Primary Human Dermal Fibroblasts Research Articles

From fabric to tissue: Recovered wool keratin/polyvinylpyrrolidone biocomposite fibers as artificial scaffold platform.

Keratin extracted from wool fibers has recently gained attention as an abundant source of renewable, biocompatible material for tissue engineering and drug delivery applications. However, keratin extraction and processing generally require a copious use of chemicals, not only bearing consequences for the environment but also possibly compromising the envisioned biological outcome. In this study, we present, for the first time, keratin-PVP biocomposite fibers obtained via an all-water co-electrospinning process and explored their properties modulation as a result of different thermal crosslinking treatments. The protein-based fibers featured homogenous morphologies and average diameters in the range of 170-290nm. The thermomechanical stability and response to a wet environment can be tuned by acting on the curing time; this can be achieved without affecting the 3D fibrous network nor the intrinsic hydrophilic behavior of the material. More interestingly, our protein-based membranes treated at 170°C for 18h successfully sustained the attachment and growth of primary human dermal fibroblasts, a cellular model which can recapitulate more faithfully the physiological human tissue conditions. Our proposed approach can be viewed as pivotal in designing tunable protein-based scaffolds for the next generation of skin tissue growth devices.

AB0167 TOFACITINIB AND NINTEDANIB MODULATE COLLAGEN FORMATION IN DERMAL FIBROBLASTS

Background:Dermal fibroblasts are responsible for the excessive extracellular matrix (ECM) formation observed in the skin of systemic sclerosis (SSc) patients and fibroblasts are therefore an obvious target for anti-fibrotic treatments. TGFβ, PDGF and IL-6 are known to be central cytokines in systemic sclerosis. Nintedanib, a tyrosine-kinase inhibitor approved for treatment of idiopathic pulmonary fibrosis, did not show effect on dermal fibrosis only on pulmonary fibrosis in SSc patients with interstitial lung disease (ILD). Tofacitinib, as Pan JAK inhibitor, has shown to inhibit dermal fibrosis in mouse models and shown positive indications in patients.Objectives:We investigated the direct effect of Nintedanib and Tofacitinib on ECM production from human dermal fibroblast using translational biomarkers of type I, III and VI collagens and fibronectin.Methods:Primary healthy human dermal fibroblasts were grown in DMEM media containing 0.4% fetal calf serum, Ficoll (to produce a crowded environment) and ascorbic acid for up to 17 days. The cells were stimulated with PDGF [3 nM] and/or TGFβ [1 nM] in combination with Nintedanib [1 nM-10 μM] treatment initiated at day 0 or 7 or Tofacitinib [3-100 nM] treatment initiated at culture start together. Media and treatments were changed twice a week. Non-activated cells (w/o) were used as control. Type I, III and VI collagen formation (PRO-C1, PRO-C3 and PRO-C6, respectively) and fibronectin (FBN-C) were evaluated by validated ELISAs (Nordic Bioscience). Statistical analysis included 1-way and 2-way ANOVA, AUC and Mann-Whitney U-test.Results:PDGF significantly increased collagen type III and VI formation and collagen type I formation minimally. PDGF did not induce changes in fibronectin levels. TGFβ increased collagen type I and VI formation but did not induce formation of collagen type III. TGFβ increased fibronectin levels, where PDGF did not.Nintedanib (≥100 nM) added either from day 0 or 7 reduced PDGF induced collagen type III and VI formation to the levels of w/o throughout the remainder of the study. In TGFβ treated fibroblasts, Nintedanib added either from day 0 or 7 reduced collagen type I and VI formation. The fibronectin levels were dose-dependently reduced by Nintedanib. The biomarker levels were at study end at the level of w/o. Nintedanib at a concentration of 1 uM and higher significantly decreased the biomarker levels. Nintedanib (≥100 nM) in fibroblasts stimulated with both TGFβ and PDGF significantly reduced collagen type I, III and VI collagen and fibronectin.A Tofacitinib concentration of 100 nM was toxic to the dermal fibroblasts as the cell viability was minimal at culture end. However, the viability of Tofacitinib (100 nM) in combination with TGFβ was decreased at study end, but only to half the viability of untreated cells. Tofacitinib dose-dependently decreased the TGFβ induced type I and III collagen formation and fibronectin in the dermal fibroblasts. Tofacitinib (100 nM) decreased the level of collagen type I and III formation to the level of w/o, where as the level of fibronectin was lowered by 80 % of TGFβ. Tofacitinib as low as 12.5 nM significantly lowered the collagen type I formation and fibronectin (both p<0.05) and Tofacitinib of 25 nM decreased collagen type III formation significantly (p<0.0001).Conclusion:Tofacitinib decreased the formation of the collagens and fibronectin. Nintedanib inhibited ECM production differently in PDGF and TGFβ induced dermal fibroblast, but in the combination of TGFβ and PDGF Nintedanib significantly decreased the ongoing fibrosis. In PDGF induced fibrosis, Nintedanib acted as an on-off switch, whereas the inhibition was dose-dependent in TGFβ induced fibrosis. This cell study indicates that Nintedanib and Tofacitinib inhibits collagen production in dermal fibroblasts.Figure:Disclosure of Interests:Anne Sofie Siebuhr Employee of: Nordic Bioscience, Morten Karsdal Shareholder of: Nordic Bioscience A/S., Employee of: Full time employee at Nordic Bioscience A/S., Pernille Juhl Employee of: Nordic Bioscience, Anne-Christine Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Full time employee at Nordic Bioscience A/S.

