Tailored Polyelectrolyte Multilayer Systems by Variation of Polyelectrolyte Composition and EDC/NHS Cross-Linking: Controlled Drug Release vs. Drug Reservoir Capabilities and Cellular Response for Improved Osseointegration.

Polyelectrolyte multilayers (PEM) are versatile tools used to investigate fundamental interactions between material-related parameters and the resulting performance in stem cell differentiation, respectively, in bone tissue engineering. In the present study, we investigate the suitability of PEMs with a varying collagen content for use as drug carriers for the human bone morphogenetic protein 2 (rhBMP-2). We use three different PEM systems consisting either of the positively charged poly-L-lysine or the glycoprotein collagen type I and the negatively charged glycosaminoglycan heparin. For a specific modification of the loading capacity and the release kinetics, the PEMs were stepwise cross-linked before loading with cytokine. We demonstrate the possibility of immobilizing significant amounts of rhBMP-2 in all multilayer systems and to specifically tune its release via cross-linking. Furthermore, we prove that the drug release of rhBMP-2 plays only a minor role in the differentiation of osteoprogenitor cells. We find a significantly higher influence of the immobilized rhBMP-2 within the collagen-rich coatings that obviously represent an excellent mimicry of the native extracellular matrix. The cytokine immobilized in its bioactive form was able to achieve an increase in orders of magnitude both in the early stages of differentiation and in late calcification compared to the unloaded layers.

Tailored Polyelectrolyte Multilayer Systems by Variation of Polyelectrolyte Composition and EDC/NHS Cross-Linking: Physicochemical Characterization and In Vitro Evaluation.

The layer-by-layer (LbL) self-assembly technique is an effective method to immobilize components of the extracellular matrix (ECM) such as collagen and heparin onto, e.g., implant surfaces/medical devices with the aim of forming polyelectrolyte multilayers (PEMs). Increasing evidence even suggests that cross-linking influences the physicochemical character of PEM films since mechanical cues inherent to the substrate may be as important as its chemical nature to influence the cellular behavior. In this study, for the first-time different collagen/heparin films have been prepared and cross-linked with EDC/NHS chemistry. Quartz crystal microbalance, zeta potential analyzer, diffuse reflectance Fourier transform infrared spectroscopy, atomic force microscopy and ellipsometry were used to characterize film growth, stiffness, and topography of different film systems. The analysis of all data proves a nearly linear film growth for all PEM systems, the efficacy of cross-linking and the corresponding changes in the film rigidity after cross-linking and an appropriate surface topography. Furthermore, preliminary cell culture experiments illustrated those cellular processes correlate roughly with the quantity of newly created covalent amide bonds. This allows a precise adjustment of the physicochemical properties of the selected film architecture regarding the desired application and target cells. It could be shown that collagen improves the biocompatibility of heparin containing PEMs and due to their ECM-analogue nature both molecules are ideal candidates intended to be used for any biomedical application with a certain preference to improve the performance of bone implants or bone augmentation strategies.

Poly-Alanine-ε-Caprolacton-Methacrylate as Scaffold Material with Tuneable Biomechanical Properties for Osteochondral Implants.

An aging population and injury-related damage of the bone substance lead to an increasing need of innovative materials for the regeneration of osteochondral defects. Biodegradable polymers form the basis for suitable artificial implants intended for bone replacement or bone augmentation. The great advantage of these structures is the site-specific implant design, which leads to a considerable improvement in patient outcomes and significantly reduced post-operative regeneration times. Thus, biomechanical and biochemical parameters as well as the rate of degradation can be set by the selection of the polymer system and the processing technology. Within this study, we developed a polymer platform based on the amino acid Alanine and ε-Caprolacton for use as raw material for osteochondral implants. The biomechanical and degradation properties of these Poly-(Alanine-co-ε-Caprolacton)-Methacrylate (ACM) copolymers can be adjusted by changing the ratio of the monomers. Fabrication of artificial structures for musculo-skeletal tissue engineering was done by Two-Photon-Polymerization (2PP), which represents an innovative technique for generating defined scaffolds with tailor-made mechanical and structural properties. Here we show the synthesis, physicochemical characterization, as well as first results for structuring ACM using 2PP technology. The data demonstrate the high potential of ACM copolymers as precursors for the fabrication of biomimetic implants for bone-cartilage reconstruction.

Bacterial nanocellulose: Reinforcement of compressive strength using an adapted Mobile Matrix Reservoir Technology and suitable post-modification strategies.

