Integrin-mediated mechanotransduction processes in TGFbeta-stimulated monolayer-expanded chondrocytes

Previous studies have demonstrated that passage in monolayer detrimentally affects the response of articular chondrocytes to the application of dynamic compression. Transforming growth factor beta (TGFbeta) is known to regulate metabolic processes in articular cartilage and can enhance the re-expression of a chondrocytic phenotype following monolayer expansion. The current study tests the hypothesis that TGFbeta also modulates the response of monolayer-expanded human chondrocytes to the application of dynamic compression, via an integrin-mediated mechanotransduction process. The data presented demonstrate that TGFbeta3 enhanced 35SO4 and [3H]thymidine incorporation and inhibited nitrite release after 48 h of culture when compared to unsupplemented constructs. Dynamic compression also enhanced 35SO4 and [3H]thymidine incorporation and inhibited nitrite release in the presence of TGFbeta3. By contrast, dynamic compression did not alter these parameters in the absence of the growth factor. The addition of the peptide, GRGDSP, which acts as a competitive ligand for the alpha5beta1 integrin, reversed the compression-induced stimulation of 35SO4 incorporation, [3H]thymidine incorporation, and suppression of nitrite release. No effect was observed when the control peptide, GRADSP, was used. The current data clearly demonstrate that the dynamic compression-induced changes observed in cell metabolism for human monolayer-expanded chondrocytes were dependent on the presence of TGFbeta3 and are integrin-mediated.     

Stimulation by concanavalin A of cartilage-matrix proteoglycan synthesis in chondrocyte cultures

The effect of concanavalin A on proteoglycan synthesis by rabbit costal and articular chondrocytes was examined. Chondrocytes were seeded at low density and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of concanavalin A to the culture medium induced a morphologic alteration of the fibroblastic cells to spherical chondrocytes and increased by 3- to 4-fold incorporation of [35S]sulfate and [3H]glucosamine into large chondroitin sulfate proteoglycan that was characteristically found in cartilage. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, as chemical analyses showed a 4-fold increase in the accumulation of macromolecules containing hexuronic acid in concanavalin A-maintained cultures. Furthermore, the effect of concanavalin A on [35S]sulfate incorporation into proteoglycans was greater than that of various growth factors or hormones. However, concanavalin A had smaller effects on [35S]sulfate incorporation into small proteoglycans and [3H]glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant glycosaminoglycans. Since other lectins tested, such as wheat germ agglutinin, lentil lectin, and phytohemagglutinin, had little effect on [35S]sulfate incorporation into proteoglycans, the concanavalin A action on chondrocytes seems specific. Although concanavalin A decreased [3H]thymidine incorporation in chondrocytes, the stimulation of proteoglycan synthesis could be observed in chondrocytes exposed to the inhibitor of DNA synthesis, cytosine arabinoside. These results indicate that concanavalin A is a potent modulator of proteoglycan synthesis by chondrocytes.     

Effect of vanadate on cartilage-matrix proteoglycan synthesis in rabbit costal chondrocyte cultures

The effect of vanadate on proteoglycan synthesis by cultured rabbit costal chondrocytes was examined. Rabbit chondrocytes were seeded at low densities and grown to confluency in medium supplemented with 10% fetal bovine serum, and then the serum concentration was reduced to 0.3%. At the low serum concentration, chondrocytes adopted a fibroblastic morphology. Addition of 4 microM vanadate to the culture medium induced a morphologic differentiation of the fibroblastic cells to spherical chondrocytes, and increased by two- to threefold incorporation of [35S]sulfate and [3H]glucosamine into large, chondroitin sulfate proteoglycans. The stimulation of incorporation of labeled precursors reflected real increases in proteoglycan synthesis, in that chemical analyses showed increases in the accumulation of macromolecules containing hexuronic acid and hexosamine in vanadate-maintained cultures. However, vanadate had only a marginal effect on [35S]sulfate incorporation into small proteoglycans and [3H]glucosamine incorporation into hyaluronic acid and chondroitinase AC-resistant material. These results provide evidence that vanadate selectively stimulates the synthesis of proteoglycans characteristically found in cartilage by rabbit costal chondrocyte cultures.     

