Preventive effect of carboxymethyl chitosan solution on postoperative peritoneal adhesions in a rat model

doi:10.3969/j.issn.2095-4344.2014.08.017

Abstract

Abstract:

BACKGROUND: The patients are suffering from peritoneal adhesions that are caused after abdominal operation. As so far, there is still no effective drug or method that can completely prevent peritoneal adhesions. Carboxymethyl chitosan is a biocompatible and biodegradable biomedical material with anti-adhesion effects, which is an ideal biomaterial for prevention of peritoneal adhesion theoretically.
OBJECTIVE: To investigate the novel anti-adhesion properties of carboxymethyl chitosan anti-adhesion solution on the prevention of postsurgical adhesion in vivo in a rat model.
METHODS: Fifty-six adult male Wistar rats were randomly divided into four groups: 0.9% normal saline solution (group A), hyaluronic acid gels (group B), medical chitosan gels (group C) and carboxymethyl chitosan anti-adhesion solution (group D). The model of postoperative intestinal adhesion was established by making cecal scratches/abdominal wall defects. All the rats were scarified after 2 or 3 weeks. Whole blood was collected by cardio-puncture, lung tissue and tissue adhesion were stripped. The incidence and degree of adhesions, histological effects, expression of transforming growth factor-β1 (TGF-β1), the amounts of hydroxyproline and white blood cells were observed.
RESULTS AND CONCLUSION: The formation of postsurgical adhesions in groups B, C and D was significantly decreased, which was lighter than that of group A (P < 0.05). Furthermore, the adhesion formation in group D was significantly decreased in comparison with group A (P < 0.01). At the same time, the levels of transforming growth factor-β1, hydroxyproline and white blood cells in group D were lighter than those of group A (P < 0.05), and the histopathological results indicated that a marked reduction in inflammatory cells and fibroblasts. Carboxymethyl
chitosan anti-adhesion solution can effectively reduce the degree and incidence of postoperative adhesion, and it is becoming a promising drug delivery system in the context of postsurgical anti-adhesion.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: biocompatible materials, chitosan, hyaluronic acid, tissue adhesions

CLC Number:

R318

Cui Juan-juan, Feng Zhan-qin, Zhang Shou-qiang, Zheng Zeng-juan, Zhang Wei-fen. Preventive effect of carboxymethyl chitosan solution on postoperative peritoneal adhesions in a rat model[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(8): 1250-1256.

Figures/Tables 3

2.1 成功建立大鼠盲肠刮伤/腹壁缺损模型生理盐水组12只大鼠，于术后2周及3周大体观察皆有肠粘连，说明实验的动物肠粘连模型制作成功。大鼠盲肠刮伤/腹壁缺损模型及参考Nair制定的粘连程度5级分类法分级(图1)。 2.2 药效测定和粘连观察在第2周，医用透明质酸组和医用几丁糖组的大鼠分别于6 d和3 d后死亡1只，尸检探查表明死于严重的腹膜炎并有肠麻痹，粘连级别均定为Ⅳ级，剩余大鼠术后均存活，缺失大鼠均予以补足。术后24 h各组大鼠一般状态差，活动量少，进食与饮水较差；术后48 h各组大鼠饮水和进食已基本接近正常。腹部术后第7天各组大鼠伤口愈合好，无切口感染。术后14 d和21 d分别处死动物，生理盐水组大鼠皆有粘连。其余3组大鼠的肠粘连数量少，且程度也较生理盐水组轻，说明3种药物均有预防肠粘连作用。不同药物处理组在同一时间点和同一药物处理组不同时间点的大鼠腹膜粘连情况通过大体观察，得到如下结果：术后2周及3周，生理盐水组12只大鼠均有粘连，可见盲肠增厚、挛缩变形，与周围肠管及肠系膜之间形成的粘连带多且较粗，需要锐性分离。大鼠粘连肠壁组织进行相关指标检测，其中4只为I级腹腔粘连，2只为Ⅱ级腹腔粘连，2只为Ⅲ级腹腔粘连，4只为Ⅳ级腹腔粘连。羧甲基壳聚糖冲洗液组发生粘连的程度比较轻，盲肠偶见增厚，无挛缩变形，形成的粘连带细小，钝性即可分离，粘连点形成的面积小，16只大鼠中有12只未出现腹腔粘连，2只大鼠为I级腹腔粘连，仅2只为IV级腹腔粘连。医用透明质酸组和医用几丁糖组未形成腹腔粘连的大鼠分别为8只和13只，医用透明质酸组有1只发生了IV级腹腔粘连(提前死亡)，医用几丁糖组有2只发生了IV级腹腔粘连(1只提前死亡)，而且粘连点形成的面积小，盲肠偶见增厚。与生理盐水组比较，"

