Primary Human Dermal Fibroblasts Research Articles

P35 Adaptation of a phenotypic screening assay to incorporate 384-well plates: opening the door to high-throughput screening

Abstract Introduction and aims High-throughput screening has significantly impacted drug discovery by increasing speed of experiments and reducing reagent and sample costs. We have previously developed several screening assays to measure myofibroblast inhibition via quantification of α-smooth muscle actin (α-SMA) expression, in a 96-well format. Currently, there are no drugs for the treatment of hypertrophic scarring, a major side-effect of burn injuries that occurs by myofibroblast transformation, leaving a huge gap in this sector. The aim for this project was to adapt our current assay for use in 384-well microplates. Methods Human primary dermal fibroblasts were transformed to myofibroblasts using 10 ng mL−1 transforming growth factor (TGF)-β1. α-SMA expression was measured using the In-Cell enzyme-linked immunosorbent assay method, with cell viability measured using DRAQ5 nuclear staining. Assay accuracy and reliability was determined by calculating the robust z-factor, with guidelines set to an average score over 0.5, with a minimum score per plate above 0.4. To further validate the assay, concentration-response curves (CRCs) for the drug vehicle [dimethyl sulfoxide (DMSO)] and our tool compound (SB-505124) were constructed at varying concentrations, in coincubation with TGF-β1. Results Optimization of well metrics was performed, producing an average robust z-factor of 0.52, confirming the assay Results are reliable and reproducible. The DMSO CRC produced no significant drop in cell viability at concentrations that are expected to be used in the screening of compounds. Our tool compound (SB-505124) produced a concentration-dependant curve for the inhibition of myofibroblast transformation. Conclusions A 384-well assay has been designed, tested and validated for the determination of myofibroblast transformation in human primary dermal fibroblasts. The assay has been shown to deliver an average robust z-factor of above 0.5, it contains a positive control and is compatible with compounds stored in up to 100% DMSO.

Development of alginate and alginate sulfate/polycaprolactone nanoparticles for growth factor delivery in wound healing therapy

Connective tissue growth factor (CTGF) holds great promise for enhancing the wound healing process; however, its clinical application is hindered by its low stability and the challenge of maintaining its effective concentration at the wound site. Herein, we developed novel double-emulsion alginate (Alg) and heparin-mimetic alginate sulfate (AlgSulf)/polycaprolactone (PCL) nanoparticles (NPs) for controlled CTGF delivery to promote accelerated wound healing. The NPs’ physicochemical properties, cytocompatibility, and wound healing activity were assessed on immortalized human keratinocytes (HaCaT), primary human dermal fibroblasts (HDF), and a murine cutaneous wound model. The synthesized NPs had a minimum hydrodynamic size of 200.25 nm. Treatment of HaCaT and HDF cells with Alg and AlgSulf2.0/PCL NPs did not show any toxicity when used at concentrations <50 µg/mL for up to 72 h. Moreover, the NPs’ size was not affected by elevated temperatures, acidic pH, or the presence of a protein-rich medium. The NPs have slow lysozyme-mediated degradation implying that they have an extended tissue retention time. Furthermore, we found that treatment of HaCaT and HDF cells with CTGF-loaded Alg and AlgSulf2.0/PCL NPs, respectively, induced rapid cell migration (76.12% and 79.49%, P<0.05). Finally, in vivo studies showed that CTGF-loaded Alg and AlgSulf2.0/PCL NPs result in the fastest and highest wound closure at the early and late stages of wound healing, respectively (36.49%, P<0.001 on day 1; 90.45%, P<0.05 on day 10), outperforming free CTGF. Double-emulsion NPs based on Alg or AlgSulf represent a viable strategy for delivering heparin-binding GF and other therapeutics, potentially aiding various disease treatments.

Antioxidant, anti-photoaging, anti-inflammatory, and skin-barrier-protective effects of Gleichenia japonica extract

Ferns native to Korea, such as Davallia mariesii, Dicranopteris pedata, and Gleichenia japonica, possess antioxidant and antibacterial properties. However, their inhibitory effects on skin photoaging have not been demonstrated. Measurement and comparison of the antioxidant activity of three types of ferns revealed that the extract from G. japonica had the best effect. This study evaluates the potential of G. japonica extract as a new functional material for preventing skin damage caused by ultraviolet radiation. G. japonica extracts showed protective effects against ultraviolet B (UVB) radiation in human epidermal keratinocyte cells; the extracts inhibited intracellular reactive oxygen species production. In addition, collagen biosynthesis increased, and matrix metalloproteinase-1 activity and protein expression level decreased in human primary dermal fibroblast irradiated with UVB. The main peak (compound 1) of the extract was separated through high-performance liquid chromatography analysis and preparative liquid chromatography. Compound 1 is strongly inferred to be the main active ingredient because it showed better antioxidant activity and UVB protection effect than G. japonica extract. These results demonstrate the physiological effects of G. japonica extract and suggest its applicability as a new functional substance for preventing skin damage caused by ultraviolet radiation.