The influence of mechanical loading on isolated chondrocytes seeded in agarose constructs

Articular cartilage is subjected to dynamic compressive loading during normal activity which influences chondrocyte metabolism through various mechanotransduction pathways. A well characterised and reproducible model system, involving chondrocytes embedded in agarose gel, has been used to investigate the effects of mechanical compression on chondrocytes, isolated from full depth cartilage or separately from the superficial and deep zone tissue. The role of nitric oxide as a mediator of mechanical-induced effects has also been studied. Chondrocytes were isolated, separately, from full depth, superficial and deep zone cartilage and seeded in 3% agarose constructs. Dynamic compressive strain was applied to the constructs using a range of frequencies (0.3, 1 and 3 Hz). Glycosaminoglycan synthesis, cell proliferation and nitrite production were assessed. In further experiments, constructs were compressed in the presence of 1 mM L-NAME or 10 microM dexamethasone. Glycosaminoglycan synthesis by full depth chondrocytes was affected by compressive strain in a frequency dependent manner. Dynamic strain at all frequencies induced an increase in [3H]-thymidine incorporation. Glycosaminoglycan synthesis by deep zone cells was affected by the strain regimes in a similar fashion to full depth cells, while superficial cells exhibited a similar proliferative response to full depth cells. Dynamic compression inhibited nitrite production, the effect being reversed by L-NAME. Compression induced stimulation of [3H]-TdR incorporation was reversed by L-NAME. These studies demonstrate that glycosaminoglycan synthesis and proliferation are influenced by the dynamic strain regimes in a distinct manner. Indeed the data suggest that these processes occur in different chondrocyte sub-populations. It may be speculated that nitric oxide acts as a mediator of mechanotransduction processes affecting proliferation primarily in the superficial cell sub-population.     

Influence of cytochalasin d-induced changes in cell shape on proteoglycan synthesis by cultured articular chondrocytes

There is growing evidence that cell shape regulates both proliferation and differentiated gene expression in a variety of cell types. We have explored the relationship between the morphology of articular chondrocytes in culture and the amount and type of proteoglycan they synthesize, using cytochalasin D to induce reversible cell rounding. When chondrocytes were prevented from spreading or when spread cells were induced to round up, 35SO4 incorporation into proteoglycan was stimulated. Incorporation into the cell layer was stimulated more than into the medium. When the cells were allowed to respread by removing cytochalasin D, proteoglycan synthesis returned to control levels. Cytochalasin D-induced stimulation of 35SO4 incorporation reflected an increase in core protein synthesis rather than lengthening of glycosaminoglycan chains, because [3H]serine incorporation into core protein was also stimulated. The observed stimulation of proteoglycan synthesis was not due to an overall stimulation of protein synthesis, to inhibition of DNA synthesis, or to accumulation of cells in one phase of the cell cycle. Cytochalasin D-treatment of cells in suspension caused no further stimulation of 35SO4 incorporation, suggesting that the observed effects were due to cell rounding rather than exposure to cytochalasin D per se; nevertheless, we cannot completely rule out other, nonspecific, effects of the drug. Fibroblasts and chondrocytes that had been passaged to stimulate dedifferentiation did not incorporate more 35SO4 when treated with cytochalasin D, suggesting that increased proteoglycan synthesis in response to rounding may itself be a differentiated property of chondrocytes.     

Inhibitory effect of Cordyceps sinensis and Cordyceps militaris on human glomerular mesangial cell proliferation induced by native LDL

Native LDL, in low concentrations, promotes proliferation of cultured human glomerular mesangial cells. LDL stimulated [(3)H]-thymidine incorporation into DNA of human glomerular mesangial cells. Increased concentrations of LDL led to increased [(3)H]-thymidine incorporation. When LDL concentrations were 5, 10 and 50 microg ml(-1), [(3)H]-thymidine incorporation was 919.5+/-216, 1106+/-132, and 1200+/-210, respectively. When Cordyceps sinensis 100, 200, 300, 400 microg ml(-1) plus LDL 10 microg ml(-1) were added, [(3)H]-thymidine incorporation was 99+/-19 and 53+/-8, respectively, P<0.01 compared with controls. With Cordyceps militaris at similar concentrations plus LDL 10 microg ml(-1), [(3)H]-thymidine incorporation was respectively 192+/-75, 168+/-66, 145+/-53 and 72+/-16, P<0.01 compared with controls. The data suggest that LDL may play a critical role in mediating mesangial cell hypertrophy or proliferation involved in the development of glomerulosclerosis. Cordyceps sinensis and Cordyceps militaris inhibited, to a certain degree, proliferation of cultured human glomerular mesangial cell induced by LDL.     

