錨定短肽組織工程骨修復大鼠股骨干缺損的成骨觀察_《中國修復重建外科雜志》

作者：

許子星 ¹ , 陳建庭 ² , 許衛紅 ¹ , 朱夏 ¹ , 王長昇 ¹ , 羅鴻斌 ¹ , 李貴雙 ¹ , 陳榮生 ¹

1 福建醫科大學附屬第一醫院脊柱外科（福州，350005）;;
2 南方醫科大學南方醫院脊柱外科;

關鍵詞：

組織工程骨消旋聚乳酸氨等離子體骨缺損大鼠

DOI：

10.7507/1002-1892.20130118

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目的探討氨等離子體錨定甘氨酸-精氨酸-甘氨酸-天冬氨酸-絲氨酸（Gly-Arg-Gly-Asp-Ser，GRGDS）短肽的消旋聚乳酸（poly-D，L-lactide acid，PDLLA）骨支架修復大鼠股骨干缺損的成骨效能。方法采用氨等離子體錨定短肽技術，在PDLLA表面以酰胺鍵錨定GRGDS活性短肽，制備錨定短肽PDLLA（peptides anchored aminated-PDLLA，PA/PDLLA）骨支架。取4只4周齡SD大鼠雙側股骨及脛骨骨髓，采用全骨髓貼壁法分離、培養BMSCs，取第3～6代成骨誘導的BMSCs分別接種至PA/PDLLA支架及PDLLA支架。45只成年雄性SD大鼠，體重350～500 g，制備右側股骨干8 mm長骨缺損模型，隨機分為3組（n=15）。骨缺損分別采用BMSCs-PA/PDLLA支架復合物（PA/PDLLA組）、BMSCs-PDLLA支架復合物（PDLLA組）填充修復，空白對照組不作處理。術后觀察大鼠一般情況，4、8、12周分別對骨缺損部位進行大體觀察、X線片檢查、組織學觀察、Micro-CT掃描及實時熒光定量PCR檢測。結果術后2只實驗動物死亡，予以補充；其余均存活至實驗完成。術后各時間點大體及X線片觀察示PA/PDLLA組骨缺損部位骨修復重建較PDLLA組快，空白對照組未見骨修復。X線片評分示各時間點PA/PDLLA組評分最高，PDLLA組次之，空白對照組最低；除術后4周空白對照組與PDLLA組比較差異無統計學意義（P gt; 0.05）外，其余各時間點組間比較，差異均有統計學意義（P lt; 0.05）；且隨術后時間延長，PDLLA組及PA/PDLLA組X線片評分均呈增加趨勢（P lt; 0.05）。組織學觀察及Micro-CT檢查示，PA/PDLLA組成骨質量最好，PDLLA組次之，空白對照組最差；骨密度、骨體積與選取感興趣區域體積比值、骨小梁數量、結構模型指數組間比較差異均有統計學意義（P lt; 0.05）。實時熒光定量PCR檢測示PA/PDLLA組成骨相關基因骨鈣素、Ⅰ型膠原、ALP、BMP-2、骨橋蛋白的表達均顯著高于其余兩組（P lt; 0.05）。結論氨等離子體錨定GRGDS短肽活性修飾的PDLLA骨支架，能夠加速SD大鼠股骨干缺損模型的骨修復重建過程。

引用本文： 許子星,陳建庭,許衛紅,朱夏,王長昇,羅鴻斌,李貴雙,陳榮生. 錨定短肽組織工程骨修復大鼠股骨干缺損的成骨觀察. 中國修復重建外科雜志, 2013, 27(5): 520-528. doi: 10.7507/1002-1892.20130118 復制

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.	許子星, 陳建庭, 尹詩衡, 等. 氨等離子體錨定短肽的消旋聚乳酸骨支架研究. 中國修復重建外科雜志, 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.

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. 許子星, 陳建庭, 尹詩衡, 等. 氨等離子體錨定短肽的消旋聚乳酸骨支架研究. 中國修復重建外科雜志, 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.

《中國修復重建外科雜志》

錨定短肽組織工程骨修復大鼠股骨干缺損的成骨觀察

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《中國修復重建外科雜志》

錨定短肽組織工程骨修復大鼠股骨干缺損的成骨觀察

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