Biocompatibility of 3D printed three-dimensional porous marine shell/cervus and cucumis polypeptide composite scaffold material

doi:10.3969/j.issn.2095-4344.1936

Abstract

Abstract:

BACKGROUND: The development of artificial bone has become a hot spot in bone tissue engineering. Clinical practice has proved that single component bone materials can not meet the clinical needs very well. Therefore, the development and application of composite scaffold materials have been paid more concern.

OBJECTIVE: To prepare three-dimensional porous marine shell/cervus and cucumis polypeptide composite scaffold material by 3D printing technology and investigate its biocompatibility.

METHODS: The porous marine shell/ cervus and cucumis polypeptide composite scaffold material was prepared by 3D printing technology. Its composition, microstructure and mechanical strength of the material were investigated. The toxicity of porous marine shell/lugua polypeptide bioscaffold materials to osteoblasts was detected by inverted microscopy and CCK-8 assay. The growth and adhesion of osteoblasts on the porous marine shell/ cervus and cucumis polypeptide composite scaffold material was determined by scanning electron microscopy. The biocompatibility of porous marine shell/cervus and cucumis polypeptide composite scaffold material was determined by acute toxicity test, muscle implantation test and bone defect implantation experiment. The study protocol was approved by the Ethics Committee of the Second Military Medical University, China.

RESULTS AND CONCLUSION: The porous marine shell/cervus and cucumis polypeptide composite scaffold material was mainly composed of calcium carbonate and biological polypeptide, with 10 MP or more compressive strength, more than 85%porosity, and 50-100 μm pore diameter. Osteoblasts grew well in the porous marine shell/cervus and cucumis polypeptide composite scaffold material, and cell viability was strong. The cytotoxicity of the scaffold material was grade 1. Osteoblasts could adhere and proliferate on the surface of the scaffold material. The scaffold material could be degraded in vivo, did not cause systemic toxicity in animals, had no muscle stimulation reaction, and could promote the repair of bone defects. These results suggest that porous marine shell/cervus and cucumis polypeptide composite scaffold material has excellent mechanical properties, three-dimensional spatial structure, good cytocompatibility and histocompatibility.

Key words: 3D printing, marine shell, cervus and cucumis polypeptide, bone repair material, biocompatibility, bone defect, osteoblast, osteoinduction

CLC Number:

R459.9','1');return false;" target="_blank">
R459.9

R-331

R318.08

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Lou Yi, Zhang Zhiwen. Biocompatibility of 3D printed three-dimensional porous marine shell/cervus and cucumis polypeptide composite scaffold material[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(34): 5497-5502.

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Figures/Tables 9

2.1 材料生物力学强度测试结果  如图1所见，初期当骨材料受纵向压力小于1 MPa时，材料的形变不明显，但随压力递增，材料的形态变化逐渐加快，当片剂压缩至原高度25%时，纵向压力已超过10 MPa。"

2.2 材料物相检测结果  多孔海洋贝壳/鹿瓜多肽复合材料的X射线衍射波形可知，碳酸钙波峰明显且规则，说明其为晶体结构；鹿瓜多肽波峰杂乱，非晶体结构，见图2；从图中3种材料波形对比，证明人工骨材料由碳酸钙、鹿瓜多肽两组物质组成，且鹿瓜多肽不能改变碳酸钙的晶体结构。"

2.3 材料扫描电镜观察结果  材料的微观结构如图3所示, 为三维多孔网状结构，表面粗糙、多孔，各孔之间相互贯穿，形状规则，大小一致，孔隙大小为50-100 µm，孔隙率超过85%，完全满足细胞贴壁、迁移、增殖及新陈代谢的空间需求[11-12]。"

2.4 材料浸提液中的细胞生长情况显微镜下观察发现成骨细胞在浸提液中生长良好，细胞活性强，虽见少量细胞凋亡，但大部分细胞贴壁生长，形态结构完好，细胞核饱满，细胞呈多角形，见图4A；3 d后细胞已成簇分布于培养皿底部，增殖旺盛，见图4B；5 d后细胞已融合成片，铺满培养皿底部，平行排列或呈漩涡状，见图4C。"

2.5 CCK-8细胞毒性实验结果  培养的第2，4，6，8天，实验组和对照组吸光度值均随着时间的推移而升高，且两组吸光度值比较差异无显著性意义(P > 0.05)，见表2，表明两组细胞在各自培养基中均能良好生长、增殖，且生物毒性趋于一致。"

2.6 材料表面的细胞黏附观察  扫描电镜观察显示种植在复合材料片剂上的成骨细胞，24 h后即散在分布并贴附于材料表面，细胞形态饱满，呈多角形，伸出伪足牢牢吸附于材料表面；随时间增长，细胞在材料的三维空间中不断增殖分化，数量明显增多，见图5。"

