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Abstract To determine the diagnostic yield of cone beam computed tomography (CBCT) compared with 3 T magnetic resonance imaging (MRI) for the evaluation of subchondral insufficiency fractures of the knee. Consecutive patients with subchondral insufficiency fractures of the knee examined by 3 T MRI and CBCT of the femoral condyles were reviewed. Two experienced raters graded the lesion severity on 3 T MRI and CBCT images: grade 1: no signs of a subchondral bone lesion; grade 2: subchondral trabecular fracture or cystic changes, but without infraction of the subchondral bone plate; grade 3: collapse of the subchondral bone plate. Ratings were repeated after six weeks to determine reliability. In addition, the bone lesion size was measured as elliptical area (mm2) and compared between CBCT and T1-weighted MRI sequences. Among 30 patients included (43.3% women; mean age: 60.9 ± 12.8 years; body mass index (BMI) 29.0 ± 12.8 kg/m2), the medial femoral condyle was affected in 21/30 patients (70%). The grading of subchondral lesions between MRI and CBCT did not match in 12 cases (40%). Based on MRI images, an underestimation (i.e., undergrading) compared with CBCT was observed in nine cases (30%), whereas overgrading occurred in three cases (10%). Compared to CBCT, routine T1-weighted 3 T sequences significantly overestimated osseus defect zones in sagittal (84.7 ± 68.9 mm2 vs. 35.9 ± 38.2 mm2, p

Bone is a dynamic tissue that is constantly remodeled throughout adult life. Recently, it has been shown that bone turnover decreases shortly after food consumption. This process has been linked to the fermentation of non-digestible food ingredients such as inulin by gut microbes, which results in the production of the short-chain fatty acids (SCFAs) acetate, propionate and butyrate. SCFAs exert various metabolic functions, which in part can be explained by activation of G protein-coupled receptors (Gpr) 41 and 43. However, the potential relevance of a SCFA-Gpr41/43 signaling axis for bone metabolism has not been established. The aim of our study is to investigate the role of Gpr41/43 in bone metabolism and osteogenic differentiation of mesenchymal stem cells. For this purpose, we analyzed the skeletal phenotype of wild type controls (WT) and Gpr41/43 double knockout (Gpr41/43 dKO) mice fed either a chow or an inulin-enriched diet. In addition, we isolated bone marrow derived mesenchymal stem cells from WT and Gpr41/43 dKO mice and differentiated them into osteoblasts in the absence or presence of acetate. MicroCT scanning of femoral bones of Gpr41/43 dKO mice revealed a significant increase of trabecular bone volume and trabecular compared to WT controls. Treatment of WT bone marrow-derived osteoblasts with acetate resulted in decreased mineralization and substantial downregulation of bone formation markers such as Phex, Ptgs2 and Col1a1. Notably, this effect was strongly attenuated in differentiated osteoblasts lacking Gpr41/43. Inversely, acetate supplementation resulted in higher levels of adipocyte marker genes including Pparg, Lpl and Adipoq in bone marrow-derived cells from WT mice, an effect blunted in differentiated cells isolated from Gpr41/43 dKO mice. Overall, these data indicate that acetate regulates bone architecture via SCFA-Gpr41/43 signaling by modulating the osteogenic versus adipogenic differentiation of mesenchymal stem cells.

Abstract Piezo proteins are mechanically activated ion channels, which are required for mechanosensing functions in a variety of cell types. While we and others have previously demonstrated that the expression of Piezo1 in osteoblast lineage cells is essential for bone-anabolic processes, there was only suggestive evidence indicating a role of Piezo1 and/or Piezo2 in cartilage. Here we addressed the question if and how chondrocyte expression of the mechanosensitive proteins Piezo1 or Piezo2 controls physiological endochondral ossification and pathological osteoarthritis (OA) development. Mice with chondrocyte-specific inactivation of Piezo1 (Piezo1 Col2a1Cre ), but not of Piezo2, developed a near absence of trabecular bone below the chondrogenic growth plate postnatally. Moreover, all Piezo1 Col2a1Cre animals displayed multiple fractures of rib bones at 7 days of age, which were located close to the growth plates. While skeletal growth was only mildly affected in these mice, OA pathologies were markedly less pronounced compared to littermate controls at 60 weeks of age. Likewise, when OA was induced by anterior cruciate ligament transection, only the chondrocyte inactivation of Piezo1, not of Piezo2, resulted in attenuated articular cartilage degeneration. Importantly, osteophyte formation and maturation were also reduced in Piezo1 Col2a1Cre mice. We further observed increased Piezo1 protein abundance in cartilaginous zones of human osteophytes. Finally, we identified Ptgs2 and Ccn2 as potentially relevant Piezo1 downstream genes in chondrocytes. Collectively, our data do not only demonstrate that Piezo1 is a critical regulator of physiological and pathological endochondral ossification processes, but also suggest that Piezo1 antagonists may be established as a novel approach to limit osteophyte formation in OA.

Abstract Mechanosensory ion channels are proteins that are sensitive to mechanical forces. They are found in tissues throughout the body and play an important role in bone remodeling by sensing changes in mechanical stress and transmitting signals to bone-forming cells. Orthodontic tooth movement (OTM) is a prime example of mechanically induced bone remodeling. However, the cell-specific role of the ion channels Piezo1 and Piezo2 in OTM has not been investigated yet. Here we first identify the expression of PIEZO1/2 in the dentoalveolar hard tissues. Results showed that PIEZO1 was expressed in odontoblasts, osteoblasts, and osteocytes, while PIEZO2 was localized in odontoblasts and cementoblasts. We therefore used a Piezo1 floxed/floxed mouse model in combination with Dmp1 cre to inactivate Piezo1 in mature osteoblasts/cementoblasts, osteocytes/cementocytes, and odontoblasts. Inactivation of Piezo1 in these cells did not affect the overall morphology of the skull but caused significant bone loss in the craniofacial skeleton. Histological analysis revealed a significantly increased number of osteoclasts in Piezo1 floxed/floxed ;Dmp1 cre mice, while osteoblasts were not affected. Despite this increased number of osteoclasts, orthodontic tooth movement was not altered in these mice. Our results suggest that despite Piezo1 being crucial for osteoclast function, it may be dispensable for mechanical sensing of bone remodeling.

