<i>In vivo</i> biological safety study of porous zinc oxide/hydroxyapatite composite materials_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LI Jingying ^1,2,3 , ZHU Bin ⁴ , ZHANG Yuqin ⁴ ,  MENG Zengdong ^2,3

1. School of Medicine, Kunming University of Science and Technology, Kunming Yunnan, 650500, P.R.China;
2. Department of Orthopedics, the First People’s Hospital of Yunnan Province, Kunming Yunnan, 650032, P.R.China;
3. Key Laboratory of Digital Orthopedics of Yunnan Province, Kunming Yunnan, 650032, P.R.China;
4. School of Materials Science and Engineering, Kunming University of Science and Technology, Kunming Yunnan, 650093, P.R.China;

Corresponding?author：

MENG Zengdong, Email: menggu7119@vip.sina.com

Keywords：

Hydroxyapatite; zinc oxide; composite material; in vivo biological safety; bone repair; rabbit

DOI：

10.7507/1002-1892.202101123

Video：

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Objective To evaluate the in vivo biological safety of porous zinc oxide (ZnO)/hydroxyapatite (HA) composite materials.Methods The porous ZnO/HA composite materials and porous HA materials were prepared by the spark plasma sintering technology. First, the materials were characterized, including scanning electron microscopy to observe the material structure, in vitro degradation experiments to detect the degradation rate of the materials, and inductively coupled plasma emission spectrometer to detect the concentration of Zn²⁺ dissolved out of the composite material degradation. Then the two kinds of material extracts were prepared for acute systemic toxicity test. Fifteen male Kunming mice were randomly divided into groups A, B, and C (n=5) and injected intraperitoneally with normal saline, HA extracts, and ZnO/HA extracts, respectively. The body mass of the mice was recorded before injection and at 24, 48, and 72 hours after injection. The liver and kidney tissues were taken at 72 hours for HE staining to evaluate the safety of the composite material. Finally, the biological safety of the material in vivo was evaluated by implantation experiment. The eighteen male New Zealand white rabbits were randomly divided into HA group and ZnO/HA group (n=9); a bilateral radius defect model (1 cm) was established, and the right forelimbs of the two groups were implanted with porous HA materials and porous ZnO/HA composite materials, respectively; the left untreated as a blank control. The general condition of the animals were observed after operation. The rabbit blood was collected at 1 day before operation and at 1 day, 1 week, 4 weeks, and 8 weeks after operation for routine blood test (inflammation-related indicators) and blood biochemistry (liver and kidney function-related indicators). X-ray films were taken at 4, 8, and 12 weeks after operation to observe the repair of bone defects.Results Material characterization showed that porous ZnO/HA composite materials had interconnected large and small pore structures with a pore size between 50 and 500 μm, which degraded faster than porous HA materials, and continuously and slowly dissolved Zn²⁺. The acute systemic toxicity test showed that the mice in each group had no abnormal performance after injection, and the body mass increased (P<0.05). HE staining showed that the cells shape and structure of liver and kidney tissue were normal. Animal implantation experiments showed that all rabbits survived until the experiment was completed; routine blood tests showed inflammation in each group (neutrophils, monocytes, and lymphocytes increased) at 1 day after operation, and all returned to normal at 8 weeks (P>0.05); compared with 1 day before operation, the content of inflammatory cells in the HA group increased at 1 day, 1 week, and 4 weeks after operation (P<0.05), and the ZnO/HA group increased at 1 day after operation (P<0.05); blood biochemistry showed that the liver and kidney function indexes were in the normal range; X-ray films showed that the ZnO/HA group had better osseointegration than the HA group at 4 weeks after operation.Conclusion The porous ZnO/HA composite material has good in vivo biological safety and good bone repair ability, which is a potential bone repair material.

Citation： LI Jingying, ZHU Bin, ZHANG Yuqin, MENG Zengdong. In vivo biological safety study of porous zinc oxide/hydroxyapatite composite materials. Chinese Journal of Reparative and Reconstructive Surgery, 2021, 35(7): 847-854. doi: 10.7507/1002-1892.202101123 Copy

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6.	廖欣宇, 王福科, 王國梁. 骨組織工程支架的進展與挑戰. 中國組織工程研究, 2021, 25(28): 4553-4560.
7.	毛克亞, 劉建恒, 崔翔. 骨組織工程材料在大段骨缺損修復中的應用進展. 武警醫學, 2020, 31(4): 277-280, 283.
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10.	朱斌, 何遠懷, 孟增東, 等. 多孔 ZnO/羥基磷灰石生物復合材料的制備與性能. 復合材料學報, 2019, 36(11): 2637-2643.
11.	張亞楠. ZnO/nHA 仿骨結構活性增強羥基磷灰石骨修復材料的構建及體外生物活性研究. 昆明: 昆明理工大學, 2020.
12.	奚廷斐. 醫療器械生物學評價. 北京: 中國標準出版社, 2012: 87-170.
13.	李燁. 具有促成骨活性的 PLGA/TCP/Mg 復合多孔支架修復骨缺損研究. 北京: 中國科學院大學, 2016.
14.	Battafarano G, Rossi M, De Martino V, et al. Strategies for bone regeneration: from graft to tissue engineering. Int J Mol Sci, 2021, 22(3): 1128. doi: 10.3390/ijms22031128.
15.	Yan Y, Chen H, Zhang H, et al. Vascularized 3D printed scaffolds for promoting bone regeneration. Biomaterials, 2019, 190-191: 97-110.
16.	嚴霞. SPS 制備多孔 SrO/nHA 骨修復材料在非人靈長類動物體內的生物相容性及成骨活性研究. 昆明: 昆明理工大學, 2019.
17.	Shen Y, Liu W, Wen C, et al. Bone regeneration: importance of local pH-strontium-doped borosilicate scaffold. J Mater Chem, 2012, 22(17): 8662-8670.
18.	Seo HJ, Cho YE, Kim T, et al. Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. Nutr Res Pract, 2010, 4(5): 356-361.
19.	Storrie H, Stupp SI. Cellular response to zinc-containing organoapatite: an in vitro study of proliferation, alkaline phosphatase activity an biomineralization. Biomaterials, 2005, 26(27): 5492-5499.
20.	Zhao C, Wu H, Hou P, et al. Enhanced corrosion resistance and antibacterial property of Zn doped DCPD coating on biodegradable Mg. Materials Letters, 2016, 180: 42-46.
21.	Bakhsheshi-Rad HR, Hamzah E, Low HT, et al. Fabrication of biodegradable Zn-Al-Mg alloy: Mechanical properties, corrosion behavior, cytotoxicity and antibacterial activities. Mater Sci Eng C Mater Biol Appl, 2017, 73: 215-219.

