SCAFFOLD FOR CARDIAC PATCH

Abstract

A biocompatible and biodegradable medical device patch actuating primarily as soft tissue structural reinforcement. The device has a layered architecture, where the primary serves as suturing layer and mechanical support to a thick porous scaffold which can be coated with a mimic-like extra cellular matrix (ECM). The device can be provided to the end user under the format of independent layers that can be cut and assembled to the specific need to the end user and patient. The layers are assembled without the need of any adhesive. Totally haemocompatible and of behavior superior to polytetrafluoroethylene used for any soft tissue repaired, the field of this invention is demonstrated for cardiovascular therapy but should not be limited to it. It is of practical relevance of vein, tendon and hernias and dermal treatments.

Claims

1. A composition comprising a hydrogel formed using one or more self-assembling peptides, wherein the self-assembling peptides: a) are 8 to 12 amino acid residues in length; b) terminates in one or more K residues; c) have an at least 5 amino acid sequence upstream of the K residue where every other amino acid residue is the same amino acid residue of a first type.

2. The composition as claimed in claim 1, wherein the amino acid residue of the first type is an F or V residue.

3. The composition as claimed in claim 2, wherein a self-assembling peptide terminates in KXK, where X is selected from F or V residues.

4. The composition as claimed in claim 1, wherein every other intervening amino acid residue is the same amino acid residue of a second type.

5. The composition as claimed in claim 4, wherein the amino acid residue of the second type is an E residue.

6. The composition as claimed in claim 1, wherein the self-assembling peptides are selected from the sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and combinations thereof.

7. The composition as claimed in claim 1, wherein the composition further comprises a biocompatible and biodegradable scaffold comprising synthetic and/or natural polymers, wherein the synthetic polymer is selected one or more of polylactic acid, glycolic acid, lactone, polyglycerol sebacate, copolymer of synthetic polymers and combinations thereof, and wherein the natural polymer is selected from one or more of chitosan, sodium alginate and cellulose.

8. The composition according to claim 7, wherein the lactone is polycaprolactone (PCL).

9. The composition according to claim 7, wherein the copolymer of synthetic polymers is L-lactide/-caprolactone, poly(lactic-co-glycolic acid) (PLGA) and/or L-lactide/-caprolactone.

10. The composition according to claim 7, wherein the synthetic polymer is PCL.

11. The composition according to claim 1, wherein the composition further comprises a support comprising polyurethane.

12. The composition according to claim 1, further comprising at least one trophic agent, preferably wherein said trophic agent is selected from BMP, IGF, VEGF and PRP.

13. The composition according to claim 1, further comprising at least one growth factor, wherein said growth factor is hepatocyte and/or insulin growth factor.

14. The composition according to claim 1, further comprising at least one drug, wherein said drug is selected from 5-azacytidine and dexamethasone.

15. The composition according to claim 1, further comprising cells.

16. The composition of claim 1, for use in therapy.

17. The composition of claim 1, for use in, or in combination with, drug delivery.

18. The composition of claim 1, for use in a method of tissue regeneration in a subject in need thereof.

19. The composition of claim 18, wherein the tissue regeneration is cardiac tissue regeneration.

20. The composition of claim 1, use in the treatment of myocardial infarction, curettage or transmural infarct treatment, preferably in the treatment of myocardial infarction.

21. A cardiac patch comprising the composition of claim 1.

22. The cardiac patch according to claim 21, for use in the treatment of myocardial infarction.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0171] As it was mentioned above, the present invention proposes a scaffold that is compatible with in-vitro cell seeding and cell culture to be used as vector for cellular therapy. Nevertheless as its primary action is of structural reinforcement, the device is totally compatible with a cellular therapy. Despite the present context wherein the present device is demonstrated is oriented towards CTE, the application of presented device should not be limited to curettage and cardiac transmural applications. Indeed such device can be used to soft tissue engineering such as but not limited to hernias, vein conduct. The scaffold composition comprises poly(-caprolactone), alginate and composite thereof with natural polymers such as chitosan, alginate fibroin. The device porosity can be tailored with a pore range of 10-250 m, hereby permitting angiogenesis and cell seeding.

