Differentiation Of Articular Chondrocytes Research Articles

Vascular Endothelial Growth Factor in Cartilage Development and Osteoarthritis

Genome wide studies indicate that vascular endothelial growth factor A (VEGF) is associated with osteoarthritis (OA), and increased VEGF expression correlates with increased disease severity. VEGF is also a chondrocyte survival factor during development and essential for bone formation, skeletal growth and postnatal homeostasis. This raises questions of how the important embryonic and postnatal functions of VEGF can be reconciled with an apparently destructive role in OA. Addressing these questions, we find that VEGF acts as a survival factor in growth plate chondrocytes during development but only up until a few weeks after birth in mice. It is also required for postnatal differentiation of articular chondrocytes and the timely ossification of bones in joint regions. In surgically induced knee OA in mice, a model of post-traumatic OA in humans, increased expression of VEGF is associated with catabolic processes in chondrocytes and synovial cells. Conditional knock-down of Vegf attenuates induced OA. Intra-articular anti-VEGF antibodies suppress OA progression, reduce levels of phosphorylated VEGFR2 in articular chondrocytes and synovial cells and reduce levels of phosphorylated VEGFR1 in dorsal root ganglia. Finally, oral administration of the VEGFR2 kinase inhibitor Vandetanib attenuates OA progression.

Rosmarinic acid induces rabbit articular chondrocyte differentiation by decreases matrix metalloproteinase-13 and inflammation by upregulating cyclooxygenase-2 expression

BackgroundMatrix metalloproteinases (MMPs) are known to play an important role in the degradation of the extracellular matrix and the pathological progression of osteoarthritis (OA). The natural polyphenolic compound rosmarinic acid (Ros. A) has been shown to suppress the inhibitory activity of matrix metalloproteinases (MMPs). However, the effects of Ros. A on OA have not been investigated.MethodsIn the current study, primary articular chondrocytes were cultured from rabbit articular cartilage and treated with Ros. A. Phenotypic characterization was performed by western blotting to assess specific markers, prostaglandin E2 (PGE2) assays, and alcian blue staining to measure sulfated-proteoglycan production.ResultsWe report that in rabbit articular chondrocytes, Ros. A increased type II collagen, sulfated-proteoglycan, cyclooxygenase-2 (COX-2), and PGE2 production in a dose- and time-dependent manner. Furthermore, Ros. A suppressed the expression of MMP-13. In addition, treatment with Ros A activated extracellular signal-regulated kinase (ERK)-1/2 and p38 kinase signaling pathways. Inhibition of MMP-13 enhanced Ros. A-induced type II collagen expression and sulfated-proteoglycan synthesis but COX-2 and PGE2 production were unchanged. Ros. A-mediated up-regulation of ERK phosphorylation was abolished by the MEK inhibitor, PD98059, which prevented induction of the associated inflammatory response. Inhibition of p38 kinase with SB203580 enhanced the increase in type II collagen expression via Ros. A-mediated down-regulation of MMP-13.ConclusionsResults suggest that ERK-1/2 regulates Ros. A-induced inflammation and that p38 regulates differentiation by inhibiting MMP-13 in rabbit articular chondrocytes.

The in vitro biocompatibility of d-(+) raffinose modified chitosan: Two-dimensional and three-dimensional systems for culturing of horse articular chondrocytes

The present study investigated the biocompatibility of chitosan films and scaffolds modified with d-(+)raffinose and their capability to support the growth and maintenance of the differentiation of articular chondrocytes in vitro. Primary equine articular chondrocytes were cultured on films and scaffolds of modified d-(+) raffinose chitosan. Their behavior was compared to that of chondrocytes grown in conventional bi- and three-dimensional culture systems, such as micromasses and alginate beads. Chitosan films maintained the phenotype of differentiated chondrocytes (typical round morphology) and sustained the synthesis of cartilaginous extracellular matrix (ECM), even at 4weeks of culture. Indeed, starting from 2weeks of culture, chondrocytes seeded on chitosan scaffolds were able to penetrate the surface pores and to colonize the internal matrix. Moreover they produced ECM expressing the genes of typical chondrocytes differentiation markers such as collagen II and aggrecan.In conclusion, chitosan modified with d-raffinose represents an ideal support for chondrocyte adhesion, proliferation and for the maintenance of cellular phenotypic and genotypic differentiation. This novel biomaterial could potentially be a reliable support for the re-differentiation of dedifferentiated chondrocytes.

