Fisetin regulates the biological effects of rat nucleus pulposus mesenchymal stem cells under oxidative stress by sirtuin-1 pathway.

BACKGROUND: Excessive oxidative stress has been accepted as one of the critical factors for intervertebral disc degeneration (IDD), which is associated with low back pain (LBP). Fisetin (Fis) is a bioactive flavonoid that possesses strong bioactive activity. In present study, we aimed to illuminate the role of Fis on nucleus pulposus mesenchymal stem cells (NPMSCs). METHODS: NPMSCs were isolated and cultured from rat NP tissues and identified by flow cytometry and multilinear differentiation. The cytotoxicity of Fis, EX-527, and hydrogen peroxide (H2 O2 ) on NPMSCs was validated using Cell Counting Kit-8 tests. Cell apoptosis was tested by flow cytometry and TUNEL assay. Inflammatory mediators were assessed by Elisa tests, RT-PCR. Extracellular matrix (ECM) metabolism was measured by Western blot analysis and RT-qPCR. The expression of the SIRT1 was evaluated by Western blot analysis. RESULTS: NPMSCs were successfully isolated and cultured from rat NP tissues, and it has been identified by flow cytometry and multilinear differentiation. The results showed that Fis attenuated H2 O2 -induced apoptosis, inflammation, and ECM degradation of NPMSCs. Moreover, the above protective effects of Fis can be inhibited by EX-527, a unique SIRT1 inhibitor, indicating that SIRT1 may involve in the mechanism of Fis in protecting NPMSCs from oxidative stress. CONCLUSIONS: As a natural compound with little cytotoxicity on NPMSCs, Fis alleviate H2 O2 -induced apoptosis, inflammation, and ECM degradation by suppressing oxidative stress, this finding may add the theoretical basis for research on new treatment of IDD based on NPMSCs.

Orientated-quantitative computed tomography study on individualized axial safety target area of femoral neck screw channel and establishment of a stable spatial coordinate system based on anterior cortex of femoral neck basilar.

BACKGROUND: Frequent in-out-in femoral neck screws were reported potential huge iatrogenic-injury risks, related to axial safe target area (ASTA) of femoral neck screws channel. However, orientated-quantitative ASTA based on stable coordinate system was unreported before. METHODS: Three-dimensional reconstruction was performed on computed tomography (CT) images of 139 intact normal hips, and the intersection area, defined as ASTA, was obtained by superimposing the axial CT images of each femoral neck. Taking anterior cortex of femoral neck basilar (AC-FNB) as landmark, a coordinate system was established to measure the anterior-posterior diameter (D-AP), the superior-inferior diameter (D-SI) and the oblique angle respectively. Each intersection was overlaid up to the axial CT images to determine the coronal location of the ASTA boundaries. RESULTS: Each ASTA presented an inclined rounded triangle with a flat anterior base coincided with AC-FNB. There were significant sex differences in D-SI (male: 33.6±2.3 vs. female: 29.4±1.9 mm) and D-AP (male: 25.3±2.1 vs. 21.9±1.9 mm), P <0.001. D-SI was found to be positively correlated with D-AP ( R2 =0.6). All fluoroscopic visible border isthmus completely matched the corresponding ASTA boundaries. The oblique angle was 5-53° (male: 28.1±10.3°, female: 27.1±8.2°) without significant difference between sexes. CONCLUSION: The intersection method was employed to conveniently acquire orientated-quantitative individualized ASTA. Under this coordinate system, x-ray data of screws could be converted to axial coordinates in CT ASTA, which could help surgeons design combined screws configuration preoperatively and evaluate quantitatively their axial position intraoperatively.

Biomechanical Analysis of Cortical Bone Trajectory Screw Versus Bone Cement Screw for Fixation in Porcine Spinal Low Bone Mass Model.

STUDY DESIGN: A prospective study of in vitro animal. OBJECTION: To compare the biomechanics of cortical bone trajectory screw (CBT) and bone cement screw (BC) in an isolated porcine spinal low bone mass model. SUMMARY OF BACKGROUND DATA: The choice of spinal fixation in patients with osteoporosis remains controversial. Is CBT better than BC? Research on this issue is lacking. METHODS: Ten porcine spines with 3 segments were treated with EDTA decalcification. After 8 weeks, all the models met the criteria of low bone mass. Ten specimens were randomly divided into groups, group was implanted with CBT screw (CBT group) and the other group was implanted with bone cement screw (BC group). The biomechanical material testing machine was used to compare the porcine spine activities of the two groups in flexion, extension, bending, and axial rotation, and then insertional torque, pull-out force, and anti-compression force of the 2 groups were compared. Independent sample t test was used for comparison between groups. RESULTS: Ten 3 segments of porcine spine models with low bone mass were established, and the bone mineral density of all models was lower than 0.75 g/cm 2 . There is no difference between the CBT and BC groups in flexion, extension, bending, and axial rotation angle, P >0.05. However, there were significant differences between the 2 groups and the control group, with P ＜0.01. The 2 groups significantly differed between the insertional torque ( P =0.03) and the screw pull-out force ( P =0.021). The anti-compression forces between the 2 groups have no significant difference between the two groups ( P =0.946). CONCLUSIONS: The insertional torque and pull-out force of the CBT were higher than those of the BC in the isolated low bone porcine spine model. The range of motion and anti-compression ability of the model was similar between the 2 fixation methods.

