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

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

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

Incorporating new approach methodologies into regulatory nonclinical pharmaceutical safety assessment

© 2023 The Author(s). ALTEX. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY), https://creativecommons.org/licenses/by/4.0/New approach methodologies (NAMs) based on human biology enable the assessment of adverse biological effects of pharmaceuticals and other chemicals. Currently, however, it is unclear how NAMs should be used during drug development to improve human safety evaluation. A series of 5 workshops with 13 international experts (regulators, preclinical scientists, and NAMs developers) was conducted to identify feasible NAMs and to discuss how to exploit them in specific safety assessment contexts. Participants generated four “maps” of how NAMs can be exploited in the safety assessment of the liver, respiratory, cardiovascular, and central nervous systems. Each map shows relevant endpoints measured and tools used (e.g., cells, assays, platforms), and highlights gaps where further development and validation of NAMs remains necessary. Each map addresses the fundamental scientific requirements for the safety assessment of that organ system, providing users with guidance on the selection of appropriate NAMs. In addition to generating the maps, participants offered suggestions for encouraging greater NAM adoption within drug development and their inclusion in regulatory guidelines. A specific recommendation was that pharmaceutical companies should be more transparent about how they use NAMs in-house. As well as giving guidance for the four organ systems, the maps provide a template that could be used for additional organ safety testing contexts. Moreover, their conversion to an interactive format would enable users to drill down to the detail necessary to answer specific scientific and regulatory questions.Peer reviewe

Incorporating new approach methodologies into regulatory nonclinical pharmaceutical safety assessment

New approach methodologies (NAMs) based on human biology enabletheassessment of adverse biological effects of pharmaceuticals and other chemicals. Currently,however, it is unclear how NAMsshould be usedduring drug development to improve human safety evaluation. A series of 5 workshops with 13 international experts (regulators, preclinical scientists and NAMs developers) were conducted to identify feasible NAMsand to discuss how to exploit them in specific safety assessmentcontexts. Participants generated four‘maps’of how NAMs can be exploited in the safety assessment ofthe liver, respiratory, cardiovascular,and central nervous systems. Each map showsrelevant end points measured, tools used (e.g.,cells, assays, platforms), and highlights gaps where furtherdevelopment and validation of NAMs remainsnecessary. Each map addresses the fundamental scientific requirements for the safety assessment of that organ system, providing users with guidance on the selection of appropriate NAMs. In addition to generating the maps, participants offered suggestions for encouraging greater NAM adoption within drug development and their inclusion in regulatory guidelines. A specific recommendation was that pharmaceutical companies should be more transparent about how they use NAMs in-house. As well as giving guidance for the fourorgan systems, the maps providea template that could be used for additional organ safety testing contexts.Moreover, their conversion to an interactive format would enable users to drill down to the detail necessary to answer specific scientific and regulatory questions. 1IntroductionExtensive nonclinical safety studies are undertaken on new pharmaceuticals prior to and alongside clinical trials. Their purpose is to identify and understand the toxic effects of thecompoundin order to determine whether its anticipated benefit versusrisk profile justifies clinical evaluation and, if so, to inform the design and monitoring of clinical studies. The nonclinical safety studies are mandated by regulatory guidelines and include a variety of safety pharmacologyand toxicology investigations.Safety pharmacology studies aimto determinewhether pharmaceuticalscause on-or off-target effects on biological processes which can affect the function of critical organ systems (e.g.,cardiovascular, respiratory, gastrointestinal,and central nervous systems)and to assess potency, which is needed to assess safety margins versushuman clinical drug exposure. Safety pharmacology studiesalso help informthe selectionof follow-on investigations that can aid human risk assessmentand may provide insight into mechanismswhich underlie any effectsthat arise in humans.Multiple leading pharmaceutical companies (e.g.,AstraZeneca, GlaxoSmithKline, Novartis,and Pfizer) have outlined the advantages provided by in vitrosafety pharmacological profiling, including early identification of off-target interactionsandthe prediction ofclinical side effects that may be missed in animalstudies, and have highlighted that these studies enable much more cost-effective and rapid profiling of large numbers of compounds than animal procedures (Bowes et al., 2012).Toxicology studies evaluate systemic organ toxicities, behavioraleffects, reproductive and developmental toxicology, genetic toxicology,eye irritancy and dermal sensitization. They include single and repeat dose studies in rodent and non-rodentanimal species, which identify target organs, assessseverity andreversibility,and define dose-response and no observed adverse effect levels. These are critical parameters which are essential for regulatory decision-makingon whether the compound can be progressed into clinical trials and if so, estimation ofa suitable starting dose,maximum dose, dose escalation regime,andany non-standard clinical safety monitoringthat may be needed.Toxicity observedinnonclinical animal safety studies is an important cause of the high attrition rate of candidate drugs prior to clinicaltrials that occurs inmultiple pharmaceutical companies(Cook et al., 2014).However, many drugs cause clinically serious adverseeffects in humans which are not detectedin animals(Bailey et al., 2015). For example, human drug induced liver injury(DILI),which is not detected in animal safety studies,is animportant cause of attrition late in clinical development, failed licensing and/or of restrictive drug labelling(Watkins, 2011). Attrition due to toxicity observed in animals and/or in humans isanimportant cause of the high failure rate of clinical drug development(Cook et al., 2014; Watkins, 2011; Thomas et al., 2021).New approach methodologies (NAMs)includemethods which predict and evaluate biological processes by which pharmaceuticals may elicit desirable pharmacological effects and/or may cause undesirable toxicity. Many different types of NAMs have been described. Theseinclude simple in vitrocell-based tests, more complex organotypic or microphysiologicalsystems (MPS)/organ-on-a-chipdevices,and whole human tissuesmaintained ex vivo. Interpretation ofthe invivorelevance of the data providedby these methods is complementedbycomputational toolswhichsimulate and predict in vivodrug disposition and kinetics, in particular physiologically based pharmacokinetic (PBPK) models. Accurate in vitroto in vivoextrapolation isfurther aided by human low-dose testing and microdosing studies (phase 0 testing), which provide precise data on systemic human drug exposure and kineticsin vivo

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