促WJ MSC骨向分化的明胶-PEG复合水凝胶的研究

发布时间：2018-01-19 07:18

本文关键词： 人脐带间充质干细胞明胶-PEG复合水凝胶体外降解预测模型成骨分化牙周骨组织工程　出处：《大连医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：背景:牙周病是危害人类口腔健康的重要疾病,目前临床上牙周病的主要治疗手段包括基础治疗、结合药物治疗、牙周引导组织再生术及植骨术、生长因子治疗等,尽管取得了一定的牙周组织再生效果,但是仍然无法满足口腔临床对牙周骨组织修复的要求。干细胞移植为牙周疾病的治疗带来了希望,然而由于病变部位微环境的改变使得移植的干细胞存活率低、活性差,治疗效果有待提高。组织工程化治疗借助生物材料能够改善干细胞的生存微环境,从而有希望进一步提高干细胞移植治疗的效果。明胶(Gelatin)水凝胶由于具有良好的生物相容性以及生物可降解性已经被广泛研究,已有研究表明聚乙二醇(Polyethylene glycol,PEG)作为增塑剂可以显著改善明胶水凝胶的机械特性,因此明胶-PEG复合水凝胶在牙周骨组织修复上可能具有一定的应用潜力。水凝胶的降解是组织工程支架的重要特性,它需要与组织修复相匹配。然而,明胶-PEG复合水凝胶的降解规律目前还不清楚,此外复合水凝胶对人脐带华顿氏胶来源的间充质干细胞(human umbilical cord Wharton’s jelly derived mesenchymal stem cells,WJ MSCs)的亲和性及成骨分化的影响还有待考察。目的:制备明胶-PEG复合水凝胶,明确主要制备参数对复合水凝胶降解的作用规律,并考察复合水凝胶对WJ MSCs粘附、生长及成骨分化的影响。方法:首先确定复合水凝胶的制备及其降解表征方法,即无菌条件下利用戊二醛(glutaraldehyde,GA)交联明胶及PEG的混合溶液,4℃交联18h,形成稳定的复合水凝胶,利用I型胶原酶溶液浸泡复合水凝胶,进行体外酶降解,之后进行真空干燥,恒重后称重,计算水凝胶的降解率。之后,采用Plackett-Burman实验设计从明胶浓度(X_1)、戊二醛浓度(X_2)、PEG浓度(X_3)中确定影响复合水凝胶降解的显著性因素。然后,利用响应面方法之Box-Behnken实验设计考察三因素X_1、X_2、X_3间交互作用对响应值Y(复合水凝胶降解率)的影响,获得复合水凝胶降解率的预测模型。之后,随机选取三个复合水凝胶制备组合条件,考察模型预测值与实际降解率之间的吻合程度,以评价模型预测的准确性。接下来,将WJ MSCs接种到不同条件的水凝胶表面,利用细胞计数法考察干细胞初始粘附数量,通过活死染色观察细胞活性及分布,使用F-actin荧光染色评价干细胞在水凝胶上的粘附和伸展,并利用CCK-8法考察干细胞在水凝胶表面的增殖。最后,使用成骨诱导培养基对水凝胶表面的干细胞进行成骨诱导,利用碱性磷酸酶(Alkaline Phosphatase,ALP)定量检测及茜素红染色考察水凝胶对WJ MSCs成骨分化的影响。显著性因素筛选和响应面设计实验分别采用Minitab和Design-Expert来进行分析,其他数据采用Origin 8.5绘图统计软件分析。结果:1.戊二醛浓度是影响明胶-PEG复合水凝胶降解的显著性因素。2.获得预测复合水凝胶降解率数学模型:Y=2.23625-0.13467*X_1-14.52844*X_2+0.061178*X_3+0.043950*X_1*X_2-0.00379432*X_1*X_3+0.23396*X_2*X_3+0.00975590*X_12+27.47820*X_22-0.023016*X_32,该方程的R2等于0.97,p值小于0.0001,表明该回归方程具有可靠的预测能力。3.实际降解率与模型预测值间的吻合度大于80%,说明该模型的可信度高。4.PEG对复合水凝胶支架降解的影响是受戊二醛浓度高低调控的,当戊二醛浓度高时,PEG的加入会显著加速水凝胶的降解;而戊二醛浓度低时,PEG会减慢水凝胶的降解过程。5.与常规无PEG的水凝胶相比,复合水凝胶对干细胞粘附、活性及增殖无显著性差异,然而复合水凝胶具有较致密的结构。6.与常规二维培养表面相比,水凝胶表面能够显著提高ALP活性并促进钙沉积,从而显著促进干细胞的成骨分化,另外还发现与常规无PEG的水凝胶相比,PEG的添加并未显著影响成骨分化的程度。结论:本研究明确了戊二醛浓度是影响明胶-PEG复合水凝胶降解的显著性因素,并建立了一个信度高的、可预测降解率的数学模型,这使得制备具有特定降解速率的复合水凝胶成为可能。另外,PEG对复合水凝胶降解的影响可能是受戊二醛及明胶浓度高低调控的,并且PEG的添加能够使得水凝胶结构更加致密。此外,复合水凝胶表面干细胞的生长、粘附及活性较好,证明复合水凝胶与干细胞有很好的亲和性,可作为干细胞移植的载体。此外,复合水凝胶能够促进干细胞向成骨细胞分化。从而本研究为明胶-PEG复合水凝胶在修复牙周骨组织缺损研究中提供了新方法、新思路。
[Abstract]:Background: periodontal disease is an important disease endangering the health of human oral cavity, the periodontal disease is mainly clinical treatments including basic treatment, combined with drug therapy, periodontal guided tissue regeneration and bone grafting, growth factor treatment, despite certain periodontal tissue regeneration effect, but still can not meet the requirements of oral clinical periodontal bone tissue repair. Stem cell transplantation for the treatment of periodontal disease has brought hope, but because the lesion microenvironment change of transplanted stem cells survival rate is low, poor activity, treatment needs to be improved. The treatment by means of tissue engineering biomaterials can improve the survival of stem cells and micro the environment, and is expected to further improve the effect of stem cell transplantation in the treatment of gelatin hydrogel (Gelatin). Due to its good biocompatibility and biodegradability have been widely studied, the existing research This study showed that the polyethylene glycol (Polyethylene glycol, PEG) as the plasticizer can significantly improve the mechanical properties of gelatin gel, therefore gelatin -PEG composite hydrogel has a certain potential possible in periodontal bone tissue repair. The degradation of the hydrogel is characteristic to tissue engineering scaffold, tissue repair and it needs to match. However, the degradation of gelatin -PEG composite hydrogel is unclear, in addition to the composite hydrogel on human umbilical cord Wharton's jelly derived mesenchymal stem cells (human umbilical cord Wharton jelly derived mesenchymal stem 's cells, WJ MSCs) affinity and osteogenic differentiation in vitro remains to be investigated. Objective: To study the preparation of gelatin -PEG composite hydrogel, clear the main preparation parameters of composite hydrogel degradation, and the effects of WJ composite hydrogel MSCs adhesion, proliferation and osteogenic differentiation. Methods: first To determine the preparation of composite hydrogel and its degrading characterization methods, namely under aseptic conditions by using glutaraldehyde (glutaraldehyde, GA) mixed solution cross-linked gelatin and PEG, 4 C crosslinked 18h composite hydrogel