Parathyroid hormone and trabectedin have differing effects on macrophages and stress fracture repair.

Stress fractures occur as a result of repeated mechanical stress on bone and are commonly found in the load-bearing lower extremities. Macrophages are key players in the immune system and play an important role in bone remodeling and fracture healing. However, the role of macrophages in stress fractures has not been adequately addressed. We hypothesize that macrophage infiltration into a stress fracture callus site promotes bone healing. To test this, a unilateral stress fracture induction model was employed in which the murine ulna of four-month-old, C57BL/6 J male mice was repeatedly loaded with a pre-determined force until the bone was displaced a distance below the threshold for complete fracture. Mice were treated daily with parathyroid hormone (PTH, 50 µg/kg/day) starting two days before injury and continued until 24 h before euthanasia either four or six days after injury, or treated with trabectedin (0.15 mg/kg) on the day of stress fracture and euthanized three or seven days after injury. These treatments were used due to their established effects on macrophages. While macrophages have been implicated in the anabolic effects of PTH, trabectedin, an FDA approved chemotherapeutic, compromises macrophage function and reduces bone mass. At three- and four-days post injury, callus macrophage numbers were analyzed histologically. There was a significant increase in macrophages with PTH treatment compared to vehicle in the callus site. By one week of healing, treatments differentially affected the bony callus as analyzed by microcomputed tomography. PTH enhanced callus bone volume. Conversely, callus bone volume was decreased with trabectedin treatment. Interestingly, concurrent treatment with PTH and trabectedin rescued the reduction observed in the callus with trabectedin treatment alone. This study reports on the key involvement of macrophages during stress fracture healing. Given these observed outcomes on macrophage physiology and bone healing, these findings may be important for patients actively receiving either of these FDA-approved therapeutics.

Matrix/mineral ratio and domain size variation with bone tissue age: A photothermal infrared study.

Atomic force microscopy-infrared spectroscopy (AFM-IR) and optical photothermal infrared spectroscopy (O-PTIR), which feature spectroscopic imaging spatial resolution down to âˆ¼ 50 nm and âˆ¼ 500 nm, respectively, were employed to characterize the nano- to microscale chemical compositional changes in bone. Since these changes are known to be age dependent, fluorescently labelled bone samples were employed. The average matrix/mineral ratio values decrease as the bone tissue matures as measured by both AFM-IR and O-PTIR, which agrees with previously published FTIR and Raman spectroscopy results. IR ratio maps obtained by AFM-IR reveal variation in matrix/mineral ratio-generating micron-scale bands running parallel to the bone surface as well as smaller domains within these bands ranging from âˆ¼ 50 to 700 nm in size, which is consistent with the previously published length scale of nanomechanical heterogeneity. The matrix/mineral changes do not exhibit a smooth gradient with tissue age. Rather, the matrix/mineral transition occurs sharply within the length scale of 100-200 nm. O-PTIR also reveals matrix/mineral band domains running parallel to the bone surface, resulting in waves of matrix/mineral ratios progressing from the youngest to most mature tissue. Both AFM-IR and O-PTIR show a greater variation in matrix/mineral ratio value for younger tissue as compared to older tissue. Together, this data confirms O-PTIR and AFM-IR as techniques that visualize bulk spectroscopic data consistent with higher-order imaging techniques such as Raman and FTIR, while revealing novel insight into how mineralization patterns vary as bone tissue ages.

Divergent effects of peripheral and global neuropeptide Y deletion.

OBJECTIVES: Neuropeptide Y (NPY) is involved in the coordination of bone mass and adiposity. However, multiple NPY sources exist and their individual contribution to the skeleton and adiposity not known. The objectives of our study were to evaluate the effects of peripheral mesenchymal derived NPY to the skeleton and adiposity and to compare them to the global NPYKO model. METHODS: To study the role of mesenchymal-derived NPY, we crossed conditional NPY (NPYfl/fl) mice with Prx1cre to generate PrxNPYKO mice. The bone phenotype was assessed using micro-CT. The skeletal phenotype of PrxNPYKO mice was subsequently compared to global NPYKO model. We evaluated body weight, adiposity and functionally assessed the feeding response of NPY neurons to determine whether central NPY signaling was altered by Prx1cre. RESULTS: We identified the increase in cortical parameters in PrxNPYKO mice with no changes to cancellous bone. This was the opposite phenotype to global NPYKO mice generated from the same conditional allele. Male NPYKOmice have increased adiposity, while PrxNPYKO mice showed no difference, demonstrating that local mesenchymal-derived NPY does not influence adiposity. CONCLUSION: NPY mediates both positive and negative effects on bone mass via separate regulatory pathways. Deletion of mesenchymal-derived NPY had a positive effect on bone mass.

