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Since the discovery of bioactive molecules sequestered in dentine, researchers have been exploring ways to harness their activities for dental regeneration. One specific area, discussed in this review, is that of dental-pulp capping. Dental-pulp caps are placed when the dental pulp is exposed due to decay or trauma in an attempt to enhance tertiary dentine deposition. Several materials are used for dental-pulp capping; however, natural biomimetic scaffolds may offer advantages over manufactured materials such as improved aesthetic, biocompatibility and success rate. The present review discusses and appraises the current evidence surrounding biomimetic dental-pulp capping, with a focus on bioactive molecules sequestered in dentine. Molecules covered most extensively in the literature include transforming growth factors (TGF-βs, specifically TGF-β1) and bone morphogenetic proteins (BMPs, specifically BMP-2 and BMP-7). Further studies would need to explore the synergistic use of multiple peptides together with the development of a tailored scaffold carrier. The roles of some of the molecules identified in dentine need to be explored before they can be considered as potential bioactive molecules in a biomimetic scaffold for dental-pulp capping. Future in vivo work needs to consider the inflammatory environment of the dental pulp in pulpal exposures and compare pulp-capping materials.

8 p.-4 fig., Small-molecule drugs targeting glycogen synthase kinase 3 (GSK3) as inhibitors of the protein kinase activity are able to stimulate reparative dentine formation. To develop this approach into a viable clinical treatment for exposed pulp lesions, we synthesized a novel, small-molecule noncompetitive adenosine triphosphate (ATP) drug that can be incorporated into a biodegradable hydrogel for placement by syringe into the tooth. This new drug, named NP928, belongs to the thiadiazolidinone (TDZD) family and has equivalent activity to similar drugs of this family such as tideglusib. However, NP928 is more water soluble than other TDZD drugs, making it more suitable for direct delivery into pulp lesions. We have previously reported that biodegradable marine collagen sponges can successfully deliver TDZD drugs to pulp lesions, but this involves in-theater preparation of the material, which is not ideal in a clinical context. To improve surgical handling and delivery, here we incorporated NP928 into a specifically tailored hydrogel that can be placed by syringe into a damaged tooth. This hydrogel is based on biodegradable hyaluronic acid and can be gelled in situ upon dental blue light exposure, similarly to other common dental materials. NP928 released from hyaluronic acid–based hydrogels upregulated Wnt/β-catenin activity in pulp stem cells and fostered reparative dentine formation compared to marine collagen sponges delivering equivalent concentrations of NP928. This drug-hydrogel combination has the potential to be rapidly developed into a therapeutic procedure that is amenable to general dental practice., The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: A. Alaohali was funded by the Medical Services Division of the Ministry of Defense in Saudi Arabia and Saudi Cultural Bureau in London. C. Salzlechner acknowledges support from the Diana Trebble Fund. E. Gentleman and C. Salzlechner received support from the Rosetrees Trust. L.K. Zaugg was funded by the Swiss National Science Foundation (Ref P300PB_167807). Funding from the NIHR GSTFT/KCL Biomedical Research Centre is acknowledged.

Aim. To determine the relevance of the application of the chlorhexidine adhesive Protocol in devital teeth.Materials and methods. A systematic review of the literature in the electronic databases Google Scholar and Pubmed was conducted. Articles related to research on the activity of matrix metalloproteinases in vital and devital teeth, as well as research on the effectiveness of the chlorhexidine Protocol, are considered and included.Results. 60 articles were reviewed during the review. After making the selection based on the exclusion criteria, the number of included studies was 36.Conclusions. According to the studied publications, it was found that MMP 2 and MMP 9 are contained not only in vital, but also in devital teeth. The use of a chlorhexidine adhesive Protocol is relevant in both clinical cases but depending on the type of MMP and the state of the pulp, the concentration of chlorhexidine is selected.

During dentin formation, odontoblast polarization ensures that odontoblasts directionally secrete dentin matrix protein, leading to tubular dentin formation; however, little is known about the major features and regulatory mechanisms of odontoblast polarization. In a study of epithelial cell polarization, β-catenin was shown to serve as a structural component of cadherin-based adherens junctions to initiate cell polarity. However, the role of β-catenin in odontoblast polarization has not been well investigated. In this study, we explored whether β-catenin participated in odontoblast polarization to regulate the secretion of mineralization proteins. We established Col1-CreErt2; β-catenin exon3fl/fl (CA-β-catenin) mice, which constitutively activate β-catenin in odontoblasts. CA-β-catenin mice exhibited disorganization and depolarization of incisor odontoblasts. Moreover, the incisor dentin was hypomineralized, and ectopic calcification was found in mouse incisor pulp. In addition, by constitutive activation of β-catenin, the expression levels of the core polarity molecule Cdc42 and its downstream polarity protein complex Par3-Par6-aPKC were decreased in the incisors of CA-β-catenin mice. These findings suggest that β-catenin plays an essential role in dentin formation by regulating odontoblast polarization.

Introduction The differentiation of dental pulp cells (DPCs) plays an important role in the repair of dental pulp injury. Bone morphogenetic protein 9 (BMP9) is one of the most effective BMPs to induce the differentiation of stem cells. However, the role of BMP9 in promoting the odontogenic differentiation of DPCs and dentinogenesis is worth knowing. Methods Fluorescence in situ hybridization and immunohistochemistry staining were performed to detect the BMP9 expression in human dental pulp. BMP9 was overexpressed in human DPCs (hDPCs), and the mineralization of hDPCs was tested by alkaline phosphatase staining and alizarin red staining. The expression of odontogenic differentiation-related genes was examined by quantitative real-time polymerase chain reaction and western blotting. The subcutaneous transplantation experiment was performed to test the odonto-induction ability of BMP9 in vivo. The rat direct pulp-capping experiment was performed to test the function of BMP9 in promoting dentin formation. Results BMP9 showed an increased expression in odontoblast layer at both the mRNA and protein levels. BMP9 enhanced the mineralization and induced the expression of odontogenic differentiation-related genes in hDPCs. More mineralized nodules, and increased expression of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP1) were detected in the beta-tricalcium phosphate scaffold/cells composites of BMP9 group compared with the control group. Meanwhile, there was thicker reparative dentin formation in the BMP9 group in the rat pulp exposure experiment. Conclusions BMP9 participates in the process of DPC differentiation and promotes DPC mineralization and dentinogenesis. BMP9 might be a potential therapeutic target in the repair of dental pulp injury.

Pulp-capping materials are commonly adopted in the clinic to form reparative dentin and thus protect dental pulp tissues from cases of deep caries, accidentally exposed pulps or partial pulpotomy. Some traditional pulp capping materials used in the clinic include calcium hydroxide and mineral trioxide aggregates. However, there are limitations to thin restorative dentin, and a long period of time is needed to cause degenerative changes in dental pulp. In this paper, injectable colloidal gels were developed to induce the formation of reparative dentin through a simple UV method from methacrylic acid functionalized gelatin loaded with notoginsenoside R1 (Gel-MA/NGR1). The results of the physicochemical property examinations showed that the prepared Gel-MA/NGR1 hydrogel possessed an appropriate interconnected porous microarchitecture with a pore size of 10.5 micrometres and suitable mechanical properties with a modulus of 50-60 kPa, enabling cell adhesion and proliferation. The hydrogel remained hydrophilic with sustained drug release performance. In addition, Gel-MA/NGR1 significantly enhanced the odontogenetic differentiation of mouse dental papilla cells by elevating the expression levels of the dentinogenic markers ALP and OCN and extracellular matrix mineralization. In vivo stimulation was carried out by injecting the precursors into the predrilled alveolar cavity of Sprague-Dawley rats followed by immediate in situ UV crosslinking. The results showed that Gel-MA/NGR1 has a strong capacity to promote reparative dentin formation. Haematoxylin& eosin, Masson, and immunohistochemical staining (DMP-1, DSPP, OCN and RUNX2) and micro-CT were employed to illustrate the effectiveness of dentinogenesis, and the relative volumes of calcification were found to have increased ~175-fold. All of the results showed that the Gel-MA/NGR1 hydrogel promoted reparative dentin formation, which suggests that this hydrogel provides great potential as a pulp-capping material to induce dentin formation.

