Primary Cultures Of Human Fibroblasts Research Articles

Impact of Combined Baricitinib and FTI Treatment on Adipogenesis in Hutchinson-Gilford Progeria Syndrome and Other Lipodystrophic Laminopathies.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease that causes premature aging symptoms, such as vascular diseases, lipodystrophy, loss of bone mineral density, and alopecia. HGPS is mostly linked to a heterozygous and de novo mutation in the LMNA gene (c.1824 C > T; p.G608G), resulting in the production of a truncated prelamin A protein called "progerin". Progerin accumulation causes nuclear dysfunction, premature senescence, and apoptosis. Here, we examined the effects of baricitinib (Bar), an FDA-approved JAK/STAT inhibitor, and a combination of Bar and lonafarnib (FTI) treatment on adipogenesis using skin-derived precursors (SKPs). We analyzed the effect of these treatments on the differentiation potential of SKPs isolated from pre-established human primary fibroblast cultures. Compared to mock-treated HGPS SKPs, Bar and Bar + FTI treatments improved the differentiation of HGPS SKPs into adipocytes and lipid droplet formation. Similarly, Bar and Bar + FTI treatments improved the differentiation of SKPs derived from patients with two other lipodystrophic diseases: familial partial lipodystrophy type 2 (FPLD2) and mandibuloacral dysplasia type B (MADB). Overall, the results show that Bar treatment improves adipogenesis and lipid droplet formation in HGPS, FPLD2, and MADB, indicating that Bar + FTI treatment might further ameliorate HGPS pathologies compared to lonafarnib treatment alone.

Targeting cannabinoid receptor 2 (CB2) limits collagen production-An in vitro study in a primary culture of human fibroblasts.

Previous studies showed that cannabinoid 2 (CB2) receptor is involved in skin inflammation, fibrogenesis and re-epithelialization in mice, indicating that this receptor may be implicated in wound healing. Thus, topical use of cannabinoids may have a role in local fibrotic and wound healing diseases such as scars or keloids. We investigate the effect of the CB2 selective receptor agonist (6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (JWH133) and the CB2 selective receptor antagonist (6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl)(4-methoxyphenyl)-methanone (AM630), on primary cultures of human fibroblasts. Primary cultures of adult human fibroblasts were obtained from abdominal human skin samples. Fibroblasts pretreated with JWH133 and/or AM630 were stimulated with TGF-β (10 ng/ml). Fibroblast activation into myofibroblasts was quantified by the expression of alpha-smooth muscle actin (α-SMA) using Immunocytochemistry and Western Blot assays. Collagen content was quantified with the Sirius red staining assay. Upon human fibroblasts stimulation with TGF-β, a significant increase on α-SMA and CB2 receptor expression was observed. In these cells, JWH133 decreased α-SMA expression and collagen content. However, this effect was not observed in resting human fibroblasts. AM630 decreased α-SMA expression and collagen content in both resting and activated fibroblasts. This effect was time- and concentration-dependent with an IC50 value of 11 μM. The CB2 receptor appears to be involved in fibroblast repair during skin wound healing in humans, as TGF-β increases CB2 receptor expression and JWH133 produces an anti-fibrotic effect in human fibroblasts. AM630 also showed an anti-fibrotic effect hypothesizing that other cannabinoid receptors, such as TRPV, may be involved in this response.

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.

Oxidative Stress Modifies the Levels and Phosphorylation State of Tau Protein in Human Fibroblasts.

Since the tau protein is closely involved in the physiopathology of Alzheimer's disease (AD), studying its behavior in cellular models might lead to new insights on understanding this devastating disease at molecular levels. In the present study, primary cultures of human fibroblasts were established and used to determine the expression and localization of the tau protein in distinct phosphorylation states in both untransfected and tau gene-transfected cells subjected to oxidative stress. Higher immunopositivity to phospho-tau was observed in cell nuclei in response to oxidative stress, while the levels of total tau in the cytosol remained unchanged. These findings were observed in both untransfected cells and those transfected with the tau gene. The present work represents a useful model for studying the physiopathology of AD at the cellular level in terms of tau protein implications.

A genetic CLOCK variant associated with cluster headache causing increased mRNA levels.

