Osteomodulin deficiency in mice causes a specific reduction of transversal cortical bone size.

Skeletal growth, modeling and remodeling are regulated by various molecules, one of them being the recently identified osteoanabolic factor WNT1. We have previously reported that WNT1 transcriptionally activates the expression of Omd, encoding Osteomodulin (OMD), in a murine mesenchymal cell line, which potentially explained the skeletal fragility of mice with mutational WNT1 inactivation, since OMD has been shown to regulate type I collagen fibril formation in vitro. In the present study we confirmed the strong induction of Omd expression in a genome-wide expression analysis of transfected cells, and we obtained further evidence for Omd being a direct target gene of WNT1. To assess the in vivo relevance of this regulation, we crossed Omd-deficient mice with a mouse line harboring an inducible, osteoblast-specific Wnt1 transgene. After induction of Wnt1 expression for 1 or 3 weeks, the osteoanabolic potency of WNT1 was not impaired despite the Omd deficiency. Since current knowledge regarding the in vivo physiological function of OMD is limited, we next focused on skeletal phenotyping of wild-type and Omd-deficient littermates, in the absence of a Wnt1 transgene. Here we did not observe an impact of Omd deficiency on trabecular bone parameters by histomorphometry and µCT either. Importantly, however, male and female Omd-deficient mice at the ages of 12 and 24 weeks displayed a slender bone phenotype with significantly smaller long bones in the transversal dimension, while the longitudinal bone growth remained unaffected. Although mechanical testing revealed no significant changes explained by impaired bone material properties, atomic force microscopy of the femoral bone surface of Omd-deficient mice revealed moderate changes at the nanostructural level, indicating altered regulation of collagen fibril formation and aggregation. Taken together, our data demonstrate that, although OMD is dispensable for the osteoanabolic effect of WNT1, its deficiency in mice specifically modulates transversal cortical bone morphology.

We explored the physiological relevance of the protein Osteomodulin (OMD) that we previously found to be induced by the osteoanabolic molecule WNT1. While other studies have shown that OMD is involved in the regulation of collagen fibril formation in vitro, its function in vivo has not been investigated. We confirmed that OMD is directly regulated by WNT1 but surprisingly, when we bred mice lacking OMD with mice engineered to highly express WNT1, we found that the osteoanabolic effect of WNT1 was unaffected by the absence of OMD. Interestingly, mice lacking OMD did show differences in the shape of their bones, particularly in their width, despite no significant changes in bone density or length. Investigation of the bone matrix of mice lacking OMD at the nanostructural level indicated moderate differences in the organization of collagen fibrils. This study provided further insights into the effect of WNT1 on bone metabolism and highlighted a specific function of OMD in skeletal morphology.

Approaching virtual osteoid volume estimation and in-depth tissue characterization in patients with tumor-induced osteomalacia.

Tumor-induced osteomalacia (TIO) poses a significant diagnostic challenge, leading to increased disease duration and patient burden also by missing clinical suspicion. Today, diagnosis of osteomalacia relies on invasive iliac crest biopsy, if needed. Therefore, a noninvasive method would be beneficial for patients with severe osteomalacia, such as TIO, to inform their clinical management and address specific needs, like estimating the regeneration capacity at high osteoid volumes (OVs) or the potential of a hungry bone syndrome after tumor removal. Furthermore, given the lack of comprehensive histological characterization of TIO, there is a need for additional tissue characterization. Therefore, our assessment encompassed iliac crest biopsies that were examined using quantitative electron backscattered microscopy, Raman spectroscopy, micro-computed tomography, and histology to analyze the biopsy tissue. Our clinical assessment encompassed DXA and high-resolution peripheral quantitative computed tomography (HR-pQCT) alongside with biochemical analyses and clinical evaluations. Combining imaging and clinical data, we established a model to predict the OV. We compared 9 TIO patients with 10 osteoporosis (OPO) patients and 10 healthy controls. Histological analyses confirmed a pronounced OV in TIO patients (OPO: 1.20% ± 1.23% vs TIO: 23.55% ± 12.23%, P < .0005), and spectroscopy revealed lower phosphate levels in TIO biopsies. By combining HR-pQCT and laboratory diagnostics, we developed a linear regression model to noninvasively predict the OV revealing significantly higher modeled OV/BVmodel values of 24.46% ± 14.22% for TIO compared to the control group (5.952% ± 3.44%, P ≤ .001). By combining laboratory diagnostics, namely, ALP and Tt.BMDRadius measured by HR-pQCT, we achieved the calculation of the virtual osteoid volume to bone volume ratio (OV/BVmodel) with a significant correlation to histology as well as reliable identification of TIO patients compared to OPO and control. This novel approach is potentially helpful for predicting OV by noninvasive techniques in diagnostic procedures and improving the clinical management of TIO.

