Search Results (133)

The sodium–citrate cotransporter (NaCT) plays a crucial role in citrate transport during amelogenesis. Mutations in the SLC13A5 gene, which encodes the NaCT, cause early infantile epileptic encephalopathy 25 and amelogenesis imperfecta. We analyzed developing pig molars and determined that the citrate concentrations in secretory- and maturation-stage enamel are both 5.3 µmol/g, with about 95% of the citrate being bound to mineral. To better understand how citrate might enter developing enamel, we developed Slc13a5^Flag reporter mice that express NaCT with a C-terminal Flag-tag (DYKDDDDK) that can be specifically and accurately recognized by commercially available anti-Flag antibodies. The 24-base Flag coding sequence was located immediately upstream of the natural translation termination codon (TAG) and was validated by Sanger sequencing. The general development, physical activities, and reproductive outcomes of this mouse strain were comparable to those of the C57BL/6 mice. No differences were detected between the Slc13a5^Flag and wild-type mice. Tooth development was extensively characterized using dissection microscopy, bSEM, light microscopy, in situ hybridization, and immunohistochemistry. Tooth formation was not altered in any detectable way by the introduction of the Flag. The Slc13a5^Flag citrate transporter was observed on all outer membranes of secretory ameloblasts (distal, lateral, and proximal), with the strongest signal on the Tomes process, and was detectable in all but the distal membrane of maturation-stage ameloblasts. The papillary layer also showed positive immunostaining for Flag. The outer membrane of odontoblasts stained stronger than ameloblasts, except for the odontoblastic processes, which did not immunostain. As NaCT is thought to only facilitate citrate entry into the cell, we performed in situ hybridization that showed Ank is not expressed by secretory- or maturation-stage ameloblasts, ruling out that ANK can transport citrate into enamel. In conclusion, we developed Slc13a5^Flag reporter mice that provide specific and sensitive localization of a fully functional NaCT-Flag protein. The localization of the Slc13a5^Flag citrate transporter throughout the ameloblast membrane suggests that either citrate enters enamel by a paracellular route or NaCT can transport citrate bidirectionally (into or out of ameloblasts) depending upon local conditions. Full article

Autophagy is a cellular process that recycles intracellular macromolecules and degrades toxic cytoplasmic material to provide the cell with nutrients and facilitate survival. Although autophagy and its role in the differentiation of osteoblasts, osteoclasts, and odontoblasts has been described, the importance of autophagy during matrix mineralization remains unaddressed. This review aims to characterize the autophagy/matrix mineralization relationship and elucidate the significance of autophagy during matrix mineralization. During the mineralization process, autophagy is important for cell survival and promotes the differentiation of osteoblasts and odontoblasts, the key cells that facilitate bone and dentin formation. Differentiation of these cells results in the synthesis of an organic proteinaceous matrix which subsequently forms the template for the deposition of calcium and phosphate to ultimately form crystalline hydroxyapatite. In bone, autophagy influences osteoblastic/osteoclastic activity and bone remodeling. In dentin, autophagy participates in odontogenic differentiation and facilitates odontoblastic secretion of dentin matrix proteins. This review aims to show that autophagy is critical for bone mineralization and tooth formation by supporting intracellular signaling pathways required for cell differentiation and subsequent matrix mineralization. Full article

Background: The dentin–pulp complex plays a vital role in tooth health. Dentin forms the main body the tooth and continues to form throughout life to maintain homeostasis and provide protection against deleterious external stimuli. However, the detailed mechanism of dentin formation remains poorly understood, and there is a need for new regenerative therapies. This study therefore investigated whether primary dental pulp cells from mice could be used to establish a new culture system. Methods: Mouse mandibles were divided along the ramus to extract dental pulp tissue containing cervical loops. The extracted tissue was cultured in an incubator to promote cell out-growth and increase the number of cells available for experimentation. Results: Cultured cells formed mineralized nodules, confirmed by Alizarin red S staining. The expression levels of dentin sialo protein, bone gamma-carboxyglutamate protein, and type I collagen mRNAs in cultured dental pulp cells on day 15 were lower than those in intact mouse dental pulp tissue, and the expression of all mRNAs was confirmed through electrophoresis. Conclusions: This study established a primary culture system using dental pulp tissue extracted from mouse mandibular incisors. The results demonstrated that dental pulp cells can differentiate into odontoblast-like cells and form dentin-like mineralized nodules, thereby offering a useful system for studying dentin formation and odontoblast differentiation. Full article

