Novel selenium and/or copper substituted hydroxyapatite-gelatin-chitosan-eggshell membrane nanocomposite scaffolds for bone tissue engineering applications.

Limitations with the majority of bone therapeutic treatments include low availability, ethical constraints and low biological compatibility. Although a number of choice materials have been exploited successfully, there has always been scope for improvement as well as development of the next-generation of materials. Herein, scaffolds - developed from gelatin, chitosan and eggshell membranes - were crosslinked using tannic acid, and further infused with selenium and/or copper substituted hydroxyapatite nanoparticles to generate a novel nanocomposite substrate. FESEM images of the nanocomposite scaffolds revealed the presence of interconnected pores, mostly spread over the whole surface of the scaffold, alongside XRD and FTIR profiling that detailed the formation of hydroxyapatite as a sole phase. Moreover, physical characterisation of the nanocomposite confirmed that the hydroxyapatite particulates and the eggshell membrane fibres were uniformly distributed and contributed to the surface roughness of the scaffold. Biocompatibility and cytotoxicity of the novel constructs were assessed using the mouse-derived osteoblastic cell line, MC3T3-E1, and standard cell culture assays. Metabolic activity assessment (i.e. MTS assay), LDH-release profiles and Live/Dead staining demonstrated good cell adhesion, viability, and proliferation rates. Accordingly, this work summarises the successful development of a novel construct which may be exploited as a clinical/therapeutic treatment for bone repair as well as a possible translational application as a novel biomaterial for the drug development pipeline.

The chicken eggshell membrane: a versatile, sustainable, biological material for translational biomedical applications.

Naturally derived materials are often preferred over synthetic materials for biomedical applications due to their innate biological characteristics, relative availability, sustainability, and agreement with conscientious end-users. The chicken eggshell membrane (ESM) is an abundant resource with a defined structural profile, chemical composition, and validated morphological and mechanical characteristics. These unique properties have not only allowed the ESM to be exploited within the food industry but has also led to it be considered for other novel translational applications such as tissue regeneration and replacement, wound healing and drug delivery. However, challenges still exist in order to enhance the native ESM (nESM): the need to improve its mechanical properties, the ability to combine/join fragments of ESM together, and the addition or incorporation of drugs/growth factors to advance its therapeutic capacity. This review article provides a succinct background to the nESM, its extraction, isolation, and consequent physical, mechanical and biological characterisation including possible approaches to enhancement. Moreover, it also highlights current applications of the ESM in regenerative medicine and hints at future novel applications in which this novel biomaterial could be exploited to beneficial use.

Selenium- and/or copper-substituted hydroxyapatite: A bioceramic substrate for biomedical applications.

Atomic substitution or doping of a bioceramic material hydroxyapatite (HA) with specific ions is an appealing approach for improving its biocompatibility and activity, as well as imparting antibacterial properties. In this study, selenium- and/or copper-substituted hydroxyapatite powders were synthesized by an aqueous precipitation method and using the freeze-drying technique. The molar concentrations of constituents were calculated based on the proposed mechanism whereby selenium (Se4+) ions partially substitute phosphorus (P5+) sites, and copper (Cu2+) ions partially substitute (Ca2+) sites in the HA lattice. Dried precipitated samples were characterized using Inductively coupled plasma optical emission spectroscopy (ICP-OES), X-ray diffraction analysis (XRD), Fourier-transform infrared spectroscopy (FTIR) and Field-emission scanning electron microscopy with energy dispersive X-ray spectroscopy (FESEM-EDX). Accordingly, substitution of Se4+ and/or Cu2+ ions took place in the crystal lattice of HA without the formation of any impurities. The presence of sulphur (S2-) ions in the hydroxyapatite was detected by ICP-OES in all samples with copper substituted in the lattice. The cytotoxicity of the powders on osteoblastic (MC3T3-E1) cells was evaluated in vitro. Selenium substituted hydroxyapatite (SeHA), at the concentration (200 µg/mL), demonstrated higher populations of the live cells than that of control (cells without powders), suggesting that selenium may stimulate the proliferation of these cells. In addition, the copper substituted hydroxyapatite (CuHA) and the selenium and copper substituted hydroxyapatite (SeCuHA) at the concentrations (200 and 300 µg/mL) and (200 µg/mL), respectively demonstrated better results than the unsubstituted HA. Antimicrobial activity was assessed using a well-diffusion method against Streptococcus mutans and Candida albicans, and superior results has obtained with SeCuHA samples. Presented findings imply that selenium and/or copper substituted modified hydroxyapatite nanoparticles, may be an attractive antimicrobial and cytocompatible substrate to be considered for use in a range of translational applications.

The eggshell membrane: A potential biomaterial for corneal wound healing.

The eggshell membrane (ESM) is an abundant resource with innate complex structure and composition provided by nature. With at least 60 million tonnes of hen eggs produced globally per annum, utilisation of this waste resource is highly attractive in positively impacting sustainability worldwide. Given the morphology and mechanical properties of this membrane, it has great potential as a biomaterials for wound dressing. However, to date, no studies have demonstrated nor reported this application. As such, the objective of this investigation was to identify and optimise a reproducible extraction protocol of the ESM and to assess the physical, chemical, mechanical and biological properties of the substrate with a view to use as a wound dressing. ESM samples were isolated by either manual peeling (ESM-strip) or via extraction using acetic acid [ESM-A0.5] or ethylenediaminetetraacetic acid, EDTA [ESM-E0.9]. Energy dispersive X-ray spectroscopy (EDS) confirmed that there were no traces of calcium residues from the extraction process. Fourier transform infrared (FTIR) spectroscopy revealed that the extraction method (acetic acid and EDTA) did not alter the chemical structures of the ESM and also clarified the composition of the fibrous proteins of the ESM. Scanning electron microscopy (SEM) analyses revealed a three-layer composite structure of the ESM: an inner layer as continuous, dense and non-fibrous (limiting membrane), a middle layer with a network of fibres (inner shell membrane) and the outer layer (outer shell membrane) of larger fibres. Material properties including optical transparency, porosity, fluid absorption/uptake, thermal stability, mechanical profiling of the ESM samples were performed and demonstrated suitable profiles for translational applications. Biological in vitro studies using SV40 immortalised corneal epithelial cells (ihCEC) and corneal mesenchymal stromal cells (C-MSC) demonstrated excellent biocompatibility. Taken together, these results document the development of a novel sustainable biomaterial that may be used for ophthalmic wounds and/or other biomedical therapies.