The transforming growth factor β (TGFβ) superfamily is a master regulator of development, adult homeostasis, and wound repair. Dysregulated TGFβ signaling can lead to cancer, fibrosis, and musculoskeletal malformations. We previously demonstrated that TGFβ receptor 2 (Tgfbr2) signaling regulates odontoblast differentiation, dentin mineralization, root elongation, and sensory innervation during tooth development. Sensory innervation also modulates the homeostasis and repair response in adult teeth. We hypothesized that Tgfbr2 regulates the neuro-pulpal responses to dentin injury. To test this, we performed a shallow dentin injury with a timed deletion of Tgfbr2 in the dental pulp mesenchyme of mice and analyzed the levels of tertiary dentin and calcitonin gene-related peptide (CGRP) axon sprouting. Microcomputed tomography imaging and histology indicated lower dentin volume in Tgfbr2cko M1s compared to WT M1s 21 days post-injury, but the volume was comparable by day 56. Immunofluorescent imaging of peptidergic afferents demonstrated that the duration of axon sprouting was longer in injured Tgfbr2cko compared to WT M1s. Thus, CGRP+ sensory afferents may provide Tgfbr2-deficient odontoblasts with compensatory signals for healing. Harnessing these neuro-pulpal signals has the potential to guide the development of treatments for enhanced dental healing and to help patients with TGFβ-related diseases.

Epigenetic modulation, including histone modification, alters gene expression and controls cell fate. Histone deacetylases (HDACs) are identified as important regulators of dental pulp cell (DPC) mineralisation processes. Currently, there is a paucity of information regarding the nature of histone modification and HDAC expression in the dentine-pulp complex during dentinogenesis. The aim of this study was to investigate post-translational histone modulation and HDAC expression during DPC mineralisation and the expression of Class I/II HDACs during tooth development and in adult teeth. HDAC expression (isoforms -1 to -6) was analysed in mineralising primary rat DPCs using qRT-PCR and Western blot with mass spectrometry being used to analyse post-translational histone modifications. Maxillary molar teeth from postnatal and adult rats were analysed using immunohistochemical (IHC) staining for HDACs (1-6). HDAC-1, -2, and -4 protein expression increased until days 7 and 11, but decreased at days 14 and 21, while other HDAC expression increased continuously for 21 days. The Class II mineralisation-associated HDAC-4 was strongly expressed in postnatal sample odontoblasts and DPCs, but weakly in adult teeth, while other Class II HDACs (-5, -6) were relatively strongly expressed in postnatal DPCs and adult odontoblasts. Among Class I HDACs, HDAC-1 showed high expression in postnatal teeth, notably in ameloblasts and odontoblasts. HDAC-2 and -3 had extremely low expression in the rat dentine-pulp complex. Significant increases in acetylation were noted during DPC mineralisation processes, while trimethylation H3K9 and H3K27 marks decreased, and the HDAC-inhibitor suberoylanilide hydroxamic acid (SAHA) enhanced H3K27me3. These results highlight a dynamic alteration in histone acetylation during mineralisation and indicate the relevance of Class II HDAC expression in tooth development and regenerative processes.

Located at the interface of the dentin-pulp complex, the odontoblasts are specialized cells responsible for dentin synthesis and nociceptive signal detection in response to external stimuli. Recent studies have shown that the mechanosensitive ion channel PIEZO1 is involved in bone formation and remodeling through the influx of calcium ions, and it is abundantly expressed in odontoblasts. However, the specific role of PIEZO1 in reactionary dentinogenesis and the underlying mechanisms remain elusive. In this study, we found intense PIEZO1 expression in the plasma membrane and cytoplasm of odontoblasts in healthy human third molars, mouse mandibular molars, and human odontoblast-like cells (hOBLCs). In hOBLCs, PIEZO1 positively regulated DSPP, DMP1, and COL1A1 expression through the Ca2+/PI3K-Akt/SEMA3A signaling pathway. In addition, exogenous SEMA3A supplementation effectively reversed reduced mineralization capacity in PIEZO1-knockdown hOBLCs. In vivo, Piezo1 expression peaked at day 7 and returned to baseline at day 21 in a wild-type mice dentin injury model, with Sema3a presenting a similar expression pattern. To investigate the specific role of PIEZO1 in odontoblast-mediated reactionary dentinogenesis, mice with a conditional knockout of Piezo1 in odontoblasts were generated, and no significant differences in teeth phenotypes were observed between the control and conditional knockout (cKO) mice. Nevertheless, cKO mice exhibited reduced reactionary dentin formation and decreased Sema3a and Dsp positive staining after dentin injury, indicating impaired dental pulp repair by odontoblasts. In summary, these findings suggest that PIEZO1 enhances the mineralization capacity of hOBLCs in vitro via the Ca2+/PI3K-Akt/SEMA3A signaling pathway and contributes to reactionary dentinogenesis in vivo.

