Recently, our group developed two different polymeric biomaterials with photodynamic antimicrobial surface activity for periodontal bone regeneration. The aim of the present study was to analyze the biocompatibility and osseointegration of these materials in vivo. Two biomaterials based on urethane dimethacrylate (BioM1) and tri-armed oligoester-urethane methacrylate (BioM2) that additionally contained ß-tricalcium phosphate and the photosensitizer mTHPC (meso-tetra(hydroxyphenyl)chlorin) were implanted in non-critical size bone defects in the femur (n = 16) and tibia (n = 8) of eight female domestic sheep. Bone specimens were harvested and histomorphometrically analyzed after 12 months. BioM1 degraded to a lower extent which resulted in a mean remnant square size of 17.4 mm², while 12.2 mm² was estimated for BioM2 (p = 0.007). For BioM1, a total percentage of new formed bone by 30.3% was found which was significant higher compared to BioM2 (8.4%, p < 0.001). Furthermore, BioM1 was afflicted by significant lower soft tissue formation (3.3%) as compared to BioM2 (29.5%). Additionally, a bone-to-biomaterial ratio of 81.9% was detected for BioM1, while 8.5% was recorded for BioM2. Implantation of BioM2 caused accumulation of inflammatory cells and led to fibrous encapsulation. BioM1 (photosensitizer-armed urethane dimethacrylate) showed favorable regenerative characteristics and can be recommended for further studies.

OBJECTIVE:Evaluation of μCT scans of bone implant complexes often shows a specific problem: if an implant material has a very similar radiopacity as the embedding medium (e.g. methacrylate resin), the implant is not visible in the μCT image. Segmentation is not possible, and especially osseointegration as one of the most important parameter for biocompatibility is not evaluable.METHODS:To ensure μCT visualisation and contrast enhancement of the evaluated materials, the embedding medium Technovit® VLC7200 was doped with an iodine monomer for higher radiopacity in different concentrations and tested regarding to handling, polymerisation, and histological preparation, and visualisation in µCT. Six different µCT devices were used and compared with regard to scan conditions, contrast, artefacts, image noise, and spatial resolution for the evaluation of the bone-implant blocks.RESULTS:Visualisation and evaluation of all target structures showed very good results in all μCT scans as well as in histology and histological staining, without negative effects caused by iodine doping. Subsequent evaluation of explants of in vivo experiments without losing important information was possible with iodine doped embedding medium.CONCLUSION:Visualisation of implants with a similar radiopacity as the embedding medium could be considerably improved. µCT scan settings should be selected with the highest possible resolution, and different implant materials should be scanned individually for optimal segmentation. µCT devices with higher resolutions should be preferred.ADVANCES IN KNOWLEDGE:Iodine doped embedding medium is a useful option to increase radiopacity for better visualisation and evaluation of special target structures in µCT.

Sustained inflammation associated with dysregulated macrophage activation prevents tissue formation and healing of chronic wounds. Control of inflammation and immune cell functions thus represents a promising approach in the development of advanced therapeutic strategies. Here we describe immunomodulatory hyaluronan/collagen (HA-AC/coll)-based hydrogels containing high-sulfated hyaluronan (sHA) as immunoregulatory component for the modulation of inflammatory macrophage activities in disturbed wound healing. Solute sHA downregulates inflammatory activities of bone marrow-derived and tissue-resident macrophages in vitro. This further affects macrophage-mediated pro-inflammatory activation of skin cells as shown in skin ex-vivo cultures. In a mouse model of acute skin inflammation, intradermal injection of sHA downregulates the inflammatory processes in the skin. This is associated with the promotion of an anti-inflammatory gene signature in skin macrophages indicating a shift of their activation profile. For in vivo translation, we designed HA-AC/coll hydrogels allowing delivery of sHA into wounds over a period of at least one week. Their immunoregulatory capacity was analyzed in a translational experimental approach in skin wounds of diabetic db/db mice, an established model for disturbed wound healing. The sHA-releasing hydrogels improved defective tissue repair with reduced inflammation, augmented pro-regenerative macrophage activation, increased vascularization, and accelerated new tissue formation and wound closure.

