Gain-of-Function of FGFR3 Accelerates Bone Repair Following Ischemic Osteonecrosis in Juvenile Mice.

Bone collapse, bone deformity, and a long treatment period are major clinical problems associated with juvenile ischemic osteonecrosis (JIO). Accelerating the process of bone repair in JIO is expected to shorten the treatment duration and better maintain morphology. We previously indicated that both bone formation and resorption were accelerated following distraction osteogenesis-mediated limb lengthening in genetically engineered mutant mice with a gain-of-function mutation in fibroblast growth factor receptor 3 (FGFR3) gene (i.e., Fgfr3 mice). The purpose of this study was to investigate the role of FGFR3 in the bone repair process following surgically induced ischemic osteonecrosis in the mutant mice. Epiphyseal deformity was less in the Fgfr3 mice compared to the wild-type mice at 6 weeks following ischemic osteonecrosis in skeletally immature age. Assessment of the morphology by micro-computed tomography (CT) revealed that the trabecular bone volume was increased in the Fgfr3 mice. Dynamic bone histomorphometry revealed increased rates of bone formation and mineral apposition in the Fgfr3 mice at 4 weeks post-surgery. The number of tartrate-resistant acid phosphatase (TRAP)-positive cells rapidly increased, and the numbers of TdT-mediated dUTP nick-end labeling (TUNEL)-positive cells rapidly decreased in the Fgfr3 mice. Vascular endothelial growth factor (VEGF) expression was increased at the earlier phase post-surgery in the Fgfr3 mice. The activation of FGFR3 signaling shortens the time needed for bone repair after ischemic osteonecrosis by accelerating revascularization, bone resorption, and new bone formation. Our findings are clinically relevant as a new potential strategy for the treatment of JIO.

A rs936306 C/T Polymorphism in the CYP19A1 Is Associated With Stress Fractures.

ABSTRACT: Kumagai, H, Miyamoto-Mikami, E, Kikuchi, N, Kamiya, N, Zempo, H, and Fuku, N. A rs936306 C/T polymorphism in the CYP19A1 is associated with stress fractures. J Strength Cond Res 36(8): 2322-2325, 2022-A stress fracture (SF) is an overuse injury, and low bone mineral density (BMD) is the risk factor for the SF. Estrogen is suggested to have a crucial role in bone metabolism, and estrogen-related genetic polymorphisms are associated with BMD. However, the possible association between SF and estrogen-related genetic polymorphisms has not been clarified yet. Therefore, we aimed to clarify whether estrogen-related genetic polymorphisms are associated with a history of SFs in Japanese athletes. A total of 1,311 (men: n = 868, women: n = 443) top-level Japanese athletes who participated in various sports and at different levels were analyzed. The history of SFs was assessed using a questionnaire, and the cytochrome P450 aromatase gene ( CYP19A1 ) rs936306 C/T and estrogen receptor α gene ( ESR1 ) rs2234693 T/C polymorphisms were analyzed using the TaqMan genotyping assay. The genotype frequency of the CYP19A1 C/T polymorphism was significantly different between the injured group and noninjured group under the C allele additive genetic model (odds ratio = 1.31, 95% confidence interval = 1.01-1.70), especially in men and in women with irregular menstruation. On the other hand, there were no significant differences with the ESR1 T/C polymorphism. This study demonstrated that the C allele in the CYP19A1 rs936306 polymorphism is a risk factor for SFs in top-level Japanese athletes.

eQTL variants in COL22A1 are associated with muscle injury in athletes.

The myotendinous junction (MTJ) is at high risk of muscle injury, and collagen XXII is strictly expressed at tissue junctions, specifically at the MTJ. We investigated the hypothesis that single-nucleotide polymorphisms (SNPs) related to collagen type XXII α-1 chain gene (COL22A1) mRNA expression are associated with susceptibility to muscle injury in athletes. History of muscle injury was assessed in 3,320 Japanese athletes using a questionnaire, and two expression quantitative trait loci (eQTL) SNPs for COL22A1 (rs11784270 A/C and rs6577958 T/C) were analyzed using the TaqMan SNP Genotyping Assay. rs11784270 [odds ratio (OR) = 1.80, 95% confidence interval (CI) = 1.27-2.62, P = 0.0006] and rs6577958 (OR = 1.45, 95% CI = 1.10-1.94, P = 0.0083) were significantly associated with muscle injury under A and T allele additive genetic models, respectively. These results suggest that the expression level of COL22A1 at the MTJ influences muscle injury risk in athletes.

Genome-wide meta-analysis and replication studies in multiple ethnicities identify novel adolescent idiopathic scoliosis susceptibility loci.

