Cristina González-García

Effect of nanoscale topography on fibronectin adsorption, focal adhesion size and matrix organisation

Phase separation of PLLA/PS (50/50, w/w) solutions during a spin-casting process gives rise to well-defined nanotopographies of 14, 29 and 45 nm deep pits depending on the concentration of the solution. Their influence on the biological activity of fibronectin (FN) was investigated. FN adsorption was quantified by radiolabelling the protein. The amount of adsorbed FN was higher on the 14 nm deep pit nanotopography than on the other two surfaces. FN distribution between valleys and peaks was investigated by AFM combined with image analysis. FN tends to adsorb preferentially on the valleys of the nanotopography only for the 14 nm system and when adsorbed from solutions of concentration lower than 10 microg/ml. Higher concentration of the FN solution leads to evenly distribution of the protein throughout the surface; moreover, there is no difference in the distribution of the protein between valleys and peaks for the other two systems (29 and 45 nm) irrespective of the concentration of the FN solution. The biological activity of the adsorbed protein layer was assessed by investigating MC3T3 osteoblast-like cells adhesion, FN reorganisation and late matrix formation on the different substrates. Even if initial cell adhesion is excellent for every substrate, the size of the focal adhesion plaques increases as the size of the pits in the nanotopography does. This is correlated to FN reorganisation, which only takes places on the 29 and 45 nm deep pits surfaces, where enhanced late matrix production was also found.

Biological Activity of the Substrate-Induced Fibronectin Network: Insight into the Third Dimension through Electrospun Fibers

Fibronectin (FN) fibrillogenesis is a cell-mediated process involving integrin activation that results in conformational changes of FN molecules and the organization of actin cytoskeleton. A similar process can be induced by some chemistries in the absence of cells, e.g., poly(ethyl acrylate) (PEA), which enhance FN-FN interactions leading to the formation of a biologically active network. Atomic force microscopy images of single FN molecules, at the early stages of adsorption on plane PEA, allow one to rationalize the process. Further, the role of the spatial organization of the FN network on the cellular response is investigated through its adsorption on electrospun fibers. Randomly oriented and aligned PEA fibers were prepared to mimic the three-dimensional organization of the extracellular matrix. The formation of the FN network on the PEA fibers but not on the supporting coverglass was confirmed. Fibroblasts aligned with oriented fibers, displayed extended morphology, developed linearly organized focal adhesion complexes, and matured actin filaments. Conversely, on random PEA fibers, cells acquired polygonal morphology with altered actin cytoskeleton but well-developed focal adhesions. Late FN matrix formation was also influenced: spatially organized FN matrix fibrils along the oriented PEA fibers and an altered arrangement on random ones.

Simple coating with fibronectin fragment enhances stainless steel screw osseointegration in healthy and osteoporotic rats.

Metal implants are widely used to provide structural support and stability in current surgical treatments for bone fractures, spinal fusions, and joint arthroplasties as well as craniofacial and dental applications. Early implant-bone mechanical fixation is an important requirement for the successful performance of such implants. However, adequate osseointegration has been difficult to achieve especially in challenging disease states like osteoporosis due to reduced bone mass and strength. Here, we present a simple coating strategy based on passive adsorption of FN7-10, a recombinant fragment of human fibronectin encompassing the major cell adhesive, integrin-binding site, onto 316-grade stainless steel (SS). FN7-10 coating on SS surfaces promoted α5β1 integrin-dependent adhesion and osteogenic differentiation of human mesenchymal stem cells. FN7-10-coated SS screws increased bone-implant mechanical fixation compared to uncoated screws by 30% and 45% at 1 and 3 months, respectively, in healthy rats. Importantly, FN7-10 coating significantly enhanced bone-screw fixation by 57% and 32% at 1 and 3 months, respectively, and bone-implant ingrowth by 30% at 3 months compared to uncoated screws in osteoporotic rats. These coatings are easy to apply intra-operatively, even to implants with complex geometries and structures, facilitating the potential for rapid translation to clinical settings.

