新型一體化纖維環-髓核雙相支架的制備與評估_《中國修復重建外科雜志》

作者：

許海委 ^1,2 , 徐寶山 ¹ , 楊強 ¹ , 李秀蘭 ³ , 馬信龍 ⁴ , 夏群 ¹ , 張春秋 ⁵ , 伍耀宏 ^1,2

天津醫院（天津，300211）1脊柱外科，3骨科研究所細胞工程室;;
2天津醫科大學研究生院;;
4天津醫科大學總醫院骨科;;
5 天津理工大學機械工程學院;

關鍵詞：

脫細胞脫鈣骨基質脫細胞髓核基質組織工程椎間盤雙相支架豬兔

DOI：

10.7507/1002-1892.20130108

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目的用脫細胞脫鈣骨基質和脫細胞髓核基質構建新型一體化纖維環-髓核雙相支架，并進行理化檢測，探討其作為組織工程椎間盤支架的可行性。方法取豬股骨近端松質骨，塑形成外徑10 mm、內徑5 mm、厚3 mm的骨環，經脫脂、脫鈣、脫細胞處理制備成纖維環相支架；取豬尾髓核組織，Triton X-100和脫氧膽酸聯合脫細胞后粉碎、離心，制備脫細胞髓核基質；將制備的脫細胞髓核基質注入纖維環相支架中央，冷凍干燥，聯合應用紫外照射和碳化二亞胺進行交聯，制備一體化纖維環-髓核雙相支架。通過大體觀察、HE染色、掃描電鏡觀察支架結構；測定支架的孔隙率、吸水率、壓縮彈性模量；MTT法檢測25%、50%、100%支架浸提液對新西蘭大白兔脂肪來源干細胞（adipose-derived stem cells，ADSCs）增殖的影響；將ADSCs接種到支架上，活/死細胞染色觀察細胞在支架上的活性。結果大體觀察支架呈乳白色；HE染色可見支架均質紅染，無細胞成分殘留；掃描電鏡可見外層纖維環相支架孔徑大小均勻一致，孔隙相互貫通，中央脫細胞髓核基質微絲相互連接，形成均勻網狀結構，交界處連接緊密。纖維環相支架孔徑為（343.00± 88.25）μm，一體化纖維環-髓核雙相支架孔隙率為82.98% ± 7.02%、吸水率為621.53% ± 53.31%，壓縮彈性模量為（89.07± 8.73）kPa。經MTT測定支架浸提液對ADSCs增殖無影響；活/死細胞染色可見細胞在支架上生長良好，無死細胞。結論用脫細胞脫鈣骨基質和脫細胞髓核基質構建的一體化纖維環-髓核雙相支架具有合適的孔徑和孔隙率且無毒，力學性能與人正常椎間盤接近，是構建組織工程椎間盤的理想支架載體。

引用本文： 許海委,徐寶山,楊強,李秀蘭,馬信龍,夏群,張春秋,伍耀宏. 新型一體化纖維環-髓核雙相支架的制備與評估. 中國修復重建外科雜志, 2013, 27(4): 475-480. doi: 10.7507/1002-1892.20130108 復制