Adolescent idiopathic scoliosis (AIS) is the most common musculoskeletal disorder of childhood development. The genetic architecture of AIS is complex, and the great majority of risk factors are undiscovered. To identify new AIS susceptibility loci, we conducted the first genome-wide meta-analysis of AIS genome-wide association studies, including 7956 cases and 88 459 controls from 3 ancestral groups. Three novel loci that surpassed genome-wide significance were uncovered in intragenic regions of the CDH13 (P-value_rs4513093 = 1.7E-15), ABO (P-value_ rs687621 = 7.3E-10) and SOX6 (P-value_rs1455114 = 2.98E-08) genes. Restricting the analysis to females improved the associations at multiple loci, most notably with variants within CDH13 despite the reduction in sample size. Genome-wide gene-functional enrichment analysis identified significant perturbation of pathways involving cartilage and connective tissue development. Expression of both SOX6 and CDH13 was detected in cartilage chondrocytes and chromatin immunoprecipitation sequencing experiments in that tissue revealed multiple HeK27ac-positive peaks overlapping associated loci. Our results further define the genetic architecture of AIS and highlight the importance of vertebral cartilage development in its pathogenesis.

COL5A1 rs12722 polymorphism is not associated with passive muscle stiffness and sports-related muscle injury in Japanese athletes.

BACKGROUND: Poor joint flexibility has been repeatedly proposed as a risk factor for muscle injury. The C-to-T polymorphism (rs12722) in the 3'-untranslated region of the collagen type V α1 chain gene (COL5A1) is reportedly associated with joint flexibility. Flexibility of a normal joint is largely determined by passive muscle stiffness, which is influenced by intramuscular collagenous connective tissues including type V collagen. The present study aimed to test the hypothesis that the COL5A1 rs12722 polymorphism influences joint flexibility via passive muscle stiffness, and is accordingly associated with the incidence of muscle injury. METHODS: In Study 1, we examined whether the rs12722 polymorphism is associated with joint flexibility and passive muscle stiffness in 363 healthy young adults. Joint flexibility was evaluated by passive straight-leg-raise and sit-and-reach tests, and passive muscle stiffness was measured using ultrasound shear wave elastography. In Study 2, the association of the rs12722 polymorphism with sports-related muscle injury was assessed in 1559 Japanese athletes. Muscle injury history and severity were assessed by a questionnaire. In both Study 1 and Study 2, the rs12722 C-to-T polymorphism in the COL5A1 was determined using the TaqMan SNP Genotyping Assay. RESULTS: Study 1 revealed that the rs12722 polymorphism had no significant effect on range of motion in passive straight-leg-raise and sit-and-reach tests. Furthermore, there was no significant difference in passive muscle stiffness of the hamstring among the rs12722 genotypes. In Study 2, rs12722 genotype frequencies did not differ between the muscle injury and no muscle injury groups. Moreover, no association was observed between rs12722 polymorphism and severity of muscle injury. CONCLUSIONS: The present study does not support the view that COL5A1 rs12722 polymorphism has a role as a risk factor for sports-related muscle injury, or that it is a determinant for passive muscle stiffness in a Japanese population.

Elevated Fibroblast Growth Factor Signaling Is Critical for the Pathogenesis of the Dwarfism in Evc2/Limbin Mutant Mice.

Ellis-van Creveld (EvC) syndrome is a skeletal dysplasia, characterized by short limbs, postaxial polydactyly, and dental abnormalities. EvC syndrome is also categorized as a ciliopathy because of ciliary localization of proteins encoded by the two causative genes, EVC and EVC2 (aka LIMBIN). While recent studies demonstrated important roles for EVC/EVC2 in Hedgehog signaling, there is still little known about the pathophysiological mechanisms underlying the skeletal dysplasia features of EvC patients, and in particular why limb development is affected, but not other aspects of organogenesis that also require Hedgehog signaling. In this report, we comprehensively analyze limb skeletogenesis in Evc2 mutant mice and in cell and tissue cultures derived from these mice. Both in vivo and in vitro data demonstrate elevated Fibroblast Growth Factor (FGF) signaling in Evc2 mutant growth plates, in addition to compromised but not abrogated Hedgehog-PTHrP feedback loop. Elevation of FGF signaling, mainly due to increased Fgf18 expression upon inactivation of Evc2 in the perichondrium, critically contributes to the pathogenesis of limb dwarfism. The limb dwarfism phenotype is partially rescued by inactivation of one allele of Fgf18 in the Evc2 mutant mice. Taken together, our data uncover a novel pathogenic mechanism to understand limb dwarfism in patients with Ellis-van Creveld syndrome.

