Human primary cells can tell body time: Dedicated to Steven A. Brown.

The field of chronobiology has advanced significantly since ancient observations of natural rhythms. The intricate molecular architecture of circadian clocks, their hierarchical organization within the mammalian body, and their pivotal roles in organ physiology highlight the complexity and significance of these internal timekeeping mechanisms. In humans, circadian phenotypes exhibit considerable variability among individuals and throughout the individual's lifespan. A fundamental challenge in mechanistic studies of human chronobiology arises from the difficulty of conducting serial sampling from most organs. The concept of studying circadian clocks in vitro relies on the groundbreaking discovery by Ueli Schibler and colleagues that nearly every cell in the body harbours autonomous molecular oscillators. The advent of circadian bioluminescent reporters has provided a new perspective for this approach, enabling high-resolution continuous measurements of cell-autonomous clocks in cultured cells, following in vitro synchronization pulse. The work by Steven A. Brown has provided compelling evidence that clock characteristics assessed in primary mouse and human skin fibroblasts cultured in vitro represent a reliable estimation of internal clock properties in vivo. The in vitro approach for studying molecular human clocks in cultured explants and primary cells, pioneered by Steve Brown, represents an invaluable tool for assessing inter-individual differences in circadian characteristics alongside comprehensive genetic, biochemical and functional analyses. In a broader context, this reliable and minimally invasive approach offers a unique perspective for unravelling the functional inputs and outputs of oscillators operative in nearly any human tissue in physiological contexts and across various pathologies.

Endogenous hydrogen sulphide deficiency and exogenous hydrogen sulphide supplement regulate skin fibroblasts proliferation via necroptosis.

An excessive proliferation of skin fibroblasts usually results in different skin fibrotic diseases. Hydrogen sulphide (H2 S) is regarded as an important endogenous gasotransmitter with various functions. The study aimed to investigate the roles and mechanisms of H2 S on primary mice skin fibroblasts proliferation. Cell proliferation and collagen synthesis were assessed with the expression of α-smooth muscle actin (α-SMA), proliferating cell nuclear antigen (PCNA), Collagen I and Collagen III. The degree of oxidative stress was evaluated by dihydroethidium (DHE) and MitoSOX staining. Mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining. Necroptosis was evaluated with TDT-mediated dUTP nick end labelling (TUNEL) and expression of receptor-interacting protein kinase 1 (RIPK1), RIPK3 and mixed lineage kinase domain-like protein (MLKL). The present study found that α-SMA, PCNA, Collagen I and Collagen III expression were increased, oxidative stress was promoted, ΔΨm was impaired and positive rate of TUNEL staining, RIPK1 and RIPK3 expression as well as MLKL phosphorylation were all enhanced in skin fibroblasts from cystathionine γ-lyase (CSE) knockout (KO) mice or transforming growth factor-ß1 (TGF-ß1, 10 ng/mL)-stimulated mice skin fibroblasts, which was restored by exogenous sodium hydrosulphide (NaHS, 50 µmol/L). In conclusion, endogenous H2 S production impairment in CSE-deficient mice accelerated skin fibroblasts proliferation via promoted necroptosis, which was attenuated by exogenous H2 S. Exogenous H2 S supplement alleviated proliferation of skin fibroblasts with TGF-ß1 stimulation via necroptosis inhibition. This study provides evidence for H2 S as a candidate agent to prevent and treat skin fibrotic diseases.

Tanshinone IIA ameliorates the development of dermal fibrosis in systemic sclerosis.

