Controlled intramyocardial release of engineered chemokines by biodegradable hydrogels as a treatment approach of myocardial infarction.

Myocardial infarction (MI) induces a complex inflammatory immune response, followed by the remodelling of the heart muscle and scar formation. The rapid regeneration of the blood vessel network system by the attraction of hematopoietic stem cells is beneficial for heart function. Despite the important role of chemokines in these processes, their use in clinical practice has so far been limited by their limited availability over a long time-span in vivo. Here, a method is presented to increase physiological availability of chemokines at the site of injury over a defined time-span and simultaneously control their release using biodegradable hydrogels. Two different biodegradable hydrogels were implemented, a fast degradable hydrogel (FDH) for delivering Met-CCL5 over 24 hrs and a slow degradable hydrogel (SDH) for a gradual release of protease-resistant CXCL12 (S4V) over 4 weeks. We demonstrate that the time-controlled release using Met-CCL5-FDH and CXCL12 (S4V)-SDH suppressed initial neutrophil infiltration, promoted neovascularization and reduced apoptosis in the infarcted myocardium. Thus, we were able to significantly preserve the cardiac function after MI. This study demonstrates that time-controlled, biopolymer-mediated delivery of chemokines represents a novel and feasible strategy to support the endogenous reparatory mechanisms after MI and may compliment cell-based therapies.

Surface-modified silicone foils for intraocular implantation.

BACKGROUND: Implantation of silicone materials like iris diaphragms into the eye can be complicated by cell migration and attachment. We studied polydimethylsiloxane (PDMS) foils coated with isocyanate terminated, star-shaped poly(ethylene glycol-stat-propylene glycol) (NCO-sP(EO-stat-PO)) equipped with heparin towards the inhibition of cell attachment without influencing cell viability. METHODS: Mouse fibroblasts L929 were cultured and seeded onto sterilized pieces of either uncoated NCO-sP(EO-stat-PO) or heparin-NCO-sP(EO-stat-PO) loaded foils. Polyvinylchloride (PVC) foils served as the positive control and biomembranes as the negative control. The cultured cells were examined after 24 h for cell viability and adhesion by fluorescence microscopy; morphological cell changes were documented after hemalaun staining. Cell density was measured and quantification of cell proliferation was assessed by a BrdU test; quantification of cell activity was analyzed by a WST-1 test. RESULTS: The fibroblasts' cell viability was excellent on all tested foils except the toxic PVC foil. NCO-sP(EO-stat-PO) coating provided significantly reduced cell activity. On heparin-loaded coatings, cells were viable and less dense but showed almost the same cell proliferation and cell activity as on the negative control. NCO-sP(EO-stat-PO) coated, heparin loaded foils proved high biocompatibility and reduced cell adhesion. CONCLUSIONS: Both NCO-sP(EO-stat-PO)-coated foils with and without heparin seemed to be a viable implantation material for less cell migration, attachment, and reduced implant complications. Conclusive we give a recommendation for further studies on the intraocular implantation in particular for the NCO-sP(EO-stat-PO)-coated foils.

Intracardiac injection of matrigel induces stem cell recruitment and improves cardiac functions in a rat myocardial infarction model.

