中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (26): 4206-4211.doi: 10.3969/j.issn.2095-4344.1361

• 材料生物相容性 material biocompatibility • 上一篇    下一篇

改性化学交联脱细胞真皮基质材料的生物相容性

李丽花1，熊  健2，曹  苹3，钟梅玲1，朱勇军1，徐炜区3，康文亭1，佘振定1，谭荣伟1
  

  1. 1深圳兰度生物材料有限公司，广东省深圳市  518107；2荆州中心医院骨科三病区，湖北省荆州市  434000；3深圳市医疗器械检测中心，广东省深圳市  518057
  • 收稿日期:2019-03-15
  • 通讯作者: 李丽花，深圳兰度生物材料有限公司，广东省深圳市 518107
  • 作者简介:李丽花，女，1986年生，海南省海口市人，汉族，2010年武汉大学化学与分子科学学院毕业，硕士，工程师，就职于深圳兰度生物材料有限公司，主要从事生物医用材料产业化研究。

Biocompatibility of modified chemically cross-linked acellular dermal matrix

Li Lihua1, Xiong Jian2, Cao Ping3, Zhong Meiling1, Zhu Yongjun1, Xu Weiqu3, Kang Wenting1, She Zhending1, Tan Rongwei1
  

  1. 1Shenzhen Lando Biomaterials Co., Ltd, Shenzhen 518107, Guangdong Province, China; 2Third Ward of Orthopedics, Jingzhou Central Hospital, Jingzhou 434000, Hubei Province, China; 3Shenzhen Medical Device Testing Center, Shenzhen 518057, Guangdong Province, China
  • Received:2019-03-15
  • Contact: Li Lihua, Shenzhen Lando Biomaterials Co., Ltd, Shenzhen 518107, Guangdong Province, China
  • About author:Li Lihua, Master, Engineer, Shenzhen Lando Biomaterials Co., Ltd, Shenzhen 518107, Guangdong Province, China

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摘要：

文章快速阅读：

 

文题释义：
脱细胞真皮基质：是去除了真皮中具有抗原性的细胞成分，而保留了原有胶原纤维结构的真皮支架。脱细胞真皮基质的免疫活性较低，不会诱发宿主组织的排斥反应，能够为组织细胞提供一个良好的支架结构。
戊二醛：是一种简单高效且应用广泛的交联剂，具有活性高、反应快等优点，但其本身具有较大的细胞毒性。
甘氨酸：是人体内一种重要的非必需氨基酸，对人体无毒副作用，甘氨酸的氨基能与戊二醛的醛基反应生成席夫碱，是有效的戊二醛中和剂。
 
 
背景：脱细胞真皮基质存在降解速率较快且不可调控、力学性能不佳等天然材料固有的缺点，对其进行戊二醛交联改性是常用到的改善措施，然而戊二醛本身具有较大的细胞毒性，会影响脱细胞真皮基质的生物相容性。
目的：利用甘氨酸中和封闭戊二醛分子中未参与反应的醛基，改善脱细胞真皮基质材料的生物相容性。
方法：对脱细胞猪真皮基质依次进行戊二醛改性、甘氨酸中和处理，作为实验组，以单纯戊二醛改性的脱细胞真皮基质为对照，利用DNA试剂盒检测实验组样品的DNA残留量。将未交联脱细胞猪真皮基质与实验组、对照组样品浸泡于胶原酶溶液中，观察材料降解情况。分别以细胞培养基、高密度聚乙烯材料浸提液、实验组样品浸提液与对照组样品浸提液培养小鼠成纤维细胞，培养24 h后，采用MTT法检测细胞增殖率。将小鼠成骨细胞分别接种到实验组与对照组样品膜表面，培养7 d后，共聚焦显微镜下观察细胞状态。将实验组与对照组样品膜片分别植入新西兰兔(深圳市医疗器械检测中心提供)皮下，2周后取试样及其周围组织，进行苏木精-伊红染色观察。动物实验经深圳市医疗器械检测中心伦理委员会审批。
结果与结论：①实验组样品DNA 残留量为(3.12±0.7) µg/g；②酶解8 h，实验组与对照组样品的失重率无显著差异，为18%-21%，未进行戊二醛交联脱细胞猪真皮基质的失重率为100%；③实验组与对照组样品浸提液中小鼠成纤维细胞的增殖率分别为98.1%，90.3%，细胞毒性均为1级；④实验组膜片表面的小鼠成骨细胞繁殖旺盛，分布较为均匀，细胞骨架铺展较为充分；对照组膜片表面的成骨细胞数量较少且细胞团聚在一起，细胞骨架未充分铺展开；⑤植入皮下2周后，两组膜片的胶原纤维结构均基本完整且清晰可见，实验组植入部位周围组织炎症反应轻微，对照组炎症反应较为严重；⑥结果表明，甘氨酸封端可在保证降解性能的前提下，提高戊二醛交联改性脱细胞真皮基质材料的生物相容性。

