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Recent research has vigorously pursued the development of tissue-engineered vascular grafts (TEVs). One of the striking points of the early phase is the instability of the artificial blood vessels and lack of vascular resistance, which is supposed to be a consequence of unstable scaffolds. Therefore, alternative biological approaches are necessary to improve the physical properties of the artificial vessel walls. We developed blood vessel-like constructs based on native spider silk as a scaffold. C2C12 and ST1.6R cells were seeded on the surface of scaffolds and cultivated under pulsatile flow (max. ~135  mmHg and min. ~90  mmHg) in a bioreactor. Constructed grafts were compared to human blood vessels and cell seeded scaffolds cultured in the absence of bioreactor. Mechanical properties, morphological structures and expression of marker genes were assessed by strength and strain experiments, SEM, histological staining, immunohistology, Western blotting and quantitative real-time PCR. The results indicate that the constructed vessel resembles native blood vessels in morphological structure as well as in function and expression of biomarkers. Spider silk scaffolds seem to provide an optimal and stable basis for vessel constructs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The benefits and risks of singular and repetitive microneedling (1 mm) have not been thoroughly investigated. The aim of this study was to evaluate the benefits and risks of singular and repetitive skin needling with a microneedling device in an animal model with and without skincare. 30 Sprague Dawley rats were randomized to five groups: control, skin-care only (Vitamin A & C), 1× needling 1 mm, 4× needling 1 mm, 4× needling 1 mm with skin-care. All animals were euthanized after 10 weeks. Skin specimens were stained with HE and Masson's trichrome. Additionally, gene expression analysis with microarray technique for various growth factors (TGFβ1-3, FGF, EGF, VEGF, TNF-α) and real time reverse transcription PCR for collagen I & III were conducted. We showed that singular microneedling matches and repetitive microneedling sessions superposition epidermal and dermal benefits such as an increase of epidermal thickness (up to 658% increase, p value 0.0008) and dermal connective tissue--even more so when combined with skin-care with vitamin A and C. Juvenile collagen I showed itself up-regulated in all groups, while collagen III was down-regulated. Singular and repetitive PCI with a microneedling device can achieve and supersede the results already shown with medical needling., (Copyright © 2013 Elsevier Ltd and ISBI. All rights reserved.)

The therapy of extensive and deep burn wounds is still a challenging task for reconstructive plastic surgery. The outcome is generally not satisfactory, neither from the functional nor from the aesthetic aspect. Several available skin substitutes are used but there is need for optimization of new skin substitutes which have to be tested in vitro as well as in vivo. Here, we show that the dorsal skin fold chamber preparation of mice is well suited for the testing of skin substitutes in vivo. Dermal skin constructs consisting of matriderm(®) covered with a collagen type I gel were inserted into full thickness skin wounds in the skin fold chambers. The skin substitutes integrated well into the adjacent skin and got epithelialized from the wound edges within 11 days. The epithelialization by keratinocytes is the prerequisite that also cell-free dermal substitutes might be used in the case of the lack of sufficient areas to gain split thickness skin grafts. Further advantage of the chambers is the lack of wound contraction, which is common but undesired in rodent wound healing. Furthermore, this model allows a sophisticated histological as well as immunohistochemical analysis. As such, we conclude that this model is well suited for the analysis of tissue engineered skin constructs. Besides epithelialization the mode and extend of neovascularization and contraction of artificial grafts may be studied under standardized conditions., (Copyright © 2012 Elsevier Ltd and ISBI. All rights reserved.)

Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a highly sensitive tool that can be used for accurate and reliable gene expression analysis; however, a critical factor for creating reliable data in relative quantification is the normalization of the expression data of the genes of interest. In this study, we demonstrate the important process of validating four muscle-specific genes (myosin, desmin, MEF2D and ADAM12) and 10 common potential reference genes (β-2-microglobulin, RPL32, RPL17, α-tubulin, CYC, ET1A, β-actin, HSPCB, SDHA and GAPDH) in engineered muscle tissues. Tissue samples were generated out of rat groin adipose tissues by myogenic induction in a perfusion bioreactor for 7, 21 and 49 days. Results of analyzed muscle-specific genes suggested that the gene expression pattern corresponding to myogenic induction observed in adequately treated rat adipose tissue was time-dependent, making the length of time in culture in myogenic medium an important factor. Our data suggest that the reference genes were expressed variably in the different samples. During engineered muscle development, β-2-microglobulin, RPL32 and RPL17 were the most stably expressed genes. The commonly used reference genes β-actin and GAPDH appeared to be too unstable for normalization of qRT-PCR expression in engineered muscle tissue. The use of β-2-microglobulin, RPL32 and RPL17 as internal standards may improve the accuracy of gene expression studies aimed at muscle tissue engineering under the proposed settings., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Ablative procedures that are used for the improvement of a degenerative process that leads to a loss of skin elasticity and integrity, injure or destroy the epidermis and its basement membrane and lead to fibrosis of the papillary dermis. It was recently shown in clinical and laboratory trials that percutaneous collagen induction (PCI) by multiple needle application is a method for safely treating wrinkles and scars and smoothening the skin without the risk of dyspigmentation. In our study, we describe the effect of PCI on epidermal thickness and the induction of genes relevant for regenerative processes in the skin in a small animal model., Methods: The purpose of this study in a rat model was to determine the effects of PCI on the skin both qualitatively and quantitatively. The epidermal and dermal changes were observed by histology and immunofluorescence. The changes in gene expression were measured by array analysis for cytokines, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF)-7, epidermal growth factor (EGF) and extracellular matrix molecules such as collagen type I and type III., Results: The present study showed that PCI with topical vitamins resulted in a 140% increase in epidermal thickness; an increase in gene and protein expression of collagen I, glycosaminoglycans (GAGs) and growth factors such as VEGF, EGF and FGF7. The collagen fibre bundles were increased, thickened, and more loosely woven in both the papillary and reticular dermis., Conclusion: We were able to show that PCI modulates gene expression in skin of those genes that are relevant for extracellular matrix remodelling., (Copyright © 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

Objective: This study aims to evaluate percutaneous collagen induction (PCI) in post-burn scarring., Background: Patients with scarring after burn frequently request help in improving the aesthetic appearance of their residual cicatricial deformity. Their scars are generally treated by tissue transfer, W- and Z-plasties, flaps, cortisone injections or ablative procedures that injure or destroy the epidermis and its basement membrane and subsequently lead to fibrosis of the papillary dermis. The ideal treatment would be to preserve the epidermis and promote normal collagen and elastin formation in the dermis., Patients: A total of 16 consecutive patients (average age: 37+/-15.5 years, average body mass index (BMI): 25.7) in Germany with post-burn scarring., Intervention: PCI using the Medical Roll-CIT (Vivida, Cape Town, South Africa). This device was designed to multiply-puncture the skin to the level of the dermal scar to institute remodelling. Patients were prepared with topical vitamin A and C cosmetic creams for a minimum of 4 weeks preoperatively to maximise collagen stimulation., Outcome Measures: The outcome was measured rating (visual analogue scale (VAS) and Vancouver Scar Scale (VSS)), histological specimen 12 months after intervention., Results: On average, patients rated their improvement as a mean of 80% better (+/-15.5) than before treatment. Histologic examination revealed considerable increase in collagen and elastin deposition 12 months postoperatively. The epidermis demonstrated 45% thickening of stratum spinosum and normal rete ridges as well as the normalisation of the collagen/elastin matrix in the reticular dermis at 1 year postoperatively., Conclusions: This pilot study shows that PCI appears to be a safe method for treating post-burn scarring without destroying the epidermis. The procedure can be repeated safely and is also applicable in regions where laser treatments and deep peels are of limited use. However, it is necessary to initiate an efficacy trial to prove the data of this pilot study., (2009 Elsevier Ltd and ISBI. All rights reserved.)

Background: About 10% of the general population report multiple and persisting physical symptoms resulting in extensive screening but with no organic explanation found., Objective: The authors sought to determine whether these somatoform-disorder patients show characteristics of the affective disorder spectrum, with the cyclothymic temperament likely showing the highest specificity for somatoform disorder., Method: This study examined temperamental traits and current mood states of 44 general-hospital inpatients diagnosed with somatoform disorder., Results: There was a higher prevalence of abnormal temperamental traits in patients with somatoform disorder., Conclusion: Based on the idea of a continuum between temperament and affective disorders, the results should trigger further research on this issue possibly leading to novel treatment options in the future.

