The classical view of the pathogenesis of osteoarthritis (OA) is that subchondral sclerosis is associated with, and perhaps causes, age-related joint degeneration. Recent observations have demonstrated that OA is associated with early loss of bone owing to increased bone remodelling, followed by slow turnover leading to densification of the subchondral plate and complete loss of cartilage. Subchondral densification is a late event in OA that involves only the subchondral plate and calcified cartilage; the subchondral cancellous bone beneath the subchondral plate may remain osteopenic. In experimental models, inducing subchondral sclerosis without allowing the prior stage of increased bone remodelling to occur does not lead to progressive OA. Therefore, both early-stage increased remodelling and bone loss, and the late-stage slow remodelling and subchondral densification are important components of the pathogenetic process that leads to OA. The apparent paradoxical observations that OA is associated with both increased remodelling and osteopenia, as well as decreased remodelling and sclerosis, are consistent with the spatial and temporal separation of these processes during joint degeneration. This Review provides an overview of current knowledge on OA and discusses the role of subchondral bone in the initiation and progression of OA. A hypothetical model of OA pathogenesis is proposed.

Prostaglandin (PG) E(2) is a potent inducer of cortical and trabecular bone formation in humans and animals. Although the bone anabolic action of PGE(2) is well documented, the cellular and molecular mechanisms that mediate this effect remain unclear. This study was undertaken to examine the effect of pharmacological inactivation of the prostanoid receptor EP(4), one of the PGE(2) receptors, on PGE(2)-induced bone formation in vivo. We first determined the ability of EP(4)A, an EP(4)-selective ligand, to act as an antagonist. PGE(2) increases intracellular cAMP and suppresses apoptosis in the RP-1 periosteal cell line. Both effects were reversed by EP(4)A, suggesting that EP(4)A acts as an EP(4) antagonist in the cells at concentrations consistent with its in vitro binding to EP(4). We then examined the effect of EP(4) on bone formation induced by PGE(2) in young rats. Five- to 6-week-old rats were treated with PGE(2) (6 mg/kg/day) in the presence or absence of EP(4)A (10 mg/kg/day) for 12 days. We found that treatment with EP(4)A suppresses the increase in trabecular bone volume induced by PGE(2). This effect is accompanied by a suppression of bone formation indices: serum osteocalcin, extent of labeled surface, and extent of trabecular number, suggesting that the reduction in bone volume is due most likely to decreased bone formation. The pharmacological evidence presented here provides strong support for the hypothesis that the bone anabolic effect of PGE(2) in rats is mediated by the EP(4) receptor.

Objective Inflammation in the bone microenvironment stimulates osteoclast differentiation, resulting in uncoupling of resorption and formation. Mechanisms contributing to the inhibition of osteoblast function in inflammatory diseases, however, have not been elucidated. Rheumatoid arthritis (RA) is a prototype of an inflammatory arthritis that results in focal loss of articular bone. The paucity of bone repair in inflammatory diseases such as RA raises compelling questions regarding the impact of inflammation on bone formation. The aim of this study was to establish the mechanisms by which inflammation regulates osteoblast activity. Methods We characterized an innovative variant of a murine model of arthritis in which inflammation is induced in C57BL/6J mice by transfer of arthritogenic K/B×N serum and allowed to resolve. Results In the setting of resolving inflammation, bone resorption ceased and appositional osteoblast-mediated bone formation was induced, resulting in repair of eroded bone. Resolution of inflammation was accompanied by striking changes in the expression of regulators of the Wnt/β-catenin pathway, which is critical for osteoblast differentiation and function. Down-regulation of the Wnt antagonists secreted frizzled-related protein 1 (sFRP1) and sFRP2 during the resolution phase paralleled induction of the anabolic and pro–matrix mineralization factors Wnt10b and DKK2, demonstrating the role of inflammation in regulating Wnt signaling. Conclusion Repair of articular bone erosion occurs in the setting of resolving inflammation, accompanied by alterations in the Wnt signaling pathway. These data imply that in inflammatory diseases that result in persistent articular bone loss, strict control of inflammation may not be achieved and may be essential for the generation of an anabolic microenvironment that supports bone formation and repair.

