Evaluation of dextran(ethylene glycol) hydrogel films for giant unilamellar lipid vesicle production and their application for the encapsulation of polymersomes.

Giant Unilamellar Vesicles (GUVs) prepared from phospholipids are becoming popular membrane model systems for use in biophysical studies. The quality, size and yield of GUVs depend on the preparation method used to obtain them. In this study, hydrogels consisting of dextran polymers crosslinked by poly(ethylene glycol) (DexPEG) were used as hydrophilic frameworks for the preparation of vesicle suspensions under physiological ionic strength conditions. A comparative study was conducted using hydrogels with varied physicochemical properties to evaluate their performance for GUV production. The prepared GUVs were quantified by flow cytometry using the Coulter Principle to determine the yield and size distribution. We find that hydrogels of lower mechanical strength, increased swellability and decreased lipid interaction favour GUV production, while their resulting size is determined by the surface roughness of the hydrogel film. Moreover, we embedded polymersomes into the crosslinked hydrogel network, creating a DexPEG - polymersome hybrid film. The re-hydration of lipids on those hybrid substrates led to the production of GUVs and the efficient encapsulation of polymersomes in the lumen of GUVs.

Reversibly Triggered Protein-Ligand Assemblies in Giant Vesicles.

External small-molecule triggers were used to reversibly control dynamic protein-ligand interactions in giant vesicles. An alcohol dehydrogenase was employed to increase or decrease the interior pH upon conversion of two different small-molecule substrates, thereby modulating the pH-sensitive interaction between a Ni-NTA ligand on the vesicle membrane and an oligohistidine-tagged protein in the lumen. By alternating the small-molecule substrates the interaction could be reversed.

Cascade reactions in multicompartmentalized polymersomes.

Enzyme-filled polystyrene-b-poly(3-(isocyano-L-alanyl-aminoethyl)thiophene) (PS-b-PIAT) nanoreactors are encapsulated together with free enzymes and substrates in a larger polybutadiene-b-poly(ethylene oxide) (PB-b-PEO) polymersome, forming a multicompartmentalized structure, which shows structural resemblance to the cell and its organelles. An original cofactor-dependent three-enzyme cascade reaction is performed, using either compatible or incompatible enzymes, which takes place across multiple compartments.

Semisynthesis of fluorescent metabolite sensors on cell surfaces.

Progress in understanding signal transduction and metabolic pathways is hampered by a shortage of suitable sensors for tracking metabolites, second messengers, and neurotransmitters in living cells. Here we introduce a class of rationally designed semisynthetic fluorescent sensor proteins, called Snifits, for measuring metabolite concentrations on the cell surface of mammalian cells. Functional Snifits are assembled on living cells through two selective chemical labeling reactions of a genetically encoded protein scaffold. Our best Snifit displayed fluorescence intensity ratio changes on living cells significantly higher than any previously reported cell-surface-targeted fluorescent sensor protein. This work establishes a generally applicable and rational strategy for the generation of cell-surface-targeted fluorescent sensor proteins for metabolites of interest.

In vitro and in vivo study on the osseointegration of BCP-coated versus uncoated nondegradable thermoplastic polyurethane focal knee resurfacing implants.

Focal knee resurfacing implants (FKRIs) are intended to treat cartilage defects in middle-aged patients. Most FKRIs are metal-based, which hampers follow-up of the joint using magnetic resonance imaging and potentially leads to damage of the opposing cartilage. The purpose of this study was to develop a nondegradable thermoplastic polyurethane (TPU) FKRI and investigate its osseointegration. Different surface roughness modifications and biphasic calcium phosphate (BCP) coating densities were first tested in vitro on TPU discs. The in vivo osseointegration of BCP-coated TPU implants was subsequently compared to uncoated TPU implants and the titanium bottom layer of metal control implants in a caprine model. Implants were implanted bilaterally in stifle joints and animals were followed for 12 weeks, after which the bone-to-implant contact area (BIC) was assessed. Additionally, 18F-sodium-fluoride (18F-NaF) positron emission tomography PET/CT-scans were obtained at 3 and 12 weeks to visualize the bone metabolism over time. The BIC was significantly higher for the BCP-coated TPU implants compared to the uncoated TPU implants (p = .03), and did not significantly differ from titanium (p = .68). Similar 18F-NaF tracer uptake patterns were observed between 3 and 12 weeks for the BCP-coated TPU and titanium implants, but not for the uncoated implants. TPU FKRIs with surface modifications could provide the answer to the drawbacks of metal FKRIs.

Polymers in Cartilage Defect Repair of the Knee: Current Status and Future Prospects.

Cartilage defects in the knee are often seen in young and active patients. There is a need for effective joint preserving treatments in patients suffering from cartilage defects, as untreated defects often lead to osteoarthritis. Within the last two decades, tissue engineering based techniques using a wide variety of polymers, cell sources, and signaling molecules have been evaluated. We start this review with basic background information on cartilage structure, its intrinsic repair, and an overview of the cartilage repair treatments from a historical perspective. Next, we thoroughly discuss polymer construct components and their current use in commercially available constructs. Finally, we provide an in-depth discussion about construct considerations such as degradation rates, cell sources, mechanical properties, joint homeostasis, and non-degradable/hybrid resurfacing techniques. As future prospects in cartilage repair, we foresee developments in three areas: first, further optimization of degradable scaffolds towards more biomimetic grafts and improved joint environment. Second, we predict that patient-specific non-degradable resurfacing implants will become increasingly applied and will provide a feasible treatment for older patients or failed regenerative treatments. Third, we foresee an increase of interest in hybrid construct, which combines degradable with non-degradable materials.