Human Fibroblast Cultures Research Articles

Macromolecular crowding agent dependent extracellular matrix deposition and growth factor retention in human corneal fibroblast cultures

The major obstacle in the commercialisation and clinical translation of tissue engineered medicines is the required for the development of implantable tissue surrogates prolonged in vitro culture. Macromolecular crowding (MMC) enhances and accelerates extracellular matrix (ECM) deposition, thus offering an opportunity to bridge the gap between research and development in tissue engineered substitutes. However, the optimal MMC agent is still elusive. Herein, we first assessed the biophysical properties of the most widely used MMC agents [κλ carrageenan (κλ CR), λ carrageenan (λ CR) and Ficoll™ cocktail (FC)] and then assessed their effect in basic cell function, ECM deposition and growth factor retention in human corneal fibroblast (hCF) cultures. Dynamic light scattering analysis revealed that both CR macromolecules had significantly lower and higher zeta potential and hydrodynamic radius, respectively, than the FC. None of the MMC agents affected hCF morphology and all induced similar hCF viability, proliferation and metabolic activity. Electrophoresis and immunofluorescence analyses made apparent that at day 10 (longest time point assessed), the FC brought about the highest fibronectin and collagen types I, III, IV, V and VI deposition. Deposited ECM pattern analysis showed that at day 10, the FC induced the lowest lacunarity and normalised end points and the highest fractal dimension and % high density matrix. Further immunofluorescence analysis revealed no significant differences between the groups in vimentin, aldehyde dehydrogenase 3 family member A1, keratocan, paired box protein 6 and α-smooth muscle actin. Importantly, at day 10, the FC resulted in the highest growth factor retention (20 molecules). Our data clearly illustrate a MMC agent dependent cell response, with the FC having the highest positive effect in hCF cultures.

Enhancement of human native skin fibroblast proliferation in natural salso-bromo-iodic mineral water added to in vitro culture.

The favorable regenerative effects of some mineral waters on wound healing have long been empirically demonstrated. The aim of this experimental study is to investigate the effects of an Italian salso-bromo-iodic mineral water (Rivanazzano, Italy) on an in vitro human native fibroblast culture model to identify any potential regenerative actions. Human native fibroblasts were cultured under different experimental conditions: - Dulbecco's modified Eagle's medium (DMEM) reconstituted with distilled water (control); - DMEM reconstituted with filtered mineral water collected from the spring; - DMEM reconstituted with filtered mineral water collected at the balneotherapy facility; - DMEM reconstituted with filtered, heated mineral water collected at the balneotherapy facility; - DMEM partially replaced with filtered mineral water collected from the spring at different concentrations (10%, 20%, 30%, 40%, 50%); - DMEM partially replaced with filtered, heated mineral water collected at the balneotherapy facility at different concentrations (10%, 20%, 30%, 40%, 50%); - DMEM partially replaced with filtered mineral water collected at the balneotherapy facility at different concentrations (10%, 20%, 30%, 40%, 50%). Cell proliferation and viability were evaluated using spectrophotometric analysis following staining with the XTT Microculture Tetrazolium Assay. Statistical analyses were performed for each experimental condition at 24, 48 and 72 h. The best outcomes were observed in fibroblasts cultured with DMEM partially replaced with filtered mineral water collected from the spring, within the range of 20-50%. Our research results showed that Rivanazzano salso-bromo-iodic mineral water has a stimulating effect on in vitro human native fibroblast cultures. This activity was most pronounced with water collected from the spring, and it decreased with water collected at the balneotherapy facilities. These findings could form the basis for clinical applications in wound healing and balneotherapy.

Development of Technology for the Synthesis of Nanocrystalline Cerium Oxide Under Production Conditions with the Best Regenerative Activity and Biocompatibility for Further Creation of Wound-Healing Agents.

