Evaluation of the reasons for preferring root canal treatment in mature permanent teeth potentially indicated for pulp preservation: a clinical case/photo-based questionnaire study.

BACKGROUND: With advances in pulp preservation procedures (PPP), indications for PPP extend to exposed pulp with symptoms in teeth with carious lesions. Scenario/text-based questionnaire studies report a high preference for PPP for exposed pulp with no pulpal symptoms. However, negative perceptions towards PPP for exposed pulp in carious teeth are prevalent among dentists. Identifying the differences in PPP preference rates in questionnaire studies and actual clinical situations is necessary to determine the current status of PPP. In this study, a clinical case/photo-based design was devised to overcome the limitations of scenario/text-based questionnaires. This study aimed to evaluate the reasons dentists prefer root canal treatment (RCT) in cases where PPP is potentially indicated. METHODS: A questionnaire containing three cases of PPP with successful results was administered to dentists. The cases were selected to elicit comprehensive responses from the dentists. Clinical photos of the pulp exposure sites were presented to dentists without describing the tooth conditions, including the extent of pulp exposure and tooth decay, pulpal surface conditions, or restorability. The questions were focused on the reasons for selecting RCT in cases where was practiced. Questionnaire data were collected using Google e-forms. Chi-squared and Fisher's exact test (P < 0.05) were used for statistical analyses. RESULTS: Pulpal diagnosis was not a dominant factor in treatment decision-making for pulp exposure during caries removal. Reasons for selecting RCT where PPP was potentially indicated included the event of pulp exposure itself and the dentists' desire to prevent post-PPP symptoms. Apart from symptomatic pulp, the tooth conditions influenced the establishment of pulpal diagnosis and selection of treatment modality. Moreover, the tooth condition and dentists' desire for good patient prognosis influenced the negative perceptions towards PPP. CONCLUSIONS: Unfavourable tooth conditions, in association with a desire for preventing post-PPP symptoms, prevent dentists from attempting PPP for pulp exposed during caries removal with no/slight symptoms. Improving negative perceptions towards PPP through accumulation of data on the high success rates of PPP is a prerequisite for achieving widespread application of PPP.

Isolation and characterization of dental follicle-derived Hertwig's epithelial root sheath cells.

OBJECTIVES: The aim of this study was the isolation and characterization of dental follicle-derived Hertwig's epithelial root sheath cells (DF-HERSCs). MATERIALS AND METHODS: DF-HERSCs were isolated from dental follicle (DF)-derived single-cell suspensions. Their epithelial phenotypes were analyzed by Western blotting, polymerase chain reaction (PCR), and quantitative polymerase chain reaction (qPCR). Epithelial-mesenchymal transition (EMT) was induced in DF-HERSCs by treatment with transforming growth factor-ß (TGF-ß) or fetal bovine serum (FBS)-added medium. Characteristics of DF-HERSCs were compared with normal human oral keratinocytes (NHOKs) and normal human epidermal keratinocytes (NHEKs). Osteogenic differentiation and mineralization of DF-HERSCs were analyzed by alkaline phosphatase (ALP) and Alizarin red staining. All experiments were conducted in triplicate. RESULTS: Primary DF-HERSCs were isolated from DF. Epithelial phenotypes of DF-HERSCs were confirmed by morphological and Western blot analysis. PCR results demonstrated that the origin of DF-HERSCs was neither endothelial nor hematopoietic. Enamel matrix derivative (EMD)-associated genes were not expressed in DF-HERSCs. Treatment with TGF-ß and FBS-added medium triggered the progression of EMT in DF-HERSCs. The acquired potency of differentiation and mineralization was shown in EMT-progressed DF-HERSCs. CONCLUSIONS: DF contains putative populations of HERSC, named DF-HERSC. DF-HERSCs shared common characteristics with NHOKs and NHEKs.

Grainyhead-like 2 regulates epithelial plasticity and stemness in oral cancer cells.

