d-Alanine metabolic pathway, a potential target for antibacterial drug designing in Enterococcus faecalis.

Enterococcus faecalis (E. faecalis) is associated with persistent root canal infection because of its biofilm and various virulence factors. However, E. faecalis exhibits extensive drug resistance. d-Alanine (D-Ala) metabolism is essential for bacterial peptidoglycan biosynthesis. d-cycloserine (DCS), a second line drug used in the treatment of Mycobacterium tuberculosis infection, can inhibit two key enzymes in D-Ala metabolism: alanine racemase and d-alanine-d-alanine ligase. The aim of this study was to evaluate the effect of D-Ala metabolism on E. faecalis growth, cell wall integrity, biofilm formation and virulence gene expression by additional DCS with or without D-Ala. The results showed that DCS inhibited the planktonic growth and biofilm formation of E. faecalis in a dose-dependent manner. Both the minimum inhibitory concentration (MIC) and minimum biofilm inhibition concentration (MBIC) of DCS against E. faecalis were 200 µg/ml, whereas 50 µg/ml of DCS could inhibit planktonic growth and biofilm formation effectively. The addition of DCS also resulted in bacterial cell wall damage, biofilm surface roughness increase and biofilm adhesion force reduction. Moreover, the treatment of DCS downregulated the expression of asa1, esp, efaA, gelE, sprE, fsrB and ace genes. However, all of these inhibitory effects of DCS could be rescued by the addition of exogenous D-Ala. Meanwhile, DCS exhibited no toxicity to HGEs and HOKs. Therefore, D-Ala metabolic pathway in E. faecalis is a potential target for drug designing.

Accumulated cytotoxicity of CDK inhibitor dinaciclib with first-line chemotherapy drugs in salivary adenoid cystic carcinoma cells.

Adenoid cystic carcinoma (ACC) is one of the most common salivary gland malignant tumors. Its treatment failure is partly due to the limitations of chemotherapeutic agents and their adverse effects. The objective of this study was to determine the potential additive anti-cancer effect of a novel CDK inhibitor dinaciclib with first-line chemotherapy drugs in ACC. Protein expression of phosphorylated CDK2 (p-CDK2) in paraffin-embedded tissue specimens of ACC from 17 patients was investigated by immunohistochemistry (IHC). Cell Counting Kit (CCK-8), clone formation assay, and flow cytometry were used to test the proliferation and apoptosis of ACC-2 cells treated with dinaciclib with or without other first-line chemotherapy drugs. Protein expression was also determined by Western blot. Interestingly, we discovered that p-CDK2 protein was expressed in both cytoplasmic and nucleus in salivary ACC tissues, which was higher than that in normal salivary tissues, indicating that agents targeting CDK2 may be potential therapeutic strategies against this type of tumor. As expected, CDK inhibitor dinaciclib significantly induced ACC-2 cells apoptosis. Moreover, it sensitized cells to the chemotherapeutic agents such as cisplatin, pemetrexed, and etoposide (VP-16), and this effect by dinaciclib may induce cell cycle arrest via abrogating CDK2 activity. Therefore, combinational therapy of CDK inhibitor dinaciclib with first-line chemotherapy drugs may be a promising strategy in the treatment of salivary ACC.

A novel mutation of adenomatous polyposis coli (APC) gene results in the formation of supernumerary teeth.

Supernumerary teeth are teeth that are present in addition to normal teeth. Although several hypotheses and some molecular signalling pathways explain the formation of supernumerary teeth, but their exact disease pathogenesis is unknown. To study the molecular mechanisms of supernumerary tooth-related syndrome (Gardner syndrome), a deeper understanding of the aetiology of supernumerary teeth and the associated syndrome is needed, with the goal of inhibiting disease inheritance via prenatal diagnosis. We recruited a Chinese family with Gardner syndrome. Haematoxylin and eosin staining of supernumerary teeth and colonic polyp lesion biopsies revealed that these patients exhibited significant pathological characteristics. APC gene mutations were detected by PCR and direct sequencing. We revealed the pathological pathway involved in human supernumerary tooth development and the mouse tooth germ development expression profile by RNA sequencing (RNA-seq). Sequencing analysis revealed that an APC gene mutation in exon 15, namely 4292-4293-Del GA, caused Gardner syndrome in this family. This mutation not only initiated the various manifestations typical of Gardner syndrome but also resulted in odontoma and supernumerary teeth in this case. Furthermore, RNA-seq analysis of human supernumerary teeth suggests that the APC gene is the key gene involved in the development of supernumerary teeth in humans. The mouse tooth germ development expression profile shows that the APC gene plays an important role in tooth germ development. We identified a new mutation in the APC gene that results in supernumerary teeth in association with Gardner syndrome. This information may shed light on the molecular pathogenesis of supernumerary teeth. Gene-based diagnosis and gene therapy for supernumerary teeth may become available in the future, and our study provides a high-resolution reference for treating other syndromes associated with supernumerary teeth.

