Utilization of a highly adaptable murine air pouch model for minimally invasive testing of the inflammatory potential of biomaterials.

Introduction: The biocompatibility of an implanted material strongly determines the subsequent host immune response. After insertion into the body, each medical device causes tissue reactions. How intense and long-lasting these are is defined by the material properties. The so-called foreign body reaction is a reaction leading to the inflammation and wound healing process after implantation. The constantly expanding field of implant technology and the growing areas of application make optimization and adaptation of the materials used inevitable. Methods: In this study, modified liquid silicone rubber (LSR) and two of the most commonly used thermoplastic polyurethanes (TPU) were compared in terms of induced inflammatory response in the body. We evaluated the production of inflammatory cytokines, infiltration of inflammatory cells and encapsulation of foreign bodies in a subcutaneous air-pouch model in mice. In this model, the material is applied in a minimally invasive procedure via a cannula and in one piece, which allows material testing without destroying or crushing the material and thus studying an intact implant surface. The study design includes short-term (6 h) and long-term (10 days) analysis of the host response to the implanted materials. Air-pouch-infiltrating cells were determined by flow cytometry after 6 h and 10 days. Inflammation, fibrosis and angiogenesis markers were analyzed in the capsular tissue by qPCR after 10 days. Results: The foreign body reaction was investigated by macroscopic evaluation and scanning electron microscopy (SEM). Increased leukocyte infiltration was observed in the air-pouch after 6 h, but it markedly diminished after 10 days. After 10 days, capsule formations were observed around the materials without visible inflammatory cells. Discussion: For biocompatibility testing materials are often implanted in muscle tissue. These test methods are not sufficiently conclusive, especially for materials that are intended to come into contact with blood. Our study primarily shows that the presented model is a highly adaptable and minimally invasive test system to test the inflammatory potential of and foreign body reaction to candidate materials and offers more precise analysis options by means of flow cytometry.

Melt blending of poly(lactic acid) with biomedically relevant polyurethanes to improve mechanical performance.

Minimally invasive surgery procedures are of utmost relevance in clinical practice. However, the associated mechanical stress on the material poses a challenge for new implant developments. In particular PLLA, one of the most widely used polymeric biomaterials, is limited in its application due to its high brittleness and low elasticity. In this context, blending is a conventional method of improving the performance of polymer materials. However, in implant applications and development, material selection is usually limited to the use of medical grade polymers. The focus of this work was to investigate the extent to which blending poly-l-lactide (PLLA) with low contents of a selection of five commercially available medical grade polyurethanes leads to enhanced material properties. The materials obtained by melt blending were characterized in terms of their morphology and thermal properties, and the mechanical performance of the blends was evaluated taking into account physiological conditions. From these data, we found that mixing PLLA with Pellethane 80A is a promising approach to improve the material's performance, particularly for stent applications. It was found that PLLA/Pellethane blend with 10% polyurethane exhibits considerable plastic deformation before fracture, while pure PLLA fractures with almost no deformation. Furthermore, the addition of Pellethane only leads to a moderate reduction in elongation at yield and yield stress. In addition, dynamic mechanical analysis for three different PLLA/Pellethane ratios was performed to investigate thermally induced shape retention and shape recovery of the blends.

Fetuin A functionalisation of biodegradable PLLA-co-PEG nonwovens towards enhanced biomineralisation and osteoblastic growth behaviour.

Therapy for large-scale bone defects remains a major challenge in regenerative medicine. In this context, biodegradable electrospun nonwovens are a promising material to be applied as a temporary implantable scaffold as their fibre diameters are in the micro- and nanometre range and possess a high surface-to-volume ratio paired with high porosity. In this work, in vitro assessment of biodegradable PLLA-co-PEG nonwovens with fetuin A covalently anchored to the surface has been performed in terms of biomineralisation and the influence on MG-63 osteoblast cell metabolic activity, biosynthesis of type I collagen propeptide and inflammatory potential. Our finding was that covalent fetuin A funtionalisation of the nonwoven material leads to a distinct increase in calcium affinity, thus enhancing biomineralisation while maintaining the distinct fibre morphology of the nonwoven. The cell seeding experiments showed that the fetuin A functionalised and subsequently in vitro biomineralised PLLA-co-PEG nonwovens did not show negative effects on MG-63 growth. Fetuin A funtionalisation and enhanced biomineralisation supported cell attachment, leading to improved cell morphology, spreading and infiltration into the material. Furthermore, no signs of increase in the inflammatory potential of the material have been detected by flow cytometry experiments. Overall, this study provides a contribution towards the development of artificial scaffolds for guided bone regeneration with the potential to enhance osteoinduction and osteogenesis.

Optimized Gingiva Cell Behavior on Dental Zirconia as a Result of Atmospheric Argon Plasma Activation.

