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Heterotopic ossifications (HOs) are the pathologic process by which bone inappropriately forms outside of the skeletal system. Despite HOs being a persistent clinical problem in the general population, there are no definitive strategies for their prevention and treatment due to a limited understanding of the cellular and molecular mechanisms contributing to lesion development. One disease in which the development of heterotopic subcutaneous ossifications (SCOs) leads to morbidity is Albright hereditary osteodystrophy (AHO). Albright hereditary osteodystrophy is caused by heterozygous inactivation of GNAS, the gene that encodes the α-stimulatory subunit (Gα_s) of G proteins. Previously, we had shown using our laboratory's AHO mouse model that SCOs develop around hair follicles. Here we show that SCO formation occurs due to inappropriate expansion and osteogenic differentiation of cells that express alpha-smooth muscle actin and that are located within the dermal sheath. We also show in AHO patients and mice that secreted frizzled related protein 2 (SFRP2) expression is upregulated in regions of SCO formation and that elimination of Sfrp2 in male AHO mice leads to earlier development, greater severity, and acceleration of formation of SCOs. These studies provide key insights into the cellular and molecular mechanisms contributing to SCO development and have implications for potential therapeutic modalities not only for AHO patients but also for patients suffering from HOs with other etiologies.

Hypophosphatasia (HPP) is an inborn error of metabolism caused by loss-of-function variants in the ALPL gene, which encodes the tissue nonspecific isozyme of alkaline phosphatase (ALP). There is no typical phenotype in adults. We used a genotyping first approach to determine whether ALPL pathogenic variants were associated with musculoskeletal symptoms, mineral metabolism abnormalities, and an impact on quality of life. We recruited individuals with a pathogenic (or likely pathogenic) variant in ALPL gene (n = 26) and their relatives (n = 44). We performed genetic tests and compared the relatives with positive (n = 20) and negative (n = 24) genetic test. We applied standard questionnaires and physical tests (Brief Pain Inventory [BPI]; Western Ontario and McMaster Universities Arthritis [WOMAC]; Modified Hypophosphatasia Impact Patient Survey; Short Form of 36 Survey [SF-36]; and the Short Physical Performance Battery). In fasting blood samples, we measured creatinine, calcium, phosphate (P), parathyroid hormone (PTH), ALP, bone ALP, 25OHD-, 1,25(OH)2D, CTX, type 1 procollagen N-terminal peptide (PINP), osteocalcin, and tartrate-resistant acid phosphatase5b (TRACP5b). Relatives with positive genetic test had lower ALP (IU/L) [32.5(12.8) vs 87.8(32.6) p < .001], bone ALP (ng/mL) [6.3(4.3, 9.8) vs 17.5 (13.12-25.7) p < .001], PTH (pg/L) [28.6(20.6, 38.1) vs 40.05(25.7, 52.3) p = .03], and higher PLP(nmol/L) [162.0 (91.75, 337.5) vs 37.5 (18.25, 60.5) p < .001] and P(mmol/L) [1.36 (0.18) vs 1.05 (0.2) p < .001]. We did not find significant differences in fractures or musculoskeletal features between the groups. Greater pain scores were observed on BPI in relatives with positive genetic tests, and bone and muscle pain were more often reported by this group, but statistical tests were not significant. No differences were found in physical performance or quality of life. In conclusion, we assessed relatives of individuals with pathogenic or likely pathogenic variants in the ALPL gene regardless of the presence of signs and symptoms. Biochemical abnormalities were more common in gene-positive relatives, but the prevalence of musculoskeletal symptoms was comparable in relatives with positive and negative genetic tests.

