Yao D, Hao D F, Zhao F, Hao X F, Feng G, Chu W L, Chen Z Q
Medical School of Chinese PLA, Beijing 100853, China.
The Third Department of Plastic Surgery and Center of Wound Repair of the Fourth Medical Center of PLA General Hospital, Beijing 100048, China.
Zhonghua Shao Shang Za Zhi. 2019 Jan 20;35(1):31-39. doi: 10.3760/cma.j.issn.1009-2587.2019.01.007.
To investigate the effects of platelet-rich plasma (PRP) combined with polylactic acid/polycaprolactone (PLA/PCL) on healing of mininature pig deep soft tissue defect caused by fragment injury. Two male Bama miniature pigs with 11 to 12 months (the same below) were selected by lottery to prepare PRP. The other twenty-seven male Bama miniature pigs were used to reproduce deep soft tissue defect caused by high-explosive ammunition fragment injury on bilateral posterior femoral region. According to the random number table, 27 pigs were divided into control group, material group, and PRP+material group, with 9 pigs in each group. After debridement, wounds of pigs in material group and PRP+material group were filled with PLA/PCL and PLA/PCL+2 mL activated PRP, respectively. Pigs in each group received suture of full-thickness skin to close the wounds. The operative duration was recorded. The length and volume of wounds of pigs in the above groups were measured immediately after surgery. In 1, 2, and 4 weeks after surgery, 3 pigs in each group were sacrificed to collect femoral wounds tissue on two sides, and PLA/PCL were collected from wounds of pigs in material group and PRP+material group for general observation of wounds tissue and degradation of the material. In 2 and 4 weeks after surgery, wounds tissue was obtained to observe the histological changes by hematoxylin-eosin staining, and expressions of transforming growth factor β (TGF-β) and vascular endothelial growth factor (VEGF), and angiogenesis were determined by immunohistochemical method. In 1, 2, and 4 weeks after surgery, wounds tissue was collected to determine mRNA expressions of TGF-β and VEGF by real-time quantitative reverse transcription polymerase chain reaction. Data were processed with analysis of variance of factorial design, one-way analysis of variance, and least significant difference- test. (1) There were no significantly statistical differences in length and volume of the wounds of pigs among the three groups (=0.336, 0.282, >0.05). The operative duration in control group [(30.9±2.1)min] was significantly shorter than that of material group [(39.7±2.2)min] and PRP+material group[(40.0±2.6)min], =-11.45, -11.88, <0.01. (2) There were respectively 10, 7, and 5 wounds tissue with infection in pigs of control group, material group, and PRP+material group. In 1, 2, 4 weeks after surgery, all of the wounds tissue of pigs was infected in control group, while none of wounds tissue of pigs was infected in material group and PRP+material group. In pigs of material group and PRP+material group, materials and tissue were easily separated in 1 week after surgery; some materials were integrated with tissue and showed a tendency of degradation in 2 weeks after surgery; materials were completely embedded with tissue in 4 weeks after surgery. (3) In pigs of control group, erythrocytes and inflammatory cells infiltration in wounds tissue were observed in 2 weeks after surgery, and necrotic tissue and inflammatory cells infiltration in wounds tissue were still observed in 4 weeks after surgery. In pigs of material group and PRP+material group, a large number of erythrocytes and inflammatory cells infiltration were observed in 2 weeks after surgery. Compared with that of material group, wounds tissue of pigs in PRP+material group had no inflammatory cells infiltration in 4 weeks after surgery. (4) Protein expressions of TGF-β in fibroblasts and multinuclear macrophagocytes, VEGF in fibroblasts and vascular endothelial cells, and blood vessel formation in wounds tissue of pigs in PRP+material group were significantly more than those of pigs in control group and material group in 2 and 4 weeks after surgery. (5) The mRNA expression of TGF-β in wounds tissue of pigs in material group was significantly higher than that in control group in 4 weeks after surgery (=-3.93, <0.01). Compared with those of pigs in control group and material group, the mRNA expression of TGF-β in wounds tissue of pigs in PRP+material group was significantly increased at each time point (=9.23, 13.81, 11.73, -7.51, -12.04, -7.80, <0.01). The mRNA expression of VEGF in wounds tissue of pigs increased significantly in material group compared with that of pigs in control group in 4 weeks after surgery (=-3.94, <0.01). Compared with those of pigs in control group and material group, the mRNA expression of VEGF in wounds tissue increased significantly in wound tissue of pigs in PRP+material group at each time point (=12.33, 3.95, 7.97, -11.36, -2.97, -4.04, <0.01). PRP combined with PLA/PCL can accelerate wound healing of deep soft tissue defect of mininature pigs caused by fragment injury by providing physical scaffold for newborn tissue growth, promoting mRNA and protein expressions of TGF-β and VEGF.
