Abstract
A permanent solution for tissue regeneration is needed for burn-injured or chronic wound patients. The scaffold-based (Top-down) tissue-engineered skin substitute approach may be a possible answer. This approach needs an improved template or scaffold which can support cell growth, proliferation, and attachment. A nanofibrous scaffold prepared as a three-dimensional network of natural biopolymers is believed to be the best template for skin tissue engineering. Electrospinning is a useful process for making three-dimensional nanofibrous scaffolds (3DENS) from biopolymers. By functionalising the scaffold with an antimicrobial agent, the antimicrobial scaffold can protect the tissue regeneration process from microorganisms which may be present in the wound bed.
Three-dimensional nanofibrous extracellular matrix-like scaffolds were fabricated using a multi spinneret electrospinning set-up as a potential candidate for tissue engineering applications. Two biopolymer solutions, namely 5% (w/w) keratin and 2% (w/w) chitosan, were used in association with 10% (w/w) poly(vinyl alcohol) (PVA) for fabrication of these 3DENS. Spectra obtained using Fourier transform infrared spectroscopy strongly suggested the formation of hydrogen bonds between PVA and keratin, and between PVA and chitosan. The thermogravimetric analysis also indicated possible interaction between the functional–OH and–NH2 groups in chitosan and keratin, and –OH groups in PVA. Improved hydrophilicity and water uptake capacity as indicated by the swelling ratio%, and enhanced porosity of 3DENS were achieved. The 3DENS prepared showed biodegradability in phosphate buffer saline. Different ratios of 5% (w/w) keratin and 2% (w/w) chitosan with 10% (w/w) PVA solution directly influenced the morphology (defect-free) and the diameter of the nanofibre as revealed by the scanning electron micrographs.
Defects in the forms of bead and spindle-like fibres appeared in 3DENS at higher volume ratios of keratin and chitosan. However, a higher content of biopolymer is desirable in 3DENS as it is associated with better biomedical performance. In order to fabricate fault-free fibre composed of the maximum volume ratio of biopolymers, biopolymer content (%) and the electrospinning process parameters including voltage (kV), flow rate (mL/h), and spinning distance (mm) were optimised using the Box-Behnken design. Three mathematical models were developed and models for fibre diameter and defect diameter fitted with experimental data with higher adjusted-R2 values. Electrospinning process parameters were optimised using these two models to obtain minimum defect diameter with fibre at nanoscale (diameter~170 nm). The optimised processing conditions resulting were: 30% biopolymer, 15.82 kV voltage, 0.25 mL/h flow rate, and 105 mm spinning distance. The developed quadratic model showed high predictability on fault-free fibre production and fibre diameter with an error of less than 4%.
The potential application of developed 3DENS as an advanced wound dressing was assessed after introducing antimicrobial agents, AgNPs, methylglyoxal, and Manuka honey in the 3DENS. The non-cytotoxic concentrations of antimicrobials for skin cell lines, HaCaT and NHDF, were determined by MTT cell assay. The minimum concentrations of antimicrobials to inhibit the growth (MIC) of a common gram-negative bacterium (E. coli) and gram-positive bacterium (S. aureus), were also determined. Non-cytotoxic concentrations of AgNPs (8 µg/mL), methylglyoxal (820 µg/mL), and Manuka honey (250 mg/mL) were higher than the MIC values of AgNPs (2 µg/mL), methylglyoxal (205 µg/mL), and Manuka honey (131.07 mg/mL). Two more concentrations for each antimicrobial, higher than their non-cytotoxic concentrations, were also incorporated in fabricating the antimicrobial 3DENS since 100 percent release of antimicrobials was considered improbable.
The release mechanisms of studied antimicrobials were identified as an exponential release with burst effect followed by exponential release due to swelling relaxation. The release behaviour of antimicrobials was studied using common kinetic models (eight models). The release behaviour of AgNPs, methylglyoxal and Manuka honey arguably fitted to the Korsmeyer-Peppas model for drug release from a polymeric film. However, only 60 percent release of a drug is considered in studying the Korsmeyer-Peppas model. Therefore, for fitting the full release data set, a new model was proposed, based on an exponential burst effect and relaxation swelling. The proposed model can fit and explain the release data reasonably well with root mean square error.
The 3DENS without antimicrobial agent did not show antimicrobial properties in the disk diffusion test although 30% of 2% (w/w) chitosan was present in the 3DENS. Incorporation of antimicrobial agents conferred antimicrobial properties to 3DENS. The highest concentrations of antimicrobial agents (200 µg/mL for AgNPs, 8.20 µg/mL for methylglyoxal, and 1.0 g/mL for Manuka honey) were detrimental in terms of cell viability (live-dead cell assay), cell proliferation (MTT cell assay), and cell attachment (SEM). The 3DENS containing AgNPs (80 µg/mL), methylglyoxal (1.64 µg/mL), and Manuka honey (0.5 g/mL) were found to be effective in preventing the growth of E. coli and S. aureus in the disk diffusion test and were biocompatible according to live-dead cell assay and MTT cell assay for 7 days. Moreover, these 3DENS enhanced the wound healing capability compared to the control. The 3DENS without an antimicrobial agent also performed better in closing the wound gap. However, these 3DENS did not support co-cultured (30 days) HaCaT and NHDF cell lines as revealed through scanning electron microscopy.