Coaxial electrospun nanofibers as drug delivery system for local treatment of periodontitis.

OBJECTIVES: The aim of the present study was to prepare resorbable polylactide fibers for periodontitis treatment using coaxial electrospinning to optimize the release of metronidazole (MNA) by reducing the initial burst effect. METHODS: Poly(L-lactide-co-D,L-lactide) (PLA) fibers mats with different distributions of metronidazole (MNA) were manufactured by coaxial electrospinning (COAX). By COAX spinning the central core of the fiber was enriched with 40% MNA (m/m), while the sheath of the fiber consisted of PLA only (test group). In contrast, fibers of the control group were prepared by conventional electrospinning with the same amount of MNA but with a homogenous drug distribution (HDD - homogenously distributed drug). The release of MNA was determined by analyzing aliquots from the fiber mats using UV-VIS spectroscopy. Agar diffusion tests were carried out to determine the antibacterial effect on periodontopathogenic bacteria. Biocompatibility was tested in direct contact to human gingival fibroblasts (HGF) for two days. RESULTS: The COAX mats showed a retarded drug release compared to the conventional HDD fibers. After 24 h, 64% of total MNA was released cumulatively from the COAX fibers while 90% of the MNA was released from the HDD fibers (controls). The antibacterial effect of COAX fibers was significantly higher after 24 h compared to the HDD fibers. Cell cultivation revealed significant higher numbers of vital cells among the COAX mats. SIGNIFICANCE: COAX fibers showed improved sustained MNA release compared to conventional fibers and can be seen as potential drug delivery systems in local periodontitis treatment.

Comparable Studies on Nanoscale Antibacterial Polymer Coatings Based on Different Coating Procedures.

The antibacterial activity of different antibiotic and metal-free thin polymer coatings was investigated. The films comprised quaternary ammonium compounds (QAC) based on a vinyl benzyl chloride (VBC) building block. Two monomeric QAC of different alkyl chain lengths were prepared, and then polymerized by two different polymerization processes to apply them onto Ti surfaces. At first, the polymeric layer was generated directly on the surface by atom transfer radical polymerization (ATRP). For comparison purposes, in a classical route a copolymerization of the QAC-containing monomers with a metal adhesion mediating phosphonate (VBPOH) monomers was carried out and the Ti surfaces were coated via drop coating. The different coatings were characterized by X-ray photoelectron spectroscopy (XPS) illustrating a thickness in the nanomolecular range. The cytocompatibility in vitro was confirmed by both live/dead and WST-1 assay. The antimicrobial activity was evaluated by two different assays (CFU and BTG, resp.,), showing for both coating processes similar results to kill bacteria on contact. These antibacterial coatings present a simple method to protect metallic devices against microbial contamination.

Glycosaminoglycan-Based Cryogels as Scaffolds for Cell Cultivation and Tissue Regeneration.

Cryogels are a class of macroporous, interconnective hydrogels polymerized at sub-zero temperatures forming mechanically robust, elastic networks. In this review, latest advances of cryogels containing mainly glycosaminoglycans (GAGs) or composites of GAGs and other natural or synthetic polymers are presented. Cryogels produced in this way correspond to the native extracellular matrix (ECM) in terms of both composition and molecular structure. Due to their specific structural feature and in addition to an excellent biocompatibility, GAG-based cryogels have several advantages over traditional GAG-hydrogels. This includes macroporous, interconnective pore structure, robust, elastic, and shape-memory-like mechanical behavior, as well as injectability for many GAG-based cryogels. After addressing the cryogelation process, the fabrication of GAG-based cryogels and known principles of GAG monomer crosslinking are discussed. Finally, an overview of specific GAG-based cryogels in biomedicine, mainly as polymeric scaffold material in tissue regeneration and tissue engineering-related controlled release of bioactive molecules and cells, is provided.

Tendon Biomimetic Electrospun PLGA Fleeces Induce an Early Epithelial-Mesenchymal Transition and Tenogenic Differentiation on Amniotic Epithelial Stem Cells.