Recent advancements in hydrogels as novel tissue engineering scaffolds for dental pulp regeneration.

Although conventional root canal treatment offers an effective therapeutic solution, it negatively affects the viability of the affected tooth. In recent years, pulp regeneration technology has emerged as a novel method for treating irreversible pulpitis due to its ability to maintain tooth vitality. The successful implementation of this technique depends on scaffolds and transplantation of exogenous stem cells or recruitment of endogenous stem cells. Accordingly, the three-dimensional structure and viscoelastic characteristics of hydrogel scaffolds, which parallel those of the extracellular matrix, have generated considerable interest. Furthermore, hydrogels support the controlled release of regenerative drugs and to load a wide variety of bioactive molecules. By integrating antibacterial agents into the hydrogel matrix and stimulating an immune response, root canal disinfection can be significantly improved and the rate of pulp regeneration can be accelerated. This review aims to provide an overview of the clinical applications of hydrogels that have been reported in the last 5 years, and offer a comprehensive summary of the different approaches that have been utilized for the optimization of hydrogel scaffolds for pulp regeneration. Advancements and challenges in pulp regeneration using hydrogels treating aged teeth are discussed.

Novel self-setting cements based on tricalcium silicate/(ß-tricalcium phosphate/monocalcium phosphate anhydrous)/hydroxypropyl methylcellulose: From hydration mechanism to biological evaluations.

Despite the clinical success of tricalcium silicate (TCS)-based materials in endodontics, the inferior handling characteristic, poor anti-washout property and slow setting kinetics hindered their wider applications. To solve these problems, an injectable fast-setting TCS/ß-tricalcium phosphate/monocalcium phosphate anhydrous (ß-TCP/MCPA) cement was developed for the first time by incorporation of hydroxypropyl methylcellulose (HPMC) and ß-TCP/MCPA. The physical-chemical characterization (setting time, anti-washout property, injectability, compressive strength, apatite mineralization and sealing property) of TCS/(ß-TCP/MCPA) were conducted. Its hydration mechanism was also investigated. Furthermore, the cytocompatibility and osteogenic/odontogenic differentiation of stem cells isolated from human exfoliated deciduous teeth (SHED) treated with TCS/ß-TCP/MCPA were studied. The results showed that HPMC could provide TCS with good anti-washout ability and injectability but slow hydration process. However, ß-TCP/MCPA effectively enhanced anti-washout characteristics and reduced setting time due to faster hydration kinetics. TCS/(ß-TCP/MCPA) obtained around 90 % of injection rate and high compressive strength whereas excessive additions of ß-TCP/MCPA compromised its injectability and compressive strength. TCS/(ß-TCP/MCPA) can induce apatite deposition and form a tight marginal sealing at the dentin-cement interface. Additionally, TCS/(ß-TCP/MCPA) showed good biocompatibility and promoted osteo/odontogenic differentiation of SHED. In general, our results indicated that TCS/(ß-TCP/MCPA) may be particularly promising as an injectable bioactive cements for endodontic treatment.

[Repair of mandibular defects with hydrogel loaded with nano-hydroxyapatite in rats].

