| [Objective] To find amplification method of bone marrow mesenchymal stem cells from Diannan Small-ear Pig,and get a better amplification method for seed cells of cartilage tissue engineering;To study cartilage differentiation of Diannan small-ear pig BMSCs, which induced in vitro with transforming growth factor beta3and bone morphogenetic protein2.[Methods](1) Bone marrow mesenchymal stem cells were harvested from Diannan Small-ear Pig,then cultured with centrifugation and adherent method.The CD29, CD44, CD45markers of BMSCs in the second generation were examined with flow cytometry. And the second generation of BMSCs were induced into bone and chondrogenic differentiation,the ability of osteoblast differentiation was examined with alizarin red,the ability of chondrogenic differentiation was examined with toluidine blue and HE staining.(2) Take the second generation BMSCs from Diannan small-ear pig and added growth factor,Then divided into four groups:Group A was transforming growth factor beta3and bone morphogenetic protein2group. Group B was transforming growth factor beta3group; Group C was bone morphogenetic protein2group; Group D was the contol group with no growth factor;2days in change the culture medium and oberserved morphological changes of BMSCs by inverted microscope and the growth curve was made. After induction, Cells was stained using type Ⅱ collagen immunohistochemistry and21days done with Western blot test to type Ⅱ collagen protein expression. The result of datas were processed and statistical analysed by SPSS17.0.[Results](1) Cells shape were spindle and fibroblast-like.(2) Flow cytometry results:P2CD29positive rate was99.41%,P2CD44positive rate was99.52%,P2CD45negative rate was99.93%.(3) alizarin red staining showed positive after BMSCs were induced2,3weeks.(4) toluidine blue showed positive after BMSCs were induced2,3weeks.(5) Type II collagen of cartilage through immunohistochemical method showed different degrees of positive expression in14and21days with three add cytokines of the experimental group and control group. The blank contol group A was negative. The grey value of type II collagen immunohistochemical with experimental group was higher than control group and blank control group, the difference was statistically significant (P<0.05). The grey value of type II collagen was showed by Western-blot in21days that experimental group was higher than control group and blank control group, the difference was statistically significant (P<0.05).[Conclusions](1) Centrifugation and adherent method could culture BMSCs from Diannan Small-ear Pig,it was an efficient method of amplification.(2) The second generation of BMSCs from Diannan Small-ear Pig with high purity and multiple differentiation potential could differentiate to chondrogenesis.(3) TGF-beta3combined with BMP-2could promote BMSCs differentiatied into cartilage cells and promote the type II collagen expression,So TGF-beta3and BMP-2couled used as factors to promote BMSCs differentiation into cartilage. [Objective] To make the slow-release microspheres which cotained the TGF-beta3and BMP-2double factor,then combined with DBM,and evaluated the drug release effect. To explore the chodrogenic effect of DBM/microspheres composite scaffolds combined with BMSCs, then provided experimental basis of scaffold materials for cartilage defects repair.[Methods](1) Demineralized bone matrix was made according to Urist’s method. The Ultra structure of DBM was observed with scanning electron microscope (SEM). The chitosan microspheres which contained TGF-beta3and BMP-2was prepared through crosslink emulsification process. Electron microscopy and Image software to observed the shape of microspheres. Enzyme-linked immunosorbent assay was used to determine the microspheres drug loadings envelopment rate and the sustained release rate in vitro. Take the chitosan microspheres which contained TGF-beta3and BMP-2was prepared and second generation of BMSCs cultured, cells were stained using type Ⅱ collagen immunohistochemistry and Western blot test type Ⅱ collagen protein expression after21days.(2) BMSCs was cultured on composite scaffold after microspheres combined with DBM, and MTT assay was used to analyze the growth curve of Bone marrow stromal cells on composite scaffold. Take the second generation of BMSCs from Diannan small-ear pig and different scafflolds were divided into three groups:Group A was (double factor microspheres/DBM composite support group); Group B was (empty microspheres/DBM support group); Group C was (simple DBM support group)2days change the culture medium, After cultivated21days, the specimens were oberserved and made in paraffin sections. All the specimens were carried out with HE staining, toluidine blue stain, and type Ⅱ collagen immunohistochemistry.[Results](1) Appearance of demineralized bone matrix scaffold material looked like white sponge, and DBM scaffold have white three-dimensional cancellous feature observed under scanning electron microscopy.(2) Average partical size of chitosan with drogel hydrogel microspher was53.38μm with good spherical、 smooth sphere and have a high rate of coating. The drug-loading rate of TGF-beta3and BMP-2are respectively58.7%,53.2%. The entrapment rate of TGF-beta3and BMP-2were respectively22.01ng/mg,17.55ng/mg.Drug release test showed that TGF-beta3and BMP-2can slow release from microspheres, and7days can accuulate to release a quantity of61%,57.5%.(3) Using ultrasonic oscillation method to pour intuo microspheres in DBM showed that microspheres can be evenly distributed in the DBM and BMSCs can grow on the composite scaffold well, and the peak of compositely culture is the6days.(4) After double factor microspheres and BMSCs composite21days,the immunohistochemical and Western blot detection of type Ⅱ collagen are all positived.(5) At21days, culture masses were observated, HE staining show that the chondrocytes have began to proliferate, caryon was blue. Alcian blue stain show that the chondrocytes proliferated and have no order, surrounded by lots of extracellar matrix. Immunohistochemical identification of Ⅱ collagen was positive, brown-yellow stained particles could be discerned in extracellar matrix. The control group dyeing were negative.[Conclusion](1) DBM scaffold had a three-dimensional structure of tissue engineering scaffold. After BMSCs in composite scafflold was compostied culture and the best time was8day.(2) The chitosan microspheres was prepared through crosslink emulsification process had good repeatability,particle size distribution and a good slow-release performance.(3) Microspheres combined with DBM/BMSCs could be an efficient alternative for articular cartilage defects repair. |
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