Normal Human Dermal Fibroblasts Research Articles

Cell Migration Chip (3DCM-chip): a New Tool Toward the Modelling of 3D Cellular Complex Systems.

3D hydrogel-based cell cultures provide models for studying cell behavior and can efficiently replicate the physiologic environment. Hydrogels can be tailored to mimic mechanical and biochemical properties of specific tissues and allow to produce gel-in-gel models. In this system, microspheres encapsulating cells are embedded in an outer hydrogel matrix, where cells are able to migrate. To enhance the efficiency of such studies, we design a lab-on-a-chip named 3D Cell Migration-chip (3DCM-chip) that offers substantial advantages over traditional methods. 3DCM-chip facilitates the analysis of biochemical and physical stimuli effects on cell migration/invasion in different cell types, including stem, normal and tumor cells. 3DCM-chip provides a smart platform for developing more complex cell co-cultures systems. Herein we investigate the impact of human fibroblasts on MDA-MB 231 breast cancer cells invasiveness. Moreover, how the presence of different cellular lines, including mesenchymal stem cells (hMSCs), normal human dermal fibroblasts (NHDFs), and human umbilical vein endothelial cells (HUVECs), affects the invasive behavior of cancer cells is investigated using 3DCM-chip and producing predictive tumoroid models with a more complex network of interactions between cells and microenvironment. 3DCM-chip moves us closer to creating in vitro systems that can potentially replicate key aspects of the physiological tumor microenvironment. This article is protected by copyright. All rights reserved.

Three-Dimensional Bio-Printed Tubular Tissue Using Dermal Fibroblast Cells as a New Tissue-Engineered Vascular Graft for Venous Replacement.

The current study was a preliminary evaluation of the feasibility and biologic features of three-dimensionally bio-printed tissue-engineered (3D bio-printed) vascular grafts comprising dermal fibroblast spheroids for venous replacement in rats and swine. The scaffold-free tubular tissue was made by the 3D bio-printer with normal human dermal fibroblasts. The tubular tissues were implanted into the infrarenal inferior vena cava of 4 male F344-rnu/rnu athymic nude rats and the short-term patency and histologic features were analyzed. A larger 3D bio-printed swine dermal fibroblast-derived prototype of tubular tissue was implanted into the right jugular vein of a swine and patency was evaluated at 4 weeks. The short-term patency rate was 100%. Immunohistochemistry analysis showed von Willebrand factor positivity on day 2, with more limited positivity observed on the luminal surface on day 5. Although the cross-sectional area of the wall differed significantly between preimplantation and days 2 and 5, suggesting swelling of the tubular tissue wall (both p < 0.01), the luminal diameter of the tubular tissues was not significantly altered during this period. The 3D bio-printed scaffold-free tubular tissues using human dermal or swine fibroblast spheroids may produce better tissue-engineered vascular grafts for venous replacement in rats or swine.

Bead-Free Electrospun Nanofibrous Scaffold Made of PVOH/Keratin/Chitosan Using a Box–Behnken Experimental Design and In Vitro Studies

Bead-free nanofibrous scaffolds composed of PVOH/keratin/chitosan were prepared using electrospinning after optimising the process parameters using a Box–Behnken experimental design. Two quadratic models were developed to optimise the fibre diameter and the diameter of fibre beads considering the voltage, flow rate, spinning distance, and amount of biopolymer as independent variables. All independent variables were found to be significant in determining responses, although not all interactions among these were significant. The models were highly effective in describing responses, with an R2 of 98.58 and 99.67%. The optimum conditions were determined to be 15.82 kV voltage, 0.25 mL/h flow rate, 105 mm spinning distance, and 30% biopolymers. The accuracy of the models was verified and found to be within an acceptable range. The bead-free nanofibrous scaffold exhibited no cytotoxicity to Human Aneuploid Immortal Keratinocyte (HaCaT) and Normal Human Dermal Fibroblast (NHDF) cell lines, enabling cell adhesion and proliferation. Both cell lines remained attached with perfect cell morphology when co-cultured on the scaffold for 30 days, indicating the scaffold’s potential for biomedical applications.

