Harnessing nano-bio-engineering tools for tissue engineering and regenerative medicine applications

Resume : Mimicking the natural extracellular matrix (ECM) has become the Holy Grail in the development of new scaffolds for regenerative medicine. However, that statement of intent turns out to be very elusive due to the complexity of the natural ECM as well as the complex relationship existing between cells and ECM. Properties of the ECM that must be taken into consideration span in a broad range from mechanical properties1 (proper elasticity) and microstructure (fibrous structure) to much more specific bioactivities such as cell adhesiveness, protease sensitiveness2, matrikine release and spatio-temporal control of released factors. Added to that, we have to keep in mind that this scaffold will be somehow eventually implanted and it will have to cope with potential rejection by immune system of the host. While, generally speaking, this task is huge and very challenging, considering the chance to create such scaffold under and injectable approach seems to be discouraging. Evidently, the possibility of having a system that is liquid and then can be injected through a fine needle to make later a solid scaffold with the adequate microstructure, mechanical properties, and desired bioactivities must rely on powerful self-assembling processes that ideally drive both the gelation of the system as well as the development of the proper microstructure. This task could be at reach by exploiting the potential of recombinant technology and the use of recombinant protein polymers or, more correctly, recombinamers as the source of the complex materials that could be the basis of truly advanced artificial ECMs. Over a precise and tailored design of the molecule, or rather the gene that codes it, it is feasible to build a recombinamer that contains all the functions required, including capacity to self-assemble and form stable gels upon injection. Among the different option within the area of recombinamers Elastin-like recombinamers (ELR) stand out. In this communication, different ELRs are being reported sowing different approaches to achieve injectable artificial ECM. The full range of ELRs designed and produced for this purpose include: • Injectable two-component ELR for covalent cross-linking. • Thermosensitive amphiphilic multiblocks for physical cross-links • Advanced beta-sheet forming ELRs for developing fibrous gels on setting. • Smart surfaces for cell harvesting The first group relies on catalyst-free click chemistry reactions. Those materials have proved exceptional performance in different applications, especially in the cardiovascular area. The second group relies on the thermal sensitiveness of the ELR. Due to their basic amphiphilic block nature and the ability to show a self-aggregating effect induced by an increase in the temperature, those materials have the chance to be liquid below body temperature and make stable gels at BT. Those injectable scaffolds have proven their efficiency in musculoskeletal applications, particularly in the total regeneration of osteochondral injuries. That is also relevant its role in designing biomimicked mineralization templates and scaffolds. The third group is based on ELRs that, along with the elastin-like main contribution and all the epitopes rendering cell-adhesiveness, protease sensitiveness, etc., included epitopes taken from silk that endow the system with a capacity to self-organize in a regular microfibrilar structure resembling that of the natural ECM proteins such as collagen. Final example demonstrate how thermal sensitiveness and cell-adhesion capabilities can be integrated to render a smart surface with the ability to switch between a cell-repellent to a cell-adherent by simply changing the temperatures within a cell-viable range Detailed description of the materials developed for the three classes and of their performance in different tissue engineering and regenerative medicine applications will be given on site.

Resume : The generation of functional surfaces via self-assembly, and the spatial control of their functionality, are currently of great interest in nanoscience and nanotechnology. In this work I present techniques based on the combined use of lithographic patterning and bio-molecular self-assembly to control the immobilization of biomolecules in arrayed nano-domains. In particular, I will show how the specific design of metal nanodot arrays, and their selective biofunctionalisation, enables the parallel monitoring of biological activity in real-time and with single-molecule resolution via conventional epi-fluorescence microscopy imaging. I will show that it is possible to simultaneously characterize hundreds of protein/DNA binding events at the single-molecule level. I will also discuss the use of our nanopatterned biomimetic surfaces to probe the importance of transmembrane proteins (integrins) clustering and geometric arrangement of binding sites in the formation of cell focal adhesions. I will further show how by a complementary nanofabrication ad surface functionalisation strategy it is possible to produce nanoaperture arrays as zero-mode waveguides for single-molecule fluorescence biological investigations. Finally I will demonstrate how the aforementioned approaches allowed us to also produce highly ordered, self-assembled arrangements of nano-objects, ranging from DNA nanostructures to bio-inorganic assemblies, for a variety of nanoscale investigations.

