LncAABR07053481 inhibits bone marrow mesenchymal stem cell apoptosis and promotes repair following steroid-induced avascular necrosis.

The osteonecrotic area of steroid-induced avascular necrosis of the femoral head (SANFH) is a hypoxic microenvironment that leads to apoptosis of transplanted bone marrow mesenchymal stem cells (BMSCs). However, the underlying mechanism remains unclear. Here, we explore the mechanism of hypoxic-induced apoptosis of BMSCs, and use the mechanism to improve the transplantation efficacy of BMSCs. Our results show that the long non-coding RNA AABR07053481 (LncAABR07053481) is downregulated in BMSCs and closely related to the degree of hypoxia. Overexpression of LncAABR07053481 could increase the survival rate of BMSCs. Further exploration of the downstream target gene indicates that LncAABR07053481 acts as a molecular "sponge" of miR-664-2-5p to relieve the silencing effect of miR-664-2-5p on the target gene Notch1. Importantly, the survival rate of BMSCs overexpressing LncAABR07053481 is significantly improved after transplantation, and the repair effect of BMSCs in the osteonecrotic area is also improved. This study reveal the mechanism by which LncAABR07053481 inhibits hypoxia-induced apoptosis of BMSCs by regulating the miR-664-2-5p/Notch1 pathway and its therapeutic effect on SANFH.

Comparison of the Wiltse Approach and Percutaneous Pedicle Screw Fixation Under O-arm Navigation for the Treatment of Thoracolumbar Fractures.

OBJECTIVES: The aim of this study was to evaluate the clinical outcomes of the Wiltse approach and percutaneous pedicle screw placement under O-arm navigation for the treatment of thoracolumbar fracture. METHODS: We enrolled a total of 54 patients with neurologically intact thoracolumbar fracture who received minimally invasive treatments between October 2014 and October 2018 in this retrospective study. Among these, 28 patients (22 males and six females, with a mean age of 48.6 ± 9.6 years) were treated with pedicle screw fixation through the Wiltse approach (WPSF), and another 26 (15 males and 11 females, with a mean age of 45.7 ± 10.6 years) received percutaneous pedicle screw fixation under O-arm navigation (OPSF). Statistical methods were used to perform a detailed comparison of clinical outcomes, radiologic findings, and complications between the two groups obtained preoperatively, postoperatively, and at last follow-up. RESULTS: All patients underwent surgery successfully and finished a follow-up of more than 12 months. No serious complications, such as infection, blood vessel injury, or spinal cord or nerve root injury occurred. Visual analog scale (VAS) scores, Oswestry disability index (ODI) scores, local Cobb angle (LCA), vertebral wedge angle (VWA), and R value were notably improved after surgery, though there was no clear discrepancy between the groups at each time point (P > 0.05). During the follow-up period, no patients developed neurological impairment or implant-related complications, and no patients underwent revision surgery. The WPSF group had a significantly shorter operation time than the OPSF group (68.1 ± 9.8 vs 76.1 ± 9.0 minutes, P = 0.005). Moreover, the WPSF group showed less cost of surgery than the WPSF group (48142.1 ± 1430.1 vs 59035.4 ± 1152.7 CNY, P < 0.001). There were no significant differences between the two groups in terms of the intraoperative bleeding, length of incision, or postoperative hospitalization time (P > 0.05). The accuracy of pedicle screw placement was 95.2% (160/168) in the WPSF group and 96.8% (151/156) in the OPSF group, with no significant difference between the groups (P = 0.432). CONCLUSION: Both WPSF and OPSF were safe and effective for the treatment of thoracolumbar fracture. Although the two groups showed favorable clinical and radiologic outcomes through to final follow-up, we recommended the minimally invasive WPSF given its shorter operation time and lower cost of surgery.

N-Acetyl cysteine (NAC)-mediated reinforcement of alpha-tricalcium phosphate/silk fibroin (α-TCP/SF) cement.

Calcium phosphate cements (CPCs) are popular bone filling materials and drug carriers. However poor mechanical properties and lack of osteoinduction restrict their clinical applications. Recent studies suggested the osteogenic properties of NAC. In our study, we incorporated NAC with α-TCP/SF. We found that the compressive strength of α-TCP/SF-NAC composites increased with increase in NAC concentration, possibly due to complex three-dimensional networks of SF induced by NAC, which was large and chemically heterogeneous and induced compact oriented growth of HA crystals. However the setting time increased slightly with the addition of NAC, due to the ruptured disulfide bonds in SF. The α-TCP/SF-NAC composites also showed decent biocompatibility in vitro. As a result, these composites hold great potential as bone filling materials for clinical applications, including minimally invasive surgeries.

