PREPARATION AND CHARACTERIZATION OF ACELLULAR ADIPOSE TISSUE MATRIX_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

FANXuejiao ^1,2 , TIANChunxiang ^1,2 , FUYuehe ^1,2 , LIXiuqun ² , DENGLi ² ,  LÜQing ¹

1. Department of Thyroid and Breast Surgery, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P. R. China;
2. Division of Stem Cell and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P. R. China;

Corresponding author：

LÜQing, Email: lqlq1963@163.com

Keywords：

Adipose tissue engineering; Acellular matrix; Human adipose-derived stem cells; Scaffold

DOI：

10.7507/1002-1892.20140084

Video：

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Objective To prepare human acellular adipose tissue matrix and to evaluate the cellular compatibility so as to explore a suitable bio-derived scaffold for adipose tissue engineering. Methods The adipose tissue was harvested from abdominal skin graft of breast cancer patients undergoing radical mastectomy or modified radical mastectomy, and then was treated with a series of decellularization processes including repeated freeze-thaw, enzyme digestion, and organic solvent extraction. The matrix was examined by histology, immunohistochemistry, DAPI fluorescence staining, and scanning electron microscopy to observe the the removal of cells and to analyze its composition of collagen type IV, laminin, and fibronectin, and microstructure. The 3rd passage human adipose-derived stem cells (hADSCs) were co-cultured with acellular adipose tissue matrix and different concentrations of extracted liquid (100%, 75%, 50%, and 25%). The cytotoxic effects of the matrix were tested by MTT. The biocompatibility of the matrix was detected by live/dead staining and scanning electron microscopy observation. Results The acellular adipose tissue matrix basically maintains intrinsical morphology. The matrix after acellular treatment consisted of extracellular matrix without any cell components, but there were abundant collagen type I; neither DNA nor lipid residual was detected. Moreover, the collagen was the main component of the matrix which was rich in laminin and fibronectin. At 1, 3, and 5 days after co-cultured with hADSCs, the cytotoxic effect of matrix was grade 0-1. The matrix displayed good cell compatibility and proliferation. Conclusion The acellular adipose tissue matrix prepared by repeated freeze-thaw, enzyme digestion, and organic solvent extraction method remains abundant extracellular matrix and has good cellular compatibility, so it is expected to be an ideal bio-derived scaffold for adipose tissue engineering.

Citation： FANXuejiao, TIANChunxiang, FUYuehe, LIXiuqun, DENGLi, LÜQing. PREPARATION AND CHARACTERIZATION OF ACELLULAR ADIPOSE TISSUE MATRIX. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(3): 377-383. doi: 10.7507/1002-1892.20140084 Copy