Resume : Organic nanotechnology is clearly a new front in the field of molecular self-assembly of new structures and composite families at the nano-scale. Our works on the mechanism of aromatic peptide self-assembly, lead to the discovery that the diphenylalanine recognition motif self-assembles into peptide nanotubes with a remarkable persistence length. Other aromatic homodipeptides (including those with non-coded amino acids as DOPA) could self-assemble in nano-spheres, nano-plates, nano-fibrils and hydrogels with nano-scale order. The modification of peptide building blocks with the Fmoc protecting group allows the formation of hydrogels with nano-scale order. We demonstrated that the peptide nanostructures have unique chemical, physical and mechanical properties including ultra-rigidity as aramides, semi-conductive, piezoelectric and non-linear optic properties. We also demonstrated the ability to use these peptide nanostructures as casting mould for the fabrication of metallic nano-wires and coaxial nano-cables. The application of the nanostructures was demonstrated in various fields including electrochemical biosensors, tissue engineering, and molecular imaging. We had developed ways for depositing of the peptide nanostructures and their organization. We had use inkjet technology as well as vapour deposition methods to coat surface and from the peptide “nano-forests”. We recently demonstrated that even a single phenylalanine amino-acid can form well-ordered fibrilar assemblies of distinct electron diffraction pattern and toxic properties. Selected References: 1. Reches, M. and Gazit, E. (2003) Casting Metal Nanowires within Discrete Self-Assembled Peptide Nanotubes. Science 300, 625-627. 2. Reches, M. and Gazit, E. (2006) Controlled Patterning of Aligned Self-Assembled Peptide Nanotubes. Nature Nanotechnology 1, 195-200. 3. Adler-Abramovich L., Aronov D., Beker P., Yevnin M., Stempler S., Buzhansky L., Rosenman G. and Gazit E. (2009) Self-Assembled Arrays of Peptide Nanotubes by Vapour Deposition. Nature Nanotechnology 4, 849-854. 4. Mahler, A., Reches, M., Rechter, M., Cohen, S. and Gazit, E. (2006) Rigid, Self-Assembled Hydrogel Composed of a Modified Aromatic Dipeptide. Advanced Materials 18, 1365-1370. 5. Adler-Abramovich, L., Vaks, L., Carny, O., Trudler, D., Frenkel, D., & Gazit, E. (2012) Phenylalanine Assembly into Toxic Fibrils Suggests Amyloid Etiology in Phenylketonuria. Nature Chem. Biol. 8, 701-706. 6. Ischakov, R., Adler-Abramovich, L., Buzhansky, L., Shekhter, T., & Gazit, E. (20013) Peptide-based Hydrogel Nanoparticles as Effective Drug Delivery Agents. Bioorg. Med. Chem. 21, 3517-3522. 7. Fichman, G., & Gazit, E. (2014) Self-Assembly of Short Peptides to Form Hydrogels: Design of Building Blocks, Physical Properties and Technological Applications. Acta Biomater. 10, 1671-1682. 8. Fichman, G., Adler-Abramovich, L., Manohar, S., Mironi-Harpaz, I., Guterman, T., Seliktar, D., Messersmith, P.B., & Gazit, E. (2014) Seamless Metallic Coating and Surface Adhesion of Self-Assembled Bioinspired Nanostructures Based on Di-(3,4-dihydroxy-l-phenylalanine) Peptide Motif. ACS Nano (in press).

Resume : In this talk, concepts of making materials, which mimic the structure and function of the biological materials through programmed self-assembly of small molecules and their applications in functional materials. The self-assembly mechanism that forms the supramolecular aggregates involves non-covalent interactions such as hydrogen bonds, electrostatic and hydrophobic interactions. Diverse functional groups were incorporated into nanostructures for functional materials applications.

