Method for preventing formation of calcified deposits and for inactivating xenoantigens in biological matrices

Abstract

To provide a method for preventing the formation of calcified deposits on or inside a biological matrix.SOLUTION: The method comprises the step of contacting an isolated biological matrix with a solution containing a mixture of phenolic compounds.SELECTED DRAWING: None

Description

Background Art

[0001] The present invention is applied to the fields of medicine, biomedicine or veterinary medicine, in particular to the production of biological and biocompatible matrices implanted in the human or animal body.

[0002] The production of bioprosthetic substitutes is currently experiencing significant market growth. Clinical improvements in surgical procedures, reduction of postoperative complications, development and management of new immunomodulatory drugs, all combined with a deeper knowledge of the interaction mechanisms between graft and host, all contribute to promoting the use of biological prostheses composed of animal or homologous tissues. In this regard, one representative sector is the cardiovascular sector, especially in terms of the social and health impacts that the established treatment of heart valve replacement can cause.

[0003] Biomedical technology makes it possible to develop and surgically apply artificial valves for replacement that can mimic the opening and closing functions of a dysfunctional native valve. A desirable bioprosthetic heart valve substitute (BHV) must enable transvalvular flow that can match the flow of a similar native healthy valve, ensure a long lifespan, and not cause the effects of hemolysis or thrombosis.

[0004] The most frequently used valve substitutes are biological prostheses derived from xenogeneic tissues, in particular valves made of pig valves, or pericardium from cows, horses or pigs (porcine).

[0005] Such artificial valves and substitutes are troubled by many serious problems that significantly limit their clinical applications.

[0006] International Patent Application WO2017 / 093147 discloses a method for inactivating xenoantigens, particularly α-Gal, in biological tissues for the manufacture of bioprostheses. Aspects regarding calcification are incidentally mentioned as being proven by the lack of experimental description to support their effectiveness.

[0007] U.S. Patent Application US2014 / 018909 discloses a method intended for application in the cardiovascular field that protects tissues from products of reactions that accumulate in diabetic patients, particularly those resulting from the oxidation of lipid components. These catabolic products, named AGEs, contribute to the mechanism of degeneration of bioprostheses such as blood vessels and heart valves in diabetic patients. The method is disclosed only in relation to pentagalloyl glucose (PGG) and is performed on a collagen matrix obtained by decellularizing porcine heart valves and an elastin matrix obtained by treating previously decellularized porcine carotid arteries with alkali.

[0008] International Patent Application WO01 / 21228 discloses the use of gallotannic acid to reduce the calcification of biological or synthetic tissues that can be used in the manufacture of heart valve replacements.

Summary of the Invention

[0009] The inventors of this patent application have surprisingly discovered a method for increasing the biocompatibility of prostheses, particularly artificial hearts. In particular, the increase in biocompatibility includes one or more of the prevention of the formation of calcification deposits, the inactivation of xenoantigens in the biological matrix, the prevention of thrombus formation in the biological matrix, the prevention of lipid infiltration into the biological matrix, the prevention of the initiation of inflammatory processes mediated by cellular components, and the prevention of the process of bioadhesion to the biological matrix.

Brief Description of the Drawings

[0010]

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Mode for Carrying Out the Invention

[0011] (Object of the Invention) In a first object, the present invention discloses a method for preventing the formation of calcific deposits in a biological matrix.

[0012] In a preferred embodiment, the method includes the step of contacting the biological matrix with a solution containing a mixture of phenolic compounds in the dark at a temperature of 35 ± 2 °C for a time less than 2 hours.

[0013] According to one aspect of the present invention, the disclosed method also inactivates heterologous antigens in the biological matrix.

[0014] According to another aspect of the present invention, the disclosed method prevents thrombus formation on the biological matrix.

[0015] According to a further aspect of the present invention, the disclosed method prevents lipid infiltration into the biological matrix.

[0016] According to still another aspect of the present invention, the disclosed method prevents the initiation of an inflammatory process mediated by cell components.

[0017] According to another further aspect of the present invention, the disclosed method prevents the formation (bioadhesion) of microbial biofilms on the biological matrix.

[0018] In a preferred embodiment, the disclosed method provides one or more of prevention of the formation of calcific deposits, inactivation of heterologous antigens in the biological matrix, prevention of thrombus formation on the biological matrix, prevention of lipid infiltration into the biological matrix, prevention of initiation of an inflammatory process mediated by cellular components, and prevention of the process of bioadhesion to the biological matrix.

[0019] In a more preferred embodiment, the disclosed method provides prevention of the formation of calcific deposits, inactivation of heterologous antigens in the biological matrix, prevention of thrombus formation on the biological matrix, prevention of lipid infiltration into the biological matrix, and prevention of initiation of an inflammatory process mediated by cellular components, and prevention of the process of bioadhesion to the biological matrix.

[0020] The biological matrix obtained by the method of the present invention is another object of the present application.

[0021] The biological matrix obtained by the method of the present invention for use in the treatment of heart diseases in the fields of medicine, biomedicine and / or veterinary medicine represents a second object of the present application.

[0022] In one aspect of the present invention, a method for the treatment of heart diseases in humans or animals is disclosed, which includes the use of a biological matrix obtained according to the method of the present invention.

[0023] A solution containing a phenolic compound or a mixture of phenolic compounds for use in the method of the present patent application represents a further object of the present invention.

[0024] According to another aspect of the present invention, a method for preparing a solution of the present invention containing a mixture of phenolic compounds is disclosed.

[0025] For a further object, the present invention discloses a kit for implementing the method of the present invention.

[0026] (Detailed description of the present invention) According to a first object, the present invention discloses a method for preventing the formation of calcified deposits in a biological matrix.

