Title: 2805 - Redox Biology-Based Preconditioning of MSCs to Assist Bone Regeneration


Jun Watanabe (Presenter)
Tohoku University Graduate School of Dentistry

Masahiro Yamada, Tohoku University Graduate School of Dentistry
Kunimichi Niibe, Tohoku University Graduate School of Dentistry
Takeru Kondo, Tohoku University Graduate School of Dentistry
Hiroshi Egusa, Tohoku University Graduate School of Dentistry


Objectives: Apoptosis of transplanted bone marrow-derived mesenchymal stem cells (BMSCs) by acute inflammation-induced oxidative stress is a critical issue in cell therapies. N-acetyl-L-cysteine (NAC) supplies cells with a major antioxidant molecule, glutathione. We hypothesized that pre-treatment of BMSCs with NAC would reinforce apoptotic resistance and enhance bone regeneration at the transplanted site. The objective of this study was to investigate the effects of pre-treatment with NAC on the apoptotic resistance and bone regeneration capability of BMSCs.

Methods: Rat femoral BMSCs were treated in growth medium with or without 5 mM NAC for 6 hours, followed by exposure to 100 μM H2O2 for 24 hours to induce oxidative stress. Apoptotic induction and oxidative stress were evaluated by annexin V-based flow cytometry, western blotting of effector caspase activation, and quantification of intracellular redox molecules. Critical-sized rat femur defects were filled with a collagen sponge containing fluorescent quantum dot-labeled autologous BMSCs with or without NAC treatment. Apoptotic induction and bone healing were evaluated by immunohistochemical and micro–computed tomography analyses.

Results: Pretreatment with NAC reduced the rate of H2O2-induced apoptosis from 75% to 14% in the BMSCs. Expression of cleaved caspase 3 induced by H2O2 was suppressed in BMSCs pretreated with NAC. Pretreatment with NAC increased the amount of cellular glutathione and alleviated the H2O2-induced elevation of intracellular reactive oxygen species. The number of apoptotic and surviving cells in the transplanted site after 3 days was significantly lower and higher in the NAC pre-treated group, respectively. By the 3th week, significantly enhanced new bone formation was observed in the NAC pre-treated group. Fluorescent cells in the NAC pre-treated group aligned near the newly formed bone.

Conclusions: Pre-treatment of BMSCs with NAC before local transplantation enhances bone regeneration via reinforced resistance to oxidative stress-induced apoptosis at the transplanted site.

This abstract is based on research that was funded entirely or partially by an outside source:
This work was supported by Grant-in-Aids for Exploratory Research (17K19742, M.Y. and H.E) and Scientific Research (C: 16K11585, M.I, M.Y. and H.E.; B: 17H04387, M.Y. and H.E.) from the Japan Society for the Promotion of Science.

Disclosure Statement:
The submitter must disclose the names of the organizations with which any author have a relationship, the nature of the relationship, and the clinical or research area involved. The following is submitted: NONE