Cannabidiol,a Major Non‐Psychotropic Cannabis Constituent Enhances Fracture Healing and Stimulates Lysyl Hydroxylase Activity in Osteoblasts

Cannabinoid ligands regulate bone mass, but skeletal effects of cannabis (marijuana and hashish) have not been reported. Bone fractures are highly prevalent, involving prolonged immobilization and discomfort. Here we report that the major non‐psychoactive cannabis constituent, cannabidiol (CBD), enhances the biomechanical properties of healing rat mid‐femoral fractures. The maximal load and work‐to‐failure, but not the stiffness, of femurs from rats given a mixture of CBD and Δ⁹‐tetrahydrocannabinol (THC) for 8 weeks were markedly increased by CBD. This effect is not shared by THC (the psychoactive component of cannabis), but THC potentiates the CBD stimulated work‐to‐failure at 6 weeks postfracture followed by attenuation of the CBD effect at 8 weeks. Using micro–computed tomography (μCT), the fracture callus size was transiently reduced by either CBD or THC 4 weeks after fracture but reached control level after 6 and 8 weeks. The callus material density was unaffected by CBD and/or THC. By contrast, CBD stimulated mRNA expression of Plod1 in primary osteoblast cultures, encoding an enzyme that catalyzes lysine hydroxylation, which is in turn involved in collagen crosslinking and stabilization. Using Fourier transform infrared (FTIR) spectroscopy we confirmed the increase in collagen crosslink ratio by CBD, which is likely to contribute to the improved biomechanical properties of the fracture callus. Taken together, these data show that CBD leads to improvement in fracture healing and demonstrate the critical mechanical role of collagen crosslinking enzymes. © 2015 American Society for Bone and Mineral Research.     

Sclerostin antibody improves skeletal parameters in a Brtl/+ mouse model of osteogenesis imperfecta

Osteogenesis imperfecta (OI) is a genetic bone dysplasia characterized by osteopenia and easy susceptibility to fracture. Symptoms are most prominent during childhood. Although antiresorptive bisphosphonates have been widely used to treat pediatric OI, controlled trials show improved vertebral parameters but equivocal effects on long‐bone fracture rates. New treatments for OI are needed to increase bone mass throughout the skeleton. Sclerostin antibody (Scl‐Ab) therapy is potently anabolic in the skeleton by stimulating osteoblasts via the canonical wnt signaling pathway, and may be beneficial for treating OI. In this study, Scl‐Ab therapy was investigated in mice heterozygous for a typical OI‐causing Gly→Cys substitution in col1a1. Two weeks of Scl‐Ab successfully stimulated osteoblast bone formation in a knock‐in model for moderately severe OI (Brtl/+) and in WT mice, leading to improved bone mass and reduced long‐bone fragility. Image‐guided nanoindentation revealed no alteration in local tissue mineralization dynamics with Scl‐Ab. These results contrast with previous findings of antiresorptive efficacy in OI both in mechanism and potency of effects on fragility. In conclusion, short‐term Scl‐Ab was successfully anabolic in osteoblasts harboring a typical OI‐causing collagen mutation and represents a potential new therapy to improve bone mass and reduce fractures in pediatric OI. © 2013 American Society for Bone and Mineral Research     

Mortality and Causes of Death in Patients With Osteogenesis Imperfecta: A Register‐Based Nationwide Cohort Study