2.7 毒性实验结果注射复合材料混悬液后1个月，实验动物生命体征平稳，未出现体质量减轻、食欲不振、活动度下降等情况，血常规检测见表3，生化指标检测见表4，实验组大鼠指标均在正常范围，与对照组大鼠对比无显著差异(P > 0.05)。将实验组大鼠处死后解剖其腹部，未见水肿、腹水及腹部脏器粘连；将心肝脾肺肾做病理切片，未见组织细胞充血、变性、凋亡等现象，组织内未见材料沉积，未见炎性细胞聚集。"

2.8 肌肉植入实验结果术后1个月，实验动物生命体征平稳，无波动，材料植入部位可见陈旧手术瘢痕，愈合良好，伤口处干洁，无红肿，局部皮温无升高，病理切片见图6，可见材料存留于肌肉组织中，部分降解，周围肌肉组织生长良好，结构正常，未见组织坏死及溶解，未见炎性细胞在材料与肌肉组织聚集。"

2.9 骨植入实验结果  所有兔手术伤口干洁，愈合良好，患肢主被动活动良好。术后6周，材料周围组织未见大量炎性细胞聚集，人工骨材料部分降解，材料与骨组织交界处骨缺损区有大量骨盐沉积，并见髓腔区域粗大的骨小梁散在分布，但骨缺损仍明显，成骨细胞活动活跃，见图7A；术后12周，骨材料完全降解，大量新生骨组织填充原骨缺损处，人工骨材料被新骨完全替代，见图7B。"

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[1]	Lu Dezhi, Mei Zhao, Li Xianglei, Wang Caiping, Sun Xin, Wang Xiaowen, Wang Jinwu. Digital design and effect evaluation of three-dimensional printing scoliosis orthosis [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(9): 1329-1334.
[2]	Zhang Tongtong, Wang Zhonghua, Wen Jie, Song Yuxin, Liu Lin. Application of three-dimensional printing model in surgical resection and reconstruction of cervical tumor [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(9): 1335-1339.
[3]	Liu Zhengpeng, Wang Yahui, Zhang Yilong, Ming Ying, Sun Zhijie, Sun He. Application of 3D printed interbody fusion cage for cervical spondylosis of spinal cord type: half-year follow-up of recovery of cervical curvature and intervertebral height [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(6): 849-853.
[4]	Hua Haotian, Zhao Wenyu, Zhang Lei, Bai Wenbo, Wang Xinwei. Meta-analysis of clinical efficacy and safety of antibiotic artificial bone in the treatment of chronic osteomyelitis [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(6): 970-976.
[5]	Zhang Bin, Sun Lihua, Zhang Junhua, Liu Yusan, Cui Caiyun. A modified flap immediate implant is beneficial to soft tissue reconstruction in maxillary aesthetic area [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(5): 707-712.
[6]	Xu Junma, Yu Yuechao, Liu Zhi, Liu Yu, Wang Feitong. Application of 3D-printed coplanar template combined with fixed needle technique in percutaneous accurate biopsy of small pulmonary nodules [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(5): 761-764.
[7]	Li Chenjie, Lü Linwei, Song Yang, Liu Jingna, Zhang Chunqiu. Measurement and statistical analysis of trabecular morphological parameters of titanium alloy peri-prosthesis under preload [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(4): 516-520.
[8]	Wu Zijian, Hu Zhaoduan, Xie Youqiong, Wang Feng, Li Jia, Li Bocun, Cai Guowei, Peng Rui. Three-dimensional printing technology and bone tissue engineering research: literature metrology and visual analysis of research hotspots [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(4): 564-569.
[9]	Li Xiaozhuang, Duan Hao, Wang Weizhou, Tang Zhihong, Wang Yanghao, He Fei. Application of bone tissue engineering materials in the treatment of bone defect diseases in vivo [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(4): 626-631.
[10]	He Jie, Chang Qi. Biological reconstruction of large bone defects after resection of malignant tumor of extremities [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(3): 420-425.
[11]	Xing Hao, Zhang Yonghong, Wang Dong. Advantages and disadvantages of repairing large-segment bone defect [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(3): 426-430.
[12]	Huang Youyi, Yuan Wei. Application of 3D printing technology in fracture and deformity of foot and ankle [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(3): 438-442.
[13]	Wei Congcong, Yao Mengxuan, Yang Meng, Li Huijie. Mechanism and treatment of osteolysis around artificial joint prosthesis [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(27): 4401-4407.
[14]	Zheng Feng, Zhang Fucai, Xu Zhe. MicroRNA-98-5p promotes osteoblast proliferation and differentiation: possibilities and mechanisms [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(26): 4112-4117.
[15]	Wang Qiufei, Gu Ye, Peng Yuqin, Xue Feng, Ju Rong, Zhu Feng, Wang Yijun, Geng Dechun, Xu Yaozeng. Effect of Wnt/beta-catenin signaling pathway on osteoblasts under the action of wear particles [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(24): 3894-3901.