OBJECTIVES: We previously revealed the role of tanshinone IIA (TAN IIA) on endothelial cells and the impact of TAN IIA on the endothelial-to-mesenchymal transition in systemic sclerosis (SSc). In this study, we sought to further determine whether TAN IIA can directly act on the skin fibroblasts of scleroderma and look into its underlying anti-fibrotic mechanisms. METHODS: Bleomycin was used to establish the SSc mouse model. After TAN IIA treatment, dermal thickness, type I collagen and hydroxyproline content were measured. Primary fibroblasts were acquired from SSc patients and cultured in vitro, and the effects of TAN IIA on proliferation, apoptosis and the cell cycle of fibroblasts were detected. RESULTS: In a bleomycin-induced SSc model, we discovered that TAN IIA significantly improved skin thickness and collagen deposition, demonstrating a potent anti-fibrotic action. TAN IIA inhibits the proliferation of skin fibroblasts derived from SSc patients by causing G2/M cell cycle arrest and promoting apoptosis. Additionally, TAN IIA downregulated extracellular matrix gene transcription and collagen protein expression in skin fibroblasts in a dose-gradient-dependent manner. Furthermore, we showed how TAN IIA can reduce the activation of the transforming growth factor-ß (TGF-ß)/Smad and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways, which are important factors in SSc. CONCLUSIONS: In summary, these data suggest that TAN IIA can reduce SSc-related skin fibrosis by modulating the TGF-ß/Smad and MAPK/ERK signalling pathways. More importantly, our results imply that TAN IIA can directly act on the skin fibroblasts of SSc, therefore, inhibiting fibrosis.

Dermal Fibroblast Cell Line from a Patient with the Huntington's Disease as a Promising Model for Studying Disease Pathogenesis: Production and Characterization.

Huntington's disease (HD) is an incurable hereditary disease caused by expansion of the CAG repeats in the HTT gene encoding the mutant huntingtin protein (mHTT). Despite numerous studies in cellular and animal models, the mechanisms underlying the biological role of mHTT and its toxicity to striatal neurons have not yet been established and no effective therapy for HD patients has been developed so far. We produced and characterized a new line of dermal fibroblasts (HDDF, Huntington's disease dermal fibroblasts) from a patient with a confirmed HD diagnosis. We also studied the growth characteristics of HDDF cells, stained them for canonical markers, karyotyped these cells, and investigated their phenotype. HDDF cells was successfully reprogrammed into induced striatal neurons via transdifferentiation. The new fibroblast line can be used as a cell model to study the biological role of mHTT and manifestations of HD pathogenesis in both fibroblasts and induced neuronal cells obtained from them by reprogramming techniques.

Wound Closure Promotion by Leucine-Based Pseudo-Proteins: An In Vitro Study.

Our research explores leucine-based pseudo-proteins (LPPs) for advanced wound dressings, focusing on their effects on wound healing in an in vitro model. We assessed three types of LPP films for their ability to enhance wound closure rates and modulate cytokine production. They all significantly improved wound closure compared to traditional methods, with the 8L6 and copolymer films showing the most pronounced effects. Notably, the latter exhibited an optimal cytokine profile: an initial burst of pro-inflammatory TNF-α, followed by a controlled release of IL-6 during the proliferative phase and a significant increase in anti-inflammatory IL-10 during remodeling. This balanced cytokine response suggests that the copolymer film not only accelerates wound closure but also supports a well-regulated healing process, potentially reducing fibrosis and abnormal scarring, underscoring the potential of copolymer LPPs as advanced wound dressing materials. Future research will aim to elucidate the specific signaling pathways activated by the copolymer LPP to better understand its mechanism of action. Overall, LPP films offer a promising approach to improving wound care and could lead to more effective treatments for complex wounds.

The Combined Anti-Aging Effect of Hydrolyzed Collagen Oligopeptides and Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells on Human Skin Fibroblasts.

