周围神经损伤(PNI)仍然是巨大的临床挑战之一,因为它们具有相当大的长期残疾潜力。出生后神经嵴衍生的多能干细胞,包括牙龈间充质干细胞(GMSCs),代表了组织工程和各种疾病的再生治疗的种子细胞的有希望的来源,包括PNI。这里,我们制备了GMSC再生神经保护剂,并在大鼠坐骨神经挤压伤模型中评价其治疗效果.
将GMSCs在甲基丙烯酸酯化胶原中混合培养48小时,允许GMSC转化为雪旺样细胞(GiSC)。通过对Schwann细胞标志物表达的荧光研究验证了GiSCs的表型。将封装在甲基丙烯酸酯化3D胶原水凝胶中的GMSC与THP-1衍生的巨噬细胞共培养,ELISA法测定上清液中抗炎细胞因子IL-10或炎性细胞因子TNF-α和IL-1β的分泌情况。此外,将混合在甲基丙烯酸酯化胶原蛋白中的GMSC填充到由去细胞小肠粘膜下细胞外基质(SIS-ECM)制成的神经保护剂中,并培养24小时,允许生成功能化的神经保护剂重新填充GiSC。我们植入神经保护剂以包裹大鼠坐骨神经的损伤部位,并在手术后4周进行功能和组织学评估。
封装在甲基丙烯酸酯化3D-胶原水凝胶中的GMSC直接转化为以S-100β表达为特征的雪旺样细胞(GiSC),p75NTR,BDNF,GDNF。体外,封装在3D胶原水凝胶中的GMSC与巨噬细胞的共培养显着增加IL-10的分泌,IL-10是促再生(M2)巨噬细胞的抗炎细胞因子特征,但强烈减少LPS刺激的TNF-1α和IL-1β的分泌,促炎(M1)巨噬细胞的两种细胞因子特征。此外,我们的结果表明,植入经GiSCs再填充的功能化神经保护剂可显著加速挤压损伤的大鼠坐骨神经的功能恢复和轴突再生,同时促再生(M2)巨噬细胞浸润增加,而促炎(M1)巨噬细胞浸润减少.
集体,这些发现表明,从GMSCs转化的雪旺样细胞通过其双重功能代表了PNI再生治疗的支持细胞的有希望的来源,神经营养作用,和促炎(M1)/促再生(M2)巨噬细胞的免疫调节。
Peripheral nerve injuries (PNIs) remain one of the great clinical challenges because of their considerable long-term disability potential. Postnatal neural crest-derived multipotent stem cells, including gingiva-derived mesenchymal stem cells (GMSCs), represent a promising source of seed cells for tissue engineering and regenerative therapy of various disorders, including PNIs. Here, we generated GMSC-repopulated nerve protectors and evaluated their therapeutic effects in a crush injury model of rat sciatic nerves.
GMSCs were mixed in methacrylated collagen and cultured for 48 h, allowing the conversion of GMSCs into Schwann-like cells (GiSCs). The phenotype of GiSCs was verified by fluorescence studies on the expression of Schwann cell markers. GMSCs encapsulated in the methacrylated 3D-collagen hydrogel were co-cultured with THP-1-derived macrophages, and the secretion of anti-inflammatory cytokine IL-10 or inflammatory cytokines TNF-α and IL-1β in the supernatant was determined by ELISA. In addition, GMSCs mixed in the methacrylated collagen were filled into a nerve protector made from the decellularized small intestine submucosal extracellular matrix (SIS-ECM) and cultured for 24 h, allowing the generation of functionalized nerve protectors repopulated with GiSCs. We implanted the nerve protector to wrap the injury site of rat sciatic nerves and performed functional and histological assessments 4 weeks post-surgery.
GMSCs encapsulated in the methacrylated 3D-collagen hydrogel were directly converted into Schwann-like cells (GiSCs) characterized by the expression of S-100β, p75NTR, BDNF, and GDNF. In vitro, co-culture of GMSCs encapsulated in the 3D-collagen hydrogel with macrophages remarkably increased the secretion of IL-10, an anti-inflammatory cytokine characteristic of pro-regenerative (M2) macrophages, but robustly reduced LPS-stimulated secretion of TNF-1α and IL-1β, two cytokines characteristic of pro-inflammatory (M1) macrophages. In addition, our results indicate that implantation of functionalized nerve protectors repopulated with GiSCs significantly accelerated functional recovery and axonal regeneration of crush-injured rat sciatic nerves accompanied by increased infiltration of pro-regenerative (M2) macrophages while a decreased infiltration of pro-inflammatory (M1) macrophages.
Collectively, these findings suggest that Schwann-like cells converted from GMSCs represent a promising source of supportive cells for regenerative therapy of PNI through their dual functions, neurotrophic effects, and immunomodulation of pro-inflammatory (M1)/pro-regenerative (M2) macrophages.