Adipose Tissue Derived Stem Cells Transferred With TGF-β1 Gene, Differentiated Into Chondrocytes, And Used To Constructed Tissue-engineering Cartilage

IntroductionThe defect and degeneration of articular cartilage are common in orthopedic field. Although artificial prothesis is the main and effective choice, high cost and complication could not be neglected. Repairing the defect of articular cartilage with tissue engineering cartilage succeeded preliminarily, but clinical application was suspended because the transplanted tissue engineering cartilage degenerated as time going. The main reason of the degeneration is that the proliferation and passage ability of chondrocyte is limited. The phenotype and activity of chondrocyte are affected, as the specific extracellular matrix (ECM) components are synthesized decreased during passage. There are some reports about application of alginate and growth factors to improve the synthesis of ECM, but there are some shortages, such as short biological half-lives, high price and repeatedly addition.Gene transfer, which means to induce aim gene into eukaryotic cells with special method, has following advantages: 1) the transferred cells are able to express aim gene continuously and effectively that can modulate the growth of themselves and other effective cells and to obtain specific functions. 2) The endogenous aim proteins produced by transferred cells have undergone authentic post-translational modification and therefore have improved potencies to combine with receptors on cells' surface as well as more biological activities. 3) The price of gene transfer is lower than that of purified recombinant proteins. Therefore, it is of great value to induce aim gene which could increase ECM synthesis into cells by employing gene transfer techniques. With these gene modified seed cells, the new gene modified tissue engineering cartilage could be constructed in order to repair the defect and degeneration of articular cartilage.The purposes of present study were to isolate and culture adipose tissue derived stem cells of rat's inguinal region, and to investigate the feasibility of oriented differentiation into osteoblast by PRP.The adipose tissue derived stem cells are transferred with TGF-β1 gene and differentiated into chondrocytes. The gene modified stem cells from adipose tissue are cultured in the three-dimensioned system of alginate gel and observed through transmission electron microscope(TEM).Heterotopic chondrogenesis of TGF-β1 gene modified stem cells loading alginate gel was observed in athymic mice.Materials and methodsMaterialsRipe male Wistar rats and 4 athymic mice (SPF) were supplied by laboratory animal department of china medical university.Methods1. ATSCs were isolated and culturedThe inguinal adipose tissue of the rats was dissected under sterile conditions, and digested by 0.25 % typeâ… collagenase at 37℃for 3h.The resulting cell suspension was diluted with D-Hank's solution and centrifuged at 1200rpm for 5 minutes. The pellet was resuspended with HG-DMEM including 15% fetal bovine serum and cultured at 37℃in a humidified atmosphere and 5% CO₂.2. CD44 immunofluorescence stainingThe second passage ATSCs grown in monolayer were fixed in acetone before histologic examination. Immunofluorescence staining procedures were performed using standard protocols.3. PRP DMEM preparation The rat's blood was taken suction from its abdominal aorta and centrifuged at 1500 rpm for 10min.The upper hematoplasma and platelet were taken suction,then were centrifuged at 3000 rpm for 10min. The pellet was PRP.1ml PRP was added into 98ml DMEM and the same time 1ml 100g/L CaCl₂ solution including 5000 U bovine thrombin was added.4. The effect of PRP on ATSCs proliferation was detected through MTT5. Histochemical staining of ALPThe twice-passaged ATSCs were cultured with PRP DMEM for 14 days. The cells were fixed with 4% paraformaldehyde for 10 min and stained according to Kaplow's method6. Detection of ALP activityThe twice-passaged ATSCs were seeded in 96-well plates at 5×10⁴/well and cultured in PRP DMEM for 7, 14, 21, 28 days respectively. After the respective cultivation, ALP activity was detected with ALP detective reageant.Simultaneously the twice-passaged ATSCs were seeded in 96-well plates at 5×10⁴/well and cultured in DMEM as controls7. Calcium nodule staining with alizarin redThe cells cultured in PRP DMEM for 14 days were washed with PBS, fixed with 95% alcohol for 5 min, stained with 2% alizarin red staining solution and washed twice with PBS.8. TGF-β1 gene transferring ATSCs for stable cellsThe twice-passaged ATSCs were plated at 5×10⁵ cells in the 35mm plate so that they will be 90% confluent at the time of transfection. ATSCs were transferred according to Lipofectamine2000 direction for use. After 6h, growth medium was replaced. Passage the cells at a 1:10 dilution into fresh growth medium after 24h.Add selective medium the following day. The cell clones formed and was plated into 24-well plate after 21 days. When the cells were confluent they were seeded into 6-well plate. Finally they were seeded in culture flask. 9.RT-PCR for TGF-β1,FN,Colâ…¡,Aggrecan,MMP-1,MMP-2,MMP-3 mRNATotal RNA of each sample were isolated by using Trizol Solution, then the concentration and purity of RNA were detected through DNA/RNA detective machine. Reverse transcription reaction tube included 10×PCR Buffer 1μl, dNTP 1μl,MgCl₂ 2μl,Random 9mers 0.5μl,RNAlμl,RNase inhibitor 0.25μl,Reverse Transcriptase 0.5μl,RNase Free dH₂O 3.75μl。