Grainyhead-like 2 (GRHL2) is one of the three mammalian homologues of Drosophila Grainyhead involved in epithelial morphogenesis. We recently showed that GRHL2 also controls normal epithelial cell proliferation and differentiation. In this study, we investigated the role of GRHL2 in oral carcinogenesis and the underlying mechanism. GRHL2 expression was elevated in cells and tissues of oral squamous cell carcinomas (OSCCs) compared with normal counterparts. Knockdown of GRHL2 resulted in the loss of in vivo tumorigenicity, cancer stemness and epithelial phenotype of oral cancer cells. GRHL2 loss also inhibited oral cancer cell proliferation and colony formation. GRHL2 regulated the expression of miR-200 family and Octamer-binding transcription factor 4 (Oct-4) genes through direct promoter DNA binding. Overexpression of miR-200 genes in the oral cancer cells depleted of GRHL2 partially restored the epithelial phenotype, proliferative rate and cancer stemness, indicating that miR-200 genes in part mediate the functional effects of GRHL2. Taken together, this study demonstrates a novel connection between GRHL2 and miR-200, and supports protumorigenic effect of GRHL2 on OSCCs.

Expression of Caveolin-1 in Periodontal Tissue and Its Role in Osteoblastic and Cementoblastic Differentiation In Vitro.

It has been previously reported that caveolin-1 (Cav-1) knockout mice exhibit increased bone size and stiffness. However, the expression and role of Cav-1 on periodontal tissue is poorly understood. The aim of this study was to investigate the immunohistochemical expression of Cav-1 in the mouse periodontium and explore the role of Cav-1 on osteoblastic and cementoblastic differentiation in human periodontal ligament cells (hPDLCs), cementoblasts, and osteoblasts. To reveal the molecular mechanisms of Cav-1 activity, associated signaling pathways were also examined. Immunolocalization of Cav-1 was studied in mice periodontal tissue. Differentiation was evaluated by ALP activity, alizarin red S staining, and RT-PCR for marker genes. Signal transduction was analyzed using Western blotting and confocal microscopy. Cav-1 expression was observed in hPDLCs, cementoblasts, and osteoblasts of the periodontium both in vivo and in vitro. Inhibition of Cav-1 expression by methyl-ß-cyclodextrin (MßCD) and knockdown of Cav-1 by siRNA promoted osteoblastic and cementoblastic differentiation by increasing ALP activity, calcium nodule formation, and mRNA expression of differentiation markers in hPDLCs, cementoblasts, and osteoblasts. Osteogenic medium-induced BMP-2 and BMP-7 expression, and phosphorylation of Smad1/5/8 were enhanced by MßCD and siRNA knockdown of Cav-1, which was reversed by BMP inhibitor noggin. MßCD and Cav-1 siRNA knockdown increased OM-induced AMPK, Akt, GSK3ß, and CREB phosphorylation, which were reversed by Ara-A, a specific AMPK inhibitor. Moreover, OM-induced activation of p38, ERK, JNK, and NF-κB was enhanced by Cav-1 inhibition. This study demonstrates, for the first time, that Cav-1 is expressed in developing periodontal tissue and in vitro in periodontal-related cells. Cav-1 inhibition positively regulates osteoblastic differentiation in hPDLCs, cementoblasts, and osteoblasts via BMP, AMPK, MAPK, and NF-κB pathway. Thus, Cav-1 inhibition may be a novel molecular target for therapeutic approaches in periodontitis or osteolytic disease.

Combined effects of mineral trioxide aggregate and human placental extract on rat pulp tissue and growth, differentiation and angiogenesis in human dental pulp cells.

