【作者】 陈鹏；

【导师】 胡懿郃；

【作者基本信息】 中南大学 ， 临床医学（专业学位）， 2022， 博士

【摘要】 背景:关节软骨损伤是严重威胁人类健康的疾病之一,既往治疗软骨损伤的方法尚不能获得长期且满意的疗效。为了迫切解决此医学难题,人工软骨材料主要通过模拟天然软骨组织特征,从而代替病变的软骨组织并恢复其功能,是一种非常有前景的软骨损伤修复方法。根据聚乙烯醇(polyvinylalcohol,PVA)水凝胶仍存在的自身缺陷,众多学者通过物理、化学或辐射交联法来提高PVA水凝胶的交联程度,或通过控制冻融循环次数等制备工艺,以增强其力学性能。并进一步通过引入羟基磷灰石(hydroxyapatite,HA),胶原等生物活性物质来提高水凝胶的细胞亲和力。本实验在PVA的基础上通过引入HA和细胞外基质组分I型胶原,来增加支架的力学强度和细胞亲和力,形成COL-HA-PVA的表面改性支架以促进接种BMSCs细胞的粘附和迁移作用,以期望加快软骨再生修复效率。目的:通过构建出孔径大小分别为1.2mm,1.4mm和1.6mm的HA-PVA水凝胶支架,并于其上添加细胞外基质组分I型胶原。构成支架孔隙结构和胶原构成比的多重仿生人工软骨材料,对其有利于接种细胞生长的物理性能进行分析评价;再将山羊骨髓间充质干细胞(goat Bone Marrow Mesenchymal Stem Cells,g BMSCs)接种于上述人工软骨材料上于体外和体内分别对其进行软骨缺陷修复能力评价,以期望制备出更有利于软骨缺损修复的仿生人工软骨材料以及对材料可提高软骨缺损修复效率的机制进行初探。方法:(1)COL-HA-PVA新型水凝胶的构建及其表征:采用CAD设计出3种不同孔径的三维模型,结合FDM(熔融沉积成型技术)制备PVA原始支架。然后,利用倒模法和反复熔融冻干法制备出3种不同孔径HA-PVA水凝胶,并通过利用将COL溶液(I型胶原)与京尼平酒精溶液交联,制备出COL-HA-PVA水凝胶支架材料。并对各支架进行组成成分、含水率、孔隙率等理化性质分析以及生物力学和弹性模量检测,以完成对材料的表征和力学性能测定。(2)COL-HA-PVA新型水凝胶体外细胞学实验研究:此部分拟通过体外实验研究对本材料修复软骨缺损的可行性及修复能力进行分析。首先,通过将从山羊骨髓中分离得到的BMSCs通过表面Maker分子和三系分化能力检测,确定其为山羊骨髓来源的BMSCs。再将其分离培养的g BMSCs定量接种于已消毒的各组人工软骨支架材料(包括1.2mm HA-PVA、1.2mm COL-HA-PVA、1.4mm COL-HA-PVA、1.6mm COL-HA-PVA)上,同时设置空白对照组(即单纯接种细胞组)经成软骨诱导(含TGF-β1)培基共培养。采用离心细胞粘附实验研究支架材料表面修饰对BMSCs粘附的影响,通过比较各组间的支架上细胞数以评价BMSCs在各组材料上黏附能力的差异。之后,分别在接种后3、7、14天对各组进行如下研究:(1)电镜及Micro-CT;(2)软骨分化标志物实时定量PCR检测(II型胶原、Aggrecan及SOX-9)基因表达差异;(3)免疫组织化学方法分析I型、II型及IV型胶原含量;(4)番红固绿、甲苯胺蓝及HE染色;(5)荧光显微镜观察FITC-鬼笔环肽染色的细胞骨架变化情况。根据实验结果,通过组织、细胞及分子水平的相关分析,比较各组材料的成软骨能力,以探讨构建符合提高软骨缺损修复效率的人工软骨材料。(3)COL-HA-PVA新型水凝胶体内动物实验研究:为全面评估支架生物体内相容性和软骨修复能力,以及在体内支架孔径和相关物理性能对软骨修复能力的影响,本研究拟对不同孔径的COL-HAPVA支架进行动物体内实验,并比较复合BMSCs的支架及未复合BMSCs的支架对软骨缺损的修复效果。为此,此部分设立了6个实验组(1.2mm COL-HA-PVA、1.4mm COL-HA-PVA、1.6mm COL-HAPVA、1.2mm COL-HA-PVA+BMSCs、1.4mm COL-HA-PVA+BMSCs、1.6mm COL-HA-PVA+BMSCs)和1个空白对照组(仅制造山羊膝关节髌骨滑槽中段缺损,不给予任何修复缺损措施)。分别将不同孔径的COL-HA-PVA支架与BMSCs(TGF-β1诱导下)共培养7天后形成的细胞支架复合物植入山羊膝关节软骨缺损内。同时,将各孔径且未复合BMSCs的COL-HA-PVA支架植入山羊膝关节软骨缺损内,并在植入4周后取出膝关节软骨修复样本进行Micro-CT观察和分析。根据Micro-CT获得的BS/BV及Cortexmean BMD等指标值,确定最优支架孔径,而后对该孔径的支架、支架+BMSCs及空白对照组分别在植入山羊体内后4周、12周取出组织样本进行下列检测:(1)电镜及Micro-CT;(2)软骨分化标志物实时定量PCR检测(II型胶原、GAG、Aggrecan及SOX-9)基因表达差异;(3)免疫组织化学方法分析I型、II型及IV型胶原含量;(4)番红固绿、甲苯胺蓝及HE染色;(5)采用DMMB分光光度法分析支架上糖胺聚糖(glycosaminoglycan,GAG)含量;(6)荧光显微镜观察FITC-鬼笔环肽染色的细胞骨架变化情况。根据实验结果,通过组织、细胞及分子水平的相关分析,比较各组材料的成软骨能力,以探讨构建符合提高软骨缺损修复效率的人工软骨材料。(4)弹性模量对BMSCs向软骨细胞分化影响及潜在作用机制研究:将预先分离培养的山羊BMSCs定量接种于已消毒的各组人工软骨材料(包括1.2mm COL-HA-PVA、1.4mm COL-HA-PVA及1.6mm COLHA-PVA)经成软骨诱导(含TGF-β1)培养在3,7,14天,通过测量、分析和比较不同弹性模量(即1.2mm、1.4mm及1.6mm孔径)的COL-HA-PVA支架上软骨分化标志物基因表达水平,以探讨弹性模量对BMSCs向软骨细胞分化效率的影响。同时,比较不同弹性模量的支架上BMSCs中整合素α5基因表达水平和核纤层蛋白A基因表达水平以及软骨分化标志物基因表达水平的差异,以初步探讨弹性模量的高低影响BMSCs向软骨细胞分化的作用机制。之后,通过抑制整合素α5的活化水平,观察对核纤层蛋白A和软骨分化成熟标志物基因表达水平的影响,以初步探讨整合素α5β1活化水平及其介导的力学信号转导通路是否对核纤层蛋白A的表达具有影响以及支架材料弹性模量的高低影响接种BMSCs转分化为软骨细胞效率的机理。结果:(1)本研究所制备的3种支架材料通过物理性能评价,发现皆具有一定的含水性和粘弹性,且多孔隙联通,这些物理性能与天然软骨相似;(2)制备的COL-HA-PVA水凝胶支架,其弹性模量的高低与孔径大小具有相关性,其中1.2mm孔径组较其他组的压缩弹性模量最小;(3)1.2mm,1.4mm,1.6mm组皆可使接种干细胞正常增殖,且各组之间的增殖能力无差别;(4)1.2mm,1.4mm,1.6mm组都具备促进接种干细胞转分化为软骨细胞的作用,其中1.2mm的促进作用最强,1.4mm和1.6mm次之;(5)1.2mm,1.4mm,1.6mm组都具备正常的细胞粘附能力,其中1.2mm支架的促进粘附作用最强;(6)COL-HA-PVA结合BMSCs修复方式和COL-HA-PVA单独修复方式皆可修复软骨缺损。但两种软骨缺损修复方式也具有差别,其中以COL-HA-PVA结合BMSCs修复软骨缺损的效率更高;(7)COL-HA-PVA结合BMSCs是一个对膝关节软骨缺损有效的修复方法。