COMPARISON OF AORTIC EXTRACELLULAR MATRIX SCAFFOLD BY DIFFERENT PROTOCOLS FOR DECELLULARIZATION_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

PULei ¹ , WUJian ¹ , MENGMingyao ² , NIHaiyan ³ , YEFei ⁴ ,  LIYaxiong ¹ ,  JIANGLihong ¹

1. Department of Cardiovascular Surgery, Kunming Yan'an Hospital, Kunming Yunnan, 650051, P. R. China;
2. Department of Central Laboratory, Kunming Yan'an Hospital, Kunming Yunnan, 650051, P. R. China;
3. Department of Pathology, Kunming Yan'an Hospital, Kunming Yunnan, 650051, P. R. China;
1. Faculty of Science, Kunming University of Science and Technology, Kunming Yan'an Hospital, Kunming Yunnan, 650051, P. R. China;

Corresponding author：

LIYaxiong, Email: liyaxiong62@aliyun.com; JIANGLihong, Email: jianglihong_yayy@163.com

Keywords：

Vessel tissue engineering; Scaffold material; Extracellular matrix; Decellularization; Porcine; Rat

DOI：

10.7507/1002-1892.20140305

Video：

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Objective To prepare the aortic extracellular matrix (ECM) scaffold by using different methods to decellularize porcine ascending aorta and to comprehensively compare the efficiency of decellularization and the damage of ECM, evaluation of biomechanical property and biocompatibil ity. Methods Thirty specimens of fresh porcine ascending aorta were randomly divided into 6 groups (n=5). The porcine ascending aorta was decellularized by 5 different protocols in groups A-E: 0.1% trypsin/0.02% ethylenediamine tetraacetic acid (EDTA)/PBS was used in group A, 1%Triton X-100/0.02% EDTA/ distilled water in group B, 1% sodium deoxycholic acid/distilled water in group C, 0.5% sodium deoxycholic acid/0.5% sodium dodecyl sulfate/distilled water in group D, and 1% deoxycholic acid/distilled water in group E; and the porcine ascending aorta was not decellularized as control in group F. The ascending aorta scaffolds were investigated by gross examination, HE staining, DNA quantitative analysis, immunohistochemistry, and scanning electron microscopy were used to observe the efficiency of decellularization, microstructure of the ECM, the damage of collagen type Ⅰ and elastin, the structure of intimal surface, and biomechanical property. The 90 Sprague Dawley rats were randomly divided into 6 groups (n=15). Each scaffold was implanted in the abdominal muscles of rats respectively to evaluate the immunogenicity and biocompatibil ity. Results HE staining and quantitative analysis of DNA showed that the cells were completely removed only in groups A and D. The expression of collagen type Ⅰ in group A was significantly lower than that in the other 5 groups (P < 0.05), and serious damage of the basement membrane and decreased beomechanical property were observed. The maximum stress and tensile strength in group A was significantly lower than those in the other groups (P < 0.05), and elongation at break was significantly higher than that in the other groups (P < 0.05). The destruction of collagen type Ⅰ was significant (P < 0.05) in group D, but the basement membrane was integrity, the biomechanical properties were close to the natural blood vessels (group F) (P > 0.05). Implantation results showed that the scaffold of group D had superior immunogenicity and histocompatibility to the scaffold of the other groups. The inflammatory reaction was gentle and the number of the inflammatory cell infiltration was lower in group D than in other groups (P < 0.05). Conclusion It is concludes that 0.5% sodium deoxycholic acid/0.5% sodium dodecyl sulfate/distilled water is more suitable for the decellularization of porcine aorta, by which the acquired ECM scaffold has the potential for constructing tissue engineered vessel.

