Alveolar Crestal Approach for Maxillary Sinus Membrane Elevation with <4 mm of Residual Bone Height: A Case Report.

INTRODUCTION: For maxillary sinus membrane elevation (MSME), the lateral window approach and crestal approach are available, and high success rates have been achieved with low residual bone height as a development of technology. OBJECTIVE: To evaluate MSME using the crestal approach with a rotary-grind bur (RGB (including reamer or sinus bur)) in patients with residual bone height of <4 mm. MATERIALS AND METHODS: Ten implants were placed in 10 patients with residual bone height of <4 mm, by sinus elevation using an RGB. The implant stability quotient (ISQ) was measured immediately after implant placement (ISQ 1) and before taking impression for the final prosthesis (ISQ 2). The extent of marginal bone loss was measured on periapical radiographs. RESULTS: The mean residual bone height before implant placement was 3.41 ± 0.53 mm; no complications, including membrane perforation, severe postoperative pain, or discomfort, occurred either during or after surgery. The mean ISQ 1 was 63.4 ± 12.1, whereas the mean ISQ 2 was 77.6 ± 5.8. The mean marginal bone resorption was 0.23 ± 0.18 mm on periapical radiographs. CONCLUSIONS: MSME using the crestal approach with an RGB is a reliable technique for implant placement in sites where available bone is insufficient.

Fucoidan-induced osteogenic differentiation promotes angiogenesis by inducing vascular endothelial growth factor secretion and accelerates bone repair.

Osteogenesis and angiogenesis, including cell-cell communication between blood vessel cells and bone cells, are essential for bone repair. Fucoidan is a chemical compound that has a variety of biological activities. It stimulates osteoblast differentiation in human mesenchymal stem cells (MSCs), which in turn induces angiogenesis. However, the mechanism by which this communication between osteoblasts and endothelial cells is mediated remains unclear. Thus, the aim of this study was to clarify the relationship between fucoidan-induced osteoblastic differentiation in MSCs and angiogenesis in endothelial cells. First, the effect was confirmed of fucoidan on osteoblast differentiation in MSCs and obtained conditioned media from these cells (Fucoidan-MSC-CM). Next, the angiogenic activity of Fucoidan-MSC-CM was investigated and it was found that it stimulated angiogenesis, demonstrated by proliferation, tube formation, migration and sprout capillary formation in human umbilical vein endothelial cells. Messenger ribonucleic acid expression and protein secretion of vascular endothelial growth factor (VEGF) were dramatically increased during fucoidan-induced osteoblast differentiation and that its angiogenic activities were reduced by a VEGF/VEGF receptor-specific binding inhibitor. Furthermore, Fucoidan-MSC-CM increased the phosphorylation of mitogen-activated protein kinase and PI3K/AKT/eNOS signalling pathway, and that its angiogenic effects were markedly suppressed by SB203580 and AKT 1/2 inhibitor. Finally, an in vivo study was conducted and it was found that fucoidan accelerated new blood vessel formation and partially promoted bone formation in a rabbit model of a calvarial bone defect. This is the first study to investigate the angiogenic effect of fucoidan-induced osteoblastic differentiation through VEGF secretion, suggesting the therapeutic potential of fucoidan for enhancing bone repair.

BST2 Mediates Osteoblast Differentiation via the BMP2 Signaling Pathway in Human Alveolar-Derived Bone Marrow Stromal Cells.

