Study on cytotoxicity of three-dimensional printed β-tricalcium phosphate loaded poly (lactide-co-glycolide) anti-tuberculosis drug sustained release microspheres and its effect on osteogenic differentiation of bone marrow mesenchymal stem cells_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

GONG Dong ^1,2 , MA Yonghai ³ , YANG Xinle ² , XIE Weiqiang ¹ , SHAO Longlong ² ,  ZHEN Ping ²

1. Gansu University of Chinese Medicine, Lanzhou Gansu, 730000, P.R.China;
2. Department of Orthopedics, Lanzhou General Hospital of Chinese PLA, Lanzhou Gansu, 730050, P.R.China;
3. Ningxia Medical University, Yinchuan Ningxia, 750000, P.R.China;

Corresponding author：

ZHEN Ping, Email: zhenpingok@163.com

Keywords：

Three-dimensional printing; β-tricalcium phosphate; bone marrow mesenchymal stem cells; cytotoxicity; osteogenic differentiation

DOI：

10.7507/1002-1892.201803067

Video：

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ObjectiveTo study the effect of three-dimensional (3D) printed β-tricalcium phosphate (β-TCP) scaffold loaded poly (lactide-co-glycolide) (PLGA) anti-tuberculosis drug sustained release microspheres on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and its cytotoxicity.MethodsIsoniazid and rifampicin/PLGA sustained release microspheres were prepared by W/O/W multiple emulsion method. The β-TCP scaffolds were prepared by 3D printing technique. The microspheres were loaded on the scaffolds by centrifugal oscillation method to prepare composite materials. The BMSCs of Sprague Dawley rat were isolated and cultured by whole bone marrow adherent method, and the third generation cells were used for the following experiments. BMSCs were co-cultured with osteogenic induction medium (group A), PLGA anti-tuberculosis drug sustained release microsphere extract (group B), 3D printed β-TCP scaffold extract (group C), and 3D printed β-TCP scaffold loaded PLGA anti-tuberculosis drug sustained release microsphere composite extract (group D), respectively. Cytotoxicity was detected by cell counting kit 8 (CCK-8) method; the calcium deposition was observed by alizarin red staining; and the mRNA expressions of alkaline phosphatase (ALP), osteocalcin (OCN), and bone sialoprotein (BSP) were detected by real-time fluorescence quantitative PCR (RT-qPCR).ResultsCCK-8 assay showed that the absorbance (A) value of groups A, B, C, and D increased gradually with the culture time prolonging. After cultured for 24, 48, and 72 hours, the A value decreased in the order of groups A, C, B, and D. There was no significant difference between groups B and D (P>0.05), but there were significant differences between other groups (P<0.05). The cytotoxicity was evaluated as grade 0-2, and the toxicity test was qualified. Alizarin red staining showed that red mineralized nodules were formed in all groups at 21 days after osteogenic induction, but the number of mineralized nodules decreased sequentially in groups C, D, A, and B. RT-qPCR test results showed that the relative expressions of OCN and BSP genes in groups A, B, C, and D increased gradually with the culture time prolonging. The relative expression of ALP gene increased at 7 and 14 days, and decreased at 21 days. After cultured for 7, 14, and 21 days, the relative expressions of ALP, OCN, and BSP genes decreased sequentially in groups C, D, A, and B; the differences were significant between groups at different time points (P<0.05).Conclusion3D printed β-TCP loaded PLGA anti-tuberculosis drug sustained release microsphere composites have no obvious cytotoxicity to BMSCs, and can promote BMSCs to differentiate into osteoblasts to a certain extent.

