Longitudinal effects of estrogen on mandibular growth and changes in cartilage during the growth period in rats.

Estrogen is a steroid hormone that induces skeletal growth and affects endochondral ossification of the long tubular bone growth plate during the growth period. However, the effects of estrogen on endochondral ossification of the mandibular condylar cartilage are unclear. In this study, ovariectomized Wistar/ST rats were used to investigate the longitudinal effects of estrogen on mandibular growth. The rats were administered different doses of estrogen. Longitudinal micro-computed tomographic scanning, histological staining and ELISA on plasma growth hormone were performed to examine the effects of estrogen on mandibular growth. The results showed that mandibular growth was suppressed throughout the growth period by estrogen in a dose-dependent manner. In addition, long-term administration of a high dose of estrogen to the rats resulted in significant increase in growth hormone throughout the growth period, significant circularization of cell nuclei in the proliferative layer, intensely staining cartilage matrix in the subchondral bone, and significant suppression of estrogen receptor (ER) alpha and beta expression in the mandibular cartilage. However, regardless of estrogen concentration, in the posterior part of the mandibular cartilage, ER expression extended to both the hypertrophic and proliferative layers. These results indicate that estrogen suppresses mandibular growth throughout the growth period. Additionally, it influences endochondral ossification via its effect on ERs.

Characteristics of the Maxillofacial Morphology in Patients with Idiopathic Mandibular Condylar Resorption.

Idiopathic mandibular condylar resorption (ICR) is a pathological condition characterized by idiopathic resorption of the mandibular condyle, resulting in a decrease in the size and height of the mandibular condyle. The purpose of this study was to characterize the maxillofacial morphology of ICR patients. Subjects were selected from patients that attended our orthodontic clinic between 1991 and 2019. Twenty-five patients were diagnosed with ICR by magnetic resonance imaging; however, growing patients were excluded. In total, 18 patients were finally selected. The control group comprised 18 healthy volunteers. Lateral and frontal cephalograms were also used. The ICR group had significantly more severe skeletal class II malocclusions than the control group, mainly due to retrusion of the mandible. In the ICR group, there was a tendency for a skeletal open bite due to a significantly larger clockwise rotation of the mandible than in the control group. There was no significant difference between the two groups in the inclination of the upper and lower central incisors or protrusion of the upper and lower central incisors and first molars. ICR patients have been suggested to exhibit skeletal open bite and maxillary protrusion with changes in maxillofacial morphology due to abnormal resorption of the mandibular condyle.

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.

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.

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.

The C-terminus of the amelogenin peptide influences the proliferation of human bone marrow mesenchymal stem cells.

BACKGROUND: Amelogenins are a family of enamel matrix proteins that are important for formation of enamel. Amelogenins may induce division of mesenchymal stem cells (MSCs), among others. Recently, the C-terminus of the amelogenin peptide (AMG-CP) has been shown to enhance the proliferation of cementoblast lineage cells. The role of the amelogenin peptide on the proliferation of human MSCs and related alterations in the intracellular signaling pathway were studied. METHODS: MSCs were exposed to AMG-CP in vitro. The MTS and 5-bromo-2'-deoxyuridine (BrdU) assays were used to determine proliferation. Expression of the amelogenin receptor, lysosomal-associated membrane protein 1 (LAMP1), was examined in MSCs with western blotting. Binding of AMG-CP to LAMP1 was inhibited with anti-LAMP1 antibody. Components of the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) pathway were studied with western blotting and enzyme-linked immunosorbent assay, and U0126, an MAPK inhibitor, was used to inhibit ERK activity. RESULTS: MSC proliferation was significantly increased in the presence of AMG-CP and significantly inhibited by anti-LAMP1 antibody or U0126. Increased phosphorylated ERK1/2 was observed in the presence of AMG-CP, and decreased phosphorylated ERK1/2 was seen in the presence of anti-LAMP1 antibody or U0126. CONCLUSION: A C-terminal amelogenin variant increased the proliferation of MSCs via an interaction with LAMP1 and the MAPK-ERK signaling pathway, indicating the possibility of using MSCs for tissue regeneration in the craniofacial region.

Effect of high-frequency near-infrared diode laser irradiation on periodontal tissues during experimental tooth movement in rats.