Osteomalacia, a bone mineralization disease, results in soft bones due to a lack of calcium or phosphate. Tumor-induced osteomalacia (TIO) is an acquired and challenging form of osteomalacia due to low serum phosphate levels that often lead to prolonged patient suffering. Current diagnosis of osteomalacia involves surgical bone biopsies, but a noninvasive approach would be beneficial, improving clinical management and addressing specific needs like estimating the bone's quality and ability to recover. We used advanced techniques like electron microscopy, spectroscopy, and high-resolution CT to study bone samples from 9 TIO patients. Additionally, we assessed their bone health through sophisticated imaging and blood analyses. Microscopy confirmed huge amounts of soft bone tissue due to a severe mineralization defect. By combining imaging and blood analysis, we developed a noninvasive method to predict the amount of soft tissue (osteoid) to understand soft bones without the need for surgical interventions. In conclusion, our innovative approach, combining blood diagnostics (alkaline phosphatase) with total BMD from high-resolution 3D clinical imaging of the lower arm, allows us to predict the osteoid amount virtually. This method can also compare TIO patients with controls or those with osteoporosis and might be helpful in the future.

Alterations in compositional and cellular properties of the subchondral bone are linked to cartilage degeneration in hip osteoarthritis.

OBJECTIVE: The subchondral bone is an emerging regulator of osteoarthritis (OA). However, knowledge of how specific subchondral alterations relate to cartilage degeneration remains incomplete. METHOD: Femoral heads were obtained from 44 patients with primary OA during total hip arthroplasty and from 30 non-OA controls during autopsy. A multiscale assessment of the central subchondral bone region comprising histomorphometry, quantitative backscattered electron imaging, nanoindentation, and osteocyte lacunocanalicular network characterization was employed. RESULTS: In hip OA, thickening of the subchondral bone coincided with a higher number of osteoblasts (controls: 3.7 ± 4.5 mm-1, OA: 16.4 ± 10.2 mm-1, age-adjusted mean difference 10.5 mm-1 [95% CI 4.7 to 16.4], p < 0.001) but a similar number of osteoclasts compared to controls (p = 0.150). Furthermore, higher matrix mineralization heterogeneity (CaWidth, controls: 2.8 ± 0.2 wt%, OA: 3.1 ± 0.3 wt%, age-adjusted mean difference 0.2 wt% [95% CI 0.1 to 0.4], p = 0.011) and lower tissue hardness (controls: 0.69 ± 0.06 GPa, OA: 0.67 ± 0.06 GPa, age-adjusted mean difference -0.05 GPa [95% CI -0.09 to -0.01], p = 0.032) were detected. While no evidence of altered osteocytic perilacunar/canalicular remodeling in terms of fewer osteocyte canaliculi was found in OA, specimens with advanced cartilage degeneration showed a higher number of osteocyte canaliculi and larger lacunocanalicular network area compared to those with low-grade cartilage degeneration. Multiple linear regression models indicated that several subchondral bone properties, especially osteoblast and osteocyte parameters, were closely related to cartilage degeneration (R2 adjusted = 0.561, p < 0.001). CONCLUSION: Subchondral bone properties in OA are affected at the compositional, mechanical, and cellular levels. Based on their strong interaction with cartilage degeneration, targeting osteoblasts/osteocytes may be a promising therapeutic OA approach. DATA AND MATERIALS AVAILABILITY: All data are available in the main text or the supplementary materials.

Piezo1 expression in chondrocytes controls endochondral ossification and osteoarthritis development.

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 (Piezo1Col2a1Cre), but not of Piezo2, developed a near absence of trabecular bone below the chondrogenic growth plate postnatally. Moreover, all Piezo1Col2a1Cre 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 Piezo1Col2a1Cre 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.

Mechanical induction of osteoanabolic Wnt1 promotes osteoblast differentiation via Plat.

