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Dermal fibroblasts are essential cells of skin tissue responsible for its normal functioning. In skin wounds, the differentiation of resident fibroblasts into myofibroblasts occurs, which is accompanied by the rearrangement of actin cytoskeleton with the expression of alpha-smooth muscle actin. This transformation is a prerequisite for a successful wound healing. At the same time, different studies indicate that extracellular matrix may be involved in regulation of this process. Since the connection between cells and matrix is provided by transmembrane integrin receptors, this work was aimed at studying the dynamics of signaling pathways associated with integrins on an in vitro model of wound healing using human skin fibroblasts. It was shown that the healing of simulated wound was accompanied by a change in the level of integrins as well as integrin-associated kinases ILK (integrin-linked kinase) and FAK (focal adhesion kinase). Pharmacological inhibition of ILK and FAK caused the suppression of p38 and Akt which proteins are involved in regulation of the actin cytoskeleton. Moreover, it resulted in an inefficient wound closure in vitro. The results of this study support the involvement of integrin-associated kinases in regulation of fibroblast-to-myofibroblast transition during wound healing. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aberrant sialylation with overexpression of the homopolymeric glycan polysialic acid (polySia) was recently reported in fibroblasts from fibrotic skin lesions. Yet, whether such a rise in polySia levels or sialylation in general may be functionally implicated in profibrotic activation of fibroblasts and their transition to myofibroblasts remains unknown. Therefore, we herein explored whether inhibition of sialylation could interfere with the process of skin fibroblast-to-myofibroblast transition induced by the master profibrotic mediator transforming growth factor β1 (TGFβ1). Adult human skin fibroblasts were pretreated with the competitive pan-sialyltransferase inhibitor 3-Fax-peracetyl-Neu5Ac (3-Fax) before stimulation with recombinant human TGFβ1, and then analyzed for polySia expression, cell viability, proliferation, migratory ability, and acquisition of myofibroblast-like morphofunctional features. Skin fibroblast stimulation with TGFβ1 resulted in overexpression of polySia, which was effectively blunted by 3-Fax pre-administration. Pretreatment with 3-Fax efficiently lessened TGFβ1-induced skin fibroblast proliferation, migration, changes in cell morphology, and phenotypic and functional differentiation into myofibroblasts, as testified by a significant reduction in FAP, ACTA2, COL1A1, COL1A2, and FN1 gene expression, and α-smooth muscle actin, N-cadherin, COL1A1, and FN-EDA protein levels, as well as a reduced contractile capability. Moreover, skin fibroblasts pre-administered with 3-Fax displayed a significant decrease in Smad3-dependent canonical TGFβ1 signaling. Collectively, our in vitro findings demonstrate for the first time that aberrant sialylation with increased polySia levels has a functional role in skin fibroblast-to-myofibroblast transition and suggest that competitive sialyltransferase inhibition might offer new therapeutic opportunities against skin fibrosis. [ABSTRACT FROM AUTHOR]

Objectives Innate immunity significantly contributes to 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 analysed, 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-1β, 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 for improving the treatment strategy for skin inflammation and fibrosis in SSc. [ABSTRACT FROM AUTHOR]

Background and purpose: Carbon nanotubes (CNTs) are a significant discovery in nanotechnology, with widespread applications in modern technology. However, there are concerns about their potential toxicity, particularly in skin cells. This study aimed to investigate the mechanisms by which CNTs induced cytotoxicity and apoptosis in mouse skin fibroblasts. Experimental approach: The mice skin fibroblasts were isolated and exposed to two types of CNTs at various concentrations and then analyzed for changes in viability, reactive oxygen species (ROS) production, the levels of Bcl-2-associated X protein (Bax), and lactate production. Findings/Results: The results demonstrated that CNTs reduced cell viability and increased ROS production in a dose-dependent manner. Additionally, the current study found that CNTs increased the protein levels of Bax, a pro-apoptotic protein, in mouse skin fibroblasts. Furthermore, it was observed a significant decrease in lactate production in cells exposed to CNTs. Conclusion and implications: The findings concluded that CNTs have the potential to be toxic substances for skin fibroblasts, which serve as the body's first line of defense. This is evidenced by their ability to increase the production of ROS and the protein levels of Bax, as well as reduce lactic acid levels. As lactic acid has been reported to have beneficial effects on skin collagen production, further studies are needed to fully understand the impact of carbon nanotube exposure on human skin health.

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

Background and purpose: Carbon nanotubes (CNTs) are a significant discovery in nanotechnology, with widespread applications in modern technology. However, there are concerns about their potential toxicity, particularly in skin cells. This study aimed to investigate the mechanisms by which CNTs induced cytotoxicity and apoptosis in mouse skin fibroblasts. Experimental approach: The mice skin fibroblasts were isolated and exposed to two types of CNTs at various concentrations and then analyzed for changes in viability, reactive oxygen species (ROS) production, the levels of Bcl-2-associated X protein (Bax), and lactate production. Findings/Results: The results demonstrated that CNTs reduced cell viability and increased ROS production in a dose-dependent manner. Additionally, the current study found that CNTs increased the protein levels of Bax, a pro-apoptotic protein, in mouse skin fibroblasts. Furthermore, it was observed a significant decrease in lactate production in cells exposed to CNTs. Conclusion and implications: The findings concluded that CNTs have the potential to be toxic substances for skin fibroblasts, which serve as the body's first line of defense. This is evidenced by their ability to increase the production of ROS and the protein levels of Bax, as well as reduce lactic acid levels. As lactic acid has been reported to have beneficial effects on skin collagen production, further studies are needed to fully understand the impact of carbon nanotube exposure on human skin health. [ABSTRACT FROM AUTHOR]

