本文選題：人工角膜 + 骨髓間充質(zhì)干細(xì)胞��； 參考：《中國人民解放軍醫(yī)學(xué)院》2014年博士論文

【摘要】：目的：對(duì)目前應(yīng)用最廣的人工角膜的材料(TI與PMMA)進(jìn)行表面HA的涂層,再復(fù)合以MSCs,體外近誘導(dǎo)成軟骨后,植入兔板層角膜中,初步評(píng)估表面改性過的復(fù)合MSCs的材料在動(dòng)物體外和體內(nèi)的安全性和有效性,以達(dá)到加固KPro與宿主界面,減少角膜融解等并發(fā)癥的目的。 方法：分三部分進(jìn)行：一采用MPS和SBF分別對(duì)鈦和PMMA表面進(jìn)行的HA涂層,SEM進(jìn)行材料表面形態(tài)學(xué)分析,并測試涂層的強(qiáng)度,分析涂層的表面性質(zhì)(EDX),模擬體液環(huán)境觀察涂層的穩(wěn)定性。二①小鼠RFP-MSCs復(fù)合于ECM支架后,2w,4w,8w倒置熒光顯微鏡下觀察標(biāo)記細(xì)胞生長；SEM觀察支架與MSCs的生長情況；觀察其支架復(fù)合細(xì)胞后的分布情況(甲苯胺藍(lán)染色)。②兔MSCs和人MSCs分別接種于HA-TI,HA-PMMA材料表面,比較兩種材料的MTT細(xì)胞生長曲線,SEM觀察細(xì)胞附著支架情況,并分別誘導(dǎo)成骨及軟骨,以茜素紅和阿爾新藍(lán)染色鑒定。三①小鼠RFP-MSCs-ECM支架植入6只眼的兔角膜板層口袋(對(duì)照組植入無細(xì)胞的支架材料),使用活體成像儀,熒光顯微鏡下觀察其細(xì)胞存活狀況,進(jìn)行光鏡下觀察(HE染色,甲苯胺藍(lán)染色)。②兔MSCs復(fù)合兩種人工角膜涂層材料,體外幾近誘導(dǎo)成軟骨時(shí),植入12只兔角膜板層中(每種材料6只眼),做臨床觀察,分別于術(shù)后2w,4W,8w取角膜組織做病理(HE染色和Masson染色),并作TEM觀察。③兔MSCs復(fù)合兩種人工角膜涂層材料植入6只兔背闊肌間,并于2w,4w,8w做HE染色和Masson染色。 結(jié)果：①兩種方法均可完成均勻連續(xù)的涂層, EDX結(jié)果分析其涂層成分為羥基磷灰石,MPS噴涂法的鈦表面涂層更為致密,在模擬體液中涂層更為穩(wěn)定；SBF進(jìn)行的PMMA材料涂層表面HA涂層強(qiáng)度略差,模擬體液中有少量鈣鹽沉積。②小鼠RFP-MSCs的ECM支架中RFP隨時(shí)間點(diǎn)呈遞增趨勢,SEM可見細(xì)胞附著支架呈網(wǎng)狀生長。兔MSCs接種于HA-TI和HA-PMMA表面后,與MSCs無涂層(對(duì)照組)相比,MTT分析其細(xì)胞生長趨勢大致一致,HA-TI與對(duì)照組無明顯差異,HA-PMMA與對(duì)照組相比有顯著性差異(P0.01), HA-TI與HA-PMMA相比有顯著性差異(P0.01), SEM可見HA-TI細(xì)胞數(shù)量明顯高于HA-PMMA材料。③人MSC接種于HA-TI和HA-PMMA表面后,MTT分析其三組間無明顯差異。④兔MSCs和人MSCs在HA涂層后的人工角膜材料表面均可誘導(dǎo)成骨及軟骨,茜素紅及阿爾新藍(lán)染色均為陽性。⑤活體成像儀可觀察到小鼠RFP-MSCs復(fù)合ECM支架在兔角膜板層內(nèi)存活(最長觀察時(shí)間8W),熒光顯微鏡下觀察細(xì)胞數(shù)量呈增長趨勢。臨床觀察及病理結(jié)果顯示炎癥輕微,無新生血管生成。⑥兔MSCs體外復(fù)合兩種涂層材料后,臨床觀察其材料均穩(wěn)定存在,角膜有新生血管生成,角膜上皮完整,病理特點(diǎn)為角膜基質(zhì)層的大量炎細(xì)胞,少量新生血管為特征的反應(yīng),可見角膜上皮細(xì)胞空泡狀變性及角膜基質(zhì)層的玻璃樣變。結(jié)論：①微束等離子噴涂技術(shù)與仿生溶液方法能夠?qū)︹伡癙MMA材料進(jìn)行表面羥基磷灰石涂層,其涂層穩(wěn)定均勻,能夠滿足作為骨髓間充質(zhì)干細(xì)胞生長支架的需要,且體外能夠成功誘導(dǎo)成骨及軟骨。②小鼠MSCs能夠在角膜板層內(nèi)環(huán)境中生長及增殖。③HA-TI體外復(fù)合MSCs的能力要優(yōu)于HA-PMMA。④ECM支架復(fù)合小鼠MSCs植入角膜板層內(nèi)僅僅表現(xiàn)為輕微的炎癥反應(yīng),支架與角膜基質(zhì)層融合生長。⑤需要更多MSCs-涂層材料植入兔角膜后的病理學(xué)研究及植入后的分化結(jié)果鑒定。
[Abstract]:Objective: to apply the coating of the most widely used artificial cornea (TI and PMMA) to the surface of the surface HA, and then compound with MSCs, and after the near induction of cartilage in vitro, it is implanted into the rabbit lamellar cornea. The safety and effectiveness of the surface modified composite MSCs materials in vitro and in vivo are preliminarily evaluated in order to strengthen the interface between the KPro and the host, and to reduce the interface between the host and the host. The purpose of complications such as corneal fusion.
Methods: the three parts were divided into three parts: (1) the HA coating on the surface of titanium and PMMA was applied to the surface of titanium and PMMA respectively. The surface morphology of the material was analyzed by SEM, the strength of the coating was tested, the surface properties of the coating (EDX) were analyzed, and the stability of the coating was observed by simulating the body fluid environment. Two 1. After the RFP-MSCs was combined with the ECM scaffold, 2W, 4W, 8W inverted fluorescence microscopy. The growth of labeled cells was observed under the microscope. The growth of the scaffold and MSCs was observed by SEM, and the distribution of the scaffold composite cells was observed (toluidine blue staining). (2) the rabbit MSCs and human MSCs were inoculated on the surface of HA-TI, HA-PMMA material, respectively, to compare the MTT cell growth curves of the two materials, and to observe the attachment of the cells by SEM, and to induce the osteogenesis of the cells respectively. And the cartilage was identified with alizarin red and alcian blue staining. Three RFP-MSCs-ECM stents were implanted in the corneal lamellar pocket of 6 eyes (the control group was implanted without cellular