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Tissue engineering of the right heart outflow tract by a biofunctionalized bioresorbable polymeric valved tube

Final Report Summary - TEH-TUBE (Tissue engineering of the right heart outflow tract by a biofunctionalized bioresorbable polymeric valved tube)

Executive Summary:
Approximately 42% of infants’ mortality in the world is related to congenital heart defects (prevalence: 8-12/1000 births). Over 1/3 require the reconstruction of the right ventricular outflow tract (RVOT) by surgical procedures which currently use inert materials without any growth potential. Consequently, multiple reoperations are often required, with their attendant high risk of mortality and morbidity. The TEH-TUBE project was designed to address these limitations by creating an innovative completely bioabsorbable polymeric valved tube device either seeded with autologous adipose tissue derived stem cells (ADSC) or functionalized by a peptidic sequence or a hydrogel triggering homing of the host cells onto the scaffold to make it a living self-populated structure. The main objectives of the project were to:
o Compare different polymers processed by electrospinning (ES) to generate a competent valved tube.
o Identify the optimal ES parameters and the optimal design of the valved scaffold to meet the mechanical specifications.
o Compare, in the selected polymer, ADSC seeding and peptide/hydrogel-based biofunctionalization using in vitro mechanical and biological tests as well as in vivo animal experiments (primarily rats).
o Validate the ultimate combination (polymer + biofunctionalization) in a clinically relevant large animal model (in this case, the growing lamb to specifically assess the regenerative and growth potential of the composite construct).

The expected final result was to develop an innovative biomaterial for the treatment of congenital heart abnormalities in children and young adults. By creating a material whose growth would keep pace with that of the patient, this product, geared to become an Advanced Therapy Medicinal Product (ATMP), should decrease the risk of re-operative surgeries, improve the quality of life and ultimately have a positive impact on healthcare costs.

The contact PI, coordinator and scientific representative of the project's coordinator and organisation was initially Dr David Kalfa (as he was still working in Europe). Dr Kalfa applied and obtained this FP7 funding as the Principal Investigator. When Dr Kalfa accepted a position of Professor at Columbia University, he had to pass on the administrative coordinating role to (AP-HP), for administrative and regulatory resasons. Dr Kalfa continued serving as the scientific co-ordinator of the THE-TUBE project, now working at the University of Columbia, USA. Working in close collaboration with the partners and the project co- ordinating partner (AP-HP), the project was exceptionally successful in achieving all of the project objectives in whole or part. The key results of the project can be stated as:

• A highly innovative biocompatible and bioresorbable polymer was produced and two patents filed with respect to the polymer.
• Design, development and optimisation of electrospinning techniques to produce an implantable bioresorbable polymeric valved tube. A patent has been filed with respect to part of the design aspects and fabrication process.
• Evaluation of the biological specifications of the innovative polymer, biofunctionalisation and sterilisation processes required for clinical application.
• In vivo evaluation in rodents confirming the biocompatibility of the polymer
• Commencement of in vivo assessment of the Teh-Tube product in growing lambs to confirm clinical utility. Initial results are very promising but due to project end date the work could not be completed to provide definitive results at this time.
• Completion of the in vivo validation of the Teh-Tube product will be completed by the consortium after the end of the project.
• Based on the results of the project future financing may be secured from USA-based investors or possibly European funds.

Although the project has achieved the development of a highly innovative technology, due to some delays at the end of the project it was only possible to initiate the final in vivo assessment. However, significant interest has been raised with respect to this technology at dissemination events. Exploitation opportunities are being considered through a range of options including USA and European funding and investment initiatives.

The potential clinical impact of the The-Tube project remains highly significant and will the successful conclusion of the current studies regulatory dossiers will be developed. These will then be used to seek approval for a first in man trial of the technology either in the USA or Europe.

Project Context and Objectives:
In the future, successful treatment of congenital cardiac abnormalities is expected to rely on ATMPs and medical devices. Around 36,000 children are born each year with a CHD; these are the most frequent congenital abnormalities (8-12/1000 births). Existing treatment involves reconstruction of the RVOT, using materials that do not grow as the patient develops. This means that patients treated for a CHD in must undergo repeat surgery (1-4 revision surgeries in their lifetime) with a high risk of mortality and morbidity.

Our aim was to combine and develop existing methodologies to produce a technology that will allow treatment of CHDs with a biomaterial that can reconstruct the heart with a single surgery without the need for re-intervention as the patient grows. This will address the currently unmet clinical need and poor clinical outcome in this patient population.

The TEH-TUBE project would ultimately improve the quality of life of patients due to improved biocompatibility and longer duration of these interventions. The improvement of quality of life is obvious, as the product would avoid revision surgeries during the growth of the child. However, the impact is not only related to the surgery but also to indirect issues such as related pathologies, permanent medication, controlled physical activity, loss of performance during studies and professional activities due to treatments, surgeries and other issues. The development of such a device that can efficiently and permanently replace the RVOT will significantly reduce all these drawbacks allowing children to lead a completely normal life. In addition to such healthcare costs directly related to surgery, there are other additional costs. Children with CHD often require numerous hospital visits, care by a multidisciplinary team of specialists, frequent imaging and other diagnostic testing, drug and device therapy, and life-long outpatient follow-up. In another study, costs were grouped as financial, emotional and family burden. Financial costs include job change or job loss (e.g. mothers unable to return to work after maternity leave due to the inability to place a child in day care) loss of earnings due to taking time off work to attend appointments.

