Final Report Summary - NANOCARD (Nanopatterned scaffolds for active myocardial implants)
Executive Summary:
NanoCARD
Nanopatterned scaffolds for active myocardial implants
The overall goal of this project was to create nanopatterned scaffolds for myocardium implants.
Cell therapy and tissue engineering are opening novel therapeutic perspectives for myocardial repair. The introduction of new myocytes into diseased areas of the heart can improve the hearts mechanical properties. The therapeutic utilities significantly hampered by the paucity of cell sources for human cardiomyocytes and by the high degree of donor cell death following cell grafting.
Therefore the fundamental different concept of NanoCARD aims to provide a biomaterial which is based on high throughput screens for identifying specific and necessary signals of extracellular matrix. These signals are activating specific cell programs such as a cell adhesion and the induction of differentiation of stem or progenitor cells to functional myocytes. The implant materials present smart nanopatterned interfaces to cells. These surfaces present chosen biomolecules in nanoscopically defined spatial patterns on biologically inert, biodegradable substrates.
A key point has been the identification of parameters required to drive cardiac tissue formation.
The scientific bases of NanoCARD is the fact, that the activity and fate of all living cells is exquisitly regulated by the cells and environments and particularly by the adhesive interaction with neighbouring cells and with the extracellular matrix. Therefore the design and use of nanostructure and specifically biofunctionalized surfaces with desirable molecular properties offers unique opportunities for programming cell responses and functions.
This biomimetic implant technology concept is based on the interaction between living cells and synthetic, nanostructured interfaces.
The economic potential is remarkable because myocardial infarction is an important disease worldwide.
Project Context and Objectives:
Since many previous attempts to engineer tissue scaffolds were based on trial and error approaches, NanoCARD aims to create a conceptually new biomimetic, nanoscopically designed implant. The implant’s highly tailored material properties will encourage the selective adhesion of stem and progenitor cells and direct their differentiation, enabling replacement myocardial tissue to be generated ex vivo.
The development of conceptually new implants is based on five pillars: (1) High-Throughput Screens for identifying specific extracellular matrix material parameters as signals for directing stem or progenitor cells towards specific cardiac tissue generation in vitro; (2) Implant synthesis by integration of the set of material parameters identified by HTS into a cell support: so-called “therapeutic surface”. The implant will be based on a microporous mulitlayered stack of “therapeutic surface”; (3) in vitro recruitment of cells and cardiac tissue formation; (4) in vivo (animal) testing of implants; (5) development of commercialization strategies for the new implant concept for cardiac tissue generation together with the companies Qiagen, Idea Bio Medical, CellMade, Evercyte and Cell System.
The in vitro HTS will reduce the number of animal experiments, which is in close agreement with the ethical aims of EU guidelines.
The biochip/HTS system will find wide application in the life sciences.
Objectives
Objective 1: Development of nanostructured, biofunctionalized and biodegradable cell supports which have the capacity to vary cell ligand type, ligand spacing, and substrate stiffness for exploring and manipulating cellular interactions with non-biological surfaces
Objective 2: High-Throughput Screens (HTS) for identifying implant material parameter: Exploration of the interaction mechanisms between cells and biomaterial by automated live cell optical microscopy
Objective 3: Characterization of the physicochemical properties of extracellular matrices in vivo and in vitro; exploring how the interaction mechanisms between selected cell types and matrix impact cell fate.
Objective 4: Translation of the set of identified cell response-specific material parameters to implants for in vivo applications
Objective 5: Transition from basic scientific understanding to the industrial market
These objectives are achieved via workpackages covering the following areas:
• Synthesis of Artificial Cell Environments on the Molecular Level by Self-Organization Principles
• Development of the BioChip
• Development of the automate optical microscope for application of High-Throughput Screens
• High-throughput screening of endothelial cells (EC) and cardiomyocytes (CM)responses to nanoengineered cell environments, Systems Biology
Analysis
• How cell-made extracellular matrix co-regulates protein expression profiles and cell fate
• Synthesis of the implant
• Directed stem cell differentiation into endothelial cells (EC) and cardiomyocytes (CM) by the implant
• In vivo assessment of therapeutic potential of the implant
• Integrative and Translational Approaches to Industrial Market
• Co-ordination and project management
• Dissemination, IPR & Knowledge Exchange - DIKE
Project Results:
1.Synthesis of Artificial Cell Environments on the Molecular Level by Self-Organization Principles
Summary
Biodegradable polymer scaffolds have been constructed, and their mechanical properties and biochemical potential have been investigated. Furthermore, the surface of the scaffold has been modified with a polymer brush that will enable their future patterning with cell adhesive ligands.
In order to examine the interaction between the synthesized scaffold and the heart a rat in-vivo model was used. Poly(lactide-co-caprolactone) (PLCL) porous and non-porous scaffolds were implanted in healthy rat hearts.
In addition, PLCL-porous scaffolds were implanted in rats after the induction of myocardial infarction.
Evaluating the biocompatibility of the polymer substrates we monitored the behavior of human bone marrow stem cells (hBMCs) in terms of cell adhesion, spreading, actin organization and focal adhesion formation using a confocal microscope. Our analysis of focal adhesion formation and actin organization on tested substrates reveals that the number of pores strongly affects cellular adhesion.
We demonstrate distinct nanotopography-induced cell phenotypes, characterized by different morphology, LOX-1 diffusivity and oligomerization state.
A model combines microfluidic flow, mass transport of the chemotractant and binding of the molecules on a model cell surface in a single model.
Description
Micro-porous films
The mechanism of the breath figure technique to create well-ordered porous films from polymers in an organic solvent has been well reported in literature.
The breath figure technique allows great control over the formation of the porous material, due to the fact that there is a number of conditions which can be varied that will have an effect of the process, such as, air flow speed, humidity content (%), organic solvent choice, polymer molecular weight and polymer solution concentration. The first scaffolds were obtained by stacking of the microporous PLCL films that were prepared with the breath figure technique. Due to the some problems with the stacking of the films, 3D scaffold preparation method was changed to the sphere-templated fabrication method (Scheme 1). Sphere-templated fabrication method allows controllable pore size and high interconnectivity between pores.
Scheme 1: Sphere-templated fabrication method for scaffold synthesis.
Biofunctionalization and nanostructuration of the biodegradable, 3D, interconnected-microporous and non-fouling polymer scaffolds were achieved. They are prepared by random copolymerization of 2-methylene-1,3-dioxepane (MDO) and 2-(hydroxyethyl) methacrylate (HEMA) monomers and a PCL-PEG-PCL crosslinker.
Mechanical Studies
Young’s modulus measurements of scaffolds were carried out by using Minimat machine with 20N cell load. Figure 1 shows the average stress-strain graph and the average Young’s Modulus of the scaffolds that were obtained with the following monomer mol ratios: HEMA/MDO/PCL-PEG-PCL : 27/5/1. This composition gives the Young’s Modulus of the scaffold as 0.33±0.07 MPa, which is close to the value of the Young’s Modulus for a heart rat tissue 0.59±0.22 MPa.
Figure 1: Avarage stress-strain graph for biodegradable scaffolds.
Biofunctionalization
Biofunctionalization of scaffolds with cell adhesive peptides (RGD) was performed by addition of RGD-labeled methacrylate monomer to the monomer mixture before polymerization (Scheme 2). The methacrylated RGD peptide is the only monomer with nitrogen atom. High resolution N1s X-Ray Photoelectron Spectroscopy (XPS) shows an increase of the nitrogen peak while increasing the feeding ratio of the RGD peptide in the monomer composition. It means that the amount of peptide integrated in the scaffold could be tuned with the amount of peptide containing monomer in the feeding ratio.
Scheme 2: Scheme for the synthesis of RGD functionalized scaffolds.
Cell Studies
In order to evaluate the cells bio-fouling property of scaffolds cell studies were carried out with different concentration of RGD peptide inserted scaffolds. RGD functionalized scaffolds were incubated with GFP expressing HeLa cells and attachment of cells was examined with confocal microscopy. Cell cytoplasm forms were observed.
The nanostructuration of scaffolds was obtained by reduction of Au+3 in sodium borohydride (NaBH4) (Scheme 3). First, scaffolds were immersed into aqueous potassium tetrachloroaurate (KAuCl4) solution and Au+3 ions were absorbed in the scaffolds after shaking for 12 hours. After that NaBH4 was added for the reduction of Au+3 ions to Au nanoparticles (Scheme 3).
Scheme 3: Schematic representation of Gold nanoparticle decoration of PHEMA scaffolds.
2.Development of the BioChip
Summary
We demonstrated the possibility to implement a microfluidic device on topographically-modified substrates yielding on-demand local 3D gradients of soluble molecules with controllable spatial and temporal profiles.
Protocols for the fabrication of biochemical chips on glass supports and on polymer support have been established. Chips are adapted and used for cell experiments within the project.
Cell tests with such substrates proved the suitability of the chip for cell culture studies.
The design of a general platform allowing for mechanical stimulation of cells by lateral stretching of an elastomer membrane is achieved.
We have developed two different microfluidic platforms that are capable of forming soluble gradients.
We showed that our systems are compatible for cell culture and cells could be maintained up to at least one week.
Description
The goal of high throughput screening (HTPS) remains the fabrication of miniaturized
laboratory reactors, called micro-arrays that can work in parallel and be
compatible with high sensitivity detection systems to monitor their outputs. Complex
analyses can be performed within a few hours with the help of microarrays or
“biochips”.
The manufacture of microarrrays and biochips benefit from the use of a great
number of nanofabrication tools. Surface molecular modification techniques include:
self-assembled mono-layers, surface spin coating with polymers or colloids) to control properties of the array interface at the molecular level (adhesion, hydrophobicity,friction) and as shown in this research, the Diblock Copolymer Micelle Nanolithography (BCML) technique.
In addition, HTPS approaches must fullfill certain criteria in order to be useful
in a research diagnostic laboratory. They must be able to perform a large number
of assays rapidly and simultaneously in a user friendly manner and be small in format.
They must be configured to provide robust and reproducible results that allow
standardization and comparison of experiments performed in different laboratories.
Figure 1 illustrates the preliminary experiments.
Figure 1: Illustration showing preliminary cell adhesion experiments. (A) Gold (B)
Gold + BSA (C) Gold and Fibronectin (D) Gold and Fibronectin + BSA (E) Gold
and RGD peptide (F) Gold and RGD peptide + BSA (G) Gold-nanodots with RGD
passivated with PEG 2000 (H) Glass surface passivated with PEG 2000
Key technology for the production of biochips is the micellar nanolithography (BCML). This technique uses self-organization principles to arrange micelles on the surface of flat glass substrates and it allows for patterning the surfaces with hexagonal arrangements of gold nanoparticle. The particles have a size of typically 8nm which is comparable to the size of cellular membrane receptors. Within the project the production of these nanoscopically-structured surfaces has been up scaled to meet the requirements of producing large numbers of surfaces for all project-relevant applications.
The method to precisely functionalize the biochip surface with Au-NP has been further extended to be applicable to more complex 3D-like environments. This is an important step to use polymers developed within WP1 and to account for more in-vivo like cell culture conditions.
The design of a general platform allowing for mechanical stimulation of cells by lateral stretching of an elastomer membrane is achieved. Several prototypes following different approaches and designed for specific purposes have been developed and manufactured. Examples are given in Fig. 7. One setup is particularly suited for live cell imaging application and allows for the observation of cells during the experiment by phase, DIC or fluorescent microscopy. Other setups use a six-well format and can apply uniaxial tensile strains to 48 wells with 8 different amplitudes or frequencies. All experimental platforms are suitable to manipulate cells over long time periods (weeks). Test runs with cells under regular culture conditions (T= 37°C, 5% CO2 and high humidity) were successful and the setups are used for screening cell reaction upon application of various mechanical stimulations (WP4).
A prototype biochip composed by a fluidic network and a textured COC substrate was fabricated and characterized. Complete biochips were realized by bonding a PDMS microfluidic network onto the grating. The microfluidic chip was aligned and mounted in close proximity to the patterned area present on the COC substrate. A fluorescein solution was then delivered through the fluidic channel to generate chemical gradients coupled to the underlying topography.
Production of surfaces for biochemical chips
Key technology for the production of the biochemical chips is the micellar nanolithography (BCML) which is illustrated in Fig. . This technique uses self-organization principles to arrange micelles on the surface of flat glass substrates and it allows for patterning the surfaces with hexagonal arrangements of gold nanoparticle. The particles have a size of typically 8 nm which is comparable to the size of cellular membrane receptors. Within the project the production of these nanoscopically-structured surfaces has been up scaled to meet the requirements of producing large numbers of surfaces for all project-relevant applications.
