Marco C. Bottino

A novel spatially designed and functionally graded electrospun membrane for periodontal regeneration

A periodontal membrane with a graded structure allows tailoring of the layer properties to design a material system that will retain its physical, chemical and mechanical characteristics for a period long enough to optimize periodontal regeneration. In this work a novel functionally graded membrane (FGM) was designed and fabricated via sequential multilayer electrospinning. The FGM consists of a core layer (CL) and two functional surface layers (SLs) interfacing with bone (nano-hydroxyapatite, n-HAp) and epithelial (metronidazole, MET) tissues. The CL comprises a neat poly(DL-lactide-co-ε-caprolactone) (PLCL) layer surrounded by two composite layers composed of a protein/polymer ternary blend (PLCL:PLA:GEL). Electrospinning parameters involved in fabrication of the individual layers (i.e. neat PLCL, ternary blend, PLA:GEL+10%n-HAp and PLA:GEL+25%MET) were optimized to obtain fibrous layers free of beads. Morphology, structure and mechanical property studies were carried out on each electrospun layer. The individual fiber morphology and roughness of the functional SLs, which are the n-HAp containing and drug-incorporating layers were evaluated by atomic force microscopy. The CL structure demonstrated higher strength (8.7 MPa) and a more elastic behavior (strain at break 357%) compared with the FGM (3.5 MPa, 297%). Incorporation of n-HAp to enhance osteoconductive behavior and MET to combat periodontal pathogens led to a novel FGM that holds promise at solving the drawbacks of currently available membranes.

Low-level laser therapy for pain caused by placement of the first orthodontic archwire: A randomized clinical trial

INTRODUCTION The purpose of this study was to clinically evaluate the effect of low-level laser therapy (LLLT) as a method of reducing pain reported by patients after placement of their first orthodontic archwires. METHODS The sample comprised 60 orthodontic patients (ages, 12-18 years; mean, 15.9 years). All patients had fixed orthodontic appliances placed in 1 dental arch (maxillary or mandibular), received the first archwire, and were then randomly assigned to the experimental (laser), placebo, or control group. This was a double-blind study. LLLT was started in the experimental group immediately after placement of the first archwire. Each tooth received a dose of 2.5 J per square centimeter on each side (buccal and lingual). The placebo group had the laser probe positioned into the mouth at the same areas overlying the dental root and could hear a sound every 10 seconds. The control group had no laser intervention. All patients received a survey to be filled out at home describing their pain during the next 7 days. RESULTS The patients in the LLLT group had lower mean scores for oral pain and intensity of pain on the most painful day. Also, their pain ended sooner. LLLT did not affect the start of pain perception or alter the most painful day. There was no significant difference in pain symptomatology in the maxillary or mandibular arches in an evaluated parameter. CONCLUSIONS Based on these findings, we concluded that LLLT efficiently controls pain caused by the first archwire.

Bioactive Nanofibrous Scaffolds for Regenerative Endodontics

Here we report the synthesis, materials characterization, antimicrobial capacity, and cytocompatibility of novel antibiotic-containing scaffolds. Metronidazole (MET) or Ciprofloxacin/(CIP) was mixed with a polydioxanone (PDS)polymer solution at 5 and 25 wt% and processed into fibers. PDS fibers served as a control. Scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), tensile testing, and high-performance liquid chromatography (HPLC) were used to assess fiber morphology, chemical structure, mechanical properties, and drug release, respectively. Antimicrobial properties were evaluated against those of Porphyromonas gingivalis/Pg and Enterococcus faecalis/Ef. Cytotoxicity was assessed in human dental pulp stem cells (hDPSCs). Statistics were performed, and significance was set at the 5% level. SEM imaging revealed a submicron fiber diameter. FTIR confirmed antibiotic incorporation. The tensile values of hydrated 25 wt% CIP scaffold were significantly lower than those of all other groups. Analysis of HPLC data confirmed gradual, sustained drug release from the scaffolds over 48 hrs. CIP-containing scaffolds significantly (p < .00001) inhibited biofilm growth of both bacteria. Conversely, MET-containing scaffolds inhibited only Pg growth. Agar diffusion confirmed the antimicrobial properties against specific bacteria for the antibiotic-containing scaffolds. Only the 25 wt% CIP-containing scaffolds were cytotoxic. Collectively, this study suggests that polymer-based antibiotic-containing electrospun scaffolds could function as a biologically safe antimicrobial drug delivery system for regenerative endodontics.