The Effect of Phenolic-Rich Extracts of Rubus fruticosus, R. ulmifolius and Morus nigra on Oxidative Stress and Caco-2 Inhibition Growth.

Currently, a clear interest has been given to berries due to their richness in active metabolites, including anthocyanins and non-coloured phenolics. Therefore, the main aim of the present work is to investigate the phenolic profile, antioxidant abilities, and antiproliferative effects on normal human dermal fibroblasts (NHDF) and human colon carcinoma cell line (Caco-2) cells of phenolic-rich extracts from three red fruits highly appreciated by consumers: two species of blackberries (Rubus fruticosus and Rubus ulmifolius) and one species of mulberry (Morus nigra). A total of 19 different phenolics were identified and quantified by HPLC-DAD-ESI/MSn and HPLC-DAD, respectively. Focusing on the biological potential of the phenolic-rich extracts, all of them revealed notable scavenging abilities. Concerning the antiproliferative properties, R. fruticosus presented a cytotoxic selectivity for Caco-2 cells compared to NHDF cells. To deeper explore the biological potential, combinations with positive controls (ascorbic acid and 5-fluorouracil) were also conducted. Finally, the obtained data are another piece of evidence that the combination of phenolic-rich extracts from natural plants with positive controls may reduce clinical therapy costs and the possible toxicity of chemical drugs.

Copper(I) complexes of acylthiourea ligands: structural insights and cytotoxic potential

The reaction of substituted acylthiourea derivatives (L1–L4) with [(PPh3)2Cu(μ‐Cl)2Cu(PPh3)] in a proper stoichiometric proportion gave the complexes (1–4) in good yields. The structures of the compounds (ligands and complexes) were advocated through spectroscopic analyses and x‐ray diffraction (XRD) studies; the latter confirmed their molecular structures. The complexes (1–4) were assessed for their cytotoxic potential through MTT assay against A549, T24, and HepG2 cancer cells, and their toxicity was evaluated using Vero and MCF‐10a normal cells. The complexes (1–4) displayed better cytotoxic action toward HepG2 cancer cells than cisplatin. Complexes 3 and 4 significantly destroyed A549 cancer cells with the IC50 values of 16.25 and 4.90 μM, respectively, which were lower than that of cisplatin (IC50 = 17.73 μM). Furthermore, the higher IC50 values in normal cells showed that the complexes were less hazardous to the normal cells except complex 3. The biocompatibility study showed that the complexes had minimal impact on normal human dermal fibroblast (NHDF) cells. The mode of cell death of complexes 3 and 4 against cancer cells was evaluated using staining assays. Both compounds demonstrated a significant reduction in live cells, an increase in early apoptotic cells, and pronounced proapoptotic effects, as evidenced by fragmented nuclei. Flow cytometry revealed a distinctive subpopulation in the subG0 phase, highlighting the compounds' ability to induce apoptosis and inhibit cell cycle progression.

A versatile high-throughput assay based on 3D ring-shaped cardiac tissues generated from human induced pluripotent stem cell-derived cardiomyocytes

We developed a 96-well plate assay which allows fast, reproducible, and high-throughput generation of 3D cardiac rings around a deformable optically transparent hydrogel (polyethylene glycol [PEG]) pillar of known stiffness. Human induced pluripotent stem cell-derived cardiomyocytes, mixed with normal human adult dermal fibroblasts in an optimized 3:1 ratio, self-organized to form ring-shaped cardiac constructs. Immunostaining showed that the fibroblasts form a basal layer in contact with the glass, stabilizing the muscular fiber above. Tissues started contracting around the pillar at D1 and their fractional shortening increased until D7, reaching a plateau at 25±1%, that was maintained up to 14 days. The average stress, calculated from the compaction of the central pillar during contractions, was 1.4±0.4 mN/mm2. The cardiac constructs recapitulated expected inotropic responses to calcium and various drugs (isoproterenol, verapamil) as well as the arrhythmogenic effects of dofetilide. This versatile high-throughput assay allows multiple in situ mechanical and structural readouts.

