Effects of two common acellular methods on the physicochemical properties of dermal acellular matrix_Journal of Biomedical Engineering

Authors：

YANG Caixian ¹ , GUO Jiqiang ^1,2 , WANG Jinghui ¹ , FAN Jiayu ¹ , XING Yanxue ¹ , ZHANG Li ² ,  AN Meiwen ¹

1. College of Biomedical Engineering, Taiyuan University of Technology, Taiyuan 030024, P.R.China;
2. Shanxi Bethune Hospital, Taiyuan 030032, P.R.China;

Corresponding author：

AN Meiwen, Email: meiwen_an@163.com

Keywords：

tissue engineering; human dermal; acellular matrix; quickly build; in vitro culture

DOI：

10.7507/1001-5515.202103019

Video：

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At present, acellular matrix is an effective replacement material for the treatment of skin damage, but there are few systematic evaluation studies on its performance. The experimental group of this study used two decellularization methods to prepare the matrix: one was the acellular matrix which sterilized with peracetic acid first (0.2% PAA/4% ethanol solution) and then treated with hypertonic saline (group A), the other was 0.05% trypsin/EDTA decellularization after γ irradiation (group B); and the control group was soaked in PBS (Group C). Then physical properties and chemical composition of the three groups were detected. Hematoxylin eosin (HE) staining showed that the acellular effect of group B was good. The porosity of group A and B were both above 84.9%. In group A, the compressive modulus of elasticity was (9.94 ± 3.81) MPa, and the compressive modulus of elasticity was (12.59 ± 5.50) MPa in group B. There was no significant difference between group A or B and group C. The total content of collagen in acellular matrix of group A and B was significantly lower than that of group C (1. 662 ± 0.229) mg/g, but there was no significant difference in the ratio of collagen Ⅰ/Ⅲ between group B and group C. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that there was no significant difference in microstructure. Qualitative detection of fibronectin and elastin in each group was basically consistent with that in group C. Therefore, acellular matrix of group B had better performance as scaffold material. The experimental results show that the acellular matrix prepared by γ-ray sterilization and decellularization of 0.05% Trypsin enzyme/EDTA could be used for the construction of tissue-engineered skin. It could also provide reference for the preparation and mounting of heterogeneous dermal acellular matrix. It was also could be used for electrostatic spinning or three-dimensional printed tissue engineered skin scaffold which could provide physical and chemical parameters for it.

Citation： YANG Caixian, GUO Jiqiang, WANG Jinghui, FAN Jiayu, XING Yanxue, ZHANG Li, AN Meiwen. Effects of two common acellular methods on the physicochemical properties of dermal acellular matrix. Journal of Biomedical Engineering, 2021, 38(5): 911-918. doi: 10.7507/1001-5515.202103019 Copy