Modulation of Notch1 signaling regulates bone fracture healing.

Fracture healing involves interactions of different cell types, driven by various growth factors, and signaling cascades. Periosteal mesenchymal progenitor cells give rise to the majority of osteoblasts and chondrocytes in a fracture callus. Notch signaling has emerged as an important regulator of skeletal cell proliferation and differentiation. We investigated the effects of Notch signaling during the fracture healing process. Increased Notch signaling in osteochondroprogenitor cells driven by overexpression of Notch1 intracellular domain (NICD1) (αSMACreERT2 mice crossed with Rosa-NICD1) during fracture resulted in less cartilage, more mineralized callus tissue, and stronger and stiffer bones after 3 weeks. Periosteal cells overexpressing NICD1 showed increased proliferation and migration in vitro. In vivo data confirmed that increased Notch1 signaling caused expansion of alpha-smooth muscle actin (αSMA)-positive cells and their progeny including αSMA-derived osteoblasts in the callus without affecting osteoclast numbers. In contrast, anti-NRR1 antibody treatment to inhibit Notch1 signaling resulted in increased callus cartilage area, reduced callus bone mass, and reduced biomechanical strength. Our study shows a positive effect of induced Notch1 signaling on the fracture healing process, suggesting that stimulating the Notch pathway could be beneficial for fracture repair.

Engraftment of skeletal progenitor cells by bone-directed transplantation improves osteogenesis imperfecta murine bone phenotype.

Osteogenesis imperfecta (OI) is a genetic disorder most commonly caused by mutations associated with type I collagen, resulting in a defective collagen bone matrix. Current treatments for OI focus on pharmaceutical strategies to increase the amount of defective bone matrix, but do not address the underlying collagen defect. Introducing healthy donor stem cells that differentiate into osteoblasts producing normal collagen in OI patients has the potential to increase bone mass and correct the mutant collagen matrix. In this study, donor bone marrow stromal cells (BMSCs, also known as bone marrow mesenchymal stem cells) expressing both αSMACreERT2/Ai9 progenitor reporter and osteoblast reporter Col2.3GFP were locally transplanted into the femur of OI murine (OIM) mice. One month post-transplantation, 18% of the endosteal surface was lined by donor Col2.3GFP expressing osteoblasts indicating robust engraftment. Long-term engraftment in the marrow was observed 3 and 6 months post-transplantation. The presence of Col1a2-expressing donor cell-derived cortical bone matrix was detected in transplanted OIM femurs. Local transplantation of BMSCs increased cortical thickness (+12%), the polar moment of inertia (+14%), bone strength (+30%), and stiffness (+30%) 3 months post-transplantation. Engrafted cells expressed progenitor markers CD51 and Sca-1 up to 3 months post-transplantation. Most importantly, 3 months post-transplantation donor cells maintained the ability to differentiate into Col2.3GFP+ osteoblasts in vitro, and in vivo following secondary transplantation into OIM animals. Locally transplanted BMSCs can improve cortical structure and strength, and persist as continued source of osteoblast progenitors in the OIM mouse for at least 6 months.

Skeletal phenotype of the neuropeptide Y knockout mouse.

Neuropeptide Y (NPY) is involved in multiple processes such as behavior, energy and bone metabolism. Previous studies have relied on global NPY depletion to examine its effects on bone. However, this approach is unable to distinguish the central or local source of NPY influencing bone. Our aim was to identify which cells within the skeleton express Npy and establish a model that will enable us to differentiate effects of NPY derived from different cell types. We have generated the NPY floxed (NPYflox) mice using CRISPR technology. By crossing the NPYflox mice with Hypoxanthine Phosphoribosyltransferase 1 (Hprt)-cre to generate a global knockout, we were able to validate and confirm loss of Npy transcript and protein in our global NPYKO. Global deletion of NPY results in a smaller femoral cortical cross-sectional area (-12%) and reduced bone strength (-18%) in male mice. In vitro, NPY-deficient bone marrow stromal cells (BMSCs) showed increase in osteogenic differentiation detected by increases in alkaline phosphatase staining and bone sialoprotein and osteocalcin expression. Despite both sexes presenting with increased adiposity, female mice had no alterations in bone mass, suggesting that NPY may have sex-specific effects on bone. In this study we identified Npy expression in the skeleton and examined the effect of global NPY depletion to bone mass. The differential impact of NPY deletion in cortical and cancellous compartments along with differences in phenotypes between in vitro and in vivo, highlights the complex nature of NPY signaling, indicative of distinct sources that can be dissected in the future using this NPYflox model.

