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Gamma irradiation, which is one of the more conventional sterilization methods, was used to induce the hydrogelation of silk fibroin in this study. The physical and chemical characteristics of the irradiation-induced silk fibroin hydrogels were investigated. Silk fibroin solution with a concentration greater than 1 wt% formed hydrogel when irradiated by gamma rays at a dose of 25 or 50 kGy. The hydrogel induced by 50 kGy of radiation was more thermally stable at 80 °C than those induced by 25 kGy of radiation. When compared to the spontaneously formed hydrogels, the irradiated hydrogels contained a greater fraction of random coils and a lower fraction of β-sheets. This finding implies that gelation via gamma irradiation occurs via other processes, in addition to crystalline β-sheet formation, which is a well-established mechanism. Our observation suggests that crosslinking and chain scission via gamma irradiation could occur in parallel with the β-sheet formation. The irradiation-induced hydrogels were obtained when the solution concentration was adequate to support the radiation crosslinking of the silk fibroin chains. This work has, therefore, demonstrated that gamma irradiation can be employed as an alternative method to produce chemical-free, random coil-rich, and sterilized silk fibroin hydrogels for biomedical applications.

Advanced wound dressings that can deliver potent antibacterial action are still much in need, especially for treating wound infections caused by drug-resistant bacteria. In this research, we utilized electron beam (EB) irradiation to develop antibacterial hydrogel sheet dressings from poly(vinyl alcohol) (PVA) and silver nanoparticles (AgNPs) in a two-step processing and evaluated their bactericidal efficacy, as well as the AgNP release. The effect of the irradiation dose on the swelling, gel fraction, network parameters, and mechanical properties of the hydrogels was first determined to establish the optimal doses for the two-step processing. The prototypic hydrogel sheets were then formed in the first EB irradiation and served as a matrix for the AgNP synthesis by the reduction of the silver nitrate precursors during the second EB irradiation. The diffusion assay showed that the minimal inhibition concentration (MIC) of the AgNP-load hydrogels was 0.25 and 0.5 mg/cm 2 against Escherichia coli and Staphylococcus aureus , respectively. At these MIC levels, the released AgNPs increased sharply before reaching the maximum, ~950 and 1800 ppb, at 24 h as analyzed by atomic absorption. Therefore, we successfully demonstrated that this two-step processing by EB irradiation provides a convenient platform to fabricate AgNP-loaded hydrogel dressings that can be further developed for wound healing.

The use of bioactive molecules derived from medicinal plants in wound healing has recently attracted considerable attention in both research and public interest. In this work, we demonstrated the first attempt to incorporate the extract from Thai red onion skins in hydrogel patches intended for transdermal delivery. The red onion skin extract (ROSE) was first prepared and evaluated for cytotoxicity by MTT assay with both L929 and human dermal fibroblast cells. Hydrogel patches with porous microstructure and high water content were fabricated from polyvinyl alcohol (PVA) by electron beam irradiation and characterized for their physical, mechanical, morphological, and cytocompatible properties prior to the loading of ROSE. After decontamination by electron beam irradiation, the in vitro release profile exhibited the burst release of extract from ROSE-coated hydrogel patches within 5 h, followed by the sustained release up to 48 h. Finally, evaluation of skin permeation using Franz cell setup with a newborn pig skin model showed that the permeation of ROSE from the hydrogel patch increased with time and reached the maximum of 262 µg/cm 2 , which was well below the cytotoxicity threshold, at 24 h. These results demonstrated that our ROSE-coated hydrogel patches could potentially be used in transdermal delivery.

DNA double-strand breaks (DSBs) are the most lethal form of damage to cells from irradiation. γ-H2AX (phosphorylated form of H2AX histone variant) has become one of the most reliable and sensitive biomarkers of DNA DSBs. However, the γ-H2AX foci assay still has limitations in the time consumed for manual scoring and possible variability between scorers. This study proposed a novel automated foci scoring method using a deep convolutional neural network based on a You-Only-Look-Once (YOLO) algorithm to quantify γ-H2AX foci in peripheral blood samples. FociRad, a two-stage deep learning approach, consisted of mononuclear cell (MNC) and γ-H2AX foci detections. Whole blood samples were irradiated with X-rays from a 6 MV linear accelerator at 1, 2, 4 or 6 Gy. Images were captured using confocal microscopy. Then, dose-response calibration curves were established and implemented with unseen dataset. The results of the FociRad model were comparable with manual scoring. MNC detection yielded 96.6% accuracy, 96.7% sensitivity and 96.5% specificity. γ-H2AX foci detection showed very good F1 scores (> 0.9). Implementation of calibration curve in the range of 0-4 Gy gave mean absolute difference of estimated doses less than 1 Gy compared to actual doses. In addition, the evaluation times of FociRad were very short (< 0.5 min per 100 images), while the time for manual scoring increased with the number of foci. In conclusion, FociRad was the first automated foci scoring method to use a YOLO algorithm with high detection performance and fast evaluation time, which opens the door for large-scale applications in radiation triage., (© 2022. The Author(s).)