In this study, we introduce a novel distribution related to the gamma distribution, referred to as the generalized incomplete gamma distribution. This new family is defined through a stochastic representation involving a linear transformation of a random variable following a distribution derived from the upper incomplete gamma function. As a result, the proposed distribution exhibits a probability density function that effectively captures data exhibiting asymmetry and both mild and high levels of kurtosis, providing greater flexibility compared to the conventional gamma distribution. We analyze the probability density function and explore fundamental properties, including moments, skewness, and kurtosis coefficients. Parameter estimation is conducted via the maximum likelihood method, and a Monte Carlo simulation study is performed to assess the asymptotic properties of the maximum likelihood estimators. To illustrate the applicability of the proposed distribution, we present two case studies involving real-world datasets related to mineral concentration and the length of odontoblasts in guinea pigs, demonstrating that the proposed distribution provides a superior fit compared to the gamma, inverse Gaussian, and slash-type distributions. Full article

Background/Objectives: Nestin is an intermediate filament protein and a marker of odontoblasts, but its function in tooth and bone formation is largely unknown. This study aimed to determine whether Nestin plays a role in postnatal tooth formation. Methods: 4-week-old Nestin knockout (KO) mice were analyzed with a range of techniques, including X-ray imaging, uCT, backscattered and acid-etched casted SEM, FITC-confocal microscopy, H&E and TRAP staining, and immunohistochemistry. Results: The KO mice had no apparent difference in crown formation compared to age-matched wild-type (WT) but showed delayed molar eruption with reduced TRAP⁺ osteoclasts. More importantly, KO mice developed expanded predentin and shorter, thinner roots with irregular and shortened dentin tubules. Additionally, the Nestin KO mice exhibited a reduced cellular cementum mass with sharp reductions in DMP1, OPN, and BSP. Conclusions: These findings suggest that Nestin plays a critical role in the postnatal development of root dentin and cellular cementum. Full article

Dental infections can disrupt root development in immature permanent teeth, making traditional endodontic treatment challenging. Apexogenesis, a regenerative approach that promotes natural root development, offers a potential solution. However, issues related to disinfection and material biocompatibility still remain. The objective of this study was to evaluate the synergistic antimicrobial and antibiofilm properties of double and triple antibiotic combinations against common oral pathogens, and to incorporate the most effective combination into a thermosensitive hydrogel, to develop an alternative intracanal medication. Antibiotics were tested alone and in combination in planktonic and biofilm conditions of oral bacteria and Candida albicans. The antibiotic combinations with potential antimicrobial synergy were tested on Enterococcus faecalis biofilms in radicular dentin by confocal microscopy. Metronidazole (ME), ciprofloxacin (CI), and fosfomycin (FO) were incorporated into poly(N-vinylcaprolactam) (PNVCL) hydrogels, and their antibiofilm activity was compared to PNVCL hydrogels containing chlorhexidine (CHX) or calcium hydroxide (CH). The cytotoxicity of the hydrogels was assessed on MDPC-23 odontoblast-like cells using metiltetrazolium assays. A statistical analysis was performed using ANOVA followed by Tukey’s test (p < 0.05). The combination of ME + CI + FO showed superior antibiofilm effects in mono- and dual-species biofilms and on biofilms inside dentinal tubules, comparable to CHX. PNVCL hydrogels with ME + CI + FO significantly reduced E. faecalis biofilms in dentinal tubules, exhibiting a higher efficacy than PNVCL + CH. Cytotoxicity tests revealed minimal effects on cell viability for both PNVCL hydrogels with and without antibiotics. In conclusion, ME + CI + FO showed potent antimicrobial synergy and, when loaded in thermosensitive PNVCL hydrogel, demonstrated significant antibiofilm activity and low cytotoxicity. These findings emphasize the potential of this formulation as an effective and biocompatible endodontic medication, especially for the treatment of immature permanent teeth. Full article

