Laschke M W, Kleer S, Scheuer C, Eglin D, Alini M, Menger M D
Institute for Clinical & Experimental Surgery, University of Saarland, 66421 Homburg/Saar,
Eur Cell Mater. 2015 Mar 20;29:190-200; discussion 200-1. doi: 10.22203/ecm.v029a14.
Adipose tissue-derived microvascular fragments represent promising vascularisation units for implanted tissue constructs. However, their reassembly into functional microvascular networks takes several days, during which the cells inside the implants are exposed to hypoxia. In the present study, we analysed whether this critical phase may be overcome by pre-cultivation of fragment-seeded scaffolds prior to their implantation. Green fluorescent protein (GFP)-positive microvascular fragments were isolated from epididymal fat pads of male C57BL/6-TgN (ACTB-EGFP) 1Osb/J mice. Nano-size hydroxyapatite particles/poly (ester-urethane) scaffolds were seeded with these fragments and cultivated for 28 days. Subsequently, these scaffolds or control scaffolds, which were freshly seeded with GFP-positive microvascular fragments, were implanted into the dorsal skinfold chamber of C57BL/6 wild-type mice to study their vascularisation and incorporation by means of intravital fluorescence microscopy, histology and immunohistochemistry over 2 weeks. Pre-cultivation of microvascular fragments resulted in the loss of their native vessel morphology. Accordingly, pre-cultivated scaffolds contained a network of individual CD31/GFP-positive endothelial cells with filigrane cell protuberances. After implantation into the dorsal skinfold chamber, these scaffolds exhibited an impaired vascularisation, as indicated by a significantly reduced functional microvessel density and lower fraction of GFP-positive microvessels in their centre when compared to freshly seeded control implants. This was associated with a deteriorated incorporation into the surrounding host tissue. These findings indicate that freshly isolated, non-cultivated microvascular fragments should be preferred as vascularisation units. This would also facilitate their use in clinical practice during intra-operative one-step procedures.
脂肪组织来源的微血管片段是植入组织构建物很有前景的血管化单元。然而，它们重新组装成功能性微血管网络需要数天时间，在此期间植入物内的细胞会暴露于缺氧环境。在本研究中，我们分析了在植入前对接种了片段的支架进行预培养是否可以克服这一关键阶段。从雄性C57BL/6-TgN (ACTB-EGFP) 1Osb/J小鼠的附睾脂肪垫中分离出绿色荧光蛋白(GFP)阳性的微血管片段。将这些片段接种到纳米级羟基磷灰石颗粒/聚(酯-聚氨酯)支架上，并培养28天。随后，将这些支架或新接种了GFP阳性微血管片段的对照支架植入C57BL/6野生型小鼠的背部皮褶腔，通过活体荧光显微镜、组织学和免疫组织化学方法在2周内研究它们的血管化和整合情况。微血管片段的预培养导致其天然血管形态丧失。因此，预培养的支架包含由具有细丝状细胞突起的单个CD31/GFP阳性内皮细胞组成的网络。植入背部皮褶腔后，与新接种的对照植入物相比，这些支架的血管化受损，表现为功能性微血管密度显著降低，且其中心GFP阳性微血管的比例较低。这与周围宿主组织的整合恶化有关。这些发现表明，应优先选择新鲜分离、未培养的微血管片段作为血管化单元。这也将便于它们在术中一步手术的临床实践中使用。
