Experimental models of osteoarthritis

Cartilage is a highly specialized tissue containing only one type of cells (chondrocytes) and characterized by the absence of blood and lymphatic vessels. Cartilage is mainly nourished by absorption from the synovial fluid. Chondrocyte metabolism is regulated by a number of soluble factors produced locally by chondrocytes and surrounding tissues. Chondrocyte function also depends on the composition of the extracellular environment (oxygen tension, ion concentration, pH, etc.), the composition of the ECM, the interaction of cells and the matrix, and physical signals. The main objective of experimental modeling is to create cultures in the extracellular environment without changing the phenotype of mature cells. The second objective is to create cultures to study the premature, delayed, short-term, or prolonged response of chondrocytes to chemical and/or physical signals. In vitro studies also provide an opportunity to study the behavior of chondrocytes in osteoarthrosis. The third objective is to develop coculture systems that allow studying the interactions of various tissues in the joint. The fourth task is the preparation of cartilage implants for subsequent transplantation. And finally, the fifth task is the study of growth factors, cytokines or therapeutic agents that are capable of stimulating reparation and/or inhibiting cartilage resorption.

Over the past decades, various models of articular cartilage cell cultures have been created, including monolayer cultures, suspended cultures, chondron cultures, explants, cocultures, and immortal cell cultures. Each culture has its own advantages and disadvantages, and each is suitable for studying one specific aspect of chondrocyte metabolism. Thus, cartilage explants are an excellent model for studying the turnover of matrix elements, which requires genuine cell surface receptors and normal cell-matrix and matrix-cell interactions. At the same time, it is recommended to study matrix deposits or mechanisms regulating chondrocyte metabolism on a culture of isolated cells. A low-density monolayer culture is necessary for studying the process of cell differentiation. Cultures suspended in a natural or synthetic matrix are a model for analyzing the adaptive response of chondrocytes to mechanical stress.

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Chondrocyte cultures

Several important points should be taken into account when selecting cartilage tissue for in vitro studies. The matrix composition and metabolic activity of chondrocytes vary among joints, and the latter also depends on the depth of chondrocyte location in the tissue. These data were obtained in several experiments in which isolated chondrocyte subpopulations from cartilage zones of different depths were studied. A number of morphological and biochemical differences were found between cultured chondrocytes located in the superficial and deep layers of articular cartilage. Superficial cells synthesize a sparse, proteoglycan-poor fibrillar matrix, whereas deeper cells produce a matrix rich in fibrils and proteoglycans. Moreover, superficial cells produce relatively more small non-aggregated proteoglycans and hyaluronic acid and relatively less aggrecan and keratan sulfate than deeper chondrocytes. Another important distinctive feature of the metabolism of chondrocytes isolated from cartilage zones of different depths is the response to an exogenous stimulus. According to M. Aydelotte et al., bovine chondrocytes from the superficial zone of cartilage were more sensitive to IL-1 than cells from the deep zone.

Cell behavior also depends on tissue location. Chondrocytes from rib and ear cartilage from the same animal respond differently to growth factors such as fibroblast growth factor (FGF) and TGF-beta. FGF increased thymidine, proline, and leucine incorporation into cultured rib but not ear chondrocytes. TGF-beta increased thymidine incorporation into rib and ear cartilage chondrocytes but had no effect on thymidine and proline incorporation into ear chondrocytes. Cartilage cells from areas of high stress differ from those from areas of low stress on the cartilage. Thus, chondrocytes of mature sheep knee joint cartilage from the central region of the articular surface of the tibia not covered by the meniscus, which bears the greatest load in vivo, synthesize less aggrecan, but more decorin than cells from the zones covered by the meniscus. The authors also emphasize the importance of using cartilage from identical joint zones when studying the synthetic function of joints.

The metabolism of chondrocytes and their response to regulatory factors also significantly depend on the age of the donor, their skeletal development, and the condition of the joints from which the cells are taken. In human chondrocytes, a significant decrease in the proliferative response with age is observed. The greatest decrease is observed in donors aged 40-50 years and over 60 years. Moreover, the severity of the proliferative response to growth factors (e.g., FGF and TGF-beta) decreases with aging. In addition to quantitative changes in chondrocyte proliferation, there are also qualitative changes. Cells from young donors (10-20 years) respond better to platelet-derived growth factor (PDGF) than to TGF-beta, while the opposite is observed in cells from adult donors. Several mechanisms are used to explain age-dependent changes in the synthetic function of chondrocytes and their response to growth factors. These include a decrease in the number and affinity of surface cell receptors, changes in the synthesis and bioactivity of growth factors and cytokines, and modification of post-receptor signals.

Pathological condition of joints also changes morphology and metabolic activity of chondrocytes. Thus, J. Kouri et al. (1996) identified three subpopulations of chondrocytes in cartilage in osteoarthrosis. Chondrocytes from the superficial and upper part of the middle of the cartilage form clusters and synthesize a greater amount of proteoglycans and collagen. TGF-beta and insulin-like growth factor (IGF) are able to stimulate the synthesis of proteoglycans by chondrocytes and partially neutralize the effects of IL-1 and TNF-a. Explants of cartilage affected by osteoarthrosis and chondrocytes isolated from the cartilage of a patient with osteoarthrosis are more sensitive to stimulation of TGF-beta than chondrocytes of healthy cartilage. These differences are most likely associated with phenotypic changes in chondrocytes in the upper layers of articular cartilage.