Citation： PULei, WUJian, MENGMingyao, NIHaiyan, YEFei, LIYaxiong, JIANGLihong. COMPARISON OF AORTIC EXTRACELLULAR MATRIX SCAFFOLD BY DIFFERENT PROTOCOLS FOR DECELLULARIZATION. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(11): 1413-1421. doi: 10.7507/1002-1892.20140305 Copy

1.	Naguib MA, Dob DP, Gatzoulis MA. A functional understanding of moderate to complex congenital heart disease and the impact of pregnancy. PartⅡ:tetralogy of Fallot, Eisenmenger's syndrome and the Fontan operation. Int J Obstet Anesth, 2010, 19(3):306-312.
2.	Kogon B. Is the extracardiac conduit the preferred Fontan approach for patients with univentricular hearts? The extracardiac conduit is the preferred Fontan approach for patients with univentricular hearts. Circulation, 2012, 126(21):2511-2515.
3.	Naito Y, Rocco K, Kurobe H, et al. Tissue engineering in the vasculature. Anat Rec (Hoboken), 2014, 297(1):83-97.
4.	Iyengar AJ, Winlaw DS, Galati JC, et al. Trends in Fontan surgery and risk factors for early adverse outcomes after Fontan surgery:The Austral ia and New Zealand Fontan Registry experience. J Thorac Cardiovasc Surg, 2014, 148(2):566-575.
5.	Patterson JT, Gilliland T, Maxfield MW, et al. Tissue-engineered vascular grafts for use in the treatment of congenital heart disease:from the bench to the cl inic and back again. Regen Med, 2012, 7(3):409-419.
6.	Shinoka T, Breuer C. Tissue-engineered blood vessels in pediatric cardiac surgery. Yale J Biol Med, 2008, 81(4):161-166.
7.	Naito Y, Shinoka T, Duncan D, et al. Vascular tissue engineering:towards the next generation vascular grafts. Adv Drug Deliv Rev, 2011, 63(4-5):312-323.
8.	Zou Y, Zhang Y. Mechanical evaluation of decellularized porcine thoracic aorta. J Surg Res, 2012, 175(2):359-368.
9.	Bader A, Steinhoff G, Strobl K, et al. Engineering of human vascular aortic tissue based on a xenogeneic starter matrix. Transplantation, 2000, 70(1):7-14.
10.	Badylak SF, Freytes DO, Gilbert TW. Extracellular matrix as a biological scaffold material:Structure and function. Acta Biomater, 2009, 5(1):1-13.
11.	Weber B, Emmert MY, Schoenauer R, et al. Tissue engineering on matrix: future of autologous tissue replacement. Semin Immunopathol, 2011, 33(3):307-315.
12.	马良龙, 黄惠民, 李建华, 等.新法制备动脉脱细胞基质的实验研究.中华小儿外科杂志, 2009, 30(7):468-471.
13.	张建, 吴英锋, 陈亮.猪主动脉脱细胞基质的简化制备及生物学评价.中国修复重建外科杂志, 2008, 22(3):364-369.
14.	黄华梅, 谢德明, 罗洁芳.应用脱细胞血管基质构建组织工程血管的初步研究.中国病理生理杂志, 2008, 24(4):792-796.
