IN VITRO EXPERIMENTAL STUDY ON INFLUENCES OF FINAL DEGRADATION PRODUCTS OF POLYACTIC ACID ON PROLIFERATION AND OSTEOBLASTIC PHENOTYPE OF OSTEOBLAST-LIKE CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

FENGTingting ¹ , LIUYanjun ¹ , XUQing ¹ , LIXiaoming ¹ ,  LUOXianglin ^1,2 ,  CHENYuanwei ¹

1. School of Polymer Science and Engineering;
2. State Key Laboratory of Polymer Materials and Engineering of China, Sichuan University, Chengdu Sichuan, 610065, P. R. China;

Corresponding author：

LUOXianglin, Email: luoxl_scu@126.com; CHENYuanwei, Email: chenyw_scu@scu.edu.cn

Keywords：

Bone tissue engineering; Scaffold material; Polylactic acid; Lactic acid; Osteoblast-l ike cells; Cell proliferation; Osteoblastic phenotype

DOI：

10.7507/1002-1892.20140330

Video：

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Objective To investigate the influences of lactic acid (LA), the final degradation product of polylactic acid (PLA) on the prol iferation and osteoblastic phenotype of osteoblast-l ike cells so as to provide theoretical basis for bone tissue engineering. Methods Ros17/2.8 osteoblast-l ike cells were harvested and divided into 3 groups. In groups A and B, the cells were cultured with the medium containing 4, 8, 16, 22, and 27 mmol/L L-LA and D, L-LA, respectively. In group C, the cells were cultured with normal medium (pH7.4). The cell prol iferation was determined with MTT method after 1, 3, and 5 days. The relative growth ratio (RGR) was calculated, and the cytotoxicity was evaluated according to national standard of China. In addition, the alkal ine phosphatase (ALP) activity of cells cultured with medium containing 4 mmol/L L-LA (group A), 4 mmol/ L D, L-LA (group B), and normal medium (group C) after 1 and 5 days were detected with ALP kits, and the relative ALP ratio (RAR) was calculated; after 21 days, the calcium nodules were tested with von Kossa staining method, and were quantitatively analyzed. Results When LA concentration was 4 mmol/L, the mean RGR of both groups A and B were all above 80%, and the cytotoxic grades were grade 0 or 1, which meant non-cytotoxicity. When LA concentration was 8 mmol/L and 16 mmol/ L, groups A and B showed cytotoxicity after 5 days and 3 days, respectively. When LA concentration was above 22 mmol/L, cell prol iferations of groups A and B were inhibited evidently after 1-day culture. At each LA concentration, RGR of group A was significantly higher than that of group B at the same culture time (P<0.05) except those at 4 mmol/L after 1-day and 3-day culture. After 1 day, the RAR of group A was significantly higher than that of group B on 1 day (144.1%±3.2% vs. 115.2%±9.8%, P<0.05) and on 5 days (129.6%±9.8% vs. 78.2%±6.9%, P<0.05). The results of von Kossa staining showed that the black gobbets in group A were obviously more than those of groups B and C. The staining area of group A (91.2%±8.2%) was significantly higher than that of groups B (50.3%±7.9%) and C (54.2%±8.6%) (P<0.05). Conclusion The concentration and composition of LA have significant effects on the cell proliferation and osteoblastic phenotype of osteoblast-l ike cells.