Inflammatory bone loss associated with MFG-E8 deficiency is rescued by teriparatide.

A prolonged increase in proinflammatory cytokines is associated with osteoporotic and autoimmune bone loss and, conversely, anti-inflammatory pathways are associated with protection against bone loss. Milk fat globule-epidermal growth factor (MFG-E)-8 is a glycoprotein that is proresolving, regulates apoptotic cell clearance, and has been linked to autoimmune disease and skeletal homeostasis. The role of MFG-E8 in the young vs. adult skeleton was determined in mice deficient in MFG-E8 (KO). In vivo, trabecular bone was similar in MFG-E8KO and wild-type (WT) mice at 6 and 16 wk, whereas 22 wk adult MFG-E8KO mice displayed significantly reduced trabecular BV/TV. The number of osteoclasts per bone surface was increased in 22-wk MFG-E8 KO vs. WT mice, and recombinant murine MFG-E8 decreased the number and size of osteoclasts in vitro. Adult MFG-E8KO spleen weight:body weight was increased compared with WT, and flow cytometric analysis showed significantly increased myeloid-derived suppressor cells (CD11bhiGR-1+) and neutrophils (CD11bhiLy6G+) in MFG-E8KO bone marrow, suggesting an inflammatory phenotype. PTH-treated MFG-E8KO mice showed a greater anabolic response (+124% BV/TV) than observed in PTH-treated WT mice (+64% BV/TV). These data give insight into the role of MFG-E8 in the adult skeleton and suggest that anabolic PTH may be a valuable therapeutic approach for autoimmune-associated skeletal disease.-Michalski, M. N., Seydel, A. L., Siismets, E. M., Zweifler, L. E., Koh, A. J., Sinder, B. P., Aguirre, J. I., Atabai, K., Roca, H., McCauley, L. K. Inflammatory bone loss associated with MFG-E8 deficiency is rescued by teriparatide.

Bone Mass Is Compromised by the Chemotherapeutic Trabectedin in Association With Effects on Osteoblasts and Macrophage Efferocytosis.

Macrophages have established roles supporting bone formation. Despite their professional phagocytic nature, the role of macrophage phagocytosis in bone homeostasis is not well understood. Interestingly, apoptosis is a pivotal feature of cellular regulation and the primary fate of osteoblasts is apoptosis. Efferocytosis (phagocytosis of apoptotic cells) is a key physiologic process for the homeostasis of many tissues, and is associated with expression of osteoinductive factors. To test effects of macrophage depletion and compromised phagocytosis on bone, 16-week-old male C57BL/6J mice were treated with trabectedin-a chemotherapeutic with established anti-macrophage effects. Trabectedin treatment reduced F4/80+ and CD68+ macrophages in the bone marrow as assessed by flow cytometry, osteal macrophages near the bone surface, and macrophage viability in vitro. Trabectedin treatment significantly reduced marrow gene expression of key phagocytic factors (Mfge8, Mrc1), and macrophages from treated mice had a reduced ability to phagocytose apoptotic mimicry beads. Macrophages cultured in vitro and treated with trabectedin displayed reduced efferocytosis of apoptotic osteoblasts. Moreover, efferocytosis increased macrophage osteoinductive TGF-ß production and this increase was inhibited by trabectedin. Long-term (6-week) treatment of 16-week-old C57BL/6J mice with trabectedin significantly reduced trabecular BV/TV and cortical BMD. Although trabectedin reduced osteoclast numbers in vitro, osteoclast surface in vivo was not altered. Trabectedin treatment reduced serum P1NP as well as MS/BS and BFR/BS, and inhibited mineralization and Runx2 gene expression of osteoblast cultures. Finally, intermittent PTH 1-34 (iPTH) treatment was administered in combination with trabectedin, and iPTH increased trabecular bone volume fraction (BV/TV) in trabectedin-treated mice. Collectively, the data support a model whereby trabectedin significantly reduces bone mass due to compromised macrophages and efferocytosis, but also due to direct effects on osteoblasts. This data has immediate clinical relevance in light of increasing use of trabectedin in oncology. © 2017 American Society for Bone and Mineral Research.