Matrigel promotes angiogenesis in the myocardium from ischemic injury and prevents remodelling of the left ventricle. We assessed the therapeutic efficacy of intracardiac matrigel injection and matrigel-mediated stem cell homing in a rat myocardial infarction (MI) model. Following MI, matrigel (250 µl) or phosphate-buffered solution (PBS) was delivered by intracardiac injection. Compared to the MI control group (MI-PBS), matrigel significantly improved left ventricular function (n= 11, P < 0.05) assessed by pressure-volume loops after 4 weeks. There is no significant difference in infarct size between MI-matrigel (MI-M; 21.48 ± 1.49%, n = 10) and MI-PBS hearts (20.98 ± 1.25%, n = 10). The infarct wall thickness of left ventricle is significantly higher (P < 0.01) in MI-M (0.72 ± 0.02 mm, n = 10) compared with MI-PBS (0.62 ± 0.02 mm, n = 10). MI-M hearts exhibited higher capillary density (border 130.8 ± 4.7 versus 115.4 ± 6.0, P < 0.05; vessels per high-power field [HPF; 400×], n = 6) than MI-PBS hearts. c-Kit(+) stem cells (38.3 ± 5.3 versus 25.7 ± 1.5 c-Kit(+) cells per HPF [630×], n = 5, P < 0.05) and CD34(+) cells (13.0 ± 1.51 versus 5.6 ± 0.68 CD34(+) cells per HPF [630×], n = 5, P < 0.01) were significantly more numerous in MI-M than in MI-PBS in the infarcted hearts (n = 5, P < 0.05). Intracardiac matrigel injection restores myocardial functions following MI, which may attribute to the improved recruitment of CD34(+) and c-Kit(+) stem cells.

Controlling pH in shake flasks using polymer-based controlled-release discs with pre-determined release kinetics.

BACKGROUND: There are significant differences in the culture conditions between small-scale screenings and large-scale fermentation processes. Production processes are usually conducted in fed-batch cultivation mode with active pH-monitoring and control. In contrast, screening experiments in shake flasks are usually conducted in batch mode without active pH-control, but with high buffer concentrations to prevent excessive pH-drifts. These differences make it difficult to compare results from screening experiments and laboratory and technical scale cultivations and, thus, complicate rational process development. In particular, the pH-value plays an important role in fermentation processes due to the narrow physiological or optimal pH-range of microorganisms. To reduce the differences between the scales and to establish a pH-control in shake flasks, a newly developed easy to use polymer-based controlled-release system is presented in this paper. This system consists of bio-compatible silicone discs embedding the alkaline reagent Na2CO3. Since the sodium carbonate is gradually released from the discs in pre-determined kinetics, it will ultimately compensate the decrease in pH caused by the biological activity of microorganisms. RESULTS: The controlled-release discs presented here were successfully used to cultivate E. coli K12 and E. coli BL21 pRSET eYFP-IL6 in mineral media with glucose and glycerol as carbon (C) sources, respectively. With glucose as the C-source it was possible to reduce the required buffer concentration in shake flask cultures by 50%. Moreover, with glycerol as the C-source, no buffer was needed at all. CONCLUSIONS: These novel polymer-based controlled-release discs allowed buffer concentrations in shake flask media to be substantially reduced or omitted, while the pH remains in the physiological range of the microorganisms during the whole cultivation time. Therefore, the controlled-release discs allow a better control of the pH, than merely using high buffer concentrations. The conditions applied here, i.e. with significantly reduced buffer concentrations, enhance the comparability of the culture conditions used in screening experiments and large-scale fermentation processes.

Directed covalent immobilization of fluorescently labeled cytokines.

Cytokines are important mediators coordinating inflammation and wound healing in response to tissue damage and infection. Therefore, immobilization of cytokines on the surface of biomaterials is a promising approach to improve biocompatibility. Soluble cytokines signal through receptors on the cell surface leading to cell differentiation, proliferation, or other effector functions. Random immobilization of cytokines on surfaces will result in a large fraction of inactive protein due to impaired cytokine--receptor interaction. We developed a strategy that combined (i) directed covalent coupling of cytokines, (ii) quantification of coupling efficiency through fluorescence detection, and (iii) a reliable protease cleavage assay to control orientation of coupling. For this purpose, fusion proteins of the SNAP-tag followed by an enterokinase recognition site, yellow fluorescent protein (YFP), and the cytokine of interest being either interleukin-6 (IL-6) or oncostatin M (OSM) were generated. The SNAP-tag is a derivative of O(6)-alkylguanine-DNA alkyltransferase that couples itself covalently to benzylguanine. Bioactivities of the SNAP-YFP-cytokines were shown to be comparable with the nontagged cytokines. Efficient coupling of SNAP-YFP-cytokines to benzylguanine-modified beads was demonstrated by flow cytometry. The fact that enterokinase treatment released most of the fluorescence from the beads is indicative for directed coupling and only marginal adsorptive binding. Cellular responses to SNAP-YFP-cytokine beads were analyzed in cellular lysates and by confocal microscopy indicating that the directionally immobilized cytokines are fully signaling competent with respect to the activation of ERK and STAT3. The strategy presented here is generally applicable for the directed covalent immobilization of fluorescently labeled proteins including the convenient and reliable control of coupling efficiency and orientation.