With this study, an innovative and convenient enrichment and detection strategy for eight clinically relevant pneumonia pathogens, namely, Acinetobacter baumannii, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Moraxella catarrhalis, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae is introduced. Bacteria were isolated from sputum samples with amine-modified particles exploiting pH-dependent electrostatic interactions between bacteria and the functionalized particle surface. Following this, an asymmetric polymerase chain reaction as well as subsequent stringent array-based hybridization with specific complementary capture probes were performed. Finally, results were visualized by an enzyme-induced silver nanoparticle deposition, providing stable endpoint signals and consequently an easy detection possibility. The assay was optimized using spiked samples of artificial sputum with different strains of the abovementioned bacterial species. Furthermore, actual patient sputum samples with S. pneumoniae were successfully analyzed. The presented approach offers great potential for the urgent need of a fast, specific, and reliable isolation and identification platform for important pneumonia pathogens, covering the complete process chain from sample preparation up to array-based detection within only 4 h.

We present a dispersion analysis of the orthorhombic peptide single crystal Z-Aib-Aib-Aib-L-Ala-OtBu (N-benzyloxycarbonyl-α-aminoisobutyryl-α-aminoisobutyryl-α-aminoisobutyryl-L-alanine tert-butyl ester, C27H42N4O7), where Z is benzyloxycarbonyl and OtBu is the tert-butylester) in the MIR spectral region by means of adapted generalized dispersion analysis, employing the naturally grown crystal faces. Based on the results we identify the orientation of the crystal axes a, b, c within the sample, and supported by a stereographic projection of the crystal, we assign the individual axes. The gained dielectric tensor function and the oscillator parameters were confirmed by forward calculation of reflection spectra of different orientations. The orientation of the crystal axes was verified by a second stereographic projection with another crystal face in the center.

Hyaluronan (HA)-based microgels generated in a microfluidic approach, containing an artificial extracellular matrix composed of collagen and high-sulfated hyaluronan (sHA3), were incorporated into a HA/collagen-based hydrogel matrix. This significantly enhanced the retention of noncrosslinked sHA3 within the gels enabling controlled sHA3 presentation. Gels containing sHA3 bound higher amounts of transforming growth factor-β1 (TGF-β1) compared to pure HA/collagen hydrogels. Moreover, the presence of sHA3-containing microgels improved the TGF-β1 retention within the hydrogels. These findings are promising for developing innovative biomaterials with adjustable sHA3 release and growth factor interaction profiles to foster skin repair, e.g., by rebalancing dysregulated TGF-β1 levels.

The crystal structure of the natural nonapeptide antibiotic helioferin has been determined and refined to 0.9 Å resolution. Helioferin consists of helioferin A and B, which contain 2-(2'-aminopropyl)aminoethanol (Apae) and 2-[(2'-aminopropyl)methylamino]ethanol (Amae) at their respective alkanolamine termini. In addition, helioferin contains the unusual amino-acid residues α-aminoisobutyric acid (Aib) and (2S,4S,6S)-2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid (Ahmod). The amino-terminus is capped with 2-methyl-n-1-octanoic acid (M8a). The peptide crystallizes with a 1:1 molar ratio of helioferin A and B in the monoclinic space group C2, with unit-cell parameters a = 34.711, b = 10.886, c = 17.150 Å, β = 93.05°. The peptide backbone folds in a regular right-handed α-helical conformation, with eight intramolecular hydrogen bonds, all but one forming 5→1 interactions. The two aliphatic chains of the fatty-acyl (M8a) and the second residue (Ahmod) extend out of the α-helical structure in opposite directions and lead to a corkscrew-like shape of the peptide molecule. Halogen anions (Cl- and F-) have been co-crystallized with the peptide molecules, implying a positive charge at the aminoalcohol end of the peptide. In the tightly packed crystal the helices are linked head to tail via the anions by electrostatic, hydrogen-bond and van der Waals interactions, forming continuous helical rods. Two nonparallel rods (forming an angle of 118°) interact directly via hydrogen bonds and via the anions, forming a double layer. Successive double layers are held together only via van der Waals contacts. The helical axes of successive double layers are also related by an angle of 118°. The structure of helioferin reported here and the previously determined structure of the homologous leucinostatin A have a total straight length of about 21 Å, indicating a different membrane-modifying bioactivity from that of long-chain, amphiphilic peptaibols.