Abstract Across species, the skeletal system shares mutual functions, including the protection of inner organs, structural basis for locomotion, and acting as an endocrine organ, thus being of pivotal importance for survival. However, insights into skeletal characteristics of marine mammals are limited, especially in the growing skeleton. Harbor seals (Phoca vitulina) are common marine mammals in the North and Baltic Seas and are suitable indicators of the condition of their ecosystem. Here, we analyzed whole-body areal bone mineral density (aBMD) by dual-energy X-ray absorptiometry (DXA) and lumbar vertebrae by high-resolution peripheral quantitative computed tomography (HR-pQCT) in neonate, juvenile, and adult harbor seals. Along skeletal growth, an increase in two-dimensional aBMD by DXA was paralleled by three-dimensional volumetric BMD by HR-pQCT, which could be attributed to an increasing trabecular thickness while trabecular number remained constant. Strong associations were observed between body dimensions (weight and length) and aBMD and trabecular microarchitecture (R2 = 0.71–0.92, all p

Abstract Pregnancy‐ and lactation‐associated osteoporosis (PLO) is a rare but clinically highly relevant condition, characterized by reduced bone mineral density (BMD) and acute onset of severe pain due to symptomatic bone marrow edema of the hip or vertebral and/or insufficiency fractures, among others. Previous reports showed a high frequency of hereditary bone disorders unmasked by PLO, predisposing for more severe forms. To date, no data on the risk for additional fractures during subsequent pregnancy in women with PLO and genetic bone disorder have been available. To address this question, we retrospectively analyzed the clinical, biochemical, and densitometric course of three women with a history of PLO and detected variants in WNT1 or LRP5 and subsequent pregnancies. Calcium homeostasis and bone turnover were optimized by basic treatment, and timely initiation of weaning was recommended. Teriparatide treatment for 12 months under strict contraception was initiated in one woman after the diagnosis of PLO. In none of the women did additional fractures or symptomatic bone marrow edemas occur, and BMD by dual‐energy X‐ray absorptiometry as bone microarchitecture by high‐resolution peripheral quantitative computed tomography remained stable. In conclusion, this report expands the understanding of this rare but severe condition and helps to improve clinical counseling and management. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Abstract Primary sclerosing cholangitis (PSC) is an idiopathic cholestatic liver disease characterized by chronic inflammation and progressive fibrosis of intra- and extrahepatic bile ducts. Osteoporosis is a frequent comorbidity in PSC, and we could previously demonstrate that IL17-dependent activation of bone resorption is the predominant driver of bone loss in PSC. Since we additionally observed an unexpected heterogeneity of bone mineral density in our cohort of 238 PSC patients, the present study focused on a comparative analysis of affected individuals with diagnosed osteoporosis (PSCOPO, n = 10) or high bone mass (PSCHBM, n = 7). The two groups were not distinguishable by various baseline characteristics, including liver fibrosis or serum parameters for hepatic function. In contrast, quantification of serum bile acid concentrations identified significant increases in the PSCOPO group, including glycoursodeoxycholic acid (GUDCA), an exogenous bile acid administered to both patient groups. Although cell culture experiments did not support the hypothesis that an increase in circulating bile levels is a primary cause of PSC-associated osteoporosis, the remarkable differences of endogenous bile acids and GUDCA in the serum of PSCOPO patients strongly suggest a yet unknown impairment of biliary metabolism and/or hepatic bile acid clearance in this patient subgroup, which is independent of liver fibrosis.

Abstract Background It is well known that skeletal integrity is influenced by the musculature. Poor muscle strength (i.e. sarcopenia) is considered a major predictor of fragility fractures. While this observation appears particularly relevant for older women with increased risk of osteoporosis, there has been no comprehensive investigation to determine the influence of muscle performance on compartment‐specific bone microarchitecture in multiple body regions. Methods We retrospectively analysed data from different muscle performance and bone microarchitecture assessments in 230 women (aged 21 to 87 years) at high risk of osteoporosis. Muscle performance tests included grip strength and chair rising test (CRT) combined with mechanography. Balance was determined by Romberg posturography. Areal bone mineral density (BMD) was measured by dual‐energy X‐ray absorptiometry (DXA) at the hip and lumbar spine. Compartment‐specific volumetric BMD, microarchitecture, and geometry were assessed by second‐generation high‐resolution peripheral quantitative computed tomography (HR‐pQCT) at multiple skeletal sites (distal radius, tibia, and fibula). Regression models were applied to test for interactions between muscle and bone parameters. Subgroups were defined to compare women with osteoporosis and osteosarcopenia regarding BMD and microarchitecture. Results While osteoporosis was diagnosed in 115/230 (50.0%) women, sarcopenia was detected in 38/230 (16.5%). Positive associations of both grip strength and CRT maximum force with cortical geometric and microarchitectural parameters were detected at all measured sites, with the strongest effect applying to CRT maximum force and tibial parameters (e.g. tibial cortical area R2 = 0.36, P

Abstract Intermittent injections of parathyroid hormone (iPTH) are applied clinically to stimulate bone formation by osteoblasts, although continuous elevation of parathyroid hormone (PTH) primarily results in increased bone resorption. Here, we identified Calca, encoding the sepsis biomarker procalcitonin (ProCT), as a novel target gene of PTH in murine osteoblasts that inhibits osteoclast formation. During iPTH treatment, mice lacking ProCT develop increased bone resorption with excessive osteoclast formation in both the long bones and axial skeleton. Mechanistically, ProCT inhibits the expression of key mediators involved in the recruitment of macrophages, representing osteoclast precursors. Accordingly, ProCT arrests macrophage migration and causes inhibition of early but not late osteoclastogenesis. In conclusion, our results reveal a potential role of osteoblast-derived ProCT in the bone microenvironment that is required to limit bone resorption during iPTH.

Abstract The recent identification of homozygous WNT1 mutations in individuals with osteogenesis imperfecta type XV (OI-XV) has suggested that WNT1 is a key ligand promoting the differentiation and function of bone-forming osteoblasts. Although such an influence was supported by subsequent studies, a mouse model of OI-XV remained to be established. Therefore, we introduced a previously identified disease-causing mutation (G177C) into the murine Wnt1 gene. Homozygous Wnt1 G177C/G177C mice were viable and did not display defects in brain development, but the majority of 24-week-old Wnt1 G177C/G177C mice had skeletal fractures. This increased bone fragility was not fully explained by reduced bone mass but also by impaired bone matrix quality. Importantly, the homozygous presence of the G177C mutation did not interfere with the osteoanabolic influence of either parathyroid hormone injection or activating mutation of LRP5, the latter mimicking the effect of sclerostin neutralization. Finally, transcriptomic analyses revealed that short-term administration of WNT1 to osteogenic cells induced not only the expression of canonical WNT signaling targets but also the expression of genes encoding extracellular matrix modifiers. Taken together, our data demonstrate that regulating bone matrix quality is a primary function of WNT1. They further suggest that individuals with WNT1 mutations should profit from existing osteoanabolic therapies.

