MAT311

Cell and Tissue Engineering

Cell Culture, Biochemistry and Mechanical Testing

Sadika Choudhury

CONTENTS
ABSTRACT

pg 3

INTRODUCTION

pg 4-6

METHOD

pg 6-8

RESULTS

pg 8-10

DISCUSSION

pg 10-13

REFERENCES

pg 14-16

Abstract: (300 words)

In this study, chondrocytes were isolated from bovine articular cartilage by enzymatic
digestion and seeded in agarose (type IX) at a final concentration of 4 x 106 cells/ml in
4% and 6% agarose over a culture period of 7 days. The aims of this study were to
determine how the biochemical and mechanical parameters of chondrocyte/agarose
constructs are influenced by the gel concentration (4% or 6%) and the time in culture at
day 0 and day 7. The objectives involved being able to measure the total amount of GAG
released by chondrocyte cultured within agarose constructs and to measure the
mechanical properties of the cell/agarose constructs. The cultures were maintained in
medium that stimulates matrix production (DMEM + 20% FCS) and showed variances in
GAG levels for chondrocyte/agarose constructs at day 0 and day 7 and dependence of
GAG levels on the mechanical properties of the agarose construct. While 6% agarose gel
showed greater stiffness than 4% at day 0, no significant change was observed in the
mechanical properties for 4% agarose concentration from day 0 to day 7. Both
concentrations showed an increase in mean GAG concentration over the culture period,
although 4% agarose concentration at day 7 showed a much steeper increase than 6%
agarose at day 7, due to enhanced porosity and better diffusion of nutrients and waste
products in the former. The absorbance of four unknown samples were measured and
their corresponding concentrations calculated by means of a GAG standard curve. Further
means of improving nutrient utilisation in constructs, preventing the oxygen/glucose
gradients and maximising anabolic signalling in constructs were investigated, with
specific attention to the use of bioreactor devices.

Introduction:
Cartilage structure/function
Intact articular cartilage is a
prerequisite for the adequate function
of joints. Reducing the coefficient of
friction and attenuating peaks of stress
it protects the joint against wear. [3] Its
primary functions include transmitting
applied loads across mobile surfaces,
lining the ends of bones and allowing
surfaces to roll or slide during motion,
causing a reduction of the friction
coefficient to 0.0025. [4] The different
types of cartilage are distinguished by
composition, microstructure and
Fig 1: Articular cartilage [3]
mechanical properties and consist of
Hyaline cartilage, Elastic cartilage and
Fibrocartilage. Hyaline cartilage is glassy-smooth and bluish-white in appearance and has
a fluid-filled wear resistance surface. Elastic cartilage, which is typically found in the
epiglottis and external auditory canal, is yellowish and opaque, and exhibits more
flexibility than Hyaline cartilage. The final type, Fibrocartilage, often exists temporarily
in fracture sites, but is permanently present in 3 major locations: the intervertebral disks
of the spine, as a covering of the mandibular condyle in the temporomandibular joint, and
in the meniscus of the knee. [1]
Frequently experiencing high stresses and excessive frictional forces, cartilage
components are arranged in a way that is maximally adapted for biomechanical functions.
Articular cartilage, whilst containing no blood vessels, lymph vessels or nerves, is
composed of Chondrocytes (~1%), Proteoglycans (15%), Collagen Type II (15%) and
water (70%) in three layers the surface, middle and deep parts and maintains
lubrication by water contained in the matrix and by the density and collagen fibre
orientation of each layer. [5] The collagen present creates a framework as a structural
protein that houses the other components of cartilage and provides tensile strength, which
is critical in the extracellular matrix. The synovial fluid containing the matrix structure is
assembled from collagen with varying orientation and density, Proteoglycan gel, which
retains a large volume of water, thick Hyaluronan and Chondroitin Sulphate, thus
functioning as a shock absorber and lubricant. [2] The structural anisotropy of each layer is
an important factor that defines the function of the cartilage.
Matrix metabolism and signalling
The extracellular matrix (ECM) provides the principal means by which mechanical
information is communicated between tissue and cellular levels of function. [6] In
addition, it sequesters a wide range of cellular growth factors, and acts as a local depot
4

