脫細胞小牛肌腱組織形態學和生物力學特性研究_《中國修復重建外科雜志》

作者：

江燕林 ¹ , 張憶 ¹ , 楊潔亮 ² , 羅靜聰 ^1,2 , 秦廷武 ^1,2

四川大學華西醫院（成都，610041）1 再生醫學研究中心;;
2 生物治療國家重點實驗室·干細胞與組織工程研究室;

關鍵詞：

脫細胞肌腱形態學組織學生物力學小牛

DOI：

10.7507/1002-1892.20130125

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目的研究反復凍融結合核酸酶處理小牛肌腱脫細胞效果以及對肌腱組織形態、結構、生化成分和力學特性的影響。方法取新鮮1日齡西門塔爾小牛跟腱48根，隨機分為3組（n=16）：A組為正常對照組，B組采用液氮冷凍/37℃復溫反復凍融5次，C組在反復凍融基礎上結合核酸酶處理24 h。每組取2根跟腱用于掃描電鏡觀察，3根用于、組織學及免疫組織化學染色觀察，3根用于DNA殘留量檢測，8根用于生物力學檢測。結果掃描電鏡觀察示A、B組膠原纖維排列致密有序，形態完整，未見斷裂；C組膠原纖維排列松散，幾乎無斷裂。阿利新藍、天狼星紅染色及免疫組織化學染色示C組糖胺聚糖、Ⅰ型膠原、Ⅲ型膠原、纖維連接蛋白大部分被保留；HE染色和DAPI染色示A、B組膠原纖維間可見較多完整的細胞核，而C組膠原纖維之間未見完整細胞核，但腱內膜上仍有部分細胞核殘留。A、B、C組的DNA殘留量分別為（0.24 ± 0.12）、（0.16 ± 0.07）、（0.05 ± 0.02）μg/mg，C組顯著低于A、B組（P lt; 0.05）；A、B組間差異無統計學意義（P gt; 0.05）。生物力學檢測顯示，A、B、C組間拉伸強度、斷裂應變、剛度和彈性模量差異均無統計學意義（P gt; 0.05）。結論反復凍融結合核酸酶處理小牛肌腱，可有效去除細胞成分，同時基本保留肌腱原始膠原纖維結構、形態、大部分細胞外基質成分和力學特性。

引用本文： 江燕林,張憶,楊潔亮,羅靜聰,秦廷武. 脫細胞小牛肌腱組織形態學和生物力學特性研究. 中國修復重建外科雜志, 2013, 27(5): 565-570. doi: 10.7507/1002-1892.20130125 復制