Single dose of bisphosphonate preserves gains in bone mass following cessation of sclerostin antibody in Brtl/+ osteogenesis imperfecta model.

Sclerostin antibody has demonstrated a bone-forming effect in pre-clinical models of osteogenesis imperfecta, where mutations in collagen or collagen-associated proteins often result in high bone fragility in pediatric patients. Cessation studies in osteoporotic patients have demonstrated that sclerostin antibody, like intermittent PTH treatment, requires sequential anti-resorptive therapy to preserve the anabolic effects in adult populations. However, the persistence of anabolic gains from either drug has not been explored clinically in OI, or in any animal model. To determine whether cessation of sclerostin antibody therapy in a growing OI skeleton requires sequential anti-resorptive treatment to preserve anabolic gains in bone mass, we treated 3week old Brtl/+ and wild type mice for 5weeks with SclAb, and then withdrew treatment for an additional 6weeks. Trabecular bone loss was evident following cessation, but was preserved in a dose-dependent manner with single administration of pamidronate at the time of cessation. In vivo longitudinal near-infrared optical imaging of cathepsin K activation in the proximal tibia suggests an anti-resorptive effect of both SclAb and pamidronate which is reversed after three weeks of cessation. Cortical bone was considerably less susceptible to cessation effects, and showed no structural or functional deficits in the absence of pamidronate during this cessation period. In conclusion, while SclAb induces a considerable anabolic gain in the rapidly growing Brtl/+ murine model of OI, a single sequential dose of antiresorptive drug is required to maintain bone mass at trabecular sites for 6weeks following cessation.

Effect of anti-sclerostin therapy and osteogenesis imperfecta on tissue-level properties in growing and adult mice while controlling for tissue age.

Bone composition and biomechanics at the tissue-level are important contributors to whole bone strength. Sclerostin antibody (Scl-Ab) is a candidate anabolic therapy for the treatment of osteoporosis that increases bone formation, bone mass, and bone strength in animal studies, but its effect on bone quality at the tissue-level has received little attention. Pre-clinical studies of Scl-Ab have recently expanded to include diseases with altered collagen and material properties such as osteogenesis imperfecta (OI). The purpose of this study was to investigate the role of Scl-Ab on bone quality by determining bone material composition and tissue-level mechanical properties in normal wild type (WT) tissue, as well as mice with a typical OI Gly➔Cys mutation (Brtl/+) in type I collagen. Rapidly growing (3-week-old) and adult (6-month-old) WT and Brtl/+ mice were treated for 5weeks with Scl-Ab. Fluorescent guided tissue-level bone composition analysis (Raman spectroscopy) and biomechanical testing (nanoindentation) were performed at multiple tissue ages. Scl-Ab increased mineral to matrix in adult WT and Brtl/+ at tissue ages of 2-4wks. However, no treatment related changes were observed in mineral to matrix levels at mid-cortex, and elastic modulus was not altered by Scl-Ab at any tissue age. Increased mineral-to-matrix was phenotypically observed in adult Brtl/+ OI mice (at tissue ages>3wks) and rapidly growing Brtl/+ (at tissue ages>4wks) mice compared to WT. At identical tissue ages defined by fluorescent labels, adult mice had generally lower mineral to matrix ratios and a greater elastic modulus than rapidly growing mice, demonstrating that bone matrix quality can be influenced by animal age and tissue age alike. In summary, these data suggest that Scl-Ab alters the matrix chemistry of newly formed bone while not affecting the elastic modulus, induces similar changes between Brtl/+ and WT mice, and provides new insight into the interaction between tissue age and animal age on bone quality.

Macrophages: Their Emerging Roles in Bone.

