Development of an antibacterial and anti-metalloproteinase dental adhesive for long-lasting resin composite restorations.

Despite all the advances in adhesive dentistry, dental bonds are still fragile due to degradation events that start during application of adhesive agents and the inherent hydrolysis of resin-dentin bonds. Here, we combined two outstanding processing methods (electrospinning and cryomilling) to obtain bioactive (antimicrobial and anti-metalloproteinase) fiber-based fillers containing a potent matrix metalloproteinase (MMP) inhibitor (doxycycline, DOX). Poly(ε)caprolactone solutions containing different DOX amounts (0, 5, 25, and 50 wt%) were processed via electrospinning, resulting in non-toxic submicron fibers with antimicrobial activity against Streptococcus mutans and Lactobacillus. The fibers were embedded in a resin blend, light-cured, and cryomilled for the preparation of fiber-containing fillers, which were investigated with antibacterial and in situ gelatin zymography analyzes. The fillers containing 0, 25, and 50 wt% DOX-releasing fibers were added to aliquots of a two-step, etch-and-rinse dental adhesive system. Mechanical strength, hardness, degree of conversion (DC), water sorption and solubility, bond strength to dentin, and nanoleakage analyses were performed to characterize the physico-mechanical, biological, and bonding properties of the modified adhesives. Statistical analyses (ANOVA; Kruskal-Wallis) were used when appropriate to analyze the data (α = 0.05). DOX-releasing fibers were successfully obtained, showing proper morphological architecture, cytocompatibility, drug release ability, slow degradation profile, and antibacterial activity. Reduced metalloproteinases (MMP-2 and MMP-9) activity was observed only for the DOX-containing fillers, which have also demonstrated antibacterial properties against tested bacteria. Adhesive resins modified with DOX-containing fillers demonstrated greater DC and similar mechanical properties as compared to the fiber-free adhesive (unfilled control). Concerning bonding performance to dentin, the experimental adhesives showed similar immediate bond strengths to the control. After 12 months of water storage, the fiber-modified adhesives (except the group consisting of 50 wt% DOX-loaded fillers) demonstrated stable bonds to dentin. Nanoleakage was similar among all groups investigated. DOX-releasing fibers showed promising application in developing novel dentin adhesives with potential therapeutic properties and MMP inhibition ability; antibacterial activity against relevant oral pathogens, without jeopardizing the physico-mechanical characteristics; and bonding performance of the adhesive.

A novel patient-specific three-dimensional drug delivery construct for regenerative endodontics.

Evoked bleeding (EB) clinical procedure, comprising a disinfection step followed by periapical tissue laceration to induce the ingrowth of undifferentiated stem cells from the periodontal ligament and alveolar bone, is currently the only regenerative-based therapeutic approach to treating pulp tissue necrosis in undeveloped (immature) permanent teeth approved in the United States. Yet, the disinfection step using antibiotic-based pastes leads to cytotoxic, warranting a biocompatible strategy to promote root canal disinfection with no or minimal side-effects to maximize the regenerative outcomes. The purpose of this investigation was to develop a tubular three-dimensional (3D) triple antibiotic-eluting construct for intracanal drug delivery. Morphological (scanning electron microscopy), chemical (Fourier transform infrared spectroscopy), and mechanical (tensile testing) characteristics of the polydioxanone-based triple antibiotic-eluting fibers were assessed. The antimicrobial properties of the tubular 3D constructs were determined in vitro and in vivo using an infected (Actinomyces naeslundii) dentin tooth slice model and a canine method of periapical disease, respectively. The in vitro data indicated significant antimicrobial activity and the ability to eliminate bacterial biofilm inside dentinal tubules. In vivo histological findings demonstrated that, using the EB procedure, the tubular 3D triple antibiotic-eluting construct allowed the formation of an appropriate environment that led to apex closure and the ingrowth of a thin layer of osteodentin-like tissue into the root canal. Taken together, these findings indicate that our novel drug delivery construct is a promising biocompatible disinfection strategy for immature permanent teeth with necrotic pulps. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1576-1586, 2019.

Antimicrobial Efficacy of Triple Antibiotic-eluting Polymer Nanofibers against Multispecies Biofilm.

The elimination of microbial flora in cases of immature permanent teeth with necrotic pulp is both key and a challenging goal for the long-term success of regenerative therapy. Recent research has focused on the development of cell-friendly intracanal drug delivery systems. This in vitro study aimed to investigate the antimicrobial action of 3-dimensional (3D) tubular-shaped triple antibiotic-eluting nanofibrous constructs against a multispecies biofilm on human dentin. Polydioxanone polymer solutions, antibiotic-free or incorporated with metronidazole, ciprofloxacin, and minocycline, were electrospun into 3D tubular-shaped constructs. A multispecies biofilm consisting of Actinomyces naeslundii, Streptococcus sanguinis, and Enterococcus faecalis was forced inside the dentinal tubules via centrifugation in a dentin slice in vitro model. The infected specimens were exposed to 2 experimental groups (ie, 3D tubular-shaped triple antibiotic-eluting constructs and triple antibiotic paste [TAP]) and 2 control groups (7-day biofilm untreated and antibiotic-free 3D tubular-shaped constructs). Biofilm elimination was quantitatively analyzed with confocal laser scanning microscopy. Confocal laser scanning microscopic (CLSM) analysis showed a dense population of viable (green) bacteria adhered to dentin and penetrated into the dentinal tubules. Upon 3D tubular-shaped triple antibiotic-eluting nanofibrous construct exposure, nearly complete elimination of viable bacteria on the dentin surface and inside the dentinal tubules was shown in the CLSM images, which was similar (P < .05) to the bacterial death promoted by the TAP group but significantly greater when compared with both the antibiotic-free 3D tubular-shaped constructs and the control (saline). The proposed 3D tubular-shaped antibiotic-eluting construct showed pronounced antimicrobial effects against the multispecies biofilm tested and therefore holds significant clinical potential as a disinfection strategy before regenerative endodontics.

