OSTEOGENIC EFFECT OF PEPTIDES ANCHORED AMINATED TISSUE ENGINEERED BONE FOR REPAIRING FEMORAL DEFECT IN RATS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

XU Zixing ¹ , CHEN Jianting ² , XU Weihong ¹ , ZHU Xia ¹ , WANG Changsheng ¹ , LUO Hongbin ¹ , LI Guishuang ¹ , CHEN Rongsheng ¹

1Department of Spinal Surgery, the First Affiliated Hospital of Fujian Medical University, Fuzhou Fujian, 350005, P.R.China;;
2Department of Spinal Surgery, Nanfang Hospital, Southern Medical University. Corresponding author: CHEN Jianting, E-mail: chenjt99@tom.com;

Corresponding author：

Keywords：

Tissue engineered bone; Poly-D, L-lactide acid; Ammonia plasma; Bone defect; Rat

DOI：

10.7507/1002-1892.20130118

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Objective To study the osteogenic effects of a new type of peptides anchored aminated-poly-D, L-lactide acid (PA/PDLLA) scaffold in repairing femoral defect in rats. Methods The PDLLA scaffolds were treated by ammonia plasma and subsequent anchor of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides via amide linkage formation. Thus PA/PDLLA scaffolds were prepared. The bone marrow was harvested from the femur and tibia of 4 4-week-old Sprague Dawley (SD) rats, and bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured by whole bone marrow adherence method. BMSCs-scaffold composites were prepared by seeding osteogenic-induced BMSCs at passages 3-6 on the PA/PDLLA and PDLLA scaffolds. The right femoral defects of 8 mm in length were prepared in 45 adult male SD rats (weighing, 350-500 g) and the rats were divided into 3 groups (n=15) randomly. BMSCs-PA/PDLLA (PA/PDLLA group) or BMSCs-PDLLA (PDLLA group) composites were used to repair defects respectively, while defects were not treated as blank control (blank control group). General state of the rats after operation was observed. At 4, 8, and 12 weeks after operation, general, radiological, histological, micro-CT observations and real-time fluorescent quantitative PCR were performed. Results Two rats died after operation, which was added; the other rats survived to the end of the experiment. At each time point after operation, general and radiological observations showed more quick and obvious restoration in PA/PDLLA group than in PDLLA group; no bone repair was observed in blank control group. The X-ray scores were the highest in PA/PDLLA group, higher in PDLLA group, and the lowest in blank control group; showing significant difference in multiple comparison at the other time (P lt; 0.05) except between blank control group and PDLLA group at 4 weeks (P gt; 0.05). The X-ray scores showed an increasing trend in PDLLA group and PA/PDLLA group with time (P lt; 0.05). Histological and micro-CT observations showed the best osteogenesis in PA/PDLLA group, better in PDLLA group, and worst in blank control group. Comparison between groups had significant differences (P lt; 0.05) in bone mineral density, bone volume/total volume of range of interest, trabecular number, and structure model index. Significant differences (P lt; 0.05) were found in the expression levels of osteogenesis-related genes, such as osteocalcin, alkaline phosphatase, collagen type I, bone morphogenetic protein 2, and osteopontin when compared PA/PDLLA group with the other groups by real-time fluorescent quantitative PCR analysis. Conclusion The PA/PDLLA scaffolds can accelerate the repair of femoral defects in rats.

Citation： XU Zixing,CHEN Jianting,XU Weihong,ZHU Xia,WANG Changsheng,LUO Hongbin,LI Guishuang,CHEN Rongsheng. OSTEOGENIC EFFECT OF PEPTIDES ANCHORED AMINATED TISSUE ENGINEERED BONE FOR REPAIRING FEMORAL DEFECT IN RATS. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(5): 520-528. doi: 10.7507/1002-1892.20130118 Copy

