Biocompatible polymeric microparticles serve as novel and reliable vehicles for exogenous hormone manipulations in passerines.

The administration of exogenous hormones emerged as an essential tool for field studies in endocrinology. However, working with wild animals remains challenging, because under field conditions not every available method meets the necessary requirements. Achieving a sustained elevation in hormone levels, while simultaneously minimising handling time and invasiveness of the procedure is a difficult task in field endocrinology. Facing this challenge, we have investigated the suitability of biocompatible polymeric microparticles, a novel method for drug-administration, as a tool to manipulate hormones in small songbirds. We chose the insulin-like growth factor-1 (IGF-1) as target hormone, because it receives great interest from the research community due to its important role in shaping life-history traits. Moreover, its short half-life and hydrophilic properties imply a major challenge in finding a suitable method to achieve a sustained, systemic long-term release. To study the release kinetics, we injected either IGF-1 loaded polylactic-co-glycolic acid (PLGA) microparticles or dispersion medium (control group) in the skin pocket of the interscapular region of captive bearded reedlings (Panurus biarmicus). We collected blood samples for 7 consecutive days plus an additional sampling period after two weeks and complemented these with an in vitro experiment. Our results show that in vitro, PLGA microparticles allowed a stable IGF-1 release for more than 15 days, following a burst release at the beginning of the measurement. In vivo, the initial burst was followed by a drop to still elevated levels in circulating IGF-1 until the effect vanished by 16 days post-treatment. This study is the first to describe the use of PLGA-microparticles as a novel tool for exogenous hormone administration in a small passerine. We suggest that this method is highly suitable to achieve the systemic long-term release of hydrophilic hormones with short half-life and reduces overall handling time, as it requires only one subcutaneous injection.

Antifouling properties of layer by layer DNA coatings.

Fouling is a major concern for solid/liquid interfaces of materials used in different applications. One approach of fouling control is the use of hydrophilic polymer coatings made from poly-anions and poly-cations using the layer-by-layer (LBL) method. The authors hypothesized that the poly-anionic properties and the poly-phosphate backbone of DNA would provide anti-biofouling and anti-scaling properties. To this end, poly(ethyleneimine)/DNA LBL coatings against microbial and inorganic fouling were developed, characterized and evaluated. DNA LBL coatings reduced inorganic fouling from tap water by 90% when incubated statically or under flow conditions mimicking surfaces in heat exchangers. The coatings also impaired biofilm formation by 93% on stainless steel from tap water, and resulted in a 97% lower adhesion force and reduced initial attachment of the human pathogens Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa on glass. This study demonstrates a proof of concept that LBL coatings with poly-anions harboring phosphate groups can address fouling in several applications.

DNA Polyelectrolyte Multilayer Coatings Are Antifouling and Promote Mammalian Cell Adhesion.

The ability of bacteria to adhere to and form biofilms on implant surfaces is the primary cause of implant failure. Implant-associated infections are difficult to treat, as the biofilm mode of growth protects microorganisms from the host's immune response and antibiotics. Therefore, modifications of implant surfaces that can prevent or delay bacterial adhesion and biofilm formation are highly desired. In addition, the attachment and spreading of bone cells are required for successful tissue integration in orthopedic and dental applications. We propose that polyanionic DNA with a negatively charged phosphate backbone could provide a dual function to repel bacterial adhesion and support host tissue cell attachment. To this end, we developed polyelectrolyte multilayer coatings using chitosan (CS) and DNA on biomaterial surfaces via a layer-by-layer technique. The assembly of these coatings was characterized. Further, we evaluated staphylococcal adhesion and biofilm growth on the coatings as well as cytotoxicity for osteoblast-like cells (SaOS-2 cells), and we correlated these to the layer structure. The CS-DNA multilayer coatings impaired the biofilm formation of Staphylococcus by ~90% on both PMMA and titanium surfaces. The presence of cationic CS as the top layer did not hinder the bacteria-repelling property of the DNA in the coating. The CS-DNA multilayer coatings demonstrated no cytotoxic effect on SaOS-2 cells. Thus, DNA polyelectrolyte multilayer coatings could reduce infection risk while promoting host tissue cell attachment on medical implants.

