Claudia Bergemann

Osteoblast response to biomimetically altered titanium surfaces

Bioinert titanium (Ti) materials are generally encapsulated by fibrous tissue after implantation into the living body. To improve the bone-bonding ability of Ti implants, we activated commercially pure titanium (cpTi) by a simple chemical pre-treatment in HCl and NaOH. Subsequently, we exposed the treated samples to simulated body fluid (SBF) for 2 (TiCT) and 14 days (TiHCA), respectively, to mimic the early stages of bone bonding and to investigate the in vitro response of osteoblasts on thus altered biomimetic surfaces. Sample surfaces were characterized by scanning electron microscopy, energy-dispersive X-ray analysis, cross-sectional transmission electron microscopy analyses, Fourier transform infrared and Raman spectroscopy. It was shown that the efflorescence consisting of sodium titanate that is present on pre-treated cpTi surfaces transformed to calcium titanate after 2 days in SBF. After 14 days in SBF a homogeneous biomimetic apatite layer precipitated. Human osteoblasts (MG-63) revealed a well spread morphology on both functionalized Ti surfaces. On TiCT, the gene expression of the differentiation proteins alkaline phosphatase (ALP) and bone sialo protein was increased after 2 days. On both TiCT and TiHCA, the collagen I and ALP expression on the protein level was enhanced at 7 and 14 days. The TiCT and the TiHCA surfaces reveal the tendency to increase the differentiated cell function of MG-63 osteoblasts. Thus, chemical pre-treatment of titanium seems to be a promising method to generate osteoconductive surfaces.

Binding of galectin-1 (gal-1) on trophoblast cells and inhibition of hormone production of trophoblast tumor cells in vitro by gal-1

Galectin-1 (gal-1), a member of the mammalian β-galactoside-binding proteins, recognizes preferentially Galβ1–4GlcNAc sequences of oligosaccharides associated with several cell surface glycoconjugates. As demonstrated histochemically, the lectin recognizes appropriate glycoepitopes on the syncytiotrophoblast and on chorionic carcinoma cells (BeWo). Freshly isolated trophoblast cells and trophoblast tumor cells Jeg3 did not bind gal-1. BeWo cells in contrast to Jeg3 form a syncytium in vitro and synthesize progesterone as well as hCG. BeWo cells were used as an approach to study the effects of gal-1 on hormone production. The lectin decreased cellular hCG and progesterone production as well as hCGβ gene transcription as measured by real-time RT-PCR. Gal-1 mediated inhibition of cellular progesterone production was reduced in the presence of a Thomsen-Friedenreich (TF)–polyacrylamide conjugate. Inhibition of cellular hCG and progesterone production was also induced by anti-TF monoclonal antibodies. The results demonstrate that ligation of Galβ1–4GlcNAc and Galβ1–3GalNAc (TF) epitopes on BeWo cells may have regulatory effects on hCG and progesterone production.

Capability of Differently Charged Plasma Polymer Coatings for Control of Tissue Interactions with Titanium Surfaces

Titanium surfaces were equipped with positively and negatively charged chemical functional groups by plasma polymerization. Their capability to influence the adhesion of human mesenchymal stem cells (hMSCs) and inflammation processes was investigated on titanium substrates, which are representative of real implant surfaces. For these purposes, titanium samples were coated with plasma polymers from allylamine (PPAAm) and acrylic acid (PPAAc). The process development was accompanied by physicochemical surface analysis using XPS, FT-IR and contact angle measurements. Very thin plasma polymer coatings were created, which are resistant to hydrolysis and delamination. Positively charged amino groups improve considerably the initial adhesion and spreading steps of hMSCs. PPAAm and PPAAc surfaces have an effect on the differentiation of hMSCs, e.g., the expression of osteogenic markers in dependence on culturing conditions. Acrylic acid groups appear to stimulate early mRNA differentiation markers (ALP, COL, Runx2) under basal conditions, whereas positively and negatively charged groups both stimulate late differentiation markers, like BSP and OCN, after 3 days of osteogenic stimulation. Long-term intramuscular implantation in rats revealed that PPAAc surfaces caused significantly stronger reactions by macrophages and antigen-presenting cells compared to untreated control (polished titanium) samples, while PPAAm films did not show a negative influence on the inflammatory reaction after implantation.

