Regeneration of Cardiac Tissue Assisted by Bioactive Implants

Project Information

RECATABI

Grant agreement ID: 229239

Project closed

Start date 1 January 2010

End date 31 December 2012

Funded under

• Specific Programme "Cooperation": Nanosciences, Nanotechnologies, Materials and new Production Technologies

Total cost € 2 929 560,87

EU contribution € 2 328 302,00

Coordinated by

INSTITUT QUIMIC DE SARRIA
Spain

Final Report Summary - RECATABI (Regeneration of Cardiac Tissue Assisted by Bioactive Implants)

Executive summary:

Heat failure (HF) is the end-stage of many cardiovascular diseases, but the leading cause is the presence of a large scar due to myocardial infarction. Acute myocardial infarction remains a major life-threatening disease for both men and women all over the world and normally happens when blood supply to the heart is interrupted. Therapeutic strategies that limit adverse post-ischemic remodeling in HF may prevent ventricular dilatation and maintain the structural support necessary for effective cardiomyocyte contraction. Adverse post ischemic remodeling is related with mitral valve regurgitation, end-stage heart failure and death. Current treatments under development consist in cellular therapy where myocardial cells or stem cells are implanted alone or encapsulated in natural scaffolds (collagens) and grafted onto infarcted ventricles with the hope that cells will contribute to the generation of new myocardial tissue. This approach seems to have a beneficial effect although it is not completely understood and optimized, yet. Moreover, most of the implanted cells died soon after transplantation partially being due the fact that the cells could not withstand the mechanical forces neither the biophysical conditions (i.e. low oxygen levels) they experience in the host ischemic tissue. One of the main mechanisms by which cellular therapy could bring functional benefits would be that the implanted cells should provide a supporting 'band-aid' scaffolding effect, which can limit the spread of the infarcted area, preventing excessive remodeling and dilatation of the ventricle.

Project Context and Objectives:
During our project the members of RECATABI consortia were involved in testing the capacity of our bioactive implant prototype to deliver adipose tissue derived stem cells (ATDSC) to the ischemic infarcted heart tissue using a small /rodent) and large (sheep) animal model. We have developed a bioactive implant (WP7) by combining the self-assembling peptide nanofibers, the elastomeric membranes developed in WP5 and stem cells (ATDSCs, WP6). Our results indicate that we were successful in developing a platform but most importantly that our bioactive patch carrying stem cells embedded in a self-assembling nanofiber network indicated good cell survival, cell distribution and most interestingly, to properly deliver cells into the ischemic tissue in a proof of concept experiment with a small animal model (mouse) of infarct developed in WP7 and WP10 by the groups of IGTP, UPVLC and IQS-URL, and with a large animal model (sheep) of infarct developed in WP7 and WP 10 by the groups of IGTP, UPVLC, Cardio-Monde and IQS-URL.

In addition, the group of IQS-URL was able to develop a new biomaterial consisted in blending self-assembling peptides with heparin moieties in order to obtain a nanofiber material with capacity of VEGF binding and release during the first 48h after incubation. This material has the ability of bind and release other heparin binding growth factors such as FGF2. In addition, the angiogenic capacity of the material was tested and demonstrated in vitro with endothelial cells.

Moreover, during the project our main goal was to see whether or not the delivered cells from the bio-implant into the affected tissues of the heart but also if these migrating cells were able to integrate into the tissue and differentiate into new functional cardiomyocites. This was analysed by using pre-cardiac stem cells carrying reporter genes (i.e. luciferase) under the transcriptional control of specific cardiac marker promoter (GPF signal from specific cTnI reporter), as we indicated in the WP7. In this way, we were able to monitor the fact that implanted cells were turning into newly cardiac-like cells. This brought good indications on the efficacy of the bio-implant platform either in the mouse as well as in the sheep model.

The work on big animal model (sheep) was carried out mainly by the group of Cardio-Monde with assistance of all the other groups. In this case, specific adipose derived adult stem cells from sheep were isolated and expanded by the group of Cardio-Monde and IGTP and used to load the bioactive implants developed by the groups of UPVLC and IQS-URL. In addition, specific readjustments were taking care in order to scale up the implant by the group of UPVLC and IQS-URL, since considerable increase on the membrane size will affect to self-assembling peptide distribution into the elastomeric membrane as well as the amount of stem cells and their survival capacity. These were real challenges at the time but we were able to succeed. It is important to mention at this time that the sheep model is closest to the human situation in terms of heart size, amount of cells and bioactive implant requirements as well as anatomically and functionally. Although RECATABI project did not intent to explore in human trials the results obtained with the sheep will tremendously help to understand the key issues to have in consideration at the time of adventuring into this next step. The functional results obtained with the sheep model indicate that the RECATABI concept was prove to be right and we conclude the successful result and accomplishment of all our tasks and deliverables.

Finally, our consortium was very successful in protecting their discoveries and platform development by obtaining patents (PCT and US patent) during the reporting period 1-36M. In addition, several manuscripts were published and more are under preparation. The members of the consortia participated in several international and national meetings, congresses and symposiums during the 36M of the project. In this way our project RECATABI was properly disseminated with the right protection in case of exploitation.

Project Results:
1. IQS-URL

During the RECATABI project, IQS-URL group was leading WP1, WP4, and WP7 and it has been involved in all WPs.

WP1: Project Coordination
During the project, the Coordinator Institution has been involved in management of activities consisting in assisting all the partners for the efficient execution of the work packages and the development of their activities including to reach the specific objectives, deliverables and time schedule. The Coordinator gave access to the available budget for each partner on time, except for the partner (Bayes) from IGTP that due to a change of Institution (from HSCSP to IGTP) its payment was delayed. The creation of a Management Support Team (MST) composed by IQS-URL (Semino), UPVLC (Monleón Padras) and IGTP (Bayes) ensured that the coordination of the project was effective and efficient.

WP2: Ethical Issues
During this period objectives were achieved by CREASPINE. During the reporting period the creation of an ethical committee has been set up. Ethical committee collected and recorded ethical certificates from laboratories involved in animal testing, with small (mice) and large animals (sheep). Moreover, extensive discussion regarding ethical issues related to implemented technologies on animal models used has been performed. In addition, the groups have exchanged essential technological platforms by several ways including student exchange among laboratories or extraordinary short meetings between two or three of the groups involved. In addition, new SOPs have been originated as to organise the documentation throughout the project, to generate consistent quality documentation and provide guidance related to the recording of critical documents and reports.

WP3: Intellectual Property
The main objectives of this workpackage were to implement a comprehensive overview of patentable research results and the alternative options to commercialize the products obtained. In order to achieve these objectives, a direct collaboration with CREASPINE was created. The main products of this workpackage were the European and American patent.

