Human Fibroblast Cell Cultures Research Articles

Preparation and Characterization of Polymeric Microparticles Based on Poly(ethylene brassylate-co-squaric Acid) Loaded with Norfloxacin.

In recent years, increasing interest has been accorded to polyester-based polymer microstructures, driven by their promising potential as advanced drug delivery systems. This study presents the preparation and characterization of new polymeric microparticles based on poly(ethylene brassylate-co-squaric acid) loaded with norfloxacin, a broad-spectrum antibiotic. Polymacrolactone was synthesised in mild conditions through the emulsion polymerization of bio-based and renewable monomers, ethylene brassylate, and squaric acid. The microparticles were obtained using the precipitation technique and subsequently subjected to comprehensive characterization. The impact of the copolymer/drug ratio on various properties of the new system was systematically evaluated, confirming the structure of the copolymer and the encapsulation of norfloxacin. The microspheres are approximately spherical and predominantly homogeneously distributed. The average hydrodynamic diameter of the microparticles falls between 400 and 2000 nm, a decrease that is observed with the increase in norfloxacin content. All samples showed good encapsulation efficiency and drug loading capacity, with the highest values obtained for microparticles synthesised using an equal ratio of copolymer and drug. In vitro drug release results disclose that norfloxacin molecules are released in a sustained biphasic manner for up to 24 h. Antimicrobial activity was also studied, with samples showing very good activity against E. coli and moderate activity against S. aureus and E. faecalis. In addition, HDFA human fibroblast cell cultures demonstrated the cytocompatibility of the microparticles.

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.

Drug Synergism of Anticancer Action in Combination with Favipiravir and Paclitaxel on Neuroblastoma Cells.

Background and Objectives: Favipiravir (FPV) is an antiviral medication and has an inhibitory effect on Cytochrome P450 (CYP2C8) protein, which is mainly involved in drug metabolism in the liver, and the expression of this gene is known to be enhanced in neuronal cells. The metabolization of Paclitaxel (PTX), a chemotherapeutic drug used in cancer patients, was analyzed for the first time in the human SH-SY5Y neuroblastoma cell line for monitoring possible synergistic effects when administered with FPV. Materials and Methods: Further, in vitro cytotoxic and genotoxic evaluations of FPV and PTX were also performed using wide concentration ranges in a human fibroblast cell culture (HDFa). Nuclear abnormalities were examined under a fluorescent microscope using the Hoechst 33258 fluorescent staining technique. In addition, the synergistic effects of these two drugs on cultured SH-SY5Y cells were determined by MTT cell viability assay. In addition, the death mechanisms that can occur in SHSY-5Y were revealed by using the flow cytometry technique. Results: Cell viability analyses on the HDFa healthy cell culture showed that both FPV and PTX have inhibitory effects at higher concentrations. On the other hand, there were no significant differences in nuclear abnormality numbers when both of the compounds were applied together. Cell viability analyses showed that FPV and PTX applications have higher cytotoxicity, which indicated synergistic toxicity against the SHSY-5Y cell line. Also, PTX exhibited higher anticancer properties against the neuroblastoma cell line when applied with FPV, as shown in both cytotoxicity and flow cytometry analyses. Conclusions: In light of our findings, the anticancer properties of PTX can be enhanced when the drug application is coupled with FPV exposure. Moreover, these results put forth that the anticancer drug dosage should be evaluated carefully in cancer patients who take COVID-19 treatment with FPV.

Influence of the Synthesis Scheme of Nanocrystalline Cerium Oxide and Its Concentration on the Biological Activity of Cells Providing Wound Regeneration.

