CHAPTER VI

SCAFFOLDS AND CELLS: PRELIMINARY BIOMECHANICAL

ANALYSIS AND RESULTS FOR THE USE OF A COLLAGEN-
GAG SCAFFOLD FOR BONE TISSUE ENGINEERING

F.J. O’Briena,b, E. Farrellb, M.A. Wallerb, I. Connellc, D. O’Mahoneyc, J.P.

McGarryc, B.P. Murphyc, P.E. McHughc, V.A. Campbellb, P.J. Prendergastb,

a
Department of Anatomy, Royal College of Surgeons in Ireland, Dublin,
Ireland. bTrinity Centre for Bioengineering, Departments of Mechanical
Engineering and Physiology, Trinity College, Dublin, Ireland cNational Centre
for Biomedical Engineering Science, National University of Ireland, Galway,
Ireland

Success in bone tissue engineering has proved elusive. In this paper we

describe an integrated set of projects that aim to provide fundamental
information allowing a scientific biomechanical basis for use of a
collagen/GAG scaffold to initiate mesenchymal stem cells into
osteogenic differentiation pathways. Experimental and numerical
methods to quantify stimuli with the scaffold are given, with
preliminary results reported in each case.

1. Introduction
Bone grafts or bone-graft substitutes are required for multiple clinical
applications. In the USA alone there are 3.1 million orthopedic
procedures performed each year, of which 500,000 are bone graft
procedures. In Ireland, there are 47,500 orthopedic procedures each year
(4). That makes bone second only to blood transfusions on the list of
transplanted materials (1). The estimated worldwide bone-graft market
is about €650 million annually. Situations that require the use of a bone
graft include: (i) to regenerate bone which is lost or damaged as a result

P.J. Prendergast and P.E. McHugh (Eds.), Topics in Bio-Mechanical Engineering, pp. 167-183.
© 2004 Trinity Centre for Bioengineering & National Centre for Biomedical Engineering Science.
167
168 Topics in Bio-Mechanical Engineering

of trauma, infection, or disease, (ii) to stimulate healing of fractures

including fresh fractures or fractures that have failed to heal after an
initial treatment attempt (non-union), (iii) to act as a bone replacement in
maxillofacial or other types of reconstructive surgery, (iv) to improve the
bone healing response and regeneration of bone tissue around surgically
implanted devices and plates and screws used to maintain bone
alignment after fracture. Present clinical therapies include autografting
(whereby bone is removed from a patient’s own body) and allografting
(whereby bone is taken from an organ donor and placed in a bone bank)
using cancellous bone, applying vascularised grafts of the fibula and iliac
crest, and other bone transport methods. However, although
commonplace in orthopaedic surgery, these treatments have a number of
limitations. Harvesting autografts, typically from the iliac crest, is
expensive, painful, constrained by anatomical limitations and associated
with donor-site morbidity due to infection and haematoma. Allografts are
not taken from the patient, so do not contain any living cells, and
therefore have fewer growth factors present to stimulate formation of
new bone and do not grow as well or as quickly as an autograft. They
are also limited by the potential risks of introducing infection or disease
while vascularised grafts are prohibitively expensive (15). The aim of
this project is to design a tissue-engineered bone graft substitute which
will reduce the need for allografts or autografts.
The goal of tissue engineering is to develop cell, construct, and living
system technologies to restore the structure and functional mechanical
properties of damaged or degenerated tissue. Tissue engineering relies
extensively on the use of porous scaffolds to provide the appropriate
environment for the regeneration of tissues and organs. These scaffolds
are typically seeded with cells and occasionally growth factors and are
either cultured in vitro to study the synthesis of tissues or are implanted
in various anatomical sites to induce regeneration of tissues or organs. A
number of considerations are important when designing a scaffold for
use in tissue engineering. The scaffold must be biocompatible to
promote cell adhesion and growth. It should also be biodegradable so as
to allow cells, over time to produce their own extracellular matrix and the
products of this degradation should be non-toxic and able to exit the
body without interference with other organs. The scaffold should have
 Scaffolds and Cells: Biomechanical Analysis 169

