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Collagen X is an extracellular matrix protein, usually found in the hypertrophic cartilage destined to be mineralized. It is intimately associated with the mineralization process of the mammalian hard tissues, and particularly, regulating the compartmentalization of matrix components. Despite the fact that the dentine of the tooth is highly mineralized, there are no previous reports to indicate the presence of collagen X in this connective tissue. Here we report, for the first time, its presence in mammalian dentine based on micromorphological and immunohistochemical data. We hypothesize that the collagen X in dentine may in the long term arrest the progression of the mineralization front towards the soft tissue components of the pulp that are not destined to be mineralized., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Background and Objectives : Regenerative dentistry aims to regenerate the pulp-dentin complex and restore those of its functions that have become compromised by pulp injury and/or inflammation. Scaffold-based techniques are a regeneration strategy that replicate a biological environment by utilizing a suitable scaffold, which is considered crucial for the successful regeneration of dental pulp. The aim of the present review is to address the main characteristics of the different scaffolds, as well as their application in dentin-pulp complex regeneration. Materials and Methods : A narrative review was conducted by two independent reviewers to answer the research question: What type of scaffolds can be used in dentin-pulp complex regeneration? An electronic search of PubMed, EMBASE and Cochrane library databases was undertaken. Keywords including "pulp-dentin regeneration scaffold" and "pulp-dentin complex regeneration" were used. To locate additional reports, reference mining of the identified papers was undertaken. Results : A wide variety of biomaterials is already available for tissue engineering and can be broadly categorized into two groups: (i) natural, and (ii) synthetic, scaffolds. Natural scaffolds often contain bioactive molecules, growth factors, and signaling cues that can positively influence cell behavior. These signaling molecules can promote specific cellular responses, such as cell proliferation and differentiation, crucial for effective tissue regeneration. Synthetic scaffolds offer flexibility in design and can be tailored to meet specific requirements, such as size, shape, and mechanical properties. Moreover, they can be functionalized with bioactive molecules, growth factors, or signaling cues to enhance their biological properties and the manufacturing process can be standardized, ensuring consistent quality for widespread clinical use. Conclusions : There is still a lack of evidence to determine the optimal scaffold composition that meets the specific requirements and complexities needed for effectively promoting dental pulp tissue engineering and achieving successful clinical outcomes.

Direct pulp capping has been established as a more conservative alternative to root canal therapy, especially in case of an iatrogenic or traumatic exposure of the dental pulp. However, recent literature focuses on regeneration therapies to develop a physiological dentin barrier. The process of tissue regeneration through stem cell therapy involves a complex interaction between stem cells, growth factors and scaffolds known as the 'tissue engineering triad'. Recent advancements in stem cell therapy focus on the re programming of stem cells, development of scaffolds and enhancement of the regenerative potential of these stem cells, using appropriate growth factors and transfer media. This article provides a summary of the current evidence regarding the use of stem cell therapy in direct pulp capping. The sources of stem cells, types of scaffolds and growth factors have been described in detail along with the limitations and future prospects.

Aim: The aim of the study was to investigate the effect of dentine conditioning agents on growth factor liberation and settlement of dental pulp progenitor cells (DPSCs) on dentine surfaces., Methodology: The agents used included ethylenediaminetetraacetic acid (EDTA; 10%, pH 7.2), phosphoric acid (37%, pH < 1), citric acid (10%, pH 1.5) and polyacrylic acid (25%, pH 3.9). Human dentine slices were conditioned for exaggerated conditioning times of 5 and 10 min, so that the growth factor liberation reached quantifiable levels above the limit of detection of the laboratory methods employed. Transforming growth factor beta-1 (TGF-β1) release and surface exposure were quantified by enzyme-linked immunosorbent assay (ELISA) and immunogold labelling. Scanning electron microscopy (SEM) was used to assess the morphology of cells and coverage by DPSCs cultured on dentine surfaces for 8 days., Results: After 5-min conditioning of dentine slices, citric acid was the most effective agent for growth factor release into the aqueous environment as measured by ELISA (Mann-Whitney U with Bonferroni correction, p < .01 compared with phosphoric and polyacrylic acid). As well as this, dentine slices treated with phosphoric acid for the same period, displayed significantly less TGF-β1 on the surface compared with the other agents used, as measured by immunogold labelling (MWU with Bonferroni correction, p < .05). After 8 days, widespread coverage by DPSCs on dentine surfaces conditioned with citric acid and EDTA were evident under SEM. On dentine surfaces conditioned with phosphoric and polyacrylic acid, respectively, less spread cells and inconsistent cell coverage were observed., Conclusions: Based on the findings of this in vitro study, a desirable biological growth factor-mediated effect may be gained when conditioning dentine by milder acidic or chelating agents such as citric acid and EDTA. The results must be interpreted in the context that the potential of the applied materials inducing a desirable biological response in DPSCs is only one consideration amongst other important ones in a clinical setting. However, it is crucial to look beyond the mere physical effects of materials and move towards biologically based treatment approaches as far as the restorative management of teeth with viable dental pulps are concerned., (© 2022 The Authors. International Endodontic Journal published by John Wiley & Sons Ltd on behalf of British Endodontic Society.)

Regenerative endodontic therapy (RET) utilizing tissue engineering approach can promote the regeneration of pulp-dentin complex to restore pulp vascularization, neuralization, immune function and tubular dentin, therefore the regenerated pulp-dentin complex will have normal function. Multiple factors may significantly affect the efficacy of RET, including stem cells, biosignaling molecules and biomaterial scaffolds. Stem cells derived from dental tissues (such as dental pulp stem cells) exhibit certain advantages in RET. Combined application of multiple signaling molecules and activation of signal transduction pathways such as Wnt/β-catenin and BMP/Smad play pivotal roles in enhancing the potential of stem cell migration, proliferation, odontoblastic differentiation, and nerve and blood vessel regeneration. Biomaterials suitable for RET include naturally-derived materials and artificially synthetic materials. Artificially synthetic materials should imitate natural tissues for biomimetic modification in order to realize the temporal and spatial regulation of pulp-dentin complex regeneration. The realization of pulp-dentin complex regeneration depends on two strategies: stem cell transplantation and stem cell homing. Stem cell homing strategy does not require the isolation and culture of stem cells in vitro , so is better for clinical application. However, in order to achieve the true regeneration of pulp-dentin complex, problems related to improving the success rate of stem cell homing and promoting their proliferation and differentiation need to be solved. This article reviews the influencing factors of pulp-dentin complex regeneration and related biological strategies, and discusses the future research direction of RET, to provide reference for clinical translation and application of RET.

Background: Bio-root regeneration is a promising treatment for tooth loss. It has been reported that dental-derived stem cells are effective seed cells for bio-root construction, but further applications are limited by their few sources. Human adipose tissues have a wide range of sources and numerous studies have confirmed the ability of adipose-derived stromal/stem cells (ASCs) in regenerative medicine. In the current study, the odontogenic capacities of ASCs were compared with dental-derived stem cells including dental follicle cells (DFCs), and stem cells from human exfoliated deciduous teeth (SHEDs)., Methods: The biological characteristics of ASCs, DFCs, and SHEDs were explored in vitro. Two-dimensional (2D) and three-dimensional (3D) cultures were compared in vitro. Odontogenic characteristics of porcine-treated dentin matrix (pTDM) induced cells under a 3D microenvironment in vitro were compared. The complexes (cell/pTDM) were transplanted subcutaneously into nude mice to verify regenerative potential. RNA sequencing (RNA-seq) was used to explore molecular mechanisms of different seed cells in bio-root regeneration., Results: 3D culture was more efficient in constructing bio-root complexes. ASCs exhibited good biological characteristics similar to dental-derived stem cells in vitro. Besides, pTDM induced ASCs presented odontogenic ability similar to dental-derived stem cells. Furthermore, 3D cultured ASCs/pTDM complex promoted regeneration of dentin-like, pulp-like, and periodontal fiber-like tissues in vivo. Analysis indicated that PI3K-Akt, VEGF signaling pathways may play key roles in the process of inducing ASCs odontogenic differentiation by pTDM., Conclusions: ASCs are potential seed cells for pTDM-induced bio-root regeneration, providing a basis for further research and application., (© 2022. The Author(s).)

