Introduction: Severe injuries, tissue ischemia and genetic defects impair skeletal muscle regeneration. Delivery of stem cells capable of differentiating into skeletal muscle and/or endothelial cells may facilitate the regeneration [1-3]. However, ischemic environment in the injured muscles significantly compromises the therapeutic efficacy, as cell survival and differentiation are limited in such an environment. In this work, we demonstrated that using hydrogels with prosurvival and proangiogenic factor basic fibroblast growth factor (bFGF) significantly augmented transplanted cell survival and differentiation, as well as promoted ischemic limb regeneration.
Materials and Methods: Hydrogel was synthesized by free radical polymerization of N-isopropylacrylamide, 2-hydroxyethyl methacrylate, and macromer Acrylic acid-polylactide (APLA). For in vitro study, mesenchymal stem cells (MSCs, 10 million/mL) were encapsulated in the hydrogel containing bFGF (50 µg/mL) and heparin (1 mg/mL). The cells were cultured under hypoxic condition (1% O2, DMEM without FBS) for a 2-week period. Cells seeded in the hydrogel without bFGF and cultured under normal and hypoxic conditions were used as controls. For in vivo study, hindlimb ischemia was first induced in mice (aged 8 to 10 weeks) by permanent ligation of femoral artery and vein. A total of 200 µL cell delivery system was then injected into muscles (4 injection points). Treatment groups included hydrogel only, hydrogel with MSCs, and hydrogel with MSCs and bFGF. Open surgery mice were used as negative control and non-operated mice as positive control.
Results and Discussion: The hydrogel had a gel-sol transition temperature around 26°C. It exhibited a Young’s modulus of 17.1± 3.4 kPa at 37oC (10% w/v in DPBS). The encapsulated bFGF was able to gradually release from hydrogel for two weeks. The released bFGF was bioactive. In vitro, the released bFGF significantly promoted MSC survival under ischemic conditions. 4 weeks after implantation, the density of surviving MSCs was significantly higher in the bFGF release group. Interestingly, some of the surviving MSCs were differentiated into skeletal muscle cells and endothelial cells and integrated with the host tissues. In addition, muscle blood perfusion was significantly improved by delivering MSCs and bFGF, with full recovery occurred after only 2 weeks of implantation.
Conclusions: A thermosensitive and biodegradable hydrogel with skeletal muscle tissue-like modulus was synthesized and used to deliver bFGF and MSCs into ischemic limb. The released bFGF significantly enhanced MSC survival under ischemic conditions. The developed cell delivery system also promoted ischemic limb regeneration.
References:
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[2] Carlson ME, et al. Aging Cell. 2007;6:371-82;
[3] Gussoni E, et al. J Clin Invest. 2002;110:807-14.