Introduction: Conventional vital pulp therapy relies primarily on calcium silicate-based cements that induce reparative dentin formation without restoring the native neurovascular architecture of the pulp tissue. Advances in tissue engineering using stem cells and biodegradable scaffolds offer the potential for true pulp regeneration rather than mere preservation. Material and Methods: Human dental pulp stem cells were isolated from healthy third molars and encapsulated within gelatin methacryloyl hydrogel constructs. In this randomized laboratory trial, 60 standardized human tooth slices were allocated into three groups: negative control (empty), positive control (Biodentine™), and test group (human dental pulp stem cells-gelatin methacryloyl construct). Cell viability, proliferation, odontogenic differentiation, and angiogenic potential were assessed using Live/Dead staining, Cell counting Kit-8 colorimetric assay, and quantitative reverse transcription polymerase chain reaction analysis of dentin sialophosphoprotein, dentin matrix acidic phosphoprotein 1, and vascular endothelial growth factor expression. Results: The quantitative reverse transcription polymerase chain reaction constructs demonstrated high cytocompatibility, with cell viability exceeding 94% at day 7. Proliferation was significantly greater in the test group compared with Biodentine at day 7 (p < 0.01). Odontogenic marker expression was comparable between the test and Biodentine groups, while vascular endothelial growth factor expression was markedly higher in the test group group, indicating superior angiogenic potential. Conclusion: Stem cell–laden gelatin methacryloyl constructs exhibit enhanced regenerative properties compared with conventional bioceramic materials in an ex vivo tooth slice model. These findings support the translational potential of hydrogel-based regenerative strategies as next-generation approaches for vital pulp therapy.

Mechanisms by Which miR-584-5p Regulates Osteogenic Differentiation and Inflammation in Human Dental Pulp Stem Cells.

To determine whether dentin sialophosphoprotein (DSPP) modulates oxaliplatin efficacy for colorectal cancer (CRC) and explore the underlying integrin αvβ3-dependent mechanism. Immunohistochemistry was used to compare the expression levels of DSPP between oxaliplatin-sensitive and oxaliplatin-resistant CRC tissues. The changes in oxaliplatin sensitivity in parental and oxaliplatin-resistant CRC cell lines after DSPP knockdown or overexpression were assessed using CCK-8 assay, and Western blotting was used to evaluate the efficacy of DSPP modulation and MAPK pathway activity. The interaction between DSPP and integrin αvβ3 was examined by immunofluorescence staining, co-immuno-precipitation, and immunohistochemistry. HE and immunofluorescence staining were used to confirm the establishment of CRC organoid models. Patient-derived xenograft (PDX) and nude mouse subcutaneous xenografts were used to evaluate the in vivo effect of targeting DSPP on oxaliplatin response. DSPP expression was significantly elevated in oxaliplatin-resistant patients and in oxaliplatin-resistant HCT8 cells. DSPP knockout significantly increased oxaliplatin sensitivity in oxaliplatin-resistant HCT8 cells and in HCT116 and SW620 cells. Co-immunoprecipitation revealed binding between DSPP and integrin αvβ3 in tumor cells, and immunofluorescence staining demonstrated their co-localization. Immunohistochemistry showed a positive correlation between DSPP expression and integrin αvβ3 expression in CRC tissues. Western blotting indicated that DSPP upregulated the phosphorylation levels of ERK and P53 in the MAPK signaling pathway, whereas the integrin αvβ3-targeted inhibitor (Cyclo) effectively abrogated this regulatory effect. In the xenograft and PDX models, targeted inhibition of DSPP or integrin αvβ3 suppressed tumor growth and improved the efficacy of oxaliplatin, for which the anti-DSPP monoclonal antibody was more effective than integrin αvβ3-targeted inhibitor. We identified a DSPP-integrin αvβ3 axis that mediates oxaliplatin resistance, and DSPP may serve as a therapeutic target to restore chemosensitivity in advanced CRC.