In the ongoing search for practical uses of rare-earth metal nanoparticles, cerium dioxide nanoparticles (nanoceria) have received special attention. The purpose of this research was to study the biomedical effects of nanocrystalline forms of cerium oxide obtained by different synthesis schemes and to evaluate the effect of different concentrations of nanoceria (from 10-2 to 10-6 M) on cells involved in the regeneration of skin cell structures such as fibroblasts, mesenchymal stem cells, and keratinocytes. Two different methods of nanoceria preparation were investigated: (1) CeO-NPs-1 by precipitation from aqueous solutions of cerium (III) nitrate hexahydrate and citric acid and (2) CeO-NPs-2 by hydrolysis of ammonium hexanitratocerate (IV) under conditions of thermal autoclaving. According to the X-ray diffraction, transmission electron microscopy, and dynamic light scattering data, CeO2-1 consists of individual particles of cerium dioxide (3-5 nm) and their aggregates with diameters of 60-130 nm. CeO2-2 comprises small aggregates of 8-20 nm in diameter, which consist of particles of 2-3 nm in size. Cell cultures of human fibroblasts, human mesenchymal stem cells, and human keratinocytes were cocultured with different concentrations of nanoceria sols (10-2, 10-3, 10-4, 10-5, and 10-6 mol/L). The metabolic activity of all cell types was investigated by MTT test after 48 and 72 h, whereas proliferative activity and cytotoxicity were determined by quantitative cell culture counting and live/dead test. A dependence of biological effects on the method of nanoceria preparation and concentration was revealed. Data were obtained with respect to the optimal concentration of sol to achieve the highest metabolic effect in the used cell cultures. Hypotheses about the mechanisms of the obtained effects and the structure of a fundamentally new medical device for accelerated healing of skin wounds were formulated. The method of nanoceria synthesis and concentration fundamentally and significantly change the biological activity of cell cultures of different types-from suppression to pronounced stimulation. The best biological activity of cell cultures was determined through cocultivation with sols of citrate nanoceria (CeO-NPs-1) at a concentration of 10-3-10-4 M.

Analysis of the Impact of Ethanol Extract of Calendula officinalis L. on Human Fibroblast Cell Cultures Using the PANsys 3000 Device for Breeding and Visualization of Cells.

Calendula officinalis L. promotes wound healing and might be effective in gingival fibroblast stimulation. The influence of different concentrations of Calendula officinalis L. ethanol extract on human gingival fibroblast was visualized using PANsys 3000-a fully automated cell culture device used for in vitro culture to study cells under conditions similar to in vivo. The human fibroblast cells were isolated from gingival tissue. The 100% brew of Calendula officinalis L., as well as 7% and 20% Calendula officinalis L. ethanol extract, were added to the cultured cells and observed for 72 h. The qualitative and quantitative composition of the volatile compounds of marigold Calendula officinalis L. flowers are presented in this study. The essential oil compounds of the decoction were isolated with solid-phase microextraction (SPME) and analyzed with gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). The presence of terpenoids, flavonoids, and other compounds was demonstrated. The composition was correlated with the fragrance properties. Observation of gingival fibroblast showed that there were no changes in cell morphology and proliferation after 100% Calendula officinalis L. brew stimulation. The growth and cell division were not inhibited. Likewise, the addition of 7% or 20% ethanol in water extract of Calendula officinalis L. stimulation did not inhibit the fibroblast proliferation. Overall, ethanol extracts of Calendula officinalis L. decrease the alcohol cytotoxic influence on gingival fibroblasts.

Human lactoferrin-clay mineral nanohybrids as emerging green biomaterials: A physicochemical characterization

Herein montmorillonite and halloysite have been exploited to prepare advanced lactoferrin-loaded nanohybrid biomaterials for potential biomedical applications. Nanohybrids were prepared following two methodologies: a traditional procedure which induces spontaneous drug-clay interactions to take place in aqueous environment and by spray drying, as alternative and preferred industrial technique for protein processing. Structural assembly of the obtained nanohybrids was assessed by solid state characterization techniques and determination of their surface charge properties. Full microscopy studies, including scanning electron microscopy and ultra-high resolution transmission electron microscopy coupled with EDS were also carried out. Cytotoxicity assays were performed to assess biocompatibility of nanohybrids towards human fibroblast cell cultures. Results showed that both intercalation-solution and spray drying procedures led to the effective formation of nanohybrids structures, with greater extent of peptide-clay mineral interactions in those obtained by spray drying. All the samples exhibited cell viability % higher than 80%, with best results for halloysite coupled with spray drying technique.

