Endothelial cell spreading on lipid bilayers with combined integrin and cadherin binding ligands.

Endothelial cells play a central role in the vascular system, where their function is tightly regulated by both cell-extracellular matrix (e.g., via integrins) and cell-cell interactions (e.g., via cadherins). In this study, we incorporated cholesterol-modified integrin and N-cadherin peptide binding ligands in fluid supported lipid bilayers. Human umbilical vein endothelial cell adhesion, spreading and vinculin localization in these cells were dependent on ligand density. One composition led to observe a higher extent of cell spreading, where cells exhibited extensive lamellipodia formation and a qualitatively more distinct N-cadherin localization at the cell periphery, which is indicative of N-cadherin clustering and a mimic of cell-cell contact formation. The results can be used to reconstitute the endothelial-pericyte interface on biomedical devices and materials.

Supramolecular Biomaterials in the Netherlands.

Synthetically designed biomaterials strive to recapitulate and mimic the complex environment of natural systems. Using natural materials as a guide, the ability to create high-performance biomaterials that control cell fate, and support the next generation of cell- and tissue-based therapeutics, is starting to emerge. Supramolecular chemistry takes inspiration from the wealth of noncovalent interactions found in natural materials that are inherently complex, and using the skills of synthetic and polymer chemistry, recreates simple systems to imitate their features. Within the past decade, supramolecular biomaterials have shown utility in tissue engineering and the progress predicts a bright future. On this 30th anniversary of the Netherlands Biomaterials and Tissue Engineering society, we briefly recount the state of supramolecular biomaterials in the Dutch academic and industrial research and development context. This review provides the background, recent advances, industrial successes and challenges, as well as future directions of the field, as we see it. Throughout this work, we notice the intricate interplay between simplicity and complexity in creating more advanced solutions. We hope that the interplay and juxtaposition between these two forces can propel the field forward. Impact statement Supramolecular biomaterials based on noncovalent interactions hold the ability to rebuild some of the complexity of natural biomaterials in synthetic systems. While still in its infancy, the field is currently vigorously moving from fundamental experiments toward applications and products in the tissue engineering and regenerative medicine arena. Herein, we review the current state of the field in the Netherlands. While supramolecular biomaterials have incredible potential, systematic studies, balancing complexity and simplicity, efficient translation, and enhanced performance are all required for success of these strategies. As we move the field toward commercial solutions for clinical patients, we must also pay homage and remember the fundamental studies that allow these jumps in innovation.

Macroscopic Supramolecular Assembly Strategy to Construct 3D Biocompatible Microenvironments with Site-Selective Cell Adhesion.

Three-dimensional (3D) scaffolds with chemical diversity are significant to direct cell adhesion onto targeted surfaces, which provides solutions to further control over cell fates and even tissue formation. However, the site-specific modification of specific biomolecules to realize selective cell adhesion has been a challenge with the current methods when building 3D scaffolds. Conventional methods of immersing as-prepared structures in solutions of biomolecules lead to nonselective adsorption; recent printing methods have to address the problem of switching multiple nozzles containing different biomolecules. The recently developed concept of macroscopic supramolecular assembly (MSA) based on the idea of "modular assembly" is promising to fabricate such 3D scaffolds with advantages of flexible design and combination of diverse modules with different surface chemistry. Herein we report an MSA method to fabricate 3D ordered structures with internal chemical diversity for site-selective cell adhesion. The 3D structure is prepared via 3D alignment of polydimethylsiloxane (PDMS) building blocks with magnetic pick-and-place operation and subsequent interfacial bindings between PDMS based on host/guest molecular recognition. The site-specific cell affinity is realized by distributing targeted building blocks that are modified with polylysine molecules of opposite chiralities: PDMS modified with films containing poly-l-lysine (PLL) show higher cell density than those with poly-d-lysine (PDL). This principle of selective cell adhesion directed simply by spatial distribution of chiral molecules has been proven effective for five different cell lines. This facile MSA strategy holds promise to build complex 3D microenvironment with on-demand chemical/biological diversities, which is meaningful to study cell/material interactions and even tissue formation.