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‐β (TGF‐β)/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‐β/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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aberrant sialylation with overexpression of the homopolymeric glycan polysialic acid (polySia) was recently reported in fibroblasts from fibrotic skin lesions. Yet, whether such a rise in polySia levels or sialylation in general may be functionally implicated in profibrotic activation of fibroblasts and their transition to myofibroblasts remains unknown. Therefore, we herein explored whether inhibition of sialylation could interfere with the process of skin fibroblast-to-myofibroblast transition induced by the master profibrotic mediator transforming growth factor β1 (TGFβ1). Adult human skin fibroblasts were pretreated with the competitive pan-sialyltransferase inhibitor 3-Fax-peracetyl-Neu5Ac (3-Fax) before stimulation with recombinant human TGFβ1, and then analyzed for polySia expression, cell viability, proliferation, migratory ability, and acquisition of myofibroblast-like morphofunctional features. Skin fibroblast stimulation with TGFβ1 resulted in overexpression of polySia, which was effectively blunted by 3-Fax pre-administration. Pretreatment with 3-Fax efficiently lessened TGFβ1-induced skin fibroblast proliferation, migration, changes in cell morphology, and phenotypic and functional differentiation into myofibroblasts, as testified by a significant reduction in FAP, ACTA2, COL1A1, COL1A2, and FN1 gene expression, and α-smooth muscle actin, N-cadherin, COL1A1, and FN-EDA protein levels, as well as a reduced contractile capability. Moreover, skin fibroblasts pre-administered with 3-Fax displayed a significant decrease in Smad3-dependent canonical TGFβ1 signaling. Collectively, our in vitro findings demonstrate for the first time that aberrant sialylation with increased polySia levels has a functional role in skin fibroblast-to-myofibroblast transition and suggest that competitive sialyltransferase inhibition might offer new therapeutic opportunities against skin fibrosis.

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.

The impaired healing of chronic wounds is often attributed to the ischemic and hypoxic microenvironment, leading to increased cell death. Ferroptosis, a novel form of cell death unveiled in recent years, could potentially be linked with the process of wound healing. In this study, we explored the significance and mechanism of ferroptosis in ischemic wounds. Using transmission electron microscopy, Western blot, flow cytometry, immunofluorescence, and glutathione (GSH) assay, we observed that the death of primary mouse skin fibroblasts induced by oxygen and glucose deprivation (OGD) was associated with ferroptosis. Specifically, we observed elevated intracellular Fe2+ and lipid peroxidation levels and decreased GSH levels in vitro, indicative of ferroptosis. Importantly, we found that ferroptosis in OGD-treated skin fibroblasts was dependent on autophagy, as the autophagy inhibitor chloroquine phosphate (CHQ) significantly reduced ferroptosis induced by OGD. Moreover, our study revealed that NCOA4-mediated ferritinophagy significantly contributed to the occurrence of ferroptosis induced by OGD in skin fibroblasts. Additionally, we identified the involvement of YAP in the regulation of ferritinophagy, with YAP suppressing NCOA4 expression in OGD-treated skin fibroblasts, thereby reducing ferroptosis. Furthermore, in ischemic wound models in mice, both inhibitors of ferroptosis and autophagy promoted wound healing, while a YAP inhibitor, verteporfin, delayed wound healing. In conclusion, these findings indicate that ferroptosis, regulated by YAP through ferritinophagy inhibition, presents a novel mechanism responsible for the delayed healing of ischemic wounds. Understanding this process could offer promising therapeutic targets to improve wound healing in ischemic conditions.

Osteogenesis imperfecta (OI) is a group of connective tissue disorders leading to abnormal bone formation, mainly due to mutations in genes encoding collagen type I (Col I). Osteogenesis is regulated by a number of molecules, including microRNAs (miRNAs), indicating their potential as targets for OI therapy. The goal of this study was to identify and analyze the expression profiles of miRNAs involved in bone extracellular matrix (ECM) regulation in patients diagnosed with OI type I caused by mutations in COL1A1 or COL1A2. Primary skin fibroblast cultures were used for DNA purification and sequence analysis, followed by analysis of miRNA expression. Sequencing analysis revealed mutations of the COL1A1 or COL1A2 genes in all OI patients, including four previously unreported. Amongst the 40 miRNAs analyzed, 9 were identified exclusively in OI cells and 26 in both OI patients and the controls. In the latter case, the expression of six miRNAs (hsa-miR-10b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, has-miR-204-5p, has-miR-216a-5p, and hsa-miR-449a) increased, while four (hsa-miR-129-5p, hsa-miR-199b-5p, hsa-miR-664a-5p, and hsa-miR-30a-5p) decreased significantly in OI cells in comparison to their expression in the control cells. The identified mutations and miRNA expression profiles shed light on the intricate processes governing bone formation and ECM regulation, paving the way for further research and potential therapeutic advancements in OI and other genetic diseases related to bone abnormality management. [ABSTRACT FROM AUTHOR]

Multiple sclerosis (MS) is the leading cause of non-traumatic disability in young adults. New avenues are needed to help predict individuals at risk for developing MS and aid in diagnosis, prognosis, and outcome of therapeutic treatments. Previously, we showed that skin fibroblasts derived from patients with MS have altered signatures of cell stress and bioenergetics, which likely reflects changes in their protein, lipid, and biochemical profiles. Here, we used Fourier transform infrared (FTIR) spectroscopy to determine if the biochemical landscape of MS skin fibroblasts were altered when compared to age- and sexmatched controls (CTRL). More so, we sought to determine if FTIR spectroscopic signatures detected in MS skin fibroblasts are disease specific by comparing them to amyotrophic lateral sclerosis (ALS) skin fibroblasts. Spectral profiling of skin fibroblasts from MS individuals suggests significant alterations in lipid and protein organization and homeostasis, which may be affecting metabolic processes, cellular organization, and oxidation status. Sparse partial least squaresdiscriminant analysis of spectral profiles show that CTRL skin fibroblasts segregate well from diseased cells and that changes in MS and ALS may be unique. Differential changes in the spectral profile of CTRL, MS, and ALS cells support the development of FTIR spectroscopy to detect biomolecular modifications in patient-derived skin fibroblasts, which may eventually help establish novel peripheral biomarkers. [ABSTRACT FROM AUTHOR]

