Personalized Preclinical Training in Dental Ergonomics and Endodontics in Undergraduate Dentistry Students (Pilot Study).

The curriculum at the Department of Pathophysiology in the Periodontal Sciences program at Okayama University includes normative preclinical training (NPT) using phantoms. NPT is given to the whole class of 5 th year students divided in groups of 8 students/instructor. In 2019, an innovative personalized preclinical training (PPT) pilot study was implemented for this group of students whereby two students, each with their own dental unit, were coached by one instructor. The main topics covered were dental ergonomics and endodontics. We aimed to evaluate the effectiveness of PPT in dental ergonomics and endodontics toward increasing the knowledge and future clinical skills of students who had already undergone NPT. A test on endodontics was taken before and after PPT. A questionnaire was completed to assess their perception of improvement regarding the above-mentioned topics. Test scores and questionnaire results both showed that the students' level of knowledge and awareness of future clinical skills was significantly higher after PPT. This pilot study demonstrated that PPT increased the students' knowledge and future clinical skills. As preclinical training forms the foundation for clinical practice, investment in future research regarding this personalized approach is likely to enhance students' understanding and clinical performance.

Establishing protein expression profiles involved in tooth development using a proteomic approach.

Various growth and transcription factors are involved in tooth development and developmental abnormalities; however, the protein dynamics do not always match the mRNA expression level. Using a proteomic approach, this study comprehensively analyzed protein expression in epithelial and mesenchymal tissues of the tooth germ during development. First molar tooth germs from embryonic day 14 and 16 Crlj:CD1 (ICR) mouse embryos were collected and separated into epithelial and mesenchymal tissues by laser microdissection. Mass spectrometry of the resulting proteins was carried out, and three types of highly expressed proteins [ATP synthase subunit beta (ATP5B), receptor of activated protein C kinase 1 (RACK1), and calreticulin (CALR)] were selected for immunohistochemical analysis. The expression profiles of these proteins were subsequently evaluated during all stages of amelogenesis using the continuously growing incisors of 3-week-old male ICR mice. Interestingly, these three proteins were specifically expressed depending on the stage of amelogenesis. RACK1 was highly expressed in dental epithelial and mesenchymal tissues during the proliferation and differentiation stages of odontogenesis, except for the pigmentation stage, whereas ATP5B and CALR immunoreactivity was weak in the enamel organ during the early stages, but became intense during the maturation and pigmentation stages, although the timing of the increased protein expression was different between the two. Overall, RACK1 plays an important role in maintaining the cell proliferation and differentiation in the apical end of incisors. In contrast, ATP5B and CALR are involved in the transport of minerals and the removal of organic materials as well as matrix deposition for CALR.

Autophagy as a potential mechanism underlying the biological effect of 1,25-Dihydroxyvitamin D3 on periodontitis: a narrative review.

The major active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D3), is known for its wide bioactivity in periodontal tissues. Although the exact mechanisms underlying its protective action against periodontitis remain unclear, recent studies have shown that 1,25D3 regulates autophagy. Autophagy is vital for intracellular pathogen invasion control, inflammation regulation, and bone metabolic balance in periodontal tissue homeostasis, and its regulation could be an interesting pathway for future periodontal studies. Since vitamin D deficiency is a worldwide health problem, its role as a potential regulator of autophagy provides new insights into periodontal diseases. Based on this premise, this narrative literature review aimed to investigate the possible connection between 1,25D3 and autophagy in periodontitis. A comprehensive literature search was conducted on PubMed using the following keywords (e.g., vitamin D, autophagy, periodontitis, pathogens, epithelial cells, immunity, inflammation, and bone loss). In this review, the latest studies on the protective action of 1,25D3 against periodontitis and the regulation of autophagy by 1,25D3 are summarized, and the potential role of 1,25D3-activated autophagy in the pathogenesis of periodontitis is analyzed. 1,25D3 can exert a protective effect against periodontitis through different signaling pathways in the pathogenesis of periodontitis, and at least part of this regulatory effect is achieved through the activation of the autophagic response. This review will help clarify the relationship between 1,25D3 and autophagy in the homeostasis of periodontal tissues and provide perspectives for researchers to optimize prevention and treatment strategies in the future.

