| 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... |
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