The State-Of-The-Art in Cartilage Bioreactors

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CHAPTER IV

THE STATE-OF-THE-ART IN CARTILAGE BIOREACTORS

L.A. McMahona, V. Barronb, A. Prina-Melloa,c, P.J. Prendergasta

a Centre for Bioengineering, Department of Mechanical and Manufacturing Engineering, Trinity College, Dublin, Ireland. b National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland. cScience Foundation Ireland Nanoscience Laboratory, Trinity College, Dublin, Ireland.

Articular cartilage has a limited capacity for self-regeneration and none of the current treatments or therapies that are used to rectify damaged cartilage are ideal or permanent. As a result, researchers are looking to tissue engineering to grow constructs in vitro with the view of implanting them into joints to replace damaged tissue. This approach would omit disadvantages associated with current treatments such as taking allografts from less weight bearing regions to be inserted into the defect or penetrating the subchondral bone to allow stem cells to migrate into the damaged area. Approaches used in cartilage tissue engineering involve the application of mechanical stimuli to cells to emulate the forces experienced by cells in vivo. This review analyses the present situation in current state-of-the-art bioreactors and looks at the various cell types and substrates used in such studies.

1. Introduction

Tissue engineering is currently one of the most promising areas of biomedical research for the regeneration of repair tissue in vitro. The goal of tissue engineering is to assist the body in producing a material that closely matches native tissue (7). At present the only successfully engineered tissue is skin. It is believed that, due to the relatively simple composition and structure of cartilage, it will be the next major tissue type to be marketed. Attempts have been made since the late 1960s to grow new cartilage from implants of isolated, living chondrocytes (20). P.J. Prendergast and P.E. McHugh (Eds.), Topics in Bio-Mechanical Engineering, pp. 94-146.© 2004 Trinity Centre for Bioengineering & National Centre for Biomedical Engineering Science.

Cartilage Bioreactors 95

Chondrocytes are responsible for the synthesis and maintenance of the extracellular matrix (ECM) of cartilage, with the primary constituents of the ECM being type II collagen and proteoglycan macromolecules. Collagen provides a high tensile strength to the matrix while proteoglycans entrap water and resist compression (21, 31). Cartilage is avascular, alymphatic and has the lowest cellular density of any tissue in the body with less than 5% cells by volume (19). There are four zones in articular cartilage (see Fig. 1). In the superficial layer (zone 1) flat chondrocytes and collagen fibres are arranged tangentially to the articular surface compared to the deep zone (zone 3) where collagen fibres and chondrons (clusters of chondrocytes) are aligned perpendicular to the subchondral plate (31).

Articular cartilage has limited reparative abilities. As a result, cartilage that is damaged due to degenerative diseases like osteoarthritis or sporting injuries is unable to regenerate and form new tissue and can ultimately lead to total joint dysfunction and severe pain. Generally, articular cartilage defects are classified as chondral (partial thickness) or osteochondral (full-thickness). Chondral defects do not penetrate the subchondral bone whereas osteochondral defects penetrate the subchondral bone, accessing vascular supplies. Current therapies and treatments range from and include; (i) inserting plugs of autografts taken from a less weight bearing region of the joint or using allograft plugs, (ii) using periosteal tissue where undifferentiated cells are induced to form

Fig. 1. Zonal organisation in articular cartilage. Collagen fibres are arranged tangentially to the articular surface and perpendicular to the subchondral bone (39).

Topics in Bio-Mechanical Engineering 96

chondrocytes and new cartilaginous tissue, (iii) transplanting extra chondrocytes into the defect and (iv) penetrating the subchondral bone by drilling to allow mesenchymal stem cells (MSCs) to migrate into the defect where they will differentiate and form new tissue (50). Presently, none of these techniques are ideal and are not highly successful. For example, the use of an autograft may require a second incision and may result in morbidity at the donor site while MSCs can differentiate in the defect to form hyaline cartilage but the mechanical properties and durability of new tissue are lower than native tissue (50). It is now believed that the application of mechanical stimuli to cells in vitro, which emulate the forces applied to tissue, will lead to the production of functional tissues (31). Mechanical stresses are an important factor of chondrocyte function as they stimulate them to increase the synthesis of ECM components. In vivo studies have reported that reduced synthesis of proteoglycans results in the degeneration of cartilage and, ultimately, joint immobilisation (20). This is due to a reduction in loading of the articular cartilage illustrating the need for mechanical forces to modulate the biochemical and biomechanical properties of the tissue (7). Many studies have shown that mechanical forces can be used to stimulate the synthesis of cartilage ECM, and may even enhance the mechanical properties of the developing tissue (45). In general, the composition, morphology and mechanical properties of articular cartilage appear more superior when grown with the application of mechanical stimuli compared to tissue grown under static conditions (31, 57). However, there is a fundamental lack of understanding as to what mechanical stimuli are necessary to promote regeneration and how such mechanical signals are interpreted at cellular level (45). Some possible mechanisms involved in the transduction of mechanical compression are listed in Table 1.

It still remains uncertain as to how cells transduce mechanical signals to the nucleus where specific genes are expressed (2). In the work of Bachrach et al. (2) it has been proposed that (i) the mechanosensitive cells transduce applied forces into biochemical signals via membrane-bound receptors or (ii) that they directly transmit stress from the ECM to intracellular structures. Both methods indicate that the chondrocytes respond to mechanical events at a local level around the cell.


Combinations of mechanical stresses are simultaneously developed during joint motion on an intermittent basis which includes cell and tissue deformation, compressive and shear force, fluid flow and changes in hydrostatic pressure. In cartilage culturing processes the main types of mechanical forces currently being investigated are:

1. hydrostatic pressure (1, 6, 20, 22, 42), 2. direct compression (3, 4, 8, 9, 11, 21, 28, 32, 36, 37, 49, 51), 3. shear environments (12, 13, 14, 16, 19, 23, 24, 25, 29, 34, 35, 36,

40, 43, 47, 48, 50, 54, 57). In order to study such forces in a 3D environment many research

studies have used biodegradable scaffolds to provide a platform to enhance matrix synthesis, deposition and organisation of a 3D cartilage construct (31). Thus, to achieve a successful 3D construct it has been investigated that an engineered cartilage implant of 5 cm in diameter and 1-5 mm thick would be needed to resurface an entire joint (50).

2. Cell Types

Several cell types that are used in tissue engineering studies are presented in this section. Chondrocytes represent about 5% of the volume of articular cartilage (19). They are essential to the tissue as they replace

Table 1. Mechanisms involved in the transduction of mechanical compression at cellular level (11, 54).

Static Compression Dynamic Compression

Cell/Tissue deformation (shape and volume) Cell/Tissue deformation Transport–related (e.g. diffusion) Altered fluid pressure Physicochemical (e.g. pH, osmotic pressure) Enhanced fluid flow (transport) Cell-matrix interaction Cell-matrix interactions Induced streaming potentials Growth factors released from

matrix binding sites or by cells


degraded matrix molecules such as collagen and proteoglycans to maintain the properties associated with the ECM of cartilage. For the purpose of tissue engineering, chondrocytes can be harvested from articular cartilage, digested and amplified through well known methods. Growth factors can be used to improve cell multiplication, redifferentiation of chondrocytes after they have differentiated during monolayer expansion, and in the production of the ECM (56). Growth factors that are used in cartilage tissue engineering studies include insulin-like growth factor I (IGF-I), transforming growth factor β (TGF- β) and fibroblast growth factor-2 (FGF-2) (56). However, it is a limited tissue in the body and with its poor capacity to repair in vivo it is not the most feasible method of obtaining such cells.

2.1 Human and Animal Articular Chondrocytes

In studies involving human articular chondrocytes, the cells are harvested from patients who have undergone knee reconstruction or from cadavers (9, 22). Chondrocytes obtained from such biopsies have a limited proliferative potential and the number of cell divisions they undergo in vitro decreases with age (5). Due to this limited availability of human chondrocytes, many research studies have been carried out using animal cells; for example, bovine and ovine cells. As with human chondrocytes, it has been discovered that chondrocytes from younger animals have an increased ability to synthesise ECM components than mature chondrocytes (1). This may be due to young cartilage having a high concentration of TGF-β, which accelerates the synthesis of the ECM (31).

2.2 Cells from Non-Articular Hyaline Cartilage

Non-articular hyaline cartilage such as nasal or rib cartilage can also yield differentiated chondrocytes. The removal procedure of such tissue from a donor would not be as invasive as to remove tissue from load bearing areas of a joint. Such tissues are not subjected to compressive forces so morbidity at the biopsy site is reduced. Kafienah et al. [25]


discovered that nasal chondrocytes proliferate around four times faster than articular chondrocytes, indicating that potentially large numbers of cells could be generated to engineer large pieces of cartilage. However, the applicability of such cell types has been questioned for load bearing cartilage repair (5).

2.3 Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) reside in bone marrow and have the ability to differentiate into various mesenchymal phenotypes such as chondrogenic and osteogenic cell types. The number of mesenchymal stem cells in fresh adult bone marrow is only around one per one-hundred-thousand nucleated cells, with this concentration decreasing with donor age. Cell culture technology is employed to isolate and mitotically expand MSCs without sacrificing their ability to differentiate regardless of donor age (41). Mitosis is a process that involves the division of the cell nucleus producing two identical daughter cells, which are identical to the original parent cells. MSCs have a higher proliferative capacity than cells from adult tissue and have the potential to be a promising source of cells for tissue engineering. The differentiation of these cells into the chondrogenic or osteogenic lineages has been performed in vitro when supplied with the correct differentiation signals from surrounding matrix and specific growth factors (54). Many studies have looked at the influences of bioactive factors such as dexamethasone, transforming growth factor-β (TGF-β) and bone morphogenetic proteins (BMPs) on MSC differentiation and proliferation, but few studies have examined the effect of mechanical factors on these cells (1, 41). Several mechanoregulation theories for the prediction of tissue differentiation have been proposed by researchers which outline that mechanical environments to which the MSCs are exposed determine the resulting tissue phenotype. For example, Pauwels (27, 59) proposed that deformation of the cell by strain or shear would result in fibrous tissue formation, compression of the cell by hydrostatic pressure would result in hyaline cartilage formation, and a combination of these stimuli would result in fibrocartilage. This hypothesis also outlined that the formation


of fibrous tissue is necessary before ossification and the formation of bone can occur.

