Biokompatibilität II: Grenzflächen

V49

Osteoklastäre Resorption Strontium-haltiger Calciumphosphatzemente

*M. Schumacher¹, A. Wagner², A. Bernhardt¹, S. Wenisch², M. Gelinsky¹

¹Technische Universität Dresden, Zentrum für translationale Knochen, Gelenk und Weichgewebeforschung, Dresden, Deutschland

²Justus-Liebig Universität Gießen, Institut für Veterinär-Anatomie,-Histologie und -Embryologie, Gießen, Deutschland

Einleitung:

Als third generation biomaterials gelten Implantatmaterialien, die aufgrund von Zell-Material Interaktionen eine spezifische zelluläre Antwort hervorrufen und damit über eine reine Biokompatibilität hinaus die Heilung von Gewebedefekten positiv beeinflussen. Dazu zählen Knochenzemente, die durch Freisetzung biologisch wirksamer Ionen, z.B. Strontium-Ionen (Sr²⁺), Einfluss auf die Knochendefektheilung nehmen. Eine Stimulation osteogener Zellen und damit der Knochenneubildung sowie eine gleichzeitige Hemmung der osteoklastären Resorption durch Sr²⁺ wurde bereits in zahlreichen Studien gezeigt¹, wird in der Osteoporosetherapie genutzt und führte zur Entwicklung Srhaltiger Calciumphosphat-Knochenzemente (Sr-CPC), deren stimulierende Wirkung auf osteogene Zellen in vitro sowie auf die Knochenneubildung in vivo nachgewiesen werden konnte.^2,3 In der vorliegenden Arbeit wurde nun der Einfluss eines Sr-CPC auf die Osteoklastogenese sowie die zelluläre Resorption in vitro untersucht.

Materialien und Methoden:

Sr-CPC wurde auf Basis eines a-Tricalciumphosphat-basierten Zementsystems (CPC, InnoTERE GmbH) durch Substitution des im Precursor-Pulver enthaltenen CaCO₃ durch SrCO₃ hergestellt. Osteoklastäre Zellen wurden aus primären humanen Monozyten durch Stimulation mit RANKL und M-CSF generiert und anhand spezifischer Marker biochemisch (TRAP, CAII) und molekulargenetisch (TRAP, CAII, CTSK, CX43, MMP9) analysiert sowie die Resorption quantifiziert (REM).

Ergebnisse und Diskussion:

Unabhängig von der Sr-Modifizierung der Zemente wurde die Bildung osteoklastärer Zellen auf den Zementen gezeigt; Sr²⁺ beeinflusste jedoch die Ausprägung osteoklastärer Marker wie TRAP und CAII. Die Resorptionsaktivität auf Sr-haltigen Zementen war gegenüber der Sr-freien Referenz signifikant reduziert (Abb. 1). Somit konnte erstmals eine direkte, inhibierende Wirkung von SrCPC auf die zelluläre Resorption nachgewiesen werden. Die beschriebenen SrCPC stellen daher eine vielversprechende Option für die Behandlung z.B. osteoporotischer Knochendefekte dar.

Referenzen

¹P. J. Marie et al., Bone, 2007

²M. Schumacher et al., Acta Biomater, 2013

³M. Schumacher et al., J Mater Chem B, 2015

Danksagung:

Diese Arbeit wurde von der Deutschen Forschungsgemeinschaft (DFG) gefördert (SFB/Transregio 79).

Abbildung 1

V50

Biomimetic materials for biophysical regulation of human hematopoietic stem cells

*C. Lee-Thedieck¹

¹Karlsruhe Institute of Technology (KIT), Institute of Functional Interfaces, Eggenstein-Leopoldshafen, Deutschland

Introduction:

The only stem cells that are nowadays used routinely in the clinics are hematopoietic stem cells (HSCs). Since more than 50 years these cells are transplanted to treat patients suffering from hematological disorders. This clinical application of HSCs is restricted by their limited availability. Therefore, a targeted proliferation of HSCs in the laboratory would be beneficial for tens of thousands of patients. The underlying problem in this purpose is the fast onset of differentiation during the in vitro culture of HSCs. In vivo HSCs are mainly found in specific stem cell niches in the red bone marrow. These niches are unique microenvironments that provide signals to the HSCs that allow their maintenance and self-renewal. During the last years it became clear that besides the biochemical composition of the environment also its physical properties such as substrate nanostructure, elasticity and architecture are able to influence cell behaviour. We aim to understand the mutual biological and physical interactions of HSCs with their niches and to use this knowledge in order to get closer to the vision of an artificial biomimetic stem cell niche that allows the targeted in vitro expansion and differentiation of HSCs.

Materials and Methods:

By using materials sciences techniques (including block copolymer micellar nanolithography and poly(ehtylene glycol) hydrogels to address biological questions we study the influence of nanostructure, mechanical properties and architecture (2D versus 3D) of the environment on HSC adhesion, migration, gene expression, differentiation and proliferation.

Results and Discussion:

We could show that HSC adhesion, signal transduction and gene expression depend on the precise nanometer-scaled distance between integrin ligands by applying nanopatterned, bio-functionalized gold nanoparticle arrays. By using PEG hydrogels with tunable mechanical properties we studied the role of substrate stiffness in the HSC niche and developed a new model how matrix stiffness could play a role as a modulatory signal during physiological processes such as egress of HSCs from the niche. The development of a biomimetic 3D scaffold allowed us to investigate how the three-dimensional architecture of the environment and the support by mesenchymal stem/stromal cells impact HSC proliferation and differentiation. In future studies we will focus on the further development of 3D cell culture systems and multifunctional nanostructured biomaterials for HSCs with the vision to gain one day the ability to design a functional biomimetic stem cell niche for HSC proliferation.