To achieve these potential impacts from the development of the TEH-TUBE project we designed the project with specific objectives and secured an outstanding consortium in order to achieve them. The Project objectives were:

• To process 3 different polymers (polydioxanone PDO, polyhydroxyalkanoate PHA and aliphatic poly(ester-urethane)ureas PEUU) via electrospinning to produce a 3-D valved bioabsorbable tube (diameter: 18mm; length: 7cm) with mechanical properties and biocompatibility relevant for in vivo implantation;
• To evaluate the mechanical properties and regenerative potential of each electrospun polymer in vitro and in vivo in a small animal model (rat)
• To determine the methods of biofunctionalization of the polymer with 2 different motifs (RGD and SDF-1 α) and evaluate biocompatibility and in situ regeneration potential of the functionalized polymers in a small animal model (rat)
• To compare the mechanical properties and potential of regeneration of bioabsorbable polymeric tubes after either ADSC preseeding or biofunctionalization by RGD or SDF-1 α motifs in a small animal model (rat) and in a limited number of a larger growing animal model (lamb) to define the best method of functionalization and validate it in the pulmonary artery anatomic position and under hemodynamic conditions relevant to the pulmonary arterial circulation.
• To create in vitro a tri-leaflet valve with one or more polymers (PDO, PHA and/or PEUU) via electrospinning, with hemodynamic competency (immediately after implantation) and long-term bioresorption potential without structural deterioration.
• To replace the native RVOT with such a tubular tri-leaflet valved bioabsorbable scaffold biofunctionalized with either ADSC or RGD/ SDF-1 α in a growing large animal model (lamb)
• To restore an autologous, living valved RVOT, displaying a valvular competence at mid- and long-term
• To demonstrate the growth potential and the absence of degeneration of such a bio-engineered device in a growing lamb model.

The expected overall endpoint of the project was to achieve the preclinical validation of this innovative biomaterial for a pilot clinical trial.

Project Results:
Work Package 1 – Polymer Processing

WP1 (Polymer processing) was concerned with using the electrospinning technique to prepare a series of increasingly complex template shapes, starting with simple flat patches and ultimately moving to the electrospinning of a complete valved tube.

Electrospinning (ES) is a technique which uses electrical energy to evaporate a volatile solvent from a polymer solution, yielding one-dimensional polymer fibres as an intertwined mesh. The process is illustrated in Annex A Fig. 1, together with a schematic of the resultant product.

In a typical experiment the polymer solution is loaded into a syringe fitted with a metal needle tip, and a syringe pump used to expel liquid at a precisely controlled rate towards a metal collector plate. A high voltage power supply is connected to the needle tip (usually +ve) and metal plate (grounded) and used to apply a large (kV) potential difference between the two. There are a range of parameters which can be varied in this technique: the applied voltage, flow rate, polymer concentration, solvent, spinneret-to-collector distance, etc. These have a profound effect on the equality of the fibre product, and thus need to be optimized for each new system studied.

We prepared a series of sinus-free electrospun tubes from polydioxanone and polyurethanes on a biomedical ES unit we purchased from NaBond Technologies, and prototyped the preparation of more complex shapes. Tubes were regularly sent to partner STAT and Partner RESC and partner APHP for analysis and feedback to permit design improvements to be made. All polymeric tubes were prepared and characterized using appropriate materials characterization techniques.

The results were not as originally anticipated for this work. Due to the screening and investigation process associated with the identification of a suitable polymer, the electro spinning and design of the tubes was delayed. The decision was made at month 18 to employ PLLA/PDLA stereocomplexes for TEH-Tube, and work continued to prepare tubes with the appropriate mechanical properties. In addition, partner HZG began development of a novel polymer based on the required specifications required for the tube. By the conclusion of work package 1, electrospinning of tubes have been undertaken using the PLLA/PDLA stereocomplexes and the technique had been successfully transferred to partner STAT so that the design could be optimised and electrospinning undertaken within a commercial environement.

Key outcomes from the work package

The work package successfully investigated a wide range of potential comemrcisl polymers that could be used for the development of the valved tube. Each polymer was assessed with respect to the specifications that would be required. In addition, electrospinning activities were performed with the polymers to determine their characteristics following eletrospinning. Following an extensive search it was determined that no commercial polymer solution could be found and partner HZG began the development of a novel polymer that would meet the project requirements.

The electrospinning was performed at UCL and following the development of initial prototype design the electrospinning parameters were transferred to partner Statice for further development within a commercial environment.

All deliverables associated with the work package were achieved and submitted to the European Commission.

Two patents were filed by partner HZG with respect to the development of the innovative polymer for the TEH-TUBE project. This has been reported to the European Commission.