3. Scaffolds

3.1 Monolayer Studies

Monolayer studies involving mechanical stimuli provide information on how the chondrocytes react and synthesise ECM under controlled conditions. However, when chondrocytes are cultured two-dimensionally they can dedifferentiate and take on a flattened spindle-like appearance, and produce type I collagen instead of type II. When seeded in three-dimensional scaffolds chondrocytes maintain their differentiated phenotype and function (50). Cells that dedifferentiate in monolayer have the capacity to redifferentiate when transferred to a 3D environment. Growth factors such as prostaglandin (F2α) and TGF-β1 can be added to the medium to enhance redifferentiation and deposition of cartilaginous matrix (9). Van Osch et al. (55) investigated the effects of TGF-β on chondrocytes that were at different stages of differentiation. Three cell populations were used in this study: (i) freshly isolated chondrocytes, (ii) partially dedifferentiated chondrocytes (after one passage in monolayer) and (iii) dedifferentiated chondrocytes (after four passages in monolayer). These cells were encapsulated in alginate gel and the aim of the study was to test the hypothesis that the effects of TGF-β depended on the differentiation stage of the chondrocytes. However, van Osch et al. (55) found that the freshly isolated chondrocytes, as well as the dedifferentiated cells, responded to the TGF-β and produced GAG. The study concluded that the effect of TGF-β on chondrocytes depended on the presence of an ECM.

3.2 Cartilage Explants

The use of cartilage explants is an approach that researchers have taken to obtain scientific knowledge regarding the regulation of cartilage metabolism during the application of mechanical stimuli. Explant


studies provide information on the behaviour of chondrocytes in their native ECM and gives an insight into normal cartilage homeostasis (45).

3.3 Three Dimensional Scaffolds

The use of three-dimensional scaffolds in the engineering of tissues is widely researched. Cells are seeded on to the scaffold and it acts as a temporary physical matrix to which the cells adhere. An ideal scaffold is three-dimensional, biodegradable, non-toxic, biocompatible, non-immunogenic and easy to manufacture (5). The designed scaffold should ideally have a high porosity, a good interconnected pore network and a high permeability to permit the diffusion of nutrients and wastes. In addition to this, the pore size should be in the range of 50-500 µm to facilitate the seeding and ingrowth of cells and the formation of tissue (26). As the cells synthesize components of the cartilaginous matrix, the biomaterial of the scaffold degrades to allow space for the developing ECM. The mechanical properties of the scaffold should be comparable to those of native cartilage to provide a replicate environment for the cells to synthesize and organise the ECM (31). The mechanical properties of the cell-scaffold construct should display similar biomechanical properties to those of the host site at the time of implantation. When cells are cultured under conditions that resemble an in vivo environment they retain their differentiated phenotype (57). Natural hydrogels such as fibrin, collagen gels and alginates are used to contain or immobilise cell suspensions to maintain the shape of the cells and to reduce dedifferentiation of chondrocytes into fibroblasts (5, 18).

Seeding density has a significant effect on the formation of a tissue. Seeding density must be high enough to allow for cell-cell and cell-matrix interactions, which play a role in the production of ECM (7). An important consideration is that not all cells attach to the scaffold during initial seeding. It has been reported by LeBaron et al. (31) that the formation of a cartilaginous material appears to be enhanced as the cell seeding density increases. Similarly, Mauck et al. (37) reports that studies have generally found an increase in glycosaminoglycans (GAG) content with an increasing cell seeding density, which indicates that a high seeding density is desirable. However, this density must not be too


high so that the mass transfer of nutrients and wastes is affected. The development of a homogeneous tissue from the lowest possible cell concentration relies on a method of uniform, efficient seeding (36). The most common seeding techniques that have been employed in studies to date include (i) static seeding and (ii) dynamic seeding involving mechanically stirred flasks. The aim of all seeding techniques is to achieve good seeding efficiencies along with a uniform distribution of cells throughout the scaffold. Recently, Wendt et al. (60) developed an oscillating perfusion technique which involves oscillating fluid containing a cell suspension through the scaffold. A study was conducted which compared this new technique to the static and stirred flask techniques. The perfusion technique shows that seeding efficiencies of up to 90% were achieved along with a good uniform distribution of cells through out the scaffold, with results showing a vast improvement on the two older techniques (60).

Various scaffolds have been used for tissue engineering approaches and these include naturally derived and synthetic polymers (50). The most commonly used natural polymer is collagen. It is reported that collagen matrices have molecular cues that can stimulate cells to synthesise collagen (50). Collagen type I is a widely available fibrillar protein, and at neutral pH and physiological temperatures, it polymerises into a stable gel. When chondrocytes are cultured in such gels, they have been shown to secrete cartilage-specific matrix components, that is, collagen type II and proteoglycans (21).

Synthetic polymers that are biodegradable and have been approved by the FDA are poly(glycolic acid) (PGA), poly(L-lactic acid) (PLLA) and their copolymer, poly (DL-lactic co-glycolic acid (PLGA) (18, 38, 50). These polymers can be sterilised to prevent infection and their degradation rates can be tailored for specific applications (38). For example, Ethisorb is a copolymer of PGA/PLLA and has a degradation rate of 3 weeks in vitro while the degradation rate of pure PLLA is 9 months (44). These degradation rates can be controlled by the amount of copolymer introduced (PGA). Scaffolds composed from poly(ethylene glycol terephtalate) (PEGT) and poly(butylenes terephtalte) (PBT) have also been used as three-dimensional constructs for chondrocytes (9). However it is difficult to compare results obtained by different


researchers as it is likely that scaffolds with different pore sizes and architectures would transduce physical signals in different ways to cells.

It is possible to modify the surface of scaffolds to promote cell adhesion, proliferation or differentiation using growth factors or supplements. For instance, Sittinger et al. (47) investigated the attachment of human articular chondrocytes to polymer meshes using various factors which included poly-L-lysine, fibronectin, collagen type II and vitronectin. Results indicated that poly-L-lysine was the most effective adhesion factor, followed by collagen type II. Tsuchiya et al. (52) carried out a more comprehensive study investigating the effects of various cell adhesion molecules on the initial cell adhesion properties of articular chondrocytes, ligament cells and MSCs. Cells were cultured for 30 minutes in culture plates coated with the different cell adhesion molecules and consequentially, the number of adhered cells was determined. This study found that type I collagen, type II collagen, fibronectin, vitronectin and poly-L-lysine promoted significantly more cell adhesion than laminin, chondroitin-4-sulfate, chondroitin-6-sulfate, aggrecan and hyaluronic acid. In all cell type groups investigated, fibronectin proved to have the most potential for cell adhesion and could be used to coat porous 3D scaffolds in tissue engineering applications.

4. Biochemical and Mechanical Indicators

Biochemical techniques are used to obtain information on the amount of proteoglycan and collagen type II found in cartilage as these are often used as experimental indicators of cartilage synthesis. Proteoglycans are molecules that are composed of polysaccharide structures called glycosaminoglycans (GAG) covalently attached to a protein core. Comparisons between studies are difficult as different studies use different methods of evaluating the histological content of engineered cartilage. Methods of evaluating cartilage content include radiolabel incorporation ([35S]sulfate and [3H]proline as measures of proteoglycan and protein synthesis), wet weight and dry weight fractions, mRNA signals and reverse transcriptase-polymerase chain reaction (RT-PCR) (4, 7, 21, 57, 61). Histology involves staining cross-sections of samples e.g. safranin O is used to stain for GAG, and then viewing the samples


under a microscope. Immunohistochemistry can be used to detect the expression of specific molecular markers (34).

In several studies the mechanical properties of constructs, as well as chemical analysis are examined to compare the structure and function of the engineered tissue to that of cartilage explants. Typical values for the mechanical properties and composition cartilage explants from 2-3 week old bovine calves are presented in Table 2. This comparison of engineered tissue to explants can be carried out by employing the following tests: equilibrium modulus, HA (MPa), dynamic stiffness, HD (MPa), hydraulic permeability, k (m4/N-s) x 1015 and streaming potential, V (mV/%) (7, 20). The equilibrium modulus can be calculated from confined chamber compression tests where samples are compressed in increments to a desired strain value. When cartilage is compressed and the applied strain is held constant (see Fig. 2) and the stress within the sample decreases exponentially. This stress reaches an equilibrium and the HA value is calculated using this equilibrium stress and the applied strain (35, 57).

Fig. 2 Stress relaxation response (39)


5. Mechanical Stimuli and Bioreactors

Many approaches are now being taken to develop articular cartilage in vitro through the use of mechanical stimuli and bioreactors, and these will be looked at in detail in the following sections. It has been shown that mechanical stimuli within physiological values can stimulate the synthesis of cartilage extracellular matrix to enhance the mechanical properties of the developing tissue (45). At present there are no standard evaluation techniques set out to allow researchers to determine the

Table 2. Typical mechanical properties and composition percentages found in bovine cartilage explants.

Native Cartilage (2-3 week old bovine calves)

Mechanical Behaviour

Aggregate Modulus (MPa) 0.949±0.021a

Hydraulic Permeability (x1015 m4/Ns) 2.72±0.641a

Dynamic Stiffness (MPa @ 1Hz) 16.8±1.14a

Construct Composition

Water (%) 82.8±0.93b

Cells content (%) wet weight dry weight

0.66±0.09b

3.79±0.50 GAG content (%) wet weight dry weight

7.05±0.56b

41.0±2.21 Total Collagen content (%) wet weight dry weight

10.7±0.91b

61.8±3.51 Type II collagen (%) of total collagen

90.3±17.9b

a – Vunjak-Novakovic et al. (57) b – Freed et al. (16)


quality of engineered constructs (7). This provides great difficulty when making comparisons among various studies. Another issue is that the ability of chondrocytes to proliferate and synthesise can vary depending on cell source, joint location and age (7).

