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.

Altered cell interactions of subchondral bone osteoblasts and articular chondrocytes in osteoarthritis is through the mediation of ERK1/2 phosphorylation

Introduction: 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 a typical 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. However, the mechanism(s) by which these changes occur during the OA development are not completely understood. Materials and Methods: ACCs and subchondral bone osteoblasts (SBOs) were harvested from OA and healthy patients for the cross-talk studies between normal and OA ACCs and SBOs. The involvement of mitogen activated protein kinase (MAPK) signalling pathway during the cell-cell interactions was analysed by zymography, ELISA and western blotting methods. Results: The direct and in-direct co-culture studies showed that OA (ACCs and SBOs) cells induced osteoarthritic changes of normal (ACC and SBOs) cells. This altered cell interaction induced by OA cells significantly aggravated the proteolytic activity, which resulted cartilage degeneration. The altered cell interaction appeared to significantly activate ERK 1/2 phosphorylation and inhibition of MAPK-ERK 1/2 pathway reversed the osteoarthrtitic phenotypic changes. Discussion and Conclusion: Our study has demonstrated that the altered bi-directional communication of SBOs and ACCs are critical for initiation and progression of OA related changes and that this process is 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 related disorders.

Cell interactions of osteoarthritic subchondral bone osteoblasts and articular chondrocytes aggravate MMP-2 and MMP-9 production through the mediation of ERK1/2 and JNK phosphorylation

Matrix Metalloproteinases (MMP) play a key role in osteoarthritis (OA) development. The aim of the present study was to investigate whether, the cross-talk between subchondral bone osteoblasts (SBOs) and articular cartilage chondrocytes (ACCs) in OA alters the expression and regulation of MMPs, and also to test the potential involvement of mitogen activated protein kinase (MAPK) signalling pathway during this process.

Aggravation of ADAMTS and MMP production and ERK1/2 pathway in the interaction of osteoarthritic subchondral bone osteoblasts and articular cartilage chondrocytes : a possible pathogenic role in osteoarthritis

Introduction: Degradative enzymes, such as A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and matrix metalloproteinases (MMPs), play key roles in osteoarthritis (OA) development. The aim of the present study was to investigate if cross-talk between subchondral bone osteoblasts (SBOs) and articular cartilage chondrocytes (ACCs) in OA alters the expression and regulation of ADAMTS5, ADAMTS4, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13, and also to test the possible involvement of mitogen activated protein kinase (MAPK) signaling pathway during this process. Methods: ACCs and SBOs were isolated from normal and OA patients. An in vitro co-culture model was developed to study the regulation of ADAMTS and MMPs under normal and OA joint cross-talk conditions. MAPK-ERK inhibitor, PD98059 was applied to delineate the involvement of specific pathway during this interaction process. Results: Indirect co-culture of OA SBOs with normal ACCs resulted in significantly increased expression of ADAMTS5, ADAMTS4, MMP-2, MMP-3 and MMP-9 in ACCs, whereas co-culture of OA ACCs led to increased MMP-1 and MMP-2 expression in normal SBOs. The upregulation of ADAMTS and MMPs under these conditions was correlated with activation of the MAPK-ERK1/2 signaling pathway and the addition of the MAPK-ERK inhibitor, PD98059, reversed the overexpression of ADAMTS and MMPs in co-cultures. Conclusion: In summary, we believe, these results add to the evidence that in human OA, altered bi-directional signals transmitted between SBOs and ACCs significantly impacts the critical features of both cartilage and bone by producing abnormal levels of ADAMTS and MMPs. Furthermore, we have demonstrated for the first time that this altered cross-talk was mediated by the phosphorylation of MAPK-ERK1/2 signaling pathway.

Male reproductive health disorders among Aboriginal and Torres Strait Islander men : a hidden problem?

OBJECTIVE: To better understand help-seeking behaviours and reproductive health disorders among Aboriginal and Torres Strait Islander men. DESIGN, SETTING AND PARTICIPANTS: A cross-sectional mixed-methods study conducted from 1 May 2004 to 30 April 2005 of 293 Aboriginal and Torres Strait Islander men aged 18 years and over from urban, rural and remote communities in the Northern Territory and Queensland. MAIN OUTCOME MEASURES: Subscale of the International Index of Erectile Function, self-reported help-seeking behaviours for erectile dysfunction (ED) and prostate disease, thematic analysis of semi-structured interviews and focus groups. RESULTS: The prevalence of moderate-to-severe ED increased across age groups, from about 10% in younger men (under 35 years) to 28% in men aged 55-74 years. Moderate-to-severe ED was strongly associated with reporting a chronic condition (odds ratio [OR], 3.67) and residing in a remote area (OR, 2.94). Aboriginal and Torres Strait Islander men aged 40-59 years showed similar low levels of help-seeking behaviours compared with non-Indigenous men from a comparable population-based study. About half of the men with ED saw a doctor or received treatment for ED in each population. While prostate cancer rates were low in both studies, testing for prostate problems was less frequent in Aboriginal and Torres Strait Islander men (11.4%) than in non-Indigenous men (34.1%, P < 0.001), despite similar levels of concern about prostate cancer. Barriers to help-seeking included shame, culturally inappropriate services and lack of awareness. CONCLUSION: This study, the first to investigate reproductive health of Aboriginal and Torres Strait Islander men, found low levels of help-seeking behaviours for reproductive health disorders, with implications for missing a predictor of chronic disease and late diagnosis of prostate disease.

