. After a week in
culture, osteoblast differentiation should begin, which indeed can be seen as the
presence of ALP bands on the gel at the 7 day time point. Interestingly, the
intensities of the ALP bands from extracts of MC3T3-E1 cells grown on all the
sample types were approximately equal. At culture day 10, primary osteoblasts
should clearly express the putative differentiation markers. At this time point,
very bright ALP bands were visualized, as expected, and again circa similarly in
all the sample types. Interestingly, on day 15 with both MC3T3-E1 cells (which
also should achieve a mineralizing phenotype by then) and primary cells, ALP
bands could still be detected in all the sample types. With primary cells, the bands
are clearly more intense, which is in agreement with the tetracycline data.
Interestingly, despite the total lack of mineralization on MHR-A, ALP is also
expressed at least on the mRNA level in cells cultured on this sample surface, as
verified by both cell types (though the band was slightly paler with primary cells
compared to the other samples). This might signify that the mineralization process
may be inhibited downstream of ALP gene expression, possibly due to some
physicochemical feature of the MHR-A surface. Still, the ALP expression level
does not always prefigure the amount of mineralization, and often ALP
expression is considered merely as a marker of a bony phenotype (Walboomers et
al. 2004). Besides the grafted coating, i.e. MHRs in this case, the properties, such
as surface microtopography, of the bulky material itself may play a role. As an
example, fetal rat calvarial osteoblasts produce more active ALP and mineralized
nodules on rougher titanium surfaces accompanied by decreased proliferation
(Boyan et al. 2002). It should also be noted that, for example, different MC3T3E1 subclones may fluctuate in e.g. ALP expression (Wang et al. 1999).
ALP enzyme activity – representing an intermediate protein-level status
between mRNA transcription and final mineralization – was investigated with
hMSCs. Only low levels of specific ALP activity were detected on culture day 3
with all the sample types, which further emphasizes the proliferative, rather than
differentiated, phase of the cells at this early time point. This also matches the
corresponding 3 d MC3T3-E1 RT-PCR results. However, at later time points, ALP
60
activity data revealed differences not apparent with the RT-PCR. At day 7, cells
grown on MHR-A did not perform as well as the other sample types; the specific
activity of ALP was lower in cells on MHR-A than in cells cultured on the other
sample types. This was also true at day 10 further indicating the inability of
MHR-A to support osteoblastic differentiation. Interestingly, the highest ALP
level was seen in cells cultured for 10 days on MHR-B, higher even than that of
cells on uncoated Ti. This difference again was not observed with the RT-PCR.
These deviations between spectrophotometric and RT-PCR outcomes may be
explained by either methodological or biological discrepancies. From a
methodological point of view, quantitative RT-PCR could possibly detect subtle
differences in the expression levels between sample types. However, this would
not change the fact that ALP mRNA is produced at least to some extent also by
cells on the MHR-A sample, which at both the enzyme activity as well as the
mineralization levels manifests the weakest biocompatibility. The biological
explanation suggests that the distinctions in differentiation capacity as well as its
timing on these sample types may become more evident at the level of protein
activity than at the level of gene expression. Furthermore, extracellular
influences, such as the presence of MHR molecules might affect the actual
mineral organization downstream of the protein level. Also the timing of the
differentiation may vary during culture. The ALP activity seems to peak at
different times on different samples, even though mineralization is equivalent by
the end of the 2-week culture time (as indicated by the tetracycline data of MHRB, AMI and Ti).
6.2
In vivo – testing of inflammatory responses
In addition to in vitro studies, further investigation of MHRs included in vivo
implantation in order to obtain preliminary indications of how these molecules are
tolerated in a living organism. Preclinical testing is always needed when a new
biomaterial is proposed to assess the possibility of actual clinical use. Also
biomaterials intended for use in hard tissue implants, are usually tested in soft
tissues for inflammatory screening (Jansen et al. 2004, Marques et al. 2005). This
is due to a greater sensitivity, freedom of implant mobility, and the ease of
measuring tissue responses in comparison to e.g. bone. Movement of the implant
may indeed play a significant role; for example, poly-L-lactic acid -coated
implants have been shown to cause different degrees of tissue response in
subcutaneous and periosteal implants (Parker et al. 2002).
61
Titanium samples were implanted into rats under the latissimus dorsi -muscle
fascia. After a 1- or 3-week follow-up period, the thicknesses of the soft tissue
capsules surrounding the implants were measured as indicative of possible
fibrosis reactions. The emergence of activated macrophages and/or FBGCs was
determined with TRACP staining.
6.2.1 Fibrous capsule formation
The amplitude of peri-implant fibrous capsule formation indicates (inflammatory)
reactivity toward an implant. A week after implantation, only the capsule
surrounding the MHR-B -coated titanium sample was statistically significantly
thicker than the capsule around uncoated Ti, as shown in figures 4 and 5.
However, after 3 weeks, the capsule thicknesses did not differ significantly
between any sample types. In general, capsules at 3 weeks were thinner than after
one week, as expected due to a decrease in the initial foreign body reactivity.
Between the two time points of parallel samples, this attenuation was
statistically significant only with MHR-B and Ti. This may indicate a weaker
initial reactivity towards MHR-A and AMI samples and/or more efficient healing
around MHR-B and Ti. Of course, in these interpretations, it should be notified
that the tissue reactivity might also vary between different implantation sites even
in the same animal.
The thicker capsule formation at 1 week with the MHR-B sample probably
resulted from the general tendency of this coating type to support cell growth;
abundant adhesion of fibroblasts would explain the observation. It has been
shown that murine fibroblasts grow well on this MHR-coating (Nagel et al.
2008). On the other hand, the in vitro -proven proclivity of MHR-A to reject
cellular adhesion could account for the temperate capsule formation around this
sample type. In fact, according to the histological sections (Fig. 4), the capsule
texture seemed to be more diffuse and disorganized around MHR-A (and AMI),
whereas it appeared as a more compact and uniform structure around MHR-B and
Ti. More detailed information on the capsule composition was obtained by
pathological interpretations and phagocyte-specific staining.
6.2.2 Consistence of the capsules
No strong or acute inflammations were detected in any of the samples at either
time point. The weak mononuclear reaction perceived 1 week after implantation
62
generally seemed to subside by the 3-week time point. Indeed, according to the
pathological interpretation, the cell layer surrounding the implant cavity appeared
to consist mainly of fibroblasts / myofibroblasts instead of inflammatory cells or
lymphocytes. To verify this, sections were stained for TRACP, a member of the
purple acid phosphatase family that is typically expressed by osteoclasts
(proteolytically processed disulfide-linked heteromeric 5b), activated
macrophages and FBGS (monomeric 5a isoform involved in the generation of
reactive oxygen species).
None of the fibrotic capsules contained any TRACP+ phagocytes. A few
TRACP+, mononuclear macrophages (but not FBGCs) were found farther away
from the peri-implant area, i.e. in connective and adipose tissue areas (mainly
perivascularly). This indicates that the fibrous capsules were formed mainly by
fibroblasts and the collagen produced by them. This was indicated by the absence
of activated inflammatory cells, which should be observed in large numbers in the
vicinity of the implant in strong inflammatory responses, especially if a chronic
inflammation was provoked. In an acute inflammation response, the levels of the
monocytes / macrophages as well as neutrophils are characteristically elevated
(Marchant et al. 1983). In these results, however, the samples at the early 1-week
time point maintained a hale appearance in this respect.
Considering the botanical origin, this surprising inertness of the MHRcoating in terms of inflammatory reactivity is a promising outcome when
evaluating their clinical potential. Especially auspicious is the attenuation of and
the absence of a foreign body reaction around MHR-B, which showed good
biocompatibility with bone cells in the in vitro experiments. It is noteworthy that
the performance of this MHR after implantation did not significantly deviate from
that of pure titanium, the gold standard of hard tissue biomaterials. However, the
MHR-B coating has been shown to promote the secretion of more proinflammatory cytokines IL-6 and TNF-α from J774.2 macrophages in vitro than
MHR-α, a fragment resembling the MHR-A while resulting in the lowest
proliferation index (Bussy et al. 2008). This offers interesting parallels with the
observations made with osteoclasts, which share the same hematopoietic origin
with macrophages. As mentioned earlier, the most abundant appearance of
osteoclast precursors was detected on MHR-B, whereas the number of terminally
differentiated cells did not differ between the MHRs.
63
6.3
General discussion and future prospects
MHR-B coatings performed nearly or even equally as well as pure titanium in
terms of compatibility with bone cells in vitro and inflammatory tolerance in vivo.
Although it is not unusual to obtain a biomaterial that is similar in performance to
titanium, the advantage of pectins is their molecular flexibility. MHRs have the
potential to be modified with multiple enzymes to produce variable types of
fragments. The results of this thesis indicate that by tailoring pectin fragments to a
more MHR-B -like structure, more osseocompatible nanocoatings could be
achieved. Screening the naturally existing sugar molecules of bone tissue could
help direct the modification of pectin fragments towards increasing
biocompatibility.
In addition to bone cells, other cell types, such as human umbilical artery
smooth muscle cells as well as murine fibroblasts, have manifested a similar
preference for MHR-B while repelling MHR-A (Morra et al. 2004, Nagel et al.
2008). Additionally, other MHRs from different plant species, such as carrot and
potato, have been tested. An apple-derived MHR-α, which has been modified
even further to the “MHR-A -like end point”, did not perform well with
fibroblasts, whereas carrot and potato MHRs were intermediate in structure and
performance between MHR-A and -α and MHR-B (Nagel et al. 2008). Thus the
cell compatibility of MHR-B seems to be a universal tendency. Moreover, pectin
fragments of the MHR-A type could have potential in non-adhesive soft tissue
applications (e.g. stents and catheters). Furthermore, the examples of the antiinflammatory effects of pectins together with our in vivo tolerance results are
intruiging and require further investigation.
6.3.1 Physicochemical features of MHR-coatings
Observations of this thesis illustrate that tailoring pectin side chains (i.e. so-called
hairs) as well as additional modifications, such as altering the methyl and acetyl
contents of MHRs, may contribute to the regulation of cellular adhesion and
behavior on device surfaces coated with these materials. It is interesting that
plant-derived macromolecules can have such a noticiable effect on the growth,
attachment, and differentiation of mammalian bone cells. This interactive
functional relationship presents an interesting possibility to utilize and connect
biological processes of very distant taxonomical kingdoms.
64
In addition to the bone cells themselves, the growth factors and cytokines
involved in bone biology should immobilize properly on implants. Schierano and
colleagues (2005) have shown that as a property of the bulk material itself, porous
titanium promotes this adsorption and subsequent osseointegration of minipig
tibial implants earlier than does a machined Ti surface (Schierano et al. 2005).
Thus the protein adsorption profiles of the MHRs are another aspect of these
materials that should be studied.
When comparing the compositions (Table I) of the two apple MHRs tested,
some deductions regarding their physicochemical effects can be made. Cell
adhesion and proliferation on a biomaterial depend greatly on the adsorption of
ECM proteins (from the surrounding fluid, i.e. interstitial fluid in vivo or the
serum of the culturing medium in vitro) onto the surface, which controls
subsequent cellular responses e.g. differentiation and even apoptosis. The length
of the RG-I molecule probably plays a role. The shorter-haired structure of MHRB may allow cellular attachment due to a lower physical hindrance of protein
adsorption. However, the long-chained MHR-A probably provides a stronger
steric repulsion thus diminishing the accumulation of the proteins (Szleifer 1997).
The most striking difference between the MHR fragments is the degree of
acetylation: the MHR-B fragment is depleted in esterified acetyl groups in
comparison to the MHR-A. Thus the acetyl group is likely to play a pivotal role in
the regulation of cell behavior. Indeed, the general importance of O-acetylation
has been reported in other cases concerning e.g. apoptotic processes (Malisan et
al. 2002, Kniep et al. 2006).