Resume : Introduction: Mimicking the biophysical and biochemical features of the extracellular matrix (ECM) in its topography and biofunctionality is imperative for the development of a functional engineered construct for musculoskeletal tissue regeneration. Recent advancements in nano-bioengineering techniques such as electrospinning have made available nano-textured-biomaterials that closely imitate the architecture of native extracellular matrix supra-molecular assemblies. To facilitate clinical translation of functionalised nano-textured scaffolds that will provide cell guidance and ultimately functional neo-tissue formation, it is essential to fully comprehend the influence of nano-fibrous hierarchical carbohydrate functionalised scaffolds on cellular response. Hyaluronic Acid is the most abundant glycosaminoglycan in the ECM of connective tissue; it provides structural support, hydration and enriches the growth factors release. Therefore combining a poly(lactic-co-glycolid acid) (PLGA) aligned scaffold and their functionlisation with carbohydrates will provide a novel therapeutic approach for musculoskeletal tissue engineering. Methods: Using electrospinning, functionalized scaffolds were designed with both hyaluronic acid and a synthetic alternative, Ficoll. Subsequently, primary human tenocyte and osteoblast attachment, alignment, metabolic activity, viability and gene expression was examined on days 4, 7, and 14 for tenocytes and on days 1, 11 and 21 for osteoblasts. Results: Scanning electron microscopy showed highly aligned fibers, which mimic the alignment of the extracellular matrix proteins in the native tissues, therefore enhancing cellular alignment for both cell types. Tenocytes and osteoblasts were also viable and metabolically active on these substrates demonstrating their proliferation over the 7 and 11 days, respectively. Conclusion: This study demonstrates electrospun nanofibrous functionalised scaffolds promote tenocyte and osteoblast cell viability and proliferation. Currently, the maintenance of cell phenotype and expression of ECM proteins is being evaluated to clearly indicate the potential of these functionalised scaffolds for musculoskeletal regeneration.

Resume : Introduction The replacement, repair and restoration of tissue function can be accomplished best by recruiting cells’ inherent proficiency to create their own tissue-specific extracellular matrix with a precision and stoichiometric efficiency. However, bereft of their tissue context, cells perform poorly; lose the therapeutic potential. Specifically to corneal tissue engineering, the cells suffer from slow deposition of extracellular matrix (ECM), and the prolonged culture time (35-84 days) results in phenotype loss (1-2). The use of low oxygen tension in keratocytes culture has been shown to protect them stress induced death and TGF-β1-induced myofibroblast transformation (3). Recent studies also have shown that macromolecular crowding (MMC); the addition of macromolecules in the medium enhances ECM deposition, and also facilitates the cell phenotype (4-5). In the present study, we describe the influence of MMC on human corneal fibroblast (HCFs) in the presence of low oxygen tension to evaluate the ECM deposition and cells phenotype restoration. Experimental Methods HCFs (Innoprot, Spain) were cultured for in the presence of 0.5, 2.0 and 20% oxygen. The medium was replaced with fresh medium containing galactose derivative macromolecules; carrageenan (75µg/ml) after 24 hours of cell seeding and incubated for 14 days as mentioned above. The stromal ECM deposition was assessed using SDS-PAGE/densitometry and immunocytochemistry. The cellular metabolism and proliferation were evaluated using alamarBlue® assay and DAPI counting method respectively. One-way ANOVA –or the Kruskal-Wallis test for non-parametric analysis, followed by post hoc tests performed for the statistics. The comprehensive genomic and proteomic studies to evaluate the cell phenotype and ECM deposition are underway. Results & Discussion MMC of HCFs significantly enhanced the deposition of ECM, notably collagen I and fibronectin, as demonstrated in SDS-PAGE/densitometry and immunocytochemistry. The collagen type I further increased in the presence of hypoxia at 2.0% oxygen tension (p<0.05). The almarBlue® assay and DAPI staining confirmed the high metabolic with good cell proliferation (>80% in all cases, p< 0.05). The phase contrast microscopy further revealed that the cell morphology remain unchanged in the presence of hypoxia and MMC up to day 14. These studies confirmed the non cytotoxic nature of MMC on HCFs. Conclusion Simultaneous application of hypoxia and MMC emulate the stromal microenvironment in HCFs culture. Thus, the application of macromolecular crowding in the presence of hypoxic conditions is a potential tool in corneal stromal regeneration. References 1. Proulx et al., Mol. Vis., 2010, 16: 2192 2. Vrana et al., Inv. Ophth Vis. Sci., 2008, 49: 5325 3. Xing and Bonnano., Mol. Vis., 2009, 15:1020 4. Satyam et al., Adv. Mat., 2014, 19: 3024 5. Zeiger AS. et al., PLoS One. 2012, 7:e37904 Acknowledgments The author would like to thank the College of Engineering and Informatics at NUI Galway and Science Foundation Ireland (09/RFP/ENM2483).

