Novel Bioactive Adhesive Monomer CMET Promotes Odontogenic Differentiation and Dentin Regeneration.

This study aimed to evaluate the in vitro effect of the novel bioactive adhesive monomer CMET, a calcium salt of 4-methacryloxyethyl trimellitate acid (4-MET), on human dental pulp stem cells (hDPSCs) and its capacity to induce tertiary dentin formation in a rat pulp injury model. Aqueous solutions of four tested materials [4-MET, CMET, Ca(OH)2, and mineral trioxide aggregate (MTA)] were added to the culture medium upon confluence, and solvent (dH2O) was used as a control. Cell proliferation was assessed using the Cell Counting Kit-8 assay, and cell differentiation was evaluated by real-time quantitative reverse transcription-polymerase chain reaction. The mineralization-inducing capacity was evaluated using alizarin red S staining and an alkaline phosphatase activity assay. For an in vivo experiment, a mechanical pulp exposure model was prepared on Wistar rats; damaged pulp was capped with Ca(OH)2 or CMET. Cavities were sealed with composite resin, and specimens were assessed after 14 and 28 days. The in vitro results showed that CMET exhibited the lowest cytotoxicity and highest odontogenic differentiation capacity among all tested materials. The favorable outcome on cell mineralization after treatment with CMET involved p38 and c-Jun N-terminal kinases signaling. The nuclear factor kappa B pathway was involved in the CMET-induced mRNA expression of odontogenic markers. Similar to Ca(OH)2, CMET produced a continuous hard tissue bridge at the pulp exposure site, but treatment with only CMET produced a regular dentinal tubule pattern. The findings suggest that (1) the evaluated novel bioactive adhesive monomer provides favorable biocompatibility and odontogenic induction capacity and that (2) CMET might be a very promising adjunctive for pulp-capping materials.

Compromised dental cells viability following teeth-whitening exposure.

This study aimed to assess the viability of dental cells following time-dependent carbamide peroxide teeth-whitening treatments using an in-vitro dentin perfusion assay model. 30 teeth were exposed to 5% or 16% CP gel (4 h daily) for 2-weeks. The enamel organic content was measured with thermogravimetry. The time-dependent viability of human dental pulp stem cells (HDPSCs) and gingival fibroblast cells (HGFCs) following either indirect exposure to 3 commercially available concentrations of CP gel using an in-vitro dentin perfusion assay or direct exposure to 5% H2O2 were investigated by evaluating change in cell morphology and by hemocytometry. The 5% and 16% CP produced a significantly lower (p < 0.001) enamel protein content (by weight) when compared to the control. The organic content in enamel varied accordingly to the CP treatment: for the 16% and 5% CP treatment groups, a variation of 4.0% and 5.4%, respectively, was observed with no significant difference. The cell viability of HDPSCs decreased exponentially over time for all groups. Within the limitation of this in-vitro study, we conclude that even low concentrations of H2O2 and CP result in a deleterious change in enamel protein content and compromise the viability of HGFCs and HDPSCs. These effects should be observed in-vivo.

Xenogeneic dentin matrix as a scaffold for biomineralization and induced odontogenesis.