Tooth/skeletal dysplasia, such as hypophosphatasia (HPP), has been extensively studied. However, there are few definitive treatments for these diseases owing to the lack of an in vitro disease model. Cells differentiated from patient-derived induced pluripotent stem cells (iPSCs) demonstrate a pathological phenotype. This study aimed to establish a method for fabricating hard tissue-forming cells derived from human iPSCs (hiPSCs) for the pathological analysis of tooth/skeletal dysplasia. Healthy (HLTH) adult-derived hiPSCs were cultured in a hard tissue induction medium (HM) with or without retinoic acid (RA) under 3D culture conditions, and mineralization and expression of dentinogenesis- and osteogenesis-related markers in 3D hiPSC constructs were evaluated. hiPSCs derived from patients with hypophosphatasia were also cultured in HM with RA. HLTH-derived hiPSCs formed mineralized 3D constructs and showed increased expression of dentinogenesis- and osteogenesis-related markers; addition of RA promoted the expression of these markers in hiPSC constructs. HPP-derived hiPSC constructs showed lower mineralization and expression of dentinogenesis- and osteogenesis-related markers than HLTH-derived hiPSCs, indicating an impaired ability to differentiate into odontoblasts and osteoblasts. This method for fabricating 3D hiPSC constructs allows for simultaneous assessment of dentinogenesis and osteogenesis, with HPP-derived hiPSC constructs recapitulating pathological phenotypes. Full article

Background: Chemotherapy (CMT) in children can disrupt dental development and calcification, causing long-term dental issues, but good dental care and habits can help improve quality of life. This case report examines permanent dental disturbances in a 7-year, 4-month-old girl undergoing CMT, explores the histology of microdontia, and outlines an oral treatment plan for CMT management. Methods: Clinical examination revealed microdontia and a groove crossing the cervical area (chronological hypoplasia), which were assessed using panoramic radiographs and histological analysis. The patient was monitored for five years, and microdontia was extracted for orthodontic reasons. A tailored treatment plan was implemented to maintain oral health during CMT. Results: Clinical and radiographic findings indicated tooth agenesis, rudimentary form, chronological hypoplasia, and microdontia. Histological analysis showed reduced odontoblast counts, abnormal dentinal tubules, thinner pre-dentin, and interglobular dentin (hypocalcification) surrounded by globular dentin (normal calcification). CMT-related microdontia caused inflammation with dilated blood vessels in the pulp. A high fever during CMT led to a groove in the enamel of all teeth, presenting as chronological hypoplasia. No new dental caries was observed over the follow-up. Conclusions: This report highlights long-term dental disturbances from CMT in permanent dentition and associated histopathological changes. It proposes an oral care protocol for managing these issues. Maintaining oral hygiene and preventing caries during the five-year follow-up reduced CMT side effects and improved the quality of the patient’s life. Full article

Patients with diabetes mellitus (DM) have an increased risk of tooth decay caused by alterations in their tooth development and their oral environment, as well as a tendency to present with pulp infection due to compromised immune response. The present study analyzed the characteristic alterations in tooth development under DM conditions using incisors from db/db type 2 diabetic mouse model (T2DM mice). In micro-CT analyses, T2DM mice showed delayed dentin and enamel formation. Through transcriptomic analyses, pre-ameloblast- and pre-odontoblast-specific genes were found to be significantly decreased in the incisors of T2DM mice, whereas major ameloblast- and mature odontoblast-specific genes were not changed. Stem cell markers were decreased in T2DM mice compared to those from the control mice, suggesting that the stemness of dental pulp cells (DPCs) is attenuated in T2DM mice. In vitro analyses demonstrated that DPCs from T2DM mice have lower colony-forming units (CFU), slower propagation, and diminished differentiation characteristics compared to the control. These data suggest that T2DM conditions could impair the differentiation property of multiple progenitor/stem cells in the tooth, resulting in delayed tooth development in T2DM mice. Full article