1.	Li CQ, Huang B, Luo G, et al. Construction of collagen II/hyaluronate/chondroitin-6-sulfate tri-copolymer scaffold for nucleus pulposus tissue engineering and preliminary analysis of its physico-chemical properties and biocompatibility. J Mater Sci Mater Med, 2010, 21(2): 741-751.
2.	Koepsell L, Zhang L, Neufeld D, et al. Electrospun nanofibrous polycaprolactone scaffolds for tissue engineering of annulus fibrosus. Macromol Biosci, 2011, 11(3): 391-399.
3.	Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues and organs. Biomaterials, 2006, 27(19): 3675-3683.
4.	Wan Y, Feng G, Shen FH, et al. Biphasic scaffold for annulus fibrosus tissue regeneration. Biomaterials, 2008, 29(6): 643-652.
5.	Mercuri JJ, Gill SS, Simionescu DT. Novel tissue-derived biomimetic scaffold for regenerating the human nucleus pulposus. J Biomed Mater Res A, 2011, 96(2): 422-435.
6.	Zhang R, Ma PX. Biomimetic polymer/apatite composite scaffolds for mineralized tissue engineering. Macromol Biosci, 2004, 4(2): 100-111.
7.	許海委, 徐寶山, 楊強, 等. 細胞共培養法誘導兔脂肪來源干細胞向軟骨細胞分化的實驗研究. 中國修復重建外科雜志, 2013, 27(2): 193-198.
8.	楊強, 彭江, 盧世璧, 等. 軟骨脫細胞基質多孔支架與骨髓基質干細胞體外構建組織工程軟骨的研究. 中華醫學雜志, 2011, 91(17): 1161-1166.
9.	李長青, 周躍. 椎間盤組織工程支架材料研究進展. 中國修復重建外科雜志, 2005, 19(12): 1033-1035.
10.	Calderon L, Collin E, Velasco-Bayon D, et al. Type II collagen-hyaluronan hydrogel—a step towards a scaffold for intervertebral disc tissue engineering. Eur Cell Mater, 2010, 20: 134-48.
11.	Wang B, Borazjani A, Tahai M, et al. Fabrication of cardiac patch with decellularized porcine myocardial scaffold and bone marrowmononuclear cells. J Biomed Mater Res A, 2010, 94(4): 1100-1110.
12.	Stapleton TW, Ingram J, Fisher J, et al. Investigation of the regenerative capacity of an acellular porcine medial meniscus for tissueengineering applications. Tissue Eng Part A, 2011, 17(1-2): 231-242.
13.	Zhao Y, Zhang Z, Wang J, et al. Abdominal hernia repair with a decellularized dermal scaffold seeded with autologous bone marrow-derived mesenchymal stem cells. Artif Organs, 2012, 36(3): 247-255.
14.	黃惠民, 馬良龍, 任宏, 等. 骨髓間質干細胞和脫細胞基質構建組織工程血管的動物實驗. 中華醫學雜志, 2007, 87(48): 3440-3442.
15.	Du L, Wu X. Development and characterization of a full-thickness acellular porcine cornea matrix for tissueengineering. Artif Organs, 2011, 35(7): 691-705.
16.	Cheng HL, Loai Y, Beaumont M, et al. The acellular matrix (ACM) for bladder tissue engineering: A quantitative magnetic resonanceimaging study. Magn Reson Med, 2010, 64(2): 341-348.
17.	Xu CC, Chan RW, Weinberger DG, et al. A bovine acellular scaffold for vocal fold reconstruction in a rat model. J Biomed Mater Res A, 2010, 92(1): 18-32.
18.	Zhuang Y, Huang B, Li CQ, et al. Construction of tissue-engineered composite intervertebral disc and preliminary morphological and biochemical evaluation. Biochem Biophys Res Commun, 2011, 407(2): 327-332.
19.	潘勇, 周躍, 郝勇, 等. 脫礦脫細胞骨基質環支架體外構建組織工程化椎間盤纖維環. 中國脊柱脊髓雜志, 2009, 19(6): 451-457.
20.	Bowles RD, Gebhard HH, Härtl R, et al. Tissue-engineered intervertebral discs produce new matrix, maintain disc height, and restore biomechanical function to the rodent spine. Proc Natl Acad Sci U S A, 2011, 108(32): 13106-13111.

