METHODS: Bone marrow-derived cells were harvested from donor mice and differentiated to macrophages (BMDM), then suspended within fibrin hydrogels to facilitate BMDM transplantation. BMDM survival was characterized in vitro. The effect of this BMDM fibrin hydrogel construct at a nerve injury site was assessed using a mouse sciatic nerve gap injury. Mice were equally distributed to \"fibrin+Mφ\" (fibrin hydrogels containing culture medium and BMDM) or \"fibrin\" hydrogel control (fibrin hydrogels containing culture medium alone) groups. Flow cytometry (n = 3/group/endpoint) and immunohistochemical analysis (n = 5/group/endpoint) of the nerve gap region were performed at days 3, 5, and 7 after repair.
RESULTS: Incorporating macrophage colony-stimulating factor (M-CSF) improved BMDM survival and expansion. Transplanted BMDM survived for at least 7 days in a nerve gap (~40% retained at day 3 and ~15% retained at day 7). From transplantation, macrophage quantities within the nerve gap were elevated when comparing fibrin+Mφ with fibrin control (~25% vs. 3% at day 3 and ~14% vs. 6% at day 7). Endothelial cells increased by about fivefold within the nerve gap, and axonal extension into the nerve gap increased almost twofold for fibrin+Mφ compared with fibrin control.
CONCLUSIONS: BMDM suspended within fibrin hydrogels at a nerve gap do not impair regeneration.
方法:从供体小鼠中收获骨髓来源的细胞,并分化为巨噬细胞(BMDM),然后悬浮在纤维蛋白水凝胶中以促进BMDM移植。在体外表征BMDM存活。使用小鼠坐骨神经间隙损伤评估该BMDM纤维蛋白水凝胶构建体在神经损伤部位的作用。将小鼠平均分配给“纤维蛋白Mφ”(含有培养基和BMDM的纤维蛋白水凝胶)或“纤维蛋白”水凝胶对照(仅含有培养基的纤维蛋白水凝胶)组。在修复后第3、5和7天进行神经间隙区域的流式细胞术(n=3/组/终点)和免疫组织化学分析(n=5/组/终点)。
结果:掺入巨噬细胞集落刺激因子(M-CSF)可改善BMDM的存活和扩增。移植的BMDM在神经间隙中存活至少7天(在第3天保留约40%,在第7天保留约15%)。从移植中,当将纤维蛋白+Mφ与纤维蛋白对照进行比较时,神经间隙内的巨噬细胞数量升高(~25%vs.3%在第3天和〜14%与6%在第7天)。内皮细胞在神经间隙内增加了约五倍,与纤维蛋白对照相比,纤维蛋白Mφ的轴突向神经间隙的延伸几乎增加了两倍。
结论:在神经间隙处悬浮在纤维蛋白水凝胶内的BMDM不损害再生。