[0172] While the importance of a biomaterial to be used as a medical device for prosthetic applications, its main function in TE is to enhance cell attachment, growth and differentiation. As such, an extension of the medical device is a functionalization with cell signaling agent capacity. The device proposed by the present invention can be functionalized with a biocompatible and biodegradable self-assembled gel. Whilst it functionalization is not necessary for the medical device to ensure its primary actuation as it is foreseen to provide the scaffold an ECM-like micro-environment, the polymeric self-assembled structure, that is composed of but not limited to peptidic or polyurethane amphiphiles, can be loaded with chemical and biological cues via entrapment method or covalently. Such cues can either be exogeneous or being PRP. Should PRP be selected, the medical device would remain a medical device and shall not fall under drug regulation.

[0173] To initiate regeneration of the host tissue, it is essential that the biomaterial encourages in-vivo revascularization as well as favours integration with the host tissue. At the same time, it should degrade at a predefined rate to enable its replacement with newly formed tissue by safely degrading at a similar rate of the new tissue formation and eventually removed from the body by natural metabolic pathways without producing toxic by-products.

[0174] The basic requirements for myocardial bioengineered constructs include robust yet flexible mechanical properties, ability to withstand contraction, electro-physiological stability and vascularization ability. In fact if it is expected that the patch will provide a temporary mechanical support to the infarcted myocardium in order to prevent negative effects on the spare tissue and to prevent aneurism formation in the infarcted area, while the regenerative processes take place. We proposed a cardiac patch adequate for both curettage and transmural application.

[0175] From the aforementioned critical review regarding existing solution for cardiac patch application, the present invention provides a cardiac patch end product based on polymeric scaffold structure of tailorable porosity and dimension/a drug delivery system/a functionalisable gel garnished with a stitch resistant biocompatible and biodegradable film support (FIG. 1).

[0176] Applications foreseen are curettage, mechanical support, pericardium contention and transmural application.

[0177] This 4 elements system is proposed as a medical device for structural reinforcement purposes. The polymeric structure is immobilized onto the film support in an adhesive less manner.

[0178] Electrospun fibers based drug delivery system is immobilized onto the film free side of the polymeric scaffold. The gel element can be immobilized onto the aforementioned architecture by impregnation, centrifugation and gravimetrically. The present statement refers to the application of a medical device for myocardial infarct treatment.

[0179] The application is foreseen for both in-vitro and in-vivo application. The drug delivery system is introduced for its complementarity with gel element of the device. Both parts of the device can be loaded with cell signaling agent, drugs for research and use. In the perspective of in-vivo application, the fiber based drug delivery system can be foreseen as a direct tool for gel functionalisation with patient blood components.

[0180] Inherent characteristics: The proposed cardiac patch is a medical device that provides structural support to the pericardium and myocardium tissues. The 4 components system mechanical properties matches those of the tissue to be replaced. The 3D hydrophobic/hydrophilic scaffold component provide a mechanical support to a gel that acts as scaffold plasticizer and provide an hydrophilic interface with the host environment. The gel can be easily functionalized with the cell signaling agent by entrapment. The gel can also collect from its immediate environment cell signaling agents. Electrospun fibers based drug delivery system can be used as a complementary tool to the gel for device loading with cell signaling agents, drugs, etc. The electrospun fibers can be used as an intrinsic element gel functionalization for in-vivo and in-vitro application. All components of the proposed cardiac patch are compatible accordingly to 10993:5 and biocompatible with cardiac and bone marrow progenitor cells.

EXAMPLES

Example 1

Natural Polymers/Plasticizers and PCL and Derivatives Thereof/Natural Polymers/Plasticizers Thick and Porous Materials

[0181] It has been defined different strategies for the development of PCL based hybrids scaffolds of tailorable thickness and porosity, production from which scaffolds should be easily amenable to mass production perspective. As presented in FIG. 2 processing routes were investigated and compatible polymeric solutions were studied.

[0182] Among the approaches and the different studied polymeric systems, salt leaching and solvent casting did not provide adequate solutions for highly porous system. Indeed in both cases studies based on a selection of aqueous or organic solvent soluble porogen and FDA approved blowing agents have led to an unsatisfactory porosity.