The molecular mechanisms controlling the differentiation of bone marrow stromal stem cells into osteoblasts remain largely unknown. In this study, we investigated whether bone marrow stromal antigen 2 (BST2) influences differentiation toward the osteoblasts lineage. BST2 mRNA expression in human alveolar-derived bone marrow stromal cells (hAD-BMSCs) increased during differentiation into osteoblasts. hAD-BMSCs differentiation into osteoblasts and the mRNA expression of the bone-specific markers alkaline phosphatase, collagen type α 1, bone sialoprotein, osteocalcin, and osterix were reduced by BST2 knockdown using siRNA. Furthermore, BST2 knockdown in hAD-BMSCs resulted in decreased RUNX2 mRNA and protein expression. We hypothesized that BST2 is involved in differentiation of into osteoblasts via the BMP2 signaling pathway. Accordingly, we evaluated the mRNA expression levels of BMP2, BMP receptors (BMPR1 and 2), and the downstream signaling molecules SMAD1, SMAD4, and p-SMAD1/5/8 in BST2 knockdown cells. BMP2 expression following the induction of differentiation was significantly lower in BST2 knockdown cells than in cells treated with a non-targeting control siRNA. Similar results were found for the knockdown of the BMP2 receptor- BMPR1A. We also identified significantly lower expression of SMAD1, SMAD4, and p-SMAD1/5/8 in the BST2 knockdown cells than control cells. Our data provide the first evidence that BST2 is involved in the osteogenic differentiation of bone marrow stromal cells via the regulation of the BMP2 signaling pathway.

Effect of nanofiber content on bone regeneration of silk fibroin/poly(ε-caprolactone) nano/microfibrous composite scaffolds.

The broad application of electrospun nanofibrous scaffolds in tissue engineering is limited by their small pore size, which has a negative influence on cell migration. This disadvantage could be significantly improved through the combination of nano- and microfibrous structure. To accomplish this, different nano/microfibrous scaffolds were produced by hybrid electrospinning, combining solution electrospinning with melt electrospinning, while varying the content of the nanofiber. The morphology of the silk fibroin (SF)/poly(ε-caprolactone) (PCL) nano/microfibrous composite scaffolds was investigated with field-emission scanning electron microscopy, while the mechanical and pore properties were assessed by measurement of tensile strength and mercury porosimetry. To assay cell proliferation, cell viability, and infiltration ability, human mesenchymal stem cells were seeded on the SF/PCL nano/microfibrous composite scaffolds. From in vivo tests, it was found that the bone-regenerating ability of SF/PCL nano/microfibrous composite scaffolds was closely associated with the nanofiber content in the composite scaffolds. In conclusion, this approach of controlling the nanofiber content in SF/PCL nano/microfibrous composite scaffolds could be useful in the design of novel scaffolds for tissue engineering.

The effects of fibrinogen concentration on fibrin/atelocollagen composite gel: an in vitro and in vivo study in rabbit calvarial bone defect.

OBJECTIVE: This study aimed to optimize the fibrinogen concentration in fibrin and atelocollagen (AT-COL) (fibrin/AT-COL) composite gel for improving bone regeneration. METHODS AND MATERIALS: The fibrin/AT-COL composite gels were fabricated using various fibrinogen concentrations, and the microstructure and mechanical properties of the resulting composite gels analyzed. The cytocompatibility of the composite gels was examined using human mesenchymal stem cell (hMSCs). Furthermore, in nine rabbits, the in vivo bone regeneration efficiency was evaluated using a rabbit calvarial defects model at 2 weeks (n = 3), 4 weeks (n = 3), and 8 weeks (n = 3). RESULTS: Scanning electron microscopy analysis revealed the formation of a fibrin layer matrix and collagen fibril networks. The composite gel containing 40 mg/ml fibrinogen showed a densely packed fibrin matrix and displayed superior mechanical properties. Cells cultured in the composite gels prepared with 5-20 mg/ml fibrinogen appeared elongated, with a spindle-like morphology. At a higher fibrinogen concentration (40 mg/ml), many cells were rounded and showed limited viability. In an in vivo study, at 8 weeks, the volume of fibrin/AT-COL gel (P = 0.02) was significantly higher than that of fibrin gel alone in the newly formed bone. Histological analysis revealed more islands of newly formed bone filling the central area of the defect in the fibrin/AT-COL gel-implanted animals. CONCLUSION: Our results demonstrate that optimization of the fibrinogen content of fibrin/AT-COL composites should be beneficial for bone tissue engineering.

