Short-term biosafety of novel absorbable nano-hydroxyapatite/collagen/poly(lactic acid) composite

doi:10.3969/j.issn.2095-4344.1279

Abstract

Abstract:

BACKGROUND: A novel nano-hydroxyapatite/collagen/poly(lactic acid) composite was manufactured in our preliminary experiments.

OBJECTIVE: To evaluate the biosafety of nano-hydroxyapatite/collagen/poly(lactic acid) composite.

METHODS: Nano-hydroxyapatite/collagen/poly(lactic acid) composite was prepared into three kinds of leaching liquor containing 100, 150 and 200 g/L poly(lactic acid), namely, nHAC/PLA1, nHAC/PLA2, nHAC/PLA3. (1) Cytotoxicity test: L929 cells were cultured with nHAC/PLA1, nHAC/PLA2, nHAC/PLA3 leaching liquor and culture medium and cell toxicity was evaluated using MTT assay. (2) Acute systemic toxicity test: nHAC/PLA1, nHAC/PLA2, nHAC/PLA3 leaching liquor and normal saline were injected into C57 mice (Laboratory Animal Center of Zhengzhou University, China) via the tail vein. At 24, 48, 72 hours after injection, the general situation of mice was observed and body weight was recorded. (3) Serum toxicity test: SD rats (Laboratory Animal Center of Zhengzhou University) were intragastrically administered nHAC/PLA1, nHAC/PLA2, nHAC/PLA3 leaching liquor and normal saline. Seven days later, serum level of interleukin-1 protein was detected by enzyme-linked immunosorbent assay. (4) Skin sensitization test: 25 guinea pigs (Laboratory Animal Center of Zhengzhou University) were randomly treated with nHAC/PLA1, nHAC/PLA2, nHAC/PLA3 leaching liquor (experimental groups), normal saline (negative control group), and 5% formaldehyde (positive control group). At 24, 48 and 72 hours after intracutaneous induction, local induction, and provocation, skin sensitization reactions, including erythema and edema, were observed in each group. (5) Hemolysis test: anti-rabbit antibodies were added into the leaching liquor of test samples, saline and distilled water to determine the rate of hemolysis. The study was approved by Ethics Committee of Zhengzhou University, China.

RESULTS AND CONCLUSION: The in vitro cytotoxicity of nano-hydroxyapatite/collagen/poly(lactic acid) composite was graded 0. Negative results were obtained in acute systemic toxicity test, serum toxicity test, skin sensitization test, acute hemolytic test, and hemolysis test of nano-hydroxyapatite/collagen/poly(lactic acid) composite. These results suggest good biosafety of nano-hydroxyapatite/ collagen/poly(lactic acid) composite.

Key words: nano-hydroxyapatite/collagen, poly(lactic acid), absorbable material, biosafety, cytotoxicity test, acute systemic toxicity test, serum toxicity test, skin sensitization test, hemolysis test

CLC Number:

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

R318.08

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Gao Li, Wang Shuyan, Wang Rui, Xu Yonghua, Gao Jingjing, Zhang Li, Liu Wentao. Short-term biosafety of novel absorbable nano-hydroxyapatite/collagen/poly(lactic acid) composite[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(22): 3530-3535.

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URL: https://www.cjter.com/EN/10.3969/j.issn.2095-4344.1279

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Figures/Tables 6

2.1 体外细胞毒性实验结果用DMEM培养基及nHAC/ PLA1、nHAC/PLA2和nHAC/PLA3浸提液培养的L-929小鼠成纤维细胞在2，4，7 d时相对增殖率在100%-105%之间，均大于100%，因此3种nHAC/PLA材料细胞毒性分级均为0级，无细胞毒性，符合生物材料的应用要求。同一时间点各组间光吸收度值比较，差异无统计学意义(F=0.851，1.172和0.833，P =0.486，0.351和0.495)，见表2。"

2.2 急性全身毒性实验结果  尾静脉注射实验组材料浸提液及生理盐水后，小鼠皮肤、黏膜未见异常，眼睑无下垂，小鼠进食、活动正常，无死亡、体质量下降、运动减少等不良反应。按毒性分级，nHAC/PLA1、nHAC/PLA2和nHAC/PLA3三种复合物属于无毒材料，各组间小鼠24，48，72 h体质量变化比较差异无显著性意义(F=0.355，0.458，2.279，P=0.786，0.715，0.119)，见表3。"

2.3 血清毒性反应实验结果获取胃饲浸提液nHAC/ PLA1、nHAC/PLA2及nHAC/PLA3实验组与空白对照组的血清白细胞介素质量浓度分别为(18.254±0.740)，(18.874±1.043)，(17.728±0.531)，(18.180±1.035) ng/L，各组比较差异无显著性意义(F=1.488，P=0.256)。 2.4 致敏实验结果   豚鼠致敏去除刺激源后，实验组24，48，72 h腹部皮肤与阴性对照组皮肤反应一致，无红斑和水肿现象；阳性对照组刺激源24 h 后豚鼠腹部皮肤出现局部点状发红、水肿反应，48 h后皮肤红斑、水肿呈片状加重，焦痂出现，72 h后红斑、水肿进一步加重，呈重度焦痂状态，见图1-3。实验组与阴性对照致敏记分均为0分。因此，nHAC/PLA材料对豚鼠皮肤无致敏性。"

"

