2.1. Synthesis of Electrocompacted Collagen Matrices
The fabrication process of traditional collagen gel (GEL), electrocompacted and crosslinked collagen (ECLC) as well as SXRGlu-conjugated ECLC (ECLCU) matrices is illustrated in Figure 1
. Type-I collagen powder (Kele Biotech Co., Chengdu, China) was dissolved in 0.5 M acetic acid at 6 mg/mL and dialyzed against ultrapure water at 4 °C for 12 h. Then the dialyzed collagen was loaded into a circular rubber washer (diameter = 1.2 cm and thickness = 2 mm) sandwiched between two oppositely charged electrodes. Upon application of an electric field of 6 volts for 15 min, a pH gradient was generated between the electrodes. Due to the amphoteric nature of the collagen molecules, the collagen molecules would be driven by the electrostatic repulsion from the electrodes and compacted along the isoelectric point thus forms a transparent “wet” collagen sheet (ECL). Then the freshly aligned collagen sheet was incubated in phosphate buffered saline (PBS) at 37 °C for 4 h for fibril formation.
To enhance the mechanical property and stability of the collagen matrices, the ECL matrices were crosslinked using 20 mM EDC and 20 mM NHS in 50 mM 2-(N
-morpholino) ethanesulfonic acid (MES) buffer for 4 h at room temperature [10
]. The crosslinked ECL matrices (ECLC) were washed thoroughly with PBS to remove unreacted chemicals in the crosslinking solution.
Traditionally fabricated collagen gels were prepared as controls by mixing the dialyzed collagen solution (8 parts) with 10 × PBS (1 part) and adjusting the pH to 7.0–7.5 using 0.1 N NaOH (1 part). The mixture was then cast into molds and allowed to gel at 37 °C for 24 h followed by crosslinking using 20 mM EDC and 20 mM NHS in 50 mM MES buffer. Strips or circle membranes were then cut from these gels and processed as mentioned above for subsequent tests. An equal amount of collagen was used for fabrication of both compacted and uncompacted sheets.
2.2. SXRGlu Conjugation to ECL Matrices
To mimic the more complex scaffold structure of skin matrices and investigate the functions of SXRGlu in wound healing applications, an SXRGlu rich extract, obtained from the cell wall of a DNA barcoded green macroalgae (PhycoTrix™, Venus Shell Systems, Bomaderry, Australia), was crosslinked to the ECL matrices by soaking the freeze-dried ECL matrices in 1% SXRGlu, 20 mM EDC and 20 mM NHS in 50 mM MES buffer for 4 h at room temperature. After that, the SXRGlu conjugated-ECL (ECLCU) were rinsed thoroughly with PBS, freeze dried and stored at 4 °C for further application.
To quantify the amount of SXRGlu in the ECLCU matrices, the ECLCU matrices were dissolved in 1 N HCl and incubated at 37 °C for 72 h. The dimethylmethylene blue (DMMB) assay [11
] was used to quantify the amount of SXRGlu in the solution which indicated the SXRGlu amount that was crosslinked to ECLCU. To visualize the distribution of SXRGlu through the ECLCU, fluorescent labelling and confocal inspection were conducted. Fluoresceinamine labelled SXRGlu (Fluo-SXRGlu) was prepared by mixing 25 mM EDC, 25 mM sulfo-NHS, 1.25 mM fluoresceinamine (Sigma-Aldrich, Castle Hill, Australia) and 5% (w
) SXRGlu into 0.1 M MES buffer (pH 6.0) and incubating under gentle stirring at room temperature for 24 h. Extra dyes were removed by dialyzing against distilled water for 7 days. Then the Fluo-SXRGlu was crosslinked to the ECL using the same procedures mentioned above.
2.7. Human Dermal Fibroblast Cell Viability, Proliferation and Morphology on Electrocompacted Collagen Matrices
Human dermal fibroblast (HDFs, Cell Applications, Inc., San Diego, CA, USA) cells at passage 8 were seeded onto the fabricated collagen scaffolds to assess the influence of electrocompaction and incorporation of SXRGlu on cell proliferation and morphology. The HDFs were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin under 5% CO2 at 37 °C. Before cell seeding, scaffolds in 24 well plate were sterilized firstly by UV irradiation for 1 h, immersion in 70% ethanol for 3 h and washing for five times with PBS for 12 h. To saturate the scaffold with cell culture medium and facilitate cell attachment onto the collagen matrices, the sterilized scaffolds were incubated in 1 mL of culture medium at 37 °C overnight before cell seeding.
The HDFs were then seeded at the density of 1.0 × 104 cells/cm2 on the pre-processed scaffolds. Cell proliferation studies over 7 days were performed using the PrestoBlue™ (Life Technologies, Mulgrave, Australia) assay according to the manufacturer’s instruction. Briefly, at day 1, 3, 5 and 7, three HDFs-scaffold constructs were incubated with PrestoBlue™ mix for 1 h at 37 °C. Following this, for each sample, 100 μL aliquots of supernatant were transferred to a 96-well plate in triplicate and measured using a microplate reader (POLAR star Omega, BMG Labtech, Offenburg, Germany).
Live/dead assays were performed to inspect the HDFs cell viability on the scaffolds. Briefly, at each time point, HDFs-scaffold complexes were incubated with Calcein AM (5 μg/mL, Life Technologies, Mulgrave, Australia) at 37 °C for 10 min. Then, after a media change, propidium iodides (PI, 5 μg/mL, Life Technologies, Mulgrave, Australia) were added into the HDF-scaffold complexes and incubated for 5 min followed by a further media change. Images were acquired using a confocal microscope (Leica TSC SP5 II, Buffalo Grove, IL, USA) and the 3D projection tools in the Leica application suite X (LAS X) software (Leica, Buffalo Grove, IL, USA) were used for depth coding.
Alexa Fluor 488-Phalloidin cell cytoskeleton staining (ThermoFisher Scientific, Waltham, MA, USA) were conducted to assess the cell morphology. To do this, collagen matrices with cells at day 7 were washed in PBS and fixed in 3.7% paraformaldehyde in PBS for 10 min at room temperature. Then, samples were washed with PBS and permeabilized with 0.1% Triton-X 100 in PBS for 5 min at room temperature. Samples were subsequently stained with Alexa Flour 488-Phalloidin (1:40 in PBS) for 1 h at room temperature, washed with PBS, and then incubated with 10 μg·mL−1 4′,6-diamidino-2-phenylindole (DAPI, ThermoFisher Scientific, Waltham, MA, USA) for 10 min at RT to visualize the nuclei. Finally, samples were washed and imaged using a confocal microscope (Leica TSC SP5 II, Buffalo Grove, IL, USA).