PURPOSE: To observe the effect of gellan gum loaded with nano-hydroxyapatite (GG/nHA) on repairing mandibular defect in rats. METHODS: Critical bone defects with 5 mm in diameter on the mandible of 16 SD rats were created and randomly divided into two groups. The bone defects in the experimental group were injected with GG/nHA and the control group were filled with absorbable gelatin sponge. The rats were sacrificed at 4 and 8 weeks after operation. The bone tissue healing was evaluated by Micro-CT. Bone tissue repairing effect was evaluated by hematoxylin-eosin (H-E) staining and Masson staining. GraphPad Prism 8.0 software package was used for statistical analysis. RESULTS: The prepared GG/nHA had a good injectability and could be delivered to the bone defect area with a syringe. Four and 8 weeks after operation, the newly formed bone and bone volume fraction (BV/TV) in the experimental group were higher than those in the control group, and the difference was statistically significant (P<0.05). Larger number of new bone were observed in the experimental group than the control group by H-E staining and Masson staining. CONCLUSIONS: GG/nHA can be injected into the mandibular defect area to promote its healing, and it is expected to be used as a novel bio-material for minimally invasive repair of oral and maxillofacial bone defects.

An injectable gellan gum-based hydrogel that inhibits Staphylococcus aureus for infected bone defect repair.

The treatment of infected bone defects in complex anatomical structures, such as oral and maxillofacial structures, remains an intractable clinical challenge. Therefore, advanced biomaterials that have excellent anti-infection activity and allow convenient delivery are needed. We fabricated an innovative injectable gellan gum (GG)-based hydrogel loaded with nanohydroxyapatite particles and chlorhexidine (nHA/CHX). The hydrogel has a porous morphology, suitable swelling ratio, and good biocompatibility. It exerts strong antibacterial activity against Staphylococcus aureus growth and biofilm formation in vitro. We successfully established an infected calvarial defect rat model. Bacterial colony numbers were significantly lower in tissues surrounding the bone in rats of the GG/nHA/CHX group after debride surgery and hydrogel implantation in the defect regions than in rats of the blank group. Rats in the GG/nHA/CHX group exhibited significantly increased new bone formation compared to those in the blank group at 4 and 8 weeks. These findings indicate that gellan gum-based hydrogel with nHA/CHX can accelerate the repair of infected bone defects.

Successful individualized endodontic treatment of severely curved root canals in a mandibular second molar: A case report.

BACKGROUND: The incidence rate of severely curved root canals in mandibular molars is low, and the root canal treatment of mandibular molars with this aberrant canal anatomy may be technically challenging. CASE SUMMARY: A 26-year-old Chinese female patient presented with intermittent and occlusal pain in the left mandibular second molar. The patient had undergone filling restoration for caries before endodontic consultation. With the aid of cone beam computed tomography (CBCT), a large periapical radiolucency was observed, and curved root canals in a mandibular second molar were confirmed, depicting a severe and curved distolingual root. Nonsurgical treatments, including novel individualized preparation skills and techniques and the use of bioceramic materials as an apical barrier, were performed, and complete healing of the periapical lesion and a satisfactory effect were achieved. CONCLUSION: A case of severely curved root canals in a mandibular second molar was successfully treated and are reported herein. The complex anatomy of the tooth and the postoperative effect were also evaluated via the three-dimensional reconstruction of CBCT images, which accurately identified the aberrant canal morphology. New devices and biomaterial applications combined with novel synthesis techniques can increase the success rate of intractable endodontic treatment.

Sequential macrophage transition facilitates endogenous bone regeneration induced by Zn-doped porous microcrystalline bioactive glass.

Macrophages play an important role in the immune microenvironment during bone healing, and sequential macrophage phenotypic transition could achieve superior osteogenic outcomes. Microcrystalline bioactive glasses (MCBGs) with osteoimmunomodulatory effects show potential in bone tissue regeneration. Zinc (Zn) has been approved to coordinate innate and adaptive immunity. Therefore, in this study, different amounts of ZnO were incorporated into microcrystalline bioactive glass to improve its immunomodulatory ability. The effect of Zn-MCBG ionic extracts on macrophage transition was studied, and the 5Zn-MCBG extracts could orchestrate sequential M1-to-M2 macrophage transition and promote the expression of proinflammatory and anti-inflammatory genes and cytokine expression to induce human bone marrow stromal cells (hBMSCs) osteogenic differentiation in vitro. Macroporous Zn-MCBG scaffolds containing mesopores were fabricated and showed good cell adhesion and feasible apatite formation when immersed in SBF in vitro. Furthermore, a rat calvarial defect model was used to confirm that the Zn-MCBG scaffold could modulate macrophage phenotypic transition and create a desirable osteogenic microenvironment to promote osteogenesis in vivo.

