Berberine regulates bone metabolism in apical periodontitis by remodelling the extracellular matrix.

OBJECTIVES: The objectives of this study were to explore the role and related mechanism of berberine in repairing bone destruction in apical periodontics (AP). MATERIALS AND METHODS: AP was established in 14 of 21 male Wistar rats (four weeks of age; 70-80 g) for 3 weeks. The canals were cleaned and administered berberine (2 mg/ml; n = 7) or calcium hydroxide (100 mg/ml; control; n = 7), followed by glass ionomer cement sealing. After 3 weeks, specimen collection followed by micro-computed tomography (µ-CT) and histological staining was performed, including haematoxylin and eosin staining, Masson's trichrome staining, tartrate-resistant acid phosphatase staining, immunohistochemistry and immunofluorescence histochemistry. RESULTS: µ-CT showed that AP lesion volume reduced in the berberine group. Histopathology showed that berberine decreased the activity and number of osteoclasts but increased the expression of proteins related to osteoblast differentiation, including alkaline phosphatase and osterix. The immune cell, T cell, dendritic cell and macrophage counts were significantly decreased in the berberine group. In the berberine group, the expression of extracellular matrix-degraded proteases, metalloproteinases, was decreased; however, that of extracellular matrix-stable proteases, lysyl oxidases, was increased. CONCLUSIONS: Berberine controlled the inflammatory response and regulated bone metabolism in AP by reducing metalloproteinase expression and increasing lysyl oxidases expression.

[The Roles of RUNX1 in the Proliferation and Osteogenic and Adipogenic Differentiation of Dental Pulp Stem Cells].

OBJECTIVE: To investigate the influence of Runt-related transcription factor 1 (RUNX1) on the proliferation, osteogenic differentiation and adipogenic differentiation of dental pulp stem cells (DPSC) in vitro. METHODS: DPSCs were transfected through lentiviral vector carrying the target gene RUNX1 and green fluorescent protein (GFP). After 48 h, transfection efficiency was determined with the fluorescent marking of GFP and Western blot. The effect of the overexpression of RUNX1 on DPSC proliferation and colony formation was determined with CCK-8 and colony formation assay; cell cycle of DPSC was detected by flow cytometry. RUNX1 siRNA was transfected into the DPSCs. After mineralized induction, the effect of RUNX1 overexpression/silencing on the osteogenetic differentiation of DPSC was tested by alkaline phosphatase (ALP) staining and alizarin red staining. After adipogenic induction, oil red O staining was done in order to observe the effect of overexpression/silencing of RUNX1 on the adipogenic differentiation of DPSC. RESULTS: RUNX1 protein was overexpressed in DPSC after lentiviral transfection. Fluorescent test showed successful transfection of lentiviral transfection and over 70% of the cells showed stable expression of GFP protein. The proliferation and colony-formation efficiency of DPSC was enhanced significantly and the proportion of DPSCs in the S phase was significantly increased in the RUNX1-overexpessed group ( P<0.05). ALP activity and mineralized nodule formation ability increased, while lipid droplets decreased in the RUNX1-overexpessed group ( P<0.05). ALP activity and mineralized nodule formation ability decreased, while lipid droplets increased in the RUNX1 knockdown group ( P<0.05) . CONCLUSION: RUNX1 promotes DPSC proliferation and osteogenic differentiation while it inhibits DPSC adipogenic differentiation.

Hemicentin-1 is an essential extracellular matrix component during tooth root formation by promoting mesenchymal cells differentiation.