The aim of this systematic review was to evaluate the application of potential therapeutic signaling molecules on complete dentin-pulp complex and pulp tissue regeneration in orthotopic and ectopic animal studies. A search strategy was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement in the MEDLINE/PubMed database. Animal studies evaluating the application of signaling molecules to pulpectomized teeth for pulp tissue or dentin-pulp complex regeneration were included. From 2530 identified records, 18 fulfilled the eligibility criteria and were subjected to detailed qualitative analysis. Among the applied molecules, basic fibroblast growth factor, vascular endothelial growth factor, bone morphogenetic factor-7, nerve growth factor, and platelet-derived growth factor were the most frequently studied. The clinical, radiographical and histological outcome measures included healing of periapical lesions, root development, and apical closure, cellular recolonization of the pulp space, ingrowth of pulp-like connective tissue (vascularization and innervation), mineralized dentin-like tissue formation along the internal dentin walls, and odontoblast-like cells in contact with the internal dentin walls. The results indicate that signaling molecules play an important role in dentin/pulp regeneration. However, further studies are needed to determine a more specific subset combination of molecules to achieve greater efficiency towards the desired tissue engineering applications.

Severe dental tissue damage induces odontoblast death, after which dental pulp stem and progenitor cells (DPSCs) differentiate into odontoblast-like cells, contributing to reparative dentin. However, the damage-induced mechanism that triggers this regeneration process is still not clear. We aimed to understand the effect of odontoblast death without hard tissue damage on dental regeneration. Herein, using a Cre/LoxP-based strategy, we demonstrated that cell-rich zone (CZ)-localizing Nestin-GFP-positive and Nestin-GFP-negative cells proliferate and differentiate into odontoblast-like cells in response to odontoblast depletion. The regenerated odontoblast-like cells played a role in reparative dentin formation. RNA-sequencing analysis revealed that the expression of odontoblast differentiation- and activation-related genes was upregulated in the pulp in response to odontoblast depletion even without damage to dental tissue. In this regenerative process, the expression of type I parathyroid hormone receptor (PTH1R) increased in the odontoblast-depleted pulp, thereby boosting dentin formation. The levels of PTH1R and its downstream mediator, i.e., phosphorylated cyclic AMP response element-binding protein (Ser133) increased in the physically damaged pulp. Collectively, odontoblast death triggered the PTH1R cascade, which may represent a therapeutic target for inducing CZ-mediated dental regeneration., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: Our goal was to define trigeminal nerve ending quantities and patterns in rat molar dentine, their responses to attrition (tooth wear), and their associated odontoblasts and connections with pulpal plexuses., Design: Trigeminal ganglia were labeled for axonal transport of 3 H-proteins to dentinal nerve endings in male rats (3-13 months old). Autoradiography detected radio-labeled dentinal tubules as indicators of nerve ending locations. Quantitative morphometry was done (ANOVA, t-tests), and littermates were compared for attrition and innervation., Results: There were six dentinal patterns, only two of which had an associated neural plexus of Raschkow and cell-free zone (Den-1, Den-2). Other nerves entered dentin from bush-like endings near elongated odontoblasts (Den-B), as single fibers (Den-X), as networks in predentine (PdN), or as single fibers in tertiary dentine at cusp tips (Den-S). There were at least 186,600 innervated dentinal tubules within the set of three right maxillary molars of the best-labeled rat, and similar densities were found in other rats. Attrition levels differed among cusps and in littermates (t-test p < 0.02-0.0001), but the matched right/left cusps per rat were similar. Innervations of tertiary and enamel-free dentine (Den-S, Den-X) were preserved in all rats. Den-B and Den-2 coronal patterns were unchanged unless displaced by dentinogenesis. Den-1 losses occurred in older cusps, while Den-2 patterns increased near cervical and intercuspal odontoblasts., Conclusions: The extensive molar dentinal innervation had unique distributions per rat per cusp that depended on region (buccal, middle, palatal) and attrition, but only two of six patterns connected to a plexus of Raschkow., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Aim: To introduce a methodology designed to simultaneously visualize dental ultrastructures, including cellular and soft tissue components, by utilizing phosphotungstic acid (PTA) as a contrast-enhancement agent., Methodology: Sound third molars were collected from healthy human adults and fixed in 4% buffered paraformaldehyde. To evaluate the impact of PTA in concentrations of 0.3%, 0.7% and 1% on dental soft and hard tissues for CT imaging, cementum and dentine-pulp sections were cut, dehydrated and stained with immersion periods of 12, 24 h, 2 days or 5 days. The samples were scanned in a high-resolution nano-CT device using pixel sizes down to 0.5 µm to examine both the cementum and pulpal regions., Results: Dental cementum and periodontium as well as odontoblasts and predentine were made visible through PTA staining in high-resolution three-dimensional nano-CT scans. Different segments of the tooth required different staining protocols. The thickness of the cementum could be computed over the length of the tooth once it was made visible by the PTA-enhanced contrast, and the attached soft tissue components of the interior of the tooth could be shown on the dentine-pulp interface in greater detail. Three-dimensional illustrations allowed a histology-like visualization of the sections in all orientations with a single scan and easy sample preparation. The segmentation of the sigmoidal dentinal tubules and the surrounding dentine allowed a three-dimensional investigation and quantitative of the dentine composition, such as the tubular lumen or the ratio of the tubular lumen area to the dentinal surface., Conclusion: The staining protocol made it possible to visualize hard tissues along with cellular layers and soft tissues in teeth using a laboratory-based nano-CT technique. The protocol depended on both tissue type and size. This methodology offers enhanced possibilities for the concomitant visualization of soft and hard dental tissues., (© 2021 The Authors. International Endodontic Journal published by John Wiley & Sons Ltd on behalf of British Endodontic Society.)

Aim: To investigate the effects of a bioactive glass with a high proportion of phosphorus (BG-hP) on the repair and regeneration of dental pulps in rats under an inflammatory microenvironment., Methodology: Human dental pulp cells (hDPCs) stimulated with 1 μg mL -1 lipopolysaccharide (LPS) were co-cultured with 0.1 mg mL -1 BG-hP. Cell proliferation was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assays. The expression of inflammation-related genes and odontogenic differentiation-related genes was determined by real-time PCR. Alizarin red staining was used to detect the formation of mineralized nodules. Coronal pulp tissues of rat molars were stimulated with 10 mg mL -1 LPS and then treated with BG-hP. The expression of inflammation-related genes in pulp tissue was determined by real-time PCR. Haematoxylin-eosin staining and Masson staining were performed to observe the inflammatory response and mineralized matrix formation, after subcutaneous implantation in nude mice, at 3 days and 4 weeks, respectively. Analysis of variance was performed to measure statistical significance (P < 0.05)., Results: BG-hP significantly reduced expression of interleukin-6 (IL-6) and IL-8 and significantly upregulated the expression of IL-10, IL-4 and transforming growth factor-β1 of the LPS-stimulated hDPCs (P < 0.05). BG-hP significantly inhibited the initial cell number (P < 0.05), but the hDPCs stimulated by LPS and co-cultured with BG-hP maintained the same proliferation rate as the untreated hDPCs. BG-hP significantly promoted the expression of dentine matrix protein-1 and dentine sialophosphoprotein and the mineralization capacity of the LPS-stimulated hDPCs (P < 0.05). Furthermore, BG-hP significantly downregulated the expression of Il-6 and reduced the inflammatory response of the LPS-stimulated pulp tissue 3 days after subcutaneous implantation (P < 0.05). Four weeks after subcutaneous implantation, BG-hP induced the formation of a continuous layer of dentine-like structure with dentinal tubules and polarizing odontoblast-like cells aligned along it in the LPS-stimulated pulp tissue., Conclusion: The present preliminarily results demonstrated that the bioactive glass with a high proportion of phosphorus inhibited the inflammatory response and promoted the formation of a pulp-dentine complex in a rat experimental model. This study provides a foundation for the construction of materials with the dual functions of exerting anti-inflammatory effects and promoting tissue regeneration to meet the needs of dental pulp repair and regeneration., (© 2021 International Endodontic Journal. Published by John Wiley & Sons Ltd.)

The dentin-pulp complex is essential for the long-term integrity and viability of teeth but it is susceptible to damage caused by external factors. Because traditional approaches for preserving the dentin-pulp complex have various limitations, there is a need for novel methods for dentin-pulp complex reconstruction. The development of stem cell-based tissue engineering has given rise to the possibility of combining dental stem cells with a tissue-reparative microenvironment to promote dentin-pulp complex regeneration. Concentrated growth factor, a platelet concentrate, is a promising scaffold for the treatment of dentin-pulp complex disorders. Given its characteristics of autogenesis, convenience, usability, and biodegradability, concentrated growth factor has gained popularity in medical and dental fields for repairing bone defects and promoting soft-tissue healing. Numerous in vitro studies have demonstrated that concentrated growth factor can promote the proliferation and migration of dental stem cells. Here, we review the current state of knowledge on the effects of concentrated growth factor on stem cells and its potential applications in dentin-pulp complex regeneration.

