Dermal Fibroblast Cultures Research Articles

Cytotoxicity of three-dimensional paper-based models from a three-dimensional paper-based printer

Objective: Our study aimed to determine the possibility of using models created with a low-cost, paper based 3D printer in an operating room. Therefore influence of different methods of sterilization on models was tested and cytotoxicity of generated models was determined. Material and methods: 30 cuboids divided into three groups were used for verification of shape stability after sterilization. Each group was sterilized either with: Ethylene oxide in temperature 55˚C, Hydrogen peroxide gas plasma in temperature 60˚C or Gamma irradiation at 21˚C, 25kGy. Each cuboid was measured using calliper three times before and three times after sterilization. Results were analysed statistically in Statgraphics Plus. Statistical significance was determined as p< 0.05. Sixty cylinders divided into six groups were used for cytotoxicity tests. Three of those groups were covered before sterilization with 2-octyl-cyanoacrylate. Each group was sterilized with one of the previously described methods. Cytotoxicity was tested by Nanostructural and Molecular Biophysics Laboratory in Technopark Lodz using normal adult human dermal fibroblasts. Survival of cells was tested using spectrophotometry with XTT and was defined as ratio of absorbency of tested probe to absorbency of control probe. Calcein/Ethidium dyeing test was performed according to LIVE/DEAD Viability/Cytotoxicity Kit protocol. Observation was done under Olympus GX71 fluorescence microscope. Results: There was no statistically significant difference for established statistical significance p=0.05 in cuboids dimensions before and after sterilization regardless of sterilization method. In XTT analysis all samples showed higher cytotoxicity against normal, human, adult dermal fibroblast culture when compared to positive control. ANOVA statistical analysis confirmed that 2-octyl cyanoacrylate coating of paper model improved biological behaviour of the material. It decreased cytotoxicity of the model independently of sterilization method. In calcein/ethidium dyeing test due to the high fluorescence of the background caused by cylinders of analysed substance it was impossible to perform the exact analysis of the number of marked cells. Conclusions: Acquired results allow to conclude that Mcor Technology Matrix 300 3D paper-based models can be used in operating room only if covered with cyanoacrylate tissue adhesive. Nemesis relevance: We found no statistically significant difference in cuboids dimensions before and after sterilization regardless of sterilization method. Three-dimensional paper-based models present with high cytotoxicity without coating.

Effect of Anisotropic Aggregates Comprising Pegylated Block Copolymers on Cell Growth and Aggregation

In recent years, effects of shape of nanomaterials on biological response has attracted attention. For example, rod or fibrous polymeric micelles show a longer circulation time in blood and preferential accumulation to tumor cells than a spherical counterpart.1) On the other hand, fibrous cationic molecular assemblies exhibit higher or/and broader spectrum of antimicrobial activity than a spherical counterpart2). In addition, nano/micro-topology of substrates for tissue engineering is now recognized as a tool of regulating cellular responses such as adhesion, extension, aggregation, and differentiation. However, there have not been active systems to control such cellular responses by shape of molecular assemblies. Previously, we have developed amphiphilic pegylated diblock copolymers conjugating poly(L-lactide) (PLLA), poly(e-caprolactone) (PCL), or poly(trimethylene carbonate) (PTMC), forming anisotropic aggregates in water by introduction of a supramolecular assembling motif in the block junction. The size and length of the anisotropic aggregates are varied depending on the type of polymer used in hydrophobic segments. In this context, we explore cell culture of normal human dermal fibroblasts (NHDFs) in the presence of these anisotropic rod/fiber-like aggregates to evaluate the effect of shape of aggregates in culture media on cellular responses. The polymer aggregates were formulated by membrane dialysis. Typically, the block copolymer (40 mg) was dissolved in DMF (1 mL) and stirred overnight at room temperature. Nile red (NR) as a fluorescent probe was dissolved in DMF to have the concentration of 0.02 mg/mL. Then, the DMF solution of NR (1 mL) was added to the block copolymer solution and stirred for 2 hours. After that, the solution was transferred to a dialysis tube (Spectra/Por®7 Dialysis Membrane) with molecular cut off (MWCO) of 2,000 and dialyzed against ultrapure water for two days. Finally, the suspension was diluted to have the polymer concentration of 10 mg/mL (1.0 wt%) NHDFs were seeded on poly(ethylene terephthalate) (PET) plates embedded in a 24-well polystyrene plate at a cell density of 0.5 × 104 cells/cm2 in a cell culture medium (950 µL). The polymer suspension (50 µL) was then added to adjust the final polymer concentration to 0.05 wt%. The cells were incubated at 37 °C in 5% CO2 for 24 h and 48 h. At a certain time of incubation, cells were washed and stained with phalloidin and DAPI to visualize actin and nuclei in the observation by a confocal laser scanning microscope (CLSM). Furthermore, as three-dimension cell culture, NHDFs were first treated with CellTrackerTM to stain cytoplasm. Then, the cell suspension with a density of 1.6 × 104 cells/well including 0.05 wt% of the polymer aggregates was loaded in PrimeSurface\U0001f12c a round-bottom 96-well plates. The cells were incubated at 37 °C in 5% CO2 for 2, 6, and 12 h. Afterwards, cells were washed, stained with DAPI, and observed on a CLSM and a phase-contrast microscope. References 1)D. Li et al., Adv. Funct. Mater., 2016, 26, 66–79. 2)K.Fukushima et al., ACS Nano, 2012, 6, 9191-9199.

