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.

Pronounced cortical porosity and sex-specific patterns of increased bone and osteocyte lacunar mineralization characterize the human distal fibula with aging.

The high occurrence of distal fibula fractures among older women suggests a potential link to impaired bone health. Here we used a multiscale imaging approach to investigate the microarchitecture, mineralization, and biomechanics of the human distal fibula in relation to age and sex. Micro-computed tomography was performed to analyze the local volumetric bone mineral density and various microarchitectural parameters of the trabecular and the cortical compartment. Bone mineral density distribution and osteocyte lacunar parameters were quantified using quantitative backscattered electron imaging in periosteal, endocortical, and trabecular regions. Additionally, cortical hardness and Young's modulus were assessed by nanoindentation. While cortical porosity strongly increased with age independent of sex, trabecular microarchitecture remained stable. Notably, nearly half of the specimens showed non-bony hypermineralized tissue located at the periosteum, similar to that previously detected in the femoral neck, with no consistent association with advanced age. Independent of this finding, cortical and trabecular mineralization, i.e., mean calcium content, as well as endocortical tissue hardness increased with age in males but not females. Importantly, we also observed mineralized osteocyte lacunae that increased with age specifically in females. In conclusion, our results indicate that skeletal aging of the distal fibula is signified not only by pronounced cortical porosity but also by an increase in mineralized osteocyte lacunae in females. These findings may provide an explanation for the increased occurrence of ankle fractures in older women.

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.

Mice heterozygous for an osteogenesis imperfecta-linked MBTPS2 variant display a compromised subchondral osteocyte lacunocanalicular network associated with abnormal articular cartilage.

Missense variants in the MBTPS2 gene, located on the X chromosome, have been associated with an X-linked recessive form of osteogenesis imperfecta (X-OI), an inherited bone dysplasia characterized by multiple and recurrent bone fractures, short stature, and various skeletal deformities in affected individuals. The role of site-2 protease, encoded by MBTPS2, and the molecular pathomechanism underlying the disease are to date elusive. This study is the first to report on the generation of two Mbtps2 mouse models, a knock-in mouse carrying one of the disease-causative MBTPS2 variants (N455S) and a Mbtps2 knock-out (ko) mouse. Because both loss-of-function variants lead to embryonic lethality in hemizygous male mutant mice, we performed a comprehensive skeletal analysis of heterozygous Mbtps2+/N455S and Mbtps2+/ko female mice. Both models displayed osteochondral abnormalities such as thinned subchondral bone, altered subchondral osteocyte interconnectivity as well as thickened articular cartilage with chondrocyte clustering, altogether resembling an early osteoarthritis (OA) phenotype. However, distant from the joints, no alterations in the bone mass and turnover could be detected in either of the mutant mice. Based on our findings we conclude that MBTPS2 haploinsufficiency results in early OA-like alterations in the articular cartilage and underlying subchondral bone, which likely precede the development of typical OI phenotype in bone. Our study provides first evidence for a potential role of site-2 protease for maintaining homeostasis of both bone and cartilage.

AXIN1 bi-allelic variants disrupting the C-terminal DIX domain cause craniometadiaphyseal osteosclerosis with hip dysplasia.

Sclerosing skeletal dysplasias result from an imbalance between bone formation and resorption. We identified three homozygous, C-terminally truncating AXIN1 variants in seven individuals from four families affected by macrocephaly, cranial hyperostosis, and vertebral endplate sclerosis. Other frequent findings included hip dysplasia, heart malformations, variable developmental delay, and hematological anomalies. In line with AXIN1 being a central component of the ß-catenin destruction complex, analyses of primary and genome-edited cells harboring the truncating variants revealed enhanced basal canonical Wnt pathway activity. All three AXIN1-truncating variants resulted in reduced protein levels and impaired AXIN1 polymerization mediated by its C-terminal DIX domain but partially retained Wnt-inhibitory function upon overexpression. Addition of a tankyrase inhibitor attenuated Wnt overactivity in the AXIN1-mutant model systems. Our data suggest that AXIN1 coordinates the action of osteoblasts and osteoclasts and that tankyrase inhibitors can attenuate the effects of AXIN1 hypomorphic variants.

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.

