Study On The Synergistic Effect Of BMP-2 And TGF-?3 On Chondrogenic Differentiation Of PBMSCs Of Pigs

Part ?Mobilization,vitro culture and identification of PBMSCs of diannan small-ear pigsObjective:To investigate the mobilization method of peripheral blood-derived mesenchymal stem cells(PBMSCs)of small-ear pigs and to explore the biological characteristics and multi-directional differentiation ability.We regard the peripheral blood-derived mesenchymal stem cells as seed cells in order to lay the foundation for subsequent experiments.Method:(1)G-CSF and AMD3100 were used to mobilize peripheral blood-derived mesenchymal stem cells of small-ear pigs and was divided into 4 groups:A(G-CSF),B(AMD3100),C(G-CSF +AMD3100),D(blank control group).(2)The density gradient centrifugation and adherent wall method were used,observe the cell morphology.Colony formation was detected by CFU-F and the percentage of CD45-CD90+ cell population was detected by flow cytometry.(3)The expression of CD44,CD90,CD34 and CD45 in P2 PBMSCs were detected by flow cytometry respectively.The P2 PBMSCs were induced into osteocytes,chondrocytes and adipocytes.Alizarin red staining,alcian blue staining and oil red O staining were used to evaluate the multidirectional ability respectively.Results:(1)There were a lot of short spindle cells and polygonal adhered cell growth in peripheral blood samples which was obtained by G-CSF+AMD3100 mobilization group after 7 days of culture,the adhered cells showed obvious colony growth after 10 days of culture,the peripheral blood samples obtained by the G-CSF mobilization group and the AMD3100 mobilization group showed only a small amount of colony formation after 10 days,and no significant colony formation was observed after 10 days of peripheral blood samples obtained from the blank control group.(2)After mobilization,the number of CFU-Fs in peripheral blood in the experimental group(G-CSF+AMD3100)was significantly increased compared with the control group(1.18±0.12vs1.25±0.24vs4.28±0.21vs0.87±0.25CFU-Fs/ml,P<0.05).The proportion of CD45-CD90+ cell population was also significantly increased.The total number of CD45-CD90+ cell population was also increased(2.28±0.15vs 6.53±0.23 vs 9.98±0.34 vs 2.23±0.12×104/ml,P<0.05).(3)Flow cytometry showed CD34,CD45 was negative,CD44,CD90 was positive.Calcium nodules formed and stained positively with alizarin red after induced osteogenic induction for 21 days,alcian blue staining was positive after induced chondrogenic induction for 21 days,lipid droplet formed and stained positively with oil red O after induced adipogenic induction for 14 days.Conclusion:G-CSF and AMD3100 can be used as an effective mobilization agent for peripheral blood mesenchymal stem cells.The mobilized stem cells had mesenchymal stem cell characteristics which can be used as seed cells for follow-up experiments.Part ? Study on the Differentiation of PBMSCs of Small-ear Pigs into Cartilage through BMP-2 and TGF-?3 induction in VitroObjective:To investigate the effect of BMP-2 and TGF-?3 on differentiation of PBMSCs into cartilage synergisticallyMethods:(1)The PBMSCs of small-ear pigs were cultured and expanded in vitro.Four groups were classified:A(BMP-2 and TGF-?3 induction group),B(BMP-2 induction group),C(TGF-?3 induction group)and D(blank control group).They were induced respectively.The morphology of each group was observed through microscope,and the growth curve was drawn.(2)The concentration of COL-2 A1 in supernatant of each group was determined by ELISA after 3,5,7,9,14,21 days of induction.(3)The expression of cartilage marker gene(COL-2A1,AC AN)mRNA was evaluated by RT-PCR after 1W,2W,3W of induction,Type ? collagen immuno histochemistry was performed at 2W and 3W.All above was investigated the effect of two-factor induced differentiation of PBMSCs into cartilage.Results:(1)The culture results showed that the morphology of cells was gradually transformed from long spindle to polygon after being induced by two factors 3-4 days,and the changes were earlier than other groups.The growth curves of the each groups were basically the same with "S",group A has the highest OD value,compared with group B,C and D,the difference was statistically significant(P<0.05).(2)The result of ELISA showed that the content of COL-2A1 in group A was the highest,compared with group B,C and D,the difference was statistically significant(P<0.05).(3)The results of RT-PCR indicated that the relative expression of COL-2A1 and ACAN in group A were higher than those in group B,C and D in 1W,2W,3W at the same time point(P<0.05).The Type ? collagen immunohistochemistry results showed that the number of staining cells and the staining strength of group A were higher than that in group B,C and D,and the difference was statistically significant(P<0.05).Conclusion:BMP-2 and TGF-?3 were more effective in synergizing the chondrog-enic differentiation of PBMSCs.Part ? The role of miR-181a in the process of BMP-2 and TGF-?3 inducing PBMSCs into chondrogenic differentiationObjective:On the basis of the part ? and previous studies,we further explore the role of miR-181a in the process of chondrogenic differentiation of PBMSCs induced by BMP-2 and TGF-?3Methods:In the previous experimental study,we induced peripheral blood-derived mesenchymal stem cells(PBMSCs)into chondrocytes through chondrogenic microenvironment in vitro.The expression of miR-181a was significantly abnormal detected by microRNA microarray and PCR.