Biohybrid elastin-like venous valve with potential for in situ tissue engineering.

Chronic venous insufficiency (CVI) is a leading vascular disease whose clinical manifestations include varicose veins, edemas, venous ulcers, and venous hypertension, among others. Therapies targeting this medical issue are scarce, and so far, no single venous valve prosthesis is clinically available. Herein, we have designed a bi-leaflet transcatheter venous valve that consists of (i) elastin-like recombinamers, (ii) a textile mesh reinforcement, and (iii) a bioabsorbable magnesium stent structure. Mechanical characterization of the resulting biohybrid elastin-like venous valves (EVV) showed an anisotropic behavior equivalent to the native bovine saphenous vein valves and mechanical strength suitable for vascular implantation. The EVV also featured minimal hemolysis and platelet adhesion, besides actively supporting endothelialization in vitro, thus setting the basis for its application as an in situ tissue engineering implant. In addition, the hydrodynamic testing in a pulsatile bioreactor demonstrated excellent hemodynamic valve performance, with minimal regurgitation (<10%) and pressure drop (<5 mmHg). No stagnation points were detected and an in vitro simulated transcatheter delivery showed the ability of the venous valve to withstand the implantation procedure. These results present a promising concept of a biohybrid transcatheter venous valve as an off-the-shelf implant, with great potential to provide clinical solutions for CVI treatment.

Antibiofilm coatings based on protein-engineered polymers and antimicrobial peptides for preventing implant-associated infections.

Implant-associated infections (IAIs) are one of the leading concerns in orthopedics and dentistry as they commonly lead to implant failure. The presence of biofilms and, increasingly frequently, drug-resistant bacteria further impairs the efficacy of conventional antibiotics. Immobilization of antimicrobial peptides (AMPs) on implant surfaces is a promising alternative to antibiotics for prevention of IAIs. In addition, the use of functional linkers for the AMP tethering enables to increase the antimicrobial potential and the bioactivities of the coating. In this study, an extracellular-matrix-mimicking system based on elastin-like recombinamers (ELRs) has been developed for the covalent anchoring of AMPs and investigated for use as a hybrid antibiofilm coating. A drip-flow biofilm reactor was used to simulate in vivo environmental dynamic conditions, thus showing that the presence of the AMPs in the hybrid coatings provided strong antibiofilm activity against monospecies and microcosm biofilm models of clinical relevance. These results, together with an excellent cytocompatibility towards primary gingival fibroblasts, encourage the use of ELRs as multivalent platforms for AMPs and open up a wide range of possibilities in the biofabrication of advanced coatings combining the antibiofilm potential of AMPs and the outstanding tunability and biomechanical properties of the ELRs.

A double safety lock tumor-specific device for suicide gene therapy in breast cancer.

The complexity and continuous evolution of cancer make the design of novel strategies of treatment a constant challenge in biomedicine. Moreover, most of cancer treatments are still not tumor-specific and provoke high systemic toxicity. Herein we have developed a novel selective nanodevice to eliminate tumor cells while leaving healthy ones intact. To achieve this objective, a polyplex carrier, comprising an elastin like-recombinamer covalently conjugated to an aptamer and complexed with therapeutic DNA, was tested. This carrier forms a double-lock multifunctional device due to specific binding to a tumor cell marker and the selective expression of therapeutic DNA inside human breast-cancer cells. Due to the stability provided by ELRs, the homogeneous population of polyplexes obtained showed selective toxicity against cancer cells in in vitro and in vivo assay. Inhibition of tumor progression was detected early being very significant at the end point, with a dose-dependent reduction in tumor mass. Histological studies revealed a specific reduction in tumor parenchyma and in specific tumor cell markers. These results represent an important step toward the rational development of an efficient, safe and more specialized gene-delivery device for tumor therapy.

Self-Assembling ELR-Based Nanoparticles as Smart Drug-Delivery Systems Modulating Cellular Growth via Akt.