Conditional Deletion of the Phd2 Gene in Articular Chondrocytes Accelerates Differentiation and Reduces Articular Cartilage Thickness

Based on our findings that PHD2 is a negative regulator of chondrocyte differentiation and that hypoxia signaling is implicated in the pathogenesis of osteoarthritis, we investigated the consequence of disruption of the Phd2 gene in chondrocytes on the articular cartilage phenotype in mice. Immunohistochemistry detected high expression of PHD2 in the superficial zone (SZ), while PHD3 and HIF-1α (target of PHD2) are mainly expressed in the middle-deep zone (MDZ). Conditional deletion of the Phd2 gene (cKO) in chondrocytes accelerated the transition of progenitors to hypertrophic (differentiating) chondrocytes as revealed by reduced SZ thickness, and increased MDZ thickness, as well as increased chondrocyte hypertrophy. Immunohistochemistry further revealed decreased levels of progenitor markers but increased levels of hypertrophy markers in the articular cartilage of the cKO mice. Treatment of primary articular chondrocytes, in vitro, with IOX2, a specific inhibitor of PHD2, promoted articular chondrocyte differentiation. Knockdown of Hif-1α expression in primary articular chondrocytes using lentiviral vectors containing Hif-1α shRNA resulted in reduced expression levels of Vegf, Glut1, Pgk1, and Col10 compared to control shRNA. We conclude that Phd2 is a key regulator of articular cartilage development that acts by inhibiting the differentiation of articular cartilage progenitors via modulating HIF-1α signaling.

MTORC1 activation downregulates FGFR3 and PTH/PTHrP receptor in articular chondrocytes to initiate osteoarthritis.

Articular chondrocyte activation, involving aberrant proliferation and prehypertrophic differentiation, is essential for osteoarthritis (OA) initiation and progression. Disruption of mechanistic target of rapamycin complex 1 (mTORC1) promotes chondrocyte autophagy and survival, and decreases the severity of experimental OA. However, the role of cartilage mTORC1 activation in OA initiation is unknown. In this study, we elucidated the specific role of mTORC1 activation in OA initiation, and identify the underlying mechanisms. Expression of mTORC1 in articular cartilage of OA patients and OA mice was assessed by immunostaining. Cartilage-specific tuberous sclerosis complex 1 (Tsc1, mTORC1 upstream inhibitor) knockout (TSC1CKO) and inducible Tsc1 KO (TSC1CKOER) mice were generated. The functional effects of mTORC1 in OA initiation and development on its downstream targets were examined by immunostaining, western blotting and qPCR. Articular chondrocyte mTORC1 was activated in early-stage OA and in aged mice. TSC1CKO mice exhibited spontaneous OA, and TSC1CKOER mice (from 2 months) exhibited accelerated age-related and DMM-induced OA phenotypes, with aberrant chondrocyte proliferation and hypertrophic differentiation. This was associated with hyperactivation of mTORC1 and dramatic downregulation of FGFR3 and PPR, two receptors critical for preventing chondrocyte proliferation and differentiation. Rapamycin treatment reversed these phenotypes in KO mice. Furthermore, invitro rescue experiments demonstrated that p73 and ERK1/2 may mediate the negative regulation of FGFR3 and PPR by mTORC1. mTORC1 activation stimulates articular chondrocyte proliferation and differentiation to initiate OA, in part by downregulating FGFR3 and PPR.

Endogenous DKK1 and FRZB Regulate Chondrogenesis and Hypertrophy in Three-Dimensional Cultures of Human Chondrocytes and Human Mesenchymal Stem Cells.

Hypertrophic differentiation occurs during in vitro chondrogenesis of mesenchymal stem cells (MSCs), decreasing the quality of the cartilage construct. Previously we identified WNT pathway antagonists Dickkopf 1 homolog (DKK1) and frizzled-related protein (FRZB) as key factors in blocking hypertrophic differentiation of human MSCs (hMSCs). In this study, we investigated the role of endogenously expressed DKK1 and FRZB in chondrogenesis of hMSC and chondrocyte redifferentiation and in preventing cell hypertrophy using three relevant human cell based systems, isolated hMSCs, isolated primary human chondrocytes (hChs), and cocultures of hMSCs with hChs for which we specifically designed neutralizing nano-antibodies. We selected and tested variable domain of single chain heavy chain only antibodies (VHH) for their ability to neutralize the function of DKK1 or FRZB. In the presence of DKK1 and FRZB neutralizing VHH, glycosaminoglycan and collagen type II staining were significantly reduced in monocultured hMSCs and monocultured chondrocytes. Furthermore, in cocultures, cells in pellets showed hypertrophic differentiation. In conclusion, endogenous expression of the WNT antagonists DKK1 and FRZB is necessary for multiple steps during chondrogenesis: first DKK1 and FRZB are indispensable for the initial steps of chondrogenic differentiation of hMSCs, second they are necessary for chondrocyte redifferentiation, and finally in preventing hypertrophic differentiation of articular chondrocytes.