This report describes the unexpected formation of root-like structures following the avulsion of immature permanent teeth without replantation. A six-year-old female patient had avulsed the four permanent mandibular incisors and the two deciduous mandibular canines. The patient was seen in an emergency health care unit but did not receive specialized treatment for tooth replantation. As follow up treatment, she received a removable prosthesis. After four years of follow-up, an image obtained by panoramic radiography showed formations similar to four root structures in the alveolus of the previously-avulsed permanent teeth. This finding was confirmed by periapical radiography and computed tomography. This case report demonstrates that in teeth with incomplete root development, even after avulsion without replantation, cells from the pulp stump may have the capacity to form mineralized structures that appear radiographically comparable to root dentin.

Relevance . Osteogenesis imperfecta is a rare hereditary disease of the connective tissue and the skeleton caused by incomplete mineralization of the bony tissue and hard dental tissue of the primary and permanent dentition. The most common oral manifestations of osteogenesis imperfecta are polymorphism, anodontia, impairment of amelogenesis and dentinogenesis. Function and esthetics are impaired in hereditary amelognenesis imperfecta and dentinogenesis imperfecta. Materials and methods . 36 children aged from 4 to 6 years with different types of osteogenesis imperfecta were examined in the clinic of the department of pediatric dentistry of the Clinical Center for Maxillofacial and Plastic Surgery and Dentistry. Morphological structure of the dental samples was studied for the first time ever using scanning electron microscopy. Results . Characteristic changes were detected by the clinical and X-ray examination. The article presents for the first time the comparison of deciduous hard dental tissue morphology in children with different types of osteogenesis imperfecta studied by the scanning electron microscopy (in vitro). Conclusions . The received data are of primary importance for prognosis and strategy of the dental treatment in children with different types of osteogenesis imperfecta.

Objectives Dental tissue-derived mesenchymal stem cell (MSC)-mediated tooth regeneration may be a useful therapeutic tool for repairing tooth loss. However, the low success rate of tooth regeneration restricts its clinical application. Identifying key factors for enhancing dentinogenesis in MSCs is crucial for promoting tooth regeneration. Materials and methods Human dental pulp stem cells (DPSCs) were transfected with retrovirus to obtain SFRP2-over-expressing DPSCs. Alkaline phosphatase (ALP) activity assay, Alizarin red staining, quantitative analysis of calcium, and dentinogenesis-related genes were detected. Additionally, transplantation in a rabbit tooth extraction model was used to explore the role of SFRP2 in dentin regeneration. Results We found SFRP2 over-expression greatly enhanced ALP activity, and mineralization in DPSCs. Real-time RT-PCR revealed SFRP2 over-expression promoted the expressions of OSX, RUNX2, DSPP, DMP1, and BSP. Moreover, Micro CT analysis showed high-density calcification occurred to a much higher extent in SFRP2 over-expressing group compared to control group in vivo. Additionally, HE staining, immmunohistochemistry staining, and scanning electron microscopy results showed much more dentin-like tissue formed in SFRP2 over-expressing group compared to control group. Conclusions Our findings revealed SFRP2 is an important regulator that enhances the dentinogenesis of DPSCs and dentin regeneration in the jaw, which may have clinical applications.

Odontoblast processes are thin cytoplasmic projections that extend from the cell body at the periphery of the pulp toward the dentin‐enamel junction. The odontoblast processes function in the secretion, assembly and mineralization of dentin during development, participate in mechanosensation, and aid in dentin repair in mature teeth. Because they are small and densely arranged, their three‐dimensional organization is not well documented. To gain further insight into how odontoblast processes contribute to odontogenesis, we used serial section electron microscopy and three‐dimensional reconstructions to examine these processes in the predentin region of mouse molars and incisors. In molars, the odontoblast processes are tubular with a diameter of ~1.8 μm. The odontoblast processes near the incisor tip are similarly shaped, but those midway between the tip and apex are shaped like plates. The plates are radially aligned and longitudinally oriented with respect to the growth axis of the incisor. The thickness of the plates is approximately the same as the diameter of molar odontoblast processes. The plates have an irregular edge; the average ratio of width (midway in the predentin) to thickness is 2.3 on the labial side and 3.6 on the lingual side. The plate geometry seems likely to be related to the continuous growth of the incisor and may provide a clue as to the mechanisms by which the odontoblast processes are involved in tooth development.

Mineralized enamel and dentin provide protection to the dental pulp, which is vital tissue rich with cells, vasculature, and nerves in the inner tooth. Dental caries left untreated threaten exposure of the dental pulp, providing facile access for bacteria to cause severe infection both in the pulp and systemically. Dental materials which stimulate the formation of a protective dentin bridge after insult are necessary to seal the pulp chamber in an effort to maintain natural dentition and prevent pulpal infection. Dental materials to date including calcium hydroxide paste, mineral trioxide aggregate, and glass ionomer resin, are used with mixed results. Herein we exploited the cell-cell communicative properties of exosomes, extracellular vesicles derived from both mineralizing primary human dental pulp stem cells (hDPSCs) and an immortalized murine odontoblast cell line (MDPC-23), to catalyze the formation of a reactionary dentin bridge by recruiting endogenous stem cells of the dental pulp, through an easy-to-handle delivery vehicle which allows for their therapeutic controlled delivery at the pulp interface. Exosomes derived from both hDPSCs and MDPCs upregulated odontogenic gene expression and increased mineralization in vitro. We designed an amphiphilic synthetic polymeric vehicle from a triblock copolymer which encapsulates exosomes by polymeric self-assembly and maintains their biologic integrity throughout release up to 8–12 weeks. The controlled release of odontogenic exosomes resulted in a reparative dentin bridge formation, superior to glass-ionomer cement alone in vivo, in a rat molar pulpotomy model after six weeks. We have developed a platform for the encapsulation and controlled, tunable release of cell-derived exosomes, which maintains their advantageous physiologic properties reflective of the donor cells. This platform is used to modulate downstream recipient cells towards a designed dentinogenic trajectory in vitro and in vivo. Additionally, we have demonstrated the utility of an immortalized cell line to produce a high yield of exosomes with cross-species efficacy.

Background Pulpitis is a common dental disease characterized by sustained inflammation and impaired pulp self-repair. Mesenchymal stem cell-based minimally invasive vital pulp therapy (MSC-miVPT) is a potential treatment method, but its application is limited by the difficulty in acquiring MSCs. We recently revealed the immunomodulatory effects of rat dental follicle stem cells (rDFSCs) on acute lung injury. The present study focused on the paracrine effects of rDFSCs on the inflammation and regeneration of rat injured dental pulp to detect whether DFSCs are a potential candidate for MSC-miVPT. Methods Conditioned medium from rDFSCs (rDFSC-CM) was applied to lipopolysaccharide (LPS)-induced inflammatory rat dental pulp cells (rDPCs). The inflammation and regeneration of rDPCs were detected by RT-qPCR, Western blotting, immunofluorescence staining, Cell Counting Kit-8 (CCK-8) assay, flow cytometry, wound-healing assay, and Masson’s staining. The effects of rDFSC-CM on inflamed rat dental pulp were further evaluated by hematoxylin-eosin and immunohistochemical staining. Results rDFSC-CM downregulated the ERK1/2 and NF-κB signaling pathways, which resulted in suppression of the expression of IL-1β, IL-6, and TNF-α and promotion of the expression of IL-4 and TGF-β, and these findings lead to the attenuation of rDPC inflammation. rDFSC-CM enhanced the in vitro proliferation, migration, and odontogenic differentiation of inflammatory rDPCs and their in vivo ectopic dentinogenesis. Furthermore, rDFSC-CM inhibited inflammatory cell infiltration in rat pulpitis and triggered Runx2 expression in some of the odontoblast-like cells surrounding the injured site, and these effects were conducive to the repair of inflamed dental pulp. Conclusions rDFSC-CM exhibits therapeutic potential by rescuing the regeneration of the inflamed rat dental pulp through an immunomodulatory mechanism, indicating the application prospects of DFSCs in biological regenerative endodontics.