Oral glucocorticoid (GC) therapy rapidly and deleteriously affects bone metabolism and blood glucose regulation. The gut microbiota regulates bone metabolism and a prior study found that Limosilactobacillus reuteri ATCC PTA6475 (L. reuteri) reduced bone loss over 12 mo in older women. Mice treated either with broad-spectrum antibiotics or with L. reuteri did not experience GC-induced trabecular bone loss. This proof-of-concept, randomized, double-blind, placebo-controlled trial aimed to investigate if daily supplementation with L. reuteri, compared with placebo, could mitigate or prevent the negative effects of oral GC on bone turnover and blood glucose regulation in healthy young adults. Twenty-one men and 29 women, aged 18-45, were randomized to either placebo or L. reuteri (1 × 10¹⁰ CFU/d) treatment for 2 wk, followed by open-label oral prednisolone 25 mg daily for 7 d. Primary outcomes were changes in blood bone status indices (osteocalcin, C-terminal telopeptide cross-links of collagen type-I (CTX), and type-I procollagen intact N-terminal propeptide [PINP]) from baseline to 7 d after starting oral GC. Secondary endpoints included changes in blood glucose levels using continuous glucose monitoring during the same period (ClinicalTrials.gov NCT04767711). Blood samples were collected from participants in the morning after overnight fasting. Forty-six participants completed the 30-d study. The L. reuteri and placebo groups were well balanced in terms of baseline characteristics (age, BMI, sex, dietary intake, and physical activity). No significant differences were found between L. reuteri vs placebo for percent changes in CTX (-0.3 [95%CI -19.2-18.7], p = .98) or PINP (4.2 [-6.3-14.8], p = .43), or in osteocalcin levels (14.2 [-7.8-36.3], p = .21), although the group-to-group difference in osteocalcin was larger. There was no effect of treatment on mean blood glucose (-0.1 [-0.3-0.1] mmol/L, p = .28). In conclusion, we failed to detect a significant effect of L. reuteri supplementation on GC-related adverse effects on bone status indices in this proof-of-concept RCT. Larger studies are needed to identify any potential smaller effects.

X-linked hypophosphatemia (XLH) is a rare inherited disorder characterized by elevated levels of FGF23, chronic hypophosphatemia, impaired bone mineralization, and chronic long-term manifestations. Treatment for XLH has been mainly focused on normalizing its biochemical abnormalities. Despite treatment, patients with XLH often present impaired physical function and decreased quality of life (QoL). We hypothesize that physical functionality and QoL are more strongly associated with chronic pain and decreased muscle mass than persistent biochemical abnormalities or exposure to conventional treatment. We conducted an observational, cross-sectional study with patients with XLH. Clinical records and biochemical parameters were assessed. QoL surveys SF36v.2 and WOMAC were applied. Functional status was measured by a physiatrist and an occupational therapist. Appendicular lean mass (ALM) was measured and compared with age and sex-matched healthy controls. Enthesopathies and osteoarthritis were evaluated. Pain was assessed using the Brief Pain Inventory, the Visual Analog Scale, and the Doleur Neuropathique-4 scales. Muscle strength was evaluated by the quadriceps muscle isometric strength (QMS) and physical performance with the 6-Minute Walk Test (6MWT) and the Functional Independence Measure (FIM) scale. A total of 30 patients were included: 21 females; median age of 32 yr. All participants had significant functional deficits, chronic pain, and reduced QoL. Limitations in daily activities were significantly associated with higher severity of pain, decreased ALM, lower QMS, and less distance in 6MWT (p < .05). Neither FIM scale, phosphate levels, FGF23, nor the lifetime exposure to conventional treatment was associated with these functional variables. In conclusion, impaired physical functionality in patients with XLH was associated with lower muscle mass, lower muscle strength, and severe chronic pain. These findings highlight the importance of, in addition to optimizing the biochemical control of the disease, expanding patient care including pain prevention and management as well as comprehensive physical therapy and rehabilitation.

Recent clinical studies have established a strong association between cigarette smoking and degenerative disc disease. Both in vitro and in vivo research indicated that cigarette smoke disrupts cellular homeostasis in the intervertebral disc (IVD), leading to spatiotemporal remodeling of the extracellular matrix, with a notable reduction in solute diffusivity within the cartilage endplate (CEP). As the CEP serves as a critical mechanical barrier and solute diffusion pathway for the IVD, both roles can be compromised by pathological changes in the tissue. This underscores the need for a more comprehensive examination of endplate remodeling during IVD degeneration, particularly in the context of cigarette smoking and cessation. The objective of this study was to perform a quantitative analysis of the structure-material property relationship changes in the endplate at tissue and cellular levels to determine how endplate mineralization progresses during IVD degeneration in the context of cigarette smoke exposure and cessation, using our previously developed Sprague-Dawley rat model. Our results indicate that cigarette smoke exposure-induced endplate remodeling is characterized by a higher CEP histological grade, increased aberrant CEP calcification level, and elevated bony endplate surface flatness score, all of which correlated with an accelerated chondrocyte cell life cycle. Smoke cessation alone was insufficient to reverse the mineralization progression in the endplate. Principal component analysis further identified alterations in endplate morphometry at the tissue level and disruptions in the chondrocyte life cycle at cellular level as key markers of degenerative remodeling. These findings establish endplate remodeling as a key indicator of smoke exposure-induced IVD degeneration and inform the development of novel therapeutic strategies aimed at preserving or improving disc health.