Impact of Wharton Jelly Peptide Р199 on fibroblast and mesenchyme skin cells functioning

Objective — to determine the effect of the sh­oligopeptide­72 on the activity and mobility of mesenchymal stem cells of the skin (MSC) and fibroblasts in vitro.Materials and methods. The primary culture of dermal fibroblasts and MSCs was obtained from healthy donors of 40 years. The metabolic and proliferative activity of cells was investigated using the MTT test and the « wound healing» test. The concentration of the polypeptide from 1 ng/ml to 1000 ng/ml was investigated.Results and discussion. The concentration range of 1—1000 ng/ml does not show any toxic or stimulating effects on differentiated fibroblasts. At the same time, MSCs of the human derma were sensitive to the action of the P199 polypeptide. The concentration of 1—2 ng/ml stimulates the metabolic activity of MSCs. Also, we have detected the differential effect of P199 on migration activity. It is shown stimulating effect on the mobility of MSC of the dermis, but not of dermal fibroblasts.Conclusions. Wharton Jelly Peptide P199 stimulates the metabolic, proliferative and migration activity of dermal MSCs. Skin fibroblasts Have not been affected.

Expression of galectin-7 in vulvar lichen sclerosus and its effect on dermal fibroblasts.

Lichen sclerosus is a chronic and inflammatory disease. Extensive studies have focused on the epidermis, with the dermis or epidermis-dermis receiving less attention. To investigate the role of galectin-7, a keratinocyte protein, in vulvar lichen sclerosus (VLS) and its potential effects on dermal fibroblasts, immunohistochemical staining was performed with VLS tissue samples and normal control samples. The expression of galectin-7 was determined by evaluating the galectin-7 integrated density analysis, and further assessed by western blot analysis. Dermal fibroblasts were isolated from the normal tissue of the female anogenital region following sexual plastic surgery. A cell viability assay was performed on isolated dermal fibroblast cells in the presence or absence of galectin-7. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine the transcriptional level of collagen I and collagen III in the response to different doses of galectin-7. In the immunohistochemical analysis, galectin-7 demonstrated a significantly elevated level in VLS, compared to control tissues, which was confirmed by western blot analysis. In the analysis of primary dermal fibroblast cells, galectin-7 significantly inhibited the viability rate of fibroblasts in a dose-dependent manner. RT-qPCR data revealed that the transcription level of collagen I and collagen III were positively associated with the galectin-7 treatment concentration. The overexpression of galectin-7 is associated with the progression of VLS in the epidermis, a high concentration of galectin-7 inhibits the viability of the primary vulvar dermal fibroblasts, and stimulates the accumulation of collagen I and collagen III in dermal fibroblast cultures, thus galectin-7 may serve as a drug target during VLS progression.

