Effects of prior osteoporosis treatment on the treatment response of romosozumab followed by denosumab in patients with postmenopausal osteoporosis.

In patients with postmenopausal osteoporosis, prior osteoporosis treatment affected the bone mineral density increase of following treatment with 12 months of romosozumab, although it did not affect that of following treatment with 12 months of denosumab after romosozumab. PURPOSE: To investigate the effects of prior osteoporosis treatment on the response to treatment with romosozumab (ROMO) followed by denosumab (DMAb) in patients with postmenopausal osteoporosis. METHODS: In this prospective, observational, multicenter study, treatment-naïve patients (Naïve; n = 55) or patients previously treated with bisphosphonates (BP; n = 37), DMAb (DMAb; n = 45) or teriparatide (TPTD; n = 17) (mean age, 74.6 years; T-scores of the lumbar spine [LS] - 3.2 and total hip [TH] - 2.6) were switched to ROMO for 12 months, followed by DMAb for 12 months. Bone mineral density (BMD) and serum bone turnover markers were evaluated for 24 months. RESULTS: A BMD increase was observed at 12 and 24 months in the following patients: Naïve (18.2% and 22.0%), BP (10.2% and 12.1%), DMAb (6.6% and 9.7%), and TPTD (10.8% and 15.0%) (P < 0.001 between the groups at both 12 and 24 months) in LS and Naïve (5.5% and 8.3%), BP (2.9% and 4.1%), DMAb (0.6% and 2.2%), and TPTD (4.3% and 5.4%) (P < 0.01 between the groups at 12 months and P < 0.001 at 24 months) in TH, respectively. The BMD increase in LS from 12 to 24 months was negatively associated with the levels of bone resorption marker at 24 months. Incidences of major fragility fractures for the respective groups were as follows: Naïve (5.5%), BP (16.2%), DMAb (11.1%), and TPTD (5.9%). CONCLUSIONS: Previous treatment affected the BMD increase of following treatment with ROMO, although it did not affect that of following treatment with DMAb after ROMO.

Prediction of pathological response to preoperative chemotherapy for pancreatic ductal adenocarcinoma using 2-[18F]-fluoro-2-deoxy-d-glucose positron-emission tomography.

AIM: To determine whether the pathological response to preoperative chemotherapy for pancreatic ductal adenocarcinoma (PDAC) can be predicted using 2-[18F]-fluoro-2-deoxy-d-glucose positron-emission tomography (F-18 FDG-PET). MATERIALS AND METHODS: Twenty-eight patients with PDAC who underwent only neoadjuvant chemotherapy (NAC) before surgery were enrolled in the study. All patients had F-18 FDG-PET examinations before NAC. The resected specimen was pathologically evaluated according to the Classification of Pancreatic Carcinoma (7th edn). Patients were categorised into a non-response group and a response group based on the pathological findings. The non-response group (Grades 1a and 1b) showed ≤50% necrosis in the specimen, while the specimens of the response group (Grades 2-3) showed >50% necrosis. The maximum standardised uptake values (SUVmax) of the tumours on F-18 FDG-PET were measured. The mean values of SUVmax were compared between the two groups. The diagnostic performance of SUVmax in distinguishing the two groups was also evaluated using receiver operating characteristic analysis. RESULTS: The mean SUVmax of the response group was higher than that of the non-response group (9.00 ± 1.78 versus 4.26 ± 2.35; p<0.001). The optimal cut-off value of SUVmax was 9.28 for distinguishing the two groups. The sensitivity, specificity, and accuracy for the prediction in the response group were 80%, 95.7%, and 92.9%, respectively. CONCLUSIONS: SUVmax on F-18 FDG-PET may be useful as a biomarker to predict the pathological response to NAC in patients with PDAC.

Chemogenetic modulation reveals distinct roles of the subthalamic nucleus and its afferents in the regulation of locomotor sensitization to amphetamine in rats.

