Community Research and Development Information Service - CORDIS

FP7

NEUROSCAFFOLDS Report Summary

Project ID: 604263
Funded under: FP7-NMP
Country: Italy

Final Report Summary - NEUROSCAFFOLDS (Rapid prototyping scaffolds for the nervous system)

Executive Summary:
Scaffolds made of different materials have been constructed for the repair of different tissues, such as bones, liver and other organs These scaffolds have been produced with a variety of different techniques including rapid prototyping (RP). However, attempts to construct scaffolds for the repair of the central nervous system have had limited success for the functional recovery of the damaged nervous system, because of the intrinsic poor regenerative properties of this tissue. The driving hypothesis of the NEUROSCAFFOLDS project is that this will be overcome by providing neurons with a scaffold containing nanomodified surfaces.
The main aim of this NEUROSCAFFOLDS project was and still is to build scaffolds for the repair of the nervous system.
The project had two main steps:
Objective 1 - Rapid Prototyping Fabrication of scaffolds for the growth and network formation of
neurons.
Objective 2 - the fabrication and implant of scaffolds for the repair of sciatic nerve and spinal cord
injuries.
To reach the identified objectives the plan consisted in carrying out the following 5 interconnected activities:
- Rapid Prototyping fabrication of 3D scaffolds for the Nervous system
- Fabrication of multichannel conduits
- Decoration of 3D Scaffolds and Multichannel conduits
- In vitro testing
- Implants

Several recent technological developments established the background of NEUROSCAFFOLDS. These developments originate from progress in tissue engineering, rapid prototyping and bionanotechnology.
The overall strategy of the project was based on 3 steps, requiring an accurate timing:
Step 1 Development and fabrication of Scaffolds
Step 2 Experimentation and testing of the formation of 3D neuronal networks
Step 3 Development and testing of scaffolds for implants
The final and most important step is the implantation of the created scaffolds in a neuronal tissue for repair.

In these 3 years, within the European Consortium we have indeed attempted to implant our scaffolds for repairing sciatic nerve injuries, a challenging aim with great clinical potentials. In Europe we have implanted these scaffolds in animal laboratories and our Chinese partners have done the same. Therefore we have made and are still making significant progress towards the effective repair of the nervous system.

The most recent experiments carried out by ULUND show good recordings 48 hours after implantation in awake animals. Hence we can state that we have proof of concept for the new technique developed to characterise neurophysiological activity in peripheral nerves. Given that, this is quite promising and as far as we know not been accomplished previously. We are continuing on this part after the end of the project.

The scientific work was divided into 6 WorkPackages:
WP 1 Fast prototyping of biocompatible 3D scaffolds; with ENS as WP Leader and SINANO as Chinese Activities Representative;
WP 2 3D scaffolds with multichannel conduits by Mask Projection Excimer laser StereoLithography (MPExSL) with IIT as WP leader and SINANO as Chinese Activities Representative;
WP 3 Integration of nanoscale topographic features or carbon nanotubes for enhanced neural growth– with ENS as WP Leader and the Chin. Ac. of Science Bejing as Chinese Activities
Representative;
WP 4 Biological testing: survival and 3D network formation –with SISSA as WP Leader and SINANO as Chinese Activities Representative;
WP 5 Long term evolution and migration –with Univ. of Trieste as WP Leader and SINANO as Chinese Activities Representative;
WP 6 Scaffolds for the repair of the peripheral nerves – with LUND University as WP Leader and Nanjing University as Chinese Activities Representative;
Moreover, this Project had other 3 WPs - i.e.: WP7, WP8 and WP9 - dedicated to the Scientific Coordination of the two Projects: the EC funded project and the China coordinated project, to the Administrative and Financial Management of the Project, to the Dissemination of the results, respectively.

The collaboration between European and Chinese institutions is very smooth and the combination of the scientific European expertise with the vibrant Chinese attitude towards innovation and clinical applications is highly promising and we greatly expect to have the opportunity to continue along these lines and have the possibility, in the near future, to start the repair of lesions in the central nervous system using new scaffolds and a variety of stem cells.

Project Context and Objectives:
The main aim of this NEUROSCAFFOLDS project was and still is to build scaffolds for the repair of the nervous system.
The project had 2 main steps:
Objective 1 - Rapid Prototyping Fabrication of scaffolds for the growth and network formation of neurons.
Objective 2 - the fabrication and implant of scaffolds for the repair of sciatic nerve and spinal cord injuries.

To reach the identified objectives the plan consisted in carrying out the following 5 interconnected activities:
- Rapid Prototyping fabrication of 3D scaffolds for the Nervous system
- Fabrication of multichannel conduits
- Decoration of 3D Scaffolds and Multichannel conduits
- In vitro testing
- Implants

Several recent technological developments established the background of NEUROSCAFFOLDS. These developments originate from progress in tissue engineering, rapid prototyping and bionanotechnology.

The overall strategy of the project was based on 3 steps, requiring an accurate timing:
Step 1 Development and fabrication of Scaffolds
Step 2 Experimentation and testing of the formation of 3D neuronal networks
Step 3 Development and testing of scaffolds for implants
The final and most important step is the implantation of the created scaffolds in a neuronal tissue for repair.
When divided per Work Package, the Objectives were the following:

WP1:
• To establish the massive and reproducible synthesis routes of 3D scaffolds based on rapid prototyping.
• To address the morphology, porosity, composition and microstructure of those resulting scaffolds.
• To develop surface decoration approaches for improving the biocompatibility of these 3D scaffolds.

