Differentiation Of Articular Chondrocytes Research Articles

Synovial joint morphogenesis requires the chondrogenic action of Sox5 and Sox6 in growth plate and articular cartilage

The mechanisms underlying synovial joint development remain poorly understood. Here we use complete and cell-specific gene inactivation to identify the roles of the redundant chondrogenic transcription factors Sox5 and Sox6 in this process. We show that joint development aborts early in complete mutants ( Sox5 −/− 6 −/− ). Gdf5 and Wnt9a expression is punctual in articular progenitor cells, but Sox9 downregulation and cell condensation in joint interzones are late. Joint cell differentiation is unsuccessful, regardless of lineage, and cavitation fails. Sox5 and Sox6 restricted expression to chondrocytes in wild-type embryos and continued Erg expression and weak Ihh expression in Sox5 −/− 6 −/− growth plates suggest that growth plate failure contribute to this Sox5 −/− 6 −/− joint morphogenesis block. Sox5/6 inactivation in specified joint cells and chondrocytes ( Sox5 fl/fl 6 fl/fl Col2Cre) also results in a joint morphogenesis block, whereas Sox5/6 inactivation in specified joint cells only ( Sox5 fl/fl 6 fl/fl Gdf5Cre) results in milder joint defects and normal growth plates. Sox5 fl/fl 6 fl/fl Gdf5Cre articular chondrocytes remain undifferentiated, as shown by continued Gdf5 expression and pancartilaginous gene downregulation. Along with Prg4 downregulation, these defects likely account for joint tissue overgrowth and incomplete cavitation in adult mice. Together, these data suggest that synovial joint morphogenesis relies on essential roles for Sox5/6 in promoting both growth plate and articular chondrocyte differentiation.

Parathyroid hormone 1–34 inhibits terminal differentiation of human articular chondrocytes and osteoarthritis progression in rats

Parathyroid hormone 1-34 (PTH[1-34]), a parathyroid hormone analog, shares the same receptor, PTH receptor 1, with parathyroid hormone-related peptide (PTHrP). This study was undertaken to address the hypothesis that PTH(1-34) inhibits terminal differentiation of articular chondrocytes and in turn suppresses the progression of osteoarthritis (OA). We studied the effect of PTH(1-34) on human articular chondrocytes with azacytidine (azaC)-induced terminal differentiation in vitro and on papain-induced OA in the knee joints of rats. In the in vitro study, we measured the levels of messenger RNA for SOX9, aggrecan, type II collagen, type X collagen, alkaline phosphatase (AP), Indian hedgehog (IHH), Bcl-2, and Bax by real-time polymerase chain reaction, levels of glycosaminoglycan (GAG) by dimethylmethylene blue assay, and rate of apoptosis by TUNEL staining. In the in vivo study, we evaluated the histologic changes in GAG, type II collagen, type X collagen, and chondrocyte apoptosis in the articular cartilage of rat knees. AzaC induced terminal differentiation of human chondrocytes, including down-regulation of aggrecan, type II collagen, and GAG and up-regulation of type X collagen, alkaline phosphatase, and IHH. Apoptosis was reversed by 3-10 days of treatment with 10 nM PTH(1-34). SOX9 expression was not changed by either azaC or PTH(1-34) treatment. Bcl-2 and Bax were up-regulated on day 10 and day 14, respectively, after azaC induction of terminal differentiation, but PTH(1-34) treatment did not reverse this effect. Furthermore, PTH(1-34) treatment reversed papain-induced OA changes (decreasing GAG and type II collagen, and increasing type X collagen and chondrocyte apoptosis) in the knee joints of rats. Our findings indicate that PTH(1-34) inhibits the terminal differentiation of human articular chondrocytes in vitro and inhibits progression of OA in rats in vivo, and may be used to treat OA.

