可注射性藻酸鍶凝膠應用于骨組織工程的體外研究_《中國修復重建外科雜志》

作者：

涂以濟 ¹ , 吳添龍 ¹ , 葉愛芳 ¹ , 徐浚懷 ¹ , 郭菲 ¹ , 程細高 ²

1 南昌大學醫學院（南昌，330000）;;
2南昌大學第二附屬醫院骨一科;

關鍵詞：

骨組織工程藻酸鍶凝膠藻酸鈣凝膠支架材料

DOI：

10.7507/1002-1892.20130329

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目的體外研究藻酸鍶凝膠作為骨組織工程支架材料的應用潛力，為可注射骨組織工程支架材料的制備提供依據。方法制備2.0wt%藻酸鈉溶液，分別與0.2 mol/L SrCl2及CaCl2膠凝液離子交聯形成藻酸鍶和藻酸鈣凝膠，掃描電鏡觀察微觀結構并測其壓縮模量、溶脹率及降解率。采用全骨髓培養法分離培養兔BMSCs并鑒定，將第5代BMSCs分別接種于藻酸鍶凝膠（實驗組）和藻酸鈣凝膠（對照組），觀察其生長、黏附及增殖情況；兩組凝膠來源的細胞成骨誘導后采用細胞化學法檢測ALP活性，熒光定量RT-PCR檢測成骨相關基因Osterix（OSX）、Ⅰ型膠原、Runx2 mRNA表達，茜素紅染色、Von Kossa染色檢測鈣鹽沉積情況，并設同法制備的藻酸鈣凝膠來源的BMSCs作為空白對照組。結果藻酸鍶與藻酸鈣凝膠微觀結構相似，均有均勻的孔隙結構；藻酸鍶及藻酸鈣凝膠壓縮模量分別為（186.53 ± 8.37）、（152.14 ± 7.45）kPa，比較差異有統計學意義（t=6.853，P=0.002）；溶脹率分別為14.32% ± 1.53%和15.25% ± 1.64%，比較差異無統計學意義（t=0.737，P=0.502）；兩種凝膠的體外降解性均較好，第20、25、30天藻酸鍶凝膠的降解率明顯低于藻酸鈣凝膠（P lt; 0.05）。BMSCs于兩組凝膠中1～4 d時呈懸浮生長，之后逐漸黏附生長并形成大量細胞集落。三維培養第21天，實驗組細胞的DNA含量為（4.38 ± 0.24）g，顯著高于對照組的（3.25 ± 0.21）g（t=8.108，P=0.001）。成骨誘導培養第12天，實驗組ALP活性為（15.28 ± 1.26）U/L，顯著高于對照組的（12.07 ± 1.12）U/L（P lt; 0.05）。實驗組OSX、Ⅰ型膠原、 Runx2 mRNA相對表達量均高于對照組，差異有統計學意義（P lt; 0.05）。成骨誘導培養第21天，兩組茜素紅染色及Von Kossa染色均可見鈣鹽沉積，實驗組的鈣結節數量及磷酸鹽沉積面積均顯著高于對照組（P lt; 0.05）。結論藻酸鍶凝膠具有優良的理化特性，對BMSCs增殖及成骨分化有顯著促進作用，可作為可注射骨組織工程支架材料。

引用本文： 涂以濟,吳添龍,葉愛芳,徐浚懷,郭菲,程細高. 可注射性藻酸鍶凝膠應用于骨組織工程的體外研究. 中國修復重建外科雜志, 2013, 27(12): 1499-1505. doi: 10.7507/1002-1892.20130329 復制

