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  • Title: [Cytocompatibility of angiogenesis-promoting acidified silk protein sponge matrices and its effects on wound healing of full-thickness skin defects in rats].
    Author: Lu QQ, Lyu GZ, Lyu J.
    Journal: Zhonghua Shao Shang Za Zhi; 2021 Jan 20; 37(1):25-33. PubMed ID: 33499566.
    Abstract:
    Objective: To analyze the mechanism of acidified silk protein sponge matrices and methanolized silk protein sponge matrices in promoting wound healing. Methods: The experimental method was conducted. Acidified silk protein sponge matrices with vascularization ability and methanolized silk protein sponge matrices without vascularization ability were prepared by improved freeze-drying method. General observation was performed. Internal morphology was observed with scanning electron microscope. The secondary structure was observed with X-ray diffractometer (XRD) and infrared spectrometer. Compressive modulus was tested by tensile machine. Two 3-week-old male Sprague-Dawley (SD) rats were used to isolate bone marrow mesenchymal stem cells (BMSCs) cultured in above-mentioned two silk protein sponge matrices, the number of cells was counted under laser scanning confocal microscope after 1, 6 days of culture. Four full-thickness skin defect wounds were made on each one of twelve 8-week-old male SD rats, which were divided into methanolized silk group (24 wounds) and acidified silk group (24 wounds) covered with the corresponding silk protein sponge matrices. On post operation day (POD) 3, 7, 10, and 14, general observation was performed and the remaining wound area was recorded. On POD 3, 7, and 14, the wounds and marginal tissue were collected for hematoxylin-eosin staining (HE) staining and Masson staining to observe growth of new tissue and collagen deposition and CD34 immunohistochemical staining to observe vascularization. Sample number of each index of each group at every time point in animal experiment was 6. Data were statistically analyzed with analysis of variance of factorial design, analysis of variance for repeated measurement, independent-samples t test, and Bonferroni correction. Results: Methanolized silk protein sponge matrices and acidified silk protein sponge matrices had the same composition and similar porous structure, with pore size of 300-500 μm. XRD showed that methanolized silk protein sponge matrices showed a significant crystallization peak, while acidified silk protein sponge matrices was mainly composed of amorphous structure. Infrared spectrometer showed that acidified silk protein sponge matrices appeared a strong absorption peak at 1 650 cm(-1), and the methanolized silk protein sponge matrices appeared a strong absorption peak at 1 630 cm(-1). Compressive modulus of methanolized silk protein sponge matrices was (23.8±1.3) kPa, which was significantly higher than (6.1±0.9) kPa of acidified silk protein sponge matrices (t=19.550, P<0.01). After one day of culture, BMSCs successfully adhered to the two kinds of silk protein sponge matrices, and the cells were not spread. After six days of culture, BMSCs were spread on the two kinds of silk protein sponge matrices, and the number of cells on the acidified silk protein sponge matrices increased significantly. On POD 3, the wounds of the 2 groups did not shrink significantly. On POD 7, the wound area in acidified silk group was significantly smaller than that in methanolized silk group, and new epithelium growth occurred at the wound edge. On