Background Cluster headache is characterized by recurrent unilateral headache attacks of severe intensity. One of the main features in a majority of patients is a striking rhythmicity of attacks. The CLOCK ( Circadian Locomotor Output Cycles Kaput) gene encodes a transcription factor that serves as a basic driving force for circadian rhythm in humans and is therefore particularly interesting as a candidate gene for cluster headache. Methods We performed an association study on a large Swedish cluster headache case-control sample (449 patients and 677 controls) screening for three single nucleotide polymorphisms (SNPs) in the CLOCK gene implicated in diurnal preference (rs1801260) or sleep duration (rs11932595 and rs12649507), respectively. We further wanted to investigate the effect of identified associated SNPs on CLOCK gene expression. Results We found a significant association with rs12649507 and cluster headache ( p = 0.0069) and this data was strengthened when stratifying for reported diurnal rhythmicity of attacks ( p = 0.0009). We investigated the effect of rs12649507 on CLOCK gene expression in human primary fibroblast cultures and identified a significant increase in CLOCK mRNA expression ( p = 0.0232). Conclusions Our results strengthen the hypothesis of the involvement of circadian rhythm in cluster headache.

Anti-ageing properties of Khelma Longevity™: treatment of human fibroblasts increases proteasome levels and decreases the levels of oxidized proteins

We have determined the putative anti-ageing properties of Khelma Longevity™, a formula based on various natural compounds from the Mediterranean area. Human primary fibroblast cultures were treated with a wide range of concentrations of Khelma Longevity™ for 1 day or 3 consecutive days. Following these treatments, two major and complementary biomarkers of ageing were measured, namely, the proteasome and the amount of oxidized proteins. It was observed that 24h of treatment with Khelma Longevity™ resulted in a maximum increase of about 41% of the total protein levels of 20S proteasome. Levels of oxidized proteins were reduced by almost 6.5-fold following longer treatments. Specifically we have observed a maximum decrease of protein carbonyls to 84.7% in comparison with nontreated control cells following 3 days of continuous treatment with Khelma Longevity™. These results support the notion that formulas rich in natural compounds from the Mediterranean area possess anti-ageing properties.

Biocompatible upconversion ink for hidden anticounterfeit labeling

Ink for rapid the application of anticounterfeit labels by means of standard printing devices has been developed based on upconversion nanoparticles (nanophosphors). Printing ink is made of an aqueous dispersion of nanoparticles with a NaYF4:YbTm/NaYF4 core–shell structure at a concentration of 0.5 mg/mL. The surface of nanoparticles is modified with amphiphilic polymers. The biosafety of ink is demonstrated in primary cultures of human fibroblasts. The hidden labeling, which is invisible in ambient lighting, is performed by the method of inkjet printing on paper. A printed image is visualized by IR laser irradiation at a wavelength of 975 nm. It is demonstrated that additional modalities of protection can be obtained by encoding the spectra and intensities of the anti-Stokes luminescence lines when combining the dopant lanthanides in nanoparticles.

Cold Atmospheric Plasma Induces a Predominantly Necrotic Cell Death via the Microenvironment.

IntroductionCold plasma is a partially ionized gas generated by an electric field at atmospheric pressure that was initially used in medicine for decontamination and sterilization of inert surfaces. There is currently growing interest in using cold plasma for more direct medical applications, mainly due to the possibility of tuning it to obtain selective biological effects in absence of toxicity for surrounding normal tissues,. While the therapeutic potential of cold plasma in chronic wound, blood coagulation, and cancer treatment is beginning to be documented, information on plasma/cell interaction is so far limited and controversial.Methods and ResultsUsing normal primary human fibroblast cultures isolated from oral tissue, we sought to decipher the effects on cell behavior of a proprietary cold plasma device generating guided ionization waves carried by helium. In this model, cold plasma treatment induces a predominantly necrotic cell death. Interestingly, death is not triggered by a direct interaction of the cold plasma with cells, but rather via a transient modification in the microenvironment. We show that modification of the microenvironment redox status suppresses treatment toxicity and protects cells from death. Moreover, necrosis is not accidental and seems to be an active response to an environmental cue, as its execution can be inhibited to rescue cells.ConclusionThese observations will need to be taken into account when studying in vitro plasma/cell interaction and may have implications for the design and future evaluation of the efficacy and safety of this new treatment strategy.

