Application of electrospun chitosan-based nanofibers as immobilization matrix for biomolecules.

Nanofiber meshes from electrospun chitosan, highly modified with biotin and arylazides, are well-suited for application as enzyme immobilization matrices. To test this, catalytically active biomolecules were immobilized onto photocrosslinked nanofibrous nonwovens consisting mainly of biotinylated fungal chitosan and a small amount (10 w%) of poly ethylene oxide. In this study, we show that over 10 µg eugenol oxidase per milligram dry polymer matrix can be loaded on UV-crosslinked chitosan nanofibers. We further demonstrate that bound enzyme activity can be fully retained for over 7 days of storage at ambient conditions in aqueous buffer. Samples loaded at maximum enzyme carrying capacity were tested in a custom-made plug-flow reactor system with online UV-VIS spectroscopy for activity determination. High wettability and durability of the hydrophilic chitosan support matrix enabled continuous oxidation of model substrate vanillyl alcohol into vanillin with constant turnover at flow rates of up to 0.24 L/h for over 6 h. This proves the above hypothesis and enables further application of the fibers as stacked microfluidic membranes, biosensors, or structural starting points for affinity crosslinked enzyme gels. KEY POINTS: • Biotinylated chitosan-based nanofibers retain enzymes via mild affinity interactions • Immobilized eugenol oxidase shows high activity and resists continuous washing • Nanofiber matrix material tolerated high flow rates in a continuous-flow setup.

Layer-by-Layer Deposition of 2D CdSe/CdS Nanoplatelets and Polymers for Photoluminescent Composite Materials.

Two-dimensional (2D) semiconductor nanoplatelets (NPLs) are strongly photoluminescent materials with interesting properties for optoelectronics. Especially their narrow photoluminescence paired with a high quantum yield is promising for light emission applications with high color purity. However, retaining these features in solid-state thin films together with an efficient encapsulation of the NPLs is a challenge, especially when trying to achieve high-quality films with a defined optical density and low surface roughness. Here, we show photoluminescent polymer-encapsulated inorganic-organic nanocomposite coatings of 2D CdSe/CdS NPLs in poly(diallyldimethylammonium chloride) (PDDA) and poly(ethylenimine) (PEI), which are prepared by sequential layer-by-layer (LbL) deposition. The electrostatic interaction between the positively charged polyelectrolytes and aqueous phase-transferred NPLs with negatively charged surface ligands is used as a driving force to achieve self-assembled nanocomposite coatings with a well-controlled layer thickness and surface roughness. Increasing the repulsive forces between the NPLs by increasing the pH value of the dispersion leads to the formation of nanocomposites with all NPLs arranging flat on the substrate, while the surface roughness of the 165 nm (50 bilayers) thick coating decreases to Ra = 14 nm. The photoluminescence properties of the nanocomposites are determined by the atomic layer thickness of the NPLs and the 11-mercaptoundecanoic acid ligand used for their phase transfer. Both the full width at half-maximum (20.5 nm) and the position (548 nm) of the nanocomposite photoluminescence are retained in comparison to the colloidal CdSe/CdS NPLs in aqueous dispersion, while the measured photoluminescence quantum yield of 5% is competitive to state-of-the-art nanomaterial coatings. Our approach yields stable polymer-encapsulated CdSe/CdS NPLs in smooth coatings with controllable film thickness, rendering the LbL deposition technique a powerful tool for the fabrication of solid-state photoluminescent nanocomposites.

TGF-ß3 Loaded Electrospun Polycaprolacton Fibre Scaffolds for Rotator Cuff Tear Repair: An in Vivo Study in Rats.