Several physico-chemical modifications have been developed to improve cell contact with prosthetic oral implant surfaces. The activation with non-thermal plasmas was one option. Previous studies found that gingiva fibroblasts on laser-microstructured ceramics were hindered in their migration into cavities. However, after argon (Ar) plasma activation, the cells concentrated in and around the niches. The change in surface properties of zirconia and, subsequently, the effect on cell behavior is unclear. In this study, polished zirconia discs were activated by atmospheric pressure Ar plasma using the kINPen®09 jet for 1 min. Surfaces were characterized by scanning electron microscopy, X-ray photoelectron spectroscopy (XPS), and water contact angle. In vitro studies with human gingival fibroblasts (HGF-1) focused on spreading, actin cytoskeleton organization, and calcium ion signaling within 24 h. After Ar plasma activation, surfaces were more hydrophilic. XPS revealed decreased carbon and increased oxygen, zirconia, and yttrium content after Ar plasma. The Ar plasma activation boosted the spreading (2 h), and HGF-1 cells formed strong actin filaments with pronounced lamellipodia. Interestingly, the cells' calcium ion signaling was also promoted. Therefore, argon plasma activation of zirconia seems to be a valuable tool to bioactivate the surface for optimal surface occupation by cells and active cell signaling.

Micro Injection Molding of Drug-Loaded Round Window Niche Implants for an Animal Model Using 3D-Printed Molds.

A novel approach for the long-term medical treatment of the inner ear is the diffusion of drugs through the round window membrane from a patient-individualized, drug-eluting implant, which is inserted in the middle ear. In this study, drug-loaded (10 wt% Dexamethasone) guinea pig round window niche implants (GP-RNIs, ~1.30 mm × 0.95 mm × 0.60 mm) were manufactured with high precision via micro injection molding (µIM, Tmold = 160 °C, crosslinking time of 120 s). Each implant has a handle (~3.00 mm × 1.00 mm × 0.30 mm) that can be used to hold the implant. A medical-grade silicone elastomer was used as implant material. Molds for µIM were 3D printed from a commercially available resin (TG = 84 °C) via a high-resolution DLP process (xy resolution of 32 µm, z resolution of 10 µm, 3D printing time of about 6 h). Drug release, biocompatibility, and bioefficacy of the GP-RNIs were investigated in vitro. GP-RNIs could be successfully produced. The wear of the molds due to thermal stress was observed. However, the molds are suitable for single use in the µIM process. About 10% of the drug load (8.2 ± 0.6 µg) was released after 6 weeks (medium: isotonic saline). The implants showed high biocompatibility over 28 days (lowest cell viability ~80%). Moreover, we found anti-inflammatory effects over 28 days in a TNF-α-reduction test. These results are promising for the development of long-term drug-releasing implants for human inner ear therapy.

Influence of PEGDA Molecular Weight and Concentration on the In Vitro Release of the Model Protein BSA-FITC from Photo Crosslinked Systems.

Novel 3D printing techniques enable the development of medical devices with drug delivery systems that are tailored to the patient in terms of scaffold shape and the desired pharmaceutically active substance release. Gentle curing methods such as photopolymerization are also relevant for the incorporation of potent and sensitive drugs including proteins. However, retaining the pharmaceutical functions of proteins remains challenging due to the possible crosslinking between the functional groups of proteins, and the used photopolymers such as acrylates. In this work, the in vitro release of the model protein drug, albumin-fluorescein isothiocyanate conjugate (BSA-FITC) from differently composed, photopolymerized poly(ethylene) glycol diacrylate (PEGDA), an often employed, nontoxic, easily curable resin, was investigated. Different PEGDA concentrations in water (20, 30, and 40 wt %) and their different molecular masses (4000, 10,000, and 20,000 g/mol) were used to prepare a protein carrier with photopolymerization and molding. The viscosity measurements of photomonomer solutions revealed exponentially increasing values with increasing PEGDA concentration and molecular mass. Polymerized samples showed increasing medium uptake with an increasing molecular mass and decreasing uptake with increasing PEGDA content. Therefore, the modification of the inner network resulted in the most swollen samples (20 wt %) also releasing the highest amount of incorporated BSA-FITC for all PEGDA molecular masses.

Detection of acoustic emission from nanofiber nonwovens under tensile strain - An ultrasonic test setup for critical medical device components.

