Nancy J. Lin

Antibacterial amorphous calcium phosphate nanocomposites with a quaternary ammonium dimethacrylate and silver nanoparticles

OBJECTIVES Calcium and phosphate ion-releasing resin composites are promising for remineralization. However, there has been no report on incorporating antibacterial agents to these composites. The objective of this study was to develop antibacterial and mechanically strong nanocomposites incorporating a quaternary ammonium dimethacrylate (QADM), nanoparticles of silver (NAg), and nanoparticles of amorphous calcium phosphate (NACP). METHODS The QADM, bis(2-methacryloyloxyethyl) dimethylammonium bromide (ionic dimethacrylate-1), was synthesized from 2-(N,N-dimethylamino)ethyl methacrylate and 2-bromoethyl methacrylate. NAg was synthesized by dissolving Ag 2-ethylhexanoate salt in 2-(tert-butylamino)ethyl methacrylate. Mechanical properties were measured in three-point flexure with bars of 2 mm×2 mm×25 mm (n=6). Composite disks (diameter=9 mm, thickness=2 mm) were inoculated with Streptococcus mutans. The metabolic activity and lactic acid production of biofilms were measured (n=6). Two commercial composites were used as controls. RESULTS Flexural strength and elastic modulus of NACP+QADM, NACP+NAg, and NACP+QADM+NAg matched those of commercial composites with no antibacterial property (p>0.1). The NACP+QADM+NAg composite decreased the titer counts of adherent S. mutans biofilms by an order of magnitude, compared to the commercial composites (p<0.05). The metabolic activity and lactic acid production of biofilms on NACP+QADM+NAg composite were much less than those on commercial composites (p<0.05). Combining QADM and NAg rendered the nanocomposite more strongly antibacterial than either agent alone (p<0.05). SIGNIFICANCE QADM and NAg were incorporated into calcium phosphate composite for the first time. NACP+QADM+NAg was strongly antibacterial and greatly reduced the titer counts, metabolic activity, and acid production of S. mutans biofilms, while possessing mechanical properties similar to commercial composites. These nanocomposites are promising to have the double benefits of remineralization and antibacterial capabilities to inhibit dental caries.

Synthesis and characterization of dimethacrylates containing quaternary ammonium functionalities for dental applications

OBJECTIVES The widespread incidence of recurrent caries highlights the need for improved dental restorative materials. The objective of this study was to synthesize low viscosity ionic dimethacrylate monomers (IDMAs) that contain quaternary ammoniums groups (antimicrobial functionalities) and are compatible with existing dental dimethacrylate-based monomers. Such monomers have the potential to copolymerize with other methacrylate monomers and produce antibacterial polymers. METHODS Two monomers (IDMA-1 and IDMA-2) were synthesized using the Menschutkin reaction and incorporated at 0-30% (by mass) into a 1:1 (by mass) bisphenol A glycerolate dimethacrylate (BisGMA):triethylene glycol dimethacrylate (TEGDMA) resin. Resin viscosity was quantified using rheology, and polymer degree of conversion (DC) and surface charge density were measured using Fourier transform infrared spectroscopy (FTIR) and fluorescein binding, respectively. Effects of IDMA-1 on initial attachment of Streptococcus mutans and on viability and metabolic activity (via reductase enzymes) of RAW 264.7 macrophage-like cells were quantified. RESULTS IDMA-1 and IDMA-2 were prepared and characterized. IDMA-1 was miscible with BisGMA:TEGDMA and slightly increased the resin viscosity and DC. As expected, polymeric surface charge density increased with increasing IDMA-1. Incorporation of 10% IDMA-1 into BisGMA:TEGDMA reduced bacterial colonization without affecting viability or metabolic activity of mammalian cells. Increasing IDMA-1 up to 30% had no additional effect on bacterial coverage, but ≥20% IDMA-1 significantly reduced macrophage density, viability, and metabolic activity. Leachables from polymers containing IDMA-1 were not cytotoxic. SIGNIFICANCE The Menschutkin reaction provides a facile, convenient means to synthesize new monomers with quaternary ammonium groups for dental and medical applications.