WP4: Nanobiomaterials
In WP4 the coordinator has been involved in developing a new class of nanobiomaterial composite, consisting of the non-covalent association of self-assembling peptide nanofibers and low molecular weight heparin moieties. Since the peptide nanofiber present total positive charge at physiological pH (7.2) the anionic association with heparin (negative charged at physiological pH due to its sulfate groups) make it very stable. In fact, it was possible to combine the peptide nanofiber and heparin without the need of synthesizing a glycopeptide, which was the original objective of the project. In this way, the platform turns to be much simple to assemble in a combinatorial way, where different heparin molecules (GMP type for human use in terms of molecular weight, level of sulphation as well as quality), can be combined at different ratios with the self-assembling peptide nanofibers. This versatility opens the possibility of testing very fast the best self-assembling peptide/heparin combination for VEGF and FGFβ delivery. Then, growth factors binding and release experiments were performed in order to test the capacity of the RAD-Heparin composite to be used as drug release hydrogel. Interestingly, it was observed that the RAD-Heparin composite continued releasing VEGF165 and FGFβ over the time as compared to the control RAD16-I.

In order to study if the presence of heparin interferes in the self-assembling peptide structure and as a consequence in the nanofibers formation, two different kinds of analysis were performed. First, samples were observed under scanning electron microscopy (SEM) to study nanofiber formation, and it was observed that nanofibers were similar in both cases. Then, circular dichroism of the self-assembling peptide RAD16-I and the composite were analyzed. As it was expected, the beta-sheet structure characteristic of the self-assembling peptide was not disrupted at the heparin working concentration. However, at higher concentrations it can be observed a negative effect of heparin in the beta-sheet structure.

To test the capacity of the nanofiber matrix for pre-cardiomyocites maintenance and biomechanical integration, the initial idea was to produce self-assembling peptide matrices functionalized with integrin-specific peptide motifs. A canonical 2 RGD tandem sequences (AcN-GPRGDSGYRGDS-GG-(RADA)4-CONH2) and a triple helix motif containing the sequence GFOGER from collagen type I (AcN-(GPP)5GFOGER(GPP)5-GG-(RADA)4-CONH2). This sequence from collagen I could not be obtained and the new self-assembling matrix was obtained by mixing RAD16-I, heparin and the RGD sequence.

Finally it was interesting to analyze the effect of these modifications (RGD and heparin moieties) in subATDPCs behavior. With this aim 3D cultures with RAD16-I peptide and the above mentioned modifications were prepared. Firstly it was verified that the gel was formed correctly. Congo Red Staining shows that all the modifications proposed allow RAD16-I peptide to gel forming β-sheet structures. Additionally, it was observed that subATDPCs remain alive encapsulated inside RAD16-I peptide with and without modifications. Additionally, MTT assay was used to analyze cell behavior along the first days of culture. Initially, it seems that modified peptides show a higher number of cells respect to unmodified peptide. Anyway, after four days of culture there are not significant differences between peptides or culture medium (two different media were tested: control medium and cardiac medium). Anyway due to diffusion these results are not concluding. In the field of protein expression GATA-4 and Connexin-43 were analyzed. Interestingly, cardiac medium highly increase both Cx43 and GATA-4 expression in contrast to control medium. Although 2D subATDPCs growing in cardiac medium show GATA-4 and Cx43 expression, 3D system highly increase their expression. It is remarkable that any of the proposed modifications increase GATA-4 or Cx43 expression in comparison with RAD16-I peptide.

WP7: Bioactive implant development
As it has been mentioned, IQS-URL group has been involved in the development of the bioactive implant in close contact with UPVLC group. UPVLC group developed different elastomeric membranes with the conditions mentioned in WP5 and in collaboration with all partners of the consortia. These elastomeric membranes were tested for cell viability, proliferation and differentiation by IQS-URL and UPVLC group. The general aim of this workpackage was to combine the nanobiomaterial (RAD16-I nanofiber peptide) with the elastomeric membranes including the cells isolated in WP6 by IGTP group. Initially the idea was to combine the RAD16-I self-assembling peptide with subATDPCs isolated in WP6 and include the mixture inside the porous of elastomeric membranes. Due to the hydrophobicity of the material this procedure did not succeed although different procedures were tested (injection, centrifugation, shake…). Therefore the initial idea was replaced for a new methodology. RAD16-I self-assembling peptide and cells were introduced inside the elastomeric membrane in two-step protocol. Firstly, the nanofiber is forced to get inside the porous of the membranes developed by UPVLC group using pressure. To assure that the nanofiber peptide was correctly introduced, it was gel induced with PBS and Congo Red staining revealed the proper β-sheet formation. Additionally, as it was observed by SEM imaging, the self-assembling peptide does not appear to fill completely the porous as it was previously assumed. RAD16-I is homogenously distributed inside the porous but absorbed on the wall of the membrane. Therefore, after injection the elastomeric membrane is superficially modified. After the preloading of the RAD16-I self-assembling peptide inside the elastomeric membrane, the cells are introduced. Two methodologies were tested: load the cells on the surface of the membrane and injection of the cells inside of the membrane. With the idea to obtain a homogeneous implant a soft shake was induced during half an hour. The best results were obtained using cell injection and soft shake. Therefore; the first prototype of the bioactive membrane (WP7) was obtained by combining the self-assembling peptide nanofibers with the elastomeric membranes developed in WP5 and injection of stem cells. Preliminary results indicate that although the initial procedure for the obtaining of the bioactive implant was not possible, we have been successful in developing the bioactive implant and most importantly that our original objectives were feasible.

It was also analyzed (as it has been done in WP4) the capacity of the bioimplant to release VEGF165 and FGFβ in a controlled manner. The experiments for VEGF165 and FGFβ binding and release experiments were performed with the membranes filled with the self-assembling peptide RAD16-I and the RAD-Heparin composite in order to demonstrate that these materials retain the capacity to release the angiogenic factors inside the membranes. It was observed that RAD-Hep composite continued releasing FGFβ during 36 hours as compared to the control RAD16-I where the growth factor was mainly released at 12 hours. For VEGF165 it was observed a high variability between samples with the same conditions. This may be due to the different quantities of gel inside each membrane which could be a consequence of the method used for filling the membranes with the gels and the variability between membranes.

Moreover, Human Dermal Microvascular Endothelial Cells (HDMECs) were cultured in the self-assembling peptide RAD16-I and the composite RAD-Heparin to test the pro-angiogenesis capacity of these materials in vitro using endothelial cells. Interestingly, when HDMECs were cultured in the media without heparin, the formation of capillary-like structures was much higher in the composite RAD-Heparin as compared to the RAD16-I where cells looked mainly rounded. These results suggest the positive effect of the heparin present in the composite on capillary-like structures formation.

