The combination of insulin-like growth factor 1 and osteogenic protein 1 promotes increased survival of and matrix synthesis by normal and osteoarthritic human articular chondrocytes

OBJECTIVE: Although growth factor therapy could be an attractive method for stimulating the repair of damaged cartilage matrix, there is evidence that with aging and/or with the development of osteoarthritis (OA), articular chondrocytes may become unresponsive to growth factor stimulation. The aim of the current study was to compare the ability of insulin-like growth factor+(IGF-1) and osteogenic protein+(OP-1), alone and in combination, to stimulate human normal and OA chondrocytes in culture. METHODS: Chondrocytes isolated by enzymatic digestion of cartilage obtained from subjects undergoing knee replacement for OA (n = 6) or from normal ankle joints of tissue donors (n = 7) were cultured in alginate beads in serum-free medium and treated for 21 days with 100 ng/ml IGF-1, 100 ng/ml OP-1, or both. Controls were treated with vehicle alone. The cultures were evaluated for cell survival, cell number by DNA analysis, matrix production by particle exclusion assay, and level of accumulated proteoglycan by dimethylmethylene blue assay. RESULTS: After 21 days in serum-free alginate culture, survival of cells from OA cartilage was 65 +/- 2% (mean +/- SEM), while survival of cells from normal cartilage was significantly greater (82 +/- 3%). Treatment with either IGF-1 or OP-1 alone minimally improved survival, while the combination IGF +OP significantly improved survival, to 87 +/- 2% for OA cells and 95+/-1% for normal cells. Cell proliferation was noted only in the IGF+OP group; this was significant for both normal and OA cells ( approximately 2-fold increase in DNA levels). Matrix production, assessed by particle exclusion and by proteoglycan accumulation, was greatest in the cells treated with IGF + OP in both normal and OA cultures. When proteoglycan levels were corrected for cell numbers (mg proteoglycan/ng DNA), a significant increase over control was noted with OP-1 alone and IGF IGF-1 alone, in both normal and OA cultures, with the greatest levels in the combination group (3-fold increase over control). CONCLUSION: OP-1 was more potent than IGF-1 in stimulating proteoglycan production in both normal and OA cells. However, the best results were obtained with the combination, suggesting that combined therapy with IGF-1 and OP-1 may be an effective strategy for treating OA cartilage damage.     

Technology insight: adult mesenchymal stem cells for osteoarthritis therapy

Despite the high prevalence and morbidity of osteoarthritis (OA), an effective treatment for this disease is currently lacking. Restoration of the diseased articular cartilage in patients with OA is, therefore, a challenge of considerable appeal to researchers and clinicians. Techniques that cause multipotent adult mesenchymal stem cells (MSCs) to differentiate into cells of the chondrogenic lineage have led to a variety of experimental strategies to investigate whether MSCs instead of chondrocytes can be used for the regeneration and maintenance of articular cartilage. MSC-based strategies should provide practical advantages for the patient with OA. These strategies include use of MSCs as progenitor cells to engineer cartilage implants that can be used to repair chondral and osteochondral lesions, or as trophic producers of bioactive factors to initiate endogenous regenerative activities in the OA joint. Targeted gene therapy might further enhance these activities of MSCs. Delivery of MSCs might be attained by direct intra-articular injection or by graft of engineered constructs derived from cell-seeded scaffolds; this latter approach could provide a three-dimensional construct with mechanical properties that are congruous with the weight-bearing function of the joint. Promising experimental and clinical data are beginning to emerge in support of the use of MSCs for regenerative applications.     

A comparative assessment of cartilage and joint fat pad as a potential source of cells for autologous therapy development in knee osteoarthritis