Mammalian cell growth versus biofilm formation on biomaterial surfaces in an in vitro post-operative contamination model.

Biomaterial-associated infections are the major cause of implant failure and can develop many years after implantation. Success or failure of an implant depends on the balance between host tissue integration and bacterial colonization. Here, we describe a new in vitro model for the post-operative bacterial contamination of implant surfaces and investigate the effects of contamination on the balance between mammalian cell growth and bacterial biofilm formation. U2OS osteosarcoma cells were seeded on poly(methyl methacrylate) in different densities and allowed to grow for 24 h in a parallel-plate flow chamber at a low shear rate (0.14 s(-1)), followed by contamination with Staphylococcus epidermidis ATCC 35983 at a shear rate of 11 s(-1). The U2OS cells and staphylococci were allowed to grow simultaneously for another 24 h under low-shear conditions (0.14 s(-1)). Mammalian cell growth was severely impaired when the bacteria were introduced to surfaces with a low initial cell density (2.5 × 10(4) cells cm(-2)), but in the presence of higher initial cell densities (8.2 × 10(4) cells cm(-2) and 17 × 10(4) cells cm(-2)), contaminating staphylococci did not affect cell growth. This study is believed to be the first to show that a critical coverage by mammalian cells is needed to effectively protect a biomaterial implant against contaminating bacteria.

Remotely Triggered Liquefaction of Hydrogel Materials.

Adaptable behavior such as triggered disintegration affords a broad scope and utility for (bio)materials in diverse applications in materials science and engineering. The impact of such materials continues to grow due to the increased importance of environmental considerations as well as the increased use of implants in medical practices. However, examples of such materials are still few. In this work, we engineer triggered liquefaction of hydrogel biomaterials in response to internal, localized heating, mediated by near-infrared light as external stimulus. This adaptable behavior is engineered into the readily available physical hydrogels based on poly(vinyl alcohol), using gold nanoparticles or an organic photothermal dye as heat generators. Upon laser light irradiation, engineered biomaterials underwent liquefaction within seconds. Pulsed laser light irradiation afforded controlled, on-demand release of the incorporated cargo, successful for small molecules as well as proteins (enzymes) in their biofunctional form.

An in vitro investigation of bacteria-osteoblast competition on oxygen plasma-modified PEEK.

Polyetheretherketone (PEEK) films were oxygen plasma treated to increase surface free energy and characterized by X-ray photoelectron microscopy, atomic force microscopy, and water contact angles. A parallel plate flow chamber was used to measure Staphylococcus epidermidis, Staphylococcus aureus, and U-2 OS osteosarcomal cell-line adhesion to the PEEK films in separate monocultures. In addition, bacteria and U-2 OS cells were cocultured to model competition between osteoblasts and contaminating bacteria for the test surfaces. Plasma treatment of the surfaces increased surface oxygen content and decreased the hydrophobicity of the materials, but did not lead to a significant difference in bacterial or U-2 OS cell adhesion in the monocultures. In the S. epidermidis coculture experiments, the U-2 OS cells adhered in greater numbers on the treated surfaces compared to the untreated PEEK and spread to a similar extent. However, in the presence of S. aureus, cell death of the U-2 OS occurred within 10 h on all surfaces. The results of this study suggest that oxygen plasma treatment of PEEK may maintain the ability of osteoblast-like cells to adhere and spread, even in the presence of S. epidermidis contamination, without increasing the risk of preoperative bacterial adhesion. Therefore, oxygen plasma-treated PEEK remains a promising method to improve implant surface free energy for osseointegration.

Soft tissue integration versus early biofilm formation on different dental implant materials.

