海藻糖凝胶对大鼠全层皮肤缺损创面及兔耳瘢痕增生的影响

Effects of trehalose gel on full-thickness skin defect wounds in rats and scar hyperplasia in rabbit ears

目的探讨海藻糖凝胶对大鼠全层皮肤缺损创面及兔耳瘢痕增生的影响。方法该研究为实验研究。制备海藻糖凝胶和卡波姆凝胶,辐照灭菌后观察其外观并检测其粘度、成模率、阻菌率、重金属含量、保湿率、水蒸气透过率、无菌性等理化特性和细胞毒性、皮内刺激、致敏性等生物相容性。取30只8~10周龄雄性SD大鼠,按照随机数字表法分为实验组、阳性对照组、阴性对照组,每组10只。在阴性对照组、阳性对照组、实验组大鼠背部制备全层皮肤缺损创面模型,分别进行常规换药、卡波姆凝胶换药、海藻糖凝胶换药处理。统计伤后6、12 d创面愈合率并记录创面愈合时间。伤后6 d,采用透射电镜观测大鼠创面组织中自噬体和自噬溶酶体数量,采用酶联免疫吸附测定法检测大鼠创面组织中微管相关蛋白轻链3Ⅰ(LC3Ⅰ)和LC3Ⅱ含量并计算其比值,采用天狼星红-苦味酸染色法检测大鼠创面组织中Ⅰ型胶原蛋白、Ⅲ型胶原蛋白占比及其比值和总胶原蛋白占比。前述实验样本数均为5。取3只3~4个月龄雄性新西兰白化模型兔,在每侧兔耳各制备3个深达软骨膜的创面,同前进行分组和处理,每组6个创面。创面愈合后30 d,大体观察并使用温哥华瘢痕量表评估兔耳瘢痕情况,采用苏木精-伊红染色检测兔耳瘢痕组织中表皮和真皮的厚度,采用天狼星红-苦味酸染色检测兔耳瘢痕组织中Ⅰ型胶原蛋白、Ⅲ型胶原蛋白占比及其比值和总胶原蛋白占比及其排布情况。样本数为6。结果辐照后的海藻糖凝胶和卡波姆凝胶均呈淡黄色透明状,无异味和杂质。海藻糖凝胶粘度、成膜率、阻菌率、保湿率均显著优于卡波姆凝胶(t值分别为4.13、3.50、4.03、5.80,P<0.05),但水蒸气透过率显著低于卡波姆凝胶(t=-4.14,P<0.05)。2种凝胶均未检出重金属和细菌。2种凝胶均无细胞毒性,皮内刺激和致敏性均为阴性。伤后6、12 d,阳性对照组大鼠创面愈合率均明显高于阴性对照组(t值分别为-6.82和-4.58,P<0.05),实验组大鼠创面愈合率均明显高于阳性对照组(t值分别为-8.90和-4.25,P<0.05)和阴性对照组(t值分别为-8.78和-4.25,P<0.05)。阳性对照组和实验组大鼠创面愈合时间分别为(20.4±2.5)、(23.4±2.5)d,均明显短于阴性对照组的(27.0±2.1)d(t值分别为2.45、-4.49,P<0.05)。伤后6 d,实验组大鼠创面组织中自噬体和自噬溶酶体数量均显著多于阳性对照组(t值分别为7.37和9.33,P<0.05)和阴性对照组(t值分别为-7.06和-8.54,P<0.05)。伤后6 d,阳性对照组大鼠创面组织中LC3Ⅱ含量及LC3Ⅱ/LC3Ⅰ均显著高于阴性对照组(t值分别为-4.48和-2.47,P<0.05);实验组大鼠创面组织中LC3Ⅱ含量及LC3Ⅱ/LC3Ⅰ均显著高于阴性对照组(t值分别为11.98和6.04,P<0.05)和阳性对照组(t值分别为-6.64和-4.17,P<0.05),LC3Ⅰ含量明显低于阴性对照组(t=2.33,P<0.05)。伤后6 d,3组大鼠创面组织中总胶原蛋白和Ⅰ型胶原蛋白占比均相近,P>0.05。伤后6 d,阳性对照组大鼠创面组织中Ⅲ型胶原蛋白占比显著高于阴性对照组(t=-3.19,P<0.05),且Ⅰ型胶原蛋白/Ⅲ型胶原蛋白显著低于阴性对照组(t=2.18,P<0.05);实验组大鼠创面组织中Ⅲ型胶原蛋白占比显著高于阴性对照组和阳性对照组(t值分别为-2.38和5.91,P<0.05),且Ⅰ型胶原蛋白/Ⅲ型胶原蛋白显著低于阴性对照组和阳性对照组(t值分别为3.08和-4.35,P<0.05)。创面愈合后30 d,可观察到阳性对照组兔耳瘢痕增生情况与阴性对照组相近,而实验组兔耳瘢痕增生情况明显减轻。创面愈合后30 d,实验组兔耳瘢痕色泽、血管、厚度、硬度评分及总分均明显低于阳性对照组(t值分别为3.80、3.80、2.39、2.71、4.84,P<0.05)和阴性对照组(t值分别为-3.81、-4.78、0.04、-2.71、-5.14,P<0.05)。创面愈合后30 d,实验组、阴性对照组和阳性对照组兔耳瘢痕组织中表皮厚度相近(P>0.05);阳性对照组兔耳瘢痕组织真皮厚度明显小于阴性对照组(t=5.42,P<0.05),实验组兔耳瘢痕组织真皮厚度明显小于阴性对照组和阳性对照组(t值分别为11.91和8.49,P<0.05)。创面愈合后30 d,3组兔耳瘢痕组织中胶原蛋白排列均紊乱,总胶原蛋白占比相近(P>0.05);实验组兔耳瘢痕组织中Ⅰ型胶原蛋白占比明显低于阳性对照组(t值分别为3.00,P<0.05),Ⅲ型胶原蛋白占比明显高于阴性对照组和阳性对照组(t值分别为-4.46和4.05,P值均<0.05),Ⅰ型胶原蛋白/Ⅲ型胶原蛋白明显低于阴性对照组和阳性对照组(t值分别为8.50和-5.25,P值均<0.05)。结论海藻糖凝胶相较于卡波姆凝胶具有更适于创面愈合的理化特性,且生物相容性良好;能基于自噬激活作用促进大鼠全层皮肤缺损创面愈合,减轻兔耳瘢痕增生。

Objective To investigate the effects of trehalose gel on full-thickness skin defect wounds in rats and scar hyperplasia in rabbit ears.Methods The study was an experimental study.The trehalose gel and carbomer gel were prepared,their appearance after irradiation sterilization,and their physicochemical characterization such as viscosity,film forming rate,bacteria resistance rate,heavy metal content,moisture retention rate,water vapor permeability,sterility,and biocompatibility such as cytotoxicity,intradermal irritation,and sensitization were observed.Thirty male Sprague-Dawley rats aged 8-10 weeks were divided into experimental group,positive control group,and negative control group using a random number table method,with 10 rats in each group.The full-thickness skin defect wound models were prepared on the back of rats in negative control group,positive control group,and experimental group,and were treated with routine dressing change,carbomer gel dressing change,and trehalose gel dressing change,respectively.The