Journal of Bone and Mineral Research最新文献

Melorheostosis is a rare disease where excessive bone overgrowth and exostoses cause deformity and pain. Somatic mutations of MAP2K1 result in hyperactivation of the ERK pathway in osteoblasts and increase in vitro mineralization. There is no effective treatment. Because melorheostosis osteoblasts show increased expression of cell cycle proliferation-related molecules compared to controls, we hypothesized that rapid progression through the cell cycle contributes to the bony overgrowth. We used an induced pluripotent stem cell (iPSCs) model from unaffected and affected regions of patient skin fibroblasts. We then differentiated iPSCs into induced mesenchymal stromal cells (iMSCs), and then into osteoblasts. Using propidium iodide (PI) mediated cell cycle assay with flow cytometry, we recorded that affected iMSCs (and primary patient osteoblasts) have a higher proportion of proliferative cells than unaffected. We noticed that affected osteoblasts and iMSCs have elevated expression of phospho-Rb-a crucial molecule for G1 to S transition. Immunofluorescence confirmed a significantly higher cell proliferation marker Ki-67 index in affected iMSCs. EdU incorporation assays validated higher percentage of S phase cells in affected populations. We applied the FDA-approved CDK4 inhibitor palbociclib to iMSCs and primary osteoblasts and found significant reduction of phospho-Rb. Palbociclib treated iMSCs & osteoblasts displayed elevated G0/G1 peak on flow cytometry and lowered EdU incorporation, thus confirming blockade of cell cycle progression by limiting S phase entry. Interestingly, palbociclib treatment for an initial 5 d of total 21 d of osteogenic stimulation restricts mineralization in affected cells to a greater extent than unaffected, suggesting that increased proliferation is contributing to the bone growth phenotype in patients. Thus, our data suggest targeting cell cycle machinery can be a potential therapeutic approach for melorheostosis patients.

Osteogenesis imperfecta (OI) is a heterogenous type 1 collagenopathy characterized by recurrent fractures, decreased bone mass, and shorter stature. Bisphosphonates reduce fracture incidence in children with OI but do not improve growth velocity. C-type natriuretic peptide (CNP) is produced in the growth plate (also in the brain and heart) and positively regulates linear bone growth; people with OI have been shown to have a reduced serum level of CNP. This pilot study evaluated whether a CNP analog combined with alendronate (ALN) improves growth and bone mineral density in oim/oim (OIM) mice, a model of moderate-to-severe Type III OI. Two-week-old OIM and WT mice received weekly ALN and one of three CNP regimens: 10 μg/kg three days/week (low), 20 μg/kg three days/week (medium), or 20 μg/kg five days/week (high). Controls received saline. Faxitron images were taken at two, eight, and 14 weeks (sacrifice) to assess fracture incidence and measure femoral length and vertebral height. Microcomputed tomography (micro-CT) was used to assess bone microstructural parameters of the femur ex vivo. The high-dose group had no fractures post-sacrifice; one fracture each was observed in the low and medium dose groups. Femoral length increased in all treated groups, with the high dose group showing the greatest increase (8.2%, significant) in OIM mice. Vertebral height increased in all treated groups; low and high dose groups had greater, comparable increases than the medium group in OIM mice. All treated groups showed increased trabecular bone mineral density (BMD). Cortical tissue mineral density (TMD), BMD, and thickness were also elevated in all treated groups compared to controls. In conclusion, CNP analog adjuvant treatment enhanced linear growth and bone quality without compromising fracture reduction, providing benefits not seen with bisphosphonates alone. These results will inform optimal dosing for future studies. A full murine study is planned to further evaluate therapeutic potential for translation to humans.