BACKGROUND AND OBJECTIVES: 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. METHODS: A nickel-titanium closed coil was mounted between the maxillary left side first molar and incisor of rats to model experimental tooth movement. The laser-irradiation and the control groups were set, and the amount of movement of the first molar on 7th and 14th days after the start of pulling of the first molar tooth on the maxillary left was measured by three-dimensional analysis of µCT. 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 TRAP staining and immunohistochemical staining for RANKL, OPG, ALP, and proliferating cell nuclear antigen (PCNA). Changes in tissue temperature following laser irradiation were also examined. RESULTS: Laser irradiation significantly increased tooth movement compared with non-irradiated controls. Histologic staining of the pressure-side mesial root in laser-irradiated rats revealed enhanced RANKL expression and increased numbers of TRAP-positive cells compared with controls. By contrast, on the tension side, laser irradiation led to increased expression of ALP and PCNA. These data indicate that high-frequency near-infrared diode laser irradiation on the pressure side upregulates RANKL expression and accelerates osteoclast differentiation, facilitating bone resorption, whereas bone formation is induced on the tension side. CONCLUSION: This study demonstrates that high-frequency near-infrared diode laser irradiation of periodontal tissue leads to metabolic activation, which ultimately increases the rate of tooth movement. Lasers Surg. Med. © 2018 Wiley Periodicals, Inc.

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.

Bovine lactoferrin reduces extra-territorial facial allodynia/hyperalgesia following a trigeminal nerve injury in the rat.

There is an urgent clinical need for an effective therapeutic agent to treat neuropathic pain. This study explored whether intrathecal administration of bovine lactoferrin (bLF), in combination with signal transduction pathway inhibition or an inflammatory cytokine production, results in reduced allodynia/hyperalgesia in the whisker pad area following mental nerve transection (MNT) in rats. Rats were intrathecally infused with bLF, lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS), an antagonist of Toll-like receptor 4 (TLR4), or interleukin (IL)-18 binding protein (BP). bLF attenuated allodynia/hyperalgesia and blocked upregulation of phosphorylated (p)-p38 mitogen-activated protein kinase (MAPK), p-nuclear factor (NF)-κB p65, p-IκB kinase, and IL-18 in the trigeminal subnucleus caudalis (Vc). Microglia expressed p-p38 and astrocytes expressed p-NF-κB p65 in the Vc following MNT. LPS-RS had the same effects as bLF, except for attenuation of p-NF-κB p65. IL-18BP attenuated allodynia/hyperalgesia and IL-18 upregulation in the Vc. These results suggest that bLF suppresses IL-18 production, which is involved in allodynia/hyperalgesia following MNT, by inhibiting TLR4-derived p38 MAPK activation in microglia. Additionally, binding of bLF to tumor necrosis factor receptor-associated factor 6 might result in inhibition of p38 MAPK and NF-κB activation. The findings suggest that bLF could serve as a potent therapeutic agent for neuropathic pain.

Effects of C-terminal amelogenin peptides on the metabolism of osteoblasts.

OBJECTIVES: Amelogenins, enamel matrix proteins secreted by ameloblasts, comprise 90% of the developing extracellular enamel matrix. Recent evidence suggests that amelogenins might induce the proliferation of various cells. However, the residues comprising the active site of amelogenin remain unclear. Therefore, this study aimed to examine the effects of a human amelogenin C-terminal peptide (amgCP) on the metabolism of osteoblasts. MATERIALS AND METHODS: Mouse calvarial osteoblastic cells (MC3T3-E1) were cultured and treated with amgCP. Cell proliferation was measured using MTS and BrdU assays. After confluence was reached, the cells were cultured in osteogenic differentiation medium and treated with 0, 100, or 1000 ng/ml amgCP. Cell differentiation activity was examined by real-time PCR, western blotting, and ALP activity. Mineralization was evaluated by Alizarin red staining. RESULTS: Cell numbers of MC3T3-E1 were significantly (P < 0.05) increased by treatment with 1000 ng/ml amgCP as compared to the control group at 4 and 6 days. In addition, the proliferative activity of MC3T3-E1 was significantly enhanced by treatment with 100 or 1000 ng/ml amgCP. The mRNA levels and protein expressions of ALP and BSP were not changed by treatment with amgCP as compared to the non-treated controls on days 7 and 14. The osteogenic differentiation of MC3T3-E1 cells was not affected by treatment with amgCP as compared with untreated controls. CONCLUSION: The C-terminus of amelogenin promotes the proliferation of MC3T3-E1 cells, indicating the possible utility of the C11 peptide in bone-tissue regeneration.

