Dentin matrix components extracted with phosphoric acid enhance cell proliferation and mineralization

Satin Salehi, Paul Cooper, Anthony Smith, Jack Ferracane

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

Objective Acids, such as those used in adhesive dentistry, have been shown to solubilize bioactive molecules from dentin. These dentin matrix components (DMC) may promote cell proliferation and differentiation, and ultimately contribute to dentin regeneration. The objective of this study was to evaluate the potential for varying concentrations of DMC extracted from human dentin by phosphoric acid of a range of pHs to stimulate proliferation and mineralization of two different cultured pulp cell populations. Methods DMC were solubilized from powdered human dentin (7 days - 4 °C) by phosphoric acid of pH 1, 3, and 5 and also, EDTA. Extracts were dialyzed for 7 days against distilled water and lyophilized. Undifferentiated mouse dental pulp cells (OD-21) and cells of the odontoblast-like cell line (MDPC-23) were seeded in six-well plates (1 × 105) and cultured for 24 h in DMEM (Dulbecco's modified Eagle's medium) containing 10% (v/v) FBS (fetal bovine serum). The cells were washed with serum-free medium and then treated with different concentrations of DMC (0.01, 0.1, 1.0 and 10.0 μg/ml) daily in serum free medium for 7 days. After 3, 5 (MDPC-23 only), and 7 days of treatment, cell proliferation was measured using 10 vol% Alamar blue solution, which was added to each well for 1 h. Cell numbers were first measured by cell counting (Trypan blue; n = 5) and Alamar blue fluorescence to validate the assay, which was then used for the subsequent assessments of proliferation. Mineralization was assessed by Alizarin Red S assay after 12 days exposure to DMC (n = 5). Controls were media-only (DMEM) and dexamethasone (DEX; positive control). Results were analysed by ANOVA/Tukey's (p ≤ 0.05). Results There was a linear correlation between cell counts and Alamar blue fluorescence (R2 > 0.96 for both cell types), verifying the validity of the Alamar blue assay for these cell types. In general, there was a dose-dependent trend for enhanced cell proliferation with higher concentration of DMC for both cell lines, especially at 10.0 μg/ml. DEX exposure resulted in significantly higher mineralization, but did not affect cell proliferation. DMC exposure demonstrated significantly greater mineralization than media-only control for 10 μg/ml for all extracts, and at lower concentrations for EDTA and pH 5 extracts. Significance Human dentin matrix components solubilized by acids at pH levels found in commercial dentin adhesives enhanced cell proliferation and mineralization of mouse and rat undifferentiated dental pulp cells when presented in adequate concentration.

Original languageEnglish (US)
Pages (from-to)334-342
Number of pages9
JournalDental Materials
Volume32
Issue number3
DOIs
StatePublished - Mar 1 2016

Fingerprint

Cell proliferation
Phosphoric acid
Dentin
Cell Proliferation
Pulp
Assays
Serum-Free Culture Media
Ethylenediaminetetraacetic acid
Cells
Edetic Acid
Adhesives
Fluorescence
Alizarin
Dentistry
Acids
Trypan Blue
Analysis of variance (ANOVA)
Dental Pulp
Eagles
Dexamethasone

Keywords

  • Cell mineralization
  • Cell proliferation
  • Dentin matrix components (DMC)
  • MDPC-23
  • OD-21 cells
  • Odontoblast cells
  • Phosphoric acid
  • Undifferentiated mouse dental pulp cells

ASJC Scopus subject areas

  • Dentistry(all)
  • Materials Science(all)
  • Mechanics of Materials

Cite this

Dentin matrix components extracted with phosphoric acid enhance cell proliferation and mineralization. / Salehi, Satin; Cooper, Paul; Smith, Anthony; Ferracane, Jack.

In: Dental Materials, Vol. 32, No. 3, 01.03.2016, p. 334-342.