WP2:
• To produce cylindrical multichannel scaffolds for in vivo experiments on sciatic nerve and spinal cord regeneration.

WP3:
• we will explore different methods to physically or biochemically decorate the prototyped 3D scaffolds and multichannel conduits.
• we will decorate these devices with a variety of molecules with specific biological actions such as controlled release
• we will jointly decide which one of these decorations will be the most promising for the planned implants in the sciatic nerve and spinal cord

WP4:
• To test the biocompatibility of constructed 3D scaffolds and multichannel conduits
• To verify and characterize neuronal migration in these scaffolds
• To measure and test the electrical activity of neurons grown inside our 3D scaffolds

WP5:
• To verify the long term properties of neurons cultivated inside our scaffolds
• To analyse the possible formation of new synapses
• To characterize the ensemble properties of neurons after 1-4 months of culture inside the scaffolds

WP6:
• To find out which of the scaffolds that should be selected for in vivo testing
• To find out which scaffold is the most efficient to support nerve regeneration
• To find out which scaffold produce a normalized activity pattern in dorsal root ganglion cells

WP7:
• co-ordinate the research teams;
• permit formal exchanges of information between the partners
• supervision of the progress on testing implants for the recovery of spinal cord injuries.

WP8:
• to carry out the management and monitoring of the project on a day-to-day basis;
• to ensure that the project works as an integrated whole.

WP9:
• to inform about and disseminate the work in progress as well as final results;
• to share with the general public the knowledge produced within the project.

Project Results:

Work progress and achievements of the NEUROSCAFFOLDS Project divided into Work Packages

WP1 Fast prototyping by SFF fabrication of biocompatible 3D scaffolds

The objective of this work package is threefold: i) to establish the massive and reproducible synthesis routes of 3D scaffolds based on rapid prototyping. ii) to address the morphology, porosity, composition and microstructure of those resulting scaffolds and iii) to develop surface decoration approaches for improving the biocompatibility of these 3D scaffolds. Accordingly, WP1 has been divided into the following tasks:
T1.1 Massive production of 3D graphene foam based on porous Ni template CVD growth
T1.2 Massive production of PEG hydrogel
T1.3 Massive production of electrospinning nanofibers
T1.4 Nanostructured super-hydrophobic scaffolds
T1.5 Massive production of microfabricated and biocompatible open scaffolds
T1.6 Morphology, porosity, composition and microstructure of those resulting scaffolds
T1.7 Final assessment of produced scaffolds (from M33 -36)

During the first period, ENS team successfully fabricated monolayer of gelatin nanofibers by electrospinging and supporting honeycomb microframe by vaccum assisted molding of PEDGA. During the second period, the fabrication method has been extended by fabricating collagen and PLGA frame, allowing manufacturing neuron patches made of biodegradable nature or synthetic polymers (T1.3). In collaboration with SISSA, ENS team has extended this approach produced PCL nanofibers on a thin layer of PDMS with high density pillar arrays, allowing modulating simultaneously the substrate stiffness and surface morphology which may result in a n improved growth of astrocytes and hippocampal neurons. ENS has also worked out a double layer nanofiber structure by electrospinning nanofibers on both sides of a through-hole membrane, showing the possibility of 3D neuronal network formation. In parallel, ENS has tested the feasibility of 3D printing of neuron scaffolds by using inks containing ultrafine particles. After dissolution of the particles, the resulted scaffolds showed imprinted porous structures which could be used for improved neuron growth (T1.2). Finally, ENS team has demonstrated an effective motor neuron differentiation of human induced pluripotent stem cells (hiPSCs) on monolayer nanofibers and plug-and-play extracellular recording of motor neurons with a commercial multi-electrode array (T1.6).

Most significant results:
The culture patch developed by ENS team is based on electrospinning and crosslinking of monolayer gelatin nanofibers on a honeycomb frame of PEGDA, allowing culturing primary neurons nicely and enhanced culture and differentiation of stem cells toward functional neurons and plug-and-play extracellular recording of neuron firing. This method is clearly advantageous over conventional ones in term of reduction of exogenous contact of neurons and increase of the cell exposure area as well as increase of the freedom of network organisation. Moreover, neurons could be obtained on the same patch by differentiating stem cells and characterized by non-invasive measurement with multi-electrode array. Finally, the resulted neuron patch can be used as a new type of scaffolds for both in vitro and in vivo assays, therefore holding high potential for a large variety of applications.

WP2 3D scaffolds with multichannel conduits by Mask Projection Excimer laser
StereoLithography (MPExSL)

In summary, 110 PPF-based conduit scaffolds have been delivered to LUND University for in-vivo experiments on the regeneration of the sciatic nerve of rats and 10 conduit scaffolds have been shipped to the Center for Regenerative Medicine, Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences Beijing for exploratory tests on the regeneration of the spinal cord of larger animals, e.g., dogs.
Following the completion of Task 2.3, a pilot investigation on the fabrication of 3D poly(ethylene glycol) diacrylate (PEGDA) scaffolds by Mask Projection Excimer laser StereoLithography has been performed.

Most significant results:
1) PPF-based conduit scaffolds for in-vivio experiments.
Deliveries were made to LUND University of the conduit scaffolds for in-vivo test on the regeneration of the sciatic nerve of rats. The actual scaffolds were delivered in December 2015 to LUND University

Such activities complete the Task 2.3 (Production of multichannel conduits for tissue regeneration experiments, M12-36), with all conduit scaffolds being shipped according to schedule and following the partner's requests, thereby achieving the objective of WP2 to produce cylindrical multichannel scaffolds for in-vivo experiments on the regeneration of peripheral nervous system via the novel 3D printing technique Mask Projection Excimer laser StereoLithography (MPExSL).

