Control Cell Behavior Research Articles

Inhibiting clathrin-mediated endocytosis of the leucine-rich G protein-coupled receptor-5 diminishes cell fitness

The leucine-rich G protein-coupled receptor-5 (LGR5) is expressed in adult tissue stem cells of many epithelia, and its overexpression is negatively correlated with cancer prognosis. LGR5 potentiates WNT/β-catenin signaling through its unique constitutive internalization property that clears negative regulators of the WNT-receptor complex from the membrane. However, both the mechanism and physiological relevance of LGR5 internalization are unclear. Therefore, a natural product library was screened to discover LGR5 internalization inhibitors and gain mechanistic insight into LGR5 internalization. The plant lignan justicidin B blocked the constitutive internalization of LGR5. Justicidin B is structurally similar to more potent vacuolar-type H+-ATPase inhibitors, which all inhibited LGR5 internalization by blocking clathrin-mediated endocytosis. We then tested the physiological relevance of LGR5 internalization blockade in vivo A LGR5-rainbow (LBOW) mouse line was engineered to express three different LGR5 isoforms along with unique fluorescent protein lineage reporters in the same mouse. In this manner, the effects of each isoform on cell fate can be simultaneously assessed through simple fluorescent imaging for each lineage reporter. LBOW mice express three different forms of LGR5, a wild-type form that constitutively internalizes and two mutant forms whose internalization properties have been compromised by genetic perturbations within the carboxyl-terminal tail. LBOW was activated in the intestinal epithelium, and a year-long lineage-tracing course revealed that genetic blockade of LGR5 internalization diminished cell fitness. Together these data provide proof-of-concept genetic evidence that blocking the clathrin-mediated endocytosis of LGR5 could be used to pharmacologically control cell behavior.

Solution blow spinning fibres: New immunologically inert substrates for the analysis of cell adhesion and motility.

The control of cell behaviour through material geometry is appealing as it avoids the requirement for complex chemical surface modifications. Significant advances in new technologies have been made to the development of polymeric biomaterials with controlled geometry and physico-chemical properties. Solution blow spinning technique has the advantage of ease of use allowing the production of nano or microfibres and the direct fibre deposition on any surface in situ. Yet, in spite of these advantages, very little is known about the influence of such fibres on biological functions such as immune response and cell migration. In this work, we engineered polymeric fibres composed of either pure poly(lactic acid) (PLA) or blends of PLA and polyethylene glycol (PEG) by solution blow spinning and determined their impact on dendritic cells, highly specialised cells essential for immunity and tolerance. We also determined the influence of fibres on cell adhesion and motility. Cells readily interacted with fibres resulting in an intimate contact characterised by accumulation of actin filaments and focal adhesion components at sites of cell-fibre interactions. Moreover, cells were guided along the fibres and actin and focal adhesion components showed a highly dynamic behaviour at cell-fibre interface. Remarkably, fibres did not elicit any substantial increase of activation markers and inflammatory cytokines in dendritic cells, which remained in their immature (inactive) state. Taken together, these findings will be useful for developing new biomaterials for applications in tissue engineering and regenerative medicine.

Engineered extracellular microenvironment with a tunable mechanical property for controlling cell behavior and cardiomyogenic fate of cardiac stem cells.

The extracellular matrix (ECM) provides a physical framework of myocardial niches in which endogenous cardiac stem cells (CSCs) reside, renew, differentiate, and replace cardiac cells. Interactions between ECM and CSCs might be critical for the maintenance of myocardial homeostasis in the adult heart. Yet most studies done so far have used irrelevant cell types and have been performed at the individual cell level, none able to reflect the in vivo situation. By the use of a chemically defined hydrogel to create a tunable 3D microenvironment, we succeeded in controlling CSC behavior at the myocardial and individual cell level and directing the cardiomyogenic fate. Our work may provide insight into the design of biomaterials for in situ myocardial regeneration as well as for tissue engineering.