Introduction: Root dentin formation is an important process in tooth development. We tried to identify potential genes that regulate root dentin formation which could be potentially used for the regeneration and repair of defective or damaged dental roots. Methods: Tissues harvested from the labial and lingual sides of mouse incisors were used for microarray analysis. Gene ontology (GO) analysis of differentially expressed genes indicated the critical role of extracellular matrix in the discrepancy of dentin formation between root and crown, for which hemicentin-1 (Hmcn1) was selected as the target gene. Single-cell RNA sequencing analysis the expression pattern of Hmcn1 at different developmental stages in mouse molars. The spatiotemporal expression of HMCN1 in mouse incisors and molars was detected by immunohistochemical staining. The functions of HMCN1 in human dental pulp cells, including proliferation, differentiation and migration, were examined in vitro by CCK8 assay, BrdU assay, wound-healing assay, ALP staining and alizarin red staining, respectively. Results: It was showed that HMCN1 expression was more pronounced in papilla-pulp on the root than crown side in mouse incisors and molars. In vitro experiments presented inhibited dentinogenesis and migration after HMCN1-knockdown in human dental pulp cells, while there was no significant difference in proliferation between the HMCN1-knockdown group and control group. Discussion: These results indicated that HMCN1 plays an important role in dentinogenesis and migration of pulp cells, contributing to root dentin formation.

Study on 3D printed MXene-berberine-integrated scaffold for photo-activated antibacterial activity and bone regeneration.

The repair of mandibular defects is a challenging clinical problem, and associated infections often hinder the treatment, leading to failure in bone regeneration. Herein, a multifunctional platform is designed against the shortages of existing therapies for infected bone deficiency. 2D Ti3C2 MXene and berberine (BBR) are effectively loaded into 3D printing biphasic calcium phosphate (BCP) scaffolds. The prepared composite scaffolds take the feature of the excellent photothermal capacity of Ti3C2 as an antibacterial, mediating NIR-responsive BBR release under laser stimuli. Meanwhile, the sustained release of BBR enhances its antibacterial effect and further accelerates the bone healing process. Importantly, the integration of Ti3C2 improves the mechanical properties of the 3D scaffolds, which are beneficial for new bone formation. Their remarkable biomedical performances in vitro and in vivo present the outstanding antibacterial and osteogenic properties of the Ti3C2-BBR functionalized BCP scaffolds. The synergistic therapy makes it highly promising for repairing infected bone defects and provides insights into a wide range of applications of 2D nanosheets in biomedicine.

Insulin-like growth factor 1 promotes neural differentiation of human stem cells from the apical papilla.

OBJECTIVE: Insulin-like growth factor 1 (IGF1) is one of the vital factors in regenerative endodontics. Previous studies have focused on the role of IGF1 in the mineralization of dental tissues. However, the role of IGF1 in the neural differentiation of dental stem cells was little discussed. DESIGN: IGF1 was overexpressed in human stem cells from the apical papilla (hSCAPs) by lentivirus and knocked down in hSCAPs by small interfering RNA. The neural differentiation level of hSCAPs was investigated histologically by HE staining and Nissl staining after neural induction for 3 days. The expression of proteins was examined by western blot and immunofluorescence. RESULTS: IGF1 promoted neural differentiation of hSCAPs, more cell processes and Nissl-positive body stained cells. IGF1 overexpression could both promote glial differentiation in hSCAPs, characterized by the increase of S100ß and GFAP proteins, and neuronal differentiation, characterized by the increase of ßIII-tubulin and functional GAD67/vGLUT1 proteins. Conversely, IGF1 knockdown suppressed both glial and neuronal differentiation. IGF1 activated AKT to regulate the early neural differentiation of hSCAPs. CONCLUSIONS: The results indicate IGF1 could promote neural differentiation of hSCAPs by activating AKT signaling and provide a cue for the candidate of induced neural seeding cells in regenerative endodontics.

Preparation and application of chitosan biomaterials in dentistry.

Chitosan is a biodegradable and biocompatible natural polysaccharide that has a wide range of applications in the field of dentistry due to its functional versatility and ease of access. Recent studies find that chitosan and its derivatives can be embedded in materials for dental adhesives, barrier membranes, bone replacement, tissue regeneration, and antimicrobial agent to better manage oral diseases. In this paper, we provide a comprehensive overview on the preparation, applications, and major breakthroughs of chitosan biomaterials. Furthermore, incorporation of chitosan additives for the modification and improvement of dental materials has been discussed in depth to promote more advanced chitosan-related research in the future.

The Expression of Semaphorin 7A in Human Periapical Lesions.