Isolation of individual chondrocytes is achieved by sequential treatment of the ECM with proteolytic enzymes. After their release from the ECM, the isolated cells are ideal for studying the de novo synthesis of matrix components. Some authors use only clostridial collagenase, while others pre-incubate the cartilage with trypsin, pronase, DNase, and/or hyaluronidase. The number of isolated cells depends on the enzymes used. Thus, when treated with collagenase alone, 1.4-10 ⁶ chondrocytes can be obtained from 1 g of tissue, while using pronase, hyaluronidase, and collagenase - 4.3-10 ⁶. When treated with collagenase, aggrecan, proteins, IL-6, and IL-8 remain in the cell culture in significantly greater quantities than with sequential treatment with different enzymes. There are several explanations for these differences between the two cell cultures:

• Cell receptors are damaged or inhibited by enzymes, TGF-beta inhibits DNA and proteoglycan synthesis in freshly isolated chondrocytes (day 1), whereas DNA and proteoglycan synthesis in chondrocytes cultured in a monolayer (7 days) are stimulated by TGF-beta. However, an adequate period is required for re-expression of these membrane components before the start of the experiment.
• Exogenous proteases can disrupt the integrin-mediated cell-matrix interaction. The integrin family promotes the attachment of chondrocytes to ECM molecules (Shakibaei M. et al., 1997). This disruption can affect the expression of matrix genes.
• The remains of matrix components can regulate the synthetic function of chondrocytes. Integrins are able to recognize the products of ECM degradation, thus playing an important role in tissue reparation after the action of proteolytic enzymes. T. Larsson et al. (1989) reported that the addition of intact or fragmented proteoglycans to cell culture stimulates the synthesis of proteins and proteoglycans. However, a high level of hyaluronic acid causes a significant decrease in the inclusion of sulfates in the synthesis of proteoglycans by chondrocytes of the chicken embryo, mature chondrocytes of the pig and rat chondrosarcoma cells. Moreover, hyaluronic acid is an inhibitor of proteoglycan release from cells even in the presence of IL-1b, TNF-a, FGF, which indicates counteraction of the first biological activity of growth factors and cytokines. The exact mechanism underlying the action of hyaluronic acid remains unclear; Chondrocytes are known to contain a receptor for hyaluronic acid associated with actin filaments of the cytosol. Binding of hyaluronic acid to its receptor stimulates protein phosphorylation. Thus, these data demonstrate modulation of chondrocyte metabolic function by fragmented or native molecules of matrix proteins through activation of cell membrane receptors.
• Rapid stimulation of matrix protein synthesis by chondrocytes by enzymes may be a consequence of changes in chondrocyte shape and/or cytoskeletal reorganization.
• Some cytokines (e.g., IL-8) and growth factors (e.g., IGF-1, TGF-β) are sequestered in the ECM. The best-known example is the binding of TGF-β by decorin, which results in a decreased ability of the former to induce cell growth in Chinese hamster ovarian cells. The finding that decorin content in cartilage increases with age suggests a decrease in TGF-β bioavailability with aging. Growth factors and cytokines may be released from matrix debris during culture and subsequently modulate chondrocyte function.

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Monolayer culture of chondrocytes

The differentiated phenotype of chondrocytes is primarily characterized by the synthesis of type II collagen and tissue-specific proteoglycans, as well as a low level of mitotic activity. There is evidence that with prolonged cell cultivation in a monolayer, as well as after several repeated cell passages, chondrocytes lose their spherical outlines and acquire an elongated, fibroblast-like shape. With such fibroblastic metaplasia, the synthetic function of the cells is also modified, characterized by a progressive decrease in the synthesis of collagens types II, IX and XI and an increase in the synthesis of collagens types I, III and Y. Small non-aggregated proteoglycans are synthesized due to functional aggrecan. The synthesis of cathepsin B and L is extremely low in differentiated cells, but increases in the process of loss of differentiation. Collagenase-1 is expressed in differentiated chondrocytes; with prolonged cultivation, its expression decreases, while the production of tissue inhibitors of metalloproteases (TIMPs) increases.

Differentiated chondrocytes re-express collagen of the differentiated phenotype when transferred from monolayer to suspended culture. The differentiation process is probably related to the cell shape. This property is regularly used by researchers studying defective grafts with autologous chondrocytes. A small number of cells obtained from biopsy material can be expanded in monolayer culture and then reintroduced into a three-dimensional matrix before transplantation. Re-expression of a specific phenotype by dedifferentiated chondrocytes transferred to agarose culture can be stimulated by TGF-β, ossein-hydroxyapatite complex, and ascorbic acid.

In response to growth factors and cytokines, chondrocytes are modified during the differentiation process. The cellular response to cytokines and growth factors differs between undifferentiated and differentiated chondrocytes. IL-1 stimulates fibroblast proliferation, whereas the growth of undifferentiated chondrocytes is inhibited by IL-1. DNA synthesis is stimulated by IGF-1 in elongated but not flattened chondrocytes. In differentiated chondrocytes, the stimulatory effects of IL-1beta and TNF-a on procollagenase production are more pronounced than in undifferentiated chondrocytes.