Nanoscale Surface Modifications of Medical Implants for Cartilage Tissue Repair and Regeneration.

Background: Natural cartilage regeneration is limited after trauma or degenerative processes. Due to the clinical challenge of reconstruction of articular cartilage, research into developing biomaterials to support cartilage regeneration have evolved. The structural architecture of composition of the cartilage extracellular matrix (ECM) is vital in guiding cell adhesion, migration and formation of cartilage. Current technologies have tried to mimic the cell’s nanoscale microenvironment to improve implants to improve cartilage tissue repair.Methods: This review evaluates nanoscale techniques used to modify the implant surface for cartilage regeneration.Results: The surface of biomaterial is a vital parameter to guide cell adhesion and consequently allow for the formation of ECM and allow for tissue repair. By providing nanosized cues on the surface in the form of a nanotopography or nanosized molecules, allows for better control of cell behaviour and regeneration of cartilage. Chemical, physical and lithography techniques have all been explored for modifying the nanoscale surface of implants to promote chondrocyte adhesion and ECM formation.Conclusion: Future studies are needed to further establish the optimal nanoscale modification of implants for cartilage tissue regeneration.

In vivo quantification of spatially varying mechanical properties in developing tissues.

The mechanical properties of the cellular microenvironment and their spatiotemporal variations are thought to play a central role in sculpting embryonic tissues, maintaining organ architecture and controlling cell behavior, including cell differentiation. However, no direct in vivo and in situ measurement of mechanical properties within developing 3D tissues and organs has yet been performed. Here we introduce a technique that employs biocompatible, magnetically responsive ferrofluid microdroplets as local mechanical actuators and allows quantitative spatiotemporal measurements of mechanical properties in vivo. Using this technique, we show that vertebrate body elongation entails spatially varying tissue mechanics along the anteroposterior axis. Specifically, we find that the zebrafish tailbud is viscoelastic (elastic below a few seconds and fluid after just 1 min) and displays decreasing stiffness and increasing fluidity toward its posterior elongating region. This method opens new avenues to study mechanobiology in vivo, both in embryogenesis and in disease processes, including cancer.

Porous Silicon-Based Cell Microarrays: Optimizing Human Endothelial Cell-Material Surface Interactions and Bioactive Release.

Porous silicon (pSi) substrates are a promising platform for cell expansion, since pore size and chemistry can be tuned to control cell behavior. In addition, a variety of bioactives can be loaded into the pores and subsequently released to act on cells adherent to the substrate. Here, we construct a cell microarray on a plasma polymer coated pSi substrate that enables the simultaneous culture of human endothelial cells on printed immobilized protein factors, while a second soluble growth factor is released from the same substrate. This allows three elements of candidate pSi scaffold materials-topography, surface functionalization, and controlled factor release-to be assessed simultaneously in high throughput. We show that protein conjugation within printed microarray spots is more uniform on the pSi substrate than on flat glass or silicon surfaces. Active growth factors are released from the pSi surface over a period of several days. Using an endothelial progenitor cell line, we investigate changes in cell behavior in response to the microenvironment. This platform facilitates the design of advanced functional biomaterials, including scaffolds, and carriers for regenerative medicine and cell therapy.

Bioadaptive Nanorod Topography of Titanium Surface to Control Cell Behaviors and Osteogenic Differentiation of Preosteoblast Cells