[0027] For the purposes of the present invention, calcification can occur on the surface of or within the biological matrix.

[0028] For the purposes of this specification, the method according to the present invention is referred to as FACTA TM and is so-called.

[0029] In particular, the method includes the step of contacting the biological matrix with a solution containing a mixture of phenolic compounds.

[0030] For the purposes of the present invention, the biological matrix is a heterologous matrix, i.e., not of human origin.

[0031] In particular, they can be of equine, porcine or bovine origin.

[0032] In a preferred embodiment of the present invention, the biological matrix should be intended as blood vessels, heart valves, tendons, ligaments, pericardium, fascia, dura mater, tympanic membrane, submucosal tissue of the intestine, cartilage, adipose tissue and bone tissue, pelvic, abdominal and breast tissue.

[0033] In a more preferred embodiment, the biological matrix is selected from the group comprising cardiovascular prostheses (heart valves) and pericardial tissue patches.

[0034] Examples of such heart valves are Trifecta TM Valve with Glide TM Technology and Epic TM Mitral Valve (Abbott / St. Jude Medical, St. Paul, MN, USA).

[0035] Other examples for illustrative purposes only are the Sapien 3, Sapien 3 Ultra and Sapien XT transcatheter heart valves (Edwards Lifesciences, Irvine, CA, USA), Inspiris Resilia, Intuity Valve System, Magna Easy, Perimount RSR and Perimount Valve (Edwards Lifesciences, Irvine, CA, USA), Avalus TM , Contegra Valved Conduit, Freestyle TM , Hancock II TM , Mosaic TM and Mosaic TM Ultra, Melody TM and CoreValve transcatheter valve replacement platforms (Medtronic, Minneapolis, MN, USA), Acurate Neo TM and Lotu Edge TM Aortic Valve System (Boston Scientific, Marlborough, MA, USA), Accufit® Transapical Mitral Valve (Sinomed, Tianjin, China), Xinli® (KingstronBio, Jiangsu, China), Bioconduit TM , Biomitral TM , Biopulmonic TM Conduit and Injectable Biopulmonic TM (BioIntegral, Mississauga, ON, Canada).

[0036] In a further preferred embodiment, the heart valve treatable according to the present invention is represented by a heart valve implantable by transcatheter valve implantation (TAVI); the valve needs to be implanted through a catheter and thus needs to be flexible to be accommodated within the catheter.

[0037] For the purposes of the present invention, the phenolic compounds in the above phenolic compound mixture are intended as phenolic compounds or polyphenolic compounds (used herein as synonyms in some cases) selected from the group comprising simple phenols, phenol aldehydes, phenolic acids, phenylamines, phenolic compounds, flavonoids, phenylpropanoids and tannins.

[0038] In particular, the phenolic compounds are selected from the group comprising vanillin, cinnamic acid, phenylalanine, coumarin, xanthone, catechin, flavononide, flavone, chalcone, flavananol, flavanol, leucocyanidin, anthocyanidin, hydroxycinnamic acid, phenylpropanoid.

[0039] More specifically, the phenolic compounds can be selected from the group comprising resveratrol, alliin, sinapine, epigallocatechin, tannic acid, caffeic acid, chlorogenic acid, hydroxytyrosol, rosmarinic acid, naringenin, gallic acid, hesperidin, quinic acid, ellagic acid, pinoresinol, luteolin, apigenin, tangeretin, isorhamnetin, kaempferol, myricetin, eriodictyol, hesperetin, naringenin, theaflavin, thearubigin, daidzein, genistein, glycitein, pterostilbene, delphinidin, malvidin, pelargonidin, peonidin, chicoric acid, ferulic acid, salicylic acid.

[0040] According to one embodiment of the present invention, curcumin can also be used.

[0041] For the purposes of the present invention, derivatives of the phenolic compounds or polyphenolic compounds disclosed above are also included; for example, salts or esters can also be used.

[0042] In one embodiment of the present invention, the solution comprises a mixture of two or more of the phenylpropanoids disclosed above.

[0043] In the solution of the present invention, each phenylpropanoid may be present at a concentration included between about 0.2 to 5 mg / ml ± 0.5 mg / ml (w / total amount of the solution).

[0044] Some solutions according to the above disclosure are shown below: [Table 1]

[0045] In particular, in the method of the present invention, the contacting step is carried out for a time less than 2 hours.

[0046] Preferably, the contacting step is carried out for about 1 hour.

[0047] In a preferred embodiment of the present invention, the contacting step continues for about 30 minutes.

[0048] In a more preferred embodiment, the first contacting step is repeated one more time for 30 minutes.

[0049] Optionally, a rinsing step may be carried out between the two contacting steps.

[0050] According to a preferred embodiment of the present invention, the method is carried out in complete darkness, i.e., avoiding exposure to light.

[0051] Regarding the temperature of the contacting step, it is preferably carried out at a temperature of about 35°C ± 2°C.

[0052] In a preferred embodiment of the present invention, after the contacting step, the matrix may be subjected to one or more washing steps.

[0053] Preferably, the washing step is carried out using a suitable buffer; for example, a suitable buffer may be represented by a phosphate buffer.

[0054] Each washing step may be carried out for about 15 minutes. [[ID=५१]]

[0055] According to one embodiment of the present invention, the method can be carried out on both natural biological matrices and previously treated biological matrices for other purposes, for example, to stabilize the structure of proteins, lipids and cells and to reduce the potential antigenic action of the host.

[0056] For the purposes of the present invention, the biological matrix can include a decellularized extracellular matrix, a partially digested matrix, and gelatin derived from animals.

