改良型富血小板纤维蛋白/壳聚糖温敏水凝胶对糖尿病大鼠全层皮肤缺损创面愈合的影响

荀浩怡; 苏小薇; 胡方超; 刘翔宇; 吴育寿; 刘甜; 孙冉; 段红杰; 迟云飞; 柴家科

doi:10.3760/cma.j.cn501225-20231020-00127

改良型富血小板纤维蛋白/壳聚糖温敏水凝胶对糖尿病大鼠全层皮肤缺损创面愈合的影响

doi: 10.3760/cma.j.cn501225-20231020-00127

1.
解放军总医院研究生院，北京　100853
2.
解放军总医院第四医学中心全军烧伤研究所，北京　100048
3.
河北北方学院，张家口　075132

基金项目:

北京市自然科学基金面上项目 7222179

详细信息

通讯作者:
柴家科，Email：cjk304@163.com

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出版历程
- 收稿日期: 2023-10-20
- 网络出版日期: 2024-04-29

Effects of advanced platelet-rich fibrin/chitosan temperature-sensitive hydrogel on full-thickness skin defect wound healing in diabetic rats

1.
Graduate School, Chinese PLA General Hospital, Beijing 100853, China
2.
Burn Institute, the Fourth Medical Center, Chinese PLA General Hospital, Beijing 100048, China
3.
Hebei North University, Zhangjiakou 075132, China

Funds:

Beijing Natural Science Foundation of China 7222179

More Information

Corresponding author: Chai Jiake, Email: cjk304@163.com

摘要

摘要: 目的制备改良型富血小板纤维蛋白（A-PRF）/壳聚糖温敏水凝胶（以下简称复合水凝胶）并探讨复合水凝胶对糖尿病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。成功制备具有多孔网状结构及温敏特性的，含质量浓度为10、15、20、50、100 g/L A-PRF的复合水凝胶。取6~8周龄雄性SD大鼠，通过腹腔注射链脲佐菌素成功制造糖尿病大鼠模型，并在每只大鼠背部制造4个全层皮肤缺损创面（最终36只大鼠成功造模）。将每只大鼠3个创面分别作为空白组（不进行药物干预）、阳性对照组（滴加重组人粒细胞巨噬细胞刺激因子凝胶）、壳聚糖水凝胶组（滴加壳聚糖水凝胶溶液）；于每只大鼠剩余的1个创面共30个创面分别滴加含10、15、20、50、100 g/L A-PRF的复合水凝胶溶液，设为10、15、20、50、100 g/L 复合水凝胶组，每组6个创面。伤后14 d，取6只其中1个创面为100 g/L 复合水凝胶组的大鼠，行苏木精-伊红（HE）染色，观察大鼠心、肝、脾、肺和肾等炎症、出血或坏死情况；伤后10 d，取6只其中1个创面为15 g/L 复合水凝胶组的大鼠，采用激光血流成像系统观测4组创面血流灌注量（样本数为6）。伤后0（即刻）、7、14 d，观察8组创面愈合情况并计算创面愈合率。伤后14 d，取8组创面组织，分别行HE、 Masson染色，观察新生上皮形成和胶原生成情况；行免疫组织化学法检测CD31、血管内皮生长因子A（VEGFA）的表达情况；行蛋白质印迹法检测CD31、VEGFA的蛋白表达；行实时荧光定量反转录PCR检测CD31、VEGFA的mRNA表达（样本数均为4）。结果伤后14 d，6只大鼠心、肝、脾、肺、肾中均未观察到明显的炎症、出血或坏死。伤后10 d，15 g/L复合水凝胶组创面血液灌注量明显多于空白组、阳性对照组、壳聚糖水凝胶组（ P值均<0.05）。伤后7、14 d，空白组创面愈合率分别为（26.0±8.9）%、（75.0±1.8）%，均分别明显低于阳性对照组、壳聚糖水凝胶组及10、15、20、50、100 g/L 复合水凝胶组的（45.8±3.2）%、（49.8±3.7）%、（51.2±2.9）%、（68.5±2.4）%、（68.8±1.5）%、（72.7±2.1）%、（75.0±3.7）%及（79.1±1.9）%、（77.2±1.7）%、（82.3±1.3）%、（89.6±1.9）%、（89.8±1.3）%、（87.3±1.1）%、（87.9±1.3）%（ P<0.05）；阳性对照组、壳聚糖水凝组、10 g/L 复合水凝胶组创面愈合率均明显低于15、20、50、100 g/L 复合水凝胶组（ P<0.05）。伤后14 d，15、20、50、100 g/L 复合水凝胶组创面上皮化程度较其他4组创面更高、新生微血管情况更好，胶原数量更多且排列更整齐。伤后14 d，阳性对照组创面CD31、VEGFA及壳聚糖水凝胶组创面VEGFA阳性面积百分比均明显高于空白组（ P<0.05），10 g/L 复合水凝胶组创面VEGFA阳性面积百分比明显高于空白组、壳聚糖水凝胶组、阳性对照组（ P值均<0.05），15、20、50、100 g/L 复合水凝胶组创面CD31、VEGFA阳性面积百分比均明显高于空白组、阳性对照组、壳聚糖水凝胶组、10 g/L 复合水凝胶组（ P<0.05）。伤后14 d，壳聚糖水凝胶组、阳性对照组、10 g/L 复合水凝胶组创面组织中CD31、VEGFA蛋白和mRNA表达均明显高于空白组（ P<0.05），10 g/L 复合水凝胶组创面组织中VEGFA蛋白表达和CD31、VEGFA mRNA表达均明显高于阳性对照组（ P<0.05），15、20、50、100 g/L复合水凝胶组创面组织中CD31、VEGFA蛋白和mRNA表达均明显高于空白组、阳性对照组、壳聚糖水凝胶组、10 g/L 复合水凝胶组（ P<0.05），壳聚糖水凝胶组创面组织中CD31、VEGFA的mRNA表达均明显低于阳性对照组（ P<0.05）。结论复合水凝胶生物安全性高，可改善创面血流灌注，有效促进创面组织中血管和胶原生成，从而促进糖尿病大鼠全层皮肤缺损创面愈合，15 g/L为复合水凝胶中A-PRF的较优使用质量浓度。
- 生物相容性材料 /
- 水凝胶 /
- 糖尿病 /
- 新生血管化，生理性 /
- 创面修复 /
- 改良型富血小板纤维蛋白
Abstract: Objective To prepare advanced platelet-rich fibrin (A-PRF)/chitosan temperature-sensitive hydrogel (hereinafter referred to as composite hydrogel) and explore the effects of composite hydrogel on full-thickness skin defect wound healing in diabetic rats. Methods This study was an experimental study. The composite hydrogel with porous mesh structure and temperature-sensitive characteristics was successfully prepared, containing A-PRF with mass concentrations of 10, 15, 20, 50, and 100 g/L. Diabetic model was successfully established in male Sprague-Dawley rats aged 6-8 weeks by intraperitoneal injection of streptozotocin, and 4 full-thickness skin defect wounds were established on the back of each rat (finally 36 rats were successfully established model). Three wounds of each rat were divided into blank group (no drug intervention), positive control group (dropping recombinant human granulocyte-macrophage stimulating factor gel), and chitosan hydrogel group (dropping chitosan hydrogel solution); the remaining one wound of each rat (totally 30 wounds) was added with composite hydrogel solution containing 10, 15, 20, 50, and 100 g/L A-PRF, respectively, and the wounds were set as 10, 15, 20, 50, and 100 g/L composite hydrogel groups, with 6 wounds in each group. On 14 d after injury, 6 rats with one wound in 100 g/L composite hydrogel group were selected for hematoxylin-eosin (HE) staining to observe the inflammation, hemorrhage, or necrosis of the heart, liver, spleen, lung, and kidney. On 10 d after injury, 6 rats with one wound in 15 g/L composite hydrogel group were selected to observe the blood flow perfusion of wounds in the four groups (with sample size of 6). On 0 (immediately), 7, and 14 d after injury, the wound healing in the eight groups was observed and the wound healing rate was calculated. On 14 d after injury, the wound tissue in the eight groups was taken for HE and