Combinatorial Chemistry & High Throughput Screening - Volume 12, Issue 6, 2009

Despite significant investment in tissue engineering research, few successful products have come to market. Hence, there is a need to accelerate tissue engineering research. One approach to accelerating development is combinatorial and high throughput (CHT) screening. Combinatorial approaches are utilized extensively for pharmaceutical research and their application to biomaterials development is growing. This special issue is a collection of primary research articles and reviews that highlight the state of the art in this exciting and burgeoning field of research. The basic premise of combinatorial and high throughput biomaterials research is the development of methods for rapidly screening cell response to libraries of biomaterials. Just as combinatorial libraries of candidate drug compounds are screened for therapeutic effects using in vitro cell culture, combinatorial libraries of biomaterials can be screened for their ability to positively influence cells. Typically, miniaturized specimens of biomaterials are fabricated in the form of gradients or arrays such that many biomaterials, compositions and material properties are present in each specimen or library. Cells are then seeded onto the library specimens and responses such as adhesion, morphology, proliferation, migration and differentiation are assessed. Instead of responding to soluble factors as is characteristic in drug screening, the cells in a biomaterials screen are responding to the properties of their adhesive biomaterial substrate. The chemical and physical properties of a material will strongly influence cell response. It is also important to keep in mind that cells rarely interact directly with a biomaterial. Proteins present in blood in vivo or serum in vitro immediately adsorb onto most materials. Thus, cell response to a biomaterial is strongly influenced (and some would say dominated) by the species, amount and conformation of proteins that adsorb onto a biomaterial. Advances in combinatorial screening of biomaterials are being made at all levels including biomaterials synthesis, library fabrication, cell screening and data analysis; and articles that focus on each of these levels have been contributed to this special issue. In addition to their applications in screening, biomaterial gradients also have potential applications as functional materials. A scaffold with a gradient in properties could serve as a template for the generation of a graded tissue. Gradients in composition and properties are common at the boundaries of most tissues and the need for “interface tissue engineering” is well-recognized. I hope you enjoy the articles in this special issue. It will be exciting to see where the innovators in this field will take us next.

Gradients and arrays have become very useful to the fields of tissue engineering and biomaterials. Both gradients and arrays make efficient platforms for screening cell response to biomaterials. Graded biomaterials also have functional applications and make useful substrates for fundamental studies of cell phenomena such as migration. This article will review the use of gradients and arrays in tissue engineering and biomaterials research, with a focus on cellular and biologic responses.

Stem cells have great potential as cell sources for regenerative medicine due to both their self-renewal and multi-lineage differentiation capacity. Despite advances in the field of stem cell biology, major challenges remain before stem cells can be widely used for therapeutic purposes. One challenge is to develop reproducible methods to control stem cell growth and differentiation. The niche in which stem cells reside is a complex, multi-factorial environment. In contrast to using cells alone, biomaterials can provide initial structural support, and allow cells to adhere, proliferate and differentiate in a three-dimensional environment. Researchers have incorporated signals into the biomaterials that can promote desired cell functions in a spatially and temporally controlled manner. Despite progress in biomaterial design and methods to modulate cellular behavior, many of the complex signal networks that regulate cell-material interactions remain unclear. Due to the vast numbers of material properties to be explored and the complexity of cell-surface interactions, it is often difficult to optimize stem cell microenvironments using conventional, iterative approaches. To address these challenges, high throughput screening of combinatorial libraries has emerged as a novel approach to achieve rapid screening with reduced materials and costs. In this review, we discuss recent research in the area of high throughput approaches for characterization and optimization of cellular interactions with their microenvironments. In contrast to conventional approaches, screening combinatorial libraries can result in the discovery of unexpected material solutions to these complex problems.

The understanding of fundamental phenomena involved in tissue engineering and regenerative medicine is continuously growing and leads to the demand for three-dimensional (3D) models that better represent tissue architecture and direct cells into the proper lineage for specific tissue repair. Porous 3D scaffolds are used in tissue engineering as templates to allow cell attachment and tissue formation. Scaffold design plays a central role in guiding cells to synthesize and maintain new tissues. While a number of techniques have been developed and are now in use for high-throughput screening of combinatorial factors involved in biotechnology in two-dimensions, high-throughput screening in 3D is still in its infancy. There is a broad interest in developing similar techniques to assess which variables are critical in designing 3D scaffolds to achieve proper and lasting tissue regeneration. We describe, herein, a number of studies adopting smart scaffold design and in vitro and in vivo analysis as the basis for 3D model systems for evaluating combinatorial factors influencing cell differentiation and tissue formation.

Chemotaxis is essential for many biological processes. Much of our understanding of the mechanisms underlying chemotaxis is based on a variety of in vitro assays. We review these assays, dividing them into groups depending on the process used to generate the gradient. We describe how each method works, its strengths and limitations, and provide some information about the kinds of cells that have been studied with each assay.

