Effects of extracellular matrix proteins in chondrocyte‐derived matrices on chondrocyte functions

Loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier to the application of chondrocytes for tissue engineering. In previous study, we showed that dedifferentiation of chondrocytes during the passage culture was delayed by matrices formed by primary chondrocytes (P0‐ECM). In this study, we investigated bovine chondrocyte functions when being cultured on isolated extracellular matrix (ECM) protein‐coated substrata and P0‐ECM. Low chondrocyte attachment was observed on aggrecan‐coated substratum and P0‐ECM. Cell proliferation on aggrecan‐ and type II collagen/aggrecan‐coated substrata and P0‐ECM was lower than that on the other ECM protein (type I collagen and type II collagen)‐coated substrata. When chondrocytes were subcultured on aggrecan‐coated substratum, decline of cartilaginous gene expression was delayed, which was similar to the cells subcultured on P0‐ECM. These results indicate that aggrecan plays an important role in the regulation of chondrocyte functions and P0‐ECM may be a good experimental control for investigating the role of each ECM protein in cartilage ECM. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1331–1336, 2013     

Regeneration of static-load-degenerated articular cartilage extracellular matrix by vitamin C supplementation

The effect of a physiological dose of vitamin C (100 mug/ml) on goat articular cartilage chondrocytes cultured in an alginate matrix and subjected to static pressurization of 2.4 MPa was investigated. Biochemical analyses of DNA, glycosaminoglycan (GAG), collagen and protease activity were carried out in various matrix fractions, i.e. cellular matrix (CM) and further removed matrix (FRM), and in culture medium. The treatment of chondrocytes with vitamin C after static pressure increased the GAG content in both CM and FRM (P < 0.03) as compared with control or vitamin C/ static load alone. The collagen content of chondrocytes treated with vitamin C alone and vitamin C after static load also increased significantly in FRM (P < 0.003) as compared with control and static load alone. The specific activity of protease in CM and FRM decreased after vitamin C supplementation both with and without static pressure relative to control (P < 0.003). Transmission electron-microscopic images showed a mixed population of spherical and elliptical chondrocytes when vitamin C was added after static load as compared with static load alone where only elliptical cells were seen. Abundant pericellular and collagen fibrils were seen in this group of chondrocytes as compared with all other groups and the control. The results thus show that, in vitro, vitamin C supplementation of chondrocytes after static loading has the potential to reduce the morphological and biochemical degeneration of chondrocytes caused by static loading, thereby improving the cellular health and functioning of articular cartilage.     

Proteoglycan production by immortalized human chondrocyte cell lines cultured under conditions that promote expression of the differentiated phenotype

Large and small proteoglycans are essential components of articular cartilage. How to induce chondrocytes to repair damaged cartilage with normal ratios of matrix components after their loss due to degenerative joint disease has been a major research focus. We have developed immortalized human chondrocyte cell lines for examining the regulation of cartilage-specific matrix gene expression. However, the decreased synthesis and deposition of cartilage matrix associated with a rapid rate of proliferation has presented difficulties for further examination at the protein level. In these studies, proteoglycan synthesis was characterized in two chondrocyte cell lines, T/C-28a2 and tsT/AC62, derived, respectively, from juvenile costal and adult articular cartilage, under culture conditions that either promoted or decreased cell proliferation. Analysis of proteo[36S]glycans by Sepharose CL-4B chromatography and SDS-PAGE showed that the large proteoglycan aggrecan and the small, leucine-rich proteoglycans, decorin and biglycan, were produced under every culture condition studied. In monolayer cultures, a high initial cell density and conditions that promoted proliferation (presence of serum for T/C-28a2 cells or permissive temperature for the temperature-sensitive tsT/AC62 cells) favored cell survival and ratios of proteoglycans expected for differentiated chondrocytes. However, the tsT/AC62 cells produced more proteoglycans at the nonpermissive temperature. Culture of cells suspended in alginate resulted in a significant decrease in proteoglycan production in all culture conditions. While the tsT/AC62 cells continued to produce a larger amount of aggrecan than small proteoglycans, the T/C-28a2 cells lost the ability to produce significant amounts of aggrecan in alginate culture. In addition, our data indicate that immortalized chondrocytes may alter their ability to retain pericellular matrix under changing culture conditions, although the production of the individual matrix components does not change. These findings provide critical information that will assist in the development of a reproducible chondrocyte culture model for the study of regulation of proteoglycan biosynthesis in cartilage.     

