Preparation of chitin/hyaluronic acid/collagen hydrogel loaded with mouse adipose-derived stem cells and its effects on wound healing of full-thickness skin defects in rats
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摘要:
目的 制备负载小鼠脂肪干细胞的甲壳素/透明质酸/胶原水凝胶并探讨其对大鼠全层皮肤缺损创面愈合的作用。 方法 该研究为实验研究。通过酸水解碱的方法提取甲壳素纳米纤维，将提取物与透明质酸和胶原混合制备甲壳素/透明质酸/胶原水凝胶（以下简称水凝胶），另制备负载小鼠脂肪干细胞的水凝胶。将30只雄性12周龄豚鼠按照随机数字表法分为阴性对照组、阳性对照组和水凝胶组（每组10只），分别在豚鼠背部两侧涂抹乙醇、4-氨基苯甲酸乙酯、前述制备的不含细胞的水凝胶进行诱导接触和激发接触，于激发接触后24、48 h观察皮肤水肿和红斑形成情况。将小鼠脂肪干细胞分为行常规培养的正常对照组和用前述制备的不含细胞的水凝胶培养的水凝胶组，培养3 d，采用蛋白质印迹法检测血小板衍生生长因子-D（PDGF-D）、胰岛素样生长因子Ⅰ（IGF-Ⅰ）、转化生长因子β 1（TGF-β 1）的蛋白表达（样本数均为3）。取8只雄性8周龄SD大鼠，在其背部两侧各制备1个圆形全层皮肤缺损创面，分别作为空白对照组（不进行任何处理）和水凝胶组（涂抹前述制备的负载脂肪干细胞的水凝胶）。伤后0（即刻）、2、4、8、10 d，观察创面愈合情况并计算伤后2、4、8、10 d的创面愈合率。取伤后10 d创面组织，行苏木精-伊红染色观察新生组织形成情况，采用酶联免疫吸附测定法检测白细胞介素1α（IL-1α）、IL-6、IL-4和IL-10的浓度，行免疫组织化学染色观察CD16、CD206阳性细胞表达并计算阳性细胞百分比。动物实验样本数均为8。 结果 激发接触后24 h，3组豚鼠皮肤均无水肿形成，仅阳性对照组7只豚鼠皮肤出现轻微红斑。激发接触后48 h，阳性对照组8只豚鼠皮肤出现红斑，4只豚鼠皮肤出现水肿；其余2组豚鼠皮肤无明显红斑或水肿。培养3 d，水凝胶组脂肪干细胞中PDGF-D、IGF-Ⅰ和TGF-β 1的蛋白表达水平均明显高于正常对照组（ t值分别为12.91、11.83、7.92， P<0.05）。伤后0~10 d，2组创面面积均逐渐缩小，水凝胶组创面于伤后10 d基本愈合。伤后4、8、10 d，水凝胶组创面愈合率分别为（38±4）%、（54±5）%、（69±6）%，分别明显高于空白对照组的（21±6）%、（29±7）%、（31±7）%（ t值分别为3.82、3.97、4.05， P值均<0.05）。伤后10 d，与空白对照组比较，水凝胶组创面表皮更加完整，毛囊、血管和其他皮肤附件增多。伤后10 d，水凝胶组创面组织中IL-1α、IL-6浓度均较空白对照组明显降低（ t值分别为8.21、7.99， P<0.05），IL-4、IL-10浓度均较空白对照组明显升高（ t值分别为6.57、9.03， P<0.05）；水凝胶组创面组织中CD16阳性细胞百分比较空白对照组明显降低（ t=8.02， P<0.05），CD206阳性细胞百分比较空白对照组明显升高（ t=7.21， P<0.05）。 结论 负载小鼠脂肪干细胞的水凝胶无致敏性，能促进脂肪干细胞中生长因子分泌，促进大鼠全层皮肤缺损创面组织中巨噬细胞向M2型极化，减轻炎症反应，从而促进创面愈合。 Abstract:Objective To prepare the chitin/hyaluronic acid/collagen hydrogel loaded with mouse adipose-derived stem cells and to explore its effects on wound healing of full-thickness skin defects in rats. Methods The research was an experimental research. Chitin nanofibers were prepared by acid hydrolysis and alkaline extraction method, and then mixed with hyaluronic acid and collagen to prepare chitin/hyaluronic acid/collagen hydrogels (hereinafter referred to as hydrogels). Besides, the hydrogels loaded with mouse adipose-derived stem cells were prepared. Thirty male 12-week-old guinea pigs were divided into negative control group, positive control group, and hydrogel group according to the random number table, with 10 guinea pigs in each group. Ethanol, 4-aminobenzoic acid ethyl ester, or the aforementioned prepared hydrogels without cells were topically applied on both sides of back of guinea pigs respectively for induced contact and stimulated contact, and skin edema and erythema formation were observed at 24 and 48 h after stimulated contact. Adipose-derived stem cells from mice were divided into normal control group cultured routinely and hydrogel group cultured with the aforementioned prepared hydrogels without cells. After 3 d of