Stem cell-derived exosomes (SC-Exos) are used as a source of regenerative medicine, but certain limitations hinder their uses. The effect of hydrolyzed collagen oligopeptides (HCOPs), a functional ingredient of SC-Exos is not widely known to the general public. We herein evaluated the combined anti-aging effects of HCOPs and exosomes derived from human umbilical cord mesenchymal stem cells (HucMSC-Exos) using a senescence model established on human skin fibroblasts (HSFs). This study discovered that cells treated with HucMSC-Exos + HCOPs enhanced their proliferative and migratory capabilities; reduced both reactive oxygen species production and senescence-associated ß-galactosidase activity; augmented type I and type III collagen expression; attenuated the expression of matrix-degrading metalloproteinases (MMP-1, MMP-3, and MMP-9), interleukin 1 beta (IL-1ß), and tumor necrosis factor-alpha (TNF-α); and decreased the expression of p16, p21, and p53 as compared with the cells treated with HucMSC-Exos or HCOPs alone. These results suggest a possible strategy for enhancing the skin anti-aging ability of HucMSC-Exos with HCOPs.

Dermal Fibroblasts as the Main Target for Skin Anti-Age Correction Using a Combination of Regenerative Medicine Methods.

This article includes the data from current studies regarding the pathophysiological mechanisms of skin aging and the regenerative processes occurring in the epidermis and dermis at the molecular and cellular level, mainly, the key role of dermal fibroblasts in skin regeneration. Analyzing these data, the authors proposed the concept of skin anti-age therapy that is based on the correction of age-related skin changes by stimulating regenerative processes at the molecular and cellular level. The main target of the skin anti-age therapy is dermal fibroblasts (DFs). A variant of the cosmetological anti-age program using the combination of laser and cellular methods of regenerative medicine is presented in the paper. The program includes three stages of implementation and defines the tasks and methods of each stage. Thus, laser technologies allow one to remodel the collagen matrix and create favorable conditions for DFs functions, whereas the cultivated autologous dermal fibroblasts replenish the pool of mature DFs decreasing with age and are responsible for the synthesis of components of the dermal extracellular matrix. Finally, the use of autological platelet-rich plasma (PRP) enables to maintenance of the achieved results by stimulating DF function. It has been shown that growth factors/cytokines contained in α-granules of platelets injected into the skin bind to the corresponding transmembrane receptors on the surface of DFs and stimulate their synthetic activity. Thus, the consecutive, step-by-step application of the described methods of regenerative medicine amplifies the effect on the molecular and cellular aging processes and thereby allows one to optimize and prolong the clinical results of skin rejuvenation.

TLR8 aggravates skin inflammation and fibrosis by activating skin fibroblasts in systemic sclerosis.

OBJECTIVES: Innate immunity significantly contributes to systemic sclerosis (SSc) pathogenesis. TLR8 is an important innate immune mediator that is implicated in autoimmunity and fibrosis. However, the expression, mechanism of action, and pathogenic role of TLR8 in SSc remain unclear. The aim of this study was to explore the roles and underlying mechanisms of TLR8 in SSc. METHODS: The expression of TLR8 was analyzed based on a public dataset and then verified in skin tissues and skin fibroblasts of SSc patients. The role of TLR8 in inflammation and fibrosis was investigated using a TLR8-overexpression vector, activator (VTX-2337), inhibitor (cu-cpt-8m), and TLR8 siRNA in skin fibroblasts. The pathogenic role of TLR8 in skin inflammation and fibrosis was further validated in a bleomycin (BLM)-induced mouse skin inflammation and fibrosis model. RESULTS: TLR8 levels were significantly elevated in SSc skin tissues and myofibroblasts, along with significant activation of the TLR8 pathway. In vitro studies showed that overexpression or activation of TLR8 by a recombinant plasmid or VTX-2337 upregulated IL-6, IL-1ß, COL I, COL III, and α-SMA in skin fibroblasts. Consistently, both TLR8-siRNA and cu-cpt-8m reversed the phenotypes observed in TLR8-activating fibroblasts. Mechanistically, TLR8 induces skin fibrosis and inflammation in a manner dependent on the MAPK, NF-κB, and SMAD2/3 pathways. Subcutaneous injection of cu-cpt-8m significantly alleviated BLM-induced skin inflammation and fibrosis in vivo. CONCLUSION: TLR8 might be a promising therapeutic target to improve the treatment strategy for SSc skin inflammation and fibrosis.

miR-1246-overexpressing exosomes suppress UVB-induced photoaging via regulation of TGF-ß/Smad and attenuation of MAPK/AP-1 pathway.