The regenerative effect of different growth factors and platelet lysate on meniscus cells and mesenchymal stromal cells and proof of concept with a functionalized meniscus implant.

Meniscus regeneration could be enhanced by targeting meniscus cells and mesenchymal stromal cells (MSCs) with the right growth factors. Combining these growth factors with the Collagen Meniscus Implant (CMI®) could accelerate cell ingrowth and tissue formation in the implant and thereby improve clinical outcomes. Using a transwell migration assay and a micro-wound assay, the effect of insulin-like growth factor-1, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor beta 1 (TGF-ß1), fibroblast growth factor, and platelet lysate (PL) on migration and proliferation of meniscus cells and MSCs was assessed. The formation of extracellular matrix under influence of the above-mentioned growth factors was assessed after 28 days of culture of both MSCs and meniscus cells. As a proof of concept, the CMI® was functionalized with a VEGF binding peptide and coated with platelet-rich plasma (PRP) for clinical application. Our results demonstrate that PDGF, TGF-ß1, and PL stimulate migration, proliferation, and/or extracellular matrix production of meniscus cells and MSCs. Additionally, the CMI® was successfully functionalized with a VEGF binding peptide and PRP which increased migration of meniscus cell and MSC into the implant. This study demonstrates proof of concept of functionalizing the CMI® with growth factor binding peptides. A CMI® functionalized with the right growth factors holds great potential for meniscus replacement after partial meniscectomy.

Magnetic Particles for CTC Enrichment.

Here, we review the characteristics and synthesis of magnetic nanoparticles (MNPs) and place these in the context of their usage in the immunomagnetic enrichment of Circulating Tumor Cells (CTCs). The importance of the different characteristics is explained, the need for a very specific enrichment is emphasized and different (commercial) magnetic separation techniques are shown. As the specificity of an MNP is in a large part dependent on the antibody coated onto the particle, different strategies in the coupling of specific antibodies as well as an overview of the available antibodies is given.

RGD-functionalized supported lipid bilayers modulate pre-osteoblast adherence and promote osteogenic differentiation.

Biomaterial integration into bone requires optimal surface conditions to promote osteoprogenitor behavior, which is affected by integrin-binding via arginine-glycine-aspartate (RGD). RGD-functionalized supported lipid bilayers (SLBs) might be interesting as biomaterial coating in bone regeneration, because they allow integration of proteins, for example, growth factors, cytokines, and/or antibacterial agents. Since it is unknown whether and how they affect osteoprogenitor adhesion and differentiation, the aim was to investigate adhesion, focal adhesion formation, morphology, proliferation, and osteogenic potential of pre-osteoblasts cultured on RGD-functionalized SLBs compared to unfunctionalized SLBs and poly-l-lysine (PLL). After 17 hr, pre-osteoblast density on SLBs without or with RGD was similar, but lower than on PLL. Cell surface area, elongation, and number and size of phospho-paxillin clusters were also similar. Cells on SLBs without or with RGD were smaller, more elongated, and had less and smaller phospho-paxillin clusters than on PLL. OPN expression was increased on SLBs with RGD compared to PLL. Moreover, after 1 week, COL1a1 expression was increased on SLBs without or with RGD. In conclusion, pre-osteoblast adhesion and enhanced differentiation were realized for the first time on RGD-functionalized SLBs, pointing to a new horizon in the management of bone regeneration using biomaterials. Together with SLBs nonfouling nature and the possibility of adjusting SLB fluidity and peptide content make SLBs highly promising as substrate to develop innovative biomimetic coatings for biomaterials in bone regeneration.

Weak Multivalent Binding of Influenza Hemagglutinin Nanoparticles at a Sialoglycan-Functionalized Supported Lipid Bilayer.