Resume : Collagen-based materials, such as tissue graft or reconstituted scaffolds, are attractive for biomedical applications due to be well-tolerated, to provide an acceptable cell support and host response. Nonetheless, the main shortfalls of collagen films are the low mechanical stability and the low resistance to enzymatic degradation. Given that native lysyl-oxidase cross-linking of collagen does not occur in vitro, exogenous cross-linking methods are under investigation to increase the mechanical and enzymatic stability1. However, chemical methods based on aldehydes, epoxides and carbodiimide are often associated with cytotoxicity, macrophage activation and foreign body reaction2. Alternative cross-linking methods, based on plant extracts and polyethylene glycol (PEG) have attracted great scientific attention the recent years, due to their low cytotoxicity3,4. Herein, it is hypothesised that collagen films can be optimally cross-linked with PEG and plant extracts to induce adequate stability, whilst avoiding toxicity and macrophage activation. The specific objective is to optimise collagen films stability modulating macrophage response without compromising cell toxicity. Collagen films were cross-linked with 0.625% glutaraldehyde, 50 mM 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), 1 mM 4-arm PEG succinimidyl glutarate MW 10,000 (4s-StarPEG), 0.625% Genipin or 0.1% Oleuropein. As control, non-cross-linked films were used. Films were assessed by SEM, mechanical testing, DSC, swelling and collagenase assays. Cytotoxicity was assessed using WS1 human skin fibroblast, and subsequent cell morphology, metabolic activity (alamarBlue™) and cell proliferation (PicoGreen®) analysis. In vitro macrophage response was assessed using human monocyte-derived THP-1 cells and subsequently by morphology, alamarBlue™, PicoGreen and multiplex assay for the main inflammatory cytokines. As control, macrophages seeded on TCP were activated by lipopolysaccharide. Glutaraldehyde, 4s-StarPEG and genipin significantly increased enzymatic resistance (p<0.001); however, only 4s-StarPEG increased mechanical properties significantly (p<0.05). Cross-linking did not show any negative impact on fibroblast response. Although multiplex cytokine analysis is not completed, preliminary results have demonstrated that all cross-linked films reduced macrophage population (p<0.05). Moreover, genipin and EDC promoted macrophage metabolic activation that may be related to the high stiffness induced by genipin, and the collagen structural variation induced by the direct addition of EDC before collagen reconstitution; structural variation was evident by the denaturation temperature decrease. REFERENCES. 1: Zeugolis D. J Biomed Mat Res. 89, 895-908, 2009. 2: Brown B. Acta Biomater. 9,4948-55, 2013. 3: Collin E. Biomaterials. 32, 2862–70, 2011. 4: Rowland C. Biomaterials. 34, 5802-12, 2013.

Resume : Introduction Tissue grafts are commonly used for tendon repair, however they lack mechanical stability and often associated with donor morbidity. Crosslinking the collagen fibres could potentially increase the mechanical properties and strength of the scaffolds. However, based on previous studies, cross-linkers such as GTA are cytotoxic for tenocytes and therefore alternative cross-linkers are required1. We hypothesise that StarPEG crosslinking of collagen fibres will optimally stabilise collagen tendon scaffold, providing sufficient mechanical stability and maintain tenocyte function, and promote tendon regeneration in vivo. Materials and Methods Collagen fibres were extruded using an established protocol. Fibres were cross-linked with 4 arm StarPEG. GTA and EDC/NHS cross-linked fibres were used as negative control and non-cross-linked fibres were used as a baseline control1. Hydrothermal stability was tested by differential scanning calorimetry. Mechanical properties were assessed by tensile tensting. Enzymatic degradation was assessed by collagenase assay and levels of free amines assessed by ninhydrin. Fibres were arranged into bundles and subsequently pre-incubated in complete media for 24 hours. Human tenocytes (passage 2-3) were seeded onto collagen fibre bundles at concentration of 10x3cells/cm2. Media changes were performed every other day. At 3, 7, 14, 21, 28 day time points in vitro, Rhodamine Phalloidin and DAPI staining were used to assess the alignment, elongation and morphology of cells on fibre bundles and alamarBlue was used to assess metabolic activity. Tenocyte markers including decorin, scleraxis, tenomodulin and tenascin-C were assessed using immunocytochemistry and gene analysis. SEM was used to examine the structure of the fibres. Results Tensile testing and DSC showed an increase in mechanical and thermal properties compared to the non-cross-linked fibres. Over time the collagen fibre bundles degraded into small fragments as assessed by collagenase assay. Over time the collagen fibre bundles degraded into small fragments as assessed by collagenase assay. Ninhydrin showed a low level of free amines in the StarPEG cross-linked fibred. Elongation of tenocytes on collagen fibre bundles was observed. Tenocytes aligned in parallel orientation along on the StarPEG collagen fibre bundles. Immunocytochemistry showed the expression of tenogenic markers specific to tenocytes. Discussion and Conclusion StarPEG crosslinking provided collagen fibres with sufficient mechanical properties and stability for use as a tendon graft substitute. In vitro assessment of tenocytes showed proliferation and alignment on StarPEG collagen fibres, indicating tenocyte function was not compromised compared to GTA and EDAC-HCL. Ongoing and future in vivo work is a sheep Achilles tendon model. Reference 1. Zeugolis, Dimitrios I., Gordon R. Paul, and Geoffrey Attenburrow. Journal of Biomedical Materials Research Part A 89.4 : 895-908.2009 2. Gough, J. E., Scotchford, CA., Downes, S. J Biomed Mater Res 61(1): 121-130.2002 Acknowledgments Teagasc, Irish Agriculture and Food Development Authority, Funding agency

