Sox9Gene Transfer Enhanced Regenerative Effect Of Bone Marrow Mesenchymal Stem Cells On The Degenerated Intervertebral Disc

Objective:1. To investigate the potential of T2mapping as a sensitive,noninvasive tool for quantitatively characterizing the process ofintervertebral disc degeneration (IDD) in a rabbit model.2. To observe the effect of Sox9gene transfer on the chondrogenicdifferentiation of monolayer BMSCs.3. To study the effect of Sox9gene transfer on the differentiation ofBMSCs encapsulated in the thermogelling chitosan gels (C/Gp).4. To investigate the efficacy and feasibility of Sox9-transducedBMSCs to repair the degenerated intervertebral disc in a rabbit model.Materials and methods:1. Disc degeneration was induced via a validated annular stab method.After T2-weighted MRI and T2mapping analysis, expression analysis ofaggrecan and type II collagen gene in nucleus pulposus (NP) wasperformed using real-time PCR. The longitudinal changes in NP T2andgene expressions as well as their correlations through the process of IDDwere studied.2. BMSCs were harvested, cultured and transduced by adenovirus-Sox9. Real-time PCR was performed for investigating the geneexpressions of monolayer BMSCs.3. The C/Gp gels was prepared. The MTT and Live/Dead assay wereperformed for the viability and proliferation of the encapsulated BMSCs.Real-time PCR was performed for investigating the gene expressions of BMSCs in three-dimension C/Gp gels.4. In vivo study was performed. The repair effect of Sox9-transducedautologous BMSCs encapsulated in C/Gp on degenerated discs wasevaluated by T2-weighted MRI, T2mapping, macroscopic appearance,histology and immunohistochemistry.Results:1. There was a strong inverse correlation between NP T2andmodified Thompson grades. The decline of L4/5NP T2through24weekswas nonlinear, the most significant decrease was observed in3weekspostoperatively. The tendency was confirmed at gene expression levels. NPT2correlated strongly with aggrecan and type II collagen geneexpressions.2. The MTT assay data showed that proliferation proceeded equally inthe Adv-Sox9and Adv-GFP groups. The expression of Sox9protein washigher in Sox9-transduced BMSCs. Sox9gene transfer significantlyup-regulated the gene expressions of Sox9, type II collagen and aggrecanin BMSCs cultured in monolayer.3. The C/Gp was liquid at room temperature and gel at37°C. TheMTT and live/dead cell staining demonstrated that the C/Gp possessessatisfactory biocompatibility. The Sox9-transduced BMSCs encapsulatedin three-dimension C/Gp showed significantly high gene expression levelsof Sox9, type II collagen and aggrecan.4. Sox9-transduced BMSCs could survive in vivo for12weeks. Ahigher T2-weighted signal intensity and T2value, better preserved NPstructure and greater amount of extracellular matrix were observed in discstreated with Sox9-transduced BMSCs compared with those withouttransduction.Conclusions:1. NP T2correlates well with aggrecan and type II collagen gene expressions. T2mapping could act as a sensitive, noninvasive tool forquantitatively evaluating IDD and characterizing the process of IDD.2. Sox9gene transduction can differentiate monolayer BMSCs into aNP-like phenotype.3. C/Gp gels possess satisfactory biocompatibility. Sox9genetransduction can differentiate BMSCs encapsulated in three-dimensionC/Gp into a NP-like phenotype.4. Sox9gene transfer could significantly enhance the repair effect ofBMSCs on the degenerated discs.