INTRODUCTION: Semaphorin 7A (SEMA7A) is a membrane-bound or secretory protein exerting multiple functions in the regulation of inflammation, neural degradation, and cancer progression. Human periapical lesions are chronic and infectious diseases mainly caused by bacteria. However, the involvement of SEMA7A in human periapical lesions is still unclear. This study aimed to explore the expression of SEMA7A in human periapical lesions accompanied by the potential association of SEMA7A with matrix metalloproteinase (MMP)-1 and MMP-3 during the progression of apical periodontitis. METHODS: Samples of periapical lesions and healthy controls were collected. Total RNA and protein were extracted respectively for quantitative real-time polymerase chain reaction and Western blot analysis. Additionally, 6 healthy samples and 27 periapical lesion samples were fixed, dehydrated, and embedded for further histologic and immunochemical analysis. The expression of SEMA7A was quantified by average integrated optical density. Immunofluorescence analysis was conducted to explore the colocalization of SEMA7A/MMP-1 and SEMA7A/MMP-3. RESULTS: Compared with healthy controls, the messenger RNA and protein expression of SEMA7A was markedly up-regulated in periapical lesions. A stronger expression of MMP-1, MMP-3, and inflammatory cytokines was exhibited in periapical lesions than in healthy groups. An increasing expression of SEMA7A can be observed in both the periapical granuloma group and the radicular cyst group compared with the normal group (P < .01). Immunofluorescence results showed the colocalization of SEMA7A with both MMP-1 and MMP-3 in vascular vessels and extracellular matrix. CONCLUSIONS: SEMA7A was up-regulated in periapical periodontitis and might be involved in the tissue destruction and infiltration of immune cells in periapical lesions.

Gap junction-mediated cell-to-cell communication in oral development and oral diseases: a concise review of research progress.

Homoeostasis depends on the close connection and intimate molecular exchange between extracellular, intracellular and intercellular networks. Intercellular communication is largely mediated by gap junctions (GJs), a type of specialized membrane contact composed of variable number of channels that enable direct communication between cells by allowing small molecules to pass directly into the cytoplasm of neighbouring cells. Although considerable evidence indicates that gap junctions contribute to the functions of many organs, such as the bone, intestine, kidney, heart, brain and nerve, less is known about their role in oral development and disease. In this review, the current progress in understanding the background of connexins and the functions of gap junctions in oral development and diseases is discussed. The homoeostasis of tooth and periodontal tissues, normal tooth and maxillofacial development, saliva secretion and the integrity of the oral mucosa depend on the proper function of gap junctions. Knowledge of this pattern of cell-cell communication is required for a better understanding of oral diseases. With the ever-increasing understanding of connexins in oral diseases, therapeutic strategies could be developed to target these membrane channels in various oral diseases and maxillofacial dysplasia.

Berberine mediates root remodeling in an immature tooth with apical periodontitis by regulating stem cells from apical papilla differentiation.

Once pulp necrosis or apical periodontitis occurs on immature teeth, the weak root and open root apex are challenging to clinicians. Berberine (BBR) is a potential medicine for bone disorders, therefore, we proposed to apply BBR in root canals to enhance root repair in immature teeth. An in vivo model of immature teeth with apical periodontitis was established in rats, and root canals were filled with BBR, calcium hydroxide or sterilized saline for 3 weeks. The shape of the roots was analyzed by micro-computed tomography and histological staining. In vitro, BBR was introduced into stem cells from apical papilla (SCAPs). Osteogenic differentiation of stem cells from apical papilla was investigated by alkaline phosphatase activity, mineralization ability, and gene expression of osteogenic makers. The signaling pathway, which regulated the osteogenesis of SCAPs was evaluated by quantitative real time PCR, Western blot analysis, and immunofluorescence. In rats treated with BBR, more tissue was formed, with longer roots, thicker root walls, and smaller apex diameters. In addition, we found that BBR promoted SCAPs osteogenesis in a time-dependent and concentration-dependent manner. BBR induced the expression of ß-catenin and enhanced ß-catenin entering into the nucleus, to up-regulate more runt-related nuclear factor 2 downstream. BBR enhanced root repair in immature teeth with apical periodontitis by activating the canonical Wnt/ß-catenin pathway in SCAPs.