Osteogenesis imperfecta (OI) is a hereditary connective tissue disease that causes frequent fractures. Little is known about causes of death and length of survival in OI. The objective of this work was to calculate the risk and cause of death, and the median survival time in patients with OI. This study was a Danish nationwide, population‐based and register‐based cohort study. We used National Patient Register data from 1977 until 2013 with complete long‐term follow‐up. Participants comprised all patients registered with the diagnosis of OI from 1977 until 2013, and a reference population matched five to one to the OI cohort. We calculated hazard ratios for all‐cause mortality and subhazard ratios for cause‐specific mortality in a comparison of the OI cohort and the reference population. We also calculated all‐cause mortality hazard ratios for males, females, and age groups (0 to 17.99 years, 18.00 to 34.99 years, 35.00 to 54.99 years, 55.00 to 74.99 years, and >75 years). We identified 687 cases of OI (379 women) and included 3435 reference persons (1895 women). A total of 112 patients with OI and 257 persons in the reference population died during the observation period. The all‐cause mortality hazard ratio between the OI cohort and the reference population was 2.90. The median survival time for males with OI was 72.4 years, compared to 81.9 in the reference population. The median survival time for females with OI was 77.4 years, compared to 84.5 years in the reference population. Patients with OI had a higher risk of death from respiratory diseases, gastrointestinal diseases, and trauma. We were limited by the lack of clinical information about phenotype and genotype of the included patients. Patients with OI had a higher mortality rate throughout their life compared to the general population. © 2016 American Society for Bone and Mineral Research.     

Compound Heterozygous Frameshift Mutations in MESD Cause a Lethal Syndrome Suggestive of Osteogenesis Imperfecta Type XX

Multiple genes are known to be associated with osteogenesis imperfecta (OI), a phenotypically and genetically heterogenous bone disorder, marked predominantly by low bone mineral density and increased risk of fractures. Recently, mutations affecting MESD, which encodes for a chaperone required for trafficking of the low-density lipoprotein receptors LRP5 and LRP6 in the endoplasmic reticulum, were described to cause autosomal-recessive OI XX in homozygous children. In the present study, whole-exome sequencing of three stillbirths in one family was performed to evaluate the presence of a hereditary disorder. To further characterize the skeletal phenotype, fetal autopsy, bone histology, and quantitative backscattered electron imaging (qBEI) were performed, and the results were compared with those from an age-matched control with regular skeletal phenotype. In each of the affected individuals, compound heterozygous mutations in MESD exon 2 and exon 3 were detected. Based on the skeletal phenotype, which was characterized by multiple intrauterine fractures and severe skeletal deformity, OI XX was diagnosed in these individuals. Histological evaluation of MESD specimens revealed an impaired osseous development with an altered osteocyte morphology and reduced canalicular connectivity. Moreover, analysis of bone mineral density distribution by qBEI indicated an impaired and more heterogeneous matrix mineralization in individuals with MESD mutations than in controls. In contrast to the previously reported phenotypes of individuals with OI XX, the more severe phenotype in the present study is likely explained by a mutation in exon 2, located within the chaperone domain of MESD, that leads to a complete loss of function, which indicates the relevance of MESD in early skeletal development. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..     

Osteoblast Malfunction Caused by Cell Stress Response to Procollagen Misfolding in α2(I)‐G610C Mouse Model of Osteogenesis Imperfecta

Glycine (Gly) substitutions in collagen Gly‐X‐Y repeats disrupt folding of type I procollagen triple helix and cause severe bone fragility and malformations (osteogenesis imperfecta [OI]). However, these mutations do not elicit the expected endoplasmic reticulum (ER) stress response, in contrast to other protein‐folding diseases. Thus, it has remained unclear whether cell stress and osteoblast malfunction contribute to the bone pathology caused by Gly substitutions. Here we used a mouse with a Gly610 to cysteine (Cys) substitution in the procollagen α2(I) chain to show that misfolded procollagen accumulation in the ER leads to an unusual form of cell stress, which is neither a conventional unfolded protein response (UPR) nor ER overload. Despite pronounced ER dilation, there is no upregulation of binding immunoglobulin protein (BIP) expected in the UPR and no activation of NF‐κB signaling expected in the ER overload. Altered expression of ER chaperones αB crystalline and HSP47, phosphorylation of EIF2α, activation of autophagy, upregulation of general stress response protein CHOP, and osteoblast malfunction reveal some other adaptive response to the ER disruption. We show how this response alters differentiation and function of osteoblasts in culture and in vivo. We demonstrate that bone matrix deposition by cultured osteoblasts is rescued by activation of misfolded procollagen autophagy, suggesting a new therapeutic strategy for OI. © 2016 American Society for Bone and Mineral Research.     

