Human Fibroblast Cultures Research Articles

Tension in the ranks: Cooperative cell contractions drive force-dependent collagen assembly in human fibroblast culture

Tension in the ranks: Cooperative cell contractions drive force-dependent collagen assembly in human fibroblast culture

Phytocannabinoids and gingival inflammation: Preclinical findings and a placebo-controlled double-blind randomized clinical trial with cannabidiol.

The aim of this study was to: (1) evaluate the anti-inflammatory effects of cannabidiol (CBD) on primary cultures of human gingival fibroblasts (HGFs) and (2) to clinically monitor the effect of CBD in subjects with periodontitis. The use of phytocannabinoids is a new approach in the treatment of widely prevalent periodontal disease. Cannabinoid receptors were analyzed by western blot and interleukin production detected using enzyme immunoassay. Activation of the Nrf2 pathway was studied via monitoring the mRNA level of heme oxygenase-1. Antimicrobial effects were determined by standard microdilution and 16S rRNA screening. In the clinical part, a placebo-control double-blind randomized study was conducted (56 days) in three groups (n = 90) using dental gel without CBD (group A) and with 1% (w/w) CBD (group B) and corresponding toothpaste (group A - no CBD, group B - with CBD) for home use to maintain oral health. Group C used dental gel containing 1% chlorhexidine digluconate (active comparator) and toothpaste without CBD. Human gingival fibroblasts were confirmed to express the cannabinoid receptor CB2. Lipopolysaccharide-induced cells exhibited increased production of pro-inflammatory IL-6 and IL-8, with deceasing levels upon exposure to CBD. CBD also exhibited antimicrobial activities against Porphyromonas gingivalis, with an MIC of 1.5 μg/mL. Activation of the Nrf2 pathway was also demonstrated. In the clinical part, statistically significant improvement was found for the gingival, gingival bleeding, and modified gingival indices between placebo group A and CBD group B after 56 days. Cannabidiol reduced inflammation and the growth of selected periodontal pathogenic bacteria. The clinical trial demonstrated a statistically significant improvement after CBD application. No adverse effects of CBD were reported by patients or observed upon clinical examination during the study. The results are a promising basis for a more comprehensive investigation of the application of non-psychotropic cannabinoids in dentistry.

Macromolecular crowding in human tenocyte and skin fibroblast cultures: A comparative analysis

Although human tenocytes and dermal fibroblasts have shown promise in tendon engineering, no tissue engineered medicine has been developed due to the prolonged ex vivo time required to develop an implantable device. Considering that macromolecular crowding has the potential to substantially accelerate the development of functional tissue facsimiles, herein we compared human tenocyte and dermal fibroblast behaviour under standard and macromolecular crowding conditions to inform future studies in tendon engineering. Basic cell function analysis made apparent the innocuousness of macromolecular crowding for both cell types. Gene expression analysis of the without macromolecular crowding groups revealed expression of tendon related molecules in human dermal fibroblasts and tenocytes. Protein electrophoresis and immunocytochemistry analyses showed significantly increased and similar deposition of collagen fibres by macromolecular crowding in the two cell types. Proteomics analysis demonstrated great similarities between human tenocyte and dermal fibroblast cultures, as well as the induction of haemostatic, anti-microbial and tissue-protective proteins by macromolecular crowding in both cell populations. Collectively, these data rationalise the use of either human dermal fibroblasts or tenocytes in combination with macromolecular crowding in tendon engineering.

Comparison between Platelet Lysate, Platelet Lysate Serum, and Fetal Bovine Serum as Supplements for Cell Culture, Expansion, and Cryopreservation.