culture, protein expressions of platelet-derived growth factor-D (PDGF-D), insulin-like growth factor-Ⅰ (IGF-Ⅰ), and transforming growth factor β 1 (TGF-β 1) were detected by Western blotting ( n=3). Eight male 8-week-old Sprague-Dawley rats were taken and a circular full-thickness skin defect wound was created on each side of the back. The wounds were divided into blank control group without any treatment and hydrogel group with the aforementioned prepared hydrogels loaded with adipose-derived stem cells applied. Wound healing was observed at 0 (immediately), 2, 4, 8, and 10 d after injury, and the wound healing rate was calculated at 2, 4, 8, and 10 d after injury. Wound tissue samples at 10 d after injury were collected, the new tissue formation was observed by hematoxylin-eosin staining; the concentrations of interleukin-1α (IL-1α), IL-6, IL-4, and IL-10 were detected by enzyme-linked immunosorbent assay method; the expressions of CD16 and CD206 positive cells were observed by immunohistochemical staining and the percentages of positive cells were calculated. The sample numbers in animal experiment were all 8. Results At 24 h after stimulated contact, no skin edema was observed in the three groups of guinea pigs, and only mild skin erythema was observed in 7 guinea pigs in positive control group. At 48 h after stimulated contact, skin erythema was observed in 8 guinea pigs and skin edema was observed in 4 guinea pigs in positive control group, while no obvious skin erythema or edema was observed in guinea pigs in the other two groups. After 3 d of culture, the protein expression levels of PDGF-D, IGF-I, and TGF-β 1 in adipose-derived stem cells in hydrogel group were significantly higher than those in normal control group (with t values of 12.91, 11.83, and 7.92, respectively, P<0.05). From 0 to 10 d after injury, the wound areas in both groups gradually decreased, and the wounds in hydrogel group were almost completely healed at 10 d after injury. At 4, 8, and 10 d after injury, the wound healing rates in hydrogel group were (38±4)%, (54±5)%, and (69±6)%, respectively, which were significantly higher than (21±6)%, (29±7)%, and (31±7)% in blank control group (with t values of 3.82, 3.97, and 4.05, respectively, Pvalues all <0.05). At 10 d after injury, compared with those in blank control group, the epidermis in wound in hydrogel group was more intact, and there were increases in hair follicles, blood vessels, and other skin appendages. At 10 d after injury, the concentrations of IL-1α and IL-6 in wound tissue in hydrogel group were significantly lower than those in blank control group (with tvalues of 8.21 and 7.99, respectively, P<0.05), while the concentrations of IL-4 and IL-10 were significantly higher than those in blank control group (with tvalues