Complement activation at the interface of wound dressings and blood does not influence keratinocyte migration/proliferation in vitro.

Recently, we reported that some wound dressings caused complement activation at the interface of wound dressing and blood. Since complement activation is associated with impaired wound healing, we investigated whether this activation of the complement cascade at the interface of wound dressings and blood does impair reepithelialization in a scratch wound healing assay. Although some samples showed higher levels of the complement activation marker SC5b-9 in our study, reepithelialization of the samples did not significantly differ from the control group. Further studies have to clarify if complement activation at the interface of wound dressings and blood plays a relevant role in the healing process especially in long-time experiments.

In vivo comparison of braided (Accero) and laser-cut intracranial stents (Acclino, Credo): evaluation of vessel responses at subacute and mid-term follow-up in a rabbit model.

This study aimed to investigate in vivo two stent technologies, with particular emphasis on thrombogenicity and inflammatory vessel remodeling processes. The micro-stents tested in this study were developed for intracranial aneurysm treatment. In our study twelve, New Zealand white rabbits were divided into two groups: 18 laser-cut stents (LCS) and 18 braided stents (BS) were impanated without admiration of antiplatelet medication. Three stents were implanted into each animal in the common carotid artery, subclavian artery, and abdominal aorta. Digital subtraction angiography was performed before and after stent implantation and at follow-up for the visualization of occurring In-stent thromboembolism or stenosis. The Stents were explanted for histopathological examination at two different timepoints, after 3 and 28 days. Angiographically neither in-stent thrombosis nor stenosis for both groups was seen. There was a progressive increase in the vessel diameter, which was more pronounced for BS than for LCS. We detected a higher number of thrombi adherent to the foreign material on day 3 for BS. On day 3, the neointima was absent, whereas the complete formation observed was on day 28. There was no significant difference between both groups regarding the thickness of the neointima. The in vivo model of our study enabled the evaluation of blood and vessel reactions for two different stent technologies. Differences in vessel dimension and tissue around the stents were observed on day 28. Histological analysis on day 3 enabled the assessment of thrombotic reactions, representing an important complementary result in long-term studies.

Publisher Correction: In vitro investigation of chemical properties and biocompatibility of neurovascular braided implants.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hemocompatibility of different burn wound dressings.

A variety of wound dressing are available for burns. Furthermore, although their impacts on wound healing have been studied sufficiently, their effects on blood remain unclear. Meanwhile, this aspect is extremely important, since blood interacts with the wound dressing, especially in extensive burn injuries. Therefore, the aim of this study is to evaluate the hemocompatibility and immunogenicity of different burn wound dressings. Accordingly, human whole blood (n = 5) was anticoagulated with heparin, treated with different wound dressings and incubated at 37°C for 30 minutes. Different parameters for coagulation and hemocompatibility were evaluated before and after incubation. Consequently, Jelonet, Xenoderm, and Matriderm showed higher TAT-III concentrations, Jelonet, Xenoderm, EZ Derm, and Matriderm were higher ß-thromboglobulin; EZ Derm and Burntec showed higher SC5b-9 concentrations after incubation with whole blood. Our ex vivo study provided initial insights into the hemocompatibility and immunogenicity of different burn wound dressings. Moreover, Xenografts (Xenoderm and EZ Derm), Jelonet and Matriderm showed a hemostyptic effect, while EZ Derm and Burntec activated the complement system. Therefore, further studies must be conducted to analyze the possible effects in vivo.

Antibacterial and antivirulence effect of 6-N-hydroxylaminopurine in Listeria monocytogenes.

The emerging development of antibiotic resistant bacteria calls for novel types of antibacterial agents. In this work we examined the putative antibacterial effect of purine analogs in Listeria monocytogenes. We show that, among several tested purine analogs, only 6-N-hydroxylaminopurine (6-N-HAP) reduces the viability of the Gram-positive pathogen Listeria monocytogenes. As in Bacillus subtilis, 6-N-HAP terminates expression at guanine riboswitches in L. monocytogenes hence preventing expression of their downstream genes. However, we show that the bacteriocidal effect of the compound was unlinked to the terminated expression at the guanine riboswitches. When further examining the antimicrobial effect, we observed that 6-N-HAP acts as a potent mutagen in L. monocytogenes, by increasing the mutation rate and inducing the SOS-response. Also, addition of 6-N-HAP decreased virulence gene expression by reducing both the levels and activity of the virulence regulator PrfA.

In vitro investigation of chemical properties and biocompatibility of neurovascular braided implants.