A versatile high-throughput assay based on 3D ring-shaped cardiac tissues generated from human induced pluripotent stem cell-derived cardiomyocytes.

We developed a 96-well plate assay which allows fast, reproducible, and high-throughput generation of 3D cardiac rings around a deformable optically transparent hydrogel (polyethylene glycol [PEG]) pillar of known stiffness. Human induced pluripotent stem cell-derived cardiomyocytes, mixed with normal human adult dermal fibroblasts in an optimized 3:1 ratio, self-organized to form ring-shaped cardiac constructs. Immunostaining showed that the fibroblasts form a basal layer in contact with the glass, stabilizing the muscular fiber above. Tissues started contracting around the pillar at D1 and their fractional shortening increased until D7, reaching a plateau at 25±1%, that was maintained up to 14 days. The average stress, calculated from the compaction of the central pillar during contractions, was 1.4±0.4 mN/mm2. The cardiac constructs recapitulated expected inotropic responses to calcium and various drugs (isoproterenol, verapamil) as well as the arrhythmogenic effects of dofetilide. This versatile high-throughput assay allows multiple in situ mechanical and structural readouts.

Exacerbated Inflammatory Gene Expression After Impaired G2/M-Checkpoint Arrest in Fibroblasts Derived From a Patient Exhibiting Severe Adverse Effects

Purpose: Recent radiotherapy (RT), such as intensity-modulated radiation therapy and particle RT, has improved the concentration of the radiation field targeting tumors. However, severe adverse effects still occur, possibly due to genetic factors in patients. We aimed to investigate the mechanism of exacerbated inflammation during RT. Methods and MaterialsDermal fibroblasts derived from a patient with severe inflammatory adverse effects during RT were compared with two normal human dermal fibroblasts. Micronuclei formation, G2/M-checkpoint arrest, DNA damage signaling and repair, and inflammatory gene expression were comprehensively examined. Results: We found greater micronuclei formation in radiation-sensitive fibroblasts (RS-Fs) after ionizing radiation (IR). Importantly, RS-Fs exhibited premature G2/M-checkpoint release after IR, which triggers micronuclei formation because RS-Fs undergo mitosis with unrepaired DNA double-strand breaks (DSBs). Additionally, we found that DSB end-resection and activation of the ATR-Chk1 pathway were impaired in RS-Fs after IR. Consistent with the increase in the formation of micronuclei, which can deliver cytosolic nucleic acids resulting in an innate immune response, the expression of genes associated with inflammatory responses was highly upregulated in RS-Fs after IR. Conclusions: Although this is a single case of RT-dependent adverse effect, our findings suggest that impaired G2/M-checkpoint arrest due to the lack of DSB end-resection and activation of the ATR-Chk1 pathway causes exacerbated inflammation during RT; therefore, genes involved in G2/M-checkpoint arrest may be a predictive marker for unexpected inflammatory responses in RT.

An innovative Fuller’s earth-based film-forming formulation for skin decontamination, through removal and entrapment of an organophosphorus compound, paraoxon-ethyl

Organophosphorus compounds (OPs), such as VX, pose a significant threat due to their neurotoxic and hazardous properties. Skin decontamination is essential to avoid irreversible effects. Fuller’s earth (FE), a phyllosilicate conventionally employed in powder form, has demonstrated decontamination capacity against OPs. The aim of this study was to develop a formulation that forms a film on the skin, with a significant OP removal capacity (>95%) coupled with sequestration capabilities, favorable drying time and mechanical properties to allow for easy application and removal, particularly in emergency context. Various formulations were prepared using different concentrations of polyvinyl alcohol (PVA), FE and surfactants. Their removal and sequestration capacity was tested using paraoxon-ethyl (POX), a chemical that simulates the behavior of VX. Formulations with removal capacity levels surpassing 95% were mechanically characterized and cell viability assays were performed on Normal Human Dermal Fibroblast (NHDF). The four most promising formulations were used to assess decontamination efficacy on pig ear skin explants. These formulations showed decontamination levels ranging from 84.4 ± 4.7% to 96.5 ± 1.3%, which is equivalent to current decontamination methods. These results suggest that this technology could be a novel and effective tool for skin decontamination following exposure to OPs.

