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  • Title: [Effects of temperature-sensitive hydroxybutyl chitosan hydrogel on wound healing of full-thickness skin defect in rats].
    Author: Chen AX, Chen YB, Jiang YF, Han Y.
    Journal: Zhonghua Shao Shang Za Zhi; 2021 Dec 20; 37(12):1166-1174. PubMed ID: 34839597.
    Abstract:
    Objective: To investigate the effects of temperature-sensitive hydroxybutyl chitosan hydrogel on wound healing of full-thickness skin defect in rats. Methods: The experimental research method was used. Fifty-one no matter male or female Sprague-Dawley rats aged 7-10 weeks were selected, and two round full-thickness skin defect wounds with a diameter of 2 cm were created on the back of each rat at a distance about 1.0 cm to the spine. The rats were divided into temperature-sensitive hydrogel group, gel group, and blank control group according to the random number table, with 17 rats and 34 wounds in each group. Wounds of rats in the first two groups were applied respectively with 0.3 mL temperature-sensitive hydroxybutyl chitosan hydrogel and carboxymethyl chitosan hydrogel immediately after injury, and the wounds of rats in blank control group received no treatment. The wounds of rats in the three groups were all covered with vaseline oil gauze. The states of temperature-sensitive hydroxybutyl chitosan hydrogel in wounds of rats in temperature-sensitive hydrogel group and carboxymethyl chitosan hydrogel in wounds of rats in gel group were observed every day when the dressings were changed, and the difficulty of vaseline oil gauze removal was recorded. On the 3rd, 7th, 10th, 14th, and 21st day after injury, the wound healing of rats in the three groups was observed and the wound healing rates were calculated. On the 3rd, 7th, 10th, 14th, and 21st day after injury, tissue from 4 wounds of 2 rats in each group was collected for the following observation and detection. The infiltration of inflammatory cells, angiogenesis, and re-epithelialization were observed by hematoxylin eosin staining. The regeneration and remodeling of collagen fibers were observed by Masson staining, and the collagen volume fraction was calculated. The expressions of interleukin-6 (IL-6), transforming growth factor β1 (TGF-β1), and matrix metalloproteinase-1 (MMP-1) were detected by enzyme-linked immunosorbent assay method. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, and Bonferroni test. Results: The carboxymethyl chitosan gel in wounds of rats in gel group was liquid gel and could flow with the body position, while the temperature-sensitive hydroxybutyl chitosan hydrogel in wounds of rats in temperature-sensitive hydrogel group was solid gel and could not flow with the body position, and the distribution of the latter was more uniform. The vaseline oil gauzes were easily removed in wounds of rats in temperature-sensitive hydrogel group, while the vaseline oil gauzes were difficult to remove in the other two groups. On the 3rd, 7th, 10th, 14th, and 21st day after injury, the wound granulation tissue of rats grew well in temperature-sensitive hydrogel group and gel group, with no obvious infection, and two rats in blank control group died of wound infection on the 3rd and 5th day after injury. On the 7th, 10th, 14th, and 21st day after injury, the wound healing rates of rats in temperature-sensitive hydrogel group and gel group were significantly higher than that in blank control group (P<0.01). On the 10th day after injury, the wound healing rate of rats in temperature-sensitive hydrogel group was significantly higher than that in gel group (P<0.05). A large number of neutrophils and lymphocytes infiltrated into the wounds of rats in the three groups on the 3rd day after injury. The infiltration of inflammatory cells was gradually reduced and the wound healed gradually in rats of temperature-sensitive hydrogel group and gel group from the 7th to 21st day after injury, and the epidermis and dermis could be seen, without hair follicles and other skin appendages. The wounds of rats in blank control group did not heal completely on 21st day after injury. From the 3rd to 10th day after injury, the newly formed collagen fibers increased gradually in the wounds of rats in the three groups. On the 14th and 21st day after injury, the collagen fibers in the wounds of rats in temperature-sensitive hydrogel group and gel group were denser and more orderly than those in blank control group. On the 10th, 14th, and 21st day after injury, the collagen volume fraction of