Work Package 2 – Scaffold designing, testing and production

The objective of work package 2 was to use computer-aided design to develop the TEH-TUBE prototype. This was undertaken to ensure the tube was designed with the optimal parameters and mechanical requirements. This development, based on the outcomes of work package 1, will allow the core theoretical underpinning and validation of the prototype. The aim of the work package was to deliver a commercial-scale prototype that had the necessary mechanical and functional specifications required for in vivo use.


Design of the valved tube

The valved tube developed in this work package includes: an inner tube acting as a valve setup placed into an outer tube with sinuses and a T-shaped tube fixed to the extremity of the outer tube according to the blood flow direction. The T-shaped tube is particularly attractive for the regeneration of pulmonary artery branches. As a proof-of-concept, a T-shaped valved tube was manufactured with the polymer P(LA-CL)-PDLA developed by partner HZG.

To ensure the functionality of the design over time, it was shown by Statice that a ring had to be sutured below the inner tube inside the outer tube. The tooling was developed to manufacture the bioresorbable ring with 2 rows of holes for suturing. Then the distance between the inner tube and the ring was investigated. It was found that the ring had to be setup very close to the inner tube to avoid a significant bending of the outer tube jeopardizing the functionality of the device.

The design of the sutures of the inner tube was investigated by Statice. It was found that the functionality was optimized when the inner tube was sutured starting 1 mm from its top. The free movement of the top of the inner tube improved the closing.

During the first in vivo implantation, blood leakage was detected where the inner tube and the ring had been sutured. Leakage occurred due to the fact that an anti-coagulant was used during surgery to prevent thrombosis. To reduce this leakage, gluing or coating of the sutures was investigated. The coating by electrospinning, developed by Statice, was the most efficient method to prevent the leakage.

Due to a change of the HZG polymer properties from batch to batch, the electrospinning and the outer and inner tubes thickness were investigated and optimized in order to manufacture the prototypes for the in vivo assays.

In order to achieve the final optimised design for the TEH-TUBE more than 100 prototypes were prepared by electrospinning during this work package. In addition, each of the prototypes were tested using specialised bench tests developed at partner Statice to monitor and ensure appropriate valve function was being seen in vitro.

The final prototype is shown in Appendix A Figure 2.

Valve functionality

In order to ensure that the tube will function appropriately in vivo it was necessary to design the trileaflet valve to ensure that it would open efficiently and close preventing any back flow of blood. To investigate functionality two bench tests were developed. An air bench test was designed to check the prototype functionality prior to sterilisation. A water bench test was used to check the functionality of the prototype over time and to investigate whether there was any leakage from the tube. The water bench test also gave the opportunity to measure the pressure below and above the valve and the flow velocities during the opening of the valve. A video camera was used to monitor the functioning of the valve during the assessments. Each prototype representing an incremental design step was tested with the water bench test. Once the final design had been optimised the water and air bench tests were used.

For the final design of the prototype made with the HZG polymer the pressure and flow values were measured. These can be seen in Annex A, Figure 3.

Prototype following sterilisation

As the ultimate product will be used during surgical procedures and expected to remain in the body until bioresorbed, it would need to be capable of sterilisation. Furthermore, sterilisation needed to minimally affect the functioning of the tube and also the characteristics of the polymer used for its manufacture. Several sterilisation processes were considered and investigated. However, it was decided that ethylene oxide (EtO) sterilisation would be most appropriate for the tube. Prototype tubes were sent to partner HZG for EtO sterilisation and the returned to partners Statice and Rescoll for analysis. During the bech test performed at Statice it was apparent that the sterilisation process did not adversely affect the functioning of the tube during the water bench test. Partner Rescoll and HZG undertook material characterisation of the polymer following the sterilisation process. The results demonstrated that although there were some modifications of the polymer strands this was not considered significant or detrimental to tube functionality.

Mechanical testing of the prototypes

During the development of the final TEH-TUBE prototype more than 45 prototypes underwent mechanical testing and analysis by partner Rescoll. Samples of the inner and outer tubes, electrospun polymer patches, and samples from prototype patches/tubes explanted from animals were analysed.

The results of the analysis demonstrated that after 3 months there was strong degradation of the polymers. This suggested that following implantation the polymer was degrading quite quickly. The implantation studies using the final prototype implanted into growing lambs would provide valuable information regarding in vivo degradation. When completed further mechanical analysis would be performed on any material remaining.

Key outcomes from the work package

This work package advanced the design of the teh-tube product and resulted in the final design optimised for use in vivo. The final design included the valve which was shown to be functionally capable of delivering blood with minimal back flow. The valve was shown to function well during bench testing and continued to function appropriately following sterilisation using EtO.

Mechanical testing demonstrated that the prototype was capable of achieving the biological parameters required to function in vivo. However, analysis of samples explanted from in vivo studies suggested that the polymer was degrading very quickly in vivo. Once the final studies are completed in the growing lambs conclusive results can be obtained and published.

All deliverables for the work package were submitted to the European Commission.

