Culture of chondrocytes in alginate and collagen carrier gels

In this in vitro study, we compared the potential of collagen and alginate gels as carriers for chondrocyte transplantation and we studied the influence of demineralized bone matrix (DBM) on chondrocytes in the gels. Chondrocytes were assessed for cell viability, phenotype (histology), proliferation rate and sulfate incorporation. Collagen gels showed a significant increase in cell numbers, but the chondrocytes dedifferentiated into fibroblast-like cells from day 6 onwards. In alginate gels, initial cell loss was found, but the cells maintained their typical chondrocyte phenotype. Although the total quantity of proteoglycans initially synthesized per cell in collagen gel was significantly higher, expressed per cell, the quantity in alginate gel eventually surpassed collagen. No effects of culturing chondrocytes in combination with DBM could be demonstrated on cell proliferation and sulfate incorporation. The collagen and alginate gels have different advantages as carriers for chondrocyte transplantation. The high proliferation rate of chondrocytes in collagen gel may be an advantage, but the preservation of the chondrocyte phenotype and the gradually increasing proteoglycan synthesis in alginate gel is a promising method for creating a hyaline cartilage implant in vitro.     

Effect of citrulline for arginine replacement on the structure and turnover of phosphopeptide substrates of protein phosphatase-1

In this study, the levels of mRNAs coding for aggrecan, decorin and biglycan in rabbit articular chondrocytes were investigated, using both monolayer and 3D-alginate cultures treated with TGF-beta 1 and IL-1 beta. The cells were shown to express higher amounts of proteoglycan messages, specially the aggrecan, in gels than in monolayers. TGF-beta 1 increased aggrecan mRNA in both systems, whereas biglycan message was elevated only in alginate. It markedly decreased decorin expression in monolayer, either in primary or passaged cultures. In contrast, IL-1 beta had a weak inhibitory effect on both decorin and biglycan expression. Subculturing induced a dramatic decrease of aggrecan mRNA, while that of decorin augmented. Biglycan expression transiently increased after two passages, whereas it declined in further subcultures. Passaged chondrocytes transferred to alginate re-expressed high levels of aggrecan, decorin and biglycan. The data point to the influence of morphology, proliferative state and environment of the articular chondrocytes on their biosynthetic responses to cytokines. Although these immature cells do not fully reflect the adult chondrocytes present in the cartilage, this study may help in understanding the behaviour of these cells in osteoarticular diseases, where the surrounding extracellular matrix is profoundly altered.     

Effect of osteogenic protein-1 on the development and mineralization of primary cultures of avian growth plate chondrocytes: modulation by retinoic acid

Osteogenic protein-1 (OP-1), a member of the TGF-beta family of proteins, induces endochondral bone formation. Here we studied the effect of OP-1 on the development of primary cultures of avian growth plate (GP) chondrocytes in either serum-free or serum-containing medium, in the absence or presence of retinoic acid (RA). OP-1 was added on day 7 of culture and continued for 7 days, or until the cultures were harvested, typically on day 21. Alone, OP-1 caused approximately 2-fold increase in proteoglycan synthesis into both the medium and the cell:matrix layer. Additionally, OP-1 caused a dosage-dependent increase in alkaline phosphatase (ALP) activity, and an increase in protein, when given from days 7-14 and examined on day 14. This stimulation was greater in cells grown in serum-free than in serum-containing media (3-5-fold vs. 2-3-fold increase in ALP; approximately 40% vs. approximately 20% increase in protein). Such stimulation of ALP activity and proteoglycan (PG) synthesis in cultured GP cells indicates that OP-1 elicits differentiation of chondrocytes. OP-1 minimally affected cell division (DNA content); however, a slight increase was seen when examined early in the culture. Alone, OP-1 increased mineral (Ca and Pi) content of the cultures by approximately 2-fold in both types of media. As early as day 14, clusters of mineral encircled many of the OP-1 treated cells. Thus, as in vivo, OP-1 strongly promoted mineral formation by the cultured GP chondrocytes. When present together, OP-1 and RA generally blocked the action of the other. Separately OP-1 and RA each stimulated protein synthesis, ALP activity, and Ca2+ deposition; together they were inhibitory to each. Also, RA blocked the stimulation of PG synthesis induced by OP-1; whereas OP-1 decreased cell division engendered by RA. Thus, this GP chondrocyte culture system is a good model for studying factors that influence differentiation and mineral deposition during bone growth in vivo.     

