Hiroyuki Inose

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.

Central control of bone remodeling by neuromedin U

Bone remodeling, the function affected in osteoporosis, the most common of bone diseases, comprises two phases: bone formation by matrix-producing osteoblasts and bone resorption by osteoclasts. The demonstration that the anorexigenic hormone leptin inhibits bone formation through a hypothalamic relay suggests that other molecules that affect energy metabolism in the hypothalamus could also modulate bone mass. Neuromedin U (NMU) is an anorexigenic neuropeptide that acts independently of leptin through poorly defined mechanisms. Here we show that Nmu-deficient (Nmu−/−) mice have high bone mass owing to an increase in bone formation; this is more prominent in male mice than female mice. Physiological and cell-based assays indicate that NMU acts in the central nervous system, rather than directly on bone cells, to regulate bone remodeling. Notably, leptin- or sympathetic nervous system–mediated inhibition of bone formation was abolished in Nmu−/− mice, which show an altered bone expression of molecular clock genes (mediators of the inhibition of bone formation by leptin). Moreover, treatment of wild-type mice with a natural agonist for the NMU receptor decreased bone mass. Collectively, these results suggest that NMU may be the first central mediator of leptin-dependent regulation of bone mass identified to date. Given the existence of inhibitors and activators of NMU action, our results may influence the treatment of diseases involving low bone mass, such as osteoporosis.

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.

Dural closure for the treatment of superficial siderosis.

Superficial siderosis (SS) of the CNS is a rare disease caused by repeated hemorrhages in the subarachnoid space. The subsequent deposition of hemosiderin in the brain and spinal cord leads to the progression of neurological deficits. The causes of bleeding include prior intradural surgery, carcinoma, arteriovenous malformation, nerve root avulsion, and dural abnormality. Recently, surgical treatment of SS associated with dural defect has been reported. The authors of the present report describe 2 surgically treated SS cases and review the literature on surgically treated SS. The patients had dural defects with fluid-filled collections in the spinal canal. In both cases, the dural defects were successfully closed, and the fluid collection was resolved postoperatively. In one case, the neurological symptoms did not progress postoperatively. In the other case, the patient had long history of SS, and the clinical manifestations partially deteriorated after surgery, despite the successful dural closure. In previously reported surgically treated cases, the dural defects were closed by sutures, patches, fibrin glue, or muscle/fat grafting. Regardless of the closing method, dural defect closure has been shown to stop CSF leakage and subarachnoid hemorrhaging. Successfully repairing the defect can halt the disease progression in most cases and may improve the symptoms that are associated with CSF hypovolemia. However, the effect of the dural closure may be limited in patients with long histories of SS because of the irreversibility of the neural tissue damage caused by hemosiderin deposition. In patients with SS, it is important to diagnose and repair the dural defect early to minimize the neurological impairments that are associated with dural defects.

MicroRNA-145 regulates osteoblastic differentiation by targeting the transcription factor Cbfb.

Osteoblastic differentiation is regulated by various factors, including hormones and transcription factors. Runt‐related transcription factor 2 (Runx2) is an essential player in osteoblastogenesis and transactivates its molecular target by creating a protein complex with its hetero‐dimeric partner core binding factor beta (Cbfb). However, the molecular regulation of Cbfb expression remains unknown. Here, we identified miR‐145 as a crucial regulator of Cbfb expression. The expression of miR‐145 increased during osteoblastogenesis, indicating that miR‐145 works as an inhibitor of osteoblastogenesis. Stable expression of miR‐145 decreased endogenous Cbfb expression and inhibited osteoblastogenesis, in cooperation with miR‐34c. Furthermore, miR‐145 decreased bone regeneration in vivo. Our results indicate that miR‐145 physiologically regulates osteoblast differentiation and bone formation via Cbfb expression by forming a regulatory microRNA network.

Modified K-line in magnetic resonance imaging predicts insufficient decompression of cervical laminoplasty.