Therapeutic interleukin-6 blockade reverses transforming growth factor-beta pathway activation in dermal fibroblasts: insights from the faSScinate clinical trial in systemic sclerosis

ObjectivesSkin fibrosis mediated by activated dermal fibroblasts is a hallmark of systemic sclerosis (SSc), especially in the subset of patients with diffuse disease. Transforming growth factor-beta (TGFβ) and interleukin-6 (IL-6)...

Effect of Fish Collagen Hydrolysates on Type I Collagen mRNA Levels of Human Dermal Fibroblast Culture.

Fish discards and subproducts may represent an important source of raw material, not only for the food industry, but for other different kind of industries, such as the nutraceutical and cosmetic industries. Collagen, which is mainly obtained from animal skins, is an important structural protein in the animal kingdom having many different applications. It is well known that fish skins constitute a significant subproduct in the fishery industry, especially in the case of some species, where fish skins may represent up to 20% of the total body weight of fish. Peptides from collagen hydrolysates have been described to be useful for preventing skin aging and osteoarthritis, however, the mechanism for these biological activities is not well known. Fibroblasts are the main cell types involved in the collagen synthesis, and in the present work, human dermal fibroblasts have been exposed to the treatment of collagen peptides of two different molecular weight ranges. Results show that higher molecular weight collagen peptides produce higher synthesis of collagen type I mRNA and, therefore, it may suggest that prior molecular weight selection may be an important step to maximize the effect of collagen hydrolysates on collagen type I synthesis by dermal fibroblasts.

Effects of genetic variants in the TSPO gene on protein structure and stability.

The 18 kDa translocator protein (TSPO) is an evolutionary conserved cholesterol binding protein localized in the outer mitochondrial membrane. Expression of TSPO is upregulated in activated microglia in various neuroinflammatory, neurodegenerative, and neoplastic disorders. Therefore, TSPO radioligands are used as biomarkers in positron emission tomography (PET) studies. In particular, a common A147T polymorphism in the TSPO gene affects binding of several high affinity TSPO radioligands. Given the relevance of TSPO as a diagnostic biomarker in disease processes, we systematically searched for mutations in the human TSPO gene by a wide array of evolution and structure based bioinformatics tools and identified potentially deleterious missense mutations. The two most frequently observed missense mutations A147T and R162H were further analysed in structural models of human wildtype and mutant TSPO proteins. The effects of missense mutations were studied on the atomic level using molecular dynamics simulations. To analyse putative effects of A147T and R162H variants on protein stability we established primary dermal fibroblast cultures from wt and homozygous A147T and R162H donors. Stability of endogenous TSPO protein, which is abundantly expressed in fibroblasts, was studied using cycloheximide protein degradation assay. Our data show that the A147T mutation significantly alters the flexibility and stability of the mutant protein. Furthermore both A147T and R162H mutations decreased the half-life of the mutant proteins by about 25 percent, which could in part explain its effect on reduced pregnenolone production and susceptibility to neuropsychiatric disorders. The present study is the first comprehensive bioinformatic analysis of genetic variants in the TSPO gene, thereby extending the knowledge about the clinical relevance of TSPO nsSNPs.

The fibronectin ED-A domain enhances recruitment of latent TGF-β-binding protein-1 to the fibroblast matrix.

Dysregulated secretion and extracellular activation of TGF-β1 stimulates myofibroblasts to accumulate disordered and stiff extracellular matrix (ECM) leading to fibrosis. Fibronectin immobilizes latent TGF-β-binding protein-1 (LTBP-1) and thus stores TGF-β1 in the ECM. Because the ED-A fibronectin splice variant is prominently expressed during fibrosis and supports myofibroblast activation, we investigated whether ED-A promotes LTBP-1-fibronectin interactions. Using stiffness-tuneable substrates for human dermal fibroblast cultures, we showed that high ECM stiffness promotes expression and colocalization of LTBP-1 and ED-A-containing fibronectin. When rescuing fibronectin-depleted fibroblasts with specific fibronectin splice variants, LTBP-1 bound more efficiently to ED-A-containing fibronectin than to ED-B-containing fibronectin and fibronectin lacking splice domains. Function blocking of the ED-A domain using antibodies and competitive peptides resulted in reduced LTBP-1 binding to ED-A-containing fibronectin, reduced LTBP-1 incorporation into the fibroblast ECM and reduced TGF-β1 activation. Similar results were obtained by blocking the heparin-binding stretch FNIII12-13-14 (HepII), adjacent to the ED-A domain in fibronectin. Collectively, our results suggest that the ED-A domain enhances association of the latent TGF-β1 by promoting weak direct binding to LTBP-1 and by enhancing heparin-mediated protein interactions through HepII in fibronectin.