Background/Objectives: The issue of effective wound healing remains highly relevant. The objective of the study is to develop an optimal method for the synthesis of nanosized cerium oxide powder obtained via the thermal decomposition of cerium carbonate precipitated from aqueous nitrate solution for the technical creation of new drugs in production conditions; the select modification of synthesis under different conditions based on the evaluation of the physicochemical characteristics of the obtained material and its biological activity, and an evaluation of the broad-spectrum effect on cells involved in the regeneration of skin structure as well as antimicrobial properties. Methods: Several modes of the industrial synthesis of cerium dioxide nanoparticles (NPs) were carried out. The synthesis stages and the chemical and physical parameters of the obtained NPs were described using transmission electron microscopy (TEM), X-ray diffraction, Raman spectroscopy, and mass spectrometry. The cell cultures of human fibroblasts and keratinocytes were cultured with different concentrations of different nanoceria variations, and the cytotoxicity and the metabolic and proliferative activity were investigated. An MTT test and cell counting were performed. The antimicrobial activity of CeO2 variations at a concentration of 0.1-0.0001 M against Pseudomonas aeruginosa was studied. Results: The purity of the synthesized nanoceria powders in all the batches was >99.99%. According to TEM data, the size of the NPs varied from 1 nm to 70 nm under different conditions and methodologies. The most optimal technology for the synthesis of the nanoceria with the maximum biological effect was selected. A method for obtaining the most bioactive NPs of optimal size (up to 10 nm) was proposed. The repeatability of the results of the proposed method of nanoceria synthesis in terms of particle size was confirmed. It was proven that the more structural defects on the surface of the CeO2 crystal lattice, the higher the efficiency of the NPs due to oxygen vacancies. The strain provided the best redox activity and antioxidant properties of the nanoceria, which was demonstrated by better regenerative potential on various cell lines. The beneficial effect of synthesized nanoceria on the proliferative and metabolic activity of the cell lines involved in skin regeneration (human fibroblasts, human keratinocytes) was demonstrated. The antimicrobial effect of synthesized nanoceria on the culture of the most-resistant-to-modern-antibiotics microorganism Pseudomonas aeruginosa was confirmed. The optimal concentrations of the nanoceria to achieve the maximum biological effect were determined (10-3 M). Conclusions: It was possible to develop a method for the industrial synthesis of nanoceria, which can be used to produce drugs and medical devices containing CeO2 NPs.

A Polyurethane Electrospun Membrane Loaded with Bismuth Lipophilic Nanoparticles (BisBAL NPs): Proliferation, Bactericidal, and Antitumor Properties, and Effects on MRSA and Human Breast Cancer Cells.

Electrospun membranes (EMs) have a wide range of applications, including use as local delivery systems. In this study, we manufactured a polyurethane Tecoflex™ EM loaded with bismuth-based lipophilic nanoparticles (Tecoflex™ EMs-BisBAL NPs). The physicochemical and mechanical characteristics, along with the antitumor and bactericidal effects, were evaluated using a breast cancer cell line and methicillin-susceptible and resistant Staphylococcus aureus (MRSA). Drug-free Tecoflex™ EMs and Tecoflex™ EMs-BisBAL NPs had similar fiber diameters of 4.65 ± 1.42 µm and 3.95 ± 1.32 µm, respectively. Drug-free Tecoflex™ EMs did not negatively impact a human fibroblast culture, indicating that the vehicle is biocompatible. Tecoflex™ EMs-BisBAL NPs increased 94% more in size than drug-free Tecoflex™ EMs, indicating that the BisBAL NPs enhanced hydration capacity. Tecoflex™ EMs-BisBAL NPs were highly bactericidal against both methicillin-susceptible S. aureus and MRSA clinical isolates, inhibiting their growth by 93.11% and 61.70%, respectively. Additionally, Tecoflex™ EMs-BisBAL NPs decreased the viability of MCF-7 tumor cells by 86% after 24 h exposure and 70.1% within 15 min. Regarding the mechanism of action of Tecoflex™ EMs-BisBAL NPs, it appears to disrupt the tumor cell membrane. In conclusion, Tecoflex™ EMs-BisBAL NPs constitute an innovative low-cost drug delivery system for human breast cancer and postoperative wound infections.

In Vitro Culture of Human Dermal Fibroblasts on Novel Electrospun Polylactic Acid Fiber Scaffolds Loaded with Encapsulated Polyepicatechin Physical Gels.

Polyepicatechin (PEC) in a hydrogel has previously shown promise in enhancing physiological properties and scaffold preparation. However, it remains unclear whether PEC-based fibers can be applied in skin tissue engineering (STE). This study aimed to synthesize and characterize electrospun PEC physical gels and polylactic acid (PLA) scaffolds (PLAloadedPECsub) for potential use as constructs with human dermal fibroblasts (HDFs). PEC was produced through enzymatic polymerization, as confirmed by Fourier transform infrared (FTIR) spectroscopy. Scanning electron microscopy (SEM) demonstrated the feasibility of producing PLAloadedPECsub by electrospinning. The metabolic activity and viability of HDFs cocultured with the scaffolds indicate that PLAloadedPECsub is promising for the use of STE.

Development and characterization of three-dimensional antibacterial nanocomposite sponges of chitosan, silver nanoparticles and halloysite nanotubes.