Physicochemical features affecting protein adsorption, cell adhesion and
blood coagulation, relate to the wettability of the surface: The availability of
hydrogen bonding of the solvent molecules is widely determined by attractive or
repulsive forces depending on the surface energy of a water-contacting
substratum (Vogler 1999). A hydrophilic surface usually allows for more protein
adsorption and cellular growth. For example, standard TCPS is hydrophobic, but
is usually converted to a hydrophilic surface for cell culturing. Micropatterned
surfaces further illustrate this phenomenon: Human gingival fibroblasts have
shown a distinctively patched growth on air plasma – treated areas of an alginatecoated substratum (Cassinelli et al. 2002).
In terms of wettability, MHRs offer a tempting candidate for the control of
hydration since this represents one of the main functions of pectins in plant cell
walls (Vincken et al. 2003). Naturally, pectins carry negative charges, but their
polarity and total charge can be altered by side chain modification. For example,
65
methyl groups neutralize negative charges, and they can be removed with enzyme
or alkali treatment. In turn, the presence of acetyl groups renders pectins more
hydrophobic. Thus the acetyl (and methyl) -poorer MHR-B chemically represents
a more hydrophilic fragment type. Interestingly, however, the obvious cellular
preference for hydrophilicity can not be directly deduced with these MHRs since
MHR-B actually produces a more hydrophobic surface than MHR-A when
grafted onto a substratum. This has been verified with water contact angle
measurements of MHR-grafted TCPS samples. This is probably due to masking
of some of the ionic, and thus water-interacting, carboxyl groups that link to the
amino groups deposited in the carbodiimide condensation process. On the other
hand, the longer-chained MHR-A may produce a more wettable hydrogel surface
in aqueous surroundings. (Morra et al. 2004)
Concerning wettability, it should also be considered that the degree of
hydrophilicity / hydrophobicity -preference may vary depending on the
conditions, proteins adsorbed, and cell types. In general, the effect of surface
modifications on the integrin receptor functionality is of primary importance
when anchorage-dependent cells, such as osteoblasts, are concerned (Stephansson
et al. 2002, Garcia & Keselowsky 2002, Keselowsky et al. 2005). Fibroblasts and
liver cells seem to prefer a highly wettable surface (Groth & Altankov 1996, De et
al. 2002), whereas in other cases, fibroblasts as well as endothelial cells favor a
moderately hydrophilic surface (Niu et al. 1990, Faucheux et al. 2004). The types
of adsorbing proteins may play an important role in determining the biological
responses. FN is usually considered as an adhesion-promoting protein, whereas
albumin has shown contrasting features (Groth & Altankov 1996, Chen et al.
1998). Indeed, the adsorption of FN onto e.g. a titanium surface promotes
osteoblastic growth (Yang et al. 2003). The wettability preference of albumin
adsorption is not clear. According to Roach and colleagues (2005), albumin
adsorbs readily onto a hydrophobic surface (Roach et al. 2005). However, some
reports show a greater affinity of albumin towards a hydrophilic substratum, such
as a wettable vitamin E -coated surface that was subsequently repelled by
MC3T3-E1 cells (Reno et al. 2005). It has also been reported that hydrophobic
chitosan-coated titanium promotes the adhesion of preosteoblasts by favoring the
adsorption of FN and albumin onto the surface (Bumgardner et al. 2003).
Wettability may also influence the conformation of the adsorbed proteins; e.g.
adsorbed albumin has displayed a less organized secondary structure on a
hydrophobic surface (Roach et al. 2005, Ganazzoli & Raffaini 2005). It has also
been demonstrated that on MHR-B, the integrin-binding RGD-sequence of the
66
adsorbed FN displays a more available conformation than on MHR-α (Nagel et
al. 2008). Interestingly, solubilized MHRs show an effect on fibroblast adhesion
opposite to that of the PS-grafted MHRs probably further reflecting the
importance of the ECM-protein binding accessibility (Nagel et al. 2008).
Different MHRs may thus cause variability in protein folding, and exploring the
tendency of the MHRs to promote or hinder the adsorption of proteins, especially
FN, would clarify this aspect.
A probable explanation for the general cellular preference for hydrophilic
surfaces involves the expression of the genes encoding integrin polypeptides. As
an example, human fetal osteoblasts have grown better on a hydrophilic surface
due to active integrin expression and subsequent FA formation (Lim et al. 2005).
According to Redey and coworkers (2000), coating biomaterials with polar
components could improve the attachment of osteoblasts and thus the
osteoconduction of the implant (Redey et al. 2000). Therefore the polarity of the
tested MHRs should also be taken into account in designing subsequent
modifications. In addition, carbohydrate moieties of the ECM proteins may
contribute to the adsorption profile (Andrade & Hlady 1987). Thus carbohydrate
interactions between the MHRs and ECM proteins, including lectins may be
significant.
6.3.2 Sugar aspects
Despite the widely recognized significance of carbohydrates in biology, the sugar
biochemistry of bone tissue and the use of sugars in the design of biomaterials
still remain relatively unfamiliar areas of glycoscience. Understanding the
interactions between plant polysaccharides and mammalian bone cells is an area
of basic research that could yield information important for hard tissue implant
applications.
Compared to the conventional polysaccharide biomaterial coatings, such as
hyaluronan, modified pectins that focus on the side chain and acetyl and methyl
groups affect the ionic properties of the molecule instead of weak hydrogen bond
-based interactions (Morra et al. 2004). For example, the degree of acetylation of
polysaccharide coating molecules, e.g. chitosan, affects the osteogenic capacity of
bone marrow stromal cells (Amaral et al. 2005). The interactions between MHRs
and osteocytes, the cells representing the final stage of osteoblastic cell
development that are trapped in mineralized bone matrix, would also be
fascinating to assess. Endochondral ossification studies could reveal whether
67
these sugar components act as stimulators, vehicles or inhibitors of bone
formation in the authentic surroundings of animal tissue.
In the bone ECM, sLRPs play a significant role in the natural differentiation
of osteoblasts as well as in the fixation of an implant into bone tissue. Thus
investigating the possible effects of MHRs with the bone ECM glycomolecules
and the “sugar language” of bone cells could offer new insights into the
compatibility of bone implants. Examples of exploitable interactions between the
glycocomponents of biomaterials and bone cells already exist: a
chitooligosaccharide molecule in a soluble form promotes the growth and
differentiation of human osteosarcoma-derived osteoblasts (Ohara et al. 2004).
Added to this, chondroitin sulfate -coated fabrics induced enhanced ALP
production of hMSCs during their differentiation into mature osteoblasts
(Wollenweber et al. 2006). Studies like these are in agreement with the premise
that the gene expression profile and maturation of osteoblasts could be affected by
polysaccharides, such as pectins.
The “sugar language” of bone cells offers another interesting carbohydrate
aspect. Understanding the mechanisms by which osteoclasts and/or the exposed
bone matrix components of a newly formed resorption pit attract osteoblasts to
the BRU site could serve as a valuable tool in implant-designing; the ability to
enhance the arrival of osteoblasts around and onto the bone implant would greatly
assist the healing process by improving contacting bone formation The
aforementioned fact that resorption pits can be specifically stained with WGA
lectin implies that osteoclastic activity probably leaves some carbohydrate signals
at the BRU site. Identification of these sugar molecules could guide the further
tailoring of MHRs to a more osteoblast-inviting format.
Lectins recognize sugar epitopes. Osteoclast inhibitory lectin (OCIL)
belongs to the C-type lectin family and is expressed on the plasma membrane of
e.g. osteoblasts in bone tissue. OCIL binds to sulfated GAGs and a natural killer
cell -associated receptor, and it inhibits the differentiation of osteoclasts that is
important for normal bone remodeling (Kartsogiannis et al. 2008). Thus in further
studies concerning pectins it would be relevant to investigate the possible effects
of different MHRs on lectin expression of bone cells. The possible interactions
between MHRs and OCILs should also be studied to obtain information about the
differentiation of osteoclasts coexisting with osteoblasts. It is noteworthy that
certain dietary pectins are capable of inhibiting galectin-3 -mediated cell-cell
interactions. For example, hemagglutination can be reduced by arabinogalactanrich pectin polysaccharides (Sathisha et al. 2007).
68
6.3.3 Immunological aspects
Pectins have been shown to be involved in various – and partially conflicting –
immunological processes. The effects of pectins on the immune system may
result from various mechanisms including the complement system, cytokine
secretion and leukocyte recruitment. For example, a soluble form pectic
arabinogalactan from Vernonia modulates B-cell proliferation and complement
fixation in vitro (Nergard et al. 2006). Dietary pectins have been reported to play
a protective role in e.g. diabetes, heart diseases, and even cancer; it has been
speculated that cancer metastasis could be inhibited with appropriate pectin
fragments blocking galectin-mediated recognition between cancer cells and their
galactose epitopes (Inohara & Raz 1994, Nangia-Makker et al. 2002, Sathisha et
al. 2007). Black currant (Ribes nigrum) seed galactans with high MWs tend to
inhibit the adhesion of Helicobacter pylori to human gastric mucosa probably by
blocking specific binding receptors on the bacterial surface (Lengsfeld et al.
2004). In turn, soluble LM apple pectin is capable of activating the apoptosis of
human colon adenocarcinoma cells (Olano-Martin et al. 2003). Pectins also
possess e.g. hypocholesterolemic and heavy metal detoxifying properties (Potter
& Steinmetz 1996, Freedman et al. 2007, Eliaz et al. 2007, Salman et al. 2008).
In some cases, pectins might excessively stimulate the immune system, which
should be taken into careful consideration in implant design. Some pectins from
the leaves of different plants, such as ginsengs (Panax ginseng C.A. Meyer) and
greater plantain (Plantago major) can activate the complement system (Kiyohara
et al. 1994, Michaelsen et al. 2000b). These results probably reflect the woundhealing effect of the leaves of some plant species. Generally plants hold a great
potential in medicine. For example, recently certain plant-derived glucocorticoid
receptor modulators have shown promising results as anti-inflammatory
components (Gossye et al. 2009).
Murine lymphocytes have been shown to be stimulated by e.g. arabinan-rich
almond (Prunus dulcis) pectin (Dourado et al. 2004b) and by root pectin
fragments of a medicinal herb, the so-called Chinese thoroughwax i.e. Bupleurum
falcatum (Sakurai et al. 1999). Interestingly, the active structure of the Bupleurum
dulcis -fragment (bupleuran 2IIc) is exactly the ramified region of the
polysaccharide containing an RG-core carrying abundant neutral sugar side
chains, which may indicate the structural immunological reactivity of pectins in
general (Sakurai et al. 1999). The immunologically relevant monosaccharide
composition of pectin fragments would also be important to resolve. For instance,
69
alginate rich in mannuronic acid residues induces stronger antibody production
than glucuronic acid -rich alginate when used as a biomaterial component in
microencapsulation of islets of Langerhans (Kulseng et al. 1999). Neither MHRA nor MHR-B contains mannose or its derivative mannuronic acid (Table I) – a
noteworthy aspect when considering the fact that macrophages carry mannose
receptors involved in the FBGC fusion process (te Velde et al. 1988, Dadsetan et
al. 2004). However, the activity of other structural details of MHRs could be
further evaluated since the immunogenicity of pectins probably is at least partly
defined by their acetyl and carboxyl group content (Wang et al. 2005). The
possibility of controlling the amounts of these chemical groups in MHRfragments with enzymes may thus provide a way to modulate their
immunological effects.
70
7
Conclusions
Study I: The cytocompatibility of apple MHRs (MHR-A and MHR-B) was tested
with both osteoclastic and osteoblastic bone cells. Due to their sensitivity,
osteoclasts are especially suitable for the preparatory screening of a biomaterial,
and their ability to differentiate on MHR-B to some extent was a promising result.
Both primary and cell line -derived bone cells proliferated well on MHR-B and
on control surfaces (TCPS and bone). However, the MHR-A coating was clearly
rejected by both cell types. The main outcome of the study was that altering the
structure of the MHRs covalently grafted onto substrate surfaces can influence
bone cell growth.