Characterisation of the kynurenine pathway in skin-derived fibroblasts and keratinocytes.

Acute UVB exposure triggers inflammation leading to the induction of indoleamine 2,3 dioxygenase (IDO1), one of the first enzymes in the kynurenine pathway (KP) for tryptophan degradation. However, limited studies have been undertaken to determine the catabolism of tryptophan within the skin. The aim of this study was two fold: (1) to establish if the administration of the proinflammatory cytokine interferon-gamma (IFN-γ) and/or UVB radiation elicits differential KP expression patterns in human fibroblast and keratinocytes; and (2) to evaluate the effect of KP metabolites on intracellular nicotinamide adenine dinucleotide (NAD(+) ) levels, and cell viability. Primary cultures of human fibroblasts and keratinocytes were used to examine expression of the KP at the mRNA level using qPCR, and at the protein level using immunocytochemistry. Cellular responses to KP metabolites were assessed by examining extracellular lactate dehydrogenase (LDH) activity and intracellular NAD(+) levels. Major downstream KP metabolites were analyzed using GC/MS and HPLC. Our data shows that the KP is fully expressed both in human fibroblasts and keratinocytes. Exposure to UVB radiation and/or IFN-γ causes significant changes in the expression pattern of downstream KP metabolites and enzymes. Exposure to various concentrations of KP metabolites showed marked differences in cell viability and intracellular NAD(+) production, providing support for involvement of the KP in the de novo synthesis of NAD(+) in the skin. This new information will have a significant impact on our understanding of the pathogenesis of UV related skin damage and the diagnosis of KP related disease states.

Abstract 2852: SNAI2 expression by cancer-associated fibroblasts is a negative prognostic factor in non-small cell lung cancer

Abstract The tumor microenvironment is increasingly recognized as a contributor to cancer development and progression since several stromal components, including fibroblasts, interact with cancer cells regulating their behavior and ultimately affecting tumor phenotype. Using primary cultures of human fibroblasts isolated from resected lung cancers we previously demonstrated that co-injection with cancer cells results in increased tumorigenicity and altered expression of ECM related genes in heterotypic tumors. To identify factors involved in the cross-talk between fibroblasts and lung cancer cells, microarray gene expression profiling was performed on 60 fibroblast lines from cancerous and normal lung tissue from primary lung cancer patients, including 25 pairs of normal (NF) and cancer-associated fibroblasts (CAF) from the same patients. Lines were also characterized for presence of the activation marker alpha-SMA and for in vivo pro-tumorigenic activity by co-injection with A549 adenocarcinoma cells. Class comparison analyses identified: (i) Factors highly expressed in cancer-associated fibroblasts (CAF) compared to normal fibroblasts (NF): RHOB, SNAI2, EDIL3, LRRN3, CHN1, CTSC, NOPE and DOCK10; (ii) Genes highly expressed in activated fibroblasts: POSTN, TNFAIP6, CLEC2B and (iii) Genes associated with an in vivo pro-tumorigenic phenotype: CNTN3, ELOVL6, MET, XPO1. To explore the potential prognostic value of these microenvironment related markers, we selected antibodies against 20 proteins differentially expressed in lung fibroblasts and evaluated their performance in immunohistochemistry on FFPE sections of 74 cases of NSCLC. Selection criteria to prioritize candidates for IHC were based on results from molecular analyses (both microarray hybridization and Real-Time PCR validation) and involvement in biological pathways identified as potentially relevant by Gene Set Enrichment Analysis (GSEA). The frequency of positive cells was evaluated on: cancer cells, cancer-associated fibroblasts (CAF) and fibroblasts in normal tissue adjacent (AF) or distant (NF) from the surgical margin. This in-depth analysis revealed heterogeneous expression of different proteins with factors predominantly expressed by cancer cells (LRRN3) and others by the stromal component (CLEC2B). Among factors expressed by fibroblasts, positivity for NOPE, MET, CTSC and CLEC2B was detected in the majority of cases (60-70%) and they were generally expressed at higher frequency by CAF compared to NF. In univariate Cox models, SNAI2 expression by CAF was shown to correlate with worst overall survival (p=0.0288) and we further identified association of CNTN3 expression by CAF with shorter disease free survival (p=0.0125).These findings strengthen the hypothesis that identification of factors responsible for proficient tumor-stroma cross-talk could be instrumental in innovative strategies for risk assessment. Citation Format: Francesca Andriani, Giorgia Leone, Elena Landoni, Federica Facchinetti, Tiziana Caputo, Erika Baldoli, Rosalba Miceli, Luigi Mariani, Ugo Pastorino, Giuseppe Pelosi, Gabriella Sozzi, Luca Roz. SNAI2 expression by cancer-associated fibroblasts is a negative prognostic factor in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2852. doi:10.1158/1538-7445.AM2014-2852