Commonly recognized mechanisms of the xenogeneic-extracellular matrix-based regenerative medicine include timely degradation, release of bioactive molecules, induced differentiation of stem cells, and well-controlled inflammation. This process is most feasible for stromal tissue reconstruction, yet unsuitable for non-degradable scaffold and prefabricated-shaped tissue regeneration, like odontogenesis. Treated dentin matrix (TDM) has been identified as a bioactive scaffold for dentin regeneration. This study explored xenogeneic porcine TDM (pTDM) for induced odontogenesis. The biological characteristics of pTDM were compared with human TDM (hTDM). To investigate its bioinductive capacities on allogeneic dental follicle cells (DFCs) in the inflammation microenvironment, pTDM populated with human DFCs were co-cultured with human peripheral blood mononuclear cells (hPBMCs), and pTDM populated with rat DFCs were transplanted into rat subcutaneous model. The results showed pTDM possessed similar mineral phases and bioactive molecules with hTDM. hDFCs, under the induction of pTDM and hTDM, expressed similar col-I, osteopontin and alkaline phosphatase (ALP) (all expressed by odontoblasts). Whereas, the expression of col-I, dentin matrix protein-1 (DMP-1) and bone sialoprotein (BSP) were down-regulated when cocultured with hPBMCs. The xenogeneic implants inevitably initiated Th1 inflammation (up-regulated CD8, TNF-α, IL-1ß, etc)in vivo. However, the biomineralization of pre-dentin and cementum were still processed, and collagen fibrils, odontoblast-like cells, fibroblasts contributed to odontogenesis. Although partially absorbed at 3 weeks, the implants were positively expressed odontogenesis-related-proteins like col-I and DMP-1. Taken together, xenogeneic TDM conserved ultrastructure and molecules for introducing allogeneic DFCs to odontogenic differentiation, and promoting odontogenesis and biomineralizationin vivo. Yet effective immunomodulation methods warrant further explorations.

mTORC1 promotes mineralization via p53 pathway.

The objectives of our study were to investigate the roles of mTORC1 in odontoblast proliferation and mineralization and to determine the mechanism by which mTORC1 regulates odontoblast mineralization. In vitro, MDPC23 cells were treated with rapamycin (10 nmol/L) and transfected with a lentivirus for short hairpin (shRNA)-mediated silencing of the tuberous sclerosis complex (shTSC1) to inhibit and activate mTORC1, respectively. CCK8 assays, flow cytometry, Alizarin red S staining, ALP staining, qRT-PCR, and western blot analysis were performed. TSC1-conditional knockout (DMP1-Cre+ ; TSC1f/f , hereafter CKO) mice and littermate control (DMP1-Cre- ; TSC1f/f , hereafter WT) mice were generated. H&E staining, immunofluorescence, and micro-CT analysis were performed. Transcriptome sequencing analysis was used to screen the mechanism of this process. mTORC1 inactivation decreased the cell proliferation. The qRT-PCR and western blot results showed that mineralization-related genes and proteins were downregulated in mTORC1-inactivated cells. Moreover, mTORC1 overactivation promoted cell proliferation and mineralization-related gene and protein expression. In vivo, the micro-CT results showed that DV/TV and dentin thickness were higher in CKO mice than in controls and H&E staining showed the same results. Mineralization-related proteins expression was upregulated. Transcriptome sequencing analysis revealed that p53 pathway-associated genes were differentially expressed in TSC1-deficient cells. By inhibiting p53 alone or both mTORC1 and p53 with rapamycin and a p53 inhibitor, we elucidated that p53 acts downstream of mTORC1 and that mTORC1 thereby promotes odontoblast mineralization. Taken together, our findings demonstrate that the role of mTORC1 in odontoblast proliferation and mineralization, and confirm that mTORC1 upregulates odontoblast mineralization via the p53 pathway.

RGD- and VEGF-Mimetic Peptide Epitope-Functionalized Self-Assembling Peptide Hydrogels Promote Dentin-Pulp Complex Regeneration.

