一种用于复合材料观察的快速病理制片方法研究
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摘要
目的探讨一种用于观察复合材料多孔结构及组分分布的简便、快捷病理制片方法。方法以聚氨酯-小肠黏膜下层(polyurethane/small intestinal submucosa,PU/SIS)复合材料为例,经OCT包埋,行冰冻切片,大体观察切片完整性。切片经水溶性伊红的醇溶液染色,明场显微镜下观察复合材料上色效果及多孔结构。将染色切片分为5组,采用不同封片方式:A组,甘油明胶湿封;B组,无水乙醇脱水、TO型生物透明剂透明、中性快干胶封片;C组,去离子水浸泡分色、风干、中性快干胶封片;D组,风干、TO型生物透明剂透明、中性快干胶封片;E组,风干、中性快干胶封片。于明场显微镜下观察复合材料形态与两种组分分布情况,选择最佳的制片方法。结果大体观察制备的PU/SIS复合材料冰冻切片厚度均为6μm,切片完整连续。明场显微镜下观察示,切片伊红染色后可观察到材料清晰轮廓与多孔结构,但无法辨认两种组分。封片后,A、B、C组材料组分仍不能辨认或溶解变形;D、E组材料形状保持,两种组分清晰可见。结论冰冻切片法制备PU/SIS复合材料标本,可通过伊红染色和中性快干胶封片的方法进行材料形态学和组分分布的表征。
Objective To explore a simple and rapid pathological slices method to observe the porous structure and the composition distribution of composite materials. Methods Taking polyurethane/small intestinal submucosa(PU/SIS) composite as an example, PU/SIS was OCT-embedded and sliced into sections by frozen section technology,after which general observation of the section integrity was carried out. After dyed with water-soluble eosin in alcoholic solution, the staining effect and the porous structure of the composite were observed under light field microscope.Sections were sealed with five different sealing methods. Group A: sealing piece using glycerogelatin method; group B:anhydrous alcohol dehydration→transparency using TO transparent reagent→sealing piece using neutral quick drying glue; group C: color separation using deionized water→air-drying→sealing piece using neutral quick drying glue; group D: air-drying→transparency using TO transparent reagent→sealing piece using neutral quick drying glue; group E: airdrying→sealing piece using neutral quick drying glue. Then, the morphology and the components distribution of the composite were observed under light field microscope, and the simple and feasible method was selected as optimum method. Results From general observation, the frozen section of the PU/SIS composite, which was 6 μm in thickness,was complete and continuous. Although the outline of the material and the porous structure in the sections could be observed clearly under light field microscope, the two components still could not be identified by using eosin staining method. After sealing piece, the material components in groups A, B, and C still could not be identified or be dissolved and deformed; the morphology of the material in groups D and E were preserved and the two components in the composite were clearly visible. Conclusion The morphology and the components distribution of PU/SIS frozen sections can be characterized after soluble eosin staining and neutral quick drying glue sealing.
Objective To explore a simple and rapid pathological slices method to observe the porous structure and the composition distribution of composite materials. Methods Taking polyurethane/small intestinal submucosa(PU/SIS) composite as an example, PU/SIS was OCT-embedded and sliced into sections by frozen section technology,after which general observation of the section integrity was carried out. After dyed with water-soluble eosin in alcoholic solution, the staining effect and the porous structure of the composite were observed under light field microscope.Sections were sealed with five different sealing methods. Group A: sealing piece using glycerogelatin method; group B:anhydrous alcohol dehydration→transparency using TO transparent reagent→sealing piece using neutral quick drying glue; group C: color separation using deionized water→air-drying→sealing piece using neutral quick drying glue; group D: air-drying→transparency using TO transparent reagent→sealing piece using neutral quick drying glue; group E: airdrying→sealing piece using neutral quick drying glue. Then, the morphology and the components distribution of the composite were observed under light field microscope, and the simple and feasible method was selected as optimum method. Results From general observation, the frozen section of the PU/SIS composite, which was 6 μm in thickness,was complete and continuous. Although the outline of the material and the porous structure in the sections could be observed clearly under light field microscope, the two components still could not be identified by using eosin staining method. After sealing piece, the material components in groups A, B, and C still could not be identified or be dissolved and deformed; the morphology of the material in groups D and E were preserved and the two components in the composite were clearly visible. Conclusion The morphology and the components distribution of PU/SIS frozen sections can be characterized after soluble eosin staining and neutral quick drying glue sealing.
引文
1Hu J, Chen B, Guo F, et al. Injectable silk fibroin/polyurethane composite hydrogel for nucleus pulposus replacement. J Mater Sci Mater Med, 2012, 23(3):711-722.