A patent associated with the manufacture/design of the teh-tube prototype was submitted by partners Statice and AH-HP. This has been reported to the European Commission.


Work Package 3 – Cell culture and characterisation of seeded scaffolds.

When the project was initiated it was thought that there would be a requirement to seed the polymeric tubes with stem cells or to biofunctionalise the polymer to enhance its biological performance in vivo. Work package 3 was designed to:

1. Optimise the culture and characterisation of human ADSC
2. Determine and validate optimal parameters for seeding the polymeric biosorbable patches with human ADSC
3. Determine cell adhesion capacities of polymeric biosorbable patches with relevant cell systems according to the in vivo environment
4. Determine the level of tissue remodelling in grafted rat tissue samples.

Following significant investigations of biofunctionalisation options for the polymer it was determined that RGD peptide biofunctionalisation would be appropriate for the polymer studies.

Patches electrospun from various polymers bio biofunctionalised or non-biofunctionalised were investigated in vitro and in vivo. For the in vitro experiments patches of the test polymers were seeded with human adipose tissue derived stem cells (hADSCs) and following 2 days of incubation the viability of the hADSCs was determined.

These studies demonstrated that the hADSCs would adhere to the patches and remain viable. Although there were some observed differences between the polymers and whether they were biofunctionalised the differences in cell viability were not significant. However, following longer incubation periods (8 days) all polymers demonstrated a reduced cell survival.

Further investigations were performed with biofunctionalised versions of the HZG polymer. The results from these investigations were positive and demonstrated that of all the polymers examined the polymer developed by partner HZG was superior. It was concluded that further work should focus on the HZG polymer.

In vitro cytotoxicity testing of the HZG polymer demonstrated that the polymer did not have any cytotoxicity at a range of concentrations relevant to the in vivo situation. Furthermore investigation of the influence of the polymer on biomarkers associated with tissue remodelling demonstrated that the HZG polymer did not significantly influence the expression of biomarkers associated with tissue remodelling.

In conclusion the in vitro results obtained during work package 3 confirmed that the polymer developed by partner HZG was superior to other polymers that had been identified. Furthermore, cell seeding and biomarker expression analysis demonstrated that the polymer was not cytotoxic, supported cell growth as a scaffold, and did not adversely affect tissue remodelling biomarker expression.

It was also concluded that biofunctionalisation of the polymer did not significantly enhance the biological effect of the polymer in supporting cell adhesion or growth.

Key outcomes from the work package

The key outcome of this work package was that biofunctionalisation of the polymer was not required in order to have the biological effect on cell adhesion and growth. Furthermore, the studies confirmed that the HZG developed polymer was the most appropriate for continued development within the project.

Work Package 4 – Polymer Biofunctionalisation.
The objectives of this work package were to provide biofunctionalized implants by:

1. Coupling peptides or adhesion peptides containing proteins such as gelatin to the surface of electrospun scaffolds made of candidate degradable polymers, using perfluorophenylazides
2. Developing a multiblock polyester urethane with defined groups available for specific peptide/protein immobilization, e.g. by click chemistry, as an alternative matrix material for the polymeric valve tube
3. Establishing the surface functionalization under GMP conform conditions in a clean room facility with a confirmed enhanced biocompatibility of functionalized implants

Polymer biofunctionalisation
During this work package partner HZG developed an innovative polymer that would meet the specifications of the required polymer for prototype development. In addition, they considered a range of approaches to support the biofunctionalisation of the polymers that had been identified as potential candidates for the project.

Following electrospinning of the polymer patches, HZG investigated a range of surface functionalisation techniques. Plasma treatment followed by peptide grating was selected as the method of choice. This required the development of processes that would support the biofunctionalisation of the polymer. In collaboration with partner UCL and Rescoll methodology was developed to allow for the peptide grafting of RGD to the polymer surface.

Gelatin biofunctionalisation was also undertaken as an alternative approach due to the cost effectiveness that could be achieved. Polymer coating was achieved by immersing the polymer in the gelatin solution. The adsorption of the gelatin onto the polymer was then assessed. This procedure resultd in efficient coating of the polymer with gelatin. The coated patches were then used for further investigation. The biofunctionalised polymer patches were sent to the partners for in vitro assessment and mechanical testing.

During the work package two methods of gelatin biofunctionalisation were developed the first is described above while the second was based on PFPA-treatment followed by gelatin coupling. The PFPA functionalisation process resulted in the covalent attachment of gelatin to the polymer structure. Polymer patches were then electrospun and sent to the partners for analysis as described in work package 3.

Partner RESCOLL also developed a process for biofunctionalisation of the polymer based on the introduction of a reactive carboxyl group through the use of low pressure plasma treatment with oxygen. Once produced the peptide RGD was grafted onto the polymer in water. The benefit of this process was that it could easily be scaled up as a GMP compliant process for polymer biofunctionalisation.

cGMP biofunctionalisation
It was recognised by the partners that biofunctionalisation of the polymer would ultimately require cGMP manufacture protocols and as such efforts were initiated at partner RESCOLL to produce a cGMP methodology that would be capable of transfer to a commercial cGMP manufacturer once the product had been developed. While this work commenced, results from work package 3 were being presented to the consortium which demonstrated that biofunctionalisation of the polymer did not significantly enhance the biological properties above that of the native polymer. As such, this work was stopped in order to focus only on the continued development of the native HZG developed polymer.