Ligneous periodontitis exacerbated by Behçet's disease in a patient with plasminogen deficiency and a stop-gained variant PLG c.1468C > T: a case report.

BACKGROUND: Plasminogen serves as the precursor to plasmin, an essential element in the fibrinolytic process, and is synthesized primarily in the liver. Plasminogen activation occurs through the action of plasminogen activator, converting it into plasmin. This conversion greatly enhances the fibrinolytic system within tissues and blood vessels, facilitating the dissolution of fibrin clots. Consequently, congenital deficiency of plasminogen results in impaired fibrin degradation. Patients with plasminogen deficiency typically exhibit fibrin deposits in various mucosal sites throughout the body, including the oral cavity, eyes, vagina, and digestive organs. Behcet's disease is a chronic recurrent systemic inflammatory disease with four main symptoms: aphthous ulcers of the oral mucosa, vulvar ulcers, skin symptoms, and eye symptoms, and has been reported worldwide. This disease is highly prevalent around the Silk Road from the Mediterranean to East Asia. We report a case of periodontitis in a patient with these two rare diseases that worsened quickly, leading to alveolar bone destruction. Genetic testing revealed a novel variant characterized by a stop-gain mutation, which may be a previously unidentified etiologic gene associated with decreased plasminogen activity. CASE PRESENTATION: This case report depicts a patient diagnosed with ligneous gingivitis during childhood, originating from plasminogen deficiency and progressing to periodontitis. Genetic testing revealed a suspected association with the PLG c.1468C > T (p.Arg490*) stop-gain mutation. The patient's periodontal condition remained stable with brief intervals of supportive periodontal therapy. However, the emergence of Behçet's disease induced acute systemic inflammation, necessitating hospitalization and treatment with steroids. During hospitalization, the dental approach focused on maintaining oral hygiene and alleviating contact-related pain. The patient's overall health improved with inpatient care and the periodontal tissues deteriorated. CONCLUSIONS: Collaborative efforts between medical and dental professionals are paramount in comprehensively evaluating and treating patients with intricate complications from rare diseases. Furthermore, the PLG c.1468C > T (p.Arg490*) stop-gain mutation could contribute to the association between plasminogen deficiency and related conditions.

Reattachment of Fractured Tooth Fragment by Multidisciplinary Treatment Approach.

Dental anterior fractures are common injuries, especially in those who practice extreme sports. This report describes a 25-year-old Bolivian patient who attended our private dental clinic in La Paz, Bolivia after experiencing an accident during downhill mountain biking. An intraoral examination revealed a fracture line on the buccal side in the middle third of the coronal portion of the right central maxillary incisor which extended towards the proximal and lingual sides. Multidisciplinary treatment, including crown lengthening, osteotomy, root canal treatment, fiberglass post insertion, and reattachment of the fracture segment was performed. A follow-up examination at 10 months later revealed that the tooth was completely reestablished both functionally and esthetically and that there was no periapical pathosis or discomfort. This outcome suggests that if a patient seeks a dental consultation soon after a complex crown-root fracture has occurred, and if the broken tooth segment is available, then reattachment offers an economical and simple treatment option which will allow immediate restoration of functionality and esthetic standards. Continued follow-up should form part of such a treatment plan to allow long-term pulp vitality and periodontal health status to be monitored.

Estimation of periodontal pocket surface area in small to medium dogs: a proof-of-concept study.

