Kengo Nakajima

Comparison of the bone regeneration ability between stem cells from human exfoliated deciduous teeth, human dental pulp stem cells and human bone marrow mesenchymal stem cells

Cleft lip and palate is the most common congenital anomaly in the orofacial region. Autogenous iliac bone graft, in general, has been employed for closing the bone defect at the alveolar cleft. However, such iliac bone graft provides patients with substantial surgical and psychological invasions. Consequently, development of a less invasive method has been highly anticipated. Stem cells from human exfoliated deciduous teeth (SHED) are a major candidate for playing a significant role in tissue engineering and regenerative medicine. The aim of this study was to elucidate the nature of bone regeneration by SHED as compared to that of human dental pulp stem cells (hDPSCs) and bone marrow mesenchymal stem cells (hBMSCs). The stems cells derived from pulp tissues and bone marrow were transplanted with a polylactic-coglycolic acid barrier membrane as a scaffold, for use in bone regeneration in an artificial bone defect of 4u202fmm in diameter in the calvaria of immunodeficient mice. Three-dimensional analysis using micro CT and histological evaluation were performed. Degree of bone regeneration with SHED relative to the bone defect was almost equivalent to that with hDPSCs and hBMSCs 12 weeks after transplantation. The ratio of new bone formation relative to the pre-created bone defect was not significantly different among groups with SHED, hDPSCs and hBMSCs. In addition, as a result of histological evaluation, SHED produced the largest osteoid and widely distributed collagen fibers compared to hDPSCs and hBMSCs groups. Thus, SHED transplantation exerted bone regeneration ability sufficient for the repair of bone defect. The present study has demonstrated that SHED is one of the best candidate as a cell source for the reconstruction of alveolar cleft due to the bone regeneration ability with less surgical invasion.

The effect of mesenchymal stem cells on chemotaxis of osteoclast precursor cells

Regeneration of tissue, including bone, using mesenchymal stem cells (MSCs) has been progressing rapidly. Regeneration of bone requires the presence of an appropriate environment and efficient chemotaxis of cells to the target site. Differentiation of MSCs into mesenchymal cells has received considerable attention, but the effect of MSCs on chemotaxis is not well understood. In this study, we investigated the effect of MSCs on chemotaxis of RAW264 cells via C-C motif chemokine ligand 2 (CCL2). Balb/c mouse bone marrow-derived MSCs and RAW264 cells, which are osteoclast precursor cells, were co-cultured without cell contact. The gene expression of CCL2 in MSCs and CC-chemokine receptor 2 (CCR2) in RAW264 cells was determined using quantitative real-time PCR. Analysis of RAW264 cell chemotaxis was performed using the Boyden chamber assay. mRNAs for CCL2 and CCR2 were significantly upregulated upon co-culture in comparison to culture of either cell type alone, and the number of chemotactic RAW264 cells was significantly increased by co-culture. MSCs enhanced the chemotaxis of RAW264 cells, possibly via CCL2-CCR2 interaction, suggesting the potential utility of MSCs for tissue regeneration.

Comparative characterization of stem cells from human exfoliated deciduous teeth, dental pulp, and bone marrow–derived mesenchymal stem cells

OBJECTIVESnMesenchymal stem cells (MSCs) are used clinically in tissue engineering and regenerative medicine. The proliferation and osteogenic differentiation potential of MSCs vary according to factors such as tissue source and cell population heterogeneity. Dental tissue has received attention as an easily accessible source of high-quality stem cells. In this study, we compared the inxa0vitro characteristics of dental pulp stem cells from deciduous teeth (SHED), human dental pulp stem cells (hDPSCs), and human bone marrow mesenchymal stem cells (hBMSCs).nnnMATERIALS AND METHODSnSEHD and hDPSCs were isolated from dental pulp and analyzed in comparison with human bone marrow (hBM)MSCs. Proliferative capacity of cultured cells was analyzed using a bromodeoxyuridine immunoassay and cell counting. Alkaline phosphatase (ALP) levels were monitored to assess osteogenic differentiation. Mineralization was evaluated by alizarin red staining. Levels of bone marker mRNA were examined by real-time PCR analysis.nnnRESULTSnSHED were highly proliferative compared with hDPSCs and hBMSCs. SHED, hDPSCs, and hBMSCs exhibited dark alizarin red staining on day 21 after induction of osteogenic differentiation, and staining of hBMSCs was significantly higher than that of SHED and hDPSCs by spectrophotometry. ALP staining was stronger in hBMSCs compared with SHED and hDPSCs, and ALP activity was significantly higher in hBMSCs compared with SHED or hDPSCs. SHED showed significantly higher expression of the Runx2 and ALP genes compared with hBMSCs, based on real-time PCR analysis. In bFGF, SHED showed significantly higher expression of the basic fibroblast growth factor (bFGF) gene compared with hDPSCs and hBMSCs.nnnCONCLUSIONnSHED exhibited higher proliferative activity and levels of bFGF and BMP-2 gene expression compared with BMMSCs and DPSCs. The ease of harvesting cells and ability to avoid invasive surgical procedures suggest that SHED may be a useful cell source for application in bone regeneration treatments.

