Reparative Dentin Research Articles

Histological analysis for pulp mineralisation after severe intrusive luxation of immature molars in rats.

Pulp mineralisation is a survival process that may occur in the pulp of immature teeth following trauma. However, the mechanism of this process remains unclear. The aim of this study was to evaluate the histological manifestations of pulp mineralisation after intrusion in immature molars of rats. Three-week-old male Sprague-Dawley rats were subjected to intrusive luxation of the right maxillary second molar by an impact force from a striking instrument through a metal force transfer rod. The left maxillary second molar of each rat was used as a control. The control and injured maxillae were collected at 3, 7, 10, 14, and 30 days after trauma (n=15 per time group) and evaluated using haematoxylin and eosin staining and immunohistochemistry. Independent two-tailed Student's t-test was used for statistical comparison of the immunoreactive area. Pulp atrophy and mineralisation were observed in 30%-40% of the animals, and no pulp necrosis occurred. Ten days after trauma, pulp mineralisation, with osteoid tissue rather than reparative dentin, formed around the newly vascularised areas in the coronal pulp. CD90-immunoreactive cells were observed in the sub-odontoblastic multicellular layer in control molars, whereas the number of these cells was decreased in the traumatised teeth. CD105 localised in cells around the pulp osteoid tissue of the traumatised teeth, whereas in control teeth, it was only expressed in the vascular endothelial cells of capillaries in the odontoblastic or sub-odontoblastic layers. In specimens with pulp atrophy at 3-10 days after trauma, hypoxia inducible factor expression and CD11b-immunoreactive inflammatory cells increased. Following intrusive luxation of immature teeth without crown fractures in rats, no pulp necrosis occurred. Instead, pulp atrophy and osteogenesis around neovascularisation with activated CD105-immunoreactive cells were observed in the coronal pulp microenvironment characterised by hypoxia and inflammation.

Tissue engineering atthe dentin-pulp interface using human treated dentin scaffolds conditioned with DMP1 or BMP2 plasmid DNA-carrying calcium phosphate nanoparticles.

Hard dental tissue pathologies, such as caries, are conventionally managed through replacement by tooth-colored inert biomaterials. Tissue engineering provides novel treatment approaches to regenerate lost dental tissues based on bioactive materials and/or signaling molecules. While regeneration in the form of reparative dentin (osteo-dentin) is feasible, the recapitulation of the tubular microstructure of ortho-dentin and its special features is sidelined. This study characterized in vitro, and in vivo human EDTA-treated, freeze-dried dentin matrices (HTFD scaffolds) conditioned with calcium phosphate nanoparticles (NPs) bearing plasmids encoding dentinogenesis-inducing factors (pBMP2/NPs or pDMP1/NPs). The uptake and transfection efficiency of the synthesized NPs on dental pulp stem cells (DPSCs) increased in a concentration- and time-dependent manner, as evaluated qualitatively by confocal laser microscopy and transmission electron microscopy, and quantitatively by flow cytometry, while, in parallel, cell viability decreased. HTFD scaffolds conditioned with the optimal transfectability-to-viability concentration at 4µg Ca/mL of each of the pBMP2/NPs or pDMP1/NPs preserved high levels of cell viability, evidenced by live/dead staining in vitro and caused no adverse reactions after implantation on C57BL6 mice in vivo. HTFD/NPs constructs induced rapid and pronounced odontogenic shift of the DPSCs, as evidenced by relevant gene expression patterns of RunX2, ALP, BGLAP, BMP-2, DMP-1, DSPP by real-time PCR, and acquirement of polarized meta-mitotic phenotype with cellular protrusions entering the dentinal tubules as visualized by scanning electron microscopy. Taken together, HTFD/NPs constitute a promising tool for customized reconstruction of the ortho-dentin/odontoblastic layer barrier and preservation of pulp vitality. STATEMENT OF SIGNIFICANCE: In clinical dentistry, the most common therapeutic approach for the reconstruction of hard dental tissue defects is the replacement by resin-based restorative materials. Even modern bioactive materials focus on reparative dentinogenesis, leading to amorphous dentin-bridge formation in proximity to the pulp. Therefore, the natural microarchitecture of tubular ortho-dentin is not recapitulated, and the sensory and defensive role of odontoblasts is sidelined. This study approaches the reconstruction at the dentin-pulp interface using a construct of human treated dentin (HTFD) scaffold and plasmid-carrying nanoparticles (NPs) encoding dentinogenic factors (DMP-1 or BMP-2) with excellent in vitro and in vivo properties. As a future perspective, the HTFD/NPs constructs could act as bio-fillings for personalized reconstruction of the dentin-pulp interface.

