Technical Reference #3229

3229.

Differentiating Dental Pulp Cells via RGD-Dendrimer Conjugates J.K. Kim; R. Shukla; L. Casagrande; C. Sedgley; J.E. Nör; J.R. Baker; Jr.; and E.E. Hill, University of Michigan School of Dentistry, Journal of Dental Researc, 89(3229), (2010)
Link To Paper

Abstract:
Traumatic dental injuries are often irreversible underscoring the need for therapies that protect dental pulp cells and enhance their regeneration. We hypothesized that generation 5 poly amido amine (PAMAM) dendrimers (G5) functionalized with fluorescein isothiocyanate (FL) and αVβ3- specific cyclic arginine-glycine-aspartic acid (RGD) peptides will bind to dental pulp cells (DPCs) and modulate their differentiation. Dental pulp cells and mouse odontoblast-like cells (MDPC-23) (±) treated with G5-FL-RGD were analyzed via Western blot RT-PCR and quantitative PCR. Transcription of dental differentiation markers was as follows: Dentin matrix protein (DMP-1) dentin sialoprotein (DSPP) and matrix extracellular phosphoglycoprotein (MEPE) as well as vascular endothelial growth factor (VEGF) all increased via the JNK pathway. Long-term G5-RGD treatment of dental pulp cells resulted in enhanced mineralization as examined via Von Kossa assay suggesting that PAMAM dendrimers conjugated to cyclic RGD peptides can increase the odontogenic potential of these cells.

Keywords:
PAMAM; nanotechnology; integrin; odontoblasts.

Materials & Methods:
Dendrimer Synthesis A commercially available generation 5 (G5) PAMAM dendrimer was partially acetylated reacted with 5 molar equivalents of FITC dissolved in dimethyl sulfoxide (DMSO) stirred ON under N2 and purified via size exclusion filtration with Sephadex beads (G-25) in PBS. The fluorescent fraction was collected with informed consent under approved guidelines set by the National Institutes of Health Office of Human Subjects Research in co-operation with the Internal Review Board (Health Sciences) of the University of Michigan (IRB ID number HUM0031704). Mouse odontoblast- like cell line (MDPC-23) and primary human dental pulp cell cultures were grown in high-glucose DMEM (GIBCO Grand Island NY USA) supplemented with 1% P/S (10000 units/ mL penicillin and 10000 μg/mL streptomycin) and 10% FBS. Cells (0.5 x 106) were seeded onto six-well culture dishes (MatTek Corp. Ashland MA USA) and cultured in 3 mL of medium at least 24 hrs before the initiation of experimental conditions. Cells were then treated for 3 hrs with 200 nM G5-FL-RGD. For the inhibition of JNK SP600125 (Calbiochem La Jolla CA USA) was used at 10 μM concentration and cells were incubated with this inhibitor for 1 hr prior to treatment with G5-FL-RGD. For the specific binding experiments free RGD peptide was used at 1.5 μM concentration and cells were incubated with this peptide for 30 min prior to treatment with G5-FL-RGD.

Microscopic Technique
Confocal microscopy

Cell Type(s)
MDPC-23 cells and dental pulp cell cultures