scaffold material). Living body imager was used to observe the survival of the cells under the fluorescence microscope (HE staining, toluidine blue staining). (2) rabbit MSCs compound two When the artificial corneal coating material was used to induce cartilage in vitro, 12 rabbits were implanted in the lamellar layer of 12 rabbits (6 eyes of each material). The corneal tissue was observed after operation (HE staining and Masson staining) and TEM observation after 2W, 4W, and 8W. (3) the rabbit MSCs compound two kinds of artificial corneal coating materials were implanted in 6 rabbit latissimus dorsi, and 2W, 4W, 8W did HE staining and Masson staining.
Results: (1) the two methods can complete uniform and continuous coating, EDX results show that the coating composition is hydroxyapatite, the MPS coating is more compact on the titanium surface coating, and the coating is more stable in the simulated body fluid; the HA coating strength of the PMMA coating on the surface of SBF is slightly worse, and there is a small amount of calcium salt in the simulated body fluid. (2) the mice RFP-MSCs In the ECM stent, RFP increased with time point, SEM visible cell attachment scaffold showed a reticular growth. Rabbit MSCs was inoculated on the surface of HA-TI and HA-PMMA, compared with MSCs without coating (control group), MTT analysis showed that the growth trend of the cells was roughly the same, HA-TI was not significantly different from the control group, HA-PMMA was significantly different from the control group (P0.01). HA-TI (P0.01), HA-TI Compared with HA-PMMA (P0.01), the number of HA-TI cells in SEM was obviously higher than that of HA-PMMA material. 3. After MSC inoculation on the surface of HA-TI and HA-PMMA, there was no significant difference between the three groups. (4) the rabbit MSCs and human MSCs were induced on the surface of the artificial corneal material after the HA coating could induce osteogenesis and cartilage, alizarin red and alxin blue staining. The living body imager could observe the survival of the mouse RFP-MSCs composite ECM scaffold in the lamina of the rabbit cornea (the longest observation time 8W), and the number of cells increased under the fluorescence microscope. The clinical observation and pathological results showed that the inflammation was slight and no angiogenesis was produced. 6. After the compound of two kinds of coating materials of rabbit MSCs in vitro, the clinical observation was made. All the materials were stable, corneal neovascularization, corneal epithelial integrity, pathological characteristics of large number of inflammatory cells in the corneal stroma, a small amount of neovascularization, and corneal epithelial cells vacuolated degeneration and vitreous change of corneal stroma. Titanium and PMMA materials are coated on the surface of hydroxyapatite. The coating is stable and uniform, which can meet the needs of bone marrow mesenchymal stem cells, and can successfully induce osteogenesis and cartilage in vitro. (2) the mice MSCs can grow and proliferate in the lamellar environment of the cornea. (3) the ability of HA-TI to compound MSCs in vitro is better than that of HA-PMMA. ECM The stent composite mouse MSCs implanted in the lamina of the cornea only showed a slight inflammatory reaction, and the scaffold fused with the corneal stroma. 5. The pathological study of the implanted rabbit cornea and the identification of the results after implantation were needed for the implantation of more MSCs- coating materials.

【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R779.6

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 付濤,憨勇,宋忠孝,李金勇,徐可為;碳/碳復(fù)合材料表面誘導(dǎo)沉積生理磷灰石層[J];無機(jī)材料學(xué)報(bào);2002年01期

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本文編號(hào)：1843211