Mouse Dental Pulp Research Articles

Localization of Both CD31- and Endomucin-Expressing Vessels in Mouse Dental Pulp

Localization of Both CD31- and Endomucin-Expressing Vessels in Mouse Dental Pulp

Single-cell RNA analysis of chromodomain-encoding genes in mesenchymal stromal cells of the mouse dental pulp.

The chromodomain helicase DNA-binding (CHD) and chromobox (CBX) families of proteins play crucial roles in cell fate decisions, differentiation, and cell proliferation in a broad variety of tissues and cell types. CHD proteins are ATP-dependent epigenetic enzymes actively engaged in transcriptional regulation, DNA replication, and DNA damage repair, whereas CBX proteins are transcriptional repressors mainly involved in the formation of heterochromatin. The pleiotropic effects of CHD and CBX proteins are largely dependent on their versatility to interact with other key components of the epigenetic and transcriptional machinery. Although the function and regulatory modes of CHD and CBX factors are well established in many cell types, little is known about their roles during osteogenic differentiation. A single-cell RNA-sequencing analysis of the mouse incisor dental pulp revealed distinct spatiotemporal expression patterns of CHD- and CBX-encoding genes within different clusters of mesenchymal stromal cells (MSCs) representing various stages of osteogenic differentiation. Additionally, genes encoding interaction partners of CHD and CBX proteins, such as subunits of the trithorax-COMPASS and polycomb chromatin remodeling complexes, exhibited differential co-expression behaviors within MSC subpopulations. Thus, CHD- and CBX-encoding genes show partially overlapping but distinct expression patterns in MSCs, suggesting their differential roles in osteogenic cell fate decisions.

The temporospatial relationship between mouse dental pulp stem cells and tooth innervation

Background/purposeDental pulp stem cells (DPSCs) exhibit versatile differentiation capabilities, including neural differentiation, prompting the hypothesis that they may be implicated in the neurodevelopment of teeth. This study aimed to explore the temporospatial dynamics between DPSCs and tooth innervation, employing immunofluorescence staining and fluorescent dye injections to investigate the distribution of DPSCs, neural stem cells (NSCs), nerve growth cones, and sensory nerves in developing mouse tooth germs at various stages. Materials and methodsImmunofluorescence staining targeting CD146, Nestin, and GAP-43, along with the injection of AM1-43 fluorescent dye, were utilized to observe the distribution of DPSCs, NSCs, nerve growth cones, and sensory nerves in mouse tooth germs at different developmental stages. ResultsPositive CD146 immunostaining was observed in microvascular endothelial cells and pericytes within and around the tooth germ. The percentage of CD146-positive cells remained consistent between 4-day-old and 8-day-old second molar tooth germs. Conversely, Nestin expression in odontoblasts and their processes decreased in 8-day-old tooth germs compared to 4-day-old ones. Positive immunostaining for GAP-43 and AM1-43 fluorescence revealed the entry of nerve growth cones and sensory nerves into the pulp in 8-day-old tooth germs, while these elements were confined to the dental follicle in 4-day-old germs. No co-localization of CD146-positive DPSCs with nerve growth cones and sensory nerves was observed. ConclusionDPSCs and NSCs were present in dental pulp tissue before nerves penetrated the pulp. The decline in NSCs after nerve entry suggests a potential role for DPSCs and NSCs in attracting neural growth and/or differentiation within the pulp.

Immunolocalization of the mechanogated ion channels PIEZO1 and PIEZO2 in human and mouse dental pulp and periodontal ligament.

PIEZO1 and PIEZO2 are essential components of mechanogated ion channels, which are required for mechanotransduction and biological processes associated with mechanical stimuli. There is evidence for the presence of PIEZO1 and PIEZO2 in teeth and periodontal ligaments, especially in cell lines and mice, but human studies are almost nonexistent. Decalcified permanent human teeth and mouse molars were processed for immunohistochemical detection of PIEZO1 and PIEZO2. Confocal laser microscopy was used to examine the co-localization of PIEZO 1 and PIEZO2 with vimentin (a marker of differentiated odontoblasts) in human teeth. In the outer layer of the human dental pulp, abundant PIEZO1- and PIEZO2-positive cells were found that had no odontoblast morphology and were vimentin-negative. Based on their morphology, location, and the absence of vimentin positivity, they were identified as dental pulp stem cells or pre-odontoblasts. However, in mice, PIEZO1 and PIEZO2 were ubiquitously detected and colocalized in odontoblasts. Intense immunoreactivity of PIEZO1 and PIEZO2 has been observed in human and murine periodontal ligaments. Our findings suggest that PIEZO1 and PIEZO2 may be mechanosensors/mechanotransducers in murine odontoblasts, as well as in the transmission of forces by the periodontal ligament in humans and mice.