Prototype sterilisation

As the ultimate product will be implanted into patients for prolonged periods of time it is essential to ensure that the product can be sterilised without the process adversely affecting the product structure or function. Consideration was given to the commercially available sterilisation processes. Due to the nature of the polymer it was evident that high temperature sterilisation was not appropriate and no longer widely used within the medical industry. Gamma irradiation is the preferred method for sterilisation. However, there were concerns that the irradiation would adversely affect the structure and biological function of the polymer. Critical CO2 sterilisation was also considered as an innovative approach to sterilising. However, as this is a relatively new process there was concern that it may not be commercially viable at the present time. It was decided to utilise ethylene oxide (EtO) sterilisation as this process would have minimal adverse effects on the polymer structure and biological activity of the polymer product.

EtO sterilisation was performed at HZG using the native and biofunctionalised patches. Analysis following sterilisation demonstrated no changes in molecular weight of the polymer; polymer composition; fibre diameter or morphology; mechanical properties; or crystallinity. Furthermore, GC-MS demonstrated that the sterilisation process did not result in EtO residue being retained as a contaminant within the polymer.

Key outcomes from the work package

• Biofunctionalisation of the polymer with RGD and gelatin was successfully achieved.
• Sterilisation using EtO was accomplished and demonstrated that no adverse effects on the polymer structure was demonstrated.
• Based on the results of work package 3, cGMP methodology development for biofunctionalisation was stopped.
• Biofunctionalisation work was not continued due to the results of work package 3 showing that the process is not significantly enhance the biological activity of the polymer over the native polymer.
• All work package deliverables have been submitted to the EC.

Work Package 5 – In vivo studies (rats).
In order to begin the process of preparing the regulatory dossiers described in work package 7, it was necessary to begin the preclinical evaluation of the polymer patches in vivo to demonstrate that there was no cytotoxicity, that the polymer was biocompatible, was bioresorbable and did not cause any adverse immunological or tissue defects. The first step in this process was the assessment of the polymer in vivo using rats. The selected polymer, both native and biofunctionalised forms were implanted in to 90 rats. The groups comprised 30 animals implanted (into the IVC) with TPU/PCL biopolymer, 30 with PLLA/PDLA biopolymer and 30 with PLLA/PDLA + gelatin. No mortality occurred following implantation and animals were then periodically sacrificed at 14 days and 90 days post implantation. The results from this study indicated:

• There was no difference in polymer appearance at 2 weeks
• All polymers demonstrated a layer of endothelial cells and extracellular matrix
• PLLA/PDLA polymer showed infiltration of inflammatory cells.
• PLLA/PDLA with gelation are not infiltrated with inflammatory cells
• No changes were observed in echocardiographic recordings

As such, the conclusion was that the polymers were not having adverse in vivo effects at 2 weeks following implantation. Further analysis was then performed at 90 days. The results after 90 days demonstrated that:

• None of the implanted patches were associated with thrombus formation
• All patches were integrated into the IVC but could still be differentiated
• Mechanical testing (where possible) showed that the polymers had undergone significant biodegradation in vivo. However, they had maintained structural integrity and supported cell growth.

The full analysis of the rat study demonstrated that there was no significant difference between the native polymer and the biofunctionalised polymer. As a result it was concluded and decided by the consortium to only progress with the development of the native polymer. Results also demonstrated that the selected native polymer was not associated with adverse biological effects when implanted.

Key outcomes from the work package

• There was no significant biological difference between the native and biofunctionalised polymers.
• Further development will only consider the use of the native polymer
• No adverse biological effects were seen for any of the polymers implanted into the rats
• The results confirmed that progress to the in vivo work using lambs can continue.
• All deliverables associated with this work package were submitted to the EC.


Work Package 6 – Pre-clinical evaluation (lambs).
As part of the preclinical assessment the prototype tubes need to be implanted in an appropriate large animal model in order to generate data to support the regulatory dossier development. Following the design and prototype optimisation and scale up undertaken during work package 8 prototypes for implantation were prepared. The tubes were sent to HZG for EtO sterilisation and then to partner AP-HP for the implantation studies in sheep. The design of the lamb study was to consider valved and non-valved tubes and also a comparison with other commercial tube products. The tubes were implanted without cell seeding or biofunctionalisation for the reasons discussed previously.

Due to the complexity of the surgical process and the necessary scheduling of the operating theatre and staff the implantations were divided into a series taking place during the final 12 months of the project. The first implantations were with non-valved tubes made from the HZG polymer and Gore-Tex tubes. Three lambs were implanted with the HZG-polymer non-valved tubes and 3 lambs with the Gore-Tex tubes (control). For the next series of implantations an 18mm Hancock tube was used with two animals but there were surgical problems and the tubes were changed to Contegra tubes (18mm) implanted into 3 lambs. As an initial study we also implanted one lamb with the HZG-polymer valved tube.