wound healing rates on 6 and 12 days after injury and the wound healing time were recorded.On 6 days after injury,the number of autophagosomes and autophagolysosomes in rat wound tissue was detected using transmission electron microscopy.The content of microtubule associated protein light chain 3Ⅰ(LC3Ⅰ)and LC3Ⅱin rat wound tissue was detected using enzyme-linked immunosorbent assay method and their ratio was calculated.The proportion of typeⅠandⅢcollagens and their ratio,as well as the total collagen proportion in rat wound tissue were detected using sirius red picric acid staining method.The number of samples in the aforementioned experiments was all 5.Three male New Zealand albino model rabbits aged 3-4 months were taken,and 3 wounds deep to the perichondrium were created on each of the rabbit ears,with six wounds in each group and being grouped and treated as mentioned above.On 30 days after wound healing,the scar tissue of the rabbit ear was observed and evaluated using the Vancouver scar scale.The thickness of the epidermis and dermis in the scar tissue of the rabbit ear was measured using hematoxylin eosin staining,and the proportion of typeⅠandⅢcollagens and their ratio,as well as the total collagen proportion and arrangement in the scar tissue of the rabbit ear were measured using sirius red picric acid staining method.The number of samples was 6.Results The irradiated trehalose gel and carbomer gel were light yellow and transparent,without odor and impurities.The viscosity,film forming rate,bacteria resistance rate,and moisture retention rate of trehalose gel were significantly better than that of carbomer gel(with t values of 4.13,3.50,4.03,and 5.80,respectively,P<0.05),but the water vapor permeability was significantly lower than that of carbomer gel(t=-4.14,P<0.05).No heavy metals or bacteria were detected in any gel.Both of the two gel had no cytotoxicity,and the intradermal irritation and sensitization were negative.On 6 and 12 days after injury,the wound healing rates of rats in positive control group were significantly higher than that in negative control group(with t values of-6.82 and-4.58,respectively,P<0.05);the wound healing rate of rats in experimental group was significantly higher than those in positive control group(with t values of-8.90 and-4.25,respectively,P<0.05)and negative control group(with t values of-8.78 and-4.25,respectively,P<0.05).The wound healing time((20.4±2.5),(23.4±2.5)d)of rats in positive control group and experimental group was significantly shorter than(27.0±2.1)d in negative control group(with t values of 2.45 and-4.49,respectively,P<0.05).On 6 days after injury,the number of autophagosomes and autophagolysosomes in wound tissue in experimental group of rats were significantly higher than those in positive control group(with t values of 7.37 and 9.33,respectively,P<0.05)and negative control group(with t values of-7.06 and-8.54,respectively,P<0.05).On 6 days after injury,the content of LC3Ⅱand LC3Ⅱ/LC3Ⅰin wound tissue in positive control group of rats were significantly higher than that in negative control group(with t values of-4.48 and-2.47,respectively,P<0.05);the content of LC3Ⅰand LC3Ⅱ/LC3Ⅰin wound tissue in experimental group of rats