Influence of Gelatin Source and Bloom Number on Gelatin Methacryloyl Hydrogels Mechanical and Biological Properties for Muscle Regeneration

Approximately half of an adult human’s body weight is made up of muscles. Thus, restoring the functionality and aesthetics of lost muscle tissue is critical. The body is usually able to repair minor muscle injuries. However, when volumetric muscle loss occurs due to tumour extraction, for instance, the body will form fibrous tissue instead. Gelatin methacryloyl (GelMA) hydrogels have been applied for drug delivery, tissue adhesive, and various tissue engineering applications due to their tuneable mechanical properties. Here, we have synthesised GelMA from different gelatin sources (i.e., porcine, bovine, and fish) with varying bloom numbers, which refers to the gel strength, and investigated for the influence of the source of gelatin and the bloom number on biological activities and mechanical properties. The results indicated that the source of the gelatin and variable bloom numbers have an impact on GelMA hydrogel properties. Furthermore, our findings established that the bovine-derived gelatin methacryloyl (B-GelMA) has better mechanical properties than the other varieties composed of porcine and fish with 60 kPa, 40 kPa, and 10 kPa in bovine, porcine, and fish, respectively. Additionally, it showed a noticeably greater swelling ratio (SR) ~1100% and a reduced rate of degradation, improving the stability of hydrogels and giving cells adequate time to divide and proliferate to compensate for muscle loss. Furthermore, the bloom number of gelatin was also proven to influence the mechanical properties of GelMA. Interestingly, although GelMA made of fish had the lowest mechanical strength and gel stability, it demonstrated excellent biological properties. Overall, the results emphasise the importance of gelatin source and bloom number, allowing GelMA hydrogels to have a wide range of mechanical and excellent biological properties and making them suitable for various muscle tissue regeneration applications

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

Lithium-loaded GelMA-Phosphate glass fibre constructs: Implications for astrocyte response

Combinations of different biomaterials with their own advantages as well as functionalization with other components have long been implemented in tissue engineering to improve the performance of the overall material. Biomaterials, particularly hydrogel platforms, have shown great potential for delivering compounds such as drugs, growth factors, and neurotrophic factors, as well as cells, in neural tissue engineering applications. In central the nervous system, astrocyte reactivity and glial scar formation are significant and complex challenges to tackle for neural and functional recovery. GelMA hydrogel-based tissue constructs have been developed in this study and combined with two different formulations of phosphate glass fibers (PGFs) (with Fe3+ or Ti2+ oxide) to impose physical and mechanical cues for modulating astrocyte cell behavior. This study was also aimed at investigating the effects of lithium-loaded GelMA-PGFs hydrogels in alleviating astrocyte reactivity and glial scar formation offering novel perspectives for neural tissue engineering applications. The rationale behind introducing lithium is driven by its long-proven therapeutic benefits in mental disorders, and neuroprotective and pronounced anti-inflammatory properties. The optimal concentrations of lithium and LPS were determined in vitro on primary rat astrocytes. Furthermore, qPCR was conducted for gene expression analysis of GFAP and IL-6 markers on primary astrocytes cultured 3D into GelMA and GelMA-PGFs hydrogels with and without lithium and in vitro stimulated with LPS for astrocyte reactivity. The results suggest that the combination of bioactive phosphate-based glass fibers and lithium loading into GelMA structures may impact GFAP expression and early IL-6 expression. Furthermore, GelMA-PGFs (Fe) constructs have shown improved performance in modulating glial scarring over GFAP regulation

A Sustainable, Green-Processed, Ag-Nanoparticle-Incorporated Eggshell-Derived Biomaterial for Wound-Healing Applications

The eggshell membrane (ESM) is a natural biomaterial with unique physical and mechanical properties that make it a promising candidate for wound-healing applications. However, the ESM’s inherent properties can be enhanced through incorporation of silver nanoparticles (AgNPs), which have been shown to have antimicrobial properties. In this study, commercially produced AgNPs and green-processed AgNPs were incorporated into ESM and evaluated for their physical, biological, and antimicrobial properties for potential dermal application. The ESM was extracted using various techniques, and then treated with either commercially produced AgNPs (Sigma-Aldrich, Poole, UK) or green-synthesized AgNPs (Metalchemy, London, UK) to produce AgNPs-ESM samples. The physical characteristics of the samples were evaluated using scanning electron microscopy (SEM), Fourier Transform Infrared (FTIR) spectroscopy, and the biological properties were assessed through in vitro studies using human dermal fibroblasts (HDFs) and BJ cells. The SEM analysis of the AgNPs-ESM samples showed localization of AgNPs on the ESM surface, and that the ESM maintained its structural integrity following AgNP incorporation. The FTIR confirmed loading of AgNPs to ESM samples. The biological studies showed that the 5 μg/mL AgNPs-ESM samples were highly biocompatible with both HDFs and BJ cells, and had good viability and proliferation rates. Additionally, the AgNPs-ESM samples demonstrated pro-angiogenic properties in the CAM assay, indicating their potential for promoting new blood vessel growth. Assessment of the antimicrobial activity of the enhanced AgNPs/ESMs was validated using the International Standard ISO 16869:2008 methodology and exploited Cladosporium, which is one of the most commonly identified fungi in wounds, as the test microorganism (≥5 × 106 cells/mL). The AgNPs-ESM samples displayed promising antimicrobial efficacy as evidenced by the measured zone of inhibition. Notably, the green-synthesized AgNPs demonstrated greater zones of inhibition (~17 times larger) compared to commercially available AgNPs (Sigma-Aldrich). Although both types of AgNP exhibited long-term stability, the Metalchemy-modified samples demonstrated a slightly stronger inhibitory effect. Overall, the AgNPs-ESM samples developed in this study exhibited desirable physical, biological, and antimicrobial properties for potential dermal wound-dressing applications. The use of green-processed AgNPs in the fabrication of the AgNPs-ESM samples highlights the potential for sustainable and environmentally friendly wound-healing therapies. Further research is required to assess the long-term biocompatibility and effectiveness of these biomaterials in vivo