Tetracycline-incorporated polymer nanofibers as a potential dental implant surface modifier.

This study investigated the antimicrobial and osteogenic properties of titanium (Ti) disks superficially modified with tetracycline (TCH)-incorporated polymer nanofibers. The experiments were carried out in two phases. The first phase dealt with the synthesis and characterization (i.e., morphology, mechanical strength, drug release, antimicrobial activity, and cytocompatibility) of TCH-incorporated fibers. The second phase was dedicated to evaluating both the antimicrobial and murine-derived osteoprecursor cell (MC3T3-E1) response of Ti-modified with TCH-incorporated fibers. TCH was successfully incorporated into the submicron-sized and cytocompatible fibers. All TCH-incorporated mats presented significant antimicrobial activity against periodontal pathogens. The antimicrobial potential of the TCH-incorporated fibers-modified Ti was influenced by both the TCH concentration and bacteria tested. At days 5 and 7, a significant increase in MC3T3-E1 cell number was observed for TCH-incorporated nanofibers-modified Ti disks when compared to that of TCH-free nanofibers-modified Ti-disks and bare Ti. A significant increase in alkaline phosphatase (ALP) levels on the Ti disks modified with TCH-incorporated nanofiber on days 7 and 14 was seen, suggesting that the proposed surface promotes early osteogenic differentiation. Collectively, the data suggest that TCH-incorporated nanofibers could function as an antimicrobial surface modifier and osteogenic inducer for Ti dental implants. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2085-2092, 2017.

Triple Antibiotic Polymer Nanofibers for Intracanal Drug Delivery: Effects on Dual Species Biofilm and Cell Function.

INTRODUCTION: Root canal disinfection and the establishment of an intracanal microenvironment conducive to the proliferation/differentiation of stem cells play a significant role in regenerative endodontics. This study was designed to (1) investigate the antimicrobial efficacy of triple antibiotic-containing nanofibers against a dual-species biofilm and (2) evaluate the ability of dental pulp stem cells (DPSCs) to adhere to and proliferate on dentin upon nanofiber exposure. METHODS: Seven-day-old dual-species biofilm established on dentin specimens was exposed for 3 days to the following: saline (control), antibiotic-free nanofibers (control), and triple antibiotic-containing nanofibers or a saturated triple antibiotic paste (TAP) solution (50 mg/mL in phosphate buffer solution). Bacterial viability was assessed using the LIVE/DEAD assay (Molecular Probes, Inc, Eugene, OR) and confocal laser scanning microscopy. For cytocompatibility studies, dentin specimens after nanofiber or TAP (1 g/mL in phosphate buffer solution) exposure were evaluated for cell adhesion and spreading by actin-phalloidin staining. DPSC proliferation was assessed on days 1, 3, and 7. Statistics were performed, and significance was set at the 5% level. RESULTS: Confocal laser scanning microscopy showed significant bacterial death upon antibiotic-containing nanofiber exposure, differing significantly (P < .05) from antibiotic-free fibers and the control (saline). DPSCs showed enhanced adhesion/spreading on dentin specimens treated with antibiotic-containing nanofibers when compared with its TAP counterparts. The DPSC proliferation rate was similar on days 1 and 3 in antibiotic-free nanofibers, triple antibiotic-containing nanofibers, and TAP-treated dentin. Proliferation was higher (9-fold) on dentin treated with antibiotic-containing nanofibers on day 7 compared with TAP. CONCLUSIONS: Triple antibiotic-containing polymer nanofibers led to significant bacterial death, whereas they did not affect DPSC attachment and proliferation on dentin.

Synthesis and characterization of CaO-loaded electrospun matrices for bone tissue engineering.

