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Title: [Influence of collagen/fibroin scaffolds containing silver nanoparticles on dermal regeneration of full-thickness skin defect wound in rat].
Author: You ZG, Zhang LP, Wang XG, Zhou HL, Guo SX, Wu P, Han CM.
Journal: Zhonghua Shao Shang Za Zhi; 2017 Feb 20; 33(2):103-110. PubMed ID: 28219143.
Abstract:
Objective: To explore the influence of collagen/fibroin scaffolds containing silver nanoparticles on dermal regeneration of full-thickness skin defect wound in rat. Methods: Eighty-one collagen/fibroin scaffolds containing silver nanoparticles (with the mass concentration of silver nanoparticles as 10 mg/L) and 81 collagen/fibroin scaffolds without silver nanoparticles were produced respectively with freeze-drying method and enrolled as silver nanoparticles scaffold group (SNS) and control scaffold group (CS). Nine scaffolds in each group were cultured with human fibroblasts. At post culture hour (PCH) 2, 12, and 24, the human fibroblasts adherent to the scaffolds (n=3) in two groups were counted. Four full-thickness skin defect wounds were reproduced on the back of each one of the 36 SD rats. The rats were divided into groups SNS (wounds were transplanted with collagen/fibroin scaffolds containing silver nanoparticles) and CS (wounds were transplanted with collagen/fibroin scaffolds without silver nanoparticles) according to the random number table, with 18 rats in each group. In post surgery week (PSW) 1, 2, and 4, 6 rats in each group were sacrificed respectively for general observation, observation of histological structure, inflammatory cell infiltration, and collagen deposition with HE staining, count of CD68 positive cells with immunohistochemical staining, and mRNA expressions of interleukin-6 (IL-6) and IL-10 with real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with analysis of variance of factorial design, t test, and Bonferroni correction. Results: (1) At PCH 2, 12, and 24, the numbers of human fibroblasts adherent to the scaffolds in the two groups were close (with t values from 1.77 to 2.60, P values above 0.05). (2) In PSW 1, no obvious symptom of infection was observed in wound or wound edge of rats in group SNS with obvious vascularization of scaffolds, while obvious symptoms of infection were observed in wounds of rats in group CS with some scaffolds exfoliated. In PSW 2, the scaffolds were firmly attached to the wounds of rats in group SNS, while obvious contracture was observed in the wounds of rats in group CS with a lot of scaffolds exfoliated. In PSW 4, the scaffolds covered the wounds of rats in group SNS with obvious epithelization on the surface of the scaffolds, while all the scaffolds exfoliated, leaving obvious contracture of residual wounds of rats in group CS. (3) In PSW 1 and 2, compared with those in group CS, more collagen secretion and tissue regeneration and less inflammatory cell infiltration in the scaffolds were observed in the wounds of rats in group SNS. In PSW 4, obvious epithelization was observed in the wounds of rats in group SNS, while inflammatory cell infiltration was observed without obvious epithelization in the wounds of rats in group CS. (4) In PSW 1, the number of CD68 positive cells in the wounds of rats in group SNS [(54±10) /mm(2)] was similar to that in group CS [(78±7) /mm(2,) t=1.52, P>0.05]. In PSW 2 and 4, the numbers of CD68 positive cells in the wounds of rats in group SNS [(154±10) and (77±7) /mm(2)] were significantly less than those in group CS [(268±16) and (136±13) /mm(2,) with t values respectively 7.31 and 3.83, P values below 0.01] respectively. (5) Except for the expression in PSW 4 (t=1.23, P>0.05), the mRNA expressions of IL-6 in the wounds of rats in group SNS in PSW 1 and 2 were significantly lower than those in group CS (with t values respectively 13.12 and 4.65, P values below 0.01). Except for the expression in PSW 1 (t=3.08, P<0.05), the mRNA expressions of IL-10 in PSW 2 and 4 in the wounds of rats in the two groups were similar (with t values respectively 2.14 and 0.49, P values above 0.05). Conclusions: Besides good biocompatibility, collagen/fibroin scaffolds containing silver nanoparticles have obvious effect in modulating inflammation, thus they can accelerate dermal regeneration induced by collagen/fibroin scaffolds for wound repair. 目的：探讨含纳米银的胶原蛋白-丝素蛋白支架对大鼠全层皮肤缺损创面真皮再生的影响。 方法：采用冷冻干燥法分别制备81个含纳米银的胶原蛋白-丝素蛋白支架(纳米银质量浓度为10 mg/L)及81个不含纳米银的胶原蛋白-丝素蛋白支架，分别归为纳米银支架组和对照支架组。每组取9个支架加入人Fb培养，于培养2、12、24 h每组取3孔支架，计数2组支架区域黏附人Fb。于36只SD大鼠背部各制作4个全层皮肤缺损创面，将大鼠按随机数字表法平均分为纳米银支架组和对照支架组，创面分别移植含纳米银的胶原蛋白-丝素蛋白支架和不含纳米银的胶原蛋白-丝素蛋白支架。于术后1、2、4周，每组分别取6只大鼠处死，对创面进行大体观察，HE染色观察组织结构、炎性细胞浸润及胶原沉积情况，免疫组织化学染色观察计数CD68阳性细胞，实时荧光定量RT-PCR法检测创面IL-6和IL-10的mRNA表达。对数据行析因设计方差分析、t检验及Bonferroni校正。 结果： (1)培养2、12、24 h，纳米银支架组和对照支架组支架区域人Fb黏附数量相近(t值为1.77～2.60，P值均大于0.05)。(2)术后1周，纳米银支架组大鼠创面及创缘未见明显感染迹象，支架血管化明显；对照支架组大鼠创面出现明显感染迹象，小部分支架脱落。术后2周，纳米银支架组大鼠创面移植支架与创面贴附牢固；对照支架组大鼠创面挛缩明显，大部分支架已脱落。术后4周，纳米银支架组大鼠创面移植支架已成功覆盖创面，支架表面已明显上皮化；对照支架组大鼠创面移植支架完全脱落，残余创面明显收缩。(3)术后1、2周，与对照支架组比较，纳米银支架组大鼠创面胶原分泌及组织再生更多，支架内部炎性细胞浸润较少。术后4周，纳米银支架组大鼠创面已明显上皮化；而对照支架组大鼠创面仍有炎性细胞浸润，未见明显上皮化。(4)术后1周，纳米银支架组大鼠创面CD68阳性细胞数为(54±10)个/mm(2)，与对照支架组的(78±7)个/mm(2)无明显差异(t＝1.52，P>0.05)。术后2、4周，纳米银支架组大鼠创面CD68阳性细胞数分别为(154±10)、(77±7)个/mm(2)，均明显少于对照支架组的(268±16)、(136±13)个/mm(2)(t值分别为7.31和3.83，P值均小于0.01)。(5)除术后4周(t＝1.23，P>0.05)外，术后1、2周纳米银支架组大鼠创面IL-6的mRNA表达量均明显低于对照支架组(t值分别为13.12和4.65，P值均小于0.01)。除术后1周(t＝3.08，P<0.05)外，术后2、4周2组大鼠创面IL-10的mRNA表达量均相近(t值分别为2.14和0.49，P值均大于0.05)。 结论：含纳米银的胶原蛋白-丝素蛋白支架不仅具有良好的生物相容性，同时具有明显的炎症调节作用，可加快胶原蛋白-丝素蛋白支架诱导真皮再生，促进创面修复。.

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