As cell culture supplements, human platelet lysate (PL) and human platelet lysate serum (PLS) are alternatives to fetal bovine serum (FBS) due to FBS-related issues such as ethical concerns, variability between batches, and the possible introduction of xenogenic contaminants. This study compared the composition and efficacy of PL, PLS, and FBS as supplements in the culture and cryopreservation of human dermal fibroblasts, Wharton's jelly-derived mesenchymal stem cells (WJ-MCS), and adipose tissue (AdMSC). Biochemical components, some growth factors, and cytokines present in each of them were analyzed; in addition, the cells were cultured in media supplemented with 5% PL, 5% PLS, and 10% FBS and exposed to different freezing and thawing solutions with the supplements under study. Biochemical parameters were found to be similar in PL and PLS compared to FBS, with some differences in fibrinogen and calcium concentration. Growth factors and cytokines were higher in PL and PLS compared to FBS. Cell proliferation and morphology showed no significant differences between the three culture media. Regarding the cryopreservation and thawing of cells, better results were obtained with PLS and FBS. In conclusion, PL and PLS are an excellent choice to replace the standard supplement of animal origin (FBS) in the media used for the culture and cryopreservation of fibroblasts, WJ-MSC, and AdMSC.

Applicability of Electron-Beam and Hybrid Plasmas for Polyethylene Terephthalate Processing to Obtain Hydrophilic and Biocompatible Surfaces.

The applicability of beam-plasma chemical reactors generating cold hybrid plasma for the production of noncytotoxic polymeric surfaces with high hydrophilicity and good biocompatibility with human fibroblast culture and human red blood cells was studied. Oxygen hybrid plasma was excited by the joint action of a continuous scanning electron beam and a capacity-coupled RF-gas discharge. Experiments showed that hybrid plasma treatment caused polar oxygen-containing functional group formation in the surface layer of poly (ethylene terephthalate) films. No thermal or radiative damage in tested polymer samples was found. The plasma-modified polymers turned out to be noncytotoxic and revealed good biocompatibility with human fibroblasts BJ-5ta as well as lower hemolytic activity than untreated poly (ethylene terephthalate). Experiments also demonstrated that no phenomena caused by the electrostatic charging of polymers occur in hybrid plasma because the electron beam component of hybrid plasma eliminates the item charge when it is treated. The electron beam can effectively control the reaction volume geometry as well as the fluxes of active plasma particles falling on the item surface. This provides new approaches to the production of abruptly structured patterns or smooth gradients of functionalities on a plane and 3D polymeric items of complicated geometry.

Enhancing wound healing and overcoming cisplatin resistance in ovarian cancer.

Ovarian cancer (OC) poses significant oncological challenges, notably impaired wound healing in the context of cisplatin (DDP) resistance. This study investigates the role of miR-200b in OC, emphasizing its impact on wound healing processes through DNMT3A/TGF-β1 pathway. The primary aim was to explore how miR-200b regulates autophagy and its consequential effects on wound healing in OC, alongside its influence on cisplatin resistance. Utilizing DDP-sensitive (A2780) and resistant (A2780/DDP) OC cell lines, along with human fibroblast cultures, the study employed an array of in vitro techniques. These included cell transfection with miR-200b mimic or inhibitor, chromatin immunoprecipitation (ChIP), dual-luciferase reporter (DLR) assays, quantitative PCR, Western blotting, MTT and particularly, wound healing assays. The research highlighted the role of miR-200b in wound healing within OC. Inhibition of miR-200b in A2780 cells and its mimic in A2780/DDP cells affected cell viability, indicating the link with DDP resistance. Crucially, miR-200b mimic significantly delayed fibroblast-mediated wound closure in assays, underscoring its impact on wound healing. Bioinformatics analysis and subsequent DLR assays confirmed miR-200b's interaction with DNMT3A, affecting TGF-β1 expression, the key factor in wound repair. Further, ChIP, quantitative PCR and Western blot analyses validated the interaction and expression changes in DNMT3A and TGF-β1. The study demonstrated that miR-200b played a pivotal role in OC by modulating autophagy, which in turn significantly affected wound healing through the DNMT3A/TGF-β1 pathway.

Lipid Metabolism Regulators Are the Possible Determinant for Characteristics of Myopic Human Scleral Stroma Fibroblasts (HSSFs).