BACKGROUND: Periodontal disease is the most common dental disease in dogs. Although the systemic effects of periodontal disease have not been clarified in veterinary science, it is necessary to evaluate the effects of periodontal disease in clinical trials in the future. There have been a few clinical attempts made, however, to assess the severity of periodontal inflammation and its impact on the systemic health of dogs. Meanwhile, in the field of dentistry for humans, the periodontal inflamed surface area (PISA) and periodontal epithelial surface area (PESA) have been used to quantitatively assess the degree of periodontal disease affecting a single tooth as well as the overall extent of periodontitis. Recent studies have also suggested the use of these assessments to examine the relationship between periodontal inflammation and systemic health. RESULTS: The estimation formula for a dog's periodontal pocket surface area (PPSA), an alternative to PISA and PESA in humans, was established using body weight and periodontal pocket depth. Actual values were measured using extracted teeth from various dog breeds and sizes (2.3-25.0 kg of body weight) to obtain universal regression equations for PPSA. Altogether, 625 teeth from 73 dogs of 16 breeds were extracted and subsequently analyzed for morphological information. PPSA was measured in 61 dogs of 10 breeds with periodontal disease using the established estimation formulas, and the correlation between PPSA and preoperative blood chemistry data was analyzed accordingly. A strong correlation was found between PPSA and serum globulin (r = 0.71) while moderate correlations were found for C-reactive protein (r = 0.54) and serum albumin (r = -0.51). CONCLUSIONS: Estimation formulas using body weight and the 6-point probing depth were established for determining PPSA. Direct correlations between PPSA and several blood test results were observed in the study sample. Taken together, these results suggest that PPSA could be useful for evaluating the effects of periodontitis on systemic conditions in dogs.

Graphene Oxide-based Endodontic Sealer: An in Vitro Study.

The failure of endodontic treatment is directly associated with microbial infection in the root canal or periapical areas. An endodontic sealer that is both bactericidal and biocompatible is essential for the success of root canal treatments. This is one of the vital issues yet to be solved in clinical dental practice. This in vitro study assessed the effectiveness of graphene oxide (GO) composites GO-CaF2 and GO-Ag-CaF2 as endodontic sealer materials. Dentin slices were coated with either the GO-based composites or commonly used root canal sealers (non-eugenol zinc oxide sealer). The coated slices were treated in 0.9% NaCl, phosphate-buffered saline (PBS), and simulated body fluid (SBF) at 37˚C for 24 hours to compare their sealing effect on the dentin surface. In addition, the radiopacity of these composites was examined to assess whether they complied with the requirements of a sealer for good radiographic visualization. Scanning electron microscopy showed the significant sealing capability of the composites as coating materials. Radiographic images confirmed their radiopacity. Mineral deposition indicated their bioactivity, especially of GO-Ag-CaF2, and thus it is potential for regenerative application. They were both previously shown to be bactericidal to oral microbes and cytocompatible with host cells. With such a unique assemblage of critical properties, these GO-based composites show promise as endodontic sealers for protection against reinfection in root canal treatment and enhanced success in endodontic treatment overall.

Use of Highly Accurate Devices for a First Lower Premolar Endodontic Treatment with Multiple Root Canals.

This case report highlights the importance of using a dental operating microscope (DOM) and ultrasonic endodontic tips (UETs) to locate all root canals in the lower first premolar. A 53-year-old woman presented to our clinic with pain in the lower right first premolar. After a detailed search using a DOM and UETs, three root canals were found, prepared with rotary HyFlex endodontic files, and obturated using the lateral condensation technique. At the five-year follow-up after treatment, the tooth was completely restored and fulfilling its function, with no signs or symptoms of any post-treatment flare-up.

Microbiome composition comparison in oral and atherosclerotic plaque from patients with and without periodontitis.

There is no conclusive evidence regarding a causal relationship between periodontitis and atherosclerosis. In this study, we examined the microbiome in the oral cavity and atheromatous plaques from atherosclerosis patients with or without periodontitis to investigate the role of oral bacteria in the formation of atheromatous plaques. We chose four patients with and without periodontitis, who had undergone carotid endarterectomy. Bacterial samples were extracted from the tongue surface, from periodontal pocket (during the oral examination), and from the atheromatous plaques (APs). We investigated the general and oral conditions from each patient and performed next-generation sequencing (NGS) analysis for all bacterial samples. There were no significant differences between both groups concerning general conditions. However, the microbiome patterns of the gingival pocket showed differences depending on the absence or presence of periodontitis, while those of the tongue surface were relatively similar. The microbiome pattern of the atheromatous plaques was entirely different from that on the tongue surface and gingival pocket, and oral bacteria were seldom detected. However, the microbiome pattern in atheromatous plaques was different in the presence or absence of periodontitis. These results suggested that oral bacteria did not affect the formation of atheromatous plaques directly.

Antimicrobial and antibiofilm effects of abietic acid on cariogenic Streptococcus mutans.