Figure: Schematic representation of the Micellar Nanolithography used for nanopatterning oft he biochip surfaces.
In order to immobilize several different peptides or other biomolecules on a chip a novel protocol was developed. Briefly, surfaces are patterned with small gold nanoparticles (Au-NP) in a hexagonal order by BMCL (BCML) as described above. The inter-particle distance can be adjusted between 30 and 200 nm. To prevent unspecific protein absorption, the surface is covered with a PEG-passivation layer. Several lengths of PEG molecules were tested. For the final chip we use typically PEG 2000 which gets immobilized on the glass (between the Au nanoparticles) via silane chemistry. This procedure provides a stable protein passivation layer on the glass but still allows the chemical modification of the Au nanoparticles with other molecules
Initial cell adhesion studies on the biochemical chip
The chips were used for cell adhesion experiments to proof their suitability for larger scale cell culture studies. In first test, rat embryonic fibroblasts were cultured on the biochips for several days. The biofunctionalization as well as the passivation of the surface between the micro-spots was demonstrated to be stable over that time period. An illustrative example of cells adhering on RGD-functionalized micro-spots is given below. The chips’ dimensions, optical and mechanically handling properties are ideally suited for high content screening microscopy within NanoCARD.
Figure 1: Rat embryonic fibroblasts on biochip surface with micro-spotted peptides (RGD). Cells adhere only on the RGD-modified spots. Within each spot the peptide is specifically bound to the Au-NP (not visible with this magnification).
Biomechanical Chip Design
Biochip Fabrication – general properties
The substrate system used in the bio chip was designed in a way that allowed for varying the biophysical and biochemical properties on the chip surface simultaneously.
Distances between the ECM mimicking peptides (biomolecules) range between 30nm and 150 nm. The size of anchor points itself is chosen not exceed a limit of about 10 nm to assure single receptor-ligand interaction. The mechanical properties of substrates were chosen to be with 3kPa and 90 kPa with additional very stiff support of 4 MPa, serving as a control substrates for the in vitro tests.
Cell tests with biomechanical chip
Initial functional test of the chips were performed with various cell lines such as normal human dermal fibroblasts (NHDF) and CCL-121, poorly differentiated fibrosarcoma cells from the acetabulum. Both cell types were trypsinized and seeded on the biofunctionalized substrates surfaces in appropriate media containing 10% fetal bovine serum (FBS). The experiments were performed after desired cell-substrate interaction time under cell-culture conditions (5 % CO2, 37o C). To optimize the measurement procedure and characterization of the cellular behavior on the biomechanical chip during different time periods, a high throughput screening (WiScanTM instrument, Idea-Bio, Israel), equipped with live cell environmental control and analysis (WiSoftTM software, Idea-Bio, Israel).
3.Development of the automate optical microscope for application of High-Throughput Screens
Summary
The WiScan family of systems to include fast acquisition and storage, accurate stage positioning, live cell conditions, large set of command functions for versatile applications, flexible sample formats, automatic sample loader and user friendly interface has been developed. The microscope is fully automatic and acquiers high-resolution images at a precise focus plane using fast auto focusing procedure.
Tools for image processing and data analysis were added to WiSoft the image analysis software.
Many biological applications were studied using the WiSoft including – GFP spot detection, cell cycle evaluation, translocation of fluorescnce protein, variation in mitochondria and ER, studies of fluorescently labeled Celegans, focal adhesion morphologicl changes, tube formation and more.
We have improved usability and stability of the software and the system has been tested extensively.
We have also been working on some simple image analysis tools and ImageJ scripts to automate processes like cell counting and analysis.
Description
Adaptation for robotic sample loading:
The WiScan® Argus system was designed to enable robotic loading and unloading of biological samples. Thus, cells can be cultured at an automatic liquid handling system and then transfered to the WiScan® Argus screening microscope automaticallly using a robotic arm.
Biochip readout:
The researchers prepared a spotted array of adhesion sites on a standard glass slide and seeded cells on the array. After fixation the cells were labeled for nuclei and for actin fibers. The biochip unique structure was defined at the WiScan® system as a spotted array, and the array was scanned using 20x/0.75 objective and two wavelengths for DAPI and FITC readout. The images were analyzed to count the number of cells in each spot and to measure the average actin fiber length (Figure 3).
Figure 3 - Biochip prototype (partial region of 36 adhesion points) scanned at WiScan® using 20x magnification. Cells are labeled with DAPI for nuclei and with phalloidin-FITC for actin. The number of cells per adhesion point and the actin fiber length was calculated using WiSoft® and these two parameters are presented for each well in color scale.
4.High-throughput screening of endothelial cells (EC) and cardiomyocytes (CM)responses to nanoengineered cell environments, Systems Biology Analysis
Summary
We tested the adhesion of rat mesenchymal stem cells to LACL and LAGA membranes. In addition, we tested the adhesion of inflammatory cells (rat splenocytes) to the synthesized surfaces.
Studies were performed on cell responses of human mesechymal stem cells (hMSC). The cells were cultured on nanostructured RGD-functionalized Biochip surfaces with different developed compliances. hMSC stem cell morphology and structure of adhesion sites and cytoskeleton depends on both parameters: ligand spacing and substrate stiffness.
All the produced surfaces were used for the induction of EC and/ or CM development, proliferation.
We have established an experimental system to test the effect of substrate rigidity on the proliferation, differentiation, and the commitment of rat cardiomyocytes.
We found that modulating surface rigidity effects rat cardiomyocyte proliferation.
Protocols for determination of cell specific markers of endothelial cells were developed using primary human endothelial cells (HUVEC and HAoEC). In addition, in order to establish reproducible and stadradizable cell models, HUVECs were immortalized using human telomerase and characterized in detail.
Description
We have selected to work, at this stage with cells of 2 origins, namely chick embryos and newborn rats. Detailed procedures for the cultivation of these two cell types were developed, optimizing the condition for reaching full differentiation into functional heart cells, namely – well organized sarcomeric organization, and beating. To study both features, we have specifically fluorescently labeled the cells, at different time points after plating, for a variety of sarcomeric proteins (e.g. actin, -actinin, myosin (cardiac)), and examined the cells for beating, using live cell microscopy.
The cells, from the same origins, were extensively examined, following labeling for multiple cytoskeletal components, enabling the visualization and quantification of multiple cellular parameters including sarcomere length, nuclear organization, sarcomere registration, cell shape and polarization, matrix adhesion structure, composition, orientation and connection to the contractile apparatus. In addition, the cells were examined by transmission electron microscopy for the organization of their contractile machinery.
Towards the development of synthetic tissue scaffolds for the regeneration of the heart muscle after myocardial infarction, we have set the experimental system, including the choice of cells for the study (primary cardiac myocytes (PCM) from chicken or rat origin, prepared as above) and characterization of the cellular response to variations in the properties of synthetic substrates.
Our preliminary finding showed selective adhesion of cardiac fibroblasts on the high-density signal hydrogel surface and higher population of cardiocycytes on the lower density signaling surfaces. These finding are based on the labeling of the cells to several sarcomeric and adhesion-associated cytoskeletal proteins, including actin and alpha actinin enabling the assessment of Z-band formation across the cardiomyocytes. In order to evaluate whether these results are differentiating outcome, or of selective adhesion, we are repeating these experiment with both the soft hydrogel surfaces and on the rigid glass surface.
Highlights of the PDMS elastomers results -
Soft PDMS substrates facilitate cardiomyocytes proliferation. We found that modulating surface rigidity effects rat cardiomyocyte proliferation. Specifically, softer PDMS substrates of 20kPa and 5kPa elevate neonatal rat cardiomyocyte proliferation by over 50%. We would perform additional experiments to further characterize the proliferating cardiomyocytes and to assess the mechanism that leads to this phenomenon.
Substrate rigidity effects sarcomeric organization of cardiomyocytes. We compared 1 day old rat cardiomyocytes cultured on either relatively rigid (2MPa), or relatively soft (5kPa) surfaces. Substrate rigidity effects the sarcomeric architecture of newborn rat cardiomyocytes, as evident by the sarcomeric markers MHC, myomesin, and Troponin T. Cardiomyocytes on the 2MPa substrates are more polarized, exhibit well organized sarcomeric structures and directionality. On the soft 5kPa substrate cardiomyocytes are radial and exhibit disorganized sarcomeric structures.
5.How cell-made extracellular matrix co-regulates protein expression profiles and cell fate
Summary
Different ECM and VEGF concentrations were investigated on their effect on tube formation of HAoEC in co-culture with HAoSMC.
Characterization of the biophysical properties of extracellular matrix that cells assemble and remodel on nanopatterned surfaces with varying ligand density and substrate rigidity.
Studies of cell assembled ECM on the biochips.
Preliminary studies whether hMSCs sense the topographical differences in the surfaces.
Fabrication of cell-derived ECM scaffolds on NanoCARD chips.
We found that the optimal conditions for vessel-network formation is low fibrin concentration (7.5 mg/ml-final concentration) and high cell density (0.3X10^6 – huvec + 0.06X10^6- fibroblasts).
Our results show that mechanical forces induce differentiation of the cells into the mesoderm direction.
Different ECM and VEGF concentrations were investigated on their effect on tube formation. Adding VEGF to the used ECM had only limited effects on tube formation in the three models.
Stem cell engineering and conditioning: Since none of the available stem cell sources can guarantee that none of the injected stem cells might differentiate into cancer cells, we started to ask how stem cells could perhaps be conditioned to minimize tumorogenesis (C. Moshfegh & V.Vogel patent filed on Oct 3, 2013).
Description
In comparison to flat surfaces, HFFs on NanoCard surfaces formed much less ECM. Also, the ECM exhibits a broader range of FRET ratios within a given matrix and even within individual fibrils, indicating a broad range of matrix strains. Although a variety of FN conformations co-exist in cell culture, our preliminary experiments show that the mean matrix tension alters with the underlying nano-topographyat a ridge width of 2 and 1.75 µm, significantly less matrix was assembled when compared to the smaller ridge width, 1.5µm and 1.25µm respectively. The trends of the mean FRET ratios are different as shown in the Figure below. The FRET ratios were highest at intermediate ridge width.
6.Synthesis of the implant
Summary
We have implanted the preliminary cardiac scaffold (LACL porous scaffold) in healthy rat hearts. Cardiac performance was measured using high frequency echocardiography in order to determine whether the implant interferes with normal cardiac function (n=4). Also, implanted hearts were evaluated by histology to detect signs of tissue inflammation or any other structural abnormality.
Description
Seeding EC and CM on implant material in vitro and investigating cardiac tissue formation.
MPI performed experiments with endothelial cells (EC) and smooth muscle cells (SMC) on biochips with different ligand spacing and peptide functionalization. Experiments demonstrate that cell adhesion of both cell types depends on the nano-scaled ligand spacing (40 vs. 90 nm) but not on the selection of one of the two different adhesive peptides (RGD and REDV).
Co culture of GFP or RFP-huvec cells and fibroblasts were grown on PLLA\PLGA in vitro for 10 days, during this time the scaffolds were monitored using confocal for the formation of vessel-like networks.
Spontaneous contraction and vessel like structure formation were observed between days 4-6.
7.Directed stem cell differentiation into endothelial cells (EC) and cardiomyocytes (CM) by the implant
Summary
We have explored endothelial-fibroblast interactions in 2D and 3D constructs and the influence of scaffold mechanical properties on formation of vessel-like structures. Moreover, we have examined the influence of scaffold patterning and topography on cell attachment, proliferation and organization.
Seeding stem cell derived cells (cardiomyocytes and endothelial progenitors and common progenitors) on biofunctionalized surfaces designed to induce specific attachment and differentiation and analysis. Histological characterization of the engineered tissue and biocompatibility tests. In addition, biosafe generation of iPSCs was tested by using recombinant Yamanaka proteins as media additives. Surprisingly, Oct4 was found to contain a functional peptide transduction domain allowing direct cellular uptake.
We used mechanical forces in purpose to differentiate embryonic stem cells (ESC) into the mesoderm germ line, as a precursor cells for both endothelial cells and cardiomyocytes.
We seeded endothelial cells and mouse ESC in defined patterns (using PDMS stamps) in purpose to mimic cardiac tissue.
Description
Effect of different RGD spacing on endothelial cells proliferation and survival
We have used three different RGD spacing (i.e. 32, 58 and 102 nm spacing between two neighboring RGD molecules) to investigate the influence of RGD spacing on EC attachment and organization.