1.	Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue:implications for cell-based therapies. Tissue Eng, 2001, 7(2):211-228.
2.	Zuk PA. The adipose-derived stem cell:looking back and looking ahead. Mol Biol Cell, 2010, 21(11):1783-1787.
3.	Morrison WA. Progress in tissue engineering of soft tissue and organs. Surgery, 2009, 145(2):127-130.
4.	Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell, 2002, 13(12):4279-4295.
5.	田春祥, 范雪娇, 陈晓禾, 等. 骨骼肌无细胞基质的制备及其生物相容性研究. 中国修复重建外科杂志, 2012, 26(6):749-754.
6.	Brown BN, Freund JM, Han L, et al. Comparison of three methods for the derivation of a biologic scaffold composed of adipose tissue extracellular matrix. Tissue Eng Part C Methods, 2011, 17(4):411-421.
7.	Flynn LE. The use of decellularized adipose tissue to provide an inductive microenvironment for the adipogenic differentiation of human adipose-derived stem cells. Biomaterials, 2010, 31(17):4715-4724.
8.	Choi JS, Yang HJ, Kim BS, et al. Human extracellular matrix (ECM) powders for injectable cell delivery and adipose tissue engineering. J Control Release, 2009, 139(1):2-7.
9.	Turner AE, Yu C, Bianco J, et al. The performance of decellularized adipose tissue microcarriers as an inductive substrate for human adipose-derived stem cells. Biomaterials, 2012, 33(18):4490-4499.
10.	Yu C, Bianco J, Brown C, et al. Porous decellularized adipose tissue foams for soft tissue regeneration. Biomaterials, 2013, 34(13):3290-3302.
11.	胡刚, 邢彬, 欧来良, 等. 动物血管脱细胞方法及细胞外基质材料评价研究. 中国生物医学工程学报, 2008, 27(6):912-921.
12.	Eto H, Suga H, Matsumoto D, et al. Characterization of structure and cellular components of aspirated and excised adipose tissue. Plast Reconstr Surg, 2009, 124(4):1087-1097.
13.	Choi JS, Kim BS, Kim JY, et al. Decellularized extracellular matrix derived from human adipose tissue as a potential scaffold for allograft tissue engineering. J Biomed Mater Res A, 2011, 97(3):292-299.
14.	Choi YC, Choi JS, Kim BS, et al. Decellularized extracellular matrix derived from porcine adipose tissue as a xenogeneic biomaterial for tissue engineering. Tissue Eng Part C Methods, 2012, 18(11):866-876.
15.	Kim BS, Choi JS, Kim JD, et al. Recellularization of decellularized human adipose-tissue-derived extracellular matrix sheets with other human cell types. Cell Tissue Res, 2012, 348(3):559-567.
16.	Young DA, Ibrahim DO, Hu D, et al. Injectable hydrogel scaffold from decellularized human lipoaspirate. Acta Biomater, 2011, 7(3):1040-1049.
17.	Turner AE, Flynn LE. Design and characterization of tissue-specific extracellular matrix-derived microcarriers. Tissue Eng Part C Methods, 2012, 18(3):186-197.
18.	Itoi Y, Takatori M, Hyakusoku H, et al. Comparison of readily available scaffolds for adipose tissue engineering using adipose-derived stem cells. J Plast Reconstr Aesthet Surg, 2010, 63(5):858-864.
19.	Lumpkins SB, Pierre N, McFetridge PS. A mechanical evaluation of three decellularization methods in the design of a xenogeneic scaffold for tissue engineering the temporomandibular joint disc. Acta Biomaterialia, 2008, 4(4):808-816.
20.	Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes. Biomaterials, 2011, 32(12):3233-3243.
21.	Lovekamp JJ, Simionescu DT, Mercuri JJ, et al. Stability and function of glycosaminoglycans in porcine bioprosthetic heart valves. Biomaterials, 2006, 27(8):1507-1518.
22.	Engler AJ, Griffin MA, Sen S, et al. Myotubes differentiate optimally on substrates with tissue-like stiffness:pathological implications for soft or stiff microenvironments. J Cell Biol, 2004, 166(6):877-887.
23.	Schenke-Layland K, Vasilevski O, Opitz F, et al. Impact of decellularization of xenogeneic tissue on extracellular matrix integrity for tissue engineering of heart valves. J Struct Biol, 2003, 143(3):201-208.
24.	Suto K, Urabe K, Naruse K, et al. Repeated freeze-thaw cycles reduce the survival rate of osteocytes in bone-tendon constructs without affecting the mechanical properties of tendons. Cell Tissue Bank, 2012, 13(1):71-80.
25.	Jung HJ, Vangipuram G, Fisher MB, et al. The effects of multiple freeze-thaw cycles on thebiomechanical properties of the human bone-patellar tendon-bone allograft. J Orthop Res, 2011, 29(8):1193-1198.
26.	Allen KD, Athanasiou KA. A surface-regional and freeze-thaw characterization of the porcine temporomandibular joint disc. Ann Biomed Eng, 2005, 33(7):951-962.
27.	Lu H, Hoshiba T, Kawazoe N, et al. Comparison of decellularization techniques for preparation of extracellular matrix scaffolds derived from three-dimensional cell culture. J Biomed Mater Res A, 2012, 100(9):2507-2516.
28.	Hausman GJ, Wright JT, Richardson RL. The influence of extracellular matrix substrata on preadipocyte development in serum-free cultures of stromal-vascular cells. J Anim Sci, 1996, 74(9):2117-2128.
29.	Patrick CW Jr, Wu X. Integrin-mediated preadipocyte adhesion and migration on laminin-1. Ann Biomed Eng, 2003, 31(5):505-514.
30.	Wu X, Patrick CW. Preadipocyte adhesion and migration dynamics on ECM proteins. Second Joint Embs-Bmes Conference 2002.[s.l.]:[s.n.], 2002.
31.	Choi JS, Yang HJ, Kim BS, et al. Fabrication of porous extracellular matrix scaffolds from human adipose tissue. Tissue Eng Part C Methods, 2010, 16(3):387-396.
32.	Sobral JM, Caridade SG, Sousa RA, et al. Three-dimensional plotted scaffolds with controlled pore size gradients:Effect of scaffold geometry on mechanical performance and cell seeding efficiency. Acta Biomater, 2011, 7(3):1009-1018.

1. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue:implications for cell-based therapies. Tissue Eng, 2001, 7(2):211-228.
2. Zuk PA. The adipose-derived stem cell:looking back and looking ahead. Mol Biol Cell, 2010, 21(11):1783-1787.
3. Morrison WA. Progress in tissue engineering of soft tissue and organs. Surgery, 2009, 145(2):127-130.
4. Zuk PA, Zhu M, Ashjian P, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell, 2002, 13(12):4279-4295.
5. 田春祥, 范雪娇, 陈晓禾, 等. 骨骼肌无细胞基质的制备及其生物相容性研究. 中国修复重建外科杂志, 2012, 26(6):749-754.
6. Brown BN, Freund JM, Han L, et al. Comparison of three methods for the derivation of a biologic scaffold composed of adipose tissue extracellular matrix. Tissue Eng Part C Methods, 2011, 17(4):411-421.
7. Flynn LE. The use of decellularized adipose tissue to provide an inductive microenvironment for the adipogenic differentiation of human adipose-derived stem cells. Biomaterials, 2010, 31(17):4715-4724.
8. Choi JS, Yang HJ, Kim BS, et al. Human extracellular matrix (ECM) powders for injectable cell delivery and adipose tissue engineering. J Control Release, 2009, 139(1):2-7.
9. Turner AE, Yu C, Bianco J, et al. The performance of decellularized adipose tissue microcarriers as an inductive substrate for human adipose-derived stem cells. Biomaterials, 2012, 33(18):4490-4499.
10. Yu C, Bianco J, Brown C, et al. Porous decellularized adipose tissue foams for soft tissue regeneration. Biomaterials, 2013, 34(13):3290-3302.
11. 胡刚, 邢彬, 欧来良, 等. 动物血管脱细胞方法及细胞外基质材料评价研究. 中国生物医学工程学报, 2008, 27(6):912-921.
12. Eto H, Suga H, Matsumoto D, et al. Characterization of structure and cellular components of aspirated and excised adipose tissue. Plast Reconstr Surg, 2009, 124(4):1087-1097.
13. Choi JS, Kim BS, Kim JY, et al. Decellularized extracellular matrix derived from human adipose tissue as a potential scaffold for allograft tissue engineering. J Biomed Mater Res A, 2011, 97(3):292-299.
14. Choi YC, Choi JS, Kim BS, et al. Decellularized extracellular matrix derived from porcine adipose tissue as a xenogeneic biomaterial for tissue engineering. Tissue Eng Part C Methods, 2012, 18(11):866-876.
15. Kim BS, Choi JS, Kim JD, et al. Recellularization of decellularized human adipose-tissue-derived extracellular matrix sheets with other human cell types. Cell Tissue Res, 2012, 348(3):559-567.
16. Young DA, Ibrahim DO, Hu D, et al. Injectable hydrogel scaffold from decellularized human lipoaspirate. Acta Biomater, 2011, 7(3):1040-1049.
17. Turner AE, Flynn LE. Design and characterization of tissue-specific extracellular matrix-derived microcarriers. Tissue Eng Part C Methods, 2012, 18(3):186-197.
18. Itoi Y, Takatori M, Hyakusoku H, et al. Comparison of readily available scaffolds for adipose tissue engineering using adipose-derived stem cells. J Plast Reconstr Aesthet Surg, 2010, 63(5):858-864.