Masson staining to observe the formation of new epithelium and collagen formation, respectively; the expressions of CD31 and vascular endothelial growth factor A (VEGFA) were detected by immunohistochemistry; the protein expressions of CD31 and VEGFA were detected by Western blotting; the mRNA expressions of CD31 and VEGFA were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction (with all sample sizes of 4). Results On 14 d after injury, no obvious inflammation, hemorrhage, or necrosis was observed in the heart, liver, spleen, lung, and kidney in the 6 rats. On 10 d after injury, the blood perfusion volume of wound in 15 g/L composite hydrogel group was significantly more than that in blank group, positive control group, and chitosan hydrogel group, respectively (with P values all <0.05). On 7 and 14 d after injury, the wound healing rates of blank group were (26.0±8.9)% and (75.0±1.8)%, which were significantly lower than those of positive control group, chitosan hydrogel group, and 10, 15, 20, 50, 100 g/L composite hydrogel group ((45.8±3.2)%, (49.8±3.7)%, (51.2±2.9)%, (68.5±2.4)%, (68.8±1.5)%, (72.7±2.1)%, (75.0±3.7)% and (79.1±1.9)%, (77.2±1.7)%, (82.3±1.3)%, (89.6±1.9)%, (89.8±1.3)%, (87.3±1.1)%, (87.9±1.3)%), P<0.05; the wound healing rates of positive control group, chitosan hydrogel group, and 10 g/L composite hydrogel group were significantly lower than those of 15, 20, 50, and 100 g/L composite hydrogel group ( P<0.05). On 14 d after injury, the wound epidermization degrees of rats in 15, 20, 50, and 100 g/L composite hydrogel groups were higher than those in the other 4 groups, and the new microvascular situation was better, and the number of collagen was more and arranged more neatly. On 14 d after injury, the percentages of CD31 and VEGFA positive areas in wounds in positive control group and the percentage of VEGFA positive area in wounds in chitosan hydrogel group were significantly higher than those in blank group ( P<0.05), the percentage of VEGFA positive area in wounds in 10 g/L composite hydrogel group was significantly higher than that in blank group, chitosan hydrogel group, and positive control group (with P values all <0.05), and the percentages of CD31 and VEGFA positive areas in wounds in 15, 20, 50, and 100 g/L composite hydrogel groups were significantly higher than those in blank group, positive control group, chitosan hydrogel group, and 10 g/L composite hydrogel group ( P<0.05). On 14 d after injury, the protein and mRNA expressions of CD31 and VEGFA in wound tissue in chitosan hydrogel group, positive control group, and 10 g/L composite hydrogel group were significantly higher than those in blank group ( P<0.05), the protein expression of VEGFA and the mRNA expressions of CD31 and VEGFA in wound tissue in 10 g/L composite hydrogel group were significantly higher than those in positive control group ( P<0.05), the protein and mRNA expressions of CD31 and VEGFA in wound tissue in 15, 20, 50, and 100 g/L composite hydrogel groups were significantly higher than those in blank group, positive control group, chitosan hydrogel group, and 10 g/L composite hydrogel group ( P<0.05), and the mRNA expressions of CD31 and VEGFA in wound tissue in chitosan hydrogel group were significantly lower than those in positive control group ( P<0.05). Conclusions The composite hydrogel has high biological safety, can improve wound blood perfusion, effectively promote the formation of blood vessels and collagen in wound tissue, thus promoting the wound healing of full-thickness skin defects in diabetic rats. 15 g/L is the optimal concentration of A-PRF in composite hydrogel.
- Biocompatible materials /
- Hydrogel /
- Diabetes mellitus /
- Neovascularization, physiologic /
- Wound repair /
- Advanced platelet-rich fibrin
荀浩怡：酝酿和设计实验、实施研究、采集数据、数据整理、统计分析、撰写论文、修改论文；苏小薇：酝酿和设计实验、分析/解释数据、统计分析、论文修改；胡方超、刘翔宇、刘甜：采集数据、修改论文；吴育寿、孙冉：采集数据、数据整理；段红杰、迟云飞、柴家科：研究指导、论文修改、经费支持