Analysis of Three-Dimensional Bone Microarchitecture of the Axis Exposes Pronounced Regional Heterogeneity Associated with Clinical Fracture Patterns.

The odontoid process (dens) of the second cervical vertebra (axis) is prone to fracture. While the importance of its skeletal integrity has been previously noted, representative three-dimensional microarchitecture analyses in humans are not available. This study aimed to determine the bone microarchitecture of the axis using high-resolution quantitative computed tomography (HR-pQCT) and to derive clinical implications for the occurrence and treatment of axis fractures. For initial clinical reference, the apparent density of the axis was determined based on clinical computed tomography (CT) images in patients without and with fractures of the axis. Subsequently, 28 human axes (female 50%) obtained at autopsy were analyzed by HR-pQCT. Analyses were performed in three different regions corresponding to zones I (tip of dens), II (base of dens), and III (corpus axis) of the Anderson and D'Alonzo classification. Lower apparent densities based on clinical CT data were detected in zone II and III compared to zone I in both the group without and with fracture. In the autopsy specimens, cortical thickness and bone volume fraction decreased continuously from zone I to zone III. Trabecular and cortical tissue mineral density was lowest in zone III, with no differences between zones I and II. In conclusion, our clinical and high-resolution ex vivo imaging data highlight a marked regional heterogeneity of bone microarchitecture, with poor cortical and trabecular properties near the dens base. These results may partly explain why zones II and III are at high risk of fracture and osteosynthesis failure.

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.

Catalysis-Independent ENPP1 Protein Signaling Regulates Mammalian Bone Mass.

Biallelic ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) deficiency induces vascular/soft tissue calcifications in generalized arterial calcification of infancy (GACI), and low bone mass with phosphate-wasting rickets in GACI survivors (autosomal hypophosphatemic rickets type-2). ENPP1 haploinsufficiency induces early-onset osteoporosis and mild phosphate wasting in adults. Both conditions demonstrate the unusual combination of reduced accrual of skeletal mineral, yet excess and progressive heterotopic mineralization. ENPP1 is the only enzyme that generates extracellular pyrophosphate (PPi), a potent inhibitor of both bone and heterotopic mineralization. Life-threatening vascular calcification in ENPP1 deficiency is due to decreased plasma PPi; however, the mechanism by which osteopenia results is not apparent from an understanding of the enzyme's catalytic activity. To probe for catalysis-independent ENPP1 pathways regulating bone, we developed a murine model uncoupling ENPP1 protein signaling from ENPP1 catalysis, Enpp1T238A mice. In contrast to Enpp1asj mice, which lack ENPP1, Enpp1T238A mice have normal trabecular bone microarchitecture and favorable biomechanical properties. However, both models demonstrate low plasma Pi and PPi, increased fibroblast growth factor 23 (FGF23), and by 23 weeks, osteomalacia demonstrating equivalent phosphate wasting in both models. Reflecting findings in whole bone, calvarial cell cultures from Enpp1asj mice demonstrated markedly decreased calcification, elevated transcription of Sfrp1, and decreased nuclear ß-catenin signaling compared to wild-type (WT) and Enpp1T238A cultures. Finally, the decreased calcification and nuclear ß-catenin signaling observed in Enpp1asj cultures was restored to WT levels by knockout of Sfrp1. Collectively, our findings demonstrate that catalysis-independent ENPP1 signaling pathways regulate bone mass via the expression of soluble Wnt inhibitors such as secreted frizzled-related protein 1 (SFRP1), whereas catalysis dependent pathways regulate phosphate homeostasis through the regulation of plasma FGF23. © 2022 American Society for Bone and Mineral Research (ASBMR).

Oral HRAS Mutation in Orofacial Nevus Sebaceous Syndrome (Schimmelpenning-Feuerstein-Mims-Syndrome): A Case Report With a Literature Survey.