Dental pulp stem cells (DPSCs) are critical for periodontal tissue regeneration, yet their therapeutic potential is limited by suboptimal osteogenic differentiation. Liraglutide (LIRA), a glucagon-like peptide-1 receptor agonist, exhibits anti-inflammatory and bone-protective properties. This study aimed to investigate the modulatory effects of LIRA on DPSCs proliferation and osteogenic differentiation. DPSCs were treated with LIRA to assess proliferation and osteogenic differentiation using Cell Counting Kit-8, 5-Ethynyl-2'-deoxyuridine staining, alkaline phosphatase (ALP), and alizarin red S (ARS) assays. Histone lactylation levels (total lactylation and H3K18la) were quantified by Western blot. Chromatin immunoprecipitation (ChIP) and dual-luciferase assays evaluated H3K18la enrichment at the dentin sialophosphoprotein (DSPP) promoter. DSPP overexpression was used in rescue experiments to validate the role of the H3K18la-DSPP axis. LIRA significantly enhanced DPSC proliferation and osteogenic differentiation, as evidenced by increased ALP activity, mineralization nodules, and upregulated osteogenic markers (DMP1, DSPP, RUNX2, OCN, OPN). LIRA elevated global lactylation and H3K18la levels, with ChIP assays showing H3K18la enrichment specifically at the DSPP promoter. Dual-luciferase and RT-qPCR confirmed LIRA-induced DSPP transcriptional activation. Oxamate reversed LIRA's effects, while Nala amplified them. DSPP overexpression rescued oxamate-mediated suppression of osteogenesis, confirming the H3K18la-DSPP regulatory mechanism. This study demonstrates that LIRA promotes DPSC osteogenesis via histone lactylation-mediated DSPP transcriptional activation. The H3K18la-DSPP axis represents a novel metabolic-epigenetic pathway for enhancing periodontal regeneration.

This study assessed the potential of vitamins K2 and B12 as dentin surface conditioners to modulate the regenerative behavior of human periodontal ligament stem cells (hPDLSCs). hPDLSCs were isolated and characterized by flow cytometry, which confirmed the mesenchymal stem cell phenotype. Cell viability assays showed no cytotoxicity at the tested concentrations; therefore, 16 µg/mL was selected for further analysis. Both vitamins significantly enhanced cell migration, attachment to dentin disks, and mineralization potential, as demonstrated by Alizarin Red staining and alkaline phosphatase activity after 14 days of osteogenic induction. Gene expression analysis revealed that vitamin B12 primarily upregulated RUNX2, whereas vitamin K2 increased the expression of osteoprotegerin, osteocalcin, dentin sialophosphoprotein, and cementum protein 1. Furthermore, both vitamins reduced the expression of nuclear factor kappa-B and interleukin-6 and increased glutathione levels, with vitamin B12 exhibiting stronger anti-inflammatory and antioxidant effects. Despite these variations, no significant differences were observed between the two vitamins in terms of cell proliferation, mineralized nodule formation, or alkaline phosphatase activity, suggesting a comparable overall regenerative potential. These findings indicate that vitamins K2 and B12 are biocompatible and promote favorable hPDLSC responses through distinct molecular pathways. They may serve as practical, chairside-applied micronutrient conditioners in regenerative endodontic procedures, potentially improving the treatment outcomes.

Variants in the dentin sialophosphoprotein (DSPP) gene are associated with dentin dysplasia type II (DD-II; OMIM # 125420) and dentinogenesis imperfecta (DI) types II (OMIM # 125490) and III (OMIM # 125500). DSPP encodes a precursor protein cleaved into three dentin matrix proteins: dentin sialoprotein (DSP), dentin phosphoprotein/phosphophoryn (DPP) and dentin glycoprotein (DGP). Exon 5 contains over 200 tandem 9-base pair repeats (DSS domain), complicating sequencing with standard methods. We studied 112 individuals (42 index cases and 70 relatives) with clinical signs of DI or DD. DNA extracted from saliva was analysed using the GenoDENT next-generation sequencing panel. For inconclusive cases, long-range PCR and Oxford Nanopore Technology (ONT) long-read sequencing were used to overcome limitations in analysing the repetitive DSPP region. Pathogenic or likely pathogenic DSPP variants were identified in 41 families, including 8 known and 14 novel variants. Most were in exon 5, causing frameshifts resulting in a -1 reading-frame shift with a hydrophobic C-terminal extension and termination at a downstream stop codon. ONT sequencing enabled detection in cases where short-read methods failed. Several variants showed familial segregation and variable expressivity. This study demonstrates the value of long-read sequencing to resolve complex DSPP regions and expands the variant spectrum. The variability in clinical presentation suggests the influence of modifier factors, warranting further genotype-phenotype studies.

