• 四川大學(xué)華西醫(yī)院（成都，610041） 1 甲狀腺乳腺外科，2 生物治療國(guó)家重點(diǎn)實(shí)驗(yàn)室· 干細(xì)胞與組織工程研究室;

目的 細(xì)胞外基質(zhì)是脂肪組織工程材料的研究熱點(diǎn)之一。通過(guò)探討骨骼肌無(wú)細(xì)胞基質(zhì)的制作方法及生物相容性，為其在脂肪組織工程中的應(yīng)用奠定基礎(chǔ)。 方法 取健康成年小香豬新鮮骨骼肌組織，橫切成厚2 ～ 3 mm 的組織塊，采用低滲- 去垢劑法脫細(xì)胞處理。處理后采用HE 染色、Masson 三色染色、免疫組織化學(xué)染色及掃描電鏡檢測(cè)骨骼肌無(wú)細(xì)胞基質(zhì)是否有細(xì)胞成分殘留，并觀察其基本結(jié)構(gòu)；應(yīng)用MTT 法檢測(cè)骨骼肌無(wú)細(xì)胞基質(zhì)細(xì)胞毒性。取乳腺癌患者自愿捐贈(zèng)脂肪組織，分離培養(yǎng)人脂肪干細(xì)胞（human adipose-derived stem cells，hADSCs），從形態(tài)學(xué)、流式細(xì)胞學(xué)和成脂、成骨分化能力方面進(jìn)行鑒定。將骨骼肌無(wú)細(xì)胞基質(zhì)與第3 代hADSCs 共培養(yǎng)，于培養(yǎng)后第1、3、5、7 天通過(guò)細(xì)胞活性檢測(cè)材料上細(xì)胞黏附、擴(kuò)散和增殖情況，了解其與細(xì)胞之間的相互作用。 結(jié)果 HE、Masson、免疫組織化學(xué)染色及掃描電鏡觀察顯示骨骼肌無(wú)細(xì)胞基質(zhì)肌纖維去除完全，無(wú)細(xì)胞核殘留，基質(zhì)結(jié)構(gòu)保留完整；大量連接成網(wǎng)狀的膠原纖維呈多孔隙樣結(jié)構(gòu)，規(guī)則排列。MTT 檢測(cè)示骨骼肌無(wú)細(xì)胞基質(zhì)細(xì)胞毒性為1 級(jí)，細(xì)胞相容性好。細(xì)胞活性檢測(cè)示hADSCs 在骨骼肌無(wú)細(xì)胞基質(zhì)上能很好地伸展，且能與周圍基質(zhì)黏附，進(jìn)入基質(zhì)內(nèi)部并相互交織。 結(jié)論 經(jīng)脫細(xì)胞處理的骨骼肌無(wú)細(xì)胞基質(zhì)具有良好生物相容性，可能作為脂肪組織工程的支架材料。

引用本文： 田春祥,范雪嬌,陳曉禾,鄧力,秦廷武,羅靜聰,李秀群,呂青. 骨骼肌無(wú)細(xì)胞基質(zhì)的制備及其生物相容性研究. 中國(guó)修復(fù)重建外科雜志, 2012, 26(6): 749-754. doi: 復(fù)制

1. Lutolf MP, Hubbell JA. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat Biotechnol, 2005, 23(1): 47-55.
2. Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues and organs. Biomaterials, 2006, 27(19): 3675-3683.
3. Borschel GH, Dennis RG, Kuzon WM Jr. Contractile skeletal muscle tissue-engineered on an acellular scaffold. Plast Reconstr Surg, 2004, 113(2): 595-602.
4. Valentin JE, Turner NJ, Gilbert TW, et al. Functional skeletal muscle formation with a biologic scaffold. Biomaterials, 2010, 31(29): 7475-7484.
5. Penolazzi L, Mazzitelli S, Vecchiatini R, et al. Human mesenchymal stem cells seeded on extracellular matrix scaffold: Viability and osteogenic potential. J Cell Physiol, 2012, 227(2): 857-866.