19. Lumpkins SB, Pierre N, McFetridge PS. A mechanical evaluation of three decellularization methods in the design of a xenogeneic scaffold for tissue engineering the temporomandibular joint disc. Acta Biomaterialia, 2008, 4(4):808-816.
20. Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes. Biomaterials, 2011, 32(12):3233-3243.
21. Lovekamp JJ, Simionescu DT, Mercuri JJ, et al. Stability and function of glycosaminoglycans in porcine bioprosthetic heart valves. Biomaterials, 2006, 27(8):1507-1518.
22. Engler AJ, Griffin MA, Sen S, et al. Myotubes differentiate optimally on substrates with tissue-like stiffness:pathological implications for soft or stiff microenvironments. J Cell Biol, 2004, 166(6):877-887.
23. Schenke-Layland K, Vasilevski O, Opitz F, et al. Impact of decellularization of xenogeneic tissue on extracellular matrix integrity for tissue engineering of heart valves. J Struct Biol, 2003, 143(3):201-208.
24. Suto K, Urabe K, Naruse K, et al. Repeated freeze-thaw cycles reduce the survival rate of osteocytes in bone-tendon constructs without affecting the mechanical properties of tendons. Cell Tissue Bank, 2012, 13(1):71-80.
25. Jung HJ, Vangipuram G, Fisher MB, et al. The effects of multiple freeze-thaw cycles on thebiomechanical properties of the human bone-patellar tendon-bone allograft. J Orthop Res, 2011, 29(8):1193-1198.
26. Allen KD, Athanasiou KA. A surface-regional and freeze-thaw characterization of the porcine temporomandibular joint disc. Ann Biomed Eng, 2005, 33(7):951-962.
27. Lu H, Hoshiba T, Kawazoe N, et al. Comparison of decellularization techniques for preparation of extracellular matrix scaffolds derived from three-dimensional cell culture. J Biomed Mater Res A, 2012, 100(9):2507-2516.
28. Hausman GJ, Wright JT, Richardson RL. The influence of extracellular matrix substrata on preadipocyte development in serum-free cultures of stromal-vascular cells. J Anim Sci, 1996, 74(9):2117-2128.
29. Patrick CW Jr, Wu X. Integrin-mediated preadipocyte adhesion and migration on laminin-1. Ann Biomed Eng, 2003, 31(5):505-514.
30. Wu X, Patrick CW. Preadipocyte adhesion and migration dynamics on ECM proteins. Second Joint Embs-Bmes Conference 2002.[s.l.]:[s.n.], 2002.
31. Choi JS, Yang HJ, Kim BS, et al. Fabrication of porous extracellular matrix scaffolds from human adipose tissue. Tissue Eng Part C Methods, 2010, 16(3):387-396.
32. Sobral JM, Caridade SG, Sousa RA, et al. Three-dimensional plotted scaffolds with controlled pore size gradients:Effect of scaffold geometry on mechanical performance and cell seeding efficiency. Acta Biomater, 2011, 7(3):1009-1018.

Chinese Journal of Reparative and Reconstructive Surgery

PREPARATION AND CHARACTERIZATION OF ACELLULAR ADIPOSE TISSUE MATRIX

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