Dental caries is a type of oral microbiome dysbiosis and biofilm infection that affects oral and systemic conditions. For healthy life expectancy, natural bacteriostatic products are ideal for daily and lifetime use as anti-oral infection agents. This study aimed to evaluate the inhibitory effects of abietic acid, a diterpene derived from pine rosin, on the in vitro growth of cariogenic bacterial species, Streptococcus mutans. The effective minimum inhibitory concentration of abietic acid was determined through observation of S. mutans growth, acidification, and biofilm formation. The inhibitory effects of abietic acid on the bacterial membrane were investigated through the use of in situ viability analysis and scanning electron microscopic analysis. Cytotoxicity of abietic acid was also examined in the context of several human cell lines using tetrazolium reduction assay. Abietic acid was found to inhibit key bacterial growth hallmarks such as colony forming ability, adenosine triphosphate activity (both planktonic and biofilm), acid production, and biofilm formation. Abietic acid was identified as bacteriostatic, and this compound caused minimal damage to the bacterial membrane. This action was different from that of povidone-iodine or cetylpyridinium chloride. Additionally, abietic acid was significantly less cytotoxic compared to povidone-iodine, and it exerted lower toxicity towards epithelial cells and fibroblasts compared to that against monocytic cells. These data suggest that abietic acid may prove useful as an antibacterial and antibiofilm agent for controlling S. mutans infection.

Molecular imaging assessment of periodontitis lesions in an experimental mouse model.

OBJECTIVE: We aimed to evaluate molecular imaging as a novel diagnostic tool for mice periodontitis model induced by ligature and Porphyromonas gingivalis (Pg) inoculation. MATERIALS AND METHODS: Twelve female mice were assigned to the following groups: no treatment as control group (n = 4); periodontitis group induced by ligature and Pg as Pg group (n = 4); and Pg group treated with glycyrrhizinic acid (GA) as Pg + GA group (n = 4). All mice were administered a myeloperoxidase (MPO) activity-specific luminescent probe and observed using a charge-coupled device camera on day 14. Image analysis on all mice was conducted using software to determine the signal intensity of inflammation. Additionally, histological and radiographic evaluation for periodontal inflammation and bone resorption at the site of periodontitis, and quantitative enzyme-linked immunosorbent assay (ELISA) were conducted on three mice for each group. Each experiment was performed three times. RESULTS: Levels of serum IgG antibody against P. gingivalis were significantly higher in the Pg than in the Pg + GA group. Histological analyses indicated that the number of osteoclasts and neutrophils were significantly lower in the Pg + GA than in the Pg group. Micro-CT image analysis indicated no difference in bone resorption between the Pg and Pg + GA groups. The signal intensity of MPO activity was detected on the complete craniofacial image; moreover, strong signal intensity was localized specifically at the periodontitis site in the ex vivo palate, with group-wise differences. CONCLUSIONS: Molecular imaging analysis based on MPO activity showed high sensitivity of detection of periodontal inflammation in mice. CLINICAL RELEVANCE: Molecular imaging analysis based on MPO activity has potential as a diagnostic tool for periodontitis.

Anti-HMGB1 Neutralizing Antibody Attenuates Periodontal Inflammation and Bone Resorption in a Murine Periodontitis Model.