mechanical properties consistent with the anatomical site into which it is

to be implanted and should have a high surface area and volume fraction
to allow for mass transport of cells within the scaffold and surrounding
host tissue and to provide space for the ingrowth of tissue and
vascularization (19). Examples of materials used as scaffolds include
sea-coral, fibrin, various polylactides, hydroxyapatite, dental plaster and
demineralized bone matrix from cadavers coated with various osteo-
inductive growth factors.
This study proposes to use collagen and collagen-glycosaminoglycan
(GAG) porous substrates to produce tissue engineered bone grafts.
Collagen provides strength and structural stability to a number of tissues
in the body including skin, tendon, blood vessels, cartilage and bone. As
such, it is an obvious candidate as a substrate on which to culture
osteoblasts to produce bone. The major disadvantage of collagen as a
scaffold for tissue-engineered bone is that it has relatively poor
mechanical properties for the bone environment (2). However, there are
a number of reasons why collagen may be an excellent substrate for
tissue engineering of bone using osteoblasts or differentiated
mesenchymal stem cells. The first is (i) that the behaviour of cells is
mediated by their extracellular environment. As a normal constituent of
bone, collagen is a more physiological substrate for osteoblasts than
materials such as polystyrene, titanium, or poly-lactic and poly-glycolic
acid (6, 17) which are commonly used as implant materials. Osteoblast-
like (Saos-2) cells grown on a collagen gel-coated dishes displayed
greater adhesion and expression of alkaline phosphatase, a marker for
osteoblast function, than those grown on untreated plastic (9). Primary
osteoblasts have also been shown to also adhere more strongly and
proliferate more rapidly on collagen-coated rather than untreated
titanium alloy (14). The second reason (ii) is that collagen templates
form part of the normal bone formation process and therefore, by using
collagen-GAG porous substrates, the normal bone formation process
may be triggered. Two mechanisms of bone formation occur during
skeletal development; the first relies on the replacement of a
cartilaginous precursor with bone (endochondral ossification). In the
second mechanism (intramembranous ossification) a collagenous
precursor to bone is produced first (osteoid), and the mineralisation of
170 Topics in Bio-Mechanical Engineering

this matrix then completes bone formation. Similarly, during the

remodeling of bone, osteoblasts first lay down the collagen component of
bone, which is then mineralized. Fracture repair too, involves the
formation of a callus which is composed of both cartilage and fibrous
(collagenous) tissue, which is remodeled and replaced by bone. This
suggests that an in vitro system consisting of osteoblasts seeded on a
three-dimensional collagen scaffold may mimic the in vivo environment
sufficiently to provide a useful model to investigate aspects of normal
bone formation.
The aim of this chapter is to report on four integrated projects that
aim to explore further the potential of collagen-GAG scaffolds for bone
tissue engineering applications. This report will provide an update on the
results to date and on the work ongoing and that which will be carried
out in the future. The four projects are:
1. To characterise the permeability of a series of collagen-GAG
scaffolds which have a constant porosity but different pore sizes.
2. To develop microscale finite element models of collagen–GAG
scaffolds which will be used to predict the effects of biophysical stimuli
on the scaffolds at a cellular level – these models will be used in the
future development of bioreactors for a related project.
3. To determine the optimal biological and chemical environment to
promote mesenchymal stem cell differentiation to osteoblasts, cell
attachment and proliferation and osteoid formation and mineralisation.
4. To develop computational models to predict the orientation
response of cells due to stretching of the substrate to which they are
attached.
However an understanding of the fabrication process of these
scaffolds is necessary to put the results of these projects into context –
this will be given in the next section.

2. Collagen-GAG Scaffold Fabrication

The scaffold used in this study is produced by a freeze-drying process.
Recent work has led to the development of a new scaffold synthesis
process which controls the rate of freezing during the fabrication
procedure (11). The modified fabrication technique has allowed the
 Scaffolds and Cells: Biomechanical Analysis 171