Background. The design of tendon biomimetic electrospun fleece with Amniotic Epithelial Stem Cells (AECs) that have shown a high tenogenic attitude may represent an alternative strategy to overcome the unsatisfactory results of conventional treatments in tendon regeneration. Methods. In this study, we evaluated AEC-engineered electrospun poly(lactide-co-glycolide) (PLGA) fleeces with highly aligned fibers (ha-PLGA) that mimic tendon extracellular matrix, their biocompatibility, and differentiation towards the tenogenic lineage. PLGA fleeces with randomly distributed fibers (rd-PLGA) were generated as control. Results. Optimal cell infiltration and biocompatibility with both PLGA fleeces were shown. However, only ha-PLGA fleeces committed AECs towards an Epithelial-Mesenchymal Transition (EMT) after 48 h culture, inducing their cellular elongation along the fibers' axis and the upregulation of mesenchymal markers. AECs further differentiated towards tenogenic lineage as confirmed by the up-regulation of tendon-related genes and Collagen Type 1 (COL1) protein expression that, after 28 days culture, appeared extracellularly distributed along the direction of ha-PLGA fibers. Moreover, long-term co-cultures of AEC-ha-PLGA bio-hybrids with fetal tendon explants significantly accelerated of half time AEC tenogenic differentiation compared to ha-PLGA fleeces cultured only with AECs. Conclusions. The fabricated tendon biomimetic ha-PLGA fleeces induce AEC tenogenesis through an early EMT, providing a potential tendon substitute for tendon engineering research.

Critical physiological factors influencing the outcome of antimicrobial testing according to ISO 22196 / JIS Z 2801.

Bactericidal materials gained interest in the health care sector as they are capable of preventing material surfaces from microbial colonization and subsequent spread of infections. However, commercialization of antimicrobial materials requires proof of their efficacy, which is usually done using in vitro methods. The ISO 22196 standard (Japanese test method JIS Z 2801) is a method for measuring the antibacterial activity of daily goods. As it was found reliable for testing the biocidal activity of antimicrobially active materials and surface coatings most of the laboratories participating in this study used this protocol. Therefore, a round robin test for evaluating antimicrobially active biomaterials had to be established. To our knowledge, this is the first report on inaugurating a round robin test for the ISO 22196 / JIS Z 2801. The first round of testing showed that analyses in the different laboratories yielded different results, especially for materials with intermediate antibacterial effects distinctly different efficacies were noted. Scrutinizing the protocols used by the different participants and identifying the factors influencing the test outcomes the approach was unified. Four critical factors influencing the outcome of antibacterial testing were identified in a series of experiments: (1) incubation time, (2) bacteria starting concentration, (3) physiological state of bacteria (stationary or exponential phase of growth), and (4) nutrient concentration. To our knowledge, this is the first time these parameters have been analyzed for their effect on the outcome of testing according to ISO 22196 / JIS Z 2801. In conclusion, to enable assessment of the results obtained it is necessary to evaluate these single parameters in the test protocol carefully. Furthermore, uniform and robust definitions of the terms antibacterial efficacy / activity, bacteriostatic effects, and bactericidal action need to be agreed upon to simplify communication of results and also regulate expectations regarding antimicrobial tests, outcomes, and materials.

Adjustable Polyurethane Foam as Filling Material for a Novel Spondyloplasty: Biomechanics and Biocompatibility.

OBJECTIVE: To investigate the biomechanics and biocompatibility of polyurethane (PU) foam with adjustable stiffness as a filling material for a novel spondyloplasty that is designed to reduce the risk of postoperative adjacent level fractures. METHODS: Sixty individual porcine lumbar vertebrae were randomly split into 4 groups: A, B, C, and D. Group A served as unmodified vertebral body controls. Groups B, C, and D consisted of hollowed vertebral bodies. Vertebrae of groups C and D were filled with adjustable PU foams of different stiffness. The compressive strength and stiffness of vertebrae from groups A-D were recorded and analyzed. 3T3 mouse fibroblasts were cultured with preformed PU foams for 4 days to test biocompatibility. RESULTS: The strength and stiffness of the hollowed groups were lower than in group A. However, the differences were not statistically significant between group A and group C (P > 0.05), and were obviously different between group A and group B or group D (P < 0.01 and <0.05, respectively). Moreover, the strength and stiffness after filling foams in group C or group D were significantly greater than in group B (P < 0.01 and <0.05, respectively). Live/dead staining of 3T3 cells confirmed the biocompatibility of the PU foam. CONCLUSIONS: The new PU foam shows adaptability regarding its stiffness and excellent cytocompatibility in vitro. The results support the clinical translation of the new PU foams as augmentation material in the development of a novel spondyloplasty.

Optimized vascular network by stereolithography for tissue engineered skin.