Disturbed bone remodelling activity varies in different stages of experimental, gradually progressive apical periodontitis in rats.

Bone remodelling keeps going through the lifespan of human by bone formation and bone resorption. In the craniofacial region, mandibles act as the main force for biting and chewing, and also become susceptible to a common bone-loss disease, namely, apical periodontitis, once infected dental pulp is not treated timely, during which bone resorption occurs from the apical foramen to the apical bone area. Although conventional root canal treatment (RCT) can remove the most of the infection, chronical apical periodontitis due to incomplete removal of dental pulp and subsequent microleakage will become refractory and more challenging, and this process has scarcely been specifically studied as a bone remodelling issue in rat models. Therefore, to study chronical and refractory apical periodontitis owing to incomplete cleaning of infected dental pulp and microleackage in vivo, we establish a modified rat model of gradually progressive apical periodontitis by sealing residual necrotic dental pulp and introducing limited saliva, which simulates gradually progressive apical periodontitis, as observed in the clinical treatment of chronical and refractory apical periodontitis. We show that bone-loss is inevitable and progressive in this case of apical periodontitis, which confirms again that complete and sound root canal treatment is crucial to halt the progression of chronical and refractory apical periodontitis and promote bone formation. Interestingly, bone remodelling was enhanced at the initial stage of apical periodontitis in this model while reduced with a high osteoblast number afterwards, as shown by the time course study of the modified model. Suggesting that the pathological apical microenvironment reserve its hard tissue formation ability to some degree but in a disturbed manner. Hopefully, our findings can provide insights for future bone regenerative treatment for apical periodontitis-associated bone loss.

Epigenetics in Odontogenesis and its Influences.

BACKGROUND: Odontogenesis is fundamentally controlled by the genome. However, epigenetic factors have indispensable effects during odontogenesis. Previous studies have shown that exogenous factors, such as the environment, that cause hypomethylation and hypermethylation in DNA may lead to dental differences in monozygotic twin pairs. In addition, abnormalities in epigenetic regulation could induce disruptions in odontogenesis, thereby causing tooth malformation or agenesis. OBJECTIVE: This review overviews the epigenetic mechanisms involved in odontogenesis with the aim of establishing a fundamental vision of tooth development, which might be useful in further research in odontogenesis and therapy for dental diseases. RESULTS: We summarized articles about epigenetics in odontogenesis. Here, we present concrete epigenetic regulation mechanisms in odontogenesis that have been reported previously, following the order of microRNA, DNA methylation and histone modification. CONCLUSION: Epigenetic factors influence the proliferation, differentiation or apoptosis of cells that play indispensable roles during the process of odontogenesis which have the ability to exquisitely regulate the tooth number, size and shape.

Aberrant activation of latent transforming growth factor-ß initiates the onset of temporomandibular joint osteoarthritis.

There is currently no effective medical treatment for temporomandibular joint osteoarthritis (TMJ-OA) due to a limited understanding of its pathogenesis. This study was undertaken to investigate the key role of transforming growth factor-ß (TGF-ß) signalling in the cartilage and subchondral bone of the TMJ using a temporomandibular joint disorder (TMD) rat model, an ageing mouse model and a Camurati-Engelmann disease (CED) mouse model. In the three animal models, the subchondral bone phenotypes in the mandibular condyles were evaluated by µCT, and changes in TMJ condyles were examined by TRAP staining and immunohistochemical analysis of Osterix and p-Smad2/3. Condyle degradation was confirmed by Safranin O staining, the Mankin and OARSI scoring systems and type X collagen (Col X), p-Smad2/3a and Osterix immunohistochemical analyses. We found apparent histological phenotypes of TMJ-OA in the TMD, ageing and CED animal models, with abnormal activation of TGF-ß signalling in the condylar cartilage and subchondral bone. Moreover, inhibition of TGF-ß receptor I attenuated TMJ-OA progression in the TMD models. Therefore, aberrant activation of TGF-ß signalling could be a key player in TMJ-OA development.