Autophagy is an intracellular self-degradation process that is essential for tissue development, cell differentiation, and survival. Nevertheless, the role of autophagy in tooth development has not been definitively identified. The goal of this study was to investigate how autophagy is involved in midkine (MK)–mediated odontoblast-like differentiation, mineralization, and tertiary dentin formation in a mouse tooth pulp exposure model. In vitro studies show that MK and LC3 have similar expression patterns during odontoblast-like cell differentiation. Odontoblast-like cell differentiation is promoted through MK-mediated autophagy, which leads to increased mineralized nodule formation. Subcutaneous transplantation of hydroxyapatite/tricalcium phosphate with rMK-treated human dental pulp cells led to dentin pulp–like tissue formation through MK-mediated autophagy. Furthermore, MK-mediated autophagy induces differentiation of dental pulp cells into odontoblast-like cells that form DSP-positive tertiary dentin in vivo. Our findings may provide 1) novel insight into the role of MK in regulating odontoblast-like differentiation and dentin formation in particular via autophagy and 2) potential application of MK in vital pulp therapy.

BACKGROUND: Caries is a destructive process leading to progressive demineralization of the inorganic part of the tooth, accompanied by enzyme disintegration of the organic component of the tooth tissue. Considering the fact that caries activity can be stopped if the demineralization process is stopped, it is clear that the objectives of caries treatment are focused toward elimination of etiological factors and stimulation of regeneration of the dental tissues. That is why in the last years treatment of the caries disease is targeted to remineralization of the initial carious lesions, as well as on the biological behavior of the pulp-dentine complex after application of certain medications. AIM: The aim of this study is to evaluate impact of the two materials for direct capping, mineral trioxide aggregate (MTA) and Biodentine®, on the expression of glycoproteins fibronectin (FN), and tenascin (TN), responsible for dentinogenesis. METHODS: In the tests were used materials MTA and Biodentine, as agents for direct capping of pulp exposure. From 60 extracted teeth included in this in vitro study, tissue cuts were made. Each of them was then analyzed on a light microscope to determine the amount of two extracellular matrix glycoproteins, FN-C, and TN-C. RESULTS: Our study show that there is an expressed immunoreactivity for FN and for TN in the fibronectin bridge under MTA and Biodentine® after 8, and even after 30 days of their application. CONCLUSION: Based on this study, we can conclude that both materials – MTA and Biodentine – may induce reparative dentinogenesis, in which FN and TN have certainly a major role in the formation of the fibronectin matrix.

The article presents the results of morphological and structural changes in the pulp using a combination drug composition for the treatment of acute focal pulpitis. The experiment included 13 teeth 5 canines, molars and premolars 8 (6 mongrel dogs between the ages of 2 to 5 years). It is found that the developed medicinal composition possesses specific properties and simultaneously pluripotent in preventing the development of inflammation, enhancing the protective properties of the pulp. In the pulp occurred relief of the inflammatory response and severe intensification of reparative processes.

OBJECTIVE: To evaluate, by means of an ex-vivo human tooth-culture model and in-vivo minipig animal study, the pulpal inflammatory reaction and reparative dentin-formation capacity of an injectable phosphopullulan-based calcium-silicate cement (GC, Tokyo, Japan) upon pulp capping, this in comparison with the commercial reference material Biodentine (Septodont). METHODS: For the ex-vivo tooth model, 9 freshly-extracted teeth from 3 different patients were pulp-capped with the experimental biomaterial (n = 3), Biodentine (n = 3) or left uncapped (control; n = 3). The teeth were kept in fresh culture medium for 4 weeks and, upon fixation three-dimensional Micro-CT and histology were performed. For the in-vivo animal study, 40 teeth from 3 minipigs were exposed and pulp capped with the experimental biomaterial containing phosphopullulan (n = 24) or Biodentine (n = 16) for 7 or 70 days. The inflammatory reaction and the tissue-regenerative potential was qualitatively and semi-quantitatively characterized using three-dimensional micro-CT and histology. RESULTS: Ex vivo, the treatment with the experimental phosphopullulan-based calcium-silicate cement and Biodentine stimulated the formation of fibrous tissue and mineralized foci. In vivo, early inflammatory reaction and regeneration of the pulp-tissue interface was promoted by both bioceramic materials after 7 and 70 days, respectively. SIGNIFICANCE: Our findings bring new insights into calcium-silicate-mediated dental pulp repair and regeneration. The novel ready-to-use and self-adhering functionalized calcium-silicate cement revealed effective pulpal repair potential. ispartof: DENTAL MATERIALS vol:36 issue:4 pages:512-526 ispartof: location:England status: published

Rhythmic incremental growth lines occur in dental hard tissues of vertebrates, and dentinogenesis in rodent incisors is suggested to be controlled by the 24-hr circadian clock. Rodent incisors continue to grow throughout the animal’s life; however, similar to human teeth, rodent molars stop growing after crown formation. This similarity suggests that the mouse molar is an excellent model to understand the molecular mechanisms underlying growth of human teeth. However, not much is known about the rhythmic dentinogenesis in mouse molars. Here, we investigated the incremental growth lines in mouse molar dentin using tetracycline as the growth marker. The incremental growth lines were observed to be generated at approximately 8-hr intervals in wild-type mice housed under 12:12 hr light-dark conditions. Moreover, the 8-hr rhythmic increments persisted in the wild-type and Bmal1−/− mice housed in constant darkness, where Bmal1−/− mice become behaviorally arrhythmic. These results revealed that the dentinogenesis in mouse molars underlie the ultradian rhythms with around 8-hr periodicity. Further, the circadian clock does not seem to be involved in this process, providing new insight into the mechanisms involved in the tooth growth.

Objectives To determine glucose transporter 1 (GLUT1) and runt-related transcription factor 2 (RUNX2) expression during reparative dentinogenesis after pulpotomy with mineral trioxide aggregate (MTA) capping. Subjects and methods Eight-week-old male Wistar rats were used. Pulp of the upper left first molar was exposed and capped with MTA. The upper right first molar of the same animal was used as a control. After collecting molars at various time points, GLUT1, RUNX2 and mammalian target of rapamycin (MTOR) were examined by immunohistochemistry. mRNA levels of Slc2a1 (encoding GLUT1), Runx2, Nestin and Mtor were determined by real-time PCR. Results Pulp exhibited progressive formation of reparative dentine lined with GLUT1- and MTOR-immunoreactive odontoblast-like cells at 5 days after pulpotomy. RUNX2 was detected in nuclei of most pulp tissue cells at day 5 after pulpotomy. Double immunofluorescence staining revealed GLUT1 immunoreactivity on odontoblast-like cells positive for Nestin or RUNX2, 5 days after pulpotomy. Slc2a1, Runx2, Nestin and Mtor mRNA levels were significantly upregulated on days 3-5 after pulpotomy. Conclusions After rat molar pulpotomy, dental pulp induced formation of reparative dentine with colocalization of GLUT1 and Nestin or RUNX2. Moreover, mRNA levels of Slc2a1, Runx2, Nestin and Mtor were significantly upregulated in pulpotomized dental pulp.

Dentin dysplasia is an autosomal-dominant genetic abnormality that occurs in humans and results in diffuse radiographic dental abnormalities and variable tooth discoloration due to an underlying defect in secondary dentinogenesis. This case report presents distinctive radiographic and histopathologic dental abnormalities in a dog that are consistent with generalized dentin dysplasia. These findings are similar to but not completely analogous to any specific clinical type of dentin dysplasia in humans. Grossly, the majority of the teeth in this case were discolored and most were determined to be vital. Dentin dysplasia should be included in the list of differential diagnoses of discolored teeth and notably this form of discoloration does not necessarily indicate loss of vitality.