[0183] On the other hand composite structures compatible with roll to roll technology of tailorable thickness and porosity have been achieved by melt processing (FIG. 3).

[0184] The nature of thermo-calorimetric behaviour of the polymers clearly dictates the selection of the system. For example, PCL is a thermo setting polymer of low glass temperature transition and therefore can be treated/processed by melt pressing at conditions compatibles with hybrids conditions. On the contrary cannot be processed by this technique.

[0185] By virtue of the end product constraint, focus was therefore drawn towards the melting and press approach and thermoplastic polymers were used for such design (FIG. 4)

[0186] Plasticizers were necessary to be introduced in such compositions due to the natural stiffness of the natural polymers and resulting hybrids. All the plasticizers used in the present work have been reported in the literature for biomaterial applications and are FDA approved. The effect of plasticizers onto the mechanical properties of the scaffold matrices were measured as exemplified in FIG. 6.

[0187] Blowing agents and glass beds (FIG. 7 and FIG. 8) were studied as pore generating agents.

[0188] Mixtures were prepared employing different combinations of natural and synthetic polymers and plasticizers (Table 3).

TABLE-US-00003 TABLE 3 Composition patches AMOUNT TOLERANCE COMPONENT ROLE (% wt.) (% wt.) COMPOSITION PATCHES PREPARED BY MELTING BLENDING PCL (or Lactide- Synthetic polymer 50 25 Caprolactone) Chitosan, sodium Natural polymer 35 25 alginate or CAB Tributyl citrate Plasticizer 20 20 or acetyl tributyl citrate Glass powder Porogen agent 40 20 (less than 160 microns) Sodium Blowing agent 4 4 bicarbonate Self-assembled Drug delivery 2 2 peptide system COMPOSITION PATCHES PREPARED BY CASTING AND DRYING PCL (or Lactide- Synthetic polymer 50 25 Caprolactone) Chitosan, sodium Natural polymer 35 25 alginate or CAB Tributyl citrate Plasticizer 20 20 or acetyl tributyl citrate Bioglass S53P4 Vascularization 40 20 agent Self-assembled Drug delivery 2 2 Peptide system

[0189] The strategies carried out to select the most suitable components for the patches are presented in FIG. 5.

[0190] Patches with the synthetic polymers (PCL, L-lactide/-caprolactone copolymer), the natural polymer (sodium salt of alginate, chitosan, CAB) and the plasticizer tributyl citrate were prepared.

[0191] Such materials were then characterized by molecular spectroscopy and calorimetrically (FIG. 9). Modification of melting point (Tm) crystalline polymer with other polymers provides information about their miscibility. Pure PCL employed for this work presents a melting point of 60.8 C. Introduction of plasticizers, natural polymer and glass could affect melting point of PCL. PCL can interact with natural polymers via hydrogen bonding.

[0192] Although the initial work was focused on polymer melting processing and further treatment with hydrofluoric acid to obtain highly porous three-dimensional scaffolds other strategies such as freeze drying and phase inversion process have been studied.

[0193] For this process the composition blends were dissolved or dispersed (in the case of chitosan and the glass powder) in dimethylsulphoxide, which presents a relatively high freezing point (18.5 C.). The studied components for the scaffolds were: PCL and L-lactide/-caprolactone as synthetic polymers, chitosan as natural polymer and bioglass S53P4 with size less than 45 microns as pore generating agent.

[0194] Advantages and drawbacks of both processing techniques (melting processing and phase inversion) are summarized in Table 4.

TABLE-US-00004 TABLE 4 Advantages and drawbacks of selected scaffold processing techniques. Processing Technique Advantages Drawbacks MELTING 1.- No solvent 1.- hydrofluoric acid PROCESSING + 2.-Introduction of (HF) treatment HF additives TREATMENT 3.-Easy mass production of thick and thin scaffolds 4.- Surface porosity: larger pores 5.- Tailorable porosity FREEZE/PHASE 1.- No HF treatment 1.- Surface porosity: INVERSION 2.- Tailorable thickness smaller pores 3.-Easier industrial implantation

[0195] The study of surface porosity by SEM shows that samples prepared by phase inversion presents smaller pores (around 20 microns) than the melt processed ones (dimensions50 microns). Moreover, porosity in melting processing samples can be controlled by the size of used glass powder (see FIG. 10).

