Scavenging of Dickkopf-1 by macromer-based biomaterials covalently decorated with sulfated hyaluronan displays pro-osteogenic effects.

Dickkopf-1 (DKK1), a Wnt inhibitor secreted by bone marrow stromal cells (MSC), is known to play an important role in long-term non-union bone fracture defects and glucocorticoid induced osteoporosis. Mitigating its effects in early bone defects could improve osteogenesis and bone defect healing. Here, we applied a biomaterial strategy to deplete a defect environment from DKK1 by scavenging the protein via a macromer-based biomaterial covalently decorated with sulfated hyaluronan (sHA3). The material consisted of cross-copolymerized three-armed macromers with a small anchor molecule. Using the glycidyl anchor, polyetheramine (ED900) could be grafted to the material to which sHA3 was efficiently coupled in a separate step. For thorough investigation of material modification, flat material surfaces were generated by fabricating them on glass discs. The binding capability of sHA3 for DKK1 was demonstrated in this study by surface plasmon resonance measurements. Furthermore, the surfaces demonstrated the ability to scavenge and inactivate pathologic amounts of DKK1 from complex media. In a combinatory approach with Wnt3a, we were able to demonstrate that DKK1 is the preferred binding partner of our sHA3-functionalized surfaces. We validated our findings in a complex in vitro setting of differentiating SaOS-2 cells and primary hMSC. Here, endogenous DKK-1 was scavenged resulting in increased osteogenic differentiation indicating that this is a consistent biological effect irrespective of the model system used. Our study provides insights in the mechanisms and efficiency of sHA3 surface functionalization for DKK1 scavenging, which may be used in a clinical context in the future.

Sustained delivery of siRNA poly- and lipopolyplexes from porous macromer-crosslinked gelatin gels.

RNA interference (RNAi) is a promising technique to treat severe diseases on a pre-protein level. We and others postulate that the release of nanoparticle-complexed small interfering RNA (siRNA) from implanted biomaterials could provide structural support for tissue repair, combined with local siRNA transfection of invading and regenerating cells. In this study, we systematically investigated cross-linked gelatin based hydrogel formulations (cGEL) as degradable controlled release matrices for siRNA. Aiming at the definition of correlations between cGEL composition, siRNA nanoparticle formulation, release kinetics of complexed siRNA and transfection efficiency, we combined five different cGEL formulations and three transfection systems, i.e. polyplexes with polyethyleneimine (PEI), PEI in combination with liposomes (lipopolyplexes) and polyplexes based on tyrosin-modified PEI (P10Y). It was found that the distribution of these poly-/lipopolyplexes, when applied onto the negatively charged hydrogels, was strongly dependent on their zeta potential. Furthermore, siRNA release from the hydrogel was a multifactorial process, as diffusion, hydrogel degradation and nanoparticle decomplexation overlapped over time. This resulted in a prolonged release of siRNA for up to 21days. In the case of PEI complexes and lipopolyplexes, release kinetics depended on the cGEL formulation. In contrast, when employing P10Y polyplexes, an initial burst release was observed with no further release thereafter. Silencing activity was determined using constitutively luciferase-expressing SKOV-3-Luc reporter cells. Surface and bulk porosity in hydrogels was introduced by addition of soluble polyethylene glycol during fabrication, leading to improved knockdown. The rapid onset of knockdown efficacy will also provide the basis for the determination of long-term effects.

The effect of nimodipine, fentanyl and remifentanil intravenous products on the stability of propofol emulsions.

Nimodipine is used parenterally to treat ischemic neurological deficits caused by subarachnoid haemorraghe. Infusion of nimodipine should be continued during anaesthesia, surgery or angiography. In this context a simultaneous administration of nimodipine, propofol and fentanyl or remifentanil could be of great advantage. So the aim of this study was to evaluate the physical stability (droplet size) of propofol emulsions in combination with nimodipine and fentanyl/remifentanil. Droplet size of intravenous emulsions is of particular relevance as the administration of larger droplets to patients may cause pulmonary embolism. So the number of oil droplets > 10 microm was determined in combinations of propofol emulsion with nimodipine and fentanyl/remifentanil immediately after mixing and after 20 hours by using microscopy. The experiments showed that all combinations of propofol (1 and 2%) with nimodipine infusion solution resulted in coalescence of oil droplets, which finally caused a visible phase separation. Macrogol (polyethylene glycol 400) was identified as the component in nimodipine infusion solution which induced the physicochemical incompatibility with propofol lipid emulsions.