Wound-healing Processes After Pulpotomy in the Pulp Tissue of Type 1 Diabetes Mellitus Model Rats.

INTRODUCTION: Patients with type 1 diabetes mellitus (DM1) tend to have delayed wound healing, even in the pulp tissue. We hypothesized that hyperglycemia affects odontoblast-like cell (OLC) differentiation and is involved in macrophage polarization. Accordingly, we evaluated dental pulp stem cell differentiation and macrophage phenotypes after pulpotomy. METHODS: After modifying DM1 rat models by streptozotocin, 8-week-old rats' upper left first molars were pulpotomized with mineral trioxide aggregate. Meanwhile, the control group was administered saline. Immunohistochemical localization of nestin, osteopontin, α-smooth muscles (α-SMAs), and CD68 (pan-macrophage marker) was conducted 7 days after pulpotomy. The OLC differentiation stage was determined using double immunofluorescence of nestin and α-SMA. Double immunofluorescence of CD68 and iNOS was counted as M1 macrophages and CD68 and CD206 as M2 macrophages. Proliferating cell nuclear antigen and Thy-1 (CD90) were evaluated by immunofluorescence. RESULTS: In DM1 rats, the reparative dentin bridge was not complete; however, the osteopontin-positive area did not differ significantly from that in controls. Proliferating cell nuclear antigen, indicative of cell proliferation, increased in positive cells in DM1 rats compared with controls. Double-positive cells for α-SMA and nestin indicated many immature OLCs in DM1. CD90 was positive only in controls. CD68-positive cells, especially M1 macrophages, were increased in DM1 rats, allowing the inflammatory stage to continue 7 days after pulpotomy. CONCLUSIONS: The condition of DM1 model rats can interfere at various stages of the wound healing process, altering OLC differentiation and macrophage polarization. These findings highlight the importance of normal blood glucose concentrations during pulp wound healing.

Preparing of Point-of-Care Reagents for Risk Assessment in the Elderly at Home by a Home-Visit Nurse and Verification of Their Analytical Accuracy.

With the rising number of older adults residing at home, there is a growing need for risk assessment and patient management in home nursing. This study aims to develop point-of-care test (POCT) reagents that can aid in risk assessment and home care, especially in settings with limited resources. Our focus was on creating a C-reactive protein (CRP) POCT, which can accurately diagnose clinically significant judgment values in home nursing. Additionally, we assessed the utility of the HemoCue WBC DIFF system in providing differential counts of white blood cells (WBC). These performances were compared with a laboratory test using blood samples from patients with pneumonia. The CRP POCT showed a comparable result to that of a laboratory method, with an average kappa index of 0.883. The leukocyte count showed good agreement with the reference method. While the correlation coefficients for both neutrophil and lymphocyte counts were deemed acceptable, it was observed that the measured values tended to be smaller in cases where the cell count was higher. This proportional error indicates a weak correlation with the neutrophil-to-lymphocyte ratio. CRP POCT and WBC counts provided reliable and accurate judgments. These tools may benefit risk management for older adults at home, patients with dementia who cannot communicate, and those living in depopulated areas.

In Vivo Assessment of the Calcium Salt-Forming Ability of a New Calcium Silicate-Based Intracanal Medicament: Bio-C Temp.

Calcium salt precipitation induced by intracanal medicaments contributes to the formation of apical hard tissue during apexification. This study compared the calcium salt-forming ability of a new calcium silicate-based intracanal medicament (Bio-C Temp) with that of two commercial calcium hydroxide pastes (Calcipex Plane II and Vitapex) in a rat subcutaneous implantation model. Polytetrafluoroethylene tubes containing each of the three materials were subcutaneously implanted in 4-week-old male Wistar rats. After 28 days, the composition and amount of calcium salts formed at the material-tissue interface were assessed using micro-Raman spectroscopy, X-ray diffraction, and elemental mapping. The tested materials produced white precipitates that had Raman spectra with peaks corresponding to hydroxyapatite and calcite. X-ray diffraction detected hydroxyapatite formation on Calcipex Plane II and Vitapex implants, as well as calcite formation on all three materials. Elemental mapping revealed that Bio-C Temp generated significantly smaller calcium- and phosphorus-rich calcified regions within the subcutaneous connective tissue than Vitapex. These results indicate that Bio-C Temp produced less calcium salt in rat subcutaneous tissue than Vitapex, although all materials formed hydroxyapatite and calcite in rat subcutaneous tissue. Bio-C Temp could be less effective than Vitapex in promoting apical hard tissue formation during apexification.