During the final months of the project it was necessary to receive an additional batch of polymer from HZG which was provided to support the production of the final valved-prototype tube for implantation in to the lambs. Unfortunately, the batch characteristics of the new batch of polymer differed from those of the previous batch (although still within the required specification limits). This caused a delay at partner Statice as they had to re-optimise the electrospinning parameters for use with the new polymer batch. This was resolved towards the end of the project but had an impact on the final lamb implantation schedule for the HZG-polymer valved tube prototype. As a result the final implantations could only be completed after the project end date. However, these studies have continued and analysis will be available during 2018 to support regulatory dossier development and project publications.

The results from the animals that have implanted demonstrated that the HZG-polymer non-valved tubes were flexible and relatively easy to surgically implant. However, two animals died during the surgery due to non-tube related effects.

Six lambs were sacrificed at 26 weeks following implantation. Histological examination showed that the Gore-Tex tube was still visable and show no signs of degradation. The HZG non-valved tube could not be differentiated from the native tissues demonstrating integration. No thrombus formation was evident and no aneurysm was noted. There was little evidence of inflammatory cell infiltration or infection.

Ecographic assessments were performed before explantation at 26 weeks on one Gore-Tex implanted and two HZG-polymer non-valved tubes. The lambs showed good right ventricular function. However, one of the HZG-polymer non-vavled tube implanted lambs showed hypokinesia and a leak.

Mechanical testing was performed by partner RESCOLL on samples of the explanted tubes. The Gore-Tex tubes demonstrated similar characteristics to the native material. The mechanical properties of the HZG-polymer non-valved tubes also had similar mechanical properties to the initial implanted material. However, it was noted that the material was degraded and new tissue/cells had infiltrated the polymer.

Histological examination confirmed that the HZG-polymer non-valved tubes were infiltrated with inflammatory cells, showed extracellular matrix secretion around the polymer and the presence of new tissue fibres.

The results from these initial studies provide some positive results for the HZG-polymer non-valved tubes. Further analysis is being undertaken to fully analyse the biological impact of the tube within the tissue/cells.

With respect to the initial implanted valved-tube made from the HZG polymer the implantation was successful and the tube functioned effectively for 2 months. However, after 2 months the valve within the tube began to demonstrate a failure. Explantation of the tube and analysis revealed that one of the valve leaflets had become non-functional. This information was incorporated into the optimisation of the final valved prototype development in work package 8.

At the point of submission of this report we have implanted 24 lambs:
• 3 lambs with Gore-Tex non-valved tubes (reported here)
• 5 lambs with HZG-polymer non-valved tubes (reported here)
• 2 lambs with Hancock tubes (reported here but discontinued)
• 6 lambs with Contegra tubes (Implanted but not analysed by project end date)
• 1 lamb with HZG-polymer valved tube (reported here)
• 8 lambs with HZG-polymer valved tubes (ongoing at the present time)

The study will continue and results will be used for the regulatory dossier development in due course and for publications.

Key outcomes from the work package

• Implantations in growing lambs has commenced
• Gor-Tex and HZG-polymer non-valved tubes were implanted for 26 weeks
• Results from the Gore-Tex and HZG-polymer non-valved tubes have been analysed and show good performance of the HZG-polymer tube.
• Implantation of a single HZG-polymer valved tube was undertaken
• At 8 weeks the prototype began to fail and analysis showed that one of the valve leaflets had failed. This information was used for the final prototype design.
• Final implantations of the HZG-polymer valved tube has been completed
• The final results and analysis will be completed post project.
• Deliverables have been submitted for this work package based on progress to date.


Work Package 7 – Quality assurance and regulatory affairs
The working package was designed to address the Regulatory and Quality aspects linked to the TEH-TUBE Technology Development. In order to facilitate the exploitation of the TEH-TUBE technology, quality requirements are essential to achieve compliance with the Regulatory requirements. Celyad would provide guidance and advice to the consortium for compliance with European and US regulatory standards. Celyad would use its expertise in the translation of ATMP and protein-based therapies in the field of cardiovascular medicine from proof of concept to phase III clinical trials on the one hand, and its expertise with the development of innovative medical devices in the very same field on the other hand.

During the project the work package was associated with gathering the necessary information required to prepare the necessary regulatory dossiers required to approach the regulatory authorities in Europe and the USA in order to secure clinical trial approval.

Celyad worked with the partners to gather information to support:
1. The classification of the device in terms of how it would be considered by the regulatory authorities.
2. The development of the technical file including the final design specifications and manufacturing protocols. Included in the document would be the preclinical data from in vitro and in vivo studies.

During the project data associated with points 1 and 2 above were gathered from the partners an reported to the EC through the work package deliverables that were submitted. However, delays in the final prototype design meant that a final design freeze did not take place until later than expected in the project. As a result, the technical file information could not be completed to the standard required. This work will continue post project as the final data is obtained from work package 5.