The emphasis in the field of orthopaedic tissue engineering is on imparting biomimetic functionality to tissue engineered bone or soft tissue grafts and enabling their translation to the clinic. A significant challenge in achieving extended graft functionality is engineering the biological fixation of these grafts with each other as well as with the host environment. Biological fixation will require re-establishment of the structure-function relationship inherent at the native soft tissue-to-bone interface on these tissue engineered grafts. To this end, strategic biomimicry must be incorporated into advanced scaffold design. To facilitate integration between distinct tissue types (e.g., bone with soft tissues such as cartilage, ligament, or tendon), a stratified or multi-phasic scaffold with distinct yet continuous tissue regions is required to pre-engineer the interface between bone and soft tissues. Using the ACL-to-bone interface as a model system, this review outlines the strategies for stratified scaffold design for interface tissue engineering, focusing on identifying the relevant design parameters derived from an understanding of the structure-function relationship inherent at the soft-to-hard tissue interface. The design approach centers on first addressing the challenge of soft tissue-to-bone integration ex vivo, and then subsequently focusing on the relatively less difficult task of bone-to-bone integration in vivo. In addition, we will review stratified scaffold design aimed at exercising spatial control over heterotypic cellular interactions, which are critical for facilitating the formation and maintenance of distinct yet continuous multi-tissue regions. Finally, potential challenges and future directions in this emerging area of advanced scaffold design will be discussed.

A recently developed technique for the measurement of cell migration on surface bound gradients was used to assay the behavior of microvascular endothelial cells on a range of fibronectin gradient slopes in the presence of soluble promoters and inhibitors of chemotaxis. Directional microvascular endothelial cell migration was shown to increase with increasing gradient slope with no significant change in cellular persistence time or random cell speed. Uniformly distributed soluble chemotactic factor in the hMEC growth media enhanced directional migration. The addition of migrationinhibiting LY294002 eliminated the directional component of cell migration at a 5 μM dosing. These experiments broaden the understanding of the directional nature of cell motion and present a reliable system for the quantitative study of cell migration in complex conditions in vitro.

Current methods for engineering immobilized, ‘solid-phase’ growth factor patterns have not addressed the need for presentation of the growth factors in a biologically-relevant context. We developed an inkjet printing methodology for creating solid-phase patterns of unmodified growth factors on native biological material substrates. We demonstrate this approach by printing gradients of fluorescently labeled bone morphogenetic protein-2 (BMP-2) and insulin-like growth factor-II (IGF-II) bio-inks on fibrin-coated surfaces. Concentration gradients were created by overprinting individual substrate locations using a dilute bio-ink to modulate the surface concentration of deposited growth factor. Persistence studies using fluorescently-labeled BMP-2 verified that the gradients retained their shape for up to 7 days. Desorption experiments performed with 125I-BMP-2 and 125I-IGF-II were used to quantify the surface concentration of growth factor retained on the substrate for up to 10 days in serum containing media after rinsing of the unbound growth factor. The inkjet method is programmable so the gradient shape can be easily modified as demonstrated by printed linear gradients with varying slopes and exponential gradients. In addition, the versatility of this method enabled combinatorial arrays of multiple growth factors to be created by printing overlapping patterns. The overlapping printing method was used to create a combinatorial square pattern array consisting of various surface concentrations of BMP-2 and fibroblast growth factor-2 (FGF-2). C2C12 myogenic precursor cells were seeded on the arrays and alkaline phosphatase staining was performed to determine the effect of FGF-2 and BMP-2 surface concentration on guiding C2C12 cells towards an osteogenic lineage. These results demonstrate the utility of inkjet printing for creating orthogonal growth factor gradients to investigate how combinations of immobilized growth factors influence cell fate.

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 instance, 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 characterization of cell response for the improved selection of biocompatible composite formulations and processing conditions.

Increasingly, combinatorial libraries are used to screen large numbers of complex chemically- and physicallypatterned polymers against cell response. There is a strong need to extract meaningful relationships between cell function and material surface features from these experiments. A novel high-throughput cell-material screening strategy, based upon local cell-feature analysis (LCFA) was applied to screen osteoblast proliferation behavior on combinatorial libraries of phase-separated PLGA and PCL. Traditional factor importance analysis, which uses summary statistical inference to identify significant variables, indicated that one controlling material surface feature was PCL diameter. However, the summary statistic analysis was unable to uncover more subtle relationships. The LCFA method, based on histograms of distances between cells and microstructures, was able to identify non-linear, discrete relationships between proliferation, PCL diameter, and cell-PCL distance. LCFA provides an advantage in that a distribution function is not assumed, but rather is developed from the data. Using these results, we propose a model for classifying the material-microstructure interactions, in which small PCL islands far from the cell nucleus act as holders for attachment and large islands close to cells act to shape the cell.