This work investigates the physicochemical properties and in vitro accuracy of a genetically engineered drug-delivery system based on elastin-like block recombinamers. The DNA recombinant techniques allowed us to create this smart complex polymer containing bioactive sequences for internalization, lysosome activation under acidic pH, and blockage of cellular growth by a small peptide inhibitor. The recombinant polymer reversibly self-assembled when the temperature was increased above 15 °C into nanoparticles with a diameter of 72 nm and negative surface charge. Furthermore, smart nanoparticles were shown to enter in the cells via clathrin-dependent endocytosis and properly blocked phosphorylation and consequent activation of Akt kinase. This system provoked apoptosis-mediated cell death in breast and colorectal cancer cells, which possess higher expression levels of Akt, whereas noncancerous cells, such as endothelial cells, fibroblasts, and mesenchymal stem cells, were not affected. Hence, we conclude that the conformational complexity of this smart elastin-like recombinamer leads to achieving successful drug delivery in targeted cells and could be a promising approach as nanocarriers with bioactive peptides to modulate multiple cellular processes involved in different diseases.

Use of proteolytic sequences with different cleavage kinetics as a way to generate hydrogels with preprogrammed cell-infiltration patterns imparted over their given 3D spatial structure.

Control over biodegradation processes is crucial to generate advanced functional structures with a more interactive and efficient role for biomedical applications. Herein, a simple, high-throughput approach is developed based on a three-dimensional (3D)-structured system that allows a preprogramed spatial-temporal control over cell infiltration and biodegradation. The 3D-structured system is based on elastin-like recombinamers (ELRs) characterized by differences in the kinetics of their peptide cleavage and consists of a three-layer hydrogel disk comprising an internal layer containing a rapidly degrading component, with the external layers containing a slow-degrading ELR. This structure is intended to invert the conventional pattern of cell infiltration, which goes from the outside to the inside of the implant, to allow an anti-natural process in which infiltration takes place first in the internal layer and later progresses to the outer layers. Time-course in vivo studies proved this hypothesis, i.e. that it is possible to drive the infiltration of cells over time in a given 3D-structured implant in a controlled and predesigned way that is able to overcome the natural tendency of conventional cell infiltration. The results obtained herein open up the possibility of applying this concept to more complex systems with multiple biological functions.

Tethering QK peptide to enhance angiogenesis in elastin-like recombinamer (ELR) hydrogels.

The development of new capillary networks in engineered constructs is essential for their survival and their integration with the host tissue. It has recently been demonstrated that ELR-based hydrogels encoding different bioactivities are able to modulate their interaction with the host after injection or implantation, as indicated by an increase in cell adhesion and the ability to trigger vascularization processes. Accordingly, the aim of this study was to increase their angiogenic ability both in vitro and in vivo using a small VEGF mimetic peptide named QK, which was tethered chemically to ELR-based hydrogels containing cell-adhesion sequences in their backbone, such as REDV and RGD, as well as a proteolytic site (VGVAPG). In vitro studies were performed using a co-culture of endothelial and fibroblast cells encapsulated into the ELR-based hydrogels in order to determine cell proliferation after 21 days of culture, as well as the number of cell-cell interactions. It was found that although the presence of this peptide does not influence the morphological and rheological properties of these hydrogels, it has an effect on cell behaviour, inducing an increase in cell proliferation and the formation of endothelial cell clusters. In vivo studies demonstrate that the QK peptide enhances the formation of prominent functional capillaries at three weeks post-injection, as confirmed by H&E staining and CD31 immunohistochemistry. The newly formed functional microvasculature ensures perfusion and connection with surrounding tissues. These results show that ELR-QK hydrogels increase capillary network formation and are therefore attractive candidates for application in tissue regeneration, for example for the treatment of cardiovascular diseases such as myocardial infarction or ischemia.

A transferrin receptor-binding mucoadhesive elastin-like recombinamer: In vitro and in vivo characterization.