The classification of the TEH-TUBE product was undertaken and it was evident that it would be viewed as a medical device which was implantable and expected to remain in the body for an extended period of time.

Consultation with regulatory experts was undertaken during this work package to identify the most appropriate approach to make to the regulatory authorities in Europe and the USA. Following these consultations it was evident that the stage of development at the end of the project was too early to make an informal approach to the authorities. However, the information gathered and the work undertaken during the project will allow all dossiers to be completed as the data from work package 5 becomes available. It is proposed to then arrange the necessary meetings with the regulatory authorities to continue the development of the TEH-TUBE product.

Key outcomes from the work package
• A clear regulatory pathway was developed for the TEH-TUBE product.
• Determination of product classification was made
• Technical dossiers were established and will be completed post project.


Work Package 8 – Clinical grade manufacture
In order to develop the final prototype for in vivo evaluation it was necessary to undertake a commercial scale production of the electrospinning within an industrial setting. Technology transfer initially took place from partner UCL to partner Statice. Following this process, Statice began the optimisation of the electrospinning for the prototype. This included the development of specialised mandrels for electrospinning the product (patent was filed in collaboration with AP-HP for this and reported to the EC). Following the development of the HZG polymer, Statice began to optimise the electrospinning process using this new polymer. At the same time, many prototypes were produced with differing designs and tested using the bench test. This was an extensive process which ultimately lead to a prototype that demonstrated very good functionality on the water and air bench tests (see Annex A figures). In all more than 100 prototypes were manufactured. The final design was achieved in July 2017 and a new batch of polymer was requested form partner HZG. Following synthesis of the new batch, sufficient to product all of the final prototypes, it was discovered that the characteristics of the new polymer batch following electrospinning was different to that of the previous batch. Following various checks it was determined that the new batch, although within parameters agreed with the consortium, was producing tubes with much higher elastic properties than previously. As such, additional work was required at Statice to optimise the electrospinning process again using the new polymer. Once optimised the final prototypes were produced and sent to partner HZG for sterilisation and then to partner AH-AP for implantation into the lambs. Unfortunately, due to the delays caused it was not possible to complete the lamb studiers before the end of the project. Final analysis will be performed post project and the data used to support regulatory dossier development.

Key outcomes from the work package

• A commercial scale electrospinning process was established for the TEH-TUBE product
• The final prototype design was established
• A patent was filed with respect to the mandrel design used for production.
• The prototype manufacture was optimised
• All prototypes were manufactured to support the in vivo studies in lambs.
• All deliverables associated with the work package were submitted to the EC


Work Package 9 – Dissemination and Exploitation
The consortium partners have been active in disseminating the results from the project. This has included presentations at a range of conferences, workshops and events. These have all been reported to the EC. In addition, during the final year of the project a major dissemination event took place at the European Association of Cardiothoracic Surgeons (EACTS). This event draws worldwide participation. During the event Dr David Kalfa gave a presentation regarding the project at the Techno-College event. This event provides an opportunity for individuals and organisations to give an elevator pitch to private sector financial organisations and companies about their innovative new technology development. Interest was received from a number of the participants at this event. Furthermore, an exhibition stand was visited by many of the delegates during the event for discussion regarding future collaborative and exploitation opportunities.

Exploitation of the project results has been managed by partner Celyad. The project has resulted in 3 patent filings which have been reported to the EC. In addition, the partners were actively engaged in discussions regarding the future development and exploitation of the project results. It was concluded by the partners that an exploitation agreement would be the most appropriate method to continue product development. It is anticipated that this agreement will be signed after the end of the project.

During the work package partner Celyad has actively engaged with the partners to determine their interest in future exploitation. In addition, Celyad has produced a SWAT analysis and business plan associated with the potential future market demand for the product. During the final year of the project Celyad engaged with selected surgeons under confidentiality agreements to consider the utility of the prototype. This feedback was extremely positive and provided a good evidence base for future commercial development of the product.

Key outcomes from the work package

This work package delivered a range of positive outcomes for the consortium including:
• Dissemination activities including the event at the EACTS which was well received by the participants
• Development of an initial business plan including SWAT analysis
• Proposals for the continued commercial development of the product
• Support for partners in determining their interests in future exploitation.


Work Package 10 – Project Management
The contact PI, coordinator and scientific representative of the project's coordinator and organisation was initially Dr David Kalfa (as he was still working in Europe). Dr Kalfa applied and obtained this FP7 funding as the Principal Investigator. When Dr Kalfa accepted a position of Professor at Columbia University, he had to pass on the administrative coordinating role to (AP-HP), for administrative and regulatory resasons. Dr Kalfa continued serving as the scientific co-ordinator of the THE-TUBE project, now working at the University of Columbia, USA. Working in close collaboration with the partners and the project co- ordinating partner (AP-HP), the project was exceptionally successful in achieving all of the project objectives in whole or part. Project management and administration was completed with the administrative support of partner Euram. This working relationship was very beneficial as it allowed the administrative project management to be undertaken allowing the partners associated with research and technology development to focus on their activities.