Cytokine-induced apoptosis inhibitor-1 causes dedifferentiation of rabbit articular chondrocytes via the ERK-1/2 and p38 kinase pathways

Cytokine-induced apoptosis inhibitor-1 (CIAPIN-1, formally named anamorsin) is a well-known regulator of apoptosis in many different cell types. Recently, it has been reported that some anti-apoptotic proteins are involved with the regulation of cell differentiation. However, relatively little is known about the role of CIAPIN-1 on rabbit articular chondrocytes differentiation. In this study, we investigated the effects of CIAPIN-1 in chondrocytes, focusing on extracellular signal-regulated kinase (ERK)-1/2 and p38 kinase signaling. CIAPIN-1 caused dedifferentiation, as determined by the inhibition of type II collagen expression and sulfated-proteoglycan synthesis. CIAPIN-1 activated ERK-1/2 and inactivated p38 kinase, as determined by the phosphorylation level of each protein. CIAPIN-1-induced ERK phosphorylation was abolished by the MEK inhibitor, PD98059, which also prevented the CIAPIN-1-induced loss of type II collagen expression. Inhibition of p38 kinase with SB203580 enhanced the decrease in type II collagen expression. Our findings collectively suggest that ERK-1/2 and p38 kinase regulate CIAPIN-1-induced dedifferentiation in rabbit articular chondrocytes.

Parathyroid hormone 1-34 reduces dexamethasone-induced terminal differentiation in human articular chondrocytes.

Intra-articular injection of dexamethasone (Dex) is occasionally used to relieve pain and inflammation in osteoarthritis (OA) patients. Dex induces terminal differentiation of chondrogenic mesenchymal stem cells in vitro and causes impaired longitudinal skeletal growth in vivo. Parathyroid hormone 1-34 (PTH 1-34) has been shown to reverse terminal differentiation of osteoarthritic articular chondrocytes. We hypothesized that Dex induces terminal differentiation of articular chondrocytes and that this effect can be mitigated by PTH 1-34 treatment. We tested the effect of Dex on terminal differentiation in human articular chondrocytes and further tested if PTH 1-34 reverses the effects. We found that Dex treatment downregulated chondrogenic-induced expressions of SOX-9, collagen type IIa1 (Col2a1), and aggrecan and reduced synthesis of cartilaginous matrix (Col2a1 and sulfated glycosaminoglycan) synthesis. Dex treatment upregulated chondrocyte hypertrophic markers of collagen type X and alkaline phosphatase at mRNA and protein levels, and it increased the cell size of articular chondrocytes and induced cell death. These results indicated that Dex induces terminal differentiation of articular chondrocytes. To test whether PTH 1-34 treatment reverses Dex-induced terminal differentiation of articular chondrocytes, PTH 1-34 was co-administered with Dex. Results showed that PTH 1-34 treatment reversed both changes of chondrogenic and hypertrophic markers in chondrocytes induced by Dex. PTH 1-34 also decreased Dex-induced cell death. PTH 1-34 treatment reduces Dex-induced terminal differentiation and apoptosis of articular chondrocytes, and PTH 1-34 treatment may protect articular cartilage from further damage when received Dex administration.