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2.5 溶血实验结果  nHAC/PLA材料溶血实验结果，见表4。实验中阳性对照组吸光度值为0.812±0.027，阴性对照组吸光度值为0.017±0.002，小于0.03，均符合实验要求。nHAC/PLA1、nHAC/PLA2和nHAC/PLA3浸提液的溶血率分别为3.7%，3.3%，3.8%，均小于5%，即认为nHAC/PLA材料无溶血反应，符合溶血实验要求。"

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Short-term biosafety of novel absorbable nano-hydroxyapatite/collagen/poly(lactic acid) composite

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[1]	Li Jie, Xu Jianzhen, Hu Ping, Lei Qiqi, Zhang Wenning, Ao Ningjian . Preparation and performance evaluation of carboxymethyl chitosan/oxidized glucomannan/Panax notoginseng compound sponge dressing for chronic wound [J]. Chinese Journal of Tissue Engineering Research, 2021, 25(16): 2528-2534.
[2]	Lei Senlin, Liu Hongyuan, Yang Hongsheng, Xiong Yan, Duan Hong. In vivo biosafety and histocompatibility of absorbable poly-D,L-lactic acid screws implanted with ultrasound-assisted technology [J]. Chinese Journal of Tissue Engineering Research, 2020, 24(34): 5454-5460.
[3]	Sun Xirao, Sun Baozhai, Zhang Zhenbao. Combination of magnesium ion with mineralized collagen intervenes osteogenic differentiation of mouse preosteoblasts [J]. Chinese Journal of Tissue Engineering Research, 2020, 24(22): 3467-3473.
[4]	Zhang Xue, Zhang Yang, Cui Fuzhai. Bioactivity of the peptide P17-bone morphogenetic protein-2/nano-hydroxyapatite-mineralized collagen composite [J]. Chinese Journal of Tissue Engineering Research, 2020, 24(22): 3547-3552.
[5]	Sun Xirao, Wang Chengyue, Zhao Yuan, Zhang Zhenbao. In vitro corrosion and in vivo biosafety of pure magnesium film [J]. Chinese Journal of Tissue Engineering Research, 2020, 24(16): 2578-2584.
[6]	Li Dongmei, Liu Xinhui,Li Qingxing. Changes of vascular endothelial growth factor after repair of pig mandibular defects by nano-hydroxyapatite/collagen composite [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(26): 4148-4153.
[7]	Wang Liang, Guo Yuxing, Huang Hua, Yuan Guangyin, Zhang Lei. Evaluation of biosafety of porous magnesium alloy scaffolds for jaw defects [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(26): 4121-4128.
[8]	Fu Na, Luo Xiaoding, Jiao Tiejun, Sui Lei. Application of electrospun polycaprolactone-polyethylene glycol-polycaprolactone fiber scaffolds in bone tissue engineering [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(22): 3445-3450.
[9]	Li Jinyu, Yu Xing, Jiang Junjie, Xu Lin, Zhao Xueqian, Sun Qi, Zheng Chenying, Bai Chunxiao, Liu Chuyin, Zhang Zhe, Jia Yusong. Osteogenic differentiation of MC3T3-E1 cells induced by osteopractic total flavone and nano-hydroxyapatite/collagen composite and the underlying mechanism [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(18): 2888-2893.
[10]	Chai Le, Quan Renfu, Lü Jianlan, Huang Xiaolong, Zhang Can, Ren Weifan, Qian Jiansheng. Physicochemical properties and cellularization of hydroxyapatite/zirconium dioxide foam ceramics [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(18): 2871-2879.
[11]	Liu Meng-yuan, Jing Miao-lei, Guan Jing, Huang Shu-jie, Yang Jian, Li Zhi-hong. Preparation of N-hexane chitosan and its cytotoxicity and coagulation property [J]. Chinese Journal of Tissue Engineering Research, 2018, 22(22): 3520-3526.
[12]	Wang Xiao, Xu Hong-guang, Xiao Liang, Liu Chen, Jin Zhong-xing, Shen Yang. Porcine decellular endplate cartilage matrix: biocompatibility assessment and establishment of a cytotoxicity evaluation system [J]. Chinese Journal of Tissue Engineering Research, 2018, 22(22): 3539-3544.
[13]	Liu Xue-jian, Liu Shi-chen, Sun Bai-chuan, Zhang Kai-hong, Meng Hao-ye, Wang Yu Huang Shao-dai, Lu Chang-feng, Wang Chong, Yu Wen, Jing Xiao-guang, Zhao Yue, Yang Jian-hua, Peng Jiang . Preparation and characterization of laser microporous acellular osteochondral scaffolds [J]. Chinese Journal of Tissue Engineering Research, 2018, 22(18): 2836-2842.
[14]	Zou Wen, Yuan Tun, Cai Yong-fu, Zheng Li-ping, Liang Jie. Biosafety evaluation strategy and practice of tissue induced biomaterials [J]. Chinese Journal of Tissue Engineering Research, 2018, 22(10): 1534-1539.
[15]	Zhou Qi-Qi, Han Xiang-zhen, Song Yan-yan, Lv Ming-fan, Hu Yang, He Hui-yu. Performance comparison of 3D printing sheep vertebral bone meal/polyvinyl alcohol scaffold, nano-hydroxyapatite/polyvinyl alcohol scaffold and sheep vertebral bone meal/polyvinyl alcohol nonporous bone plate [J]. Chinese Journal of Tissue Engineering Research, 2016, 20(52): 7851-7857.