High-throughput screening of Hansenula polymorpha clones in the batch compared with the controlled-release fed-batch mode on a small scale.

Most large-scale production processes in biotechnology are performed in fed-batch operational mode. In contrast, the screenings for microbial production strains are run in batch mode, which results in the microorganisms being subjected to different physiological conditions. This significantly affects strain selection. To demonstrate differences in ranking during strain selection depending on the operational mode, screenings were performed in batch and fed-batch modes. Two model populations of the methylotrophic yeast Hansenula polymorpha RB11 with vector pC10-FMD (P(FMD)-GFP) (220 clones) and vector pC10-MOX (P(MOX)-GFP) (224 clones) were applied. For fed-batch cultivations in deep-well microtiter plates, a controlled-release system made of silicone elastomer discs containing glucose was used. Three experimental set-ups were investigated: batch cultivation with (1) glucose as a substrate, which catabolite represses product formation, and (2) glycerol as a carbon source, which is partially repressing, respectively, and (3) fed-batch cultivation with glucose as a limiting substrate using the controlled-release system. These three experimental set-ups showed significant variations in green fluorescent protein (GFP) yield. Interestingly, screenings in fed-batch mode with glucose as a substrate resulted in the selection of yeast strains different from those cultivated in batch mode with glycerol or glucose. Ultimately, fed-batch screening is considerably better than screening in batch mode for fed-batch production processes with glucose as a carbon source.

Functionalization of electrospun fibers of poly(epsilon-caprolactone) with star shaped NCO-poly(ethylene glycol)-stat-poly(propylene glycol) for neuronal cell guidance.

Microfibers produced with electrospinning have recently been used in tissue engineering. In the development of artificial implants for nerve regeneration they are of particular interest as guidance structures for cell migration and axonal growth. Using electrospinning we produced parallel-orientated biocompatible fibers in the submicron range consisting of poly(epsilon-caprolactone) (PCL) and star shaped NCO-poly(ethylene glycol)-stat-poly(propylene glycol) (sPEG). Addition of the bioactive peptide sequence glycine-arginine-glycine-aspartate-serine (GRGDS) or the extracellular matrix protein fibronectin to the electrospinning solution resulted in functionalized fibers. Surface characteristics and biological properties of functionalized and non-functionalised fibers were investigated. Polymer solutions and electrospinning process parameters were varied to obtain high quality orientated fibers. A polymer mixture containing high molecular weight PCL, PCL-diol, and sPEG permitted a chemical reaction between hydroxyl groups of the diol and isocyanante groups of the sPEG. Surface analysis demonstrated that sPEG at the fiber surface minimized protein adhesion. In vitro experiments using dorsal root ganglia explants showed that the cell repellent property of pure PCL/sPEG fibers was overcome by functionalization either with GRGDS peptide or fibronectin. In this way cell migration and axonal outgrowth along fibers were significantly increased. Thus, functionalized electrospun PCL/sPEG fibers, while preventing non-specific protein adsorption, are a suitable substrate for biological and medical applications.

Equalizing growth in high-throughput small scale cultivations via precultures operated in fed-batch mode.

