Polymeric scaffold for myocardial regeneration and protection

Abstract

Polymeric scaffold (10) for myocardial regeneration and protection, comprising a support structure (20) comprising at least a first and a second support layers (30a; 30b) and at least one inner layer (33) interposed between said first and second support layers (30a; 30b), wherein said first and second support layers (30a; 30b) comprise at least one polymeric material selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), poly(dioxanone) (PDS), polycaprolactone (PCL), and polyhydroxyalkanoates (PHA), preferably poly(3-hydroxybutyric-co-3-hydroxyvaleric acid), optionally in combination with at least one biological polymer. At least one of said support layers (30a; 30b) further comprises at least one organic and biodegradable semiconducting material, preferably an organic and biodegradable semiconducting peptide. The at least one inner layer (33) comprises a hydrogel, preferably containing an organic and biodegradable semiconducting material, more preferably an organic and biodegradable semiconducting peptide. The support layers (30a; 30b) furthermore comprise a surface sculpture, said surface sculpture comprising a plurality of cavities (41) arranged according to a regular frame, said cavities (41) being defined by an array of longitudinal linear projections (40a, 40b) and an array of transverse linear projections (40c) intersecting each other respectively, said first and second support layers (30a; 30b) having cavities (41) respectively facing opposite sides of the support structure (20).

Description

[0001] “Polymeric scaffold for myocardial regeneration and protection”

[0002] Field of the invention

[0003] This disclosure generally refers to a device for use in tissue engineering. More specifically, the disclosure refers to a polymeric scaffold which, by virtue of its particular geometry and chemical composition, proves to be highly effective in the cardiac tissue regeneration and protection, in particular following myocardial infarction.

[0004] Background of the invention

[0005] The leading cause of death from cardiovascular disease (CVD) is coronary heart disease which accounts for 43% of CVD deaths (WORLD HEALTH STATISTICS 2015, https: / / www.who.int / docs / default-source / gho-documents / world-health-statistics- reports / world-health-statistics-2015.pdf). Current therapy for patients with CVD who develop acute myocardial infarction (approximately 1 million patients / year in Europe) is based on the administration of drugs, coronary artery bypass grafting and angioplasty. These therapies, however, are not able to restore the complete functionality of the damaged myocardial tissue.

[0006] An effective intervention in such a context could instead derive from a therapeutic approach capable of stimulating autonomous myocardial regeneration and, at the same time, protecting against post-infarction ventricular myocardial remodeling. The process defined as “ventricular remodeling” consists of a morphological and functional alteration which is achieved through a progressive increase in the cavity volumes of the left ventricle, accompanied by modifications of the global and segmental geometry of the chamber itself. This can progress to heart failure, a clinical condition in which the heart is unable to pump an adequate amount of blood to meet the body’s needs.

[0007] The engineering of myocardial tissue is considered one of the most promising strategies to efficiently treat cardiac pathologies, in particular with reference to the use of biomaterials suitable for implantation directly in the cardiac site to be treated. In this regard and as an example, it is estimated that, in the year 2017, the patients candidate for a surgical treatment who could have benefited from a scaffold for myocardial regeneration and protection were over 200,000 in Europe alone (Health at a glance. Europe 2018. European Commission). Globally, more than 1.5 million potential patients can be estimated to be targeted by this technology each year in countries with sufficient purchasing capacity (Heart Disease and Stroke Statistics-2020 Update: A Report From the American Heart Association. Circulation 2020; 141 : el39-e596).

[0008] A crucial role played by a cardiac scaffold is to provide mechanical support to damaged myocardial tissue and at the same time instruct resident or circulating cells to correct morphogenesis. Although a large variety of polymeric materials have been explored, the optimal scaffold for engineering cardiac muscle tissue has not yet been identified; the ideal cardiac scaffold has a broad repertoire of requirements that a system designed to replace cardiac extracellular matrix (ECM) structure and function must possess. Although in recent years many biophysical and biochemical signals have been studied to obtain better control over cellular processes (cell adhesion, migration, division and differentiation), some important questions associated with this strategy still remain open (Xu, Bioactive Materials, 2016).

[0009] Considering a market analysis, “cellularized” products are based on an “ / / / vitro approach” which requires a cell culture phase and the use of allogeneic stem cells. However, the use of cellularized scaffolds presents some critical issues related in particular to potential risks of rejection following implantation in the host. Furthermore, compared to “acellularized” scaffolds, the production processes of cellularized scaffolds are more complex due to the cell culture phase before implantation and the more difficult methods of conservation and transport of the scaffold itself. Furthermore, these scaffolds are not designed for cardioprotection or to counteract cardiac remodeling.

[0010] The scenario of acellularized cardiac scaffolds is mainly characterized by products whose constitutive materials are not biologically active and only offer mechanical support, such as the non-biodegradable polymeric ones used in the treatment of ventricular aneurysm to prevent the rupture of the thinned ventricular wall in the lesion area. A subsequent progress occurred with the use of acellularized biological scaffolds of animal origin, produced from the extracellular matrix (ECM) of bovine or porcine pericardium, or pig intestine. Their main area of application is congestive heart failure and they are implanted during surgery. They are marketed as structural supports, although recently some experimental data suggest that they may represent scaffolds biologically favoring cellular incorporation. These completely biological scaffolds have the limitation of poor mechanical robustness; in fact, during the decellularization process, the ECM becomes fragile and is therefore unsuitable for supporting the mechanical stress of an epicardial implant. Other important limitations of biological matrices are the potential immunogenicity, the tendency to calcifications, the possibility of contamination by toxic residues derived from the decellularization process. Finally, these scaffolds do not offer any protective effect against post-infarction myocardial remodeling.

[0011] Other microfabricated bioartificial polymeric scaffolds that imitate the anisotropic structure and mechanical properties of the myocardium capable of communicating, at a molecular level, both with mesenchymal stem cells and with cardiac progenitors and nonmyocyte cardiac cells in a precise and controlled way, such as to reproduce the interactions existing between cells and native ECM, are disclosed in the document W02014108814A1. The results obtained in vitro with these scaffolds can allow a concrete technological transfer on industrial scale; the clinical success of these devices, however, depends on their ability to effectively recruit resident precursors, including vascular ones, and to induce their complete differentiation in order to guarantee electromechanical coupling with the surrounding tissue and reduce ventricular remodeling.

[0012] In light of the aforementioned evidence, there is a need to develop new polymeric scaffolds for the cardiac tissue regeneration and protection that overcome the critical issues and limitations associated with known devices.

[0013] Summary of the invention

[0014] The object of this disclosure is to provide a polymeric scaffold effective in the cardiac tissue regeneration and protection, in particular following myocardial infarction.

[0015] This scaffold has improved mechanical and electrically conductive capabilities compared to known scaffolds; it is also characterized by an optimized angiogenic, cardioinductive and cell recruitment effect and by the ability to limit ventricular remodelling. It can be used to promote the cardiac tissue regeneration and revascularization both after acute and chronic infarction.

[0016] Furthermore, this scaffold can be used for a controlled release of active ingredients (drugs) which not only have cardioprotective activity against reperfusion damage or which are able to promote cell recruitment, but which can also perform anti -fibrotic, antiinflammatory, anti-apoptotic / anti-necrotic, pro-angiogenic and pro-regenerative functions, which improve the contractile capacity of the myocardium and limit tissue remodeling, also in order to treat a series of other genetic or acquired pathologies that alter the cardiac tissue. The aforementioned purpose is achieved thanks to the subject-matter specifically referred to in the following claims, which are intended as an integral part of this disclosure.

[0017] In particular, this disclosure refers to a polymeric scaffold for myocardial regeneration and protection, comprising a support structure which comprises at least a first and a second support layers and at least one inner layer interposed between said first and second support layers,

[0018] - wherein said first and second support layers comprise at least one polymeric material selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), poly(dioxanone) (PDS), polycaprolactone (PCL), polyhydroxyalkanoates (PHA), among the polyhydroxyalkanoates (PHA) preferably poly(3-hydroxybutyric-co-3- hydroxyvaleric acid), optionally in combination with at least one biological polymer,

[0019] - wherein at least one of said support layers further comprises at least one organic and biodegradable semiconducting material, preferably an organic and biodegradable semiconducting peptide,

[0020] - wherein said at least one inner layer comprises a hydrogel, preferably comprising at least one organic and biodegradable semiconducting material, more preferably one organic and biodegradable semiconducting peptide.

[0021] The support layers comprise a surface sculpture which includes a plurality of cavities arranged according to a regular frame, said cavities being defined by an array of longitudinal linear projections and an array of transversal linear projections intersecting each other respectively, said first and second support layers having cavities respectively facing opposite sides of the support structure.