Dental caries demineralises the enamel and dentine of the teeth, and as infection progresses it can lead to pulpal inflammation, infection and severe pain. To determine and compare the level of mRNA expression of Toll-like receptors ((TLR)-2, TLR-4 and TLR-9), tumour necrosis factor (TNF)-α, interleukins ((IL)-1α, IL-1β, IL-4, IL-6, IL-8, IL-17 and IL-23) as well as markers of dentinogenic (dentine matrix protein (DMP)-1, dentine sialophosphoprotein (DSPP)), regulatory (nuclear factor-kappa B (NF-κB1), mitogen activated protein kinase (MAPK1)), proliferative (mitogen activated protein kinase (MKi)) and stemness (sex determining region Y-box2 (SOX2)) between non-carious and carious dental pulp tissues. This study undertook a comprehensive analysis of inflammatory markers including TLR-2, TLR-4, TLR-9, TNF-α, IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-17 and IL-23, as well as markers of dentinogenic DMP-1, DSPP, NF-κB1, MAPK1, proliferative MKi and stemness SOX2 processes in healthy and carious pulp tissues using quantitative real-time reverse-transcription polymerase chain reaction. We found higher levels of TLR-2, TLR-4, IL-6, IL-8, IL-17A, IL-23A, along with NF-κB1 and MKi67 in the carious pulps (p < 0.05). The concurrent upregulation of IL-17A and IL-23A may suggest the activation of the IL-23/IL-17 signalling axis in the carious pulps, a point underreported in the literature. These findings highlight the crucial role of the immune system in pulpal inflammation and potential implications in developing targeted molecular treatments, supporting the need for further translational research.

Dental pulp extracellular matrix (DPEM), as a naturally derived scaffold material for dental pulp regeneration, exhibits excellent biocompatibility. This study aims to develop a novel photo-cross-linked hydrogel composed of methacry- lated gelatin (GelMA) loaded with DPEM for application in pulp regeneration. In vitro experiments demonstrated that the GelMA–DPEM hydrogel, fabricated by mixing a 10 mg mL − 1 DPEM solution with a 10% (w/v) GelMA solution at a ratio of 1:1, formed a stable cross-linked network with a swelling ratio of approx- imately 343%. Frequency-sweep rheology indicated that incorporation of DPEM increased the storage modulus of the hydrogel compared with GelMA alone, and the composite hydrogel significantly promoted cell migration and angiogenic ca- pacity. RT–qPCR analysis revealed that the GelMA–DPEM hydrogel markedly upregulated the expression levels of neuroepithelial stem cell protein (Nestin), dentin sialophosphoprotein (DSPP), platelet endothelial cell adhesion molecule-1 (CD31), and runt-related transcription factor 2 (Runx2). Moreover, ectopic trans- plantation in nude mice indicated that, when combined with human dental pulp stem cells (hDPSCs), the GelMA–DPEM hydrogel preserved the DPEM’s capa- bility to support the formation of a cell-rich, vascularized pulp-like tissue con- sisting of collagen-rich connective tissue and CD31-positive blood vessels. In conclusion, the GelMA–DPEM hydrogel showed favorable cytocompatibility and preliminary pro-angiogenic effects in vitro and supported pulp-like tissue forma- tion in a small-animal ectopic treated dentin matrix (TDM) model. These findings suggest that GelMA–DPEM is a promising candidate scaffold for dental pulp re- generation.