The subthalamic nucleus (STN) is a key node in cortico-basal-ganglia thalamic circuits, guiding behavioral output through its position as an excitatory relay of the striatal indirect pathway and its direct connections with the cortex. There have been conflicting results regarding the role of the STN in addiction-related behavior to psychostimulants, and little is known with respect to the role of STN afferents. To address this, we used viral vectors to express DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) in the STN of rats in order to bidirectionally manipulate STN activity during the induction of amphetamine sensitization. In addition, we used a Cre-recombinase dependent Gi/o-coupled DREADD approach to transiently inhibit afferents from ventral pallidum (a subcomponent of the striatal indirect pathway) or the prelimbic cortex (a subcomponent of the cortico-STN hyperdirect pathway). Despite inducing mild hyperactivity in non-drug controls, stimulation of STN neurons with Gq-DREADDs blocked the development and persistence of amphetamine sensitization as well as conditioned responding. In contrast, inhibition of STN neurons with Gi/o-DREADDs enhanced the induction of sensitization without altering its persistence or conditioned responding. Chemogenetic inhibition of afferents from ventral pallidum had no effect on amphetamine sensitization but blocked conditioned responding whereas chemogenetic inhibition of afferents from prelimbic cortex attenuated the persistence of sensitization as well as conditioned responding. These results suggest the STN and its afferents play complex roles in the regulation of amphetamine sensitization and highlight the need for further characterization of how integration of inputs within STN guide behavior.

In vitro evidence of propagation of superoxide dismutase-1 protein aggregation in canine degenerative myelopathy.

Canine degenerative myelopathy (DM) is a progressive and fatal neurodegenerative disorder that has been linked to mutations in the superoxide dismutase 1 (SOD1) gene. The accumulation of misfolded protein aggregates in spinal neurons and astrocytes is implicated as an important pathological process in DM; however, the mechanism of protein aggregate formation is largely unknown. In human neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), cell-to-cell propagation of disease-relevant proteins has been demonstrated. Therefore, in this study, propagation of aggregation-forming property of mutant SOD1 protein in DM in vitro was investigated. This study demonstrated that aggregates composed of canine wild type SOD1 protein were increased by co-transfection with canine mutant SOD1 (E40K SOD1), indicating intracellular propagation of SOD1 aggregates. Further, aggregated recombinant SOD1 proteins were released from the cells, taken up by other cells, and induced further aggregate formation of normally folded SOD1 proteins. These results suggest intercellular propagation of SOD1 aggregates. The hypothesis of cell-to-cell propagation of SOD1 aggregates proposed in this study may underly the progressive nature of DM pathology.

Disease-modifying effects of COX-2 selective inhibitors and non-selective NSAIDs in osteoarthritis: a systematic review.

Osteoarthritis (OA) is a potentially disabling disease whose progression is dependent on several risk factors. OA management usually involves the use of non-steroidal anti-inflammatory drugs (NSAIDs) that are the primary pharmacological treatments of choice. However, NSAIDs have often been associated with unwanted side effects. Cyclooxygenase (COX)-2 specific inhibitors, such as celecoxib, have been successfully used as an alternative in the past for OA treatment and have demonstrated fewer side effects. While abundant data are available for the clinical efficacy of drugs used for OA treatment, little is known about the disease-modifying effects of these agents. A previous review published by Zweers et al. (2010) assessed the available literature between 1990 and 2010 on the disease-modifying effects of celecoxib. In the present review, we aimed to update the existing evidence and identify evolving concepts relating to the disease-modifying effects of not just celecoxib, but also other NSAIDs. We conducted a review of the literature published from 2010 to 2016 dealing with the effects, especially disease-modifying effects, of NSAIDs on cartilage, synovium, and bone in OA patients. Our results show that celecoxib was the most commonly used drug in papers that presented data on disease-modifying effects of NSAIDs. Further, these effects appeared to be mediated through the regulation of prostaglandins, cytokines, and direct changes to tissues. Additional studies should be carried out to assess the disease-modifying properties of NSAIDs in greater detail.

Results of a multi-institutional, randomized, non-inferiority, phase III trial of accelerated fractionation versus standard fractionation in radiation therapy for T1-2N0M0 glottic cancer: Japan Clinical Oncology Group Study (JCOG0701).