No association between MTHFR A1298C and MTRR A66G polymorphisms, and MS in an Australian cohort

Multiple sclerosis (MS) is a complex neurological disease that affects the central nervous system (CNS) resulting in debilitating neuropathology. Pathogenesis is primarily defined by CNS inflammation and demyelination of nerve axons. Methionine synthase reductase (MTRR) is an enzyme that catalyzes the remethylation of homocysteine (Hcy) to methionine via cobalamin and folate dependant reactions. Cobalamin acts as an intermediate methyl carrier between methylenetetrahydrofolate reductase (MTHFR) and Hcy. MTRR plays a critical role in maintaining cobalamin in an active form and is consequently an important determinant of total plasma Hcy (pHcy) concentrations. Elevated intracellular pHcy levels have been suggested to play a role in CNS dysfunction, neurodegenerative, and cerebrovascular diseases. Our investigation entailed the genotyping of a cohort of 140 cases and matched controls for MTRR and MTHFR, by restriction length polymorphism (RFLP) techniques. Two polymorphisms: MTRR A66G and MTHFR A1298C were investigated in an Australian age and gender matched case-control study. No significant allelic frequency difference was observed between cases and controls at the α = 0.05 level (MTRR χ2 = 0.005, P = 0.95, MTHFR χ2 = 1.15, P = 0.28). Our preliminary findings suggest no association between the MTRR A66G and MTHFR A1298C polymorphisms and MS

Role of dentin matrix protein 1 in cartilage redifferentiation and osteoarthritis

Objective The aim of this study was to test the possible involvement, relevance and significance of dentin matrix protein 1 (DMP1) in chondrocyte redifferentiation and OA. Methods To examine the function of DMP1 in vitro, bone marrow stromal cells (BMSCs) and articular chondrocytes (ACs) were isolated and differentiated in micromasses in the presence or absence of DMP1 small interfering RNA and analysed for chondrogenic phenotype. The association of DMP1 expression with OA progression was analysed time dependently in the OA menisectomy rat model and in grade-specific OA human samples. Results It was found that DMP1 was strongly related to chondrogenesis, which was evidenced by the strong expression of DMP1 in the 14.5-day mouse embryonic cartilage development stage and in femoral heads of post-natal days 0 and 4. In vitro chondrogenesis in BMSCs and ACs was accompanied by a gradual increase in DMP1 expression at both the gene and protein levels. In addition, knockdown of DMP1 expression led to decreased chondrocyte marker genes, such as COL2A1, ACAN and SOX9, and an increase in the expression of COL10A and MMP13 in ACs. Moreover, treatment with IL-1β, a well-known catabolic culprit of proteoglycan matrix loss, significantly reduced the expression of DMP1. Furthermore, we also observed the suppression of DMP1 protein in a grade-specific manner in knee joint samples from patients with OA. In the menisectomy-induced OA model, an increase in the Mankin score was accompanied by the gradual loss of DMP1 expression. Conclusion Observations from this study suggest that DMP1 may play an important role in maintaining the chondrogenic phenotype and its possible involvement in altered cartilage matrix remodelling and degradation in disease conditions like OA.

Genetic analysis for a shared biological basis between migraine and coronary artery disease

Objective: To apply genetic analysis of genome-wide association data to study the extent and nature of a shared biological basis between migraine and coronary artery disease (CAD). Methods: Four separate methods for cross-phenotype genetic analysis were applied on data from 2 large-scale genome-wide association studies of migraine (19,981 cases, 56,667 controls) and CAD (21,076 cases, 63,014 controls). The first 2 methods quantified the extent of overlapping risk variants and assessed the load of CAD risk loci in migraineurs. Genomic regions of shared risk were then identified by analysis of covariance patterns between the 2 phenotypes and by querying known genome-wide significant loci. Results: We found a significant overlap of genetic risk loci for migraine and CAD. When stratified by migraine subtype, this was limited to migraine without aura, and the overlap was protective in that patients with migraine had a lower load of CAD risk alleles than controls. Genes indicated by 16 shared risk loci point to mechanisms with potential roles in migraine pathogenesis and CAD, including endothelial dysfunction (PHACTR1) and insulin homeostasis (GIP). Conclusions: The results suggest that shared biological processes contribute to risk of migraine and CAD, but surprisingly this commonality is restricted to migraine without aura and the impact is in opposite directions. Understanding the mechanisms underlying these processes and their opposite relationship to migraine and CAD may improve our understanding of both disorders.