Chondrocyte Cultivation

Cultivation of chondrocytes in suspension in a liquid medium or in a natural or synthetic three-dimensional matrix stabilizes the chondrocyte phenotype. The cells retain their spherical shape and synthesize tissue-specific proteins. Suspended culture of chondrocytes is usually recommended for studying the formation of a new pericellular matrix. Cultures of chondrocytes in synthetic or natural absorbent polymers are used for implantation of cells into cartilage defects to stimulate regeneration of joint cartilage tissue. The synthetic or natural medium for implanted cells must meet a number of requirements:

• implants must have a porous structure for cell adhesion and growth,
• neither the polymer itself nor its degradation products should cause inflammation or toxic reactions when implanted in vivo,
• the graft carrier must have the ability to bond to adjacent cartilage or subchondral bone,
• the natural or synthetic matrix must have the ability to absorb, its degradation must be balanced by tissue regeneration,
• to facilitate cartilage repair, the chemical structure and pore architecture of the matrix must facilitate the maintenance of the cellular phenotype and the synthesis of tissue-specific proteins by the chondrocytes placed in it,
• During in vivo implantation, it is necessary to study the mechanical properties of the synthetic or natural matrix.

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Suspension of chondrocytes in liquid phase

Attachment of cells to plastic vessels in which chondrocytes are cultured can be prevented by coating their walls with a solution of methylcellulose, agarose, hydrogel (poly-2-hydroxyethyl methacrylate), or a collagen-agarose mixture. Under these conditions, chondrocytes form clusters and synthesize mainly aggrecan and tissue-specific collagens (II, IX, XI types). Two types of cells are usually found. The cells located in the center retain a spherical shape and are surrounded by a well-developed ECM, which is confirmed by histochemical and ultrastructural studies. At the periphery, chondrocytes have discoid outlines and are surrounded by a sparse ECM; little is known about the functional characteristics of such cells.

It is possible to cultivate chondrocytes on microcarriers maintained in suspension; dextran beads (cytodex), collagen-coated dextran beads (cytodex III), and non-porous microspheres of type I collagen (cellagen) are used as microcarriers. Under these culturing conditions, chondrocytes attach to the surface of the microcarrier, retain their spherical shape, and produce a matrix-like material. Moreover, the use of cellagen promotes chondrocyte proliferation and re-expression of the normal phenotype. Therefore, culturing chondrocytes on cellagen microspheres can be used to restore the cell phenotype before transplantation.

Another method of culturing chondrocyte suspension in a liquid medium is their cultivation in the form of dense balls consisting of cells (0.5-1 * 10 ^b ), obtained by centrifugation. Such chondrocytes are capable of producing a matrix containing a large amount of proteoglycans, collagen type II, but not collagen type I, which is confirmed by histological, immunohistochemical and quantitative methods.

Suspension of chondrocytes in natural ECM

Chondrocytes can be cultured in suspension in a three-dimensional matrix (soft agar, agarose, collagen gel or sponge, hyaluronic acid, fibrin glue, alginate beads).

Chondrocytes cultured in agarose retain their normal phenotype and synthesize type II collagen and tissue-specific aggrecan aggregates. When cultured in agarose, proteoglycans synthesized by the cell are released into the medium for 50 days. For comparison, in a monolayer culture, the cellular phase is overfilled with glycosaminoglycans already in the first 5-6 days of cultivation; when cultured in the medium, after increased synthesis and release of glycosaminoglycans in the first 8-10 days, their time-dependent decrease occurs. Nevertheless, the behavior of chondrocytes when cultured in agarose differs from that in vivo. In agarose, a large number of synthesized aggrecan aggregates contain smaller molecules and fewer molecules than in vivo. TGF-β stimulates proteoglycan synthesis in the explant, but reduces aggrecan synthesis in agarose.

Alginate is a linear polysaccharide obtained from brown seaweed. In the presence of divalent cations, such as Ca ²⁺ ions, this polymer becomes a gel. Each chondrocyte trapped in alginate is surrounded by a matrix of negatively charged polysaccharides, the pores of which are comparable to those in hyaline cartilage. The matrix formed by chondrocytes in alginate beads consists of two compartments - a thin layer of cell-associated matrix corresponding to the pericellular and territorial matrix of articular cartilage and a more distant matrix equivalent to the interterritorial in native tissue. On the 30th day of culture, the relative and absolute volume occupied by the cells and each of the two compartments in an alginate bead are almost completely identical to those in native cartilage. For almost 30 days, chondrocytes retain their spherical shape and produce aggrecan, the hydrodynamic properties of which are similar to those of aggrecan molecules in the articular cartilage matrix, as well as collagen molecules of types II, IX, and XI. At the same time, like other suspension cultures, flattened cells are present on the surface of alginate beads, which produce a small amount of collagen molecules of type I, which are directly released into the medium and are not incorporated into the ECM. Moderate proliferation of chondrocytes is observed in alginate beads. After 8 months of cultivation in alginate gel, mature chondrocytes do not lose metabolic activity and continue to synthesize tissue-specific collagen type II and aggrecan.

H. Tanaka et al. (1984) investigated the diffusion properties of various natural molecules in alginate and found that molecules larger than 70 kDa did not diffuse through alginate. Thus, cell culture in alginate is suitable for studying the regulation of matrix biosynthesis and ECM organization. The availability of cells cultured in alginate allows one to study the action of peptide regulatory factors and pharmacological agents at the transcriptional, post-transcriptional, and translational levels.

