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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Abstract Full text Figures/Tables Video References Cited by

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.	范志強, 劉雯恩, 周艷芳, 等. 脫細胞生物支架的研究進展. 醫學綜述, 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.	陸瑩瑩, 許爭, 溫東朋, 等. 脫細胞真皮基質修復組織缺損研究進展. 國際生物醫學工程雜志, 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, 敖強. 制備細胞外基質材料組織脫細胞方法的研究與熱點. 中國組織工程研究, 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.	許潔, 金冰慧, 趙應征. 大鼠子宮去細胞支架及其細胞外基質凝膠的制備. 生物醫學工程學雜志, 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.	韓巧芳. 胰蛋白酶的作用機理. 生物學教學, 2007, 264(12): 63.
17.	王鵬, 張剛, 李毅, 等. 去細胞組織工程異種瓣膜支架制備方法的比較研究. 中國組織工程研究與臨床康復, 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.	田廣招, 楊振, 查康康, 等. 脫細胞軟骨基質對巨噬細胞極化的調控. 中國組織工程研究, 2021, 951(22): 107-112.
20.	付勇, 廖斌, 于風旭, 等. 脫細胞腸系膜制備生物膜支架的實驗研究. 醫用生物力學, 2020, 35(1): 96-100.
21.	鄭建明, 顧漢卿. 三種常用脫細胞技術的比較研究. 透析與人工器官, 2006, 17(2): 38-41.
22.	李寶興, 馬紹英, 李寶明. 一種同種脫細胞真皮基質制備方法: 201110134511.9. 2011-10-19.
23.	胡欣欣. γ射線照射對蛋白質結構和功能影響的研究. 濟南: 山東大學, 2015.
24.	孫建宇. ⁶⁰Co-γ射線輻照滅菌在中藥及制劑中的應用研究. 中國藥師, 2006, 9(5): 464.
25.	王思穎. 輻照消毒與納米微囊化對骨形態發生蛋白活性的影響研究. 天津: 天津大學, 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, 張怡, 李艷, 等. 一種新型的脫細胞組織工程血管支架的構建和評價. 生物醫學工程學雜志, 2007, 24(2): 379-384.
28.	張華, 周團鋒, 楊雪, 等. 口腔內 3 種合金共存下的離子析出及表面粗糙度的改變. 上海口腔醫學, 2020, 29(1): 46-51.
29.	關燕清, 鐘輝珍, 王笑春, 等. 腫瘤壞死因子-α的光化學修飾及其固定化研究. 生物醫學工程學雜志, 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, 艾凡榮. 海藻酸鈉/納米氧化鋅/生物活性玻璃多孔支架的制備及表征. 硅酸鹽通報, 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.	姜濤, 張愛君, 李雪陽, 等. 人新型脫細胞真皮制備及性狀分析. 中國組織工程研究, 2016, 20(7): 1006-1012.
35.	王靈平. 豬脫細胞真皮基質的制備與性能研究. 濟南: 山東大學, 2011.
36.	孫偉. 新型豬膀胱脫細胞基質的制備與理化性能表征. 濟南: 山東大學, 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, 榮艷華, 沈佘明, 等. 正常皮膚與增生性瘢痕中Ⅰ型和Ⅲ型膠原的含量與比例. 山東大學學報: 醫學版, 2016, 54(11): 64-67.
39.	宋維旭, 伍津津, 魯元剛, 等. 天狼猩紅-偏振光檢測組織工程皮膚中膠原的觀察. 重慶醫學, 2007, 36(5): 450-453.
40.	劉建國, 王素華, 鄭姍姍, 等. 稀土氧化釹粉塵染毒大鼠不同時間點Ⅰ型和Ⅲ型膠原纖維含量及 IL-12 表達的研究. 環境衛生學雜志, 2015, 5(3): 189-193, 201.
41.	劉策勵, 趙雄飛, 黎鰲, 等. 燒傷后不同類型膠原在增生性瘢痕發生中的作用研究. 中國組織工程研究, 2001, 5(10): 36-37.
42.	邱林. 不同年齡增生性瘢痕的膠原構成及影響因素的研究. 重慶: 重慶醫科大學, 2002.
43.	王曉麗. 彈性蛋白的改性研究. 天津: 天津大學, 2007.

