Isolating human dermal fibroblasts using serial explant culture

Nejaddehbashi, Fereshteh; Bayati, Vahid; Mashali, Leila; Hashemitabar, Mahmoud; Abbaspour, Mohammadreza; Moghimipour, Eskandar; Orazizadeh, Mahmoud

doi:10.21037/sci.2019.08.05

Cited by 19 publications

(11 citation statements)

References 28 publications

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“…Our study describedfor the first time the pathogenesis of S. haemolyticus isolated from patients with DFUs on PHSF cells.Staphylococci, including S. haemolyticus ,arewidely distributed in hospital environment and are important causative agents of DFUs [ 23 , 24 ]. We employed an explant culture method for the isolation of the PHSF cells from human skin, which is a simple, reliable andinexpensive methodfor isolation of skin fibroblasts [ 25 ]. Keratinocytes did not attach to tissue culture plates and were removed by washing with PBSbecause keratinocytes need additional nutritional supplements and growth factors that were not included in our media [ 26 ].…”

Section: Discussionmentioning

confidence: 99%

Pathogenesis of Staphylococcus haemolyticus on primary human skin fibroblast cells

et al. 2020

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Staphylococcus haemolyticus ( S. haemolyticus ) is one of the Coagulase-negative staphylococci (CoNS) that inhabits the skin as a commensal. It is increasingly implicated in opportunistic infections, including diabetic foot ulcer (DFU) infections. In contrast to the abundance of information available for S. aureus and S. epidermidis , little is known about the pathogenicity of S. haemolyticus , despite the increased prevalence of this pathogen in hospitalized patients. We described, for the first time, the pathogenesis of different clinical isolates of S. haemolyticus isolated from DFU on primary human skin fibroblast (PHSF) cells. Virulence-related genes were investigated, adhesion and invasion assays were carried out using Giemsa stain, transmission electron microscopy (TEM), MTT and flowcytometry assays. Our results showed that most S. haemolyticus carried different sets of virulence-related genes. S. haemolyticus adhered to the PHSF cells to variable degrees. TEM showed that the bacteria were engulfed in a zipper-like mechanism into a vacuole inside the cell. Bacterial internalization was confirmed using flowcytometry and achieved high intracellular levels. PHSF cells infected with S.haemolyticus suffered from amarked decrease in viability and increased apoptosis when treated with whole bacterial suspensions or cell-free supernatants but not with heat-treated cells. After co-culture with PBMCs, S. haemolyticus induced high levels of pro-inflammatory cytokines. This study highlights the significant development of S. haemolyticus , which was previously considered a contaminant when detected in cultures of clinical samples. Their high ability to adhere, invade and kill the PHSF cells illustrate the severe damage associated with DFU infections. Abbreviations CoNS, coagulase-negative staphylococci; DFU, diabetic foot ulcer; DM, diabetes mellitus; DMEM, Dulbecco’s Modified Eagle Medium; MTT, 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide; PBMCs,peripheral blood mononuclear cells; PHSF, primary human skin fibroblast; CFU, colony-forming unit.

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Section: Discussionmentioning

confidence: 99%

Pathogenesis of Staphylococcus haemolyticus on primary human skin fibroblast cells

et al. 2020

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“…The osteoblastic phenotype of the isolated cells was verified by the extracellular matrix (ECM) mineralization potential of confluent cell cultures at day 28 as described by Perpétuo et al 65 . Immunofluorescent staining of vimentin, a fibroblast specific marker 66 , 67 , was used to verify the fibroblastic phenotype. Osteoblasts and fibroblasts were passaged after reaching a confluence of approximately 80%.…”

Section: Methodsmentioning

confidence: 99%

Human osteoblast and fibroblast response to oral implant biomaterials functionalized with non-thermal oxygen plasma