[0022] In one or more embodiments, the support structure can further comprise at least one intermediate layer, said intermediate layer comprising at least one polymeric material selected from the group consisting of polydioxanone (PDS), polyhydroxyalkanoates (PHA), and polycaprolactone (PCL), and optionally at least one organic and biodegradable semiconducting material, preferably an organic and biodegradable semiconducting peptide.

[0023] The polymeric material of the at least one support layer and of the at least one intermediate layer is a biodegradable material.

[0024] The intermediate layer can comprise at least one polymeric material that biodegrades more slowly than the polymeric material contained in at least one of the first and second support layers. In particular, for example, the at least one support layer can comprise PLGA optionally in combination with a biological polymer and the intermediate layer can comprise a polymer which has a lower degradation rate than PLGA, preferably at least one polymer selected from polydioxanone (PDS), polycaprolactone (PCL), polyhydroxyalkanoates (PHA), preferably poly(3-hydroxybutyric-co-3 -hydroxy valeric acid). In this way, the intermediate layer will degrade more slowly than at least one or both support layers, so that the scaffold will release active ingredients / MIPs gradually.

[0025] In one or more embodiments, the support structure can comprise a first and a second inner layers, and the intermediate layer can be interposed between the first and second inner layers.

[0026] The at least one biological polymer optionally included in at least one of the two support layers can be selected from the group consisting of gelatin, hyaluronic acid, collagen, gellan.

[0027] The hydrogel of the at least one inner layer can comprise at least one biological polymer which can be selected from the group consisting of gelatin, hyaluronic acid, collagen, gellan.

[0028] The organic and biodegradable semiconducting material can be selected from the group consisting of self-assembling N-fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) peptides, self-assembling N-fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) peptides functionalized with carbohydrate moieties, Fmoc-FF-glucosamine- 6 sulfate (Fmoc-GlcS) or Fmoc-FF-glucosamine-6 phosphate (Fmoc-GlcP), combinations thereof.

[0029] Advantageously, the support structure further comprises molecularly imprinted particles included in at least one of the two support layers and / or in at least one intermediate layer. These particles can be accommodated in one or more of the cavities of the support layers and / or be located on the projections of the support structure.

[0030] Such molecularly imprinted particles can be selective for at least one enzyme belonging to the metalloprotease (MMP) family, preferably selected from the group consisting of MMP-9, MMP-8, MMP -2, MMP-14 or TGF[3, thrombospondins (TSP-1, TSP-2, TSP-3).

[0031] Furthermore, such molecularly imprinted particles can comprise at least one water- soluble polymer preferably selected from the group consisting of polyvinylpyrrolidone (PVP), poly-n-isopropylacrylamide copolymers (PNIPAAm). These particles can have a diameter between 20 nm and 2 pm, preferably between 80 nm and 700 nm.

[0032] In one or more embodiments, the scaffold can further comprise at least one active ingredient which can be included in at least one of the support layers and / or in the at least one inner layer, and / or in the at least one intermediate layer.

[0033] The active ingredient may be encapsulated in particles which can comprise at least one biodegradable polymer preferably selected from the group consisting of polyhydroxybutyrate (PHB), poly-3 -hydroxybutyrate (P3HB) and related copolymers. The diameter of the particles can be between 20 nm and 2 pm, preferably between 90 nm and 700 nm. Such particles can be accommodated in one or more of the cavities of the support layers or on at least one projection of the support layers.

[0034] Furthermore, in one or more embodiments, the at least one intermediate layer may comprise at least one preferably anti-fibrotic active ingredient and at least one molecularly imprinted particle.

[0035] Preferably, the scaffold of this disclosure may be an acellular scaffold. The absence of an in vitro culture phase presents a series of advantages that make this specific solution extremely attractive and more rapidly applicable for transfer to a biomedical industry, in particular since: 1) the use of the scaffold is independent from the individuality of the patient; 2) once produced, the scaffold can be immediately implanted (the time for in vitro culture of the cells is not necessary); 3) transport and storage are simpler than those of cellularized scaffolds; 4) in the absence of cellular, therefore vital, components, the reproducibility of the product in an industrial context is significantly favored.

[0036] The results of a physico-chemical, morphological, mechanical and functional characterization have demonstrated for the scaffold of this disclosure a mechanical anisotropy typical of a cardiac tissue, good mechanical properties, an optimization in terms of conductivity, a controlled release of active ingredients included in the layers of the assembled system and / or within microfibers and micro-nanoparticles and the possibility of incorporating molecularly imprinted micro-nanoparticles (MIPs) selective for molecules and enzymes involved in the alteration of the matrix leading to ventricular remodeling.

[0037] Advantageously, the scaffold of this disclosure has a different composition of its layers and consequently a different degradation rate thereof, as described below.

[0038] Brief description of the figures

[0039] One or more embodiments will be described, purely by way of non-limiting example with reference to the attached figures, listed below.

[0040] - Figure 1 is a top view of a polymeric scaffold according to embodiments of this disclosure;

[0041] - Figure 2 is a cross-sectional view of a polymeric scaffold according to embodiments of this disclosure;

[0042] - Figure 3 is a cross-section view of a further embodiment of the polymeric scaffold of this disclosure;

[0043] - Figure 4 schematically represents the molecular mechanisms of action of a polymeric scaffold according to the described embodiments;

[0044] - Figure 5 shows the cellular adhesion of non-myocyte cardiac cells (NMC) on the scaffold of this disclosure;

[0045] - Figure 6 shows the cytocompatibility of cells (H9c2 and hMSC) cultured in the presence of nanoparticles (MIP-PVP or PHB) and in the presence of scaffolds of this disclosure;

[0046] - Figure 7 shows the migration of NMCs to the scaffolds of this disclosure;

[0047] - Figure 8 shows the changes in gene expression of NMCs cultured on scaffolds for 7 days;

[0048] - Figure 9 shows the changes in gene expression of iPSCs cultured on scaffolds for 7 days;

[0049] - Figure 10 shows the colonization of the scaffold of this disclosure by the cell sheets;

[0050] - Figure 11 shows the synchronization of the beating of the iPSC-CMs on the scaffold of this disclosure compared to the control;

[0051] - Figure 12 shows the quantification of troponin and connexin 43 in hearts explanted from rats subjected to the different experimental protocols;

[0052] - Figure 13 shows the density of vessels in hearts explanted from rats subjected to the different experimental protocols;

[0053] - Figure 14 is relative to FT-ir Chemical Imaging. Optical image of the surface of PLGA / Gelatin / Fmoc-FF (A.) and its corresponding Average spectrum (B.); - Figure 15 shows a phase-contrast imaging of hiPS-CMs seeded on either PLGA / gelatin or PLGA / gelatin / Fmoc-FF patches for 30 days;

[0054] - Figure 16 refers to a cell elongation analysis. The percentage of elongation of H9C2 cardiomyoblasts cultured on either PLGA / gelatin or PLGA / gelatin / Fmoc-FF patches is shown (mean ± SEM, n=50, **p<0.01; ***p<0.001). Data are normalized to the 2D control (dashed line);

[0055] - Figure 17 refers to cardioinductivity analysis. Fold change gene expression of the sternness marker c-KIT and the cardiomyogenic transcription factors. GATA4, MEF2C and NKX2.5 was assessed through real-time PCR experiments in hMSCs cultured for two weeks on either PLGA / gelatin or PLGA / gelatin / Fmoc-FF patches (**p<0.01 between the two scaffold types; ##p<0.01, ###p<0.001 with respect to 2D control). Data are normalized against the 2D control.

[0056] Detailed description

[0057] In the following description, numerous specific details are provided to permit a complete understanding of the embodiments. The embodiments may be practiced without one or more specific details, or with other processes, components, materials, etc. In other cases, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments.

[0058] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular function, structure, or feature described in connection with the embodiment is included in at least one embodiment. Therefore, the appearances of the phrases “in one embodiment” or “in an embodiment” at various points throughout this specification do not necessarily refer entirely to the same embodiment. Furthermore, the particular functions, structures or features may be combined in any suitable manner in one or more embodiments. The headlines are provided herein for convenience only and do not construe the scope or meaning of the embodiments.

[0059] The polymeric scaffold disclosed in this disclosure derives from the implementation of a scaffold previously developed, that is the subject-matter of the document W02014108814A1 referred to in the previous sections.

[0060] The scaffold of this disclosure combines key elements (such as the geometry and specific components of its layers) in a single device for the complex control of the regenerative process of cardiac tissue and post-infarction remodelling. The use of the materials described below and the immediate applicability of the device which avoids delays due to cell culture represent the main advantages of the scaffold.