Age-related differences in the accumulation and size of hyaluronan in alginate culture

The alginate bead culture system has unique properties that make it possible to study the accumulation and turnover of macromolecules in two distinct matrix compartments of the cartilage matrix: the cell-associated matrix (CM) and the further removed matrix (FRM). Taking advantage of this culture system, the purpose of this study was to examine age-related changes in the metabolism of hyaluronan (HA) in these two compartments. Bovine chondrocytes, isolated from fetal, young adult, and old adult articular cartilage, were cultured in alginate beads. On Days 7 and 14 of culture, the alginate gel was solubilized, the CM and FRM were separated and macromolecules in both compartments were analyzed. When compared to the cells from fetal and old adult animals, the young adult cells proliferated at the fastest rate. Fetal cells produced a more abundant CM that was richer in proteoglycans (PGs) than the CM of young or old adult cells. With increasing age, there was an increased tendency for PG, collagen, and HA to escape incorporation into the CM and to become immobilized in the FRM. Very striking changes also were observed in the ratio of HA to PG, which increased markedly with age, and in the size of the HA molecules, which decreased markedly with age. The results suggest that the metabolism of HA in cartilage undergoes pronounced age-related changes, some of which are retained during culture in alginate gel. The findings also suggest that the previously documented age-related decrease in the size of HA in native bovine cartilage reflects, at least in part, a biochemical process occurring at the time or at least soon after the glycosaminoglycan chain is synthesized. It does not appear to simply be the result of age-related changes occurring slowly with time after synthesis, as was previously suggested to be the case for human articular cartilage.     

DIFFERENTIATION OF MESENCHYMAL LIMB BUD CELLS TO CHONDROCYTES IN ALGINATE BEADS

Manyin vitromodels of embryonic material used for the cultivation of chondrocytes yield mixed cultures consisting of chondrocytes and fibroblast-like cells. For the optimization of cartilage cell cultures, alginate, a semisolid medium, was employed to obtain pure chondrocyte cultures. Isolated mesenchymal cells from 12-day-old mouse limb buds were grown in alginate for up to 4 weeks. A sub-population of the cells differentiated to chondrocytes and exhibited a stable phenotype until the end of the culture period. After 3 to 4 days a cartilage-specific matrix started to develop. Fibroblast-like cells from this mixed culture did not survive; they became necrotic. When alginate was later on dissolved by chelating agents, only chondrocytes were isolated. During dissolution of alginate and centrifugation, chondrocytes did not lose their contact with their new matrix present on their surfaces. Cultivation of these chondrocytes or chondrones in mass culture yields a pure chondrocyte population. Immunoelectron microscopic investigations revealed collagen type II, fibronectin, decorin and chondroitin sulfate-proteoglycans in the chondrocyte capsules and in mass culture.     

Chondrocyte-alginate constructs with or without TGF-β1 produces superior extracellular matrix expression than monolayer cultures

Tissue engineering approaches often require expansion of cell numbers in vitro to accelerate tissue regenerative processes. Although several studies have used this technique for therapeutic purposes, a major concern involving the use of isolated chondrocyte culture is the reduction of extracellular matrix (ECM) protein expressed due to the transfer of cells from the normal physiological milieu to the artificial 2D environment provided by the cell culture flasks. To overcome this issue, the use of alginate hydrogel beads as a substrate in chondrocyte cultures has been suggested. However, the resultant characteristics of cells embedded in this bead is elusive. To elucidate this, a study using chondrocytes isolated from rabbit knee articular cartilage expanded in vitro as monolayer and chondrocyte-alginate constructs was conducted. Immunohistochemical evaluation and ECM distribution was examined with or without transforming growth factor (TGF-β1) supplement to determine the ability of cells to express major chondrogenic proteins in these environments. Histological examination followed by transmission electron microscopy and scanning electron microscopy was performed to determine the morphology and the ultrastructural characteristics of these cells. Results demonstrated a significant increase in glycosaminoglycan/mg protein levels in chondrocyte cultures grown in alginate construct than in monolayer cultures. In addition, an abundance of ECM protein distribution surrounding chondrocytes cultured in alginate hydrogel was observed. In conclusion, the current study demonstrates that the use of alginate hydrogel beads in chondrocyte cultures with or without TGF-β1 supplement provided superior ECM expression than monolayer cultures.     

beta-Integrin-collagen interaction reduces chondrocyte apoptosis.