The development of mucoadhesive materials is of great interest and is also a major challenge. Being adsorption sites, mucosae are suitable targets for drug delivery, but as defensive barriers they are complex biological surfaces to interact with, mainly due to their protective mucus layer. As such, first- and second-generation mucoadhesives focused on material-mucus interactions, whereas the third generation of mucoadhesives introduced structural motifs that are able to interact with the cells beneath the mucus layer. The combination of different prerequisites (water solubility, soft gel formation at body temperature and able to interact with the mucus) in a single molecule is easily achieved using elastin-like recombinamers (ELRs) given their multiple block design. Moreover, we have been able to introduce a short amino-acid sequence known as T7 that is able to bind to transferrin receptors in the epithelial cell layer. The T7 sequence enhances the cell-binding properties of the mucoadhesive ELR (MELR), as demonstrated using a Caco-2 epithelial cell model. In vivo experiments confirmed the mucoadhesive properties found in vitro. STATEMENT OF SIGNIFICANCE: The development of a mucoadhesive material is a major challenge. Mucosae are suitable targets for drug delivery, but as defense barriers, they are complex surfaces to interact with. In this work we report the first ELR that combines different functional blocks, in a single molecule, which provide it with the properties of soft-gel forming at body temperature and being able of efficiently adhering to the mucus layer of mucosas, as well as to the underlying epithelial cell layer, as demonstrated in vitro and in vivo. The rationally designed materials presented in this work sets the basis for developing ELR-based, mucosa-directed drug delivery systems, which could improve patient's compliance, enhancing drug retention at the mucosal site.

A novel lipase-catalyzed method for preparing ELR-based bioconjugates.

Herein we present a novel one-pot method for the chemical modification of elastin-like recombinamers (ELRs) in a mild and efficient manner involving enzymatic catalysis with Candida antarctica lipase B. The introduction of different functionalities into such ELRs could open up new possibilities for the development of advanced biomaterials for regenerative medicine and, specifically, for controlled drug delivery given their additional ability to respond to stimuli other than pH or temperature, such as glucose concentration or electromagnetic radiation. Candida antarctica lipase B immobilized on a macroporous acrylic resin (Novozym 435) was used to enzymatically couple different aminated substrates to a recombinamer containing carboxylic groups along its amino acid chain by way of an amidation reaction. A preliminary study of the kinetics of this amidation in response to different reaction conditions, such as solvent, temperature or reagent ratio, was carried out using a phenylazobenzene derivative (azo-NH2) as a model. The optimal amidation conditions were used to couple other amine reagents, such as phenylboronic acid (FB-NH2) or polyethylene glycol (PEG-NH2), thus allowing us to obtain photoresponsive, glucose-responsive or PEGylated ELRs that could potentially be useful as sensors in devices for controlled drug delivery.

Tuning the Stiffness of Surfaces by Assembling Genetically Engineered Polypeptides with Tailored Amino Acid Sequence.

We introduce elastin-like recombinamers (ELRs) as polypeptides with precise amino acid positioning to generate polypeptide coatings with tunable rigidity. Two ELRs are used: V84-ELR, a hydrophobic monoblock, and EI-ELR, an amphiphilic diblock. Both were modified with the amine-reactive tetrakis (hydroxymethyl) phosphonium chloride compound. We evaluated the affinity, conformation, and dissipative behavior of ELRs assembled on alkanethiol self-assembled coatings by quartz crystal microbalance with dissipation monitoring, multiparametric surface plasmon resonance, and atomic force microscopy. The thickness of the polypeptide coatings showcases the preferential affinity of ELRs to NH2- and CH3-terminated surfaces. We demonstrate that V84-ELR strongly bonded to the substrate and reorganizes into an extended and more hydrated layer as the adsorbed amount increases, whereas EI-ELR has a less dissipative behavior. The results suggest that ELR adsorption depends on the amino acid sequence and the substrate chemistry, ultimately influencing the stiffness of the polypeptide coatings.