Synergistic enhancement of human bone marrow stromal cell proliferation and osteogenic differentiation on BMP-2-derived and RGD peptide concentration gradients

Rational design of bioactive tissue engineered scaffolds for directing bone regeneration in vivo requires a comprehensive understanding of cell interactions with the immobilized bioactive molecules. In the current study, substrates possessing gradient concentrations of immobilized peptides were used to measure the concentration-dependent proliferation and osteogenic differentiation of human bone marrow stromal cells. Two bioactive peptides, one derived from extracellular matrix protein (ECM), GRGDS, and one from bone morphogenic protein-2 (BMP-2), KIPKASSVPTELSAISTLYL, were found to synergistically enhance cell proliferation, up-regulate osteogenic mRNA markers bone sialoprotein (BSP) and Runt-related transcription factor 2, and produce mineralization at densities greater than 130 pmol cm(-2) (65 pmol cm(-2) for each peptide). In addition, COOH-terminated self-assembled monolayers alone led to up-regulated BSP mRNA levels at densities above 200 pmol cm(-2) and increased cell proliferation from day 3 to day 14. Taking further advantage of both the synergistic potentials and the concentration-dependent activities of ECM and growth-factor-derived peptides on proliferative activity and osteogenic differentiation, without the need for additional osteogenic supplements, will enable the successful incorporation of the bioactive species into biorelevant tissue engineering scaffolds.

Antibacterial and physical properties of calcium-phosphate and calcium-fluoride nanocomposites with chlorhexidine ,

OBJECTIVES Previous studies have developed calcium phosphate and fluoride releasing composites. Other studies have incorporated chlorhexidine (CHX) particles into dental composites. However, CHX has not been incorporated in calcium phosphate and fluoride composites. The objectives of this study were to develop nanocomposites containing amorphous calcium phosphate (ACP) or calcium fluoride (CaF(2)) nanoparticles and CHX particles, and investigate Streptococcus mutans biofilm formation and lactic acid production for the first time. METHODS Chlorhexidine was frozen via liquid nitrogen and ground to obtain a particle size of 0.62 μm. Four nanocomposites were fabricated with fillers of: nano ACP; nano ACP+10% CHX; nano CaF(2); nano CaF(2)+10% CHX. Three commercial materials were tested as controls: a resin-modified glass ionomer, and two composites. S. mutans live/dead assay, colony-forming unit (CFU) counts, biofilm metabolic activity, and lactic acid were measured. RESULTS Adding CHX fillers to ACP and CaF(2) nanocomposites greatly increased their antimicrobial capability. ACP and CaF(2) nanocomposites with CHX that were inoculated with S. mutans had a growth medium pH>6.5 after 3 d, while the control commercial composites had a cariogenic pH of 4.2. Nanocomposites with CHX reduced the biofilm metabolic activity by 10-20 folds and reduced the acid production, compared to the controls. CFU on nanocomposites with CHX were three orders of magnitude less than that on commercial composite. Mechanical properties of nanocomposites with CHX matched a commercial composite without fluoride. SIGNIFICANCE The novel calcium phosphate and fluoride nanocomposites could be rendered antibacterial with CHX to greatly reduce biofilm formation, acid production, CFU and metabolic activity. The antimicrobial and remineralizing nanocomposites with good mechanical properties may be promising for a wide range of tooth restorations with anti-caries capabilities.

The use of immobilized osteogenic growth peptide on gradient substrates synthesized via click chemistry to enhance MC3T3-E1 osteoblast proliferation

In this study, we report the use of surface immobilized peptide concentration gradient technology to characterize MC3T3-E1 osteoblast cell response to osteogenic growth peptide (OGP), a small peptide found naturally in human serum at mumol/L concentrations. OGP was coupled to oxidized self assembled monolayer (SAM) gradients by a polyethylene oxide (PEO) linker using click chemistry. After 4h incubation with MC3T3-E1 cells, OGP functionalized surfaces had higher cell attachment at low peptide concentrations compared to control gradients. By day 3, OGP gradient substrates had higher cell densities compared to control gradients at all concentrations. MC3T3-E1 cell doubling time was 35% faster on OGP substrates relative to SAM gradients alone, signifying an appreciable increase in cell proliferation. This increase in cell proliferation, or decrease in doubling time, due to OGP peptide was reduced by day 7. Hence, immobilized OGP increased cell proliferation from 0 days to 3 days at all densities indicating it may be useful as a proliferative peptide that can be used in tissue engineering substrates.