Biological factors such as TGF-ß3 are possible supporters of the healing process in chronic rotator cuff tears. In the present study, electrospun chitosan coated polycaprolacton (CS-g-PCL) fibre scaffolds were loaded with TGF-ß3 and their effect on tendon healing was compared biomechanically and histologically to unloaded fibre scaffolds in a chronic tendon defect rat model. The biomechanical analysis revealed that tendon-bone constructs with unloaded scaffolds had significantly lower values for maximum force compared to native tendons. Tendon-bone constructs with TGF-ß3-loaded fibre scaffolds showed only slightly lower values. In histological evaluation minor differences could be observed. Both groups showed advanced fibre scaffold degradation driven partly by foreign body giant cell accumulation and high cellular numbers in the reconstructed area. Normal levels of neutrophils indicate that present mast cells mediated rather phagocytosis than inflammation. Fibrosis as sign of foreign body encapsulation and scar formation was only minorly present. In conclusion, TGF-ß3-loading of electrospun PCL fibre scaffolds resulted in more robust constructs without causing significant advantages on a cellular level. A deeper investigation with special focus on macrophages and foreign body giant cells interactions is one of the major foci in further investigations.

Impact of sterilization by electron beam, gamma radiation and X-rays on electrospun poly-(ε-caprolactone) fiber mats.

Biodegradable polymers such as polycaprolactone (PCL) are increasingly used for electrospinning substrates for tissue engineering. These materials offer great advantages such as biocompatibility and good mechanical properties. However, in order to be approved for human implantation they have to be sterilized. The impact of commonly used irradiation sterilization methods on electrospun PCL fiber mats was investigated systematically. Electron beam (ß-irradiation), gamma and X-ray irradiation with two different doses (25 and 33 kGy) were investigated. To determine the impact on the fiber mats, mechanical, chemical, thermal properties and crystallinity were investigated. Irradiation resulted in a significant decrease in molecular weight. At the same time, crystallinity of fiber mats increased significantly. However, the mechanical properties did not change significantly upon irradiation, mostly likely because effects of a lower molecular weight were balanced with the higher degree of crystallinity. The irradiation effects were dose dependent, a higher irradiation dose led to stronger changes. Gamma irradiation seemed to be the least suited method, while electron beams (ß irradiation) had a lower impact. Therefore, ß irradiation is recommended as sterilization method for electrospun PCL fiber mats.

Introducing a semi-coated model to investigate antibacterial effects of biocompatible polymers on titanium surfaces.

Peri-implant infections from bacterial biofilms on artificial surfaces are a common threat to all medical implants. They are a handicap for the patient and can lead to implant failure or even life-threatening complications. New implant surfaces have to be developed to reduce biofilm formation and to improve the long-term prognosis of medical implants. The aim of this study was (1) to develop a new method to test the antibacterial efficacy of implant surfaces by direct surface contact and (2) to elucidate whether an innovative antimicrobial copolymer coating of 4-vinyl-N-hexylpyridinium bromide and dimethyl(2-methacryloyloxyethyl) phosphonate (VP:DMMEP 30:70) on titanium is able to reduce the attachment of bacteria prevalent in peri-implant infections. With a new in vitro model with semi-coated titanium discs, we were able to show a dramatic reduction in the adhesion of various pathogenic bacteria (Streptococcus sanguinis, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis), completely independently of effects caused by soluble materials. In contrast, soft tissue cells (human gingival or dermis fibroblasts) were less affected by the same coating, despite a moderate reduction in initial adhesion of gingival fibroblasts. These data confirm the hypothesis that VP:DMMEP 30:70 is a promising antibacterial copolymer that may be of use in several clinical applications.

Hydrophobic spacers enhance the helicity and lectin binding of synthetic, pH-responsive glycopolypeptides.

The influence of different hydrophobic spacers on the structural and lectin binding properties of well-defined glycopolypeptides decorated with galactose moieties was investigated. All glycopolypeptides were prepared from a poly(α,l-glutamic acid) (PGA) precursor via a polymer-analogous aqueous amide coupling reaction. Thereby, two alkyl spacers of different length (C6 and C11) as well as an aromatic spacer were introduced between the backbone and the galactose moieties, as confirmed by (1)H NMR spectroscopy. The secondary structure was investigated as a function of the sugar density and the pH by circular dichroism (CD) spectroscopy. It was found that the helicity in acidic medium and thus the typical coil-to-helix transition is strongly enhanced by the hydrophobic spacers. Preliminary lectin binding tests via turbidimetric assay revealed that the spacers also significantly enhance the interaction of the glycopolypeptides with the lectin RCA120.

