Abstract

Preliminary data from in-vivo investigations (rat model) suggest that a nanofiber prosthetic device of FGF-2-modified nanofibers can correctly guide regenerating axons across an injury gap with aligned functional recovery. Scanning Probe Recognition Microscopy with auto-tracking of individual nanofibers is used for investigation of the key nanoscale properties of the nanofiber prosthetic device for spinal cord tissue engineering and repair.

I. Introduction

Scanning Probe Microscopy (SPM) is one of the ideal techniques for nanoscale science. Its great advantages are its direct investigative capability and its inherent resolution which easily and reliably reaches the nanometer level. But SPM has limitations in several aspects. One limitation is speed of imaging, which may be due to overall system performance but may also be due to scanning outside the true region of interest. Another limitation is artifact restriction on large aspect ratio surfaces. While this limitation is typically associated with the vertical walls prevalent in semiconductor processing, another important subset is the nanoscale curved surfaces found in nanobiology.

The present investigations will use atomic force microscopy operated in a new mode, Scanning Probe Recognition Microscopy (SPRM), as a powerful investigative tool. Scanning Probe Recognition Microscopy was developed by our group in collaborative partnership with Veeco Instruments (Woodbury, NY) [1] [2] [3] . An SPRM system is given the ability to auto-track on regions of interest through incorporation of recognitionbased tip control. The recognition capability is realized using algorithms and techniques from computer vision, pattern recognition and signal processing fields. Adaptive learning and prediction are also implemented to make the detection and recognition procedure quicker and more reliable. The integration of recognition makes the SPRM system more powerful and flexible in investigating specific properties of samples. In the present investigations, SPRM auto-tracking along individual nanofibers enables measurements of scaffold topography, mechanics, and surface chemistry that are improved in three ways: 1) reliable data points are identified using deconvolution algorithms, 2) statistically meaningful numbers of reliable data points are extracted providing ranges and distributions, and 3) all data is extracted using an automatic procedure that maintains uniformity of experimental conditions. An example of SPRM auto-tracking along individual nanofibers within a tissue scaffold is shown in Fig. 1 , clockwise (a)-(d). A conventional atomic force microscopy image of the same region is shown on the left for comparison. When in SPRM mode, a coarse-step scan is initiated within the red-box the region. When a nanofiber is recognized, the instrument switches automatically into a fine-step raster scan: trace, retrace, down along the full length of the nanofiber. When the end of the nanofiber (defined by the red bounding box) is reached, the tip returns to approximately the same location where it left the coarsestep mode, re-enters coarse-step mode, recognizes and steps over the first nanofiber and continues until it recognizes a second nanofiber and begins the fine-step raster scan along its length again. An SPRM investigation of key nanoscale properties of a nanofiber prosthetic device for neural cell tissue engineering and repair is the focus of the present research. The brain and spinal cord neural cell system includes, amongst others, oligodendrocytes, macrophages, neurons, astrocytes, and endothelial cells. Neurons are vital for such functions as cognition, memory, and emotion, which are largely facilitated through neurotransmission between axons on pre-synaptic cells and dendrites or cell somas on post-synaptic cells. Axons and dendrites are collectively referred to as neurites, and a major goal of the proposed research is to identify fundamental factors that promote neurite generation and outgrowth. Astrocytes are key in this role and, in addition to provision of nutrients for and removal of wastes from neurons, provide mechanical support and express extracellular matrix molecules (e.g. laminin-1, fibronectin, tenascin-C [4] and growth factors (e.g. fibroblast growth factor-2 (FGF-2), nerve growth factor (NGF) [5] ) that can promote, inhibit, or direct neurite outgrowth.