2) PEGDA scaffolds by MPExSL.
In order to assess further the capability of the newly developed 3D printing system, a study was started to investigate the photocuring process of PEGDA by Excimer laser radiation towards the fabrication of 3D scaffolds by MPExSL. Specifically, the curing depth of PEGDA with and without photoinitiator (Irgacure 2959) was systematically investigated using both 248 nm laser light (KrF excimer laser) and 308 nm laser light (XeCl Excimer laser). Significantly, it was found that curing of pure PEGDA can be achieved even without the use of any phototinitiator molecules by using Excimer laser light. This is possible through the absorption of the energetic photons tha fall within the UV absorption band of the acrylic group, thereby activating the carbon double bond possibly through internal conversion and inter-system crossing. It is noted that no other stereolithography method (based either on single photon absorption or on the more sophisticated two-photon absorption version) has yet demonstrated the possibility to produce photoinitiator-free 3D scaffolds. Further biological, mechanical and FT-IR studies are necessary to fully characterize the photoinitiator-free scaffolds, optimize the fabrication process and proceed towards TE applications of these novel scaffolds.

Additional details in D2.4:
During this second period, towards the accomplishment of D2.3 3D scaffolds for in vivo tissue regeneration, UniTS team contributed to this deliverable by developing two novel scaffolds for use in the central nervous system (CNS): multiwall carbon nanotubes (CNT) were used for the creation of 3D frames of neural-compatible composite scaffolds (A) and PDMS scaffolds interlaid with a CNT meshwork (B). These findings allow us to further develop and exploit the outstanding properties of carbon-based nanomaterials in novel devices for neural tissue engineering purposes.

WP3 Decoration of 3D scaffolds and multichannel conduits

The objective of work package is threefold: i) to explore different methods physically or biochemically decorate the prototyped 3D scaffolds and multichannel conduits, ii) to decorate these devices with a variety of molecules with specific biological actions such as controlled release and iii) to jointly decide which one of these decorations will be the most promising for the planned implants in the sciatic nerve and spinal cord (Milestone 2). Accordingly, WP3 has been divided into the following five tasks:
Task 3.1: Decoration plasma etching, incubation or calcinations
Task 3.2: Decoration with vesicles with a controlled release
Task 3.3: Decoration with new nano-composites
Task 3.4: Decoration with nanostructures
Task 3.5: Surface decoration approaches

ENS team has explored several scaffold decoration techniques which improved the performance of the scaffolds. Firstly, high resolution surface patterning of a thin layer of PCL could be done by hot embossing, which chould used as biodegrable neuron scaffolds (T3.4). Secondly, PCL nanofibers could be deposited on PDMA pillars to spontaneously improve the substrate stiffness and surface morphology for neuron culture (T3.4). Thirdly, gelatin nanofibers could be surface functionalized by VEGF growth factor via an intermediate surface coating of chitosan and heparin, followed by immunological coupling of the factor and heparin. Long-team release of the growth factors from the fibres could be achieved over a couple of weeks (T3.2). Finally, proteins could be integrated into gelatin nanofibers by electrospinning with a solution of nanogel and gelatin (T3.5).

The UniTS group has been working towards D.3.1 Methodologies for decoration with selected roughness: Report on methodologies for decoration with selected roughness, was accomplished at month 24. They implemented PDMS-based scaffolds with conductivity and roughness at the nanoscale by decorating the scaffold with pure MWCNT or graphene flakes and D3.2 Decoration of vesicles with controlled release: Report on decoration of vesicles with controlled release accomplished at month 24, by developing suspended graphene oxide flaxes of controlled lateral size able to regulate exocytosis of vesicles in neurons and glial cells. UniTS has further nanostructured the polymer based-scaffold by means of multi walled carbon nanotubes (MWCNTs), implementing the polymer with novel nano-topographies and controlling the dimension of the 3D skeleton supporting tissue reconstruction.

UniTS team deviated from the planned decoration of 3D scaffolds with lipid vesicles, containing selected molecules able to drive and control neuronal presynaptic terminal growth and function, that is able to control the release of neurotransmitters (D3.2). While investigating the potential use of graphene flakes in decorating 3D PDMS scaffolds with nanomaterials, we observed the ability of graphene oxide flakes with small lateral dimension (s-GO; 200 nm) to alter excitatory synapse formation and/or function without affecting cell survival or the global network size. Thus we measured the kinetics of synaptic vesicle release by real-time imaging of vesicles labelled with FM dye to monitor the rate of presynaptic vesicle recycling from hippocampal neurons treated or untreated with s-GO. After staining with the lipophilic dye FM1-43 clusters of presynaptic terminals were visible as bright fluorescence spots. In s-GO treated cells, upon high-K+-loading protocol, we detected a significant reduction in the raw fluorescence intensity of FM1-43-positive hippocampal terminals, suggesting that chronic incubation with s-GO decreased the recycling vesicle pool. When analysing the decay time constant (τ) of the FM1-43 fluorescence de-staining profiles during vesicle exocytosis, we observed a significant difference in the kinetic displayed by control and s-GO. In a parallel approach, when testing the occurrence of evoked inhibitory PSCs by pair recordings of mono-synaptically coupled neurons we observed that s-GO apparently did not interfere with the inhibitory mediated connections. Taken together, these results support the specific ability of s-GO flakes to reduce the amount of excitatory synaptic contacts and to interfere with presynaptic vesicle recycling, thus providing an additional tool to control synaptic release in synthetic scaffolds.