1.	Carey JL, Dunn WR, Dahm DL, et al. A systematic review of anterior cruciate ligament reconstruction with autograft compared with allograft. J Bone Joint Surg (Am), 2009, 91(9): 2242-2250.
2.	Whitlock PW, Smith TL, Poehling GG, et al. A naturally derived, cytocompatible, and architecturally optimized scaffold for tendon and ligament regeneration. Biomaterials, 2007, 28(29): 4321-4329.
3.	Stone KR, Walgenbach AW, Turek TJ, et al. Anterior cruciate ligament reconstruction a porcinexenograft: a serologic, histologic, and biomechanical study in primates. Arthroscopy, 2007, 23(4): 411-419.
4.	Stone KR, Abdel-Motal UM, Walgenbach AW, et al. Replacement of human anterior cruciate ligaments with pig ligaments: a model for anti-non-gal antibody response in long-term xenotransplantation. Transplantation, 2007, 83(2): 211-219.
5.	Randall KL, Booth BA, Miller AJ, et al. Use of an acellular regenerative tissue matrix in combination with vacuumassisted closure therapy for treatment of a diabetic foot wound. J Foot Ankle Surg, 2008, 47(5): 430-433.
6.	Pridgen BC, Woon CY, Kim K, et al. Flexor tendon tissue engineering: acellularization of human flexor tendons with preservation of biomechanical properties and biocompatibility. Tissue Eng Part C Methods, 2011, 17(8): 819-828.
7.	Harrison RD, Gratzer PF. Effect of extraction protocols and epidermal growth factor on the cellular repopulation of decellularized anterior cruciate ligament allografts. J Biomed Mater Res A, 2005, 75(4): 841-854.
8.	Ning LJ, Zhang Y, Chen XH, et al. Preparation and characterization of decellularized tendon slices for tendon tissue engineering. J Biomed Mater Res A, 2012, 100(6): 1448-1456.
9.	Badylak SF, Freytes DO, Gilbert TW. Extracellular matrix as a biological scaffold material: Structure and function. Acta Biomater, 2009, 5(1): 1-13.
10.	Azuma C, Tohyama H, Nakamura H, et al. Antibody neutralization of TGF-beta enhances the deterioration of collagen fascicles in a tissue-cultured tendon matrix with ex vivo fibroblast infiltration. J Biomech, 2007, 40(10): 2184-2190.
11.	Ponce Márquez S, Martínez VS, McIntosh Ambrose W, et al. Decellularization of bovine corneas for tissue engineering applications. Acta Biomater, 2009, 5(6): 1839-1847.
12.	Aurora A, McCarron J, Iannotti JP, et al. Commercially available extracellular matrix materials for rotator cuff repairs: state of the art and future trends. J Shoulder Elbow Surg, 2007, 16(5 Suppl): S171-178.
13.	Yang JJ, Jang EC, Lee JS, et al. The Effect of amniotic membrane transplantation on tendon-healing in a rabbit Achilles tendon model. Tissue Engineering and Regenerative Medicine, 2010, 7(3): 323-329.
14.	Gilbert TW, Stewart AM, Simmons BA, et al. Degradation and remodeling of small intestinal submucosa in canine Achilles tendon repair. J Bone Joint Surg (Am), 2007, 89(6): 621-630.
15.	Androgens C, Spragg RK, Derwin KA. Mechanical conditioning of cell-seeded small intestine submucosa: a potential tissue-engineering strategy for tendon repair. Tissue Eng, 2007, 13(2): 233-240.
16.	Derwin KA, Badylak SF, Steinmann SP, et al. Extracellular matrix scaffold devices for rotator cuff repair. J Shoulder Elbow Surg, 2010, 19(3): 467-476.
17.	Vavken P, Joshi S, Murray MM. Triton-X is most effective among three decellularization agents for ACL tissue engineering. J Orthop Res, 2009, 27(12): 1612-1618.
18.	Cartmell JS, Dunn MG. Effect of chemical treatments on tendon cellularity and mechanical properties. J Biomed Mater Res, 2000, 49(1): 134-140.
19.	Deeken CR, White AK, Bachman SL, et al. Method of preparing a decellularized porcine tendon using tributyl phosphate. J Biomed Mater Res B Appl Biomater, 2011, 96(2): 199-206.
20.	Minami A, Ishii S, Ogino T, et al. Effect of the immunological antigenicity of the allogenaic tendon grafting. Hand, 1982, 14(2): 111-119.
21.	Qin TW, Chen QS, Sun YL, et al. Mechanical characteristics of native tendon slices for tissue engineering scaffold. J Biomed Mater Res B, 2012, 100(3): 752-758.