Abstract Background Tissue-nonspecific alkaline phosphatase (TNSALP) encoded by the ALPL gene is of particular importance for bone mineralization. Mutation in the ALPL gene can lead to persistent low ALP activity resulting in the rare disease Hypophosphatasia (HPP) that is characterized by disturbed bone and dental mineralization. While severe forms are extremely rare with an estimated prevalence of 1/100.000, recent studies suggest that moderate form caused by heterozygous mutations are much more frequent with an estimated prevalence of 1/508. The purpose of this study was to estimate the prevalence of low AP levels in the population based on laboratory measurements. Methods In this study, the prevalence of low AP activity and elevated pyridoxal-5-phosphate (PLP) levels was analyzed in 6.918.126 measurements from 2011 to 2016 at a single laboratory in northern Germany. Only laboratory values of subjects older than 18 years of age were included. Only the first measurement was included, all repeated values were excluded. Results In total, 8.46% of the measurements of a total of 6.918.126 values showed a value

Mucopolysaccharidosis VI (MPS VI) is a hereditary lysosomal storage disease caused by the absence of the enzyme arylsulfatase B (ARSB). Craniofacial defects are common in MPS VI patients and manifest as abnormalities of the facial bones, teeth, and temporomandibular joints. Although enzyme replacement therapy (ERT) is the treatment of choice for MPS VI, the effects on the craniofacial and dental structures are still poorly understood. In this study, we used an Arsb-deficient mouse model (Arsbm/m) that mimics MPS VI to investigate the effects of ERT on dental and craniofacial structures and compared these results with clinical and radiological observations from three MPS VI patients. Using micro-computed tomography, we found that the craniofacial phenotype of the Arsbm/m mice was characterized by bone exostoses at the insertion points of the masseter muscles and an overall increased volume of the jaw bone. An early start of ERT (at 4 weeks of age for 20 weeks) resulted in a moderate improvement of these jaw anomalies, while a late start of ERT (at 12 weeks of age for 12 weeks) showed no effect on the craniofacial skeleton. While teeth typically developed in Arsbm/m mice, we observed a pronounced loss of tooth-bearing alveolar bone. This alveolar bone loss, which has not been described before in MPS VI, was also observed in one of the MPS VI patients. Interestingly, only an early start of ERT led to a complete normalization of the alveolar bone in Arsbm/m mice. The temporomandibular joints in Arsbm/m mice were deformed and had a porous articular surface. Histological analysis revealed a loss of physiological cartilage layering, which was also reflected in an altered proteoglycan content in the cartilage of Arsbm/m mice. These abnormalities could only be partially corrected by an early start of ERT. In conclusion, our results show that an early start of ERT in Arsbm/m mice achieves the best therapeutic effects for tooth, bone, and temporomandibular joint development. As the MPS VI mouse model in this study resembles the clinical findings in MPS VI patients, our results suggest enzyme replacement therapy should be started as early as possible.

Intact mineralization of the auditory ossicles - the smallest bones in the body - is essential for sound transmission in the middle ear, while ossicular hypomineralization is associated with conductive hearing loss. Here, we performed a high-resolution analysis of the ossicles in vitamin D receptor deficient mice (Vdr-/-), which are characterized by hypocalcemia and skeletal mineralization defects, and investigated whether local hypomineralization can be prevented by feeding a calcium-rich rescue diet (Vdr-/- res). In Vdr-/- mice fed a regular diet (Vdr-/- reg), quantitative backscattered electron imaging (qBEI) revealed an increased void volume (porosity, p0.05 vs. WT) and partial rescue of porosity and CaWidth (p=0.02 and p=0.04 vs. WT) were observed. Compared with Hyp mice, a model of X-linked hypophosphatemic rickets, Vdr-/- reg mice showed a lower osteoid volume in the ossicles (p=0.0002), but similar values in the lumbar spine. These results are consistent with later postnatal impairment of mineral homeostasis in Vdr-/- mice than in Hyp mice, underscoring the importance of intact mineral homeostasis for ossicle mineralization during development. In conclusion, we revealed a distinct phenotype of hypomineralization in the auditory ossicles of Vdr-/- mice that can be partially prevented by a rescue diet. Since a positive effect of a calcium-rich diet on ossicular mineralization was demonstrated, our results open new treatment strategies for conductive hearing loss. Future studies should investigate the impact of improved ossicular mineralization on hearing function.

Abstract Degradation at the modular head-neck interface in total hip arthroplasty (THA) is predominately expressed in the form of corrosion and fretting, potentially causing peri-prosthetic failure by adverse reactions to metal debris. This retrieval study aimed to quantify variations in stem taper surface topographies and to assess the influence on the formation of corrosion and/or fretting in titanium alloy stem tapers combined with metal and ceramic heads. Four hip stem designs (Alloclassic, CLS, Bicontact and SL-Plus) were characterized using high-resolution 3D microscopy, and corrosion and fretting were rated using the Goldberg scoring scheme. Quantification of the taper surface topographies revealed a high variability in surface characteristics between threaded stem tapers: Alloclassic and CLS tapers feature deeply threaded trapezoid-shaped profiles with thread heights over 65 µm. The sawtooth-shaped Bicontact and triangular SL-Plus taper are characterized by low thread heights below 14 µm. Significantly lower corrosion and fretting scores were observed in lightly threaded compared to deeply threaded tapers in ceramic head combinations. No significant differences in corrosion or fretting scores with thread height were found in pairings with metal heads. Understanding the relationship between stem taper surface topography and the formation of corrosion and fretting could help to improve the performance of modern THAs and lead to longer-lasting clinical results.

Abstract Severe skeletal alterations are common symptoms in patients with mucolipidosis type II (MLII), a rare lysosomal storage disorder of childhood. We have previously reported that progressive bone loss in a mouse model for MLII is caused by an increased number of bone-resorbing osteoclasts, which is accompanied by elevated expression of the cytokine interleukin-6 (IL-6) in the bone microenvironment. In the present study we addressed the question, if pharmacological blockade of IL-6 can prevent the low bone mass phenotype of MLII mice. Since the cellular IL-6 response can be mediated by either the membrane-bound (classic signaling) or the soluble IL-6 receptor (trans-signaling), we first performed cell culture assays and found that both pathways can increase osteoclastogenesis. We then crossed MLII mice with transgenic mice expressing the recombinant soluble fusion protein sgp130Fc, which represents a natural inhibitor of IL-6 trans-signaling. By undecalcified histology and bone-specific histomorphometry we found that high circulating sgp130Fc levels do not affect skeletal growth or remodeling in wild-type mice. Most importantly, blockade of IL-6 trans-signaling did neither reduce osteoclastogenesis, nor increase bone mass in MLII mice. Therefore, our data clearly demonstrate that the bone phenotype of MLII mice cannot be corrected by blocking the IL-6 trans-signaling.