In Vivo Assessment of the Apatite-Forming Ability of New-Generation Hydraulic Calcium Silicate Cements Using a Rat Subcutaneous Implantation Model.

Hydroxyapatite formation on endodontic hydraulic calcium silicate cements (HCSCs) plays a significant role in sealing the root canal system and elevating the hard-tissue inductivity of the materials. This study evaluated the in vivo apatite-forming ability of 13 new-generation HCSCs using an original HCSC (white ProRoot MTA: PR) as a positive control. The HCSCs were loaded into polytetrafluoroethylene tubes and implanted in the subcutaneous tissue of 4-week-old male Wistar rats. At 28 days after implantation, hydroxyapatite formation on the HCSC implants was assessed with micro-Raman spectroscopy, surface ultrastructural and elemental characterization, and elemental mapping of the material-tissue interface. Seven new-generation HCSCs and PR had a Raman band for hydroxyapatite (v1 PO43- band at 960 cm-1) and hydroxyapatite-like calcium-phosphorus-rich spherical precipitates on the surfaces. The other six HCSCs with neither the hydroxyapatite Raman band nor hydroxyapatite-like spherical precipitates did not show calcium-phosphorus-rich hydroxyapatite-layer-like regions in the elemental mapping. These results indicated that 6 of the 13 new-generation HCSCs possessed little or no ability to produce hydroxyapatite in vivo, unlike PR. The weak in vivo apatite-forming ability of the six HCSCs may have a negative impact on their clinical performance.

Prostaglandin E2-Transporting Pathway and Its Roles via EP2/EP4 in Cultured Human Dental Pulp.

INTRODUCTION: Prostaglandin E2 (PGE2) exerts biological actions through its transport pathway involving intracellular synthesis, extracellular transport, and receptor binding. This study aimed to determine the localization of the components of the PGE2-transporting pathway in human dental pulp and explore the relevance of PGE2 receptors (EP2/EP4) to angiogenesis and dentinogenesis. METHODS: Protein localization of microsomal PGE2 (mPGES)synthase, PGE2 transporters (multidrug resistance-associated protein-4 [MRP4] and prostaglandin transporter [PGT]), and EP2/EP4 was analyzed using double immunofluorescence staining. Tooth slices from human third molars were cultured with or without butaprost (EP2 agonist) or rivenprost (EP4 agonist) for 1 week. Morphometric analysis of endothelial cell filopodia was performed to evaluate angiogenesis, and real-time polymerase chain reaction was performed to evaluate angiogenesis and odontoblast differentiation markers. RESULTS: MRP4 and PGT were colocalized with mPGES and EP2/EP4 in odontoblasts and endothelial cells. Furthermore, MRP4 was colocalized with mPGES and EP4 in human leukocyte antigen-DR-expressing dendritic cells. In the tooth slice culture, EP2/EP4 agonists induced significant increases in the number and length of filopodia and mRNA expression of angiogenesis markers (vascular endothelial growth factor and fibroblast growth factor-2) and odontoblast differentiation markers (dentin sialophosphoprotein and collagen type 1). CONCLUSIONS: PGE2-producing enzyme (mPGES), transporters (MRP4 and PGT), and PGE2-specific receptors (EP2/EP4) were immunolocalized in various cellular components of the human dental pulp. EP2/EP4 agonists promoted endothelial cell filopodia generation and upregulated angiogenesis- and odontoblast differentiation-related genes, suggesting that PGE2 binding to EP2/EP4 is associated with angiogenic and dentinogenic responses.

SVCT2-GLUT1-mediated ascorbic acid transport pathway in rat dental pulp and its effects during wound healing.

