• 四川大學(xué)華西醫(yī)院（成都，610041）1 骨科，2 生物治療國家重點(diǎn)實(shí)驗(yàn)室?干細(xì)胞與組織工程研究室;

【摘要】　目的　 以脫細(xì)胞牛軟骨基質(zhì)（acellular cartilaginous matrix，ACM）作支架體外構(gòu)建組織工程軟骨，了解其作為軟骨組織工程支架的可行性。　方法　 2003年1月-2005年12月聯(lián)合應(yīng)用凍干-反復(fù)凍融-酶消化法對牛軟骨基質(zhì)行脫細(xì)胞處理。將體外培養(yǎng)擴(kuò)增的2~5代兔軟骨細(xì)胞接種在材料上，體外培養(yǎng)3周，觀察軟骨細(xì)胞在支架材料上的生長分布情況。　結(jié)果　 軟骨細(xì)胞在制備的ACM上可較好地黏附生長，并且分泌大量Ⅱ型膠原和葡萄糖胺聚糖；但軟骨細(xì)胞不能長入ACM內(nèi)部，只能在表層生長，少量軟骨細(xì)胞分布在ACM孔隙中。　結(jié)論　 ACM支架材料具有良好的細(xì)胞相容性和活性，并且能促進(jìn)軟骨細(xì)胞增殖和維持軟骨細(xì)胞表型。
【Abstract】　Objective　 To investigate the feasibility of applying the acellular cartilaginous matrix (ACM) as the scaffold in constructing tissue engineering cartilage in vitro.　Methods　 We processed the bovine cartilaginous matrix by combination of lyophilization (freeze dry) and repeated freeze-thaw (12 cycles) and enzyme digestion in order to remove the cell component. The 2-5 passage chondrocytes were seeded onto the ACM, and had been cultured for 3 weeks. The cell growth, attachment and distribution were detected by histochemical stain, immunohistochemical stain, and scan electron microscope.　Results　 The chondrocytes could adhere and grow well on the matrix surface, and synthesize plenty of glycosaminoglycanand type Ⅱ collagen. However, the chondrocytes grew only on the surface and superficial layer of the scaffold, and they did not move into the inner part of the scaffold.　Conclusion　 ACM has good cellular compatibility without cytotoxicity and provide temporary substrate to which these anchorage-dependent cells can adhere, and stimulate the chondrocytes anchored on the scaffold to proliferate and keep differentiated phenotype.

引用本文： 高博,孔清泉,項(xiàng)舟,楊志明,羅靜聰,李秀群. 以脫細(xì)胞牛軟骨基質(zhì)為支架體外構(gòu)建組織工程軟骨的實(shí)驗(yàn)研究. 華西醫(yī)學(xué), 2010, 25(11): 2028-2031. doi: 復(fù)制