Stem cell therapy is widely employed for the treatment of skin diseases, especially in skin rejuvenation. Exosomes derived from stem cells have been demonstrated to possess anti-photoaging effects; however, the precise components within exosomes that are responsible for this effect remain unknown. Previously, miR-1246 was found to be one of the most abundant nucleic acids in adipose-derived stem cells (ADSCs)-derived exosomes. This study examined whether miR-1246 was the major therapeutic agent employed by ADSCs to protect against UVB-induced photoaging. Lentivirus infection was used to obtain miR-1246-overexpressing ADSCs and exosomes. We then determined the anti-photoaging effects of miR-1246-overexpressing exosomes (OE-EX) on both UVB-irradiated human skin fibroblasts (HSFs) and Kunming mice. The results showed that OE-EX could significantly decrease MMP-1 by inhibiting the MAPK/AP-1 signaling pathway. Meanwhile, OE-EX markedly increased procollagen type I secretion by activating the TGF-ß/Smad pathway. OE-EX also exhibited an anti-inflammatory effect by preventing the UVB-induced degradation of IκB-α and NF-κB overexpression. Animal experiments demonstrated that OE-EX could reduce UVB-induced wrinkle formation, epidermis thickening, and the loss of collagen fibers reduction in Kunming mice. The combined results suggested that miR-1246 is the key component within ADSCs-derived exosomes that protects against UVB-induced skin photoaging.

Photoprotective properties of new derivatives of kinetin.

The chronic exposure of skin to ultraviolet (UV) radiation causes adverse dermal reactions, such as erythema, sunburn, photoaging, and cancer, by altering several signalling pathways associated with oxidative stress, inflammation, and DNA damage. One of the possible UV light protection strategies is the use of dermal photoprotective preparations. The plant hormone kinetin (N6-furfuryladenine; KIN) exhibits antioxidant and anti-senescent effects in human cells. Topically applied KIN also reduced some of the clinical signs of photodamaged skin. To improve the biological activities of KIN, several derivatives have been recently prepared and their beneficial effects on cell viability of skin cells exposed to UVA and UVB light were screened. Two potent candidates, 6-(tetrahydrofuran-2-yl)methylamino-9-(tetrahydrofuran-2-yl)purine (HEO) and 6-(thiophen-2-yl)methylamino-9-(tetrahydrofuran-2-yl)purine (HEO6), were identified. Here the effects of KIN, its N9-substituted derivatives the tetrahydropyran-2-yl derivative of KIN (THP), tetrahydrofuran-2-yl KIN (THF), HEO and HEO6 (both THF derivatives) on oxidative stress, apoptosis and inflammation in UVA- or UVB-exposed skin cell was investigated. Human primary dermal fibroblasts and human keratinocytes HaCaT pre-treated with the tested compounds were then exposed to UVA/UVB light using a solar simulator. All compounds effectively prevented UVA-induced ROS generation and glutathione depletion in both cells. HEO6 was found to be the most potent. All compounds also reduced UVB-induced caspase-3 activity and interleukin-6 release. THP and THF exhibited the best UVB protection. In conclusion, our results demonstrated the UVA- and UVB-photoprotective potential of KIN and its derivatives. From this point of view, they seem to be useful agents for full UV spectrum protective dermatological preparations.

An atypical expression of core α-Dystroglycan and Laminin-α2 in skin fibroblasts of patients with congenital muscular dystrophies.