Bacterial nanocellulose (BNC) is a highly interesting biomaterial due to some outstanding properties especially when used in medical therapeutics and diagnostics. BNC is absolutely bioinert and is characterised by intrinsic properties such as high tensile stiffness and elasticity, high porosity, exceptional water uptake and swelling capacity. Furthermore, these properties can be adjusted in a very defined way by specifically changing the cultivation conditions or performing post-modifications such as crosslinking, functionalisation with additives, dehydration or drying. Especially the high tensile strength of the nanofibrillar material has been the subject of many investigations in the past couple of years. Nevertheless, the enormous tensile strength and elasticity of BNC is contrary to an almost purely viscous behaviour under compressive load. In the present study, different methods to influence the mechanical behaviour under compression with respect to load bearing applications of BNC are systematically investigated. The possibilities and limitations of the variable layer-by-layer cultivation known as Mobile Matrix Reservoir Technology (MMR-Tech) as well as the effect of different post-modification strategies of BNC are thoroughly investigated. Beside of commonly used indentation tests for characterising the mechanical properties of BNC, we introduce a novel evaluation methodology based on mechanical relaxation measurements and an evolutionary regression algorithm for the derivation of a viscoelastic material law, which for the first time allows standardised, comparative viscoelastic investigations of soft-matter biomaterials to be performed independently of the measurement setup. Using this methodology, we are able to show, that cultivation conditions for BNC and suitable post-modifications can result in different effects on the viscoelastic behaviour of the fabricated composites. We show that the cultivation conditions for BNC primarily affect the height of dispersion and the frequency of the relaxation centre which corresponds roughly to the mean value of the logarithmic distributed relaxation times, and that these effects could be enhanced by post-modifications. However, we also identify parameters, such as the width of the relaxation region, which corresponds roughly to the standard deviation of the logarithmic distributed relaxation times, on which the type of cultivation obviously shows no influence but which can be influenced exclusively by post-modifications. Our methodology enables for the first time a clear identification of those parameters which represent a significant factor of influence to the viscoelastic material behaviour, which should enable a more targeted and application-relevant development of BNC composites in the future.

Label-Free Detection and Characterization of Heparin-Induced Thrombocytopenia (HIT)-like Antibodies.

Heparin-induced thrombocytopenia (HIT) antibodies (Abs) can mediate and activate blood cells, forming blood clots. To detect HIT Abs, immunological assays with high sensitivity (≥95%) and fast response are widely used, but only about 50% of these tests are accurate as non-HIT Abs also bind to the same antigens. We aim to develop biosensor-based electrical detection to better differentiate HIT-like from non-HIT-like Abs. As a proof of principle, we tested with two types of commercially available monoclonal Abs including KKO (inducing HIT) and RTO (noninducing HIT). Platelet factor 4/Heparin antigens were immobilized on gold electrodes, and binding of antibodies on the chips was detected based on the change in the charge transfer resistance (R ct). Binding of KKO on sensors yielded a significantly lower charge transfer resistance than that of RTO. Bound antibodies and their binding characteristics on the sensors were confirmed and characterized by complementary techniques. Analysis of thermal kinetics showed that RTO bonds are more stable than those of KKO, whereas KKO exhibited a higher negative ζ potential than RTO. These different characteristics made it possible to electrically differentiate these two types of antibodies. Our study opens a new avenue for the development of sensors for better detection of pathogenic Abs in HIT patients.

Controlling Surface-Induced Platelet Activation by Agarose and Gelatin-Based Hydrogel Films.

Platelet-surface interaction is of paramount importance in biomedical applications as well as in vitro studies. However, controlling platelet-surface activation is challenging and still requires more effort as they activate immediately when contacting with any nonphysiological surface. As hydrogels are highly biocompatible, in this study, we developed agarose and gelatin-based hydrogel films to inhibit platelet-surface adhesion. We found promising agarose films that exhibit higher surface wettability, better controlled-swelling properties, and greater stiffness compared to gelatin, resulting in a strong reduction of platelet adhesion. Mechanical properties and surface wettability of the hydrogel films were varied by adding magnetite (Fe3O4) nanoparticles. While all of the films prevented platelet spreading, films formed by agarose and its nanocomposite repelled platelets and inhibited platelet adhesion and activation stronger than those of gelatin. Our results showed that platelet-surface activation is modulated by controlling the properties of the films underneath platelets and that the bioinert agarose can be potentially translated to the development of platelet storage and other medical applications.

Two-Photon Polymerized Poly(2-Ethyl-2-Oxazoline) Hydrogel 3D Microstructures with Tunable Mechanical Properties for Tissue Engineering.

