Osteocyte dysfunction promotes osteoarthritis through MMP13-dependent suppression of subchondral bone homeostasis.

Osteoarthritis (OA), long considered a primary disorder of articular cartilage, is commonly associated with subchondral bone sclerosis. However, the cellular mechanisms responsible for changes to subchondral bone in OA, and the extent to which these changes are drivers of or a secondary reaction to cartilage degeneration, remain unclear. In knee joints from human patients with end-stage OA, we found evidence of profound defects in osteocyte function. Suppression of osteocyte perilacunar/canalicular remodeling (PLR) was most severe in the medial compartment of OA subchondral bone, with lower protease expression, diminished canalicular networks, and disorganized and hypermineralized extracellular matrix. As a step toward evaluating the causality of PLR suppression in OA, we ablated the PLR enzyme MMP13 in osteocytes while leaving chondrocytic MMP13 intact, using Cre recombinase driven by the 9.6-kb DMP1 promoter. Not only did osteocytic MMP13 deficiency suppress PLR in cortical and subchondral bone, but it also compromised cartilage. Even in the absence of injury, osteocytic MMP13 deficiency was sufficient to reduce cartilage proteoglycan content, change chondrocyte production of collagen II, aggrecan, and MMP13, and increase the incidence of cartilage lesions, consistent with early OA. Thus, in humans and mice, defects in PLR coincide with cartilage defects. Osteocyte-derived MMP13 emerges as a critical regulator of cartilage homeostasis, likely via its effects on PLR. Together, these findings implicate osteocytes in bone-cartilage crosstalk in the joint and suggest a causal role for suppressed perilacunar/canalicular remodeling in osteoarthritis

Sostdc1 deficiency accelerates fracture healing by promoting the expansion of periosteal mesenchymal stem cells

Loss of Sostdc1, a growth factor paralogous to Sost, causes the formation of ectopic incisors, fused molars, abnormal hair follicles, and resistance to kidney disease. Sostdc1 is expressed in the periosteum, a source of osteoblasts, fibroblasts and mesenchymal progenitor cells, which are critically important for fracture repair. Here, we investigated the role of Sostdc1 in bone metabolism and fracture repair. Mice lacking Sostdc1 (Sostdc1−/−) had a low bone mass phenotype associated with loss of trabecular bone in both lumbar vertebrae and in the appendicular skeleton. In contrast, Sostdc1−/− cortical bone measurements revealed larger bones with higher BMD, suggesting that Sostdc1 exerts differential effects on cortical and trabecular bone. Mid-diaphyseal femoral fractures induced in Sostdc1−/− mice showed that the periosteal population normally positive for Sostdc1 rapidly expands during periosteal thickening and these cells migrate into the fracture callus at 3 days post fracture. Quantitative analysis of mesenchymal stem cell (MSC) and osteoblast populations determined that MSCs express Sostdc1, and that Sostdc1−/− 5 day calluses harbor > 2-fold more MSCs than fractured wildtype controls. Histologically a fraction of Sostdc1-positive cells also expressed nestin and α-smooth muscle actin, suggesting that Sostdc1 marks a population of osteochondral progenitor cells that actively participate in callus formation and bone repair. Elevated numbers of MSCs in D5 calluses resulted in a larger, more vascularized cartilage callus at day 7, and a more rapid turnover of cartilage with significantly more remodeled bone and a thicker cortical shell at 21 days post fracture. These data support accelerated or enhanced bone formation/remodeling of the callus in Sostdc1−/− mice, suggesting that Sostdc1 may promote and maintain mesenchymal stem cell quiescence in the periosteum

Conditional Deletion of Sost in MSC‐derived lineages Identifies Specific Cell Type Contributions to Bone Mass and B Cell Development