The Liaoning cashmere goat is a unique breed in China. It has an excellent germplasm resource with high villus yield, fine villus fibre quality, stable genetic performance and significant economic benefits.In this study, the effects of different concentrations of 1.25(OH)2D3 on the proliferation of skin fibroblasts was detected via CCK8 assay, while the expression of genes and proteins related to villus growth in Liaoning cashmere goat were detected by qRT-PCR and Western Blot. Treatment of Liaoning cashmere goat skin fibroblasts with 0.01 ng/L of 1.25(OH)2D3 for 72 h significantly promoted the proliferation of Liaoning cashmere goat skin fibroblasts, increased the expression of VDR, WNT10B, LEF1 and β-catenin genes, and decreased the expression of BMP4 and BMPRIB genes, indicating that 1.25(OH)2D3 plays a positive role in regulating hair follicle growth and development through the WNT and BMP signaling pathway, which positively regulates the growth and development of hair follicles. VDR interacting proteins were screened using Co-IP combined mass spectrometry. After treatment of cells with 0.01 ng/L of 1.25(OH)2D3 for 72 h, VDR interacting proteins were enriched to PI3K-AKT signaling pathways associated with hair follicle growth and development. Moreover, mitogen-activated protein kinase, protein S100, protein disulfide bond isomerase protein, FLII actin remodeling protein and multifunctional proteoglycan protein related to the growth and development of hair follicles were upregulated, indicating that a positive regulatory effect of 1.25(OH)2D3 on hair follicle growth and development. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

BackgroundOlder aged adults and those with pre-existing conditions are at highest risk for severe COVID-19 associated outcomes.MethodsUsing a large dataset of genome-wide RNA-seq profiles derived from human dermal fibroblasts (GSE113957) we investigated whether age affects the expression of pattern recognition receptor (PRR) genes and ACE2, the receptor for SARS-CoV-2.ResultsExtremes of age are associated with increased expression of selected PRR genes, ACE2 and four genes that encode proteins that have been shown to interact with SAR2-CoV-2 proteins.ConclusionsAssessment of PRR expression might provide a strategy for stratifying the risk of severe COVID-19 disease at both the individual and population levels.

The aim of the study was to research the international experience in quality assurance of the products based on skin cells in order to identify the features of the quality control strategy in the development, production, as well as during an expert quality assessment as a part of the state registration procedure in the Russian Federation.Materials and methods. The article provides an analysis of the materials presented in the assessment reports of the USA and Japanese regulatory authorities, as well as on the official websites of manufacturers, in review and scientific papers on the study of the structure and properties of tissue-engineered skin analogs.Results. The manufacture of products containing human skin cells is associated with such risks as the possibility of contamination of the preparation with infective agents transmitted by materials of the animal origin, feeder cells, donor cells, or during the manufacturing process; a small amount of biopsy materials; a complexity of a three-dimensional product structure when combining cells with a scaffold; continuity of the manufacture process and a short product expiry date. The raw materials and reagents control, the creation of cell banks, using animal feeder cells only from qualified cell banks, an in-process control and release testing in accordance with the requirements of the finished product specification, make it possible to obtain a preparation with a reproducible quality. The specification should contain information about the identity, safety and potency of the product. For each preparation, the choice of approaches for assessing the quality is individual and depends on its composition and mode of action.Conclusion. The features of the quality control strategy for the drugs based on human skin cells, consist in the implementation of control measures in order to obtain a proper quality of cellular (viability, sterility, identity, potency, et al) and non-cellular (physico-chemical scaffold properties) components or the whole graft (bioburden, barrier properties). The approaches and methods for determining the potency should be selected individually for each product and reflect the number, viability and identity of cells, a proliferative activity and secretable ability of the cellular component.

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

Multiple sclerosis (MS) is the leading cause of non-traumatic disability in young adults. New avenues are needed to help predict individuals at risk for developing MS and aid in diagnosis, prognosis, and outcome of therapeutic treatments. Previously, we showed that skin fibroblasts derived from patients with MS have altered signatures of cell stress and bioenergetics, which likely reflects changes in their protein, lipid, and biochemical profiles. Here, we used Fourier transform infrared (FTIR) spectroscopy to determine if the biochemical landscape of MS skin fibroblasts were altered when compared to age- and sex-matched controls (CTRL). More so, we sought to determine if FTIR spectroscopic signatures detected in MS skin fibroblasts are disease specific by comparing them to amyotrophic lateral sclerosis (ALS) skin fibroblasts. Spectral profiling of skin fibroblasts from MS individuals suggests significant alterations in lipid and protein organization and homeostasis, which may be affecting metabolic processes, cellular organization, and oxidation status. Sparse partial least squares-discriminant analysis of spectral profiles show that CTRL skin fibroblasts segregate well from diseased cells and that changes in MS and ALS may be unique. Differential changes in the spectral profile of CTRL, MS, and ALS cells support the development of FTIR spectroscopy to detect biomolecular modifications in patient-derived skin fibroblasts, which may eventually help establish novel peripheral biomarkers.

Objective To explore the function and mechanism of tamoxifen, an estrogen receptor modulator, in the pathogenesis of hyperplatic scar. Methods Primary human fibroblasts were isolated and cultured for 4 generations and then aliquated into 3 groups: control group, TGF-β1 group and TGF-β1+ tamoxifen group. The effects of tamoxifen on human fibrogenic factor production, cell proliferation, migration and differentiation of TGF-β1-induced skin fibroblasts were detected by real-time quantitative polymerase chain reaction (RT-qPCR), MTS, cell scratch test, Western blot and immunofluorescence assay. Results Tamoxifen (10 μmol/L) inhibited the production of collagen 1, collagen 3 and α-SMA induced by TGF-β1 in fibroblasts. MTS results showed that the proliferation activity of TGF-β1 group was higher than that of tamoxifen group (P＜0.05). Wound healing test verified that the migration ability of cells was reduced by tamoxifen intervention. Tamoxifen inhibited collagen secretion and differentiation of skin fibroblasts into muscle fibroblasts. Conclusions The estrogen receptor modulator tamoxifen inhibits pathological proliferation of fibroblasts, so it may be a potential novel strategy for the treatment of hyperplastic scar.