Finally, the incorporation (MTS test and SEM imaging) and possible cardiomyocyte differentiation (immunohistochemistry, RT-PCR and Western Blot) of the pre-cardiomyocytes was analyzed. MTS test cannot give as an idea of cell proliferation due to diffusion problems but it assures that there are live cells inside the bioimplant. The distribution of these cells were confirmed by SEM imaging observing that RAD16-I peptide facilitates cell connectivity. Studies on subATDPCs gene and protein profile were performed in vitro in PEA elastomeric membrane bioactive implant. It was observed that a-actinin was clearly expressed at protein level which was corroborated by RT-PCR analysis. It was also observed Tbx5, MEF2C and cTnT gene expression. Western blot results show no expression of GATA-4 or Cx-43 while confirm Tbx5 expression.

In resume, we obtained a bioactive patch carrying stem cells embedded in a self-assembling nanofiber network that indicated good cell survival, excellent cell distribution and, most interestingly, seems to be functional in a proof of concept experiment with a small animal model of infarct developed in WP7 by the group of IGTP.

2. FRAUNHOFER

WP6: Cells
Standardized protocols were established for isolation of bone marrow (mouse, rat and sheep origin) and adipose tissue-derived (rat and sheep origin) mesenchymal stem cells (MSCs). Further protocols were established to characterize the obtained cells by flow cytometry and the generation of growth curves over several passages. For the assessment of their cardiomyogenic potential (WP 6) mouse MSCs were analyzed and compared to mouse embryonic stem cells as positive control. These 2D culture experiments did not result in a significant increase in the expression of cardiac markers in mMSCs after different culture conditions and times. Therefore, experiments were started in a small-scale bioreactor providing a 3D microenvironment and mechanical shear stress in order to facilitate differentiation of the adult stem cells towards a cardiac fate (WP 8). The survival of mouse and sheep MSCs, however, was very low under different inoculation densities, stirring rates and oxygen concentrations. Consequently, characterization of cardiomyogenic differentiation under 3D conditions has not been started. Further experiments will be necessary to identify optimal microenvironment and possibly crucial growth factors. These experiments will also include the inoculation of 'floating discs' and submersed (fixated) patches in the bioreactor as proposed. The successful preparation of the first prototype of the bioactive membrane by embedding stem cells in the elastomeric membranes combined with the self-assembling peptide provides the basis for these experiments. So far, protocols have been established to seed and culture cells in the peptide nanofiber scaffold as well as to characterize cell survival and death (WP 8).

3. UPVLC

WP5: Elastomeric Membranes
Two different classes of bioactive patches were developed (WP5), according to their non-degradable (biostable) or semidegradable nature. Both classes were designed bearing in mind the general requirements imposed by the preclinical character of the surgery in the big animal (sheep) model. Basically, these may be summarized as follows:
(i) the patches should act as a cell supply vehicle, in the hope that ATSCs will improve, through different direct and indirect mechanisms, myocardial regeneration (revascularization, motility recovery, remission of scar fibrosis, etc). Thus, a design objective was the capability to host a large number (100 million) of large-sized cells (mesenchymal ATSCs).
(ii) The patches had to cover a significant area of the induced infarct to lend valuable results for human application. Thus, large-area (25 square centimeters) membrane scaffolds had to be developed.
(iii) The above-mentioned amount of cells had to be seeded one day before implantation and cultured within the patch in vitro, and the patches had to ensure viability of the seeded cells once implanted. This cannot be fulfilled unless the patch is immediately invaded by capillaries which make the cells within the scaffold viable; the patch must be capable of immediate vascularization of its inner structure. These requirements summed altogether meant that the patch had to consist of a matrix with large-sized pores with a good interconnectivity controlled to a high degree. Thus, the option for a porogenic-template manufacturing technique was adopted. Sintered microbeads with a narrow distribution of diameters were employed as a polymerization template, and eliminated after polymerization of the matrix precursors in its interstices. Thus, porous structures of high inner regularity, with controlled pore and throat sizes in the desired range could be produced in different chemistries. The procedure had to be repeatedly adjusted and modified to reach a scaled-up production of patches of the desired, unusual sizes, with an adequate quality control ensuring uniformity of their properties to minimize the risk of failure of the animal experiments. Further additional requirements were taken into account:
(iv) good mechanical properties to allow for manipulability in situ during the surgery, and to match as close as possible the elasticity of the living tissue, in order not to impose on the myocardial surface a mechanically strange body which could result in undesired frictional motion. Thus, the option for elastomer, macromolecular-network materials as matrices was adopted. And finally,
(v) the porous membranes thus manufactured had to be filled with the SAP solution, and later seeded with the ATSCs. These requirements were faced by analyzing several variants and choosing on that basis the best options for the filling-and-gelling of the SAP solution within the patch and for the cell-seeding procedure. These choices were fixed in detailed protocols, accurately followed prior to each surgery.

A series of non-degradable patches fulfilling the above requirements was manufactured from poly(ethyl acrylate), PEA, crosslinked polymer.

Non-degradable elastomer patches were developed with the following rationale: first, permanent structures may lend a durable protective microenvironment for the grafted ATSCs, impeding the action on them of macrophages and thus increasing their survival, thus improving over the performance of other frequently used matrices employed for analogous purposes, which degrade too rapidly; second, a stable elastomer membrane, perfectly integrated with the host tissue, may have advantageous restraining effects on ventricle dilation.

The elastomeric PEA polymer was chosen owing to its excellent cell-compatibility properties, its being an elastomer at body temperature, and its capability of yielding scaffolds with the porogen-template technique. PEA scaffolds with interconnected spherical pores whose diameter is about 120 microns were fabricated. The preparation procedure briefly consisted in the polymerization of the monomeric mixture in a porogen template previously obtained by sintering spheres of poly(ethyl methacrylate), PMMA, as porogen. After polymerization, the porogen was dissolved by rinsing in acetone, next exchanged to water, and the scaffolds were dried under vacuum.

Besides the non-degradable ones, a series of semi-degradable patches fulfilling the above general requirements was manufactured with the rationale of having materials which, while initially providing protective environment to the grafted cells, could in time be partially resorbed, leaving a permanent skeleton for mechanical support.

The experience acquired during the surgery of the first animals suggested the development of a material which was more soft, flexible and adaptable to the curvature of the heart surface. These objectives could be fulfilled with the concept of a patch made from interpenetrating polymer networks (IPNs) of crosslinked hyaluronic acid (HA) and crosslinked poly(caprolactone methacryloiloxy ethyl ester), PCLMA; these matrices, abbreviated HA-i-CLMA, were produced in the form of porous membranes with the described sizes and inner structure as explained previously.