Titanium (Ti) nanorods fabricated using selective corrosion of Ti substrate by anodic technology show better biocompatibility with pre-osteoblast cells. The current study investigated the response of the murine pre-osteoblast cell MC3T3-E1 on Ti nanorod topography and untreated Ti surfaces by means of examination of the morphology and osteogenic differentiation responsible for the pre-osteoblast reaction. The morphology of MC3T3-E1 cells was observed using scanning electron microscopy, and alkaline phosphatase (ALP) activity was measured using a colorimetric assay after incubation for 7, 14, and 21 days. The expression of three osteogenic differentiation markers including ALP, osteocalcin (OCN), and collagen type 1A1 (COL1A1) and two transcription factors including runt related transcription factor 2 (Runx2) and osterix (Osx) at different time points was detected using real-time polymerase chain reaction analysis in both groups. Osx was used to confirm the protein level. The results showed that Ti nanorod surfaces provided prolonged higher levels of ALP activity compared with unmodified Ti surface on the 14th and 21st days. Gene expression analysis of ALP, OCN, and COL1A1 showed significant upregulation with modified nanorod topography after incubation for 14 and 21 days. Osteogenic transcription factors of Runx2 and Osx exhibited changes consistent with the osteogenic differentiation markers, and this may contribute to the persistently active differentiation of MC3T3-E1 cells in the Ti nanorod group. These results demonstrated that the current nanostructured surface may be considered bioadaptive topography to control cellular behaviors and osteoblast differentiation. The in vivo performance and applicability are further required to investigate osseointegration between implant and host bone in the early stages for prevention of aseptic implant loosening.

Structural hydrogels

Research on hydrogel systems with structural features has aroused great interests due to its wide range of applications, including actuators, microfluidic units, synthetic adhesives, transplantable tissue organs, and cell scaffolds. This review article describes, with special emphasis, the design and fabrication of structural hydrogels with diversified geometric shapes and dimensions. These hydrogel structures include gradient gel, anisotropic gel, gel patterns, gel wrinkle, gel arrays and gel tubes. Several typical applications of such structural hydrogels are introduced, such as for controlling cell behavior, developing responsive actuators, and designing nature inspired bio-lubrication surface. Some perspectives on future development of structural hydrogel systems are provided.

Synthetic Biology Platform for Sensing and Integrating Endogenous Transcriptional Inputs in Mammalian Cells

SummaryOne of the goals of synthetic biology is to develop programmable artificial gene networks that can transduce multiple endogenous molecular cues to precisely control cell behavior. Realizing this vision requires interfacing natural molecular inputs with synthetic components that generate functional molecular outputs. Interfacing synthetic circuits with endogenous mammalian transcription factors has been particularly difficult. Here, we describe a systematic approach that enables integration and transduction of multiple mammalian transcription factor inputs by a synthetic network. The approach is facilitated by a proportional amplifier sensor based on synergistic positive autoregulation. The circuits efficiently transduce endogenous transcription factor levels into RNAi, transcriptional transactivation, and site-specific recombination. They also enable AND logic between pairs of arbitrary transcription factors. The results establish a framework for developing synthetic gene networks that interface with cellular processes through transcriptional regulators.

Protein Adsorption as a Key Mediator in the Nanotopographical Control of Cell Behavior.

Surface nanotopography is widely employed to control cell behavior and in particular controlled disorder has been shown to be important in cell differentiation/maturation. However, extracellular matrix proteins, such as fibronectin (FN), initially adsorbed on a biomaterial surface are known to mediate the interaction of synthetic materials with cells. In this work, we examine the effect of nanotopography on cell behavior through this adsorbed layer of adhesive proteins using a nanostructured polycarbonate surface comprising 150 nm-diameter pits originally defined using electron beam lithography. We address the effect of this nanopitted surface on FN adsorption and subsequently on cell morphology and behavior using C2C12 myoblasts. Wettability measurements and atomic force microscopy imaging showed that protein is adsorbed both within the interpits spaces and inside the nanopits. Cells responded to this coated nanotopography with the formation of fewer but larger focal adhesions and by mimicking the pit patterns within their cytoskeleton, nanoimprinting, ultimately achieving higher levels of myogenic differentiation compared to a flat control. Both focal adhesion assembly and nanoimprinting were found to be dependent on cell contractility and are adversely affected by the use of blebbistatin. Our results demonstrate the central role of the nanoscale protein interface in mediating cell-nanotopographical interactions and implicate this interface as helping control the mechanotransductive cascade.

