Plasma polymerized allylamine-the unique cell-attractive nanolayer for dental implant materials

Biomaterials should be bioactive in stimulating the surrounding tissue to accelerate the ingrowth of permanent implants. Chemical and topographical features of the biomaterial surface affect cell physiology at the interface. A frequently asked question is whether the chemistry or the topography dominates the cell-material interaction. Recently, we demonstrated that a plasma-chemical modification using allylamine as a precursor was able to boost not only cell attachment and cell migration, but also intracellular signaling in vital cells. This microwave plasma process generated a homogenous nanolayer with randomly distributed, positively charged amino groups. In contrast, the surface of the human osteoblast is negatively charged at −15 mV due to its hyaluronan coat. As a consequence, we assumed that positive charges at the material surface—provoking electrostatic interaction forces—are attractive for the first cell encounter. This plasma-chemical nanocoating can be used for several biomaterials in orthopedic and dental implantology like titanium, titanium alloys, calcium phosphate scaffolds, and polylactide fiber meshes produced by electrospinning. In this regard, we wanted to ascertain whether plasma polymerized allylamine (PPAAm) is also suitable for increasing the attractiveness of a ceramic surface for dental implants using Yttria-stabilized tetragonal zirconia

Gas-discharge plasma-assisted functionalization of titanium implant surfaces

A crucial factor for in-growth of metallic implants in the bone stock is the rapid cellular acceptance whilst prevention of bacterial adhesion on the surface. Such contradictorily adhesion events could be triggered by surface properties. There already exists fundamental knowledge about the influence of physicochemical surface properties like roughness, titanium dioxide modifications, cleanness, and (mainly ceramic) coatings on cell and microbial behavior in vitro and in vivo. The titanium surface can be equipped with antimicrobial properties by plasma-based copper implantation, which allows the release and generation of small concentrations of copper ions during contact with water-based biological liquids. Additionally, the titanium surface was equipped with amino groups by the deposition of an ultrathin plasma polymer. This coating on the one hand does not significantly reduce the generation of copper ions, and on the other hand improves the adhesion and spreading of osteoblast cells. The process development was accompanied by physicochemical surface analyses like XPS, FTIR, contact angle, SEM, and AFM. Very thin modified layers were created, which are resistant to hydrolysis and delamination. These titanium surface functionalizations were found to have either an antimicrobial activity or cell-adhesive properties. Intramuscular implantation of titanium samples coated with the cell-adhesive plasma polymer in rats revealed a reduced inflammation reaction compared to uncoated titanium. © (2010) Trans Tech Publications

Differenzierte Untersuchung der lokalen und systemischen Entzündungsreaktionen nach Implantation von Niedertemperatur- Plasmapolymer-modifizierten Titanplättchen im Tiermodell Ratte