Radiation-induced DNA double-strand breaks in cortisol exposed fibroblasts as quantified with the novel foci-integrated damage complexity score (FIDCS)

Without the protective shielding of Earth’s atmosphere, astronauts face higher doses of ionizing radiation in space, causing serious health concerns. Highly charged and high energy (HZE) particles are particularly effective in causing complex and difficult-to-repair DNA double-strand breaks compared to low linear energy transfer. Additionally, chronic cortisol exposure during spaceflight raises further concerns, although its specific impact on DNA damage and repair remains unknown. This study explorers the effect of different radiation qualities (photons, protons, carbon, and iron ions) on the DNA damage and repair of cortisol-conditioned primary human dermal fibroblasts. Besides, we introduce a new measure, the Foci-Integrated Damage Complexity Score (FIDCS), to assess DNA damage complexity by analyzing focus area and fluorescent intensity. Our results show that the FIDCS captured the DNA damage induced by different radiation qualities better than counting the number of foci, as traditionally done. Besides, using this measure, we were able to identify differences in DNA damage between cortisol-exposed cells and controls. This suggests that, besides measuring the total number of foci, considering the complexity of the DNA damage by means of the FIDCS can provide additional and, in our case, improved information when comparing different radiation qualities.

Downregulation of PLIN2 in human dermal fibroblasts impairs mitochondrial function in an age-dependent fashion and induces cell senescence via GDF15.

Perilipin 2 (PLIN2) is a lipid droplet (LD)-coating protein playing important roles in lipid homeostasis and suppression of lipotoxicity in different tissues and cell types. Recently, a role for PLIN2 in supporting mitochondrial function has emerged. PLIN2 dysregulation is involved in many metabolic disorders and age-related diseases. However, the exact consequences of PLIN2 dysregulation are not yet completely understood. In this study, we knocked down (KD) PLIN2 in primary human dermal fibroblasts (hDFs) from young (mean age 29 years) and old (mean age 71 years) healthy donors. We have found that PLIN2 KD caused a decline of mitochondrial function only in hDFs from young donors, while mitochondria of hDFs from old donors (that are already partially impaired) did not significantly worsen upon PLIN2 KD. This mitochondrial impairment is associated with the increased expression of the stress-related mitokine growth differentiation factor 15 (GDF15) and the induction of cell senescence. Interestingly, the simultaneous KD of PLIN2 and GDF15 abrogated the induction of cell senescence, suggesting that the increase in GDF15 is the mediator of this phenomenon. Moreover, GDF15 KD caused a profound alteration of gene expression, as observed by RNA-Seq analysis. After a more stringent analysis, this alteration remained statistically significant only in hDFs from young subjects, further supporting the idea that cells from old and young donors react differently when undergoing manipulation of either PLIN2 or GDF15 genes, with the latter being likely a downstream mediator of the former.

Factors Affecting the Evaluation of Collagen Deposition and Fibrosis In Vitro.

