Alvaro Mata

Self-Assembly of Large and Small Molecules into Hierarchically Ordered Sacs and Membranes

We report here the self-assembly of macroscopic sacs and membranes at the interface between two aqueous solutions, one containing a megadalton polymer and the other, small self-assembling molecules bearing opposite charge. The resulting structures have a highly ordered architecture in which nanofiber bundles align and reorient by nearly 90° as the membrane grows. The formation of a diffusion barrier upon contact between the two liquids prevents their chaotic mixing. We hypothesize that growth of the membrane is then driven by a dynamic synergy between osmotic pressure of ions and static self-assembly. These robust, self-sealing macroscopic structures offer opportunities in many areas, including the formation of privileged environments for cells, immune barriers, new biological assays, and self-assembly of ordered thick membranes for diverse applications.

A self-assembly pathway to aligned monodomain gels

Aggregates of charged amphiphilic molecules have been found to access a structure at elevated temperature that templates alignment of supramolecular fibrils over macroscopic scales. The thermal pathway leads to a lamellar plaque structure with fibrous texture that breaks upon cooling into large arrays of aligned nanoscale fibres and forms a strongly birefringent liquid. By manually dragging this liquid crystal from a pipette onto salty media, it is possible to extend this alignment over centimetres in noodle-shaped viscoelastic strings. Using this approach, the solution of supramolecular filaments can be mixed with cells at physiological temperatures to form monodomain gels of aligned cells and filaments. The nature of the self-assembly process and its biocompatibility would allow formation of cellular wires in situ that have any length and customized peptide compositions for use in biological applications.

Bone Regeneration Mediated by Biomimetic Mineralization of a Nanofiber Matrix

Rapid bone regeneration within a three-dimensional defect without the use of bone grafts, exogenous growth factors, or cells remains a major challenge. We report here on the use of self-assembling peptide nanostructured gels to promote bone regeneration that have the capacity to mineralize in biomimetic fashion. The main molecular design was the use of phosphoserine residues in the sequence of a peptide amphiphile known to nucleate hydroxyapatite crystals on the surfaces of nanofibers. We tested the system in a rat femoral critical-size defect by placing pre-assembled nanofiber gels in a 5mm gap and analyzed bone formation with micro-computed tomography and histology. We found within 4 weeks significantly higher bone formation relative to controls lacking phosphorylated residues and comparable bone formation to that observed in animals treated with a clinically used allogenic bone matrix.

Fabrication of multi-layer SU-8 microstructures

The fabrication of multi-level SU-8 microstructures using multiple coating and exposure steps and a single developing step has been achieved for up to six layers of SU-8. Alternating layers of SU-8 2010 (thin) and SU-8 2100 (thick) photoresist films were spin coated, followed by soft-bake, ultraviolet (UV) exposure and post-exposure bake steps. The multiple SU-8 layers were simultaneously developed to create patterned microstructures with overall thicknesses of up to 500 µm and minimum lateral feature size of 10 µm. The use of a single developing step facilitated fabrication of complex multi-level SU-8 microstructures that might be difficult, or even impossible, to achieve by sequential processing of multiple SU-8 layers that are individually coated, baked, exposed and developed.

Bioactive nanofibers instruct cells to proliferate and differentiate during enamel regeneration.

During tooth development, ectoderm‐derived ameloblast cells create enamel by synthesizing a complex protein mixture serving to control cell to matrix interactions and the habit of hydroxyapatite crystallites. Using an in vitro cell and organ culture system, we studied the effect of artificial bioactive nanostructures on ameloblasts with the long‐term goal of developing cell‐based strategies for tooth regeneration. We used branched peptide amphiphile molecules containing the peptide motif Arg‐Gly‐Asp, or “RGD” (abbreviated BRGD‐PA), known to self‐assemble in physiologic environments into nanofibers that display on their surfaces high densities of this biological signal. Ameloblast‐like cells (line LS8) and primary enamel organ epithelial (EOE) cells were cultured within PA hydrogels, and the PA was injected into the enamel organ epithelia of mouse embryonic incisors. The expression of amelogenin, ameloblastin, integrin α5, and integrin α6 was detected by quantitative real‐time PCR and immunodetection techniques. We performed cell proliferation assay using BrdU labeling and a biomineralization assay using Alizarin red S staining with quantitative Ca2+ measurements. In the cell culture model, ameloblast‐like cells (LS8) and primary EOE cells responded to the BRGD‐PA nanostructures with enhanced proliferation and greater amelogenin, ameloblastin, and integrin expression levels. At the site of injection of the BRGD‐PA in the organ culture model, we observed EOE cell proliferation with differentiation into ameloblasts as evidenced by their expression of enamel specific proteins. Ultrastructural analysis showed the nanofibers within the forming extracellular matrix, in contact with the EOE cells engaged in enamel formation and regeneration. This study shows that BRGD‐PA nanofibers present with enamel proteins participate in integrin‐mediated cell binding to the matrix with delivery of instructive signals for enamel formation.