Ziel der Arbeit war die Untersuchung der lokalen und systemischen Entzündungsreaktionen nach intramuskulärer Implantation von Niedertemperatur-Plasmapolymer-modifizierten Titanplättchen (Ti) im Tiermodell Ratte. Ausgangspunkt dafür waren vorherige Zellkultur-Untersuchungen zu Ti-Proben mit einer positiv geladenen Schicht aus plasma-polymerisiertem Allylamin (PPAAm) beziehungsweise einer negativ geladenen Schicht aus plasma-polymerisierter Acrylsäure (PPAAc). Diese In-vitro-Studien ergaben für eine Beschichtung mit PPAAm positive Effekte auf das Wachstum von Osteoblasten sowie für eine Beschichtung mit PPAAc auf die osteogene Differenzierung humaner mesenchymaler Stammzellen. Für die darauf aufbauenden in dieser Arbeit durchgeführten In-vivo-Untersuchungen war es zunächst notwendig, eine Methode zur quantitativen histologischen Untersuchung der lokalen Gewebsreaktionen nach Implantation von Ti-Proben zu etablieren. Deren Evaluierung zeigte eine gute Reproduzierbarkeit der Ergebnisse und damit die Eignung des Bildanalyse-Verfahrens für die weiteren Untersuchungen. Anschließend erfolgten unter Anwendung dieser Methode morphometrische immunhistochemische Untersuchungen der lokalen Entzündungsantwort nach intramuskulärer Implantation von PPAAc- und PPAAm-beschichteten Ti-Plättchen. Hierzu wurden in einer ersten Studie für beide Beschichtungen die Gesamt-Monozyten und -Makrophagen sowie die MHC-Klasse-II-positiven antigen-präsentierenden Zellen im Periimplantatgewebe nach 56 Tagen Implantationsdauer im Vergleich zu unbeschichteten Kontrollproben untersucht. Dabei ergab sich für die PPAAc-beschichteten Ti-Plättchen im Vergleich zu PPAAm-beschichteten Implantaten und Kontrollen eine verstärkte chronische Entzündungsreaktion. Aufgrund dieser Ergebnisse wurden in einer anschließenden zweiten Studie für drei verschiedene PPAAm-Beschichtungen, die sich hinsichtlich der Plasmaprozessparameter im Duty cycle unterschieden, die Gewebsreaktionen im Kurz- und Langzeitverlauf analysiert. Dafür erfolgte eine Untersuchung der Gesamt-Monozyten und -Makrophagen, der gewebsständigen Makrophagen, der T Lymphozyten und der MHC-Klasse-II-positiven antigen-präsentierenden Zellen 7, 14 und 56 Tage nach Implantation für diese drei PPAAm-Varianten im Vergleich zu Kontrollen. Im Ergebnis waren die lokalen Gewebsreaktionen für die zwei PPAAm-Varianten mit dem höheren Duty cycle im Langzeitverlauf schwächer ausgeprägt als für die PPAAm-Schicht mit dem geringen Duty cycle und die Kontrollen. Dieses Ergebnis stand im Einklang mit entsprechenden Unterschieden in den physikochemischen Schichteigenschaften wie zum Beispiel der Schichtdicke, der Aminogruppendichte und der Proteinadsorption. Darüber hinaus wurden das Serumprofil und die Korrelationen der pro-inflammatorischen Zytokine IL-2 und IFNγ sowie der anti-inflammatorischen Zytokine IL-4 und IL-10 vor sowie wöchentlich für 56 Tage nach Implantation von PPAAc-und PPAAm-beschichteten Implantaten sowie Kontrollen analysiert. Diese Untersuchungen ergaben für die PPAAc-Gruppe in der Spätphase einen gegensätzlichen Verlauf von IL-4 und IL-10 sowie abweichende Korrelationen des IL-10 mit den anderen untersuchten Zytokinen, während in der PPAAm-Gruppe die systemischen Reaktionen und die Korrelationen zwischen den untersuchten Zytokinen mit den Befunden in der Kontrollgruppe vergleichbar waren. Die Gesamtbetrachtung der in dieser Arbeit erhobenen In-vivo-Ergebnisse mit den vorherigen In-Vitro-Befunden zeigt, dass eine positiv geladene PPAAm-Beschichtung einen vielversprechenden Ansatz zur Erzeugung von zelladhäsiven Implantatoberflächen mit dem Ziel einer Verbesserung des Einwachsens von Ti-Implantaten darstellt. Darüber hinaus konnte für die PPAAm-Beschichtung gezeigt werden, dass Variationen in den Plasmaprozessparametern zu Unterschieden in den physikochemischen Eigenschaften und den daraus resultierenden In-vivo-Gewebsreaktionen führen. Die Ergebnisse der Arbeit wurden in vier wissenschaftlichen Fachzeitschriften veröffentlicht (Walschus et al. 2011 J Microsc 242:94–99; Schröder et al. 2010 J Adh Sci Technol 24:1191–1205; Hoene et al. 2010 Acta Biomater 6:676–683; Walschus et al. 2012 J Mater Sci Mater Med 23:1299–1307).The aim of the thesis was an examination of the local and systemic inflammatory reactions following intramuscular implantation of titanium (Ti) plates modified with low-temperature plasma-polymer processes. This originated from previous cell culture experiments with Ti samples coated either with a positively charged plasma-polymerized allylamine film (PPAAm) or a negatively charged plasma-polymerized acrylic acid film (PPAAc). These in vitro studies demonstrated positive effects for a coating with PPAAm regarding the growth of osteoblasts and for a coating with PPAAc regarding the differentiation of human mesenchymal stem cells. For the in vivo evaluation performed in this thesis, it was basically necessary to establish a method for quantitative histological examination of the local tissue reactions after implantation of Ti samples. Its evaluation showed good reproducibility of the obtained results and thus the suitability of this digital image procedure for the subsequent investigations. Using this method, a morphometric immunohistological examination of the local inflammatory tissue response after intramuscular implantation of PPAAc- and PPAAm-coated Ti plates was performed. In a first study, the total monocytes and macrophages as well as MHC class II antigen-presenting cells were examined in the peri-implant tissue after 56 days for both coatings in comparison to uncoated control samples. The results demonstrated a stronger chronic inflammatory response for PPAAc-coated Ti plates compared to PPAAm-coated samples and controls. Based on these findings, the short- and long-term tissue reactions were analysed in a second study for three different PPAAm coatings varying in their plasma process parameters regarding the duty cycle. Total monocytes and macrophages, tissue macrophages, T lymphocytes and MHC class II antigen-presenting cells were evaluated for these PPAAm variants after 7, 14 and 56 days of implantation in comparion to controls. It was found that the long term local tissue reactions following implantation of PPAAm-coated Ti plates were lower for the two PPAAm variations which had a higher duty cycle than for the PPAAm variant with the low duty cycle and the controls. This result was in accordance with differences in the physico-chemical film properties such as layer thickness, amino group density and protein adsorption. Additionally, the serum profile and correlations of the pro-inflammatory cytokines IL-2 and IFNγ and the anti-inflammatory cytokines IL-4 and IL-10 after implantation of PPAAc- and PPAAm-coated Ti samples as well as controls were quantified before and weekly for 56 days after implantation. These experiments revealed for the PPAAc group contrary changes in the time course of IL-4 and IL-10 in the late phase as well as divergent correlations of IL-10 with the other examined cytokines, while the PPAAm group was comparable to the control group regarding the systemic reactions and the correlations between the different cytokines. Taken together with the previous in vitro data, these in vivo results indicate that a positively charged PPAAm film is a promising approach for generation of cell-adhesive implant surfaces aimed at improving the ingrowth of Ti implants. Furthermore, it could be demonstrated for the PPAAm coating that variations of the plasma process parameters cause differences of the physico-chemical properties and the resulting in vivo tissue reactions. The results of this thesis have been published in four scientific journals (Walschus et al. 2011 J Microsc 242:94–99; Schröder et al. 2010 J Adh Sci Technol 24:1191–1205; Hoene et al. 2010 Acta Biomater 6:676–683; Walschus et al. 2012 J Mater Sci Mater Med 23:1299–1307)