High mobility group box 1 (HMGB1) is a non-histone DNA-binding protein that is secreted into the extracellular milieu in response to inflammatory stimuli. The secreted HMGB1 mediates various inflammatory diseases, including periodontitis; however, the underlying mechanisms of HMGB1-induced periodontal inflammation are not completely understood. Here, we examined whether anti-HMGB1 neutralizing antibody inhibits periodontal progression and investigated the molecular pathology of HMGB1 in vitro and in vivo. In vitro analysis indicated that HMGB1, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-1ß (IL-1ß) were secreted in response to tumor necrosis factor-α (TNF-α) stimuli in human gingival epithelial cells (HGECs) and human monocytic leukemia cells (THP-1) treated with phorbol myristate acetate. Increased levels of GM-CSF and IL-1ß were observed in the conditioned media from TNF-α-stimulated HGECs and THP-1 in vitro Simultaneous stimulation with TNF-α and anti-HMGB1 antibody significantly decreased TNF-α-induced inflammatory cytokine secretion. Experimental periodontitis was induced in mice using Porphyromonas gingivalis-soaked ligatures. The extracellular translocation was confirmed in gingival epithelia in the periodontitis model mice by immunofluorescence analysis. Systemic administration of anti-HMGB1 neutralizing antibody significantly inhibited translocation of HMGB1. The anti-HMGB1 antibody inhibited periodontal inflammation, expression of IL-1ß and C-X-C motif chemokine ligand 1 (CXCL1), migration of neutrophils, and bone resorption, shown by bioluminescence imaging of myeloperoxidase activity, quantitative reverse transcription-PCR (RT-PCR), and micro-computed tomography analysis. These findings indicate that HMGB1 is secreted in response to inflammatory stimuli caused by periodontal infection, which is crucial for the initiation of periodontitis, and the anti-HMGB1 antibody attenuates the secretion of a series of inflammatory cytokines, consequently suppressing the progression of periodontitis.

HMGB1-induced inflammatory response promotes bone healing in murine tooth extraction socket.

High mobility group box 1 (HMGB1) is a non-histone DNA-binding protein that is secreted into the extracellular milieu in response to inflammatory stimuli. The secreted HMGB1 has been suggested to mediate various inflammatory diseases. However, it is still unknown whether HMGB1 is involved in a healing process in the tooth extraction socket, the tissue containing gingival epithelium, and alveolar bone that is exposed to oral bacteria. In this study, we constructed a murine tooth extraction model with anti-HMGB1 neutralization antibody administration and observed the inflammatory response and bone healing process in tooth extraction sockets by molecular imaging of myeloperoxidase (MPO) activity, histological analysis, and quantitative RT-PCR. The translocation of HMGB1 from the nucleus to the cytoplasm in gingival epithelial cells and inflammatory cells was inhibited by anti-HMGB1 antibody administration. The MPO activity around the tooth extraction socket was significantly reduced, and the numbers of CD31- and CD68-positive cells were significantly lower in the anti-HMGB1 antibody treatment samples than in the control samples. The TRAP-positive cells, osteocalcin positive cells, and the neoplastic bone area were significantly lower in anti-HMGB1 antibody treatment samples than in control samples. The expression levels of IL-1ß and VEGF-A were also decreased in anti-HMGB1 antibody treatment samples compared to that in control samples. Secreted HMGB1 induced initial acute inflammation and inflammatory cells recruitment after tooth extraction. HMGB1 was associated with angiogenesis and bone remodeling by osteoclast and osteoblast activation and promoted bone healing in the tooth extraction socket.

Effects of Lectins on initial attachment of cariogenic Streptococcus mutans.

Oral bacteria initiate biofilm formation by attaching to tooth surfaces via an interaction of a lectin-like bacterial protein with carbohydrate chains on the pellicle. This study aimed to find naturally derived lectins that inhibit the initial attachment of a cariogenic bacterial species, Streptococcus mutans (S. mutans), to carbohydrate chains in saliva in vitro. Seventy kinds of lectins were screened for candidate motifs that inhibit the attachment of S. mutans ATCC 25175 to a saliva-coated culture plate. The inhibitory effect of the lectins on attachment of the S. mutans to the plates was quantified by crystal violet staining, and the biofilm was observed under a scanning electron microscope (SEM). Surface plasmon resonance (SPR) analysis was performed to examine the binding of S. mutans to carbohydrate chains and the binding of candidate lectins to carbohydrate chains, respectively. Moreover, binding assay between the biotinylated-lectins and the saliva components was conducted to measure the lectin binding. Lectins recognizing a salivary carbohydrate chain, Galß1-3GalNAc, inhibited the binding of S. mutans to the plate. In particular, Agaricus bisporus agglutinin (ABA) markedly inhibited the binding. This inhibition was confirmed by SEM observation. SPR analysis indicated that S. mutans strongly binds to Galß1-3GalNAc, and ABA binds to Galß1-3GalNAc. Finally, the biotinylated Galß1-3GalNAc-binding lectins including ABA demonstrated marked binding to the saliva components. These results suggest that ABA lectin inhibited the attachment of S. mutans to Galß1-3GalNAc in saliva and ABA can be useful as a potent inhibitor for initial attachment of oral bacteria and biofilm formation.