Considerable amount of research has been focused on dentin mineralization, odontoblast differentiation, and their application in dental tissue engineering. However, very little is known about the differential role of functionally and spatially distinct types of dental epithelium during odontoblast development. Here we show morphological and functional differences in dentin located in the crown and roots of mouse molar and analogous parts of continuously growing incisors. Using a reporter (DSPP-cerulean/DMP1-cherry) mouse strain and mice with ectopic enamel (Spry2(+/-);Spry4(-/-)), we show that the different microstructure of dentin is initiated in the very beginning of dentin matrix production and is maintained throughout the whole duration of dentin growth. This phenomenon is regulated by the different inductive role of the adjacent epithelium. Thus, based on the type of interacting epithelium, we introduce more generalized terms for two distinct types of dentins: cementum versus enamel-facing dentin. In the odontoblasts, which produce enamel-facing dentin, we identified uniquely expressed genes (Dkk1, Wisp1, and Sall1) that were either absent or downregulated in odontoblasts, which form cementum-facing dentin. This suggests the potential role of Wnt signalling on the dentin structure patterning. Finally, we show the distribution of calcium and magnesium composition in the two developmentally different types of dentins by utilizing spatial element composition analysis (LIBS). Therefore, variations in dentin inner structure and element composition are the outcome of different developmental history initiated from the very beginning of tooth development. Taken together, our results elucidate the different effects of dental epithelium, during crown and root formation on adjacent odontoblasts and the possible role of Wnt signalling which together results in formation of dentin of different quality. (c) 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Direct pulp capping is an effective treatment for preserving dental pulp against carious or traumatic pulp exposure via the formation of protective reparative dentin by odontoblast-like cells. Reparative dentin formation can be stimulated by several signaling molecules, therefore, we investigated the effects of secreted frizzled-related protein (SFRP) 1 that was reported to be strongly expressed in odontoblasts of newborn molar tooth germs on odontoblastic differentiation and reparative dentin formation. In developing rat incisors, cells in the dental pulp, cervical loop, and inner enamel epithelium, as well as ameloblasts and preodontoblasts, weakly expressed Sfrp1, however, Sfrp1 was strongly expressed in mature odontoblasts. Human dental pulp cells (hDPCs) showed stronger expression of SFRP1 compared with periodontal ligament cells and gingival cells. SFRP1 knockdown in hDPCs abolished calcium chloride-induced mineralized nodule formation and odontoblast-related gene expression and decreased BMP-2 gene expression. Conversely, SFRP1 stimulation enhanced nodule formation and expression of BMP-2. Direct pulp capping treatment with SFRP1 induced the formation of a considerable amount of reparative dentin that has a structure similar to primary dentin. Our results indicate that SFRP1 is crucial for dentinogenesis and is important in promoting reparative dentin formation in response to injury.

Background: Dental bleaching agents show the ability to permeate through dental hard tissues, which may lead to pulp tissue changes. This systematic review (PROSPERO register: CRD42020213767) is aimed at understanding the effects of bleaching agents on the process of mineralization of the pulp tissue.Methods: Only in vitro studies evaluating the influence of hydrogen peroxide (HP) on mineralization in dental pulp cells were included. Studies without a non-bleached control group or cells after co-treatment with a bleaching agent other than HP and/or carbamide peroxide were excluded. The primary outcomes evaluated were alkaline phosphatase (ALP) activity and mineralized nodule deposition. The mineralization markers analysis in dental pulp cells and the cell viability were considered secondary outcomes. Two independent authors conducted a systematic search (PubMed/MEDLINE, Scopus, Embase, Cochrane Library, and OpenGrey until January 2021) with no language restrictions and performed data extraction. The quality assessment was appraised according to a modified Joanna Briggs Institute critical appraisal checklist.Results: The search resulted in 473 studies, and 11 were considered eligible. Overall, a reduction in the process of mineralization was observed among pulp cells after bleaching. A reduction in the ALP activity was reported in the mostly bleached groups using different protocols and analysis periods of nine studies. Regarding mineralized nodule deposition, 6 studies reported a significant reduction from 7 to 21 days among bleached groups. Of those three studies that investigated other mineralization markers, two found a reduction in the expression of dentin matrix acidic phosphoprotein (DMP)-1, dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE) among some bleaching gel concentrations. In contrast, one study showed a greater expression of osteopontin (OPN) and osteocalcin (OCN) in 100 μmol/L HP after 5 or 10 min of exposure, and another study showed significant induction of DSPP in concentrations of up to 0.5 mmol/L HP.Conclusion: Especially, high concentrations of bleaching gel reduce the potential of mineralization in pulp cells in in vitro studies; however, different HP concentrations, bleaching protocols, and analysis periods can influence this outcome.

Background: Wnt/beta-Catenin signalling and DMP1 have key roles in tertiary dentinogenesis. Aim: To compare the relationship between remaining dentine thickness (RDT), tertiary dentine thickness (TDT), beta-catenin and dentine matrix protein 1 (DMP1) in carious second primary molar teeth with normal (SPM) and hypomineralised enamel (HSPM). Design: Extracted carious SPM and HSPM were fixed, sectioned (5 mu m) and stained with haematoxylin and eosin or with indirect immunofluorescence for beta-catenin and DMP1. Image analysis was performed to determine RDT, TDT, beta-catenin and DMP1 intensity in the odontoblast layer and dentine-pulp complex. Results: Carious SPM (n = 11; mean RDT = 1536.1 mu m) and HSPM (n = 12; mean RDT = 1179.9 mu m) had mean TDT 248.6 mu m and 518.1 mu m, respectively (P = .02). There were no significant differences in intensity values in the odontoblast layer and dentine-pulp complex for beta-catenin and DMP1 for both groups. Conclusion: There was no observable variation in Wnt/beta-catenin and DMP1 expression between HSPM and SPM despite a statistically significant twofold increased TDT in HSPM compared with SPM that had similar RDT. Thus, the observed increased TDT in HSPM is more likely due to an earlier onset of repair processes rather than an amplified response to caries.

Wnt/β-catenin signaling is known to play essential roles in odontoblast differentiation and reparative dentin formation. Various Wnt activators including LiCl have been increasingly studied for their effectiveness to induce repair of the dentin-pulp complex. LiCl is a simple salt thought to activate Wnt/β-catenin signaling by inhibiting GSK3β. Previous in vitro and in vivo studies showed that LiCl increased odontoblast differentiation and enhanced reparative dentin formation. However, the underlying molecular and cellular mechanisms by which LiCl regulates odontoblast and osteoblast differentiation during reparative dentinogenesis are not well-understood. Our in vitro studies show that exposure of early dental pulp progenitors to LiCl increased the survival and the pool of αSMA+ progenitors, leading to enhanced odontoblast and osteoblast differentiation. The positive effects of LiCl in the differentiation of osteoblasts and odontoblasts from αSMA+ progenitors are mediated by Wnt/β-catenin signaling. Our results also showed that continuous and late exposure of dental pulp cells to LiCl increased the expression of odontoblast markers through Wnt/β-catenin signaling, and the number of odontoblasts expressing DMP1-Cherry and DSPP-Cerulean transgenes. However, unlike the early treatment, both continuous and late treatments decreased the expression of Bsp and the expression of BSP-GFPtpz transgene. These observations suggest that prolonged treatment with LiCl in more mature cells of the dental pulp has an inhibitory effect on osteoblast differentiation. The inhibitory effects of LiCl on osteogenesis and Bsp were not mediated through Wnt/β-catenin signaling. These observations suggest that the effects of LiCl, and GSK3β antagonists on reparative dentinogenesis involve multiple pathways and are not specific to Wnt/β-catenin signaling.

Dentin hypersensitivity commonly occurs due to opened dentinal tubules for many reasons. In our previous study, copine 7 (CPNE7) could induce dentin formation for an indirect pulp-capping model in vivo. This study aims to investigate the formation of tertiary dentin when CPNE7 is applied to intentionally exposed dentin with nothing over it in vivo, whether it affects microleakage of the teeth, and the penetration ability of CPNE7 molecules through dentinal tubules in vitro. Cervical dentin areas of 6 maxillary incisors of 5 beagles were exposed to a class V–like lesion, and 1 side of 3 maxillary incisors was adapted with recombinant CPNE7 protein for 5 min as the experimental group. The other side was the control group, and there was no treatment of ethylenediaminetetraacetic acid (EDTA) and CPNE7 after preparation. The defects were exposed without any restorations, and all beagles were sacrificed after 4 wk. The fluid penetration of exposed dentin areas was investigated by a microleakage-testing device and confocal laser scanning microscope. Tertiary dentin formation was confirmed with histological scanning electronic microscopic analysis. Tertiary dentin formation reduces dentinal fluid flow due to occluded tubules or discontinuity with primary or secondary dentin. The in vivo hypersensitivity model with the anterior teeth of beagle dogs showed newly formed tertiary dentin at the dentin-pulp boundary in recombinant CPNE7–treated teeth when compared with the untreated control group in histologic analysis. Scanning electronic microscopic analysis revealed occluded sites with mineral deposition of intratubular dentin. In the permeability test, the mean microleakage value of the CPNE7-treated group was significantly lower than that of the control group ( P < 0.05). The tubular penetration of rhodamine B–combined CPNE7 was confirmed under confocal laser scanning microscope. CPNE7 induces formation of tertiary dentin through shallowly exposed dentinal tubules, which decreases dentin permeability.