INTRODUCTION: Cell-based tissue engineering is a promising method for dentin-pulp complex (DPC) regeneration. The challenges associated with DPC regeneration include the generation of a suitable microenvironment that facilitates the complete odontogenic differentiation of dental pulp stem cells (DPSCs) and the rapid induction of angiogenesis. Thus, the survival and subsequent differentiation of DPSCs are limited. Extracellular matrix (ECM)-like biomimetic hydrogels composed of self-assembling peptides (SAPs) were developed to provide an appropriate microenvironment for DPSCs. For functional DPC regeneration, the most important considerations are to provide an environment that promotes the adequate attachment of DPSCs and rapid vascularization of the regenerating pulp. Morphogenic signals in the form of growth factors (GFs) have been incorporated into SAPs to promote productive DPSC behaviors. However, the use of GFs has several drawbacks. We envision using a scaffold with SAPs coupled with long-term factors to increase DPSC attachment and vascularization as a method to address this challenge. METHODS: In this study, we developed synthetic material for an SAP-based scaffold with RGD- and vascular endothelial growth factor (VEGF)-mimetic peptide epitopes with the dual functions of dentin and pulp regeneration. DPSCs and human umbilical vein endothelial cells (HUVECs) were used to evaluate the biological effects of SAP-based scaffolds. Furthermore, the pulpotomized molar rat model was employed to test the reparative and regenerative effects of SAP-based scaffolds. RESULTS: This scaffold simultaneously presented RGD- and VEGF-mimetic peptide epitopes and provided a 3D microenvironment for DPSCs. DPSCs grown on this composite scaffold exhibited significantly improved survival and angiogenic and odontogenic differentiation in the multifunctionalized group in vitro. Histological and functional evaluations of a partially pulpotomized rat model revealed that the multifunctionalized scaffold was superior to other options with respect to stimulating pulp recovery and dentin regeneration in vivo. CONCLUSION: Based on our data obtained with the functionalized SAP scaffold, a 3D microenvironment that supports stem cell adhesion and angiogenesis was generated that has great potential for dental pulp tissue engineering and regeneration.

Drug Repurposing in Dentistry; towards Application of Small Molecules in Dentin Repair.

One of the main goals of dentistry is the natural preservation of the tooth structure following damage. This is particularly implicated in deep dental cavities affecting dentin and pulp, where odontoblast survival is jeopardized. This activates pulp stem cells and differentiation of new odontoblast-like cells, accompanied by increased Wnt signaling. Our group has shown that delivery of small molecule inhibitors of GSK3 stimulates Wnt/ß-catenin signaling in the tooth cavity with pulp exposure and results in effective promotion of dentin repair. Small molecules are a good therapeutic option due to their ability to pass across cell membranes and reach target. Here, we investigate a range of non-GSK3 target small molecules that are currently used for treatment of various medical conditions based on other kinase inhibitory properties. We analyzed the ability of these drugs to stimulate Wnt signaling activity by off-target inhibition of GSK3. Our results show that a c-Met inhibitor, has the ability to stimulate Wnt/ß-catenin pathway in dental pulp cells in vitro at low concentrations. This work is an example of drug repurposing for dentistry and suggests a candidate drug to be tested in vivo for natural dentin repair. This approach bypasses the high level of economical and time investment that are usually required in novel drug discoveries.

Near-infrared NIR irradiation and sodium hypochlorite: An efficacious association to counteract the Enterococcus faecalis biofilm in endodontic infections.

New strategies are necessary for the prevention of endodontic infections caused by Enterococcus faecalis, a common resistant pathogen and biofilm producer. Aim of the present study was to compare the effects of Near-Infrared (NIR) Light-Emitting Diode (LED) irradiation and different concentrations of sodium hypochlorite (NaOCl) alone or combined to each other on the E. faecalis biofilm, on artificial and human dentin surfaces. E. faecalis ATCC 29212 preformed biofilms, on polystyrene wells and on dentin discs, were treated with 880 nm NIR irradiation and NaOCl at 4%, 2.5%, 1% and 0.5% alone and combined to each other (NIR irradiation plus NaOCl 1% or 0.5%) at 5 and 10 min. Treated biofilms were compared to the controls for (i) biofilm biomass evaluation, (ii) CFU count for the quantification of cultivable cells and (iii) cells viability. All the detected experimental conditions displayed a significant reduction of biofilm biomass (p < 0.001) and CFUs/mL (p < 0.01) in respect to the controls on both tested surfaces. The effects on the E. faecalis biomass, colony count and cell viability were not time-dependent except for NaOCl 2.5% and 1% in the biofilm biomass reductions on human dentin discs. NIR-LED irradiation alone showed a reduction of E. faecalis aggregates without interfering with cell viability whereas NaOCl alone expressed a killing effect in a concentration dependent way. The combination of NIR-LED irradiation with NaOCl 1% and 0.5% displayed a double effect of cluster disaggregation and cell killing. In particular, NIR-LED irradiation combined with NaOCl 0.5% displayed an anti-biofilm activity major than those expressed by NaOCl 0.5% alone (p = 0.001) with a reduction of biomass 93% vs 71% and 97% vs 25% after 10 min, on polystyrene wells and human dentin discs, respectively. The innovative use of NIR-LED irradiation combined at short times with low concentration of NaOCl (1% and 0.5%) is capable to reach a significant effect on E. faecalis biofilm, especially on human dentin discs.