Resume : Recently, Mayor et al. [1] investigated the charge transport properties of seven rigid rod dithiol-terminated molecules between gold leads. In the same vein, the charge transport in field-effect transistors (FETs) has been investigated both experimentally and computationally. [2, 3, 4, 5] In these materials, the charge transport occurs mainly in the first molecular monolayer at the vicinity of the dielectric interface. This makes self-assembled monolayer FETs (SAMFETs) an ideal device to study charge transport in organic materials. These progress in building new organic molecular devices make it essential to understand the charge transport not only through the molecular junction but also at the interface between the organic compound and the inorganic electrode. This requires insight that atomistic resolution quantum calculations provide. The present work involves studying by computational means the charge transport through the pi-system of an aromatic junction between gold leads. Particular attention is paid to the organic-inorganic bond and to the surface states of the lead. This is in view of applications for new electronic devices and in broader fashion to potential applications for the fine control of the adsorption/ desorption of proteins on the surface of organic flms self-assembled on metal surfaces, which could provide alternatives to hydrogels for truly nanoengineered electro-responsive scaffolds for tissue engineering.

Resume : Following implantation of a recording or stimulating neural electrode, glial scar formation at the electrode–tissue interface accelerates neural loss and increases electrical signal impedance, compromising the efficiency of the stimulating system1. Studies with conducting polymers (CPs) as functional electrode coatings have shown to enhance tissue/electrode integration and electrode performance in situ through reduced impedance and the presentation of neurotropic moeties2. Electrodeposition is routinely employed for the formation of poly(3,4ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT:PSS) conducting polymer electrode coatings. However, electrodeposition parameters and their influence on the electrical, morphological, chemical and cytocompatibility properties of electrode films needs to be addressed. This study developed PEDOT:PSS polymeric films with altered roughness, thickness and conducting profiles by altering the current density of film deposition. Furthermore, the adhesion and proliferation of SH-5YSY cells on the surface of these films formed at different current densities indicated the cytocompatibility of this material and no toxicity was detected. In addition, neurospecific differentiation was achieved as cells began to show neuritic outgrowth and cell elongation up to period of fourteen days in culture. Designing an optimized electrode coating material will require a trade-off between desired electrical, morphological, chemical and biological properties. This work was funded through Science Foundation Ireland 11/SIRG/B2135. Thanks to Dr Donal Leech and the Biomolecular Electronics Research Laboratory, Ms Sarah Burke, Ms Caroline Tachet, and Dr Leo Quinlan. REFERENCES [1] Durand DM, Ghovanloo M, et al. J Neural Eng. 2014;11:020201. [2] Green RA, Lovell NH, et al. Biomaterials. 2008;29:3393-9.