Effects of C-Terminal Amelogenin Peptide on Proliferation of Human Cementoblast Lineage Cells.

BACKGROUND: Extracts of enamel matrix proteins are used to regenerate periodontal tissue; amelogenin, the most abundant enamel protein, plays an important role in this regeneration. Studies have demonstrated that amelogenin fragments promote tissue regeneration, but the bioactive site of amelogenin remains unclear. This study explores the functional domain of amelogenin by investigating effects of four amelogenin species on cementoblast proliferation. METHODS: Four amelogenin species based on amelogenin cleavage products were investigated: 1) recombinant human full-length amelogenin (rh174); 2) amelogenin cleavage product lacking the C-terminal (rh163); 3) amelogenin cleavage product lacking the N-terminal (rh128); and 4) the C-terminal region of rh174 (C11 peptide), which was synthesized and purified. Human cementoblast-like cell line (HCEM) cells were cultured and treated with rh174, rh163, rh128, or C11 peptide. Cell proliferation was evaluated using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium assay and cell proliferation enzyme-linked immunosorbent assay. Mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) (MAPK-ERK) pathway was examined by Western blot analysis. RESULTS: Proliferation of HCEM cells was significantly enhanced on treatment with rh174, rh128, or C11 peptide. However, rh163 had no effect compared with the untreated control group. Western blot analysis revealed enhanced phosphorylated ERK1/2 signaling after addition of rh128 or C11 peptide and reduced phosphorylated ERK1/2 signaling after blocking with a specific MAPK inhibitor (U0126). CONCLUSION: C-terminal amelogenin cleavage product increased proliferation of HCEM through MAPK-ERK signaling pathway, indicating possible application of C11 peptide for periodontal tissue regeneration.

Experimental Tooth Movement Into New Bone Area Regenerated by Use of Bone Marrow-Derived Mesenchymal Stem Cells.

OBJECTIVE: The aim of this study was to examine experimental tooth movement into regenerated bone in alveolar cleft with mesenchymal stem cells and a granulated carbonated hydroxyapatite scaffold. DESIGN: An artificial bone defect was created bilaterally in upper incisor regions of beagle dogs to simulate alveolar clefts in patients with cleft palate. The mesenchymal stem cells derived from the iliac bone marrow were cultured and transplanted with carbonated hydroxyapatite into the bone defect area. Carbonated hydroxyapatite alone was transplanted on the control side. Six months after the transplantation, multi-bracket appliances were attached to the lateral incisors and canines on both sides of the maxilla to exert an orthodontic force of 100 × g using an elastic chain. The distance between lateral incisor and canine was measured, and standardized x-ray images were taken every month. The tissue after tooth movement was evaluated by histological observation. RESULTS: The experimental tooth movement, accompanied by resorption of regenerated bone and new bone formation, was achieved on the experimental and control sides. Although there was no difference in the amount of tooth movement obtained on the experimental and control sides during the 6-month experimental period, the rate of tooth movement varied on the control side; whereas, the rate was consistent on the experimental side. Root resorption of the tooth was observed on the control side in one dog. CONCLUSION: It is suggested that mesenchymal/carbonated hydroxyapatite transplantation therapy has great potential as a new treatment modality for bone regeneration in patients with cleft palate.

Bone regeneration in artificial jaw cleft by use of carbonated hydroxyapatite particles and mesenchymal stem cells derived from iliac bone.

Objectives of the Study. Cleft lip and palate (CLP) is a prevalent congenital anomaly in the orofacial region. Autogenous iliac bone grafting has been frequently employed for the closure of bone defects at the jaw cleft site. Since the related surgical procedures are quite invasive for patients, it is of great importance to develop a new less invasive technique. The aim of this study was to examine bone regeneration with mesenchyme stem cells (MSCs) for the treatment of bone defect in artificially created jaw cleft in dogs. Materials and Methods. A bone defect was prepared bilaterally in the upper incisor regions of beagle dogs. MSCs derived from iliac bone marrow were cultured and transplanted with carbonated hydroxyapatite (CAP) particles into the bone defect area. The bone regeneration was evaluated by standardized occlusal X-ray examination and histological observation. Results. Six months after the transplantation, perfect closure of the jaw cleft was achieved on the experimental side. The X-ray and histological examination revealed that the regenerated bone on the experimental side was almost equivalent to the original bone adjoining the jaw cleft. Conclusion. It was suggested that the application of MSCs with CAP particles can become a new treatment modality for bone regeneration for CLP patients.