BACKGROUND/AIM: The aim of this study was to present the long-term course of a patient with nevus sebaceous syndrome (NSS). Recent genetic studies place the syndrome in the emerging group of so-called RASopathies. The focus of the report is on surgical treatment and morphological and genetic findings of the face and oral cavity. CASE REPORT: A female patient was treated for congenital alterations of facial skin and oral mucosa. The oral lesions were removed repeatedly. Eruption of teeth on the lesion sites was made easier by the measures taken. However, after repeated ablation of the affected gingiva, the periodontal papillomatous epithelium re-differentiated into the same reddish, conspicuous, hyperplastic epithelium. The teeth in the affected region showed noticeable changes in position, surface, and shape. A HRAS mutation was detected only in the regions of altered oral epithelia and not in adjacent soft tissues. CONCLUSION: Reports on NSS rarely address oral manifestations. The recorded alterations of oral soft and hard tissues in NSS indicate a topographical relationship between the development of oral mucosa and teeth as well as the long-lasting impact of a sporadic mutation on organ development at this site.

Impaired bone quality in the superolateral femoral neck occurs independent of hip geometry and bone mineral density.

Skeletal adaptation is substantially influenced by mechanical loads. Osteocytes and their lacuno-canalicular network have been identified as a key player in load sensation and bone quality regulation. In the femoral neck, one of the most common fracture sites, a complex loading pattern with lower habitual loading in the superolateral neck and higher compressive stresses in the inferomedial neck is present. Variations in the femoral neck-shaft angle (NSA), i.e., coxa vara or coxa valga, provide the opportunity to examine the influence of loading patterns on bone quality. We obtained femoral neck specimens of 28 osteoarthritic human subjects with coxa vara, coxa norma and coxa valga during total hip arthroplasty. Bone mineral density (BMD) was assessed preoperatively by dual energy X-ray absorptiometry (DXA). Cortical and trabecular microstructure and three-dimensional osteocyte lacunar characteristics were assessed in the superolateral and inferomedial neck using ex vivo high resolution micro-computed tomography. Additionally, BMD distribution and osteocyte lacunar characteristics were analyzed by quantitative backscattered electron imaging (qBEI). All groups presented thicker inferomedial than superolateral cortices. Furthermore, the superolateral site exhibited a lower osteocyte lacunar density along with lower lacunar sphericity than the inferomedial site, independent of NSA. Importantly, BMD and corresponding T-scores correlated with microstructural parameters at the inferomedial but not superolateral neck. In conclusion, we provide micromorphological evidence for fracture vulnerability of the superolateral neck, which is independent of NSA and BMD. The presented bone qualitative data provide an explanation why DXA may be insufficient to predict a substantial proportion of femoral neck fractures. STATEMENT OF SIGNIFICANCE: The femoral neck, one of the most common fracture sites, is subject to a complex loading pattern. Site-specific differences (i.e., superolateral vs. inferomedial) in bone quality influence fracture risk, but it is unclear how this relates to hip geometry and bone mineral density (BMD) measurements in vivo. Here, we examine femoral neck specimens using a variety of high-resolution imaging techniques and demonstrate impaired bone quality in the superolateral compared to the inferomedial neck. Specifically, we found impaired cortical and trabecular microarchitecture, mineralization, and osteocyte properties, regardless of neck-shaft angle. Since BMD correlated with bone quality of the inferomedial but not the superolateral neck, our results illustrate why bone densitometry may not predict a substantial proportion of femoral neck fractures.

The WNT1G177C mutation specifically affects skeletal integrity in a mouse model of osteogenesis imperfecta type XV.

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 Wnt1G177C/G177C mice were viable and did not display defects in brain development, but the majority of 24-week-old Wnt1G177C/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.

Allograft Chip Incorporation in Acetabular Reconstruction: Multiscale Characterization Revealing Osteoconductive Capacity.

BACKGROUND: Impacted bone-grafting with morselized allograft chips is commonly used to reconstruct acetabular bone defects in revision total hip arthroplasty (THA). While the overall clinical outcome of this procedure is described to be excellent, the microstructural basis and histological determinants of allograft incorporation remained to be further elucidated. METHODS: The acetabula of 23 individuals with documented previous use of allograft chips during revision THA were explanted post mortem. The time that the allografts were in situ averaged 10.3 ± 4.5 years (range, 1.2 to 19.8 years). The host bone (HB)-allograft bone (AB) interface was characterized using a suite of high-resolution (HR) imaging techniques including HR-peripheral quantitative computed tomography (HR-pQCT), histological analysis, cellular histomorphometry, and scanning electron microscopy. RESULTS: AB could be identified in 16 of the 23 cases. The HB and AB showed overlap (i.e., ingrowth) in 91.3% of the total interface. The mean ingrowth was 2.2 ± 1.0 mm with a maximum of 4.7 ± 2.1 mm. The periphery of the AB showed a tight interconnection with the HB associated with increased bone remodeling indices and increased trabecular thickness. While no association between the time in situ and the ingrowth was observed, the bone defect area was positively associated with the thickness of a fibrosis layer separating the ingrowth zone from the AB. CONCLUSIONS: Allograft chips in revision THA form an adequate osseous foundation with successful incorporation through ingrowth of the HB (i.e., osteoconduction). While complete remodeling was not observed, larger defects were associated with fibrosis formation, which may compromise stability. CLINICAL RELEVANCE: Our study provides the first systematic, multiscale long-term evaluation of chip allograft incorporation in revision THA to underscore its successful clinical use. As larger defects were associated with fibrous ingrowth, structural allografts may be superior for larger defects in terms of long-term outcomes.