Tissue-engineering-based Strategies for Regenerative Endodontics

Stemming from in vitro and in vivo pre-clinical and human models, tissue-engineering-based strategies continue to demonstrate great potential for the regeneration of the pulp-dentin complex, particularly in necrotic, immature permanent teeth. Nanofibrous scaffolds, which closely resemble the native extracellular matrix, have been successfully synthesized by various techniques, including but not limited to electrospinning. A common goal in scaffold synthesis has been the notion of promoting cell guidance through the careful design and use of a collection of biochemical and physical cues capable of governing and stimulating specific events at the cellular and tissue levels. The latest advances in processing technologies allow for the fabrication of scaffolds where selected bioactive molecules can be delivered locally, thus increasing the possibilities for clinical success. Though electrospun scaffolds have not yet been tested in vivo in either human or animal pulpless models in immature permanent teeth, recent studies have highlighted their regenerative potential both from an in vitro and in vivo (i.e., subcutaneous model) standpoint. Possible applications for these bioactive scaffolds continue to evolve, with significant prospects related to the regeneration of both dentin and pulp tissue and, more recently, to root canal disinfection. Nonetheless, no single implantable scaffold can consistently guide the coordinated growth and development of the multiple tissue types involved in the functional regeneration of the pulp-dentin complex. The purpose of this review is to provide a comprehensive perspective on the latest discoveries related to the use of scaffolds and/or stem cells in regenerative endodontics. The authors focused this review on bioactive nanofibrous scaffolds, injectable scaffolds and stem cells, and pre-clinical findings using stem-cell-based strategies. These topics are discussed in detail in an attempt to provide future direction and to shed light on their potential translation to clinical settings.

A novel three‐dimensional scaffold for regenerative endodontics: materials and biological characterizations

An electrospun nanocomposite fibrous material holds promise as a scaffold, as well as a drug‐delivery device to aid in root maturogenesis and the regeneration of the pulp–dentine complex. A novel three‐dimensional (3D) nanocomposite scaffold composed of polydioxanone (PDS II®) and halloysite nanotubes (HNTs) was designed and fabricated by electrospinning. Morphology, structure, mechanical properties and cell compatibility studies were carried out to evaluate the effects of HNTs incorporation (0.5–10 wt% relative to PDS w/w). Overall, a 3D porous network was seen in the different fabricated electrospun scaffolds, regardless of the HNT content. The incorporation of HNTs at 10 wt% led to a significant (p < 0.0001) fibre diameter increase and a reduction in scaffold strength. Moreover, PDS–HNTs scaffolds supported the attachment and proliferation of human‐derived pulp fibroblast cells. Quantitative proliferation assay performed with human dental pulp‐derived cells as a function of nanotubes concentration indicated that the HNTs exhibit a high level of biocompatibility, rendering them good candidates for the potential encapsulation of distinct bioactive molecules. Collectively, the reported data support the conclusion that PDS–HNTs nanocomposite fibrous structures hold potential in the development of a bioactive scaffold for regenerative endodontics. Copyright

Y-TZP ceramic processing from coprecipitated powders: A comparative study with three commercial dental ceramics

