CYTOCOMPATIBILITY AND PREPARATION OF BONE TISSUE ENGINEERING SCAFFOLD BY COMBINING LOW TEMPERATURE THREE DIMENSIONAL PRINTING AND VACUUM FREEZE-DRYING TECHNIQUES_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

LIDong ^1,2,3 , ZHANGZhenhui ^1,2,3 , ZHENGChengcheng ⁴ , ZHAOBin ² , SUNKai ^1,2,3 , NIANZhenghao ^1,2,3 , ZHANGXizheng ² ,  LIRuixin ² ,  LIHui ³

1. Graduate School of Tianjin Medical University, Tianjin, 300070, P.R.China;
2. Military Academy of Medical Sciences Institute of Medical Equipment;
3. Department of Orthopedics, the General Hospital of Tianjin Medical University;
4. Department of Hematology, Xuanwu Hospital of Capital Medical University;

Corresponding author：

LIRuixin, Email: limxinxin@163.com; LIHui, Email: ortholivea@126.com

Keywords：

Bone tissue engineering; Scaffold material; Low temperature three dimensional printing technique; Vacuum freeze-drying technique; Cytocompatibility

DOI：

10.7507/1002-1892.20160058

Video：

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Objective To study the preparation and cytocompatibility of bone tissue engineering scaffolds by combining low temperature three dimensional (3D) printing and vacuum freeze-drying techniques. Methods Collagen (COL)and silk fibroin (SF) were manufactured from fresh bovine tendon and silkworm silk. SolidWorks2014 was adopted to design bone tissue engineering scaffold models with the size of 9 mm×9 mm×3 mm and pore diameter of 500μm. According to the behavior of composite materials that low temperature 3D printing equipment required, COL, SF, and nano-hydroxyapatite (nHA)at a ratio of 9:3:2 and low temperature 3D printing in combination with vacuum freeze-drying techniques were accepted to build COL/SF/nHA composite scaffolds. Gross observation and scanning electron microscope (SEM) were applied to observe the morphology and surface structures of composite scaffolds. Meanwhile, compression displacement, compression stress, and elasticity modulus were measured by mechanics machine to analyze mechanical properties of composite scaffolds. The growth and proliferation of MC3T3-E1 cells were evaluated using SEM, inverted microscope, and MTT assay after cultured for 1, 7, 14, and 21 days on the composite scaffolds. The RT-PCR and Western blot techniques were adopted to detect the gene and protein expressions of COL I, alkaline phosphatase (ALP), and osteocalcin (OCN) in MC3T3-E1 cells after 21 days. Results COL/SF/nHA composite scaffolds were successfully prepared by low temperature 3D printing technology and vacuum freeze-drying techniques; the SEM results showed that the bionic bone scaffolds were 3D polyporous structures with macropores and micropores. The mechanical performance showed that the elasticity modulus was (344.783 07±40.728 55) kPa; compression displacement was (0.958 41±0.000 84) mm; and compression stress was (0.062 15±0.007 15) MPa. The results of inverted microscope, SEM, and MTT method showed that a large number of cells adhered to the surface with full extension and good cells growth inside the macropores, which demonstrated a satisfactory proliferation rate of the MC3T3-E1 cells on the composite scaffolds. The RT-PCR and Western blot electrophoresis revealed gene expressions and protein synthesis of COL I, ALP, and OCN in MC3T3-E1 cells. Conclusion Low temperature 3D printing in combination with vacuum freeze-drying techniques could realize multi-aperture coexisted bionic bone tissue engineering scaffolds and control the microstructures of composite scaffolds precisely that possess good cytocompatibility. It was expected to be a bone defect repair material, which lays a foundation for further research of bone defect.