This study aimed to assess the association between genetic polymorphisms in BMP2 (rs1005464 and rs235768), BMP4 (rs17563), SMAD6 (rs2119261 and rs3934908) and RUNX2 (rs59983488 and rs1200425) and pulp stones (PS). A total of 117 participants, consisting of 63 individuals with PS and 54 without PS, were included. Digital radiographs and a demographic/clinical questionnaire were used. Genomic DNA from salivary cells was genotyped via real-time polymerase chain reaction. Statistical analyses, including Chi-Square, Fisher\'s exact tests, Poisson regression and dimensionality reduction, were conducted. The rs2119261 polymorphism in the SMAD6 gene showed an association with genotype distribution in the recessive model (p = 0.049). The T-T haplotype in the SMAD6 gene (rs2119261 and rs3934908) was more prevalent in the control group and significantly linked with PS (p = 0.029). No associations were found between PS risk and genetic polymorphisms in BMP2, BMP4 and RUNX2. Polymorphisms in the SMAD6 gene were associated with PS.

BACKGROUND: The regeneration of pulp tissue is crucial for true regenerative endodontic treatment, which requires a reduction in osteogenic differentiation. Garcinol, a histone acetyltransferase inhibitor, is a natural regulator that is known to suppress the osteogenic differentiation of dental pulp stem cells. In this study, the inhibitory effect of garcinol on the osteogenic differentiation of human dental pulp stem cells (hDPSCs) was evaluated using three-dimensional culture under in vitro and in vivo conditions.
METHODS: hDPSCs were obtained from caries-free third molars and cultured with 10 μM garcinol for 7 days in an ultra-low attachment plate. The cell stemness and expression of osteogenic differentiation-related genes were analyzed using reverse transcription-polymerase chain reaction and single-cell analysis. A transplantation experiment was performed in mice to investigate whether garcinol-treated hDPSCs showed restrained osteogenic differentiation.
RESULTS: hDPSCs cultured in the U-shaped ultra-low attachment plate showed the highest expression of stemness-related genes. Garcinol-treated hDPSCs demonstrated downregulation of osteogenic differentiation, with lower expression of bone sialoprotein, which is related to bone formation, and higher expression of dentin sialophosphoprotein, which is related to dentin formation. However, the garcinol-treated hDPSCs did not show any alterations in their stemness. Consistent results were observed in the transplantation experiment in mice.
CONCLUSIONS: Garcinol reduced the osteogenic differentiation of hDPSCs, which can contribute to true regenerative endodontic treatment.

The current development of microfluidics-based microphysiological systems (MPSs) will rapidly lead to a paradigm shift from traditional static 2-dimensional cell cultivation towards organized tissue culture within a dynamic cellular milieu. Especially organs-on-a-chip (OoCs) can very precisely re-create the mechanical and unique anatomical structures of the oral environment. This review provides an introduction to such technology, from commonly used chip materials and fabrication methods to the application of OoC in in vitro culture. OoCs are advantageous because of their small-scaled culture environment, the highly controlled dynamic experimental conditions, and the likeness to the in vivo structure. We specifically focus on current chip designs in dental, oral, and craniofacial (DOC) research. Also, future perspectives are discussed, like model standardization and the development of integrated platforms with advanced read-out functionality. By doing so, it will be possible for OoCs to serve as an alternative for animal testing and to develop highly predictive human models for clinical experiments and even personalized medicine.

Pericytes and glial cells are known to collaborate in dental pulp tissue repair. Cell-based therapies that stimulate these stromal components may be of therapeutic relevance for partially vital dental pulp conditions. This study aimed to examine the early effect of photobiomodulation (PBM) in pericytes from experimentally injured pulp tissue. To accomplish this, we used the Nestin-GFP/NG2-DsRed mice, which could allow the identification of distinct pericyte phenotypes. We discovered the presence of two pericytes subsets within the dental pulp, the Nestin + NG2+ (type-2) and Nestin- NG2+ (type-1). Upon injury, PBM treatment led to a significant increase in Nestin+ cells and pericytes. This boost was mainly conferred by the more committed pericyte subset (NestinNG2+ ). PBM also stimulated terminal blood vessels sprouting adjacent to the injury site while maintaining signs of pulp vitality. In vitro, PBM induced VEGF upregulation, improved dental pulp cells proliferation and migration, and favored their mineralization potential. Herein, different subsets of perivascular cells were unveiled in the pulp tissue. PBM enhanced not only NG2+ cells but nestin-expressing progenitors in the injured dental pulp.