Inclusion of calcium phosphate does not further improve in vitro and in vivo osteogenesis in a novel, highly biocompatible, mechanically stable and 3D printable polymer

At a time of unpredictable challenges for health, one trend is certain: there is an exceedingly high demand for functional implants, particularly bone grafts. This has encouraged the emergence of bone tissue engineering substitutes as an alternative method to conventional bone grafts. However, the current approaches in the field face several limitations that have prevented the ultimate translation into clinical settings. As a result, many attempts have been made to fabricate synthetic bone implants that can offer suitable biological and mechanical properties.Light curable methacrylate-based polymers have ideal properties for bone repair. These materials are also suitable for 3D printing which can be applicable for restoration of both function and aesthetics. The main objective of this research was to investigate the role of calcium phosphate (CaP) incorporation in a mechanically stable, biologically functional and 3D printable polymer for the reconstruction of complex craniofacial defects. The experimental work initially involved the synthesis of (((((((((((3R,3aR,6S,6aR)- hexahydrofuro[3,2-b]furan-3,6-diyl)bis(oxy))bis(ethane-2,1- 48 diyl))bis(oxy))bis(carbonyl))bis(azanediyl))bis(3,3,5-trimethylcyclohexane-5,1- 49 diyl))bis(azanediyl))bis(carbonyl))bis(oxy))bis(ethane-2,1-diyl) bis(2-methylacrylate) referred to as CSMA and fabrication of composite discs via a Digital Light Printing (DLP) method. The flow behaviour of the polymer as a function of CaP addition, surface remineralisation potential, in vitro cell culture, using MC3T3 and Adipose-Derived Mesenchymal Stem Cells (ADSCs) and ex ovo angiogenic response was assessed. Finally, in vivo studies were carried out to investigate neo-bone formation at 4- and 8-weeks post-implantation. Quantitative micro-CT and histological evaluation did not show a higher rate of bone formation in CaP filled CSMA composites compared to CSMA itself. Therefore, such polymeric systems hold promising features by allowing more flexibility in designing a 3D printed scaffold targeted at the reconstruction of maxillofacial defects

Therapeutic Application of an Ag-Nanoparticle-PNIPAAm-Modified Eggshell Membrane Construct for Dermal Regeneration and Reconstruction

Current therapeutic treatments for the repair and/or replacement of damaged skin following disease or traumatic injury is severely limited. The chicken eggshell membrane (ESM) is a unique material: its innate physical and mechanical characteristics offer optimal barrier properties and, as a naturally derived extract, it demonstrates inherent biocompatibility/biodegradability. To further enhance its therapeutic and clinical potential, the ESM can be modified with the thermo-responsive polymer, poly(N-isopropylacrylAmide) (PNIPAAm) as well as the incorporation of (drug-loaded) silver nanoparticles (AgNP); essentially, by a simple change in temperature, the release and delivery of the NP can be targeted and controlled. In this study, ESM samples were isolated using a decellularization protocol, and the physical and mechanical characteristics were profiled using SEM, FT-IR, DSC and DMA. PNIPAAm was successfully grafted to the ESM via amidation reactions and confirmed using FT-IR, which demonstrated the distinctive peaks associated with Amide A (3275 cm−1), Amide B (2970 cm−1), Amide I (1630 cm−1), Amide II (1535 cm−1), CH2, CH3 groups, and Amide III (1250 cm−1) peaks. Confirmation of the incorporation of AgNP onto the stratified membrane was confirmed visually with SEM, qualitatively using FT-IR and also via changes in absorbance at 380 nm using UV-Vis spectrophotometry during a controlled release study for 72 h. The biocompatibility and cytotoxicity of the novel constructs were assessed using human dermal fibroblast (HDFa) and mouse dermal fibroblast (L929) cells and standard cell culture assays. Metabolic activity assessment (i.e., MTS assay), LDH-release profiles and Live/Dead staining demonstrated good attachment and spreading to the samples, and high cell viability following 3 days of culture. Interestingly, longer-term viability (>5 days), the ESM-PNIPAAm and ESM-PNIPAAm (AgNP) samples showed a greater and sustained cell viability profile. In summary, the modified and enhanced ESM constructs were successfully prepared and characterized in terms of their physical and mechanical profiles. AgNP were successfully loaded into the construct and demonstrated a desirable release profile dependent on temperature modulation. Fibroblasts cultured on the extracted ESM samples and ESM-PNIPAAm demonstrated high biocompatibility in terms of high cell attachment, spreading, viability and proliferation rates. As such, this work summarizes the development of an enhanced ESM-based construct which may be exploited as a clinical/therapeutic wound dressing as well as a possible application as a novel biomaterial scaffold for drug development