Citation： GONG Dong, MA Yonghai, YANG Xinle, XIE Weiqiang, SHAO Longlong, ZHEN Ping. Study on cytotoxicity of three-dimensional printed β-tricalcium phosphate loaded poly (lactide-co-glycolide) anti-tuberculosis drug sustained release microspheres and its effect on osteogenic differentiation of bone marrow mesenchymal stem cells. Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(9): 1131-1136. doi: 10.7507/1002-1892.201803067 Copy

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8.	Oliveira WF, Silva PMS, Silva RCS, et al. Staphylococcus aureus and Staphylococcus epidermidis infections on implants. J Hosp Infect, 2018, 98(2): 111-117.
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11.	Danhier F, Ansorena E, Silva JM, et al. PLGA-based nanoparticles: an overview of biomedical applications. J Control Release, 2012, 161(2): 505-522.
12.	Ashizawa N, Tsuji Y, Kawago K, et al. Successful treatment of methicillin-resistant Staphylococcus aureus osteomyelitis with combination therapy using linezolid and rifampicin under therapeutic drug monitoring. J Infect Chemother, 2016, 22(5): 331-334.
13.	Gómez-Barrena E, Rosset P, Lozano D, et al. Bone fracture healing: cell therapy in delayed unions and nonunions. Bone, 2015, 70: 93-101.
14.	Moshiri A, Shahrezaee M, Shekarchi B, et al. Three-Dimensional Porous Gelapin-Simvastatin Scaffolds Promoted Bone Defect Healing in Rabbits. Calcif Tissue Int, 2015, 96(6): 552-564.
15.	Tayton E, Purcell M, Smith JO, et al. The scale-up of a tissue engineered porous hydroxyapatite polymer composite scaffold for use in bone repair: an ovine femoral condyle defect study. J Biomed Mater Res A, 2015, 103(4): 1346-1356.
16.	Inzana JA, Olvera D, Fuller SM, et al. 3D printing of composite calcium phosphate and collagen scaffolds for bone regeneration. Biomaterials, 2014, 35(13): 4026-4034.
17.	Hosseinpour S, Ghazizadeh Ahsaie M, Rezai Rad M, et al. Application of selected scaffolds for bone tissue engineering: a systematic review. Oral Maxillofac Surg, 2017, 21(2): 109-129.
18.	Seidenstuecker M, Kerr L, Bernstein A, et al. 3D Powder Printed Bioglass and β-Tricalcium Phosphate Bone Scaffolds. Materials (Basel), 2017, 11(1): pii: E13.
19.	Kasten P, Vogel J, Luginbühl R, et al. Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier. Biomaterials, 2005, 26(29): 5879-5889.
20.	王晓庆, 仲照东, 陈智超, 等. 全骨髓贴壁法培养人骨髓间充质干细胞的改良研究. 中国实验血液学杂志, 2014, 22(2): 496-502.
21.	Milhan NVM, Carvalh ICS, Prado RFD, et al. Analysis of indicators of osteogenesis, cytotoxicity and genotoxicity of an experimental β-TCP compared to other bones substitutes. Acta Scientiarum Health Sciences, 2017, 39(1): 97-105.
22.	Rodan GA, Noda M. Gene expression in osteoblastic cells. Crit Rev Eukaryot Gene Expr, 1991, 1(2): 85-98.