1. Kozusko SD, Riccio C, Goulart M, et al. Chitosan as a bone scaffold biomaterial. J Craniofac Surg, 2018, 29(7): 1788-1793.
2. Ginebra MP, Espanol M, Maazouz Y, et al. Bioceramics and bone healing. EFORT Open Rev, 2018, 3(5): 173-183.
3. Fox K, Tran PA, Tran N. Recent advances in research applications of nanophase hydroxyapatite. Chemphyschem, 2012, 13(10): 2495-2506.
4. 王麗萍. 微量氟、鋅、鍶摻雜羥基磷灰石晶體結構及生物學效應研究. 上海: 中國科學院大學(中國科學院上海硅酸鹽研究所), 2018.
5. Tamai N, Myoui A, Tomita T, et al. Novel hydroxyapatite ceramics with an interconnective porous structure exhibit superior osteoconduction in vivo. J Biomed Mater Res, 2002, 59(1): 110-117.
6. 廖欣宇, 王福科, 王國梁. 骨組織工程支架的進展與挑戰. 中國組織工程研究, 2021, 25(28): 4553-4560.
7. 毛克亞, 劉建恒, 崔翔. 骨組織工程材料在大段骨缺損修復中的應用進展. 武警醫學, 2020, 31(4): 277-280, 283.
8. Su Y, Cockerill I, Wang Y, et al. Zinc-based biomaterials for regeneration and therapy. Trends Biotechnol, 2019, 37(4): 428-441.
9. Ferrone E, Araneo R, Notargiacomo A, et al. ZnO nanostructures and electrospun ZnO-polymeric hybrid nanomaterials in biomedical, health, and sustainability applications. Nanomaterials (Basel), 2019, 9(10): 1449. doi: 10.3390/nano9101449.
10. 朱斌, 何遠懷, 孟增東, 等. 多孔 ZnO/羥基磷灰石生物復合材料的制備與性能. 復合材料學報, 2019, 36(11): 2637-2643.
11. 張亞楠. ZnO/nHA 仿骨結構活性增強羥基磷灰石骨修復材料的構建及體外生物活性研究. 昆明: 昆明理工大學, 2020.
12. 奚廷斐. 醫療器械生物學評價. 北京: 中國標準出版社, 2012: 87-170.
13. 李燁. 具有促成骨活性的 PLGA/TCP/Mg 復合多孔支架修復骨缺損研究. 北京: 中國科學院大學, 2016.
14. Battafarano G, Rossi M, De Martino V, et al. Strategies for bone regeneration: from graft to tissue engineering. Int J Mol Sci, 2021, 22(3): 1128. doi: 10.3390/ijms22031128.
15. Yan Y, Chen H, Zhang H, et al. Vascularized 3D printed scaffolds for promoting bone regeneration. Biomaterials, 2019, 190-191: 97-110.
16. 嚴霞. SPS 制備多孔 SrO/nHA 骨修復材料在非人靈長類動物體內的生物相容性及成骨活性研究. 昆明: 昆明理工大學, 2019.
17. Shen Y, Liu W, Wen C, et al. Bone regeneration: importance of local pH-strontium-doped borosilicate scaffold. J Mater Chem, 2012, 22(17): 8662-8670.
18. Seo HJ, Cho YE, Kim T, et al. Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. Nutr Res Pract, 2010, 4(5): 356-361.
19. Storrie H, Stupp SI. Cellular response to zinc-containing organoapatite: an in vitro study of proliferation, alkaline phosphatase activity an biomineralization. Biomaterials, 2005, 26(27): 5492-5499.
20. Zhao C, Wu H, Hou P, et al. Enhanced corrosion resistance and antibacterial property of Zn doped DCPD coating on biodegradable Mg. Materials Letters, 2016, 180: 42-46.
21. Bakhsheshi-Rad HR, Hamzah E, Low HT, et al. Fabrication of biodegradable Zn-Al-Mg alloy: Mechanical properties, corrosion behavior, cytotoxicity and antibacterial activities. Mater Sci Eng C Mater Biol Appl, 2017, 73: 215-219.

Chinese Journal of Reparative and Reconstructive Surgery

In vivo biological safety study of porous zinc oxide/hydroxyapatite composite materials

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