Most significant results:

ENS: In addition to the biochemical properties, the stiffness and the surface morphology of the substrate plays a very important rule in neuron growth and neuron network formation. To be capable to modulate both stiffness and the surface morphology of the substrate, high density and high aspect ratio PDMS pillars were obtained, which provided a low effective Young’s modulus of the substrate (down to 1 kPa) whereas PLGA nanofibres electrospun on the PDMS pillars provided ECM-like surface morphology. Such a material and structure combination should meet the requirement of the stiffness and ECM surface morphology of neurons and in particular astrocytes. Our primary results showed clearly an improvement of primary astrocyte culture. After optimizing the medium, the culture performance of neurons and neuron networks should also be improved.

T3.5: Surface decoration approaches (SISSA)
We tested different coating protocols in order to decorate the 3D graphene scaffolds. Graphene foams were cut into small pieces (5x5mm squares) and attached with PDMS to round glass coverslips of 12 or 15 mm diameter. The samples were then sterilized with steps of 100%, 50% ethanol and then MilliQ H2O followed by 20min UV exposure. The coating was done with polyornithine overnight. The next day polyornithine was removed and neuron medium was added overnight. Before plating the cells, Matrigel was added and incubated for 30 min. This coating protocol gave the best results. We tested different procedures such as treatment with Oxygen plasma to hydrophilize the scaffold, but without significant improvement. We also tested the condition in which coating with polyornithine and Matrigel was omitted; however, cell survival was dramatically reduced.
The cultures were maintained for several weeks in culture and the neuronal activity was probed with Calcium Imaging using Fluo-4-AM calcium indicator. For control experiments, neurons were plated on glass coverslips.

We also performed immunocytochemistry in which we stained neurons for microtubule-associated protein 2 (MAP2) that localize in cell bodies and dendrites and a presynaptic marker synaptophysin. Nuclei were marked with Hoechst 33342. Neurons adhered to the scaffold and also crossed the pores connecting to each other.

WP4 Biological testing: survival and 3D network formation

The objective of work package is threefold: i) to test the biocompatibility of constructed 3D scaffolds and multichannel conduits, ii) to verify and characterize neuronal migration in these scaffolds and iii) to measure and test the electrical activity of neurons grown inside our 3D scaffolds to find out which of the scaffolds is most promising for in vivo implants.

Accordingly, for the second period’s activity, IIT, SISSA and UNITS have been concentrated on the following tasks:

T4.3 Measurement and characterization of electrical properties of neurons grown inside 3D scaffolds (M12-24)
T4.4 Testing the response to guidance molecules and growth factors (M18-30)
T4.5 Symposium on biological testing DEVIATION: the FINAL MEETING was held in China and there was also a symposium organized as Final Joint Event: the 2016 Suzhou Symposium on Material-Cell Interfaces

The optimal experimental conditions for favouring neuronal migration inside 3D scaffolds (T4.1) and morphological characterization (T4.2) were done and described in D4.1 and D4.2 respectively. Next, we investigated electrical properties of neuronal networks grown inside 3D scaffolds with calcium imaging (T4.3) using Fluo-4-AM calcium indicator on cultures after 8-9 days in vitro (DIV), 15-16 DIV and some experiments were performed on long-term cultures (more than 21 DIV, see also WP5).

Most significant results:
We used dissociated rat postnatal hippocampal networks grown on 3D graphene foams (3D-GFs). Glass coverslips and 2D graphene films (2D G) were used as controls. After 1 week of culture, on 3D scaffolds the cells formed a network that covered entirely the scaffold backbone. In particular, almost 80% of glial cells extended processes while on the 2D cultures the majority (>60%) of the glial cells were lacking processes. Neurons projected their neurites in all three dimensions increasing the network connectivity. These observations suggest that 3D scaffolds promote a more differentiated and in vivo-like morphology.

After one week of culture, we analyzed the electrical properties of neurons grown inside 3D scaffolds. Cells were loaded with Fluo-4-AM calcium indicator and spontaneous calcium transients from both 2D and 3D networks were recorded. Neuronal activity is significantly different for 3D cultures: firstly, it has a higher frequency, as shown in where the cumulative count of the Inter Events Interval (IEI) shows a significant difference (p<0,01) in the frequency among the two conditions.
Secondly, 2D and 3D cultures had a different degree of synchrony, with 3D cultures appearing consistently more synchronous. We analyzed the degree of synchrony of 2D and 3D neuronal networks by computing the mean correlation coefficient (cc). The value of cc for calcium transients in 2D Glass and 2D-G cultures was significantly lower (0,53 ± 0,006 and 0,61 ± 0,007 respectively) than that of 3D-GF (0,82 ± 0,005) (Ulloa et al, 2016).