A new way to prepare gold nanoparticles by sputtering - Sterilization, stability and other properties.

We have developed a novel simple method for effective preparing gold nanoparticles (AuNPs) intended for utilization in biomedicine. The method is based on gold sputtering into liquid poly(ethylene glycol) (PEG). The PEG was used as a basic biocompatible stabilizer of the AuNP colloid. In addition, two naturally occurring polysaccharides - Chitosan (Ch) and Methylcellulose (MC) - were separately diluted into the PEG base with the aims to enhance the yield of the sputtering without changing the sputtering parameters, and to further improve the stability and the biocompatibility of the colloid. The colloids were sterilized by steam, and their stability was measured before and after the sterilization process by dynamic light scattering and UV-Vis spectrophotometry. The results indicated a higher sputtering yield in the colloids containing the polysaccharides. The colloids were also characterized by atomic absorption spectroscopy (AAS) to reveal the composition of the prepared nanoparticles by transmission electron microscopy (TEM) to visualize the nanoparticles and to evaluate their size and clustering, and by rheometry to estimate the viscosity of the colloids. The zeta-potential of the AuNPs was also determined as an important parameter indicating the stability and the biocompatibility of the colloid. In addition, in vitro tests of antimicrobial activity and cytotoxicity were carried out to estimate the biological activity and the biocompatibility of the colloids. Antimicrobial tests were performed by a drip test on two bacterial strains - Gram-positive Staphylococcus epidermidis and Gram-negative Escherichia coli. AuNP with chitosan proved to possess the highest antibacterial activity, especially towards the Gram-positive S. epidermidis. In vitro tests on eukaryotic cells, i.e. human osteoblastic cell line SAOS-2 and primary normal human dermal fibroblasts (NHDF), were performed after a 7-day cultivation to determine the effect and the toxic dose of the colloids on human cells. The studied colloid concentrations were in the range from 0.6 μg/ml to 6 μg/ml. Toxicity of the colloids started to reappear at a concentration of 4.5 μg/ml, especially with chitosan in the colloid, where the colloid with a concentration of 6 μg/ml proved to be the most toxic, especially towards the SAOS-2 cell line. However, the PEG and PEG-MC containing colloids proved to be relatively non-toxic, even at the highest concentration, but with a slowly decreasing tendency of the cell metabolic activity.

Preparation and characterization of chitosan/pectin/ZnO porous films for wound healing

Three-dimensional (3D) porous films based on chitosan/pectin/ZnO nanoparticles (NPs) were prepared for wound healing by the freeze-drying method. The chemical nature, composition and morphology of these films were revealed by FTIR, XRD, EDX, SEM and BET analysis. SEM micrographs showed a decrease in the pore size and porosity of chitosan/pectin/ZnO films when increasing the content of ZnO NPs. The developed films presented the swelling degree and water retention ability in the range of 189–465 and 230–390%, respectively. Moreover, they showed an improved compression strength and controlled degradation in the lysozyme-containing medium in comparison with control. MTT assay demonstrated the biocompatibility of chitosan/pectin/ZnO films against the primary human dermal fibroblast cells (HFCs). Among the developed chitosan/pectin/ZnO films, CPZnO-2 films presented the increased rate of cell proliferation and migration. Also, they exhibited antimicrobial activity against the gram-positive and gram-negative bacteria and fungi. These results suggested that chitosan/pectin/ZnO films could be safe, convenient and effective for wound healing.