A three dimensional scaffold with precise micro-architecture and surface micro-textures

A three-dimensional (3D) structure comprising precisely defined micro-architecture and surface micro-textures, designed to present specific physical cues to cells and tissues, may provide an efficient scaffold in a variety of tissue engineering and regenerative medicine applications. We report a fabrication technique based on microfabrication and soft lithography that permits for the development of 3D scaffolds with both precisely engineered architecture and tailored surface topography. The scaffold fabrication technique consists of three key steps starting with microfabrication of a mold using an epoxy-based photoresist (SU-8), followed by dual-sided molding of a single layer of polydimethylsiloxane (PDMS) using a mechanical jig for precise motion control; and finally, alignment, stacking, and adhesion of multiple PDMS layers to achieve a 3D structure. This technique was used to produce 3D Texture and 3D Smooth PDMS scaffolds, where the surface topography comprised 10 microm diameter/height posts and smooth surfaces, respectively. The potential utility of the 3D microfabricated scaffolds, and the role of surface topography, were subsequently investigated in vitro with a combined heterogeneous population of adult human stem cells and their resultant progenitor cells, collectively termed connective tissue progenitors (CTPs), under conditions promoting the osteoblastic phenotype. Examination of bone-marrow derived CTPs cultured on the 3D Texture scaffold for 9 days revealed cell growth in three dimensions and increased cell numbers compared to those on the 3D Smooth scaffold. Furthermore, expression of alkaline phosphatase mRNA was higher on the 3D Texture scaffold, while osteocalcin mRNA expression was comparable for both types of scaffolds.

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.

Analysis of Connective Tissue Progenitor Cell Behavior on Polydimethylsiloxane Smooth and Channel Micro-Textures

Growth of human connective tissue progenitor cells (CTPs) was characterized on smooth and microtextured polydimethylsiloxane (PDMS) surfaces. Human bone marrow derived cells were cultured for nine days under conditions promoting osteoblastic differentiation on Smooth PDMS and PDMS Channel microtextures (11 μm high, 45 μm wide channels, and separated by 5 μm wide ridges). Glass tissue culture dish surfaces were used as controls. Cell numbers per colony, cell density within colonies, alignment of cells, area of colonies, and colony shapes were determined as a function of substrate surface topography. An alkaline phosphatase stain was used as a marker for osteoblastic phenotype. CTPs attached, proliferated, and differentiated on all surfaces with cell process lengths of up to 80 μm. Cells on the Smooth PDMS and control surfaces spread and proliferated as colonies in proximity to other cells and migrated in random directions creating colonies that covered significantly larger areas (0.96 and 1.05 mm2, respectively) than colonies formed on PDMS Channel textures (0.64 mm2). In contrast, cells on PDMS Channel textures spread, proliferated, aligned along the channel axis, and created colonies that were more dense, and with lengths of longest colony axes that were significantly longer (3252 μm) than those on the Smooth PDMS (1265 μm) and control surfaces (1319 μm). Cells on PDMS Channel textures were aligned at an angle of 14.44° relative to the channel axis, and the resulting colonies exhibited a significantly higher aspect ratio (13.72) compared to Smooth PDMS (1.57) and control surfaces (1.51).

Co-assembly, spatiotemporal control and morphogenesis of a hybrid protein–peptide system

Controlling molecular interactions between bioinspired molecules can enable the development of new materials with higher complexity and innovative properties. Here we report on a dynamic system that emerges from the conformational modification of an elastin-like protein by peptide amphiphiles and with the capacity to access, and be maintained in, non-equilibrium for substantial periods of time. The system enables the formation of a robust membrane that displays controlled assembly and disassembly capabilities, adhesion and sealing to surfaces, self-healing and the capability to undergo morphogenesis into tubular structures with high spatiotemporal control. We use advanced microscopy along with turbidity and spectroscopic measurements to investigate the mechanism of assembly and its relation to the distinctive membrane architecture and the resulting dynamic properties. Using cell-culture experiments with endothelial and adipose-derived stem cells, we demonstrate the potential of this system to generate complex bioactive scaffolds for applications such as tissue engineering.

Osteoblast attachment to a textured surface in the absence of exogenous adhesion proteins

The present study investigated whether osteoblasts could attach to a culture substratum through a surface texture-dependent mechanism. Four test groups were used: (A) untextured, and three texture groups with maximum feature sizes of (B) <0.5 /spl mu/m, (C) 2 /spl mu/m, and (D) 4 /spl mu/m, respectively. All surfaces were coated with the nonadhesive protein bovine serum albumin (BSA). Osteoblasts were allowed to adhere in serum-free medium for either 1 or 4 h, at which time nonadherent cells were removed. At 4 h, untextured surface A exhibited no cell attachment, while textured surfaces B, C, and D exhibited 9%, 32%, and 16% cell adhesion, respectively. At 16 h of incubation, adherent osteoblasts on textured surface C exhibited focal adhesion contacts and microfilament stress-fiber bundles. These results indicate that microtextured surfaces in the absence of exogenous adhesive proteins can facilitate osteoblast adhesion.