Citation： LIDong, ZHANGZhenhui, ZHENGChengcheng, ZHAOBin, SUNKai, NIANZhenghao, ZHANGXizheng, LIRuixin, LIHui. CYTOCOMPATIBILITY AND PREPARATION OF BONE TISSUE ENGINEERING SCAFFOLD BY COMBINING LOW TEMPERATURE THREE DIMENSIONAL PRINTING AND VACUUM FREEZE-DRYING TECHNIQUES. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(3): 292-297. doi: 10.7507/1002-1892.20160058 Copy

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7.	Wei G, Li C, Fu Q, et al. Preparation of PCL/silk fibroin/collagen electrospun fiber for urethral reconstruction. Int Urol Nephrol, 2015, 47 (1): 95-99.
8.	陆史俊, 左保齐, 刘洪臣.丝素蛋白生物支架材料在骨组织工程中的应用进展.中国修复重建外科杂志, 2014, 28(10): 1307-1310.
9.	蓝旭, 许建中, 刘雪梅, 等.丝素蛋白/羟基磷灰石复合材料细胞相容性的研究.实用骨科杂志, 2013, 19(3): 226-229.
10.	Li ZY, Mi WY, Wang HL, et al. Nano-hydroxyapatite/polyacrylamide composite hydrogels with high mechanical strengths and cell adhesion properties. Colloids and Surfaces B: Biointerfaces, 2014, 123: 959-964.
11.	徐水, 张胡静, 张海萍, 等.高强度力学性能丝素/羟基磷灰石多孔支架材料的初步制备.高分子材料科学与工程, 2011, 27(10): 150-153.
12.	Meinel L, Fajardo R, Hofmann S, et al. Silk implants for the healing of critical size bone defects. Bone, 2005, 37(5): 688-698.
13.	Vachiraroj N, Ratanavaraporn J, Damrongsakkul S, et al. A comparison of Thai silk fibroin-based and chitosan-based materials on in vitro biocompatibility for bone substitutes. Int J Biol Macromol, 2009, 45(5): 470-477.
14.	Wu SL, Liu XM, Yeung KWK, et al. Biomimetic porous scaffolds for bone tissue engineering. Materials Science and Engineering R, 2014, 80: 1-36.
15.	Sun F, Zhou H, Lee J. Various preparation methods of highly porous hydroxyapatite/polymer nanoscale biocomposites for bone regeneration. Acta Biomater, 2011, 7(11): 3813-3828.
16.	Zhao C, Tan A, Pastorin G, et al. Nanomaterial scaffolds for stem cell proliferation and differentiation in tissue engineering. Biotechnol Adv, 2013, 31(5): 654-668.
17.	Arahira T, Maruta M, Matsuya S, et al. Development and characterization of a novel porousβ-TCP scaffold with a three-dimensional PLLA network structure for use in bone tissue engineering. Materials Letters, 2015, (152): 148-150.
18.	Domingos M, Intranuovo F, Gloria A, et al. Improved osteoblast cell affinity on plasma-modified 3-D extruded PCL scaffolds. Acta Biomaterialia, 2013, 9(4): 5997-6005.
19.	Yoon YJ, Moon SK, Hwang JH. 3D printing as an efficient way for comparative study of biomimetic structures-trabecular bone and honeycomb. Journal of Mechanical Science and Technology, 2014, 28(11): 4635-4640.
20.	Korpela J, Kokkari A, Korhonen H, et al. Biodegradable and bioactive porous scaffold structures prepared using fused deposition modeling. J Biomed Mater Res B Appl Biomater, 2012, 101(4): 610-619.
21.	Bhumiratana S, Grayson W, Castaneda A, et al. Nucleation and growth of mineralized bone matrix on silk-hydroxyapatite composite scaffolds. Biomaterials, 2011, 32 (11): 2812-2820.
22.	年争好, 李晖, 李瑞欣, 等.纳米羟基磷灰石/胶原蛋白/丝素蛋白复合骨组织工程支架材料的生物相容性.中国组织工程研究, 2015, 19(8): 1149-1154.

