HDF morphology on PCL fibres.

<p>Nucleus (DAPI in blue) and cytoskeleton (phalloidin-TRITC in red) of HDFs seeded on random and aligned scaffolds. HDF morphology was visualised at day 7 (top) and 21 (bottom) imaged at different depths (5–40 µm). Cells on random fibres were generally attached in multiple directions while cells on aligned fibres were spindle shaped and aligned in the fibre direction. White arrows indicate direction of scaffold fibre alignment. Scale bars are 50 µm.</p

Electrospun PCL fibres with a random orientation or highly aligned.

<p>Random (A) and aligned (B) fibres were formed from a PCL (80 kDa) 15 w% in DCM solution. Conditions were 4 ml/h, 11 kV, 20 cm Wd, 200 rpm (A) and 2000 rpm (B). SEM micrographs (A–B) show different magnifications (insert scale bar is 5 µm). Fibre diameter distribution (C–D) and frequency of fibre orientation (E–F) were calculated from 150 fibres (n = 3).</p

Fibroblasts and secreted collagen grow into constructs and align with scaffold fibre orientation.

<p>Fibroblast-secreted collagen and scaffold fibres were visualised using SHG emissions collected at depths of 10–40 µm (A). Yellow arrow heads indentify PCL fibres of the scaffold. Fibroblast-seeded constructs were fixed and visualised via SHG emissions (collagen and scaffold fibres in yellow) and DAPI staining (cell nucleus in blue) at depths of 10–40 µm (B). Fixed constructs were also visualised for fibroblast cytoskeleton (phalloidin-TRITC in red) proximity to secreted collagen (SHG in green) (C). SHG was obtained using 800 nm illumination and all images were collected after 21 days of culture. White arrows indicate scaffold fibre orientation.</p

HDF viability and migration on PCL fibres.

<p>HDFs seeded into the centre of random and aligned fibres and assayed with MTT at days 3, 7 and 12 to observe cell migration and viability. Cells on random fibres migrated equally in all directions whereas cells on aligned fibres migrated along the direction of the fibre. Data is mean ± SD (n = 6), <i>*p<0.05</i>.</p

Physical and mechanical properties of random and aligned electrospun PCL fibres.

<p>Parallel refers to values obtained from aligned fibres in the longitudinal direction and across refers to values obtained from aligned fibres perpendicular to the fibre direction. Values are quoted as mean ± SD (n = 6), *<i>p<0.05</i> for random versus both aligned orientations.</p

3D images of collagen SHG from HDF-seeded constructs at day 21.

<p>Images of HDF deposited-collagen on random and aligned scaffolds were compiled from 35 µm z-stacks (2.5 µm slices). A rotated 3D view from the top and bottom of the constructs are shown along with 2D images in the z-plane taken from both the horizontal and longitudinal edges.</p

Collagen SHG from HDF-seeded constructs at day 14.

<p>SHG images of collagen matrix deposited on random and aligned scaffolds were obtained at day 14 taken from different depths. An increase in SHG intensity suggests an increase in collagen production and/or a more organised collagen fibrous network. Arrow indicates direction of scaffold fibre alignment. Scale bars are 50 µm.</p

Tensile testing performed on blank and HDF-seeded constructs of different fibre orientations.

<p>Scaffold properties were observed at day 0 (blank-no cells) and days 2, 7, 14, and 21 (with cells). Notice the difference in the y-axis scale for each scaffold orientation (parallel > random > perpendicular). SEM images indicate scaffold orientation with arrow showing direction of tensile force applied. Data is mean ± SD (n = 6: random and parallel or n = 3: perpendicular), <i>*p<0.05</i> verses previous time point tested.</p

Total DNA and SR staining of HDFs on PCL fibres.

<p>HDFs seeded on random and aligned electrospun PCL fibres were assayed for total DNA by Pico Green and collagen deposition by SR staining. Total DNA (A) and total SR stain (B) was quantified at days 7, 14 and 21. SR was normalised to total DNA (C) at each time point. SR staining can be visualised (D). Data is mean ± SD (n = 6–9), <i>*p<0.05</i>.</p

Explanation for the area distribution shape given the uneven surface of the insert.

<p>A) Scanning electron microscope image showing the uneven surface of a sterile insert; B) Schematic of the uneven insert surface with biofilm growth, HDPE roughness height is commonly taken in modelling to be 20 μm, 80 μm was the greatest depth measured in Day 28 biofilms. Broken red lines indicate parts of an example area distribution curve and the corresponding cut through position of the biofilm.</p