formation, stability, using type I collagenase solution composite hydrogel, enzymatic degradation in vitro, after vacuum drying, constant weight after weighing the degradation rate of hydrogel, calculated by Plackett-Burman. After the experimental design from the concentration of gelatin (X_1), the concentration of glutaraldehyde (X_2), PEG concentration (X_3) in determining the significance of influence factors of composite hydrogel degradation. Then, the response surface method of Box-Behnken experimental design of three factors of X_1, using X_2, X_3 interactions on the the response value of Y (composite hydrogel degradation rate) influence, obtained composite hydrogel degradation rate prediction model. Then, three randomly selected composite hydrogel preparation conditions, effects of model prediction and The degree of correspondence between the actual degradation rate, with an accuracy evaluation model prediction. Then, the WJ MSCs was inoculated into the hydrogel surface in different conditions, by using cell counting method was used to investigate the amount of stem cell initial adhesion, observe cell activity and distribution by live / dead staining, staining of adherent cells in the evaluation of dry hydrogel and extension using F-actin fluorescence. And the effects of stem cell proliferation in the hydrogel surface by CCK-8 method. Finally, the use of osteogenic medium on the surface of the hydrogel stem cells by osteogenic induction, alkaline phosphatase (Alkaline Phosphatase, ALP) quantitative detection and alizarin red staining of hydrogel on osteoblastic differentiation of WJ MSCs. The significant factor screening and response design experiment using Minitab and Design-Expert to analyze other data analysis using Origin 8.5 statistical software. Results: 1. drawing two amyl aldehyde concentration is Influencing factors of gelatin -PEG composite hydrogel degradation of.2. composite hydrogel degradation rate prediction mathematical model of the equation: Y=2.23625-0.13467*X_1-14.52844*X_2+0.061178*X_3+0.043950*X_1*X_2-0.00379432*X_1*X_3+0.23396*X_2*X_3+0.00975590*X_12+27.47820*X_22-0.023016*X_32, R2 = 0.97, P value is less than 0.0001, indicated that the regression equation has the ability of.3. to predict actual degradation rate and forecasting model of reliable value of the degree of agreement is greater than 80%, indicating the influence of the credibility of the model high.4.PEG of composite hydrogel scaffold degradation by glutaraldehyde concentration control, when the concentration of glutaraldehyde is high, the addition of PEG can significantly accelerate the degradation of hydrogels; while the glutaraldehyde concentration is low, PEG will slow down the degradation process of.5. hydrogel and conventional PEG hydrogel composite hydrogel compared to stem cell adhesion, proliferation activity and no significant difference However, the structure of.6. composite hydrogel with dense surface compared with conventional two-dimensional culture, the hydrogel surface can significantly improve ALP activity and promote calcium deposition, thus significantly promote the osteogenic differentiation of stem cells, it is also found that compared with the conventional PEG hydrogels, the addition of PEG did not significantly affect the osteogenic differentiation degree. Conclusion this study defined the glutaraldehyde concentration are significant factors that influenced the degradation of gelatin -PEG composite hydrogel, and the establishment of a reliable, predictable mathematical model of the degradation rate, which makes the composite hydrogel prepared with specific degradation rate as possible. In addition, the effect of PEG on the composite hydrogel degradation may be affected by glutaraldehyde and gelatin concentration control, and the addition of PEG can make the structure more compact hydrogel composite hydrogel surface. In addition, stem cell growth, adhesion and good activity, Proof of composite hydrogel and stem cells have good compatibility, can be used as carrier of stem cell transplantation. In addition, the composite hydrogel can promote stem cells to differentiate into osteoblasts. Thus the research of gelatin -PEG composite hydrogel provides a new method in the study of repairing periodontal bone defects in new ideas.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R781.4

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