其可以在表面改性支架修复缺损的基础上,以细胞再生治疗的方式来有效提高关节软骨缺损的修复效率;(8)本实验中1.2mm孔径支架对所接种的干细胞向软骨细胞转分化的促进能力最强,其弹性模量最低,更有利于促进干细胞向软骨细胞分化和成熟;(9)软骨修复材料的力学性质在调控干细胞的生物学行为方面具有决定性作用,其中弹性模量是一个决定干细胞分化方向的重要因素;(10)本研究制备的不同孔径COL-HA-PVA支架材料因其弹性模量差异,导致修复软骨缺损的效率差异的机制为材料本身弹性模量的高低,可通过刺激BMSCs转分化为软骨细胞的细胞膜上整合素活化,导致相应细胞信号级联通路诱导细胞核膜上核纤层蛋白的表达出现差异,而影响指导干细胞向软骨细胞分化的信号分子调控网络出现表达效率差异,导致最终的软骨细胞形成效率差异。结论:(1)本研究所制备的COL(I型胶原)-HA-PVA水凝胶多孔支架材料复合山羊骨髓间充质干细胞修复关节软骨缺损是一种很好的细胞复合材料再生医学的治疗方法。(2)HA-PVA水凝胶有效的仿生了软骨组织的力学和支撑应力作用,且其上附着的I型胶原作为一种细胞外基质成分能够有效的为接种的g BMSCs提供良好的生长微环境,并且可以提高g BMSCs转分化为软骨细胞的效率。(3)1.2mm孔径支架相比于其他孔径支架,具备最低的弹性模量。这种特性同样能够提高接种g BMSCs转分化为软骨细胞的效率。(4)具备一定弹性模量的表面改性水凝胶支架复合骨髓间充质干细胞转分化为软骨细胞为软骨缺损再生治疗提供了全新的治疗策略。图61幅,表19个,参考文献148篇更多还原

【Abstract】 Backgrounds:Articular cartilage injury is one of the diseases that seriously threaten human health.The previous methods of treating cartilage injury cannot obtain long-term and satisfactory curative effect.In order to solve this medical problem urgently,artificial cartilage material mainly simulates the characteristics of natural cartilage tissue,so as to replace the diseased cartilage tissue and restore its function.It is a very promising repair method of cartilage injury.According to the inherent defects of polyvinyl alcohol(PVA)hydrogels,many scholars have improved the crosslinking degree of PVA hydrogels by physical,chemical or radiation crosslinking methods,or by controlling the number of freeze-thaw cycles and other preparation processes to enhance their mechanical properties.The cell affinity of the hydrogel was further improved by introducing bioactive substances such as hydroxyapatite(HA)and collagen.Based on PVA,hydroxyapatite(HA)and extracellular matrix component type I collagen were introduced to increase the mechanical strength and cell affinity of the scaffold.The surface modified scaffolds of COL(type I collagen)-HA-PVA were formed to promote the adhesion and migration of inoculated BMSCs cells,in order to accelerate the efficiency of cartilage regeneration and repair.Objectives:HA-PVA hydrogel scaffolds with pore sizes of 1.2mm,1.4mm and 1.6mm were constructed and added with extracellular matrix component type I collagen.Multiple biomimetic artificial cartilage materials composed of scaffold pore structure and collagen composition ratio,and their physical properties conducive to the growth of inoculated cells were analyzed and evaluated;Then,g BMSCs were inoculated on the above artificial cartilage materials,and their cartilage defect repair ability was evaluated in vitro and in vivo,in order to prepare bionic artificial cartilage materials more conducive to cartilage defect repair and explore the mechanism that the materials can improve the efficiency of cartilage defect repair.Methods:(1)Construction and characterization of COL-HA-PVA hydrogel:three dimensional models with different pore sizes were designed by CAD,and the original PVA scaffolds were prepared by FDM(melt deposition molding).Then,three kinds of HA-PVA hydrogels with different pore sizes were prepared by reverse mold method and repeated melt freeze-drying method.After that,COL-HA-PVA hydrogel scaffold material was prepared by crosslinking COL-I solution with genipin alcohol solution.The composition,moisture content,porosity and other physical and chemical properties of each scaffold were analyzed,and the biomechanics and elastic modulus were tested to complete the characterization and mechanical properties of the material.