Esophageal cancer related gene 4 (ECRG4) is a marker of articular chondrocyte differentiation and cartilage destruction

With the aim of identifying novel genes regulating cartilage development and degeneration, we screened a cartilage-specific expressed sequence tag database. Esophageal cancer related gene 4 (ECRG4) was selected, based on the criteria of ‘chondrocyte-specific’ and ‘unknown function.’ ECRG4 expression was particularly abundant in chondrocytes and cartilage, compared to various other mouse tissues. ECRG4 is a secreted protein that undergoes cleavage after secretion. The protein is specifically expressed in chondrocytes in a manner dependent on differentiation status. The expression is very low in mesenchymal cells, and dramatically increased during chondrogenic differentiation. The ECRG4 level in differentiated chondrocytes is decreased during hypertrophic maturation, both in vitro and in vivo, and additionally in dedifferentiating chondrocytes induced by interleukin-1β or serial subculture, chondrocytes of human osteoarthritic cartilage and experimental mouse osteoarthritic cartilage. However, ectopic expression or exogenous ECRG4 treatment in a primary culture cell system does not affect chondrogenesis of mesenchymal cells, hypertrophic maturation of chondrocytes or dedifferentiation of differentiated chondrocytes. Additionally, cartilage development and organization of extracellular matrix are not affected in transgenic mice overexpressing ECRG4 in cartilage tissue. However, ectopic expression of ECRG4 reduced proliferation of primary culture chondrocytes. While the underlying mechanisms of ECRG4 expression and specific roles remain to be elucidated in more detail, our results support its function as a marker of differentiated articular chondrocytes and cartilage destruction.

Impact of 3D-culture on the expression of differentiation markers and hormone receptors in growth plate chondrocytes as compared to articular chondrocytes

In this study we compared the expression of hormone receptors and markers of differentiation in growth plate chondrocytes (GPC) and articular chondrocytes (AC) under different culture conditions by real-time PCR and immunofluorescence. After one week of monolayer culture, porcine GPC and AC of 6-8-week-old piglets were either seeded in alginate beads or further grown in monolayer culture up to 5 weeks. During monolayer culture the expression of Col2, Col1, Col10, aggrecan, ERalpha and ERbeta decreased dramatically, whereas culture in alginate beads restored an expression pattern similar to native tissue. The receptors IGF-1R, IGF-2R and GHR were, however, increased in monolayer culture compared to alginate culture or native tissue. The relative proportion of ERalpha and ERbeta expression was different in GPC when compared to AC. All of our results on mRNA expression during culture in alginate beads were also verified to be translated to the protein level. Thus, the expression of hormone rereptors and differentiation markers were found to be highly influenced by culture conditions. Moreover, the porcine model is promising as defined by tissue availability, expression of relevant hormonal receptors and comparability to human conditions, in particular in the area of basic growth research.

Regulation of articular chondrocyte proliferation and differentiation by indian hedgehog and parathyroid hormone–related protein in mice

Chondrocytes of the epiphyseal growth zone are regulated by the Indian hedgehog (IHH)-parathyroid hormone-related protein (PTHrP) axis. In weight-bearing joints, this growth zone comes to be subdivided by the secondary ossification center into distinct articular and growth cartilage structures. The purpose of this study was to explore the cells of origin, localization, regulation of expression, and putative functions of IHH and PTHrP in articular cartilage in the mouse. We assessed IHH and PTHrP expression in an allelic PTHrP-LacZ-knockin mouse and several versions of PTHrP-null mice. Selected joints were unloaded surgically to examine load-induction of PTHrP and IHH. The embryonic growth zone appears to serve as the source of PTHrP-expressing proliferative chondrocytes that populate both the forming articular cartilage and growth plate structures. In articular cartilage, these cells take the form of articular chondrocytes in the midzone. In PTHrP-knockout mice, mineralizing chondrocytes encroach upon developing articular cartilage but appear to be prevented from mineralizing the joint space by IHH-driven surface chondrocyte proliferation. In growing and adult mice, PTHrP expression in articular chondrocytes is load-induced, and unloading is associated with rapid changes in PTHrP expression and articular chondrocyte differentiation. We conclude that the IHH-PTHrP axis participates in the maintenance of articular cartilage. Dysregulation of this system might contribute to the pathogenesis of arthritis.