An often underestimated problem when working with different clones in microtiter plates and shake flask screenings is the non-parallel and non-equal growth of batch cultures. These growth differences are caused by variances of individual clones regarding initial biomass concentration, lag-phase or specific growth rate. Problems arising from unequal growth kinetics are different induction points in expression studies or uneven cultivation periods at the time of harvest. Screening for the best producing clones of a library under comparable conditions is thus often impractical or even impossible. A new approach to circumvent the problem of unequal growth kinetics of main cultures is the application of fed-batch mode in precultures in microtiter plates and shake flasks. Fed-batch operation in precultures is realized through a slow-release system for glucose. After differently growing cultures turn to glucose-limited growth, they all consume the same amount of glucose due to the fixed feed profile of glucose provided by the slow-release system. This leads to equalized growth. Inherent advantages of this method are that it is easy to use and requires no additional equipment like pumps. This new technique for growth equalization in high-throughput cultivations is simulated and verified experimentally. The growth of distinctly inoculated precultures in microtiter plates and shake flasks could be equalized for different microorganisms such as Escherichia coli and Hansenula polymorpha.

Process development in Hansenula polymorpha and Arxula adeninivorans, a re-assessment.

A range of industrial H. polymorpha-based processes exist, most of them for the production of pharmaceuticals. The established industrial processes lean on the use of promoters derived from MOX and FMD, genes of the methanol metabolism pathway. In Hansenula polymorpha these promoters are de-repressed upon depletion of a range of carbon sources like glucose and glycerol instead of being induced by methanol as reported for other methylotrophs. Due to these characteristics screening and fermentation modes have been defined for strains harbouring such expression control elements that lean on a limited supplementation of glycerol or glucose to a culture medium. For fermentation of H. polymorpha a synthetic minimal medium (SYN6) has been developed. No industrial processes have been developed so far based on Arxula adeninivorans and only a limited range of strong promoter elements exists, suitable for heterologous gene expression. SYN6 originally designed for H. polymorpha provided a suitable basis for the initial definition of fermentation conditions for this dimorphic yeast. Characteristics like osmo- and thermotolerance can be addressed for the definition of culture conditions.

Topographical control of human macrophages by a regularly microstructured polyvinylidene fluoride surface.

In this study we investigated the influence of surface topography on the inflammatory response of human macrophages. We generated different polyvinylidene fluoride (PVDF) surfaces including (i) a smooth surface of PVDF spherulites as a control, (ii) a randomly nanotextured surface with alumina particles, and (iii) a microstructure using laser ablation. The identical chemistry of all PVDF surfaces was demonstrated by X-ray photoelectron spectroscopy. The topography was evaluated by white light interferometry and X-profile analysis. Macrophages were cultured on the different surfaces including lipopolysaccharide (LPS) treatment as an inflammatory activator. Our results demonstrate that the microstructured surface but not the nanotexured significantly affects the activation of primary human macrophages by inducing a specific cytokine and gene expression pattern. This activation resulted in a subtype of macrophages with pro- but also anti-inflammatory properties. Interestingly, the response on the topography differed from that triggered by LPS, pointing to a different activation state of the cells. Our data clearly show that a particular topography induces an inflammatory response. This suggests that the modification of topography could influence the inflammatory potency of a biomaterial and hence could affect the biocompatibility of implants.

Control of protein adsorption on functionalized electrospun fibers.