(2)In vitro cytological experimental study of COL-HA-PVA new hydrogel: this part intends to analyze the feasibility and repair ability of this material to repair cartilage defects through in vitro experimental study.Firstly,BMSCs isolated from goat bone marrow were determined to be goat bone marrow-derived BMSCs through the detection of surface maker molecule and three-line differentiation ability.Then the isolated and cultured goat BMSCs were quantitatively inoculated on the sterilized artificial cartilage scaffold materials of each group(including 1.2mm HAPVA,1.2mm COL-HA-PVA,1.4mm COL-HA-PVA and 1.6mm COLHA-PVA).At the same time,the blank control group(i.e.,the simple inoculation cell group)was set for chondrogenesis induction(contain TGF-β1)medium co culture.Centrifugal cell adhesion experiment was used to study the effect of scaffold material surface modification on the adhesion of BMSCs.The adhesion ability of BMSCs on each group was evaluated by comparing the number of cells on the scaffold between each group.Then,the following studies were carried out in each group on3,7 and 14 days after inoculation:(1)electron microscope and micro CT;(2)The gene expression differences of cartilage differentiation markers(type II collagen,aggrecan and Sox-9)were detected by real-time quantitative PCR;(3)The contents of type I,type II and type IV collagen were analyzed by immunohistochemistry;(4)Safranine solid green,toluidine blue and HE staining;(5)The changes of cytoskeleton stained with FITC ghost pen cyclic peptide were observed by fluorescence microscope.According to the experimental results,through the correlation analysis of tissue,cell and molecular level,compare the chondrogenic ability of each group of materials,so as to explore the construction of artificial cartilage materials that can improve the repair efficiency of cartilage defects.(3)In vivo animal experimental study of COL-HA-PVA new hydrogel: To comprehensively evaluate the biocompatibility and cartilage repair ability of the scaffold,as well as the pore size and phase of the scaffold in vivo.Considering the influence of physical properties on cartilage repair ability,this study intends to carry out in vivo experiments on COL-HA-PVA scaffolds with different pore sizes,and compare the repair effects of scaffolds with and without BMSCs on cartilage defects.For this purpose,six experimental groups(1.2mm COL-HA-PVA,1.4mm COL-HA-PVA,1.6mm COL-HA-PVA,1.2mm COL-HA-PVA+BMSCs,1.4mm COL-HA-PVA+BMSCs and 1.6mm COL-HA-PVA+BMSCs)and a blank control group(only the defect of the middle part of the patellar chute of goat knee joint is made,and no defect repair measures are given).COL-HA-PVA scaffolds with different pore sizes were compared with BMSCs(contain TGF-β 1)the cell scaffold complex formed after 7 days of co culture was implanted into the cartilage defect of goat knee joint.At the same time,COL-HA-PVA scaffolds with various apertures and without BMSCs were implanted into goat knee cartilage defects.After 4 weeks of implantation,the repaired samples of knee cartilage were taken