scholarly journals Folate bioavailability in reconstructed skin models and effects of folate in a monolayer wound healing assay. Approaches on topic application.

2021 ◽  
Author(s):  
Dirk Dressler ◽  
Martin Ulmann ◽  
Gerd Wiesler
Keyword(s):  
Wound Healing ◽  
Skin Penetration ◽  
Human Keratinocytes ◽  
Primary Human Keratinocytes ◽  
Scratch Assay ◽  
Reconstructed Skin ◽  
Metabolic Role ◽  
Folate Bioavailability ◽  
Enhance Wound Healing

In chronic and degenerative diseases impacting the skin folates play an important metabolic role improving wound healing and reducing skin irritations. In contrast to systemic folate administration little is known on skin penetration of folates after topical application. Here the penetration of simple aqueous solutions of reduced folates have been investigated with in-vitro reconstructed skin models mimicking the barrier of native human skin. For up to 24 h, penetration of the epidermis by newly developed folate salts and formulations were investigated. Aqueous and lipophilic solutions of L-formyltetrahydrofolate and L-mefolinate salts were able to penetrate the epidermis. Even more importantly, the skin model revealed the metabolic conversion of L-folinate to L-methyltetrahydrofolate. Exemplarily the effects of these new folate salts have been tested on wound healing in a scratch assay with primary human keratinocytes. All folates applied were able to enhance wound healing compared to the control.

scholarly journals Achyrocline satureioides (Lam.) DC (Asteraceae) Extract-Loaded Nanoemulsions as a Promising Topical Wound Healing Delivery System: In Vitro Assessments in Human Keratinocytes (HaCaT) and HET-CAM Irritant Potential

Pharmaceutics ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1241
Author(s):  
Lucélia Albarello Balestrin ◽  
Tainá Kreutz ◽  
Flávia Nathiely Silveira Fachel ◽  
Juliana Bidone ◽  
Nicolly Espindola Gelsleichter ◽  
...  
Keyword(s):  
Wound Healing ◽  
Hacat Cell ◽  
Chorioallantoic Membrane ◽  
Human Keratinocytes ◽  
Scratch Assay ◽  
Hen’S Egg ◽  
And Migration ◽  
Achyrocline Satureioides ◽  
Proliferation And Migration

Achyrocline satureioides (Lam.) DC Asteraceae extracts (ASEs) have been investigated for the treatment of various skin disorders. This study reports the effects of ASE-loaded nanoemulsions (NEASE) on the cellular viability, death by necrosis, and migration of immortalized human keratinocytes (HaCaT cell line), as well as the irritant potential through the hen’s egg chorioallantoic membrane test (HET-CAM). NEASE exhibited a polydispersity index above 0.12, with a droplet size of 300 nm, ζ-potential of −40 mV, and content of flavonoids close to 1 mg/mL. No cytotoxicity of the ASE was observed on HaCaT by MTT assay (up to 10 µg/mL). A significant increase of HaCaT viability was observed to NEASE (up to 5 μg/mL of flavonoids), compared to treatment with the ASE. The necrosis death evaluation demonstrated that only NEASE did not lead to cell death at all the tested concentrations. The scratch assay demonstrated that NEASE was able to increase the cell migration at low flavonoid concentrations. Finally, the HET-CAM test proved the non-irritative potential of NEASE. Overall, the results indicate the potential of the proposed formulations for topical use in wound healing, in view of their promising effects on proliferation and migration in keratinocytes, combined with an indication of the absence of cytotoxicity and non-irritating potential.

IL-6 as a corneal wound healing mediator in an in vitro scratch assay

2014 ◽  
Vol 125 ◽  
pp. 183-192 ◽  
Author(s):  
Isabel Arranz-Valsero ◽  
Laura Soriano-Romaní ◽  
Laura García-Posadas ◽  
Antonio López-García ◽  
Yolanda Diebold
Keyword(s):  
Wound Healing ◽  
Scratch Assay ◽  
Corneal Wound Healing ◽  
Corneal Wound

scholarly journals HaCaT Cells as a Reliable In Vitro Differentiation Model to Dissect the Inflammatory/Repair Response of Human Keratinocytes

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Irma Colombo ◽  
Enrico Sangiovanni ◽  
Roberta Maggio ◽  
Carlo Mattozzi ◽  
Stefania Zava ◽  
...  
Keyword(s):  
Cell Density ◽  
Skin Diseases ◽  
Inflammatory Responses ◽  
Human Keratinocytes ◽  
Suitable Model ◽  
Hacat Cells ◽  
Primary Human Keratinocytes ◽  
Proinflammatory Mediators ◽  
Cytokines And Chemokines

