| Objective：The aim of this study is to provide experimential data for intervertebraldisc degeneration stem cell therapy through comparing of the proliferationand differentiation ability of the rabbit bone marrow mesenchymal stem cells(BMSCs) and adipose-derived stem cells (ADSCs) in vitro and the effects ofdegenerative disc nucleus pulposus repairment with this two stem cellstransplantation in vivo.Methods:1. Nucleus pulposus cells (NPCs) of2-weeks old Japanese rabbit wereisolated and cultured in vitro. NPCs were identified by histology,immunohistochemical and immunofluorescence staining.2. BMSCs and ADSCs of2-weeks old Japanese rabbit were isolated andcultured respectively in vitro. The proliferation ability of two kinds of stemcells was compared through growth curve and the culture doubling time.3. The two kinds of stem cells were induced to chondrocytes-like cellsrespectively with the TGF-β1in vitro. Differentiation abilities were comparedthrough histochemical staining, collagenⅡ protein and gene expression.4. The intervertebral discs degeneration animal model was induced bythe nucleus pulposus aspirated method. NP tissue (5-8mg wet weight) wasaspirated from the IVDs at regions L3-4, L4-5and L5-6, with a21-gauge needle on a10ml syringe.5. Forty-five Japanese rabbits (male or female,5-months) were randomlydivided into5groups:(1) The normal control group (n=9);(2) The NPCstransplantation group (n=9);(3) The BMSCs transplantation group (n=9);(4)The ADSCs transplantation group (n=9);(5) The DMEM group (n=9).Experimental discs in each group were L3-4, L4-5and L5-6discs.6. The samples were harvested at4th,8th and12th week after cellstransplanted. The disc height index (%DHI) and the standard T2weightedindex (%ST2WI) were calculated and statistical analyzed according to theX-ray and MRI examination.7. The discs harvested at the pre-determined time point were stained byHE and Safranin-O staining. Then degeneration scores were calculatedthrough histologic evaluation standard. The repairment for the degenerativedisc was observed by the degeneration scores.8. The collagenⅡand aggrecan expression levels were evaluated throughimmunochemical staining and real-time PCR.9. The relative contents of sulfated glycosaminoglycan (sGAG) innucleus pulposus were measured at each time point. The sGAG content wasmeasured with1,9-dimethylmethylene blue-chloride (DMMB) dye and theDNA content was measured with bisbenzimide Hoechst33258DNAquantitation kit. Then the sGAG/DNA was calculated to assess the nucleuspulposus extracellular matrix relative content.Results：1. The rabbit nucleus pulposus tissue could be successfully cultured invitro, and it is well kept of the cell phenotype and its specific extracellularmatrix collagenⅡ and aggrecan secretion. However, the cells proliferated slowly in vitro and the first cell number doubling time was about96hours.2. The growth curves showed that the first cell number doubling time ofADSCs was about36hours and46hours in BMSCs, which indicated that theproliferation rate ofADSCs is more quickly than that of BMSCs.3. The results of the differentiation to the chondrocyte-like cells showedthat the extracellular matrix secretion and the collagenⅡ expression level ofBMSCs were higher than those of ADSCs, which suggested that thedifferentiation ability of BMSCs is better than ADSCs in vitro.4. The%DHI and%ST2WI in the DMEM group was significantly lowerthan those in the other four groups12weeks post-operation (P<0.05). And thetwo indexes in the NPCs and BMSCs groups were both higher than those inthe ADSCs group (P<0.05). But there were no differences between theBMSCs group and NPCs group.5. The degeneration scores calculated by histologic evaluation standardin the control group was lower than those in the other four groups12weekspost-operation (P<0.05). And the score in the DMEM group was significantlyhigher than that in the NPCs group, BMSCs group or ADSCs group (P<0.05).However, there were no differences among the NPCs group, BMSCs groupand ADSCs group.6. The immunohistochemical and immunofluorescence staining in allgroups were strong positive except the DMEM group. The staining in theDMEM group was also positive at the beginning, however, the positive resultsgradually weakened with time. The real-time PCR showed that the mRNAexpression levels of collagenⅡand aggrecan in the DMEM group wereweaker than those in the other four groups12weeks post-operation (P<0.05).The expression levels in the NPCs group and BMSCs group were stronger than that in the ADSCs group (P<0.05). And there were no differencesbetween the BMSCs group and NPCs group. From these results we canconcluded that the mRNA expression levels of the collagenⅡand aggrecan inthe BMSCs group were stronger than those in the ADSCs group.7. The sGAG/DNA level of the nucleus pulposus in the DMEM groupwas lower than those in the other four groups12weeks post-operation(P<0.05). And the levels in the NPCs group and BMSCs group weresignificantly higher than the ADSCs group (P<0.05). And there were nodifferences between the NPCs group and BMSCs group.Conclusion：1. The ADSCs could proliferate more quickly than the BMSCs in vitro,which could provide a lots of cells for cell transplantation in short time.However, the differentiation ability of BMSCs was better than that ofADSCs.2. The stem cell transplantation studies showed that BMSCs and ADSCscould restrain the intervertebral disc degeneration in vivo, but BMSCs couldbe more effectively than ADSCs. |
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