Dentin sialophosphoprotein (DSPP) is an extracellular matrix protein, which is highly expressed in odontoblasts and transiently expressed in presecretory ameloblasts. DSPP mutations were related to dentinogenesis imperfecta (DGI), some of which can be accompanied by amelogenesis imperfecta (AI). However, the mechanism underlying DGI and AI caused by DSPP mutations is still unclarified. This study aimed to reveal the molecular pathogenesis in a Chinese family with both DGI and AI caused by the DSPP splicing mutation. One Chinese family with DGI and AI was recruited. Whole-exome sequencing and Sanger sequencing were performed to identify mutations in candidate genes. Minigene splicing assays were performed to analyze the mutation effects on mRNA splicing alteration. Furthermore, point mutation (named KI) and exon 3 knocked out (named KO) mouse models were generated to understand the in vivo consequences. HE staining and microCT analysis were performed to observe the morphological changes. RNA sequencing and quantitative real-time PCR were conducted to explore the pathogenic molecular mechanism. The dentitions of the proband exhibited an opalescent color with severe attrition. Additionally, pitted enamel can be observed in the crown. A splicing mutation (NM_014208.3: c.135 + 3 A > G) of DSPP can be detected in the proband and her father. Minigene splicing assay revealed that this mutation could cause partial exon 3 of DSPP (c.246-c.255) skipping. Phenotypic analysis of mutated mice revealed enlarged pulp cavities in younger mice, and narrowed pulp chamber and canals in older mutants. Enamel abnormalities were exclusively in KI mice. RNA sequencing and quantitative real-time PCR suggested that the splicing mutation of Dspp might downregulate the expression of secreted phosphoprotein 1 (Spp1) and cartilage oligomeric matrix protein (Comp) gene, implicating the ECM-receptor interaction and focal adhesion signaling pathways in the pathogenesis of tooth abnormalities. In this study, we identified a splicing mutation in DSPP, which caused both DGI and AI. This research enhances our understanding of pathologic mechanisms of DSPP splicing mutations in tooth defects through genetic and molecular lens.

Complex tooth injuries, including dental caries, require the reestablishment of tissue architecture and functionality through the regeneration of cellular populations that enable tertiary dentin formation and the reestablishment of vascular and neural components. Among these cellular populations, the odontoblasts play a critical role, as they are responsible for dentin formation and maintenance, and can differentiate from stem cells of the dental pulp and apical papilla. We previously demonstrated that Histatin-1, a salivary peptide with wound-healing properties, enhances the mineralizing activity of primary mesenchymal cells from immature permanent teeth. Here, we demonstrate that Histatin-1 upregulates odontoblastic differentiation markers, including dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP1), together with odontoblast-like cellular features. Immunohistochemistry and tissue immunofluorescence revealed increased DSPP and DMP1 expression in Histatin-1-treated apical papilla explants, and to a lesser extent, dental pulp explants. These findings were corroborated in primary cultures of these tissues, which also showed upregulation of DSPP and β-catenin. Histatin-1 further promoted primary ciliogenesis and Golgi-polarization. Moreover, Histatin-1 stimulated in vitro mineralization and cell migration in a VEGFR2-dependent manner, as confirmed by pharmacological inhibition and a VEGFR2-binding-deficient mutant of Histatin-1. Collectively, these results suggest that Histatin-1 drives odontoblastic differentiation, opening new avenues for dental regenerative medicine.

Osteopontin deficiency disturbs dentin bridge formation after direct pulp capping with mineral trioxide aggregate.