Chondrocytes are also cultured in a matrix of collagen fibers of types I and II. S. Nehrer et al. (1997) compared the functioning of canine chondrocytes in porous collagen-proteoglycan polymer matrices containing collagens of different types. They found important differences in the morphology of the biosynthetic function of chondrocytes cultured in collagen matrices containing collagen types I and II. Cells in a matrix of collagen type II retained their spherical shape, while in collagen type I they had a fibroblast-like morphology. Moreover, in a matrix of collagen type II, chondrocytes produced a greater amount of glycosaminoglycans. J. van Susante et al. (1995) compared the properties of chondrocytes cultured in alginate and collagen (type I) gel. The authors found a significant increase in the number of cells in the collagen gel, but from the 6th day of cultivation the cells lost their characteristic phenotype, turning into fibroblast-like cells. In the alginate gel, a decrease in the number of cells was observed, but chondrocytes retained their normal phenotype. In the collagen gel, the number of proteoglycans per cell was significantly higher than in alginate, but in the gel a decrease in the synthesis of matrix elements was observed, starting from the 6th day of cultivation, while in alginate the synthesis continued to increase.

Solid three-dimensional fibrin matrix is a natural substance that supports chondrocytes suspended in it in a differentiated phenotype. Three-dimensional fibrin matrix can also be used as a carrier in chondrocyte transplantation. The advantages of fibrin are the absence of cytotoxicity, the ability to fill space, and adhesive capacity. Histological and biochemical studies, autoradiography, and electron microscopy have shown that chondrocytes in fibrin gel retain their morphology, proliferate, and produce matrix even after 2 weeks of cultivation. However, G. Homminga et al. (1993) reported that fibrin disintegration begins after 3 days of cultivation, and chondrocyte dedifferentiation progresses.

Suspension of chondrocytes in artificial (synthetic) ECM

Cartilage implants for reconstructive or orthopaedic surgery can be obtained by growing isolated chondrocytes in vitro in a synthetic biocompatible matrix.

Chondrocytes cultured in polyglycolic acid proliferate and maintain normal morphology and phenotype for 8 weeks. The chondrocyte-polyglycolic acid complex consists of cells, glycosaminoglycans, collagens, and has an external collagen capsule. However, such implants contain two types of collagen molecules - I and II. Implants from chondrocytes dedifferentiated by a series of passages have a greater amount of glycosaminoglycans and collagens than implants from primarily undifferentiated chondrocytes.

L. Freed et al. (1993b) compared the behavior of human and bovine chondrocyte cultures in fibrous polyglycolic acid (FPGA) and porous polylactic acid (PPLA). After 6-8 weeks of culturing bovine chondrocytes in FPGA or PPLA, the authors observed cell proliferation and regeneration of the cartilage matrix. In FPGA, chondrocytes had a spherical shape and were located in lacunae surrounded by a cartilage matrix. After 8 weeks of in vitro culturing, the regenerated tissue contained up to 50% dry matter (4% cellular mass, 15% glycosaminoglycans, and 31% collagens). In PPLA, the cells had a spindle-shaped shape and a small amount of glycosaminoglycans and collagen. In FPGA, cell growth was 2 times more intense than in PPLA. In vivo, chondrocytes grown in VPGK and PPLC produced tissue histologically similar to cartilage within 1-6 months. The implants contained glycosaminoglycans, collagens type I and II.

Bovine fetal chondrocytes were cultured in porous high-density hydrophobic and hydrophilic polyethylene. After 7 days of incubation in both substrates, the cells retained a spherical shape and contained mainly type II collagen. After 21 days of cultivation, the hydrophilic matrix was found to contain more type II collagen than the hydrophobic matrix.

Cartilage tissue can also be obtained by culturing in a monolayer on Millicell-CM filters. Pre-coating of the filters with collagen is necessary for the attachment of chondroitins. Histological examination of the culture demonstrates the accumulation of chondrocytes in the ECM containing proteoglycans and type II collagen. Type I collagen was not detected in such a culture. Chondrocytes in the obtained cartilage tissue are spherical in shape, but on the surface of the tissue they are somewhat flattened. The thickness of the newly formed tissue increased with time and depended on the initial density of the cell monolayer. Under optimal culturing conditions, the thickness of the cartilage tissue reached 110 μm, the organization of its cells and collagen into superficial and deep layers is similar to that of articular cartilage. The ECM contains approximately 3 times more collagen and proteoglycans. After 2 weeks of cultivation, matrix accumulation was observed, allowing the tissue to be extracted from the filter and used for transplantation.

Sims et al. (1996) studied the cultivation of chondrocytes in polyethylene oxide gel, an encapsulated polymer matrix that allows the transfer of large numbers of cells by injection. Six weeks after injection into the subcutaneous tissue of athymic mice, new cartilage was formed, which was morphologically characterized by a white opalescence similar to hyaline cartilage. Histological and biochemical data indicated the presence of actively proliferating chondrocytes producing ECM.

Explantation

Explantation of cartilage tissue is used to study the processes of ana- and catabolism in it, maintenance of homeostasis, resorption and repair. Chondrocytes in cartilage explants maintain a normal phenotype and ECM composition similar to those in articular cartilage in vivo. After 5 days of culturing in the presence of serum, a constant level of synthesis and natural degradation processes is achieved. Tissue resorption can be accelerated in the main culture and in culture with the addition of serum using a number of agents, such as IL-IB, TNF-a, bacterial lipopolysaccharides, retinoic acid derivatives or active oxygen radicals. To study cartilage reparation, its damage is induced by soluble inflammatory mediators (H ₂ O ₂, IL-1, TNF-a) or physical rupture of the matrix.

