Despite the growing use of nanofiber scaffolds for tissue engineering applications, there is not a validated, readily available, free solution for rapid, automated analysis of nanofiber diameter from scanning electron microscope (SEM) micrographs. Thus, the goal of this study was to create a user friendly ImageJ/FIJI plugin that would analyze SEM micrographs of nanofibers to determine nanofiber diameter on a desktop computer within 60 seconds. Additional design goals included 1) compatibility with a variety of existing segmentation algorithms, and 2) an open source code to enable further improvement of the plugin. Using existing algorithms for centerline determination, Euclidean distance transforms and a novel pixel transformation technique, a plugin called “DiameterJ” was created for ImageJ/FIJI. The plugin was validated using 1) digital synthetic images of white lines on a black background and 2) SEM images of nominally monodispersed steel wires of known diameters. DiameterJ analyzed SEM micrographs in 20 seconds, produced diameters not statistically different from known values, was over 10-times closer to known diameter values than other open source software, provided hundreds of times the sampling of manual measurement, and was hundreds of times faster than manual assessment of nanofiber diameter. DiameterJ enables users to rapidly and thoroughly determine the structural features of nanofiber scaffolds and could potentially allow new insights to be formed into fiber diameter distribution and cell response.
Stem cell response to a library of scaffolds with varied 3D structures was investigated. Microarray screening revealed that each type of scaffold structure induced a unique gene expression signature in primary human bone marrow stromal cells (hBMSCs). Hierarchical cluster analysis showed that treatments sorted by scaffold structure and not by polymer chemistry suggesting that scaffold structure was more influential than scaffold composition. Further, the effects of scaffold structure on hBMSC function were mediated by cell shape. Of all the scaffolds tested, only scaffolds with a nanofibrous morphology were able to drive the hBMSCs down an osteogenic lineage in the absence of osteogenic supplements. Nanofiber scaffolds forced the hBMSCs to assume an elongated, highly branched morphology. This same morphology was seen in osteogenic controls where hBMSCs were cultured on flat polymer films in the presence of osteogenic supplements (OS). In contrast, hBMSCs cultured on flat polymer films in the absence of OS assumed a more rounded and less-branched morphology. These results indicate that cells are more sensitive to scaffold structure than previously appreciated and suggest that scaffold efficacy can be optimized by tailoring the scaffold structure to force cells into morphologies that direct them to differentiate down the desired lineage.
Cells are known to sense and respond to the physical properties of their environment and those of tissue scaffolds. Optimizing these cell-material interactions is critical in tissue engineering. In this work, a simple and inexpensive combinatorial platform was developed to rapidly screen three-dimensional (3D) tissue scaffolds and was applied to screen the effect of scaffold properties for tissue engineering of bone. Differentiation of osteoblasts was examined in poly(ethylene glycol) hydrogel gradients spanning a 30-fold range in compressive modulus (≈ 10 kPa to ≈ 300 kPa). Results demonstrate that material properties (gel stiffness) of scaffolds can be leveraged to induce cell differentiation in 3D culture as an alternative to biochemical cues such as soluble supplements, immobilized biomolecules and vectors, which are often expensive, labile and potentially carcinogenic. Gel moduli of ≈ 225 kPa and higher enhanced osteogenesis. Furthermore, it is proposed that material-induced cell differentiation can be modulated to engineer seamless tissue interfaces between mineralized bone tissue and softer tissues such as ligaments and tendons. This work presents a combinatorial method to screen biological response to 3D hydrogel scaffolds that more closely mimics the 3D environment experienced by cells in vivo.
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