Research output: Contribution to journalArticle

Salehi, Satin ; Cooper, Paul ; Smith, Anthony ; Ferracane, Jack. / Dentin matrix components extracted with phosphoric acid enhance cell proliferation and mineralization. In: Dental Materials. 2016 ; Vol. 32, No. 3. pp. 334-342.
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abstract = "Objective Acids, such as those used in adhesive dentistry, have been shown to solubilize bioactive molecules from dentin. These dentin matrix components (DMC) may promote cell proliferation and differentiation, and ultimately contribute to dentin regeneration. The objective of this study was to evaluate the potential for varying concentrations of DMC extracted from human dentin by phosphoric acid of a range of pHs to stimulate proliferation and mineralization of two different cultured pulp cell populations. Methods DMC were solubilized from powdered human dentin (7 days - 4 °C) by phosphoric acid of pH 1, 3, and 5 and also, EDTA. Extracts were dialyzed for 7 days against distilled water and lyophilized. Undifferentiated mouse dental pulp cells (OD-21) and cells of the odontoblast-like cell line (MDPC-23) were seeded in six-well plates (1 × 105) and cultured for 24 h in DMEM (Dulbecco's modified Eagle's medium) containing 10{\%} (v/v) FBS (fetal bovine serum). The cells were washed with serum-free medium and then treated with different concentrations of DMC (0.01, 0.1, 1.0 and 10.0 μg/ml) daily in serum free medium for 7 days. After 3, 5 (MDPC-23 only), and 7 days of treatment, cell proliferation was measured using 10 vol{\%} Alamar blue solution, which was added to each well for 1 h. Cell numbers were first measured by cell counting (Trypan blue; n = 5) and Alamar blue fluorescence to validate the assay, which was then used for the subsequent assessments of proliferation. Mineralization was assessed by Alizarin Red S assay after 12 days exposure to DMC (n = 5). Controls were media-only (DMEM) and dexamethasone (DEX; positive control). Results were analysed by ANOVA/Tukey's (p ≤ 0.05). Results There was a linear correlation between cell counts and Alamar blue fluorescence (R2 > 0.96 for both cell types), verifying the validity of the Alamar blue assay for these cell types. In general, there was a dose-dependent trend for enhanced cell proliferation with higher concentration of DMC for both cell lines, especially at 10.0 μg/ml. DEX exposure resulted in significantly higher mineralization, but did not affect cell proliferation. DMC exposure demonstrated significantly greater mineralization than media-only control for 10 μg/ml for all extracts, and at lower concentrations for EDTA and pH 5 extracts. Significance Human dentin matrix components solubilized by acids at pH levels found in commercial dentin adhesives enhanced cell proliferation and mineralization of mouse and rat undifferentiated dental pulp cells when presented in adequate concentration.",
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AU - Salehi, Satin