The spacing between the RGD molecules plays a key role in cell attachment and organization.
Effect of different RGD spacing on fibroblasts proliferation and survival
When fetal fibroblasts were seeded on PEG gels, we observed some morphological changes between the different surfaces. The fibroblasts are more elongated and able to create cell-to-cell interactions with the increase in RGD spacing.
Fibroblasts seeded on higher density and low spacing (i.e. 33 nm) cover glasses proliferated more than fibroblasts seeded on lower density and high spacing (i.e. 74 nm).
We can see that in the of 33 nm spacing, we have approximately 50% of proliferating cells, while in the 74 nm spacing, we have only 10% proliferating cells.
Effect of different RGD spacing on endothelial-fibroblasts organization
When EC and fibroblasts were seeded together on RGD cover glasses no significant difference was observed between low (i.e. 74 nm spacing) and high (i.e. 33 nm spacing) density RGD.
Non Canonical Wnt Pathway:
In order to evaluate the effect of Wnt signaling through the non canonical Wnt pathway we evaluated the effect of Wnt-11 and Wnt 5a conditioned media on hESC cardiac differentiation. Wnt 11 conditioned media applied at equal volumes with culturing media during the initial 8 days of differentiation resulted in a 2-fold increase in the percentage of contracting areas.
We aimed at evaluating whether non canonical Wnt signaling effected cardiac differentiation during early hESC differentiation (day 1-4) suggesting a potential role mesoderm induction or late hESC differentiation (days 5-8) suggesting influence on commitment to the cardiac lineage. While early exposure to Wnt5a conditioned media resulted in significant upregulation of cardiac markers late exposure to Wnt5a conditioned media did not result in upregulation of cardiac markers.
Activation of the non-canonical WNT pathway is known to induce cardiac specification through protein kinase C (PKC) and Jun N-terminal Kinase (JNK) activation in the xenopus laveis model.
Selective JNK inhibition did not significantly affected the proportion of the contracting areas significantly (p=0.70 n=150). However, PKC inhibition by bisindolylmalmeimide I reduced significantly the proportion of contracting areas by 67%.
Canonical WNT Signaling
To evaluate the effect of activation of canonical Wnt signaling on differentiating hESC, we applied Wnt3A conditioned media during the first 8 days of differentiation.
In conclusion, we have showed that: 1) Non-canonical Wnt signaling promotes human embryonic stem cell differentiation into the cardiac lineage through both Wnt11 and Wnt5a. 2) The time frame in which non-canonical Wnt signaling exert its cardiogenic effect is between days 1-4 of hESC differentiation. 3) Among the suggested signaling pathways through which non canonical Wnt is known to mediate its effect, activation of PKC but not JNK results in enhancement of cardiogenesis in hESC. 4) Activation of canonical Wnt signaling during the suspension period inhibits hESC differentiation to the cardiac lineage.
In order to examine mESCs differentiation under the different mechanical conditions real time PCR was used for examining representative genes of the three germ layers. We started by examining the genes expression after two days of oscillations
One can notice that under static stretch significant change couldn’t be observed in the three germ layers vs. control. However, by using oscillatory stretching significant change in oscillations vs. control was observed both in bry, pax6 and nestin, representative genes of the mesoderm and ectoderm layers.
Establishment of an efficient hiPSCs cardiomyocyte differentiation system. Since the initial demonstration that beating cardiomyocytes can be generated from both hESC and hiPSCs using the spontaneous, but relatively inefficient, serum-dependent embryoid-body (EB) differentiation system, several important improvements were made. In this part of the project we evaluated a number of strategies in an attempt to establish a well-defined, scalable, serum-free, directed hiPSCs cardiomyocyte differentiation system. These different suggested methods were inspired from lessons learned from embryology and are based on sequential manipulation of the BMP, Activin/nodal, and Wnt pathways. Comparing the different strategies we noted that in our hands the most effective, reproducible, and cost-effective differentiation system was a modification of the method suggested by Lian et al. (PNAS 2012).
This method is based on the use of small molecules to manipulate a single signaling-pathway (the canonical Wnt pathway). This method is based on initial activation of the Wnt pathway by CHIR-99021, a GSK3 inhibitor, to facilitate mesoendoderm formation followed by Wnt inhibition by IWP-2 or IWP-4 to induce cardio-mesoderm formation. Optimization of this strategy in a monolayer approach resulted in a highly-efficient cardiomyocyte differentiating system.
Induction of anisotropic cardiomyocyte alignment by nano-patterened films.
Ordered alignment of cardiomyocyte cells, similar to the anisotropic in-vivo cardiomyocyte alignment, is one of the major challenges in myocardial tissue engineering. Anisotropic alignment may allow achieving tissues with better force forming capabilities and lower risk for arrhythmogenicity. To this aim we collaborated with Marco Cechinni’s group (Pisa, Italy) and seeded nano-patterned scaffolds containing grooves with a variety of geometries. Scaffolds were generated from COC and PDMS, the unpatterened borders of the scaffolds were used as controls. Both neonatal rat ventricular cardiomyocytes and hiPSCs derived cardiomyocytes were seeded on the scaffolds. Scaffolds were covered prior to seeding with fibronectin to improve cell attachment and were followed microscopically for two weeks. Additionally, time-lapse imaging was generated to allow real-time follow up of the alignment kinetics. Seeding of both neonatal rat ventricular cardiomyocytes(NRVC) and human induced pluripotent cardiomyocytes (hiPSC-CMs) demonstrated that the cells can attach to the surface. Most of the cells survived following seeding and the ordered alignment process was started within hours following seeding. Following 24 hours, most cell in all groups (COC/NRVCMs, COC/hIPSCs, PDMS/NRVCMs, PDMS/hiPSCs) were generally aligned to the direction of the nano-patterned grooves.
8.In vivo assessment of therapeutic potential of the implant
Summary
We have selected a preliminary list of 7 candidate ligands known to affect the migration of adult stem cells, and tested the migration of rat mesenchymal stem cells towards these ligands.
We tested the ability of the same candidate ligands to induce migration of inflammatory cells.
We tested the safety, as well as the therapeutic potential of PLC scaffolds implanted to rat heart after the induction of myocardial infarction. PLC scaffolds were implanted either in single layer or in 4 stacked layers.
There was no indication of cardiac or systemic toxicity of the implanted scaffolds.
However, no positive effect was seen on cardiac function.
Description
We have previously implanted tissue constructs seeded with human embryonic stem cells derived cardiomyocytes (hESC-CM), endothelial cells (HUVECs) and mouse embryonic fibroblasts (mEFs) onto rat myocardium. The cells were seeded in PLLA/PLGA scaffolds and grown for two weeks in-vitro pre-implantation and then for an additional 3 weeks in-vivo. We have shown that the engineered constructs beat synchronously and form endothelial vessel networks (1,2). We have demonstrated vascularization of the grafts and organization of functional blood vessels within the scaffold. We also have showed that hESC-CM seeded with HUVECs and mEFs are able to contract synchronously and express cardiac markers (1,2).
Following the development of spontaneously synchronized beating areas, the engineered muscle constructs were engrafted to the left ventricular surface of immunosupressed rats. Two weeks after transplantation, histological analysis revealed the presence of vascularized cardiac tissue.
Following these experiments we then proceeded to infarct rat model. We have seeded PLLA/PLGA scaffolds with HUVECs, human foreskin fibroblasts (hff) and hESC-CM. One week later we induced infarct in rats. The infarct was confirmed by Ultra-Sound Electrocardiography (U.S-EKG). One week after infarct induction, we tried to implant our scaffolds onto the infracted area; unfortunately the scaffolds didn't attach to the infracted area. Ongoing studies are trying to establish a suitable protocol for implanting the scaffold onto the infracted area and to test biofunctionalized surfaces.
We have implanted tissue constructs containing skeletal myoblasts, EC and human neonatal dermal fibroblasts (HNDF) around the femoral artery of mice to enhance angiogenesis in the engineered muscle tissue. The cells were seeded in PLLA/PLGA scaffolds and grown for 10 days in-vitro pre-implantation and then an additional 4-12 days in-vivo. We have shown that the engineered constructs have blood perfusion and promoted angiogenesis.
To explore the ability to establish a 3D supportive environment for generation of a vascularized engineered tissue which can induce angiogenesis in the implantation arae we used PLLA (50%)/PLGA (50%) biodegradable scaffolds. We evaluated 3 cell culture combinations: (1) scaffolds seeded with myoblasts alone; (2) a co- culture of neonat fibroblasts and EC and (3) a tri-culture of myoblasts, fibroblasts and EC.
We tested the safety, as well as the therapeutic potential of PLC scaffolds implanted to rat heart after the induction of myocardial infarction. PLC scaffolds were implanted either in single layer or in 4 stacked layers.
There was no indication of cardiac or systemic toxicity of the implanted scaffolds. In addition, implantation of 4 stacked scaffolds resulted in attenuation of the end-diastolic diameters which can indicate improved cardiac remodeling. Also, implantation of 4 stacked scaffolds resulted in reduction of left ventricular fibrosis, as indicated by reduced infarct size.
However, no positive effect was seen on cardiac function.
9.Integrative and Translational Approaches to Industrial Market
A monitor has been sent to all NANOCard groups (see below) in which they could indicate achievements that they thought are possibly interesting for further development by industry. These techniques will be evaluated together with the industrial partners within the project for suitability for commercial use in the future. None of the partners indicated techniques or other achievements possibly transferable to industrial partners or ready for commercialization.
11.Dissemination IPR & Knowledge Exchange - DIKE
See next section for details
Potential Impact:
I. Scientific and technological impact and actual NanoCARD context
Heart failure affects over 14 million people worldwide and is a leading cause of death. Since cardio-myocytes have very little regenerative capacity, therapies are limited at present. The introduction of exogenous stem-cell-derived cardico-myocytes holds promises but there are up to date several challenges remaining: That includes the delivery, integration, rejection and cellular maturation. Reprogramming adult fibroblasts into induced pluripotent stem cells (iPSCs) that are similar to embryonic stem cells addresses some issues, but others, including efficient directed differentiation into CMs and effective delivery, remains a challenge. Novel surface technologies to supporting heart muscle cell growth as proposed in NanoCARD may offer new and unique tools.
There were several reviews published in the last two years giving an up-to-date overview of tissue engineering approaches for cardiac repair. Several of the publications follow similar strategies as proposed in the NanoCARD project. An overview of such strategies is given in (1).
A general understanding of several publications is that the engineering of heart tissue requires an mulit-parametric approach by providing an environment to the cells which considers, next to the
chemistry, the micro and nanostructure of the scaffolds, the mechanics and also active mechanical and/or electrical stimulations ((2), (3)).
1. Cell sources for heart muscle regeneration
There are several key publications demonstrating the potential use of different cell types for repairing infarcted heart tissue. The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types promises new pathways for regenerative medicine in this area. Idea et al. reported previously that cardiac fibroblasts, which represent 50% of the cells in the mammalian heart, can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT) ((4)). Quian used a similar approach to reprogram in vivo (mouse) resident nonmyocytes of the murine heart can into cardiomyocyte-like cells ((5)). This in vivo delivery of GMT decreased infarct size and modestly attenuated cardiac dysfunction up to 3 months after coronary ligation. Delivery of the pro-angiogenic and fibroblast-activating
peptide, thymosin b4, along with GMT, resulted in further improvements in scar area and cardiac function. These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes. A recent review list stem cell sources for regenerative treatment of ischemic heart muscle tissue and typical markers of these cells (6). In addition, there is an overview about clinical trials and stem cell types used with these. Most of the human clinical trials performed to date used blood- and bone marrow-derived stem cells that are defined by different characteristics. According to Brenner et al., two encouraging trials utilizing cardiac stem cells from heart biopsies have been launched and are still recruiting; first results are expected soon ((6)). Most of these trials involved, however, only the administration of bone marrow-derived stem cells after myocardial infarction and did not use scaffold materials or specifically designed surfaces for cell differentiation or expansion. As such they followed a different approach compared to the NanoCARD strategy.
The reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) opens new possibility in regenerative medicine. A large pool of fibroblasts exists in the postnatal heart and Ieda et al. reported recently that a combination of three developmental transcription factors (Gata4, Mef2c, and Tbx5) quickly and effectively reprogrammed post-natal cardiac or dermal fibroblasts into differentiated cardiomyocyte-like cells ((7)). These directly induced cardio-myocytes expressed
cardiac-specific markers and showed spontaneous contractions in vitro. Fibroblasts transplanted into mouse hearts one day after transduction of the three factors also differentiated into cardiomyocyte-like cells in vivo.