Many physical and chemical factors can influence the Nrf2 signaling pathway, and its deregulation has been associated with various disease states. Nrf2 and Keap1 molecules are key elements that regulate the response under cell stress conditions. Hence, the aim of the research was to assess the impact of a static magnetic field that was generated by permanent magnets on the transcriptional activity of the NFE2L2 and KEAP1 genes in a human dermal fibroblast cell line that had been cultured with fluoride ions and in an environment without them. Additionally, our research aimed to answer the question of whether the regulatory RNA molecules—miRNAs—are involved in these effects. Experiments were performed on an NHDF cell line that had been treated with fluoride ions, along with simultaneous exposure to a moderate-intensity static magnetic field. The RT-qPCR method was used to determine the mRNA level of the NFE2L2 and KEAP1 genes and the miRNA level of miR-28-3p, miR-29b-1-5p, miR-141-3p and miR-365a-3p. Our results indicate that fluoride ions influence the Nrf2 signaling pathway and that miRNAs are involved in regulating it. Moreover, the static magnetic field had a beneficial effect on the cells with fluoride-induced oxidative stress due to stimulating the antioxidant defense. [ABSTRACT FROM AUTHOR]

Objective To explore the effect and preliminary mechanism of stathmin on skin fibroblast migration in the early inflammatory reaction of wound healing mimicked by TNF-α stimulation. Methods Human dermal fibroblast was stimulated by TNF-α to establish a cell model of inflammation at the early stage of wound healing. The effect of TNF-α on filopodium formation was observed by immunofluorescence assay, cell migration was detected by scratch wound healing, and stathmin expression level under TNF-α stimulation was measured by immunofluorescence assay and Western blotting. After cytochalasin B stimulation and small interfering RNA were used to interfere stathmin expression, respectively, immunofluorescence assay and scratch wound healing were performed again to observe the changes of filopodia and cell migration. Results TNF-α treatment promoted the formation of filopodia in human dermal fibroblasts, enhanced cell migration (P < 0.05) and increased stathmin expression (P < 0.05). Cytochalasin B treatment reduced the formation of filopodia and cell migration (P < 0.05); and interference of stathmin expression suppressed the formation of filopodia and reduced cell migration (P < 0.05). Conclusion In early inflammation, stathmin mediates skin fibroblast migration by promoting the formation of filopodia.

Osteogenesis imperfecta (OI) is a group of connective tissue disorders leading to abnormal bone formation, mainly due to mutations in genes encoding collagen type I (Col I). Osteogenesis is regulated by a number of molecules, including microRNAs (miRNAs), indicating their potential as targets for OI therapy. The goal of this study was to identify and analyze the expression profiles of miRNAs involved in bone extracellular matrix (ECM) regulation in patients diagnosed with OI type I caused by mutations in COL1A1 or COL1A2. Primary skin fibroblast cultures were used for DNA purification and sequence analysis, followed by analysis of miRNA expression. Sequencing analysis revealed mutations of the COL1A1 or COL1A2 genes in all OI patients, including four previously unreported. Amongst the 40 miRNAs analyzed, 9 were identified exclusively in OI cells and 26 in both OI patients and the controls. In the latter case, the expression of six miRNAs (hsa-miR-10b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, has-miR-204-5p, has-miR-216a-5p, and hsa-miR-449a) increased, while four (hsa-miR-129-5p, hsa-miR-199b-5p, hsa-miR-664a-5p, and hsa-miR-30a-5p) decreased significantly in OI cells in comparison to their expression in the control cells. The identified mutations and miRNA expression profiles shed light on the intricate processes governing bone formation and ECM regulation, paving the way for further research and potential therapeutic advancements in OI and other genetic diseases related to bone abnormality management.

Hibiscus sabdariffa L. (HS) has a long history of edible and medicinal uses. In this study, the biological activities of the extracts, chromatographic fractions, and hibiscus acid obtained from HS were evaluated for their potential bioactivities. Their ability to promote extracellular matrix synthesis in skin fibroblasts was evaluated by enzyme-linked immunosorbent assays. Their anti-inflammatory activity was evaluated in a nitric oxide (NO)–Griess inflammatory experiment. Furthermore, hibiscus acid was found to have a strong anti-oxidative stress effect through the establishment of an oxidative stress model induced by hydrogen peroxide. Several assays indicated that hibiscus acid treatment can effectively reduce extracellular adenosine triphosphate (ATP) secretion and carbonyl protein production, as well as maintain a high level of reduced/oxidized glutathione (GSH/GSSG) in skin cells, thus providing a possible mechanism by which hibiscus acid can counter antioxidative stress. The present study is the first to explore the reversing skin aging potential and the contributory component of HS. [ABSTRACT FROM AUTHOR]

Osteogenesis imperfecta (OI) is a heterogeneous connective tissue disease mainly caused by structural mutations in type I collagen. Mutant collagen accumulates intracellularly, causing cellular stress that has recently been shown to be phenotype-related. Therefore, the aim of the study was to search for potential drugs reducing collagen accumulation and improving OI fibroblast homeostasis. We found that rosemary extract (RE), which is of great interest to researchers due to its high therapeutic potential, at concentrations of 50 and 100 µg/mL significantly reduced the level of accumulated collagen in the fibroblasts of four patients with severe and lethal OI. The decrease in collagen accumulation was associated with RE-induced autophagy as was evidenced by an increase in the LC3-II/LC3-I ratio, a decrease in p62, and co-localization of type I collagen with LC3-II and LAMP2A by confocal microscopy. The unfolded protein response, activated in three of the four tested cells, and the level of pro-apoptotic markers (Bax, CHOP and cleaved caspase 3) were attenuated by RE. In addition, the role of RE-modulated proteasome in the degradation of unfolded procollagen chains was investigated. This study provides new insight into the beneficial effects of RE that may have some implications in OI therapy targeting cellular stress. [ABSTRACT FROM AUTHOR]