Topographically and Chemically Enhanced Textile Polycaprolactone Scaffolds for Tendon and Ligament Tissue Engineering.

The use of tissue engineering to address the shortcomings of current procedures for tendons and ligaments is promising, but it requires a suitable scaffold that meets various mechanical, degradation-related, scalability-related, and biological requirements. Macroporous textile scaffolds made from appropriate fiber material have the potential to fulfill the first three requirements. This study aimed to investigate the biocompatibility, sterilizability, and functionalizability of a multilayer braided scaffold. These macroporous scaffolds with dimensions similar to those of the human anterior cruciate ligament consist of fibers with appropriate tensile strength and degradation behavior melt-spun from Polycaprolactone (PCL). Two different cross-sectional geometries resulting in significantly different specific surface areas and morphologies were used at the fiber level, and a Chitosan-graft-PCL (CS-g-PCL) surface modification was applied to the melt-spun substrates for the first time. All scaffolds elicited a positive cell response, and the CS-g-PCL modification provided a platform for incorporating functionalization agents such as drug delivery systems for growth factors, which were successfully released in therapeutically effective quantities. The fiber geometry was found to be a variable that could be manipulated to control the amount released. Therefore, scaled, surface-modified textile scaffolds are a versatile technology that can successfully address the complex requirements of tissue engineering for ligaments and tendons, as well as other structures.

Evaluation of the Immune Response to Chitosan-graft-poly(caprolactone) Biopolymer Scaffolds.

Biomimetic scaffolds recreating key elements of the architecture and biological activity of the extracellular matrix have enormous potential for soft tissue engineering applications. Combining appropriate mechanical properties with select biological cues presents a challenge for bioengineering, as natural materials are most bioactive but can lack mechanical integrity, while synthetic polymers have strength but are often biologically inert. Blends of synthetic and natural materials, aiming to combine the benefits of each, have shown promise but inherently require a compromise, diluting down favorable properties in each polymer to accommodate the other. Here, we electrospun a material comprising chitosan, a natural polysaccharide, and polycaprolactone (PCL), one of the most widely studied synthetic polymers used in materials engineering. In contrast to a classical blend, here PCL was chemically grafted onto the chitosan backbone to create chitosan-graft-polycaprolactone (CS-g-PCL) and then combined further with unmodified PCL to generate scaffolds with discreet chitosan functionalization. These small amounts of chitosan led to significant changes in scaffold architecture and surface chemistry, reducing the fiber diameter, pore size, and hydrophobicity. Interestingly, all CS-g-PCL-containing blends were stronger than control PCL, though with reduced elongation. In in vitro assessments, increasing the CS-g-PCL content led to significant improvements in in vitro blood compatibility compared to PCL alone while increasing fibroblast attachment and proliferation. In a mouse subcutaneous implantation model, a higher CS-g-PCL content improved the immune response to the implants. Macrophages in tissues surrounding CS-g-PCL scaffolds decreased proportionately to the chitosan content by up to 65%, with a corresponding decrease in pro-inflammatory cytokines. These results suggest that CS-g-PCL is a promising hybrid material comprising natural and synthetic polymers with tailorable mechanical and biological properties, justifying further development and in vivo evaluation.

Thermoresponsive Glycopolypeptide Containing Block Copolymers, Particle Formation, and Lectin Interaction.

Amphiphilic block copolymers with a thermoresponsive poly(N-isopropylacrylamide) block and a glycopeptide block are synthesized and particle formation as well as interaction of the glyco-corona with lectins is investigated. The synthetic route comprises the preparation of block copolymers by N-carboxyanhydride polymerization and subsequent deprotection to obtain pH- and thermoresponsive poly(l-glutamic acid)-b-poly(N-isopropylacrylamide) (pGA-b-pNIPAM), which is then further modified with different amino sugars by a versatile coupling method with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium chloride (DMT-MM). The glycosylated pGA-b-pNIPAM block copolymers are investigated with regard to cloud point temperatures (Tcp ), particle size, and stability. The morphology of the particles is visualized using cryo-SEM. Zeta potential measurements are indicating that the saccharide moieties are located on the surface of the particles. This assumption is further substantiated by quantitative lectin interaction assays with nonaggregated and aggregated glycosylated pGA-b-pNIPAM. The interaction of the model lectin ConA with the block copolymers is independent of the degree of substitution in the nonaggregated state at room temperature. However, at 37 °C, when particles of pGA-b-pNIPAM are present, the interaction becomes stronger with increasing degree of substitution. This interaction with lectins can be used for targeting saccharide-modified particles in drug delivery.