V51

Enhanced adhesion of endothelial cells and osteoblasts by surface immobilized matrix proteins onto titanium using plasma polymerization

*M. Heller¹, P. W. Kämmerer², B. Al-Nawas^1,3, M.- A. Luzspinski^1,4, R. Förch⁵, J. Brieger^1,4

¹Universitätsmedizin Mainz, Biomatics, Mainz, Deutschland

²Universität, MKG Chirurgie , Rostock, Deutschland

³Universitätsmedizin, MKG Chirurgie, Mainz, Deutschland

⁴Universitätsmedizin, HNO-Klinik, Mainz, Deutschland

⁵Frauenhofer, ICT-IMM, Mainz, Deutschland

Introduction - Biomimetic surface modifications are regarded as promising approach to stimulate cellular behavior at the interface of implant materials. Aim of the study was an evaluation of the cellular response of human umbilical cord cells (HUVECS) and human osteoblasts (HOBS) on titanium covalently coated with the extracellular matrix (ECM)-proteins fibronectin, collagen, laminin and osteopontin.

Material and Methods - For the surface modification titanium discs were first amino-functionalized by plasma polymerization of allylamine. The ECM protein conjugation was performed using the linker molecule α, ω-bis-N-hydroxysuccinimide polyethylene glycol (Di-NHS linker). For surface characterization, infrared spectroscopy and fluoresceineisothiocyanate staining (FITC) were utilized in order to evaluate the presence and distribution of primary amines in the plasma polymer film. Real-time analyses of the respective protein conjugation processes were performed via SPR kinetic measurements. For the evaluation of the biological functionality of the immobilized ECM proteins, the cellular response of HUVECS and HOBS was analyzed quantitatively calculating the surface coverage via imageJ software.

Results and Discussion - All ECM-proteins were immobilized successfully. Furthermore, the biological functionality of the conjugated factors fibronectin and collagen could be proven as they led to a distinct stimulation of cell adhesion of HUVECS and HOBS when compared to the control group. The highest cell coverage of HUVECS was observed on fibronectin-modified surfaces with approximately 35% and on collagen with 33% after 24 hours (PT: 9,4%). For laminin, no additional effect was observed, and for osteopontin only a slight enhancement of cell adhesion was found. A similar, cell stimulating tendency of fibronectin and collagen was seen as well after 3 and 7 days. Biomimetic surface modification via plasma polymerization is a powerful method for biomolecule conjugation with a high retention of biological functionality and offer promising clinical perspectives.

V52

Heparin-based coatings on polymer surfaces, coupling of cell adhesion peptides, and surface characterization in terms of endothelial cell adhesion and blood compatibility

*K. Borchers¹, M. Dettling¹, K. Kübrich², M. Schandar³, K. Linke³, A. Wenz³

¹Fraunhofer IGB, Grenzflächentechnologie und Materialwissenschaft, Stuttgart, Deutschland

²Universität Stuttgart, Stuttgart, Deutschland

³Fraunhofer IGB, Zellsysteme, Stuttgart, Deutschland

Introduction:

Synthetic materials that come into contact with blood have to be optimized in terms of hemocompatibility. Therefore the potential of biofunctionalization of surfaces with endothelial cells is currently being investigated in order to decrease the thrombogenicity, inflammation reactions, coagulation potential of artificial surfaces.

We have developed biofunctional coatings for polymeric materials which are based on heparin and cell recognition peptides in order to achieve surfaces which promote stable adhesion of endothelial cells.

Materials and Methods:

Two methods of coupling heparin to polymeric surfaces, in particular to polymethylpenten PMP, have been evaluated: Layer-by-layer coating with albumin and heparin (method which is already approved for medicinal application) and light-induced covalent coupling of benzophenon-modified heparin. Benzophenon-modified heparin was synthezised and characterized by ¹H-NMR. The effectiveness of heparin-coating was tested by staining and X-ray photoelectron spectroscopy (XPS). Coupling of peptides to the albumin-heparin-coated surfaces was triggered by 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimid (EDC). Peptide functionalization of benzophenon-modified heparin was achieved by UV-irradiation. Peptide detection was performed using fluorescently labelled peptides. We used a range of peptide sequences, which are discussed to be general cell recognition sites (RGD), endothelial cell specific (REDV), and of low platelet activation level (EILDVPST). We used human microvascular endothelial cells for cell adhesion assays. Dynamic hemocompatibility testing was performed using the Chandler loop technique.

Results and Discussion:

We will present our results on the synthesis of a photo-reactive heparin-derivative, the coupling and stability of heparin-based coatings onto PMP surfaces, and the coupling of peptide sequences to the heparin-coated surfaces. We will show and discuss our investigations on cell adhesion and endothelial layer formation within culture in flow conditions with regard to different cell adhesion peptide sequences. We will present the results on comparative testing of the hemocompatibility of the various coatings in a dynamic setting.

Acknowledgement:

Our work was funded by the European ommission (GA 304932 “AmbuLung”), Fraunhofer and the University of Stuttgart. The dynamic hemocompatibility assay was performed at Klinisches Forschungslabor Thorax-, Herz- und Gefäßchirurgie, Universität Tübingen. We thank C. Schmidt from Hochschule Reutlingen for lab assistance.