Objectives. The utility of autologous chondrocytes for cartilagerepair strategies in older subjects with osteoarthritis (OA)may be limited by both age-related and disease-associated declinein chondrogenesis. The aim of this work was to assess OA Hoffa'sfat pad as an alternative source of autologous chondroprogenitorcells and to compare it with OA chondrocytes derived from differentareas of cartilage. Methods. Cartilage and fat pad tissue digests were obtainedfrom 26 subjects with knee OA and compared with normal bonemarrow (BM) mesenchymal stem cells (MSCs) with respect to theirin vitro colony-forming potential, growth kinetics, multipotentialityand clonogenicity. Flow cytometry was used to investigate theirMSC marker phenotype. Results. Expanded cultures derived from eroded areas of cartilagewere slightly more chondrogenic than those derived from macroscopicallynormal cartilage or chondro-osteophytes; however, all cartilage-derivedcultures failed to maintain their chondrogenic potency followingextended expansion. In contrast, OA fat pads contained highlyclonogenic and multipotential cells with stable chondrogenicpotency in vitro, even after 16 population doublings. Standardcolony-forming assays failed to reflect the observed functionaldifferences between the studied tissues whereas flow cytometryrevealed higher levels of a putative MSC marker low-affinitygrowth factor receptor (LNGFR) on culture expanded fat pad-derived,but not cartilage-derived, MSCs. Conclusions. In contrast to OA cartilage from three differentsites, OA Hoffa's fat pad contains clonogenic cells that meetthe criteria for MSCs and produce multipotential cultures thatmaintain their chondrogenesis long term. These findings havebroad implications for future strategies aimed at cartilagerepair in OA. KEY WORDS: Mesenchymal Stem Cells, Osteoarthritis, Cartilage Submitted 7 February 2007; revised version accepted 17 July 2007.     

Reduced chondrogenic and adipogenic activity of mesenchymal stem cells from patients with advanced osteoarthritis

OBJECTIVE: Mesenchymal stem cells (MSCs) are resident in the bone marrow throughout normal adult life and have the capacity to differentiate along a number of connective tissue pathways, among them bone, cartilage, and fat. To determine whether functionally normal MSC populations may be isolated from patients with advanced osteoarthritis (OA), we have compared cells from patients undergoing joint replacement with cells from normal donors. Cell populations were compared in terms of yield, proliferation, and capacity to differentiate. METHODS: MSCs were prepared from bone marrow aspirates obtained from the iliac crest or from the tibia/femur during joint surgery. In vitro chondrogenic activity was measured as glycosaminoglycan and type II collagen deposition in pellet cultures. Adipogenic activity was measured as the accumulation of Nile Red O-positive lipid vacuoles, and osteogenic activity was measured as calcium deposition and by von Kossa staining. RESULTS: Patient-derived MSCs formed colonies in primary culture that were characteristically spindle-shaped with normal morphology. The primary cell yield in 36 of 38 cell cultures from OA donors fell within the range found in cultures from normal donors. However, the proliferative capacity of patient-derived MSCs was significantly reduced. There was a significant reduction in in vitro chondrogenic and adipogenic activity in cultures of patient-derived cells compared with that in normal cultures. There was no significant difference in in vitro osteogenic activity. There was no decline in chondrogenic potential with age in cells obtained from individuals with no evidence of OA. CONCLUSION: These results raise the possibility that the increase in bone density and loss of cartilage that are characteristic of OA may result from changes in the differentiation profile of the progenitor cells that contribute to the homeostatic maintenance of these tissues.     

Up-regulation of CD44 in rheumatoid chondrocytes

The adhesion molecule CD44 is thought to play an important role in the inflammatory process. To identify the expression of CD44 in articular chondrocytes in rheumatoid arthritis (RA), monoclonal anti-CD44 antibodies were immunohistochemically used to react with articular cartilage specimens of 15 patients with RA, 9 with osteoarthritis (OA), and 6 with femoral neck fracture (FF). The proportion of CD44-positive chondrocytes in RA was 93 +/- 2% (N=16), which was significantly higher than that in OA (59 +/- 7%, N=9, p<0.001) and FF (46 +/- 5%, N=6, p<0.001). Among CD44 isoforms examined, the hemopoietic form was dominant in chondrocytes in RA. Therefore, up-regulation of CD44 on chondrocytes may play a significant role in cartilage degeneration in RA.     

Clonal precursor of bone,cartilage, and hematopoietic niche stromal cells

Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells.     

Enumeration and phenotypic characterization of synovial fluid multipotential mesenchymal progenitor cells in inflammatory and degenerative arthritis