Quantification of the multivalent interactions of influenza viruses binding at interfaces may provide ways to tackle key biological questions regarding influenza virulence and zoonoses. Yet, the deconvolution of the contributions of molecular and interfacial parameters, such as valency, interaction area, and receptor density, to the binding of whole viruses is hindered by difficulties in the direct determination of these parameters. We report here a chemical platform technology to study the binding of multivalent recombinant hemagglutinin (rHA) nanoparticles at artificial sialoglycan cell receptor-presenting interfaces in which all these parameters can be derived, thus allowing the desired full and quantitative binding analysis. SiO2 substrates were functionalized with supported lipid bilayers containing a targeted and tunable fraction of a biotinylated lipid, followed by the adsorption of streptavidin and biotinylated polyvalent 2,3- or 2,6-sialyl lactosamine (SLN). rHA nanoparticles were used as a virus mimic to provide a good prediction of the number of interactions involved in binding. Low nanomolar affinities and selectivities for binding at the 2,6-SLN platforms were observed for rHA particles from three different virus variants. When fitting the data to a multivalency model, the nanomolar overall affinity appears to be achieved by 6-9 HA-sugar molecular interaction pairs, which individually present a rapid association/dissociation behavior. This dynamic behavior may be an essential biological attribute in the functioning of the influenza virus.

Microwell Scaffolds Using Collagen-IV and Laminin-111 Lead to Improved Insulin Secretion of Human Islets.

IMPACT STATEMENT: This research deals with finding a proper bioengineering strategy to improve the outcome of islets transplantation for treatment of type 1 diabetes. It is focused on the mimicking of islet extracellular matrix niche in microwell islet delivery devices to improve their endocrine function.

Multivalency in Heteroternary Complexes on Cucurbit[8]uril-Functionalized Surfaces: Self-assembly, Patterning, and Exchange Processes.

The spatial confinement of multivalent azopyridine guest molecules mediated by cucurbit[8]urils is described. Fluorescent dye-labelled multivalent azopyridine molecules were attached to preformed methyl viologen/cucurbit[8]uril inclusion complexes in solution and at surfaces. The formation of the resulting heteroternary host-guest complexes was verified in solution and on gold substrates. Surface binding constants of the multivalent ligands were two orders of magnitude higher than that of the monovalent one. Poly-l-lysine grafted with oligo(ethylene glycol) and maleimide moieties was deposited on cyclic olefin polymer surfaces and further modified with thiolated methyl viologen and cucurbit[8]uril. Defined micrometer-sized patterns were created by soft lithographic techniques. Supramolecular exchange experiments were performed on these surface-bound heterocomplexes, which allowed the creation of cross-patterns by taking advantage of the molecular valency, which led to the substitution of the monovalent guest by the multivalent guests but not vice versa.

Lipid bilayers cushioned with polyelectrolyte-based films on doped silicon surfaces.

Silicon semiconductors with a thin surface layer of silica were first modified with polyelectrolytes (polyethyleneimine, polystyrene sulfonate and poly(allylamine)) via a facile layer-by-layer deposition approach. Subsequently, lipid vesicles were added to the preformed polymeric cushion, resulting in the adsorption of intact vesicles or fusion and lipid bilayer formation. To study involved interactions we employed optical reflectometry, electrochemical impedance spectroscopy and fluorescent recovery after photobleaching. Three phospholipids with different charge of polar head groups, i.e. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used to prepare vesicles with varying surface charge. We observed that only lipid vesicles composed from 1:1 (mole:mole) mixture of DOPC/DOPS have the ability to fuse onto an oppositely charged terminal layer of polyelectrolyte giving a lipid bilayer with a resistance of >100 kΩ. With optical reflectometry we found that the vesicle surface charge is directly related to the amount of mass adsorbed onto the surface. An interesting observation was that zwitterionic polar head groups of DOPC allow the adsorption on both positively and negatively charged surfaces. As found with fluorescent recovery after photobleaching, positively charged surface governed by the presence of poly(allylamine) as the terminal layer resulted in intact DOPC lipid vesicles adsorption whereas in the case of a negatively charged silica surface formation of lipid bilayers was observed, as expected from literature.

