Vinicius Rosa

Dental Pulp Tissue Engineering in Full-length Human Root Canals

The clinical translation of stem-cell-based dental pulp regeneration will require the use of injectable scaffolds. Here, we tested the hypothesis that stem cells from exfoliated deciduous teeth (SHED) can generate a functional dental pulp when injected into full-length root canals. SHED survived and began to express putative markers of odontoblastic differentiation after 7 days when mixed with Puramatrix™ (peptide hydrogel), or after 14 days when mixed with recombinant human Collagen (rhCollagen) type I, and injected into the root canals of human premolars in vitro. Roots of human premolars injected with scaffolds (Puramatrix™ or rhCollagen) containing SHED were implanted subcutaneously into immunodeficient mice (CB-17 SCID). We observed pulp-like tissues with odontoblasts capable of generating new tubular dentin throughout the root canals. Notably, the pulp tissue engineered with SHED injected with either Puramatrix™ or rhCollagen type I presented similar cellularity and vascularization when compared with control human dental pulps. Analysis of these data, collectively, demonstrates that SHED injected into full-length human root canals differentiate into functional odontoblasts, and suggests that such a strategy might facilitate the completion of root formation in necrotic immature permanent teeth.

Tissue engineering: From research to dental clinics

UNLABELLED Tissue engineering is an interdisciplinary field that combines the principles of engineering, material and biological sciences toward the development of therapeutic strategies and biological substitutes that restore, maintain, replace or improve biological functions. The association of biomaterials, stem cells, growth and differentiation factors has yielded the development of new treatment opportunities in most of the biomedical areas, including Dentistry. The objective of this paper is to present the principles underlying tissue engineering and the current scenario, the challenges and the perspectives of this area in Dentistry. SIGNIFICANCE The growth of tissue engineering as a research field has provided a novel set of therapeutic strategies for biomedical applications. Indeed, tissue engineering may lead to new strategies for the clinical management of patients with dental and craniofacial needs in the future.

Graphene: A Versatile Carbon-Based Material for Bone Tissue Engineering

The development of materials and strategies that can influence stem cell attachment, proliferation, and differentiation towards osteoblasts is of high interest to promote faster healing and reconstructions of large bone defects. Graphene and its derivatives (graphene oxide and reduced graphene oxide) have received increasing attention for biomedical applications as they present remarkable properties such as high surface area, high mechanical strength, and ease of functionalization. These biocompatible carbon-based materials can induce and sustain stem cell growth and differentiation into various lineages. Furthermore, graphene has the ability to promote and enhance osteogenic differentiation making it an interesting material for bone regeneration research. This paper will review the important advances in the ability of graphene and its related forms to induce stem cells differentiation into osteogenic lineages.

Regenerative endodontics in light of the stem cell paradigm

Stem cells play a critical role in development and in tissue regeneration. The dental pulp contains a small sub-population of stem cells that are involved in the response of the pulp to caries progression. Specifically, stem cells replace odontoblasts that have undergone cell death as a consequence of the cariogenic challenge. Stem cells also secrete factors that have the potential to enhance pulp vascularisation and provide the oxygen and nutrients required for the dentinogenic response that is typically observed in teeth with deep caries. However, the same angiogenic factors that are required for dentine regeneration may ultimately contribute to the demise of the pulp by enhancing vascular permeability and interstitial pressure. Recent studies focused on the biology of dental pulp stem cells revealed that the multipotency and angiogenic capacity of these cells could be exploited therapeutically in dental pulp tissue engineering. Collectively, these findings suggest new treatment paradigms in the field of endodontics. The goal of this review is to discuss the potential impact of dental pulp stem cells to regenerative endodontics.

Effect of acid etching of glass ionomer cement surface on the microleakage of sandwich restorations