RvE1 Promotes Axin2+ Cell Regeneration and Reduces Bacterial Invasion

Vital pulp therapy and root canal therapy (RCT) are the dominant treatment for irreversible pulpitis. While the success rate of these procedures is favorable, they have some limitations. For instance, RCT leads to removing significant dentin in the coronal third of the tooth that increases root-fracture risk, which forces tooth removal. The ideal therapeutic goal is dental pulp regeneration, which is not achievable with RCT. Specialized proresolving mediators (SPMs) are well known for inflammatory resolution. The resolution of inflammation and tissue restoration or regeneration is a dynamic and continuous process. SPMs not only have potent immune-modulating functions but also effectively promote tissue homeostasis and regeneration. Resolvins have been shown to promote dental pulp regeneration. The purpose of this study was to explore further the cellular target of Resolvin E1 (RvE1) therapy in dental pulp regeneration and the impact of RvE1 in infected pulps. We investigated the actions of RvE1 on experimentally exposed pulps with or without microbial infection in an Axin2 Cre-Dox ;Ai14 genetically defined mouse model. Our results showed RvE1 promoted Axin2-tdTomato+ cell expansion and odontoblastic differentiation after direct pulp capping in the mouse, which we used to mimic reversible pulpitis cases in the clinic. In cultured mouse dental pulp stem cells (mDPSCs), RvE1 facilitated Axin2-tdTomato+ cell proliferation and odontoblastic differentiation and also rescued impaired functions after lipopolysaccharide stimulation. In infected pulps exposed to the oral environment for 24 h, RvE1 suppressed inflammatory infiltration, reduced bacterial invasion in root canals, and prevented the development of apical periodontitis, while its proregenerative impact was limited. Collectively, topical treatment with RvE1 facilitated dental pulp regenerative properties by promoting Axin2-expressing cell proliferation and differentiation. It also modulated the resolution of inflammation, reduced infection severity, and prevented apical periodontitis, presenting RvE1 as a novel therapeutic for treating endodontic diseases.

Vascular and lymphatic heterogeneity and age-related variations of dental pulps

ObjectivesDental pulp tissue is highly vascularized. However, age-related vascular changes of the dental pulp in mice and humans remain poorly understood. We modified a novel tissue clearing method, mapped the vasculature, pericytes, and perivascular matrix in the dental pulp via high-resolution 3D imaging. MethodsWe isolated young and aged pulps from mouse teeth, and mapped vasculature through a high-resolution thick frozen sections imaging method and a modified tissue clearing method. Human dental pulps were also mapped for vasculature studying. Furthermore, young and aged human dental pulps were collected and were compared with mouse pulps through RNA- sequencing. ResultsFive vascular subtypes of blood vessels were found in the mouse dental pulp, which constituted the arterioles-capillaries-venules network. The density of capillaries and venules of molars declined obviously in aged mice. Among the age-dependent changes in the perivascular pulp matrix, the perivascular macrophages remarkably increased, lymphatic capillaries increased, while the nerves and extracellular matrix remained unchanged. Furthermore, the vascular patterns of human formed a complex vascular network. Both mouse and human dental pulps exhibited an inflammaging state. TNF pathway and Rap1 pathway might become promising targets for combating inflammaging and promoting angiogenesis. ConclusionsFive subtypes of blood vessels were identified within the dental pulp of mice. Notably, the density of capillaries and venules in pulps of aged mice was reduced. Furthermore, partial similarities were observed in the vascular patterns between the dental pulps of humans and mice. RNA-sequencing analysis revealed that both mouse and human dental pulps exhibit indications of an inflammaging state. Clinical SignificanceThis study may contribute to unraveling potential therapeutic targets in the pulp regeneration and treatment of relevant diseases in the elderly.