Effects of fibrinogen concentration on fibrin glue and bone powder scaffolds in bone regeneration.

Fibrin polymers are widely used in the tissue engineering field as biomaterials. Although numerous researchers have studied the fabrication of scaffolds using fibrin glue (FG) and bone powder, the effects of varied fibrinogen content during the fabrication of scaffolds on human mesenchymal stem cells (hMSCs) and bone regeneration remain poorly understood. In this study, we formulated scaffolds using demineralized bone powder and various fibrinogen concentrations and analyzed the microstructure and mechanical properties. Cell proliferation, cell viability, and osteoblast differentiation assays were performed. The ability of the scaffold to enhance bone regeneration was evaluated using a rabbit calvarial defect model. Micro-computed tomography (micro-CT) showed that bone powders were uniformly distributed on the scaffolds, and scanning electron microscopy (SEM) showed that the fibrin networks and flattened fibrin layers connected adjacent bone powder particles. When an 80 mg/mL fibrinogen solution was used to formulate scaffolds, the porosity decreased 41.6 ± 3.6%, while the compressive strength increased 1.16 ± 0.02 Mpa, when compared with the values for the 10 mg/mL fibrinogen solution. Proliferation assays and SEM showed that the scaffolds prepared using higher fibrinogen concentrations supported and enhanced cell adhesion and proliferation. In addition, mRNA expression of alkaline phosphatase and osteocalcin in cells grown on the scaffolds increased with increasing fibrinogen concentration. Micro-CT and histological analysis revealed that newly formed bone was stimulated in the scaffold implantation group. Our results demonstrate that optimization of the fibrinogen content of fibrin glue/bone powder scaffolds will be beneficial for bone tissue engineering.

Intracellular delivery of cell-penetrating peptide-transcriptional factor fusion protein and its role in selective osteogenesis.

Protein-transduction technology has been attempted to deliver macromolecular materials, including protein, nucleic acids, and polymeric drugs, for either diagnosis or therapeutic purposes. Herein, fusion protein composed of an arginine-rich cell-penetrating peptide, termed low-molecular-weight protamine (LMWP), and a transcriptional coactivator with a PDZ-binding motif (TAZ) protein was prepared and applied in combination with biomaterials to increase bone-forming capacity. TAZ has been recently identified as a specific osteogenic stimulating transcriptional coactivator in human mesenchymal stem cell (hMSC) differentiation, while simultaneously blocking adipogenic differentiation. However, TAZ by itself cannot penetrate the cells, and thus needs a transfection tool for translocalization. The LMWP-TAZ fusion proteins were efficiently translocalized into the cytosol of hMSCs. The hMSCs treated with cell-penetrating LMWP-TAZ exhibited increased expression of osteoblastic genes and protein, producing significantly higher quantities of mineralized matrix compared to free TAZ. In contrast, adipogenic differentiation of the hMSCs was blocked by treatment of LMWP-TAZ fusion protein, as reflected by reduced marker-protein expression, adipocyte fatty acid-binding protein 2, and peroxisome proliferator-activated receptor-γ messenger ribonucleic acid levels. LMWP-TAZ was applied in alginate gel for the purpose of localization and controlled release. The LMWP-TAZ fusion protein-loaded alginate gel matrix significantly increased bone formation in rabbit calvarial defects compared with alginate gel matrix mixed with free TAZ protein. The protein transduction of TAZ fused with cell-penetrating LMWP peptide was able selectively to stimulate osteogenesis in vitro and in vivo. Taken together, this fusion protein-transduction technology for osteogenic protein can thus be applied in combination with biomaterials for tissue regeneration and controlled release for tissue-engineering purposes.

Growth and osteogenic differentiation of alveolar human bone marrow-derived mesenchymal stem cells on chitosan/hydroxyapatite composite fabric.