Bacterial nanocellulose loaded with bromelain and nisin as a promising bioactive material for wound debridement

The bacterial nanocellulose (BnC) membranes were produced extracellularly by a novel aerobic acetic acid bacterium Komagataeibacter melomenusus. The BnC was modified in situ by adding carboxymethyl cellulose (CMC) into the culture media, obtaining a BnC-CMC product with denser fibril arrangement, improved rehydration ratio and elasticity in comparison to BnC. The proteolytic enzyme bromelain (Br) and antimicrobial peptide nisin (N) were immobilized to BnC matrix by ex situ covalent binding and/or adsorption. The optimal Br immobilization conditions towards the maximized specific proteolytic activity were investigated by response surface methodology as factor variables. At optimal conditions, i.e., 8.8 mg/mL CMC and 10 mg/mL Br, hyperactivation of the enzyme was achieved, leading to the specific proteolytic activity of 2.3 U/mg and immobilization efficiency of 39.1 %. The antimicrobial activity was observed against Gram-positive bacteria (S. epidermidis, S. aureus and E. faecalis) for membranes with immobilized N and was superior when in situ modified BnC membranes were used. N immobilized on the BnC or BnC-CMC membranes was cytocompatible and did not cause changes in normal human dermal fibroblast cell morphology. BnC membranes perform as an efficient carrier for Br or N immobilization, holding promise in wound debridement and providing antimicrobial action against Gram-positive bacteria, respectively.

Merging Modular Molecular Design with High Throughput Screening of Cell Adhesion on Antimicrobial Supramolecular Biomaterials.

A polymer microarray based on the supramolecular ureido-pyrimidinone (UPy) moiety was fabricated to screen antimicrobial materials for their ability to support cell adhesion. UPy-functionalized additives, either cell-adhesive, antimicrobial or control peptides, were used, and investigated in different combinations at different concentrations, resulting in a library of 194 spots. These were characterized on composition and morphology to evaluate the microarray fabrication. Normal human dermal fibroblasts were cultured on the microarrays and cell adhesion to the spots was systematically analyzed. Results demonstrated enhanced cell adhesion on spots with combinations including the antimicrobial peptides. This study clearly proves the power of the high throughput approach in combination with supramolecular molecules, to screen additive libraries for desired biological response. This article is protected by copyright. All rights reserved.

Abstract 5232: Placental circulating T cells expressing CD16 in combination with trastuzumab demonstrate robust anti-tumor antibody-dependent cellular cytotoxicity (ADCC) against gastric cancer

