Stem cells derived from human exfoliated deciduous teeth-based media in a rat root resorption model.

OBJECTIVE: Root resorption may occur during orthodontic treatment. Herein, we investigated the effect of a culture supernatant of stem cells derived from human exfoliated deciduous teeth on root resorption. DESIGN: Twelve 8-week-old male Sprague-Dawley rats were used, and their maxillary first molars were pulled with excessive orthodontic force to induce root resorption. On days 1 and 7 after traction initiation, stem cells derived from human exfoliated deciduous teeth and alpha minimum essential medium (control group) were administered. After 14 days, the maxillary bone was evaluated for tooth movement. The expression of osteoprotegerin, receptor activator of nuclear factor κB ligand, tumor necrosis factor α, interleukin 1ß, interleukin 6, and interleukin 17 was evaluated on the compression side and tension side. RESULTS: No significant difference in tooth movement was observed between the two groups. Root resorption decreased in the group administered the culture supernatant compared with in the control. Immunohistochemical staining revealed increased osteoprotegerin expression and decreased receptor activators for nuclear factor κB ligand, tumor necrosis factor α, interleukin 1ß, interleukin 6, and interleukin 17 on the compression side and tension side. CONCLUSIONS: Administration of stem cells derived from human exfoliated deciduous teeth affected the expression of osteoprotegerin, receptor activator of nuclear factor κB ligand, tumor necrosis factor α, interleukin 1ß, interleukin 6 and interleukin 17; hence, these stem cells may inhibit root resorption by regulating their expression.

Effect of CD146+ SHED on bone regeneration in a mouse calvaria defect model.

OBJECTIVE: Stem cells from human exfoliated deciduous teeth (SHED) have bone regeneration ability and potential therapeutic applications. CD146, a cell adhesion protein expressed by vascular endothelial cells, is involved in osteoblastic differentiation of stem cells. The effect of CD146 on SHED-mediated bone regeneration in vivo remains unknown. We aimed to establish efficient conditions for SHED transplantation. MATERIALS AND METHODS: SHED were isolated from the pulp of an extracted deciduous tooth and cultured; CD146-positive (CD146+ ) and CD146-negative (CD146- ) populations were sorted. Heterogeneous populations of SHED and CD146+ and CD146- cells were transplanted into bone defects generated in the skulls of immunodeficient mice. Micro-computed tomography was performed immediately and 4 and 8 weeks later. Histological and immunohistochemical assessments were performed 8 weeks later. RESULTS: Bone regeneration was observed upon transplantation with CD146+ and heterogeneous populations of SHED, with significantly higher bone regeneration observed with CD146+ cells. Bone regeneration was higher in the CD146- group than in the control group, but significantly lower than that in the other transplant groups at 4 and 8 weeks. Histological and immunohistochemical assessments revealed that CD146+ cells promoted bone regeneration and angiogenesis. CONCLUSION: Transplantation of CD146+ SHED into bone defects may be useful for bone regeneration.

Comparison of Orthodontic Tooth Movement of Regenerated Bone Induced by Carbonated Hydroxyapatite or Deproteinized Bovine Bone Mineral in Beagle Dogs.

Orthodontic treatments often involve tooth movement to improve dental alignment. In this study, we aimed to compare tooth movement in regenerated bone induced by two different bone fillers, carbonated hydroxyapatite (CAP) and deproteinized bovine bone mineral (DBBM). Four beagle dogs were used in this comparative study. The first, second, and fourth lower mandibular premolars (P1, P2, and P4) on both sides of the mouth were extracted, and CAP was implanted into the extraction site on the left side and DBBM into the right side. Following regenerative bone healing, orthodontic devices were attached to perform orthodontic tooth movement of the lower third mandibular premolar (P3) on both sides. X-ray examination, intraoral scan, and histological analysis were performed. The Mann-Whitney U test was used for statistical analysis, and p < 0.05 was considered significant. Bone regeneration and orthodontic tooth movement were observed in the CAP and DBBM groups. Histologically, normal periodontal tissue remodeling was observed on the compression and tension sides of CAP and DBBM. No statistical difference was observed in the number of osteoclasts around the periodontal ligament and the root resorption area. Orthodontic tooth movement of regenerated bone induced by CAP and DBBM was therefore achieved.

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.

Effects of baicalin on the proliferation and expression of OPG and RANKL in human cementoblast-lineage cells.