Physical activity-induced mechanical stimuli play a crucial role in preserving bone mass and structure by promoting bone formation. While the Wnt pathway is pivotal for mediating the osteoblast response to loading, the exact mechanisms are not fully understood. Here, we found that mechanical stimulation induces osteoblastic Wnt1 expression, resulting in an upregulation of key osteogenic marker genes, including Runx2 and Sp7, while Wnt1 knockdown using siRNA prevented these effects. RNAseq analysis identified Plat as a major target through which Wnt1 exerts its osteogenic influence. This was corroborated by Plat depletion using siRNA, confirming its positive role in osteogenic differentiation. Moreover, we demonstrated that mechanical stimulation enhances Plat expression, which, in turn leads to increased expression of osteogenic markers like Runx2 and Sp7. Notably, Plat depletion by siRNA prevented this effect. We have established that Wnt1 regulates Plat expression by activating ß-Catenin. Silencing Wnt1 impairs mechanically induced ß-Catenin activation, subsequently reducing Plat expression. Furthermore, our findings showed that Wnt1 is essential for osteoblasts to respond to mechanical stimulation and induce Runx2 and Sp7 expression, in part through the Wnt1/ß-Catenin/Plat signaling pathway. Additionally, we observed significantly reduced Wnt1 and Plat expression in bones from ovariectomy (OVX)-induced and age-related osteoporotic mouse models compared with non-OVX and young mice, respectively. Overall, our data suggested that Wnt1 and Plat play significant roles in mechanically induced osteogenesis. Their decreased expression in bones from OVX and aged mice highlights their potential involvement in post-menopausal and age-related osteoporosis, respectively.

Age-related changes in the midpalatal suture: Comparison between CBCT staging and bone micromorphology.

The age-related maturation of the human midpalatal suture is challenging to predict, but critical for successful non-surgical rapid maxillary expansion (RME). While cone-beam computed tomography (CBCT) can be used to categorize the suture into stages, it remains unclear how well the stages predict the actual micromorphology of the palate. To address this clinically relevant question, we used CBCT together with three-dimensional micro-computed tomography (µCT) analysis on 24 human palate specimens from individuals aged 14-34 years. We first classified the specimens into stages (A-E) using CBCT images and then correlated the results with our comprehensive µCT analysis. Our analysis focused on several factors, including bone volume fraction (BV/TV), sutural width, volume, interdigitation, ossification, and their associations with age, CBCT stage, and sex. Our µCT analysis revealed a decrease in sutural width and volume after the age of 20 years, accompanied by sutural closure beginning in the palatal segment. The overall rate of ossification remained low but increased after the age of 20 years. No significant differences were found between males and females. Importantly, we also found no correlation between individual age and CBCT stages. Furthermore, there was no association between CBCT stages and patalal suture volume, ossification and interdigitation. Taken together, our findings cast doubt on the reliability of CBCT stage as a means of predicting skeletal maturity of the palatal suture, as it appears to lack the precision required to accurately assess the true micromorphology of the palatal suture. Future investigations should explore whether alternative CBCT parameters may be more useful in addressing the challenging question of whether RME requires surgical bone weakening.

Cholesteatoma Severely Impacts the Integrity and Bone Material Quality of the Incus.

Cholesteatoma can lead to progressive destruction of the auditory ossicles along with conductive hearing loss but precise data on the microstructural, cellular, and compositional aspects of affected ossicles are not available. Here, we obtained incus specimens from patients who had cholesteatoma with conductive hearing loss. Incudes were evaluated by micro-computed tomography, histomorphometry on undecalcified sections, quantitative backscattered electron imaging, and nanoindentation. Results were compared with two control groups taken from patients with chronic otitis media as well as from skeletally intact donors at autopsy. The porosity of incus specimens was higher in cholesteatoma than in chronic otitis media, along with a higher osteoclast surface per bone surface. Histomorphometric assessment revealed higher osteoid levels and osteocyte numbers in cholesteatoma incudes. Incudes affected by cholesteatoma also showed lower matrix mineralization compared with specimens from healthy controls and chronic otitis media. Furthermore, the modulus-to-hardness ratio was higher in cholesteatoma specimens compared with controls. Taken together, we demonstrated increased porosity along with increased osteoclast indices, impaired matrix mineralization, and altered biomechanical properties as distinct features of the incus in cholesteatoma. Based on our findings, a possible impact of impaired bone quality on conductive hearing loss should be further explored.