Aqueous Processable One-Dimensional Polypyrrole Nanostructured by Lignocellulose Nanofibril: A Conductive Interfacing Biomaterial.

One-dimensional (1D) nanomaterials of conductive polypyrrole (PPy) are competitive biomaterials for constructing bioelectronics to interface with biological systems. Synergistic synthesis using lignocellulose nanofibrils (LCNF) as a structural template in chemical oxidation of pyrrole with Fe(III) ions facilitates surface-confined polymerization of pyrrole on the nanofibril surface within a submicrometer- and micrometer-scale fibril length. It yields a core-shell nanocomposite of PPy@LCNF, wherein the surface of each individual fibril is coated with a thin nanoscale layer of PPy. A highly positive surface charge originating from protonated PPy gives this 1D nanomaterial a durable aqueous dispersity. The fibril-fibril entanglement in the PPy@LCNFs facilely supported versatile downstream processing, e.g., spray thin-coating on glass, flexible membranes with robust mechanics, or three-dimensional cryogels. A high electrical conductivity in the magnitude of several to 12 S·cm-1 was confirmed for the solid-form PPy@LCNFs. The PPy@LCNFs are electroactive and show potential cycling capacity, encompassing a large capacitance. Dynamic control of the doping/undoping process by applying an electric field combines electronic and ionic conductivity through the PPy@LCNFs. The low cytotoxicity of the material is confirmed in noncontact cell culture of human dermal fibroblasts. This study underpins the promises for this nanocomposite PPy@LCNF as a smart platform nanomaterial in constructing interfacing bioelectronics.

Biocompatibility of crab shell-derived graft sterilized by ultraviolet light towards human gingival fibroblast cell culture

Background: Portunus pelagicus crab species meat is generally taken, and their shells that is thrown away becoming untapped waste. Their shells contain so many useful minerals such as calcium carbonate ­which can be processed into hydroxyapatite (HA). HA has been extensively used in the medical field for bone grafting, to repair damaged in bone structures. Purpose: To determine the biocompatibility of hydroxyapatite from Portunus pelagicus crab’s shells extract after sterilized using UV on human gingival fibroblast cell. Methods: This research is an experimental laboratory research using post-test only control group design. The shells were first heated using furnace. Once hydroxyapatite was obtained, it was sterilized using ultraviolet (UV) with certain concentration (25 ppm, 50 ppm, and 100 ppm) to kill the microorganisms. MTT assay was done to test the biocompatibility of the sterilized hydroxyapatite on human gingival fibroblast cell. Results: The highest viability of fibroblast cells was the group with 50 ppm UV (98.10%), followed by 100 ppm UV (97.93%), and 25 ppm (93.28%). Conclusion: Hydroxyapatite from Portunus pelagicus crab’s shells extract sterilized using UV is biocompatible towards human gingival fibroblast cell.

Skin Beautification with an ImmunatuRNA® Complex: A Natural Complex Based on Yeast RNA and Marine Derivatives on the Human Skin Immunity and Microbiome Composition

