Pretreatment of nucleus pulposus mesenchymal stem cells with appropriate concentration of HO enhances their ability to treat intervertebral disc degeneration.

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 a low ability to resist the harsh microenvironment of the degenerated intervertebral disc. There is an urgent need to explore feasible methods to enhance the therapeutic efficacy of NPMSCs transplantation.

OBJECTIVE

To identify the optimal concentration for NPMSCs pretreatment with hydrogen peroxide (HO) and explore the therapeutic efficacy of NPMSCs transplantation using HO pretreatment in IVDD.

METHODS

Rat NPMSCs were pretreated with different concentrations (range from 25 to 300 μM) of HO. The proliferation, reactive oxygen species (ROS) level, and apoptosis of NPMSCs were detected by cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, and flow cytometry in vitro. The underlying signalling pathways were explored utilizing Western blotting. A rat needle puncture-stimulated IVDD model was established. X-ray, histological staining, and a multimode small animal live imaging system were used to evaluate the therapeutic effect of HO-pretreated NPMSCs in vivo.

RESULTS

NPMSCs pretreated with 75 μM HO demonstrated the strongest elevated cell proliferation by inhibiting the Hippo pathway (P < 0.01). Meanwhile, 75 μM HO-pretreated NPMSCs exhibited significantly enhanced antioxidative stress ability (P < 0.01), which is related to downregulated Brd4 and Keap1 and upregulated Nrf2. NPMSCs pretreated with 75 μM HO also exhibited distinctly decreased apoptosis (P < 0.01). In vivo experiments verified that 75 μM HO-pretreated NPMSCs-transplanted rats exhibited an enhanced disc height index (DHI% = 90.00 ± 4.55, P < 0.01) and better histological morphology (histological score = 13.5 ± 0.5, P < 0.01), which means 75 μM HO-pretreated NPMSCs can better adapt to the environment of degenerative intervertebral discs and promote the repair of IVDD.

CONCLUSIONS

Pretreatment with 75 μM HO was the optimal concentration to improve the proliferation, antioxidative stress, and antiapoptotic ability of transplanted NPMSCs, which is expected to provide a new feasible method to improve the stem cell therapy efficacy of IVDD.