CaCO3 from Anadara granosa shell as reparative dentin inducer in odontoblast pulp cells: In-vivo study.

Anadara granosa (blood clam) shell contained 98.7% of calcium carbonate (CaCO3). This material has bio-properties that able to induced the dentin regeneration. This study is expected to reveal the nuclear factor kappa beta (NF-kB), transforming growth factor beta (TGF-β1), and vascular endothelial growth factor A (VEGF-A) expression in dental pulp after application of CaCO3 from Anadara granosa shell. The thirty Rattus norvegicus strain Wistar used as model. The maxillary first molar was preparation using 0.84mm low-speed diamond bur to made cavity. The cavity then applied glass ionomer cement (as control group) and other group applied CaCO3 from Anadara granosa shell. The teeth in each group were extracted after 1st, 3rd and 7th days of application for immunohistochemistry analysis for NF-kB, TGF-β1, and VEGF-A expression. The NF-kB expression in the group with CaCO3 from Anadara granosa shell lower than control after 1st, 3rd and 7th days (p<0.05). In other hand, the TGF-β1 and VEGF-A expression in the group with CaCO3 from Anadara granosa shell higher than control after 1st, 3rd and 7th days (p<0.05). CaCO3 from Anadara granosa shell able to stimulate the TGF-β1 and VEGF-A and suppress the NF-kB expression in the dental pulp. This material able to develop as dentin-pulp material restoration.

Clinical and Radiographic Evaluation of Combined Acemannan and Periodontal Surgery Induced-Periodontal Regeneration: 5-Year Follow-up Case Report

Background and Objective: Acemannan, a polysaccharide extracted from aloe vera gel, accelerates oral wound healing, reparative dentin formation, and bone formation in extraction sockets. In this report, we evaluated the efficacy of combined acemannan and periodontal surgery on periodontal regeneration in severe periodontitis cases requiring periodontal surgery. Case Presentation and Discussion: Three chronic periodontitis patients with probing pocket depths of at least 6 mm and two- or three-walled vertical bone defects after initial periodontal therapy were included. The patients underwent minimally invasive periodontal surgery with acemannan sponges placed in the defect. Maintenance recall was scheduled every 6 months for 5 years. Clinical and radiographic evaluations were performed to assess the effects of the combined acemannan and periodontal surgery on periodontal regeneration. The patients demonstrated improved clinical parameters and increased radiographic bone fill at the 5-year follow-ups. The percentage bone fill in the three-walled defect, combined two- and three-walled defect, and two-walled defect cases were 70%, 60%, and 20%, respectively. The underlying mechanism of action of acemannan in periodontal regeneration was also discussed. Conclusion: Acemannan sponges can be used as an adjunct to periodontal surgery for periodontal regeneration.

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.

Wnt3a promotes odonto/osteogenic differentiation in vitro and tertiary dentin formation in a rat model.