Hypophosphatasia is a rare genetic disease caused by deficient alkaline phosphatase activity. In adults, this causes functional limitations, substantial disability with pain and reduced quality of life. This Phase 1b, single-center, open-label trial investigated ilofotase alfa, a fully human recombinant protein intended as enzyme replacement therapy, in adults with hypophosphatasia. Changes in plasma levels of alkaline phosphatase substrates inorganic pyrophosphate and pyridoxal 5'-phosphate were evaluated. Participants were randomized 1:1 to receive at 0.8 or 3.2 mg/kg ilofotase alfa intravenously over one hour. Twelve participants were enrolled and completed the trial. At baseline, all participants had reduced alkaline phosphatase activity and elevated pyridoxal 5'-phosphate. The greatest reduction in inorganic pyrophosphate and pyridoxal 5'-phosphate occurred two hours after start of dosing in both treatment groups. Across the 10-day follow-up period, inorganic pyrophosphate values returned to baseline levels more rapidly in the 0.8 mg/kg group compared with the 3.2 mg/kg group. Mean circulating alkaline phosphatase activity peaked 24 hours after dosing and subsequently declined but remained above the lower limit of normal throughout the study. A dose-proportional increase in ilofotase alfa was observed, reaching peak concentration one-hour post-infusion. Eight treatment-emergent adverse events occurred, all classified as mild. These data demonstrate that single-dose ilofotase alfa enhances alkaline phosphatase activity and results in dose-dependent reductions in primary disease-specific biomarkers without undesired effects on mineral homeostasis.

The postnatal growth plate undergoes dynamic morphogenetic changes essential for endochondral bone formation. While morphogen signaling in this context is well studied, microRNA-mediated post-transcriptional control is poorly understood. Here, we identify miR-433-3p (miR-433) as a key regulator of chondrocyte proliferation and hypertrophy, acting in part through direct targeting of vital chondrocyte genes. miR-433 is evolutionarily conserved and prominently expressed in precursor chondrocytes embryonically and in the proliferating zone of the growth plate postnatally. To interrogate miR-433 function in vivo, we generated a conditional miR-433 tough decoy (competitive inhibitor) mouse model to decrease endogenous miR-433 activity in a lineage-restricted manner. Male and female mice expressing miR-433 tough decoy in Prrx1-expressing skeletal progenitors and their progeny exhibited shortened and narrower femurs, while significantly decreased trabecular bone volume was only apparent in males. Male miR-433 decoy mice had disorganized growth plates with fewer resting zone cells, abnormal hypertrophic-like cells in the proliferative zone and delayed secondary ossification center development. These defects were accompanied by elevated expression of Sox9, Ihh, PTHrP, Bmpr1a, as well as increased expression of validated miR-433 targets Runx2, Hdac6, and Hif1a. Tempering miR-433 activity increased proliferation in the resting zone at one and three weeks of age, and intensified SOX9 immunofluorescence throughout growth plate, including the hypertrophic zone. The miR-433 target RUNX2 was ectopically expressed within the proliferating zone and showed increased expression in the hypertrophic zone, consistent with premature hypertrophic transition. Luciferase assays confirmed direct targeting of Bmpr1a and Ihh by miR-433. Given that BMP signaling induces Sox9 and IHH promotes Runx2 expression, miR-433 may act as a molecular brake on both BMP and Hedgehog signaling axes, contributing to the spatial restriction of transcriptional programs driving chondrocyte maturation, thereby safeguarding orderly chondrocyte differentiation and bone elongation.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a devastating ultrarare genetic premature aging disease resulting in early atherosclerosis and death during adolescence due to heart failure. Structures of mesenchymal origin, including bone, fat, and muscle, create a progressive skeletal dysplasia, lifelong failure to thrive, and unique bone phenotype. Characterizing the interaction between muscle and bone has emerged as a powerful tool for defining drivers of bone disease in other conditions but has not been previously explored in HGPS. We examined the "muscle-bone unit" using radial pQCT in youth with HGPS aged 2 to 18 years before and after treatment with lonafarnib, a farnesyltransferase inhibitor that extends HGPS lifespan. Untreated radii displayed highly abnormal shapes in 70% of individuals spanning all ages. Compared to controls, HGPS forearm muscle and radial area were lower (p<0.001) and grew more slowly (muscle β=1.4 cm2/year vs. 0.3 cm2/year in HGPS; radius β=5.8 mm2/year vs. 0.5 mm2/year in HGPS). Fat area decreased with age (β=-0.2 cm2/year, p<0.001) and muscle area, normalized for either BMI or radial length, was reduced in HGPS (p=0.02 and p=<0.001, respectively). These normalized outcomes were similar to controls at younger ages but diverged as patients aged. Radial architectural changes were present even before changes in muscle area and represent a pattern distinct from the normal aging process and other muscle-wasting pediatric conditions. Lonafarnib therapy did not normalize the muscle-bone phenotype after 24 months, although some individuals (25%) had partial normalization of radial shape. These results demonstrate that the muscle-bone unit is uncoupled in children with HGPS. Normal muscle mass for body size at younger ages implies that there is an opportunity for early treatment to avoid impending pathology. New strategies are needed to ameliorate this phenotype in HGPS, and this study provides a benchmark for gauging future therapies.