The organotypic culture method is a model for in vitro studies of the effects of isolated external factors on chondrocytes and the surrounding matrix. In vivo, chondrocytes are sparsely located in the ECM and do not contact each other. The articular cartilage explant culture preserves this structural organization, as well as the specific interactions between chondrocytes and the surrounding extracellular environment. This model is also used to study the effects of mechanical stress, pharmacological agents, growth factors, cytokines, and hormones on cartilage metabolism.

Another advantage of cartilage tissue explantation is the absence of damage to chondrocytes under the action of proteolytic enzymes or mechanical factors, which is inevitable when isolating cells. Receptors and other membrane proteins and glycoproteins are protected from damaging factors.

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Chondron culture

Chondron is a structural, functional and metabolic unit of articular cartilage consisting of a chondrocyte, its pericellular matrix and compact filamentous capsule and responsible for matrix homeostasis. Chondrons are mechanically extracted from cartilage and collected using several successive low-speed homogenizations. Chondrons isolated from zones of different cartilage depths can be divided into four categories: single chondron, paired chondrons, multiple (three or more) linearly arranged chondrons (chondron columns), and chondron clusters.

Single chondrons are usually found in the middle layers of intact cartilage, paired chondrons are found at the border of the middle and deep layers, linearly arranged multiple chondrons are typical of the deep layers of intact cartilage. Finally, chondron clusters consist of randomly organized groups of single and paired chondrons, which retain an aggregated state after homogenization. Chondron clusters are large fragments of cartilage, usually containing several chondrons and radially arranged collagen fibrils, i.e., a typical organization characteristic of the deep layers of the matrix. Chondrons are immobilized in transparent agarose, which allows for studies of their structure, molecular composition, and metabolic activity. The chondron-agarose system is considered a micromodel of cartilage, which differs from the traditional chondrocyte-agarose system in that the natural microenvironment is preserved, and there is no need to synthesize and assemble it. Chondron culture is a model for studying the interactions of cells and matrix in articular cartilage under normal and pathological conditions.

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Culture of immortal chondrocytes

Recombinant DNA or oncogene-containing viruses capable of making a cell "immortal" are used to create permanent cell lines. Immortal chondrocytes have the ability to proliferate endlessly while maintaining a stable phenotype. F. Mallein-Gerin et al. (1995) showed that the SV40T oncogene induces the proliferation of mouse chondrocytes, which continue to stably express collagen types II, IX, and XI, as well as articular aggrecan and binding protein. However, such a cell line acquires the ability to synthesize collagen type I when culturing it in a monolayer culture or in an agarose gel.

W. Horton et al. (1988) described a line of immortal cells with a low level of expression of type II collagen mRNA. These cells were obtained by their transformation with a mouse retrovirus containing I-myc- and y-ra-oncogenes. This type of cells represents a unique model for studying the interactions of the articular matrix in the absence of type II collagen, as well as the regulation of type II collagen synthesis.

Culture of chondroprites with mutated or deleted genes is a convenient model for studying their physiological function. This model is especially suitable for studying the role of specific molecules in the organization of the cartilage matrix or for investigating the effects of various regulatory factors on cartilage metabolism. Chondrocytes with a deleted gene for type IX collagen synthesize collagen fibrils that are wider than normal, indicating that type IX collagen regulates the diameter of the fibrils. As noted in Chapter 1, a mutation in the COLAI gene encoding type II collagen has recently been discovered in families with primary generalized osteoarthritis. To study the effect of mutant type II collagen on the articular matrix, R. Dharmrvaram et al. (1997) transfected (infected with foreign nucleic acid) defective COL ₂ AI (arginine at position 519 is replaced by cysteine) into human fetal chondrocytes in vitro.

Coculture system. In the joint, cartilage interacts with other types of cells contained in the synovial membrane, synovial fluid, ligaments, and subchondral bone. The metabolism of chondrocytes can be affected by various soluble factors synthesized by the listed cells. Thus, in arthritis, articular cartilage is destroyed by proteolytic enzymes and free radicals produced by synovial cells. Therefore, models have been developed to study complex interactions between cartilage and surrounding tissues, which are called cocultures.

S. Lacombe-Gleise et al. (1995) cultured rabbit chondrocytes and osteoblasts in a coculture system (COSTAR) in which the cells were separated by a microporous membrane (0.4 μm) allowing exchange between the two cell types without any direct contacts. This study demonstrated the ability of osteoblasts to stimulate chondrocyte growth via soluble mediators.

A.M. Malfait and co-authors (1994) studied the relationship between peripheral blood monocytes and chondrocytes. This model is convenient for studying cytokine-mediated processes in inflammatory arthropathies (rheumatoid arthritis, seronegative spondyloarthritides, etc.). The authors of the model separated the cells with a protein-binding membrane with pores of 0.4 μm in diameter. The study showed that monocytes stimulated with lipopolysaccharide produced IL-1 and TNF-a, which inhibited the synthesis of aggrecan by chondrocytes and contributed to the degradation of already synthesized aggrecan aggregates.

K. Tada et al. (1994) created a coculture model in which endothelial cells in a collagen (type I) gel were placed in an inner chamber separated from the outer chamber with chondrocytes placed in it by a filter with a pore size of 0.4 μm. In a state of complete isolation from the outer chamber, human endothelial cells formed tubes in a collagen gel in the presence of EGF or TGF-a. When both types of cells were cultured simultaneously, TGF-a-dependent tube formation by endothelial cells was inhibited. Inhibition of this process by chondrocytes was partially eliminated by anti-TGF-beta antibodies. It can be assumed that TGF-beta produced by chondrocytes inhibits vascularization of the cartilage itself.