Spatially and Temporally Controllable BMP-2 and TGF-ß3 Double Release From Polycaprolactone Fiber Scaffolds via Chitosan-Based Polyelectrolyte Coatings.

Temporally and spatially controlled growth factor release from a polycaprolactone fiber mat, which also provides a matrix for directional cell colonization and infiltration, could be a promising regenerative approach for degenerated tendon-bone junctions. For this purpose, polycaprolactone fiber mats were coated with tailored chitosan-based nanogels to bind and release the growth factors bone morphogenetic protein 2 (BMP-2) and transforming growth factor-ß3 (TGF-ß3), respectively. In this work we provide meaningful in vitro data for the understanding of the drug delivery performance and sterilizability of novel implant prototypes in order to lay the foundation for in vivo testing. ELISA-based in vitro release studies were used to investigate the spatial and temporal control of release, as well as the influence of radiation sterilization on protein activity and release behavior. Layer-by-layer coatings based on BMP-2-containing chitosan tripolyphosphate nanogel particles and negatively charged alginate showed a good sustainment of BMP-2 release from chemically modified polycaprolactone fiber mats. Release control improved with increasing layer numbers. The approach of controlling the release via a barrier of cross-linked chitosan azide proved less promising. By using a simple, partial immersion-based dip-coating process, it was possible to apply opposing gradients of the growth factors BMP-2 and TGF-ß3. Final radiation sterilization of the growth factor-loaded implant prototypes resulted in a radiation dose-correlated degradation of the growth factors, which could be prevented by lyophilization into protective matrices. For the manufacture of sterile implants, the growth factor loading step must probably be carried out under aseptic conditions. The layer-by-layer coated implant prototypes provided sustained release from opposing gradients of the growth factors BMP-2 and TGF-ß3 and thus represent a promising approach for the restoration of tendon-bone defects.

Development of Neuronal Guidance Fibers for Stimulating Electrodes: Basic Construction and Delivery of a Growth Factor.

State-of-the-art treatment for sensorineural hearing loss is based on electrical stimulation of residual spiral ganglion neurons (SGNs) with cochlear implants (CIs). Due to the anatomical gap between the electrode contacts of the CI and the residual afferent fibers of the SGNs, spatial spreading of the stimulation signal hampers focused neuronal stimulation. Also, the efficiency of a CI is limited because SGNs degenerate over time due to loss of trophic support. A promising option to close the anatomical gap is to install fibers as artificial nerve guidance structures on the surface of the implant and install on these fibers drug delivery systems releasing neuroprotective agents. Here, we describe the first steps in this direction. In the present study, suture yarns made of biodegradable polymers (polyglycolide/poly-ε-caprolactone) serve as the basic fiber material. In addition to the unmodified fiber, also fibers modified with amine groups were employed. Cell culture investigations with NIH 3T3 fibroblasts attested good cytocompatibility to both types of fibers. The fibers were then coated with the extracellular matrix component heparan sulfate (HS) as a biomimetic of the extracellular matrix. HS is known to bind, stabilize, modulate, and sustainably release growth factors. Here, we loaded the HS-carrying fibers with the brain-derived neurotrophic factor (BDNF) which is known to act neuroprotectively. Release of this neurotrophic factor from the fibers was followed over a period of 110 days. Cell culture investigations with spiral ganglion cells, using the supernatants from the release studies, showed that the BDNF delivered from the fibers drastically increased the survival rate of SGNs in vitro. Thus, biodegradable polymer fibers with attached HS and loaded with BDNF are suitable for the protection and support of SGNs. Moreover, they present a promising base material for the further development towards a future neuronal guiding scaffold.