Simvastatin induces differentiation of rabbit articular chondrocytes via the ERK-1/2 and p38 kinase pathways

Statins are competitive inhibitors of hydroxy-methyl-glutaryl Coenzyme A (HMG-CoA) reductase, a key enzyme involved in the conversion of HMG-CoA to the cholesterol precursor mevalonate. Some statins, such as simvastatin (simvastatin), have been shown to have anti-cancer and anti-inflammatory effects, reducing cartilage degradation in osteoarthritic rabbits in vivo. However, the regulatory mechanisms undergirding simvastatin mediated chondrocyte differentiation have not been well elucidated. Thus, we investigated the action and mechanism of simvastatin on differentiation of rabbit articular chondrocytes through western blot analyses, RT-PCR, and immunohistochemical (IHC) and immunofluorescence (IF) staining. Simvastatin treatment was found to induce type II collagen expression and sulfated-proteoglycan synthesis in a dose- and time-dependent manner. Indeed, RT-PCR revealed increased expression of type II collagen on treatment with simvastatin. Both IHC and IF staining indicated differentiation of chondrocytes. Simvastatin treatment reduced activation of ERK-1/2 and stimulated activation of p38 kinase. Inhibition of ERK-1/2 with PD98059 enhanced simvastatin induced differentiation, whereas inhibition of p38 kinase with SB203580 inhibited simvastatin induced differentiation. Simvastatin treatment also inhibits loss of type II collagen in serial monolayer culture. Collectively, our results indicate that ERK-1/2 and p38 kinase regulate simvastatin-induced differentiation of chondrocytes in opposing manners. Thus, these findings suggest that simvastatin may be a potential therapeutic drug for osteoarthritis.

DKK1 AND FRZB are necessary for chondrocyte (RE)differentiation and prevention of cell hypertrophy in 3D cultures of human chondrocytes and human mesenchymal stem cells

Purpose: Autologous chondrocytes and mesenchymal stem cells (MSCs) in either monoculture or co-culture, for the enhancement of chondrogenesis, show great promise for treatment of cartilage disorders. However, maintaining the chondrogenic phenotype and avoiding hypertrophy of chondrogenic differentiating MSCs remain a big challenge in these cell-based strategies. Previously, we identified Dickkopf 1 homolog (DKK1) and frizzled-related protein (FRZB) as key factors in controlling the articular chondrocyte phenotype. DKK1 and FRZB are naturally occurring antagonists of the WNT signaling pathway. The expression of DKK1 and FRZB is low in cultured human mesenchymal stem cells (hMSCs) and high in cultured human articular chondrocytes (hChs). We have previously shown that addition of either DKK1 or FRZB blocks hypertrophy in chondrogenically differentiating hMSCs and that expression of DKK1 and FRZB is lost in OA. We hypothesized that DKK1 and FRZB are necessary for chondrogenesis and for preventing cell hypertrophy in chondrocyte terminal differentiation. Methods: To block DKK1 and FRZB the variable domain of single chain heavy chain only antibodies (VHH) was used to neutralize DKK1 and FRZB. We tested our hypothesis using 3D pellet cultures of three relevant human cell based systems: isolated hChs, isolated hMSCs and co-culture of hChs with hMSCs. The effects were analyzed using real-time quantitative polymerase chain reaction (qPCR), histology, and immunohistochemistry (IHC). Results: In the presence of DKK1 and FRZB neutralizing VHH, glycosaminoglycan (GAG) deposition and Collagen type II staining were significantly reduced in redifferentiating chondrocytes (Figure 1 A and B) and in chondrogenic differentiating hMSCs, indicating loss of chondrogenic potential. Upon neutralization of DKK1 and FRZB in co-cultures, the cells in pellets showed hypertrophic differentiation as indicated by matrix mineralization, apoptosis and significantly increased expression of the hypertrophic markers, COL10A1, MMP13 and RUNX2. This is indicative of terminal differentiation. Conclusions: DKK1 and FRZB are necessary for multiple steps during chondrogenesis: first DKK1 and FRZB are necessary for the initial steps of chondrogenic differentiation of hMSCs and in redifferentiation of cultured chondrocytes, and secondly in preventing chondrocyte hypertrophy in terminal differentiation of articular chondrocytes (Figure 2).Figure 2. DKK1 and FRZB are necessary for multiple steps during chondrogenesis to ensure chondrogenesis and to prevent cell hypertrophy in terminal differentiation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Carbon nanotubes: a promising tissue engineering approach for in vitro cultivation & differentiation of primary canine articular chondrocytes