6. Zhu WD, Xu YM, Feng C, et al. Different bladder defects reconstructed with bladder acellular matrix grafts in a rabbit model. Urologe A, 2011, 50(11): 1420-1425.
7. Zhang X, Yang J, Li Y, et al. Functional neovascularization in tissue engineering with porcine acellular dermal matrix and human umbilical vein endothelial cells. Tissue Eng Part C Methods, 2011, 17(4): 423-433.
8. 張建, 吳英鋒, 陳亮. 豬主動(dòng)脈脫細(xì)胞基質(zhì)的簡(jiǎn)化制備及生物學(xué)評(píng)價(jià). 中國(guó)修復(fù)重建外科雜志, 2008, 22(3): 364-369.
9. Wainwright JM, Hashizume R, Fujimoto KL, et al. Right ventricular outflow tract repair with a cardiac biologic scaffold. Cells Tissues Organs, 2012, 195(1-2): 159-170.
10. Soto-Gutierrez A, Zhang L, Medberry C, et al. A whole organ regenerative medicine approach for liver replacement. Tissue Eng Part C Methods, 2011, 17(6): 677-686.
11. Uygun BE, Soto-Gutierrez A, Yagi H, et al. Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat Med, 2010, 16(7): 814-820.
12. Petersen TH, Calle EA, Zhao L, et al. Tissue-engineered lungs for in vivo implantation. Science, 2010, 329(5991): 538-541.
13. Karabekmez FE, Duymaz A, Moran SL. Early clinical outcomes with the use of decellularized nerve allograft for repair of sensory defects within the hand. Hand (N Y), 2009, 4(3): 245-249.
14. Vindigni V, Mazzoleni F, Rossini K, et al. Reconstruction of ablated rat rectus abdominis by muscle regeneration. Plast Reconstr Surg, 2004, 114(6): 1509-1515.
15. Conconi MT, De Coppi P, Bellini S, et al. Homologous muscle acellular matrix seeded with autologous myoblasts as a tissue-engineering approach to abdominal wall-defect repair. Biomaterials, 2005, 26(15): 2567-2574.
16. 薛輝, 陳東, 張秀英, 等. 化學(xué)去細(xì)胞肌肉組織工程支架與大鼠脊髓的生物相容性. 吉林大學(xué)學(xué)報(bào): 醫(yī)學(xué)版, 2009, 35(5): 801-804.
17. Zhang XY, Xue H, Liu JM, et al. Chemically extracted acellular muscle: A new potential scaffold for spinal cord injury repair. J Biomed Mater Res A, 2012, 100(3): 578-587.
18. Wang B, Borazjani A, Tahai M, et al. Fabrication of cardiac patch with decellularized porcine myocardial scaffold and bone marrow mononuclear cells. J Biomed Mater Res A, 2010, 94(4): 1100-1110.
19. Flynn L, Semple JL, Woodhouse KA. Decellularized placental matrices for adipose tissue engineering. J Biomed Mater Res A, 2006, 79(2): 359-369.
20. Flynn L, Woodhouse KA. Adipose tissue engineering with cells in engineered matrices. Organogenesis, 2008, 4(4): 228-235.
21. Gomillion CT, Burg KJ. Stem cells and adipose tissue engineering. Biomaterials, 2006, 27(36): 6052-6063.
22. Cherubino M, Marra KG. Adipose-derived stem cells for soft tissue reconstruction. Regen Med, 2009, 4(1): 109-117.
23. Huss FR, Kratz G. Adipose tissue processed for lipoinjection shows increased cellular survival in vitro when tissue engineering principles are applied. Scand J Plast Reconstr Surg Hand Surg, 2002, 36(3): 166-171.
24. Beahm EK, Walton RL, Patrick CW Jr. Progress in adipose tissue construct development. Clin Plast Surg, 2003, 30(4): 547-558, viii.