3D cultures because of their dimension have many connections among distant neurons leading to small-world networks and their characteristic dynamics. Indeed, the higher connectivity of 3D networks leads to highly synchronized (HS) regime of activity, that was never observed in 2D networks. Moreover, we observed neurites able to cross the pores and bridge distances of 100–200 μm in the scaffold. Clear calcium transients that originated from crossing neurites were recorded, and these transients were correlated with those that originated from neighbouring neurons. In some preparations, holes were not only filled with crossing neurites but also with the soma of neurons and glial cells, which appeared to be hanging in the pore. 3D-GFs with a high degree of connectivity, as indicated by the presence of the soma of neurons inside the holes and many crossing neurites, had calcium transients almost completely synchronous, defining this state as highly synchronous (HS). This higher synchrony (cc = 0.93 ± 0.004; n = 483 couples of neurons) is attributed to a more extensive connectivity associated to the presence of crossings (or shortcuts) across the holes and the long neurites extending in 3D along the scaffold backbone. Neuronal cultures grown on 3D-GFs often exhibited clear transitions between the MS and HS regimes .

Taken together, these results show that the dynamics of 3D neuronal networks differ from those of 2D neuronal networks and better recapitulate what is observed in vivo.
These results were published in Scientific Reports:

Francesco Paolo Ulloa Severino, Jelena Ban, Qin Song, Mingliang Tang, Ginestra Bianconi, Guosheng Cheng and Vincent Torre. “The role of dimensionality in neuronal network dynamics“, Scientific Reports. 2016 6, 29640; DOI: 10.1038/srep29640

Towards D4.3 Electrical properties of neurons grown inside 3D scaffolds, UniTS engineered PDMS supports to permit, as shown by our previous report, a genuine 3D growth of percolating neurons and neuroglia, which allows linking the emerging activity of living networks to the mere 2D or 3D interactions among their constitutive elements. The 2D and 3D networks display basically an equal size and are similarly integrated to glial GFAP-positive cells, but differ for their being projected in 2D or in 3D. We observed differences in activity patterns when recorded by calcium imaging in the two culturing conditions. Certainly, in both networks, episodes of calcium activity, whose neuronal and synaptic nature was supported by TTX experiments and fluorescence microscopy inspection of the recorded fields, were reflecting spontaneous synaptic activity, due to synchronous firing, typical of these preparations, without a resolution at the single action potential or individual post synaptic current levels. However, such bursts are an alternative and recognised measure of network state and dynamics. We speculate that 3D networks, besides displaying a similar degree of active cells when compared to 2D ones, display a higher rate of bursting, under both spontaneous and disinhibited conditions, due to an improved efficiency of neurons and neuronal connections in synchronizing their activity. We favour the hypothesis that the ability to distribute connections in 3D is the main responsible for such behaviours, as outlined by our mathematical modelling. When interfaced neurons to MWCNTs in 3D constructs we replicated the ability of carbon nanotubes in improving robustness of signal transfer and synchronization in cortical cultured circuits. This novel finding sustains the exploitation of scaffolds, engineered at the nanoscale by carbon nanotubes that, by virtue of the acquired physical features may guide different biological responses. Overall, our approach could provide a versatile platform with the potential to contribute with exciting new possibilities both in in vitro and in vivo applications. For example, our 3D system can be exploited in analysing functional interactions among region-specific brain cells, or can represent a biocompatible cellular scaffold to be engineered for tissue formation, setting the stage for the development of novel hybrid materials.
These results have been published in Bosi et al Scientific Reports 5, Article number: 9562 (2015) doi:10.1038/srep09562

Response to guidance molecules and growth factors of neurons grown inside 3D scaffolds
IIT tested the ability of SH-PCL supports to provide a prolonged and consistent release of bioactive BDNF in culture medium. Enzyme-linked immunosorbent assay results indicate that BDNF-coated SH-PCL can deliver BDNF over a 3 week period.
Binding of BDNF to TrkB and p75 receptors elicits various intracellular signaling pathways, including mitogenactivated protein kinase/extracellular signal-regulatedprotein kinase (MAPK/ERK) and transcriptional regulation of several genes, including Synaptotagmin 1 (Syt1), Synapsin 1 (Syn 1), glutamate receptor, ionotropic, AMPA 2 (GluA2) , glutamate receptor, ionotropic, AMPA 1 (GluA1), glutamate receptor, ionotropic, NMDA2A (NR2A) and glutamate receptor, ionotropic, NMDA2B (NR2B). Therefore, we have analyzed the expression of markers known to be regulated by BDNF activation in neurons , grown on BDNF coated SH-PCL scaffolds, confirming the activation of BDFN signaling pathway.

Response to guidance molecules and growth factors of neurons grown inside 3D scaffolds
The viability of neural networks grown on flat substrates was comparable to what observed under standard culture conditions. On the contrary, increased cell viability was apparent in cultures grown on pillared SH-PCL devices. Experimental data demonstrate that pillared SH-PCL scaffolds are biocompatibile and promote neuronal adhesion and viability. We have also tested the ability of SH-PCL scaffolds to work as drug carrier. Flat and pillared SH-PCL scaffolds have been incubated in medium containg BDNF ( 300 ng/ ml) for 14 days. Media were collected and replaced at specific points. BDNF concentrations were measured by ELISA assay.
Results indicate that pillared SH-PCL substrates can deliver BDNF over a 3 week period and at higher level compare to flat SH-PCL supports.
The activity of SH-PCL -released BDNF was investigated by analyzing the expression of mRNAs known to be regulated by BDNF and the phosphorilation level of ERK1/2 in neuronal cultures.
We plated cortical neurons on poly-D-lysine-coated flat and nanopatterned supports, preincubated with BDNF, let the cultures grow and processed them for real time PCR and western blotting analysis.
Real time analysis indicates that BDNF regulated mRNAs are significantly upregulated in neurons grown on BDNF –coated pillared SH-PCL scaffolds compare to flat SH-PCL supports after 7 and 21days in cultures. Similarly, Erk1/2 phosphorilationa level is increased in BDNF –coated pillared SH-PCL scaffolds compare to flat SH-PCL surface.
In addition we analysed the expression vGLUT1 and vGAT mRNAs. Indeed, neurons grown on BDNF-SH – PCL exhibited an significant increase of density of inhibitory GABAergic and excitatory glutamatergic synapses.

