Recent efforts in the field of regenerative medicine have focused on the fabrication of scaffolds capable of promoting a repertoire of cellular responses. These scaffolds are designed to be mimetic in structure and function to the extracellular matrix/basement membrane, which serves as the physiological support for cells within tissues. Understanding the nature of the physical interactions of cells within these biomimetic structures and deriving information that would correlate geometric properties of the scaffolds with the promotion of specific cellular responses would have a major impact on their design and utility. In this paper, we introduce the use of a new and powerful form of atomic force microscopy developed by our group, termed Scanning Probe Recognition Microscopy (SPRM). SPRM is used to examine the physical interactions of protrusions emanating from NIH 3T3 fibroblasts with the surfaces of both 2D planar and 3D nanofibrillar cell culture surfaces. This technique provides the means to maintain focus on user defined regions of contact (in the nanometer range) between the cell protrusions and the 2D and 3D surfaces. Differences in the number and shape of contact regions between the cell protrusions and the two types of surface were observed using SPRM. These observations were supported by similar imaging results obtained, albeit at significantly lower resolution, using phase contrast and bright field microscopy.
The use of scaffolds composed of biocompatible materials as vehicles to deliver or promote growth/differentiation of cells within damaged tissues of the body is an exciting new tool for the field of regenerative medicine. To optimize the performance of these scaffolds for each type of tissue or application, new analytical methods must be developed that can examine the interaction of these cells with the surfaces of these scaffolds in vitro under a variety of biochemical and physical conditions at a resolution in the nanometer range.
In a situation that reproduces its natural biological environment, a cell will extend protrusions towards the scaffold. These physical interactions initiate a cascade of events that effect the cytoskeletal organization, signaling pathways, and cell-cell interactions. The initial attachment of the cell with scaffold surface is triggered through a complex interaction of chemical and mechanical receptors at the leading edge of the protrusion. Actin-based cells develop the protrusions through a dynamic cycle of assembly and disassembly of intracelluar actin filaments.. Recent research has provided new insight into the internal signalling cascades that promote the reorganization of actin filaments into aligned and branched internal structures that result in the extension of two different types of protrusions, narrow filopodia and broad lamellipodia [1, 2] . Surface characteristics that have been demonstrated to effect these actin rearrangements are surface roughness  , nano-patterning  , elasticity  and curvature  .
In this work, we evaluate the interaction of the tips of protrusions emanating from NIH 3T3 fibroblasts with 2D planar and 3D nanofibrillar surfaces.
NIH 3T3 fibroblast cells were cultured for 24 hours on two different surfaces, planar (2D) tissue culture plastic and amine coated nanofibers. The amine coated nanofibers were randomly oriented polyamide nanofibers (a continuous fiber that collects as a nonwoven fabric) electrospun by Donaldson Co., Inc. (Minneapolis, MN). They were electrospun from a blend of two polymers [(C28O4N4H47)n and (C27O4.4N4H50)n] onto plastic coverslips. The polymeric nanofiber mat was crosslinked in the presence of an acid catalyst and formed a dense network of filaments 50-80 nm in diameter with minimal porosity. The nanofibers were covalently coated with a proprietary polyamine polymer by Surmodics, Inc. (Eden Prairie, MN).
Phase Contrast Microscopy
Phase Contrast Microscopy was performed on live cells using an Olmpus 1X70 inverted microscope. The images were captured by using IP lab scientific imaging software.
Cell Fixation And Optical Microscopy
Samples for optical and AFM microscopy were fixed with 2.5% glutaraldehyde in 0.1% phosphate buffer for 15 minutes, briefly rinsed with 0.1M phosphate buffer and washed 3 times with triple distilled water, . The samples were then permitted to desiccate in air for 48 hours prior to optical, AFM and SPRM-AFM imaging. The desiccated samples were optically examined during AFM imaging using a Sony XC-999P CCD color video camera microscope with a VCL-12S12XM lens (f = 12 mm).
Sprm Enhanced Atomic Force Microscopy
Scanning Probe Recognition Microscopy (SPRM) is a new scanning probe microscopy technique developed by our group which allows us to adaptively follow and investigate specific regions of interest using any scanning probe microscopy technique [7, 8] The SPM system itself is given the ability to auto-focus on regions of interest through incorporation of recognitionbased tip control. The recognition capability is realized using techniques in pattern recognition and image processing fields. Adaptive learning and prediction are also implemented to make detection and recognition procedures quicker and more reliable. In the present work, SPRMenhanced atomic force microscopy (SPRM-AFM) was used to investigate the curvature and surface roughness of individual nanofibers, and details of the cell-substrate interfaces.
SPRM is implemented as an adaptation of a Veeco Instruments Nanoscope IIIA system. The system was operated in AFM and SPRM-AFM contact mode in ambient air. Other instrument parameters included the use of a J scanner with a maximum 125×125 square micron x-y scan range and silicon nitride tips with a nominal 20 nm tip radius of curvature.