Nanofiber Alignment Regulates NIH3T3 Cell Orientation and Cytoskeletal Gene Expression on Electrospun PCL+Gelatin Nanofibers

<div><p>To examine the influence of substrate topology on the behavior of fibroblasts, tissue engineering scaffolds were electrospun from polycaprolactone (PCL) and a blend of PCL and gelatin (PCL+Gel) to produce matrices with both random and aligned nanofibrous orientations. The addition of gelatin to the scaffold was shown to increase the hydrophilicity of the PCL matrix and to increase the proliferation of NIH3T3 cells compared to scaffolds of PCL alone. The orientation of nanofibers within the matrix did not have an effect on the proliferation of adherent cells, but cells on aligned substrates were shown to elongate and align parallel to the direction of substrate fiber alignment. A microarray of cyotoskeleton regulators was probed to examine differences in gene expression between cells grown on an aligned and randomly oriented substrates. It was found that transcriptional expression of eight genes was statistically different between the two conditions, with all of them being upregulated in the aligned condition. The proteins encoded by these genes are linked to production and polymerization of actin microfilaments, as well as focal adhesion assembly. Taken together, the data indicates NIH3T3 fibroblasts on aligned substrates align themselves parallel with their substrate and increase production of actin and focal adhesion related genes.</p></div

Proliferation assay of NIH 3T3 cells growing on various substrates.

<p>A) The average amount of DNA for cells grown on each substrate at 0 and 4 days. The DNA content is directly proportional to the number of cells. B) The cellular growth rate on various substrates as determined by the ratio of DNA content on days 0 and 4. This data indicates that orientation of the fibrous substrate does not influence the initial attachment or growth rate of NIH3T3 cells. However, the addition of gelatin does a substantially increase the growth rate over 100% PCL matrices (P<0.05, 2-way ANOVA, Tukey post-hoc). Error bars are ±SD.</p

Polymer Characterization.

<p>A) Fourier Transform Infrared Spectroscopy (FTIR) of samples of electrospun gelatin, PCL, and PCL+Gel blend. The data PCL+Gel shows a characteristic N-H stretch peak for amines at 3300 1/cm. This confirms the presence of gelatin within the electrospun matrix. B) Differential Scanning Calorimetry (DSC) of samples of electrospun gelatin, PCL, and PCL+Gel blend. The data indicates that the addition of gelatin to the PCL does not drastically change the crystallinity or melting point of the PCL.</p

Electrospinning Apparatus and Resulting Nanofibers.

<p>A) Diagram of the electrospinning set-up: A polymer solution is extruded from a syringe into a high-voltage electric field towards a grounded cylindrical collector. When the collector is rotating at high RPM, aligned fibers are collected; when the collector is rotating at low RPM, randomly oriented fibers are collected. B) Example SEM images of nanofibers collected under various conditions. Scale bars are 10 microns.</p