25. Patrick CW Jr. Tissue engineering strategies for adipose tissue repair. Anat Rec, 2001, 263(4): 361-366.
26. 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.
27. Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes. Biomaterials, 2011, 32(12): 3233-3243.
28. 卿泉, 秦廷武. 大鼠骨骼肌脫細(xì)胞處理方法的優(yōu)化研究. 中國(guó)修復(fù)重建外科雜志, 2009, 23(7): 836-839.
29. Ciapetti G, Cenni E, Pratelli L, et al. In vitro evaluation of cell/biomaterial interaction by MTT assay. Biomaterials, 1993, 14(5): 359-364.
30. 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.
31. 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.
32. Gimble JM, Guilak F. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy, 2003, 5(5): 362-369.
33. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res, 2007, 100(9): 1249-1260.
34. Dobson DE, Kambe A, Block E, et al. 1-Butyryl-glycerol: a novel angiogenesis factor secreted by differentiating adipocytes. Cell, 1990, 61(2): 223-230.
35. Rodriguez AM, Pisani D, Dechesne CA, et al. Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse. J Exp Med, 2005, 201(9): 1397-1405.
36. McIntosh K, Zvonic S, Garrett S, et al. The immunogenicity of human adipose-derived cells: temporal changes in vitro. Stem Cells, 2006, 24(5): 1246-1253.
  1. 1. Lutolf MP, Hubbell JA. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat Biotechnol, 2005, 23(1): 47-55.
  2. 2. Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues and organs. Biomaterials, 2006, 27(19): 3675-3683.
  3. 3. Borschel GH, Dennis RG, Kuzon WM Jr. Contractile skeletal muscle tissue-engineered on an acellular scaffold. Plast Reconstr Surg, 2004, 113(2): 595-602.
  4. 4. Valentin JE, Turner NJ, Gilbert TW, et al. Functional skeletal muscle formation with a biologic scaffold. Biomaterials, 2010, 31(29): 7475-7484.
  5. 5. Penolazzi L, Mazzitelli S, Vecchiatini R, et al. Human mesenchymal stem cells seeded on extracellular matrix scaffold: Viability and osteogenic potential. J Cell Physiol, 2012, 227(2): 857-866.
  6. 6. Zhu WD, Xu YM, Feng C, et al. Different bladder defects reconstructed with bladder acellular matrix grafts in a rabbit model. Urologe A, 2011, 50(11): 1420-1425.
  7. 7. Zhang X, Yang J, Li Y, et al. Functional neovascularization in tissue engineering with porcine acellular dermal matrix and human umbilical vein endothelial cells. Tissue Eng Part C Methods, 2011, 17(4): 423-433.
  8. 8. 張建, 吳英鋒, 陳亮. 豬主動(dòng)脈脫細(xì)胞基質(zhì)的簡(jiǎn)化制備及生物學(xué)評(píng)價(jià). 中國(guó)修復(fù)重建外科雜志, 2008, 22(3): 364-369.
  9. 9. Wainwright JM, Hashizume R, Fujimoto KL, et al. Right ventricular outflow tract repair with a cardiac biologic scaffold. Cells Tissues Organs, 2012, 195(1-2): 159-170.
  10. 10. Soto-Gutierrez A, Zhang L, Medberry C, et al. A whole organ regenerative medicine approach for liver replacement. Tissue Eng Part C Methods, 2011, 17(6): 677-686.
  11. 11. Uygun BE, Soto-Gutierrez A, Yagi H, et al. Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat Med, 2010, 16(7): 814-820.
  12. 12. Petersen TH, Calle EA, Zhao L, et al. Tissue-engineered lungs for in vivo implantation. Science, 2010, 329(5991): 538-541.
  13. 13. Karabekmez FE, Duymaz A, Moran SL. Early clinical outcomes with the use of decellularized nerve allograft for repair of sensory defects within the hand. Hand (N Y), 2009, 4(3): 245-249.
