The Study Of In Vitro Chondrogenesis Of Adipose Tissue-derived Stromal Cells Loading On Chitosan-collagen-chondroitin Sulfate Scaffold

ObjectiveThe aim of the study was to produce a novel porous chitosan-ollagen-chondroitin sulfate scaffold by Freeze-drying method,and then loading with rat adipose tissuederived stromal cells on the scaffold.in the chondrogenic condition,we explore the feasibility of this tricopolymer as a new biomimetic biodegradable polymer scaffold for the articular cartilage tissue engineering;and and provide the experimental basis for the repair of articular cartilage defectsMethodPreparation of the scaffold 2%chitosan was fully mixed with 5%collagen with volume ratio 7:3.first pre-frozen 2 hours under -80℃,and then freeze-dried for 12 hours.we cut the scaffold into the cylinder pieces with 5mm in diameter and 2mm in high.Cross-linked with 50mmol / L2-Morpholine ethane sulfonic acid,50mmol/L Carbodiimide and 50 mmol/L N-hydroxy succinimide,2%chondroitin sulfate ethanol in 40%crosslinking at room temperature 6 hours.Incubated at room temperature for 1.5h by 0.1mol/L Na₂HPO4(pH 7.4),then washed 3 times by 40%ethanol,Double distilled water repeatedly washed to neutral,then freeze-dried again.2 the Cell isolation and amplification SD rat adipose tissue was resected from the renal region of seven adult male rats,The obtained tissue was washed with phosphate-buffered saline(PBS), chopped into small pieces of about 50 mm3,and the extracellular matrix was digested for 20 min at 37_C with 0.1%collagenase and 0.25%trypsin.The cell suspension was centrifuged at 800g for 8 min,and the pellet was resuspended in culture medium,which was composed of Dulbecco's modified Eagle's medium(D-MEM,Gibco,Paisley,UK) supplemented with 10%fetal bovine serum The stromal cells were then plated in tissue culture flasks.The primary cells(P0) were cultured until fusion,after which they were harvested by treatment with trypsin(0.25%)/EDTA.3 the cells plantation and experimental grouping the passages 3 cells were resuspended at concentration of 2×10⁶/ml,and seeded in the scaffold.the Experimental group was induced in chondrogenic medium; the control group was culture with the same medium but exclude TGF-β1.4 Relevant target detection(1) The morphology of the ADSC is observed by microscope The CD29,CD34 and CD44 CD45 expressions of the ADSC cells were detected by flow cytometry.(2) The porosity and water content of the scaffold is detected.(3) The microstructure of the scaffold and cell-scaffold construction was observed via scanning electron microscopy and HE staining;(4) The differentiation of the ADSCs into the chondrocytes was identified by the histology-ical,immunohistochemical staining RT-PCR and western blot methods.ResultThe results of scanning electron microscopy and HE staining showed the inside of scaffold was porous strcture similar to sponginess,the scaffold is uniform pore size,and the pore size is about 100-120 microns.the SEM images of the scaffolds seeded with ADSC and cultured for24 h showed thecells spread Well.after 3 weeks In all the scaffolds the chondrocytes spread Well and produced much extra-cell matrix.The porosity of the scaffold was 92.23±1.68%and the pore size was 100-130um.The morphological characteristics of ADSC,It can be seen 24 hours after inoculation there is a small number of adherent cells,form small round,spindle and polygonal short,The cells is similar to fibroblast-like about nine days and they are fusion more than 90%..Flow cytometry characterization of ADSCs.ADSCs were stained with surface markers CD2980.2%,C34430.2%,CD34,CD45.Immunohisochemical analysis of collagenâ…¡expression in paraffin section of the cell/scaffold construct after culturing 3 weeks,showed typeâ…¡collagen was positive in experimental group but negative in control group.The expression of aggrecan,collagenâ…¡was analyzed at day 21 by RT-PCR. RT-PCR showed that Col2al and Agc mRNA were expressed in the experimental group, but not in the control group. Western-blot detection confirmed positive collagenâ…¡expression in experimental group,but not in the control group.ConlusionThese results show that the chitosan- collagen-chondroitin sulfate copolymer scaffold have an appropriate porosity,the scaffold can conducive to transport the necessary nutrients and metabolites for cell growth,while conducive to cell adhesion and proliferation,It can p rovide an appropriate environment for the generation of cartilage-like tissues and have a potential application in the cartilage tissue engineering scaffold field.