Highly efficient intracellular transduction in three-dimensional gradients for programming cell fate

Fundamental behaviour such as cell fate, growth and death are mediated through the control of key genetic transcriptional regulators. These regulators are activated or repressed by the integration of multiple signalling molecules in spatio-temporal gradients. Engineering these gradients is complex but considered key in controlling tissue formation in regenerative medicine approaches. Direct programming of cells using exogenously delivered transcription factors can by-pass growth factor complexity but there is still a requirement to deliver such activity spatio-temporally. We previously developed a technology termed GAG-binding enhanced transduction (GET) to efficiently deliver a variety of cargoes intracellularly using GAG-binding domains to promote cell targeting, and cell penetrating peptides (CPPs) to allow cell entry. Herein we demonstrate that GET can be used in a three dimensional (3D) hydrogel matrix to produce gradients of intracellular transduction of mammalian cells. Using a compartmentalised diffusion model with a source-gel-sink (So-G-Si) assembly, we created gradients of reporter proteins (mRFP1-tagged) and a transcription factor (TF, myogenic master regulator MyoD) and showed that GET can be used to deliver molecules into cells spatio-temporally by monitoring intracellular transduction and gene expression programming as a function of location and time. The ability to spatio-temporally control the intracellular delivery of functional proteins will allow the establishment of gradients of cell programming in hydrogels and approaches to direct cellular behaviour for many regenerative medicine applications. Statement of SignificanceRegenerative medicine aims to reform functional biological tissues by controlling cell behaviour. Growth factors (GFs) are soluble cues presented to cells in spatio-temporal gradients and play important roles programming cell fate and gene expression. The efficient transduction of cells by GET (Glycosaminoglycan-enhanced transducing)-tagged transcription factors (TFs) can be used to by-pass GF-stimulation and directly program cells. For the first time we demonstrate diffusion of GET proteins generate stable protein transduction gradients. We demonstrated the feasibility of creating spatio-temporal gradients of GET-MyoD and show differential programing of myogenic differentiation. We believe that GET could provide a powerful tool to program cell behaviour using gradients of recombinant proteins that allow tissue generation directly by programming gene expression with TFs.

New connections: Role of 4EBPs in controlling cell behavior

The regulation of protein synthesis by 4EBP proteins underlies memory and tumor drug resistance.

Machine grinding as an alternative method for creating functional surfaces for controlling cell behaviour

There is extensive evidence to show that certain cellular behaviours including cell proliferation, migration and adhesion can be controlled by culturing cells on surfaces containing different micro-metre- and nanometre-scale features. This paper will introduce the use of machine grinding to generate surfaces with micro-sized features and their ability to affect cell behaviour. Results are presented which show that polyurethane castings of the ground surfaces can promote cell adhesion and migration. This study demonstrates the usefulness of surface grinding as a cost-effective method for generating functional surfaces for modifying cell behaviour.

3D Printed Silicone-Hydrogel Scaffold with Enhanced Physicochemical Properties.

Scaffolds with multiple functionalities have attracted widespread attention in the field of tissue engineering due to their ability to control cell behavior through various cues, including mechanical, chemical, and electrical. Fabrication of such scaffolds from clinically approved materials is currently a huge challenge. The goal of this work was to fabricate a tissue engineering scaffold from clinically approved materials with the capability of delivering biomolecules and direct cell fate. We have used a simple 3D printing approach, that combines polymer casting with supercritical fluid technology to produce 3D interpenetrating polymer network (IPN) scaffold of silicone-poly(2-hydroxyethyl methacrylate)-co-poly(ethylene glycol) methyl ether acrylate (pHEMA-co-PEGMEA). The pHEMA-co-PEGMEA IPN materials were employed to support growth of human mesenchymal stem cells (hMSC), resulting in high cell viability and metabolic activity over a 3 weeks period. In addition, the IPN scaffolds support 3D tissue formation inside the porous scaffold with well spread cell morphology on the surface of the scaffold. As a proof of concept, sustained doxycycline (DOX) release from pHEMA-co-PEGMEA IPN was demonstrated and the biological activity of released drug from IPN was confirmed using a DOX regulated green fluorescent reporter (GFP) gene expression assay with HeLa cells. Given its unique mechanical and drug releasing characteristics, IPN scaffolds may be used for directing stem cell differentiation by releasing various chemicals from its hydrogel network.