A loss of organs or the destruction of tissue leaves wounds to which organisms and living things react differently. Their response depends on the extent of damage, the functional impairment and the biological potential of the organism. Some can completely regenerate lost body parts or tissues, whereas others react by forming scars in the sense of a tissue repair. Overall, the regenerative capacities of the human body are limited and only a few tissues are fully restored when injured. Dental tissues may suffer severe damage due to various influences such as caries or trauma; however, dental care aims at preserving unharmed structures and, thus, the functionality of the teeth. The dentin-pulp complex, a vital compound tissue that is enclosed by enamel, holds many important functions and is particularly worth protecting. It reacts physiologically to deleterious impacts with an interplay of regenerative and reparative processes to ensure its functionality and facilitate healing. While there were initially no biological treatment options available for the irreversible destruction of dentin or pulp, many promising approaches for endodontic regeneration based on the principles of tissue engineering have been developed in recent years. This review describes the regenerative and reparative processes of the dentin-pulp complex as well as the morphological criteria of possible healing results. Furthermore, it summarizes the current knowledge on tissue engineering of dentin and pulp, and potential future developments in this thriving field.

Objective: To study the features of reparative dentinogenesis when using biomaterials during accidental opening of the pulp in an experiment., Material and Methods: 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., Results: 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., Conclusion: According to morphological data the studied biomaterials (Aurosil and Biodentin) contribute to dentin regeneration equally.

Introduction: Biomaterials designed for tissue engineering should be nontoxic and nonimmunogenic and should achieve their intended functions. Treated dentin matrix (TDM), a bioactive extracellular matrix, is promising for tooth regeneration. However, the effect of sterilization on the surface properties of allogenous TDM in the animal model is unclear., Methods: The biological characteristics and influences of dental pulp stem cells (DPSCs) with autoclaved TDM (a-TDM) were studied using scanning electron microscopy, immunofluorescence microscopy, immunohistochemistry, and reverse transcription polymerase chain reaction in vitro. In addition, a-TDM was implanted in a mouse model for 6 weeks and was a substrate with DPSCs for tooth reconstruction in a goat animal model in vivo., Results: Allogenous a-TDM induced and supported DPSCs to develop new dentin pulp-like tissues, enamel dental pulp, and cementum periodontal complexes. Immunohistochemistry data showed that the markers dentin sialoprotein, βⅢ-tubulin, dentin matrix protein 1, amelogenin, VIII factors, type I collagen, cementum-derived attachment protein, and scleraxis transcription factor were positive stained in toothlike tissue., Conclusions: Allogenous a-TDM served as an effective scaffold enabling DPSCs to proliferate and differentiate into a broad array of resident cells including odontoblasts, fibroblasts, vascular cells, and neural endings. Allogenous a-TDM with DPSCs can provide an ideal biomaterial for optimizing the regeneration of tooth material., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Stem cell-mediated regenerative endodontics has reached the human clinical trial phase; however, many issues still exist that prevent such technology to be a widely used clinical practice. These issues are not straightforward and are complicated. They should be because pulp regeneration is dealing with a small dead-end space. In addition, when regeneration is needed, the space is often heavily infected. The true standard of pulp regeneration should be everything except generation of some fibrous connective tissue and amorphous mineral deposit. As of now, we are still far short of reaching the standard of complete vascularized and innervated pulp regeneration with newly formed tubular dentin in all types of teeth. Thus, we need to go back to the bench and use established animal models or create new animal models to tackle those issues. This article will address several key issues including the possibility of pulp regeneration in small canals of molar teeth by enhancing the neovascularization, and whether the organized tubular dentin can be generated on the canal walls. Data from our semi-orthotopic tooth fragment mouse model have shown that complete pulp regeneration using dental pulp stem cells (DPSCs) in small canal has been inconsistent because of limited blood supply. This inconsistency is similar in our orthotopic miniature swine model, although in some cases vascularized pulp-like tissue can be formed throughout the canal space after DPSC transplantation. Furthermore, no tubular dentin was observed in the orthotopic pulp regeneration, despite the fact that DPSCs have the capacity to generate some tubular dentin-like structure in the hydroxyapatite/tricalcium phosphate-mediated ectopic pulp/dentin formation model in mice. Potential strategies to be tested to address these regeneration issues are discussed herein., (Copyright © 2020 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

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.

The aim of this study was to develop a highly porous calcium-containing chitosan scaffold suitable for dentin regeneration. A calcium hydroxide (Ca[OH] 2 ) suspension was used to modulate the degree of porosity and chemical composition of chitosan scaffolds. The chitosan solution concentration and freezing protocol were adjusted to optimize the porous architecture using the phase-separation technique. Scanning electron microscopy/energy-dispersive spectroscopy demonstrated the fabrication of a highly porous calcium-linked chitosan scaffold (CH-Ca), with a well-organized and interconnected porous network. Scaffolds were cross-linked on glutaraldehyde (GA) vapor. Following a 28-day incubation in water, cross-linked CH scaffold had no changes on humid mass, and CH-Ca featured a controlled degradability profile since the significant humid mass loss was observed only after 21 (26.0%) and 28 days (42.2%). Fourier-transform infrared spectroscopy indicated the establishment of Schiff base on cross-linked scaffolds, along with calcium complexation for CH-Ca. Cross-linked CH-Ca scaffold featured a sustained Ca 2+ release up to 21 days in a humid environment. This porous and stable architecture allowed for human dental pulp cells (HDPCs) to spread throughout the scaffold, with cells exhibiting a widely stretched cytoplasm; whereas, the cells seeded onto CH scaffold were organized in clusters. HDPCs seeded onto CH-Ca featured significantly higher ALP activity, and gene expressions for ALP, Col1, DMP-1, and DSPP in comparison to CH, leading to a significant 3.5 times increase in calcium-rich matrix deposition. In sum, our findings suggest that CH-Ca scaffolds are attractive candidates for creating a highly porous and bioactive substrate for dentin tissue engineering., (© 2020 Wiley Periodicals, Inc.)

Introduction: This study aimed to use nanobioactive glass (nBG) to guide the directional migration of stem cells and odontogenic differentiation on primary dentin, which are important for the functional regeneration of pulp-dentin tissue., Methods: Human bone marrow stem cells (BMSCs) were cocultured with 0.5 mg/mL nBG. The cell-biomaterial interaction was monitored using the IncuCyte S3 live cell imaging system (Essen BioScience, Ann Arbor, MI). The adhesion and morphology of BMSCs growing on nBG-coated dentin were assessed at 2 hours and 3 days. The chemotaxis effect of nBG-coated dentin on BMSCs was tested using a 3-dimensional collagen gel model. Subcutaneous transplantation of nBG-treated dentin slices into nude mice was used to investigate cell homing and odontogenic differentiation in vivo., Results: nBG particles showed good biocompatibility, and they were gradually degraded and relocated during interactions with BMSCs. BMSCs had better initial attachment to an nBG-coated dentin surface than to an untreated dentin surface. Cell migration assays showed that nBG-coated dentin induced significantly more cell migration than untreated dentin. An in vivo study revealed that nBG-coated dentin slices facilitated recellularization and revascularization in the root canal and that dentin sialophosphoprotein-positive cells were detected on the surface of the primary dentin., Conclusions: nBG recruits stem cells to move toward dentin and further promotes cell adhesion and odontogenic differentiation on primary dentin, which help regenerate the biomimetic structure of pulp-dentin tissue., (Copyright © 2019 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: This study aims to develop and characterize the regenerative potential of an atelopeptidized treated dentin matrix xenograft using in vitro and in vivo models., Methods: Freshly extracted bovine dentin was pulverized into 250- to 500-μm particles and demineralized with 17% EDTA for 1, 7, and 13 days. The samples were atelopeptidized with pepsin. The degree of demineralization and the effect of atelopeptidization were assessed using field emission scanning electron microscopy combined with energy-dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy, respectively. The expression of dentin matrix acidic phosphoprotein 1, dentin sialophosphoprotein, and osteopontin was evaluated in dental pulp stem cells using quantitative real-time polymerase chain reaction. The samples were then implanted intramuscularly in rats for 30 days, and the inflammatory cells were quantified histologically., Results: Field emission scanning electron microscopy combined with energy-dispersive X-ray spectroscopy revealed an exposed tubular structure of dentin after 1 and 7 days of demineralization. Fourier transform infrared spectroscopy confirmed the absence of amide peaks at 1260 to 1640/cm after atelopeptidization. The dental pulp stem cell expression of dentin matrix acidic phosphoprotein 1 and dentin sialophosphoprotein increased in all compared with the untreated control group (P < .05). The maximum expression rates were observed for the 1-day demineralized and atelopeptidized group. The 1-day demineralized group elicited the highest inflammatory response compared with the 7- or 13-day demineralized groups (P < .001). Atelopeptidization significantly decreased the inflammatory response only in the 1-day demineralized dentin group (P < .05)., Conclusions: Atelopeptidization of 1-day demineralized dentin xenograft preserved the collagen structure, minimized the immune reaction, and provided sufficient regenerative potential., (Copyright © 2019 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objectives: The aim of this study was to compare the effects of different bulk-fill resin composites, polimerization modes, and the thickness of remaining dentin on the increase of intrapulpal temperature., Methods: Human-extracted upper premolar teeth ( n  = 10) were used to design a single-tooth model with remaining dentin thicknesses of 1 mm and 0.5 mm. Estelite Bulk-fill Flow (Tokuyama, Japan), Surefil SDR™ Flow (Dentsply Caulk, Brazil), Filtek Bulk-Fill Posterior (3M, USA), and SonicFill™ 2 Bulk-fill (Kerr, USA) composites were applied according to the manufacturer's instructions. The standard and high modes of a light emitted diode (LED) light curing unit (LCU) (VALO™ Utradent, USA), were used for polymerization. In order to mimic the in vivo conditions of pulpal circulation, digital flowmetry (SK-600II, SK Medical, China) was used. Intrapulpal temperature rise was measured using K type thermocoupling (CEM DT 610B, Robosem Engineering, China). Data were analyzed using three-way variance analysis (ANOVA) and the independent t -test., Results: No significant statistical differences in intrapulpal temperature rise between low viscosity bulk-fill composites (SDR and Estelite) were found. The lowest intrapulpal temperature rise was found in groups which used the Filtek Bulk-fill composite. Decreases in the remaining dentin thickness increased the intrapulpal temperature rise., Significance: This study demonstrated that remaining dentin thickness, filler ratio of bulk-fill composites, and power and application time of the LED-LCU may affect intrapulpal temperature rise., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 Serdar Akarsu and Sultan Aktuğ Karademir.)