Study II: After obtaining preliminary data from TCPS-grafted MHRs,
osteblast differentiation on similarly coated titanium samples was tested. This
process is of prime significance in osseointegration. In concordance with previous
observations of osteoblastic proliferation, MHR-B again outperformed MHR-A
by providing a substratum suitable for differentiation of both the osteoblastic cell
line and primary osteoblasts that was comparable with pure Ti, the gold standard
material in hard tissue implantations. Thus this study together with the previous
one illustrate the potential of the MHR-B fragment to support bone cell
proliferation and differentiation, and suggest that future modifications of MHRs
should be modeled after MHR-B.
Study III: Investigating the in vivo responses to MHR-coated and control
titanium samples indicated that these pectin fragments are immunologically
relatively well-tolerated since neither of them provoked significantly stronger
inflammatory reactions in soft tissue surroundings than the pure, widely-used Ti.
The slightly thicker fibrous capsule formed around the MHR-B implant after 1
week subsided after 3 weeks. Thus none of the sample types caused a severe,
acute inflammatory reaction, as verified by the absence of activated macrophages
and / or FBGS in the tissue capsules. This surprisingly good tolerance of
mammalian tissue towards these plant-derived polysaccharides indicates MHRs
hold promise for implant coatings.
Study IV: The effects of in vivo -modified MHRs isolated from transgenic
potato tubers were ascertained by screening the ability of preosteoblasts to
proliferate on them. It was found that reducing the galactose moieties on MHRs
affected the FA formation of the cells. On the other hand, the performance of
MHRs with an arabinan deficiency was similar to the wild type MHR, which was
the most biocompatible potato pectin fragment tested. However, in contrast to the
71
apple MHRs, none of the potato samples exceeded the performance of the control
TCPS surface. Both the transgenic and wild type potato MHRs were less suitable
for the growth of osteoblasts than the apple MHR-B, but were still better than the
apple MHR-A. The results of this study indicate that osteoblastic growth can be
affected by genetically engineered potato pectin fragments.
72
References
Aarden EM, Burger EH & Nijweide PJ (1994) Function of osteocytes in bone. J Cell
Biochem 55: 287–299.
Abercrombie M, Heaysman JE & Pegrum SM (1971) The locomotion of fibroblasts in
culture. IV. Electron microscopy of the leading lamella. Exp Cell Res 67: 359–367.
Adams JC & Watt FM (1993) Regulation of development and differentiation by the
extracellular matrix. Development 117: 1183–1198.
Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwinski MJ, Fine DL, Abbott BJ,
Mayo JG, Shoemaker RH & Boyd MR (1988) Feasibility of drug screening with
panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res
48: 589–601.
Alzubaydi TL, Alameer SS, Ismaeel T, Alhijazi AY & Geetha M (2008) In vivo studies of
the ceramic coated titanium alloy for enhanced osseointegration in dental applications.
J Mater Sci Mater Med: Jul 1.
Amaral IF, Lamghari M, Sousa SR, Sampaio P & Barbosa MA (2005) Rat bone marrow
stromal cell osteogenic differentiation and fibronectin adsorption on chitosan
membranes: the effect of the degree of acetylation. J Biomed Mater Res A 75: 387–
397.
Andersen TL, Sondergaard TE, Skorzynska KE, gnaes-Hansen F, Plesner TL, Hauge EM,
Plesner T & Delaisse JM (2009) A physical mechanism for coupling bone resorption
and formation in adult human bone. Am J Pathol 174: 239–247.
Anderson HC (1984) Mineralization by matrix vesicles. Scan Electron Microsc: 953–964.
Anderson JM (1988) Inflammatory response to implants. ASAIO Trans 34: 101–107.
Andrade JD & Hlady V (1987) Plasma protein adsorption: the big twelve. Ann N Y Acad
Sci 516: 158–172.
Aubin JE, Liu F, Malaval L & Gupta AK (1995) Osteoblast and chondroblast
differentiation. Bone 17: 77S-83S.
Bagno A, Piovan A, Dettin M, Chiarion A, Brun P, Gambaretto R, Fontana G, Di BC, Palu
G & Castagliuolo I (2007) Human osteoblast-like cell adhesion on titanium substrates
covalently functionalized with synthetic peptides. Bone 40: 693–699.
Baron R, Neff L, Brown W, Courtoy PJ, Louvard D & Farquhar MG (1988) Polarized
secretion of lysosomal enzymes: co-distribution of cation-independent mannose-6phosphate receptors and lysosomal enzymes along the osteoclast exocytic pathway. J
Cell Biol 106: 1863–1872.
Bauer TW & Schils J (1999a) The pathology of total joint arthroplasty. I. Mechanisms of
implant fixation. Skeletal Radiol 28: 423–432.
Bauer TW & Schils J (1999b) The pathology of total joint arthroplasty.II. Mechanisms of
implant failure. Skeletal Radiol 28: 483–497.
Beck GRJr, Zerler B & Moran E (2001) Gene array analysis of osteoblast differentiation.
Cell Growth Differ 12: 61–83.
73
Best SM, Duer MJ, Reid DG, Wise ER & Zou S (2008) Towards a model of the mineralorganic interface in bone: NMR of the structure of synthetic glycosaminoglycan- and
polyaspartate-calcium phosphate composites. Magn Reson Chem 46: 323–329.
Bi Y, Seabold JM, Kaar SG, Ragab AA, Goldberg VM, Anderson JM & Greenfield EM
(2001) Adherent endotoxin on orthopedic wear particles stimulates cytokine
production and osteoclast differentiation. J Bone Miner Res 16: 2082–2091.
Blanquaert F, Barritault D & Caruelle JP (1999) Effects of heparan-like polymers
associated with growth factors on osteoblast proliferation and phenotype expression. J
Biomed Mater Res 44: 63–72.
Boudreau NJ & Jones PL (1999) Extracellular matrix and integrin signalling: the shape of
things to come. Biochem J 339 (Pt 3): 481–488.
Boyan BD, Bonewald LF, Paschalis EP, Lohmann CH, Rosser J, Cochran DL, Dean DD,
Schwartz Z & Boskey AL (2002) Osteoblast-mediated mineral deposition in culture is
dependent on surface microtopography. Calcif Tissue Int 71: 519–529.
Braconnot H (1825a) Nouvelles observations sur l`acide pectique. Annales de Chimie et de
Physique 30: 96–102
Braconnot H (1825b) Recherches sur un nouvel acide universellement répandu dans tous
les végétaux. Annales de Chimie et de Physique 28: 173–178
Brunold C, Deters A, Knoepfel-Sidler F, Hafner J, Muller B & Hensel A (2004)
Polysaccharides from Hibiscus sabdariffa flowers stimulate proliferation and
differentiation of human keratinocytes. Planta Med 70: 370–373.
Bumgardner JD, Wiser R, Elder SH, Jouett R, Yang Y & Ong JL (2003) Contact angle,
protein adsorption and osteoblast precursor cell attachment to chitosan coatings
bonded to titanium. J Biomater Sci Polym Ed 14: 1401–1409.
Bussy C, Verhoef R, Haeger A, Morra M, Duval JL, Vigneron P, Bensoussan A,
Velzenberger E, Cascardo G, Cassinelli C, Schols H, Knox JP & Nagel MD (2008)
Modulating in vitro bone cell and macrophage behavior by immobilized enzymatically
tailored pectins. J Biomed Mater Res A 86: 597–606.
Butler WT (1989) The nature and significance of osteopontin. Connect Tissue Res 23:
123–136.
Butler WT, Ritchie HH & Bronckers AL (1997) Extracellular matrix proteins of dentine.
Ciba Found Symp 205: 107–115.
Campo McKnight DA, Sosnoski DM, Koblinski JE & Gay CV (2006) Roles of osteonectin
in the migration of breast cancer cells into bone. J Cell Biochem 97: 288–302.
Cassinelli C, Morra M, Carpi A, Giardino R & Fini M (2002) Controlling the lateral
distribution and alignment of human gingival fibroblasts by micropatterned
polysaccharide surfaces. Biomed Pharmacother 56: 325–331.
Chambers TJ (1985) The pathobiology of the osteoclast. J Clin Pathol 38: 241–252.
Chambers TJ & Fuller K (1985) Bone cells predispose bone surfaces to resorption by
exposure of mineral to osteoclastic contact. J Cell Sci 76: 155–165.
Chambers TJ & Hall TJ (1991) Cellular and molecular mechanisms in the regulation and
function of osteoclasts. Vitam Horm 46: 41–86.
74
Chambers TJ, Revell PA, Fuller K & Athanasou NA (1984) Resorption of bone by isolated
rabbit osteoclasts. J Cell Sci 66: 383–399.
Chang C, Wang ZC, Quan CY, Cheng H, Cheng SX, Zhang XZ & Zhuo RX (2007)
Fabrication of a novel pH-sensitive glutaraldehyde cross-linked pectin nanogel for
drug delivery. J Biomater Sci Polym Ed 18: 1591–1599.
Chang EJ, Kim HH, Huh JE, Kim IA, Seung KJ, Chung CP & Kim HM (2005) Low
proliferation and high apoptosis of osteoblastic cells on hydrophobic surface are
associated with defective Ras signaling. Exp Cell Res 303: 197–206.
Chen CH, Sheu MT, Chen TF, Wang YC, Hou WC, Liu DZ, Chung TC & Liang YC
(2006) Suppression of endotoxin-induced proinflammatory responses by citrus pectin
through blocking LPS signaling pathways. Biochem Pharmacol 72: 1001–1009.
Chen G, Imanishi Y & Ito Y (1998) Effect of protein and cell behavior on pattern-grafted
thermoresponsive polymer. J Biomed Mater Res 42: 38–44.
Chen XD, Bian X, Teslovich TM, Stephan DA & Young MF (2005) Dissection of the sets
of genes that control the behavior of biglycan-deficient pre-osteoblasts using
oligonucleotide microarrays. Bone 37: 192–203.
Chen XD, Fisher LW, Robey PG & Young MF (2004) The small leucine-rich proteoglycan
biglycan modulates BMP-4-induced osteoblast differentiation. FASEB J 18: 948–958.
Chentoufi J, Hott M, Lamblin D, Buc-Caron MH, Marie PJ & Kellermann O (1993)
Kinetics of in vitro mineralization by an osteogenic clonal cell line (C1) derived from
mouse teratocarcinoma. Differentiation 53: 181–189.
Chow JW, Wilson AJ, Chambers TJ & Fox SW (1998) Mechanical loading stimulates
bone formation by reactivation of bone lining cells in 13-week-old rats. J Bone Miner
Res 13: 1760–1767.
Colognato H, Galvin J, Wang Z, Relucio J, Nguyen T, Harrison D, Yurchenco PD &
Ffrench-Constant C (2007) Identification of dystroglycan as a second laminin receptor
in oligodendrocytes, with a role in myelination. Development 134: 1723–1736.
Cortes W & Gosain AK (2006) Recurrent ectopic calcification involving the maxillofacial
skeleton: A potential harbinger of Albright's osteodystrophy. J Craniofac Surg 17: 21–
27.
Crean SM, Meneski JP, Hullinger TG, Reilly MJ, DeBoever EH & Taichman RS (2004)
N-linked sialyated sugar receptors support haematopoietic cell-osteoblast adhesions.
Br J Haematol 124: 534–546.
Daas PJ, Voragen AG & Schols HA (2000) Characterization of non-esterified galacturonic
acid sequences in pectin with endopolygalacturonase. Carbohydr Res 326: 120–129.
Dadsetan M, Jones JA, Hiltner A & Anderson JM (2004) Surface chemistry mediates
adhesive structure, cytoskeletal organization, and fusion of macrophages. J Biomed
Mater Res A 71: 439–448.
Dalton BA, McFarland CD, Underwood PA & Steele JG (1995) Role of the heparin
binding domain of fibronectin in attachment and spreading of human bone-derived
cells. J Cell Sci 108 (Pt 5): 2083–2092.
