N-Acetyl Cysteine Modulates the Inflammatory and Oxidative Stress Responses of Rescued Growth-Arrested Dental Pulp Microtissues Exposed to TEGDMA in ECM.

Dental pulp is exposed to resin monomers leaching from capping materials. Toxic doses of the monomer, triethyleneglycol dimethacrylate (TEGDMA), impact cell growth, enhance inflammatory and oxidative stress responses, and lead to tissue necrosis. A therapeutic agent is required to rescue growth-arrested tissues by continuing their development and modulating the exacerbated responses. The functionality of N-Acetyl Cysteine (NAC) as a treatment was assessed by employing a 3D dental pulp microtissue platform. Immortalized and primary microtissues developed and matured in the extracellular matrix (ECM). TEGDMA was introduced at various concentrations. NAC was administered simultaneously with TEGDMA, before or after monomer addition during the development and after the maturation stages of the microtissue. Spatial growth was validated by confocal microscopy and image processing. Levels of inflammatory (COX2, NLRP3, IL-8) and oxidative stress (GSH, Nrf2) markers were quantified by immunoassays. NAC treatments, in parallel with TEGDMA challenge or post-challenge, resumed the growth of the underdeveloped microtissues and protected mature microtissues from deterioration. Growth recovery correlated with the alleviation of both responses by decreasing significantly the intracellular and extracellular levels of the markers. Our 3D/ECM-based dental pulp platform is an efficient tool for drug rescue screening. NAC supports compromised microtissues development, and immunomodulates and maintains the oxidative balance.

The self-renewal dental pulp stem cell microtissues challenged by a toxic dental monomer.

Dental pulp stem cells (DPSCs) regenerate injured/diseased pulp tissue and deposit tertiary dentin. DPSCs stress response can be activated by exposing cells to the monomer triethyleneglycol dimethacrylate (TEGDMA) and inducing the DNA-damage inducible transcript 4 (DDIT4) protein expression. The goal of the present study was to determine the impact of TEGDMA on the ability of DPSCs to maintain their self-renewal capabilities, develop and preserve their 3D structures and deposit the mineral. Human primary and immortalized DPSCs were cultured in extracellular matrix/basement membrane (ECM/BM) to support stemness and to create multicellular interacting layers (microtissues). The microtissues were exposed to the toxic concentrations of TEGDMA (0.5 and 1.5 mmol/l). The DPSCs spatial architecture was assessed by confocal microscopy. Mineral deposition was detected by alizarin red staining and visualized by stereoscopy. Cellular self-renewal transcription factor SOX2 was determined by immunocytochemistry. The microtissue thicknesses/vertical growth, surface area of the mineralizing microtissues, the percentage of area covered by the deposited mineral, and the fluorescence intensity of the immunostained cells were quantified ImageJ. DDIT4 expression was determined by a single molecule RNA-FISH technique and the cell phenotype was determined morphologically. DDIT4 expression was correlated with the cytotoxic phenotype. TEGDMA affected the structures of developing and mature microtissues. It inhibited the deposition of the mineral in the matrix while not affecting the SOX2 expression. Our data demonstrate that DPSCs retained their self-renewal capacity although their other functions were impeded. Since the DPSCs pool remained preserved, properties effected by the irritant should be restored by a proper rescue therapy.

Physicochemical, Mechanical, and Antimicrobial Properties of Novel Dental Polymers Containing Quaternary Ammonium and Trimethoxysilyl Functionalities.