Introduction: Odontoblasts produce dentin throughout life and in response to trauma. The purpose of this study was to identify the roles of endogenous Wnt signaling in regulating the rate of dentin accumulation., Methods: Histology, immunohistochemistry, vital dye labeling, and histomorphometric assays were used to quantify the rate of dentin accumulation as a function of age. Two strains of Wnt reporter mice were used to identify and follow the distribution and number of Wnt-responsive odontoblasts as a function of age. To show a causal relationship between dentin secretion and Wnt signaling, dentin accumulation was monitored in a strain of mice in which Wnt signaling was aberrantly elevated., Results: Dentin deposition occurs throughout life, but the rate of accumulation slows with age. This decline in dentin secretion correlates with a decrease in endogenous Wnt signaling. In a genetically modified strain of mice, instead of tubular dentin, aberrantly elevated Wnt signaling resulted in accumulation of reparative dentin or osteodentin secreted from predontoblasts., Conclusions: Wnt signaling regulates dentin secretion by odontoblasts, and the formation of reparative or osteodentin is the direct consequence of elevated Wnt signaling. These preclinical data have therapeutic implications for the development of a biologically based pulp capping medicant., (Copyright © 2019 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Dentin is a suitable scaffold for tooth tissue engineering and can provide the appropriate combinations and concentrations of bioactive proteins to accommodate odontogenic cells. Freeze drying is a possible strategy to treat dentin because it ensures long-term stability of biopharmaceutical products and can increase the stability of dentin for improved distribution and storage. We hypothesized that freeze-dried dentin matrix (FDDM) constitutes a novel bioinstructive scaffold for tooth tissue engineering., Methods: This study used a modified technique to prepare an FDDM that preserved the mechanical and biological properties of dentin. The compression resistance and microhardness of FDDM were determined. Similarly, various biological characteristics, including cell morphology, cell proliferation, collagen secretion, alkaline phosphatase activity, and gene and protein expression, were investigated. To assess the inductive capacity of FDDM in vivo, FDDM and human dental pulp stem cell (DPSC) sheets were subcutaneously implanted in the dorsal pockets of nude mice. At 8 weeks postimplantation, the transplants were removed and histologically studied., Results: The results show that FDDM has mechanical and biological characteristics similar to those of dentin (P > .05). DPSCs cultured on FDDM and dentin showed superior attachment, growth, viability, and collagen secretion capacity but decreased mineral capability compared with DPSCs cultured with alpha minimum essential medium or hydroxyapatite (P < .05). The histologic results show that FDDM, which is similar to dentin, supported dentin/pulplike tissue regeneration in vivo as shown by the related expression levels of dentin markers, such as dentin sialoprotein and alkaline phosphatase., Conclusions: These results suggest that FDDM constitutes a novel superior scaffold for tooth tissue engineering., (Copyright © 2019 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Impact Statement: This research has been conducted with the aim to contribute to the development of treatment modalities for the reconstruction of lost/damaged mineralized tissues. Currently, determining the most appropriate stromal cell population and signaling cues stands at the core of developing effective treatments. We provide new insights into the effect of innate inductive cues found in human dentin matrix components, on the osteogenic differentiation of various human stromal cell types. The effects of dentin extracellular matrix components on umbilical cord mesenchymal stromal cells have not been investigated before. The findings of this study could underpin translational research based on the development of techniques for mineralized tissue engineering and will be of great interest for the readership of Tissue Engineering Part A.

The aim of this study was to evaluate the ability of spectral-domain optical coherence tomography (SD-OCT) to display the roof of the pulp chamber and to estimate the residual dentin thickness (RDT) of the pulp complex. The roots of 20 extracted human molars were embedded in epoxy resin, and crowns were longitudinally sectioned in the mesial-distal direction, exposing the pulp chamber. The coronal part of the crown was removed up to an RDT to the pulp chamber roof of 2 mm. Samples were imaged by SD-OCT from coronal view and by light microscopy (LM) in the sagittal plane. Using a microtome, dentin was subsequently removed in four levels from the occlusal aspect in steps of 250 μm. At each level, RDT was documented and measured by both methods. The data were compared (Spearman's rho correlation coefficient, Wilcoxon signed-rank test). Using OCT, the roof of the pulp chamber was first displayed at a maximum RDT of 1.94 mm. The minimal RDT that could be imaged by OCT was 0.06 mm. Values from both methods were strongly correlated (r, 0.83-0.95; p i  ≤ 0.05) and differed significantly for large RDTs (dentin levels 1, 2; p i  < 0.05) but not for small RDTs (levels 3, 4; p i  ≥ 0.226). The roof of the dental pulp chamber could be already visualized by SD-OCT with a RDT of 1.94 mm. Therefore, the method could be a useful diagnostic tool during the preparation of deep dentin cavities and might help to preserve the integrity of the pulp chamber.