Braiding of Nitinol micro wires is an established technology for the manufacturing of fine-meshed neurovascular implants for tortuous vessel geometries. Electropolishing of wires before the braiding process has the potential to improve the in vitro behaviour in terms of thrombogenicity and endothelial cell proliferation. In this study, we present the first in vitro investigation of braided electropolished/blue oxide Nitinol samples in a blood flow loop, showing a significantly lower activation of the coagulation pathway (represented by the TAT III marker) and a tendency towards reduced platelet adhesion. Furthermore, we applied the same surface treatment on flat disks and measured protein adhesion as well as endothelial cell proliferation. We compared our results to non-electropolished samples with a native oxide surface. While platelet deposition was reduced on electropolished/blue oxide surface, a significant increase of endothelial cell seeding was observed. Investigation of inflammatory marker expression in endothelial cells provided divergent results depending on the marker tested, demanding closer investigation. Surface analysis using Auger electron spectroscopy revealed a thin layer mainly consisting of titanium oxynitride or titanium oxide + titanium nitride as a potential cause of the improved biological performance. Translated to the clinical field of intracranial aneurysm treatment, the improved biocompatibility has the potential to increase both safety (low thrombogenicity) and effectiveness (aneurysm neck reconstruction).

Preventing Surgical Site Infections Using a Natural, Biodegradable, Antibacterial Coating on Surgical Sutures.

Surgical site infections (SSIs) are one of the most common nosocomial infections, which can result in serious complications after surgical interventions. Foreign materials such as implants or surgical sutures are optimal surfaces for the adherence of bacteria and subsequent colonization and biofilm formation. Due to a significant increase in antibiotic-resistant bacterial strains, naturally occurring agents exhibiting antibacterial properties have great potential in prophylactic therapies. The aim of this study was to develop a coating for surgical sutures consisting of the antibacterial substance totarol, a naturally occurring diterpenoid isolated from Podocarpustotara in combination with poly(lactide-co-glycolide acid) (PLGA) as a biodegradable drug delivery system. Hence, non-absorbable monofilament and multifilament sutures were coated with solutions containing different amounts and ratios of totarol and PLGA, resulting in a smooth, crystalline coating. Using an agar diffusion test (ADT), it became evident that the PLGA/totarol-coated sutures inhibited the growth of Staphylococcus aureus over a period of 15 days. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the coated sutures were not cytotoxic to murine fibroblasts. Overall, the data indicates that our innovative, biodegradable suture coating has the potential to reduce the risk of SSIs and postoperative biofilm-formation on suture material without adverse effects on tissue.

Hypercoagulant Abdominal Swabs in Cardiac Surgery: Potential Problems and Background.

Background During cardiac surgery with heart-lung-machine support, abdominal swabs are routinely used to adsorb blood from the operation field. In part, abdominal swabs exhibit procoagulant activity, which is usually considered harmless. However, coagulation induction and abnormal clot formation on the surface of abdominal swabs in the operation field may, if the blood is retransfused into the extracorporeal circuit, lead to severe thromboembolic complications. The aim of the present study was to elucidate the origin of the unexpected blood clotting upon contact with hypercoagulant swabs. Methods The coagulant properties of three abdominal swabs were characterized using a simple clotting test and human whole blood, which was anticoagulated with different heparin concentrations. Eluates prepared from the abdominal swabs and the color stabilizer polydiallyamine (PDAA) were incubated with blood and blood clotting was investigated. Furthermore, the effects of the abdominal swabs on blood clotting time and on heparin were investigated. Results Our data show that the three abdominal swabs as well as the respective eluates exhibit distinctive coagulant properties. The abdominal swab with the highest hypercoagulant effect significantly reduced blood clotting time and also led to a reduction in free heparin. PDAA does not induce activation of the coagulation system. Conclusion The data indicate that the hypercoagulant swab reduces the clotting time and the concentration of free heparin. Abdominal swabs used during complex cardiac surgery with heart-lung-machine support should definitely be tested for their coagulant properties using appropriate tests before clinical applications, as it cannot be specified what leads to their hypercoagulant property.

Hemocompatibility of styrenic block copolymers for use in prosthetic heart valves.