1.	Scott TG, Blackburn G, Ashley M, et al. Advances in bionanomaterials for bone tissue engineering. J Nanosci Nanotechnol, 2013, 13(1): 1-22.
2.	Jakob F, Ebert R, Ignatius A, et al. Bone tissue engineering in osteoporosis. Maturitas, 2013, 75(2): 118-124.
3.	Honda H, Tamai N, Naka N, et al. Bone tissue engineering with bone marrow-derived stromal cells integrated with concentrated growth factor in Rattus norvegicus calvaria defect model. J Artif Organs, 2013. [Epub ahead of print].
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6.	Drury JL, Boontheekul T, Mooney DJ. Cellular cross-linking of peptide modified hydrogels. J Biomech Eng, 2005, 127(2): 220-228.
7.	Ohsumi H, Hirata H, Nagakura T, et al. Enhancement of perineurial repair and inhibition of nerve adhesion by viscous injectable pure alginate sol. Plast Reconstr Surg, 2005, 116(3): 823-830.
8.	Singh B, Sharma DK, Gupta A. The controlled and sustained release of a fungicide from starch and alginate beads. J Environ Sci Health B, 2009, 44(2): 113-122.
9.	Poldervaart MT, Wang H, van der Stok J, et al. Sustained release of BMP-2 in bioprinted alginate for osteogenicity in mice and rats. PLoS One, 2013, 8(8): e72610.
10.	Reyes R, Delgado A, Sánchez E, et al. Repair of an osteochondral defect by sustained delivery of BMP-2 or TGFβ1 from a bilayered alginate-PLGA scaffold. J Tissue Eng Regen Med, 2012. [Epub ahead of print].
11.	Park Y, Sugimoto M, Watrin A, et al. BMP-2 induces the expression of chondrocyte-specific genes in bovine synovium-derived progenitor cells cultured in three-dimensional alginate hydrogel. Osteoarthritis Cartilage, 2005, 13(6): 527-536.
12.	Pors Nielsen S. The biological role of strontium. Bone, 2004, 35(3): 583-588.
13.	Augst AD, Kong HJ, Mooney DJ. Alginate hydrogels as biomaterials. Macromol Biosci, 2006, 6(8): 623-633.
14.	Cowan CM, Soo C, Ting K, et al. Evolving concepts in bone tissue engineering. Curr Top Dev Biol, 2005, 66: 239-285.
15.	Paige KT, Cima LG, Yaremchuk MJ, et al. De novo cartilage generation using calcium alginate-chondrocyte constructs. Plast Reconstr Surg, 1996, 97(1): 168-180.
16.	Xia Y, Mei F, Duan Y, et al. Bone tissue engineering using bone marrow stromal cells and an injectable sodium alginate/gelatin scaffold. J Biomed Mater Res A, 2012, 100(4): 1044-1050.
17.	Smidsrød O, Skjåk-Braek G. Alginate as immobilization matrix for cells. Trends Biotechnol, 1990, 8(3): 71-78.
18.	Wee S, Gombotz WR. Protein release from alginate matrices. Adv Drug Deliv Rev, 1998, 31(3): 267-285.
19.	Peng S, Zhou G, Luk KD, et al. Strontium promotes osteogenic differentiation of mesenchymal stem cells through the Ras/MAPK signaling pathway. Cell Physiol Biochem, 2009, 23(1-3): 165-174.
20.	Capuccini C, Torricelli P, Sima F, et al. Strontium-substituted hydroxyapatite coatings synthesized by pulsed-laser deposition: in vitro osteoblast and osteoclast response. Acta Biomater, 2008, 4(6): 1885-1893.
21.	Barzegar-Jalali M, Hanaee J, Omidi Y, et al. Preparation and evaluation of sustained release calcium alginate beads and matrix tablets of acetazolamide. Drug Res (Stuttg), 2013, 63(2): 60-64.
22.	Ashton RS, Banarjee A, Punyani S, et al. Scaffolds based on degradable alginate hydrogels and poly (lactide-co-glycolide) microspheres for stem cell culture. Biomaterials, 2007, 28(36): 5518-5525.
23.	Mitalipova MM, Rao RR, Hoyer DM, et al. Preserving the genetic integrity of human embryonic stem cells. Nat Biotechnol, 2005, 23(1): 19-20.