Ascorbic acid (AA; vitamin C) plays a crucial role in the biosynthesis and secretion of collagen to produce the organic matrix of hard tissues. Nevertheless, the detailed mechanism by which AA induces reparative dentinogenesis is still unknown. This study aimed to investigate the pathway and function of AA during wound healing in a rat pulpotomy model. Sodium-dependent vitamin C transporter (SVCT) 2 and glucose transporter (GLUT) 1 were detected in odontoblasts, endothelial cells, and nerve fibers in normal pulp tissues. SVCT2 and GLUT1 were also expressed in odontoblast-like cells in pulpotomized tissues of Wistar rats, and immunopositive cells of SVCT2 were significantly increased at 5 days after pulpotomy (p < 0.05). By contrast, osteogenic disorder Shionogi (ODS) rats, which cannot generate AA, also expressed SVCT2 and GLUT1 in normal and wound healing conditions. However, in ODS rats, when compared with the AA-addition group, the formation of dentin bridges in the AA-loss group was not evident, a layer of osteopontin was significantly increased beneath the wound surface (p < 0.05), and alpha smooth muscle actin at the odontoblast-like cells observed along this layer was significantly increased (p < 0.05), but not Nestin. Moreover, the amounts of type 1 collagen generated in the reparative dentin and beneath the wound healing site were significantly diminished (p < 0.05). Macrophages expressing CD68 and CD206 increased beneath the wound site. Hence, AA may be involved in odontoblast-like cell differentiation and anti-inflammatory response during dental pulp wound healing. Our results provide new insights into the function of AA through SVCT2 and GLUT1 in reparative dentinogenesis and may help in developing new therapeutic targets for dental pulpal disease.

Efficacy of Combining an Extraoral High-Volume Evacuator with Preprocedural Mouth Rinsing in Reducing Aerosol Contamination Produced by Ultrasonic Scaling.

The coronavirus disease pandemic has afforded dental professionals an opportunity to reconsider infection control during treatment. We investigated the efficacy of combining extraoral high-volume evacuators (eHVEs) with preprocedural mouth rinsing in reducing aerosol contamination by ultrasonic scalers. A double-masked, two-group, crossover randomized clinical trial was conducted over eight weeks. A total of 10 healthy subjects were divided into two groups; they received 0.5% povidone-iodine (PI), essential oil (EO), or water as preprocedural rinse. Aerosols produced during ultrasonic scaling were collected from the chest area (PC), dentist's mask, dentist's chest area (DC), bracket table, and assistant's area. Bacterial contamination was assessed using colony counting and adenosine triphosphate assays. With the eHVE 10 cm away from the mouth, bacterial contamination by aerosols was negligible. With the eHVE 20 cm away, more dental aerosols containing bacteria were detected at the DC and PC. Mouth rinsing decreased viable bacterial count by 31-38% (PI) and 22-33% (EO), compared with no rinsing. The eHVE prevents bacterial contamination when close to the patient's mouth. Preprocedural mouth rinsing can reduce bacterial contamination where the eHVE is positioned away from the mouth, depending on the procedure. Combining an eHVE with preprocedural mouth rinsing can reduce bacterial contamination in dental offices.

Comparison of calcium and hydroxyl ion release ability and in vivo apatite-forming ability of three bioceramic-containing root canal sealers.

