Preparation of an acellular cartilage matrix scaffold

doi:10.3969/j.issn.2095-4344.2015.16.007

Abstract

Abstract:

BACKGROUND: Acellular matrix which is decellularized but retains the matrix components in the nature materials can reduce the immunogenicity of natural materials effectively and keep the material mechanical strength.
OBJECTIVE: To prepare the natural biological scaffold material by using elution method to remove the cells in the rabbit costicartilage matrix.
METHODS: After removal of the surrounding tissues, the costicartilage samples from New Zealand white rabbits were randomly divided them into three groups: costicartilages untreated as normal control group, detergent-enzyme digestion for 48 hours as 48-hour treatment group, detergent-enzyme digestion for 96 hours as 96-hour treatment group. Hematoxylin-eosin staining and electron microscope were used to observe decellularized effects. At 7 days of induction, bone marrow mesenchymal stem cells, 3×109/L, were collected and co-cultured with allogeneic acellular costicartilage matrix in vitro. At 3 and 7 days of co-culture, the composite was taken for observation of cell growth on the cell-free substrate surface under electron microscope.
RESULTS AND CONCLUSION: Two or three cartilage cells were closely packed in each cartilage pit, began to depigment using the detergent-nuclease digestion method, and then completely depigmented at 96 hours after treatment. At 3 days of co-culture, there were a large amount of polygon-shaped adherent cells with pseudopodias observed on the acellular matrix surface in vitro, and cells could proliferate and divide in partial regions. At 7 days of co-culture, adherent cells spread mostly throughout the acellular matrix surface, these cells were flat-shaped and extended with multiple interconnected processes. Mass of secreted excellular matrixes were deposited on the matrix surface and showed frost-like changes, indicating the prepared acellular matrix has favorable cytocompatibility.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: Tissue Engineering, Stem Cells, Chondrocytes

CLC Number:

R318

Sun Liang, Li Pi-bao, Luan Bao-hua. Preparation of an acellular cartilage matrix scaffold [J]. Chinese Journal of Tissue Engineering Research, 2015, 19(16): 2494-2499.

Figures/Tables 2

2.1 大体观察新鲜肋软骨去除多余组织充分冲洗后，标本色淡黄，弹性良好(图1)。经脱细胞处理后，标本变成乳白色，软骨弹性良好(图2)。 2.2 组织学观察正常未经脱细胞处理的新鲜肋软骨苏木精-伊红染色切片，可见每个软骨陷窝内均有排列紧密的二三个软骨细胞，其细胞基质中的胶原红染，分布较为均一，细胞核明显蓝染(图3A)。随着消化处理时间延长，软骨陷窝内细胞逐渐脱失(图3B)，至96 h时，标本软骨陷窝内的细胞完全脱失(图3C)。 2.3 干细胞生长及诱导后染色观察原代骨髓间充质干细胞培养5 d后可见细胞集落单位形成(图4A)；诱导14 d后可见Ⅱ型胶原阳性细胞较多，阳性位置位于胞浆内，呈棕黄色至棕褐色细密颗粒，在胞核周围更加明显(图4B)，大部分细胞胞浆被染成蓝色，分布均匀一致，而胞核呈蓝黑色，异染明显(图4C)。 2.4 微观结构观察扫描电镜下可见，新鲜肋软骨标本软骨陷窝内软骨细胞生长，无脱失(图5A)；化学消化48 h组标本可见软骨细胞脱失随消化时间增长而增多(图5B)，至化学消化96 h仅见塌陷的细胞基底存在，软骨表面较新鲜标本光滑，软骨细胞脱失，软骨陷窝结构清晰可见(图5C)。 2.5 骨髓间充质干细胞与脱细胞软骨基质的联合培养第3天电镜下可见脱细胞基质表面有大量细胞成分分布，细胞为多角形，有伪足伸出，锚定在基质表面(图6A)，部分区域可见细胞在基质表面增殖分裂(图6B)；第7天时，脱细胞基质表面大部分均为细胞成分覆盖，细胞呈扁平状，有多个足突充分伸展，细胞之间互相连接，三四层细胞成层排列，局部可见6-8层，分泌大量细胞外基质沉积在基质表面，呈冰霜样改变(图6C)。"