75
Damsky CH & Werb Z (1992) Signal transduction by integrin receptors for extracellular
matrix: cooperative processing of extracellular information. Curr Opin Cell Biol 4:
772–781.
Davies JE (2007) Bone bonding at natural and biomaterial surfaces. Biomaterials 28:
5058–5067.
De BL, Morelli S, Bader A & Drioli E (2002) Evaluation of cell behaviour related to
physico-chemical properties of polymeric membranes to be used in bioartificial
organs. Biomaterials 23: 2485–2497.
Dee KC, Andersen TT & Bizios R (1998) Design and function of novel osteoblastadhesive peptides for chemical modification of biomaterials. J Biomed Mater Res 40:
371–377.
DeFife KM, Jenney CR, McNally AK, Colton E & Anderson JM (1997) Interleukin-13
induces human monocyte/macrophage fusion and macrophage mannose receptor
expression. J Immunol 158: 3385–3390.
DeJong (1926). La substance minérale dans les os. Recl Trav Chim Pays-Bas Belg 45,
445–448
Deters AM, Lengsfeld C & Hensel A (2005) Oligo- and polysaccharides exhibit a
structure-dependent bioactivity on human keratinocytes in vitro. J Ethnopharmacol
102: 391–399.
Dettin M, Bagno A, Gambaretto R, Iucci G, Conconi MT, Tuccitto N, Menti AM, Grandi
C, Di BC, Licciardello A & Polzonetti G (2009) Covalent surface modification of
titanium oxide with different adhesive peptides: surface characterization and
osteoblast-like cell adhesion. J Biomed Mater Res A 90: 35–45.
Dettin M, Conconi MT, Gambaretto R, Pasquato A, Folin M, Di BC & Parnigotto PP
(2002) Novel osteoblast-adhesive peptides for dental/orthopedic biomaterials. J
Biomed Mater Res 60: 466–471.
Ding K, Fang JN, Dong T, Tsim KW & Wu H (2003) Characterization of a
rhamnogalacturonan and a xyloglucan from neriumindicum and their activities on
PC12 pheochromocytoma cells. J Nat Prod 66: 7–10.
Doco T, Williams P, Vidal S & Pellerin P (1997) Rhamnogalacturonan II, a dominant
polysaccharide in juices produced by enzymic liquefaction of fruits and vegetables.
Carbohydr Res 297: 181–186.
Donahue HJ, McLeod KJ, Rubin CT, Andersen J, Grine EA, Hertzberg EL & Brink PR
(1995) Cell-to-cell communication in osteoblastic networks: cell line-dependent
hormonal regulation of gap junction function. J Bone Miner Res 10: 881–889.
Doty SB (1981) Morphological evidence of gap junctions between bone cells. Calcif
Tissue Int 33: 509–512.
Dourado F, Madureira P, Carvalho V, Coelho R, Coimbra MA, Vilanova M, Mota M &
Gama FM (2004b) Purification, structure and immunobiological activity of an
arabinan-rich pectic polysaccharide from the cell walls of Prunus dulcis seeds.
Carbohydr Res 339: 2555–2566.
76
Dourado F, Madureira P, Carvalho V, Coelho R, Coimbra MA, Vilanova M, Mota M &
Gama FM (2004a) Purification, structure and immunobiological activity of an
arabinan-rich pectic polysaccharide from the cell walls of Prunus dulcis seeds.
Carbohydr Res 339: 2555–2566.
Ducy P, Zhang R, Geoffroy V, Ridall AL & Karsenty G (1997) Osf2/Cbfa1: a
transcriptional activator of osteoblast differentiation. Cell 89: 747–754.
Dumbleton J & Manley MT (2004) Hydroxyapatite-coated prostheses in total hip and knee
arthroplasty. J Bone Joint Surg Am 86-A: 2526–2540.
Efstratiadis T & Moss DW (1985a) Tartrate-resistant acid phosphatase in human alveolar
macrophages. Enzyme 34: 140–143.
Efstratiadis T & Moss DW (1985b) Tartrate-resistant acid phosphatase of human lung:
apparent identity with osteoclastic acid phosphatase. Enzyme 33: 34–40.
Eliaz I, Weil E & Wilk B (2007) Integrative medicine and the role of modified citrus
pectin/alginates in heavy metal chelation and detoxification--five case reports. Forsch
Komplementmed 14: 358–364.
Everts V, Delaisse JM, Korper W, Jansen DC, Tigchelaar-Gutter W, Saftig P & Beertsen
W (2002) The bone lining cell: its role in cleaning Howship's lacunae and initiating
bone formation. J Bone Miner Res 17: 77–90.
Faucheux N, Schweiss R, Lutzow K, Werner C & Groth T (2004) Self-assembled
monolayers with different terminating groups as model substrates for cell adhesion
studies. Biomaterials 25: 2721–2730.
Ferreira L, Rafael A, Lamghari M, Barbosa MA, Gil MH, Cabrita AM & Dordick JS
(2004) Biocompatibility of chemoenzymatically derived dextran-acrylate hydrogels. J
Biomed Mater Res A 68: 584–596.
Flitsch SL & Ulijn RV (2003) Sugars tied to the spot. Nature 421: 219–220.
Freedman ND, Park Y, Subar AF, Hollenbeck AR, Leitzmann MF, Schatzkin A & Abnet
CC (2007) Fruit and vegetable intake and esophageal cancer in a large prospective
cohort study. Int J Cancer 121: 2753–2760.
Frost H (1960) In vivo osteocyte death. J Bone Joint Surg Am 42-A: 138–143
Frost H (1973) Bone remodeling and its relationship to metabolic bone disease. Springfield
IL, Charles C Thomas.
Furth ME, Atala A & Van Dyke ME (2007) Smart biomaterials design for tissue
engineering and regenerative medicine. Biomaterials 28: 5068–5073.
Ganazzoli F & Raffaini G (2005) Computer simulation of polypeptide adsorption on model
biomaterials. Phys Chem Chem Phys 7: 3651–3663.
Gange CT, Quinn JM, Zhou H, Kartsogiannis V, Gillespie MT & Ng KW (2004)
Characterization of sugar binding by osteoclast inhibitory lectin. J Biol Chem 279:
29043–29049.
Garcia AJ & Keselowsky BG (2002) Biomimetic surfaces for control of cell adhesion to
facilitate bone formation. Crit Rev Eukaryot Gene Expr 12: 151–162.
Gorbet MB & Sefton MV (2005) Endotoxin: the uninvited guest. Biomaterials 26: 6811–
6817.
77
Gorski JP, Liu FT, Artigues A, Castagna LF & Osdoby P (2002) New alternatively spliced
form of galectin-3, a member of the beta-galactoside-binding animal lectin family,
contains a predicted transmembrane-spanning domain and a leucine zipper motif. J
Biol Chem 277: 18840–18848.
Gossye V, Elewaut D, Van BK, Dewint P, Haegeman G & De BK (2009) A plant-derived
glucocorticoid receptor modulator attenuates inflammation without provoking ligandinduced resistance. Ann Rheum Dis: Feb 9.
Goto T, Yoshinari M, Kobayashi S & Tanaka T (2004) The initial attachment and
subsequent behavior of osteoblastic cells and oral epithelial cells on titanium. Biomed
Mater Eng 14: 537–544.
Gristina AG & Costerton JW (1985) Bacterial adherence to biomaterials and tissue. The
significance of its role in clinical sepsis. J Bone Joint Surg Am 67: 264–273.
Gronthos S, Stewart K, Graves SE, Hay S & Simmons PJ (1997) Integrin expression and
function on human osteoblast-like cells. J Bone Miner Res 12: 1189–1197.
Groth T & Altankov G (1996) Studies on cell-biomaterial interaction: role of tyrosine
phosphorylation during fibroblast spreading on surfaces varying in wettability.
Biomaterials 17: 1227–1234.
Haapala J, Arokoski JP, Hyttinen MM, Lammi M, Tammi M, Kovanen V, Helminen HJ &
Kiviranta I (1999) Remobilization does not fully restore immobilization induced
articular cartilage atrophy. Clin Orthop Relat Res: 218–229.
Hallab NJ, Anderson S, Stafford T, Glant T & Jacobs JJ (2005) Lymphocyte responses in
patients with total hip arthroplasty. J Orthop Res 23: 384–391.
Hausser HJ & Brenner RE (2004) Low doses and high doses of heparin have different
effects on osteoblast-like Saos-2 cells in vitro. J Cell Biochem 91: 1062–1073.
Haynes DR, Crotti TN & Zreiqat H (2004) Regulation of osteoclast activity in peri-implant
tissues. Biomaterials 25: 4877–4885.
Heino TJ, Kurata K, Higaki H & Väänänen HK (2009) Evidence for the role of osteocytes
in the initiation of targeted remodeling. Technol Health Care 17: 49–56.
Heiskanen A, Hirvonen T, Salo H, Impola U, Olonen A, Laitinen A, Tiitinen S, Natunen S,
Aitio O, Miller-Podraza H, Wuhrer M, Deelder AM, Natunen J, Laine J, Lehenkari P,
Saarinen J, Satomaa T & Valmu L (2009) Glycomics of bone marrow-derived
mesenchymal stem cells can be used to evaluate their cellular differentiation stage.
Glycoconj J 26: 367–384.
Heiskanen A, Satomaa T, Tiitinen S, Laitinen A, Mannelin S, Impola U, Mikkola M,
Olsson C, Miller-Podraza H, Blomqvist M, Olonen A, Salo H, Lehenkari P, Tuuri T,
Otonkoski T, Natunen J, Saarinen J & Laine J (2007) N-glycolylneuraminic acid
xenoantigen contamination of human embryonic and mesenchymal stem cells is
substantially reversible. Stem Cells 25: 197–202.
Hinz SW, Verhoef R, Schols HA, Vincken JP & Voragen AG (2005) Type I
arabinogalactan contains beta-D-Galp-(1-->3)-beta-D-Galp structural elements.
Carbohydr Res 340: 2135–2143.
78
Hirata M, Kobayashi M, Takita M, Matsumoto C, Miyaura C & Inada M (2009)
Hyaluronan inhibits bone resorption by suppressing prostaglandin E synthesis in
osteoblasts treated with interleukin-1. Biochem Biophys Res Commun 381: 139–143.
Horbett TA (1981) Adsorption of proteins from plasma to a series of hydrophilichydrophobic copolymers. II. Compositional analysis with the prelabeled protein
technique. J Biomed Mater Res 15: 673–695.
Horbett TA & Weathersby PK (1981) Adsorption of proteins from plasma to a series of
hydrophilic-hydrophobic copolymers. I. Analysis with the in situ radioiodination
technique. J Biomed Mater Res 15: 403–423.
Hu YS, Zhou H, Myers D, Quinn JM, Atkins GJ, Ly C, Gange C, Kartsogiannis V, Elliott
J, Kostakis P, Zannettino AC, Cromer B, McKinstry WJ, Findlay DM, Gillespie MT
& Ng KW (2004) Isolation of a human homolog of osteoclast inhibitory lectin that
inhibits the formation and function of osteoclasts. J Bone Miner Res 19: 89–99.
Huang J, Best SM, Brooks RA, Rushton N & Bonfield W (2008) In vitro evaluation of
nanosized carbonate-substituted hydroxyapatite and its polyhydroxyethylmethacrylate
nanocomposite. J Biomed Mater Res A 87: 598–607.
Huhtala P, Humphries MJ, McCarthy JB, Tremble PM, Werb Z & Damsky CH (1995)
Cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins regulates
metalloproteinase gene expression in fibroblasts adhering to fibronectin. J Cell Biol
129: 867–879.
Hynes RO (1987) Integrins: a family of cell surface receptors. Cell 48: 549–554.
Ichibouji,T, Miyazaki T, Ishida E, Ashizuka M, Sugino A, Ohtsuki C, Kuramoto K
(2008). Evaluation of apatite-forming ability and mechanical property of pectin
hydrogels. Journal of the Ceramic Society of Japan 116, 74–78
Ichibouji,T, Miyazaki,T, Ishida,E, Sugino,A, & Ohtsuki,C (2009). Apatite mineralization
abilities and mechanical properties of covalently cross-linked pectin hydrogels.