Surface mobility regulates skeletal stem cell differentiation

A family of polymer substrates which consists of a vinyl backbone chain with the side groups -COO(CH(2))(x)H, with x = 1, 2, 4, was prepared. Substrates with similar chemical groups but decreasing stiffness, characterized by their elastic modulus at 37 °C, as well as surface mobility, characterized by the glass transition temperature, were obtained. We have investigated whether these subtle variations in polymer chemistry lead to alterations in fibronectin (FN) adsorption and mesenchymal stem cell response. The same FN density was adsorbed on every substrate (∼450 ng cm(-2)) although the supramolecular organization of the protein at the material interface, as obtained with AFM, was different for x = 1 and the other two surfaces (x = 2, 4). Consequently, this allows one to investigate the effect of physical properties of the matrix on stem cell differentiation after ruling out any influence of protein activity. Cell adhesion was quantified by calculating the size distribution of focal adhesions. Mesenchymal stem cell differentiation to the osteoblastic lineage was determined by quantifying protein levels for osteocalcin, osteopontin and Runx2, in the absence of any additional osteogenic soluble factors in the culture media, but as a direct effect of material properties. The findings indicate the potential to modulate skeletal progenitor cell commitment to the osteoblastic lineage through surface mobility of the underlying material surface.

Subtle variations in polymer chemistry modulate substrate stiffness and fibronectin activity

A family of polymer substrates which consists of a vinyl backbone chain with the side groups –COO(CH2)xCH3, with x = 0, 1, 3, 5 was prepared. Substrates with decreasing stiffness, characterised by the elastic modulus at 37 °C, and similar chemical groups were obtained. Firstly, we have investigated whether these minute variations in polymer chemistry lead to differences in fibronectin (FN) adsorption: the same FN density was obtained on every substrate (450 ng cm−2) but the supramolecular organisation of the protein at the material interface, as obtained with AFM, was different for x = 0 and the other surfaces (x = 1, 3, 5). Consequently, this allows one to use a set of substrates (x = 1, 3, 5) to investigate the effect of substrate stiffness on cell behavior as the unique physical parameter, i.e. after ruling out any influence of the length of the side group on protein conformation. Moreover, the importance of investigating the intermediate layer of proteins at the cell-material interface is stressed: the effect of x = 0 and x = 1 on cell behavior cannot be ascribed to the different stiffness of the substrate anymore, since the biological activity of the protein on the material surface was also different. Afterwards, initial cellular interaction was investigated using MC3T3-E1 osteoblasts-like cells and focusing on actin cytoskeleton development, focal adhesion formation and the ability of cells to reorganize the adsorbed FN layer on the different substrates. Image analysis was used to quantify the frequency distribution of the focal plaques, which revealed broader distributions on the stiffer substrates, with formation of larger focal plaques revealing that cells exert higher forces on stiffer substrates.

Material-driven fibronectin assembly for high-efficiency presentation of growth factors.

Researchers develop a simple technique to enhance the activity of growth factors during tissue healing. Growth factors (GFs) are powerful signaling molecules with the potential to drive regenerative strategies, including bone repair and vascularization. However, GFs are typically delivered in soluble format at supraphysiological doses because of rapid clearance and limited therapeutic impact. These high doses have serious side effects and are expensive. Although it is well established that GF interactions with extracellular matrix proteins such as fibronectin control GF presentation and activity, a translation-ready approach to unlocking GF potential has not been realized. We demonstrate a simple, robust, and controlled material-based approach to enhance the activity of GFs during tissue healing. The underlying mechanism is based on spontaneous fibrillar organization of fibronectin driven by adsorption onto the polymer poly(ethyl acrylate). Fibrillar fibronectin on this polymer, but not a globular conformation obtained on control polymers, promotes synergistic presentation of integrin-binding sites and bound bone morphogenetic protein 2 (BMP-2), which enhances mesenchymal stem cell osteogenesis in vitro and drives full regeneration of a nonhealing bone defect in vivo at low GF concentrations. This simple and translatable technology could unlock the full regenerative potential of GF therapies while improving safety and cost-effectiveness.