[0061] The scaffold of the present invention can appear as an acellularized scaffold and this means that the scaffold may not contain stem or adult cells (which would require cultivation phases of the same), thus favoring industrial applicability. However, in order to allow cardiomyocyte differentiation of resident cells or cells recalled in situ, and to increase the probability of cardiac tissue regeneration, the scaffold was designed considering the need for spatial and temporal control involving various elements, such as stem cells and biochemical signals of cellular differentiation, but also the cardiac extracellular matrix (ECM) and this in order to restore healthy tissue after a pathological change so as to make the ventricular remodeling processes reversible.

[0062] In order to stimulate the process of cellular differentiation by the scaffold, the Inventors of the present application have combined a series of elements such as for example i) the use of an organic and biodegradable semiconducting material, for example a semiconducting and biodegradable peptide (for example Fmoc-FF); ii) the use of gradual release active ingredients such as pleiotropic molecules, for example apelin-13, neuregulin-1 (as detailed below).

[0063] The Inventors have also observed that optimal results are obtained when such molecules of active ingredients are incorporated into particles (possibly in addition to being loaded directly into one or more layers of the scaffold) in order to increase their long-term bioavailability and counteract their degradation with loss of activity.

[0064] A further objective achieved by the scaffold of this disclosure concerns the need to reduce the remodeling that occurs after the infarction. In fact, after an infarction, an altered balance between some metalloproteinases (MMPs), such as MMP-9, MMP-2, and their inhibitors is observed in the cardiac ECM. Current therapies use drugs that reduce in vivo concentrations of MMPs but in a non-specific manner. The Inventors of the present application have used molecular imprinting nanotechnology for a more specific and selective in vivo removal of MMPs at the cardiac ECM. In particular, the system selected for molecular imprinting to specific molecular signals (e.g. MMP-9) capable of preventing left ventricular remodeling was based on the polymerization of methacrylic acid in the presence of polyvinylpyrrolidone (PVP), a synthetic polymer having hydrophilic properties and structure similar to the biological protein component, as described below.

[0065] A further molecularly imprinted system was based on the polymerization of methacrylic acid in the presence of the thermoreversible and bioresorbable copolymer poly-(N isopropylacrylamide-2-hydroxyethyl methacrylate-6-hydroxyhexanoate) to the same molecular signals. Both PVP -based and PNIPAAm-based nanoparticles can be obtained both in the form of nanospheres (with a dense structure) and in the form of nanocapsules (with a hollow structure) in order to simultaneously release an active ingredient (drug, nucleic acid or peptide) encapsulated inside.

[0066] In Figure 1, the reference number 10 indicates a polymeric scaffold according to the invention seen from a top view. In the figure, a support structure indicated with the reference number 20 which includes the support layer 30a made of polymeric material and the particular geometric characteristics which - in combination with the specific chemical composition of the layers - make the scaffold 10 surprisingly effective in cardiac tissue protection and regeneration, can be seen.

[0067] As illustrated in Figure 2, in a cross-sectional view of a scaffold of this disclosure, (for example along the II-II plane of the embodiment of the polymeric scaffold 10 of Figure 1) the support structure 20 includes in addition to the first support layer 30a, a second support layer 30b and an inner layer 33 interposed between the first and second support layers 30a and 30b.

[0068] The support layers 30a and 30b include a surface sculpture characterized by the presence of a plurality of cavities 41 which are arranged according to a regular frame. These cavities are defined by an array of longitudinal linear projections 40a, 40b and an array of transverse linear projections 40c intersecting each other respectively, as illustrated for example in Figure 1. The two support layers have the cavities 41 facing respectively towards opposite sides of the support structure 20, in particular towards the outside of the scaffold. The two support layers are preferably the same in geometric structure and composition.

[0069] In one or more embodiments, the total thickness of the single support layer can be between 40 pm and 250 pm.

[0070] The cavities 41 have a longitudinal dimension and a transversal dimension, which in Figure 1 are respectively indicated by L and Wi. Preferably, the cavities 41 have a longitudinal dimension L included in the range between 350 pm and 600 pm and a transversal dimension Wi included in the range between 30 gm and 150 gm. The cavities 41 are characterized by a depth which is preferably included in the range between 20 pm and 100 pm. These dimensions are preferred because they easily accommodate the particles described below.

[0071] The cavities 41 have a substantially rectangular shape. However, the scope of the invention includes alternative embodiments, which are not specifically illustrated in the figures, in which the cavities 41 have different geometric shapes, which can be substantially regular or even irregular.

[0072] As it can be clearly seen in Figure 1, the cavities 41 are defined by an array of longitudinal linear projections 40a, 40b and an array of transverse linear projections 40c, respectively intersecting with each other. According to the embodiment illustrated in Figure 1, there are two different types of longitudinal projections: wide projections 40b and narrow projections 40a, which are arranged in an alternating succession. The wide projections 40b are characterized by a transverse dimension W2 which is preferably but not limited to the range of 50 gm- 160 gm; the narrow projections 40a are characterized by a transversal dimension W3 which is preferably but not limited to the range of 20 gm- 120 gm, with the condition that W2 is greater than W3. For example, in the alternating succession of wide projections 40b and narrow projections 40a, one wide projection 40b can be provided for every two narrow projections 40a.

[0073] In addition to comprising two support layers 30a and 30b and at least one inner layer 33, the scaffold according to one or more embodiments can also comprise at least one further layer, the intermediate layer 34.

[0074] As illustrated in Figure 3, in the cross-sectional view of the scaffold according to one or more embodiments, the support structure 20 includes two support layers 30a and 30b, two inner layers 33a;33b and an intermediate layer 34 which is interposed between these inner layers. The two inner layers 33a;33b preferably have the same composition.

[0075] With reference to the chemical composition of the scaffold of this disclosure, the polymeric material of the scaffold can be biodegradable.

[0076] The first and second support layers 30a;30b comprise at least one polymeric material selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), poly(dioxanone) (PDS), polycaprolactone (PCL), polyhydroxyalkanoates (PHA), preferably poly(3-hydroxybutyric-co-3 -hydroxy valeric acid) among the polyhydroxyalkanoates, optionally in combination with at least one biological polymer.

[0077] The biological polymer can be selected from the group consisting of gelatin, hyaluronic acid, collagen, gellan. Preferably, the support layers comprise a combination of PLGA and gelatin.

[0078] Advantageously, moreover, at least one of the support layers 30a; 30b comprises an organic and biodegradable semiconducting material, preferably an organic and biodegradable semiconducting peptide.

[0079] With reference to the inner layer for example illustrated with reference number 33 in Figure 2 (or with reference to the two inner layers 33a and 33b illustrated in Figure 3), it comprises a hydrogel. The hydrogel may comprise a biological polymer, wherein the biological polymer may be selected from the group consisting of gelatin, hyaluronic acid, collagen, gellan.

[0080] The hydrogel can comprise a liquid phase (water) in a quantity by weight between 3% and 90%, preferably between 5% and 40%.

[0081] In one or more embodiments, the inner layer hydrogel may further comprise at least one organic and biodegradable semiconducting material, preferably an organic and biodegradable semiconducting peptide.

[0082] The concentration of the hydrogel comprising the semiconducting material can be between 1 mM and 100 mM.

[0083] The inner layer comprising the hydrogel has multiple functions which may include: 1) it can function as a reserve of drugs and active ingredients; 2) it can present an electrically conductive capacity in the entire section of the scaffold; 3) it can promote the adhesion of the inner layers and the intermediate layer 34 for the assembly of the entire scaffold.

[0084] The intermediate layer 34 (illustrated for example in Figure 3) comprises at least one polymeric material selected from the group consisting of polydioxanone (PDS), polyhydroxyalkanoates (PHA), and polycaprolactone (PCL) and optionally at least one organic and biodegradable semiconducting material, preferably a peptide organic and biodegradable semiconducting.

[0085] In one or more embodiments, the intermediate layer may degrade more slowly than at least one of the first and second support layers. For example, at least one of the support layers and the intermediate layer can have a different polymer composition; in particular, at least one support layer may have a polymeric composition that degrades faster than the polymeric composition of the intermediate layer.

[0086] In particular, for example, the at least one support layer can comprise PLGA optionally in combination with a biological polymer and the intermediate layer can comprise at least one polymer which has a lower degradation rate than PLGA, preferably selected from polydioxanone (PDS), and polycaprolactone (PCL), polyhydroxyalkanoates (PHA), preferably poly(3-hydroxybutyric-co-3 -hydroxy valeric acid).

[0087] The polymeric material of the intermediate layer 34 can be presented in a dense form or in the form of microfibers, optionally with microporosity on the surface. The diameter of the microfibers can be between 0.5 m and 250 pm. Surface microporosities can be between 0.05 pm and 20 pm. The intermediate layer 34, whether in dense form or in the form of microfibers, can include for example particles comprising at least one active ingredient, molecularly imprinted particles, drugs and / or other active ingredients such as those mentioned above.