OBJECTIVES (1) To synthesize 3mol% yttria-stabilized zirconia (3Y-TZP) powders via coprecipitation route, (2) to obtain zirconia ceramic specimens, analyze surface characteristics, and mechanical properties, and (3) to compare the processed material with three reinforced dental ceramics. METHODS A coprecipitation route was used to synthesize a 3mol% yttria-stabilized zirconia ceramic processed by uniaxial compaction and pressureless sintering. Commercially available alumina or alumina/zirconia ceramics, namely Procera AllCeram (PA), In-Ceram Zirconia Block (CAZ) and In-Ceram Zirconia (IZ) were chosen for comparison. All specimens (6mmx5mmx5mm) were polished and ultrasonically cleaned. Qualitative phase analysis was performed by XRD and apparent densities were measured on the basis of Archimedes principle. Ceramics were also characterized using SEM, TEM and EDS. The hardness measurements were made employing Vickers hardness test. Fracture toughness (K(IC)) was calculated. Data were analyzed using one-way analysis of variance (ANOVA) and Tukeys test (alpha=0.05). RESULTS ANOVA revealed that the Vickers hardness (p<0.0001) and fracture toughness (p<0.0001) were affected by the ceramic materials composition. It was confirmed that the PA ceramic was constituted of a rhombohedral alumina matrix, so-called alpha-alumina. Both CAZ and IZ ceramics presented tetragonal zirconia and alpha-alumina mixture of phases. The SEM/EDS analysis confirmed the presence of aluminum in PA ceramic. In the IZ and CAZ ceramics aluminum, zirconium and cerium in grains involved by a second phase containing aluminum, silicon and lanthanum were identified. PA showed significantly higher mean Vickers hardness values (H(V)) (18.4+/-0.5GPa) compared to vitreous CAZ (10.3+/-0.2GPa) and IZ (10.6+/-0.4GPa) ceramics. Experimental Y-TZP showed significantly lower results than that of the other monophased ceramic (PA) (p<0.05) but it showed significantly higher fracture toughness (6.0+/-0.2MPam(1/2)) values when compared to the other tested ceramics (p<0.05). SIGNIFICANCE The coprecipitation method used to synthesize zirconia powders and the adopted ceramic processing conditions led to ceramics with mechanical properties comparable to commercially available reinforced ceramic materials.

Full-contour Y-TZP ceramic surface roughness effect on synthetic hydroxyapatite wear

OBJECTIVE To investigate the effects of polishing techniques on the surface roughness of Y-TZP ceramic and on the wear behavior of synthetic hydroxyapatite (HA). METHODS Thirty-two full-contour Y-TZP (Diazir(®)) sliders (φ=2 mm × 1.5 mm in height) were manufactured using CAD/CAM, embedded in acrylic resin using brass holders, and randomly allocated into four groups (n=8): according to the finishing/polishing procedure: G1-as-machined, G2-glazed, G3-diamond bur finishing and G4-G3+OptraFine(®) polishing kit. Thirty-two sintered HA disks (φ=13 mm × 2.9 mm in height) were similarly mounted in brass holders. Y-TZP sliders baseline surface roughness values (Ra and Rq, in μm) were recorded using a non-contact profilometer (Proscan 2000). A two-body pin-on-disc wear test was performed. HA height (μm) and volume (mm(3)) losses were measured. Y-TZP height loss was measured using a digital micrometer. One-way ANOVA was used to determine the effect of the polishing techniques on the surface roughness. Comparisons between groups for differences in antagonist height loss/volume, and slider height loss were performed using one-way ANOVA. Statistical significance was set at α=0.05. RESULTS Roughness measurements showed significant differences (p=0.0001) among the surface treatments with G1 (Ra=0.84, Rq=1.13 μm) and G3 (Ra=0.89, Rq=1.2 μm) being the roughest, and G2 (Ra=0.42, Rq=0.63 μm) the smoothest (p=0.0001). Y-TZP slider height loss was highest for the glazed group (35.39 μm), and was lowest for the polished group (6.61 μm) (p=0.0001). Antagonist volume and height losses for groups (G1-G3) were similar, while the polished group (1.3 mm(3), 14.7 μm) showed significant lower values (p=0.0001). SIGNIFICANCE Although glazed zirconia provides an initially smooth surface, significantly increased antagonist wear was observed compared to the polished Y-TZP zirconia surface.

Freeze-dried acellular dermal matrix graft: effects of rehydration on physical, chemical, and mechanical properties.