Fractures and pseudofractures cause considerable morbidity in adults with X-linked hypophosphatemia (XLH). They frequently occur in cortically-enriched bones of the lower extremities. Burosumab, a neutralizing antibody to FGF23, heals fractures in adults with XLH, presumably by healing osteomalacia. Histomorphometry has documented healing of osteomalacia in trabecular bone. The effects of burosumab on cortical bone have not been reported. Therefore, 3D-DXA measurements of the proximal femur were used to examine the impact of 1 yr of burosumab therapy on cortical and trabecular bone in symptomatic adults with XLH. Twenty volunteers from the registration trial for burosumab were separately consented for this study. DXA scans of the hip were obtained before and 6, 12, and 18-24 mo after drug therapy (1 mg/kg every 4 wk). 3D-DXA analyses were performed using 3D-Shaper software (3D-Shaper Medical). Changes in FN areal BMD (aBMD), TH aBMD, TH trabecular volumetric BMD (vBMD), FN trabecular vBMD, TH cortical surface BMD (sBMD), FN cortical sBMD, and FN cross-sectional moment of inertia (CSMI) were analyzed. Both TH and FN trabecular vBMD showed significant increases over the course of therapy (13.6% and 14.1% respectively at the end of treatment compared to baseline; p < .0001 for each). Fractures and pseudofractures often occur in the cortically-enriched FN in XLH. Burosumab induced a significant increase in FN cortical sBMD between 6 and 12 mo (p < .05) and between 6 and 18-24 mo of drug treatment (p < .05). The FN CSMI, an indicator of FN strength, also significantly increased at 12 and 18-24 mo when compared to baseline; p < .05 and p < .01, respectively. These data demonstrate that burosumab increases both cortical and trabecular bone in the hip, a site of frequent fracture in XLH.

Endochondral ossification is a highly coordinated process involving distinct progenitor cell populations within the mesenchymal condensation and subsequent cartilage anlage and perichondrium, all of which drive skeletal formation. Cell-type specific lineage tracing conducted to understand fetal bone development has revealed various fates of early skeletal cells. However, the underlying continuous and precise cellular dynamics of fetal skeletal cells, particularly along the dorsoventral axis, remain unclear. Here, we show that spatiotemporally specific skeletal progenitor cells in the early developmental stage contribute to the dorsal-ventral axis in a manner that is strictly determined during initial developmental stages. Lineage-tracing experiments using Fgfr3-creER and Dlx5-creER lines revealed that Fgfr3+ cells in mesenchymal condensation exclusively contributed to hypertrophic chondrocytes and the dorsal side of the resting and proliferating zones within the cartilage anlage. These cells made dorsal-restricted contributions to skeletal development, including growth plate chondrocytes, trabecular and cortical osteoblasts, and bone marrow stromal cells. Functional ablation of Fgfr3+ cells using the Rosa26iDTA (inducible diphtheria toxin fragment A) allele during the mesenchymal condensation stage caused severe disruption in long-bone development, underscoring its indispensable role in initiating skeletal growth. Collectively, these findings suggest that the condensation stage is pivotal for the formation of skeletal progenitors and dorsoventral patterning during bone development. Understanding these mechanisms will provide insight into skeletal growth disorders and therapeutic strategies for bone regeneration.