OBJECTIVES: This study aims to synthesize and characterize biodegradable polymer-based matrices loaded with CaO nanoparticles for osteomyelitis treatment and bone tissue engineering. MATERIALS AND METHODS: Poly(ε-caprolactone) (PCL) and PCL/gelatin (1:1, w/w) solutions containing CaO nanoparticles were electrospun into fibrous matrices. Scanning (SEM) and transmission (TEM) electron microscopy, Fourier transformed infrared (FTIR), energy dispersive X-ray spectroscopy (EDS), contact angle (CA), tensile testing, and antibacterial activity (agar diffusion assay) against Staphylococcus aureus were performed. Osteoprecursor cell (MC3T3-E1) response (i.e., viability and alkaline phosphatase expression/ALP) and infiltration into the matrices were evaluated. RESULTS: CaO nanoparticles were successfully incorporated into the fibers, with the median fiber diameter decreasing after CaO incorporation. The CA decreased with the addition of CaO, and the presence of gelatin made the matrix very hydrophilic (CA = 0°). Increasing CaO concentrations progressively reduced the mechanical properties (p ≤ 0.030). CaO-loaded matrices did not display consistent antibacterial activity. MC3T3-E1 cell viability demonstrated the highest levels for CaO-loaded matrices containing gelatin after 7 days in culture. An increased ALP expression was consistently seen for PCL/CaO matrices when compared to PCL and gelatin-containing counterparts. CONCLUSIONS: Despite inconsistent antibacterial activity, CaO nanoparticles can be effectively loaded into PCL or PCL/gelatin fibers without negatively affecting the overall performance of the matrices. More importantly, CaO incorporation enhanced cell viability as well as differentiation capacity, as demonstrated by an increased ALP expression. CLINICAL SIGNIFICANCE: CaO-loaded electrospun matrices show potential for applications in bone tissue engineering.

Effects of Novel 3-dimensional Antibiotic-containing Electrospun Scaffolds on Dentin Discoloration.

INTRODUCTION: Although intracanal application of the triple antibiotic paste (TAP) may offer advantages (eg, disinfection), this practice has been associated with significant drawbacks, including tooth discoloration. In this study, the color change of dentin was monitored during treatment with distinct TAP pastes and novel tubular-shaped 3-dimensional electrospun scaffolds containing minocycline (MINO) or doxycycline (DOX). METHODS: Two TAP pastes (TAPMINO [MINO, metronidazole, and ciprofloxacin] and TAPDOX [DOX, metronidazole, and ciprofloxacin]), 4 scaffold-based groups containing MINO or DOX at distinct concentrations, 1 antibiotic-free scaffold, and 1 untreated group (control) were investigated. Human canines were sectioned at the cementoenamel junction and tubular-shaped scaffolds or paste were placed into the root canals and sealed. Color measurements (CIEL(*)a(*)b(*) parameters) were performed at baseline and after 1, 3, 7, 14, 21, and 28 days. Color changes were expressed as ΔE(*) values. In addition, scanning electron microscopy and energy-dispersive X-ray spectroscopy were also performed on the specimens after treatment. Data were analyzed using repeated measures analysis of variance (alpha = 0.05). RESULTS: All antibiotic-containing groups led to greater discoloration than the antibiotic-free groups. A severe discoloration occurred after 1 day. At the end of the experiment, antibiotic-treated samples exhibited crusts/agglomerates over the dentin surface, which totally or partially obliterated the dentinal tubules. The presence of MINO resulted in a greater color change than DOX. CONCLUSIONS: Scaffolds containing MINO or DOX produced similar color change to dentin when compared with their respective TAP systems, although DOX-related discoloration was less pronounced.

Antimicrobial Effects of Novel Triple Antibiotic Paste-Mimic Scaffolds on Actinomyces naeslundii Biofilm.

INTRODUCTION: Actinomyces naeslundii has been recovered from traumatized permanent teeth diagnosed with necrotic pulps. In this work, a triple antibiotic paste (TAP)-mimic scaffold is proposed as a drug-delivery strategy to eliminate A. naeslundii dentin biofilm. METHODS: Metronidazole, ciprofloxacin, and minocycline were added to a polydioxanone (PDS) polymer solution and spun into fibrous scaffolds. Fiber morphology, mechanical properties, and drug release were investigated by using scanning electron microscopy, microtensile testing, and high-performance liquid chromatography, respectively. Human dentin specimens (4 × 4 × 1 mm(3), n = 4/group) were inoculated with A. naeslundii (ATCC 43146) for 7 days for biofilm formation. The infected dentin specimens were exposed to TAP-mimic scaffolds, TAP solution (positive control), and pure PDS (drug-free scaffold). Dentin infected (7-day biofilm) specimens were used for comparison (negative control). Confocal laser scanning microscopy was done to determine bacterial viability. RESULTS: Scaffolds displayed a submicron mean fiber diameter (PDS = 689 ± 312 nm and TAP-mimic = 718 ± 125 nm). Overall, TAP-mimic scaffolds showed significantly (P ≤ .040) lower mechanical properties than PDS. Within the first 24 hours, a burst release for all drugs was seen. A sustained maintenance of metronidazole and ciprofloxacin was observed over 4 weeks, but not for minocycline. Confocal laser scanning microscopy demonstrated complete elimination of all viable bacteria exposed to the TAP solution. Meanwhile, TAP-mimic scaffolds led to a significant (P < .05) reduction in the percentage of viable bacteria compared with the negative control and PDS. CONCLUSIONS: Our findings suggest that TAP-mimic scaffolds hold significant potential in the eradication/elimination of bacterial biofilm, a critical step in regenerative endodontics.

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.