‘Stealth’ nanoparticles evade neural immune cells but also evade major brain cell populations: Implications for PEG-based neurotherapeutics

Surface engineering to control cell behavior is of high interest across the chemical engineering, drug delivery and biomaterial communities. Defined chemical strategies are necessary to tailor nanoscale protein interactions/adsorption, enabling control of cell behaviors for development of novel therapeutic strategies. Nanoparticle-based therapies benefit from such strategies but particle targeting to sites of neurological injury remains challenging due to circulatory immune clearance. As a strategy to overcome this barrier, the use of stealth coatings can reduce immune clearance and prolong circulatory times, thereby enhancing therapeutic capacity. Polyethylene glycol (PEG) is the most widely-used stealth coating and facilitates particle accumulation in the brain. However, once within the brain, the mode of handling of PEGylated particles by the resident immune cells of the brain itself (the ‘microglia’) is unknown. This is a critical question as it is well established that microglia avidly sequester nanoparticles, limiting their bioavailability and posing a major translational barrier. If PEGylation can be proved to promote evasion of microglia, then this information will be of high value in developing tailored nanoparticle-based therapies for neurological applications. Here, we have conducted the first comparative study of uptake of PEGylated particles by all the major (immune and non-immune) brain cell types. We prove for the first time that PEGylated nanoparticles evade major brain cell populations — a phenomenon which will enhance extracellular bioavailability. We demonstrate changes in protein coronas around these particles within biological media, and discuss how surface chemistry presentation may affect this process and subsequent cellular interactions.

A nanopatterning platform to study the mechanisms of nanoscale sensing of focal adhesions

Event Abstract Back to Event A nanopatterning platform to study the mechanisms of nanoscale sensing of focal adhesions Stefania Di Cio1, 2* and Julien Gautrot1, 2* 1 Queen Mary University of London, Institute of Bioengineering, United Kingdom 2 Queen Mary University of London , School of Engineering and Material Science, United Kingdom Tissue engineering and regenerative medicine aim to develop materials that mimic human tissues with the purpose of replacing them when they are compromised, as for degenerative diseases. Important to this aim is the understanding of processes occurring at the cell-biomaterials interface. Cells can sense and respond to chemical and physical stimuli coming from their surroundings (the extracellular matrix -ECM) ultimately deciding whether to adhere or not to them [1]. Cell adhesion is a complex process which involves recruiting of molecules to focal adhesion clusters and coupling of shear stress at the cell membrane to the actin cytoskeleton, and which activates a cascade of signalling pathways that control cell behaviour and, in the case of stem cells, cell fate [2]. Scientists have modified the surface of materials at the nanoscale via either chemical [3] or topographical [4]-[6] patterning techniques to assess how cells sense these features. Here we developed a novel technique (fibre mask lithography) to produce fibre-shaped quasi 2D nanopatterns with varying sizes and controlled chemistry on large scales. This constrained geometry will help assessing the early stages of the cell adhesion process and cytoskeleton dynamics and giving insight in cell response to extracellular stimuli. The nanopatterning technique we developed is based on electrospinning of polymeric fibres (which constitute the "mask") and reproduction of this geometry via surface-initiated polymerisation [7]. After the mask removal, the fibre shaped pattern left is selectively functionalised with a cell adhesive protein (fibronectin in this case) while the background presents a non-fouling thin layer of polymer brushes [8]. Cells were then seeded on the pattern and their phenotype studied. Fibre diameters ranging from 200 nm to 1000 nm were obtained in order to study the adhesion process at different stages, from nascent adhesions (composed of few molecules) to more organised structures composed of hundreds of molecules (focal adhesions), and reorganisation of the cell cytoskeleton. Cells were effectively adhering on the patterned area only after functionalization with fibronectin, proving the effectiveness of the non-fouling background generated (polymer brushes). Cell spreading was found to depend strongly on fibre size, with cells struggling to adhere and spread on the smallest topographies while forming mature adhesions and stress fibres on the wider topographies. It is clear that recruiting of molecules to the adhesion site is essential for cell cytoskeleton assembly and cell shape. When adhesion complexes are not allowed to mature, due to geometrical constraints, stress fibre formation is prevented, resulting in abnormal morphologies and restricted cell spreading. Fig.1 Confocal microscopy image of cell adhering on 500nm fibres diameter pattern: actin (red), vinculin (green) and dapi (blue). Fig.2 Confocal microscopy image (reflection mode) of cell on pattern. The development of a novel and scalable nanopattern with a controlled topography and chemistry allowed us to assess how cells behave depending on the constrained geometry. This work showed an effective dependence of cell morphologies on patter size. Future directions of this project will investigate further the fundamental processes underlying cell adhesion to structured surfaces with possible application for tissue engineering and regenerative medicine.