Study Design. A retrospective single-center study. Objective. To clarify preoperative factors predicting unsatisfactory indirect decompression after laminoplasty in patients with cervical spondylotic myelopathy. Summary of Background Data. Many authors have shown that inadequate indirect decompression after laminoplasty can inhibit neural recovery and should be considered a complication. We previously demonstrated that residual anterior compression of the spinal cord (ACS) impaired recovery of upper extremity motor function. Although the K-line has been established as a predictive index indicating that laminoplasty is required in patients with ossification of the posterior longitudinal ligament, it remains unclear what preoperative factors can predict insufficient posterior cord decompression in patients with cervical spondylotic myelopathy. Methods. Forty-six consecutive patients who underwent laminoplasty for the treatment of cervical spondylotic myelopathy at our hospital were reviewed. A modified K-line was defined as the line connecting the midpoints of the spinal cord at C2 and C7 on a T1-weighted sagittal magnetic resonance image. We also determined the minimum interval between the tip of local kyphosis and a line connecting the midpoint of the cord at the level of the inferior endplates of C2 and C7 (INTmin) on the midsagittal image. Data analysis involved logistic regression and receiver operating characteristic curve analysis to select the most valuable index for predicting postoperative ACS. Results. Ten patients had ACS immediately after laminoplasty. Logistic regression analysis showed that INTmin was a significant predictive factor for the occurrence of postoperative ACS (odds ratio = 0.485; 95% confidence interval = 0.29–0.81; P = 0.02). Receiver operating characteristic curve analysis showed an area under the curve of 0.871. A cutoff of 4.0 mm had a sensitivity of 80% and a specificity of 80.6% for prediction of postoperative ACS. Conclusion. The parameter INTmin correlated with the occurrence of postoperative ACS. A cutoff point of 4.0 mm is most appropriate for alerting spine surgeons to a high likelihood of postoperative ACS.

Warning thresholds on the basis of origin of amplitude changes in transcranial electrical motor-evoked potential monitoring for cervical compression myelopathy

Study Design. A retrospective analysis of prospectively collected data from consecutive patients undergoing transcranial electrical motor-evoked potential (TCE-MEP: compound muscle action potentials) monitoring during cervical spine surgery. Objective. To divide the warning threshold of TCE-MEP amplitude changes on the basis of origin into the spinal tract and spinal segments and decide warning thresholds for each. Summary of Background Data. The parameter commonly used for the warning threshold in TCE-MEP monitoring is wave amplitude, but amplitude changes have not been examined by anatomical origin. Methods. Intraoperative TCE-MEP amplitude changes were reviewed for 357 patients with cervical myelopathy. Most of the patients were monitored by transcranial electrical stimulated spinal-evoked potential combined with TCE-MEP. The warning threshold of TCE-MEP was taken as waveform disappearance. For each patient, amplitude changes were separated, according to origin, into the spinal tract and spinal segments and compared with clinical outcome. Results. Assessable TCE-MEP waves were obtained in 350 cases. Disappearance of TCE-MEP waves, which were innervated by the spinal levels exposed to the surgical invasion, was seen in 11 cases. Disappearance of TCE-MEPs, which were innervated by the spinal levels inferior to them, was seen in 43 cases. There was no postoperative motor deficit in those cases. However, such deficits caused by spinal segment injury were seen in 2 cases, which showed that intraoperative amplitude decreased to 4.5% and 27%. Conclusion. If we had established the warning threshold as 30% of the control amplitude, we would likely have prevented both cases of postoperative motor deficits, but 106 (30.3%) cases would have become positive cases. If we had established the warning threshold separately as wave disappearance for the spinal tract and 30% of the control amplitude for the spinal segments, sensitivity and specificity would have been 100% and 83.7%, respectively. Dividing the warning threshold on the basis of origin of amplitude changes could reduce false-positive cases and prevent intraoperative injuries.

Modified K-line in magnetic resonance imaging predicts clinical outcome in patients with nonlordotic alignment after laminoplasty for cervical spondylotic myelopathy.