In our previous work, we established an in vivo coronal pulp regeneration model in which biodegradable hydrogel-made scaffolds carrying rat bone marrow mesenchymal stem cells (BM-MSCs) were implanted in the coronal pulp chamber of pulpotomized rat maxillary first molars. In this study, we investigated the in vivo fate of LacZ-labeled BM-MSCs in our coronal pulp regeneration model. BM-MSCs were nucleofected with pVectOZ-LacZ plasmid encoding β-galactosidase 1 day before implantation, and the LacZ-transfected BM-MSCs were implanted into the pulpotomized pulp chamber with biodegradable preformed scaffold-hydrogel constructs. Empty vector was used as a control. After 3 and 14 days, the molars were retrieved and subjected to β-galactosidase staining. At 3 days, β-galactosidase-expressing cells with a round profile were located mainly around the scaffold. At 14 days, when the pulp-like tissue had been generated, the majority of β-galactosidase-expressing cells were detected under the newly formed dentin bridge-like structure, where nestin-expressing odontoblast-like cells were arranged. Immunoreactivity for dentin sialoprotein, a marker of mature odontoblasts, was strongly detected under the original dentin. No β-galactosidase staining was observed in the control group. Thus, we demonstrated that BM-MSCs survived for 2 weeks after implantation and colonized within the site of potential cytodifferentiation. Our findings indicated that BM-MSCs could differentiate into cells involved in mineralized tissue formation in the functionally relevant region., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Dental pulp has been revealed as an accessible and a rich source of mesenchymal stem cells (MSCs) and its biological potential is currently under intense investigation. MSCs from dental pulp stem cells (DPSCs) have been indicated as a heterogeneous population oriented not only in repairing dentine but also in maintaining vascular and nervous homeostasis of the teeth. We sought to verify the phenotype of cells isolated from dental pulp of young donors and to investigate in vitro their role as pericyte-like cells. Specifically, we evaluated how culture conditions can modulate expression of pericyte markers in DPSCs and their capacity to stabilize endothelial tubes in vitro. DPSCs cultured in standard conditions expressed MSC markers and demonstrated to contain a population expressing the pericyte marker NG2. These DPSCs were associated with low sprouting capacity in extra-cellular (EC) Matrix and limited ability in retaining tubes formed by endothelial cells in a coculture angiogenesis model. When cultured in endothelial growth medium (EGM)-2, DPSCs significantly upregulated NG2, and partially alpha-smooth muscle actin. The resulting population conserved the stem marker CD73, but was negative for calponin and endothelial markers. EGM-2-conditioned DPSCs showed a higher sprouting ability in EC Matrix and efficient association with human umbilical vein endothelial cells allowing the partial retention of endothelial tubes for several days. Among growth factors contained in EGM-2 we identified basic fibroblast growth factor (bFGF) as mainly responsible for NG2 upregulation and long-term stabilization of endothelial tubes. According to the in vitro analysis, DPSCs represent an effective source of pericytes and the appropriate culture conditions could result in a population with a promising ability to stabilize vessels and promote vascular maturation.

The aim of this study was to investigate the effect of prolonged release of iloprost, a prostacyclin analog, on angiogenesis and dental pulp healing in a rat model of mechanical pulp exposure. The profile of iloprost release from poly (lactic-co-glycolic) acid (PLGA) microspheres was evaluated, and expression of vascular endothelial growth factor (VEGF) mRNA was determined. The molars of rats were subjected to mechanical pulp exposure and 5 different forms of treatment: Ca(OH) 2 , PLGA (blank), iloprost, and iloprost/PLGA. Blood flow was determined at 0, 3, and 7 days using laser Doppler flowmetry. After 30 days, the tooth specimens were collected, and subjected to micro-CT and immunohistological analysis. The results showed that iloprost release from the microspheres was prolonged for 4 days, and that the treatment increased tooth blood flow for up to 7 days. At 30 days, an increase of mineralized tissue formation and dentin bridge formation was observed in the iloprost and iloprost/PLGA microsphere groups. VEGF expression was significantly increased in the iloprost/PLGA microsphere group relative to the other groups. In conclusion, this PLGA microsphere iloprost delivery system significantly increased dental pulp blood flow in a prolonged manner and increased tertiary dentin formation in this rat pulp injury model. Prolonged prostacyclin release could be a potentially useful approach for regeneration of dental pulp.

Lesions on tissues and organs critically affect quality of life, due to severe tissue defects that are threatening. Tissue repair and functional reconstruction are concurrent challenges in modern medicine. Tissue engineering brings hope for tissue and organ regeneration. Scaffolds provide a microenvironment for cell growth, proliferation and differentiation. Moreover, scaffolds influence the size and morphology of regenerated tissues. Dentin matrix, which is a natural bioactive and biocompatible scaffold, has become a research hotspot in recent years and has been widely used in tissue engineering. Studies on the use of dentin matrix as scaffolds have made a series of important progress in tooth root, periodontal, dental pulp and bone regeneration. This review demonstrates the biological characteristics of dentin matrix as bioactive scaffolds, describes the application of dentin matrix in tissue regeneration and provides a theoretical basis for the use of a dentin matrix in clinical applications.

The preservation of vital dental pulp with vasculature and nerve components remains one of the most significant challenges in modern dentistry. Due to the immense potential for neurovascularization, mesenchymal stem cell (MSC) transplantation has shown emerging promise in regenerative medicine and dental translational practice. Actually, pulp mesenchymal stem cells, including postnatal dental pulp stem cells (from permanent teeth) and stem cells from human exfoliated deciduous teeth, possess unique properties based on their origins from neural crest or glial cells. Furthermore, they reside in a neurovascular niche and have the potential for neurogenesis, angiogenesis, and neurovascular inductive activity. According to current pulp regeneration strategies, pulp stem cell-mediated approaches to regeneration have demonstrated convincing evidence that they can rebuild the complex histologic structure of native pulp in situ with highly organized physiologic patterns or even achieve de novo regeneration of complete dental pulp tissues. More importantly, recent clinical studies emphasized in situ neurovascularization outcomes in successful regeneration of vitalized pulp via pulp stem cell transplantation. In this review, we summarize recent breakthroughs in pulp stem cell-mediated pulp regeneration, emphasizing the crucial achievement of neurovascularization. This functional pulp regeneration represents an innovative and promising approach for future regenerative endodontics.

With the rapid development of cell biology and biological materials, the dentine-pulp complex regeneration research has gone further. Scaffolds play important roles in the construction of tissue engineered dentine-pulp complex. At present, scaffolds used in dentine-pulp complex regeneration include not only natural biological materials and synthetic biomaterials, but also various composite materials and cell- or body-based carrier materials. In this paper, the latest research status of various scaffolds for dentine-pulp complex regeneration were reviewed. The advantages and problems of these scaffolds were analyzed. The future development direction was predicted.

The objective of our experiments was to identify new therapeutic strategies to stimulate dentin formation in an adult tooth. To address this objective, we evaluated dentin production in 2 acute trauma models: one involving a pulp exposure and the other involving a superficial dentin injury. Molecular, cellular, and histologic analyses revealed that in response to a severe injury, where the pulp is exposed to the oral cavity, cell death is rampant and the repair response initiates from surviving pulp cells and, to a lesser extent, surviving odontoblasts. When an injury is superficial, as in the case of a dentin injury model, then disturbances are largely confined to pulp tissue immediately underneath the damaged dentin tubules. We found that the pulp remained vital and innervated; primary odontoblasts upregulated HIF1α; and the rate of mineralization was significantly increased. A tamoxifen-inducible Axin2 CreERT2/+ ; R26R mTmG/+ reporter strain was then used to demonstrate that a population of long-lived Wnt-responsive odontoblasts, which secreted dentin throughout the life of the animal, were responsible for depositing new dentin in response to a superficial injury. Amplifying Wnt signaling in the pulp stimulates dentin secretion, and in the dentin injury model, we show that a liposomal formulation of human WNT3A protein passes through dentinal tubules and is capable of upregulating Wnt signaling in the pulp. These data provide strong proof of concept for a therapeutic pulp-capping material to stimulate Wnt signaling in odontoblasts and thus improve the pulp repair response.

Introduction: The improvement of biomaterials capable of driving the regeneration of the pulp-dentin complex mediated by resident cells is the goal of regenerative dentistry. In the present investigation, a chitosan scaffold (CHSC) that released bioactive concentrations of simvastatin (SIM) was tested, aimed at the development of a cell-free tissue engineering system., Methods: First, we performed a dose-response assay to select the bioactive dose of SIM capable of inducing an odontoblastic phenotype in dental pulp cells (DPCs); after which we evaluated the synergistic effect of this dosage with the CHSC/DPC construct. SIM at 1.0 μmol/L (CHSC-SIM1.0) and 0.5 μmol/L were incorporated into the CHSC, and cell viability, adhesion, and calcium deposition were evaluated. Finally, we assessed the biomaterials in an artificial pulp chamber/3-dimensional culture model to simulate the cell-free approach in vitro., Results: SIM at 0.1 μmol/L was selected as the bioactive dose. This drug was capable of strongly inducing an odontoblastic phenotype on the DPC/CHSC construct. The incorporation of SIM into CHSC had no deleterious effect on cell viability and adhesion to the scaffold structure. CHSC-SIM1.0 led to significantly higher calcium-rich matrix deposition on scaffold/dentin disc assay compared with the control (CHSC). This biomaterial induced the migration of DPCs from a 3-dimensional culture to its surface as well as stimulated significantly higher expressions of alkaline phosphatase, collagen type 1 alpha 1, dentin matrix acidic phosphoprotein 1, and dentin sialophosphoprotein on 3-dimensional-cultured DPCs than on those in contact with CHSC., Conclusions: CHSC-SIM1.0 scaffold was capable of increasing the chemotaxis and regenerative potential of DPCs., (Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

In order to regenerate the dental pulp, many strategies have been developed as phototherapy. In the pulp repair, we do not know if gallium-aluminum-arsenide (GaAlAs) laser preserves the primary odontoblasts or stimulates the formation of more dentin matrix when dental pulp is damaged. The aim of the present study was to examine the effect of laser phototherapy (λ780 nm) on vascularization, inflammation, density of the primary odontoblast layer, and formation of reactionary and reparative dentin in the dental pulp by provoking extrusion of the rat incisor. The upper incisors were extruded 3 mm and then repositioned into their original sockets followed by a laser irradiation of the palatal mucosa (λ = 780 nm; p = 70 mW; CW; 4.2 J/cm 2 ; 60 s) every 48 h. Non-traumatized and/or non-irradiated incisors were used as the controls. At 8 and 30 days after surgery, incisors were processed for histological and histomorphometric analysis. Morphological analysis revealed no differences in vascularization between groups, but showed discrete inflammation in some non-irradiated and injured specimens, which correlated with a more irregular reparative dentin. The density of primary odontoblasts in the groups treated with lasers was higher when compared to non-irradiated groups, but no statistically significant difference between groups (p > 0.05). The thickness of the tertiary dentin was increased in both traumatized groups with no statistically significant difference between non-irradiated and irradiated groups (p > 0.05).The present findings revealed that the GaAlAs laser induced small changes on dentin-pulp complex, with more regular dentin matrix in the irradiated dental pulps.