OBJECTIVE: The aim of this study was to evaluate the combined effects of mineral trioxide aggregate (MTA) and human placental extract (HPE) on cell growth, differentiation and in vitro angiogenesis of human dental pulp cells (HDPCs) and to identify underlying signal transduction mechanisms. In vivo dental pulp responses in rats for a pulp-capping agent were examined. MATERIALS AND METHODS: MTS assay. ALP activity test, alizarin red S staining and RT-PCR for marker genes were carried out to evaluate cell growth and differentiation. HUVEC migration, mRNA expression and capillary tube formation were measured to evaluate angiogenesis. Signal transduction was analysed using Western blotting and confocal microscopy. The pulps of rat maxillary first molars were exposed and capped with either MTA or MTA plus HPE. Histologic observation and scoring were performed. RESULTS: Compared to treatment of HDPCs with either HPE or MTA alone, the combination of HPE and MTA increased cell growth, ALP activity, mineralized nodules and expression of marker mRNAs. Combination HPE and MTA increased migration, capillary tube formation and angiogenic gene expression compared with MTA alone. Activation of Akt, mammalian target of rapamycin (mTOR), p38, JNK and ERK MAPK, Akt, and NF-κB were significantly increased by combining HPE and MTA compared with MTA alone. Pulp capping with MTA plus HPE in rats showed superior dentin bridge formation, odontoblastic layers and dentinal tubules and lower inflammatory cell response, compared to the MTA alone group. CONCLUSIONS: This study demonstrates for the first time that the use of MTA with HPE promotes cell growth, differentiation and angiogenesis in HDPCs, which were associated with mTOR, MAPK and NF-κB pathways. Direct pulp capping with HPE plus MTA showed superior results when compared with MTA alone. Thus, the combination of MTA and HPE may be useful for regenerative endodontics.

Increased osteogenic differentiation of periodontal ligament stem cells on polydopamine film occurs via activation of integrin and PI3K signaling pathways.

BACKGROUND/AIMS: Mussel-inspired polydopamine (PDA) is known to be an effective bioadhesive and bioactive material for controlling stem cell fate, which is important in stem cell-based regenerative medicine; however, the effect of PDA on osteogenic differentiation of periodontal ligament stem cells (PDLSCs) is not fully understood. In this study, we investigated the osteoinductive effect of PDA on PDLSCs and examined how this phenomenon is encouraged. METHODS: Osteogenic induction of PDLSCs was established by culturing cells on PDA film or on an uncoated polystyrene surface as a control. Osteogenic differentiation of PDLSCs was assessed by measurement of intracellular calcium levels and alkaline phosphatase (ALP) activity as well as by evaluation of protein expression of osteocalcin (OCN), osterix (OSX), and runt-related transcription factor 2 (RUNX2). RESULTS: The PDLSCs cultured on PDA film showed higher osteogenic activity than those on the control surface. Moreover, PDLSCs on PDA film expressed increased levels of the integrin adhesion receptors integrin α5 and ß1 compared to control cells. Expression of one isoform of the intracellular signaling protein phosphatidylinositol-3-kinase (PI3K), p110γ, was increased in PDLSCs on PDA film in a PDA dose-dependent manner. This signaling protein was found to interact with integrin ß1, demonstrating integrin-linked PI3K activation in response to PDA. Finally, the blockage of PI3K reduced the PDA-induced osteogenic activity of PDLSCs. CONCLUSION: our findings suggest that the bioadhesive PDA stimulates osteogenic differentiation of PDLSCs via activation of the integrin α5/ß1 and PI3K signaling pathways.

Isolation of Epithelial Cells from Human Dental Follicle.

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while α-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells. The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

Role of Protein Phosphatase 1 in Angiogenesis and Odontoblastic Differentiation of Human Dental Pulp Cells.

INTRODUCTION: The aims of this study were to examine the immunolocalization of protein phosphatase 1 (PP1) in developing mouse pulp tissue and to explore the role of PP1 in odontoblastic differentiation and in vitro angiogenesis in human dental pulp cells (HDPCs). METHODS: Immunolocalization of PP1 was assessed in developing mouse pulp tissue. Odontogenic differentiation was examined by alkaline phosphatase activity, alizarin red staining, and reverse transcriptase polymerase chain reaction. Angiogenesis was evaluated by endothelial cell migration and capillary tube formation. Signaling pathways were analyzed by Western blotting and confocal immunofluorescence. RESULTS: PP1 expression was detected in preodontoblasts, odontoblasts, dental pulp cells, and endothelial cells within pulp tissue during the crown formed, root formation, and root completion stages. PP1 messenger RNA (mRNA) and protein levels were up-regulated at the late mineralization stage during odontogenic differentiation of HDPCs. The PP1 activator C2 ceramide increased alkaline phosphatase activity, mineralized nodule formation, and mRNA expression of dentin matrix protein 1 and dentin sialophosphoprotein. In contrast, knockdown by PP1 small interfering RNA inhibited odontoblastic differentiation. Moreover, PP1 activator up-regulated mRNA expression of angiogenic genes in HDPCs and increased the migration and capillary tube formation of endothelial cells, whereas PP1 small interfering RNA showed opposite effects. C2 ceramide increased levels of bone morphogenetic protein 2, phosphorylation of Smad 1/5/8, and mRNA expression of runt-related transcription factor 2 and osterix. CONCLUSIONS: This study provides the first evidence that PP1 might be a potent regulator of developing pulp tissue in vivo and odontoblastic differentiation and angiogenesis in HDPCs in vitro and may have clinical implications for pulp/dentin regeneration or reparative dentinogenesis.