Single-cell Transcriptome Landscape of DNA Methylome Regulators Associated with Orofacial Clefts in the Mouse Dental Pulp.

Significant evidence links epigenetic processes governing the dynamics of DNA methylation and demethylation to an increased risk of syndromic and nonsyndromic cleft lip and/or cleft palate (CL/P). Previously, we characterized mesenchymal stem/stromal cells (MSCs) at different stages of osteogenic differentiation in the mouse incisor dental pulp. The main objective of this research was to characterize the transcriptional landscape of regulatory genes associated with DNA methylation and demethylation at a single-cell resolution. We used single-cell RNA sequencing (scRNA-seq) data to characterize transcriptome in individual subpopulations of MSCs in the mouse incisor dental pulp. The biomedical research institution. This study did not include patients. This study collected and analyzed data on the single-cell RNA expssion in the mouse incisor dental pulp. Molecular regulators of DNA methylation/demethylation exhibit differential transcriptional landscape in different subpopulations of osteogenic progenitor cells. scRNA-seq analysis revealed that genes encoding DNA methylation and demethylation enzymes (DNA methyltransferases and members of the ten-eleven translocation family of methylcytosine dioxygenases), methyl-DNA binding domain proteins, as well as transcription factors and chromatin remodeling proteins that cooperate with DNA methylation machinery are differentially expressed within distinct subpopulations of MSCs that undergo different stages of osteogenic differentiation. These findings suggest some mechanistic insights into a potential link between epigenetic alterations and multifactorial causes of CL/P phenotypes.

Identification of GPI-anchored protein LYPD1 as an essential factor for odontoblast differentiation in tooth development

Lipid rafts are membrane microdomains rich in cholesterol, sphingolipids, glycosylphosphatidylinositol-anchored proteins (GPI-APs), and receptors. These lipid raft components are localized at the plasma membrane and are essential for signal transmission and organogenesis. However, few reports have been published on the specific effects of lipid rafts on tooth development. Using microarray and single-cell RNA sequencing methods, we found that a GPI-AP, lymphocyte antigen-6/Plaur domain-containing 1 (Lypd1), was specifically expressed in preodontoblasts. Depletion of Lypd1 in tooth germ using an exvivo organ culture system and in mouse dental pulp (mDP) cells resulted in the inhibition of odontoblast differentiation. Activation of bone morphogenetic protein (BMP) signaling by BMP2 treatment in mDP cells promoted odontoblast differentiation via phosphorylation of Smad1/5/8, while this BMP2-mediated odontoblast differentiation was inhibited by depletion of Lypd1. Furthermore, we created a deletion construct of the C terminus containing the omega site in LYPD1; this site is necessary for localizing GPI-APs to the plasma membrane and lipid rafts. We identified that this site is essential for odontoblast differentiation and morphological change of mDP cells. These findings demonstrated that LYPD1 is a novel marker of preodontoblasts in the developing tooth; in addition, they suggest that LYPD1 is important for tooth development and that it plays a pivotal role in odontoblast differentiation by regulating Smad1/5/8 phosphorylation through its effect as a GPI-AP in lipid rafts.

Single-Cell Transcriptome Analysis Defines Expression of Kabuki Syndrome-Associated KMT2D Targets and Interacting Partners.

Objectives. Kabuki syndrome (KS) is a rare genetic disorder characterized by developmental delay, retarded growth, and cardiac, gastrointestinal, neurocognitive, renal, craniofacial, dental, and skeletal defects. KS is caused by mutations in the genes encoding histone H3 lysine 4 methyltransferase (KMT2D) and histone H3 lysine 27 demethylase (KDM6A), which are core components of the complex of proteins associated with histone H3 lysine 4 methyltransferase SET1 (SET1/COMPASS). Using single-cell RNA data, we examined the expression profiles of Kmt2d and Kdm6a in the mouse dental pulp. In the incisor pulp, Kmt2d and Kdm6a colocalize with other genes of the SET1/COMPASS complex comprised of the WD-repeat protein 5 gene (Wdr5), the retinoblastoma-binding protein 5 gene (Rbbp5), absent, small, and homeotic 2-like protein-encoding gene (Ash2l), nuclear receptor cofactor 6 gene (Ncoa6), and Pax-interacting protein 1 gene (Ptip1). In addition, we found that Kmt2d and Kdm6a coexpress with the downstream target genes of the Wingless and Integrated (WNT) and sonic hedgehog signaling pathways in mesenchymal stem/stromal cells (MSCs) at different stages of osteogenic differentiation. Taken together, our results suggest an essential role of KMT2D and KDK6A in directing lineage-specific gene expression during differentiation of MSCs.