The HA-i-CLMA IPNs contain around a 30% wt of PCLMA, which itself is a synthetic elastomer having a non-degradable main chain backbone and a hydrolytically degradable caprolactone (ester) side-chain; HA is a naturally occurring hydrogel, a main component of the extracellular matrix of many tissues, completely degradable by enzymatic action, whose low-molecular degradation products stimulate angiogenesis. In order to combine the essentially hydrophobic CLMA monomer with the highly hydrophilic HA chains a special methodology had to be developed. Moreover, HA binds to CD44+ cells as are ATSCs, which resulted in an excellent cell colonization of the patches manufactured from these IPNs.

The scaffolds obtained were characterized by scanning electron microscopy (SEM), density, porosity, swelling measurements in physiological medium and mechanical tests.

WP7: Bioactive implant development
In order to fill the hydrophobic PEA scaffolds with the self-assembling peptide, SAP, RAD16-I to obtain constructs where the adipose tissue derived stem cells were to be seeded, a loading protocol had to be developed (WP7). The effectiveness of this filling procedure and the gelled state of the SAP gel once inside the scaffold pores was assessed by cryoSEM, by congo red staining and macroscopical observation.

The HA-based semi-degradable patches were easily filled with the SAP solution and seeded with the ATSCs, which rapidly proliferate within the patch in vitro. The bioactive patches possess improved flexibility compared with the biostable, hydrophobic PEA patches, and adapted much better to the heart's surface curvature.

Next, a procedure was set up to assemble the bioactive implants consisting of the scaffold-membranes with pores filled with the self-assembling peptide (SAP) gel entrapping adipose stem cells (ATSCs), to be later implanted in small and big animals.

Preliminary cultures with commercial L929 mouse fibroblasts were employed to check working hypothesis on the assembly conditions and loading of the SAP solution and cell seeding, to ensure the best invasibility and homogeneous cell distribution within the membrane's pores. In these experiments, the pre-loaded SAP solution was found to act as a diffusion medium for cell invasion of the pores of the scaffolds than mere culture medium, and a mechanically-assisted seeding was found to improve the uniformity of cells within the pores.

Next, a set of experiments was designed to refine the optimal parameters for the assembly of the biohybrids, employing adipose-derived stem cells (ATSCs) obtained from biopsies in WP6. The influence of the peptide solution as a cell diffusion medium was checked comparing a prior SAPs loading within the scaffold versus a simultaneous SAPs and cells loading. The seeding was mechanically assisted by different techniques. Immunocytochemistry techniques and fluorescence microscopy were employed to analyze the cell invasion and early distribution before the scale up of the protocol to large biohybrids.

The assembly protocol giving the best results in terms of invasion and homogeneous distribution of the cells in small biohybrids was translated into a protocol for large PEA and HA-i-CLMA membranes for implantation in sheep (WP10). The scaffolds were pre-loaded with the peptide solution following the previously established protocol, seeded internally (100•106 cells per membrane), smoothly shaken and incubated for 24 h. Immunocytochemistry techniques and confocal and scanning electron microscopy were employed to confirm the viability and uniform distribution of cells in implants of PEA and HA-i-CLMA scaffolds.

While providing excellent cell-lodging properties, the HA-i-CLMA IPNs, owing to their high water sorption, were too soft for manipulation with the instruments during the surgical act. This problem was solved with the concept of an external reinforcing grid, made from the same semi-degradable CLMA chemistry as the matrix' second component. This reinforcing grid, or mesh, may represent in a future design an improved variant of the restraining socket concept for ventricular contention. Furthermore, in order to facilitate the positioning of the bioactive patch avoiding premature undesired adherences, a biocompatible poly(caprolactone) envelope was designed and manufactured to contain the bioactive patch while the surgeon manoeuvres with the instruments through the pericardial incision. With its help the patch is approached to the desired placement, and the envelope is then withdrawn leaving the patch in place.

Degradation tests of these materials have been performed in vitro, in different media, and in vivo, in subcutaneous implants in rabbits. Mechanical property testing has been described in deliverables D5.1 and D5.2. The incorporation into the membranes of the SAP gel, and the cell-seeding procedure has been described in deliverables D7.1 (gel filling) and D7.2 D7.3 (cell seeding). These operations were translated into protocols to ensure repeatability. The patches incorporating the peptide gel in the pores permitted a much faster and uniform colonization by the seeded cells than was the case with the same materials without the SAP gel, thus establishing the usefulness of the RECATABI concept.

Thus, the definitive design of the bioactive patches resulted from the interplay of options dictated both by the biological and mechanical requirements of the intended application and by the concrete needs of the surgical technique.

4. IGTP

During the RECATABI project, IGTP group was leading WP3 and WP6 and it has been involved in WP3, WP6, WP8 and WP10.

WP3: Intellectual Property
The main objectives of this workpackage were to implement a comprehensive overview of patentable research results and the alternative options to commercialize the products obtained. In order to achieve these objectives, a direct collaboration with CREASPINE was created. The main products of this work package were the European and American patent.

WP6: Cells
IGTP group is the leader participant of WP6 and has been working in the biological component of the bioactive implant (human and sheep cells).

Main objectives have been achieved in this work package:
- Preparation and validation of standardized protocols for adipose tissue-derived progenitor cell (ATDPC) and bone-marrow mesenchymal stem cell (bmMSC) isolation and culture.
- Compilation of data from superficial antigen profile and proliferative properties analysis.
- Compilation of data from cardiac-specific genes, establishment of a gene profile and protein analysis.
- Creation of a cell bank for use by the consortium.

To date, we have successfully collected 428 samples of subcutaneous adipose tissue from different patients, and 15 samples of mediastinal adipose tissue from sheep undergoing myocardial infarct surgery. Cells were isolated by collagenase digestion and adhered cells were grown in a-MEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin and cultured to subconfluence under standard conditions (37ºC and 5%CO2). Subcutaneous human ATDPCs have a duplication time of 3.1 days, bmMSCs of 4.4 days and sheep mediastinal ATDPCs of 0.7 days.

Immunocytofluorescence, flow cytometry and real-time PCR were performed on human subcutaneous ATDPCs and bmMSCs for superficial and cardiogenic markers characterization. Immunophenotypical characterization of both cell populations revealed a mesenchymal stem cell (MSC)-like pattern. Moreover, RNA and protein analysis in basal conditions showed a slight expression of few cardiac markers.