  14. 14. Vindigni V, Mazzoleni F, Rossini K, et al. Reconstruction of ablated rat rectus abdominis by muscle regeneration. Plast Reconstr Surg, 2004, 114(6): 1509-1515.
  15. 15. Conconi MT, De Coppi P, Bellini S, et al. Homologous muscle acellular matrix seeded with autologous myoblasts as a tissue-engineering approach to abdominal wall-defect repair. Biomaterials, 2005, 26(15): 2567-2574.
  16. 16. 薛輝, 陳東, 張秀英, 等. 化學(xué)去細(xì)胞肌肉組織工程支架與大鼠脊髓的生物相容性. 吉林大學(xué)學(xué)報(bào): 醫(yī)學(xué)版, 2009, 35(5): 801-804.
  17. 17. Zhang XY, Xue H, Liu JM, et al. Chemically extracted acellular muscle: A new potential scaffold for spinal cord injury repair. J Biomed Mater Res A, 2012, 100(3): 578-587.
  18. 18. Wang B, Borazjani A, Tahai M, et al. Fabrication of cardiac patch with decellularized porcine myocardial scaffold and bone marrow mononuclear cells. J Biomed Mater Res A, 2010, 94(4): 1100-1110.
  19. 19. Flynn L, Semple JL, Woodhouse KA. Decellularized placental matrices for adipose tissue engineering. J Biomed Mater Res A, 2006, 79(2): 359-369.
  20. 20. Flynn L, Woodhouse KA. Adipose tissue engineering with cells in engineered matrices. Organogenesis, 2008, 4(4): 228-235.
  21. 21. Gomillion CT, Burg KJ. Stem cells and adipose tissue engineering. Biomaterials, 2006, 27(36): 6052-6063.
  22. 22. Cherubino M, Marra KG. Adipose-derived stem cells for soft tissue reconstruction. Regen Med, 2009, 4(1): 109-117.
  23. 23. Huss FR, Kratz G. Adipose tissue processed for lipoinjection shows increased cellular survival in vitro when tissue engineering principles are applied. Scand J Plast Reconstr Surg Hand Surg, 2002, 36(3): 166-171.
  24. 24. Beahm EK, Walton RL, Patrick CW Jr. Progress in adipose tissue construct development. Clin Plast Surg, 2003, 30(4): 547-558, viii.
  25. 25. Patrick CW Jr. Tissue engineering strategies for adipose tissue repair. Anat Rec, 2001, 263(4): 361-366.
  26. 26. 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.
  27. 27. Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes. Biomaterials, 2011, 32(12): 3233-3243.
  28. 28. 卿泉, 秦廷武. 大鼠骨骼肌脫細(xì)胞處理方法的優(yōu)化研究. 中國(guó)修復(fù)重建外科雜志, 2009, 23(7): 836-839.
  29. 29. Ciapetti G, Cenni E, Pratelli L, et al. In vitro evaluation of cell/biomaterial interaction by MTT assay. Biomaterials, 1993, 14(5): 359-364.
  30. 30. 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.
  31. 31. 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.
  32. 32. Gimble JM, Guilak F. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy, 2003, 5(5): 362-369.
  33. 33. Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res, 2007, 100(9): 1249-1260.
  34. 34. Dobson DE, Kambe A, Block E, et al. 1-Butyryl-glycerol: a novel angiogenesis factor secreted by differentiating adipocytes. Cell, 1990, 61(2): 223-230.
  35. 35. Rodriguez AM, Pisani D, Dechesne CA, et al. Transplantation of a multipotent cell population from human adipose tissue induces dystrophin expression in the immunocompetent mdx mouse. J Exp Med, 2005, 201(9): 1397-1405.
  36. 36. McIntosh K, Zvonic S, Garrett S, et al. The immunogenicity of human adipose-derived cells: temporal changes in vitro. Stem Cells, 2006, 24(5): 1246-1253.