Comparison of meshes, gels and ceramic for cartilage tissue engineering in vitro

Full-thickness articular cartilage defects lack the capacity of healing because of lack of blood supply and lack of chondrocyte proliferation around the injury site. These factors contribute to the difficulty of getting good healing of cartilage defects. Autologous chondrocyte transplantation has been proposed as a method for treating cartilage defects using chondrocytes grown in vitro, which are then transplanted into the defects using a periosteal flap to retain the cells at the defect site. Studies that followed have attempted to refine this technique by using a cell matrix to support the chondrocytes. The reason for adding a resorbable cell matrix support that acts as a temporary scaffold until the chondrocytes are capable of producing extracellular matrix. Moreover, such a matrix may help in maintaining chondrocyte differentiation and phenotype. In this study, we have investigated the biocompatibility between human chondrocytes and biomaterials that could be used as matrix implants. It is a comparative study in vitro that involves assessing the proliferation and differentiation of human articular chondrocytes cultured on different resorbable biomaterials. Human chondrocytes were isolated from collagenase digest of articular cartilage provided by patients undergoing total knee replacements for osteoarthritis from the non-involved areas of the knee. The chondrocytes were then allowed to proliferate in vitro to increase the number of cells available for study. After adequate multiplication, the cells were seeded onto different biomaterials and allowed to from a cell biomaterial construct. The biomaterials used in this study were collagen I, calcium alginate, agarose, polyglycolic acid and Bioglass 45S5. The cell–biomaterial constructs were then collected at specific time points 3, 7, 14 and 21 days for histological and biochemical studies. The assessment includes studying proliferation, differentiation and extracellular matrix production. This was performed by immunostaining for collagen I and II production and histochemistry staining for glycosaminoglycans. Chondrocyte proliferation was more effective on 3D gels compared to ceramics and mesh. Cells on Bioglass expressed the same collagen type and at the same proportion as that expressed by freshly isolated cells. Moreover, Bioglass has induced cells to re-differentiate after they lost their differentiation in monolayer culture. Overall, however, there was no clear relationship between the cell morphology and type of collagen produced. Bioactive glass seems to behave as a suitable material for chondrocyte tissue engineering because it can maintain a chondrocyte phenotype.

I-27 TARGETING NF-KAPPAB SIGNALING IN OA: IS IT ONLY ABOUT INFLAMMATION?

I-27 TARGETING NF-KAPPAB SIGNALING IN OA: IS IT ONLY ABOUT INFLAMMATION?

Endochondral bone tissue engineering using embryonic stem cells

Embryonic stem cells can provide an unlimited supply of pluripotent cells for tissue engineering applications. Bone tissue engineering by directly differentiating ES cells (ESCs) into osteoblasts has been unsuccessful so far. Therefore, we investigated an alternative approach, based on the process of endochondral ossification. A cartilage matrix was formed in vitro by mouse ESCs seeded on a scaffold. When these cartilage tissue-engineered constructs (CTECs) were implanted s.c., the cartilage matured, became hypertrophic, calcified, and was ultimately replaced by bone tissue in the course of 21 days. Bone aligning hypertrophic cartilage was observed frequently. Using various chondrogenic differentiation periods in vitro, we demonstrated that a cartilage matrix is required for bone formation by ESCs. Chondrogenic differentiation of mesenchymal stem cells and articular chondrocytes showed that a cartilage matrix alone was not sufficient to drive endochondral bone formation. Moreover, when CTECs were implanted orthotopically into critical-size cranial defects in rats, efficient bone formation was observed. We report previously undescribed ESC-based bone tissue engineering under controlled reproducible conditions. Furthermore, our data indicate that ESCs can also be used as a model system to study endochondral bone formation.