Human subcutaneous ATDPCs and sheep mediastinal ATDPCs were labelled with fluorescent reporters for further monitor survival and differentiation. Thus, human subATDPCs and sheep mediastinal ATDPCs were transduced using CMV:RLuc:RFP:ttk concentrated lentiviral stock (2x106 transduction units/mL, MOI=21) for 48 hours, as a reporter of cell number. In addition, human subATDPCs were also transduced with lentiviral vector pLox:hcTnIp:PLuc:GFP, as a reporter of cardiomyogenic differentiation. The highest RFP expressing cells were selected by FACS and used for implantation. Cells were then expanded and loaded into the PEA or CLMA membranes as explained in WP7.

WP8: In vitro models
IGTP group has been working on different tasks of WP8 involving in vitro models and equipment development.

Main goals have been accomplished in this work package:
- Non-destructive characterization of bioactive implants during electrocomechanical stimulation protocols using electrical impedance spectroscopy (EIS) techniques.
- Supply of primary cultures of stem cells for testing designed cardiomyogenic protocols in 2D cultures, 3D constructs and bioactive scaffolds.
- Design of electromechanical protocols and enhancement of cell differentiation.

Firstly, our bioengineering partner from the Catalonia Polytechnic University (UPC), designed and implemented a fast EIS multichannel characterization system for in vitro applications (multiple well plate or simultaneous individual culture plates) with a measurement technique based on broadband signals, which allow determining the impedance spectrum in the range of 50O to 10kO from 1 kHz to 1MHz in 1ms. UPC also developed custom software to provide control and user interface functions to the EIS system. This device will allow the in vitro experiments in which test the electrical and/or mechanical stimulation.

Secondly, IGTP tested some in vitro cardiomyogenic differentiation protocols on bmMSCs and subATDPCs. bmMSCs cultured with 5-azacitidine increased Cx43 and β-MHC expression; alternatively, the co-culture of subATDPCs with neonatal rat cardiomyocytes showed no change on gene or protein expression of cardiac markers. Comparison of both cell populations showed subATDPCs as a better candidate for their use in the bioactive implant, as IQS group observed some cardiomyogenic markers increase (GATA-4 and cTnI) in subATDPCs 3D constructs cultured with cardiogenic inductive media (10 µM 5-azacitidine during 15 days).

Thirdly, IGTP developed in vitro electrical, mechanical and electromechanical protocols to test with subATDPCs. Deepen in the effects, electrical stimulation of subcutaneous ATDPCs improved cell alignment and increased important cardiac markers, also appreciated in electrostimulated 3D cultures; on the other hand, cell mechanical stimulation modulated their organization and enhanced few cardiac markers expression as well. Both stimulations evidenced that cell distribution is pattern-dependent and could facilitate intercellular connections.

Finally, some preliminary experiments of electromechanical stimulation were carried out and relevant genetic changes were appreciated in subcutaneous ATDPCS. Latest results are promising, but still preliminary.

In conclusion, electrical, mechanical and electromechanical stimulation achieved some degree of cardiodifferentiation on subATDPCs, making them more suitable for cardiac regeneration approaches. Thus, it seems advisable to use biophysical cell training before delivery as a cell suspension or within engineered tissue.

WP10: Animal models
The aim of WP10 was to assess the efficacy of the bioactive implant in two animal models, mouse and sheep. As far as small animal model is concern, we first evaluated the cardiac function in infarcted mice treated with the bioactive implant and compared them with controls. The purpose of this analysis was to determine if the administration of the bioactive implant (combinations of subATDPCs loaded into the CLMA membrane and peptide hydrogel) could modulate the cardiac function of infarcted mice. Echocardiogram analysis revealed no statistically significant differences in ejection fraction values hence, application of the bioactive implant in the small animal model of myocardial infarction did not improve their cardiac function. Moreover, although histological examination of heart cross sections showed that bioactive implant was nicely attached to the myocardium, morphometric analyses revealed that the scar size was not reduced in animals treated with the bioactive implant compared to controls. Thus, differences in scar size were concordant with cardiac function results. However, our results on cardiac function and morphometry showed that the bioactive implant did not have a detrimental effect on infarcted animals.

An important aspect to evaluate the effect of the bioactive implant in the infarcted animals was the revascularization and repopulation with new cardiomyocytes of the damaged myocardium. To investigate whether subATDPCs promote angiogenesis when administered through a bioactive implant, vessel density at the border of the scar and at distal areas were measured and compared between groups (sham with bioactive implant, control MI, control MI-CLMA and test MI-bioimplant). By using fluorescent isolectin to stain endothelial cells, we found that vessel density was higher in subjacent myocardium tissue sections from hearts with myocardial infarction compared to sham and independent to bioactive implant presence. Thus, the results suggested that the bioactive implant did not promote angiogenesis. Moreover, immunostaining against RFP showed presence of subATDPCs in the bioactive implant 4 weeks post-implantation in all cases. However, migration of subATDPCs into the myocardium was not detected in infarcted or sham animals. Interestingly, presence of human cells expressing cTnI was observed in the bioactive implant. Therefore, the bioactive implant is an appropriated environment for cardiac differentiation.

In our study, we also wanted to determine if subATDPCs administrated through the bioactive implant were able to promote cardiomyocytes' proliferation and thus, induce regeneration of the damaged myocardium. As a result, no proliferating cardiomyocytes were found in any of the sections analysed so; subATDPCs did not present a proliferation induction of cardiac cells. However, we also found interesting to analyse the cells in the bioactive implant. Although we were able to detect presence of cells inside the membrane at 4 weeks post-implantation, their proliferation rate was very low, finding only few diving subATDPCs. Thus, the implanted cells did not proliferate in the bioactive implant and did not promote the proliferation and regeneration of the host cells.

Cardiac and endothelial differentiation of subATDPCs into the bioactive implant after 4 weeks of implantation was also evaluated. To monitor survival and cardiomyogenic lineage differentiation, subATDPCs were transduced with lentiviral vector CMV:RLuc:RFP:ttk for expression of the RLuc:RFP reporter regulated by the CMV promoter, a reporter of cell number. Positively transduced cells expressing RLuc-RFP were selected by FACS and transduced with lentiviral vector pLox:hcTnIp:PLuc:GFP for expression of the PLuc:GFP reporter regulated by the human cTnI promoter, as a reporter of cardiomyogenic differentiation. Transduced cells were then loaded into the bioactive implant and delivered over the infarcted area. The bioluminescent signal allowed an in vivo non-invasive monitoring of the cells, and fluorescence detection was used for cell differentiation analysis at histological level. BLI quantifications showed de novo and progressive increase of cTnI expression in MI and sham-operated animals, being the levels similar in both groups. Hence, the cardiac environment, rather than injure signals, exerted a differentiation effect over the implanted cells. cTnI expression was confirmed at the protein level by immunofluorescence staining. Bioactive implant was fully vascularized and functional vessels traversing the myocardium-bioimplant interphase were also found. Thus, the cells were able to survive into the bioactive implant throughout the experiment. However, subATDPCs were rarely integrated into bioactive implant vessels. As a conclusion, the engineered bioactive implant used in our study confers a suitable environment for subATDPCs to de novo express cTnI when implanted over a mouse infarcted myocardium, being a promising tool for cardiac regeneration purposes.