Do calcium and zinc ions influence matrix molecule synthesis of chondrocytes?

The experiments described here tested the effect of various calcium (Ca) and Zinc (Zn) concentrations on cell proliferation and matrix molecule synthesis of fetal and adult bovine chondrocytes in monolayer cultures. Levels of Ca < 0.2 mM in a culture medium or the addition of Zn (0.1-50 microM) selectively promoted the production of collagen but did not affect significantly synthesis of proteoglycans. No change in proliferation of fetal and adult chondrocytes could be observed. In contrast 10 mM Ca promoted the hypertrophic differentiation of chondrocytes (e.g. expression of collagen type X). The results are related to calcium channel configurations in chondrocytes in the discussion.     

Metabolism of the extracellular matrix formed by intervertebral disc cells cultured in alginate

STUDY DESIGN: Cells from normal rabbit nucleus pulposus (NP) and anulus fibrosus (AF) were cultured in alginate beads for as long as 14 days to allow them to reform a matrix made up of two compartments: the cell-associated matrix (CM) and further removed matrix (FRM). At different time points, the CM and FRM made by each cell population were analyzed using histologic, biochemical, and immunologic assays. OBJECTIVES: To study the metabolism of normal rabbit NP and AF cells in alginate by characterizing the CM and FRM formed by each cell population, and to identify metabolic properties that may shed light on mechanisms at play in disc degeneration. SUMMARY OF BACKGROUND DATA: Little is known about the metabolism of intervertebral disc cells, in part because of the lack of microculture systems appropriate for the study of these cells in vitro. In recent studies from our laboratories, it was suggested that articular chondrocytes cultured in alginate beads remain phenotypically stable and reform a matrix similar to the one they populate in vivo. This culture system appears ideally suited for the study of intervertebral cells available only in limited numbers. METHODS: Rabbit NP and AF cells released from the matrix by sequential enzyme digestion were encapsulated in alginate beads (20,000 cells/bead) and cultured for as long as 14 days. At selected time points, beads were solubilized with calcium chelating agents, and the CM and FRM were isolated. The rate of 35S-sulfate incorporation into proteoglycans, and the contents of various extracellular matrix molecules (total sulfated proteoglycans, antigenic keratan sulfate, hyaluronan, collagen, and pyridinium crosslinks) were measured. RESULTS: Both NP and AF cells remained phenotypically stable in the alginate gel throughout the culture period and reestablished a matrix composed of CM and FRM compartments. The two cell populations exhibited numerous differences in their metabolic activities in vitro. Nucleus pulposus cells synthesized fewer proteoglycan and collagen molecules and were less effective in incorporating these into the CM than AF cells. CONCLUSIONS: Intervertebral disc cells, especially NP cells, are extremely sluggish in reforming a CM, a protective shell rich in proteoglycans and collagen molecules. This may help explain why damage to the NP often is accompanied by progressive degeneration of the disc in vivo.     

Genetic and clinical features of sensorineural hearing loss associated with the 1555 mitochondrial mutation

