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Promoting neovascularization is a prerequisite for many tissue engineering applications and the treatment of cardiovascular disease. Delivery of a pro-angiogenic stimulus via acellular materials offers several benefits over biological therapies but has been hampered by interaction of the implanted material with the innate immune response. However, macrophages, a key component of the innate immune response, release a plurality of soluble factors that can be harnessed to stimulate neovascularization and restore blood flow to damaged tissue. This study investigates the ability of biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres to restore tissue perfusion in a hind limb model of ischaemia. Microspheres exhibiting a hierarchical porous structure are associated with an increase in blood flow at day 21 post-implantation compared with solid microspheres composed of the same polymer. This corresponds with an increase in blood vessel density in the surrounding tissue. In vitro simulation of the foreign body response observed demonstrates M2-like macrophages incubated with the porous microspheres secreted increased amounts of vascular endothelial growth factor (VEGF) compared with M1-like macrophages providing a potential mechanism for the increased neovascularization. The results from this study demonstrate implantable biodegradable porous microspheres provide a novel approach for increasing neovascularization that could be exploited for therapeutic applications., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Cell therapy is a potential novel treatment for cardiac regeneration and numerous studies have attempted to transplant cells to regenerate the myocardium lost during myocardial infarction. To date, only minimal improvements to cardiac function have been reported. This is likely to be the result of low cell retention and survival following transplantation. This study aimed to improve the delivery and engraftment of viable cells by using an injectable microcarrier that provides an implantable, biodegradable substrate for attachment and growth of cardiomyocytes derived from induced pluripotent stem cells (iPSC). We describe the fabrication and characterisation of Thermally Induced Phase Separation (TIPS) microcarriers and their surface modification to enable iPSC-derived cardiomyocyte attachment in xeno-free conditions is described. The selected formulation resulted in iPSC attachment, expansion, and retention of pluripotent phenotype. Differentiation of iPSC into cardiomyocytes on the microcarriers is investigated in comparison with culture on 2D tissue culture plastic surfaces. Microcarrier culture is shown to support culture of a mature cardiomyocyte phenotype, be compatible with injectable delivery, and reduce anoikis. The findings from this study demonstrate that TIPS microcarriers provide a supporting matrix for culturing iPSC and iPSC-derived cardiomyocytes in vitro and are suitable as an injectable cell-substrate for cardiac regeneration., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

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 Fe 3+ or Ti 2+ 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., (© 2024 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Targeted delivery and retention are essential requirements for implantable tissue-engineered products. Non-invasive imaging methods that can confirm location, retention, and biodistribution of transplanted cells attached to implanted tissue engineering scaffolds will be invaluable for the optimization and enhancement of regenerative therapies. To address this need, an injectable tissue engineering scaffold consisting of highly porous microspheres compatible with transplantation of cells is modified to contain the computed tomography (CT) contrast agent barium sulphate (BaSO 4 ). The trackable microspheres show high x-ray absorption, with contrast permitting whole-body tracking. The microspheres are cellularized with GFP+ Luciferase+ mesenchymal stem cells and show in vitro biocompatibility. In vivo, cellularized BaSO 4 -loaded microspheres are delivered into the hindlimb of mice where they remain viable for 14 days. Co-registration of 3D-bioluminescent imaging and µCT reconstructions enable the assessment of scaffold material and cell co-localization. The trackable microspheres are also compatible with minimally-invasive delivery by ultrasound-guided transthoracic intramyocardial injections in rats. These findings suggest that BaSO 4 -loaded microspheres can be used as a novel tool for optimizing delivery techniques and tracking persistence and distribution of implanted scaffold materials. Additionally, the microspheres can be cellularized and have the potential to be developed into an injectable tissue-engineered combination product for cardiac regeneration., (© 2024 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Introduction: Among gene mutations and variants linked to an increased risk of PD, mutations of leucine-rich repeat kinase 2 gene (LRRK2) are among the most frequently associated with early- and late-onset PD. Clinical and neuropathological characteristics of idiopathic-PD (iPD) and LRRK2-PD are similar, and these similarities suggest that the pathomechanisms between these two conditions are shared. LRRK2 mutations determine a gain-of-function and yield higher levels of lrrk2 across body tissues, including brain. On another side, recent animal studies supported the potential use of low dose radiation (LDR) to modify the pathomechanisms of diseases such as Alzheimer's disease (AD)., Methods: We assessed if a single total-body LDR (sLDR) exposure in normal swine could alter expression levels of the following PD-associated molecules: alpha-synuclein (α-syn), phosphorylated-α-synuclein (pα-syn), parkin, tyrosine hydroxylase (th), lrrk2, phosphorylated-lrrk2 (pS935-lrrk2), and some LRRK2 substrates (Rab8a, Rab12) across different brain regions. These proteins were measured in frontal cortex, hippocampus, striatum, thalamus/hypothalamus, and cerebellum of 9 radiated (RAD) vs. 6 sham (SH) swine after 28 days from a sLDR of 1.79Gy exposure., Results: Western Blot analyses showed lowered lrrk2 levels in the striatum of RAD vs. SH swine (p < 0.05), with no differences across the remaining brain regions. None of the other protein levels differed between RAD and SH swine in any examined brain regions. No lrrk2 and p-lrrk2 (S935) levels differed in the lungs of RAD vs. SH swine., Conclusions: These findings show a specific striatal lrrk2 lowering effect due to LDR and support the potential use of LDR to interfere with the pathomechanisms of PD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Chitosan-triphosphate (TPP) nanogels are widely studied drug delivery carrier systems, typically prepared via a simple mixing process. However, the effects of the processing factors on nanogel production have not been extensively explored, despite the importance of understanding and standardising such factors to allow upscaling and commercial usage. This study aims to systematically evaluate the effects of various fabrication and processing factors on the properties of nanogels using a Design of Experiment approach. Hydrodynamic size, polydispersity index (PDI), zeta potential, and encapsulation efficiency were determined as the dependent factors. The temperature, stirring rate, chitosan grade, crosslinker choice, and the interaction term between temperature and chitosan grade were found to have a significant effect on the particle size, whereas the effect of temperature and the addition rate of crosslinker on the PDI was also noteworthy. Moreover, the addition rate of the crosslinker and the volume of the reaction vessel were found to impact the encapsulation efficiency. The zeta potential of the nanogels was found to be governed by the chitosan grade. The optimal fabrication conditions for the development of medium molecular weight chitosan and TPP nanogels included the following: the addition rate for TPP solution was set at 2 mL/min, while the solution was then stirred at a temperature of 50 °C and a stirring speed of 600 rpm. The volume of the glass vial used was 28 mL, while the stirrer size was 20 mm. The second aim of the study was to evaluate the potential for scaling up the nanogels. Size and PDI were found to increase from 128 nm to 151 nm and from 0.232 to 0.267, respectively, when the volume of the reaction mixture was increased from 4 to 20 mL and other processing factors were kept unchanged. These results indicate that caution is required when scaling up as the nanogel properties may be significantly altered with an increasing production scale.