Stimulation of hCG protein and mRNA in first trimester villous cytotrophoblast cells in vitro by glycodelin A

Abstract Aim: Human chorionic gonadotropin (hCG) is produced by fetal trophoblast cells and secreted into maternal circulation mainly in the first trimester of pregnancy. Another glycoprotein, glycodelin A, is one of the main products of the maternal decidua during this period. The purpose of this study was to investigate the effect of glycodelin A on hCG release by isolated cytotrophoblast cells in vitro. Methods: Cytotrophoblast cells were prepared from human first trimester placenta and incubated with varying concentrations of glycodelin A. Supernatants were assayed for hCG protein concentrations, and quantification of beta hCG mRNA was carried out by RT-PCR. Expression of hCG was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry. Results: Glycodelin A induces a dose-dependent increase of hCG production. An increase of hCG expression was measured at 100 and 200 μg/mL glycodelin-A treatment in trophoblast cell culture by TaqMan assay on mRNA level. We found a moderate staining of hCG in control trophoblast cells, whereas a strong hCG staining was seen in glycodelin A-treated trophoblast cells. Conclusions: HCG is a marker for the differentiation process of trophoblast cells. Our results suggest that glycodelin A secreted by the decidualized endometrium is involved in the regulation of hormones produced by the trophoblast.

Persistent effectivity of gas plasma-treated, long time-stored liquid on epithelial cell adhesion capacity and membrane morphology.

Research in plasma medicine includes a major interest in understanding gas plasma-cell interactions. The immediate application of gas plasma in vitro inhibits cell attachment, vitality and cell-cell contacts via the liquid. Interestingly, in our novel experiments described here we found that the liquid-mediated plasma effect is long-lasting after storage up to seven days; i. e. the liquid preserves the characteristics once induced by the argon plasma. Therefore, the complete Dulbeccos Modified Eagle cell culture medium was argon plasma-treated (atmospheric pressure, kINPen09) for 60 s, stored for several days (1, 4 and 7 d) at 37°C and added to a confluent mouse hepatocyte epithelial cell (mHepR1) monolayer. Impaired tight junction architecture as well as shortened microvilli on the cell membrane could be observed, which was accompanied by the loss of cell adhesion capacity. Online-monitoring of vital cells revealed a reduced cell respiration. Our first time-dependent analysis of plasma-treated medium revealed that temperature, hydrogen peroxide production, pH and oxygen content can be excluded as initiators of cell physiological and morphological changes. The here observed persisting biological effects in plasma-treated liquids could open new medical applications in dentistry and orthopaedics.

Glycodelin A and differentiation of first trimester trophoblast cells in vitro

AimThe glycoprotein, glycodelin A (GdA) is a main product of the maternal decidua in the first trimester of pregnancy and is secreted into the amniotic fluid. The purpose of this study was to investigate the effect of GdA on secretion and surface markers of isolated first trimester trophoblasts in vitro.MethodsCytotrophoblasts were prepared from human first trimester placentae and incubated with varying concentrations of GdA or transfected separately with the expression plasmid of GdA. Supernatants were assayed for human chorionic gonadotropin (hCG) protein concentrations. Expression of human placental lactogen (hPL), mucin 1 (MUC1) and the Thomsen–Friedenreich (TF) epitope was analysed in stimulated trophoblast cells and in unstimulated controls by immunocytochemistry.ResultsGlycodelin A induced a reduced expression of hPL compared with unstimulated controls. Expression of MUC1 was not affected by GdA. Freshly isolated trophoblast cells showed no TF expression but became positive for this antigen after 96 h of cultivation. GdA-stimulated trophoblast cells inhibited TF expression after 96 h of cultivation. GdA plasmids induced a significantly higher hCG production in transfected cells than in cells transfected with the empty plasmid.ConclusionsThe results obtained in this study suggest that GdA is involved in the differentiation of trophoblast cells. The treatment of GdA plasmid transfected trophoblast cells stimulated hCG production in isolated trophoblast cells and inhibited hPL and TF expression, suggesting a functional link between hCG and GdA.