Scaffolds can be used for tissue engineering because they can serve as templates for cell adhesion and proliferation for tissue repair. In this study, chitosan/hydroxyapatite (CS/HAp) composites were prepared by coprecipitation synthesis. Then, CS and CS/HAp fabrics were prepared by wet spinning. CS fibers with a diameter of 15 ± 1.3 µm and CS/HAp fibers with a diameter of 22 ± 1.2 µm were successfully produced; incorporation of HAp into the CS/HAp fibers was confirmed by X-ray diffraction analysis. Biological in vitro evaluations showed that human mesenchymal stem cells (hMSCs) cultured on CS/HAp fabric showed increased proliferation compared to those cultured on pure CS fabric, which was observed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, DNA content assay, and [(3) H] thymidine incorporation assay. Neither the CS nor CS/HAp scaffold exhibited any cytotoxicity to hMSCs, as shown by viability staining and cytotoxicity fluorescence image assays. After 10 days of culturing, the attachment of cells onto the scaffold was observed by scanning electron microscopy. Furthermore, under osteogenic differentiation conditions, alkaline phosphatase (ALP) activity and calcium accumulation was higher in cells cultured on the CS/HAp scaffold than in cells cultured on the CS scaffold. The mRNA expression of osteoblast markers, including ALP, osteocalcin, Co1Ia1, and runt-related transcription factor 2, was higher in cells cultured on CS/HAp than in cells cultured on the CS fabric. The results of this study indicate that the CS/HAp composite fabric may serve as a good scaffold for bone tissue engineering applications.

IFITM1 increases osteogenesis through Runx2 in human alveolar-derived bone marrow stromal cells.

The exact molecular mechanisms governing the differentiation of bone marrow stromal stem/progenitor cells (BMSCs) into osteoblasts remain largely unknown. In this study, a highly expressed protein that had a high degree of homology with interferon-induced transmembrane protein 1 (IFITM1) was identified using differentially expressed gene (DEG) screening. We sought to determine whether IFITM1 influenced osteoblast differentiation. During differentiation, IFITM1 expression gradually increased from 5 to 10days and subsequently decreased at 15 days in culture. Analysis of IFITM1 protein expression in several cell lines as well as in situ studies on human tissues revealed its selective expression in bone cells and human bone. Proliferation of human alveolar-derived bone marrow stromal cells (hAD-BMSCs) was significantly inhibited by IFITM1 knockdown by using short hairpin RNA, as were bone specific markers such as alkaline phosphatase, collagen type I α 1, bone sialoprotein, osteocalcin, and osterix were decreased. Calcium accumulation also decreased following IFITM1 knockdown. Moreover, IFITM1 knockdown in hAD-BMSCs was associated with inhibition of Runx2 mRNA and protein expression. Collectively, the present data provide evidence for the role of IFITM1 in osteoblast differentiation. The exact mechanisms of IFITM1's involvement in osteoblast differentiation are still under investigation.

Cellular attachment and osteoblast differentiation of mesenchymal stem cells on natural cuttlefish bone.

The purpose of this study was to describe an approach that aims to provide fundamental information for the application of natural cuttlefish bone. Before applying cuttlefish bone as a bone defect filling material, we evaluated proliferation, adhesion, and cell viability of human mesenchymal stem cells (hMSCs) cultured on cuttlefish bone. Cuttlefish bone was separated into two parts (dorsal shield and lamellar region) and each part was used. Cell proliferation and viability were assessed using the MTS assay and live/dead fluorescence staining method. The morphology was observed using scanning electron microscopy (SEM). hMSCs were stimulated with osteogenic medium and osteoblast differentiation was evaluated. The fluorescence images showed that the seeded cells grew well and that cell distribution was in accordance with the surface morphology of the cuttlefish bone. Compared with the dorsal shield, cells penetrated deeper into the three-dimensional inner space of the lamellar part. Furthermore, under osteogenic differentiation conditions, alkaline phosphatase activity increased and the mRNA expression of ALP, runt-related transcription factor 2, and collagen type I α1 was increased in hMSCs cultured on both the dorsal shield and lamellar block. These results indicate the potential of cuttlefish bone as an ideal scaffold for bone regenerative materials.