Upregulation of Runx2 and Osterix during in vitro chondrogenesis of human adipose-derived stromal cells

The aim of this study was to create a gene expression profile to better define the phenotype of human adipose-derived stromal cells (HADSCs) during in vitro chondrogenesis, osteogenesis and adipogenesis. A novel aspect of this work was the analysis of the same subset of genes during HADSC differentiation into all three lineages. Chondrogenic induction resulted in increased mRNA expression of Sox transcription factors, COL2A1, COL10A1, Runx2, and Osterix. This is the first report demonstrating significant upregulation in expression of osteogenesis-related transcription factors Runx2 and Osterix by TGF-β3 induction of HADSCs during in vitro chondrogenesis. These findings suggest that the commonly-used chondrogenic induction reagents promote differentiation suggestive of hypertrophic chondrocytes and osteoblasts. We conclude that alternative strategies are required to induce efficient articular chondrocyte differentiation in order for HADSCs to be of clinical use in cartilage tissue engineering.

Chondrogenic Differentiation of Human Articular Chondrocytes Differs in Biodegradable PGA/PLA Scaffolds

Cartilage tissue engineering is applied clinically to cover and regenerate articular cartilage defects. Two bioresorbable nonwoven scaffolds, polyglycolic acid (PGA) and poly(lactic-co-glycolic acid) (PLGA) (90/10 copolymer of L-lactide and glycolide), were seeded with human chondrocytes after initial progeny in a monolayer with a serum-free medium. Two subgroups of nontreated and plasma-treated (using low-pressure plasma technique) scaffolds were investigated. The constructs were cultivated after seeding in six-well plates with serum-free medium for 7 days and implanted subcutaneously into nude mice for 6 and 12 weeks. Chondrogenic differentiations were investigated using immunhistology and reverse transcriptase-polymerase chain reaction. Cell adhesion only differed from 50% to 65% without a significant difference between the groups. During further cultivation for 7 days, the aggrecan synthesis of the seeded constructs was always higher in the PGA groups (p < 0.05). The mRNA gene expression for collagen type II was significantly higher in the PGA groups after 6 and 12 weeks (p < 0.05). A decrease in the expression of collagen type I was investigated in all groups. The expression for collagen type X and cartilage oligomeric matrix protein (COMP) increased in all groups over time. After cell proliferation in serum-free medium, the long-term chondrogenic differentiation in PGA scaffolds in vitro is cartilage specific and may be utilized in cartilage tissue engineering applications.

Altered Gene Expression in Articular Chondrocytes of Smad3ex8/ex8 Mice, Revealed by Gene Profiling Using Microarrays

It has been previously reported that small mother against decapentaplegic 3 ( Smad3) gene knockout ( Smad3 ex8/ex8) mice displays phenotypes similar to human osteoarthritis, as characterized by abnormal hypertrophic differentiation of articular chondrocytes. To further clarify the crucial target genes that mediate transformation growth factor-β (TGF-β)/Smad3 signals on articular chondrocytes differentiation and investigate the underlying molecular mechanism of osteoarthritis, microarrays were used to perform comparative transcriptional profiling in the articular cartilage between Smad3 ex8/ex8 and wild-type mice on day five after birth. The gene profiling results showed that the activity of bone morphogenetic protein (BMP) and TGF-β/cell division cycle 42 (Cdc42) signaling pathways were enhanced in Smad3 ex8/ex8 chondrocytes. Moreover, there was altered gene expression in growth hormone/insulin-like growth factor 1 (Igf1) axis and fibroblast growth factor (Fgf) signaling pathway. Notably, protein synthesis related genes and electron transport chain related genes were upregulated in Smad3 ex8/ex8 chondrocytes, implying that accelerated protein synthesis and enhanced cellular respiration might contribute to hypertrophic differentiation of articular chondrocytes and the pathogenesis of osteoarthritis.

