Haisong Yang

MicroRNA-320a Regulates the Osteogenic Differentiation of Human Bone Marrow- Derived Mesenchymal Stem Cells by Targeting HOXA10

Background/Aims: Human bone marrow-derived mesenchymal stem cells (hMSCs) are a promising cell source for bone engineering owing to their high potential to differentiate into osteoblasts. The bone morphogenetic protein-inducible gene homeobox a10 (HOXA10) is a critical regulator of osteogenesis. The objective of the present study was to identify microR-NAs (miRNAs) targeting HOXA10 and examine the effects on the osteogenic differentiation of hMSCs. Methods: Based on in silico analysis, HOXA10-targeting miRNAs were selected and their regulatory roles in osteoblast differentiation were investigated. Results: Six HOXA10-targeting miRNAs were identifIed by computational analysis, of which miR-320a was selected for further analysis because it was downregulated during osteogenic induction. Overexpression of miR-320a downregulated HOXA10 and significantly inhibited osteogenesis in hMSCs, as determined by the downregulation of the osteogenic markers Runx2, ALP, and OC and the inhibition of ALP activity and matrix mineralization, whereas miR-320a inhibition had the opposite effects. Furthermore, ectopic expression of HOXA10 (not including 3′-UTR) rescued the effects of miR-320a on osteogenic differentiation. Conclusion: These results suggest that miR-320a acts as a critical regulator of osteogenic differentiation of hMSCs by repressing its target HOXA10.

Connexin 43 promotes ossification of the posterior longitudinal ligament through activation of the ERK1/2 and p38 MAPK pathways

Although cervical ossification of the posterior longitudinal ligament (OPLL) is one of the most common spinal diseases, the pathogenic mechanism is still not fully understood. Abnormal mechanical stress distribution is believed to be one of the main causes of OPLL. We have previously found that mechanical stress can up-regulate connexin 43 (Cx43) expression in ligament fibroblasts; this transduces mechanical signals to promote osteoblastic differentiation. In the present study, in order to explore further the intracellular mechanisms of Cx43-induced osteoblast differentiation of ligament fibroblasts, we investigate the potential roles of the osteogenic signaling pathway components ERK1/2, p38 MAPK and JNK in Cx43-mediated mechanical signal transduction. We first confirm higher Cx43 levels in both in vivo ligament tissue from OPLL patients and in vitro cultured OPLL cells. We find that ERK1/2, p38 MAPK and the JNK pathway are all activated both in vivo and in vitro. The activation of these signals was dependent upon Cx43, as its knock-down resulted in diminished mechanical effects and reduced signaling. Moreover, its knock-down almost reversed the osteogenic effect of mechanical stress on ligament fibroblasts and the blocking of the ERK1/2 and p38 MAPK pathways but not the JNK pathway, partly diminished this effect. Therefore, Cx43, which is up-regulated by mechanical stress, seems to function partly via the activation of ERK1/2 and p38 MAPK signals to promote the osteoblastic differentiation of ligament fibroblasts.

MiR-101 Targets the EZH2/Wnt/β-Catenin the Pathway to Promote the Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

Mounting evidence indicates that microRNAs (miRNAs) are involved in multiple processes of osteogenic differentiation. MicroRNA-101 (miR-101), identified as a tumor suppressor, has been implicated in the pathogenesis of several types of cancer. However, the expression of miR-101 and its roles in the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) remain unclear. We found that the miR-101 expression level was significantly increased during the osteogenic differentiation of hBMSCs. MiR-101 depletion suppressed osteogenic differentiation, whereas the overexpression of miR-101 was sufficient to promote this process. We further demonstrated that enhancer of zeste homolog 2 (EZH2) was a target gene of miR-101. EZH2 overexpression and depletion reversed the promoting or suppressing effect of osteogenic differentiation of hBMSCs, respectively, caused by miR-101. In addition, we showed that miR-101 overexpression promoted the expression of Wnt genes, resulting in the activation of the Wnt/β-catenin signaling pathway by targeting EZH2, while the activity of β-catenin and the Wnt/β-catenin signaling pathway was inhibited by ICG-001, a β-Catenin inhibitor, which reversed the promoting effect of miR-101. Finally, miR-101 also promotes in vivo bone formation by hBMSCs. Collectively, these data suggest that miR-101 is induced by osteogenic stimuli and promotes osteogenic differentiation at least partly by targeting the EZH2/Wnt/β-Catenin signaling pathway.