S. Groot et al. (1994) simultaneously cultured chondrocytes from the hypertrophic and proliferative zones of bone of a 16-day-old mouse fetus with pieces of brain tissue. After 4 days of cultivation, transdifferentiation of chondrocytes into osteoblasts and the beginning of osteoid formation were observed. After 11 days of cultivation, part of the cartilage was replaced by bone tissue and the bone matrix was partially calcified. Some neuropeptides and neurotransmitters produced by brain tissue affect the metabolism of osteoblasts or have receptors for them. Among them are norepinephrine, vasoactive intestinal peptide, calcitonin gene-related peptide, substance P and somatostatin. Pieces of brain tissue cultured together with chondrocytes can produce some of the listed factors capable of inducing the process of transdifferentiation of chondrocytes into osteoblasts.

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The influence of external factors on chondrocyte culture

The influence of oxygen tension on chondrocyte metabolism

In most cases, chondrocyte cultures develop under conditions of atmospheric oxygen tension. However, it is well known that in vivo chondrocytes exist under hypoxic conditions and oxygen tension varies under various pathological conditions. During the maturation process, significant changes in the blood supply to the epiphyses are observed. Since vascularization varies in different zones of the growth plate, the oxygen tension in them also varies. C. Brighton and R. Heppenstall (1971) demonstrated that in the tibial plate of rabbits, the oxygen tension in the hypertrophic zone is lower than in the surrounding cartilage. Measurements of some metabolic parameters showed that chondrocytes are able to quickly respond to local changes in oxygen concentration. First of all, at low oxygen tension, its consumption by chondrocytes decreases. With a decrease in oxygen tension from 21 to 0.04%, glucose utilization increases, the activity of glycolytic enzymes and the synthesis of lactic acid increase. Even at low oxygen tension, the absolute amount of ATP, ADP and AMP remains stable. These data indicate that chondrocyte metabolism is aimed at maximal energy conservation. However, synthetic activity, and therefore reparation processes, change under hypoxic conditions.

High oxygen tension also affects chondrocyte metabolism, causing a decrease in proteoglycan and DNA synthesis and degradation of the cartilage matrix. These effects are usually accompanied by the production of free oxygen radicals.

The influence of ion concentration and osmotic pressure of the environment on chondrocyte function

In native cartilage, the concentration of ions differs significantly from that in other tissues: the sodium content in the extracellular medium is 250-350 mmol, and its osmolarity is 350-450 mosmol. When chondrocytes are isolated from the ECM and incubated in standard media (DMEM (Dulbecco's Minimal Essential Medium), osmolarity is 250-280.7 mosmol), the environment surrounding the cells changes dramatically. In addition, the concentration of calcium and potassium in standard media is significantly lower than in native tissue, and the concentration of anions is significantly higher.

Addition of sucrose to the medium increases its osmolarity and induces a transient intracellular increase in the concentration of H ⁺ and calcium anions in the cytosol. Such intracellular changes can affect the processes of chondrocyte differentiation and their metabolic activity. J. Urban et al. (1993) found that the incorporation of ³⁵ 8-sulfate and ³ H-proline by isolated chondrocytes incubated in standard DMEM for 2-4 h was only 10% of that in native tissue. The intensity of synthesis reached a maximum at an osmolarity of the extracellular medium of 350-400 mosmol both in freshly isolated chondrocytes and in cartilage tissue explants. Moreover, the volume of chondrocytes increased by 30-40% after placing the isolated cells in standard DMEM of the specified osmolarity. However, when culturing chondrocytes under conditions of non-physiological osmolarity for 12-16 hours, the cells adapt to the new conditions, reducing the intensity of biosynthesis in proportion to the shift in osmolarity of the extracellular environment.

P. Borgetti et al. (1995) studied the effect of extracellular medium osmolarity on the growth, morphology, and biosynthesis of pig chondrocytes. The authors demonstrated similar biochemical and morphological features of chondrocytes cultured in media with osmolarity of 0.28 and 0.38 mosmol. At a medium osmolarity of 0.48 mosmol, a decrease in cell proliferation and protein synthesis was observed during the first 4-6 hours of cultivation, but these parameters subsequently recovered and eventually reached control values. When chondrocytes were cultivated in a medium with an osmolarity of 0.58 mosmol, the cells lost the ability to maintain the physiological intensity of proliferative processes, and after 6 days the number of chondrocytes was significantly reduced. At a medium osmolarity of 0.58 mosmol, profound inhibition of protein synthesis was observed. In addition, when cultured in media with an osmolarity of 0.28-0.38 mOsm, chondrocytes retain their physiological phenotype; at higher osmolarity (0.48-0.58 mOsm), significant changes in cell morphology occur, which is manifested by the loss of the characteristic phenotype, the transformation of chondrocytes into fibroblast-like cells, and the loss of the ability of cells to assemble matrix proteoglycans. The results of this study indicate the ability of chondrocytes to respond to limited fluctuations in the osmolarity of the extracellular environment.