The development of dependable in vitro models that replicate the pulp-dentin complex is important for regenerative endodontics, biomaterials testing, and disease modelling. However, most existing approaches concentrate on isolated techniques, providing limited guidance regarding their comparative performance, practical limitations, or translational applicability. Furthermore, they often present only the final optimized method without elaborating on the decision-making process, including the challenges faced and the rationale for not pursuing alternative approaches. This study aimed to address this gap by evaluating multiple three-dimensional (3D) strategies for the development of pulp-dentin models. Two principal assembly routes were explored: manual and digital. Manual assemblies used natural dentin rings combined with different 3D culture strategies to generate the pulp core. Scaffold-free spheroids were formed using ultra-low attachment plates (ULA) and non-adherent overlays (Agarose and Matrigel), while scaffold-based systems employed Matrigel encapsulation. These were manually integrated within dentin rings to establish pulp-dentin interfaces. Digital assemblies utilized extrusion-based 3D bioprinting to fabricate composite constructs composed of dentin powder-reinforced Gelatin Methacryloyl (GelMA) or alginate outer rings and collagen or alginate-based cellular cores. All constructs were evaluated for structural stability, reproducibility, cellular organization, and interaction with the dentin interface, primarily through morphological and histological analyses. Both manual and digital assembly strategies successfully produced 3D pulp-dentin constructs with distinct characteristics. In the manual assemblies, scaffold-free and scaffold-based approaches enabled spheroid formation and matrix-supported tissue organization, respectively. When integrated with natural dentin rings, these cultures established localized pulp-dentin interfaces with Dentin Sialophosphoprotein (DSPP) positive cells, indicating odontogenic differentiation. However, construct uniformity and stability were influenced by spheroid size, hydrogel degradation, and dentin ring geometry. Digital bioprinting enabled precisefabrication of biphasic constructs comprising a dentin powder-reinforced outer phase and acell-laden inner core. Stable and reproducible dentin-mimetic outer rings were achieved atdentin powder concentrations ≤20% (w/w), with GelMA exhibiting slower degradation andgreater mass retention than alginate. The inner pulp-like core was bioprinted using cell-ladenbioinks containing 5 × 10⁵ cells, with LifeInk 220 collagen providing consistent print fidelity andhomogeneous cell distribution. The resulting dual-layered architecture enhanced the structuraland biological resemblance of the model to native pulp-dentin tissue, with dentin powderincorporation contributing to dentin-like features and supporting odontogenic cell organization. This study establishes a methodological framework for developing in vitro pulp-dentin models by comparing manual and digital assembly strategies. Rather than identifying a single optimal approach, the work highlights how each method contributes unique advantages and challenges. Documenting both successful outcomes and technical limitations provides valuable guidance for optimizing 3D pulp-dentin constructs and advancing their application in regenerative endodontics, biomaterial evaluation, and translational research.

Bioengineered Gelatin Hydrogels Functionalized with VEGF/BMP2 Mimetic Peptides for Advancing Regenerative Endodontics.

BACKGROUND: Direct pulp capping (DPC) aims to preserve pulp vitality but requires agents that are both bioactive and biocompatible. Calcium hydroxide (CaOH2) is widely used, however its long-term success is limited, and it may cause adverse effects. Natural biomaterials such as chitosan and Aloe vera have shown potential, but their combined regenerative effects are still not well understood. This study was conducted to evaluate the efficacy of chitosan–Aloe vera composites in promoting stem cell activation, odontoblast differentiation, and reparative dentinogenesis in a rat model of reversible pulpitis.METHODS: Twenty-four Wistar rats with mechanically induced reversible pulpitis were divided into six groups: normal control, reversible pulpitis, CaOH2, and chitosan–Aloe vera pastes at 20%, 30%, and 40% (CA20, CA30, CA40). Pulp capping was performed following standardized pulp exposure. After 28 days, reparative dentin thickness and dentin bridge formation were assessed histologically, and STRO-1 and dentine sialophosphoprotein (DSPP) expression were analyzed immunohistochemically.RESULTS: Dentin bridge was observed in the CA40 group, presenting the thickest dentin formation (113.5±13.5 μm). STRO-1 and DSPP were significantly higher in all chitosan–Aloe vera combination groups compared with reversible pulpitis group (p&lt;0.01), with DSPP in CA30 and CA40 also higher than CaOH2 group. Both biomarkers demonstrated a positive correlation, and reparative dentin thickness showed a strong positive correlation with DSPP level (r=0.786, p&lt;0.001).CONCLUSION: Chitosan–Aloe vera combination showed encouraging biological activity in this 28-day preclinical model of reversible pulpitis. Although higher concentrations enhanced stem cell activation, odontoblast differentiation, and reparative dentin formation, these results should be interpreted cautiously due to the small sample size and study design limitations.KEYWORDS: pulp capping, STRO-1, DSPP, dentin bridge, CaOH2, natural biomaterial, endodontics