Connexin 43 Affects Osteogenic Differentiation of the Posterior Longitudinal Ligament Cells via Regulation of ERK Activity by Stabilizing Runx2 in Ossification.

Aims: Connexin 43 is one of the most potent gap junction proteins related to osteoblast differentiation and bone formation. We hypothesized that Connexin 43 is a significant factor in osteogenic differentiation in the posterior longitudinal ligament through the regulation of extracellular signal-regulated kinases (ERK) activity by converging on Runt-related transcription factor 2 (Runx2) activity. In this study, we mapped the activity of Connexin 43 to ERK and Runx2 by extracting longitudinal ligament cell for culture and silencing Connexin expression in addition to dexamethasone treatment in vitro. Methods: qRT-PCR, Western Blot, and Runx2-responsive Luciferase Reporter Assay were performed to detect the activity of ERK, Runx2 and the expression levels of osseous genes under Connexin 43 modification. Results: Downregulation of Connexin 43 resulted in suppression of dexamethasone-induced osteogenic differentiation, inhibition of the ERK and Runx2 activity, and reduction of osseous gene expression. Conclusion: these data support that Connexin 43 significantly regulates osteogenic differentiation in the cells from posterior longitudinal ligament by altering the activity of ERK, and subsequently causing the modification of Runx2.

Ossification of the posterior ligament is mediated by osterix via inhibition of the β-catenin signaling pathway

Ossification of the posterior longitudinal ligament (OPLL) involves ectopic calcification of the spinal ligament preferentially at the cervical spine. OPLL is associated with different diseases and occurs by endochondral ossification, which is associated with the activity of different transcription factors. However, the pathogenesis of OPLL remains unclear. Here, we investigated the role of osterix (Osx), a transcription factor that functions downstream of Runx2 and is an important regulator of osteogenesis, in the process of OPLL in a dexamethasone (Dex)-induced model of spinal ligament ossification. Our results showed that Osx is upregulated in patients with OPLL and during the ossification of ligament cells in parallel with the upregulation of osteogenic markers including osteocalcin (OCN), alkaline phosphatase (ALP) and collagen-1 (Col-1). Dex-induced ossification of ligament cells was associated with the downregulation and inactivation of β-catenin, and these effects were offset by Osx knockdown. Activation of β-catenin signaling abolished the effect of Dex on ossification and the upregulation of osteogenic markers. Taken together, our results suggest that OPLL is mediated by Osx via a mechanism involving the Wnt/β-catenin signaling pathway, providing a basis for further research to identify potential targets for the treatment of OPLL.

In Situ Decompression to Spinal Cord During Anterior Controllable Antedisplacement Fusion Treating Degenerative Kyphosis with Stenosis: Surgical Outcomes and Analysis of C5 Nerve Palsy Based on 49 Patients

OBJECTIVE To observe outcomes of anterior controllable antedisplacement fusion (ACAF) in treatment of degenerative kyphosis with stenosis (DKS) and analyze probability of C5 nerve palsy. METHODS From 2016 to 2017, a consecutive cohort of adults with DKS underwent ACAF. All patients underwent cervical radiography, computed tomography, and magnetic resonance imaging. Operative duration, blood loss, and hospital stay were estimated. Radiologic assessment included kyphotic correction, decompression width, and spinal canal area. Postoperative curvature of spinal cord was observed on sagittal magnetic resonance imaging. Japanese Orthopaedic Association score was used to evaluate neurologic status. C5 nerve palsy and other complications were recorded. RESULTS The study included 49 patients. There was significant kyphosis correction postoperatively (-19.4° vs. 3.5°, P < 0.01). On cross-sectional computed tomography, mean decompression width was 19.0 mm, and spinal canal area was 218.5 mm2. On sagittal magnetic resonance imaging, spinal cord curvature was classified into 5 types: type I, lordosis; type II, straight with no shifting; type III, straight with shifting; type IV, sigmoid; and type V, kyphosis. After ACAF, the spinal cord was maintained in good curvature with no shifting in all patients. No patient presented with C5 nerve palsy. Mean postoperative Japanese Orthopaedic Association score was significantly better than preoperatively (14.9 points vs. 9.0 points, P < 0.01), with mean improvement rate of 79.8%. CONCLUSIONS ACAF provides in situ decompression and good curvature to the spinal cord. Good neurologic recovery is obtained with lower incidence of C5 nerve palsy when ACAF is used to treat DKS.