Resume : The physical properties of a cell's environment are important factors in determining cell behavior and ultimately, phenotype. Spatial organization of extracellular matrix (ECM) molecules and matrix rigidity, in particular, have been associated with major changes in cell morphology, function and behavior. In order to understanding how cells sense these factors at the nanoscale, and how these factors affect cell function, we have developed new nanofabricated surfaces in which these physical characteristics of a cell’s environment are simulated. The first type of surface combines electron beam and nanoimprint lithography and self-aligned pattern transfer to create metallic nanodot anchors ~ 2 – 10 nm in size, to which a ligand of choice can be selective bound. Virtually any geometric arrangement is possible, ranging from individual molecular binding sites to small clusters and extended hexagonal close packed arrays, with spacings ranging from 40 nm and below to 1 μm and above. In order to probe the geometric requirements for cellular adhesion, we functionalized the nanodots with integrin binding peptides. Cell spreading and motility assays performed using 3T3 fibroblasts on arrays in which binding site spacing, density and number were independently varied. Cell spreading efficiency was markedly enhanced for clusters comprising at least 4 liganded sites spaced ≤ 60 nm apart, with no noticeable dependence on global density. This points to the existence of a minimal matrix adhesion unit defined in space and stoichiometry. We recently employed a variation on this platform to study the geometric requirements for T-cell stimulation. By systematically varying the spacing and cluster size of T-cell receptor (TCR) binding sites, we were able to determine the minimal conditions that support T-cell signaling. Enhanced T-cell stimulation was observed below a threshold spacing ~ 60 nm; TCR signaling increased with decreasing spacing below this threshold. For clusters with these spacings the formation of the stereotypic “bullseye” geometry that characterizes the immune synapse became evident. A second type of surface consists of elastomeric substrates with locally variable rigidity. We have found that exposure of polydimethyl siloxane (PDMS) to an electron beam alters the rigidity of the elastomer, with the modulus of the exposed regions increasing with the applied electron dose. Finite element analysis of nanoindentation measurements performed on irradiated PDMS films show that in a thin layer near the film surface, where approximately 90% of the electron energy is absorbed, the Young’s modulus of the elastomer undergoes a significant increase as a function of electron beam dose. Human skeletal stem cells plated on soft PDMS surfaces patterned in this manner displayed a distinct preference for the more rigid, exposed regions, forming focal adhesion nearly exclusively there. Furthermore, focal adhesion formation diminished significantly as the size of the exposed features was reduced below 1 μm, indicating that there is a length scale for cellular rigidity sensing, with the critical length in the range of a few hundred nanometers. Interestingly, for T-cells plated on these surfaces, a threshold for adhesion between 1 and 2 μm was observed. T-cell stimulation was also enhanced on surfaces patterned with heterogeneous rigidity. By adapting the tools of nanomanufacturing to cellular systems, we are able to define important physical parameters that can control aspects of cell function and behavior and will help identify conditions under which these functions may be altered. Potential applications range from therapeutic treatments that block metastasis to the development of new adoptive immunotherapies, as well as the development of new guidelines for the design of tissue scaffolds that can optimize healing without scarring.

Resume : Bone is the second most common transplantation tissue after blood. While the use of bone grafts remains the optimum choice, the problems associated with them has made the use of synthetic implants ever more popular. Over the last decade, there has been a lot research into the development of engineered new bone, to replace damaged tissue. An important part of this research effort has gone into the development of three-dimensional porous scaffolds, to support and guide the new cells. Here, we describe our research into the fabrication and evaluation as bone scaffolds of 3D biodegradable structures made using Direct fs Laser Writing (DLW). The material we use is a photostructurable polylactide-based material (PLA) synthesized for this purpose [1]. We test its degradation in vitro in PBS and we show that the material looses one third of its weight after six weeks, therefore allowing the slow release of an implanted scaffold. We demonstrate the fabrication of artificial scaffolds with precisely controlled geometries and different pore sizes and we test them for up to eight weeks using the mouse pre-osteoblastic cell line MC3T3-E1 [2]. Our results show good cell adhesion and a preference to scaffolds with 86% porosity, compare to other porosities studied. Our study shows that DLW is a suitable technique for the fabrication of 3D biodegradable scaffolds for bone repair and other tissue engineering applications. References 1 V. Melissinaki, A.A. Gill, I. Ortega, M. Vamvakaki, A. Ranella, J.W. Haycock, C. Fotakis, M. Farsari, F. Claeyssens, “Direct laser writing of 3D scaffolds for neural tissue engineering applications”, Biofabrication, 3 045005 (2011) [2] K. Terzaki, M. Kissamitaki, A. Skarmoutsou, C. Fotakis, C.A. Charitidis, M. Farsari, M. Vamvakaki, M. Chatzinikolaidou, “Pre-osteoblastic cell response on three-dimensional, organic-inorganic hybrid material scaffolds for bone tissue engineering” J Biomed Mater Res Part A, DOI:1083689 (2013)