Determination of Glutamic Acid Decarboxylase (GAD65) in Pancreatic Islets and Its In Vitro and In Vivo Degradation Kinetics in Serum Using a Highly Sensitive Enzyme Immunoassay

Glutamic acid decarboxylase GAD65 autoantibodies (GADA) are an established marker for autoimmune diabetes. Recently, the autoantigen GAD65 itself was proposed as biomarker of beta-cell loss for prediction of autoimmune diabetes and graft rejection after islet transplantation. Therefore, the GAD65 content in pancreatic islets of different species and its serum degradation kinetics were examined in this study using a sensitive immunoassay. GAD65 was found in quantities of 78 (human), 43.7 (LEW.1A rat) and 37.4 (BB/OK rat) ng per 1,000 islets, respectively, but not in mouse islets. The in vitro half-life of porcine GAD65 and human recombinant GAD65 ranged from 1.27 to 2.35 hours at 37°C in human serum, plasma and blood, and was unaffected by presence of GAD65 autoantibodies. After injecting 2,000 ng recombinant human GAD65 into LEW.1A rats, the in vivo half-life was 2.77 hours. GAD65 was undetectable after 24 hours in these animals, and for up to 48 hours following diabetes induction by streptozotocin in LEW.1A rats. Estimated from these data, at least 13 islets in rat and 1,875 in human must be simultaneously destroyed to detect GAD65 in circulation. These results should be taken into consideration in further studies aimed at examining the diagnostic relevance of GAD65