The skin is the largest organ of human body implicated in vital physiological and immunological processes, including protection against injury, infections, and environmental exposures. Nevertheless, a number of negative external and internal factors, including increased pollution along with modern lifestyle can significantly influence on the skin immunity, promote aging and reduce regenerative capabilities. Cosmetic formulations based on natural ingredients, including plant and marine/algae extracts, and microbial-derived bio-actives may help to maintain healthy skin condition and protect its beautiful appearance. The ImmunatuRNA®* (*The trademark ImmunatuRNA® is a registered trademark in the European Union EUTM 018365297 and Great Britain UK00003630172. Any reference to the registration of the ImmunatuRNA® trademark applies to the designated territories), a signature complex (VRFD SA/VERDILAB Switzerland) based on the Natural RNA molecules obtained from yeasts, with added marine exopolysaccharides and natural hyaluronic acid with potential benefits for antibacterial and antiradical defense and maintaining skin detox homeostasis. Therefore, the main aim of this study is to experimentally assess the ImmunatuRNA® complex capabilities to enhance natural skin antioxidant defenses and protection against oxidative stress, by using human fibroblast cell culture. Additionally, this study will investigate the effects of the skincare products with added ImmunatuRNA® complex on the skin microbiome composition in the group of healthy adults with various skin types. The results obtained from the in vitro studies consistently demonstrated that treatment with an ImmunatuRNA® complex protects human fibroblasts against exposure to pro-oxidants, while at the same time enhances anti-oxidative defenses of these cells, demonstrated as reduced formation of free radicals and upregulation of SIRT-1 protein. Furthermore, the cosmetic intervention carried on the group of 10 healthy human volunteers with different skin types (oily, dry, combined, normal) aged between 23 and 65 years old demonstrated that using interchangeably of two cosmetic products-a Natural Moisturizer Probiotic Cream and Natural Detox Replenishing Face Mask containing the ImmunatuRNA® complex, for 4-weeks led to favorable changes in the composition of skin microbiome observed as a reduction of relative abundance of adverse microorganisms, previously linked to the skin inflammation and infections, such as Citrobacter koseri, Bacillus spp., Enterococcus spp., Alcaligenes spp., Pantoea spp. and Staphylococcus aureus. Based on following results, the use of skincare products containing the ImmunatuRNA® complex can help maintain a healthy skin microbiome while possibly preventing against infections and negative environmental exposures, in particularly against oxidative stress in healthy adults with all types of skin with age range 23-65 years old.

Assessment of the effects of hydrogen water on human gingival fibroblast cell culture in patients with chronic periodontitis.

Activated inflammatory cells produce reactive oxygen species (ROS) to eliminate pathogens. Under normal conditions, the pathogens are taken care of, and tissues are repaired. However, in periodontal disease, persistent inflammation causes increased ROS release and impaired healing. Therefore, removal of overproduced ROS using antioxidants is necessary. Hydrogen water has an antioxidative effect on cells and impedes oxidative stress-related disorders. To study the effect of hydrogen water on cell viability, migration, and its antioxidative potential in fibroblasts obtained from chronic periodontitis patients. The gingival tissue samples were obtained from 26 subjects (13 periodontally healthy individuals and 13 chronic periodontitis patients) and processed. The human gingival fibroblasts were cultured and the assays were commenced once adequate growth was detected. The effect of hydrogen water on cell viability was checked by neutral red assay, while the migration potential was assessed by transwell migration assay. The antioxidative potential of hydrogen water was evaluated by CUPRAC assay. Intergroup comparison was done using Mann-Whitney U-test. Intragroup comparison was done using Wilcoxon signed-rank test. Hydrogen water was nontoxic to the fibroblasts at 24 h and 48 h. The intergroup comparison of the cell viability between hydrogen water-treated periodontally healthy gingival fibroblasts (HF) and fibroblasts from patients with chronic periodontitis (CF) showed a statistically significant (P = 0.00) difference at 24 h and 48 h. Hydrogen water also positively influenced the migratory capacity. Hydrogen water-treated fibroblasts obtained from chronic periodontitis patients showed more migration in comparison to the healthy group (P = 0.00). Hydrogen water showed an antioxidative potential. The maximum potential was seen in relation to the fibroblasts obtained from chronic periodontitis patients at 48 h. Hydrogen water was nontoxic, increased the migratory capacity, and showed an antioxidative potential on human fibroblasts obtained from periodontally healthy individuals and patients with chronic periodontitis.

The Effects of the Action of Chromium, Aluminum, Nickel and Iron on Human Fibroblast and Stem Cell Cultures

This review is a collection of general information about chromium, aluminum, nickel and iron. We tried to include not only the data about absorption, metabolism, interactions with other elements and the importance of those microelements in the human body but also their toxic and mutagenic effects. Moreover, we examined their effect on human fibroblast and stem cell cultures which may be important due to abuse of supplementation in the population nowadays.