A Sustainable, Green-Processed, Ag-Nanoparticle-Incorporated Eggshell-Derived Biomaterial for Wound-Healing Applications

The eggshell membrane (ESM) is a natural biomaterial with unique physical and mechanical properties that make it a promising candidate for wound-healing applications. However, the ESM’s inherent properties can be enhanced through incorporation of silver nanoparticles (AgNPs), which have been shown to have antimicrobial properties. In this study, commercially produced AgNPs and green-processed AgNPs were incorporated into ESM and evaluated for their physical, biological, and antimicrobial properties for potential dermal application. The ESM was extracted using various techniques, and then treated with either commercially produced AgNPs (Sigma-Aldrich, Poole, UK) or green-synthesized AgNPs (Metalchemy, London, UK) to produce AgNPs-ESM samples. The physical characteristics of the samples were evaluated using scanning electron microscopy (SEM), Fourier Transform Infrared (FTIR) spectroscopy, and the biological properties were assessed through in vitro studies using human dermal fibroblasts (HDFs) and BJ cells. The SEM analysis of the AgNPs-ESM samples showed localization of AgNPs on the ESM surface, and that the ESM maintained its structural integrity following AgNP incorporation. The FTIR confirmed loading of AgNPs to ESM samples. The biological studies showed that the 5 μg/mL AgNPs-ESM samples were highly biocompatible with both HDFs and BJ cells, and had good viability and proliferation rates. Additionally, the AgNPs-ESM samples demonstrated pro-angiogenic properties in the CAM assay, indicating their potential for promoting new blood vessel growth. Assessment of the antimicrobial activity of the enhanced AgNPs/ESMs was validated using the International Standard ISO 16869:2008 methodology and exploited Cladosporium, which is one of the most commonly identified fungi in wounds, as the test microorganism (≥5 × 106 cells/mL). The AgNPs-ESM samples displayed promising antimicrobial efficacy as evidenced by the measured zone of inhibition. Notably, the green-synthesized AgNPs demonstrated greater zones of inhibition (~17 times larger) compared to commercially available AgNPs (Sigma-Aldrich). Although both types of AgNP exhibited long-term stability, the Metalchemy-modified samples demonstrated a slightly stronger inhibitory effect. Overall, the AgNPs-ESM samples developed in this study exhibited desirable physical, biological, and antimicrobial properties for potential dermal wound-dressing applications. The use of green-processed AgNPs in the fabrication of the AgNPs-ESM samples highlights the potential for sustainable and environmentally friendly wound-healing therapies. Further research is required to assess the long-term biocompatibility and effectiveness of these biomaterials in vivo

Poly-ε-Caprolactone/Fibrin-Alginate Scaffold: A New Pro-Angiogenic Composite Biomaterial for the Treatment of Bone Defects

We hypothesized that a composite of 3D porous melt-electrowritten poly-ε-caprolactone (PCL) coated throughout with a porous and slowly biodegradable fibrin/alginate (FA) matrix would accelerate bone repair due to its angiogenic potential. Scanning electron microscopy showed that the open pore structure of the FA matrix was maintained in the PCL/FA composites. Fourier transform infrared spectroscopy and differential scanning calorimetry showed complete coverage of the PCL fibres by FA, and the PCL/FA crystallinity was decreased compared with PCL. In vitro cell work with osteoprogenitor cells showed that they preferentially bound to the FA component and proliferated on all scaffolds over 28 days. A chorioallantoic membrane assay showed more blood vessel infiltration into FA and PCL/FA compared with PCL, and a significantly higher number of bifurcation points for PCL/FA compared with both FA and PCL. Implantation into a rat cranial defect model followed by microcomputed tomography, histology, and immunohistochemistry after 4-and 12-weeks post operation showed fast early bone formation at week 4, with significantly higher bone formation for FA and PCL/FA compared with PCL. However, this phenomenon was not extrapolated to week 12. Therefore, for long-term bone regeneration, tuning of FA degradation to ensure syncing with new bone formation is likely necessary.</p