Effect of different decellularizing methods on cell-secreted extracellular matrix in vitro

doi:10.3969/j.issn.2095-4344.2014.30.017

Abstract

Abstract:

BACKGROUND: Compared with single or composite biomolecular materials, decellulazied matrices are more biomimetic to natural ectocytic surroundings. So cell-secreted extracellular matrix is paid more and more attention in the field of tissue engineering, and the composition of these matrices are influenced by decellulezired preparation methods more or less. But there are few studies about the biological effect of different decellulazried methods on the extracellular matrix.
OBJECTIVE: To investigate the composition of cell-secreted extracellular matrices in vitro by different decellularizing methods and their effects as surface modification on cytobiological reaction.
METHODS: After the treatment of different decellularizing methods (freeze/thaw cycles, trypsin, weak alkal, detergents), the extracellular matrix was obtained and grouped into four kinds. The biological composition of the extracellular matrix was determined by ELISA assay. Then osteoblasts were seeded onto the four kinds of extracellular matrix surfaces. Cells cultured normally served as controls. The effect of extracellular matrix coatings on cell growth and differentiation were determined by MTT test and alkaline phosphatase activity test.
RESULTS AND CONCLUSION: The residual components were the most in the freeze-thaw group, followed by the detergent and weak alkal groups, and the least in the trypsin group. Compared with the control group, the absorbance value of cells were lower in the freeze-thaw and detergent groups at days 3, 5, 7 after inoculation (both P < 0.05); the alkaline phosphatase activity was lower in the trypsin group at days 5, 7 after inoculation (P < 0.05), in the weak alkal group at 7 days after inoculation (P < 0.05), and in the detergent group at days 3, 5, 7 after inoculation (P < 0.05). Therefore, we can harvest more extracellular matrices by the freeze-thaw method, and the extracellular matrix coating synthesized by the freeze-thaw method is more helpful for cell growth than others.

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: osteoblasts, extracellular matrix

CLC Number:

R318

Fan Li-mei, Xia Rong, Dou Xiao-chen . Effect of different decellularizing methods on cell-secreted extracellular matrix in vitro[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(30): 4863-4867.