Chip technologies, Materials, Scaffolds and chip for heart tissue regeneration
Independent of the cell source major challenges of heart muscle repair remain to be the cell survival, cell fate determination and engraftment as well as vascularization after transplantation. Several strategies of tissue-engineering combining scaffolds and different cell types have been developed and were partially adapted for specific application to enhance stem cell function. Different approaches and scaffold techniques for cardiac cell therapy are described by Karam et al. in a recent review ((9)). Several advancements in the field of biomaterials for heart muscle scaffolds are discussed. Examples are given for 2D studies and 3D supports for the cells also
mimicking the structural architecture of the heart. In some studies hydrogels of different types were used to control the biophysical and biochemical microenvironments of transplanted cells. Interestingly, stacks of cells-seeded polymeric sheets, as similarly proposed in NanoCARD, were already suggested in 2002 by Shimizu et al. ((10)).
Several studies of biomaterial scaffolds for heart muscle regeneration or tissue engineering, cell types and stimulation signals are documented in a recent review ((1)).
Several approached have been proposed to use micro- or nanofabrications techniques to modify surfaces of scaffold polymers in order to direct stem or progenitor cells to cardio-myocytes.
For example, polymeric scaffold surfaces have been modified by specific recognition sites for cells using technologies like the Molecular Imprinting ((11)). The authors of this publication claim that this technique is extremely suitable to produce intelligent matrices usable in the field of heart tissue engineering characterized by their particular capacity for molecular recognition of extracellular proteins (collagen, fibronectin, laminin, vitronectin, etc.) that favor cellular adhesion or cell functions. It is proposed that the polymeric matrices will show a capacity for increasing the adhesion and growth characteristics of the cells on the scaffolds thanks to the presence of nanosites that are complementary and selective towards specific peptide sequences presented by ECM proteins and with which specific integrins interact.
Other approaches followed the strategy to incorporate resilience-imparting protein found in all elastic human tissues into hydrogels to form a highly elastic scaffold (12). These hydrogels, with
photocrosslinked methacryl-tropoelastin, facilitated the attachment, adhesion, alignment, function, and intercellular communication of cardiomyocytes by providing an elastic mechanical support mimicking mechanical in vivo properties.
Considering that MI cannot be used directly on biodegradable polymers, which are a fundamental requisite for tissue engineered scaffolds, biostable polymers in the form of nanoparticles will be realized. They will be used in small quantities to modify degradable materials, so that these will retain their ability to serve as temporary scaffolds.
There are several publications reporting on the impact of surface modification of culture systems on stem cell differentiation or expansion. Focus is often on nanotopography of polymer support but also on the functionalization of the surface with peptides. To the later approach there is one publication reporting a similar technique for nanopatterning by loaded block-copolymers as commonly used in the NanoCARD consortium. The group demonstrates, next to variation in adhesion of mesenchymal stem cells (MSC), that MSC osteogenesis is reduced on surfaces with increased lateral RGD spacing while adipogenic differentiation is increased ((13)).
Many more publication report on the behavior of various stem cells controlled or directed by topographic surface features. Zouani et al., for example, demonstrated that the depth (on a nanometric scale) of micro-patterned surface structures allowed increased adhesion of human mesenchymal stem cells (hMSCs) with specific differentiation into osteoblasts in the absence of osteogenic medium (14). There have been also reports on the screening of stem cell responses to surface featured by a chip approach.
Generally, the approach of using chip technologies and high content screenings to explore the complex interaction of stem cells with cues from their environment is well accepted and widely followed(16-18).
Figure 11: Schematic representation of high throughput approaches in stem cell applications. Hydrogel engineering combined with robotics can generate several hundreds to a thousand printed artificial niche candidates in one experiment (from (16)).
Devices for mechanical cell manipulations in regenerative medicine
Mechanical forces are ubiquitous in our body and greatly affect the development and functional homeostasis of tissues. In particular, in vivo mechanical stimuli within muscles and blood vessel walls may play an important role in differentiation of stem cells to various phenotypes. Previous work has shown that mechanical strain has an effect on proliferation and differentiation on many cell types also on MSC, including up-regulation of various SMC contractile markers (8–10), and the cells aligned perpendicularly to the axis of strain (11). Cyclic stretching has been used for improved tissue engineering on heart muscles for more than one decade. Zimmermann at al. reported, for example, first results by using simple mechanical devices (19,20). There are also several reports on stretching devices within the last 3 years (21,22), some of them trying to mimic specific in vivo conditions for tissue engineering (23). Parallel to the development of a stretching
device for live cell microscopy within NanoCARD designs for similar devices have been reported (24-26).
References
1. Ye KY, Black LD. Strategies for Tissue Engineering Cardiac Constructs to Affect Functional Repair Following Myocardial Infarction. J of Cardiovasc Trans Res. 2011 Aug 5;4(5):575–91.
2. Pereira MJN, Carvalho IF, Karp JM, Ferreira LS. Sensing the Cardiac Environment: Exploiting Cues for Regeneration. J of Cardiovasc Trans Res. 2011 Jul 7;4(5):616–30.
3. Godier-Furnémont AFG, Duan Y, Maidhof R, Vunjak-Novakovic G. Regenerating the Heart. Cohen IS, Gaudette GR, editors. Totowa, NJ: Humana Press; 2011. 33 p.
4. Ieda M, Fu J, Delgado-Olguin P, Vedantham V. ScienceDirect.com - Cell – Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by Defined Factors. Cell. 2010.
5. Qian L, Huang Y, Spencer CI, Foley A, Vedantham V, Liu L, et al. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature. 2012 Apr 18.
6. Brenner C, Franz W-M. The use of stem cells for the repair of cardiac tissue in ischemic heart disease. Expert Rev Med Devices. 2011 Mar;8(2):209–25.
7. Ieda M, Fu J-D, Delgado-Olguin P, Vedantham V, Hayashi Y, Bruneau BG, et al. Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by Defined Factors. Cell. 2010 Aug;142(3):375–86.
8. Passier R, Mummery C. Getting to the heart of the matter: direct reprogramming to cardiomyocytes. Cell Stem Cell. 2010 Aug 6;7(2):139–41.
9. Karam J-P, Muscari C, Montero-Menei CN. Combining adult stem cells and polymeric devices for tissue engineering in infarcted myocardium. Biomaterials. 2012 Aug;33(23):5683–95.
10. Shimizu T, Yamato M, Isoi Y, Akutsu T, Setomaru T, Abe K, et al. Fabrication of Pulsatile Cardiac Tissue Grafts Using a Novel 3-Dimensional Cell Sheet Manipulation Technique and Temperature Responsive Cell Culture Surfaces. 2002.
11. Rosellini E, Cristallini C, Barbani N, Giusti P. Studies in Mechanobiology, Tissue Engineering and Biomaterials. Boccaccini AR, Harding SE, editors. Berlin, Heidelberg: Springer Berlin Heidelberg; 2010. 28 p.
12. Annabi N, Tsang K, Mithieux SM, Nikkhah M, Ameri A, Khademhosseini A, et al. Highly Elastic Micropatterned Hydrogel for Engineering Functional Cardiac Tissue. Adv Funct Mater. 2013 Apr 26;23(39):4950–9.
13. Frith JE, Mills RJ, Cooper-White JJ. Lateral spacing of adhesion peptides influences human mesenchymal stem cell behaviour. Journal of Cell Science. 2012 Jan 15;125(Pt 2):317–27.
14. Zouani OF, Chanseau C, Brouillaud B, Bareille R, Deliane F, Foulc MP, et al. Altered nanofeature size dictates stem cell differentiation. Journal of Cell Science. 2012 Feb 2.
15. Unadkat HV, Hulsman M, Cornelissen K, Papenburg BJ, Truckenmüller RK, Post GF, et al. An algorithm-based topographical biomaterials library to instruct cell fate. Proceedings of the National Academy of Sciences. National Acad Sciences; 2011;108(40):16565–70.
16. Marx V. Where stem cells call home. Nat Meth. Nature Publishing Group; 2013;10(2):111–5.
17. Nikkhah M, Edalat F, Manoucheri S, Khademhosseini A. Engineering microscale topographies to control the cell–substrate interface. Biomaterials. 2012 Jul;33(21):5230–46
18. Ranga A, Lutolf MP. High-throughput approaches for the analysis of extrinsic regulators of stem cell fate. Current Opinion in Cell Biology. 2012 Apr;24(2):236–44.
19. Zimmermann WH. Tissue Engineering of a Differentiated Cardiac Muscle Construct. Circulation Research. 2001 Dec 13;90(2):223–30.
20. Eschenhagen T. Engineering Myocardial Tissue. Circulation Research. 2005 Dec 9;97(12):1220–31.
21. Huang Y, Nguyen N-T. A polymeric cell stretching device for real-time imaging with optical microscopy. Biomed Microdevices. 2013 Jul 19;15(6):1043–54.
22. Yung YC, Vandenburgh H, Mooney DJ. Cellular strain assessment tool (CSAT): Precision-controlled cyclic uniaxial tensile loading. Journal of Biomechanics. 2009 Jan;42(2):178–82.
23. Zhou J, Niklason LE. Microfluidic artificial “vessels” for dynamic mechanical stimulation of mesenchymal stem cells. Integr Biol. 2012;4(12):1487.
24. Shao Y, Tan X, Novitski R, Muqaddam M, List P, Williamson L, et al. Uniaxial cell stretching device for live-cell imaging of mechanosensitive cellular functions. Rev Sci Instrum. 2013 Nov;84(11):114304.
25. Tremblay D, Chagnon-Lessard S, Mirzaei M, Pelling AE, Godin M. A microscale anisotropic biaxial cell stretching device for applications in mechanobiology. Biotechnol Lett. 2013 Oct 16.
26. Wang D, Xie Y, Yuan B, Xu J, Gong P, Jiang X. A stretching device for imaging real-time molecular dynamics of live cells adhering to elastic membranes on inverted microscopes during the entire process of the stretch. Integr Biol. Royal Society of Chemistry; 2010;2(5-6):288–93.
I. Dissemination
1. After the periodic project commitment discussions at the consortium meetings to explore commercialization opportunities, the dissemination in high impact scientific journals has been launched.
2. NanoCARD has been producing a lot of high impact scientific publications.
A lot of publications are in preparation
3. Several international conference contributions have been made (see below).
4. Several exhibitions the products have been shown (see below).
The exhibitions were:
1. High content analysis SF, USA
2. Lab automation SF, USA
3. Medica, Germany
4. Israel life imaging forum, Israel
II. Intellectual property rights
Project participants are working closely with suitable consultants to safeguard their intellectual property rights and to ensure the best technology transfer from the project. Therefore the participation of Qiagen and other industrial partners at the meetings have been very helpful, especially to maximalist control over the project outcome. To guarantee the IPR protection the coordinator is discussing with experts before publishing any results the possibility of the protection of the results. After this check of the manuscript by the coordinator whether a protectable result has been achieved or not, the results were published in high impact scientific journals.
The coordinator has been organizing an industrial workshop to achieve the best possible exploitation of results.
III. Knowledge exchange
a. Public website of the project has been established. All informations
related to the scientific goals and achievements of the project have been put into the wiki. http://www.is.mpg.de/NanoCARD
b. An eternal project web page has been created for the exchange of protocols, methods, results and specific organizational questions as well as making the whole details of NanoCARD available for the different participants.
c. Every scientific group has been participating at the NanoCARD meetings. The different groups have been giving talks, so that the very lively knowledge exchange has been guaranteed. Different labs offered training courses to use the different microscopical material and cellular systems.
d. An important instrument for the knowledge exchange has been the PhD- and Postdoc Workshops as well as the principal investigator meetings.
NanoCARD Meetings/PhD Workshops
• Kick off meeting 12.-13. Januar 2010, Stuttgart
• NanoCARD Meeting 06.-08. June 2010, Rehovot
• Brussels Meeting 31.01.2011 with the European Commission
• NanoCARD Meeting 05.-07. April 2011, MPI Stuttgart
• Midterm review in Brussels 30.06.-01.07.2011with the European Commission
• Principal Investigator Meeting 13.09.-14.09.2012 Stuttgart
• Final Meeting 17.10.-18.10.2013 MPI Stuttgart
e. Regular meetings with the industrial partners for early identification of potential protection development opportunities for the European industry have been taken place.
List of Websites:
http://www.mf.mpg.de/NanoCARD/
NanoCARD
Nanopatterned scaffolds for active myocardial implants
The overall goal of this project was to create nanopatterned scaffolds for myocardium implants.