OBJECTIVE: Dental implants anchor in bone through a tight fit and osseo-integratable properties of the implant surfaces, while a protective soft tissue seal around the implants neck is needed to prevent bacterial destruction of the bone-implant interface. This tissue seal needs to form in the unsterile, oral environment. We aim to identify surface properties of dental implant materials (titanium, titanium-zirconium alloy and zirconium-oxides) that determine the outcome of this "race-for-the-surface" between human-gingival-fibroblasts and different supra-gingival bacterial strains. METHODS: Biofilms of three streptococcal species or a Staphylococcus aureus strain were grown in mono-cultures on the different implant materials in a parallel-plate-flow-chamber and their biovolume evaluated using confocal-scanning-laser-microscopy. Similarly, adhesion, spreading and growth of human-gingival-fibroblasts were evaluated. Co-culture experiments with bacteria and human-gingival-fibroblasts were carried out to evaluate tissue interaction with bacterially contaminated implant surfaces. Implant surfaces were characterized by their hydrophobicity, roughness and elemental composition. RESULTS: Biofilm formation occurred on all implant materials, and neither roughness nor hydrophobicity had a decisive influence on biofilm formation. Zirconium-oxide attracted most biofilm. All implant materials were covered by human-gingival-fibroblasts for 80-90% of their surface areas. Human-gingival-fibroblasts lost the race-for-the-surface against all bacterial strains on nearly all implant materials, except on the smoothest titanium variants. SIGNIFICANCE: Smooth titanium implant surfaces provide the best opportunities for a soft tissue seal to form on bacterially contaminated implant surfaces. This conclusion could only be reached in co-culture studies and coincides with the results from the few clinical studies carried out to this end.

Conditions of lateral surface confinement that promote tissue-cell integration and inhibit biofilm growth.

Surfaces with cell adhesiveness modulated at micro length scales can exploit differences between tissue/bacterial cell size, membrane/wall plasticity, and adhesion mechanisms to differentially control tissue-cell/material and bacteria/material interactions. This study explores the short-term interactions of Staphylococcus aureus and osteoblast-like cells with surfaces consisting of cell-adhesive circular patches (1-5 µm diameter) separated by non-adhesive electron-beam patterned poly(ethylene glycol) hydrogel thin films at inter-patch distances of 0.5-10 µm. Osteoblast-like U2OS cells both bind to and spread on the modulated surfaces, in some cases when the cell-adhesive area comprises only 9% of the total surface and in several cases at least as well as on the continuously adhesive control surfaces. In contrast, S. aureus adhesion rates are 7-20 times less on the modulated surfaces than on the control surfaces. Furthermore, the proliferation of those bacteria that do adhere is inhibited by the lateral confinement imposed by the non-adhesive boundaries surrounding each patch. These findings suggest a new approach to create biomaterial surfaces that may promote healing while simultaneously reducing the probability of infection.

Influence of prophylactic antibiotics on tissue integration versus bacterial colonization on poly(methyl methacrylate).

PURPOSE: Biomaterial-associated infections (BAI) remain a major concern in modern health care. BAI is difficult to treat and often results in implant replacement or removal. Pathogens can be introduced on implant surfaces during surgery and compete with host cells attempting to integrate the implant. Here we studied the influence of prophylactically given cephatholin in the competition between highly virulent Staphylococcus aureus and human osteoblast-like cells (U-2 OS, ATCC HTB-94) for a poly(methyl methacrylate) surface in vitro using a peri-operative contamination model. METHOD: S. aureus was seeded on the acrylic surface in a parallel plate flow chamber prior to adhesion of U-2 OS cells. Next, S. aureus and U-2 OS cells were allowed to grow simultaneously under shear (0.14 1/s) in a modified culture medium containing cephatholin for 8 h, the time period this drug is supposed to be active in situ. Subsequently, the flow was continued with modified culture medium for another 64 h. RESULTS: In the absence of cephatholin, highly virulent S. aureus caused U-2 OS cell death within 18 h. In contrast, the presence of cephatholin for 8 h resulted in survival of U-2 OS cell up to 72 h during simultaneous growth of U-2 OS cells and bacteria. Not all adhering bacteria were killed however, but they showed a delayed growth. CONCLUSIONS: These findings are in line with the recalcitrance of biofilms against antibiotic treatment observed clinically, and represent another support for the use of in vitro co-culture models in mimicking the clinical situation.