1. Carey JL, Dunn WR, Dahm DL, et al. A systematic review of anterior cruciate ligament reconstruction with autograft compared with allograft. J Bone Joint Surg (Am), 2009, 91(9): 2242-2250.
2. Whitlock PW, Smith TL, Poehling GG, et al. A naturally derived, cytocompatible, and architecturally optimized scaffold for tendon and ligament regeneration. Biomaterials, 2007, 28(29): 4321-4329.
3. Stone KR, Walgenbach AW, Turek TJ, et al. Anterior cruciate ligament reconstruction a porcinexenograft: a serologic, histologic, and biomechanical study in primates. Arthroscopy, 2007, 23(4): 411-419.
4. Stone KR, Abdel-Motal UM, Walgenbach AW, et al. Replacement of human anterior cruciate ligaments with pig ligaments: a model for anti-non-gal antibody response in long-term xenotransplantation. Transplantation, 2007, 83(2): 211-219.
5. Randall KL, Booth BA, Miller AJ, et al. Use of an acellular regenerative tissue matrix in combination with vacuumassisted closure therapy for treatment of a diabetic foot wound. J Foot Ankle Surg, 2008, 47(5): 430-433.
6. Pridgen BC, Woon CY, Kim K, et al. Flexor tendon tissue engineering: acellularization of human flexor tendons with preservation of biomechanical properties and biocompatibility. Tissue Eng Part C Methods, 2011, 17(8): 819-828.
7. Harrison RD, Gratzer PF. Effect of extraction protocols and epidermal growth factor on the cellular repopulation of decellularized anterior cruciate ligament allografts. J Biomed Mater Res A, 2005, 75(4): 841-854.
8. Ning LJ, Zhang Y, Chen XH, et al. Preparation and characterization of decellularized tendon slices for tendon tissue engineering. J Biomed Mater Res A, 2012, 100(6): 1448-1456.
9. Badylak SF, Freytes DO, Gilbert TW. Extracellular matrix as a biological scaffold material: Structure and function. Acta Biomater, 2009, 5(1): 1-13.
10. Azuma C, Tohyama H, Nakamura H, et al. Antibody neutralization of TGF-beta enhances the deterioration of collagen fascicles in a tissue-cultured tendon matrix with ex vivo fibroblast infiltration. J Biomech, 2007, 40(10): 2184-2190.
11. Ponce Márquez S, Martínez VS, McIntosh Ambrose W, et al. Decellularization of bovine corneas for tissue engineering applications. Acta Biomater, 2009, 5(6): 1839-1847.
12. Aurora A, McCarron J, Iannotti JP, et al. Commercially available extracellular matrix materials for rotator cuff repairs: state of the art and future trends. J Shoulder Elbow Surg, 2007, 16(5 Suppl): S171-178.
13. Yang JJ, Jang EC, Lee JS, et al. The Effect of amniotic membrane transplantation on tendon-healing in a rabbit Achilles tendon model. Tissue Engineering and Regenerative Medicine, 2010, 7(3): 323-329.
14. Gilbert TW, Stewart AM, Simmons BA, et al. Degradation and remodeling of small intestinal submucosa in canine Achilles tendon repair. J Bone Joint Surg (Am), 2007, 89(6): 621-630.
15. Androgens C, Spragg RK, Derwin KA. Mechanical conditioning of cell-seeded small intestine submucosa: a potential tissue-engineering strategy for tendon repair. Tissue Eng, 2007, 13(2): 233-240.
16. Derwin KA, Badylak SF, Steinmann SP, et al. Extracellular matrix scaffold devices for rotator cuff repair. J Shoulder Elbow Surg, 2010, 19(3): 467-476.
17. Vavken P, Joshi S, Murray MM. Triton-X is most effective among three decellularization agents for ACL tissue engineering. J Orthop Res, 2009, 27(12): 1612-1618.
18. Cartmell JS, Dunn MG. Effect of chemical treatments on tendon cellularity and mechanical properties. J Biomed Mater Res, 2000, 49(1): 134-140.
19. Deeken CR, White AK, Bachman SL, et al. Method of preparing a decellularized porcine tendon using tributyl phosphate. J Biomed Mater Res B Appl Biomater, 2011, 96(2): 199-206.
20. Minami A, Ishii S, Ogino T, et al. Effect of the immunological antigenicity of the allogenaic tendon grafting. Hand, 1982, 14(2): 111-119.
21. Qin TW, Chen QS, Sun YL, et al. Mechanical characteristics of native tendon slices for tissue engineering scaffold. J Biomed Mater Res B, 2012, 100(3): 752-758.

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脫細胞小牛肌腱組織形態學和生物力學特性研究

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