Rabel

Kohal

Steinberg

et al. 2021

Sci Rep

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Plasma-treatment of oral implant biomaterials prior to clinical insertion is envisaged as a potential surface modification method for enhanced implant healing. To investigate a putative effect of plasma-functionalized implant biomaterials on oral tissue cells, this investigation examined the response of alveolar bone osteoblasts and gingival fibroblasts to clinically established zirconia- and titanium-based implant surfaces for bone and soft tissue integration. The biomaterials were either functionalized with oxygen-plasma in a plasma-cleaner or left untreated as controls, and were characterized in terms of topography and wettability. For the biological evaluation, the cell adhesion, morphogenesis, metabolic activity and proliferation were examined, since these parameters are closely interconnected during cell-biomaterial interaction. The results revealed that plasma-functionalization increased implant surface wettability. The magnitude of this effect thereby depended on surface topography parameters and initial wettability of the biomaterials. Concerning the cell response, plasma-functionalization of smooth surfaces affected initial fibroblast morphogenesis, whereas osteoblast morphology on rough surfaces was mainly influenced by topography. The plasma- and topography-induced differential cell morphologies were however not strong enough to trigger a change in proliferation behaviour. Hence, the results indicate that oxygen plasma-functionalization represents a possible cytocompatible implant surface modification method which can be applied for tailoring implant surface wettability.

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“…Consistent with previous research, the vimentin was a fibroblast-specific marker. 21 Immunofluorescence staining showed that the nuclei of fibroblasts were stained blue and the cytoplasm of fibroblasts was stained green because a vimentin-specific marker was regarded as positively stained cell. The proportion of positively stained cells was nearly 100 percent (Fig.…”

Section: Resultsmentioning

confidence: 99%

USP15 Enhances the Proliferation, Migration, and Collagen Deposition of Hypertrophic Scar–Derived Fibroblasts by Deubiquitinating TGF-βR1 In Vitro

Lin

Huang

et al. 2021

Plastic &Amp; Reconstructive Surgery

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Background: Hypertrophic scar is a fibroproliferative disorder caused by skin injury. The incidence of hypertrophic scar following trauma or burns is 40 to 70 percent or 70 percent, respectively. It has been shown that transforming growth factor (TGF) β1/Smad signaling plays a crucial role in hypertrophic scar, and that USP15 can regulate the activity of TGFβ1/Smad signaling to affect the progression of the disease. However, the underlying mechanism of USP15 in hypertrophic scar remains unclear. The authors hypothesized that USP15 was up-regulated and enhanced the proliferation, migration, invasion, and collagen deposition of hypertrophic scar–derived fibroblasts by deubiquitinating TGF-β receptor I (TβRI) in vitro. Methods: Fibroblasts were isolated from human hypertrophic scars in vitro. The knockdown and overexpression of USP15 in hypertrophic scar–derived fibroblasts were performed using lentivirus infection. The effect of USP15 on hypertrophic scar–derived fibroblast proliferation, migration, and invasion, and the expression of TβRI , Smad2 , Smad3 , α- SMA , COL1 , and COL3 , were detected by Cell Counting Kit-8, scratch, invasion, quantitative real-time polymerase chain reaction, and Western blot assays. The interaction between USP15 and TβRI was detected by co-immunoprecipitation and ubiquitination assays. Results: The authors demonstrated that USP15 knockdown significantly inhibited the proliferation, migration, and invasion of hypertrophic scar–derived fibroblasts in vitro and down-regulated the expression of TβRI , Smad2 , Smad3 , α- SMA , COL1 , and COL3 ; in addition, USP15 overexpression showed the opposite trends ( p < 0.05). Co-immunoprecipitation and ubiquitination assays revealed that USP15 interacted with TβRI and deubiquitinated TβRI. Conclusion: USP15 enhances the proliferation, migration, invasion, and collagen deposition of hypertrophic scar–derived fibroblasts by deubiquitinating TβRI in vitro.

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Isolating human dermal fibroblasts using serial explant culture

Cited by 19 publications

References 28 publications

Pathogenesis of Staphylococcus haemolyticus on primary human skin fibroblast cells

Pathogenesis of Staphylococcus haemolyticus on primary human skin fibroblast cells

Human osteoblast and fibroblast response to oral implant biomaterials functionalized with non-thermal oxygen plasma

USP15 Enhances the Proliferation, Migration, and Collagen Deposition of Hypertrophic Scar–Derived Fibroblasts by Deubiquitinating TGF-βR1 In Vitro

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