Materials Science and Engineering C In press.
Illes T, Dubousset J, Szendroi M & Fischer J (1992) Characterization of bone forming
cells in posttraumatic myositis ossificans by lectins. Pathol Res Pract 188: 172–176.
Illes T & Fischer J (1989) Distribution of lectin binding glycoprotein in osteoclasts.
Histochemistry 91: 55–59.
Inkinen RI, Lammi MJ, Lehmonen S, Puustjärvi K, Kääpä E & Tammi MI (1998) Relative
increase of biglycan and decorin and altered chondroitin sulfate epitopes in the
degenerating human intervertebral disc. J Rheumatol 25: 506–514.
Inohara H & Raz A (1994) Effects of natural complex carbohydrate (citrus pectin) on
murine melanoma cell properties related to galectin-3 functions. Glycoconj J 11: 527–
532.
Ishii T & Matsunaga T (2001) Pectic polysaccharide rhamnogalacturonan II is covalently
linked to homogalacturonan. Phytochemistry 57: 969–974.
Ivaska KK, Käkönen SM, Gerdhem P, Obrant KJ, Pettersson K & Väänänen HK (2005)
Urinary osteocalcin as a marker of bone metabolism. Clin Chem 51: 618–628.
79
Jansen K, van der Werff JF, van Wachem PB, Nicolai JP, de Leij LF & van Luyn MJ
(2004) A hyaluronan-based nerve guide: in vitro cytotoxicity, subcutaneous tissue
reactions, and degradation in the rat. Biomaterials 25: 483–489.
Jones SJ, Boyde A & Ali NN (1984) The resorption of biological and non-biological
substrates by cultured avian and mammalian osteoclasts. Anat Embryol (Berl) 170:
247–256.
Jones SJ, Boyde A & Ali NN (1986) The interface of cells and their matrices in
mineralized tissues: a review. Scan Electron Microsc: 1555–1569.
Jortikka MO, Inkinen RI, Tammi MI, Parkkinen JJ, Haapala J, Kiviranta I, Helminen HJ &
Lammi MJ (1997) Immobilisation causes longlasting matrix changes both in the
immobilised and contralateral joint cartilage. Ann Rheum Dis 56: 255–261.
Kadoya Y, al-Saffar N, Kobayashi A & Revell PA (1994) The expression of osteoclast
markers on foreign body giant cells. Bone Miner 27: 85–96.
Kapanen A, Ryhänen J, Danilov A & Tuukkanen J (2001) Effect of nickel-titanium shape
memory metal alloy on bone formation. Biomaterials 22: 2475–2480.
Kartsogiannis V, Sims NA, Quinn JM, Ly C, Cipetic M, Poulton IJ, Walker EC, Saleh H,
McGregor NE, Wallace ME, Smyth MJ, Martin TJ, Zhou H, Ng KW & Gillespie MT
(2008) Osteoclast inhibitory lectin, an immune cell product that is required for normal
bone physiology in vivo. J Biol Chem 283: 30850–30860.
Keselowsky BG, Collard DM & Garcia AJ (2005) Integrin binding specificity regulates
biomaterial surface chemistry effects on cell differentiation. Proc Natl Acad Sci U S A
102: 5953–5957.
Keuren JF, Wielders SJ, Willems GM, Morra M, Cahalan L, Cahalan P & Lindhout T
(2003) Thrombogenicity of polysaccharide-coated surfaces. Biomaterials 24: 1917–
1924.
Kim SH, Hoshiba T & Akaike T (2003) Effect of carbohydrates attached to polystyrene on
hepatocyte morphology on sugar-derivatized polystyrene matrices. J Biomed Mater
Res A 67: 1351–1359.
Kiviranta I, Tammi M, Jurvelin J, Arokoski J, Säämänen AM & Helminen HJ (1994)
Articular cartilage thickness and glycosaminoglycan distribution in the young canine
knee joint after remobilization of the immobilized limb. J Orthop Res 12: 161–167.
Kiyohara H, Hirano M, Wen XG, Matsumoto T, Sun XB & Yamada H (1994)
Characterisation of an anti-ulcer pectic polysaccharide from leaves of Panax ginseng
C.A. Meyer. Carbohydr Res 263: 89–101.
Klinger MM, Rahemtulla F, Prince CW, Lucas LC & Lemons JE (1998) Proteoglycans at
the bone-implant interface. Crit Rev Oral Biol Med 9: 449–463.
Kniep B, Kniep E, Ozkucur N, Barz S, Bachmann M, Malisan F, Testi R & Rieber EP
(2006) 9-O-acetyl GD3 protects tumor cells from apoptosis. Int J Cancer 119: 67–73.
Kölliker A (1873) Die normale resorption des knochengewebes und ihre bedeutung für die
entstehung der typischen knochenformen. Leipzig, F C W Vogel.
Konttinen YT, Zhao D, Beklen A, Ma G, Takagi M, Kivelä-Rajamäki M, Ashammakhi N
& Santavirta S (2005) The microenvironment around total hip replacement prostheses.
Clin Orthop Relat Res: 28–38.
80
Kram V, Zcharia E, Yacoby-Zeevi O, Metzger S, Chajek-Shaul T, Gabet Y, Muller R,
Vlodavsky I & Bab I (2006) Heparanase is expressed in osteoblastic cells and
stimulates bone formation and bone mass. J Cell Physiol 207: 784–792.
Kreja L, Liedert A, Hasni S, Claes L & Ignatius A (2008) Mechanical regulation of
osteoclastic genes in human osteoblasts. Biochem Biophys Res Commun 368: 582–
587.
Kulseng B, Skjak-Braek G, Ryan L, Andersson A, King A, Faxvaag A & Espevik T (1999)
Transplantation of alginate microcapsules: generation of antibodies against alginates
and encapsulated porcine islet-like cell clusters. Transplantation 67: 978–984.
Kurata K, Heino TJ, Higaki H & Väänänen HK (2006) Bone marrow cell differentiation
induced by mechanically damaged osteocytes in 3D gel-embedded culture. J Bone
Miner Res 21: 616–625.
Kusano K, Miyaura C, Inada M, Tamura T, Ito A, Nagase H, Kamoi K & Suda T (1998)
Regulation of matrix metalloproteinases (MMP-2, -3, -9, and -13) by interleukin-1 and
interleukin-6 in mouse calvaria: association of MMP induction with bone resorption.
Endocrinology 139: 1338–1345.
Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero
A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A,
Qian YX, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J & Boyle WJ
(1998) Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation
and activation. Cell 93: 165–176.
Lai CF & Cheng SL (2005) Alphavbeta integrins play an essential role in BMP-2 induction
of osteoblast differentiation. J Bone Miner Res 20: 330–340.
Laitala-Leinonen T, Howell ML, Dean GE & Väänänen HK (1996) Resorption-cycledependent polarization of mRNAs for different subunits of V-ATPase in boneresorbing osteoclasts. Mol Biol Cell 7: 129–142.
Lakkakorpi PT, Horton MA, Helfrich MH, Karhukorpi EK & Väänänen HK (1991)
Vitronectin receptor has a role in bone resorption but does not mediate tight sealing
zone attachment of osteoclasts to the bone surface. J Cell Biol 115: 1179–1186.
Lakkakorpi PT & Väänänen HK (1991) Kinetics of the osteoclast cytoskeleton during the
resorption cycle in vitro. J Bone Miner Res 6: 817–826.
Lamoureux F, Picarda G, Garrigue-Antar L, Baud'huin M, Trichet V, Vidal A, MiotNoirault E, Pitard B, Heymann D & Redini F (2009) Glycosaminoglycans as potential
regulators of osteoprotegerin therapeutic activity in osteosarcoma. Cancer Res 69:
526–536.
Lane JM (1979) Biochemistry of fracture healing. AAOS Monterey Seminar, Chicago. Am
Acad Orthop Surg: 141-165.
Lee S & Voros J (2005) An aqueous-based surface modification of poly(dimethylsiloxane)
with poly(ethylene glycol) to prevent biofouling. Langmuir 21: 11957–11962.
Lengsfeld C, Deters A, Faller G & Hensel A (2004) High molecular weight
polysaccharides from black currant seeds inhibit adhesion of Helicobacter pylori to
human gastric mucosa. Planta Med 70: 620–626.
81
Lentino JR (2003) Prosthetic joint infections: bane of orthopedists, challenge for infectious
disease specialists. Clin Infect Dis 36: 1157–1161.
Lickorish D, Guan L & Davies JE (2007) A three-phase, fully resorbable,
polyester/calcium phosphate scaffold for bone tissue engineering: Evolution of
scaffold design. Biomaterials 28: 1495–1502.
Lim JY, Taylor AF, Li Z, Vogler EA & Donahue HJ (2005) Integrin expression and
osteopontin regulation in human fetal osteoblastic cells mediated by substratum
surface characteristics. Tissue Eng 11: 19–29.
Lossdorfer S, Schwartz Z, Wang L, Lohmann CH, Turner JD, Wieland M, Cochran DL &
Boyan BD (2004) Microrough implant surface topographies increase osteogenesis by
reducing osteoclast formation and activity. J Biomed Mater Res A 70: 361–369.
Lounev VY, Ramachandran R, Wosczyna MN, Yamamoto M, Maidment AD, Shore EM,
Glaser DL, Goldhamer DJ & Kaplan FS (2009) Identification of progenitor cells that
contribute to heterotopic skeletogenesis. J Bone Joint Surg Am 91: 652–663.
Lynch MP, Stein JL, Stein GS & Lian JB (1995) The influence of type I collagen on the
development and maintenance of the osteoblast phenotype in primary and passaged rat
calvarial osteoblasts: modification of expression of genes supporting cell growth,
adhesion, and extracellular matrix mineralization. Exp Cell Res 216: 35–45.
Ma L, Feng XY, Cui BL, Law F, Jiang XW, Yang LY, Xie QD & Huang TH (2005)
Human umbilical cord Wharton's Jelly-derived mesenchymal stem cells
differentiation into nerve-like cells. Chin Med J (Engl ) 118: 1987–1993.
Magnusson P & Farley JR (2002) Differences in sialic acid residues among bone alkaline
phosphatase isoforms: a physical, biochemical, and immunological characterization.
Calcif Tissue Int 71: 508–518.
Malisan F, Franchi L, Tomassini B, Ventura N, Condo I, Rippo MR, Rufini A, Liberati L,
Nachtigall C, Kniep B & Testi R (2002) Acetylation suppresses the proapoptotic
activity of GD3 ganglioside. J Exp Med 196: 1535–1541.
Manton KJ, Haupt LM, Vengadasalam K, Nurcombe V & Cool SM (2007a)
Glycosaminoglycan and growth factor mediated murine calvarial cell proliferation. J
Mol Histol 38: 415–424.
Manton KJ, Leong DF, Cool SM & Nurcombe V (2007b) Disruption of heparan and
chondroitin sulfate signaling enhances mesenchymal stem cell-derived osteogenic
differentiation via bone morphogenetic protein signaling pathways. Stem Cells 25:
2845–2854.
Marchant R, Hiltner A, Hamlin C, Rabinovitch A, Slobodkin R & Anderson JM (1983) In
vivo biocompatibility studies. I. The cage implant system and a biodegradable
hydrogel. J Biomed Mater Res 17: 301–325.
Marcus R (1987) Normal and abnormal bone remodeling in man. Annu Rev Med 38: 129–
141.
Marques AP, Reis RL & Hunt JA (2005) An in vivo study of the host response to starchbased polymers and composites subcutaneously implanted in rats. Macromol Biosci 5:
775–785.
Martin RB (2000) Toward a unifying theory of bone remodeling. Bone 26: 1–6.
82
Matkovic V, Jelic T, Wardlaw GM, Ilich JZ, Goel PK, Wright JK, Andon MB, Smith KT
& Heaney RP (1994) Timing of peak bone mass in Caucasian females and its
implication for the prevention of osteoporosis. Inference from a cross-sectional model.