1. 范志強, 劉雯恩, 周艷芳, 等. 脫細胞生物支架的研究進展. 醫學綜述, 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. 陸瑩瑩, 許爭, 溫東朋, 等. 脫細胞真皮基質修復組織缺損研究進展. 國際生物醫學工程雜志, 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, 敖強. 制備細胞外基質材料組織脫細胞方法的研究與熱點. 中國組織工程研究, 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. 許潔, 金冰慧, 趙應征. 大鼠子宮去細胞支架及其細胞外基質凝膠的制備. 生物醫學工程學雜志, 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. 韓巧芳. 胰蛋白酶的作用機理. 生物學教學, 2007, 264(12): 63.
17. 王鵬, 張剛, 李毅, 等. 去細胞組織工程異種瓣膜支架制備方法的比較研究. 中國組織工程研究與臨床康復, 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. 田廣招, 楊振, 查康康, 等. 脫細胞軟骨基質對巨噬細胞極化的調控. 中國組織工程研究, 2021, 951(22): 107-112.
20. 付勇, 廖斌, 于風旭, 等. 脫細胞腸系膜制備生物膜支架的實驗研究. 醫用生物力學, 2020, 35(1): 96-100.
21. 鄭建明, 顧漢卿. 三種常用脫細胞技術的比較研究. 透析與人工器官, 2006, 17(2): 38-41.
22. 李寶興, 馬紹英, 李寶明. 一種同種脫細胞真皮基質制備方法: 201110134511.9. 2011-10-19.
23. 胡欣欣. γ射線照射對蛋白質結構和功能影響的研究. 濟南: 山東大學, 2015.
24. 孫建宇. ⁶⁰Co-γ射線輻照滅菌在中藥及制劑中的應用研究. 中國藥師, 2006, 9(5): 464.
25. 王思穎. 輻照消毒與納米微囊化對骨形態發生蛋白活性的影響研究. 天津: 天津大學, 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, 張怡, 李艷, 等. 一種新型的脫細胞組織工程血管支架的構建和評價. 生物醫學工程學雜志, 2007, 24(2): 379-384.
28. 張華, 周團鋒, 楊雪, 等. 口腔內 3 種合金共存下的離子析出及表面粗糙度的改變. 上海口腔醫學, 2020, 29(1): 46-51.
29. 關燕清, 鐘輝珍, 王笑春, 等. 腫瘤壞死因子-α的光化學修飾及其固定化研究. 生物醫學工程學雜志, 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, 艾凡榮. 海藻酸鈉/納米氧化鋅/生物活性玻璃多孔支架的制備及表征. 硅酸鹽通報, 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. 姜濤, 張愛君, 李雪陽, 等. 人新型脫細胞真皮制備及性狀分析. 中國組織工程研究, 2016, 20(7): 1006-1012.
35. 王靈平. 豬脫細胞真皮基質的制備與性能研究. 濟南: 山東大學, 2011.
36. 孫偉. 新型豬膀胱脫細胞基質的制備與理化性能表征. 濟南: 山東大學, 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, 榮艷華, 沈佘明, 等. 正常皮膚與增生性瘢痕中Ⅰ型和Ⅲ型膠原的含量與比例. 山東大學學報: 醫學版, 2016, 54(11): 64-67.
39. 宋維旭, 伍津津, 魯元剛, 等. 天狼猩紅-偏振光檢測組織工程皮膚中膠原的觀察. 重慶醫學, 2007, 36(5): 450-453.
40. 劉建國, 王素華, 鄭姍姍, 等. 稀土氧化釹粉塵染毒大鼠不同時間點Ⅰ型和Ⅲ型膠原纖維含量及 IL-12 表達的研究. 環境衛生學雜志, 2015, 5(3): 189-193, 201.
41. 劉策勵, 趙雄飛, 黎鰲, 等. 燒傷后不同類型膠原在增生性瘢痕發生中的作用研究. 中國組織工程研究, 2001, 5(10): 36-37.
42. 邱林. 不同年齡增生性瘢痕的膠原構成及影響因素的研究. 重慶: 重慶醫科大學, 2002.
43. 王曉麗. 彈性蛋白的改性研究. 天津: 天津大學, 2007.

Journal of Biomedical Engineering

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

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