1. 1
Abstract
Cartilage is an avascular, alymphatic and aneural tissue in the body.It consists of a extracellular
matrix in which chondrocytes are dispersed at low densities. It plays an important role in maintaining
shape and giving support to body structure. It is necessary to protect the ends of bones from rubbing
together. Damage of these cartilage caused by direct blow , stress on joint for prolonged period or lack
of movement of joint. Deep cartilage defects can be treated by autologous cell transplantation. For
this, biopsy is collected from the outside the affected area. Chondrocytes are liberated from the matrix
by an enzymatic treatment and allowed to grow in cell culture. The cells are then harvested and
introduced in affected area. Main objective of the study is to examine the mechanical and biochemical
properties under various culture environment . Different concentrations of agarose gel used to culture
chondrocytes and hence their mechanical properties are noted. Strength of the engineered tissue can
be determined by measurement of total amount of GAG released by chondrocytes cultured within
agarose constructs .Stress and strain analysis help to develop the final model for cartilage tissue.
Analysis of the cell constructs shows that total GAG synthesis is more for a 4% concentration of
agarose gel at day 7. It has explained the detail in further discussion how it affect the mechanical
strength of the tissue. In mechanical loading test , graphs has been plotted for dynamic loading
applied to the cell constructs it is found as the concentration increases; the peak load stress achieved
by the sample shows lower value. This journal report describes the methods of isolation of cells, their
maintenance in cell culture and factors influencing the mechanical properties and mention ideas to
improve further nutrient utilization with maximum signalling events in chondrocyte/ agarose
constructs.
2. 2
INTRODUCTION
Cartilage is a flexible connective tissue observed in bodies of human being and other animals. It is
the fundamental part of body which contributes to shape, strength and protection of bone in body.
Cartilage is composed of specialized cells called chondrocytes which shows different properties in
three different types of cartilage.
Table1: Types of cartilage with different location
Cartilage Type Location
Hyaline
Cartilage
Joints, rib cartilage, nose, trachea and larynx
Elastic
Cartilage
Ear, epiglottis, and larynx
Fibrous
Cartilage
Intervertebrataldiscs.
Amount of water content in cartilage varies with age, 90 per cent in young and decreases to 65 -75 per
cent in adult with the aging.
Collagen occupies 50 per cent of dry weight of cartilage. It composed of 3 aplha-I chains and differ
genetically from collagen of skin and bone. Its concentration is high in superficial zone and low in the
base of radial zone[28].
Cartilage proteoglycan composed of different concentration of glycosaminoglycans, chondroitin
sulphates 4 and 6, and keratin sulphate ,attached to protein core and aggregated on hyaluronic acid
molecules to form a proteoglycan aggregate of high molecular weight capable of binding larger
quantities of water and occupying large spatial volume. Concentration of proteoglycan is less in
superficial and higher in deeper zones[28].
Cell signalling:
It is molecular mechanism by which cell communicate. It is initiated by generation of ligand ;a
molecular entity generated by sending cell to bring about change in physiology of responding cell.
Multiple growth factors and hormones involves in activation of variety of signal transduction pathway
such as BMP/ TGF β family, Wnt family, Indian Hedgehog (IHh), Parathyroid Hormone related
protein( PTHLH),Fibroblast growth factor (FGF). TGF β invariantly used in chondrogenic culture
media inhibits chondrocyte differentiation and simultaneously stimulates production of chondrogenic
extracellular matrix. FGF2 is also potent inhibitor of chondrogenic proliferation[29].
In pathophysiology, destruction of articular cartilage is induced by wide array of cytokines expressed
by mononuclear cells and chondrocytes. These cytokines induce expression of matrix
metalloproteases and activate osteoblasts, both of which stimulate matrix decay.
Evaluation of cell signalling pathway involved in formation articular cartilage wiil be advantageous
for cartilage tissue engineering. It will allow to use such strategies to recapitulate events during
implantation.