This study aimed to develop, characterize, and evaluate antibacterial and cytotoxic properties of novel fullerene derivative composed of C60 fullerenol and standard aminoglycoside antibiotic–gentamicin (C60 fullerenol-gentamicin conjugate). The successful introduction of gentamicin to fullerenol was confirmed by X-ray photoelectron spectroscopy which together with thermogravimetric and spectroscopic analysis revealing the formula of the composition as C60(OH)12(GLYMO)11(Gentamicin)0.8. The dynamic light scattering (DLS) revealed that conjugate possessed ability to form agglomerates in water (size around 115 nm), while Zeta potential measurements demonstrated that such agglomerates possessed neutral character. In vitro biological assays indicated that obtained C60 fullerenol-gentamicin conjugate possessed the same antibacterial activity as standard gentamicin against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli, which proves that combination of fullerenol with gentamicin does not cause the loss of antibacterial activity of antibiotic. Moreover, cytotoxicity assessment demonstrated that obtained fullerenol-gentamicin derivative did not decrease viability of normal human fibroblasts (model eukaryotic cells) compared to control fibroblasts. Thus, taking into account all of the results, it can be stated that this research presents effective method to fabricate C60 fullerenol-gentamicin conjugate and proves that such derivative possesses desired antibacterial properties without unfavorable cytotoxic effects towards eukaryotic cells in vitro. These promising preliminary results indicate that obtained C60 fullerenol-gentamicin conjugate could have biomedical potential. It may be presumed that obtained fullerenol may be used as an effective carrier for antibiotic, and developed fullerenol-gentamicin conjugate may be apply locally (i.e., at the wound site). Moreover, in future we will evaluate possibility of its applications in inter alia tissue engineering, namely as a component of wound dressings and implantable biomaterials. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Background Older aged adults and those with pre-existing conditions are at highest risk for severe COVID-19 associated outcomes. Methods Using a large dataset of genome-wide RNA-seq profiles derived from human dermal fibroblasts (GSE113957) we investigated whether age affects the expression of pattern recognition receptor (PRR) genes and ACE2, the receptor for SARS-CoV-2. Results Extremes of age are associated with increased expression of selected PRR genes, ACE2 and four genes that encode proteins that have been shown to interact with SAR2-CoV-2 proteins. Conclusions Assessment of PRR expression might provide a strategy for stratifying the risk of severe COVID-19 disease at both the individual and population levels.

Prolonged exposure to sun radiation may result in harmful skin photoaging. Therefore, discovering novel anti-photoaging treatment modalities is critical. An active component isolated from Salvia miltiorrhiza (SM), Salvianolic acid B (Sal-B), is a robust antioxidant and anti-inflammatory agent. This investigation aimed to discover the therapeutic impact and pathways of salvianolic acid B for UVB-induced skin photoaging, an area that remains unexplored. We conducted in vitro experiments on human dermal fibroblasts (HDFs) exposed to UVB radiation, assessing cellular senescence, superoxide dismutase (SOD) activity, cell viability, proliferation, migration, levels of reactive oxygen species (ROS), and mitochondrial health. The potential mechanism of Sal-B was analyzed using RNA sequencing, with further validation through Western blotting, PCR, and nuclear factor erythroid 2-related factor 2 (NRF2) silencing methods. In vivo , a model of skin photoaging induced by UVB in nude mice was employed. The collagen fiber levels were assessed utilizing hematoxylin and eosin (H&E), Masson, and Sirus red staining. Additionally, NRF2 and related gene and protein expression levels were identified utilizing PCR and Western blotting. Sal-B was found to significantly counteract photoaging in UVB-exposed skin fibroblasts, reducing aging-related decline in fibroblast proliferation and an increase in apoptosis. It was observed that Sal-B aids in protecting mitochondria from excessive ROS production by promoting NRF2 nuclear translocation. NRF2 knockdown experiments established its necessity for Sal-B's anti-photoaging effects. The in vivo studies also verified Sal-B's anti-photoaging efficacy, surpassing that of tretinoin (Retino-A). These outcomes offer novel insights into the contribution of Sal-B in developing clinical treatment modalities for UVB-induced photodamage in skin fibroblasts. In this investigation, we identified the Sal-B protective impact on the senescence of dermal fibroblasts and skin photoaging induced by radiation of UVB. The outcomes suggest Sal-B as a potential modulator of the NRF2 signaling pathway. 1). Salvianolic acid B protects from skin aging caused by UVB via NRF2 activation in vitro and in vivo. 2). NRF2 is necessary for salvianolic acid B to exert anti-photoaging effects. Created with BioRender.com [Display omitted] [ABSTRACT FROM AUTHOR]

In this study, we investigated the protective effects of low-molecular-weight fish collagen from tilapia against melanogenesis in melanocytes, ultraviolet B (UVB)-irradiated Hs27 skin fibroblasts, and hairless mice. We observed collagen production-related pathways in UVB-irradiated Hs27 skin fibroblasts and hairless mice, and the melanogenesis-related pathways in melanocyte and UVB-irradiated hairless mice. The collagen production-related pathways were activated in the UVB-irradiated Hs27 skin fibroblasts and hairless mice. In addition, UVB exposure stimulated the melanogenesis-related pathways in melanocytes and hairless mice. However, treatment with low-molecular-weight fish collagen significantly increased the messenger RNA expressions of collagen production-related factors and significantly decreased the production of cytokines. Furthermore, treatment with low-molecular-weight fish collagen suppressed melanogenesis by inhibiting glutathione synthesis and downregulating melanocyte-inducing transcription factor expression through the suppression of cyclic AMP/protein kinase A/cAMP-responsive binding protein signaling and nitric oxide production. Low-molecular-weight fish collagen exerts protective effects against UVB-induced photoaging, through anti-inflammatory, antioxidant, and anti-melanogenesis activities and could be used for developing effective natural anti-photoaging products. [ABSTRACT FROM AUTHOR]