Hematopoietic acute radiation syndrome (H-ARS) involves injury to multiple organ systems following total body irradiation (TBI). Our laboratory demonstrated that captopril, an angiotensin-converting enzyme inhibitor, mitigates H-ARS in Göttingen minipigs, with improved survival and hematopoietic recovery, as well as the suppression of acute inflammation. However, the effects of captopril on the gastrointestinal (GI) system after TBI are not well known. We used a Göttingen minipig H-ARS model to investigate captopril's effects on the GI following TBI ( 60 Co 1.79 or 1.80 Gy, 0.42-0.48 Gy/min), with endpoints at 6 or 35 days. The vehicle or captopril (0.96 mg/kg) was administered orally twice daily for 12 days, starting 4 h post-irradiation. Ilea were harvested for histological, protein, and RNA analyses. TBI increased congestion and mucosa erosion and hemorrhage, which were modulated by captopril. GPX-4 and SLC7A11 were downregulated post-irradiation, consistent with ferroptosis at 6 and 35 days post-irradiation in all groups. Interestingly, p21/waf1 increased at 6 days in vehicle-treated but not captopril-treated animals. An RT-qPCR analysis showed that radiation increased the gene expression of inflammatory cytokines IL1B , TNFA , CCL2, IL18 , and CXCL8 , and the inflammasome component NLRP3 . Captopril suppressed radiation-induced IL1B and TNFA . Rectal microbiome analysis showed that 1 day of captopril treatment with radiation decreased overall diversity, with increased Proteobacteria phyla and Escherichia genera. By 6 days, captopril increased the relative abundance of Enterococcus, previously associated with improved H-ARS survival in mice. Our data suggest that captopril mitigates senescence, some inflammation, and microbiome alterations, but not ferroptosis markers in the intestine following TBI.

There is a growing interest in low dose radiation (LDR) to counteract neurodegeneration. However, LDR effects on normal brain have not been completely explored yet. Recent analyses showed that LDR exposure to normal brain tissue causes expression level changes of different proteins including neurodegeneration-associated proteins. We assessed the proteomic changes occurring in radiated vs. sham normal swine brains. Due to its involvement in various neurodegenerative processes, including those associated with cognitive changes after high dose radiation exposure, we focused on the hippocampus first. We observed significant proteomic changes in the hippocampus of radiated vs. sham swine after LDR (1.79Gy). Mass spectrometry results showed 190 up-regulated and 120 down-regulated proteins after LDR. Western blotting analyses confirmed increased levels of TPM1, TPM4, PCP4 and NPY (all proteins decreased in various neurodegenerative processes, with NPY and PCP4 known to be neuroprotective) in radiated vs. sham swine. These data support the use of LDR as a potential beneficial tool to interfere with neurodegenerative processes and perhaps other brain-related disorders, including behavioral disorders., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

The risk of exposure to high levels of ionizing radiation from nuclear weapons or radiological accidents is an increasing world concern. Partial- or total-body exposure to high doses of radiation is potentially lethal through the induction of acute radiation syndrome (ARS). Hematopoietic cells are sensitive to radiation exposure; white blood cells primarily undergo apoptosis while red blood cells (RBCs) undergo hemolysis. Several laboratories demonstrated that the rapid hemolysis of RBCs results in the release of acellular iron into the blood. We recently demonstrated using a murine model of ARS after total-body irradiation (TBI) and the loss of RBCs, iron accumulated in the bone marrow and spleen, notably between 4-21 days postirradiation. Here, we investigated iron accumulation in the bone marrow and spleens from TBI nonhuman primates (NHPs) using histological stains. We observed trends in increased intracellular and extracellular brown pigmentation in the bone marrow after various doses of radiation, especially after 4-15 days postirradiation, but these differences did not reach significance. We observed a significant increase in Prussian blue-staining intracellular iron deposition in the spleen 13-15 days after 5.8-8.5 Gy of TBI. We observed trends of increased iron in the spleen after 30-60 days postirradiation, with varying doses of radiation, but these differences did not reach significance. The NHP model of ARS confirms our earlier findings in the murine model, showing iron deposition in the bone marrow and spleen after TBI., (©2023 by Radiation Research Society. All rights of reproduction in any form reserved.)

Fecal incontinence, although not life-threatening, has a high impact on the economy and patient quality of life. So far, available treatments are based on both surgical and nonsurgical approaches. These can range from changes in diet, to bowel training, or sacral nerve stimulation, but none of which provides a long-term solution. New regenerative medicine-based therapies are emerging, which aim at regenerating the sphincter muscle and restoring continence. Usually, these consist of the administration of a suspension of expanded skeletal-derived muscle cells (SkMDCs) to the damaged site. However, this strategy often results in a reduced cell viability due to the need for cell harvesting from the expansion platform, as well as the non-native use of a cell suspension to deliver the anchorage-dependent cells. In this study, we propose the proof-of-concept for the bioprocessing of a new cell delivery method for the treatment of fecal incontinence, obtained by a scalable two-step process. First, patient-isolated SkMDCs were expanded using planar static culture systems. Second, by using a single-use PBS-MINI Vertical-Wheel ® bioreactor, the expanded SkMDCs were combined with biocompatible and biodegradable (i.e., directly implantable) poly(lactic- co -glycolic acid) microcarriers prepared by thermally induced phase separation. This process allowed for up to 80% efficiency of SkMDCs to attach to the microcarriers. Importantly, SkMDCs were viable during all the process and maintained their myogenic features (e.g., expression of the CD56 marker) after adhesion and culture on the microcarriers. When SkMDC-containing microcarriers were placed on a culture dish, cells were able to migrate from the microcarriers onto the culture surface and differentiate into multinucleated myotubes, which highlights their potential to regenerate the damaged sphincter muscle after administration into the patient. Overall, this study proposes an innovative method to attach SkMDCs to biodegradable microcarriers, which can provide a new treatment for fecal incontinence.

Brain radiation has been medically used to alter the metabolism of cancerous cells and induce their elimination. Rarely, though, brain radiation has been used to interfere with the pathomechanisms of non-cancerous brain disorders, especially neurodegenerative disorders. Data from low-dose radiation (LDR) on swine brains demonstrated reduced levels of phosphorylated-tau (CP13) and amyloid precursor protein (APP) in radiated (RAD) versus sham (SH) animals. Phosphorylated-tau and APP are involved in Alzheimer's disease (AD) pathogenesis. We determined if the expression levels of hyperphosphorylated-tau, 3R-tau, 4R-tau, synaptic, intraneuronal damage, and DNA damage/oncogenic activation markers were altered in RAD versus SH swine brains. Quantitative analyses demonstrated reduced levels of AT8 and 3R-tau in hippocampus (H) and striatum (Str), increased levels of synaptophysin and PSD-95 in frontal cortex (FCtx), and reduced levels of NF-L in cerebellum (CRB) of RAD versus SH swine. DNA damage and oncogene activation markers levels did not differ between RAD and SH animals, except for histone-H3 (increased in FCtx and CRB, decreased in Str), and p53 (reduced in FCtx, Str, H and CRB). These findings confirm the region-based effects of sLDR on proteins normally expressed in larger mammalian brains and support the potential applicability of LDR to beneficially interfere against neurodegenerative mechanisms., (© 2023. The Author(s).)

