The combination of forskolin and VPA increases gene expression efficiency to the hypoxia/neuron-specific system.

BACKGROUND: Spinal cord injury (SCI) tends to damage neural tissue and generate a hypoxic environment. Studies have confirmed that single therapy with gene or stem cells is inefficient, but research into combining stem cells and gene therapy in treating tissue damage has been undertaken to overcome the related limitations, which include low gene delivery efficiency and therapeutic outcome. Thus, a combination of stem cells, gene therapy, and a hypoxia-specific system may be useful for the reconstruction of SCI. METHODS: To synergistically treat SCI, a combined platform using a hypoxia/neuron-inducible gene expression system (HNIS) and human induced-neural stem cells (hiNSCs) produced by direct reprogramming was designed. Sox2- or nestin-positive hiNSCs were differentiated to Tuj1-, MAP2-, or NeuN-positive neurons. RESULTS: HNIS showed consistent hypoxia/neuron-specific gene expression in hiNSCs cultured under hypoxia. In particular, the HNIS-hiNSC combined platform revealed a complex pattern with higher gene expression compared with a single platform. In addition, we found that an optimal combination of small molecules, such as CHIR99021, valproic acid (VPA), glycogen synthase kinase-3ß (GSK3ß), and histone deacetylase (HDAC) inhibitors, could significantly enhance gene expression with HNIS-hiNSCs in the hypoxic environment. CONCLUSIONS: This experiment demonstrated that HNIS-hiNSCs combined with GSK3 and HDAC inhibitors may present another promising strategy in the treatment of SCI.

Postoperative Cervical Sagittal Realignment Improves Patient-Reported Outcomes in Chronic Atlantoaxial Anterior Dislocation.

BACKGROUND: Chronic atlantoaxial anterior dislocation (AAD) not only results in myelopathy, but dislocation-related kyphosis also results in cervical malalignment, which permanently affects neck function and patient-reported outcomes (PROs). OBJECTIVE: To investigate the effect of kyphotic correction on realigning cervical spine and independent cervical alignment parameters, which may be correlated with an improvement of PROs. METHODS: The study included 21 patients with chronic AAD-related kyphosis who underwent C1-2 reduction and correction surgery. Radiographic parameters were measured to assess cervical realignment preoperatively and postoperatively. Neck disability index (NDI), short form 12 physical component summary (SF-12 PCS), and Japanese Orthopaedic Association (JOA) scores were recorded to reveal changes in PROs. The independent parameters correlated with the improvements of PROs were analyzed. RESULTS: Of the radiographic parameters, the C1-2 Cobb angle, the C2-7 Cobb angle, thoracic inlet angle, cervical tilt, and T1 slope were significantly changed from -4.0° ± 16.2°, -29.2° ± 11.2°, 73.1° ± 13.3°, 30.4° ± 8.5°, and 29.1° ± 8.8° preoperatively to -13.5° ± 8.1° (P = .005), -18.0° ± 12.0° (P < .001), 67.1° ± 11.6° (P = .042), 23.1° ± 10.3° (P = .007), and 24.0° ± 7.0° (P = .011) at last follow-up, respectively. NDI, JOA, and SF-12 PCS scores were significantly improved postoperatively. The C1-2 Cobb angle was an independent parameter correlated with the improvements in SF-12 PCS, NDI, and JOA scores. CONCLUSION: Correction and reduction surgery can realign cervical spine in chronic AAD patients. The C1-2 Cobb angle was an independent parameter correlated with the improvements of PROs.

Débridement and Reconstruction Improve Postoperative Sagittal Alignment in Kyphotic Cervical Spinal Tuberculosis.