OBJECTIVE: Bioceramic-containing root canal sealers promote periapical healing via Ca2+ and OH- release and apatite formation on the surface. This study aimed to compare Ca2+ and OH- release and in vivo apatite formation of three bioceramic-containing root canal sealers: EndoSequence BC sealer (Endo-BC), MTA Fillapex (MTA-F), and Nishika Canal Sealer BG (N-BG). MATERIALS AND METHODS: Polytetrafluoroethylene tubes filled with sealers were immersed in distilled water for 6 and 12 h and for 1, 7, 14, and 28 days to measure Ca2+ and OH- release. Additionally, tubes filled with sealers were implanted in the backs of rats for 28 days, and in vivo apatite formation was analyzed using an electron probe microanalyzer. RESULTS: Endo-BC released significantly more Ca2+ than the other sealers at 6 and 12 h and 1 day. Ca2+ release was significantly lower from N-BG than from Endo-BC and MTA-F at 14 and 28 days. OH- release was significantly higher from Endo-BC than from the other sealers throughout the experiment, except at 1 day. OH- release was lower from N-BG than from MTA-F at 6 h and 7 days. Only Endo-BC implants exhibited apatite-like calcium-, phosphorus-, oxygen-, and carbon-rich spherulites and apatite layer-like calcium- and phosphorus-rich, but radiopaque element-free, surface regions. CONCLUSIONS: Ca2+ and OH- release is ranked as follows: Endo-BC > MTA-F > N-BG. Only Endo-BC demonstrated in vivo apatite formation. CLINICAL RELEVANCE: Endo-BC could promote faster periapical healing than MTA-F and N-BG.

Effect of a resin-modified calcium silicate cement on inflammatory cell infiltration and reparative dentin formation after pulpotomy in rat molars.

Resin monomers and polymerisation initiators have been shown to be cytotoxic for pulp cells and to disturb odontoblast differentiation. This study aimed to compare the effect of a resin-modified calcium silicate cement (TheraCal LC; TC) and a resin-free calcium silicate cement (ProRoot MTA; PR) on pulpal healing after pulpotomy. Pulpotomy was performed on the maxillary first molars of 8-week-old rats using either PR or TC. After 1, 3, 7, 14 and 28 days, pulpal responses were assessed by micro-computed tomography, haematoxylin-eosin staining and immunostaining against CD68, which is a pan-macrophage marker. The results showed that pulpotomy with TC induced persistent infiltration of inflammatory cells, including CD68-positive macrophages, and delayed the formation of reparative dentin as compared with that with PR, although both materials allowed pulpal healing over the long term. Therefore, resin-modified TC was not as biocompatible nor bioinductive as resin-free PR when applied on the healthy pulp of rat molars.

Laminin Isoforms in Human Dental Pulp: Lymphatic Vessels Express Laminin-332, and Schwann Cell-Associated Laminin-211 Modulates CD163 Expression of M2-like Macrophages.

Laminin, a basement membrane heterotrimeric glycoprotein composed of α/ß/γ subunits, has important tissue-specific functions in the control of cellular behavior. Our recent study showed the colocalization of CD163+ M2-like macrophages with Schwann cells in human dental pulp, leading us to hypothesize that the laminin isoform of Schwann cells is associated with CD163 expression. The present study investigated the distribution of laminin isoforms in human dental pulp and the underlying mechanisms that affect macrophage phenotypes. Immunofluorescence analysis indicated that blood vessels were exclusively positive for laminin α4 and α5, whereas laminin α2 was associated with Schwann cells. Unexpectedly, laminin α3/laminin-332 (α3ß3γ2) was detected on lymphatic vessels. In intact and carious teeth, CD163+ cells were associated with laminin α2, whereas CD206 single-positive cells were present inside, outside, and along blood vessels. In vitro incubation of THP-1 macrophages in plates coated with laminin-211/511 or its functionally analogous E8 fragments of α-chain (E8-α) indicated that cell shapes differed between macrophages grown on laminin-211/E8-α2 and macrophages grown on laminin-511/E8-α5. Laminin-211/E8-α2-coated plates upregulated CD163 expression, compared with laminin-511/E8-α5-coated plates. Integrin α3- and integrin α6-neutralizing Abs altered the shape of THP-1 macrophages and upregulated mRNA levels of CD206 and CD163 in macrophages grown on laminin-511; the neutralizing Abs did not affect macrophages grown on laminin-211. These findings suggest that laminin isoforms differentially regulate macrophage behavior via distinct integrin-laminin affinities. Of note, laminin-332 is expressed by pulpal lymphatic vessels, the existence of which has been debated; laminin-211 might have a role in maintaining CD163 expression on macrophages.

Periodontal Pathogens Inhabit Root Caries Lesions Extending beyond the Gingival Margin: A Next-Generation Sequencing Analysis.

