生物3D打印丹酚酸B⁃海藻酸钠⁃明胶皮肤支架促进糖尿病大鼠创面愈合
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江苏省中医药科技发展计划面上项目(MS2021056);常州市应用基础研究项目(CJ20220258)


3D bioprinted salvianolic acid B ⁃ sodium alginate ⁃ gelatin skin scaffold promotes wound healing in diabetic rats
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    摘要:

    目的:探讨生物 3D 打印丹酚酸 B(salvianolic acid B,SAB)-海藻酸钠-明胶皮肤支架对糖尿病大鼠创面的治疗作用。方法:按照SAB占海藻酸钠与明胶总重的百分比制成含0%、0.5%、1.0%、1.5%SAB的生物墨水,采用生物3D打印技术制备不同规格的皮肤支架。扫描电镜观察微观结构,高效液相色谱测定SAB体外累积释放浓度。皮肤支架用于治疗2型糖尿病大鼠创面,分为空白对照组、凡士林纱布组以及皮肤支架组(0%SAB、0.5%SAB、1.0%SAB、1.5%SAB组)。于第7、14天观察创面愈合、渗出情况,同时取创面组织进行HE、Masson、活性氧(reactive oxygen species,ROS)染色,并检测超氧化物歧化酶(super- oxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-PX)、过氧化氢酶(catalase,CAT)及丙二醛(malondial- dehyde,MDA)水平。结果:扫描电镜下,4组皮肤支架均呈网孔状立体结构,孔隙分布均匀。SAB累积释放量随时间延长逐渐增加。各组创面均愈合良好,1.0%SAB 组创面修复优于其他各组。第 7 天时,1.0%SAB 组 ROS 水平均低于其他各组(P < 0.05);第14天时,1.0% SAB组ROS水平低于除1.5% SAB组外的其他各组(P < 0.01),1.0% SAB组和1.5% SAB组ROS表达水平差异无统计学意义(P=0.136)。第7天时,1.0%SAB组SOD、GSH-PX及CAT水平均高于其他各组(P < 0.05),MDA水平低于空白对照组、凡士林纱布组及0%SAB组(P < 0.05);第14天时,1.0%SAB组SOD、GSH-PX及CAT水平高于空白对照组、凡士林纱布组和0% SAB组(P < 0.05),MDA水平低于空白对照组、凡士林纱布组(P < 0.05)。HE染色显示,各组创面皮肤均修复较好,1.0%SAB组新生组织较多。Masson染色显示,第7、14天时,0.5%、1.0%、1.5%SAB组创面胶原蛋白沉积多于其他各组(P < 0.05)。结论:1.0%SAB-海藻酸钠-明胶皮肤支架在第7、14天时不仅可以抑制ROS的产生,并上调各种抗氧化酶的表达,抑制创面组织内氧化应激反应,还可以促进胶原沉积,最终促进糖尿病大鼠的创面愈合。

    Abstract:

    Objective:To investigate the therapeutic effect of 3D bioprinted salvianolic acid B(SAB)-sodium alginate-gelatin skin scaffold on diabetic rat wounds. Methods:The 0%,0.5%,1.0% and 1.5% of bioinks were prepared according to the percentage of SAB in the total weight of sodium alginate and gelatin. Skin scaffolds with different types were produced by biological 3D printing technology. The microstructure was observed by scanning electron microscopy,and the concentration of SAB in vitro was determined by high-performance liquid chromatography. Skin scaffolds were used to treat wounds in type 2 diabetic rats that were divided into the control group,the vaseline gauze group,and the skin scaffold groups(0%SAB,0.5%SAB,1.0%SAB,1.5%SAB groups). The healing and exudation of the wounds were observed on day 7 and 14. Meanwhile,the wound tissues wete taken for HE and Masson staining,as well as for reactive oxygen species(ROS)detection,and the levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-PX), catalase(CAT)and malondialdehyde(MDA)were detected. Results:Skin scaffolds in the four groups showed a mesh-like 3D structure with uniform pore distribution. The cumulative release of SAB increased gradually over time. General observations showed that wounds healed well in each group and healed better in 1.0% SAB group than in other groups. On day 7,ROS Levels were lower in the 1.0% SAB group than in other groups(P < 0.05). On day 14,ROS levels were lower in the 1.0% SAB group than in all groups except the 1.5% SAB group(P < 0.01),and there was no significant difference in ROS levels between the 1.0% SAB group and the 1.5% SAB group(P=0.136). On day 7,the levels of SOD,GSH-PX and CAT were higher in the 1.0%SAB group than in other groups (P < 0.05),MDA level was lower in the 1.0% SAB group than in the control group,vaseline gauze group and 0% SAB group(P < 0.05). On day 14,the levels of SOD,GSH-PX and CAT were higher in the 1.0%SAB group than in the control group,vasolin gauze group and the 0% SAB group(P < 0.05),MDA level was lower in the 1.0% SAB group than in the control group and vaseline gauze group(P < 0.05). HE staining showed that the wounds were well repaired in each group and more new tissues were formed in the 1.0% SAB group than in other groups. Masson staining showed that on day 7 and 14,collagen deposition in the 0.5%,1.0%,1.5% SAB groups was more than that in other groups(P < 0.05). Conclusion:The 1.0% SAB-sodium alginate-gelatin skin scaffold on the day 7 and 14 may not only inhibits the generation of ROS and upregulates the expression of various antioxidant enzymes,thereby inhibiting oxidative stress response in wound tissues,but also promotes collagen deposition,ultimately enhancing wound healing in diabetic rat.

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秦立昊,李静燕,张嘉伟,柏益飞,刘婷婷,唐蛰雨,薛同庆,贾中芝.生物3D打印丹酚酸B⁃海藻酸钠⁃明胶皮肤支架促进糖尿病大鼠创面愈合[J].南京医科大学学报(自然科学版),2024,(3):297-304,328

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  • 收稿日期:2023-10-10
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  • 在线发布日期: 2024-03-07
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