Esther Tejeda-Montes

Integrating top-down and self-assembly in the fabrication of peptide and protein-based biomedical materials

The capacity to create an increasing variety of bioactive molecules that are designed to assemble in specific configurations has opened up tremendous possibilities in the design of materials with an unprecedented level of control and functionality. A particular challenge involves guiding such self-assembling interactions across scales, thus precisely positioning individual molecules within well-organized, highly-ordered structures. Such hierarchical control is essential if peptides and proteins are to serve as both structural and functional building blocks of biomedical materials. To achieve this goal, top-down techniques are increasingly being used in combination with self-assembling systems to reproducibly manipulate, localize, orient and assemble peptides and proteins to form organized structures. In this tutorial review we provide insight into how both standard and novel top-down techniques are being used in combination with peptide or protein self-assembly to create a new generation of functional materials.

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.

Engineering membrane scaffolds with both physical and biomolecular signaling

We report on the combination of a top-down and bottom-up approach to develop thin bioactive membrane scaffolds based on functional elastin-like polymers (ELPs). Our strategy combines ELP cross-linking and assembly, and a variety of standard and novel micro/nanofabrication techniques to create self-supporting membranes down to ∼500 nm thick that incorporate both physical and biomolecular signals, which can be easily tailored for a specific application. In this study we used an ELP that included the cell-binding motif arginine-glycine-aspartic acid-serine (RGDS). Furthermore, fabrication processes were developed to create membranes that exhibited topographical patterns with features down to 200 nm in lateral dimensions and up to 10 μm in height on either one or both sides, uniform and well-defined pores, or multiple ELP layers. A variety of processing parameters were tested in order to optimize membrane fabrication, including ELP and cross-linker concentration, temperature, reaction time and ambient humidity. Membrane micro/nanopatterning, swelling and stiffness were characterized by atomic force microscopy, nanoindentation tests and scanning electron microscopy. Upon immersion in phosphate-buffered saline and an increase in temperature from 25 to 40°C, membranes exhibited a significant increase in surface stiffness, with the reduced Youngs modulus increasing with temperature. Finally, rat mesenchymal stem cells were cultured on thin RGDS-containing membranes, which allowed cell adhesion, qualitatively enhanced spreading compared to membranes without RGDS epitopes and permitted proliferation. Furthermore, cell morphology was drastically affected by topographical patterns on the surface of the membranes.

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 Youngs 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.

Design of biomolecules for nanoengineered biomaterials for regenerative medicine.

An important goal in the development of highly functional organic materials is to design self-assembling molecules that can reproducibly display chemical signals across length scales. Within the biomedical field, biomolecules are highly attractive candidates to serve as bioactive building blocks for the next generation of biomaterials. The peptide amphiphiles (PAs) developed by the Stupp Laboratory at Northwestern University generated a highly versatile self-assembly code to create well-defined bioactive nanofibers that have been proven to be very effective at signaling cells in vitro and in vivo. Here, we describe the basic steps necessary for synthesis and assembly of PA molecules into functional nanostructures.

Protein disorder–order interplay to guide the growth of hierarchical mineralized structures

A major goal in materials science is to develop bioinspired functional materials based on the precise control of molecular building blocks across length scales. Here we report a protein-mediated mineralization process that takes advantage of disorder–order interplay using elastin-like recombinamers to program organic–inorganic interactions into hierarchically ordered mineralized structures. The materials comprise elongated apatite nanocrystals that are aligned and organized into microscopic prisms, which grow together into spherulite-like structures hundreds of micrometers in diameter that come together to fill macroscopic areas. The structures can be grown over large uneven surfaces and native tissues as acid-resistant membranes or coatings with tuneable hierarchy, stiffness, and hardness. Our study represents a potential strategy for complex materials design that may open opportunities for hard tissue repair and provide insights into the role of molecular disorder in human physiology and pathology.There is evidence that disordered proteins play a role in the mineralization process. Here, the authors report on the development of elastin-like recombinant protein membranes using disordered-ordered interplay to investigate and guide mineralization.