In this work, we developed novel nanocomposite three-dimensional (3D) scaffolds composed of chitosan (CTS), halloysite nanotubes (HNTs) and silver nanoparticles (AgNPs) with enhanced antimicrobial activity and fibroblast cell compatibility for their potential use in wound dressing applications. A stock CTS-HNT solution was obtained by mixing water-dispersed HNTs with CTS aqueous-acid solution, and then, AgNPs, in different concentrations, were synthesized in the CTS-HNT solution via a CTS-mediated in situ reduction method. Finally, freeze-gelation was used to obtain CTS-HNT-AgNP 3D porous scaffolds (sponges). Morphology analysis showed that synthesized AgNPs were spherical with an average diameter of 11 nm. HNTs' presence did not affect the AgNPs morphology or size but improved the mechanical properties of the scaffolds, where CTS-HNT sponges exhibited a 5 times larger compression stress than bare-CTS sponges. AgNPs in the scaffolds further increased their mechanical strength in correlation to the AgNP concentration, and conferred them improved antibacterial activity against Gram-negative and Gram-positive bacteria, inhibiting the planktonic proliferation and adhesion of bacteria in a AgNP concentration depending on manner. In vitro cell viability and immunofluorescence assays exhibited that human fibroblast (HF) culture was supported by the sponges, where HF retained their phenotype upon culture on the sponges. Present CTS-HNT-AgNP sponges showed promising mechanical, antibacterial and cytocompatibility properties to be used as potential scaffolds for wound dressing applications.

Exosomes Released from Senescent Cells and Circulatory Exosomes Isolated from Human Plasma Reveal Aging-associated Proteomic and Lipid Signatures.

Senescence emerged as a significant mechanism of aging and age-related diseases, offering an attractive target for clinical interventions. Senescent cells release a senescence-associated secretory phenotype (SASP), including exosomes that may act as signal transducers between distal tissues, propagating secondary or bystander senescence and signaling throughout the body. However, the composition of exosome SASP remains underexplored, presenting an opportunity for novel unbiased discovery. Here, we present a detailed proteomic and lipidomic analysis of exosome SASP using mass spectrometry from human plasma from young and older individuals and from tissue culture of senescent primary human lung fibroblasts. We identified ~1,300 exosome proteins released by senescent fibroblasts induced by three different senescence inducers causing most exosome proteins to be differentially regulated with senescence. In parallel, a human plasma cohort from young and old individuals revealed over 1,350 exosome proteins and 171 plasma exosome proteins were regulated when comparing old vs young individuals. Of the age-regulated plasma exosome proteins, we observed 52 exosome SASP factors that were also regulated in exosomes from the senescent fibroblasts, including serine protease inhibitors (SERPINs), Prothrombin, Coagulation factor V, Plasminogen, and Reelin. In addition, 247 lipids were identified with high confidence in all exosome samples. Following the senescence inducers, a majority of the identified phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin species increased significantly indicating cellular membrane changes. The most notable categories of significantly changed proteins were related to extracellular matrix remodeling and inflammation, both potentially detrimental pathways that can damage surrounding tissues and even induce secondary or bystander senescence. Our findings reveal mechanistic insights and potential senescence biomarkers, enabling a better approach to surveilling the senescence burden in the aging population and offering promising therapeutic targets for interventions.

Mono- and Bi-specific Nanobodies Targeting the CUB Domains of PCPE-1 Reduce the Proteolytic Processing of Fibrillar Procollagens

The excessive deposition of fibrillar collagens is a hallmark of fibrosis. Collagen fibril formation requires proteolytic maturations by Procollagen N- and C-proteinases (PNPs and PCPs) to remove the N- and C-propeptides which maintain procollagens in the soluble form. Procollagen C-Proteinase Enhancer-1 (PCPE-1, a glycoprotein composed of two CUB domains and one NTR domain) is a regulatory protein that activates the C-terminal processing of procollagens by the main PCPs. It is often up-regulated in fibrotic diseases and represents a promising target for the development of novel anti-fibrotic strategies.Here, our objective was to develop the first antagonists of PCPE-1, based on the nanobody scaffold. Using both an in vivo selection through the immunization of a llama and an in vitro selection with a synthetic library, we generated 18 nanobodies directed against the CUB domains of PCPE1, which carry its enhancing activity. Among them, I5 from the immune library and H4 from the synthetic library have a high affinity for PCPE-1 and inhibit its interaction with procollagens. The crystal structure of the complex formed by PCPE-1, H4 and I5 showed that they have distinct epitopes and enabled the design of a biparatopic fusion, the diabody diab-D1. Diab-D1 has a sub-nanomolar affinity for PCPE-1 and is a potent antagonist of its activity, preventing the stimulation of procollagen cleavage in vitro. Moreover, Diab-D1 is also effective in reducing the proteolytic maturation of procollagen I in cultures of human dermal fibroblasts and hence holds great promise as a tool to modulate collagen deposition in fibrotic conditions.