Strontium Ranelate Reduces the Fracture Incidence in a Growing Mouse Model of Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a genetic bone dysplasia characterized by brittle bones with increased fracture risk. Although current treatment options to improve bone strength in OI focus on antiresorptive bisphosphonates, controlled clinical trials suggest they have an equivocal effect on reducing fracture risk. Strontium ranelate (SrR) is a promising therapy with a dual mode of action that is capable of simultaneously maintaining bone formation and reducing bone resorption, and may be beneficial for the treatment of OI. In this study, SrR therapy was investigated to assess its effects on fracture frequency and bone mass and strength in an animal model of OI, the oim/oim mouse. Three‐week‐old oim/oim and wt/wt mice were treated with either SrR or vehicle (Veh) for 11 weeks. After treatment, the average number of fractures sustained by SrR‐treated oim/oim mice was significantly reduced compared to Veh‐treated oim/oim mice. Micro–computed tomographic (μCT) analyses of femurs showed that both trabecular and cortical bone mass were significantly improved with SrR treatment in both genotypes. SrR significantly inhibited bone resorption, whereas bone formation indices were maintained. Biomechanical testing revealed improved bone structural properties in both oim/oim and wild‐type (wt/wt) mice under the treatment, whereas no significant effects on bone brittleness and material quality were observed. In conclusion, SrR was able to effectively reduce fractures in oim/oim mice by improving bone mass and strength and thus represents a potential therapy for the treatment of pediatric OI. © 2015 American Society for Bone and Mineral Research.     

A Transgenic Mouse Model of OI Type V Supports a Neomorphic Mechanism of the IFITM5 Mutation

Osteogenesis imperfecta (OI) type V is characterized by increased bone fragility, long bone deformities, hyperplastic callus formation, and calcification of interosseous membranes. It is caused by a recurrent mutation in the 5' UTR of the IFITM5 gene (c.‐14C > T). This mutation introduces an alternative start codon, adding 5 amino acid residues to the N‐terminus of the protein. The mechanism whereby this novel IFITM5 protein causes OI type V is yet to be defined. To address this, we created transgenic mice expressing either the wild‐type or the OI type V mutant IFITM5 under the control of an osteoblast‐specific Col1a1 2.3‐kb promoter. These mutant IFITM5 transgenic mice exhibited perinatal lethality, whereas wild‐type IFITM5 transgenic mice showed normal growth and development. Skeletal preparations and radiographs performed on E15.5 and E18.5 OI type V transgenic embryos revealed delayed/abnormal mineralization and skeletal defects, including abnormal rib cage formation, long bone deformities, and fractures. Primary osteoblast cultures, derived from mutant mice calvaria at E18.5, showed decreased mineralization by Alizarin red staining, and RNA isolated from calvaria showed reduced expression of osteoblast differentiation markers such as Osteocalcin, compared with nontransgenic littermates and wild‐type mice calvaria, consistent with the in vivo phenotype. Importantly, overexpression of wild‐type Ifitm5 did not manifest a significant bone phenotype. Collectively, our results suggest that expression of mutant IFITM5 causes abnormal skeletal development, low bone mass, and abnormal osteoblast differentiation. Given that neither overexpression of the wild‐type Ifitm5, as shown in our model, nor knock‐out of Ifitm5, as previously published, showed significant bone abnormalities, we conclude that the IFITM5 mutation in OI type V acts in a neomorphic fashion. © 2014 American Society for Bone and Mineral Research.     