Levan is a high molecular weight fructose-based biotechnologically available polysaccharide with a range of interesting properties qualifying this molecule for applications in biomedicine. In this study, new levan derivatives containing methacrylate groups attached either via ester or urethane linkages to the fructan backbone could be synthesized and structurally characterized by conventional analytical techniques. The photochemical crosslinking of these substances applying different photoinitiator systems and reaction conditions resulted in hydrogels of diverse properties which were investigated with regard to mechanical behaviour, hydrolytic degradability, and cytocompatibility. It was found that crosslinkable levan derivatives represent a new class of promising biopolymer-based macromers broadening the spectrum of available biomaterials to facilitate the adaption to the requirements of specific applications.

Bergofungin is a peptide antibiotic that is produced by the ascomycetous fungus Emericellopsis donezkii HKI 0059 and belongs to peptaibol subfamily 2. The crystal structure of bergofungin A has been determined and refined to 0.84 Å resolution. This is the second crystal structure of a natural 15-residue peptaibol, after that of samarosporin I. The amino-terminal phenylalanine residue in samarosporin I is exchanged to a valine residue in bergofungin A. According to agar diffusion tests, this results in a nearly inactive antibiotic peptide compared with the moderately active samarosporin I. Crystals were obtained from methanol solutions of purified bergofungin mixed with water. Although there are differences in the intramolecular hydrogen-bonding scheme of samarosporin I, the overall folding is very similar for both peptaibols, namely 310-helical at the termini and α-helical in the middle of the molecules. Bergofungin A and samarosporin I molecules are arranged in a similar way in both lattices. However, the packing of bergofungin A exhibits a second solvent channel along the twofold axis. This latter channel occurs in the vicinity of the N-terminus, where the natural substitution resides.

OBJECTIVE:Our study was performed with the aim of preparing electrospun polylactide fibers with a combination of ampicillin (AMP) and metronidazole (MNZ) and investigating their drug release behavior and the antibacterial effect on Aggregatibacter actinomycetemcomitans and other oral pathogens. METHODS:AMP and MNZ were integrated as a combination in two separate fibers (dual fiber mats - DFW mix) of electrospun PLA fiber mats by means of multijet electrospinning and in a single fiber (single fiber mats - SFW mix). HPLC (high-performance liquid chromatography) was used to measure the released drug quantities. Agar diffusion tests were used to determine the antibacterial effect of the eluates on A. actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis and Enterococcus faecalis. The neutral red test was made to examine the cytocompatibility of the eluates with human gingival fibroblasts (hGFs). RESULTS:The release of the active agents varied with the antibiotic concentrations initially used in the fiber mats, but also with the distribution of the active agents in one or two fibers. Of the total quantity of MNZ (AMP), the SFW mix fiber mats released >60% (>70%) within a span of 5min, and 76% (71%) after 96h. With these drug concentrations released by the fiber mats (≥5m%), an antibacterial effect was achieved on A. actinomycetemcomitans and on all other species tested. Fiber mats and their eluates have no cytotoxic influence on human gingival fibroblasts (hGFs). SIGNIFICANCE:Electrospun AMP/MNZ-loaded polymer fibers are a potential drug delivery system for use in periodontal and endodontic infections.

The fabrication of 3D hydrogel microarrays for DNA analytics that allow simple visual signal readout for on-site applications is described. A convenient one-step polymerization of the hydrogel including in situ capture oligonucleotide immobilization is accomplished by using N,N'-dimethylacrylamide/polyethylene glycol (PEG1900 )-bisacrylamide monomers. The implementation of an acylphosphine-oxide photoinitiator even allows polymerization at daylight, whereas other approaches require exposure with light in the UV-range. This minimizes the risk of UV-caused DNA damages within the capture DNA-strand that could adversely affect the subsequent hybridization step. The porous network of these gel segments allows DNA as well as protein penetration. Thus, the successful in-gel DNA hybridization is monitored by the deposition of silver nanoparticles. These metal particles allow naked eye signal readout.

This study focuses on the development of novel biocompatible macroporous cryogels by electron-beam assisted free-radical crosslinking reaction of polymerizable dextran and hyaluronan derivatives. As a main advantage this straightforward approach provides highly pure materials of high porosity without using additional crosslinkers or initiators. The cryogels were characterized with regard to their morphology and their basic properties including thermal and mechanical characteristics, and swellability. It was found that the applied irradiation dose and the chemical composition strongly influence the material properties of the resulting cryogels. Preliminary cytotoxicity tests illustrate the excellent in vitro-cytocompatibility of the fabricated cryogels making them especially attractive as matrices in tissue regeneration procedures.