To investigate the effect of Wnt3a on odonto/osteogenic differentiation of stem cells isolated from human exfoliated deciduous teeth (SHEDs) and reparative dentine formation in a rat model. Stem cells isolated from human exfoliated deciduous teeth were cultured in media with Wnt3a (50-200 ng/ml). Wnt activation was confirmed by β-catenin immunocytochemistry. Colony-forming unit assay (normalized percentage area), osteogenic gene expression analysis by real-time polymerase chain reaction and mineralization assays measured by the absorption at 540 nm were performed. Tertiary dentine formation in vivo was evaluated using 8-week-old, male Wistar rats. Cavities with pinpoint pulp exposure by a sharp instrument were prepared at the mesial surface of the first molars. Teeth were divided into (n=6): (1) distilled water (negative control), (2) phosphate-buffered saline (PBS), (3) lithium chloride in DI (20 μM), and (4) Wnt3a in PBS (200 ng/ml). Collagen sponge was used as a scaffold. The cavity was sealed with glass ionomer restoration. Four weeks later, animals were euthanized by sodium pentobarbital (120 mg/kg body weight). Hard tissue formation was evaluated using micro-computerized tomography. Sixty consecutive slides from the initial plane were analysed and calculated as bone/dentine volume per total tissue volume. Paraffin sections (2μm) were stained with haematoxylin and eosin and Masson's trichrome for morphological evaluation. Data are presented as the mean ± standard error. Mann-Whitney U test was used for two-group comparison. Kruskal Wallis followed by pairwise comparison was employed for three or more group comparisons. Statistical analysis was performed using GraphPad Prism 7. Differences were considered significant at p < .05. Wnt3a decreased SHEDs colony formation and increased OSX, BMP2, and DMP1 expression, corresponding to an increase in mineralization. Additionally, a significant increase in dentine/bone volume per total tissue volume was observed in Wnt3a treated defects. Dentine bridge formation at the exposure sites treated with Wnt3a demonstrated, while fibrous tissues were observed in the control. Wnt3a suppressed proliferation, increased osteogenic differentiation of SHEDs and promotes tertiary dentine formation. Wnt3a could be utilized as biological molecule for vital pulp therapy.

Immunohistochemical localization of CD146 and alpha-smooth muscle actin during dentin formation and regeneration.

Cluster of differentiation 146 (CD146) is known to localize in stem cells and precursor cells of various tissues. In this study, to analyze the function of CD146 in odontoblast differentiation, immunohistochemical localization of CD146 was examined during rat molar tooth development and after cavity preparation. At the cap and bell stages, many CD146-positive cells were visible around the blood vessels in the dental papillae. On Postnatal day 2, osterix-positive odontoblasts were arranged in the dentin sialoprotein (DSP)-positive predentin, and many CD146-positive cells were observed near these odontoblasts with blood vessels. Some perivascular CD146-positive cells overlapped with Smad4-positive cells. However, the immunoreactivity for alpha-smooth muscle actin (α-SMA), one of the markers for undifferentiated cells, was negligible. Furthermore, the number of these cells decreased in the dental pulp on Postnatal day 28. On Day 4 after cavity preparation, Osterix-positive odontoblasts appeared lining the reparative dentin. Most of the blood vessels near the reparative dentin showed immunoreactivities for CD146. Reparative odontoblasts actively formed DSP-positive dentin matrix because these cells were positive for Smad4 and Osterix, but not for α-SMA. After 7 days, the number of CD146-positive cells near blood vessels decreased in the dental pulp beneath the cavity. These results suggest that the CD146 is expressed in the perivascular area of the dental pulp and induces vascularization in the vicinity of dentin formation, and some CD146-positive cells are activated by the bone morphogenetic protein signaling pathway and differentiate into odontoblasts in the early stages of dentin formation and repair.

Substrate stiffness promotes dentinogenesis via LAMB1-FAK-MEK1/2 signaling axis.