(1)Four groups were classified like part ?:A(BMP-2 and TGF-?3 induction group),B(BMP-2 induction group),C(TGF-?3 induction group)and D(blank control group),the results of microRNA chip were further verified by PCR after 7?14?21 days induction of PBMSCs respectively.The expression of cartilage marker genes BMP-2,COL-2A1 and aggrecan(AGR)in PBMSCs induced by BMP-2 and TGF-?3 were further detected by Western blot.(2)transfected miR-181a inhibitor and NC inhibitor,CCK-8 were used to detect the effect of miR-181a inhibitor on cell proliferation.The ability of chondrocyte differentiation was assayed by alcian blue staining.MiR-181a and the expression of BMP-2,COL-2 A1 and aggrecan(AGR)were detected by PCR,Western blot,immunohistochemistry 3,7 and 14 days after transfection.(3)The target genes of miR-181a were predicted by bioinformatics software miRBase and miRDB.The dual luciferase reporter gene method was used to further predict the target gene of miR-181 a preliminary validation.Results:(1)PCR showed that miR-181a was highly expressed during the chondrogenic differentiation at 7,14 and 21 days in the two-factor-induced group compared other groups(P<0.05).Western blot indicated BMP-2,COL-2A1 and aggrecan(AGR)of group A were highly expressed at the same time.(2)CCK-8 and alcian blue staining showed that down-regulated miR-181a inhibited cell proliferation and differentiation after transfection with miR-181a inhibitor.PCR showed that the expression of miR-181a in miR-181a inhibitor group was significantly lower than that in NC inhibitor group at 3,7 and 14 days after transfection(P<0.05);Western blot showed that the expression of BMP-2,COL-2A1 and aggrecan(AGR)in miR-181a inhibitor group was significantly lower than NC inhibitor group(P<0.05).Immunofluorescence showed that the intensity of immunofluorescence staining of BMP-2 and COL-2A1 in miR-181a inhibitor group was significantly lower than that in NC inhibitor group(P<0.05).(3)Bioinformatics software suggested that miR-181a had potential binding with 3'UTR of GREM1 and CRIM1.Western blot showed that the expression of GREM1 and CRIM1 in PBMSCs induced by chondrogenic differentiation was significantly lower than that in the blank control group(P<0.05)on the 3,7 and 14 days after being induced by two-factors group,the GREM1 and CRIM1 levels in miR-181a inhibitor group were significantly higher than those in NC inhibitor group at 3,7 and 14 days after being transfected.(4)The result of dual luciferase reporter assay showed that GREM1 MiR-181a mimics co-transfected with the CRIM1 wild-type plasmid vector binding site,the intracellular luciferase activity was significantly decreased compared with the NC group,while the GRIM1 and CRIM1 mutant plasmid vector binding sites were co-transfected after miR-181a mimics.Compared with the NC group,intracellular luciferase activity did not change significantly.Conclusion:MiR-181a is highly expressed in the process of chondrogenic differentiation of PBMSCs induced by BMP-2 andTGF-?3.Complementary base sequence exists in miR-181a and its potential target genes GREM1 and CRIM13'UTR.MiR-181a in cells can merge into the 3'UTR in the GREM1 and CRIM1 genes within a specific region.Part ? Study on the repair of articular cartilage defects in pigs with BMP-2 and TGF-?3 chitosan sustained release microspheres/DBM composite PBMSCsObjective:To construct a new type of composite tissue engineered cartilage,and investigate the effect of the new tissue-engineered cartilage in repairing the articular cartilage defect.Methods:(1)The composite scaffolds carrying BMP-2 and TGF-P3 chitosan sustained release microspheres/DBM were prepared by emulsion cross-linking and Urist method,SEM observation were performed.(2)Constructed new tissue engineering cartilage after composited PBMSCs.SEM was used to observe the growth and proliferation of cells on the scaffold.(3)Meanwhile,the model of cartilage defect in the piglets of small-ear pigs was divided into 4 groups(group ?:blank defect group,group ?:DBM alone,group ?:double factors microspheres + PBMSCs,and group?:double factors microspheres/DBM+PBMSCs).The animals in each group were taken to be observed at 4,8 and 12 weeks after operation.Gross observation,histological examination,immunohistochemistry and O'driscoll cartilage histomor-phometry were performed.Results:(1)The appearance of composite scaffolds was yellow-white,the surface is rough and has a porous structure.SEM showed that the microspheres were uniformly distributed in DBM,there was no significant change in morphology.(2)SEM of the new tissue engineered cartilage showed that PBMSCs were evenly grown on the surface of the scaffold,microspheres morphology had no obvious change.(3)In vivo experiments in pigs showed that the cartilage tissue filled in defect area of group? was better than that of group ?,? and ?,and close to the normal cartilage tissue(P<0.05).The HE staining showed the typical cartilaginous structure of the repaired tissue in the defect area.In group ? collagen ? immunohistochemical staining was positive,the staining was better than that in ?,?,? group,which is close to normal surrounding cartilage tissue(P<0.05).The scores of O'driscoll in group ? were higher than which in groups ?,? and ?,there was obvious difference(P<0.05).Conclusion:The new tissue engineering cartilage constructed by two-factor sustained-release microspheres/DBM composite PBMSCs,could repair the articular cartilage defect in diannan small-ear pigs well.