5. CARDIO-MONDE Assoc.

WP10: Animal Models
The general objective of this workpackage is the assessment of the efficacy of the bioactive implant in animal models.
Large animal model experiments will be carried out in Cardio-Monde and will be reduced and carefully re-designed in order to test an animal model more physiological and biomechanical-similar to humans (to be more predictive to future human use).

Background
Dilated cardiomyopathy produced by myocardial infarction induces geometric remodeling of ventricular cavities, which change from a natural elliptical (conical) shape to a spherical shape. Ventricular chamber dilatation and spherical deformation are important causes of morbidity and mortality of patients with congestive heart failure.

State of the art
Results of cardiac cell therapy showed that cell bio-retention and engraftment within infarct is low and that extracellular matrix degradation contributes to adverse LV remodelling.

Ventricular restraint therapy using polyester mesh wrap and nitinol devices have been used for heart failure patients who develop oversized, dilated hearts due to increased filling pressures. These constraint devices failed to demonstrate clear clinical benefits. Adverse effects like diastolic function restriction and lack of improvement of systolic function, without evidence of myocardial healing open the door to associate a new approach based on biological regenerative therapy and myocardial tissue engineering

Goal of the Study
The goal of this experimental study was to evaluate 'Bioactive Implants' grafted with autologous stem cells in myocardial infarction sheep model. These implants are Bio-Hybrid tissue engineered materials.

MATERIALS AND METHODS
In female 'Ile de France sheep' weighing 36.2 ± 5.3 Kg, through median sternotomy left ventricular (LV) myocardial infarctions were created by surgical occlusion of left coronary artery branches. During the procedure mediastinal fat tissue was removed for isolation and expansion of autologous Adipose Tissue Derived Progenitor Cells (ATDPCs).

Two months later through left thoracotomy the infarcted LV wall was treated either with non degradable or with semidegradable patches seeded with ATDPCs. A 3rd control group underwent myocardial infarction without treatment.

All experiments were performed in accordance with the principles in the use of animals for scientific research of the French National Institute of Health and Medical Research (INSERM), and received care in compliance with the European Conventions. This study was approved by the ethical committee for animal research of the University of Paris Descartes (registered report n° CEEA34.JCC.102.12).

SURGICAL PROCEDURES
1st Surgical Procedure: Experimental myocardial infarction
After preoperative medication with Vetranquil 1 mg/kg (Acepromazine, Ceva, Libourne, France) and induction of anesthesia with Diprivan 6 mg/kg (Propofol, AstraZeneca, Rueil-Malmaison, France), animals were intubated and mechanically ventilated with an Aestiva 5 system (Datex-Ohmeda, Helsinki, Finland). Anaesthesia was maintained with oxygen inhalation of Forene 2-3% (Isoflurane, Abbot, Rungis, France). EKG was monitored during operation, a central venous line was placed through the external jugular vein and blood pressure was assessed using an arterial line from the animal ear. These parameters were recorded using a Philips MP50 IntelliVue Patient Monitor (Philips Medical Systems, Eindhoven, Netherlands).

Median sternotomy was performed and the pericardial sac was opened to expose the heart. To reduce the risk of ventricular fibrillation, a continuous IV infusion (2 mg/kg per hour) of Xylocaine 1% (Lidocaine, AstraZeneca) was performed during the entire surgical procedure.
In all animals an LV myocardial infarction was surgically created by ligation of diagonal branches of the left coronary artery using 5-0 non-absorbable Prolene sutures. Significant EKG changes, including widening of the QRS complex and elevation of the ST segment, and colour and kinetics changes of the area at risk were considered indicative of coronary occlusion. Echocardiography was performed before and after infarction to assess ventricular dimensions and function and to identify the myocardial infarct area. The sterile transducer was placed on the surface of ventricles.

Treatment Groups
After myocardial infarction animals were randomized into 3 groups:

Group 1 (n=5): myocardial infarction without treatment (control group).
Group 2 (n=5): implantation of PEA patch seeded with stem cells onto the infarct.
Group 3 (n=5): implantation of CLMA patch seeded with stem cells onto the infarct.

2nd Surgical Procedure: Treatment with bioactive implants (2 months post-infarct)
General anaesthesia was performed using the same protocol applied for the creation of myocardial infarction. A left thoracotomy was performed at the level of the 5th intercostal space. The pericardium was opened to expose the lateral and anterior left ventricular walls. Pericardial adhesions from the previous operation were divided. Echocardiography was performed to assess ventricular dimensions and function and to identify the myocardial infarct scar.

To reduce the risk of ventricular fibrillation, a continuous IV infusion (2 mg/kg per hour) of Xylocaine 1% (Lidocaine, AstraZeneca) was performed during the entire surgical procedure.

Preparation of Bioactive Implants (scaffold membranes with stem cells)

1st Step: Isolation, culture and transduction of mediastinal ATDPCs from sheep (Cooperation with IGTP, Badalona, SP)

We have successfully collected samples of mediastinal adipose tissue from sheep undergoing myocardial infarct surgery. Cells were isolated by collagenase digestion and adhered cells were grown in a-MEM supplemented with 10% foetal bovine serum and 1% penicillin-streptomycin and cultured to subconfluence under standard conditions (37ºC and 5%CO2). Sheep mediastinal ATDPCs have a duplication time of 0.7 days. Sheep mediastinal ATDPCs were labelled with fluorescent reporters for further histology tracking. Thus, sheep mediastinal ATDPCs were transduced using CMV:RLuc:RFP:ttk concentrated lentiviral stock (2x106 transduction units/mL, MOI=21) for 48 hours. The highest 11% RFP expressing cells were selected by FACS and used for implantation. Cells were then expanded and loaded into the PEA or CLMA membranes.

2nd Step: Preparation of biomaterials and stem cell seeding (Cooperation with UPV and IQS, Spain)

Two different classes of bioactive patches were developed, according to their non-degradable (biostable) or semidegradable nature. The patches consisted in a matrix with large-sized pores with a good interconnectivity controlled to a high degree, obtained by a porogenic-template manufacturing technique. Sintered microbeads with a narrow distribution of diameters were employed as a polymerization template, and eliminated after polymerization of the matrix precursors in its interstices. Thus, porous structures of high inner regularity, with controlled pore and throat sizes in the desired range could be produced in different chemistries.