医用透明质酸组、医用几丁糖及羧甲基壳聚糖冲洗液组腹腔内粘连的发生率和粘连程度差异有显著性意义 (P < 0.05)，说明医用透明质酸、医用几丁糖与羧甲基壳聚糖冲洗液均能明显减轻术后腹腔粘连的范围和程度；而医用透明质酸组、医用几丁糖组与羧甲基壳聚糖冲洗液组组间症状评分两两比较，差异无显著性意义(P > 0.05)，表明羧甲基壳聚糖冲洗液能有效防止腹腔粘连。然而，2周时间点羧甲基壳聚糖冲洗液组与生理盐水组粘连水平比较差异有非常显著性意义(P < 0.01)，防粘连效果较医用透明质酸组、医用几丁糖组为优。不同时间点各组大鼠术后腹膜粘连分级评分结果见表1。于第3周解剖大鼠时发现羧甲基壳聚糖冲洗液组多数动物无腹腔粘连，切口愈合良好，表明羧甲基壳聚糖冲洗液在减轻粘连的同时并未影响切口及创面的愈合。 2.3 组织学观察各组病理观察结果(图2)：生理盐水组术后2周时纤维结缔组织明显增生、粘连，胶原纤维排列致密，有渗出及各型粒细胞和巨噬细胞较多；术后3周时炎性细胞浸润，主要为淋巴细胞，形成的炎性组织有渗出物，纤维组织及血管增生。医用透明质酸组术后2周时腹膜炎性细胞浸润明显；术后3周时腹壁增厚，有少量炎性细胞。医用几丁糖组术后2周时可见炎性细胞浸润，纤维结缔组织明显增生；术后3周时为炎性细胞浸润，且多数为淋巴细胞，无纤维组织增生。羧甲基壳聚糖冲洗液组术后2周时纤维结缔组织增生，形成肉芽，可见巨细胞；术后3周时有炎性细胞浸润，但程度较轻，纤维细胞较少。于术后2周时从各组选取肠壁组织进行转化生长因子β1的免疫组织化学染色测定，转化生长因子β1免疫组化染色阳性部位主要位于粘连组织内成纤维细胞，可见细胞质内出现棕黄色颗粒，未见炎症细胞染色。转化生长因子β1对成纤维细胞具有化学趋化作用，且促进细胞增殖、分化、血管形成，引起许多组织发生纤维化及在引起动物实验腹膜粘连的形成中起关键作用。图3显示生理盐水组可见有较多的棕黄色颗粒，其余3组染色强度较生理盐水组弱。因此，羧甲基壳聚糖冲洗液与医用透明质酸和医用几丁糖皆有降低转化生长因子β1表达的作用，从而达到减少腹膜粘连形成的程度和范围的作用。 2.4 血常规指标测定测定血液白细胞总数及分类，比较不同药物在预防粘连的同时对血液指标是否产生影响，验证羧甲基壳聚糖外科用手术防粘连冲液是否比其他药物具有较好的组织相容性。对不同时间点肠粘连大鼠的血液分析测定见表2。术后2周结果显示：①白细胞：医用透明质酸、羧甲基壳聚糖冲洗液组与生理盐水组比较差异有显著性意义(P < 0.05)，医用几丁糖组与生理盐水组比较差异无显著性意义(P > 0.05)，而医用几丁糖组与医用透明质酸组比较差异有显著性意义(P < 0.05)。②中性粒细胞和淋巴细胞：医用透明质酸组与生理盐水组差异有显著性意义(P < 0.05)。③血红蛋白、红细胞和血小板：医用透明质酸组、医用几丁糖组、羧甲基壳聚糖冲洗液组与生理盐水组比较差异无显著性意义(P > 0.05)。医用透明质酸组和羧甲基壳聚糖冲洗液组的白细胞明显低于生理盐水组，说明羧甲基壳聚糖冲洗液与透明质酸一样具有很好的抗炎作用；羧甲基壳聚糖冲洗液组血红蛋白、红细胞、中性粒细胞、淋巴细胞及血小板与生理盐水组比较差异均无显著性意义，"

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