of 6.57 and 9.03, respectively, P<0.05). The percentage of CD16 positive cells in wound tissue in hydrogel group was significantly lower than that in blank control group ( t=8.02, P<0.05), while the percentage of CD206 positive cells was significantly higher than that in blank control group ( t=7.21, P<0.05). Conclusions The hydrogel loaded with mouse adipose-derived stem cells is non-allergenic, can promote the secretion of growth factors in adipose-derived stem cells, promote the polarization of macrophages to M2 phenotype in wound tissue in rats with full-thickness skin defects, and alleviate inflammatory reaction, thereby promoting wound healing. -
Key words:
- Biocompatible materials /
- Hydrogel /
- Inflammation /
- Macrophages /
- Adipose-derived stem cells /
- Wound repair
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1 伤后各时间点2组全层皮肤缺损大鼠创面愈合情况。1A、1B、1C、1D、1E.分别为空白对照组伤后0（即刻）、2、4、8、10 d创面情况；1F、1G、1H、1I、1J.分别为水凝胶组伤后0、2、4、8、10 d创面情况，图1H、1I、1J创面面积分别小于图1C、1D、1E
注：空白对照组创面不进行任何处理，水凝胶组创面涂抹负载小鼠脂肪干细胞的甲壳素/透明质酸/胶原水凝胶
2 伤后10 d 2组全层皮肤缺损大鼠创面新生组织情况 苏木精-伊红×100。2A.空白对照组皮肤附件较少；2B.水凝胶组皮肤附件较图2A增多，新生表皮较图2A更完整
注：空白对照组创面不进行任何处理，水凝胶组创面涂抹负载小鼠脂肪干细胞的甲壳素/透明质酸/胶原水凝胶；图2B中箭头指示皮肤附件，2条虚线间为新生表皮
3 2组全层皮肤缺损大鼠伤后10 d创面组织中CD16和CD206阳性细胞情况 二氨基联苯胺-苏木精×400。3A.空白对照组CD16阳性表达多；3B.水凝胶组CD16阳性表达，较图3A明显减少；3C.空白对照组CD206阳性表达少；3D.水凝胶组CD206阳性表达，较图3C增多
注：空白对照组创面不进行任何处理，水凝胶组创面涂抹负载小鼠脂肪干细胞的甲壳素/透明质酸/胶原水凝胶；CD16和CD206阳性表达均为棕色
表1 培养3 d的2组小鼠脂肪干细胞中3种生长因子的蛋白表达比较（
表1. Comparison of protein expressions of three growth factors in two groups of mouse adipose-derived stem cells after 3 d of culture
组别 样本数 PDGF-D IGF-Ⅰ TGF-β 1 正常对照组 3 0.58±0.11 0.18±0.09 0.29±0.13 水凝胶组 3 1.23±0.09 0.73±0.04 0.72±0.11 t值 12.91 11.83 7.92 P值 <0.001 <0.001 <0.001 注：正常对照组细胞进行常规培养，水凝胶组细胞采用甲壳素/透明质酸/胶原水凝胶培养；PDGF-D为血小板衍生生长因子-D，IGF-Ⅰ为胰岛素样生长因子Ⅰ，TGF-β 1为转化生长因子β 1 
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表2 伤后各时间点2组全层皮肤缺损大鼠创面愈合率比较（%，
表2. Comparison of wound healing rates in two groups of rats with full-thickness skin defects at various time points after injury
组别 样本数 2 d 4 d 8 d 10 d 空白对照组 8 18±5 21±6 29±7 31±7 水凝胶组 8 18±3 38±4 54±5 69±6 t值 0.88 3.82 3.97 4.05 P值 0.351 0.003 0.001 <0.001 注：空白对照组创面不进行任何处理，水凝胶组创面涂抹负载小鼠脂肪干细胞的甲壳素/透明质酸/胶原水凝胶；处理因素主效应， F=103.37， P<0.001；时间因素主效应， F=171.90， P<0.001；两者交互作用， F=54.61， P<0.001
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表3 伤后10 d 2组全层皮肤缺损大鼠创面组织中炎症因子浓度比较（pg/mL，
表3. Comparison of concentrations of inflammatory factors in wound tissue of two groups of rats with full-thickness skin defects at 10 d after injury
组别 样本数 IL-1α IL-6 IL-4 IL-10 空白对照组 8 26.7±2.3 39.4±1.7 19.1±1.9 55.1±8.7 水凝胶组 8 17.1±2.1 18.7±1.1 28.9±1.3 89.8±1.1 t值 8.21 7.99 6.57 9.03 P值 <0.001 <0.001 <0.001 <0.001 注：空白对照组创面不进行任何处理，水凝胶组创面涂抹负载小鼠脂肪干细胞的甲壳素/透明质酸/胶原水凝胶；IL为白细胞介素
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