Accelerated Wound Closure - Differently Organized Nanofibers Affect Cell Migration and Hence the Closure of Artificial Wounds in a Cell Based In Vitro Model.

Nanofiber meshes holds great promise in wound healing applications by mimicking the topography of extracellular matrix, hence providing guidance for crucial cells involved in the regenerative processes. Here we explored the influence of nanofiber alignment on fibroblast behavior in a novel in vitro wound model. The model included electrospun poly-ε-caprolactone scaffolds with different nanofiber orientation. Fibroblasts were cultured to confluency for 24h before custom-made inserts were removed, creating cell-free zones serving as artificial wounds. Cell migration into these wounds was evaluated at 0-, 48- and 96h. Cell morphological analysis was performed using nuclei- and cytoskeleton stainings. Cell viability was assessed using a biochemical assay. This study demonstrates a novel in vitro wound assay, for exploring of the impact of nanofibers on wound healing. Additionally we show that it's possible to affect the process of wound closure in a spatial manner using nanotopographies, resulting in faster closure on aligned fiber substrates

Stainings for nuclei (blue) and actin filaments (red).

<p>A) flat- B) random fiber- C) aligned fiber surfaces. Cells have formed confluent monolayers on all surfaces, and cellular morphologies are thus difficult to analyze. Scale bar = 20 μm.</p

Resulting static water contact angles for the two types of PCL scaffolds.

<p>The angle was measured both parallel- and perpendicular to the aligned fibers to exclude any possible discrepancies.</p

The electrospinning process parameters used to produce random- and aligned nanofiber scaffolds.

<p>The electrospinning process parameters used to produce random- and aligned nanofiber scaffolds.</p

Cell respiratory activity.

<p>The percentage of reduced alamarBlue reagent has visibly increased between 48- and 96 h on all topographies. This is to be expected as cells are given serum to keep proliferating throughout the experiment duration. The extent to which they reduce the reagent differs minimally at both time points.</p

Results from characterization of both nanofiber scaffold types.

<p>Results from characterization of both nanofiber scaffold types.</p

Nuclei analyzes.

<p>A) Cell nuclei size differs significantly between cells cultured on different substrates. B) The aspect ratio of the nuclei is however unaffected by the surface and displays a slight elongation on all topographies. C) The orientation of the nuclei long axes shows a clear trend to orient along the aligned nanofibers, which is not displayed on the other topographies.</p

Closing of artificial wounds.

<p>A) To visualize the wound closing process, nuclei stainings (bisBenzimide) were used. B) The cell free area of each wound was measured at the different time points. C) Additionally, the aspect ratio <i>i</i>.<i>e</i>. shape of the wound areas was calculated for each time point and wounds on aligned nanofiber scaffolds appeared to adopt an increasingly elliptical shape over time.</p

Accelerated Wound Closure - Differently Organized Nanofibers Affect Cell Migration and Hence the Closure of Artificial Wounds in a Cell Based <i>In Vitro</i> Model - Fig 1

<p><b>SEM images of A) random- and B) aligned PCL nanofiber scaffolds</b>. The histograms display a typical skewed right distribution of fiber diameters for both C) random- and D) aligned scaffolds. The electrospun PCL fibers had similar distribution with median diameters around 750 nm. The random and aligned fiber structures show distinct differences regarding fiber morphology.</p

The design of the different parts comprising the custom cell stopper inserts.

<p>A) The main part of the insert onto which the scaffold is mounted. B) The toothed ring holding the scaffold in place. C) A removable part with four 1.5 mm pillars acting as cell stoppers. D) All parts assembled into the final insert design. E) The final device as printed in PLA.</p