关键词: 引导骨组织再生, 屏障膜, 脱细胞真皮基质, 交联, 戊二醛, 甘氨酸, 中和, 降解性能, 生物相容性

Abstract:

BACKGROUND: Acellular dermal matrix has the disadvantages of natural materials such as rapid and unregulated degradation and poor mechanical properties. It is a commonly used improvement measure for glutaraldehyde cross-linking modification. However, glutaraldehyde itself has a high cytotoxicity, which affects the biocompatibility of acellular dermal matrix.
OBJECTIVE: To improve the biocompatibility of the glutaraldehyde cross-linked acellular dermal matrix by using glycine to neutralize remaining aldehyde groups.
METHODS: As an experimental group, the acellular porcine dermal matrix was cross-linked by glutaraldehyde and then neutralized by glycine. The control group was that acellular dermal matrix which was cross-linked by glutaraldehyde but not neutralized by glycine. The residual exogenous DNA of materials of the experimental group was detected by DNA kit. The uncrosslinked acellular porcine dermal matrix and the samples of the experimental group and control group were immersed in collagenase solution to observe degradation performance. Mouse fibroblasts were cultured in culture medium, high-density polyethylene extracts, sample extracts of the experimental group and control group, respectively. After 24 hours of culture, cell proliferation rate was determined by MTT assay. Mouse osteoblasts were seeded onto the membrane surface of the experimental group and the control group and cultured for 7 days. The cell status was observed under confocal microscope. The membrane materials of the experimental group and the control group were respectively implanted beneath the skin of New Zealand rabbits, and the samples with surrounding tissues were removed at 2 weeks later for hematoxylin-eosin staining observation.
RESULTS AND CONCLUSION: (1) The amount of DNA residues in samples of the experimental group was (3.12±0.7) µg/g. (2) After enzymolysis for 8 hours, there was no significant difference in the weight loss rate of samples between the experimental group and the control group (18%-21%), while the weight loss rate of samples without glutaraldehyde cross-linking was 100%. (3) After culture for 24 hours, the proliferation rates of fibroblasts cultured in the leaching liquor of samples in the experimental and the control groups were 98.1% and 91.3%, respectively. The results showed the level of cytotoxicity was both grade 1. (4) Osteoblasts spread evenly and multiplied vigorously on the membrane surface in the experimental group, while curled up and were few in number in the control group. (5) After membranes implanted beneath the skin of the rabbit for 2 weeks, tissues around the implant site of the experimental group showed a slight inflammatory response, while those in the control group showed severe inflammatory response. (6) These results suggest that glycine neutralizing remaining aldehyde groups could improve the biocompatibility of glutaraldehyde cross-linked acellular dermal matrix material on the premise of guaranteeing the degradation performance.

Key words: guided bone tissue regeneration, barrier membrane, acellular dermal matrix, cross-linking, glutaraldehyde, glycine, neutralizing, degradation performance, biocompatibility

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