Immune responses to biomedical implants, wound healing, and diseased tissues often involve collagen deposition by fibroblasts and other stromal cells. Dysregulated collagen deposition can lead to complications, such as biomaterial fibrosis, cardiac fibrosis, desmoplasia, liver fibrosis, and pulmonary fibrosis, which can ultimately result in losses of organ function or failure of biomedical implants. Current in vitro methods to induce collagen deposition include growing the cells under macromolecular crowding conditions or on fibronectin-coated surfaces. However, the majority of these methods have been demonstrated with a single cell line, and the combined impacts of culture conditions and postculture processing on collagen deposition have not been explored in detail. In this work, the effects of macromolecular crowding versus fibronectin coating, fixation with methanol versus fixation with paraformaldehyde, and use of plastic substrates versus glass substrates were evaluated using the WI-38 human lung fibroblast cell line. Fibronectin coating was found to provide enhanced collagen deposition under macromolecular crowding conditions, while a higher plating density led to improved collagen I deposition compared with macromolecular crowding. Collagen deposition was found to be more apparent on plastic substrates than on glass substrates. The effects of primary cells versus cell lines, and mouse cells versus human cells, were evaluated using WI-38 cells, primary human lung fibroblasts, primary human dermal fibroblasts, primary mouse lung fibroblasts, primary mouse dermal fibroblasts, and the L929 mouse fibroblast cell line. Cell lines exhibited enhanced collagen I deposition compared with primary cells. Furthermore, collagen deposition was quantified with picrosirius red staining, and plate-based drug screening through picrosirius red staining of decellularized extracellular matrices was demonstrated. The results of this study provide detailed conditions under which collagen deposition can be induced in vitro in multiple cell types, with applications including material development, development of potential antifibrotic therapies, and mechanistic investigation of disease pathways. Impact Statement This study demonstrated the effects of cell type, biological conditions, fixative, culture substrate, and staining method on in vitro collagen deposition and visualization. Further the utility of plate-based picrosirius red staining of decellularized extracellular matrices for drug screening through collagen quantification was demonstrated. These results should provide clarity and a path forward for researchers who aim to conduct in vitro experiments on collagen deposition.

Pulsed Radiofrequency Electromagnetic Fields as Modulators of Inflammation and Wound Healing in Primary Dermal Fibroblasts of Ulcers.

Venous leg ulcers are one of the most common nonhealing conditions and represent an important clinical problem. The application of pulsed radiofrequency electromagnetic fields (PRF-EMFs), already applied for pain, inflammation, and new tissue formation, can represent a promising approach for venous leg ulcer amelioration. This study aims to evaluate the effect of PRF-EMF exposure on the inflammatory, antioxidant, cell proliferation, and wound healing characteristics of human primary dermal fibroblasts collected from venous leg ulcer patients. The cells' proliferative and migratory abilities were evaluated by means of a BrdU assay and scratch assay, respectively. The inflammatory response was investigated through TNFα, TGFβ, COX2, IL6, and IL1β gene expression analysis and PGE2 and IL1β production, while the antioxidant activity was tested by measuring GSH, GSSG, tGSH, and GR levels. This study emphasizes the ability of PRF-EMFs to modulate the TGFβ, COX2, IL6, IL1β, and TNFα gene expression in exposed ulcers. Moreover, it confirms the improvement of the proliferative index and wound healing ability presented by PRF-EMFs. In conclusion, exposure to PRF-EMFs can represent a strategy to help tissue repair, regulating mediators involved in the wound healing process.

Attenuation of fibroblast activation and fibrosis by adropin in systemic sclerosis.

Fibrotic diseases impose a major socioeconomic challenge on modern societies and have limited treatment options. Adropin, a peptide hormone encoded by the energy homeostasis-associated (ENHO) gene, is implicated in metabolism and vascular homeostasis, but its role in the pathogenesis of fibrosis remains enigmatic. Here, we used machine learning approaches in combination with functional in vitro and in vivo experiments to characterize adropin as a potential regulator involved in fibroblast activation and tissue fibrosis in systemic sclerosis (SSc). We demonstrated consistent down-regulation of adropin/ENHO in skin across multiple cohorts of patients with SSc. The prototypical profibrotic cytokine TGFβ reduced adropin/ENHO expression in a JNK-dependent manner. Restoration of adropin signaling by therapeutic application of bioactive adropin34-76 peptides in turn inhibited TGFβ-induced fibroblast activation and fibrotic tissue remodeling in primary human dermal fibroblasts, three-dimensional full-thickness skin equivalents, mouse models of bleomycin-induced pulmonary fibrosis and sclerodermatous chronic graft-versus-host-disease (sclGvHD), and precision-cut human skin slices. Knockdown of GPR19, an adropin receptor, abrogated the antifibrotic effects of adropin in fibroblasts. RNA-seq demonstrated that the antifibrotic effects of adropin34-76 were functionally linked to deactivation of GLI1-dependent profibrotic transcriptional networks, which was experimentally confirmed in vitro, in vivo, and ex vivo using cultured human dermal fibroblasts, a sclGvHD mouse model, and precision-cut human skin slices. ChIP-seq confirmed adropin34-76-induced changes in TGFβ/GLI1 signaling. Our study characterizes the TGFβ-induced down-regulation of adropin/ENHO expression as a potential pathomechanism of SSc as a prototypical systemic fibrotic disease that unleashes uncontrolled activation of profibrotic GLI1 signaling.