Type I collagen induces expression of bone morphogenetic protein receptor type II

The extracellular matrix regulates many fundamental cellular processes such as proliferation, migration, and differentiation. Among the ECM components, type I collagen induces endothelial tube formation in vitro. By analysing genes participating in this event, the bone morphogenetic protein receptor-II (BMPR-II) was detected to be upregulated in cells cultured on or within fibrillar type I collagen. Furthermore, the basement membrane type IV collagen or amorphous type I collagen did not show an induction of BMPR-II. Addition of the BMPR-II specific ligands, BMP2 and BMP4, in the culture medium of the endothelial cells seeded on type I collagen increased [(3)H]-thymidine incorporation into cellular DNA, indicating that endothelial cells were able to form a functional receptor. In addition, in the chick chorioallantoic membrane (CAM), an in vivo angiogenesis model, BMPR-II and BMPR-I were upregulated in the growing phase and ceased in the mature CAM.     

Effect of the neuropeptide substance P on the rat bone marrow-derived osteogenic cells in vitro

Substance P containing, thin, sensory nerve fibres have been demonstrated in bone and bone marrow. However the role of substance P in bone tissue is not fully understood. We investigated the effects of substance P on the growth and development of rat bone marrow-derived osteogenic cells in vitro. To examine this, the marrow-derived osteogenic cells were treated from 3rd to 6th day of subculture with substance P at concentrations 10(-10), 10(-9) and 10(-8)M. [(3)H]-thymidine, L-2,3-[(3)H]-proline incorporation, protein accumulation, alkaline phosphatase activity, and calcium deposition were measured in cultures. Substance P slightly stimulated [(3)H]-thymidine incorporation at 10(-10) M. Protein accumulation and L-2,3-[(3)H]-proline incorporation were enhanced in a dose dependent manner. Simultaneous application of spantide, a substance P receptor antagonist, could not block substance P-induced L-2,3-[(3)H]-proline incorporation probably because of statistically significant effect of spantide itself. Calcium deposits were significantly lower (about 30%) in cultures treated with SP. This effect was probably due in part by the fall in alkaline phosphatase activity which in substance P treated cultures was decreased about 17%. Our results indicate that substance P could be one of the factors modulating bone metabolism.     

Lithium chloride-induced primary cilia recovery enhances biosynthetic response of chondrocytes to mechanical stimulation

Mechanical stimulation is commonly used in cartilage tissue engineering for enhancing tissue formation and improving the mechanical properties of resulting engineered tissues. However, expanded chondrocytes tend to dedifferentiate and lose expression of their primary cilia, which is necessary for chondrocyte mechanotransduction. As treatment with lithium chloride (LiCl) can restore passaged chondrocytes in monolayer, in this study, we investigated whether this approach would be effective in 3D culture and restore chondrocyte mechanosensitivity. Chondrocytes at different passages (P0 to P2) were treated with 0–50 mM LiCl for 24 h, with different pre-culture durations (0 to 4 days). The primary cilia incidence and length were measured in α-tubulin-stained images. Treated chondrocytes were cultured with or without dynamic compression to evaluate the effect of LiCl-induced primary cilia expression on matrix synthesis by mechanically stimulated chondrocytes. LiCl treatment of chondrocytes in 3D agarose culture increased primary cilia incidence and length, with significant increases in incidence and length using 50 mM LiCl compared to other concentrations (P?<?0.05). This effect was further optimized by including a 4-day pre-culture prior to the 24-h 50 mM LiCl treatment. Importantly, LiCl-induced primary cilia expression increased chondrocyte mechanosensitivity. When stimulated with dynamic compression, LiCl-treated P1 chondrocytes increased collagen (1.4-fold, P?<?0.1) and proteoglycan (1.5-fold, P?<?0.05) synthesis compared to untreated, unstimulated cells. The LiCl treatment method described here can be used to restore primary cilia in passaged chondrocytes, transforming them into a mechanosensitive cell source for cartilage tissue engineering.