Chemomechanical caries removal (CMCR) offers a non-invasive alternative to conventional drilling techniques; however, the potential cytotoxic and genotoxic effects of CMCR agents on the dental pulp complex remain underexplored. This study aimed to evaluate the effects of enzymatic-based CMCR on the proliferation and osteogenic differentiation of dental pulp stem cells (DPSCs). MTT assay, scratch assay, and gene expression analysis were conducted to assess cell proliferation (viability percentage), migration (wound healing percentage), and osteogenic differentiation (RT-qPCR). The viability of DPSCs after direct exposure to enzymatic-based CMCR (0.5%, 1%, and 2% in DMEM) was measured at 24 and 48h post-exposure. Indirect exposure was also tested using sound mineralized (0.5mm and 1mm) and demineralized (0.5mm) dentin discs. Scratch assay was performed for control, direct, and indirect groups at 0, 24, and 48h, followed by fixation and crystal violet staining at 48h. Dentin sialophosphoprotein (DSPP) gene expression was quantified using RT-qPCR after 48h. Two-way ANOVA of proliferation and migration assays revealed that both indirect (0.5mm demineralized dentin) and direct (0.5% DMEM) exposure groups showed the lowest cytotoxic effect after 24h (82.75 ± 3.37 and 68.11 ± 4.99, respectively). After 48h, a recovery trend in DPSC viability was observed, with both groups exhibiting the highest proliferation (93.60 ± 4.67 and 78.25 ± 4.88, respectively). These groups also showed the highest wound closure percentages (78.78 ± 0.89 for direct and 74.19 ± 1.03 for indirect exposure). One-way ANOVA of RT-qPCR results demonstrated significant upregulation of DSPP gene expression in the indirect (0.5mm demineralized dentin) group (5.35 ± 2.01, p < 0.05). Enzymatic-based CMCR exhibits favorable biocompatibility, particularly when applied directly on DPSCs at low concentrations and when used indirectly through dentin discs. These findings suggest that this enzymatic caries removal approach is potentially safe, effective, and minimally invasive, making it suitable for conservative restorative dentistry.

Biomaterial-based strategies offer therapeutic potential for inherited disorders such as Dentinogenesis imperfecta type II (DGI-II). DGI-II, a hereditary dental disorder caused by dentine sialophosphoprotein (Dspp) gene mutations, results in fragile, discoloured teeth susceptible to wear and decay. This study investigates the structural alterations in the dentine matrix induced by mutant DSPP and the underlying molecular mechanisms. Furthermore, we evaluated a therapeutic strategy combining nanofibrous substrates with molecular activation of the Wnt5a-Cdc42 pathway, aiming to mitigate these structural defects and restore dentine integrity. A maxillary first premolar extracted from a patient with dentinogenesis imperfecta type II (DGI-II) carrying a known DSPP frameshift mutation was analysed using micro-computed tomography and scanning electron microscopy. MDPC-23 mouse dental pulp pre-odontoblasts were cultured invitro on electrospun nanofibrous scaffolds with tunable fibre diameters. Dspp expression was silenced to investigate its regulatory role in cell-cell contact formation, while the Wnt5a-Cdc42 pathway was activated via recombinant Wnt5a protein and a small molecule. Data were analysed using Student's t-test or one-way ANOVA with a significance threshold of p < 0.05. Decalcified or freeze-fractured samples exhibited thick fibres, sparse dentinal tubules, and a disorganised, non-layered dentine matrix, indicating that disrupted cell-cell junctions impair directional appositional dentine formation. Silencing Dspp expression in dental pulp cells led to reduced expression of junctional proteins, Zona occludens-1 (ZO-1) and Connexin43 (Cx43), which are essential for vectorial matrix apposition. Modulating fibre thickness and activating the Wnt5a-Cdc42 signalling axis restored both the expression and cellular localisation of ZO-1 and Cx43, thereby re-establishing cell-cell junctions and paracellular permeability in Dspp-silenced dental pulp cells. The results provide evidence that Dspp contributes to the structural integrity of dentine by modulating ZO-1 and Cx43 expression and suggest that biomaterial and molecular interventions may offer supportive strategies to restore dentine structure and function in DGI-affected teeth, though they do not address the underlying genetic defect.