Macroporous click-elastin-like hydrogels for tissue engineering applications.

Elastin is a key extracellular matrix (ECM) protein that imparts functional elasticity to tissues and therefore an attractive candidate for bioengineering materials. Genetically engineered elastin-like recombinamers (ELRs) maintain inherent properties of the natural elastin (e.g. elastic behavior, bioactivity, low thrombogenicity, inverse temperature transition) while featuring precisely controlled composition, the possibility for biofunctionalization and non-animal origin. Recently the chemical modification of ELRs to enable their crosslinking via a catalyst-free click chemistry reaction, has further widened their applicability for tissue engineering. Despite these outstanding properties, the generation of macroporous click-ELR scaffolds with controlled, interconnected porosity has remained elusive so far. This significantly limits the potential of these materials as the porosity has a crucial role on cell infiltration, proliferation and ECM formation. In this study we propose a strategy to overcome this issue by adapting the salt leaching/gas foaming technique to click-ELRs. As result, macroporous hydrogels with tuned pore size and mechanical properties in the range of many native tissues were reproducibly obtained as demonstrated by rheological measurements and quantitative analysis of fluorescence, scanning electron and two-photon microscopy images. Additionally, the appropriate size and interconnectivity of the pores enabled smooth muscle cells to migrate into the click-ELR scaffolds and deposit extracellular matrix. The macroporous structure together with the elastic performance and bioactive character of ELRs, the specificity and non-toxic character of the catalyst-free click-chemistry reaction, make these scaffolds promising candidates for applications in tissue regeneration. This work expands the potential use of ELRs and click chemistry systems in general in different biomedical fields.

Bioactive scaffolds based on elastin-like materials for wound healing.

Wound healing is a complex process that, in healthy tissues, starts immediately after the injury. Even though it is a natural well-orchestrated process, large trauma wounds, or injuries caused by acids or other chemicals, usually produce a non-elastic deformed tissue that not only have biological reduced properties but a clear aesthetic effect. One of the main drawbacks of the scaffolds used for wound dressing is the lack of elasticity, driving to non-elastic and contracted tissues. In the last decades, elastin based materials have gained in importance as biomaterials for tissue engineering applications due to their good cyto- and bio-compatibility, their ease handling and design, production and modification. Synthetic elastin or elastin like-peptides (ELPs) are the two main families of biomaterials that try to mimic the outstanding properties of natural elastin, elasticity amongst others; although there are no in vivo studies that clearly support that these two families of elastin based materials improve the elasticity of the artificial scaffolds and of the regenerated skin. Within the next pages a review of the different forms (coacervates, fibres, hydrogels and biofunctionalized surfaces) in which these two families of biomaterials can be processed to be applied in the wound healing field have been done. Here, we explore the mechanical and biological properties of these scaffolds as well as the different in vivo approaches in which these scaffolds have been used.

Biocompatible ELR-Based Polyplexes Coated with MUC1 Specific Aptamers and Targeted for Breast Cancer Gene Therapy.

The search for new and biocompatible materials with high potential for improvement is a challenge in gene delivery applications. A cell type specific vector made of elastin-like recombinamer (ELR) and aptamers has been specifically designed for the intracellular delivery of therapeutic material for breast cancer therapy. A lysine-enriched ELR was constructed and complexed with plasmid DNA to give positively charged and stable polyplexes. Physical characterization of these polyplexes showed a particle size of around 140 nm and a zeta potential of approximately +40 mV. The incorporation of MUC1-specific aptamers into the polyplexes resulted in a slight decrease in zeta potential but increased cell transfection specificity for MCF-7 breast cancer cells with respect to a MUC1-negative tumor line. After showing the transfection ability of this aptamer-ELR vector which is facilitated mainly by macropinocytosis uptake, we demonstrated its application for suicide gene therapy using a plasmid containing the gene of the toxin PAP-S. The strategy developed in this work about using ELR as polymeric vector and aptamers as supplier of specificity to deliver therapeutic material into MUC1-positive breast cancer cells shows promising potential and continues paving the way for ELRs in the biomedical field.

