A hydrogel scaffold that maintains viability and supports differentiation of dental pulp stem cells

Abstract

Objectives

The clinical translation of stem cell-based Regenerative Endodontics demands further development of suitable injectable scaffolds. Puramatrix™ is a defined, self-assembling peptide hydrogel which instantaneously polymerizes under normal physiological conditions. Here, we assessed the compatibility of Puramatrix™ with dental pulp stem cell (DPSC) growth and differentiation.

Methods

DPSC cells were grown in 0.05–0.25% Puramatrix™. Cell viability was measured colorimetrically using the WST-1 assay. Cell morphology was observed in 3D modeling using confocal microscopy. In addition, we used the human tooth slice model with Puramatrix™ to verify DPSC differentiation into odontoblast-like cells, as measured by expression of DSPP and DMP-1.

Results

DPSC survived and proliferated in Puramatrix™ for at least three weeks in culture. Confocal microscopy revealed that cells seeded in Puramatrix™ presented morphological features of healthy cells, and some cells exhibited cytoplasmic elongations. Notably, after 21 days in tooth slices containing Puramatrix™, DPSC cells expressed DMP-1 and DSPP, putative markers of odontoblastic differentiation.

Significance

Collectively, these data suggest that self-assembling peptide hydrogels might be useful injectable scaffolds for stem cell-based Regenerative Endodontics.

Introduction

The focus on conservative strategies for treatment of diseased dental pulps has led Endodontics to advance in the fields of stem cell biology, genetics, and tissue engineering. The new field of Regenerative Endodontics has emerged around the premise that it might be possible to generate a new dental pulp to treat necrotic teeth . The discovery of dental pulp stem cells (DPSC) capable of differentiating into many cell types has provided a significant boost to this field. Dental pulp stem cells contain specific sub-populations of specific progenitor cells making them ideally suitable to the engineering of dental tissues . Of specific interest for Regenerative Endodontics, these cells can differentiate into both functional, dentin-making odontoblasts and vascular endothelial cells . These features are critically important, since adequate vascularization is vital for the regeneration of the dentin–pulp complex.

Despite the excitement around the clinical application of dental pulp stem cells, there are still several challenges that have to be overcome before regenerative approaches can be used routinely in Endodontics. An area of considerable interest now is the interaction between dental pulp stem cells and three-dimensional (3D) scaffolds. Scaffolds provide cell adhesion and enables cell proliferation, mimicking the microenvironment observed in natural tissues and organs . In the context of dental pulp tissue engineering, scaffolds should have a relatively fast setting time, and provide adequate root canal modeling and adaptation . Notably, at the time that this manuscript was prepared there were no commercially available scaffolds developed for dental pulp tissue engineering . Puramatrix™ is a self-assembling peptide hydrogel that has been tested in differentiated primary cells and stem cells . It supports the regeneration of functional bone in murine calvaria . The hydrogel comprises a 16-mer peptide in aqueous solution, which instantly polymerizes forming a biodegradable scaffold when introduced to physiological salt conditions. This capacity renders it ideal for clinical situations requiring both biocompatibility and rapid matrix formation. Here, we hypothesized that DPSC cultured in Puramatrix™ will survive, proliferate and differentiate into odontoblasts.

Materials and methods

Dental pulp stem cells (DPSC) were kindly provided by Dr. Songtao Shi (University of Southern California) and cultured as previously described . Briefly, cells from 4th to 8th passage were grown in α-MEM medium (Invitrogen, Grand Island, NY, USA), supplemented by 20% FBS and incubated at 37 °C in 5% CO 2 .