Changes in the concentration of other ions can also affect the biosynthesis processes in chondrocytes. Thus, the degree of ³⁵ S (sulfate) incorporation increases by half with an increase in the concentration of potassium ions from 5 mmol (the concentration in the standard DM EM medium) to 10 mmol (the concentration in the ECM in vivo). Calcium concentrations below 0.5 mmol promoted collagen production by mature bovine chondrocytes, while a concentration of 1-2 mmol (corresponding to the concentration in the standard DM EM medium) caused a significant decrease in collagen synthesis. A moderate increase in biosynthesis was observed at high calcium levels (2-10 mmol). Various cations participate in the attachment of chondrocytes to ECM proteins. Thus, magnesium and manganese ions provide attachment to fibronectin and type II collagen, while calcium ions do not participate in the attachment of chondrocytes to proteins. Thus, the results of the described studies indicate the influence of changes in extracellular ions of potassium, sodium, calcium and osmolarity of the medium on the biosynthetic function of chondrocytes incubated in standard media.

Effect of mechanical stress on chondrocyte metabolism

Joint immobilization causes reversible cartilage atrophy, which indicates the need for mechanical stimuli for normal metabolic processes in the ECM. In most cases, the cell culture models used exist under normal atmospheric pressure. M. Wright et al. (1996) showed that the mechanical environment affects chondrocyte metabolism, the cell response depends on the intensity and frequency of compressive loading. Experiments with loading on explants of intact articular cartilage in vitro demonstrated a decrease in the synthesis of proteins and proteoglycans under the action of static loading, while dynamic loading stimulates these processes. The exact mechanisms of the effect of mechanical loading on cartilage are complex and are probably associated with cell deformation, hydrostatic pressure, osmotic pressure, electrical potential and surface cellular receptors for matrix molecules. To study the effect of each of these parameters, it is necessary to create a system in which one parameter can be varied independently. For example, explant culture is not suitable for studying cell deformation, but it can be used to study the general effect of pressure on the metabolic activity of chondrocytes. Compression of cartilage leads to cell deformation and is also accompanied by the emergence of a hydrostatic pressure gradient, electrical potential, fluid flow, and changes in such physicochemical parameters as the water content in the matrix, electrical charge density, and osmotic pressure level. Cell deformation can be studied using isolated chondrocytes immersed in agarose or collagen gel.

Several systems have been developed to study the effect of mechanical stimulation on chondrocyte culture. Some researchers use systems in which pressure is applied to the cell culture through the gaseous phase. Thus, JP Veldhuijzen et al. (1979), using pressure 13 kPa above atmospheric with a low frequency (0.3 Hz) for 15 min, observed an increase in the synthesis of cAMP and proteoglycans and a decrease in DNA synthesis. R. Smith et al. (1996) showed that intermittent exposure of a culture of primary bovine chondrocytes to hydrostatic pressure (10 MPa) with a frequency of 1 Hz for 4 h caused an increase in the synthesis of aggrecan and type II collagen, whereas constant pressure did not affect these processes. Using a similar system, Wright et al. (1996) reported that cyclic pressure on cell culture is associated with hyperpolarization of the chondrocyte cell membrane and activation of Ca ²⁺ -dependent potassium channels. Thus, the effects of cyclic pressure are mediated by stretch-activated ion channels in the chondrocyte membrane. The response of chondrocytes to hydrostatic pressure depends on the cell culture conditions and the frequency of the applied load. Thus, cyclic hydrostatic pressure (5 MPa) decreases sulfate incorporation into the chondrocyte monolayer at a frequency of 0.05, 0.25, and 0.5 Hz, whereas at a frequency greater than 0.5 Hz, sulfate incorporation into the cartilage explant increases.

M. Bushmann et al. (1992) reported that chondrocytes in agarose gels alter biosynthesis in response to static and dynamic mechanical loading in the same way as the cultured intact organ. The authors found that mechanical loading generates a hyperosmotic stimulus with a subsequent decrease in pH in chondrocytes.

The effect of mechanical stretching can be studied on a cell culture immersed in a gel. The stretching force can be created using a computer-controlled vacuum. When the system is under a certain degree of vacuum, the bottom of the Petri dish with the cell culture is extended by a known amount, the deformation is maximum at the edges of the bottom of the dish and minimum in the center. The stretch is also transmitted to the chondrocytes cultured in the Petri dish. Using this method, K. Holm-vall et al. (1995) showed that in chondrosarcoma cells cultured in a collagen (type II) gel, the expression of mRNA of a 2 -integrin is increased _{. a 2 βintegrin} is _able to bind to type II collagen. It is considered a mechanoreceptor, since it interacts with actin-binding proteins, thus connecting the ECM and the cytoskeleton.

The effect of pH on chondrocyte metabolism

The pH of the interstitial fluid of the ECM of cartilage tissue is more acidic than in other tissues. A. Maroudas (1980) determined the pH of the articular cartilage matrix at 6.9. B. Diamant et al. (1966) found a pH of 5.5 under pathological conditions. It is known that chondrocytes live at low PO2, which indicates the important role of glycolysis (95% of all glucose metabolism) in the metabolism of these cells; glycolysis is accompanied by the production of a large amount of lactic acid.