This article describes the modulation, by extracellular collagen, of DNA and proteoglycan synthesis in articular chondrocytes stimulated with transforming growth factor-beta 1. Type-I and type-II collagen, heat-denatured type-II collagen, and bovine serum albumin were each incorporated into alginate in increasing concentrations. Bovine articular chondrocytes were isolated and were resuspended in the alginate, yielding alginate beads with final extracellular protein concentrations of 0-1.5% (wt/vol) for the collagens and 0-2.5% (wt/vol) for bovine serum albumin. Cultures of beads were maintained for 7 days in basal Dulbecco's modified Eagle medium or in medium supplemented with 10 ng/ml transforming growth factor-beta 1. Subsequently, the synthesis of DNA and proteoglycan was measured by radiolabel-incorporation methods with [35S]sulfate and [3H]thymidine, and the values were normalized to the DNA content. Transforming growth factor-beta 1 stimulated the synthesis of both DNA and proteoglycan in a bimodal fashion. The presence of extracellular type-II collagen increased the rate of DNA and proteoglycan synthesis in a dose-dependent fashion in cultures stimulated by transforming growth factor-beta 1, whereas heat-inactivated type-II collagen abrogated the effects observed with type-II collagen for synthesis of both DNA and proteoglycan. In contrast, the presence of extracellular type-I collagen caused a dose-dependent inhibition of synthesis of both DNA and proteoglycan in cultures stimulated with transforming growth factor-beta 1. Extracellular bovine serum albumin brought about a limited increase in synthesis rates, presumably by blocking nonspecific cytokine binding. These results suggest that type-II collagen has a specific role in chondrocyte regulation and serves to mediate the response of chondrocytes to transforming growth factor-beta 1.     

Effects of cyclopiazonic acid on Ca2+ regulation by the sarcoplasmic reticulum in saponin-permeabilized skeletal muscle fibres

Slices of lapine meniscus produced large amounts of nitric oxide after stimulation with interleukin-1, tumor necrosis factor alpha, or a mixture of lapine synovial cytokines known as chondrocyte-activating factors. Monolayer cultures of meniscal cells produced from the proteolysis of meniscal tissue contained a mixed population of chondrocytic and fibroblastic cells. These cultures also produced large amounts of nitric oxide in response to cytokines. Monolayer cultures of meniscal cells produced by the explant method, in contrast, were uniformly fibroblastic and did not produce nitric oxide in response to cytokines. We conclude that menisci contain two populations of cells, one fibroblastic and the other chondrocytic. The chondrocytic cells are responsible for generating most of the nitric oxide in response to cytokines. Endogenously generated nitric oxide suppressed the synthesis of collagen and proteoglycan by menisci but protected proteoglycan from the catabolic effects of interleukin-1. The inhibitory effect of nitric oxide on collagen synthesis occurred without greatly altering the abundance of mRNAs encoding the various collagen alpha chains. During further investigation, arginine was unexpectedly found to stimulate the synthesis of collagen and, to a lesser degree, of noncollagenous proteins but not of proteoglycans. Fragments of meniscus, but not meniscal cells in monolayer culture, increased their production of matrix metalloproteinases, lactate, and, especially, prostaglandin E2 in response to interleukin-1. Inhibition of nitric oxide production with NG-monomethyl-L-arginine enhanced production of matrix metalloproteinases but had little effect on the synthesis of lactate or prostaglandin E2.     

Transforming growth factor-beta 1 stimulates articular chondrocyte proteoglycan synthesis and induces osteophyte formation in the murine knee joint

BACKGROUND: High concentrations of active transforming growth factor-beta (TGF-beta) have been found in synovial fluids from arthritic joints. TGF-beta stimulates articular cartilage proteoglycan synthesis and suppresses proteoglycan degradation in vitro. In an earlier study, we found no effect on cartilage proteoglycan metabolism shortly after a single intra-articular injection of TGF-beta 1. In the present study, we used multiple intra-articular injections and a longer time-scale. EXPERIMENTAL DESIGN: TGF-beta 1 was injected into the murine knee joint to gain insight in the consequences of its overproduction in joint diseases. This was evaluated using histologic sections of the whole knee joint and measurements of articular cartilage proteoglycan synthesis and content. RESULTS: At 6 hours after a single TGF-beta 1 injection, recruitment of polymorphonuclear leukocytes (PMNs) was observed. After 24 hours, the amount of inflammatory cells had already decreased. Multiple TGF-beta 1 injections induced synovial hyperplasia and synovitis predominantly consisting of cells of the macrophage/monocyte lineage. Both single and multiple TGF-beta 1 injections induced strong and long-lasting stimulation of articular cartilage proteoglycan synthesis. This in vivo stimulation of proteoglycan synthesis was similar in cartilage of young (3 months) and old mice (18 months). Multiple TGF-beta 1 injections resulted in an increased GAG content in patellar cartilage. After triple TGF-beta 1 injections, impressive osteophyte formation was noted at specific sites. The size and the localization of osteophytes was identical in young and old mice. Interestingly, the localization of TGF-beta 1-induced osteophytes was very similar to that of osteophytes observed in experimental arthritis and osteoarthritis models, suggesting a role for endogenous TGF-beta in osteophyte formation during joint pathology. CONCLUSIONS: Our data indicate that TGF-beta 1 injection into a normal joint induces inflammation, synovial hyperplasia, osteophyte formation, and prolonged elevation of proteoglycan synthesis and content in articular cartilage.     