Cultured primary human keratinocytes are frequently employed for studies of immunological and inflammatory responses; however, interpretation of experimental data may be complicated by donor to donor variability, the relatively short culture lifetime, and variations between passages. To standardize the in vitro studies on keratinocytes, we investigated the use of HaCaT cells, a long-lived, spontaneously immortalized human keratinocyte line which is able to differentiate in vitro, as a suitable model to follow the release of inflammatory and repair mediators in response to TNFα or IL-1β. Different treatment conditions (presence or absence of serum) and differentiation stimuli (increase in cell density as a function of time in culture and elevation of extracellular calcium) were considered. ELISA and Multiplex measurement technologies were used to monitor the production of cytokines and chemokines. Taken together, the results highlight that Ca2+ concentration in the medium, cell density, and presence of serum influences at different levels the release of proinflammatory mediators by HaCaT cells. Moreover, HaCaT cells maintained in low Ca2+ medium and 80% confluent are similar to normal keratinocytes in terms of cytokine production suggesting that HaCaT cells may be a useful model to investigate anti-inflammatory interventions/therapies on skin diseases.

scholarly journals Human Keratinocyte Growth and Differentiation on Acellular Porcine Dermal Matrix in relation to Wound Healing Potential

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Robert Zajicek ◽  
Vaclav Mandys ◽  
Ondrej Mestak ◽  
Jan Sevcik ◽  
Radana Königova ◽  
...  
Keyword(s):  
Wound Healing ◽  
Human Keratinocytes ◽  
Dermal Matrix ◽  
Keratinocyte Proliferation ◽  
Proliferation And Differentiation ◽  
A Cell ◽  
Cell Culture Assay ◽  
Air Liquid Interface

A number of implantable biomaterials derived from animal tissues are now used in modern surgery. Xe-Derma is a dry, sterile, acellular porcine dermis. It has a remarkable healing effect on burns and other wounds. Our hypothesis was that the natural biological structure of Xe-Derma plays an important role in keratinocyte proliferation and formation of epidermal architecturein vitroas well asin vivo. The bioactivity of Xe-Derma was studied by a cell culture assay. We analyzed growth and differentiation of human keratinocytes culturedin vitroon Xe-Derma, and we compared the results with formation of neoepidermis in the deep dermal wounds treated with Xe-Derma. Keratinocytes cultured on Xe-Derma submerged in the culture medium achieved confluence in 7–10 days. After lifting the cultures to the air-liquid interface, the keratinocytes were stratified and differentiated within one week, forming an epidermis with basal, spinous, granular, and stratum corneum layers. Immunohistochemical detection of high-molecular weight cytokeratins (HMW CKs), CD29, p63, and involucrin confirmed the similarity of organization and differentiation of the cultured epidermal cells to the normal epidermis. The results suggest that the firm natural structure of Xe-Derma stimulates proliferation and differentiation of human primary keratinocytes and by this way improves wound healing.

scholarly journals Inhibition of Calcineurin/NFAT Signaling Blocks Oncogenic H-Ras Induced Autophagy in Primary Human Keratinocytes

2021 ◽  
Vol 9 ◽  
Author(s):  
Shuangshuang Wang ◽  
Hua Qian ◽  
Liwei Zhang ◽  
Panpan Liu ◽  
Dexuan Zhuang ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Human Keratinocytes ◽  
Primary Human Keratinocytes ◽  
Activated T Cells ◽  
Ras Pathway ◽  
Ras Family ◽  
Interfering Rna

Mutations of H-Ras, a member of the RAS family, are preferentially found in cutaneous squamous cell carcinomas (SCCs). H-Ras has been reported to induce autophagy, which plays an essential role in tissue homeostasis in multiple types of cancer cells and in fibroblasts, however, the potential role of H-Ras in regulating autophagy in human keratinocytes has not been reported. In this study, we found that the stable expression of the G12V mutant of H-RAS (H-RasG12V) induced autophagy in human keratinocytes, and interestingly, the induction of autophagy was strongly blocked by inhibiting the calcineurin/nuclear factor of activated T cells (NFAT) pathway with either a calcineurin inhibitor (Cyclosporin A) or a NFAT inhibitor (VIVIT), or by the small interfering RNA (siRNA) mediated knockdown of calcineurin B1 or NFATc1 in vitro, as well as in vivo. To characterize the role of the calcineurin/NFAT pathway in H-Ras induced autophagy, we found that H-RasG12V promoted the nuclear translocation of NFATc1, an indication of the activation of the calcineurin/NFAT pathway, in human keratinocytes. However, activation of NFATc1 either by the forced expression of NFATc1 or by treatment with phenformin, an AMPK activator, did not increase the formation of autophagy in human keratinocytes. Further study revealed that inhibiting the calcineurin/NFAT pathway actually suppressed H-Ras expression in H-RasG12V overexpressing cells. Finally, chromatin immunoprecipitation (ChIP) assays showed that NFATc1 potentially binds the promoter region of H-Ras and the binding efficiency was significantly enhanced by the overexpression of H-RasG12V, which was abolished by treatment with the calcineurin/NFAT pathway inhibitors cyclosporine A (CsA) or VIVIT. Taking these data together, the present study demonstrates that the calcineurin/NFAT signaling pathway controls H-Ras expression and interacts with the H-Ras pathway, involving the regulation of H-Ras induced autophagy in human keratinocytes.