BACKGROUND: Cervical spinal tuberculosis is relatively common in some developing countries. It erodes vertebrae and discs, which sometimes results in cervical kyphosis and myelopathy. However, to our knowledge, no studies have evaluated improvements to patient-reported outcomes among patients who undergo surgical cervical sagittal realignment after kyphotic cervical spinal tuberculosis has been treated by débridement and reconstruction. QUESTIONS/PURPOSES: (1) Can a spine with kyphotic cervical spinal tuberculosis be returned to normal alignment and fused successfully? (2) Will patient-reported outcomes be improved with this intervention? (3) Are patient-reported outcomes correlated with realignment? METHODS: Forty-six patients with kyphotic cervical spinal tuberculosis were evaluated in this retrospective study. We generally performed surgery on patients with this condition when patients with cervical spinal tuberculosis presented with cervical kyphosis with or without neurologic deficits. Patients who did not meet these criteria were treated with other surgical procedures during the study period. Study patients were evaluated with cervical imaging, patient-reported outcomes questionnaires (Neck Disability Index [NDI], and the Japanese Orthopaedic Association [JOA] score), and physical examinations. Scores were collected by fellows preoperatively and at followup. No patient died during the followup. The mean followup was 26.8 months (range, 20-35 months). Preoperative and 2-year followup radiologic parameters were measured, including C0-2 Cobb angle, C2-7 Cobb angle, C2-7 sagittal vertical axis, center of gravity (CG) to C7 sagittal vertical axis (CG-C7 sagittal vertical axis), thoracic inlet angle, T1 slope, and neck tilt. The correlations between cervical alignment and the NDI and JOA score were analyzed. Factors correlated with the NDI and JOA score improvements were identified by multiple stepwise regression analysis. CT was used to assess bone fusion after surgery. RESULTS: All 46 patients showed bone fusion on CT scans. The preoperative C0-2 Cobb angle improved after surgery (mean difference, 5.0°; 95% CI, 2.3°-7.7°; p = 0.0068), as did C2-7 Cobb angle (mean difference, -33°; 95% CI, -35° to -31°; p = 0.0074), C2-7 sagittal vertical axis (mean difference, -28 mm; 95% CI, -30 mm to -26 mm; p = 0.0036), CG-7 sagittal vertical axis (mean difference, -26 mm; 95% CI, -28 mm to -24 mm; p = 0.0049), T1 slope (mean difference, 6.0°; 95% CI, 3.7°-8.3°; p = 0.0053) and the thoracic inlet angle (mean difference, 8.0°; 95% CI, 3.7°-12°; p = 0.0072). With the numbers available, the neck tilt angle did not improve (mean difference, -0.2°; 95% CI, -1.0° to 0.6°; p = 0.079). The preoperative NDI of 34 ± 5.1 decreased to 17 ± 4.6 (p = 0.0096) at followup. Improvements in NDI were correlated with the magnitude of correction of the cervical deformities, including C0-2 Cobb angle (r = -0.357, p = 0.007), C2-7 Cobb angle (r = 0.410, p = 0.002), T1 slope (r = -0.366, p = 0.006, thoracic inlet angle (r = -0.376, p = 0.005), C2-7 sagittal vertical axis (r = 0.450, p = 0.001), and CG-C7 sagittal vertical axis (r = 0.361, p = 0.007). The JOA score improved to 13 ± 2.6 from 7.2 ± 1.9, which did not correlate with postoperative cervical realignment. After controlling for potential confounding variables like Cobb angles and T1 slope, we found C2-7 sagittal vertical axis was the most influential factor correlated with NDI improvement (r = 0.450, p = 0.002). CONCLUSION: When treating kyphotic cervical spinal tuberculosis by débridement, decompression, and reconstruction, more attention should be drawn to realigning the cervical spine, in particular to restoring the C2-7 sagittal vertical axis. However, how best to restore the C2-7 sagittal vertical axis and cervical alignment in a kyphotic cervical spine needs further study. LEVEL OF EVIDENCE: Level III, therapeutic study.

Increased sorbitol levels in the hypertrophic ligamentum flavum of diabetic patients with lumbar spinal canal stenosis.