Amphiphilic Elastin-Like Block Co-Recombinamers Containing Leucine Zippers: Cooperative Interplay between Both Domains Results in Injectable and Stable Hydrogels.

Many biological processes are regulated by reversible binding events, with these interactions between macromolecules representing the core of dynamic chemistry. As such, any attempt to gain a better understanding of such interactions, which would pave the way to the extrapolation of natural designs to create new advanced systems, is clearly of interest. This work focuses on the development of a leucine zipper-elastin-like recombinamer (ZELR) in order to elucidate the behavior of such domains when coexisting along the same molecule and to engineer reversible, injectable and stable hydrogels. The unique propensity of the Z-moiety selected to dimerize, together with the thermosensitive behavior of the ELR, which has been constructed as a thermosensitive amphiphilic tetrablock, has been engineered into a single recombinant molecule. In this molecular design, the Z-moieties are unable to form a network, while the ELR is below its Tt, thus, guaranteeing the liquid-like state of the system. However, this situation changes rapidly as the temperature increases above Tt, where a stable hydrogel is formed, as demostrated by rheological tests. The inability of the ELR molecule (without Z-domains) to form such a stable hydrogel above Tt clearly points to a positive cooperative effect between these two domains (Z and EL), and no conformational changes in the former are involved, as demonstrated by circular dichroism analysis. AFM shows that Z-motifs seem to induce the aggregation of micelles, which supports the enhanced stability displayed by ZELRs when compared to ELR at the macroscale level. To the best of our knowledge, this is the first time that such an interplay between these two domains has been reported. Furthermore, the cytocompatibility of the resulting hydrogels opens the door to their use in biomedical applications.

Self-organized ECM-mimetic model based on an amphiphilic multiblock silk-elastin-like corecombinamer with a concomitant dual physical gelation process.

Although significant progress has been made in the area of injectable hydrogels for biomedical applications and model cell niches, further improvements are still needed, especially in terms of mechanical performance, stability, and biomimicry of the native fibrillar architecture found in the extracellular matrix (ECM). This work focuses on the design and production of a silk-elastin-based injectable multiblock corecombinamer that spontaneously forms a stable physical nanofibrillar hydrogel under physiological conditions. That differs from previously reported silk-elastin-like polymers on a major content and predominance of the elastin-like part, as well as a more complex structure and behavior of such a part of the molecule, which is aimed to obtain well-defined hydrogels. Rheological and DSC experiments showed that this system displays a coordinated and concomitant dual gelation mechanism. In a first stage, a rapid, thermally driven gelation of the corecombinamer solution takes place once the system reaches body temperature due to the thermal responsiveness of the elastin-like (EL) parts and the amphiphilic multiblock design of the corecombinamer. A bridged micellar structure is the dominant microscopic feature of this stage, as demonstrated by AFM and TEM. Completion of the initial stage triggers the second, which is comprised of a stabilization, reinforcement, and microstructuring of the gel. FTIR analysis shows that these events involve the formation of ß-sheets around the silk motifs. The emergence of such ß-sheet structures leads to the spontaneous self-organization of the gel into the final fibrous structure. Despite the absence of biological cues, here we set the basis of the minimal structure that is able to display such a set of physical properties and undergo microscopic transformation from a solution to a fibrous hydrogel. The results point to the potential of this system as a basis for the development of injectable fibrillar biomaterial platforms toward a fully functional, biomimetic, artificial extracellular matrix, and cell niches.

Mineralization and bone regeneration using a bioactive elastin-like recombinamer membrane.

