Acquisition Of Rat Lumbar Cartilage Endplate Stem Cells And Preparation Of Decellular Endplate Cartilage Matrix Tissue Scaffold

Objective:to Isolation and identification of rat lumbar cartilage endplate stem cells,prepare the Decellular cartilage endplate matrix(DCEM)to explore it's biocompatibility and influence on the differentiation of rat lumbar cartilage endplate stem cells(CESCs),The tissue scaffold was prepared by freeze-drying DCEM combined with chitosan to explore the proliferation and apoptosis of CESCs on the scaffold.Methods:The rat lumbar cartilage endplate was isolated and the primary chondrocytes were obtained by enzyme digestion,After inoculation of chondrocytes at a certain density,crystal violet staining was performed to calculate the percentage of cartilage stem cell colony formation.Trypsinizes colonies and performs expansion culture,then adding of osteogenic,adipogenic and chondrogenic induced medium,staining by alizarin red,oil red O staining and Safranin and multidirectional differentiation observed,Flow cytometry was used to detect the expression of CD44 and CD90 and negative marker CD45 on stem cell surface.After the porcine cartilage was crushed,the DCEM was obtained by trypsin combined with ribozyme and TritonX-100,the decellularization effect of cartilage tissue was observed by DAPI staining,The morphology and components of the DCEM was observed by electron microscopy and by Fourier transform infrared spectroscopy,respectively.After dissolving the DCEM in acetic acid,an Decellular matrix membrane was prepared and its general characteristics were observed by Inverted phase contrast microscope,The obtained CESCs were planted on the membrane,the growth morphology was observed,the proliferation was detected by CCK-8.Introducing the concept of relative cell proliferation rate(RGR)and establishing a new evaluation system for evaluating biocompatibility from cytology,what's more,The micro-syringe was pipetted with 1 ml of acellular matrix solution and acetic acid,injected into the skin of SD rats,respectively.The redness around the dermis was observed,The body temperature and the diameter of the cumulus were measured at the time of injection and at 12h,24h and 36h after injection.evaluate its biocompatibility from a living perspective.In addition,P3 generation endplate chondrocytes were planted in a perforated plate with cartilage matrix,and then induced by osteogenic,adipose and chondrogenic differentiation.After 2 weeks,alizarin red,oil red O and saffron stained,while Real-Time PCR detects the relative expression levels of osteogenesis,adipogenic and chondrogenic genes Runx2,Col-2a1 and PPAR?in each group,and analyzes the differentiation tendency of stem cells on cartilage matrix.Finally,the obtained matrix and chitosan were mixed in a certain ratio,and then freeze-dried and scanned under electron microscope,and to observe the proliferation and apoptosis of CESCs in the tissue scaffold.Results:The CESCs were inoculated by different densities,and cell colony formation was observed in the culture for 3 to 5 days,the morphology of the cells in the colonies was fusiform.The colony formation rate was calculated,including(4.87%±0.30%)in100/cm~2 group,(4.12%±0.42%)in 200/cm~2 group,(2.43%±0.12%)in 300/cm~2group.and one-way analysis of variance.,P=0.0002,the results are statistically significant.After induction of multi-directional differentiation,CESCs can be stained with alizarin red,oil red O and safranin,indicating that they have osteogenesis,adipogenic and chondrogenic differentiation.The surface expression of CD44,CD90,and CD45 were 90.75%,99.7%,and 1.36%,respectively.The proliferation of EPSCs is stronger than that of EPCs by RTCA.The prepared DCEM has a white enamel shape,the membrane is white and transparent and is composed of staggered collagen fibers under electron microscope,it's also has good biocompatibility.The DCEM can promote the differentiation of CESCs into osteogenic direction,inhibiting chondrogenic and adipogenic differentiation.Real-Time PCR detected the osteogenic gene Runx2 in the DEPM group was higher than the other two groups,while the Col-2a1 and PPAR?gene expression was lower.The matrix combined with the chitosan scaffold has a milky white"sponge"-like structure,and the cartilage stem cells have good proliferative ability and less apoptosis.Conclusion:There are CESCs with multi-directional differentiation ability,monoclonal colony forming ability and high expression of CD44 and CD90 in cartilage tissue,and their proliferative ability is much stronger than that of chondrocytes.The prepared matrix promotes osteogenic differentiation of cartilage stem cells and has good biocompatibility;after mixing chitosan,the prepared freeze-dried scaffold has less cytotoxicity and meets the selection criteria of tissue engineering scaffolds.