Comparison of Motion Grading in 1,000 Patients by First- and Second-Generation HR-pQCT: A Propensity Score Matched Cohort Study.

In-vivo bone microstructure measured by high-resolution peripheral quantitative computed tomography (HR-pQCT) is gaining importance in research and clinical practice. Second-generation HR-pQCT (XCT2) shows improved image quality and shorter measurement duration compared to the first generation (XCT1). Predicting and understanding the occurrence of motion artifacts is crucial for clinical practice. We retrospectively analyzed data from HR-pQCT measurements at the distal radius and tibia of 1,000 patients (aged 20 to 89) evenly distributed between both generations of HR-pQCT. Motion artifacts were graded between 1 (no motion) and 5 (severe motion), with grades greater 3 considered unusable. Additionally, baseline characteristics and patients' muscle performance and balance were measured. Various group comparisons between the two generations of HR-pQCT and regression analyses between patient characteristics and motion grading were performed. The study groups of XCT1 and XCT2 did not differ by age (XCT1: 64.9 vs. XCT2: 63.8 years, p = 0.136), sex (both 74.5% females, p > 0.999), or BMI (both 24.2 kg/m2, p = 0.911) after propensity score matching. XCT2 scans exhibited significantly lower motion grading in both extremities compared to XCT1 (Radius: p < 0.001; Tibia: p = 0.002). In XCT2 motion-corrupted scans were more than halved at the radius (XCT1: 35.3% vs. XCT2: 15.5%, p < 0.001), and at the tibia the frequency of best image quality scans was increased (XCT1: 50.2% vs. XCT2: 63.7%, p < 0.001). The strongest independent predictor for motion-corrupted images is the occurrence of high motion grading at the other scanning site during the same consultation. The association between high motion grading in one scan and a corresponding high motion grading in another scan within the same session suggests a non-resting patient. Additionally, aged, female, and patients with smaller stature tend towards higher motion grading, requiring special attention to a correct extremity fixation.

Dynamics of Skeletal Status under Optimized Management during Subsequent Pregnancy in Three Women with a History of Pregnancy- and Lactation-Associated Osteoporosis Carrying pathogenic Variants in WNT1 and LRP5.

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.

Do Clinical Parameters Reflect Local Bone Metabolism in Heterotopic Ossification After Septic or Aseptic THA?