Abstract Introduction: Celularity is developing PT-CD16VS as a novel platform for use in combination with approved monoclonal antibodies for the potential treatment of multiple cancers. PT-CD16VS is an allogeneic cell therapy derived from human postpartum placental circulating T (P-T) cells that are genetically modified to express a proprietary CD16 variant and endogenous T cell receptor (TCR) knockout. Here we report the characterization and preclinical evaluation of PT-CD16VS against HER2+ gastric cancer cells in combination with Trastuzumab. In vitro safety studies evaluating the potential for on-target/off-tumor activity of PT-CD16VS against HER2 low expressing lung epithelial cells (LEC) and human dermal fibroblasts (HDF) are also highlighted. Methods: P-T cells were activated and transduced with a lentiviral vector containing a CD16 construct expressing a high affinity CD16 variant, CD16VS. Following transduction, P-T cells were transfected to knock out the TCR. In vitro, the functional activity of PT-CD16VS cells in combination with Trastuzumab was assessed against the HER2+ gastric cancer NCI-N87 cell line in cytotoxicity, cytokine release, and proliferation assays. For on-target/off-tumor studies, similar assays were utilized to assess activity against the HER2 low expressing LEC and HDF cell lines. In vivo, PT-CD16VS with Trastuzumab was evaluated in a subcutaneous NCI-N87 xenograft model in NSG mice. Tumor volume was measured and tumor samples were evaluated for PT-CD16VS infiltration. Results: High CD16 transduction efficiency (&amp;gt;60%) was achieved across donors. In vitro, PT-CD16VS plus Trastuzumab demonstrated potent cytotoxicity against NCI-N87 cells at low E:T ratios (0.625:1) and at 12 hours of co-culture with moderate cytokine secretion at 24-hour of co-culture. PT-CD16VS proliferated following 48-hour incubation with NCI-N87 cells and Trastuzumab, as compared to an IgG1 control. PT-CD16VS in combination with Trastuzumab did not elicit on-target/off-tumor cytotoxicity or cytokine release in response to low HER2 expression on normal LEC or HDF cells. In vivo, PT-CD16VS with Trastuzumab demonstrated significant reduction in tumor volume compared to vehicle, Trastuzumab alone, and the positive control Enhertu groups. In addition, PT-CD16VS infiltration into the tumor was shown to be Trastuzumab dependent, with infiltrating cells expressing high levels of CD16 and Ki67. Conclusions: Our results show that PT-CD16VS has robust in vitro and in vivo ADCC activity against gastric cancer tumor cells when combined with Trastuzumab. Furthermore, PT-CD16VS activity is specific to tumor HER2 antigen expression and does not target low HER2 expression on normal healthy cells. PT-CD16VS is being developed in combination with various monoclonal antibodies for the treatment of multiple cancers. Citation Format: Kathy Karasiewicz-Mendez, Joseph Gleason, Kristina Tess, Mansour Djedaini, Shengchen Lin, Chenfei Huang, Shuyang He, Robert Hariri, Adrian Kilcoyne. Placental circulating T cells expressing CD16 in combination with trastuzumab demonstrate robust anti-tumor antibody-dependent cellular cytotoxicity (ADCC) against gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5232.

Rosemary extract and rosmarinic acid accelerate elastic fiber formation by increasing the expression of elastic fiber components in dermal fibroblasts.

Ultraviolet (UV)-induced skin photoaging is caused by qualitative and quantitative degradation of dermal extracellular matrix components such as collagen and elastic fibers. Elastic fibers are important for maintaining cutaneous elasticity, despite their small amount in the skin. Previously, microfibril-associated protein 4 (MFAP-4), which is downregulated in photoaging dermis, has been found to be essential for elastic fiber formation by interaction with both fibrillin-1 and elastin, which are core components of elastic fiber. In addition, enhanced cutaneous MFAP-4 expression in a human skin-xenografted murine photoaging model protects against UV-induced photodamage accompanied by the prevention of elastic fiber degradation and aggravated elasticity. We therefore hypothesized that the upregulation of MFAP-4 in dermal fibroblasts may more efficiently accelerate elastic fiber formation. We screened botanical extracts for MFAP-4 expression-promoting activity in normal human dermal fibroblasts (NHDFs). We found that rosemary extract markedly promotes early microfibril formation and mature elastic fiber formation along with a significant upregulation of not only MFAP-4 but also fibrillin-1 and elastin in NHDFs. Furthermore, rosmarinic acid, which is abundant in rosemary extract, accelerated elastic fiber formation via upregulation of transforming growth factor β-1. This was achieved by the induction of cAMP response element-binding protein phosphorylation, demonstrating that rosmarinic acid represents one of the active ingredients in rosemary extract. Based on the findings in this study, we conclude that rosemary extract and rosmarinic acid represent promising materials that exert a preventive or ameliorative effect on skin photoaging by accelerating elastic fiber formation.