Abstract 6383: Targeting radiation-induced fibrosis: Exploring promising therapeutic avenues and innovative assessment methods

Abstract Introduction: Radiation therapy is a vital cancer treatment for nearly 50% of patients, yet it poses a significant challenge in the form of radiation-induced fibrosis (RF). RF affects about 60% of those undergoing radiation, leading to painful scarring from excessive collagen accumulation. In head and neck cancer patients, as an example, RF reduces tissue flexibility, causing limited neck mobility and impaired swallowing. Furthermore, approximately 30% of patients also face disease recurrence post-radiation, with RF increasing surgical complications. Our laboratory previously identified the role of reduced fatty acid oxidation in RF (Zhao et al; Nat Metab;1(1):147 2019), offering a promising avenue for future drug development. We described the potential therapeutic benefit of caffeic acid phenethyl ester (CAPE) in countering pro-fibrotic metabolic changes. Our current objective is to develop more effective lead compounds to mitigate RF. Methods: Collagen expression, utilized as a surrogate marker for fibrosis, was quantified using ELISA, Western blot analysis, and RT-qPCR. Metabolic changes were assessed by examining the expression levels of PPARG and CD36. CAPE-like analogs were derived from an in-house collection, a commercially available library, and structure-activity relationship (SAR) analyses. In vivo, RF was induced in the hindlimbs of mice through exposure to 40 Gy, followed by weekly X-ray evaluations of hindlimb angles for 16 weeks. During X-ray assessments, both hindlimbs were subjected to a 5 g weight to enable measurement of the joint angle between the femur and tibia (wherein higher acute angles were a measure of worsening fibrosis). Skin samples from irradiated and unirradiated limbs were collected for the evaluation of collagen deposition, utilizing Picro Sirius staining followed by polarized microscopy. Results: Amongst 200 newly synthesized analogs, approximately one-third successfully reduced collagen secretion in human primary dermal fibroblasts. The most promising compounds exhibited a half-maximum inhibition concentration (IC50) for collagen secretion from 1 to 5µM. They also decreased intracellular collagen production and gene expression, associated with upregulation of PPARG and CD36 transcripts. The joint angle measurements in vivo revealed three distinct phases in the development of dermal fibrosis: 1) inflammatory; 2) recovery; and 3) fibrotic phases. Conclusions: We have successfully synthesized novel and potent analogs of CAPE which were able to reduce secretion of collagen. Furthermore, a novel method for the in vivo assessment of fibrosis was also developed. This innovative approach will facilitate the detailed assessments of future compounds capable of reducing collagen deposition in vivo, thereby providing an innovative avenue to mitigate one of the most pressing late normal tissue toxicities of radiation therapy. Citation Format: Pierre-Antoine Bissey, Leonardo Massignan, Ross Mancini, Wei Shi, Justin Williams, Mark Reed, Kenneth W. Yip, Fei-Fei Liu. Targeting radiation-induced fibrosis: Exploring promising therapeutic avenues and innovative assessment methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6383.

Transfection of hypoxia-inducible factor-1α mRNA upregulates the expression of genes encoding angiogenic growth factors

Hypoxia-Inducible Factor-1α (HIF-1α) has presented a new direction for ischemic preconditioning of surgical flaps to promote their survival. In a previous study, we demonstrated the effectiveness of HIF-1a DNA plasmids in this application. In this study, to avoid complications associated with plasmid use, we sought to express HIF-1α through mRNA transfection and determine its biological activity by measuring the upregulation of downstream angiogenic genes. We transfected six different HIF-1a mRNAs–one predominant, three variant, and two novel mutant isoforms–into primary human dermal fibroblasts using Lipofectamine, and assessed mRNA levels using RT-qPCR. At all time points examined after transfection (3, 6, and 10 h), the levels of HIF-1α transcript were significantly higher in all HIF-1α transfected cells relative to the control (all p < 0.05, unpaired Student’s T-test). Importantly, the expression of HIF-1α transcription response genes (VEGF, ANG-1, PGF, FLT1, and EDN1) was significantly higher in the cells transfected with all isoforms than with the control at six and/or ten hours post-transfection. All isoforms were transfected successfully into human fibroblast cells, resulting in the rapid upregulation of all five downstream angiogenic targets tested. These findings support the potential use of HIF-1α mRNA for protecting ischemic dermal flaps.