[0196] The mechanical properties of patches prepared by phase inversions show to be with suitable elasticity (191% for PCL and 257% for and copolymer L-lactide/-caprolactone patches) and tensile strength for cardiac patch application (see FIG. 11).

Example 2

PCL/Natural Polymers (Chitosan, Cellulose Acetyl Butyrate, Alginic Acid)/FEFEFKFK (PERA-UniMa-PPI)

[0197] In order to facilitate CP end product production and acceptability by the end user, a multi layered architecture where laminas of distinctive but complementary materials have been proposed, where a biocompatible elastomer such as but not limited to polyurethane serves as a base to a thick porous scaffold composed of but not limited to composites of poly(-caprolactone) with natural polymers such as but not limited to alginate and chitosan. While the micro and macro structural features are compatible with angiogenesis, cell seeding, cross-structural nutrient transport, a self-assembled peptide is used to coat this central element to provide an ECM like feature to the device. Device functionalization with chemical and biological cues is achieved through the functionalisation such assemble assembly of peptides. The structure permits the instauration of concentration gradient thereby, facilitating cell differentiation, expansion and proliferation.

[0198] A stichable or sewable medical device will be composed of 3 elements: a melt pressed, freeze-thaw, freeze-dried scaffold with impregnated or supporting a self-assemble polypeptide gel. In such configuration, the synthetic-natural polymer will provide a support for an easily functionalisable peptidic gels. The argument relies on the easy chemical and biological cue entrapment within a gel allied to a synthetic structure of mechanical properties. While such edifice should be place in lieu of the scar tissue for host tissue regeneration therapy, a biocompatible and biodegradable polyurethane support is used to immobilise the patch onto the cardiovascular tissue (FIG. 12).

[0199] Among all the evaluated combinations, best results are obtained with composite structures of low surface charges (low chitosan or alginic acid content) or with neutral polymers such as the PCL/Cellulose acetyl butyrate (FIG. 13)

[0200] Scaffolds prepared as aforementioned are the base of three components patch concept. Components beside scaffold are polyurethane layer for sewing of patch, scaffold and gel for release of growth factor or other possible entrapped agents. These entrapped agents could be but are not limited to: [0201] Bioactive molecules promoting stem cell recruitment, adhesion, proliferation and differentiation: IGF-1 (insulin-like growth factor-1), HGF (hepatocyte growth factor), 5-azacytidine, etc. [0202] Bioactive molecules promoting angiogenesis: VEGF-A (vascular endothelial growth factor A), HGF. [0203] Plasma Enriched Platelets can also been prepared from patient own blood components and entrapped within the gel formulation so as to provide a medical device functionalised with endogeneous chemical and biological cues.

[0204] FIG. 14 shows a schematic representation of three components patch concept. In the following examples (Example 3 and 4) the development of three components system is presented

[0205] The device can be further functionalized with drug delivery system such as but not limited to poly(-caprolactone), and composites thereof where natural polymers are second component of the formulated drug delivery device (FIG. 15).

Example 3

[0206] Preparation of Cardiac Patch

[0207] To facilitate the suture of cardiac patch, polyurethane sewable support was immobilized onto PCL based scaffolds. FIG. 16 represent step by step procedure for the preparation of stitchable cardiac patch.

Example 4

Functionalization of Patches

Polypeptide Impregnation

[0208] Self assembled peptide such as but not limited to FEFEFKFK (SEQ ID NO: 1) polypeptide gel have been immobilized onto/into PCL/chitosan, PCL/alginate and PCL/cellulose acetyl butyrate (CAB) patches. Gels of polypeptides were introduced in the patches by centrifugation and heating. These can also be applied by lamination with wire bar or application blade application technique. Chromatography analysis reveals that all patches tested contains polypeptides. Gel content of patches does not depend on the preparation method or the gel concentration. It depends on the scaffold porosity and natural polymer structure. Best results were obtained for neutral polymers such as CAB (FIG. 13)

Polyurethane Gel Impregnation

[0209] Two types of polyurethane gel are prepared: [0210] Physical crosslinked gel: after 24 hours in water increase its volume 20-25 times. [0211] Thermoreversible gel: polyurethane hydrogel with low LCST (lower critical solution temperature) and a gelification time of 62 seconds at 28 C.