Thymosin Beta-4 Suppresses Osteoclastic Differentiation and Inflammatory Responses in Human Periodontal Ligament Cells.

BACKGROUND: Recent reports suggest that thymosin beta-4 (Tß4) is a key regulator for wound healing and anti-inflammation. However, the role of Tß4 in osteoclast differentiation remains unclear. PURPOSE: The purpose of this study was to evaluate Tß4 expression in H2O2-stimulated human periodontal ligament cells (PDLCs), the effects of Tß4 activation on inflammatory response in PDLCs and osteoclastic differentiation in mouse bone marrow-derived macrophages (BMMs), and identify the underlying mechanism. METHODS: Reverse transcription-polymerase chain reactions and Western blot analyses were used to measure mRNA and protein levels, respectively. Osteoclastic differentiation was assessed in mouse bone marrow-derived macrophages (BMMs) using conditioned medium (CM) from H2O2-treated PDLCs. RESULTS: Tß4 was down-regulated in H2O2-exposed PDLCs in dose- and time-dependent manners. Tß4 activation with a Tß4 peptide attenuated the H2O2-induced production of NO and PGE2 and up-regulated iNOS, COX-2, and osteoclastogenic cytokines (TNF-α, IL-1ß, IL-6, IL-8, and IL-17) as well as reversed the effect on RANKL and OPG in PDLCs. Tß4 peptide inhibited the effects of H2O2 on the activation of ERK and JNK MAPK, and NF-κB in PDLCs. Furthermore, Tß4 peptide inhibited osteoclast differentiation, osteoclast-specific gene expression, and p38, ERK, and JNK phosphorylation and NF-κB activation in RANKL-stimulated BMMs. In addition, H2O2 up-regulated Wnt5a and its cell surface receptors, Frizzled and Ror2 in PDLCs. Wnt5a inhibition by Wnt5a siRNA enhanced the effects of Tß4 on H2O2-mediated induction of pro-inflammatory cytokines and osteoclastogenic cytokines as well as helping osteoclastic differentiation whereas Wnt5a activation by Wnt5a peptide reversed it. CONCLUSION: In conclusion, this study demonstrated, for the first time, that Tß4 was down-regulated in ROS-stimulated PDLCs as well as Tß4 activation exhibited anti-inflammatory effects and anti-osteoclastogenesis in vitro. Thus, Tß4 activation might be a therapeutic target for inflammatory osteolytic disease, such as periodontitis.

Comparative study of the dentin bond strength of a new universal adhesive.

This study compared the dentin bond strength of a new universal adhesive with that of contemporary multi-step dentin adhesives. Six experimental groups were prepared according to the adhesives used and their application modes: Optibond FL (OB), Adper Single Bond Plus (SB), One-Step Plus (OS), Clearfil SE Bond (CS), All-Bond Universal using etch-and-rinse mode (ABE), and AllBond Universal using self-etch mode (ABS). Micro-tensile bond strength (µTBS) and failure mode were evaluated for each group. The bonded interface was analyzed using transmission electron microscopy (TEM). As a result, µTBS of 6 experimental groups was followed as: OB=ABE=SE=ABS>SB>OS group. TEM micrographs of ABE and ABS groups revealed a homogenous adhesive layer formation. In conclusion, a new universal adhesive can make reliable bond to dentin, regardless of the application mode.

Magnetic nanofiber scaffold-induced stimulation of odontogenesis and pro-angiogenesis of human dental pulp cells through Wnt/MAPK/NF-κB pathways.