Certain styrenic thermoplastic block copolymer elastomers can be processed to exhibit anisotropic mechanical properties which may be desirable for imitating biological tissues. The ex-vivo hemocompatibility of four triblock (hard-soft-hard) copolymers with polystyrene hard blocks and polyethylene, polypropylene, polyisoprene, polybutadiene or polyisobutylene soft blocks are tested using the modified Chandler loop method using fresh human blood and direct contact cell proliferation of fibroblasts upon the materials. The hemocompatibility and durability performance of a heparin coating is also evaluated. Measures of platelet and coagulation cascade activation indicate that the test materials are superior to polyester but inferior to expanded polytetrafluoroethylene and bovine pericardium reference materials. Against inflammatory measures the test materials are superior to polyester and bovine pericardium. The addition of a heparin coating results in reduced protein adsorption and ex-vivo hemocompatibility performance superior to all reference materials, in all measures. The tested styrenic thermoplastic block copolymers demonstrate adequate performance for blood contacting applications.

Temperature-driven differential gene expression by RNA thermosensors.

Many prokaryotic genes are organized in operons. Genes organized in such transcription units are co-transcribed into a polycistronic mRNA. Despite being clustered in a single mRNA, individual genes can be subjected to differential regulation, which is mainly achieved at the level of translation depending on initiation and elongation. Efficiency of translation initiation is primarily determined by the structural accessibility of the ribosome binding site (RBS). Structured cis-regulatory elements like RNA thermometers (RNATs) can contribute to differential regulation of individual genes within a polycistronic mRNA. RNATs are riboregulators that mediate temperature-responsive regulation of a downstream gene by modulating the accessibility of its RBS. At low temperature, the RBS is trapped by intra-molecular base pairing prohibiting translation initiation. The secondary structure melts with increasing temperature thus liberating the RBS. Here, we present an overview of different RNAT types and specifically highlight recently discovered RNATs. The main focus of this review is on RNAT-based differential control of polycistronic operons. Finally, we discuss the influence of temperature on other riboregulators and the potential of RNATs in synthetic RNA biology. This article is part of a Special Issue entitled: Riboswitches.

Delayed targeting of CD39 to activated platelet GPIIb/IIIa via a single-chain antibody: breaking the link between antithrombotic potency and bleeding?

The ecto-nucleoside triphosphate diphosphohydrolase CD39 represents a promising antithrombotic therapeutic. It degrades adenosine 5'-diphosphate (ADP), a main platelet activating/recruiting agent. We hypothesized that delayed enrichment of CD39 on developing thrombi will allow for a low and safe systemic concentration and thus avoid bleeding. We use a single-chain antibody (scFv, specific for activated GPIIb/IIIa) for targeting CD39. This should allow delayed enrichment on growing thrombi but not on the initial sealing layer of platelets, which do not yet express activated GPIIb/IIIa. CD39 was recombinantly fused to an activated GPIIb/IIIa-specific scFv (targ-CD39) and a nonfunctional scFv (non-targ-CD39). Targ-CD39 was more effective at preventing ADP-induced platelet activation than non-targ-CD39. In a mouse carotid artery thrombosis model, non-targ-CD39, although protective against vessel occlusion, was associated with significant bleeding on tail transection. In contrast, targ-CD39 concentrated at the thrombus site; hence, a dose ∼10 times less of CD39 prevented vessel occlusion to a similar extent as high-dose non-targ-CD39, without prolonged bleeding time. An equimolar dose of non-targ-CD39 at this low concentration was ineffective at preventing vessel occlusion. Thus, delayed targeting of CD39 via scFv to activated platelets provides strong antithrombotic potency and yet prevents bleeding and thereby promotes CD39 toward clinical use.

Simple clotting test to detect procoagulant abdominal swabs.

During surgical procedures, abdominal swabs are routinely used to adsorb blood from the operation field and for the retention of tissues and organs. Due to the material characteristics, abdominal swabs exhibit a slight procoagulant activity, which is usually desirable and mostly harmless. However, during cardiac surgery with heart-lung machine (HLM) support, abnormal clot formation may result in life-threatening thromboembolic complications. Therefore, a simple clotting test (SCT) allowing in vitro detection of abdominal swabs with elevated hypercoagulant potency in the presence of heparinized human blood was developed and validated. In order to establish a SCT, heparinized human blood from 100 donors was incubated with five different cotton abdominal swabs for 30 min at 37 °C and then macroscopically analyzed. In a second study, 10 other swabs were screened with the established SCT (n=11) to confirm its suitability. Scanning electron microscopy, measurements of activated clotting times and thrombin-antithrombin were further performed. In the SCT, the results are dichotomized as negative (no detectable blood clot) and positive (blood clot formation). In the first study, three of the five tested abdominal swabs exhibited hypercoagulant potency in at least 25% of the donors. Calculations using the binomial distribution showed that blood of 11 donors is needed for routine testing with the SCT, which was confirmed in the second study using another 10 swabs. The established SCT can be used for detection of abdominal swabs with an elevated procoagulant potency, thereby minimizing the risk of thromboembolic complications during cardiac surgery with HLM support.