BACKGROUND: Heterotopic ossification (HO) is a common complication after THA. Although current research primarily focuses on treatment and prevention, little is known about the local bone metabolism of HO and clinical contributing factors. QUESTIONS/PURPOSES: We aimed to assess bone remodeling processes in HO using histomorphometry, focusing on the effects of inflammation and prior NSAID treatment. Specifically, we asked: (1) Are HO specimens taken from patients with periprosthetic joint infection (PJI) more likely to exhibit active bone modeling and remodeling than specimens taken at the time of revision from patients without infection? (2) Do clinical or inflammatory serum and synovial parameters reflect the microstructure of and remodeling in both HO entities? (3) Is NSAID treatment before revision surgery associated with altered local bone mineralization or remodeling properties? METHODS: Between June 2021 and May 2022, we screened 395 patients undergoing revision THA at two tertiary centers in Germany. Of those, we considered all patients with radiographic HO as potentially eligible. Based on that, 21% (83 of 395) were eligible; a further 43 were excluded because of an inability to remove the implant intraoperatively (16 patients), insufficient material (11), comorbidities with a major effect on bone metabolism (10), or bone-specific drugs (six), leaving 10% (40) for analysis in this retrospective, comparative study. HO specimens were collected during aseptic (25 patients: 18 male, seven female, mean age 70 ± 11 years, mean BMI 29 ± 4 kg/m 2 ) and septic (15 patients: 11 male, four female, mean age 69 ± 9 years, mean BMI 32 ± 9 kg/m 2 ) revision THA at a mean of 6 ± 7 years after primary implantation and a mean age of 70 ± 9 years at revision. Septic origin (PJI) was diagnosed based on the 2018 International Consensus Meeting criteria, through a preoperative assessment of serum and synovial parameters. To specify the local bone microstructure, ossification, and cellular bone turnover, we analyzed HO specimens using micro-CT and histomorphometry on undecalcified sections. Data were compared with those of controls, taken from femoral neck trabecular bone (10 patients: five female, five male, mean age 75 ± 6 years, mean BMI 28 ± 4 kg/m 2 ) and osteophytes (10 patients: five female, five male, mean age 70 ± 10 years, mean BMI 29 ± 7 kg/m 2 ). The time between primary implantation and revision (time in situ), HO severity based on the Brooker classification, and serum and synovial markers were correlated with HO microstructure and parameters of cellular bone turnover. In a subgroup of specimens of patients with NSAID treatment before revision, osteoid and bone turnover indices were evaluated and compared a matched cohort of specimens from patients without prior NSAID treatment. RESULTS: Patients with aseptic and septic HO presented with a higher bone volume (BV/TV; aseptic: 0.41 ± 0.15, mean difference 0.20 [95% CI 0.07 to 0.32]; septic: 0.43 ± 0.15, mean difference 0.22 [95% CI 0.08 to 0.36]; femoral neck: 0.21 ± 0.04; both p < 0.001), lower bone mineral density (aseptic: 809 ± 66 mg HA/cm 3 , mean difference -91 mg HA/cm 3 [95% CI -144 to -38]; septic: 789 ± 44 mg HA/cm 3 , mean difference -111 mg HA/cm 3 [95% CI -169 to -53]; femoral neck: 899 ± 20 mg HA/cm 3 ; both p < 0.001), and ongoing bone modeling with endochondral ossification and a higher proportion of woven, immature bone (aseptic: 25% ± 17%, mean difference 25% [95% CI 9% to 41%]; septic: 37% ± 23%, mean difference 36% [95% CI 19% to 54%]; femoral neck: 0.4% ± 0.5%; both p < 0.001) compared with femoral neck specimens. Moreover, bone surfaces were characterized by increased osteoblast and osteoclast indices in both aseptic and septic HO, although a higher density of osteocytes was detected exclusively in septic HO (aseptic: 158 ± 56 1/mm 2 versus septic: 272 ± 48 1/mm 2 , mean difference 114 1/mm 2 [95% CI 65 to 162]; p < 0.001). Compared with osteophytes, microstructure and turnover indices were largely similar in HO. The Brooker class was not associated with any local bone metabolism parameters. The time in situ was negatively associated with bone turnover in aseptic HO specimens (osteoblast surface per bone surface: r = -0.46; p = 0.01; osteoclast surface per bone surface: r = -0.56; p = 0.003). Serum or synovial inflammatory markers were not correlated with local bone turnover in septic HO. Specimens of patients with NSAID treatment before revision surgery had a higher osteoid thickness (10.1 ± 2.1 µm versus 5.5 ± 2.6 µm, mean difference -4.7 µm [95% CI -7.4 to -2.0]; p = 0.001), but there was no difference in other osteoid, structural, or cellular parameters. CONCLUSION: Aseptic and septic HO share phenotypic characteristics in terms of the sustained increase in bone metabolism, although differences in osteocyte and adipocyte numbers suggest distinct homeostatic mechanisms. These results suggest persistent bone modeling or remodeling, with osteoblast and osteoclast indices showing a moderate decline with the time in situ in aseptic HO. Future studies should use longitudinal study designs to correlate our findings with clinical outcomes (such as HO growth or recurrence). In addition, the molecular mechanisms of bone cell involvement during HO formation and growth should be further investigated, which may allow specific therapeutic and preventive interventions. CLINICAL RELEVANCE: To our knowledge, our study is the first to systematically investigate histomorphometric bone metabolism parameters in patients with HO after THA, providing a clinical reference for evaluating modeling and remodeling activity. Routine clinical, serum, and synovial markers are not useful for inferring local bone metabolism.

Zinc and vitamin D deficiency and supplementation in hypophosphatasia patients - A retrospective study.