In the biomedical field, nanofiber materials are gaining increasing application. For material characterization of nanofiber fabrics, tensile testing and scanning electron microscopy (SEM) are established standards. However, tensile tests provide information about the entire sample without information about single fibers. Conversely, SEM images examine individual fibers, but cover only a small section near the surface of the sample. To gain information on failure at the fiber level under tensile stress, recording of acoustic emission (AE) is a promising method, but challenging due to weak signal intensity. Using AE recording, beneficial findings can be obtained even on "invisible" material failure without affecting tensile tests. In this work, a technology for recording weak ultrasonic AE of tearing nanofiber nonwovens is presented, which uses a highly sensitive sensor. Functional proof of the method using biodegradable PLLA nonwoven fabrics is provided. The potential benefit is demonstrated by unmasking significant AE intensity in an almost imperceptible bend in the stress-strain curve of a nonwoven fabric. AE recording has not yet been performed on standard tensile tests of unembedded nanofiber material intended for safety-related medical applications. The technology has the potential to enrich the spectrum of testing methods, even those not confined to medical field.

The Use of Zwitterionic Methylmethacrylat Coated Silicone Inhibits Bacterial Adhesion and Biofilm Formation of Staphylococcus aureus.

In recent decades, biofilm-associated infections have become a major problem in many medical fields, leading to a high burden on patients and enormous costs for the healthcare system. Microbial infestations are caused by opportunistic pathogens which often enter the incision already during implantation. In the subsequently formed biofilm bacteria are protected from the hosts immune system and antibiotic action. Therefore, the development of modified, anti-microbial implant materials displays an indispensable task. Thermoplastic polyurethane (TPU) represents the state-of-the-art material in implant manufacturing. Due to the constantly growing areas of application and the associated necessary adjustments, the optimization of these materials is essential. In the present study, modified liquid silicone rubber (LSR) surfaces were compared with two of the most commonly used TPUs in terms of bacterial colonization and biofilm formation. The tests were conducted with the clinically relevant bacterial strains Staphylococcus aureus and Staphylococcus epidermidis. Crystal violet staining and scanning electron microscopy showed reduced adhesion of bacteria and thus biofilm formation on these new materials, suggesting that the investigated materials are promising candidates for implant manufacturing.

Influence of Drug Incorporation on the Physico-Chemical Properties of Poly(l-Lactide) Implant Coating Matrices-A Systematic Study.

Local drug delivery has become indispensable in biomedical engineering with stents being ideal carrier platforms. While local drug release is superior to systemic administration in many fields, the incorporation of drugs into polymers may influence the physico-chemical properties of said matrix. This is of particular relevance as minimally invasive implantation is frequently accompanied by mechanical stresses on the implant and coating. Thus, drug incorporation into polymers may result in a susceptibility to potentially life-threatening implant failure. We investigated spray-coated poly-l-lactide (PLLA)/drug blends using thermal measurements (DSC) and tensile tests to determine the influence of selected drugs, namely sirolimus, paclitaxel, dexamethasone, and cyclosporine A, on the physico-chemical properties of the polymer. For all drugs and PLLA/drug ratios, an increase in tensile strength was observed. As for sirolimus and dexamethasone, PLLA/drug mixed phase systems were identified by shifted drug melting peaks at 200 °C and 240 °C, respectively, whereas paclitaxel and dexamethasone led to cold crystallization. Cyclosporine A did not affect matrix thermal properties. Altogether, our data provide a contribution towards an understanding of the complex interaction between PLLA and different drugs. Our results hold implications regarding the necessity of target-oriented thermal treatment to ensure the shelf life and performance of stent coatings.

Evaluation of the suitability of a fluidized bed process for the coating of drug-eluting stents.

Drug-eluting stents are often coated using single-stent coating techniques. In pharmaceutical industry, single-tablet coating is unthinkable. Instead large batches of tablets are coated in fluidized bed apparatuses or pan coaters. Therefore, it was the aim of this work to evaluate whether stents can be coated using a fluidized bed process. For this purpose stents were coated with the model fluorescent drug triamterene embedded in ammonium methacrylate copolymer. Different stent lengths as well as different coating yields were assessed and also a drug-free topcoat was evaluated. The coated stents were analysed regarded coating layer mass, drug content, surface structure, coating thickness and drug release. Furthermore, coating yield and stent defect rate were examined. Except for one stent configuration good results were obtained without optimization of process parameters which indicates the suitability of the method to coat large amounts of stents simultaneously in principle. Drug release was tuneable over a wide range of time spans and a wide range of drug loadings was produced. Further work will be necessary to transform the results of this study from a model stent to a clinically relevant product.

In vitro study of sirolimus release from a drug-eluting stent: Comparison of the release profiles obtained using different test setups.