Evaluating the antioxidant effects of human hair protein extracts

The intrinsically high cysteine content in human hair keratins and keratin associated proteins confer hair its outstanding mechanical strength through the formation of strong intermolecular disulfide bonds. In addition, these proteins offer the potential to be exploited as potent antioxidants. This report presents our findings on the antioxidant effects of human hair protein extracts and their consequent protective role against oxidative stress in human dermal fibroblast (HDF) cultures. Protein extracts were obtained from human hair using sodium sulfide as the reducing agent, and characterized using SDS-PAGE, Western blotting, MALDI-ToF mass spectrometry and amino acid analysis. Cysteine was found to account for 11.2 mol % in the extracted fractions. By measuring 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging activity, the hair protein fractions were shown to possess significant antioxidant ability (IC50 = 16.22 μM). As a supplement in cell culture media, the extracts protected HDFs from H2O2 induced oxidative stress, which was demonstrated by the maintenance of cell viability and reduced reactive oxygen species production. Besides offering mechanical support as a scaffolding material, the unique antioxidizing ability of human hair protein extracts may also be exploited in biomedical applications.

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts.

Quiescence is a temporary, reversible state in which cells have ceased cell division, but retain the capacity to proliferate. Multiple studies, including ours, have demonstrated that quiescence is associated with widespread changes in gene expression. Some of these changes occur through changes in the level or activity of proliferation-associated transcription factors, such as E2F and MYC. We have demonstrated that mRNA decay can also contribute to changes in gene expression between proliferating and quiescent cells. In this protocol, we describe the procedure for establishing proliferating and quiescent cultures of human dermal foreskin fibroblasts. We then describe the procedures for inhibiting new transcription in proliferating and quiescent cells with Actinomycin D (ActD). ActD treatment represents a straightforward and reproducible approach to dissociating new transcription from transcript decay. A disadvantage of ActD treatment is that the time course must be limited to a short time frame because ActD affects cell viability. Transcript levels are monitored over time to determine transcript decay rates. This procedure allows for the identification of genes and isoforms that exhibit differential decay in proliferating versus quiescent fibroblasts.

Modulation of Oxidative Status by Normoxia and Hypoxia on Cultures of Human Dermal Fibroblasts: How Does It Affect Cell Aging?

Reactive oxygen species (ROS) production in the skin is among the highest compared to other organs, and a clear correlation exists between ROS production and skin aging. Many attempts are underway to reduce oxidative stress in the skin by topical treatment or supplementation with antioxidants/cosmeceuticals, and cultures of human dermal fibroblasts (HDF) are widely used for these studies. Here, we examined the influence of oxygen tension on cell aging in HDF and how this impacted ROS production, the enzymatic and nonenzymatic antioxidant response system, and the efficacy of this defense system in limiting DNA damage and in modulating gene expression of proteins involved in the extracellular matrix, linked to skin aging. We investigated a selection of parameters that represent and reflect the behavior of cellular responses to aging and oxygen tension. Serial passaging of HDF under normoxia (21%) and hypoxia (5%) leads to cell aging as confirmed by β-galactosidase activity, p16 expression, and proliferation rate. However, in HDF under 21% O2, markers of aging were significantly increased compared to those under 5% O2 at matched cell passages despite having lower levels of intracellular ROS and higher levels of CoQ10, total GSH, SOD1, SOD3, and mitochondrial superoxide anion. miRNA-181a, which is known to be upregulated in HDF senescence, was also analyzed, and indeed, its expression was significantly increased in old cells at 21% O2 compared to those at 5% O2. Upregulation of MMP1 and downregulation of COL1A1 along with increased DNA damage were also observed under 21% O2 vs 5% O2. The data highlight that chronic exposure to atmospheric 21% O2 is able to trigger hormetic adaptive responses in HDF that however fail, in the long term, to prevent cellular aging. This information could be useful in further investigating molecular mechanisms involved in adaptation of skin fibroblasts to oxidative stress and may provide useful hints in addressing antiaging strategies.