A quick, reliable, and reproducible biological assay to distinguish individuals with possible life-threatening risk following radiological or nuclear incidents remains a quest in biodosimetry. In this paper, we examined the use of a γ-H2AX assay as an early dose estimation for rapid triage based on both flow cytometry and image analyses. In the experiment, whole blood from 11 donors was irradiated ex vivo inside a water phantom by gamma rays from Co-60 at 0.51 Gy/min. After the lysis of red blood cells, the white blood cells were collected for immunofluorescence labeling of γ-H2AX, CD45, and nuclear stained for signal collection and visualization. Analysis by flow cytometry showed that the relative γ-H2AX intensities of lymphocytes and granulocytes increased linearly with absorbed doses from 0 to 6 Gy with a large variation among individuals observed above 2 Gy. The relative γ-H2AX intensities of lymphocytes assessed by two different laboratories were highly correlated (ICC = 0.979). Using confocal microscopic images, γ-H2AX foci were observed to be discretely distributed inside the nuclei and to increase proportionally with doses from 0 to 2 Gy, whereas large plagues of merged foci appeared at 4 and 6 Gy, resulting in the saturation of foci counts above 4 Gy. The number of total foci per cell as well as the number of foci per plane were significantly different at 0 vs 1 and 2 vs 4 Gy doses (p < 0.01). Blind tests at 0.5 Gy and 1 Gy doses showed that dose estimation by flow cytometry had a mean absolute difference of less than 0.5 Gy from the actual value. In conclusion, while flow cytometry can provide a dose estimation with an uncertainty of 0.5 Gy at doses ≤ 1 Gy, foci counting can identify merged foci that are prominent at doses ≥ 4 Gy., Competing Interests: The authors have declared that no competing interests exist.

Proliferation and migration of keratinocytes and fibroblasts play an important role in cutaneous wound healing, while oral mucosal squamous cell proliferation and migration are crucial for oral wound healing. In this study, the phytochemical profile of Pluchea indica branch ethanolic extract was characterized. The bioactive compound of Pluchea indica branch ethanolic extract was identified and analyzed by the validated HPLC method. The nanoparticles of P. indica branch extract were formulated by solvent displacement method to increase the solubility and the colloidal stability of the extract. The stability of the nanoparticles was investigated by using the dynamic light scattering technique. Effects of P. indica crude extract and nanoparticles on cell viability, proliferation and migration of primary epidermal keratinocytes, human dermal fibroblasts, and oral mucosal keratinocyte cells were investigated by MTT assay and scratch assay, respectively. The results showed that P. indica branch extract contained a high content of total phenolic and total flavonoids. The HPLC analysis revealed that the main compound in the extract was 4,5- O -dicaffeoylquinic acid. The cell viability of the extract and nanoparticles decreased when cells were exposed to a high concentration of extract and nanoparticles. These results demonstrate that P. indica branch extract and extract nanoparticles at specific concentrations possess in vitro wound healing activity and they may be possibly used to treat different types of wounds including dermal and oral mucosal wounds.

The uniform alignment of endothelial cells inside small-diameter synthetic grafts can be directed by surface topographies such as microgrooves and microfibers to recapitulate the flow-induced elongation and alignment of natural endothelium. These surface micropatterns may also promote directional migration and potentially improve anastomotic ingrowth of endothelial cells inside the synthetic grafts. In this paper, we developed electrospinning and spin casting techniques to pattern the luminal surface of small-diameter polyurethane (PU) grafts with microfibers and microgrooves, respectively, and evaluated endothelial cell orientation on these surface micropatterns. Tracks of circumferentially oriented microfibers were generated by electrospinning PU onto a mandrel rotated at high velocity, whereas longitudinal tracks of microgrooves were generated by spin casting PU over a rotating poly(dimethylsiloxane) mold. We found that both PU grafts possessed longitudinal Young's moduli in the range of 0.43 ± 0.04 to 2.00 ± 0.40 MPa, comparable with values obtained from native artery. Endothelial cells seeded onto the grafts formed confluent monolayers with individual cells exhibiting elongated morphology parallel to the micropatterns. The cells were phenotypically similar to natural endothelium as assessed by the expression of the endothelial cell-specific marker, vascular endothelial cell cadherin. In addition, the cells were also responsive to stimulation with the pro-inflammatory cytokine tumor necrosis factor-α as assessed by the inducible expression of intercellular adhesion molecule-1. These results demonstrate that our micropatterned PU grafts possessed longitudinal Young's moduli in the same range as native vascular tissue and were capable of promoting the formation of aligned and cytokine-responsive endothelial monolayers., (Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

To mimic the uniformly elongated endothelium in natural linear vessels, bovine aortic endothelial cells (BAECs) are cultured on micro- to nanogrooved, model poly(dimethylsiloxane) (PDMS) substrates preadsorbed with about 300 ng/cm(2) of fibronectin. BAEC alignment, elongation, and projected area were investigated for channel depths of 200 nm, 500 nm, 1 microm, and 5 microm, as well as smooth surfaces. Except for the 5 microm case, the ridge and channel widths were held nearly constant about 3.5 microm. With increasing channel depth, the percentage of aligned BAECs increased by factors of 2, 2, 1.8, and 1.7 for 1, 4, 24, and 48 h. Maximum alignment, about 90%, was observed for 1 microm deep channels at 1 h. The alignment of BAECs on grooved PDMS was maintained at least until cells reached near confluence. F-actin and vinculin at focal adhesions also aligned with channel direction. Analysis of confocal microscopy images showed that focal adhesions localized at corners and along the sidewalls of 1-microm deep channels. In contrast, focal adhesions could not form on the bottom of the 5-microm deep channels. Cell proliferation was similar on grooved and smooth substrates. In summary, PDMS substrates engraved with micro- and nanochannels provide a powerful method for investigating the interplay between topography and cell/cytoskeletal alignment., ((c) 2005 Wiley Periodicals, Inc.)