A new culture system to study the metabolism of the intervertebral disc in vitro

STUDY DESIGN: This study determined whether entrapment of a rabbit intervertebral disc in alginate gel helped to promote the retention of normal metabolic activities by the nucleus pulposus and anulus fibrosus in tissue culture. OBJECTIVES: To establish an in vitro culture system to study the metabolism of the intervertebral disc as a whole integral organ. SUMMARY OF BACKGROUND DATA: In vitro studies of the metabolism of intervertebral discs have been scarce because of the difficulties involved in maintaining the integrity of the tissues, especially that of the nucleus pulposus, in culture medium. METHODS: Rabbit intervertebral discs were embedded in alginate gel and maintained in culture for as long as 1 month. At weekly intervals, experiments were performed to measure the rate of proteoglycan synthesis and to characterize proteoglycans newly synthesized by cells in the anulus fibrosus and nucleus pulposus. In addition, at these same time intervals, the contents of sulfated proteoglycans, antigenic keratan sulfate, hyaluronan, and collagen in these two intervertebral disc tissues were measured to evaluate tissue integrity. Intervertebral discs cultured in medium alone were used as controls and analyzed in parallel. RESULTS: The anulus fibrosus and nucleus pulposus of intervertebral discs cultured in alginate gel sustained a higher rate of proteoglycan synthesis and maintained a higher content of extracellular matrix components than the respective controls at all times. CONCLUSIONS: This new alginate tissue culture system should prove useful for studying the metabolism of whole intervertebral discs.     

Inhibition of proteoglycan biosynthesis decreases the calcification of chondrocyte cultures

In order to determine the role of proteoglycan in the calcification of cartilage, the effects on calcifying chondrocyte cultures of treatments that disrupt proteoglycan biosynthesis have been studied. Treatment of secondary cultures of embryonic chick chondrocytes with non-toxic concentrations of the beta-xyloside p-nitrophenyl beta-D-xylopyranoside (PNPX) resulted in dose-dependent inhibition of both proteoglycan and mineral deposition. Based on the expression of Type X collagen, however, PNPX is also a potent inhibitor of chondrocyte differentiation. Under-sulfation of proteoglycans was effected by growth of chondrocyte cultures in sulfate-depleted medium. Growth in low-sulfate medium did not significantly affect the growth or differentiation of these cultures, but caused an approximate two-fold decrease in mineral content compared to cultures grown in normal medium. These findings indicate that disruption of proteoglycan biosynthesis in chondrocyte cultures results in decreased levels of calcification. Therefore, proteoglycans appear to function as promoters of chondrocyte calcification.     

Phenotypic diversity of neoplastic chondrocytes and extracellular matrix gene expression in cartilaginous neoplasms

Chondrocyte differentiation is characterized by distinct cellular phenotypes, which can be identified by specific extracellular matrix gene expression profiles. By applying in situ analysis on the mRNA and protein level in a series of benign and malignant human chondrogenic neoplasms, we were able to identify for the first time different phenotypes of neoplastic chondrocytes in vivo: 1) mature chondrocytes, which synthesized the characteristic cartilaginous extracellular tumor matrix, 2) cells resembling hypertrophic chondrocytes of the fetal growth plate, 3) cells resembling so-called dedifferentiated chondrocytes, and 4) well differentiated chondrocytic cells, which expressed type I collagen, indicating the presence of post-hypertrophic differentiated neoplastic chondrocytes. Chondrocytes exhibiting a range of phenotypes were found to be present in the same neoplasm. The different observed phenotypes, including the dedifferentiated phenotype, were in contrast to the anaplastic cells of high-grade chondrosarcomas. Comparison of expression data with tumor morphology revealed a relationship between the cellular phenotypes, the tumor matrix composition, and the matrix and cell morphology within the neoplasms. The distinctly different phenotypes of neoplastic chondrocytes are the basis of the characteristic high biochemical and morphological heterogeneity of chondroid neoplasms and shed light on their biological and clinical behavior.     

