IADR Abstract Archives
Dentin Regeneration by DMP-1/BMP-2-Plasmid-Modified Nanoparticle-Treated DPSCs on Dentin Scaffolds
Objectives: Investigation of the potential of Calcium Phosphate Nanoparticles (CaP NPs), carrying plasmid DNA encoding Dentin Matrix Protein 1 or Bone Morphogenetic Protein 2 (DMP-1/BMP-2-CaP NPs), combined with human Freeze-Dried Dentin Scaffolds (hFDDSs) to promote odontogenic differentiation by Dental Pulp Stem Cells (DPSCs).
Methods: DPSCs were isolated, characterized and seeded on EDTA-treated hFDDSs. CaP NPs were synthesized and characterized [colloidally, by Dynamic Light Scattering; microscopically (SEM) for particle size; and spectroscopically (UV-vis) for functionalization]. Control CaP NPs conjugated with Cy-5 fluorescent dye or carrying the plasmid encoding green fluorescent protein were tested at a range of concentrations (0.5-8μg Ca/ml) for 24h-uptake and 48h-transfection efficiency, respectively (Confocal Microscopy, Flow Cytometry). The effect of CaP NPs on cell viability was assessed by MTT assay. Two most favorable for viability/transfectability concentrations of each DMP-1 or BMP-2-CaP NPs were selected to evaluate cell morphology on hFDDSs (SEM), viability (Live/Dead Staining), and gene expression of odontogenic markers, including DMP-1, BMP-2, DSPP, RunX2, Osterix, MSX-1 and MSX-2 (real-time PCR).
Results: Increasing concentrations of CaP NPs reduced cell viability, while increased uptake and transfection efficiency by DPSCs in a dose- and time-dependent manner. The uptake efficiency reached 51% of the total cell population at the concentration of 8μg/ml, while the transfection efficiency was 37%. Considering the transfectability/viability balance, the concentrations of 1 and 4μg/ml causing reduction of cell viability that did not exceed 45% for the DMP-1 NPs and 38% for the BMP-2 NPs, 72h post-treatment, were further evaluated in DPSC-seeded hFDDSs. Real-time PCR results showed a NP-specific, time-dependent upregulation of odontogenic markers (BMP-2, DMP-1 and DSPP), with downregulation of the transcription factors (Osterix, RunX2, MSX-1, MSX-2). Studies are in progress to evaluate in vitro biomineralization leading to dentin-like tissue formation.
Conclusions: The combination of DMP-1/BMP-2-CaP NPs and DPSCs on hFDDSs presents a promising approach in dentin tissue engineering.
Methods: DPSCs were isolated, characterized and seeded on EDTA-treated hFDDSs. CaP NPs were synthesized and characterized [colloidally, by Dynamic Light Scattering; microscopically (SEM) for particle size; and spectroscopically (UV-vis) for functionalization]. Control CaP NPs conjugated with Cy-5 fluorescent dye or carrying the plasmid encoding green fluorescent protein were tested at a range of concentrations (0.5-8μg Ca/ml) for 24h-uptake and 48h-transfection efficiency, respectively (Confocal Microscopy, Flow Cytometry). The effect of CaP NPs on cell viability was assessed by MTT assay. Two most favorable for viability/transfectability concentrations of each DMP-1 or BMP-2-CaP NPs were selected to evaluate cell morphology on hFDDSs (SEM), viability (Live/Dead Staining), and gene expression of odontogenic markers, including DMP-1, BMP-2, DSPP, RunX2, Osterix, MSX-1 and MSX-2 (real-time PCR).
Results: Increasing concentrations of CaP NPs reduced cell viability, while increased uptake and transfection efficiency by DPSCs in a dose- and time-dependent manner. The uptake efficiency reached 51% of the total cell population at the concentration of 8μg/ml, while the transfection efficiency was 37%. Considering the transfectability/viability balance, the concentrations of 1 and 4μg/ml causing reduction of cell viability that did not exceed 45% for the DMP-1 NPs and 38% for the BMP-2 NPs, 72h post-treatment, were further evaluated in DPSC-seeded hFDDSs. Real-time PCR results showed a NP-specific, time-dependent upregulation of odontogenic markers (BMP-2, DMP-1 and DSPP), with downregulation of the transcription factors (Osterix, RunX2, MSX-1, MSX-2). Studies are in progress to evaluate in vitro biomineralization leading to dentin-like tissue formation.
Conclusions: The combination of DMP-1/BMP-2-CaP NPs and DPSCs on hFDDSs presents a promising approach in dentin tissue engineering.