We performed a comprehensive microbiome analysis of root caries lesions using 22 teeth extracted from patients with severe periodontitis. The carious lesions were mechanically collected and cryo-pulverized following tooth extraction. Differences in the microbiome were compared between independent lesions at the supragingival site (SG) and lesions extending beyond the gingival margin (GCB). DNA was extracted and the microbiome was characterized on the basis of the V3-V4 hypervariable region of the 16S rRNA gene using paired-end sequencing on an Illumina MiSeq device. The microbiota in root caries lesions showed compositionally distinct microbiota depending on the location. The most abundant OTUs in the SG group were Streptococcus (26.0%), Actinomyces (10.6%), and Prevotella (7.6%). GCB presented Prevotella (11.1%) as the most abundant genus, followed by Fusobacterium (9.6%) and Actinomyces (8.7%). The SG group showed a lack of uniformity in microbiota compared with the GCB group. The bacterial profiles of GCB varied considerably among patients, including periodontal pathogens such as Porphyromonas, Selenomonas, Filifactor, Peptococcus, and Tannerella. Periodontal pathogens inhabit root caries lesions that extend beyond the gingival margin. This study provides a new perspective for elucidating the microbial etiology of root caries.

A Repeated State of Acidification Enhances the Anticariogenic Biofilm Activity of Glass Ionomer Cement Containing Fluoro-Zinc-Silicate Fillers.

This study aimed to evaluate the anticariogenic biofilm activity of a novel zinc-containing glass ionomer cement, Caredyne Restore (CR), using a flow-cell system that reproduces Stephan responses. Streptococcus mutans biofilms were cultured on either CR or hydroxyapatite (HA) discs mounted on a modified Robbins device. The media were allowed to flow at a speed of 2 mL/min for 24 h while exposed to an acidic buffer twice for 30 min to mimic dietary uptake. Acid exposure enhanced biofilm inhibition in the CR group, which showed 2.6 log CFU/mm2 in viable cells and a 2 log copies/mL reduction in total cells compared to the untreated group after 24 h of incubation, suggesting enhanced anticariogenic activity due to the release of fluoride and zinc ions. However, there was no difference in the number of viable and total cells between the two experimental groups after 24 h of incubation in the absence of an acidic environment. The anticariogenic biofilm activity of CR occurs in acidic oral environments, for example in the transient pH drop following dietary uptake. CR restorations are recommended in patients at high risk of caries due to hyposalivation, difficulty brushing, and frequent sugar intake.

Impact of remnant healthy pulp and apical tissue on outcomes after simulated regenerative endodontic procedure in rat molars.

When regenerative endodontic procedures (REPs) are performed on immature teeth diagnosed with pulp necrosis and apical periodontitis, various healing patterns occur. Furthermore, infected immature teeth with endodontic disorders often exhibit some remnant pulp and apical tissue. Therefore, this study investigated the impact of remnant healthy or fully functional pulp and apical tissue on healing patterns after REPs. Simulated REPs were performed on non-infected immature rat molars with different amounts of remnant pulp and apical tissue. Healing patterns in these teeth were assessed after 28 days. Teeth with 0.81-0.91 mm of remnant pulp healed with pulp-like tissue, dentin, and osteodentin-like dentin-associated mineralized tissue (OSD-DAMT); teeth with 0.60-0.63 mm of remnant pulp healed with pulp-like tissue and OSD-DAMT; teeth with 0.13-0.43 mm of remnant pulp healed with periodontal ligament (PDL)-like tissue, OSD-DAMT, and cementum-like dentin-associated mineralized tissue (CEM-DAMT); and teeth with disorganization of pulp and apical tissues at 0.15-0.38 mm beyond the root apex healed with PDL-like tissue, CEM-DAMT, and intracanal bone (IB). Loss of Hertwig's epithelial root sheath was observed with IB formation. These results showed that four distinct healing patterns occurred after REPs, depending on the preoperative amount of remnant healthy pulp and apical tissue.

Immunohistochemistry and gene expression of GLUT1, RUNX2 and MTOR in reparative dentinogenesis.