Repurposing Drugs for Senotherapeutic Effect: Potential Senomorphic Effects of Female Synthetic Hormones.

Repurposing previously approved drugs may fast track the route to the clinic for potential senotherapeutics and improves the inefficiency of the clinical drug development pipeline. We performed a repurposing screen of 240 clinically approved molecules in human primary dermal fibroblasts for their effects on CDKN2A expression. Molecules demonstrating effects on CDKN2A expression underwent secondary screening for senescence-associated beta galactosidase (SAB) activity, based on effect size, direction, and/or molecule identity. Selected molecules then underwent a more detailed assessment of senescence phenotypes including proliferation, apoptosis, DNA damage, senescence-associated secretory phenotype (SASP) expression, and regulators of alternative splicing. A selection of the molecules demonstrating effects on senescence were then used in a new bioinformatic structure-function screen to identify common structural motifs. In total, 90 molecules displayed altered CDKN2A expression at one or other dose, of which 15 also displayed effects on SAB positivity in primary human dermal fibroblasts. Of these, 3 were associated with increased SAB activity, and 11 with reduced activity. The female synthetic sex hormones-diethylstilboestrol, ethynyl estradiol and levonorgestrel-were all associated with a reduction in aspects of the senescence phenotype in male cells, with no effects visible in female cells. Finally, we identified that the 30 compounds that decreased CDKN2A activity the most had a common substructure linked to this function. Our results suggest that several drugs licensed for other indications may warrant exploration as future senotherapies, but that different donors and potentially different sexes may respond differently to senotherapeutic compounds. This underlines the importance of considering donor-related characteristics when designing drug screening platforms.

Natural cellulosic biofunctional textiles from onion (Allium cepa L.) skin extracts: A sustainable strategy for skin protection

Cellulosic fabrics made of cotton, linen, bamboo, hemp, and nettle, were selected for the eco-sustainable production of colored biofunctional textiles, using natural dyes from onion skin of the Dorata di Parma (Allium cepa L.) variety. Dyeing experiments were performed in net water with and without pre-treatment with alum, tin chloride, and tannic acid as mordants. The color and the UV-protection factor (UPF) were evaluated through UV–visible spectroscopy. The treatment with onion skin extract induced a relevant increase of UPF in all investigated fabrics, promoting the protection category from insufficient (UPF<15) to good for linen (UPF = 21–26), up to very good and excellent for cotton (UPF = 35–66) and bamboo (UPF = 48–56), respectively. To mimic the direct contact with the skin, textile samples were immersed in artificial sweat where dyed cotton, hemp, and nettle were found to promote higher phenolic release (0.28–0.50 mg gallic acid equivalents/g textile) than linen and bamboo (0.12–0.22 mg gallic acid equivalents/g textile). The obtained “biofunctional sweat” did not impact on the survival of primary human dermal fibroblasts (HDF) and skin keratinocytes (NCTC2455), while protected these cells against heat shock and reduced reactive oxygen species levels upon exposure to pro-oxidant and inflammatory agents as H2O2 and lipopolysaccharide, respectively. Increased activity levels of the H2O2-scavenging enzyme Catalase (CAT) were also observed, thus demonstrating enhanced stress response and homeostatic capability of these cells. This study provides evidence that extracts from onion waste can be used to functionalize cellulosic fabrics conferring properties as natural shield against UV radiation, and antioxidant and anti-inflammatory materials thus holding potential in skin protection and prevention of dermatological diseases.

Inhibiting IL11RA to mitigate hepatic metastasis in skin cutaneous melanoma: Comprehensive insights from in vitro and in vivo investigations.