The main task of tissue engineering (TE) is to reproduce, replicate, and mimic all kinds of tissues in the human body. Nowadays, it has been proven useful in TE to mimic the natural extracellular matrix (ECM) by an artificial ECM (scaffold) based on synthetic or natural biomaterials to regenerate the physiological tissue/organ architecture and function. Hydrogels have gained interest in the TE community because of their ability to absorb water similar to physiological tissues, thus mechanically simulating the ECM. In this work, we present a novel hydrogel platform based on poly(2-ethyl-2-oxazoline)s, which can be processed to 3D microstructures via two-photon polymerization (2PP) with tunable mechanical properties using monomers and crosslinker with different degrees of polymerization (DP) for future applications in TE. The ideal parameters (laser power and writing speed) for optimal polymerization via 2PP were obtained using a specially developed evaluation method in which the obtained structures were binarized and compared to the computer-aided design (CAD) model. This evaluation was performed for each composition. We found that it was possible to tune the mechanical properties not only by application of different laser parameters but also by mixing poly(2-ethyl-2-oxazoline)s with different chain lengths and variation of the crosslink density. In addition, the swelling behavior of different fabricated hydrogels were investigated. To gain more insight into the viscoelastic behavior of different fabricated materials, stress relaxation tests via nanoindentation experiments were performed. These new hydrogels can be processed to 3D microstructures with high structural integrity using optimal laser parameter settings, opening a wide range of application properties in TE for this material platform.

Modulation of Platelet-Surface Activation: Current State and Future Perspectives.

Modulation of platelet-surface activation is important for many biomedical applications such as in vivo performance, platelet storage, and acceptance of an implant. Reducing platelet-surface activation is challenging because they become activated immediately after short contact with nonphysiological surfaces. To date, controversies and open questions in the field of platelet-surface activation still remain. Here, we review state-of-the-art approaches in inhibiting platelet-surface activation, mainly focusing on modification, patterning, and methodologies for characterization of the surfaces. As a future perspective, we discuss how the combination of biochemical and physiochemical strategies together with the topographical modulations would assist in the search for an ideal nonthrombogenic surface.

Biomimetic Designer Scaffolds Made of D,L-Lactide-ɛ-Caprolactone Polymers by 2-Photon Polymerization.

IMPACT STATEMENT: In tissue engineering (TE), the establishment of cell targeting materials, which mimic the conditions of the physiological extracellular matrix (ECM), seems to be a mission impossible without advanced materials and fabrication techniques. With this in mind we established a toolbox based on (D,L)-lactide-ɛ-caprolactone methacrylate (LCM) copolymers in combination with a nano-micromaskless lithography technique, the two-photon polymerization (2-PP) to mimic the hierarchical structured and complex milieu of the natural ECM. To demonstrate the versatility of this toolbox, we choose two completely different application scenarios in bone and tumor TE to show the high potential of this concept in therapeutic and diagnostic application.

Influence of the Microstructure and Silver Content on Degradation, Cytocompatibility, and Antibacterial Properties of Magnesium-Silver Alloys In Vitro.

Implantation is a frequent procedure in orthopedic surgery, particularly in the aging population. However, it possesses the risk of infection and biofilm formation at the surgical site. This can cause unnecessary suffering to patients and burden on the healthcare system. Pure Mg, as a promising metal for biodegradable orthopedic implants, exhibits some antibacterial effects due to the alkaline pH produced during degradation. However, this antibacterial effect may not be sufficient in a dynamic environment, for example, the human body. The aim of this study was to increase the antibacterial properties under harsh and dynamic conditions by alloying silver metal with pure Mg as much as possible. Meanwhile, the Mg-Ag alloys should not show obvious cytotoxicity to human primary osteoblasts. Therefore, we studied the influence of the microstructure and the silver content on the degradation behavior, cytocompatibility, and antibacterial properties of Mg-Ag alloys in vitro. The results indicated that a higher silver content can increase the degradation rate of Mg-Ag alloys. However, the degradation rate could be reduced by eliminating the precipitates in the Mg-Ag alloys via T4 treatment. By controlling the microstructure and increasing the silver content, Mg-Ag alloys obtained good antibacterial properties in harsh and dynamic conditions but had almost equivalent cytocompatibility to human primary osteoblasts as pure Mg.

Biomimetic surface modification with bolaamphiphilic archaeal tetraether lipids via liposome spreading.