Identification of Dental Stem Cells Similar to Skeletal Stem Cells

Stem and progenitor cells play important roles in the development and maintenance of teeth and bone. Surface markers expressed in bone marrow–derived mesenchymal stem cells are also expressed in dental tissue–derived stem cells. Mouse skeletal stem cells (mSSCs, CD45−Ter119−Tie2−CD51+Thy−6C3−CD105−CD200+) and human skeletal stem cells (hSSCs, CD45−CD235a−TIE2−CD31−CD146−PDPN+CD73+CD164+) have been identified in bone and shown to play important roles in skeletal development and regeneration. However, it is unclear whether dental tissues also harbor mSSC or hSSC populations. Here, we employed rainbow tracers and found that clonal expansion occurred in mouse dental tissues similar to that in bone. We sorted the mSSC population from mouse periodontal ligament (mPDL) tissue and mouse dental pulp (mDP) tissue in the lower incisors by fluorescence-activated cell sorting (FACS). In addition, we demonstrated that mPDL-derived skeletal stem cells (mPDL-SSCs) and mDP-derived skeletal stem cells (mDP-SSCs) have similar clonogenic capacity, as well as cementogenic and odontogenic potential, but not adipogenic potential, similar to the characteristics of mSSCs. Moreover, we found that the dental tissue–derived mSSC population plays an important role in repairing clipped incisors. Importantly, we sorted the hSSC population from human periodontal ligament (hPDL) and human dental pulp (hDP) tissue in molars and identified its stem cell characteristics. Finally, hPDL-like and hDP-like structures were generated after transplanting hPDL-SSCs and hDP-SSCs beneath the renal capsules. In conclusion, we demonstrated that mouse and human PDL and DP tissues harbor dental stem cells similar to mSSCs and hSSCs, respectively, providing a precise stem cell population for the exploration of dental diseases.

The chromatin accessibility landscape in the dental pulp of mouse molars and incisors.

The dental pulp is a promising source of progenitor cells for regenerative medicine. The natural function of dental pulp is to produce odontoblasts to generate reparative dentin. Stem cells within the pulp tissue originate from the migrating neural crest cells and possess mesenchymal stem cell properties with the ability to differentiate into multiple lineages. To elucidate the transcriptional control mechanisms underlying cell fate determination, we compared the transcriptome and chromatin accessibility in primary dental pulp tissue derived from 5-6-day-old mice. Using RNA sequencing and assay for transposase-accessible chromatin using sequencing (ATAC-seq), we correlated gene expression with chromatin accessibility. We found that the majority of ATAC-seq peaks were concentrated at genes associated with development and cell differentiation. Most of these genes were highly expressed in the mouse dental pulp. Surprisingly, we uncovered a group of genes encoding master transcription factors that were not expressed in the dental pulp but retained open chromatin states. Within this group, we identified key developmental genes important for specification of the neural crest, adipocyte, neural, myoblast, osteoblast and hepatocyte lineages. Collectively, our results uncover a complex relationship between gene expression and the chromatin accessibility landscape in the mouse dental pulp.

Leukotriene B4 loaded in microspheres regulate the expression of genes related to odontoblastic differentiation and biomineralization by dental pulp stem cells

