Osteoarthritic cartilage chondrocytes alter subchondral bone osteoblast differentiation via MAPK signalling pathway involving ERK1/2

Osteoarthritic subchondral bone is characterized by abnormal bone density and enhanced production of bone turnover markers, an indication of osteoblast dysfunction. Several studies have proposed that pathological changes in articular cartilage influence the subchondral bone changes, which are typical of the progression of osteoarthritis; however, direct evidence of this has yet to be reported. The aim of the present study was to investigate what effects articular cartilage cells, isolated from normal and osteoarthritic joints, may have on the subchondral bone osteoblast phenotype, and also the potential involvement of the mitogen activated protein kinase (MAPK) signalling pathway during this process. Our results suggest that chondrocytes isolated from a normal joint inhibited osteoblast differentiation, whereas chondrocytes isolated from an osteoarthritic joint enhanced osteoblast differentiation, both via a direct and indirect cell interaction mechanisms. Furthermore, the interaction of subchondral bone osteoblasts with osteoarthritic chondrocyte conditioned media appeared to significantly activate ERK1/2 phosphorylation. On the other hand, conditioned media from normal articular chondrocytes did not affect ERK1/2 phosphorylation. Inhibition of the MAPK–ERK1/2 pathways reversed the phenotype changes of subchondral bone osteoblast, which would otherwise be induced by the conditioned media from osteoarthritic chondrocytes. In conclusion, our findings provide evidence that osteoarthritic chondrocytes affect subchondral bone osteoblast metabolism via an ERK1/2 dependent pathway.

Expression of chondromodulin-1 in the temporomandibular joint condylar cartilage and disc

BACKGROUND: The temporomandibular joint (TMJ) cartilage consists of condylar cartilage and disc and undergoes continuous remodeling throughout post-natal life. To maintain the integrity of the TMJ cartilage, anti-angiogenic factors play an important role during the remodeling process. In this study, we investigated the expression of the anti-angiogenic factor, chondromodulin- 1 (ChM-1), in TMJ cartilage and evaluate its potential role in TMJ remodeling. METHODS: Eight TMJ specimens were collected from six 4-month-old Japanese white rabbits. Safranin-O staining was performed to determine proteoglycan content. ChM-1 expression in TMJ condylar cartilage and disc was determined by immunohistochemistry. Three human perforated disc tissue samples were collected for investigation of ChM-1 and vascular endothelial growth factor (VEGF) distribution in perforated TMJ disc. RESULTS: Safranin-O stained weakly in TMJ compared with tibial articular and epiphyseal cartilage. In TMJ, ChM-1 was expressed in the proliferative and hypertrophic zone of condylar cartilage and chondrocyte-like cells in the disc. No expression of ChM-1 was observed in osteoblasts and subchondral bone. ChM-1 and VEGF were both similarly expressed in perforated disc tissues. CONCLUSIONS: ChM-1 may play a role in the regulation of TMJ remodeling by preventing blood vessel invasion of the cartilage, thereby maintaining condylar cartilage and disc integrity.

ERK-1/2 and p38 in the Regulation of Hypertrophic Changes of Normal Articular Cartilage Chondrocytes Induced by Osteoarthritic Subchondral Osteoblasts

Objective. Previous studies have shown the influence of subchondral bone osteoblasts (SBOs) on phenotypical changes of articular cartilage chondrocytes (ACCs) during the development of osteoarthritis (OA). The molecular mechanisms involved during this process remain elusive, in particular, the signal transduction pathways. The aim of this study was to investigate the in vitro effects of OA SBOs on the phenotypical changes in normal ACCs and to unveil the potential involvement of MAPK signaling pathways during this process. Methods. Normal and arthritic cartilage and bone samples were collected for isolation of ACCs and SBOs. Direct and indirect coculture models were applied to study chondrocyte hypertrophy under the influence of OA SBOs. MAPKs in the regulation of the cell–cell interactions were monitored by phosphorylated antibodies and relevant inhibitors. Results. OA SBOs led to increased hypertrophic gene expression and matrix calcification in ACCs by means of both direct and indirect cell–cell interactions. In this study, we demonstrated for the first time that OA SBOs suppressed p38 phosphorylation and induced ERK-1/2 signal phosphorylation in cocultured ACCs. The ERK-1/2 pathway inhibitor PD98059 significantly attenuated the hypertrophic changes induced by conditioned medium from OA SBOs, and the p38 inhibitor SB203580 resulted in the up-regulation of hypertrophic genes in ACCs. Conclusion. The findings of this study suggest that the pathologic interaction of OA SBOs and ACCs is mediated via the activation of ERK-1/2 phosphorylation and deactivation of p38 phosphorylation, resulting in hypertrophic differentiation of ACCs.