In addition to acidification of the environment by glycolysis products, the matrix components themselves are of great importance. The large amount of fixed negative charge on proteoglycans modifies the extracellular ionic composition: a high concentration of free cations (e.g., H ⁺, Na ⁺, K ⁺ ) and a low concentration of anions (e.g., O2, HCO3 ) are observed. In addition, under the influence of mechanical loading, water is expelled from the ECM, which leads to an increase in the concentration of fixed negative charges and the attraction of more cations into the matrix. This is accompanied by a decrease in the pH of the extracellular environment, which affects the intracellular pH, thereby modifying the metabolism of chondrocytes. R. Wilkin and A. Hall (1995) studied the effect of the pH of the extracellular and intracellular environment on the biosynthesis of the matrix by isolated bovine chondrocytes. They observed a dual modification of matrix synthesis with a decrease in pH. A slight decrease in pH (7.4of 35 SO ₄ and ³ H-proline into chondrocytes, whereas deeper acidification of the medium (pH<7.1) inhibited synthesis by 75% compared to the control. Creating a low pH (6.65) using ammonium ions caused a decrease in matrix synthesis by only 20%. The results obtained indicate that the modification of the pH of the extracellular medium of matrix synthesis cannot be explained only by changes in the pH of the intracellular medium. Moreover, chondrocytes have the ability to regulate intracellular pH using the Na ⁺, H ⁺ -exchanger, the Ka ⁺ -dependent Cl ^_ -НСОС3 -transporter, and H ⁺ /ATPase.

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The influence of the composition of the culture medium on the metabolism of chondrocytes

The medium for culturing chondrocytes must correspond to the experimental conditions. In recent years, calf serum has been used to optimize the culturing conditions. However, when using serum, a number of important points must be taken into account:

• outward growth of cells from the periphery of tissue in organ cultures,
• variability in the composition of serums of different series,
• the presence of unknown components in them,
• increased risk of interference and artifacts when studying the influence of various biological factors on the metabolic activity of cells.

An example of the latter is a study of the effect of EGF on cartilage chondrocytes in rats. EGF stimulated the incorporation of ³ H-thymidine and an increase in DNA content in the culture. This effect was more pronounced at low serum concentrations (<1%), but at high concentrations (>7.5%) the effect disappeared.

It is well known that the levels of synthesis and degradation in DMEM supplemented with calf serum are significantly increased compared to in vivo conditions. Differences between in vivo and in vitro metabolism may be due to differences between the synovial fluid and the medium in which the cells are cultured. Lee et al. (1997) cultured young bovine chondrocytes in agarose using a nutrient medium containing DMEM supplemented with 20% calf serum and a large amount of normal allogeneic synovial fluid. The presence of synovial fluid in the medium induced an increase in the amount of proteoglycans, up to 80% of the total amount of synovial fluid. These results indicate that synovial fluid in culture induces a level of metabolism similar to that in vivo, with a high level of glycosaminoglycan synthesis and a low level of cell division.

G. Verbruggen et al. (1995) showed that ³⁵ S-arrpeKaHa synthesis by human chondrocytes cultured in agarose in serum-free DMEM was 20-30% of the level of synthesis observed in DMEM supplemented with 10% calf serum. The authors determined the extent to which IGF-1, IGF-2, TGF-R, or insulin restored aggrecan production in serum-free medium. The authors concluded that 100 ng/ml insulin, IGF-1, or IGF-2 partially restored aggrecan synthesis to 39-53% of the control level. No synergism or cumulation was observed with a combination of the listed factors. At the same time, 10 ng/ml TGF-R in the presence of 100 ng/ml insulin stimulated aggrecan synthesis to 90% or more of the reference level. Finally, human serum transferrin, alone or in combination with insulin, did not affect aggrecan synthesis. When calf serum was replaced with bovine serum albumin, the content of aggrecan aggregates decreased significantly. Enrichment of the culture medium with insulin, IGF, or TGF-R partially restored the ability of cells to produce aggrecan aggregates. Moreover, IGF-1 and insulin are able to maintain homeostasis in cell cultures. After 40 days of cultivation in a medium enriched with 10-20 ng/ml IGF-1, proteoglycan synthesis was maintained at the same level or even higher compared to the medium containing 20% calf serum. Catabolic processes proceeded more slowly in the medium enriched with IGF-1 than in the medium enriched with 0.1% albumin solution, but somewhat faster in the medium enriched with 20% serum. In long-lived cultures, 20 ng/ml IGF-1 maintains a stable state of cells.

D. Lee et al. (1993) compared the effect of the culture medium composition (DMEM, DMEM+20% calf serum, DMEM+20 ng/ml IGF-1) on DNA synthesis in a cartilage tissue explant culture, a monolayer culture, and in agarose suspension. When culturing in agarose in the presence of serum, the authors observed a tendency for chondrocytes to group into large clusters. Cells cultured without serum or with IGF-1 retained a round shape in agarose, collected into small groups, but did not form large aggregates. In a monolayer, DNA synthesis was significantly higher in the serum-containing medium than in the medium enriched with IGF-1; DNA synthesis in the latter was significantly higher than in the unenriched medium. No differences in DNA synthesis were found when chondrocytes were cultured in agarose suspension in a non-enriched medium and in a medium with IGF-1. At the same time, culturing chondrocyte suspensions in agarose in a serum-enriched medium was accompanied by increased incorporation of the radionucleotide ³ H-thymidine compared to other media.

Vitamin C is necessary for the activation of enzymes involved in the formation of a stable helical structure of collagen fibrils. Chondrocytes deficient in ascorbic acid synthesize underhydroxylated non-helical collagen precursors, which are secreted slowly. Administration of ascorbic acid (50 μg/ml) causes hydroxylation of collagen types II and IX and their secretion in normal quantities. Addition of vitamin C did not affect the level of proteoglycan synthesis. Therefore, collagen secretion is regulated independently of proteoglycan secretion.

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