Activin A-Mediated Regulation of XT-I in Human Skin Fibroblasts.

Fibrosis is a fundamental feature of systemic sclerosis (SSc) and is characterized by excessive accumulation of extracellular matrix components like proteoglycans (PG) or collagens in skin and internal organs. Serum analysis from SSc patients showed an increase in the enzyme activity of xylosyltransferase (XT), the initial enzyme in PG biosynthesis. There are two distinct XT isoforms—XT-I and XT-II—in humans, but until now only XT-I is associated with fibrotic remodelling for an unknown reason. The aim of this study was to identify new XT mediators and clarify the underlying mechanisms, in view of developing putative therapeutic anti-fibrotic interventions in the future. Therefore, we used different cytokines and growth factors, small molecule inhibitors as well as small interfering RNAs, and assessed the cellular XT activity and XYLT1 expression in primary human dermal fibroblasts by radiochemical activity assays and qRT-PCR. We identified a new function of activin A as a regulator of XYLT1 mRNA expression and XT activity. While the activin A-induced XT-I increase was found to be mediated by activin A receptor type 1B, MAPK and Smad pathways, the activin A treatment did not alter the XYLT2 expression. Furthermore, we observed a reciprocal regulation of XYLT1 and XYLT2 transcription after inhibition of the activin A pathway components. These results improve the understanding of the differential expression regulation of XYLT isoforms under pathological fibroproliferative conditions.

Electroporation does not affect human dermal fibroblast proliferation and migration properties directly but indirectly via the secretome

Aesthetic wound healing is often experienced by patients after electrochemotherapy. We hypothesized that pulsed electric fields applied during electrochemotherapy (ECT) or gene electrotransfer (GET) protocols could stimulate proliferation and migration of human cutaneous cells, as described in protocols for electrostimulation of wound healing. We used videomicroscopy to monitor and quantify in real time primary human dermal fibroblast behavior when exposed in vitro to ECT and GET electric parameters, in terms of survival, proliferation and migration in a calibrated scratch wound assay. Distinct electric field intensities were applied to allow gradient in cell electropermeabilization while maintaining reversible permeabilization conditions, in order to mimic in vivo heterogeneous electric field distribution of complex tissues. Neither galvanotaxis nor statistical modification of fibroblast migration were observed in a calibrated scratch wound assay after application of ECT and GET parameters. The only effect on proliferation was observed under the strongest GET conditions, which drastically reduced the number of fibroblasts through induction of mitochondrial stress and apoptosis. Finally, we found that 24 h-conditioned cell culture medium by electrically stressed fibroblasts tended to increase the migration properties of cells that were not exposed to electric field. RT-qPCR array indicated that several growth factor transcripts were strongly modified after electroporation.

Lily steroidal glycoalkaloid promotes early inflammatory resolution in wounded human fibroblasts

Ethnopharmacological relevanceThe bulbs and flowers of plants from the Lilium genus have historically been used in Asian and Greco-Roman medicine to treat burns and promote skin healing. Aim of the studyTo evaluate a steroidal glycoalkaloid isolated from Easter lily bulbs for its potential wound healing promoting properties. Materials and methodsA lily-derived steroidal glycoalkaloid (LSGA), (22R, 25R)-spirosol-5-en-3β-yl O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→4)-β-D-glucopyranoside, was isolated from Easter lily bulbs, and its structure was confirmed by LC-MS and NMR spectrometry. LSGA effects on wound scratch closure were evaluated in a primary human dermal fibroblast cell culture, and the changes in gene expression profiles were quantitated using an 84 wound-related gene qPCR microarray. ResultsLSGA promoted migration of dermal fibroblasts into the wounded area. The treatment was associated with a rapid upregulation of early inflammatory (CD40LG, CXCL11, IFNG, IL10, IL2 and IL4), cell growth (CSF3 and TNF) and remodeling (CTSG, F13A1, FGA, MMP and PLG) genes both in the wounded and unwounded cells treated with LSGA. A selective decrease in gene expression profiles associated with inflammatory (CXCL2 and CCL7) and remodeling (MMP7 and PLAT) phases was observed in wounded cells treated with LSGA, in contrast to the wounded cells (control). ConclusionThis study demonstrates that a glycoalkaloid present in lilies promoted fibroblast migration in vitro and affected inflammatory, remodeling and growth factor gene expression. The decreases in expression of key genes may impact the wound healing process, possibly contributing to an earlier end of the inflammatory response and shortening the early phases of model tissue reconstitution. The results of this preliminary investigation may provide a basis for the historical use of lily bulbs to promote dermal healing after injury.