The purpose of the current investigation was to elucidate what kinds of responsible mechanisms induce elongation of the sclera in myopic eyes. To do this, two-dimensional (2D) cultures of human scleral stromal fibroblasts (HSSFs) obtained from eyes with two different axial length (AL) groups, <26 mm (low AL group, n = 2) and >27 mm (high AL group, n = 3), were subjected to (1) measurements of Seahorse mitochondrial and glycolytic indices to evaluate biological aspects and (2) analysis by RNA sequencing. Extracellular flux analysis revealed that metabolic indices related to mitochondrial and glycolytic functions were higher in the low AL group than in the high AL group, suggesting that metabolic activities of HSSF cells are different depending the degree of AL. Based upon RNA sequencing of these low and high AL groups, the bioinformatic analyses using gene ontology (GO) enrichment analysis and ingenuity pathway analysis (IPA) of differentially expressed genes (DEGs) identified that sterol regulatory element-binding transcription factor 2 (SREBF2) is both a possible upstream regulator and a causal network regulator. Furthermore, SREBF1, insulin-induced gene 1 (INSIG1), and insulin-like growth factor 1 (IGF1) were detected as upstream regulators, and protein tyrosine phosphatase receptor type O (PTPRO) was detected as a causal network regulator. Since those possible regulators were all pivotally involved in lipid metabolisms including fatty acid (FA), triglyceride (TG) and cholesterol (Chol) biosynthesis, the findings reported here indicate that FA, TG and Chol biosynthesis regulation may be responsible mechanisms inducing AL elongation via HSSF.

Biological Effects of "Inflammageing" on Human Oral Cells: Insights into a Potential Confounder of Age-Related Diseases.

The term "inflammageing" describes the process of inflammation-induced aging that leads living cells to a state of permanent cell cycle arrest due to chronic antigenic irritation. This in vitro study aimed to shed light on the mechanisms of "inflammageing" on human oral cells. Primary cultures of human gingival fibroblasts (hGFs) were exposed to variable pro-inflammatory stimuli, including lipopolysaccharide (LPS), Tumor Necrosis Factor-alpha (TNFa), and gingival crevicular fluid (GCF) collected from active periodontal pockets of systemically healthy patients. Inflammageing was studied through two experimental models, employing either late-passage ("aged") cells (p. 10) that were exposed to the pro-inflammatory stimuli or early-passage ("young") cells (p. 1) continuously exposed during a period of several passages (up to p. 10) to the above-mentioned stimuli. Cells were evaluated for the expression of beta-galactosidase activity (histochemical staining), senescence-associated genes (qPCR analysis), and biomarkers related to a Senescence-Associated Secretory Phenotype (SASP), through proteome profile analysis and bioinformatics. A significant increase (p < 0.05) in beta-galactosidase-positive cells was observed after exposure to each pro-inflammatory stimulus. The senescence-associated gene expression included upregulation for CCND1 and downregulation for SUSD6, and STAG1, a profile typical for cellular senescence. Overall, pro-inflammatory priming of late-passage cells caused more pronounced effects in terms of senescence than long-term exposure of early-passage cells to these stimuli. Proteomic analysis showed induction of SASP, evidenced by upregulation of several pro-inflammatory proteins (IL-6, IL-10, IL-16, IP-10, MCP-1, MCP-2, M-CSF, MIP-1a, MIP-1b, TNFb, sTNF-RI, sTNF-RII, TIMP-2) implicated in cellular aging and immune responses. The least potent impact on the induction of SASP was provoked by LPS and the most pronounced by GCF. This study demonstrates that long-term exposure of hGFs to various pro-inflammatory signals induced or accelerated cellular senescence with the most pronounced impact noted for the late-passage cells. The outcome of these analyses provides insights into oral chronic inflammation as a potential confounder of age-related diseases.

Achatina fulica Mucus Ameliorates UVB-induced Human Dermal Fibroblast Photoaging via the TGF-β/Smad Pathway