OBJECTIVES:We aimed to achieve detailed biomaterials characterization of a drug delivery system for local periodontitis treatment based on electrospun metronidazole-loaded resorbable polylactide (PLA) fibers. METHODS:PLA fibers loaded with 0.1-40% (w/w) MNA were electrospun and were characterized by SEM and DSC. HPLC techniques were used to analyze the release profiles of metronidazole (MNA) from these fibers. The antibacterial efficacy was determined by measuring inhibition zones of drug-containing aliquots from the same electrospun fiber mats in an agar diffusion test. Three pathogenic periodontal bacterial strains: Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were studied. Cytotoxicity testing was performed with human gingival fibroblasts by: (i) counting viable cells via live/dead staining methods and (ii) by exposing cells directly onto the surface of MNA-loaded fibers. RESULTS:MNA concentration influenced fiber diameters and thus w/w surface areas: diameter being minimal and area maximal at 20% MNA. HPLC showed that these 20% MNA fibers had the fastest initial MNA release. From the third day, MNA release was slower and nearly linear with time. All fiber mats released 32-48% of their total drug content within the first 7 days. Aliquots of media taken from the fiber mats inhibited the growth of all three bacterial strains. MNA released up to the 28th day from fiber mats containing 40% MNA significantly decreased the viability of F. nucleatum and P. gingivalis and up to the 2nd day also for the resistant A. actinomycetemcomitans. All of the investigated fibers and aliquots showed excellent cytocompatibility. SIGNIFICANCE:This study shows that MNA-loaded electrospun fiber mats represent an interesting class of resorbable drug delivery systems. Sustained drug release properties and cytocompatibility suggest their potential clinical applicability for the treatment of periodontal diseases.

Objectives. Inflammatory periodontal diseases are accompanied by destruction of periodontal tissue and alveolar bone. Infrabony lesions can be regenerated with adequate bone substitutes, which require high biocompatibility of the material.Methods. To rate the biocompatibility of nine polymeric periodontal bone substitutes (Bio 1-Bio 9), cell viability and cytotoxicity assays were performed. For viability, human gingival fibroblasts (HGFs) and MC3T3 osteoblasts were cultured on the bone substitutes. For cytotoxicity, biomaterial extracts were prepared by incubation with culture medium for maximally 28 days, and cells were exposed to the extracts for 1 day. Polymers Bio 1 to Bio 5 were prepared by solvent casting, Bio 6 to Bio 9 by photopolymerization of the monomers at wavelengths of 400-500 nm in the presence of a suitable photoinitiation system.Results. Bio 1, Bio 3, Bio 4, Bio 5, and Bio 7 showed moderate to excellent cytocompatibility for both HGFs and osteoblasts in viability tests. Together with the results of the cytotoxicity assays, four of the nine tested polymers were considered cytocompatible: Bio 1 (poly(vinyl butyral-co-vinyl alcohol-co-vinyl acetate; PVB)), Bio 4 and Bio 5 (functionalized oligolactones), and, to a limited degree, Bio 7 (urethane methacrylate). Except for Bio 7, the cytocompatible polymers showed intermediate water contact angles (74-85.) and therefore moderate to low hydrophilicity.Significance. The non-cross-linked polymers Bio 1, Bio 4, or Bio 5, and the photopolymerized polymeric network Bio 7 display good/excellent cytocompatibility and are therefore potential candidates for tissue engineering in alveolar bone substitution. (C) 2012 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

In order to investigate the significance of antibiotics for the producing organism(s) in the natural habitat, we screened specimens of the polyporicolous fungus Hypocrea pulvinata growing on its natural hosts Piptoporus betulinus and Fomitopsis pinicola. Results showed that a particular group of nonribosomally biosynthesised antibiotic polypeptides, the peptaibiotics, which contain the nonproteinogenic marker amino acid α-aminoisobutyric acid (Aib), was produced in the natural habitat by the fungicolous producer and, consequently, released into the host. Using liquid chromatography coupled to electrospray high-resolution mass spectrometry we detected especially 19-, but also 11-, 18-, and 20-residue peptaibiotics in the five infected specimens analysed. Structures of peptaibiotics found were confirmed by analysing the peptaibiome of pure agar cultures obtained by single-ascospore isolation from the specimens. The 19-residue peptaibols were determined as deletion sequences of the trichosporins B lacking the Aib residue in position 6. Notably, 26 of the 28 peptaibiotics sequenced were novel; therefore the name 'hypopulvins' was introduced. Considering not only the ubiquity of both the two host species but also the highly specific association between H. pulvinata and P. betulinus/F. pinicola, and the abundance of this fungicolous species in north temperate regions of the world, a decisive role for the peptaibiotics detected in this study is predicted, which may act as mediators of the complex interactions between the basidiomycetous host and its fungicolous ascomycete 'partner'. Structural analogies of the hypopulvins, particularly with other 18-, 19-, and 20-residue peptaibiotics, suggest that the hypopulvins are forming transmembrane ion channels and could thus support the hypothesis of a parasitic lifestyle of the fungicolous producer.