Creating hierarchical porosity hydroxyapatite scaffolds with osteoinduction by three-dimensional printing and microwave sintering.

Hierarchical porosity, which includes micropores and macropores in scaffolds, contributes to important multiple biological functions for tissue regeneration. This paper introduces a two-step method of combining three-dimensional printing (3DP) and microwave sintering to fabricate two-level hierarchical porous scaffolds. The results showed that 3D printing made the macroporous structure well-controlled and microwave sintering generated micropores on the macropore surface. The resulting hierarchical macro/microporous hydroxyapatite scaffold induced bone formation following intramuscular implantation. Moreover, when comparing the hierarchical macro/microporous hydroxyapatite scaffold to the non-osteoinductive hydroxyapatite scaffolds (either 3D printed or H2O2 foamed) subjected to muffle sintering which do not have micropores, the critical role of micropores in material-driven bone formation was shown. The findings presented herein could be useful for the further optimization of bone grafting materials for bone regeneration.

Expression and Function of MicroRNAs in Enamel Development.

MicroRNAs (miRNAs) are short (~21nt), noncoding, and single-stranded RNAs that can negatively regulate gene expression by binding to 3'UTRs of target mRNAs sequence-specifically to affect their translation and/or stability. MiRNAs are involved in multiple developmental events in various tissues and organs. Such events include dental enamel development. This review focuses on the expression and functions of miRNAs regulated in enamel development. This study further discusses the possible participation of signaling pathways affected by miRNAs during stem cell proliferation or renewal, cell differentiation, and cusp patterning formation. Research on the enamel developmental process and miRNA regulation mechanisms can facilitate better understanding of clinical enamel malformation and provide potential therapeutic schemes.

BMP7 and EREG Contribute to the Inductive Potential of Dental Mesenchyme.

Odontogenesis is accomplished by reciprocal signaling between the epithelial and mesenchymal compartments. It is generally accepted that the inductive mesenchyme is capable of inducing the odontogenic commitment of both dental and non-dental epithelial cells. However, the duration of this signal in the developing dental mesenchyme and whether adult dental pulp tissue maintains its inductive capability remain unclear. This study investigated the contribution of growth factors to regulating the inductive potential of the dental mesenchyme. Human oral epithelial cells (OEs) were co-cultured with either human dental mesenchymal/papilla cells (FDPCs) or human dental pulp cells (ADPCs) under 2-dimensional or 3-dimensional conditions. Odontogenic-associated genes and proteins were detected by qPCR and immunofluorescence, respectively, and significant differences were observed between the two co-culture systems. The BMP7 and EREG expression levels in FDPCs were significantly higher than in ADPCs, as indicated by human growth factor PCR arrays and immunofluorescence analyses. OEs co-cultured with ADPCs supplemented with BMP7 and EREG expressed ameloblastic differentiation genes. Our study suggests that BMP7 and EREG expression in late bell-stage human dental papilla contributes to the inductive potential of dental mesenchyme. Furthermore, adult dental pulp cells supplemented with these two growth factors re-established the inductive potential of postnatal dental pulp tissue.

MicroRNA 224 Regulates Ion Transporter Expression in Ameloblasts To Coordinate Enamel Mineralization.

Enamel mineralization is accompanied by the release of protons into the extracellular matrix, which is buffered to regulate the pH value in the local microenvironment. The present study aimed to investigate the role of microRNA 224 (miR-224) as a regulator of SLC4A4 and CFTR, encoding the key buffering ion transporters, in modulating enamel mineralization. miR-224 was significantly downregulated as ameloblasts differentiated, in parallel with upregulation of SLC4A4 and CFTR. Overexpression of miR-224 downregulated SLC4A4 and CFTR expression in cultured human epithelial cells. A microRNA luciferase assay confirmed the specific binding of miR-224 to the 3' untranslated regions (UTRs) of SLC4A4 and CFTR mRNAs, thereby inhibiting protein translation. miR-224 agomir injection in mouse neonatal incisors resulted in normal enamel length and thickness, but with disturbed organization of the prism structure and deficient crystal growth. Moreover, the enamel Ca/P ratio and microhardness were markedly reduced after miR-224 agomir administration. These results demonstrate that miR-224 plays a pivotal role in fine tuning enamel mineralization by modulating SLC4A4 and CFTR to maintain pH homeostasis and support enamel mineralization.