Development and synthesis of innovative approaches to the surgical assessment of articular cartilage degeneration

This research has established, through ultrasound, near infrared spectroscopy and biomechanics experiments, parameters and parametric relationships that can form the framework for quantifying the integrity of the articular cartilage-on-bone laminate, and objectively distinguish between normal/healthy and abnormal/degenerated joint tissue, with a focus on articular cartilage. This has been achieved by: 1. using traditional experimental methods to produce new parameters for cartilage assessment; 2. using novel methodologies to develop new parameters; and 3. investigating the interrelationships between mechanical, structural and molec- ular properties to identify and select those parameters and methodologies that can be used in a future arthroscopic probe based on points 1 and 2. By combining the molecular, micro- and macro-structural characteristics of the tissue with its mechanical properties, we arrive at a set of critical benchmarking parameters for viable and early-stage non-viable cartilage. The interrelationships between these characteristics, examined using a multivariate analysis based on principal components analysis, multiple linear regression and general linear modeling, could then to deter- mine those parameters and relationships which have the potential to be developed into a future clinical device. Specifically, this research has found that the ultrasound and near infrared techniques can subsume the mechanical parameters and combine to characterise the tissue at the molecular, structural and mechanical levels over the full depth of the cartilage matrix. It is the opinion in this thesis that by enabling the determination of the precise area of in uence of a focal defect or disease in the joint, demarcating the boundaries of articular cartilage with dierent levels of degeneration around a focal defect, better surgical decisions that will advance the processes of joint management and treatment will be achieved. Providing the basis for a surgical tool, this research will contribute to the enhancement and quanti�cation of arthroscopic procedures, extending to post- treatment monitoring and as a research tool, will enable a robust method for evaluating developing (particularly focalised) treatments.

Diffusion tensor of water in model articular cartilage

We used Monte Carlo simulations of Brownian dynamics of water to study anisotropic water diffusion in an idealised model of articular cartilage. The main aim was to use the simulations as a tool for translation of the fractional anisotropy of the water diffusion tensor in cartilage into quantitative characteristics of its collagen fibre network. The key finding was a linear empirical relationship between the collagen volume fraction and the fractional anisotropy of the diffusion tensor. Fractional anisotropy of the diffusion tensor is potentially a robust indicator of the microstructure of the tissue because, in the first approximation, it is invariant to the inclusion of proteoglycans or chemical exchange between free and collagen-bound water in the model. We discuss potential applications of Monte Carlo diffusion-tensor simulations for quantitative biophysical interpretation of MRI diffusion-tensor images of cartilage. Extension of the model to include collagen fibre disorder is also discussed.

Cross-talk of subchondral bone osteoblasts and articular cartilage chondrocytes : a new insight in understanding osteoarthritis pathogenesis

Osteoarthritis (OA) is the most common musculoskeletal disorder and represents a major health burden to society. In the course of the pathological development of OA, articular cartilage chondrocytes (ACCs) undergo atypical phenotype changes characterized by the expression of hypertrophic differentiation markers. Also, the adjacent subchondral bone shows signs of abnormal mineral density and enhanced production of bone turnover markers, indicative of osteoblast dysfunction. Collectively these findings indicate that the pathological changes typical of OA, involve alterations of the phenotypic properties of cells in both the subchondral bone and articular cartilage. However, the mechanism(s) by which these changes occur during OA development are not completely understood. The purpose of this project was to address the question of how subchondral bone osteoblasts (SBOs) and ACCs interact with each other with respect to regulation of respective cells’ phenotypic properties and in particular the involvement of mitogen activated protein kinase (MAPK) signalling pathways under normal and OA joint condition. We also endeavoured to test the influence of cross-talk between SBOs and ACCs isolated from normal and OA joint on matrix metalloproteinase (MMP) expression. For this purpose tissues from the knees of OA patients and normal controls were collected to isolate SBOs and ACCs. The cellular cross-talk of SBOs and ACCs were studied by means of both direct and indirect co-culture systems, which made it possible to identify the role of both membrane bound and soluble factors. Histology, immunohistochemistry, qRT-PCR, zymography, ELISA and western blotting were some of the techniques applied to distinguish the changes in the co-cultured vs. non co-cultured cells. The MAPK signalling pathways were probed by using targeted MAPK inhibitors, and their activity monitored by western blot analysis using phospho MAPK specific antibodies. Our co-culture studies demonstrated that OA ACCs enhanced the SBOs differentiation compared to normal ACCs. We demonstrated that OA ACCs induced these phenotypic changes in the SBOs via activating an ERK1/2 signalling pathway. The findings from this study thus provided clear evidence that OA ACCs play an integral role in altering the SBO phenotype. In the second study, we tested the influence of normal SBOs and OA SBOs on ACCs phenotype changes. The results showed that OA SBOs increased the hypertrophic gene expression in co-cultured ACCs compared to normal SBOs, a phenotype which is considered as pathological to the health and integrity of articular cartilage. It was demonstrated that these phenotype changes occurred via de-activation of p38 and activation of ERK1/2 signaling pathways. These findings suggest that the pathological interaction of OA SBOs with ACCs is mediated by cross-talking between ERK1/2 and p38 pathways, resulting in ACCs undergoing hypertrophic differentiation. Subsequent experiments to determine the effect on MMP regulation, of SBOs and ACCs cross-talk, revealed that co-culturing OA SBOs with ACCs significantly enhanced the proteolytic activity and expression of MMP-2 and MMP-9. In turn, co-culture of OA ACCs with SBOs led to abundant MMP-2 expression in SBOs. Furthermore, we showed that the addition of ERK1/2 and JNK inhibitors reversed the elevated MMP-2 and MMP-9 production which otherwise resulted from the interactions of OA SBOs-ACCs. Thus, this study has demonstrated that the altered interactions between OA SBOs-ACCs are capable of triggering the pathological pathways leading to degenerative changes seen in the osteoarthritic joint. In conclusion, the body of work presented in this dissertation has given clear in vitro evidence that the altered bi-directional communication of SBOs and ACCs may play a role in OA development and that this process was mediated by MAPK signalling pathways. Targeting these altered interactions by the use of MAPK inhibitors may provide the scientific rationale for the development of novel therapeutic strategies in the treatment and management of OA.