BackgroundLeukotriene B4 (LTB4) is a potent lipid mediator that stimulate the immune response. Because dental pulp inflammation and dentin repair are intrinsically related responses, the aim of this research was to investigate the potential of LTB4 in inducing differentiation of dental pulp stem cells.MethodsMicrospheres (MS) loaded with LTB4 were prepared using an oil emulsion solvent extraction evaporation process and sterility, characterization, efficiency of LTB4 encapsulation and in vitro LTB4 release assay were investigated. Mouse dental pulp stem cells (OD-21) were stimulated with soluble LTB4 or MS loaded with LTB4 (0.01 and 0.1 μM). Cytotoxicity and cell viability was determined by lactate dehydrogenase and methylthiazol tetrazolium assays. Gene expression were measured by quantitative reverse transcription polymerase chain reaction after 3, 6, 24, 48 and 72 h. Mineralized nodule formation was assessed after 28 days of OD-21 cell stimulation with LTB4 in mineralized media or not. Groups were compared using one-way ANOVA test followed by Dunnett’s post-test (α = 0.05).ResultsTreatment with LTB4 or MS loaded with LTB4 (0.01 and 0.1 µm-μM) were not cytotoxic to OD-21 cells. Treatment with LTB4 modulated the expression of the Ibsp (integrin binding sialoprotein) and Runx2 (runt-related transcription factor 2) genes differently depending on the experimental period analyzed. Interestingly LTB4 loaded in microspheres (0.1 μM) allowed long term dental pulp cell differentiation and biomineralization.ConclusionLTB4, soluble or loaded in MS, were not cytotoxic and modulated the expression of the Ibsp and Runx2 genes in cultured OD-21 cells. When LTB4 was incorporated into MS, odontoblast differentiation and mineralization was induced in long term culture.

Preparation of Absorption-Resistant Hard Tissue Using Dental Pulp-Derived Cells and Honeycomb Tricalcium Phosphate.

In recent years, there has been increasing interest in the treatment of bone defects using undifferentiated mesenchymal stem cells (MSCs) in vivo. Recently, dental pulp has been proposed as a promising source of pluripotent mesenchymal stem cells (MSCs), which can be used in various clinical applications. Dentin is the hard tissue that makes up teeth, and has the same composition and strength as bone. However, unlike bone, dentin is usually not remodeled under physiological conditions. Here, we generated odontoblast-like cells from mouse dental pulp stem cells and combined them with honeycomb tricalcium phosphate (TCP) with a 300 μm hole to create bone-like tissue under the skin of mice. The bone-like hard tissue produced in this study was different from bone tissue, i.e., was not resorbed by osteoclasts and was less easily absorbed than the bone tissue. It has been suggested that hard tissue-forming cells induced from dental pulp do not have the ability to induce osteoclast differentiation. Therefore, the newly created bone-like hard tissue has high potential for absorption-resistant hard tissue repair and regeneration procedures.

Development of an Organic-Inorganic Nanostructured Hybrid Dental Biocomposite.

Dental pathologies such as caries is one of the most prevalent diseases worldwide. Dental pulp contains stem cells capable of regenerating the dentine in the tooth, consequently, healthy dental pulp is essential for long term tooth survival. The aim of this study was to incorporate a variety of polymers that provide strength, an antibacterial substance and a protein-based polymer to provide cell support. These components were combined into a triphasic hybrid dental biocomposite (3HB), that together could provide regenerative properties for the pulp tissue. The 3HB biocomposite was incorporated into Organic-inorganic nanostructured materials such as Mineral Trioxide Aggregate (MTA) as a base to assemble a hybrid dental biocomposite. The effects of the 3HB on cytotoxicity was examined in mouse dental pulp cells, MDPC-23. In vitro studies showed that 3HB supported the proliferative growth of the cells significantly more than the no treatment control. 3HB also caused little stress to the cells and supported cell viability. Fourier transform infrared (FTIR) spectra confirmed the presence of polymer functional groups within the 3HB biocomposite. Therefore, 3HB compound has the potential to be applied as a pulp wound dressing providing superior cytocompatibility than the present options but also may be indispensable for the regeneration of dental pulp.

MicroRNA-875-5p plays critical role for mesenchymal condensation in epithelial-mesenchymal interaction during tooth development