Resume : A platform technology that brings together the toughness of cellulose nano-fibers from the plant kingdom, the remarkable elasticity and resilience of resilin that enables flees to jump as high as 400 times their height from the insect kingdom, and the adhesion power of DOPA, the functional molecule of mussels that enable it to bind tightly under water to organic and inorganic matter from the marine kingdom. Resilin is a polymeric rubber-like protein secreted by insects to specialized cuticle regions, in areas where high resilience and low stiffness are required. Resilin binds to the cuticle polysaccharide chitin via a chitin binding domain and is further polymerized through oxidation of the tyrosine residues resulting in the formation of dityrosine bridges and assembly of a high-performance protein-carbohydrate composite material. Plant cell walls also present durable composite structures made of cellulose, other polysaccharides, and structural proteins. Plant cell wall composite exhibit extraordinary strength exemplified by their ability to carry the huge mass of some forest trees. Inspired by the remarkable mechanical properties of insect cuticle and plant cell walls we have developed novel composite materials of resilin and Nano-Crystalline Cellulose (resiline-NCC) that display remarkable mechanical properties combining strength and elasticity. We produced a novel resilin protein with affinity to cellulose by genetically engineering a cellulose binding domain into the resilin. This CBD-Resilin enable, interfacing at the nano-level between the resilin; the elastic component of the composite, to the cellulose, the tough component. Furthermore, chemical and enzymatic modifications of the composite are developed to produce DOPA- Resiline-NCC which confers adhesive and sealant properties to the composite.

Resume : Research in our laboratory is dedicated to understanding and applying basic cell biological principles in the field of biomedical engineering, in particular in the regeneration of bone tissue. The research program is characterized by a holistic approach to both discovery and application, aiming at combining high throughput technologies, computational modeling and experimental cell biology to streamline the wealth of biological knowledge to real clinical applications. In my seminar I will discuss our recent work on signal transduction in mesenchymal stem cells and osteoblast, induced by either candidate small molecules or molecules extracted from small molecule screens. Enhanced performance of the cells in vitro led to increased tissue formation in vivo. I will also discuss the interaction of cells with biomaterials. For instance, we are interested in the bone-inducing properties of a subset of porous calcium phosphate ceramics and show how through reverse engineering, we are uncovering an interesting and complex response of cells to materials. Inspired by this, we have started to design high throughput screening strategies of biomaterials libraries, and in particular libraries of surface topographies. Using a design algorithm, we have generated numerous different patterns, which can first be reproduced on a silicon mold and then imprinted onto polymers using microfabrication. After cell seeding, we use quantitative high content imaging and machine learning algorithms to characterize the response of the cells to the thousands of different surfaces and learn more about the relation between surface topography and cell response. For instance, we have screened for surfaces which stimulate osteogenic differentiation of mesenchymal stem cells, surfaces which could potentially be applied on the surface of materials used in orthopedic research.