The purposes of this study were to evaluate the sealing ability of different glass ionomer cements (GICs) used for sandwich restorations and to assess the effect of acid etching of GIC on microleakage at GIC-resin composite interface. Forty cavities were prepared on the proximal surfaces of 20 permanent human premolars (2 cavities per tooth), assigned to 4 groups (n=10) and restored as follows: Group CIE – conventional GIC (CI) was applied onto the axial and cervical cavity walls, allowed setting for 5 min and acid etched (E) along the cavity margins with 35% phosphoric acid for 15 s, washed for 30 s and water was blotted; the adhesive system was applied and light cured for 10 s, completing the restoration with composite resin light cured for 40 s; Group CIN – same as Group CIE, except for acid etching of the CI surface; Group RME – same as CIE, but using a resin modified GIC (RMGIC); Group RMN – same as Group RME, except for acid etching of the RMGIC surface. Specimens were soaked in 1% methylene blue dye solution at 24°C for 24 h, rinsed under running water for 1 h, bisected longitudinally and dye penetration was measured following the ISO/TS 11405-2003 standard. Results were statistically analyzed by Kruskal-Wallis and chi-square tests (α=0.05). Dye penetration scores were as follow: CIE – 2.5; CIN – 2.5; RME – 0.9; and RMN – 0.6. The results suggest that phosphoric acid etching of GIC prior to the placement of composite resin does not improve the sealing ability of sandwich restorations. The RMGIC was more effective in preventing dye penetration at the GIC-resin composite- dentin interfaces than CI.

Subcritical crack growth and in vitro lifetime prediction of resin composites with different filler distributions

OBJECTIVES Verify the influence of different filler distributions on the subcritical crack growth (SCG) susceptibility, Weibull parameters (m and σ(0)) and longevity estimated by the strength-probability-time (SPT) diagram of experimental resin composites. METHODS Four composites were prepared, each one containing 59 vol% of glass powder with different filler sizes (d(50)=0.5; 0.9; 1.2 and 1.9 μm) and distributions. Granulometric analyses of glass powders were done by a laser diffraction particle size analyzer (Sald-7001, Shimadzu, USA). SCG parameters (n and σ(f0)) were determined by dynamic fatigue (10(-2) to 10(2) MPa/s) using a biaxial flexural device (12 × 1.2 mm; n=10). Twenty extra specimens of each composite were tested at 10(0) MPa/s to determine m and σ(0). Specimens were stored in water at 37°C for 24 h. Fracture surfaces were analyzed under SEM. RESULTS In general, the composites with broader filler distribution (C0.5 and C1.9) presented better results in terms of SCG susceptibility and longevity. C0.5 and C1.9 presented higher n values (respectively, 31.2 ± 6.2(a) and 34.7 ± 7.4(a)). C1.2 (166.42 ± 0.01(a)) showed the highest and C0.5 (158.40 ± 0.02(d)) the lowest σ(f0) value (in MPa). Weibull parameters did not vary significantly (m: 6.6 to 10.6 and σ(0):170.6 to 176.4 MPa). Predicted reductions in failure stress (P(f)=5%) for a lifetime of 10 years were approximately 45% for C0.5 and C1.9 and 65% for C0.9 and C1.2. Crack propagation occurred through the polymeric matrix around the fillers and all the fracture surfaces showed brittle fracture features. SIGNIFICANCE Composites with broader granulometric distribution showed higher resistance to SCG and, consequently, higher longevity in vitro.

Effect of ion exchange on strength and slow crack growth of a dental porcelain

OBJECTIVES To determine the effect of ion exchange on slow crack growth (SCG) parameters (n, stress corrosion susceptibility coefficient, and sigma(f0), scaling parameter) and Weibull parameters (m, Weibull modulus, and sigma(0), characteristic strength) of a dental porcelain. METHODS 160 porcelain discs were fabricated according to manufacturers instructions, polished through 1 microm and divided into two groups: GC (control) and GI (submitted to an ion exchange procedure using a KNO3 paste at 470 degrees C for 15 min). SCG parameters were determined by biaxial flexural strength test in artificial saliva at 37 degrees C using five constant stress rates (n=10). 20 specimens of each group were tested at 1 MPa/s to determine Weibull parameters. The SPT diagram was constructed using the least-squares fit of the strength data versus probability of failure. RESULTS Mean values of m and sigma(0) (95% confidence interval), n and sigma(f0) (standard deviation) were, respectively: 13.8 (10.1-18.8) and 60.4 (58.5-62.2), 24.1 (2.5) and 58.1 (0.01) for GC and 7.4 (5.3-10.0) and 136.8 (129.1-144.7), 36.7 (7.3) and 127.9 (0.01) for GI. Fracture stresses (MPa) calculated using the SPT diagram for lifetimes of 1 day, 1 year and 10 years (at a 5% failure probability) were, respectively, 31.8, 24.9 and 22.7 for GC and 71.2, 60.6 and 56.9 for GI. SIGNIFICANCE For the porcelain tested, the ion exchange process improved strength and resistance to SCG, however, the materials reliability decreased. The predicted fracture stress at 5% failure probability for a lifetime of 10 years was also higher for the ion treated group.