Abstract Coffin–Lowry–Syndrome (CLS) is a X-linked mental retardation characterized by skeletal dysplasia and premature tooth loss. We and others have previously demonstrated that the ribosomal S6 kinase RSK2, mutated in CLS, is essential for bone and cementum formation; however, it remains to be established whether RSK2 plays also a role in mechanically induced bone remodeling during orthodontic tooth movement (OTM). We, therefore, performed OTM in wild-type (WT) mice and Rsk2-deficient mice using Nitinol tension springs that were fixed between the upper left molars and the incisors. The untreated contralateral molars served as internal controls. After 12 days of OTM, the jaws were removed and examined by micro-computed tomography (µCT), decalcified histology, and immunohistochemistry. Our analysis of the untreated teeth confirmed that the periodontal phenotype of Rsk2-deficient mice is characterized by alveolar bone loss and hypoplasia of root cementum. Quantification of OTM using µCT revealed that OTM was more than two-fold faster in Rsk2-deficient mice as compared to WT. We also observed that OTM caused alveolar bone loss and root resorptions in WT and Rsk2-deficient mice. However, quantification of these orthodontic side effects revealed no differences between WT and Rsk2-deficient mice. Taken together, Rsk2 loss-of-function accelerates OTM in mice without causing more side effects.

Harbour porpoises are under pressure from increasing human activities. This includes the detonation of ammunition that was dumped in large amounts into the sea during and after World War II. In this context, forty-two British ground mines from World War II were cleared by means of blasting in the period from 28 to 31 August 2019 by a NATO unit in the German Exclusive Economic Zone within the marine protected area of Fehmarn Belt in the Baltic Sea, Germany. Between September and November 2019, 24 harbour porpoises were found dead in the period after those clearing events along the coastline of the federal state of Schleswig-Holstein and were investigated for direct and indirect effects of blast injury. Health evaluations were conducted including examinations of the brain, the air-filled (lungs and gastrointestinal tract) and acoustic organs (melon, acoustic fat in the lower jaw, ears and their surrounding tissues). The bone structure of the tympano-periotic complexes was examined using high-resolution peripheral quantitative computed tomography (HR-pQCT). In 8/24 harbour porpoises, microfractures of the malleus, dislocation of middle ear bones, bleeding, and haemorrhages in the melon, lower jaw and peribullar acoustic fat were detected, suggesting blast injury. In addition, one bycaught animal and another porpoise with signs of blunt force trauma also showed evidence of blast injury. The cause of death of the other 14 animals varied and remained unclear in two individuals. Due to the vulnerability and the conservation status of harbour porpoise populations in the Baltic Sea, noise mitigation measures must be improved to prevent any risk of injury. The data presented here highlight the importance of systematic investigations into the acute and chronic effects of blast and acoustic trauma in harbour porpoises, improving the understanding of underwater noise effects and herewith develop effective measures to protect the population level.

Doses of irradiation above 25 ​kGy are known to cause irreversible mechanical decay in bone tissue. However, the impact of irradiation doses absorbed in a clinical setting on the mechanical properties of bone remains unclear. In daily clinical practice and research, patients and specimens are exposed to irradiation due to diagnostic imaging tools, with doses ranging from milligray to Gray. The aim of this study was to investigate the influence of irradiation at these doses ranges on the mechanical performance of bone independent of inter-individual bone quality indices.Therefore, cortical bone specimens (n ​= ​10 per group) from a selected organ donor were irradiated at doses of milligray, Gray and kilogray (graft tissue sterilization) at five different irradiation doses. Three-point bending was performed to assess mechanical properties in the study groups.Our results show a severe reduction in mechanical performance (work to fracture: 50.29 ​± ​11.49 Nmm in control, 14.73 ​± ​1.84 Nmm at 31.2 ​kGy p ​≤ ​0.05) at high irradiation doses of 31.2 ​kGy, which correspond to graft tissue sterilization or synchrotron imaging. In contrast, no reduction in mechanical properties were detected for doses below 30 ​Gy. These findings are further supported by fracture surface texture imaging (i.e. more brittle fracture textures above 31.2 ​kGy).Our findings show that high radiation doses (≥31.2 ​kGy) severely alter the mechanical properties of bone. Thus, irradiation of this order of magnitude should be taken into account when mechanical analyses are planned after irradiation. However, doses of 30 ​Gy and below, which are common for clinical and experimental imaging (e.g., radiation therapy, DVT imaging, CT imaging, HR-pQCT imaging, DXA measurements, etc.), do not alter the mechanical bending-behavior of bone.

Inactivation of the tumor suppressor p53 (encoded by the Trp53 gene) is relevant for development and growth of different cancers, including osteosarcoma, a primary bone tumor mostly affecting children and young adolescents. We have previously shown that deficiency of the ribosomal S6 kinase 2 (Rsk2) limits osteosarcoma growth in a transgenic mouse model overexpressing the proto-oncogene c-Fos. Our initial aim for the present study was to address the question, if Rsk2 deficiency would also influence osteosarcoma growth in another mouse model. For that purpose, we took advantage of Trp53fl/fl mice, which were crossed with Runx2Cre transgenic mice in order to inactivate p53 specifically in osteoblast lineage cells. However, since we unexpectedly identified Runx2Cre-mediated recombination also in the thymus, the majority of 6-month-old Trp53fl/fl;Runx2-Cre (thereafter termed Trp53Cre) animals displayed thymic lymphomas, similar to what has been described for Trp53-deficient mice. Since we did not detect osteosarcoma formation at that age, we could not follow our initial aim, but we studied the skeletal phenotype of Trp53Cre mice, with or without additional Rsk2 deficiency. Here we unexpectedly observed that Trp53Cre mice display a unique accumulation of trabecular bone in the midshaft region of the femur and the humerus, consistent with its previously established role as a negative regulator of osteoblastogenesis. Since this local bone mass increase in Trp53Cre mice was significantly reduced by Rsk2 deficiency, we isolated bone marrow cells from the different groups of mice and analyzed their behavior ex vivo. Here we observed a remarkable increase of colony formation, osteogenic differentiation and proliferation in Trp53Cre cultures, which was unaffected by Rsk2 deficiency. Our data thereby confirm a critical and tumorigenesis-independent function of p53 as a key regulator of mesenchymal cell differentiation.