Background: We assessed the non-inferiority of accelerated fractionation (AF) (2.4 Gy/fraction) compared with standard fractionation (SF) (2 Gy/fraction) regarding progression-free survival (PFS) in patients with T1-2N0M0 glottic cancer (GC). Patients and methods: In this multi-institutional, randomized, phase III trial, patients were enrolled from 32 Japanese institutions. Key inclusion criteria were GC T1-2N0M0, age 20-80, Eastern Cooperative Oncology Group performance status of 0-1, and adequate organ function. Patients were randomly assigned to receive either SF of 66-70 Gy (33-35 fractions), or AF of 60-64.8 Gy (25-27 fractions). The primary end point was the proportion of 3-year PFS. The planned sample size was 360 with a non-inferiority margin of 5%. Results: Between 2007 and 2013, 370 patients were randomized (184/186 to SF/AF). Three-year PFS was 79.9% (95% confidence interval [CI] 73.4-85.4) for SF and 81.7% (95% CI 75.4-87.0) for AF (difference 1.8%, 91% CI-5.1% to 8.8%; one-sided P = 0.047 > 0.045). The cumulative incidences of local failure at 3 years for SF/AF were 15.9%/10.3%. No significant difference was observed in 3-year overall survival (OS) between SF and AF. Grade 3 or 4 acute and late toxicities developed in 22 (12.4%)/21 (11.5%) and 2 (1.1%)/1 (0.5%) in the SF/AF arms. Conclusion: Although the non-inferiority of AF was not confirmed statistically, the similar efficacy and toxicity of AF compared with SF, as well as the practical convenience of its fewer treatment sessions, suggest the potential of AF as a treatment option for early GC. Clinical trials registration: UMIN Clinical Trial Registry, number UMIN000000819.

Toward classification criteria for early osteoarthritis of the knee.

OBJECTIVE: To propose draft classification criteria for early stage osteoarthritis (OA) of the knee for use in a primary care setting. METHODS: A group of basic scientists, physician-scientists, rheumatologists, orthopedic surgeons, and physiotherapists in a workshop setting discussed potential classification criteria for early osteoarthritis of the knee. The workshop was divided into sessions around relevant topics with short state of the art presentations followed by breakout sessions, consensus discussions, and consolidation into a consensus document. RESULTS: Three classes of criteria were agreed: (1) Pain, symptoms/signs, self-reported function, and quality of life using tools such as KOOS: scoring ≤85% in at least 2 out of these 4 categories; (2) Clinical examination: at least 1 present out of joint line tenderness or crepitus; (3) Knee radiographs: Kellgren & Lawrence (KL) grade of 0 or 1. MRI is at present not recommended as an aid to identify or define early OA in routine clinical practice or primary care, in light of the absence of validated consensus criteria and the high population prevalence of structural joint changes detected by this method. Biomarkers may have future utility in early OA classification, but no individual or set of biomarkers is yet robust enough. CONCLUSION: Based on our consensus proposal, draft classification criteria for early OA of the knee for use in clinical studies should include patient reported outcomes such as pain and function, together with clinical signs and KL grade 0-1 on radiographs.

Changes in axial alignment of the ipsilateral hip and knee after total hip arthroplasty.

AIMS: We investigated changes in the axial alignment of the ipsilateral hip and knee after total hip arthroplasty (THA). PATIENTS AND METHODS: We reviewed 152 patients undergoing primary THA (163 hips; 22 hips in men, 141 hips in women) without a pre-operative flexion contracture. The mean age was 64 years (30 to 88). The diagnosis was osteoarthritis (OA) in 151 hips (primary in 18 hips, and secondary to dysplasia in 133) and non-OA in 12 hips. A posterolateral approach with repair of the external rotators was used in 134 hips and an anterior approach in 29 hips. We measured changes in leg length and offset on radiographs, and femoral anteversion, internal rotation of the hip and lateral patellar tilt on CT scans, pre- and post-operatively. RESULTS: The mean internal rotation increased by 11° (-15° to 46°) and was associated with underlying disease (OA), pre-operative range of internal rotation, gender, surgical approach, leg lengthening, and change of femoral anteversion (adjusted R(2) : 0.253, p < 0.001). The mean lateral patellar tilt increased by 4° (-5° to 14°) and was associated with age, leg lengthening, and increment of hip internal rotation (adjusted R(2): 0.193, p < 0.001). CONCLUSION: Both internal rotation of the hip at rest and lateral patellar tilt are increased after THA. Changes in rotation after THA may affect gait, daily activities, the rate of dislocation of the hip, and ipsilateral knee pain. TAKE HOME MESSAGE: Internal rotation of the hip at rest and lateral patellar tilt increase after THA.

Corticostriatal Afferents Modulate Responsiveness to Psychostimulant Drugs and Drug-Associated Stimuli.

The medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) are both integral components of the corticobasal ganglia-thalamic circuitry that regulates addiction-related behaviors. However, the role of afferent inputs from mPFC to NAc in these behaviors is unclear. To address this, we used a Cre-recombinase-dependent viral vector approach to express G(i/o)-coupled DREADDs (designer receptors exclusively activated by designer drugs) selectively in mPFC neurons projecting to the NAc and examined the consequences of attenuating activity of these neurons on the induction of amphetamine sensitization and on drug taking and drug seeking during cocaine self-administration. Surprisingly, decreasing mPFC afferent activity to the NAc only transiently reduced locomotor sensitization and had no effect on drug taking during cocaine self-administration. However, inhibiting corticostriatal afferent activity during sensitization subsequently enhanced conditioned responding. In addition, this manipulation during drug self-administration resulted in slower rates of extinction and increased responding during drug prime-induced reinstatement-an effect that was normalized by inhibiting these corticostriatal afferents immediately before the drug prime. These results suggest that dampening cortical control over the NAc during drug exposure may lead to long-term changes in the ability of drugs and associated stimuli to drive behavior that has important implications for guiding treatments to prevent relapse.

IL-1ß-induced matrix metalloproteinase-3 regulates cell proliferation in rat dental pulp cells.

OBJECTIVE: We previously reported that matrix metalloproteinase-3(MMP-3) accelerates wound healing following dental pulp injury. In this study, we tested the hypothesis that induction of MMP-3 activity by interleukin-1ß would promote proliferation and apoptosis of dental pulp cells. MATERIALS AND METHODS: Dental pulp cells were isolated from rat incisors and subjected to interleukin-1ß. Matrix metalloproteinase-3 mRNA and protein expression were assessed using reverse transcription-polymerase chain reaction and Western blotting, respectively. Matrix metalloproteinase-3 activity was measured using fluorescence. Dental pulp cell proliferation and apoptosis were determined using enzyme-linked immunosorbent assays (ELISA) for BrdU and DNA fragmentation, respectively. siRNA was used to reduce MMP-3 transcripts in these cells. RESULTS: Treatment with interleukin-1ß increased MMP-3 mRNA and protein levels as well as its activity in dental pulp cells. Cell proliferation was also markedly increased, with no changes in apoptosis observed. Treatment with siRNA against MMP-3 potently suppressed this interleukin-1ß-induced increase in MMP-3 expression and activity, and also suppressed cell proliferation but unexpectedly increased apoptosis in these cells (P < 0.05). This siRNA-mediated increase in apoptosis could be reversed with exogenous MMP-3 stimulation (P < 0.05). CONCLUSIONS: Interleukin-1ß induces MMP-3-regulated cell proliferation and suppresses apoptosis in dental pulp cells.

Similar in vitro effects and pulp regeneration in ectopic tooth transplantation by basic fibroblast growth factor and granulocyte-colony stimulating factor.

OBJECTIVES: Granulocyte-colony stimulating factor (G-CSF) has been shown to have combinatorial trophic effects with dental pulp stem cells for pulp regeneration. The aim of this investigation is to examine the effects of basic fibroblast growth factor (bFGF) in vitro and in vivo compared with those of G-CSF and to assess the potential utility of bFGF as an alternative to G-CSF for pulp regeneration. MATERIALS AND METHODS: Five different types of cells were examined in the in vitro effects of bFGF on cell migration, proliferation, anti-apoptosis, neurite outgrowth, angiogenesis, and odontogenesis compared with those of G-CSF. The in vivo regenerative potential of pulp tissue including vasculogenesis and odontoblastic differentiation was also compared using an ectopic tooth transplantation model. RESULTS: Basic fibroblast growth factor was similar to G-CSF in high migration, proliferation and anti-apoptotic effects and angiogenic and neurite outgrowth stimulatory activities in vitro. There was no significant difference between bFGF and G-CSF in the regenerative potential in vivo. CONCLUSIONS: The potential utility of bFGF for pulp regeneration is demonstrated as a homing/migration factor similar to the influence of G-CSF.

Pt-free solar driven photoelectrochemical hydrogen fuel generation using 1T MoS2 co-catalyst assembled CdS QDs/TiO2 photoelectrode.

The solar-to-hydrogen generation from the TiO2-CdS-ZnS-MoS2 (TCZM) heterointerface was demonstrated. We found that a Pt-free CdS quantum dot-sensitized TiO2 mesoporous electrode with a metallic-type 1T MoS2 co-catalyst resulted in 0.11 ml cm(-2) h(-1) H2 fuel generation in unassisted potential mode, which was strikingly improved to 1.47 ml cm(-2) h(-1) under 1 V applied potential.

Cyclic compressive loading on 3D tissue of human synovial fibroblasts upregulates prostaglandin E2 via COX-2 production without IL-1ß and TNF-α.