Polycaprolactone-Based 3D-Printed Scaffolds as Potential Implant Materials for Tendon-Defect Repair.

Chronic tendon ruptures are common disorders in orthopedics. The conventional surgical methods used to treat them often require the support of implants. Due to the non-availability of suitable materials, 3D-printed polycaprolactone (PCL) scaffolds were designed from two different starting materials as suitable candidates for tendon-implant applications. For the characterization, mechanical testing was performed. To increase their biocompatibility, the PCL-scaffolds were plasma-treated and coated with fibronectin and collagen I. Cytocompatibility testing was performed using L929 mouse fibroblasts and human-bone-marrow-derived mesenchymal stem cells. The mechanical testing showed that the design adaptions enhanced the mechanical stability. Cell attachment was increased in the plasma-treated specimens compared to the control specimens, although not significantly, in the viability tests. Coating with fibronectin significantly increased the cellular viability compared to the untreated controls. Collagen I treatment showed an increasing trend. The desired cell alignment and spread between the pores of the construct was most prominent on the collagen-I-coated specimens. In conclusion, 3D-printed scaffolds are possible candidates for the development of tendon implants. Enhanced cytocompatibility was achieved through surface modifications. Although adaptions in mechanical strength still require alterations in order to be applied to human-tendon ruptures, we are optimistic that a suitable implant can be designed.

Adhesion Forces of Oral Bacteria to Titanium and the Correlation with Biophysical Cellular Characteristics.

Bacterial adhesion to dental implants is the onset for the development of pathological biofilms. Reliable characterization of this initial process is the basis towards the development of anti-biofilm strategies. In the present study, single-cell force spectroscopy (SCFS), by means of an atomic force microscope connected to a microfluidic pressure control system (FluidFM), was used to comparably measure adhesion forces of different oral bacteria within a similar experimental setup to the common implant material titanium. The bacteria selected belong to different ecological niches in oral biofilms: the commensal pioneers Streptococcus oralis and Actinomyces naeslundii; secondary colonizer Veillonella dispar; and the late colonizing pathogens Porphyromonas gingivalis as well as fimbriated and non-fimbriated Aggregatibacter actinomycetemcomitans. The results showed highest values for early colonizing pioneer species, strengthening the link between adhesion forces and bacteria's role in oral biofilm development. Additionally, the correlation between biophysical cellular characteristics and SCFS results across species was analyzed. Here, distinct correlations between electrostatically driven maximum adhesion force, bacterial surface elasticity and surface charge as well as single-molecule attachment points, stretching capability and metabolic activity, could be identified. Therefore, this study provides a step towards the detailed understanding of oral bacteria initial adhesion and could support the development of infection-resistant implant materials in future.

A new liquid chromatography/electrospray ionization mass spectrometry method for the analysis of underivatized aliphatic long-chain polyamines: application to diatom-rich sediments.

Natural polyamines are found in all three domains of life and long-chain polyamines (LCPAs) play a special role in silicifying organisms such as diatoms and sponges where they are actively involved in the complex formation and nanopatterning of siliceous structures. With chain lengths extending up to 20 N-methylated propylamine repeat units, diatom LCPAs constitute the longest natural polyamines. Mixtures of natural LCPAs are typically purified in bulk using ion-exchange, size-exclusion and dialysis and then analyzed either by direct infusion mass spectrometry or by MALDI-TOF. Here, we describe a novel ion-pairing liquid chromatographic method that allows baseline separation, detection and structural elucidation of underivatized aliphatic methylated and non-methylated LCPAs with a wide range of chain lengths. Complete separation of synthetic mixtures of LCPA species differing by either a propylamine or an N-methylpropylamine unit is achievable using this method and chromatographic separation of natural, diatom frustule bound LCPAs extracted from sediment core samples is greatly improved. Using electrospray ionization mass spectrometry (ESI-MS), we detected singly [M+H](+) and multiply [M+nH](n+) charged protonated ions. The abundance of multiply charged LCPA species increased linearly as a function of LCPA chain length (N) and multiprotonated ions [M+nH](n+) were more abundant for longer chain polyamines. The abundance of multiply charged LCPAs along with the concomitant disappearance of the singly charged protonated molecular ion significantly increases the complexity of the MS spectra, justifying the need for good chromatographic separation of complex LCPA mixtures. This analytical procedure will likely constitute a powerful tool for the characterization, quantification, as well as the purification of individual LCPAs in natural and synthetic samples for studies of silica precipitation as well as nitrogen and carbon isotopic analysis used in paleoceanographic studies.

