A method for the fixative treatment of biological tissue, which does not include the use of glutaraldehyde

Abstract

The present invention relates to a method for the fixation of biological tissues, which does not require the use of glutaraldehyde or other analogous or derivative fixating agents.

Description

[0001] “A method for the fixative treatment of biological tissue, which does not include the use of glutaraldehyde” DESCRIPTION

[0002] Since 1995, a possible immunological cause in the degeneration of bioprosthetic heart valves (BHVs) has been suspected. In 1996, researchers found that the administration of steroids, which suppress the immune response, significantly reduced bioprosthetic calcification in aortic valve replacement [1], In 1998, another study in the same animal model showed that a glutaraldehyde- fixed (GA) xenogeneic tissue calcified 35 times more than an autologous one [2], A direct relationship between the presence of specific antibodies against the BHV tissue and its relative calcification was reported in 2001 through the implantation of commercial porcine BHV tissue samples in an animal model with 3 weeks of follow-up [3], Studies conducted in the early 2000s revealed that the residual presence of aGal epitopes in BHV tissue can lead to an increase in human anti-galactose titers. This increase is specifically directed towards the Gal alpha-1 , 3-Gal terminal starting from day 10 after the BHV implantation with a peak concentration at 3 months for both anti-Gal IgM and IgG isotypes. [4- 6].

[0003] Studies have shown that the rise in anti-Gal antibodies [4-6] persists for up to five years after BHV implantation [7], which suggests constant antibody stimulation due to the exposure of previously covered aGal antigens. This continuous exposure led to valve degeneration, especially in younger patients with a more active immune system. This aspect is potentially very alarming as it suggests constant antibody stimulation over time, probably due to the depolymerization of GA with the consequent availability of previously covered aGal antigens [8], Consensus guidelines caution the use of BHVs in patients younger than 65, as this predisposition to immunological hyper-reactivity is linked to more frequent valve degeneration phenomena [9],

[0004] Notwithstanding GA is considered the preferred fixative and sterilizing agent for many commercial BHVs, its well-known chemical instability is further implicated in the exposure of potential calcium-binding sites (residual aldehydes, acids, Schiff bases). As a result of the interaction between tissue amino-acid residues and GA, negatively charged carboxylic groups can be created which can electrostatically interact with positively Ca2+charged ions, becoming a high-affinity calcium attraction site. To decrease the influence on the epitope exposure and the calcification process, several improvements in the GA fixation protocols have been proposed by the BHVs manufacturers, including the addition of novel steps, aimed at the stabilization of the reactive chemical groups [4,5], Moreover, following the cytotoxic action of GA, BHVs are rendered non-viable and, by definition, are not capable of extracellular matrix (ECM) regeneration and remodeling. Any changes in the ECM, resulting from cyclic loading (delamination, structural rearrangements, and destruction), are considered irreversible damage. Prolonged accelerated wear testing cycles highlighted a decrease in radial extensibility, due to the stiffening of the effective collagen fiber network. The stiffening of the leaflets over time, causing anomalies in the distribution of mechanical stress, leads to stress concentrations, especially in the bending and sutured areas around the stent.

[0005] A fundamental generational change has occurred over the past 20 years in patients with valve disease: 1) patients are choosing xenograft tissue valves over mechanical valves in all age groups, and 2) patients choose transcatheter valve replacement over surgery. Like the surgical BHVs, transcatheter bioprosthetic heart valves are manufactured using xenogeneic pericardium treated with GA, thus initiating a humoral immune response with the formation of lgG3 antibodies against aGal, neutrophil granulocyte, and C3a complement activation and the secretion of the decoy receptor sST2 in the context of an inflammatory reaction [9], The widespread presence of aGal in BHVs and the confirmation of the trigger effect towards degenerative phenomena encouraged the development of technologies for the inactivation of such xenoantigens

[0026] and for the chemical stabilization of the GA. At present, there is no commercially available solution to ensure such needs in bioprosthetic tissues.

[0006] Summary of the Invention

[0007] The present invention addresses the problem of avoiding the use of GA for fixation, immunological tolerance, and mechanical stability of biological tissue. The invention describes the use of a polyphenol-based crosslinking capable of replacing the action of GA while giving the tissue unprecedented and stable xenoantigen masking over time together with correct biomechanical stability. The tissue treated in this way is also protected from the formation of thrombi, calcium precipitation, and microbial infections.

[0008] Object of the invention An object of the invention is to provide a method for the fixation of biological tissue with a polyphenol compound or of a mixture of polyphenolic compounds.

[0009] The method does not comprise the use of traditional cross-linking agents (also known as fixative agents) such as glutaraldehyde, formaldehyde, or similar or derivatives thereof.

[0010] Another object of the invention is to provide a method for the inactivation of xenoantigens and to ensure adequate biomechanical resistance to the treated biological tissue.

