Effects of platelet-rich plasma combined with polylactic acid/polycaprolactone on healing of pig deep soft tissue defect caused by fragment injury
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摘要: 目的 探讨富血小板血浆(PRP)联合聚乳酸/聚己内酯(PLA/PCL)对小型猪破片伤深部软组织缺损愈合的影响。 方法 采用抽签法选取2只雄性巴马小型猪(11~12个月龄,下同)制备PRP。取27只雄性巴马小型猪,于每只猪双侧后肢股部各制造1处高爆弹破片伤深部软组织缺损,按照随机数字表法分为对照组、材料组、PRP+材料组,每组9只。创面清创后,材料组、PRP+材料组猪用PLA/PCL填塞伤道,PRP+材料组同时向伤道内注入2 mL激活的PRP,3组猪均缝合皮肤全层封闭创口。记录各组猪手术时间,术后即刻测量伤道长度和体积。术后1、2、4周,每组各处死3只猪,取两侧股部伤道组织及材料组、PRP+材料组猪PLA/PCL,行伤道大体观察和材料降解情况观察。术后2、4周,取各组猪伤道组织,苏木精-伊红染色观察组织形态学变化,免疫组织化学法检测伤道组织转化生长因子β(TGF-β)、血管内皮生长因子(VEGF)蛋白表达和血管生成。取术后1、2、4周伤道组织,实时荧光定量反转录PCR法检测伤道组织TGF-β、VEGF mRNA表达。对数据行析因设计方差分析、单因素方差分析及LSD-
t 检验。 结果 (1)3组猪伤道长度及体积比较,差异无统计学意义(F =0.336、0.282,P >0.05)。对照组猪手术时间[(30.9±2.1)min]明显短于材料组[(39.7±2.2)min]和PRP+材料组[(40.0±2.6)min,t =-11.45、-11.88,P <0.01]。(2)对照组、材料组及PRP+材料组猪伤道感染数分别为10、7、5个。对照组猪术后1、2、4周伤道组织均发生感染。材料组及PRP+材料组猪伤道组织均未发生感染,术后1周材料与组织易分离;术后2周部分材料与组织融合,部分材料有降解趋势;术后4周材料与组织完全融合。(3)对照组猪伤道组织术后2周有红细胞及炎性细胞浸润,术后4周仍可见坏死组织及炎性细胞浸润。材料组及PRP+材料组猪伤道组织术后2周有大量红细胞及炎性细胞浸润;术后4周,与材料组比较,PRP+材料组猪伤道组织无炎性细胞浸润。(4)术后2、4周,PRP+材料组猪伤道组织成纤维细胞(Fb)、多核巨噬细胞TGF-β蛋白表达和Fb、血管内皮细胞VEGF蛋白表达及血管形成明显多于对照组和材料组。(5)术后4周,材料组猪伤道组织TGF-β mRNA表达水平明显高于对照组(t =-3.93,P <0.01)。与对照组及材料组比较,PRP+材料组猪术后1、2、4周伤道组织TGF-β mRNA表达水平均明显升高(t =9.23、13.81、11.73,-7.51、-12.04、-7.80,P <0.01)。术后4周,材料组猪伤道组织VEGF mRNA表达水平明显高于对照组(t =-3.94,P <0.01)。与对照组及材料组比较,PRP+材料组猪术后1、2、4周伤道组织的VEGF mRNA表达水平均明显升高(t =12.33、3.95、7.97,-11.36、-2.97、-4.04,P <0.01)。 结论 PRP联合PLA/PCL可为新生组织长入提供物理支架,同时能促进创面局部TGF-β、VEGF的mRNA和蛋白表达,从而促进小型猪破片伤深部软组织缺损愈合。Abstract: Objective 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. Methods 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-t test. Results (1) There were no significantly statistical differences in length and volume of the wounds of pigs among the three groups (F =0.336, 0.282,P >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],t =-11.45, -11.88,P <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 (t =-3.93,P <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 (t =9.23, 13.81, 11.73, -7.51, -12.04, -7.80,P <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 (t =-3.94,P <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 (t =12.33, 3.95, 7.97, -11.36, -2.97, -4.04,P <0.01). Conclusions 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.-
Key words:
- Soft tissue injuries /
- Platelet-rich plasma /
- Biocompatible materials
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