out for micro-CT observation and analysis.According to the index values of BS / BV and cortex mean BMD obtained by micro CT,the optimal stent aperture was determined,and then the tissue samples of the stent,stent + BMSCs and blank control group were taken out 4 weeks and 12 weeks after implantation in goats for the following tests:(1)Electron microscope and micro-CT;(2)The gene expression differences of cartilage differentiation markers(type II collagen,gag,aggrecan and Sox-9)were detected by real-time quantitative PCR;(3)The contents of type I,type II and type IV collagen were analyzed by immunohistochemistry;(4)Safranine solid green,toluidine blue and HE staining;(5)The content of glycosaminoglycan(GAG)on scaffolds was analyzed by DMMD spectrophotometry;(6)The changes of cytoskeleton stained with FITC ghost pen cyclic peptide were observed by fluorescence microscope.According to the experimental results,through the correlation analysis of tissue,cell and molecular level,compare the chondrogenic ability of each group of materials,so as to explore the construction of artificial cartilage materials that can improve the repair efficiency of cartilage defects.(4)Effect of elastic modulus on chondrocyte differentiation of BMSCs and its potential mechanism: goat BMSCs isolated and cultured in advance were quantitatively inoculated into sterilized artificial cartilage materials(including 1.2mm COL-HA-PVA,1.4mm COL-HAPVA and 1.6mm COL-HA-PVA)for chondrogenesis induction(contain TGF-β 1)cultured for 3,7 and 14 days,the gene expression levels of cartilage differentiation markers on COL-HA-PVA scaffolds with different elastic modulus(i.e.,1.2mm,1.4mm and 1.6mm pore diameter)were measured,analyzed and compared to explore the effect of elastic modulus on the differentiation efficiency of BMSCs into chondrocytes.At the same time,the integrins in BMSCs on scaffolds with different elastic modulus were compared α 5 gene expression level,lamin A gene expression level and cartilage differentiation marker gene expression level,in order to preliminarily explore the mechanism of elastic modulus affecting the differentiation of BMSCs into chondrocytes.Then,by inhibiting integrinα5 to observe the effect on the gene expression of lamin A and cartilage differentiation and maturation markers,so as to explore the integrinα5β1 whether the activation level and its mediated mechanical signal transduction pathway have an impact on the expression of lamin A and the mechanism of the elastic modulus of scaffold material affecting the efficiency of BMSCs trans-differentiation into chondrocytes.Results:(1)Through the evaluation of physical properties,it is found that the three scaffold materials prepared in this study have certain water content,viscoelasticity and multi porosity.These physical properties are similar to natural cartilage;(2)The elastic modulus of the prepared COL-HA-PVA hydrogel scaffold is correlated with the pore size,and the compression elastic modulus of the 1.2 mm pore size group is the smallest than that of other groups;(3)The 1.2mm,1.4mm and 1.6mm groups could make the inoculated stem cells proliferate normally,and there was no difference in the proliferation ability between the groups;(4)The 