The efficacy of cell therapy is limited by low retention and survival of transplanted cells in the target tissues. In this work, we hypothesize that pharmacological preconditioning with celastrol, a natural potent antioxidant, could improve the viability and functions of mesenchymal stromal cells (MSC) encapsulated within an injectable scaffold. Bone marrow MSCs from rat (rMSC) and human (hMSC) origin were preconditioned for 1 hour with celastrol 1 μM or vehicle (DMSO 0.1% v), then encapsulated within a chitosan-based thermosensitive hydrogel. Cell viability was compared by alamarBlue and live/dead assay. Paracrine function was studied first by quantifying the proangiogenic growth factors released, followed by assessing scratched HUVEC culture wound closure velocity and proliferation of HUVEC when cocultured with encapsulated hMSC. In vivo, the proangiogenic activity was studied by evaluating the neovessel density around the subcutaneously injected hydrogel after one week in rats. Preconditioning strongly enhanced the viability of rMSC and hMSC compared to vehicle-treated cells, with 90% and 75% survival versus 36% and 58% survival, respectively, after 7 days in complete media and 80% versus 64% survival for hMSC after 4 days in low serum media (). Celastrol-treated cells increased quantities of proangiogenic cytokines compared to vehicle-pretreated cells, with a significant 3.0-fold and 1.8-fold increase of VEGFa and SDF-1α, respectively (). The enhanced paracrine function of preconditioned MSC was demonstrated by accelerated growth and wound closure velocity of injured HUVEC monolayer () in vitro. Moreover, celastrol-treated cells, but not vehicle-treated cells, led to a significant increase of neovessel density in the peri-implant region after one week in vivo compared to the control (blank hydrogel). These results suggest that combining cell pretreatment with celastrol and encapsulation in hydrogel could potentiate MSC therapy for many diseases, benefiting particularly ischemic diseases.

中文翻译：

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药理学预处理提高了水凝胶包裹的间充质基质细胞的活力和促血管生成旁分泌功能

细胞疗法的功效受到目标组织中移植细胞的低保留和存活率的限制。在这项工作中，我们假设用天然强效抗氧化剂 celastrol 进行药理学预处理可以提高封装在可注射支架内的间充质基质细胞 (MSC) 的活力和功能。来自大鼠 (rMSC) 和人 (hMSC) 的骨髓 MSC 用 celastrol 1  μ M 或载体 (DMSO 0.1%v)，然后封装在基于壳聚糖的热敏水凝胶中。通过 alamarBlue 和活/死测定比较细胞活力。首先通过量化释放的促血管生成生长因子来研究旁分泌功能，然后评估划伤的 HUVEC 培养伤口闭合速度和 HUVEC 在与封装的 hMSC 共培养时的增殖。在体内，通过评估大鼠皮下注射水凝胶一周后的新血管密度来研究促血管生成活性。与载体处理的细胞相比，预处理显着增强了 rMSC 和 hMSC 的存活率，在完全培养基中 7 天后分别具有 90% 和 75% 的存活率与 36% 和 58% 的存活率，4 天后 hMSC 的存活率分别为 80% 和 64%低血清培养基中的天数（）。与载体预处理的细胞相比，Celastrol 处理的细胞增加了促血管生成细胞因子的数量，其中 VEGFa 和 SDF-1 α分别显着增加了 3.0 倍和 1.8 倍。）。受损 HUVEC 单层的加速生长和伤口闭合速度证明了预处理 MSC 的旁分泌功能增强。)体外。此外，与对照（空白水凝胶）相比，在体内一周后，celastrol 处理的细胞（而非载体处理的细胞）导致种植体周围区域的新血管密度显着增加。这些结果表明，将细胞预处理与 celastrol 相结合并封装在水凝胶中可以增强 MSC 对许多疾病的治疗，特别是对缺血性疾病有好处。