Biomimetic interfaces to trigger tissue restoration

Event Abstract Back to Event Biomimetic interfaces to trigger tissue restoration Ennio Tasciotti1, Francesca Taraballi1, Alessandro Parodi1, Jonathan O. Martinez1, Silvia Minardi1, Naama E. Toledano Furman1, Roberto Molinaro1, Fernando Cabrera1, Laura Pandolfi1, Bruna Corradetti1, Ciro Chiappini1, Jeffrey Van Eps1, Anna Tampieri1 and Bradley K. Weiner1 1 Houston Methodist Research Institute, Regenerative Medicine, United States Introduction: Cells interact and locally interface with the surrounding environment at the macroscale (whole organ), microscale (tissue), and nanoscale (extracellular matrix). Advances in micro and nano-engineering allow the synthesis of materials with features that allow to control and direct cell behavior at multiple scales. By tuning the physical, chemical and biological properties of the materials to match those of native tissues and of cellular processes, we generated biomimetic platforms able to overcome the biological barriers involved in drug delivery and tissue repair. By using the design principles of biomimicry, we exploited the nano-bio interface as a way to negotiate the restoration of tissues function. Materials and Methods: We developed two main classes of biomimetic materials: implantables and injectables. In both cases we applied fabrication strategies based on use of biomaterials able to mimic the material-cell interface for composition, architecture, function, assembly, and biochemical environment. In fact, in the synthesis of our multi-functional platforms was performed using exclusively the components typically found in the body: cellular membranes, proteins, or natural extracellular components (minerals and sugars). Results and Discussion: To overcome the barriers encountered during systemic administration, we developed a drug delivery system comprised of purified leukocyte membranes grafted onto the surface of a biodegradable core (i.e., leuko-like vectors, LLV). The proteolipid material transferred to LLV functioned as a biomimetic camouflage that allowed the inhibition of opsonization and reticulo-endothelial internalization; promoted the increased adherence to inflamed vasculature (Fig 1A), and the activation of intracellular pathways that resulted in the avoidance of endolysosomal entrapment and an increase in vascular permeability (Fig 1B)[1]. To gain access to the cytosol and increase the delivery of biologically labile payloads (nucleic acids), we developed biodegradable nanoneedles capable of directing interfacing with the cytosol through the penetration of the cellular membrane (i.e., nanoinjection). This nano-bio interface did not induce any toxic response in the cell by prompting the rearrangement of the nuclear and plasma membrane (Fig 1C,D)[2],[3], and allowed the in vivo delivery of genes to locally trigger increased neovascularization2 (Fig 1E-G). In tissue regeneration we widely exploited the idea of actively targeting the stem cell niche using different routes. We mimicked the regenerative niche in pore size, stiffness and overall architecture (Fig 2 A, B, C)[4]. Using nanoengineering, we tuned the release profiles of the embedded growth factors to control their local concentration to resemble the kinetics observed in physiological conditions (Fig 2 D, E, F, H)[5],[6]. All these strategies were successfully applied to functionally regenerate both hard and soft tissues. Conclusion: Biomimetic nanomaterials represent a powerful solution to interact with cells and guide their ultimate fate. By engineering surfaces to bestow them with the ability to instruct cells towards a regenerative outcome we achieved superior functional recovery and therapeutic outcome through the activation of the natural processes of cell and tissue healing.