production of collagen-GAG scaffolds with a more homogeneous

structure characterised by less variation in mean pore size throughout the
scaffold compared to an original version of the scaffold (20) which
received FDA approval in 1996 for use as dermal replacement in burns
patients. The pores produced using the new technique are also more
equiaxed, compared with those in scaffolds produced using the original
technique. Chondroitin 6-sulfate is the glycosaminoglycan used, this is
mixed with acetic acid and fibrillar collagen type I from bovine tendon.
Freeze-drying the mixture at a temperature of -40ºC forces the co-
precipitate into the spaces between the growing ice crystals to form a
continuous interpenetrating network of ice, and the co-precipitate.
Sublimation of the ice crystals, leads to formation of a highly porous
sponge. Following the freeze-drying process, each collagen-GAG
scaffold is cross-linked in a vacuum oven at a temperature of 105oC
under a vacuum of 50mTorr for 24 hours in order to stiffen the collagen
network. This process also sterilises the scaffold. The volume fraction
and size of the pores can be controlled by the volume fraction of the
precipitate and the freezing temperature respectively. Typical porosities
of these scaffolds are above 99% while if the freezing temperature is
increased above -40ºC, scaffolds containing larger mean pore sizes are
obtained. These composite scaffolds have been used successfully to
regenerate partial skin in burn patients, the conjunctiva in an animal
model, and has significantly increased the quality of peripheral nerve
regeneration (19).

3. Permeability Analysis of Collagen–GAG Scaffolds

Permeability, also referred to as hydraulic conductivity, relates the ease
by which a fluid passes through a medium. In tissue engineering, this
material property is important for a number of reasons: it affects the
diffusion of nutrients and waste in and out of the scaffold, and the
amount of pressure built up inside the scaffold. Both of these, in turn,
influence cellular activity and therefore the overall bioactivity of the
scaffold. While permeability has been characterized for a variety of
biological tissues including cartilage and bone and in scaffolds used for
tissue engineering applications including various ceramics and synthetic
172 Topics in Bio-Mechanical Engineering

polymers, it has not been characterised for a natural polymer scaffold,

such as the collagen-GAG scaffold.

3.1 Methods for Measuring Permeability

Collagen-GAG scaffolds were fabricated using the technique described

above. However, the final temperature of freezing was varied (-10, -20, -
30, -40°C) to produce a series of scaffolds with constant composition
(dry porosity 99.5%) but with four distinct mean pore sizes (95, 110,
121, 150 microns). A device was constructed to measure Darcy’s
permeability (k = Qlµ/∆PA) at different scaffold compression levels
(Fig. 1). 13 mm2 samples were cut from each of the scaffolds containing
varying mean pore size, and submerged in saline solution for 24 hours
prior to testing. The scaffold sample was clamped down with a silicone
spacer (10 mm diameter) onto a stainless steel mesh. The mesh
supported the scaffold over the 8 mm diameter brass tube, through which
the fluid flowed. Permeability was measured at compressions of 0, 14,
29, and 40% for each of the four mean pore sizes (95 – 150 µm).
Pressure head
(saline solution)

Brass plates, with

stainless steel mesh Silicone spacer/clamp
over 8mm dia tube

8mm diameter

Measure flow rate

Fig. 1. Apparatus constructed for measuring the permeability of the collagen-GAG

scaffolds.
 Scaffolds and Cells: Biomechanical Analysis 173

3.2 Permeability Results

Fig. 2 shows the results to date. The collagen-GAG scaffolds was found
to be highly permeable with a mean value in the order of 10-10 m4/Ns.
Permeability was found to increase with increasing pore size, and
decrease with increasing compression. It is expected that this high
permeability will prove highly beneficial for cell seeding and tissue
formation, allowing the cells to diffuse into the centre of the scaffold and
will provide space for the ingrowth of tissue and subsequent
vascularisation. Future work will involve establishing a better
understanding between permeability and pore size so as a pore size could
be determined to optimise biosynthesis rates. As standard mathematical
models will not work with such a highly porous material, the scaffold’s
hydrated 3-D microstructure will be examined using confocal reflection
microscopy. Based on the geometry, a computational fluids model will
examine the pressures inside scaffolds of different pore sizes. These data
will be used in the future development of bioreactors to optimise the
tissue engineering of bone.
2.5
0% compression
14% compression
29% compression
2
40% compression
k *10 ^ -10 (m^4/Ns)

1.5

0.5

0
96 110 121 150
Pore Size (microns)
Fig. 2. Permeability (m4/Ns) of the collagen-GAG scaffold as a function of pore size and
compression.
174 Topics in Bio-Mechanical Engineering

4. Microscale Finite Element Models of Collagen–GAG Scaffolds

The aim of this project is to develop microscale finite element models of
collagen-GAG scaffolds which will be used to predict the effects of
biophysical stimuli on the scaffolds at a cellular level, these models will
be used in the future development of bioreactors which are being
developed to optimise the tissue engineering of bone.