[0057] For example, before treating the biological matrix according to the method of the present invention, it can be subjected to treatment with glutaraldehyde, formaldehyde and quercetin.

[0058] According to one aspect of the present invention, the disclosed method also inactivates heterologous antibodies in the biological matrix.

[0059] The term "heterologous antigen" is intended to refer to an animal origin that can be recognized by the immune system and induce an antibody / immune-mediated / inflammatory response in a human host organism; herein, the terms "heterologous antigen", "antigen", "xenoantigen", "epitope" and "critical antigen" can have the same meaning and can be used together or in place of each other.

[0060] For the purposes of the present invention, "heterologous antigen" refers to the α-Gal epitope.

[0061] According to another aspect of the present invention, the disclosed method prevents thrombosis and blood clots on and in the biological matrix.

[0062] According to a further aspect of the present invention, the disclosed method prevents lipid infiltration into the biological matrix.

[0063] According to yet another aspect of the present invention, the disclosed method prevents the initiation of an inflammatory process mediated by cell components.

[0064] According to another further aspect of the present invention, the disclosed method prevents the formation (bioadhesion) of microbial biofilms on a biological matrix.

[0065] In particular, the method of the present invention has been proven to prevent the formation of biofilms formed by Staphylococcus aureus by reducing the adhesion ability of the prosthesis surface.

[0066] The method of the present invention is effective against both Gram-negative and Gram-positive cells.

[0067] The biological matrix obtained by the method of the present invention is another object of the present application.

[0068] In particular, the biological matrix is represented by the cardiovascular bioprosthesis obtained by the disclosed method, which has significant resistance to the formation of calcium deposits, thrombus structuring, lipoprotein infiltration, and bacterial adhesion and resulting tissue colonization.

[0069] According to a second object, the present invention discloses a biological matrix obtained by the above method for use in the treatment of heart diseases in the field of medicine or veterinary medicine.

[0070] As a further object of the present invention, a solution containing a mixture of phenolic compounds for use in the method of the present patent application is disclosed.

[0071] In particular, the solution is prepared by mixing two or more of the above phenolic compounds.

[0072] More specifically, the solution can be prepared in a suitable buffer.

[0073] For example, sodium phosphate buffer can be used.

[0074] Alternatively, the mixture of phenolic compounds can be prepared in a solution of NaOH.

[0075] The solution of the present invention may be added with a suitable enzyme imparted with oxidase activity; for example, tyrosinase, L-gulonolactone oxidase, laccase, etc. may be used.

[0076] The solution is preferably prepared in the dark, i.e., avoiding exposure to light.

[0077] According to a third object of the present invention, there is disclosed a kit for carrying out the method disclosed above.

[0078] In particular, the kit includes: A container containing a suitable buffer, A container containing an appropriate amount of a mixture of phenolic compounds to be dissolved, One or more containers holding a washing buffer, An instruction manual including an explanation of the timing and manner of application of the procedure and includes.

[0079] In one aspect of the present invention, the phenolic compound may be present in a powder form to be dissolved in a buffer.

[0080] The present invention will be further described in relation to the following experimental section.

Example

[0081] (Experimental section) In the following experimental section, the term "α-Gal antigen knockout animal" refers to an animal in which the gene encoding the α-galactosyltransferase enzyme is silenced. Such an enzyme plays a role in attacking the membrane glycoproteins and lipoproteins of the α-Gal epitope. Its absence completely lacks the epitope in question and causes the formation of tissue that is completely comparable to human tissue in this regard. In the present invention, the α-Gal antigen knockout animal vascular tissue was used as an absolute negative control.

[0082] Inactivation of Heterologous Antigens Commercially available Trifecta GTTM The bioprosthetic aortic heart valve was removed from the package and washed twice with phosphate buffer for 15 minutes each. Various solutions were prepared based on the phenolic mixture shown above and filtered through a 0.2 μm sterile membrane. Trifecta GT TM The valve was incubated with each single solution at room temperature for 25 ± 5 minutes twice in the dark with moderate but constant stirring.

[0083] At the end of the incubation, the treated bioprosthetic heart valve was washed twice with phosphate buffer for 15 minutes each.

[0084] Original Trifecta GT TM A set of valves was adopted as an untreated off-the-shelf product for comparison.

[0085] The evaluation of the presence of epitopes still active on the surface of the treated samples is based on a modification of the method exemplified by the inventors and is described in Italian Patent No. 0001409783 and EP2,626,701.

[0086] ]> Briefly, treated and untreated Trifecta TM Tissue samples from the valve were placed in test tubes, phosphate buffer was added to make a final volume of 1000 μL - 1500 μL.

[0087] Thereafter, a monoclonal mouse antibody against the α-Gal epitope (in this example, this is an IgM clone called M86) was added at a preferred concentration of [1:50] v / v, and the whole was incubated at 37 ± 2 °C for 120 ± 10 minutes with constant but moderate stirring.

[0088] Finally, the samples were centrifuged at 14,750 × g for 30 ± 5 minutes at ambient temperature.

[0089] During incubation with the M86 antibody, a 96-well plate was prepared with the bottom of the well covered with 5 μg / ml α-Gal / serum albumin in 100 μL of phosphate buffer per well. The plate thus prepared was preferably stabilized at 37°C for all, but was incubated at a temperature of 30°C to 40°C for 60 ± 10 minutes.

[0090] Subsequently, washing was performed 3 times at 300 μL per well with phosphate buffer at ambient temperature. The first wash was allowed to act for 5 minutes, and then the next 2 washes were performed for 3 minutes.

[0091] Blocking was performed at 300 μL per well using serum albumin and then incubated at ambient temperature in the dark for 60 ± 10 minutes. Subsequently, washing was performed 3 times as described above.