J Clin Invest 93: 799–808.
Matsuura T, Tsubaki S, Tsuzuki T, Duarte WR, Yamauchi M & Sato H (2005) Differential
gene expression of collagen-binding small leucine-rich proteoglycans and lysyl
hydroxylases, during mineralization by MC3T3-E1 cells cultured on titanium implant
material. Eur J Oral Sci 113: 225–231.
Matsuzaki K, Udagawa N, Takahashi N, Yamaguchi K, Yasuda H, Shima N, Morinaga T,
Toyama Y, Yabe Y, Higashio K & Suda T (1998) Osteoclast differentiation factor
(ODF) induces osteoclast-like cell formation in human peripheral blood mononuclear
cell cultures. Biochem Biophys Res Commun 246: 199–204.
Mattila RH, Laurila P, Rekola J, Gunn J, Lassila LV, Mantyla T, Aho AJ & Vallittu PK
(2009) Bone attachment to glass-fibre-reinforced composite implant with porous
surface. Acta Biomater 5: 1639–1646.
McCann MC, Bush M, Milioni D, Sado P, Stacey NJ, Catchpole G, Defernez M, Carpita
NC, Hofte H, Ulvskov P, Wilson RH & Roberts K (2001) Approaches to
understanding the functional architecture of the plant cell wall. Phytochemistry 57:
811–821.
McCarthy AD, Etcheverry SB, Bruzzone L, Lettieri G, Barrio DA & Cortizo AM (2001a)
Non-enzymatic glycosylation of a type I collagen matrix: effects on osteoblastic
development and oxidative stress. BMC Cell Biol 2: 16.
McCarthy AD, Etcheverry SB & Cortizo AM (1999) Advanced glycation endproductspecific receptors in rat and mouse osteoblast-like cells: regulation with stages of
differentiation. Acta Diabetol 36: 45–52.
McCarthy AD, Etcheverry SB & Cortizo AM (2001b) Effect of advanced glycation
endproducts on the secretion of insulin-like growth factor-I and its binding proteins:
role in osteoblast development. Acta Diabetol 38: 113–122.
McCarthy AD, Uemura T, Etcheverry SB & Cortizo AM (2004) Advanced glycation
endproducts interefere with integrin-mediated osteoblastic attachment to a type-I
collagen matrix. Int J Biochem Cell Biol 36: 840–848.
McEver RP (2002) Selectins: lectins that initiate cell adhesion under flow. Curr Opin Cell
Biol 14: 581–586.
McNally AK, DeFife KM & Anderson JM (1996) Interleukin-4-induced macrophage
fusion is prevented by inhibitors of mannose receptor activity. Am J Pathol 149: 975–
985.
Mercer N, Ahmed H, McCarthy AD, Etcheverry SB, Vasta GR & Cortizo AM (2004)
AGE-R3/galectin-3 expression in osteoblast-like cells: regulation by AGEs. Mol Cell
Biochem 266: 17–24.
Michaelsen TE, Gilje A, Samuelsen AB, Hogasen K & Paulsen BS (2000b) Interaction
between human complement and a pectin type polysaccharide fraction, PMII, from the
leaves of Plantago major L. Scand J Immunol 52: 483–490.
83
Michaelsen TE, Gilje A, Samuelsen AB, Hogasen K & Paulsen BS (2000a) Interaction
between human complement and a pectin type polysaccharide fraction, PMII, from the
leaves of Plantago major L. Scand J Immunol 52: 483–490.
Micheli F (2001) Pectin methylesterases: cell wall enzymes with important roles in plant
physiology. Trends Plant Sci 6: 414–419.
Miranti CK, Ohno S & Brugge JS (1999) Protein kinase C regulates integrin-induced
activation of the extracellular regulated kinase pathway upstream of Shc. J Biol Chem
274: 10571–10581.
Mochida Y, Duarte WR, Tanzawa H, Paschalis EP & Yamauchi M (2003) Decorin
modulates matrix mineralization in vitro. Biochem Biophys Res Commun 305: 6–9.
Morin P, Sagne C & Gasnier B (2004) Functional characterization of wild-type and mutant
human sialin. EMBO J 23: 4560–4570.
Morko JP, Söderström M, Säämänen AM, Salminen HJ & Vuorio EI (2004) Up regulation
of cathepsin K expression in articular chondrocytes in a transgenic mouse model for
osteoarthritis. Ann Rheum Dis 63: 649–655.
Morra M (2005) Engineering of biomaterials surfaces by hyaluronan. Biomacromolecules
6: 1205–1223.
Morra M (2006) Biochemical modification of titanium surfaces: peptides and ECM
proteins. Eur Cell Mater 12: 1–15.
Morra M & Cassinelli C (2000) Force measurements on cell repellant and cell adhesive
alginic acid coated surfaces. Colloids Surf B Biointerfaces 18: 249–259.
Morra M, Cassinelli C, Cascardo G, Cahalan P, Cahalan L, Fini M & Giardino R (2003a)
Surface engineering of titanium by collagen immobilization. Surface characterization
and in vitro and in vivo studies. Biomaterials 24: 4639–4654.
Morra M, Cassinelli C, Cascardo G, Nagel MD, Della VC, Siboni S, Maniglio D, Brugnara
M, Ceccone G, Schols HA & Ulvskov P (2004) Effects on interfacial properties and
cell adhesion of surface modification by pectic hairy regions. Biomacromolecules 5:
2094–2104.
Morra M, Cassinelli C, Pavesio A & Renier D (2003b) Atomic force microscopy
evaluation of aqueous interfaces of immobilized hyaluronan. J Colloid Interface Sci
259: 236–243.
Moursi AM, Globus RK & Damsky CH (1997) Interactions between integrin receptors and
fibronectin are required for calvarial osteoblast differentiation in vitro. J Cell Sci 110
(Pt 18): 2187–2196.
Muhonen V, Kujala S, Vuotikka A, Ääritalo V, Peltola T, Areva S, Närhi T & Tuukkanen
J (2009) Biocompatibility of sol-gel-derived titania-silica coated intramedullary NiTi
nails. Acta Biomater 5: 785–793.
Mulari MT, Qu Q, Härkönen PL & Väänänen HK (2004) Osteoblast-like cells complete
osteoclastic bone resorption and form new mineralized bone matrix in vitro. Calcif
Tissue Int 75: 253–261.
Mundy G & Roodman G (1987) Osteoclast ontogeny and function. In: Peck WA (ed) Bone
and Mineral Research. Elsevier, Amsterdam: 209–279.
84
Mutter M, Beldman G, Pitson SM, Schols HA & Voragen AG (1998a)
Rhamnogalacturonan alpha-d-galactopyranosyluronohydrolase. An enzyme that
specifically removes the terminal nonreducing galacturonosyl residue in
rhamnogalacturonan regions of pectin. Plant Physiol 117: 153–163.
Mutter M, Colquhoun IJ, Beldman G, Schols HA, Bakx EJ & Voragen AG (1998b)
Characterization of recombinant rhamnogalacturonan alpha-L-rhamnopyranosyl-(1,4)alpha-D-galactopyranosyluronide lyase from Aspergillus aculeatus. An enzyme that
fragments rhamnogalacturonan I regions of pectin. Plant Physiol 117: 141–152.
Mutter M, Colquhoun IJ, Schols HA, Beldman G & Voragen AG (1996)
Rhamnogalacturonase B from Aspergillus aculeatus is a rhamnogalacturonan alpha-Lrhamnopyranosyl-(1-->4)-alpha-D-galactopyranosyluronide lyase. Plant Physiol 110:
73–77.
Nagahata M, Tsuchiya T, Ishiguro T, Matsuda N, Nakatsuchi Y, Teramoto A, Hachimori
A & Abe K (2004) A novel function of N-cadherin and Connexin43: marked
enhancement of alkaline phosphatase activity in rat calvarial osteoblast exposed to
sulfated hyaluronan. Biochem Biophys Res Commun 315: 603–611.
Nagel MD, Verhoef R, Schols H, Morra M, Knox JP, Ceccone G, Della VC, Vigneron P,
Bussy C, Gallet M, Velzenberger E, Vayssade M, Cascardo G, Cassinelli C, Haeger
A, Gilliland D, Liakos I, Rodriguez-Valverde M & Siboni S (2008) Enzymaticallytailored pectins differentially influence the morphology, adhesion, cell cycle
progression and survival of fibroblasts. Biochim Biophys Acta 1780: 995–1003.
Nangia-Makker P, Conklin J, Hogan V & Raz A (2002) Carbohydrate-binding proteins in
cancer, and their ligands as therapeutic agents. Trends Mol Med 8: 187–192.
Narbaitz R, Stumpf WE, Sar M, Huang S & DeLuca HF (1983) Autoradiographic
localization of target cells for 1 alpha, 25-dihydroxyvitamin D3 in bones from fetal
rats. Calcif Tissue Int 35: 177–182.
Nergard CS, Kiyohara H, Reynolds JC, Thomas-Oates JE, Matsumoto T, Yamada H, Patel
T, Petersen D, Michaelsen TE, Diallo D & Paulsen BS (2006) Structures and
structure-activity relationships of three mitogenic and complement fixing pectic
arabinogalactans from the malian antiulcer plants Cochlospermum tinctorium A. Rich
and Vernonia kotschyana Sch. Bip. ex Walp. Biomacromolecules 7: 71–79.
Nguyen DH, Tangvoranuntakul P & Varki A (2005) Effects of natural human antibodies
against a nonhuman sialic acid that metabolically incorporates into activated and
malignant immune cells. J Immunol 175: 228–236.
Niikura K, Nakajima S, Takano M & Yamazaki H (2007) FR177995, a novel vacuolar
ATPase inhibitor, exerts not only an inhibitory effect on bone destruction but also
anti-immunoinflammatory effects in adjuvant-induced arthritic rats. Bone 40: 888–
894.
Niu S, Matsuda T & Oka T (1990) Endothelialization on various segmented polyurethanes:
cellular behavior and its substrate dependency. ASAIO Trans 36: M164-M168.
85
Ohara N, Hayashi Y, Yamada S, Kim SK, Matsunaga T, Yanagiguchi K & Ikeda T (2004)
Early gene expression analyzed by cDNA microarray and RT-PCR in osteoblasts
cultured with water-soluble and low molecular chitooligosaccharide. Biomaterials 25:
1749–1754.
Ohnishi T, Arakaki N, Nakamura O, Hirono S & Daikuhara Y (1991) Purification,
characterization, and studies on biosynthesis of a 59-kDa bone sialic acid-containing
protein (BSP) from rat mandible using a monoclonal antibody. Evidence that 59-kDa
BSP may be the rat counterpart of human alpha 2-HS glycoprotein and is synthesized
by both hepatocytes and osteoblasts. J Biol Chem 266: 14636–14645.
Olano-Martin E, Rimbach GH, Gibson GR & Rastall RA (2003) Pectin and pecticoligosaccharides induce apoptosis in in vitro human colonic adenocarcinoma cells.
Anticancer Res 23: 341–346.
Oldberg A, Franzen A & Heinegard D (1986) Cloning and sequence analysis of rat bone
sialoprotein (osteopontin) cDNA reveals an Arg-Gly-Asp cell-binding sequence. Proc
Natl Acad Sci U S A 83: 8819–8823.
Ortega N, Behonick DJ, Colnot C, Cooper DN & Werb Z (2005) Galectin-3 is a
downstream regulator of matrix metalloproteinase-9 function during endochondral
bone formation. Mol Biol Cell 16: 3028–3039.
Owen TA, Aronow M, Shalhoub V, Barone LM, Wilming L, Tassinari MS, Kennedy MB,
Pockwinse S, Lian JB & Stein GS (1990) Progressive development of the rat
osteoblast phenotype in vitro: reciprocal relationships in expression of genes
associated with osteoblast proliferation and differentiation during formation of the
bone extracellular matrix. J Cell Physiol 143: 420–430.