Graphene oxide-based substrate: physical and surface characterization, cytocompatibility and differentiation potential of dental pulp stem cells

OBJECTIVE The aim of this study was to evaluate the cytotoxicity and differentiation potential of a graphene oxide (GO)-based substrate using dental pulp stem cell (DPSC). METHODS GO was obtained via chemical exfoliation of graphite using the modified Hummers method and dispersed in water-methanol solution. 250μL of 1.5mg/mL solution were added to a cover slip and allowed to dry (25°C, 24h). GO-based substrate was characterized by Raman spectroscopy, AFM and contact angle. DPSC were seeded on GO and glass (control). Cell attachment and proliferation were evaluated by polymeric F-actin staining, SEM and MTS assay for five days. mRNA expression of MSX-1, PAX-9, RUNX2, COL I, DMP-1 and DSPP were evaluated by qPCR (7 and 14 days). Statistical analyses were performed by either Mann-Whitney, one or two-way Anova followed by and Tukeys post hoc analysis (α=0.05). RESULTS Peaks at 1587cm(-1) and 1340cm(-1) (G and D band) and ID/IG of 0.83 were observed for GO with Raman. AFM showed that GO was randomly deposited and created a rougher surface comparing to the control. Cells successfully adhered on both substrates. There was no difference in cell proliferation after 5 days. Cells on GO presented higher expression for all genes tested except MSX-1 and RUNX2 for 7 days. SIGNIFICANCE GO-based substrate allowed DPSC attachment, proliferation and increased the expression of several genes that are upregulated in mineral-producing cells. These findings open opportunities to the use of GO alone or in combination with dental materials to improve their bioactivity and beyond.

Modulation of Dental Pulp Stem Cell Odontogenesis in a Tunable PEG-Fibrinogen Hydrogel System

Injectable hydrogels have the great potential for clinical translation of dental pulp regeneration. A recently developed PEG-fibrinogen (PF) hydrogel, which comprises a bioactive fibrinogen backbone conjugated to polyethylene glycol (PEG) side chains, can be cross-linked after injection by photopolymerization. The objective of this study was to investigate the use of this hydrogel, which allows tuning of its mechanical properties, as a scaffold for dental pulp tissue engineering. The cross-linking degree of PF hydrogels could be controlled by varying the amounts of PEG-diacrylate (PEG-DA) cross-linker. PF hydrogels are generally cytocompatible with the encapsulated dental pulp stem cells (DPSCs), yielding >85% cell viability in all hydrogels. It was found that the cell morphology of encapsulated DPSCs, odontogenic gene expression, and mineralization were strongly modulated by the hydrogel cross-linking degree and matrix stiffness. Notably, DPSCs cultured within the highest cross-linked hydrogel remained mostly rounded in aggregates and demonstrated the greatest enhancement in odontogenic gene expression. Consistently, the highest degree of mineralization was observed in the highest cross-linked hydrogel. Collectively, our results indicate that PF hydrogels can be used as a scaffold for DPSCs and offers the possibility of influencing DPSCs in ways that may be beneficial for applications in regenerative endodontics.

Influence of shade and irradiation time on the hardness of composite resins

This study tested the following hypotheses: 1. increasing light irradiation time (IT) produces greater values of superficial hardness on different depths (0 and 3 mm); and 2. a dark shade composite (A3) needs longer IT than a light shade composite (A1) to produce similar hardness. Disk-shaped specimens (n=24 per shade) were fabricated using a 3-mm-thick increment of composite resin (Z100). Specimens were randomly assigned to 3 groups (n=8) according to the IT (400 mW/cm2) at the upper (U) surface: A1-10 and A3-10: 10 s; A1-20 and A3-20: 20 s; A1-40 and A3-40: 40 s. Specimens were stored in black lightproof containers at 37 masculineC for 24 h before indentation in a hardness tester. Three Vickers indentations were performed on the U and lower (L) surfaces of each specimen. The indent diagonals were measured and the hardness value calculated. The results were analyzed statistically by ANOVA and Tukeys test (alpha=0.05). Statistically significant differences were found between U and L surfaces of each composite shade-IT combination (p=0.0001) and among the ITs of same shade-surface combination (p=0.0001), except between groups A1-20U and A1-40U, confirming the study hypothesis 1 and partially rejecting the hypothesis 2.