Effects of nicotine on proliferation and osteoblast differentiation in human alveolar bone marrow-derived mesenchymal stem cells.

AIMS: Nicotine is a risk factor for various diseases, including osteoporosis, oral cancer, and periodontal disease. Numerous studies have elucidated the effects of nicotine on cell proliferation and differentiation. The purpose of this study was to determine the effects of nicotine on the proliferation and osteoblast differentiation of human alveolar bone marrow-derived mesenchymal stem cells (hABMMSCs). MAIN METHODS: In this study, we treated hABMMSCs with different doses (1 µM to 5 mM) of nicotine. The survival and proliferation of hABMMSCs were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay and crystal violet assay. TUNEL and propidium iodide (PI) double staining assay were also performed. The effect of nicotine on osteoblast differentiation of hABMMSCs was determined by measuring calcium accumulation using alizarin red-sulfate (AR-S) staining, measurement of alkaline phosphatase (ALP) activity, and semi-quantitative PCR of osteoblast markers. KEY FINDINGS: The survival and proliferation of hABMMSCs did not differ when they were exposed to nicotine at concentrations ranging from 1 µM to 100 µM; however, cell proliferation increased when the cells were exposed to nicotine at concentrations of 1-2 mM and decreased significantly when exposed to 5mM of nicotine. A number of cells were stained by PI but not by TUNEL, and membrane vacuolization was observed in hABMMSCs treated with 5mM nicotine. Calcium accumulation; ALP activity; and mRNA levels of ALP, bone sialoprotein (BSP), collagen type I α 1 (Col1αI), and runt-related transcription factor 2 (Runx2) were significantly decreased by treatment with 2mM of nicotine, while osteocalcin transcripts decreased by treatment with 1 to 2 mM of nicotine. SIGNIFICANCE: These results suggest that nicotine has a bimodal effect on the proliferation and osteoblast differentiation in hABMMSCs.

N-formyl-methionyl-leucyl-phenylalanine (fMLP) promotes osteoblast differentiation via the N-formyl peptide receptor 1-mediated signaling pathway in human mesenchymal stem cells from bone marrow.

Binding of N-formyl-methionyl-leucyl-phenylalanine (fMLP) to its specific cell surface receptor, N-formyl peptide receptor (FPR), triggers different cascades of biochemical events, eventually leading to cellular activation. However, the physiological role of fMLP and FPR during differentiation of mesenchymal stem cells is unknown. In this study, we attempted to determine whether fMLP regulates differentiation of mesenchymal stem cells derived from bone marrow. Analysis by quantitative-PCR and flow cytometry showed significantly increased expression of FPR1, but not FPR2 and FPR3, during osteoblastic differentiation. fMLP, a specific ligand of FPR1, promotes osteoblastic commitment and suppresses adipogenic commitment under differentiation conditions. Remarkably, fMLP-stimulated osteogenesis is associated with increased expression of osteogenic markers and mineralization, which were blocked by cyclosporine H, a selective FPR1 antagonist. In addition, fMLP inhibited expression of peroxisome proliferator-activated receptor-γ1, a major regulator of adipocytic differentiation. fMLP-stimulated osteogenic differentiation was mediated via FPR1-phospholipase C/phospholipase D-Ca(2+)-calmodulin-dependent kinase II-ERK-CREB signaling pathways. Finally, fMLP promoted bone formation in zebrafish and rabbits, suggesting its physiological relevance in vivo. Collectively, our findings provide novel insight into the functional role of fMLP in bone biology, with important implications for its potential use as a therapeutic agent for treatment of bone-related disorders.