Background: Aging is associated with progressive structural and functional changes in dentin, reducing its mechanical integrity and increasing vulnerability to damage. Among the most important regulators of dentin physiology are extracellular matrix proteins from the SIBLING family, including Dentin Matrix Protein 1 (DMP-1), Dentin Sialophosphoprotein (DSPP), and Osteopontin (OPN). These proteins are essential for dentin mineralization, collagen organization, and tissue remodeling. Despite their critical role, knowledge about their age-related distribution and correlation with dentin structure and morphology remains limited. Aim: To examine age-dependent changes in the expression of SIBLING proteins (DMP-1, DSPP, OPN) in human dentin and to evaluate their relationship with collagen structure and ultramorphology using polarized light microscopy (PLM), immunohistochemistry (IHC), and scanning electron microscopy (SEM). Materials and Methods: Ninety extracted human teeth were categorized into young (≤17 years), mature (18–50 years), and old (>51 years) groups. IHC was applied to detect protein distribution, PLM to assess collagen organization, and SEM to evaluate dentinal morphology. Results and Conclusions: Aging was associated with increased expression of DMP-1 and OPN and a reduction in DSPP, which is particularly evident in peritubular dentin. Older samples showed collagen disorganization, reduced birefringence, and extensive intratubular mineralization. These findings suggest that age-related alterations in SIBLING proteins contribute to structural changes in dentin, providing new insights relevant to dental care in elderly patients.

IntroductionInositol-requiring enzyme 1 alpha (IRE1α), encoded by endoplasmic reticulum (ER) to nucleus signaling 1 (Ern1) gene, is the most conserved sensor of ER stress. IRE1α-initiated signaling pathways contribute to functional maturation of secretory cells and have been implicated in various human diseases. In this study, we examined the roles of IRE1α in odontoblast development and dentin formation in wild-type mice as well as in DsppP19L mutant mice, which express a pathogenic variant of dentin sialophosphoprotein (P19L-DSPP) and exhibit a dentinogenesis imperfecta (DGI)-like phenotype.MethodsWestern-blotting and stains-all staining analyses were used to assess whether secretion of mutant P19L-DSPP was impaired in dental pulp cells containing odontoblasts from DsppP19L/P19L mice compared with Dspp+/+ controls. Immunohistochemistry and reverse-transcription PCR were performed to examine changes in IRE1α and its downstream target X-box binding protein 1 (XBP1) in P19L-DSPP mutant mice. To further investigate the roles of IRE1α in tooth development, we generated 2.3 Col1-Cre;Ern1fl/fl and compound 2.3 Col1-Cre;Ern1fl/fl;DsppP19L/+ mice. Structural and histological changes in mandibular molars were analyzed using plain X-ray radiography, micro-computed tomography (µCT), and histology. Additionally, in situ hybridization, quantitative real-time PCR, and immunohistochemistry were performed to compare molecular changes among these mice and Ern1fl/fl and Ern1fl/fl;DsppP19L/+ controls.ResultsWestern-blotting and stains-all staining analyses support that mutant P19L-DSPP protein was not efficiently secreted into dentin matrix and was accumulated within odontoblasts. Further, immunostaining signals for phosphorylated IRE1α and total XBP1 were dramatically increased in odontoblasts and other dental pulp cells of DsppP19L/+ and DsppP19L/P19L mice, in comparison with Dspp+/+ mice. Consistently, there was a small increase in spliced XBP1S protein and Xbp1s mRNA levels in P19L-DSPP mutant mice. Moreover, loss of IRE1α function reduced dentin formation in 2.3 Col1-Cre;Ern1fl/fl mice and exacerbated the dental defects of P19L-DSPP mutant mice. Notably, IRE1α deficiency did not restore the Dspp mRNA levels in the mutant mice but normalized the increased thickness of the dental pulp chamber floor dentin.ConclusionThese findings underscore the essential role of IRE1α in odontoblast function and dentinogenesis. Moreover, they reveal a context-dependent pathogenic role of IRE1α, providing new insights into ER stress in dental tissue development and disease.