RNA-based gene circuits for cell regulation.

A major goal of synthetic biology is to control cell behavior. RNA-mediated genetic switches (RNA switches) are devices that serve this purpose, as they can control gene expressions in response to input signals. In general, RNA switches consist of two domains: an aptamer domain, which binds to an input molecule, and an actuator domain, which controls the gene expression. An input binding to the aptamer can cause the actuator to alter the RNA structure, thus changing access to translation machinery. The assembly of multiple RNA switches has led to complex gene circuits for cell therapies, including the selective killing of pathological cells and purification of cell populations. The inclusion of RNA binding proteins, such as L7Ae, increases the repertoire and precision of the circuit. In this short review, we discuss synthetic RNA switches for gene regulation and their potential therapeutic applications.

Nanotemplated and fibronectin based-polyelectrolytes films : different ways for bioactivation

Event Abstract Back to Event Nanotemplated and fibronectin based-polyelectrolytes films : different ways for bioactivation Adeline Gand1, Coline Chat1, Mathilde Hindie1, Paul Van Tassel2 and Emmanuel Pauthe1 1 University of Cergy-Pontoise, Biomaterial for Health Group, ERRMECe, Department of Biology, France 2 Yale University, Department of Chemical and Environmental Engineering, United States Introduction: Engineering multilayer polyelectrolyte thin films to direct cell behavior represents a significant challenge but also an excellent opportunity toward a variety of cell contacting applications. Here we propose two new bioactive thin film biomaterials, one that contains a reservoir for biomolecules, such as growth factors, for an efficient local and controlled release, and one that is formed with biomolecules from the extracellular matrix, such as Fibronectin (Fn), in order to mimic the extracellular environment and promote cell behavior. Materials and Methods: Thin films are formed by the Layer-by-Layer assembly of poly(L-lysine) and poly(L-glutamic acid) or Fn. Carboxy-functionalized latex nanoparticles (25 nm) are incorporated as template. Films are cross-linked (EDC/NHS) to increase mechanical rigidity. Tetrahydrofuran (THF) is used to dissolve nanoparticles and create porous films to enable loading of bioactive species, as BMP2. Results and Discussion: We describe here two strategies to control cell behavior. First, a nanotemplating strategy toward porous, polyelectrolyte-based thin films capable of controlled biomolecular loading and release is presented. Films are formed via the Layer-by-Layer assembly of charged polymers and nanoparticles, chemically cross-linked to increase mechanical rigidity and stability, and finally exposed to tetrahydrofuran to dissolve the NP and create an intra-film porous network[1]. We report here on the loading and release of the growth factor bone morphogenetic protein 2 (BMP-2), and the influence of BMP-2 loaded films on contacting murine C2C12 myoblasts. We observe nanotemplating to enable stable BMP-2 loading throughout the thickness of the film, and find the nanotemplated film to exhibit comparable cell adhesion, and enhanced cell differentiation, compared to a non-porous cross-linked film (where BMP-2 loading is mainly confined to the film surface)[2]. The second strategy consist in the direct incorporation of Fn as the anionic polymer during film assembly in order to generate a thin film enriched in Fn that could mimic the extracellular environment and influence the cell behavior. We investigate the influence of Fn on the film construction, physicochemical and mechanical properties as well as the influence on pre-osteoblastic cells. We observe that Fn-based thin films present a novel growth regime with a terminated growth, and are capable of enhancing cell adhesion, spreading and proliferation compared to a Fn monolayer. The Fn present in the films is reorganized into fibrils by contacting cells. Conclusion: We engineer, design and construct bioactive thin films biomaterials able to direct the behavior of contacting cells. Nanotemplated polyelectrolyte films can be employed to spatially and temporally release bioactive molecules, such as BMP2. Fn incorporation within Layer-by-Layer films leads to Fn-rich films, which enhance cell adhesion, spreading and proliferation compared to a standard 2D Fn layer.