[0088] The thickness of the intermediate layer can be between 0.10 mm and 0.80 mm.

[0089] The organic and biodegradable semiconducting material contained in the layers of the scaffold (in particular in at least one of the support layers, optionally in the at least one inner layer and optionally in the intermediate layer 34) is selected from the group consisting of self-assembling N-fluorenylmethoxycarbonyl-diphenylalanine peptides (Fmoc-FF), self-assembling N-fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) peptides functionalized with carbohydrate moieties, Fmoc-FF-glucosamine-6 sulfate (Fmoc-GlcS) or Fmoc-FF-glucosamine-6 phosphate (Fmoc-GlcP).

[0090] In a preferred embodiment, at least one support layer, preferably both support layers 30a;30b comprise a combination of PLGA, Fmoc-FF, gelatin.

[0091] Advantageously, the support structure 20 of the scaffold can further comprise molecularly imprinted particles 25a, 25b. References 25a and 25b indicate types of molecularly imprinted particles distributed in different layers of the scaffold. Molecularly imprinted particles employed in different layers of the scaffold can be molecularly imprinted particles to a signal overexpressed in late cardiac remodeling (25b) or to a signal overexpressed in early cardiac remodeling (25a). As illustrated in Figures 2 and 3 for example, these particles 25a can be included in at least one of the two support layers 30a; 30b, preferably in both layers 30a;30b and, optionally also in the intermediate layer 34 (25b).

[0092] The molecularly imprinted particles 25a, 25b can be selective for at least one enzyme belonging to the metalloprotease family (MMP, indicated with reference number 24 in Figure 2), preferably selected from the group consisting of MMP-9, MMP-8, MMP- 2, MMP-14 or TGF[3, thrombospondins (TSP-1, TSP-2, TSP-3).

[0093] Such molecularly imprinted particles can comprise at least one water-soluble polymer preferably selected from the group consisting of polyvinylpyrrolidone (PVP) or poly-n-isopropylacrylamide copolymers (PNIPAAm). Such particles can further comprise at least one polymer deriving from the polymerization of acrylic monomers, preferably selected from acrylic acid, methacrylic acid, 2-hydroxyethyl methacrylate, 2- hydroxyethyl methacrylate-6-hydroxyhexanoate and as cross-linking agents, preferably trimethylolpropane trimethacrylate (TRIM), pentaerythritol triacrylate (PETRA).

[0094] The diameter of the particles 25a, 25b can be between 20 nm and 2 pm, preferably between 80 nm and 700 nm. They can be accommodated in the cavities 41 or be present on the projections of the support structure 20, preferably in a density between 1 pg / cm2and 500 pg / cm2.

[0095] In one or more embodiments, the scaffold 10 can further comprise at least one active ingredient (indicated in the figures generically with reference number 27); it can be included in at least one of the support layers 30a; 30b and / or in said at least one inner layer 33, and / or in said at least one intermediate layer 34.

[0096] Furthermore, the active ingredient can be encapsulated in particles 23 which preferably comprise at least one biodegradable polymer preferably selected from the group consisting of polyhydroxybutyrate (PHB), poly-3 -hydroxybutyrate (P3HB) and related copolymers. These particles 23 can have a diameter between 20 nm and 2 pm. Furthermore, they can be present on the support layer in a density between 0.1 pg / cm2and 100 pg / cm2.

[0097] In one or more embodiments, the active ingredient 27 can be selected from the group consisting of preferably pleiotropic cardioprotective molecules such as neuregulin- 1 and ligands for the APJ receptor, preferably apelin; anti-fibrotic and / or antiinflammatory molecules, preferably nintedanib, verteporfin, pirfenidone; molecules for the improvement of ventricular systolic and diastolic function, preferably milrinone, levosimedan; anti-apoptotic and / or anti-necrotic molecules; pro-regenerative molecules; nucleic acids, preferably mRNA, miRNA; pro-angiogenic molecules; and chemoattractant molecules, preferably SDF-1.

[0098] Figure 2 shows particles 23 including an active ingredient 27 therein, for example apelin-13, accommodated inside the cavities 41 of the polymeric scaffold 10 of the invention which, consequently, once implanted, will be able to carry out mechanisms regulating the migration and recruitment of resident and circulating endogenous stem cells. Apelin-13 has also proven to be a powerful stimulator of cardiac contractility and improves cardiac output, stimulates neoangiogenesis, reduces blood pressure by inducing vasodilation, protects against reperfusion damage, decreases infarct size in vivo and increases heart rate, counteracts cardiac dysfunction and pathological remodeling.

[0099] In the embodiment of the scaffold illustrated in Figure 3, a preferably anti-fibrotic and positive inotropic / lusitropic active ingredient 27 together with at least one molecularly imprinted particle to a signal overexpressed in late cardiac remodeling 25b are included in the intermediate layer 34. While the outer component (support layers 30a, 30b) performs mechanical, positive inotropic / lusitropic, angiogenic, anti-fibrotic, antiinflammatory, pro-regenerative, recruitment, cardioprotection, cardioinduction functions, mainly in the first two post-infarction phases, in a third phase, when this outer component is biodegraded, the intermediate layer 34 interfaces directly with the tissue so as to continue to perform the mechanical support, positive inotropic / lusitropic and control functions over the fibrotic cardiac tissue.

[0100] The use of PHB as the constitutive polymer of the particles 23 containing the active ingredient 27 compared to particles comprising PLGA, which are the most used as release systems, provides a series of advantages: i) the PLGA can be a main component of the support layers and it was advantageous to use a different material for the particles 23 to be deposited on the surface or to be incorporated into the cavities 41 of the support layers; ii) PHB has degradation kinetics slower than PLGA; after a rather rapid release in the first two hours after implantation of the scaffold, a continuous and gradual release phase follows. In the specific case of the active ingredient apelin-13, it was observed that the gradual release allows the active ingredient to exert a cardioprotective effect in the first hours and act as a factor for the recruitment of progenitor cells and induction of vascularization over time, maintaining a prolonged cardioprotective effect on the myocardium; iii) the amount of active ingredient, for example apelin-13, released from particles comprising PHB is according to the effective doses demonstrated in the literature; iv) PHB is more resistant to the use of solvents used at the time of deposition of the particles on the surface of the scaffold.

[0101] Particles 23 containing active ingredients and / or molecularly imprinted particles 25a can be accommodated in the cavities 41 as indicated in the previous sections. A further deposition can provide for the accommodation of the particles comprising active ingredients 23 preferentially in the cavities 41 and the accommodation of the molecularly imprinted particles 25a, to the overexpressed signals of the ECM, on the projections 40a and 40b which imitate the structure of the ECM fibers.

[0102] The polymeric scaffold 10 will be able to behave as a “drug reservoir”, capable of releasing the pharmaceutical ingredient in situ according to the desired times and quantities, and therefore will be able to exert cardioprotective effects at the same time as anti-inflammatory, pro-angiogenic, chemoattractant, positive inotropic / lusitropic and cardioregenerative effects.

[0103] Even these further active ingredients may not be directly included in the polymeric matrix of the scaffold but rather encapsulated within particles 23 comprising biodegradable polymers which are in turn included in the support layers 30a and 30b and / or in the inner layer 33. This embodiment allows to achieve a release of the active pharmaceutical ingredient prolonged over time.

[0104] The particles (23 and 25a, 25b) can both contain and release and in turn recognize cardioprotective molecules, preferably pleiotropic molecules such as adenosine, neuregulin-1, ligands for the APJ receptor such as apelin, anti-fibrotic and / or antiinflammatory molecules (such as inhibitors of pathways involved in cardiac fibrosis such as nintedanib, verteporfin, pirfenidone), molecules that can improve ventricular function (such as positive inotropic and lusitropic molecules capable of both increasing the contractility of the heart and promoting relaxation of the ventricle, thus limiting its contracture, such as milrinone and levosimedan), anti-apoptotic / anti-necrotic, and pro- regenerative molecules including nucleic acids for cellular reprogramming and / or gene therapy (e.g. mRNA, miRNA) for a controlled release to reduce the impact of remodeling and to promote cardioregeneration in patients suffering from heart failure, chemoattractant (e.g. SDF-1) and pro-angiogenic molecules. The molecules released can be selected on the basis of the patient’s clinical history, allowing them to be adopted in personalized medicine and not limited to the treatment of myocardial infarction but also for the treatment of acquired or genetic pathologies that alter the cardiac tissue.

[0105] Figure 2 further illustrates molecularly imprinted nanoparticles 25a to signals mainly overexpressed in the early stages of cardiac remodeling e.g. MMP-9 (reference number 24).

[0106] With the aim of obtaining completely biodegradable devices that meet the required requirements including, mainly, controlled porosity, homogeneity in chemical terms and satisfactory mechanical properties, the polymeric scaffolds of the present invention were prepared using two different preparation techniques: phase inversion by immersion in a non-solvent and phase inversion by controlled evaporation of solvent mixtures.

