Abstract
Engineering of tissue constructs is highly dependent on scaffolding material to support cell growth. Scaffolds must provide the correct physical, mechanical, and biochemical features necessary for tissue repair. Decellularisation methods enable isolation of native extracellular matrix (ECM) from animal tissue, providing biocompatible scaffolds for tissue engineering. Decellularised plants provide cellulose matrices with similarities to ECM, representing an abundant, ethical, low-cost alternative. However, current methods of decellularisation are reliant on chemical use, compromising their sustainability. This study aimed to develop a sustainable alternative to chemical protocols used to produce scaffolds from the brown seaweed Ecklonia radiata. Pulsed Electric Field (PEF) technology, which applies an electric field to biological materials resulting in electroporation and removal of cellular contents, was hypothesised to promote E. radiata decellularisation and reduce chemical usage.
A chemical decellularisation protocol utilising a 1% Triton X-100/2% bleach solution was applied to intact E. radiata fronds for 14 days. This chemical decellularisation protocol was compared to PEF treatment with or without chemical post-treatment. Histology and scanning electron microscopy allowed for the visualisation of scaffold structures. The degree of scaffold decellularisation was gauged by measuring the DNA content and pigment clearance of the scaffolds. Biocompatibility with dermal fibroblasts was assessed for up to 7 days using SEM and confocal microscopy.
Structural characterisation revealed that both chemical and PEF scaffolds were structurally similar to the native material, with an inner layer of cellulose fibres flanked by two porous layers (8-10 pores deep). Pigment and DNA content within chemical-treated scaffolds were reduced by 95% and 83%, respectively, compared to the native material, confirming successful decellularisation. Moderate to high PEF treatment was able to achieve comparable results, reducing pigment by 93% and DNA content by 88%. By implementing PEF, the time to ascertain a ready-to-use scaffold was reduced from 14 days to ~3 days. Chemical-treated scaffolds were stable under culture conditions and supported human BJ/5Ta dermal fibroblast growth for 7 days, with SEM and confocal microscopy revealing dense populations throughout the inner fibrous layer of the scaffold. Experiments are on-going with the PEF-treated scaffolds to ascertain whether their biocompatibility with human cells is comparable or improved relative to chemical-treated scaffolds.
These results present PEF as a compelling novel alternative to chemical decellularisation approaches, and further work will elucidate the exact role that PEF may be able to play in the production of decellularised matrices for tissue engineering.