猪小肠黏膜下层脱细胞支架的制备及组织学评价

王付燕 杜立群

解剖学报 ›› 2016, Vol. 47 ›› Issue (6) : 824-828.

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解剖学报 ›› 2016, Vol. 47 ›› Issue (6) : 824-828. DOI: 10.16098/j.issn.0529-1356.2016.06.018
组织学胚胎学发育生物学

猪小肠黏膜下层脱细胞支架的制备及组织学评价

  • 王付燕 杜立群*
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Preparation and histological evaluation of the decellularized scaffold for porcine small intestinal submucosa

  • WANG Fu-yan DU Li-qun*
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摘要

目的 评价不同浓度的十二烷基磺酸钠(SDS)处理猪小肠黏膜下层(SIS)的脱细胞效果,筛选最佳脱细胞浓度,为组织工程化角膜上皮的制备提供支架材料。方法 配制0.1%、0.2%、0.3%、0.5% SDS随机分成A、B、C、D 4组,分别脱细胞处理SIS 15min、30min、1h、2h(n=20),同时观察SIS的大体形态变化,HE和4,6-二脒基-2-苯基吲哚(DAPI)染色观察脱细胞前后SIS的生物学特性,并对脱细胞支架进行基因组 DNA 分析(n=5)。 结果 脱细胞SIS肉眼观察呈乳白色、半透明的膜状物,具有一定的弹性、韧性及透光性;HE和DAPI染色光学显微镜观察结果显示,A组及B组处理30min时SIS生物支架无细胞及DNA残留,组织结构保留完整,胶原纤维间孔隙率增加;A组及B组处理30min脱细胞率可达90%以上。 结论 0.1%及0.2%SDS处理30min条件下脱细胞效果较好,能更好地降低SIS的免疫原性,是SDS脱细胞处理SIS比较理想的浓度时间条件,为组织工程化角膜上皮的构建奠定理论基础。

Abstract

Objective To evaluate the different concentrations of sodium dodecyl sulfonate(SDS)on efficiency of cell removal from porcine small intestinal submucosa(SIS)and optimize the best concentration to produce an acellular porcine SIS scaffold for use in developing a tissueengineered corneal epithelium. Methods Fresh porcine SIS were decellularized with SDS (concentration of 0.1%, 0.2%, 0.3%, and 0.5% for 15 minutes, 30 minutes, 1 hour, and 2 hours) and randomly divided into 4 groups (A, B, C, D, 20 pieces in each group ) and the decellularization process was observed at the same time.After decellularization,the scaffolds were examined through the HE, 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining and the genomic DNA contect analysis to observe the biological characteristics of SIS. Results A cellular SIS was shown ivory, translucent and had certain transmittance. HE and DAPI staining revealed that when treatment with the time of 30 minutes in group A and B SIS were decellularized completely and preserved the overall tissue histoarchitecture, the collagen fiber porosity was increased, the acellular rate was more than 90%. Conclusion The acellular matrix treatment effect of 0.1% and 0.2% SDS for a period of 30min is satisfactory. The immunogenicity of the SIS can be reduced, therefore, it is an ideal acellular concentration for SIS and provides a theoretical basis for the construction of tissue engineering corneal epithelium.

关键词

组织工程 / 支架材料 / 脱细胞 / 小肠黏膜下层 / HE染色 / DAPI染色

Key words

Tissue engineering / Scaffold / Decellularization / Small intestinal submucosa / HE staining / DAPI staining

引用本文

导出引用
王付燕 杜立群. 猪小肠黏膜下层脱细胞支架的制备及组织学评价[J]. 解剖学报. 2016, 47(6): 824-828 https://doi.org/10.16098/j.issn.0529-1356.2016.06.018
WANG Fu-yan DU Li-qun. Preparation and histological evaluation of the decellularized scaffold for porcine small intestinal submucosa[J]. Acta Anatomica Sinica. 2016, 47(6): 824-828 https://doi.org/10.16098/j.issn.0529-1356.2016.06.018

参考文献

[1]Wirostko B, Rafii MJ, Sullivan DA, et al. Novel therapy to treat corneal epithelial defects: a hypothesis with growth hormone[J]. Ocul Surf, 2015, 13(3):204-212.
[2]Menzel-Severing J, Kruse FE, Schlǒtzer-Schrghardt U. Stem cell-based therapy for corn ealepithelial reconstruction: present and future[J]. Can J Ophthalmol, 2013, 48(1):13-21.