Bone is remodelled constantly through a balance of bone formation and resorption. This process can be affected by various factors such as hormones, vitamins, nutrients and environmental factors, which can create an imbalance resulting in systemic or local bone alteration. The aim of the present study was to analyse the changes in bone mineral density (BMD) over time in skulls of ringed seals (Pusa hispida) from the Baltic and Greenland using museum samples. Overall, 303 skulls (102 Male, 89 Female, 112 unknown) were used for bone investigations and were divided into three periods according to collection year: before 1958 (n = 167), between 1958 and 1989 (n = 40) and after 1994 up to 2019 (n = 96). All skulls were examined by dual-energy X-ray absorptiometry to obtain the BMD. Skull BMD of the Baltic seals was positively correlated with the historical polychlorinated biphenyls (PCB) contamination having potential effects on the constitution of bones. BMD fluctuated between the three study periods (LM: p-value

Background: Impaired fracture healing represents an ongoing clinical challenge, as treatment options remain limited. Calcitonin gene-related peptide (CGRP), a neuropeptide targeted by emerging anti-migraine drugs, is also expressed in sensory nerve fibres innervating bone tissue. Method: Bone healing following a femoral osteotomy stabilized with an external fixator was analysed over 21 days in αCGRP-deficient and WT mice. Bone regeneration was evaluated by serum analysis, µCT analysis, histomorphometry and genome-wide expression analysis. Bone-marrow-derived osteoblasts and osteoclasts, as well as the CGRP antagonist olcegepant were employed for mechanistic studies. Findings: WT mice with a femoral fracture display increased CGRP serum levels. αCGRP mRNA expression after skeletal injury is exclusively induced in callus tissue, but not in other organs. On protein level, CGRP and its receptor, calcitonin receptor-like receptor (CRLR) complexing with RAMP1, are differentially expressed in the callus during bone regeneration. On the other hand, αCGRP-deficient mice display profoundly impaired bone regeneration characterised by a striking reduction in the number of bone-forming osteoblasts and a high rate of incomplete callus bridging and non-union. As assessed by genome-wide expression analysis, CGRP induces the expression of specific genes linked to ossification, bone remodeling and adipogenesis. This suggests that CGRP receptor-dependent PPARγ signaling plays a central role in fracture healing. Interpretation: This study demonstrates an essential role of αCGRP in orchestrating callus formation and identifies CGRP receptor agonism as a potential approach to stimulate bone regeneration. Moreover, as novel agents blocking CGRP or its receptor CRLR are currently introduced clinically for the treatment of migraine disorders, their potential negative impact on bone regeneration warrants clinical investigation. Funding: This work was funded by grants from the Else-Kröner-Fresenius-Stiftung (EKFS), the Deutsche Forschungsgemeinschaft (DFG), and the Berlin Institute of Health (BIH).

Mutations in the gene ANO5, encoding for the transmembrane protein Anoctamin 5 (Ano5), have been identified to cause gnathodiaphyseal dysplasia (GDD) in humans, a skeletal disorder characterized by sclerosis of tubular bones, increased fracture risk and fibro-osseous lesions of the jawbones. To better understand the pathomechanism of GDD we have generated via Crispr/CAS9 gene editing a mouse model harboring the murine equivalent (Ano5 p.T491F) of a GDD-causing ANO5 mutation identified in a previously reported patient. Skeletal phenotyping by contact radiography, μCT and undecalcified histomorphometry was performed in male mice, heterozygous and homozygous for the mutation, at the ages of 12 and 24 weeks. These mice did not display alterations of skeletal microarchitecture or mandible morphology. The results were confirmed in female mice and animals derived from a second, independent clone. Finally, no skeletal phenotype was observed in mice lacking ~40% of their Ano5 gene due to a frameshift mutation. Therefore, our results indicate that Ano5 is dispensable for bone homeostasis in mice, at least under unchallenged conditions, and that these animals may not present the most adequate model to study the physiological role of Anoctamin 5.

Abstract Background Impaired bone quality is associated with poor outcome of spinal surgery. The aim of the study was to assess the bone mineral status of patients scheduled to undergo spinal surgery and to report frequencies of bone mineral disorders. Methods We retrospectively analyzed the bone mineral status of 144 patients requiring spinal surgery including bone mineral density by dual-energy X-ray absorptiometry (DXA) as well as laboratory data with serum levels of 25-hydroxyvitamin D (25-OH-D), parathyroid hormone, calcium, bone specific alkaline phosphate, osteocalcin, and gastrin. High-resolution peripheral quantitative computed tomography (HR-pQCT) was additionally performed in a subgroup of 67 patients with T-Score below − 1.5 or history of vertebral fracture. Results Among 144 patients, 126 patients (87.5%) were older than 60 years. Mean age was 70.1 years. 42 patients (29.1%) had suffered from a vertebral compression fracture. 12 previously undiagnosed vertebral deformities were detected in 12 patients by vertebral fracture assessment (VFA). Osteoporosis was present in 39 patients (27.1%) and osteopenia in 63 patients (43.8%). Only 16 patients (11.1%) had received anti-osteoporotic therapy, while 54 patients (37.5%) had an indication for specific anti-osteoporotic therapy but had not received it yet. The majority of patients had inadequate vitamin D status (73.6%) and 34.7% of patients showed secondary hyperparathyroidism as a sign for a significant disturbed calcium homeostasis. In a subgroup of 67 patients, severe vertebral deformities were associated with stronger deficits in bone microarchitecture at the distal radius compared to the distal tibia. Conclusions This study shows that bone metabolism disorders are highly prevalent in elderly patients scheduled for spinal surgery. Vertebral deformities are associated with a predominant deterioration of bone microstructure at the distal radius. As impaired bone quality can compromise surgical outcome, we strongly recommend an evaluation of bone mineral status prior to operation and anti-osteoporotic therapy if necessary.

The bone matrix is constantly remodeled by the coordinated activities of bone-forming osteoblasts and bone-resorbing osteoclasts. Whereas type I collagen is the most abundant bone matrix protein, there are several other proteins present, some of them specifically produced by osteoblasts. In a genome-wide expression screening for osteoblast differentiation markers we have previously identified two collagen-encoding genes with unknown function in bone remodeling. Here we show that one of them, Col22a1, is predominantly expressed in bone, cultured osteoblasts, but not in osteoclasts. Based on this specific expression pattern we generated a Col22a1-deficient mouse model, which was analyzed for skeletal defects by µCT, undecalcified histology and bone-specific histomorphometry. We observed that Col22a1-deficient mice display trabecular osteopenia, accompanied by significantly increased osteoclast numbers per bone surface. In contrast, cortical bone parameters, osteoblastogenesis or bone formation were unaffected by the absence of Col22a1. Likewise, primary osteoblasts from Col22a1-deficient mice did not display a cell-autonomous defect, and they did not show altered expression of Rankl or Opg, two key regulators of osteoclastogenesis. Taken together, we provide the first evidence for a physiological function of Col22a1 in bone remodeling, although the molecular mechanisms explaining the indirect influence of Col22a1 deficiency on osteoclasts remain to be identified.