BACKGROUND: Congenital muscular dystrophies (CMDs) result from genetically inherited defects in the biosynthesis and/or the posttranslational modification (glycosylation) of laminin-α2 and α-dystroglycan (α-DG), respectively. The interaction between both proteins is responsible for the stability and integrity of the muscle cell. We aimed to study the expression profiles of both proteins in two classes of CMDs. SUBJECTS AND METHODS: Whole-exome sequencing (WES) was done for four patients with neuromuscular manifestations. The expression of core α-DG and laminin-α2 subunit in skin fibroblasts and MCF-7 cells was assessed by western blot. RESULTS: WES revealed two cases with nonsense mutations; c.2938G > T and c.4348 C > T, in LAMA2 encodes laminin-α2. It revealed also two cases with mutations in POMGNT1 encode protein O-mannose beta-1,2-N-acetylglucosaminyltransferase mutations. One patient had a missense mutation c.1325G > A, and the other had a synonymous variant c.636 C > T. Immunodetection of core α-DG in skin fibroblasts revealed the expression of truncated forms of core α-DG accompanied by reduced expression of laminin-α2 in POMGNT1-CMD patients and one patient with LAMA2-CMD. One patient with LAMA2-CMD had overexpression of laminin-α2 and expression of a low level of an abnormal form of increased molecular weight core α-DG. MCF-7 cells showed truncated forms of core α-CDG with an absent laminin-α2. CONCLUSION: A correlation between the expression pattern/level of core α-DG and laminin-α2 could be found in patients with different types of CMD.

Effects of lncRNA MTC on protein expression in skin fibroblasts of Liaoning Cashmere goat based on iTRAQ technique.

Existing experiments have found a new intergenic lncRNA activated by melatonin, which is called lncRNA MTC. However, the regulatory mechanism of lncRNA MTC in Liaoning Cashmere goat skin fibroblasts has not been clarified. Specific knockdown of lncRNA MTC inhibits cell proliferation and increases apoptosis. iTRAQ reagent was used for relative and absolute quantification of proteins in lncRNA MTC-KD and NC groups to evaluate changes in protein expression during dermal fibroblast development following lncRNA MTC deletion. A total of 5931 proteins were found in Liaoning Cashmere goat skin fibroblasts, of which 123 were differentially expressed, including 32 up-regulated proteins and 91 down-regulated proteins. Of the 91 down-regulated proteins, 32 act mainly through related pathways (e.g., cell cycle, mitochondrial function, ribosomal structure, vesicular transport, cytoskeletal components and skin morphogenesis). LncRNA MTC facilitates the proliferation of Liaoning Cashmere goat skin fibroblasts by regulating ITGB5, TlN2, CTSS, POLG, RAP1B, CHAF1A, CDCA8 and other proteins involved in cell proliferation. The results of this study provide some candidate proteins for the in-depth investigation of the molecular mechanism of lncRNA MTC, which facilitates hair growth in cashmere goats and provides more insights into their regulatory networks and biochemical pathways.

Biological Activities of Ceratonia siliqua Pod and Seed Extracts: A Comparative Analysis of Two Cretan Cultivars.

Ceratonia siliqua L., commonly known as the carob tree, appears in most Mediterranean countries, often cultivated for the collection of its fruits to be used as food for humans and animals. This study was aimed at the phytochemical characterization of two common Cretan C. siliqua cultivars and the biological evaluation of deseeded pod and seed extracts regarding their putative use in cosmetics. Gas and liquid chromatographic techniques were used to assess their essential oil, fatty acid, and carbohydrate profiles. Cell-free assays, including free-radical scavenging; the inhibition of tyrosinase and collagenase; the blocking of advanced glycation end product (AGE) formation; along with assays in human skin fibroblast cultures, i.e., reactive oxygen species suppression, glutathione stimulation, and protection from oxidative stress and from ultraviolet (UVB) radiation, were also used. Extracts from both cultivars were found to possess antioxidant capacity, tyrosinase- and collagenase-inhibitory activities, an ability to block glucose-induced AGEs, and in certain cases, UVB absorbance and photoprotective activities. Seed extracts were in general more active, while the use of 30% aqueous methanol seemed to be more efficient than n-hexane for extraction. Serial partition of the most active extracts resulted in fractions with enriched biological activities. These properties make Cretan carob extracts and their fractions suitable candidates for use in cosmetics.