[0107] Both preparation techniques were optimized by evaluating the effect of a wide repertoire of parameters (including composition, concentrations of the polymeric solutions, nature and quantity of solvent and non-solvent, temperatures and duration of the individual steps) on the characteristics of the final products. In the experimental part that follows, several preparation examples of polymeric scaffolds falling within the scope of the present invention are provided, using the two preparation techniques mentioned above. Of course, the technical teachings provided in these specific preparation examples are generally applicable to the preparation of any polymeric scaffold falling within the scope of the invention.

[0108] The following experimental part also illustrates the preparation of different embodiments of the polymeric scaffold of the invention as well as the preparation and use of nanoparticles designed to encapsulate the active ingredient and release it gradually. The results of mechanical, chemi cal -physical and morphological characterization studies conducted on various embodiments of the polymeric scaffold of the invention, as well as the results of biological tests carried out with the aim of verifying their biocompatibility, cardioregenerative capabilities and cardioprotective, are also described. This experimental part is provided for purely illustrative purposes and should in no way be construed as limiting the scope of the invention as defined by the attached claims.

[0109] EXAMPLES

[0110] Method for producing a first polymeric scaffold according to the invention

[0111] The scaffold produced as described below is a scaffold comprising a first and a second support layers and an inner layer interposed between these support layers, as illustrated for example in Figure 2. The two support layers are microstructured as described in the preceding sections and have cavities respectively facing opposite sides of the support structure. The two microstructured outer support layers comprise poly(lactic-co-glycolic acid) (PLGA), gelatin and Fmoc-FF molecules. The inner layer comprises an Fmoc-FF hydrogel containing apelin-13 as the active ingredient. Two classes of nanoparticles based on polyhydroxybutyrate (PHB) containing apelin-13 and based on PVP and polymethacrylic acid (PVP-PMAA) with a molecular imprint to MMP- 9 were deposited on both outer surfaces of the support layers.

[0112] Step 1) Preparation of the two support layers

[0113] The two microstructured PLGA / gelatin / Fmoc-FF support layers were obtained with predefined geometry in polydimethylsiloxane (PDMS) molds following the steps described below. i) Dissolution of gelatin in double-distilled water at a concentration of 10% (weight / volume; w / v) under stirring for 45 min at 50°C; ii) dissolution of PLGA in di chloromethane at a concentration of 10% (w / v); iii) once the complete dissolution of the PLGA in di chloromethane was reached, an acetone-based solution was added in which the electrically conductive oligomer (Fmoc-FF and / or Fmoc-GlcP and / or Fmoc- GlcS and their combinations) had previously been dissolved at concentration 0.025% (w / v).

[0114] The mixture obtained was stirred for 5 minutes, and the temperature was raised to 34°C for 2 min; a predefined volume of gelatin was added to the PLGA / Fmoc- FF / acetone / dichloromethane-based mixture in order to obtain a final solution in which the PLGA / gelatin weight ratio was equal to 70:30 (weight / weight, w / w). The final solution was kept under stirring for 2 minutes at 34°C. 2 ml of final solution were deposited on each PDMS mold (sized 4.5 cm x 4.5 cm), which were dried for 24 hours under a ventilated hood. Each individual microstructured layer was then removed from the mold.

[0115] Step 2) Preparation of the inner hydrogel layer including Fmoc-FF and loading with the active ingredient

[0116] The hydrogel was produced following a protocol that involves the formation of a gel following a change in pH; firstly, a volume of NaOH sufficient to reach a pH of approximately 12 was added to a solution of double-distilled water, in which the Fmoc- FF peptide had previously been dissolved, to make the oligomer soluble in water. Subsequently, sonication was performed (for 15 minutes) and a volume ofHCl was added such as to adjust the pH of the solution to 8-9. The hydrogel thus obtained was stored at a temperature of approximately 4°C (in a refrigerator). A volume of 250 pL of 0.05 mM apelin-13 solution was introduced into 1 mL of hydrogel.

[0117] Step 3) Preparation of the scaffold

[0118] To make the final structure of the scaffold, the inner layer (1 ml of hydrogel, loaded with apelin-13) was deposited on one of the support layers (non-microstructured side); subsequently the second support layer was placed on top of the inner layer (over the hydrogel) exposing the microstructured side towards the outside. The assembly was carried out by fixing the edges of the scaffold using a system with sharp blades (specially designed with varying dimensions) which are pre-heated and allow cutting and welding to be carried out simultaneously.

[0119] Step 4) Synthesis ofPHB nanoparticles and loading with the active ingredient

[0120] The nanoparticles were obtained using the nanoprecipitation technique: 20 mg of preformed polymer, polyhydroxybutyrate (PHB), were dispersed in 1 ml of acetone. The solution was then added dropwise into 8 ml of 0.1% (w / v) aqueous polyvinyl alcohol solution, under gentle stirring for 16 h at 800 rpm.

[0121] The nanoparticles were centrifuged for 15 minutes at 11000 rpm. At least 3 washes were performed in double-distilled water to eliminate residual solvents. The nanoparticles were dried under a ventilated hood until the residual supernatant had completely evaporated.

[0122] Subsequently, absorption loading was carried out by placing 3 mg of nanoparticles in contact with a solution of the active ingredient (0.05 mM for 2 hours), apelin-13. After centrifugation, the particles were dispersed in a waterethanol solution (in volume ratio 30:70), sonicated for 20 minutes and deposited by atomization on both microstructured support sides of the scaffold until reaching a deposition density equal to 3.4 pg / cm2.

[0123] Step 5) Synthesis of molecularly imprinted nanoparticles (MIPs)

[0124] The technique used for the synthesis was high dilution radical polymerization of monomer by precipitation, using a non-covalent molecular template approach.

[0125] An example of a synthesis procedure for the production of MIPs in dense form is described below. The following solutions were prepared:

[0126] - solution a): 0.054 g of potassium persulfate were dissolved in 4 ml of PBS at pH 7.4;

[0127] - solution b): 0.222 g of polyvinylpyrrolidone (PVP, with molecular weight (MW) 20000) was dissolved in 4 ml of PBS to which 160 pL of methacrylic acid (MAA) and 160 pL of trimethylolpropane trimethacrylate (TRIM) were added;

[0128] - solution c): the MMP-9 enzyme was dissolved in 2 ml of PBS at a concentration of 50 ng / ml.

[0129] Solution b) was introduced into a glass flask heated to a temperature of 60°C, degassing was carried out with inert nitrogen, solution c) and solution a) were added, keeping the system under gentle agitation for 2 hours.

[0130] Finally, the synthesis product was removed and subjected to at least 3 washes in order to eliminate the unpolymerized monomer and excess PVP. The washing solutions used are the following: 1) acetic acid in water 0.1% (vol / vol; v / v) and methanol in a ratio of 30:70 (v / v) at 70°C for 1 h, cooling and subsequent centrifugation (15 minutes at 11000 rpm); water-ethanol solution 30 / 70 (v / v) for 10 minutes at 11000 rpm and centrifugation; 3) double-distilled water and centrifugation. The MIP nanoparticles were subsequently deposited, by atomization, onto the scaffold, onto the support layers, to achieve a MIP density on the scaffold of approximately 10 pg / cm2.

[0131] The particles were also prepared in hollow form using, as a sacrificial inner core, polymethylmethacrylate (PMMA)-based nanoparticles obtained by emulsion polymerization of methylmethacrylate (MMA) in the presence of sodium dodecyl sulfate (SDS) as a surfactant in ethanol or in a mixture of water / ethanol (60 / 40 v / v) in the presence of radical initiator. The second step consisted of the polymerization of MAA in the presence of PVP, a radical initiator as described before for dense particles. Next, the nucleus was extracted by dispersing the particles in chloroform.

[0132] The MIP particles, both in dense and hollow form, have been loaded with the active ingredients (cardioprotective agent or nucleic acid) by adsorption at concentrations in a range between 0.2 and 5 mg / ml and the in vitro release ability thereof has been shown.

[0133] A second example of a synthesis procedure for the production of PNIPAAm-based MIPs, in dense form, is described below.

[0134] A preweighed amount (68 mg) of poly-(N isopropylacrylamide-2-hydroxyethyl methacrylate-6-hydroxyhexanoate) copolymer was dissolved in 6 mL of double-distilled water at 22 °C. After 15 minutes of stirring, 50 pl of MAA were added and after 10 min, 38.2 pl of TRIM were added. After 5 min, the enzyme, previously dispersed in solution, was introduced. After 2 minutes, 1 ml of initiator solution was added to the reaction mixture. The temperature was gradually increased until reaching the temperature of 60°C for 2 hours. The particles were recovered by ultracentrifugation and enzyme extraction was performed as previously described for PVP -based nanoparticles.

