羟基磷灰石涂层复合骨髓间充质干细胞增加人工角膜生物整合性的实验研究

发布时间：2018-05-04 13:53

本文选题：人工角膜 + 骨髓间充质干细胞　；参考：《中国人民解放军医学院》2014年博士论文

【摘要】：目的：对目前应用最广的人工角膜的材料(TI与PMMA)进行表面HA的涂层,再复合以MSCs,体外近诱导成软骨后,植入兔板层角膜中,初步评估表面改性过的复合MSCs的材料在动物体外和体内的安全性和有效性,以达到加固KPro与宿主界面,减少角膜融解等并发症的目的。方法：分三部分进行：一采用MPS和SBF分别对钛和PMMA表面进行的HA涂层,SEM进行材料表面形态学分析,并测试涂层的强度,分析涂层的表面性质(EDX),模拟体液环境观察涂层的稳定性。二①小鼠RFP-MSCs复合于ECM支架后,2w,4w,8w倒置荧光显微镜下观察标记细胞生长；SEM观察支架与MSCs的生长情况；观察其支架复合细胞后的分布情况(甲苯胺蓝染色)。②兔MSCs和人MSCs分别接种于HA-TI,HA-PMMA材料表面,比较两种材料的MTT细胞生长曲线,SEM观察细胞附着支架情况,并分别诱导成骨及软骨,以茜素红和阿尔新蓝染色鉴定。三①小鼠RFP-MSCs-ECM支架植入6只眼的兔角膜板层口袋(对照组植入无细胞的支架材料),使用活体成像仪,荧光显微镜下观察其细胞存活状况,进行光镜下观察(HE染色,甲苯胺蓝染色)。②兔MSCs复合两种人工角膜涂层材料,体外几近诱导成软骨时,植入12只兔角膜板层中(每种材料6只眼),做临床观察,分别于术后2w,4W,8w取角膜组织做病理(HE染色和Masson染色),并作TEM观察。③兔MSCs复合两种人工角膜涂层材料植入6只兔背阔肌间,并于2w,4w,8w做HE染色和Masson染色。结果：①两种方法均可完成均匀连续的涂层, EDX结果分析其涂层成分为羟基磷灰石,MPS喷涂法的钛表面涂层更为致密,在模拟体液中涂层更为稳定；SBF进行的PMMA材料涂层表面HA涂层强度略差,模拟体液中有少量钙盐沉积。②小鼠RFP-MSCs的ECM支架中RFP随时间点呈递增趋势,SEM可见细胞附着支架呈网状生长。兔MSCs接种于HA-TI和HA-PMMA表面后,与MSCs无涂层(对照组)相比,MTT分析其细胞生长趋势大致一致,HA-TI与对照组无明显差异,HA-PMMA与对照组相比有显著性差异(P0.01), HA-TI与HA-PMMA相比有显著性差异(P0.01), SEM可见HA-TI细胞数量明显高于HA-PMMA材料。③人MSC接种于HA-TI和HA-PMMA表面后,MTT分析其三组间无明显差异。④兔MSCs和人MSCs在HA涂层后的人工角膜材料表面均可诱导成骨及软骨,茜素红及阿尔新蓝染色均为阳性。⑤活体成像仪可观察到小鼠RFP-MSCs复合ECM支架在兔角膜板层内存活(最长观察时间8W),荧光显微镜下观察细胞数量呈增长趋势。临床观察及病理结果显示炎症轻微,无新生血管生成。⑥兔MSCs体外复合两种涂层材料后,临床观察其材料均稳定存在,角膜有新生血管生成,角膜上皮完整,病理特点为角膜基质层的大量炎细胞,少量新生血管为特征的反应,可见角膜上皮细胞空泡状变性及角膜基质层的玻璃样变。结论：①微束等离子喷涂技术与仿生溶液方法能够对钛及PMMA材料进行表面羟基磷灰石涂层,其涂层稳定均匀,能够满足作为骨髓间充质干细胞生长支架的需要,且体外能够成功诱导成骨及软骨。②小鼠MSCs能够在角膜板层内环境中生长及增殖。③HA-TI体外复合MSCs的能力要优于HA-PMMA。④ECM支架复合小鼠MSCs植入角膜板层内仅仅表现为轻微的炎症反应,支架与角膜基质层融合生长。⑤需要更多MSCs-涂层材料植入兔角膜后的病理学研究及植入后的分化结果鉴定。
[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.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R779.6

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