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1 A-PRF的形貌特征观察。1A.A-PRF富含纤维蛋白、白细胞和血小板苏木精-伊红×40；1B.A-PRF由细小的纤维蛋白组成松散排列的大网状结构，间质中散在分布着正常或变形的白细胞和血小板扫描电子显微镜×3 000

注：A-PRF为改良型富血小板纤维蛋白；白色箭头指示纤维蛋白，黄色箭头指示白细胞，红色箭头指示血小板

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2 壳聚糖水凝胶和含15 g/L A-PRF的复合水凝胶在不同温度下的形态及微观形貌。2A、2B.分别为壳聚糖水凝胶及复合水凝胶，在25 ℃时均为液态；2C、2D.分别为壳聚糖水凝胶及复合水凝胶，在37 ℃水浴10 min后均为凝胶状态；2E、2F.分别为壳聚糖水凝胶及复合水凝胶的微观形貌，均为多孔网状结构，但复合水凝胶网状结构更加致密、均匀扫描电子显微镜×3 000

注：复合水凝胶为改良型富血小板纤维蛋白（A-PRF）/壳聚糖水凝胶

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3 其中1个创面为100 g/L复合水凝胶组创面的全层皮肤缺损糖尿病大鼠伤后14 d主要脏器情况苏木精-伊红×200。3A、3B、3C、3D、3E.分别为心、肝、脾、肺、肾的情况，均未观察到明显的炎症、出血或坏死

注：100 g/L复合水凝胶组创面滴加含100 g/L改良型富血小板纤维蛋白（A-PRF）的A-PRF/壳聚糖水凝胶溶液

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4 4组全层皮肤缺损糖尿病大鼠伤后10 d创面血流灌注情况。4A、4B、4C、4D.分别为空白组、阳性对照组、壳聚糖水凝胶组、15 g/L复合水凝胶组创面血流灌注情况，图4D血流灌注量明显多于图4A、4B、4C

注：空白组创面不进行药物干预，于阳性对照组、壳聚糖水凝胶组创面滴加重组人粒细胞巨噬细胞刺激因子凝胶、壳聚糖水凝胶，15 g/L复合水凝胶组创面滴加含15 g/L改良型富血小板纤维蛋白（A-PRF）的A-PRF/壳聚糖水凝胶溶液；黑色表示无血流灌注，绿色表示血流灌注少，红色表示血流灌注充足

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5 8组全层皮肤缺损糖尿病大鼠伤后14 d创面愈合情况。5A、5B、5C、5D、5E、5F、5G、5H.分别为空白组、壳聚糖水凝胶组、阳性对照组及10、15、20、50、100 g/L复合水凝胶组，图5E、5F、5G、5H创面面积均明显小于图5A、5B、5C、5D

注：空白组创面不进行药物干预，于阳性对照组、壳聚糖水凝胶组创面滴加重组人粒细胞巨噬细胞刺激因子凝胶、壳聚糖水凝胶溶液，于5个复合水凝胶组创面滴加含相应质量浓度改良型富血小板纤维蛋白（A-PRF）的A-PRF/壳聚糖水凝胶溶液；创面外的白色圆环为防收缩环

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6 8组全层皮肤缺损糖尿病大鼠伤后14 d创面组织中CD31、VEGFA阳性表达情况二氨基联苯胺-苏木精×400。6A、6B、6C、6D、6E、6F、6G、6H.分别为空白组、阳性对照组、壳聚糖水凝胶组及10、15、20、50、100 g/L复合水凝胶组创面CD31表达情况，图6E、6F、6G、6H CD31表达情况高于图6A、6B、6C、6D；6I、6J、6K、6L、6M、6N、6O、6P.分别为空白组、阳性对照组、壳聚糖水凝胶组及10、15、20、50、100 g/L 复合水凝胶组创面VEGFA表达情况，图6M、6N、6O、6P VEGFA表达高于图6I、6J、6K、6L