4.1 Development of the Model

The microstructure of the collagen-GAG scaffolds is comprised of a

random three-dimensional network of interconnected struts. In this
study, initial attempts at developing finite element analysis (FEA)
models of the scaffold were carried out by modelling the microstructure
of the scaffold in two dimensions as a repeating array of hexagonal unit
cells as shown in Fig. 3. By taking advantage of symmetry, only one
quadrant of this unit cell was considered when modelling the
microstructure. In this model, the elements defining the scaffold strut
material were defined as a linear elastic material. The elastic modulus
used in these simulations was calculated from compression test data on
hydrated specimens (7) using established formulae (Eqn. 1) for open
cellular solids (5):
2
E  ρ 
=   (Eqn.1)
E S  ρ S 
E and ES are the elastic modulus of the scaffold and strut material
respectively and the ratio ρ / ρ S is the relative density of the scaffold.

Fig. 3. Idealised 2D microstructure of collagen-GAG scaffold comprising a repeating

array of hexagonal unit cells. A single unit is shaded.
 Scaffolds and Cells: Biomechanical Analysis 175

4.2 Calculated Stress/Strain Behaviour

Fig. 4 shows a linear stress-strain curve calculated by considering the

deformation, in response to a uni-axial tensile load of the unit cell. It also
shows a linear stress strain curve for the strut material calculated using
volume-averaging techniques. The elastic modulus calculated by
considering the stress-strain response of the entire unit cell is 300 kPa.
This value compares well with values reported in literature for collagen
scaffolds in the hydrated state subjected to tensile loading (3). Figure 4
also shows the stress-strain response for an individual collagen strut. It
can be seen that they are significantly stiffer that the unit cell, with a
modulus of 5 MPa. These results clearly show that the response of the
scaffold measured macroscopically is not a good indication of how the
individual struts, which supply the physical support structure for cells,
respond to the same loading. As well as giving insight into the
biophysical stimuli that are imposed on cells during mechanical loading
of these scaffolds, they also highlight the need for accurate material
properties for the strut material. While the strut material in this
simulation was considered to be linear elastic, in reality only a very small
portion of the experimental curves measured for hydrated scaffolds is
linear and it would be necessary to define the strut material as a hyper
and/or viscoelastic material. This would likely give a better
approximation to the actual strut material behaviour.
0.6

0.5
Volume averaged ( Struts)
Stress (MPa)

0.4 Unit cell Deformation

0.3

0.2
Stress = (0.3441) Strain
0.1

0
0.000 0.050 0.100 0.150 0.200 0.250 0.300
Strain
Fig.4. Stress strain response of the entire unit cell and strut material.
176 Topics in Bio-Mechanical Engineering

4.3 Future Development of the Finite Element Model

While the 2D unit cell modelling approach presented here highlights the
need for microscale modelling of the collagen-GAG scaffolds in order to
determine the biophysical stimuli imposed on cells, it is certainly an
over-simplification of the scaffold microstructure. In reality collagen-
GAG scaffolds are random three dimensional structures and ideally
should be modelled as such. The pores in the scaffold are often partially
or fully filled with fluid which will impose a further mechanical stimulus
on cells attached to the scaffold and so must also be included in the
models. Work is ongoing investigating the strains and surface pressures
between cells and scaffold as it is deformed in two dimensions. Fluids
are also being introduced to investigate the additional stimuli induced on
the cells as a result of flow through the pores. Future work will involve
development of three dimensional tetrakaidecahedral unit cell models
that incorporate all the aspects of the two dimensional models. These
tetrakaidecahedral unit cells (fourteen-sided polyhedrons that pack to fill
space) have previously been used to model the geometry of the porous
collagen-GAG scaffold in order to determine the relationship between
specific surface area, ligand density and pore size in the scaffolds (12).

5. Mesenchymal Stem Cell Behaviour on Collagen-GAG Scaffolds

In order to stimulate mesenchymal stem cell (MSC) differentiation to
osteoblasts and subsequent formation of osteoid and eventual
mineralisation, a suitable biological and chemical environment is
required. This project aims to determine the optimal biological and
chemical environment required to promote mesenchymal stem cell
differentiation to osteoblasts, cell attachment and proliferation and
osteoid formation and mineralisation.