Gas foamed scaffolds as smart 3D structures in skin tissue engineering

Globally, wound care is a major problem that poses big issues for national healthcare systems. Consequently, the development of wound dressings, systems applied over a wound to prevent complications and speed up recovery, is receiving increasing attention. Electrospun nanofibrous scaffolds gained great interest in the medical field due to their many advantages and beneficial characteristics. However, they are formed by of densely packed fiber layers, which render the available space between adjacent fibers minimal. In this study, nanometric fibers from pullulan polymeric blends were developed using cricket powder as novel component, due to its high chitin and chitosan content. Hydroxyapatite was also added due to its biocompatibility and capability to interact with the host tissues. The 2D mats based on electropsun fibers in membrane were converted to a 3D structure by gas foaming, involving NaBH4. The obtained 3D systems were characterized for their morphology and composition, and mechanical properties. The scaffolds biocompatibility was tested on normal human dermal fibroblasts and mesenchymal stem cells, and cell adhesion was assessed. TNF-α secretion was thereafter characterized to evaluate the anti-inflammatory effects of the scaffolds on macrophages, and their antioxidant activity was also investigated. Finally, an evaluation of the scaffolds’ safety and efficacy in vivo on a murine incisional and burn model was performed. The results obtained not only revealed greater structural and mechanical integrity of the gas-foamed scaffolds containing cricket powder, but also higher cytocompatibility towards different cell lines together with anti-inflammatory and antioxidant properties, thereby making them innovative tools to improve the process of wound healing.

Sustainable whey proteins-nanostructured zinc oxide-based films for the treatment of chronic wounds: New insights from biopharmaceutical studies

Chronic wounds represent silent epidemic affecting a large portion of the world population, especially the elders; in this context, the development of advanced bioactive dressings is imperative to accelerate wound healing process, while contrasting or preventing infections. The aim of the present work was to provide a deep characterization of the functional and biopharmaceutical properties of a sustainable thin and flexible films, composed of whey proteins alone (WPI) and added with nanostructured zinc oxide (WPZ) and intended for the management of chronic wounds. The potential of whey proteins-based films as wound dressings has been confirmed by their wettability, hydration properties, elastic behavior upon hydration, biodegradation propensity and, when added with nanostructured zinc oxide, antibacterial efficacy against both Gram-positive and Gram-negative pathogens, i.e. Staphylococcus aureus and Escherichia coli. In-vitro experiments, performed on normal human dermal fibroblasts, confirmed film cytocompatibility, also revealing the possible role of Zn2+ ions in promoting fibroblast proliferation. Finally, in-vivo studies on rat model confirmed film suitability to act as wound dressing, since able to ensure a regular healing process while providing effective protection from infections. In particular, both films WPI and WPZ are responsible for the formation in the wound bed of a continuous collagen layer similar to that of healthy skin.

Argon plasma-modified bacterial nanocellulose: Cell-specific differences in the interaction with fibroblasts and endothelial cells