Electrospun fibers that are protein resistant and functionalized with bioactive signals were produced by solution electrospinning amphiphilic block copolymers. Poly (ethylene glycol)-block-poly(D,L-lactide) (PEG-b-PDLLA) was synthesized in two steps, with a PEG segment of 10 kDa, while the PDLLA block ranged from 20 to 60 kDa. Depending on the PEG and PDLLA segment ratio, as well as solvent selection, the hydrophilicity and protein adsorption could be altered on the electrospun mesh. Furthermore, an alpha-acetal PEG-b-PDLLA was synthesized that allowed the conjugation of active molecules, resulting in surface functionalization of the electrospun fiber. Electrospun material with varying morphologies and diameter were electrospun from 10, 20, and 30 wt.% solutions. Sessile drop measurements showed a reduction in the contact angle from 120 degrees for pure poly(D,L-lactide) with increasing PEG/PDLLA ratio. All electrospun block PEG-b-PDLLA fibers had hydrophilic properties, with contact angles below 45 degrees . The fibers were collected onto six-arm star-poly(ethylene glycol) (star-PEG) coated silicon wafers and incubated with fluorescently labeled proteins. All PEG-b-PDLLA fibers showed no detectable adsorption of bovine serum albumin (BSA) independent of their composition while a dependence between hydrophobic block length was observed for streptavidin adsorption. Fibers of block copolymers with PDLLA blocks smaller than 39 kDa showed no adsorption of BSA or streptavidin, indicating good non-fouling properties. Fibers were surface functionalized with N(epsilon)-(+)-biotinyl-L-lysine (biocytin) or RGD peptide by attaching the molecule to the PEG block during synthesis. Protein adsorption measurements, and the controlled interaction of biocytin with fluorescently labeled streptavidin, showed that the electrospun fibers were both resistant to protein adsorption and are functionalized. Fibroblast adhesion was contrasting between the unfunctionalized and RGD-coupled electrospun fabrics, confirming that the surface of the fibers was functionalized. The PEG-b-PDLLA surface functionalized electrospun fibers are promising substrates for controlling cell-material interactions, particularly for tissue-engineering applications.

Influence of different ECM mimetic peptide sequences embedded in a nonfouling environment on the specific adhesion of human-skin keratinocytes and fibroblasts on deformable substrates.

Mechanical stress is a decisive factor for the differentiation, proliferation, and general behavior of cells. However, the specific signaling of mechanotransduction is not fully understood. One basic problem is the clear distinction between the different extracellular matrix (ECM) constituents that participate in cellular adhesion and their corresponding signaling pathways. Here, a system is proposed that enables mechanical stimulation of human-skin-derived keratinocytes and human dermal fibroblasts that specifically interact with peptide sequences immobilized on a non-interacting but deformable substrate. The peptide sequences mimic fibronectin, laminin, and collagen type IV, three major components of the ECM. To achieve this, PDMS is activated using ammonia plasma and coated with star-shaped isocyanate-terminated poly(ethylene glycol)-based prepolymers, which results in a functional coating that prevents unspecific cell adhesion. Specific cell adhesion is achieved by functionalization of the layers with the peptide sequences in different combinations. Moreover, a method that enables the decoration of deformable substrates with cell-adhesion peptides in extremely defined nanostructures is presented. The distance and clustering of cell adhesion molecules below 100 nm has been demonstrated to be of utmost importance for cell adhesion. Thus we present a new toolbox that allows for the detailed analysis of the adhesion of human-skin-derived cells on structurally and biochemically decorated deformable substrates.

Guidance of glial cell migration and axonal growth on electrospun nanofibers of poly-epsilon-caprolactone and a collagen/poly-epsilon-caprolactone blend.

Our long-term goal is to develop an artificial implant as a conduit for axonal regeneration after peripheral nerve injury. In this study, biodegradable, aligned poly-epsilon-caprolactone (PCL) and collagen/PCL (C/PCL) nanofibers designed as guidance structures were produced by electrospinning and tested in cell culture assays. We compared fibers of 100% PCL with fibers consisting of a 25:75% C/PCL blend. To test their biocompatibility, assays of cell adhesion, survival, migration, effects on cell morphology, axonal growth and axonal guidance were performed. Both types of eletrospun fibers supported oriented neurite outgrowth and glial migration from dorsal root ganglia (DRG) explants. Schwann cell migration, neurite orientation, and process formation of Schwann cells, fibroblasts and olfactory ensheathing cells were improved on C/PCL fibers, when compared to pure PCL fibers. While the velocity of neurite elongation from DRG explants was higher on PCL fibers, analysis of isolated sensory neurons showed significantly better axonal guidance by the C/PCL material. The data demonstrate that electrospun fibers composed of a collagen and PCL blend represent a suitable substrate for supporting cell proliferation, process outgrowth and migration and as such would be a good material for artificial nerve implants.

BMP-7 loaded microspheres as a new delivery system for the cultivation of human chondrocytes in a collagen type-I gel.