Microbiota-Derived Short-Chain Fatty Acids Promote BMP Signaling by Inhibiting Histone Deacetylation and Contribute to Dentinogenic Differentiation in Murine Incisor Regeneration.

Microbiota and their metabolites short-chain fatty acids (SCFAs) have important roles in regulating tissue regeneration and mesenchymal stem cell (MSC) differentiation. In this study, we explored the potential effects of SCFAs on murine incisor regeneration and dental MSCs. We observed that SCFA deficiency induced by depletion of microbiota through antibiotic treatment led to lower renewal rate and delayed dentinogenesis in mice incisors. Supplementation with SCFAs in drinking water during antibiotic treatment can rescue the renewal rate and dentinogenesis effectively. In vitro, stimulation with SCFAs could promote differentiation of dental MSCs to odontoblasts. We further found that SCFAs could contribute to dentinogenic differentiation of dental MSCs by increasing bone morphogenetic protein (BMP) signal activation. SCFAs could inhibit deacetylation and increase BMP7 transcription of dental MSCs, which promoted BMP signaling. Our results suggested that SCFAs were required for incisor regeneration as well as differentiation of dental MSCs. Microbiota and their metabolites should be concerned as important factors in the tissue renewal and regeneration.

Current evidence of tissue engineering for dentine regeneration in animal models: a systematic review.

Aim: The aim of this study is to verify the type of scaffold effect on tissue engineering for dentine regeneration in animal models. Materials & methods: Strategic searches were conducted through MEDLINE/PubMed, Web of Science and Scopus databases. The studies were included with the following eligibility criteria: studies evaluating dentine regeneration, and being an in vivo study. Results: From 1392 identified potentially relevant studies, 15 fulfilled the eligibility criteria. All studies described characteristics of neoformed dentine, being that the most reported reparative dentine formation. Most of included studies presented moderate risk of bias. Conclusion: Up to date scientific evidence shows a positive trend to dentine regeneration when considering tissue engineering in animal models, regardless the type of scaffolds used.

The adverse effects of radiotherapy on the structure of dental hard tissues and longevity of dental restoration.

Purpose: The main goal of this study was to evaluate the impact of different ionizing radiation doses on the mineral (carbonate/phosphate ratio, crystallinity index [CI]) and organic (amide III/phosphate, amide I sub-band ratios) structures, as well as the microhardness, of enamel and dentin, along with their influence on the bonding strength stability of the etch-and-rinse (ER) and self-etch (SE) dental adhesive strategies.Materials and methods: Enamel and dentin human tissue specimens were irradiated (with 0, 20, 40, and 70 Gy radiation doses, respectively) and sectioned to perform an attenuated total reflection-Fourier transform IR spectroscopy assay (ATR-FTIR) and the Vickers microhardness (VHN) test to conduct a biochemical and biomechanical evaluation of the tissues. Regarding the adhesive properties, restored enamel and dentin specimens exposed to the same radiation doses were submitted to microshear bond strength (µSBS) tests for enamel in immediate time (IM) and to microtensile bond strength (µTBS) tests after for IM and 12-month (12 M) period of time, Mann-Whitney U tests were implemented, using the ATR-FTIR data for significant differences (α < 0.05), and three- and two-way analyses of variance, along with post-testing, were performed on the µTBS and µSBS data (MPa), respectively (Tukey post hoc test at α = 0.05).Results: The ATR-FTIR results showed a significant decrease (p < .05) in the amide III/phosphate ratio after 20 Gy for the enamel and after 40 Gy for the dentin. The CI was significantly reduced for both tissues after a dose of 70 Gy (p < .05). All radiation doses significantly decreased microhardness values, relative to the respective enamel and dentin controls (p < .05). In both tissues and adhesive strategies, the decrease in bond strength was influenced by ionizing radiation starting from 40 Gy. The ER strategy showed high percentages of enamel cohesive failure. In general, ER in both tissues showed greater and more stable bond strength than SE against increased radiation doses and long term.Conclusions: It is possible to conclude that structural alterations of enamel and dentin are generated by all radiation doses, decreasing the microhardness of dental hard tissues and influencing bond strength over time, starting at 40 Gy radiation dose. The etch-and-rinse strategy demonstrates better adhesive performance but generates cohesive fractures in the enamel.