References

[1] 李春根,曲戈,叶超,等.组织工程软骨与柚皮苷联合修复兔膝关节软骨缺损的组织学证实[J].中国组织工程研究,2014,18(20): 3165-3171.
[2] Jungebluth P,Bader A,Baiguera S,et al.The concept of in vivo airway tissue engineering. Biomaterials. 2012;33(17): 4319-4326.
[3] Zang M,Zhang Q,Chang EI,et al.Decellularized Tracheal Matrix Scaffold for Tissue Engineering.Plast Reconstr Surg. 2012; 130(3):532-540.
[4] Haag J,Baiguera S,Jungebluth P,et al.Biomechanical and angiogenic proprties of tissue-engineered rat trachea using genipin cross-linked decellularized tissue.Biomaterials. 2012; 33(3):780-789.
[5] Ott LM,Weatherly RA,Detamore MS.Overiew of tracheal tissue engineering;clinical need drives the laboratory approach.Ann Biomed Eng.2011;39:2091-2113.
[6] Hoshiba T,Lu H,Kawazoe N,et al.Decellularized matrices for tissue engineer.Expert Opin Biol Ther.2011;10:1717-1728.
[7] 孙飞,潘枢,史宏灿,等.去污剂-联合酶法行兔脱细胞组织工程气管制备及性能评价[J].中华胸心血管外科杂志,2014,30(1): 38-41.
[8] 章方彪,史宏灿,张卫东,等.兔脱细胞同种异体气管基质衍生材料的形态特征及其生物力学[J].中华实验外科杂志,2014,31(3): 573-576.
[9] 李炜,赁可,周艳荣,等.制备犬胸主动脉脱细胞血管支架的新方法[J].中国心血管病研究,2014,12(4):337-384.
[10] Badylak SF,Weiss DJ,Caplan A,et al.Engineered whole organs and complex tissues.Lancet.2012;379:943-952.
[11] Crapo PM,Gilbert TW,Badylak SF.An overview of tissue and whole organ decellularization processes.Biomaterials. 2011; 32:3233-3243.
[12] Elliott MJ,De Coppi P,Speggiorin S,et al.Stem-cell-based, tissue engineered tracheal replacement iin a child:a2-year follow-up study.Lancet.2012;380:994-1000.
[13] Partington L,Mordan NJ, Mason C,et al.Biochemical changes caused by decellularization may compromise mechanical integrity of tracheal scaffolds.Acta Biomater.2013;9: 5251-5261.
[14] Baiiguera S,Del Gaudio C,Jaus MO,et al.Long-term changes to in vitro preserbed bioengineered human trachea and their implications for decellularized tissues.Biomaterials. 2012; 33:3662-3672.
[15] 张新.不同材料构建组织工程软骨及支架的应用[J].中国组织工程研究与临床康复,2012,16(8):1491-1495.
[16] 陈哲峰,范卫民,刘峰.利用PLA、PGA、PLGA三种支架再生兔关节软骨[J].江苏医学,2005,31(8):599-601.
[17] Breitbait S,Graride DA.Tissue engineerde bone repair of calvarial defects using cultured periosteal cells.Plast Reconstr Surg.1998;101(3):567-572.
[18] Cook AD,Harkach JS.Characterization and development of RGD-pe ptide-modified poly(Lactic acidcolysine)as an interactive resorbable biomaterial. Biomed Mater Res.1997; 35(4):513-518．
[19] 周长忍,丁珊,李立华.可降解材料的制备及用研究进展[J].中华创伤骨科杂志,2000,12(4):317-319.
[20] 韦益毅.不同生物材料复合支架对关节软骨缺损的修复评价[J].中国组织工程研究与临床康复,2011,15(25):4723-4725.
[21] Wakitani S,Goto T,Young RG,et al.Repair of large fullthickness articular cartilage defectswith allograft articular chondrocytes embedded in a collagen gel.Tissue Eng.1998;4(4):429-444.
[22] Lee CR,Grodzinsky AJ,Spector M.Biosynthetic response of passaged chondrocytes in a type II collagen scaffold to mechanical compression.J Biomed Mater Res.2003;64(3): 560-569.
[23] Van Susante JL,Buma P,Homminga GN,et al.Chondrocyte seeded hydroxyapatite for repair of large articular cartilage defects：A pilot study in the goat.Biomaterials.1998; 19(24): 2367-2374．
[24] Wainwright DJ.Use of an acellular allograft dermal matrix (AlloDerm) in the management of full-thickness burns.Burns. 1995;21(4):243-248.
[25] Martin ND,Schaner PJ,Tulenko TN,et al.In vivo behavior of decellularized vein allograft.J Surg Res.2005;129(1):17-23.
[26] Ng KW,Khor HL,Hutmacher DW.In vitro characterization of natural and synthetic dermal matrices cultured with human dermal fibroblasts.Biomaterials.2004;25(14):2807-2818.
[27] Jernigan TW,Croce MA,Cagiannos C,et al.Small intestinal submucosa for vascular reconstruction in the presence of gastrointestinal contamination.Ann Surg.2004;239(5): 733-738.
[28] Cebotari S,Mertsching H,Kallenbach K,et al.Construction of autologous human heart valves based on an acellular allograft matrix.Circulation.2002;106(12 Suppl 1):163-168.
[29] Courtman DW,Pereira CA,Kashef V,et al.Development of a pericardial acellular matrix biomaterial: biochemical and mechanical effects of cell extraction.J Biomed Mater Res. 1994;28(6):655-666.
[30] 徐斌,王浩,徐洪港,等.同种异体脱钙骨复合骨髓间充质干细胞在兔膝关节腔内培养组织工程软骨的研究[J].中华实验外科杂志, 2011,28(6):979-982.