Fibronectin and Collagen IV Microcontact Printing Improves Insulin Secretion by INS1E Cells.

IMPACT STATEMENT: This research deals with finding a proper bioengineering strategy for the creation of improved ß-cell replacement therapy in type 1 diabetes. It specifically deals with the microenvironment of ß-cells and its relationship to their endocrine function.

Re- and Preconfigurable Multistable Visible Light Responsive Surface Topographies.

Light responsive materials that are able to change their shape are becoming increasingly important. However, preconfigurable bistable or even multi-stable visible light responsive coatings have not been reported yet. Such materials will require less energy to actuate and will have a longer lifetime. Here, it is shown that fluorinated azobenzenes can be used to create rewritable and pre-configurable responsive surfaces that show multi-stable topographies. These surface structures can be formed and removed by using low intensity green and blue light, respectively. Multistable preconfigured surface topographies can also be created in the absence of a mask. The method allows for full control over the surface structures as the topographical changes are directly linked to the molecular isomerization processes. Preliminary studies reveal that these light responsive materials are suitable as adaptive biological surfaces.

Bioactive Tape With BMP-2 Binding Peptides Captures Endogenous Growth Factors and Accelerates Healing After Anterior Cruciate Ligament Reconstruction.

BACKGROUND: The anterior cruciate ligament (ACL) has poor regenerative capacity, and an injury leads to loss of function, limiting quality of life and increasing the incidence of osteoarthritis. Surgical interventions can stabilize the joint and improve functional recovery. The delivery of growth factors (GFs) enhances the healing process; however, this is complex in its regulation, is high in costs, has side effects, and can only be accomplished with supraphysiological concentrations and thus is currently not clinically feasible. However, the immobilization of a patient's endogenous GFs in biomaterials can overcome these problems. PURPOSE: To develop a method to capture endogenous bone morphogenetic protein-2 (BMP-2) and ultimately show enhanced ACL healing in vivo using this novel methodology. STUDY DESIGN: Controlled laboratory study. METHODS: BMP-2 binding peptides were synthetized, purified, and immobilized on polycaprolactone (PCL) films. The affinity between the peptide and human BMP-2 (hBMP-2) was confirmed with immunofluorescence and enzyme-linked immunosorbent assay. The C2C12 Luc reporter cell line was used to confirm the bioactivity of immobilized BMP-2. For in vivo experiments, the same functionalization technology was applied to the commercially available Polytape, and the functionalized tape was sutured together with the graft used for ACL reconstruction in rats. Each animal underwent reconstruction with either native Polytape (n = 3) or Polytape with BMP-2 binding peptides (n = 3). At 2 and 6 weeks after surgery, the graft was assessed by histology and micro-computed tomography. RESULTS: The covalent immobilization of the peptide in PCL was successful, allowing the peptide to capture hBMP-2, which remained bioactive and led to the osteogenic differentiation of C2C12. In vivo experiments confirmed the potential of the Polytape functionalized with the BMP-2 binding peptide to capture endogenous BMP-2, leading to enhanced bone formation inside the femoral and tibial tunnels and ultimately improving the graft's quality. CONCLUSION: The incorporation of BMP-2 binding peptides into materials used for ACL reconstruction can capture endogenous hBMP-2, which enhances the healing process inside the bone tunnels. CLINICAL RELEVANCE: These results demonstrate the potential of using synthetic peptides to endow biomaterials with novel biological functions, namely to capture and immobilize endogenous GFs.

Hydrolytically Labile Linkers Regulate Release and Activity of Human Bone Morphogenetic Protein-6.