[0092] 100 μL of the supernatant collected after centrifugation from each treated sample was added to each well.

[0093] The samples were loaded onto the plate and each type of sample occupied the entire column of the well. Subsequently, the plate was preferably stabilized at 37°C for all, but was incubated at a temperature of 30°C to 40°C for 120 ± 10 minutes.

[0094] Subsequently, washing was performed 3 times as described above, and 100 μL of a solution of a secondary antibody (rabbit polyclonal anti-mouse) conjugated with peroxidase enzyme was added per well (the ideal solutions for such antibodies are known to be [1:1000], [1:500], and [1:100], and preferably the intermediate [1:500] was adopted).

[0095] Subsequently, the plate was preferably stabilized at 37°C for all, but was incubated again at a temperature of 30°C to 40°C for 60 ± 10 minutes.

[0096] Subsequently, the above washing was performed three times. 100 μL of the peroxidase enzyme development solution was added per well, and then the plate was incubated in the dark for 5 ± 1 minutes.

[0097] Subsequently, 50 μL of the stop solution composed of 2M H2SO4 was added per well, and then the plate was read with a plate reader at 450 nm.

[0098] The overall analysis of the various polyphenol mixtures employed showed that it was possible to obtain 80 ± 2% to 95 ± 0.8% inactivation of α-Gal antigen with treatment Trifecta compared to untreated samples. TM valve as compared to untreated samples.

[0099] The same type of treated and untreated valves were subjected to the following in vitro and in vivo analyses.

[0100] In particular, the treatment was carried out using the solution according to the present invention.

[0101] FACTA TM Evaluation of the Stability of Treatment over Time FACTA TM The treatment is based on the action of a mixture containing specific polyphenols according to the present invention. However, a specific dose of polyphenol is toxic (LD50 5 g / Kg) and can accumulate in the liver and kidneys. FACTA TM To confirm the stability of the treatment, the release of phenolic residues over time was evaluated. FACTA TMAfter treatment, the commercial BHV valve leaflets were left in the storage solution as supplied by the manufacturer. The amount of phenolic compounds released from the tissue was measured at various time points of 7 and 14 days as well as 1, 3, and 9 months (n = 9 samples at each time point). As a control, a set of untreated commercial BHV valve leaflets was analyzed at the same time points. The total polyphenol content present in the solution was evaluated using a procedure based on the reaction between the phenolic compounds and the diazonium salt present in the final storage solution under alkaline pH conditions. The reaction product is a stable chromophore and can be detected and quantified by absorbance analysis at a wavelength of 480 nm. The concentration of phenol present in the samples was quantified by external calibration using a catechin standard and expressed as millimolar concentration (mM) of catechin equivalents.

[0102] The results are shown in Figure 1.

[0103] In total, 160 ppm of phenolic compounds were released from the treated tissue over 9 months. The release occurs mostly during the first 14 days after treatment. With regard to the potential for toxicity, it was confirmed that the total amount of polyphenol leakage from the treated tissue does not pose a health risk; in fact, it is lower than the polyphenol concentration present in human plasma after a meal intake.

[0104] In Vitro Mineralization Assay Untreated (B) and FACTA TM Valve leaflets from commercial BHV of untreated (B) and FACTA TM Treated native porcine aortic valve leaflets (WT T) and α-Gal knockout porcine aortic valve leaflets (KO) were incubated at 37 °C for 14 days in pooled normal human serum (Innovative Research, Peary Court Novi, MI) containing 2% penicillin and streptomycin. As a control, a set of samples was incubated in PBS under the same conditions. After incubation, the samples were washed twice in PBS for 10 minutes and subjected to acid hydrolysis in HNO3 at 110 °C for 12 hours. The evaluation of calcium was performed on the hydrolyzed samples by inductively coupled plasma according to the instructions of the EPA6010D method, μgCa2+ It is expressed as the dry degreased weight (d.d.w.) per mg.

[0105] The results are shown in Figure 2.

[0106] FACTA TM [[ID=I1]]Treatment BHV (BT) shows a 90% (B vs. BT, p < 0.05) and 44.4% (BT vs. KO, p < 0.05) reduction in calcification tendency compared to untreated BHV (B) and α-Gal knockout tissue (KO), respectively. The knockout tissue is regarded as the biological support with the least calcification and is used as an absolute negative control in the xenograft-induced calcification test. FACTA TM Treatment BHV has been shown to have approximately 50% less calcification than the KO standard. FACTA TM When the method is applied to natural tissue (WT T, not fixed with glutaraldehyde), the anti-calcification effect compared to the reference tissue KO increases to 85% (WT T vs. KO, p < 0.05).

[0107] Hydrodynamic Evaluation The hydrodynamic performance of the valve was evaluated under simulated pulsatile flow in a Vivitro® Pulse Duplicator System (Vivitro Labs Inc., Victoria, BC, Canada). The left side of the heart was modeled using a flow simulator. The test valve was attached to a custom-made silicon holder (Figure 3) and placed at the aortic position of the pulsatile flow system. A 25-mm Bjork-Shiley tilting disc valve was used as the reference valve at the mitral valve position. The tests were carried out in 0.9% (w / v) NaCl at room temperature. The valve was tested under five pulsatile flow conditions (Table 1) according to ISO 5840-3. The flow conditions corresponded to cardiac outputs of 2.5 to 9 l / min. During the test, the diastolic / systolic blood pressure was set at 80 / 120 mmHg, but the systolic duration occupied 35% of the cardiac cycle. At each flow condition, the valve was pre-treated for 5 minutes, and then the blood pressure and flow signals were recorded for 10 cycles at each flow condition for each valve group (FACTA TM (Treatment BHV, n = 3; untreated BHV, n = 2).