Response of the ENPP1-Deficient Skeletal Phenotype to Oral Phosphate Supplementation and/or Enzyme Replacement Therapy: Comparative Studies in Humans and Mice.

Inactivating mutations in human ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) may result in early-onset osteoporosis (EOOP) in haploinsufficiency and autosomal recessive hypophosphatemic rickets (ARHR2) in homozygous deficiency. ARHR2 patients are frequently treated with phosphate supplementation to ameliorate the rachitic phenotype, but elevating plasma phosphorus concentrations in ARHR2 patients may increase the risk of ectopic calcification without increasing bone mass. To assess the risks and efficacy of conventional ARHR2 therapy, we performed comprehensive evaluations of ARHR2 patients at two academic medical centers and compared their skeletal and renal phenotypes with ENPP1-deficient Enpp1asj/asj mice on an acceleration diet containing high phosphate treated with recombinant murine Enpp1-Fc. ARHR2 patients treated with conventional therapy demonstrated improvements in rickets, but all adults and one adolescent analyzed continued to exhibit low bone mineral density (BMD). In addition, conventional therapy was associated with the development of medullary nephrocalcinosis in half of the treated patients. Similar to Enpp1asj/asj mice on normal chow and to patients with mono- and biallelic ENPP1 mutations, 5-week-old Enpp1asj/asj mice on the high-phosphate diet exhibited lower trabecular bone mass, reduced cortical bone mass, and greater bone fragility. Treating the Enpp1asj/asj mice with recombinant Enpp1-Fc protein between weeks 2 and 5 normalized trabecular bone mass, normalized or improved bone biomechanical properties, and prevented the development of nephrocalcinosis and renal failure. The data suggest that conventional ARHR2 therapy does not address low BMD inherent in ENPP1 deficiency, and that ENPP1 enzyme replacement may be effective for correcting low bone mass in ARHR2 patients without increasing the risk of nephrocalcinosis. © 2021 American Society for Bone and Mineral Research (ASBMR).

Bacterial adhesion characteristics on implant materials for intervertebral cages: titanium or PEEK for spinal infections?

PURPOSE: Surgical intervention with intercorporal stabilisation in spinal infections is increasingly needed. Our aim was to compare titanium and polyetheretherketon (PEEK) cages according to their adhesion characteristics of different bacteria species in vitro. METHODS: Plates made from PEEK, polished titanium (Ti), two-surface-titanium (TiMe) (n = 2-3) and original PEEK and porous trabecular structured titanium (TiLi) interbody cages (n = 4) were inoculated in different bacterial solutions, S.aureus (MSSA, MRSA), S.epidermidis and E.coli. Growth characteristics were analysed. Biofilms and bacteria were visualised using confocal- and electron microscopy. RESULTS: Quantitative adherence of MSSA, MRSA, S.epidermidis and E.coli to Ti, TiMe and PEEK plates were different, with polished titanium being mainly advantageous over PEEK and TiMe with significantly less counts of colony forming units (CFU) for MRSA after 56 h compared to TiMe and at 72 h compared to PEEK (p = 0.04 and p = 0.005). For MSSA, more adherent bacteria were detected on PEEK than on TiMe at 32 h (p = 0.02). For PEEK and TiLi cages, significant differences were found after 8 and 72 h for S.epidermidis (p = 0.02 and p = 0.008) and after 72 h for MSSA (p = 0.002) with higher bacterial counts on PEEK, whereas E.coli showed more CFU on TiLi than PEEK (p = 0.05). Electron microscopy demonstrated enhanced adhesion in transition areas. CONCLUSION: For S.epidermidis, MSSA and MRSA PEEK cages showed a higher adherence in terms of CFU count, whereas for E.coli PEEK seemed to be advantageous. Electron microscopic visualisation shows that bacteria did not adhere at the titanium mesh structure, but at the border zones of polished material to rougher parts.