Oxenboll SS, Pauly M, Bush M, Skjot M, McCann MC, Borkhardt B & Ulvskov P (2000)
Pectin engineering: modification of potato pectin by in vivo expression of an endo1,4-beta-D-galactanase. Proc Natl Acad Sci U S A 97: 7639–7644.
Palokangas H, Mulari M & Väänänen HK (1997) Endocytic pathway from the basal
plasma membrane to the ruffled border membrane in bone-resorbing osteoclasts. J
Cell Sci 110 (Pt 15): 1767–1780.
Parisuthiman D, Mochida Y, Duarte WR & Yamauchi M (2005) Biglycan modulates
osteoblast differentiation and matrix mineralization. J Bone Miner Res 20: 1878–
1886.
Park JY, Gemmell CH & Davies JE (2001) Platelet interactions with titanium: modulation
of platelet activity by surface topography. Biomaterials 22: 2671–2682.
Parker JA, Walboomers XF, Von den Hoff JW, Maltha JC & Jansen JA (2002) The effect
of bone anchoring and micro-grooves on the soft tissue reaction to implants.
Biomaterials 23: 3887–3896.
Peter B, Pioletti DP, Laib S, Bujoli B, Pilet P, Janvier P, Guicheux J, Zambelli PY, Bouler
JM & Gauthier O (2005) Calcium phosphate drug delivery system: influence of local
zoledronate release on bone implant osteointegration. Bone 36: 52–60.
Potter JD & Steinmetz K (1996) Vegetables, fruit and phytoestrogens as preventive agents.
IARC Sci Publ: 61–90.
86
Price PA, Parthemore JG & Deftos LJ (1980) New biochemical marker for bone
metabolism. Measurement by radioimmunoassay of bone GLA protein in the plasma
of normal subjects and patients with bone disease. J Clin Invest 66: 878–883.
Puleo DA, Kissling RA & Sheu MS (2002) A technique to immobilize bioactive proteins,
including bone morphogenetic protein-4 (BMP-4), on titanium alloy. Biomaterials 23:
2079–2087.
Qin C, Brunn JC, Jones J, George A, Ramachandran A, Gorski JP & Butler WT (2001) A
comparative study of sialic acid-rich proteins in rat bone and dentin. Eur J Oral Sci
109: 133–141.
Qu Q, Perälä-Heape M, Kapanen A, Dahllund J, Salo J, Väänänen HK & Härkönen P
(1998) Estrogen enhances differentiation of osteoblasts in mouse bone marrow
culture. Bone 22: 201–209.
Raisz LG (1988) Local and systemic factors in the pathogenesis of osteoporosis. N Engl J
Med 318: 818–828.
rana-Chavez VE & Bradaschia-Correa V (2009) Clastic cells: mineralized tissue resorption
in health and disease. Int J Biochem Cell Biol 41: 446–450.
Redey SA, Nardin M, Bernache-Assolant D, Rey C, Delannoy P, Sedel L & Marie PJ
(2000) Behavior of human osteoblastic cells on stoichiometric hydroxyapatite and
type A carbonate apatite: role of surface energy. J Biomed Mater Res 50: 353–364.
Redondo-Nevado J, Moyano E, Medina-Escobar N, Caballero JL & Munoz-Blanco J
(2001) A fruit-specific and developmentally regulated endopolygalacturonase gene
from strawberry (Fragaria x ananassa cv. Chandler). J Exp Bot 52: 1941–1945.
Reijnders CM, Bravenboer N, Holzmann PJ, Bhoelan F, Blankenstein MA & Lips P
(2007) In vivo mechanical loading modulates insulin-like growth factor binding
protein-2 gene expression in rat osteocytes. Calcif Tissue Int 80: 137–143.
Renkonen J, Tynninen O, Häyry P, Paavonen T & Renkonen R (2002) Glycosylation
might provide endothelial zip codes for organ-specific leukocyte traffic into
inflammatory sites. Am J Pathol 161: 543–550.
Reno F, Aina V, Gatti S & Cannas M (2005) Effect of vitamin E addition to poly(D,L)lactic acid on surface properties and osteoblast behaviour. Biomaterials 26: 5594–
5599.
Ripamonti U & Reddi AH (1992) Growth and morphogenetic factors in bone induction:
role of osteogenin and related bone morphogenetic proteins in craniofacial and
periodontal bone repair. Crit Rev Oral Biol Med 3: 1–14.
Roach P, Farrar D & Perry CC (2005) Interpretation of protein adsorption: surface-induced
conformational changes. J Am Chem Soc 127: 8168–8173.
Robey PG, Young MF, Flanders KC, Roche NS, Kondaiah P, Reddi AH, Termine JD,
Sporn MB & Roberts AB (1987) Osteoblasts synthesize and respond to transforming
growth factor-type beta (TGF-beta) in vitro. J Cell Biol 105: 457–463.
87
Rohde H, Burandt EC, Siemssen N, Frommelt L, Burdelski C, Wurster S, Scherpe S,
Davies AP, Harris LG, Horstkotte MA, Knobloch JK, Ragunath C, Kaplan JB &
Mack D (2007) Polysaccharide intercellular adhesin or protein factors in biofilm
accumulation of Staphylococcus epidermidis and Staphylococcus aureus isolated from
prosthetic hip and knee joint infections. Biomaterials 28: 1711–1720.
Roman-Roman S, Garcia T, Jackson A, Theilhaber J, Rawadi G, Connolly T, SpinellaJaegle S, Kawai S, Courtois B, Bushnell S, Auberval M, Call K & Baron R (2003)
Identification of genes regulated during osteoblastic differentiation by genome-wide
expression analysis of mouse calvaria primary osteoblasts in vitro. Bone 32: 474–482.
Rosen SD (2004) Ligands for L-selectin: homing, inflammation, and beyond. Annu Rev
Immunol 22: 129–156.
Ruggieri M, Pavone V, Smilari P, Rizzo R & Sorge G (1995) Primary osteoma cutis-multiple cafe-au-lait spots and woolly hair anomaly. Pediatr Radiol 25: 34–36.
Ruoslahti E & Pierschbacher MD (1986) Arg-Gly-Asp: a versatile cell recognition signal.
Cell 44: 517–518.
Ruoslahti E & Pierschbacher MD (1987) New perspectives in cell adhesion: RGD and
integrins. Science 238: 491–497.
Säämänen AM, Puustjarvi K, Ilves K, Lammi M, Kiviranta I, Jurvelin J, Helminen HJ &
Tammi M (1993) Effect of running exercise on proteoglycans and collagen content in
the intervertebral disc of young dogs. Int J Sports Med 14: 48–51.
Sackstein R, Merzaban JS, Cain DW, Dagia NM, Spencer JA, Lin CP & Wohlgemuth R
(2008) Ex vivo glycan engineering of CD44 programs human multipotent
mesenchymal stromal cell trafficking to bone. Nat Med 14: 181–187.
Sakurai MH, Matsumoto T, Kiyohara H & Yamada H (1999) B-cell proliferation activity
of pectic polysaccharide from a medicinal herb, the roots of Bupleurum falcatum L.
and its structural requirement. Immunology 97: 540–547.
Salgado AJ, Coutinho OP, Reis RL & Davies JE (2007) In vivo response to starch-based
scaffolds designed for bone tissue engineering applications. J Biomed Mater Res A
80: 983–989.
Salman H, Bergman M, Djaldetti M, Orlin J & Bessler H (2008) Citrus pectin affects
cytokine production by human peripheral blood mononuclear cells. Biomed
Pharmacother 62: 579–582.
Salminen-Mankonen H, Säämänen AM, Jalkanen M, Vuorio E & Pirilä L (2005)
Syndecan-1 expression is upregulated in degenerating articular cartilage in a
transgenic mouse model for osteoarthritis. Scand J Rheumatol 34: 469–474.
Salo J, Lehenkari P, Mulari M, Metsikkö K & Väänänen HK (1997) Removal of osteoclast
bone resorption products by transcytosis. Science 276: 270–273.
Saltel F, Destaing O, Bard F, Eichert D & Jurdic P (2004) Apatite-mediated actin
dynamics in resorbing osteoclasts. Mol Biol Cell 15: 5231–5241.
Sastry SK & Burridge K (2000) Focal adhesions: a nexus for intracellular signaling and
cytoskeletal dynamics. Exp Cell Res 261: 25–36.
88
Sathisha UV, Jayaram S, Harish Nayaka MA & Dharmesh SM (2007) Inhibition of
galectin-3 mediated cellular interactions by pectic polysaccharides from dietary
sources. Glycoconj J 24: 497–507.
Schierano G, Canuto RA, Navone R, Peirone B, Martinasso G, Pagano M, Maggiora M,
Manzella C, Easton M, Davit A, Trombetta A, Amedeo S, Biolatti B, Carossa S &
Preti G (2005) Biological factors involved in the osseointegration of oral titanium
implants with different surfaces: a pilot study in minipigs. J Periodontol 76: 1710–
1720.
Schneider G & Burridge K (1994) Formation of focal adhesions by osteoblasts adhering to
different substrata. Exp Cell Res 214: 264–269.
Schneider GB, Perinpanayagam H, Clegg M, Zaharias R, Seabold D, Keller J & Stanford
C (2003) Implant surface roughness affects osteoblast gene expression. J Dent Res 82:
372–376.
Schols HA, Vierhuis E, Bakx EJ & Voragen AG (1995) Different populations of pectic
hairy regions occur in apple cell walls. Carbohydr Res 275: 343–360.
Schols,H, Voragen A.G.J., Colquhoun I.J. (1994). Isolation and characterization of
rhamnogalacturonan oligomers, liberated during degradation of pectic hairy regions
by rhamnogalacturonase. Carbohydr Res 256, 97–111
Schwartz Z, Amir D, Weinberg H & Sela J (1987) Extracellular matrix vesicle distribution
in primary mineralization two weeks after injury to rat tibial bone (ultrastructural
tissue morphometry). Eur J Cell Biol 45: 97–101.
Searle-van Leuween M, van den Brock L, Schols H, Beldman G, Voragen A (1992)
Rhamnogalacturonan acetylesterase: A novel enzyme from Aspergillus aculeatus,
specific for the deacetylation of hairy (ramified) regions of pectins. Appl Microbiol
Biotechnol 38: 347–349
Selander K, Lehenkari P & Väänänen HK (1994) The effects of bisphosphonates on the
resorption cycle of isolated osteoclasts. Calcif Tissue Int 55: 368–375.
Sharp CA, Linder C & Magnusson P (2007) Analysis of human bone alkaline phosphatase
isoforms: comparison of isoelectric focusing and ion-exchange high-performance
liquid chromatography. Clin Chim Acta 379: 105–112.
Shriver Z, Raguram S & Sasisekharan R (2004) Glycomics: a pathway to a class of new
and improved therapeutics. Nat Rev Drug Discov 3: 863–873.
Siebers MC, ter Brugge PJ, Walboomers XF & Jansen JA (2005) Integrins as linker
proteins between osteoblasts and bone replacing materials. A critical review.
Biomaterials 26: 137–146.
Silve CM, Hradek GT, Jones AL & Arnaud CD (1982) Parathyroid hormone receptor in
intact embryonic chicken bone: characterization and cellular localization. J Cell Biol
94: 379–386.
Skjøt, Pauly M, Bush MS, Borkhardt B, McCann MC & Ulvskov P (2002) Direct
interference with rhamnogalacturonan I biosynthesis in Golgi vesicles. Plant Physiol
129: 95–102.
Somerman MJ, Fisher LW, Foster RA & Sauk JJ (1988) Human bone sialoprotein I and II
enhance fibroblast attachment in vitro. Calcif Tissue Int 43: 50–53.
89
Song SJ, Cool SM & Nurcombe V (2007) Regulated expression of syndecan-4 in rat
calvaria osteoblasts induced by fibroblast growth factor-2. J Cell Biochem 100: 402–
411.