A series of non-degradable patches was manufactured from poly(ethyl acrylate), PEA, crosslinked polymer. Non-degradable elastomer patches may lend a durable protective microenvironment for the grafted ATSCs, impeding the action on them of macrophages and thus increasing their survival, thus improving over the performance of other frequently used matrices employed for analogous purposes, which degrade too rapidly; moreover, a stable elastomer membrane, perfectly integrated with the host tissue, may have advantageous restraining effects on ventricle dilation.

The PEA polymer was chosen owing to its excellent cell-compatibility properties and mechanical properties. PEA is a hydrophobic elastomer with very good cell-adhesion properties. In order to fill it with the SAP solution (Puramatrix) a gentle vacuum had to be applied. Afterwards, cell seeding was easy and efficient throughout the patch.

Besides the non-degradable ones, a series of semi-degradable patches was manufactured with the rationale of having materials which, while initially providing protective environment to the grafted cells, could in time be partially resorbed, leaving a permanent skeleton for mechanical support. These patches were made from interpenetrating polymer networks (IPNs) of crosslinked hyaluronic acid (HA) and crosslinked poly(caprolactone methacryloiloxy ethyl ester), PCLMA; these matrices, abbreviated HA-i-CLMA, were produced in the form of porous membranes with the described sizes and inner structure as explained previously. The HA-i-CLMA IPNs contain around a 30% wt of PCLMA, which itself is a synthetic elastomer having a non-degradable main chain backbone and a hydrolytically degradable caprolactone (ester) side-chain. These semi-degradable patches were easily filled with the SAP solution and seeded with the ATSCs, which rapidly proliferate within the patch in vitro.

The bioactive CLMA patches possess improved flexibility compared with the biostable, hydrophobic PEA patches, and adapted much better to the heart's surface curvature. While providing excellent cell-lodging properties, these IPNs, owing to their high water sorption, were too soft for manipulation with the instruments during the surgical act. This problem was solved with the concept of an external reinforcing grid, made from the same semi-degradable CLMA chemistry as the matrix' second component. Furthermore, in order to facilitate the positioning of the bioactive patch avoiding premature undesired adherences, a biocompatible poly(caprolactone) envelope was designed and manufactured to contain the bioactive patch while the surgeon maneuvers with the instruments through the pericardial incision. With its help the patch is approached to the desired placement, and the envelope is then withdrawn leaving the patch in place.

3rd Step: Surgical implantation of biomaterials onto the myocardial infarct scar
The scaffolds membranes measuring 45x45 mm and charged with the stem cells were placed onto the left ventricular surfaces. The infarct regions were largely covered by the patches, which were fixed to the heart with interrupted superficial sutures (Prolene 5-0) placed at the periphery of the implants. Afterwards the pericardial sac was closed using interrupted biodegradable sutures (PDF Ethicon).

At the end of the operation, a chest drain was placed into the pleural cavity. Thoracotomy was then closed and the sheep ventilated until recovery. The drain was removed as soon as the sheep started spontaneous respiration. Postoperative sheep treatment consisted in cefazolin antibiotic injected intramuscularly at 1 g per day over 5 days.
3rd Surgical Procedure: Final evaluation at 6 months post-infarct (echocardiography, MRI magnetic resonance imaging, histopathology)

General anaesthesia was performed using the same protocol described for the creation of infarcts. A right thoracotomy was performed at the level of the 5th intercostal space. The heart was exposed after division of pleural and mediastinal adhesions from the previous operations. Echocardiography was then performed with the transducer positioned onto the heart and patch surfaces. Complementary MRI studies were performed; an intravenous injection of gadolinium solution at the dose of 0.4 ml/kg was performed 15 minutes before heart removal to visualize the infarcted myocardial tissue. Thereafter animals were sacrificed by lethal IV injection of Dolethal (Pentobarbitone sodium 200 mg/L, solution for injection for euthanasia). Heart harvesting was the last procedure.

EVALUATION
Echocardiography Images Acquisition
A MyLab25Gold Cardiovascular ultrasound system (Esaote-Kontron SPA, Firenze, Italy) equipped with a phased-array transducer set to 1-5 MHz was used to acquire echocardiographic images. The transducer was placed on the heart surface. Ultrasound scans for each sheep included: 1) apical 2-chamber and 4-chamber views for measurements of LV cardiac output, end-diastolic volume (EDV), end-systolic volume, stroke volume, and ejection fraction (EF); 2) Doppler spectral flow for peak early (E-wave) and atria (A-wave) filling velocities; and 3) tissue Doppler for peak velocities of the mitral annulus in early (E') phases of LV filling (measured at the basal medial portion of the mitral annulus), and the dimensionless ratio E/E' was computed. For each scan, 1 cardiac cycle was acquired at a frame rate of 60-70 Hz. To assess the distortion of ventricular wall after infarction we evaluated longitudinal, radial and circumferential strain.

Magnetic Resonance Imaging (MRI)
Magnetic resonance imaging techniques were used to assess accurately infarcted myocardial areas. Acute and chronic myocardial infarctionshave been demonstrated as hyperenhanced regions on delayed contrast-enhanced MRI (DE-MRI). Extracellular MR contrast-media agents have been used for the diagnostic of infarcted myocardium. Gadolinium produces hyperenhancement on the scar tissue, producing high signals in white tonality. A BioSpec 47/40 USR system (Bruker Corp., Karlsruhe, Germany) was used to perform MRI studies. Immediately after autopsy, the isolated sheep hearts underwent MRI to evaluate the size and evolution of the ischemic lesions, the integration of bioactive implants onto the ventricular wall and to assess the infarct size related to myocardial mass (gadolinium white marked areas).

Histological Studies
At postinfaction month-6, all sheep were sacrificed after echocardiography assessment and MRI studies. The site of myocardial injury was identified and dissected. Four to 5 specimens were used, and each of these was fixed in 10% formalin, embedded in paraffin and sectioned to yield 10-µm-thick slices. The sections were stained with hematoxylin, eosin and saffron. For the identification of grafted stem cells into the patches and the myocardium, heart slices were tested with fluorescence microscopy and DAPI staining. Scaffolds membranes were evaluated with confocal and electron microscopy.

Statistical analysis
Results were analyzed and reported as percentage or mean ± standard deviation (SD). Student's t-test was used to compare the groups. A value of p less than 0.05 was considered to be statistically significant.

RESULTS
One animal died during the infarct procedure due to irreversible ventricular fibrillation. Two animals were excluded from the study due to skin and mediastinal infections. In summary, 15 sheep survived the procedures (5 for each study group) and were evaluated at long term.