To improve the survival of implanted construct, it is necessary to focus on the nutrient supply to
engineered tissues. Cells within the engineered tissue all rely on the supply of nutrients and transport
of waste products mainly by diffusion. Insufficient nutrient to the centre of the construct leads to loss
3. 3
in cell viability. Oxygen as a nutrient exhibits low solubility in culture medium and thus it needs to be
constantly replenished. Also it is found that oxygen diffusion is relatively slow, in comparison to
other major nutrients such as glucose. Studies on dependency of glucose uptake and lactate production
on oxygen(O2) concentration shows that a low O2 level shown to increase or decrease glycolysis in
articular chondrocytes[15]. Hence proteoglycan synthesis is affected by nutrition, pH and O2. Due to
improved mixing in bioreactor systems followed by increased fluid flow around and through
construct result in improved mass transfer. Bioreactor are design to establish spatially uniform
concentrations of cells seeded onto clinically sized biomaterial scaffolds, to maintain culture
conditions such as temperature,pH, osmolarity, levels of oxygen, nutrients and also to provide
physiologically relevant signals which includes fluid flow, shear, pressure,compression, stretch and
able to facilitate mass transfer between the cells and the culture environment. Improvement in cell
survival can be achieved by cultivating construct in culture medium in spinner flasks. Also, in order to
enhance external mass transfer under laminar flow conditions , tissue engineered constructs can be
cultivated in rotating vessels.
High cell density incorporated with high cellular activity help to promote matrix synthesis and to
obtain sufficient quantities of organized extracellular matrix. Depending on culture conditions, large
gradients in cell viability and matrix deposition can arises between periphery and centre of the
construct leading to inferior functional properties such as stiffness and permeability[13]. However,
cell viability is affected by cell culture parameters. Culture medium should maintain following
contents in order to maintain cell viability in medium:
Bulk ions- Na, K, Ca,Mg, Cl, P
Sugar- Glucose
Serum – which contains large number of growth promoting activities such as buffering toxic
nutrients by binding them, neutralizes trypsin and other proteases,has undefined effect on
interaction between cells and substrate and contains peptide hormone growth factor that
promote healthy growth.
Freshly thawed hES (Human Embryonic Stem cells)incubated within 37°C incubator shows decrease
in cell viability over time. Studies concluded that apoptosis is major mechanism for loss of cell
viability of cryopreserved cells during freezing with slow cooling protocol. Also it is demonstrated
that cell viability could be maintain by adding selected cytokines to the culture concluded by research
conducted on Mouse natural killer (NK) cells culture[17]. Further studies on porcine cartilage
explants shows that glycosaminoglycan loss can result chondrocyte death. During subsequent culture,
cell death spreads via apoptotic mechanisms[16]. To study relationship between cell nutrition,
nutrient concentration and cell viability, in vitro model has been developed by Horner and
urban(2001).It has observed that some cell types are able to remain viable at very low oxygen tensions
and have low oxygen requirements. Therefore,it can be said that oxygen levels within tissue-
engineered constructs might not always directly relate to the survival of the cells. This model revealed
that glucose is a critical nutrient for cell survival , whereas some cells found to survive in anoxic
conditions for period of 13 days.
Purification of alginate may improve biocompatibility of the chondrocytes constructs. However,
chondrocytes in purified alginate culture shows higher cell viability than those in non purified
alginate.
4. 4
Overall, successfulexperiment in performed to study influence of concentration of culture medium
and time period of culture on GAG synthesis of chondrocytes with accurate measurement of
mechanical properties.
MATERIALS ANDMETHODS
Preparation of chondrocyte- agarose construct:
In order to prepare construct articular cartilage removed from bovine metacarpalphalangeal joint.
Chondrocytes were isolated from extracellular matrix with enzymes, pronase and collagenase. After
this, washed cells resuspended in 10ml of culture medium consisted of Dulbecco’s Minimal Essential
Medium(DMEM) and 20% Foetal calf serum(FCS). 50ul volumeof cell suspension diluted in 100 ul
of trypan blue. Ultre low gelling temperature agarose suspension in Earls Balanced Salt Solution is
prepared. Autoclaved agarose is cooled to 37°C followed by swirling. Using 3 ml Pasteur pipette cell-
agarose solution poured into holes of mould which is then placed into petri dish and gel at 4°C for 45
minutes. Ready specimen is removed with pipette tip. Petridishes were labelled with date of
preparation, concentration of agarose,day0 and day7. At completion of day7, specimens with cell
density of 4 x 10⁶cell/ml with 4 % and 6% agarose type IX were assessed for mechanicalproperties at
day7 and total GAG synthesis at day0 and day 7.