Objectives: To evaluate the effect of intrinsic water permeation on the microtensile bond strengths of different adhesive systems to dentine and the quality of resin-dentine interfaces., Methods: Ninety-six non-carious human third molars were divided into 4 groups: Clearfil S 3 Bond Plus (CSBP; Kuraray); Clearfil S 3 Bond (C3S; Kuraray); iBond Self-Etch (IB; Heraeus-Kulzer) and Prime&Bond NT (PB, control etch-and-rinse adhesive, Dentply-Sirona). For each adhesive, specimens from one subgroup (N=10) were bonded using zero pulpal pressure, while specimens from the other subgroup (N=10) were bonded using 15cm water pressure (PP). Each bonded tooth was sectioned into 1×1mm sticks and stressed to failure. Data were analysed using two-way ANOVA and Holm-Sidak pairwise comparisons to examine the effects of "adhesive", "pulpal pressure" and their interaction on bond strength (α=0.05). Representative fractured sticks were examined by SEM. The remaining tooth slabs in each subgroup were used for TEM and CLSM., Results: Microtensile bond strengths (mean±SD; in MPa) were: 33.4±6.9 (CSBP), 33.2±4.7 (CSBP-PP), 35.0±8.6 (C3S), 25.5±7.3 (C3S-PP), 18.4±4.0 (IB), 16.5±6.9 (IB-PP), 28.2±5.5 (PB), 20.5±7.2 (PB-PP). "Adhesive-type" (P<0.001), "pulpal-pressure" (P<0.001) and their interactions (P<0.001) significantly affected bond strength results. No difference between no-PP and PP subgroups was found for CSBP and IB (P>0.05). Water droplets were identified along the resin-dentine interface for IB, IB-PP and C3S-PP., Conclusion: IB exhibits water sensitivity when bonding is performed with/without pulpal pressure. C3S exhibits water sensitivity when bonding is performed with pulpal pressure. CSBP does not exhibit water sensitivity when bonding is performed with/without pulpal pressure., Clinical Significance: Intrinsic water permeation during bonding procedures significantly affects bond strength results and the resin-dentine interface of contemporary single-bottle self-etch dentine adhesive systems., (Copyright © 2017. Published by Elsevier Ltd.)

Objectives: The aim of this paper is to determine the interrelationship between the microstructure - in terms of chemical composition and crystallinity - to the microhardness of coronal dentin., Methods: Dentin microhardness was tested by a novel reference point indenter and compared to the traditional Knoop hardness method. Micro-Raman spectroscopy was used to determine the chemical composition and crystallinity of dentin., Results: From the occlusal groove to the border of the coronal pulp chamber, dentin hardness decreased from superficial dentin (SD) to deep dentin (DD). Mineral/organic matrix ratios (phosphate/CH and phosphate/amide I) also decreased from SD to DD; however, this change was significant (P<0.05) in the phosphate/amide I ratio only. The phosphate/carbonate ratio decreased significantly by varying position from SD to DD. The degree of the crystallinity, as measured by the full width at half maximum (FWHM) of the peak at 960cm -1 , decreased significantly going from superficial to deep dentin., Significance: For the first time, the interrelationship between the microstructure and the mechanical properties of coronal dentin was determined by using the novel reference point indentation technique and micro-Raman spectroscopy. We hypothesize that the decrease in hardness from superficial to deep dentin can potentially be explained by decreased mineral content and increased carbonate content, which is also associated with decreased crystallinity. Collectively, there is a positive association between dentin hardness and mineral content and a negative association between dentin hardness and carbonate content., (Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Dental pulp is a highly specialized tissue that preserves teeth. It is important to maintain the capabilities of dental pulp before a pulpectomy by creating a local restoration of the dentin-pulp complex from residual dental pulp. The articles identified were selected by two reviewers based on entry and exit criteria. All relevant articles indexed in PubMed, Springer, Science Direct, and Scopus with no limitations from 1961 to 2016 were searched. Factors investigated in the selected articles included the following key words: Dentin-Pulp Complex, Regeneration, Tissue Engineering, Scaffold, Stem Cell, and Growth Factors. Of the 233 abstracts retrieved, the papers which were selected had evaluated the clinical aspects of the application of dentin-pulp regeneration. Generally, this study has introduced a new approach to provoke the regeneration of the dentin-pulp complex after a pulpectomy, so that exogenous growth factors and the scaffold are able to induce cells and blood vessels from the residual dental pulp in the tooth root canal. This study further presents a new strategy for local regeneration therapy of the dentin-pulp complex. This review summarizes the current knowledge of the potential beneficial effects derived from the interaction of dental materials with the dentin-pulp complex as well as potential future developments in this exciting field., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: The present study examined the effect of a quaternary ammonium silane (QAS) cavity disinfectant on the viability of human dental pulp cells, dentine bond durability and nanoleakage of simplified etch-and-rinse adhesives., Methods: Etched dentine surface of third molars were randomly divided into two adhesive groups, Adper™ Single Bond 2 and Prime & Bond ® NT™. For each adhesive, the teeth were randomly assigned to five cavity disinfectant groups (N=6): Group 1: deionised water (control); Group 2: 2% chlorhexidine (CHX); Group 3: 2% QAS; Group 4: 5% QAS and Group 5: 10% QAS. The cavity disinfectants were applied on etched dentine surfaces for 20s, followed by adhesive application. The bonded teeth were sectioned for bond strength testing at 24h, 6 months and 12 months. Viability of human dental pulpal cells was examined using MTT assay. Bond strength data were analysed using 3-way ANOVA and Tukey test. Interfacial nanoleakage was evaluated after 24h and 12 months and analysed using Kruskal-Wallis test., Results: Significant differences in bond strength were observed for the factors disinfectants (p<0.001) and time (p<0.001); while the factor, adhesive, was not significantly different (p=0.203). The 2% QAS cavity disinfectant preserved bond strength of both adhesives and reduced interfacial nanoleakage after 12 months. Cell viability was the lowest for 2% CHX, followed by 2% QAS and the control., Conclusions: The 2% QAS cavity disinfectant demonstrated greater cell viability compared to 2% CHX, with no adverse effect on immediate bond strength and preserved bond stability over time., Clinical Significance: Incorporation of 2% quaternary ammonium silane cavity disinfectant in the resin-dentine bonding protocol enhances the success rate of bonded restorations., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: To investigate the proliferation, odontogenic differentiation and mineralization of human dental pulp cells (HDPCs) on bioactive glass(BG) and extracted dentin proteins(EDP)., Methods: Primary HDPCs were isolated from third molars by enzyme digestion and were cultured in Dulbecco's minimum essential medium (DMEM). Then the 4th generation of HDPCs was cultured with DMEM, which contained BG-EDP, BG, and EDP, respectively. Meanwhile HDPCs were cultured in DMEM as control group. Proliferation of HDPCs was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) colorimetric assay. Odontogenic differentiation was determined by alkaline phosphatase (ALP) activity assay and real-time PCR. Mineralization was investigated by Alizarin red staining and cetylpyridinium chloride (CPC) assay., Results: The proliferation of HDPCs was increased significantly in BG-EDP group on 3,7,and 9 d (optical density value: 1.36±0.06, 2.52±0.20, 2.72±0.29) compared with BG (optical density value: 1.20±0.26, 2.33±0.26, 2.50±0.30), EDP(optical density value: 1.13±0.15, 2.10±0.13, 2.38±0.22) and control group (optical density value: 0.84±0.17, 1.84±0.18, 1.95±0.19), P<0.05. After 7 days, ALP activity of BG-EDP group had no statistical difference compared with EDP group and control group; the expression of odontogenic differentiation genes (DSPP, DMP-1) showed no difference among all the groups(P>0.05). After 14 days, ALP activity of BG-EDP group (56.67±1.83) was significantly upregulated compared with EDP group (41.98±9.71) and control group (30.82±6.70), P<0.05, but had no statistical difference compared with BG group (56.29±6.20), P>0.05; DSPP gene expression was upregulated significantly in BG-EDP group (5.79±1.94) compared with the other groups (P<0.05); DMP-1 gene expression of BG-EDP group (3.87±1.87) increased but had no statistical difference compared with the other groups (P>0.05). The alizarin red staining showed more mineral nodules in BG-EDP group, the cetylpyridinium chloride semi-quantification presented higher calcification in BG-EDP group (0.27±0.01) compared with the other groups (P<0.05)., Conclusion: Compared with either BG or EDP, BG-EDP significantly promotes the proliferation, odontogenic differentiation and mineralization of HDPCs.