Stein GS, Lian JB, Gerstenfeld LG, Shalhoub V, Aronow M, Owen T & Markose E (1989)
The onset and progression of osteoblast differentiation is functionally related to
cellular proliferation. Connect Tissue Res 20: 3–13.
Steinberg J, Neumann AW, Absolom DR & Zingg W (1989) Human erythrocyte adhesion
and spreading on protein-coated polymer surfaces. J Biomed Mater Res 23: 591–610.
Stenman M, Ainola M, Valmu L, Bjartell A, Ma G, Stenman UH, Sorsa T, Luukkainen R
& Konttinen YT (2005) Trypsin-2 degrades human type II collagen and is expressed
and activated in mesenchymally transformed rheumatoid arthritis synovitis tissue. Am
J Pathol 167: 1119–1124.
Stephansson SN, Byers BA & Garcia AJ (2002) Enhanced expression of the osteoblastic
phenotype on substrates that modulate fibronectin conformation and integrin receptor
binding. Biomaterials 23: 2527–2534.
Stock M, Schafer H, Stricker S, Gross G, Mundlos S & Otto F (2003) Expression of
galectin-3 in skeletal tissues is controlled by Runx2. J Biol Chem 278: 17360–17367.
Suda RK, Billings PC, Egan KP, Kim JH, Carrick-Walmsley R, Glaser DL, Porter DL,
Shore EM & Pignolo RJ (2009) Circulating Osteogenic Precursor Cells in Heterotopic
Bone Formation. Stem Cells.
Suda T, Takahashi N & Martin TJ (1992) Modulation of osteoclast differentiation. Endocr
Rev 13: 66–80.
Szleifer I (1997) Protein adsorption on surfaces with grafted polymers: a theoretical
approach. Biophys J 72: 595–612.
Takahata M, Iwasaki N, Nakagawa H, Abe Y, Watanabe T, Ito M, Majima T & Minami A
(2007) Sialylation of cell surface glycoconjugates is essential for osteoclastogenesis.
Bone 41: 77–86.
Takano-Yamamoto T & Rodan GA (1990) Direct effects of 17 beta-estradiol on trabecular
bone in ovariectomized rats. Proc Natl Acad Sci U S A 87: 2172–2176.
te Velde AA, Klomp JP, Yard BA, de Vries JE & Figdor CG (1988) Modulation of
phenotypic and functional properties of human peripheral blood monocytes by IL-4. J
Immunol 140: 1548–1554.
Termine JD, Kleinman HK, Whitson SW, Conn KM, McGarvey ML & Martin GR (1981)
Osteonectin, a bone-specific protein linking mineral to collagen. Cell 26: 99–105.
Tsai AT, Rice J, Scatena M, Liaw L, Ratner BD & Giachelli CM (2005) The role of
osteopontin in foreign body giant cell formation. Biomaterials 26: 5835–5843.
Turhani D, Cvikl B, Watzinger E, Weissenbock M, Yerit K, Thurnher D, Lauer G & Ewers
R (2005) In vitro growth and differentiation of osteoblast-like cells on hydroxyapatite
ceramic granule calcified from red algae. J Oral Maxillofac Surg 63: 793–799.
Turner CH, Warden SJ, Bellido T, Plotkin LI, Kumar N, Jasiuk I, Danzig J & Robling AG
(2009) Mechanobiology of the skeleton. Sci Signal 2: t3.
90
Tuukkanen J, Koivukangas A, Jämsä T, Sundquist K, Mackay CA & Marks SC, Jr. (2000)
Mineral density and bone strength are dissociated in long bones of rat osteopetrotic
mutations. J Bone Miner Res 15: 1905–1911.
U.S.Department of Health and Human Services,OotSG (2004). Bone Health and
Osteoporosis:
A
Report
of
the
Surgeon
General.
http://www.surgeongeneral.gov/library/bonehealth/chapter_2.html (read 5.11.2009)
Väänänen HK (1993) Mechanism of bone turnover. Ann Med 25: 353–359.
Väänänen KK, Malmi R, Tuukkanen J, Sundquist K & Harkonen P (1986) Identification of
osteoclasts by rhodamine-conjugated peanut agglutinin. Calcif Tissue Int 39: 161–
165.
Vääräniemi J, Halleen JM, Kaarlonen K, Ylipahkala H, Alatalo SL, Andersson G, Kaija H,
Vihko P & Väänänen HK (2004) Intracellular machinery for matrix degradation in
bone-resorbing osteoclasts. J Bone Miner Res 19: 1432–1440.
van Alebeek GJ, Christensen TM, Schols HA, Mikkelsen JD & Voragen AG (2002) Mode
of action of pectin lyase A of Aspergillus niger on differently C(6)-substituted
oligogalacturonides. J Biol Chem 277: 25929–25936.
van Alebeek GJ, van SK, Beldman G, Schols HA & Voragen AG (2003) Partially
esterified oligogalacturonides are the preferred substrates for pectin methylesterase of
Aspergillus niger. Biochem J 372: 211–218.
Varki A (1997) Sialic acids as ligands in recognition phenomena. FASEB J 11: 248–255.
Vincken JP, Schols HA, Oomen RJ, McCann MC, Ulvskov P, Voragen AG & Visser RG
(2003) If homogalacturonan were a side chain of rhamnogalacturonan I. Implications
for cell wall architecture. Plant Physiol 132: 1781–1789.
Vogler EA (1999) Water and the acute biological response to surfaces. J Biomater Sci
Polym Ed 10: 1015–1045.
Voragen AGJ, Pilnik W, Thibault J-F, Axelos MAV & Renard CMGC (1995) Pectins. In:
Stephen AM (ed) Food Polysaccharides and Their Applications. New York, Marcel
Dekker Inc: 287–339.
Walboomers XF, Habraken WJ, Feddes B, Winter LC, Bumgardner JD & Jansen JA
(2004) Stretch-mediated responses of osteoblast-like cells cultured on titanium-coated
substrates in vitro. J Biomed Mater Res A 69: 131–139.
Walker DG (1973) Nicolas Andry award for 1973. Experimental osteopetrosis. Clin
Orthop Relat Res 97: 158–174.
Wan C, Yang Q, Deng L, Shen W, He C & Qi J (2002) Osteogenic potential of rabbit
marrow stromal stem cells cultured in vitro: a histochemical and scanning electron
microscopic study. Chin J Traumatol 5: 374–379.
Wang D, Christensen K, Chawla K, Xiao G, Krebsbach PH & Franceschi RT (1999)
Isolation and characterization of MC3T3-E1 preosteoblast subclones with distinct in
vitro and in vivo differentiation/mineralization potential. J Bone Miner Res 14: 893–
903.
Wang J, de BJ & de GK (2008) Proliferation and differentiation of MC3T3-E1 cells on
calcium phosphate/chitosan coatings. J Dent Res 87: 650–654.
91
Wang NL, Kiyohara H, Matsumoto T, Otsuka H, Hirano M & Yamada H (1994)
Polyclonal antibody against a complement-activating pectin from the roots of
Angelica acutiloba. Planta Med 60: 425–429.
Wang XS, Dong Q, Zuo JP & Fang JN (2003) Structure and potential immunological
activity of a pectin from Centella asiatica (L.) Urban. Carbohydr Res 338: 2393–2402.
Wang,XS, Liu,L, Fang,JN (2005). Immunological activities and structure of pectin from
Centella asiatica. Carbohydrate Polymers 60, 95–101
Wiktor-Jedrzejczak W, Bartocci A, Ferrante AW, Jr., hmed-Ansari A, Sell KW, Pollard
JW & Stanley ER (1990) Total absence of colony-stimulating factor 1 in the
macrophage-deficient osteopetrotic (op/op) mouse. Proc Natl Acad Sci U S A 87:
4828–4832.
Willats WG, McCartney L, Mackie W & Knox JP (2001) Pectin: cell biology and
prospects for functional analysis. Plant Mol Biol 47: 9–27.
Williams DC & Frolik CA (1991) Physiological and pharmacological regulation of
biological calcification. Int Rev Cytol 126: 195–292.
Williams DF (2008) On the mechanisms of biocompatibility. Biomaterials 29: 2941–2953.
Wollenweber M, Domaschke H, Hanke T, Boxberger S, Schmack G, Gliesche K,
Scharnweber D & Worch H (2006) Mimicked bioartificial matrix containing
chondroitin sulphate on a textile scaffold of poly(3-hydroxybutyrate) alters the
differentiation of adult human mesenchymal stem cells. Tissue Eng 12: 345–359.
Wu RC, Wang Z, Liu MJ, Chen DF & Yue XS (2004) beta2-integrins mediate a novel
form of chemoresistance in cycloheximide-induced U937 apoptosis. Cell Mol Life Sci
61: 2071–2082.
Yang Y, Cavin R & Ong JL (2003) Protein adsorption on titanium surfaces and their effect
on osteoblast attachment. J Biomed Mater Res A 67: 344–349.
Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Goto M, Mochizuki SI,
Tsuda E, Morinaga T, Udagawa N, Takahashi N, Suda T & Higashio K (1999) A
novel molecular mechanism modulating osteoclast differentiation and function. Bone
25: 109–113.
You L, Temiyasathit S, Lee P, Kim CH, Tummala P, Yao W, Kingery W, Malone AM,
Kwon RY & Jacobs CR (2008) Osteocytes as mechanosensors in the inhibition of
bone resorption due to mechanical loading. Bone 42: 172–179.
Zhao Y, Guan H, Liu SF, Wu RC & Wang Z (2005) Overexpression of QM induces cell
differentiation and mineralization in MC3T3-E1. Biol Pharm Bull 28: 1371–1376.
Zheng F, Liang H, Liu R, Quan JX, Li XX, Dai CL, Guo G, Zhang JY & Wang BL (2009)
Parathyroid hormone-related protein regulates osteoclast inhibitory lectin expression
via multiple signaling pathways in osteoblast-like cells. Endocrine 35: 47–56.
Zhou H, Kartsogiannis V, Hu YS, Elliott J, Quinn JM, McKinstry WJ, Gillespie MT & Ng
KW (2001) A novel osteoblast-derived C-type lectin that inhibits osteoclast formation.
J Biol Chem 276: 14916–14923.
92
Original publications
I
Kokkonen HE, Ilvesaro J, Morra M, Schols HA & Tuukkanen J (2007) Effect
of Modified Pectin Molecules on the Growth of Bone Cells.
Biomacromolecules 8(2): 509–15.
II Kokkonen H, Cassinelli C, Morra M, Schols HA & Tuukkanen J (2008)
Differentiation
of
Osteoblasts
on
Pectin-Coated
Titanium.
Biomacromolecules 9(9): 2369–76.
III Kokkonen H, Niiranen H, Schols HA, Morra M, Stenbäck F & Tuukkanen J
(2009) Pectin-Coated Titanium Implants are Well-Tolerated in vivo. Journal
of Biomedical Materials Research: Part A. In press.
IV Kokkonen H, Verhoef R, Kauppinen K, Muhonen V, Jørgensen B, Damager I,
Schols H, Morra M, Ulvskov P & Tuukkanen J (2009) Proliferation of
Osteoblastic Cells on In vivo Engineered Potato Pectin Fragments.
Manuscript.
Reprinted with permissions from American Chemical Society (I, II) and John
Wiley & Sons Inc. (III).
Original publications are not included in the electronic version of the dissertation.
93
94
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familial breast cancer
1020. Niinimäki, Jaakko (2009) Diffusion-weighted MRI and delayed contrast
enhancement of degenerated intervertebral disc
1021. Habjanic, Ana (2009) Quality of institutional elderly care in Slovenia
1022. Hugg, Timo (2009) Exposure to environmental tobacco smoke, animals and
pollen grains as determinants of atopic diseases and respiratory infections
1023. Sormunen, Erja (2009) Repetitive work in the cold. Work ability, musculoskeletal
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Pekka
(2009)
Keuhkoahtaumataudin
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1034. Sipola, Annina (2009) Effects of vascular endothelial growth factor (VEGF-A) and
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