Vitamin D deficiency is common across the world. However, the standard clinical biomarker for vitamin D, 25OHD, may be a poor marker of vitamin D status, as most of circulating vitamin D is protein bound and not bioavailable. Free (unbound) vitamin D may therefore be a better marker of vitamin D status. We evaluated the relationship of free vitamin D with incident cardiovascular disease (CVD), heart failure (HF), kidney function decline (KFD) and fracture, among 786 participants in the Health Aging and body composition study. We used sequential models to assess hazard ratios (HRs) of each outcome that adjusted for age, sex, race, season of blood sampling, and study site, kidney function, serum calcium and phosphate, FGF 23, PTH, BMI, and vitamin D supplementation. The mean age of the 786 participants was 75 ± 3 yr, 53% were women, and 40% were Black. The median free vitamin D concentration was 5.3 (interquartile range 4.1-6.7) pg/mL. There were 157 cases of incident CVD, 123 cases of incident HF, 382 cases of incident KFD, and 178 fractures over 11 yr of follow-up. In fully adjusted models, a 2-fold greater free vitamin D was associated with lower risk of incident HF [HR 0.75, 95%CI,0.58-0.96 ] and greater risk KFD [1.25(1.03-1.52)]. We found no association between free vitamin D and incident CVD or fracture. We did not find evidence that free vitamin D was a superior marker of clinical outcomes compared to total 25OHD alone. Further studies are needed to elucidate the relationship of free vitamin D with clinical outcomes.

Mechanical loading is required for bone health and results in skeletal adaptation to optimize strength. Local nerve axons, particularly within the periosteum, may respond to load-induced biomechanical and biochemical cues. However, their role in the bone anabolic response remains controversial. We hypothesized that spatial alignment of periosteal nerves with sites of load-induced bone formation would clarify this relationship. To achieve this, we developed RadialQuant, a custom tool for spatial histomorphometry. Tibiae of control and neurectomized (sciatic/femoral nerve cut) pan-neuronal Baf53b-tdTomato reporter mice were loaded for 5 days. Bone formation and periosteal nerve axon density were then quantified simultaneously in non-decalcified sections of the mid-diaphysis using RadialQuant. In control animals, anabolic loading induced maximal periosteal bone formation at the site of peak compression, as has been reported previously. By contrast, loading did not significantly change overall periosteal nerve density. Neurectomy depleted ~90% of all periosteal axons, with near-total depletion on load-responsive surfaces. Neurectomy alone also caused de novo bone formation on the lateral aspect of the mid-diaphysis. However, neurectomy did not inhibit load-induced increases in periosteal bone area, mineralizing surface, or bone formation rate. Rather, neurectomy spatially redistributed load-induced bone formation toward the lateral tibial surface with a reduction in periosteal bone formation at the posterolateral apex (-63%) and enhancement at the lateral surface (+1360%). Altogether, this contributed to comparable load-induced changes in cortical bone area fraction. Our results show that local skeletal innervation modulates but is not required for skeletal adaptation to applied load in our model. This supports the continued use of loading and weight-bearing exercise as an effective strategy to increase bone mass, even in settings of peripheral nerve damage or dysfunction.