Cell therapy and tissue engineering are opening novel therapeutic perspectives for myocardial repair. The introduction of new myocytes into diseased areas of the heart can improve the hearts mechanical properties. The therapeutic utilities significantly hampered by the paucity of cell sources for human cardiomyocytes and by the high degree of donor cell death following cell grafting.
Therefore the fundamental different concept of NanoCARD aims to provide a biomaterial which is based on high throughput screens for identifying specific and necessary signals of extracellular matrix. These signals are activating specific cell programs such as a cell adhesion and the induction of differentiation of stem or progenitor cells to functional myocytes. The implant materials present smart nanopatterned interfaces to cells. These surfaces present chosen biomolecules in nanoscopically defined spatial patterns on biologically inert, biodegradable substrates.
A key point has been the identification of parameters required to drive cardiac tissue formation.
The scientific bases of NanoCARD is the fact, that the activity and fate of all living cells is exquisitly regulated by the cells and environments and particularly by the adhesive interaction with neighbouring cells and with the extracellular matrix. Therefore the design and use of nanostructure and specifically biofunctionalized surfaces with desirable molecular properties offers unique opportunities for programming cell responses and functions.
This biomimetic implant technology concept is based on the interaction between living cells and synthetic, nanostructured interfaces.
The economic potential is remarkable because myocardial infarction is an important disease worldwide.
Project Context and Objectives:
Since many previous attempts to engineer tissue scaffolds were based on trial and error approaches, NanoCARD aims to create a conceptually new biomimetic, nanoscopically designed implant. The implant’s highly tailored material properties will encourage the selective adhesion of stem and progenitor cells and direct their differentiation, enabling replacement myocardial tissue to be generated ex vivo.
The development of conceptually new implants is based on five pillars: (1) High-Throughput Screens for identifying specific extracellular matrix material parameters as signals for directing stem or progenitor cells towards specific cardiac tissue generation in vitro; (2) Implant synthesis by integration of the set of material parameters identified by HTS into a cell support: so-called “therapeutic surface”. The implant will be based on a microporous mulitlayered stack of “therapeutic surface”; (3) in vitro recruitment of cells and cardiac tissue formation; (4) in vivo (animal) testing of implants; (5) development of commercialization strategies for the new implant concept for cardiac tissue generation together with the companies Qiagen, Idea Bio Medical, CellMade, Evercyte and Cell System.
The in vitro HTS will reduce the number of animal experiments, which is in close agreement with the ethical aims of EU guidelines.
The biochip/HTS system will find wide application in the life sciences.
Objectives
Objective 1: Development of nanostructured, biofunctionalized and biodegradable cell supports which have the capacity to vary cell ligand type, ligand spacing, and substrate stiffness for exploring and manipulating cellular interactions with non-biological surfaces
Objective 2: High-Throughput Screens (HTS) for identifying implant material parameter: Exploration of the interaction mechanisms between cells and biomaterial by automated live cell optical microscopy
Objective 3: Characterization of the physicochemical properties of extracellular matrices in vivo and in vitro; exploring how the interaction mechanisms between selected cell types and matrix impact cell fate.
Objective 4: Translation of the set of identified cell response-specific material parameters to implants for in vivo applications
Objective 5: Transition from basic scientific understanding to the industrial market
These objectives are achieved via workpackages covering the following areas:
• Synthesis of Artificial Cell Environments on the Molecular Level by Self-Organization Principles
• Development of the BioChip
• Development of the automate optical microscope for application of High-Throughput Screens
• High-throughput screening of endothelial cells (EC) and cardiomyocytes (CM)responses to nanoengineered cell environments, Systems Biology
Analysis
• How cell-made extracellular matrix co-regulates protein expression profiles and cell fate
• Synthesis of the implant
• Directed stem cell differentiation into endothelial cells (EC) and cardiomyocytes (CM) by the implant
• In vivo assessment of therapeutic potential of the implant
• Integrative and Translational Approaches to Industrial Market
• Co-ordination and project management
• Dissemination, IPR & Knowledge Exchange - DIKE
Project Results:
1.Synthesis of Artificial Cell Environments on the Molecular Level by Self-Organization Principles
Summary
Biodegradable polymer scaffolds have been constructed, and their mechanical properties and biochemical potential have been investigated. Furthermore, the surface of the scaffold has been modified with a polymer brush that will enable their future patterning with cell adhesive ligands.
In order to examine the interaction between the synthesized scaffold and the heart a rat in-vivo model was used. Poly(lactide-co-caprolactone) (PLCL) porous and non-porous scaffolds were implanted in healthy rat hearts.
In addition, PLCL-porous scaffolds were implanted in rats after the induction of myocardial infarction.
Evaluating the biocompatibility of the polymer substrates we monitored the behavior of human bone marrow stem cells (hBMCs) in terms of cell adhesion, spreading, actin organization and focal adhesion formation using a confocal microscope. Our analysis of focal adhesion formation and actin organization on tested substrates reveals that the number of pores strongly affects cellular adhesion.
We demonstrate distinct nanotopography-induced cell phenotypes, characterized by different morphology, LOX-1 diffusivity and oligomerization state.
A model combines microfluidic flow, mass transport of the chemotractant and binding of the molecules on a model cell surface in a single model.
Description
Micro-porous films
The mechanism of the breath figure technique to create well-ordered porous films from polymers in an organic solvent has been well reported in literature.
The breath figure technique allows great control over the formation of the porous material, due to the fact that there is a number of conditions which can be varied that will have an effect of the process, such as, air flow speed, humidity content (%), organic solvent choice, polymer molecular weight and polymer solution concentration. The first scaffolds were obtained by stacking of the microporous PLCL films that were prepared with the breath figure technique. Due to the some problems with the stacking of the films, 3D scaffold preparation method was changed to the sphere-templated fabrication method (Scheme 1). Sphere-templated fabrication method allows controllable pore size and high interconnectivity between pores.
Scheme 1: Sphere-templated fabrication method for scaffold synthesis.
Biofunctionalization and nanostructuration of the biodegradable, 3D, interconnected-microporous and non-fouling polymer scaffolds were achieved. They are prepared by random copolymerization of 2-methylene-1,3-dioxepane (MDO) and 2-(hydroxyethyl) methacrylate (HEMA) monomers and a PCL-PEG-PCL crosslinker.
Mechanical Studies
Young’s modulus measurements of scaffolds were carried out by using Minimat machine with 20N cell load. Figure 1 shows the average stress-strain graph and the average Young’s Modulus of the scaffolds that were obtained with the following monomer mol ratios: HEMA/MDO/PCL-PEG-PCL : 27/5/1. This composition gives the Young’s Modulus of the scaffold as 0.33±0.07 MPa, which is close to the value of the Young’s Modulus for a heart rat tissue 0.59±0.22 MPa.
Figure 1: Avarage stress-strain graph for biodegradable scaffolds.
Biofunctionalization
Biofunctionalization of scaffolds with cell adhesive peptides (RGD) was performed by addition of RGD-labeled methacrylate monomer to the monomer mixture before polymerization (Scheme 2). The methacrylated RGD peptide is the only monomer with nitrogen atom. High resolution N1s X-Ray Photoelectron Spectroscopy (XPS) shows an increase of the nitrogen peak while increasing the feeding ratio of the RGD peptide in the monomer composition. It means that the amount of peptide integrated in the scaffold could be tuned with the amount of peptide containing monomer in the feeding ratio.
Scheme 2: Scheme for the synthesis of RGD functionalized scaffolds.
Cell Studies
In order to evaluate the cells bio-fouling property of scaffolds cell studies were carried out with different concentration of RGD peptide inserted scaffolds. RGD functionalized scaffolds were incubated with GFP expressing HeLa cells and attachment of cells was examined with confocal microscopy. Cell cytoplasm forms were observed.
The nanostructuration of scaffolds was obtained by reduction of Au+3 in sodium borohydride (NaBH4) (Scheme 3). First, scaffolds were immersed into aqueous potassium tetrachloroaurate (KAuCl4) solution and Au+3 ions were absorbed in the scaffolds after shaking for 12 hours. After that NaBH4 was added for the reduction of Au+3 ions to Au nanoparticles (Scheme 3).
Scheme 3: Schematic representation of Gold nanoparticle decoration of PHEMA scaffolds.
2.Development of the BioChip
Summary
We demonstrated the possibility to implement a microfluidic device on topographically-modified substrates yielding on-demand local 3D gradients of soluble molecules with controllable spatial and temporal profiles.
Protocols for the fabrication of biochemical chips on glass supports and on polymer support have been established. Chips are adapted and used for cell experiments within the project.
Cell tests with such substrates proved the suitability of the chip for cell culture studies.
The design of a general platform allowing for mechanical stimulation of cells by lateral stretching of an elastomer membrane is achieved.
We have developed two different microfluidic platforms that are capable of forming soluble gradients.
We showed that our systems are compatible for cell culture and cells could be maintained up to at least one week.
Description
The goal of high throughput screening (HTPS) remains the fabrication of miniaturized
laboratory reactors, called micro-arrays that can work in parallel and be
compatible with high sensitivity detection systems to monitor their outputs. Complex
analyses can be performed within a few hours with the help of microarrays or
“biochips”.
The manufacture of microarrrays and biochips benefit from the use of a great
number of nanofabrication tools. Surface molecular modification techniques include:
self-assembled mono-layers, surface spin coating with polymers or colloids) to control properties of the array interface at the molecular level (adhesion, hydrophobicity,friction) and as shown in this research, the Diblock Copolymer Micelle Nanolithography (BCML) technique.
In addition, HTPS approaches must fullfill certain criteria in order to be useful
in a research diagnostic laboratory. They must be able to perform a large number
of assays rapidly and simultaneously in a user friendly manner and be small in format.
They must be configured to provide robust and reproducible results that allow
standardization and comparison of experiments performed in different laboratories.
Figure 1 illustrates the preliminary experiments.
Figure 1: Illustration showing preliminary cell adhesion experiments. (A) Gold (B)
Gold + BSA (C) Gold and Fibronectin (D) Gold and Fibronectin + BSA (E) Gold
and RGD peptide (F) Gold and RGD peptide + BSA (G) Gold-nanodots with RGD
passivated with PEG 2000 (H) Glass surface passivated with PEG 2000
Key technology for the production of biochips is the micellar nanolithography (BCML). This technique uses self-organization principles to arrange micelles on the surface of flat glass substrates and it allows for patterning the surfaces with hexagonal arrangements of gold nanoparticle. The particles have a size of typically 8nm which is comparable to the size of cellular membrane receptors. Within the project the production of these nanoscopically-structured surfaces has been up scaled to meet the requirements of producing large numbers of surfaces for all project-relevant applications.
The method to precisely functionalize the biochip surface with Au-NP has been further extended to be applicable to more complex 3D-like environments. This is an important step to use polymers developed within WP1 and to account for more in-vivo like cell culture conditions.
The design of a general platform allowing for mechanical stimulation of cells by lateral stretching of an elastomer membrane is achieved. Several prototypes following different approaches and designed for specific purposes have been developed and manufactured. Examples are given in Fig. 7. One setup is particularly suited for live cell imaging application and allows for the observation of cells during the experiment by phase, DIC or fluorescent microscopy. Other setups use a six-well format and can apply uniaxial tensile strains to 48 wells with 8 different amplitudes or frequencies. All experimental platforms are suitable to manipulate cells over long time periods (weeks). Test runs with cells under regular culture conditions (T= 37°C, 5% CO2 and high humidity) were successful and the setups are used for screening cell reaction upon application of various mechanical stimulations (WP4).
A prototype biochip composed by a fluidic network and a textured COC substrate was fabricated and characterized. Complete biochips were realized by bonding a PDMS microfluidic network onto the grating. The microfluidic chip was aligned and mounted in close proximity to the patterned area present on the COC substrate. A fluorescein solution was then delivered through the fluidic channel to generate chemical gradients coupled to the underlying topography.
Production of surfaces for biochemical chips
Key technology for the production of the biochemical chips is the micellar nanolithography (BCML) which is illustrated in Fig. . This technique uses self-organization principles to arrange micelles on the surface of flat glass substrates and it allows for patterning the surfaces with hexagonal arrangements of gold nanoparticle. The particles have a size of typically 8 nm which is comparable to the size of cellular membrane receptors. Within the project the production of these nanoscopically-structured surfaces has been up scaled to meet the requirements of producing large numbers of surfaces for all project-relevant applications.
Figure: Schematic representation of the Micellar Nanolithography used for nanopatterning oft he biochip surfaces.