The aims of this study were to evaluate the physicochemical and mechanical properties, antimicrobial (AM) functionality, and cytotoxic potential of novel dental polymers containing quaternary ammonium and trimethoxysilyl functionalities (e.g., N-(2-(methacryloyloxy)ethyl)-N,N-dimethyl-3-(trimethoxysilyl)propan-1-aminium iodide (AMsil1) and N-(2-(methacryloyloxy)ethyl)-N,N-dimethyl-11-(trimethoxysilyl)undecan-1-aminium bromide (AMsil2)). AMsil1 or AMsil2 were incorporated into light-cured (camphorquinone + ethyl-4-N,N-dimethylamino benzoate) urethane dimethacrylate (UDMA)/polyethylene glycol-extended UDMA/ethyl 2-(hydroxymethyl)acrylate (EHMA) resins (hereafter, UPE resin) at 10 or 20 mass %. Cytotoxic potential was assessed by measuring viability and metabolic activity of immortalized mouse connective tissue and human gingival fibroblasts in direct contact with monomers. AMsil-UPE resins were evaluated for wettability by contact angle measurements and degree of vinyl conversion (DVC) by near infra-red spectroscopy analyses. Mechanical property evaluations entailed flexural strength (FS) and elastic modulus (E) testing of copolymer specimens. The AM properties were assessed using Streptococcus mutans (planktonic and biofilm forms) and Porphyromonas gingivalis biofilm. Neither AMsil exhibited significant toxicity in direct contact with cells at biologically relevant concentrations. Addition of AMsils made the UPE resin more hydrophilic. DVC values for the AMsil-UPE copolymers were 2%-31% lower than that attained in the UPE resin control. The mechanical properties (FS and E) of AMsil-UPE specimens were reduced (11%-57%) compared to the control. Compared to UPE resin, AMsil1-UPE and AMsil2-UPE (10% mass) copolymers reduced S. mutans biofilm 4.7- and 1.7-fold, respectively (p ≤ 0.005). Although not statistically different, P. gingivalis biofilm biomass on AMsil1-UPE and AM AMsil2-UPE copolymer disks were lower (71% and 85%, respectively) than that observed with a commercial AM dental material. In conclusion, the AM function of new monomers is not inundated by their toxicity towards cells. Despite the reduction in mechanical properties of the AMsil-UPE copolymers, AMsil2 is a good candidate for incorporation into multifunctional composites due to the favorable overall hydrophilicity of the resins and the satisfactory DVC values attained upon light polymerization of AMsil-containing UDMA/PEG-U/EHMA copolymers.

Morphological and kinetic study of oral keratinocytes assembly on reconstituted basement membrane: Effect of TEGDMA.

OBJECTIVE: Open wounds of oral cavity require rapid healing. The cytotoxic monomer, triethylene glycol dimethacrylate (TEGDMA) can leach out from dental restoratives, reach the oral epithelial barrier and trigger an immune response. It is speculated that low and moderate concentrations of TEGDMA (0.5 and 1.5 mmol/L, respectively) influence the assembly kinetics and morphology of the keratinocyte layers overlying the extracellular matrix (ECM) in vivo. A three-dimensional cell system composed of immortalized oral keratinocytes (iMOK) cultured on reconstituted basement membrane (ECM) was used to investigate the development of epithelial layers upon exposure to TEGDMA. METHODS: Adherence and opposing movement of adjacent keratinocytes using actin protrusions (lamellipodia and filopodia) to create spheroids, and their fusion capacity to establish subsequent layers were tested at different time points. Fluorescent, confocal, differential interference contrast microscopy and image processing were employed to quantify the morphological modifications over time. RESULTS: Increasing concentrations of TEGDMA decreased the number of viable cells that utilized the actin protrusions and led to a delay in the communication/interaction among cells. Consequently, cells assembly was affected and the formation of more than a single layer prevented. Areas of basal-like proliferating cells were replaced with the increasing areas of non-replicating large cell population and extended gaps. CONCLUSIONS: These findings suggest that TEGDMA may prevent rapid sealing of open wounds by keratinocytes and suppress the establishment of a resistant and impermeable barrier against pathogen internalization. The iMOK-ECM-based platform facilitated the validation and quantification of solubilized dental materials impact on the reconstitution of epithelial layer.

Assessing the effect of triethyleneglycol dimethacrylate on tissue repair in 3D organotypic cultures.

Leachables from dental restoratives induce toxicity in gingival and pulp tissues and affect tissue regeneration/healing. Appropriate testing of these materials requires a platform that mimics the in vivo environment and allows the architectural self-assembly of cells into tissue constructs. In this study, we employ a new 3D model to assess the impact of triethyleneglycol dimethacrylate (TEGDMA) on early organization and advanced recruitment/accumulation of immortalized mouse gingival fibroblasts (GFs) and dental papilla mesenchymal cells (DPMCs) in extracellular matrix. We hypothesize that TEGDMA (1) interferes with the developmental architecture of GFs and DPMCs, and (2) inhibits the deposition of mineral. To test these hypotheses, GFs and DPMCs were incubated with the soluble TEGDMA at concentrations (0-2.5) mmol/L. Diameter and thickness of the constructs were determined by microscopic analysis. Cell differentiation was assessed by immunocytochemistry and the secreted mineral detected by alizarin-red staining. TEGDMA interfered with the development of GFs and/or DPMCs microtissues in a dose-dependent manner by inhibiting growth of inter-spherical cell layers and decreasing spheroid size (four to six times). At low/moderate TEGDMA levels, GFs organoids retained their structures while reducing thickness up to 21%. In contrast, at low TEGDMA doses, architecture of DPMC organoids was altered and thickness decreased almost twofold. Overall, developmental ability of TEGDMA-exposed GFs and DPMCs depended on TEGDMA level. GFs constructs were more resistant to structural modifications. The employed 3D platform was proven as an efficient tool for quantifying the effects of leachables on tissue repair capacities of gingiva and dental pulp.