AU - Cooper, Paul

AU - Smith, Anthony

AU - Ferracane, Jack

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N2 - Objective Acids, such as those used in adhesive dentistry, have been shown to solubilize bioactive molecules from dentin. These dentin matrix components (DMC) may promote cell proliferation and differentiation, and ultimately contribute to dentin regeneration. The objective of this study was to evaluate the potential for varying concentrations of DMC extracted from human dentin by phosphoric acid of a range of pHs to stimulate proliferation and mineralization of two different cultured pulp cell populations. Methods DMC were solubilized from powdered human dentin (7 days - 4 °C) by phosphoric acid of pH 1, 3, and 5 and also, EDTA. Extracts were dialyzed for 7 days against distilled water and lyophilized. Undifferentiated mouse dental pulp cells (OD-21) and cells of the odontoblast-like cell line (MDPC-23) were seeded in six-well plates (1 × 105) and cultured for 24 h in DMEM (Dulbecco's modified Eagle's medium) containing 10% (v/v) FBS (fetal bovine serum). The cells were washed with serum-free medium and then treated with different concentrations of DMC (0.01, 0.1, 1.0 and 10.0 μg/ml) daily in serum free medium for 7 days. After 3, 5 (MDPC-23 only), and 7 days of treatment, cell proliferation was measured using 10 vol% Alamar blue solution, which was added to each well for 1 h. Cell numbers were first measured by cell counting (Trypan blue; n = 5) and Alamar blue fluorescence to validate the assay, which was then used for the subsequent assessments of proliferation. Mineralization was assessed by Alizarin Red S assay after 12 days exposure to DMC (n = 5). Controls were media-only (DMEM) and dexamethasone (DEX; positive control). Results were analysed by ANOVA/Tukey's (p ≤ 0.05). Results There was a linear correlation between cell counts and Alamar blue fluorescence (R2 > 0.96 for both cell types), verifying the validity of the Alamar blue assay for these cell types. In general, there was a dose-dependent trend for enhanced cell proliferation with higher concentration of DMC for both cell lines, especially at 10.0 μg/ml. DEX exposure resulted in significantly higher mineralization, but did not affect cell proliferation. DMC exposure demonstrated significantly greater mineralization than media-only control for 10 μg/ml for all extracts, and at lower concentrations for EDTA and pH 5 extracts. Significance Human dentin matrix components solubilized by acids at pH levels found in commercial dentin adhesives enhanced cell proliferation and mineralization of mouse and rat undifferentiated dental pulp cells when presented in adequate concentration.

AB - Objective Acids, such as those used in adhesive dentistry, have been shown to solubilize bioactive molecules from dentin. These dentin matrix components (DMC) may promote cell proliferation and differentiation, and ultimately contribute to dentin regeneration. The objective of this study was to evaluate the potential for varying concentrations of DMC extracted from human dentin by phosphoric acid of a range of pHs to stimulate proliferation and mineralization of two different cultured pulp cell populations. Methods DMC were solubilized from powdered human dentin (7 days - 4 °C) by phosphoric acid of pH 1, 3, and 5 and also, EDTA. Extracts were dialyzed for 7 days against distilled water and lyophilized. Undifferentiated mouse dental pulp cells (OD-21) and cells of the odontoblast-like cell line (MDPC-23) were seeded in six-well plates (1 × 105) and cultured for 24 h in DMEM (Dulbecco's modified Eagle's medium) containing 10% (v/v) FBS (fetal bovine serum). The cells were washed with serum-free medium and then treated with different concentrations of DMC (0.01, 0.1, 1.0 and 10.0 μg/ml) daily in serum free medium for 7 days. After 3, 5 (MDPC-23 only), and 7 days of treatment, cell proliferation was measured using 10 vol% Alamar blue solution, which was added to each well for 1 h. Cell numbers were first measured by cell counting (Trypan blue; n = 5) and Alamar blue fluorescence to validate the assay, which was then used for the subsequent assessments of proliferation. Mineralization was assessed by Alizarin Red S assay after 12 days exposure to DMC (n = 5). Controls were media-only (DMEM) and dexamethasone (DEX; positive control). Results were analysed by ANOVA/Tukey's (p ≤ 0.05). Results There was a linear correlation between cell counts and Alamar blue fluorescence (R2 > 0.96 for both cell types), verifying the validity of the Alamar blue assay for these cell types. In general, there was a dose-dependent trend for enhanced cell proliferation with higher concentration of DMC for both cell lines, especially at 10.0 μg/ml. DEX exposure resulted in significantly higher mineralization, but did not affect cell proliferation. DMC exposure demonstrated significantly greater mineralization than media-only control for 10 μg/ml for all extracts, and at lower concentrations for EDTA and pH 5 extracts. Significance Human dentin matrix components solubilized by acids at pH levels found in commercial dentin adhesives enhanced cell proliferation and mineralization of mouse and rat undifferentiated dental pulp cells when presented in adequate concentration.

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KW - Cell proliferation

KW - Dentin matrix components (DMC)

KW - MDPC-23

KW - OD-21 cells

KW - Odontoblast cells

KW - Phosphoric acid

KW - Undifferentiated mouse dental pulp cells

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