Cartilage acidic protein 1 promotes increased cell viability, cell proliferation and energy metabolism in primary human dermal fibroblasts

Cartilage acidic protein 1 (CRTAC1) is an extracellular matrix protein of human chondrogenic tissue that is also present in other vertebrates, non-vertebrate eukaryotes and in some prokaryotes. The function of CRTAC1 remains unknown but the protein’s structure indicates a role in cell-cell or cell-matrix interactions and calcium-binding. The aim of the present study was to evaluate the in vitro effects of hCRTAC1-A on normal human dermal fibroblasts (NHDF). A battery of in vitro assays (biochemical and PCR), immunofluorescence and a biosensor approach were used to characterize the protein’s biological activities on NHDF cells in a scratch assay. Gene expression analysis revealed that hCRTAC1-A protein is associated with altered levels of expression for genes involved in the processes of cell proliferation (CXCL12 and NOS2), cell migration (AQP3 and TNC), and extracellular matrix-ECM regeneration and remodeling (FMOD, TIMP1, FN1) indicating a role for hCRTAC1-A in promoting these activities in a scratch assay. In parallel, the candidate processes identified by differential gene transcription were substantiated and extended using Electric cell-substrate impedance sensing (ECIS) technology, immunofluorescence and cell viability assays. Our findings indicate that hCRTAC1-A stimulated cell proliferation, migration and ECM production in primary human fibroblasts in vitro.

In Vitro Aging of Human Skin Fibroblasts: Age-Dependent Changes in 4-Hydroxynonenal Metabolism.

Evidence suggests that the increased production of free radicals and reactive oxygen species lead to cellular aging. One of the consequences is lipid peroxidation generating reactive aldehydic products, such as 4-hydroxynonenal (HNE) that modify proteins and form adducts with DNA bases. To prevent damage by HNE, it is metabolized. The primary metabolic products are the glutathione conjugate (GSH-HNE), the corresponding 4-hydroxynonenoic acid (HNA), and the alcohol 1,4-dihydroxynonene (DHN). Since HNE metabolism can potentially change during in vitro aging, cell cultures of primary human dermal fibroblasts from several donors were cultured until senescence. After different time points up to 30 min of incubation with 5 µM HNE, the extracellular medium was analyzed for metabolites via liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI-MS). The metabolites appeared in the extracellular medium 5 min after incubation followed by a time-dependent increase. But, the formation of GSH-HNL and GSH-DHN decreased with increasing in vitro age. As a consequence, the HNE levels in the cells increase and there is more protein modification observed. Furthermore, after 3 h of incubation with 5 µM HNE, younger cells showed less proliferative capacity, while in older cells slight increase in the mitotic index was noticed.

Pechiche (Vitex Cymosa Berteo ex Speng), a Nontraditional Fruit from Ecuador, is a Dietary Source of Phenolic Acids and Nutrient Minerals, in Addition to Efficiently Counteracting the Oxidative-Induced Damage in Human Dermal Fibroblasts.

Pechiche fruits (Vitex cymosa Berteo ex Speng) from Ecuador were studied to determine their phenolic acid profile, nutrient minerals and capacity to protect primary human dermal fibroblasts (HDFa) against oxidative-induced damage. Up to five phenolic acids were identified, with homovanillic acid as the main one. Vitamin C, β-carotene and lutein were also determined. Phosphorus and potassium were the main macrominerals, while iron was the principal micromineral. HDFa were preincubated with a crude pechiche extract (PCext) and then subjected to oxidative stress. The activity of five antioxidant enzymes, intracellular reactive oxygen species (ROS) and ATP levels and lipid peroxidation and protein oxidation were used as markers of oxidative damage. Preincubation with PCext for 24 h allowed for the significant reduction of intracellular ROS levels, improved the intracellular ATP levels and protected lipids and proteins against oxidative damage (p < 0.05). Additionally, preincubation with PCext was also able to significantly (p < 0.05) improve the activity of the antioxidant enzymes catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase and glutathione transferase, compared to the stressed group without pretreatment. The results obtained in this study suggest the potential of pechiche as a source of bioactive compounds, as well as its beneficial effect against oxidative stress.