OBJECTIVE: Magnetic biomaterials have recently gained great attention due to their some intriguing cell and tissue responses. However, little attention has been given to the fields of dental tissue regeneration. In this sense, we aim to investigate the effects of magnetic nanofiber scaffolds on the human dental pulp cell (HDPC) behaviors and to elucidate the underlying signaling mechanisms in the events. METHODS: Magnetic nanofiber scaffolds incorporating magnetic nanoparticles at varying contents were prepared into nanofibrous matrices to cultivate cells. Cell growth by MTS assay, odontoblastic differentiation by alkaline phosphatase (ALP) activity, mineralization, and the mRNA expression of differentiation-related genes of HDPCs, in vitro angiogenesis by migration and capillary tube formation in endothelial cells on the conditioned medium obtained from HDPSCs in the presence or absence of scaffolds. Western blot analysis and confocal immunofluorescene were used to asses signaling pathways. RESULTS: The growth of HDPCs was significantly enhanced on the magnetic scaffolds with respect to the non-magnetic counterpart. The odontogenic differentiation of cells was significantly up-regulated by the culture with magnetic scaffolds. Furthermore, the magnetic scaffolds promoted the HDPC-induced angiogenesis of endothelial cells. The expression of signaling molecules, Wnt3a, phosphorylated GSK-3ß and nuclear ß-catenin, was substantially stimulated by the magnetic scaffolds; in parallel, the MAPK and NF-κB were highly activated when cultured on the magnetic nanofiber scaffolds. SIGNIFICANCE: This study is the first to demonstrate that magnetic nanofiber scaffolds stimulate HDPCs in the events of growth, odontogenic differentiation, and pro-angiogenesis, and the findings imply the novel scaffolds can be potentially useful as dentin-pulp regenerative matrices.

Surgical endodontic management of infected lateral canals of maxillary incisors.

This case report presents surgical endodontic management outcomes of maxillary incisors that were infected via the lateral canals. Two cases are presented in which endodontically-treated maxillary central incisors had sustained lateral canal infections. A surgical endodontic treatment was performed on both teeth. Flap elevation revealed vertical bone destruction along the root surface and infected lateral canals, and microscopy revealed that the lateral canals were the origin of the lesions. After the infected lateral canals were surgically managed, both teeth were asymptomatic and labial fistulas were resolved. There were no clinical or radiographic signs of surgical endodontic management failure at follow-up visits. This case report highlights the clinical significance and surgical endodontic management of infected lateral canal of maxillary incisor. It is important to be aware of root canal anatomy variability in maxillary incisors. Maxillary central incisors infected via the lateral canal can be successfully managed by surgical endodontic treatment.

Odontoblastic Differentiation, Inflammatory Response, and Angiogenic Potential of 4 Calcium Silicate-based Cements: Micromega MTA, ProRoot MTA, RetroMTA, and Experimental Calcium Silicate Cement.

INTRODUCTION: The aim of this study was to analyze the effects of different calcium silicate-based cements (CSCs) for pulp capping materials including MicroMega MTA (MMTA; MicroMega, Besanchon, France), RetroMTA (RMTA; BioMTA, Seoul, Korea), ProRoot MTA (PMTA; Dentsply, Tulsa, OK), and experimental CSC (ECSC) on odontoblastic differentiation, in vitro angiogenesis, and the inflammatory response in human dental pulp cells. METHODS: Differentiation was evaluated by alkaline phosphatase activity, alizarin red staining, and reverse-transcriptase polymerase chain reaction (RT-PCR) for the marker genes. The levels of inflammatory mediators and cytokines were measured by RT-PCR and an enzyme-linked immunosorbent assay. In vitro angiogenesis was assessed by RT-PCR for angiogenic genes and an endothelial tube formation assay. RESULTS: PMTA, MMTA, and ECSC increased the alkaline phosphatase activity and mineralization nodule formation and up-regulated messenger RNA (mRNA) expression of odontoblastic markers compared with RMTA. In addition, PMTA, MMTA, and ECSC up-regulated the mRNA of angiogenic genes in human dental pulp cells and increased the capillary tube formation of endothelial cells compared with RMTA. However, all CSCs showed similar expression levels of inducible nitric oxide synthase and cyclooxygenase-2 protein as well as proinflammatory mediators such as nitric oxide, prostaglandin E2, tumor necrosis factor alpha, interleukin (IL)-1ß, IL-6, and IL-8 mRNA. CONCLUSIONS: Taken together, our experimental results suggest that all CSCs are favorable materials for pulp capping, but PMTA, MMTA, and ECSC may be recommended over RMTA.