Diabetic nephropathy in Type 1 diabetes mellitus (T1DM) is associated with altered expression of genes regulating TGF-Beta signalling, fibrosis, apoptosis and cell cycle. Studies in primary cultures of human fibroblasts

Diabetic nephropathy in Type 1 diabetes mellitus (T1DM) is associated with altered expression of genes regulating TGF-Beta signalling, fibrosis, apoptosis and cell cycle. Studies in primary cultures of human fibroblasts

Naïve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin (1-4). The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory (5, 6). These early studies described a multipotent precursor cell population from adult mammalian dermis (5). These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons (5). Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers (6). Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies. Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies (7). The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable (7). These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures (7). We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

The clinical efficacy of primary culture of human fibroblasts in gingival augmentation procedures—A preliminary report

The treatment of mucogingival problems is one of the main objectives of periodontal treatment. The insufficient or absent gingival attachment increases the risk of development of gingival recessions. Multiple gingival augmentation techniques of varying level of success are known. The aim of this study has been to clinically and histologically evaluate the integration process of scaffolds composed of primary human fibroblasts derived from keratinized gingiva on collagenous carriers. Ten patients exhibiting a mucogingival problem and gingival inflammation of the related teeth were included in the study. In total, 34 teeth in the anterior part of the maxilla and the mandible underwent treatment. Clinically, over a 6 month period of observation, a significant decrease in the distance from cemento-enamel junction to mucogingival junction (CEJ-MGJ) was revealed. Histologically, mature connective tissue covered by keratinized epithelium was found after 12 weeks. No specimens revealed an inflammatory response. A complete clinical healing was observed after 2 weeks in eight patients [early healing index (EHI)=I]. The results of clinical evaluation indicate that the method of primary culture of human fibroblasts on a collagenous carrier for gingival augmentation is an esthetic and effective method of mucogingival complex repair. The scaffolds were substituted and completely re-epithelialized within 12 weeks according to histologic results.

Extracellular low pH affects circadian rhythm expression in human primary fibroblasts

Circadian rhythm is a fundamental biological system involved in the regulation of various physiological functions. However, little is known about a nature or function of circadian clock in human primary cells. In the present study, we have applied in vitro real time circadian rhythm monitoring to study human clock properties using primary skin fibroblasts. Among factors that affect human physiology, slightly lower extracellular pH was chosen to test its effects on circadian rhythm expression. We established human primary fibroblast cultures obtained from three healthy subjects, stably delivered a circadian reporter gene Bmal1-luciferase, and recorded circadian rhythms in the culture medium at pH 7.2 and 6.7. At pH 7.2, robust and sustained circadian rhythms were observed with average period length 24.47±0.03h. Such rhythms were also found at pH 6.7; however, period length was significantly shortened to 22.60±0.20, amplitude was increased, and damping rate was decreased. The effect of exposure to low pH on the period length was reversible. The shortened period was unlikely caused by factors affecting cell viability because cell morphology and MTT assay showed no significant difference between the two conditions. In summary, our results showed that the circadian rhythm expression is affected at pH 6.7 in human primary fibroblasts without affecting cell viability.