Can proteoglycan change in articular cartilage be detected by ultrasound evaluation?

This paper assesses the capacity of high-frequency ultrasonic waves for detecting changes in the proteoglycan (PG) content of articular cartilage. 50 cartilage-on-bone samples were exposed to ultrasonic waves via an ultrasound transducer at a frequency of 20MHz. Histology and ImageJ processing were conducted to determine the PG content of the specimen. The ratios of the reflected signals from both the surface and the osteochondral junction (OCJ) were determined from the experimental data. The initial results show an inconsistency in the capacity of ultrasound to distinguish samples with severe proteoglycan loss (i.e. >90% PG loss) from the normal intact sample. This lack of clear distinction was also demonstrated at for samples with less than 60% depletion, while there is a clear differentiation between the normal intact sample and those with 55-70% PG loss.

Near infrared for non-destructive testing of articular cartilage

The concept of non-destructive testing (NDT) of materials and structures is of immense importance in engineering and medicine. Several NDT methods including electromagnetic (EM)-based e.g. X-ray and Infrared; ultrasound; and S-waves have been proposed for medical applications. This paper evaluates the viability of near infrared (NIR) spectroscopy, an EM method for rapid non-destructive evaluation of articular cartilage. Specifically, we tested the hypothesis that there is a correlation between the NIR spectrum and the physical and mechanical characteristics of articular cartilage such as thickness, stress and stiffness. Intact, visually normal cartilage-on-bone plugs from 2-3yr old bovine patellae were exposed to NIR light from a diffuse reflectance fibre-optic probe and tested mechanically to obtain their thickness, stress, and stiffness. Multivariate statistical analysis-based predictive models relating articular cartilage NIR spectra to these characterising parameters were developed. Our results show that there is a varying degree of correlation between the different parameters and the NIR spectra of the samples with R2 varying between 65 and 93%. We therefore conclude that NIR can be used to determine, nondestructively, the physical and functional characteristics of articular cartilage.

Subchondral bone osteoblasts can induce chondrocyte mineralization during osteoarthritis and this process is relevant to cartilage degradation

Pathological mineralization of articular cartilage is a characteristic feature of osteoarthritis (OA); however, the underlying mechanisms, and their relevance to cartilage degeneration, are not clear. The involvement of subchondral bone changes in OA have been reported previously with the characterization of abnormal subchondral bone mineral density (BMD), osteiod volume, altered bone mechanical parameters and an increase in bone turnover markers. A number of osteoarthritic animal models have demonstrated that subchondral bone changes often precede cartilage degeneration. In this study site specific localization of mineralization markers were detected in the OA cartilage. Chondrocytes and osteoblasts derived from OA cartilage and subchondral bone showed a significant increase in the mRNA expressions of mineralization markers. Interestingly, osteoblasts from OA subchondral bone could significantly decrease cartilage matrix expression; whereas, increase mineralization of chondrocytes (Figure 1). Osteogenic factors, such as CBFA1, ALP, and type X collagen (Col-X), were detected in chondrocytes under mineralization conditions (Figure 2). Furthermore, chondrocyte mineralization was followed by increased mRNA and protein levels of MMP-2, MMP-9 and MMP-13, all of which are detrimental to cartilage integrity in vivo. The data reported here suggests that the upregulation of subchondral bone-mineralization, typical of OA progression, causes cartilage mineralization, and that the mineralization of chondrocytes induce increased MMP levels with a subsequent degradation of the articular cartilage.