In order to immobilize several different peptides or other biomolecules on a chip a novel protocol was developed. Briefly, surfaces are patterned with small gold nanoparticles (Au-NP) in a hexagonal order by BMCL (BCML) as described above. The inter-particle distance can be adjusted between 30 and 200 nm. To prevent unspecific protein absorption, the surface is covered with a PEG-passivation layer. Several lengths of PEG molecules were tested. For the final chip we use typically PEG 2000 which gets immobilized on the glass (between the Au nanoparticles) via silane chemistry. This procedure provides a stable protein passivation layer on the glass but still allows the chemical modification of the Au nanoparticles with other molecules
Initial cell adhesion studies on the biochemical chip
The chips were used for cell adhesion experiments to proof their suitability for larger scale cell culture studies. In first test, rat embryonic fibroblasts were cultured on the biochips for several days. The biofunctionalization as well as the passivation of the surface between the micro-spots was demonstrated to be stable over that time period. An illustrative example of cells adhering on RGD-functionalized micro-spots is given below. The chips’ dimensions, optical and mechanically handling properties are ideally suited for high content screening microscopy within NanoCARD.
Figure 1: Rat embryonic fibroblasts on biochip surface with micro-spotted peptides (RGD). Cells adhere only on the RGD-modified spots. Within each spot the peptide is specifically bound to the Au-NP (not visible with this magnification).
Biomechanical Chip Design
Biochip Fabrication – general properties
The substrate system used in the bio chip was designed in a way that allowed for varying the biophysical and biochemical properties on the chip surface simultaneously.
Distances between the ECM mimicking peptides (biomolecules) range between 30nm and 150 nm. The size of anchor points itself is chosen not exceed a limit of about 10 nm to assure single receptor-ligand interaction. The mechanical properties of substrates were chosen to be with 3kPa and 90 kPa with additional very stiff support of 4 MPa, serving as a control substrates for the in vitro tests.
Cell tests with biomechanical chip
Initial functional test of the chips were performed with various cell lines such as normal human dermal fibroblasts (NHDF) and CCL-121, poorly differentiated fibrosarcoma cells from the acetabulum. Both cell types were trypsinized and seeded on the biofunctionalized substrates surfaces in appropriate media containing 10% fetal bovine serum (FBS). The experiments were performed after desired cell-substrate interaction time under cell-culture conditions (5 % CO2, 37o C). To optimize the measurement procedure and characterization of the cellular behavior on the biomechanical chip during different time periods, a high throughput screening (WiScanTM instrument, Idea-Bio, Israel), equipped with live cell environmental control and analysis (WiSoftTM software, Idea-Bio, Israel).
3.Development of the automate optical microscope for application of High-Throughput Screens
Summary
The WiScan family of systems to include fast acquisition and storage, accurate stage positioning, live cell conditions, large set of command functions for versatile applications, flexible sample formats, automatic sample loader and user friendly interface has been developed. The microscope is fully automatic and acquiers high-resolution images at a precise focus plane using fast auto focusing procedure.
Tools for image processing and data analysis were added to WiSoft the image analysis software.
Many biological applications were studied using the WiSoft including – GFP spot detection, cell cycle evaluation, translocation of fluorescnce protein, variation in mitochondria and ER, studies of fluorescently labeled Celegans, focal adhesion morphologicl changes, tube formation and more.
We have improved usability and stability of the software and the system has been tested extensively.
We have also been working on some simple image analysis tools and ImageJ scripts to automate processes like cell counting and analysis.
Description
Adaptation for robotic sample loading:
The WiScan® Argus system was designed to enable robotic loading and unloading of biological samples. Thus, cells can be cultured at an automatic liquid handling system and then transfered to the WiScan® Argus screening microscope automaticallly using a robotic arm.
Biochip readout:
The researchers prepared a spotted array of adhesion sites on a standard glass slide and seeded cells on the array. After fixation the cells were labeled for nuclei and for actin fibers. The biochip unique structure was defined at the WiScan® system as a spotted array, and the array was scanned using 20x/0.75 objective and two wavelengths for DAPI and FITC readout. The images were analyzed to count the number of cells in each spot and to measure the average actin fiber length (Figure 3).
Figure 3 - Biochip prototype (partial region of 36 adhesion points) scanned at WiScan® using 20x magnification. Cells are labeled with DAPI for nuclei and with phalloidin-FITC for actin. The number of cells per adhesion point and the actin fiber length was calculated using WiSoft® and these two parameters are presented for each well in color scale.
4.High-throughput screening of endothelial cells (EC) and cardiomyocytes (CM)responses to nanoengineered cell environments, Systems Biology Analysis
Summary
We tested the adhesion of rat mesenchymal stem cells to LACL and LAGA membranes. In addition, we tested the adhesion of inflammatory cells (rat splenocytes) to the synthesized surfaces.
Studies were performed on cell responses of human mesechymal stem cells (hMSC). The cells were cultured on nanostructured RGD-functionalized Biochip surfaces with different developed compliances. hMSC stem cell morphology and structure of adhesion sites and cytoskeleton depends on both parameters: ligand spacing and substrate stiffness.
All the produced surfaces were used for the induction of EC and/ or CM development, proliferation.
We have established an experimental system to test the effect of substrate rigidity on the proliferation, differentiation, and the commitment of rat cardiomyocytes.
We found that modulating surface rigidity effects rat cardiomyocyte proliferation.
Protocols for determination of cell specific markers of endothelial cells were developed using primary human endothelial cells (HUVEC and HAoEC). In addition, in order to establish reproducible and stadradizable cell models, HUVECs were immortalized using human telomerase and characterized in detail.
Description
We have selected to work, at this stage with cells of 2 origins, namely chick embryos and newborn rats. Detailed procedures for the cultivation of these two cell types were developed, optimizing the condition for reaching full differentiation into functional heart cells, namely – well organized sarcomeric organization, and beating. To study both features, we have specifically fluorescently labeled the cells, at different time points after plating, for a variety of sarcomeric proteins (e.g. actin, -actinin, myosin (cardiac)), and examined the cells for beating, using live cell microscopy.
The cells, from the same origins, were extensively examined, following labeling for multiple cytoskeletal components, enabling the visualization and quantification of multiple cellular parameters including sarcomere length, nuclear organization, sarcomere registration, cell shape and polarization, matrix adhesion structure, composition, orientation and connection to the contractile apparatus. In addition, the cells were examined by transmission electron microscopy for the organization of their contractile machinery.
Towards the development of synthetic tissue scaffolds for the regeneration of the heart muscle after myocardial infarction, we have set the experimental system, including the choice of cells for the study (primary cardiac myocytes (PCM) from chicken or rat origin, prepared as above) and characterization of the cellular response to variations in the properties of synthetic substrates.
Our preliminary finding showed selective adhesion of cardiac fibroblasts on the high-density signal hydrogel surface and higher population of cardiocycytes on the lower density signaling surfaces. These finding are based on the labeling of the cells to several sarcomeric and adhesion-associated cytoskeletal proteins, including actin and alpha actinin enabling the assessment of Z-band formation across the cardiomyocytes. In order to evaluate whether these results are differentiating outcome, or of selective adhesion, we are repeating these experiment with both the soft hydrogel surfaces and on the rigid glass surface.
Highlights of the PDMS elastomers results -
Soft PDMS substrates facilitate cardiomyocytes proliferation. We found that modulating surface rigidity effects rat cardiomyocyte proliferation. Specifically, softer PDMS substrates of 20kPa and 5kPa elevate neonatal rat cardiomyocyte proliferation by over 50%. We would perform additional experiments to further characterize the proliferating cardiomyocytes and to assess the mechanism that leads to this phenomenon.
Substrate rigidity effects sarcomeric organization of cardiomyocytes. We compared 1 day old rat cardiomyocytes cultured on either relatively rigid (2MPa), or relatively soft (5kPa) surfaces. Substrate rigidity effects the sarcomeric architecture of newborn rat cardiomyocytes, as evident by the sarcomeric markers MHC, myomesin, and Troponin T. Cardiomyocytes on the 2MPa substrates are more polarized, exhibit well organized sarcomeric structures and directionality. On the soft 5kPa substrate cardiomyocytes are radial and exhibit disorganized sarcomeric structures.
5.How cell-made extracellular matrix co-regulates protein expression profiles and cell fate
Summary
Different ECM and VEGF concentrations were investigated on their effect on tube formation of HAoEC in co-culture with HAoSMC.
Characterization of the biophysical properties of extracellular matrix that cells assemble and remodel on nanopatterned surfaces with varying ligand density and substrate rigidity.
Studies of cell assembled ECM on the biochips.
Preliminary studies whether hMSCs sense the topographical differences in the surfaces.
Fabrication of cell-derived ECM scaffolds on NanoCARD chips.
We found that the optimal conditions for vessel-network formation is low fibrin concentration (7.5 mg/ml-final concentration) and high cell density (0.3X10^6 – huvec + 0.06X10^6- fibroblasts).
Our results show that mechanical forces induce differentiation of the cells into the mesoderm direction.
Different ECM and VEGF concentrations were investigated on their effect on tube formation. Adding VEGF to the used ECM had only limited effects on tube formation in the three models.
Stem cell engineering and conditioning: Since none of the available stem cell sources can guarantee that none of the injected stem cells might differentiate into cancer cells, we started to ask how stem cells could perhaps be conditioned to minimize tumorogenesis (C. Moshfegh & V.Vogel patent filed on Oct 3, 2013).
Description
In comparison to flat surfaces, HFFs on NanoCard surfaces formed much less ECM. Also, the ECM exhibits a broader range of FRET ratios within a given matrix and even within individual fibrils, indicating a broad range of matrix strains. Although a variety of FN conformations co-exist in cell culture, our preliminary experiments show that the mean matrix tension alters with the underlying nano-topographyat a ridge width of 2 and 1.75 µm, significantly less matrix was assembled when compared to the smaller ridge width, 1.5µm and 1.25µm respectively. The trends of the mean FRET ratios are different as shown in the Figure below. The FRET ratios were highest at intermediate ridge width.
6.Synthesis of the implant
Summary
We have implanted the preliminary cardiac scaffold (LACL porous scaffold) in healthy rat hearts. Cardiac performance was measured using high frequency echocardiography in order to determine whether the implant interferes with normal cardiac function (n=4). Also, implanted hearts were evaluated by histology to detect signs of tissue inflammation or any other structural abnormality.
Description
Seeding EC and CM on implant material in vitro and investigating cardiac tissue formation.
MPI performed experiments with endothelial cells (EC) and smooth muscle cells (SMC) on biochips with different ligand spacing and peptide functionalization. Experiments demonstrate that cell adhesion of both cell types depends on the nano-scaled ligand spacing (40 vs. 90 nm) but not on the selection of one of the two different adhesive peptides (RGD and REDV).
Co culture of GFP or RFP-huvec cells and fibroblasts were grown on PLLA\PLGA in vitro for 10 days, during this time the scaffolds were monitored using confocal for the formation of vessel-like networks.
Spontaneous contraction and vessel like structure formation were observed between days 4-6.
7.Directed stem cell differentiation into endothelial cells (EC) and cardiomyocytes (CM) by the implant
Summary
We have explored endothelial-fibroblast interactions in 2D and 3D constructs and the influence of scaffold mechanical properties on formation of vessel-like structures. Moreover, we have examined the influence of scaffold patterning and topography on cell attachment, proliferation and organization.
Seeding stem cell derived cells (cardiomyocytes and endothelial progenitors and common progenitors) on biofunctionalized surfaces designed to induce specific attachment and differentiation and analysis. Histological characterization of the engineered tissue and biocompatibility tests. In addition, biosafe generation of iPSCs was tested by using recombinant Yamanaka proteins as media additives. Surprisingly, Oct4 was found to contain a functional peptide transduction domain allowing direct cellular uptake.
We used mechanical forces in purpose to differentiate embryonic stem cells (ESC) into the mesoderm germ line, as a precursor cells for both endothelial cells and cardiomyocytes.
We seeded endothelial cells and mouse ESC in defined patterns (using PDMS stamps) in purpose to mimic cardiac tissue.
Description
Effect of different RGD spacing on endothelial cells proliferation and survival
We have used three different RGD spacing (i.e. 32, 58 and 102 nm spacing between two neighboring RGD molecules) to investigate the influence of RGD spacing on EC attachment and organization.
The spacing between the RGD molecules plays a key role in cell attachment and organization.
Effect of different RGD spacing on fibroblasts proliferation and survival
When fetal fibroblasts were seeded on PEG gels, we observed some morphological changes between the different surfaces. The fibroblasts are more elongated and able to create cell-to-cell interactions with the increase in RGD spacing.