     

Endothelin-1 regulates proliferative responses, both alone and synergistically with PDGF, in rat tracheal smooth muscle cells.

The peptide, endothelin-1 (ET-1) regulates proliferative responses in numerous cell types. Recently, a dual ET receptor antagonist was shown to prevent the increase in airway smooth muscle cell (SMC) proliferation that accompanies airway smooth muscle remodeling in a rat model of experimental asthma. Thus, we used [(3)H]-thymidine incorporation assays and western immunoblotting to identify signaling pathways that regulate proliferative responses in cultured rat tracheal SMC. Our data indicate that ET-1 activation of the ET A receptor subtype induced [(3)H]-thymidine incorporation and activation of ERK 1/2 in primary rat tracheal SMC. ET-1-induced [(3)H]-thymidine incorporation and activation of ERK 1/2 were inhibited by pretreatment of SMC with pertussis toxin or down regulation of phorbol ester responsive isoforms of PKC. While ET- 1-induced ERK 1/2 activation was unaffected following inhibition of Rho kinase, ET-1-induced [(3)H]-thymidine incorporation was abrogated. ET-1 also potentiated [(3)H]-thymidine incorporation as well as cell proliferation of SMC stimulated with PDGF-BB and this response did not appear to be regulated by ERK1/ 2. These data demonstrate that ET-1 induces activation of multiple G proteins that regulate rat tracheal SMC proliferative responses, likely through signaling pathways downstream of ERK1/2 and Rho kinase.     

The effects of 1-hydroxyethane-1,1-diphosphonate and dichloromethanediphosphonate on collagen synthesis by rabbit articular chondrocytes and rat bone cells.

Investigations were performed to assess the effects of dichloromethanediphosphonate on the synthesis of collagen by (1) isolated rabbit articular chondrocytes, (2) isolated rat calvaria bone cells and (3) bone explants from rats treated with the diphosphonates. The studies showed that dichloromethanediphosphonate, but not 1-hydroxyethane-1,1-diphosphonate, causes articular chondrocytes to increase net collagen biosynthesis, both when measured as 3H-labelled or as non-radioactive material, in a dose-related fashion. The increment in collagen synthesis was still evident with cells that were exposed continuously to the diphosphonate in primary as well as secondary culture; however, it declined with cells in tertiary culture and was absent after the fourth subculture. The type of collagen was not affected by the diphosphonate. The synthesis of collagen by bone cells was likewise increased with dichloromethanediphosphonate. No effects were detected with 1-hydroxyethane-1,1-diphosphonate was tested. Finally, when calvaria and tibiae from diphosphonate-treated rats were cultured in vitro, the positive effect of dichloromethanediphosphonate on collagen synthesis was also evident. 1-Hydroxyethane-1,1-diphosphonate, on the other hand, decreased the incorporation of [3H]proline into the collagen of calvaria and osseous tibial shafts and showed no effect on the collagen synthesis of the cartilaginous tibial heads.     

Human meniscus cells express hypoxia inducible factor-1alpha and increased SOX9 in response to low oxygen tension in cell aggregate culture

