| Objective: To investigate the methods of integrating TGF-β1 gene eukaryotic expression plasmid pCMV6-TGF-β1-GFP into the collagen / chitosan scaffold and made into gene activated matrix. To explore the effect of gene activated matrix on the differentiation of adipose derived stem cells into chondrocytes, providing experimental basis for the repair of cartilage defects.Methods:1 The production of gene activation of matrix:2% collagen and 3% chitosan acetic acid solution was mixed at 7:3 ratio, poured into the mold, pre-frozened for 10 hours at -20℃, then lyophilized for 24 hours. After that, the scaffold was added pCMV6-TGF-β1-GFP plasmid into it, lyophilized again, crosslinked at room temperature for 2 hours and was made gene activated matrix. Detect the pore size and porosity of gene activated matrix by SEM and liquid displacement method.2 Isolation, culture and passage of Adipose derived stem cells:Under sterile conditions, bilateral inguinal fat was taken for about 10 ml. After removing the blood vessels, this fat was completely cut into small pieces, then was digested by 0.1% type I collagenase for 1.5 hours at 37℃, under the oscillation. The digestion was terminated by the LG-DMEM medium without fetal bovine serum. Cell suspension was centrifuged, the supernatant discarded, then resuspended, counted and seeded in culture flasks at 37℃, saturated humidity, 5% CO2 in incubator. First exchange of medium after 48 hours, then the medium was changed once every 3 days. The first passage in 7-10 days to reach about 80% confluence. then each next passage at about 80% confluence.3 Gene activated matrix composites Adipose derived stem cells in vitro: Take the 4th generation adipose derived stem cells as seed cells, loaded onto Gene activated matrix. Setting the experimental group A and group B: Group A use GAM as scaffold; Group B use collagen/chitosan without plasmid as scaffold. The complex of cells and scaffolds was placed into 12-well plate, cultured in LG-DMEM containing 10% fetal bovine serum at 37℃, saturated humidity, 5% CO2 in incubator. Medium was changed once every 3 days. To detect the cell growth on scaffolds by MTT.4 To detect the effect of gene transfer:After cultured for 24 hours, the complex was observed by fluorescence microscopy to see if there is green fluorescent. After cultured for 7 days, the specimens were cracked, homogenized in the EP tube and then added 1ml of Trizol, Total RNA was extracted according to the instructions. Expression of TGF-β1 was detected by RT-PCR. 1,4,7,10,13 days after inoculation, the medium was detected by ELISA for the TGF-β1 protein content.5 Detection of the complex by SEM and immunohistochemistry:Cultured for 3 days, 7 days, 14 days, cell morphology and growth was observed by SEM. Samples were taken on day 14,detected by immunohistochemistry and related biological methods.Results:1 The aperture of GAM is about 100-150um, at an average of 120um, with good communication between pores. The porosity is (86.7±1.5)%.2 Morphological observation of Adipose stem cells: Primary Adipose derived stem cells adhered within 24 hours, cells showed spindle or polygonal. Clone-like growth began to show from day 3 and 80-90% confluence was reached in 8-10 days. Passage cells showed a significantly accelerate of growth, a typical spiral-like growth and reached 80-90% confluence in 5-7 day.3 Transfection effect of GAM:24 hours after inoculation, Fluorescence microscopy showed green fluorescence emitted by the experimental group scaffold, This fluorescence phenomenon continued until the 14 days, No such phenomenon in the control group. Cultured for 7 days, taking the total extracted RNA of complex, detected the expression of TGF-β1 by RT-PCR. The results showed that the experimental group expressed TGF-β1, but no expression in the control group. 3,6,9,12,15 days after inoculation, the TGF-β1 protein content in medium was detected by ELISA, Results showed that the experimental group was detected at the highest level at day 7, and still have expression at day 14, whereas no expression in the control group.4 Scanning electron microscopy and immunohistochemistry:48 hours after the composition of ADSCs and GAM, the Scanning electron microscopy showed that the surface of GAM was covered with spindle-shaped cells, some pores were filled with cells which were colony-like growing. After 2 weeks of culture, scanning electron microscopy showed that the cell shape transformed into polygonal or irregular, the volume increases, and more extracellular matrix appeared. Most cells of the control group showed a fibrous cell growth, and the cell numbers and ECM are less.Conclusion:GAM has good biocompatibility and porosity. ADSCs can be loaded on it and transfected by TGF-β1 gene. Then these cells can express TGF-β1 protein and promote their differentiation into chondrocytes. GAM can be used as a promising scaffold for cartilage tissue engineering.
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