本文選題：牙周組織工程　切入點(diǎn)：骨髓基質(zhì)細(xì)胞　出處：《山西醫(yī)科大學(xué)》2013年碩士論文

【摘要】：目的 本實(shí)驗(yàn)通過(guò)對(duì)殼聚糖-絲素蛋白（chitosan-silk fibroinCS-SF）支架與殼聚糖-絲素蛋白-磷酸三鈣（chitosan-silk fibroin-tricalcium CS-SF-TCP）支架的基本性能、力學(xué)性能及異位成骨能力進(jìn)行研究，尋求更適合牙周組織工程的支架。 方法 采用二次凍干技術(shù)制備兩種支架，，液體替代法、冰凍切片、INSTRON5544拉伸實(shí)驗(yàn)機(jī)對(duì)其一般性能及力學(xué)性能進(jìn)行檢測(cè)、全髓貼壁法培養(yǎng)骨髓基質(zhì)細(xì)胞（BMSCs）。將裸鼠24只、雄性、體重20～25g，采用完全隨機(jī)法分為4組，每組6只，CS-SF組、CS-SF+BMSCs組、CS-SF-TCP組、CS-SF-TCP+BMSCs組，所有的植入物均植入裸鼠背部皮下。每只裸鼠背部植入3塊。每組分別于術(shù)后4、8周各處死動(dòng)物3只。通過(guò)大體觀察、組織學(xué)與Ⅰ型膠原免疫組織化學(xué)染色觀察兩種支架的成骨能力。 結(jié)果 1、支架的一般性能及力學(xué)性能的觀察 CS-SF與CS-SF-TCP支架大體外觀呈白色疏松多孔海綿樣物質(zhì)，有一定的彈性與脆性。兩種支架的孔隙率約為70%-80%，孔徑值為100-200μm。置于無(wú)水乙醇中有溶脹現(xiàn)象但未發(fā)生溶解。冰凍切片觀察支架的內(nèi)部結(jié)構(gòu)，可見(jiàn)支架孔徑較均勻且彼此相連。力學(xué)性能的結(jié)果顯示：CS-SF支架的壓縮強(qiáng)度為0.44±0.15MPa，CS-SF-TCP支架的壓縮強(qiáng)度為1.44±0.96MPa。 2、大體及組織學(xué)結(jié)果 所有的植入物均被一層致密的纖維薄膜包繞，并發(fā)生肉眼可見(jiàn)的吸收現(xiàn)象，觸之稍硬。組織學(xué)染色顯示：4周時(shí)，CS-SF組與CS-SF-TCP組僅見(jiàn)支架的邊緣有纖維膜包繞，支架發(fā)生吸收斷裂，支架的中心并未見(jiàn)細(xì)胞的長(zhǎng)入及膠原樣物質(zhì)生成。CS-SF+BMSCs組與CS-SF-TCP+BMSCs組支架的邊緣有纖維膜包繞，支架的中心可見(jiàn)細(xì)胞及膠原樣物質(zhì)生成。8周時(shí)，CS-SF組與CS-SF-TCP組支架的吸收更明顯，仍未見(jiàn)細(xì)胞長(zhǎng)入。CS-SF+BMSCs組與CS-SF-TCP+BMSCs組可見(jiàn)細(xì)胞樣物質(zhì)增多,膠原樣物質(zhì)增多增粗。所有組均未見(jiàn)有明顯的骨及類骨質(zhì)樣物質(zhì)生成。 3、Ⅰ型膠原免疫組織化學(xué)染色 所有組4、8周時(shí)均可見(jiàn)Ⅰ型膠原陽(yáng)性表達(dá)，CS-SF組與CS-SF-TCP組陽(yáng)性表達(dá)主要在支架的邊緣，CS-SF+BMSCs組與CS-SF-TCP+BMSCs組陽(yáng)性表達(dá)主要在支架的孔隙表面及中央。4、8周時(shí)CS-SF-TCP+BMSCs的平均光密度值均高于CS-SF+BMSCs組，差別有統(tǒng)計(jì)學(xué)意義（P0.05）。CS-SF組的平均光密度值最低，與其他組差別有統(tǒng)計(jì)學(xué)意義（P0.05）。 結(jié)論 CS-SF-TCP支架能更好的誘導(dǎo)骨髓基質(zhì)細(xì)胞向成骨細(xì)胞分化的能力，故更適合于牙周組織工程。
[Abstract]:Purpose. In this experiment, the basic properties, mechanical properties and ectopic osteogenesis of chitosan-silk fibroin CS-SF scaffolds and chitosan-silk fibroin-tricalcium CS-SF-TCP-based scaffolds were studied in order to find a more suitable scaffold for periodontal tissue engineering. Method. Two kinds of scaffolds were prepared by secondary freeze-drying technique, liquid substitution method, frozen section INSTRON5544 tensile test machine to test their general and mechanical properties, bone marrow stromal cells were cultured by whole pulp adherent method, 24 nude mice were male, The body weight was 20 ~ 25 g, and was randomly divided into 4 groups with 6 rats in each group, CS-SF BMSCs group, CS-SF-TCP BMSCs group, CS-SF-TCP group, CS-SF-TCP group, CS-SF-TCP group, CS-SF-TCP BMSCs group, and CS-SF-TCP BMSCs group. All the implants were implanted subcutaneously into the back of the nude mice. The osteogenic ability of the two scaffolds was observed by histological and immunohistochemical staining. Results. 1. General and mechanical properties of the scaffold. The appearance of CS-SF and CS-SF-TCP scaffolds was white and porous sponge. There is some elasticity and brittleness. The porosity of the two kinds of scaffolds is about 70-80 and the pore size is 100-200 渭 m. There is swelling phenomenon in anhydrous ethanol, but no dissolution. Frozen sections are used to observe the internal structure of the scaffolds. The results of mechanical properties show that the compression strength of the W CS-SF stent is 0.44 鹵0.15MPA and the compression strength of CS-SF-TCP scaffold is 1.44 鹵0.96MPa. 2. Gross and histological results. All the implants were wrapped in a dense fibrous film, and the absorption phenomenon was visible to the naked eye, and it was slightly hard to be touched. Histological staining showed that in the CS-SF group and the CS-SF-TCP group at 4 weeks, only the fibrous membrane was wrapped around the edge of the scaffold, and the scaffold was absorbed and broken. There was no cell-growth and collagen-like substance formation in the center of the scaffold. The scaffolds in CS-SF BMSCs group and CS-SF-TCP BMSCs group had fibrous membrane wrapping around the edge, and the absorption of scaffold in CS-SF group and CS-SF-TCP group was more obvious at 8 weeks after the formation of collagen like substance and cells in the center of the scaffold. In the CS-SF BMSCs group and CS-SF-TCP BMSCs group, the cell like substance increased and the collagen-like substance increased and thickened. No bone and bone-like substances were found in all the groups. Immunohistochemical staining of type 3, type I collagen. Positive expression of type I collagen was found in all groups at 48 weeks. The positive expression of type 鈪

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