Citation： FENGTingting, LIUYanjun, XUQing, LIXiaoming, LUOXianglin, CHENYuanwei. IN VITRO EXPERIMENTAL STUDY ON INFLUENCES OF FINAL DEGRADATION PRODUCTS OF POLYACTIC ACID ON PROLIFERATION AND OSTEOBLASTIC PHENOTYPE OF OSTEOBLAST-LIKE CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(12): 1525-1529. doi: 10.7507/1002-1892.20140330 Copy

1.	Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials, 2000, 21(24):2529-2543.
2.	杨志明. 组织工程. 北京:化学工业出版社, 2002:286.
3.	Eldesoqi K, Henrich D, El-Kady AM, et al. Safety evaluation of a bioglass-polylactic acid composite scaffold seeded with progenitor cells in a rat skull critical-size bone defect. PLoS One, 2014, 9(2):e87642.
4.	Datta P, Chatterjee J, Dhara S. Phosphate functional ized and lactic acid containing graft copolymer:synthesis and evaluation as biomaterial for bone tissue engineering applications. J Biomater Sci Polym Ed, 2013, 24(6):696-713.
5.	Weiler A, Hell ing HJ, Kirch U, et al. Foreign-body reaction and the course of osteolysis after polyglycolide implants for fracture fixation-Experimental study in sheep. J Bone Joint Surg (Br), 1996, 78(3):369-376.
6.	Taylor MS, Daniels AU, Andriano KP, et al. Six bioabsorbable polymers: in vitro acute toxicity of accumulated degradation products. J Appl Biomat, 1994, 5(2):151-157.
7.	Ignatius AA, Claes LE. In vitro biocompatibility of bioresorbable polymers: Poly(L, DL-lactide) and poly (L-lactide-co-glycolide). Biomaterials, 1996, 17(8):831-839.
8.	Cordewene FW, van Geffen MF, Joziasse CA, et al. Cytotoxicity of poly(96L/4D-lactide):the influence of degradation and steril ization. Biomaterials, 2000, 21(23):2433-2442.
9.	Brandao-Burch A, Utting JC, Orriss IR, et al. Acidosis inhibits bone formation by osteoblasts by preventing mineralization. Calcif Tissue Int, 2005, 77(3):167-174.
10.	Kohn DH, Sarmadi M, Helman JI, et al. Effects of pH on human bone marrow stromal cells in vitro:Implications for tissue engineering of bone. J Biomed Mater Res, 2002, 60(2):292-299.
11.	Thiede MA, Yoon K, Golub EE, et al. Structure and expression of rat osteosarcoma (ROS17/2.8) alkal ine phosphatase:product of a single copy gene. Proc Natl Acad Sci U S A, 1988, 85(2):319-323.
12.	Mosmann T. Rapid colorimetric assay of cellular growth and survival: application to proliferation and cytotoxicity assay. J Immunol Methods, 1983, 65(1-2):55-63.
13.	国家标准GB/T16175-2008医用有机硅材料生物学评价试验方法. 北京:中国标准出版社, 2008.
14.	Meloan SN, Puchtler H. Chemical mechanisms of staining methods: von Kossa's technique. What von Kossa really wrote and a modified reaction for selective demonstration of inorganic phosphate. J Histotechol, 1985, 8(1):11-13.
15.	Wang CJ, Zhou ZG, Holmqvist A, et al. Survivin expression quantified by image Pro-Plus compared with visual assessment. Appl Immunohistochem Mol Morphol, 2009, 17(6):530-535.
16.	司徒镇强, 吴军正. 细胞培养. 西安:世界图书出版公司, 2004:62.
17.	Xiong M, Elson G, Legarda D, et al. Production of vascular endothelial growth factor by murine macrophages:Regulation by hypoxia, lactate and inducible nitric oxide synthase pathway. Am J Pathol, 1998, 153(2):587-598.
18.	Burns PA, Wilson DJ. Angiogenesis mediated by metabolite is dependent on vascular endothelial growth factor. Angiogenesis, 2003, 6(1): 73-77.
19.	Kumar VB, Viji RI, Kiran MS, et al. Endothelial cell response to lactate: implication of PAR modification of VEGF. J Cell Physiol, 2007, 211(2): 477-485.