WP5 Long term evolution and migration

The objectives of the work package are: i) to verify the long-term properties of neurons cultivated inside the scaffolds, ii) to analyse the possible formation of new synapses and iii) to characterize the ensemble properties of neurons after 1-4 months of culture inside the scaffolds.

Accordingly, the activity has been divided into the following tasks:

T5.1 Histochemical characterization of long term survival and properties of neurons grown inside 3D scaffolds.
T5.2 Long term development of electrical properties of neurons grown inside 3D scaffolds.
T5.3 Testing the response to signaling molecules.
T5.4 Long term changes of mechanical properties of neurons grown inside 3D scaffolds.
T5.5 Symposium on long term survival DEVIATION: the FINAL MEETING was held in China and there was also a symposium organized as Final Joint Event: the 2016 Suzhou Symposium on Material-Cell Interfaces

In relation to
T5.1 Histochemical characterization of long term survival and properties of neurons grown inside 3D scaffolds.
T5.2 Long term development of electrical properties of neurons grown inside 3D scaffolds.

SISSA worked on the following:

In order to obtain long term neuronal cultures, we tested different culture protocols and compared morphology and function of hippocampal cultures grown in the presence of three different media: i- neuronal medium supplemented with FBS (currently used in SISSA for this project), ii- Neurobasal® medium supplemented with B27® and iii- astrocytes conditioned medium (ACM). We have selected the latter since there is an increasing body of evidence that the role of astrocytes is fundamental in maintaining the homeostasis of neuronal cells, both in vivo and in vitro. The culturing protocol should therefore provide optimal conditions for growth, differentiation and maturation of both neurons and glial cells.
At 1 day in vitro (DIV), neurons cultured with ACM had larger growth cones, higher degree of branching and survived better, compared to both B27- and FBS-containing cultures. Calcium imaging experiments performed after 1 week showed that ACM cultures had higher activity and synchrony resulting in overall more mature morphology of both neurons and glia.

The functional and morphological improvement of ACM was confirmed also after 2 weeks of cultures. Moreover, ACM cultures were active also after almost 6 weeks, while the other two methods showed progressively lower (FBS) or no activity (B27).

These results will be implemented in the generation of long-term 3D neuronal network on scaffolds that will be used for in vivo transplantation testing their biocompatibility, integration in the existing brain networks and hopefully for the improved function in case of neurodegenerative disease animal models.

Another paper in preaparation is the following:
“Morphometric and functional characterization of neuronal cell cultures grown in astrocyte – conditioned medium", Pozzi D., Ban J. (coauthors), Iseppon F., Torre V.

Most significant results:
Towards D5.1 Histochemical characterization
UniTS used immunofluorescence techniques and confocal microscopy to compare neurons grown on 2D control substrates (named 2D-PDMS) with those grown on 3D scaffolds (named 3D-PDMS). To prove the formation in vitro of 3D cultures, we image by immunofluorescence the specific cytoskeletal components β-tubulin III, to visualize neurons, and glial fibrillary acidic protein (GFAP) to visualize astrocytes. These 3D reconstructions reveal that neurons and neuronal processes are not constrained by the pre-build scaffold infrastructure and processes are also directly bridging one pore to the other. It is also possible to visualise β-tubulin III positive cell bodies suspended on glial cells with only their neuronal processes providing the necessary anchorage to the scaffold.

These results have been published in Bosi et al Scientific Reports 5, Article number: 9562 (2015) doi:10.1038/srep09562

Towards D5.2 Long term development of electrical properties of neurons
UniTS: Hippocampal neurons were developed for 1 months embedded into PDMS 3D scaffolds manufactured as described in Bosi et al (Scientific Reports 5, 9562, 2015). SEM images and confocal microscopy (below some samples) demonstrate that neurons and glial cells invaded the 3D sponge generating a 400 μm thick artificial tissue construct. SEM images on long term cultures were obtained by IIT collaborators, note the presence of neurons (framed area magnified in bottom micrographs) deep in the structure after long term growth (3 weeks in vitro).

We detected neuronal activity that after 3-4 weeks in vitro was organized in patterns different from those detected in sister cultures in 2D. The histograms summarize recordings performed after long-term growth of neurons in 3D scaffolds of PDMS, compared to 2D standard control at matched ages in vitro. Note the similar % of active cells in each field on the contrary the occurrence of spontaneous calcium events is significantly higher in 3D, either in standard Krebs and in the presence of bicuculline to pharmacologically remove inhibitory synaptic activity.
Notably, induction of synaptic plasticity by glutamate exposure highlighted different plasticity induction profile and time- course in 3D in respect to 2D. In addition, also the contribution of NMDA glutamate receptors subtype to the network activity and to the circuit response to glutamate application differed when comparing 2D and PDMS 3D. These results provide the first evidence of long term growth of primary neurons in 3D scaffolds. Neuronal circuits once grown for 3-4 weeks display the ability to perform glutamate-dependent synaptic plasticity such as long term potentiation, also in 3D construct. However, functional networks reconstructed in 3D display different properties in terms of synchronization, event occurrence and induction of plasticity.