Mechanical characterization of nacre as an ideal-model for innovative new endoprosthesis materials.

INTRODUCTION: To mimic the impressive mechanical behavior of natural ceramics for technical or biomedical applications, interest has been focused on nacre, a natural composite consisting of imbricated aragonite platelets embedded in a protein matrix. Nacre is an ideal model material for implants, since it possesses favorable strength and toughness properties compared to the component materials of which it is composed. The focus of the present study was to test standardized parameters which are good indicators of the material's suitability as an implant material. MATERIALS AND METHODS: A three-point bending test was performed on polished nacre samples according to international standards for Young's modulus, bending strength and fracture toughness. A total of 60 nacre samples were tested, with 5 samples each in 4 states of hydration (dry, distilled water, 0.9% NaCl and sea water). As a basis for comparison, 10 samples of a newly developed bioceramic material were tested for fracture toughness. RESULTS: The fracture toughness of nacre tended to be higher for specimens conditioned in 0.9% NaCl than for dry specimens (5.3 ± 0.6 vs. 4.3 ± 0.7 MPam(1/2), p = 0.061). The fracture toughness of the bioceramic investigated was observed to be somewhat higher than nacre (5.8 ± 0.4 vs. 4.3 ± 0.7 MPam(1/2), p ≤ 0.001). DISCUSSION AND CONCLUSION: The increase in fracture toughness of hydrated nacre was not as large as would be expected based on the difference in stiffness of the matrix material after hydration that has been reported. Modulus and toughness were similar to published values and the fracture toughness observed was somewhat higher than reported for alumina implant ceramics, which are in use in total hip arthroplasty. In a direct comparison, we found that a newly developed alumina bioceramic material can in fact match nature in terms of fracture toughness.

Coating of titanium implants with copolymer supports bone regeneration: a comparative in vivo study in rabbits.

PURPOSE: In modern orthopedics aseptic loosening caused by the formation of micro-wear particles remains a problem for endoprosthetic joint replacements as revision surgery is necessary with corresponding costs and exertions by patients. This study is devoted to the question of how the osseous ingrowth of implants can be supported. It was investigated whether the developed copolymer, p-VBP-co-GMA, coated on the surface of the implants, supports bone healing. In addition, it was analyzed whether covalent linkage of Bone Morphogenetic Protein 2 (BMP-2) to the copolymer layer enhances bone formation. METHODS: Eight adult New Zealand White Rabbits were implanted with four different foils (control, copolymer, copolymer + BMP-2, control + BMP-2) each. The histomorphometric analysis of all samples was made 28 days after implantation. RESULTS: The copolymer had a positive effect on bone remodeling compared to the control group. We observed that the copolymer group had a significantly increased bone volume per tissue volume ratio and bone density to the control group. In contrast, this in-vivo study showed that the immobilization of BMP-2 onto the copolymer layer did not enhance bone healing. The bone volume per tissue volume ratio was decreased as well as the bone density compared to control + BMP-2 group. CONCLUSION: The analysis showed that the bone remodeling process in the copolymer + BMP-2-group is in an early phase comparable to the control group. These results suggest that the coating with the developed copolymer has major potential for medical use as it enhances bone mass around the implant.

Optimization of Critical Parameters for Carbodiimide Mediated Production of Highly Modified Chitosan.