1. Li CQ, Huang B, Luo G, et al. Construction of collagen II/hyaluronate/chondroitin-6-sulfate tri-copolymer scaffold for nucleus pulposus tissue engineering and preliminary analysis of its physico-chemical properties and biocompatibility. J Mater Sci Mater Med, 2010, 21(2): 741-751.
2. Koepsell L, Zhang L, Neufeld D, et al. Electrospun nanofibrous polycaprolactone scaffolds for tissue engineering of annulus fibrosus. Macromol Biosci, 2011, 11(3): 391-399.
3. Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues and organs. Biomaterials, 2006, 27(19): 3675-3683.
4. Wan Y, Feng G, Shen FH, et al. Biphasic scaffold for annulus fibrosus tissue regeneration. Biomaterials, 2008, 29(6): 643-652.
5. Mercuri JJ, Gill SS, Simionescu DT. Novel tissue-derived biomimetic scaffold for regenerating the human nucleus pulposus. J Biomed Mater Res A, 2011, 96(2): 422-435.
6. Zhang R, Ma PX. Biomimetic polymer/apatite composite scaffolds for mineralized tissue engineering. Macromol Biosci, 2004, 4(2): 100-111.
7. 許海委, 徐寶山, 楊強, 等. 細胞共培養法誘導兔脂肪來源干細胞向軟骨細胞分化的實驗研究. 中國修復重建外科雜志, 2013, 27(2): 193-198.
8. 楊強, 彭江, 盧世璧, 等. 軟骨脫細胞基質多孔支架與骨髓基質干細胞體外構建組織工程軟骨的研究. 中華醫學雜志, 2011, 91(17): 1161-1166.
9. 李長青, 周躍. 椎間盤組織工程支架材料研究進展. 中國修復重建外科雜志, 2005, 19(12): 1033-1035.
10. Calderon L, Collin E, Velasco-Bayon D, et al. Type II collagen-hyaluronan hydrogel—a step towards a scaffold for intervertebral disc tissue engineering. Eur Cell Mater, 2010, 20: 134-48.
11. Wang B, Borazjani A, Tahai M, et al. Fabrication of cardiac patch with decellularized porcine myocardial scaffold and bone marrowmononuclear cells. J Biomed Mater Res A, 2010, 94(4): 1100-1110.
12. Stapleton TW, Ingram J, Fisher J, et al. Investigation of the regenerative capacity of an acellular porcine medial meniscus for tissueengineering applications. Tissue Eng Part A, 2011, 17(1-2): 231-242.
13. Zhao Y, Zhang Z, Wang J, et al. Abdominal hernia repair with a decellularized dermal scaffold seeded with autologous bone marrow-derived mesenchymal stem cells. Artif Organs, 2012, 36(3): 247-255.
14. 黃惠民, 馬良龍, 任宏, 等. 骨髓間質干細胞和脫細胞基質構建組織工程血管的動物實驗. 中華醫學雜志, 2007, 87(48): 3440-3442.
15. Du L, Wu X. Development and characterization of a full-thickness acellular porcine cornea matrix for tissueengineering. Artif Organs, 2011, 35(7): 691-705.
16. Cheng HL, Loai Y, Beaumont M, et al. The acellular matrix (ACM) for bladder tissue engineering: A quantitative magnetic resonanceimaging study. Magn Reson Med, 2010, 64(2): 341-348.
17. Xu CC, Chan RW, Weinberger DG, et al. A bovine acellular scaffold for vocal fold reconstruction in a rat model. J Biomed Mater Res A, 2010, 92(1): 18-32.
18. Zhuang Y, Huang B, Li CQ, et al. Construction of tissue-engineered composite intervertebral disc and preliminary morphological and biochemical evaluation. Biochem Biophys Res Commun, 2011, 407(2): 327-332.
19. 潘勇, 周躍, 郝勇, 等. 脫礦脫細胞骨基質環支架體外構建組織工程化椎間盤纖維環. 中國脊柱脊髓雜志, 2009, 19(6): 451-457.
20. Bowles RD, Gebhard HH, Härtl R, et al. Tissue-engineered intervertebral discs produce new matrix, maintain disc height, and restore biomechanical function to the rodent spine. Proc Natl Acad Sci U S A, 2011, 108(32): 13106-13111.

《中國修復重建外科雜志》

新型一體化纖維環-髓核雙相支架的制備與評估

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《中國修復重建外科雜志》

新型一體化纖維環-髓核雙相支架的制備與評估

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