A tricistronic heat shock operon is important for stress tolerance of Pseudomonas putida and conserved in many environmental bacteria.

Small heat shock proteins (sHsps) including the well-studied IbpA protein from Escherichia coli are molecular chaperones that bind to non-native proteins and prevent them from aggregation. We discovered an entirely unexplored tricistronic small heat shock gene cluster in Pseudomonas putida. The genes pp3314, pp3313 and pp3312 (renamed to hspX, hspY and hspZ respectively) are transcribed in a single transcript. In addition to σ(32) -dependent transcriptional control, translation of the first and second gene of the operon is controlled by RNA thermometers with novel architectures. Biochemical analysis of HspY, HspZ and P. putida IbpA demonstrated that they assemble into homo-oligomers of different sizes whose quaternary structures alter in a temperature-dependent manner. IbpA and HspY are able to prevent the model substrate citrate synthase from thermal aggregation in vitro. Increased stress sensitivity of a P. putida strain lacking HspX, HspY and HspZ revealed an important role of these sHsps in stress adaptation. The hspXYZ operon is conserved among metabolically related bacteria that live in hostile environments including polluted soils. This heat shock operon might act as a protective system to promote survival in such ecological niches.

Bacterial interactions with proteins and cells relevant to the development of life-threatening endocarditis studied by use of a quartz-crystal microbalance.

Implant-related infections are a major challenge in clinical routine because of severe complications, for example infective endocarditis (IE). The purpose of this study was to investigate the real-time interaction of S. gordonii with proteins and cells important in the development of IE, in a flow system, by use of a quartz-crystal microbalance (QCM). Acoustic sensors were biologically modified by preconditioning with sterile saliva, platelet-poor plasma (PPP), or platelet-rich plasma (PRP), followed then by perfusion of a bacterial suspension. After perfusion, additional fluorescence and scanning electron microscopic (SEM) studies were performed. The surface structure of S. gordonii was analyzed by atomic force microscopy (AFM). Compared with S. gordonii adhesion on the abiotic sensor surface following normal mass loading indicated by a frequency decrease, adhesion on saliva, PPP, or PRP-conditioned sensors resulted in an increase in frequency. Furthermore, adhesion induced slightly increased damping signals for saliva and PPP-coated sensors but a decrease upon bacterial adhesion to PRP, indicating the formation of a more rigid biofilm. Microscopic analysis confirmed the formation of dense and vital bacterial layers and the aggregation of platelets and bacteria. In conclusion, our study shows that the complex patterns of QCM output data observed are strongly dependent on the biological substrate and adhesion mechanisms of S. gordonii. Overall, QCM sheds new light on the pathways of such severe infections as IE.

Highly sensitive pyrogen detection on medical devices by the monocyte activation test.

Pyrogens are components of microorganisms, like bacteria, viruses or fungi, which can induce a complex inflammatory response in the human body. Pyrogen contamination on medical devices prior operation is still critical and associated with severe complications for the patients. The aim of our study was to develop a reliable test, which allows detection of pyrogen contamination on the surface of medical devices. After in vitro pyrogen contamination of different medical devices and incubation in a rotation model, the human whole blood monocyte activation test (MAT), which is based on an IL-1ß-specific ELISA, was employed. Our results show that when combining a modified MAT protocol and a dynamic incubation system, even smallest amounts of pyrogens can be directly detected on the surface of medical devices. Therefore, screening of medical devices prior clinical application using our novel assay, has the potential to significantly reduce complications associated with pyrogen-contaminated medical devices.

Surface-Mediated Modulation of Different Biological Responses on Anatase-Coated Titanium.

Various surface modification strategies are being developed to endow dental titanium implant surfaces with micro- and nano-structures to improve their biocompatibility, and first of all their osseointegration. These modifications have the potential to address clinical concerns by stimulating different biological processes. This study aims to evaluate the biological responses of ananatase-modified blasted/etched titanium (SLA-anatase) surfaces compared to blasted/acid etched (SLA) and machined titanium surfaces. Using unipolar pulsed direct current (DC) sputtering, a nanocrystalline anatase layer was fabricated. In vitro experiments have shown that SLA-anatase discs can effectively promote osteoblast adhesion and proliferation, which are regarded as important features of a successful dental implant with bone contact. Furthermore, anatase surface modification has been shown to partially enhance osteoblast mineralization in vitro, while not significantly affecting bacterial colonization. Consequently, the recently created anatase coating holds significant potential as a promising candidate for future advancements in dental implant surface modification for improving the initial stages of osseointegration.