BACKGROUND: Ultraviolet B (UVB) induces skin photoaging by reducing collagen deposition via impairment of the TGF-β/Smad signaling pathway. Achatina fulica mucus (AFM) is a native medicine acting as vehicle of anti-aging ingredients. The present investigation examined the effect of AFM on UVB-induced fibroblast photoaging by assessing TGF-β, Smad3, and Smad7 mRNA expressions.METHODS: AFM was extracted from A. fulica using electrical shock and freeze-dried into a powder. Normal human dermal fibroblast (NHDF) cultures were irradiated with/without 100 mJ/cm2 UVB and treated with/without 10% platelet-rich plasma or different concentrations of AFM: 3.9 μg/mL in AF3 group; 15.625 μg/mL in AF15 group, and 62.5 μg/mL in AF62 group. The mRNA expressions of TGF-β, Smad3, and Smad7 in NHDF were evaluated by quantitative polymerase chain reaction.RESULTS: TGF-β mRNA expressions in the AF3 (0.85±0.01), AF15 (0.94±0.02) and AF62 (1.64±0.03) groups were significantly higher (p&lt;0.05) compared with that in the UVB group (0.55±0.04). Moreover, Smad3 expressions in the AF3 (1.42±0.25), AF15 (1.89±0.13), and AF62 (2.50±0.31) groups were significantly higher (p&lt;0.05) compared with that in the UVB group (0.57±0.08). Furthermore, Smad7 expressions in the AF3 (1.57±0.18), AF15 (0.87±0.03), and AF62 (0.25±0.09) groups were significantly lower (p&lt;0.05) than that in the UVB group (2.57±0.06).CONCLUSION: AFM ameliorates UVB-induced fibroblast photoaging by upregulating the TGF-β/Smad3 expressions and downregulating Smad7 expression.KEYWORDS: Achatina fulica, TGF-β, Smad, collagen, UVB, fibroblast, photoaging

Ascorbic Acid Enhances the Metabolic Activity, Growth and Collagen Production of Human Dermal Fibroblasts Growing in Three-dimensional (3D) Culture

Tissue engineering (TE) enables the development of functional synthetic substitutes to be replaced with damaged tissues and organs instead of the use of auto or allografts. A wide range of biomaterials is currently in use as TE scaffolds. Among these materials, naturally sourced ones are favorable due to being highly biocompatible and supporting cell growth and function, whereas synthetic ones are advantageous because of the high tunability on mechanical and physical properties as well as being easy to process. Alongside the advantages of synthetic polymers, they mostly show hydrophobic behavior that limits biomaterial-cell interaction and, consequently, the functioning of the developed TE constructs. In this study, we assessed the impact of L-Ascorbic acid 2-phosphate (AA2P) on improving the culture conditions of human dermal fibroblasts (HDFs) growing on a three-dimensional (3D) scaffold made of polycaprolactone (PCL) using emulsion templating. Our results demonstrated that AA2P enhances the metabolic activity and growth of HDFs as well as collagen deposition by them when supplemented in their growth medium at 50 µg/mL concentration. It showed a great potential to be used as a growth medium supplement to circumvent the disadvantages of culturing human cells on a synthetic biomaterial that is not favored in default. AA2P's potential to improve cell growth and collagen deposition may prove an effective way to culture human cells on 3D PCL PolyHIPE scaffolds for various TE applications.

Antifibrotic properties of hyaluronic acid crosslinked polyisocyanide hydrogels

Fibrosis is characterized by the formation of fibrous connective tissue in response to primary injury. As a result, an affected organ may lose part of its functionality due to chronic, organ-specific tissue damage. Since fibrosis is a leading cause of death worldwide, targeting fibrotic diseases with antifibrotic hydrogels can be a lifesaving therapeutic strategy. This study developed a novel hybrid antifibrotic hydrogel by combining the synthetic polyisocyanide (PIC) with hyaluronic acid (HA). Gels of PIC are highly tailorable, thermosensitive, and strongly biomimetic in architecture and mechanical properties, whereas HA is known to promote non-fibrotic fetal wound healing and inhibits inflammatory signaling. The developed HA-PIC hybrids were biocompatible with physical properties comparable to those of the PIC gels. The antifibrotic nature of the gels was assessed by 3D cultures of human foreskin fibroblasts in the presence (or absence as control) of TGFβ1 that promotes differentiation into myofibroblasts, a critical step in fibrosis. Proliferation and macroscopic contraction assays and studies on the formation of stress fibers and characteristic fibrosis markers all indicate a strong antifibrotic nature of HA-PIC hydrogel. We showed that these effects originate from both the lightly crosslinked architecture and the presence of HA itself. The hybrid displaying both these effects shows the strongest antifibrotic nature and is a promising candidate for use as in vivo treatment for skin fibrosis.