Three-dimensional microstructured scaffolds provide a means for cells to be cultured in vitro in a way that resembles natural conditions more closely than flat tissue culture polystyrene. In the presented work, two-photon polymerization (2PP) is applied as a tool for the engineering of high-resolution 3D scaffold structures with a well defined microarchitecture made of biocompatible photo resins. 2PP is a novel photolithographic technique using femtosecond laser pulses which enables free 3D microstructuring of liquid photo resins due to the relationship of the axial and lateral spatial confinement of the photoreaction to the focal volume of a focused laser beam. A set of photo resins were tested with regard to 2PP processability and three different classes of methacrylated photopolymerizable monomers (methacrylated oligolactones, urethane dimethacrylate, poly(ethylene glycol diacrylate)) were found to be efficient 2PP materials. 3D microstructures based on computer models were produced and tested for biocompatibility. The initial cell adhesion and the viability of bovine chondrocytes on the polymeric scaffolds were evaluated morphologically by confocal laser scanning microscopy (CLSM) after three-day culture on 2PP derived microstructures. 2PP derived scaffolds were fabricated in different sizes and geometries, starting from the 100 mu m-range reaching out to the cm-range showing the actual possibilities to produce large volume scaffolds even for implantation purposes.

Poly(ethyleneimine) (PEI) is used as an initiating base layer or intermediate layer for layer‐by‐layer (LBL) technology. Since PEI is potentially cytotoxic in solution, it is necessary to investigate the influence of the physical properties of PEI layers on osteoblast cells. Therefore, the first aim of this study was to test the hypothesis that there is no statistically significant difference of cell adhesion, proliferation, and viability in response to nanometer thin polymer layers of PEI compared to other well‐known polymers. The second aim of this study was to test the hypothesis that the amine‐substituted dextran (aminodextran (AMD)) adheres well on Ti and can be used as a polymer coating in biomaterials applications. Titanium samples were coated with PEI, poly‐L‐lysine (PLL), or AMD using dip coating. The polymer films were investigated with ellipsometry, atomic force microscopy, contact angle measurements, and X‐ray‐photoelectron spectroscopy (XPS). Cell adhesion, proliferation, and viability tests were carried out using osteoblast‐like cells (CAL‐72). Thin polymer layers below 10 nm layer thickness were found on the Ti surfaces. It was shown for the first time that base layers of PEI with nanometer thickness do not affect the bone cell reaction negatively. PEI, therefore, can be used as a functional base layer for LBL functionalization of metallic implants. AMD can be used in biomedical applications but does not adhere on titanium without an adhesion promoter.

Two-photon polymerization (2-PP) is a promising new photolithographic technique to fabricate three-dimensional (3D), micro-and nano-structured tissue engineering scaffolds from photopolymerizable monomers. Although various photo resins are known for the use in 2-PP, there is currently a need for photo-curable monomers processable by 2-PP to generate biocompatible 3D-structured hydrogel materials for soft or cartilage tissue regeneration. In the present work hydrophilic methacrylate monomers and macromers based on synthetic poly(glycerine) and poly(ethylene glycol) urethanes as well as on the biopolymers dextran and hyaluronan is prepared. The photopolymerization behavior of these substances are investigated and formed hydrogel networks are studied with regard to their mechanical properties, cytocompatibility, and hydrolytic degradation. Based on these examinations simple 3D model structures are fabricated from these photo-curable monomers and macromers by 2-PP. It is shown that both the synthetic monomers and the dextran methacrylate macromer are efficient 2-PP starting materials whereas the hyaluronan methacrylate can be used for 2-PP only in combination with suitable water-soluble co-monomers. No cytotoxic effects are found in preliminary chondrocyte cultivation experiments on 2-PP-fabricated scaffolds but initial cell adhesion on the hydrophilic scaffold surfaces is rather low and has to be further improved to apply these structures in tissue engineering.