for them. Changes in physiological conditions can trigger protease activities that cause
local release of such depots. This allows the rapid and local growth factor-mediated
activation of cellular functions. [7] Bissell et al [8] suggested the idea of 'dynamic
reciprocity' of the ECM with the cytoskeleton and nuclear matrix. Cell-surface receptors
interacting with ECM molecules would transmit signals across the cell membrane to
molecules in the cytoplasm, initiating a series of events via the cytoskeleton into the
nucleus, culminating in the expression of specific genes that, in turn, would affect the
ECM.
Transmembrane receptors for specific sequences on ECM molecules are known to bind to
three categories of cell-surface receptors: integrins, cell-surface proteoglycans and other
receptors, for instance CD 36, which binds collagen and thrombospondin to monocytes,
endothelial cells and some types of epithelial cells. [9] Integrins make up a family of
heterodimeric transmembrane proteins composed of and subunits, and though
structurally unrelated, they maintain 40-50% homology within each subunit. The
cytoplasmic domain of integrins interacts with the cytoskeleton, making transduction
ECM signalling via the cytoskeletal elements possible. [10] [11] Of the proteoglycan
receptors that have been isolated and characterized are: syndecans, CD44 and
thrombomodulin. The former two bind via Chondroitin and Heparan-sulphate
glycosaminoglycans, while Thrombomodulin is expressed in endothelial cells and its
biological activity can be regulated either by its protein or glycosaminoglycan
components. [12]
Bioreactors and Nutrient Utilisation
Loosely defined as devices that
provide the transport system for
nutrients to cultured cells and
allow the efficient withdrawal of
toxic wastes and inhibitory
metabolic by-products, bioreactors
play an invaluable role in enabling
reproducible and controlled
changes in specific environmental
factors whilst providing an
understanding of specific
biochemical and mechanical
interactions through technical
means.

Fig 2: Bioreactor device

Bioreactors are used for different purposes such as cell proliferation on a small scale or
generation of 3D constructs on a large scale. The high rate of nutrient utilization
associated with high cell density and increased cellular activity (for matrix synthesis and
sufficient quantities of organized ECM) requires an adequate nutrient supply. [13] [14] This
can lead to large gradients in cell viability and consequently inadequate functional
properties. Previous experiments have shown that a close relationship between matrix
5

synthesis and energy metabolism of chondrocytes exists, [15-19] and that Proteoglycan
synthesis is affected by nutrition, pH, and oxygen. [13]
Various studies show the large concentration dependencies that cellular regulation
mechanisms hold on nutrient utilization. For example, studies on the effect of oxygen
concentration on glucose uptake and lactate production have shown that depending on the
culture conditions, a low oxygen level has been shown to either increase [20] [21] or
decrease [16] [19] Glycolysis in articular chondrocytes. Similarly, an investigation on
different concentrations of glucose medium on chondrocyte nutrient utilisation showed
marked differences in oxygen uptake and glucose utilisation, with the high glucose (HG)
medium showing an up-regulation in oxygen uptake before a sudden decrease after a
certain point in time, [22] which is explained by the high lactate levels reached in the HG
case at high cell densities. The lactate accumulation was related to a decreased viability
observed in HG, resulting in a decrease in overall oxygen uptake. [23]
This study looks to explore how the culture condition and properties of scaffold material
will influence the rate at which cells produce glycosaminoglycans (GAGs) and the
mechanical properties of the cells seeded in construct. Bovine chondrocytes are seeded in
agarose gel and cultured for 7 days in medium

Method:

Chondrocyte isolation
1. Cut away connective tissue and ligament in metacarpal-phalangeal joint.
2. Using sharpest point of autoclaved blade, remove superficial layer of articular
cartilage.
3. Isolate chondrocytes from extracellular matrix by digestion with 2 enzymes,
Pronase and Collagenase.

Preparation of chondrocyte/agarose constructs

1. Using 20l chondrocyte suspension diluted in 20l Trypan Blue, count total nos.
cells and % viability using Trypan Blue exclusion method.
2. Calculate how much cell/agarose can be prepared at 4x106 cells/ml
3. Resuspend cells at 8x106 cells/ml in 10ml DMEM+20%FCS
4. Prepare ultra-low gelling temperature agarose suspension 10ml Earls Balanced
Salt Solution (EBBS).
5. Autoclave agarose then cool to 37C in oven
6. Mix cells into agarose by swirling carefully
7. Assemble mould under sterile conditions 5mm height x 5mm diameter
8. Using 3ml Pasteur Pipette, pipette cell/agarose solution into holes
9. Place moulds in Petri dish and gel at 4C for 45minutes
10. Remove specimens with pipette tip
11. Place specimens in universal tubes and 1ml of medium

12. Label Petri dish with: Date of preparation, agarose concentration (4% or 6%), Day
0 or Day 7.

Biochemical analysis of GAGs

1. Label eleven Eppindorf tubes, 1-11
2. Dilute 1mg/ml stock solution of bovine chondroitin-4-sulphate standard to
100g/ml by taking 1800l water + 200l standard
3. Prepare series of standard dilutions between 0-50g/ml as follows:
Tube Nos.