Epithelial-mesenchymal interaction has critical roles for organ development including teeth, during which epithelial thickening and mesenchymal condensation are initiated by precise regulation of the signaling pathway. In teeth, neural crest-derived mesenchymal cells expressed PDGF receptors migrate and become condensed toward invaginated epithelium. To identify the molecular mechanism of this interaction, we explored the specific transcriptional start sites (TSSs) of tooth organs using cap analysis of gene expression (CAGE). We identified a tooth specific TSS detected in the chromosome 15qD1 region, which codes microRNA-875 (mir875). MiR875-5p is specifically expressed in dental mesenchyme during the early stage of tooth development. Furthermore, PRRX1/2 binds to the mir875 promoter region and enhances the expression of mir875. To assess the role of miR875-5p in dental mesenchyme, we transfected mimic miR875-5p into mouse dental pulp (mDP) cells, which showed that cell migration toward dental epithelial cells was significantly induced by miR875-5p via the PDGF signaling pathway. Those results also demonstrated that miR875-5p induces cell migration by inhibiting PTEN and STAT1, which are regulated by miR875-5p as part of post-transcriptional regulation. Together, our findings indicate that tooth specific miR875-5p has important roles in cell condensation of mesenchymal cells around invaginated dental epithelium and induction of epithelial-mesenchymal interaction.

Prostaglandin E2 Induces Expression of Mineralization Genes by Undifferentiated Dental Pulp Cells.

Prostaglandin E2 (PGE2) is a lipid mediator usually released during inflammation. This study aimed to investigate the potential of soluble or microsphere-loaded PGE2 on inducing differentiation of dental pulp stem cells. PGE2-loaded microspheres (MS) were prepared using an oil-in-water emulsion solvent extraction-evaporation process and were characterized. Mouse dental pulp stem cells (OD-21) were stimulated with soluble or PGE2-loaded MS (0.01 and 0.1 µM). Cell viability was determined by MTT colorimetric assay. Ibsp, Bmp2 and Runx2 expression was measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) after 3, 6, and 24 h. The results showed that the soluble PGE2 reduced dental pulp stem cells viability after 24 h of stimulation whereas PGE2-loaded MS did not. Soluble PGE2 up-regulated Ibsp and Bmp2 at 3 h, differently from PGE2-loaded MS. On the other hand, PGE2-MS induced Bmp2 and Runx2 at 6 h and Ibsp at 24 h. In conclusion, our in vitro results show that PGE2, soluble or loaded in MS are not cytotoxic and modulateIbsp,Bmp2, andRunx2gene expression in cultured OD-21 cells.

Knockdown of NRAGE induces odontogenic differentiation by activating NF-κB signaling in mouse odontoblast-like cells

ABSTRACTPurpose: Neurotrophin receptor-interacting MAGE homologue (Nrage) plays an important role in bone development and the metabolism of normal skeletal structures. Our previous study showed that Nrage inhibited the odontogenic differentiation of mouse dental pulp cells. However, the potential roles and mechanism of Nrage in regulating odontogenic differentiation are unknown. The aim of this study was to investigate the molecular mechanism of Nrage in odontogenic differentiation of mouse odontoblast-like cells.Materials and methods: Endogenous expression of Nrage was stably downregulated by lentivirus-mediated shRNA. Mineralized nodules formation was detected by alizarin red S staining. Dmp-1, Dspp, and ALP mRNA and protein levels were detected by qRT-PCR and western blotting, respectively. In addition, ALPase activity was detected. Confocal microscopy and co-immunoprecipitation (co-IP) were used to analyze the interactions between NRAGE and NF-κB signaling molecules. An IKK inhibitor was also used in the study.Results: NRAGE expression in odontoblasts was downregulated during mouse first maxillary molar development. Moreover, NRAGE expression was downregulated during odontogenic differentiation of odontoblast-like cells. NRAGE knockdown significantly upregulated DMP1 and DSP expression, increased ALPase activity, and promoted mineralized nodule formation. In addition, NRAGE knockdown increased the translocation of NF-κB1 to the nucleus and phosphorylation levels of p65. Co-IP results showed that NRAGE bound to IKKβ. Most importantly, the promoting effect of Nrage knockdown on odontoblastic differentiation was reduced after treatment with an IKK inhibitor.Conclusions: Our data confirmed that NRAGE is an important regulator of odontogenic differentiation of odontoblasts by inhibiting the NF-κB signaling pathway through binding to IKKβ.Abbreviations: Nrage: neurotrophin receptor-interacting MAGE homologue; DSP: dentin sialophospho protein; DMP-1: dentin matrix protein-1; BMP: bone morphogenetic protein; Wnt: wingless; NF-κB: nuclear factor of activated B cells; DAPI: 4′,6-diamidino-2-phenylindole; KO: knockout; DPCs: dental pulp cells; AA: ascorbic acid; β-Gly: β-glycerophosphate; Dex: dexamethasone; co-IP: co-immunoprecipitation; IκB: inhibitor of NF-κB; IKK: IκB kinase

Mice dental pulp and periodontal ligament endothelial cells exhibit different proangiogenic properties.