2 Kuppan P, Sethuraman S, Krishnan UM. PCL and PCL-gelatin nanofibers as esophageal tissue scaffolds:optimization,characterization and cell-matrix interactions. J Biomed Nanotechnology, 2013, 9(9):1540-1555.
3Jwo SC, Chin CH, Tang SJ, et al. Tubular scaffolds of gelatin and poly(ε-caprolactone)-block-poly(γ-glutamic acid)blending hydrogel for the proliferation of the primary intestinal smooth muscle cells of rats. Biomed Mater, 2013, 8(6):065002.
4李瑞,王青山.生物材料生物相容性的评价方法和发展趋势.中国组织工程研究, 2011, 15(29):5471-5474.
5Fan MR, Gong M, Da LC, et al. Tissue engineered esophagus scaffold constructed with porcine small intestinal submucosa and synthetic polymers. Biomed Mater, 2014, 9(1):015012.
6D’Amore A, Yoshizumi T, Luketich SK, et al. Bi-layered polyurethane-Extracellular matrix cardiac patch improves ischemic ventricular wall remodeling in a rat model. Biomaterials, 2016, 107:1-14.
7Da LC, Gong M, Chen A, et al. Composite elastomeric polyurethane scaffolds incorporating small intestinal submucosa for soft tissue engineering. Acta Biomater, 2017, 59:45-57.
8李静,孙保存,刘增辉,等. 2839例术中冰冻切片诊断与术后石蜡切片诊断的对比研究.天津医科大学学报, 2013, 19(6):471-473.
9杨文英.病理技术HE染色在病理诊断中的应用研究.大家健康(学术版), 2014, 8(10):50-51.
10王巧. HE染色方法在临床病理诊断中的应用.临床合理用药杂志, 2014, 7(32):118-119.
11Ning LJ, Jiang YL, Zhang CH, et al. Fabrication and characterization of a decellularized bovine tendon sheet for tendon reconstruction. J Biomed Mater Res A, 2017, 105(8):2299-2311.
12Huang YZ, Wang JJ, Huang YC, et al. Organic composite-mediated surface coating of human acellular bone matrix with strontium.Mater Sci Eng C Mater Biol Appl, 2018, 84:12-20.
13Xiong BJ, Tan QW, Chen YJ, et al. The effects of platelet-rich plasma and adipose-derived stem cells on neovascularization and fat graft survival. Aesthetic Plast Surg, 2018, 42(1):1-8.
14Yang JL, Yao X, Qing Q, et al. An engineered tendon/ligament bioscaffold derived from decellularized and demineralized cortical bone matrix. J Biomed Mater Res A, 2017, 106(2):468-478.
15Tan QW, Zhang Y, Luo JC, et al. Hydrogel derived from decellularized porcine adipose tissue as a promising biomaterial for soft tissue augmentation. J Biomed Mater Res A, 2017, 105(6):1756-1764.
16晁宁宁,邓雪琴,王玮,等.可注射CSCl-GP-HEC/SIS微粒温敏复合材料软组织填充初步实验.全国组织工程与再生医学大会,2013.
17吴江锋,李红军.制作冰冻切片的体会.右江民族医学院学报,2011, 33(6):819-820.
18胡垚.富含脂肪及淋巴组织的冰冻切片制作体会.哈尔滨医药,2015, 35(3):200-202.
19陈敬文,王天娲,赖美娜.碳蜡包埋技术在脂肪染色中的应用研究.实用医技杂志, 2016, 23(10):1133-1134.
20王小晓.水溶性伊红Y不同配置法对HE染色效果的影响.临床与实验病理学杂志, 2013, 29(5):575-576.
21张婉仪,陈哲.两种伊红染色液在HE染色中的应用比较.现代诊断与治疗, 2015, 26(12):2725-2726.
22田玉旺,朱红艳,朱培双,等.介绍一种改良的伊红染色液.中国组织化学与细胞化学杂志, 2013, 22(4):328-329.
23Wei RQ, Tan B, Tan MY, et al. Grafts of porcine small intestinal submucosa with cultured autologous oral mucosal epithelial cells for esophageal repair in a canine model. Exp Biol Med(Maywood),2009, 234(4):453-461.
24刘宏银,冯喆,柏林,等.雪旺细胞纯化培养及体外复合猪小肠黏膜下层的实验研究.中国修复重建外科杂志, 2013, 27(6):743-748.
25 邹仲之,李继承.组织学与胚胎学. 8版.北京:人民卫生出版社,2013:4.