Adhesion and invasion ofCandida albicansfrom periodontal pockets of patients with chronic periodontitis and diabetes to gingival human fibroblasts

The objectives of this study were to evaluate clinical isolates of Candida albicans, particularly their adhesion to and invasion of gingival human fibroblasts in culture and to measure nitric oxide concentration (NO) produced by fibroblasts in the presence of these yeasts. Sixteen strains of C. albicans isolated from patients with chronic periodontitis and diabetes mellitus type II were divided on the basis of phenotypic tests into two groups, i.e., highly or weakly hydrophobic. Primary cultures of human fibroblasts were isolated from gingival biopsies and after subsequent subcultures, the cells were seeded into culture plates and incubated for 24 h. C. albicans strains were inoculated into these plates and maintained for 2 and 4 h to assess their adhesion and invasion, respectively. The number of adherent or invasive yeasts was evaluated by assessing colony-forming units (CFU). The production of NO by fibroblasts was also quantified. The results showed that strains with high hydrophobicity had a greater ability to adhere and invade fibroblasts (p < 0.05, ANOVA and Tukey). The production of NO was higher for the most hydrophobic strains, but did not reach statistical difference with the weakly hydrophobic isolates. These data indicated that the hydrophobicity may play a role in the adhesion and invasion of C. albicans in fibroblast cultures.

The Aryl Hydrocarbon Receptor Ligand ITE Inhibits TGFβ1-Induced Human Myofibroblast Differentiation

Fibrosis can occur in any human tissue when the normal wound healing response is amplified. Such amplification results in fibroblast proliferation, myofibroblast differentiation, and excessive extracellular matrix deposition. Occurrence of these sequelae in organs such as the eye or lung can result in severe consequences to health. Unfortunately, medical treatment of fibrosis is limited by a lack of safe and effective therapies. These therapies may be developed by identifying agents that inhibit critical steps in fibrotic progression; one such step is myofibroblast differentiation triggered by transforming growth factor-β1 (TGFβ1). In this study, we demonstrate that TGFβ1-induced myofibroblast differentiation is blocked in human fibroblasts by a candidate endogenous aryl hydrocarbon receptor (AhR) ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Our data show that ITE disrupts TGFβ1 signaling by inhibiting the nuclear translocation of Smad2/3/4. Although ITE functions as an AhR agonist, and biologically persistent AhR agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, cause severe toxic effects, ITE exhibits no toxicity. Interestingly, ITE effectively inhibits TGFβ1-driven myofibroblast differentiation in AhR(-/-) fibroblasts: Its ability to inhibit TGFβ1 signaling is AhR independent. As supported by the results of this study, the small molecule ITE inhibits myofibroblast differentiation and may be useful clinically as an antiscarring agent.

Proliferation of normal and tumor cells in presence of serum of patients with breast carcinoma after phototherapy with visible and near infrared light

Simultaneous low-intensity visible (VIS) and near infrared (nIR) irradiation from laser and non-laser sources was used for treatment of complications developing in cancer patients after surgical tumor resection, chemo- and radiation therapy. However, the question remains about the impact of this physiotherapeutic method on proliferative activity of the patients' tumor cells and cells involved in wound healing, fibroblasts (FB) and keratinocytes (KC). In this paper, we studied the effect blood serum obtained from the patients with breast cancer after the course of irradiation with visible and NI light (480--3400 nm, 95 % polarization, 40 mW/cm2, 12 J/cm2) in postoperative period on the proliferative activity of primary cultures of human FB and KC, and of several human tumor cell lines (BT-474, HBL-100, Hs578T and A431). Seven-day course of phototherapy increase proliferation of FB (as compared to the initial level) and KC (as compared to postoperative level) by 22 and 28 %, respectively. The tumor cells BT-474, Hs578T and A431 showed statistically significant decrease in proliferative activity compared with the preoperative (initial) level by 31.5, 8.97 and 6.47%, respectively, whereas the cells BT-474, HBL-100, Hs578T and A431 also reduced their proliferative activity by 32,16, 8.65 and 6.26%, respectively, as compared with postperative level. The results obtained demonstrate the safety of the phototherapy with the visible and NI light for BC patients in the postoperative period.