OBJECTIVES To test the effect of rehydration time over the range prescribed in the manufacturers protocol on (1) the biomechanical properties and on (2) the recovery and stabilization of the collagenous matrix of AlloDerm. METHODS A sterile dish containing warm saline solution was prepared, and samples rehydrated for 5 min. Subsequently, three other dishes with the solution were prepared and samples assigned into three groups according to the total rehydration time: 10 min (G1), 20 min (G2), and 40 min (G3). Uni-axial tensile testing was used to assess the biomechanical properties of the different groups and the control (dry condition). Physico-chemical properties were examined by Fourier transform infrared spectroscopy (FT-IR), and differential scanning calorimetry (DSC) as a function of rehydration time. RESULTS ANOVA revealed a significant change in tensile strength (p=0.0269) and in elastic modulus (p=0.0306) for AlloDerm following different rehydration times. The lowest tensile strength was in the dry condition, whereas the highest was achieved after a 40 min rehydration. The shortest rehydration periods did not result in a statistically significant (p>0.05) change in elastic modulus. However, after 40 min the elastic modulus increased significantly when compared to the shortest periods. FT-IR confirmed the protein backbone recovery of the graft matrix after rehydration. DSC scans of rehydrated samples showed visible shifts in the denaturation temperature to higher values compared to as-received sample (dry) suggesting stronger polymer-water bridge formation, supporting the increase in the biomechanical properties. SIGNIFICANCE The current study suggests that there are major changes on the biomechanical properties of the collagenous graft as rehydration time increases, which were also structurally confirmed by the physico-chemical analyses. Clinicians must be aware that the rehydration times of the manufacturers protocol result in a significant range in mechanical and physico-chemical properties. Therefore, a rehydration time of at least 20 min guarantees not only better handling and mechanical properties but, most importantly, supplies a material that closely resembles the natural tissue.

Polishing methods of an alumina-reinforced feldspar ceramic

The purpose of this study was to test the hypothesis that mechanical polishing methods of ceramic surfaces allow similar superficial roughness to that of glazed surfaces. Twenty-five Vitadur Alpha ceramic discs (5 mm x 2 mm) were prepared according to the manufacturers specifications. All specimens were glazed and randomly assigned to 5 groups (n=5), according to finishing and polishing protocols: G1: glazed (control); G2: diamond bur finishing; G3: G2 + silicon rubber tip polishing; G4: G3 + felt disc/diamond polishing paste; G5: G3 + felt disc impregnated with fine-particle diamond paste. Next, surface roughness means (Ra - microm) were calculated. Qualitative analysis was made by scanning electron microscopy. Surface roughness data were submitted to ANOVA and Tukeys test at 5% significance level. G1 and G4 were statistically similar (p>0.05). G2 presented the highest roughness means (p<0.05) followed by groups G3, G5, G4 and G1 in a decreasing order. The hypothesis was partially confirmed as only the mechanical polishing (G4) produced similar superficial roughness to that of surface glazing, although finishing and polishing are technically critical procedures.

Effects of ciprofloxacin-containing scaffolds on enterococcus faecalis biofilms.

INTRODUCTION Antibiotic-containing polymer-based nanofibers (hereafter referred to as scaffolds) have demonstrated great potential for their use in regenerative endodontics from both an antimicrobial and cytocompatibility perspective. This study sought to evaluate in vitro the effects of ciprofloxacin (CIP)-containing polymer scaffolds against Enterococcus faecalis biofilms. METHODS Human mandibular incisors were longitudinally sectioned to prepare radicular dentin specimens. Sterile dentin specimens were distributed in 24-well plates and inoculated with E. faecalis for biofilm formation. Infected dentin specimens were exposed to 3 groups of scaffolds, namely polydioxanone (PDS) (control), PDS + 5 wt% CIP, and PDS + 25 wt% CIP for 2 days. Colony-forming units (CFU/mL) (n = 10) and scanning electron microscopy (SEM) (n = 2) were performed to quantitatively and qualitatively assess the antimicrobial effectiveness, respectively. RESULTS PDS scaffold containing CIP at 25 wt% showed maximum bacteria elimination with no microbial growth, differing statistically (P < .05) from the control (PDS) and from PDS scaffold containing CIP at 5 wt%. Statistical differences (P < .05) were also seen for the CFU/mL data between pure PDS (5.92-6.02 log CFU/mL) and the PDS scaffold containing CIP at 5 wt% (5.39-5.87 log CFU/mL). SEM images revealed a greater concentration of bacteria on the middle third of the dentin specimen after 5 days of biofilm formation. On scaffold exposures, SEM images showed similar results when compared with the CFU/mL data. Dentin specimens exposed to PDS + 25 wt% CIP scaffolds displayed a practically bacteria-free surface. CONCLUSIONS On the basis of the data presented, newly developed antibiotic-containing electrospun scaffolds hold promise as an intracanal medicament to eliminate biofilm/infection before regenerative procedures.