Tooth development is regulated by sequential and reciprocal epithelium-mesenchymal interactions and their related molecular signaling pathways, such as bone morphogenetic proteins (BMPs). Among the 14 types of BMPs, BMP9 (also known as growth differentiation factor 2) is one of the most potent BMPs to induce osteogenic differentiation of mesenchymal stem cells. The purpose of this study was to examine potential roles of BMP9 signaling in tooth development. First, we detected the expression pattern of BMP9 in tooth germ during postnatal tooth development, and we found that BMP9 was widely expressed in odontoblasts, ameloblasts, dental pulp cells, and osteoblasts in alveolar bones. Then, we established a BMP9-KO mouse model. Gross morphological examination revealed that the tooth cusps of BMP9-KO mice were significantly abraded with shorter roots. Micro-computed tomography and three-dimensional reconstruction analysis indicated that the first molars of the BMP9-KO mice exhibited a reduced thickness dentin, enlarged pulp canals, and shortened roots, resembling the phenotypes of the common hereditary dental disease dentinogenesis imperfecta. Further, the alveolar bone of the BMP9-KO mutants was found to be shorter and had a decreased mineral density and trabecular thickness and bone volume fraction compared with that of the wild-type control. Mechanistically, we demonstrated that both dentin sialophosphoprotein and dentin matrix protein 1 were induced in dental stem cells by BMP9, whereas their expression was reduced when BMP9 was silenced. Further studies are required to determine whether loss of or decreased BMP9 expression is clinically associated with dentinogenesis imperfecta. Collectively, our results strongly suggest that BMP9 may play an important role in regulating dentinogenesis and tooth development. Further research is recommended into the therapeutic uses of BMP9 to regenerate traumatized and diseased tissues and for the bioengineering of replacement teeth.

Objectives: The development of teeth is affected by genetic and environmental factors. Amoxicillin is a widely prescribed semi-synthetic antibiotic. Its most frequent side effects are gastrointestinal disorders and hypersensitivity reactions. The purpose of this study was to determine the effect produced by amoxicillin administration on dental enamel and dentin in Wistar rats. Materials and Methods: Twelve pregnant adult Wistar rats were equally divided into four different groups. Negative controls were prescribed with a saline solution. Positive controls were prescribed with tetracycline (130 mg/kg). The other two groups were treated with amoxicillin doses of 50 and 100 mg/kg (every 8 hours), respectively. The treatments were daily administered by oral gavage from the 13th gestation day to the end of gestation. After birth, the offspring also received the same treatment as their mothers from day one to day twelve. After 24 hours, the newborns were sacrificed, the jaws were dissected, and the first molar teeth were collected. The samples were fixed in 10% for­maldehyde solution and were histomorphologically and histopathologically observed to determine enamel and dentin abnormalities. Results: The mean ameloblastic layer thickness, enamel thickness, odontoblastic layer thickness, and dentin thickness were significantly different in the tetracycline group and the amoxicillin 50 and 100 mg/kg groups compared to the control group. Also, dentin hypomineralization and vacuolization of the odontoblastic layer were observed in the tetracycline- and amoxicillin- treated groups. Conclusion: This study showed that amoxicillin interferes with amelogenesis and dentinogenesis and reduces enamel and dentin thickness.

Introduction Neurotrophic factors play a significant role in the innervation of the pulp-dentin complex during and after organogenesis. There have been numerous bioactive molecules identified in the dentin extracellular matrix; however, the expression of neurotrophic factors in the dentin matrix and their biological activity are largely unknown. The purpose of this study was to characterize the relative expression of neurotrophic factors in human dentin matrix proteins (DMPs) and their effect on neurite outgrowth of trigeminal (TG) neurons. Methods Dentin was powdered in liquid nitrogen from noncarious human third molar teeth. DMPs were solubilized through an EDTA extraction method, dialyzed, and lyophilized until use. The relative expression of nerve growth factor, brain-derived neurotrophic factor, glial cell−line derived neurotrophic factor, neurotrophin 3, and neurotrophin 4/5 was determined by the enzyme-linked immunosorbent assay. Rat TG neurons were cultured and exposed to different concentrations of DMPs (1–105 ng/mL) or vehicle, and a quantitative neurite outgrowth assay was performed. Results Human DMPs contained all of the tested neurotrophic factors, with glial cell−line derived neurotrophic factor and neurotrophin 4/5 found at the highest levels. DMPs were able to promote the neurite outgrowth of rat TG neurons at an optimum concentration of 10–102 ng/mL, whereas the effect was partially inhibited at higher concentrations (>103 ng/mL). Conclusions The human dentin extracellular matrix is a rich reservoir for neurotrophic factors that are key components for neuronal homeostasis, differentiation, and regeneration. These data suggest that neurotrophins in DMPs could play an important role as signaling molecules for the innervation of the pulp-dentin complex during the processes of tooth formation, repair, and regeneration.

Interactions between the ectodermal and mesenchymal tissues are the basis of the central mechanism regulating tooth development. Based on this epithelial-mesenchymal interaction (EMI), we demonstrated that copine-7 (CPNE7) is secreted by preameloblasts and regulates the differentiation of mesenchymal cells of dental or non-dental origin into odontoblasts. However, the precise expression patterns of CPNE7 in the stages of tooth development have not yet been elucidated. The aim of the present study was to establish the spatiotemporal expression pattern of CPNE7 during mouse tooth development. To examine the spatiotemporal expression patterns of CPNE7 during mouse tooth development, we investigate the distribution of CPNE7 in the embryonic and postnatal developing mouse tooth. Immunohistochemistry, in situ hybridization, real-time PCR, and western blot analysis are performed to investigate the CPNE7 expression pattern during tooth development of the mandibular mouse first molar. During the initiation stage (bud stage), CPNE7 protein expression is observed in the dental epithelium but not yet in the dental mesenchyme. At E18 (bell stage), expression of CPNE7 protein and mRNA is primarily observed in ectomesenchymal cells of dental papilla. At P7 (crown formation stage), CPNE7 is localized in differentiating odontoblasts but weak expression is detected in mature ameloblasts. These findings suggest that CPNE7 secreted by dental epithelium induces the differentiation of ectomesenchymal cells into preodontoblast in concert with EMI. CPNE7 is clearly expressed in differentiating odontoblasts and the odontoblast process during dentinogenesis, but is no longer expressed in fully differentiated odontoblasts. Furthermore, CPNE7 is expressed in the Hertwig's epithelial root sheath (HERS) and in the facing preodontoblasts during root dentin formation. Taken together, these results illustrate the dynamic expression of CPNE7 during tooth development and suggest its important function in entire stages of tooth development.

Introduction To investigate the physiological function of leptin in human dental pulp, and to determine the specific pathways implicated in its effect. Methods Twenty-seven dental pulp samples were obtained from human third molars. Pulp samples were treated with or without human recombinant leptin. Leptin functional effect was analyzed in terms of regulation of the synthesis levels of DSPP and DMP-1, determined by immunoblot. Results Leptin stimulated DMP-1 and DSPP synthesis in all human dental pulp specimens. The stimulatory effect of leptin on DMP-1 and DSPP synthesis was partially prevented by blocking mitogen-activated protein kinase (MAPK 1/3) and phosphatidylinositol 3 kinase (PI3K) pathways, respectively. Conclusions The present study demonstrates the functional effect of leptin in human dental pulp stimulating the expression of DMP-1 and DSPP, both proteins implicated in dentinogenesis. Leptin stimulates DSPP expression via PI3K pathway and DMP-1 synthesis via MAPK 1/3 pathway. These results support the role of leptin in pulpal reparative response, opening a new research line that could have translational application to the clinic in vital pulp therapy procedures.