Impact of Genetic and Pharmacologic Inhibition of Myostatin in a Murine Model of Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a genetic connective tissue disorder characterized by compromised skeletal integrity, altered microarchitecture, and bone fragility. Current OI treatment strategies focus on bone antiresorptives and surgical intervention with limited effectiveness, and thus identifying alternative therapeutic options remains critical. Muscle is an important stimulus for bone formation. Myostatin, a TGF-β superfamily myokine, acts through ActRIIB to negatively regulate muscle growth. Recent studies demonstrated the potential benefit of myostatin inhibition with the soluble ActRIIB fusion protein on skeletal properties, although various OI mouse models exhibited variable skeletal responses. The genetic and clinical heterogeneity associated with OI, the lack of specificity of the ActRIIB decoy molecule for myostatin alone, and adverse events in human clinical trials further the need to clarify myostatin's therapeutic potential and role in skeletal integrity. In this study, we determined musculoskeletal outcomes of genetic myostatin deficiency and postnatal pharmacological myostatin inhibition by a monoclonal anti-myostatin antibody (Regn647) in the G610C mouse, a model of mild–moderate type I/IV human OI. In the postnatal study, 5-week-old wild-type and +/G610C male and female littermates were treated with Regn647 or a control antibody for 11 weeks or for 7 weeks followed by a 4-week treatment holiday. Inhibition of myostatin, whether genetically or pharmacologically, increased muscle mass regardless of OI genotype, although to varying degrees. Genetic myostatin deficiency increased hindlimb muscle weights by 6.9% to 34.4%, whereas pharmacological inhibition increased them by 13.5% to 29.6%. Female +/mstn +/G610C (Dbl.Het) mice tended to have similar trabecular and cortical bone parameters as Wt showing reversal of +/G610C characteristics but with minimal effect of +/mstn occurring in male mice. Pharmacologic myostatin inhibition failed to improve skeletal bone properties of male or female +/G610C mice, although skeletal microarchitectural and biomechanical improvements were observed in male wild-type mice. Four-week treatment holiday did not alter skeletal outcomes. © 2020 American Society for Bone and Mineral Research (ASBMR).     

Targeting the LRP5 Pathway Improves Bone Properties in a Mouse Model of Osteogenesis Imperfecta

The cell surface receptor low‐density lipoprotein receptor‐related protein 5 (LRP5) is a key regulator of bone mass and bone strength. Heterozygous missense mutations in LRP5 cause autosomal dominant high bone mass (HBM) in humans by reducing binding to LRP5 by endogenous inhibitors, such as sclerostin (SOST). Mice heterozygous for a knockin allele (Lrp5^p.A214V) that is orthologous to a human HBM‐causing mutation have increased bone mass and strength. Osteogenesis imperfecta (OI) is a skeletal fragility disorder predominantly caused by mutations that affect type I collagen. We tested whether the LRP5 pathway can be used to improve bone properties in animal models of OI. First, we mated Lrp5^+/p.A214V mice to Col1a2^+/p.G610C mice, which model human type IV OI. We found that Col1a2^+/p.G610C;Lrp5^+/p.A214V offspring had significantly increased bone mass and strength compared to Col1a2^+/p.G610C;Lrp5^+/+ littermates. The improved bone properties were not a result of altered mRNA expression of type I collagen or its chaperones, nor were they due to changes in mutant type I collagen secretion. Second, we treated Col1a2^+/p.G610C mice with a monoclonal antibody that inhibits sclerostin activity (Scl‐Ab). We found that antibody‐treated mice had significantly increased bone mass and strength compared to vehicle‐treated littermates. These findings indicate increasing bone formation, even without altering bone collagen composition, may benefit patients with OI. © 2014 American Society for Bone and Mineral Research.     