Lipoic Acid Conjugated Boron Hybrids Enhance Wound Healing and Antimicrobial Processes.

Complications of chronic non-healing wounds led to the emergence of nanotechnology-based therapies to enhance healing, facilitate tissue repair, and prevent wound-related complications like infections. Here, we design alpha lipoic acid (ALA) conjugated hexagonal boron nitride (hBN) and boron carbide (B4C) nanoparticles (NPs) to enhance wound healing in human dermal fibroblast (HDFa) cell culture and characterize its antimicrobial properties against Staphylococcus aureus (S. aureus, gram positive) and Escherichia coli (E. coli, gram negative) bacterial strains. ALA molecules are integrated onto hBN and C4B NPs through esterification procedure, and molecular characterizations are performed by using transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and UV-vis spectroscopy. Wound healing and antimicrobial properties are investigated via the use of cell viability assays, scratch test, oxidative stress, and antimicrobial activity assays. Based on our analysis, we observe that ALA-conjugated hBN NPs have the highest wound-healing feature and antimicrobial activity compared to ALA-B4C. On the other hand, hBN, ALA-B4C, and ALA compounds showed promising regenerative and antimicrobial properties. Also, we find that ALA conjugation enhances wound healing and antimicrobial potency of hBN and B4C NPs. We conclude that the ALA-hBN conjugate is a potential candidate to stimulate regeneration process for injuries.

Effect of recombinant Sox9 protein on the expression of cartilage-specific genes in human dermal fibroblasts cell culture

Introduction: Damage to the hyaline layer of large joints resulting from injuries or age-related changes restricts their mobility. The repair of these disorders is an actual issue in medicine. One of the promising therapies is the usage of cell engineering constructs based on a biodegradable scaffold and a modified cell culture. A frequently used method to modify the proliferation of cell culture for tissue engineering of hyaline cartilage, which makes it possible to introduce an experimental technique into clinical practice, is the application of recombinant proteins that affect chondrogenesis and lead to increase synthesis of extracellular matrix proteins. The goal of this work was to elucidate the effect of the key transcription factor in the chondrogenesis process – Sox9 protein – on the expression of genes responsible for chondrogenesis (Tgfβ3, Sox9, Acan, Comp, Col2a1).
 Materials and methods: Human dermal fibroblasts were used as a cell culture; recombinant Sox9 was added at each change of medium; the modification was carried out for 21 days, and difference in gene expression was determined by real-time PCR and -ΔΔCt method.
 Results and discussion: To assess the effectiveness of fibroblast modification, we analyzed the changing of expression of genes responsible for chondrogenesis (Tgfß3, Sox9, Col2a1, Acan, Comp). We studied the direct effect of different concentrations of the recombinant Sox9 protein on the proliferation of dermal fibroblasts in the chondrogenic direction. We showed that the addition of the recombinant Sox9 protein in various concentration did not significantly change the expression of both the genes encoding proteins of the extracellular matrix of hyaline cartilage (Acan, Col2a1, Comp) and the genes encoding chondrogenesis inducers (Tgfß3, Sox9).
 Conclusion: As a result of the experiments, it was shown that the recombinant Sox9 protein has practically no effect on chondrogenic differentiation and does not significantly change the expression of chondrogenesis genes.

Benzalkonium Chloride, Even at Low Concentrations, Deteriorates Intracellular Metabolic Capacity in Human Conjunctival Fibroblasts.