In previous work we demonstrated that the matrix-forming phenotype of cultured human cells from whole meniscus was enhanced by hypoxia (5% oxygen). Because the meniscus contains an inner region that is devoid of vasculature and an outer vascular region, here we investigate, by gene expression analysis, the separate responses of cells isolated from the inner and outer meniscus to lowered oxygen, and compared it with the response of articular chondrocytes. In aggregate culture of outer meniscus cells, hypoxia (5% oxygen) increased the expression of type II collagen and SOX9 (Sry-related HMG box-9), and decreased the expression of type I collagen. In contrast, with inner meniscus cells, there was no increase in SOX9, but type II collagen and type I collagen increased. The articular chondrocytes exhibited little response to 5% oxygen in aggregate culture, with no significant differences in the expression of these matrix genes and SOX9. In both aggregate cultures of outer and inner meniscus cells, but not in chondrocytes, there was increased expression of collagen prolyl 4-hydroxylase (P4H)alpha(I) in response to 5% oxygen, and this hypoxia-induced expression of P4H alpha(I) was blocked in monolayer cultures of meniscus cells by the hypoxia-inducible factor (HIF)-1alpha inhibitor (YC-1). In fresh tissue from the outer and inner meniscus, the levels of expression of the HIF-1alpha gene and downstream target genes (namely, those encoding P4H alpha(I) and HIF prolyl 4-hydroxylase) were significantly higher in the inner meniscus than in the outer meniscus. Thus, this study revealed that inner meniscus cells were less responsive to 5% oxygen tension than were outer meniscus cells, and they were both more sensitive than articular chondrocytes from a similar joint. These results suggest that the vasculature and greater oxygen tension in the outer meniscus may help to suppress cartilage-like matrix formation.     

Extracellular matrix synthesis by articular chondrocytes and synovial fibroblasts in long-term monolayer culture

Synthesis of collagen and proteoglycan by rabbit articular chondrocytes and synovial fibroblasts has been studied over a 12-week period in primary monolayer culture. Chondrocytes, but not fibroblasts, accumulate large quantities of proteoglycan over the culture period studied. Radiolabeling studies with [35S]sulfate have shown that the major proteoglycan synthesized by cultured chondrocytes is similar to the proteoglycan of cartilage matrix. Chondrocytes also synthesize a smaller dermatan sulfate proteoglycan, which is apparently the only proteoglycan species produced by synovial fibroblasts. Collagen synthesis was studied by radiolabeling with [3H]proline. Cultured chondrocytes produce mainly Type II collagen, with lesser amounts of Type I, whereas synovial fibroblasts produce Type I collagen and some low molecular weight collagenous species. Therefore, long-term monolayer culture permits the production of extensive chondroid matrix by chondrocytes, but not fibroblasts.     

Differing in vitro biology of equine,ovine, porcine and human articular chondrocytes derived from the knee joint: an immunomorphological study

For lack of sufficient human cartilage donors, chondrocytes isolated from various animal species are used for cartilage tissue engineering. The present study was undertaken to compare key features of cultured large animal and human articular chondrocytes of the knee joint. Primary chondrocytes were isolated from human, porcine, ovine and equine full thickness knee joint cartilage and investigated flow cytometrically for their proliferation rate. Synthesis of extracellular matrix proteins collagen type II, cartilage proteoglycans, collagen type I, fibronectin and cytoskeletal organization were studied in freshly isolated or passaged chondrocytes using immunohistochemistry and western blotting. Chondrocytes morphology, proliferation, extracellular matrix synthesis and cytoskeleton assembly differed substantially between these species. Proliferation was higher in animal derived compared with human chondrocytes. All chondrocytes expressed a cartilage-specific extracellular matrix. However, after monolayer expansion, cartilage proteoglycan expression was barely detectable in equine chondrocytes whereby fibronectin and collagen type I deposition increased compared with porcine and human chondrocytes. Animal-derived chondrocytes developed more F-actin fibers during culturing than human chondrocytes. With respect to proliferation and extracellular matrix synthesis, human chondrocytes shared more similarity with porcine than with ovine or equine chondrocytes. These interspecies differences in chondrocytes in vitro biology should be considered when using animal models.     

The in vitro cell culture age and cell density of articular chondrocytes alter sulfated-proteoglycan biosynthesis