5.1 Cell Culture and Staining

Mesenchymal stem cells were obtained from the bone marrow of young
adult Wistar rat tibiae and femora. These cells were plated out, separated
from haematopoietic cells, and expanded in culture. Cells were either
 Scaffolds and Cells: Biomechanical Analysis 177

maintained in a 2-D environment by plating onto glass coverslips or were

seeded onto 3-D samples of the collagen-GAG scaffold. The MSCs were
incubated with culture media supplemented with ß-glycerophosphate and
ascorbic acid-2-phosphate to promote osteogenesis. Control studies were
carried out with no osteogenic growth factors added. The samples were
maintained in culture for two and three week time periods. For MSCs
grown in 2-D, western immunoblotting was used to measure osteocalcin
expression, a late stage marker of osteoblast formation, and compared to
cells cultured in control medium (without the osteoinductive factors).
Von Kossa staining for calcium and Alizarin Red S staining for calcium
phosphate was used to determine whether mineralisation of the MSC
seeded scaffolds had taken place.

5.2 Results and Discussion

The significant increase in osteocalcin expression (P<0.01, n=10) for

MSCs grown in 2-D indicates that the environment provided has
successfully caused cells to differentiate along an osteogenic route. In
order to understand the mechanism behind this differentiation, it was
hypothesised that the mitogen activated protein kinase ERK
(extracellular signal-regulated kinase) might be involved, as it has a role
in cell survival and differentiation. To establish this, cells were cultured
in a 2-D environment with osteoinductive factors for two weeks in the
presence of U0126, an inhibitor of ERK activity. These results show that
the induction of osteocalcin expression was absent when cells were
treated with the inhibitor, U0126. This demonstrates that ERK is
involved in the differentiation of MSCs along the osteogenic route.
When the degree of mineralisation was analysed, seeded-scaffold
samples maintained in culture for two weeks, showed no sign of
mineralisation. They did however show positive immunofluorescent
staining for bone-specific osteocalcin in the areas where the cells were
located. Following three weeks in culture, cells had fully penetrated the
scaffolds, had differentiated to osteoblasts as indicated by osteocalcin
expression and most promisingly, mineralisation was evidenced by both
staining techniques (Alizarin Red S and Von Kossa). Figure 5 shows the
positive results indicating that dexamethasone, β-glycerophosphate (β-
178 Topics in Bio-Mechanical Engineering

GPh) and ascorbic acid (AA) successfully caused MSC differentiation,

osteoid formation and subsequent mineralisation.

Control. Dex + β-GPh + AA

Scalebar= 200 µm Scalebar= 200 µm

(d)

Fig. 5. Effect of dexamethasone (dex) (10 nM), β-glycerophosphate (β-GPh) (10 mM)
and ascorbic acid (AA) (0.5 mM) on matrix mineralisation. MSCs (n = 6) were seeded
onto collagen GAG scaffolds for 21 days in the absence or presence of osteo-inductive
factors. Cells were stained using the Alizarin Red S method. The numerous dense dark
patches (two patches indicated by arrows) indicate mineralisation.

6. Computational Predictions for Bioreactors

The aim of this project is to develop computational models to predict the
orientation response of individual cells. This will reflect experimental
work to be carried out in a bioreactor currently under development. The
orientation response to a strain stimulus is important for two reasons.
Firstly, the mechanism responsible for orientation may alter mRNA and
protein synthesis. Secondly, cell reorientation alters the deformation they
experience. The loads imposed on cells alters their remodeling behavior
and abnormal tissue conditions are therefore thought to result from
irregular loading of cells. Experimental testing carried out on human
aortic endothelial cells grown on a deformable silicone substrate reveal a
very specific pattern of cell reorientation in response to cyclic stretching
of the substrate. In cases where lateral deformation of the substrate was
prohibited, cells were found to elongate perpendicular to the stretch
 Scaffolds and Cells: Biomechanical Analysis 179

direction. However, if the substrate is laterally unrestrained cells were

found to elongate at an angle to the stretch direction (10, 18).