Effect of high-frequency near-infrared diode laser irradiation on periodontal tissues during experimental tooth movement in rats: HIGH FREQUENCY LASER ACCELERATES TOOTH MOVEMENT

Tooth movement during orthodontic treatment is associated with bone neoplasticity and bone resorption on the tension and pressure sides. Previous clinical studies have suggested that low‐power laser irradiation can accelerate tooth movement during orthodontic treatment, although the underlying mechanism remains unclear. In this study, we used a high‐frequency near‐infrared diode laser that generates less heat and examined the histologic changes in periodontal tissue during experimental tooth movement with laser irradiation.

Effects of Nd:YAG low-level laser irradiation on cultured human osteoblasts migration and ATP production: in vitro study

Low-level laser therapy has become one of the fastest growing fields of medicine in recent years. Many in vivo and in vitro studies have shown that laser irradiation activates a range of cellular processes in a variety of cell types and can promote tissue repair. However, few in vitro experiments have evaluated the effects of laser irradiation on cells in real time. The purpose of this study was to examine the effects of neodymium-doped yttrium aluminum garnet (Nd:YAG) laser irradiation on the migration of cultured human osteoblasts. A dedicated 96-well plate was used, and confluent cultures of the human osteoblast-like cell line, Saos-2, were injured with a wound maker. The wounded cells were then exposed to the Nd:YAG laser (wavelength of 1064xa0nm) for 60xa0s at 0.3xa0W (10xa0pps, 30xa0mJ). The total energy density was about 10.34xa0J/cm2. Images of the wounds were automatically acquired inside the CO2 incubator by the IncuCyte ZOOM™ software. In addition, after laser irradiation, the production of adenosine triphosphate (ATP) was measured using the CellTiter-Glo™ Luminescent Cell Viability Assay. Migration of cells from the border of the original scratch zone was accelerated by laser irradiation. In addition, compared with the control group, significant enhancement of ATP production was observed in the irradiated group. The present study showed that Nd:YAG laser irradiation (wavelength of 1064xa0nm, 0.3xa0W, 10 pps, 30xa0mJ, 10.34xa0J/cm2, irradiation time 60xa0s) may contribute to the regeneration of bone tissues owing to enhanced osteoblast cell migration.

Effects of high-frequency near-infrared diode laser irradiation on the proliferation and migration of mouse calvarial osteoblasts

Laser irradiation activates a range of cellular processes and can promote tissue repair. Here, we examined the effects of high-frequency near-infrared (NIR) diode laser irradiation on the proliferation and migration of mouse calvarial osteoblastic cells (MC3T3-E1). MC3T3-E1 cells were cultured and exposed to high-frequency (30xa0kHz) 910-nm diode laser irradiation at a dose of 0, 1.42, 2.85, 5.7, or 17.1xa0J/cm2. Cell proliferation was evaluated with BrdU and ATP concentration assays. Cell migration was analyzed by quantitative assessment of wound healing using the Incucyt® ZOOM system. In addition, phosphorylation of mitogen-activated protein kinase (MAPK) family members including p38 mitogen-activated protein kinase (p38), stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK), and extracellular signal-regulated protein kinase (ERK)1/2) after laser irradiation was examined with western blotting. Compared to the control, cell proliferation was significantly increased by laser irradiation at a dose of 2.85, 5.7, or 17.1xa0J/cm2. Laser irradiation at a dose of 2.85xa0J/cm2 induced MC3T3-E1 cells to migrate more rapidly than non-irradiated control cells. Irradiation with the high-frequency 910-nm diode laser at a dose of 2.85xa0J/cm2 induced phosphorylation of MAPK/ERK1/2 15 and 30xa0min later. However, phosphorylation of p38 MAPK and SAPK/JNK was not changed by NIR diode laser irradiation at a dose of 2.85xa0J/cm2. Irradiation with a high-frequency NIR diode laser increased cell division and migration of MT3T3-E1 cells, possibly via MAPK/ERK signaling. These observations may be important for enhancing proliferation and migration of osteoblasts to improve regeneration of bone tissues.