Rapid isolation and expansion of skin-derived precursor cells from human primary fibroblast cultures

ABSTRACTSkin-derived precursor (SKP) cells have self-renewal and multipotent abilities and are found in the dermis. SKP cells have been isolated previously from pre-established dermal fibroblast cultures. In these procedures, long-term culture and low yield remain the crucial aspects requiring improvement. In this study, we exposed pre-established dermal fibroblasts to 30-min acid stress prior to isolating SKP cells (termed pH-SKP) and compared the yield to the previously published trypsin- and no-stress methods. Spheroid formation was confirmed and analyzed at days 3, 5 and 7. Stemness was investigated by immunohistochemistry for the stem cell markers Nestin, CD9, vimentin and NG2. Multipotency was investigated by differentiation into adipocytes, smooth muscle cells and fibroblasts. The pH-SKP spheroid yield at day 5 was four- and threefold higher than those obtained using trypsin- and no-stress methods, respectively. The expression of stem cell markers Nestin, CD9, vimentin and NG2 were significantly expressed in pH-SKPs compared to the fibroblast origin. Successful pH-SKP spheroid formation and differentiation were achieved and validated in 11 distinct human primary fibroblast lines. These results demonstrate that acute acidic stress treatment of dermal fibroblast cultures greatly improves SKP isolation, growth, yield and multipotency compared to previous methods.

DETERMINATION OF BIOCOMPATIBILITY AND CYTOTOXICITY OF POROUS TITANIUM-BASED MATERIALS IN EXPERIMENT

Aim - to evaluate the proliferative activity of dermal fibroblast cultures in the presence of composite materials based on titanium silicides in vitro. Materials and methods. To assess the proliferative activity of dermal fibroblasts in vitro, the following materials were used: titanium silicide, titanium carbosilicide oxidized in vacuum and without vacuum, titanium VT-00 (comparison group). Testing of proliferative activity was carried out by the direct contact method. The proliferation index, the doubling time and the number of culture doubling during the cultivation period were calculated. Attachment of dermal fibroblasts to the surface of the test materials and their presence on it during cultivation was assessed by scanning electron microscopy. Results. The study of the morphofunctional characteristics of dermal fibroblasts cultured in the presence of the test samples of material showed that during the entire experiment no major changes occurred in any of the series, the cells retained the monolayer growth characteristic of fibroblasts, preferably spindleshaped with 2-4 shoots. Moreover, all cultures of dermal fibroblasts underwent the same number of doublings during the experiment and reached saturation density 7 days after sowing, which indicates good proliferative activity of cells in the presence of test materials. The results of scanning electron microscopy demonstrate the high affinity of human dermal fibroblasts for both titanium silicide and titanium carbosilicides. Conclusion. Absence of morphofunctional changes in dermal fibroblasts and active proliferation testify to the absence of cytotoxicity of the investigated alloys, and the ability of cells to adhere to the surface of materials indicates their good biocompatibility.

Effects of Prisma® Skin dermal regeneration device containing glycosaminoglycans on human keratinocytes and fibroblasts