Formation of promutagenic methylation damage in tissue-DNA of mice treated with antischistosomal agents

BACKGROUND: Cultured bovine corneal endothelial cells (CEC) synthesize heparan sulfate and dermatan sulfate containing proteoglycans and distribute them between different compartments. METHODS AND RESULTS: [35S]sulfate labelled proteoglycans are found associated with the cell layer, secreted into the culture medium and deposited into the underlaying extracellular matrix. In the presence of basic fibroblast growth factor (bFGF)-a strong mitogen for CEC-subconfluent cells incorporate [35S]sulfate into the sulfated proteoglycans at a rate three times higher as compared with the proteoglycans of CEC in the absence of bFGF. The enhanced proteoglycan synthesis is accompanied with a shift in the proteoglycan distribution pattern. While in control cells the cell-associated heparan sulfate accounts for about 30% of the total glycosaminoglycans under the influence of bFGF the HS percentage increases to approximately 60%. CONCLUSIONS: CEC synthesize and deposit endogenous bFGF into the extracellular matrix. Heparitinase treatment of the extracellular matrix releases bFGF activity which is able to stimulate the 35S incorporation into proteoglycans in a comparable manner as exogenous bFGF but does not influence the proteoglycan distribution pattern. Pretreatment of the matrix-bound bFGF activity with polyclonal antibodies against bFGF abolishes its stimulating activity.     

Regulation of matrix synthesis rates by the ionic and osmotic environment of articular chondrocytes

Chondrocytes in cartilage are embedded in a matrix containing a high concentration of proteoglycans and hence of fixed negative charges. Their extracellular ionic environment is thus different from that of most cells, with extracellular Na+ being 250-350 mM and extracellular osmolality 350-450 mOsm. When chondrocytes are isolated from the matrix and incubated in standard culture medium (DMEM; osmolality 250-280 mOsm), their extracellular environment changes sharply. We incubated isolated bovine articular chondrocytes and cartilage slices in DMEM whose osmolality was altered over the range 250-450 mOsm by Na+ or sucrose addition. 35S-sulphate and 3H-proline incorporation rates were at a maximum when the extracellular osmolality was 350-400 mOsm for both freshly isolated chondrocytes and for chondrocytes in cartilage. The incorporation rate per cell of isolated chondrocytes was only 10% that of chondrocytes in situ both 4 and 24 hours after isolation. For freshly isolated chondrocytes, the rate increased 30-50% in DMEM to which NaCl or sucrose had been added to increase osmolality. In chondrocytes incubated overnight in DMEM, the rate was greatest in DMEM of normal osmolality and fell from the maximum in proportion to the change in osmolality. The effects of sucrose addition on incorporation rates were similar but not identical to those of Na+ addition. Changes in cell volume might be linked to changes in synthesis rates since the cell volume of chondrocytes (measured by Coulter-counter) increased 30-40% when the cells were removed from their in situ environment into DMEM. Synthesis rates can thus be partly regulated by changes in extracellular osmolality, which in cartilage is controlled by proteoglycan concentration. This provides a mechanism by which the chondrocytes can rapidly respond to changes in extracellular matrix composition.     

