Isaac Jordão de Souza Araújo

Previously, our in vitro tube model, simulating the root canal space, demonstrated that human dental pulp stem cell (DPSC)–derived endothelial-like cells (ECs) in fibrin gel can form vascular-like structures. In the present study, we extended to an in vivo tube model and developed a tooth fragment dorsal skinfold window chamber (t-SWC) in vivo model to investigate vascularization and blood perfusion from the host vasculature. In the tube model, we observed significant variation in the extension of red blood cell (RBC)–present vasculature from the host tissue into the tube. There was a demarcation between the RBC-present vessels and RBC-absent vessels in the tissue within the tube. Cells were labeled with green fluorescent protein (GFP) and traced with anti-GFP antibodies via immunohistochemistry. RBC-present vessels were made of both GFP ⁺ and GFP ^– cells, indicating the ingrowth of the mouse vascular system via angiogenesis. Cells within the RBC-absent vessel region exhibited robust growth, and morphologically odontoblast-like cells were observed at the coronal end in some samples. DPSC-ECs appeared to contribute to RBC-containing vessel formation, although they were insufficient to independently generate fully mature, functional vasculature capable of providing blood supply. Freshly prepared mineral trioxide aggregate exhibited cytotoxic effects on implanted cells within the tube, and this toxicity was not mitigated by the in vivo environment. The t-SWC model allowed real-time and direct observation of the in vivo neovascularization. Under this testing model, blood perfusion within the canal space was not detected during up to 50 d of observation. These qualitative observations suggest that, in the evaluable specimens, DPSC-EC–derived vascular-like structures did not show detectable functional maturation into perfused vessels. The limitations of the ectopic models may play a role in the findings, as subcutaneous tissue is different from the periapical environment. Regardless, the 2 models provided experimental platforms for evaluating host–construct vascular interactions and neovascularization in the canal space.

Canonical and noncanonical WNT signaling pathways are tissue specific and play roles in mineralized tissue development and repair. Here we characterize the activity of noncanonical WNT5B and canonical WNT10B in dental pulp stem cells (DPSCs) in osteo-/odontogenesis and target dentin–pulp complex regeneration using rWNT-loaded hydrogels. DPSCs in monolayers were treated with recombinant WNT10B (rWNT10B, 50 ng/mL), rWNT5B (50 ng/mL), or the canonical WNT pan-activator CHIR 99021 (10 nM). Gene expression of osteo-/odontogenic markers and Alizarin Red assay were performed. Collagen-based hydrogels were prepared from bovine type I collagen and loaded with rWNT10B, rWNT5B, or CHIR. A control group with no treatment was included. WNT release from hydrogels in phosphate-buffered saline was performed up to 14 d. The DPSC response to hydrogels was characterized by viability, morphology, gene expression, and alkaline phosphatase (ALP) activity. In vivo investigation was conducted using an ectopic model of pulp exposure in human tooth fragments implanted in mice for 4 wk. Hematoxylin and eosin, trichrome staining, and immunohistochemistry were performed. Statistical analysis was performed using analysis of variance and Tukey post hoc (α = 0.05). Our in vitro studies showed that rWNT5B significantly upregulated ALP , RUNX2 , and DMP1 after 7 d. rWNTs and CHIR significantly enhanced mineralization. rWNTs were almost completely released from the hydrogels in 14 d, with 2 release peaks at 6 h and 4 d. rWNT-loaded hydrogels did not affect cell viability and morphology. rWNT10B-loaded hydrogels significantly increased the expression of ALP and RUNX2 . The rWNT-loaded hydrogels significantly increased ALP activity. Our in vivo histological data indicate the presence of gel in the exposure area, the formation of pulp-like tissue in contact with the hydrogels, new blood vessels, and polarized cells adjacent to dentin tubules. Canonical WNT10B and noncanonical WNT5B appear to play specific roles in DPSC differentiation. WNT-loaded hydrogels are cytocompatible, increase the expression of osteo-/odontogenic markers and ALP activity, and induce pulp-like tissue formation.