[0135] PNIPAAm-based particles can also be produced in hollow form and can be loaded with the active ingredients as previously described for PVP -based particles.

[0136] Different deposition strategies on the profiles or in the cavities of the microstructure were also evaluated.

[0137] Method for producing a second polymeric scaffold according to the invention

[0138] The scaffold produced as described below includes - compared to the scaffold described in the previous example - an intermediate layer interposed between two inner layers, as for example illustrated in Figure 3. In particular it includes a first and a second support layers, two inner layers interposed between said support layers and an intermediate layer interposed between said inner layers. The two support layers are microstructured as described in the previous sections and have cavities respectively facing opposite sides of the support structure.

[0139] In this case, the intermediate layer is a polymeric, bioartificial layer, loaded with i) at least one active ingredient with anti-fibrotic action and ii) molecularly imprinted nanoparticles to enzymes overexpressed in the late post-infarction remodeling phase (for example MMP -8, TGF-b, TSP). For the production of the scaffold, the assembly of the two outer microstructured support layers based on PLGA / gelatin and including a semiconducting peptide (for example Fmoc-FF), modified on the surface with MIP nanoparticles to MMP-9, with the first inner layer, based on a gelatin and conducting peptide hydrogel, loaded, after inclusion in nanoparticles or directly, with active ingredients and with the addition of a biodegradable, possibly bioartificial (mixture with biological polymer) and conducting polymer layer, consisting of the third polymeric component (e.g. PDS, PCL, PHAs), is provided.

[0140] The second inner layer can for example be inserted inside the first inner layer which will consequently be divided into two further layers, as schematically illustrated in Figure 3. To this end, the technique of microfabrication, phase inversion, electrospinning or dryspinning according to a predefined geometry.

[0141] Examples of intermediate layer production

[0142] Electrospinning method. PHBHV microfibers were produced starting from PHBHV dissolved in chloroform or in a mixture of chloroform / methanol (90: 10 v / v) at a concentration of 10-15% under stirring of 100-300 rpm for at least 12 hours at room temperature. The fibers loaded with the active ingredients and the nanoparticles were obtained by introducing these into the PHBHV solution. The polymer solution, possibly functionalized, was loaded into a glass syringe with a blunt stainless steel needle and inserted into a syringe pump. A potential of 30 kV was applied and the solution was injected at a constant flow rate of O.OOlml / min. The collector was placed 15 cm away from the tip of the needle. The fibers were electrospinned in order to obtain a net of homogeneously interconnected fibers. Before assembling the intermediate layer, the fibers were subjected to removal of any traces of solvent.

[0143] Dryspinning method. PCL microfibers were produced in organic solvent, dichloromethane (DCM), at 20-30% w / w. The polymer was dissolved at room temperature under gentle and constant stirring for 24 hours to obtain a homogeneous solution. After one hour, the solution was pushed through a needle with a diameter of 0.50-0.80 mm and a spinning speed of 5-8 cm / s. The fiber is formed by evaporation of the solvent in the air gap between the spinneret and the pickup coil.

[0144] The microfibers can be loaded with the MIPs possibly loaded with the active ingredient; pre-weighed MIPs were introduced into the PCL solution in DCM (MIP / PCL 0.5-5% w / w). After gentle dispersion, the fibers were produced as previously indicated.

[0145] The intermediate layer can be loaded with an anti-fibrotic active ingredient and / or with MIP nanoparticles to MMP-8, TGFp or other overexpressed factors of the cardiac ECM in order to selectively reduce the overexpression of these factors and re-establish their correct balance in an attempt to prevent / limit post-infarction fibrosis. If the inner layer is in the form of fibers, the particles can possibly be loaded inside the fibers themselves. While the outer component carries out its mechanical, recruitment, cardioinduction functions and the function of protection from reperfusion damage mainly in the first two post-infarction phases, in the third phase the outer component is biodegraded and the inner membrane interfaces directly with the tissue so as to continue to perform the functions of mechanical support and control over the fibrotic cardiac tissue.

[0146] Figure 4 schematically illustrates the mechanisms of action of a polymeric scaffold implanted in the host according to embodiments of this disclosure where the reference number 34 indicates the intermediate layer and the reference number 27 generically indicates active ingredients with properties such as for example anti-fibrotic and positive inotropic / lusitropic properties. The changes that can occur over time after scaffold implantation are attributable to three main phases (indicated with 1., 2. and 3. in Figure 4) i.e. the early post-infarction reparative phase (1.), the early remodeling in the granulation phase (2.), the late remodeling (3.). During phase 1, the actions carried out by the scaffold are: i) cardioprotection, cell recruitment, cardioinductivity due to the release of loaded and unloaded active ingredients 27 within biodegradable particles and the combination of composition and microstructure of the reticular structure 20, ii) mechanical compliance due to the mechanical behavior of the scaffold comprising overlapping layers, iii) protection from ventricular remodeling thanks to the presence of MIP 25a to the molecules and enzymes overexpressed in the cardiac ECM during phases 1 and 2. The intermediate layer 34, includes polymers with a slower degradation compared to the polymers contained in the support layers) (e.g. PDS, PHAs, PCL) in dense form or in microfibers, and in turn contains active ingredients 27 and MIP 25b to enzymes and molecules involved in the late remodeling phase. During phase 2, the support layers 30a and 30b and the inner layers 33a and 33b undergo degradation and complete release of active ingredients 27 while the innermost intermediate layer remains almost unchanged. The actions provided in this phase are mechanical compliance, cardioinductivity and cardioprotective action. In phase 3, the intermediate layer 34 remains predominantly comprising active ingredients 27 and MIP 25b, from which the anti-fibrotic drugs will be released and the molecularly imprinted MIP 25b will act to the enzymes and molecules expressed in high quantities in the late remodeling phase

[0147] Analysis of the properties of polymeric scaffolds of the invention

[0148] A series of chemical-physical, mechanical, morphological, functional and biological evaluations were performed on polymeric scaffolds obtained as described in the previous sections.

[0149] The FT-IR infrared analysis made it possible to verify a homogeneous distribution of the different components of the scaffold. The viscoelastic properties of the samples were evaluated, before degradation, through dynamic-mechanical analysis, testing the samples along the two main directions, longitudinal and transversal, with respect to the surface micropatterning. The results obtained are consisted with those reported in the literature for ovine and porcine myocardial samples. The mechanical anisotropy was also confirmed by Young’s modulus values in both dry and wet conditions. The suture resistance test of the scaffold was performed to evaluate the maximum stress required to break a sutured specimen. The suture resistance strength was always above the threshold value compared to the calculated minimum force specification (2 N). The breakout resistance test of the scaffold was performed according to the ISO 7198 standard relating to cardiovascular implants. The maximum force recorded for each sample is approximately 15 N. This value is consisted with that reported in the literature for porcine myocardial samples.

[0150] Degradation analysis of the final assembled scaffold was also performed: the degradation properties were studied for up to one month by evaluating the degradation rate and changes in molecular weight over time using chromatographic analysis. A slow and gradual degradation kinetics is observed which reaches almost 15% after 30 days of incubation.

[0151] Conductivity analysis was performed on the final scaffold using an impedance spectrometer; there are no significant differences in terms of electrical conductivity between the three different peptides. The results showed that the Fmoc-FF peptide increased the conductivity of the scaffold. In the case of affixing the scaffold with surgical glue instead of suture, the evaluation performed showed only a small reduction in the conductivity of the scaffold after its complete covering with glue.

[0152] A series of in vitro and in vivo analyzes were conducted on PVP -based MIP nanoparticles (a polymer approved by the FDA for biomedical use). Thermogravimetric analysis showed the presence of interactions between the enzyme and the nanoparticles and improved thermal stability. The ability of MIPs to specifically recognize and bind the MMP-9 enzyme was demonstrated by HPLC. The selectivity of MIPs to MMP-9 was also evaluated with respect to MMP-2, another enzyme belonging to the metalloproteinase family. The drug release tests were performed by HPLC and showed a more gradual and prolonged release of apelin-13 compared to scaffolds functionalized with PHB particles in comparison with scaffolds loaded with active ingredients dispersed within the hydrogel.

[0153] The PVP -based MIP nanoparticles, both in hollow and dense forms, were also loaded with nucleic acid (DNA), showing an encapsulation capacity between 20 and 40% and a DNA release capacity, initially more rapid and then gradual over time, especially for the hollow particles which continued to release the nucleic acid after 4 weeks. The PVP -based MIP nanoparticles were also loaded with the cardioprotective drug Adenosine at concentrations ranging from 1 to 5 mg / ml. The particles in both hollow and dense forms showed an encapsulation capacity greater than 50% and a controlled release capacity with values between 20 and 30% after 100 hours of in vitro release regardless of the shape. The PVP -based nanoparticles loaded with Adenosine were in turn loaded inside the PCL microfibers which can constitute the intermediate layer, these microfibers are dense in section and microporous on the surface. The nanoparticles loaded inside the microfibers are distributed mainly within the surface porosities.