6.1 Development of the Model

In this project computational models were developed in order to

investigate cell-substrate contact during cyclic loading. Three-
dimensional cohesive zone formulations were used to simulate cell
adhesion to and de-bonding from the substrate surface. Homogeneous
visco-elastic behaviour was assumed for the cell (16). Cyclic substrate
stretching for the case of both unrestrained and restrained lateral
deformation was modelled (i.e. perpendicular to the applied load).

6.2 Predicted Cell Reorientation

In the unrestrained mode of deformation, the mode of cell debonding was

accompanied by an accumulation of compressive strain leading to
additional de-bonding of the cell perpendicular to the direction of
stretching. The resultant cell-substrate contact area was found to be
aligned at 70o to the direction of substrate stretching. This can be seen
on the left hand image of Fig. 6, note the orientation of the centre part of
the cell. In the restrained mode of deformation, it was found that cell
debonding resulted from an accumulation of tensile strain due to cell
viscoelasticity leading to a final cell-substrate contact area aligned at 90o
to the direction of stretching (see the right hand image of Fig. 6). These
results are consistent with the experimental observations of Wang et al.
(18) who used endothelial cells grown on a deformable silicone
substrate. These results are shown in Fig. 7. Here again the cells on the
left are grown on a substrate unrestrained perpendicular to the applied
load, while those on the right are not.

7. Discussion and Conclusions

The results to date have laid a firm basis for future work. The collagen-
glycosaminoglycan scaffolds have proven to be a suitable substrate on
180 Topics in Bio-Mechanical Engineering

Fig. 6. Computational prediction of cell reorientation on cyclically deformed substrate.

The left hand images are for a substrate that is unrestrained perpendicular to the applied
load, with the cells aligning at 70 degrees to the load. The substrate on the right hand
side is restrained perpendicular to the applied load, with the cells perpendicular to the
applied load.

Fig. 7. Images of cell re-orientation from Wang et al. (18), corresponding to the cases
modelled in Fig. 6.

which to culture mesenchymal stem cells with the aim of ultimately

producing bone. They have provided a biological environment which
has allowed large numbers of mesenchymal stem cells to attach and
proliferate and with the addition of growth factors to successfully
differentiate to osteoblasts. Developing a seeding technique and
determining the quantities and types of growth factors necessary to cause
this differentiation is a laborious process but the technique developed has
proven to be very successful. Perhaps, the most positive result to date
has been the subsequent mineralisation of the scaffolds by the
 Scaffolds and Cells: Biomechanical Analysis 181

differentiated stem cells (Fig. 5). It had previously been envisaged that
this was a process that may not occur without the addition of biophysical
stimuli in the form of a bioreactor.
The two projects which are aiming to provide scientific data which
will facilitate the design of bioreactors have produced interesting results.
The permeability tests have indicated that, at constant porosity, the pore
size can be used to control permeability. This in turn can be used to
control fluid flow and pressure on the scaffold which, theoretically at
least (8, 13) might control MSC differentiation pathways. In addition,
the finite element modelling may have the potential to allow precise
calculation of the cell-level strain field acting to stimulate differentiation.
It is envisaged that these bioreactors will allow MSC differentiation to
osteoblasts (without the addition of chemical growth factors) and
subsequent formation of osteoid and eventual mineralisation by
providing a mechanical environment with appropriate biophysical
stimuli.
The extension of the present work developing microscale finite
element models of the scaffolds will be a major contributor to the
bioreactor design project. The present two-dimensional models will be
enhanced with the development of three dimensional tetrakaidecahedral
unit cell models which will provide a better understanding of the
scaffolds at a microstructural level. As anticipated, the scaffolds were
found to be highly permeable. The degree of permeability was found to
increase with increasing pore size, and decrease with increasing
compression. This data will be used initially to establish a theoretical
understanding between permeability and pore size. This mathematical
model will be extended to include other material properties of the
scaffold which will allow for a more accurate prediction of the levels of
strain and fluid-generated shear stress to which a cell-seeded scaffold
should be subjected in a bioreactor to encourage cell differentiation and
subsequent bone formation.

Acknowledgements
This study was funded by the Higher Education Authority under Cycle 3
of the Programme for Research in Third Level Institutions (PRTLI).
182 Topics in Bio-Mechanical Engineering

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