The effect of cell culture age and concomitant changes in cell density on the biosynthesis of sulfated-proteoglycan by rabbit articular chondrocytes in secondary monolayer culture was studied. Low density (LD, 2 d), middle density (MD, 5-7 d), and high density (HD, 12-15 d) cultures demonstrated changes in cellular morphology and rates of DNA synthesis. DNA synthesis was highest at LD to MD densities, but HD cultures continued to incorporate [3H]-thymidine. LD cultures incorporated 35SO4 into sulfated-proteoglycans at a higher rate than MD or LD cultures. The qualitative nature of the sulfated-proteoglycans synthesized at the different culture ages were analyzed by assessing the distribution of incorporated 35SO4 in associative and dissociative CsCl density gradients and by elution profiles on Sepharose CL-2B. Chondrocytes deposited into the extracellular matrix (cell-associated fraction) 35SO4-labeled proteoglycan aggregate. More aggregated proteoglycan was found in the MD and HD cultures than at LD. A 35SO4-labeled aggregated proteoglycan of smaller hydrodynamic size than that found in the cell-associated fraction was secreted into the culture medium at each culture age. The proteoglycan monomer (A1D1) of young and older cultures had similar hydrodynamic sizes at all cell culture ages and cell densities. The glycosaminoglycan chains of A1D1 were hydrodynamically larger in the younger LD cultures than in the older HD cultures and consisted of only chondroitin 6 and 4 sulfate chains. A small amount of chondroitin 4,6 sulfate was detected, but no keratan sulfate was measured. The A1D2 fractions of young LD cultures contained measurable amounts of dermatan sulfate; no dermatan sulfate was found in older MD or HD cultures. These studies indicated that chondrocytes at LD synthesized a proteoglycan monomer with many of the characteristics of young immature articular cartilage of rabbits. These results also indicated that rapidly dividing chondrocytes were capable of synthesizing proteoglycans which form aggregates with hyaluronic acid. Culture age and cell density appears primarily to modulate the synthesis of glycosaminoglycan types and chain length. Whether or not these glycosaminoglycans are found on the same or different core proteins remains to be determined.     

Dermal fibroblasts respond to mechanical conditioning in a strain profile dependent manner

Fibroblasts within tissues are exposed to a dynamic mechanical environment, which influences the structural integrity of both healthy and healing soft tissues. Various systems have been proposed to subject such cells to mechanical stimulation in culture. However the diverse nature of the studies, in terms of the strain profiles and the cell types, makes direct comparisons almost impossible. The present study addresses this issue by examining the metabolic response of two cell types subjected to three well defined strain profiles.A young fibroblast cell population, represented by HuFFs, showed both greater cell proliferation and collagen production than adult dermal fibroblasts under unstrained conditions. The three strain profiles produced differing effects on both cell types. Uniaxial strains enhanced [(3)H]-thymidine incorporation for both cell types, whilst biaxial strains either inhibited or had no effect on its incorporation. In contrast, [(3)H]-proline incorporation was inhibited under biaxial and uniaxial strains for the adult fibroblasts, whilst the HuFF cells showed a small increase in proline incorporation under non-uniform and uniaxial strains.     

Hydrostatic fluid pressure enhances matrix synthesis and accumulation by bovine chondrocytes in three-dimensional culture

Monolayer cell cultures and cartilage tissue fragments have been used to examine the effects of hydrostatic fluid pressure (HFP) on the anabolic and catabolic functions of chondrocytes. In this study, bovine articular chondrocytes (bACs) were grown in porous three-dimensional (3-D) collagen sponges, to which constant or cyclic (0.015 Hz) HFP was applied at 2.8 MPa for up to 15 days. The effects of HFP were evaluated histologically, immunohistochemically, and by quantitative biochemical measures. Metachromatic matrix accumulated around the cells within the collagen sponges during the culture period. There was intense intracellular, pericellular, and extracellular immunoreactivity for collagen type II throughout the sponges in all groups. The incorporation of [(35)S]-sulfate into glycosaminoglycans (GAGs) was 1.3-fold greater with constant HFP and 1.4-fold greater with cyclic HFP than in the control at day 5 (P < 0.05). At day 15, the accumulation of sulfated-GAG was 3.1-fold greater with constant HFP and 2.7-fold with cyclic HFP than the control (0.01). Quantitative immunochemical analysis of the matrix showed significantly greater accumulation of chondroitin 4-sulfate proteoglycan (C 4-S PG), keratan sulfate proteoglycan (KS PG), and chondroitin proteoglycan (chondroitin PG) than the control (P < 0.01). With this novel HFP culture system, 2.8 MPa HFP stimulated synthesis of cartilage-specific matrix components in chondrocytes cultured in porous 3-D collagen sponges.