BACKGROUND/PURPOSE: Baicalin, a natural bioactive flavonoid extracted from Scutellaria baicalensis Georgi, mediates bone metabolism, and recent studies have revealed that it has cell signaling properties. However, its biological functions in cementoblasts still remain unclear. This study therefore aimed to investigate the effects of baicalin on bone resorption markers, including osteoprotegerin (OPG) and receptor activator of nuclear factor-κß ligand (RANKL), in human cementoblast-lineage cells, as well as their proliferation ability. MATERIALS AND METHODS: Human cementoblast cell line (HCEM) cells were cultured and treated with 0, 0.01, 0.1, or 1 µM of baicalin. The proliferative capacity of cultured HCEM cells was analyzed using bromodeoxyuridine immunoassay and cell counting. The baicalin effect on OPG and RANKL expression was determined using quantitative polymerase chain reaction (qPCR) and western blotting. Furthermore, OPG expression was measured in 1 µM baicalin-treated HCEM cells in the presence or absence of the Wnt signaling pathway inhibitor, Dickkopf (Dkk)-1, using qPCR and western blotting. RESULTS: The addition of 0.01, 0.1, and 1 µM of baicalin did not significantly change the proliferative capacity of cultured HCEM cells. Compared with the non-supplemented group, baicalin increased and suppressed OPG and RANKL gene and protein expression, respectively, in a concentration-dependent manner. OPG mRNA and protein expression levels were increased by 1 µM baicalin, which was suppressed by Dkk-1 addition. CONCLUSION: Baicalin enhanced OPG expression in HCEM cells through the Wnt/beta-catenin signaling pathway, which could contribute to periodontal tissue regeneration.

Examination of the effect of combined use of Er:YAG laser irradiation and mechanical force loading on bone metabolism using primary human gingival fibroblasts.

Prolonged treatment and painful tooth movement are major problems for patients undergoing orthodontic treatment. Accelerating the movement of teeth leads to shortening of the treatment period, so various studies on the movement of teeth have been conducted in the field of orthodontics. In previous studies, we performed a fiber incision-like fiberotomy using an Er:YAG laser in rats and confirmed acceleration of tooth movement. Therefore, in this study, the effect of Er:YAG laser irradiation on human gingival fibroblasts was investigated in vitro. Human gingival fibroblasts (2.0 × 105 cells) were seeded in a 6-well plate and reached 80% confluence 24 h later. A control group not undergoing any irradiation and 3 groups undergoing laser irradiation at 0.6 W, 1.0 W, and 1.2 W were investigated. Laser irradiation was performed 24 h after cell seeding. The cells were then recovered 24 h later, and the cyclooxygenase-2 (COX-2), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), bone morphogenetic protein-2 (BMP-2), and BMP-4 genes were confirmed by PCR. In addition, a control group not undergoing any procedures, a group undergoing only Er:YAG laser irradiation, a group undergoing only centrifugal loading, and a group undergoing both Er:YAG laser irradiation and centrifugal force loading were investigated. After 24 h, cells were collected and PCR was performed. Twenty-four hours after laser irradiation, gene expressions were examined by quantitative RT-PCR, which showed that the gene expressions of COX-2, IL-1ß, TNF-α, BMP-2, and BMP-4 increased depending on the amount of irradiation energy, with the largest value at 1.2 W. Gene expressions of COX-2, IL-1ß, TNF-α, BMP-2, and BMP-4 were significantly higher in the laser with centrifugal load group than in the load group. These results suggest that genes related to bone metabolism are activated in human gingival fibroblasts when mechanical stimulation and laser irradiation are combined. This helps to elucidate the effects of Er:YAG laser irradiation during tooth movement.

Baicalin inhibits root resorption during tooth movement in a rodent model.

OBJECTIVE: Baicalin mediates bone metabolism and has shown protective activity against periodontal tissue damage in a rat model of periodontitis. Therefore, we hypothesized that baicalin may inhibit the root resorption that occurs during orthodontic tooth movement and examined its effect on the histological changes in periodontal tissue that occur during tooth movement. METHODS: First molars of rats were subjected to traction using excessive orthodontic force to produce a root resorption model. Rats in the baicalin group received baicalin for 3 weeks during tooth movement, and the amount of first molar movement on day 21 after the initiation of traction was measured by three-dimensional micro-computed tomography analysis. After tooth movement, tissue samples from the mesial and tension sides were collected, and successive horizontal sections were prepared and examined using hematoxylin-eosin and tartrate-resistant acid phosphatase (TRAP) staining and immunohistochemical staining for the receptor activator of NF-kB ligand (RANKL) and osteoprotegerin (OPG). The severity of root resorption was also determined by histological analysis. RESULTS: There was no significant intergroup difference in tooth movement during the experimental exaggerated tooth movement. In comparison with the control group, the baicalin-treated group showed increased OPG expression, suppressed RANKL expression, and significantly fewer TRAP-positive cells in the first molars. The root resorption area was significantly smaller in the baicalin group. CONCLUSIONS: Treatment with baicalin prevented root resorption without preventing tooth movement. Baicalin may be useful for the management of root resorption during orthodontic treatment.