Antioxidant, Anti-Inflammatory Effects and Ability to Stimulate Wound Healing of a Common-Plantain Extract in Alginate Gel Formulations.

Our study aimed to investigate the biological effects of a common-plantain (Plantago major L.) extract, encapsulated in alginate, on dermal human fibroblast cultures in vitro, in view of its potential use as a wound healing adjuvant therapy. Common-plantain extracts were obtained by infusion and ultrasound extraction, and their total polyphenolic content and antioxidant capacity were determined by spectrophotometry. The best extract, which was obtained by infusion, was further encapsulated in sodium alginate in two different formulations. Fourier Transform Infrared Spectroscopy (FTIR) was used to demonstrate the existing interactions in the obtained common-plantain extract in the alginate formulations. The encapsulation efficiency was evaluated based on the total polyphenol content. These alginate gel formulations were further used in vitro to determine their biocompatibility and antioxidant and anti-inflammatory effects by spectrophotometry and ELISA, as well as their ability to stimulate fibroblast migration (scratch test assay) at different time points. In addition, the collagen 1 and 3 levels were determined by Western blot analysis. The data showed that the microencapsulated plantain extract formulations induced an antioxidant, anti-inflammatory effect, enhanced collagen production and increased wound closure in the first 8 h of their application. These results are encouraging for the use of this alginate plantain extract formulation as an adjuvant for skin wound healing.

3-D culture of human lung fibroblasts decreases proliferative and increases extracellular matrix remodeling genes.

Fibroblasts are the main producers of extracellular matrix (ECM) responsible for ECM maintenance and repair, a process often disrupted in chronic lung diseases. The accompanying mechanical changes adversely affect resident cells and overall lung function. Numerous models have been used to elucidate fibroblast behavior that are now evolving toward complex three-dimensional (3-D) models incorporating ECM, aiming to replicate the cells' native environment. Little is known about the cellular changes that occur when moving from two-dimensional (2-D) to 3-D cell culture. This study compared the gene expression profiles of primary human lung fibroblasts from seven subjects with normal lung function, that were cultured for 24 h on 2-D collagen I-coated tissue culture plastic and in 3-D collagen I hydrogels, which are commonly used to mimic ECM in various models, from contraction assays to intricate organ-on-a-chip models. Comparing 3-D with 2-D cell culture, 6,771 differentially expressed genes (2,896 up, 3,875 down) were found; enriched gene sets within the downregulated genes, identified through Gene Set Enrichment Analysis and Ingenuity Pathway Analysis, were involved in the initiation of DNA replication which implied downregulation of fibroblast proliferation in 3-D. Observation of cells for 72 h in 2-D and 3-D environments confirmed the reduced progression through the cell cycle in 3-D. A focused analysis, examining the Hippo pathway and ECM-associated genes, showed differential patterns of gene expression in the 3-D versus 2-D culture. Altogether, the transcriptional response of fibroblasts cultured in 3-D indicated inhibition of proliferation, and alterations in Hippo and ECM pathways indicating a complete switch from proliferation to ECM remodeling.NEW & NOTEWORTHY With the introduction of complex three-dimensional (3-D) lung models, comes a need for understanding cellular behavior in these models. We compared gene expression profiles of human lung fibroblasts grown on two-dimensional (2-D) collagen I-coated surfaces with those in 3-D collagen I hydrogels. RNA sequencing and subsequent pathway analyses showed decreased proliferation, increased extracellular matrix (ECM) remodeling, and altered Hippo signaling and ECM deposition-related gene signatures. These findings highlight unique responses of fibroblasts in 3-D models.

Retraction Note to: Glycosaminoglycans reduce oxidative damage induced by copper (Cu+2), iron (Fe+2) and hydrogen peroxide (H2O2) in human fibroblast cultures.

Retraction Note to: Glycosaminoglycans reduce oxidative damage induced by copper (Cu+2), iron (Fe+2) and hydrogen peroxide (H2O2) in human fibroblast cultures.

