卵磷脂、多聚赖氨酸二次包埋新型支架材料的亲水性及对人鼻中隔软骨细胞的吸附力(英文)

背景: 甲壳胺是一种天然生物材料, 具有良好的生物相容性、可降解吸收性及优良的力学性能, 但在软骨组织工程中可否成为一种较理想的细胞支架?目的: 观察以卵磷脂和多聚赖氨酸共同包埋新型支架- 聚丙交酯- 乙交酯共聚物 / 甲壳胺无纺布, 对支架亲水性和对人鼻中隔软骨细胞吸附力的改变, 及对人鼻中隔软骨细胞功能的影响。设计: 空白对照实验。单位: 解放军南京军区南京总医院耳鼻咽喉- 头颈外科。材料: 实验于 2005- 10/2006- 06 于解放军南京军区总医院耳鼻咽喉-头颅外科实验室完成。甲壳胺无纺布( 海南欣龙公司) , 主要技术参数为: 脱乙酰度≥90%, 相对分子质量(2～5)×105; 聚丙交酯- 乙交酯共聚物 / 甲壳胺无纺布支架为南京军区南京总医院耳鼻咽喉- 头颈外科与中山大学高分子研究所合作自行制备, 其主要技术参数为: 单体丙交酯和乙交酯的摩尔比为 75∶25, 孔隙率 82%～86%, 孔径 100～300 μm, 剪切强度为 48 MPa, 厚度约 1.5 mm, 完全降解时间 14～18 周。人鼻中隔软骨细胞为无菌条件下取鼻中隔偏曲手术患者的鼻中隔软骨。方法: 将 PLGA/ 甲壳胺无纺布支架剪成 1.5 cm×1.5 cm 大小的片状, 体积分数为 0.01 的卵磷脂无水乙醇及 1 g/L 多聚赖氨酸中浸泡 6 h, 干燥后紫外线照射 1 h, 体积分数为 0.75 乙醇中浸泡 24 h, 大量 Hanks 液冲洗后孵育 24 h。得到含不同成分的 2 种类型支架: ①单纯支架; ②卵磷脂 多聚赖氨酸包埋的支架。取第 3 代的人鼻中隔软骨细胞制成细胞悬液, 将细胞悬液种于 2 种支架上, 通过观察人鼻中隔软骨细胞悬液在支架上扩散程度来判断其亲水性大小, 在相差显微镜下观察人鼻中隔软骨细胞悬液在支架上是否飘动来判断细胞与支架的吸附情况; 同时观察细胞生长繁殖及基质产生情况。主要观察指标: ①两种支架材料亲水性( 人鼻中隔软骨细胞悬液在支架上扩散程度) 、对细胞的吸附力(人鼻中隔软骨细胞悬液在支架上是否飘动)。②细胞生长繁殖及基质产生情况。结果: ①单纯支架加入人鼻中隔软骨细胞悬液后悬液先呈球型附在支架表面, 然后缓慢地扩散到支架空隙内。培养期间相差显微镜观察见细胞成团地附在支架纤维表面, 晃动培养皿可见细胞团漂浮不定[黏附率为(21±3.7)%], 随着培养时间延长无基质产生。②卵磷脂 多聚赖氨酸包埋的支架加入人鼻中隔软骨细胞悬液后悬液迅速均匀地扩散到支架空隙内[黏附率为(89±5.6)%, 高于单纯支架组], 表明支架有较强的亲水性; 相差显微镜下见软骨细胞以单个或群落状均匀分布于支架纤维之间及吸附在支架纤维表面, 晃动培养皿细胞团无飘动, 散落在培养皿底部的细胞较少, 表明支架对细胞有较强的吸附力; 培养 1 周, 支架纤维间有呈蜘蛛网状基质产生, 随着培养时间延长, 基质产生旺盛。结论: 以卵磷脂和多聚赖氨酸共同包埋的聚丙交酯- 乙交酯共聚物 / 甲壳胺无纺布的新型生物支架, 亲水性良好, 对人鼻中隔软骨细胞的吸附力明显增强, 并可促进软骨细胞分泌基质。

Hydrophilicity and adsorptivity of a novel scaffold twice embedded by lecithin and poly-L-lysine to human nasal septum chondrocytes