This study aimed to investigate the role of Interleukin-11 receptor alpha (IL11RA) in skin cutaneous melanoma (SKCM) metastasis to the liver. Human SKCM cell lines (A375, A375-MA2, SK-MEL-28, RPMI-7951) and primary dermal fibroblasts (HDFa) were utilized to assess IL11RA expression. IL11RA siRNA was transfected into RPMI-7951 and A375-MA2 cells for Wound healing and Transwell invasion assays. Il11ra knockout (KO) mice and wild-type (WT) mice were injected with B16-F10 cells into the spleen to evaluate hepatic melanoma metastasis. Correlation between IL11RA and MMP family genes was explored using online databases, including LinkedOmics, TIMER (Tumor Immune Estimation Resource), and GEPIA (Gene Expression Profiling Interactive Analysis). RT-qPCR and Western blotting were performed for expression analysis of Mmp2 and Mmp9 in liver tissues of mice. The impact of IL11RA on the STAT3 pathway was investigated in vitro and in vivo. Elevated expression of IL11RA was observed in SKCM cell lines compared to normal cells. IL11RA downregulation significantly inhibited migratory and invasive capabilities of A375-MA2 and RPMI-7951 in vitro. Il11ra gene knockout in mice demonstrated a substantial reduction in hepatic melanoma metastasis. Correlation analyses revealed associations between IL11RA and MMP2/MMP8. Il11ra gene knockout significantly decreased Mmp2 expression while increasing Mmp8 in liver tissues. IL11RA correlated positively with STAT3, and its inhibition led to a suppressed STAT3 pathway in SKCM cells and mouse liver tissue. IL11RA plays a crucial role in SKCM metastasis, affecting migratory and invasive abilities. Targeting IL11RA may offer a promising avenue for therapeutic interventions in cutaneous melanoma progression.

3D modeling of normal skin and cutaneous squamous cell carcinoma. A comparative study in 2D cultures, spheroids, and 3D bioprinted systems.

The current cancer research and drug testing are primarily based on 2D cell cultures and animal models. However, these methods have limitations and yield distinct drug response patterns. This study addressed this gap by developing an innovativein vitrohuman three-dimensional (3D) normal skin model and a multicellular model of human cutaneous squamous cell carcinoma (cSCC) using 3D bioprinting technology. Comparative analyzes were performed between bioprinted 3D-cSCC model, consisting of HaCaT keratinocytes, primary normal human dermal fibroblasts and A431 cancer cells (tricellular), bioprinted 3D-A431 model composed of A431 cancer cells only (monocellular), A431 cancer cell spheroids, and conventional 2D models. The models were structurally characterized by light microscopy, immunofluorescence (LIVE/DEAD assay, confocal microscopy) and immunohistochemistry (hematoxylin/eosin, p63, vimentin, Ki67, epidermal growth factor receptor stainings). The spatial arrangement of the 3D models was analyzed using the ARIVIS scientific image analysis platform. All models were also functionally assessed by cetuximab (CTX) response testing with the MTS assay. 3D-cSCC models were maintained for up to 16 weeks. Morphological and histological examinations confirmed the presence of skin-like layers in the bioprinted 3D models of normal skin, and the intricate and diverse features of the bioprinted skin cancer model, replicating the criticalin vivocharacteristics. In both mono- and tricellular bioprinted tumor constructs, there was a gradual formation and continuous growth of spheroid-like clusters of cancer cells, significantly influencing the morphology of the models. Cancer cells in the 3D bioprinted constructs showed reduced sensitivity to CTX compared to spheroids and 2D cultures. This study underscores the potential of 3D multicellular models in elucidating drug responses and gaining a better understanding the intricate interplay of cellular components within the tumor microenvironment. Developing the multicellular 3D tumor model paves the way for new research critical to advancing fundamental cancer research and future clinical applications, particularly drug response testing.

Hydroxypyridone anti-fungals selectively induce myofibroblast apoptosis in an in vitro model of hypertrophic scars

Hypertrophic scars are a common complication of burn injuries, yet there are no medications to prevent their formation. During scar formation, resident fibroblasts are transformed to myofibroblasts which become resistant to apoptosis. Previously, we have shown that hydroxypyridone anti-fungals can inhibit transformation of fibroblasts, isolated from hypertrophic scars, to myofibroblasts. This study aimed to investigate if these drugs can also target myofibroblast persistence.Primary human dermal fibroblasts, derived from burn scar tissue, were exposed to transforming growth factor beta-1 (TGF-β1) for 72 h to induce myofibroblast transformation. The cells were then incubated with three hydroxypyridone anti-fungals (ciclopirox, ciclopirox ethanolamine and piroctone olamine; 0.03–300 μM) for a further 72 h. The In-Cell ELISA method was utilised to quantify myofibroblast transformation by measuring alpha-smooth muscle actin (α-SMA) expression and DRAQ5 staining, to measure cell viability. TUNEL staining was utilised to assess if the drugs could induce apoptosis.When given to established myofibroblasts, the three hydroxypyridones did not reverse myofibroblast transformation, but instead elicited a concentration-dependent decrease in cell viability. TUNEL staining confirmed that the hydroxypyridone anti-fungals induced apoptosis in established myofibroblasts.This is the first study to show that hydroxypyridone anti-fungals are capable of inducing apoptosis in established myofibroblasts. Together with our previous results, we suggest that hydroxypyridone anti-fungals can prevent scar formation by preventing the formation of new myofibroblasts and by reducing the number of existing myofibroblasts.