1.
2. 　Ciorba A, Martini A. Tissue engineering and cartilage regeneration for auricular reconstruction[J]. Inter J Pediatr Otorhinol, 2006, 70: 1507-1515.
3. 　Badylak SF. Xenogeneic extracellular matrix as a scaffold for tissue reconstruction[J]. Transpl Immunol, 2004, 12(3-4): 367-377.
4. 　Schlote N, Wefer J, Sievert KD. Acellular matrix for functional reconstruction of the urogenital tract. Special form of "tissue engineering"[J]? Urologe A, 2004, 43(10): 1209-1212.
5. 　Sharma B, Elisseeff JH. Engineering structurally organized cartilage and bone tissues[J]. Ann Biomed Eng, 2004, 32(1): 148-159.
6. 　Capito RM, Spector M. Scaffold-based articular cartilage repair. IEEE Eng Med Biol Mag, 2003, 22(5): 42-50.
7. 　Kleinman HK, Philp D, Hoffman MP. Role of the extracellular matrix in morphogenesis[J]. Curr Opin Biotechnol, 2003, 14(5): 526-532.
8. 　Takezawa T. A strategy for the development of tissue engineering scaffolds that regulate cell behavior[J]. Biomaterials, 2003, 24(13): 2267-2275.
9. 　Badylak SF. The extracellular matrix as a scaffold for tissue reconstruction[J]. Semin Cell Dev Biol, 2002, 13(5): 377-383.
10. 　Abenavoli FM, Corelli R. About AlloDerm[J]. Plast Reconstr Surg, 2001, 108(7): 2175-2176.
11. 　Sclafani AP, Romo T 3rd, Jacono AA, et al. Evaluation of acellular dermal graft in sheet (AlloDerm) and injectable (micronized AlloDerm) forms for soft tissue augmentation. Clinical observations and histological analysis[J]. Arch Facial Plast Surg, 2000, 2(2): 130-136.
12. 　張晨, 王者生, 高景恒. 軟骨脫細(xì)胞基質(zhì)生物性的實(shí)驗(yàn)研究[J]. 實(shí)用美容整形外科雜志, 2003, 14(2): 59-61.
13. 　張晨, 景士兵, 楊琨, 等. 軟骨脫細(xì)胞基質(zhì)支架材料的軟骨組織工程實(shí)驗(yàn)研究[J]. 中國修復(fù)重建外科雜志, 2008, 22(7): 846-850.
14. 　Peretti GM, Randolph MA, Villa MT, et al. Cell-based tissue-engineered allogeneic implant for cartilage repair[J]. Tissue Eng, 2000, 6(5): 567-576.
15. 　康紅軍, 盧世避, 孫明學(xué), 等. 利用脂肪干細(xì)胞構(gòu)建組織工程軟骨修復(fù)兔膝關(guān)節(jié)軟骨缺損[J]. 中華創(chuàng)傷骨科雜志, 2007, 9(4): 326-330.
16. 　Kelley TF, Sutton FM, Wallace, et al. Chondrocyte repopulation of allograft cartilage: a preliminary investigation and strategy for developing cartilage matrices for reconstruction[J]. Otolaryngol Head Neck Surg, 2002, 127(4): 265-270.
17. 　Zaleske D, Peretti G, Allemann F, et al. Engineering a joint: a chimeric construct with bovine chondrocytes in a devitalized chick knee[J]. Tissue Eng, 2003, 9(5): 949-956.
  1. 1.
  2. 2. 　Ciorba A, Martini A. Tissue engineering and cartilage regeneration for auricular reconstruction[J]. Inter J Pediatr Otorhinol, 2006, 70: 1507-1515.
  3. 3. 　Badylak SF. Xenogeneic extracellular matrix as a scaffold for tissue reconstruction[J]. Transpl Immunol, 2004, 12(3-4): 367-377.
  4. 4. 　Schlote N, Wefer J, Sievert KD. Acellular matrix for functional reconstruction of the urogenital tract. Special form of "tissue engineering"[J]? Urologe A, 2004, 43(10): 1209-1212.
  5. 5. 　Sharma B, Elisseeff JH. Engineering structurally organized cartilage and bone tissues[J]. Ann Biomed Eng, 2004, 32(1): 148-159.
  6. 6. 　Capito RM, Spector M. Scaffold-based articular cartilage repair. IEEE Eng Med Biol Mag, 2003, 22(5): 42-50.
  7. 7. 　Kleinman HK, Philp D, Hoffman MP. Role of the extracellular matrix in morphogenesis[J]. Curr Opin Biotechnol, 2003, 14(5): 526-532.
  8. 8. 　Takezawa T. A strategy for the development of tissue engineering scaffolds that regulate cell behavior[J]. Biomaterials, 2003, 24(13): 2267-2275.
  9. 9. 　Badylak SF. The extracellular matrix as a scaffold for tissue reconstruction[J]. Semin Cell Dev Biol, 2002, 13(5): 377-383.
  10. 10. 　Abenavoli FM, Corelli R. About AlloDerm[J]. Plast Reconstr Surg, 2001, 108(7): 2175-2176.
  11. 11. 　Sclafani AP, Romo T 3rd, Jacono AA, et al. Evaluation of acellular dermal graft in sheet (AlloDerm) and injectable (micronized AlloDerm) forms for soft tissue augmentation. Clinical observations and histological analysis[J]. Arch Facial Plast Surg, 2000, 2(2): 130-136.
  12. 12. 　張晨, 王者生, 高景恒. 軟骨脫細(xì)胞基質(zhì)生物性的實(shí)驗(yàn)研究[J]. 實(shí)用美容整形外科雜志, 2003, 14(2): 59-61.
  13. 13. 　張晨, 景士兵, 楊琨, 等. 軟骨脫細(xì)胞基質(zhì)支架材料的軟骨組織工程實(shí)驗(yàn)研究[J]. 中國修復(fù)重建外科雜志, 2008, 22(7): 846-850.
  14. 14. 　Peretti GM, Randolph MA, Villa MT, et al. Cell-based tissue-engineered allogeneic implant for cartilage repair[J]. Tissue Eng, 2000, 6(5): 567-576.
  15. 15. 　康紅軍, 盧世避, 孫明學(xué), 等. 利用脂肪干細(xì)胞構(gòu)建組織工程軟骨修復(fù)兔膝關(guān)節(jié)軟骨缺損[J]. 中華創(chuàng)傷骨科雜志, 2007, 9(4): 326-330.
  16. 16. 　Kelley TF, Sutton FM, Wallace, et al. Chondrocyte repopulation of allograft cartilage: a preliminary investigation and strategy for developing cartilage matrices for reconstruction[J]. Otolaryngol Head Neck Surg, 2002, 127(4): 265-270.
  17. 17. 　Zaleske D, Peretti G, Allemann F, et al. Engineering a joint: a chimeric construct with bovine chondrocytes in a devitalized chick knee[J]. Tissue Eng, 2003, 9(5): 949-956.