Study On Rabbit Nucleus Pulposus Cells Induces The Differentiation Of Mesenchymal Stem Cells By Co-cultured In A 3-dimensional Environment

PartⅠCulture of rabbit nucleus pulposus cells in alginate beads andstudy the biological characteristicsObjective To culture rabbit nucleus pulposus cells using alginate beads and plates, thendetected the biological character in vitro.Method We got the primary nucleus pulposus cells (NPCs) from 6 healthy New Zealandrabbits of 4-month-old, and then divided the cells into the experiment group (culture inalginate beads) and the control group (culture on plates) after passage. Cell morphology ofNPCs was observed by inverted phase contrast microscope. MTT was used to determine thecells proliferation, and RT-PCR was applied to detect the expression of collagenⅡandaggrecan in NPCs.Results After two weeks cultivation, there was no significant difference in the cellsproliferation rate of NPCs between the experiment group and the control group. But theexpression of collagenⅡand aggrecan in the experiment group was superior to thecontrol(P＜0.05).Conclusion Culturing NPCs in alginate beads can promote the expression of collagenⅡand aggrecan, it was better than that on plates to maintain phenotypic stability of NPCs.PartⅡExperimental study on differentiation of the rabbit mesenchymal stem cells labeled by green fluorescent protein into osteoblasts andlipoblasts in vitroObjective To label rabbit mesenchymal stem cells (MSCs) by green fluorescentprotein(GFP), and then observe the potency of multi-directional differentiation byosteoblast and lipoblast formation induction.Method We got six New Zealand rabbits aged 4 months after birth, of either sex. Thedisposition of the rabbits met the ethics standards. MSCs were isolated from the femurs ofrabbits, then they were purified and cultured in vitro by density gradient centrifugation andadherent culture. MSCs were evaluated by morphology and surface marker. MSCs culturewas observed with an inverted microscope. Green fluorescent protein plasmid wastransfected into mesenchymal stem cells using Lipofectamine. The stable transfection cellswere obtained using G418 selection after transfection. The stable transfection MSCs wereinduced to osteoblasts and lipoblasts by osteogenic inductor and adipogenic inductor. Oilred O staining and alizarin red staining were used to determine the differentiation.Results The MCSs were mostly fusiform in shape, to be similar to fibroblast cell aftercultivation of primary and subcultured. The flow cytometer analysis showed that themembrane mark CD44 was positive, CD34 was negative. MSCs labeled by greenfluorescent protein were apparent calcium mineralization after 21 days osteogenic induction.Alizarin red staining showed positive with red. Intra-cellular lipid droplet appeared after 3days adipogenic induction. The quantity of lipid droplet increased and were fused eachother 2 weeks later. The morphous of fusiform shape became round or polygon, and oil redO staining showed a great quantity lipid deposited.Conclusion MSCs can be isolated and cultured by the method of density gradientcentrifugation and adherent culture in vitro. MSCs labeled by green fluorescent proteinusing Lipofectamine have the potency of multi-directional differentiation after induction. PartⅢStudy on rabbit nucleus pulposus cells induces thedifferentiation of mesenchymal stem cells byco-cultured in a 3-dimensional environmentObjective To investigate the changes of mesenchymal stem cells phenotype afterco-cultured with nucleus pulposus cells in a 3-dimensional environment by alginate Beadsand find a new source of seed cells for constructing tissue engineered intervertebral disc.Method We got the primary nucleus pulposus cells (NPCs) and mesenchymal stem cells(MSCs) from healthy New Zealand rabbits. After passage and purification, the MSCs werelabeled by transduction with green fluorescent protein (GFP) and divided into theexperiment group and the control group. After transduction MSCs were cultured eitheralone (controls) or at a NPCs-to-MSCs ratio of 1:1 in alginate beads. The beads weredissolved after 14 days co-culture. Fluorescence-activated cell sorting was used to collectthe green-MSCs, and then RT-PCR and immunohistochemical staining were applied todetect the expression of collagenⅡand aggrecan in MSCs.Results The RT-PCR showed that the experiment group MSCs expressed collagen-Ⅱand aggrecan after 14 days co-culture with NPCs while the control group MSCs did not.Immunohistochemical staining of the experiment group MSCs for collagen-Ⅱwerepositive and the control group was negative.Conclusion MSC differentiation toward NP-like cells in vitro can be induced by co-culturewith NPCs in a 3-dimensional environment. These data support the use of undifferentiatedMSC for stem cell therapy for intervertebral disc degeneration treatment.