Sporadic inclusion body myositis (sIBM) is an idiopathic inflammatory myopathy with invasion of CD8 T cells in muscle and aggregation of proteins in the sarcoplasm. TDP-43 and p62 are two proteins that aggregate in affected muscle, and have been suggested as specific markers for sIBM over other inflammatory myopathies. TDP-43 is also mislocalised from the nucleus to the sarcoplasm in sIBM. It is not clear if inflammation precedes protein aggregation in sIBM. This study investigated if exposure to cytotoxic inflammatory cells caused TDP-43 and p62 aggregation or TDP-43 mislocalisation in cultured myotubes. TALL-104 coculture was highly cytotoxic to myotubes after 24 h. Secretion of IFNγ and TNFα were higher in cocultures compared to monocultured TALL-104 cells, indicating activation. TALL-104 cells attached to and infiltrated myotubes. There was no effect of TALL-104 coculture on TDP-43 or p62 sarcoplasmic aggregate size or frequency. However, there was decreased localisation of TDP-43 to the nucleus with TALL-104 coculture compared to control. In an in vitro setting, cytotoxic immune cells did not cause TDP-43 or p62 sarcoplasmic aggregation, suggesting cellular cytotoxicity may not trigger aggregation of these proteins. However TALL-104 coculture influenced TDP-43 localisation, suggesting cytotoxic immune cells may contribute to TDP-43 localisation shifts which is observed in sIBM., (© 2023. Springer Nature Limited.)

Skeletal muscle of patients with sporadic inclusion body myositis (sIBM) presents with inflammation, including upregulation of inflammatory cytokines such as interferon γ (IFN γ ). Non-inflammatory features are also observed, like the sarcoplasmic accumulation of proteins including TDP-43 and p62. This study aimed to investigate the effect of IFN γ and interleukin 1- β (IL-1 β ) on TDP-43 and p62 aggregation in vitro. Primary human myotubes were treated with IL-1 β (20 ng/mL) and IFN γ (750 ng/mL) separately or combined for 48 hr. Sarcoplasmic TDP-43 aggregates and p62 puncta were assessed using image analysis for size, frequency, and colocalization with each other. Total protein expression of TDP-43, p62 and LC3 was assessed using western blotting. The subcellular localization of TDP-43 was also analyzed using image analysis. Combined IL-1 β and IFN γ treatment increased puncta size of p62 compared to control (0.49 ± 0.13  µ m 2 versus 0.28 ± 0.06  µ m 2 ), without affecting puncta frequency or p62 expression but with an increased LC3II/LC3I ratio, suggesting autophagic alterations. IL-1 β or IFN γ did not alter p62 puncta size or frequency, suggesting a combined insult of multiple inflammatory mediators is necessary to cause p62 alterations. IL-1 β increased p62 protein expression in an autophagy-independent manner. None of the cytokine treatments affected TDP-43 protein expression, size, or frequency of TDP-43 aggregates or localization, suggesting IL-1 β and IFN γ may influence TDP-43 processing in human skeletal muscle cells. TDP-43 was localized to the sarcoplasm under control conditions, suggesting this may not be a pathological feature. Overall, sIBM-like TDP-43/p62 features were not triggered by IL-1 β and/or IFN γ ., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Bryony McCord and Richard M. Day.)

Skeletal muscle-derived cells (SMDC) hold tremendous potential for replenishing dysfunctional muscle lost due to disease or trauma. Current therapeutic usage of SMDC relies on harvesting autologous cells from muscle biopsies that are subsequently expanded in vitro before re-implantation into the patient. Heterogeneity can arise from multiple factors including quality of the starting biopsy, age and comorbidity affecting the processed SMDC. Quality attributes intended for clinical use often focus on minimum levels of myogenic cell marker expression. Such approaches do not evaluate the likelihood of SMDC to differentiate and form myofibres when implanted in vivo, which ultimately determines the likelihood of muscle regeneration. Predicting the therapeutic potency of SMDC in vitro prior to implantation is key to developing successful therapeutics in regenerative medicine and reducing implementation costs. Here, we report on the development of a novel SMDC profiling tool to examine populations of cells in vitro derived from different donors. We developed an image-based pipeline to quantify morphological features and extracted cell shape descriptors. We investigated whether these could predict heterogeneity in the formation of myotubes and correlate with the myogenic fusion index. Several of the early cell shape characteristics were found to negatively correlate with the fusion index. These included total area occupied by cells, area shape, bounding box area, compactness, equivalent diameter, minimum ferret diameter, minor axis length and perimeter of SMDC at 24 h after initiating culture. The information extracted with our approach indicates live cell imaging can detect a range of cell phenotypes based on cell-shape alone and preserving cell integrity could be used to predict propensity to form myotubes in vitro and functional tissue in vivo., Competing Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: D.D. is an employee of King’s College London and an employee of bit.bio. D.D. declares no other affiliations with or involvement in any organization or entity with any financial or non-financial interest in the subject matter or materials discussed in this manuscript., (© The Author(s) 2023.)

In recent years, extracellular vesicles (EVs) have emerged as promising biomarkers, cell-free therapeutic agents, and drug delivery carriers. Despite their great clinical potential, poor yield and unscalable production of EVs remain significant challenges. When using 3D culture methods, such as scaffolds and bioreactors, large numbers of cells can be expanded and the cell environment can be manipulated to control the cell phenotype. This has been employed to successfully increase the production of EVs as well as to enhance their therapeutic effects. The physiological relevance of 3D cultures, such as spheroids, has also provided a strategy for understanding the role of EVs in the pathogenesis of several diseases and to evaluate their role as tools to deliver drugs. Additionally, 3D culture methods can encapsulate EVs to achieve more sustained therapeutic effects as well as prevent premature clearance of EVs to enable more localised delivery and concentrated exosome dosage. This review highlights the opportunities and drawbacks of different 3D culture methods and their use in EV research.

Low dose-rate radiation exposure can occur in medical imaging, as background from environmental or industrial radiation, and is a hazard of space travel. In contrast with high dose-rate radiation exposure that can induce acute life-threatening syndromes, chronic low-dose radiation is associated with Chronic Radiation Syndrome (CRS), which can alter environmental sensitivity. Secondary effects of chronic low dose-rate radiation exposure include circulatory, digestive, cardiovascular, and neurological diseases, as well as cancer. Here, we investigated 1-2 Gy, 0.66 cGy/h, 60 Co radiation effects on primary human mesenchymal stem cells (hMSC). There was no significant induction of apoptosis or DNA damage, and cells continued to proliferate. Gene ontology (GO) analysis of transcriptome changes revealed alterations in pathways related to cellular metabolism (cholesterol, fatty acid, and glucose metabolism), extracellular matrix modification and cell adhesion/migration, and regulation of vasoconstriction and inflammation. Interestingly, there was increased hypoxia signaling and increased activation of pathways regulated by iron deficiency, but Nrf2 and related genes were reduced. The data were validated in hMSC and human lung microvascular endothelial cells using targeted qPCR and Western blotting. Notably absent in the GO analysis were alteration pathways for DNA damage response, cell cycle inhibition, senescence, and pro-inflammatory response that we previously observed for high dose-rate radiation exposure. Our findings suggest that cellular gene transcription response to low dose-rate ionizing radiation is fundamentally different compared to high-dose-rate exposure. We hypothesize that cellular response to hypoxia and iron deficiency are driving processes, upstream of the other pathway regulation.