Modulation of IL-1-induced cartilage injury by NO synthase inhibitors: a comparative study with rat chondrocytes and cartilage entities

Nitric oxide (NO) is produced in diseased joints and may be a key mediator of IL-1 effects on cartilage. Therefore, we compared the potency of new [aminoguanidine (AG), S-methylisothiourea (SMT), S-aminoethylisothiourea (AETU)] and classical [Nomega-monomethyl-L-arginine (L-NMMA), Nomega-nitro-L-arginine methyl ester (L-NAME)] NO synthase (NOS) inhibitors on the inhibitory effect of recombinant human interleukin-1beta (rhIL-1beta) on rat cartilage anabolism. Three different culture systems were used: (1) isolated chondrocytes encapsulated in alginate beads; (2) patellae and (3) femoral head caps. Chondrocyte beads and cartilage entities were incubated in vitro for 48 h in the presence of rhIL-1beta with a daily change of incubation medium to obtain optimal responses on proteoglycan synthesis and NO production. Proteoglycan synthesis was assessed by incorporation of radiolabelled sodium sulphate [Na2(35)SO4] and NO production by cumulated nitrite release during the period of study. Chondrocytes and patellae, as well as femoral head caps, responded concentration-dependently to IL-1beta challenge (0 to 250 U ml(-1) and 0 to 15 U ml(-1) respectively) by a large increase in nitrite level and a marked suppression of proteoglycan synthesis. Above these concentrations of IL-1beta (2500 U ml(-1) and 30 U ml(-1) respectively), proteoglycan synthesis plateaued whereas nitrite release still increased thus suggesting different concentration-response curves. When studying the effect of NOS inhibitors (1 to 1000 microM) on NO production by cartilage cells stimulated with IL-1beta (25 U ml(-1) or 5 U ml(-1)), we observed that: (i) their ability to reduce nitrite level decreased from chondrocytes to cartilage samples, except for L-NMMA and AETU; (ii) they could be roughly classified in the following rank order of potency: AETU > L-NMMA > or = SMT > or = AG > or = L-NAME and (iii) AETU was cytotoxic when used in the millimolar range. When studying the effect of NOS inhibitors on proteoglycan synthesis by cartilage cells treated with IL-1beta, we observed that: (i) they had more marked effects on proteoglycan synthesis in chondrocytes than in cartilage samples; (ii) they could be roughly classified in the following rank order of potency: L-NAME > or = L-NMMA > > AG > SMT > > AETU and (iii) potentiation of the IL-1 effect by AETU was consistent with cytotoxicity in the millimolar range. D-isomers of L-arginine analog inhibitors (1000 microM) were unable to correct nitrite levels or proteoglycan synthesis in IL-1beta treated cells. L-arginine (5000 microM) tended to reverse the correcting effect of L-NMMA (1000 microM) on proteoglycan synthesis, thus suggesting a NO-related chondroprotective effect. However, data with L-NAME and SMT argued against a general inverse relationship between nitrite level and proteoglycan synthesis. Dexamethasone (0.1 to 100 microM) (i) failed to inhibit NO production in femoral head caps and chondrocytes beads whilst reducing it in patellae (50%) and (ii) did not affect or worsened the inhibitory effect of IL-1beta on proteoglycan synthesis. Such results suggested a corticosteroid-resistance of rat chondrocyte iNOS. Data from patellae supported a possible contribution of subchondral bone in NO production. In conclusion, our results suggest that (i) NO may account only partially for the suppressive effects of IL-1beta on proteoglycan synthesis, particularly in cartilage samples; (ii) the chondroprotective potency of NOS inhibitors can not be extrapolated from their effects on NO production by joint-derived cells and (iii) L-arginine analog inhibitors are more promising than S-substituted isothioureas for putative therapeutical uses.     

The global ethics of latex gloves: reflections on natural resource use in healthcare

The collagens produced by chick embryo chondrocytes cultured in alginate beads were investigated both biochemically and ultrastructurally. The cartilage phenotype is maintained for at least 14 days, as indicated by the production of the cartilage-specific collagens II, IX, and XI and the absence of collagen I. There were differences in the distributions of collagens among the three different compartments analyzed (cells and their associated matrix, further-removed matrix (released by alginate solubilization), and culture medium), with large amounts of collagen IX (mainly in proteoglycan form) in the culture medium. Inhibition of lysyl oxidase activity by beta-aminopropionitrile led to an overall decrease in collagen production. In contrast to the biochemical observations, collagen ultrastructure in the extracellular matrix of alginate cultures was not in the form of the expected 64-nm banded fibrils, but rather in the form of segment-long-spacing-like crystallites. This abnormal structure is likely to be a result of alginate disrupting normal assembly. We conclude that, in this system, the native fibrillar structure of the collagenous matrix is not essential for the maintenance of the differentiated phenotype of chondrocytes.     