1. Pigrau-Serrallach C, Rodríguez-Pardo D. Bone and joint tuberculosis. Eur Spine J, 2013, 22 Suppl 4: 556-566.
2. Yuan J, Zhen P, Zhao H, et al. The preliminary performance study of the 3D printing of a tricalcium phosphate scaffold for the loading of sustained release anti-tuberculosis drugs. Journal of Materials Science, 2015, 50(5): 2138-2147.
3. Mauffrey C, Giannoudis PV, Conway JD, et al. Masquelet technique for the treatment of segmental bone loss have we made any progress? Injury, 2016, 47(10): 2051-2052.
4. 黄进成, 刘曦明, 蔡贤华, 等. Masquelet技术治疗感染性骨缺损的研究进展. 中国矫形外科杂志, 2017, 25(20): 1867-1871.
5. Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet, 2001, 358(9276): 135-138.
6. 王步祥, 杨铁翼, 赵振群, 等. 组织工程技术在感染性骨缺损治疗中的应用及优势. 中国组织工程研究, 2017, 21(28): 4543-4549.
7. 张迟, 李梅, 赵基源. 细胞外基质材料在骨组织工程中的研究进展. 中国生物医学工程学报, 2017, 36(1): 103-108.
8. Oliveira WF, Silva PMS, Silva RCS, et al. Staphylococcus aureus and Staphylococcus epidermidis infections on implants. J Hosp Infect, 2018, 98(2): 111-117.
9. 孟磊, 甄平, 梁晓燕. 3D打印多孔β-磷酸三钙负载聚乳酸-羟基乙酸共聚物抗结核药物缓释微球复合材料: 构建及细胞毒性评价. 中国组织工程研究, 2016, 20(25): 3750-3756.
10. Lee JH, Ryu MY, Baek HR, et al. Fabrication and evaluation of porous beta-tricalcium phosphate/hydroxyapatite (60/40) composite as a bone graft extender using rat calvarial bone defect model. Scientific World Journal, 2013, 2013: 481789.
11. Danhier F, Ansorena E, Silva JM, et al. PLGA-based nanoparticles: an overview of biomedical applications. J Control Release, 2012, 161(2): 505-522.
12. Ashizawa N, Tsuji Y, Kawago K, et al. Successful treatment of methicillin-resistant Staphylococcus aureus osteomyelitis with combination therapy using linezolid and rifampicin under therapeutic drug monitoring. J Infect Chemother, 2016, 22(5): 331-334.
13. Gómez-Barrena E, Rosset P, Lozano D, et al. Bone fracture healing: cell therapy in delayed unions and nonunions. Bone, 2015, 70: 93-101.
14. Moshiri A, Shahrezaee M, Shekarchi B, et al. Three-Dimensional Porous Gelapin-Simvastatin Scaffolds Promoted Bone Defect Healing in Rabbits. Calcif Tissue Int, 2015, 96(6): 552-564.
15. Tayton E, Purcell M, Smith JO, et al. The scale-up of a tissue engineered porous hydroxyapatite polymer composite scaffold for use in bone repair: an ovine femoral condyle defect study. J Biomed Mater Res A, 2015, 103(4): 1346-1356.
16. Inzana JA, Olvera D, Fuller SM, et al. 3D printing of composite calcium phosphate and collagen scaffolds for bone regeneration. Biomaterials, 2014, 35(13): 4026-4034.
17. Hosseinpour S, Ghazizadeh Ahsaie M, Rezai Rad M, et al. Application of selected scaffolds for bone tissue engineering: a systematic review. Oral Maxillofac Surg, 2017, 21(2): 109-129.
18. Seidenstuecker M, Kerr L, Bernstein A, et al. 3D Powder Printed Bioglass and β-Tricalcium Phosphate Bone Scaffolds. Materials (Basel), 2017, 11(1): pii: E13.
19. Kasten P, Vogel J, Luginbühl R, et al. Ectopic bone formation associated with mesenchymal stem cells in a resorbable calcium deficient hydroxyapatite carrier. Biomaterials, 2005, 26(29): 5879-5889.
20. 王晓庆, 仲照东, 陈智超, 等. 全骨髓贴壁法培养人骨髓间充质干细胞的改良研究. 中国实验血液学杂志, 2014, 22(2): 496-502.
21. Milhan NVM, Carvalh ICS, Prado RFD, et al. Analysis of indicators of osteogenesis, cytotoxicity and genotoxicity of an experimental β-TCP compared to other bones substitutes. Acta Scientiarum Health Sciences, 2017, 39(1): 97-105.
22. Rodan GA, Noda M. Gene expression in osteoblastic cells. Crit Rev Eukaryot Gene Expr, 1991, 1(2): 85-98.

Chinese Journal of Reparative and Reconstructive Surgery

Study on cytotoxicity of three-dimensional printed β-tricalcium phosphate loaded poly (lactide-co-glycolide) anti-tuberculosis drug sustained release microspheres and its effect on osteogenic differentiation of bone marrow mesenchymal stem cells

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