The search for alternative therapies to improve bone regeneration continues to be a major challenge for the medical community. Here we report on the enhanced mineralization, osteogenesis, and in vivo bone regeneration properties of a bioactive elastin-like recombinamer (ELR) membrane. Three bioactive ELRs exhibiting epitopes designed to promote mesenchymal stem cell adhesion (RGDS), mineralization (DDDEEKFLRRIGRFG), and both cell adhesion and mineralization were synthesized using standard recombinant protein techniques. The ELR materials were then used to fabricate membranes comprising either a smooth surface (Smooth) or channel microtopographies (Channels). Mineralization and osteoblastic differentiation of primary rat mesenchymal stem cells (rMSCs) were analyzed in both static and dynamic (uniaxial strain of 8% at 1 Hz frequency) conditions. Smooth mineralization membranes in static condition exhibited the highest quantity of calcium phosphate (Ca/P of 1.78) deposition with and without the presence of cells, the highest Young's modulus, and the highest production of alkaline phosphatase on day 10 in the presence of cells growing in non-osteogenic differentiation medium. These membranes were tested in a 5 mm-diameter critical-size rat calvarial defect model and analyzed for bone formation on day 36 after implantation. Animals treated with the mineralization membranes exhibited the highest bone volume within the defect as measured by micro-computed tomography and histology with no significant increase in inflammation. This study demonstrates the possibility of using bioactive ELR membranes for bone regeneration applications.

Hybrid nanotopographical surfaces obtained by biomimetic mineralization of statherin-inspired elastin-like recombinamers.

Modification of surfaces mimicking unique chemical and physical features of mineralized tissues is of major interest for obtaining biomaterials for replacing and regenerating biological tissues. Here, human salivary statherin-inspired genetically engineered recombinamers (ELRs, HSS) on biomedical surfaces regulates mineralization to form an amorphous-calcium-phosphate (ACP) layer that reproduces the original substrate nanotopography. The HSS-ELRs carry a statherin-derived peptide with high affinity to tooth enamel. They are tethered to nanorough surfaces and mineralized using an enzyme-directed process. A homogeneous layer of ACP-minerals forms on HSS-coated surfaces retaining the original nanotopography of the substrate. In contrast, biomineralization of control surfaces results in uncontrolled growth of minerals. This suggest the statherin-inspired ELRs have ability to induce and control growth of the minerals on the biofunctional surfaces. Likely, the HSS-ELR coating have similar bioactivity to that of statherin in human saliva. The hybrid nanorough surfaces improve adhesion and differentiation of preosteoblasts and show potential for dental and orthopedic implants integration. This method enables the combination and tailoring of nanotopographical and biochemical cues to design functionalized surfaces to investigate and potentially direct the stem cell fate.

Effect of surfactants on the self-assembly of a model elastin-like block corecombinamer: from micelles to an aqueous two-phase system.

Recent advances in genetic engineering now allow the synthesis of protein-based block corecombinamers derived from elastin-like peptide sequences with complete control of chemistry and molecular weight, thereby resulting in unique physical and biological properties. The individual blocks of the elastin-like block corecombinamers (ELbcR's) display different phase behaviors in aqueous solution, which leads to the thermally triggered self-assembly of nano-objects ranging from micelles to vesicles. Herein, the interaction of cationic surfactant dodecyl trimethylammonium bromide (DTAB), anionic surfactant dodecyl sodium sulfate (SDS), and nonionic surfactant octyl-ß-glucopyranoside (OG) with an ELbcR has been investigated by dynamic light scattering (DLS), the ζ potential and cryo-transmission electron microscopy (cryo-TEM). At 65 °C and neutral pH in aqueous solution, the ELbcR (E50A40) is associated into micelles with a diameter of 150 nm comprising a hydrophobic (A) core and a hydrophilic (E) anionic (from the glutamic acid residues) corona. The size of these self-assemblies can be controlled by adjusting the cosurfactant concentrations. Although the effects of surfactants on the self-assembly behavior of ELbcR's depend on the hydrocarbon chain length and headgroup of the surfactants, a general tendency to increase in size, which in some cases leads to flocculation and a phase-separated state, is observed. These results support the use of surfactants as a highly interesting means of controlling the self-assembly of ELbcR's in aqueous solution as well as their use in drug delivery and purification processes.