Characterization of Primary Human Dermal Fibroblasts to Ensure for Instance EMF Exposure Experiments under Comparable Cell Culture Condition

HDFa (human dermal fibroblasts) are used as cellular models for EMF exposure. To ensure reproducible in vitro experiments, comparable proliferation and differentiation cell conditions must exist, and different donors, passage numbers, culture time, and growth media must be considered. In this study, the authors cultured fibroblasts in DMEM or 106 medium. Growth curves, vitality, morphology, and gene expression of genes coding for proliferation (PCNA, CDKN2A, CDKN1A, SFN), differentiation (PDGFRA, TGM2, ACTA2, PDPN, NTN1, MGP, PPP1R14), and SFN target genes (TP63, MMP1, MMP3) were examined in both media and passage numbers 3-4, 5-6 and >6. At passages 3-4, proliferating cells can be observed in both media. While cells cultured in DMEM proliferate over the passages, from passage 5, cells in 106 medium persisted around the seeded number. TGM2 down-regulation over all passages in both media and cells morphology suggest papillary-type fibroblasts. Downregulation of SFN (negative regulator of mitotic translation and cell differentiation) coincided with proliferating fibroblasts over all examined conditions. Downstream SFN target genes in proliferating cells appeared upregulated (TP63) and downregulated (MMP1/MMP3), suggestive for a status characterized by increased stemnesses (upregulated TP63) and wound healing capacity (downregulated MMP1, MMP3). Resting cells (SFN control values) were associated with control values of TP63 and MMP1/MMP3 expression, suggesting a reduced stemness and wound healing capacity. In conclusion, a set of markers related to proliferation (SFN), differentiation (TGM2), stemnesses (TP63), and wound healing (MMP1/MMP3) allow a culture characterization so that cells under two different conditions can be exposed, thus enabling reproducible EMF experiments or experiments with other exposures.

Transient receptor potential vanilloid-3 (TRPV3) channel induces dermal fibrosis via the TRPV3/TSLP/Smad2/3 pathways in dermal fibroblasts

BackgroundExcessive wound healing can lead to hypertrophic scars, which are not only a cosmetic issue but could also be itchy or painful. Previously, we reported that, in comparison with normal tissue, thymic stromal lymphopoietin (TSLP) expression was increased in skin burn scars when the activation of transient receptor potential vanilloid-3 (TRPV3) of keratinocytes was increased. However, the functional role of TRPV3 in dermal fibrosis remains unclear. ObjectiveWe aimed to determine whether TRPV3 affects the collagen production of human primary dermal fibroblasts (HPDFs) and to investigate the mechanism involved. MethodHuman primary dermal fibroblasts were cultured and transformed into myofibroblasts using TSLP and carvacrol. Expression levels of α-SMA, fibronectin, and COL1A1 were determined using qPCR, western blotting, and immunofluorescence staining. Ca2+ influx was measured using a calcium-sensitive fluorescent dye, Fura3-AM. Nuclear factor of activated T-cells (NFAT) and phosphorylated-Smad2/3 were determined by western blotting. Silencing of TRPV3 with TRPV3-specific small interference RNA was evaluated using qPCR and western blotting. ResultsThe expression levels of α-SMA, fibronectin, COL1A1, and TSLP were significantly increased in carvacrol-treated HPDFs. The expression levels of α-SMA, fibronectin, and COL1A1 were significantly increased by TSLP. The expression levels of TSLP and COL1A1 were significantly blocked by TRPV3 silencing in HPDFs. Regulation of Ca2+ influx and the expression levels of NFAT and p-Smad2/3 were significantly increased in carvacrol-treated HPDFs. ECM productions induced via the TRPV3/TSLP/Smad2/3 pathways. ConclusionsThe activation of the TRPV3 channels regulates dermal fibrosis by reducing extracellular matrix production via the TRPV3/TSLP/Smad2/3 pathways in dermal fibroblasts.