Table 3 lists the mechanical stimuli that have been investigated by researchers to date. These stimuli have been individually looked at in experiments but recent studies have employed combined stimuli systems to benefit from the advantages of each individual stimulus.

Bioreactors should be capable of regulating the following parameters:

Temperature Medium pH Exchange of gases Mechanical stimuli pO2 pCO2 Humidity Easy to sterilise Manufactured from non toxic materials

Bioreactors can incorporate each of the above parameters into one system, but most studies involve placing an apparatus controlling the mechanical stimuli into an incubator which regulates the temperature, pH and gaseous exchange.

Perfusion is another important factor when designing bioreactors or systems to apply mechanical stimuli to cell to ensure that all cells cultured in monolayer or in 3D structures receive a constant supply of nutrients and that waste products are removed efficiently. A perfusion system should ideally be present in a system when long-term studies are carried out. Bioreactors without perfusion systems can successfully grow tissue but medium must be manually changed every few days and this increases the risk of contamination. A detailed account of the perfusion systems that have been employed to date is discussed in Section 5.5.1. It has also been suggested that the complex organisation of articular cartilage is not an intrinsic property of articular chondrocytes but may arise when constructs are subjected to physiological mechanical loading in vivo (25).


5.1 Compression

Articular cartilage continuously undergoes periodic loading from compression during everyday activities such as walking, running and standing. It has been proven that immobilised joints that are not subjected to such loading regimes loose mechanical integrity as the cartilage reduces the synthesis of proteoglycans and degrades (19). When the tissue is compressed a hydrostatic pressure gradient develops and the fluid primarily absorbs the shock. This fluid is forced out of the matrix, energy is dissipated and the load is cushioned by the ECM without damaging cells or matrix. Numerous studies have been performed investigating the effect of static and dynamic compression on cartilage synthesis.

Different loading configurations used in studies (confined or unconfined compression and/or permeable or impermeable platens) induce different strain amplitudes and fluid flows in constructs during static or dynamic compression (11). In unconfined compression the cartilage is placed between two platens and can bulge radially when compressed (see Fig. 3). Confined compression surrounds the cartilage on all sides but one, so that radial bulging does not occur. A permeable

Table 3. Types of Mechanical Stimuli used by various researchers in Cartilage Tissue Engineering.

Mechanical Stimulus Journal Reference

Static Compression 4, 11, 21, 28, 49*, 51

Dynamic Compression 3, 4, 8, 9, 11, 28, 32, 36, 37, 51

Tension 10, 17, 33, 46*, 53

Hydrostatic Pressure 1*, 6, 20, 22, 42

Strain-Induced Shear 13, 23, 24, 54

Fluid-Induced Shear 12, 14, 16, 19, 25, 29, 34, 35, 36, 40, 43, 47, 48, 50, 57

* = stem cells used in the study


platen is used to compress the sample so that fluid can escape during compression. Although in vivo strain levels are still unknown it is possible that unconfined compression tests result in larger matrix deformations than that experienced by cartilage in vivo (51).

5.1.1 Static Compression

Static compression of cartilage has been found to decrease cartilage-specific gene expression and matrix biosynthesis of proteoglycans and proteins, compared to dynamic compression (21, 28). This method of compression results in compaction of the solid matrix reducing pore size and fluid flow. Cartilage matrix is negatively charged and a decrease in hydration caused by compaction of the matrix increases the negative fixed charge density (FCD), attracts ions from the medium causing a decrease in intratissue pH and an increase in osmotic pressure of the intratissue fluid, and ultimately a decrease in biosynthesis (28, 51). The reduction in pore size as a result of tissue compaction may limit the

Fig. 3. Schematic illustrating a uniaxial loading chamber in unconfined compression (51).


diffusion or mass transport of nutrients, growth factors and wastes to and from cells in the matrix (see Fig. 4) (7, 28).

Static loading is reported to inhibit cell proliferation and matrix synthesis and this has been backed up by numerous experiments (4, 11, 21, 28, 51). Bushmann et al. (4) and Kim et al. (28) carried out experiments on chondrocyte/agarose disks and calf cartilage explants, respectively. These studies involved uniaxial unconfined compression between two impermeable platens, and found that static compression reduced synthesis of matrix molecules. Buschmann et al. (4) reports a decrease in [35S]sulphate and [3H]proline incorporation of disks under static compression compared to uncompressed disks. This change was also dependant on the amount of matrix that had accumulated in the disk prior to compression. Static compression had little effect on disks with little matrix whereas constructs with increased amounts of matrix

Fig. 4. Schematic of changes that occur in cartilage during static compression.


showed increased inhibition in biosynthesis. Kim et al. (28) reports that when compression was removed from explant disks after 60 hours of free-swelling recovery, the biosynthetic rates recovered to that of free-swelling controls indicating that no cell damage occurred.

Davisson et al. (11) performed static compression experiments on constructs of fibrous, non-woven PGA scaffolds (porosity of 97% and density of 45 mg/cm3) seeded with chondrocytes from 3 week old bovine calf cartilage and cultured free-swelling for 3 weeks so that the constructs would contain appreciable amounts of matrix. Compression of 10%, 30% and 50% was carried out using porous platens in a uniaxial confined environment. Static compression at 10% and 30% had no effect on total protein or GAG synthesis whereas 50% compression diminished total protein synthesis by 35% and total GAG by 57% when compared to uncompressed controls.

The above studies, which use cartilage explants or chondrocytes harvested from articular cartilage, highlight that static loading results in a reduction in cell proliferation and matrix biosynthesis. However, a study carried out by Takahashi et al. (49) found that static compressive forces of 1 kPa, 1.5 kPa and 2 kPa, applied to mouse embryonic limb bud mesenchymal cells embedded in a 3D collagen gel, promoted chondrogenesis. The mesenchymal cells used in the study were acquired prior to chondrogenesis in vivo. By using RT-PCR, the study found that the accelerated chondrogenesis was due to the up-regulation a transcriptional activator of type II collagen, Sox9, and down-regulation of a repressor of type II collagen and GAG, IL-1β. The expression of type II collagen was also found to be proportional to the magnitude of the force applied; a compressive force of 2 kPa resulted in 3.8-fold increase compared to levels detected in samples subjected to 1 kPa of force. It is interesting to note this contrasting response of undifferentiated mesenchymal cells and mature chondrocytes to static loading.


5.1.2 Dynamic Compression

Dynamic compression has delivered more positive results than static compression experiments. During dynamic compression, physical phenomena such as hydrostatic pressure gradients, intra-tissue fluid flow and streaming potentials within the tissue can be induced (24). It is difficult to assess the individual effects of each of these stimuli as they all occur simultaneously, but dynamic loading at certain amplitudes and frequencies has been shown to increases biosynthesis. Various methods and devices have been developed to carry out dynamic compression. Three main parameters are considered for experimental testing (7):

1. frequency of the applied load 2. strain or force used 3. duration of the experiment.

These have been shown to range between minimum and maximum values as shown in Table 4.

Buschmann et al. (4) reports an increase of 15% to 25% in sulphate incorporation after dynamic loading of 3 mm thick chondrocyte/agarose cultures in unconfined compression of ~3% amplitude (30 µm displacement amplitude) at 0.01 Hz to 1.0 Hz, superimposed on a static offset of ~25% (0.73 µm displacement). Stimulation was found in samples that were cultured for longer periods where more ECM was present. The agarose mimicked the ECM and maintained normal cell morphology by preventing loss of the differentiated phenotype. It is

Table 4. Range of frequency, strain and load applied during dynamic compression tests carried out to date; from Darling and Athanasiou (7).

Stimulus Min Value Max Value

Frequency (Hz) 0.0001 3

Load (MPa) 0.1 24

Strain (%) 0.1 25

Duration Hours Weeks


therefore hypothesised that the ECM is involved in the transduction of signals at the cell-matrix interface.

Kim et al. (28) also report an increase in biosynthesis as a result of dynamic compression of calf cartilage explants. Dynamic compression is reported to induce a nonuniform radial biosynthesis that is influenced by changing frequency. In explant disks of 3 mm-diameter and 1 mm-thick, low frequencies of 0.01 Hz stimulated biosynthesis throughout the sample, whereas a frequency of 0.1 Hz limited synthesis to the radial periphery (dynamic strains of 0.63% to 10.4% were employed). The resulting radial distribution, which varied with the frequency of compression, can be compared to cell deformation and the profiles of hydrostatic pressure, fluid flow and streaming potential which occur during dynamic compression. When constructs are subjected to compression, tissue deformation and fluid flow vary radially. Fluid flow and the associated streaming potential gradient would be (i) highest at the outer radial periphery where pressure would be lowest and (ii) lower at the centre of the construct where the pressure would be highest, as illustrated in Figure 5. Compared to samples compressed at lower frequencies it is suggested that these radial gradients would be quite steep when a frequency of 0.1Hz is subjected on a 3mm-diameter disk resulting in an increase in synthesis at the periphery. Buschmann et al. (4) also report on greater stimulation in the outer ring compared to the centre in chondrocyte/agarose disks which increased with culture time and the deposition of ECM.

Davisson et al. (11) carried out dynamic uniaxial confined compression experiments at frequencies of 0.001 Hz and 0.1 Hz on 10 mm-diameter and 2 mm-thick PGA scaffolds, seeded with chondrocytes harvested from the patellofemoral groove of bovine calves. Dynamic compression of 5% amplitude superimposed on a static offset of 10% or 50% generally increased total synthesis of protein and GAG when compared to statically compressed results. The amplitude of the static compression offset and the frequency of loading had significant effects on biosynthesis. A loading regime of 0.1 Hz frequency and 5% strain superimposed on a static compression offset of 50% produced the most dramatic results with increases of 99% and 179% in protein and GAG synthesis, respectively, when compared to uncompressed controls.