The importance of connexin hemichannels during chondroprogenitor cell differentiation in hydrogel versus microtissue culture models

Appropriate selection of scaffold architecture is a key challenge in cartilage tissue engineering. Gap junction-mediated intercellular contacts play important roles in precartilage condensation of mesenchymal cells. However, scaffold architecture could potentially restrict cell-cell communication and differentiation. This is particularly important when choosing the appropriate culture platform as well as scaffold-based strategy for clinical translation, that is, hydrogel or microtissues, for investigating differentiation of chondroprogenitor cells in cartilage tissue engineering. We, therefore, studied the influence of gap junction-mediated cell-cell communication on chondrogenesis of bone marrow-derived mesenchymal stromal cells (BM-MSCs) and articular chondrocytes. Expanded human chondrocytes and BM-MSCs were either (re-) differentiated in micromass cell pellets or encapsulated as isolated cells in alginate hydrogels. Samples were treated with and without the gap junction inhibitor 18-α glycyrrhetinic acid (18αGCA). DNA and glycosaminoglycan (GAG) content and gene expression levels (collagen I/II/X, aggrecan, and connexin 43) were quantified at various time points. Protein localization was determined using immunofluorescence, and adenosine-5'-triphosphate (ATP) was measured in conditioned media. While GAG/DNA was higher in alginate compared with pellets for chondrocytes, there were no differences in chondrogenic gene expression between culture models. Gap junction blocking reduced collagen II and extracellular ATP in all chondrocyte cultures and in BM-MSC hydrogels. However, differentiation capacity was not abolished completely by 18αGCA. Connexin 43 levels were high throughout chondrocyte cultures and peaked only later during BM-MSC differentiation, consistent with the delayed response of BM-MSCs to 18αGCA. Alginate hydrogels and microtissues are equally suited culture platforms for the chondrogenic (re-)differentiation of expanded human articular chondrocytes and BM-MSCs. Therefore, reducing direct cell-cell contacts does not affect in vitro chondrogenesis. However, blocking gap junctions compromises cell differentiation, pointing to a prominent role for hemichannel function in this process. Therefore, scaffold design strategies that promote an increasing distance between single chondroprogenitor cells do not restrict their differentiation potential in tissue-engineered constructs.

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.

Adaptive and phase transition behavior in performance of discrete multi-articular actions by degenerate neurobiological systems

The identification of attractors is one of the key tasks in studies of neurobiological coordination from a dynamical systems perspective, with a considerable body of literature resulting from this task. However, with regards to typical movement models investigated, the overwhelming majority of actions studied previously belong to the class of continuous, rhythmical movements. In contrast, very few studies have investigated coordination of discrete movements, particularly multi-articular discrete movements. In the present study, we investigated phase transition behavior in a basketball throwing task where participants were instructed to shoot at the basket from different distances. Adopting the ubiquitous scaling paradigm, throwing distance was manipulated as a candidate control parameter. Using a cluster analysis approach, clear phase transitions between different movement patterns were observed in performance of only two of eight participants. The remaining participants used a single movement pattern and varied it according to throwing distance, thereby exhibiting hysteresis effects. Results suggested that, in movement models involving many biomechanical degrees of freedom in degenerate systems, greater movement variation across individuals is available for exploitation. This observation stands in contrast to movement variation typically observed in studies using more constrained bi-manual movement models. This degenerate system behavior provides new insights and poses fresh challenges to the dynamical systems theoretical approach, requiring further research beyond conventional movement models.

The ultra-low friction of the articular surface is pH-dependent and is built on a hydrophobic underlay including a hypothesis on joint lubrication mechanism

In this study, the influence of pH on interfacial energy distributed over the phospholipids-bilayer surface model and the effect of hydrophobicity on coefficient of friction (f) were investigated by using microelectrophoresis. An important clinical implication of deficiency in hydrophobicity is the loss of phospholipids that is readily observed in osteoarthritis joints. This paper establishes the influence of pH on interfacial energy upon an increase f, which might be associated with a decrease of hydrophobicity of the articular surface.

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.