OBJECTIVE: To evaluate synovial fluid (SF) for the presence of mesenchymal progenitor cells (MPCs), to compare SF MPCs with bone marrow (BM) MPCs, and to enumerate these cells in both inflammatory arthritis and osteoarthritis (OA). METHODS: SF from 100 patients with arthritis (53 rheumatoid arthritis [RA], 20 OA, and 27 other arthropathies) was evaluated. To establish multipotentiality, polyclonal and single cell-derived cultures of SF fibroblasts were examined by standard and quantitative differentiation assays. Their phenotype before and after expansion was determined by multiparameter flow cytometry. A colony-forming unit-fibroblast assay was used for SF MPC enumeration. RESULTS: Regardless of the nature of the arthritis, both polyclonal and single cell-derived cultures of SF fibroblasts possessed trilineage mesenchymal differentiation potentials. The number of MPCs in a milliliter of SF was higher in OA (median 37) than in RA (median 2) (P < 0.00001). No significant differences in MPC numbers were found between early and established RA (median 3 and 2 cells/ml, respectively). Culture-expanded SF and BM MPCs had the same phenotype (negative for CD45 and positive for D7-FIB, CD13, CD105, CD55, and CD10). Rare, uncultured SF fibroblasts were CD45(low) and expressed low-affinity nerve growth factor receptor, similar to in vivo BM MPCs. CONCLUSION: Our findings prove the presence of rare tripotential MPCs, at the single-cell level, in the SF of patients with arthritis. SF MPCs are clonogenic and multipotential fibroblasts that, despite the pathologic environment within a diseased joint, have a phenotype similar to that of uncultured BM MPCs. The higher prevalence of MPCs in OA SF suggests their likely origin from disrupted joint structures. These findings could determine the role of MPCs in the pathogenesis of inflammatory arthritis, together with their role in attempted joint regeneration in degenerative arthritis, which has yet to be established.     

Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9

OBJECTIVE: Human osteoarthritis (OA) is characterized by a pathologic shift in articular cartilage homeostasis toward the progressive loss of extracellular matrix (ECM). The purpose of this study was to investigate the ability of rAAV-mediated SOX9 overexpression to restore major ECM components in human OA articular cartilage. METHODS: We monitored the synthesis and content of proteoglycans and type II collagen in 3-dimensional cultures of human normal and OA articular chondrocytes and in explant cultures of human normal and OA articular cartilage following direct application of a recombinant adeno-associated virus (rAAV) SOX9 vector in vitro and in situ. We also analyzed the effects of this treatment on cell proliferation in these systems. RESULTS: Following SOX9 gene transfer, expression levels of proteoglycans and type II collagen increased over time in normal and OA articular chondrocytes in vitro. In situ, overexpression of SOX9 in normal and OA articular cartilage stimulated proteoglycan and type II collagen synthesis in a dose-dependent manner. These effects were not associated with changes in chondrocyte proliferation. Notably, expression of the 2 principal matrix components could be restored in OA articular cartilage to levels similar to those in normal cartilage. CONCLUSION: These data support the concept of using direct, rAAV-mediated transfer of chondrogenic genes to articular cartilage for the treatment of OA in humans.     

MicroRNA‐140 is expressed in differentiated human articular chondrocytes and modulates interleukin‐1 responses

Objective

MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue‐specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA‐140 (miR‐140).

Methods

To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR‐140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin‐1β (IL‐1β) on miR‐140 expression. Double‐stranded miR‐140 (ds–miR‐140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA.

Results

Microarray analysis showed that miR‐140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR‐140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR‐140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL‐1β suppressed miR‐140 expression. Transfection of chondrocytes with ds–miR‐140 down‐regulated IL‐1β–induced ADAMTS5 expression and rescued the IL‐1β–dependent repression of AGGRECAN gene expression.

Conclusion

This study shows that miR‐140 has a chondrocyte differentiation–related expression pattern. The reduction in miR‐140 expression in OA cartilage and in response to IL‐1β may contribute to the abnormal gene expression pattern characteristic of OA.

     

Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells

OBJECTIVE: There is an increased interest in rheumatology in mesenchymal progenitor/stem cells (MPCs) and their roles in rheumatic diseases, but little is known about the phenotype of these cells in vivo. The aim of this study was to isolate and characterize human bone marrow (BM) MPCs. METHODS: Fluorescence microscopy was used to identify putative MPCs among adherent BM cells. To purify them, a positive selection with antifibroblast microbeads was used, combined with fluorescence-activated cell sorting (FACS) for microbead+,CD45(low) cells. A more detailed phenotype of these cells was determined using 4-color flow cytometry, and standard chondrogenic, osteogenic, and adipogenic assays were used to investigate their differentiation potentials. RESULTS: Putative MPCs microscopically identified as large, fibroblast-like, D7-FIB+ cells were purified using positive selection with D7-FIB-conjugated (antifibroblast) microbeads followed by FACS for specifically bound microbead+,CD45(low) cells. These cells represented 0.01% of mononuclear cells in the BM. They were uniformly positive for CD105, LNGFR, HLA-DR, CD10, CD13, CD90, STRO-1, and bone morphogenetic protein receptor type IA (BMPRIA) and were negative for CD14, CD34, CD117, and CD133. Only cells with this phenotype could proliferate and produce adherent cell monolayers capable of chondrogenic, osteogenic, and adipogenic differentiation. D7-FIB- cells in the BM lacked any MPC activity. Uncultured skin fibroblasts had a phenotype similar to that of BM MPCs, but were negative for LNGFR, STRO-1, HLA-DR, and BMPRIA. CONCLUSION: This study shows the distinct phenotype, morphology, and method of isolation of BM MPCs. The findings may have implications for defining the physiologic roles of MPCs in arthritis, bone diseases, and joint regeneration.     