Lysophosphatidic Acid Modulates TGF-β2-Induced Biological Phenotype in Human Conjunctival Fibroblasts

Background: Although lysophosphatidic acid (LPA) is known to have multiple pathophysiological roles, its contributions to ocular tissues, especially conjunctival fibrogenesis, remain to be elucidated. Methods: To study this issue, the effects of LPA on transforming growth factor-β2 (TGF-β2)-induced fibrogenesis of two-dimensional (2D) and three-dimensional (3D) cultures of human conjunctival fibroblasts (HconF) were examined by the following analyses: (1) planar proliferation determined by transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran permeability measurements, (2) real-time metabolic analyses, (3) measurements of the size and stiffness of 3D spheroids, and (4) mRNA expression of extracellular matrix (ECM) molecules and their modulators. Results: LPA had no effect on TGF-β2-induced increase in the planar proliferation of HconF cells. LPA induced a more quiescent metabolic state in 2D HconF cells, but this metabolic suppression by LPA was partially blunted in the presence of TGF-β2. In contrast, LPA caused a substantial decrease in the hardness of 3D HconF spheroids independently of TGF-β2. In agreement with these different LPA-induced effects between 2D and 3D cultured HconF cells, mRNA expressions of ECM and their modulators were differently modulated. Conclusion: The findings that LPA induced the inhibition of both TGF-β2-related and -unrelated subepithelial proliferation of HconF cells may be clinically applicable.

Senescent glia link mitochondrial dysfunction and lipid accumulation

Senescence is a cellular state linked to ageing and age-onset disease across many mammalian species1,2. Acutely, senescent cells promote wound healing3,4 and prevent tumour formation5; but they are also pro-inflammatory, thus chronically exacerbate tissue decline. Whereas senescent cells are active targets for anti-ageing therapy6–11, why these cells form in vivo, how they affect tissue ageing and the effect of their elimination remain unclear12,13. Here we identify naturally occurring senescent glia in ageing Drosophila brains and decipher their origin and influence. Using Activator protein 1 (AP1) activity to screen for senescence14,15, we determine that senescent glia can appear in response to neuronal mitochondrial dysfunction. In turn, senescent glia promote lipid accumulation in non-senescent glia; similar effects are seen in senescent human fibroblasts in culture. Targeting AP1 activity in senescent glia mitigates senescence biomarkers, extends fly lifespan and health span, and prevents lipid accumulation. However, these benefits come at the cost of increased oxidative damage in the brain, and neuronal mitochondrial function remains poor. Altogether, our results map the trajectory of naturally occurring senescent glia in vivo and indicate that these cells link key ageing phenomena: mitochondrial dysfunction and lipid accumulation.

СВЯЗЬ МЕЖДУ КЛЕТОЧНЫМ СТАРЕНИЕМ И ИЗМЕНЕНИЯМИ КОЛИЧЕСТВА И РАЗМЕРОВ ФОКУСОВ ФОСФОРИЛИРОВАННОГО ГИСТОНА Н2AX В ОБЛУЧЕННЫХ ФИБРОБЛАСТАХ ЧЕЛОВЕКА

Purpose: To analyze the relationship between cellular aging and changes in the number and size of phosphorylated histone H2AX (γH2AX) foci in human fibroblasts and their descendants (up to 15 passages) after exposure to low and high doses of X-ray radiation. Material and methods: The work was performed on a culture of human skin fibroblasts. Cells were irradiated in the exponential growth phase on an X-ray biological unit RUB RUST-M1 (Russia), equipped with two X-ray emitters, at a dose rate of 40 mGy/min (dose 100 mGy) or 850 mGy/min (doses 2000 and 5000 mGy) and temperature 4˚C. Immunocytochemical staining was used to assess the number and size of γH2AX foci and the proportion of proliferating cells using antibodies to γH2AX and Ki67 (a cell proliferation marker protein), respectively. To assess cellular senescence, the proportion of cells positive for senescence-associated β-galactosidase (CA-β-gal(+)) was analyzed. Statistical and mathematical analysis of the obtained data was carried out using the statistical software package Statistica 8.0 (StatSoft). Results: The studies showed that irradiation of cultured human fibroblasts at a low dose (100 mGy) does not lead to statistically significant changes in the number and size of γH2AX foci, as well as the proportion of non-proliferating and senescent cells in the progenies of irradiated cells up to the 15th passage after irradiation. The phenomenon of aging-associated persistence of an increased number and size of γH2AX foci in passages of cells irradiated at a dose of 5000 mGy was discovered. Mathematical analysis of the relationship between changes in the proportion of CA-β-gal(+) cells, the number and size of γH2AX foci in populations of irradiated cells indicates that radiation-induced cellular aging is more associated with the size, rather than the number, of γH2AX foci.