GAG concentration
Volume of standard
Volume of water
(g/ml)
(l)
(l)
1
0
0
200
2
5
10
190
3
10
20
180
4
15
30
170
5
20
40
160
6
25
50
150
7
30
60
140
8
35
70
130
9
40
80
120
10
45
90
110
11
50
100
100
4. Vortex samples. Pipette 40l of standard into 96-well plate as shown:
1
2
Blank Blank
Blank Blank
Un1
Un2
Un1
Un2

A
B
C
D

3
5
5
Un3
Un3

4
10
10
Un4
Un4

5
15
15

6
20
20

7
25
25

8
30
30

9
35
35

10
40
40

11
45
45

12
50
50

5. Add 250l of 1-9-dimethylmethylene blue to wells containing standard

6. Transfer to plate reader, select protocol and press start. Ascent software plots
concentration versus absorbance.

Mechanical testing of chondrocyte/agarose constructs

1.
2.
3.
4.

Mount cell/agarose construct in MTS machine and align centrally to platens

Load cell platen moves down until it detects 0.003N load on core
Hydrate core with EBBS
Crosshead moves at 0.0167mm/s to compression of 20% strain and holds position
to measure stress relaxation
5. Load data acquired at 10Hz in compression, 1Hz in relaxation
6. Remove specimen, clean platens. Repeat procedure with new specimen.
7. Test each specimen once-repeat tests of same specimen have variances due to
viscoelastic properties.
7

Statistical analysis
1. Convert data values into stress and strain data, where:
Strain = Specimen Displacement (mm) / Original height (mm)
Stress (Nm2) = Load (N) / Cross-sectional Area (m2)
2. Calculate mean values for each set of data and standard deviations
3. Perform Student t-tests to enable comparisons between different gel
concentrations and culture period for both mechanical and GAG data at 5%
significance level.
4. Calculate, using GAG standard curve and measured absorbance, the concentration
values for 4 unknown samples.

Results:

GAG production in 4% and 6% cell/agarose constructs cultured up to 7 days

Increased glycosaminoglycan (GAG) concentration showed a relatively lower mean

value (when compared to the construct with lower concentration) after 7 days of cell
culture. Mean GAG concentration at 4% on day 0 was found to be 8.221g/ml and
increased by ~77% when measured on day 7. In contrast, 6% concentration of agarose gel
only showed an increase of ~64% in mean GAG concentration of chondrocyte/agarose
construct.

4% Day 0
4% Day 7
6% Day 0
6% Day 7

Mean GAG conc.

(g/ml)
8.221
35.226
11.423
31.519

Standard
Deviation
8.504654
6.902542
17.58293
13.61388

Student T-test
Result
7.3823
5.7692

At 5% level
Distributions are:
>2.179
DIFFERENT
>2.179
DIFFERENT

Table 1: Comparing Mean GAG production

The student t-tests comparing GAG production over the 7-day culture period proved that
distributions were, when analysed at 5% level, widely different. This is to be somewhat
expected, considering that cells would be undergoing matrix synthesis over this period.
The varying concentration of the agarose gel brings to question how the porosity of the
constructs may have affected the mechanical properties. With the lower concentration of
4% agarose gel, a higher porosity would lead to greater diffusion and nutrient utilisation,
consequently leading to more proteoglycan and ECM synthesis. For a construct with
greater density i.e. 6% agarose concentration, cells at centre of scaffold have reduced
viability due to hindered diffusion of nutrients and waste products and consequently
shows a lower mean GAG concentration.

Mean data for Group A

60

GAG ( g/ml)

50
40
30
20
10
0
Day 0 (4%)

Day 0 (6%)

Day 7 (4%)

Day 7 (6%)

Graph 1: Mean GAG concentration over culture period

Mechanical properties of 4% and 6% chondrocyte/agarose constructs

15% Tangent Modulus of chondrocyte/agarose construct at 4% agarose concentration

showed very little change, with the measured value of ~54KPa falling to ~53KPa at day 7
of the culture period. This could be suggesting that almost no change took place at this
concentration over the culture period, but bearing in mind the differences in mean GAG
concentration from day 0 to day 7 under said conditions, it is known that matrix synthesis
did occur, as cells metabolised the nutrients available.
The lack in change from day 0 to day 7 at 4% agarose concentration may be explained by
one set of data quite anomalous from the rest. Specimen 2 at the said concentration on
day 7, showed a mean stress of only ~4KPa and consequently a 15% Tangent Modulus of
29KPa. This is remarkably lower than the remaining specimen tested for that data set,
with values exhibiting ~60KPa Tangent Modulii at 15% Strain.