In the progress of the project UniTS, after month 24 UniTS tested the scaffolds developed in D2.3 and D3.1 in the following experimental settings: i. in vitro long term settings by interfacing these structures to long-term co-cultured pairs of mouse organotypic spinal cord explants that allow developing motor networks for extended periods. Co-cultured transversal slices will be separated by a distance known to impair their functional reconnection in basal conditions (>300 μm). This experimental paradigm allows testing the regeneration of intrinsic interneuron projections crucial in recovering coherent motor outputs. Control slices will be grown embedded in a gelified protein-rich plasma clot (i.e. fibrin glue) while those supported by the carbon nanotubes three-dimensional frame will be interfaced to the 3D structures and included in the same plasma gel. Functional and histological analysis will measure the formation of biohybrids; ii. Proper placement and understanding of environmental interaction is necessary to the efficient and effective study of the functionality of a nanomaterial and the tissue response to the implant in vivo. Ongoing studies are designed to test the immunoreaction of cortical tissue surrounding implanted 3D scaffolds in adult male rats visual cortex and sacrificed up to 8 weeks post-implantation.
The production of novel scaffolds for use in the central nervous system benefits from the integration of advancements in material sciences and fabrication and a thorough understanding of the biological milieu of the nervous system and its cells. We are exploring carbon-based nanomaterials such as graphene and multiwall carbon nanotubes (CNT) for the creation of 3D frames of neural-compatible composite scaffolds. CNT, in particular, provide an ideal material basis for engineering in the nervous system because of their unique physical and electrical properties. CNT meshworks contain the structure and dimensions that mimic the extracellular matrix environment. In addition, we have repeatedly demonstrated that CNT improve neuronal activity and signalling in a variety of experimental settings, including three-dimensional scaffolds. We have also shown that neural cells and processes infiltrate and create complex morphological networks within these materials. However, we must also consider the in vivo biocompatibility of such material scaffolds. Therefore, in the progress of the proposal, following extensive in vitro experimentation and characterization, we will implant CNT scaffolds into adult rat visual cortex and analyse the surrounding tissue for incorporation into the brain and any signs of adverse immunoreactivity.

From month 24 to 36 UniTS accomplished the planned Long Term evolution and migration within WP5 by the use of long term tissue explant cultures (spinal organotypic slices) and in vivo assessment. We focused on the generation of in vitro hybrids growing functional neuronal fibers within carbon nanotube based 3D scaffolds for weeks, and we tested tissue integration to the same structures implanted in vivo in the cortex for up to 8 weeks. Results regarding these experiemnts are detailed within the following Deliverables D5.3 (Response to Signalling Molecules) and D5.4 (Long Term Changes of Mechanical Properties of Neurons) and have been published in Science Advances 15 Jul 2016: Vol. 2, no. 7, e1600087 DOI: 10.1126/sciadv.1600087

WP6 Scaffolds for the repair of the peripheral nerves

the activities for WP6 were related to the following Tasks:
Task 6.2 In vivo testing of regeneration through the scaffolds. (M12-36)
Task 6.3 Functional recovery (M18-36)

T6.4 Workshop on the repair of the nervous system (M33 -36). DEVIATION: the FINAL MEETING was held in China and there was also a symposium organized as Final Joint Event: the 2016 Suzhou Symposium on Material-Cell Interfaces

This part of the project aimed at identifying the best performing scaffold for regeneration of the sciatic nerve in a standard rat model. Furthermore, in the next period of time the regeneration will be studied with novel implantable electrodes. Three differently designed fumarate based scaffolds, manufactured using a rapid prototyping method at IIT in Genua were tested. In addition, we developed a gelatin scaffold in Lund with a similar hole dimension as one of the fumarate scaffolds. Both materials are biodegradable and hence possible candidates for future clinical applications.

Most significant results:
The in vitro test was performed using organ cultured dorsal root ganglia, cultured on or in close proximity of the different scaffolds. The axonal regeneration was found to be generally hampered by the fumarate based scaffolds, while the other material showed regeneration comparable to controls with no scaffold material. Nevertheless, the positive indication of fumarate based scaffolds in an early in vivo pilot study qualified those for further in vivo testing.
The in vivo testing was performed in the standard sciatic nerve rat model, using 23 SD female rats. In short, the sciatic nerve was cut and the scaffold of interest was placed to bridge the injury. After three weeks the animals where sacrificed, the nerve and scaffold dissected before the regeneration was analyzed.

Both Schwann cells and axons migrated/regenerated successfully into the different scaffolds, which is a crucial finding for further work within the project. This result confirmed the pilot study previously reported in Milestone 1 and most importantly, we saw no general toxic signs caused by the scaffolds. However, a high frequency of autopsy occurred in some groups.

The initial gelatin prototype scaffolds needs to be improved and more evaluated, but the total absence of autopsy within this group indicates that the material as such is a good candidate for further experiments. In conclusion the single conduit fumarate scaffold exhibited the best regeneration but on the other hand increased the risk for autopsy. A further study with 400 µm channels (to rule out inadvertent contamination of the scaffolds during manufacturing) and the gelatin-based scaffolds is ongoing.