探讨富血小板血浆(PRP)联合聚乳酸/聚己内酯(PLA/PCL)对微型猪弹片伤所致深部软组织缺损愈合的影响。通过抽签选取2只11至12月龄的雄性巴马微型猪(下同)制备PRP。选取另外27只雄性巴马微型猪,在双侧股后区制造高爆弹药弹片伤所致深部软组织缺损。根据随机数字表,将27只猪分为对照组、材料组和PRP+材料组,每组9只。清创后,材料组和PRP+材料组猪的伤口分别填充PLA/PCL和PLA/PCL+2 mL活化PRP。每组猪均行全层皮肤缝合以闭合伤口。记录手术时长。术后立即测量上述各组猪伤口的长度和体积。术后1、2和4周,每组处死3只猪,采集双侧股部伤口组织,从材料组和PRP+材料组猪的伤口收集PLA/PCL,进行伤口组织大体观察及材料降解情况观察。术后2和4周,获取伤口组织,采用苏木精-伊红染色观察组织学变化,采用免疫组化法检测转化生长因子β(TGF-β)和血管内皮生长因子(VEGF)的表达及血管生成情况。术后1、2和4周,收集伤口组织,采用实时定量逆转录聚合酶链反应检测TGF-β和VEGF的mRNA表达。数据采用析因设计方差分析、单因素方差分析和最小显著差检验进行处理。(1)三组猪伤口的长度和体积差异无统计学意义(=0.336,0.282,>0.05)。对照组的手术时长[(30.9±2.1)min]显著短于材料组[(39.7±2.2)min]和PRP+材料组[(40.0±2.6)min],=-11.45,-11.88,<0.01。(2)对照组、材料组和PRP+材料组猪分别有10、7和5处伤口组织发生感染。术后1、2、4周,对照组猪的所有伤口组织均发生感染,而材料组和PRP+材料组猪的伤口组织均未发生感染。材料组和PRP+材料组猪术后1周材料与组织易于分离;术后2周部分材料与组织融合并呈降解趋势;术后4周材料完全埋入组织。(3)对照组猪术后2周伤口组织可见红细胞和炎性细胞浸润,术后4周伤口组织仍可见坏死组织和炎性细胞浸润。材料组和PRP+材料组猪术后2周可见大量红细胞和炎性细胞浸润。与材料组相比,PRP+材料组猪术后4周伤口组织无炎性细胞浸润。(4)术后2和4周,PRP+材料组猪伤口组织中成纤维细胞和多核巨噬细胞TGF-β的蛋白表达、成纤维细胞和血管内皮细胞VEGF的蛋白表达及血管生成均显著多于对照组和材料组。(5)术后4周,材料组猪伤口组织TGF-β的mRNA表达显著高于对照组(=-3.93,<0.01)。与对照组和材料组相比,PRP+材料组猪伤口组织TGF-β的mRNA表达在各时间点均显著升高(=9.23,13.81,11.73,-7.51,-12.04,-7.80,<0.01)。术后4周,材料组猪伤口组织VEGF的mRNA表达较对照组显著升高(=-3.94,<0.01)。与对照组和材料组相比,PRP+材料组猪伤口组织VEGF的mRNA表达在各时间点均显著升高(=12.33,3.95,7.97,-11.36,-2.97,-4.04,<0.01)。PRP联合PLA/PCL可为新生组织生长提供物理支架,促进TGF-β和VEGF的mRNA及蛋白表达,从而加速微型猪弹片伤所致深部软组织缺损的伤口愈合。