Mechanical Testing:
Well prepared constructs were mounted on plate of loading MTS machine configured with crosshead
speed of 0.0167mm/second to compression of 20 %. Construct were hydrated with EBSS. Crosshead
is moved and load data acquired at 10 Hz during compression phase and 1 Hz during relaxation phase.
Precaution has been taken in order to avoid viscoelastic effect of specimens as each specimen was
tested only once. For data analysis machine interfaced with Student-t-test ANOVA (analysis of
variance). So displacement was converted into stress and strain percentage values and using resulted
graph of stress-strain the mean 15% Tangent Modului and standard deviations have been calculated.
Total GAG determination:
Series of standard solution between 0 to 50 ug/ml is prepared using set of Eppindorf tubes labelled 1
to 11.Volume of standard from 0 to 100ul and volume of water from 200 to 100ul were poured as per
GAG standard table. 96- well plate pattern is filled with 40ul of standard by following plate layout
given for standard. 250 ul of DMB (1-9-dimethylmethylene blue) reagent added to wells containing
standard. Plate transferred to plate reader and with Ascent software typical standard curve for
concentration versus absorbance was obtained. Using standard curve, absolute concentration of
sulphated GAG( ug/ml) is calculated. For statistical analysis, graph of mean GAG (ug/ml) versus
culture period ( day 0 or day 7) plotted and statistical difference between day 0 and day 7 for 4 % and
6 % gel was calculated.
7. 7
Effect of mechanical loading :
Loading data is recorded at 10 Hz in compression phase and 1 Hz in relaxation phase. Excel data
sheet is prepared with extension (mm) and load data (N) for each specimen and stress-strain
relationship is plotted as illustrated in following figure.
Fig 2: Representative stress-strain curves obtained for 4% and 6% agarose chondrocyte
constructs subjected to uniaxial compression of20% strain and load data is recorded using 50
N load cell
It is observed that constructs with 6 % at day 7 shows highest peak value than other constructs. It
means that it exhibits more strength than other samples. Following table illustrates that modulus of
elasticity has significantly improved in constructs with 6% concentration from day 0 to day 7.
Table 5 : Tangent modulus calculated for specimens cultured in 4% and 6% agarose for a
period of7 days subjected to compressive load of20 % strain.
This data clearly explains the influence of mechanical loading on agarose-chondrocyte model.
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
0 5 10 15 20 25
Stress(N)
Percentage Strain (%)
Representative graphfor the incorporation of compressive
dynamic loadingconfiguredfor 20% strain subjectedto
chondrocyte constructs culturedin agarose mediumfor 7 days
day 0 (4%)
Day7 (4%)
Day 7 (6%)
Day0 (6%)
Day 0 (4%) Day0 (6%) Day7(4%) Day 7 (6%)
Tangent Modulus(kPa) 636.94 283.08 254.75 849.44
8. 8
Discussion
The current study explores the enhancements of matrix synthesis and mechanical properties of
chondrocytes seeded agarose- hydrogel model cultured for 7 days and with application of dynamic
loading within physiological range. Frequent dynamic loading has a key role in maintaining cartilage
thickness as well as normal cartilage integrity. A number of previous studies published by authors
have demonstrated that compressive strain applied in static or dynamic manner will affect metabolic
responses of chondrocytes cultured for in agarose constructs. The three parameters were investigated
namely the synthesis of glycosaminoglycan, the DNA and protein which were influenced by strain
regimens during the experiment performed to examine influence of mechanical loading on
signalling mechanism in chondrocytes[6]. In current study, we prefer to use agarose-chondrocyte
model on behalf of its simplicity, reproducibility and capacity to apply physiological levels of all
strain which make ideal system for investigating effect of compressive strain on chondrocytes.