Table 2

[0108] The hydrodynamic performance of the valve was evaluated in terms of the mean pressure drop (MPD) and peak pressure drop (PPD) across the entire valve, the root mean square forward jet flow (Q RMS ) through the valve, the peak forward jet flow (Q ピーク ) through the valve, the valve effective orifice area (EOA), the backflow through the valve during valve closure and while the valve was fully closed, and the valve energy loss. As defined in ISO 5840-3, the EOA was measured using only the volume flow rate and the pressure difference across the entire valve during the positive pressure interval of the forward jet flow period. The results of each test were averaged by the number of cycles recorded (n = 10). The data were analyzed using Microsoft Excel (registered trademark) and Prism (registered trademark) 7 for Windows (v7.03, GraphPad Software lnc., California) and presented as mean ± standard deviation (SD). A two-sided t-test without correction was used to evaluate the significant differences between the treated and untreated groups at a confidence level of 0.05.

[0109] The MPD-Q RMS profiles of the untreated and treated valves averaged over 10 cycles at each flow condition are shown in FIGS. 4A and 4B, respectively. Both valve groups showed excellent reproducibility and similar Q RMS -PD profiles between them.

[0110] The mean EOA of the treated and untreated valves calculated at each flow condition is shown in FIG. 5. Except for the flow condition 100 / 80 (bpm / sv), there was no statistically significant difference in EOA between the untreated and treated groups, and the treated group showed a significantly smaller (p < 0.05) EOA compared to the untreated group.

[0111] The PPD-Q ピーク profiles of the untreated and treated valves averaged over 10 cycles at each condition are shown in FIGS. 6A and 6B, respectively. Both groups showed similar PPD-Q ピーク profiles.

[0112] The average dynamic and static regurgitation volumes of the untreated and treated groups under each flow condition are shown in Fig. 7. The dynamic regurgitation (closure volume) and static regurgitation (closed volume) for both groups were negative, representing regurgitation through the valve. Generally, the regurgitation volume was more prominent at lower flow conditions and increased with increasing flow velocity. No statistically significant difference in the regurgitation volume was observed between the untreated and treated groups.

[0113] The average energy losses of the treated and untreated groups are shown in Fig. 8. As the flow condition increased, opposite patterns were observed for the antegrade bleeding flow and the energy loss during the closed period. The minimum energy loss during the antegrade bleeding flow was observed at the low flow condition (60 / 60) and increased with increasing flow velocity. In contrast, the maximum energy loss during the closed period was observed at the low flow condition and decreased with increasing flow velocity. No significant difference in energy loss was observed between the untreated and treated groups at any of the tested flow conditions.

[0114] Biomechanical Evaluation The uniaxial tensile load at low strain rate until failure was performed on an Instron® tensile testing machine (5967 Dual Column Series) equipped with a 100 N load cell. One valve tip was excised from each valve and used to isolate test specimens with test dimensions of 6 × 3 mm (length × width). The thickness of the specimens was measured at three points along the length of the specimen using a Mitutoyo® thickness gauge and averaged. During the test, each specimen was pre-loaded to 0.01 N at a strain rate of 20 mm / min and then loaded at the same rate until failure. The recorded load-elongation response of each specimen was converted to engineering stress-engineering strain. Using the stress-strain curve, the gradients of the elastic and collagen phases, transition stress and strain, failure strain, and ultimate tensile strength were calculated. The data were analyzed in Excel and presented as mean ± SD. A two-sided t-test without pairing was used to evaluate the significant difference between the treated and untreated groups at a confidence level of 0.05.

[0115] FACTA TMThe average biomechanical parameters of the treatment group and the untreated group are shown in Fig. 9. For the fracture strain, a significant difference was found between the treatment group and the untreated group (p<0.05). FACTA TM No significant differences were found in any of the other parameters between the treatment group and the untreated group. For the elastic phase gradient (Fig. 9A) and transition strain (Fig. 9C) between the two groups, no statistical significance was found, but these parameters were shown to increase (elastic phase gradient) or decrease (transition strain) in the treatment group compared to the untreated group. Higher elastic phase and lower transition strain indicate a stiffer and less extensible material under low strain, respectively.

[0116] In Vivo Study in a Porcine Animal Model FACTA TM Commercially available Trifecta GT BHV (St. Jude Medical, USA) with treatment (n = 5) and without treatment (n = 3) were implanted at the lung position in a porcine animal model. After 1 month of follow-up, the BHV was removed for examination. The explanted BHV was washed twice in sterile PBS at 4°C in 15-minute steps and immediately photographed for macroscopic evaluation (Fig. 10). Subsequently, the valve leaflets were excised individually from each valve. The valve leaflets for histological and immunohistochemical tests were immediately embedded in OCT (optimal cutting temperature) compound, and the remaining valve leaflets were subjected to α-Gal quantification by ELISA test. Non-implanted Trifecta BHV was used as a reference control.

[0117] Gross Examination As can be seen from Fig. 10, the untreated commercially available BHV shows significant fibrous deposits and blood clots on the ventricular surface compared to those treated with FACTA TM These specific characteristics of the untreated BHV can be detected better in Fig. 11, together with the formation of a homogeneous and thick pannus both around the suture ring on the aortic surface and on the valve leaflets.

[0118] Histological Analysis The tissue was embedded in OCT compound (Tissue Tek; Sakura Finetek, Tokyo, Japan), cryocooled with liquid nitrogen, and cut into 6-mm cryosections. Histological analysis was performed using a commercially available kit from Bio-Optica (Milan, Italy) according to the instructions provided by the manufacturer. The histological kits used are shown in Table 2.