Synthesis and characterization of PLGA-PEG-PLGA based thermosensitive polyurethane micelles for potential drug delivery.

Polyurethane nanomicelle is a promising functional drug delivery system. In this work, the polyurethane (P3-PU) was synthesized from PLGA1200-PEG1450-PLGA1200 (P3, a thermosensitive and biodegradable triblock copolymer) and L-lysine ester diisocyanate (LDI). Then, reactive benzaldehyde was further imported to terminate P3-PU to obtain benzaldehyde modified polyurethane (P3-PUDA). The micelles, temperature-sensitive P3-PU nanomicelle and P3-PUDA nanomicelle, were systematically investigated, including the size, stability, temperature sensitivity, drug loading and release behavior, cytotoxic on human hepatocytes (L02), and inhibitory effect on human hepatocellular carcinoma cells (HepG2). The results show the thermosensitive behavior of the micelles can be adjusted by the terminal group. The polyurethane micelles with a uniform size between 20 nm and 30 nm showed excellent stability and good biocompatibility to L02 cells. Besides, in vitro experiments showed that Dox-loaded P3-PUDA micelles exhibited faster and higher release rate at 37 °C and better inhibitory effect on HepG2 than the Dox-loaded P3-PU micelles. Moreover, the achieved benzaldehyde modified polyurethanes also provides various possibilities to adjust further to enlarge its applications. Therefore, the polyurethane micelles will have great potential in the field of drug carriers.

Preparation and characterisation of a gellan gum-based hydrogel enabling osteogenesis and inhibiting Enterococcus faecalis.

Infections are the leading cause of failure of osteogenic material implantation. Antibiotic treatment, treatment with bone cement, or collagen sponge placement can result in drug resistance and difficulties in operation. To address this, gellan gum (GG) was selected in this study and prepared as an injectable hydrogel containing chlorhexidine (CHX) and nanohydroxyapatite (nHA) that overcomes these intractable problems. Scanning electron microscopy and micro-computed tomography revealed a three-dimensional polymeric network of the hydrogel. The hydrogel had excellent biocompatibility, as detected by cell counting kit-8 and Live/Dead assay. Bone marrow mesenchymal stem cells could be encapsulated into the network, showing that the structure was suitable for cell growth. Additionally, loading the hydrogel with nHA improved its mechanical, biodegradable, and osteogenic properties. Quantitative alkaline phosphatase and Alizarin Red S staining validated its osteogenic ability. Furthermore, antibacterial activity assessment showed that the hydrogel loaded with 50 µg/mL CHX inhibited Enterococcus faecalis in a concentration-dependent manner. Thus, we report an injectable GG-based hydrogel with superior antibacterial effect against E. faecalis and osteogenesis, which holds promise for treating infectious bone defects caused by refractory periradicular periodontitis.

Research progress on the effect of new disinfection methods on the clearance of Enterococcus faecalis in the root canal / 口腔疾病防治

@#Enterococcus faecalis has been confirmed to be closely related to dental pulp and periapical disease in recent years. Enterococcus faecalis is one of the important bacteria causing persistent or secondary root canal infection and root canal treatment failure. Traditional root canal disinfection drugs such as sodium hypochlorite, chlorhexidine and calcium hydroxide can not completely remove Enterococcus faecalis in the root canal because of the concentration limitation of the drug and the complexity of the root canal system. Therefore, how to effectively resist the Enterococcus faecalis infection in the root canal has become one of the important research directions in the treatment of periodontal pulp periapical disease. In recent years, some new antimicrobial agents and disinfection methods have emerged due to the drug resistance and pathogenicity of Enterococcus faecalis, such as laser, photodynamic, ultrasonic irrigation and ozone therapy. Their combination with traditional root canal irrigation drugs can significantly enhance the ability of traditional drugs to remove Enterococcus faecalis in the root canal. In addition, the emergence of new disinfection methods such as chlorine dioxide, nano-magnesia, superoxidized water and N-acetylcysteine have been shown to have a unique killing effect on Enterococcus faecalis in root canals. At present, most of the new disinfection methods described above are in the in vitro experimental stage, and their stimulation and damage to normal tissue still lack relevant clinical data support; thus, these outcomes need to be further studied.