ABSTRACTPrisma® Skin is a new pharmaceutical device developed by Mediolanum Farmaceutici S.p.a. It includes alginates, hyaluronic acid and mainly mesoglycan. The latter is a natural glycosaminoglycan preparation containing chondroitin sulfate, dermatan sulfate, heparan sulfate and heparin and it is used in the treatment of vascular disease. Glycosaminoglycans may contribute to the re-epithelialization in the skin wound healing, as components of the extracellular matrix. Here we describe, for the first time, the effects of Prisma® Skin in in vitro cultures of adult epidermal keratinocytes and dermal fibroblasts. Once confirmed the lack of cytotoxicity by mesoglycan and Prisma® Skin, we have shown the increase of S and G2 phases of fibroblasts cell cycle distribution. We further report the strong induction of cell migration rate and invasion capability on both cell lines, two key processes of wound repair. In support of these results, we found significant cytoskeletal reorganization, following the treatments with mesoglycan and Prisma® Skin, as confirmed by the formation of F-actin stress fibers. Additionally, together with a significant reduction of E-cadherin, keratinocytes showed an increase of CD44 expression and the translocation of ezrin to the plasma membrane, suggesting the involvement of CD44/ERM (ezrin-radixin-moesin) pathway in the induction of the analyzed processes. Furthermore, as showed by immunofluorescence assay, fibroblasts treated with mesoglycan and Prisma® Skin exhibited the increase of Fibroblast Activated Protein α and a remarkable change in shape and orientation, two common features of reactive stromal fibroblasts. In all experiments Prisma® Skin was slightly more potent than mesoglycan. In conclusion, based on these findings we suggest that Prisma® Skin may be able to accelerate the healing process in venous skin ulcers, principally enhancing re-epithelialization and granulation processes.

Generation of Xeno-Free, cGMP-Compliant Patient-Specific iPSCs from Skin Biopsy.

This unit describes protocols for the generation of clinical-grade patient-specific induced pluripotent stem cell (iPSC)-derived retinal cells from patients with inherited retinal degenerative blindness. Specifically, we describe how, using xeno-free reagents in an ISO class 5 environment, one can isolate and culture dermal fibroblasts, generate iPSCs, and derive autologous retinal cells via 3-D differentiation. The universal methods described herein for the isolation of dermal fibroblasts and generation of iPSCs can be employed regardless of disease, tissue, or cell type of interest. © 2017 by John Wiley & Sons, Inc.

Fluorinated Methacrylamide Chitosan Hydrogels Enhance Cellular Wound Healing Processes.

Low availability of oxygen can lead to stalled wound healing processes and chronic wounds. To address local hypoxia and to better understand direct cellular benefits, a perfluorocarbon conjugated chitosan (MACF) hydrogel that delivers oxygen was created and applied for the first time to in vitro cultures of human dermal fibroblasts and human epidermal keratinocytes under both normoxic (21% O2) and hypoxic (1% O2) environments. Results revealed that local application of MACF provided 233.8±9.9mmHg oxygen partial pressure at 2h andmaintained equilibrium oxygen levels that were approximately 17mmHg partial pressure greater than untreated controls. Cell culture experiments showed that MACF oxygenating gels improved cellular functions involved in wound healing such as cell metabolism, total DNA synthesis and cell migration under hypoxia in both fibroblasts and keratinocytes. Adenosine triphosphate (ATP) quantification also revealed that MACF treatments improved cellular ATP levels significantly over controls under both normoxia and hypoxia (p<0.005). In total, these studies provide new data to indicate that supplying local oxygen via MACF hydrogels under hypoxic environments improves key wound healing cellular functions.

Photobiomodulation of human dermal fibroblasts in vitro: decisive role of cell culture conditions and treatment protocols on experimental outcome

Photobiomodulation-based (LLLT) therapies show tantalizing promise for treatment of skin diseases. Confidence in this approach is blighted however by lamentable inconsistency in published experimental designs, and so complicates interpretation. Here we interrogate the appropriateness of a range of previously-reported treatment parameters, including light wavelength, irradiance and radiant exposure, as well as cell culture conditions (e.g., serum concentration, cell confluency, medium refreshment, direct/indirect treatment, oxygen concentration, etc.), in primary cultures of normal human dermal fibroblasts exposed to visible and near infra-red (NIR) light. Apart from irradiance, all study parameters impacted significantly on fibroblast metabolic activity. Moreover, when cells were grown at atmospheric O2 levels (i.e. 20%) short wavelength light inhibited cell metabolism, while negligible effects were seen with long visible and NIR wavelength. By contrast, NIR stimulated cells when exposed to dermal tissue oxygen levels (approx. 2%). The impact of culture conditions was further seen when inhibitory effects of short wavelength light were reduced with increasing serum concentration and cell confluency. We conclude that a significant source of problematic interpretations in photobiomodulation reports derives from poor optimization of study design. Further development of this field using in vitro/ex vivo models should embrace significant standardization of study design, ideally within a design-of-experiment setting.