Table 3

[0119] Generally, the leaflets from untreated valves show the presence of a homogeneous fibrotic pannus on the ventricular surface, as indicated by the black arrows in Figure 12 (Boxes A and E). Foreign body reactions are clearly seen in all explanted leaflets of both untreated (Figure 12) and commercially available BHV after FACTA TM treatment (Figure 13). Note that in untreated commercially available valves, cellular components can penetrate the matrix (Figure 12, Boxes D and H).

[0120] FACTA TM treatment is thought to exert a barrier effect that counteracts the penetration of inflammatory cell components (Figure 13, Box C) into the interstitium, which causes subsequent degeneration of the extracellular matrix.

[0121] In untreated valves, it is possible to observe the presence of abundant lipid infiltration in the region intrinsic to the fibrotic pannus and adjacent to the cell layer, as shown in Figure 14 (Boxes A, B, C, and D), probably due to the action of monocytes that are unable to process LDL. Monocytes take up lipid droplets but cannot degrade them, becoming foam cells and subsequently functioning as sites for calcium deposition. FACTA TM treatment ensures the preservation of the tissue from this critical state (Figure 14, Boxes E, F, G, and H, no lipid infiltration).

[0122] As can be predicted from the large presence of lipid infiltration, untreated commercial BHV in pigs is significantly calcified after 1 month of follow-up, with significant calcium accumulation particularly at the level of the external interface of the valve leaflets (Figure 15). However, where large calcium deposits are not evident, an extensive spread of microcalcifications affecting the interior of the stroma is shown (Figure 15, boxes D, E, G, and H).

[0123] As can be seen from Figure 16, FACTA TM Treat commercial BHV optimally from calcium deposition.

[0124] As shown in Figure 17, in addition to the development of fibrous pannus, untreated commercial valves showed a very strong tendency for structured thrombi to develop on the ventricular surface of the valve leaflets.

[0125] In particular, Figure 18 shows a panel demonstrating the presence of fibrous pannus leading to structured thrombosis.

[0126] With Mallory trichrome staining, FACTA TM Treated commercial BHV does not show the presence of abnormal fibrosis such as pannus or structured thrombi (Figure 19). It is important to note the presence of pink / violet streaks within the collagen matrix in some of the explanted samples corresponding to elastic fibers (Figure 19, boxes E and F, G and H). Interestingly, such elastic fibers are not seen at all in the explants of untreated commercial BHV.

[0127] Commercially available BHV not implanted in the animal model was histologically treated as a reference control. The tests revealed that this tissue did not show a foreign body reaction (Figure 20, boxes A and B), and as a result, was not affected by the development of fibrous pannus or thrombi (Figure 20, boxes E and F). There are also no lipid infiltrations or calcified deposits (Figure 20, boxes C and D, G and H respectively). Elastic fibers are FACTA TMNote that, as seen in the commercially available BHV of the treatment (Boxes E and F, G and H in Figure 19), it is well represented by Mallory staining (Boxes E and F in Figure 20).

[0128] Figure 21 shows FACTA TM shows different histological stains of the commercially available BHV of the treatment. This panel is FACTA TM summarizes all the improvements that the treatment provides to the bioprosthesis tissue. The barrier effect exerted by the treatment is clearly shown; in fact, the host cell population cannot penetrate into the matrix (Boxes B and D). This barrier effect is further confirmed by the absence of lipid infiltration, as shown in Box E. Finally, there is no calcium deposition in the tissue (Box F).

[0129] Resistance to Bacterial Adhesion FACTA TM The anti-adhesive bacterial activity against treated and untreated commercially available valves was evaluated with the bacterial species Staphylococcus aureus ATCC 6538 (Gram-positive). The bacteria were grown overnight at 37 °C in tryptic soy broth (TSB). The total bacterial amount was evaluated by 10-fold serial dilutions (10-1 to 10-7) in TSB, seeded onto Petri dishes containing the appropriate selective medium (MSA - mannitol selective agar), and kept in an incubator overnight. At the end of the incubation, the colony-forming units (CFU) were counted to determine the effective concentration of the microorganisms. Furthermore, the optical density (OD600) values at 600 nm were determined from each serial dilution to verify the linearity between the latter and the effective microbial mass of the broth. Commercially available FACTA TM Treated and untreated BHV valve tips were cut with a biopsy punch (3 mm in diameter) to obtain the same useful surface for bacterial adhesion. The tissue punches thus obtained were washed with PBS and incubated overnight at room temperature in PBS + gentamicin (300 μg / mL) under moderate but constant stirring.

[0130] After overnight incubation, the commercially available BHV tissue punches were thoroughly washed with PBS to remove the remaining antibiotics. Subsequently, FACTATM The treated and untreated samples were exposed to a bacterial suspension of S. aureus (bacterial load 1×10 7 CFU / mL) at room temperature for 90 minutes with moderate but constant stirring. At the end of the incubation, the tissue samples were subjected to three moderate vortex mixings to facilitate the separation of loosely bound bacteria.

[0131] Subsequently, different tissue punches were homogenized by Ultraturrax, the resulting homogenate was serially diluted, and plated onto Petri dishes containing appropriate selective growth media. Finally, after incubation at 37 °C for 24 hours, colony forming units were counted for each type of sample. The anti-adhesion activity of the bacteria was calculated using the following formula:

Equation

[0132] As shown in Figure 22, the FACTA TM treatment significantly limits the ability of S. aureus to adhere to the tissue surface (a 96±4% decrease).