Development of biocompatible materials has great potential in biomedical engineering both for in vitro studies as well as for in vivo applications. Two- and three-dimensional carbon nanotube (CNT) substrates imitating and providing an extracellular matrix-like structure are promising constructs as cell-supporting scaffolds. Lately, they have received considerable interest in tissue engineering; however, cellular responses to nanoscale stimuli need to be better understood. Here, we present the preliminary results on the effect of CNT-based scaffolds on the proliferation and arrangement of primary canine chondrocytes (PCCs). We aim to develop scaffolding materials for the in vitro cultivation of normal and neoplastic cells with the ultimate objective of using them for applications such as tissue implants in cartilage repair and tissue regeneration after surgical intervention. In the proposed studies we aim to use an aerogel network of CNTs that has been drawn from a vertically aligned array as a synthetic substrate for the growth and alignment of primary canine chondrocytes. This aerogel consists of CNTs that are aligned parallel to the major axis of the CNTs; they have exceptionally low densities, are electrically and thermally conductive whilst maintaining very high tensile strength and elasticity. We are studying the cell growth, adhesion, morphology, viability and metabolism of cells seeded onto CNT substrates. Preliminary results to date have revealed that PCCs are capable of proliferating on CNT-based scaffolds, although the cell viability seems to be slightly decreased in comparison to the conventional 2D cell culture. Moreover, our nanosubstrates are able to induce directional cell growth of PCCs via aligning cells along CNTs. The latter is essential for in vivo application of nanosubstrates in tissue regeneration.

Dynamic Regulation ofTWIST1Expression During Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

Human bone marrow-derived mesenchymal stem cells (BMSCs) are clinically promising to repair damaged articular cartilage. This study investigated TWIST1, an important transcriptional regulator in mesenchymal lineages, in BMSC chondrogenesis. We hypothesized that downregulation of TWIST1 expression is required for in vitro chondrogenic differentiation. Indeed, significant downregulation of TWIST1 was observed in murine skeletal progenitor cells during limb development (N = 3 embryos), and during chondrogenic differentiation of culture-expanded human articular chondrocytes (N = 3 donors) and isolated adult human BMSCs (N = 7 donors), consistent with an inhibitory effect of TWIST1 expression on chondrogenic differentiation. Silencing of TWIST1 expression in BMSCs by siRNA, however, did not improve chondrogenic differentiation potential. Interestingly, additional investigation revealed that downregulation of TWIST1 in chondrogenic BMSCs is preceded by an initial upregulation. Similar upregulation is observed in non-chondrogenic BMSCs (N = 5 donors); however, non-chondrogenic cells fail to downregulate TWIST1 expression thereafter, preventing their chondrogenic differentiation. This study describes for the first time endogenous TWIST1 expression during in vitro chondrogenic differentiation of human BMSCs, demonstrating dynamic regulation of TWIST1 expression whereby upregulation and then downregulation of TWIST1 expression are required for chondrogenic differentiation of BMSCs. Elucidation of the molecular regulation of, and by, TWIST1 will provide targets for optimization of BMSC chondrogenic differentiation culture.

PEP-1-SIRT2 causes dedifferentiation and COX-2 expression via the MAPK pathways in rabbit articular chondrocytes

SIRT2 is a member of the mammalian sirtuin protein family, primarily found in the cytoplasm. It regulates numerous cellular processes including aging, DNA repair, cell cycle, and survival under stress conditions. However, the biological function and mechanism of the SIRT2 protein was not well understood in normal cells such as primary chondrocytes. In this study, we examined the effects of SIRT2 on differentiation and inflammation in rabbit articular chondrocytes by using a cell-permeative PEP-1-SIRT2 protein. Our results indicate that PEP-1-SIRT2-induced a loss of type II collagen and decreased sulfate proteoglycan levels in a dose- and time-dependent manner, as examined by Western blotting, alcian blue staining, and immunohistochemistry. Furthermore, PEP-1-SIRT2 caused an inflammatory response by inducing the expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2). In addition, after treatment with PEP-1-SIRT2, phosphorylation of both p38 and ERK was observed. Inhibition of ERK with PD98059 (PD) suppressed PEP-1-SIRT2-induced dedifferentiation and COX-2 expression. Reduction in PEP-1-SIRT2-induced inflammatory response was observed upon inhibition of p38 by SB203580 (SB). The same pattern was demonstrated in PEP-1-SIRT2-induced dedifferentiation and inflammatory response during culture with serial passages. During expansion to four passages, levels of type II collagen decreased, whereas levels of COX-2 and SIRT2 increased and activated ERK and p38. Furthermore, PEP-1-SIRT2 enhances dedifferentiation through the ERK pathway and inflammatory response through the ERK and p38 pathways in rabbit chondrocytes in vitro. These findings suggest that PEP-1-SIRT2 induces dedifferentiation via the ERK pathway and inflammation through the p38 and ERK pathways in rabbit articular chondrocytes.