POD 14, the wounds of acidified silk group basically healed, and the wounds of methanolized silk group were dry and shrinked significantly. Remaining wound area of acidified silk group on POD 3, 7, 10, and 14 were significantly smaller compared with that in methanolized silk group ( t=7.782, 10.620, 3.707, 6.830, P<0.05 or P<0.01). HE staining, Masson staining, and CD34 immunohistochemical staining showed on POD 3, new tissue growing into silk protein sponge matrices of wounds of acidified silk group was more than that in methanolized silk group, the former group secreted a small amount of collagen, collagen formation was not observed in the latter group, the number of vascular endothelial cells migrated into the matrices were more in the former group than the latter group; on POD 7, the area of new tissue covering matrices hole of wounds of acidified silk group was larger than that in methanolized silk group, collagen in the former group was more than that in the latter group and was evenly distributed, the number of blood vessels in the former group was more than that on POD 3, and the new blood vessels in the latter group were scattered; on POD 14, the new tissue in acidified silk group was similar in structure to normal skin tissue and formed a certain thickness, the new tissue in methanolized silk group basically grew into the matrices, the former group had rich collagen deposition, the latter group had scattered collagen, and blood vessels in the former group distributed uniformly and density of blood vessels was significantly higher than that in the latter group ((55.7±6.0) and (34.1±1.0) pieces/mm(2), respectively, t=9.042, P<0.01). Conclusions: Angiogenesis-promoting acidified silk protein sponge matrices have good cytocompatibility, which can facilitate the rapid formation of vascular network in wound area, providing sufficient blood supply to accelerate the tissue regeneration and collagen deposition, thereby promoting wound healing and improving healing quality, these effects are better than methanolized silk protein sponge matrices. 目的： 分析酸化丝蛋白海绵敷料和甲醇化丝蛋白海绵敷料在促进创面愈合方面的机制。 方法： 采用实验研究方法。采用改良冻干法制备具有促血管化能力的酸化丝蛋白海绵敷料和不具备促血管化能力的甲醇化丝蛋白海绵敷料，行大体观察、扫描电镜观察内部形貌，X线衍射仪(XRD)、红外光谱仪观察二级结构，拉力机测定压缩模量。于2只3周龄雄性SD大鼠分离培养骨髓间充质干细胞(BMSC)并接种在上述2种丝蛋白海绵敷料上，培养1、6 d激光扫描共聚焦显微镜下计数细胞。将12只8周龄雄性SD大鼠每只造成4个全层皮肤缺损创面，分为甲醇化丝蛋白组(24个创面)和酸化丝蛋白组(24个创面)，并应用对应丝蛋白海绵敷料覆盖。术后3、7、10、14 d，行大体观察并记录剩余创面面积。术后3、7、14 d，收集创面及创缘组织行苏木精-伊红(HE)染色和Masson染色观察新生组织生长及胶原沉积情况，CD34免疫组织化学染色观察血管化情况。动物实验每组各指标各时间点样本数为6。对数据行析因设计方差分析、重复测量方差分析、独立样本t检验、Bonferroni校正。 结果： 甲醇化丝蛋白海绵敷料、酸化丝蛋白海绵敷料具有相同的成分、类似的多孔结构，孔径为300～500 μm。XRD显示，甲醇化丝蛋白海绵敷料表现出显著的结晶峰，酸化丝蛋白海绵敷料则主要由非晶结构组成。红外光谱仪显示，酸化丝蛋白海绵敷料在1 650 cm(-1)处出现强吸收峰，甲醇化丝蛋白海绵敷料则在1 630 cm(-1)处表现出强吸收峰。甲醇化丝蛋白海绵敷料的压缩模量为(23.8±1.3)kPa，明显高于酸化丝蛋白海绵敷料的(6.1±0.9)kPa，t＝19.550，P<0.01。培养1 d，BMSC成功黏附在2种丝蛋白海绵敷料上，细胞未铺展开。培养6 d，BMSC铺展在2种丝蛋白海绵敷料上，其中酸化丝蛋白海绵敷料上细胞数量显著增多。术后3 d，2组创面无明显收缩；术后7 d，酸化丝蛋白组创面面积较甲醇化丝蛋白组明显缩小，创缘有新生上皮生长；术后14 d，酸化丝蛋白组创面已经基本愈合，甲醇化丝蛋白组创面干燥、收缩明显。与甲醇化丝蛋白组比较，酸化丝蛋白组术后3、7、10、14 d剩余创面面积明显缩小，t＝7.782、10.620、3.707、6.830，P<0.05或P<0.01。HE染色、Masson染色、CD34免疫组织化学染色显示：术后3 d，酸化丝蛋白组创面新生组织长入丝蛋白海绵敷料多于甲醇化丝蛋白组，前组分泌少量胶原蛋白，后组未见胶原蛋白形成，前组向敷料内游走的血管内皮细胞数量多于后组；术后7 d，酸化丝蛋白组创面新生组织覆盖敷料孔壁面积大于甲醇化丝蛋白组，前组胶原蛋白多于后组且分布均匀，前组血管数量多于术后3 d，后组新生血管散在分布；术后14 d，酸化丝蛋白组新生组织与正常皮肤组织结构相似且形成一定厚度，甲醇化丝蛋白组新生组织已基本长入敷料内，前组胶原沉积丰富，后组胶原散在分布，前组血管分布均匀且密度明显高于后组[分别为(55.7±6.0)、(34.1±1.0)个/mm(2), t＝9.042, P<0.01]。 结论： 具有促血管化能力的酸化丝蛋白海绵敷料细胞相容性好，可实现创面部位血管网络的快速形成，提供较为充足血供加快新生组织长入速度、胶原沉积，从而促进创面愈合、改善愈合质量，效果优于甲醇化丝蛋白海绵敷料。.
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