Contrastingly, when an offset of 10% was employed, a frequency of 0.001 Hz resulted in higher increases in protein and GAG compared to synthesis from 0.1 Hz frequency. The increase in biosynthesis at a frequency of 0.1 Hz is believed to be as a result of increased fluid velocities, whereas tissue and cell strain are expected to influence synthesis at the lower frequency of 0.001 Hz.

Comparing the reports of Kim et al. (28) and Davisson et al. (11) it can be noted that both experiments saw beneficial effects of dynamic compression compared to static compression. However, while Davisson et al. (11) obtained most significant results at a frequency of 0.1Hz, Kim et al. (28) reports that, at this frequency, biosynthesis was increased at the periphery of the explant compared to the centre, unlike lower frequencies where biosynthesis was stimulated throughout the disks. It must be noted that there are two major differences between both experimental set-ups. Kim et al. (28) used cartilage explants subjected to 23 hours of uniaxial unconfined compression between two impermeable platens whereas Davisson et al. (11) used bovine chondrocytes seeded on PGA scaffolds and subjected them to 24 hours of uniaxial confined

Fig. 5. Normalised profiles of hydrostatic pressure and interstitial fluid velocity (relative to the (ECM) as a function of the radial position within a disk, with r=0 at the axial centre and r=1 corresponding to the radial periphery; as reported by Kim et al. (28).


compression between two porous platens. These distinct differences between experimental set-ups make it difficult to compare results and to define exactly what loading conditions are necessary to invoke increased rates of ECM synthesis.

Torzilli et al. (51) carried out unconfined compression experiments using full-thickness cartilage explants removed from the subchondral bone of adult canine humeral heads and found that both static and cyclic loading always inhibited proteoglycan biosynthesis under stress conditions ranging between 0.5 – 24 MPa load at a frequency of 1 Hz. It is difficult to establish if the frequency had an effect on the results as the frequency was not varied in the study. This inhibition in biosynthesis, compared to other studies, may be due to:

1. the type of cartilage used i.e. full thickness as opposed to partial thickness

2. the loading conditions employed – a porous loading-platen rather than a nonporous platen.

Lee et al. (32) investigated how chondrocytes harvested from different full depth, superficial or deep zones of cartilage explants and seeded in 3% agarose responded to compression. Unconfined uniaxial compression of 15% was employed using fluid impermeable platens for 48 hours at frequencies of 0.3 Hz, 1 Hz and 3 Hz. Results were normalised to those of unstrained control samples. Unstrained controls showed that increased proliferation and GAG synthesis occurred in deep cells compared to superficial cells. Cells from the deep zone exhibited a frequency dependent response to 15% strain. Constructs subjected to compression showed that 0.3 Hz inhibited GAG synthesis, 1Hz induced a 50% increase in synthesis and 3Hz showed no change, with these trends being similar to those obtained for full depth cells. This suggested that there may be an optimum frequency level which gains an increased response from cells. Contrastingly, superficial cells subjected to the full range of frequencies all demonstrated reduced amounts of GAG synthesis. When superficial cells were mechanically stimulated their proliferative potential increased as did full depth cells. Deep cells demonstrated a good proliferative potential in unstrained controls with no change being recorded when mechanically stimulated. An overview


of these results in terms of GAG synthesis and cell proliferation is presented in Table 5.

Recently, a computer-controlled bioreactor has been designed by Démarteau et al. (9) to expose constructs to compressive deformation at various strains and frequencies, along with simultaneous medium stirring at 30 rpm (see Fig. 6) (8, 9, 36). Martin et al. (36) used this system to study the effects of compressive deformation on human adult nasal cartilage cells seeded onto hyaluronic acid-derived polymer meshes. Constructs were maintained uncompressed, or were exposed to 25% static compression or 20% sinusoidally compressed superimposed on a 25% strain offset for 2, 14 or 24 hours. Results show that dynamic compression for 2 hours induced significant increases in collagen type II (9.1-fold), collagen type I (8.3-fold) and aggrecan (6.4-fold) synthesis

Table 5. Illustration of the response of superficial, deep and full depth cells subjected to 15% compression at a range of frequencies (0.3Hz, 1Hz and 3Hz). GAG synthesis and cell proliferation values were all normalized to unstrained control results (32).

0.3Hz 1Hz 3Hz

GAG

GAG

GAG

Superficial Cells

Proliferation

Proliferation

Proliferation

GAG

50% GAG

GAG

Deep Cells

Proliferation

Proliferation

Proliferation

GAG

40% GAG

GAG

Full Depth Cells

Proliferation

Proliferation

Proliferation

= increase = decrease = no change


compared to free-swelling controls. After 14 and 24 hours of culture there was no significant difference between free swelling, static and dynamically compressed constructs. It is believed that the cells desensitised to the loading applied due to the unchanged synthesis of components over increased time. As a result, further investigations are to be carried out involving intermittent loading regimes for long periods of time that would simulate the normal intermittent in vivo regime.

Démarteau et al. (9) used this system culture human adult chondrocytes harvested from post-mortem knee articular surfaces of four individuals, seeded on PEGT/PBT scaffolds (porosity of 75% and pore size of 400-600 µm). The study was carried out to investigate the effect of dynamic compression on GAG metabolism, imposed on constructs that were cultured for either 3 or 14 days. Growth factors were added to the medium during expansion to enhance the capacity of the cells to proliferate and redifferentiate when seeded onto scaffolds. Constructs were cultured for 3 or 14 days with 30 rpm orbital mixing and were further supplemented with prostaglandin F2α, to enhance the redifferentiation and deposition of cartilaginous matrix. Constructs were then cultured for an additional three days, with or without dynamic compression. Intermittent compression was applied for a total of three days and involved cycles of 2 hours of sinusoidal deformation followed by 10 hours without deformation. A frequency of 0.1 Hz was employed with 5% strain amplitude superimposed on a 5% strain offset. The peak stress of 20 kPa measured during loading was similar to cell-free scaffolds indicating that the mechanical properties of the scaffold provided the stiffness of the construct. The DNA content did not change within the time frame of the experiment indicating that no change in cell proliferation occurred. GAG synthesis, accumulation and release responded positively to cyclic loading. However, it was found that compression stimulated GAG accumulation only if the GAG content prior to compression was sufficiently high (i.e. in constructs that were initially cultured for 14 days). This indicates that the ECM plays a role in (i) modulating the response of cells to mechanical loading and (ii) in retaining newly synthesised matrix components. The development of the ECM was also found to vary with different donor cells.


Fig. 6. Compression chamber incorporating medium mixing to improve mass transfer. A: (I) compression chambers, (II) magnetic bar for medium stirring, (III) inlet/outlet for medium change. B: (I) cover lid to maintain sterility, (II) micrometer screw to establish contact between the plungers and each specimen. C: (I) tissue culture unit comprising of two culture chambers, (II) two magnetic stirrers, (III) a positioning table, (IV) a load cell and (V) a stepper motor (9).

Bonassar et al. (3) took a different approach to determine the effect

of dynamic compression on articular cartilage explants by using a biochemical regulator, insulin-like growth factor-I (IGF-I). IGF-I stimulates chondrocyte proliferation and the synthesis of cartilage matrix components, collagen and proteoglycans, in vivo. It is present in biologically active concentrations in synovial fluid and in articular cartilage and is believed to help induce articular cartilage repair. The experimental set-up involved a uniaxial unconfined compression system with fluid-impermeable plates. As well as mechanical stimuli influencing chondrocyte metabolic activity, it has been proven that biochemical factors within the joint also regulate how chondrocytes maintain the ECM. An increase of 180% in protein synthesis and 290%


in proteoglycan synthesis was obtained when dynamic compression of 3% strain and 0.1Hz frequency was used in conjunction with the growth factor IGF-I. It is possible that dynamic compression improved the transport of IGF-I into the matrix to the chondrocytes inducing a greater response by the cells to produce matrix molecules.

Mauck et al. (37) investigated the effect of seeding density (10, 20 or 60x106 cells/ml) on the development of chondrocyte-seeded agarose hydrogels subjected to unconfined dynamic compression between two impermeable platens. The loading employed 10% peak-to-peak compressive strain amplitude at a frequency of 1 Hz, for three daily cycles of 1hr on/1hr off, 5 days a week for 4 weeks. In free-swelling cultures, higher seeding densities increased the aggregate modulus (HA), Young’s modulus (EY) and GAG content. When dynamic loading was applied, constructs seeded at 20x106 cells/ml enhanced both biochemical content by ~150% and mechanical properties by ~three fold. However, a seeding density of 60x106 cells/ml exhibited no change in mechanical properties compared to free-swelling controls. In this group, collagen content was significantly higher than controls. It is most likely that at the higher seeding density, the increase in mass transfer normally associated with dynamic compression was diminished with insufficient amounts of nutrients reaching the cells.

5.2 Tension

Tension may also be experienced by cartilage in vivo, and some studies have investigated the effects of it on cells. Lee et al. (33) seeded chondrocytes harvested from 15-day-old chick embryo sternae onto elastin membranes of adult bovine thoracic aortas. After 4 days of culture membranes were (i) subjected to a 10% sinusoidal stretch at 1Hz for 8 hours, or were (ii) agitated in the medium by connecting the two clamps together so that the membrane, kept at a fixed length, was moved in the medium at 1Hz. It was found that chondrocytes did not bind directly to the elastin fibres but adhered indirectly through the ECM. Both methods of stimulating the cells invoked an increase in GAG synthesis rates compared to control samples. Stretching and agitation produced a 2-fold and 3-fold increase in GAG, respectively. Synthesis


of protein and collagen did not show a significant change, but did tend towards decreased rates in stimulated samples compared to controls. Deformation of the cell cytoskeleton may be important in the type of biosynthetic response.