Abstract Although vertebral bone microarchitecture has been studied in various tetrapods, limited quantitative data are available on the structural and compositional changes of vertebrae in marine mammals. Whales exhibit exceptional swimming and diving behaviour, and they may not be immune to diving-associated bone pathologies. Lumbar vertebral bodies were analysed in three toothed whale species: the sperm whale (Physeter macrocephalus), orca (Orcinus orca) and harbour porpoise (Phocoena phocoena). The bone volume fraction (BV/TV) did not scale with body size, although the trabeculae were thicker, fewer in number and further apart in larger whale species than in the other two species. These parameters had a negative allometric scaling relationship with body length. In sperm whales and orcas, the analyses revealed a central ossification zone (“bone-within-bone”) with an increased BV/TV and trabecular thickness. Furthermore, a large number of empty osteocyte lacunae was observed in the sperm whales. Quantitative backscattered electron imaging showed that the lacunae were significantly smaller and less densely packed. Our results indicate that whales have a unique vertebral bone morphology with an inside-out appearance and that deep diving may result in a small number of viable osteocytes because of diving depth-related osteocyte death.

Osteoid osteoma (OO) is a benign bone tumor producing non-mineralized bone matrix (i.e., osteoid). While peritumoral edema is commonly found in OO, extensive bone marrow edema has been reported less frequently. Furthermore, the micro-morphological characteristics of the nidus and its central calcification remain unclear. In this study, a consecutive series of four patients suffering from extensive bone marrow edema triggered by intra-articular osteoid osteoma underwent clinical examination, magnetic resonance imaging (MRI) and computed tomography (CT) as well as dual-energy X-ray absorptiometry (DXA) and laboratory bone turnover analyses. The obtained resection specimens were processed by undecalcified histology and were subsequently analyzed by light microscopy and quantitative backscattered electron imaging (qBEI). We report an entity of intra-articular osteoid osteoma in the knee and foot, in which an extensive and persistent bone marrow edema syndrome masked the correct diagnosis. While metabolic bone diseases were excluded in all cases, the reassessment of the patients’ clinical history including pain characteristics (nocturnal, aspirin sensitivity) led us to perform additional CT, where the tumor was diagnosed. The micro-morphological analysis of the OO biopsies revealed that the nidus was surrounded by hyperosteoidosis, while central mineralization was detected in all cases. This mineralized area showed a significantly higher mineralization heterogeneity than the surrounding trabecular bone and more disorganized collagen fibers detected by qBEI and polarized light microscopy, respectively. Taken together, our results indicate that osteoid osteoma should be considered when persistent and extensive, peri-articular bone marrow edema is diagnosed. The central calcification that is found inside the nidus in conventional imaging was mirrored by bone matrix with a heterogeneous mineralization pattern. Keywords: Osteoid osteoma, Bone marrow edema, Joint pain, Bone tumor, MRI, qBEI

ABSTRACT The response to teriparatide has been described in very few cases of hypophosphatasia (HPP). In this cross‐sectional study, we report the prevalence of symptomatic bone marrow edema (BME) and fracture healing complications in a large cohort of childhood and adult HPP patients and discuss the results of teriparatide treatment in four cases. From 2016 to 2018, 51 patients with a diagnosis of HPP were seen at our institution. The diagnosis of HPP was established by low serum alkaline phosphatase (ALP), elevated serum pyridoxal‐5‐phosphate (PLP), at least one typical clinical symptom of HPP and supported by ALPL mutation analysis. In this study cohort, 28 (56%) and 14 (27%) patients had a history of fracture or a history of BME, respectively. Four patients, including middle‐aged to elderly women and men who all presented with persistent symptomatic BME or fracture healing complications, were treated with teriparatide. DXA was performed prior to treatment and laboratory values were measured on a regular basis during treatment. Treatment with teriparatide showed variable effects in terms of clinical and biochemical response. Although all four patients displayed a temporary increase in ALP activity, only two patients with a mild form of adult HPP and moderately increased PLP levels showed definite clinical and radiological improvement after teriparatide treatment. In conclusion, fracture healing complications and BME occur frequently in HPP patients. Teriparatide shows variable clinical and biochemical effects depending on the severity of the disease. PLP levels and the number of ALPL alleles might be good parameters to predict treatment outcomes. © 2019 The Authors. JBMR Plus Published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research

Abstract The osseous sword of a swordfish (Xiphias gladius) is specialized to incapacitate prey with stunning blows. Considering the sword's growth and maturation pattern, aging from the sword's base to the tip, while missing a mechanosensitive osteocytic network, an in‐depth understanding of its mechanical properties and bone quality is lacking. Microstructural, compositional, and nanomechanical characteristics of the bone along the sword are investigated to reveal structural mechanisms accounting for its exceptional mechanical competence. The degree of mineralization, homogeneity, and particle size increase from the base toward the tip, reflecting aging along its length. Fracture experiments reveal that crack‐growth toughness vastly decreases at the highly and homogeneously mineralized tip, suggesting the importance of aging effects. Initiation toughness, however, is unchanged suggesting that aging effects on this hierarchical level are counteracted by constant mineral/fibril interaction. In conclusion, the sword of the swordfish provides an excellent model reflecting base‐to‐tip‐wise aging of bone, as indicated by increasing mineralization and decreasing crack‐growth toughness toward the tip. The hierarchical, structural, and compositional changes along the sword reflect peculiar prerequisites needed for resisting high mechanical loads. Further studies on advanced teleosts bone tissue may help to unravel structure–function relationships of heavily loaded skeletons lacking mechanosensing cells.

Albeit osteoporosis is one of the most prevalent disorders in the aged population, treatment options stimulating the activity of bone-forming osteoblasts are still limited. We and others have previously identified sphingosine-1-phosphate (S1P) as a bone remodeling coupling factor, which is released by bone-resorbing osteoclasts to stimulate bone formation. Moreover, S1pr3, encoding one of the five known S1P receptors (S1P3), was found differentially expressed in osteoblasts, and S1P3 deficiency corrected the moderate high bone mass phenotype of a mouse model (deficient for the calcitonin receptor) with increased S1P release from osteoclasts. In the present study we addressed the question, if S1P3 deficiency would also influence the skeletal phenotype of mice lacking S1P-lyase (encoded by Sgpl1), which display markedly increased S1P levels due to insufficient degradation. Consistent with previous reports, the majority of Sgpl1-deficient mice died before or shortly after weaning, and this lethality was not influenced by additional S1P3 deficiency. At 3 weeks of age, Sgpl1-deficient mice displayed increased trabecular bone mass, which was associated with enhanced osteoclastogenesis and bone resorption, but also with increased bone formation. Most importantly however, none of the skeletal parameters assessed by μCT, histomorphometry and serum analyses were significantly influenced by additional S1P3 deficiency. Taken together, our findings fully support the concept that S1P is a potent osteoanabolic molecule, although S1P3 is not the sole receptor mediating this influence. Since S1P receptors are considered excellent drug targets, it is now required to screen for the impact of other family members on bone formation.