Unique properties of bacterial nanocelullose (BNC), such as high purity, water retention, nanofibrillar structure and non-cytotoxicity, allow its application in regenerative medicine, particularly for active wound healing and for skin tissue engineering. However, its biological activity is not fully sufficient to be considered a good cell scaffold. This can be enhanced by physicochemical modifications, particularly plasma treatment. In this work, air-dried (AD) or lyophilized (L) BNC samples were modified in a direct current Ar+ plasma discharge (PM). BNC_AD samples showed higher surface roughness, tensile strength and Young's modulus but a lower swelling ratio than BNC_L samples. The swelling ratio of BNC_L samples further increased after PM. The samples were subjected to six-day in vitro cell culture tests with normal human dermal fibroblasts (NHDFs) or human umbilical vein endothelial cells (HUVECs), i.e. cell types important for skin defect healing. NHDFs adhered in higher numbers, by a larger cell spreading area and reached higher cell population densities on PM samples than on unmodified samples. However, in HUVECs, this effect of PM on cell growth was less pronounced or even opposite, especially on mechanically weaker and more swellable BNC_L. At the same time, both cell types preferred mechanically stronger BNC_AD for their adhesion and growth, which was more apparent in HUVECs. The expression of mRNA for markers of cell adhesion and phenotypic maturation in NHDFs (talin, vinculin, CD90) was generally similar on BNC_PM and on tissue culture polystyrene. In HUVECs, the expression of vinculin and PECAM-1 on all BNC samples was lower than on polystyrene, but the expression of KDR (VEGF receptor 2) was higher, especially on BNC_PM. These results indicate cell type-specific differences in the response to various BNC modifications, which must be properly balanced to accommodate various cell types needed for wound healing and skin tissue engineering.

Elevated expression of interleukin-6 (IL-6) and serum amyloid A (SAA) in the skin and the serum of recessive dystrophic epidermolysis bullosa: Skin as a possible source of IL-6 through Toll-like receptor ligands and SAA.

The effect of persistent skin inflammation on extracutaneous organs and blood is not well studied. Patients with recessive dystrophic epidermolysis bullosa (RDEB), a severe form of the inherited blistering skin disorder, have widespread and persistent skin ulcers, and they develop various complications including anaemia, hyperglobulinaemia, hypoalbuminaemia and secondary amyloidosis. These complications are associated with the bioactivities of IL-6, and the development of secondary amyloidosis requires the persistent elevation of serum amyloid A (SAA) level. We found that patients with RDEB had significantly higher serum levels of IL-6 and SAA compared to healthy volunteers and patients with psoriasis or atopic dermatitis. Both IL-6 and SAA were highly expressed in epidermal keratinocytes and dermal fibroblasts of the skin ulcer lesions. Keratinocytes and fibroblasts surrounding the ulcer lesions are continuously exposed to Toll-like receptor (TLR) ligands, pathogen-associated and damage-associated molecular pattern molecules. Invitro, TLR ligands induced IL-6 expression via NF-κB in normal human epidermal keratinocytes (NHEKs) and dermal fibroblasts (NHDFs). SAA further induced the expression of IL-6 via TLR1/2 and NF-κB in NHEKs and NHDFs. The limitation of this study is that NHEKs and NHDFs were not derived from RDEB patients. These observations suggest that TLR-mediated persistent skin inflammation might increase the risk of IL-6-related systemic complications, including RDEB.

Simvastatin Abrogates TGF-β Induced Stimulation of Normal Human Dermal Fibroblast Collagen Gene Expression in Culture

A potent stimulation of fibroblast collagen production is one of the crucial pleotropic effects of transforming growth factor β (TGF-β) and has been considered to play a crucial role in the pathogenesis of fibrotic diseases including Systemic Sclerosis and Pulmonary Fibrosis. This complex process involves numerous intracellular reactions mediated by canonical Smad-dependent or non-canonical pathways that transduce the extracellular stimuli into the nucleus. Here, we demonstrated that Simvastatin, a widely used statin, induces a potent inhibition of TGF-β1 profibrotic effects in cultured normal human dermal fibroblasts, and studied the molecular mechanisms involved in these effects. We also examined Simvastatin modulation of TGF-β1 induced fibroblast to myofibroblast transition. Normal human dermal fibroblasts were cultured with various concentrations of Simvastatin in the presence or absence of TGF-β1 (10ng/ml) for 24, 48, and 72 h. The effects of Simvastatin on TGF-β1 stimulation of COL1A1 expression and type 1 collagen production were examined. Assessment of Smad2/3 and Erk1/2 phosphorylation, chromatin immunoprecipitation assays for Sp1 transcription factor binding to the COL1A1 proximal promoter, siRNA-mediated RhoA knockdown, and F-actin immunofluorescence microscopy was performed to examine the molecular mechanisms involved.