Silanization improves biocompatibility of graphene oxide.

Evaluation of the biological properties of silanized graphene oxide is important in the context of biomedical applications of the material. In this study, we have evaluated the toxicity, immunogenicity and other biological properties like osteogenicity of silanized graphene oxide (SiGO). Graphene oxide (GO) was silanized using a common silanizing agent namely (3-aminopropyl) triethoxysilane (APTES). Silanization was confirmed through infrared spectroscopy and elemental mapping. Post-silanization, we did not observe any significant changes in the morphology of GO. Silanization leads to an increase in the interlayer distance and disorder in the lattice. Study of in vitro toxicity of SiGO on three different cell lines namely primary human dermal fibroblast, murine embryonic fibroblast and human osteosarcoma cell lines revealed that toxicity of SiGO was significantly less than GO. We further showed that in vitro immune activation of macrophage was less in the case of SiGO in comparison to GO. Profiling of osteogenic differentiation of human mesenchymal stem cell revealed that SiGO is less osteogenic than GO. Study of acute toxicity in the murine model indicated that GO was hepatotoxic at experimental concentration whereas SiGO did not show any significant toxicity. This study implied that SiGO is a better biocompatible material than GO.

Progenitor Biological Bandages: An Authentic Swiss Tool for Safe Therapeutic Management of Burns, Ulcers, and Donor Site Grafts.

Clinical experience gathered over two decades around therapeutic use of primary human dermal progenitor fibroblasts in burn patient populations has been at the forefront of regenerative medicine in Switzerland. Relative technical simplicity, ease of extensive serial multitiered banking, and high stability are major advantages of such cell types, assorted to ease of safety and traceability demonstration. Stringent optimization of cell source selection and standardization of biobanking protocols enables the safe and efficient harnessing of the considerable allogenic therapeutic potential yielded by primary progenitor cells. Swiss legal and regulatory requirements have led to the procurement of fetal tissues within a devised Fetal Progenitor Cell Transplantation Program in the Lausanne University Hospital. Proprietary nonenzymatic isolation of primary musculoskeletal cell types and subsequent establishment of progeny tiered cell banks under cGMP standards have enabled safe and effective management of acute and chronic cutaneous affections in various patient populations. Direct off-the-freezer seeding of viable dermal progenitor fibroblasts on a CE marked equine collagen scaffold is the current standard for delivery of the therapeutic biological materials to patients suffering from extensive and deep burns. Diversification in the clinical indications and delivery methods for these progenitor cells has produced excellent results for treatment of persistent ulcers, autograft donor site wounds, or chronic cutaneous affections such as eczema. Herein we describe the standard operating procedures for preparation and therapeutic deployment of the progenitor biological bandages within our translational musculoskeletal regenerative medicine program, as they are routinely used as adjuvants in our Burn Center to treat critically ailing patients.

Effect of silica nano-spheres on adhesion of oral bacteria and human fibroblasts

Objective This study investigated the effect of surface nano-patterning on adhesion of an oral early commensal colonizer, Streptococcus mitis and the opportunistic pathogen Staphylococcus aureus and human fibroblasts (HDFa) in a laminar flow cell. Methods Nanostructured surfaces were made by functionalizing glass substrates with 40 nm SiO2 nanoparticles. Gradients in nanoparticle surface coverage were fabricated to study the effect of nanoparticle spacing within a single experiment. Bacterial adhesion was investigated after 5 min of contact time by subjecting surfaces to a flow in a laminar flow cell. In addition, to examine the particles effect on human cells, the establishment of focal adhesion and spreading of primary human dermal fibroblasts (HDFa) were investigated after 4 and 24 h. Results Adhesion of both S. aureus and S. mitis decreased on surfaces functionalized with nanoparticles and coincided with higher nanoparticle surface coverage on the surface. Both strains were tested on three separate surfaces. The regression analysis showed that S. mitis was influenced more by surface modification than S. aureus. The establishment of focal adhesions in HDFa cells was delayed on the nanostructured part of the surfaces after both 4 and 24 h of culturing. Significance In the current manuscript, we have used a flow cell to investigate the effect of nanotopographies on S. aureus and S. mitis adhesion. The present findings are of relevance for design of future implant and prostheses surfaces in order to reduce adhesion of bacteria.