[0154] From a biological point of view, apelin-13 released from the scaffold has demonstrated its effectiveness in improving the ability of cardiac non-myocyte cells (NMCs) to adhere to scaffolds and also to maintain a markedly elongated cell shape for a prolonged period of time (Figure 5).

[0155] Biocompatibility was demonstrated for both MIP and PHB -comprising nanoparticles (Figure 6 a,b) and on the scaffold functionalized with both particles (Figure 6 c). Cytocompatibility results performed on various cell types (NMCs, human mesechymal stem cells (hMSCs), iPSC-derived cardiomyocytes and H9c2 cardiomyoblasts) show cells capable of adhering to and colonizing the entire scaffold structure (Figure 5c). Furthermore, the results of migration assays showed that the final scaffold (electrically conductive and functionalized with apelin-13) improved the recruitment of NMCs (Figure 7) and hMSCs. The cardioinductivity study highlighted how for the NMC cells grown for 7 days on the final scaffold there was a reduced expression of the sternness marker c-kit and an increased expression of the genes of an initial cardiac differentiation, as well as of the genes that code for the sarcomeric proteins and the main proteins (connexins) assembled to form the gap junctions typical of cardiac cells and essential for contractile function (Figure 8). Furthermore, for cardiomyocytes derived from iPSCs cultured for 7 days on the final scaffold, an increase in the expression of the various genes encoding for cardiac differentiation markers is observed (Figure 9). Ex vivo cell sheet-based technology was explored to achieve complete maintenance of cell-cell and cell -ECM junctions. It was possible to create cell sheets in vitro that mimic the native structure of the myocardium, composed of 70% NMC and 30% H9C2 cells. These cell sheets were able to adhere to and colonize the scaffold, allowing for the formation of a three-dimensional tissue-like structure (Figure 10). Furthermore, the cell sheets seeded on the scaffold maintained extraordinary viability.

[0156] The cardiomyocytes derived from iPSCs seeded on the scaffolds arrange themselves on the structure maintaining their synchronous beating, important for the functional recovery of the damaged area. Electrical conductivity was also assessed by measurements of the ratiometric concentration of calcium ions in the cytosol, essential for driving synchronous cell beating. Good synchronicity was induced by the 3D structure of the final scaffold, making it capable of promoting good cell-cell interaction (Figure H).

[0157] In vivo experiments were conducted on rats in which infarction was induced by ligation of the left descending coronary artery and in which the acellularized scaffold was positioned on the infarcted area. 5 experimental groups were considered: infarction + final scaffold (IR+P), infarction + non-functionalized scaffold (IR+Pn), infarction (IR), control (SHAM), control + final scaffold (SHAM+P). The animals after infarction and scaffold implantation remained alive for the entire duration of the experiments. The health status of the animals was monitored by ECG and MRI analysis. The analyzes of the ECG traces performed at the end of each week, apart from a certain variability in the animals in which only the infarction was induced, do not show alterations in the other groups, including the one with infarction and treated with the scaffold. The parameters obtained with MRI indicate an increase in the ejection fraction for infarcted animals treated with the scaffold, compared to those with only infarction, and also an increase in the volume of blood released into circulation at each heartbeat, highlighting how the scaffold on infarcted animals does not have a disturbing effect, but rather improves performance compared to controls.

[0158] After 4 weeks, the animals were sacrificed and the hearts recovered and analyzed by various techniques performing histological and immunohistochemical analyses, evaluation of vascularization, cell recruitment and evaluation of cardioprotection. From the morphological and infrared analysis it emerged that the scaffold at 4 weeks was well adhered and largely degraded, covered by a matrix which from FT-IR analysis highlighted spectra referable to the extracellular matrix and cellular elements. Histological analysis with eosin and hematoxylin showed an interaction between scaffold and the underlying myocardial tissue. A space filled by ECM is observed at the interface. The pericardial membrane incorporates the scaffold in a continuum.

[0159] The images derived from immunofluorescence (IF) analyzes in the infarcted tissue treated with the scaffold highlight how the staining for both troponin C (TnC, a typical protein of the sarcomere of cardiomyocytes) and for connexin 43 (a protein present in the gap junctions essential for the transmission of cell-cell impulses) is more similar to sham tissue than that of infarcted tissue, indicating how the scaffold restores a more normal morphology of cardiac tissue after infarction. The quantitative results obtained from the study with IF confirm how the quantity of both the sarcomeric protein and connexin 43 in the treated tissue are significantly higher than the values measured in the group with only infarction (Figure 12). These results are therefore indicative of the fact that the scaffold can have a cardioprotective effect, promote the differentiation of cells to the cardiac phenotype and increase the transmission of signals between cells.

[0160] Furthermore, both the analysis with hematoxylin-eosin and the Mallory’s triple stain highlighted the presence of elongated cells characteristic of the presence of stem cells within the scaffold. This indicates that the scaffold has begun to recruit undifferentiated progenitor cells into its structure. IF analysis was used to analyze the c- kit marker within the scaffold and showed the presence of c-kit positive cells in the scaffold both in the one positioned on the infarcted tissue and in the one on the sham tissue. Furthermore, cells positive for Gata-4, an early differentiation factor, were also highlighted, confirming the recruitment effect of the scaffold as can be seen from the values which are significantly higher than in IR and sham tissues in the absence of the scaffold; this ability was also highlighted to a lesser extent in the scaffold without apelin- 13. In vivo evidence of cell recruitment is recorded due to the presence of abundant immature cells both on the scaffold and in the area immediately below, indicating an ongoing repair.

[0161] The IF investigation with alpha-actin of smooth muscle cells and with endothelial markers (CD31 and von Willebrand factor) allowed us to highlight the presence of new vessels both inside the scaffold and in the underlying myocardium. The quantitative analysis highlighted an increased number of vessels in the infarcted tissue treated with the final scaffold compared to the other groups, but above all there was an increase in small vessels confirming the hypothesis that the final scaffold is able to promote formation of new vessels in the myocardium under the scaffold and in the infarcted area (Figure 13). Staining with hematoxylin-eosin also made it possible to demonstrate that the scaffold promotes the formation of functional vessels (presence of red blood cells inside) to a greater extent for the one in contact with the infarcted tissue at 4 weeks postischemia, confirming that the scaffold has a chemoattractant action on the cell precursors which differentiate giving rise to new functional vessels.

[0162] The quantitative analysis of the percentage of fibrosis extension conducted by histological analysis indicated a reduction in the percentage of fibrosis in the presence of the scaffold, indicating a protective effect on the infarcted tissue after 4 weeks of ischemia.

[0163] The FT-IR Chemical Imaging analysis also allowed an analysis of the protein structure of the tissue sections underlying the scaffold, suggesting how, overall, it has both a protective and regenerative effect in the area where the infarction occurred.

[0164] With reference to the scaffold comprising a first and second inner layers, it has been verified that the additional, slower-degrading layer is biocompatible and capable of promoting the alignment of the cells along the lines of the structure. The in vitro degradation and mechanical tests confirm a lower degradation-erosion percentage compared to the outer layers and above all the maintenance of the mechanical properties even after 15 days, allowing the viscoelasticity, suturability and breakout resistance parameters to be obtained even after 15 days of degradation at 37°C.

[0165] The effects exerted by the biodegradable semiconducting material (Fmoc-FF peptide) in the scaffold - comparative analysis

[0166] The Inventors of the instant application have also realized, against all expectations already disclosed to date, that the presence of a semiconducting material, in particular the Fmoc-FF peptide, also produced some unexpected effects from mechanical, degradation and physico-chemical homogeneity points of view.

[0167] Despite the low percentage of Fmoc-FF compared to the other components, it has in fact determined a decrease in the stiffness of the scaffold making it more flexible and compatible with the characteristics of the cardiac tissue. Table 1 below refers to comparative analysis performed on the scaffold herein disclosed (comprising a first and a second support layer formed of PLGA as polymeric material and gelatin, an inner hydrogel layer, the support layers having a surface structure comprising a plurality of cavities defined by an array of longitudinal linear projections and an array of transverse linear projections intersecting each other) with and without Fmoc-FF.

[0168] Table 1

[0169] The results shown in Table 1 provide the evidence that in the scaffold comprising Fmoc-FF the value of the storage modulus, E’, is lower than the value observed for the scaffold without the semiconducting material.