Scleraxis-Lineage Cells Contribute to Ectopic Bone Formation in Muscle and Tendon.

The pathologic development of heterotopic ossification (HO) is well described in patients with extensive trauma or with hyperactivating mutations of the bone morphogenetic protein (BMP) receptor ACVR1. However, identification of progenitor cells contributing to this process remains elusive. Here we show that connective tissue cells contribute to a substantial amount of HO anlagen caused by trauma using postnatal, tamoxifen-inducible, scleraxis-lineage restricted reporter mice (Scx-creERT2/tdTomatofl/fl ). When the scleraxis-lineage is restricted specifically to adults prior to injury marked cells contribute to each stage of the developing HO anlagen and coexpress markers of endochondral ossification (Osterix, SOX9). Furthermore, these adult preinjury restricted cells coexpressed mesenchymal stem cell markers including PDGFRα, Sca1, and S100A4 in HO. When constitutively active ACVR1 (caACVR1) was expressed in scx-cre cells in the absence of injury (Scx-cre/caACVR1fl/fl ), tendons and joints formed HO. Postnatal lineage-restricted, tamoxifen-inducible caACVR1 expression (Scx-creERT2/caACVR1fl/fl ) was sufficient to form HO after directed cardiotoxin-induced muscle injury. These findings suggest that cells expressing scleraxis within muscle or tendon contribute to HO in the setting of both trauma or hyperactive BMP receptor (e.g., caACVR1) activity. Stem Cells 2017;35:705-710.

Generation of Evc2/Limbin global and conditional KO mice and its roles during mineralized tissue formation.

Ellis-van Creveld (EvC) syndrome (OMIM 225500) is an autosomal recessive disease characterized with chondrodysplastic dwarfism in association with abnormalities in oral cavity. Ciliary proteins EVC and EVC2 have been identified as causative genes and they play an important role on Hedgehog signal transduction. We have also identified a causative gene LIMBIN for bovine chondrodysplastic dwarfism (bcd) that is later identified as the bovine ortholog of EVC2. Here, we report generation of conventional and conditional mutant Evc2/Limbin alleles that mimics mutations found in EvC patients and bcd cattle. Resulted homozygous mice showed no ciliary localization of EVC2 and EVC and displayed reduced Hedgehog signaling activity in association with skeletal and oral defects similar to the EvC patients. Cartilage-specific disruption of Evc2/Limbin resulted in similar but milder skeletal defects, whereas osteoblast-specific disruption did not cause overt changes in skeletal system. Neural crest-specific disruption of Evc2/Limbin resulted in defective incisor growth similar to that seen in conventional knockouts; however, differentiation of amelobolasts was relatively normal in the conditional knockouts. These results showcased functions of EVC2/LIMBIN during formation of mineralized tissues. Availability of the conditional allele for this gene should facilitate further detailed analyses of the role of EVC2/LIMBIN in pathogenesis of EvC syndrome. genesis 53:612-626, 2015. © 2015 Wiley Periodicals, Inc.

Development of a mouse model of ischemic osteonecrosis.