Acute and Chronic Local Inflammatory Reaction after Implantation of Different Extracellular Porcine Dermis Collagen Matrices in Rats

Two cross-linked acellular porcine dermal collagen matrices (Permacol and NRX) were implanted into rats and the acute and chronic local inflammatory tissue reactions were investigated after 7, 14, 28, and 112 days. Both membranes were stable in vivo for up to 112 days. All investigated immune cell populations (CD68+ macrophages, CD163+ macrophages, T lymphocytes, MHC class II positive cells, mast cells, and NK cells) were present. Their amount decreased significantly over time compared to day 7 after implantation. A change from an acute to a chronic inflammation and an associated shift from proinflammatory M1-like to anti-inflammatory M2-like macrophages were observed. In the early phase there was a significant correlation of T cells to CD68+ (M1-like) macrophages, whereas in the chronic phase T lymphocytes were positively correlated with CD163+ (M2-like) macrophages. The material NRX showed an enhanced inflammatory reaction in comparison to Permacol possibly caused by material characteristics such as a twofold higher thickness of the membrane, roughness, and water absorption capacity. Nevertheless, a more pronounced regenerative process as, for example, indicated by nestin expression demonstrated its possible suitability for applications as wound repair material

Local Inflammatory Response after Intramuscularly Implantation of Anti-Adhesive Plasma-Fluorocarbon-Polymer Coated Ti6AI4V Discs in Rats

Orthopaedic implants and temporary osteosynthesis devices are commonly based on Titanium (Ti). For short-term devices, cell-material contact should be restricted for easy removal after bone healing. This could be achieved with anti-adhesive plasma-fluorocarbon-polymer (PFP) films created by low-temperature plasma processes. Two different PFP thin film deposition techniques, microwave (MW) and radiofrequency (RF) discharge plasma, were applied to receive smooth, hydrophobic surfaces with octafluoropropane (C3F8) or hexafluorohexane (C6F6) as precursors. This study aimed at examining the immunological local tissue reactions after simultaneous intramuscular implantation of four different Ti samples, designated as MW-C3F8, MW-C6F6, RF-C3F8 and Ti-controls, in rats. A differentiated morphometric evaluation of the inflammatory reaction was conducted by immunohistochemical staining of CD68+ macrophages, CD163+ macrophages, MHC class II-positive cells, T lymphocytes, CD25+ regulatory T lymphocytes, NK cells and nestin-positive cells in cryosections of surrounding peri-implant tissue. Tissue samples were obtained on days 7, 14 and 56 for investigating the acute and chronical inflammation (n = 8 rats/group). Implants with a radiofrequency discharge plasma (RF-C3F8) coating exhibited a favorable short- and long-term immune/inflammatory response comparable to Ti-controls. This was also demonstrated by the significant decrease in pro-inflammatory CD68+ macrophages, possibly downregulated by significantly increasing regulatory T lymphocytes

Surface-Coated Polylactide Fiber Meshes as Tissue Engineering Matrices with Enhanced Cell Integration Properties

Poly(L-lactide-co-D/L-lactide)-based fiber meshes resembling structural features of the native extracellular matrix have been prepared by electrospinning. Subsequent coating of the electrospun fibers with an ultrathin plasma-polymerized allylamine (PPAAm) layer after appropriate preactivation with continuous O2/Ar plasma changed the hydrophobic nature of the polylactide surface into a hydrophilic polymer network and provided positively charged amino groups on the fiber surface able to interact with negatively charged pericellular matrix components. In vitro cell experiments using different human cell types (epithelial origin: gingiva and uroepithelium; bone cells: osteoblasts) revealed that the PPAAm-activated surfaces promoted the occupancy of the meshes by cells accompanied by improved initial cell spreading. This nanolayer is stable in its cell adhesive characteristics also after γ-sterilization. An in vivo study in a rat intramuscular implantation model demonstrated that the local inflammatory tissue response did not differ between PPAAm-coated and untreated polylactide meshes