Regional contribution of proteoglycans to the fracture toughness of the dentin extracellular matrix.

This study investigated the contribution of small leucine rich proteoglycans (SLRPs) to the fracture toughness of the dentin extracellular matrix (ECM) by enzymatically-assisted selective removal of glycosaminoglycan chains (GAGs) and proteoglycans (PGs) core protein. We adapted the Mode III trouser tear test to evaluate the energy required to tear the dentin ECM. Trouser-shaped dentin specimens from crown and root were demineralized. Depletion of GAGs and PGs followed enzymatic digestion using chondroitinase ABC (c-ABC) and matrix metalloproteinase 3 (MMP-3), respectively. The legs from specimen were stretched under tensile force and the load at tear propagation was determined to calculate the tear energy (T, kJ/m2). SLRPs decorin and biglycan were visualized by immunohistochemistry and ECM tear pattern was analyzed in SEM. Results showed T of crown ECM was not affected by PGs/GAGs depletion (p = 0.799), whereas the removal of PGs significantly reduced T in root dentin ECM (p = 0.001). Root dentin ECM exhibited higher T than crown (p < 0.03), however no regional difference are present after PG depletion (p = 0.480). Immunohistochemistry confirmed removal of GAGs and PGs. SEM images showed structural modifications after PGs/GAGs removal such as enlargement of dentinal tubules, increased interfibrillar spaces and presence of untwisted fibrils with increased diameter. Findings indicate that the capacity of the PGs to unfold and untwist contribute to the dentin ECM resistance to tear, possibly influencing crack growth propagation. The regional differences are likely an evolutionary design to increase tooth survival, that undergoes repetitive mechanical loading and load stress dissipation over a lifetime of an individual.

Histological Review of Demineralized Dentin Matrix as a Carrier of rhBMP-2.

In 2007, recombinant human bone morphogenetic protein-2 (rhBMP-2) was approved for use in humans at a concentration of 1.5 mg/mL with absorbable collagen sponges as an alternative to autogenous bone grafts for alveolar ridge augmentation, defects associated with extraction sockets, and sinus augmentation. However, the use of supraphysiological doses and the insufficient retention of rhBMP-2, when delivered through collagen sponge, result in dose-dependent side effects related to off-label use. Demineralized dentin matrix (DDM), an osteoinducing bone substrate, has been used as an rhBMP-2 carrier since 1998. In addition, DDM has both microparticle and nanoparticle structures, which do not undergo remodeling, unlike bone. In vitro, DDM is a suitable carrier for BMP-2, with the continued release over 30 days at concentrations sufficient to stimulate osteogenic differentiation. In this review, we discuss the histological outcomes of DDM loaded with rhBMP-2 to highlight the biological functions of exogenous rhBMP-2 associated with the DDM carrier in clinical applications in implant dentistry. Impact Statement Demineralized dentin matrix (DDM) has been used as an recombinant human bone morphogenetic protein (rhBMP-2) carrier and osteo-inducing bone substrate to facilitate continued release and stimulate osteogenic differentiation. In this review, we discuss the histological outcomes of DDM loaded with rhBMP-2 in order to highlight the biological functions of exogenous rhBMP-2 associated with the DDM carrier in clinical applications in implant dentistry.