Feasibility and limitations of cultured skin fibroblasts for germline genetic testing in hematologic disorders.

To avoid acquired variants found in the blood, cultured skin fibroblasts are a recommended DNA source for germline genetic testing in patients with hematologic disorders, but data are lacking regarding practicality and limitations. We conducted a retrospective cohort study of 350 subjects with hematologic disorders who underwent skin fibroblast culture for germline genetic testing. We analyzed next-generation sequencing data from the targeted capture of 144 inherited cancer and bonemarrow failure genes to identify variants at heterozygous and subclonal variant allele frequencies. Sixteen (5%) biopsies failed to culture. Culture failure was more likely in samples with delays in culture initiation (OR = 4.3; p < 0.01) or a pathogenic variant in a telomere gene (OR = 42.6; p < 0.01). Median culture time was 28 days (IQR 22-29 days). Culture time was longer for subjects with prior allogeneic stem cell transplantation (+10.7%; p = 0.02) and shorter in subjects with a heterozygous pathogenic variant (-11.9%; p < 0.01), larger biopsy size (-10.6%; p < 0.01), or lymphoid malignancy (-8.4%; p < 0.01). Subclonal variants were identified in 10 (4%) and confirmed in five (56%) of eight with alternate samples available. Subclonal and discordant variants illustrate that germline testing from cultured skin fibroblasts requires phenotypic correlation and, in rare cases, follow-up studies for optimal interpretation.

Bovine skin fibroblasts mediated immune responses to defend against bovine Acinetobacter baumannii infection.

Acinetobacter baumannii (A. baumannii) is an opportunistic pathogen which can cause pneumonia, sepsis and infections of skin and soft tissue. The host mostly relies on innate immune responses to defend against the infection of A. baumannii. Currently, it has been confirmed that fibroblasts involved in innate immune responses. Therefore, to explore how bovine skin fibroblasts mediated immune responses to defend against A. baumannii infection, we analyzed the differential transcripts data of bovine skin fibroblasts infected with bovine A. baumannii by RNA-sequencing (RNA-seq). We found that there were 3014 differentially expressed genes (DEGs) at 14h with bovine A. baumannii infection, including 1940 up-regulated genes and 1074 down-regulated genes. Gene Ontology (GO) enrichment showed that ubiquitin protein ligase binding, IL-6 receptor complex, ERK1 and ERK2 cascade terms were mainly enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that innate immune pathways were significantly enriched, such as TNF, IL-17, NLR, MAPK, NF-κB, endocytosis, apoptosis and HIF-1 signaling pathways. Furthermore, Gene Set Enrichment Analysis (GSEA) revealed that GO terms such as chemokine receptor binding and Th17 cell differentiation and KEGG pathways such as TLR and cytokine-cytokine receptor interaction pathways were up-regulated. In addition, CASP3 and JUN were the core functional genes of apoptosis, while IL-6, ERBB2, EGFR, CHUK and MAPK8 were the core functional genes of immunity by Protein-Protein Interaction (PPI) analysis. Our study provided an in-depth understanding of the molecular mechanisms of fibroblasts against A. baumannii infection. It also lays the foundation for the development of new therapeutic targets for the diseases caused by A. baumannii infection and formulates effective therapeutic strategies for the prevention and control of the diseases caused by A. baumannii.

miR-24-3p obstructs the proliferation and migration of human skin fibroblasts after thermal injury by targeting PPAR-ß and positively regulated by NF-κB.

