Redox Biology-Based Preconditioning of MSCs to Assist Bone Regeneration

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.