OBJECTIVE: Excessive mechanical stress on synovial joints causes osteoarthritis (OA) and results in the production of prostaglandin E2 (PGE2), a key molecule in arthritis, by synovial fibroblasts. However, the relationship between arthritis-related molecules and mechanical stress is still unclear. The purpose of this study was to examine the synovial fibroblast response to cyclic mechanical stress using an in vitro osteoarthritis model. METHOD: Human synovial fibroblasts were cultured on collagen scaffolds to produce three-dimensional constructs. A cyclic compressive loading of 40 kPa at 0.5 Hz was applied to the constructs, with or without the administration of a cyclooxygenase-2 (COX-2) selective inhibitor or dexamethasone, and then the concentrations of PGE2, interleukin-1ß (IL-1ß), tumour necrosis factor-α (TNF-α), IL-6, IL-8 and COX-2 were measured. RESULTS: The concentrations of PGE2, IL-6 and IL-8 in the loaded samples were significantly higher than those of unloaded samples; however, the concentrations of IL-1ß and TNF-α were the same as the unloaded samples. After the administration of a COX-2 selective inhibitor, the increased concentration of PGE2 by cyclic compressive loading was impeded, but the concentrations of IL-6 and IL-8 remained high. With dexamethasone, upregulation of PGE2, IL-6 and IL-8 was suppressed. CONCLUSION: These results could be useful in revealing the molecular mechanism of mechanical stress in vivo for a better understanding of the pathology and therapy of OA. Cite this article: Bone Joint Res 2014;3:280-8.

Avaliação da acessibilidade à assistência farmacêutica básica no município de Várzea Grande (Mato Grosso) / Study on the accessibility of basic pharmaceutical services to the public in the city of Várzea Grande (Mato Grosso, Brazil)

In order to gauge the accessibility of essential medicines in the service network pertaining to the Brazilian Public Health System (SUS) in the city of Várzea Grande (MT), we performed a case study with multiple levels of analysis. We identified public access as an important dimension of pharmaceutical services in the city after carrying out an exploratory analysis (or feasibility study) on how to assess their implementation. We designed a logical model of pharmaceutical services in the city, from which was derived a matrix containing the questions and criteria for assessment. This matrix was validated by a consensus of experts and with information gathered from semi-structured interviews, document analysis and observation. The findings show that the National Policy for Pharmaceutical Service has been implemented only to a limited extent (34.7%) in this city, given that problems were found in all the component parts of the cycle of pharmaceutical service. Only geographical accessibility was ranked as at an advanced stage (89.5%). We conclude with recommendations for improvements related to criteria assessed as still at an early stage, especially with respect to the components of organizational accessibility. Finally, we note that the obstacles to be overcome not only call for specific measures related to pharmaceutical service itself, but also concern more general issues regarding health care organization...

Com o objetivo de avaliar a acessibilidade a medicamentos essenciais na rede de serviços pertencentes ao Sistema Único de Saúde (SUS) no município de Várzea Grande-MT, foi realizado um estudo de caso com múltiplos níveis de análise. A identificação do acesso como dimensão importante para avaliação da Assistência Farmacêutica no município foi identificada após a realização de uma análise exploratória ou estudo de avaliabilidade. Foi elaborado um modelo lógico da Assistência Farmacêutica no município do qual foi derivada uma matriz contendo as dimensões e critérios para avaliação. Essa matriz foi validada por meio de consenso de experts e as informações obtidas por meio de entrevistas semiestruturadas, análise documental e observação. Os achados mostram que a Política Nacional de Assistência Farmacêutica está implantada de forma incipiente (34,7%), no município estudado, tendo em vista que foram encontrados problemas em todos os componentes do ciclo de Assistência Farmacêutica. Apenas a acessibilidade geográfica foi classificada como em avançada (89,5%). Os autores concluem com recomendações relacionadas ao aperfeiçoamento dos critérios avaliados como em estágio incipiente, principalmente no que diz respeito aos componentes da acessibilidade organizacional. Registram ainda que a superação dos obstáculos encontrados relaciona-se não apenas com medidas específicas relacionadas à Assistência Farmacêutica propriamente dita, mas também com aspectos de ordem mais geral relacionados a organização da atenção a saúde...

Synergistic metal-metal oxide nanoparticles supported electrocatalytic graphene for improved photoelectrochemical glucose oxidation.

