中国组织工程研究

中国组织工程研究 ›› 2016, Vol. 20 ›› Issue (52): 7765-7772.doi: 10.3969/j.issn.2095-4344.2016.52.002

• 组织工程骨及软骨材料 tissue-engineered bone and cartilage materials • 上一篇    下一篇

复合N-乙酰半胱氨酸丝素蛋白磷酸钙骨水泥的理化性能及细胞毒性

冯  焘，皮  斌，姜  磊，李  斌，陆英杰，杨惠林，朱雪松
  

  1. 苏州大学附属第一医院骨科，江苏省苏州市  215006
  • 收稿日期:2016-09-26 出版日期:2016-12-16 发布日期:2016-12-16
  • 通讯作者: 朱雪松，苏州大学附属第一医院骨科，江苏省苏州市 215006
  • 作者简介:冯焘，男，1989年生，江苏省苏州市人，汉族，2016年苏州大学毕业，硕士，医师，主要从事脊柱外科基础和临床研究。 并列第一作者：皮斌，男，1985年生，湖南省常德市人，汉族，2012年中南大学毕业，博士，医师，主要从事脊柱外科生物材料研究。 通讯作者：朱雪松，苏州大学附属第一医院骨科，江苏省苏州市 215006
  • 基金资助:

    国家863计划(2015AA020316)；国家自然科学基金资助项目(81171689、81301559)；江苏省自然科学基金资助项目(SBK201341421)

Physiochemical properties and cytotoxicity of silk fibroin based calcium phosphate cement incorporated with N-acetylcysteine

Feng Tao, Pi Bin, Jiang Lei, Li Bin, Lu Ying-jie, Yang Hui-lin, Zhu Xue-song
  

  1. Department of Orthopaedics, the First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China
  • Received:2016-09-26 Online:2016-12-16 Published:2016-12-16
  • Contact: Zhu Xue-song, Department of Orthopedics, the First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China
  • About author:Feng Tao, Master, Physician, Department of Orthopedics, the First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China Pi Bin, M.D., Physician, Department of Orthopedics, the First Affiliated Hospital of Soochow University, Suzhou 215006, Jiangsu Province, China Feng Tao and Pi Bin contributed equally to this work.
  • Supported by:

    the National 863 Program of China, No. 2015AA020316; the National Natural Science Foundation of China, No. 81171689, 81301559; the Natural Science Foundation of Jiangsu Province, No. SBK201341421

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文题释义：
磷酸钙骨水泥：相对于聚甲基丙烯酸甲酯具有优势，因其具有一定的骨传导性，无细胞毒性，并且最终水化产物为骨的主要无机成分羟基磷灰石，因此可用于年轻患者骨缺损的修复。此外，磷酸钙骨水泥体系可作为缓释系统，负载多种药物，如抗生素、抗肿瘤药物及成骨活性药物(如骨形态发生蛋白2等)，然而磷酸钙骨水泥的生物力学性能欠佳，使得磷酸钙骨水泥的临床应用限制于非负重部位骨缺损的填充。
丝素蛋白：作为天然高分子纤维蛋白，已被证实具有良好生物相容性，并且能够促进间充质干细胞增殖及向成骨细胞分化的能力。前期研究同样表明，丝素蛋白能够通过分子内羟基、羰基等负电荷基团与Ca 2+的静电作用，为羟基磷灰石的规律沉积提供模板，提高磷酸三钙的力学强度。

背景：磷酸钙骨水泥具有骨传导性、可降解等显著优点，其水化产物羟基磷灰石为骨骼主要无机成分，然而其力学性能不足限制了在负重区域骨缺损修复的应用。
目的：研制具有良好生物力学性能及生物相容性的磷酸钙复合骨水泥。
方法：①配置含有不同浓度(0，10，25 mmol/L)N-乙酰半胱氨酸的6%丝素蛋白溶液，作为固化液；以α-磷酸三钙粉末为固体，按液固比0.4 mL：1 g制备含有不同浓度N-乙酰半胱氨酸的丝素蛋白磷酸钙骨水泥，同时设置α-磷酸三钙粉末与ddH2O混合为对照组。测定复合骨水泥的抗压强度、凝固时间，通过X射线衍射仪检测骨水泥晶体成分，扫描电镜观察骨水泥微观形貌；②将MC3T3-E1细胞接种于各组材料表面，24 h后扫描电镜观察细胞形态；③以各组材料浸提液培养MC3T3-E1细胞，1，3，5，7 d后检测细胞增殖，1，3 d后检测细胞培养基中乳酸脱氢酶含量。
结果与结论：①X射线衍射和扫描电镜检测结果表明，各组材料中α-磷酸三钙的水化反应产物均为羟基磷灰石；②当N-乙酰半胱氨酸浓度为25 mmol/L时，材料的抗压强度最大，达(49.39±1.68) MPa，材料的初凝时间为(21.77±1.07) min，终凝时间为(31.88±1.69) min；③细胞在含有不同浓度N-乙酰半胱氨酸的丝素蛋白磷酸钙骨水泥表面的形态无明显差异；④含有不同浓度N-乙酰半胱氨酸的丝素蛋白磷酸钙骨水泥可促进细胞增殖，抑制乳酸脱氢酶分泌；⑤结果表明，N-乙酰半胱氨酸可显著提高丝素蛋白磷酸钙骨水泥的力学强度，具有良好的细胞相容性。

关键词: 生物材料, 骨生物材料, 磷酸钙骨水泥, 丝素蛋白, N-乙酰半胱氨酸, 抗压强度, 细胞毒性, 国家自然科学基金

Abstract:

BACKGROUND: Calcium phosphate cements (CPCs) possess the bio-degradation and osteoconduction, and its final hydration product, hydroxyapatite, is the main inorganic constituent of bones. However, its poor mechanical property makes it unable to be used for repairing weight-bearing bone defects.
OBJECTIVE: To develop a kind of bioactive bone cements with decent biomechanical property and biocompatibility.
METHODS: 6% silk fibroin aqueous solutions containing different concentrations of N-acetylcysteine (0, 10 and 25 mmol/L) were prepared. Each cement sample was prepared by mixing the curing liquid and α-tricalcium phosphate powder with the ratio of 0.4 mL: 1 g; α-tricalcium phosphate powder mixed with ddH2O as control group. The compressive strength, setting time of the cements were measured. The crystal components of the cements were characterized using X-ray diffraction and the microstructure was observed using scanning electron microscope. MC3T3-E1 cells were seeded onto the material in each group, and cell morphology was observed under scanning electron microscope at 24 hours. MC3T3-E1 cells were cultured in the extract of each material, cell proliferation was detected at 1, 3, 5 and 7 days, and the lactate dehydrogenase level was detected at 1 and 3 days.
RESULTS AND CONCLUSION: X-ray diffraction and scanning electron microscope showed that the final hydration products of α-tricalcium phosphate in all specimens were hydroxyapatite. When the concentration of N-acetylcysteine was 25 mmol/L, the compressive strength of the material reached (49.39±1.68) MPa, with the initial setting time of (21.77±1.07) minutes and the final setting time of (31.88±1.69) minutes. There was no significant difference in cell morphology among cements. These results suggest that the cement containing N-acetylcysteine exhibites good biocompatibility and high mechanical strength.

Key words: Calcium Phosphates, Silk, Cysteamine, Tissue Engineering

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