A Dual Role of Copper on the Surface of Bone Implants

To stimulate bone regeneration, the design of bioactive implants is a great challenge in current orthopedic research. We reasoned that implants should be suitable both to stimulate osteogenic differentiation of mesenchymal stem cells and prevent infections at the site of implantation. Therefore, we focus on copper ions, which are known to exert antimicrobial effects. On the other hand, copper is essential for the cell physiology, including the formation of the extracellular matrix. We studied the influence of copper ions on mesenchymal stem cells at various concentrations and identified the limits of copper concentrations for cell survival. Below the critical concentration for cell survival we analysed proliferation and osteogenic differentiation of the cells in the presence of copper ions. We found that copper stimulated the proliferation of the mesemchymal stem cells at 0.1 mM. Osteogenic differentiation decreased after 14 days at a concentration of 0.05 - 0.1 mM copper ions in osteogenic medium measured by the expression of osteogenic proteins, like alkaline phosphatase (ALP), bone sialoprotein (BSP) and collagen I (COL). We argue that at the implant surface a higher concentration of copper could prevent biofilm formation of bacteria and physiological concentrations in the vicinity of the implant would stimulate stem cell expansion. Together, copper is an interesting agent to control both bacteria and stem cells in the field of implant technology.

Osteoblast Behavior In Vitro in Porous Calcium Phosphate Composite Scaffolds, Surface Activated with a Cell Adhesive Plasma Polymer Layer

Synthetic materials such as bone substitutes are permanently under development for applications in orthopedic and trauma surgery. Our porous scaffolds were produced from ß-tricalcium phosphate (TCP) using the three dimensional (3D)-printing technology. After sintering the porosity and the pore size of the 3D printed scaffolds reached nearly 50 % and 500 µm, respectively. TCP scaffolds were additionally stabilized by infiltration with polylactic acid (PLA). Because PLA usually impeded cell adhesion we activated the composite surface with plasma polymerized allylamine in a low temperature plasma process. For cell investigations inside the scaffold we used a module system, where two porous discs can be horizontally fixed within a clamping ring. Thereby a 3D cell culture module with four levels and a maximal height of 10 mm was generated. Human MG-63 osteoblasts (ATCC) were seeded apically and placed in serum-containing DMEM. After 14 days of a static cell culture the cell ingrowth and mobility was analyzed by scanning electron microscopy. Osteoblasts initial adhesion and short time occupation of the surface is significantly improved on plasma polymer activated TCP surfaces, which could be a precondition for an enhanced colonization inside a calcium phosphate scaffold. Interestingly, the plasma functionalization of the pure TCP scaffold was possible and successful concerning cell acceptance.

A novel modular device for 3-D bone cell culture and non-destructive cell analysis.

Synthetic materials have emerged as bone substitutes for filling bone defects of critical sizes. Because bone healing requires a mechanically resistant matrix (scaffold) attractive to osteogenic cells and must allow revascularization for nutrient and oxygen supply, scaffold-based strategies focus on the further development of chemical and physical qualities of the material. Cellular ingrowth towards the scaffold center is critical; therefore selective information from inner regions, in particular from the central part, is essential. In this paper we introduce a novel modular in vitro system for three-dimensional (3-D) in vitro bone cell cultures. This 3-D system is developed exclusively for in vitro research purposes, with special emphasis on the geometrical scaffold design (pore size, pore design). The system is composed of a stack of titanium slices which are mounted on a clamp and which enable the separate monitoring of cell growth patterns on every single slice of the slide stack. In this way we are able to gain selective information about the regulation of the cell physiology in the inner part of the 3-D construct which can be used for the development of an optimized scaffold design for orthopedic implants.

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.