BFGF influences human articular chondrocyte differentiation

The possible functional role of basic fibroblast growth factor (bFGF) in regulating the mitotic and metabolic activity of primary human articular chondrocytes was investigated. [EF1]Chondrocytes were enzymatically isolated from femoral head cartilage, and were cultured in vitro in monolayer. bFGF-dependent cell proliferation, production of collagen type II and aggrecan were monitored 10 days after isolation. Furthermore, effect of bFGF on cell cycle, cell morphology, and mRNA expression of integrins and chondrogenic markers determined by real time PCR were analyzed. bFGF concentrations in supernatants of primary human articular chondrocytes peaked immediately after isolation and then declined. In a dose-dependent manner, bFGF enhanced cell amplification and viability. BFGF induced a decrease in the apoptotic cell population, while the number of proliferating cells remained unchanged. Supplementation of cell culture with bFGF reduced collagen type II mRNA by 49%, but increased expression of the integrin alpha(2) by 70%. bFGF did not significantly regulate the integrins alpha(1), alpha(5), alpha(10), alpha(v) and type I collagen. bFGF reduced the amount of collagen type II by 53%, which was correlated with diminished mRNA production. Monolayer cultured chondrocytes secreted significant amounts of aggrecan that decreased over time. Secretion of this cartilage-specific marker was further reduced by the addition of bFGF. These findings highlight the potential role of bFGF as an endogenous chondrocyte mediator that can enhance cell amplification and regulate cell differentiation.

Markedly different effects of hyaluronic acid and chondroitin sulfate‐A on the differentiation of human articular chondrocytes in micromass and 3‐D honeycomb rotation cultures

A source of morphologically and functionally available human cartilagenous tissue for implantation is required in the field of tissue engineering. To achieve this goal, we evaluated the effects of hyaluronic acid (HA-810 and 1680 kDa), and chondroitin sulfate (CS-A 16 and C-34 kDa) on human articular chondrocytes (HC) in micromass and rotation culture conditions. Cell proliferation was increased by CS-A 16 kDa under micromass and rotation cultures, while cell differentiation was increased under rotation but not micromass conditions. Proliferation and differentiation due to CS-C 34 kDa were very similar to the control under both culture conditions. With HA, cell proliferation was increased depending on the molecular weight under micromass and rotation conditions. In contrast, chondrocyte differentiation was enhanced under rotation conditions, but decreased under micromass conditions depending on the molecular weight of HA. In both culture conditions, aggrecan gene was continuously expressed. However, the collagen type II gene was more weakly expressed in rotation than the micromass culture conditions. Thus, the chemical structures of polysaccharides, and the culture condition, rotation or micromass, caused differences in chondrogenesis.

Differentiation of human mesenchymal stem cells and articular chondrocytes: Analysis of chondrogenic potential and expression pattern of differentiation‐related transcription factors

Mesenchymal stem cells (MSCs) are a candidate for replacing chondrocytes in cell-based repair of cartilage lesions. However, it has not been clarified if these cells can acquire the hyaline phenotype, and whether chondrocytes and MSCs show the same expression patterns of critical control genes in development. In order to study this, articular chondrocytes and iliac crest derived MSCs were allowed to differentiate in pellet mass cultures. Gene expression of markers for the cartilage phenotype, helix-loop-helix (HLH) transcription factors, and chondrogenic transcription factors were analyzed by real-time PCR. Matrix production was assayed using biochemical analysis for hydroxyproline, glycosaminoglycans, and immunohistochemistry for collagen types I and II. Significantly decreased expression of collagen type I was accompanied by increased expression of collagen types IIA and IIB during differentiation of chondrocytes, indicating differentiation towards a hyaline phenotype. Chondrogenesis in MSCs on the other hand resulted in up-regulation of collagen types I, IIA, IIB, and X, demonstrating differentiation towards cartilage of a mixed phenotype. Expression of HES1 increased significantly during chondrogenesis in chondrocytes while expression in MSCs was maintained at a low level. The HLH gene HES5 on the other hand was only detected in chondrocytes. Expression of ID1 decreased significantly in chondrocytes while the opposite was seen in MSCs. These findings suggest that chondrocytes and MSCs differentiated and formed different subtypes of cartilage, the hyaline and a mixed cartilage phenotype, respectively. Differentially regulated HLH genes indicated the possibility for HLH proteins in regulating chondrogenic differentiation. This information is important to understand the potential use of MSCs in cartilage repair.

