Microcurrent stimulates cell proliferation and modulates cytokine release in fibroblast cells

Aims: To analyse the effects of microcurrent on L929 fibroblast cell culture. Methods: Cells were cultivated in six-well plates at densities of 5×104, 1×105, 3×105 and 5×105 cells/well to determine the best plating density. Subsequently, two methods of current application were tested: with a paper cone coupled to the electrode (M1) and with the electrode directly inside the well (M2). Then, streams of 60µA (G60), 100µA (G100), 500µA (G500) and 900µA (G900) were applied to the cells (n=3) once a day for three minutes, for a period of one (T1), two (T2) and three days (T3). The MTT assay method was used to evaluate cell proliferation. For the quantification of the inflammatory markers by flow cytometry, the group and time that presented the best results were selected. Results: The ideal plating density was established as 1x105 cells/well and M2 as the best application method. An increase in cell viability was observed at all intensities from T1 to T2, but with no significant differences. From T2 to T3, there was a decrease in viability in all groups, with a significant difference only in G500 (p<0.05). Flow cytometry was performed in the GC and G900 groups at T2. It was possible to observe an increase of 0.56pg/ml in Interleukin (IL)-17 and a decrease of 5.45pg/ml in IL-2. Conclusion: This study showed that two applications of microcurrent increases cell proliferation and modulates the inflammatory response, aiding tissue regeneration and playing a key role in rehabilitation.

Levels of Cyclooxygenase 2, Interleukin-6, and Tumour Necrosis Factor-α in Fibroblast Cell Culture Models after Photobiomodulation at 660 nm

Chemicals and signaling molecules released by injured cells at the beginning of wound healing prompt inflammation. In diabetes, prolonged inflammation is one of the probable causes for delayed wound healing. Increased levels of cyclooxygenase-2 (cox-2), interleukin–6 (IL-6), and tumour necrosis factor-alpha (TNF-α) are associated with the inflammatory response and in diabetes, and increased levels of these contribute to chronic wounds that do not heal. Rising levels of cox-2, IL-6, and TNF-α have also been associated with increased oxidative stress. Photobiomodulation (PBM) may impact wound healing processes by affecting the signaling pathways and molecules pertinent to tissue repair. In the present study, the effect of PBM (wavelength: 660 nm; energy density: 5 J/cm2) on levels of cox-2, IL-6, and TNF-α was determined in fibroblast cell culture models. Four WS1 models (normal, normal wounded, diabetic, and diabetic wounded) were irradiated at 660 nm, and the culture media was collected at 0, 24, and 48 h postirradiation. Cells that were not irradiated (0 J/cm2) served as the controls. The following parameters were determined postirradiation: cell morphology using light microscopy, cell viability using the Trypan Blue exclusion assay, and levels of the inflammatory markers cox-2, IL-6, and TNF-α were measured using ELISA. Cell migration increased in the wounded groups over the 48 h interval after PBM; viability improved postirradiation in the diabetic wounded groups at 0 and 24 h ( P ≤ 0.05 and P ≤ 0.01 , respectively); levels of cox-2 decreased in normal and diabetic wounded groups at 0 h ( P ≤ 0.001 ) and increased in the diabetic and diabetic wounded groups at 48 h postirradiation ( P ≤ 0.05 and P ≤ 0.01 , respectively), while levels of IL-6 decreased in the normal ( P ≤ 0.01 ), diabetic ( P ≤ 0.05 ), and diabetic wounded ( P ≤ 0.001 ) groups at 24 h and in the diabetic and diabetic wounded groups at 48 h ( P ≤ 0.05 ) postirradiation. TNF-α was decreased in the normal wounded groups ( P ≤ 0.05 ) at 48 h. Through its effect on decreased IL-6 levels in diabetic cell models, PBM at 660 nm may be successful at decreasing oxidative stress; however, the present study also found an increase in cox-2 levels at 48 h postirradiation.

Development of nanostructured bioplastic material for wound healing

The development of new biomaterials whose characteristics are as close as possible to the properties of living human tissues is one of the most promising areas of regenerative medicine. This work aimed at creating a bioplastic material based on collagen, elastin and hyaluronic acid and studying its structure and properties to assess the prospects for further use in clinical practice. Bioplastic material was obtained by mixing collagen, hyaluronic acid and elastin in predetermined proportions with distilled water. We treated the material with photochemical crosslinking to stabilize biofilm in a liquid medium and form a nanostructured scaffold. A commercial human skin fibroblast cell culture was used to assess the biomaterial cytotoxicity and biocompatibility. The visualization and studies of the biomaterial structure were performed using light and scanning electron microscopy. It has been shown that the obtained biomaterial is characterized by high resilience; it has also a high porosity. The co-culturing of the bioplastic material and human fibroblasts did not reveal any of its cytotoxic effects on cells in culture. It was shown that the biomaterial samples could maintain physical properties in the culture medium for more than 10 days, while the destruction of the matrix was observed 3–4 weeks after the beginning of incubation. Thus, the created biomaterial can be used on damaged skin areas due to its physical properties and structure. The use of the developed biomaterial provides effective conditions for good cell proliferation, which allows us to consider it as a promising wound cover for use in clinical practice.