BACKGROUND: The aim of this study was to compare the levels of different A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS; ie, ADAMTS-1, -4, and -9]) related to the breakdown of the proteoglycans and tissue inhibitor of metalloproteinase 3 (TIMP-3) levels that inhibit ADAMTS in the extracellular matrix of healthy pulp and symptomatic irreversible pulpitis.
METHODS: Pulp tissue samples diagnosed with healthy and symptomatic irreversible pulpitis were collected from 48 participants. Healthy and inflamed pulps were directly transferred to Eppendorf tubes (Labosel, Istanbul, Turkey) and stored at -80°C (Nüve-DF490; Nüve, Ankara, Turkey) until further use. Routine root canal treatment procedures for the teeth were performed, after which the treatment process was completed. The levels of ADAMTS-1, -4, and -9 and TIMP-3 were measured in supernatants of human dental pulp tissue extracts using enzyme-linked immunosorbent assay kits (USCN, Wuhan, China). The independent sample t test and 1-way analysis of variance were used for parametric data, and the Kruskal-Wallis and Mann-Whitney U tests were used for nonparametric data.
RESULTS: There was a statistically significant difference between ADAMTS-1 and -9 and TIMP-3 levels of the healthy pulp and pulpitis groups (P < .05), but there was no significant difference for ADAMTS-4 levels (P > .05). It was found that the levels of ADAMTS-1 and -9 were higher in the pulpitis group compared with the healthy pulp group, whereas the TIMP-3 level decreased (P < .05).
CONCLUSIONS: As a result of this study, it was concluded that ADAMTS-1 and -9 and TIMP-3 might have a role in pulpal inflammation, whereas ADAMTS-4 was not related to pulpal inflammation.

BACKGROUND: During pulp inflammation, recruited macrophages can differentiate into 2 phenotypes: proinflammatory M1 and anti-inflammatory M2. Pulp fibroblasts have previously been shown to regulate pulp inflammation via cytokine and growth factor secretion. We hypothesized that upon carious injury, pulp fibroblasts interact with macrophages and modulate their differentiation.
METHODS: Cultures of pulp fibroblasts were physically injured and incubated with lipoteichoic acid (LTA) to mimic the pulp environment underlying a carious lesion. Physical injuries without LTA were performed on cultured fibroblasts to simulate the surrounding pulp tissue. Fibroblast supernatants were collected and added to undifferentiated macrophages to study their differentiation into M1 or M2 phenotypes by investigating cytokine secretion profiles and phagocytosis capacity. Histologic staining and immunofluorescence were performed on healthy and carious human tooth sections to localize the 2 macrophage phenotypes.
RESULTS: LTA-stimulated fibroblasts induced macrophage differentiation into the M1 phenotype with a significant increase both in tumor necrosis factor alpha secretion and phagocytosis capacity. By contrast, injured fibroblasts without LTA led to M2 differentiation with a significant increase in interleukin 10 secretion and low phagocytosis capacity. In carious teeth, M1 macrophages were detected mainly in the pulp zone underlying caries, whereas M2 macrophages were detected in the peripheral inflammatory zone.
CONCLUSIONS: Fibroblasts induced macrophage differentiation to proinflammatory M1 with high bacteria phagocytosis capacity to control infection at the carious front. Fibroblasts located at the periphery of the inflammatory zone induced macrophage differentiation to anti-inflammatory M2. The fine balance between the 2 phenotypes may represent a prerequisite for initiating the healing process.

Autophagy plays important roles in odontogenic differentiation of dental pulp cells (DPCs) in the developmental stage of tooth bud. Few studies have reported the role of autophagy during reparative dentin formation process. The objective of this study was to discover gene expression pattern correlated to autophagy and their role during odontogenic differentiation process in DPCs.
After tooth cavities were prepared on the mesial surface of lower first molar crown of rats. Odontogenic differentiation and reparative dentin formation were assessed based on detection of morphology change with hematoxylin and eosin staining.
After tooth cavities were prepared on the mesial surface of lower first molar crown of rats, odontogenic differentiation and reparative dentin formation were assessed based on detection of morphology change with hematoxylin and eosin staining and dentin sialophosphoprotein (DSPP), whereas autophagy inhibitor 3-methyladenine (3MA) reversed. Results of quantitative polymerized chain reaction array of autophagosome formation related genes revealed that GABARAPL2 was prominently upregulated while expression of other ATG8 family members were moderately increased after tooth cavity preparation. In addition, human DPCs incubated in differentiation medium predominantly upregulated MAP1LC3C, which selectively decreased by 3MA but not by autophagy enhancer trehalose. Knock-down of MAP1LC3C using shRNA resulted in strong downregulation of dentin matrix protein 1 and DSPP as well-known odontogenic marker compared to knock-down of MAP1LC3B during odontogenic differentiation process of human DPCs.
Our results suggest that MAP1LC3C plays a crucial role in odontogenic differentiation of human DPCs via regulating autophagic flux.