Functionalized self-assembling peptide RADKPS hydrogels promote regenerative repair of degenerated intervertebral discs.

Objective: the aim of this study was to investigate whether the functionalized self-assembling peptide hydrogel RADKPS is safe and effective for regenerative repair of degenerative intervertebral discs. Methods: an in vitro degenerative model of human nucleus pulposus cells was constructed by serum starvation culture, and their proliferation, apoptosis and viability were examined after three-dimensional culture with the RADKPS hydrogel. An in vivo degenerative model of the rabbit intervertebral disc was constructed by annulus fibrosus puncture, and the degeneration of the intervertebral disc was evaluated by imaging, histology, immunohistochemistry, and biomechanics after RADKPS hydrogel intervention. Results: through in vitro cell experiments it is shown that human degenerated nucleus pulposus cells after three-dimensional culture with the RADKPS hydrogel still exhibited better proliferation, viability, and low apoptosis rate. Through in vivo animal experiments we found that rabbit degenerated intervertebral discs intervened with the RADKPS hydrogel had higher water content, better histological morphology, more extracellular matrix synthesis, and better biomechanical properties. It is demonstrated that the RADKPS hydrogel may initiate the endogenous repair process through the sustained recruitment and enrichment of nucleus pulposus progenitor cells. Conclusion: it is verified from both in vitro cellular experiments and in vivo animal experiments that the regenerative repair effect of RADKPS, a functionalized self-assembling peptide hydrogel, on degenerated intervertebral discs is safe and effective. It is shown that it would be a new therapeutic approach for the regenerative repair action of intervertebral discs.

Intradiscal Injection of Autologous Discogenic Cells in Patients with Discectomy: A Prospective Clinical Study of Its Safety and Feasibility.

The treatment of intervertebral disc degeneration (IVDD) is still a huge challenge for clinical updated surgical techniques and basic strategies of intervertebral disc regeneration. Few studies have ever tried to combine surgery and cell therapy to bridge the gap between clinical and basic research. A prospective clinical study with a 72-month follow-up was conducted to assess the safety and feasibility of autologous discogenic cells transplantation combined with discectomy in the treatment of lumbar disc herniation (LDH) and to evaluate the regenerative ability of discogenic cells in IVDD. Forty patients with LDH who were scheduled to have discectomy enrolled in our study and were divided into the observed group (transplantation of autologous discogenic cells after discectomy) and control group (only-discectomy). Serial MRI and X-ray were used to evaluate the degenerative extent of index discs, and clinical scores were used to determine the symptomatic improvement. No adverse events were observed in the observed group, and seven patients in the control group underwent revisions. Both groups had significant improvement of all functional scores post-operatively, with the observed group improving more considerably at 36-month and 72-month follow-up. The height and water content of discs in both groups decreased significantly since 36 months post-op with the control group decreased more obviously. Discectomy combined with autologous discogenic cells transplantation is safe and feasible in the treatment of LDH. Radiological analysis demonstrated that discogenic cells transplantation could slow down the further degeneration of index discs and decrease the complications of discectomy.

NAT10 Promotes Osteogenic Differentiation of Mesenchymal Stem Cells by Mediating N4-Acetylcytidine Modification of Gremlin 1.

OBJECTIVE: To investigate the function of NAT10 in mesenchymal stem cell (MSC) osteogenic differentiation and study the mechanism by which NAT10 affects MSC osteogenesis by mediating Gremlin 1 N4-acetylcytidine (ac4C) modification. METHODS: Osteogenic differentiation of MSCs was induced, and the osteogenic ability was evaluated with alizarin red S (ARS) and alkaline phosphatase (ALP) assays. The NAT10 expression level during MSC osteogenesis was measured by western blot (WB). MSCs were transfected with lentiviruses to inhibit (Sh-NAT10) or overexpress NAT10 (Over-NAT10), and the osteogenic differentiation ability was assessed by ARS, ALP, and osteogenic gene marker assays. ß-Catenin, Akt, and Smad signaling pathway component activation levels were assessed, and the expression levels of key Smad signaling pathway molecules were determined by PCR and WB. The Gremlin 1 mRNA ac4C levels were analyzed using RIP-PCR, and the Gremlin 1 mRNA degradation rate was determined. Sh-Gremlin 1 was transfected to further investigate the role of NAT10 and Gremlin 1 in MSC osteogenesis. RESULTS: During MSC osteogenesis, NAT10 expression, ARS staining, and the ALP level gradually increased. Decreasing NAT10 expression inhibited, and increasing NAT10 expression promoted MSC osteogenic differentiation. NAT10 affected the BMP/Smad rather than the Akt and ß-Catenin signaling pathway activation by regulating Gremlin 1 expression. The Gremlin 1 mRNA ac4C level was positively regulated by NAT10, which accelerated Gremlin 1 degradation. Sh-Gremlin 1 abolished the promotive effect of NAT10 on MSC osteogenic differentiation. CONCLUSION: NAT10 positively regulated MSC osteogenic differentiation through accelerating the Gremlin 1 mRNA degradation by increasing its ac4C level. These results may provide new mechanistic insight into MSC osteogenesis and bone metabolism in vivo.