Cell growth in Puramatrix™

For all Puramatrix™ (BD, Franklin Lakes, NJ, USA) preparations, cells were washed in 10% (isotonic) sucrose solution to remove salts. Compatibility of Puramatrix™ with DPSC growth was assessed by proliferation assay. Cells were suspended in 0.2% Puramatrix™ and added to the wells of a 96-well plate then gelation was induced by careful addition of an equal volume of complete culture medium to give final densities as indicated in the text. The 0.2% concentration was chosen based on injection assays performed with syringes to determine suitability for endodontic use. After gelation, assessed by microscopic examination, complete culture medium was layered onto the gel (100 μl) and the cells were finally incubated at 37 °C, 5% CO 2 for indicated times. Cell growth curves were performed for up to 72 hours, analyzing different cell densities from 100,000 to 800,000 cells/ml of gel. In studies investigating the effects of gel concentration on cell growth, the cells (200,000 cells/ml) were suspended in varying dilutions (0.05–0.25%) of Puramatrix in sucrose, and left to grow for 72 hours. After suspension samples were treated as described above.

At indicated times relative cell density was assessed by addition of WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) reagent to a 1:10 final concentration. WST-1 (Roche, Indianapolis, IN, USA) is a soluble tetrazolium salt converted to a deep red colored product by mitochondrial activity. Samples were incubated for 3 hours at 37 °C in 5% CO 2 to allow reagent color development and absorbance was measured using microplate reader at 420 nm (Tecan, Austria). Here, and throughout this manuscript, experiments were performed in triplicate wells per concentration and time point. Three independent experiments were performed to verify reproducibility of the data. Data were analyzed by one-way ANOVA followed by recommended post hoc tests using the SigmaStat 2.0 software (SPSS, Chicago, IL, USA). Significance was determined at a p < 0.05.

3D modeling

Cells were suspended in 0.2% Puramatrix™ and cell bearing beads were formed and carefully dropping the cell suspension, using a manual pipette, into complete medium where the cell suspension instantaneously polymerized. Hydrogel beads were immobilized within a coverslip base 30 mm culture dish by setting in collagen gel (Invitrogen). Samples were fixed using paraformaldehyde (10%) then probed for actin using Alexafluor 488 phalloidin (Invitrogen) as indicated by the manufacturer. To prevent drying and to stain nuclei, Prolong Gold plus DAPI (Invitrogen) was diluted 1:3 in PBS then layered over the gel beads.

Cell differentiation using Puramatrix™

Previous studies of solid polymer scaffolds have shown that differentiation of DPSC may be stimulated by the presence of a tooth slice . Thus in the present study, cells were seeded in the tooth slice model at 1 × 10 5 cells/ml, in Puramatrix™ at 0.2%. After 21 days of culture, changing the media every other day, the matrix was removed from the tooth slices for RNA extraction by Trizol (Sigma/Aldrich), as indicated by manufacturer. cDNA from the samples were used in a reverse transcriptase polymerase chain reaction (SuperScript II Platinum ® , Invitrogen). The human-specific primers were designed according to published cDNA sequences of GenBank, as follows: DSPP sense 5′-gaccccttcattgacctcaact-3′, antisense 5′-tgccatttgctgtgatgttt-3′; DMP-1 sense 5′-caggagcacaggaaaaggag-3′, antisense 5′-ctggtggtatcttgggcact-3′; GAPDH sense 5′-gaccccttcattgacctcaact-3′, antisense 5′-caccaccttcttgatgtcatc-3′. The following PCR protocol was used: denaturation, 94 °C for 45 seconds; annealing, 57 °C for 45 seconds; and extension, 72 °C for 60 seconds, for 35 cycles, then 72 °C for 5 minutes and held at 4 °C. The PCR products were separated by 1.5% agarose gel electrophoresis, stained with ethidium bromide and digital images were taken under ultraviolet illumination.

Materials and methods

Dental pulp stem cells (DPSC) were kindly provided by Dr. Songtao Shi (University of Southern California) and cultured as previously described . Briefly, cells from 4th to 8th passage were grown in α-MEM medium (Invitrogen, Grand Island, NY, USA), supplemented by 20% FBS and incubated at 37 °C in 5% CO 2 .