This paper demonstrates the essential and efficient methods to design, and fabricate optimal vascular network for tissue engineering structures based on their physiological conditions. Comprehensive physiological requirements in both micro and macro scales were considered in developing the optimisation design for complex vascular vessels. The optimised design was then manufactured by stereolithography process using materials that are biocompatible, elastic and surface bio-coatable. The materials are self-developed photocurable resin consist of BPA-ethoxylated-diacrylate, lauryl acrylate and isobornylacrylate with Irgacure® 184, the photoinitiator. The optimised vascular vessel offers many advantages: 1) it provides the maximum nutrient supply; 2) it minimises the recirculation areas and 3) it allows the wall shear stress on the vessel in a healthy range. The stereolithography manufactured vascular vessels were then embedded in the hydrogel seeded with cells. The results of in vitro studies show that the optimised vascular network has the lowest cell death rate compared with a pure hydrogel scaffold and a hydrogel scaffold embedded within a single tube in day seven. Consequently, these design and manufacture routes were shown to be viable for exploring and developing a high range complex and specialised artificial vascular networks.

Formation of new, cytocompatible hydrogels based on photochemically crosslinkable levan methacrylates.

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.

In situ foamable, degradable polyurethane as biomaterial for soft tissue repair.

Degradable foams which can be inserted endoscopically as liquid or pasty mixtures into soft tissue defects possess a promising potential for the surgical treatment of such defects. The defects can be sealed under in situ foaming and simultaneous material expansion. We developed an in situ foamable (l-lactide-co-ε-caprolactone)-based, star-shaped prepolymer by ring opening polymerization of l-lactide and ε-caprolactone in the presence of meso-erythritol as starter. By conversion of the terminal hydroxyl groups of the formed oligoester with lysine diisocyanate ethyl ester (LDI) an isocyanate-endcapped, reactive prepolymer has been received. Foaming can be initiated by addition of 1,4-diazabicyclo[2,2,2]octane (DABCO), water, LDI and DMSO. By varying the composition of these additives, the foaming and curing time could be varied within a clinically acceptable range. A porosity of approximately 90%, and an average tensile strength of 0.3MPa with elongations of 90% were determined for the foams. In vitro cytotoxicity on cured foams was assayed on 3T3 fibroblasts and demonstrated an excellent cytocompatibility. This was also confirmed in an in vivo study using an established rat model, where prefabricated foams and in situ hardening material were inserted into subdermal skin incisions in parallel. The feature of chronic inflammation was only weakly developed in both groups and slightly more pronounced and persisted for longer time in the group of in situ foamed material. In both groups the foreign materials were vascularized, degraded and substituted by connective tissue. The results encourage to proceed with trials where the materials are used to fill more heavily loaded defects.

Bactericidal Effect of a Photoresponsive Carbon Monoxide-Releasing Nonwoven against Staphylococcus aureus Biofilms.

Staphylococcus aureus is a leading pathogen in skin and skin structure infections, including surgical and traumatic infections that are associated with biofilm formation. Because biofilm formation is accompanied by high phenotypic resistance of the embedded bacteria, they are almost impossible to eradicate by conventional antibiotics. Therefore, alternative therapeutic strategies are of high interest. We generated nanostructured hybrid nonwovens via the electrospinning of a photoresponsive carbon monoxide (CO)-releasing molecule [CORM-1, Mn2(CO)10] and the polymer polylactide. This nonwoven showed a CO-induced antimicrobial activity that was sufficient to reduce the biofilm-embedded bacteria by 70% after photostimulation at 405 nm. The released CO increased the concentration of reactive oxygen species (ROS) in the biofilms, suggesting that in addition to inhibiting the electron transport chain, ROS might play a role in the antimicrobial activity of CORMs on S. aureus The nonwoven showed increased cytotoxicity on eukaryotic cells after longer exposure, most probably due to the released lactic acid, that might be acceptable for local and short-time treatments. Therefore, CO-releasing nonwovens might be a promising local antimicrobial therapy against biofilm-associated skin wound infections.

Biocompatible polysaccharide-based cryogels.

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.

Light-triggered CO release from nanoporous non-wovens.

The water insoluble and photoactive CO releasing molecule dimanganese decacarbonyl (CORM-1) has been non-covalently embedded into poly(l-lactide-co-d/l-lactide) fibers via electrospinning to enable bioavailability and water accessibility of CORM-1. SEM images of the resulting hybrid non-wovens reveal a nanoporous fiber morphology. Slight CO release from the CORM-1 in the electrospinning process induces nanoporosity. IR spectra show the same set of carbonyl bands for the CORM-1 precursor and the non-woven. When the material was exposed to light (365-480 nm), CO release from the incorporated CORM-1 was measured via heterogeneous myoglobin assay, a portable CO electrode and an IR gas cuvette. The CO release rate was wavelength dependent. Irradiation at 365 nm resulted in four times faster release than at 480 nm. 3.4 µmol of CO per mg non-woven can be generated. Mouse fibroblast 3T3 cells were used to show that the hybrid material is non-toxic in the darkness and strongly photocytotoxic when light is applied.