Human macrophage immunometabolism regulator (MACIR) in patients with periodontitis

ObjectivePeriodontitis is a local inflammatory reaction caused by bacterial infection in which immune cells, including macrophages, are involved. Recent studies have shown that an important regulator of macrophage function is the human macrophage immunometabolism regulator (MACIR). This gene has been shown to play a key role in modulating the immune response by affecting the activity of fibroblasts and macrophages.In this study, we investigated the expression of MACIR in the gingival tissues of patients with periodontal disease, as well as the effect of IL-1β and TNF-α on the expression of MACIR gene and protein in human gingival fibroblasts. MethodsMACIR mRNA expression in gingival tissue samples was determined using Real-time PCR. Expression of MACIR protein was determined using immunofluorescent staining and western blotting. ResultsThe MACIR mRNA expression in gingival tissue samples in patients with periodontitis was statistically significantly lower than in gingival tissue samples from healthy controls (p = 0.009). The stimulation of human gingival fibroblasts with IL-1β and TNF-α resulted in a statistically significant decrease of MACIR gene mRNA expression. In western blotting and immunofluorescent analysis, we confirmed that the stimulation of the primary culture of human gingival fibroblasts by both IL-1β and TNF-α decreases the expression of MACIR protein. ConclusionThe results of the study suggest that MACIR is an important regulator of the inflammatory process in patients with periodontitis. Decreased expression of the MACIR gene may activate macrophages to secrete mediators that increase inflammation and cause periodontal tissue destruction.

Features of the functioning of the innate and adaptive immunity system in patients with COVID-19 of the older age group

Goal. Characteristics of innate, cellular and adaptive immunity in patients of the older age group with COVID19.Materials and methods. Blood leukocytes were induced by Newcastle disease virus (α-interferon), phytohemagglutinin (γ-interferon), SARS CoV 2: RBD antigens and S-protein; interferon activity in human fibroblast culture and enzyme immunoassay were evaluated. In serum, IgG antibodies to SARS CoV2 and autoantibodies to interferon and to the endothelium of blood vessels were determined using a mono-layer of human umbilical vein cells. Statistical processing was performed in Excel 2016.Results. A decrease in the production of α-interferon and γ-interferon was revealed: 1 week -74.2±15.1; 3 week-144.0±35.7 (p=0.01); control – 266.6 ±82 (relative to 3 weeks p=0.004) and IFN γ: 1 week -6.8±2; 3 week – 14.4 ±3.5 (p=0.03); control – 28.87.15 (relative to 3 weeks (p=0.007). Decreased production of γ-interferon by leukocytes of patients with induction by SARS CoV2 RBD and S-trimer anti-gens was revealed. Antibodies to SARS CoV2 were detected starting from the 2nd week of the disease, a large spread of indicators was noted. Autoantibodies to α2-interferon and to vascular surface antigens were detected.Conclusion. The state of innate immunity in patients of the older age group with severe and moderate COVID-19 was characterized by a decrease in the activity of the interferon system. Decreased activity of cellular immunity to SARS CoV2 antigens was noted. Adaptive immunity was characterized by the development of an imbalance in the form of the appearance of autoantibodies to α-interferon and vascular endothelium.

Comparative analysis of KnockOut serum replacement and fetal bovine serum for in vitro culture of human dermal fibroblasts