Effect of Stratified Culture Compared to Confluent Culture in Monolayer on Proliferation and Differentiation of Human Articular Chondrocytes

Effect of Stratified Culture Compared to Confluent Culture in Monolayer on Proliferation and Differentiation of Human Articular Chondrocytes

Effect of Stratified Culture Compared to Confluent Culture in Monolayer on Proliferation and Differentiation of Human Articular Chondrocytes

With conventional tissue culture of cells, it is generally assumed that when the available 2D substrate is fully occupied, growth ceases or is greatly reduced.However, in nature wound repair mostly involves proliferation of cells that are attracted to the defect site in a 3D environment.Hence, proliferation continues in 3D until the defect site is filled with cells contributing to repair tissue. With this in mind,we examined the growth behavior of human articular chondrocytes during stratified culture as opposed to routine culture to confluency. Additionally, we studied the influence of growth factors on proliferation during stratified culture and differentiation thereafter. Chondrocytes were cultured in monolayer on tissue culture plastic to confluency or stratified for an additional 7 days. Culture medium was based on DMEM with 10% serum and either supplemented with high concentrations of nonessential amino acids (NEAA) and ascorbic acid (AsAP), or instead with basic fibroblastic growth factor (bFGF), platelet-derived growth factor (PDBF-BB), and/or transforming growth factor beta1 (TGF-beta). After expansion, cells were harvested, counted, and their differentiation capacity was examined in pellet culture assay. It was shown that chondrocytes, cultured stratified proliferate exponentially for up to an additional 4 days and that cell yield increased 5-fold. Furthermore, during stratified culture the number of cells increased further in the presence of bFGF, PDBF-BB, and TGFbeta1 or high concentrations of NEAA and AsAP. Depending on donor variation and factors supplemented the cell yield ranged from 0.06 up to 1.1 million cells/cm2 at the second passage. During stratified culture in the presence of either bFGF and PDGF or high concentrations of NEAA and AsAP, exponential growth continued for up to 7 days. Finally, cells maintained their differentiation capacity when cultured stratified with or without growth factors (bFGF, TGF-beta, and PDGF), but not when cultured with high levels of AsAP and NEAA. In contrast to other 3D culture techniques like microcarrier or suspension culture, nutrient consumption remained the same as with conventional expansion. Because this allows culturing of clinically relevant amounts of chondrocytes without increasing the amount of serum, chondrocytes can be fully expanded in the presence autologous serum, avoiding the risk of viral and/or prion disease transmission associated with the use of animal-derived serum or serum replacers with animal-derived constituents.

Proliferative and re-defferentiative effects of photo-immobilized micro-patterned hyaluronan surfaces on chondrocyte cells

A photo-immobilisation procedure was utilised to create two different micro-patterned surfaces (tracks 25 and 5 μm wide) of hyaluronan (Hyal) on polyethylene-terephthalate (PET) previously plasma activated. Aim of the study was to investigate the proliferation and re-differentiation capacity of articular chondrocytes cultured on micro-patterned Hyal, compared to homogeneous Hyal and plain plasma-treated (pt-)PET substrates. Cytotoxicity, cell proliferation, activation and differentiation of articular knee cartilage chondrocytes (Mongrel sheep) were evaluated after 14 days of culture. It was found that micro-patterned Hyal surfaces induced the adhesion, migration and alignment of chondrocytes, as shown by light and scanning electron microscopy. Furthermore, the same surfaces induced chondrocyte differentiation, with a significant increase of aggrecan and collagen type II production, while homogeneous Hyal and pt-PET surfaces did not.