trichothecenes and their derivatives have recently attracted much attention of researchers with respect of their potential role in medicine, including for the treatment of different types of cancer. The purpose of the study was to investigate the cytotoxic effect of Fusarium trichothecene on human breast cancer cells, human skin fibroblasts and embryonic kidney cells in vitro. Material and methods. Based on the Mtt assay, the cytotoxic effect of trichothecene on cell cultures was determined. Evaluation of morphological changes in cell cultures under the influence of trichothecene was performed by light microscopy. Results. Fusarium trichothecene was found to exhibit a dose-dependent toxic effect on cell lines in the range 1 nM to 1000 nM. the most pronounced cytotoxic effect of trichothecene was observed in human breast cancer cell line (IС50 94.72 ± 4.1 нМ). Lower doses of trichothecene led to a change in the size, shape of human breast cancer cells, human skin fibroblasts and embryonic kidney cells, and loss of contact between them and their isolation. the degradation of cell membranes, formation of unformed cell aggregates and fragments were observed in higher doses of trichothecene. Conclusion. Fusarium trichothecen is a biologically active compound and is less toxic to the normal than to the cancer cell lines, therefore, further studies of this agent are needed.

Many physical and chemical factors can influence the Nrf2 signaling pathway, and its deregulation has been associated with various disease states. Nrf2 and Keap1 molecules are key elements that regulate the response under cell stress conditions. Hence, the aim of the research was to assess the impact of a static magnetic field that was generated by permanent magnets on the transcriptional activity of the NFE2L2 and KEAP1 genes in a human dermal fibroblast cell line that had been cultured with fluoride ions and in an environment without them. Additionally, our research aimed to answer the question of whether the regulatory RNA molecules—miRNAs—are involved in these effects. Experiments were performed on an NHDF cell line that had been treated with fluoride ions, along with simultaneous exposure to a moderate-intensity static magnetic field. The RT-qPCR method was used to determine the mRNA level of the NFE2L2 and KEAP1 genes and the miRNA level of miR-28-3p, miR-29b-1-5p, miR-141-3p and miR-365a-3p. Our results indicate that fluoride ions influence the Nrf2 signaling pathway and that miRNAs are involved in regulating it. Moreover, the static magnetic field had a beneficial effect on the cells with fluoride-induced oxidative stress due to stimulating the antioxidant defense.

In recent years, natural product's research gained momentum, fueled by technological advancement and open availability of research data. To date, sea buckthorn (Hippophae rhamnoides L. [ Elaeagnaceae ]) plant parts, especially berries, are well characterized and repeatedly tested for antioxidant activity and regenerative properties, in various cell types and tissues. However, fatty acids (FA) have been less investigated in term of biological effects, although, they are important bioactive components of the sea buckthorn fruit and oil. The aim of our work was to determine whether sea buckthorn seed oil is a suitable source of FA with regenerative properties on normal skin cells. Using high-performance liquid chromatography (HPLC) and liquid chromatography – mass spectrometry (LC-MS), we purified and characterized four fractions enriched in saturated (palmitic) and non-saturated (linoleic, alfa-linolenic, oleic) FA, which were tested for cytotoxicity, cytokine and growth factor production, and regenerative effect on normal keratinocytes and skin fibroblasts. Evidence is presented that the palmitic acid enriched fraction was a suitable sea buckthorn seed oil derived product with cell proliferation properties on both skin cell types. [ABSTRACT FROM AUTHOR]

Fluoride can weaken the protective role of melatonin in reducing cellular damage. A static magnetic field is a physical factor that can counteract the negative effect of fluoride. Hence, the main objective of the study was to analyze the transcriptional activity of the genes that are associated with the activity of melatonin in human skin fibroblasts that have been co-exposed to fluoride and a moderate-strength static magnetic field. The expression of the melatonin-associated genes in human fibroblasts that had simultaneously been exposed to F− and a static magnetic field was determined using an oligonucleotide microarray and RT-qPCR techniques. The concentration of oxidative damage markers was also measured. In NaF and static magnetic field-treated cells, there was a tendency to compensate for the expression of the differentiating genes (IL27RA, NR1D1, RRP7A, YIPF1, HIST1H2BD) that had been modified by the presence of fluoride. It has been also shown that the oxidative damage marker concentration was statistically lower in the cells that had simultaneously been exposed to fluoride and a static magnetic field compared to the F-treated cells. In conclusion, the protective role of a moderate-strength static magnetic field on human dermal fibroblasts that had been exposed to fluoride was demonstrated, and its mechanism of action is associated with the melatonin-dependent pathways. [ABSTRACT FROM AUTHOR]

Osteogenesis imperfecta (OI) is a heterogeneous connective tissue disease mainly caused by structural mutations in type I collagen. Mutant collagen accumulates intracellularly, causing cellular stress that has recently been shown to be phenotype-related. Therefore, the aim of the study was to search for potential drugs reducing collagen accumulation and improving OI fibroblast homeostasis. We found that rosemary extract (RE), which is of great interest to researchers due to its high therapeutic potential, at concentrations of 50 and 100 µg/mL significantly reduced the level of accumulated collagen in the fibroblasts of four patients with severe and lethal OI. The decrease in collagen accumulation was associated with RE-induced autophagy as was evidenced by an increase in the LC3-II/LC3-I ratio, a decrease in p62, and co-localization of type I collagen with LC3-II and LAMP2A by confocal microscopy. The unfolded protein response, activated in three of the four tested cells, and the level of pro-apoptotic markers (Bax, CHOP and cleaved caspase 3) were attenuated by RE. In addition, the role of RE-modulated proteasome in the degradation of unfolded procollagen chains was investigated. This study provides new insight into the beneficial effects of RE that may have some implications in OI therapy targeting cellular stress.