Estrogenic, Antiestrogenic and Antiproliferative Activities of Euphorbia bicolor (Euphorbiaceae) Latex Extracts and Its Phytochemicals.

Estrogen receptor antagonists are effective in breast cancer treatment. However, the side effects of these treatments have led to a rise in searching for alternative therapies. The present study evaluated the estrogenic, antiestrogenic, and antiproliferative activities of Euphorbia bicolor (Euphorbiaceae), a plant native to south-central USA. Estrogenic and antiestrogenic activities of latex extract and its phytochemicals were evaluated with a steroid-regulated yeast system expressing the human estrogen receptor α and antiproliferative properties were assessed in the ER-positive MCF-7 and T47-D and triple-negative MDA-MB-231 and MDA-MB-469 breast carcinomas. Genistein and coumestrol identified in the latex extract induced higher estrogenic and antiestrogenic activities compared to diterpenes and flavonoids. The latex extract, resiniferatoxin (RTX) and rutin induced antiproliferative activities in all cell lines in a dose-dependent manner, but not in human normal primary dermal fibroblast cultures. A biphasic effect was observed with MDA-MB-468 breast carcinoma in which the latex extract at low concentrations increased and at high concentrations decreased cell proliferation. Treatments with latex extract in combination with RTX or rutin reduced even more the proliferation of MCF-7 breast carcinoma compared to the individual latex, RTX, and rutin treatments. E. bicolor latex phytochemicals could contribute to developing commercial therapeutic agents for breast cancer treatment.

Gold nanoparticles synthesised by flavonoid tricetin as a potential antibacterial nanomedicine to treat respiratory infections causing opportunistic bacterial pathogens

In this study, flavonoid tricetin was used as a reducing and capping agent for the synthesis of gold nanoparticles (AuNPs). Further, the antibacterial efficacy of the synthesised AuNPs was evaluated against the opportunistic bacterial pathogens that cause respiratory infections. The optimum levels for the synthesis of AuNPs were found to be pH 8, temperature 30 °C, tricetin 125 μM and chloroauric acid 250 μM. The tricetin synthesised AuNPs exhibited in spherical shape with an average size of 12 nm. FT-IR results confirmed that the hydroxyl (OH) and carbonyl (CO) groups of tricetin were mainly participated in the synthesis of AuNPs. The opportunistic bacterial pathogens isolated from immunocompromised patients suffering with different respiratory infections were identified as Staphylococcus aureus, Enterobacter xiangfangensis, Bacillus licheniformis, Escherichia fergusonii, Acinetobacter pittii, Pseudomonas aeruginosa, Aeromonas enteropelogenes and Proteus mirabilis. The antibacterial studies confirmed the broad-spectrum antibacterial activity of AuNPs against the tested Gram-positive and Gram-negative bacteria. The synthesised AuNPs showed high biocompatibility on primary normal human dermal fibroblast (NHDF-c) cells up to 50 μM mL−1. Best of our knowledge, this is the first report on the synthesis of AuNPs using tricetin, which may be a potential antibacterial nanomedicine to treat bacterial infections.

Influence of Different Cell Types and Sources on Pre-Vascularisation in Fibrin and Agarose–Collagen Gels

ABSTRACTVascularisation is essential for the development of tailored, tissue-engineered organs and tissues due to diffusion limits of nutrients and the lack of the necessary connection to the cardiovascular system. To pre-vascularize, endothelial cells and supporting cells can be embedded in the scaffold to foster an adequate nutrient and oxygen supply after transplantation. This technique is applied for tissue engineering of various tissues, but there have been few studies on the use of different cell types or cells sources. We compare the effect of supporting cells from different sources on vascularisation. Fibrin gels and agarose-collagen hydrogels were used as scaffolds. The supporting cells were primary human dermal fibroblasts (HDFs), human nasal fibroblasts (HNFs), human mesenchymal stem cells from umbilical cord’s Wharton’s jelly (WJ MSCs), adipose-derived MSCs (AD MSCs) and femoral bone marrow-derived MSCs (BM MSCs). The tissue constructs were incubated for 14 days and analyzed by two-photon laser scanning microscopy. Vascularisation was supported by all cell types, forming branched networks of tubular vascular structures in both hydrogels. In general, fibrin gels present a higher angiogenic promoting environment compared to agarose-collagen hydrogels and fibroblasts show a high angiogenic potential in co-culture with endothelial cells. In agarose-collagen hydrogels, vascular structures supported by AD MSCs were comparable to our HDF control in terms of volume, area and length. BM MSCs formed a homogeneous network of smaller structures in both hydrogels. This study provides data toward understanding the pre-vascularisation properties of different supporting cell types and sources for tissue engineering of different organs and tissues.