Synovial fluid concentration of gremlin1 but not frizzled-related protein is increased in patients after acute knee injury

Purpose: Degenerative joint diseases are characterized by a disturbance in joint homeostasis, which is poorly understood at the molecular level. Previously, we have identified the secreted WNT antagonists dickkopf 1 homolog (Xenopus laevis) (DKK1) and frizzled-related protein (FRZB) and the BMP antagonists Gremlin1 (GREM1) as regulators of cartilage homeostasis by preventing hypertrophic differentiation of articular chondrocytes, consistent with their role in articular cartilage homeostasis. In this study we tested the hypothesis that health status of the joint is reflected by the expression levels of FRZB and GREM1 in synovial fluid. To test this hypothesis, we performed a cross sectional analysis of synovial fluid samples collected from patients with acute and chronic knee injury and compared these with synovial fluid samples from knee healthy controls. Methods: Synovial fluid was aspirated (Lund University, Sweden) from patients with acute (n =106, mean age 30 years [range 17-57], 0-77 days from injury) and chronic (n = 50, mean age 38 years [18-70] 1-37 years after injury) knee injuries, and from knee healthy subjects (n = 8, mean age 28 years [17-85]). Synovial fluid concentrations of FRZB and GREM1 were determined using ELISA (FRZB, RD GREM1, Bio-connect diagnostics). Concentration of cartilage biomarkers in synovial fluid; ARGS-aggrecan (in-house), cartilage oligomeric matrix protein (COMP; BioVendor) and collagen epitope C2C (IBEX), and proinflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α; Meso Scale Discovery) were analyzed by immunoassays. Mann-Whitney tests for group comparisons and Spearman’s rank test (rS) for correlation analysis was used. Results: Synovial fluid concentration of FRZB in the acute injury group was no different from the level in the reference group (p = 0.99), but was 2-fold (based on median concentrations) higher than the level of the chronic injury group (p = 0.023) (Fig. 1a). Synovial fluid concentrations of GREM1 in the acute and chronic injury groups were both higher than the level of the reference group, 2-fold (p = 0.003) and 1.8-fold (p = 0.031), respectively (Fig. 1b); also, in these patients the GREM1 concentration was negatively correlated to time after injury (rS = -0.224, p = 0.005). The FRZB and GREM1 concentrations of the acute and chronic injury groups were not influenced by gender (p = 0.550, p = 0.825) or by age (rS =-0.146, p = 0.068; rS = -0.137, p = 0.089), although, the FRZB concentration was significantly decreased in knee healthy subjects with age (rS= -0.833, p = 0.005). In the acute and chronic injury groups, there was no correlation between FRZB and GREM1 concentrations in synovial fluid (rS = 0.04, p = 0.585). Also, for a portion of the acute injury group (n = 52) synovial fluid FRZB concentration were correlated with COMP levels (rS = 0.284, p = 0.041) but not with other cartilage markers (C2C p = 0.55, ARGS-aggrecan p = 0.311) nor with any cytokine levels (p> 0.05 for all cytokines); the synovial fluid GREM1 concentration did not correlated with any cartilage marker nor with cytokine levels (p > 0.05 for all markers). Conclusions: Compared to chronic injured knees, acute injured knees have higher synovial fluid concentrations of FRZB and GREM1. Their levels were not influenced by gender or age, indicating that the differences of FRZB and GREM1 concentrations between acute and chronic injured knee joints depend on differences in disease conditions. Persistent synovial fluid levels of FRZB in acute injured knee joints might be a way for cells trying to protect the cartilage from degradation by inhibiting the Wnt signaling pathway. Similarly, high synovial fluid levels of GREM1 in injured knee joints (acute and chronic) suggests of an effort to maintain cartilage homeostasis by inhibiting BMP signaling.

Human platelet releasates combined with polyglycolic acid scaffold promote chondrocyte differentiation and phenotypic maintenance.

In the present study, we aimed to demonstrate the differentiating properties of platelet-rich plasma releasates (PRPr) on human chondrocytes seeded on a polygtlycolic acid (PGA) 3D scaffold. Gene expression and biochemical analysis were carried out to assess the improved quality of our PGA-based cartilage constructs supplemented with PRPr. We observed that the use of PRPr as cell cultures supplementation to PGA-chondrocyte constructs may promote chondrocyte differentiation, and thus may contribute to maintaining the chondrogenic phenotype longer than conventional supplementation by increasing high levels of important chondrogenic markers (e.g. sox9, aggrecan and type II collagen), without induction of type I collagen. Moreover, our constructs were analysed for the secretion and deposition of important ECM molecules (sGAG, type II collagen, etc.). Our results indicate that PRPr supplementation may synergize with PGA-based scaffolds to stimulate human articular chondrocyte differentiation, maturation and phenotypic maintenance.