During the project there were monthly teleconferences arranged to ensure that project progress and management issues could considered and consortium meetings were arranged every six months. These meetings have enabled the project to progress effectively and risks associated with the project to be quickly identified and resolved.

During the project there were 4 amendments to the description of work. Three of these were associated with work programme changes and budget alterations. One was associated with a change to the contact personnel at the co-ordinating institution.

Deliverables for the project have been achieved and submitted to the EC. Most of these have been completed, however, for the in vivo work package (work package 6) the reports are progress reports to the end of the project due to the unexpected delays with the polymer batch at the end of the project.

Milestones for the project were achieved and overall the project achieved most of the objectives that were anticipated. In terms of future plans the consortium have confirmed that they will complete the final in vivo studies and use the data to continue the product development. While it is too early to consider commercial exploitation, there is a willingness to make further private and public sector funding applications from EU and USA organisations to continue the support for product development.

Each of the partners has completed their activities to a very high standard of work and initial analysis of the financial expenditure associated with each of the partners demonstrate that they have adhered to their budget. Staff effort levels have increased with respect to some of the work packages due to the technical difficulties that were encountered at the start and end of the project.
Potential Impact:
In the future, successful treatment of congenital cardiac abnormalities is expected to rely on ATMPs and medical devices. Around 36,000 children are born each year with a CHD; these are the most frequent congenital abnormalities (8-12/1000 births). Existing treatment involves reconstruction of the RVOT, using materials that do not grow as the patient develops. This means that patients treated for a CHD in must undergo repeat surgery (1-4 revision surgeries in their lifetime) with a high risk of mortality and morbidity. Our aim wasto combine and develop existing methodologies to produce a technology that would allow treatment of CHDs with a biomaterial that can reconstruct the heart with a single surgery without the need for re-intervention as the patient grows. This would address the currently unmet clinical need and poor clinical outcome in this patient population. The TEH-TUBE project would improve the quality of life of patients due to improved biocompatibility and longer duration of these interventions. The improvement of quality of life is obvious, as the product would avoid revision surgeries during the growth of the child. However, the impact is not only related to the surgery but also to indirect issues such as related pathologies, permanent medication, controlled physical activity, loss of performance during studies and professional activities due to treatments, surgeries and other issues. The development of such a device that can efficiently and permanently replace the RVOT will significantly reduce all these drawbacks allowing children to lead a completely normal life. In addition to such healthcare costs directly related to surgery, there are other additional costs. Children with CHD often require numerous hospital visits, care by a multidisciplinary team of specialists, frequent imaging and other diagnostic testing, drug and device therapy, and life-long outpatient follow-up. In another study, costs were grouped as financial, emotional and family burden. Financial costs include job change or job loss (e.g. mothers unable to return to work after maternity leave due to the inability to place a child in day care) loss of earnings due to taking time off work to attend appointments. Emotional impact was most commonly reported to be substantial stress in immediate family but also extended family who assist in taking time off work for childcare. In addition, due to repeated surgeries with cardiopulmonary bypass and their potential morbidity, some children/adolescents/adults display lower cognitive function and a decreased quality of life, with an impact on social function. Secondary clinical impacts are numerous. The technology can be applied to all patients with an abnormality of the left ventricular outflow tract, heart valve disease, patients requiring a vascular surgical reconstruction, or cardiac reconstruction using a bioabsorbable patch. Secondary impacts and applications include polymeric meshes for abdominal wall reconstruction, polymeric films to decrease pericardial adhesions after cardiac surgery, biofunctionalized reconstruction scaffold in laryngofacial and plastic surgery, biofunctionalized bioabsorbable tubes for pediatric urologic surgery, hemodialysis access shunts. Finally, TEH-TUBE will support the EU2020 Strategy and health policies within the group of patients who experience CHD to:
• Improve health
• Promote sustainability in EU healthcare systems by decreasing the economic burden of healthcare costs
• Reduce numbers of deaths due to major/chronic disease
• Increase employment within the patient group
• Promote social cohesion.

This also aligns with aspirations of the EU2020 programme to support the Innovation Union in the development of knowledge and producing an innovative product concerning health and the aging population. Together, these will help to promote a sustainable, more competitive economy.

While there a significant amount of future development required before the TEH-TUBE could enter the market, our results demonstrate that our approach is feasible. In addition, our dissemination activities have raised significant interest within the academic, clinical and industrial community with respect to this technology. Raising the profile of European capabilities in this area is of significant importance and will continue to provide future opportunities for collaboration between the consortium partners.

List of Websites:
Project Website URL: www.teh-tube.eu

David Kalfa, MD, PhD,
Assistant Professor of Surgery, Section of Pediatric and Congenital Cardiac Surgery
Director, Pediatric Heart Valve Center
Assistant Director, Cardiothoracic Research Lab
Columbia University College of Physicians & Surgeons
New York-Presbyterian Morgan Stanley Children's Hospital
3959 Broadway, CHN-274
New York, NY 10032
davidkalfa@gmail.com