Antimicrobial Monomers for Polymeric Dental Restoratives: Cytotoxicity and Physicochemical Properties.

A trend for the next generation of polymeric dental restoratives is to incorporate multifunctional capabilities to regulate microbial growth and remineralize tooth surfaces. Polymerizable 2-(methacryloyloxy)-N-(2-(methacryloyloxy)ethyl)-N,N-dimethylethan-1-aminium bromide (IDMA1) and N,N'-([1,1'-biphenyl]-2,2'-diylbis(methylene))bis(2-(methacryloyloxy)-N,N-dimethylethan-1-aminium) bromide (IDMA2), intended for utilization in bi-functional antimicrobial and remineralizing composites, were synthesized, purified with an ethanol-diethyl ether-hexane solvent system, and validated by nuclear magnetic resonance (¹H and 13C NMR) spectroscopy, mass spectrometry, and Fourier-transform infrared spectroscopy. When incorporated into light-curable urethane dimethacrylate (UDMA)/polyethylene glycol-extended UDMA (PEG-U)/ethyl 2-(hydroxymethyl)acrylate (EHMA) (assigned UPE) resins, IDMAs did not affect the overall resins' hydrophilicity/hydrophobicity balance (water contact angle: 60.8-65.5°). The attained degrees of vinyl conversion (DVC) were consistently higher in both IDMA-containing copolymers and their amorphous calcium phosphate (ACP) composites (up to 5% and 20%, respectively) reaching 92.5% in IDMA2 formulations. Notably, these high DVCs values were attained without an excessive increase in polymerization stress. The observed reduction in biaxial flexure strength of UPE-IDMA ACP composites should not prevent further evaluation of these materials as multifunctional Class V restoratives. In direct contact with human gingival fibroblasts, at biologically relevant concentrations, IDMAs did not adversely affect cell viability or their metabolic activity. Ion release from the composites was indicative of their strong remineralization potential. The above, early-phase biocompatibility and physicochemical tests justify further evaluation of these experimental materials to identify formulation(s) suitable for clinical testing. Successful completion is expected to yield a new class of restoratives with well-controlled bio-function, which will physicochemically, mechanically, and biologically outperform the conventional Class V restoratives.

Effects of protein-coated nanofibers on conformation of gingival fibroblast spheroids: potential utility for connective tissue regeneration.

Deep wounds in the gingiva caused by trauma or surgery require a rapid and robust healing of connective tissues. We propose utilizing gas-brushed nanofibers coated with collagen and fibrin for that purpose. Our hypotheses are that protein-coated nanofibers will: (i) attract and mobilize cells in various spatial orientations, and (ii) regulate the expression levels of specific extracellular matrix (ECM)-associated proteins, determining the initial conformational nature of dense and soft connective tissues. Gingival fibroblast monolayers and 3D spheroids were cultured on ECM substrate and covered with gas-blown poly-(DL-lactide-co-glycolide) (PLGA) nanofibers (uncoated/coated with collagen and fibrin). Cell attraction and rearrangement was followed by F-actin staining and confocal microscopy. Thicknesses of the cell layers, developed within the nanofibers, were quantified by ImageJ software. The expression of collagen1α1 chain (Col1α1), fibronectin, and metalloproteinase 2 (MMP2) encoding genes was determined by quantitative reverse transcription analysis. Collagen- and fibrin- coated nanofibers induced cell migration toward fibers and supported cellular growth within the scaffolds. Both proteins affected the spatial rearrangement of fibroblasts by favoring packed cell clusters or intermittent cell spreading. These cell arrangements resembled the structural characteristic of dense and soft connective tissues, respectively. Within three days of incubation, fibroblast spheroids interacted with the fibers, and grew robustly by increasing their thickness compared to monolayers. While the ECM key components, such as fibronectin and MMP2 encoding genes, were expressed in both protein groups, Col1α1 was predominantly expressed in bundled fibroblasts grown on collagen fibers. This enhanced expression of collagen1 is typical for dense connective tissue. Based on results of this study, our gas-blown, collagen- and fibrin-coated PLGA nanofibers are viable candidates for engineering soft and dense connective tissues with the required structural characteristics and functions needed for wound healing applications. Rapid regeneration of these layers should enhance healing of open wounds in a harsh oral environment.