Evaluation of wound healing activity of ethanol extract of Annona reticulata L. leaf both in vitro and in diabetic mice model

Background and aimThe leaves of AnnonareticulataLinn (牛心果niú x ınguǒ; Bullock’s heart), a member of Annonaceae family, have been used extensively in folk medicine; however, its wound healing potential is yet to be explored. Our aim was to investigate the wound healing ability of A. reticulataleaf extract in vitro and in streptozotocin induced diabetic mice model. Material and methodsWe observed the plant extract induced proliferation and migration of primary human dermal fibroblast (HDF), human skin fibroblast cell line (GM00637) and human keratinocyte cell line (HACAT). The expression of transforming growth factor beta (TGF-β), connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), alpha smooth muscle actin (α-SMA), matrix metalloproteinases (MMP-2, MMP-9), collagen-1, collagen-3, focal adhesion kinase (FAK) were evaluated by Western blot and gelatin zymography. Excisional diabetic wound model was used for in vivo wound healing assay. Furthermore, we processed wound tissue for histological and immunohistochemical study. ResultA. reticulata L. leaf extract stimulates proliferation and migration of HDF, skin fibroblast and keratinocyte significantly in a dose dependent manner; expression of TGF-β, CTGF, VEGF, α-SMA, MMP-2, MMP-9, collagen-1, collagen-3, FAK increased. Additionally, an enhanced expression of phospho-SMAD2, phospho-SMAD3 in the treated cells indicated the activation of TGF-β signal transduction pathway, similarly increased expression of phospho-AkT suggested activation of PI3/AkT pathway. Expression of CTGF and α-SMA was also increased significantly in wound tissue. Mass spectrometric analysis revealed that mainly two compounds to be present in the extract: quercetin and β-sitosterol. ConclusionCollective data suggest that A.reticulata leaf extract may have a stimulatory effect in diabetic wound healing.

Biocompatible Aloe vera and Tetracycline Hydrochloride Loaded Hybrid Nanofibrous Scaffolds for Skin Tissue Engineering.

Aloe vera (AV) and tetracycline hydrochloride (TCH) exhibit significant properties such as anti-inflammatory, antioxidant and anti-bacterial activities to facilitate skin tissue engineering. The present study aims to develop poly-ε-caprolactone (PCL)/ AV containing curcumin (CUR), and TCH loaded hybrid nanofibrous scaffolds to validate the synergistic effect on the fibroblast proliferation and antimicrobial activity against Gram-positive and Gram-negative bacteria for wound healing. PCL/AV, PCL/CUR, PCL/AV/CUR and PCL/AV/TCH hybrid nanofibrous mats were fabricated using an electrospinning technique and were characterized for surface morphology, the successful incorporation of active compounds, hydrophilicity and the mechanical property of nanofibers. SEM revealed that there was a decrease in the fiber diameter (ranging from 360 to 770 nm) upon the addition of AV, CUR and TCH in PCL nanofibers, which were randomly oriented with bead free morphology. FTIR spectra of various electrospun samples confirmed the successful incorporation of AV, CUR and TCH into the PCL nanofibers. The fabricated nanofibrous scaffolds possessed mechanical properties within the range of human skin. The biocompatibility of electrospun nanofibrous scaffolds were evaluated on primary human dermal fibroblasts (hDF) by MTS assay, CMFDA, Sirius red and F-actin stainings. The results showed that the fabricated PCL/AV/CUR and PCL/AV/TCH nanofibrous scaffolds were non-toxic and had the potential for wound healing applications. The disc diffusion assay confirmed that the electrospun nanofibrous scaffolds possessed antibacterial activity and provided an effective wound dressing for skin tissue engineering.