were significantly higher than those in negative control group(with t values of 11.98 and 6.04,respectively,P<0.05)and positive control group(with t values of-6.64 and-4.17,respectively,P<0.05),the content of LC3Ⅰwas significantly lower than that in negative control group(t=2.33,P<0.05).On 6 days after injury,the proportions of total collagen and typeⅠcollagen in wound tissue of rats in the three groups were similar,P>0.05.On 6 days after injury,the proportion of typeⅢcollagen in wound tissue of rats in positive control group was significantly higher than that in negative control group(t=-3.19,P<0.05),and the typeⅠcollagen/typeⅢcollagen was significantly lower than that in negative control group(t=2.18,P<0.05);the proportion of typeⅢcollagen in the wound tissue of rats in experimental group was significantly higher than those in negative control group and positive control group(with t values of-2.38 and 5.91,respectively,P<0.05),and typeⅠcollagen/typeⅢcollagen was significantly lower than those in negative control group and positive control group(with t values of 3.08 and-4.35,respectively,P<0.05).On 30 days after wound healing,it was observed that the rabbit ear scar proliferation in positive control group was similar to that in negative control group,while the rabbit ear scar proliferation in experimental group was significantly reduced.On 30 days after wound healing,the color,blood vessels,thickness,hardness score,and total score of rabbit ear scars in experimental group were significantly lower than those in positive control group(with t values of 3.80,3.80,2.39,2.71,and 4.84,respectively,P<0.05)and negative control group(with t values of-3.81,-4.78,0.04,-2.71,and-5.14,respectively,P<0.05).On 30 days after wound healing,there was no significant difference in the epidermal thickness of rabbit ear scar tissue among experimental group,negative control group,and positive control group(P>0.05);the dermal thickness of rabbit ear scar tissue in positive control group was significantly smaller than that in negative control group(t=5.42,P<0.05),while the dermal thickness of rabbit ear scar tissue in experimental group was significantly smaller than those in negative control group and positive control group(with t values of 11.91 and 8.49,respectively,P<0.05).On 30 days after wound healing,the collagen protein arrangement of scar tissue of rabbits in the three groups was disordered,and the total collagen proportion was similar(P>0.05).The proportion of typeⅠcollagen of scar tissue in experimental group was significantly lower than that in positive control group(t=3.00,P<0.05),the content of typeⅢcollagen was significantly higher than those in negative control group and positive control group(with t values of-4.46 and 4.05,respectively,P<0.05),and the typeⅠcollagen/typeⅢcollagen was significantly lower than those in negative control group and positive control group(with t values of 8.50 and-5.25,respectively,P<0.05).Conclusions Compared with carbomer gel,trehalose gel has a more suitable physicochemical characterization for wound healing,and has good biocompatibility.It can promote the wound healing of full-thickness defects in rats and reduce scar hyperplasia in rabbit ears based on autophagy activation.