Hypophosphatasia (HPP) is characterized by severe skeletal symptoms including mineralization defects, insufficiency fractures, and delayed facture healing or non-unions. HPP is caused by mutations of the tissue non-specific alkaline phosphatase (TNSALP). Zinc is a cofactor of TNSALP and vitamin D an important regulator of bone matrix mineralization. Data from this retrospective study indicates that deficiencies in zinc or vitamin D occur in HPP patients with a similar frequency as in the general population. While guidelines for repletion of these micronutrients have been established for the general population, the transferability of the efficacy and safety of these regiments to HPP patients still needed to be determined. We filtered for variant classification (ACMG 3-5, non-benign) and data completeness from a total cohort of 263 HPP patients. 73.5 % of this sub-cohort were vitamin D deficient while 27.2 % were zinc deficient. We retrospectively evaluated the effect of supplementation according to general guidelines in 10 patients with zinc-deficiency and 38 patients with vitamin d-deficiency. The treatments significantly raised serum zinc or vitamin D levels respectively. All other assessed disease markers (alkaline phosphatase, pyrodoxal-5-phosphate) or bone turnover markers (phosphate, calcium, parathyroid hormone, bone specific alkaline phosphatase, creatinine, desoxypyridinoline) remained unchanged. These results highlight that general guidelines for zinc and vitamin D repletion can be successfully applied to HPP patients in order to prevent deficiency symptoms without exacerbating the disease burden or causing adverse effects due to changes in bone and calcium homeostasis.

Mechanical-induced bone remodeling does not depend on Piezo1 in dentoalveolar hard tissue.

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 Piezo1floxed/floxed mouse model in combination with Dmp1cre 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 Piezo1floxed/floxed;Dmp1cre 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.

Radial HR-pQCT and Finite Element Analysis in HPP Patients are Superior in Identifying Susceptibility to Fracture-Associated Skeletal Affections Compared to DXA and Laboratory Tests.

Hypophosphatasia (HPP) is an inborn disease that causes a rare form of osteomalacia, a mineralization disorder affecting mineralized tissues. Identification of patients at high risk for fractures or other skeletal manifestations (such as insufficiency fractures or excessive bone marrow edema) by bone densitometry and laboratory tests remains clinically challenging. Therefore, we examined two cohorts of patients with variants in the ALPL gene grouped by bone manifestations. These groups were compared by means of bone microarchitecture using high-resolution peripheral quantitative computed tomography (HR-pQCT) and simulated mechanical performance utilizing finite element analysis (FEA). Whereas the incidence of skeletal manifestations among the patients could not be determined by dual energy X-ray absorptiometry (DXA) or laboratory assessment, HR-pQCT evaluation showed a distinct pattern of HPP patients with such manifestations. Specifically, these patients had a pronounced loss of trabecular bone mineral density, increased trabecular spacing, and decreased ultimate force at the distal radius. Interestingly, the derived results indicate that the non-weight-bearing radius is superior to the weight-bearing tibia in identifying deteriorated skeletal patterns. Overall, the assessment by HR-pQCT appears to be of high clinical relevance due to the improved identification of HPP patients with an increased risk for fractures or other skeletal manifestations, especially at the distal radius.

Bone mineral density and microarchitecture change during skeletal growth in harbor seals (Phoca vitulina) from the German coast.

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 < 0.001). To validate the results of the DXA measurement (i.e., the standard method used worldwide to diagnose osteoporosis in humans), we performed linear regression analyses with the three-dimensional measurements from the HR-pQCT method, which revealed strong associations between the two imaging techniques (e.g., aBMD and Tb.Th: R2 = 0.96, p < 0.0001). Taken together, our findings highlight the importance of systematic skeletal investigations in marine mammals during growth, illustrating the high accuracy of DXA in this context. Regardless of the limited sample size, the observed trabecular thickening is likely to represent a distinct pattern of vertebral bone maturation. As differences in nutritional status, among other factors, are likely to affect skeletal health, it appears essential to routinely perform skeletal assessments in marine mammals. Placing the results in the context of environmental exposures may allow effective measures to protect their populations.

Osteocyte apoptosis and cellular micropetrosis signify skeletal aging in type 1 diabetes.

Bone fragility is a profound complication of type 1 diabetes mellitus (T1DM), increasing patient morbidity. Within the mineralized bone matrix, osteocytes build a mechanosensitive network that orchestrates bone remodeling; thus, osteocyte viability is crucial for maintaining bone homeostasis. In human cortical bone specimens from individuals with T1DM, we found signs of accelerated osteocyte apoptosis and local mineralization of osteocyte lacunae (micropetrosis) compared with samples from age-matched controls. Such morphological changes were seen in the relatively young osteonal bone matrix on the periosteal side, and micropetrosis coincided with microdamage accumulation, implying that T1DM drives local skeletal aging and thereby impairs the biomechanical competence of the bone tissue. The consequent dysfunction of the osteocyte network hampers bone remodeling and decreases bone repair mechanisms, potentially contributing to the enhanced fracture risk seen in individuals with T1DM. STATEMENT OF SIGNIFICANCE: Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease that causes hyperglycemia. Increased bone fragility is one of the complications associated with T1DM. Our latest study on T1DM-affected human cortical bone identified the viability of osteocytes, the primary bone cells, as a potentially critical factor in T1DM-bone disease. We linked T1DM with increased osteocyte apoptosis and local accumulation of mineralized lacunar spaces and microdamage. Such structural changes in bone tissue suggest that T1DM speeds up the adverse effects of aging, leading to the premature death of osteocytes and potentially contributing to diabetes-related bone fragility.