注：CD31、血管内皮生长因子A（VEGFA）阳性表达均为棕色；空白组创面不进行药物干预，于阳性对照组、壳聚糖水凝胶组创面滴加重组人粒细胞巨噬细胞刺激因子凝胶、壳聚糖水凝胶溶液，于5个浓度复合水凝胶组创面滴加含相应质量浓度改良型富血小板纤维蛋白（A-PRF）的A-PRF/壳聚糖水凝胶溶液

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7 蛋白质印迹法检测的8组全层皮肤缺损糖尿病大鼠伤后14 d创面组织中CD31、VEGFA蛋白表达

注：1、2、3、4、5、6、7、8分别为不进行药物干预的空白组、滴加重组人粒细胞巨噬细胞刺激因子凝胶的阳性对照组、滴加壳聚糖水凝胶溶液的壳聚糖水凝胶组、滴加含相应质量浓度改良型富血小板纤维蛋白（A-PRF）的10、15、20、50、100 g/L A-PRF/壳聚糖水凝胶组；VEGFA为血管内皮生长因子A，GAPDH为3-磷酸甘油醛脱氢酶

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表1 8组全层皮肤缺损糖尿病大鼠伤后各时间点创面愈合率比较（%， x ¯ ± s ）

表1. Comparison of wound healing rates at each time point after injury in diabetic rats with full-thickness skin defects

组别	样本数	7 d	14 d
空白组	6	26.0±8.9	75.0±1.8
阳性对照组	6	45.8±3.2 ^a	79.1±1.9 ^a
壳聚糖水凝胶组	6	49.8±3.7 ^a	77.2±1.7 ^a
10 g/L 复合水凝胶组	6	51.2±2.9 ^a	82.3±1.3 ^a
15 g/L 复合水凝胶组	6	68.5±2.4 ^abcd	89.6±1.9 ^abcd
20 g/L复合水凝胶组	6	68.8±1.5 ^abcd	89.8±1.3 ^abcd
50 g/L 复合水凝胶组	6	72.7±2.1 ^abcd	87.3±1.1 ^abcd
100 g/L 复合水凝胶组	6	75.0±3.7 ^abcd	87.9±1.3 ^abcd
F值		82.63	66.78
P值		P<0.001	P<0.001
注：空白组创面不进行药物干预，于阳性对照组、壳聚糖水凝胶组创面滴加重组人粒细胞巨噬细胞刺激因子凝胶、壳聚糖水凝胶溶液，于5个复合水凝胶组创面滴加含相应质量浓度改良型富血小板纤维蛋白（A-PRF）的A-PRF/壳聚糖水凝胶溶液；与空白组比较， ^a P<0.05；与阳性对照组比较， ^b P<0.05；与壳聚糖水凝胶组比较， ^c P<0.05；与10 g/L 复合水凝胶组比较， ^d P<0.05

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表2 8组全层皮肤缺损糖尿病大鼠伤后14 d 创面组织中CD31、VEGFA阳性面积百分比比较（%， x ¯ ± s ）

表2. Comparison of positive area percentages of CD31 and VEGFA in tissue of 8 groups of wounds in diabetic rats with full-thickness skin defects on 14 d after injury

组别	样本量	CD31	VEGFA
空白组	6	26.1±1.6	25.2±2.0
阳性对照组	6	31.5±3.4 ^a	32.5±1.2 ^a
壳聚糖水凝胶组	6	28.9±1.9	30.7±1.7 ^a
10 g/L 复合水凝胶组	6	29.2±1.6	38.7±2.1 ^abc
15 g/L 复合水凝胶组	6	39.3±2.4 ^abcd	46.1±2.4 ^abcd
20 g/L复合水凝胶组	6	41.1±2.1 ^abcd	42.8±1.2 ^abcd
50 g/L 复合水凝胶组	6	40.3±1.6 ^abcd	45.3±1.3 ^abcd
100 g/L 复合水凝胶组	6	40.4±1.2 ^abcd	45.8±2.3 ^abcd
F值		57.57	108.80
P值		P<0.001	P<0.001
注：空白组创面不进行药物干预，于阳性对照组、壳聚糖水凝胶组创面滴加重组人粒细胞巨噬细胞刺激因子凝胶、壳聚糖水凝胶溶液，于5个复合水凝组胶创面滴加含相应质量浓度改良型富血小板纤维蛋白（A-PRF）的A-PRF/壳聚糖水凝胶溶液；VEGFA为血管内皮生长因子A；与空白组相比， ^a P<0.05；与阳性对照组相比， ^b P<0.05；与壳聚糖水凝胶组相比， ^c P<0.05；与10 g/L 复合水凝胶组相比， ^d P<0.05