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8.	Karaoglu S, Baktir A, Kabak S, et al. Experimental repair of segmental bone defects in rabbits by demineralized allograft covered by free autogenous periosteum. Injury, 2002, 33(8): 679-683.
9.	Tsuchida H, Hashimoto J, Crawford E, et al. Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats. J Orthop Res, 2003, 21(1): 44-53.
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12.	Megas P. Classification of non-union. Injury, 2005, 36 Suppl 4: S30-S37.
13.	Perren SM. Evolution of the internal fixation of long bone fractures. The scientific basis of biological internal fixation: choosing a new balance between stability and biology. J Bone Joint Surg (Br), 2002, 84(8): 1093-1110.
14.	Wang S, Cui W, Bei J. Bulk and surface modifications of polylactide. Anal Bioanal Chem, 2005, 381(3): 547-556.
15.	Shekaran A, García AJ. Extracellular matrix-mimetic adhesive biomaterials for bone repair. J Biomed Mater Res A, 2011, 96(1): 261-272.
16.	Barros RR, Novaes AB Jr, Papalexiou V, et al. Effect of biofunctionalized implant surface on osseointegration: a histomorphometric study in dogs. Braz Dent J, 2009, 20(2): 91-98.
17.	Shim JH, Moon TS, Yun MJ, et al. Stimulation of healing within a rabbit calvarial defect by a PCL/PLGA scaffold blended with TCP using solid freeform fabrication technology. J Mater Sci Mater Med, 2012, 23(12): 2993-3002.
18.	Thomsen JS, Laib A, Koller B, et al. Stereological measures of trabecular bone structure: comparison of 3D micro computed tomography with 2D histological sections in human proximal tibial bone biopsies. J Microsc, 2005, 218(Pt 2): 171-179.
19.	Cowan CM, Aghaloo T, Chou YF, et al. MicroCT evaluation of three-dimensional mineralization in response to BMP-2 doses in vitro and in critical sized rat calvarial defects. Tissue Eng, 2007, 13(3): 501-512.
20.	Patel M, Dunn TA, Tostanoski S, et al. Cyclic acetal hydroxyapatite composites and endogenous osteogenic gene expression of rat marrow stromal cells. J Tissue Eng Regen Med, 2010, 4(6): 422-436.
21.	Huang W, Carlsen B, Wulur I, et al. BMP-2 exerts differential effects on differentiation of rabbit bone marrow stromal cells grown in two-dimensional and three-dimensional systems and is required for in vitro bone formation in a PLGA scaffold. Exp Cell Res, 2004, 299(2): 325-334.
22.	Navarro M, Michiardi A, Castaño O, et al. Biomaterials in orthopaedics. J R Soc Interface, 2008, 5(27): 1137-1158.
23.	Rezwan K, Chen QZ, Blaker JJ, et al. Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials, 2006, 27(18): 3413-3431.
24.	Cai K, Yao K, Yang Z, et al. Surface modification of three-dimensional poly(d, l-lactic acid) scaffolds with baicalin: a histological study. Acta Biomater, 2007, 3(4): 597-605.
25.	Middleton JC, Tipton AJ. Synthetic biodegradable polymers as orthopedic devices. Biomaterials, 2000, 21(23): 2335-2346.

