Institute for Clinical and Experimental Surgery, Saarland University, D-66421 Homburg/Saar, Germany.matthias.laschke@ uks.eu.
Eur Cell Mater. 2018 May 9;35:268-280. doi: 10.22203/eCM.v035a19.
The seeding of tissue constructs with adipose tissue-derived microvascular fragments (ad-MVF) is an emerging pre-vascularisation strategy. Ad-MVF rapidly reassemble into new microvascular networks after in vivo implantation. Herein it was analysed whether this process was improved by erythropoietin (EPO). Ad-MVF were isolated from green fluorescent protein (GFP)+ as well as wild-type C57BL/6 mice and cultivated for 24 h in medium supplemented with EPO (20 IU/mL) or vehicle. Freshly isolated, non-cultivated ad-MVF served as controls. Protein expression, cell viability and proliferation of ad-MVF were assessed by proteome profiler array and fluorescence microscopy. GFP+ ad-MVF were seeded on collagen-glycosaminoglycan matrices, which were implanted into dorsal skinfold chambers of C57BL/6 mice, to analyse their vascularisation over 14 d by intravital fluorescence microscopy, histology and immunohistochemistry. Cultivation up-regulated the expression of pro- and anti-angiogenic factors within both vehicle- and EPO-treated ad-MVF when compared with non-cultivated controls. Moreover, EPO treatment suppressed cultivation-associated apoptosis and significantly increased the number of proliferating endothelial cells in ad-MVF when compared with vehicle-treated and non-cultivated ad-MVF. Accordingly, implanted matrices seeded with EPO-treated ad-MVF exhibited an improved vascularisation, as indicated by a significantly higher functional microvessel density. The matrices of the three groups contained a comparably large fraction of GFP+ microvessels originating from the ad-MVF, whereas the tissue surrounding the matrices seeded with EPO-treated ad-MVF exhibited a significantly increased microvessel density when compared with the other two groups. These findings indicated that EPO represents a promising cytokine to further boost the excellent vascularisation properties of ad-MVF in tissue-engineering applications.
脂肪组织来源的微血管片段（ad-MVF）种植是一种新兴的预血管化策略。ad-MVF 在体内植入后迅速重新组装成新的微血管网络。在此，分析了促红细胞生成素（EPO）是否改善了这一过程。从绿色荧光蛋白（GFP）+以及野生型 C57BL/6 小鼠中分离出 ad-MVF，并在补充 EPO（20IU/mL）或载体的培养基中培养 24 小时。新鲜分离的未培养的 ad-MVF 作为对照。通过蛋白质组芯片分析和荧光显微镜评估 ad-MVF 的蛋白表达、细胞活力和增殖。将 GFP+ad-MVF 种植在胶原-糖胺聚糖基质上，将其植入 C57BL/6 小鼠的背部皮褶室中，通过活体荧光显微镜、组织学和免疫组织化学分析 14 天的血管生成情况。与未培养的对照组相比，培养上调了载体和 EPO 处理的 ad-MVF 中促血管生成和抗血管生成因子的表达。此外，与载体处理和未培养的 ad-MVF 相比，EPO 处理显著抑制了培养相关的细胞凋亡，并显著增加了 ad-MVF 中增殖的内皮细胞数量。因此，用 EPO 处理的 ad-MVF 种植的植入物基质表现出改善的血管生成，这表现为功能微血管密度显著增加。三组基质均含有大量源自 ad-MVF 的 GFP+微血管，而与其他两组相比，用 EPO 处理的 ad-MVF 种植的基质周围组织的微血管密度显著增加。这些发现表明，EPO 是一种有前途的细胞因子，可以进一步增强 ad-MVF 在组织工程应用中的优异血管生成特性。