Surface micropatterning with calcium phosphate ceramics by micromoulding in capillaries

Event Abstract Back to Event Surface micropatterning with calcium phosphate ceramics by micromoulding in capillaries David Barata1, 2, Daniel De Melo Pereira1, 2, Alessandro Resmini3, Sjöerd A. Veldhuis3, Clemens A. Van Blitterswijk1, 2, J E. Ten Elshof3 and Pamela Habibovic1, 2 1 MIRA Institute for Miomedical Technology and Technical Medicine, University of Twente, Department of Tissue Regeneration, Netherlands 2 MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Instructive Biomaterials Engineering, Netherlands 3 MESA+ Institute for Nanotechnology, University of Twente, Inorganic Materials Science, Netherlands Introduction: Current design of biomaterials for regenerative medicine is progressing towards greater control over the biological response in vivo, along with the evolution of the concept of biocompatibility from bioinert to bioactive[1]. Surface patterning is a promising method to achieve spatial control over cell behaviour by physico-chemical cues such as surface chemistry or topography[2]. While a variety of techniques exist to develop patterns of polymers or biomolecules, patterning ceramic materials is explored to a lesser extent and requires further research[3]. This is particularly interesting for the field of bone regeneration, where calcium phosphate (CaP) ceramics are widely used for their close resemblance to bone mineral and associated bioactivity in terms of osteoconduction and osteoinduction[4]. Here we present an approach to micropatterning of surfaces with CaPs by employing micromoulding in capillaries (MIMIC), a method previously shown suitable for patterning zirconia[5]. Materials and Methods: MIMIC was employed to create patterns of dibasic calcium phosphate anhydrous (DCPA) on a flat silicon substrate. Briefly, a polydimethylsiloxane (PDMS) mould with grooves was placed on the silicon substrate to create channels. After air plasma, CaP solution was infiltrated and controlled heating facilitated nucleation and crystal growth inside the microchannels. After completion, the PDMS mould was removed. Selected samples were further annealed to convert DCPA into β-tricalcium phosphate (β-TCP). Substrates were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS). Biocompatibility was assessed by culturing MG-63 osteoblast-like cells on the Si-CaP substrates and analysing cell morphology, metabolic activity and proliferation over a period of 7 days. Results and Discussion: CaP bioceramic patterns were successfully formed by nucleation and growth of crystals in the microchannels. The SEM (Figure 1) and the EDS analyses revealed patterns that were confined to the channels used for patterning. The patterns consisted of flower-like crystals varying in size between 5 and 25 μm. XRD pattern showed peaks characteristic of DCPA (before annealing) and of β-TCP (after annealing). The analysis of cell morphology on the patterns showed the effect of the patterns on cell orientation, an effect that was dependent on the pattern size. Metabolic activity of MG-63 cells showed an increasing trend during the 7-day culture, as did the total DNA amount, confirming the biocompatibility of these materials. The results demonstrated that MIMIC is a suitable technique to fabricate micropatterns of CaP against a chemically different background, and that the created micropatterns can be used to spatially control cell behaviour. Conclusion: MIMIC is a promising technique for patterning of CaP ceramics. Further improvements on the patterning method will focus on obtaining different CaP phases, improving control over the morphology and homogeneity of the CaP pattern, and increasing the complexity of the patterns. DB gratefully acknowledges the financial support of the NIRM (Netherlands Institute of Regenerative Medicine); This research has been in part made possible with the support of the Dutch Province of Limburg