Hypophosphatasia (HPP) is an inherited error in metabolism resulting from loss-of-function variants in the ALPL gene, which encodes tissue-nonspecific alkaline phosphatase (TNAP). TNAP plays a crucial role in biomineralization of bones and teeth, in part by reducing levels of inorganic pyrophosphate (PP_i), an inhibitor of biomineralization. HPP onset in childhood contributes to rickets, including growth plate defects and impaired growth. In adulthood, osteomalacia from HPP contributes to increased fracture risk. HPP also affects oral health. The dentoalveolar complex, that is, the tooth and supporting connective tissues of the surrounding periodontia, include 4 unique hard tissues: enamel, dentin, cementum, and alveolar bone, and all can be affected by HPP. Premature tooth loss of fully rooted teeth is pathognomonic for HPP. Patients with HPP often have complex oral health issues that require multidisciplinary dental care, potentially involving general or pediatric dentists, periodontists, prosthodontists, and orthodontists. The scientific literature to date has relatively few reports on dental care of individuals with HPP. Animal models to study HPP included global Alpl knockout mice, Alpl mutation knock-in mice, and mice with tissue-specific conditional Alpl ablation, allowing for new studies on pathological mechanisms and treatment effects in dental and skeletal tissues. Enzyme replacement therapy (ERT) in the form of injected, recombinant mineralized tissue-targeted TNAP has been available for nearly a decade and changed the prognosis for those with HPP. However, effects of ERT on dental tissues remain poorly defined and limitations of the current ERT have prompted exploration of gene therapy approaches to treat HPP. Preclinical gene therapy studies are promising and may contribute to improved oral health in HPP.

Bariatric surgeries such as Roux-en-Y gastric bypass (RYGB) and adjustable gastric banding (AGB) lead to long-term deficits in bone density but are also accompanied by decreased weight, which may lower the impact force with falls. The aim of this study was to compare the long-term skeletal impact of RYGB and AGB using a biomechanical evaluation of load-to-strength ratio at the distal radius as a surrogate for wrist fracture risk. We conducted a cross-sectional study evaluating bone microarchitectural parameters and bone turnover in adults who received either RYGB or AGB surgery ≥10 yr ago (RYGB: n = 22; AGB: n = 23). Bone strength at the distal radius was estimated by microfinite element analysis from HR-pQCT. We used a single-spring biomechanical model to estimate impact force and then calculated load-to-strength ratio as a ratio of impact force to bone strength, with higher load-to-strength ratios representing a higher susceptibility to fracture. In multivariable analyses, the RYGB group had higher bone resorption marker C-telopeptide (CTX) levels, lower volumetric bone density, and worse cortical and trabecular microarchitectural parameters than the AGB group. Furthermore, estimated bone strength at the radius was lower in the RYGB group (3725 ± 139 N vs 4141 ± 157 N, p = .030), and the load-to-strength ratio was higher in RYGB group as compared with AGB (0.84 ± 0.04 vs 0.72 ± 0.05, p = .035), suggestive of higher propensity for wrist fracture. Taken together, these results indicate the long-term deleterious skeletal effects are more concerning with RYGB than AGB.

Biomineralization in bones and teeth is a highly regulated extracellular event. In the skeleton, mineralization at the tissue level is controlled within the collagenous extracellular matrix by both circulating and local factors. While systemic regulation of mineral ion homeostasis has been well-studied over many decades, much less is known about the regulation of mineralization at the local level directly within the extracellular matrix. Some local regulators have been identified, such as tissue-nonspecific alkaline phosphatase (TNAP), phosphate-regulating endopeptidase homolog X-linked (PHEX), pyrophosphate, and osteopontin, and others are currently under investigation. Dysregulation of the actions of enzyme-inhibitor substrate pairs engaged in mineralization (as we describe by the Stenciling Principle for extracellular matrix mineralization) leads to osteomalacic "soft bone" diseases, such as hypophosphatasia (HPP) and X-linked hypophosphatemia (XLH). This review addresses how advances in 3D imaging tools and software now allow contextual and correlative viewing and interpretation of mineralized tissue structure across most length scales. Contextualized and integrated 3D multiscale data obtained from these imaging modalities have afforded an unprecedented structural biology view of bone from the macroscale to the nanoscale. Such correlated volume imaging data is highly quantitative, providing not only an integrated view of the skeleton in health, but also a means to observe alterations that occur in disease. In the context of the many hierarchical levels of skeletal organization, here we summarize structural features of bone over multiple length scales, with a focus on nano- and microscale features as viewed by X-ray and electron tomography imaging methods (submicron μCT and FIB-SEM). We additionally summarize structural changes observed after dysregulation of the mineralization pathway, focusing here on the Hyp mouse model for XLH. More specifically, we summarize how mineral patterns/packs at the microscale (3D crossfibrillar mineral tessellation), and how this is defective in Hyp mouse bone and Hyp enthesis fibrocartilage.