Anterior Controllable Antedisplacement Fusion as a Choice for Degenerative Cervical Kyphosis with Stenosis: Preliminary Clinical and Radiologic Results

OBJECTIVE The optimal surgical procedure for degenerative cervical kyphosis with stenosis (DCKS) remains controversial. The purpose of this study is to describe the preliminary clinical and radiologic results of anterior controllable antedisplacement fusion (ACAF) as a surgical technique for DCKS. METHODS In the period from 2016 through 2017, a consecutive cohort of adults with degenerative cervical kyphosis (30° ≥ Cobb angles ≥5°) and stenosis (anteroposterior diameter of the spinal canal <12 mm) underwent ACAF, a technique making the vertebrae move forward in a hoisting manner. We retrospectively reviewed these patients, and primary outcomes of interest included operation duration, blood loss, hospital stay, kyphosis correction, decompression width, anteroposterior diameter of the spinal canal, morphology of the spinal cord, Japanese Orthopaedic Association (JOA) scores, and complications. RESULTS Thirty patients were included in the study. All patients were followed for a mean of 12 months. Mean operation duration and estimated blood loss were 104.8 minutes and 221.6 mL, respectively, and the mean length of hospital stay was 5.6 days. The decompression width reached 17.7 mm. The postoperative anteroposterior diameter of the spinal canal was 14.8 mm, with a significant improvement compared with preoperation (P < 0.01). There was also significant kyphosis correction after operation (20.4° vs. 4.8°, P < 0.01). The mean JOA score was significantly better than preoperation (15.0 vs. 9.3 points; P < 0.01). There were no surgery-related complications. CONCLUSIONS ACAF, correcting cervical kyphosis and simultaneously enlarging the volume of the spinal canal, is a good choice for DCKS.

Surgical results and complications of anterior controllable antedisplacement fusion as a revision surgery after initial posterior surgery for cervical myelopathy due to ossification of the posterior longitudinal ligament

Posterior surgery has been widely used as an initial surgery for cervical ossification of the posterior longitudinal ligament (OPLL). However, some patients require revision surgery because of failure of symptom relief or late neurological deterioration after posterior surgery. The aim of this study is to retrospectively investigated the surgical results and complications of anterior controllable antedisplacement fusion (ACAF) as a revision surgical technique after initial posterior surgery for OPLL. The present study concluded 13 patients. The operation duration, blood loss and hospital stay was estimated. Radiologic assessment included type and extent of OPLL, decompression width and antero-posterior (AP) diameter of the spinal cord. The JOA scoring system was used to evaluate the neurological status. Surgery- and implant-related complications such as cerebrospinal fluid (CSF) leakage, spinal cord or nerve injury, subsidence and pseudoarthrosis were all recorded. The results showed that nine patients undergoing revision ACAF because of residual stenosis after initial posterior surgery, two because of OPLL progression, one because of lamina closure, one because of a blind man requiring better neurological function of both hands. The mean decompression width and AP diameter of the spinal cord was 20.3 ± 2.1 mm and 5.8 ± 1.1 mm respectively. The mean postoperative JOA score at last follow-up was significantly better than preoperation (14.8 ± 2.5 vs. 8.5 ± 2.7 points, P < 0.01), with a mean improvement rate of 75.3% ± 12.2%. No complication such as CSF leakage, spinal cord or nerve injury, subsidence and pseudoarthrosis occurred. In conclusion, ACAF is a well choice for revision surgery after initial posterior surgery for OPLL.

Corrigendum to "Ossification of the posterior ligament is mediated by osterix via inhibition of the β-catenin signaling pathway" [Exp. Cell Res. 349 (2016) 53-59].

Corrigendum to “Ossification of the posterior ligament is mediated by osterix via inhibition of the β-catenin signaling pathway” [Exp. Cell Res. 349 (2016) 53–59] Lei Shi, Guodong Cai, Jiangang Shi, Yongfei Guo, Dechun Chen, Deyu Chen, Haisong Yanga, a Department of Orthopedics, Changzheng Hospital, Second Military Medical University, No. 415, Fengyang Road, Shanghai 200003, China b Department of Orthopedics, Affiliated Hospital of Taishan Medical University, 706 Taishan Street, Tai’an 271000, Shangdong Province, China