Functionalized, biocompatible coating for superparamagnetic nanoparticles by controlled polymerization of a thioglycosidic monomer.

It is demonstrated that water-soluble, glucosylated poly(pentafluorostyrene) derivatives revealed favorable coating material properties for magnetic iron oxide nanoparticles. To prepare the coating material in high reproducibility and purity as well as in sufficient amounts, a new route of synthesis is established. The preparation and characterization of the glucosylated, tetrafluorostyryl monomer, by thiol-para-fluorine "click" reaction, and its polymerization, via nitroxide-mediated radical process, is presented in detail. In addition, the coating material and the resulting particle properties are investigated by means of XPS, DLS, TGA, TEM, and cryo-TEM as well as flow cytometry. The glycopolymer acts as an appropriate stabilizing agent for the superparamagnetic nanoparticles by the formation of an approximately 10 nm thick shell, as shown by the XPS analysis. Furthermore, the application of FITC-labeled glycopolymer yielded fluorescent, superparamagnetic nanoparticles, which can be used for monitoring cell-carbohydrate interactions, because these particles show no cytotoxicity toward 3T3 fibroblasts.

Adhesion of preosteoblasts and fibroblasts onto poly(pentafluorostyrene)-based glycopolymeric films and their biocompatibility.

An efficient and metal-catalyst free method of glycopolymer synthesis via thiol/para-fluorine "click" reaction was used to graft acetylated 1-thio-ß-D-glucopyranose and 1-thio-ß-D-galactopyranose onto a homopolymer of pentafluorostyrene (PFS) as well as onto a block copolymer of styrene and PFS. Subsequent deprotection of the carbohydrate moieties yielded well-defined, sugar-modified polymers (PDI < 1.2). The prepared polymers were not cytotoxic against 3T3 fibroblasts and MC3T3-E1 preosteoblasts. Furthermore, the water-insoluble copolymers were drop-cast and examined as synthetic biocompatible coatings on poly(propylene) substrates for culturing the investigated cell types. Both fibro- and preosteoblasts showed stable adhesion and proliferation on the glycopolymer-coated surfaces.

Electrospun poly(D/L-lactide-co-L-lactide) hybrid matrix: a novel scaffold material for soft tissue engineering.

Electrospinning is a long-known polymer processing technique that has received more interest and attention in recent years due to its versatility and potential use in the field of biomedical research. The fabrication of three-dimensional (3D) electrospun matrices for drug delivery and tissue engineering is of particular interest. In the present study, we identified optimal conditions to generate novel electrospun polymeric scaffolds composed of poly-D/L-lactide and poly-L-lactide in the ratio 50:50. Scanning electron microscopic analyses revealed that the generated poly(D/L-lactide-co-L-lactide) electrospun hybrid microfibers possessed a unique porous high surface area mimicking native extracellular matrix (ECM). To assess cytocompatibility, we isolated dermal fibroblasts from human skin biopsies. After 5 days of in vitro culture, the fibroblasts adhered, migrated and proliferated on the newly created 3D scaffolds. Our data demonstrate the applicability of electrospun poly(D/L-lactide-co-L-lactide) scaffolds to serve as substrates for regenerative medicine applications with special focus on skin tissue engineering.

Degradable phosphate glass fiber reinforced polymer matrices: mechanical properties and cell response.

The development of biodegradable materials for internal fracture fixation is of great interest, as they would both eliminate the problem of stress shielding and obviate the need for a second operation to remove fixation devices. Preliminary investigations for the production of degradable fiber reinforced polymer composite materials are detailed. Composites were produced of phosphate invert glass fibers of the glass system P(2)O(5)-CaO-MgO-Na(2)O-TiO(2), which showed a low solubility in previous work. The fibers were embedded into a matrix of a degradable organic polymer network based on methacrylate-modified oligolactide. Fracture behavior, bending strength and elastic modulus were evaluated during 3-point bending tests and the fracture surface of the composites was investigated using a scanning electron microscope. Short-term biocompatibility was tested in an FDA/EtBr viability assay using MC3T3-E1 murine pre-osteoblast cells and showed a good cell compatibility of the composite materials. Results suggested that these composite materials are biocompatible and show mechanical properties which are of interest for the production of degradable bone fixation devices.

Dextran and hyaluronan methacrylate based hydrogels as matrices for soft tissue reconstruction.