A simple two dimensional culture method to study the hypertrophic differentiation of rat articular chondrocytes

Chondrocytes hypertrophy is a physiological process observed in endochondral ossification during development until adolescence in human. It can also be observed during pathophysiological conditions such as osteoarthritis. Hypertrophic chondrocytes synthesise collagen X and express matrix metalloproteinase 13 and alkaline phosphatase. The cellular models available to study this process are either not convenient, they might lead to a rapid dedifferentiation of chondrocytes, or they are far from the physiological conditions. The objective of this study was to design an user-friendly 2D-primary cell culture of young articular chondrocytes of rat able to follow the terminal differentiation process. After confluence, chondrocytes were cultured according to 4 differentiation protocols. Protocol 1 contained DMEM/F12 supplemented with 10% foetal bovine serum (FBS) and 2 μg/ml insulin. Protocol 2 contained alpha-MEM supplemented with 5% FBS and 2 μg/ml insulin. Protocol 3 contained 2% FBS and 2 μg/ml insulin. Protocol 4 contained DMEM/F12 supplemented with 2% FBS in absence or in presence of 2 μg/ml insulin and 37.5 μg/ml ascorbate. The cell morphology was observed by phase-contrast microscopy and the expression of markers specific of mature and hypertrophic chondrocytes were assessed by RT-qPCR. The effect of a decrease in nutrient quality of the culture medium after confluence was tested using protocols 1, 2 and 3. Protocol 1 did not allow the maintenance of chondrocyte phenotype more than one week, because cells became fibroblastic. A decrease in Sox9 mRNA expression, in collagen II/collagen I and in aggrecan/versican mRNA ratios was also found with protocol 1. Protocol 3 was the best when compared with protocols 1 and 2. It allowed chondrocytes to adopt a hypertrophic morphology. Cells also expressed the collagen X specific hypertrophic marker, and presented an increase in collagen II/I and aggrecan/versican ratios after 15 days of culture post-confluence. The effect of the insulin/ascorbate supplementation was studied using protocol 4. The insulin/ascorbate supplementation allowed an earlier chondrocytes conversion to terminal differentiation with a prolonged effect till 3 weeks post-confluence, compared to control without insulin/ascorbate. Finally, the profile of chondrocyte differentiation was checked during 5 successive sub-cultures. Only the first passage could be used to study hypertrophy. A convenient protocol to study chondrocyte hypertrophy is proposed. Protocol 4 offers the possibility to study this differentiation phenotype which is crucial for the development of articular diseases such as osteoarthritis. Our model could also be used in tissue engineering for cartilage repair strategies in which hypertrophic differentiation of chondrocyte should be avoided.

Subchondral bone influences chondrogenic differentiation and collagen production of human bone marrow-derived mesenchymal stem cells and articular chondrocytes.

IntroductionOsteoarthritis (OA) is characterized by an imbalance in cartilage and underlying subchondral bone homeostasis. We hypothesized that signals from the subchondral bone may modulate production of matrix components, alter chondrogenic differentiation potential of cocultured bone marrow-derived mesenchymal stem cells (BMSC) and induce a phenotypic shift in differentiated OA chondrocytes.MethodsWe established a novel coculture model between BMSC, mixed cultures (BMSC and chondrocytes) and chondrocytes embedded in fibrin gel with OA and normal subchondral bone explants (OAB and NB). Tissues and cells were either derived from OA or trauma patients. In addition, we used adipose-derived stem cells (ASC) from liposuction. With gene expression analysis, biochemical assays, immunofluorescence and biomechanical tests we characterized the properties of newly generated extracellular matrix (ECM) from chondrocytes and chondrogenically differentiating BMSC cocultured with OAB or NB in comparison with monocultures (cultures without bone explants).ResultsOverall, gene expression of collagens of OAB and NB cocultured cells was reduced compared to monocultures. Concomitantly, we observed significantly lower collagen I, II and III and glycosaminoglycan (GAG) production in OAB cocultured cell lysates. In parallel, we detected increased concentrations of soluble GAGs and basic fibroblast growth factor (bFGF), interleukin (IL)-6 and IL-8 in supernatants of OAB and NB cocultures mainly at early time points. IL-1ß concentration was increased in supernatants of OAB cocultures, but not in NB cocultures. Cell-free NB or OAB explants released different amounts of IL-1ß, bFGF and soluble GAG into cell culture supernatants. In comparison to cocultures, monocultures exhibited higher Young’s modulus and equilibrium modulus. Stimulation of monocultures with IL-1ß led to a downregulation of aggrecan (ACAN) gene expression and in general to induced matrix metalloprotease (MMP)2, MMP3 and MMP-13 gene expression while IL-6 and IL-8 stimulation partly reduced ACAN, MMP3 and MMP-13 gene expression.ConclusionsOur results suggest an alteration of molecular composition and mechanical properties of the newly formed ECM in subchondral bone cocultures. We suggest that soluble factors, that is interleukins and bFGF, released in cocultures exert inhibitory effects on collagen and temporary effects on proteoglycan production, which finally results in a reduction of mechanical strength of newly formed fibrillar networks.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-014-0453-9) contains supplementary material, which is available to authorized users.