In recent years, interest in chondrocyte cultures for transplantation has gained increasing attention. We investigated the use of PGLA microspheres as a new delivery system for BMP-7 and the effects on human chondrocytes cultivated in a 3D collagen gel culture. In an in vitro study, human chondrocytes obtained from osteoarthritic knee joints were released, transferred into a collagen type-I gel, and cultivated up to 14 days. In the treatment group PGLA microspheres loaded with human recombinant BMP-7 protein were added to the matrix. After the cultivation period, histological and immunohistochemical investigations were performed. In addition, the aggrecan core protein and type-II collagen mRNA concentrations were measured by real-time PCR. Histological staining for proteoglycan and collagen type-II protein and quantification via digital image processing revealed a significantly higher content in the samples cultivated with BMP-7 loaded microspheres in comparison to the control samples. Moreover, the collagen gel scaffold was partially remodeled by the chondrocytes and replaced by newly synthesized extracellular matrix. Cellular proliferation as well as apoptosis were low. In conclusion, we consider the PGLA microsphere system to be a functional device for the delivery of growth factors during the cultivation of articular chondrocytes leading to an increased content of type-II collagen and proteoglycan in the extracellular matrix.

Targeting In-Stent-Stenosis with RGD- and CXCL1-Coated Mini-Stents in Mice.

Atherosclerotic lesions that critically narrow the artery can necessitate an angioplasty and stent implantation. Long-term therapeutic effects, however, are limited by excessive arterial remodeling. We here employed a miniaturized nitinol-stent coated with star-shaped polyethylenglycole (star-PEG), and evaluated its bio-functionalization with RGD and CXCL1 for improving in-stent stenosis after implantation into carotid arteries of mice. Nitinol foils or stents (bare metal) were coated with star-PEG, and bio-functionalized with RGD, or RGD/CXCL1. Cell adhesion to star-PEG-coated nitinol foils was unaltered or reduced, whereas bio-functionalization with RGD but foremost RGD/CXCL1 increased adhesion of early angiogenic outgrowth cells (EOCs) and endothelial cells but not smooth muscle cells when compared with bare metal foils. Stimulation of cells with RGD/CXCL1 furthermore increased the proliferation of EOCs. In vivo, bio-functionalization with RGD/CXCL1 significantly reduced neointima formation and thrombus formation, and increased re-endothelialization in apoE-/- carotid arteries compared with bare-metal nitinol stents, star-PEG-coated stents, and stents bio-functionalized with RGD only. Bio-functionalization of star-PEG-coated nitinol-stents with RGD/CXCL1 reduced in-stent neointima formation. By supporting the adhesion and proliferation of endothelial progenitor cells, RGD/CXCL1 coating of stents may help to accelerate endothelial repair after stent implantation, and thus may harbor the potential to limit the complication of in-stent restenosis in clinical approaches.

Development of a temperature sensitive drug release system for polymeric implant devices.

A low density polyethylene (LDPE) model surface was coated with poly(amino-p-xylylene) (amino-ppx) via chemical vapour deposition (CVD) polymerization. The functional surface was used to immobilize a polymeric drug release system consisting of poly(N-isopropylacrylamide) (NIPAAm)-co-poly(acrylic acid) (AAc). The coupled drug release system was used to incorporate the thrombin inhibitor r-hirudin. For the investigation of the concentration of incorporated r-hirudin and the release profile over a given period of time r-hirudin was labelled with 123I as well as fluorescein isothiocyanate (FITC). Using an incubation solution concentration of 0.2% at pH 5 and an ionic strength of 0.7 M a maximum concentration of 2.21 (+/- 0.11) nmol/cm2 of r-hirudin was detected. FITC-r-hirudin was almost quantitatively (2.08 +/- 10 nmol/cm2; 94%) released from the surface coating within a period of 14 days.

Surface modification of poly(vinylidenefluoride) to improve the osteoblast adhesion.