IS1598 (IsPg4) distributed to abscess-forming strains of Porphyromonas gingivalis may enhance virulence through upregulation of nrdD-like gene expression.

An insertion sequence, IS1598 (IsPg4) has been found in virulent strains of Porphyromonas gingivalis in a murine abscess model. The present study was performed to investigate the effects of genetic rearrangements by IS1598 on the phenotypic characteristics of the virulent strains. For this purpose, we searched for a common insertion site of IS1598 among the virulent strains. Through cloning and database search, a common insertion site was identified beside an nrdD-like gene in the virulent FDC 381, W83 and W50 strains. In this region, predicted promoters of the nrdD-like gene and IS1598 are located in tandem, and accumulation of nrdD-like gene mRNA was 5-fold higher in virulent strains (W83, W50, FDC 381) than avirulent strains (ATCC33277, SU63, SUNY1021, ESO59 without IS1598). The role of the nrdD-like gene in virulence of P. gingivalis was investigated by constructing a nrdD-deficient mutant. In the murine abscess model, the parental W83 strain produced necrotic abscesses, while the nrdD-deficient mutant had almost lost this ability. Insertion of IS1598 into the nrdD-like gene promoter region may be related to the phenotypic differences in virulence among P. gingivalis strains through upregulation of the expression of this gene.

Expression of optineurin isolated from rat-injured dental pulp and the effects on inflammatory signals in normal rat kidney cells.

We previously isolated rat 14.7K-interacting protein-2 (rFIP-2) from the rat-wounded pulp. The protein, homologous to human FIP-2, is known as optineurin and was initially identified as a novel tumor necrosis factor-α (TNF-α)-inducible protein, and more recently, as an autophagy receptor. However, the biological role of optineurin in dental pulp remains elusive. We hypothesized that optineurin has a crucial role in regulating molecular processes during pulp inflammatory responses induced by TNF-α. We examined the kinetics of optineurin expression in pulp inflammation. Optineurin localization and expression were examined using rat pulp fibroblasts. The cells were treated with pharmacological inhibitors for TNF-α-induced inflammatory signals or with hydrogen peroxide as apoptotic stimuli. Stable optineurin-knockdown cells (OPTN-KD cells) were established by transfecting normal rat kidney cells with a vector expressing optineurin-specific small interfering RNA. Cell proliferation and the profiles of cytokines and intracellular signaling molecules were examined using OPTN-KD cells stimulated by TNF-α. Optineurin was localized in the cytoplasm and then translocated into the nucleus upon apoptotic stimuli. Optineurin expression was increased by TNF-α and decreased by a specific inhibitor of c-Jun N-terminal kinase. The OPTN-KD cells secreted smaller amounts of monocyte chemotactic protein-1 (MCP-1) and intracellular MCP-1 mRNA, and cell proliferation was significantly increased. Apoptosis-related signaling molecules were downregulated in OPTN-KD cells. These results demonstrated that optineurin is a crucial molecule mediated by TNF-α, which induces the production of inflammatory factors and apoptosis signaling, suggesting the presence of signaling interactions between optineurin and a transcription factor for MCP-1.

Correction to: Expression of optineurin isolated from rat-injured dental pulp and the effects on inflammatory signals in normal rat kidney cells.

In the original publication of the article, one of the author name was published incorrectly as "Keisuke Yamashairo" and correct name should be "Keisuke Yamashiro".

Aggregatibacter actinomycetemcomitans regulates the expression of integrins and reduces cell adhesion via integrin α5 in human gingival epithelial cells.