In vivo, the principal function of mechanosensitive odontoblasts is to synthesize and secrete the matrix which then calcifies and forms reactive dentin after exposure to appropriate stimuli. This study aims to develop the influence of mechanical factors on dentinogenesis based on odontoblasts, which contribute to reparative dentin formation. We fabricated polydimethylsiloxane with different stiffnesses and seeded 17IIA11 odontoblast-like cells on the substrates in different stiffnesses. Cell morphology was detected by scanning electron microscope, and the mineralization phenotype was detected by alkaline phosphatase staining and alizarin red staining, while expression levels of dentinogenesis-related genes (including Runx2, Osx, and Alp) were assayed by qPCR. To explore mechanism, protein distribution and expression levels were detected by immunofluorescent staining, Western blotting, and immunoprecipitation. In our results, during dentinogenesis, 17IIA11 odontoblast-like cells appeared better extension on stiffer substrates. The binding between LAMB1 and FAK contributed to converting mechanical stimuli into biochemical signaling, thereby controlling mitogen-activated protein kinase kinase 1/2 activity in stiffness-driven dentinogenesis. The present study suggests odontoblast behaviors can be directly regulated by mechanical factors at cell-material interfaces, which offers fundamental mechanism in remodeling cell microenvironment, thereby contributing to physiological phenomena explanation and tissue engineering progress.

The Effect of Hydrogel Hyaluronic Acid on Dentine Sialophosphoprotein Expression of Human Dental Pulp Stem Cells.

Hyaluronic acid (HA) is glycosaminoglycan and one of important factors in extracellular matrix. In an inflamed pulp, when niche biology is conducive, the recruitment of human dental pulp stem cells (hDPSCs) will take place and differentiate into odontoblast like cell, creating reparative dentine and expressing dentine sialophosphoprotein (DSPP). Therefore, the purpose of this study was to analyze the potential of hydrogel HA in various concentration towards hDPSCs differentiation via DSPP expression at day 7 and 14. After hDPSCs incubation reaching 80% confluence, cells were then starved for 24 hours. Then, culture media were supplemented with osteogenic media. hDPSCs planted into 96 well plate and HA 10 μg/mL, 20 μg/mL, and 30 μg/mL were added. DSPP expression was analysed using elisa reader at day 7 and 14, qualitative result was analysed using alizarin red at day 21. Data was analysed using one-way ANOVA. At day 7, there was a statistically significant different potential of HA conditioned media in various concentration (p<0.05) towards hDPSCs differentiation via expression of DSPP with HA 30 μg/mL being the most potential concentration to increase DSPP expression. HA have the potential to increase odontoblast differentiation process via expression of DSPP, with HA 30 μg/mL being the optimum concentration for hDPSCs. (EEJ-2022-12-169).

ASSESSMENT OF BIODENTINE PULPOTOMY AS AN ALTERNATIVE TO CONVENTIONAL ROOT CANAL TREATMENT FOR PERMANENT TEETH IN CHILDREN-AN IN VIVO STUDY

A vital pulp tissue is responsible for supporting the tooth structure through reparative dentin production. Preserving pulp vitality is essential in maintaining vascularization and nutrition to the tooth that eventually will support tooth structure &amp; reduce teeth mortality. Root canal treatment for permanent teeth in children is a complex procedure requiring lengthy appointments and multiple visits and often require a full coverage restoration. on the other hand vital pulpotomy requires shorter appointments and usually accomplished in one visit. Vital pulp therapy can help to retain vital permanent teeth that are able to withstand normal function. Vital pulp therapy (VPT) has been increasingly considered as a minimally invasive approach for the management of teeth with inflamed pulps compared to the conventional approach of root canal treatment. Aim To assess the outcome of Biodentine in complete coronal pulpotomy in permanent teeth with symptomatic reversible pulpitis. Methodology In the present study 30 healthy children of age below 12 years requiring pulpotomy were selected after clinical and radiographic assessment. Pulpotomy technique was carried out after local anaesthesia and rubber dam application. After exposure, pulp was amputated to the level of canal orifices (full pulpotomy) using a sterile spoon excavator and high-speed round bur under water coolant. Hemostasias is achieved by cotton pellet moistened with 3 % NaOCl for 2 minutes and repeat as needed, after haemostasis achieved Biodentine was mixed according to the manufacturer’s instructions and gently placed over the pulp to a thickness of 2-3 mm and after that a permanent restoration was placed. All treated patients were followed up at 3-month,6-month,9-month interval for clinical and radiographic evaluation. Resulting data were tabulated and statistically analyzed. Results. Out of the 30 study samples,2 patient cases revealed abnormal clinical and radiographical changes at the end of 3 months and one more patient reported with pain and tenderness to percussion at the end of 6 months. At the end of 9 months follow up, clinical and radiographical success rate was 90.0% respectively. Conclusion Biodentine as a pulpotomy agent has a high successful rate and fulfilled all requirements of an ideal pulpotomy medicament.