Aim To investigate pulp responses after pulpotomy and EDTA conditioning of pulp chamber dentinal walls with or without the placement of a collagenous scaffold in the experimental model of miniature swine teeth. Methodology Forty-two fully developed permanent premolars and molars of healthy miniature swines were used. After preparation of pulp exposures through Class I cavities, the tissue of the pulp chamber was completely removed. The haemorrhage was controlled, and the root pulp was protected using a polyurethane film. The circumpulpal pulp chamber dentine was treated for 3 min with normal saline (group 1), or 17% EDTA solution (groups 2 and 3). The film was removed, and the pulp chamber cavities were left empty (groups 1 and 2), or filled with swine collagenous sponge (group 3). The access cavities were restored with a Teflon disc and glass ionomer. Teeth were evaluated histo-morphologically after 10 weeks. Data were compared using the nonparametric Fisher's exact test. Results Teeth after treatment of dentine with saline (group 1) were associated with no or only traces of hard tissue formation along the root canal walls. Atubular tertiary dentine deposition in the form of matrix deposition along root canal walls, or dentine bridge formation at the orifice of root canals or complete pulp canal obliteration, was found after treatment of dentine with EDTA in both experiments (groups 2 and 3). Significantly different types of mineralization in the root canals of groups 2 and 3 were seen (P = 0.001). Tissue changes in the pulp cavity, characterized by soft tissue growth and osteodentine or atubular tertiary dentine formation, were only seen after EDTA conditioning of dentine, in 6.2% of the teeth without scaffold and 64.7% of the teeth with scaffold application. Newly deposited mineralized matrix in the pulp chamber was always in continuation with hard tissue deposited in the root canals. Conclusions The EDTA conditioning of pulp cavity dentinal walls after pulpotomy induced dentinogenic events in the root pulp. Application of collagenous scaffold in the pulp chamber enhanced soft tissue growth and mineralized tissue formation along the treated circumpulpal dentine.

Dentin is a major component of teeth that protects dental pulp and maintains tooth health. Bone morphogenetic protein (BMP) signaling is required for the formation of dentin. Mice lacking a BMP type I receptor, activin A receptor type 1 (ACVR1), in the neural crest display a deformed mandible. Acvr1 is known to be expressed in the dental mesenchyme. However, little is known about how BMP signaling mediated by ACVR1 regulates dentinogenesis. To explore the role of ACVR1 in dentin formation in molars and incisors in mice, Acvr1 was conditionally disrupted in Osterix-expressing cells (designated as cKO). We found that loss of Acvr1 in the dental mesenchyme led to dentin dysplasia in molars and osteodentin formation in incisors. Specifically, the cKO mice exhibited remarkable tooth phenotypes characterized by thinner dentin and thicker predentin, as well as compromised differentiation of odontoblasts in molars. We also found osteodentin formation in the coronal part of the cKO mandibular incisors, which was associated with a reduction in the expression of odontogenic gene Dsp and an increase in the expression of osteogenic gene Bsp, leading to an alteration of cell fate from odontoblasts to osteoblasts. In addition, the expressions of WNT antagonists, Dkk1 and Sost, were downregulated and B-catenin was up-regulated in the cKO incisors, while the expression levels were not changed in the cKO molars, compared with the corresponding controls. Our results indicate the distinct and critical roles of ACVR1 between incisors and molars, which is associated with alterations in the WNT signaling related molecules. This study demonstrates for the first time the physiological roles of ACVR1 during dentinogenesis.

Calcium and phosphate are the major components of hydroxyapatite crystals that form the inorganic portion of the teeth. Apart from these, certain elements are present in little amounts in enamel and dentin of the human teeth. Although they are required in minute quantities, their absence may alter healthy development of enamel and dentin and may result in developmental tooth defects as well as dental caries. Furthermore, excessive intake of some trace elements may inversely affect tooth development and health. The exact of effects that trace elements have on teeth and oral health is still an unexplored territory. The present paper reviews the presence of trace elements in teeth and their role in tooth health and development.

Dental pulp vitality is a desideratum for preserving the health and functionality of the tooth. In certain clinical situations that lead to pulp exposure, bioactive agents are used in direct pulp-capping procedures to stimulate the dentin-pulp complex and activate reparative dentinogenesis. Hydraulic calcium-silicate cements, derived from Portland cement, can induce the formation of a new dentin bridge at the interface between the biomaterial and the dental pulp. Odontoblasts are molecularly activated, and, if necessary, undifferentiated stem cells in the dental pulp can differentiate into odontoblasts. An extensive review of literature was conducted on MedLine/PubMed database to evaluate the histological outcomes of direct pulp capping with hydraulic calcium-silicate cements performed on animal models. Overall, irrespective of their physico-chemical properties and the molecular mechanisms involved in pulp healing, the effects of cements on tertiary dentin formation and pulp vitality preservation were positive. Histological examinations showed different degrees of dental pulp inflammatory response and complete/incomplete dentin bridge formation during the pulp healing process at different follow-up periods. Calcium silicate materials have the ability to induce reparative dentinogenesis when applied over exposed pulps, with different behaviors, as related to the animal model used, pulpal inflammatory responses, and quality of dentin bridges.

AIM: Our study aimed to measure (1) the flexural strength, (2) shear bond strength to dentin, (3) pH, and (4) calcium (Ca) release of a series of innovative resin-modified calcium-silicate pulp-capping cements (Rm-CSCs). Using an ex-vivo human vital tooth-culture model, we additionally assessed (5) their pulp-healing initiation when brought in direct contact with human dental pulp tissue. METHODOLOGY: Three experimental Rm-CSCs, being referred to 'Exp_HEAA', 'Exp_GDM' and 'Exp_HEAA/GDM', contained either 20 wt% N-(2-hydroxyethyl) acrylamide (HEAA), 20 wt% glycerol dimethacrylate (GDM) or 10 wt% HEAA plus 10 wt% GDM, added to a common base composition consisting of 25 wt% urethane dimethacrylate (UDMA), 10 wt% 4-methacryloxyethyl trimellitate anhydride (4-MET), and 5 wt% N,N'-{[(2-acrylamido-2-[(3-acrylamidopropoxy)methyl] propane-1,3-diyl)bis(oxy)]bis-(propane-1,3-diyl)}diacrylamide (FAM-401). As Ca source and radiopacifier, 37 wt% tricalcium silicate powder (TCS) and 3 wt% zirconium oxide (ZrO 2) were respectively added. RESULTS: All three experimental Rm-CSCs revealed a significantly higher flexural strength and shear bond strength to dentin (p

Tooth root morphogenesis involves two biological processes, root elongation and dentinogenesis, which are guaranteed by downgrowth of Hertwig’s epithelial root sheath (HERS) and normal odontoblast differentiation. Ubiquitin-dependent protein degradation has been reported to precisely regulate various physiological processes, while its role in tooth development is still elusive. Here we show ubiquitin-specific protease 34 (USP34) plays a pivotal role in root formation. Deletion of Usp34 in dental mesenchymal cells leads to short root anomaly, characterized by truncated roots and thin root dentin. The USP34-deficient dental pulp cells (DPCs) exhibit decreased odontogenic differentiation with downregulation of nuclear factor I/C (NFIC). Overexpression of NFIC partially restores the impaired odontogenic potential of DPCs. These findings indicate that USP34-dependent deubiquitination is critical for root morphogenesis by stabilizing NFIC.