The objective of this study was to clarify the effects of benzalkonium chloride (BAC) on two-dimensional (2D) and three-dimensional (3D) cultures of human conjunctival fibroblast (HconF) cells, which are in vitro models replicating the epithelial barrier and the stromal supportive functions of the human conjunctiva. The cultured HconF cells were subjected to the following analyses in the absence and presence of 10−5% or 10−4% concentrations of BAC; (1) the barrier function of the 2D HconF monolayers, as determined by trans-endothelial electrical resistance (TEER) and FITC dextran permeability, (2) real-time metabolic analysis using an extracellular Seahorse flux analyzer, (3) the size and stiffness of 3D HconF spheroids, and (4) the mRNA expression of genes that encode for extracellular matrix (ECM) molecules including collagen (COL)1, 4 and 6, and fibronectin (FN), α-smooth muscle actin (α-SMA), ER stress related genes including the X-box binding protein-1 (XBP1), the spliced XBP1 (sXBP1) glucose regulator protein (GRP)78, GRP94, and the CCAAT/enhancer-binding protein homologous protein (CHOP), hypoxia inducible factor 1α (HIF1α), and Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α). In the presence of BAC, even at low concentrations at 10−5% or 10−4%, the maximal respiratory capacity, mitochondrial respiratory reserve, and glycolytic reserve of HconF cells were significantly decreased, although the barrier functions of 2D HconF monolayers, the physical properties of the 3D HconF spheroids, and the mRNA expression of the corresponding genes were not affected. The findings reported herein highlight the fact that BAC, even such low concentrations, may induce unfavorable adverse effects on the cellular metabolic capacity of the human conjunctiva.

Anti-Inflammatory Activity of Soluble Epoxide Hydrolase Inhibitors Based on Selenoureas Bearing an Adamantane Moiety.

The inhibitory potency of the series of inhibitors of the soluble epoxide hydrolase (sEH) based on the selenourea moiety and containing adamantane and aromatic lipophilic groups ranges from 34.3 nM to 1.2 μM. The most active compound 5d possesses aliphatic spacers between the selenourea group and lipophilic fragments. Synthesized compounds were tested against the LPS-induced activation of primary murine macrophages. The most prominent anti-inflammatory activity, defined as a suppression of nitric oxide synthesis by LPS-stimulated macrophages, was demonstrated for compounds 4a and 5b. The cytotoxicity of the obtained substances was studied using human neuroblastoma and fibroblast cell cultures. Using these cell assays, the cytotoxic concentration for 4a was 4.7–18.4 times higher than the effective anti-inflammatory concentration. The genotoxicity and the ability to induce oxidative stress was studied using bacterial lux-biosensors. Substance 4a does not exhibit genotoxic properties, but it can cause oxidative stress at concentrations above 50 µM. Put together, the data showed the efficacy and safety of compound 4a.

All Trans-Retinoic Acids Facilitate the Remodeling of 2D and 3D Cultured Human Conjunctival Fibroblasts.

Vitamin A derivative, all-trans-retinoic acid (ATRA), is known to be a potent regulator of the growth and differentiation of various types of cells. In the present study, the unidentified effects of ATRA on superficial and vertical spreading conjunctival scarring were examined. The study involved the use of two-dimensional (2D) and three-dimensional (3D) cultures of human conjunctival fibroblast (HconF) cells in the presence or absence of TGF-β2. The effects of ATRA (1 μM) on superficial or vertical spreading conjunctival scarring were evaluated by the barrier function by trans-endothelial electrical resistance (TEER) and FITC dextran permeability measurements and real-time metabolic analysis, as well as the physical properties, namely, the size and stiffness, of 3D spheroids, respectively. In addition, the expressions of several related molecules, including extracellular matrix (ECM) molecules, ECM modulators including a tissue inhibitor of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs), and ER stress-related factors, were examined. ATRA significantly induced (1) an increase in TEER values and a decrease in FITC dextran permeability, respectively, in the 2D monolayers, and (2) relatively and substantially increased the size and stiffness, respectively, of the 3D spheroids. These ATRA-induced effects were further enhanced in the TGF-β2-treated cells, whereas the TGF-β2-induced enhancement in glycolytic capacity was canceled by the presence of ATRA. Consistent with these physical and morphological effects, the mRNA expressions of several molecules were significantly but differently induced between 2D and 3D cultures by ATRA, although the presence of TGF-β2 did not substantially affect these gene expression levels. The findings reported in this study indicate that ATRA may exacerbate both superficial and vertical conjunctival fibrosis spreading independently of TGF-β2-induced changes.