BACKGROUNDHuman dermal fibroblast (HDF) cultures can be used as a regenerative agent for wound healing. Fetal bovine serum (FBS) as a culture supplement is derived from animals, therefore not being constant in composition, causes variations in research results, thus requiring a substitute such as KnockOut serum replacement (KOSR). This study evaluated the defined KOSR as FBS substitute for HDF culture by measuring the relative expression of basic fibroblast growth factor (bFGF) and keratinocyte growth factor (KGF) messenger RNA (mRNA), HDF cell proliferation, and HDF migration. METHODSHuman dermal fibroblast culture was divided into 2 intervention groups receiving KOSR 5% and KOSR 10%, respectively, and a control group receiving FBS 10%. Reverse transcription polymerase chain reaction (RT-PCR) was used for bFGF and KGF mRNA relative expression at the fifth passage (P5). Cell counting kit-8 (CCK-8) reagent was used for the HDF cell proliferation assay at P5 and the scratch assay was used for HDF cell migration at P6. Data were analyzed using dependent t-test, One-way ANOVA, or Kruskal-Wallis test. RESULTSThere were no significant differences in bFGF and KGF mRNA relative expression and HDF migration velocity between the intervention and control groups (p&gt;0.05 and p&gt;0.05, respectively). The doubling time of the KOSR 5% group showed no significant difference (p&gt;0.05), but KOSR 10% and FBS 10% showed significant differences between treatment days 2-6 and treatment days 6-10 (p&lt;0.05). CONCLUSIONSThe KOSR 10% was comparable to FBS 10% in supporting bFGF and KGF mRNA relative expression, HDF cell proliferation, and HDF cell migration in HDF culture.

Exploration of SUMO2/3 Expression Levels and Autophagy Process in Fragile X-Associated Tremor/Ataxia Syndrome: Addressing Study Limitations and Insights for Future Research.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder that appears in adult FMR1 premutation carriers. The neuropathological hallmark of FXTAS is an intranuclear inclusion in neurons and astrocytes. Nearly 200 different proteins have been identified in FXTAS inclusions, being the small ubiquitin-related modifier 2 (SUMO2), ubiquitin and p62 the most highly abundant. These proteins are components of the protein degradation machinery. This study aimed to characterize SUMO2/3 expression levels and autophagy process in human postmortem brain samples and skin fibroblast cultures from FXTAS patients. Results revealed that FXTAS postmortem brain samples are positive for SUMO2/3 conjugates and supported the idea that SUMO2/3 accumulation is involved in inclusion formation. Insights from RNA-sequencing data indicated that SUMOylation processes are significantly upregulated in FXTAS samples. In addition, the analysis of the autophagy flux showed the accumulation of p62 protein levels and autophagosomes in skin fibroblasts from FXTAS patients. Similarly, gene set analysis evidenced a significant downregulation in gene ontology terms related to autophagy in FXTAS samples. Overall, this study provides new evidence supporting the role of SUMOylation and autophagic processes in the pathogenic mechanisms underlying FXTAS.

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.

Development of a Self-Assembled Dermal Substitute from Human Fibroblasts Using Long-term Three-Dimensional Culture.

Skin substitutes have emerged as an alternative to autografts for the treatment of skin defects. Among them, scaffold-based dermal substitutes have been extensively studied; however, they have certain limitations, such as delayed vascularization, limited elasticity, and the inability to achieve permanent engraftment. Self-assembled, cell-based dermal substitutes are a promising alternative that may overcome these shortcomings but have not yet been developed. In this study, we successfully developed a cell-based dermal substitute (cultured dermis) through the long-term culture of human dermal fibroblasts using the net-mold method, which enables three-dimensional cell culture without the use of a scaffold. Spheroids prepared from human dermal fibroblasts were poured into a net-shaped mold and cultured for 2, 4, or 6 months. The dry weight, tensile strength, collagen and glycosaminoglycan levels, and cell proliferation capacity were assessed and compared among the 2-, 4-, and 6-month culture periods. We found that collagen and glycosaminoglycan levels decreased over time, while the dry weight remained unchanged. Tensile strength increased at 4 months, suggesting that remodeling had progressed. In addition, the cell proliferation capacity was maintained, even after a 6-month culture period. Unexpectedly, the internal part of the cultured dermis became fragile, resulting in the division of the cultured dermis into two collagen-rich tissues, each of which had a thickness of 400 μm and sufficient strength to be sutured during in vivo analysis. The divided 4-month cultured dermis was transplanted to skin defects of immunocompromised mice and its wound healing effects were compared to those of a clinically available collagen-based artificial dermis. The cultured dermis promoted epithelialization and angiogenesis more effectively than the collagen-based artificial dermis. Although further improvements are needed, such as the shortening of the culture period and increasing the size of the cultured dermis, we believe that the cultured dermis presented in this study has the potential to be an innovative material for permanent skin coverage.