1. Horner EA, Kirkham J, Wood D, et al. Long bone defect models for tissue engineering applications: criteria for choice. Tissue Eng Part B Rev, 2010, 16(2): 263-271.
2. 许子星, 陈建庭, 尹诗衡, 等. 氨等离子体锚定短肽的消旋聚乳酸骨支架研究. 中国修复重建外科杂志, 2010, 24(11): 1376-1385.
3. Xu ZX, Li T, Zhong ZM, et al. Amide-linkage formed between ammonia plasma treated poly (D, L-lactide acid) scaffolds and bio-peptides: enhancement of cell adhesion and osteogenic differentiation in vitro. Biopolymers, 2011, 95(10): 682-694.
4. Kraus KH, Kadiyala S, Wotton H, et al. Critically sized osteo-periosteal femoral defects: a dog model. J Invest Surg, 1999, 12(2): 115-124.
5. Zeng Q, Li X, Beck G, et al. Growth and differentiation factor-5 (GDF-5) stimulates osteogenic differentiation and increases vascular endothelial growth factor (VEGF) levels in fat-derived stromal cells in vitro. Bone, 2007, 40(2): 374-381.
6. Livak K J, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods, 2001, 25(4): 402-408.
7. Cook SD, Salkeld SL, Patron LP, et al. Healing course of primate ulna segmental defects treated with osteogenic protein-1. J Invest Surg, 2002, 15(2): 69-79.
8. Karaoglu S, Baktir A, Kabak S, et al. Experimental repair of segmental bone defects in rabbits by demineralized allograft covered by free autogenous periosteum. Injury, 2002, 33(8): 679-683.
9. Tsuchida H, Hashimoto J, Crawford E, et al. Engineered allogeneic mesenchymal stem cells repair femoral segmental defect in rats. J Orthop Res, 2003, 21(1): 44-53.
10. Betz OB, Betz VM, Nazarian A, et al. Direct percutaneous gene delivery to enhance healing of segmental bone defects. J Bone Joint Surg (Am), 2006, 88(2): 355-365.
11. Vögelin E, Jones NF, Huang JI, et al. Healing of a critical-sized defect in the rat femur with use of a vascularized periosteal flap, a biodegradable matrix, and bone morphogenetic protein. J Bone Joint Surg (Am), 2005, 87(6): 1323-1331.
12. Megas P. Classification of non-union. Injury, 2005, 36 Suppl 4: S30-S37.
13. Perren SM. Evolution of the internal fixation of long bone fractures. The scientific basis of biological internal fixation: choosing a new balance between stability and biology. J Bone Joint Surg (Br), 2002, 84(8): 1093-1110.
14. Wang S, Cui W, Bei J. Bulk and surface modifications of polylactide. Anal Bioanal Chem, 2005, 381(3): 547-556.
15. Shekaran A, García AJ. Extracellular matrix-mimetic adhesive biomaterials for bone repair. J Biomed Mater Res A, 2011, 96(1): 261-272.
16. Barros RR, Novaes AB Jr, Papalexiou V, et al. Effect of biofunctionalized implant surface on osseointegration: a histomorphometric study in dogs. Braz Dent J, 2009, 20(2): 91-98.
17. Shim JH, Moon TS, Yun MJ, et al. Stimulation of healing within a rabbit calvarial defect by a PCL/PLGA scaffold blended with TCP using solid freeform fabrication technology. J Mater Sci Mater Med, 2012, 23(12): 2993-3002.
18. Thomsen JS, Laib A, Koller B, et al. Stereological measures of trabecular bone structure: comparison of 3D micro computed tomography with 2D histological sections in human proximal tibial bone biopsies. J Microsc, 2005, 218(Pt 2): 171-179.
19. Cowan CM, Aghaloo T, Chou YF, et al. MicroCT evaluation of three-dimensional mineralization in response to BMP-2 doses in vitro and in critical sized rat calvarial defects. Tissue Eng, 2007, 13(3): 501-512.
20. Patel M, Dunn TA, Tostanoski S, et al. Cyclic acetal hydroxyapatite composites and endogenous osteogenic gene expression of rat marrow stromal cells. J Tissue Eng Regen Med, 2010, 4(6): 422-436.
21. Huang W, Carlsen B, Wulur I, et al. BMP-2 exerts differential effects on differentiation of rabbit bone marrow stromal cells grown in two-dimensional and three-dimensional systems and is required for in vitro bone formation in a PLGA scaffold. Exp Cell Res, 2004, 299(2): 325-334.
22. Navarro M, Michiardi A, Castaño O, et al. Biomaterials in orthopaedics. J R Soc Interface, 2008, 5(27): 1137-1158.
23. Rezwan K, Chen QZ, Blaker JJ, et al. Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials, 2006, 27(18): 3413-3431.
24. Cai K, Yao K, Yang Z, et al. Surface modification of three-dimensional poly(d, l-lactic acid) scaffolds with baicalin: a histological study. Acta Biomater, 2007, 3(4): 597-605.
25. Middleton JC, Tipton AJ. Synthetic biodegradable polymers as orthopedic devices. Biomaterials, 2000, 21(23): 2335-2346.

Chinese Journal of Reparative and Reconstructive Surgery

OSTEOGENIC EFFECT OF PEPTIDES ANCHORED AMINATED TISSUE ENGINEERED BONE FOR REPAIRING FEMORAL DEFECT IN RATS

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