Piezo1 Inactivation in Chondrocytes Impairs Trabecular Bone Formation.

The skeleton is a dynamic tissue continuously adapting to mechanical stimuli. Although matrix-embedded osteocytes are considered as the key mechanoresponsive bone cells, all other skeletal cell types are principally exposed to macroenvironmental and microenvironmental mechanical influences that could potentially affect their activities. It was recently reported that Piezo1, one of the two mechanically activated ion channels of the Piezo family, functions as a mechanosensor in osteoblasts and osteocytes. Here we show that Piezo1 additionally plays a critical role in the process of endochondral bone formation. More specifically, by targeted deletion of Piezo1 or Piezo2 in either osteoblast (Runx2Cre) or osteoclast lineage cells (Lyz2Cre), we observed severe osteoporosis with numerous spontaneous fractures specifically in Piezo1Runx2Cre mice. This phenotype developed at an early postnatal stage and primarily affected the formation of the secondary spongiosa. The presumptive Piezo1Runx2Cre osteoblasts in this region displayed an unusual flattened appearance and were positive for type X collagen. Moreover, transcriptome analyses of primary osteoblasts identified an unexpected induction of chondrocyte-related genes in Piezo1Runx2Cre cultures. Because Runx2 is not only expressed in osteoblast progenitor cells, but also in prehypertrophic chondrocytes, these data suggested that Piezo1 functions in growth plate chondrocytes to ensure trabecular bone formation in the process of endochondral ossification. To confirm this hypothesis, we generated mice with Piezo1 deletion in chondrocytes (Col2a1Cre). These mice essentially recapitulated the phenotype of Piezo1Runx2Cre animals, because they displayed early-onset osteoporosis with multiple fractures, as well as impaired formation of the secondary spongiosa with abnormal osteoblast morphology. Our data identify a previously unrecognized key function of Piezo1 in endochondral ossification, which, together with its role in bone remodeling, suggests that Piezo1 represents an attractive target for the treatment of skeletal disorders. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Accelerated tooth movement in Rsk2-deficient mice with impaired cementum formation.

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.

Symptomatic Intraosseous Vascular Malformation of Infraorbital Rim: A Case Report With Literature Survey.

BACKGROUND/AIM: Intraosseous orbital hemangiomas or vascular malformations (VM) are rare. This report is intended to complement the experience of diagnosing and treating a rare vascular lesion at this site. Special attention is paid to three-dimensional imaging and the morphological distinction between the two entities in this location. CASE REPORT: A 54-year-old female was examined and surgically treated for an exophytic firm mass of the infraorbital, which had become palpable as a hard mass due to growth in size. At first, a bone tumor, for example, an osteoma, was suspected. Intraoperatively, an osseous expansion with distinct fenestrations of the newly grown bone's surface, was detected. The lesion was firmly attaching to the orbital rim. The densely vascularized tumor was well defined to the soft tissues but had grown in continuity from the orbital floor and rim. Vascularized cavities caused the tumor to have a slightly reddish color. The histological examination confirmed the suspicion of the lesion's vascular origin. The lesion's immunohistochemical expression profile approved the final diagnosis of intraosseous VM. CONCLUSION: The symptoms of intraosseous vascular lesions of the orbit are determined by location and size. Modern imaging techniques facilitate the estimation of tumor-like expansion of lesions. However, the imaging characteristics of intraosseous vascular lesions are very variable. The symptoms of the patient presented herein show that growth phases of a vascular orbital malformation can occur in later stages of life and are initially indistinguishable from a neoplasm. In individual cases, patient care necessitates advanced diagnostic measures to establish the diagnosis and determine surgical therapy.

Gnathodiaphyseal dysplasia is not recapitulated in a respective mouse model carrying a mutation of the Ano5 gene.

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.