Traditional bone mechanical testing techniques require excised bone and destructive sample preparation. Recently, a cyclic-microindentation technique, reference-point indentation (RPI), was described that allows bone to be tested in a clinical setting, permitting the analysis of changes to bone material properties over time. Because this is a new technique, it has not been clear how the measurements generated by RPI are related to the material properties of bone measured by standard techniques. In this paper, we describe our experience with the RPI technique, and correlate the results obtained by RPI with those of traditional mechanical testing, namely 3-point bending and axial compression. Using different animal models, we report that apparent bone material toughness obtained from 3-point bending and axial compression is inversely correlated with the indentation distance increase (IDI) obtained from RPI with r2 values ranging from 0.50 to 0.57. We also show that conditions or treatments previously shown to cause differences in toughness, including diabetes and bisphosphonate treatment, had significantly different IDI values compared to controls. Collectively these results provide a starting point for understanding how RPI relates to traditional mechanical testing results.

Raloxifene is an FDA approved agent used to treat bone loss and decrease fracture risk. In clinical trials and animal studies, raloxifene reduces fracture risk and improves bone mechanical properties, but the mechanisms of action remain unclear because these benefits occur largely independent of changes to bone mass. Using a novel experimental approach, machined bone beams, both from mature male canine and human male donors, were depleted of living cells and then exposed to raloxifene ex vivo. Our data show that ex vivo exposure of non-viable bone to raloxifene improves intrinsic toughness, both in canine and human cortical bone beams tested by 4-point bending. These effects are cell-independent and appear to be mediated by an increase in matrix bound water, assessed using basic gravimetric weighing and sophisticated ultrashort echo time magnetic resonance imaging. The hydroxyl groups (−OH) on raloxifene were shown to be important in both the water and toughness increases. Wide and small angle x-ray scattering patterns during 4-pt bending show that raloxifene alters the transfer of load between the collagen matrix and the mineral crystals, placing lower strains on the mineral, and allowing greater overall deformation prior to failure. Collectively, these findings provide a possible mechanistic explanation for the therapeutic effect of raloxifene and more importantly identify a cell-independent mechanism that can be utilized for novel pharmacological approaches for enhancing bone strength.

Diabetes detrimentally affects the musculoskeletal system by stiffening the collagen matrix due to increased advanced glycation end products (AGEs). In this study, tibiae and tendon from Zucker diabetic Sprague-Dawley (ZDSD) rats were compared to Sprague-Dawley derived controls (CD) using Atomic Force Microscopy. ZDSD and CD tibiae were compared using Raman Spectroscopy and Reference Point Indentation (RPI). ZDSD bone had a significantly different distribution of collagen D-spacing than CD (p = 0.015; ZDSD n = 294 fibrils; CD n = 274 fibrils) which was more variable and shifted to higher values. This shift between ZDSD and CD D-spacing distribution was more pronounced in tendon (p < 0.001; ZDSD n = 350; CD n = 371). Raman revealed significant increases in measures of bone matrix mineralization in ZDSD (PO43− ν1/Amide I p = 0.008; PO43− ν1/CH2 wag p = 0.047; n = 5 per group) despite lower bone mineral density (aBMD) and ash fraction indicating diabetes may preferentially reduce the Raman signature of collagen. Decreased indentation distance increase (p = 0.010) and creep indentation distance (p = 0.040) measured by RPI (n = 9 per group) in ZDSD rats suggest a matrix more resistant to indentation under the high stresses associated with RPI at this length scale. There were significant correlations between Raman and RPI measurements in the ZDSD population (n = 18 locations) but not the CD population (n = 16 locations) indicating that while RPI is relatively unaffected by biological noise, it is sensitive to disease-induced compositional changes. In conclusion, diabetes in the ZDSD rat causes changes to the nanoscale morphology of collagen that result in compositional and mechanical effects in bone at the microscale.