Figures/Tables 3

References

[1] Morra M. Biochemical Modification of titanium surfaces: peptide and ECM proteins. Eur Cell Mater.2006;12:1-15.
[2] Kim S,Myung WC,Lee JS,et al.The effect of fibronectin-coated implant on canine osseointegration.J Periodontal Implant Sci.2011;41(5):242-247.
[3] Lamberg A,Schmidmaier G,Soballe K.Locally delivered TGF-beta1 and IGF-1 enhance the fixation of titanium implants: a study in dogs.Acta Orthopaedica.2006;77(5): 799-805.
[4] Hennessy KM,Pollot BE,Clem WC,et al.The effect of collagen I mimetic peptides on mesenchymal stem cell adhesion and differentiation, and on bone formation at hydroxyapatite surfaces.Biomaterials.2009;30(10):1898-1909.
[5] Culpepper BK,Bonvallet PP,ReddyM S,et al.Polyglutamate directed coupling of bioactive peptides for the delivery of osteoinductive signals on allograft bone. Biomaterials. 2013; 34(5):1506-1513.
[6] Shekaran A,García AJ.Extracellular matrix-mimetic adhesive biomaterials for bone repair.J Biomed Mater Res A. 2011; 96(1):261-272.
[7] Baas J,Jakobsen T,Elmengaard B,et al.The effect of adding an equine bone matrix protein lyophilisate on fixation and osseointegration of HA-coated Ti implants. J Biomed Mater Res A.2012;100(1):188-194.
[8] 毛晶晶,何家才,郑先雨,等.富血小板纤维蛋白在犬上颌窦底提升中促骨缺损修复的作用[J].安徽医科大学学报, 2010,45(2): 176-179
[9] 孔丽,唐旭炎,李全利.新型纳米复合材料对犬下颌牙槽骨缺损的修复效果[J].安徽医科大学学报,2011,46(12):1245-1248.
[10] 周允芝,唐旭炎,宁天云,等.类釉原蛋白寡肽仿生修饰钛表面的研究[J].安徽医科大学学报,2013,48(2):124-127.
[11] 杨全全,何家才,杨瑞,等.富血小板纤维蛋白修复牙种植体周围骨缺损的研究[J].安徽医科大学学报,2012,47(5):581-584.
[12] 陈永吉,曾宪涛,解龙川,等.异种脱细胞真皮基质在牙种植术中的临床应用[J].临床口腔医学杂志,2011,27(4):240-242.
[13] 邹嘉平,杨契超.异种脱细胞真皮基质修复膜修复鼓膜穿孔的临床应用[J].南京医科大学学报:自然科学版, 2011,31(1):94-95, 122.
[14] 刘广毅,杨军成.脱细胞异体真皮基质修复口腔组织缺损的疗效观察[J].四川医学,2012,33(7):1206-1207.
[15] 张毓姣,沈国良,林伟,等.异种脱细胞真皮基质与薄自体皮复合移植用于深度烧伤创面治疗[J].浙江临床医学, 2012,14(12): 1519-1520.
[16] 余勇,赵慧,许瑾,等.脱细胞异体真皮支架与自体皮片复合移植的临床应用[J].蚌埠医学院学报,2012,37(10):1189-1190.
[17] 刘立强,马莲.异种脱细胞真皮基质用于牙槽嵴裂植骨术的效果观察[J].北京口腔医学,2012,20(6):338-340.
[18] 王乾锋,刘宏伟.脱细胞真皮基质作为GTR屏障膜治疗Ⅱ度根分叉缺损的实验研究[J].口腔颌面外科杂志, 2013,23(5): 347-351.
[19] 周瑜,叶茂昌,李容新,等.应用改良型异种脱细胞真皮基质修复重建小型舌鳞癌切除术后缺损[J].中国口腔颌面外科杂志, 2014, 12(1):56-60.
[20] 王永前,李森恺.脱细胞膀胱黏膜下基质异种移植的实验研究[J].军事医学,2013,37(9):696-699.
[21] 韦殿闯.磨痂术联合DR脱细胞异种皮移植术在四肢深Ⅱ度烧伤创面的运用[J].广西医科大学学报,2013,30(5):793-794.
[22] Datta N,Pham QP,Sharma U,et al.In vitro generated extracellularmatrix and fluid shear stress synergistically enhance 3D osteoblastic differentiation.Proc Natl Acad Sci U S A.2006;103(8):2488-2493.
[23] Pham QP,Kasper FK,Scott BL,et al.The influence of an in vitro generated bone-like extracellular matrix on osteoblastic gene expression of marrow stromal cells. Biomaterials. 2008; 29(18): 2729-2739.
[24] 范利梅,唐旭炎,李全利,等.骨髓基质细胞在胞外基质涂层钛表面的黏附和铺展[J].中国组织工程研究与临床康复, 2009,13(21): 4029-4032.
[25] 范利梅,唐旭炎,李全利,等.钛表面胞外基质涂层对骨髓基质细胞增值和分化的影响[J].安徽医科大学学报, 2009,44(5): 551-553.
[26] Thibault RA,Scott Baggett L,Mikos AG,et al.Osteogenic differentiation of mesenchymal stem cells on pregenerated extrcellular matrix scaffolds in the absence uf osteogenic cell culture supplements.Tissue Eng A.2010;16(2)431-440.
[27] Decaris ML,Mojadedi A,Bhat A,et al.Transferable cell-secreted extracellular matrices enhance osteogenic differentiation.Acta Biomater.2012;8(2):744-752.
[28] Decaris ML,Leach JK.Design of experiments approach to engineer cell-secreted matrices for direcring osteogenic differentiation.Ann Biomed Eng.2011;39(4):1174-1185.
[29] Prewitz MC,Seib FP,von Bonin M,et al.Tightly anchored tissue-mimetic matrices as instructive stem cell microenviroments.Nat Methods.2013;10(8):788-794.
[30] 孙亨,王艳霞,吴江.组织脱细胞方法的研究进展[J].四川解剖学杂志,2011,19(4):24-30.
[31] 陈博,彭代智.组织和器官脱细胞方法的研究进展[J].中华损伤与修复杂志:电子版, 2012,7(6):58-63.
[32] 赵宇,于淼,柏树令.脱细胞技术及其在组织工程中的应用研究进展[J].中国修复重建外科杂志,2013,27(8):950-954.
[33] 张红霞,翟万银,张红锋.适用于不同尺寸血管的脱细胞方法研究[J].华东师范大学学报:自然科学版,2012,58(4):50-60.
[34] 阴洪,任先军,蒋涛.脱细胞技术在同种异体组织工程支架中的研究进展[J].中国矫形外科杂志,2012,20(11):1010-1012.
[35] 陈博,彭代智,左海斌,等.新生小牛皮肤的形态学和生物力学特点及脱细胞方法探索[J].重庆医学,2012,41(11):1043-1046,封2.
[36] 杨召,马信龙,李秀兰,等.不同脱细胞方法对周围神经生物力学性能的影响[J].中国生物医学工程学报,2011,30(1):155-159.
[37] 贾桦,王莹,佟雷,等.不同脱细胞方法制备的AECM成分分析及比较[J].牡丹江医学院学报,2013,34(4):1-4.
[38] 许海委,徐宝山,杨强,等.不同脱细胞方法对猪尾纤维环生物力学特性及组织结构的影响[J].医用生物力学, 2013,28(4): 448-453.
[39] 范雪梅,李胜平,徐惠成.交联剂对脱细胞膀胱基质生物力学性能的影响[J].北京生物医学工程,2013,32(4):370-374.
[40] 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): 38-41,55.
[41] 唐孝明,万伦,刘仲前,等.改良组织块法大鼠成骨细胞培养的研究[J].四川医学,2004,25(5):45-45,245.
[42] 胡静,郑洪新.改良成骨细胞体外培养和鉴定方法[J].中国老年学杂志,2006,26(1):76-78.
[43] 李晓峰,赵劲民,苏伟,等.大鼠成骨细胞的原代培养和鉴定[J].中国组织工程研究与临床康复,2011,15(6):990-994.
[44] 王双利,查振刚,刘宁,等.改进酶消化法培养SD大鼠成骨细胞及其鉴定[J].实用医学杂志,2008,24(6):915-918.
[45] 程浩,张延芳,许巍.新生大鼠成骨细胞原代培养与鉴定[J].中国组织工程研究,2013,17(41):7199-7204.