Objective: The present study aims to investigate whether reparative dentinogenesis could be guided at central pulpal sites or at a distance from the amputated pulp of miniature pig teeth, by using set calcium silicate-based carriers containing human recombinant bioactive molecules., Design: Pulp exposures were performed in 72 permanent teeth of 4 healthy miniature swine. The teeth were capped with pre-manufactured implants of set calcium silicate-based material containing BMP-7, TGFβ1 or WnT-1, for 3 weeks. Conical-shaped intrapulpal implants were exposed in the central pulp core, while disc-shaped extrapulpal implants were placed at a distance from the amputated pulp. Implants without bioactive molecules were used as controls. Thickness and forms of new matrix mineralized deposition were assessed histologically at post-operative periods of 3 weeks by light microscopy., Results: Intrapulpal applications: Calcified structures composed of osteodentine were found in contact with the BMP-7 implants. An inhomogeneous calcified tissue matrix was found around the WnT-1 carriers. A two-zone calcified structure composed of osteodentine and a thicker tubular matrix zone was seen at the TGFβ1 carrier-pulp interface. Extrapulpal applications: The space between WnT-1 implants and pulp periphery had been invaded by soft tissue with traces of calcified foci. Thick calcified structures composed of osteodentine were found surrounding pulp exposure sites in response to application of BMP-7. Spindle-shaped cells associated with atubular calcified matrix or elongated polarized cells associated with tubular dentine-like matrix were found along the cut dentinal walls of the TGFβ1 group., Conclusion: The present experiments indicated that set calcium silicate could be used as carrier for biologically active molecules. TGFβ1 was shown to be an effective bioactive molecule in guiding tertiary dentine formation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: This study aimed to investigate the potential of Mg-based bioceramic scaffolds combined with human treated-dentin matrices (hTDMs) and dentinogenesis-related morphogens to promote odontogenic differentiation and dentin-like tissue formation by Dental Pulp Stem Cells-DPSCs., Methods: DPSC cultures were established and characterized by flow cytometry. Experimental cavities were prepared inside crowns of extracted teeth and demineralized by EDTA (hTDMs). Zn-doped, Mg-based bioceramic scaffolds, synthesized by the sol-gel technique, were hosted inside the hTDMs. DPSCs were spotted inside the hTDMs/scaffold constructs with/without additional exposure to DMP-1 or BMP-2 (100ng/ml, 24h). Scanning Electron Microscopy-SEM, live/dead fluorescence staining and MTT assay were used to evaluate cell attachment and viability; Real time PCR for expression of osteo/odontogenic markers; Inductively Coupled Plasma-Atomic Emission Spectrometry-ICP/AES for scaffold elemental release analysis; ELISA for hTDM growth factor release analysis; SEM and X-ray Diffraction-XRD for structural/chemical characterization of the regenerated tissues., Results: Scaffolds constantly released low concentrations of Mg(2+), Ca(2+), Zn(2+) and Si(4+), while hTDMs growth factors, like DMP-1, BMP-2 and TGFβ-1. hTDMs/scaffold constructs supported DPSC viability, inducing their rapid odontogenic shift, indicated by upregulation of DSPP, BMP-2, osteocalcin and osterix expression. Newly-formed Ca-P tissue overspread the scaffolds partially transforming into bioapatite. Exposure to DMP-1 or BMP-2 pronouncedly enhanced odontogenic differentiation phenomena., Significance: This is the first study to validate that combining the bioactivity and ion releasing properties of bioceramic materials with growth factor release by treated natural dentin further supported by exogenous addition of key dentinogenesis-related morphogens (DMP-1, BMP-2) can be a promising strategy for targeted dentin regeneration., (Copyright © 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.)

Objective: The fracture toughness of dentin is critical to the prevention of tooth fracture. Within the tooth crown, the mechanical properties of dentin are influenced by spatial variations in the density and diameter of the dentin tubules with distance from the pulp. There are also relevant changes to the microstructure of dentin with age. In this investigation the importance of tubule density to the fracture toughness of dentin was evaluated in "young" and "old" age groups., Methods: The variations in microstructure (density and diameter of tubules) from young and old donor teeth were studied by means of optical microscopy., Results: A reduction in the density and diameter of tubules was identified to occur with aging. An approach previously proposed to study the mechanical behavior of porous materials was used to model the fracture toughness of coronal dentin in terms of the tubule characteristics. Results were then compared with published results from previous studies., Conclusions: The model predictions were consistent with experimental results for the fracture toughness of dentin from young donor teeth, but overestimated the values that have been reported for "old" dentin., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Objective: To investigate the effect of epigallocatechin-3-gallate (EGCG) on biomodification of demineralized dentine substrate, in its permeability, hydrophobicity, and inhibition ability to collagen enzymatic degradation., Methods: The dentine substrates were treated with simulated pulpal pressure created by mixtures of 0.02%, 0.1% EGCG/bovine serum albumin (BSA) in acidic environment (pH4.4) for 48 h. A fluid-transport model was used to measure the fluid permeability through demineralized dentine substrate. Positive replicas of dentine substrate were fabricated before and after being subjected to acidic environment for scanning electron microscope (SEM) examination. The blank group contained no EGCG and the positive group were treated with Gluma desensitizer. Static contact angle measurements on demineralized dentin and 0.1% EGCG primed dentin were performed by contact angle analyzer. The priming time were 60 s, 120 s, 0.5 h, 1 h. Dentine specimens bonded with Adper single bond 2 were subjected to 100 mg/L collagenase and observed under SEM. Resin-bonded specimens (with 0.02%, 0.1%, 0.5% EGCG priming, or without EGCG priming) were created for micro-tensile bond strength evaluation (MTBS). Resin-bonded specimens after thermol cycling were created for MTBS evaluation., Results: The fluid permeability in the blank control group increased ([151.3±22.3]%), the fluid permeability in 0.1% EGCG/BSA group decreased ([23.7±6.3]%). Compared to the blank control group, the contact angle of 120 s, 0.5 h, 1 h groups increased by 31.0%, 53.5%, 57.8% in deep dentin and 37.4%, 59.3%, 62.4% in shallow dentin. The SEM examination showed that 0.1% and 0.5% EGCG priming for 120 s significantly increased dentin collagen's resistance to collagenase. The immediate MTBS of 0.1% and 0.5% EGCG groups were (29.4±4.8) and (19.8± 4.9) MPa. After thermol cycling, the MTBS of 0.1% and 0.5% EGCG groups were (19.9±5.1) and (15.3± 6.3) MPa., Conclusions: Under acidic environment (pH4.4), the 0.1% EGCG can reduce dentine permeability under acidic environment. The 0.1% EGCG can increase hydrophobicity of dentin substrate, and strengthen dentin substrate's resistance to collagenase hydrolysis, thus increased the resin-dentin bonding durability.