Pulsed electromagnetic fields used in regenerative medicine: An in vitro study of the skin wound healing proliferative phase.

Numerous studies have demonstrated the efficacy of extremely low frequency-pulsed electromagnetic fields (ELF-PEMF) in accelerating the wound healing process in vitro and in vivo. Our study focuses specifically on ELF-PEMF applied with the Magnomega® device and aims to assess their effect during the main stages of the proliferative phase of dermal wound closure, in vitro. Thus, after the characterization of the EMFs delivered by the Magnomega® unit, primary culture of human dermal fibroblasts (HDFs) were exposed, or not for the control culture, to 10-12and 100 Hz ELF-PEMF. These parameters are used in clinical practice by physiotherapists in order to enhance healing of dermal lesions in patients. HDFs proliferation was first assessed and revealed an increase in the expression of one of the two genetic markers of cell proliferation tested (PCNA and MKI67), after initial exposure of the cells to 10-12 Hz PEMF. Next, migration of HDFs was investigated by performing scratch assays on HDF layers. The observed wound closure kinetics corroborate the early organization of actin stress fibers that was revealed in the cytoplasm of HDFs exposed to 100 Hz ELF-PEMF. Also, maturation of HDFs into myofibroblasts was significantly increased in cells exposed to 10-12or to 100 Hz PEMF. The present study is the first to demonstrate, in vitro, an early stimulation of HDFs, after their exposure to ELF-PEMF delivered by the Magnomega® device, which could contribute to an acceleration of the wound healing process.

Senescence and Mitochondrial Dysfunction in Lung Trisomy 21 Fibroblast

Rationale: Lung associated diseases are one of the leading causes of death in Down syndrome/Trisomy21 (DS/T21). DS is often considered a progeroid syndrome, associated with increased cellular senescence and oxidative stress in dermal T21 fibroblasts. Senescence plays a crucial role in development and organogenesis. We recently demonstrated that T21 lung anomalies are initiated in utero. We aim to better understand the role of senescence in lung disease in T21. Methods: Fresh T21 and, age- and sex-matched non-T21 human fetal lungs were collected, dissociated, and human fetal lung fibroblast (HFLF) cultures were established using differential adhesion. Baseline senescence levels were determined in whole tissue and isolated fibroblasts using TAF staining for tissue only and RT-qPCR and immunofluorescence (IF) for both. β-galactosidase, CellROX/MitoSOX, and calcium release (FURA2/AM imaging) assays were performed on T21 and non-T21 fibroblasts, cultured in the absence or presence of an ER stress inhibitor (4-PBA, 2mM) or a mitochondriogenesis stimulator (PQQ, 20μM). Results: Whole tissue sections demonstrated increased TAF and γ-H2AFX staining in T21 vs non-T21. RT-qPCR demonstrated significantly increased expression of several senescence markers including CDKN1A, CDKN2A, TP53, CDKN2B and PAI1 (p<0.05; n=6) at baseline in T21 HFLF as compared to non-T21. IF for γ-H2AFX and P21 were also significantly increased in T21 cultures vs non-T21 (p<0.01, n=6). Upregulated senescence was further confirmed in T21 HFLF by increased β-galactosidase activity (6-fold vs non-T21, p<0.05, n=4) as well as increased ER and mitochondria stress markers (CellROX and mitoSOX, p<0.05, n=6) in T21 cultures. Fura2/AM assay revealed that T21 HFLF exhibit greater intracellular calcium release (p<0.01, n=3) than non-T21 HFLF at both baseline and post Acetylcholine (10uM) challenge, an indicator of increased ER stress. In addition, several mitochondria morphology genes ( FIS1, DNML1 and MFN1) increased in T21 HFLF (p<0.05, n=6). Both 4-PBA or PQQ treatments reversed senescence in T21 HFLF as shown by decreased β-galactosidase staining (p<0.01; n = 4). No differences were observed in non-T21 HFLF after treatment. However, gene expression analyses of CDKN1A, CDKN2A, TP53 and CDKN2B demonstrated attenuated expression in T21 HFLF only following PQQ treatment but not 4-PBA (p<0.05, n=4). Conclusion: In this study, we established that fibroblasts from T21 lungs present with both ER and mitochondria-associated senescence. However, only PQQ, the mitochondriogenesis stimulator, was able to completely reverse the senescence observed at baseline in T21 HFLF. Understanding this increase in senescence in the lungs of individuals with Down Syndrome may help address the respiratory issues that arise after birth. CIRM (EDUC4-12837) Postdoctoral Training Grant (RB) NIH/NHLBI R01HL141856 (DA); NIH/NHLBI Offce of The Director, National Institutes of Health (OD) R01HL155104 (SD); NIH/NHLBI R21HL165411 (SD and DA). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