Synergistic action of transforming growth factor-beta and insulin-like growth factor-I induces expression of type II collagen and aggrecan genes in adult human articular chondrocytes

Reexpression of aggrecan and type II collagen genes in dedifferentiated adult human articular chondrocytes (AHAC) in suspension culture varied widely depending on the specific lot of bovine serum used to supplement the culture medium. Some lots of serum provided strong induction of aggrecan and type II collagen expression by AHAC while others did not stimulate significant production of these hyaline cartilage extracellular matrix molecules even following several weeks in culture. Addition of 50 ng/ml insulin-like growth factor-I (IGF-I) to a deficient serum lot significantly enhanced its ability to induce aggrecan and type II collagen mRNA. Given this observation, IGF-I and other growth factors were tested in defined serum-free media for their effects on the expression of these genes. Neither IGF-I nor insulin nor transforming growth factor beta (TGF-beta) alone stimulated induction of aggrecan or type II collagen production by dedifferentiated AHAC. However, TGF-beta 1 or TGF-beta 2 combined with IGF-I or insulin provided a strong induction as demonstrated by RNase protection and immunohistochemical assays. Interestingly, type I collagen, previously shown to be downregulated in serum supplemented suspension cultures of articular chondrocytes, persisted for up to 12 weeks in AHAC cultured in defined medium supplemented with TGF-beta and IGF-I.     

Effects of injurious compression on matrix turnover around individual cells in calf articular cartilage explants

The effects of mechanical injury on the metabolism of cartilage matrix are of interest for understanding the pathogenesis of osteoarthrosis and the development of strategies for cartilage repair. The purpose of the present study was to examine the effects of injury on matrix turnover in a calf articular cartilage explant system for which the effects of mechanical loading on cell activity and the cell-mediated pathways of matrix metabolism are already well characterized. New methods of quantitative autoradiography were used in combination with established biochemical and biomechanical techniques for the analysis of cell and matrix responses to acute mechanical injury, with particular attention to the processes of localized matrix turnover in the cell-associated matrices of individual chondrocytes. Matrix deposition and turnover around cells in control explants was spatially dependent, with the highest rates of proteoglycan deposition and turnover and the lowest rates of collagen deposition (as indicated by [3H]proline autoradiography) occurring in the pericellular matrix. Injurious compression was associated with (a) an abrupt decrease in the tensile load-carrying capacity of the collagen matrix, apparently associated with mechanical failure of the tissue, (b) a considerable but subtotal decrease in cell viability, marked by the emergence of an apparently inactive cell population interspersed within catabolically active but abnormally large cells, and (c) sustained, elevated rates of proteoglycan turnover, particularly in the cell-associated matrices of apparently viable cells, which involved the increased release of aggregating species in addition to a spectrum of degradation fragments that were also in controls. These results may represent an in vitro model for the responses of chondrocytes and the cartilage extracellular matrix to mechanical injury.     

Chondrogenic potential of skeletal cell populations: selective growth of chondrocytes and their morphogenesis and development in vitro