BACKGROUND:Chitosan is a kind of natural biomaterial and is characterized by great biocompatibility, progressive degeneration and absorption and excellent mechanical property; however, whether it may become an ideal cytoskeleton in the engineering of cartilage tissue or not should be researched further.OBJECTIVE: To observe the hydrophilicity and adsorptivity to human nasal septum chondrocytes and the effect of its function of a novel scaffold made by [poly (dl-lactide-co-glycolide)] (PLGA)/chitosan nonwoven cloth embedded with lecithin (LEC) and poly-L-lysine (PLYS).DESIGN: Blank control study.SETTING: Department of Otolaryngology-Head and Neck Surgery, Nanjing General Hospital of Nanjing Military Area Command of Chinese PLA.MATERIALS: The experiment was carried out in the Department of Otolaryngology-Head and Neck Surgery, Nanjing General Hospital of Nanjing Military Area Command of Chinese PLA from October 2005 to June 2006. Chitosan nonwoven cloth was provided by Hainan Xinlong Company. The mainly technical parameters were detailed as the follows: degree of deacetylation ≥ 90% and relative molecular weight 2-5 × 105. PLGA/chitosan nonwoven cloth scaffold was made in Department of Otolaryngology-Hean and Neck Surgery, Nanjing General Hospital of Nanjing Military Area Command of Chinese PLA and High Polymer Institute of Sun Yat-sen University. The mainly technical parameters were detailed as the follows: mole ratio between monome lactide and glycolide 75:25, porosity 82%-86%, pore diamater 100-300 μm, shear strength 48 MPa, depth 1.5 mm and completely degenerated duration 14-18 weeks. Human nasal septum chondrocytes were the septal cartilage of nose which was derived from operated patients with deflection of nasal septum under sterile condition.METHODS: PLGA/chitosan nonwoven cloth was sheared into pieces with the size of 1.5 cm × 1.5 cm, dipped in 0.01 volume fraction of LEC anhydrous alcohol and 1 g/L PLYS for 6 hours, dealt with ultraviolet radiation after dehydration for 1hour, dipped in 0.75 volume fraction of ethanol for 24 hours, washed with Hanks solution, and incubated for 24 hours. After operations mentioned above, two kinds of novel scaffolds containing various components were obtained, and they were simple scaffold and scaffold embedded with LEC and PLYS. Cells derived from the third generation of human nasal septum chondrocytes were used to make suspension. In addition, cell suspension was grown on those two scaffolds to determine the degree of hydrophilicity through observing diffused degree of cell suspension. Whether cell suspension of human nasal septum chondrocytes was wafted on scaffolds or not were observed under phase contrast microscope so as to determine adsorptivity between cells and scaffolds; meanwhile, growth of cells and production of matrix were also observed.MAIN OUTCOME MEASURES: ① Hydrophilicity of those two kinds of scaffolds (diffused degree of cell suspension of human nasal septum chondrocytes on scaffold) and adsorptivity to cells (whether cell suspension of human nasal septum chondrocytes was wafted on scaffolds or not); ② growth of cells and production of matrix.RESULTS: ① When simple scaffold was put in cell suspension of human nasal septum chondrocytes, cell suspension showing like balls attached to the surface of scaffold, and then, scattered into space of scaffold gradually. During the period of culture, phase contrast microscope indicated that masses of cells attached to the surface of fiber of scaffold.When the petri dish was shaken, cell groups drifted irregularly. The adherent rate was (21±3.7)%. With the cultured time passing by, matrix was not produced. ② When scaffold embedded with LEC and PLYS was put in cell suspension of human nasal septum chondrocytes, cell suspension scattered into space of scaffold rapidly. The adherent rate was (89±5.6)%, which was higher than that of single scaffold group. This suggested that scaffold showed a strong hydrophilicity.Phase contrast microscope indicated that chondrocytes as the form of monome or community were distributed between scaffold and fiber averagely and attached to the surface of fiber of scaffold. When the petri dish was shaken, cell groups did not drift irregularly, and only a few of cells were scarred at the bottom of petri dish. This suggested that scaffold had a strong adsorptivity to cells. One week after culture, matrix showing like cobweb was produced among fibers of scaffold.With the cultured time passing by, matrix was produced abundantly.CONCLUSION: The novel scaffold of PLGA/chitosan nonwoven cloth embedded with LEC and PLYS is characterized by an excellent hydrophilicity and adsorptivity to human nasal septum chondrocytes; meanwhile, it also can secrete matrix.