Exposure to ionizing radiation can occur during medical treatments, from naturally occurring sources in the environment, or as the result of a nuclear accident or thermonuclear war. The severity of cellular damage from ionizing radiation exposure is dependent upon a number of factors including the absorbed radiation dose of the exposure (energy absorbed per unit mass of the exposure), dose rate, area and volume of tissue exposed, type of radiation (e.g., X-rays, high-energy gamma rays, protons, or neutrons) and linear energy transfer. While the dose, the dose rate, and dose distribution in tissue are aspects of a radiation exposure that can be varied experimentally or in medical treatments, the LET and eV are inherent characteristics of the type of radiation. High-LET radiation deposits a higher concentration of energy in a shorter distance when traversing tissue compared with low-LET radiation. The different biological effects of high and low LET with similar energies have been documented in vivo in animal models and in cultured cells. High-LET results in intense macromolecular damage and more cell death. Findings indicate that while both low- and high-LET radiation activate non-homologous end-joining DNA repair activity, efficient repair of high-LET radiation requires the homologous recombination repair pathway. Low- and high-LET radiation activate p53 transcription factor activity in most cells, but high LET activates NF-kB transcription factor at lower radiation doses than low-LET radiation. Here we review the development, uses, and current understanding of the cellular effects of low- and high-LET radiation exposure.

N-acetyl-seryl-aspartyl-lysyl proline (Ac-SDKP) is a tetrapeptide possessing anti-fibrotic, angiogenic, anti-inflammatory, anti-apoptotic, and immunomodulatory properties. Currently, the main method to quantify the peptide is liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA), both of which are labour intensive and require expensive equipment and consumables. Furthermore, these techniques are generally utilised to detect very low or trace concentrations, such as in biological samples. The use of high concentrations of analyte might overload the extraction column or the separation column in LC-MS/MS or the ELISA plates, so the response could be a non-linear relationship at high analyte concentrations. Thus, they are not ideal for formulation development where detection of dose-equivalent concentrations is typically required. Therefore, a cost-effective, simple, and accurate quantification method for the peptide at a higher concentration needs to be developed. In this study, a simple and novel HPLC-UV method is proposed and validated using an Analytical Quality by Design (AQbD) approach. The method is first screened and optimised using chromatographic responses including capacity factor, resolution, tailing factor, and theoretical plate counts, fulfilling the International Council for Harmonisation (ICH) Q2 (R1) guidelines. The resultant optimised chromatography conditions utilised 10 mM phosphate buffer at pH 2.5 and acetonitrile as mobile phases, starting at 3% (v/v) acetonitrile and 97% (v/v) buffer and increasing to 9.7% (v/v) acetonitrile and 90.3% (v/v) buffer over 15 min at a flow rate of 1 mL/min at the column temperature of 25 °C. The injection volume is set at 10 μL and the VWD detector wavelength is 220 nm. The method established is suitable for detecting the peptide at a relatively high concentration, with a quantifiable range from 7.8 μg/mL to 2.0 mg/mL. In addition, the use of a relatively simple HPLC-UV approach could significantly reduce costs and allow easier access to quantify the peptide concentration. A limitation of this method is lower sensitivity compared with using LC-MS/MS and ELISA methods but running costs are lower and the methodology is simpler. The method is capable to quantify the peptide in various tested matrix solutions, with successful quantitation of the peptide in samples obtained from in vitro drug release study in PBS and from a chitosan-TPP nanogels formulation. Therefore, the method developed here offers a complementary approach to the existing quantification methods, quantifying this peptide at increased concentrations in simple to intermediately complex matrix solutions, such as HBSS, DMEM and FluoroBrite cell culture media., (Copyright © 2022. Published by Elsevier Inc.)

Total body irradiation (TBI) can result in death associated with hematopoietic insufficiency. Although radiation causes apoptosis of white blood cells, red blood cells (RBC) undergo hemolysis due to hemoglobin denaturation. RBC lysis post-irradiation results in the release of iron into the plasma, producing a secondary toxic event. We investigated radiation-induced iron in the spleens of mice following TBI and the effects of the radiation mitigator captopril. RBC and hematocrit were reduced ~7 days (nadir ~14 days) post-TBI. Prussian blue staining revealed increased splenic Fe 3+ and altered expression of iron binding and transport proteins, determined by qPCR, western blotting, and immunohistochemistry. Captopril did not affect iron deposition in the spleen or modulate iron-binding proteins. Caspase-3 was activated after ~7-14 days, indicating apoptosis had occurred. We also identified markers of iron-dependent apoptosis known as ferroptosis. The p21/Waf1 accelerated senescence marker was not upregulated. Macrophage inflammation is an effect of TBI. We investigated the effects of radiation and Fe 3+ on the J774A.1 murine macrophage cell line. Radiation induced p21/Waf1 and ferritin, but not caspase-3, after ~24 h. Radiation ± iron upregulated several markers of pro-inflammatory M1 polarization; radiation with iron also upregulated a marker of anti-inflammatory M2 polarization. Our data indicate that following TBI, iron accumulates in the spleen where it regulates iron-binding proteins and triggers apoptosis and possible ferroptosis.

Drug deposition into the intrapericardial space is favourable for achieving localised effects and targeted cardiac delivery owing to its proximity to the myocardium as well as facilitating optimised pharmacokinetic profiles and a reduction in systemic side effects. Access to the pericardium requires invasive procedures but the risks associated with this have been reduced with technological advances, such as combining transatrial and subxiphoid access with different guidance methods. A variety of introducer devices, ranging from needles to loop-catheters, have also been developed and validated in pre-clinical studies investigating intrapericardial delivery of therapeutic agents. Access techniques are generally well-tolerated, self-limiting and safe, although some rare complications associated with certain approaches have been reported. This review covers these access techniques and how they have been applied to the delivery of drugs, cells, and biologicals, demonstrating the potential of intrapericardial delivery for treatments in cardiac arrhythmia, vascular damage, and myocardial infarction., Competing Interests: Declaration of Competing Interest No conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The physical properties of nanoparticles may affect the uptake mechanism, biodistribution, stability, and other physicochemical properties of drug delivery systems. This study aimed to first develop a model exploring the factors controlling the nanogel physical properties using a single drug (propranolol), followed by an evaluation of whether these models can be applied more generally to a range of drugs. Size, polydispersity, ζ potential, and encapsulation efficiency were investigated using a design of experiment (DOE) approach to optimize formulations by systematically identifying the effects of, and interactions between, parameters associated with nanogel formulation and drug loading. Three formulation factors were selected, namely, chitosan concentration, the ratio between the chitosan and cross-linker─sodium triphosphate─and the ratio between the chitosan and drug. The results indicate that the DOE approach can be used not only to model but also to predict the size and polydispersity index (PDI). To explore the application of these prediction models with other drugs and to identify the relationship between the drug structure and nanogel properties, nanogels loaded with 12 structurally distinct drugs and 6 structurally similar drugs were fabricated at the optimal condition for propranolol in the model. The measured size, PDI, and ζ potential of the nanogels could not be modeled using distinct DOE parameters for dissimilar drugs, indicating that each drug requires a separate analysis. Nevertheless, for drugs with structural similarities, various linear and nonlinear trends were observed in the size, PDI, and ζ potential of nanogels against selected molecular descriptors, indicating that there are indeed relationships between the drug molecular structure and the performance outcomes, which may be modeled and predicted using the DOE approach. In conclusion, the study demonstrates that DOE models can be applied to model and predict the influence of formulation and drug loading on key performance parameters. While distinct models are required for structurally unrelated drugs, it was possible to establish correlations for the drug series investigated, which were based on polarity, hydrophobicity, and polarizability, thereby elucidating the importance of the interactions between the drug and the nanogels based on the nanogel properties and thus deepening the understanding of the drug-loading mechanisms in nanogels.