Finally, endothelial cells with special adaptations form central nervous system capillaries. An ultra-dense basement membrane surrounds each capillary and contributes to the interstitial fluid-tight blood-brain barrier. Fig. 2(a) shows an immunocytohistochemical image of a capillary (rat model) at the blood-brain barrier. The capillary is enclosed by the ultra-dense extracellular matrix (ECM) as discussed above. Fluorescent imaging has been used to identify FGF-2 macromolecules (white dots with arrows) within the ECM along the length of the capillary. Investigations by our group and others indicate that the slowly biodegradable synthetic polyamide nanofibrillar matrix shown in The neural cell prosthetic device under investigation incorporates nanofibrillar matrices composed of a nonwoven fabric of biodegradable polyamide nanofibers prepared via electrospinning into a prosthetic for injury repair. The aim of the present SPRM research is to quantify the material properties and organization of the nanofibers to understand how it mimics the fibrillar organization of the ECM that forms a network for neuronal attachment and axonal growth and guidance.

Ii. Results And Discussion

The key properties under SPRM investigation include nanofiber stiffness and surface roughness, nanofiber curvature, nanofiber mesh density and porosity, and growth factor presentation and distribution. Each of these factors has been demonstrated to have global effects on cell morphology, function, proliferation, morphogenesis, migration, and differentiation. The present work will focus on elasticity and surface roughness. Unmodified nanofibrillar matrices (-X-linked FGF-2) were investigated to quantify the physical properties elasticity and surface roughness. Nanofibrillar matrices covalently modified with FGF-2 (+X-linked FGF-2), which promotes cell survival, angiogenesis and axonal regeneration were also investigated. Importantly, research by Meiners’ group has demonstrated that FGF-2 is far more potent and retains biological activity significantly longer when immobilized on nanofibers than when presented to cells as a soluble molecule [6] . SPRM was used to determine how the physical properties elasticity and surface roughness are changed by FGF-2 modification.

I. Elasticity

Several research groups have recently produced evidence that mechanical properties may strongly influence cell attachment and motility [7] . SPRM was used to directly evaluate elasticity along individual nanofibers within the nanofibrillar matrix for unmodified and FGF-2 modified samples. SPRM-based nano-indentation is performed [2] by first using trace between recognized boundaries, then nanoindentation during re-trace at the nanofiber midpoint. A new fundamental point that emerged from this investigation was the importance of mathematical definition of the region of interest (ROI) appropriate to the physics of the problem. The curved cylindrical nanofiber surface has a highly restricted ROI in mechanical property measurements by nano-indentation, which is the single median point per trace/retrace where the tip is normal to the sample surface.

A Hertz model was used directly to quantify the contact part of force curve. The Hertz model describes the elastic deformation of two surfaces touching under a load force. The general force-indentation relation can be expressed as (1) where F is the loading force and δ is the indentation depth. The term λ and exponent β are parameters decided by the two touching surfaces. For normal nano-indentation achieved through SPRM, an individual nanofiber surface can be considered as a flat soft sample. Since the tip is much harder than the sample, the elastic deformation of the sample can be related to its Young’s modulus in the contact part of the force curve. When the tip geometry is described as a sphere of radius R (estimated by tip characterization), the Hertz model is defined as:

(2) where F sphere is the measured force, E is the Young’s modulus, µ is the Poisson’s ratio and δ is the indention depth. The indentation δ is calculated as follows:

(3) where z is the sample height, z 0 is the contact point where tip touches the sample, d is the cantilever deflection and d 0 is the deflection of the free cantilever when the cantilever is far from the sample surface. To simply the problem, the nonlinear force (F sphere ) and indentation (δ) equation is converted to linear equation for (F sphere ) 2/3 and δ by substituting equation 3to equation 2: (4) In equation 4, F sphere , z, d and d 0 can be obtained from the experimental force curves. Typically, the Poisson’s ration can be assumed as 0.5 for soft materials. The unknown parameters are Young’s modulus E and the tip-sample contact point position z 0 . In this work, we use a least squares estimation procedure to determine E and z 0 by minimizing the error in equation 4with respect to unknown parameters E and z 0 (5) Sigma is the RMS error, is the measured force in contact region and is the force calculated using equation (4) .