We have observed that the spent culture media in suspended chondrocyte cultures is essential for the survival of the cells, since complete change of the spent media induces severe programmed cell death (apoptosis). Moreover, we showed that extracellular matrix (ECM) molecules in the culture media provide vital chondrocyte-matrix interactions; when media are changed, cells are deprived of matrix molecules and undergo apoptosis. In this paper we report that interaction with collagen, a ubiquitous extracellular matrix molecule, is essential for chondrocyte survival. Such an interaction causes chondrocyte aggregation and reduces the level of chondrocyte apoptosis. Hyaluronan, an abundant ECM molecule, can influence the effects of collagen by preventing chondrocyte aggregation. Degradation of hyaluronan with hyaluronidase results in chondrocyte aggregation, and this reduces the level of chondrocyte apoptosis. Experiments with an antibody to integrin beta1 suggest that the collagen-chondrocyte interactions are mediated through integrin beta1, and these interactions may protect chondrocytes from apoptosis. We hypothesize that hyaluronan binds aggrecan and link protein, forming stable ternary complexes, which interact with the chondrocyte surface, perhaps via CD44, and thus maintains a stable chondrocyte-matrix network.     

Stimulation of type II collagen biosynthesis and secretion in bovine chondrocytes cultured with degraded collagen

The functional integrity of articular cartilage is dependent on the maintenance of the extracellular matrix (ECM), a process which is controlled by chondrocytes. The regulation of ECM biosynthesis is complex and a variety of substances have been found to influence chondrocyte metabolism. In the present study we have investigated the effect of degraded collagen on the formation of type II collagen by mature bovine chondrocytes in a cell culture model. The culture medium was supplemented with collagen hydrolysate (CH) and biosynthesis of type II collagen by chondrocytes was compared to control cells treated with native type I and type II collagen and a collagen-free protein hydrolysate. The quantification of type II collagen by means of an ELISA technique was confirmed by immunocytochemical detection as well as by the incorporation of (14)C-proline in the ECM after a 48 h incubation. Chondrocytes in the control group were maintained in the basal medium for 11 days. The presence of extracellular CH led to a dose-dependent increase in type II collagen secretion. However, native collagens as well as a collagen-free hydrolysate of wheat proteins failed to stimulate the production of type II collagen in chondrocytes. These results clearly indicate a stimulatory effect of degraded collagen on the type II collagen biosynthesis of chondrocytes and suggest a possible feedback mechanism for the regulation of collagen turnover in cartilage tissue.     

Use of porous alginate sponges for substantial chondrocyte expansion and matrix production: effects of seeding density

Autologous cell transplantation is a promising approach for cartilage repair, but the expansion of chondrocytes in a monolayer, a common approach to amplifying the cell number, inevitably leads to cell de-differentiation. To explore whether porous alginate sponges could be utilized for chondrocyte expansion and investigate the effects of seeding densities, the porcine chondrocytes were seeded to porous alginate sponges at low (5 x 10(5) cells per 40 sponges), medium (5 x 10(6) cells per 40 sponges), or high (2 x 10(7) cells per 40 sponges) density. After 4-week perfusion culture, all three groups resulted in chondrocyte proliferation, maintenance of chondrocytic gene (collagen II, Sox 9 and aggrecan) expression, and formation of cell clusters resembling cartilaginous tissues. The higher the seeding density, the higher the final cell density and GAGs production and, accordingly, the larger the cell clusters. Strikingly, the cumulative expansion ratios achieved by the low-density group ( approximately 150-fold) significantly exceeded those achieved by the medium (approximately 21-fold) and high (approximately 4.7-fold) density groups, as well as those achieved using other scaffolds. In conclusion, seeding chondrocytes to the alginate sponges at a low density, combined with perfusion culture, represents a drastic improvement in expanding autologous chondrocytes.     

The role of hydrogel structure and dynamic loading on chondrocyte gene expression and matrix formation

Crosslinked poly(ethylene glycol) (PEG) hydrogels are attractive scaffolds for cartilage tissue engineering because of their ability to mimic the aqueous environment and mechanical properties of native cartilage. In this study, hydrogel crosslinking density was varied to study the influence of gel structure and the application of dynamic loading (continuous, 1 Hz, 15% amplitude strain) on chondrocyte gene expression over 1 week culture. Gene expression was quantified using real-time RT-PCR for collagen II and aggrecan, the major cartilage extracellular matrix (ECM) components, and collagen I, an indicator of chondrocyte de-differentiation. When chondrocytes were encapsulated in PEG gels with low or high crosslinking, a high collagen II expression compared to collagen I expression (1000 or 100,000:1, respectively) indicated the native chondrocyte phenotype was retained. In the absence of loading, relative gene expression for collagen II and aggrecan was significantly higher (e.g., 2-fold and 4-fold, respectively, day 7) in the low crosslinked gels compared to gels with higher crosslinking. Dynamic loading, however, showed little effect on ECM gene expression in both crosslinked systems. To better understand the cellular environment, ECM production was qualitatively assessed using an in situ immunofluorescent technique and standard histology. A pericellular matrix (PCM) was observed as early as day 3 post-encapsulation and the degree of formation was dependent on gel crosslinking. These results suggest the PCM may protect the cells from sensing the applied loads. This study demonstrates that gel structure has a profound effect on chondrocyte gene expression, while dynamic loading has much less of an effect at early culture times.     

