壳聚糖-藻酸钙-锂皂石纳米片止血复合膜的制备及其对口腔内创面的治疗作用

doi:10.19439/j.sjos.2022.06.002

上海口腔医学 ›› 2022, Vol. 31 ›› Issue (6): 569-575.doi: 10.19439/j.sjos.2022.06.002

壳聚糖-藻酸钙-锂皂石纳米片止血复合膜的制备及其对口腔内创面的治疗作用

吴靖, 邹多宏, 张志愿

上海交通大学医学院附属第九人民医院口腔外科,上海交通大学口腔医学院,国家口腔医学中心,国家口腔疾病临床医学研究中心,上海市口腔医学重点实验室,口腔颌面再生医学创新单元,上海市口腔医学研究所,上海 200011

收稿日期:2021-01-23 修回日期:2021-09-01 发布日期:2022-12-29
通讯作者: 张志愿,E-mail: zhzhy@sjtu.edu.cn.
作者简介:吴靖（1994-）,男,硕士,E-mail: wujingsjtu2021@163.com
基金资助:
国家自然科学基金（31870969）; 中国医学科学院医学与健康科技创新工程项目（2019-I2M-5-037）; 上海交通大学医学院“双百人”人才计划（20191816）

The hemostasis effect and cell compatibility of a chitosan-calcium alginate-laponite nanosheet composite membrane: in vitro and in vivo evaluation

WU Jing, ZOU Duo-hong, ZHANG Zhi-yuan

Department of Oral Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology; Research Unit of Oral and Maxillofacial Regenerative Medicine, Chinese Academy of Medical Sciences; Shanghai Research Institute of Stomatology. Shanghai 200011, China

Received:2021-01-23 Revised:2021-09-01 Published:2022-12-29

摘要/Abstract

摘要： 目的: 利用自蒸发与冷冻干燥相结合技术,构建壳聚糖-藻酸钙-锂皂石纳米片止血复合膜 (止血复合膜),体外检测促凝血效果及细胞毒性,体内验证止血效果与黏膜黏附效果,探讨其作为新型口腔止血膜的可行性。方法: 分层构建双层结构止血复合膜,利用自蒸发法制备下层壳聚糖部分,结合冷冻干燥技术制备上层藻酸钙-锂皂石纳米片海绵部分。采用扫描电镜与透射电镜观察复合膜的微观结构,X射线衍射分析其成分。使用凝血板法分析止血复合膜组、医用纱布组、海壳生^®复合膜组以及空白对照组的体外凝血时间。培养NIH/3T3细胞,与止血复合膜浸提液（实验组）、壳聚糖-藻酸钙浸提液（阴性对照组）及细胞培养基（空白对照组）共培养24、48 h,以CCK-8法检测细胞增殖情况。构建比格犬口腔颊黏膜创面与下颌前磨牙拔牙创模型,体内验证凝血复合膜的止血性能及口腔黏膜黏附效果。采用SPSS 18.0软件包对数据进行统计学分析。结果: 止血复合膜呈双层结构,上层为包含锂皂石纳米片的海绵样结构,下层为均质壳聚糖膜,X射线衍射分析证实锂皂石纳米片的存在。体外凝血实验中,止血复合膜组的体外凝血时间显著低于医用纱布组、海壳生组^®及空白对照组 (P<0.001)。CCK-8检测显示,实验组、阴性对照组及空白对照组之间的细胞增殖差异无统计学意义 (P>0.05)。动物实验证实,止血复合膜具有良好的黏膜黏附性及止血效果。结论: 制备的止血复合膜具有较好的止血作用,无明显细胞毒性,具有作为口腔止血膜的潜能。

关键词: 壳聚糖, 藻酸钙, 锂皂石纳米片, 口腔止血膜, 凝血

Abstract: PURPOSE: To explore the feasibility of a chitosan-calcium alginate-laponite nanosheet composite membrane being used as a new hemostatic membrane for wounds in oral cavity. METHODS: The composite membrane was prepared in a layered fashion: the lower layer of chitosan membrane was made through self-evaporation and the upper layer of calcium alginate-laponite nanosheet sponge was made via freeze-drying. The microstructure of the composite membrane was observed under scanning electron microscopy (SEM) and transmission electron microscopy (TEM). X-ray diffraction was used as a means of identifying the compounds. Clotting time of composite membrane, medical gauze and Chitin dressing in vitro was measured by the plate method for blood coagulation. Cytotoxicity tests were quantified through co-culturing NIH/3T3 cells with chitosan-calcium alginate extract, composite hemostatic membrane extract and DMEM. Superficial buccal mucosal wound models and tooth extraction models were created on beagles dogs, the hemostatic effect and adhesion to oral mucosa were evaluated on these models. Statistical analysis was performed using SPSS 18.0 software package. RESULTS: The composite hemostatic membrane consisted of double-layer in microstructure, the upper layer was a foam layer consisting of calcium alginate and laponite nanosheet, the substratum was formed by uniform chitosan film. X-ray diffraction results showed that laponite nanosheet can be found in the composite membrane. Coagulation test showed that the composite hemostatic membrane group significantly shortened clotting time in vitro compared to pure calcium alginate group, commercial hemostatic membrane and blank control group(P<0.001). CCK-8 test of NIH/3T3 cell showed that there was no significant difference in absorbance between the experimental group, negative control group and blank control group (P>0.05). In addition, composite hemostatic membrane displayed a good hemostatic effect and strong adhesion to oral mucosa in animal models. CONCLUSIONS: The composite hemostatic membrane showed great hemostatic effects and had no significant cytotoxicity, which has the potential for clinical application as hemostatic membrane for wounds in oral cavity.

Key words: Chitosan, Calcium alginate, Laponite nanosheet, Hemostatic membrane for oral mucosal wounds, Coagulation

中图分类号:

R783.1

吴靖, 邹多宏, 张志愿. 壳聚糖-藻酸钙-锂皂石纳米片止血复合膜的制备及其对口腔内创面的治疗作用[J]. 上海口腔医学, 2022, 31(6): 569-575.

WU Jing, ZOU Duo-hong, ZHANG Zhi-yuan. The hemostasis effect and cell compatibility of a chitosan-calcium alginate-laponite nanosheet composite membrane: in vitro and in vivo evaluation[J]. Shanghai Journal of Stomatology, 2022, 31(6): 569-575.

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壳聚糖-藻酸钙-锂皂石纳米片止血复合膜的制备及其对口腔内创面的治疗作用

The hemostasis effect and cell compatibility of a chitosan-calcium alginate-laponite nanosheet composite membrane: in vitro and in vivo evaluation

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