Evaluation of NACA and diNACA in human cystinosis fibroblast cell cultures as potential treatments for cystinosis

BackgroundCystinosis is a rare autosomal recessive lysosomal storage disease, associated with high morbidity and mortality. Mutations in the CTNS gene disable a membrane protein responsible for the transport of cystine out of the lysosome. Loss of transporter function leads to intralysosomal cystine accumulation and long-term damage to various tissues and organs, including the kidneys, eyes, liver, muscles, pancreas, and brain. The only cystine-depletion therapy for treatment of cystinosis is cysteamine which requires frequent administration of high doses and often causes gastrointestinal pain as well as pungent sulfurous odor in patients. The current in vitro study evaluated antioxidants, N-acetylcysteine amide (NACA; NPI-001) and (2R,2R′)-3,3′-disulfanediyl bis(2-acetamidopropanamide) (diNACA; NPI-002), as potential treatments for cystinosis.MethodsCytotoxicity of cysteamine, NACA and diNACA was evaluated in cultured human cystinotic fibroblasts (HCFs). HCFs were cultured in 96 well plates incubated for 0–72 h in the presence of 25, 50 or 75 μM each of either cysteamine, NACA or diNACA along with an untreated control. Media was removed and cell viability assessed. Next, cystine-depleting activities of cysteamine, NACA and diNACA were screened in HCFs cell culture utilizing an inexpensive, proven colorimetric assay. HCFs were seeded and allowed to reach approximately 80% confluence before the addition of the test articles: 50 μM of either cysteamine, NACA or diNACA in media along with an untreated control. HCFs were incubated, harvested, and cystine was reduced to cysteine, the concentration of which was then determined per quantity of protein compared to a cysteine standard. Statistically significant cystine depletion was determined by paired t-test versus untreated control (p < 0.05).ResultsNeither cysteamine, NACA nor diNACA at 25, 50 or 75 μM caused cytotoxicity in HCFs. Treatment with all tested concentrations (25, 50 or 75 µM) of either NACA or diNACA at 48 or 72 h resulted in statistically significant increases in cell viability, relative to untreated control, whereas the higher concentrations (50 or 75 µM) of cysteamine achieved statistical significance at both timepoints but not the lowest concentration (25 µM). All test articles depleted cystine from HCFs compared to control. NACA depletion of cystine was statistically superior to cysteamine at 6, 24 and 48 h and numerically greater at 72 h. DiNACA depletion of cystine was statistically superior to cysteamine at 6 and 48 h, slightly numerically greater at 24 h and slightly less at 72 h.ConclusionsNACA and diNACA were non cytotoxic to HCFs and significantly increased cell viability. Cystine reduction was determined as percent of control after incubation with 50 µM of NACA, diNACA or cysteamine in HCFs cell culture for 6, 24, 48 and 72 h. Of the three test articles, NACA exhibited most rapid and greatest potency in cystine reduction. Rank order potency for cystine reduction over time was observed, NACA > diNACA ≥ cysteamine. Therefore, further study of NACA and diNACA as potential treatments for cystinosis is warranted.

Primary results of replacement the experimental hyaline cartilage defect with human fibroblast cell culture

The hyaline cartilage damage problem is actual, but existing recovery methods are not fully sufficient. A promising solution of this problem can be the implantation of a tissue-engineered structure (TEC), consisting of a cell culture and a biodegradable scaffold. In this experiment we created a construct consisting of a polylactide scaffold and a human dermal fibroblasts culture. After implantation of TEC in the modeled defect area we analyzed the experimental group regeneration results in comparison to the control group.

