Combinatorial Chemistry & High Throughput Screening, 2009, 12, 619-625 619 1386-2073/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd. Quantification of Cell Response to Polymeric Composites Using a Two- Dimensional Gradient Platform Nancy J. Lin *,1 , Haiqing Hu 2 , Lipin Sung 2 and Sheng Lin-Gibson *,1 1 Polymers Division, National Institute of Standards and Technology, Gaithersburg, MD 20899-8543, USA 2 Materials and Construction Research Division, National Institute of Standards and Technology, Gaithersburg, MD 20899-8615, USA Abstract: A simple and straightforward screening process to assess the toxicity and corresponding cell response of dental composites would be useful prior to extensive in vitro or in vivo characterization. To this end, gradient composite samples were prepared with variations in filler content/type and in degree of conversion (DC). The DC was determined using near infrared spectroscopy (NIR), and the surface morphology was evaluated by laser scanning confocal microscopy (LSCM). RAW 264.7 macrophage-like cells were cultured directly on the composite gradient samples, and cell viability, density, and area were measured at 24 h. All three measures of cell response varied as a function of material properties. For in- stance, compositions with higher filler content had no reduction in cell viability or cell density, even at low conversions of 52%, whereas significant decreases in viability and density were present when the filler content was 35% or below (by mass). The overall results demonstrate the complexity of the cell-material interactions, with properties including DC, filler type, filler mass ratio, and surface morphology influencing the cell response. The combinatorial approach described herein enables simultaneous screening of multiple compositions and material properties, providing a more thorough characteriza- tion of cell response for the improved selection of biocompatible composite formulations and processing conditions. Keywords: Biocompatibility, cell spreading, cell viability, combinatorial, dental composites, degree of conversion, surface roughness, cell-material interaction. INTRODUCTION Polymeric composites have steadily gained a broader acceptance as dental restorative materials largely due to their superior aesthetics [1]. Most dental composites are com- prised of a photopolymerizable dimethacrylate matrix rein- forced with filler particles. Although several properties have improved over the past few years, particularly with respect to strength and wear resistance, many other properties critical to clinical longevity remain less than optimal [2]. In addition to efforts focused on improving various physical and me- chanical properties, there exists a renewed interest in the biocompatibility of this class of materials [3-5]. An appro- priate biological response for a given application is essential for successful clinical use of new and existing dental materi- als. Materials with poor biocompatibility may result in toxic- ity, mutagenicity, sensitization, inflammation, and a loss of pulp vitality [6], and often lead to restoration failure and replacement. Many of the deleterious biological effects of dental materials are due to the leaching of various chemical species. In addition, dental materials can also affect tissues in the oral and maxillofacial environment via direct contact. For instance, some restorations may directly contact the gin- gival tissue, and most endodontic materials are typically in direct contact with the pulp chamber. Moreover, as dental materials become increasingly bioactive and interactive with the oral tissues, a thorough characterization of the biological response is critical. *Address correspondence to these authors at the Polymers Division, Na- tional Institute of Standards and Technology, Gaithersburg, MD 20899- 8543, USA; E-mail: nancy.lin@nist.gov, slgibson@nist.gov A large amount of work has been devoted to improving the clinical performance of dental composites by incorporat- ing new chemistries or improving existing formulations. Multiple chemical and processing parameters have been shown to affect physical and mechanical properties as well as biocompatibility [7]. The matrix alone is typically com- prised of a binary or ternary resin mixture in which the mate- rial properties can be easily adjusted by chemical and com- positional changes and a large number of processing parame- ters. The addition of the filler phase, typically comprised of different filler types, sizes, shapes, and surface treatments, further complicates the formulation. The filler loading is often determined by the desired application. For example, flowable composites such as those used in dental adhesives are lightly filled. On the other hand, dental restoratives tar- geted for posterior tooth restorations are more highly filled to withstand greater forces of mastication. Depending on the desired properties, different filler sizes (among other pa- rameters) are selected. Larger fillers generally permit higher filler loading, which in turn increases the composite strength and reduces the overall composite shrinkage. A nano-filler is often incorporated to improve the wear resistance and some- times to improve the stability of the composite paste. Thus, many of the dental composites commercially available today are dual-filled systems to achieve both high filler loading and enhanced wear resistance [1]. Combinatorial and high throughput (C&HT) methods, which are useful tools in chemistry [8, 9] and biology [10, 11], have become increasingly popular in materials discov- ery, characterization, and optimization and are ideal for studying complex systems with multiple parameters [12]. Advantages for utilizing combinatorial methods include faster data acquisition, more thorough examination of the