An inhibitor of the stretch-activated cation receptor exerts a potent effect on chondrocyte phenotype

Rat chondrosarcoma (RCS) cells are unusual in that they display a stable chondrocyte phenotype in monolayer culture. This phenotype is reflected by a rounded cellular morphology with few actin-containing stress fibers and production of an extracellular matrix rich in sulfated proteoglycans, with high-level expression of aggrecan, COMP, Sox9, and collagens type II, IX, and XI. Additionally, these cells do not express collagen type I. Here it is shown that in the absence of any mechanical stimulation, treatment of RCS cells with gadolinium chloride (Gd3+), a stretch-activated cation channel blocker, caused the cells to undergo de-differentiation, adopting a flattened fibroblast phenotype with the marked appearance of actin stress fibers and vinculin-containing focal contacts. This change was accompanied by a dramatic reduction in the expression of aggrecan, Sox9, collagen types II, IX, and XI, with a corresponding increase in the expression of collagen type I and fibronectin. These effects were found to be reversible by simple removal of Gd3+ from the medium. Gd3+ also had a similar effect on expression of chondrocyte marker genes in freshly isolated human chondrocytes. These data suggest that mechanoreceptor signaling plays a key role in maintenance of the chondrocyte phenotype, even in the absence of mechanical stimulation. Further, treatment of RCS cells with Gd3+ provides a tractable system for assessing the molecular events underlying the reversible differentiation of chondrocytes.     

Rapid regulation of collagen but not metalloproteinase 1, 3, 13, 14 and tissue inhibitor of metalloproteinase 1, 2, 3 expression in response to mechanical loading of cartilage explants in vitro

This study analyzes the molecular response of articular chondrocytes to short-term mechanical loading with a special focus on gene expression of molecules relevant for matrix turnover. Porcine cartilage explants were exposed to static and dynamic unconfined compression and viability of chondrocytes was assessed to define physiologic loading conditions. Cell death in the superficial layer correlated with mechanical loading and occurred at peak stresses >or=6 MPa and a cartilage compression above 45%. Chondrocytes in native cartilage matrix responded to dynamic loading by rapid and highly specific suppression of collagen expression. mRNA levels dropped 11-fold (collagen 2; 6 MPa, P=0.009) or 14-fold (collagen 1; 3 and 6 MPa, P=0.009) while levels of aggrecan, tenascin-c, matrix metalloproteinases (MMP1, 3, 13, 14), and their inhibitors (TIMP1-3) did not change significantly. Thus, dynamic mechanical loading rapidly shifted the balance between collagen and aggrecan/tenascin/MMP/TIMP expression. A better knowledge of the chondrocyte response to mechanical stress may improve our understanding of mechanically induced osteoarthrits.     

Gene expression by human articular chondrocytes cultured in alginate beads.

Culture of articular chondrocytes in alginate beads offers several advantages over culture in monolayer; cells retain their phenotype for 8 months or longer. Earlier studies of chondrocytes cultured in alginate concentrated on collagen and proteoglycan synthesis. However, gene expression by in situ hybridization (ISH) has not been investigated. The purposes of the present study on human chondrocytes were (a) to modify the ISH procedure for the alginate beads to examine the mRNA expression of alpha1 (II) procollagen, aggrecan, and two matrix metalloproteinases (MMP-3 and MMP-8) thought to be involved in cartilage matrix degradation, and (b) to compare expression in cultured chondrocytes with that in chondrocytes of intact human cartilage. The modifications made for ISH include the presence of CaCl2 and BaCl2 in the fixation and washing steps and exclusion of cetyl pyridinium chloride. By ISH we show that aggrecan, MMP-3, and MMP-8 are continuously expressed during 8 months of culture. The alpha1 (II) procollagen gene is expressed only during the first 2 months of culture and after 3 months its expression is undetectable, which is consistent with its absence in adult articular cartilage. By Western blotting, Type II collagen protein had been synthesized and deposited in both the cell-associated and further-removed matrix compartments at 7 and 14 days of culture. These data indicate that chondrocytes cultured in alginate beads could be preserved for immunohistochemistry and ISH and that culture of human chondrocytes in alginate beads may serve as a good model for studying cartilage-specific phenotype as well as factors that influence cartilage matrix turnover.     