Sclerostin (Sost) is a negative regulator of bone formation and blocking its function via antibodies has shown great therapeutic promise by increasing both bone mass in humans and animal models. Sclerostin deletion in Sost knockout mice (Sost‐/‐) causes high bone mass (HBM) similar to Sclerosteosis patients. Sost‐/‐ mice have been shown to display an up to 300% increase in bone volume/total volume (BV/TV), relative to aged matched controls, and it has been postulated that the main source of skeletal Sclerostin is the osteocyte. To understand the cell‐type specific contributions to the HBM phenotype described in Sost‐/‐ mice, as well as to address the endocrine and paracrine mode of action of sclerostin, we examined the skeletal phenotypes of conditional Sost loss‐of‐function (SostiCOIN/iCOIN) mice with specific deletions in (1) the limb mesenchyme (Prx1‐Cre; targets osteoprogenitors and their progeny); (2) mid‐stage osteoblasts and their progenitors (Col1‐Cre); (3) mature osteocytes (Dmp1‐Cre) and (4) hypertrophic chondrocytes and their progenitors (ColX‐Cre). All conditional alleles resulted in significant increases in bone mass in trabecular bone in both the femur and lumbar vertebrae, but only Prx1‐Cre deletion fully recapitulated the amplitude of the HBM phenotype in the appendicular skeleton and the B cell defect described in the global knockout. Despite wildtype expression of Sost in the axial skeleton of Prx1‐Cre deleted mice, these mice also had a significant increase in bone mass in the vertebrae, but the Sclerostin released in circulation by the axial skeleton did not affect bone parameters in the appendicular skeleton. Also, both Col1 and Dmp1 deletion resulted in a similar 80% significant increase in trabecular bone mass, but only Col1 and Prx1 deletion resulted in a significant increase in cortical thickness. We conclude that several cell types within the Prx1‐osteoprogenitor derived lineages contribute significant amounts of Sclerostin protein to the paracrine pool of Sost, in bone

Investigating Osteocytic Perilacunar/Canalicular Remodeling.

Purpose of reviewIn perilacunar/canalicular remodeling (PLR), osteocytes dynamically resorb, and then replace, the organic and mineral components of the pericellular extracellular matrix. Given the enormous surface area of the osteocyte lacuna-canalicular network (LCN), PLR is important for maintaining homeostasis of the skeleton. The goal of this review is to examine the motivations and critical considerations for the analysis of PLR, in both in vitro and in vivo systems.Recent findingsMorphological approaches alone are insufficient to elucidate the complex mechanisms regulating PLR in the healthy skeleton and in disease. Understanding the role and regulation of PLR will require the incorporation of standardized PLR outcomes as a routine part of skeletal phenotyping, as well as the development of improved molecular and cellular outcomes. Current PLR outcomes assess PLR enzyme expression, the LCN, and bone matrix composition and organization, among others. Here, we discuss current PLR outcomes and how they have been applied to study PLR induction and suppression in vitro and in vivo. Given the role of PLR in skeletal health and disease, integrated analysis of PLR has potential to elucidate new mechanisms by which osteocytes participate in skeletal health and disease

Mechanosensitive Control of Articular Cartilage and Subchondral Bone Homeostasis in Mice Requires Osteocytic Transforming Growth Factor β Signaling

ObjectiveTransforming growth factor β (TGFβ) signaling plays a complex tissue-specific and nonlinear role in osteoarthritis (OA). This study was conducted to determine the osteocytic contributions of TGFβ signaling to OA.MethodsTo identify the role of osteocytic TGFβ signaling in joint homeostasis, we used 16-week-old male mice (n = 9-11 per group) and female mice (n = 7-11 per group) with an osteocyte-intrinsic ablation of TGFβ receptor type II (TβRIIocy-/- mice) and assessed defects in cartilage degeneration, subchondral bone plate (SBP) thickness, and SBP sclerostin expression. To further investigate these mechanisms in 16-week-old male mice, we perturbed joint homeostasis by subjecting 8-week-old mice to medial meniscal/ligamentous injury (MLI), which preferentially disrupts the mechanical environment of the medial joint to induce OA.ResultsIn all contexts, independent of sex, genotype, or medial or lateral joint compartment, increased SBP thickness and SBP sclerostin expression were spatially associated with cartilage degeneration. Male TβRIIocy-/- mice, but not female TβRIIocy-/- mice, had increased cartilage degeneration, increased SBP thickness, and higher levels of SBP sclerostin compared with control mice (all P < 0.05), demonstrating that the role of osteocytic TGFβ signaling on joint homeostasis is sexually dimorphic. With changes in joint mechanics following injury, control mice had increased SBP thickness, subchondral bone volume, and SBP sclerostin expression (all P < 0.05). TβRIIocy-/- mice, however, were insensitive to subchondral bone changes with injury, suggesting that mechanosensation at the SBP requires osteocytic TGFβ signaling.ConclusionOur results provide new evidence that osteocytic TGFβ signaling is required for a mechanosensitive response to injury, and that osteocytes control SBP homeostasis to maintain cartilage health, identifying osteocytic TGFβ signaling as a novel therapeutic target for OA

