Pretreatment of NPMSCs with Appropriate Concentration of H2O2 Enhances its Ability to Treat IVDD

Background: Nucleus pulposus mesenchymal stem cells (NPMSCs) transplantation is a promising treatment for intervertebral disc degeneration (IVDD). However, the transplanted NPMSCs exhibited weak cell proliferation, high cell apoptosis, and low ability to resist the harsh microenvironment of the degenerated intervertebral disc. There is an urgent need in exploring feasible methods to enhance the therapeutic efficacy of NPMSCs transplantation.Objective: To identify the optimal concentration for NPMSCs pretreatment with hydrogen peroxide (H2O2) and explore the therapeutic efficacy of NPMSCs transplantation using H2O2 pretreatment in IVDD.Methods: Rat NPMSCs were pretreated with different concentrations of H2O2. The proliferation, ROS level, and apoptosis of NPMSCs were detected by CCK-8 test, EdU staining, and flow cytometry in vitro. The underlying signaling pathways were explored utilizing western blot. A rat needle-puncture stimulated IVDD model was established. X-ray, histological staining, and multi-mode small animal live imaging system were performed to evaluate the therapeutic effect of H2O2-pretreated NPMSCs in vivo.Results: 75 μM H2O2 pretreated NPMSCs demonstrated the strongest elevated cell proliferation by inhibiting the Hippo pathway. Meanwhile, 75 μM H2O2 pretreated NPMSCs exhibited significantly enhanced anti-oxidative stress ability, which is related to downregulated Brd4, Keap1, and upregulated Nrf2. 75 μM H2O2 pretreated NPMSCs also exhibited distinctly declined apoptosis. In vivo experiments results verified that 75 μM H2O2 pretreated NPMSCs-transplanted rats exhibited enhanced disc height index (DHI%) and better histological morphology, which means 75 μM H2O2 pretreated NPMSCs can better adapt to the environment of degenerative intervertebral discs and promote the repair of IVDD.Conclusions: Pretreatment with 75 μM H2O2 was the optimal concentration to improve the ability of proliferation, anti-oxidative stress, and anti-apoptosis of transplanted NPMSCs, which is expected to provide a new feasible method to improve the stem cell therapy efficacy of IVDD.