The vascular system is sensitive to radiation injury, and vascular damage is believed to play a key role in delayed tissue injury such as pulmonary fibrosis. However, the response of endothelial cells to radiation is not completely understood. We examined the response of primary human lung microvascular endothelial cells (HLMVEC) to 10 Gy (1.15 Gy/min) X-irradiation. HLMVEC underwent senescence (80-85%) with no significant necrosis or apoptosis. Targeted RT-qPCR showed increased expression of genes CDKN1A and MDM2 (10-120 min). Western blotting showed upregulation of p2/waf1, MDM2, ATM, and Akt phosphorylation (15 min-72 h). Low levels of apoptosis at 24-72 h were identified using nuclear morphology. To identify novel pathway regulation, RNA-seq was performed on mRNA using time points from 2 to 24 h post-irradiation. Gene ontology and pathway analysis revealed increased cell cycle inhibition, DNA damage response, pro- and anti- apoptosis, and pro-senescence gene expression. Based on published literature on inflammation and endothelial-to-mesenchymal transition (EndMT) pathway genes, we identified increased expression of pro-inflammatory genes and EndMT-associated genes by 24 h. Together our data reveal a time course of integrated gene expression and protein activation leading from early DNA damage response and cell cycle arrest to senescence, pro-inflammatory gene expression, and endothelial-to-mesenchymal transition., (© 2021. The Author(s).)

Glial cell alignment in tissue engineered constructs is essential for achieving functional outcomes in neural recovery. While gelatin methacrylate (GelMA) hydrogel offers superior biocompatibility along with permissive structure and tailorable mechanical properties, phosphate glass fibers (PGFs) can provide physical cues for directionality of neural growth. Aligned PGFs were fabricated by a melt quenching and fiber drawing method and utilized with synthesized GelMA hydrogel. The mechanical properties of GelMA and biocompatibility of the GelMA-PGFs composite were investigated in vitro using rat glial cells. GelMA with 86% methacrylation degree were photo-crosslinked using 0.1%wt photo-initiator (PI). Photocrosslinking under UV exposure for 60 s was used to produce hydrogels (GelMA-60). PGFs were introduced into the GelMA before crosslinking. Storage modulus and loss modulus of GelMA-60 was 24.73 ± 2.52 and 1.08 ± 0.23 kN/m 2 , respectively. Increased cell alignment was observed in GelMA-PGFs compared with GelMA hydrogel alone. These findings suggest GelMA-PGFs can provide glial cells with physical cues necessary to achieve cell alignment. This approach could further be used to achieve glial cell alignment in bioengineered constructs designed to bridge damaged nerve tissue., (© 2021 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Our laboratory has demonstrated that captopril, an angiotensin converting enzyme inhibitor, mitigates hematopoietic injury following total body irradiation in mice. Improved survival in mice is correlated with improved recovery of mature blood cells and bone marrow, reduction of radiation-induced inflammation, and suppression of radiation coagulopathy. Here we investigated the effects of captopril treatment against radiation injuries in the Göttingen mini pig model of Hematopoietic-Acute Radiation Syndrome (H-ARS). Minipigs were given captopril orally (0.96 mg/kg) twice daily for 12 days following total body irradiation (60Co 1.79 Gy, 0.42-0.48 Gy/min). Blood was drawn over a time course following irradiation, and tissue samples were collected at euthanasia (32-35 days post-irradiation). We observed improved survival with captopril treatment, with survival rates of 62.5% in vehicle treated and 87.5% in captopril treated group. Additionally, captopril significantly improved recovery of peripheral blood mononuclear cells, and a trend toward improvement in recovery of red blood cells and platelets. Captopril significantly reduced radiation-induced expression of cytokines erythropoietin and granulocyte-macrophage colony-stimulating factor and suppressed radiation-induced acute-phase inflammatory response cytokine serum amyloid protein A. Using quantitative-RT-PCR to monitor bone marrow recovery, we observed significant suppression of radiation-induced expression of redox stress genes and improved hematopoietic cytokine expression. Our findings suggest that captopril activities in the Göttingen minipig model of hematopoietic-acute radiation syndrome reflect findings in the murine model., Competing Interests: KOG is currently employed by Leidos Biomedical Research Inc. KOG was not employed by Leidos Biomedical Research Inc at the time this study was conducted. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The increasing potential for radiation exposure from nuclear accidents or terrorist activities has intensified the need to develop pharmacologic countermeasures against injury from total body irradiation (TBI). Many initial experiments to develop and test these countermeasures utilize murine irradiation models. Yet, the route of drug administration can alter the response to irradiation injury. Studies have demonstrated that cutaneous injuries can exacerbate damage from radiation, and thus surgical implantation of osmotic pumps for drug delivery could adversely affect the survival of mice following TBI. However, daily handling and injections to administer drugs could also have negative consequences. This study compared the effects of subcutaneous needlesticks with surgical implantation of osmotic pumps on morbidity and mortality in a murine model of hematopoietic acute radiation syndrome (H-ARS). C57BL/6 mice were sham irradiated or exposed to a single dose of 7.7 Gy 60 Co TBI. Mice were implanted with osmotic pumps containing sterile saline seven days prior to irradiation or received needlesticks for 14 days following irradiation or received no treatment. All irradiated groups exhibited weight loss. Fewer mice with osmotic pumps survived to 30 days post irradiation (37.5%) than mice receiving needlesticks or no treatment (70% and 80%, respectively), although this difference was not statistically significant. However, mice implanted with the pump lost significantly more weight than mice that received needlesticks or no treatment. These data suggest that surgical implantation of a drug-delivery device can adversely affect the outcome in a murine model of H-ARS.

The lung is sensitive to radiation and exhibits several phases of injury, with an initial phase of radiation-induced pneumonitis followed by delayed and irreversible fibrosis. The angiotensin-converting enzyme inhibitor captopril has been demonstrated to mitigate radiation lung injury and to improve survival in animal models of thoracic irradiation, but the mechanism remains poorly understood. Here we investigated the effect of captopril on early inflammatory events in the lung in female CBA/J mice exposed to thoracic X-ray irradiation of 17-17.9 Gy (0.5-0.745 Gy min-1). For whole-body + thoracic irradiation, mice were exposed to 7.5 Gy (0.6 Gy min-1) total-body 60Co irradiation and 9.5 Gy thoracic irradiation. Captopril was administered orally (110 mg kg-1 day-1) in the drinking water, initiated 4 h through to150 days post-irradiation. Captopril treatment increased survival from thoracic irradiation to 75% at 150 days compared with 0% survival in vehicle-treated animals. Survival was characterized by a significant decrease in radiation-induced pneumonitis and fibrosis. Investigation of early inflammatory events showed that captopril significantly attenuated macrophage accumulation and decreased the synthesis of radiation-induced interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) pro-inflammatory cytokines in the lungs of irradiated mice. Suppression of IL-1β and TNF-α correlated with an increase of the anti-inflammatory cytokine IL-10 in the spleen with captopril treatment. We also found that captopril decreased markers for radiation-induced accelerated senescence in the lung tissue. Our data suggest that suppression of inflammation and senescence markers, combined with an increase of anti-inflammatory factors, are a part of the mechanism for captopril-induced survival in thoracic irradiated mice., (© The Author(s) 2021. Published by Oxford University Press on behalf of The Japanese Radiation Research Society and Japanese Society for Radiation Oncology.)