Thermal injury repair is a complex process during which the maintenance of the proliferation and migration of human skin fibroblasts (HSFs) exert a crucial role. MicroRNAs have been proven to exert an essential function in repairing skin burns. This study delves into the regulatory effects of miR-24-3p on the migration and proliferation of HSFs that have sustained a thermal injury, thereby, providing deeper insight into thermal injury repair pathogenesis. The PPAR-ß protein expression level progressively increased in a time-dependent manner on the 12th, 24th and 48th hour following the thermal injury of the HSFs. The knockdown of PPAR-ß markedly suppressed the proliferation of and migration of HSF. Following thermal injury, the knockdown also promoted the inflammatory cytokine IL-6, TNF-α, PTGS-2 and P65 expression. PPAR-ß contrastingly exhibited an opposite trend. A targeted relationship between PPAR-ß and miR-24-3p was predicted and verified. miR-24-3p inhibited thermal injured HSF proliferation and migration and facilitated inflammatory cytokine expression through the regulation of PPAR-ß. p65 directly targeted the transcriptional precursor of miR-24 and promoted miR-24 expression. A negative correlation between miR-24-3p expression level and PPAR-ß expression level in rats' burnt dermal tissues was observed. Our findings reveal that miR-24-3p is conducive to rehabilitating the denatured dermis, which may be beneficial in providing effective therapy of skin burns.

Uptake of nanoparticles from sunscreen physical filters into cells arising from increased environmental microwave radiation: increased potential risk of the use of sunscreens to human health.

This study examines the microwave chemical risks posed by photocatalysts present in sunscreens (physical filters) against the increasing use of microwaves (radio waves) in the environment, sometimes referred to as electronic smog. Specifically, the study assesses the damage caused by silica-coated physical filters (photocatalysts, TiO2⋅ and/or ZnO) contained in commercially available sunscreens and fresh silica-coated ZnO for sunscreens to mouse skin fibroblasts cells (NIH/3T3) evaluated in vitro by the life/death of cells using two types of electromagnetic waves: UV light and microwave radiation, and under simultaneous irradiation with both UV light and microwaves. Conditions of the electromagnetic waves were such as to be of lower light irradiance than that of UVA/UVB radiation from incident sunlight, and with microwaves near the threshold power levels that affect human health. The photocatalytic activity of the physical filters was investigated by examining the degradation of the rhodamine B (RhB) dye in aqueous media and by the damage caused to DNA plasmids from E. coli. Compared to the photocatalytic activity of ZnO and TiO2 when irradiated with UV light alone, a clear enhanced photocatalytic activity was confirmed upon irradiating these physical filters concurrently with UV and microwaves. Moreover, the uptake of these metal oxides into the NIH/3T3 cells led to the death of these cells as a result of the enhanced photocatalytic activity of the metal oxides on exposure to microwave radiation.

Effect of the flavonoids quercetin and taxifolin on UVA-induced damage to human primary skin keratinocytes and fibroblasts.

The ultraviolet (UV) part of solar radiation can permanently affect skin tissue. UVA photons represent the most abundant UV component and stimulate the formation of intracellular reactive oxygen species (ROS), leading to oxidative damage to various biomolecules. Several plant-derived polyphenols are known as effective photoprotective agents. This study evaluated the potential of quercetin (QE) and its structurally related flavonoid taxifolin (TA) to reduce UVA-caused damage to human primary dermal fibroblasts (NHDF) and epidermal keratinocytes (NHEK) obtained from identical donors. Cells pre-treated with QE or TA (1 h) were then exposed to UVA light using a solar simulator. Both flavonoids effectively prevented oxidative damage, such as ROS generation, glutathione depletion, single-strand breaks formation and caspase-3 activation in NHDF. These protective effects were accompanied by stimulation of Nrf2 nuclear translocation, found in non-irradiated and irradiated NHDF and NHEK, and expression of antioxidant proteins, such as heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 and catalase. For most parameters, QE was more potent than TA. On the other hand, TA demonstrated protection within the whole concentration range, while QE lost its protective ability at the highest concentration tested (75 µM), suggesting its pro-oxidative potential. In summary, QE and TA demonstrated UVA-protective properties in NHEK and NHDF obtained from identical donors. However, due to the in vitro phototoxic potential of QE, published elsewhere and discussed herein, further studies are needed to evaluate QE safety in dermatological application for humans as well as to confirm our results on human skin ex vivo and in clinical trials.