[0133] Calcium mitigation properties in a wild-type mouse animal model FACTA TM To evaluate the calcium mitigation properties of the FACTA TM technology, treated (F) and untreated (C) pericardial bioprosthetic heart valve (BHV) leaflets from the Trifecta GT TM model (Abbott / St. Jude) were implanted subcutaneously on the backs of C57Bl / 6 wild-type mice. A total of 30 mice were enrolled and each animal received one sample. The tissue samples were explanted after 1, 2, and 4 months of follow-up. As a control sample, calcium quantification was also performed on the original Trifecta GT of the off-the-shelf productThe tip was performed with the label UN (not implanted). The calcium content was evaluated by inductively coupled plasma (ICP - a specific type of mass spectrometry with a calcium quantification threshold of 0.048 μg / mg of tissue) and expressed as μg per mg of dry defatted weight (d.d.w.).

[0134] To further confirm the calcium quantification performed by ICP analysis, FACTA after 4 - month follow - up TM Histological preparations of the treated sample (F) and the untreated sample (C) were made. The histological preparations were analyzed by von Kossa staining, a specific technique for highlighting calcium deposition in biological tissues.

[0135] The results of calcium quantification by ICP analysis are shown in Figure 23.

[0136] The original untreated sample (C) showed a significant tendency to calcify just 1 month after implantation. This tendency continued significantly during the follow - up, resulting in a calcium deposition of 7.17 μg per mg of d.d.w. quantified at the end of 4 months of implantation (Table 1). On the other hand, the FACTA TM treated sample (F) showed only a very small amount of calcium even after 4 - month follow - up. Notably, the calcium content of the F sample did not significantly exceed the total calcium detected by UN, and it was confirmed that there was no calcium uptake over time (Table 1).

Table 4

[0137] The results of von Kossa tissue staining are shown in Figure 24.

[0138] Calcium deposits can be recognized as black spots (indicated by arrows if present). According to ICP analysis, FACTA TMThe treated sample (F) does not show a significant presence of calcium deposition after 4 months of follow-up in the subcutaneous tissue of the mouse. On the other hand, the original untreated control sample (C) shows the presence of various calcium aggregates, some of considerable size (C4 box).

[0139] Calcium mitigation properties in an α-Gal knockout mouse animal model The purpose of the study was to evaluate calcium uptake in leaflets excised from the treated counterparts of commercially available Trifecta GT TM BHV and its FACTA TM when implanted in an animal model of α-Gal knockout (KO) mice. The KO mice are a specially developed BCI-bearing mouse model genetically engineered to suppress the expression of the α-Gal xenoantigen. α-Gal KO mice are characterized by having a human-like immune response mechanism in which the α-Gal antigen stimulates the production of specific anti-Gal antibodies. Leaflets were implanted subcutaneously on the backs of C57BI / 6 α-Gal KO mice.

[0140] Calcium content was evaluated by inductively coupled plasma (ICP - a specific type of mass spectrometry) and expressed as μg per mg of dry defatted weight (d.d.w.). FACTA excised from a commercially available Trifecta GT TM treated leaflets (F) and original leaflets (C) were implanted subcutaneously on the backs of wild-type (WT) and knockout (KO) α-Gal mice. Each animal received one sample.

[0141] As a control sample, calcium quantification was also performed on the original Trifecta GT TM leaflets of the off-the-shelf product, labeled UN (not implanted).

[0142] The results are shown in Figure 25.

[0143] The amount of calcium determined in the F sample at 1 and 2 months follow-up was very low as it was below the ICP quantification threshold (0.048 μg / mg). The calcium content in the F sample did not exceed the total calcium detected by the UN, and it was confirmed that there was no calcium uptake at 1 and 2 months follow-up. On the other hand, the C specimens embedded in the KO showed a significant and homogeneous presence of calcium, indicating an effect of promoting lime deposition. The results show a clear role of α-Gal in promoting calcium deposition in KO mice, similar to what occurs in humans.

[0144] FACTA TM Treatment has been proven effective in neutralizing calcification even in important physiological systems such as the α-Gal KO model.

[0145] The advantages provided by the present invention are immediately apparent to those skilled in the art from the above disclosure.

[0146] For example, the method of the present invention can limit the deposition of calcium salts and, therefore, prevent the formation of episodes of calcific dystrophy of the valve.

[0147] Also, the method of the present invention has been shown to protect treated cardiovascular bioprostheses against the formation of blood clots and structured thrombi.

[0148] The method of the present invention makes it possible to avoid the infiltration of lipoproteins from the blood circulation and, as a result, the initiation of a cell-mediated inflammatory tissue response in the treated cardiovascular bioprosthesis.

[0149] Finally, a method has been developed according to the present invention to avoid bacterial colonization and protect cardiovascular tissue from the occurrence of endocarditis.

[0150] The method disclosed in the present invention has been proven to be extremely stable and safe.

[0151] The collected evidence indicates that it did not significantly change the hydrodynamic and biomechanical integrity of the treatment valve. Nevertheless, the values of the hydrodynamic parameters obtained for the treatment valve are within the range of values indicated in the ISO 5840-3 standard.

[0152] Also, the method has been proven to be able to inhibit the formation of calcific deposits in both in vitro and in vivo studies. FACTA TM The BHV treated with FACTA was clearly not thrombogenic and was protected from cell and lipid infiltration.

[0153] Finally, and importantly, the present invention has devised a method that can be implemented with conventional devices and machines.

[0154] Regarding the kit of the present invention, it aims at the autonomous treatment of a bioprosthesis already prepared by the method according to the present invention, which is useful in medical facilities such as clinics and hospitals.

[0155] The present invention is susceptible to many modifications and changes, all of which are within the scope of the claims.