Tissue engineering of stratified articular cartilage from chondrocyte subpopulations

Objective: To test if subpopulations of chondrocytes from different cartilage zones could be used to engineer cartilage constructs with features of normal stratification. Design: Chondrocytes from the superficial and middle zones of immature bovine cartilage were cultured in alginate, released, and seeded either separately or sequentially to form cartilage constructs. Constructs were cultured for 1 or 2 weeks and were assessed for growth, compressive properties, and deposition, and localization of matrix molecules and superficial zone protein (SZP). Results: The cartilaginous constructs formed from superficial zone chondrocytes exhibited less matrix growth and lower compressive properties than constructs from middle zone chondrocytes, with the stratified superficial-middle constructs exhibiting intermediate properties. Expression of SZP was highest at the construct surfaces, with the localization of SZP in superficial-middle constructs being concentrated at the superficial surface. Conclusions: Manipulation of subpopulations of chondrocytes can be useful in engineering cartilage tissue with a biomimetic approach, and in fabricating constructs that exhibit stratified features of normal articular cartilage. (C) 2003 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Synthesis of proteoglycan 4 by chondrocyte subpopulations in cartilage explants, monolayer cultures, and resurfaced cartilage cultures

Objective: To quantify the levels of proteoglycan 4 (PRG4) expression by subpopulations of chondrocytes from superficial, middle, and deep layers of normal bovine calf cartilage in various culture systems. Methods: Bovine calf articular cartilage discs or isolated cells were used in I of 3 systems of chondrocyte culture: explant, monolayer, or transplant, for 1-9 days. PRG4 expression was quantified by enzyme-linked immunosorbent assay of spent medium and localized by immunohistochemistry at the articular surface and within chondrocytes in explants and cultured cells. Results: Superficial chondrocytes secreted much more PRG4 than did middle and deep chondrocytes in all cultures. The pattern of PRG4 secretion into superficial culture medium varied with the duration of culture, decreasing with time in explant culture (from similar to25 mug/cm(2)/day on days 0-1 to similar to3 mug/cm(2)/day on days 5-9), while increasing in monolayer culture (from similar to1 pg/cell/day on days 0-1 to similar to7 pg/cell/day on days 7-9) and tending to increase in transplant culture (reaching similar to2 mug/cm(2)/day by days 7-9). In all of the culture systems, inclusion of ascorbic acid stimulated PRG4 secretion, and the source of PRG4 was immunolocalized to superficial cells. Conclusion: The results described here indicate that the phenotype of PRG4 secretion by chondrocytes in culture is generally maintained, in that PRG4 is expressed to a much greater degree by chondrocytes from the superficial zone than by those from the middle and deep zones. The marked up-regulation of PRG4 synthesis by ascorbic acid may have implications for cartilage homeostasis and prevention of osteoarthritic disease. Transplanting specialized cells that secrete PRG4 to a surface may impart functional lubrication and be generally applicable to many tissues in the body.

Inhibition of integrative cartilage repair by proteoglycan 4 in synovial fluid

To determine the effects of the articular cartilage surface, as well as synovial fluid (SF) and its components, specifically proteoglycan 4 (PRG4) and hyaluronic acid (HA), on integrative cartilage repair in vitro. Methods. Blocks of calf articular cartilage were harvested, some with the articular surface intact and others without. Some of the latter types of blocks were pretreated with trypsin, and then with bovine serum albumin, SF, PRG4, or HA. Immunolocalization of PRG4 on cartilage surfaces was performed after treatment. Pairs of similarly treated cartilage blocks were incubated in partial apposition for 2 weeks in medium supplemented with serum and 3 H-proline. Following culture, mechanical integration between apposed cartilage blocks was assessed by measuring adhesive strength, and protein biosynthesis and deposition were determined by incorporated 3 H-proline. Results. Samples with articular surfaces in apposition exhibited little integrative repair compared with samples with cut surfaces in apposition. PRG4 was immunolocalized at the articular cartilage surface, but not in deeper, cut surfaces (without treatment). Cartilage samples treated with trypsin and then with SF or PRG4 exhibited an inhibition of integrative repair and positive immunostaining for PRG4 at treated surfaces compared with normal cut cartilage samples, while samples treated with HA exhibited neither inhibited integrative repair nor PRG4 at the tissue surfaces. Deposition of newly synthesized protein was relatively similar under conditions in which integration differed significantly. Conclusion. These results support the concept that PRG4 in SF, which normally contributes to cartilage lubrication, can inhibit integrative cartilage repair. This has the desirable effect of preventing fusion of apposing surfaces of articulating cartilage, but has the undesirable effect of inhibiting integrative repair.