2'-O-ribose methylation levels of ribosomal RNA distinguish different types of growth arrest in human dermal fibroblasts.

The 2'-O-methylation (2'-O-Me) of ribosomal RNA (rRNA) shows plasticity that is potentially associated with cell phenotypes. We used RiboMeth-seq profiling to reveal growth arrest-specific 2'-O-Me patterns in primary human dermal fibroblasts from three different donors. We exposed cells to hydrogen peroxide to induce cellular senescence and to high cell densities to promote quiescence by contact inhibition. We compared both modes of cell cycle arrest to proliferating cells and could indeed distinguish these conditions by their overall 2'-O-Me patterns. Methylation levels at a small fraction of sites showed plasticity and correlated with the expression of specific small nucleolar RNAs (snoRNAs) but not with expression of fibrillarin. Moreover, we observed subtle senescence-associated alterations in ribosome biogenesis. Knockdown of the snoRNA SNORD87, which acts as a guide for modification of a hypermethylated position in non-proliferating cells, was sufficient to boost cell proliferation. Conversely, depletion of SNORD88A, SNORD88B and SNORD88C, which act as guides for modification of a hypomethylated site, caused decreased proliferation without affecting global protein synthesis or apoptosis. Taken together, our findings provide evidence that rRNA modifications can be used to distinguish and potentially influence specific growth phenotypes of primary cells.

The Development and Characterisation of a P(3HB-co-4HB)-Bioactive Glass-Graphene Hydrogel as a Potential Formulation for Biomedical and Therapeutical Translation.

The clinical management of wounds is known to be a significant challenge: not only does the dressing need to ensure and provide the appropriate barrier and healing characteristics, but consideration of patient compliance concerning comfort, functionality, and practicality also needs to be included. The poly(3-hydroxybutyrate-co-4-hydroxubutyrate) (P(3HB-co-4HB)) copolymer, isolated from Cupriavidus malaysiensis USM1020 (C. malaysiensis USM1020), was produced in the presence of excess carbon sources (1,4-butanediol and 1,6-hexanediol) using either a shake flask cultivation process or a bioreactor fermentation system. P(3HB-co-4HB) is widely known to be biodegradable and highly biocompatible and contains a tuneable 4HB monomer molar fraction, which is known to affect the final physicochemical properties of the intracellular copolymer. In this paper, we describe not only the fabrication of the polymeric gel but also its optimised profiling using a range of physical and mechanical techniques, i.e., SEM, FTIR, DMA, DSC, and WCA. The further enhancement of the gel through additional functionalisation with sol-gel-derived bioactive glass and liquid-exfoliated graphene was also investigated. The biocompatibility and biological characterisation of the substrates was assessed using murine osteoblasts (MC3T3), human primary dermal fibroblasts (HDFs), human fibroblast (BJ) cells, and standard cell culture assays (i.e., metabolic activity, LDH release, and live/dead staining). In short, P(3HB-co-4HB) was successfully isolated from the bacteria, with the defined physico-chemical profiles dependent on the culture substrate and culturing platform used. The additional enhancement of the copolymer with bioactive glass and/or graphene was also demonstrated by varying the combination loading of the materials, i.e., graphene resulted in an increase in tensile strength (~11 MPa) and the wettability increased following the incorporation of bioactive glass and 0.01 wt% graphene (WCA ~46.3°). No detrimental effects in terms of biocompatibility were noticed during the 7 days of culture in the primary and established cell lines. This study demonstrates the importance of optimising each of the individual components within the biocomposite and their relationship concerning the fine-tuning of the material's properties, thus targeting and impacting the endpoint application.