Release of growth factors while simultaneously maintaining their full biological activity over a period of days to weeks is an important issue in controlled drug delivery and in tissue engineering. In addition, the selected strategy to immobilize growth factors largely determines their biological activity. Silica surfaces derivatized with glycidyloxy propyl trimethoxysilane and poly(glycidyl methacrylate) brushes yielded epoxide-functionalized surfaces onto which human bone morphogenetic protein-6 (hBMP-6) was immobilized giving stable secondary amine bonds. The biological activity of hBMP-6 was unleashed by hydrolysis of the surface siloxane and ester bonds. We demonstrate that this type of labile bonding strategy can be applied to biomaterial surfaces with relatively simple and biocompatible chemistry, such as siloxane, ester, and imine bonds. Our data indicates that the use of differential hydrolytically labile linkers is a versatile method for functionalization of biomaterials with a variety of growth factors providing control over their biological activity.

About Chemical Strategies to Fabricate Cell-Instructive Biointerfaces with Static and Dynamic Complexity.

Properly functioning cell-instructive biointerfaces are critical for healthy integration of biomedical devices in the body and serve as decisive tools for the advancement of our understanding of fundamental cell biological phenomena. Studies are reviewed that use covalent chemistries to fabricate cell-instructive biointerfaces. These types of biointerfaces typically result in a static presentation of predefined cell-instructive cues. Chemically defined, but dynamic cell-instructive biointerfaces introduce spatiotemporal control over cell-instructive cues and present another type of biointerface, which promises a more biomimetic way to guide cell behavior. Therefore, strategies that offer control over the lateral sorting of ligands, the availability and molecular structure of bioactive ligands, and strategies that offer the ability to induce physical, chemical and mechanical changes in situ are reviewed. Specific attention is paid to state-of-the-art studies on dynamic, cell-instructive 3D materials. Future work is expected to further deepen our understanding of molecular and cellular biological processes investigating cell-type specific responses and the translational steps toward targeted in vivo applications.

Compartmentalized supramolecular hydrogels based on viral nanocages towards sophisticated cargo administration.

Introduction of compartments with defined spaces inside a hydrogel network brings unique features, such as cargo quantification, stabilization and diminishment of burst release, which are all desired for biomedical applications. As a proof of concept, guest-modified cowpea chlorotic mottle virus (CCMV) particles and complementary guest-modified hydroxylpropyl cellulose (HPC) were non-covalently cross-linked through the formation of ternary host-guest complexes with cucurbit[8]uril (CB[8]). Furthermore, CCMV based virus-like particles (VLPs) loaded with tetrasulfonated zinc phthalocyanine (ZnPc) were prepared, with a loading efficiency up to 99%, which are subsequently successfully integrated inside the supramolecular hydrogel network. It was shown that compartments provided by protein cages not only help to quantify the loaded ZnPc cargo, but also improve the water solubility of ZnPc to avoid undesired aggregation. Moreover, the VLPs together with ZnPc cargo can be released in a controlled way without an initial burst release. The photodynamic effect of ZnPc molecules was retained after encapsulation of capsid protein and release from the hydrogel. This line of research suggests a new approach for sophisticated drug administration in supramolecular hydrogels.

Photo-responsive Bioactive Surfaces Based on Cucurbit[8]uril-Mediated Host-Guest Interactions of Arylazopyrazoles.

A photoswitchable arylazopyrazole (AAP) derivative binds with cucurbit[8]uril (CB[8]) and methylviologen (MV2+ ) to form a 1:1:1 heteroternary host-guest complex with a binding constant of Ka =2×103 m-1 . The excellent photoswitching properties of AAP are preserved in the inclusion complex. Irradiation with light of a wavelength of 365 and 520 nm leads to quantitative E- to Z- isomerization and vice versa, respectively. Formation of the Z-isomer leads to dissociation of the complex as evidenced using 1 H NMR spectroscopy. AAP derivatives are then used to immobilize bioactive molecules and photorelease them on demand. When Arg-Gly-Asp-AAP (AAP-RGD) peptides are attached to surface bound CB[8]/MV2+ complexes, cells adhere and can be released upon irradiation. The heteroternary host-guest system offers highly reversible binding properties due to efficient photoswitching and these properties are attractive for designing smart surfaces.