[0156] Furthermore, all components can be replaced with other technically equivalent components.

Claims

**Claim 1** A method for preventing the formation of calcified deposits on or within an isolated biological matrix, comprising the step of contacting the biological matrix with a solution containing a mixture of phenolic compounds. **Claim 2** The method of claim 1, wherein the contacting is carried out in the dark at a temperature of 35 ± 2 °C for a time of less than 2 hours. **Claim 3** The method according to claim 1 or 2, wherein the solution containing the mixture of phenolic compounds comprises a mixture of phenylpropanoids selected from the group consisting of simple phenols, phenol aldehydes, phenolic acids, phenylamines, phenolic compounds, flavonoids, phenylpropanoids and tannins. **Claim 4** The method according to claim 1 or 2, wherein the solution containing the mixture of phenolic compounds comprises a mixture of phenylpropanoids selected from the group consisting of resveratrol, alloin, cinnarin, epigallocatechin, tannic acid, caffeic acid, chlorogenic acid, hydroxytyrosol, rosmarinic acid, naringenin, gallic acid, hesperidin, quinic acid, elenolic acid, pinoresinol, luteolin, apigenin, tangeretin, isorhamnetin, kaempferol, myricetin, eriodictyol, hesperetin, naringenin, theaflavin, thearubigin, daidzein, genistein, glycitein, pterostilbene, delphinidin, malvidin, pelargonidin, peonidin, chicoric acid, ferulic acid, salicylic acid. **Claim 5** The method according to any one of claims 1 to 4, wherein the solution of phenolic compounds further comprises a suitable buffer. **Claim 6** The method according to any one of claims 1 to 5, wherein in each solution, the phenylpropanoid is contained at a concentration of about 0.2 to 5 mg / ml ± 0.5 mg / ml. **Claim 7** The solution comprises one of the following combinations of phenylpropanoids: 【Table 1】 The method according to any one of claims 1 to 6. **Claim 8** The method according to any one of claims 1 to 7, wherein the isolated biological matrix is represented by blood vessels, heart valves, tendons, ligaments, pericardium, fascia, dura mater, tympanic membrane, intestinal submucosa, cartilage, adipose tissue and bone tissue, pelvic, abdominal and breast tissues, cardiovascular prostheses such as heart valves and heart tissue patches. **Claim 9** The method according to any one of claims 1 to 8, wherein the isolated biological matrix is represented by a transcatheter aortic valve implantation (TAVI) prosthesis or a surgical artificial valve. **Claim 10** The method according to any one of claims 1 to 9, which provides one or more of inactivation of a heterologous antigen in a biological matrix, prevention of thrombosis on or within the biological matrix, prevention of lipid infiltration into the biological matrix, prevention of initiation of an inflammatory process mediated by cell components, and prevention of a bioadhesion process onto the biological matrix.

11. An isolated biological matrix obtained according to the method according to any one of claims 1 to 10.

12. An isolated biological matrix obtained according to the method according to any one of claims 1 to 10, for use in the treatment of heart diseases in the fields of medicine, biomedicine and / or veterinary medicine.

13. A solution containing a mixture of phenolic compounds for use in preventing the formation of calcified deposits in an isolated biological matrix.

14. The solution containing a mixture of phenolic compounds for use in preventing the formation of calcified deposits in the isolated biological matrix according to claim 13, wherein the solution contains a mixture of phenylpropanoids selected from the group consisting of simple phenols, phenol aldehydes, phenolic acids, phenylamines, phenolic compounds, flavonoids, phenylpropanoids and tannins.

15. The mixture of phenolic compounds contains a mixture of phenylpropanoids selected from the group consisting of resveratrol, aloetin, sinapine, epigallocatechin, tannic acid, caffeic acid, chlorogenic acid, hydroxytyrosol, rosmarinic acid, naringenin, gallic acid, hesperidin, quinic acid, ellagic acid, pinoresinol, luteolin, apigenin, tangeretin, isorhamnetin, kaempferol, myricetin, eriodictyol, hesperetin, naringenin, theaflavin, thearubigin, daidzein, genistein, glycitein, pterostilbene, delphinidin, malvidin, pelargonidin, peonidin, chicoric acid, ferulic acid, salicylic acid, the solution according to claim 13 or 14.

16. The solution according to any one of claims 13 to 15, wherein the solution of phenolic compounds further contains a suitable buffer.

17. The solution according to any one of claims 13 to 16, wherein in each solution, the phenylpropanoid is contained at a concentration of about 0.2 to 5 mg / ml ± 0.5 mg / ml.

18. The solution comprises one of the following combinations of phenylpropanoids: 【Table 2】 The solution according to any one of claims 13 to 17, comprising one of them.

19. An isolated biological matrix obtained according to the method of any one of claims 1 to 10 for use in the treatment of heart disease in the fields of medicine, biomedical and / or veterinary medicine.

20. The isolated biological matrix according to claim 19, represented by a transcatheter aortic valve implantation (TAVI) prosthesis or a surgical artificial valve.

21. A method for the treatment of heart disease in humans or animals, comprising the use of an isolated biological matrix obtained according to the method of any one of claims 1 to 10.

22. The method for the treatment of heart disease in humans or animals according to claim 21, wherein the biological matrix is represented by a transcatheter aortic valve implantation (TAVI) prosthesis or a surgical artificial valve.

23. A container containing a suitable buffer, A container containing an appropriate amount of the solution according to any one of claims 13 to 18 to be dissolved in the buffer One or more containers containing a washing buffer, Instructions for use including an explanation of the timing and manner for carrying out the method A kit for carrying out the method of any one of claims 1 to 10, comprising the same.

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