Resume : The control of the outgrowth of neuronal cultured cells is of critical importance in a wide spectrum of neuroscience applications including tissue engineering scaffolds and neural electrodes. However, the study of neuron cell outgrowth on more complex topographies remains limited. Phenotype alteration of stem cells and differentiated neuronal cells cultured on traditional flat substrates that lack structural cues, emphasize the necessity to shift from 2D to 3D cell culture models. The aim of the present study was to investigate the cellular response to topographical cues both at the micro and the nanoscale. In particular, we have previously reported that the artificial surfaces obtained by direct femtosecond laser texturing of solid surfaces in reactive gas atmosphere exhibit roughness at both micro- and nano-scales that mimics the hierarchical morphology of natural surfaces [1]. Variation of the laser fluence, alters the surface morphology, while the respective patterned substrates exhibit different roughness ratios and wettabilities. Cells with nerve cell phenotype were cultured on the substrates. Results on the culture of PC12 cells showed that the morphology of microspiked surfaces alone can be used for directional cytoskeletal rearrangement and subsequent differentiation into a neuronal phenotype. Besides this, the experiments with DRG/SCG nerve cells showed a good attachment, outgrowth and network formation and depending on the substrate morphology there was a differential orientation of the cells. In particular, cells were randomly oriented on low roughness surfaces, whereas there was a trend for parallel alignment on the intermediate and high roughness substrates. Our results indicate a method to tune cell responses by proper selection of the surface free energy of the substrate and may be promising for the design of cell culture platforms with controlled differentiation environment. [1] V. Zorba, E. Stratakis, M. Barberoglou, E. Spanakis, P. Tzanetakis, S. H. Anastasiadis and C. Fotakis, Advanced Materials 20, (2008), 4049.

Resume : Abstract: Stimuli responsive matrices are the epitome of "smart" biomaterials, and as such they are the subject of a great deal of research in the musculoskeletal repair field. The ability to control at the micro- and nano-scales, spatially and temporally, the three-dimensional (3D) environments in which cells or drugs are encapsulated allow for deciphering the influence of chemical, structural and mechanical cues in defined conditions, and potentially design instructive mimicking matrices for cellular therapies as well as effective drug delivery systems. Of particular interest are the injectable polymeric solutions able to increase their stiffness upon temperature rise. Their combination with hyaluronic acid, also commonly referred to as hyaluronan, is a promising approach to synergise the polymers respective properties. In fact, hyaluronan is a shear thinning biopolymer with ubiquitous biological roles, non-immunogenic. In addition, it is a natural component of the extracellular matrix with a central role in wound healing and the regeneration of connective tissues. Thus, synthetic strategies to create thermoreversible derivatives of hyaluronan will be reported together with the ability to biofunctionalise hyaluronan with bioactive, biomimetic, multifunctional, and biodegradable well define nanostructures. The potential of thermoreversible hyaluronan-based hydrogels to modulate the microenvironment of cells in the context of musculoskeletal repair will be reported as well as the ability to deliver growth factors and small molecules.

Resume : Surface topography is well known to influence the fate of stem cells. There are many ways to generate topographies but electron beam lithography, a technology used in the semiconductor industry, has proven particularly useful to make very specific patterns with a high degree of control, e.g. dimensions or geometric arrangement. Specifically we have shown that highly regular nanopatterns are capable of maintaining multipotency of mesenchymal stem cells in vitro. Thus this unique topography mimics the natural niche in which the cells normally reside. On the other hand, if a small amount of disordered is added to the pattern, without total disorder, the stem cells will specifically different onto bone forming cells (osteogenic). This delicate control can only be achieved by electron beam lithography. The master patterns are manufactured on semiconductor substrates which is not the ideal material for neither in vitro nor in vivo testing. To this end we apply a replication process into polymeric substrates by injection moulding. To accelerate the discovery of influential patterns, we have constructed a high-content screening platform where we systematically can design and fabricate 100s of different patterns. Traditionally, such array platforms are manufactured in a serial and discrete manner, however, we have developed an alternative manufacturing route where such arrays can be made in a single step. The platform is also compatible with our injection moulding process and thus can provide us with large quantities of samples. The process enables us to make fully patterned microscope slides in 15 sec and with an extremely high reproducibility between the parts. This high reproducibility and rapid manufacturing rate means it provides an ideal means for translation of our findings to medical devices.

Resume : To explore the most dynamic cross field of nanotechnology and life science, and to find a more practical method of fabricating nanodevices on a very large scale with advantages of low manufacturing cost, high reliability and reproducibility, we successfully fabricated a submicron IrO2 nanowire array biosensor platform by conventional complementary metal-oxide-semiconductor (CMOS) process. Single crystal IrO2 nanowire array was grown uniformly on a 6-in. wafer surface by chemical vapor deposition (CVD) method and patterned into submicron array clusters. The obtained clusters were positioned in a designed pattern similar to a multiple electrode array (MEA) format, and each was individually addressed. The fabrication method was found to be reliable, low cost and robust. The final chip showed excellent transparency, functionality and durability. These chips have been used for neuron cell stimulation and protein sensing, the experiment results will be presented.