Objectives: Calcium silicate cements are biocompatible dental materials applicable in contact with vital tissue. The novel tricalcium silicate cement Biodentine™ offers properties superior to commonly used mineral trioxide aggregate (MTA). Objective of this study was to evaluate its cytocompatibility and ability to induce differentiation and mineralization in three-dimensional cultures of dental pulp stem cells after direct contact with the material., Materials and Methods: Test materials included a new tricalcium silicate (Biodentine™, Septodont, Saint-Maur-des-Fossés, France), MTA (ProRoot® MTA, DENSPLY Tulsa Dental Specialities, Johnson City, TN, USA), glass ionomer (Ketac™ Molar Aplicap™, 3M ESPE, Seefeld, Germany), human dentin disks and polystyrene. Magnetic activated cell sorting for to the surface antigen STRO-1 was performed to gain a fraction enriched with mesenchymal stem cells. Samples were allowed to set and dental pulp stem cells in collagen carriers were placed on top. Scanning electron microscopy of tricalcium silicate cement surfaces with and without cells was conducted. Cell viability was measured for 14 days by MTT assay. Alkaline phosphatase activity was evaluated (days 3, 7, and 14) and expression of mineralization-associated genes (COL1A1, ALP, DSPP, and RUNX2) was quantified by real-time quantitative PCR. Nonparametric statistical analysis for cell viability and alkaline phosphatase data was performed to compare different materials as well as time points (Mann-Whitney U test, α = 0.05)., Results: Cell viability was highest on tricalcium silicate cement, followed by MTA. Viability on glass ionomer cement and dentin disks was significantly lower. Alkaline phosphatase activity was lower in cells on new tricalcium silicate cement compared to MTA, whereas expression patterns of marker genes were alike., Conclusions: Increased cell viability and similar levels of mineralization-associated gene expression in three-dimensional cell cultures on the novel tricalcium silicate cement and mineral trioxide aggregate indicate that the material is cytocompatible and bioactive., Clinical Relevance: The tested new tricalcium silicate cement confirms its suitability as an alternative to MTA in vital pulp therapy.

Dental pulp/dentine complex regeneration is indispensable to the construction of biotissue-engineered tooth roots and represents a promising approach to therapy for irreversible pulpitis. We used a tissue-engineering method based on odontogenic stem cells to design a three-dimensional (3D) and scaffold-free stem-cell sheet-derived pellet (CSDP) with the necessary physical and biological properties. Stem cells were isolated and identified and stem cells from root apical papilla (SCAPs)-based CSDPs were then fabricated and examined. Compact cell aggregates containing a high proportion of extracellular matrix (ECM) components were observed, and the CSDP culture time was prolonged. The expression of alkaline phosphatase (ALP), dentine sialoprotein (DSPP), bone sialoprotein (BSP) and runt-related gene 2 (RUNX2) mRNA was higher in CSDPs than in cell sheets (CSs), indicating that CSDPs have greater odonto/osteogenic potential. To further investigate this hypothesis, CSDPs and CSs were inserted into human treated dentine matrix fragments (hTDMFs) and transplanted into the subcutaneous space in the backs of immunodeficient mice, where they were cultured in vivo for 6 weeks. The root space with CSDPs was filled entirely with a dental pulp-like tissue with well-established vascularity, and a continuous layer of dentine-like tissue was deposited onto the existing dentine. A layer of odontoblast-like cells was found to express DSPP, ALP and BSP, and human mitochondria lined the surface of the newly formed dentine-like tissue. These results clearly indicate that SCAP-CSDPs with a mount of endogenous ECM have a strong capacity to form a heterotopic dental pulp/dentine complex in empty root canals; this method can be used in the fabrication of bioengineered dental roots and also provides an alternative treatment approach for pulp disease., (Copyright © 2013 John Wiley & Sons, Ltd.)

The aim of this study was to evaluate the effects of simulated pulpal pressure (SPP) on the variation of intrapulpal temperature (ΔT) and microtensile bond strength (μTBS) to dentin submitted to an adhesive technique using laser irradiation. One hundred sound human molars were randomly divided into two groups (n = 50), according to the presence or absence of SPP (15 cm H2O). Each group was divided into five subgroups (n = 10) according to Nd:YAG laser energy (60, 80, 100, 120, 140 mJ/pulse). The samples were sequentially treated with the following: 37 % phosphoric acid, adhesive (Scotchbond Universal), irradiation with Nd:YAG laser (60 s), and light curing (10 s). ΔT was evaluated during laser irradiation using a type K thermocouple. Next, a composite resin block was build up onto the irradiated area. After 48 h, samples were submitted to microtensile test (10 kgf load cell, 0.5 mm/min). Data were analyzed by two-way ANOVA and Tukey tests (p = 0.05). ANOVA revealed significant differences for ΔT and TBS in the presence of SPP. For ΔT, the highest mean (14.3 ± 3.23 °C)(A) was observed in 140 mJ and without SPP. For μTBS, the highest mean (33.4 ± 4.15 MPa)(A) was observed in 140 mJ and without SPP. SPP significantly reduced both ΔT and μTBS during adhesive procedures, lower laser energy parameters resulted in smaller ΔT, and the laser parameters did not influence the μTBS values.

Regeneration of dentin tissues in the pulp space of teeth serves the ultimate goal of preserving teeth via endodontic approaches. In recent times, many studies suggested that human dentin scaffolds combined with dental stem cells was a potential strategy for the complete dentin tissue regeneration. In this study, human dental pulp stem cells (DPSCs) were isolated and cultured. Dentin specimens were prepared from human third molars and treated with ethylene diamine tetra-acetic acid and citric acid to remove the smear layer. Then, DPSCs were cultured onto human treated dentin (hTD) and implanted in mouse model for 4, 6 and 8 weeks. The resulting grafts were assessed by hematoxylin and eosin stain and immunohistochemical stains. As a result, DPSCs were supported and induced to regenerate of dentin-like tissues which expressed specific dentin markers such as dentin sialophosphoprotein and dentin matrix protein 1 by combination with hTD in vivo. Furthermore, cells existed in the newly-formed dentin-like tissues also expressed typical human mitochondria antibodies, demonstrated that new tissues originated from human. In conclusion, the obtain results extend hopefully newly-established therapy to apply in endodontics and traumatic dental hard tissues.

Synthetic octacalcium phosphate (OCP) has been suggested to be a useful biomaterial for the regeneration of hard tissues, including bone. However, it remains unknown whether OCP induces dentine formation by dental pulp. We investigated biomineralization of dental pulp exposed to synthetic OCP in vitro and in vivo. When dental pulp was exposed directly to OCP, rapid formation of reparative dentine (RD) was induced and expression of dentine sialoprotein synthesis was observed in dental pulp adjacent to newly synthesized RD. OCP inhibited the proliferation of rat pulp cells and also promoted their odontoblastic differentiation in vitro, as alkaline phosphatase activity, mineralization of pulp cells and the expression level of dentine sialophosphoprotein were enhanced. Direct contact between OCP and pulp cells is required for OCP to exhibit its effects in vitro. The expression level of Runx2, a transcription factor whose downregulation is closely related to odontoblast differentiation, was downregulated in pulp cells cultured with OCP. Structural changes of OCP during culture were determined by Fourier transform infrared spectroscopy. OCP tended to be converted to carbonate hydroxyapatite after incubation with or without pulp cells, which may be analogous to biological apatite crystals. Taken together, our data suggest that synthetic OCP supports RD formation by dental pulp and downregulation of Runx2 may be involved in that stimulatory activity. Furthermore, OCP-apatite conversion is involved in this stimulatory capacity of OCP., (Copyright © 2013 John Wiley & Sons, Ltd.)

ATP is involved in neurosensory processing, including nociceptive transduction. Thus, ATP signaling may participate in dentin hypersensitivity and dental pain. In this study, we investigated whether pannexins, which can form mechanosensitive ATP-permeable channels, are present in human dental pulp. We also assessed the existence and functional activity of ecto-ATPase for extracellular ATP degradation. We further tested if ATP is released from dental pulp upon dentin mechanical or thermal stimulation that induces dentin hypersensitivity and dental pain and if pannexin or pannexin/gap junction channel blockers reduce stimulation-dependent ATP release. Using immunofluorescence staining, we demonstrated immunoreactivity of pannexin 1 and 2 in odontoblasts and their processes extending into the dentin tubules. Using enzymatic histochemistry staining, we also demonstrated functional ecto-ATPase activity within the odontoblast layer, subodontoblast layer, dental pulp nerve bundles, and blood vessels. Using an ATP bioluminescence assay, we found that mechanical or cold stimulation to the exposed dentin induced ATP release in an in vitro human tooth perfusion model. We further demonstrated that blocking pannexin/gap junction channels with probenecid or carbenoxolone significantly reduced external dentin stimulation-induced ATP release. Our results provide evidence for the existence of functional machinery required for ATP release and degradation in human dental pulp and that pannexin channels are involved in external dentin stimulation-induced ATP release. These findings support a plausible role for ATP signaling in dentin hypersensitivity and dental pain., (© International & American Associations for Dental Research 2015.)