Histological and developmental insights into the herbivorous dentition of tapinocephalid therapsids.

Tapinocephalids were one of the earliest therapsid clades to evolve herbivory. In acquiring derived tooth-to-tooth occlusion by means of an exaggerated heel and talon crown morphology, members of this family have long been considered herbivorous, yet little work has been done to describe their dentition. Given the early occurrence of this clade and their acquisition of a dentition with several derived features, tapinocephalids serve as an important clade in understanding adaptations to herbivory as well as macroevolutionary patterns of dental trait acquisition. Here we describe the histology of tapinocephalid jaws and incisors to assess adaptations to herbivory. Our results yield new dental characters for tapinocephalids including a peculiar enamel structure and reduced enamel deposition on the occlusal surface. These traits are convergent with other specialized herbivorous dentitions like those found in ornithischian dinosaurs and ungulates. Furthermore, these results demonstrate that while acquiring some specializations, tapinocephalids also retained plesiomorphic traits like alternate, continuous replacement. We interpret these findings as an example of how different combinations of traits can facilitate a derived and specialized dentition and then discuss their implications in the acquisition of a mammal-like dentition.

Laser-assisted biomineralization on human dentin for tooth surface functionalization.

A technique for tooth surface modification with biocompatible calcium phosphate (CaP) has huge potential in dental applications. Recently, we achieved a facile and area-specific CaP coating on artificial materials by a laser-assisted biomimetic process (LAB process), which consists of pulsed laser irradiation in a supersaturated CaP solution. In this study, we induced the rapid biomineralization on the surface of human dentin by using the LAB process. A human dentin substrate was immersed in a supersaturated CaP solution, then its surface was irradiated with weak pulsed laser light for 30 min (LAB process). Ultrastructural analyses revealed that the pristine substrate had a demineralized collagenous layer on its surface due to the previous EDTA surface cleaning. After the LAB process, this collagenous layer disappeared and was replaced with a submicron-thick hydroxyapatite layer. We believe that the laser irradiation induced pseudo-biomineralization through the laser ablation of the collagenous layer, followed by CaP nucleation and growth at the dentin-liquid interface. The mineralized layer on the dentin substrate consisted of needle-like hydroxyapatite nanocrystals, whose c-axes were weakly oriented along the direction perpendicular to the substrate surface. This LAB process would offer a new tool enabling tooth surface modification and functionalization through the in situ pseudo-biomineralization.

Dentin tubule orientation determines odontoblastic differentiation in vitro: A morphological study.

Odontoblasts are post-mitotic cells responsible for maintenance of the dentin, and are therefore important for dental health. In some cases, irreversible pulpitis leads to necrosis and consequently death of odontoblasts. Regenerative endodontics (RE) uses the concept of tissue engineering to restore the root canals to a healthy state, allowing for continued development of the root and surrounding tissue. Human dental pulp stem cells (hDPSCs) have been successfully used in RE to restore odontoblast function. Surface microgeometry is one of the most important factors involved in the induction of differentiation of hDPSCs into odontoblast-like cells. Although different authors have demonstrated the importance of a dentin-like surface with accessible dentin tubules to induce differentiation of hDPSCs, the ultrastructural characteristics of the cells and the secreted extracellular matrix have not been studied in depth. Here, we used an acellular dentin scaffold containing dentin tubules in different spatial geometries, which regulated their accessibility to cells. hDPSCs were cultured on the scaffolds for up to 6 weeks. Systematic characterization of differentiated cells was performed using both optical (hematoxylin and eosin, Masson trichrome, and immunohistochemical determination of dentin sialoprotein [DSSP]) and transmission electron microscopy. The results presented here indicated that cells grown on the dentin surface containing accessible dentin tubules developed a characteristic odontoblastic phenotype, with cellular processes similar to native odontoblasts. The cell organization and characteristics of secreted extracellular matrix were also similar to those of native dentin tissue. Cells grown on non-accessible dentin tubule surfaces secreted a more abundant and dense extracellular matrix, and developed a different phenotype consisting of secretory flat cells organized in layers. Cells grown far from the scaffold, i.e., directly on the culture well surface, developed a secretory phenotype probably influenced by biochemical factors released by the dentin scaffold or differentiated cells. The results presented here support the use of hDPSCs to regenerate dentin and show the utility of scaffold microgeometry for determining the differentiation and secretory phenotype of cultured cells.