Cell adhesion to biomaterials is mediated primarily by the interaction between surface bound proteins and corresponding receptors on the membrane of the cells. The attachment of fibronectin onto poly(vinylidenefluoride) (PVDF) surface and the application of PVDF as biomaterial in bone contact was the subject of our study. PVDF is a biomaterial established for soft tissue applications. Surface modifications of PVDF were performed by plasma induced graft copolymerisation of acrylic acid or CVD polymerisation of 4-amino[2.2]paracyclophane. The provided functionalised PVDF surface was used to immobilise fibronectin using different techniques. All modification steps were verified by means of X-ray photoelectron spectroscopy (XPS), attenuated total reflection infrared spectroscopy (IR-ATR) and contact angle measurements. Surface topology was studied by atomic force measurements (AFM). Protein adsorption was controlled by enzyme linked immunosorbent assay (ELISA). Cell attachment was enhanced if physically adsorbed fibronectin was used, while enhanced attachment and proliferation were induced by covalently binding fibronectin to the surface modified PVDF.

Lipase catalyzed copolymerization of 3(S)-isopropylmorpholine-2,5-dione and D,L-lactide.

The polymerization and copolymerization of 3(S)-isopropylmorpholine-2,5-dione (IPMD) and D,L-lactide (DLLA) were carried out in the presence of Porcine pancreatic lipase type II (PPL) as a catalyst at 100 degrees C for 168 h. Homopolymers and random copolymers of various compositions were obtained with a carboxylic acid group at one end and a hydroxyl group at the other end. The glass transition temperature of the copolymers decreases with increasing mole fraction of DLLA residue in the copolymers.

Dexamethasone released from cochlear implant coatings combined with a protein repellent hydrogel layer inhibits fibroblast proliferation.

The insertion of cochlear implants into the inner ear often causes inflammation and fibrosis inside the scala tympani and thus growth of fibrous tissue on the implant surface. This deposition leads to the loss of function in both electrical and laser-based implants. The design of this study was to realize fibroblast growth inhibition by dexamethasone (Dex) released from the base material of the implant [polydimethylsiloxane (PDMS)]. To prevent cell and protein adhesion, the PDMS was coated with a hydrogel layer [star-shaped polyethylene glycol prepolymer (sPEG)]. Drug release rates were studied over 3 months, and surface characterization was performed. It was observed that the hydrogel slightly smoothened the surface roughened by the Dex crystals. The hydrogel coating reduced and prolonged the release of the drug over several months. Unmodified, sPEG-coated, Dex-loaded, and Dex/sPEG-equipped PDMS filaments were cocultivated in vitro with fluorescent fibroblasts, analyzed by fluorescent microscopy, and quantified by cell counting. Compared to the unmodified PDMS, cell growth on all modified filaments was averagely 95% ±standard deviation (SD) less, while cell growth on the bottom of the culture dishes containing Dex-loaded filaments was reduced by 70% ±SD. Both, Dex and sPEG prevented direct cell growth on the filament surfaces, while drug delivery was maintained for the duration of several months.

The RGD coupling strategy determines the inflammatory response of human primary macrophages in vitro and angiogenesis in vivo.

Surface modifications of implants are frequently done using bioactive peptides. However, immune cells such as macrophages might evoke a rejection of an implant due to an undesired activation by the materials. Here, the influence of different strategies for peptide immobilization onto (poly)-vinylidene fluoride (PVDF) on inflammation and angiogenesis is studied. The inflammatory response of human primary macrophages is investigated by analyzing inflammatory cytokine expression. Surface roughness and adsorptive coupling have only minor effects on macrophage activation. Acrylic acid (AAc)-based covalent RGD-coupling leads to the most favorable cellular reaction, indicated by increased VEGF release. Chemical vapor deposition treated surfaces are inert, but additional covalent coupling of RGD induces a pronounced proinflammatory reaction. An in vivo angiogenesis study reveals that covalent coupling of RGD results in delayed but increased angiogenesis. It is concluded that for implant decoration with peptides, the substrate material has to be selected carefully to prevent inflammatory immune responses.