Wnt1 Boosts Fracture Healing by Enhancing Bone Formation in the Fracture Callus.

Despite considerable improvement in fracture care, 5%-10% of all fractures still heal poorly or result in nonunion formation. Therefore, there is an urgent need to identify new molecules that can be used to improve bone fracture healing. One activator of the Wnt-signaling cascade, Wnt1, has recently gained attention for its intense osteoanabolic effect on the intact skeleton. The aim of the present study was to investigate whether Wnt1 might be a promising molecule to accelerate fracture healing both in skeletally healthy and osteoporotic mice that display a diminished healing capacity. Transgenic mice for a temporary induction of Wnt1 specifically in osteoblasts (Wnt1-tg) were subjected to femur osteotomy. Non-ovariectomized and ovariectomized Wnt1-tg mice displayed significantly accelerated fracture healing based on a strong increase in bone formation in the fracture callus. Transcriptome profiling revealed that Hippo/yes1-associated transcriptional regulator (YAP)-signaling and bone morphogenetic protein (BMP) signaling pathways were highly enriched in the fracture callus of Wnt1-tg animals. Immunohistochemical staining confirmed increased activation of YAP1 and expression of BMP2 in osteoblasts in the fracture callus. Therefore, our data indicate that Wnt1 boosts bone formation during fracture healing via YAP/BMP signaling both under healthy and osteoporotic conditions. To further test a potential translational application of Wnt1, we applied recombinant Wnt1 embedded into a collagen gel during critical-size bone-defect repair. Mice treated with Wnt1 displayed increased bone regeneration compared to control mice accompanied by increased YAP1/BMP2 expression in the defect area. These findings are of high clinical relevance because they indicate that Wnt1 could be used as a new therapeutic agent to treat orthopedic complications in the clinic. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Effects of Infantile Hypophosphatasia on Human Dental Tissue.

Hypophosphatasia (HPP) is an inherited, systemic disorder, caused by loss-of-function variants of the ALPL gene encoding the enzyme tissue non-specific alkaline phosphatase (TNSALP). HPP is characterized by low serum TNSALP concentrations associated with defective bone mineralization and increased fracture risk. Dental manifestations have been reported as the exclusive feature (odontohypophosphatasia) and in combination with skeletal complications. Enzyme replacement therapy (asfotase alfa) has been shown to improve respiratory insufficiency and skeletal complications in HPP patients, while its effects on dental status have been understudied to date. In this study, quantitative backscattered electron imaging (qBEI) and histological analysis were performed on teeth from two patients with infantile HPP before and during asfotase alfa treatment and compared to matched healthy control teeth. qBEI and histological methods revealed varying mineralization patterns in cementum and dentin with lower mineralization in HPP. Furthermore, a significantly higher repair cementum thickness was observed in HPP compared to control teeth. Comparison before and during treatment showed minor improvements in mineralization and histological parameters in the patient when normalized to matched control teeth. HPP induces heterogeneous effects on mineralization and morphology of the dental status. Short treatment with asfotase alfa slightly affects mineralization in cementum and dentin. Despite HPP being a rare disease, its mild form occurs at higher prevalence. This study is of high clinical relevance as it expands our knowledge of HPP and dental involvement. Furthermore, it contributes to the understanding of dental tissue treatment, which has hardly been studied so far.

Early enzyme replacement therapy prevents dental and craniofacial abnormalities in a mouse model of mucopolysaccharidosis type VI.

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 (Arsb m/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 Arsb m/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 Arsb m/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 Arsb m/m mice. The temporomandibular joints in Arsb m/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 Arsb m/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 Arsb m/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.

Increased concentrations of conjugated bile acids are associated with osteoporosis in PSC patients.

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.