EFFICIENCY OF PHOTON CAPTURE BEAM TECHNOLOGY AND PHOTODYNAMIC IMPACT ON MALIGNANT AND NORMAL HUMAN CELLS IN VITRO.

to investigate the structural and morphofunctional changes in test systems of malignant (cell lineA549) and normal (stem fibroblasts) human cells exposed to X-rays in the presence of gadoliniumcontaining photon capture agent «Dotavist» and optical light (red spectrum) in combination with «Fotolon» photosensitizer. The continuous cell culture of normal human fibroblasts and malignant human cells technology, X-ray and red light exposure, cytological and statistical methods. Effects of the two binary radiation technologies, namely the photon capture impact on malignant cells(human nonsmall cell lung cancer cells i.e. line A-549) and normal cells (human stem fibroblasts) when incubated with gadoliniumcontaining photon capture agent «Dotavist» and photodynamic effect in the presence of «Fotolon» photosensitizer applied separately and in combination were studied in a comparative mode. Proceeding from morphofunctional characteristics (growth kinetics, proliferative and mitotic activity) of the abovementioned test systems, peculiarities of the effect on malignant and normal cells were established. Irradiation with X-rays to the 1.0, 5.0, and 10.0 Gy doses resulted in inactivation of respectively 10 %, 46 %, and 80% of the A-549 line malignant cells.Cellular irradiation to a 1.0 Gy dose in the presence of the photon capture agent «Dotavist» (10 μl/ml concentration) inhibited cell proliferation by 50 %, suppressing their mitotic activity. At a dose of 10.0 Gy in the presence of «Dotavist» the inhibition by 93 % of the growth and division of malignant cells occurred, indicating the high efficiency of binary radiation technology. The effect of two binary radiation technologies on malignant human cells (A-549 line), namely the combination of red light with «Fotolon» (0.05 mg/ml concentration) and X-ray exposure in the above doses with «Dotavist» (10 μl/ ml concentration) resulted in the death of respectively 64 %, 86 %, and 99% malignant cells. The culture of normal fibroblasts was found being more sensitive to the influence of a complex of binary radiation impact, as exposure to a dose of 10.0 Gy in the presence of «Dotavist» and «Fotolon» inactivated 100 % of cells. The obtained results provide basis of preclinical evaluation of effectiveness of the combined impact of two binary technologies and drugs used in the photon capture technology and photodynamic therapy i.e. the photon capture agent «Dotavist» and «Fotolon» photosensitizer respectively.

Exploring the Effects of 630 nm Wavelength of Light-Emitting Diode Irradiation on the Proliferation and Migration Ability of Human Biceps Tendon Fibroblast Cells.

Light-emitting diode (LED)-based photobiomodulation is used as an inducer of cell regeneration. Although numerous in vitro and in vivo orthopedic studies have been conducted, the ideal LED wavelength range for tendon healing has not yet been determined. This study, thus, focused on the effects of LED of a 630 nm wavelength on the cell viability, proliferation, and migration of human biceps tendon fibroblast cells. Human tendon fibroblast cell culture was performed using the biceps tendon of patients who had undergone biceps tenodesis. Human biceps tendon fibroblasts from two patients (male, aged 42 and 69 years) were isolated and cultured. The cell type was confirmed by a morphological analysis and using tendon and fibroblast specific markers. They were then split into three groups, with each receiving a different irradiation treatment: no LED treatment (control), 630 nm LED, and 630 nm + 880 nm LED for 20 minutes each. After the LED treatment, cell viability, proliferation, and migration assays were performed, and the results were compared between the groups. Twenty-four hours after LED treatment, cell viability and proliferation were significantly increased in the 630 nm LED and 630 nm + 880 nm LED treatment groups compared to that in the control group (p < 0.05). Under the same conditions, compared with the control group, the 630 nm LED alone treatment group showed a 3.06 ± 0.21 times higher cell migration rate (p < 0.05), and the 630 nm + 880 nm LED combination treatment group showed a 2.88 ± 0.20 times higher cell migration rate (p < 0.05) in three-dimensional migration assay. In human tendon fibroblast cells, 20 minutes of LED treatment at 630 nm and 630 nm + 880 nm exhibited significant effects on cell proliferation and migration. Our findings suggest the potential of LED therapy as an adjuvant treatment for tendon healing, and hence, further research is warranted to standardize the various parameters to further develop and establish this as a reliable treatment regimen.