Hibiscus sabdariffa L. (HS) has a long history of edible and medicinal uses. In this study, the biological activities of the extracts, chromatographic fractions, and hibiscus acid obtained from HS were evaluated for their potential bioactivities. Their ability to promote extracellular matrix synthesis in skin fibroblasts was evaluated by enzyme-linked immunosorbent assays. Their anti-inflammatory activity was evaluated in a nitric oxide (NO)–Griess inflammatory experiment. Furthermore, hibiscus acid was found to have a strong anti-oxidative stress effect through the establishment of an oxidative stress model induced by hydrogen peroxide. Several assays indicated that hibiscus acid treatment can effectively reduce extracellular adenosine triphosphate (ATP) secretion and carbonyl protein production, as well as maintain a high level of reduced/oxidized glutathione (GSH/GSSG) in skin cells, thus providing a possible mechanism by which hibiscus acid can counter antioxidative stress. The present study is the first to explore the reversing skin aging potential and the contributory component of HS.

Objective: The aim of this study was to investigate the molecular mechanism of human bone marrow mesenchymal stem cells (hMSCs) secreting miR-26a exosomes on the function of skin fibroblasts. Methods: Exosomes from hMSCs were extracted and identified by transmission electron microscopy, particle size was analyzed and protein markers were detected. Then, the exosomes were co-cultured with human skin fibroblasts (BJ). CCK-8, Annexin V/P and Transwell assays were used to detect the proliferation, apoptosis, and migration of BJ cells. In addition, the expressions of miR-26a, related proteins, and related inflammatory factors were detected by qRT-PCR, western blotting, and ELISA. Results: Compared with the high glucose group, the proliferation rate, migration rate, and the expression of α-SMA, bcl-2, TLR4, NF-κB, TNF-α, IL-6, IL- and IL-1 were significantly decreased in the high glucose + MSC-Exo-miR-26a mimics group, while the apoptosis rate and the expression of miR-26a, cleaved-caspase 3, cleaved-caspase 9 and Bax were significantly increased. The results of the high glucose + MSC-Exo-miR-26a inhibitor group were the opposite. Conclusion: These results suggest that hMSCs cells secreting miR-26a exosomes inhibited the proliferation, migration, and transdifferentiation of high glucose-induced BJ cells, and promoted cell apoptosis, which may be related to the TLR4/NF-κB signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract Biomarkers of aging can be used to assess the health of individuals and to study aging and age-related diseases. We generate a large dataset of genome-wide RNA-seq profiles of human dermal fibroblasts from 133 people aged 1 to 94 years old to test whether signatures of aging are encoded within the transcriptome. We develop an ensemble machine learning method that predicts age to a median error of 4 years, outperforming previous methods used to predict age. The ensemble was further validated by testing it on ten progeria patients, and our method is the only one that predicts accelerated aging in these patients.

：Objective To observe and compare the growth factor release levels of platelet-rich fibrin (PRF) prepared under different centrifugation conditions and its effect on the proliferation and migration of fibroblasts. Methods The whole blood of SD rats was obtained by cardiac blood sampling，and the leukocyte-rich PRF (L-PRF)，advanced PRF (A-PRF)，and advanced PRF+（A-PRF+）were obtained by three centrifugal procedures of 708 g 12 min，208 g 14 min, and 208 g 8 min. The PRF diaphragm was pressed and placed in DMEM/F12 medium which was then collected at 1h，6h, 1 d, 3 d，and 5 d，respectively. The platelet-derived growth factor (PDGF-Ab)，transforming growth factor β (TGFβ)，and vascular endothelial growth factor（VEGF）in the culture medium were detected by ELISA. Skin fibroblasts were isolated and cultured from the dorsal skin of SD rats，and then were divided into the L-PRF group，A-PRF group, A-PRF+ group, and blank group, which were cultured in L-PRF，A-PRF，and A-PRF+ conditioned medium and 10% FBS medium, respectively. CCK-8 cell proliferation assay was used to detect cell proliferation，and Transwell chamber assay was used to detect cell migration. Results The overall release levels of PDGF-Ab，TGF-β and VEGF in A-PRF and A-PRF+ medium were higher than those in L-PRF medium within 5 days after preparation（all P<0. 05）. The cell proliferation abili‐ ties of the A-PRF+ group，A-PRF group，and L-PRF group were higher than that of blank group on day 3 and 5，and the cell proliferation abilities of the A-PRF+ group and A-PRF group were higher than that of L-PRF group on day 3（all P< 0. 05）. The numbers of transmembrane cells in the L-PRF group, A-PRF group，and A-PRF+ group were higher than that in the blank group，and the numbers of transmembrane cells in the A-PRF group and A-PRF+ group were higher than that in the L-PRF group（all P<0. 05). Conclusion The effects of A-PRF+ and A-PRF prepared by reducing the relative cen‐ trifugal force on the growth factor release and promoting the proliferation and migration of fibroblasts were significantly stron‐ ger than that of L-PRF，and the effects of A-PRF+ obtained by appropriately shorting the centrifugal time were stronger. [ABSTRACT FROM AUTHOR]

We have created entirely biological tissue-engineered vascular grafts (TEVGs) using sheets of cell-assembled extracellular matrix (CAM) produced by human fibroblasts in vitro. A large animal TEVG would allow long-term pre-clinical studies in a clinically relevant setting (graft size and allogeneic setting). Therefore, canine, porcine, ovine, and human skin fibroblasts were compared for their ability to form CAM sheets. Serum sourcing greatly influenced CAM production in a species-dependent manner. Ovine cells produced the most homogenous and strongest animal CAM sheets but remained ≈3-fold weaker than human sheets despite variations of serum, ascorbate, insulin, or growth factor supplementations. Key differences in cell growth dynamics, tissue development, and tissue architecture and composition were observed between human and ovine. This study demonstrates critical species-to-species differences in fibroblast behavior and how they pose a challenge when attempting to substitute animal cells for human cells during the development of tissue-engineered constructs that require long-term cultures. [ABSTRACT FROM AUTHOR]

