仿生型α-半水硫酸钙/磷酸八钙/透明质酸钠复合人工骨材料的细胞毒性实验
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摘要
目的:通过进行体外细胞毒性实验研究,探讨α-半水硫酸钙/磷酸八钙/透明质酸钠(α-CSH/OCP/SH)复合人工材料的体外细胞毒性作用情况。方法:提取并纯化兔骨髓间充质干细胞(BMSCs);制备α-半水硫酸钙/磷酸八钙/透明质酸钠复合人工骨材料的浸提液;分别以培养基、0. 64%苯酚溶液为阴性/阳性对照组,25%、50%、100%浓度的材料浸提液为实验组;分别于第1、3、5、7天观察BMSCs生长状况、检测各组的吸光度(A)、计算细胞相对增殖率(RGR),并对材料的细胞毒性进行分级。结果:各实验组及阴性对照组中兔BMSCs均生长良好,呈典型长梭形;MTT实验结果表明各实验组阳性对照组之间吸光度(A)值的差异均有统计学意义(P<0. 05),且在相应时间节点上25%浓度实验组与50%、100%浓度实验组之间的A值也具有明显统计学差异(P<0. 05)。结论:仿生型α-半水硫酸钙/磷酸八钙/透明质酸钠对兔BMSCs无细胞毒性,且低浓度的材料浸提液对兔BMSCs增殖有利。
Objective: To investigate cytotoxicity of the bionic composite artificial bone material of alpha-calcium sulfate hemihydrate/octacalcium phosphate/sodium hyaluronate(α-CSH/OCP/SH) on BMSCs in vitro.Methods: The BMSCs of rabbit were extracted and purified, the leaching solution of α-CSH/OCP/SH was prepared then; cells treated with normal culture media and 0. 64% phenol solution respectively were considered as negative control group and positive control group, respectively, and25%, 50% and 100% α-CSH/OCP/SH treated BMSCs were set as the experimental groups, respectively. The growth status of BMSCs was observed, the absorbance of each group was detected, the relative proliferation rate was calculated and the cytotoxicity was graded on the first day, third day,fifth day, and seventh day after cultivation.Results: BMSCs grew well in the experimental groups and negative control group, and there was no significant difference in cell morphology between these groups. The result of MTT test showed that the differences of absorbance between the all experimental groups and the positive control group were statistically significant(P<0. 05), and there was also a significant difference of absorbance between the experimental group of 25% concentration and the experimental group of 50% and 100% concentration at the corresponding time node(P<0. 05).Conclusion: The bionic composite artificial bone material of α-CSH/OCP/SH had no cytotoxicity to rabbit BMSCs, and the low concentration of material extract was beneficial to the proliferation of rabbit BMSCs. Alpha-CSH/OCP/SH is a potential material for bone tissue engineering.
Objective: To investigate cytotoxicity of the bionic composite artificial bone material of alpha-calcium sulfate hemihydrate/octacalcium phosphate/sodium hyaluronate(α-CSH/OCP/SH) on BMSCs in vitro.Methods: The BMSCs of rabbit were extracted and purified, the leaching solution of α-CSH/OCP/SH was prepared then; cells treated with normal culture media and 0. 64% phenol solution respectively were considered as negative control group and positive control group, respectively, and25%, 50% and 100% α-CSH/OCP/SH treated BMSCs were set as the experimental groups, respectively. The growth status of BMSCs was observed, the absorbance of each group was detected, the relative proliferation rate was calculated and the cytotoxicity was graded on the first day, third day,fifth day, and seventh day after cultivation.Results: BMSCs grew well in the experimental groups and negative control group, and there was no significant difference in cell morphology between these groups. The result of MTT test showed that the differences of absorbance between the all experimental groups and the positive control group were statistically significant(P<0. 05), and there was also a significant difference of absorbance between the experimental group of 25% concentration and the experimental group of 50% and 100% concentration at the corresponding time node(P<0. 05).Conclusion: The bionic composite artificial bone material of α-CSH/OCP/SH had no cytotoxicity to rabbit BMSCs, and the low concentration of material extract was beneficial to the proliferation of rabbit BMSCs. Alpha-CSH/OCP/SH is a potential material for bone tissue engineering.