Development of nanostructured bioplastic material for wound healing

The development of new biomaterials whose characteristics are as close as possible to the properties of living human tissues is one of the most promising areas of regenerative medicine. This work aimed at creating a bioplastic material based on collagen, elastin and hyaluronic acid and studying its structure and properties to assess the prospects for further use in clinical practice. Bioplastic material was obtained by mixing collagen, hyaluronic acid and elastin in predetermined proportions with distilled water. We treated the material with photochemical crosslinking to stabilize biofilm in a liquid medium and form a nanostructured scaffold. A commercial human skin fibroblast cell culture was used to assess the biomaterial cytotoxicity and biocompatibility. The visualization and studies of the biomaterial structure were performed using light and scanning electron microscopy. It has been shown that the obtained biomaterial is characterized by high resilience; it has also a high porosity. The co-culturing of the bioplastic material and human fibroblasts did not reveal any of its cytotoxic effects on cells in culture. It was shown that the biomaterial samples could maintain physical properties in the culture medium for more than 10 days, while the destruction of the matrix was observed 3–4 weeks after the beginning of incubation. Thus, the created biomaterial can be used on damaged skin areas due to its physical properties and structure. The use of the developed biomaterial provides effective conditions for good cell proliferation, which allows us to consider it as a promising wound cover for use in clinical practice.

MTT-BASED CYTOTOXIC EVALUATION OF AMBONESE BANANA STEM SAP (Musa paradisiaca var. sapientum (L.) Kuntze) ON FIBROBLAST CELLS

Traditional herbal remedies are naturally occurring, plant-derived substances with limited to no chemical processing and have been used in local or national healing traditions to treat illness. In global health debates, traditional herbal medicines are gaining considerable attention. Many hope new research into herbal medicine will play a vital role in global health. Countries like China, India, Nigeria, USA, and also the World Health Organization (WHO) made large investments in ancient herbal medicines. Currently, the use of the Indonesian plant has dramatically improved the medical and dentistry field. The dental and oral care is often related to wounds, and the Ambonese banana stem has been proven as an effective treatment for these injuries. This study aimed to evaluate the effectiveness and safety of the Ambonese banana stem sap through cytotoxicity tests on the fibroblast cell culture of Baby Hamster Kidney-21 (BHK-21). This study was carried out on three BHK-21 fibroblast cell culture, namely, the media and cell control, and the Ambonese banana stem sap with a concentration of 10%, 20%, 30%, 40%, 50%, 60 %, 70%, 80%, 90%, and 100% incubated for 24 hours at 37ºC and 5% CO2. Then, MTT was evenly dispersed on the media to obtain accurate optical density value. All quantitative data were statistically analyzed using one-way ANOVA and Tukey's HSD Test. The result showed significant differences in optical density values between groups with p = 0.000 (p 0.05). There was no significant difference between the cell control and the Ambonese banana stem sap group with concentrations of 90%, 80%, 70%, 60%, 50%, 30%, 20%, and 10%. It was also observed that the Ambonese banana stem sap is nontoxic to fibroblast cells since its viability value was more than 60%.

Description of a Novel Mechanism Possibly Explaining the Antiproliferative Properties of Glucocorticoids in Duchenne Muscular Dystrophy Fibroblasts Based on Glucocorticoid Receptor GR and NFAT5

Glucocorticoids are drugs of choice in Duchenne muscular dystrophy (DMD), prolonging patients’ ambulation. Their mode of action at the protein level is not completely understood. In DMD, muscle tissue is replaced by fibrotic tissue produced by fibroblasts, reducing mobility. Nuclear factor of activated T-cells 5 (NFAT5) is involved in fibroblast proliferation. By treating one DMD fibroblast cell culture and one of unaffected skeletal muscle fibroblasts with methylprednisolone (MP) or hydrocortisone (HC) for 24 h or 12 d, the antiproliferative properties of glucocorticoids could be unraveled. NFAT5 localization and expression was explored by immunocytochemistry (ICC), Western blotting (WB) and RT-qPCR. NFAT5 and glucocorticoid receptor (GR) colocalization was measured by ImageJ. GR siRNA was used, evaluating GR’s influence on NFAT5 expression during MP and HC treatment. Cell proliferation was monitored by IncuCyte ZOOM. In DMD fibroblasts, treatment with MP for 24 h induced dots (ICC) positive for NFAT5 and colocalizing with GR. After 12 d of MP or HC in DMD fibroblasts, NFAT5 expression was decreased (RT-qPCR and WB) and growth arrest was observed (Incucyte ZOOM), whereas NFAT5 expression and cell growth remained unchanged in unaffected skeletal muscle fibroblasts. This study may help understand the antiproliferative properties of glucocorticoids in DMD fibroblasts.

Fibroblasts as the subject of proliferative activity research in vitro

This review presents the data devoted to anatomical and functional diversity of fibroblasts, peculiarities of metabolic processes and energy exchange in these cells. In particular, the changes in fibroblast proliferative activity depending on various factors are discussed. The review shows the influence of the malate dehydrogenase shuttle system on the activity of metabolic processes and the life span of fibroblastsin vitro. The increase of cell cultivation timein vitrois associated with the cytosolic isoform of this enzyme. The stability of fibroblast cell culture to the activation of free-radical processes and peroxidation with addition of biologically active compounds is described and followed by a discussion of the role of separate metabolites in providing free-radical protection and maintenance of the proliferative potential of cells.