In Vitro Conditioning Determines the Capacity of Dental Pulp Stem Cells to Function as Pericyte-Like Cells.

Dental pulp has been revealed as an accessible and a rich source of mesenchymal stem cells (MSCs) and its biological potential is currently under intense investigation. MSCs from dental pulp stem cells (DPSCs) have been indicated as a heterogeneous population oriented not only in repairing dentine but also in maintaining vascular and nervous homeostasis of the teeth. We sought to verify the phenotype of cells isolated from dental pulp of young donors and to investigate in vitro their role as pericyte-like cells. Specifically, we evaluated how culture conditions can modulate expression of pericyte markers in DPSCs and their capacity to stabilize endothelial tubes in vitro. DPSCs cultured in standard conditions expressed MSC markers and demonstrated to contain a population expressing the pericyte marker NG2. These DPSCs were associated with low sprouting capacity in extra-cellular (EC) Matrix and limited ability in retaining tubes formed by endothelial cells in a coculture angiogenesis model. When cultured in endothelial growth medium (EGM)-2, DPSCs significantly upregulated NG2, and partially alpha-smooth muscle actin. The resulting population conserved the stem marker CD73, but was negative for calponin and endothelial markers. EGM-2-conditioned DPSCs showed a higher sprouting ability in EC Matrix and efficient association with human umbilical vein endothelial cells allowing the partial retention of endothelial tubes for several days. Among growth factors contained in EGM-2 we identified basic fibroblast growth factor (bFGF) as mainly responsible for NG2 upregulation and long-term stabilization of endothelial tubes. According to the in vitro analysis, DPSCs represent an effective source of pericytes and the appropriate culture conditions could result in a population with a promising ability to stabilize vessels and promote vascular maturation.

Effect of grape seed extract (GSE) on functional activity and mineralization of OD-21 and MDPC-23 cell lines.

Recent studies on functional tissue regeneration have focused on substances that favor cell proliferation and differentiation, including the bioactive phenolic compounds present in grape seed extract (GSE). The aim of this investigation was to evaluate the stimulatory potential of GSE in the functional activity of undifferentiated pulp cells and odontoblast-like cells. OD-21 and MDPC-23 cell lines were cultivated in odontogenic medium until subconfluence, seeded in 24-well culture plates in a concentration of 2x104/well and divided into: 1) OD-21 without GSE; 2) OD-21+10 µg/mL of GSE; 3) MDPC-23 without GSE; 4) MDPC-23+10 µg/mL of GSE. Cell proliferation, in situ detection of alkaline phosphatase (ALP) and total protein content were assessed after 3, 7 and 10 days, and mineralization was evaluated after 14 days. The data were analyzed by ANOVA statistical tests set at a 5% level of significance. Results revealed that cell proliferation increased after 10 days, and protein content, after 7 days of culture in MDPC-23 cells. In situ ALP staining intensity was higher in undifferentiated pulp cells and odontoblast-like cells after 7 and 10 days, respectively. A discrete increase in MDPC-23 mineralization after GSE treatment was observed despite OD-21 cells presenting a decrease in mineralized nodule deposits. Data suggest that GSE favors functional activity of differentiated cells more broadly than undifferentiated cells (OD-21). More studies with different concentrations of GSE must be conducted to confirm its benefits to cells regarding dentin regeneration.

In vitro performance of a nanobiocomposite scaffold containing boron-modified bioactive glass nanoparticles for dentin regeneration.