wounds of rats in temperature-sensitive hydrogel group and gel group was significantly higher than that in blank control group (P<0.01). On the 14th day after injury, the collagen volume fraction of wounds of rats in temperature-sensitive hydrogel group was significantly higher than that in gel group (P<0.01). On the 3rd, 7th, and 10th day after injury, the expressions of IL-6 in wounds of rats in temperature-sensitive hydrogel group were significantly higher than those in gel group and blank control group (P<0.01), and the expressions of IL-6 in wounds of rats in gel group were significantly lower than those in blank control group (P<0.01). On the 3rd, 7th, and 10th day after injury, the expressions of TGF-β1 in wounds of rats in temperature-sensitive hydrogel group were significantly higher than those in gel group and blank control group (P<0.01). The expressions of TGF-β1 in wounds of rats in gel group were significantly lower than those in blank control group on the 3rd and 7th day after injury (P<0.01), and the expression of TGF-β1 in wounds of rats in gel group was significantly higher than that in blank control group on the 10th day after injury (P<0.01). On the 14th day after injury, the expression of TGF-β1 in wounds of rats in gel group was significantly higher than that in temperature-sensitive hydrogel group and blank control group (P<0.01). On the 21st day after injury, the expression of TGF-β1 in wounds of rats in temperature-sensitive hydrogel group was significantly lower than that in gel group and blank control group (P<0.01), and the expression of TGF-β1 in wounds of rats in gel group was significantly lower than that in blank control group (P<0.01). On the 7th day after injury, the expression of MMP-1 in wounds of rats in gel group was significantly higher than that in temperature-sensitive hydrogel group and blank control group (P<0.01). On the 10th, 14th, and 21st day after injury, the expressions of MMP-1 in wounds of rats in temperature-sensitive hydrogel group were significantly higher than those in gel group and blank control group (P<0.01). On the 10th day after injury, the expression of MMP-1 in wounds of rats in gel group was significantly lower than that in blank control group (P<0.01). On the 14th and 21st day after injury, the expressions of MMP-1 in wounds of rats in gel group were significantly higher than those in blank control group (P<0.01). Conclusions: Temperature-sensitive hydroxybutyl chitosan hydrogel can promote the healing of full-thickness skin defect wounds in rats by increasing the expressions of IL-6, TGF-β1, and MMP-1, regulating the wound healing environment, inhibiting inflammatory reaction, improving the strength of tissue repair, and promoting collagen synthesis or decomposition. 目的： 探讨温敏性羟丁基壳聚糖水凝胶对大鼠全层皮肤缺损创面愈合的作用。 方法： 采用实验研究方法。取51只7~10周龄SD大鼠，雌雄不限，在大鼠背部距脊柱约1.0 cm处，制造2个直径2 cm的全层皮肤缺损圆形创面。将大鼠按随机数字表法分为温敏性水凝胶组、凝胶组和空白对照组，每组17只大鼠（34个创面），前2组大鼠创面伤后即刻分别涂抹0.3 mL温敏性羟丁基壳聚糖水凝胶和羧甲基壳聚糖凝胶，空白对照组大鼠创面不行任何处理，3组大鼠创面均外用凡士林油纱。每天换药时观察温敏性水凝胶组大鼠创面温敏性羟丁基壳聚糖水凝胶及凝胶组大鼠创面羧甲基壳聚糖凝胶状态，记录去除凡士林油纱的难易程度。伤后3、7、10、14、21 d，观察3组大鼠创面愈合情况，并计算创面愈合率。伤后3、7、10、14、21 d，每组各取2只大鼠的4个创面组织，行苏木精-伊红染色观察炎症细胞浸润、血管新生、再上皮化情况；Masson染色观察胶原纤维再生和重塑情况，并计算胶原容积分数；酶联免疫吸附测定法检测白细胞介素6（IL-6）、转化生长因子β1（TGF-β1）、基质金属蛋白酶1（MMP-1）的表达。对数据行析因设计方差分析、单因素方差分析及Bonferroni检验。 结果： 伤后换药时，凝胶组大鼠创面羧甲基壳聚糖凝胶呈液态凝胶状，可随体位流动；而温敏性水凝胶组大鼠创面温敏性羟丁基壳聚糖水凝胶呈固态凝胶状，不随体位变化而流动，且分布更均匀；温敏性水凝胶组大鼠创面凡士林油纱较易去除，而其余2组凡士林油纱不易去除。伤后3、7、10、14、21 d，温敏性水凝胶组和凝胶组大鼠创面肉芽组织生长好，未见明显感染；空白对照组伤后3、5 d各有1只大鼠因创面感染而死亡。伤后7、10、14、21 d，温敏性水凝胶组和凝胶组大鼠创面愈合率明显高于空白对照组（P<0.01）；伤后10 d，温敏性水凝胶组大鼠创面愈合率明显高于凝胶组（P<0.05）。伤后3 d，3组大鼠创面均有大量中性粒细胞和淋巴细胞浸润。伤后7~21 d，温敏性水凝胶组和凝胶组大鼠创面炎症细胞浸润逐渐减少，创面逐渐愈合，可见表皮层和真皮层，未见毛囊等皮肤附件；空白对照组大鼠创面伤后21 d未完全愈合。伤后3~10 d，3组大鼠创面新生胶原纤维组织逐渐增多；伤后14、21 d，与空白对照组比较，温敏性水凝胶组和凝胶组大鼠创面胶原纤维更加致密，排列整齐有序。伤后10、14、21 d，温敏性水凝胶组和凝胶组大鼠创面胶原容积分数显著高于空白对照组（P<0.01）；伤后14 d，温敏性水凝胶组大鼠创面胶原容积分数显著高于凝胶组（P<0.01）。伤后3、7、10 d，温敏性水凝胶组大鼠创面IL-6的表达显著高于凝胶组和空白对照组（P<0.01），凝胶组大鼠创面IL-6的表达显著低于空白对照组（P<0.01）。伤后3、7、10 d，温敏性水凝胶组大鼠创面TGF-β1的表达显著高于凝胶组及空白对照组（P<0.01）；伤后3、7 d，凝胶组大鼠创面TGF-β1的表达显著低于空白对照组（P<0.01）；伤后10 d，凝胶组大鼠创面TGF-β1的表达显著高于空白对照组（P<0.01）；伤后14 d，凝胶组大鼠创面TGF-β1的表达明显高于温敏性水凝胶组和空白对照组（P<0.01）；伤后21 d，温敏性水凝胶组大鼠创面TGF-β1的表达显著低于凝胶组和空白对照组（P<0.01），凝胶组大鼠创面TGF-β1的表达显著低于空白对照组（P<0.01）。伤后7 d，凝胶组大鼠创面MMP-1的表达显著高于温敏性水凝胶组和空白对照组（P<0.01）；伤后10、14、21 d，温敏性水凝胶组大鼠创面MMP-1的表达显著高于凝胶组及空白对照组（P<0.01）；伤后10 d，凝胶组大鼠创面MMP-1的表达显著低于空白对照组（P<0.01）；伤后14、21 d，凝胶组大鼠创面MMP-1的表达显著高于空白对照组（P<0.01）。 结论： 温敏性羟丁基壳聚糖水凝胶可通过增加IL-6、TGF-β1和MMP-1的表达，调节创面愈合环境，抑制炎症反应，提高组织修复强度，促进胶原的合成/分解，从而促进大鼠全层皮肤缺损创面的愈合。.
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