De Witt et al. (10) used a slightly different approach in researching the effect of tension on chondrocytes. Chick epiphyseal chondrocytes cultured for 14 days formed a 5-8 cell thick tissue with a tough matrix. Instead of using a substrate during the application of the tensile stretch, the tissue was directly subjected to a strain of 5.5% at a frequency of 0.2Hz for periods of up to 2 weeks (see Fig. 7). It was found that DNA synthesis increased with mechanical loading. Protein and collagen synthesis remained unaffected and an increase in GAG was reported, with these trends similar to those reported by Lee et al. (33). When cultures were mechanically stretched for periods greater than 24h an increase in protein synthesis and collagen synthesis was observed (10).

From the studies present in literature it has been shown that the application of mechanical tension over short periods alters the metabolism of chondrocytes. The increased synthesis of proteoglycan is important to the structure and function of the ECM.

Other methods of applying tensile forces to cells have been used and involved:

1. seeding cells onto a cover slip and deforming it (53) and 2. seeding cells onto a flexible membrane and applying an

equibiaxial mechanical strain using vacuum to deform the cells (17).

Both studies report increases in GAG/proteoglycan synthesis. Fukuda et al. (17) reports that lower stress levels of 5% elongation at a rate of 10 cycles per hour stimulated this increase in GAG, however high stress levels of 17% elongation at a rate of 10 cycles per minute reduced synthesis. Contrastingly, this higher stress level enhanced DNA synthesis.

Recently, an experiment was carried out by Simmons et al. (46) to investigate the effect of cyclic substrate deformation on the proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs). The study also investigated the hypothesis that the hMSCs respond to mechanical stimuli through mitogen-activated protein kinase


(MAPK) pathways. A Flexercell system was employed to apply a 3% equibiaxial cyclic strain at 0.25Hz for up to 16 days on cells plated on silicone rubber bottoms and maintained in an osteogenic medium. Cyclic strain was found to inhibit the rate of proliferation but stimulated a 2.3-fold increase in matrix mineralisation (calcium deposition) after 16 days strain. It was found that strain-induced mineralisation was mediated by the ERK1/2 pathway as inhibition of this pathway reduced calcium deposition by 55%. Inhibition of the p38 pathway resulted in a more mature osteogenic phenotype which suggests that this pathway plays an inhibitory role in the modulation of strain-induced differentiation. The study shows that mechanical signals such as equibiaxial cyclic strain can regulate hMSC function and resulting matrix deposition, indicating a requirement for the stimulation of cells to induce tissue formation. This adds to the theory that mechanical loading of cells results in a more functional engineered tissue. It is likely that substrate deformation would also induce MSCs to proliferate and differentiate down the chondrogenic lineage.

Fig. 7. Picture of tension system employed by De Witt et al. (10); (A) central rotating axle, (B) stationary clamp, (C) moving clamp, (D) clamp shaft, (E) eccentric Teflon clamp, and (F) coupling arm.


5.3 Hydrostatic Pressure – Intermittent and Cyclic

Hydrostatic pressure is a significant component of the mechanical loading environment in vivo and is experienced by chondrocytes in cartilage during periods of loading via the compression of fluid (7, 22). The pressure does not induce fluid flow or tissue deformation and acts uniformly on the chondrocytes in the matrix. Compared to direct tissue compression, the structural integrity of the tissue is maintained when hydrostatic pressure is applied as the matrix is not subjected to any stretching or shear forces (7, 42). Physiological levels of hydrostatic pressure are reported to fall within the range of 5-15 MPa, with 5 MPa being the estimated pressure level in the knee during walking (7, 22, 42).

Parkkinen et al. (42) carried out cyclic hydrostatic pressure experiments on both primary chondrocyte cell cultures and bovine cartilage explants and found proteoglycan synthesis rates were increased in both culture systems, and depended on both the duration and magnitude of loading. A pressure of 5 MPa was employed with loading times and frequency of loading being the variables. Short-term pressurisation of 1.5 hours produced varied results with proteoglycan synthesis stimulated in cartilage explants but inhibited in chondrocyte cultures. Long-term experiments of 20 hours on chondrocyte cultures stimulated proteoglycan synthesis when subjected to 0.25 Hz and 0.5 Hz, but inhibited synthesis at 0.0167 Hz. This indicates that chondrocytes respond differently depending on the duration and magnitude of loading. Compared to cartilage explants cultured chondrocytes are more flattened in shape and are not surrounded by an organised matrix. Parkkinen et al. (42) hypothesises that the presence of an established ECM for cell-matrix interactions and the shape of the chondrocytes subjected to the pressure are important in regulating the synthesis of proteoglycans.

Ikenoue et al. (22) applied intermittent hydrostatic pressure (IHP) of 1, 5 and 10 MPa to chondrocytes harvested from adult human cartilage in high-density monolayer culture at a frequency of 1 Hz. Results indicated that collagen and aggrecan are independently regulated by IHP as a short duration and low magnitude environment (4 h, 1 MPa) did not stimulate collagen mRNA signal levels but increased aggrecan mRNA expression.


Carver and Heath (6) developed a system that consisted of semicontinuous medium perfusion and intermittent hydrostatic pressure to investigate the effects of intermittent pressurisation levels and age of cells. Chondrocytes from foals and adult horses were seeded onto PGA scaffolds (porosity of 97%) and subjected to pressures of 500 psi (3.44 MPa) and 1000 psi (6.87 MPa). A 20 minute intermittent cyclic pressure regime involving 5 seconds pressurisation and 15 seconds depressurisation was applied every 4 hours for 5 weeks. Prior to the pressurisation regime, 20 mL of medium was perfused into the vessel over a period of 3 minutes. Although there was some variation in the results, the study concluded that intermittent pressurisation reduced cell proliferation and increased the amount of GAG and collagen synthesis in constructs compared to non-pressurised controls (see Table 6). The level of pressurisation and age of the cells affected GAG synthesis with juvenile cells and high pressure producing the highest GAG concentration. Juvenile constructs contained fractions of GAG that resembled native cartilage. It was discovered that a minimum level of pressure is required to induce collagen synthesis. Juvenile constructs produced higher concentrations of collagen than adult constructs, but none of the constructs reached native levels (6, 20).

In the intermittent hydrostatic pressure experiment carried out by Carver and Heath (6) small constructs were produced due to mass

Table 6. Illustration of GAG and collagen results obtained by Heath for foal and adult chondrocytes seeded in PGA scaffold and subjected to 5 weeks of intermittent pressurisation (20).

Intermittent Pressure

GAG Concentration (mg/g tissue)

Foal Adult

Collagen Concentration (mg/g tissue)

Foal Adult Native Cartilage 40 - 120 80 - 120 100 - 150 120 - 180 Control (no pressure)

26.0±24.4 2.0±1.0 6.3±1.6 0.5±0.3

500psi 89.3±31.4 5.7±1.0 6.7±1.9 3.0±0.3 1000psi 133.7±38.5 3.5±1.4 11.9±2.7 7.3±0.5


transfer limitations when applying fluid flow conditions of 6.67 mL/min occurring for 18 mins daily. This fluid flow was not turbulent and the fluid remained still at all other times. It is possible that longer periods of fluid flow at higher velocities would increase the mass transfer of nutrients to the inner regions of the construct. As reported by Ikenoue et al. (22), it is possible that collagen and GAG synthesis have a different signalling mechanism as they responded differently to the intermittent pressure (20).

As discussed in Section 2.3, much attention has been given to the effect of bioactive factors on MSC differentiation and proliferation, with few studies investigating the effect of mechanical factors on MSCs. Angele et al. (1) examined the influence of cyclic hydrostatic pressure on bone marrow-derived human mesenchymal progenitor cells that were cultured in a medium to induce chondrogenic differentiation. Bone marrow-derived human mesenchymal progenitor cells were cultured and then spun down to form cell clusters or aggregates. These aggregates were then placed in polypropylene tubes, filled with chondrogenic medium. The tubes were sealed using aluminium caps with a flexible rubber seal in the middle of the cap. The tubes were placed in a custom-built hydrostatic pressure chamber (see Fig. 8) and located in a temperature-controlled incubator.

A 1 Hz sinusoidal cyclic hydrostatic pressure waveform with a minimum pressure of 0.55 MPa and a peak pressure of 5.03 MPa was applied to the samples for 4 hours per day. Loading occurred at various time points: single (on day 1 or on day 3) or multiple (days 1-7). After each daily loading period aggregates were maintained in standard conditions. Aggregates were cultured for either 14 or 28 days to allow for extensive differentiation and matrix formation. Only multi-day loading influenced the matrix production of the differentiating progenitor cells, indicating that the effect of loading is cumulative. Multi-day loading resulted in statistically significant increases in proteoglycan and collagen contents at both 14 and 28 days of culture. Compared to non-loaded controls and single-day loaded samples after 14 and 28 days in culture, multi-day loaded aggregates had a qualitative increase in ECM production with increased staining and a greater distance between cells. At days 14 and days 28, no statistical difference in DNA content was


found between loaded constructs and non-loaded controls. It was unclear from the study if cyclic hydrostatic pressure directly influenced differentiation. Chondrogenic medium, which promotes the differentiation of MSCs to chondrocytes, was used in the study. As a result the direct influence of hydrostatic pressure on chondrogenic differentiation was not explored. However the study does indicate that physiological loading does have a positive effect on matrix formation during chondrogenesis.

5.4 Strain-Induced Shear

Articular cartilage provides a low-friction interface between contacting surfaces of a joint. Physiological loading induces a complex loading environment within cartilage which includes direct shear stresses in the superficial zone as the gliding surfaces move over each other. Section 5.5 outlines the various fluid-induced shear conditions have been employed to induce biosynthesis within monolayer or 3D constructs. Recently, studies have investigated the effect of dynamic shear strain on chondrocyte biosynthesis.

It is reported that the positive results obtained from dynamic compression is associated with intratissue fluid flow, cell/tissue deformations and pressure gradients. Dynamic shear strain does induce deformation of cells and matrix. Minimal volumetric changes occur during loading and minimal intratissue fluid flow and pressure gradients

Fig. 8. Diagram of cyclic hydrostatic pressure device (1).


are developed. This allows studies to examine the effects of tissue deformations that are independent of fluid flow and pressure gradients (13, 23, 24, 58).

