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

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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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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.
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7.	張遲, 李梅, 趙基源. 細胞外基質材料在骨組織工程中的研究進展. 中國生物醫學工程學報, 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, 梁曉燕. 3D打印多孔β-磷酸三鈣負載聚乳酸-羥基乙酸共聚物抗結核藥物緩釋微球復合材料: 構建及細胞毒性評價. 中國組織工程研究, 2016, 20(25): 3750-3756.
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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.	王曉慶, 仲照東, 陳智超, 等. 全骨髓貼壁法培養人骨髓間充質干細胞的改良研究. 中國實驗血液學雜志, 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. 黃進成, 劉曦明, 蔡賢華, 等. Masquelet技術治療感染性骨缺損的研究進展. 中國矯形外科雜志, 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, 楊鐵翼, 趙振群, 等. 組織工程技術在感染性骨缺損治療中的應用及優勢. 中國組織工程研究, 2017, 21(28): 4543-4549.
7. 張遲, 李梅, 趙基源. 細胞外基質材料在骨組織工程中的研究進展. 中國生物醫學工程學報, 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, 梁曉燕. 3D打印多孔β-磷酸三鈣負載聚乳酸-羥基乙酸共聚物抗結核藥物緩釋微球復合材料: 構建及細胞毒性評價. 中國組織工程研究, 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. 王曉慶, 仲照東, 陳智超, 等. 全骨髓貼壁法培養人骨髓間充質干細胞的改良研究. 中國實驗血液學雜志, 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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