Followed by the conditions: 10min at 30℃, 30min at 42℃,5min at 99℃and 5min at 5℃.PCR amplification was performed on a PCR thermal cycler. PCR reaction conditions included initial denaturation for 2min at 95℃, followed by 30 cycles of denaturation of 95℃for 1min and annealing at 72℃for 1min, then extension at 72℃for 7min. The efficiency were detected byβ-actin. PCR products were loaded onto a 8% agarose gels electrophoresis containing ethidium bromide and the bands were visualized under ultraviolet light, then ascertained with the comparison to Makers (TaKaRa Co).10. Western-blot analysisThe samples were lysed in 200μ1 RIPA buffer. The protein concentration was measured according to the Lowry method;The protein was loaded onto polyacrylamide gel eletrophoresis;After 2h the product was transferred to PVDF membrane,the blot was probed with rabbit multiclonal anti-TGF-β1 antibody (1: 400, Santa Cruz,USA) and rabbit multiclonal anti-β-actin antibody (1:400, Santa Cruz,USA),incubated at 4℃for 12h,incubated with the secondary goat anti-rabbit monoclonal antibody conjugated with alkaline phosphatase for 2h at room temperature, stained with alkaline phosphatase coloration solution for 15min.The positive blot were scanned and then measured for intensity by using the UPV gel imaging-analyzing system (Chemi Imager 5500, USA).11. Observe the stable cells growing in alginate gel through SEMThe stable cells at 1×10⁶个/ml was blended with 1.25 % algin solution, dropped into 102M Cacl₂ solution and stirred so that alginate gel formed. The complex was cultured in 15% FBS DMEM for a week. The alginate gel without stable cells was made control. Observe the stable cells growing in alginate gel through SEM12.Observe complex of the stable cells and alginate gel through toluidine blue stainingThe complex was cultured in 15% FBS DMEM for a week, made stressed plate and stained with blue toluidine solution.13. Construction of tissue engineering cartilageThe cells of TGF-β1 gene stable transfection at 5×10⁶个/ml was blended with 1.25 % algin solution, dropped into 102M Cacl₂ solution and stirred at the same time so that alginate gel formed. Then the complex was injected into athymic mice subcutaneouly. The complexes were dissected from the athymic mice respectively after 4, 8 weeks, made paraffin sections and stained with respect to HE,Masson. and immunohistochemistry.14. The data were analyzed by the SPSS12.0 software.ResultsThe ATSCs of primary culture adhered to the plate the second day and were confluent between 7 to 10 days. They exhibited fibroblast-like phenotype, without obvious variation among passages. To confirm ATSCs, expression of CD44 was determined by immunofluorescence. MTT demonstrated experimental groups were different from control groups significantly (P＜0.01).The PRP inducing ATSCs expressed positive after 14 days by ALP staining. Detection of ALP activity demonstrated experimental groups differentiated with control groups significantly (P＜0.01). Alizarin red staining expressed that the PRP inducing ATSCs formed calcium nodules after 14 days.RT-PCR demonstrated that TGFβ1,FN,Colâ…¡,Aggrecan,MMP-2 mRNA of experimental groups increased more evidently than control groups (P＜0.01). MMP-1mRNA of experimental groups decreased more evidently than control groups (P＜0.01), at the same time MMP-3 mRNA of experimental groups also declined more evidently than control groups(P＜0. 05).Western blot demonstrated that TGFβ1 protein of experimental groups increase more evidently than control groups significantly (P＜0.01)Alginate gel was observed high porosity, and that TGFβ1 modified ATSCs grew up well and secrete extracellular matrix by scanning electronic microscope. TGF-β1 modified ATSCs cultured in alginate gel expressed positive toluidine blue staining and splitting phase of the nucleus was observed.Histologic findings demonstrated that the constructs included many cartilage-like cells and extracellular matrix, were similar to cartilage tissue. Many cells located in lacunes. Extracellular matrix was loose and minority of alginate gel was not absorbed at 4 weeks after injection. Extracellular matrix demonstrated blue and enclosed cartilage-like cells by Masson staining. At 8 weeks after injection, alginate gel was completely absorbed, simultaneously cartilage-like cells located in lacunes and extracellular matrix was stained blue by Masson staining. The conducts at 4 and 8 weeks after injection expressed collagenâ…¡through immunohistochemistry.Conclusions1. ATSCs of Wistar rats could be separated and cultured successfully and their expression of CD44 was positive2. Platelet-rich plasma (PRP) induced ATSCs to differentiate into osteoblast3. TGF-β1 gene was transferred into ATSCs at the first time in our country. The specific extracellular matrix of chondrocyte differentiation increased evidently. The results demonstrated TGF-β1 gene could induce ATSCs into chondrocyte.4. TGF-β1 gene promoted MMP-2 to increase and promoted MMP-1,3 to decrease. The decrease of MMP-1,3 was one of reasons that the specific extracellular matrix of chondrocyte differentiation increased.5. TGF-β1 gene modified ATSCs-alginate gel complex successfully constructed tissue-engineering cartilage...