INVESTIGATION OF ENZYMATIC ACTIVITY IN HUMAN DERMAL FIBROBLASTS DURING VARIOUS CULTIVATION PERIODS

Aim. Research into the change in enzymatic indicators of cell activity during the aging of human dermal fibroblasts in culture from 3 to 15 passages to determine the most optimal terms for cell transplantation to patients for further cell therapy. Methods: Dermal fibroblasts of donors A, B, and C aged from 40 to 60 years were cultivated in сulture medium containing FBS, antibiotics, bFGF. To determine the cell cycle, cells were fixed with formalin, stained with propidium iodide/RNase buffer and analyzed on a flow cytometer. To study the enzymatic activity of mitochondria, fibroblasts were seeded in microplate, MTT was added, followed by DMSO and glycine. To determine the activity of lysosomal enzymes, fibroblasts were fixed with formalin, stained with X-Gal and photographed on a microscope. A statistical analysis of the results was carried out. Results: Dermal fibroblasts retain their mitotic activity from early to late passages. The average percentage of mitotic cells was higher than the average value of the cells at the interphase. Optical density did not reveal significant changes with the change in the cultivation term. The increase in formazan level corresponds to a percentage of cells in mitosis. Studying microphotographs of early and late passages to detect cells with enhanced β-galactosidase secretion, no signs of aging of dermal fibroblasts of donors were noticed. Conclusions: Using various cytochemical methods, it has been proven that the culture of human dermal fibroblasts from donors of the age group from 40 to 60 years maintains stability during their cultivation from 3 to 15 passages.

Rose Bengal Photodynamic Therapy (RB-PDT) Modulates the Inflammatory Response in LPS-Stimulated Human Corneal Fibroblasts By Influencing NF-κB and p38 MAPK Signaling Pathways

Purpose To investigate the effect of rose bengal photodynamic therapy on lipopolysaccharide-induced inflammation in human corneal fibroblasts. Furthermore, to analyze potential involvement of the mitogen-activated protein kinase and nuclear factor kappa B signaling pathways in this process. Methods Human corneal fibroblast cultures underwent 0–2.0 µg/mL lipopolysaccharide treatment, and 24 h later rose bengal photodynamic therapy (0.001% RB, 565 nm wavelength illumination, 0.17 J/cm2 fluence). Interleukin-6, interleukin-8, intercellular adhesion molecule-1, interferon regulatory factor-3, interferon α2, and interferon β1 gene expressions were determined by quantitative PCR. Interleukin-6, interleukin-8, and C-C motif chemokine ligand-4 concentrations in the cell culture supernatant were measured by enzyme-linked immunosorbent assays and intercellular adhesion molecule-1 protein level in human corneal fibroblasts by western blot. In addition, the nuclear factor kappa B and mitogen-activated protein kinase signaling pathways were investigated by quantitative PCR and phosphorylation of nuclear factor kappa B p65 and p38 mitogen-activated protein kinase by western blot. Results Rose bengal photodynamic therapy in 2.0 µg/mL lipopolysaccharide-stimulated human corneal fibroblasts triggered interleukin-6 and interleukin-8 mRNA (p < .0001) and interleukin-6 protein increase (p < .0001), and downregulated intercellular adhesion molecule-1 expression (p < .001). C-C motif chemokine ligand-4, interferon regulatory factor-3, interferon α2, and interferon β1 expressions remained unchanged (p ≥ .2). Rose bengal photodynamic therapy increased IκB kinase subunit beta, nuclear factor kappa B p65, extracellular signal-regulated kinases-2, c-Jun amino terminal kinase, and p38 transcription (p ≤ .01), and triggered nuclear factor kappa B p65 and p38 mitogen-activated protein kinase phosphorylation (p ≤ .04) in lipopolysaccharide treated human corneal fibroblasts. Conclusion Rose bengal photodynamic therapy of lipopolysaccharide-stimulated human corneal fibroblasts can modify the inflammatory response by inducing interleukin-6 and interleukin-8 expression, and decreasing intercellular adhesion molecule-1 production. C-C motif chemokine ligand-4, interferon regulatory factor-3, and interferon α and β expressions are not affected by rose bengal photodynamic therapy in these cells. The underlying mechanisms may be associated with nuclear factor kappa B and p38 mitogen-activated protein kinase pathway activation.