Every year, many dental restoration methods are carried out in the world and most of them do not succeed. High cost of these restorations and rejection possibility of the implants are main drawbacks. For this reason, a regenerative approach for repairing the damaged dentin-pulp complex or generating a new tissue is needed. In this study, the potential of three-dimensional cellulose acetate/oxidized pullulan/gelatin-based dentin-like constructs containing 10 or 20% bioactive glass nanoparticles was studied to explore their potential for dentin regeneration. Three-dimensional nano biocomposite structures were prepared by freeze-drying/metal mold pressing methods and characterized by in vitro degradation analysis, water absorption capacity and porosity measurements, scanning electron microscopy, in vitro biomineralization analysis. During one-month incubation in phosphate buffered saline solution at 37°C, scaffolds lost about 25-30% of their weight and water absorption capacity gradually decreased with time. Scanning electron microscopy examinations showed that mean diameter of the tubular structures was about 420 µm and the distance between walls of the tubules was around 560 µm. Calcium phosphate precipitates were formed on scaffolds surfaces treated with simulated body fluid, which was enhanced by boron-modified bioactive glass addition. For cell culture studies human dental pulp stem cells were isolated from patient teeth. An improvement in cellular viability was observed for different groups over the incubation period with the highest human dental pulp stem cells viability on B7-20 scaffolds. ICP-OES analysis revealed that concentration of boron ion released from the scaffolds was between 0.2 and 1.1 mM, which was below toxic levels. Alkaline phosphatase activity and intracellular calcium amounts significantly increased 14 days after incubation with highest values in B14-10 group. Von Kossa staining revealed higher levels of mineral deposition in these groups. In this work, results indicated that developed dentin-like constructs are promising for dentin regeneration owing to presence of boron-modified bioactive glass nanoparticles.

A Bilayered Poly (Lactic-Co-Glycolic Acid) Scaffold Provides Differential Cues for the Differentiation of Dental Pulp Stem Cells.

IMPACT STATEMENT: In this article we used an FDA-approved biodegradable biomaterial, poly (lactic-co-glycolic acid) (PLGA 75:25) to generate a bilayered scaffold with the capacity to induce differential, layer-specific dentinogenic differentiation of dental pulp stem cells (DPSCs) in vitro. We surmise that such a scaffold can be used in conjunction with current regenerative endodontic procedures to help regenerating a physiologic dentin-pulp complex in vivo. We hypothesize that our scaffold in conjunction with DPSCs will advance current regenerative endodontics by restoring dentin and initiating the innervation and revascularization of the pulp.

Effect of grape seed extract (GSE) on functional activity and mineralization of OD-21 and MDPC-23 cell lines

Abstract Recent studies on functional tissue regeneration have focused on substances that favor cell proliferation and differentiation, including the bioactive phenolic compounds present in grape seed extract (GSE). The aim of this investigation was to evaluate the stimulatory potential of GSE in the functional activity of undifferentiated pulp cells and odontoblast-like cells. OD-21 and MDPC-23 cell lines were cultivated in odontogenic medium until subconfluence, seeded in 24-well culture plates in a concentration of 2x104/well and divided into: 1) OD-21 without GSE; 2) OD-21+10 µg/mL of GSE; 3) MDPC-23 without GSE; 4) MDPC-23+10 µg/mL of GSE. Cell proliferation, in situ detection of alkaline phosphatase (ALP) and total protein content were assessed after 3, 7 and 10 days, and mineralization was evaluated after 14 days. The data were analyzed by ANOVA statistical tests set at a 5% level of significance. Results revealed that cell proliferation increased after 10 days, and protein content, after 7 days of culture in MDPC-23 cells. In situ ALP staining intensity was higher in undifferentiated pulp cells and odontoblast-like cells after 7 and 10 days, respectively. A discrete increase in MDPC-23 mineralization after GSE treatment was observed despite OD-21 cells presenting a decrease in mineralized nodule deposits. Data suggest that GSE favors functional activity of differentiated cells more broadly than undifferentiated cells (OD-21). More studies with different concentrations of GSE must be conducted to confirm its benefits to cells regarding dentin regeneration.