Enhanced effects of antagomiR-3074-3p-conjugated PEI-AuNPs on the odontogenic differentiation by targeting FKBP9

The odontogenic differentiation of dental pulp stem cells (DPSCs), which is vital for tooth regeneration, was regulated by various functional molecules. In recent years, a growing body of research has shown that miRNAs play a crucial role in the odontogenic differentiation of human dental pulp stem cells (hDPSCs). However, the mechanisms by which miRNAs regulated odontogenic differentiation of hDPSCs remained unclear, and the application of miRNAs in reparative dentin formation in vivo was also rare. In this study, we first discovered that miR-3074-3p had an inhibitory effect on odontogenic differentiation of hDPSCs and antagomiR-3074-3p-conjugated PEI-AuNPs effectively promoted odontogenic differentiation of hDPSCs in vitro. AntagomiR-3074-3p-conjugated PEI-AuNPs was further applied to the rat pulp-capping model and showed the increased formation of restorative dentin. In addition, the results of lentivirus transfection in vitro suggested that FKBP9 acted as the key target of miR-3074-3p in regulating the odontogenic differentiation of hDPSCs. These findings might provide a new strategy and candidate target for dentin restoration and tooth regeneration.

The Influence of Pulp Capping Procedures on the Long-Term Prognosis and Survival of Teeth

Pulp capping is a dental procedure to treat deep caries and preserve the vitality of affected teeth. It involves placing a biocompatible material directly on the exposed pulp (direct pulp capping) or using a medicated liner to protect the pulp (indirect pulp capping). The choice of biomaterials, such as calcium hydroxide or mineral trioxide aggregate (MTA), significantly impacts the success of pulp capping. Patient-related factors, including age, oral hygiene, and systemic health conditions, influence treatment outcomes. Younger patients generally have better results due to higher pulp vitality and regenerative capacity. Maintaining good oral hygiene and managing systemic health conditions are crucial for long-term success. Complications and treatment failure can occur in pulp capping, depending on factors like cavity size, infection, and initial pulp condition. Early detection and intervention are important to prevent further issues. Success in pulp capping is assessed based on factors like continuous root development, preserved pulp vitality, minimal inflammation, and formation of reparative dentin. Success rates may decrease over time, and further research is needed to understand why. Understanding procedural techniques, biomaterial selection, patient-related factors, and complications are vital for informed decision-making, optimizing outcomes, and increasing the longevity of pulp-capped teeth

Genome wide analysis of dexamethasone stimulated mineralization in human dental pulp cells by RNA sequencing.

Human dental pulp cells (hDPCs) contain mesenchymal stem cells and are therefore indispensible for reparative dentin formation. Here, we present a pilot study of transcriptomic profiles of mineralized hDPCs isolated from sound human maxillary third molars. We observed altered gene expression of hDPCs between control (dexamethasone free) and experimental (dexamethasone 1nm) groups. Differential expression analysis revealed up-regulation of several inflammation and mineralization-related genes in the experimental group. After a Gene Ontology analysis for predicting genes involved in biological process, cellular component and molecular function, we found enrichment of genes related to protein binding. Based on the results of Kyoto Encylopedia of Genes and Genomes pathway analysis, it is suggested up-regulated genes in mineralized hDPCs were mostly enriched in the mitogen-activated protein kinase (MAPK) signaling pathway, fluid shear stress and the atherosclerosis signaling pathway, etc. Importantly, Gene Set Enrichment Analysis revealed dexamethasone was positively related to the Janus kinase/signal transducer and activator of transcription, MAPK and Notch signaling pathway. Moreover, it was suggested that dexamethasone regulates signaling pathway in pluripotency of stem cells. Collectively, our work highlights transcriptome level gene regulation and intercellular interactions in mineralized hDPCs. The database produced in the present study paves the way for further investigations looking to explore genes that are involved in dental pulp cells mineralization.