Recurrence of prostate cancer after radical prostatectomy is a consequence of incomplete tumor resection. Systemic chemotherapy after surgery is associated with significant toxicity. Improved delivery methods for toxic drugs capable of targeting positive resection margins can reduce tumor recurrence and avoid their known toxicity. This study evaluates the effectiveness and toxicity of docetaxel (DTX) release from highly porous biodegradable microparticles intended for delivery into the tissue cavity created during radical prostatectomy to target residual tumor cells. The microparticles, composed of poly(dl-lactide- co -glycolide) (PLGA), are processed using thermally induced phase separation (TIPS) and loaded with DTX via antisolvent precipitation. Sustained drug release and effective toxicity in vitro are observed against PC3 human prostate cells. Peritumoral injection in a PC3 xenograft tumor model results in tumor growth inhibition equivalent to that achieved with intravenous delivery of DTX. Unlike intravenous delivery of DTX, implantation of DTX-TIPS microparticles is not accompanied by toxicity or elevated systemic levels of DTX in organ tissues or plasma. DTX-TIPS microparticles provide localized and sustained release of nontoxic therapeutic amounts of DTX. This may offer novel therapeutic strategies for improving management of patients with clinically localized high-risk disease requiring radical prostatectomy and other solid cancers at high risk of positive resection margins., Competing Interests: A patent application has been submitted by UCL Business covering the combination of DTX and TIPS microspheres., (© 2020 The Authors. Advanced Therapeutics published by Wiley‐VCH GmbH.)

Brain radiation can occur from treatment of brain tumors or accidental exposures. Brain radiation has been rarely considered, though, as a possible tool to alter protein levels involved in neurodegenerative disorders. We analyzed possible molecular and neuropathology changes of phosphorylated-Tau (pTau), all-Tau forms, β-tubulin, amyloid precursor protein (APP), glial fibrillary acidic protein (GFAP), ionized calcium binding adaptor molecule 1 (IBA-1), myelin basic protein (MBP), and GAP43 in Frontal Cortex (FC), Hippocampus (H) and Cerebellum (CRB) of swine brains following total-body low-dose radiation (1.79 Gy). Our data show that radiated-animals had lower levels of pTau in FC and H, APP in H and CRB, GAP43 in CRB, and higher level of GFAP in H versus sham-animals. These molecular changes were not accompanied by obvious neurohistological changes, except for astrogliosis in the H. These findings are novel, and might open new perspectives on brain radiation as a potential tool to interfere with the accumulation of specific proteins linked to the pathogenesis of various neurodegenerative disorders.

The molecule 3,3'-diindolylmethane (DIM) is small, a major bioactive metabolite of indole-3 carbinol (13C), and a phytochemical compound from cruciferous vegetables released upon exposure to the gut acid environment. DIM is a proposed anti-cancer agent and was previously demonstrated to prevent radiation damage in the bone marrow and the gastrointestinal tract. Here we investigated the effect of DIM on radiation-induced injury to the lung in a murine model through untargeted metabolomics and gene expression studies of select genes. CBA mice were exposed to thoracic irradiation (17.5 Gy). Mice were treated with vehicle or DIM (250 mg kg, subcutaneous injection) on days -1 pre-irradiation through +14 post-irradiation. DIM induced a significant improvement in survival by day 150 post-irradiation. Fibrosis-related gene expression and metabolomics were examined using lung tissue from days 15, 45, 60, 90, and 120 post-irradiation. Our qRT-PCR experiments showed that DIM treatment reduced radiation-induced late expression of collagen Iα and the cell cycle checkpoint proteins p21/waf1 (CDKN1A) and p16ink (CDKN2A). Metabolomic studies of lung tissue demonstrated a significant dampening of radiation-induced changes following DIM treatment. Metabolites associated with pro-inflammatory responses and increased oxidative stress, such as fatty acids, were suppressed by DIM treatment compared to irradiated samples. Together these data suggest that DIM reduces radiation-induced sequelae in the lung.

The recent rise of adoptive T cell therapy (ATCT) as a promising cancer immunotherapy has triggered increased interest in therapeutic T cell bioprocessing. T cell activation is a critical processing step and is known to be modulated by physical parameters, such as substrate stiffness. Nevertheless, relatively little is known about how biophysical factors regulate immune cells, such as T cells. Understanding how T cell activation is modulated by physical and biochemical cues may offer novel methods to control cell behavior for therapeutic cell processing. Inspired by T cell mechanosensitivity, we developed a multiwell, reusable, customizable, two-dimensional (2D) polyacrylamide (PA) hydrogel-integrated culture device to study the physicochemical stimulation of Jurkat T cells. Substrate stiffness and ligand density were tuned by concentrations of the hydrogel cross-linker and antibody in the coating solution, respectively. We cultured Jurkat T cells on 2D hydrogels of different stiffnesses that presented surface-immobilized stimulatory antibodies against CD3 and CD28 and demonstrated that Jurkat T cells stimulated by stiff hydrogels (50.6 ± 15.1 kPa) exhibited significantly higher interleukin-2 (IL-2) secretion, but lower proliferation, than those stimulated by softer hydrogels (7.1 ± 0.4 kPa). In addition, we found that increasing anti-CD3 concentration from 10 to 30 μg/mL led to a significant increase in IL-2 secretion from cells stimulated on 7.1 ± 0.4 and 9.3 ± 2.4 kPa gels. Simultaneous tuning of substrate stiffness and stimulatory ligand density showed that the two parameters synergize (two-way ANOVA interaction effect: p < 0.001) to enhance IL-2 secretion. Our results demonstrate the importance of physical parameters in immune cell stimulation and highlight the potential of designing future immunostimulatory biomaterials that are mechanically tailored to balance stimulatory strength and downstream proliferative capacity of therapeutic T cells.