BACKGROUND: Availability of a reliable mouse model of ischemic osteonecrosis could accelerate the development of novel therapeutic strategies to stimulate bone healing after ischemic osteonecrosis; however, no mouse model of ischemic osteonecrosis is currently available. QUESTIONS/PURPOSES: To develop a surgical mouse model of ischemic osteonecrosis, we asked, (1) if the blood vessels that contribute to the blood supply of the distal femoral epiphysis are cauterized, can we generate an osteonecrosis mouse model; (2) what are the histologic changes observed in this mouse model, and (3) what are the morphologic changes in the model. METHODS: We performed microangiography to identify blood vessels supplying the distal femoral epiphysis in mice, and four vessels were cauterized using microsurgical techniques to induce ischemic osteonecrosis. Histologic assessment of cell death in the trabecular bone was performed using terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) and counting the empty lacunae in three serial sections. Quantitation of osteoclast and osteoblast numbers was performed using image analysis software. Morphologic assessments of the distal femoral epiphysis for deformity and for trabecular bone parameters were performed using micro-CT. RESULTS: We identified four blood vessels about the knee that had to be cauterized to induce total ischemic osteonecrosis of the distal femoral epiphysis. Qualitative assessment of histologic sections of the epiphysis showed a loss of nuclear staining of marrow cells, disorganized marrow structure, and necrotic blood vessels at 1 week. By 2 weeks, vascular tissue invasion of the necrotic marrow space was observed with a progressive increase in infiltration of the necrotic marrow space with the vascular tissue at 4 and 6 weeks. TUNEL staining showed extensive cell death in the marrow and trabecular bone 24 hours after the induction of ischemia. The mean percent of TUNEL-positive osteocytes in the trabecular bone increased from 2% ± 1% in the control group to a peak of 98% ± 3% in the ischemic group 1 week after induction of ischemia (mean difference, 96%; 95% CI, 81%-111%; p < 0.0001). The mean percent of empty lacunae increased from 1% ± 1% in the control group to a peak of 78% ± 15% in the ischemic group at 4 weeks (mean difference, 77%; 95% CI, 56%-97%; p < 0.0001). Quantitative analysis showed that the mean number of osteoclasts per bone surface was decreased in the ischemic group at 1, 2, and 4 weeks (p < 0.0001, < 0.0001, and p = 0.02, respectively) compared with the control group. The mean number of osteoclasts increased to a level similar to that of the control group at 6 weeks (p = 0.23). The numbers of osteoblasts per bone surface were decreased in the ischemic group at 1, 2 and 4 weeks (p < 0.0001 for each) compared with the numbers in the control group. The mean number of osteoblasts also increased to a level similar to that of the control group at 6 weeks (p = 0.91). Mean bone volume percent assessed by micro-CT was lower in the ischemic group compared with the control group from 2 to 6 weeks. The mean differences in the percent bone volume between the control and ischemic groups at 2, 4, and 6 weeks were 5.5% (95% CI, 0.9%-10.2%; p = 0.01), 5.3% (95% CI, 0.6%-9.9%; p = 0.02), and 6.0% (95% CI, 1.1%-10.9%; p = 0.008), respectively. A deformity of the distal femoral epiphysis was observed at 6 weeks with the mean epiphyseal height to width ratio of 0.74 ± 0.03 in the control group compared with 0.66 ± 0.06 in the ischemic group (mean difference, 0.08; 95% CI, 0.00-0.16; p = 0.03). CONCLUSION: We developed a novel mouse model of ischemic osteonecrosis that produced extensive cell death in the distal femoral epiphysis which developed a deformity with time. CLINICAL RELEVANCE: The new mouse model may be a useful tool to test potential therapeutic strategies to improve bone healing after ischemic osteonecrosis.

Lactoferrin activates BMP7 gene expression through the mitogen-activated protein kinase ERK pathway in articular cartilage.

Rheumatoid arthritis (RA) is a chronic inflammatory disease leading to destruction of cartilage in joints. Neutrophil granulocytes are the predominant cell type in the synovial fluid of affected joints. Neutrophils release stimulants that alter the chondrocyte metabolism. Lactoferrin (LF) is a marker of neutrophil granulocyte activation. Local concentrations of LF in synovial fluid are much higher in the joints of RA patients. However, the effect of LF on articular cartilage of joints is not well understood. Effect of LF on gene expression in primary chondrocytes of articular cartilage was investigated in this study. We found that LF preferentially activated BMP7 expression rather than BMP2 or BMP4, and that LF activated BMP7 expression in a dose-dependent and time-dependent manner. Interestingly, a specific mitogen-activated protein kinase ERK inhibitor U0126, but not JNK kinase inhibitor SP600125, abrogated LF activation of BMP7 gene expression. LF-induced increase in BMP7 protein level was in parallel with the phosphorylation of ERK in primary chondrocytes. Taken together, we provide the first evidence to demonstrate that LF activates BMP7 expression through the mitogen-activated protein kinase ERK pathway in primary chondrocytes of articular cartilage. Since BMP7 is important for the maintenance of homeostasis in articular cartilage, we speculate that there is a protective function of LF at the site of joint inflammation.

Interleukin-6 receptor blockade improves bone healing following ischemic osteonecrosis in adolescent mice.

Objective: Juvenile ischemic osteonecrosis (JIO) of the femoral head is one of the most serious hip disorders causing a permanent deformity of the femoral head in childhood. We recently reported that interleukin 6 (IL-6) is significantly increased in the hip synovial fluid of patients with JIO and that articular chondrocytes are primary source of IL-6. Adolescent JIO is particularly challenging to treat and has poor outcome. This study determined if IL-6 receptor blockade prevents bone loss and improves the bone healing in adolescent JIO. Method: Adolescent mice (12-week-old) surgically induced with JIO were treated with either saline or MR16-1, an IL-6 receptor blocker. Results: Micro-CT assessment showed significantly increased bone volume (p â€‹< â€‹0.001, Cohen's d â€‹= â€‹2.0) and trabecular bone thickness (p â€‹< â€‹0.001, d â€‹= â€‹2.3) after the MR16-1 treatment. Histomorphometric assessment showed significantly increased osteoblast number (p â€‹< â€‹0.01, d â€‹= â€‹2.3), bone formation rate (p â€‹< â€‹0.01, d â€‹= â€‹4.3), and mineral apposition rate (p â€‹< â€‹0.01, d â€‹= â€‹4.1) after the MR16-1 treatment. The number of osteoclasts was unchanged. Histologic assessment showed significantly increased revascularization (p â€‹< â€‹0.01) and restoration of the necrotic marrow with new hematopoietic bone marrow (p â€‹< â€‹0.01). Vascular endothelial growth factor (VEGF) expression was increased in the revascularized area and the articular cartilage, and in the cultured chondrocytes treated with IL-6 receptor inhibitor. Conclusion: IL-6 blockade in adolescent mice with JIO enhanced bone formation and revascularization. The findings suggest IL-6 receptor blocker as a potential medical therapy for adolescent JIO.

Increased BMP-Smad signaling does not affect net bone mass in long bones.

Bone morphogenetic proteins (BMPs) have been used for orthopedic and dental application due to their osteoinductive properties; however, substantial numbers of adverse reactions such as heterotopic bone formation, increased bone resorption and greater cancer risk have been reported. Since bone morphogenetic proteins signaling exerts pleiotropic effects on various tissues, it is crucial to understand tissue-specific and context-dependent functions of bone morphogenetic proteins. We previously reported that loss-of-function of bone morphogenetic proteins receptor type IA (BMPR1A) in osteoblasts leads to more bone mass in mice partly due to inhibition of bone resorption, indicating that bone morphogenetic protein signaling in osteoblasts promotes osteoclast function. On the other hand, hemizygous constitutively active (ca) mutations for BMPR1A (caBmpr1a wt/+ ) in osteoblasts result in higher bone morphogenetic protein signaling activity and no overt skeletal changes in adult mice. Here, we further bred mice for heterozygous null for Bmpr1a (Bmpr1a +/- ) and homozygous mutations of caBmpr1a (caBmpr1a +/+ ) crossed with Osterix-Cre transgenic mice to understand how differences in the levels of bone morphogenetic protein signaling activity specifically in osteoblasts contribute to bone phenotype. We found that Bmpr1a +/- , caBmpr1a wt/+ and caBmpr1a +/+ mice at 3 months of age showed no overt bone phenotypes in tibiae compared to controls by micro-CT and histological analysis although BMP-Smad signaling is increased in both caBmpr1a wt/+ and caBmpr1a +/+ tibiae and decreased in the Bmpr1a +/- mice compared to controls. Gene expression analysis demonstrated that slightly higher levels of bone formation markers and resorption markers along with levels of bone morphogenetic protein-Smad signaling, however, there was no significant changes in TRAP positive cells in tibiae. These findings suggest that changes in bone morphogenetic protein signaling activity within differentiating osteoblasts does not affect net bone mass in the adult stage, providing insights into the concerns in the clinical setting such as high-dose and unexpected side effects of bone morphogenetic protein application.

Incidence of anterior cruciate ligament injury patterns in Japanese judo players from a nationwide insurance database.

Objective: This study aimed to characterize the age- and sex-specific Anterior cruciate ligament (ACL) injury rates and related injury patterns in judo players in Japan using the nationwide insurance database. Methods: This was a descriptive epidemiological study. We examined a total of 2142 adolescents with anterior cruciate ligament injuries registered in the insurance system of the Japan Sports Council between January 2009 and December 2018. The age- and sex-specific incidences were estimated for the levels of 7th, 8th, and 9th grades of junior high school and 10th, 11th, and 12th grades of high school. The anterior cruciate ligament injury circumstances were classified into three patterns based on the impact to the involved knee: high-impact valgus force, low-impact trunk displacement, or no-impact knee twisting. Results: The incidence of anterior cruciate ligament injury from the 7th to 12th grades were 0.5, 0.9, 0.9, 6.9, 8.6, and 6.1 per 1000 athlete-years in male players and 1.3, 3.8, 3.4, 16.8, 19.5, and 13.6 per 1000 athlete-years in female players. The most prevalent injury pattern was a low-impact contact injury (42.6%) with Osoto-gari, followed by a high-impact contact injury (29.8%). The concomitant medial collateral ligament (MCL) injury rate was 18.1%, which was correlated with a high-impact contact injury (p = 0.005) by multiple regression analysis. Conclusions: The highest incidence of age- and sex-specific anterior cruciate ligament injury was 19.5 per 1000 athlete-years in female high school students in the 11th grade. The most frequent injury pattern was low-impact contact injury with trunk displacement, indicating that trunk stabilization training could help prevent anterior cruciate ligament injury in judo.

Genetic polymorphisms related to muscular strength and flexibility are associated with artistic gymnastic performance in the Japanese population.

This study aimed to examine how genetic polymorphisms related to muscular strength and flexibility influence artistic gymnastic performance in an attempt to identify a novel polymorphism associated with flexibility. In study 1, the passive straight-leg-raise (PSLR) score and aromatase gene CYP19A1 rs936306 polymorphism, a key enzyme for estrogen biosynthesis, were assessed in 278 individuals. In study 2, athletes (281 gymnasts and 1908 other athletes) were asked about their competition level, and gymnasts were assessed using the difficulty score (D-score) for each event. Muscular strength- (ACTN3 R577X rs1815739 and ACE I/D rs4341) and flexibility-related (ESR1 rs2234693 T/C and CYP19A1 rs936306 C/T) genetic polymorphisms were analyzed. In study 1, males with the CYP19A1 CT + TT genotype showed significantly higher PSLR scores than those with the CC genotype. In study 2, male gymnasts with the R allele of ACTN3 R577X showed a correlation with the floor, rings, vault, and total D-scores. In addition, male gymnasts with the C allele of ESR1 T/C and T allele of CYP19A1 C/T polymorphisms were correlated with the pommel horse, parallel bars, horizontal bar, and total D-scores. Furthermore, genotype scores of these three polymorphisms correlated with the total D-scores and competition levels in male gymnasts. In contrast, no such associations were observed in female gymnasts. Our findings suggest that muscular strength- and flexibility-related polymorphisms play important roles in achieving high performance in male artistic gymnastics by specifically influencing the performance of events that require muscular strength and flexibility, respectively.HighlightsEstrogen-related CYP19A1 polymorphism is a novel determinant of flexibility in males.Muscular strength- and flexibility-related polymorphisms play important roles in high performance in male artistic gymnastics.Genotypes of ACTN3 R577X, ESR1 rs2234693, and CYP19A1 rs936306 may contribute to training plan optimization and event selection in artistic gymnastics.

Genetic variants within the COL5A1 gene are associated with ligament injuries in physically active populations from Australia, South Africa, and Japan.

Previous small-scale studies have shown an association between the COL5A1 gene and anterior cruciate ligament (ACL) injury risk. In this larger study, the genotype and allele frequency distributions of the COL5A1 rs12722 C/T and rs10628678 AGGG/deletion (AGGG/-) indel variants were compared between participants: (i) with ACL injury in independent and combined cohorts from South-Africa (SA) and Australia (AUS) vs controls (CON), and (ii) with any ligament (ALL) or only ACL injury in a Japanese (JPN) cohort vs CON. Samples were collected from SA (235 cases; 232 controls), AUS (362 cases; 80 controls) and JPN (500 cases; 1,403 controls). Genomic DNA was extracted and genotyped. Distributions were compared, and inferred haplotype analyses performed. No independent associations were noted for rs12722 or rs10628678 when the combined SA + AUS cohort was analysed. However, the C-deletion (rs12722-rs10628678) inferred haplotype was under-represented (p = 0.040, OR = 0.15, CI = 0.04-0.56), while the T-deletion inferred haplotype was over-represented in the female SA + AUS ACL participants versus controls (p < 0.001, OR = 4.74, CI = 1.66-13.55). Additionally, the rs12722 C/C genotype was under-represented in JPN CON vs ACL (p = 0.039, OR = 0.52, 0.27-1.00), while the rs10628678 -/- genotype was associated with increased risk of any ligament injuries (p = 0.035, OR = 1.31, CI = 1.02-1.68) in the JPN cohort. Collectively, these results highlight that a region within the COL5A1 3'-UTR is associated with ligament injury risk. This must be evaluated in larger cohorts and its functional relevance to the structure and capacity of ligaments and joint biomechanics be explored.Highlights The COL5A1 T-deletion inferred haplotype (rs12722-rs10628678) was associated with an increased risk of ACL rupture in the combined SA and AUS female participants.The COL5A1 C-deletion inferred haplotype (rs12722-rs10628678) was associated with a decreased risk of ACL rupture in the combined SA and AUS female participants.The COL5A1 rs12722 C/C and rs10628678 -/- genotypes were associated with increased risk of ACL rupture and of ligament injuries in JPN, respectively.A region within the COL5A1 3'-UTR is associated with risk of ligament injury, including ACL rupture, and therefore the functional significance of this region on ligament capacity and joint biomechanics requires further exploration.

Loss-of-function of ACVR1 in osteoblasts increases bone mass and activates canonical Wnt signaling through suppression of Wnt inhibitors SOST and DKK1.