Gerodermia osteodysplastica (GO) is characterized by skin laxity and early-onset osteoporosis. GORAB, the responsible disease gene, encodes a small Golgi protein of poorly characterized function. To circumvent neonatal lethality of the GorabNull full knockout, Gorab was conditionally inactivated in mesenchymal progenitor cells (Prx1-cre), pre-osteoblasts (Runx2-cre), and late osteoblasts/osteocytes (Dmp1-cre), respectively. While in all three lines a reduction in trabecular bone density was evident, only GorabPrx1 and GorabRunx2 mutants showed dramatically thinned, porous cortical bone and spontaneous fractures. Collagen fibrils in the skin of GorabNull mutants and in bone of GorabPrx1 mutants were disorganized, which was also seen in a bone biopsy from a GO patient. Measurement of glycosaminoglycan contents revealed a reduction of dermatan sulfate levels in skin and cartilage from GorabNull mutants. In bone from GorabPrx1 mutants total glycosaminoglycan levels and the relative percentage of dermatan sulfate were both strongly diminished. Accordingly, the proteoglycans biglycan and decorin showed reduced glycanation. Also in cultured GORAB-deficient fibroblasts reduced decorin glycanation was evident. The Golgi compartment of these cells showed an accumulation of decorin, but reduced signals for dermatan sulfate. Moreover, we found elevated activation of TGF-β in GorabPrx1 bone tissue leading to enhanced downstream signalling, which was reproduced in GORAB-deficient fibroblasts. Our data suggest that the loss of Gorab primarily perturbs pre-osteoblasts. GO may be regarded as a congenital disorder of glycosylation affecting proteoglycan synthesis due to delayed transport and impaired posttranslational modification in the Golgi compartment.

Fracture healing is impaired in aged and osteoporotic individuals. Because adequate mechanical stimuli are able to increase bone formation, one therapeutical approach to treat poorly healing fractures could be the application of whole-body vibration, including low-magnitude high-frequency vibration (LMHFV). We investigated the effects of LMHFV on fracture healing in aged osteoporotic mice. Female C57BL/6NCrl mice (n=96) were either ovariectomised (OVX) or sham operated (non-OVX) at age 41 weeks. When aged to 49 weeks, all mice received a femur osteotomy that was stabilised using an external fixator. The mice received whole-body vibrations (20 minutes/day) with 0.3 g peak-to-peak acceleration and a frequency of 45 Hz. After 10 and 21 days, the osteotomised femurs and intact bones (contra-lateral femurs, lumbar spine) were evaluated using bending-testing, micro-computed tomography (μCT), histology and gene expression analyses. LMHFV disturbed fracture healing in aged non-OVX mice, with significantly reduced flexural rigidity (−81%) and bone formation (−80%) in the callus. Gene expression analyses demonstrated increased oestrogen receptor β (ERβ, encoded by Esr2) and Sost expression in the callus of the vibrated animals, but decreased β-catenin, suggesting that ERβ might mediate these negative effects through inhibition of osteoanabolic Wnt/β-catenin signalling. In contrast, in OVX mice, LMHFV significantly improved callus properties, with increased flexural rigidity (+1398%) and bone formation (+637%), which could be abolished by subcutaneous oestrogen application (0.025 mg oestrogen administered in a 90-day-release pellet). On a molecular level, we found an upregulation of ERα in the callus of the vibrated OVX mice, whereas ERβ was unaffected, indicating that ERα might mediate the osteoanabolic response. Our results indicate a major role for oestrogen in the mechanostimulation of fracture healing and imply that LMHFV might only be safe and effective in confined target populations.

Key metabolic hormones, such as insulin, leptin, and adiponectin, have been studied extensively in obesity, however the pathophysiologic relevance of the calcitonin family of peptides remains unclear. This family includes calcitonin (CT), its precursor procalcitonin (PCT), and alpha calcitonin-gene related peptide (αCGRP), which are all encoded by the gene Calca. Here, we studied the role of Calca-derived peptides in diet-induced obesity (DIO) by challenging Calcr-/- (encoding the calcitonin receptor, CTR), Calca-/-, and αCGRP-/- mice and their respective littermates with high-fat diet (HFD) feeding for 16 weeks. HFD-induced pathologies were assessed by glucose tolerance, plasma cytokine and lipid markers, expression studies and histology. We found that DIO in mice lacking the CTR resulted in impaired glucose tolerance, features of enhanced nonalcoholic steatohepatitis (NASH) and adipose tissue inflammation compared to wildtype littermates. Furthermore, CTR-deficient mice were characterized by dyslipidemia and elevated HDL levels. In contrast, mice lacking Calca were protected from DIO, NASH and adipose tissue inflammation, and displayed improved glucose tolerance. Mice exclusively lacking αCGRP displayed a significantly less improved DIO phenotype compared to Calca-deficient mice. In summary, we demonstrate that the CT/CTR axis is involved in regulating plasma cholesterol levels while Calca, presumably through PCT, seems to have a detrimental effect in the context of metabolic disease. Our study provides the first comparative analyses of the roles of Calca-derived peptides and the CTR in metabolic disease.

The anaphylatoxin receptor C5aR1 plays an important role not only in innate immune responses, but also in bone metabolism and fracture healing, being highly expressed on immune and bone cells, including osteoblasts and osteoclasts. C5aR1 induces osteoblast migration, cytokine generation and osteoclastogenesis, however, the exact role of C5aR1-mediated signaling in osteoblasts is not entirely known. Therefore, we hypothesized that osteoblasts are essential target cells for C5a and that fracture healing should be disturbed in mice with an osteoblast-specific C5aR1 overexpression (Col1a1-C5aR1). Osteoblast activity in vitro, bone phenotype and fracture healing after isolated osteotomy and after combined osteotomy with additional thoracic trauma were analyzed. The systemic and local inflammatory reactions were analyzed by determining C5a and IL-6 concentrations in blood, bronchoalveolar lavage fluid and fracture callus and the recruitment of immune cells. In vitro, osteoblast proliferation and differentiation were similar to wildtype cells, and phosphorylation of p38 and expression of IL-6 and RANKL were increased in osteoblasts derived from Col1a1-C5aR1 mice. Bone phenotype and the inflammatory reaction were unaffected in Col1a1-C5aR1 mice. Fracture healing was significantly impaired as demonstrated by significantly reduced bone content, bone mineral density and flexural rigidity, possibly due to significantly increased osteoclast numbers. C5aR1 signaling in osteoblasts might possibly affect RANKL/OPG balance, leading to increased bone resorption. Additional trauma significantly impaired fracture healing, particularly in Col1a1-C5aR1 mice. In conclusion, the data indicate that C5aR1 signaling in osteoblasts plays a detrimental role in bone regeneration after fracture.