[0170] An ordered P-turn type structure of the peptide is maintained in the microstructured layer even after mixing with the synthetic polymer in organic solvents as highlighted by the FT-IR Chemical Imaging analysis performed on the scaffold. In Figure 14 is reported the average spectrum of a scaffold comprising the first and the second support layer comprising PLGA and gelatin, the inner hydrogel layer, the support layers having a surface structure comprising a plurality of cavities defined by an array of longitudinal linear projections and an array of transverse linear projections intersecting each other with Fmoc-FF in the supporting layers analyzed in second derivative (1700-1550 cm'1) to better detect the peptide structure. The second derivative points out the presence of the bands at 1690 cm'1and 1636 cm'1associated to P -turn and random coil conformation, respectively, indicative of the conductive oligomer presence and the maintenance of its secondary structure.

[0171] In addition, the mass loss over time is limited by the presence of the conductive peptide which makes the scaffold more stable (after 30 days of incubation the mass loss on average decreases from 30% in the absence to 15% in the presence of the electroconductive component).

[0172] This result was also confirmed by in vitro biological tests, where hiPSC-CMs were seeded and cultured onto the same scaffolds comprising and not comprising Fmoc-FF for 30 days. The presence of Fmoc-FF showed the ability to maintain the integrity of the scaffold throughout the entire culture period, as shown in Figure 15.

[0173] A longitudinal study of cardiomyoblasts cultured onto the same scaffolds comprising and not comprising Fmoc-FF revealed that the integrity of the scaffold microstructure provided by Fmoc-FF incorporation also favors cell elongation (Figure 16), which is a pre-requisite for cellular differentiation. Accordingly, a direct comparison of cardioinductive ability of the same scaffolds comprising and not comprising Fmoc-FF on hMSCs revealed that the latter induced more evident downregulation of the sternness marker c-KIT as well as evident upregulation of the cardiomyogenic transcription factor GATA4, thus resulting more cardioinductive (Figure 17).

[0174] References

[0175] WORLD HEALTH STATISTICS 2015, htps: / / www.who.int / docs / default- source / gho-documents / world-health-statistic-reports / world-health-statistics-2015.pdf

[0176] Heart Disease and Stroke Statistics-2020 Update: A Report From the American Heart Association. Circulation 2020;141 :el39-e596

[0177] Xu Y, Guan J. Biomaterial property-controlled stem cell fates for cardiac regeneration. Bioact Mater. 2016; 1(1): 18-28. doi: 10.1016 / j.bioactmat.2016.03.002.

[0178] Schellings MW, van Almen GC, Sage EH, Heymans S. Thrombospondins in the heart: potential functions in cardiac remodeling. J Cell Commun Signal. 2009; 3(3- 4):201-13. doi: 10.1007 / sl2079-009-0070-6.

Claims

CLAIMS1. Polymeric scaffold (10) for myocardial regeneration and protection, comprising a support structure (20) comprising at least a first and second support layers (30a; 30b) and at least one inner layer (33) interposed between said first and second support layers (30a; 30b),- wherein said first and second support layers (30a; 30b) comprise at least one polymeric material selected from the group consisting of poly(lactic-co-glycolic acid) (PLGA), poly(dioxanone) (PDS), polycaprolactone (PCL) and polyhydroxyalkanoates (PHA), preferably poly(3-hydroxybutyric-co-3-hydroxyvaleric acid) among PHA, optionally in combination with at least one biological polymer,- wherein at least one of said first and second support layers (30a; 30b) further comprises at least one organic and biodegradable semiconducting material, preferably an organic and biodegradable semiconducting peptide,- wherein said at least one inner layer (33) comprises a hydrogel, preferably containing at least one an organic and biodegradable semiconducting material, more preferably an organic and biodegradable semiconducting peptide,- wherein said first and second support layers (30a; 30b) comprise a surface sculpture, said surface sculpture comprising a plurality of cavities (41) arranged according to a regular frame, said cavities (41) being defined by an array of longitudinal linear projections (40a, 40b) and an array of transverse linear projections (40c) intersecting with each other respectively, said first and second support layers (30a; 30b) having cavities (41) respectively facing opposite sides of the support structure (20).

2. A polymeric scaffold (10) for myocardial regeneration and protection according to claim 1, wherein said support structure (20) further comprises at least one intermediate layer (34), said intermediate layer (34) comprising at least one polymeric material selected from the group consisting of polydioxanone (PDS), polyhydroxyalkanoates (PHA), and polycaprolactone (PCL) and optionally at least one organic and biodegradable semiconducting material, preferably an organic and biodegradable semiconducting peptide.

3. Polymeric scaffold (10) for myocardial regeneration and protection according to claim 2, wherein the intermediate layer (34) is degradable more slowly than at least onebetween the first and second support layers (30a; 30b).

4. Polymeric scaffold (10) for myocardial regeneration and protection according to claim 3, wherein the at least one support layer (30a; 30b) comprises PLGA preferably in combination with at least one biological polymer, and wherein the intermediate layer (34) comprises at least one polymer having a lower degradation rate than PLGA preferably selected in the group consisting of poly(dioxanone, PDS), polycaprolactone (PCL) and polyhydroxyalkanoates (PHA), preferably poly(3-hydroxybutyric-co-3 -hydroxy valeric acid) among PHA.

5. Polymeric scaffold (10) for myocardial regeneration and protection according to claim 2, wherein said support structure (20) comprises a first and second of said inner layer (33a;33b), and wherein the intermediate layer (34) is interposed between said first inner layer (33a) and said second inner layer (33b).

6. Polymeric scaffold (10) for myocardial regeneration and protection according to any one of the preceding claims, wherein the hydrogel of the at least one inner layer (33) comprises at least one biological polymer.

7. Polymeric scaffold (10) for myocardial regeneration and protection according to any one of the preceding claims, wherein said at least one biological polymer is selected in the group consisting of gelatin, hyaluronic acid, collagen, gellan.

8. Polymeric scaffold (10) for myocardial regeneration and protection according to any one of the preceding claims, wherein the at least one organic, biodegradable semiconducting material is selected in the group consisting of self-assembling N- fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) peptides, self-assembling N- fluorenylmethoxycarbonyl-diphenylalanine (Fmoc-FF) peptides functionalized with carbohydrate moieties, Fmoc-FF-glucosamine-6 sulfate (Fmoc-Glcs) or Fmoc-FF- glucosamine-6 phosphate (Fmoc-GlcP).

9. A polymeric scaffold (10) for myocardial regeneration and protection according to any one of the preceding claims, wherein said support structure (20) further comprises molecularly imprinted particles (25a, 25b), said molecularly imprinted particles being included in at least one of said support layers (30a; 30b) and / or in said at least one intermediate layer (34).

10. Polymeric scaffold (10) for myocardial regeneration and protection according to claim 9, wherein said molecularly imprinted particles (25a, 25b) are selective for atleast one enzyme belonging to the metalloprotease (MMP) family, preferably selected from the group consisting of MMP-9, MMP-8, MMP -2, MMP-14 or TGFp, trombospondine (TSP-1, TSP-2, TSP-3).

11. Polymeric scaffold (10) for myocardial regeneration and protection according to claim 9 or claim 10, wherein said molecularly imprinted particles (25a, 25b) comprise at least one water-soluble polymer preferably selected from the group consisting of polyvinylpyrrolidone (PVP), poly-n-isopropylacrylamide copolymers (PNIPAAm).

12. Polymeric scaffold (10) for myocardial regeneration and protection according to any one of claims 9 to 11, wherein said particles (25a, 25b) have a diameter between 20 nm and 2 pm.

13. Polymeric scaffold (10) for myocardial regeneration and protection according to any one of the preceding claims, further comprising at least one active ingredient (27).

14. Polymeric scaffold (10) for myocardial regeneration and protection according to claim 13, wherein said active ingredient (27) is included in at least one of said support layers (30a; 30b) and / or in said at least one inner layer (33), and / or in said at least one intermediate layer (34).

15. Polymeric scaffold (10) for myocardial regeneration and protection according to any one of claims 13 or 14, wherein said active ingredient (27) is encapsulated in particles (23).

16. Polymeric scaffold (10) for myocardial regeneration and protection according to claim 15, wherein said particles (23) comprise at least one biodegradable polymer preferably selected in the group consisting of polyhydroxybutyrate (PHB), poly-3- hydroxybutyrate (P3HB) and copolymers thereof.

17. Polymeric scaffold (10) for myocardial regeneration and protection according to claim 15 or claim 16, wherein said particles (23) have a diameter between 20 nm and 2 pm.

18. Polymeric scaffold (10) for myocardial regeneration and protection according to any one of the preceding claims, wherein the at least one intermediate layer (34) comprises at least one anti-fibrotic active ingredient and at least one molecularly imprinted particle (25b).

19. Polymeric scaffold (10) for myocardial regeneration and protection according to any one of the preceding claims, said scaffold being acellularized.

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