Dynamic imaging of the effect of mesenchymal stem cells on osteoclast precursor cell chemotaxis for bone defects in the mouse skull

Background/purpose Mesenchymal stem cells (MSCs) transplantation has previously been used in the field of regenerative medicine. Although bone regeneration is known to occur through the interaction between osteoblasts and osteoclasts, the effect of MSCs on osteoclasts is unknown. Therefore, the purpose of this study was to investigate the effect of MSCs on the chemotaxis of osteoclast precursor cells (RAW264 macrophage cells). Materials and methods Bone defects were created in mice skulls, and MSCs and a scaffold of carbonated hydroxyapatite were transplanted into the bone defects. RAW264 cells were then transplanted into the mouse tail vein, and their dynamics were observed by an in vivo imaging system. Results The fluorescent intensity of the MSCs transplant group at the bone defect region was significantly higher on days 3, 5, and 7 compared with the MSCs non-transplant group. Conclusion Increased RAW264 chemotaxis to the bone defect region occurred following the simultaneous implantation of MSCs in the skull defect.

A comparison of proliferation of human mesenchymal stem cells derived from adipose tissue treated with human full-length amelogenin and C-terminal amelogenin peptide

Amelogenins are enamel matrix proteins that play crucial roles in enamel formation. Previous studies have indicated that amelogenin and amelogenin C-terminal peptides have cell-signaling functions. Recently, adipocyte-derived mesenchymal stem cells (ADSCs) have received attention as a potential source of stem cells for use in regeneration therapy. In this study, we examined the effects of human full-length amelogenin (rh174) and amelogenin C-terminal peptide (amgCP) on the proliferation of ADSCs. ADSCs were cultured in the presence of amgCP or rh174. Cell proliferation was analyzed using BrdU immunoassay and MTS assay. Cell migration was evaluated by ELISA. The MAPK-ERK pathway was examined by phospho-p44/42 MAPK (Thr202/Tyr204) sandwich ELISA and western blotting. A specific MAPK inhibitor, U0126, was used to block ERK activity. ADSC proliferation and migration were significantly (P < 0.05) increased in the presence of rh174 or amgCP compared to non-treated control cells. The increased proliferation of ADSCs induced by rh174 or amgCP was significantly (P < 0.05) inhibited in the presence of 2 µg/ml U0126. The pERK/tERK ratio was significantly (P < 0.05) increased upon treatment with rh174 or amgCP compared to non-treated ADSCs, while this increase was significantly (P < 0.05) suppressed by the addition of U0126. Similar results were found by western blot analysis. In conclusion, amgCP and rh174 increase ADSC proliferation via the MAPK-ERK signaling pathway, and ADSCs may be useful for tissue regeneration in the orofacial region.

Quantitative Endoscopic Phototransducer Investigation of Normal Velopharyngeal Physiology.

PURPOSEnThe purpose of this research was to learn the extent to which healthy individuals vary in their ability to achieve velopharyngeal closure for speech.nnnMETHODnTwenty healthy adult volunteers (10 women, 10 men) were tested using an endoscopic phototransducer system that tracks variations in velopharyngeal closure during speech production. Each speaker produced multiple repetitions of three utterances that differed in phonetic content. The data were amplitude normalized and averaged for each speaker.nnnRESULTSnAverage phototransducer measurements were similar across subjects for utterances containing only oral phonemes. Average percentage of velopharyngeal closure varied considerably among subjects when producing utterances containing both oral and nasal phonemes (54%-95%). Average percentage of velopharyngeal closure levels were significantly lower (p < .05) for utterances that included nasal consonants.nnnCONCLUSIONSnPhototransducer measurements of velopharyngeal closure for speech are sensitive to nasal phoneme content. The findings suggest that motor programming that accomplishes rapid oral-nasal velopharyngeal valving for speech may differ among healthy subjects. However, such variations in motor programming may not perceptually affect typical speakers. If present in individuals with abnormal velopharyngeal mechanisms, these variations may help explain variations among speakers in speech outcomes after physical and behavioral management.