Sclerostin antibody treatment improves fracture outcomes in a Type I diabetic mouse model

AbstractType 1 diabetes mellitus (T1DM) patients have osteopenia and impaired fracture healing due to decreased osteoblast activity. Further, no adequate treatments are currently available that can restore impaired healing in T1DM; hence a significant need exists to investigate new therapeutics for treatment of orthopedic complications. Sclerostin (SOST), a WNT antagonist, negatively regulates bone formation, and SostAb is a potent bone anabolic agent. To determine whether SOST antibody (SostAb) treatment improves fracture healing in streptozotocin (STZ) induced T1DM mice, we administered SostAb twice weekly for up to 21days post-fracture, and examined bone quality and callus outcomes at 21days and 42days post-fracture (11 and 14weeks of age, respectively). Here we show that SostAb treatment improves bone parameters; these improvements persist after cessation of antibody treatment. Markers of osteoblast differentiation such as Runx2, collagen I, osteocalcin, and DMP1 were reduced, while an abundant number of SP7/osterix-positive early osteoblasts were observed on the bone surface of STZ calluses. These results suggest that STZ calluses have poor osteogenesis resulting from failure of osteoblasts to fully differentiate and produce mineralized matrix, which produces a less mineralized callus. SostAb treatment enhanced fracture healing in both normal and STZ groups, and in STZ+SostAb mice, also reversed the lower mineralization seen in STZ calluses. Micro-CT analysis of calluses revealed improved bone parameters with SostAb treatment, and the mineralized bone was comparable to Controls. Additionally, we found sclerostin levels to be elevated in STZ mice and β-catenin activity to be reduced. Consistent with its function as a WNT antagonist, SostAb treatment enhanced β-catenin activity, but also increased the levels of SOST in the callus and in circulation. Our results indicate that SostAb treatment rescues the impaired osteogenesis seen in the STZ induced T1DM fracture model by facilitating osteoblast differentiation and mineralization of bone

Altered canalicular remodeling associated with femur fracture in mice

We previously showed that femur fracture in mice caused a reduction in bone volume at distant skeletal sites within 2 weeks post-fracture. Osteocytes also have the ability to remodel their surrounding bone matrix through perilacunar/canalicular remodeling (PLR). If PLR is altered systemically following fracture, this could affect bone mechanical properties and increase fracture risk at all skeletal sites. In this study, we investigated whether lacunar-canalicular microstructure and the rate of PLR are altered in the contralateral limb following femoral fracture in mice. We hypothesized that femoral fracture would accelerate PLR by 2 weeks postfracture, followed by partial recovery by 4 weeks. We used histological evaluation and high-resolution microcomputed tomography to quantify the morphology of the lacunar-canalicular network at the contralateral tibia, and we used quantitative real-time polymerase chain reaction (RT-PCR) and RNA-seq to measure the expression of PLR-associated genes in the contralateral femur. We found that at both 2 and 4 weeks postfracture, canalicular width was significantly increased by 18.6% and 16.6%, respectively, in fractured mice relative to unfractured controls. At 3 days and 4 weeks post-fracture, we observed downregulation of PLR-associated genes; RNA-seq analysis at 3 days post-fracture showed a deceleration of bone formation and mineralization in the contralateral limb. These data demonstrate notable canalicular changes following fracture that could affect bone mechanical properties. These findings expand our understanding of systemic effects of fracture and how biological and structural changes at distant skeletal sites may contribute to increased fracture risk following an acute injury

Sost and its paralog Sostdc1 coordinate digit number in a Gli3-dependent manner.

WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emerging as key regulatory proteins with great promise as therapeutic agents for bone disorders. Here we show that Sost and its paralog Sostdc1 emerged through ancestral genome duplication and their expression patterns have diverged to delineate non-overlapping domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme. While Sostdc1(-/-) mice lack any obvious limb or skeletal defects, Sost(-/-) mice recapitulate the hand defects described for Sclerosteosis patients. However, elevated WNT signaling in Sost(-/-); Sostdc1(-/-) mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and preaxial polydactyly in a Gli1- and Gli3-dependent manner. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost(-/-) and Sost(-/-); Sostdc1(-/-) mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanism and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling