Koji Fujita

A microRNA regulatory mechanism of osteoblast differentiation

Growing evidence shows that microRNAs (miRNAs) regulate various developmental and homeostatic events in vertebrates and invertebrates. Osteoblast differentiation is a key step in proper skeletal development and acquisition of bone mass; however, the physiological role of non-coding small RNAs, especially miRNAs, in osteoblast differentiation remains elusive. Here, through comprehensive analysis of miRNAs expression during osteoblast differentiation, we show that miR-206, previously viewed as a muscle-specific miRNA, is a key regulator of this process. miR-206 was expressed in osteoblasts, and its expression decreased over the course of osteoblast differentiation. Overexpression of miR-206 in osteoblasts inhibited their differentiation, and conversely, knockdown of miR-206 expression promoted osteoblast differentiation. In silico analysis and molecular experiments revealed connexin 43 (Cx43), a major gap junction protein in osteoblasts, as a target of miR-206, and restoration of Cx43 expression in miR-206-expressing osteoblasts rescued them from the inhibitory effect of miR-206 on osteoblast differentiation. Finally, transgenic mice expressing miR-206 in osteoblasts developed a low bone mass phenotype due to impaired osteoblast differentiation. Our data show that miRNA is a regulator of osteoblast differentiation.

Sema3A regulates bone-mass accrual through sensory innervations

Semaphorin 3A (Sema3A) is a diffusible axonal chemorepellent that has an important role in axon guidance. Previous studies have demonstrated that Sema3a−/− mice have multiple developmental defects due to abnormal neuronal innervations. Here we show in mice that Sema3A is abundantly expressed in bone, and cell-based assays showed that Sema3A affected osteoblast differentiation in a cell-autonomous fashion. Accordingly, Sema3a−/− mice had a low bone mass due to decreased bone formation. However, osteoblast-specific Sema3A-deficient mice (Sema3acol1−/− and Sema3aosx−/− mice) had normal bone mass, even though the expression of Sema3A in bone was substantially decreased. In contrast, mice lacking Sema3A in neurons (Sema3asynapsin−/− and Sema3anestin−/− mice) had low bone mass, similar to Sema3a−/− mice, indicating that neuron-derived Sema3A is responsible for the observed bone abnormalities independent of the local effect of Sema3A in bone. Indeed, the number of sensory innervations of trabecular bone was significantly decreased in Sema3asynapsin−/− mice, whereas sympathetic innervations of trabecular bone were unchanged. Moreover, ablating sensory nerves decreased bone mass in wild-type mice, whereas it did not reduce the low bone mass in Sema3anestin−/− mice further, supporting the essential role of the sensory nervous system in normal bone homeostasis. Finally, neuronal abnormalities in Sema3a−/− mice, such as olfactory development, were identified in Sema3asynasin−/− mice, demonstrating that neuron-derived Sema3A contributes to the abnormal neural development seen in Sema3a−/− mice, and indicating that Sema3A produced in neurons regulates neural development in an autocrine manner. This study demonstrates that Sema3A regulates bone remodelling indirectly by modulating sensory nerve development, but not directly by acting on osteoblasts.

Vitamin E decreases bone mass by stimulating osteoclast fusion.

Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. Osteoclasts are multinucleated cells that are formed by mononuclear preosteoclast fusion. Fat-soluble vitamins such as vitamin D are pivotal in maintaining skeletal integrity. However, the role of vitamin E in bone remodeling is unknown. Here, we show that mice deficient in α-tocopherol transfer protein (Ttpa(-/-) mice), a mouse model of genetic vitamin E deficiency, have high bone mass as a result of a decrease in bone resorption. Cell-based assays indicated that α-tocopherol stimulated osteoclast fusion, independent of its antioxidant capacity, by inducing the expression of dendritic-cell-specific transmembrane protein, an essential molecule for osteoclast fusion, through activation of mitogen-activated protein kinase 14 (p38) and microphthalmia-associated transcription factor, as well as its direct recruitment to the Tm7sf4 (a gene encoding DC-STAMP) promoter. Indeed, the bone abnormality seen in Ttpa(-/-) mice was rescued by a Tm7sf4 transgene. Moreover, wild-type mice or rats fed an α-tocopherol-supplemented diet, which contains a comparable amount of α-tocopherol to supplements consumed by many people, lost bone mass. These results show that serum vitamin E is a determinant of bone mass through its regulation of osteoclast fusion.Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. Osteoclasts are multinucleated cells that are formed by mononuclear preosteoclast fusion. Fat-soluble vitamins such as vitamin D are pivotal in maintaining skeletal integrity. However, the role of vitamin E in bone remodeling is unknown. Here, we show that mice deficient in α-tocopherol transfer protein (Ttpa−/− mice), a mouse model of genetic vitamin E deficiency, have high bone mass as a result of a decrease in bone resorption. Cell-based assays indicated that α-tocopherol stimulated osteoclast fusion, independent of its antioxidant capacity, by inducing the expression of dendritic-cell–specific transmembrane protein, an essential molecule for osteoclast fusion, through activation of mitogen-activated protein kinase 14 (p38) and microphthalmia-associated transcription factor, as well as its direct recruitment to the Tm7sf4 (a gene encoding DC-STAMP) promoter. Indeed, the bone abnormality seen in Ttpa−/− mice was rescued by a Tm7sf4 transgene. Moreover, wild-type mice or rats fed an α-tocopherol–supplemented diet, which contains a comparable amount of α-tocopherol to supplements consumed by many people, lost bone mass. These results show that serum vitamin E is a determinant of bone mass through its regulation of osteoclast fusion.

Runx1 and Runx2 cooperate during sternal morphogenesis

Chondrocyte differentiation is strictly regulated by various transcription factors, including Runx2 and Runx3; however, the physiological role of Runx1 in chondrocyte differentiation remains unknown. To examine the role of Runx1, we generated mesenchymal-cell-specific and chondrocyte-specific Runx1-deficient mice [Prx1 Runx1f/f mice and α1(II) Runx1f/f mice, respectively] to circumvent the embryonic lethality of Runx1-deficient mice. We then mated these mice with Runx2 mutant mice to obtain mesenchymal-cell-specific or chondrocyte-specific Runx1; Runx2 double-mutant mice [Prx1 DKO mice and α1(II) DKO mice, respectively]. Prx1 Runx1f/f mice displayed a delay in sternal development and Prx1 DKO mice completely lacked a sternum. By contrast, α1(II) Runx1f/f mice and α1(II) DKO mice did not show any abnormal sternal morphogenesis or chondrocyte differentiation. Notably, Runx1, Runx2 and the Prx1-Cre transgene were co-expressed specifically in the sternum, which explains the observation that the abnormalities were limited to the sternum. Histologically, mesenchymal cells condensed normally in the prospective sternum of Prx1 DKO mice; however, commitment to the chondrocyte lineage, which follows mesenchymal condensation, was significantly impaired. In situ hybridization analyses demonstrated that the expression of α1(II) collagen (Col2a1 — Mouse Genome Informatics), Sox5 and Sox6 in the prospective sternum of Prx1 DKO mice was severely attenuated, whereas Sox9 expression was unchanged. Molecular analyses revealed that Runx1 and Runx2 induce the expression of Sox5 and Sox6, which leads to the induction of α1(II) collagen expression via the direct regulation of promoter activity. Collectively, these results show that Runx1 and Runx2 cooperatively regulate sternal morphogenesis and the commitment of mesenchymal cells to become chondrocytes through the induction of Sox5 and Sox6.

Potential pathogenic mechanism for stress fractures of the bowed femoral shaft in the elderly : mechanical analysis by the CT-based finite element method

INTRODUCTIONnStress fractures of the bowed femoral shaft (SBFs) may be one of the causes of atypical femoral fractures (AFFs). The CT-based finite element method (CT/FEM) can be used to structurally evaluate bone morphology and bone density based on patient DICOM data, thereby quantitatively and macroscopically assessing bone strength. Here, we clarify the pathogenic mechanism of SBFs and demonstrate this new understanding of AFFs through mechanical analysis by CT/FEM.nnnPATIENTS AND METHODSnA prospective clinical study was performed from April 2012 to February 2014. We assembled two study groups, the bowed AFF group (n=4 patients; mean age, 78.0 years) including those with a prior history of AFF associated with bowing deformity and the thigh pain group (n=14 patients; mean age, 78.6 years) comprising outpatients with complaints of thigh pain and tenderness. Stress concentration in the femoral shaft was analysed by CT/FEM, and the visual findings and extracted data were assessed to determine the maximum principal stress (MPS) and tensile stress-strength ratio (TSSR). In addition, we assessed femoral bowing, bone density, and bone metabolic markers. Wilcoxons rank sum test was used for statistical analysis.nnnRESULTSnAll patients in the bowed AFF group showed a marked concentration of diffuse stress on the anterolateral surface. Thirteen patients in the thigh pain group had no significant findings. However, the remaining 1 patient had a finding similar to that observed in the bowed AFF group, with radiographic evidence of bowing deformity and a focally thickened lateral cortex. Patients were reclassified as having SBF (n=5) or non-SBF (n=13). Statistical analysis revealed significant differences in MPS (p=0.0031), TSSR (p=0.0022), and femoral bowing (lateral, p=0.0015; anterior, p=0.0022) between the SBF and non-SBF groups, with no significant differences in bone density or bone metabolic markers.nnnCONCLUSIONSnSignificant tensile stress due to bowing deformity can induce AFFs. SBFs should be considered a novel subtype of AFF, and patients with complaints of thigh pain and femoral shaft bowing deformity must be considered at high risk for AFFs. This project (Ref: AOTAP 13-13) was supported by AOTrauma Asia Pacific.

Autotransplantation combined with orthodontic treatment in adult patients

Abstract Autotransplantation has been a reliable approach to replace the missing tooth. However, some undesirable prognoses are still remaining especially in those teeth with complete root development. This case report presents the treatment of three adult patients with missing teeth, and autotransplantation was carried out to reconstruct their dentition. In order to avoid dentoalveolar ankylosis and inflammatory root resorption of the transplanted teeth, the period for splinting was limited to a minimum and the following orthodontic treatments were performed with Ti–Ni alloy wires which possess stable light forces. Neither ankylosis nor root resorption was evident during the treatment, and the conditions of the transplanted teeth were excellent in more than 4 years after transplantation.

Fall-related deterioration of subjective symptoms in patients with cervical myelopathy

Study design. Retrospective multi-center study. Objective. This study was conducted to clarify the incidence and neurological outcomes of fall-related deterioration of subjective symptoms in patients undergoing surgical treatment. Summary of Background Data. The evidence that minor trauma, including falls, increases the risk of worsening cervical myelopathy is insufficient. Methods. A retrospective analysis of patients who had undergone surgery for cervical myelopathy at 12 participating institutes was conducted. Patients who had undergone surgery for symptomatic cervical myelopathy from January 2012 to December 2013 and completed at least 1-year follow-up were included in this study. Data were collected by chart review and a questionnaire that included numbers of recalled falls during the last preoperative year and first postoperative year, circumstances of falls, and whether the patient had experienced fall-related deterioration of subjective symptoms. Results. A total of 360 eligible patients were recruited into the study. Of these, 177 (49%) reported at least one fall during the last preoperative year, and 105 (29%) experienced fall-related deterioration of subjective symptoms. Forty (11%) reported deterioration of numbness in the arms or legs, and 65 (18%) reported deterioration of motor deficits. Incidences of falls and fall-related deterioration of symptoms decreased significantly after surgery. Patients who experienced fall-related deterioration of motor deficits showed significantly worse surgical outcomes as assessed by Japanese Orthopaedic Association (JOA) score compared with those who did not experience deterioration. The optimal cut-off for preoperative JOA score in predicting an increased risk of fall-related deterioration in motor deficits was 8. Conclusion. Patients with cervical myelopathy commonly experienced preoperative fall-related deterioration of subjective symptoms, associated with significantly worse neurological outcomes. Surgical treatment significantly reduced the incidence of both falls and fall-related deterioration of subjective symptoms. Level of Evidence: 4

Location of atypical femoral fracture can be determined by tensile stress distribution influenced by femoral bowing and neck-shaft angle: a CT-based nonlinear finite element analysis model for the assessment of femoral shaft loading stress

INTRODUCTIONnLoading stress due to individual variations in femoral morphology is thought to be strongly associated with the pathogenesis of atypical femoral fracture (AFF). In Japan, studies on AFF regarding pathogenesis in the mid-shaft are well-documented and a key factor in the injury is thought to be femoral shaft bowing deformity. Thus, we developed a CT-based finite element analysis (CT/FEA) model to assess distribution of loading stress in the femoral shaft.nnnPATIENTS AND METHODSnA multicenter prospective study was performed at 12 hospitals in Japan from August 2015 to February 2017. We assembled three study groups-the mid-shaft AFF group (n=12), the subtrochanteric AFF group (n=10), and the control group (n=11)-and analyzed femoral morphology and loading stress in the femoral shaft by nonlinear CT/FEA.nnnRESULTSnFemoral bowing in the mid-shaft AFF group was significantly greater (lateral bowing, p<0.0001; anterior bowing, p<0.01). Femoral neck-shaft angle in the subtrochanteric AFF group was significantly smaller (p<0.001). On CT/FEA, both the mid-shaft and subtrochanteric AFF group showed maximum tensile stress located adjacent to the fracture site. Quantitatively, there was a correlation between femoral bowing and the ratio of tensile stress, which was calculated between the mid-shaft and subtrochanteric region (lateral bowing, r=0.6373, p<0.0001; anterior bowing, r=-0.5825, p<0.001).nnnCONCLUSIONSnCT/FEA demonstrated that tensile stress by loading stress can cause AFF. The location of AFF injury could be determined by individual stress distribution influenced by femoral bowing and neck-shaft angle.

Intramuscular Tendon of the Adductor Pollicis and Underlying Capsule of the Metacarpophalangeal Joint: An Anatomical Study With Possible Implications for the Stener Lesion

PURPOSEnTo identify the layered relationship anatomically between the musculotendinous structures of the adductor pollicis, the ulnar collateral ligament, and the capsule of the metacarpophalangeal joint in terms of understanding the pathomechanism of a Stener lesion.nnnMETHODSnWe macroscopically analyzed 37 cadaveric thumbs to identify the intramuscular tendon of the adductor pollicis and bony attachments of the joint capsule including the ulnar collateral ligament. In addition, we histologically analyzed 3 thumbs and made a 3-dimensional image of 3 other thumbs, using micro-computed tomography.nnnRESULTSnThe adductor pollicis has 3 components of an intramuscular tendon (dorsal, palmar, and distal), which connect to form a lambda shape. The dorsal part inserts into the joint capsule dorsal to the ulnar sesamoid. The palmar part inserts into the ulnar sesamoid. The distal part inserts into the lateral tubercle of the proximal phalanx. The thickened and cord-like part of the joint capsule, which has generally been referred to as the proper ulnar collateral ligament, has a distinct bony attachment on the proximal slope of the lateral tubercle of the proximal phalanx separate from the adductor pollicis insertion.nnnCONCLUSIONSnOf the 3 components of the intramuscular tendon of the adductor pollicis muscle, the dorsal part inserted into not only the aponeurosis but also the joint capsule.nnnCLINICAL RELEVANCEnThe results of the current study suggest the anatomic basis for a possible pathomechanism of the Stener lesion.

Complications of Intramedullary Fixation for Distal Radius Fractures in Elderly Patients: A Retrospective Analysis Using McKay’s Complication Checklist

BACKGROUNDnIntramedullary fixation for distal radius fractures is reported to be free of hardware irritation and less invasive than other fixation methods. Some specific complications associated with intramedullary fixation, such as radial nerve sensory neuritis, have been reported, but no study has focused on the complication rates of intramedullary fixation for distal radius fractures in the elderly population. Furthermore, no studies have analyzed common complications, such as carpal tunnel syndrome and flexor tenosynovitis including trigger finger, among patients with distal radius fractures treated by intramedullary fixation based on a comprehensive complication checklist.nnnMETHODSnWe reviewed the medical records of 52 elderly patients with distal radius fractures treated with intramedullary nail fixation. We investigated the postoperative complications in these patients using McKays complication checklist.nnnRESULTSn5 patients experienced radial nerve sensory disorder, and one patients developed carpal tunnel syndrome. All neurological symptoms resolved spontaneously, and these neurological complications were categorized as mild. Further, 3 patients developed trigger finger at the A1 pulley and needed triamcinolone injections for symptomatic relief. There were no tendinous complications around the implanted hardware. All tendinous complications were categorized as moderate complications and resolved with steroid injection therapy. Among skeletal complications, 1 case of postoperative volar displacement resolved with good functional outcome without the need for corrective osteotomy. This was considered a mild complication. The total complication rate was 19.2%. All complications were categorized as mild or moderate, and no patients experienced severe complications that needed further surgery such as hardware removal.nnnCONCLUSIONSnIntramedullary fixation for distal radius fractures was free from tendinous complications such as tenosynovitis and tendon ruptures around the implant, which are frequently caused by volar locking plate fixation. However, this less invasive technique could not avoid common complications such as trigger finger and carpal tunnel syndrome associated with distal radius fractures.