To study the features of reparative dentinogenesis when using biomaterials during accidental opening of the pulp in an experiment.In an experiment on 3 dwarf pigs of the Svetlogorsk breed a traumatic opening of the tooth cavity was carried out in 18 teeth (6 molars, 6 premolars, 6 incisors), then Biodentin (9 teeth) and Aurosil (9 teeth) were applied on the opened pulp horn. After 14, 30 and 60 days, dwarf pigs were euthanized, and jaw blocks taken. Histological examination of the teeth was carried out using the method of embedding in methyl methacrylate (OsteoBead, Sigma, USA). Thin sections of 35-50 microns were made from the samples and stained using the «Heavenly Trichrome» method. Documentation was carried out using a Leica DM100 microscope and an EC3 digital camera in reflected and transmitted light.Histological examination of the thin sections of teeth stained according to the «Heavenly Trichrome» method revealed that 2 weeks after the pulp opening and applying of Aurosil and Biodentin, comparable processes occurred in the tooth cavity. Thus, the proliferation of odontoblasts was noted on the lateral surfaces of the chamber. New dentin layers and odontoblasts spread over the lower surface of the material with the formation of dentinal bridges by 4 weeks of observation in both groups. By week 12, there was a decrease in the proliferative activity of odontoblasts and dentin maturation in all cases. At the initial stages of the experiment, some pulp edema and moderate plethora were noted, which was no longer observed at 4 weeks. By 12 weeks, because of the proliferation of odontoblasts, the volume of the tooth cavity was reduced by a third. By the 6th month in the experiment, no significant changes in the structure of dentin and pulp were revealed.According to morphological data the studied biomaterials (Aurosil and Biodentin) contribute to dentin regeneration equally.Изучить особенности репаративного дентиногенеза при применении биоматериалов при случайном вскрытии пульпы в эксперименте.В эксперименте на 3 карликовых свиньях светлогорской породы проведено травматическое вскрытие полости зуба, в области 18 зубов (из них 6 моляров, 6 премоляров, 6 резцов) на вскрытый рог пульпы наносили препарат Биодентин (9 зубов) и Ауросил (9). Через 14, 30 и 60 дней карликовые свиньи подвергались эвтаназии, был проведен забор блоков челюстей. Гистологическое исследование зубов карликовых свиней проводили с использованием метода заливки в метилметакрилат (OsteoBead, «Sigma», США). Из образцов изготавливали шлифы толщиной 35—50 мкм и окрашивали по методу «Небесный трихром». Документирование осуществлялось с помощью микроскопа Leica DM100 и цифровой камеры EC3 в отраженном и проходящем свете.Гистологическое исследование шлифов зубов, окрашенных по методу «небесный трихром», выявило, что через 2 нед после вскрытия пульпы и нанесения биоактивных цементов в полости зуба происходили сопоставимые процессы. Так, на боковых поверхностях камеры отмечали пролиферацию одонтобластов. Напластования нового дентина и одонтобласты распространялись по нижней поверхности материала с образованием дентинных мостиков к 4 нед наблюдения в обеих группах. К 12 нед отмечали снижение пролиферативной активности одонтобластов и созревание дентина во всех случаях. На начальных этапах эксперимента отмечали некоторый отек пульпы и умеренное полнокровие, которое уже не наблюдалось на сроке 4 нед. К 12 нед в результате пролиферации одонтобластов произошло сокращение объема полости зуба наПо данным морфологического исследования установлено, что биоматериалы Ауросил и Биодентин способствуют регенерации дентина в равной степени.

The formation of dentin is a highly regulated and well-controlled process, in which odontoblasts form a massive dentin tubular system with one main stalk. These tubules secrete collagenous proteins followed by the deposition of inorganic calcium phosphate in the form of mineral crystals. Two non-collagenous matrix proteins (dentin matrix protein 1, DMP1; and dentin sialophosphoprotein, DSPP) play an important role in the control of mineralization. The current theory, named “mineralization front,” is that minerals are deposited at the edge of the dental pulp and the dentin. In this chapter, we briefly introduced an overall background on dentin mineralization and key roles of DMP1 and DSPP. We then focus on two new findings in researches of dentin mineralization: a recent breakthrough in mineralization mechanism studies because of applications of mineral labeling in a cell lineage-tracing line (the Gli1-creERT2; R26RtdTomato) and the 2.3 Col1a1-GFP reporter line; and discoveries of a novel function of bone morphogenetic protein 1 (BMP1) and tolloid-like 1 (TLL1) proteinase in control of DMP1 and DSPP during dentinogenesis. These findings challenge the current dogma and will shed new light on our understanding of dentin structure and function as well as the mechanisms of mineralization.

Dentin dysplasia (DD) and dentinogenesis imperfecta (DGI) patients have abnormal structure, morphology, and function of dentin. DD-II, DGI-II, and DGI-III are caused by heterozygous mutations in the dentin sialophosphoprotein (DSPP) gene in humans. Evidences have shown that loss of function of DSPP in Dspp knockout mice leads to phenotypes similar to DGI-III, and that the abnormal dentinogenesis is associated with decreased levels of DSPP, indicating that DSPP haploinsufficiency may play a role in dentinogenesis. Thus, to testify the haploinsufficiency of Dspp, we used a Dspp heterozygous mouse model to observe the phenotypes in the teeth and the surrounding tissues. We found that Dspp heterozygous mice displayed dentin phenotypes similar to DD-II at the ages of 12 and 18 months, which was characterized by excessive attrition of the enamel at the occlusal surfaces, thicker floor dentin of the pulp chamber, decreased pulp volume, and compromised mineralization of the dentin. In addition, the periodontium was also affected, exhibiting apical proliferation of the junctional epithelium, decreased height and width of the alveolar bone, and infiltration of the inflammatory cells, leading to the destruction of the periodontium. Both the dental and periodontal phenotypes were age-dependent, which were more severe at 18 months old than those at 12 months old. Our report is the first to claim the haploinsufficiency of Dspp gene and a DD-II mouse model, which can be further used to study the molecular mechanisms of DD-II.

The interaction between immune cells and stem cells is important during tissue repair. Macrophages have been described as being crucial for limb regeneration and in certain circumstances have been shown to affect stem cell differentiation in vivo. Dentine is susceptible to damage as a result of caries, pulp infection and inflammation all of which are major problems in tooth restoration. Characterising the interplay between immune cells and stem cells is crucial to understand how to improve natural repair mechanisms. In this study, we used an in vivo damage model, associated with a macrophage and neutrophil depletion model to investigate the role of immune cells in reparative dentine formation. In addition, we investigated the effect of elevating the Wnt/β-catenin pathway to understand how this might regulate macrophages and impact upon Wnt receiving pulp stem cells during repair. Our results show that macrophages are required for dental pulp stem cell activation and appropriate reparative dentine formation. In addition, pharmacological stimulation of the Wnt/β-catenin pathway via GSK-3β inhibitor small molecules polarises macrophages to an anti-inflammatory state faster than inert calcium silicate-based materials thereby accelerating stem cell activation and repair. Wnt/β-catenin signalling thus has a dual role in promoting reparative dentine formation by activating pulp stem cells and promoting an anti-inflammatory macrophage response.

FUSE binding protein 1 (Fubp1), a regulator of the c-Myc transcription factor and a DNA/RNA-binding protein, plays important roles in the regulation of gene transcription and cellular physiology. In this study, to reveal the precise developmental function of Fubp1, we examined the detailed expression pattern and developmental function of Fubp1 during tooth morphogenesis by RT-qPCR, in situ hybridization, and knock-down study using in vitro organ cultivation methods. In embryogenesis, Fubp1 is obviously expressed in the enamel organ and condensed mesenchyme, known to be important for proper tooth formation. Knocking down Fubp1 at E14 for two days, showed the altered expression patterns of tooth development related signalling molecules, including Bmps and Fgf4. In addition, transient knock-down of Fubp1 at E14 revealed changes in the localization patterns of c-Myc and cell proliferation in epithelium and mesenchyme, related with altered tooth morphogenesis. These results also showed the decreased amelogenin and dentin sialophosphoprotein expressions and disrupted enamel rod and interrod formation in one- and three-week renal transplanted teeth respectively. Thus, our results suggested that Fubp1 plays a modulating role during dentinogenesis and amelogenesis by regulating the expression pattern of signalling molecules to achieve the proper structural formation of hard tissue matrices and crown morphogenesis in mice molar development.

Odontoblast differentiation is a complex and multistep process regulated by signaling pathways, including the Wnt/β-catenin signaling pathway. Both positive and negative effects of Wnt/β-catenin signaling on dentinogenesis have been reported, but the underlying mechanisms of these conflicting results are still unclear. To gain a better insight into the role of Wnt/β-catenin in dentinogenesis, we used dental pulp cells from a panel of transgenic mice, in which fluorescent protein expression identifies cells at different stages of odontoblast and osteoblast differentiation. Our results showed that exposure of pulp cells to WNT3a at various times and durations did not induce premature differentiation of odontoblasts. These treatments supported the survival of undifferentiated cells in dental pulp and promoted the formation of 2.3GFP+ preodontoblasts and their rapid transition into differentiated odontoblasts expressing DMP1-Cherry and DSPP-Cerulean transgenes. WNT3a also promoted osteogenesis in dental pulp cultures. These findings provide critical information for the development of improved treatments for vital pulp therapy and dentin regeneration.