Examination of the Effect of the Combined Use of Nd: YAG Laser Irradiation and Mechanical Force Loading on Bone Metabolism Using Cultured Human Osteoblasts.

Introduction: In recent years, laser irradiation in the near-infrared ray (NIR) area has been reported to promote bone healing. There are also reports that laser irradiation accelerates orthodontic tooth movement. In this study, we investigated the effect of NIR laser irradiation and mechanical stimulation on osteoblasts. Methods: We seeded osteoblast-like cells and laser irradiation was performed 24 hours after cell seeding. In addition, a control group not receiving anything, a group receiving only Nd: YAG (neodymium-doped yttrium aluminum garnet) laser irradiation, a group receiving only centrifugal loading, and a group receiving both Nd: YAG laser irradiation and centrifugal force loading were set, and after 24 hours and after 48 hours, cells were collected and quantitative real-time polymerase chain reaction (PCR) was performed. Results: 24 hours after laser irradiation, the gene expression of alkaline phosphatase (ALP), the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) was significantly higher in the 2.0 W group than in the control group. In addition, the RANKL/OPG ratio was higher in the 2.0 W group than in the control group. Also, in the group using laser irradiation and centrifugal loading in combination, 24 hours after laser irradiation, ALP and OPG showed significantly higher values than those in the centrifugal load only group. Furthermore, the RANKL/OPG ratio also showed high values. Conclusion: These results suggest that osteoblast-like cells activate genes related to bone metabolism by combining mechanical stimulation and laser irradiation. This helps to elucidate the influence of laser irradiation during tooth movement.

Bone Regeneration in a Canine Model of Artificial Jaw Cleft Using Bone Marrow-Derived Mesenchymal Stem Cells and Carbonate Hydroxyapatite Carrier.

OBJECTIVE: Cleft lip and palate (CLP) is a common anomaly of the orofacial region. Mesenchymal stem cell (MSC) transplantation has been a focus of regenerative medicine, and its application to the repair of bone defects in patients with CLP is highly anticipated. This study investigated the potential for using MSCs to regenerate bone in a jaw cleft as well as the survival of transplanted MSCs using a canine model of CLP. DESIGN: Mesenchymal stem cells collected from the bone marrow of beagle dogs were transplanted along with carbonate hydroxyapatite into jaw clefts in beagle dogs. Mesenchymal stem cells labeled with fluorescent silica nanoparticles were also transplanted, and a histological analysis was performed 3 months later to evaluate MSC survival. RESULTS: Carbonate hydroxyapatite regeneration into bone was enhanced by cotransplantation of MSCs. The survival rate of MSCs transplanted after 3 months was 5.7%. CONCLUSIONS: Transplanted MSCs promote bone regeneration, although their survival rate is low.

Stem cell-derived conditioned media from human exfoliated deciduous teeth promote bone regeneration.

OBJECTIVES: Cleft lip and palate (CL/P) are common congenital orofacial anomalies. Autogenous iliac bone grafting closes alveolar cleft defects but requires surgical intervention. Mesenchymal stem cell culture supernatant can regenerate tissues via paracrine activity. However, little is known about the bone-regenerative effects of stem cells from human exfoliated deciduous teeth (SHED) and conditioned media (CM). Our aim was to address this. MATERIALS AND METHODS: Stem cells were isolated from primary tooth pulp and cultured. Defects were made in calvariae of immunodeficient mice and implanted with stem cell- or CM-containing atelocollagen. Regenerated bone was analysed by microcomputed tomography, haematoxylin-eosin and Masson's trichrome staining. Vascular endothelial growth factor, CD31 and CD34 expression were confirmed by immunohistochemistry, and the presence of several proteins and growth factors was verified in SHED-CM. RESULTS: Bone regeneration was enhanced in defects treated with stem cells and CM compared to that in controls 8 weeks after transplantation. Mature bone formation and angiogenesis were confirmed with CM but not with stem cells or in controls. Secretome analysis using multiple cytokine assays revealed that SHED-CM contained tissue-regenerating factors with roles in angiogenesis and osteogenesis. CONCLUSION: CM non-invasively regenerate bone and might be effective to reconstruct alveolar clefts in CL/P patients.

Success rates in isolating mesenchymal stem cells from permanent and deciduous teeth.