Macrophages are present in nearly all tissues and are critical for development, homeostasis, and regeneration. Resident tissue macrophages of bone, termed osteal macrophages, are recently classified myeloid cells that are distinct from osteoclasts. Osteal macrophages are located immediately adjacent to osteoblasts, regulate bone formation, and play diverse roles in skeletal homeostasis. Genetic or pharmacological modulation of macrophages in vivo results in significant bone phenotypes, and these phenotypes depend on which macrophage subsets are altered. Macrophages are also key mediators of osseous wound healing and fracture repair, with distinct roles at various stages of the repair process. A central function of macrophages is their phagocytic ability. Each day, billions of cells die in the body and efferocytosis (phagocytosis of apoptotic cells) is a critical process in both clearing dead cells and recruitment of replacement progenitor cells to maintain homeostasis. Recent data suggest a role for efferocytosis in bone biology and these new mechanisms are outlined. Finally, although macrophages have an established role in primary tumors, emerging evidence suggests that macrophages in bone support cancers which preferentially metastasize to the skeleton. Collectively, this developing area of osteoimmunology raises new questions and promises to provide novel insights into pathophysiologic conditions as well as therapeutic and regenerative approaches vital for skeletal health.

Rapidly growing Brtl/+ mouse model of osteogenesis imperfecta improves bone mass and strength with sclerostin antibody treatment.

Osteogenesis imperfecta (OI) is a heritable collagen-related bone dysplasia, characterized by brittle bones with increased fracture risk that presents most severely in children. Anti-resorptive bisphosphonates are frequently used to treat pediatric OI and controlled clinical trials have shown that bisphosphonate therapy improves vertebral outcomes but has little benefit on long bone fracture rate. New treatments which increase bone mass throughout the pediatric OI skeleton would be beneficial. Sclerostin antibody (Scl-Ab) is a potential candidate anabolic therapy for pediatric OI and functions by stimulating osteoblastic bone formation via the canonical Wnt signaling pathway. To explore the effect of Scl-Ab on the rapidly growing OI skeleton, we treated rapidly growing 3week old Brtl/+ mice, harboring a typical heterozygous OI-causing Gly→Cys substitution on col1a1, for 5weeks with Scl-Ab. Scl-Ab had anabolic effects in Brtl/+ and led to new cortical bone formation and increased cortical bone mass. This anabolic action resulted in improved mechanical strength to WT Veh levels without altering the underlying brittle nature of the material. While Scl-Ab was anabolic in trabecular bone of the distal femur in both genotypes, the effect was less strong in these rapidly growing Brtl/+ mice compared to WT. In conclusion, Scl-Ab was able to stimulate bone formation in a rapidly growing Brtl/+ murine model of OI, and represents a potential new therapy to improve bone mass and reduce fracture risk in pediatric OI.

Impact of proteoglycan-4 and parathyroid hormone on articular cartilage.

Proteoglycan-4 (Prg4) protects synovial joints from arthropathic changes by mechanisms that are incompletely understood. Parathyroid hormone (PTH), known for its anabolic actions in bone, increases Prg4 expression and has been reported to inhibit articular cartilage degeneration in arthropathic joints. To investigate the effect of Prg4 and PTH on articular cartilage, 16-week-old Prg4 mutant and wild-type mice were treated with intermittent PTH (1-34) or vehicle control daily for six weeks. Analyses included histology of the knee joint, micro-CT of the distal femur, and serum biochemical analysis of type II collagen fragments (CTX-II). Compared to wild-type littermates, Prg4 mutant mice had an acellular layer of material lining the surfaces of the articular cartilage and menisci, increased articular cartilage degradation, increased serum CTX-II concentrations, decreased articular chondrocyte apoptosis, increased synovium SDF-1 expression, and irregularly contoured subchondral bone. PTH-treated Prg4 mutant mice developed a secondary deposit overlaying the acellular layer of material lining the joint surfaces, but PTH-treatment did not alter signs of articular cartilage degeneration in Prg4 mutant mice. The increased joint SDF-1 levels and irregular subchondral bone found in Prg4 mutant mice introduce novel candidate mechanisms by which Prg4 protects articular cartilage.

The effects of zoledronic acid in the bone and vasculature support of hematopoietic stem cell niches.