Study Design. Retrospective single-center study. Objective. To investigate whether a preoperative index predicts clinical outcome after laminoplasty for cervical spondylotic myelopathy. Summary of Background Data. This is the first study using the modified K-line, which connects the midpoints of the spinal cord at the C2 and C7 levels on midsagittal magnetic resonance imaging, to assess the relationship between postoperative clinical outcome and anticipated degree of spinal cord shifting. Methods. Sixty-one consecutive patients who underwent laminoplasty for the treatment of cervical spondylotic myelopathy between 2000 and 2011 at our hospital were retrospectively reviewed. The interval between the preoperative mK-line and the anterior structure of the spinal canal at each segment of the C3 to C6 levels (INTn, n = 3–6) were measured on sagittal T1-weighted magnetic resonance imaging, and the sum of the INTn (INTsum) was then calculated. The degree of posterior cord shift was defined as follows: %Csum = &Sgr;Cn; Cn = (Bn−An) × 100/An (n = 3–6; An and Bn represent the preoperative and postoperative intervals between the midpoint of the spinal cord and the anterior impingement at each segment on sagittal T1-weighted magnetic resonance imaging, respectively). In addition, we defined INTmin as the minimum interval of the INTn in each patient. All patients were divided into lordotic and nonlordotic groups on the basis of lateral neutral radiography. The Japanese Orthopaedic Association (JOA) scoring system and recovery rate of the JOA score for cervical myelopathy was evaluated as clinical outcomes. Results. The recovery rate of the JOA score was 48.1%. The lordotic and nonlordotic groups contained 38 and 23 patients, respectively. Linear regression analysis revealed that INTmin was significantly correlated with the recovery rate of the patients in the nonlordotic group, whereas INTsum was not associated with recovery of the JOA score. Conclusion. We identified INTmin as a predictive factor for clinical outcomes in patients with nonlordotic alignment after laminoplasty. Level of Evidence: 4

Runx2 haploinsufficiency ameliorates the development of ossification of the posterior longitudinal ligament.

Ossification of the Posterior Longitudinal Ligament (OPLL) is a disease that is characterized by the ectopic calcification of the ligament; however, the pathogenesis of OPLL remains to be investigated. We attempted to identify the in vivo role of Runx2, a master regulator of osteoblast differentiation and skeletal mineralization, in the pathogenesis of OPLL. The expression of Runx2 in the ligament was examined using in situ hybridization and immunohistochemistry and by monitoring the activity of a LacZ gene that was inserted into the Runx2 gene locus. To investigate the functional role of Runx2, we studied ENPP1ttw/ttw mice, a mouse model of OPLL, that were crossed with heterozygous Runx2 mice to decrease the expression of Runx2, and we performed histological and quantitative radiological analyses using 3D-micro CT. Runx2 was expressed in the ligament of wild-type mice. The induction of Runx2 expression preceded the development of ectopic calcification in the OPLL-like region of the ENPP1ttw/ttw mice. Runx2 haploinsufficiency ameliorated the development of ectopic calcification in the ENPP1ttw/ttw mice. Collectively, this study demonstrated that Runx2 is expressed in an OPLL-like region, and its elevation is a prerequisite for developing the complete OPLL-like phenotype in a mouse model of OPLL.

Porous/dense Composite Hydroxyapatite for Anterior Cervical Discectomy and Fusion

Study Design. A prospective analysis Objective. Our aim was to investigate the efficacy of new synthetic porous/dense composite hydroxyapatite (HA) for use in anterior cervical discectomy and fusion (ACDF). Summary of Background Data. Iliac crest bone graft (ICBG) has been traditionally used as the “gold standard” for ACDF. The significant complication rate associated with harvesting tricortical ICBG, however, has encouraged development of alternative graft substitutes. Methods. The morphology of the porous/dense HA was observed by scanning electron microscopy (SEM), and the in vitro compressive strength of the composite HA was measured. From April 2005, 51 consecutive patients underwent 81 levels of ACDF using the composite HA with percutaneously harvested trephine bone chips. Clinical and radiological evaluation was performed during the postoperative hospital stay and at follow-up. Furthermore, the outcomes in ACDF using the composite HA were compared with those using tricortical ICBG. Results. The SEM images demonstrated 100- to 300-&mgr;m pores (approximately 40% of porosity) in the porous layers of the HA. The compressive strength of the composite HA was 203.1 ± 4.1 MPa. In the clinical study, the demographic data of the patients were similar in HA and ICBG groups. The fusion rates in HA group were comparable with those in ICBG group. The cervical lordosis was enhanced postoperatively in both groups and well preserved at 2-year follow-up without significant differences between the groups. The intraoperative blood loss in HA group was significantly lesser than that in ICBG group. Donor site complications were found in 29.2% of the patients in ICBG group, whereas no donor site morbidity was found in HA group. No major collapse or fragmentation of the composite HA was found. Conclusion. The hybrid graft of composite HA and percutaneously harvested trephine chips seemed promising as a graft substitute for ACDF. Level of Evidence: 4