Stem cells from human exfoliated deciduous teeth (SHED) and human dental pulp stem cells (hDPSCs) have emerged as attractive cell sources for bone regeneration. However, the specific teeth and the conditions most suitable for stem cell isolation remain unclear. Therefore, the success rate of SHED and hDPSCs isolation, the patient age and remaining root length in deciduous teeth were evaluated. Successful isolation was defined as when the cell culture was maintained up to the third passage without any contamination or other issues. Remaining tooth length was calculated using the root-to-crown ratio from patient X-rays and compared to the norm value from the literature. The overall successful isolation rate of SHED and hDPSCs was 82% and 70%. The average patient ages at extraction of the deciduous teeth and permanent teeth were 11 years and 9 months, and 22 years and 10 months respectively. In the successful SHED group, the average remaining root length of the anterior deciduous teeth was 71.4%, and that of the deciduous molars was 61.4%. Successful isolation appears to be associated with patient age, length of the remaining root, and also mechanical stress and other factors. Tooth selection criteria need to be identified to improve the success rate.

The effect of mesenchymal stem cells on osteoclast precursor cell differentiation.

Transplantation of mesenchymal stem cells (MSCs) has been extensively studied in the field of regenerative medicine. Bone regeneration is achieved via the interaction of osteoblasts and osteoclasts. However, the influence of MSCs on osteoclasts is unknown. The purpose of this study was to investigate the effect of MSCs on the expression of genes for osteoclast differentiation factors using qPCR after indirect co-culture of MSCs and RAW264 cells. The numbers of osteoclasts after addition of soluble receptor activator of nuclear factor kappa B (NF-κB) ligand (sRANKL) were also compared. Expression of osteoprotegerin (OPG) by MSCs was significantly elevated in co-culture over time. The differentiation of RAW264 cells into mature osteoclasts following addition of sRANKL was significantly inhibited by co-culture with MSCs. Expression of RANK, colony stimulating factor 1 receptor, NF-κB, and nuclear factor of activated T-cell cytoplasmic 1 in RAW264 cells was significantly inhibited by co-culture with MSCs. Expression of OPG protein was higher in co-culture with RAW264 cells than in MSCs alone, and the expression level was clearly higher than that of RANKL. MSCs appeared to inhibit osteoclast differentiation via expression of OPG.

Effects of Human Full-length Amelogenin and C-terminal Amelogenin Peptide on the Proliferation of Human Mesenchymal Stem Cells Derived from Adipose Tissue.

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.

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.

OBJECTIVES: Mesenchymal 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 in vitro characteristics of dental pulp stem cells from deciduous teeth (SHED), human dental pulp stem cells (hDPSCs), and human bone marrow mesenchymal stem cells (hBMSCs). MATERIALS AND METHODS: SEHD 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. RESULTS: SHED 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. CONCLUSION: SHED 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.

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 4 mm 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.

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 (30 kHz) 910-nm diode laser irradiation at a dose of 0, 1.42, 2.85, 5.7, or 17.1 J/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 17.1 J/cm2. Laser irradiation at a dose of 2.85 J/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 2.85 J/cm2 induced phosphorylation of MAPK/ERK1/2 15 and 30 min later. However, phosphorylation of p38 MAPK and SAPK/JNK was not changed by NIR diode laser irradiation at a dose of 2.85 J/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.

Baicalin Promotes Osteogenic Differentiation of Human Cementoblast Lineage Cells Via the Wnt/ß Catenin Signaling Pathway.

BACKGROUND: Baicalin constitutes a natural bioactive flavonoid extracted from Scutellaria baicalensis Georgi that mediates bone formation. However, the biological functions of baicalin in cementoblasts remain unclear. The purpose of this study was to examine the effects of baicalin on osteogenic differentiation of human cementoblast (HCEM) cells. METHODS: HCEM cells were cultured and treated with 0, 0.01, 0.1 or 1 µM baicalin. Alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2) mRNA and protein levels were examined by real-time polymerase chain reaction and western blot analysis, respectively. Cell mineralization was assessed using Alizarin red staining. Glycogen synthase kinase-3 beta (GSK3ß) phosphorylation was measured in 1 µM baicalin-treated HCEM cells with or without the Wnt signaling pathway inhibitor, DKK-1 using ELISA and western blotting. RESULTS: The protein levels of ALP and Runx2 and the intensity of Alizarin red staining were enhanced by baicalin in a dose-dependent manner compared to that of the non-treated control. The ratio of phosphorylated to total GSK3ß increased in the presence of baicalin but was reduced by the addition of DKK-1. Treatment of HCEMs with baicalin up-regulated mRNA levels of ALP and Runx2, which were reduced by DKK-1. In addition, the protein levels of ALP and Runx2, ALP activity, and calcium deposition were also enhanced by baicalin, and these parameters were inhibited by DKK-1. CONCLUSION: Baicalin enhanced osteogenic differentiation of HCEM cells through the Wnt/beta catenin signaling pathway which may be useful for periodontal tissue regeneration.

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.