Fibroblasts seeded on higher density and low spacing (i.e. 33 nm) cover glasses proliferated more than fibroblasts seeded on lower density and high spacing (i.e. 74 nm).
We can see that in the of 33 nm spacing, we have approximately 50% of proliferating cells, while in the 74 nm spacing, we have only 10% proliferating cells.
Effect of different RGD spacing on endothelial-fibroblasts organization
When EC and fibroblasts were seeded together on RGD cover glasses no significant difference was observed between low (i.e. 74 nm spacing) and high (i.e. 33 nm spacing) density RGD.
Non Canonical Wnt Pathway:
In order to evaluate the effect of Wnt signaling through the non canonical Wnt pathway we evaluated the effect of Wnt-11 and Wnt 5a conditioned media on hESC cardiac differentiation. Wnt 11 conditioned media applied at equal volumes with culturing media during the initial 8 days of differentiation resulted in a 2-fold increase in the percentage of contracting areas.
We aimed at evaluating whether non canonical Wnt signaling effected cardiac differentiation during early hESC differentiation (day 1-4) suggesting a potential role mesoderm induction or late hESC differentiation (days 5-8) suggesting influence on commitment to the cardiac lineage. While early exposure to Wnt5a conditioned media resulted in significant upregulation of cardiac markers late exposure to Wnt5a conditioned media did not result in upregulation of cardiac markers.
Activation of the non-canonical WNT pathway is known to induce cardiac specification through protein kinase C (PKC) and Jun N-terminal Kinase (JNK) activation in the xenopus laveis model.
Selective JNK inhibition did not significantly affected the proportion of the contracting areas significantly (p=0.70 n=150). However, PKC inhibition by bisindolylmalmeimide I reduced significantly the proportion of contracting areas by 67%.
Canonical WNT Signaling
To evaluate the effect of activation of canonical Wnt signaling on differentiating hESC, we applied Wnt3A conditioned media during the first 8 days of differentiation.
In conclusion, we have showed that: 1) Non-canonical Wnt signaling promotes human embryonic stem cell differentiation into the cardiac lineage through both Wnt11 and Wnt5a. 2) The time frame in which non-canonical Wnt signaling exert its cardiogenic effect is between days 1-4 of hESC differentiation. 3) Among the suggested signaling pathways through which non canonical Wnt is known to mediate its effect, activation of PKC but not JNK results in enhancement of cardiogenesis in hESC. 4) Activation of canonical Wnt signaling during the suspension period inhibits hESC differentiation to the cardiac lineage.
In order to examine mESCs differentiation under the different mechanical conditions real time PCR was used for examining representative genes of the three germ layers. We started by examining the genes expression after two days of oscillations
One can notice that under static stretch significant change couldn’t be observed in the three germ layers vs. control. However, by using oscillatory stretching significant change in oscillations vs. control was observed both in bry, pax6 and nestin, representative genes of the mesoderm and ectoderm layers.
Establishment of an efficient hiPSCs cardiomyocyte differentiation system. Since the initial demonstration that beating cardiomyocytes can be generated from both hESC and hiPSCs using the spontaneous, but relatively inefficient, serum-dependent embryoid-body (EB) differentiation system, several important improvements were made. In this part of the project we evaluated a number of strategies in an attempt to establish a well-defined, scalable, serum-free, directed hiPSCs cardiomyocyte differentiation system. These different suggested methods were inspired from lessons learned from embryology and are based on sequential manipulation of the BMP, Activin/nodal, and Wnt pathways. Comparing the different strategies we noted that in our hands the most effective, reproducible, and cost-effective differentiation system was a modification of the method suggested by Lian et al. (PNAS 2012).
This method is based on the use of small molecules to manipulate a single signaling-pathway (the canonical Wnt pathway). This method is based on initial activation of the Wnt pathway by CHIR-99021, a GSK3 inhibitor, to facilitate mesoendoderm formation followed by Wnt inhibition by IWP-2 or IWP-4 to induce cardio-mesoderm formation. Optimization of this strategy in a monolayer approach resulted in a highly-efficient cardiomyocyte differentiating system.
Induction of anisotropic cardiomyocyte alignment by nano-patterened films.
Ordered alignment of cardiomyocyte cells, similar to the anisotropic in-vivo cardiomyocyte alignment, is one of the major challenges in myocardial tissue engineering. Anisotropic alignment may allow achieving tissues with better force forming capabilities and lower risk for arrhythmogenicity. To this aim we collaborated with Marco Cechinni’s group (Pisa, Italy) and seeded nano-patterned scaffolds containing grooves with a variety of geometries. Scaffolds were generated from COC and PDMS, the unpatterened borders of the scaffolds were used as controls. Both neonatal rat ventricular cardiomyocytes and hiPSCs derived cardiomyocytes were seeded on the scaffolds. Scaffolds were covered prior to seeding with fibronectin to improve cell attachment and were followed microscopically for two weeks. Additionally, time-lapse imaging was generated to allow real-time follow up of the alignment kinetics. Seeding of both neonatal rat ventricular cardiomyocytes(NRVC) and human induced pluripotent cardiomyocytes (hiPSC-CMs) demonstrated that the cells can attach to the surface. Most of the cells survived following seeding and the ordered alignment process was started within hours following seeding. Following 24 hours, most cell in all groups (COC/NRVCMs, COC/hIPSCs, PDMS/NRVCMs, PDMS/hiPSCs) were generally aligned to the direction of the nano-patterned grooves.
8.In vivo assessment of therapeutic potential of the implant
Summary
We have selected a preliminary list of 7 candidate ligands known to affect the migration of adult stem cells, and tested the migration of rat mesenchymal stem cells towards these ligands.
We tested the ability of the same candidate ligands to induce migration of inflammatory cells.
We tested the safety, as well as the therapeutic potential of PLC scaffolds implanted to rat heart after the induction of myocardial infarction. PLC scaffolds were implanted either in single layer or in 4 stacked layers.
There was no indication of cardiac or systemic toxicity of the implanted scaffolds.
However, no positive effect was seen on cardiac function.
Description
We have previously implanted tissue constructs seeded with human embryonic stem cells derived cardiomyocytes (hESC-CM), endothelial cells (HUVECs) and mouse embryonic fibroblasts (mEFs) onto rat myocardium. The cells were seeded in PLLA/PLGA scaffolds and grown for two weeks in-vitro pre-implantation and then for an additional 3 weeks in-vivo. We have shown that the engineered constructs beat synchronously and form endothelial vessel networks (1,2). We have demonstrated vascularization of the grafts and organization of functional blood vessels within the scaffold. We also have showed that hESC-CM seeded with HUVECs and mEFs are able to contract synchronously and express cardiac markers (1,2).
Following the development of spontaneously synchronized beating areas, the engineered muscle constructs were engrafted to the left ventricular surface of immunosupressed rats. Two weeks after transplantation, histological analysis revealed the presence of vascularized cardiac tissue.
Following these experiments we then proceeded to infarct rat model. We have seeded PLLA/PLGA scaffolds with HUVECs, human foreskin fibroblasts (hff) and hESC-CM. One week later we induced infarct in rats. The infarct was confirmed by Ultra-Sound Electrocardiography (U.S-EKG). One week after infarct induction, we tried to implant our scaffolds onto the infracted area; unfortunately the scaffolds didn't attach to the infracted area. Ongoing studies are trying to establish a suitable protocol for implanting the scaffold onto the infracted area and to test biofunctionalized surfaces.
We have implanted tissue constructs containing skeletal myoblasts, EC and human neonatal dermal fibroblasts (HNDF) around the femoral artery of mice to enhance angiogenesis in the engineered muscle tissue. The cells were seeded in PLLA/PLGA scaffolds and grown for 10 days in-vitro pre-implantation and then an additional 4-12 days in-vivo. We have shown that the engineered constructs have blood perfusion and promoted angiogenesis.
To explore the ability to establish a 3D supportive environment for generation of a vascularized engineered tissue which can induce angiogenesis in the implantation arae we used PLLA (50%)/PLGA (50%) biodegradable scaffolds. We evaluated 3 cell culture combinations: (1) scaffolds seeded with myoblasts alone; (2) a co- culture of neonat fibroblasts and EC and (3) a tri-culture of myoblasts, fibroblasts and EC.
We tested the safety, as well as the therapeutic potential of PLC scaffolds implanted to rat heart after the induction of myocardial infarction. PLC scaffolds were implanted either in single layer or in 4 stacked layers.
There was no indication of cardiac or systemic toxicity of the implanted scaffolds. In addition, implantation of 4 stacked scaffolds resulted in attenuation of the end-diastolic diameters which can indicate improved cardiac remodeling. Also, implantation of 4 stacked scaffolds resulted in reduction of left ventricular fibrosis, as indicated by reduced infarct size.
However, no positive effect was seen on cardiac function.
9.Integrative and Translational Approaches to Industrial Market
A monitor has been sent to all NANOCard groups (see below) in which they could indicate achievements that they thought are possibly interesting for further development by industry. These techniques will be evaluated together with the industrial partners within the project for suitability for commercial use in the future. None of the partners indicated techniques or other achievements possibly transferable to industrial partners or ready for commercialization.
11.Dissemination IPR & Knowledge Exchange - DIKE
See next section for details
Potential Impact:
I. Scientific and technological impact and actual NanoCARD context
Heart failure affects over 14 million people worldwide and is a leading cause of death. Since cardio-myocytes have very little regenerative capacity, therapies are limited at present. The introduction of exogenous stem-cell-derived cardico-myocytes holds promises but there are up to date several challenges remaining: That includes the delivery, integration, rejection and cellular maturation. Reprogramming adult fibroblasts into induced pluripotent stem cells (iPSCs) that are similar to embryonic stem cells addresses some issues, but others, including efficient directed differentiation into CMs and effective delivery, remains a challenge. Novel surface technologies to supporting heart muscle cell growth as proposed in NanoCARD may offer new and unique tools.
There were several reviews published in the last two years giving an up-to-date overview of tissue engineering approaches for cardiac repair. Several of the publications follow similar strategies as proposed in the NanoCARD project. An overview of such strategies is given in (1).
A general understanding of several publications is that the engineering of heart tissue requires an mulit-parametric approach by providing an environment to the cells which considers, next to the
chemistry, the micro and nanostructure of the scaffolds, the mechanics and also active mechanical and/or electrical stimulations ((2), (3)).
1. Cell sources for heart muscle regeneration
There are several key publications demonstrating the potential use of different cell types for repairing infarcted heart tissue. The reprogramming of adult cells into pluripotent cells or directly into alternative adult cell types promises new pathways for regenerative medicine in this area. Idea et al. reported previously that cardiac fibroblasts, which represent 50% of the cells in the mammalian heart, can be directly reprogrammed to adult cardiomyocyte-like cells in vitro by the addition of Gata4, Mef2c and Tbx5 (GMT) ((4)). Quian used a similar approach to reprogram in vivo (mouse) resident nonmyocytes of the murine heart can into cardiomyocyte-like cells ((5)). This in vivo delivery of GMT decreased infarct size and modestly attenuated cardiac dysfunction up to 3 months after coronary ligation. Delivery of the pro-angiogenic and fibroblast-activating
peptide, thymosin b4, along with GMT, resulted in further improvements in scar area and cardiac function. These findings demonstrate that cardiac fibroblasts can be reprogrammed into cardiomyocyte-like cells in their native environment for potential regenerative purposes. A recent review list stem cell sources for regenerative treatment of ischemic heart muscle tissue and typical markers of these cells (6). In addition, there is an overview about clinical trials and stem cell types used with these. Most of the human clinical trials performed to date used blood- and bone marrow-derived stem cells that are defined by different characteristics. According to Brenner et al., two encouraging trials utilizing cardiac stem cells from heart biopsies have been launched and are still recruiting; first results are expected soon ((6)). Most of these trials involved, however, only the administration of bone marrow-derived stem cells after myocardial infarction and did not use scaffold materials or specifically designed surfaces for cell differentiation or expansion. As such they followed a different approach compared to the NanoCARD strategy.
The reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) opens new possibility in regenerative medicine. A large pool of fibroblasts exists in the postnatal heart and Ieda et al. reported recently that a combination of three developmental transcription factors (Gata4, Mef2c, and Tbx5) quickly and effectively reprogrammed post-natal cardiac or dermal fibroblasts into differentiated cardiomyocyte-like cells ((7)). These directly induced cardio-myocytes expressed
cardiac-specific markers and showed spontaneous contractions in vitro. Fibroblasts transplanted into mouse hearts one day after transduction of the three factors also differentiated into cardiomyocyte-like cells in vivo.
