In the present study, a scaffold-free tissue construct was developed as an approach for the regeneration of tissue defects, which produced good outcomes. We fabricated a scaffold-free tissue construct from human dental pulp stem cells (hDPSCs construct), and examined the characteristics of the construct. For its fabrication, basal sheets prepared by 4-week hDPSCs culturing were subjected to 1-week three-dimensional culture, with or without osteogenic induction, whereas hDPSC sheets (control) were fabricated by 1-week culturing of basal sheets on monolayer culture. The hDPSC constructs formed a spherical structure and calcified matrix that are absent in the control. The expression levels for bone-related genes in the hDPSC constructs were significantly upregulated compared with those in the control. Moreover, the hDPSC constructs with osteogenic induction had a higher degree of calcified matrix formation, and higher expression levels for bone-related genes, than those for the hDPSC constructs without osteogenic induction. These results suggest that the hDPSC constructs with osteogenic induction are composed of cells and extracellular and calcified matrices, and that they can be a possible scaffold-free material for bone regeneration.

Dental pulp stem cells (DPSCs) are an attractive cell source for use in cell-based therapy, regenerative medicine, and tissue engineering because DPSCs have a high cell proliferation ability and multidifferentiation capacity. However, several problems are associated with the collection and preservation of DPSCs for use in future cell-based therapy. In particular, the isolation of DPSCs for cryopreservation is time consuming and expensive. In this study, we developed a novel cryopreservation method (NCM) for dental pulp tissues to isolate suitable DPSCs after thawing cryopreserved tissue. Using the NCM, dental pulp tissues were cultured on adhesion culture dishes for 5 days and then cryopreserved. After thawing, the cryopreserved dental pulp tissue fragments exhibited cell migration. We evaluated each property of DPSCs isolated using the NCM (DPSCs-NCM) and the explant method alone without cryopreservation (DPSCs-C). DPSCs-NCM had the same proliferation capacity as DPSCs-C. Flow cytometry (FACS) analysis indicated that both DPSCs-NCM and DPSCs-C were positive for mesenchymal stem cell markers at the same level but negative for hematopoietic cell markers. Moreover, both DPSCs-NCM and DPSCs-C could differentiate into osteogenic, chondrogenic, and adipogenic cells during culture in each induction medium. These results suggest that DPSCs-NCM may be mesenchymal stem cells. Therefore, our novel method might facilitate the less expensive cryopreservation of DPSCs, thereby providing suitable DPSCs for use in patients in future cell-based therapies.

The purpose of this study is to evaluate the usefulness of 405 nm monochromatic laser irradiation as an alternative management for prevention and/or treatment of endodontic infections. A monochromatic laser-emitting device equipped with a 405-nm laser diode was developed. Using this device, the effect of 405 nm laser irradiation on the growth of Porphyromonas gingivalis, Prevotella intermedia, Enterococcus faecalis, and Candida albicans, which are microorganisms associated with persistent endodontic infections, was evaluated by viable colony counting. As a result, the irradiation with a 405 nm laser had a significant bactericidal/fungicidal effect on P. gingivalis, P. intermedia, and C. albicans, whereas the growth of E. faecalis was not affected by the irradiation. The inhibition rate in P. gingivalis and P. intermedia was ∼60% and ∼80%, respectively, following irradiation at 0.2 W for 300 sec. The inhibition rate in C. albicans was ∼90% following irradiation at 0.2 W for 1200 sec. These results indicate that 405 nm monochromatic laser irradiation exerts a bactericidal/fungicidal effect on these microorganisms. The present study clearly demonstrates that 405 nm laser irradiation is a promising alternative management strategy for prevention and/or treatment of endodontic infections.

This study aimed to investigate the cleansing effects of grapefruit seed extract (GSE) on biofilms of Candida albicans ( C . albicans ) formed on denture-base resin and the influence of GSE on the mechanical and surface characteristics of the resin. GSE solution diluted with distilled water to 0.1% (0.1% GSE) and 1% (1% GSE) and solutions with Polident® denture cleansing tablet dissolved in distilled water (Polident) or in 0.1% GSE solution (0.1% G+P) were prepared as cleansing solutions. Discs of acrylic resin were prepared, and the biofilm of C . albicans was formed on the discs. The discs with the biofilm were treated with each solution for 5 min at 25°C. After the treatment, the biofilm on the discs was analyzed using a colony forming unit (CFU) assay, fluorescence microscopy, and scanning electron microscopy (SEM). In order to assess the persistent cleansing effect, the discs treated with each solution for 5 min were aerobically incubated in Yeast Nitrogen Base medium for another 24 h. After incubation, the persistent effect was assessed by CFU assay. Some specimens of acrylic resin were immersed in each solution for 7 days, and changes in surface roughness (Ra), Vickers hardness (VH), flexural strength (FS), and flexural modulus (FM) were evaluated. As a result, the treatment with 1% GSE for 5 min almost completely eliminated the biofilm formed on the resin; whereas, the treatment with 0.1% GSE, Polident, and 0.1% G+P for 5 min showed a statistically significant inhibitory effect on biofilms. In addition, 0.1% GSE and 0.1% G+P exerted a persistent inhibitory effect on biofilms. Fluorescence microscopy indicated that Polident mainly induced the death of yeast, while the cleansing solutions containing at least 0.1% GSE induced the death of hyphae as well as yeast. SEM also revealed that Polident caused wrinkles, shrinkage, and some deep craters predominantly on the cell surfaces of yeast, while the solutions containing at least 0.1% GSE induced wrinkles, shrinkage, and some damage on cell surfaces of not only yeasts but also hyphae. No significant changes in Ra, VH, FS, or FM were observed after immersion in any of the solutions. Taken together, GSE solution is capable of cleansing C . albicans biofilms on denture-base resin and has a persistent inhibitory effect on biofilm development, without any deteriorations of resin surface.

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