Magnetic Nanocomposite Scaffold-Induced Stimulation of Migration and Odontogenesis of Human Dental Pulp Cells through Integrin Signaling Pathways.

Magnetism is an intriguing physical cue that can alter the behaviors of a broad range of cells. Nanocomposite scaffolds that exhibit magnetic properties are thus considered useful 3D matrix for culture of cells and their fate control in repair and regeneration processes. Here we produced magnetic nanocomposite scaffolds made of magnetite nanoparticles (MNPs) and polycaprolactone (PCL), and the effects of the scaffolds on the adhesion, growth, migration and odontogenic differentiation of human dental pulp cells (HDPCs) were investigated. Furthermore, the associated signaling pathways were examined in order to elucidate the molecular mechanisms in the cellular events. The magnetic scaffolds incorporated with MNPs at varying concentrations (up to 10%wt) supported cellular adhesion and multiplication over 2 weeks, showing good viability. The cellular constructs in the nanocomposite scaffolds played significant roles in the stimulation of adhesion, migration and odontogenesis of HDPCs. Cells were shown to adhere to substantially higher number when affected by the magnetic scaffolds. Cell migration tested by in vitro wound closure model was significantly enhanced by the magnetic scaffolds. Furthermore, odontogenic differentiation of HDPCs, as assessed by the alkaline phosphatase activity, mRNA expressions of odontogenic markers (DMP-1, DSPP,osteocalcin, and ostepontin), and alizarin red staining, was significantly stimulated by the magnetic scaffolds. Signal transduction was analyzed by RT-PCR, Western blotting, and confocal microscopy. The magnetic scaffolds upregulated the integrin subunits (α1, α2, ß1 and ß3) and activated downstream pathways, such as FAK, paxillin, p38, ERK MAPK, and NF-κB. The current study reports for the first time the significant impact of magnetic scaffolds in stimulating HDPC behaviors, including cell migration and odontogenesis, implying the potential usefulness of the magnetic scaffolds for dentin-pulp tissue engineering.

Generation of functionalized polymer nanolayer on implant surface via initiated chemical vapor deposition (iCVD).

Initiated chemical vapor deposition (iCVD) was utilized to generate a 200nm thick, uniform, functionalized polymer nanolayer comprised of glycidyl methacrylate (GMA) on the surface of titanium implants as a means to improve cellular attachment. Dot-patterned GMA-coated specimens were prepared as well as fully coated specimens. In vitro cellular responses, including cell morphology, protein adsorption, cell proliferation assays, alkaline phosphate activity (ALP) assays, and calcium deposition assays were studied using adipose derived stem cells. The mechanical stability of the thin film was investigated by XPS and FE-SEM analysis of the GMA-coated implant after implantation to an extracted bone from a pig. The GMA-coated specimens displayed increased protein adsorption, higher alkaline phosphatase activities, and higher calcium deposition as compared to control sample with no cytotoxicity. Additionally, no defect was observed in the test of mechanical stability. Notably, dot-patterned GMA-coated samples displayed higher alkaline phosphatase activities than others. Functionalized polymer nanolayer deposition via iCVD is a flexible and robust technique capable of mass production of biocompatible layers. These properties make this technique very suitable for implant applications in a variety of ways.

Osteo-/odontogenic differentiation of induced mesenchymal stem cells generated through epithelial-mesenchyme transition of cultured human keratinocytes.

