Research Highlights of Polymers Division

Metrologies for Tissue Engineering

Tissue engineering is a rapidly growing field that presents a host of new measurement problems for producers and regulators. Tissue engineered medical products are typically hybrid devices that incorporate materials and cells in order to regenerate damaged tissue. The safety and efficacy of both materials and cells as well as their interactions must be qualified in order for these to gain acceptance, and we are developing a host of in vitro tests to assist in this process.

Events at the cell-material interface determine the performance of these devices but the complexity of the interactions has prevented development of a more rational approach to tissue engineering. In the Polymers Division, we are extending the established program in combinatorial methods to biomaterial interfaces. This will permit rapid screening of material variables and evaluation of their effects on cells.

The extracellular matrix (ECM) to which cells adhere can initiate healing, direct cell migration, control cell cycle progression, and determine cell differentiation. The scientific community is just beginning to understand intracellular signaling networks but it is clear that external stimuli participate in the cycle of cell activity. The challenge of tissue engineering is to control cellular behavior through the rational design of materials and culturing conditions. The industry is years away from meeting this challenge due, in part, to a void in the measurement infrastructure at the cell-biomaterial interface. Efforts within the Polymers Division to fill this void fall under three general topics: developing material libraries, expanding imaging capabilities, and managing and analyzing data.

Developing Material Libraries

We are developing a three-tiered material library for investigating cell-material interactions; each tier represents an increased level of complexity. The first tier comprises polymeric material surfaces designed to mimic bulk material interfaces currently found in industrial applications. The second tier contains material surfaces designed to mimic the ECM through incorporation of cell-signaling molecules such as peptides and oligosaccharides. While the first two tiers describe pseudo-two-dimensional phenomena, the third tier includes three-dimensional materials.

Current industrial applications, and those of the near future rely on relatively simple materials such as poly(lactic acid) and poly(dimethylsiloxane). Even so, variables such as composition and processing conditions can drastically affect physical properties. Particular properties of interest include: nanometer- and micrometer-scale topography, hydrophilicity, charge, and mechanical rigidity. The Polymers Division has established methods for preparing gradient material surfaces encompassing this parameter space. As these methods are transferred to biomaterial systems, the first tier of our library will develop. From this tier, we will identify and develop SRMs that will assist industry in developing products, assessing quality control, and gaining regulatory approval.

The next generation of tissue engineered medical products will incorporate cell-signaling molecules such as peptides and oligosaccharides. These materials will be engineered to interact with specific receptors and elicit selective cellular responses. Doing so requires fine-tuning the stimuli offered to cells by presenting appropriate combinations of signaling molecules. The cells then receive signals capable of initiating more complex cascades ultimately leading to the desired cellular response. This chain of events would emulate that which occurs during natural tissue formation. A high-throughput approach is uniquely qualified to characterize this complex, multi-dimensional space of ECM cues and cellular responses. As methods for preparing such materials are developed the second tier of our library will emerge. From this tier, we will contribute to the general understanding of intracellular signaling networks, groundwork necessary for the development of new tissue engineered medical products.

Real tissues are inherently three-dimensional and realistic measurement tools will need to be as well. We are currently developing three-dimensional materials and associated measurement methods to begin a metrology system complementary to the two-dimensional systems. Similar material issues will be faced with the added complication of three-dimensional architecture and its influence on cellular response. Such is the scope of the third tier of our library. In addition, other issues related to tissue engineered medical products will be investigated with a goal of creating necessary measurement capabilities. The devices being developed by tissue engineering companies have multiple requirements in order for them to be safe and effective. They have stringent mechanical requirements, which depend on the tissue being engineered; they must allow infiltration by the necessary cells; and the inflammatory responses they evoke must be minimized. Toward this end we are developing in vitro testing protocols for these performance requirements that will be used in screening tissue engineered medical products.

Expanding Imaging Capabilities

The described three-tiered material library only has value if cell response across the library can be well characterized. A host of strategies exist for characterizing cell response; many fall beyond the scope of the Polymers Division and are pursued through external collaborators. On site, we have targeted visual imaging of cells through conventional microscopy, fluorescence microscopy, confocal microscopy, and optical coherence tomography (OCT). The OCT image below shows pores in red and tissue scaffolding in blue.

The very nature of the combinatorial methodology will require that image acquisition be well documented and unbiased in order to correlate cell response to material properties. We envision a streamlined process where cells are seeded onto a material, incubated, fixed, and stained following an established experimental protocol. An array of images would be collected and the entire surface mapped. Images would then be reduced to quantitative measures of cell response.

Managing and Analyzing Data

The task of accurately overlaying data maps presents a significant challenge. For example, a thin film could be prepared from a blend of two polymers. The film might consist of a composition gradient along one axis and might be annealed (processed) under a temperature gradient orthogonal to the composition gradient. Characterization of this film would begin by mapping processing conditions, i.e., composition and annealing temperature as a function of position. An array of AFM images may be taken to map the surface topography. An array of contact angles may be measured to map the surface energy. Finally, an array of images would be collected and analyzed to measure cell response. In order to identify correlations each of these data maps must be in alignment.

Once data maps are aligned, a rigorous statistical analysis is necessary to quantify correlations and make meaningful hypotheses of causality. Material parameters can be analyzed following traditional engineering statistics. Data from living organisms requires an alternative strategy. Whether measuring general, morphological parameters such as cell area upon spreading or more specific parameters such as the regulation of gene expression, the cell response will fall within a distribution defined by the cell line. Unlike measured material parameters, variations from the mean may not be random, so reducing data to a mean and standard deviation can result in a loss of critical information.

As such, images of cells are collected, analyzed, and reported in a manner that maintains the fullest description of a population distribution. Furthermore, when investigating the cell response to a variety of material scaffolds, evaluations are based on a statistical comparison between corresponding cell populations.

For more information on this topic:

Please contact: Newell Washburn, Scott Kennedy, Eric Amis

Niklason LE, Langer R. "Prospects for Organ and Tissue Replacement" JAMA, February 7, 2001.