Frank et al. (13) developed a biaxial loading device capable of applying both compressive and shear forces to study the biosynthetic response of chondrocytes to dynamic deformation. The apparatus (see Fig. 9) applied the dynamic shear deformation as the bottom half of the chamber was rotated with respect to the top half. Cartilage explants (3 mm diameter and 1.1 mm thick) were obtained from the femoropatellar grove of 1 to 2 week old calves. The disks were statically compressed to a thickness of 1 mm and were subjected to cyclic 1% shear deformation at 0.1 Hz for 12 or 24 hours. Results indicated an increase in proteoglycan and protein content of 25% and 41%, respectively, when compared to control disks held at the static compressive offset. The study also looked at radial distribution of matrix molecules as previously reported by Kim et al. (28) during compressive loading. It was found that there was no significant difference between the core and the outer ring in the dynamically sheared disks.

Fig. 9. Shear/Compression apparatus: (A) axial linear stepper motor, (B) bearing/carriage assembly, (C) sample chamber, (L) load cell, (T) torque cell, (R) rotary position table. The adjustable plate may be moved to accommodate other fixtures. The LVDT shown to the left of chamber (C) senses rotational displacement. Rotary table (R) is driven by a stepper motor behind the table (13).


Jin et al. (23) extended the work reported on by Frank et al. (13). Cartilage explants from calves were subjected to shear loading of 1-3% strain at frequencies of 0.01 Hz, 0.1 Hz and 1.0 Hz for 24 hours using the apparatus pictured in Figure 9. Shear deformation caused an increase in protein (~50%) and proteoglycan (~25%) synthesis compared to controls held at the same static compressive offset. The increased synthesis did not significantly depend on the frequency range or strains applied. Shear deformation caused a twofold stimulation of protein synthesis compared to proteoglycan synthesis. No radial distribution was found in samples, as was reported by Frank et al. (13). It is believed that the stimulation of biosynthesis was as a result of ECM deformation, resulting in the activation of transduction pathways associated with integrin-cytoskeleton machinery.

The experimental set-up and findings reported on by Jin et al. (23) was developed to investigate the combined effects of dynamic tissue shear deformation and insulin-growth factor I (IGF-I) on chondrocyte biosynthesis (24). Cartilage explants were maintained at 0% offset compression and were subjected to sinusoidal shear strain amplitudes of 0.5 to 6.0% at 0.1 Hz for 24 hours. The separate effects of tissue shear and IGF-I on matrix synthesis were tested with increases in protein and proteoglycan synthesis found in both sets of tests. Increased biosynthesis as a result of IGF-I incorporation was found to be dose dependent. When tissue shear and IGF-I were combined the increase in matrix synthesis was close to the sum of each individual stimulus. However, analysis showed that there was no interaction between the two stimuli, unlike the findings of Bonassar et al. (3) who found that dynamic compression and associated intratissue fluid flow enhanced the transport of IGF-I into cartilage explants, as discussed in Section 5.1.2. Dynamic tissue shear does not induce fluid flows in the tissue so the combined effect of IGF-I and dynamic shear is believed to be as a result of specific signal transduction machinery that are sensitive to deformation of chondrocytes and matrix.

As shear deformation studies were carried out over short durations of 24 hours, Waldman et al. (58) carried out long-term intermittent cyclic shear deformation studies where constructs were stimulated every second day for one week to investigate if there would be an increase in ECM


accumulation and if the mechanical properties of the developing tissue would be improved. Chondrocytes harvested from 6-9 month old bovine animals seeded in porous calcium polyphosphate substrates were used in the study. A dual axis Mach-1TM mechanical tester housed in a standard incubator and was used to subject the constructs to shear stresses. A 5% static compressive preload was applied to prevent slippage of the sample during shear loading. Constructs were subjected to shearing amplitudes of 2%, 6% or 12% of the constructs height for 400 or 2000 cycles per day at a frequency of 1 Hz. Results showed that low amplitudes of shear stimulation for short periods, i.e. 2% strain at 1Hz for 400 cycles, returned the largest increases in both collagen (23%) and proteoglycan (20%) synthesis compared to controls after one week. Compared to controls, no change in DNA content was found. Long-term stimulation was investigated by stimulating constructs for the final four weeks of an eight-week culture period using the optimal conditions of 2% strain for 400 cycles per day. Results demonstrated an increase in the accumulation of collagen and proteoglycans by 40% and 35%, respectively. Constructs that were subjected to four weeks of intermittent cyclic shear loading also had a 3-fold increase in load-bearing capacity and a 6-fold increase in stiffness compared to unstimulated controls. This can be linked to the increased synthesis of proteoglycan and collagen matrix molecules.

5.5 Fluid-Induced Shear

As stated in Section 5.4 above, shear stresses are found in articular cartilage during relative motion between two bones. However cartilage-on-cartilage loading also creates shear stresses in the tissue due to internal fluid flow. Researchers have attempted to emulate these forces by stimulating fluid to either flow across or flow through cell-seeded scaffolds (7). Various rigs have been designed to apply both high and low fluid-induced shear forces to constructs:

direct perfusion mechanically stirred flasks rotating wall.


5.5.1 Perfusion

Mass transfer is a critical consideration when culturing cells in scaffolds to ensure adequate nutrient and pH levels are maintained and to eliminate wastes from the construct. Direct perfusion bioreactors have been developed to culture cell/scaffold constructs to improve mass transfer rates.

Sittinger et al. (47) used a perfusion culture system to investigate the engineering of cartilage from chondrocytes harvested from human femoral heads seeded in bioresorbable co-polymer fleeces, which gave temporary artificial intercellular matrix stability. The surfaces of the polymer fleeces were coated with various attachment factors to investigate cell adhesion with poly-L-lysine proving to be the most efficient. The constructs were encapsulated by agarose to (i) mimic the in vivo environment where nutrients only reach cartilage cells by diffusion and to (ii) reduce the permeability for the diffusion of synthesised matrix components into the medium from the matrix. The agarose also allows for wastes to diffuse out of the construct. The perfusion culture system delivered 1 ml/L of fresh medium through the culture chamber with a pump operating on a 30 min on/30 min off cycle. Medium exited the chamber through a second port and emptied into a waste vessel (see Fig. 10). It must be noted that in this experiment the perfusion system was used to introduce nutrients and oxygen into the chamber and to remove wastes, and not to apply mechanical stimuli to cells. Results indicated that the chondrocytes maintained their phenotypic properties when seeded in 3-D polymer fleeces with the accumulation of collagen and proteoglycan molecules inside the construct.

Perfusion systems can also be used to apply mechanical stimuli to cells as well as simultaneously supplying nutrients and oxygen. As fluid flows through a construct shear forces are generated and it is possible that ECM is produced in response to this force (7). Pazzano et al. (43) used a perfusion bioreactor system to culture chondrocytes harvested from bovine calves seeded onto PGA/PLLA disks. Media was forced through the constructs at a fluid velocity of 1 µm/s, as this has been previously shown to stimulate cartilage matrix biosynthesis (29). It was


found that the axial flow through the constructs resulted in columns of chondrocytes and ECM that were aligned in the direction of the flow. Therefore, the ability of fluid flow to create aligned tissues may be useful in the engineering of articular cartilage as demonstrated in the work by Pazzano et al. (43).

The effect of perfusion at various velocities, times and durations was investigated in a study by Davisson et al. (12). Ovine chondrocytes were seeded onto PGA scaffold (porosity of 97% and density of 45 mg/cm3) disks and cultured for 1 day or 1 week, either statically or in a perfusion of 10 µm/s. Constructs were then placed into bioreactors that supplied continuous perfusion for 48 hours at flows of either 0.05 mL/min or 0.8 mL/min (fluid velocities of 10 µm/s and 170 µm/s, respectively). It was found that three days perfusion suppressed the deposition and release of GAG in constructs whereas perfusion for 9 days was found to increase synthesis under certain conditions. Such conditions involved perfusion of 10µm/s for the first 7 days and 170 µm/s for the next two days which resulted in increases in the deposition and release of GAG by 60% and 136%, respectively, compared to static controls. Cell content was also investigated in this study. Compared to constructs subjected to perfusion at velocities of 10 µm/s, higher fluid velocities of 170 µm/s at early time points in culture decreased cell content. Matrix content is minimal in early periods of culture with GAG and collagen content increasing

Fig. 10. Perfusion culture system. An agarose coat separates the cell-polymer tissue from the medium flow (47)


dramatically during initial days of culture. As a result, it is possible that a high perfusion rate would enable fluid shear to detach or force cells off the scaffold. However, in this study cell content in the medium was not measured so this theory was not proven. The effects of perfusion also depend on the changing environment within a construct as the tissue grows. As ECM is synthesised and accumulated in a construct, the permeability of the construct decreases and this results in changes in the mechanical stimuli experienced by cells. Therefore, more mature constructs are likely to experience different amplitudes of strain and pressures than young constructs.

A limitation with direct perfusion bioreactors is the difference in mechanical stress between one side of a scaffold and the other. The cells at the end of the scaffold where the flow enters are subjected to higher mechanical shear stresses due to oncoming flow whereas cells at the opposite end are subjected to weaker forces. This is due to a reduction in the energy of the fluid as it dissipates through the scaffold (7). However, Wendt et al. (60) are currently using an oscillating perfusion system that was developed to improve seeding efficiencies (as discussed in Section 3), to investigate the effects of oscillating perfusion on cell culture after seeding. This closed system facilitates the seeding and culturing of constructs in a single system allowing for a reproducible, controlled system which reduces the risk of contamination.

It is possible to use a perfusion system to nourish cells in conjunction with other mechanical forces like compression or hydrostatic pressure to obtain positive results. Carver and Heath (6, 20) used this approach where perfusion was used to nourish cells that were mechanically stimulated by intermittent pressure, as discussed in Section 5.3.