OBJECTIVES: To determine glucose transporter 1 (GLUT1) and runt-related transcription factor 2 (RUNX2) expression during reparative dentinogenesis after pulpotomy with mineral trioxide aggregate (MTA) capping. SUBJECTS AND METHODS: Eight-week-old male Wistar rats were used. Pulp of the upper left first molar was exposed and capped with MTA. The upper right first molar of the same animal was used as a control. After collecting molars at various time points, GLUT1, RUNX2 and mammalian target of rapamycin (MTOR) were examined by immunohistochemistry. mRNA levels of Slc2a1 (encoding GLUT1), Runx2, Nestin and Mtor were determined by real-time PCR. RESULTS: Pulp exhibited progressive formation of reparative dentine lined with GLUT1- and MTOR-immunoreactive odontoblast-like cells at 5 days after pulpotomy. RUNX2 was detected in nuclei of most pulp tissue cells at day 5 after pulpotomy. Double immunofluorescence staining revealed GLUT1 immunoreactivity on odontoblast-like cells positive for Nestin or RUNX2, 5 days after pulpotomy. Slc2a1, Runx2, Nestin and Mtor mRNA levels were significantly upregulated on days 3-5 after pulpotomy. CONCLUSIONS: After rat molar pulpotomy, dental pulp induced formation of reparative dentine with colocalization of GLUT1 and Nestin or RUNX2. Moreover, mRNA levels of Slc2a1, Runx2, Nestin and Mtor were significantly upregulated in pulpotomized dental pulp.

Glucose Transporter 2 and 4 Are Involved in Glucose Supply during Pulpal Wound Healing after Pulpotomy with Mineral Trioxide Aggregate in Rat Molars.

INTRODUCTION: Pulp capping materials allow healing of injured pulp with a layer of reparative dentin. Glucose is needed to cure the injured area. Glucose is transported by glucose transporter (Glut) 2 and Glut4, which are transmembrane proteins that act as gatekeepers. We hypothesized that the transport of glucose via Glut2/Glut4 might contribute to the production of a dentin bridge during wound healing. Therefore, we explored Glut2 and Glut4 expression during reparative dentinogenesis after mineral trioxide aggregate capping. METHODS: The upper left first molar of 8-week-old Wistar rats underwent pulpotomy with mineral trioxide aggregate. At 1, 3, 5, 7, and 14 days after treatment, localization and colocalization of Glut2, Glut4, nestin (odontoblast marker), and antiendothelial cell antigen 1 (RECA-1; endothelial cell marker) were analyzed with immunohistochemical staining. Messenger RNA expression levels of Slc2a2 (encoding Glut2), Slc2a4 (encoding Glut4), Igf-1r (encoding insulinlike growth factor 1 receptor), and nestin were analyzed in the extracted teeth using real-time polymerase chain reaction. RESULTS: Glut2 and Glut4 were localized within odontoblasts and endothelial cells in normal control teeth. Three days after pulpotomy, Glut2- and Glut4-positive cells were detected; 7 days after pulpotomy, immunoreactivity for Glut2 and Glut4 was confined to newly differentiated odontoblastlike cells arranged beneath reparative dentin. Messenger RNA expression levels of Slc2a2 and Slc2a4 were significantly up-regulated after pulpotomy. CONCLUSIONS: Glut2 and Glut4 regulate glucose transport during wound healing beneath the injured area. This may contribute to the development of new vital pulp therapy for patients with deep caries.

Effects of pulpotomy using mineral trioxide aggregate on prostaglandin transporter and receptors in rat molars.

Mineral trioxide aggregate (MTA) is a commonly used dental pulp-capping material with known effects in promoting reparative dentinogenesis. However, the mechanism by which MTA induces dentine repair remains unclear. The aim of the present study was to investigate the role of prostaglandin E2 (PGE2) in dentine repair by examining the localisation and mRNA expression levels of its transporter (Pgt) and two of its receptors (Ep2 and Ep4) in a rat model of pulpotomy with MTA capping. Ep2 expression was detected in odontoblasts, endothelial cells, and nerve fibres in normal and pulpotomised tissues, whereas Pgt and Ep4 were immunolocalised only in the odontoblasts. Moreover, mRNA expression of Slco2a1 (encoding Pgt), Ptger2 (encoding Ep2), and Ptger4 (encoding Ep4) was significantly upregulated in pulpotomised dental pulp and trigeminal ganglia after MTA capping. Our results provide insights into the functions of PGE2 via Pgt and Ep receptors in the healing dentine/pulp complex and may be helpful in developing new therapeutic targets for dental disease.