Most vertebrate embryonic and post-embryonic skeletal tissue formation occurs through the endochondral process in which cartilage serves a transitory role as the anlage for the bone structure. The differentiation of chondrocytes during this process in vivo is characterized by progressive morphological changes associated with the hypertrophy of these cells and is defined by biochemical changes that result in the mineralization of the extracellular matrix. The mechanisms, which, like those in vivo, promote both chondrogenesis in presumptive skeletal cell populations and endochondral progression of chondrogenic cells, may be examined in vitro. The work presented here describes mechanisms by which cells within presumptive skeletal cell populations become restricted to a chondrogenic lineage as studied within cell populations derived from 12-day-old chicken embryo calvarial tissue. It is found that a major factor associated with selection of chondrogenic cells is the elimination of growth within serum-containing medium. Chondrogenesis within these cell populations appears to be the result of permissive conditions which select for chondrogenic proliferation over osteogenic cell proliferation. Data suggest that chondrocyte cultures produce autocrine factors that promote their own survival or proliferation. The conditions for promoting cell growth, hypertrophy, and extracellular matrix mineralization of embryonic chicken chondrocytes in vitro include ascorbic acid supplementation and the presence of an organic phosphate source. The differentiation of hypertrophic chondrocytes in vitro is associated with a 10-15-fold increase in alkaline phosphatase enzyme activity and deposition of mineral within the extracellular matrix. Temporal studies of the biochemical changes coincident with development of hypertrophy in vitro demonstrate that proteoglycan synthesis decreases 4-fold whereas type X collagen synthesis increases 10-fold within the same period. Ultrastructural examination reveals cellular and extracellular morphology similar to that of hypertrophic cells in vivo with chondrocytes embedded in a well formed extracellular matrix of randomly distributed collagen fibrils and proteoglycan. Mineral deposition is seen in the interterritorial regions of the matrix between the cells and is apatitic in nature. These characteristics of chondrogenic growth and development are very similar in vivo and in vitro and they suggest that studies of chondrogenesis in vitro may provide a valuable model for the process in vivo.     

Yohimbine, erectile capacity, and sexual response in men

In this study we investigated the hypothesis that cartilage from defined regions of ovine stifle joints, which were subjected to differing mechanical stresses, contained phenotypically distinct chondrocyte populations. Chondrocyte phenotypes were identified by the relative biosynthesis of the proteoglycans (PGs) aggrecan, biglycan and decorin. Articular cartilage (AC) from adult and neonatal ovine stifle joints were examined. Cells were cultured as both full-depth AC explants and in alginate beads after their isolation from the AC matrix. When chondrocytes from the various topographical regions of adult ovine knee joints were cultured as explants they demonstrated a consistent difference with regard to the metabolism of aggrecan and decorin. Significantly, this topographically-dependent phenotypic expression of PGs was preserved when the chondrocytes were cultured in alginate beads. In adult joints, chondrocytes from the central region of the tibial plateau not covered by the meniscus, which is subjected to high mechanical loads in-vivo, synthesized less aggrecan but more decorin than cells from regions covered by the meniscus. When chondrocytes from identical AC regions of neonatal ovine joints were cultured as explants, no topographical difference in aggrecan nor decorin metabolism could be detected. The results of this study, in association with the existing literature, lead us to propose that post-natal mechanical loading of AC could select for chondrocyte clones or induce a lasting modulation of chondrocyte phenotypic expression in different joint regions. Such cellular changes could result in the synthesis of PG populations that confer properties to AC most suited to resist the variable mechanical stresses in the different joint regions. This study serves to emphasize the importance of using cartilage from identical joint areas when examining PG metabolism by chondrocytes. Further investigation into the relationship between mechanical loading, regional chondrocyte phenotype selection and the response of these cells to anabolic and catabolic factors may provide important insights into the focal nature of AC degeneration in osteoarthritis.     

Alternatives to platelet transfusion]

It is well established that a full-thickness articular cartilage defect is repaired with a fibrocartilaginous tissue, cells of which are derived from undifferentiated mesenchymal stem cells in the bone marrow. To characterize the repair cells biochemically, full-thickness defects were created in rabbit knee joints and the repair tissues taken at 3, 6, and 12 weeks after surgery. The repair cells were cultured and examined biochemically to investigate the effects of four exogenous growth factors with regard to the metabolism of type II collagen and proteoglycans. A significant increase of carboxy-terminal type II procollagen peptide production was observed in the conditional medium of the repair cells, especially taken at 6 weeks after surgery, in the presence of each growth factor. Glycosaminoglycan content was also increased and proteoglycan synthesis stimulated. The repair cells taken at the early stage of the repair process could originally have more activity of type II collagen synthesis, and the growth factors used could enhance the differentiation of the repair cells in vitro.