[0212] PU hydrogel absorption in scaffolds has been measured. This value goes from 0.5% wt. to 5.70% wt depending on porosity and composition of scaffold and porosity of polyurethane immobilized layer.

Electrospun Functionalized Nanofibres

[0213] Besides gel impregnation, functionalized fibers have been introduced in the patches for topical drug/trophic agent release (4 components system: polyurethane layer, biopolymer patch, electrospun fibers based drug delivery system and a functionalisable self-assembling peptide). Introduction of fibers could also be a vehicle to load the patches with cell signaling agents or grow factors. has deposited electro spun fibres onto polycaprolactone patches by coaxial electro spinning technique (FIG. 17).

[0214] Coaxial electrospinning technique allows preparing the core of the nano-fiber with one material and the shell with a different one. Using this technique, liposomes and growth factors were encapsulated in the core of the fibre in a PVA solution and the shell of the fibre was made of PCL. Drug profile of growth factors entrapped in coaxially electrospun fibers has been determined. These nanofibers showed good adhesion to the material and were suitable for further use in combination with bioactive substances. According on the results coaxial electro-spinning seems adequate for functionalization of scaffold. Moreover this technique allows the gel immobilization.

Example 5

Characterisation

Swelling Test

[0215] PCL does not show a significant swelling in water or cell mediums such as phosphate buffered saline medium (PBS) due to the hydrophobic nature of this polymer. Blends of PCL with natural polymers present swelling in PBS. PCL/CAB blends, increasing of CAB produce higher swelling: up to 10-12.5% for sample with 30 w % PCL (FIG. 18).

[0216] The swelling in PBS of L-lactide/-caprolactone copolymer blends has been studied. Copolymer/chitosan blends presents more swelling than PCL due to higher hydrophilicity of L-lactide/-caprolactone. Blends with percentage of 20% chitosan presents 16% weight gain for PCL blend and until 42% for L-lactide/-caprolactone (FIG. 19).

Example 6

Biological Evaluation

Biodegradation/Biocompatibility (Long Term Toxicity) Tests

[0217] Tests were carried out in accordance with the supplied protocol.

[0218] Protocol: The in vitro degradation properties were evaluated in three different solutions: [0219] phosphate buffer solution (PBS); [0220] cell culture medium, composed by Dulbecco's modified Eagle's medium with high glucose, 10% fetal bovine serum, 2 mM glutamine, penicillin (100 U/ml) and streptomycin (100 g/ml); [0221] PBS with collagenase (16 U/ml).

[0222] Scaffolds weight losses during degradation are measured by changes in dry weight after incubation for specified time periods. All the experiments should be done in triplicates; the results are the mean (SE) of three determinations.

[0223] 1) Materials samples, cut into squares of 1 cm.sup.2, are dried in oven at 37 C. up to constant weight;

[0224] 2) The starting dry weight, W.sub.0, is determined for each sample;

[0225] 3) Samples are introduced in 10 ml of solution and maintained into an agitating bath at 37 C. for specified time periods (e.g. 1, 3, 7, 14, 21, 30, 45, 60 . . . days);

[0226] 4) At appointed times, samples are removed from solution, rinsed in bi-distilled water and dried in oven at 37 C. up to constant weight;

[0227] 5) The dry weight at time t of degradation, W.sub.t, is determined for each sample;

[0228] 6) Percentage weight loss is evaluated according to the following equation:

(W.sub.oW.sub.t)/W.sub.o100.

[0229] In some cases, the following additional aspects were taken into consideration: [0230] Quantitative determination of the degradation products, using HPLC or UV methods; [0231] Physicochemical characterization of the degradation products, as well as the degraded samples, through FT-IR, DSC, GPC; [0232] Variations of pH in the degradation solution.

[0233] Said protocol has been used with the following modifications: (1) in order to increase the surface area, samples were cut at 3 cm squared (3 cm2); (2) in order to improve the drying process, samples were dried at 45 deg. C,; (3) in order to prevent microbial contamination, samples were sterilized by dipping them in ethanol at each sampling time for about 30 seconds and dried in air to remove excess ethanol; and (4) pH variations have been monitored. Tests were carried out in phosphate saline buffer (PBS), Dulbecco's Modified Eagle Medium DMEM and PBS/Collagenase.