Background: Dental pulp (DP) is a connective tissue composed of various cell types, including fibroblasts, neurons, adipocytes, endothelial cells, and odontoblasts. It contains a rich supply of pluripotent stem cells, making it an important resource for cell-based regenerative medicine. However, current stem cell collection methods rely heavily on the enzymatic digestion of dissected DP tissue to isolate and propagate primary cells, which often results in low recovery rates and reduced cell survival, particularly from deciduous teeth. Methods: We developed a novel and efficient method to obtain a sufficient number of cells through a one-step cultivation process of isolated DP. After the brief digestion of DP with proteolytic enzymes, it was scratched onto a culture dish and cultured in a suitable medium. By day 2, the cells began to spread radially from DP, and by day 10, they reached a semi-confluent state. Cells harvested through trypsinization consistently yielded over 1 million cells, and after re-cultivation, the cells could be propagated for more than ten passages. Results: The proliferative and differentiation capacities of the cells after the 10th passage were comparable to those from the first passage. The cells expressed alkaline phosphatase as an undifferentiation marker. Similarly, they also maintained the constitutive expression of stem cell-specific markers and differentiation-related markers, even after the 10th passage. Conclusions: This method, termed “scratch-based isolation of primary cells from human dental pulps (SCIP)”, enables the efficient isolation of a large number of DP cells with minimal equipment and operator variability, while preserving cell integrity. Its simplicity, high success rate, and adaptability for patients with genetic diseases make it a valuable tool for regenerative medicine research and clinical applications. Full article

As dental pulp contains the stem cells necessary for regeneration, the tooth should hold the intrinsic capacity for self-repair. A triphasic hybrid dental biocomposite (3HB) composed of biocompatible biopolymers to provide strength, antibacterial properties and protein-based cell support could provide a conducive microenvironment for the regeneration of dental structures. 3HB was incorporated into Mineral Trioxide Aggregate (ProRoot MTA) to construct a malleable injectable implant. Human tooth pulp cells (hDPCs) significantly increased proliferation in the presence of 3HB+MTA compared to 3HB or MTA alone. Cell viability decreased with MTA alone but increased with 3HB and 3HB+MTA. 3HB+MTA was implanted into the residual tooth of drilled Wistar rat M2 molars for up to 45 days. Stereological analysis from micro-CT images showed the volume of the tooth remaining. Histologically, regenerative pulpal architecture was seen invading 3HB. A continuous odontoblastic profile lined a deposit of dentin-like material suggesting reparative dentinogenesis. Overall, no infection or encapsulation was seen. Immunohistochemically, odontoblasts were seen along the margins of the wounded tooth undergoing repair. Mesenchymal cells (MSCs) were seen at the base of the drilled tooth and by 21 days had translocated into the implant itself. Cells stimulating remineralization were highly expressed in the tooth undergoing repair. CD146-positive MSCs were seen in the center of the implant, possibly stimulating remineralization. In conclusion, behavior of 3HB⁺ in vitro and in vivo provided a promising start as 3HB+MTA may serve as a viable regenerative scaffold for pulp regeneration; however, this should be further studied before clinical use can be considered. Full article