Hematopoietic stem cells (HSC) are maintained in a tightly regulated bone microenvironment constituted by a rich milieu of cells. Bone cells such as osteoblasts are associated with niche maintenance as regulators of the endosteal microenvironment. Bone remodeling also plays a role in HSC mobilization although it is poorly defined. The effects of zoledronic acid (ZA), a potent bisphosphonate that inhibits bone resorption, were investigated on bone marrow cell populations focusing on HSCs, and the endosteal and vascular niches in bone. ZA treatment significantly increased bone volume and HSCs in both young and adult mice (4 week and 4 month old, respectively). ZA increased vessel numbers with no overall change in vascular volume in bones of young and had no effect on vasculature in adult mice. Since both young and adult mice had increased HSCs and bone mass with differing vasculature responses, this suggests that ZA indirectly supports HSCs via the osteoblastic niche and not the vascular niche. Additionally, gene expression in Lin- cells demonstrated increased expression of self-renewal-related genes Bmi1 and Ink4a suggesting a role of ZA in the modulation of cell commitment and differentiation toward a long-term self-renewing cell. Genes that support the osteoblastic niche, BMP2 and BMP6 were also augmented in ZA treated mice. In conclusion, ZA-induced HSC expansion occurs independent of the vascular niche via indirect modulation of the osteoblastic niche.

Sclerostin antibody improves skeletal parameters in a Brtl/+ mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a genetic bone dysplasia characterized by osteopenia and easy susceptibility to fracture. Symptoms are most prominent during childhood. Although antiresorptive bisphosphonates have been widely used to treat pediatric OI, controlled trials show improved vertebral parameters but equivocal effects on long-bone fracture rates. New treatments for OI are needed to increase bone mass throughout the skeleton. Sclerostin antibody (Scl-Ab) therapy is potently anabolic in the skeleton by stimulating osteoblasts via the canonical wnt signaling pathway, and may be beneficial for treating OI. In this study, Scl-Ab therapy was investigated in mice heterozygous for a typical OI-causing Gly→Cys substitution in col1a1. Two weeks of Scl-Ab successfully stimulated osteoblast bone formation in a knock-in model for moderately severe OI (Brtl/+) and in WT mice, leading to improved bone mass and reduced long-bone fragility. Image-guided nanoindentation revealed no alteration in local tissue mineralization dynamics with Scl-Ab. These results contrast with previous findings of antiresorptive efficacy in OI both in mechanism and potency of effects on fragility. In conclusion, short-term Scl-Ab was successfully anabolic in osteoblasts harboring a typical OI-causing collagen mutation and represents a potential new therapy to improve bone mass and reduce fractures in pediatric OI.

Proteoglycan 4: a dynamic regulator of skeletogenesis and parathyroid hormone skeletal anabolism.

Proteoglycan 4 (Prg4), known for its lubricating and protective actions in joints, is a strong candidate regulator of skeletal homeostasis and parathyroid hormone (PTH) anabolism. Prg4 is a PTH-responsive gene in bone and liver. Prg4 null mutant mice were used to investigate the impact of proteoglycan 4 on skeletal development, remodeling, and PTH anabolic actions. Young Prg4 mutant and wild-type mice were administered intermittent PTH(1-34) or vehicle daily from 4 to 21 days. Young Prg4 mutant mice had decreased growth plate hypertrophic zones, trabecular bone, and serum bone formation markers versus wild-type mice, but responded with a similar anabolic response to PTH. Adult Prg4 mutant and wild-type mice were administered intermittent PTH(1-34) or vehicle daily from 16 to 22 weeks. Adult Prg4 mutant mice had decreased trabecular and cortical bone, and blunted PTH-mediated increases in bone mass. Joint range of motion and animal mobility were lower in adult Prg4 mutant versus wild-type mice. Adult Prg4 mutant mice had decreased marrow and liver fibroblast growth factor 2 (FGF-2) mRNA and reduced serum FGF-2, which were normalized by PTH. A single dose of PTH decreased the PTH/PTHrP receptor (PPR), and increased Prg4 and FGF-2 to a similar extent in liver and bone. Proteoglycan 4 supports endochondral bone formation and the attainment of peak trabecular bone mass, and appears to support skeletal homeostasis indirectly by protecting joint function. Bone- and liver-derived FGF-2 likely regulate proteoglycan 4 actions supporting trabeculae formation. Blunted PTH anabolic responses in adult Prg4 mutant mice are associated with altered biomechanical impact secondary to joint failure.