Agarose hydrogels are suitable to provide a three dimensional environment to investigate cell
mechanobiology[19]. Sample size is maintained between 5mm height and 5mm diameter in order to
assure diffusion of nutrients to the centre and thus influence the viability. In order to maintain the
viability of cell , cell concentration and incubation time are critically important. During the initial
period there is no significance difference in the glycosaminoglycans synthesis by two constructs with
different agarose concentration , 4% and 6% respectively. But with the culture period of 7 days ,
construct with 4% agarose found to synthesise more amount of glycosaminoglycan than construct
with 6%. This result suggest that culture conditions will have influence on the GAG synthesis and in
turn chondrocyte metabolic activities. This result resemblance with previously done experiment on
chondrocyte-seeded agarose hydrogels to examine effect of temperature and concentration of gel on
tissue development. It was found that by 28 day of culture period, 2% chondrocyte seeded agarose
hydrogels showed higher value of young’s modulus than 3% chondrocyte hydrogels. But the result
related to GAG synthesis were in contrast to our findings. In agreement with reports of the other
authors ,gel concentration has significant influence on GAG synthesis as demonstrated by Conor
Buckley. Chondrocytes seeded in different gel concentrations of 2%, 4% and 6% shown to have
different amount of GAG synthesis and thus exhibit different mechanical properties[6].
Mechanical characterization of the constructs demonstrated that stiffness of the constructs were
enhanced by the increasing concentration of the constructs. This conclusion suggest a link between
content of the matrix and the distribution of the nutrients within the constructs. It has found that
stiffness of the resultant sample increases with increase in the concentration. These current findings
are consistent with the previous studies reporting cyclic tensile strain leads to the downregulation of
the catabolic genes. This results in the suppression of nitric oxide (NO) and prostaglandin E2 (PGE2)
levels in the tissue. Ultimately, increase in the agrrecan and collagen type II synthesis and GAG
synthesis was noticed[7,8]. Positive impact of the strain on cartilage also supported by the studies on
hamster by Julkunen concluded about the long term effects of the applied mechanical load
.ArticularCartilage samples(n=191) were harvested from tibia of hamsters of different ages subjected
to physical activities such as running and some group to sedentary activities[9]. It suggested that
physical exercises at the young age enhance the cartilage maturation and alters the depth-wise
cartilage structure and composition which can be beneficial. But exercises at young age demonstrated
adverse effects on cartilage at a later age with increase in osteoarthritis.
However,cartilage damage occurs if it subjected to injurious or impact loading. Previous studies
reported that it leads to increased fibronectin synthesis, MMP-3 gene expression which in turn result
in collagen damage and proteoglycan breakdown in cartilage explants. Excess loading may also result
in upregulation of inducible nitric oxide synthesis (Inos) and cyclo-oxygenase-2(cox-2) enzymes. It
9. 9
forms increased cytokine production and ROS production. Finally, it leads to apoptis of the
cartilage[5,6]. Experiment performed to examine role of cytoskeleton in viscoelastic properties of
human articular chondrocytes concluded that static compression exhibit disruption of the golgi
apparatus and cytoskeletal proteins which associated with loss of mechanical strength with reduction
in collagen gene II expression[1].
As a part of chondrocyte metabolism, oxygen tension is a important parameter in order to determine
the nutrient concentration within the tissue. Oxygen tension has influence on the endogeneous
production of NO and PEG2 and significantly affect the induction of mediators in response to
mechanical compression. Composition of culture medium and method influence the oxygen
consumption by the articular cartilage. The previous study on isolated articular cartilages to examine
the effect of medium osmolarity, concentration of glucose in medium on oxygen consumption by
chondrocytes published by David Lee illustrates the precise example. It was found that medium
osmolarity has less effect on the oxygen consumption[20]. However,it was noticed that oxygen
consumption was sensitive to glucose concentration in the medium. Upregulation of the oxygen is
associated with the inhibition of glycolysis is in correlation with the Crabtree effect. It describes that
chondrocytes exhibit oxidative energy metabolism to provide basic ATP demands from limited
glucose supply. Thus , oxygen consumption by cells results in gradients in the oxygen concentration.
In order to determine the oxygen gradient in the construct, mathematical model was developed which
measures diffusion coefficient of cartilage, water and construct. Model concluded that polymer
constructs cultured for period of 27 days have low oxygen requirements than chondrocytes in the
native tissue.Recent studies have shown a beneficial effect of low oxygen tension mimicking in vivo
environment. Low oxygen culture has been recognised as a effective means to controloxidative stress
and to increase the proliferation potential and biosynthesis of chondrocytes. Hence chondrocytes
cultured under low oxygen tension (usually 5% O2) better maintain their phenotype and synthesize
more cartilage-specific molecules compared with chondrocytes cultured under normoxic
conditions[21]. Thus, combination of low oxygen tension and mechanical loading would be the useful
tool to preserve the chondrocytic phenotype and to enhance matrix production with adequate nutrient
supply.
Safe and reproducible production of tissue engineered constructs requires the efficient bioreactor
system. Bioreactors are important to maintain engineering aspects such as medium perfusion, uniform
and efficient mixing, shear stress,gravitational effect,reliability, reproducibility,scalability as well as
biological aspects such as sterility and aseptic conditions, oxygen tension and perfusion through
culture medium, consequent addition of fresh medium and removal of waste products. Perfusion is the
important factor in designing of bioreactor to apply mechanical stimuli to cell to ensure that all cell
cultured in 3D structures receive constant supply of nutrients and that waste products are removed
satisfactorily. According to previously published by David Lee ,bioreactor was developed to perfuse
fluid through the cylindrical constructs. To determine compatibility of bioreactor for transport
enhancement, transport and distribution of dextran molecules (FD-4,FD-500,FD-2000) within the
agarose were investigated. Small culture dishes such as petri dishes,12-well plates,T- flasks are used
for the purpose of cell expansion. These devices increase cell number by a factor of 10 which
demands more severe subcultivation[23]. Recent studies have shown that microcarrier cultures
performed in well- mixed bioreactor systems can significantly increase the cell expansion[24].
Bioreactors allow for different process strategies including the batch, fed-batch or continuous
cultivation. On the other hand, a bioreactor system becomes more complex as additional features such
as feeding pumps, vessels for fresh and spent medium, and control strategies are required particularly
in the case of mechanical stimulation. The bioreactor system has to be integrated into the entire
10. 10
cultivation scheme including biopsy, proliferation, cell seeding, tissue formation and delivery to the
site of application (e.g.,hospital). In many cases,the bioreactor itself is only used for tissue
formation. In many cases,the bioreactor itself is only used for tissue formation. However,for a
wholistic approach from biopsy to the implantation of tissue, the entire procedure should be
coordinated to decrease the number of steps, risk of contamination, and labour costs among others.
This is particularly important with respect to the manufacture of engineered tissue constructs for
clinical applications, in which good manufacturing practice (GMP) requirements have to be met[25].
In many cases,the culture systems and bioreactors were not optimized in this respect. Parameterssuch
as perfusion rate,flow conditions, shear stress,and compression magnitude were varied, quite often
by a trial-and-error approach. Furthermore, different conditions have to be examined accurately with
regard to their effect. For example, hydrostatic pressure applied during cartilage culture can lead to an
improved mass transfer of small and large molecules into the cartilage matrix, but can also induce a
mechanical stimulation of embedded cells. These two effects have to be examined separately.
Incorporation of biodegradable material also holds a set of benefits to the tissue engineered construct.
For example, biodegradable polymer holds advantage of porosity, hydrophobicity, degradation time,
improved mechanical properties which can be varied accordingly to the requirement of diverse
clinical application.
In summary, the efficient approach with improved culture parameters accompanied by the well
developed bioreactor design will lead the aim perfect tissue engineered construct which would match
native tissue criteria.
11. 11
References
1. M.O. Jortikka,J.J.Parkkinen,R.I.Inkinen et al., “ The role of microtubules in the regulation of
proteoglycan synthesis in chondrocytes under hydrostatic pressure”, Archives of Biochemistry
and biophysics,vol374,pp-172-180,2000
2. W.R.Trickey,T.P.Vail,F.Guilak, “The role of cytoskeleton in the viscoelastic properties of
human articular chondrocytes”, Journal Orthopaedic research,vol22,pp 131-139,2004
3. M.Lotz,Hashimoto and K.Kuhn, “ Mechanism of chindrocytes apoptis”, Osteoarthritis and
cartilage,vol7,pp 389-391,1999
4. Y.Henrotin,B.kurz and T.Aigner, “Oxygen and reactive oxygen species in cartilage
degradation: friends and foes?”,osteoarthritis and cartilage,vol13, pp 643-654,2005
5. P.M.Lin,C.T.C. Chen, “Increased stromelysin-1(MMP-3),proteoglycan degradation and
collagen damage in cyclically load-injured articular cartilage”,osteoarthritis and
cartilage,vol12,pp 485-496,2004
6. J. Steinmeyer and B. Ackermann,“ Effect of continuously applied cyclic mechanical loading
on the fibronectin metabolism of articular cartilage explants” , Research in Experimental
medicine,vol 198, pp 247-260,1999
7. R. Gassner,M.J. Buckley, S. Agarwal,“Cyclic tensile strain acts as an antagonist of IL-1 beta
actions in chondrocytes”,Journal of immunology,vol-165,pp 453-460,2000
8. P.Long, R. Gassner and S .Agarwal, “ Tumor necrosis factor alpha –dependent
proinflammatory gene induction is inhibited by cyclic tensile strain in articular chondrocytes
in vitro”,Arthritis and rheumatism, vol44, 2311-2319,2001.
9. Julkunen.P,Halmesmaki. EP,“ Effect of growth and exercise on composition , structural
maturation and appearance of osteoarthritis in articular cartilage of hamster”, Journal of
Anatomy,vol 14, 2010
10. Fermor.B,Weinberg.JB, “ Influence of oxygen tension on the induction of nitric oxide and
prostaglandin E2 by mechanical stress in articular cartilage, Osteoarthritis and cartilage,
vol12,pp 145-153, 2000
11. David Lee,Dan Bader,“ Nutrient utilisation by Bovine articular cartilage: A combine
experiment and theory”, Journal of biomechanical engineering
12. Otte.P,“ Basic cell metabolism of articular cartilage”,1991
13. Razaq.S, Wikin R.J and Urban , “ The effect of extracellular pH on matrix turnover by cells
of Bovine nucleus pilposus”, 2003
14. Martin .I,Obradovic.B, “ Method for Quantitative Analysis of GAG distribution in cultured
Natural and Engineered cartilage”, 1999
15. Omen CW , Senqeurs. BG. “ Computational study of culture conditions and nutrient supply in
cartilage tissue engineering” , Biotechnology progress,2005
16. Otsuki.S, Brinson DC, “ Effect of GAG loss on chondrocyte viability”, Arthritis and
rheumatism,2008
17. Pamela Hebert,Stephen .B.P,“ Selected loss of viability of mouse NK cells in culture”,in-
vitro and molecular toxicology, 2001
18. Boon Chin Heng, Abdul Jalil, E.H.Lee,“Loss of viability on freeze”, Jounal of Biomedical
Science,2006
19. Conor Bucklet, Stephen Thope, “ Effect of concentration , thermal history and cell seeding
density on the initial mechanical properties of agarose hydrogels”, Journal of mechanical
behaviourof Biomedical material, vol-2, 2009
20. D .A .Lee,Dan Blader, “ Rate of Oxygen consumption by isolated chondrocyte is sensitive to
medium glucose concentration”, Journal of cellular physiology
12. 12
21. Kurz, “ Tissue engineering of articular cartilageunder influence of collagen I/III membrane
and low oxygen tension”, Tissue Engineering vol-10,pp 1277-1280,2004
22. J. Malda, J .Riesle, “ O 2 gradient in Tissue engineered pegt/ pbt cartilaginous construct:
measurement and modelling”, Biotechnology and bioengineering,vol-86, 2004
23. J. Malda, P. Meeuwse ,“ Effect of oxygen tension on adult articular chondrocyte in
microcarrier bioreactor culture”, Tissue Engineering,vol10, pp 987-994, 2001
24. C. Bardouille, J. Lehmann, “ Growth and differentiation of permanent and secondary mouse
myogenic cell lines in microcarriers”, Application in Microbiology and Biotechnology,vol-
55,pp 556-562, 2001
25. G.K. Naughton, “ From Lab bench to market: critical issues in Tissue Engineering”, Annual
Acad. Sci.(961). pp 372-385, 2002
26. Hannah .K.H. , Dan Blader, “ glucose availability regulates oxygentension within tissue
engineered constructs”, Journal of cellular physiology,vol-206, pp 402-410, 2006
27. M.M.Knight, D.A.Lee,“ Activation of chondrocyte Calcium signalling by dynamic
compression is indepent of number of cycles”,2005
13. 13
Abstract
Cartilage is an avascular, alymphatic and aneural tissue in the body.It consists of a extracellular
matrix in which chondrocytes are dispersed at low densities. It plays an important role in maintaining
shape and giving support to body structure. It is necessary to protect the ends of bones from rubbing
together. Damage of these cartilage caused by direct blow , stress on joint for prolonged period or lack
of movement of joint. Deep cartilage defects can be treated by autologous cell transplantation. For
this, biopsy is collected from the outside the affected area. Chondrocytes are liberated from the matrix
by an enzymatic treatment and allowed to grow in cell culture. The cells are then harvested and
introduced in affected area. Main objective of the study is to examine the mechanical and biochemical
properties under various culture environment . Different concentrations of agarose gel used to culture
chondrocytes and hence their mechanical properties are noted. Strength of the engineered tissue can
be determined by measurement of total amount of GAG released by chondrocytes cultured within
agarose constructs .Stress and strain analysis help to develop the final model for cartilage tissue.