High performance dental resin composites with hydrolytically stable monomers.

OBJECTIVE: The objectives of this project were to: 1) develop strong and durable dental resin composites by employing new monomers that are hydrolytically stable, and 2) demonstrate that resin composites based on these monomers perform superiorly to the traditional bisphenol A glycidyl dimethacrylate/triethylene glycol dimethacrylate (Bis-GMA/TEGDMA) composites under testing conditions relevant to clinical applications. METHODS: New resins comprising hydrolytically stable, ether-based monomer, i.e., triethylene glycol divinylbenzyl ether (TEG-DVBE), and urethane dimethacrylate (UDMA) were produced via composition-controlled photo-polymerization. Their composites contained 67.5wt% of micro and 7.5wt% of nano-sized filler. The performances of both copolymers and composites were evaluated by a battery of clinically-relevant assessments: degree of vinyl conversion (DC: FTIR and NIR spectroscopy); refractive index (n: optical microscopy); elastic modulus (E), flexural strength (F) and fracture toughness (KIC) (universal mechanical testing); Knoop hardness (HK; indentation); water sorption (Wsp) and solubility (Wsu) (gravimetry); polymerization shrinkage (Sv; mercury dilatometry) and polymerization stress (tensometer). The experimental UDMA/TEG-DVBE composites were compared with the Bis-GMA/TEGDMA composites containing the identical filler contents, and with the commercial micro hybrid flowable composite. RESULTS: UDMA/TEG-DBVE composites exhibited n, E, Wsp, Wsu and Sv equivalent to the controls. They outperformed the controls with respect to F (up to 26.8% increase), KIC (up to 27.7% increase), modulus recovery upon water sorption (full recovery vs. 91.9% recovery), and stress formation (up to 52.7% reduction). In addition, new composites showed up to 27.7% increase in attainable DC compared to the traditional composites. Bis-GMA/TEGDMA controls exceeded the experimental composites with respect to only one property, the composite hardness. Significantly, up to 18.1% lower HK values in the experimental series (0.458GPa) were still above the clinically required threshold of approx. 0.4GPa. SIGNIFICANCE: Hydrolytic stability, composition-controlled polymerization and the overall enhancement in clinically-relevant properties of the new resin composites make them viable candidates to replace traditional resin composites as a new generation of strong and durable dental restoratives.

Utility of Amorphous Calcium Phosphate-Based Scaffolds in Dental/Biomedical Applications.

Calcium phosphate (CaP) materials are important inorganic constituents in biological hard tissue. CaPs, including amorphous calcium phosphate (ACP) have been widely applied in dental and biomedical applications, such as tissue engineering. Scaffold constructs are commonly used as templates to create a biomimetic environment. This review considers ACP scaffold fabrication techniques, including tissue-engineered constructs with intrinsic incorporation of ACP as well as scaffolds formed via precipitation of mineralized solutions on a substrate. Attention is given to the approaches used to assess cellular and molecular responses elicited by ACP scaffolds, such as biocompatibility, cell conductivity, cell adhesion, cell differentiation, phenotypic profiles, and gene expression. Bioactivity of composite ACP scaffolds can be enhanced by incorporating biomolecules to create multi-functional properties. Herein we summarize the use of antibiotics, growth factors, and gene delivery systems to create multi-functional ACP scaffolds. Inasmuch as CaP materials have been investigated as drug delivery systems for many years, we briefly consider the potential of integrating these systems with existing ACP scaffold constructs and the potential for precision medicine.

Spatial development of gingival fibroblasts and dental pulp cells: Effect of extracellular matrix.