Healing pattern of the mucous membrane after tooth extraction in the maxillary sinus.

PURPOSE: To investigate the healing pattern of the mucous membrane after tooth extraction necessitated by periodontal disease in the maxillary sinus. METHODS: One hundred and three patients with 119 maxillary sinuses were investigated. Before implant placement, cone-beam computed tomography (CT) scanning was performed. The causes of extraction, the time elapsed since extraction, smoking, periodontal disease in adjacent teeth, and gender were recorded. In addition, the thickness of the mucous membrane of the maxillary sinus and the height of residual alveolar bone at the extracted area were calculated from CT images. RESULTS: The thickness of the mucous membrane in the periodontal disease group (3.05±2.71 mm) was greater than that of the pulp disease group (1.92±1.78 mm) and the tooth fracture group (1.35±0.55 mm; P<0.05). The causes of extraction, the time elapsed since extraction, and gender had relationships with a thickening of the mucous membrane of the maxillary sinus (P<0.05). In contrast, the height of the residual alveolar bone at the extracted area, periodontal disease in adjacent teeth, and smoking did not show any relation to the thickening of the mucous membrane of the maxillary sinus. CONCLUSIONS: The present study revealed distinct differences in healing patterns according to the causes of extraction in the maxillary sinus, especially periodontal disease, which resulted in more severe thickening of the mucous membrane.

Effects of the dichloromethane fraction of Dipsaci Radix on the osteoblastic differentiation of human alveolar bone marrow-derived mesenchymal stem cells.

Dipsaci Radix is the dried root of Dipsacus asper Wall. It has been used in Korean herbal medicine to treat bone fractures. In this study, we examined the effect of the dichloromethane fraction of Dipsaci Radix (DR(DM)) on the osteoblastic differentiation of human alveolar bone marrow-derived MSCs (ABM-MSCs). The ABM-MSCs were isolated from healthy subjects and cultured in vitro, followed by phenotypic characterization. They showed a fibroblast-like morphology and expressed CD29, CD44, CD73, and CD105, but not CD34. Calcified nodules were generated in response to both dexamethasone (DEX) and DR(DM). There was a significant increase in the alkaline phosphatase (ALP) activity and protein expression of bone sialoprotein (BSP) and osteocalcin (OC) in response to DEX and DR(DM) as compared to control. These results provide evidence for the osteogenic potential of cultured ABM-MSCs in response to DR(DM). Also, an active single compound was additionally isolated from DR(DM). The single compound (hederagenin 3-O-(2-O-acetyl)-α-L-arabinopyranoside) also significantly increased ALP activity and the level of protein expression of BSP and OC. These results highlight the possible clinical applications of DR(DM) and hederagenin 3-O-(2-O-acetyl)-α-L-arabinopyranoside in bone regeneration.

Effects of gangliosides on the differentiation of human mesenchymal stem cells into osteoblasts by modulating epidermal growth factor receptors.

Gangliosides are sialic acid-conjugated glycosphingolipids that are believed to regulate cell differentiation as well as the signals of several signal molecules, including epidermal growth factor receptors (EGFR). These compounds are localized in a glycosphingolipid-enriched microdomain on the cell surface and regulated by the glycosphingolipid composition. However, the role that gangliosides play in osteoblastogenesis is not yet clearly understood, therefore, in this study, the relationship between gangliosides and EGFR activation was investigated during osteoblast differentiation in human mesenchymal stem cells (hMSCs). The results of high-performance thin-layer chromatography (HPTLC) showed that ganglioside GM3 expression was decreased, whereas ganglioside GD1a expression was increased during the differentiation of hMSCs into osteoblasts. In addition, an increase in the activation of alkaline phosphatase (ALP) was observed in response to treatment with EGF (5 ng/ml) and GD1a (1 microM) (p<0.05). The activation of ALP was significantly elevated in response to treatment of ganglioside GD1a with EGF when compared to control cells (p<0.01). However, treatment with GM3 (1muM) resulted in decreased ALP activation (p<0.01), and treatment of hMSCs with a chemical inhibitor of EGFR, AG1478, removed the differential effect of the two gangliosides. Moreover, incubation of the differentiating cells with GD1a enhanced the phosphorylation of EGFR, whereas treatment with GM3 reduced the EGFR phosphorylation. However, AG1478 treatment inhibited the effect of ganglioside GD1a elicitation on EGFR phosphorylation. Taken together, these results indicate that GD1a promotes osteoblast differentiation through the enhancement of EGFR phosphorylation, but that GM3 inhibits osteoblast differentiation through reduced EGFR phosphorylation, suggesting that GM3 and GD1a are essential molecules for regulating osteoblast differentiation in hMSCs.