Temporospatial Expression of Neuropeptide Substance P in Dental Pulp Stem Cells During Odontoblastic Differentiation in Vitro and Reparative Dentinogenesis in Vivo

IntroductionSubstance P (SP) is a neuropeptide released from the nervous fibers in response to injury. In addition to its association with pain and reactions to anxiety and stress, SP exerts various physiological functions by binding to the neurokinin-1 receptor (NK1R). However, the expression and role of SP in reparative dentinogenesis remain elusive. Here, we explored whether SP is involved in odontoblastic differentiation during reparative dentinogenesis. MethodsDental pulp stem cells (DPSCs) were isolated from healthy human dental pulp tissues and subjected to odontoblastic differentiation. The expression of SP and NK1R during odontoblastic differentiation was investigated in vitro. The effects of SP on odontoblastic differentiation of DPSCs were evaluated using alizarin red staining, alkaline phosphatase staining, and real-time polymerase chain reaction. After direct pulp capping with mineral trioxide aggregate, the expression of SP and NK1R during reparative dentin formation in rats were identified using histological and immunohistochemical staining. ResultsSP and NK1R expression increased during the odontoblastic differentiation of DPSCs. SP translocated to the nucleus when DPSCs were exposed to differentiation medium. NK1R was always present in the nuclei of DPSCs and odontoblast-like cells. Additionally, we discovered that 10−8 M SP marginally enhanced the odontoblastic differentiation of DPSCs, and that these effects could be impaired by the NK1R antagonist. Furthermore, SP and NK1R were expressed in odontoblast-like and dental pulp cells during reparative dentin formation in vivo. ConclusionsSP contributes to odontoblastic differentiation during reparative dentin formation by binding to the NK1R.

Combination of Dental Capping Agents With LowLevel Laser Therapy Increases the Cell Viability Percent of Stem Cells From Apical Papilla (SCAPs).

Introduction: Dental pulp capping is a technique that is highly applicable in dental restorations. In this technique, a material is directly placed over the exposed pulp tissue, which promotes pulp healing and generates reparative dentin. Herein, we aimed to investigate the combined effect of different pulp capping agents, including mineral trioxide aggregate (MTA), Emdogain (EMD), calcium-enriched mixture (CEM), and low-level laser therapy (LLLT), on enhancing viability and proliferation of stem cells from apical papilla (SCAPs). Methods: SCAPs were isolated from two immature third molar teeth through collagenase type I enzymatic activity. Isolated stem cells were then cultured with DMEM and α-MEM media enriched with 15% and 10% FBS respectively. After reaching 70%-80% confluency, the cells were seeded in a 96-well plate. Cell viability percent was assessed using the MTT assay after treatment with MTA, EMD, CEM and LLLT (λ=630 nm, 5 mW, 4 J/cm2 ) alone and in combination for 24, 48 and 168 hours. Results: Combination of MTA, CEM, EMD and LLLT resulted in significantly increased SCAPs viability as compared with other treatment groups. Increased SCAPs proliferation and viability were also observed in groups treated with the combination of MTA and CEM with EMD. However, the SCAPs survival rate in all defined time spans was reduced after treatment with MTA and CEM alone. Conclusion: LLLT can be a stimulator of SCAPs cell viability when applied in combination with dental capping agents such as MTA, EMD and CEM, providing a therapeutic option for stem cell-based dental regeneration.

Caffeic Acid Phenethyl Ester Induces Vascular Endothelial Growth Factor Production and Inhibits CXCL10 Production in Human Dental Pulp Cells.