Assessment of the toxic effect of the medical glue «Sulfacrylate» in M-22 cell culture

Sulfacrylate is a medical glue used in general and vascular surgery, traumatology, maxillofacial surgery, thoracic and abdominal surgery, and dentistry. To connect tissues in plastic ophthalmic surgery, the toxic properties of Sulfacrylate should be studied.&#x0D; AIM: To evaluate the toxic effect of Sulfacrylate in human cell culture in vitro.&#x0D; MATERIAL AND METHODS: In vitro studies were conducted on human fibroblast cultures of the M-22 line from the 20th passage. Sulfacrylate was used in the present study (registration certificate for a medical device no. FSR 2010/09805; December 31, 2010). A lyophilized bandage based on human type 1 collagen was used to fix the glue. The total number of cells at the bottom of wells, cell density at the bottom of wells and on collagen dressings (1 thousand cells per cm2), structural integrity of cells and their morphology, integrity of cell membranes were evaluated.&#x0D; RESULTS: The toxic effect of collagen dressings with Sulfacrylate was limited by the size of the bandage. Collagen dressings with Sulfacrylate did not affect the viability of cells not in direct contact with the dressings and did not cause a pronounced decrease in the proliferative activity of cells. The minimum optimal dosage of adhesive for plastic ophthalmic surgery was 1–3 µkl.&#x0D; CONCLUSION: The use of Sulfacrylate in plastic ophthalmic surgery is feasible; however, the volume used should be minimized.

Gums as Macromolecular Crowding Agents in Human Skin Fibroblast Cultures.

Even though tissue-engineered medicines are under intense academic, clinical, and commercial investigation, only a handful of products have been commercialised, primarily due to the costs associated with their prolonged manufacturing. While macromolecular crowding has been shown to enhance and accelerate extracellular matrix deposition in eukaryotic cell culture, possibly offering a solution in this procrastinating tissue-engineered medicine development, there is still no widely accepted macromolecular crowding agent. With these in mind, we herein assessed the potential of gum Arabic, gum gellan, gum karaya, and gum xanthan as macromolecular crowding agents in WS1 skin fibroblast cultures (no macromolecular crowding and carrageenan were used as a control). Dynamic light scattering analysis revealed that all macromolecules had negative charge and were polydispersed. None of the macromolecules affected basic cellular function. At day 7 (the longest time point assessed), gel electrophoresis analysis revealed that all macromolecules significantly increased collagen type I deposition in comparison to the non-macromolecular crowding group. Also at day 7, immunofluorescence analysis revealed that carrageenan; the 50 µg/mL, 75 µg/mL, and 100 µg/mL gum gellan; and the 500 µg/mL and 1000 µg/mL gum xanthan significantly increased both collagen type I and collagen type III deposition and only carrageenan significantly increased collagen type V deposition, all in comparison to the non-macromolecular crowding group at the respective time point. This preliminary study demonstrates the potential of gums as macromolecular crowding agents, but more detailed biological studies are needed to fully exploit their potential in the development of tissue-engineered medicines.

FOXO1 regulates wound-healing responses in human gingival fibroblasts.