Stem Cell cultures: The results of in-vitro cell expansion allowed obtaining 100+/-7 million ATDPCs for each animal.

Echocardiography
LV function and dimensions were quantified the day of myocardial infarction (at baseline and after infarct), at 2 months during the implantation of the patch and at 6 months of infarction.

At baseline and after infarction, the mean LV DV (diastolic volume) and EF (ejection fraction) were similar in all three groups. Over the course of 6 months, each group increased their LV end diastolic dimension compared with their baseline value. A statistically significant attenuation of LV dilation was demonstrated with the implantation of PEA and CLMA patches compared with the non-treated control group (not shown). Functional results at 6 months showed significantly greater benefits in the hearts treated with CLMA patches compared with those receiving PEA patches or without treatment. Diastolic function measured by Doppler-derived mitral deceleration time (DT) improved in the CLMA group but not in the other groups.

Histopathology
Ventricular Wall: The surgical coronary artery ligation model used for this study results in transmural necrosis of the LV wall, with central fibrosis, intermediate borderzone, all surrounded by healthy myocardium. In the bioactive patch treated sheep the necrotic areas were much less prominent, mainly in the group treated with CLMA. It was found fluorescent marked cells into the patch and into infarct scars as well as focuses of angiogenesis (capillary network). These findings seem to indicate that adipose progenitor stem cells growing in bioactive implants and migrating to the nearby cardiac tissue can be involved in a regenerative process, promoting angiogenesis and possibly cardiomyogenesis.

Interface: At the level of the epicardium we found minimal fibrosis interface without inflammation between both bioactive implants and the heart surface. The CLMA patch was completely anchored and integrated to the nearest infarct area and the myocardium. In contrast, the PEA patches showed poor adhesions to the heart and the infarct scar.

Magnetic Resonance Imaging
Left ventricular myocardial mass and infarct size (in white gadolinium stain) were evaluated using longitudinal and transversal MRI sections. The volume of the infarct scars related with the ventricular mass was calculated (data not shown). CLMA patches were more adherent and integrated to the ventricular wall compared with PEA patches. Specimens treated with bioactive implants showed no inflammatory activity.

CONCLUSIONS
We demonstrated the feasibility and safety of Bioactive Implants for the treatment of myocardial infarction in large animal models. Bioactive implants improved systolic and diastolic functions, reducing adverse cardiac dilatation. Better results were obtained using semidegradable materials (CLMA), no inflammatory reaction was observed after long-term implantation of both materials. Grafted marked stem cells were observed into bioactive implants and into infarcted myocardium after 4 months of implantation.

PERSPECTIVES and CLINICAL IMPLICATIONS
Bioactive molecules and nanobiotechnologies may contribute for the creation of a new 'Bioprosthesis' for ventricular support and myocardial regeneration. Future research should include the creation of semi-degradable 'ventricular support bioprostheses' designed with the concept of helical myocardial bands and models for left or right ventricle diseases with controlled degradation in response to the physiology of the left or right heart. Ventricular bioprostheses derived from bioactive implants may reduce the risk of death or heart failure (HF) progression. Long-term effectiveness studies of bioactive implants evaluating the efficacy to prevent HF progression are needed to guide future clinical translation.

6. CREASPINE

WP11: Dissemination/Exploitation
During the project Creaspine was focused on 3 items: 1- define and potentially execute an IP strategy 2- develop tools and plans to disseminate the relevant information generated by the consortium members and 3- organise the ethical work for the study. As a very first step, Creaspine members spent some time at Cardio-Monde to update/complete their understanding of the issue to be addressed. Thanks to their previous patenting experiences and with the professional guidance of the expert patent attorney, Creaspine members developed strategy options to optimize the protection of the outcome of the research program, exposed them and executed the option selected by the group, including the design and execution of a Joint Ownership Agreement that will survive beyond the period of the program. Creaspine was involved as well in the design of a website considered as the most appropriate tool in the early phase for communication within the consortium and outwards.

Thanks to previous experience, Creaspine handled the organization of the ethical surveillance of the research work and designed simple SOPs for the consortium. As to anticipate on future tasks, Creaspine attended a training session on ATMPs in order to draft an early Regulatory strategy and to educate the RECATABI members on this critical issue that may influence later decision and study protocols. As the IP looks secured, Creaspine members started promoting the concept to the Industry community.

Potential Impact:
Heart failure (HF) is a major public health problem in developed countries. In the United States, approximately 5 million patients have HF, and more than 550,000 patients are diagnosed with HF for the first time each year. The incidences of HF approaches 10% of the population after age 65 and approximately 80% of patients hospitalized with HF are older than 65 years. Furthermore, HF is the most common Medicare diagnosis-related group (i.e. hospital discharge diagnosis), and more money is spent for the diagnosis and treatment of HF than for any other diagnosis. Patients with heart failure, particularly those after a myocardial infarction, have a poor prognosis. Initial small recovery due to scaring shortly results in chronic increase of blood pressure eventually leading to death.

The therapeutic success of the project was accomplish by developing beyond- state-of-the-art nanoscale engineered biomaterials and scaffolds to obtain a smart bioactive implant with capacity of triggering functional recovery at the local tissue environment, thereby guiding changes in cellular responses at the molecular level. Basically, this was based in the hypothesis that induction of angiogenic processes and delivery of trained stem cells (biophysically and biomechanically) at the necrotic tissue would enhance intrinsic tissue neo-formation. Surprisingly, the local treatment of the scar in a large animal model (sheep) induced enhanced functional recovery at the entire ventricle evidenced by monitoring during treatment time three main important parameters in heart function, ventricle dilatation, ejection fraction and de-acceleration time. This clearly indicates that the single patch treatment was good enough to induce a tissue response promoting better heart performance.

Moreover, involving excellent Research Centres, Hospitals and a Company in the consortium we ensured the integration and dissemination of the produced patents with the clear outcome of bringing a solution for recovery to the most prevalent cause of disability and death in Europe. We believe that we were able to develop with RECATABI the platform of a novel therapeutic option for patients with chronic myocardial postinfaction scars. The bioactive implanted was able to integrate in the damaged myocardium, contract synchronously with the adjacent healthy heart assisting to increase in the contractile forces in a large animal model. Future research should include the development of semi-degradable 'ventricular support bioprostheses' derived from our bioactive implants, designed with the concept of helical myocardial bands and models for left or right ventricle diseases with controlled degradation in response to the physiology of the left or right heart. We expect that our platform once in human patients would provide better quality of life and longer life expectancy.

List of Websites:
http://www.recatabi.com/

Related documents

• Final Report - RECATABI (Regeneration of Cardiac Tissue Assisted by Bioactive Implants)