Cells sensing changes in their microenvironmental stiffness and composition alter their responses, accordingly. This study determines whether gingival fibroblasts (GFs) and dental pulp mesenchymal stem cells (DPMSCs) support the formation of continuous layers in vitro by mimicking the stiffness and protein composition of their native extracellular matrix (ECM). Immortalized cells were incubated with (i) 0-100% Matrigel-ECM (M-ECM) for 7-28d, and with (ii) collagen and fibrin matrices for 14d. Cultures were analyzed by phase-contrast, fluorescence and confocal microscopies. The diameters and surface areas were measured via ImageJ. Self-renewal markers were detected by RT-PCR and immunocytochemistry assays. GFs and DPMSCs developed spheroids interconnected by elongated cell bundles or layers, respectively, expressing the self-renewal markers. Increased matrix stiffness resulted in spheroids replacement by the interconnecting cells/layers. Both cells required 100% M-ECM to reduce their spheroid diameter. However, it reduced the surface area of the interconnecting layers. Those differences led to extended, spindle-shaped GFs vs. compact, ring-shaped DPMSCs constructs. Collagen and fibrin matrices developed continuous layers of tightly connected cells vs. distinctive scattered cell aggregates, respectively. The ability of GFs and DPMSCs to create tissue-like multicellular layers at various matrix conditions may be imprinted by cells' adaptation to mechanical forces and composition in vivo.

Biophysical characterization of functionalized titania nanoparticles and their application in dental adhesives.

It is demonstrated that carboxylic acid-functionalized titanium dioxide (TiO2) NPs produce significantly higher levels of reactive oxygen species (ROS) after visible light irradiation (400-800nm, 1600mW/cm2) in comparison to nonfunctionalized TiO2 NPs. The level of ROS produced under these irradiation conditions was not capable of generating oxidatively induced DNA damage in a cell-free system for TiO2 concentrations of 0.5mg/L or 5mg/L. In addition, specific incorporation of the acrylic acid-functionalized TiO2 NPs into dental composites allowed us to utilize the generated ROS to enhance photopolymerization (curing and degree of vinyl conversion (DC)) of resin adhesives and create mechanically superior and biocompatible materials for dental applications. Incorporation of the TiO2 NPs into selected dental composites increased the mean DC values by ≈7%. The modified TiO2 materials and dental composite materials were extensively characterized using thermogravimetric analysis, electron microscopy, Fourier transform infrared spectroscopy, and electron paramagnetic resonance. Notably, dental adhesives incorporated with acrylic acid-functionalized TiO2 NPs produced stronger bonds to human teeth following visible light curing in comparison to traditional dental adhesives not containing NPs with an increase in the shear bond strength of ≈29%. In addition, no leaching of the incorporated NPs was detectable from the dental adhesives after 2500 thermal cycles using inductively coupled plasma-optical emission spectroscopy, indicating that biocompatibility of the adhesives was not compromised after extensive aging. These findings suggest that NP-induced ROS may be useful to produce enhanced nanocomposite materials for selected applications in the medical device field. STATEMENT OF SIGNIFICANCE: Titanium dioxide nanoparticles (TiO2 NPs) have unique photocatalytic, antibacterial and UV-absorbing properties that make them beneficial additives in adhesives and composites. However, there is concern that the reactive oxygen species (ROS) produced by photoactivated TiO2 NPs might pose toxicological risks. We demonstrate that it is possible to incorporate acid-functionalized TiO2 NPs into dental resins which can be applied as dental adhesives to human teeth. The ROS generated by these NPs through visible-light irradiation may be utilized to increase the degree of vinyl conversion of resins, leading to adhesives that have an enhanced shear-bond strength to human teeth. Investigation into the potential genotoxicity of the NPs and their potential for release from dental composites indicated a low risk of genotoxic effects.

Bioactive Polymeric Materials for Tissue Repair.

Bioactive polymeric materials based on calcium phosphates have tremendous appeal for hard tissue repair because of their well-documented biocompatibility. Amorphous calcium phosphate (ACP)-based ones additionally protect against unwanted demineralization and actively support regeneration of hard tissue minerals. Our group has been investigating the structure/composition/property relationships of ACP polymeric composites for the last two decades. Here, we present ACP's dispersion in a polymer matrix and the fine-tuning of the resin affects the physicochemical, mechanical, and biological properties of ACP polymeric composites. These studies illustrate how the filler/resin interface and monomer/polymer molecular structure affect the material's critical properties, such as ion release and mechanical strength. We also present evidence of the remineralization efficacy of ACP composites when exposed to accelerated acidic challenges representative of oral environment conditions. The utility of ACP has recently been extended to include airbrushing as a platform technology for fabrication of nanofiber scaffolds. These studies, focused on assessing the feasibility of incorporating ACP into various polymer fibers, also included the release kinetics of bioactive calcium and phosphate ions from nanofibers and evaluate the biorelevance of the polymeric ACP fiber networks. We also discuss the potential for future integration of the existing ACP scaffolds into therapeutic delivery systems used in the precision medicine field.