Characterization of Dental Pulp Myofibroblasts in Rat Molars after Pulpotomy.

INTRODUCTION: Myofibroblasts express alpha smooth muscle actin (α-SMA) and play a critical role in wound healing. Myofibroblast differentiation is controlled by the joint actions of transforming growth factor beta 1 (TGF-ß1) and the extradomain A fibronectin splice variant (EDA-FN). Currently, the contribution of myofibroblasts to dental pulp healing is unknown. Therefore, we analyzed expressional characteristics of α-SMA-positive cells and investigated TGF-ß1, EDA-FN, and α-SMA expression levels after pulpotomy to better understand dental pulp healing. METHODS: The maxillary first molars of 8-week-old Wistar rats were pulpotomized with mineral trioxide aggregate. After 1 to 14 days, localization and colocalization of α-SMA, rat endothelial cell antigen-1 (as a marker of endothelial cells), neuron-glial antigen 2 (as a marker of perivascular cells), prolyl-4-hydroxylase (P4H, as an additional marker of myofibroblasts), and EDA-FN were analyzed using immunohistochemistry and double immunofluorescence. Time-course changes in the messenger RNA expression levels of TGF-ß1, EDA-FN, and α-SMA were evaluated using quantitative real-time polymerase chain reaction analysis. RESULTS: Spindle-shaped α-SMA-positive cells transiently appeared after pulpotomy. These cells initially emerged in the pulp core on day 3 and then accumulated at the wound site by day 5. These cells were isolated from rat endothelial cell antigen-1 positive cells and did not express neuron-glial antigen 2 but did express P4H. The messenger RNA levels of TGF-ß1, EDA-FN, and α-SMA were significantly up-regulated after pulpotomy. EDA-FN and α-SMA were colocalized at the wound sites on day 5. CONCLUSIONS: In association with up-regulation of TGF-ß1 and EDA-FN expression, α-SMA and P4H double-positive cells accumulated at the wound sites after pulpotomy. This suggests that myofibroblasts participate in dental pulp healing.

Correlation between Fibrillin-1 Degradation and mRNA Downregulation and Myofibroblast Differentiation in Cultured Human Dental Pulp Tissue.

Myofibroblasts and extracellular matrix are important components in wound healing. Alpha-smooth muscle actin (α-SMA) is a marker of myofibroblasts. Fibrillin-1 is a major constituent of microfibrils and an extracellular-regulator of TGF-ß1, an important cytokine in the transdifferentiation of resident fibroblasts into myofibroblasts. To study the correlation between changes in fibrillin-1 expression and myofibroblast differentiation, we examined alterations in fibrillin-1 and α-SMA expression in organotypic cultures of dental pulp in vitro. Extracted healthy human teeth were cut to 1-mm-thick slices and cultured for 7 days. In intact dental pulp, fibrillin-1 was broadly distributed, and α-SMA was observed in pericytes and vascular smooth muscle cells. After 7 days of culture, immunostaining for fibrillin-1 became faint concomitant with a downregulation in its mRNA levels. Furthermore, fibroblasts, odontoblasts and Schwann cells were immunoreactive for α-SMA with a significant increase in α-SMA mRNA expression. Double immunofluorescence staining was positive for pSmad2/3, central mediators of TGF-ß signaling, and α-SMA. The administration of inhibitors for extracellular matrix proteases recovered fibrillin-1 immunostaining; moreover, fibroblasts lost their immunoreactivity for α-SMA along with a downregulation in α-SMA mRNA. These findings suggest that the expression of α-SMA is TGF-ß1 dependent, and fibrillin-1 degradation and downregulation might be implicated in the differentiation of myofibroblasts in dental pulp wound healing.