15.	Yang M, Chen CZ, Wang XN, et al. Favorable effects of the detergent and enzyme extraction method for preparing decellularized bovine pericardium scaffold for tissue engineered heart valves. J Biomed Mater Res B Appl Biomater, 2009, 91(1):354-361.
16.	Cebotari S, Tudorache I, Jaekel T, et al. Detergent decellularization of heart valves for tissue engineering:toxicological effects of residual detergents on human endothelial cells. Artif Organs, 2010, 34(3):206-210.
17.	Boer U, Lohrenz A, Klingenberg M, et al. The effect of detergent-based decellularization procedures on cellular proteins and immunogenicity in equine carotid artery grafts. Biomaterials, 2011, 32(36):9730-9737.
18.	Cebotari S, Tudorache I, Ciubotaru A, et al. Use of fresh decellularized allografts for pulmonary valve replacement may reduce the reoperation rate in children and young adults:early report. Circulation, 2011, 124(11 Suppl):S115-S123.
19.	Bloch O, Erdbrugger W, Volker W, et al. Extracellular matrix in deoxycholic acid decellularized aortic heart valves. Med Sci Monit, 2012, 18(12):BR487-R492.
20.	Honge JL, Funder J, Hansen E, et al. Recellularization of aortic valves in pigs. Eur J Cardiothorac Surg. 2011, 39(6):829-834.
21.	Tudorache I, Cebotari S, Sturz G, et al. Tissue engineering of heart valves:biomechanical and morphological properties of decellularized heart valves. J Heart Valve Dis. 2007, 16(5):567-573, 574.
22.	Badylak SF. The extracellular matrix as a biologic scaffold material. Biomaterials, 2007, 28(25):3587-3593.
23.	Knight RL, Wilcox HE, Korossis SA, et al. The use of acellular matrices for the tissue engineering of cardiac valves. Proc Inst Mech Eng H, 2008, 222(1):129-143.
24.	Galil i U. Theα-Gal epitope (Galα1-3Galβ1-4GlcNAc-R) in xenotransplantation. Biochimie, 2001, 83(7):557-563.
25.	Schoen FJ, Levy RJ. Calcification of tissue heart valve substitutes:progress toward understanding and prevention. Ann Thorac Surg, 2005, 79(3):1072-1080.
26.	Cicha I, Ruffer A, Cesnjevar R, et al. Early obstruction of decellularized xenogenic valves in pediatric patients:involvement of inflammatory and fibroproliferative processes. Cardiovasc Pathol, 2011, 20(4):222-231.
27.	Brown BN, Ratner BD, Goodman SB, et al. Macrophage polarization: an opportunity for improved outcomes in biomaterials and regenerative medicine. Biomaterials, 2012, 33(15):3792-3802.
28.	Badylak SF, Gilbert TW. Immune response to biologic scaffold materials. Semin Immunol, 2008, 20(2):109-116.

