共聚比不同的两种聚乳酸-聚羟基乙酸共聚物材料生物相容性观测

背景近年来,α-羟基酸及其衍生物合成的脂肪族聚酯,如聚乳酸、聚羟基乙酸等已广泛用于周围神经组织工程的支架材料研究,这种支架有可能克服因自体神经移植缺乏及供区的永久性失神经支配以及供受区神经匹配等问题,提高神经诱导作用.目的对共聚比为(8515)及(5050)的聚乳酸-聚羟基乙酸共聚物材料的生物相容性及神经再生诱导作用进行对比观测.设计分组观察对比实验.单位解放军第三军医大学附属新桥医院骨科.材料清洁级健康成年Wistar大鼠66只,雌雄不拘,体质量180～200 kg;共聚比为(8515)及(5050)的聚乳酸-聚羟基乙酸共聚物.方法实验于2001-11/2002-12在第三军医大学大坪医院野战外科研究所6室,创伤、烧伤与复合伤国家重点实验室完成.①许旺细胞的培养及与聚乳酸-聚羟基乙酸共聚物膜的共培养观察将培养的许旺细胞接种于聚乳酸-聚羟基乙酸共聚物膜上,应用扫描电镜观察细胞在共聚物膜上的生长情况.②聚乳酸-聚羟基乙酸共聚物膜材料肌肉包埋组织学观察取Wistar大鼠15只,实验动物按随机数字法分以1,2,4,8和12周为时相点分为5组,每组3只.将共聚物材料(8515)裁剪成10 mm×5 mm×0.3 mm的膜,植入大鼠椎旁肌内,各时相点切取包含聚乳酸-聚羟基乙酸共聚物膜在内的周围组织进行苏木精-伊红染色.③聚乳酸-聚羟基乙酸共聚物管材料样品桥接大鼠坐骨神经缺损预实验取Wistar大鼠51只,分为共聚物8515管组、共聚物5050管组和硅胶管组,各组以2,4.6,8和12周为观察时相点,除12周为5只大鼠外,其余各时相点均为3只大鼠.于各时相点对各组材料进行大体观察及12周对大鼠进行再生神经运动传导速度测定,并取材桥接管中新生神经中段进行甲苯胺蓝染色,光镜观察.主要观察指标主要结局①大鼠肌肉包埋条件下聚乳酸-聚羟基乙酸共聚物组织学观察.②坐骨神经缺损桥接大鼠实验模型应用组织学及神经电生理学检测聚乳酸-聚羟基乙酸共聚物管神经诱导作用.次要结局许旺细胞和共聚物共培养条件下许旺细胞的生长行为观察.结果66只大鼠均进入结果分析.①大鼠肌肉包埋条件下聚乳酸-聚羟基乙酸共聚物组织学观察结果聚乳酸-聚羟基乙酸共聚物早期诱发以淋巴细胞及成纤维细胞为主的轻度的非特异性炎性反应,持续到10～12周时基本消退.②许旺细胞的培养及与聚乳酸-聚羟基乙酸共聚物膜的共培养观察结果许旺细胞在聚乳酸-聚羟基乙酸共聚物膜上能良好的生长并发生增殖.③大鼠体内神经桥接实验结果大体观察硅胶管促发有明显的纤维组织增生,而形成包囊包裹于管壁,而两种聚乳酸-聚羟基乙酸共聚物管组未见类似现象;12周再生神经运动传导速度聚乳酸-聚羟基乙酸共聚物8515管组与硅胶管组无显著差别(17.03±0.66),(17.15±0.76)m/s,P＞0.05];共聚物8515管组髓鞘厚度、神经纤维直径、轴突数及神经组织面积比与硅胶管组均无显著差别(0.45±0.16)μm,(3.96±1.73)μm,(10 135±1 053)个/mm2,(23.4±2.7)%比(0.45±0.19)μm,(4.07±1.86)μm,(9 879±1 491)个/mm2,(23.6±3.1)%,P＞0.05];而聚乳酸-聚羟基乙酸共聚物(5050)管体内神经桥接4周时即出现破裂,不能为坐骨神经桥接提供良好的支持作用.结论与硅胶管及聚乳酸-聚羟基乙酸共聚物(5050)相比,聚乳酸-聚羟基乙酸共聚物(8515)是一种异物反应小、生物相容性佳,生物降解速率合适的周围神经组织工程材料.