1. Hutmacher DW. Scaffolds in tissue engineering bone and cartilage. Biomaterials, 2000, 21(24):2529-2543.
2. 杨志明. 组织工程. 北京:化学工业出版社, 2002:286.
3. Eldesoqi K, Henrich D, El-Kady AM, et al. Safety evaluation of a bioglass-polylactic acid composite scaffold seeded with progenitor cells in a rat skull critical-size bone defect. PLoS One, 2014, 9(2):e87642.
4. Datta P, Chatterjee J, Dhara S. Phosphate functional ized and lactic acid containing graft copolymer:synthesis and evaluation as biomaterial for bone tissue engineering applications. J Biomater Sci Polym Ed, 2013, 24(6):696-713.
5. Weiler A, Hell ing HJ, Kirch U, et al. Foreign-body reaction and the course of osteolysis after polyglycolide implants for fracture fixation-Experimental study in sheep. J Bone Joint Surg (Br), 1996, 78(3):369-376.
6. Taylor MS, Daniels AU, Andriano KP, et al. Six bioabsorbable polymers: in vitro acute toxicity of accumulated degradation products. J Appl Biomat, 1994, 5(2):151-157.
7. Ignatius AA, Claes LE. In vitro biocompatibility of bioresorbable polymers: Poly(L, DL-lactide) and poly (L-lactide-co-glycolide). Biomaterials, 1996, 17(8):831-839.
8. Cordewene FW, van Geffen MF, Joziasse CA, et al. Cytotoxicity of poly(96L/4D-lactide):the influence of degradation and steril ization. Biomaterials, 2000, 21(23):2433-2442.
9. Brandao-Burch A, Utting JC, Orriss IR, et al. Acidosis inhibits bone formation by osteoblasts by preventing mineralization. Calcif Tissue Int, 2005, 77(3):167-174.
10. Kohn DH, Sarmadi M, Helman JI, et al. Effects of pH on human bone marrow stromal cells in vitro:Implications for tissue engineering of bone. J Biomed Mater Res, 2002, 60(2):292-299.
11. Thiede MA, Yoon K, Golub EE, et al. Structure and expression of rat osteosarcoma (ROS17/2.8) alkal ine phosphatase:product of a single copy gene. Proc Natl Acad Sci U S A, 1988, 85(2):319-323.
12. Mosmann T. Rapid colorimetric assay of cellular growth and survival: application to proliferation and cytotoxicity assay. J Immunol Methods, 1983, 65(1-2):55-63.
13. 国家标准GB/T16175-2008医用有机硅材料生物学评价试验方法. 北京:中国标准出版社, 2008.
14. Meloan SN, Puchtler H. Chemical mechanisms of staining methods: von Kossa's technique. What von Kossa really wrote and a modified reaction for selective demonstration of inorganic phosphate. J Histotechol, 1985, 8(1):11-13.
15. Wang CJ, Zhou ZG, Holmqvist A, et al. Survivin expression quantified by image Pro-Plus compared with visual assessment. Appl Immunohistochem Mol Morphol, 2009, 17(6):530-535.
16. 司徒镇强, 吴军正. 细胞培养. 西安:世界图书出版公司, 2004:62.
17. Xiong M, Elson G, Legarda D, et al. Production of vascular endothelial growth factor by murine macrophages:Regulation by hypoxia, lactate and inducible nitric oxide synthase pathway. Am J Pathol, 1998, 153(2):587-598.
18. Burns PA, Wilson DJ. Angiogenesis mediated by metabolite is dependent on vascular endothelial growth factor. Angiogenesis, 2003, 6(1): 73-77.
19. Kumar VB, Viji RI, Kiran MS, et al. Endothelial cell response to lactate: implication of PAR modification of VEGF. J Cell Physiol, 2007, 211(2): 477-485.

Chinese Journal of Reparative and Reconstructive Surgery

IN VITRO EXPERIMENTAL STUDY ON INFLUENCES OF FINAL DEGRADATION PRODUCTS OF POLYACTIC ACID ON PROLIFERATION AND OSTEOBLASTIC PHENOTYPE OF OSTEOBLAST-LIKE CELLS

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