Alteration in the Extent of Collagen I Hydroxylation,Isolated from Femoral Heads of Women with a Femoral Neck Fracture Caused by Osteoporosis

The aim of this study was to investigate the extent of lysyl and prolyl hydroxylation of collagen I in osteoporosis and compare it with collagen I from ``bone healthy' individuals. Collagen I was isolated from femoral heads of osteoporotic women, from women suffering from osteoarthrosis of the hip, and from healthy women 60–85 years of age. The femoral heads were dissected into compact and trabecular bone of the neck region and from trabecular bone of the head region, and collagen I was extracted by limited pepsin digestion. The amino acid analysis of individual α-chains showed a remarkably higher degree of hydroxylation of lysine residues both in the α1(I)- and in the α2(I)-chains in osteoporotic bone compared with osteoarthrotic and ``normal' bone, whereas the prolyl hydroxylation was nearly unchanged. The lysyl overhydroxylation was observed in the compact as well as in the trabecular bone of osteoporotic femoral heads. These biochemical alterations may play a crucial role in the pathogenesis of osteoporosis. Received: 2 May 1996 / Accepted: 31 December 1996     

Mechanical Loading Stimulates Differentiation of Periodontal Osteoblasts in a Mouse Osteoinduction Model: Effect on Type I Collagen and Alkaline Phosphatase Genes

The effects of mechanical loading on the osteoblast phenotype remain unclear because of many variables inherent to the current experimental models. This study reports on utilization of a mouse tooth movement model and a semiquantitative video image analysis of in situ hybridization to determine the effect of mechanical loading on cell-specific expression of type I collagen (collagen I) and alkaline phosphatase (ALP) genes in periodontal osteoblasts, using nonosseous cells as an internal standard. The histomorphometric analysis showed intense osteoid deposition after 3 days of treatment, confirming the osteoinductive nature of the mechanical signal. The results of in situ hybridization showed that in control periodontal sites both collagen I and ALP mRNAs were expressed uniformly across the periodontium. Treatment for 24 hours enhanced the ALP mRNA level about twofold over controls and maintained that level of stimulation after 6 days. In contrast, collagen I mRNA level was not affected after 24 hours of treatment, but it was stimulated 2.8-fold at day 6. This increase reflected enhanced gene expression in individual osteoblasts, since the increase in osteoblast number was small. These results indicate that (1) the mouse model and a semiquantitative video image analysis are suitable for detecting osteoblast-specific gene regulation by mechanical loading; (2) osteogenic mechanical stress induces deposition of bone matrix primarily by stimulating differentiation of osteoblasts, and, to a lesser extent, by an increase in number of these cells; (3) ALP is an early marker of mechanically-induced differentiation of osteoblasts. (4) osteogenic mechanical stimulation in vivo produces a cell-specific 2.8-fold increase in collagen gene expression in mature, matrix-depositing osteoblasts located on the bone surface and within the osteoid layer. Received: 9 August 1999 / Accepted: 4 February 2000     

A New Subtype of Multiple Synostoses Syndrome Is Caused by a Mutation in GDF6 That Decreases Its Sensitivity to Noggin and Enhances Its Potency as a BMP Signal

Growth and differentiation factors (GDFs) are secreted signaling molecules within the BMP family that have critical roles in joint morphogenesis during skeletal development in mice and humans. Using genetic data obtained from a six‐generation Chinese family, we identified a missense variant in GDF6 (NP_001001557.1; p.Y444N) that fully segregates with a novel autosomal dominant synostoses (SYNS) phenotype, which we designate as SYNS4. Affected individuals display bilateral wrist and ankle deformities at birth and progressive conductive deafness after age 40 years. We find that the Y444N variant affects a highly conserved residue of GDF6 in a region critical for binding of GDF6 to its receptor(s) and to the BMP antagonist NOG, and show that this mutant GDF6 is a more potent stimulator of the canonical BMP signaling pathway compared with wild‐type GDF6. Further, we determine that the enhanced BMP activity exhibited by mutant GDF6 is attributable to resistance to NOG‐mediated antagonism. Collectively, our findings indicate that increased BMP signaling owing to a GDF6 gain‐of‐function mutation is responsible for loss of joint formation and profound functional impairment in patients with SYNS4. More broadly, our study highlights the delicate balance of BMP signaling required for proper joint morphogenesis and reinforces the critical role of BMP signaling in skeletal development. © 2015 American Society for Bone and Mineral Research.     