Targeting inflammation and mineralization responses in a controlled manner for vital pulp therapies is a challenging task. Here, we prepared dexamethasone-β-cyclodextrin (DEX/ICD) inclusion complex-loaded hydrogels for pulp–dentin complex regeneration. The formation of the DEX/ICD inclusion complex was verified using Fourier-transformed infrared spectroscopy (FTIR) and X-ray diffraction (XRD). Pristine dexamethasone (DEX) and DEX/ICD were added to methacrylated gelatin (GelMA) hydrogels at a concentration of 1%. Pristine GelMA serves as the control. Drug-loaded hydrogels were evaluated for mechanical properties ( n = 10/group), swelling and enzymatic degradation ( n = 5/group), and drug release ( n = 3/group). Cell proliferation, cell morphology, and mineralization assays were conducted using SHEDs. Cell proliferation was assessed on days 1, 3, and 7 using the MTS assay ( n = 6/group). Cell morphology was analyzed via fluorescence microscopy using phalloidin and DAPI. A mineralization assay was performed using Alizarin Red at 14 and 21 days ( n = 4/group). One-way ANOVA and Tukey’s ( α = 0.05) post-hoc tests were applied for statistical analysis. FTIR and XRD data confirmed the formation of the DEX/ICD complex. No statistical differences were found in mechanical properties, swelling, and degradation after incorporating DEX or DEX/ICD. DEX/ICD-loaded hydrogels consistently released dexamethasone, peaking at day 14, and maintained stability until day 21. DEX-loaded GelMA release decreased after day 7. DEX/ICD-loaded GelMA exhibited higher cell proliferation than DEX-loaded GelMA on day 7 ( p < 0.05), with no statistical differences observed for days 1 and 3 (p > 0.05). The hydrogels did not compromise cell morphology. DEX/ICD-loaded GelMA significantly enhanced mineralization potential ( p < 0.05). The β-CD inclusion complex optimized dexamethasone delivery, and DEX/ICD-containing GelMA enhances the proliferation and mineralization capacity of dental stem cells. DEX/ICD-loaded GelMA is a promising injectable material for halting inflammation and could potentially induce mineralized tissue formation in pulp capping strategies.

The present in vitro study evaluated the effect of titanium dioxide nanotubes (nTiO₂) incorporated into glass ionomer cement (GIC) on the growth and viability of Lactobacillus acidophilus (L. acidophilus). GIC (Ketac Molar EasyMix^® = KM) was added to concentrations of 0%, 3%, 5%, 7% by weight of nTiO₂ (20 nm in size). L. acidophilus strains (1x10⁸ CFU/mL) were cultivated on GIC discs with or without the addition of nTiO₂ for 1, 3 and 7 days, and the following parameters were evaluated: inhibition zone (mm) (n = 6); cell viability (Live/Dead) (n = 6); cell morphology (SEM) (15 KV, 2000X, n = 3). The data were submitted to ANOVA and the Tukey and Dunnett tests (α = 0.05). Regarding the agar diffusion test, there was no difference between GIC and the groups containing nTiO₂ (p > 0.05). As for bacterial viability, the percentage of viable bacteria was lower for GIC+7% nTiO₂ (p ≤ 0.05). There was no difference in the percentage of non-viable bacteria (p > 0.05). In addition, the morphology of L. acidophilus did not change in the presence of nTiO₂. It can be concluded that the incorporation of titanium dioxide nanotubes into GIC, particularly at 5%, reduced L. acidophilus viability, and might hence interfere negatively with the initial colonization process of the bacterial biofilm.