1. Naguib MA, Dob DP, Gatzoulis MA. A functional understanding of moderate to complex congenital heart disease and the impact of pregnancy. PartⅡ:tetralogy of Fallot, Eisenmenger's syndrome and the Fontan operation. Int J Obstet Anesth, 2010, 19(3):306-312.
2. Kogon B. Is the extracardiac conduit the preferred Fontan approach for patients with univentricular hearts? The extracardiac conduit is the preferred Fontan approach for patients with univentricular hearts. Circulation, 2012, 126(21):2511-2515.
3. Naito Y, Rocco K, Kurobe H, et al. Tissue engineering in the vasculature. Anat Rec (Hoboken), 2014, 297(1):83-97.
4. Iyengar AJ, Winlaw DS, Galati JC, et al. Trends in Fontan surgery and risk factors for early adverse outcomes after Fontan surgery:The Austral ia and New Zealand Fontan Registry experience. J Thorac Cardiovasc Surg, 2014, 148(2):566-575.
5. Patterson JT, Gilliland T, Maxfield MW, et al. Tissue-engineered vascular grafts for use in the treatment of congenital heart disease:from the bench to the cl inic and back again. Regen Med, 2012, 7(3):409-419.
6. Shinoka T, Breuer C. Tissue-engineered blood vessels in pediatric cardiac surgery. Yale J Biol Med, 2008, 81(4):161-166.
7. Naito Y, Shinoka T, Duncan D, et al. Vascular tissue engineering:towards the next generation vascular grafts. Adv Drug Deliv Rev, 2011, 63(4-5):312-323.
8. Zou Y, Zhang Y. Mechanical evaluation of decellularized porcine thoracic aorta. J Surg Res, 2012, 175(2):359-368.
9. Bader A, Steinhoff G, Strobl K, et al. Engineering of human vascular aortic tissue based on a xenogeneic starter matrix. Transplantation, 2000, 70(1):7-14.
10. Badylak SF, Freytes DO, Gilbert TW. Extracellular matrix as a biological scaffold material:Structure and function. Acta Biomater, 2009, 5(1):1-13.
11. Weber B, Emmert MY, Schoenauer R, et al. Tissue engineering on matrix: future of autologous tissue replacement. Semin Immunopathol, 2011, 33(3):307-315.
12. 马良龙, 黄惠民, 李建华, 等.新法制备动脉脱细胞基质的实验研究.中华小儿外科杂志, 2009, 30(7):468-471.
13. 张建, 吴英锋, 陈亮.猪主动脉脱细胞基质的简化制备及生物学评价.中国修复重建外科杂志, 2008, 22(3):364-369.
14. 黄华梅, 谢德明, 罗洁芳.应用脱细胞血管基质构建组织工程血管的初步研究.中国病理生理杂志, 2008, 24(4):792-796.
15. Yang M, Chen CZ, Wang XN, et al. Favorable effects of the detergent and enzyme extraction method for preparing decellularized bovine pericardium scaffold for tissue engineered heart valves. J Biomed Mater Res B Appl Biomater, 2009, 91(1):354-361.
16. Cebotari S, Tudorache I, Jaekel T, et al. Detergent decellularization of heart valves for tissue engineering:toxicological effects of residual detergents on human endothelial cells. Artif Organs, 2010, 34(3):206-210.
17. Boer U, Lohrenz A, Klingenberg M, et al. The effect of detergent-based decellularization procedures on cellular proteins and immunogenicity in equine carotid artery grafts. Biomaterials, 2011, 32(36):9730-9737.
18. Cebotari S, Tudorache I, Ciubotaru A, et al. Use of fresh decellularized allografts for pulmonary valve replacement may reduce the reoperation rate in children and young adults:early report. Circulation, 2011, 124(11 Suppl):S115-S123.
19. Bloch O, Erdbrugger W, Volker W, et al. Extracellular matrix in deoxycholic acid decellularized aortic heart valves. Med Sci Monit, 2012, 18(12):BR487-R492.
20. Honge JL, Funder J, Hansen E, et al. Recellularization of aortic valves in pigs. Eur J Cardiothorac Surg. 2011, 39(6):829-834.
21. Tudorache I, Cebotari S, Sturz G, et al. Tissue engineering of heart valves:biomechanical and morphological properties of decellularized heart valves. J Heart Valve Dis. 2007, 16(5):567-573, 574.
22. Badylak SF. The extracellular matrix as a biologic scaffold material. Biomaterials, 2007, 28(25):3587-3593.
23. Knight RL, Wilcox HE, Korossis SA, et al. The use of acellular matrices for the tissue engineering of cardiac valves. Proc Inst Mech Eng H, 2008, 222(1):129-143.
24. Galil i U. Theα-Gal epitope (Galα1-3Galβ1-4GlcNAc-R) in xenotransplantation. Biochimie, 2001, 83(7):557-563.
25. Schoen FJ, Levy RJ. Calcification of tissue heart valve substitutes:progress toward understanding and prevention. Ann Thorac Surg, 2005, 79(3):1072-1080.
26. Cicha I, Ruffer A, Cesnjevar R, et al. Early obstruction of decellularized xenogenic valves in pediatric patients:involvement of inflammatory and fibroproliferative processes. Cardiovasc Pathol, 2011, 20(4):222-231.
27. Brown BN, Ratner BD, Goodman SB, et al. Macrophage polarization: an opportunity for improved outcomes in biomaterials and regenerative medicine. Biomaterials, 2012, 33(15):3792-3802.
28. Badylak SF, Gilbert TW. Immune response to biologic scaffold materials. Semin Immunol, 2008, 20(2):109-116.

Chinese Journal of Reparative and Reconstructive Surgery

COMPARISON OF AORTIC EXTRACELLULAR MATRIX SCAFFOLD BY DIFFERENT PROTOCOLS FOR DECELLULARIZATION

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