[0234] (PCL or L-lactide/-caprolactone) alginic acid based cardiac patch did lead to any pH change.

[0235] Cytotoxicity tests have been carried out by the University of Pisa. Additionally cell culture tests were also performed. FIG. 20 represents rCPC (Rat Cardiac Progenitor Cells) and BMC (Bone Marrow Progenitor Cells) cell culture after 7 days of culture supporting short and long term non-toxicity of (PCL or L-lactide/-caprolactone) alginic acid based cardiac patch.

Cell Seeding Culture Test of the Poly(-Caprolactone):Chitosan Scaffold Device

[0236] poly(-caprolactone):chitosan was seeded with rat aortic endothelial cells (rAoECs) and rat cardiac cell progenitors (rCPCs), 45.10.sup.3 cells/cm.sup.2 respectively. FIGS. 21 and 22 represent cell survival under long term and chronic toxicity biological evaluation. The results show that the scaffold supports and promotes proliferation of rAoECs and rCPCs (FIG. 23).

Example 7

In-Vivo Studies

[0237] The seeded polyurethane(support)/poly(-caprolactone):chitosan base structure of the cardiac patch were implanted in vivo and sutured onto the on the Left Ventricular (LV) free wall after cryoinjury on the rat heart (FIG. 24).

[0238] At day of implant +4 and +10 days respectively, the animal was sacrificed for immunohistochemical analysis.

[0239] FIG. 25 demonstrates the stability of the proposed device post sacrifice.

[0240] FIGS. 26 and 27 demonstrate the adequateness of the device for soft tissue structural support/ acellular therapy (FIG. 26), cellular therapy without being associated to any inflammatory process.

[0241] From FIG. 27, in-vitro data support static biological evaluation, which qualified the scaffold material as non-toxic and able to support stem cell culture. Furthermore, this supports the claims of the proposed device:

[0242] a. provide a structural support to the damage tissue

[0243] b. can be used with cellular and cell sheet therapy.

[0244] c. Cells seeded onto the scaffold can be expanded in vitro and implanted in-vivo.

[0245] As migration occurs in-wards towards the zone of the lesion, the proposed scaffold is also adequate for transmural applications.

Example 8

Mechanical Properties

[0246] Mechanical properties of the cardiac patches have been determined as a function of natural polymer content in dry and wet conditions showing the adequate mechanical properties of the proposed polymeric scaffolds (Table 4)

TABLE-US-00005 TABLE 4 the tensile properties of selected PCL and L- lactide/-caprolactone based cardiac patches. ELASTIC MODULUS (MPa) Sample: L-lactide- ELASTIC MODULUS (MPa) Inmersion caprolactone/chitosan/ Sample: Time TBC/glass PCL/chitosan/TBC/glass In PBS (125-160 m)/NaHCO.sub.3 (125-160 m)/NaHCO.sub.3 (hours) treated 10 min HF treated 10 min HF 0 13.14 1.41 31.94 8.35 48 4.40 2.22 28.19 2.84 120 5.04 1.53 44.44 58.63

BRIEF DESCRIPTION OF THE FIGURES

[0247] FIG. 1: Schematic representation of Cardiac Patch medical device

[0248] FIG. 2: Polymer processing technologies studied for the production of size and porosity controllable biocompatible and biodegradable polymers for cardiac patch applications

[0249] FIG. 3 Selected examples of scaffolds obtained from PCL and PVA composites by A) solvent casting/particle leaching; B) lyophilisation and C) melt press approach.

[0250] FIG. 4: schematic representation of the Cardiac Patch compositions

[0251] FIG. 5: Schematic representation of chronological experimental work carried out for patches preparation

[0252] FIG. 6: Elongation at maximum load of patches with different concentrations of CAB and plasticizers: acetyl tributyl citrate, epoxidized soybean oil and tributyl citrate.

[0253] FIG. 7: SEM photographs of PCL+chitosan+glass patches treated 5 and 10 minutes with hydrofluoric acid.