The heparin-binding growth and differentiation factor midkine (Mdk) is proposed to negatively regulate osteoblast activity and bone formation in the adult skeleton. As Mdk-deficient mice were protected from ovariectomy (OVX)-induced bone loss, this factor may also play a role in the pathogenesis of postmenopausal osteoporosis. We have previously demonstrated that Mdk negatively influences bone regeneration during fracture healing. Here, we investigated whether the inhibition of Mdk using an Mdk-antibody (Mdk-Ab) improves compromised bone healing in osteoporotic OVX-mice. Using a standardized femur osteotomy model, we demonstrated that Mdk serum levels were significantly enhanced after fracture in both non-OVX and OVX-mice, however, the increase was considerably greater in osteoporotic mice. Systemic treatment with the Mdk-Ab significantly improved bone healing in osteoporotic mice by increasing bone formation in the fracture callus. On the molecular level, we demonstrated that the OVX-induced reduction of the osteoanabolic beta-catenin signaling in the bony callus was abolished by Mdk-Ab treatment. Furthermore, the injection of the Mdk-Ab increased trabecular bone mass in the skeleton of the osteoporotic mice. These results implicate that antagonizing Mdk may be useful for the therapy of osteoporosis and osteoporotic fracture-healing complications.

Bone grafts, i.e., autologous, allogeneic or synthetic bone substitute materials play an increasing role in reconstructive orthopedic surgery. While the indications and materials differ, it is important to understand the cellular mechanisms regarding their integration and remodeling, which are discussed in this review article. Osteoconductivity describes the new bone growth on the graft, while osteoinductivity represents the differentiation of undifferentiated cells into bone forming osteoblasts. The best case is that both mechanisms are accompanied by osteogenesis, i.e., bone modeling and remodeling of the graft material. Graft incorporation is mediated by a number of molecular pathways that signal the differentiation and activity of osteoblasts and osteoclasts (e.g., parathyroid hormone (PTH) and receptor activator of nuclear factor κβ ligand (RANKL), respectively). Direct contact of the graft and host bone as well as the presence of a mechanical load are a prerequisite for the successful function of bone grafts. Interestingly, while bone substitutes show good to excellent clinical outcomes, their histological incorporation has certain limits that are not yet completely understood. For instance, clinical studies have shown contrasting results regarding the complete or incomplete resorption and remodeling of allografts and synthetic grafts. In this context, a foreign body response can lead to complete material degradation via phagocytosis, however it may also cause a fibrotic reaction to the bone substitute. Finally, the success of bone graft incorporation is also limited by other factors, including the bone remodeling capacities of the host, the material itself (e.g., inadequate resorption, toxicity) and the surgical technique or preparation of the graft.

Osteosarcoma (OS), a highly aggressive primary bone tumor, belongs to the most common solid tumors in growing children. Since specific molecular targets for OS treatment remain to be identified, surgical resection combined with multimodal (neo-)adjuvant chemotherapy is still the only way to help respective individuals. We have previously identified the protein tyrosine phosphatase Rptpζ as a marker of terminally differentiated osteoblasts, which negatively regulates their proliferation in vitro. Here we have addressed the question if Rptpζ can function as a tumor suppressor protein inhibiting OS development in vivo. We therefore analyzed the skeletal phenotype of mice lacking Ptprz1, the gene encoding Rptpζ on a tumor-prone genetic background, i.e. Trp53-heterozygosity. By screening a large number of 52 week old Trp53-heterozygous mice by contact radiography we found that Ptprz1-deficiency significantly enhanced OS development with 19% of the mice being affected. The tumors in Ptprz1-deficient Trp53-heterozygous mice were present in different locations (spine, long bones, ribs), and their OS nature was confirmed by undecalcified histology. Likewise, cell lines derived from the tumors were able to undergo osteogenic differentiation ex vivo. A comparison between Ptprz1-heterozygous and Ptprz1-deficient cultures further revealed that the latter ones displayed increased proliferation, a higher abundance of tyrosine-phosphorylated proteins and resistance towards the influence of the growth factor Midkine. Our findings underscore the relevance of Rptpζ as an attenuator of proliferation in differentiated osteoblasts and raise the possibility that activating Rptpζ-dependent signaling could specifically target osteoblastic tumor cells.

Although articular cartilage degeneration represents a major public health problem, the underlying molecular mechanisms are still poorly characterized. We have previously utilized genome-wide expression analysis to identify specific markers of porcine articular cartilage, one of them being Thrombospondin-4 (Thbs4). In the present study we analyzed Thbs4 expression in mice, thereby confirming its predominant expression in articular cartilage, but also identifying expression in other tissues, including bone. To study the role of Thbs4 in skeletal development and integrity we took advantage of a Thbs4-deficient mouse model that was analyzed by undecalcified bone histology. We found that Thbs4-deficient mice do not display phenotypic differences towards wildtype littermates in terms of skeletal growth or bone mass acquisition. Since Thbs4 has previously been found over-expressed in bones of Phex-deficient Hyp mice, we additionally generated Thbs4-deficient Hyp mice, but failed to detect phenotypic differences towards Hyp littermates. With respect to articular cartilage we found that Thbs4-deficient mice display transient thinning of articular cartilage, suggesting a protective role of Thbs4 for joint integrity. Gene expression analysis using porcine primary cells revealed that Thbs4 is not expressed by synovial fibroblasts and that it represents the only member of the Thbs gene family with specific expression in articular, but not in growth plate chondrocytes. In an attempt to identify specific molecular effects of Thbs4 we treated porcine articular chondrocytes with human THBS4 in the absence or presence of conditioned medium from porcine synovial fibroblasts. Here we did not observe a significant influence of THBS4 on proliferation, metabolic activity, apoptosis or gene expression, suggesting that it does not act as a signaling molecule. Taken together, our data demonstrate that Thbs4 is highly expressed in articular chondrocytes, where its presence in the extracellular matrix is required for articular cartilage integrity.