Background: The present study evaluated the histological outcomes of two dental restorative materials, polyacid-modified composite resin (compomer) and conventional composite resin, in the primary molars of puppies. Materials and Methods: Twenty sound primary molars in four puppies were used. The puppies were rendered unconscious using general anesthesia. Similar cylindrical Class V cavities were prepared in 16 of the 20 selected primary molars. The teeth were divided into three groups: Group I: Eight cavities were restored with compomer; Group II: Eight cavities were restored with conventional composite resin; Group III: Four teeth remained untreated and were used as controls. In Groups I and II, four teeth were examined histologically after 2 weeks and the other four after 6 weeks. The histological findings were analyzed and compared to determine the effects of each type of resin material on the dentine and the pulp. Results: At 6 weeks, the specimens tested for compomer showed obvious destructive changes in the central region and the region of the pulp adjacent to the cavity. The specimens tested for conventional composite resin revealed, at 6 weeks, massive destruction of the pulp tissues and abscess formation was observed. All the specimens tested in the control group showed normal cellularity, normal vascularity, and proper alignment of odontoblast cells. Conclusions: The teeth restored with compomer demonstrated more favorable pulpal reactions when compared with the teeth restored with conventional composite resin after 6 weeks. Full article

Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine–phosphate–guanine (CpG) motifs (CpG-ODNs) are ligand molecules for Toll-like receptor 9 (TLR9), which is expressed by odontoblasts in vitro and dental pulp cells. This study determined the effects of CpG-ODNs on pulpal immunomodulatory response and repair following injury. Briefly, the upper right first molars of three-week-old mice were extracted, immersed in Type A (D35) or B (K3) CpG-ODN solutions (0.1 or 0.8 mM) for 30 min, and then replanted. Pulpal healing and immunomodulatory activity were assessed by hematoxylin–eosin and AZAN staining, as well as immunohistochemistry. One week following the operation, inflammatory reactions occurred in all of the experimental groups; however, re-revascularization and newly formed hard tissue deposition were observed in the pulp chamber of all groups at week 2. A positive trend in the expression of immune cell markers was observed toward the CpG-ODN groups at 0.1 mM. Our data suggest that synthetic CpG-ODN solutions at low concentrations may evoke a long-lasting macrophage–TLR9-mediated pro-inflammatory, rather than anti-inflammatory, response in the dental pulp to modulate the repair process and hard tissue formation. Further studies are needed to determine the effects of current immunomodulatory agents in vitro and in vivo and develop treatment strategies for dental tissue regeneration. Full article

Putatively, tooth agenesis was attributed to the initiation failure of tooth germs, though little is known about the histological and molecular alterations. To address if constitutively active FGF signaling is associated with tooth agenesis, we activated Fgf8 in dental mesenchyme with Osr-cre knock-in allele in mice (Osr2-cre^KI; Rosa26R-Fgf8) and found incisor agenesis and molar microdontia. The cell survival assay showed tremendous apoptosis in both the Osr2-cre^KI; Rosa26R-Fgf8 incisor epithelium and mesenchyme, which initiated incisor regression from cap stage. In situ hybridization displayed vanished Shh transcription, and immunostaining exhibited reduced Runx2 expression and enlarged mesenchymal Lef1 domain in Osr2-cre^KI; Rosa26R-Fgf8 incisors, both of which were suggested to enhance apoptosis. In contrast, Osr2-cre^KI; Rosa26R-Fgf8 molar germs displayed mildly suppressed Shh transcription, and the increased expression of Ectodin, Runx2 and Lef1. Although mildly smaller than WT controls prenatally, the Osr2-cre^KI; Rosa26R-Fgf8 molar germs produced a miniature tooth with impaired mineralization after a 6-week sub-renal culture. Intriguingly, the implanted Osr2-cre^KI; Rosa26R-Fgf8 molar germs exhibited delayed odontoblast differentiation and accelerated ameloblast maturation. Collectively, the ectopically activated Fgf8 in dental mesenchyme caused incisor agenesis by triggering incisor regression and postnatal molar microdontia. Our findings reported tooth agenesis resulting from the regression from the early bell stage and implicated a correlation between tooth agenesis and microdontia. Full article

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 Tgfbr2^cko 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 Tgfbr2^cko 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. Full article