In situ formation of silver nanoparticles in photocrosslinking polymers

Nanocomposites of cross-linked methacrylate polymers with silver nanoparticles have been synthesized by coupling photoinitiated free radical polymerization of dimethacrylates with in situ silver ion reduction. A polymerizable methacrylate bearing a secondary amino functional group was used to increase the solubility of the silver salt in the hydrophobic resin system. Fourier transform infrared spectroscopy (FTIR) revealed that the silver ion reduction had no significant effect on the degree of vinyl conversion of the methacrylate. X-ray photoelectron spectroscopy (XPS) measurements showed an increased silver concentration at the composite surface compared to the expected concentration based on the total amount of silver salt added. Furthermore, the surface silver concentration leveled off when the silver salt mass fractions were 0.08% or greater. Composites with low concentrations of silver salt (< 0.08% by mass) exhibited comparable mechanical properties to those containing no silver. Transmission electron microscopy (TEM) confirmed that the silver nanoparticles formed within the polymer matrix were nanocrystalline in nature and primarily ≈ 3 nm in diameter, with some large particle aggregates. Composites containing silver nanoparticles were shown to reduce bacterial colonization with as little as 0.03% (by mass) silver salt, while additional amounts of silver salt did not further decrease their surface colonization. With a substantial effect on bacterial growth and minimal effects on mechanical properties, the in situ formation of silver nanoparticles within methacrylate materials is a promising technique for synthesizing antibacterial nanocomposites for biomedical applications.

Effect of amorphous calcium phosphate and silver nanocomposites on dental plaque microcosm biofilms

A dental composite containing amorphous calcium phosphate nanoparticles (NACP) was developed that released calcium (Ca) and phosphate (PO(4)) ions and possessed acid-neutralization capability. There has been little study on incorporation of antibacterial agents into calcium phosphate composites. The objective of this study was to investigate the effect of silver nanoparticle (NAg) mass fraction in NACP nanocomposite on mechanical properties and dental plaque microcosm biofilm for the first time. NACP nanoparticles of 116 nm were synthesized via a spray-drying technique. NAg nanoparticles were synthesized using Ag 2-ethylhexanoate and 2-(tert-butylamino)ethyl methacrylate, yielding NAg of particle size of 2.7 nm that were well-dispersed in the resin. Five NACP nanocomposites were fabricated with NAg mass fractions of 0, 0.028, 0.042, 0.088, and 0.175%, respectively. Mechanical properties of NACP nanocomposites containing 0-0.042% of NAg matched those of a commercial composite without antibacterial activity. Live/dead assay of dental plaque microcosm biofilms showed complete coverage with live bacteria on commercial composite. However, there were increasingly more dead bacteria with higher NAg content in the NACP nanocomposite. Colony-forming unit (CFU) counts for total microorganisms, total streptococci, and mutans streptococci for NACP nanocomposite with 0.042% NAg were about 1/4 those of commercial composite. Lactic acid production on NACP nanocomposite with 0.042% NAg was 1/3 that on commercial composite. In conclusion, novel NACP-NAg nanocomposites were developed which possessed good mechanical properties and potent antibacterial properties, with substantially reduced biofilm viability and lactic acid production. Hence, the NACP-NAg nanocomposites are promising for dental restorations with remineralizing and antibacterial capabilities.

Thermodynamic underpinnings of cell alignment on controlled topographies.