Large‐scale extraction and characterization of CD271+ multipotential stromal cells from trabecular bone in health and osteoarthritis: Implications for bone regeneration strategies based on uncultured or minimally cultured multipotential stromal cells

Objective

To test the hypothesis that CD45^lowCD271+ bone marrow multipotential stromal cells (MSCs) are abundant in the trabecular bone niche and to explore their functional “fitness” in health and osteoarthritis (OA).

Methods

Following enzymatic extraction, MSC release was evaluated using colony‐forming unit–fibroblast (CFU‐F) and colony‐forming unit–osteoblast assays, flow cytometry, and confocal microscopy. CD45^lowCD271+ cells isolated by fluorescence‐activated cell sorting were enumerated and expanded under standard and clonal conditions. Their proliferative and osteogenic potencies were assessed in relation to donor age and compared with those of aspirated CD45^lowCD271+ cells. In vitro and in vivo MSC “aging” was measured using quantitative polymerase chain reaction–based telomere length analysis, and standard differentiation assays were utilized to demonstrate multipotentiality.

Results

Cellular isolates from trabecular bone cavities contained ∼65‐fold more CD45^lowCD271+ cells compared with aspirates (P < 0.0001) (median 1.89% [n = 39] and 0.029% [n = 46], respectively), concordant with increased CFU‐F release. Aspirated and enzymatically released CD45^lowCD271+ cells had identical MSC phenotypes (∼100% CD73+CD105+CD13+, ∼50–60% CD146+CD106+CD166+) and contained large proportions of highly clonogenic multipotential cells. In vitro osteogenic potency of freshly isolated CD45^lowCD271+ cells was comparable with, and often above, that of early‐passage MSCs (8–14%). Their frequency and in vivo telomere status in OA bone were similar to those in bone from age‐matched controls.

Conclusion

Our findings show that CD45^lowCD271+ MSCs are abundant in the trabecular bone cavity and indistinguishable from aspirated CD45^lowCD271+ MSCs. In OA they display aging‐related loss of proliferation but no gross osteogenic abnormality. These findings offer new opportunities for direct study of MSCs in musculoskeletal diseases without the requirement for culture expansion. They are also relevant for direct therapeutic exploitation of prospectively isolated, minimally cultured MSCs in trauma and OA.

     

人胎肺间充质干细胞的分离

目的 探索分离人胎肺间充质干细胞（MSCs）的有效方法.方法 在无菌条件下采集人工流产胎儿的肺组织,分别以组织块培养法和酶消化法分离细胞;通过相差显微镜观察细胞形态,流式细胞学方法检测细胞表型、成脂和成骨分化的多向分化潜能并证实其MSCs特征.结果 两种分离方法所得细胞,在原代培养初期的形态有所不同,但培养后期和传代后均为长梭形的贴壁细胞.流式细胞学检测显示,两种方法分离的细胞均表达MSCs标志物CD13、CD29、CD44、CD90、CD105、CD166及HLA-ABC,但不表达造血细胞系的标志物CD45、CD34、CD14、CD38、CD133和内皮相关抗原CD31,也不表达CD41a、CD42b、CD49d、CD106、CD61和HLA-DR.成脂诱导3～5d后,部分细胞转变为肥大、扁平的多角形细胞;1周后可见细胞内有囊泡状脂滴积聚;2周后脂滴增多、变大,并可被红O着色.成骨诱导者细胞内逐渐出现钙盐沉着,经诱导2周后大部分细胞内可见因钙盐沉着被茜素红S着色.结论 组织块培养法和胶原酶消化法均为获得人胎肺MSCs的有效方法.     