Wound healing activity of Ullucus tuberosus, an Andean tuber crop

ObjectiveThis study was designed to investigate the wound healing activity of aqueous extracts of Ullucus tuberosus (U. tuberosus) using in vitro models. MethodsLyophilized pulp and acetone extracts of U. tuberosus were produced using ultrasound extraction. The capacity for collagenase activation was evaluated using fluorescence detection of the enzymatic activity. Then, the influence of U. tuberosus extracts on cell proliferation, cell migration and synthesis of the extracellular matrix (ECM) proteins, metalloproteinase (MMP-1) and pro-collagen was analyzed using human dermal fibroblasts in culture. ResultsAn increase in collagenase activity of 12% supports the utility of U. tuberosus as an agent for scar treatment. In addition, the extracts showed an increase in the proliferation and migration of human dermal fibroblasts and the production of pro-collagen and MMP-1 after treatment with U. tuberosus extracts. The increase in proliferation, migration and pro-collagen levels positively influenced the regeneration of scarless tissue during the proliferation phase, whereas the increase in MMP-1 may have favored the wound healing process during the remodeling and cellular differentiation phases. ConclusionThe results of this study show for first time that U. tuberosus is a promising candidate to support scarless tissue regeneration.

The Role of &lt;i&gt;L. plantarum&lt;/i&gt; as an Immunomodulator Secretion of &lt;i&gt;Transforming Growth Factor&lt;/i&gt;-β1, &lt;i&gt;Transforming Growth Factor&lt;/i&gt;-β3, and &lt;i&gt;Interferon&lt;/i&gt;-α Macrophages and Dermal Fibroblasts

This research aims to see the effect of L.plantarum in modulating the secretion of Transforming Growth Factor-TGFβ1, TGFβ3 macrophages and fibroblasts, Interferon-IFNα macrophages, and to analyze the possibility of L.plantarum potency in supporting the process of scarless wound healing. The culture of peritoneal macrophages was treated with L.plantarum for 24 hours, while another macrophage was S.aureus stimulated for 6 hours before treatment of L.plantarum for 24 hours. The formed supernatant was separated and centrifuged to serve as a treatment on the culture of rat dermal fibroblasts for 24 hours. The supernatant was then separated and centrifuged; its cytokine level was measured with enzyme-linked immunosorbent assay-ELISA. Treatment of L.plantarum with medium and high doses increased the secretion of IFNa macrophages compared with the control; all L.plantarum doses can stimulate the secretion of TGFβ1 fibroblast and TGFβ3 macrophage significantly, but it does not affect the secretion of TGFβ1 macrophages. It can be concluded that L.plantarum increased the secretion of IFNα macrophages higher than the treatment preceded by S.aureus stimulation. The secretion of TGFβ1 fibroblasts and TGFβ3 fibroblasts also increased, but it was not as high as L.plantarum treatment stimulated by S.aureus. Therefore, the application of L.plantarum to support the process of wound healing, prophylactic of the excessive scar and fibrosis can be researched further.

Differential decrease in soluble and DNA-bound telomerase in senescent human fibroblasts.

The role of telomere shortening in the induction of replicative cellular senescence (CS) is well known and as a result, the involvement of telomerase and in particular its catalytic subunit, the telomerase reverse transcriptase (TERT) in CS has also been investigated. However, the majority of studies were conducted on cells that generally express high levels of TERT (cancer and immortalized cells) while the role of telomerase in CS in normal cells has been investigated to a much lesser extent. In particular, it was reported that active TERT is expressed in early passages of cultured human keratinocytes but rapidly diminished towards entry to CS, without telomere shortening. With the putative importance of TERT/telomerase in CS and the aging process in mind, we investigated the expression of TERT and telomerase activity in primary cultures of adult human dermal fibroblasts (HDFs) in the in vitro model of replicative CS. We found that (i) HDFs expressed active TERT; (ii) TERT protein levels and telomerase activity were markedly decreased in senescent HDFs; and (iii) the reduction of TERT in the soluble fraction was more pronounced than in the DNA-bound one. The results suggest the importance of the non-canonical (telomere-unrelated) functions of TERT in cellular senescence.