The survival rate of root non-vital teeth is lower than that of vital teeth. Therefore, to preserve the dental pulp is very important. The vascular endothelial growth factor (VEGF) is the most potent angiogenic factor involved in the vitality of dental pulp including reparative dentin formation. Caffeic acid phenethyl ester (CAPE) is a physiologically active substance of propolis and has some bioactivities such as anti-inflammatory effects. However, there are no reports on the effects of CAPE on dental pulp inflammation. In this study, we investigated the effects of CAPE on VEGF and inflammatory cytokine production in human dental pulp cells (HDPCs) to apply CAPE to an ideal dental pulp protective agent. We found that CAPE induced VEGF production from HDPCs. Moreover, CAPE induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases (ERK), and stress-activated protein kinase/c-Jun N-terminal kinase (SAP/JNK) in HDPCs. Furthermore, CAPE inhibited C-X-C motif chemokine ligand 10 (CXCL10) production in Pam3CSK4- and tumor necrosis factor-alpha (TNF-α)-stimulated HDPCs. In conclusion, these results suggest that CAPE might be useful as a novel biological material for vital pulp therapy by exerting the effects of VEGF production and anti-inflammatory activities.

Efficacy of Calcium Hydroxide and Mineral Trioxide Aggregate in the Formation of Dentin Bridge – A Randomized Controlled Trial

OBJECTIVE: This study is to compare the dentin bridge thickness achieved using calcium hydroxide and MTA using radiographs. METHODOLOGY: Single blinded randomized controlled trial conducted in the Operative Dentistry department at Fatima Jinnah Dental College and Hospital, Karachi. A total of 100 premolar and molar teeth with class I and II cavities were included in this study. The study participants were assigned into two groups, A and B of 50 participants each. Under local anesthesia, Group A was indirectly pulp capped with Calcium hydroxide (Dycal) and Group B received Mineral Trioxide Aggregate as an indirect pulp capping material. Both groups were then restored with Glass Ionomer Cement. Radiographic follow up was carried out at three and six months to determine mean dentin thickness of reparative dentin bridge. RESULTS: Statistical analysis was performed using SPSS v 23. Independent Sample t-test was applied to evaluate the formation of dentin bridge formation using Ca(OH)2 and MTA at 3 months &amp; 6 months, the outcomes were highly significant (p-value&lt;0.001). Paired sample t-test was applied to evaluate the difference in dentin bridge formation at three months and 6 months, the results were highly significant (p-value &lt;0.001). CONCLUSION: Statistically significant difference was observed in the dentin thickness of reparative dentin bridge amongst the two groups after three months and six months. A greater success rate was noted in the MTA group as compared to the Ca(OH)2 group after 6 months. KEYWORDS: Dentin bridge, Endodontic treatment, Indirect pulp capping, Reparative dentin, randomized controlled trial.

Involvement of M1/M2 Macrophage Polarization in Reparative Dentin Formation.

In cases in which dental pulp tissue is accidentally exposed, direct pulp capping is often performed to induce reparative dentin formation. Although macrophages are essential for the inflammatory response and tissue repair, the emergence pattern and the role of macrophages in dental pulp tissue have not been clarified. Here, we investigated the emergence of M1/M2 macrophages in dental pulp tissue after a direct pulp capping and the effects of M2 macrophages on odontoblastic differentiation of the dental pulp stem cell (DPSC) clones. The emergence of macrophages in dental pulp tissue was investigated using a rat direct pulp capping model. Alizarin Red S staining and quantitative RT-PCR was performed to examine the effect of M2 macrophages on the mineralization and odontoblastic differentiation of DPSC clones. Immunohistochemical staining revealed that M1 macrophages were detected in dental pulp tissue after treatment and increased in number at three days after treatment. However, M2 macrophages gradually increased in number in dental pulp tissue after treatment, with the highest level recorded at seven days post-operation. Additionally, conditioned medium from M2 macrophages induced odontoblast-like differentiation of DPSC clones. These results suggest that macrophages play a role in the inflammatory response and reparative dentin formation after dental pulp exposure.