We report the fabrication of graphene-WO3-Au hybrid membranes and evaluate their photocatalytic activity towards glucose oxidase mediated enzymatic glucose oxidation. The dual-functionality of gold nanoparticles in the reinforcement of visible light activity of graphene-WO3 membranes and improving the catalytic activity of immobilized enzymes for unique photoelectrochemical sensing application is demonstrated. This work provides new insights into the fabrication of light-sensitive hybrid materials and facilitates their application in future.

Aetiology, incidence and morphology of the C-shaped root canal system and its impact on clinical endodontics.

The C-shaped root canal constitutes an unusual root morphology that can be found primarily in mandibular second permanent molars. Due to the complexity of their structure, C-shaped root canal systems may complicate endodontic interventions. A thorough understanding of root canal morphology is therefore imperative for proper diagnosis and successful treatment. This review aims to summarize current knowledge regarding C-shaped roots and root canals, from basic morphology to advanced endodontic procedures. To this end, a systematic search was conducted using the MEDLINE, BIOSIS, Cochrane Library, EMBASE, Google Scholar, Web of Science, PLoS and BioMed Central databases, and many rarely cited articles were included. Furthermore, four interactive 3D models of extracted teeth are introduced that will allow for a better understanding of the complex C-shaped root canal morphology. In addition, the present publication includes an embedded best-practice video showing an exemplary root canal procedure on a tooth with a pronounced C-shaped root canal. The survey of this unusual structure concludes with a number of suggestions concerning future research efforts.

Cytokines induce MMP-3-regulated proliferation of embryonic stem cell-derived odontoblast-like cells.

OBJECTIVES: Matrix metalloproteinase (MMP)-3 expression increases after pulpectomy and accelerates angiogenesis in rat dental pulp by an uncharacterised mechanism. Odontoblasts, a major component of dental pulp, could represent a therapeutic target. We investigated whether MMP-3 activity is induced by cytokines and/or is associated with cell proliferation and apoptosis in embryonic stem cell-derived odontoblast-like cells. MATERIALS AND METHODS: We used reverse transcriptase polymerase chain reaction, western blotting, an MMP-3 activity assay, a BrdU-cell proliferation enzyme-linked immunosorbent assay and DNA fragmentation analysis to evaluate siRNA-mediated downregulation of MMP-3 expression and activity, and any changes in the proliferative and apoptotic responses associated with this reduced expression. RESULTS: Pro-inflammatory cytokines (interleukin-1ß, tumour necrosis factor-α and interferon-γ, at relatively low concentrations) induced MMP-3 mRNA and protein expression, and increased MMP-3 activity and cell proliferation, but not apoptosis. MMP-3 silencing produced a potent and significant suppression of cytokine-induced MMP-3 expression and activity, decreased cell proliferation and increased apoptosis. These effects were rescued by application of exogenous MMP-3. CONCLUSIONS: Our results suggest that pro-inflammatory cytokines induce MMP-3-regulated cell proliferation and anti-apoptosis effects in odontoblast-like cells derived from embryonic stem cells, in addition to their well-documented destructive role in inflammation.

Mouse ES cells have a potential to differentiate into odontoblast-like cells using hanging drop method.

OBJECTIVES: We examined whether mouse embryonic stem (ES) cells can differentiate into odontoblast-like cells without epithelial-mesenchymal interaction. MATERIALS AND METHODS: Cells were cultured by the 'hanging drop' method using a collagen type-I scaffold (CS) combined with bone morphogenetic protein (BMP)-4 (CS/BMP-4). Expression of odontoblast-related mRNA and protein, and cell proliferation were performed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and WST-1 assay, respectively. RESULTS: Cells potently expressed odontoblast-related cell marker mRNAs following induction of odontoblastic differentiation. Dentin sialophosphoprotein, a marker of mature odontoblasts, was strongly expressed in differentiated ES cells. The cells also acquired an odontoblast-like functional phenotype, as evidenced by the appearance of alkaline phosphatase activity and calcification. The cell-surface expression of α2, α6, αV and αVß3 integrin proteins was rapidly upregulated in differentiated cells. Finally, anti-α2 integrin antibody suppressed the expression of odontoblastic markers in cells grown using this culture system, suggesting that α2 integrin expression in ES cells triggers their differentiation into odontoblast-like cells. CONCLUSIONS: Mouse ES cells cultured by the 'hanging drop' method are able to differentiate into cells with odontoblast-specific physiological functions and cell-surface integrin protein expression.