Expression and immunohistochemical localization of the Gβγ‐activated and calcineurin‐inhibited adenylyl cyclase isoforms in rat articular chondrocytes (922.9)

Aims: Cross‐talk between signal transduction pathways is essential to maintain normal homeostasis. cAMP mediated pathway is archetypical in mammalian cells and adenylyl cyclase (AC) is the effector enzyme that converts ATP into cAMP. We believe that AC/cAMP mediated pathway is involved in homeostasis of the articular cartilage. In this study we report the expression of Gβγ‐activated AC isoforms 2, 4, and 7 and calcineurin‐inhibited AC isoform 9 in rat articular chondrocytes.Main methods: Fresh slices of articular cartilage were taken from various synovial joints of rat and the expression of AC isoforms was determined by RT‐PCR. Immunohistochemistry was performed to localize these isoforms in articular chondrocytes.Key findings: All AC isoforms studied were found differentially expressed in different zones of the rat articular cartilage. Generally, expression of all AC isoforms increased with age.Significance: These data add to the information about signaling cascades possibly involved in articular chondrocytes. Presence of AC isoforms 2, 4, 7, and 9 suggest a role for the cAMP mediated 2nd messenger signaling cascades in articular chondrocytes. Further studies, such as measuring the AC and cAMP levels during development and differentiation of articular chondrocytes would confirm our results and help in understanding the signaling mechanisms in articular chondrocytes.Grant Funding Source: Supported by Seed money grant from the FHS‐Research Committee of the Aga Khan University, Karachi

GSK3β inhibition induces terminal differentiation and extracellular matrix remodelling in human osteoarthritic articular chondrocytes

GSK3β inhibition induces terminal differentiation and extracellular matrix remodelling in human osteoarthritic articular chondrocytes

A novel autologous plasma based viscosupplementation

Purpose: Combined long acting viscosupplement and autologous cartilage promoting factors for the treatment of osteoarthritis. Methods: RegenoGel-SP is a novel elastomer made of fibrinogen enriched plasma chemically linked to high molecular weight Hyaluronic acid (HA). Plasma fibrinogen-HA conjugate solution polymerized by CaCl2 was seeded with primary human articular chondrocytes. Cell viability and potency in the 3D constructs were evaluated using Alamar blue, DMMB for sulphated glycosaminoglycans (sGAGs) and quantitative rtPCR for chondro-specific markers. Results: In the last OARSI meeting we presented a novel Fibrin-HA conjugate as a long acting viscosupplement and as a carrier for the disease modifying agent FGF18 or its FGF receptor selective variants. To further enhance the biological properties of the hydrogel, a similar chemical approach was implemented using whole human plasma conjugated to high molecular weight Hyaluronic acid. This self plasma based hydrogel designated Regenogel-SP demonstrated remarkable physical endurance and biocompatibility to diverse primary cell types. Specifically, Regenogel-SP demonstrated superior capacity to support cell proliferation, viability and differentiation of human articular chondrocytes from both normal and OA cartilage. Conclusions: We have previously reported on the regenerative and cell supportive activity of Fibrin-HA compositions combined with FGF18 variants. The newly developed Regenogel-SP provides a novel and safe approach for delivering autologous plasma rich constituents in a stable, long acting, viscous support for cartilage preservation and repair in OA.