1.	范志强, 刘雯恩, 周艳芳, 等. 脱细胞生物支架的研究进展. 医学综述, 2019, 25(14): 2788-2793.
2.	Crapo P M, Gilbert T W, Badylak S F. An overview of tissue and whole organ decellularization processes. Biomaterials, 2011, 32(12): 3233-3243.
3.	Matuska A M, McFetridge P S. The effect of terminal sterilization on structural and biophysical properties of a decellularized collagen-based scaffold; implications for stem cell adhesion. J Biomed Mater Res B Appl Biomater, 2015, 103(2): 397-406.
4.	陆莹莹, 许争, 温东朋, 等. 脱细胞真皮基质修复组织缺损研究进展. 国际生物医学工程杂志, 2017, 40(1): 58-61.
5.	Daugs A, Hutzler B, Meinke M, et al. Detergent-based decellularization of bovine carotid arteries for vascular tissue engineering. Ann Biomed Eng, 2017, 45(11): 2683-2692.
6.	Floy M E, Mateyka T D, Foreman K L, et al. Human pluripotent stem cell-derived cardiac stromal cells and their applications in regenerative medicine. Stem Cell Res, 2020, 45: 101831.
7.	何晶, 敖强. 制备细胞外基质材料组织脱细胞方法的研究与热点. 中国组织工程研究, 2020, 24(927): 7-14.
8.	Wojciech L, Glik J, Agnieszka K, et al. Atomic force microscopy in the production of a biovital skin graft based on human acellular dermal matrix produced in house and in vitro cultured human fibroblasts. J Biomed Mater Res B Appl Biomater, 2018, 106(2): 726-733.
9.	许洁, 金冰慧, 赵应征. 大鼠子宫去细胞支架及其细胞外基质凝胶的制备. 生物医学工程学杂志, 2018, 35(2): 237-243.
10.	Lehr E J, Rayat G R, Chiu B, et al. Decellularization reduces immunogenicity of sheep pulmonary artery vascular patches. J Thorac Cardiovasc Surg, 2011, 141(4): 1056-1062.
11.	Yang B, Zhang Y, Zhou L, et al. Development of a porcine bladder acellular matrix with well-preserved extracellular bioactive factors for tissue engineering. Tissue Eng Part C Methods, 2010, 16(5): 1201-1211.
12.	Ott H C, Clippinger B, Conrad C, et al. Regeneration and orthotopic transplantation of a bioartificial lung. Nat Med, 2010, 16(8): 927-933.
13.	Reing J E, Brown B N, Daly K A, et al. The effects of processing methods upon mechanical and biologic properties of porcine dermal extracellular matrix scaffolds. Biomaterials, 2010, 31(33): 8626-8633.
14.	Du L, Wu X, Pang K, et al. Histological evaluation and biomechanical characterisation of an acellular porcine cornea scaffold. Br J Ophthalmol, 2010, 95(3): 410-414.
15.	Badylak S F, Freytes D O, Gilbert T W. Reprint of: Extracellular matrix as a biological scaffold material: Structure and function. Acta Biomaterialia, 2015, 23(Suppl): S17-S26.
16.	韩巧芳. 胰蛋白酶的作用机理. 生物学教学, 2007, 264(12): 63.
17.	王鹏, 张刚, 李毅, 等. 去细胞组织工程异种瓣膜支架制备方法的比较研究. 中国组织工程研究与临床康复, 2007, 13(5): 3041-3044.
18.	Gorschewsky O, Puetz A, Riechert K, et al. Quantitative analysis of biochemical characteristics of bone-patellar tendon-bone allografts. Biomed Mater Eng, 2005, 15(6): 403-411.
19.	田广招, 杨振, 查康康, 等. 脱细胞软骨基质对巨噬细胞极化的调控. 中国组织工程研究, 2021, 951(22): 107-112.
20.	付勇, 廖斌, 于风旭, 等. 脱细胞肠系膜制备生物膜支架的实验研究. 医用生物力学, 2020, 35(1): 96-100.
21.	郑建明, 顾汉卿. 三种常用脱细胞技术的比较研究. 透析与人工器官, 2006, 17(2): 38-41.
22.	李宝兴, 马绍英, 李宝明. 一种同种脱细胞真皮基质制备方法: 201110134511.9. 2011-10-19.
23.	胡欣欣. γ射线照射对蛋白质结构和功能影响的研究. 济南: 山东大学, 2015.
24.	孙建宇. ⁶⁰Co-γ射线辐照灭菌在中药及制剂中的应用研究. 中国药师, 2006, 9(5): 464.
25.	王思颖. 辐照消毒与纳米微囊化对骨形态发生蛋白活性的影响研究. 天津: 天津大学, 2015.
26.	Islam M M, Sharifi R, Mamodaly S, et al. Effects of gamma radiation sterilization on the structural and biological properties of decellularized corneal xenografts. Acta Biomaterialia, 2019, 96: 330-344.
27.	涂秋芬, 张怡, 李艳, 等. 一种新型的脱细胞组织工程血管支架的构建和评价. 生物医学工程学杂志, 2007, 24(2): 379-384.
28.	张华, 周团锋, 杨雪, 等. 口腔内 3 种合金共存下的离子析出及表面粗糙度的改变. 上海口腔医学, 2020, 29(1): 46-51.
29.	关燕清, 钟辉珍, 王笑春, 等. 肿瘤坏死因子-α的光化学修饰及其固定化研究. 生物医学工程学杂志, 2006, 23(2): 346-349.
30.	Feyza G, Bukem T, Etinkaya A, et al. The effect of thickness on surface structure of rf sputtered TiO₂ thin films by XPS, SEM/EDS, AFM and SAM. Vacuum, 2020, 182: 109766.
31.	Javid A, Hekmatshoar M, Hedayatifar L, et al. Effect of surface roughness on electrical conductivity and hardness of silver plated copper. Mater Res Express, 2019, 6(3): 036407.
32.	魏坤, 辛勇, 艾凡荣. 海藻酸钠/纳米氧化锌/生物活性玻璃多孔支架的制备及表征. 硅酸盐通报, 2019, 38(278): 200-206.
33.	Dhasmana A, Singh L, Roy P, et al. Silk fibroin protein modified acellular dermal matrix for tissue repairing and regeneration. Mater Sci Eng C Mater Biol Appl, 2019, 97: 313-324.
34.	姜涛, 张爱君, 李雪阳, 等. 人新型脱细胞真皮制备及性状分析. 中国组织工程研究, 2016, 20(7): 1006-1012.
35.	王灵平. 猪脱细胞真皮基质的制备与性能研究. 济南: 山东大学, 2011.
36.	孙伟. 新型猪膀胱脱细胞基质的制备与理化性能表征. 济南: 山东大学, 2019.
37.	Sneddon I N. The relation between load and penetration in the axisymmetric boussinesq problem for a punch of arbitrary profile. Int J Eng Sci, 1965, 3(1): 47-57.
38.	王成, 荣艳华, 沈佘明, 等. 正常皮肤与增生性瘢痕中Ⅰ型和Ⅲ型胶原的含量与比例. 山东大学学报: 医学版, 2016, 54(11): 64-67.
39.	宋维旭, 伍津津, 鲁元刚, 等. 天狼猩红-偏振光检测组织工程皮肤中胶原的观察. 重庆医学, 2007, 36(5): 450-453.
40.	刘建国, 王素华, 郑姗姗, 等. 稀土氧化钕粉尘染毒大鼠不同时间点Ⅰ型和Ⅲ型胶原纤维含量及 IL-12 表达的研究. 环境卫生学杂志, 2015, 5(3): 189-193, 201.
41.	刘策励, 赵雄飞, 黎鳌, 等. 烧伤后不同类型胶原在增生性瘢痕发生中的作用研究. 中国组织工程研究, 2001, 5(10): 36-37.
42.	邱林. 不同年龄增生性瘢痕的胶原构成及影响因素的研究. 重庆: 重庆医科大学, 2002.
43.	王晓丽. 弹性蛋白的改性研究. 天津: 天津大学, 2007.