scholarly journals A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis

2017 ◽  
Author(s):  
Ajay Mishra ◽  
Angela Oliveira Pisco ◽  
Benedicte Oules ◽  
Tony Ly ◽  
Kifayathullah Liakath-Ali ◽  
...  
Keyword(s):  
Stem Cell ◽  
Terminal Differentiation ◽  
Human Keratinocytes ◽  
Cell Renewal ◽  
Stable States ◽  
Primary Human Keratinocytes ◽  
Network Modelling ◽  
Epidermal Homeostasis

AbstractEpidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation1,2. While progress has been made in characterising the stem and differentiated cell compartments3, the transition between the two cell states, termed commitment4, is poorly understood. Here we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension for up to 12h. We have previously shown that commitment begins at approximately 4h and differentiation is initiated by 8h5. We find that cell detachment induces a network of protein phosphatases. The pro-commitment phosphatases – including DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA – promote terminal differentiation by negatively regulating ERK MAPK and positively regulating key API transcription factors. Their activity is antagonised by concomitant upregulation of the anti-commitment phosphatase DUSP10. The phosphatases form a dynamic network of transient positive and negative interactions, with DUSP6 predominating at commitment. Boolean network modelling identifies a mandatory switch between two stable states (stem cell and differentiated cell) via an unstable (committed) state. In addition phosphatase expression is spatially regulated relative to the location of stem cells, both in vivo and in response to topographical cues in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment.

In-vitro Wound Healing Properties of Commiphora swynnertonii Resinous Extracts

2021 ◽  
Vol 38 ◽  
pp. 32-37
Author(s):  
G.G. Bakari ◽  
S.A. Mshamu ◽  
M.H. Ally ◽  
R.A. Max ◽  
H. Bai
Keyword(s):  
Wound Healing ◽  
Herbal Remedies ◽  
3T3 Cells ◽  
Scratch Assay ◽  
Sequence Of Events ◽  
Nih 3T3 ◽  
Wound Scratch Assay ◽  
Commiphora Swynnertonii ◽  
Nih 3T3 Cells

Wound healing is a complex multicellular process involving many cell types which include; inflammatory cells, endothelial cells, fibroblasts and keratinocytes. The process involves an orderly sequence of events with four overlapping phases namely; haemostasis, inflammatory, proliferation and remodeling phases.  The process can be facilitated by the use of wound healing agents including herbal remedies from plants. In this study the main objective was to evaluate the in vitro wound healing activity of the resin obtained from Commiphora swynnertonii (C.swynnertonii). First the NIH -3T3 cells viability were evaluated using (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl Tetrazolium Bromide (MTT) assay. Then the wound scratch assay model was used to evaluate cellular proliferation, closure of the wound and release of matrix metalloproteinase enzymes. Results indicate differences in mean cell viability between different concentrations within 24 hours of incubation. The highest viability was recorded at the concentration of 1% (v/v). The in-vitro wound scratch assay showed positive NIH - 3T3 cells proliferation on the wound area and cells migration when compared with control group (without treatment) at 0 and 24 hours. In addition, C. swynnertonii was able to stimulate secretion of MMP-2 release from NIH - 3T3 cells. MMP-2 is an important enzyme for extracellular matrix remodeling during wound healing suggesting that C. swynnertonii promotes wound healing by stimulating cell proliferation and production of MMP-2 in a mechanism that is currently not known.

scholarly journals Thiolated Carboxymethyl-Hyaluronic-Acid-Based Biomaterials Enhance Wound Healing in Rats, Dogs, and Horses

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Guanghui Yang ◽  
Glenn D. Prestwich ◽  
Brenda K. Mann
Keyword(s):  
Wound Healing ◽  
Veterinary Medicine ◽  
Hyaluronic Acid ◽  
Wound Closure ◽  
Keratinocyte Proliferation ◽  
Accelerate Wound Healing ◽  
Multiple Species ◽  
Enhance Wound Healing

The progression of wound healing is a complicated but well-known process involving many factors, yet there are few products on the market that enhance and accelerate wound healing. This is particularly problematic in veterinary medicine where multiple species must be treated and large animals heal slower, oftentimes with complicating factors such as the development of exuberant granulation tissue. In this study a crosslinked-hyaluronic-acid (HA-) based biomaterial was used to treat wounds on multiple species: rats, dogs, and horses. The base molecule, thiolated carboxymethyl HA, was first found to increase keratinocyte proliferation in vitro. Crosslinked gels and films were then both found to enhance the rate of wound healing in rats and resulted in thicker epidermis than untreated controls. Crosslinked films were used to treat wounds on forelimbs of dogs and horses. Although wounds healed slower compared to rats, the films again enhanced wound healing compared to untreated controls, both in terms of wound closure and quality of tissue. This study indicates that these crosslinked HA-based biomaterials enhance wound healing across multiple species and therefore may prove particularly useful in veterinary medicine. Reduced wound closure times and better quality of healed tissue would decrease risk of infection and pain associated with open wounds.

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