In pet clinics, the number of cases using trauma drugs accounts for >10% of the total number of cases, and most wounds are healing by second intention. The prolongation of wound healing time causes inconvenience and burden to pets and pet owners. Therefore, how to reduce wound healing time and achieve maximum recovery of tissue function and aesthetics is one of the focuses of veterinary clinical practice. Wound suppuration caused by Staphylococcus aureus and Pseudomonas aeruginosa is the main cause of delaying wound healing. Clinically, available antimicrobial treatments are almost exhausted due to the production of large numbers of resistant bacteria. At present, there are no bacteria resistant to traditional Chinese medicine (TCM), which makes TCM have the potential to become an effective drug for the treatment of bacterial infections, so the use of TCM in the treatment of traumatic infections has broad prospects. Based on the characteristics of infection syndrome, three different prescriptions were formulated in our laboratory, and the most effective prescription and dosage form was screened and named Lianrong Healing Cream (LRHC). The results showed that LRHC regulated the expression of fibroblast growth factor-2 (FGF-2), epidermal growth factor-1 (EGF-1), transforming growth factor-β (TGF-β) and vascular endothelial growth factor-1 (VEGF-1) genes in wound tissues and fibroblasts, thereby accelerating wound healing and repairing wound appearance and function. The results of this study may be help to develop TCM formulation for traumatic infections., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Abstract Background Systemic sclerosis (SSc) is characterized by vasculopathy and progressive fibrosis. CTLA4-Ig (abatacept) is able to interact with the cell surface costimulatory molecule CD86 and downregulate the target cell. The aim of this study was to evaluate the in-vitro effects of CTLA4-Ig treatment on circulating fibrocytes and skin fibroblasts isolated from the same SSc patient. Methods Circulating fibrocytes and skin fibroblasts were obtained from eight SSc patients with “limited” cutaneous involvement and from four healthy subjects (HSs). Samples were analyzed by fluorescence-activated cell sorter analysis (FACS) at baseline (T0) and after 8 days of culture (T8) for CD45, collagen type I (COL I), CXCR4, CD14, CD86, and HLA-DRII expression. Circulating fibrocytes were treated for 3 h and skin fibroblasts for 24/48 h with CTLA4-Ig (10, 50, 100, 500 μg/ml). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for CD86, COL I, FN, TGFβ, αSMA, S100A4, CXCR2, CXCR4, CD11a, and Western blotting was performed for COL I and FN. Results Using qRT-PCR, the T8-cultured SSc circulating fibrocytes which had not been treated with CTLA4-Ig showed higher gene expression for CD86, αSMA, S100A4, TGFβ, and COL I compared with HS circulating fibrocytes. Interestingly, αSMA/COL I gene expression was significantly lower only in the SSc circulating fibrocytes treated with CTLA4-Ig for 3 h (p

Abstract Background Myofibroblasts contribute to fibrosis through the overproduction of extracellular matrix (ECM) proteins, primarily type I collagen (COL-1) and fibronectin (FN), a process which is mediated in systemic sclerosis (SSc) by the activation of fibrogenic intracellular signaling transduction molecules, including extracellular signal-regulated kinases 1 and 2 (Erk1/2) and protein kinase B (Akt). Selexipag is a prostacyclin receptor agonist synthesized for the treatment of pulmonary arterial hypertension. The study investigated the possibility for selexipag and its active metabolite (ACT-333679) to downregulate the profibrotic activity in primary cultures of SSc fibroblasts/myofibroblasts and the fibrogenic signaling molecules involved. Methods Fibroblasts from skin biopsies obtained with Ethics Committee (EC) approval from patients with SSc, after giving signed informed consent, were cultured until the 3rd culture passage and then either maintained in normal growth medium (untreated cells) or independently treated with different concentrations of selexipag (from 30 μM to 0.3 μM) or ACT-333679 (from 10 μM to 0.1 μM) for 48 h. Protein and gene expressions of α-smooth muscle actin (α-SMA), fibroblast specific protein-1 (S100A4), COL-1, and FN were investigated by western blotting and quantitative real-time PCR. Erk1/2 and Akt phosphorylation was investigated in untreated and ACT-333679-treated cells by western botting. Results Selexipag and ACT-333679 significantly reduced protein synthesis and gene expression of α-SMA, S100A4, and COL-1 in cultured SSc fibroblasts/myofibroblasts compared to untreated cells, whereas FN was significantly downregulated at the protein level. Interestingly, ACT-333679 significantly reduced the phosphorylation of Erk1/2 and Akt in cultured SSc fibroblasts/myofibroblasts. Conclusions Selexipag and mainly its active metabolite ACT-333679 were found for the first time to potentially interfere with the profibrotic activity of cultured SSc fibroblasts/myofibroblasts at least in vitro, possibly through the downregulation of fibrogenic Erk1/2 and Akt signaling molecules.

Sirtuin 3 (SIRT3) plays a vital role in several dermatological diseases. However, the role and detailed mechanism of SIRT3 in diabetic wound healing are unknown well yet. To explore possible involvement of SIRT3 and necroptosis in diabetic skin wound healing, SIRT3 knockout (KO) mice and 129S1/SvImJ wild‐type (WT) mice were injected with streptozotocin (STZ), and mice skin fibroblasts were exposed to high glucose (HG). It was found that SIRT3 expression decreased in the skin of diabetic patients. SIRT3 deficiency delayed healing rate, reduced blood supply and vascular endothelial growth factor expression, promoted superoxide production, increased malondialdehyde (MDA) levels, decreased total antioxidant capacity (T‐AOC), reduced superoxide dismutase (SOD) activity and aggravated ultrastructure disorder in skin wound of diabetic mice. SIRT3 deficiency inhibited mice skin fibroblasts migration with HG stimulation, which was restored by SIRT3 overexpression. SIRT3 deficiency also suppressed α‐smooth muscle actin (α‐SMA) expression, enhanced superoxide production but decreased mitochondrial membrane potential with HG stimulation after scratch. SIRT3 deficiency further elevated receptor‐interacting protein kinase 3 (RIPK3), RIPK1 and caspase 3 expression both in vitro and in vivo. Collectively, SIRT3 deficiency delayed skin wound healing in diabetes, the mechanism might be related to impaired mitochondria function, enhanced oxidative stress and increased necroptosis. This may provide a novel therapeutic target to accelerate diabetic skin wound healing. [ABSTRACT FROM AUTHOR]