The meteoric rise of cancer immunotherapy in the past decade has led to promising treatments for a number of hard-to-treat malignancies. In particular, adoptive T cell therapy has recently reached a major milestone with two products approved by the US FDA. However, the inherent complexity of cell-based immunotherapies means that their manufacturing time, cost, and controllability limit their effectiveness and geographic reach. One way to address these issues may lie in complementing the dominant, reductionistic mentality in modern medicine with complex systems thinking. In this opinion article, we identify key concepts from complexity theory to address manufacturing challenges in cell-based immunotherapies and raise the possibility of a unifying framework upon which future bioprocessing strategies may be designed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Peripheral arterial disease and critical limb ischemia are common symptoms of cardiovascular disease. Vascular surgery is used to create a bypass around occluded blood vessels to improve blood flow to ischemic muscle, thus avoiding the need for amputation. Attempts to vascularize tissues by therapeutic angiogenesis using delivery of exogenous angiogenic agents are underwhelming. A material-based approach that provides an endogenous stimulus capable of promoting angiogenesis and increased tissue perfusion would provide a paradigm shift in treatment options available. It is reported here that microporous biodegradable films produced using thermally induced phase separation provide a localized biophysical stimulus of proangiogenic genes in vivo that is associated with increased blood vessel density and restoration of blood flow to ischemic tissue. These findings show, for the first time, that acellular, nonfunctionalized biodegradable biomaterials can provide an innovative, material-based approach for therapeutic angiogenesis to enhance tissue reperfusion in vivo., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mesenchymal stromal cells (MSC) hold significant potential for tissue engineering applications. Modular tissue engineering involves the use of cellularized "building blocks" that can be assembled via a bottom-up approach into larger tissue-like constructs. This approach emulates more closely the complexity associated hierarchical tissues compared with conventional top-down tissue engineering strategies. The current study describes the combination of biodegradable porous poly(DL-lactide-co-glycolide) (PLGA) TIPS microcarriers with canine adipose-derived MSC (cAdMSC) for use as implantable conformable building blocks in modular tissue engineering applications. Optimal conditions were identified for the attachment and proliferation of cAdMSC on the surface of the microcarriers. Culture of the cellularized microcarriers for 21 days in transwell insert plates under conditions used to induce either chondrogenic or osteogenic differentiation resulted in self-assembly of solid 3D tissue constructs. The tissue constructs exhibited phenotypic characteristics indicative of successful osteogenic or chondrogenic differentiation, as well as viscoelastic mechanical properties. This strategy paves the way to create in situ tissue engineered constructs via modular tissue engineering for therapeutic applications., (Copyright © 2020 Simitzi, Vlahovic, Georgiou, Keskin-Erdogan, Miller and Day.)

Adipose-derived mesenchymal stromal cells (AdMSC) release numerous soluble factors capable of stimulating angiogenesis. Improved methods for delivering these cells to maximize their potency are now sought that ideally they retain viable cells in the target tissue while promoting the secretion of angiogenic factors. Substrate surface topography is a parameter that can be used to manipulate the behavior of AdMSC but challenges exist with translating this parameter into materials compatible with minimally invasive delivery into tissues for in situ delivery of the angiogenic secretome. The current study investigates three compositions of hierarchically structured, porous biodegradable microcarriers for the culture of AdMSC and the influence of their surface topographies on the angiogenic secretome. All three compositions perform well as cell microcarriers in xeno-free conditions. The attached AdMSC retain their capacity for subsequent trilineage differentiation. The secretome of AdMSC attached to the microcarriers consists of multiple proangiogenic factors, including significantly elevated levels of vascular endothelial growth factor, which stimulates angiogenesis in vitro. The unique properties of hierarchically structured, porous biodegradable microcarriers investigated in this study offer a radically transformative approach for achieving targeted in vivo delivery of AdMSC and enhancing the potency of their proangiogenic activity to induce neovascularization in ischemic tissue., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Exposure to high-dose total body irradiation (TBI) can result in hematopoietic acute radiation syndrome (H-ARS), characterized by leukopenia, anemia, and coagulopathy. Death from H-ARS occurs from hematopoietic insufficiency and opportunistic infections. Following radiation exposure, red blood cells (RBCs) undergo hemolysis from radiation-induced hemoglobin denaturation, causing the release of iron. Free iron can have multiple detrimental biological effects, including suppression of hematopoiesis. We investigated the impact of radiation-induced iron release on the bone marrow following TBI and the potential impact of the ACE inhibitor captopril, which improves survival from H-ARS. C57BL/6J mice were exposed to 7.9 Gy, 60 Co irradiation, 0.6 Gy/min (LD 70-90/30 ). RBCs and reticulocytes were significantly reduced within 7 days of TBI, with the RBC nadir at 14-21 days. Iron accumulation in the bone marrow correlated with the time course of RBC hemolysis, with an ∼10-fold increase in bone marrow iron at 14-21 days post-irradiation, primarily within the cytoplasm of macrophages. Iron accumulation in the bone marrow was associated with increased expression of genes for iron binding and transport proteins, including transferrin, transferrin receptor 1, ferroportin, and integrin αMβ2. Expression of the gene encoding Nrf2, a transcription factor activated by oxidative stress, also increased at 21 days post-irradiation. Captopril did not alter iron accumulation in the bone marrow or expression of iron storage genes, but did suppress Nrf2 expression. Our study suggests that following TBI, iron is deposited in tissues not normally associated with iron storage, which may be a secondary mechanism of radiation-induced tissue injury., Competing Interests: Conflict of interest disclosure All authors confirm that there are no financial or nonfinancial conflicts of interest., (Published by Elsevier Inc.)

Due to their homogeneity, tuneable properties, low cost and ease of manufacture, thermally induced phase separation (TIPS) polymeric microparticles are emerging as an exciting class of injectable device for the treatment of damaged tissue or complex diseases, such as cancer. However, relatively little work has explored enhancing surface functionalisation of this system. Herein, we present the functionalisation of TIPS microparticles with both small molecules and an antibody fragment of Herceptin™, via a heterobifunctional pyridazinedione linker capable of participating in SPAAC "click" chemistry, and compare it to the traditional method of preparing active-targeted microparticle systems, that is, physisorption of antibodies to the microparticle surface. Antigen-binding assays demonstrated that functionalisation of microparticles with Herceptin Fab, via a pyridazinedione linker, provided an enhanced avidity to HER2+ when compared to traditional physisorption methods.

Prostate cancer is a leading cause of death in men and despite improved surgical procedures that aid tumor resection, the risk of recurrence after surgery as a result of positive resection margins remains significant. Adjuvant chemotherapy is often required but this is associated with toxicity. Improved ways of delivering highly toxic chemotherapeutic drugs in a more controlled and targeted manner after the prostate has been removed during surgery could reduce the risk of recurrence and avoid systemic toxicity. The aim of this study was to develop a novel drug-device combination tissue scaffold that can be used to deliver the chemotherapeutic agent, docetaxel, into the tissue cavity that is created following radical prostatectomy. The device component investigated consisted of highly porous, poly(dl-lactide-co-glycolide) microparticles made using thermally induced phase separation. A facile method was established for loading docetaxel with high efficiency within one hour. Sustained drug release was observed from the microparticles when placed into a dynamic system simulating tissue perfusion. The drug released from the microparticles into perfusates collected at regular time intervals inhibited colony formation and exhibited sustained cytotoxicity against 3D spheroids of PC3 prostate cancer cells over 10 days. In conclusion, this study demonstrates the concept of combining docetaxel with the biodegradable microparticles at the point of care is technically feasible for achieving an effective drug-device combination tissue scaffold. This approach could provide an effective new approach for delivering adjuvant chemotherapy following radical prostatectomy.

