Evidence for the dysregulated expression of TWIST1, TGFß1 and SMAD3 in differentiating osteoblasts from primary hip osteoarthritis patients.

OBJECTIVE: This study compared human primary osteoblasts derived from hip osteoarthritis (OA) cases against controls (CTLs) to investigate candidate OA disease genes, twist homologue 1 (TWIST1), wingless MMTV integration site family member 5B (WNT5B), transforming growth factor-ß (TGFß1) and SMAD family member 3 (SMAD3), during osteoblast differentiation, relative to calcium apposition and elemental mineral composition. MATERIALS & METHODS: Primary osteoblast cultures were generated from intertrochanteric trabecular bone samples from five female primary hip OA cases and five age-matched female CTLs. During a 42-day differentiation time-course, alizarin red stains, energy-dispersive X-ray spectroscopy and real-time RT-polymerase chain reaction (PCR) were used to quantify calcium, elemental composition and gene expression, respectively. Data were analysed using linear mixed effects models and Pearson correlation matrices. RESULTS: Significant differences, correlations and associations were found in OA and CTL osteoblasts between gene and mineral measures. The calcium: phosphorous (Ca:P) ratio was significantly more varied in OA compared to CTL. Calcium apposition, mineral composition as well as TWIST1 and TGFß1 mRNA expression changed significantly over time. TWIST1 mRNA expression was elevated and correlated with SMAD3 mRNA levels in the OA cohort during the time-course. Associations were observed between tissue non-specific alkaline phosphatase (TNAP), osteocalcin (OCN), TWIST1, TGFß1, SMAD3 mRNA levels and mineral measures in OA against CTL. Temporal differences between SMAD3 mRNA expression and mineral composition were also found in OA. CONCLUSIONS: Dysregulated expression of TWIST1, TGFß1 and SMAD3 mRNA observed in OA bone is reflected in the functionality of the osteoblast when these cells are cultured ex vivo. The results presented here are consistent with at least part of the aetiology of primary hip OA deriving from altered intrinsic properties of the osteoblast.

Critical molecular regulators, histomorphometric indices and their correlations in the trabecular bone in primary hip osteoarthritis.

OBJECTIVE: This study examined differential gene expression, histomorphometric indices and relationships between these, in femoral trabecular bone from osteoarthritis (OA) patients and control (CTL) subjects, with the aim of identifying potential molecular drivers consistent with changes in structural and remodelling indices in the OA pathology. MATERIALS AND METHODS: Bone samples from the intertrochanteric (IT) region were obtained from age and sex-matched cohorts of 23 primary hip OA patients and 21 CTL subjects. Real-time polymerase chain reaction (PCR) and histomorphometric analysis were performed on each sample and correlations between gene expression and histomorphometric variables determined. RESULTS: Alterations in gene expression, structural indices and correlations between these were found in OA bone compared to CTL. In OA bone, expression of critical regulators of osteoblast differentiation (TWIST1) and function (PTEN, TIMP4) were decreased, while genes associated with inflammation (SMAD3, CD14) were increased. Bone structural and formation indices (BV/TV, Tb.N, OS/BS) were increased, whereas resorption indices (ES/BS, ES/BV) were decreased. Importantly, significant correlations in CTL bone between CTNNB1 expression and formation indices (OS/BS, OS/BV, OV/BV) were absent in OA bone, indicating altered WNT/ß-catenin signalling. TWIST1 expression and BV/TV were correlated in CTL bone, but not in OA bone, consistent with altered osteoblastogenesis in OA. Matrix metalloproteinase 25 (MMP25) expression and remodelling indices (ES/BS, ES/BV, ES/TV) were correlated only in OA pointing to aberrant bone remodelling in this pathology. CONCLUSIONS: These findings indicate an altered state of osteoblast differentiation and function in OA driven by several key molecular regulators. In association with this differential gene expression, an altered state of both trabecular bone remodelling and resulting microarchitecture were also observed, further characterising the pathogenesis of primary hip OA.