Periodontitis and severe trauma are major causes of damage to the periodontal ligament (PDL). Repairing the native conditions of the PDL is essential for the stability of the tissue and its interfaces. Bioprinting periodontal ligament stem cells (PDLSCs) is an interesting approach to guide the regeneration of PDL and interfacial integration. Herein, a collagen-based bioink mimicking the native extracellular matrix conditions and carrying PDLSCs was tested to guide the periodontal ligament organization. The bioink was tested at two different concentrations (10 and 15 mg/mL) and characterized by swelling and degradation, microstructural organization, and rheological properties. The biological properties were assessed after loading PDLSCs into bioinks for bioprinting. The characterization was performed through cell viability, alizarin red assay, and expression for ALP, COL1A1, RUNX2, and OCN. The in vivo biocompatibility of the PDLSC-laden bioinks was verified using subcutaneous implantation in mice. Later, the ability of the bioprinted PDLSC-laden bioinks on dental root fragments to form PDL was also investigated in vivo in mice for 4 and 10 weeks. The bioinks demonstrated typical shear-thinning behavior, a porous microstructure, and stable swelling and degradation characteristics. Both concentrations were printable and provided suitable conditions for a high cell survival, proliferation, and differentiation. PDLSC-laden bioinks demonstrated biocompatibility in vivo, and the bioprinted scaffolds on the root surface evidenced PDLSC alignment, organization, and PDLSC migration to the root surface. The versatility of collagen-based bioinks provides native ECM conditions for PDLSC proliferation, alignment, organization, and differentiation, with translational applications in bioprinting scaffolds for PDL regeneration.

The aim of this study was to evaluate physical-mechanical properties, degree of conversion, and chemical stability of a nanohybrid composite containing diatomite as filler. Degree of conversion (DC%) of diatomite-containing composite (Zirconfill®) was performed using FTIR immediately, and 1-, and 7-days post-curing. SEM was conducted to evaluate the surface of the resin after curing and measure particles size. Also, elemental characterization was performed to verify the major components of the composite through EDS. Mechanical characterization using 3-point bending test was performed prior and after thermo-cycling (10000 cycles) (n=10). Knoop microhardness (KHN) was used to characterize mechanical stability after chemical solutions aggression (water, juice, coffee, coke) up to 28 days (n=10/solution). After data normality evaluation using Shapiro-Wilk, One-way ANOVA and Tukey post hoc were conducted to verify differences between groups for DC% and mechanical properties. Split-plot ANOVA was used to compare groups for microhardness characterization (α=0.05). Immediate DC was 60% and significantly increased up to 80% at 7 days (p0.05). All the dietary solutions affected the KHN of the composite up to 21 days. For 28 days, the KHN evidenced and stabilization regarding all the solutions. Diatomite-containing composites present good degree of conversion and relevant mechanical properties and demonstrate time-dependent stability against chemical degradation.

After bleaching, enamel surfaces are damaged, contributing to erosion and tooth sensitivity. Although fluoride is used after bleaching to try and revert alterations, it is not capable of repairing tooth structure. This study compared the effect of a self-assembly peptide (P₁₁-4), with and without fluoride, and sodium fluoride (NaF 2%) on the Knoop microhardness (KHN) and surface roughness (Ra (μm)) of bleached enamel with an in-office bleaching regimen. Enamel blocks of bovine teeth (5 × 5 × 2 mm) with standardized surface hardness were bleached with 35% carbamide peroxide, following the manufacturer’s instructions. The teeth were randomly divided into the following groups (n = 7) according to post-bleaching treatment: no treatment (negative control) (C-); 2% NaF (NaF); Curodont™ Repair (Repair); and Curodont™ Protect (Protect). Specimens were stored in artificial saliva at 37 °C. To evaluate the effect of the post-bleaching treatments, KHN and Ra were measured before bleaching (baseline) and 24 h and 7 days after bleaching. Data were submitted to repeated measures ANOVA and Bonferroni tests (α = 0.05). There were significant interactions between the study factors (p = 0.001). After 7 days, Repair (572.50 ± 79.04) and Protect (583.00 ± 74.76) specimens showed increased surface KHN, with values higher than the NaF (465.50 ± 41.50) and C- (475.22 ± 58.95) baseline values. There was no significant difference in KHN at 24 h among groups (p = 0.587). At 24 h after bleaching, Repair was significantly different from all groups (p < 0.05). Repair showed the lowest Ra (μm) values (0.133 ± 0.035). After seven days, there was no significant difference in Ra values among groups when compared to the baseline. The use of P₁₁-4-based materials after bleaching resulted in the fastest recovery to baseline enamel properties.