Electrophysiological work
During the second year we did extensive work to enable recordings of nerve activity during regeneration from dorsal root ganglia in vivo with the aim to be able to monitor ongoing activity during regeneration. This is not a trivial task, since these cells are encapsulated in a thick fibrous sheath and the movements between vertebrae during daily life are substantial. During the first 6 months of this part of the project, the experiments have been focused on developing a useful implantation technique that enable long term recordings from single dorsal root ganglion cells. This includes techniques to secure the implanted electrodes in a nearby vertebra (L4) and techniques allowing the implanted electrodes to be reversibly connected to an external computer. We have also begun the process of modifying our patented superflexible neural interface to be implanted in the ganglion.
From month 24 to 36 ULUND has accomplished the planned evaluation of differently shaped fumarate based scaffolds for nerve regeneration in vivo within WP6 but has focused on developing new techniques, including adaptation of previously developed ultraflexible neural interfaces and introducing new surgical procedures, to allow functional evaluation of nerve regeneration. In specific, we have focused on overcoming the considerable challenge involved in recording neuronal signals from dorsal root ganglion cells in awake freely moving animals, which is key to allow an assessment of the precision with which regenerating fibers find their previous targets after nerve lesions. Results regarding these experiments are detailed within Deliverable D6.3 "Biological assessments of rapid prototype scaffolds in the peripheral nervous system".

The most recent experiments show good recordings 48 hours after implantation in awake animals. Hence we can state that we have proof of concept for the new technique developed to characterise neurophysiological activity in peripheral nerves.

Given that, this is quite promising and as far as we know not been accomplished previously. We will continue this part after the project has ended.

Potential Impact:
As already mentioned the collaboration between European and Chinese institutions is very promising and the combination of the scientific European expertise with the vibrant Chinese attitude towards innovation and clinical applications is highly promising and we greatly expect to have the opportunity to continue along these lines and have the possibility, in the near future, to start the repair of lesions in the central nervous system using new scaffolds and a variety of stem cells.
In relation to the consortium, the exchanges that have taken place in the three years of the project have now led to a plan for the creation of a joint research center in Suzhou which could be initially SISSA-Suzhou (Sinano) but that in the future could quickly expand to something bigger, including the other partners and become a link between EU and China.

Possible joint initiatives in Suzhou
1 – Industrial activities
1.1 Soya Based for Wound Skin Repair
Brighton Wound Care (BWC) is a “spin-out” from the University of Brighton. Its founder, Prof Matteo Santin is the leader of the Brighton Cluster for Regenerative Medicine and has 25 years-experience in the field of biomaterials for medical devices and tissue engineering. BWC offers a range of proprietary, novel, low-cost wound dressing products for the regeneration of skin following its damage by either disease or trauma. The products’ characteristics respond to the clinical demands for highly performing and cost-effective biodegradable materials.
1.2 Nanofabrication of cantilever tips for AFM
In collaboration with the German high-tech company JPK we could design, fabricate and test novel cantilever tips for AFM imaging and experimentation. These tips could be commercialized in China by a Chinese Company already active in Suzhou and JPK would offer them to their customers worldwide. These cantilever tips could be fabricated at the SINANO facility in Suzhou.
1.3 Fabrication of tapered optical fibers for biomedical applications
In collaboration with the German high-tech company Rapp-OptoElectronic we could design and fabricate novel optical fibers with reproducible properties and covering a wide range of biomedical applications. These optical fibers could be commercialized in China by a Chinese Company which has already shown interest in this endeavor and outside of China by Rapp-OptoElectronic. These optical fibers could be fabricated at the SINANO facility in Suzhou.

2 – Scientific activities with short term industrial applications
Scientific collaborations between SISSA and the SIP companies
• Suzhou InnoMed Medical Device Co., Ltd.
• Suzhou Natong BioMed Co., Ltd.
• China Heart Biomedical, Inc.
• Suzhou Albert Biomedical Engineering Co. Ltd.
• Suzhou Soundtech oceanic instrument Ltd.
For the design and optimization of mechanical components based on the solution of aerodynamic and hydrodynamic problems, based on the expertise of the MathLab in SISSA.

3 – Scientific activities with medium term industrial applications
3.1 Scientific activities on Regenerative Medicine for the repair of the nervous system in collaboration with Guosheng Cheng of SINANO, SISSA and CNR. These activities will involve stem cell technology, nanofabrication, functional testing and possible clinical applications.
3.2 Scientific activity for the development of nanotools dedicated to in-vitro early diagnostic. These tools, targeting both intra and extracellular fluid, will be designed at SISSA/SINANO and their prototyping and characterization will be realized at SINANO. A combination of multiphysics computational systems, nanofabrication, advanced spectroscopy and cell technology will be required.

In relation to the main dissemination results, we can say that the participation in scientific events in which our progress has been promoted are many.

We think it is more significant to indicate the events organized by the Consortium Partners on the meetings occasions:

The Symposium on "Tissue Engineering for Nervous System Repair and Regeneration" was organized in June 2014 in Genova, Italy.
The symposium on “Integrated Cell-Material Sciences” was organized in Paris in October 2015.
A final symposium was organized in May 2016, this time in Suzhou, and the title was “2016 Suzhou Symposium on Material-Cell Interfaces”.

List of Websites:
www.neuroscaffolds.eu

The website has a private and a public section and it is regularly updated with news and information related to the project.

A second longer video has been produced in 2016 and published on the Neuroscaffolds website.

Related information

Contact

Gabriele Rizzetto, (Secretary General)
Tel.: +39 0403787201
Fax: +39 040 3787249
E-mail
Record Number: 191874 / Last updated on: 2016-11-15