Aqueous distillate of mature leaves of Vernonia zeylanica (L.) Less. and Mallotus repandus (Rottler) Müll. Arg. cued from traditional medicine exhibits rapid wound healing properties

Ethnopharmacological relevanceSri Lankan traditional medicine uses Vernonia zeylanica and Mallotus repandus broadly for the treatment of a multitude of disease conditions, including wound healing. Aim of the studyWe aimed to scientifically validate the safety and efficacy of wound healing of an aqueous distillate of Vernonia zeylanica and Mallotus repandus (ADVM) mature leaves, tested on primary human dermal fibroblasts. Materials and methodsHuman dermal fibroblasts isolated from clinical waste from circumcision surgery were characterized by flowcytometry and trilineage differentiation. The MTT dye reduction assay, and the ex vivo wound healing scratch assay established wound healing properties of ADVM using the primary human dermal fibroblast cell line. Upregulation of genes associated with wound healing (MMP3, COL3A1, TGFB1, FGF2) were confirmed by RT qPCR. GC-MS chromatography evaluated the phytochemical composition of ADVM. ResultsCompared to the synthetic stimulant, β fibroblast growth factor, ADVM at 0.25% concentration on the primary dermal fibroblast cell line exhibited significant ex vivo, (i) 1.7-fold % cell viability (178.7% vs 304.3 %, p < 0.001), (ii) twofold greater % wound closure (%WC) potential (47.74% vs 80.11%, p < 0.001), and (iii) higher rate of % WC (3.251 vs 3.456 % WC/h, p < 0.05), sans cyto-genotoxicity. Up regulated expression of FGF2, TGFB1, COL3A1 and MMP3, genes associated with wound healing, confirmed effective stimulation of pathways of the three overlapping phases of wound healing (P < 0.05). GC-MS profile of ADVM characterized four methyl esters, which may be posited as wound healing phytochemicals. ConclusionsExceeding traditional medicine claims, the exvivo demonstration of rapid skin regeneration, reiterated by upregulated expression of genes related to wound healing pathways, sans cytotoxicity, propounds ADVM, cued from traditional medicine, as a potential safe and effective natural stimulant for rapid wound-healing. Additionally, it may serve as an effective proliferative stimulant of dermal fibroblasts for cell therapy, with potential in reparative and regenerative therapy of skin disorders.

Vutiglabridin Alleviates Cellular Senescence with Metabolic Regulation and Circadian Clock in Human Dermal Fibroblasts.

The process of cellular senescence, which is characterized by stable cell cycle arrest, is strongly associated with dysfunctional cellular metabolism and circadian rhythmicity, both of which are reported to result from and also be causal to cellular senescence. As a result, modifying any of them-senescence, metabolism, or the circadian clock-may affect all three simultaneously. Obesity accelerates aging by disrupting the homeostasis of reactive oxygen species (ROS) via an increased mitochondrial burden of fatty acid oxidation. As a result, if senescence, metabolism, and circadian rhythm are all linked, anti-obesity treatments may improve metabolic regulation while also alleviating senescence and circadian rhythm. Vutiglabridin is a small molecule in clinical trials that improves obesity by enhancing mitochondrial function. We found that chronic treatment of senescent primary human dermal fibroblasts (HDFs) with vutiglabridin alleviates all investigated markers of cellular senescence (SA-β-gal, CDKN1A, CDKN2A) and dysfunctional cellular circadian rhythm (BMAL1) while remarkably preventing the alterations of mitochondrial function and structure that occur during the process of cellular senescence. Our results demonstrate the significant senescence-alleviating effects of vutiglabridin, specifically with the restoration of cellular circadian rhythmicity and metabolic regulation. These data support the potential development of vutiglabridin against aging-associated diseases and corroborate the intricate link between cellular senescence, metabolism, and the circadian clock.

Cellulose nanocrystals from marine algae Cladophora glomerata by using microwave-assisted extraction

Algae of the order Cladophorales are the source of a unique nanocellulose with high crystallinity and a large aspect ratio, enabling broad surface modification. Cellulose nanocrystals (CNCs) are obtained via acid hydrolysis of nanocellulose, which is highly crystalline. However, the production of CNCs from Cladophorales algae is limited and still uses a conventional heating method. Thus, this study aimed to develop a microwave-assisted extraction (MAE) method for fast and efficient extraction of CNCs from Cladophora glomerata algae. Additionally, we replaced the use of hypochlorite with H2O2, which is more environmentally friendly, and compared the CNCs obtained from the conventional methods with our new method. The functional structure of CNCs was confirmed by Fourier-transform infrared spectroscopy. Single-step H2O2 bleaching with MAE yielded the smallest-sized CNCs. Our developed method resulted in the production of CNCs with a high crystallinity index, high thermal stability, and high purity of native cellulose. Additionally, none of the CNCs were toxic to primary normal human dermal fibroblasts. The properties of the isolated CNCs may make them useful materials in pharmaceutical and cosmetic formulations.