H /H o Surface topography is an important environmental cue for controlling cellular responses such as morphology, adhesion, alignment, migration, and gene expression. [ 1–7 ] Surface topographies with feature sizes covering the range of cell and cell components, i.e., from a few nanometers to tens of micrometers, have been broadly investigated with respect to effects on cell contact guidance (CG). [ 2 , 8 ] Despite the signifi cant work done to date, there has not been a satisfactory general explanation for the phenomenon, although many hypothesize that it is related to a biological response. In this paper, we fabricate a platform with precisely controlled surface topography, and use it to perform systematic cell studies that lead us to a new mechanistic understanding of CG under these conditions, which indicates that the response is rapid and largely physical rather than biological in nature. Below, we describe a two-step approach to fabricate submicrometer polymer gratings with continuous variations in grating height ( H ). First, large-area uniform gratings consisting of equally spaced lines were generated via nanoimprint lithography [ 9 , 10 ] on polystyrene (PS) and polymethylmethacrylate (PMMA). For each polymer, two sets of gratings were created with one-to-one line-to-space ratios, each with a pitch ( Λ ) of approximately 420 and 800 nm. Next, the uniformly patterned area was transformed to a continuous gradient in height by annealing on a thermal gradient stage for a fi xed time (see Supporting Information for details). A sketch of an annealed pattern with a height gradient is shown in the inset of Figure 1 . As indicated, the direction of the gradient is parallel to that of the polymer lines. Figure 1 shows position-dependent grating heights for two PS gratings ( Λ = 420 and 800 nm). The grating heights were characterized by atomic force microscopy (AFM) and are normalized in Figure 1 by the maximum height, H 0 , at x = 0.

Exploring cellular contact guidance using gradient nanogratings.

Nanoscale surface features that mimic extracellular matrix are critical environmental cues for cell contact guidance and are vital in advanced medical devices in order to manipulate cell behaviors. Among them, nanogratings (line-and-space gratings) are common platforms to study geometric effects on cell contact guidance, especially cell alignment, but generally are one pattern height per platform. In this study, we developed a strategy to fabricate controlled substrates with a wide range of pattern shapes and surface chemistries and to separate surface chemistry and topography effects. As a demonstration of this strategy, six nanograting platforms on three materials were fabricated and applied to examine and differentiate the effects of surface topography and surface chemistry on cell contact guidance of murine preosteoblasts. All of the six platforms contained the same gradient in pattern height (0 to ≈350 nm). They were prepared using nanoimprint lithography and annealing for thermoplastic materials (low molecular weight polystyrene (PS) and polymethylmethacrylate (PMMA)) and photoimprint for a thermoset material (a cross-linked dimethacrylate (DMA)). Each material contains two platforms that are only different in line-and-space pitch (420 or 800 nm). The DMA nanogratings had a reverse line-and-space profile to those of the PS and PMMA nanogratings. Using these platforms, a full range of cell alignment, from randomly orientated to completely parallel to the grating direction was achieved. Results from focal adhesion assays and scanning electronic microscopy indicated a change in cell-substrate contact from a noncomposite state (full contact) to a composite state (partial contact between cell and substrate) as pattern height increased. These gradient platforms allowed for the separation of surface chemistry and surface topography to provide insight into the mechanisms responsible for cell contact guidance on nanopatterned surfaces.

Effects of filler type and content on mechanical properties of photopolymerizable composites measured across two-dimensional combinatorial arrays.

Multicomponent formulations coupled with complex processing conditions govern the final properties of photopolymerizable dental composites. In this study, a single test substrate was fabricated to support multiple formulations with a gradient in degree of conversion (DC), allowing the evaluation of multiple processing conditions and formulations on one specimen. Mechanical properties and damage response were evaluated as a function of filler type/content and irradiation. DC, surface roughness, modulus, hardness, scratch deformation and cytotoxicity were quantified using techniques including near-infrared spectroscopy, laser confocal scanning microscopy, depth-sensing indentation, scratch testing and cell viability. Scratch parameters (depth, width, percent recovery) were correlated to composite modulus and hardness. Total filler content, nanofiller and irradiation time/intensity all affected the final properties, with the dominant factor for improved properties being a higher DC. This combinatorial platform accelerates the screening of dental composites through the direct comparison of properties and processing conditions across the same sample.