5.5.2 Mechanically Stirred Flasks

The spinner/mixed flask is the most common and one of the simplest bioreactor designs. Scaffolds, seeded with cells, are suspended from needles from the stopper of the flask and are covered with culture medium (7, 50). An impeller at the base of the flask is used to mix nutrients and oxygen throughout the medium. The mixed flask can operate on a dual role of both seeding the scaffolds with cells and then


culturing them. Under constant mixing conditions, the turbulent flow of medium enhances mass transfer of nutrients and gases but induces a high shear force over the surface of the construct (57). Equations have been developed to determine the hydrodynamic conditions (shear intensity and fluid flow) within the mixed flask, which allows for controlled experiments (7). Mixed flasks have also been effectively used in experiments to uniformly distribute cells through scaffolds (16, 25, 34, 36, 40, 57).

The first reported studies of the effects of fluid flow on chondrocyte growth and matrix formation were carried out by Freed et al. (14, 19). Experiments involved the use of a mixed flask to culture chondrocytes seeded on fibrous PGA scaffolds (porosity of 92-96% and density of 1.6 g/cm3) for 6 weeks. Stirred cultures produced higher rates of cell growth and matrix production in when compared to static cultures, indicating that the environment in the flask was able to simulate the type of fluid motion found in tissues in vivo (14, 19).

Vunjak-Novakovic et al. (57) carried out 6 week experiments investigating the effect of turbulent flow conditions in a mixed flask on chondrocytes harvested form the femoropatellar groove (FPG) of 2-3 week old calves and seeded on to fibrous PGA scaffolds (porosity of 97% and density of 62 mg/cm3). The stirring rate of 0.83 s-1 generated a turbulence at an intensity below those previously reported to cause cell damage. Constructs grown in mixed flasks generated more GAG in the inner tissue phase compared to statically grown constructs. In static constructs, GAG accumulated in a 1mm outer layer which indicates that mixed flasks increased the mass transfer of nutrients inside the constructs. Collagen synthesis was higher in mixed constructs compared to constructs grown statically. The turbulent mixing also created an outer fibrous capsule, 100-400 µm thick, on the surface of the construct. This study also cultivated constructs in a low shear rotating wall bioreactor (rotating wall vessel is discussed in detail in Section 5.5.4), which produced higher amounts of collagen and GAG than either mixed flask constructs or static controls. Scaffolds were initially seeded with 5 million cells and this increased to 9.66±0.06, 16.6±3.3 and 14.4±2.0 million cells per construct in the static control flask, mixed flasks and the rotating wall vessels, respectively (57). However, the 6-week constructs


from each group (i.e. static, mixed and rotating) all produced tissue that was structurally and functionally inferior to cartilage explants.

The use of mixed flasks leads to the formation of inferior tissue. The fibrous capsule induced by the turbulent environment is composed mainly of type I collagen which is undesirable in articular cartilage. Constructs in mixed flasks are subjected to nonuniform mechanical stimuli such as non-uniform mass transfer rates, nutrient and pH gradients and shear gradients. The force at the surface of the impeller is higher than at any other point in the bioreactor so cells situated closer to the impeller have an increased rate of cell injury due to high shear levels (7).

5.5.3 Oribital Shaker and Cone Viscometer

Other methods of mechanically stirring medium include the orbital shaker and the cone viscometer. The orbital shaker can slowly mix the contents of a petri dish with minimal turbulence. Kafienah et al. (25) used this device to culture PGA scaffolds seeded with human adult nasal and adult articular chondrocytes to compare the chondrogenic capacity of the cells. The human nasal cells were found to proliferate four times faster than articular cells and could be engineered into a cartilage-like tissue. The articular chondrocytes had a slow ECM synthesis rate as the scaffold dissolved before a cartilaginous matrix was deposited, with the synthesised matrix being confined to the outer perimeter of the scaffold. Nasal constructs contained higher levels of type II collagen and proteoglycan than the articular constructs. Due to the differences between nasal and articular cartilage in their organisation and function, it is not known if nasal chondrocytes can be used to repair articular cartilage defects. Tissue engineered from articular chondrocytes also lacks complex zonal organisation but it is thought that the tissue may remodel under normal loading conditions in vivo.

Martin et al. (34) also used an orbital shaker while investigating the differentiation of chick embryo bone marrow stromal cells into cartilaginous and bone-like tissue. Bone marrow stromal cells were expanded in monolayer and seeded onto biodegradable PGA scaffolds (porosity of 96%). The constructs were placed in dishes coated with 1%


agarose and were cultivated on the orbital shaker at 75 rpm. Results show that bone marrow stromal cells can be differentiated in vitro in the presence of fibroblast growth factor-2 (FGF-2) and a three-dimensional polymer scaffold, to form a cartilaginous tissue with an ECM rich in GAG. After 4 weeks of culture, constructs had GAG, DNA and collagen fractions comparable to avian epiphyseal cartilage. The addition of beta-glycerophosphate and dexamethasone to the culture medium during cultivation produced a more bone-like tissue with a mineralised matrix at the periphery and reduced amounts of GAG and collagen.

The cone viscometer has been used to apply uniformly distributed laminar shear forces across the surface of cells in monolayer. Smith et al. (48) used chondrocytes plated at high density in monolayer from normal and osteoarthritic human cartilage and from normal bovine cartilage for experiments. Cultures were exposed to shear forces of 1.6Pa and after 48 and 72 hours of exposure to fluid-induced shear, individual chondrocytes elongated and aligned tangential to the direction of cone rotation. Results indicated a 2-fold increase in GAG synthesis, but an inflammatory response was also detected with a 10-20-fold increase in prostaglandin E2 and a 9-fold increase in metalloproteinase tissue inhibitor. The fluid-induced shear stress also increased osteoarthritic markers interleukin-6 10-fold (7, 48). These results indicate that the use of a cone viscometer to induce shear stresses may have an overall negative effect on chondrogenesis.

5.5.4 Rotating Wall Bioreactor

A rotating wall bioreactor was developed by NASA (see Fig. 11) and has proved to be successful in the culturing of cells. This system simulates a microgravity environment and operates on the principle of a low-shear, high diffusion bioreactor. It consists of two concentric cylinders that can rotate independently at the same speed or at different speeds depending on the desired shear environment (7). The inner cylinder is covered with a silicone membrane to allow for the perfusion of gases. Culture medium is contained in the space between the two cylinders in which pre-seeded scaffolds are placed (57). As the bioreactor is rotated along its horizontal axis a laminar rotational flow field is created. By adjusting


the speed of rotation of the vessel through cultivation, constructs are kept in a state of constant free-fall as the centrifugal force balances the forces of gravity and drag. The rotational speed is increased during the cultivation period as the constructs grow in order to keep them suspended within the vessel (16, 57). Freed et al. (16) used the rotating wall vessel to study chondrogenesis in cell-polymer constructs in vitro. Chondrocytes were harvested from full-thickness articular cartilage from the femoropatellar groove of 2 to 3-week-old bovine calves and were seeded in to fibrous PGA disks using a mixed flask. After 6 days, constructs were transferred to the bioreactor where they were cultured at intervals between 4 and 40 days. The bioreactor was rotated at rates between 15 and 30 rpm to maintain the constructs suspended in the medium as they increased in mass. Cell densities, GAG and collagen type II were higher at the construct periphery in 4- and 12-day constructs than at the centre. When constructs were cultured for 40 days cells were uniformly distributed across the construct and the ECM resembled cartilaginous tissue over the cross-sectional area (16). When results were compared to those of native cartilage, 40-day constructs contained similar fraction of cells, 68% GAG and 33% collagen type II per gram wet weight. In earlier constructs the

Fig. 11. The Rotating Wall Bioreactor developed by NASA, after Obradovic et al. (40)


higher cell densities at the periphery may be due to higher cell proliferation rates at the surface due to superior nutrient and gas transfer rates or to preferential seeding at the periphery. This continued synthesis and deposition of the ECM may allow for a tissue comparable to native cartilage to be obtained with increased cultivation time.

Vunjak-Novakovic et al. (57) and Martin et al. (35) carried out experiments using PGA disks seeded with chondrocytes harvested from 2-3 week old calves in three different environments: static flask, mixed flask (as mentioned in Section 5.5.2) and rotating wall vessels. Relationships between the composition and mechanical properties of engineered cartilage were studied. In the rotating vessels the constructs were dynamically suspended in the rotating laminar flow field. The flat circular areas of the constructs aligned perpendicular to the direction of flow. Constructs cultured in the rotating wall vessel produced the most significant results in both composition and mechanical function, with the constructs being continuously cartilaginous over the cross-sections after 6 weeks. After this culture period, GAG synthesis was comparable to cartilage explants and was approximately 2-fold higher than that in constructs from static or mixed flasks. Total collagen was comparable to mixed flask results, significantly higher than in static flasks but was significantly lower than explant values.

Overall constructs from rotating vessels after 6 weeks of culture contained 78% as much GAG and 43% as much collagen as explants (57). Mechanically these constructs also had the highest equilibrium modulus, dynamic stiffness and streaming potential and the lowest permeability (35, 57). However constructs from all three environments proved to be structurally and functionally inferior to native cartilage. It was therefore proposed that the accumulation of GAG and collagen in constructs precedes their assembly into functional ECM or that the ECM assembly in the constructs differs from that in natural cartilage. Constructs were also cultured for 7 months in rotating wall vessels by Vunjak-Novakovic et al. (57) who reports that this extra cultivation time led to constructs that were comparable to cartilage in that GAG levels were increased and they had better mechanical properties than 6 week constructs.