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表3 8组全层皮肤缺损糖尿病大鼠伤后14 d创面组织中CD31、VEGFA蛋白表达比较（ x ¯ ± s ）

表3. Comparison of the protein expressions of CD31 and VEGFA in tissue of 8 groups of wounds in diabetic rats with full-thickness skin defects on 14 d after injury

组别	样本数	CD31	VEGFA
空白组	4	0.23±0.05	0.147±0.033
阳性对照组	4	0.53±0.07 ^a	0.379±0.053 ^a
壳聚糖水凝胶组	4	0.62±0.06 ^a	0.480±0.024 ^a
10 g/L 复合水凝胶组	4	0.71±0.05 ^a	0.555±0.031 ^ab
15 g/L 复合水凝胶组	4	0.96±0.05 ^abcd	0.800±0.061 ^abcd
20 g/L复合水凝胶组	4	1.06±0.04 ^abcd	0.852±0.040 ^abcd
50 g/L 复合水凝胶组	4	1.05±0.07 ^abcd	0.866±0.050 ^abcd
100 g/L 复合水凝胶组	4	1.06±0.07 ^abcd	0.830±0.065 ^abcd
F值		92.89	126.80
P值		P<0.001	P<0.001
注：空白组创面不进行药物干预，于阳性对照组、壳聚糖水凝胶组创面滴加重组人粒细胞巨噬细胞刺激因子凝胶、壳聚糖水凝胶溶液，于5个复合水凝胶组创面滴加含相应质量浓度改良型富血小板纤维蛋白（A-PRF）的A-PRF/壳聚糖水凝胶溶液；VEGFA为血管内皮生长因子A；与空白组相比， ^a P<0.05；与阳性对照组相比， ^b P<0.05；与壳聚糖水凝胶组相比， ^c P<0.05；与10 g/L 复合水凝胶组相比， ^d P<0.05

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表4 8组全层皮肤缺损糖尿病大鼠伤后14 d创面组织中CD31、VEGFA mRNA表达比较（ x ¯ ± s ）

表4. Comparison of the mRNA expressions of CD31 and VEGFA in tissue of 8 groups of wounds in diabetic rats with full-thickness skin defects on 14 d after injury

组别	样本数	CD31	VEGFA
空白组	4	0.99±0.05	0.993±0.023
阳性对照组	4	2.11±0.09 ^a	1.556±0.012 ^a
壳聚糖水凝胶组	4	1.63±0.05 ^ab	1.429±0.044 ^ab
10 g/L 复合水凝胶组	4	2.34±0.04 ^abc	1.904±0.035 ^abc
15 g/L 复合水凝胶组	4	2.93±0.04 ^abcd	2.446±0.062 ^abcd
20 g/L复合水凝胶组	4	2.93±0.05 ^abcd	2.393±0.048 ^abcd
50 g/L 复合水凝胶组	4	2.96±0.04 ^abcd	2.436±0.039 ^abcd
100 g/L 复合水凝胶组	4	3.01±0.03 ^abcd	2.437±0.017 ^abcd
F值		697.00	852.70
P值		P<0.001	P<0.001
注：空白组创面不进行药物干预，于阳性对照组、壳聚糖水凝胶组创面滴加重组人粒细胞巨噬细胞刺激因子凝胶、壳聚糖水凝胶溶液，于5个复合水凝胶组创面滴加含相应质量浓度改良型富血小板纤维蛋白（A-PRF）的A-PRF/壳聚糖水凝胶溶液；与空白组相比， ^a P<0.05；与阳性对照组相比， ^b P<0.05；与壳聚糖水凝胶组相比， ^c P<0.05；与10 g/L复合水凝胶组相比， ^d P<0.05

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