Polysaccharide hydrogels have become increasingly studied as matrices in soft tissue engineering because of their known cytocompatibility. In this work cross-linkable dextran methacrylates and hyaluronan methacrylate were synthesized and their transformation into stable hydrogels was studied. The in vitro degradation behaviour of the formed hydrogels could be controlled by the polysaccharide structure and the cross-linking density. Under in vitro conditions, the formed gels had no cytotoxic effects against fibroblasts, but cells could adhere only inefficiently in long term experiments. The use of composite gels improved the adherence of cells. Different scaffold architectures were studied including porous structures and perforated gel layers. Selected hydrogels were examined in an in vivo pilot study using a rabbit model to evaluate their biocompatibility, stability and degradation. No signs of inflammation were seen and with prolonged duration the material was degraded and lacunas were formed by immigrating or ingrowing cells. Optimizing their mechanical properties, the formed hydrogels represent promising candidates as matrices for soft tissue reconstruction.

Development of a new calcium phosphate powder-binder system for the 3D printing of patient specific implants.

A key requirement for three-dimensional printing (3-DP) of medical implants is the availability of printable and biocompatible powder-binder systems. In this study we developed a powder mixture comprising tetracalcium phosphate (TTCP) as reactive component and beta-tricalcium phosphate (beta-TCP) or calcium sulfate as biodegradable fillers, which can be printed with an aqueous citric acid solution. The potential of this material combination was demonstrated printing various devices with intersecting channels and filigree structures. Two post-processing procedures, a sintering and a polymer infiltration process were established to substantially improve the mechanical properties of the printed devices. Preliminary examinations on relevant application properties including in vitro cytocompatibility testing indicate that the new powder-binder system represents an efficient approach to patient specific ceramic bone substitutes and scaffolds for bone tissue engineering.

Fabrication and in vitro characterization of porous biodegradable composites based on phosphate glasses and oligolactide-containing polymer networks.

Degradable porous composite materials for use as temporary bone replacement or tissue engineering scaffolds were produced using a methacrylate-modified oligolactide polymer network and phosphate invert glasses in the system P2O5-CaO-MgO-Na2O-(TiO2). Porous glasses with an open interconnective porosity were produced by a salt sintering process. Compressive strengths were significantly enhanced by polymer coating of the inner surface of the porous glasses or by fabrication of glass powder-reinforced porous polymer specimens. In vitro degradation in simulated body fluid showed a degradation pattern of the composites which could be modulated by the composition and resulting solubility of the incorporated glass phase. Cytocompatibility of the composites was investigated in a FDA/EtBr viability assay using an MC3T3-E1 osteoblast-like cell line and showed good biocompatibility of the materials in vitro.

Four distinct chondrocyte populations in the fetal bovine growth plate: highest expression levels of PTH/PTHrP receptor, Indian hedgehog, and MMP-13 in hypertrophic chondrocytes and their suppression by PTH (1-34) and PTHrP (1-40).

Differentiation and growth of chondrocytes in fetal growth plates of vertebrate long bones and ribs appear to occur in a gradual, continuous manner between the resting zone through the proliferation zone, maturation zone, and upper and lower hypertrophic zones, with a continuous increase in cell size up to 10-fold of the volume of a resting chondrocyte. Here we provide evidence, however, that after centrifugation through a continuous Percoll gradient growth plate chondrocytes separate into four distinct cell populations (B1 to B4) which differ markedly in density, size, and gene expression. These populations collect in the absence of any phase borders in the gradient which might serve as concentration barriers. Fractions B1 and B2 contained the largest cells with the lowest buoyant density and showed the highest expression levels for type X collagen (Col X), but only the B1 population expressed high levels of matrix metalloproteinase-13 (collagenase 3). Cells in fraction B3 were significantly smaller and expressed little Col X, while cells in fraction B4 were of similar size to cells in the resting zone without significant Col X expression. The highest levels of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR-1), and Indian hedgehog (Ihh) expression were also found in the hypertrophic fractions B1 and B2 and not in the prehypertrophic fraction B3, as expected from in situ hybridization data on PTHR-1 expression in fetal rodent or chicken growth plates. Incubation of fractions B1 to B3 with the amino-terminal fragments PTH (1-34) or PTHrP (1-40) suppressed the expression of Col X and PTHR-1 by more than 50% and the expression of Ihh nearly completely. In contrast, the mid-regional PTH fragment PTH (28-48) and PTH (52-84) consistently stimulated the expression of PTHR-1 by 10-20% in fractions B1 to B3. These findings confirm the existence of distinct differentiation stages within chondrocytes of the growth plate and support the hypothesis proposed by Vortkamp et al. (Science 273(1996)613) of a regulatory feedback loop of Ihh and PTH/PTHrP fragments controlling the differentiation of proliferating to prehypertrophic chondrocytes, but extend the ability to respond to PTH/PTHrP hypertrophic chondrocytes.