引文
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[11] Suzuki O, Nakamura M, Miyasaka Y, et al. Maclura pomifera agglutinin-binding glycoconjugates on converted apatite from synthetic octacalcium phosphate implanted into subperiosteal region of mouse calvaria[J]. Bone Miner, 1993,20(2):151-166.
[12] Yamakoshi Y. Dentin sialophophoprotein(DSPP)and dentin[J]. Journal of Oral Biosciences, 2008, 50(1):33-44.
[13] Kikawa T, Kashimoto O, Imaizumi H, et al. Intramembranous bone tissue response to biodegradable octacalcium phosphate implant[J]. Acta Biomaterialia,2009,5(5):1 756-1 766.
[14] Santis RD, Gloria A, Russo T, et al. A basic approach toward the development of nanocomposite magnetic scaffolds for advanced bone tissue engineering[J].Journal of Applied Polymer Science, 2011,122(6):3 599-3 605.
[2]薛双丽,包崇云.骨诱导性双相磷酸钙陶瓷的研究与应用[J].中国组织工程研究,2013,17(47):8 235-8 241.Xue SL, Bao CY. Osteoinductive properties of biphasic calcium phosphates[J]. Chinese Journal of Tissue Engineering Research, 2013,17(47):8 235-8 241
[3] Liu Y, Lim J, Teoh SH. Review:Development of clinically relevant scaffolds for vascularised bone tissue engineering[J]. Biotechnol Adv, 2013,31(5):688-705.
[4]王小红.骨修复材料的研究进展[J].生物医学工程学杂志,2001,18(4):647-652.Wang XH, Ma JB, Wang YN, et al. Progress in the research of bone substitutes[J]. J Biomed Eng, 2001,18(4):647-652.
[5]刘晓阳,李广润,刘洪涛,等.硫酸钙人工骨/骨髓间充质干细胞构建组织工程化骨诱导脊柱融合[J].中国组织工程研究,2014,18(21):3 281-3 286.Liu XY, Li GR, Liu HT, et al. Construction of calcium sulfate/bone marrow mesenchymal stem cells tissueengineered bone for spinal fusion[J]. Journal of Clinical Rehabilitative Tissue Engineering Research, 2014, 18(21):3 281-3 286.
[6] Wang Z, Li M, Yu B, et al. Nanocalcium-deficient hydroxyapatite–poly(?-caprolactone)–polyethylene glycol–poly(?-caprolactone)composite scaffolds[J]. International Journal of Nanomedicine, 2012, 7:3 123-3 131.
[7] Hu G, Xiao L, Fu H, et al. Study on injectable and degradable cement of calcium sulphate and calcium phosphate for bone repair[J]. J Mater Sci Mater Med,2010,21(2):627-634.
[8] Pandit N, Sharma A, Jain A, et al. The use of nanocrystalline and two other forms of calcium sulfate in the treatment of infrabony defects:A clinical and radiographic study[J]. Journal of Indian Society of Periodontology, 2015,19(5):545-553.
[9] Hughes EAB, Grover LM. Characterisation of a novel poly(ether ether ketone)/calcium sulphate composite for bone augmentation[J]. Biomaterials Research,2017,21(1):7.
[10] Suzuki O. Octacalcium phosphate:osteoconductivity and crystal chemistry[J]. Acta Biomaterialia, 2010, 6(9):3 379-3 387.
[11] Suzuki O, Nakamura M, Miyasaka Y, et al. Maclura pomifera agglutinin-binding glycoconjugates on converted apatite from synthetic octacalcium phosphate implanted into subperiosteal region of mouse calvaria[J]. Bone Miner, 1993,20(2):151-166.
[12] Yamakoshi Y. Dentin sialophophoprotein(DSPP)and dentin[J]. Journal of Oral Biosciences, 2008, 50(1):33-44.
[13] Kikawa T, Kashimoto O, Imaizumi H, et al. Intramembranous bone tissue response to biodegradable octacalcium phosphate implant[J]. Acta Biomaterialia,2009,5(5):1 756-1 766.
[14] Santis RD, Gloria A, Russo T, et al. A basic approach toward the development of nanocomposite magnetic scaffolds for advanced bone tissue engineering[J].Journal of Applied Polymer Science, 2011,122(6):3 599-3 605.