Cellular uptake of multilayered capsules produced with natural and genetically engineered biomimetic macromolecules.

Multilayered microcapsules of chitosan and biomimetic elastin-like recombinamers (ELRs) were prepared envisaging the intracellular delivery of active agents. Two ELRs containing either a bioactive RGD sequence or a scrambled non-functional RDG were used to construct two types of functionalized polymeric microcapsules, both of spherical shape ∼4µm in diameter. Cell viability studies with human mesenchymal stem cells (hMSCs) were performed using microcapsule/cell ratios between 5:1 and 100:1. After 3 and 72h of co-incubation, no signs of cytotoxicity were found, but cells incubated with RGD-functionalized microcapsules exhibited higher viability values than RDG cells. The internalization efficacy and bioavailability of encapsulated DQ-ovalbumin were assessed by monitoring the fluorescence changes in the cargo. The data show that surface functionalization did not significantly influence internalization by hMSCs, but the bioavailability of DQ-ovalbumin degraded faster when encapsulated within RGD-functionalized microcapsules. The microcapsules developed show promise for intracellular drug delivery with increased drug efficacy.

A bioactive elastin-like recombinamer reduces unspecific protein adsorption and enhances cell response on titanium surfaces.

We present the immobilization on synthetic substrates of elastin-like recombinamers (ELR) that combine a bioactive motif for cell adhesion with protein antifouling properties. Physical adsorption of the recombinamers and covalent-grafting through organosilane chemistry were investigated. The biochemically-modified surfaces were thoroughly characterized and tested for protein absorption in serum by fluorescence-labelling, XPS, Ellipsometry, and OWLS. The ELR were successfully grafted and stable, even upon mechanical stresses; being the covalent bonding favourable over physical adsorption. The coated metal surfaces exhibited excellent reduction of serum protein adsorption (9 ng/cm(2)) compared to the bare metal surface (310 ng/cm(2)). Non-specific protein adsorption may mask the introduced bioactive motifs; therefore, the bioactivated surfaces should display serum-protein antifouling properties. Finally, improved hMSCs response was assessed on the bioactivated substrates. In summary, the coatings simultaneously displayed anti-fouling and bioactive properties. These studies investigated key factors to enhance tissue material interactions fundamental for the design of bioactive devices and future biomedical applications.

Bioactive membranes for bone regeneration applications: effect of physical and biomolecular signals on mesenchymal stem cell behavior.

This study focuses on the in vitro characterization of bioactive elastin-like recombinamer (ELR) membranes for bone regeneration applications. Four bioactive ELRs exhibiting epitopes designed to promote mesenchymal stem cell adhesion (RGDS), endothelial cell adhesion (REDV), mineralization (HAP), and both cell adhesion and mineralization (HAP-RGDS) were synthesized using standard recombinant protein techniques. The materials were then used to fabricate ELR membranes incorporating a variety of topographical micropatterns including channels, holes and posts. Primary rat mesenchymal stem cells (rMSCs) were cultured on the different membranes and the effects of biomolecular and physical signals on cell adhesion, morphology, proliferation, and differentiation were evaluated. All results were analyzed using a custom-made MATLAB program for high throughput image analysis. Effects on cell morphology were mostly dependent on surface topography, while cell proliferation and cell differentiation were largely dependent on the biomolecular signaling from the ELR membranes. In particular, osteogenic differentiation (evaluated by staining for the osteoblastic marker osterix) was significantly enhanced on cells cultured on HAP membranes. Remarkably, cells growing on membranes containing the HAP sequence in non-osteogenic differentiation media exhibited significant up-regulation of the osteogenic marker as early as day 5, while those growing on fibronectin-coated glass in osteogenic differentiation media did not. These results are part of our ongoing effort to develop an optimized molecularly designed periosteal graft.