The pathomechanism of the ligamentum flavum (LF) hypertrophy in diabetic patients with lumbar spinal canal stenosis (LSCS) remains unclear. A cross-sectional study was undertaken to investigate the mechanism of LF hypertrophy in these patients. Twenty-four diabetic and 20 normoglycemic patients with LSCS were enrolled in the study. The structure of the LF in the study subjects was evaluated using histological and immunohistochemical methods, and the levels of sorbitol, pro-inflammatory cytokines, and the fibrogenic factor, TGF-ß1, in the LF were analyzed. In vitro experiments were performed using NIH3T3 fibroblasts to evaluate the effect of high-glucose conditions and an aldose reductase inhibitor on the cellular production of sorbitol, pro-inflammatory factors, and TGF-ß1. We found that the LF of diabetic patients exhibited significantly higher levels of sorbitol and pro-inflammatory cytokines, TGF-ß1 and of CD68-positive staining than that of the normoglycemic subjects. The diabetic LF was significantly thicker than that of the controls, and showed evidence of degeneration. The high glucose-cultured fibroblasts exhibited significantly higher levels of sorbitol, pro-inflammatory factors, and TGF-ß1 compared to the low glucose-cultured cells, and these levels were dose-dependently reduced by treatment with the aldose reductase inhibitor. Taken together, our data suggests that increased sorbitol levels in the LF of diabetic patients results in increased production of pro-inflammatory and fibrogenic factor, which contribute to LF hypertrophy, and could increase the susceptibility of diabetic patients to LSCS. Furthermore, aldose reductase inhibition effectively reduced the levels of sorbitol and sorbitol-induced pro-inflammatory factor expression in high glucose-cultured fibroblasts. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1058-1066, 2017.

The Protective Effect of Bafilomycin A1 Against Cobalt Nanoparticle-Induced Cytotoxicity and Aseptic Inflammation in Macrophages In Vitro.

Co ions released due to corrosion of Co nanoparticles (CoNPs) in the lysosomes of macrophages may be a factor in the particle-induced cytotoxicity and aseptic inflammation accompanying metal-on-metal (MOM) hip prosthesis failure. Here, we show that CoNPs are easily dissolved under a low pH, simulating the acidic lysosomal environment. We then used bafilomycin A1 to change the pH inside the lysosome to inhibit intracellular corrosion of CoNPs and then investigated its protective effects against CoNP-induced cytotoxicity and aseptic inflammation on murine macrophage RAW264.7 cells. XTT {2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide} assays revealed that bafilomycin A1 can significantly decrease CoNP-induced cytotoxicity in RAW264.7 cells. Enzyme-linked immunosorbent assays showed that bafilomycin A1 can significantly decrease the subtoxic concentration of CoNP-induced levels of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-1ß, and interleukin-6), but has no effect on anti-inflammatory cytokines (transforming growth factor-ß and interleukin-10) in RAW264.7 cells. We studied the protective mechanism of bafilomycin A1 against CoNP-induced effects in RAW264.7 cells by measuring glutathione/oxidized glutathione (GSH/GSSG), superoxide dismutase, catalase, and glutathione peroxidase levels and employed scanning electron microscopy, transmission electron microscopy, and energy dispersive spectrometer assays to observe the ultrastructural cellular changes. The changes associated with apoptosis were assessed by examining the pAKT and cleaved caspase-3 levels using Western blotting. These data strongly suggested that bafilomycin A1 can potentially suppress CoNP-induced cytotoxicity and aseptic inflammation by inhibiting intracellular corrosion of CoNPs and that the reduction in Co ions released from CoNPs may play an important role in downregulating oxidative stress in RAW264.7 cells.

hBMP-2 and hTGF-ß1 expressed in implanted BMSCs synergistically promote the repairing of segmental bone defects.

OBJECTIVE: To evaluate the effects of co-expressing hBMP-2 and hTGF-ß1 in BMSCs (bone marrow-derived mesenchymal stem cells) on the repairing process of radial segmental defects in rats. METHODS: BMSCs were infected with a high titer recombinant adenovirus carrying hTGF-ßl and/or hBMP-2 genes. Expression of exogenous genes in BMSCs was confirmed by RT-PCR and ELISA assays. In vitro effects of exogenous genes were assessed by MTT and ALP activity tests. A left radial defect model was created using 120 SD rats. Genetically modified or unmodified BMSCs were implanted with collagen sponge scaffolds into the 5-mm radial defect. The bone repair process was systematically monitored and evaluated by X-ray examinations, gross anatomic examinations, histological analyses, and biomechanical tests. RESULTS: Expression of hBMP-2 and hTGF-ß1 showed synergistic effects on promoting BMSC proliferation and enhancing ALP activity in vitro. Bone repair assays showed that hBMP-2 and hTGF-ß1 promoted the production of chondrocytes and osteoblasts. Implanted BMSCs transfected with both hBMP-2 and hTGF-ß1 led to the best bone repair outcome. CONCLUSION: hBMP-2 and hTGF-ß1 can synergistically improve the bone repair process. Our results suggest a potential clinical value of combining hBMP-2 and hTGF-ß1 in repairing bone defects.