Notochordal Cells Induce Mesenchymal Stem Cells To Differentiate Toward Nucleus Pulposus Phenotype And Restraining Degeneration Of Punctured Intervertebral Discs By Transplantation Of Rabbit NCs And MSCs

Partâ… Isolation and characterization of notochordal cells(NCs) and mesenchymal stem cells(MSCs)Objective To isolate and co-culture notochordal cells(NC) and mesenchymal stem cells(MSCs), to assess the cell characteristic of notochordal cells and mesenchymal stem cells.Methods Notochordal cell were obtained from immature NP of 4 New Zealand rabbits(4-6 week-old) and purified by discontinuous gradient density centrifugation, MSCs were released from femur bone marrow and purified by discontinuous gradient density centrifugation. Cells culture expanded to passage 3, Investigate expression of CD29, CD34, CD44, CD45 in MSCs by flow cytometry (FCM). Results NCs were round or oval, the diameter between 20-30um, filled with vesicles, the cells aggregated commonly. Observed by electron microscope, there are many different size vesicles, but a few organelles in NCs, glycogen on the surface of membrane. MSCs were spindle shape or triangular, the original cells covered the flask for 7-10 days, grown rapidly by passage. The surface molecules of MSC were detected by FCM, the expression of CD29 and CD45 was positive compare to the negative expression of CD34 and CD44 that provided the evidence for identification of MSC.Conclusions Notochordal cell were obtained from immature NP and purified by discontinuous gradient density centrifugation, MSCs were released from femur bone marrow and purified by discontinuous gradient density centrifugation.Partâ…¡Notochordala cell stimulates proliferation and differention of mesenchymal stem cell toward nucleus pulposus phenotype Objective:To isolate and co-culture notochordal cells(NC) and mesenchymal stem cells(MSCs) from New Zealand rabbit immature nucleus pulposus(NP), to assess the contribution of notochordal cells to proliferation and differention of mesenchymal stem cells.Methods:Notochordal cell were got from immature NP of 4 New Zealand rabbits(4-6 week-old) and purified by discontinuous gradient density centrifugation, MSCs were released from femur bone marrow and purified by discontinuous gradient density centrifugation. MSCs were cultured alone and co-cultured with NC(1:1/1:2/2:1), evaluated cell proliferation by CCK8-kit and expressions of collagenâ…¡and proteoglycan by toluidine blue and immunocytochemistry staining. Assessed the expression of collagenâ…¡and proteoglycan in gene and protein level.Results:NCs and MSCs were isolated and purified. NC were with diameter of 20-30un, had abundant intracytoplasmic vesicles and poor proliferation. MSCs were adherent growth with fusiform, arrayed with whirlpool. By co-cultured, the group of cell ration at 1:1(NC:MSC) increased significantly in proliferation compared with other groups. After co-culture, MSCs from the group co-culture were observed expression of collagenâ…¡and proteoglycan compared with negative expression in the group of MSCs cultured alone. Conclusion:By coculture, NC can stimulate MSCs'proliferation that will be significant at cell ration 1:1, and differention toward nucleus pulposus cell.Part IIIInducing mesenchymal stem cells to differentiate toward nucleus pulposus phenotype with indirect co-cultureObjective To isolate and co-culture notochordal cells(NC) and mesenchymal stem cells(MSCs) from New Zealand rabbit immature nucleus pulposus(NP), to assess the contribution of notochordal cells to differentiation of mesenchymal stem cells.Methods Notochordal cell were got from immature NP of 8 New Zealand rabbits (4-6 week-old) and purified by discontinuous gradient density centrifugation, MSCs were released from femur bone marrow and purified by discontinuous gradient density centrifugation. MSCs were cultured alone and non-contact co-cultured with NC(1:1), evaluated expressions of collagenâ…¡and proteoglycan by toluidine blue and immunocytochemistry staining. Assessed the expression of collagenâ…¡and proteoglycan in gene and protein level. Results NCs and MSCs were isolated and purified. NC were with diameter of 20-30un, had abundant intracytoplasmic vesicles and poor proliferation. MSCs were adherent growth with fusiform, arrayed with whirlpool. After indirect co-cultured, MSCs from the group co-culture were observed expression of collagenâ…¡and proteoglycan compared with negative expression in the group of MSCs cultured alone.Conclusion By indirect coculture, NC can stimulate MSCs'proliferation and differentiation toward nucleus pulposus cell, These findings maybe supply a new choice to cell therapy strategy of intervertebral disc regeneration.Part IVRestraining Degeneration of Punctured Intervertebral Discs by Transplantation of Rabbit notochordal cells and Marrow Stroma CellsObjective:To assess the ability of transplanted notochordal cells (NCs) and marrowstroma cells (MSCs) in restraining the degeneration of punctured intervertebral discs in rabbits.Methods The first passage NCs and third passage MSCs were harvested for transplantation. Twelve New Zealand white rabbits were invited in this experimentation. The L3/4, L4/5, L5/6, and L6/7 discs of the rabbits were stabbed and punctured using a 18G needle. After two weeks,The L3/4 discs were then untreated, L4/5 discs were treated by transplantation NCs and MSCs, L5/6 discs treated by transplantation NCs, L6/7 discs were treated by transplantation MSCs. Magnetic Resonance Images of the lumbar vertebral T2 weighed signals Were collected after 2 weeks by the operations. The DHI and standardized T2WI(ST2wI) were Measured using Image-proplus6.0 and Mergee Film Workstation. Nucleus pulposus were evaluated expressions of collagenâ…¡and proteoglycan by toluidine blue and immunocytochemistry staining. Assessed the expression of collagen II and collagen I in protein level.Results:Animals survived above 2 weeks after been transplanted. The transplantation effectively restrained the degeneration of the punctured discs. The L4/5 discs had higher DHI in 2 weeks by operation than other discs (P<0.05). The L4/5 discs demonstrated significantly positive compared with control by stained with toluidine blue and in Immunocytochemistry. The L4/5 discs were observed expression of collagen II compared with less expression in the L5/6, L6/7 disc.Conclusion:Transplantation of Rabbit NCs and MSCs restrain the degeneration of punctured discs. This cell therapy have shown stronger potential of extracellular matrixsynthesis, and height and water content Recovery of discs.