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 The Biomaterials Group is contributing significant effort into developing measurement methods to characterize the interactions associated with specific cellular behavior, high-throughput techniques capable of measuring cell response over very small physico-chemical increments, and analytical methods that preserve the full descriptive power of population distributions on orthogonal gradient substrates. Our contributions to RESBIO can be broken down into 4 subgroups. 1) Effects of Processing Conditions on Bioresponse 2) Continuous Variable Gradient Substrates 3) Structural and Chemical Imaging 4) Design and Fabrication of Micro-reservoir Arrays Effects of Processing Conditions on Bioresponse A series of discrete blends were made to outline small areas of physical parameter space to probe whether more significant differences in cell response existed and could be measured. In each of the annealed blend compositions, two distinct phases, which correspond predominately to DTE and DTO are seen. In this instance, a change in temperature initiated phase separation where the two blend components spontaneously demixed from their initially homogeneous state. The size and spacing are tunable by varying the composition of the blend. The discrete DTE/DTO blends form phase-separated domains under in vacuo, 105 °C annealing conditions. The atomic force microscopy (AFM) images of the annealed polycarbonate homopolymers yielded very smooth amorphous thin films that were largely indistinguishable by optical or AFM methods. Prior to the annealing process, the blends were smooth homogeneous films slightly rougher than the homopolymers materials. However, during the annealing process and the accompanied phase separation, the blends become significantly rougher.  )) In the five 5 mm × 5 mm AFM images shown on the right, the amount of DTO is increasing. Immuno-fluorescent staining for actin and vinculin and overlays of MC3T3-E1 osteoblasts showing the cytoskeleton and focal adhesion contact formation 16 h after seeding on each of the respective homopolymers and blends. Upon close examination of the vinculin staining the extension and distortion of the lamellapodia increase in the blend samples with increasing DTO content. Also, the cells appear to spread less with increasing DTO content as seen in the distribution of the fibrilar actin. Publication LO Bailey, ML Becker, JS Stephens, ND Gallant, CM Mahoney, NR Washburn, A Rege, J Kohn, EJ Amis, “Cellular Responses to Phase-separated Blends of Tyrosine-derived Polycarbonates.” Journal of Biomedical Materials Research Part A, 2006, in press. )) Gene copy numbers of interleukin-1b and fibronectin after 24 h of surface exposure on the respective homopolymers and blends on MC 3T3 bone osteoblasts exhibited compositionally dependent gene expression profiles that were significantly different than the respective homopolymers films. The physico-chemical parameters that led to the upregulation of FN in each of the blend compositions are under investigation. While the roughness in the blend samples as compared to the homopolymers certainly contributed significantly to the upregulation in both gene expression markers, they cannot entirely explain the statistically significant differences between the respective compositions. Continuous Variable Gradient Substrates )) We are using a gradient library approach – single samples containing a continuous variation in a physical or chemical parameter along one or two orthogonal directions – as a measurement tool for biomaterials research and a means for systematically reducing the amount of physical parameter space for further analysis. Surface characteristics such as hydrophobicity, morphology, surface charge, and chemical functionality each play key roles in governing cell adhesion and proliferation. These factors, not withstanding new chemical and processing methodologies, are continually increasing the physical parameter space, making gradient sample fabrication methods very attractive. Orthogonal gradient methods developed in the NIST Combinatorial Materials Center (NCMC) produce well-defined materials that afford simultaneous coverage of multidimensional chemical, composition, and physical property parameter space. Our initial efforts have focused on developing in vitro assays for the measurement of inflammation and extracellular matrix (ECM) gene regulation, which we anticipate will provide preliminary assessments of in vivo material performance. Structural and Chemical Imaging The various effects of thermal processing are being scrutinized closely to characterize any structural changes that may be occurring during annealing and sterilization )) The question of whether the scaffolds would lose their fine microstructure was addressed initially via SEM characterization. Core S fabricated and characterized salt-leached homopolymer and blend scaffolds per our request for this investigation. The scaffolds were prepared using a rapid quench protocol that traps everything in an amorphous state to yield a well-defined microstructure. The SEMs revealed that annealing with the salt in vacuo at 105 °C did not significantly alter the scaffold structure. We are continuing to analyze these materials to further confirm that this processing step do not alter the integrity of the scaffolds. )) Several of the tyrosine-derived homopolymer and blend scaffolds have been examined using Optical Coherence Microscopy (OCM) in the NIST Polymer Laboratory. OCM uses interferometry rather than a pinhole to reject out-of-focus light and is 6 to 7 orders of magnitude more sensitive than traditional confocal microscopy, which translates into deeper imaging depth. This method provides a means of non-destructive, high resolution, in-situ imaging to gather structural information of a 3D scaffold. Initial results show that the DTE scaffold is highly interconnected. The image on the right is 1 ? mm ? 1mm ? 430 μm.Design and Fabrication of Micro-reservoir Arrays The frontal photo-polymerization technique was established previously within the NIST Combinatorial Methods Center (NCMC). This rapid prototyping technique in conjunction with a microfluidic device
)) affords easily adjustable arrays that can be used in high-throughput cell experimentation. The interesting expression profiles seen in the discrete blend work led to discussions with Core B on adapting the Micro-reservoir Arrays to monitoring cell migration using two–photon confocal microscopy. The rapid prototyping method approach was not only feasible but also provided a convenient way for fluid handling devices in making small array of compositional blends for near simultaneous monitoring of cell migration. Several slides were transferred to the Core B lab for proof-of-concept with the cell migration studies underway. NIST Contributors Eric Amis, Division Chief Lori Henderson, Group Leader Matthew Becker, Project Leader Joy Dunkers Marc Cicerone Nathan Gallant Jean Stephens Tak Kee Carl Simon Christine Mahoney |
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