We aim to examine the effects of a newly developed peptide derived from CPNE7 (Cpne7-DP) in tertiary dentin formation and peritubular space occlusion, and comprehensively evaluate its potential as a bioactive therapeutic agent. Human dental pulp cells (HDPCs) and a mouse pre-odontoblast cell line, MDPC-23, were chosen for in vitro studies to characterize lineage-specific cell responses after Cpne7-DP treatment. Whether Cpne7-DP reproduces the dentin regenerative potential of CPNE7 was tested using a beagle dog model by generating dentinal defects of various degrees in vivo. Peritubular space occlusion was further examined by scanning electron microscopy and microleakage test, while overall mineralization capacity of Cpne7-DP was tested ex vivo. CPNE7 promotes tubular dentin formation under both shallow and deep dentinal defects, and the functional peptide Cpne7-DP induces odontoblast-like differentiation in vitro, mineralization ex vivo, and tubular dentin formation in in vivo beagle dog dentin exposure and pulp exposure models. Moreover, Cpne7-DP leads to peritubular space occlusion and maintains stability under different conditions. We show that CPNE7 and its derivative functional peptide Cpne7-DP promotes dentin regeneration in dentinal defects of various degrees and that the regenerated hard tissue demonstrates the characteristics of true dentin. Limitations of the current dental materials including post-operative hypersensitivity make biological repair of dentin a field of growing interest. Here, we suggest that the dual functions of Cpne7-DP in tubular dentin formation and peritubular space occlusion are promising for the treatment of dentinal loss and sensitivity.

Odontoblasts and pulp stroma cells are embedded within supramolecular networks of extracellular matrix (ECM). Fibrillin microfibrils and associated proteins are crucial constituents of these networks, serving as contextual scaffolds to regulate tissue development and homeostasis by providing both structural and mechanical properties and sequestering growth factors of the TGF-β superfamily. EMILIN-1, -2, and -3 are microfibril-associated glycoproteins known to modulate cell behaviour, growth factor activity, and ECM assembly. So far their expression in the various cells of the dentin-pulp complex during development, in the adult stage, and during inflammation has not been investigated. Confocal immunofluorescence microscopy and western blot analysis of developing and adult mouse molars and incisors revealed an abundant presence of EMILINs in the entire dental papilla, at early developmental stages. Later in development the signal intensity for EMILIN-3 decreases, while EMILIN-1 and -2 staining appears to increase in the pre-dentin and in the ECM surrounding odontoblasts. Our data also demonstrate new specific interactions of EMILINs with fibulins in the dentin enamel junction. Interestingly, in dentin caries lesions the signal for EMILIN-3 was significantly increased in inflamed odontoblasts. Overall our findings point for the first time to a role of EMILINs in dentinogenesis, pulp biology, and inflammation.

FUSE binding protein 1 (Fubp1), a regulator of the c-Myc transcription factor and a DNA/RNA-binding protein, plays important roles in the regulation of gene transcription and cellular physiology. In this study, to reveal the precise developmental function of Fubp1, we examined the detailed expression pattern and developmental function of Fubp1 during tooth morphogenesis by RT-qPCR, in situ hybridization, and knock-down study using in vitro organ cultivation methods. In embryogenesis, Fubp1 is obviously expressed in the enamel organ and condensed mesenchyme, known to be important for proper tooth formation. Knocking down Fubp1 at E14 for two days, showed the altered expression patterns of tooth development related signalling molecules, including Bmps and Fgf4. In addition, transient knock-down of Fubp1 at E14 revealed changes in the localization patterns of c-Myc and cell proliferation in epithelium and mesenchyme, related with altered tooth morphogenesis. These results also showed the decreased amelogenin and dentin sialophosphoprotein expressions and disrupted enamel rod and interrod formation in one- and three-week renal transplanted teeth respectively. Thus, our results suggested that Fubp1 plays a modulating role during dentinogenesis and amelogenesis by regulating the expression pattern of signalling molecules to achieve the proper structural formation of hard tissue matrices and crown morphogenesis in mice molar development.

This chapter describes the formation of dentin, dentinogenesis. Since most biochemical studies have been performed on dentin from cow and rat, those data concern mainly these species. Relatively little has, in fact, been done on the biochemistry of human dentin. Initially, a brief outline of dentin morphology is also given, this bearing mostly upon human dentin. Dentin, the most voluminous mineralized tissue of the tooth, may be looked upon as a connective tissue, the extracellular matrix of which has been modified to contain a mineral phase in order to fulfill the functional requirements on the issue. On an average, the mineral content of human dentin is somewhat higher than that of bone. The rate of tertiary dentin formation and its structure vary according to the intensity and character of the stimulus. The morphology is distinct and no resorptive processes occur in the tissue simultaneously with calcification.

Microbiota and their metabolites short-chain fatty acids (SCFAs) have important roles in regulating tissue regeneration and mesenchymal stem cell (MSC) differentiation. In this study, we explored the potential effects of SCFAs on murine incisor regeneration and dental MSCs. We observed that SCFA deficiency induced by depletion of microbiota through antibiotic treatment led to lower renewal rate and delayed dentinogenesis in mice incisors. Supplementation with SCFAs in drinking water during antibiotic treatment can rescue the renewal rate and dentinogenesis effectively. In vitro, stimulation with SCFAs could promote differentiation of dental MSCs to odontoblasts. We further found that SCFAs could contribute to dentinogenic differentiation of dental MSCs by increasing bone morphogenetic protein (BMP) signal activation. SCFAs could inhibit deacetylation and increase BMP7 transcription of dental MSCs, which promoted BMP signaling. Our results suggested that SCFAs were required for incisor regeneration as well as differentiation of dental MSCs. Microbiota and their metabolites should be concerned as important factors in the tissue renewal and regeneration.

Objective Evaluate the dentinogenesis in the offspring of rats submitted to gestational protein restriction (GPR). Design: The offspring were evaluated at the 21st day of gestation (21 dG). Assessments were made of morphological parameters and the RANKL/OPG system - bone tissue maturation markers - in the upper incisor tooth germ. Pregnant 10-week-old female Wistar rats were divided into normal protein (NP, 17% casein, n = 5) and low protein (LP, 6% casein, n = 5) diet groups. At 21 dG, the offspring maxillae were collected for histomorphometric and immunohistochemical analyses. Results: The LP group showed decreased thickness of the dentin and odontoblast cell layers on the tooth germ. GPR led to decreased OPG expression and increased RANKL expression in the incisor germ. Conclusion: The results suggested that gestational protein restriction altered odontoblast RANKL/OPG expression and decreased dentin matrix deposition and thickness in tooth development.

With increasing life expectancy, demands for dental tissue and whole-tooth regeneration are becoming more significant. Despite great progress in medicine, including regenerative therapies, the complex structure of dental tissues introduces several challenges to the field of regenerative dentistry. Interdisciplinary efforts from cellular biologists, material scientists, and clinical odontologists are being made to establish strategies and find the solutions for dental tissue regeneration and/or whole-tooth regeneration. In recent years, many significant discoveries were done regarding signaling pathways and factors shaping calcified tissue genesis, including those of tooth. Novel biocompatible scaffolds and polymer-based drug release systems are under development and may soon result in clinically applicable biomaterials with the potential to modulate signaling cascades involved in dental tissue genesis and regeneration. Approaches for whole-tooth regeneration utilizing adult stem cells, induced pluripotent stem cells, or tooth germ cells transplantation are emerging as promising alternatives to overcome existing in vitro tissue generation hurdles. In this interdisciplinary review, most recent advances in cellular signaling guiding dental tissue genesis, novel functionalized scaffolds and drug release material, various odontogenic cell sources, and methods for tooth regeneration are discussed thus providing a multi-faceted, up-to-date, and illustrative overview on the tooth regeneration matter, alongside hints for future directions in the challenging field of regenerative dentistry.

Tooth formation can be affected by various factors, such as oral disease, drug administration, and systemic illness, as well as internal conditions including dentin formation. Dyslipidemia is an important lifestyle disease, though the relationship of aberrant lipid metabolism with tooth formation has not been clarified. This study was performed to examine the effects of dyslipidemia on tooth formation and tooth development. Dyslipidemia was induced in mice by giving a high-fat diet (HFD) for 12 weeks. Additionally, LDL receptor-deficient (Ldlr−/−) strain mice were used to analyze the effects of dyslipidemia and lipid metabolism in greater detail. In the HFD-fed mice, incisor elongation was decreased and pulp was significantly narrowed, while histological findings revealed disappearance of predentin. In Ldlr−/− mice fed regular chow, incisor elongation showed a decreasing trend and pulp a narrowing trend, while predentin changes were unclear. Serum lipid levels were increased in the HFD-fed wild-type (WT) mice, while Ldlr−/− mice given the HFD showed the greatest increase. These results show important effects of lipid metabolism, especially via the LDL receptor, on tooth homeostasis maintenance. In addition, they suggest a different mechanism for WT and Ldlr−/− mice, though the LDL receptor pathway may not be the only factor involved.