1. 范志强, 刘雯恩, 周艳芳, 等. 脱细胞生物支架的研究进展. 医学综述, 2019, 25(14): 2788-2793.
2. Crapo P M, Gilbert T W, Badylak S F. An overview of tissue and whole organ decellularization processes. Biomaterials, 2011, 32(12): 3233-3243.
3. Matuska A M, McFetridge P S. The effect of terminal sterilization on structural and biophysical properties of a decellularized collagen-based scaffold; implications for stem cell adhesion. J Biomed Mater Res B Appl Biomater, 2015, 103(2): 397-406.
4. 陆莹莹, 许争, 温东朋, 等. 脱细胞真皮基质修复组织缺损研究进展. 国际生物医学工程杂志, 2017, 40(1): 58-61.
5. Daugs A, Hutzler B, Meinke M, et al. Detergent-based decellularization of bovine carotid arteries for vascular tissue engineering. Ann Biomed Eng, 2017, 45(11): 2683-2692.
6. Floy M E, Mateyka T D, Foreman K L, et al. Human pluripotent stem cell-derived cardiac stromal cells and their applications in regenerative medicine. Stem Cell Res, 2020, 45: 101831.
7. 何晶, 敖强. 制备细胞外基质材料组织脱细胞方法的研究与热点. 中国组织工程研究, 2020, 24(927): 7-14.
8. Wojciech L, Glik J, Agnieszka K, et al. Atomic force microscopy in the production of a biovital skin graft based on human acellular dermal matrix produced in house and in vitro cultured human fibroblasts. J Biomed Mater Res B Appl Biomater, 2018, 106(2): 726-733.
9. 许洁, 金冰慧, 赵应征. 大鼠子宫去细胞支架及其细胞外基质凝胶的制备. 生物医学工程学杂志, 2018, 35(2): 237-243.
10. Lehr E J, Rayat G R, Chiu B, et al. Decellularization reduces immunogenicity of sheep pulmonary artery vascular patches. J Thorac Cardiovasc Surg, 2011, 141(4): 1056-1062.
11. Yang B, Zhang Y, Zhou L, et al. Development of a porcine bladder acellular matrix with well-preserved extracellular bioactive factors for tissue engineering. Tissue Eng Part C Methods, 2010, 16(5): 1201-1211.
12. Ott H C, Clippinger B, Conrad C, et al. Regeneration and orthotopic transplantation of a bioartificial lung. Nat Med, 2010, 16(8): 927-933.
13. Reing J E, Brown B N, Daly K A, et al. The effects of processing methods upon mechanical and biologic properties of porcine dermal extracellular matrix scaffolds. Biomaterials, 2010, 31(33): 8626-8633.
14. Du L, Wu X, Pang K, et al. Histological evaluation and biomechanical characterisation of an acellular porcine cornea scaffold. Br J Ophthalmol, 2010, 95(3): 410-414.
15. Badylak S F, Freytes D O, Gilbert T W. Reprint of: Extracellular matrix as a biological scaffold material: Structure and function. Acta Biomaterialia, 2015, 23(Suppl): S17-S26.
16. 韩巧芳. 胰蛋白酶的作用机理. 生物学教学, 2007, 264(12): 63.
17. 王鹏, 张刚, 李毅, 等. 去细胞组织工程异种瓣膜支架制备方法的比较研究. 中国组织工程研究与临床康复, 2007, 13(5): 3041-3044.