Study on Long-Term Tracing of Fibroblasts on Three-Dimensional Tissue Engineering Scaffolds Based on Graphene Quantum Dots.

In order to find a convenient and stable way to trace human skin fibroblasts (HSFs) in three-dimensional tissue engineering scaffolds for a long time, in this experiment, Graphene Oxide Quantum Dots (GOQDs), Amino Graphene Quantum Dots (AGQDs) and Carboxyl Graphene Quantum Dots (CGQDs) were used as the material source for labeling HSFs. Exploring the possibility of using it as a long-term tracer of HSFs in three-dimensional tissue engineering scaffolds, the contents of the experiment are as follows: the HSFs were cultured in a cell-culture medium composed of three kinds of Graphene Quantum Dots for 24 h, respectively; (1) using Cell Counting Kit 8 (CCK8), Transwell migration chamber and Phalloidin-iFlior 488 to detect the effect of Graphene Quantum Dots on the biocompatibility of HSFs; (2) using a living cell workstation to detect the fluorescence labeling results of three kinds of Graphene Quantum Dots on HSFs, and testing the fluorescence attenuation of HSFs for 7 days; (3) the HSFs labeled with Graphene Quantum Dots were inoculated on the three-dimensional chitosan demethylcellulose sodium scaffold, and the living cell workstation was used to detect the spatial distribution of the HSFs on the three-dimensional scaffold through the fluorescence properties of the HSFs.. Experimental results: (1) the results of CCK8, Transwell migration, and FITC-Phalloidin cytoskeleton test showed that the three kinds of Graphene Quantum Dots had no effect on the biological properties of HSFs (p < 0.05); (2) the results of the fluorescence labeling experiment showed that only AGQDs could make HSFs fluorescent, and cells showed orange−red fluorescence; (3) the results of long-range tracing of HSFs which were labeled by with AGQDs showed that the fluorescence life of the HSFs were as long as 7 days; (4) The spatial distribution of HSFs can be detected on the three-dimensional scaffold based on their fluorescence properties, and the detection time can be up to 7 days.

Human Vascular Endothelial Cells Promote the Secretion of Vascularization Factors and Migration of Human Skin Fibroblasts under Co-Culture and Its Preliminary Application.

The good treatment of skin defects has always been a challenge in the medical field, and the emergence of tissue engineering skin provides a new idea for the treatment of injured skin. However, due to the single seed cells, the tissue engineering skin has the problem of slow vascularization at the premonitory site after implantation into the human body. Cell co-culture technology can better simulate the survival and communication environment of cells in the human body. The study of multicellular co-culture hopes to bring a solution to the problem of tissue engineering. In this paper, human skin fibroblasts (HSFs) and human vascular endothelial cells (HVECs) were co-cultured in Transwell. The Cell Counting Kit 8 (CCK8), Transwell migration chamber, immunofluorescence, Western blot (WB), and real time quantitative PCR (RT-qPCR) were used to study the effects of HVECs on cell activity, migration factor (high mobility group protein 1, HMGB1) and vascularization factor (vascular endothelial growth factor A, VEGFA and fibroblast growth factor 2, FGF2) secretion of HSFs after co-cultured with HVECs in the Transwell. The biological behavior of HSFs co-cultured with HVECs was studied. The experimental results are as follows: (1) The results of cck8 showed that HVECS could promote the activity of HSFs. (2) HVECs could significantly promote the migration of HSFs and promote the secretion of HMGB1. (3) HVECs could promote the secretion of VEGFA and FGF2 of HSFs. (4) The HVECs and HSFs were inoculated on tissue engineering scaffolds at the ratio of 1:4 and were co-cultured and detected for 7 days. The results showed that from the third day, the number of HSFs was significantly higher than that of the control group without HVECs.