Adenoviral transduction supports matrix expression of alginate cultured articular chondrocytes

The present study examines the effects of adenoviral (Ad) transduction of human primary chondrocyte on transgene expression and matrix production. Primary chondrocytes were isolated from healthy articular cartilage and from cartilage with mild osteoarthritis (OA), transduced with an Ad vector and either immediately cultured in alginate or expanded in monolayer before alginate culture. Proteoglycan production was measured using dimethylmethylene blue (DMMB) assay and matrix gene expression was quantified by real-time PCR. Viral infection of primary chondrocytes results in a stable long time transgene expression for up to 13 weeks. Ad transduction does not significantly alter gene expression and matrix production if chondrocytes are immediately embedded in alginate. However, if expanded prior to three dimension (3D) culture in alginate, chondrocytes produce not only more proteoglycans compared to non-transduced controls, but also display an increased anabolic and decreased catabolic activity compared to non-transduced controls. We therefore suggest that successful autologous chondrocyte transplantation (ACT) should combine adenoviral transduction of primary chondrocytes with expansion in monolayer followed by 3D culture. Future studies will be needed to investigate whether the subsequent matrix production can be further improved by using Ad vectors bearing genes encoding matrix proteins.     

Strategies for enhancing the accumulation and retention of extracellular matrix in tissue-engineered cartilage cultured in bioreactors

Production of tissue-engineered cartilage involves the synthesis and accumulation of key constituents such as glycosaminoglycan (GAG) and collagen type II to form insoluble extracellular matrix (ECM). During cartilage culture, macromolecular components are released from nascent tissues into the medium, representing a significant waste of biosynthetic resources. This work was aimed at developing strategies for improving ECM retention in cartilage constructs and thus the quality of engineered tissues produced in bioreactors. Human chondrocytes seeded into polyglycolic acid (PGA) scaffolds were cultured in perfusion bioreactors for up to 5 weeks. Analysis of the size and integrity of proteoglycans in the constructs and medium showed that full-sized aggrecan was being stripped from the tissues without proteolytic degradation. Application of low (0.075 mL min(-1)) and gradually increasing (0.075-0.2 mL min(-1)) medium flow rates in the bioreactor resulted in the generation of larger constructs, a 4.0-4.4-fold increase in the percentage of GAG retained in the ECM, and a 4.8-5.2-fold increase in GAG concentration in the tissues compared with operation at 0.2 mL min(-1). GAG retention was also improved by pre-culturing seeded scaffolds in flasks for 5 days prior to bioreactor culture. In contrast, GAG retention in PGA scaffolds infused with alginate hydrogel did not vary significantly with medium flow rate or pre-culture treatment. This work demonstrates that substantial improvements in cartilage quality can be achieved using scaffold and bioreactor culture strategies that specifically target and improve ECM retention.     

Latrunculin and cytochalasin decrease chondrocyte matrix retention.

The proteoglycan-rich extracellular matrix (ECM) directly associated with the cells of articular cartilage is anchored to the chondrocyte plasma membrane via interaction with the hyaluronan receptor CD44. The cytoplasmic tail of CD44 interacts with the cortical cytoskeleton. The objective of this study was to determine the role of the actin cytoskeleton in CD44-mediated matrix assembly by chondrocytes and cartilage matrix retention and homeostasis. Adult bovine articular cartilage tissue slices and isolated chondrocytes were treated with latrunculin or cytochalasin. Tissues were processed for histology and chondrocytes were examined for CD44 expression and pericellular matrix assembly. Treatments that disrupt the actin cytoskeleton reduced chondrocyte pericellular matrix assembly and the retention of proteoglycan within cartilage explants. There was enhanced detection of a neoepitope resulting from proteolysis of aggrecan. Cytoskeletal disruption did not reduce CD44 expression, as monitored by flow cytometry, but detergent extraction of CD44 was enhanced and hyaluronan binding was decreased. Thus, disruption of the cytoskeleton reduces the anchorage of CD44 in the chondrocyte membrane and the capacity of CD44 to bind its ligand. The results suggest that cytoskeletal disruption within cartilage uncouples chondrocytes from the matrix, resulting in altered metabolism and deleterious changes in matrix structure.