4% Day
0
4% Day
7
6% Day
0
6% Day
7

Mean Stress
(Nm2)
8159.32

Strain Standard
(%) Deviation
15
8417.86

Mean 15% Tangent

Modulus (Nm2)
54395.47

Approx. Tangent
Modulus (KPa)
54

7914.84

15

13206.59

52765.60

53

8464.91

15

10703.54

56432.73

56

11093.01

15

21007.24

73953.40

74

Table 2: Mechanical Properties of chondrocyte/agarose constructs

The change in mean Tangent Modulus at a strain of 15% is comparatively much more
marked, showing and increase from ~56KPa to ~74KPa, indicating a construct with
9

enhanced mechanical properties in terms of stiffness at the end of the culture period. The
student t-test carried out on mechanical testing data showed that at the 5% level,
distributions were the same for 4% agarose gel from day 0 to day 7, whereas the
distribution was very different for 6% agarose gel over the same period i.e. a greater
change in mechanical properties was observed in 6% chondrocyte/agarose constructs.

Student T-test

4% Day 0, 4%
Day 7
6% Day 0, 6%
Day 7
4% Day 0, 6%
Day 0
4% Day 7, 6%
Day 7

Standard Deviation of
15% Tangent Modulus
4550.9

T-test Result

At 5% level,
Distributions are:
<2.306 SAME

0.358140148

2556.32

6.853856325

6089.77

0.334534802

<2.306 SAME

11097.01

1.909320619

<2.306 SAME

>2.306 DIFFERENT

Table 3: Student T-test data Comparing Mechanical Properties of Constructs

Four unknown samples were also included in the GAG data, and their absorbance values
measured. Using the GAG standard curve, the mean concentrations of the unknowns
were calculated to be (in g/ml) as follows: Unknown 1: -1.651, Unknown 2: 2.648,
Unknown 3: 9.0495, Unknown 4: 12.8155

Discussion:

Further studies on how to improve nutrient utilisation

A study on scaffold degradation showed increased construct collagen content and

dynamic mechanical properties in engineered articular cartilage. [24] The mechanisms
credited were increased nutrient transport, increased space for collagen fibril formation,
and cellular response to the loss of GAG with agarase treatment. Results showed that
over a 91-day culture period, agarase-treated constructs possessed ~25% more DNA,
~60% more collagen, and ~40% higher dynamic modulus compared to untreated controls.
Further studies on controlling cell biomechanics have explored the optimisation of
mechano-transduction pathways for successful tissue engineering. Focusing primarily on
the effects of cellsubstrate adhesion, attenuation of voltage-operated calcium channels
(VOCC) activation states and biological conditioning of cell-scaffold constructs utilising
bioreactors, [26] increased matrix protein synthesis was observed. By manipulating
scaffold properties by varying the type of material utilised, the shape and size of pores in
which cells are located, the mechanical integrity of the construct and substrate coating, [26]
it is possible to promote cell adhesion and incorporate chemicals and growth factors that
will optimise tissue formation and function and improve nutrient utilisation within the
construct.

10

An investigation into the use of different media for chondrocyte culture involved
harvesting autologous auricular cartilage in swine model, with three categories: Group A,
10% foetal calf serum; Group B, supplemented with 10% autologous serum and Group C,
supplemented with growth factors. [37] Results showed that although the cells in all the
three groups exhibited normal chondrocyte morphology, there was a statistically
significant difference in the number of cells between Group A and the two other groups
(p < 0.05). By day 12, both Groups A and C demonstrated greater cell number as
compared to Group B (p < 0.05), proving that choice of medium significantly affects cell
viability and can be attributed to varying nutrient utilisation in each group. The study
concluded that either an autologous serum enriched and/or a growth factor enriched
culture medium can be utilized successfully for the multiplication of tissue-engineered
chondrocytes. [37]

Fig 3: Increase in the cell numbers in the three groups at different culture time. At early time points there
was a statistically significant difference in the number of cells between Group A and the two other groups.
By day 12, both Groups A and C demonstrated greater cell number as compared to Group B. [37]

Prevent oxygen/glucose gradients

Bioreactors control the biochemical and biomechanical environment for a tissueengineered construct. Transport of nutrients and waste to and from cells control the
biochemical environment, whilst biomechanical stimuli can be controlled through
variable shear stresses, axial compression of the cell-seeded scaffold or tensile forces,
depending on the bioreactor used. [26] Changing the scaffolds circulation regime is one
way to solve the issue of mass transfer of nutrients and degradation products and prevent
poor cell viability and activity due to limited nutrient diffusion. Fluid flow in 3-D, for
instance, spinner flasks, rotating wall vessels and perfusion systems are all means of
improving nutrient diffusion and promoting cell proliferation throughout the constructs.
[31] [32]