Effects of Dentin Phosphophoryn-Derived RGD Peptides on the Differentiation and Mineralization of Human Dental Pulp Stem Cells In Vitro

The purposes of this study were to investigate the in vitro effects of arginine-glycine-aspertic acid (RGD) peptides derived from human dentin phosphophoryn (DPP) on human dental pulp stem cell-proliferation, differentiation and mineralization, and to explore the mechanism of the peptides' function. The 1 M concentration of soluble DPP-derived RGD peptides, RGD-1, RGD-2 and RGD-3 were coated onto non-tissue-culture polystyrene plates, and human dental pulp stem cells (hDPSCs) were cultured on them to examine the effects of the peptides on hDPSCs. In addition, 1 M arginine-alanine-aspertic acid (RAD) peptides were used as the control. Cell proliferation of hDPSCs was promoted by all three RGD peptides. All three RGD peptides had significantly higher alkaline phosphatase (ALP) activity compared to the control. RGD-3 induced the highest ALP activity compared to the control. RGD-3 also significantly promoted the mRNA expression of the following genes: 1.69-fold in dentine matrix protein-1 (DMP-1), 1.99-fold in dentine sialophosphoprotein (DSPP), 1.51-fold in ALP, and 2.31-fold in bone sialoprotein (BSP), as compared to the control group. Mineralization of hDPSCs was accelerated by all three RGD peptides, RGD-3 in particular. The MAPK p38 inhibitor SB202190 inhibited the effect of RGD-3 to a level comparable to the control, observed in both ALP activity assay and Arizarin red S (ARS) staining. It suggests that the p38 pathway may be responsible for eliciting the differentiation and mineralization effects of DPP-derived RGD peptides in the hDPSCs. The mRNA expression levels of the integrins ITGA1-5, ITGA7, ITGB1 and ITGB3 were significantly upregulated. Among them, expression of ITGA5 was promoted 1.9-fold, ITGA7 1.58-fold, ITGB1 1.75-fold and ITGB3 1.9-fold compared to the control. It suggests the possible involvement of these integrin channels in different subunit combinations facilitating signal transduction for differentiation of hDPSCs into odontoblasts. As conclusions, human DPP-derived RGD peptides RGD-1, RGD-2 and RGD-3 promoted the proliferation, differentiation and mineralization of hDPSCs in vitro. Among the three peptides, RGD-3 had the most significant effects. It is also suggested that RGD-3 binds to integrin receptors on the surface of hDPSCs and regulates the odontogenic gene expression and differentiation via activation of p38 of MAPK pathway. DPP-derived RGD-3 may be a promising choice in the formulation of a novel material for vital pulp therapy to induce dental pulp stem cells into odontoblasts and form reparative dentin on the exposed pulp tissue.

Role of osteopontin in the process of pulpal healing following tooth replantation in mice.

IntroductionThe role of osteopontin (OPN) following severe injury remains to be elucidated, especially its relationship with type I collagen (encoded by the Col1a1 gene) secretion by newly-differentiated odontoblast-like cells (OBLCs). In this study, we examined the role of OPN in the process of reparative dentin formation with a focus on reinnervation and revascularization after tooth replantation in Opn knockout (KO) and wild-type (WT) mice.MethodsMaxillary first molars of 2- and 3-week-old-Opn KO and WT mice (Opn KO 2W, Opn KO 3W, WT 2W, and WT 3W groups) were replanted, followed by fixation 3–56 days after operation. Following micro-computed tomography analysis, the decalcified samples were processed for immunohistochemistry for Ki67, Nestin, PGP 9.5, and CD31 and in situ hybridization for Col1a1.ResultsAn intense inflammatory reaction occurred to disrupt pulpal healing in the replanted teeth of the Opn KO 3W group, whereas dental pulp achieved healing in the Opn KO 2W and WT groups. The tertiary dentin in the Opn KO 3W group was significantly decreased in area compared with the Opn KO 2W and WT groups, with a significantly low percentage of Nestin-positive, newly-differentiated OBLCs during postoperative days 7–14. In the Opn KO 3W group, the blood vessels were significantly decreased in area and pulp healing was disturbed with a failure of pulpal revascularization and reinnervation.ConclusionsOPN is necessary for proper reinnervation and revascularization to deposit reparative dentin following severe injury within the dental pulp of erupted teeth with advanced root development.