1. Scott TG, Blackburn G, Ashley M, et al. Advances in bionanomaterials for bone tissue engineering. J Nanosci Nanotechnol, 2013, 13(1): 1-22.
2. Jakob F, Ebert R, Ignatius A, et al. Bone tissue engineering in osteoporosis. Maturitas, 2013, 75(2): 118-124.
3. Honda H, Tamai N, Naka N, et al. Bone tissue engineering with bone marrow-derived stromal cells integrated with concentrated growth factor in Rattus norvegicus calvaria defect model. J Artif Organs, 2013. [Epub ahead of print].
4. 參考文獻.
5. Haidar ZS, Hamdy RC, Tabrizian M. Delivery of recombinant bone morphogenetic proteins for bone regeneration and repair. Part B: Delivery systems for BMPs in orthopaedic and craniofacial tissue engineering. Biotechnol Lett, 2009, 31(12): 1825-1835.
6. Drury JL, Boontheekul T, Mooney DJ. Cellular cross-linking of peptide modified hydrogels. J Biomech Eng, 2005, 127(2): 220-228.
7. Ohsumi H, Hirata H, Nagakura T, et al. Enhancement of perineurial repair and inhibition of nerve adhesion by viscous injectable pure alginate sol. Plast Reconstr Surg, 2005, 116(3): 823-830.
8. Singh B, Sharma DK, Gupta A. The controlled and sustained release of a fungicide from starch and alginate beads. J Environ Sci Health B, 2009, 44(2): 113-122.
9. Poldervaart MT, Wang H, van der Stok J, et al. Sustained release of BMP-2 in bioprinted alginate for osteogenicity in mice and rats. PLoS One, 2013, 8(8): e72610.
10. Reyes R, Delgado A, Sánchez E, et al. Repair of an osteochondral defect by sustained delivery of BMP-2 or TGFβ1 from a bilayered alginate-PLGA scaffold. J Tissue Eng Regen Med, 2012. [Epub ahead of print].
11. Park Y, Sugimoto M, Watrin A, et al. BMP-2 induces the expression of chondrocyte-specific genes in bovine synovium-derived progenitor cells cultured in three-dimensional alginate hydrogel. Osteoarthritis Cartilage, 2005, 13(6): 527-536.
12. Pors Nielsen S. The biological role of strontium. Bone, 2004, 35(3): 583-588.
13. Augst AD, Kong HJ, Mooney DJ. Alginate hydrogels as biomaterials. Macromol Biosci, 2006, 6(8): 623-633.
14. Cowan CM, Soo C, Ting K, et al. Evolving concepts in bone tissue engineering. Curr Top Dev Biol, 2005, 66: 239-285.
15. Paige KT, Cima LG, Yaremchuk MJ, et al. De novo cartilage generation using calcium alginate-chondrocyte constructs. Plast Reconstr Surg, 1996, 97(1): 168-180.
16. Xia Y, Mei F, Duan Y, et al. Bone tissue engineering using bone marrow stromal cells and an injectable sodium alginate/gelatin scaffold. J Biomed Mater Res A, 2012, 100(4): 1044-1050.
17. Smidsrød O, Skjåk-Braek G. Alginate as immobilization matrix for cells. Trends Biotechnol, 1990, 8(3): 71-78.
18. Wee S, Gombotz WR. Protein release from alginate matrices. Adv Drug Deliv Rev, 1998, 31(3): 267-285.
19. Peng S, Zhou G, Luk KD, et al. Strontium promotes osteogenic differentiation of mesenchymal stem cells through the Ras/MAPK signaling pathway. Cell Physiol Biochem, 2009, 23(1-3): 165-174.
20. Capuccini C, Torricelli P, Sima F, et al. Strontium-substituted hydroxyapatite coatings synthesized by pulsed-laser deposition: in vitro osteoblast and osteoclast response. Acta Biomater, 2008, 4(6): 1885-1893.
21. Barzegar-Jalali M, Hanaee J, Omidi Y, et al. Preparation and evaluation of sustained release calcium alginate beads and matrix tablets of acetazolamide. Drug Res (Stuttg), 2013, 63(2): 60-64.
22. Ashton RS, Banarjee A, Punyani S, et al. Scaffolds based on degradable alginate hydrogels and poly (lactide-co-glycolide) microspheres for stem cell culture. Biomaterials, 2007, 28(36): 5518-5525.
23. Mitalipova MM, Rao RR, Hoyer DM, et al. Preserving the genetic integrity of human embryonic stem cells. Nat Biotechnol, 2005, 23(1): 19-20.

《中國修復重建外科雜志》

可注射性藻酸鍶凝膠應用于骨組織工程的體外研究

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《中國修復重建外科雜志》

可注射性藻酸鍶凝膠應用于骨組織工程的體外研究

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