5-Azacytidine-treated human mesenchymal stem/progenitor cells derived from umbilical cord, cord blood and bone marrow do not generate cardiomyocytes in vitro at high frequencies

Background and Objectives   Mesenchymal stem/progenitor cells (MSCs) are multipotent progenitors that differentiate into such lineages as bone, fat, cartilage and stromal cells that support haemopoiesis. Bone marrow MSCs can also contribute to cardiac repair, although the mechanism for this is unclear. Here, we examine the potential of MSCs from different sources to generate cardiomyocytes in vitro , as a means for predicting their therapeutic potential after myocardial infarction.
Materials and Methods   Mesenchymal stem/progenitor cells were isolated from the perivascular tissue and Wharton's jelly of the umbilical cord and from cord blood. Their immunophenotype and differentiation potential to generate osteoblasts, chondrocytes, adipocytes and cardiomyoxcytes in vitro was compared with those of bone marrow MSCs.
Results   Mesenchymal stem/progenitor cells isolated from umbilical cord and cord blood were phenotypically similar to bone marrow MSCs, the exception being in the expression of CD106, which was absent on umbilical cord MSCs, and CD146 that was highly expressed in cord blood MSCs. They have variable abilities to give rise to osteoblasts, chondrocytes and adipocytes, with bone marrow MSCs being the most robust. While a small proportion (~0·07%) of bone marrow MSCs could generate cardiomyocyte-like cells in vitro, those from umbilical cord and cord blood did not express cardiac markers either spontaneously or after treatment with 5-azacytidine.
Conclusion   Although MSCs may be useful for such clinical applications as bone or cartilage repair, the results presented here indicate that such cells do not generate cardiomyocytes frequently enough for cardiac repair. Their efficacy in heart repair is likely to be due to paracrine mechanisms.     

Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow

Human mesenchymal stem/progenitor cells (MSCs) have been identified in adult bone marrow, but little is known about their presence during fetal life. MSCs were isolated and characterized in first-trimester fetal blood, liver, and bone marrow. When 10(6) fetal blood nucleated cells (median gestational age, 10(+2) weeks [10 weeks, 2 days]) were cultured in 10% fetal bovine serum, the mean number (+/- SEM) of adherent fibroblastlike colonies was 8.2 +/- 0.6/10(6) nucleated cells (69.6 +/- 10/microL fetal blood). Frequency declined with advancing gestation. Fetal blood MSCs could be expanded for at least 20 passages with a mean cumulative population doubling of 50.3 +/- 4.5. In their undifferentiated state, fetal blood MSCs were CD29(+), CD44(+), SH2(+), SH3(+), and SH4(+); produced prolyl-4-hydroxylase, alpha-smooth muscle actin, fibronectin, laminin, and vimentin; and were CD45(-), CD34(-), CD14(-), CD68(-), vWF(-), and HLA-DR(-). Fetal blood MSCs cultured in adipogenic, osteogenic, or chondrogenic media differentiated, respectively, into adipocytes, osteocytes, and chondrocytes. Fetal blood MSCs supported the proliferation and differentiation of cord blood CD34(+) cells in long-term culture. MSCs were also detected in first-trimester fetal liver (11.3 +/- 2.0/10(6) nucleated cells) and bone marrow (12.6 +/- 3.6/10(6) nucleated cells). Their morphology, growth kinetics, and immunophenotype were comparable to those of fetal blood-derived MSCs and similarly differentiated along adipogenic, osteogenic, and chondrogenic lineages, even after sorting and expansion of a single mesenchymal cell. MSCs similar to those derived from adult bone marrow, fetal liver, and fetal bone marrow circulate in first-trimester human blood and may provide novel targets for in utero cellular and gene therapy.     