MiR-146a is a critical target associated with multiple biological pathways of skin aging.

Introduction: The skin is the largest organ of the human body and fulfills protective, immune, and metabolic functions. Skin function and barrier integrity are actively regulated through circadian rhythm-associated genes and epigenetic mechanisms including DNA methylation/demethylation, histone acetylation/deacetylation, and microRNAs. MicroRNA-146a-5p (miR-146a) has been associated with immune activation and skin inflammation; however, the role of miR-146a in regulating skin aging is an open question. This study investigated the role of miR-146a in fibroblasts obtained from different donors in the context of aging, and a potential association of this miRNA with circadian rhythm. Methods: Normal human dermal fibroblasts (NHDFs) from 19y, 27y, 40y, and 62y old donors were used to analyze for miR-146a expression. Expression of miR-146a was downregulated with the hsa-mirVana miR-146a inhibitor, and upregulated with an extract from Adansonia digitata. Effects on markers of skin aging, including cell proliferation, production of Collagen-1 and inflammatory cytokines were assessed. Results: We show that the expression of miR-146a decreases with age in dermal fibroblasts and inhibition of miR-146a in 19y and 62y old NHDFs induced significant changes in essential clock genes indicating an association with circadian rhythm control. Furthermore, downregulation of miR-146a results in a reduction of cellular proliferation, Collagen-1 production, as well as an increase in DNA damage and pro-inflammatory markers. Activation of miR-146a with the Adansonia digitata extract reduced the deleterious effects seen during miR-146a inhibition and increased miR-146a transport through exosome transfer. Conclusion: miR-146a interacts with multiple biological pathways related to skin aging, including circadian rhythm machinery, cell-to-cell communication, cell damage repair, cell proliferation, and collagen production and represents a promising target to fight skin aging. Adansonia digitata extract can promote miR-146a expression and therefore support skin cells' health.

3D modeling of normal skin and cutaneous squamous cell carcinoma. A comparative study in 2D cultures, spheroids, and 3D bioprinted systems.

The current cancer research and drug testing are primarily based on 2D cell cultures and animal models. However, these methods have limitations and yield distinct drug response patterns. This study addressed this gap by developing an innovativein vitrohuman three-dimensional (3D) normal skin model and a multicellular model of human cutaneous squamous cell carcinoma (cSCC) using 3D bioprinting technology. Comparative analyzes were performed between bioprinted 3D-cSCC model, consisting of HaCaT keratinocytes, primary normal human dermal fibroblasts and A431 cancer cells (tricellular), bioprinted 3D-A431 model composed of A431 cancer cells only (monocellular), A431 cancer cell spheroids, and conventional 2D models. The models were structurally characterized by light microscopy, immunofluorescence (LIVE/DEAD assay, confocal microscopy) and immunohistochemistry (hematoxylin/eosin, p63, vimentin, Ki67, epidermal growth factor receptor stainings). The spatial arrangement of the 3D models was analyzed using the ARIVIS scientific image analysis platform. All models were also functionally assessed by cetuximab (CTX) response testing with the MTS assay. 3D-cSCC models were maintained for up to 16 weeks. Morphological and histological examinations confirmed the presence of skin-like layers in the bioprinted 3D models of normal skin, and the intricate and diverse features of the bioprinted skin cancer model, replicating the criticalin vivocharacteristics. In both mono- and tricellular bioprinted tumor constructs, there was a gradual formation and continuous growth of spheroid-like clusters of cancer cells, significantly influencing the morphology of the models. Cancer cells in the 3D bioprinted constructs showed reduced sensitivity to CTX compared to spheroids and 2D cultures. This study underscores the potential of 3D multicellular models in elucidating drug responses and gaining a better understanding the intricate interplay of cellular components within the tumor microenvironment. Developing the multicellular 3D tumor model paves the way for new research critical to advancing fundamental cancer research and future clinical applications, particularly drug response testing.