Fibroblast Growth Factor (FGF) 18 Signals through FGF Receptor 3 to Promote Chondrogenesis

Signaling by fibroblast growth factor (FGF) 18 and FGF receptor 3 (FGFR3) have been shown to regulate proliferation, differentiation, and matrix production of articular and growth plate chondrocytes in vivo and in vitro. Notably, the congenital absence of either FGF18 or FGFR3 resulted in similar expansion of the growth plates of fetal mice and the addition of FGF18 to human articular chondrocytes in culture enhanced proliferation and matrix production. Based on these and other experiments it has been proposed that FGF18 signals through FGFR3 to promote cartilage production by chondrocytes. Its role in chondrogenesis remains to be defined. In the current work we used the limb buds of FGFR3(+/+) and FGFR3(-/-) embryonic mice as a source of mesenchymal cells to determine how FGF18 signaling affects chondrogenesis. Confocal laser-scanning microscopy demonstrated impaired cartilage nodule formation in the FGFR3(-/-) cultures. Potential contributing factors to the phenotype were identified as impaired mitogenic response to FGF18, decreased production of type II collagen and proteoglycan in response to FGF18 stimulation, impaired interactions with the extracellular matrix resulting from altered integrin receptor expression, and altered expression of FGFR1 and FGFR2. The data identified FGF18 as a selective ligand for FGFR3 in limb bud mesenchymal cells, which suppressed proliferation and promoted their differentiation and production of cartilage matrix. This work, thus, identifies FGF18 and FGFR3 as potential molecular targets for intervention in tissue engineering aimed at cartilage repair and regeneration of damaged cartilage.

Collaborative action of M-CSF and CTGF/CCN2 in articular chondrocytes: Possible regenerative roles in articular cartilage metabolism

It is known that expression of the macrophage colony-stimulating factor (M-CSF) gene is induced in articular chondrocytes upon inflammation. However, the functional role of M-CSF in cartilage has been unclear. In this study, we describe possible roles of M-CSF in the protection and maintenance of the articular cartilage based on the results of experiments using human chondrocytic cells and rat primary chondrocytes. Connective tissue growth factor (CTGF/CCN2) is known to be a potent molecule to regenerate damaged cartilage by promoting the growth and differentiation of articular chondrocytes. Here, we uncovered the fact that M-CSF induced the mRNA expression of the ctgf/ccn2 gene in those cells. Enhanced production of CTGF/CCN2 protein by M-CSF was also confirmed. Furthermore, M-CSF could autoactivate the m-csf gene, forming a positive feed-back network to amplify and prolong the observed effects. Finally, promotion of proteoglycan synthesis was observed by the addition of M-CSF. These findings taken together indicate novel roles of M-CSF in articular cartilage metabolism in collaboration with CTGF/CCN2, particularly during an inflammatory response. Such roles of M-CSF were further supported by the distribution of M-CSF producing chondrocytes in experimentally induced rat osteoarthritis cartilage in vivo.

Calcium crystal deposition diseases: lessons from histochemistry.

Recent progress in molecular biology and biochemistry has enabled researchers to identify possible key players in physiologic and pathologic calcification. However, important lessons from immunohistochemical studies have contributed greatly to our current understanding of the pathogenesis of calcium crystal deposition disease. Histologic findings led to the hypothesis of the important role of hypertrophic differentiation of articular chondrocytes in calcium crystal deposition. In addition, histologic studies have confirmed the importance of individual proteins that may have direct or indirect roles in calcium crystal formation. Future studies will determine whether in vitro data showing key roles for certain factors in mineralization and calcification in cartilage are relevant to crystal deposition disease in humans.