18. Gorschewsky O, Puetz A, Riechert K, et al. Quantitative analysis of biochemical characteristics of bone-patellar tendon-bone allografts. Biomed Mater Eng, 2005, 15(6): 403-411.
19. 田广招, 杨振, 查康康, 等. 脱细胞软骨基质对巨噬细胞极化的调控. 中国组织工程研究, 2021, 951(22): 107-112.
20. 付勇, 廖斌, 于风旭, 等. 脱细胞肠系膜制备生物膜支架的实验研究. 医用生物力学, 2020, 35(1): 96-100.
21. 郑建明, 顾汉卿. 三种常用脱细胞技术的比较研究. 透析与人工器官, 2006, 17(2): 38-41.
22. 李宝兴, 马绍英, 李宝明. 一种同种脱细胞真皮基质制备方法: 201110134511.9. 2011-10-19.
23. 胡欣欣. γ射线照射对蛋白质结构和功能影响的研究. 济南: 山东大学, 2015.
24. 孙建宇. ⁶⁰Co-γ射线辐照灭菌在中药及制剂中的应用研究. 中国药师, 2006, 9(5): 464.
25. 王思颖. 辐照消毒与纳米微囊化对骨形态发生蛋白活性的影响研究. 天津: 天津大学, 2015.
26. Islam M M, Sharifi R, Mamodaly S, et al. Effects of gamma radiation sterilization on the structural and biological properties of decellularized corneal xenografts. Acta Biomaterialia, 2019, 96: 330-344.
27. 涂秋芬, 张怡, 李艳, 等. 一种新型的脱细胞组织工程血管支架的构建和评价. 生物医学工程学杂志, 2007, 24(2): 379-384.
28. 张华, 周团锋, 杨雪, 等. 口腔内 3 种合金共存下的离子析出及表面粗糙度的改变. 上海口腔医学, 2020, 29(1): 46-51.
29. 关燕清, 钟辉珍, 王笑春, 等. 肿瘤坏死因子-α的光化学修饰及其固定化研究. 生物医学工程学杂志, 2006, 23(2): 346-349.
30. Feyza G, Bukem T, Etinkaya A, et al. The effect of thickness on surface structure of rf sputtered TiO₂ thin films by XPS, SEM/EDS, AFM and SAM. Vacuum, 2020, 182: 109766.
31. Javid A, Hekmatshoar M, Hedayatifar L, et al. Effect of surface roughness on electrical conductivity and hardness of silver plated copper. Mater Res Express, 2019, 6(3): 036407.
32. 魏坤, 辛勇, 艾凡荣. 海藻酸钠/纳米氧化锌/生物活性玻璃多孔支架的制备及表征. 硅酸盐通报, 2019, 38(278): 200-206.
33. Dhasmana A, Singh L, Roy P, et al. Silk fibroin protein modified acellular dermal matrix for tissue repairing and regeneration. Mater Sci Eng C Mater Biol Appl, 2019, 97: 313-324.
34. 姜涛, 张爱君, 李雪阳, 等. 人新型脱细胞真皮制备及性状分析. 中国组织工程研究, 2016, 20(7): 1006-1012.
35. 王灵平. 猪脱细胞真皮基质的制备与性能研究. 济南: 山东大学, 2011.
36. 孙伟. 新型猪膀胱脱细胞基质的制备与理化性能表征. 济南: 山东大学, 2019.
37. Sneddon I N. The relation between load and penetration in the axisymmetric boussinesq problem for a punch of arbitrary profile. Int J Eng Sci, 1965, 3(1): 47-57.
38. 王成, 荣艳华, 沈佘明, 等. 正常皮肤与增生性瘢痕中Ⅰ型和Ⅲ型胶原的含量与比例. 山东大学学报: 医学版, 2016, 54(11): 64-67.
39. 宋维旭, 伍津津, 鲁元刚, 等. 天狼猩红-偏振光检测组织工程皮肤中胶原的观察. 重庆医学, 2007, 36(5): 450-453.
40. 刘建国, 王素华, 郑姗姗, 等. 稀土氧化钕粉尘染毒大鼠不同时间点Ⅰ型和Ⅲ型胶原纤维含量及 IL-12 表达的研究. 环境卫生学杂志, 2015, 5(3): 189-193, 201.
41. 刘策励, 赵雄飞, 黎鳌, 等. 烧伤后不同类型胶原在增生性瘢痕发生中的作用研究. 中国组织工程研究, 2001, 5(10): 36-37.
42. 邱林. 不同年龄增生性瘢痕的胶原构成及影响因素的研究. 重庆: 重庆医科大学, 2002.
43. 王晓丽. 弹性蛋白的改性研究. 天津: 天津大学, 2007.

Journal of Biomedical Engineering

Effects of two common acellular methods on the physicochemical properties of dermal acellular matrix

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