Introduction: The interaction of the CNS and the immune system is well known. A parasympathetic anti-inflammatory pathway has recently been described. Both electrical and pharmacological parasympathetic stimulation attenuate proinflammatory mediator generation. Burn induces abacterial cytokine generation and we sought to evaluate whether parasympathetic stimulation after experimental burn decreases cardiodepressive mediator generation., Material and Methods: A 30% TBSA full-thickness rat burn model was used. After microsurgical preparation of the cervical portion of the vagus nerve, we performed electric vagus nerve stimulation. Serum was harvested and organ samples of heart and liver were homogenized. Samples were subjected to sandwich-ELISA specific for TNF-alpha, IL-1beta and IL-6. Heart rate measurements were done using left ventricular microcatheterization. Statistical analysis was done using Student's t-tests and analysis of variance (ANOVA)., Results: Burn induced a significant rise of TNF-alpha, IL-1beta and IL-6 in organ homogenates and serum. After cervical vagal electrostimulation, serum and organ homogenate levels of proinflammatory cytokines were markedly reduced compared to burn controls. Left ventricular microcatheter assessment demonstrated no cardiodepressive effect of the vagal stimulation itself., Conclusion: Our results encourage further research regarding the neuroimmunologic background of burn, possibly leading to the development of a novel therapeutic approach to burn-induced organ dysfunction and immunodysregulation.

The secreted isoform of fibroblast growth factor 3 (FGF3) induces a mitogenic cell response, while the nuclear form inhibits cell proliferation. Recently, we identified a nucleolar FGF3-binding protein which is implicated in processing of pre-rRNA as a possible target of nuclear FGF3 signalling. Here, we report a second candidate protein identified by a yeast two-hybrid screen for nuclear FGF3 action, ribosomal protein S2, rpS2. Recombinant rpS2 binds to in vitro translated FGF3 and to nuclear FGF3 extracted from transfected COS-1 cells. Characterization of the FGF3 binding domain of rpS2 showed that both the Arg-Gly-rich N-terminal region and a short carboxyl-terminal sequence of rpS2 are necessary for FGF3 binding. Mapping the S2 binding domains of FGF3 revealed that these domains are important for both NoBP and rpS2 interaction. Transient co-expression of rpS2 and nuclear FGF3 resulted in a reduced nucleolar localization of the FGF. These findings suggest that the nuclear form of FGF3 inhibits cell proliferation by interfering with ribosomal biogenesis.

Ribosomal proteins must be imported into the nucleus after being synthesized in the cytoplasm. Since the rpS2 amino acid sequence does not contain a typical nuclear localization signal, we used deletion mutant analysis and rpS2-beta-galactosidase chimeric proteins to identify the nuclear targeting domains in rpS2. Nuclear rpS2 is strictly localized in the nucleoplasm and is not targeted to the nucleoli. Subcellular localization analysis of deletion mutants of rpS2-beta-galactosidase chimeras identified a central domain comprising 72 amino acids which is necessary and sufficient to target the chimeric beta-galactosidase to the nucleus. The nuclear targeting domain shares no significant similarity to already characterized nuclear localization signals in ribosomal proteins or other nuclear proteins. Although a Nup153 fragment containing the importinbeta binding site fused to VP22 blocks nuclear import of rpS2-beta-galactosidase fusion proteins, nuclear uptake of rpS2 could be mediated by several import receptors since it binds to importinalpha/beta and transportin.

Respiratory syncytial virus (RSV) is the most important cause of severe lower respiratory tract infections of infants in industrial nations. In addition, the participation of RSV in the genesis of asthma is under discussion. The RSV glycoproteins F and G have key positions in the viral pathogenesis. At present no satisfactory protein purification protocols are available for these proteins. The methods published for the G protein using preparative SDS-PAGE or immunoaffinity chromatography yield only small amounts of purified G protein that has partially lost its antigenicity. We describe a three-step purification protocol for these glycoproteins. RSV-infected HEp-2 cells were lysed by a Triton X-100 containing buffer. The viral proteins were captured by QAE-Sephadex A-50 material in a batch procedure. A first elution with 100 mM NaCl led to a crude F protein fraction, and a second elution with 300 mM NaCl led to a crude G protein fraction. The F protein was further purified on a Lentil-lectin Sepharose 4B column and finally polished using a Resource Isopropyl column. Lentil-lectin Sepharose 4B was also used to purify the G protein from the crude fraction, but polishing of the G protein was carried out on a Resource Q column. Homogenous RSV-F and RSV-G proteins were obtained by this protein purification protocol. No loss of antigenicity could be observed during this procedure as the highly purified viral proteins remain detectable by a set of monoclonal antibodies and specific antisera. The G protein was isolated as a 90000 monomer, whereas the purified F protein was recovered as a functional homodimer of 140000.