An optimization of the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and hydroxy benzotriazole mediated conjugation of the polysaccharide chitosan with functional carboxylic acids was shown. Optimal parameters that enable resource-efficient synthesis of highly functionalized chitosan were identified. In particular, use of only catalytic instead of stoichiometric amounts of hydroxy benzotriazole and tight control of pH in reaction mixture resulted in highly efficient incorporation of the desired moieties as side chains in chitosan. As a result, the model reactant 4-azidobenzoic acid was incorporated resulting in a degree of substitution of over 30% with very high coupling efficacy of up to 90%. Similar results were obtained with other carboxylic acids such as methacrylic acid, 3-(2-furyl) propionic acid and 3-maleimido propionic acid, highlighting the broad applicability of our findings for the functionalization of chitosan.

Enzyme-Responsive Nanoparticles and Coatings Made from Alginate/Peptide Ciprofloxacin Conjugates as Drug Release System.

Infection-controlled release of antibacterial agents is of great importance, particularly for the control of peri-implant infections in the postoperative phase. Polymers containing antibiotics bound via enzymatically cleavable linkers could provide access to drug release systems that could accomplish this. Dispersions of nanogels were prepared by ionotropic gelation of alginate with poly-l-lysine, which was conjugated with ciprofloxacin as model drug via a copper-free 1,3-dipolar cycloaddition (click reaction). The nanogels are stable in dispersion and form films which are stable in aqueous environments. However, both the nanogels and the layers are degraded in the presence of an enzyme and the ciprofloxacin is released. The efficacy of the released drug against Staphylococcus aureus is negatively affected by the residues of the linker. Both the acyl modification of the amine nitrogen in ciprofloxacin and the sterically very demanding linker group with three annellated rings could be responsible for this. However the basic feasibility of the principle for enzyme-triggered release of drugs was successfully demonstrated.

ELISA- and Activity Assay-Based Quantification of BMP-2 Released In Vitro Can Be Biased by Solubility in "Physiological" Buffers and an Interfering Effect of Chitosan.

Chitosan nanogel-coated polycaprolactone (PCL) fiber mat-based implant prototypes with tailored release of bone morphogenic protein 2 (BMP-2) are a promising approach to achieve implant-mediated bone regeneration. In order to ensure reliable in vitro release results, the robustness of a commercially available ELISA for E. coli-derived BMP-2 and the parallel determination of BMP-2 recovery using a quantitative biological activity assay were investigated within a common release setup, with special reference to solubility and matrix effects. Without bovine serum albumin and Tween 20 as solubilizing additives to release media buffed at physiological pH, BMP-2 recoveries after release were notably reduced. In contrast, the addition of chitosan to release samples caused an excessive recovery. A possible explanation for these effects is the reversible aggregation tendency of BMP-2, which might be influenced by an interaction with chitosan. The interfering effects highlighted in this study are of great importance for bio-assay-based BMP-2 quantification, especially in the context of pharmaceutical release experiments.

Varying the sustained release of BMP-2 from chitosan nanogel-functionalized polycaprolactone fiber mats by different polycaprolactone surface modifications.

Polycaprolactone (PCL) fiber mats with different surface modifications were functionalized with a chitosan nanogel coating to attach the growth factor human bone morphogenetic protein 2 (BMP-2). Three different hydrophilic surface modifications were compared with regard to the binding and in vitro release of BMP-2. The type of surface modification and the specific surface area derived from the fiber thickness had an important influence on the degree of protein loading. Coating the PCL fibers with polydopamine resulted in the binding of the largest BMP-2 quantity per surface area. However, most of the binding was irreversible over the investigated period of time, causing a low release in vitro. PCL fiber mats with a chitosan-graft-PCL coating and an additional alginate layer, as well as PCL fiber mats with an air plasma surface modification boundless BMP-2, but the immobilized protein could almost completely be released. With polydopamine and plasma modifications as well as with unmodified PCL, high amounts of BMP-2 could also be attached directly to the surface. Integration of BMP-2 into the chitosan nanogel functionalization considerably increased binding on all hydrophilized surfaces and resulted in a sustained release with an initial burst release of BMP-2 without detectable loss of bioactivity in vitro.