Matrix metalloproteinase and proinflammatory cytokine production by chondrocytes of human osteoarthritic cartilage: associations with degenerative changes

OBJECTIVE: To examine by immunohistochemistry the relative distributions of 6 matrix metalloproteinases (MMPs 1, 2, 3, 8, 9, and 13) and the 2 proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) in osteoarthritic (OA) cartilage compared with normal, age-matched articular cartilage. METHODS: Articular cartilage samples were obtained from the tibial plateau of OA knees removed at arthroplasty and from normal, nonarthritic, knees obtained at autopsy. Specimens were promptly fixed in Carnoy's fixative, processed, embedded in paraffin, sectioned, and examined by immunohistochemistry for MMP and cytokine production. In addition, human articular chondrocytes (HAC) were treated in vitro with either IL-1beta, TNFalpha, or phorbol myristate acetate (PMA) to assess their potential to produce each of the MMPs, as determined by Western blotting and gelatin zymography. RESULTS: Immunodetection of the collagenases (MMPs 1, 8, and 13) and stromelysin 1 (MMP-3) was demonstrated in a proportion of chondrocytes in the superficial zone of almost all of the OA specimens that had degenerative matrix changes. The gelatinases (MMPs 2 and 9) were also demonstrated by immunohistochemistry but were not so prominent. IL-1beta- and TNFalpha-positive chondrocytes were also observed in a proportion of cells in the superficial zones of OA specimens. Much less immunostaining for MMPs and cytokines was observed in the deep zone of all OA specimens, where the cartilage matrix and chondrocyte morphology appeared normal. In contrast, full-thickness normal cartilage specimens showed virtually no immunostaining for these MMPs or cytokines. Confirmation that chondrocytes can produce these 6 MMPs was obtained from HAC cultures treated with either IL-1beta, TNFalpha, or PMA; conditioned medium from activated HAC contained all the MMPs demonstrated by immunohistochemistry. Dual immunolocalization studies of OA cartilage specimens demonstrated the coexpression of IL-1 with MMP-8 by individual chondrocytes in situ. CONCLUSION: These results indicate that the superficial zone of OA cartilage specimens, which is characterized by fibrillations, chondrocyte clusters, and degenerative matrix changes, contains a variable proportion of cells that immunostain for IL-1beta, TNFalpha, and 6 different MMPs. These observations support the concept that cytokine-MMP associations reflect a modified chondrocyte phenotype and an intrinsic process of cartilage degradation in OA.     

Copper modulation of extracellular matrix synthesis by human articular chondrocytes

OBJECTIVE: The effects of Cu2+ on human articular chondrocytes, arising from both N (normal) and OA (osteoarthritic) cartilage, were investigated "in vitro". METHODS: Chondrocytes, cultured in high density, were incubated with copper chloride (0.01-0.25 microM/mL). Proteoglycan and collagen were assessed by incorporation of [35S]-Sulfate and [3H]-Proline. SDS-PAGE analysis was performed to quantify the ratio of type II to type I collagen. RESULTS: Cu2+ neither increased proteoglycan synthesis by chondrocytes. of origin N or OA, nor influenced their proliferation rate. Collagen synthesis was increased. This effect is time and concentration dependant: in cultures treated for 12 days, collagen synthesis stimulation was +20% and +26% (P < 0.02) in N and OA cultures respectively, the ratio of type II to type I collagen was slightly increased. This effect was more obvious in OA cell lines than in N ones. CONCLUSION: The observations suggest that Cu2+ upregulates collagen anabolism in human articular chondrocytes.     

FAS/FAS ligand expression and induction of apoptosis in chondrocytes

Objective. To examine the expression of Fas/Fas ligand and the role of this ligand/receptor interaction in the regulation of apoptosis in normal human articular chondrocytes and in osteoarthritis (OA) cartilage. Methods. Normal and OA human knee cartilage and cells isolated from these tissues were tested for Fas expression by flow cytometry. Induction of apoptosis by antibody to Fas was analyzed by DAPI staining and electron microscopy. Results. Treatment of freshly isolated normal human articular chondrocytes with an agonistic Fas antibody induced apoptosis in a subpopulation (-20%) of the cells. Apoptosis induced by anti-Fas was not dependent on nitric oxide (NO), and anti-Fas also did not induce NO production. Analysis of isolated cells demonstrated similar levels of Fas expression on normal and OA chondrocytes (28% and 32%, respectively). In normal articular cartilage, Fas-positive cells were located mainly in the superficial and midzones. In contrast, in fibrillated OA cartilage, surface layers were partially absent and Fas-expressing cells were also detected in the deeper layers. Fas ligand messenger RNA was not detectable in resting or activated normal or OA chondrocytes. Analysis by electron microscopy showed the nuclear and cytoplasmic changes typical of apoptosis in cultures treated with antibody to Fas. Conclusion. A subpopulation of chondrocytes expresses Fas and is susceptible to Fas-induced apoptosis. Fas-mediated chondrocyte apoptosis may contribute to cartilage degradation in arthritis.