Application of a New Serum Assay for Type I Collagen Cross-Linked N-Telopeptides: Assessment of Diurnal Changes in Bone Turnover With and Without Alendronate Treatment

Biochemical markers of bone turnover are finding increased application in the investigation and management of skeletal diseases such as osteoporosis. The present study assessed for the first time the diurnal variation of serum type I collagen cross-linked N-telopeptides (NTx), a new serum-based marker of bone resorption, and the effect of antiresorptive therapy with alendronate on this marker in elderly osteopenic women. The concentrations of serum NTx were monitored over 24 hours in a randomly selected subset of 38 women (placebo n = 13, 69 ± 3 (SD) year; alendronate n = 25, 69 ± 3 year), who had completed 12–15 months of a larger (n = 120) randomized, double-blind, parallel group, placebo-controlled trial with alendronate 5 mg/day. Blood was obtained every 4 hours for measurement of serum NTx using a new chemiluminescent-based immunoassay. There was a significant diurnal variation of serum NTx (p = 0.001) in both the placebo and alendronate groups. Mean peak levels occurred at ∼0504 h with a mean nadir at ∼1320 h in the placebo group, with no significant difference on alendronate. Serum NTx was ∼25% lower in the alendronate group over the entire 24-hour period. Mean (SE) daytime (0800–2000) and nighttime (2200–0800) serum NTx values were 6.40 ± 0.30 versus 8.45 ± 0.58 nmol BCE/liter, and 7.42 ± 0.23 versus 10.01 ± 0.53 nmol BCE/liter for alendronate versus placebo, respectively (P≤ 0.003 for both comparisons). Combining the data of both treatment groups, serum NTx was significantly (P < 0.05) correlated with serum osteocalcin (r = 0.753) and urine NTx (r = 0.628) measurements previously obtained over the entire 24-hour period. Serum NTx has a significant diurnal variation and is responsive to antiresorptive therapy with alendronate. Alendronate reduces the amplitude but maintains the pattern of the 24-hour serum NTx profile. These data suggest that serum NTx may be a useful new marker of bone resorption. Received: 13 March 1997 / Accepted: 15 October 1997     

Abnormal Response to Physical Activity in Femurs after Heterozygous Inactivation of One Allele of the Col2a1 Gene for Type II Collagen in Mice

The objective of this study was to evaluate the influence of heterozygous inactivation of one allele of the type II collagen gene (Col2a1) on biomechanical properties and mineral density of bone under physical loading conditions. C57BL/6−TGN mice with heterozygous knockout (HZK) inactivation of Col2a1 gene and their nontransgenic littermate controls were housed in individual cages with running wheels for 9 and 15 months. The running activity of each mouse was monitored continuously throughout the experiment. Bone mineral density (BMD) of mice femora was measured using dual-energy X-ray absorptiometry (DXA) and peripheral quantitative computerized tomography (pQCT). Biomechanical properties were determined using three-point bending tests. Vertebral bone samples were prepared for quantitative polarized light microscopy and digital densitometry of proteoglycans. The concentration of total collagen and collagen cross-links were analyzed using high-performance liquid chromatograpy (HPLC). The average daily running distance was shorter for the HZK mice between the age of 4 and 15 months as compared with normal runners (P < 0.05). The ultimate breaking force was 14.8% and 23.6% (9 vs. 15 months) lower in HZK-runners than in wild-type runners. BMD of the femur was 6.1% lower in HZK-runners at the age of 9 months (P < 0.05). Physical activity increased cortical BMD in wild-type runners but not in the HZK runners at the age of 9 months. The collagen network of the HZK mice was less organized. There were only minor changes in BMD and mechanical and structural properties between sedentary HZK mice and their wild-type controls. Increased physical activity induced significantly lower bone density, mechanical properties, and organization of collagen fibers in male HZK mice. However, there were no major differences in biomechanical parameters between sedentary HZK and wild-type male mice. This suggests an important guiding role of collagen type II in bone remodelling and maturation.