Dental pulp is a highly vascularized tissue with a high regenerative capacity. This is attributed to its unique blood supply and the presence of progenitor or postnatal dental pulp stem cells. Here we aimed to isolate and compare the angiogenic properties of endothelial cells (EC) prepared from mouse dental pulp and periodontal ligament (PDL). EC were isolated from 4-week-old wild type immorto mice. Mice were sacrificed and after mandible isolation, the molar and incisor teeth and the PDL from molar teeth were dissected. EC were prepared by collagenase digestion of tissues and affinity purification using magnetic beads coated with platelet/endothelial cell adhesion molecule-1 (PECAM-1/CD31) antibody. EC prepared from incisor and molar pulps and PDL were examined for expression of appropriate markers by fluorescence-activated cell sorting (FACS) analysis. The proliferation, migration, and capillary morphogenesis of EC were evaluated. Ex vivo sprouting angiogenesis from various tissues was also compared. Data were analyzed at the level of significance of P<0.05. Pulp EC prepared from incisors proliferated and migrated significantly faster than molar and PDL EC (P<0.05). In addition, molar and PDL EC formed a more extensive capillary network when plated on Matrigel. This is consistent with the lower proliferative and migratory characteristics of these cells compared with incisor EC (P<0.05). However, PDL tissue showed significantly more sprouting area than molar and incisor pulp tissues (P<0.05). Thus, pulp EC from molar and incisor and PDL EC present different proangiogenic properties. Collectively our results suggest that EC from different tooth tissue have unique characteristics related to their target tissue and function.

Enhanced differentiation of dental pulp cells cultured on microtubular polymer scaffolds in vitro.

Regenerative engineering of diseased or traumatized tooth structure could avoid the deficiencies of traditional dental restorative (filling) materials. Cells in the dental pulp have the potential to differentiate to dentin-producing odontoblast cells. Furthermore, cell-supporting scaffolds that mimic a natural extracellular matrix (ECM) are known to influence behavior of progenitor cells. Accordingly, we hypothesized that a dentin-like microtubular scaffold would enhance differentiation of dental pulp cells. The hypothesis was proven true and differentiation to odontoblasts increased with increasing density of the microtubules. However, mineralization was suppressed, possibly due to a low density of cells. The results demonstrate the potential benefits of a microtubular scaffold design to promote odontoblast cells for regeneration of dentin.

Relationship between localization of proteoglycans and induction of neurotrophic factors in mouse dental pulp

ObjectivesDental pulp is composed of an odontoblastic layer, a subodontoblastic layer, and a central part. Although extracellular matrices affect the cells residing in these regions, the precise role of proteoglycans in dental pulp is incompletely understood. Hence, we investigated the immunolocalization of glucosaminoglycans, such as heparan sulfate and chondroitin sulfate, as well as core proteins of proteoglycans, such as perlecan and versican, in adult mouse dental pulp. MethodsLocalization of glucosaminoglycans and proteoglycans in dental pulp was evaluated immunohistochemically. Dental pulp cells cultured with or without fibroblast growth factor 2 (FGF2) and heparin were stained with Alizarin red S to visualize calcified nodules. Real-time PCR was used to assess the levels of gene expression of neurotrophic factors in cultured cells. ResultsHeparan sulfate and perlecan were mainly localized in the subodontoblastic layer, whereas chondroitin sulfate and versican were restricted to the center of dental pulp. Similar to perlecan, Erk1/2, a signaling transducer of FGF, was localized in the subodontoblastic layer. Moreover, we show that simultaneous treatment of cultured dental pulp cells with recombinant human FGF2 and heparin induced the expression of glial cell-derived neurotrophic factor (GDNF). ConclusionsThese data suggest that heparan sulfate proteoglycans, such as perlecan, enhance the expression of GDNF by facilitating FGF-induced Erk1/2 signaling in the subodontoblastic layer.