Dynamic changes of gangliosides expression during the differentiation of embryonic and mesenchymal stem cells into neural cells.

Stem cells are used for the investigation of developmental processes at both cellular and organism levels and offer tremendous potentials for clinical applications as an unlimited source for transplantation. Gangliosides, sialic acid-conjugated glycosphingolipids, play important regulatory roles in cell proliferation and differentiation. However, their expression patterns in stem cells and during neuronal differentiation are not known. Here, we investigated expression of gangliosides during the growth of mouse embryonic stem cells (mESCs), mesenchymal stem cells (MSCs) and differentiated neuronal cells by using high-performance thin-layer chromatography (HPTLC). Monosialoganglioside 1 (GM1) was expressed in mESCs and MSCs, while GM3 and GD3 were expressed in embryonic bodies. In the 9-day old differentiated neuronal cells from mESCs cells and MSCs, GM1 and GT1b were expressed. Results from immunostaining were consistent with those observed by HPTLC assay. These suggest that gangliosides are specifically expressed according to differentiation of mESCs and MSCs into neuronal cells and expressional difference of gangliosides may be a useful marker to identify differentiation of mESCs and MSCs into neuronal cells.

Effects of aqueous extract of Sophora flavescens on the expression of cell cycle regulatory proteins in human oral mucosal fibroblasts.

Sophorae Radix, the dried roots of Sophora flavescens AITON (Leguminosae), has been used in Oriental traditional medicine for treatment of skin and mucosal ulcers, sores, gastrointestinal hemorrhage, diarrhea, inflammation and arrhythmia. In the present study, we examine the effect of the aqueous extract of Sophorae Radix (AESR) on cell proliferation and cell cycle regulation in human oral mucosal fibroblasts (HOMFs). To study the molecular mechanisms of cell cycle regulation by AESR, we also measured the intracellular levels of cell cycle regulatory proteins such as cyclin D, cyclin-dependent kinases (CDK)-4, CDK-6, cyclin E, CDK-2, p53, p21WAF1/CIP1 and p16INK4A. Cell proliferation was increased in the presence of 10 approximately 500 microg/ml of AESR. Maximal growth stimulation was observed in those cells exposed to 100 microg/ml of AESR. Exposure of HOMFs to 100 microg/ml of AESR resulted in an increase of cell cycle progression. The levels of cyclin E and CDK-2 were increased in HOMFs after 100 microg/ml of AESR treatment, but the levels of cyclin D, CDK-4, and CDK-6 were unchanged. After exposure to 100 microg/ml of AESR, the protein levels of p16INK4A and p53 were decreased as compared to that of the control group, but the level of p21WAF1/CIP1 was similar in the cells treated with 100 microg/ml of AESR and untreated cells. The results suggest that AESR may increase cell proliferation and cell cycle progression in HOMFs, which is linked to increased cellular levels of cyclin E and CDK-2 and decreased cellular levels of p53 and p16INK4A. Further studies are necessary to clarify the active constituents of AESR responsible for such biomolecular activities.