Remineralizing amorphous calcium phosphate based composite resins: the influence of inert fillers on monomer conversion, polymerization shrinkage, and microhardness.

AIM: To determine if the addition of inert fillers to a bioactive dental restorative composite material affects its degree of conversion (DC), polymerization shrinkage (PS), and microhardness (HV). METHODS: Three amorphous calcium phosphate (ACP)-based composite resins: without added fillers (0-ACP), with 10% of barium-glass fillers (Ba-ACP), and with 10% of silica fillers (Si-ACP), as well as commercial control (Ceram•X, Dentsply DeTrey) were tested in laboratory conditions. The amount of ACP (40%) and the composition of the resin mixture (based on ethoxylated bisphenol A dimethacrylate) was the same for all ACP materials. Fourier transform infrared spectroscopy was used to determine the DC (n=40), 20 min and 72 h after polymerization. Linear PS and Vickers microhardness (n=40) were also evaluated. The results were analyzed by paired samples t test, ANOVA, and one-way repeated measures ANOVA with Student-Newman-Keuls or Tukey's post-hoc test (P=0.05). RESULTS: The addition of barium fillers significantly increased the DC (20 min) (75.84±0.62%) in comparison to 0-ACP (73.92±3.08%), but the addition of silica fillers lowered the DC (71.00±0.57%). Ceram•X had the lowest DC (54.93±1.00%) and linear PS (1.01±0.24%) but the highest HV (20.73±2.09). PS was significantly reduced (P<0.010) in both Ba-ACP (1.13±0.25%) and Si-ACP (1.17±0.19%) compared to 0-ACP (1.43±0.21%). HV was significantly higher in Si-ACP (12.82±1.30) than in 0-ACP (10.54±0.86) and Ba-ACP (10.75±0.62) (P<0.010). CONCLUSION: Incorporation of inert fillers to bioactive remineralizing composites enhanced their physical-mechanical performance in laboratory conditions. Both added fillers reduced the PS while maintaining high levels of the DC. Silica fillers additionally moderately improved the HV of ACP composites.

Structural and recovery mechanisms of 3D dental pulp cell microtissues challenged with Streptococcusmutans in extracellular matrix environment.

Cariopathogen Streptococcus mutans exists in infected dental pulp of deciduous teeth and is frequently linked with heart diseases. Organotypic (3D) dental pulp stem cell (DPSC) cultures/microtissues, developed to mimic the physiological conditions in vivo, were utilized to assess the bacterial impact on their (i) 3D structural configuration and (ii) recovery mechanisms. The cultures, developed in extracellular matrix (ECM) bio-scaffold (Matrigel™), interacted with WT and GFP-tagged bacterial biofilms by permitting their infiltration through the ECM. Challenged cell constructs were visualized by F-actin/nuclei staining. Their pluripotency (Sox2) and differentiation (osteocalcin) markers were assessed by immunocytochemistry. Secreted mineral was detected by alizarin red, and 3D structural arrangements were analysed by epi-fluorescence and confocal scanning microscopy. Bacterial biofilm/ECM-embedded DPSC interactions appeared in distinct areas of the microtissues. Bacterial attachment to the cell surface occurred without evidence of invasion. Surface architecture of the challenged versus unchallenged microtissues was apparently unaltered. However, significant increases in thickness (138.42 vs 106.51 µm) and bacterial penetration were detected in challenged structures causing canal-like microstructures with various diameters (12.94 -42.88 µm) and average diameter of 20.66 to 33.42 µm per microtissue. Challenged constructs expressed pluripotency and differentiation markers and secreted the mineral. Presented model shows strong potential for assessing pulp-pathogen interactions in vivo. S. mutans infiltrated and penetrated the microtissues but did not invade the cells or compromise major cell repair mechanisms. These findings would suggest reexamining the role of S. mutans as an endodontic pathogen and investigating DPSC resistance to its pathogenicity.

Tuning photo-catalytic activities of TiO2 nanoparticles using dimethacrylate resins.