Forkhead box-O 1 (FOXO1) is a transcription factor actively involved in oral wound healing at the epithelial barrier. However, less is known regarding the role of FOXO1 during the tissue repair response in the connective tissue compartment. This study explored the involvement of FOXO1 in the modulation of fibroblast activity related to wound healing. Primary cultures of human gingival fibroblasts were obtained from four healthy young donors. Myofibroblastic differentiation, collagen gel contraction, cell migration, cell spreading, and integrin activation were evaluated in the presence or absence of a FOXO1 inhibitor (AS1842856). Variations in mRNA and proteins of interest were evaluated through qRT-PCR and western blot, respectively. Distribution of actin, α-smooth muscle actin, and β1 integrin was evaluated using immunofluorescence. FOXO1 and TGF-β1 expression in gingival wound healing was assessed by immunohistochemistry in gingival wounds performed in C57BL/6 mice. Images were analyzed using ImageJ/Fiji. ANOVA or Kruskal-Wallis test followed by Tukey's or Dunn's post-hoc test was performed. All data are expressed as mean ± SD. p < .05 was considered statistically significant. FOXO1 inhibition caused a decrease in the expression of the myofibroblastic marker α-SMA along with a reduction in fibronectin, type I collagen, TGF-β1, and β1 integrin mRNA level. The FOXO1 inhibitor also caused decreases in cell migration, cell spreading, collagen gel contraction, and β1 integrin activation. FOXO1 and TGF-β1 were prominently expressed in gingival wounds in fibroblastic cells located at the wound bed. The present study indicates that FOXO1 plays an important role in the modulation of several wound-healing functions in gingival fibroblast. Moreover, our findings reveal an important regulatory role for FOXO1 on the differentiation of gingival myofibroblasts, the regulation of cell migration, and collagen contraction, all these functions being critical during tissue repair and fibrosis.

Experimental evidence for Parthanatos-like mode of cell death of heat-damaged human skin fibroblasts in a cell culture-based in vitro burn model

The cellular mechanisms of burn conversion of heat damaged tissue are center of many studies. Even if the molecular mechanisms of heat-induced cell death are controversially discussed in the current literature, it is widely accepted that caspase-mediated apoptosis plays a central role. In the current study we wanted to develop further information on the nature of the mechanism of heat-induced cell death of fibroblasts in vitro. We found that heating of human fibroblast cultures (a 10 s rise from 37 °C to 67 °C followed by a 13 s cool down to 37 °C) resulted in the death of about 50% of the cells. However, the increase in cell death started with a delay, about one hour after exposure to heat, and reached the maximum after about five hours. The lack of clear evidence for an active involvement of effector caspase in the observed cell death mechanism and the lack of observation of the occurrence of hypodiploid nuclei contradict heat-induced cell death by caspase-mediated apoptosis. Moreover, a dominant heat-induced increase in PARP1 protein expression, which correlated with a time-delayed ATP synthesis inhibition, appearance of double-strand breaks and secondary necrosis, indicate a different type of cell death than apoptosis. Indeed, increased translocation of Apoptosis Inducing Factor (AIF) and Macrophage Migration Inhibitory Factor (MIF) into cell nuclei, which correlates with the mentioned enhanced PARP1 protein expression, indicate PARP1-induced, AIF-mediated and MIF-activated cell death. With regard to the molecular actors involved, the cellular processes and temporal sequences, the mode of cell death observed in our model is very similar to the cell death mechanism via Parthanatos described in the literature.

Bioresorbable molybdenum temporary epicardial pacing wires

Cardiac pacing with temporary epicardial pacing wires (TEPW) is used to treat rhythm disturbances after cardiac surgery. Occasionally, TEPW cannot be mechanically extracted and remain in the thorax, where they may rarely cause serious complications like migration and infection. We aim to develop bioresorbable TEPW that will dissolve over time even if postoperative removal is unsuccessful. In the present study, we demonstrate a completely bioresorbable design using molybdenum (Mo) as electric conductor and the resorbable polymers poly(D, L-lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) for electrically insulating double-coating. We compared the pacing properties of these Mo TEPW demonstrators to conventional steel TEPW in Langendorff-perfused rat hearts and observed similar functionality. In vitro, static immersion tests in simulated body fluid for up to 28 days elucidated the degradation behaviour of uncoated Mo strands and the influence of polymer coating thereon. Degradation was considerably reduced in double-coated Mo TEPW compared to the uncoated and the PLGA-coated condition. Furthermore, we confirmed good biocompatibility of Mo degradation products in the form of low cytotoxicity in cell cultures of human cardiomyocytes and cardiac fibroblasts. Statement of significanceTemporary pacing wires are routinely implanted on the heart surface to treat rhythm disturbances in the days following cardiac surgery. Subsequently, these wires are to be removed. When removal attempts are unsuccessful, wires are cut at skin level and the remainders are left inside the chest. Retained fragments may migrate within the body or become a centre of infection. These complications may be prevented using resorbable pacing wires. We manufactured completely resorbable temporary pacing wires using molybdenum as electrical conductor and assessed their function, degradation and biological compatibility. Our study represents an important step in the development of a safer approach to the treatment of rhythm disturbances after cardiac surgery.