1. Dimitriou R, Mataliotakis GI, Angoules AG, et al. Complications following autologous bone graft harvesting from the iliac crest and using the RIA: a systematic review. Injury, 2011, 42 Suppl 2: S3-S15.
2. 张仁坤, 涂悦, 赵明亮, 等. 3-D打印胶原蛋白-硫酸肝素仿生脊髓支架的研究.中国修复重建外科杂志, 2015, 29(8): 1028-1033.
3. 魏学磊, 董福慧.计算机辅助成型技术制备骨组织工程支架的研究进展.中国修复重建外科杂志, 2011, 25(12): 1508-1512.
4. Liu FH. Fabrication of bioceramic bone scaffolds for tissue engineering. Journal of Materials Engineering and Performance, 2014, 23 (10): 3762-3769.
5. 孙凯, 年争好, 徐成, 等.丝素蛋白复合胶原蛋白支架的制备及性能研究.中国修复重建外科杂志, 2014, 28(7): 903-908.
6. Liu Y, Lim J, Teoh SH. Review: development of clinically relevant scaffolds for vascularised bone tissue engineering. Biotechnol Adv, 2013, 31(5): 688-705.
7. Wei G, Li C, Fu Q, et al. Preparation of PCL/silk fibroin/collagen electrospun fiber for urethral reconstruction. Int Urol Nephrol, 2015, 47 (1): 95-99.
8. 陆史俊, 左保齐, 刘洪臣.丝素蛋白生物支架材料在骨组织工程中的应用进展.中国修复重建外科杂志, 2014, 28(10): 1307-1310.
9. 蓝旭, 许建中, 刘雪梅, 等.丝素蛋白/羟基磷灰石复合材料细胞相容性的研究.实用骨科杂志, 2013, 19(3): 226-229.
10. Li ZY, Mi WY, Wang HL, et al. Nano-hydroxyapatite/polyacrylamide composite hydrogels with high mechanical strengths and cell adhesion properties. Colloids and Surfaces B: Biointerfaces, 2014, 123: 959-964.
11. 徐水, 张胡静, 张海萍, 等.高强度力学性能丝素/羟基磷灰石多孔支架材料的初步制备.高分子材料科学与工程, 2011, 27(10): 150-153.
12. Meinel L, Fajardo R, Hofmann S, et al. Silk implants for the healing of critical size bone defects. Bone, 2005, 37(5): 688-698.
13. Vachiraroj N, Ratanavaraporn J, Damrongsakkul S, et al. A comparison of Thai silk fibroin-based and chitosan-based materials on in vitro biocompatibility for bone substitutes. Int J Biol Macromol, 2009, 45(5): 470-477.
14. Wu SL, Liu XM, Yeung KWK, et al. Biomimetic porous scaffolds for bone tissue engineering. Materials Science and Engineering R, 2014, 80: 1-36.
15. Sun F, Zhou H, Lee J. Various preparation methods of highly porous hydroxyapatite/polymer nanoscale biocomposites for bone regeneration. Acta Biomater, 2011, 7(11): 3813-3828.
16. Zhao C, Tan A, Pastorin G, et al. Nanomaterial scaffolds for stem cell proliferation and differentiation in tissue engineering. Biotechnol Adv, 2013, 31(5): 654-668.
17. Arahira T, Maruta M, Matsuya S, et al. Development and characterization of a novel porousβ-TCP scaffold with a three-dimensional PLLA network structure for use in bone tissue engineering. Materials Letters, 2015, (152): 148-150.
18. Domingos M, Intranuovo F, Gloria A, et al. Improved osteoblast cell affinity on plasma-modified 3-D extruded PCL scaffolds. Acta Biomaterialia, 2013, 9(4): 5997-6005.
19. Yoon YJ, Moon SK, Hwang JH. 3D printing as an efficient way for comparative study of biomimetic structures-trabecular bone and honeycomb. Journal of Mechanical Science and Technology, 2014, 28(11): 4635-4640.
20. Korpela J, Kokkari A, Korhonen H, et al. Biodegradable and bioactive porous scaffold structures prepared using fused deposition modeling. J Biomed Mater Res B Appl Biomater, 2012, 101(4): 610-619.
21. Bhumiratana S, Grayson W, Castaneda A, et al. Nucleation and growth of mineralized bone matrix on silk-hydroxyapatite composite scaffolds. Biomaterials, 2011, 32 (11): 2812-2820.
22. 年争好, 李晖, 李瑞欣, 等.纳米羟基磷灰石/胶原蛋白/丝素蛋白复合骨组织工程支架材料的生物相容性.中国组织工程研究, 2015, 19(8): 1149-1154.

Chinese Journal of Reparative and Reconstructive Surgery

CYTOCOMPATIBILITY AND PREPARATION OF BONE TISSUE ENGINEERING SCAFFOLD BY COMBINING LOW TEMPERATURE THREE DIMENSIONAL PRINTING AND VACUUM FREEZE-DRYING TECHNIQUES

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