Gingival epithelial cells form a physiological barrier against bacterial invasion. Excessive bacterial invasion destroys the attachment between the tooth surface and the epithelium, resulting in periodontitis. Integrins play a significant role in cell attachment; therefore, we hypothesized that bacterial infection might decrease the expressions of these integrins in gingival epithelial cells, resulting in reduced cell adhesion. Immortalized human gingival epithelial cells were co-cultured with Aggregatibacter actinomycetemcomitans Y4 (Aa Y4), and the gene expression levels of IL-8, proliferating cell nuclear antigen (PCNA), and integrins (α2, α3, α5, ß4, and ß6) were measured using quantitative reverse transcription polymerase chain reaction. Expression of PCNA and integrins, except integrin α5, was significantly downregulated, while expression of IL-8 and integrin α5 was significantly upregulated in the cells co-cultured with Aa Y4. The number of adherent cells significantly decreased when co-cultured with Aa Y4, as determined using cell adhesion assays. In the cells co-cultured with Aa Y4 and an integrin α5 neutralizing antibody, there was no effect on the expression of IL-8 and PCNA, while the expressions of integrins α2, α3, ß4, and ß6, and the number of adherent cells did not decrease. The number of invading bacteria in the cells was reduced in the presence of the antibody and increased in the presence of TLR2/4 inhibitor. Therefore, integrin α5 might be involved in Aa Y4 invasion into gingival epithelial cells, and the resulting signal transduction cascade reduces cell adhesion by decreasing the expression of integrins, while the TLR2/4 signaling cascade regulates IL-8 expression.

Rho-kinase regulates extracellular matrix-mediated osteogenic differentiation of periodontal ligament cells.

The periodontal ligament (PDL) cells contain heterogeneous mesenchymal cell populations, which have the ability to differentiate into cells that produce adjacent mineralized tissues and abundant extracellular matrix (ECM). ECM is essential not only for the homeostasis of the periodontal tissue, but also for controlling the differentiation of the PDL cells. The process of differentiation involves mechanotransduction, which links the ECM to the cytoskeleton. The present study investigated the roles of Rho-associated coiled-coil containing protein kinase (ROCK) signaling, a crucial regulator of the cytoskeleton, during ECM-mediated osteogenic differentiation of PDL cells in vitro. The PDL cells were isolated from human periodontal ligaments of extracted teeth and cultured in osteogenic medium with or without Y-27632, a pharmacological inhibitor of ROCK. ECM-coated plates were used for ECM-mediated differentiation. The osteogenic phenotype was evaluated at different time points by real-time RT-PCR for the gene encoding alkaline phosphatase (ALP) and an ALP activity assay. The effects of ROCK on cytoskeletal changes and ECM synthesis were examined by immunofluorescence analysis. Y-27632 significantly inhibited ALP at the mRNA and protein activity levels in the late stage of differentiation; concomitantly, the actin filament content and the extracellular levels of collagen-I and fibronectin were markedly decreased by Y-27632. Exogenous collagen-I and fibronectin temporally increased ALP activity, with fibronectin showing a more pronounced effect. Importantly, ECM-mediated differentiation was almost completely inhibited by Y-27632. These findings indicated that ECM-mediated differentiation is dependent on ROCK signaling, and ROCK signaling contributes to the establishment of the ECM microenvironment for PDL cell differentiation.

Construction and evaluation of pH-sensitive immunoliposomes for enhanced delivery of anticancer drug to ErbB2 over-expressing breast cancer cells.

1,5-Dihexadecyl N,N-diglutamyl-lysyl-L-glutamate (GGLG) liposomes were previously developed to enhance drug delivery efficiency in tumor cells owing to its pH-responsive properties. Herein, we report the modification of GGLG liposomes by conjugating a Fab' fragment of an ErbB2 antibody to the terminus of PEG (polyethylene glycol)-lipid (Fab'-GGLG liposomes). The conjugation of Fab' fragments did not affect the antibody activity, drug (doxorubicin, DOX) encapsulation efficiency, stability during storage or pH-sensitivity. However, the binding affinity of Fab'-GGLG liposomes was enhanced to ErbB2-overexpressing HCC1954 cells specifically, and the cell association increased 10-fold in comparison to GGLG liposomes. Consequently, intracellular DOX delivery was enhanced, with an increased cytotoxicity in HCC1954 cells (i.e., IC50 of 1.17 and 3.08 µg/mL for Fab'-GGLG-DOX and GGLG-DOX liposomes, respectively). Further, a significantly enhanced tumor growth inhibition was obtained in an ErbB2-overexpressing breast cancer-bearing mouse model. Therefore, a potent anticancer drug delivery system was constructed by the immunological modification of pH-sensitive liposomes.