In this study drug release from the CYPHER™ stent, the gold standard in drug-eluting stent therapy until the end of its marketing in 2011/2012, was systematically evaluated using different in vitro release tests. The test systems included incubations setups, the reciprocating holder apparatus (USP7), the flow-through cell apparatus (USP4) and the vessel-simulating flow-through cell (vFTC) specifically designed for stent testing. The results obtained show a large variability regarding the fractions released into the media after 7d ranging from 38.6% ± 4.5% to 74.6% ± 1.2%. The lowest fraction released was observed in the vFTC and the highest in an incubation setup with frequently changed media of a volume of 2 mL. Differences were even observed when using fairly similar and simple incubations setups with mere changes of the media volume, under maintenance of sink conditions, and of the vessel geometry. From these data it can be concluded, that in vitro release even from a slow releasing drug-eluting stent is greatly influenced by the experimental conditions and care must be taken when choosing a suitable setup. Comparison of the obtained in vitro release profiles to published in vivo data did not result in a distinct superiority of any of the tested methods regarding the predictability for the situation in vivo due to large differences in the reported in vivo data. However, this comparison yielded that the release observed in vitro using the 2 mL incubation setup and the reciprocating holder apparatus may be faster than the reported in vivo release. The results of this study also emphasize the necessity to use highly standardized release tests when comparisons between results from different experiments or even different labs are to be performed. In this context, the compendial methods are most likely offering the highest degree of standardization.

Implant-associated local drug delivery systems based on biodegradable polymers: customized designs for different medical applications.

Implants providing controlled, local release of active substances are of interest in different medical applications. Therefore, the focus of the present article is the development of implant-associated diffusion- or chemically controlled local drug delivery (LDD) systems based on biodegradable polymeric drug carriers. In this context, we provide new data and review our own recently published data concerning the drug release behavior of diffusion-controlled LDD systems in relation to the kind of polymer, drug content, coating mass/thickness, and layer composition. We demonstrate that polymers allow a wide range of control over the drug release characteristics. In this regard, we show that the glass transition temperature of a polymer has an impact on its drug release. Additionally, the blending of hydrophobic, semicrystalline polymers with amorphous polymers leads to an increase in the rate of drug release compared with the pure semicrystalline polymer. Moreover, the percentage loading of the embedded drug has a considerable effect on the rate and duration of drug release. Furthermore, we discuss chemically controlled LDD systems designed for the release of biomolecules, such as growth factors, as well as nanoparticle-mediated LDD systems. With our own published data on drug-eluting stents, microstents, and cochlear implants, we highlight exemplary implant-associated LDD systems designed to improve implant performance through the reduction of undesirable effects such as in-stent restenosis and fibrosis.

In vitro study of dual drug-eluting stents with locally focused sirolimus and atorvastatin release.

Within the context of novel stent designs we developed a dual drug-eluting stent (DDES) with an abluminally focussed release of the potent anti-proliferative drug sirolimus and a luminally focussed release of atorvastatin with stabilizing effect on atherosclerotic deposits and stimulating impact on endothelial function, both from biodegradable poly(L-lactide)-based stent coatings. With this concept we aim at simultaneous inhibition of in-stent restenosis as a result of disproportionally increased smooth muscle cell proliferation and migration as well as thrombosis due to failed or incomplete endothelialisation. The especially adapted spray-coating processes allowed the formation of smooth form-fit polymer coatings at the abluminal and luminal side with 70% respectively 90% of the drug/polymer solution being deposited at the intended stent surface. The impacts of tempering, sterilization, and layer composition on drug release are thoroughly discussed making use of a semi-empirical model. While tempering at 80 °C seems to be necessary for the achievement of adequate and sustained drug release, the coating sequence for DDES should be rather abluminal-luminal than luminal-abluminal, as reduction of the amount of sirolimus eluted luminally could then potentially minimize the provocation of endothelial dysfunction. In vitro proliferation and viability assays with smooth muscle and endothelial cells underline the high potential of the developed DDES.

Examination of drug release and distribution from drug-eluting stents with a vessel-simulating flow-through cell.

The recently introduced vessel-simulating flow-through cell offers new possibilities to examine the release from drug-eluting stents in vitro. In comparison with standard dissolution methods, the additional compartment allows for the examination of distribution processes and creates dissolution conditions which simulate the physiological situation at the site of implantation. It was shown previously that these conditions have a distinct influence on the release rate from the stent coating. In this work, different preparation techniques were developed to examine the spatial distribution within the compartment simulating the vessel wall. These methods allowed for the examination of diffusion depth and the distribution resulting in the innermost layer of the compartment simulating the vessel wall. Furthermore, the in vitro release and distribution examined experimentally were modelled mathematically using finite element (FE) methods to gain further insight into the release and distribution behaviour. The FE modelling employing the experimentally determined diffusion coefficients yielded a good general description of the experimental data. The results of the modelling also provided important indications that inhomogeneous coating layer thicknesses around the strut may result from the coating process which influence release and distribution behaviour. Taken together, the vessel-simulating flow-through cell in combination with FE modelling represents a unique method to analyse drug release and distribution from drug-eluting stents in vitro with particular opportunities regarding the examination of spatial distributions within the vessel-simulating compartment.