Raloxifene treatment has been shown previously to positively affect bone mechanical properties following 1 year of treatment in skeletally mature dogs. Reference point indentation (RPI) can be used for in vivo assessment of mechanical properties and has been shown to produce values that are highly correlated with properties derived from traditional mechanical testing. The goal of this study was to use RPI to determine if raloxifene-induced alterations in mechanical properties occurred after 6 months of treatment. Twelve skeletally mature female beagle dogs were treated for 6 months with oral doses of saline vehicle (VEH, 1 ml/kg/day) or a clinically relevant dose of raloxifene (RAL, 0.5 mg/kg/day). At 6 months, all animals underwent in vivo RPI (10 N force, 10 cycles) of the anterior tibial midshaft. RPI data were analyzed using a custom MATLAB program, designed to provide cycle-by-cycle data from the RPI test and validated against the manufacturer-provided software. Indentation distance increase (IDI), a parameter that is inversely related to bone toughness, was significantly lower in RAL-treated animals compared to VEH (− 16.5%), suggesting increased bone toughness. Energy absorption within the first cycle was significantly lower with RAL compared to VEH (− 21%). These data build on previous work that has documented positive effects of raloxifene on material properties by showing that these changes exist after 6 months.

Human osteoblasts produce PGD 2 , which acts on the DP receptor to decrease osteoprotegerin production and on the CRTH2 receptor to decrease RANKL expression and to induce osteoblast chemotaxis. These results indicate that activation of CRTH2 may lead to an anabolic response in bone.Introduction: Whereas the actions of prostaglandin (PG)E 2 as a modulator of bone and osteoblast function are relatively well characterized, little is known about PGD 2 and bone metabolism. The objectives of this study were to determine if human osteoblasts can produce PGD 2 , which prostaglandin D 2 synthases are implicated in this synthesis, to identify the PGD 2 receptors (DP and CRTH2) on these cells and to characterize the biological effects resulting from their activation. Materials and Methods:RT-PCR analysis and immunohistochemistry were used to detect PGD 2 receptor and synthases in cultured human osteoblasts. Immunohistochemistry was used to identify the synthases and receptors in human bone tissue. Intracellular cAMP and calcium levels were determined to verify receptor activation. The cells were stimulated with PGD 2 or the specific agonists BW 245C (DP) and DK-PGD 2 (CRTH2), and the resulting effects on osteoprotegerin (OPG) secretion, RANKL expression, and chemotaxis were determined. Osteoblast production of PGD 2 was evaluated by measuring PGD 2 in the culture supernatants after stimulation with interleukin (IL)-1, TNF-␣, PTH, vascular endothelial growth factor (VEGF), and insulin-like growth factor I (IGF-I). Results: Human osteoblasts in culture generated PGD 2 when stimulated. Both osteoblasts in culture and in situ present the lipocalin-type PGD 2 synthase only. Both DP and CRTH2 receptors were present in human osteoblasts in culture and in situ. Stimulation of DP resulted in an increase in cAMP, whereas CRTH2 increased the intracellular calcium level. OPG production was reduced by 60% after DP receptor stimulation, whereas CRTH2 receptor stimulation decreased RANKL expression on human osteoblasts. As reported for other cell types, CRTH2 was a potent inducer of chemotaxis for human osteoblasts in culture. Conclusions: Human osteoblasts in culture produce PGD 2 under biologically relevant stimuli through the lipocalin-type PGD 2 synthase (L-PGDS) pathway. As an autacoid, PGD 2 can act on DP and CRTH2 receptors, both present on these cells. Specific activation of CRTH2 could lead directly and indirectly to an anabolic response in bone.