[0254] FIG. 8: SEM photographs of PCL/cellulose/TBC/glass powder (diameters of 125 m and 160 m)/NaHCO.sub.3 patches treated with hydrofluoric acid.

[0255] FIG. 9: DSC thermograms of A) PCL pure; B) PCL+30% chitosan 30%, C) PCL+40% chitosan and D) PCL+60% chitosan. Blends B, C and D are prepared with tributyl citrate/glass powder (125 and 160 microns particle size)/NaHCO3 without treatment.

[0256] FIG. 10. SEM photographs of patches prepared by melt processing and freeze/phase inversion.

[0257] FIG. 11. Stress-strain curves: A) PCL-3 (PCL+chitosan+plasticizer+bioglass) and B) copolymer 3 (L-lactide/-caprolactone chitosan+plasticizer+bioglass).

[0258] FIG. 12: Supported cardiac patch concept: 1) a biodegradable film of PEUUs; 2) primer adhesive of poly(hydroxyurethane); 3) PCL based composites structures with impregnated self assemble peptide gels.

[0259] FIG. 13: Evaluation of FEFEFKFK content in PCL/CAB systems

[0260] FIG. 14: schematic representation of a 3 components cardiac patch

[0261] FIG. 15: schematic representation of a 4 component cardiac patch, where numbering represent sites of chemical and/or biological cues.

[0262] FIG. 16: Step by step representation of the preparation of stich able cardiac patch with 3D scaffold polyurethane support and bio-polyurethane adhesive.

[0263] FIG. 17: SEM photographs of patch before and after deposition of electrospun nano fibres and release profile of cardiac patch+coaxial nano fibres PCL/PVA charged with FITC dextran.

[0264] FIG. 18. Percentage weight gain of PCL/cellulose acetyl butyrate blends in PBS medium: [0265] A) (.square-solid.) PCL+10% CAB, (.circle-solid.) PCL+20% CAB, (.box-tangle-solidup.) PCL+30% CAB. Three blend series were prepared with 20% tributyl citrate, 10% glass powder 125 microns, 10% glass powder 160 microns and 3% NaHCO3 and then treated with HF during 10 minutes. [0266] B) (.square-solid.) PCL+10% CAB, (.circle-solid.) PCL+20% CAB, (.box-tangle-solidup.) PCL+30% CAB. Three blend series were prepared with 20% tributyl citrate, 20% glass powder 125 microns, 20% glass powder 160 microns and 3% NaHCO3 and then treated with HF during 10 minutes.

[0267] FIG. 19: Percentage weight gain of L-lactide--caprolactone/chitosan blends in PBS medium: (.square-solid.) 40% chitosan and (.circle-solid.) 20% chitosan. Both blends series were prepared with 20% tributyl citrate, 20% glass powder 125 microns, 20% glass powder 160 microns and 3% NaHCO3 and then treated with HF during 10 minutes.

[0268] FIG. 20: confocal analysis of 7 days culture of rat Cardiac progenitor Cells (CPC) isolated from the heart and Bone Marrow Progenitor Cells (BMC) form EGFP transgenic rats on (PCL or L-lactide/-caprolactone) alginic acid based cardiac patch. Data kindly supplied by Prof. Quaini, University of Parma

[0269] FIG. 21: Fluorescent imaging of rAoEC seeded onto poly(-caprolactone):chitosan using DIL and eGFP as fluroscent cell tracker.

[0270] FIG. 22: Fluorescent imaging of rCPC seeded onto poly(-caprolactone):chitosan using DIL and eGFP as fluorescent cell tracker

[0271] FIG. 23: In Vitro rAoECs/rCPCs survival on proposed scaffold, central element of the cardiac patch

[0272] FIG. 24: Open chest picture of implanted cardiac patch onto rat animal model at the time of sacrifice.

[0273] FIG. 25: pictures of rat heart post sacrifice and pre-immunohistochemical characterisation

[0274] FIG. 26: tissue imaging of injury formation on animal model following cryogenisation of LV, surgical removal of scare tissue and implantation of scaffold (unseeded)

[0275] FIG. 27: Immunochemical staining of CPCs that were seed onto a poly(-caprolactone):chitosan cardiac patch.