OBJECTIVE: The unique photo-catalytic activities (PCAs) of titanium dioxide nanoparticles (TiO2 NPs) made them attractive in many potential applications in medical devices. The objective of this study is to optimize the benefits of PCAs of TiO2 NPs through varying chemical structures of dimethacrylate resins. METHODS: TiO2 NPs were functionalized to improve the PCAs and bonding to the resins. The PCAs of TiO2 NPs were evaluated using electron paramagnetic resonance (EPR) and UV-vis spectroscopy to determine the amount of the radicals generated and the energy required for their production, respectively. The beneficial effects of the radicals were assessed through: (1) the improvement of degree of vinyl conversion (DC) and (2) modification of resin hydrophilicity. One-way ANOVA with a 95% confidence interval was used to indicate the significant differences between the experimental groups. RESULTS: EPR and UV-vis results clearly showed that the functionalization of TiO2 NPs enhanced PCAs in terms of generating radicals under visible light irradiation. The presence of hydroxyl and carboxylic acid functionalities played an important role in DC enhancement and hydrophilicity modification. The DC could be increased up to 22% by adding only 0.1wt% TiO2 NPs. Viscosity of the resins had minimal or no role in DC improvement through TiO2 NPs. In resins with abundant hydroxyl groups, radicals were more effective in making the resin more hydrophilic. SIGNIFICANCE: Knowledge learned from this study will help formulating nano-composites with optimized use of TiO2 PCAs as co-initiators for photo-polymerization, additives for making super-hydrophilic materials and/or antibacterial agents.

Polymeric dental composites based on remineralizing amorphous calcium phosphate fillers.

For over two decades we have systematically explored structure-composition-property relationships of amorphous calcium phosphate (ACP)-based polymeric dental composites. The appeal of these bioactive materials stems from their intrinsic ability to prevent demineralization and/or restore defective tooth structures via sustained release of remineralizing calcium and phosphate ions. Due to the compositional similarity of the ACP to biological tooth mineral, ACP-based composites should exhibit excellent biocompatibility. Research described in this article has already yielded remineralizing sealants and orthodontic adhesives as well as a prototype root canal sealer. Our work has also contributed to a better understanding on how polymer matrix structure and filler/matrix interactions affect the critical properties of these polymeric composites and their overall performance. The addition of antimicrobial compounds to the formulation of ACP composites could increase their medical and dental regenerative treatment applications, thereby benefiting an even greater number of patients.

Airbrushed composite polymer Zr-ACP nanofiber scaffolds with improved cell penetration for bone tissue regeneration.

Electrospun polymer nanofibers have multiple applications in the tissue engineering field despite limited cell penetration within the scaffolds and slow synthesis rates. Airbrushing, a proposed alternative to traditional electrospinning, is a technique capable of synthesizing open structure nanofiber scaffolds at high rates. In this study, three biocompatible polymers-poly-D,L-lactic acid (P-DL-LA), polycaprolactone (PCL), and poly(methyl methacrylate) (PMMA), were airbrushed to form networks for bone tissue regeneration. All three polymers were loaded with up to 20% (w/w) zirconium-modified amorphous calcium phosphate (Zr-ACP). A simple one-step mix and straightforward material deposition yielded open structure networks with well-distributed Zr-ACP. Cell penetration within the airbrushed scaffolds was found to be more than twice the cell penetration within conventional electrospun networks. The airbrushed polymer network supported cell growth and differentiation. Cells grown on the Zr-ACP in P-DL-LA fibers exhibited improved levels of osteocalcin protein with an increase in the Zr-ACP content by day 16. This airbrushing method promises to be a viable and attractive alternative to currently used electrospinning techniques in the formation of composite 3D nanofiber scaffolds for tissue engineering applications.

Structural and dynamical studies of acid-mediated conversion in amorphous-calcium-phosphate based dental composites.