A balance of biocompatibility and antibacterial capability of 3D printed PEEK implants with natural totarol coating.

OBJECTIVE: Polyetheretherketone (PEEK), a biomaterial with appropriate bone-like mechanical properties and excellent biocompatibility, is widely applied in cranio-maxillofacial and dental applications. However, the lack of antibacterial effect is an essential drawback of PEEK material and might lead to infection and osseointegration issues. This study aims to apply a natural antibacterial agent, totarol coating onto the 3D printed PEEK surface and find an optimized concentration with balanced cytocompatibility, osteogenesis, and antibacterial capability. METHODS: In this study, a natural antibacterial agent, totarol, was applied as a coating to fused filament fabrication (FFF) 3D printed PEEK surfaces at a series of increasing concentrations (1 mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml, and 20 mg/ml). The samples were then evaluated for cytocompatibility with L929 fibroblast and SAOS-2 osteoblast using live/dead staining and CCK-8 assay. The antibacterial capability was assessed by crystal violet staining, live/dead staining, and scanning electron microscopy (SEM) utilizing the oral primary colonizer S. gordonii and isolates of mixed oral bacteria in a stirring system simulating the oral environment. The appropriate safe working concentration for totarol coating is selected based on the results of the cytocompatibility and antibacterial test. Subsequently, the influence on osteogenic differentiation was evaluated by alkaline phosphatase (ALP) and alizarin red staining (ARS) analysis of pre-osteoblasts. RESULTS: Our results showed that the optimal concentration of totarol solution for promising antibacterial coating was approximately 10 mg/ml. Such surfaces could play an excellent antibacterial role by inducing a contact-killing effect with an inhibitory effect against biofilm development without affecting the healing of soft and hard tissues around FFF 3D printed PEEK implants or abutments. SIGNIFICANCE: This study indicates that the totarol coated PEEK has an improved antibacterial effect with excellent biocompatibility providing great clinical potential as an orthopedic/dental implant/abutment material.

Thermozymes: Synthetic RNA thermometers based on ribozyme activity.

Synthetic biology approaches often combine natural building blocks to generate new cellular activities. Here, we make use of two RNA elements to design a regulatory device with novel functionality. The system is based on a hammerhead ribozyme (HHR) that cleaves itself to generate a liberated ribosome-binding site and, thus, permits expression of a downstream gene. We connected a temperature-responsive RNA hairpin to the HHR and, thus, generated a temperature-controlled ribozyme that we call thermozyme. Specifically, a Salmonella RNA thermometer (RNAT) known to modulate small heat shock gene expression by temperature-controlled base-pairing and melting was fused to the ribozyme. Following an in vivo screening approach, we isolated two functional thermozymes. In vivo expression studies and in vitro structure probing experiments support a mechanism in which rising temperatures melt the thermometer structure impairing the self-cleavage reaction of the ribozyme. Since RNA cleavage is necessary to liberate the RBS, these engineered thermozymes shut off gene expression in response to a temperature increase and, thus, act in a reverse manner as the natural RNAT. Our results clearly emphasize the highly modular nature and biotechnological potential of ribozyme-based RNA thermometers.

Biological Performance of Titanium Surfaces with Different Hydrophilic and Nanotopographical Features.

The micro- and nanostructures, chemical composition, and wettability of titanium surfaces are essential for dental implants' osseointegration. Combining hydrophilicity and nanostructure has been shown to improve the cell response and to shorten the healing time. This study aimed to investigate the biological response to different wettability levels and nanotopographical modifications in aged and non-aged titanium surfaces. By plasma etching titanium surfaces with the fluorine gas 2,3,3,3-tetrafluoropropene (R1234yF), additional nanostructures were created on the sample surfaces. Furthermore, this treatment resulted in sustained superhydrophilicity and fluoride accumulation. We examined the effect of various nanostructuring processes and aging using scanning electron microscopy, roughness analyses, and wettability measurement. In addition, all the surface modifications were tested for their effects on fibroblast adhesion, proliferation, and viability as well as osteoblast differentiation. Our study indicates that the plasma etching, with 2,3,3,3-tetrafluoropropene, of the machined and SLA surface neither favored nor had an adverse effect on the biological response of the SAOS-2 osteoblast cell line. Although the fluorine-plasma-etched surfaces demonstrated improved fibroblast cell viability, they did not lead to improved early osseointegration. It is still unclear which surface properties mainly influence fibroblast and osteoblast adhesion. Further physiochemical aspects, such as electrostatic interaction and surface tension, are crucial to be analyzed along with wettability and roughness.