BMPs (Bone morphogenetic proteins) such as BMP2 and BMP7 have been used about one decade as bone anabolic agents in orthopaedics. The BMP receptor ACVR1, which is a key receptor of BMP7, is expressed in bone. The pathological role of ACVR1 in humans has been reported: a point mutation in ACVR1 can cause fibrodysplasia ossificans progressiva (FOP) in which ectopic ossification occurs in skeletal muscles and deep connective tissues. The physiological function of ACVR1 in bone, however, is totally unknown. The purpose of this study is to investigate the endogenous role of ACVR1 in osteoblasts, one of the most dominant cell-types in bone. We generated Acvr1-null mice in an osteoblast-specific manner using an inducible Cre-loxP system. Surprisingly, we found that bone mass was increased in the Acvr1-null mice. Interestingly, canonical Wnt signaling was increased and expression levels of Wnt inhibitors Sost and Dkk1 were both suppressed in the null bones during the developmental stages. In addition, we confirmed that expression levels of both Sost and Dkk1 were upregulated by BMP7 dose-dependently in vitro. These results suggest that the Acvr1-deficiency can increase bone mass by activating Wnt signaling in which both Sost and Dkk1 expression levels are diminished. This study leads to a new concept of the BMP7-ACVR1-SOST/DKK1 axis in osteoblasts, in which BMP7 signaling through ACVR1 can reduce Wnt signaling via SOST/DKK1 and then inhibits osteogenesis. Although this concept is beyond the current known function of BMP7, it can explain the varied outcomes of BMP7 treatment. We believe BMP signaling can exhibit multifaceted effects by context and cell type.

Elevation of Proinflammatory Cytokine HMGB1 in the Synovial Fluid of Patients With Legg-Calvé-Perthes Disease and Correlation With IL-6.

Legg-Calvé-Perthes disease (LCPD) is a childhood ischemic osteonecrosis (ON) of the femoral head associated with the elevation of proinflammatory cytokine interleukin-6 (IL-6) in the synovial fluid. Currently, there is no effective medical therapy for patients with LCPD. In animal models of ischemic ON, articular chondrocytes produce IL-6 in response to ischemic ON induction and IL-6 receptor blockade improves bone healing. High-mobility group box 1 (HMGB1) is a damage-associated molecular pattern released from dying cells. In addition, extracellular HMGB1 protein is a well-known proinflammatory cytokine elevated in the synovial fluid of patients with rheumatoid arthritis and osteoarthritis. The purpose of this study was to investigate IL-6-related proinflammatory cytokines, including HMGB1, in the synovial fluid of patients with LCPD. Our working hypothesis was that HMGB1, produced by articular chondrocytes following ischemic ON, plays an important role in IL-6 upregulation. Here, HMGB1 protein levels were significantly higher in the synovial fluid of patients with LCPD by threefold compared with controls (p < 0.05), and were highly correlated with IL-6 levels (Pearson correlation coefficient 0.94, p < 0.001, R 2 = 0.87). In the mouse model of ischemic ON, both HMGB1 gene expression and protein levels were elevated in the articular cartilage. In vitro studies revealed a significant elevation of HMGB1 and IL-6 proteins in the supernatants of human chondrocytes exposed to hypoxic and oxidative stresses. Overexpressed HMGB1 protein in the supernatants of chondrocytes synergistically increased IL-6 protein. Silencing HMGB1 RNA in human chondrocytes significantly repressed inteleukin-1ß (IL-1ß) gene expression, but not IL-6. Further, both IL-1ß and tumor necrosis factor-α (TNF-α) protein levels in the synovial fluid of patients with LCPD were significantly correlated with IL-6 protein levels. Taken together, these results suggest that proinflammatory cytokines, HMGB1, tumor necrosis factor-α (TNF-α), and IL-1ß, are significantly involved with IL-6 in the pathogenesis of LCPD. This study is clinically relevant because the availability of multiple therapeutic targets may improve the development of therapeutic strategy for LCPD. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Development of a murine model of ischemic osteonecrosis to study the effects of aging on bone repair.

Age at onset is one of the most important predictors of outcome following ischemic osteonecrosis (ON). Currently, there is no well-established animal model to study the effects of age on the repair process following ischemic ON. The purpose of this study was to further advance a murine model of ischemic ON using four age groups of mice to determine the effects of aging on revascularization and bone repair following ischemic ON. Ischemia was surgically induced in the distal femoral epiphysis of four age groups of skeletally immature and mature mice; juvenile (5 weeks), adolescent (12 weeks), adult (22 weeks), and middle age (52 weeks). Mice were euthanized at 2 days or 4 weeks post-ischemia surgery to evaluate the extent of ON, revascularization, and bone repair. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining showed extensive cell death in the epiphysis of all four age groups at 2 days post-ischemia surgery. At 4 weeks, the juvenile mice followed by the adolescent mice had significantly greater revascularization and repair of the necrotic marrow space, increased osteoblast and osteoclast numbers, and increased bone formation rates compared to the adult and middle-age mice. Faster revascularization and bone healing were observed in the skeletally immature mice compared to the skeletally mature mice following ischemic ON. The findings resemble the clinical observation of aging on bone repair following ischemic ON. The mouse model may serve as a useful tool to investigate the mechanisms underlying the age-related impairment of bone repair in adolescent and adult ON and to develop novel therapeutic strategies.