Chip technologies, Materials, Scaffolds and chip for heart tissue regeneration
Independent of the cell source major challenges of heart muscle repair remain to be the cell survival, cell fate determination and engraftment as well as vascularization after transplantation. Several strategies of tissue-engineering combining scaffolds and different cell types have been developed and were partially adapted for specific application to enhance stem cell function. Different approaches and scaffold techniques for cardiac cell therapy are described by Karam et al. in a recent review ((9)). Several advancements in the field of biomaterials for heart muscle scaffolds are discussed. Examples are given for 2D studies and 3D supports for the cells also
mimicking the structural architecture of the heart. In some studies hydrogels of different types were used to control the biophysical and biochemical microenvironments of transplanted cells. Interestingly, stacks of cells-seeded polymeric sheets, as similarly proposed in NanoCARD, were already suggested in 2002 by Shimizu et al. ((10)).
Several studies of biomaterial scaffolds for heart muscle regeneration or tissue engineering, cell types and stimulation signals are documented in a recent review ((1)).
Several approached have been proposed to use micro- or nanofabrications techniques to modify surfaces of scaffold polymers in order to direct stem or progenitor cells to cardio-myocytes.
For example, polymeric scaffold surfaces have been modified by specific recognition sites for cells using technologies like the Molecular Imprinting ((11)). The authors of this publication claim that this technique is extremely suitable to produce intelligent matrices usable in the field of heart tissue engineering characterized by their particular capacity for molecular recognition of extracellular proteins (collagen, fibronectin, laminin, vitronectin, etc.) that favor cellular adhesion or cell functions. It is proposed that the polymeric matrices will show a capacity for increasing the adhesion and growth characteristics of the cells on the scaffolds thanks to the presence of nanosites that are complementary and selective towards specific peptide sequences presented by ECM proteins and with which specific integrins interact.
Other approaches followed the strategy to incorporate resilience-imparting protein found in all elastic human tissues into hydrogels to form a highly elastic scaffold (12). These hydrogels, with
photocrosslinked methacryl-tropoelastin, facilitated the attachment, adhesion, alignment, function, and intercellular communication of cardiomyocytes by providing an elastic mechanical support mimicking mechanical in vivo properties.
Considering that MI cannot be used directly on biodegradable polymers, which are a fundamental requisite for tissue engineered scaffolds, biostable polymers in the form of nanoparticles will be realized. They will be used in small quantities to modify degradable materials, so that these will retain their ability to serve as temporary scaffolds.
There are several publications reporting on the impact of surface modification of culture systems on stem cell differentiation or expansion. Focus is often on nanotopography of polymer support but also on the functionalization of the surface with peptides. To the later approach there is one publication reporting a similar technique for nanopatterning by loaded block-copolymers as commonly used in the NanoCARD consortium. The group demonstrates, next to variation in adhesion of mesenchymal stem cells (MSC), that MSC osteogenesis is reduced on surfaces with increased lateral RGD spacing while adipogenic differentiation is increased ((13)).
Many more publication report on the behavior of various stem cells controlled or directed by topographic surface features. Zouani et al., for example, demonstrated that the depth (on a nanometric scale) of micro-patterned surface structures allowed increased adhesion of human mesenchymal stem cells (hMSCs) with specific differentiation into osteoblasts in the absence of osteogenic medium (14). There have been also reports on the screening of stem cell responses to surface featured by a chip approach.
Generally, the approach of using chip technologies and high content screenings to explore the complex interaction of stem cells with cues from their environment is well accepted and widely followed(16-18).
Figure 11: Schematic representation of high throughput approaches in stem cell applications. Hydrogel engineering combined with robotics can generate several hundreds to a thousand printed artificial niche candidates in one experiment (from (16)).
Devices for mechanical cell manipulations in regenerative medicine
Mechanical forces are ubiquitous in our body and greatly affect the development and functional homeostasis of tissues. In particular, in vivo mechanical stimuli within muscles and blood vessel walls may play an important role in differentiation of stem cells to various phenotypes. Previous work has shown that mechanical strain has an effect on proliferation and differentiation on many cell types also on MSC, including up-regulation of various SMC contractile markers (8–10), and the cells aligned perpendicularly to the axis of strain (11). Cyclic stretching has been used for improved tissue engineering on heart muscles for more than one decade. Zimmermann at al. reported, for example, first results by using simple mechanical devices (19,20). There are also several reports on stretching devices within the last 3 years (21,22), some of them trying to mimic specific in vivo conditions for tissue engineering (23). Parallel to the development of a stretching
device for live cell microscopy within NanoCARD designs for similar devices have been reported (24-26).
References
1. Ye KY, Black LD. Strategies for Tissue Engineering Cardiac Constructs to Affect Functional Repair Following Myocardial Infarction. J of Cardiovasc Trans Res. 2011 Aug 5;4(5):575–91.
2. Pereira MJN, Carvalho IF, Karp JM, Ferreira LS. Sensing the Cardiac Environment: Exploiting Cues for Regeneration. J of Cardiovasc Trans Res. 2011 Jul 7;4(5):616–30.
3. Godier-Furnémont AFG, Duan Y, Maidhof R, Vunjak-Novakovic G. Regenerating the Heart. Cohen IS, Gaudette GR, editors. Totowa, NJ: Humana Press; 2011. 33 p.
4. Ieda M, Fu J, Delgado-Olguin P, Vedantham V. ScienceDirect.com - Cell – Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by Defined Factors. Cell. 2010.
5. Qian L, Huang Y, Spencer CI, Foley A, Vedantham V, Liu L, et al. In vivo reprogramming of murine cardiac fibroblasts into induced cardiomyocytes. Nature. 2012 Apr 18.
6. Brenner C, Franz W-M. The use of stem cells for the repair of cardiac tissue in ischemic heart disease. Expert Rev Med Devices. 2011 Mar;8(2):209–25.
7. Ieda M, Fu J-D, Delgado-Olguin P, Vedantham V, Hayashi Y, Bruneau BG, et al. Direct Reprogramming of Fibroblasts into Functional Cardiomyocytes by Defined Factors. Cell. 2010 Aug;142(3):375–86.
8. Passier R, Mummery C. Getting to the heart of the matter: direct reprogramming to cardiomyocytes. Cell Stem Cell. 2010 Aug 6;7(2):139–41.
9. Karam J-P, Muscari C, Montero-Menei CN. Combining adult stem cells and polymeric devices for tissue engineering in infarcted myocardium. Biomaterials. 2012 Aug;33(23):5683–95.
10. Shimizu T, Yamato M, Isoi Y, Akutsu T, Setomaru T, Abe K, et al. Fabrication of Pulsatile Cardiac Tissue Grafts Using a Novel 3-Dimensional Cell Sheet Manipulation Technique and Temperature Responsive Cell Culture Surfaces. 2002.
11. Rosellini E, Cristallini C, Barbani N, Giusti P. Studies in Mechanobiology, Tissue Engineering and Biomaterials. Boccaccini AR, Harding SE, editors. Berlin, Heidelberg: Springer Berlin Heidelberg; 2010. 28 p.
12. Annabi N, Tsang K, Mithieux SM, Nikkhah M, Ameri A, Khademhosseini A, et al. Highly Elastic Micropatterned Hydrogel for Engineering Functional Cardiac Tissue. Adv Funct Mater. 2013 Apr 26;23(39):4950–9.
13. Frith JE, Mills RJ, Cooper-White JJ. Lateral spacing of adhesion peptides influences human mesenchymal stem cell behaviour. Journal of Cell Science. 2012 Jan 15;125(Pt 2):317–27.
14. Zouani OF, Chanseau C, Brouillaud B, Bareille R, Deliane F, Foulc MP, et al. Altered nanofeature size dictates stem cell differentiation. Journal of Cell Science. 2012 Feb 2.
15. Unadkat HV, Hulsman M, Cornelissen K, Papenburg BJ, Truckenmüller RK, Post GF, et al. An algorithm-based topographical biomaterials library to instruct cell fate. Proceedings of the National Academy of Sciences. National Acad Sciences; 2011;108(40):16565–70.
16. Marx V. Where stem cells call home. Nat Meth. Nature Publishing Group; 2013;10(2):111–5.
17. Nikkhah M, Edalat F, Manoucheri S, Khademhosseini A. Engineering microscale topographies to control the cell–substrate interface. Biomaterials. 2012 Jul;33(21):5230–46
18. Ranga A, Lutolf MP. High-throughput approaches for the analysis of extrinsic regulators of stem cell fate. Current Opinion in Cell Biology. 2012 Apr;24(2):236–44.
19. Zimmermann WH. Tissue Engineering of a Differentiated Cardiac Muscle Construct. Circulation Research. 2001 Dec 13;90(2):223–30.
20. Eschenhagen T. Engineering Myocardial Tissue. Circulation Research. 2005 Dec 9;97(12):1220–31.
21. Huang Y, Nguyen N-T. A polymeric cell stretching device for real-time imaging with optical microscopy. Biomed Microdevices. 2013 Jul 19;15(6):1043–54.
22. Yung YC, Vandenburgh H, Mooney DJ. Cellular strain assessment tool (CSAT): Precision-controlled cyclic uniaxial tensile loading. Journal of Biomechanics. 2009 Jan;42(2):178–82.
23. Zhou J, Niklason LE. Microfluidic artificial “vessels” for dynamic mechanical stimulation of mesenchymal stem cells. Integr Biol. 2012;4(12):1487.
24. Shao Y, Tan X, Novitski R, Muqaddam M, List P, Williamson L, et al. Uniaxial cell stretching device for live-cell imaging of mechanosensitive cellular functions. Rev Sci Instrum. 2013 Nov;84(11):114304.
25. Tremblay D, Chagnon-Lessard S, Mirzaei M, Pelling AE, Godin M. A microscale anisotropic biaxial cell stretching device for applications in mechanobiology. Biotechnol Lett. 2013 Oct 16.
26. Wang D, Xie Y, Yuan B, Xu J, Gong P, Jiang X. A stretching device for imaging real-time molecular dynamics of live cells adhering to elastic membranes on inverted microscopes during the entire process of the stretch. Integr Biol. Royal Society of Chemistry; 2010;2(5-6):288–93.
I. Dissemination
1. After the periodic project commitment discussions at the consortium meetings to explore commercialization opportunities, the dissemination in high impact scientific journals has been launched.
2. NanoCARD has been producing a lot of high impact scientific publications.
A lot of publications are in preparation
3. Several international conference contributions have been made (see below).
4. Several exhibitions the products have been shown (see below).
The exhibitions were:
1. High content analysis SF, USA
2. Lab automation SF, USA
3. Medica, Germany
4. Israel life imaging forum, Israel
II. Intellectual property rights
Project participants are working closely with suitable consultants to safeguard their intellectual property rights and to ensure the best technology transfer from the project. Therefore the participation of Qiagen and other industrial partners at the meetings have been very helpful, especially to maximalist control over the project outcome. To guarantee the IPR protection the coordinator is discussing with experts before publishing any results the possibility of the protection of the results. After this check of the manuscript by the coordinator whether a protectable result has been achieved or not, the results were published in high impact scientific journals.
The coordinator has been organizing an industrial workshop to achieve the best possible exploitation of results.
III. Knowledge exchange
a. Public website of the project has been established. All informations
related to the scientific goals and achievements of the project have been put into the wiki. http://www.is.mpg.de/NanoCARD
b. An eternal project web page has been created for the exchange of protocols, methods, results and specific organizational questions as well as making the whole details of NanoCARD available for the different participants.
c. Every scientific group has been participating at the NanoCARD meetings. The different groups have been giving talks, so that the very lively knowledge exchange has been guaranteed. Different labs offered training courses to use the different microscopical material and cellular systems.
d. An important instrument for the knowledge exchange has been the PhD- and Postdoc Workshops as well as the principal investigator meetings.
NanoCARD Meetings/PhD Workshops
• Kick off meeting 12.-13. Januar 2010, Stuttgart
• NanoCARD Meeting 06.-08. June 2010, Rehovot
• Brussels Meeting 31.01.2011 with the European Commission
• NanoCARD Meeting 05.-07. April 2011, MPI Stuttgart
• Midterm review in Brussels 30.06.-01.07.2011with the European Commission
• Principal Investigator Meeting 13.09.-14.09.2012 Stuttgart
• Final Meeting 17.10.-18.10.2013 MPI Stuttgart
e. Regular meetings with the industrial partners for early identification of potential protection development opportunities for the European industry have been taken place.
List of Websites:
http://www.mf.mpg.de/NanoCARD/