Martin et al. (35) carried out a more extensive investigation of constructs cultured for 7 months. Such constructs reached or exceeded physiological levels of GAG content. Total collagen reached 39% of native cartilage at week 6 and remained at this level. Equilibrium moduli and hydraulic permeabilities were similar to native cartilage, compared to 6 week constructs where the equilibrium modulus was 18% of native cartilage and the permeabilities were 4-fold higher. Dynamic stiffness and streaming potentials were highest in 7-month constructs but were still significantly lower than native cartilage at 46% and 28%, respectively. These studies indicate that there is a relationship between the composition of tissue engineered cartilage and its mechanical properties with GAG content, equilibrium moduli and permeability similar to native cartilage, and collagen content, dynamic stiffness and streaming potentials being significantly lower. By changing cultivation conditions and time, tissue compositions and mechanical properties can be controlled and studied. The effects of fluid flow due to the different hydrodynamic conditions are also highlighted in this study with the laminar, low shear environment proving most effective.

Such results can be compared to those obtained by Carver and Heath (6, 20) who employed a semicontinuous medium perfusion and intermittent hydrostatic pressure system, as discussed in Section 5.2. It must be noted that the semicontinuous medium perfusion was not used to stimulate cells but solely to introduce fresh medium into the system. Like the results for GAG and collagen reported by Vunjak-Novakovic et al. (57) and Martin et al. (35) as discussed above, Carver and Heath (6, 20) found that chondrocytes harvested from foals and seeded on PGA scaffolds produced quantities of GAG similar to that found in native foal cartilage. Collagen synthesis was increased in these constructs compared to the response of chondrocytes harvested from adult horses, but no constructs reach native levels. This is interesting as it illustrated that cell harvested from two different types of animal and two different systems, one employing fluid shear and the other employing hydrostatic pressure, produce similar trends.

A study was also undertaken by Freed et al. (15) to study the effects of microgravity in space on the tissue engineering of cartilage. Articular chondrocytes, harvested from 2-3 week old bovine calves were seeded on


PGA scaffold discs and grown in rotating wall vessels for 3 months on Earth and then for a further 4 months under pure microgravity conditions on the Mir Space Station and on Earth. The constructs from Mir and Earth were then tested for construct composition and mechanical behaviour. Results found that cells maintained their viability and differentiated phenotype in both environments. Constructs grown on earth maintained their initial discoid shape as the discs aligned their flat circular area perpendicular to the direction of the flow, whereas those grown on Mir became more spherical as they were exposed to uniform shear and mass transfer at all surfaces which resulted in tissue growing equally in all directions. Constructs grown on Mir and Earth had fractions of type II collagen that were comparable to each other at 78% and 75.3% total collagen, respectively. A fibrous outer capsule, 0.15-0.45mm thick, was found on both sets of constructs. It was also found that the mechanical properties of constructs grown on Mir were inferior to those grown on Earth, with compressive moduli of 0.313±0.045MPa and 0.932±0.049MPa, respectively. It is believed that the lower modulus of Mir-grown constructs is due to the low GAG content.

The rotating wall bioreactor was also used in a study by Obradovic et al. (40) to investigate the integration of engineered cartilage with native cartilage. In mixed flasks, disc-shaped PGA scaffolds were dynamically seeded with chondrocytes harvested from the femoropatellar groove of 2-3 week old bovine calves and cultured for 5 days or 5 weeks. These constructs were then press-fit into cartilage explant rings and cultured for 1-8 weeks in the rotating bioreactor. The composites were evaluated biochemically, histologically and mechanically. The adhesive strength of the disk-ring interface of the composites was determined as the stress required to fracture the integration site. The highest adhesive strength was obtained from composites of immature construct disks and trypsin treated explants. This would indicate that engineered cartilage is only required to be cultured to a certain stage in vitro before it is transplanted in vivo. The scaffold would have a desired compressive stiffness that would initially act as the load bearing material until a sufficient ECM is produced and the construct integrates into the defective site.

The trypsin treatment removed some GAG from the integration interface of the explant rings as proteoglycans inhibit cell adhesion. The


immature construct disks containing proliferating cells within an immature matrix would migrate to the trypsin treated explant ring, where the permeability is increased due to the removal of protelglycans. These cells, capable of synthesising ECM, would have filled in the gaps in the explant interface increasing the adhesive strength of the composite (40). These findings have important implications for the integration of tissue engineered cartilage with native cartilage in vivo.

6. Discussion and Conclusion

It is now evident that a wide range of systems have been developed for subjecting both MSCs and chondrocytes to mechanical stimuli for the purpose of producing tissue engineered cartilage. We can see different that experimental set-ups and evaluation techniques make it difficult to compare results; however it must be highlighted that all tissue engineered cartilage to date has been inferior to that of native cartilage, in both composition and function. Experiments have produced varying results suggesting that a particular combination of frequency, duration of loading and sample type is required to elicit a positive response from cells. Many types of mechanical stimuli have been analysed in this review with most stimuli producing positive results when compared to static control samples. It can be seen that most studies show an overall increase in synthesis however these increases are of varying magnitudes and not all groups of data within each of the studies produced such positive results. Some trends obtained from various systems used by researchers are outlined in Table 7.

The most successful engineered cartilage constructs have been obtained from the low shear, low turbulent rotating wall vessel and from intermittently applied hydrostatic pressure with simultaneous medium perfusion. In these studies GAG levels were similar to native cartilage values. However, it is interesting that both studies produced inferior collagen type II fractions compared to native values. This highlights the hypothesis that GAG and collagen have different signalling mechanisms. Researchers are now beginning to combine mechanical stimuli (6, 9, 57) to benefit from the advantages of each individual stimulus in one system.


Table 7: Comparison of results obtained from various mechanical environments, all relative to static controls.

Dynamic

Compressiona Hydrostatic Pressureb

Tensionc Mixed Flaskd

Rotating Wall

Vessele

Mass transfer

α

Proteoglycan /GAG

Total Protein/ Collagen

Cell Proliferation

Other β

γ

δ

= increase = decrease = no change aSee Buschmann et al. (4) α - Via combined perfusion bSee Carver et al. (6) β -Radial Biosynthesis cSee DeWitt et al. (10) γ - Acts uniformly on cells, No tissue

deformation or fluid flow dSee Vunjak-Novakovic et al. (57) δ - Formation of fibrous capsule eSee Martin et al. (35)

It is this approach that will be used to develop future systems in the attempt to produce an optimised bioreactor to develop engineered tissue. Ultimately, the aim is to apply physical loading to cells that mimic the physiological environment to develop tissue constructs that can undergo mechanical forces in vivo. However as discussed by Martin [36], the specific physical stimuli required to induce a specific response from chondrocytes is still unclear. As discussed cells in different tissue


engineered systems experience biochemical and mechanical environments that are significantly different from those encountered in vivo.

The specific cellular mechanotransduction signalling pathways are still unknown, but may involve cell deformation, hydrostatic pressure, or changes in the osmotic pressure, pH or streaming potentials associated with deformation of the ECM (30). Studies have shown the requirement of an ECM surrounding cells within a construct prior to the application of mechanical stimuli to induce biosynthesis. This indicates a requirement to culture constructs for a period of time prior to the application of mechanical stimuli. Collagen type II and GAG synthesis are believed to have different signalling mechanism as they responded differently to the intermittent pressure as reported by both Carver et al.(6) and Ikenoue et al. (22).

The zonal organisation associated with articular cartilage is not likely to be obtained during in vitro cultivation. It is proposed that the zonal organisation of articular cartilage into superficial, transitional, deep and calcified zones may develop when tissue engineered constructs are implanted in vivo (14). Kafienah et al (25) suggests that the complex organisation of articular cartilage may arise when constructs are subjected to physiological loading in vivo and is not an intrinsic property of articular chondrocytes.

As discussed in Section 2.3, mesenchymal stem cells (MSCs) are undifferentiated cells that have the potential to differentiate into numerous cell phenotypes, including chondrocytes. As a result, MSCs may provide a useful source of cells for cartilage tissue engineering. Few studies have examined the effects of biophysical stimuli on MSCs even though scientists, such as Pauwels (27, 55), have proposed mechanoregulation theories for the prediction of MSC differentiation into various phenotypes as a result of applied mechanical stimuli.

Angele et al. (1) examined the influence of cyclic hydrostatic pressure on bone marrow-derived human mesenchymal progenitor cells that were cultured in a medium to induce chondrogenic differentiation. It is not known from this study if the hydrostatic pressure directly influenced the differentiation of the stem cells into chondrocytes as a chondrogenic medium was used. However, it does indicate that


hydrostatic pressure enhanced cartilaginous matrix formation. No study has looked at the possibility of using mechanical stimuli alone to induce the differentiation of MSCs into the chondrocyte or osteoblast lineage. If this was found to be possible great advances would be made in tissue engineering studies, as the harvesting of differentiated cells from donors would no longer be required.

It must also be noted that in all tissue engineering studies that have been performed to date, the stress and strains that cells experience when seeded in 3D scaffolds and exposed to mechanical loading has not yet been characterised. There is still a fundamental lack of understanding of the type of environment that is created around individual cells when subjected to various stimuli of different frequencies, loads and durations. Computational techniques such as finite element modelling are now being employed to calculate the mechanical stimuli that cells experience in 3D environments. Such models are used to predict the differentiation of MSCs into various cell types such as fibroblasts, chondrocytes and osteoblasts, and the corresponding tissue phenotypes these cells produce, depending on the mechanical stimuli that the cells are subjected to. For example, Kelly and Prendergast (27) developed a mechano-regulation model to predict how stem cells would differentiate in osteochondral defects. In this model, the differentiation process was controlled by the biophysical stimuli (shear strain and fluid velocity) that were developed during loading. Such models could be applied to in vitro tissue engineering where cells are seeded in scaffolds and subjected to specific mechanical stimuli. If experimental results matched the computational predictions, finite element modelling could be employed to aid in the design of an optimised bioreactor that would apply specific mechanical stimuli to constructs to elicit specific cell responses.

Acknowledgements Ms. Louise McMahon is funded by an Embark Scholarship from the Irish Research Council for Science, Engineering and Technology. Funding for this project is also provided by the Programme for Research in Third Level Institutions (PRTLI) Cycle III, Administered by the Higher Education Authority.


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The State-Of-The-Art in Cartilage Bioreactors

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