Electron Transfer Processes in Ferrocene-Modified Poly(ethylene glycol) Monolayers on Electrodes.

Electrochemistry is a powerful tool to study self-assembled monolayers. Here, we modified cystamine-functionalized electrodes with different lengths of linear poly(ethylene glycol) (PEG) polymers end-functionalized with a redox-active ferrocene (Fc) group. The electron transport properties of the Fc probes were studied using cyclic voltammetry. The Fc moiety attached to the shortest PEG (Mn = 250 Da) behaved as a surface-confined species, and the homogeneous electron transfer rate constants were determined. The electron transfer of the ferrocene group on the longer PEGs (Mn = 3.4, 5, and 10 kDa) was shown to be driven by diffusion. For low surface densities, where the polymer exists in the mushroom conformation, the diffusion coefficients (D) and rate constants were increasing with polymer length. In the loose brush conformation, where the polymers are close enough to interact with each other, the thickness of the layers (e) was unknown and a parameter D1/2/e was determined. This parameter showed no dependence on surface density and an increase with polymer length.

Controlling Protein Surface Orientation by Strategic Placement of Oligo-Histidine Tags.

We report oriented immobilization of proteins using the standard hexahistidine (His6)-Ni2+:NTA (nitrilotriacetic acid) methodology, which we systematically tuned to give control of surface coverage. Fluorescence microscopy and surface plasmon resonance measurements of self-assembled monolayers (SAMs) of red fluorescent proteins (TagRFP) showed that binding strength increased by 1 order of magnitude for each additional His6-tag on the TagRFP proteins. All TagRFP variants with His6-tags located on only one side of the barrel-shaped protein yielded a 1.5 times higher surface coverage compared to variants with His6-tags on opposite sides of the so-called ß-barrel. Time-resolved fluorescence anisotropy measurements supported by polarized infrared spectroscopy verified that the orientation (and thus coverage and functionality) of proteins on surfaces can be controlled by strategic placement of a His6-tag on the protein. Molecular dynamics simulations show how the differently tagged proteins reside at the surface in "end-on" and "side-on" orientations with each His6-tag contributing to binding. Also, not every dihistidine subunit in a given His6-tag forms a full coordination bond with the Ni2+:NTA SAMs, which varied with the position of the His6-tag on the protein. At equal valency but different tag positions on the protein, differences in binding were caused by probing for Ni2+:NTA moieties and by additional electrostatic interactions between different fractions of the ß-barrel structure and charged NTA moieties. Potential of mean force calculations indicate there is no specific single-protein interaction mode that provides a clear preferential surface orientation, suggesting that the experimentally measured preference for the end-on orientation is a supra-protein, not a single-protein, effect.

Light-Responsive Hierarchically Structured Liquid Crystal Polymer Networks for Harnessing Cell Adhesion and Migration.

Extracellular microenvironment is highly dynamic where spatiotemporal regulation of cell-instructive cues such as matrix topography tightly regulates cellular behavior. Recapitulating dynamic changes in stimuli-responsive materials has become an important strategy in regenerative medicine to generate biomaterials which closely mimic the natural microenvironment. Here, light responsive liquid crystal polymer networks are used for their adaptive and programmable nature to form hybrid surfaces presenting micrometer scale topographical cues and changes in nanoscale roughness at the same time to direct cell migration. This study shows that the cell speed and migration patterns are strongly dependent on the height of the (light-responsive) micrometer scale topographies and differences in surface nanoroughness. Furthermore, switching cell migration patterns upon in situ temporal changes in surface nanoroughness, points out the ability to dynamically control cell behavior on these surfaces. Finally, the possibility is shown to form photoswitchable topographies, appealing for future studies where topographies can be rendered reversible on demand.