OBJECTIVE: To investigate the complex structural and dynamical conversion process of the amorphous-calcium-phosphate (ACP)-to-apatite transition in ACP based dental composite materials. METHODS: Composite disks were prepared using zirconia hybridized ACP fillers (0.4 mass fraction) and photo-activated Bis-GMA/TEGDMA resin (0.6 mass fraction). We performed an investigation of the solution-mediated ACP-to-apatite conversion mechanism in controlled acidic aqueous environment with in situ ultra-small angle X-ray scattering based coherent X-ray photon correlation spectroscopy and ex situ X-ray diffraction, as well as other complementary techniques. RESULTS: We established that the ACP-to-apatite conversion in ACP composites is a two-step process, owing to the sensitivity to local structural changes provided by coherent X-rays. Initially, ACP undergoes a local microstructural rearrangement without losing its amorphous character. We established the catalytic role of the acid and found the time scale of this rearrangement strongly depends on the pH of the solution, which agrees with previous findings about ACP without the polymer matrix being present. In the second step, ACP is converted to an apatitic form with the crystallinity of the formed crystallites being poor. Separately, we also confirmed that in the regular Zr-modified ACP the rate of ACP conversion to hydroxyapatite is slowed significantly compared to unmodified ACP, which is beneficial for targeted slow release of functional calcium and phosphate ions from dental composite materials. SIGNIFICANCE: For the first time, we were able to follow the complete solution-mediated transition process from ACP to apatite in this class of dental composites in a controlled aqueous environment. A two-step process, suggested previously, was conclusively identified.

Reinforcement of experimental composite materials based on amorphous calcium phosphate with inert fillers.

OBJECTIVES: The aim of this study was to examine the influence of the addition of glass fillers with different sizes and degrees of silanization percentages to remineralizing composite materials based on amorphous calcium phosphate (ACP). METHODS: Four different materials were tested in this study. Three ACP based materials: 0-ACP (40 wt% ACP, 60 wt% resin), Ba-ACP (40 wt% ACP, 50 wt% resin, 10 wt% barium-glass) and Sr-ACP (40 wt% ACP, 50 wt% resin, 10 wt% strontium-glass) were compared to the control material, resin modified glass ionomer (Fuji II LC capsule, GC, Japan). The fillers and composites were characterized using scanning electron microscopy. Flexural strength and modulus were determined using a three-point bending test. Calcium and phosphate ion release from ACP based composites was measured using inductively coupled plasma atomic emission spectroscopy. RESULTS: The addition of barium-glass fillers (35.4 (29.1-42.1) MPa) (median (25-75%)) had improved the flexural strength in comparison to the 0-ACP (24.8 (20.8-36.9) MPa) and glass ionomer control (33.1 (29.7-36.2) MPa). The admixture of strontium-glass (20.3 (19.5-22.2) MPa) did not have any effect on flexural strength, but significantly improved its flexural modulus (6.4 (4.8-6.9) GPa) in comparison to 0-ACP (3.9 (3.4-4.1) GPa) and Ba-ACP (4.6 (4.2-6.9) GPa). Ion release kinetics was not affected by the addition of inert fillers to the ACP composites. SIGNIFICANCE: Incorporation of barium-glass fillers to the composition of ACP composites contributed to the improvement of flexural strength and modulus, with no adverse influence on ion release profiles.

Effect of silanized nanosilica addition on remineralizing and mechanical properties of experimental composite materials with amorphous calcium phosphate.

OBJECTIVES: Experimental composite resins with amorphous calcium phosphate (ACP) have the potential to regenerate demineralized tooth structures. The aim of the study was to investigate the effect of the addition of silanized silica nanofillers to the ACP-based composites on their mechanical properties and the kinetics of calcium and phosphate release. MATERIALS AND METHODS: The test materials comprised 5 wt% (5-ACP) or 10 wt% (10-ACP) of silanized silica admixed to the 40 wt% ACP and 50 or 55 wt% resin. The ACP control (0-ACP) contained 40 wt% ACP and 60 wt% resin. Additionally, composite material CeramX (Dentsply, Germany) was included as control. Three-point bending test was performed to calculate flexural strength and modulus of elasticity. Inductively coupled plasma atomic emission spectroscopy was used for measurement of ion release. The micromorphology of calcium phosphate depositions on composite samples has been qualitatively evaluated using a scanning electron microscope. The results were analyzed using Mann-Whitney and Wilcoxon rank sum tests (α < 0.05). RESULTS: Ion release was enhanced by the silica fillers, when compared to the 0-ACP. Although not statistically significant, flexural strength of 10-ACP was improved by 46 % compared to 0-ACP. Flexural modulus of 5-ACP was significantly higher than 0-ACP. CONCLUSIONS: The admixture of silanized fillers seems to be a promising approach for the improvement of mechanical and remineralizing properties of ACP composite resins. CLINICAL RELEVANCE: ACP-based composite resins with modified composition could serve as an effective remineralizing aid as base materials in restorative dental medicine.