Restor Dent Endod.  2016 Nov;41(4):296-303. 10.5395/rde.2016.41.4.296.

Effects of proanthocyanidin, a crosslinking agent, on physical and biological properties of collagen hydrogel scaffold

• Choi Y
• Kim HJ
• Min KS

• KMID: 2356006
• DOI: http://doi.org/10.5395/rde.2016.41.4.296

Abstract

OBJECTIVES
The purpose of the present study was to evaluate the effects of proanthocyanidin (PAC), a crosslinking agent, on the physical properties of a collagen hydrogel and the behavior of human periodontal ligament cells (hPDLCs) cultured in the scaffold.
MATERIALS AND METHODS
Viability of hPDLCs treated with PAC was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The physical properties of PAC treated collagen hydrogel scaffold were evaluated by the measurement of setting time, surface roughness, and differential scanning calorimetry (DSC). The behavior of the hPDLCs in the collagen scaffold was evaluated by cell morphology observation and cell numbers counting.
RESULTS
The setting time of the collagen scaffold was shortened in the presence of PAC (p < 0.05). The surface roughness of the PAC-treated collagen was higher compared to the untreated control group (p < 0.05). The thermogram of the crosslinked collagen exhibited a higher endothermic peak compared to the uncrosslinked one. Cells in the PAC-treated collagen were observed to attach in closer proximity to one another with more cytoplasmic extensions compared to cells in the untreated control group. The number of cells cultured in the PAC-treated collagen scaffolds was significantly increased compared to the untreated control (p < 0.05).
CONCLUSIONS
Our results showed that PAC enhanced the physical properties of the collagen scaffold. Furthermore, the proliferation of hPDLCs cultured in the collagen scaffold crosslinked with PAC was facilitated. Conclusively, the application of PAC to the collagen scaffold may be beneficial for engineering-based periodontal ligament regeneration in delayed replantation.

Keyword

Collagen; Crosslinking; Periodontal ligament cell; Proanthocyanidin; Proliferation

MeSH Terms

Calorimetry, Differential Scanning
Cell Count
Collagen*
Cytoplasm
Humans
Hydrogel*
Periodontal Ligament
Regeneration
Replantation
Collagen
Hydrogel

Figure

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  Figure 1 Effects of PAC on cell viability measured by the MTT assay. *Significant difference compared with the control (p < 0.05). MTT assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; OD, optical density; PAC, proanthocyanidin.

• 

  Figure 2 Setting times of uncrosslinked and crosslinked collagen as determined by the tilting method. *Significant difference compared with the control (p < 0.05). CON, control; PAC, proanthocyanidin.

• 

  Figure 3 Effect of PAC on the surface roughness of the collagen scaffolds. AFM images of (a) untreated collagen and (b) PAC-treated collagen. (c) Quantitative evaluation of the surface roughness. *Significant difference compared with control (p < 0.05). CON, control; PAC, proanthocyanidin; AFM, atomic force microscopy.

• 

  Figure 4 Differential scanning calorimetric curves of uncrosslinked and crosslinked collagen scaffolds. PAC, proanthocyanidin; TD, denaturation temperature; ΔH, denaturation enthalpy.

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  Figure 5 SEM images of hPDLCs incubated on (a) uncrosslinked and (b) crosslinked collagen (×1,000) for three days.

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  Figure 6 Proliferation analyses of hPDLCs cultured in the collagen scaffolds. Optical microscope images of hPDLCs cultured in (a) untreated and (b) PAC-treated collagen matrices (×200). Representative fluorescence microscopy images (×400) showing hPDLCs (stained with DAPI) growing in the collagen scaffolds after three days of culture in the (c) absence or (d) presence of PAC. (e) Effects of PAC-induced crosslinking on the proliferation of hPDLCs cultured in collagen. *Significant difference compared with control (p < 0.05). hPDLC, human periodontal ligament cells; OM, optical microscope; CM, confocal laser scanning microscope; COL, collagen without PAC; COL/PAC, collagen treated with PAC, PAC, proanthocyanidin.

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