Enhanced connexin 43 expression delays intra‐mitoitc duration and cell cycle traverse independently of gap junction channel function

Connexins (Cxs) and gap junction (GJ)-mediated communication have been linked with the regulation of cell cycle traverse. However, it is not clear whether Cx expression or GJ channel function are the key mediators in this process or at what stage this regulation may occur. We therefore tested the hypothesis that enhanced Cx expression could alter the rate of cell cycle traverse independently of GJ channel function. Sodium butyrate (NaBu) or anti-arrhythmic peptide (AAP10) were used to enhance Cx expression in HeLa cells stably expressing Cx43 (HeLa-43) and primary cultures of human fibroblasts (HFF) that predominantly express Cx43. To reduce GJ-mediated communication, 18-alpha-glycyrrhetinic acid (GA) was used. In HeLa-43 and HFF cells, NaBu and AAP10 enhanced Cx43 expression and increased channel function, while GA reduced GJ-mediated communication but did not significantly alter Cx43 expression levels. Timelapse microscopy and flow cytometry of HeLa-WT (wild-type, Cx deficient) and HeLa-43 cells dissected cell cycle traverse and enabled measurements of intra-mitotic time and determined levels of G1 arrest. Enhanced Cx43 expression increased mitotic durations corresponding with a G1 delay in cell cycle, which was linked to an increase in expression of the cell cycle inhibitor p21(waf1/cip1) in both HeLa-43 and HFF cells. Reductions in Cx43 channel function did not abrogate these responses, indicating that GJ channel function was not a critical factor in reducing cell proliferation in either cell type. We conclude that enhanced Cx43 expression and not GJ-mediated communication, is involved in regulating cell cycle traverse.

Use of primary culture of human fibroblasts in gingiva augmentation procedure

The aim of this study was to assess gingival aesthetic after usage of an own method of primary culture of human fibroblasts derived from the connective tissue of oral cavity keratinized gingiva on collagenous carrier in gingival augmentation procedures. Procedures were performed on 10 patients (7 females, 3 males) aged 18-35 years. In total, 34 teeth in the anterior part of the maxilla and the mandible underwent treatment. The protocol consisted of (1) preparing the patient for tissue biopsy, (2) biopsy of keratinized tissue, (3) laboratory tissue culture, (4) application of expanded cells into the recipient site, and (5) post-procedure management. Aesthetic index, pocket depth (PD), and plaque (PI1) and after surgery complications were examined. There was post-procedure aesthetic improvement in all 34 cases compared with the pre-procedure condition (grade 1), and furthermore a significant decrease in PD and PI1 were revealed. Use of own method of primary culture of human fibroblasts on a collagenous carrier for gingival augmentation is an aesthetic method of mucogingival complex repair.

Versatile Nanocomposite Coatings with Tunable Cell Adhesion and Bactericidity

AbstractTiO2‐Ag nanocomposites are known for their bactericidal effect during exposure to appropriate UV radiation. While involving hazardous radiation, and limited to accessible areas, the bactericidity of these coatings is not persistent in the absence of UV light, which impedes their commercial application. Herein it is shown that TiO2‐Ag nanocomposites can be made highly bactericidal without the need of irradiation. Beyond this, bactericidity can even be mitigated in the presence of pre‐irradiated coatings. Biocompatibility and cell adhesion are also negligibly small for the as‐processed, non‐irradiated coatings, and become fairly high when the coatings are irradiated prior to testing. This opens the possibility to pattern the coatings into areas with high and low cell adhesion properties. Indeed by irradiating the coating through a mechanical mask it is shown that fibroblast cell adherence is sharply confined to the irradiated area. These properties are achieved using TiO2‐Ag thin films with high silver loadings of 50 wt%. The films are processed on stainless steel substrates using solution deposition. Microstructural characterization by means of X‐ray diffraction, Raman, and X‐ray photoelectron spectroscopy, high‐resolution scanning electron microscopy, and atomic force microscopy show a highly amorphous TiO2‐AgxO nanocomposite matrix with scattered silver nanoparticles. UV irradiation of the films results in the precipitation of a high density of silver nanoparticles at the film surface. Bactericidal properties of the films are tested on α‐haemolyzing streptococci and in‐vitro biocompatibility is assessed on primary human fibroblast cultures. The results mentioned above as to the tunable bactericidity and biocompatibility of the TiO2‐Ag coatings developed herein, are amenable to silver ion release, to catalytic effects of silver nanoparticles, and to specific wettabilities of the surfaces.