2 Kuppan P, Sethuraman S, Krishnan UM. PCL and PCL-gelatin nanofibers as esophageal tissue scaffolds:optimization,characterization and cell-matrix interactions. J Biomed Nanotechnology, 2013, 9(9):1540-1555.
3Jwo SC, Chin CH, Tang SJ, et al. Tubular scaffolds of gelatin and poly(ε-caprolactone)-block-poly(γ-glutamic acid)blending hydrogel for the proliferation of the primary intestinal smooth muscle cells of rats. Biomed Mater, 2013, 8(6):065002.
4李瑞,王青山.生物材料生物相容性的评价方法和发展趋势.中国组织工程研究, 2011, 15(29):5471-5474.
5Fan MR, Gong M, Da LC, et al. Tissue engineered esophagus scaffold constructed with porcine small intestinal submucosa and synthetic polymers. Biomed Mater, 2014, 9(1):015012.
6D’Amore A, Yoshizumi T, Luketich SK, et al. Bi-layered polyurethane-Extracellular matrix cardiac patch improves ischemic ventricular wall remodeling in a rat model. Biomaterials, 2016, 107:1-14.
7Da LC, Gong M, Chen A, et al. Composite elastomeric polyurethane scaffolds incorporating small intestinal submucosa for soft tissue engineering. Acta Biomater, 2017, 59:45-57.
8李静,孙保存,刘增辉,等. 2839例术中冰冻切片诊断与术后石蜡切片诊断的对比研究.天津医科大学学报, 2013, 19(6):471-473.
9杨文英.病理技术HE染色在病理诊断中的应用研究.大家健康(学术版), 2014, 8(10):50-51.
10王巧. HE染色方法在临床病理诊断中的应用.临床合理用药杂志, 2014, 7(32):118-119.
11Ning LJ, Jiang YL, Zhang CH, et al. Fabrication and characterization of a decellularized bovine tendon sheet for tendon reconstruction. J Biomed Mater Res A, 2017, 105(8):2299-2311.
12Huang YZ, Wang JJ, Huang YC, et al. Organic composite-mediated surface coating of human acellular bone matrix with strontium.Mater Sci Eng C Mater Biol Appl, 2018, 84:12-20.
13Xiong BJ, Tan QW, Chen YJ, et al. The effects of platelet-rich plasma and adipose-derived stem cells on neovascularization and fat graft survival. Aesthetic Plast Surg, 2018, 42(1):1-8.
14Yang JL, Yao X, Qing Q, et al. An engineered tendon/ligament bioscaffold derived from decellularized and demineralized cortical bone matrix. J Biomed Mater Res A, 2017, 106(2):468-478.
15Tan QW, Zhang Y, Luo JC, et al. Hydrogel derived from decellularized porcine adipose tissue as a promising biomaterial for soft tissue augmentation. J Biomed Mater Res A, 2017, 105(6):1756-1764.
16晁宁宁,邓雪琴,王玮,等.可注射CSCl-GP-HEC/SIS微粒温敏复合材料软组织填充初步实验.全国组织工程与再生医学大会,2013.
17吴江锋,李红军.制作冰冻切片的体会.右江民族医学院学报,2011, 33(6):819-820.
18胡垚.富含脂肪及淋巴组织的冰冻切片制作体会.哈尔滨医药,2015, 35(3):200-202.
19陈敬文,王天娲,赖美娜.碳蜡包埋技术在脂肪染色中的应用研究.实用医技杂志, 2016, 23(10):1133-1134.
20王小晓.水溶性伊红Y不同配置法对HE染色效果的影响.临床与实验病理学杂志, 2013, 29(5):575-576.
21张婉仪,陈哲.两种伊红染色液在HE染色中的应用比较.现代诊断与治疗, 2015, 26(12):2725-2726.
22田玉旺,朱红艳,朱培双,等.介绍一种改良的伊红染色液.中国组织化学与细胞化学杂志, 2013, 22(4):328-329.
23Wei RQ, Tan B, Tan MY, et al. Grafts of porcine small intestinal submucosa with cultured autologous oral mucosal epithelial cells for esophageal repair in a canine model. Exp Biol Med(Maywood),2009, 234(4):453-461.
24刘宏银,冯喆,柏林,等.雪旺细胞纯化培养及体外复合猪小肠黏膜下层的实验研究.中国修复重建外科杂志, 2013, 27(6):743-748.
25 邹仲之,李继承.组织学与胚胎学. 8版.北京:人民卫生出版社,2013:4.