[3]Yuan S, Fan G. Stem cell-based therapy of corneal epithelial and endothelial diseases.[J]. Regen Med, 2015, 10(4):495-504.
[4]Andrée B, B?r A, Haverich A, et al. Small intestinal submucosa segments as matrix for tissue engineering: review.[J]. Tissue Eng Part B Rev, 2013, 19(4):279-291.
[5]Jia XJ.Preliminary study on construction of corneal posterior lamellar with small intestinal submueosa compound of rabbit corneal endothelial cells in vitro[D].Guangzhou: jinan university,2011.(in Chinese)
贾晓静. 复合兔角膜内皮细胞的猪小肠粘膜下层体外构建角膜后板层的初步研究[D]. 广州:暨南大学, 2011.
[6]Goulle F. Use of porcine small intestinal submucosa for corneal reconstruction in dogs and cats: 106 cases[J]. J Small Anim Pract, 2012, 53(1):34-43.
[7]Lin XF,Shao YK,Wang H,et al.Preparation and identification of the pancreatic decellularized bio-derived scaffold in isolated rats[J].Acta Anatomica Sinica,2012, 43(5):717-722.(in Chinese)
林贤丰, 邵营宽, 王辉,等. 离体大鼠胰腺去细胞生物支架的制备与鉴定[J]. 解剖学报, 2012, 43(5):717-722.
[8]Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues and organs[J]. Biomaterials, 2006, 27(19):3675-3683.
[9]Du L, Wu X, Pang K, et al. Histological evaluation and biomechanical characterisation of an acellular porcine cornea scaffold.[J]. Br J Ophthalmol, 2011, 95(3):410-414.
[10]Oliveira AC, Garzón I, Ionescu AM, et al. Evaluation of small intestine grafts decellularization methods for corneal tissue engineering.[J]. PLoS One, 2013, 8(6): e38566.
[11]Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes[J]. Biomaterials, 2011, 32(12):3233-3243.
[12]Du L, Wu X. Development and characterization of a full-thickness acellular porcine cornea matrix for tissue engineering[J]. Artif Organs, 2011, 35(7):691-705.
[13]Bayyoud T, Thaler S, Hofmann J, et al. Decellularized bovine corneal posterior lamellae as carrier matrix for cultivated human corneal endothelial cells.[J]. Curr Eye Res, 2012, 37(3):179-186.
[14]Yoeruek E, Bayyoud T, Maurus C, et al. Decellularization of porcine corneas and repopulation with human corneal cells for tissue-engineered xenografts[J]. Acta Ophthalmol, 2012, 90(2):e125-e131.
[15]Nakayama KH, Batchelder CA, Lee CI, et al. Decellularized rhesus monkey kidney as a three-dimensional scaffold for renal tissue engineering.[J]. Tissue Eng Part A, 2010, 16(7):2207-2216.
[16]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[J]. Acta Biomater, 2008, 4(4):808-816.
[17]Wilson SL, Sidney LE, Dunphy SE, et al. Keeping an eye on decellularized corneas: a review of methods, characterization and applications[J]. J Funct Biomater, 2013, 4(3):114-161.
[18]Bo W, Tedder ME, Perez CE, et al. Structural and biomechanical characterizations of porcine myocardial extracellular matrix[J]. J Mater Sci Mater Med, 2012, 23(8):1835-1847.[19]Schaner PJ, Martin ND, Tulenko TN, et al. Decellularized vein as a potential scaffold for vascular tissue engineering [J]. J Vasc Surg, 2004, 40(1):146-153.

基金

山东省自然科学基金面上项目;山东省优秀中青年科学家科研奖励基金


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