Analysis of the cell constructs shows that total GAG synthesis is more for a 4% concentration of
agarose gel at day 7. It has explained the detail in further discussion how it affect the mechanical
strength of the tissue. In mechanical loading test , graphs has been plotted for dynamic loading
applied to the cell constructs it is found as the concentration increases; the peak load stress achieved
by the sample shows lower value. This journal report describes the methods of isolation of cells, their
maintenance in cell culture and factors influencing the mechanical properties and mention ideas to
improve further nutrient utilization with maximum signalling events in chondrocyte/ agarose
constructs.
![2
INTRODUCTION
Cartilage is a flexible connective tissue observed in bodies of human being and other animals. It is
the fundamental part of body which contributes to shape, strength and protection of bone in body.
Cartilage is composed of specialized cells called chondrocytes which shows different properties in
three different types of cartilage.
Table1: Types of cartilage with different location
Cartilage Type Location
Hyaline
Cartilage
Joints, rib cartilage, nose, trachea and larynx
Elastic
Cartilage
Ear, epiglottis, and larynx
Fibrous
Cartilage
Intervertebrataldiscs.
Amount of water content in cartilage varies with age, 90 per cent in young and decreases to 65 -75 per
cent in adult with the aging.
Collagen occupies 50 per cent of dry weight of cartilage. It composed of 3 aplha-I chains and differ
genetically from collagen of skin and bone. Its concentration is high in superficial zone and low in the
base of radial zone[28].
Cartilage proteoglycan composed of different concentration of glycosaminoglycans, chondroitin
sulphates 4 and 6, and keratin sulphate ,attached to protein core and aggregated on hyaluronic acid
molecules to form a proteoglycan aggregate of high molecular weight capable of binding larger
quantities of water and occupying large spatial volume. Concentration of proteoglycan is less in
superficial and higher in deeper zones[28].
Cell signalling:
It is molecular mechanism by which cell communicate. It is initiated by generation of ligand ;a
molecular entity generated by sending cell to bring about change in physiology of responding cell.
Multiple growth factors and hormones involves in activation of variety of signal transduction pathway
such as BMP/ TGF β family, Wnt family, Indian Hedgehog (IHh), Parathyroid Hormone related
protein( PTHLH),Fibroblast growth factor (FGF). TGF β invariantly used in chondrogenic culture
media inhibits chondrocyte differentiation and simultaneously stimulates production of chondrogenic
extracellular matrix. FGF2 is also potent inhibitor of chondrogenic proliferation[29].
In pathophysiology, destruction of articular cartilage is induced by wide array of cytokines expressed
by mononuclear cells and chondrocytes. These cytokines induce expression of matrix
metalloproteases and activate osteoblasts, both of which stimulate matrix decay.
Evaluation of cell signalling pathway involved in formation articular cartilage wiil be advantageous
for cartilage tissue engineering. It will allow to use such strategies to recapitulate events during
implantation.
To improve the survival of implanted construct, it is necessary to focus on the nutrient supply to
engineered tissues. Cells within the engineered tissue all rely on the supply of nutrients and transport
of waste products mainly by diffusion. Insufficient nutrient to the centre of the construct leads to loss](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)