Biocompatibility of two binds of poly (DL-lactide-co-glycolide)conduits with different copolymer ratio of lactide

BACKGROUND: Recently, α-hydroxy-acid and aliphatic series polyester synthetized with its derivates such as polylactide and polyglycolide have been studied extensively for scaffold of peripheral nerve tissue engineering.These materials might improve the effect of nerve guidance through overcoming several shortcomings of autografts, including permanent denervation of donor site, tissue deficiency, and potential differences in tissue structure and size.OBJECTIVE: To investigate the biocompatibility of poly (DL-lactide-coglycolide) (PLGA). Copolymer ratio of lactide: glycolide was 85:15 or 50:50)and evaluate its effect on nerve guidance.DESIGN: A comparative study and observation.SETTING: Department of Orthopaedics, Xinqiao Hospital of Third Military Medical University of Chinese PLA.MATERIALS: A total of 66 Wistar rats of either sex and clean grade,weighting 180-200 g, were involved; the 85:15 or 50:50 copolymer of poly (lactide-co-glycolde) (PLGA).METHODS: The experiment was carried out in Field Surgery Institute No. 6; State Key Laboratory of Trauma, Burns and Compound Injury, Daping Hospital of Third Military Medical University, from November 2001 to December 2002. ① Co-culture of Schwann cells with PLGA film: Growth of Schwann cells co-cultured with PLGA film was observed under scanning electron microscope. ② Histological observation of PLGA film: Fifteen Wistar rats were randomly divided into five groups (according to 1, 2, 4, 8and 12 weeks time point postoperatively) with 3 rats in each group. PLGA was trimmed to pieces of film with the size of 10.0 mm × 5.0 mm × 0.3 mmand implanted into the rat dorsal muscles under aseptic condition. Hematoxylin/eosin staining was used to evaluate inflammatory reaction. ③Bridging of sciatic nerve defect in rats with PLGA conduits: Fifty-one Wistar rats were divided into PLGA (85:15) conduit group, PLGA (50:50) conduit group and silicone conduit group, each of which was further divided into 5 subgroups according to different time points (2, 4, 6, 8, and 12weeks postoperatively) with 3 rats in each subgroup, except for 12-week time point subgroup which used 5 rats. Gross observation and electrophysiological studies (only 12 weeks time point postoperatively) were performed.The midconduit regenerated nerves were removed and then stained with toluidine blue. Histological observation of regenerated nerves was performed under the optical microscope.MAIN OUTCOME MEASURES: Primary outcome: ① Histological observation of PLGA film implanted in rat muscles. ② Electrophysiological and morphological evaluation of nerve guidance effect of PLGA conduits.Secondary outcome: Growth behavior of Schwann cells when co-cultured with PLGA films.RESULTS: All the 66 rats entered the result analysis. ① Histological observation of PLGA film implanted in rat muscles: The PLGA implants caused only minimal non-specific inflammatory reaction at an early time,as characterized by a low concentration of lymphocytes and fibroblasts,which was not present 10-12 weeks postoperatively. ② Cell attachment and morphological observation by scanning election microscopy: Schwann cells grew and proliferated well when co-cultured with PLGA film. ③Bridging of sciatic nerve defect in rats with PLGA conduits: Gross observation: Silicone induced significant proliferation of fibre tissues, characterized by encapsulation, when bridging sciatic nerve defects in rats, but the similar response could not be seen in PLGA in the same situation. At week 12 after bridging sciatic nerve defects in rats, motor nerve conduction velocity in PLGA (85:15) group was 17.03±0.66 m/s; it was 17.15±0.76 m/sin silicone group, with no significant difference between the two groups (P ＞ 0.05). Quantitative evaluation of regenerated nerves in PLGA group showed that the thickness of myelin sheath was (0.45±0.16) μm, fiber width was (3.96±1.73) μm, axon/mm2 10 135±1 053, and area percentage of neural tissues (%) was 23.4±2.7; in silicone group the corresponding data were (0.45±0.19) μm, (4.07±1.86) μm, (9879±1491) mm2 and (23.6±3.1)%, respectively, and there was no significant difference between the two groups (P ＞ 0.05). Since PLGA (50:50) conduit collapsed at an early time in the same animal model, corresponding indexes couldnot be detected in PLGA (50:50) group.CONCLUSION: In contrast to PLGA (50:50) and silicone, PLGA (85:15)is a good material for peripheral nerve tissue engineering because of its good biocompatibility and proper biodegradation rate.