With donor organs not readily available, the need for a tissue-engineered oesophagus remains high, particularly for congenital childhood conditions such as atresia. Previous attempts have not been successful, and challenges remain. Small intestine submucosa (SIS) is an acellular matrix material with good biological properties; however, as is common with these types of materials, they demonstrate poor mechanical properties. In this work, electrospinning was performed to mechanically reinforce tubular SIS with polylactic-co-glycolic acid (PLGA) nanofibres. It was hypothesised that if attachment could be achieved between the two materials, then this would (i) improve the SIS mechanical properties, (ii) facilitate smooth muscle cell alignment to support directional growth of muscle cells and (iii) allow for the delivery of bioactive molecules (VEGF in this instance). Through a relatively simple multistage process, adhesion between the layers was achieved without chemically altering the SIS. It was also found that altering mandrel rotation speed affected the alignment of the PLGA nanofibres. SIS-PLGA scaffolds performed mechanically better than SIS alone; yield stress improvement was 200% and 400% along the longitudinal and circumferential directions, respectively. Smooth muscle cells cultured on the aligned fibres showed resultant unidirectional alignment. In vivo the SIS-PLGA scaffolds demonstrated limited foreign body reaction judged by the type and proportion of immune cells present and lack of fibrous encapsulation. The scaffolds remained intact at 4 weeks in vivo, and good cellular infiltration was observed. The incorporation of VEGF within SIS-PLGA scaffolds increased the blood vessel density of the surrounding tissues, highlighting the possible stimulation of endothelialisation by angiogenic factor delivery. Overall, the designed SIS-PLGA-VEGF hybrid scaffolds might be used as a potential matrix platform for oesophageal tissue engineering. In addition to this, achieving improved attachment between layers of acellular matrix materials and electrospun fibre layers offers the potential utility in other applications. STATEMENT OF SIGNIFICANCE: Because of its multi-layered nature and complex structure, the oesophagus tissue poses several challenges for successful clinical grafting. Therefore, it is promising to utilise tissue engineering strategies to mimic and form structural compartments for its recovery. In this context, we investigated the use of tubular small intestine submucosa (SIS) reinforced with polylactic-co-glycolic acid (PLGA) nanofibres by using electrospinning and also, amongst other parameters, the integrity of the bilayered structure created. This was carried out to facilitate smooth muscle cell alignment, support directional growth of muscle cells and allow the delivery of bioactive molecules (VEGF in this study). We evaluated this approach by using in vitro and in vivo models to determine the efficacy of this new system., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

There are no FDA-approved drugs that can be administered prior to ionizing radiation exposure to prevent hematopoietic-acute radiation syndrome (H-ARS). A suspension of synthetic genistein nanoparticles was previously shown to be an effective radioprotectant against H-ARS when administered prior to exposure to a lethal dose of total body radiation. Here we aimed to determine the time to protection and the duration of protection when the genistein nanosuspension was administered by intramuscular injection, and we also investigated the drug's mechanism of action. A single intramuscular injection of the genistein nanosuspension was an effective radioprotectant when given prophylactically 48 h to 12 h before irradiation, with maximum effectiveness occurring when administered 24 h before. No survival advantage was observed in animals administered only a single dose of drug after irradiation. The dose reduction factor of the genistein nanosuspension was determined by comparing the survival of treated and untreated animals following different doses of total body irradiation. As genistein is a selective estrogen receptor beta agonist, we also explored whether this was a central component of its radioprotective mechanism of action. Mice that received an intramuscular injection of an estrogen receptor antagonist (ICI 182,780) prior to administration of the genistein nanosuspension had significantly lower survival following total body irradiation compared with animals only receiving the nanosuspension (P < 0.01). These data define the time to and duration of radioprotection following a single intramuscular injection of the genistein nanosuspension and identify its likely mechanism of action., (© Crown copyright 2019.)

The increasing potential for accidental radiation exposure from either nuclear accidents or terrorist activities has escalated the need for radiation countermeasure development. We previously showed that a 30-day course of high-dose captopril, an ACE inhibitor, initiated 1-4 h after total body irradiation (TBI), improved Hematopoietic Acute Radiation Syndrome (H-ARS) and increased survival in mice. However, because of the time likely required for the deployment of a stockpiled radiation countermeasure to a radiation mass casualty site, there is a need for therapies that can be administered 24-48 hours after initial exposure. Using C57BL/6 mice exposed to an LD 50-80/30 of 60 Co TBI (7.75-7.9 Gy, 0.615 Gy/min), we show that low-dose captopril administration, initiated as late as 48 h post-TBI and continued for 14 days, significantly enhanced overall survival similarly to high-dose, rapid administration. Captopril treatment did not affect radiation-induced cell cycle arrest genes or the immediate loss of hematopoietic precursors. Reduced mortality was associated with the recovery of bone marrow cellularity and mature blood cell recovery at 21-30 days post-irradiation. Captopril reduced radiation-induced cytokines EPO, G-CSF, and SAA in the plasma. Finally, delayed captopril administration mitigated brain micro-hemorrhage at 21 days post-irradiation. These data indicate that low dose captopril administered as late as 48 h post-TBI for only two weeks improves survival that is associated with hematopoietic recovery and reduced inflammatory response. These data suggest that captopril may be an ideal countermeasure to mitigate H-ARS following accidental radiation exposure.

Radiotherapy is a common modality for treatment of brain cancers, but it can induce long-term physiological and cognitive deficits. The responses of normal human brain cells to radiation is not well understood. Astrocytes have been shown to have a variety of protective mechanisms against oxidative stress and have been shown to protect neurons. We investigated the response of cultured normal human astrocytes (NHAs) to X-ray irradiation. Following exposure to 10 Gy X-irradiation, NHAs exhibited DNA damage as indicated by the formation of γ-H2AX foci. Western blotting showed that NHAs displayed a robust increase in expression of non-homologous end joining DNA repair enzymes within 15 min post-irradiation and increased expression of homologous recombination DNA repair enzymes ~2 h post-irradiation. The cell cycle checkpoint protein p21/waf1 was upregulated from 6-24 h, and then returned to baseline. Levels of DNA repair enzymes returned to basal ~48 h post-irradiation. NHAs re-entered the cell cycle and proliferation was observed at 6 days. In contrast, normal human mesenchymal stem cells (MSCs) failed to upregulate DNA repair enzymes and instead displayed sustained upregulation of p21/waf1, a cell cycle checkpoint marker for senescence. Ectopic overexpression of Ku70 was sufficient to protect MSCs from sustained upregulation of p21/waf1 induced by 10 Gy X-rays. These findings suggest that increased expression of Ku70 may be a key mechanism for the radioresistance of NHAs, preventing their accelerated senescence from high-dose radiation. These results may have implications for the development of novel targets for radiation countermeasure development.

Allogeneic stem cell transplantation is currently the only curative therapy for primary myelofibrosis (MF), while the JAK2 inhibitor, ruxolitinib. Has been approved only for palliation. Other therapies are desperately needed to reverse life-threatening MF. However, the cell(s) and cytokine(s) that promote MF remain unclear. Several reports have demonstrated that captopril, an inhibitor of angiotensin-converting enzyme that blocks the production of angiotensin II (Ang II), mitigates fibrosis in heart, lung, skin and kidney. Here, we show that captopril can mitigate the development of MF in the Gata1 low mouse model of primary MF. Gata1 low mice were treated with 79 mg/kg/d captopril in the drinking water from 10 to 12 months of age. At 13 months of age, bone marrows were examined for fibrosis, megakaryocytosis and collagen expression; spleens were examined for megakaryocytosis, splenomegaly and collagen expression. Treatment of Gata1 low mice with captopril in the drinking water was associated with normalization of the bone marrow cellularity; reduced reticulin fibres, splenomegaly and megakaryocytosis; and decreased collagen expression. Our findings suggest that treating with the ACE inhibitors captopril has a significant benefit in overcoming pathological changes associated with MF., (© 2018 Virginia Commonwealth University. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)