Through investigations of the self-assembly behavior of three different tetraether lipids, the authors successfully established a solid supported, biomimetic tetraether lipid membrane via liposome spreading. These bolaamphiphilic lipids are the main compound in membranes of archaea, extremophile microorganisms, which underwent an enormous adaptation to extreme conditions in their natural environment with regard to temperature, pH, and high salt concentrations. Starting from a mathematical point of view, the authors calculated hydrophilic-lipophilic balance values for each lipid and recognized a wide difference in self-assembly potentials relying on size and hydrophilic properties of the lipid head groups. These results were in good accordance with data generated by lipid experiments at the air-water interface applying a Langmuir-Blodgett film balance so that the self-assembly potential of two different tetraether lipids was found to be sufficient to form stable liposomes in aqueous media. Liposomes composed of the main phospholipid of the archaea strain Sulfolobus acidocaldarius fused covalently on silanized glass substrates and formed a monomolecular lipid layer with upright standing molecules at film consistent thicknesses of approximately 5 nm determined by ellipsometry and atomic force microscopy. This work can be considered as a basic strategy to find optimized lipid properties in terms of liposome formation and spreading in water, and it is the first report about archaeal liposome fusing on surfaces to establish a solid supported lipid monolayer.

Investigations on the secondary structure of polypeptide chains in polyelectrolyte multilayers and their effect on the adhesion and spreading of osteoblasts.

Inspired by the composition of the native extracellular matrix, biomimetic polyelectrolyte multilayers were assembled from polypeptides and the glycosaminoglycan chondroitin sulfate (CS). To investigate whether peptide conformation imposes an effect on the cell biological functions of osteoblasts, the secondary structure was analyzed by in situ infra-red and circular dichroism spectroscopy. Multilayers composed of polypeptides and CS reveal a predominantly random coiled conformation and impede osteoblast spreading. On the contrary, polypeptide chains in assemblies of poly-L-lysine and poly-L-glutamic acid (PGA) primarily adopt an intermolecular ß sheet structure and reveal an increased area of spread, which consequently supports the proliferation of osteoblasts. When CS is replaced by PGA in mixed multilayers, we observe a structural rearrangement from random coils to ß sheets with a concomitant improved cell response. We conclude that polypeptide conformation in biomimetic multilayer assemblies affects osteoblast response by altering the stiffness of the multilayer.

Biomimetic assembly of polyelectrolyte multilayers containing phosvitin monitored with reflectometric interference spectroscopy.

Coatings of biomaterials or implants that facilitate biomineralization possess a great potential for applications focused to the replacement, augmentation, and regeneration of bone tissue. Biomimetic approaches utilize biomolecules for either templating or supporting the crystallization process. One of these promising biomolecules is phosvitin (PV), an egg yolk protein known to transport and store inorganic phosphates and calcium ions. The incorporation of PV into polyelectrolyte multilayers is favorable due to PVs high degree of phosphorylation and thus a high acidity. Utilizing the reflectometric interference spectroscopy, the adsorption kinetics of this novel polyelectrolyte system composed of poly-L-lysine and the heavily phosphorylated phosvitin were monitored. The results demonstrate an unexpected nonregular growth regime called overshoot. Effective measures of shifting this irregular polyelectrolyte adsorption process back to a regular multilayer growth regime are reported in this paper.

Colloidal force spectroscopy and cell biological investigations on biomimetic polyelectrolyte multilayer coatings composed of chondroitin sulfate and heparin.

To promote osteoblast adhesion and proliferation on (bio)material surfaces, biomimetic coatings resembling the natural extracellular matrix (ECM) are desirable. The glycosamino glycans (GAGs) chondroitin sulfate (CS) and heparin (HEP) are promising candidates for a biomimetic coating since they are two of the most prevalent noncollagenous biomolecules constituting the ECM. Coatings containing CS and HEP were prepared employing the "layer by layer" technique yielding polyelectrolyte multilayers (PEMs). Physicochemical and mechanical characterization of the coatings were performed by means of streaming potential measurements and colloidal force spectroscopy. The capability of the coatings to support cell adhesion, spreading, proliferation, and maintenance of an osteoblastic phenotype was assessed with SaOS osteosarcoma cells. We demonstrate that PEMs constructed from CS as the polyanion display a low Young's modulus correlated with poorly supported cell adhesion and proliferation. When the CS was adsorbed onto a stiffer polypeptide PEM basis, the Young's modulus increased, and the cell response was significantly improved. For HEP coatings an intermediate Young's modulus and moderate cell adhesion and spreading were observed. No significant changes in stiffness or cell response were detected when HEP was adsorbed onto the polypeptide film.