Bioactivity of porous biphasic calcium phosphate enhanced by recombinant human bone morphogenetic protein 2/silk fibroin microsphere.

To prepare a bioactive bone substitute, which integrates biphasic calcium phosphate (BCP) and rhBMP-2/silk fibroin (SF) microsphere, and to evaluate its characteristics. Hydroxyapatite and ß-tricalcium phosphate were integrated with a ratio of 60­40%. RhBMP-2/SF (0.5 µg/1 mg) microsphere was prepared, and its rhBMP-2-release kinetics was assed. After joining pore-forming agent (Sodium chloride, NaCl), porous BCP/rhBMP-2/SF were manufactured, and its characteristics and bioactivity in vitro were evaluated. Mean diameter of rhBMP-2/SF microsphere was 398.7 ± 99.86 nm, with a loading rate of 4.53 ± 0.08%. RhBMP-2 was released in a dual-phase pattern, of which fast-release (nearly half of protein released) focused on the initial 3 days, and slow-release sustained more than 28 days. With the increase in concentration of NaCl, greater was porosity and pore size, but smaller mechanical strength of BCP/rhBMP-2/SF. Material with 150% (w/v) NaCl had an optimal performance, with a porosity of 78.83%, pore size of 293.25 ± 42.77µm and mechanical strength of 31.03 MPa. Proliferation of human placenta-derived mesenchymal stem cells (hPMSCs) on leaching extract medium was similar to the normal medium (P = 0.89), which was better than that on control group (P = 0.03). Activity of alkaline phosphatase on BCP/rhBMP-2/SF surface was higher than on pure BCP at each time point except at 1 day (P < 0.05). RhBMP-2 has a burst release on early times and a sustaining release on later times. BCP/rhBMP-2/SF with 150% (w/v) pore-forming agent has excellent porosity, pore size and mechanical strength. The biomaterial induces proliferation and differentiation hPMSCs effectively.

Cannabinoid receptor-2 selective antagonist negatively regulates receptor activator of nuclear factor kappa B ligand mediated osteoclastogenesis.

BACKGROUND: The cannabinoid receptor-2 (CB2) is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist (AM630) on receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7. METHODS: RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase (TRAP) staining using a commercial kit. Total ribonucleic acid (RNA) was isolated and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was done to examine the expression of RANK, cathepsin K (CPK) and nuclear factor kappa B (NF-κB). The extracellular signal-regulated kinase (ERK), phosphorylation of ERK (P-ERK) and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. RESULTS: AM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of ≥ 100 nmol/L could reduce TRAP positive cells to the levels that were significantly lower than the control. AM630 suppressed the expression of genes associated with osteoclast differentiation and activation, such as RANK and CPK. An analysis of a signaling pathway showed that AM630 inhibited the RANKL-induced activation of ERK, but not NF-κB. CONCLUSION: AM630 could inhibit the osteoclastogenesis from RAW264.7 induced with RANKL.

[Construction of lentiviral vector containing SOX9 gene and its expression in bone marrow mesenchymal stem cells].

OBJECTIVE: To construct the lentiviral vector containing SOX9 gene and to detect its expression in MSCs derived from rabbit bone marrow. METHODS: Human sox-9 gene coding region fragment was obtained by RT-PCR (reverse transcription-polymerase chain reaction) and then cloned into the plasmid of Pwpxl-MOD2 to form Pwpxl-MOD2/SOX9. Pwpxl-MOD2/SOX9, pRsv-REV, pMDlg-Prre and pMD2G were co-transfected into 293T cells to obtain recombinant virus containing SOX9 gene. Meanwhile, Pwpxl-MOD2, pRsv-REV, pMDlg-pRRE and pMD2G were transfected into another group of 293T cells as a control group packing into blank lentiviral vector. Then the packed lentiviral vector was transfected into MSCs which derived from rabbit bone marrow. The expression of SOX9 was detected by both RT-PCR and Western blot. Identification and proliferation of MSCs was determined by MTT after transfection. RESULTS: The sequencing and restriction analysis showed that SOX9 gene fragment was correctly connected and cloned into the plasmid Pwpxl-MOD in lentiviral vectors. After transfection, the expression of SOX9 gene in MSCs was confirmed by RT-PCR and Western blot. MTT showed the growth of MSCs had no significant effect after transfection with lentiviral vector. CONCLUSION: Lentiviral vector carrying SOX9 gene has been successfully constructed. There is a stable expression in transfected MSCs. Thus it will facilitate the exploratory development of gene and biological therapy for intervertebral disc degeneration.