Cell growth in Puramatrix™

For all Puramatrix™ (BD, Franklin Lakes, NJ, USA) preparations, cells were washed in 10% (isotonic) sucrose solution to remove salts. Compatibility of Puramatrix™ with DPSC growth was assessed by proliferation assay. Cells were suspended in 0.2% Puramatrix™ and added to the wells of a 96-well plate then gelation was induced by careful addition of an equal volume of complete culture medium to give final densities as indicated in the text. The 0.2% concentration was chosen based on injection assays performed with syringes to determine suitability for endodontic use. After gelation, assessed by microscopic examination, complete culture medium was layered onto the gel (100 μl) and the cells were finally incubated at 37 °C, 5% CO 2 for indicated times. Cell growth curves were performed for up to 72 hours, analyzing different cell densities from 100,000 to 800,000 cells/ml of gel. In studies investigating the effects of gel concentration on cell growth, the cells (200,000 cells/ml) were suspended in varying dilutions (0.05–0.25%) of Puramatrix in sucrose, and left to grow for 72 hours. After suspension samples were treated as described above.

At indicated times relative cell density was assessed by addition of WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) reagent to a 1:10 final concentration. WST-1 (Roche, Indianapolis, IN, USA) is a soluble tetrazolium salt converted to a deep red colored product by mitochondrial activity. Samples were incubated for 3 hours at 37 °C in 5% CO 2 to allow reagent color development and absorbance was measured using microplate reader at 420 nm (Tecan, Austria). Here, and throughout this manuscript, experiments were performed in triplicate wells per concentration and time point. Three independent experiments were performed to verify reproducibility of the data. Data were analyzed by one-way ANOVA followed by recommended post hoc tests using the SigmaStat 2.0 software (SPSS, Chicago, IL, USA). Significance was determined at a p < 0.05.

3D modeling

Cells were suspended in 0.2% Puramatrix™ and cell bearing beads were formed and carefully dropping the cell suspension, using a manual pipette, into complete medium where the cell suspension instantaneously polymerized. Hydrogel beads were immobilized within a coverslip base 30 mm culture dish by setting in collagen gel (Invitrogen). Samples were fixed using paraformaldehyde (10%) then probed for actin using Alexafluor 488 phalloidin (Invitrogen) as indicated by the manufacturer. To prevent drying and to stain nuclei, Prolong Gold plus DAPI (Invitrogen) was diluted 1:3 in PBS then layered over the gel beads.

Cell differentiation using Puramatrix™

Previous studies of solid polymer scaffolds have shown that differentiation of DPSC may be stimulated by the presence of a tooth slice . Thus in the present study, cells were seeded in the tooth slice model at 1 × 10 5 cells/ml, in Puramatrix™ at 0.2%. After 21 days of culture, changing the media every other day, the matrix was removed from the tooth slices for RNA extraction by Trizol (Sigma/Aldrich), as indicated by manufacturer. cDNA from the samples were used in a reverse transcriptase polymerase chain reaction (SuperScript II Platinum ® , Invitrogen). The human-specific primers were designed according to published cDNA sequences of GenBank, as follows: DSPP sense 5′-gaccccttcattgacctcaact-3′, antisense 5′-tgccatttgctgtgatgttt-3′; DMP-1 sense 5′-caggagcacaggaaaaggag-3′, antisense 5′-ctggtggtatcttgggcact-3′; GAPDH sense 5′-gaccccttcattgacctcaact-3′, antisense 5′-caccaccttcttgatgtcatc-3′. The following PCR protocol was used: denaturation, 94 °C for 45 seconds; annealing, 57 °C for 45 seconds; and extension, 72 °C for 60 seconds, for 35 cycles, then 72 °C for 5 minutes and held at 4 °C. The PCR products were separated by 1.5% agarose gel electrophoresis, stained with ethidium bromide and digital images were taken under ultraviolet illumination.

Only gold members can continue reading. Log In or Register to continue

Nov 28, 2017 | Posted by in Dental Materials | Comments Off on A hydrogel scaffold that maintains viability and supports differentiation of dental pulp stem cells
Premium Wordpress Themes by UFO Themes