Improvement of Accuracy in Flow Immunosensor System by Introduction of Poly-2-[3-(methacryloylamino)propylammonio]ethyl 3-aminopropyl Phosphate

In order to improve the accuracy of immunosensor systems, poly-2-[3-(methacryloylamino)propylammonio]ethyl 3-aminopropyl phosphate (poly-3MAm3AP), which includes both phosphorylcholine and amino groups, was synthesized and applied to the preparation of antibody-immobilized beads. Acting as an antibody-immobilizing material, poly-3MAm3AP is expected to significantly lower nonspecific adsorption due to the presence of the phosphorylcholine group and recognize large numbers of analytes due to the increase in antibody-immobilizing sites. The elimination of nonspecific adsorption was compared between the formation of a blocking layer on antibody-immobilized beads and the introduction of a material to combine antibody with beads. Determination with specific and nonspecific antibodies was then investigated for the estimation of signal-to-noise ratio. Signal intensities with superior signal-to-noise ratios were obtained when poly-3MAm3AP was introduced. This may be due to the increase in antibody-immobilizing sites and the extended space for antigen-antibody interaction resulting from the electrostatic repulsion of poly-3MAm3AP. Thus, the application of poly-3MAm3AP coatings to immunoassay beads was able to improve the accuracy of flow immunosensor systems

Bone Differentiation Ability of CD146-Positive Stem Cells from Human Exfoliated Deciduous Teeth

Regenerative therapy for tissues by mesenchymal stem cell (MSCs) transplantation has received much attention. The cluster of differentiation (CD)146 marker, a surface-antigen of stem cells, is crucial for angiogenic and osseous differentiation abilities. Bone regeneration is accelerated by the transplantation of CD146-positive deciduous dental pulp-derived mesenchymal stem cells contained in stem cells from human exfoliated deciduous teeth (SHED) into a living donor. However, the role of CD146 in SHED remains unclear. This study aimed to compare the effects of CD146 on cell proliferative and substrate metabolic abilities in a population of SHED. SHED was isolated from deciduous teeth, and flow cytometry was used to analyze the expression of MSCs markers. Cell sorting was performed to recover the CD146-positive cell population (CD146+) and CD146-negative cell population (CD146-). CD146 + SHED without cell sorting and CD146-SHED were examined and compared among three groups. To investigate the effect of CD146 on cell proliferation ability, an analysis of cell proliferation ability was performed using BrdU assay and MTS assay. The bone differentiation ability was evaluated using an alkaline phosphatase (ALP) stain after inducing bone differentiation, and the quality of ALP protein expressed was examined. We also performed Alizarin red staining and evaluated the calcified deposits. The gene expression of ALP, bone morphogenetic protein-2 (BMP-2), and osteocalcin (OCN) was analyzed using a real-time polymerase chain reaction. There was no significant difference in cell proliferation among the three groups. The expression of ALP stain, Alizarin red stain, ALP, BMP-2, and OCN was the highest in the CD146+ group. CD146 + SHED had higher osteogenic differentiation potential compared with SHED and CD146-SHED. CD146 contained in SHED may be a valuable population of cells for bone regeneration therapy

Effect of Er: YAG Laser Irradiation on Bone Metabolism-Related Factors Using Cultured Human Osteoblasts: Examination of the effect of Er: YAG laser irradiation using osteoblasts

Introduction: A variety of laser treatments have been applied in numerous medical fields. In dentistry, laser treatments are used for caries, root canals, and periodontal disease, as well as surgical resection. Numerous reports have recently been published on the use of lasers for bone regeneration. If laser irradiation is found to promote the activation of bone metabolism, it might also be effective for periodontal treatment, peri implantitis, and bone regeneration. Therefore, the present in vitro study aimed to elucidate the mechanisms underlying the effects of erbium-doped yttrium aluminum garnet (Er: YAG) laser irradiation on the bone using osteoblast-like cells.Methods: Osteoblast-like Saos 2 cells (5.0×104 cells) were seeded in 24-well plates. 24 hours after being seeded, the cells were subjected to 0.3 W, 0.6 W, and 2.0 W Er: YAG laser irradiation and then allowed to recover for 48 hours. The expression levels of bone metabolism-related factors alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteoprotegerin (OPG) were then evaluated using reverse transcription–quantitative polymerase chain reaction and western blot analyses.Results: Saos 2 cells subjected to Er: YAG laser irradiation at 0.3 W, 0.6 W, and 2.0 W showed normal growth. When the Er: YAG laser irradiation and control groups were compared after 48 hours, increases were observed in ALP, BSP, and OPG gene and protein expression in the 2.0 W group. Similar results were obtained in the western blot analysis.Conclusion: These findings suggest that the Er: YAG laser irradiation of osteoblast-like cells is effective for activating bone metabolism factors

Effects of Human Deciduous Dental Pulp-Derived Mesenchymal Stem Cell-Derived Conditioned Medium on the Metabolism of HUVECs, Osteoblasts, and BMSCs

In this study, we assessed the effects of human deciduous dental pulp-derived mesenchymal stem cell-derived conditioned medium (SHED-CM) on the properties of various cell types. The effects of vascular endothelial growth factor (VEGF) in SHED-CM on the luminal architecture, proliferative ability, and angiogenic potential of human umbilical vein endothelial cells (HUVECs) were determined. We also investigated the effects of SHED-CM on the proliferation of human-bone-marrow mesenchymal stem cells (hBMSCs) and mouse calvarial osteoblastic cells (MC3T3-E1) as well as the expression of ALP, OCN, and RUNX2. The protein levels of ALP were examined using Western blot analysis. VEGF blockade in SHED-CM suppressed the proliferative ability and angiogenic potential of HUVECs, indicating that VEGF in SHED-CM contributes to angiogenesis. The culturing of hBMSCs and MC3T3-E1 cells with SHED-CM accelerated cell growth and enhanced mRNA expression of bone differentiation markers. The addition of SHED-CM enhanced ALP protein expression in hBMSCs and MT3T3-E1 cells compared with that of the 0% FBS group. Furthermore, SHED-CM promoted the metabolism of HUVECs, MC3T3-E1 cells, and hBMSCs. These findings indicate the potential benefits of SHED-CM in bone tissue regeneration

Combination of Carbonate Hydroxyapatite and Stem Cells from Human Deciduous Teeth Promotes Bone Regeneration by Enhancing BMP-2, VEGF and CD31 Expression in Immunodeficient Mice

The objective of this study was to clarify the efficiency of a combination of stem cells from human deciduous teeth and carbonate apatite in bone regeneration of calvarial defects. Immunodeficient mice (n = 5 for each group/4 groups) with artificial calvarial bone defects (5 mm in diameter) were developed, and stem cells from human deciduous teeth (SHEDs) and carbonate hydroxyapatite (CAP) granules were transplanted with an atelocollagen sponge as a scaffold. A 3D analysis using microcomputed tomography, and 12 weeks after transplantation, histological and immunohistochemical evaluations of markers of bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), and cluster of differentiation (CD) 31 were performed. In the 3D analysis, regenerated bone formation was observed in SHEDs and CAP, with the combination of SHEDs and CAP showing significantly greater bone regeneration than that in the other groups. Histological and immunohistochemical evaluations showed that combining SHEDs and CAP enhanced the expression of BMP-2, VEGF, and CD31, and promoted bone regeneration. This study demonstrates that the combination of SHEDs and CAP transplantation may be a promising tool for bone regeneration in alveolar defects