Anti-Interleukin-6 Therapy Decreases Hip Synovitis and Bone Resorption and Increases Bone Formation Following Ischemic Osteonecrosis of the Femoral Head.

Legg-Calvé-Perthes disease (LCPD) is a juvenile form of ischemic femoral head osteonecrosis, which produces chronic hip synovitis, permanent femoral head deformity, and premature osteoarthritis. Currently, there is no medical therapy for LCPD. Interleukin-6 (IL-6) is significantly elevated in the synovial fluid of patients with LCPD. We hypothesize that IL-6 elevation promotes chronic hip synovitis and impairs bone healing after ischemic osteonecrosis. We set out to test if anti-IL-6 therapy using tocilizumab can decrease hip synovitis and improve bone healing in the piglet model of LCPD. Fourteen piglets were surgically induced with ischemic osteonecrosis and assigned to two groups: the no treatment group (n = 7) and the tocilizumab group (15 to 20 mg/kg, biweekly intravenous injection, n = 7). All animals were euthanized 8 weeks after the induction of osteonecrosis. Hip synovium and femoral heads were assessed for hip synovitis and bone healing using histology, micro-CT, and histomorphometry. The mean hip synovitis score and the number of synovial macrophages and vessels were significantly lower in the tocilizumab group compared with the no treatment group (p < .0001, p = .01, and p < .01, respectively). Micro-CT analysis of the femoral heads showed a significantly higher bone volume in the tocilizumab group compared with the no treatment group (p = .02). The histologic assessment revealed a significantly lower number of osteoclasts per bone surface (p < .001) in the tocilizumab group compared with the no treatment group. Moreover, fluorochrome labeling showed a significantly higher percent of mineralizing bone surface (p < .01), bone formation rate per bone surface (p < .01), and mineral apposition rate (p = .04) in the tocilizumab group. Taken together, tocilizumab therapy decreased hip synovitis and osteoclastic bone resorption and increased new bone formation after ischemic osteonecrosis. This study provides preclinical evidence that tocilizumab decreases synovitis and improves bone healing in a large animal model of LCPD. © 2020 American Society for Bone and Mineral Research (ASBMR).

Female Athletes Genetically Susceptible to Fatigue Fracture Are Resistant to Muscle Injury: Potential Role of COL1A1 Variant.

PURPOSE: We aimed to investigate the hypothesis that type I collagen plays a role in increasing bone mineral density (BMD) and muscle stiffness, leading to low and high risks of fatigue fracture and muscle injury, respectively, in athletes. As a potential mechanism, we focused on the effect of the type I collagen alpha 1 chain gene (COL1A1) variant associated with transcriptional activity on bone and skeletal muscle properties. METHODS: The association between COL1A1 rs1107946 and fatigue fracture/muscle injury was evaluated in Japanese athletes. Effects of the polymorphism on tissue properties (BMD and muscle stiffness) and type I collagen α1/α2 chain ratios in muscles were examined in Japanese nonathletes. RESULTS: The C-allele carrier frequency was greater in female athletes with fatigue fracture than in those without (odds ratio = 2.44, 95% confidence interval [CI] = 1.17-5.77) and lower in female athletes with muscle injury than in those without (odds ratio = 0.46, 95% CI = 0.24-0.91). Prospective validation analysis confirmed that in female athletes, muscle injury was less frequent in C-allele carriers than in AA genotype carriers (multivariable-adjusted hazard ratio = 0.27, 95% CI = 0.08-0.96). Among female nonathletes, the C-allele of rs1107946 was associated with lower BMD and lower muscle stiffness. Muscle biopsy revealed that C-allele carriers tended to have a larger type I collagen α1/α2 chain ratio than AA genotype carriers (2.24 vs 2.05, P = 0.056), suggesting a higher proportion of type I collagen α1 homotrimers. CONCLUSION: The COL1A1 rs1107946 polymorphism exerts antagonistic effects on fatigue fracture and muscle injury among female athletes by altering the properties of these tissues, potentially owing to increased levels of type I collagen α1 chain homotrimers.