Department of Plastic & Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Medical College of Jiao Tong University, 639 Zhizaoju Road, Shanghai 200011, PR China.
J Plast Reconstr Aesthet Surg. 2010 Mar;63(3):474-81. doi: 10.1016/j.bjps.2008.11.076. Epub 2008 Dec 30.
Flap pre-fabrication is dependent on the eventual re-vascularisation of the implanted vascular carrier and the presence of a desirable, donor-skin site. However, insufficient neo-vascularisation and subsequent necrosis is an obstacle for this technique. A recent discovery demonstrated that endothelial progenitor cells (EPCs) augment post-natal neo-vascularisation in ischaemic tissues. As a result, we examined whether transplantation of bone-marrow-derived EPCs (BM-EPCs) increases neo-vascularisation and augments the survival areas of pre-fabricated flap in a rat model.
Rat bone-marrow-derived mononuclear cells (BM-MNCs) were isolated by density gradient centrifugation and cultured in EGM-2MV. The EPCs derived from BM-MNCs were identified by surface makers such as CD34, KDR, CD133 and double-positive staining with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labelled acetylated low-density lipoprotein (Dil-Ac-LDL) and FITC-labelled Ulex europaeus agglutinin-1 (FITC-UEA-1). Pre-fabricated flaps were created by ligating the right femoral vascular pedicle and implanting it underneath the abdominal flap. Forty-five rats were randomly divided into three equal groups. The implantation site around the pedicle was injected subcutaneously with fluorescence-labelled BM-EPCs in group I (n=15), with vascular endothelium growth factor (VEGF) protein in group II (n=15) and with phosphate-buffered saline (PBS) in control group III. Four weeks after injection, the abdominal island flap was elevated and sutured back. Then, neo-vascularisation and flap viability was evaluated on day 7. The labelled EPCs were examined by fluorescence microscopy.
After 7 days of culture, the attached cells were spindle shaped and expressed CD34, KDR and CD133. These cells incorporated DiI-Ac-LDL and bound FITC-UEA-1. Greater augmentation of flap survival (87.26+/-10.13% vs. 66.13+/-9.9% and 55.59+/-13.06%, P<0.001), higher capillary density (38.67+/-9.52 capillaries per mm(2) vs. 25.83+/-6.34 capillaries per mm(2) and 26.5+/-5.61 capillaries per mm(2), P<0.05) and larger vascular territories on the microangiogram were observed in the EPCs-treated group relative to the other two groups. The labelled cells formed new vessel structures and expressed von Willebrand factor (vWF) in the pre-fabricated flap.
Local transplantation of BM-EPCs may be a useful strategy for increasing the survival of pre-fabricated flaps, which is consistent with 'therapeutic vasculogenesis'. EPCs are superior to VEGF in their neo-vascularisation ability.
皮瓣预制依赖于植入血管载体的最终再血管化和理想的供体皮肤部位的存在。然而，新生血管不足和随后的坏死是该技术的一个障碍。最近的一项发现表明，内皮祖细胞（EPCs）增强了缺血组织的后天新生血管化。因此，我们研究了骨髓来源的内皮祖细胞（BM-EPCs）移植是否会增加预制皮瓣的新生血管化并增加其存活面积。
通过密度梯度离心从大鼠骨髓中分离出单核细胞（BM-MNCs），并在 EGM-2MV 中培养。通过表面标志物如 CD34、KDR、CD133 和 1,1'-二辛基-3,3,3',3'-四甲基吲哚碳酰基乙酰化低密度脂蛋白（Dil-Ac-LDL）和 FITC 标记的欧洲越橘凝集素-1（FITC-UEA-1）的双阳性染色来鉴定源自 BM-MNC 的 EPCs。通过结扎右股血管蒂并将其植入腹部皮瓣下，创建预制皮瓣。45 只大鼠随机分为三组。在 I 组（n=15）中，将荧光标记的 BM-EPCs 皮下注射到蒂部周围，在 II 组（n=15）中注射血管内皮生长因子（VEGF）蛋白，在对照组 III 组（n=15）中注射磷酸盐缓冲盐水（PBS）。注射后 4 周，抬起腹部岛状皮瓣并缝合回原处。然后在第 7 天评估新生血管化和皮瓣存活情况。通过荧光显微镜检查标记的 EPCs。
培养 7 天后，贴壁细胞呈纺锤形，表达 CD34、KDR 和 CD133。这些细胞摄取 DiI-Ac-LDL 并结合 FITC-UEA-1。与对照组相比，皮瓣存活面积增加更明显（87.26+/-10.13% vs. 66.13+/-9.9%和 55.59+/-13.06%，P<0.001），毛细血管密度更高（38.67+/-9.52 个毛细血管/平方毫米 vs. 25.83+/-6.34 个毛细血管/平方毫米和 26.5+/-5.61 个毛细血管/平方毫米，P<0.05），微血管造影上的血管区域更大，在 EPCs 处理组观察到的。标记的细胞在预制皮瓣中形成新的血管结构并表达血管性血友病因子（vWF）。
局部移植 BM-EPCs 可能是增加预制皮瓣存活的一种有用策略，这与“治疗性血管生成”一致。EPCs 在新生血管化能力方面优于 VEGF。
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