Quantitation of zoledronic acid in murine bone by liquid chromatography coupled with tandem mass spectrometry.

An in vitro method for extraction and quantification of zoledronic acid (ZA) from murine bone was developed. Whole mouse bones were incubated in ZA solutions with predetermined concentrations and bound ZA was subsequently extracted from bone with phosphoric acid and derivatized using trimethylsilyl diazomethane (TMS-DAM). ZA tetra-methyl phosphonate was quantified by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS). This resulted in a sensitive, accurate, and precise method that was linear over three orders of magnitude (0.0250-50.0µg/mL ZA). For quality control (QC) samples, intra-and inter-day coefficients of variance were calculated and were less than 10%. This method was then applied to an in vivo model to quantitate ZA from the femur and mandible of three mice treated with ZA for two weeks. The mean ZA extracted from the mandible was four fold higher than that extracted from the femur (3.06±0.52 vs. 0.76±0.09ng/mg, respectively) indicating that ZA did not distribute equally in the skeleton and had a preference to the mandible. In conclusion, a highly sensitive method to measure ZA from mouse skeleton was developed, which can be easily adapted to multiple mammalian models including humans receiving ZA treatment.

Effect of Ni(II) on inflammatory gene expression in THP1 monocytic cells.

Nickel-containing alloys are in common use for dental restorations, but tend to corrode and release Ni(II) in service. Ni(II) increases secretion of several inflammatory cytokines from activated monocytic cells, suggesting that nickel alloys may exaggerate inflammatory responses in adjacent periodontal tissues. In this work, the effects of Ni(II) on expression of inflammatory cytokine and receptor genes as well as nuclear factor-kappa B (NFκB)-related genes were assessed using quantitative real-time polymerase chain reaction (PCR) and PCR-based arrays in the human THP1 monocytic cell line pre-exposed to Ni(II) for 72 h, then activated by lipopolysaccharide. The expression of 10 inflammatory genes was down-regulated ≥50% by Ni(II) versus non-Ni(II) controls, whereas some genes like IL8 were up-regulated significantly by Ni(II). Expression of seven NFκB-related genes was up-regulated by Ni(II) by ≥50%, and HMOX1 expression, a redox protein regulated by NRF2, was increased by >500%. The current results suggest that Ni(II) has diverse effects on inflammatory gene expression, which may partly account for previous reports of Ni(II)-induced changes in inflammatory cytokine secretion from monocytes and alterations in NFκB regulation. Further work is needed to verify these effects in primary cells and to ascertain how Ni(II) alters gene expression.

Laser capture microdissection enables cellular and molecular studies of tooth root development.

Epithelial-mesenchymal interactions (EMIs) are critical for tooth development. Molecular mechanisms mediating these interactions in root formation is not well understood. Laser capture microdissection (LCM) and subsequent microarray analyses enable large scale in situ molecular and cellular studies of root formation but to date have been hindered by technical challenges of gaining intact histological sections of non-decalcified mineralized teeth or jaws with well-preserved RNA. Here,we describe a new method to overcome this obstacle that permits LCM of dental epithelia,adjacent mesenchyme,odontoblasts and cementoblasts from mouse incisors and molars during root development. Using this method,we obtained RNA samples of high quality and successfully performed microarray analyses. Robust differences in gene expression,as well as genes not previously associated with root formation,were identified. Comparison of gene expression data from microarray with real-time reverse transcriptase polymerase chain reaction (RT-PCR) supported our findings. These genes include known markers of dental epithelia,mesenchyme,cementoblasts and odontoblasts,as well as novel genes such as those in the fibulin family. In conclusion,our new approach in tissue preparation enables LCM collection of intact cells with well-preserved RNA allowing subsequent gene expression analyses using microarray and RT-PCR to define key regulators of tooth root development.