Biomaterial-associated infection: locating the finish line in the race for the surface.

Biomaterial-associated infections occur on both permanent implants and temporary devices for restoration or support of human functions. Despite increasing use of biomaterials in an aging society, comparatively few biomaterials have been designed that effectively reduce the incidence of biomaterial-associated infections. This review provides design guidelines for infection-reducing strategies based on the concept that the fate of biomaterial implants or devices is a competition between host tissue cell integration and bacterial colonization at their surfaces.

In vitro interactions between bacteria, osteoblast-like cells and macrophages in the pathogenesis of biomaterial-associated infections.

Biomaterial-associated infections constitute a major clinical problem that is difficult to treat and often necessitates implant replacement. Pathogens can be introduced on an implant surface during surgery and compete with host cells attempting to integrate the implant. The fate of a biomaterial implant depends on the outcome of this race for the surface. Here we studied the competition between different bacterial strains and human U2OS osteoblast-like cells (ATCC HTB-94) for a poly(methylmethacrylate) surface in the absence or presence of macrophages in vitro using a peri-operative contamination model. Bacteria were seeded on the surface at a shear rate of 11 1/s prior to adhesion of U2OS cells and macrophages. Next, bacteria, U2OS cells and macrophages were allowed to grow simultaneously under low shear conditions (0.14 1/s). The outcome of the competition between bacteria and U2OS cells for the surface critically depended on bacterial virulence. In absence of macrophages, highly virulent Staphylococcus aureus or Pseudomonas aeruginosa stimulated U2OS cell death within 18 h of simultaneous growth on a surface. Moreover, these strains also caused cell death despite phagocytosis of adhering bacteria in presence of murine macrophages. Thus U2OS cells are bound to loose the race for a biomaterial surface against S. aureus or P. aeruginosa, even in presence of macrophages. In contrast, low-virulent Staphylococcus epidermidis did not cause U2OS cell death even after 48 h, regardless of the absence or presence of macrophages. Clinically, S. aureus and P. aeruginosa are known to yield acute and severe biomaterial-associated infections in contrast to S. epidermidis, mostly known to cause more low-grade infection. Thus it can be concluded that the model described possesses features concurring with clinical observations and therewith has potential for further studies on the simultaneous competition for an implant surface between tissue cells and pathogenic bacteria in presence of immune system components.

Microbial biofilm growth versus tissue integration on biomaterials with different wettabilities and a polymer-brush coating.

Biomaterials-associated infections (BAI) constitute a major clinical problem and often necessitate implant replacement. In this study, the race for the surface between Staphylococcus epidermidis ATCC 35983 and U2OS osteosarcoma cells is studied on biomaterials with different wettabilities and on a polymer-brush coating. S. epidermidis was deposited on the different surfaces in a parallel plate flow chamber and then U2OS cells were seeded. Subsequently, staphylococci and U2OS cells were allowed to grow simultaneously on the surfaces for 48 h under low flow conditions. The presence of staphylococci reduced cell growth on all surfaces, but adhering cells spread equally well in the absence and presence of staphylococci. A hydrophilic polymer-brush coating discouraged bacterial and cellular adhesion and growth. Thus, whereas the biomaterials evaluated support both biofilm formation and tissue integration, polymer-brush coatings support neither. Therewith, the outcome of the race for the surface on these surfaces remains uncertain, emphasizing the need for biofunctionalized surfaces that discourage biofilm formation and support tissue growth at the same time.