INTRODUCTION: Revascularization of necrotic pulp has been successful in the resolution of periradicular inflammation; yet, several case studies suggest the need for cell-based therapies using mesenchymal stem cells (MSCs) as an alternative for de novo pulp regeneration. Because the availability of MSCs may be limited, especially in an aged population, the current study reports an alternative approach in generating MSCs from epidermal keratinocytes through a process called epithelial-mesenchymal transition (EMT). METHODS: We induced EMT in primary normal human epidermal keratinocytes (NHEKs) by transient transfection of small interfering RNA targeting the p63 gene. The resulting cells were assayed for their mesenchymal marker expression, proliferation capacities as a monolayer and in a 3-dimensional collagen scaffold, and differentiation capacities. RESULTS: Transient transfection of p63 small-interfering RNA successfully abolished the expression of endogenous p63 in NHEKs and induced the expression of mesenchymal markers (eg, vimentin and fibronectin), whereas epithelial markers (eg, E-cadherin and involucrin) were lost. The NHEKs exhibiting the EMT phenotype acquired extended replicative potential and an increased telomere length compared with the control cells. Similar to the established MSCs, the NHEKs with p63 knockdown showed attachment onto the 3-dimensional collagen scaffold and underwent progressive proliferation and differentiation. Upon differentiation, these EMT cells expressed alkaline phosphatase activity, osteocalcin, and osteonectin and readily formed mineralized nodules detected by alizarin S red staining, showing osteo-/odontogenic differentiation. CONCLUSIONS: The induction of EMT in primary NHEKs by means of transient p63 knockdown allows the generation of induced MSCs from autologous sources. These cells may be used for tissues engineering purposes, including that of dental pulp.

Inhibition of osteoclast differentiation by gold nanoparticles functionalized with cyclodextrin curcumin complexes.

Gold nanoparticles (GNPs) have been previously reported to inhibit osteoclast (OC) formation. However, previous research only confirmed the osteoclastogenesis inhibitory effect under in vitro conditions. The aim of this study was to develop a therapeutic agent for osteoporosis based on the utilization of GNPs and confirm their effect both in vitro and in vivo. We prepared ß-cyclodextrin (CD) conjugated GNPs (CGNPs), which can form inclusion complexes with curcumin (CUR-CGNPs), and used these to investigate their inhibitory effects on receptor activator of nuclear factor-κb ligand (RANKL)-induced osteoclastogenesis in bone marrow-derived macrophages (BMMs). The CUR-CGNPs significantly inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells in BMMs without inducing cytotoxicity. The mRNA expressions of genetic markers of OC differentiation including c-Fos, nuclear factor of activated T cells 1 (NFATc1), TRAP, and osteoclast associated receptor (OSCAR) were significantly decreased in the presence of CUR-CGNPs. In addition, the CUR-CGNPs inhibited OC differentiation of BMMs through suppression of the RANKL-induced signaling pathway. Additionally, CUR-CGNPs caused a decrease in RANKL-induced actin ring formation, which is an essential morphological characteristic of OC formation allowing them to carry out bone resorption activity. Furthermore, the in vivo results of an ovariectomy (OVX)-induced osteoporosis model showed that CUR-CGNPs significantly improved bone density and prevented bone loss. Therefore, CUR-CGNPs may prove to be useful as therapeutic agents for preventing and treating osteoporosis.

Efavirenz induces autophagy and aberrant differentiation in normal human keratinocytes.

Although efavirenz (EFV) is efficacious as an antiretroviral therapy when combined with other antiretroviral drugs, it may cause adverse clinical effects, including skin and mucosal eruptions, central nervous system complications, hepatotoxicity, renal failure and pulmonary complications. The present study investigated the phenotypic alterations caused by EFV in normal human keratinocytes (NHKs) and determined the cell death pathways leading to the lack of epithelial proliferation and regeneration. Replication kinetics, cellular morphology, and protein and mRNA levels of cell cycle regulatory genes and cell death markers were compared between the EFV-exposed cells and the untreated control. EFV treatment led to cell proliferation arrest and cell death of the NHKs by inducing autophagy mediated by proteasome-dependent degradation of p53. EFV also reduced the levels of mTOR and active ERK signaling in NHKs. Chemical inhibition of p53 degradation with a proteasome inhibitor led to reduced autophagic response of NHKs to EFV. In addition, EFV triggered terminal differentiation of NHKs by inducing the expression of involucrin, filaggrin, loricrin and genes involved in cornified envelope formation. Inhibition of autophagy in the EFV-treated NHKs with 3-methylalanine reduced the levels of involucrin and the extent of cell death. Our data indicate that EFV elicits cytotoxic effects on NHKs in part through induction of autophagy and aberrant differentiation of cells.