Decellularized tissue scaffolds mimic the native pulp-dentin microenvironment and support the odontogenic development of stem cells. This study investigated the dentinogenic effect of Wharton's Jelly Mesenchymal Stem Cells (WJMSCs) in decellularized human dental pulp (DHDP) with bone morphogenic protein-7 (BMP-7) at three concentrations: 0 ng/mL (control), 25 ng/mL, and 50 ng/mL. The effects of BMP-7 were evaluated by histological examination, WJMSC viability using AlamarBlue, dentinogenic gene expression by qPCR, and dentinogenic protein expression by ELISA. By day 21, all three groups exhibited cell distribution along the pore surfaces of DHDP, followed by the presence of a collagen matrix in the tissue. WJMSC viability treated with 25 ng/mL and 50 ng/mL showed a statistically significant increase on days 7, 14, and 21 compared to the control group (p < 0.05). Gene expression analysis of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1), and odontogenic marker (Runx2) revealed 25 ng/mL BMP-7 resulted in significantly higher expression levels for DMP-1 and Runx2 on day 21 compared to control and 50 ng/mL BMP-7 group (p < 0.05). DSPP and DMP-1 protein expressions also showed trends similar to those of gene expressions. BMP-7 (25 ng/mL) can maintain cell viability and promote dentinogenic effects of WJMSC in the DHDP scaffold.

To investigate the clinical characteristics and genetic etiology of a Chinese pedigree affected with Hereditary dentin dysplasia type II (DD-II) due to variant of dentin sialophosphoprotein (DSPP) gene. A child diagnosed with DD- II at the Third Clinical Division of Peking University Hospital of Stomatology in December 2021 and her family members were selected as study subjects. Clinical data were retrospectively analyzed. Saliva samples were collected from the proband, her parents and sister for genomic DNA extraction. Whole exome sequencing (WES) was carried out. Candidate variant was verified by Sanger sequencing and TOPO-TA cloning sequencing. The candidate variant was also subjected to bioinformatics analysis using Mutation Taster v2021. Secondary and tertiary structures of the wild-type and variant DSPP proteins were predicted with psipred v4.0 and PyMOL v2.3 software, respectively. The pathogenicity of the variant was classified based on the guidelines from American College of Medical Genetics and Genomics (ACMG). This study was approved by the Medical Ethics Committee of Peking University Hospital of Stomatology (Ethics No.: PKUSSIRB-202162021). The proband and her mother and sister had all exhibited typical clinical manifestations of hereditary DD-II. The primary dentition of the proband displayed yellowish brown discoloration, wear, and obliteration in the chamber and root canal, while the permanent teeth of the proband's sister and mother appeared nearly normal in both color and appearance, though with obliteration in the chamber and root canal. Her father showed normal dentition. WES identified a heterozygous c.1915_1918delAAGT, p.(Lys639Glnfs*674) frameshift variant in the DSPP gene. Sanger sequencing and TOPO-TA cloning sequencing confirmed the presence of this variant in the proband, the proband's sister, and the mother, while the proband's father was negative for the variant, indicating an autosomal dominant inheritance pattern. The variant was predicted to be pathogenic by Mutation Taster v2021. Prediction of the secondary structure of the DSPP protein showed that the variant has changed it from coil to helix. The tertiary structure prediction of the DSPP protein showed change of the spatial structure of the variant DSPP, with the loops in the variant region replaced by helices at multiple sites. Based on the guidelines from the ACMG, the variant was classified as pathogenic (PVS1+PM2_Supporting+PP1+PP4). Phenotypic analysis and genetic testing of this family has clarified the clinical diagnosis of hereditary DD- II. The c.1915_1918delAAGT variant probably underlay the pathogenesis of DD-II in this family. Above results have expanded the phenotypic spectrum of the disease and may contribute to further clinical and genetic research on this disease.