Fibronectin distribution on demixed nanoscale topographies

Purpose It is known that surface nanotopography influences cell adhesion and differentiation. Our aim is to analyze the effect of nanoscale topography on fibronectin adsorption and, afterwards, on cell adhesion in order to rationalize the cell-material interaction by focusing on the state of the intermediate layer of adsorbed fibronectin at the material interphase. Methods Nanotopographic surfaces were produced by demixing of thin film polymer blends - PLLA and PS - during a high speed spin-casting process. Fibronectin (FN) was adsorbed on the different nanotopographies and the protein distribution was directly observed by atomic force microscopy (AFM). The fraction of the surface covered by the protein was quantified by image analysis, as well as the distribution of FN between peaks and valleys. Focal adhesion protein –vinculin- was immunostained and quantified by image analysis on the different nanoscale surfaces. Results Different nanoscale domains were obtained by changing the composition of the system within a height range of 3 nm to 30 nm. FN tends to adsorb on the peaks of nanoisland topographies, especially in compositions that did not enhance cell adhesion. Moreover, protein distribution between valleys and peaks alters the size of focal adhesion plaques, which grew larger on surfaces with an even distribution of fibronectin. Conclusions Our results suggest that the surface nanotopography is a key material property capable of influencing protein adsorption. Additionally, the distribution of the protein on the different samples was correlated to the initial ability of cells to adhere in terms of the size of the focal plaques.

Crosslinked fibrin gels for tissue engineering: Two approaches to improve their properties

Fibrin is a protein that can be used as an ideal scaffolding material to promote tissue regeneration. In order to enhance its physical properties in this study a natural crosslinker, genipin (GP), was employed with the aim to obtain a hydrogel with tuneable properties for tissue engineering applications. The fibrin gel was crosslinked by two different methods using four concentrations of GP to get a stable hydrogel network. Crosslinking density, mechanical properties, swelling, and enzymatic degradation of the hydrogels were tested for each GP content and method employed. The method I: Crosslinking after gel formation promotes a high crosslinking and retains the gel shape for long term whilst the method II: Simultaneous gel formation and crosslinking improves the mechanical properties of the gel. This study confirms the use of GP at different concentrations as a suitable crosslinker of fibrin that promotes the cellular viability of L929 for 21 days of in vitro culture.

Vitronectin alters fibronectin organization at the cell-material interface

Cells assemble fibronectin (FN) into fibrils in a process mediated by integrins. For this process to occur, it is known that the presence of other serum proteins is necessary. However, the individual effect of these proteins on FN fibrillogenesis has not been addressed so far. In this study, the effect of vitronectin (VN), an ECM adhesion protein, on material-driven FN fibrillogenesis and cell-mediated FN reorganization is investigated. Poly(ethyl acrylate), PEA, which has previously shown the ability to induce the organization of FN into well-developed physiological-like networks upon adsorption, was employed as a material substrate. FN adsorption, cell adhesion and cellular FN reorganization in the presence or absence of VN were studied. Both FN surface density, quantified via western blot, and its distribution on PEA surfaces, determined via atomic force microscopy, were altered when FN was adsorbed competitively with VN at certain compositions. Moreover, the presence of VN on the material surfaces enhanced cell-mediated FN reorganization and secretion, in comparison with the process which took place in the presence of serum proteins.

Molecular assembly and biological activity of a recombinant fragment of fibronectin (FNIII7–10) on poly(ethyl acrylate)

Fibronectin (FN) fibrillogenesis is a cell-mediated process involving integrin activation that results in conformational changes of FN molecules and the organization of actin cytoskeleton. A similar process can be induced by some particular chemistries in the absence of cells, e.g., poly(ethyl acrylate) (PEA), which enhance FN-FN interactions leading to the formation of a biologically active network on the material surface. We have investigated the organization of a recombinant fragment of fibronectin (FNIII(7-10)) upon adsorption on this particular chemistry, PEA. Atomic force microscopy (AFM) was used to identify individual molecules of the fragment after adsorption, as well as the evolution of the distribution of adsorbed molecules on the surface of the material as the concentration of the adsorbing solution increased. Globular molecules that turn into small aggregates were found as a function of solution concentration. Above a threshold concentration of the adsorbing solution (50 microg/mL) an interconnected network of the FNIII(7-10) fragment is obtained on the material surface. The bioavailability of specific cell adhesion domains, including RGD, within the molecules was higher on PEA than on the control glass. The biological activity of the fragment was further investigated by evaluating focal adhesion formation and actin cytoskeleton for MC3T3-E1 osteoblast-like cells. Well-developed focal adhesion complexes and insertions of actin stress fibers were found on PEA in a similar way as it happens in the control SAM-OH. Moreover, increasing the hydrophilicity of the surface by incorporating -OH groups led to globular molecules of the fragment homogeneously distributed throughout the surface; and the cell-material interaction is reduced as depicted by the lack of well-developed focal plaques and actin cytoskeleton.