| Objective:To seek a series of effective means of intervention to promote proliferation and differentiation to mature chondrocyte of the tissue engeneering seed cells, prebartilagious stem cells(PSCs), in vitro. we investigate the different effects of transforming growth factor subtypes:GF-β1,TGF-βand TGF-β3, which have been proved that paly the key role in reagulation the diiferentiation of stem cells to mature chondrocytes, on the activities of proliferation and differentiation of PSCs to choose an ideal cell factor, furthermore transfect the gen of this cell factor into the PSCs the make this cell auto-secret the cell factor to self-induce their proliferation and differentiation. We study the effects of pulsed electromagnetic fields on diiferentiation of precartilaginous stem cells, to find a economics and effective means of intervention. We compare the difference of transfection efficency between the culturing condition of 3-D in self-asembly peptide nano scaffold of KLD-12 and 2-D by the means of nano transfection reagent to find a high-efficency and safy tranfection method. Finally we plant the tranfected PSCs into the nano scaffold of KLD-12, then detect their ability of proliferation and differentiation in vitro and transplant to cartilage defect of SD rat to evaluate the treatment effects. To find a new way to repair cartilage deffect by the means of tissue engineering.Methods:(1) obtain the PSCs by the ways of Immun-Magnetic activated cell sorting after seperation and purifcation. Culture PSCs in the medium with the concentration of lOng/ml of different TGF-pisfroms.Proliferation and DNA synthesis of PScs in response to TGF-(3 isoforms was examined by MTS and BrdU/PI double-reference method at the time of 0,3,6,9d after be cultured. Observe the level of the gens expression of Col II,Aggrecan at the time of 0,3,6,9 d by means PT-PCR and expresion of SOX9 by means Immunohistochemistry at the time of 2w to study the effects of TGF-β1, TGF-β2 and TGF-β3 on chondrogenic differentiation.(2) exposed the PSCs of Experimental group in PEMF(1mT,50MHz)for 30min twice a day and continue to stimulate the PSCs for 2days,4days and 6days, control group do not receive the stimulation of PEMF. Collect the PSCs of each groups, then detect the gene expression levels of Collagen-Ⅱ(COL-Ⅱ) and aggrecan by the method of RT-PCR and and use the technology of Western-blot to determine protein expression levels of ihh and PTHrp in each group.(3) transplant the cells in the self-assembled scaffold of KLD-12,transfect the gene of h TGF-β3 in the PSCs cultured in self-assembled scaffold and common tow-dimension medium by using the nano-transfection reagent(xfect stem), after 48h,then detect the gene expression levels of h TGF-β3 by the method of immunofluorescence and RT-PCR, and detect the concentration of TGF-β3 in the Supernatant of medium in different groups and different time after transfecion by the method of ELISA.(4) PSCs transfected with hTGF-beta3 gene which was study group was embeded and cultured in the self-assembly peptide nano gel of KLD-12 and control group use PSCs which was not transfect with exogenous gene. After 1 week, detect the level of extracellular matrix,type II collagen by means of immunohistochemistry,and Using Western-Blot to detect the protein expression of Aggrecan and SOX9 after 1 w,2w and 3w. Dectect proliferation of each group at the time of 3d and 5d by means of MTS and BrdU/PI double-reference method. Use these self-assembly peptide nano gel with Transgenic PSCs and untransgenic PSCs to repair cartilage defect of rats, and evaluate their treatment effect after 6w and compare it with contrl group which use merely self-assembly peptide nano gel. Results:(1) After 3 d cultrued the activity of proliferation in group of TGF-β1 decreased significantly (P＜0.05) but it dose not appear in the groups of TGF-β2 and TGF-β3 (P>0.05), after 6 d the activity of proliferation in group of TGF-β1 decreased significantly comparing with the control group, but the group of TGF-β2 and TGF-β3 show increasing effect on the proliferation of PSCs (P<0.05). RT-PCR shows:With the extension of time, the gen exprssions of ColⅡin each group of TGF-βhave the increasing inclinations, the gens exprssions ofAggrecan also appears increasing in the groups of TGF-β1 and TGF-β3, but these gens expression show no difference in TGF-β2 group. Immunohistochemistry shows the synthesis of SOX9 protein in groups of TGF-β1,TGF-β3 are higer than the control group (P<0.05).while TGF-β2 show no difference comparing with the control group (P>0.05)(2) The result of RT-PCR show with the time of exposure to PEMF prolonging, the gene expression levels of Collagen-Ⅱand aggrecan increase. comparing with the control group, except the group of 2 days exposure in which the gene expression level of aggrecan has Statistically difference (P<0.05), all the experimental groups have Statistically significant difference (P<0.01). Western-blot also shows that protein expression levels of ihh and PTHrp increase with the time prolonging, but comparing with the control group, the expression of ihh in all the experimental groups have Statistically significant difference (P<0.01),but the expression level of PTHrp has no Statistically difference (P>0.05) in the group of 2 days exposure, the level of 4 days group has Statistically difference (P<0.05) and the level of 6 days has Statistically significant difference (P＜0.01).(3) Transplant the cells in the self-assembled scaffold of KLD-12,transfect the gene of h TGF-β3 in the PSCs cultured in self-assembled scaffold and common tow-dimension medium by using the nano-transfection reagent(xfect stem), after 48h,then detect the gene expression levels of h TGF-β3 by the method of immunofluorescence and RT-PCR, and detect the concentration of TGF-β3 in the Supernatant of medium in different groups and different time after transfecion by the method of ELISA.(4) PSCs transfected with hTGF-beta3 gene which was study group was embeded and cultured in the self-assembly peptide nano gel of KLD-12 and control group use PSCs which was not transfect with exogenous gene. After 1 week, detect the level of extracellular matrix,type II collagen by means of immunohistochemistry,and Using Western-Blot to detect the protein expression of Aggrecan and SOX9 after 1 w,2w and 3w. Dectect proliferation of each group at the time of 3d and 5d by means of MTS and BrdU/PI double-reference method. Use these self-assembly peptide nano gel with Transgenic PSCs and untransgenic PSCs to repair cartilage defect of rats, and evaluate their treatment effect after 6w and compare it with contrl group which use merely self-assembly peptide nano gel.Conclusions:The promoting effect of TGF-β3 on perolifertion, synthesizing extracellular matrix and differentiation to mature chondrocyte in PSCs is higher than that of TGF-β1 and TGF-β2, because of that it could be the ieal intervention factor in tissue engineering. Pulsed electromagnetic fields also has the ability to induce PSCs differntiation, so it coould be a subsidiary physical means of intervention.Our study show the Transfection efficency of PSCs with TGF-β3 gene cultured in 3-D self-assembly peptide nano scaffold of KLD-12 is higer than cells cultured in 2-D, PSCs transfected with TGF-β3 gene cultured in 3-D self-assembly peptide nano scaffold have higher activities of proliferation and differentiation, and the mixture could treat cartilage defect in SD rat. In summary,the tissue egineering cartilage composed of KLD-12 scaffold and PSCs transfected with TGF-β3 gene has good prospect to treat cartilage defect.
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