1.2mm,1.4mm and 1.6mm groups can promote the trans differentiation of inoculated stem cells into chondrocytes,of which1.2mm has the strongest promoting effect,followed by 1.4mm and1.6mm;(5)The 1.2mm,1.4mm and 1.6mm groups all had normal cell adhesion ability,and the 1.2mm scaffold had the strongest effect on promoting adhesion;(6)Both COL-HA-PVA combined with BMSCs and COL-HA-PVA alone can repair cartilage defects.However,there are also differences between the two methods of cartilage defect repair,in which the efficiency of repairing cartilage defect with COL-HA-PVA combined with BMSCs is higher;(7)COL-HA-PVA combined with BMSCs is an effective method to repair knee cartilage defects.On the basis of repairing defects with surface modified scaffolds,it can effectively improve the repair efficiency of articular cartilage defects by means of cell regeneration therapy;(8)In this experiment,the 1.2mm aperture scaffold has the strongest ability to promote the transdifferentiating of inoculated stem cells into chondrocytes,and its elastic modulus is the lowest,which is more conducive to promote the differentiation and maturation of stem cells into chondrocytes;(9)The mechanical properties of cartilage repair materials play a decisive role in regulating the biological behavior of stem cells,and the elastic modulus is an important factor determining the differentiation direction of stem cells;(10)Due to the different elastic modulus of COL-HA-PVA scaffolds with different pore sizes prepared in this study,the mechanism leading to the difference in the efficiency of repairing cartilage defects is the elastic modulus of the material itself.It can stimulate the activation of integrin on the cell membrane of BMSCs transdifferentiated into chondrocytes,resulting in the difference in the expression of lamin on the cell nuclear membrane induced by the corresponding cell signal level communication pathway,However,there are differences in the expression efficiency of the signal molecular regulatory network that affects the differentiation of stem cells into chondrocytes,resulting in the final difference in the formation efficiency of chondrocytes.Conclusions:COL(type I collagen)-HA-PVA hydrogel porous scaffold material combined with goat bone marrow mesenchymal stem cells can repair articular cartilage defects.It is a good method for cell composite regenerative medicine.Its HA-PVA hydrogel effectively mimic the mechanical and supporting stress of cartilage tissue,and the I collagen attached to it as an extracellular matrix component can effectively provide a good growth environment for g BMSCs inoculation,and can improve the efficiency of transdifferentiating of goat bone marrow mesenchymal stem cells into chondrocytes.And the 1.2mm aperture support has the lowest elastic modulus compared with other aperture supports.This characteristic can also improve the efficiency of transforming goat bone marrow mesenchymal stem cells into chondrocytes.The surface modified hydrogel scaffolds with a certain modulus of elasticity and bone marrow mesenchymal stem cells can be transformed into chondrocytes to provide a new treatment strategy for cartilage defect regeneration.61 figures,19 tables and 148 references更多还原