11

In large-scale bioreactors gradients often occur due to imperfect mixing, which in turn
complicates the design and control of large-scale bioreactors. A solution is to model
gradients in the oxygen concentration with a two-compartment model of the liquid phase,
[33]
where the aqueous phase of the reactor is conceptually divided into an aerated and a
non-aerated section, both ideally mixed and exchange of oxygen from either
compartment is via a circulation flow. In contrast to this, research on bioartificial livers
(BAL) have suggested that oxygen transfer limitations could hinder the efficient
functioning of hollow fibre BAL reactors, giving rise to ideas for a radial flow hepatocyte
bioreactor. Oxygen permeable flat membranes or hollow fibres are just two of the ideas
being explored as oxygenation techniques to be incorporated into newer BAL designs. [34]
It is universally maintained that oxygen, although one of the most important nutrients for
cells, and playing a major actor in aerobic metabolic cycles, can often also be the limiting
nutrient in successful tissue growth in vitro, the reason being the poor oxygen solubility
in culture media, which leads to difficulty in bringing sufficient amounts of oxygen to the
surface of the cells. Oxygen solubility is lower than the availability of glucose and
consequently, medium must be continually circulated and re-oxygenated by passing
through an in-line gas-exchanger, without inducing cytotoxic presence of free-radicals. [35]
A study into bioreactors for tissue mass culture submitted that low oxygen tension
(~40mmHg) and low pH (~6.7) led to more anaerobic conditions, which in turn
suppressed chondrogenesis in 3-D tissues. Aerobic conditions at higher oxygen tension
(~80mmHg) and higher pH (~7.0) resulted in larger constructs containing higher amounts
of cartilaginous tissue components. [36] Thus, in cell culture systems, oxygen
concentrations are usually maintained at between 20% and 100% air saturation to
maintain a certain balance between oxygen needs and tolerance to free radicals of the
cells. [35]

Maximise anabolic (GAGs) signalling events in chondrocyte/agarose constructs

Surface properties, for instance surface chemistry, hydrophilicity or surface tension and
topography are known to influence cellsubstrate interactions. [27] According to one study,
collagen was found to play a pivotal role in the delivery of mechanical signals to cells via
adhesive interactions between the cell and its substrate. Bone cells seeded on collagencoated surfaces were more responsive to mechanical load in comparison to their uncoated
control counterparts, [27] with the ECM components of the protein serving both as a
scaffold for cell attachment, and also as a mechanism for transferring mechanical signals
to cells. Alongside surface modifications, recent studies feature polymers and their
composites for producing tissue-engineering scaffolds, incorporated with bioactive
agents. [28] [29] Improved cell response and tissue formation are explored, through
optimising substrate biocompatibility and cell adhesion. [30]
Moreover, current research on progression of chondrocyte signalling depicted how
mechanical stimulation of 3-D chondrocyte cultures increased extracellular matrix
(ECM) production and mechanical stiffness in regenerating cartilage. [25] Disruption of
v3 and 51 integrins interaction with the ECM influenced proteoglycan synthesis in
distinct pathways and v3 more specifically was seen to influence the mechanical
12

response. Comparatively, In vitro and in vivo studies on bone have shown that
parathyroid hormone (PTH) directly activates survival signalling in osteoblasts and also
led to an increase in osteoblast number beyond that needed to replace the bone removed
by osteoclasts during bone remodelling. [38]
Another factor to be considered is the construct properties and the effect they would have
on anabolic signalling events. Hydrogels are a promising type of biomaterial for articular
cartilage constructs since they have been shown to enable encapsulated chondrocytes to
express their predominant phenotypic marker, type II collagen. [39] Results from this
particular study suggest that cell density and alginate concentration at high cell density
can significantly affect the endogenous Insulin-like Growth Factor-1 expression by
chondrocytes, therefore indicating that construct properties can impact chondrocyte gene
expression and should be considered in order to create a proper, engineered articular
cartilage construct. [39]

Fig 4: Percentage of chondrocyte viability when encapsulated in 0.8%, 1.2%, and 2.0% w/v alginate bead
concentrations for 25,000 and 100,000 cell density over the 8 d study. [39]

Conclusively, it can be seen that many factors can affect both mechanical and
biochemical properties of chondrocyte/agarose constructs, but it is only with further
investigation that better relationships can be drawn.

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13

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