[0011] A further object is to provide a method to ensure an effective protective effect against thrombosis, calcification, and bacterial infections.

[0012] A still further object is to provide a method to ensure the immunological tolerance of the treated tissue in the case of homologous, autologous, allogeneic, isogenic and xenogenic transplants.

[0013] The present invention discloses a method for the treatment of a biological tissue to be contacted with biological fluids.

[0014] As per an embodiment, the biological tissue may be a biological surface.

[0015] As per a preferred aspect, said biological surface is the surface of a medical device.

[0016] As per a more preferred aspect, said biological surface is the surface of a biological prosthesis, which can be a cardiac prosthesis.

[0017] In a second object, the present invention discloses a biological tissue obtained with the method of the invention.

[0018] The medical device, the biological prosthesis, and the cardiac prosthesis comprising the biological tissue or the surface according to the disclosed method do represent further objects of the invention.

[0019] Brief description of the figures

[0020] Figure 1 shows the bovine pericardial samples locked inside bespoke frames.

[0021] Figure 2 shows the pericardial strips prepared for biomechanical testing.

[0022] Figure 3 shows the results of the biomechanical assays of the sample obtained according to Example 1 vs GA samples.

[0023] Figure 4 shows the results of the biomechanical assays of the sample obtained according to Example 2 vs GA samples. Figure 5 shows the bovine pericardial strips prepared for the bending test.

[0024] Detailed description of the invention

[0025] According to the first object, the present invention discloses a method for the fixation of a biological tissue.

[0026] As per the present invention, the method for the treatment of a biological tissue is a crosslinking method.

[0027] For the purposes of the present invention, in the following description, a “biological tissue” shall also comprise or be read as a “biological surface”.

[0028] A biological tissue may be represented by of human or animal origin.

[0029] In particular, biological tissues of animal origin may have an equine, porcine, or bovine origin and preferably have a porcine or bovine origin; such surfaces may be considered as biological matrices.

[0030] In particular, said biological tissue may be or may be part of a medical device.

[0031] Medical devices according to the present invention may be represented by: cardiac valve, tendon, ligament, pericardium, muscular fasciae, dura mater, tympanic membrane, intestinal submucosa, cartilage, adipose and bone tissue, pelvic, abdominal, breast, and dermal tissue.

[0032] As per another aspect of the invention, said biological tissue is or is part of a biological prosthesis.

[0033] A biological prosthesis according to the invention may be represented by: a vessel, a cardiac valve, a tendon, a ligament, pericardium, muscular fasciae, cornea and crystalline intraocular lens, dura mater, tympanic membrane, intestinal submucosa, cartilage, adipose and bone tissue, pelvic, abdominal, breast, and dermal tissue.

[0034] In a preferred embodiment, the biological prosthesis according to the invention is represented by a cardiovascular prosthesis, such as a cardiac valve, or a pericardial tissue patch.

[0035] In a more preferred embodiment, a cardiac valve that can be treated according to the present invention is represented by a surgical heart valve.

[0036] In an even more preferred embodiment, the cardiac valve which can be treated according to the present invention is represented by a trans-catheter implantable heart valve; said valve requires to be implanted through a catheter and is folded to be housed within the catheter. According to the present invention, the biological tissue is to be in contact with a biological fluid.

[0037] A biological fluid within the purposes of the present invention is represented by blood, serum, plasma, vitreous gel, tears, urine, saliva, and feces; including synovial, peritoneal, pericardial, pleural, and amniotic fluid.

[0038] According to the method of the invention, the disclosed biological tissue is contacted with a solution comprising a phenolic compound or a mixture of phenolic compounds.

[0039] For the purposes of the present invention, a phenolic compound shall be intended as a phenolic or polyphenolic compound (in some instances they both are referred to as "phenolic" or "polyphenolic", only) used here as synonyms selected from the group comprising: phenols, phenolic aldehydes, phenolic acids, phenylamines, phenol compounds, flavonoids, phenylpropanoids and tannins.

[0040] In particular, a phenolic compound is selected in the group comprising: vanillin, cinnamic acids, phenylalanine, coumarins, xanthones, catechins, flavonons, flavones, chaicones, flavanonols, flavonols, leucoanthocyanidin, anthocyanidin, hydroxycinnamic acids.

[0041] More, in particular, a phenolic compound can be selected in the group comprising: resveratrol, aloin, cynarin, epigallocatechin, tannic acid, caffeic acid, chlorogenic acid, hydroxytyrosol, rosmarinic acid, narigenin, gallic acid, hesperitin, quinic acid, eleonolic acid, pinoresinol, luteolin, apigenin, tangeritin, isorhamnetin, kaempferol, myricetin, eriodictyol, hesperetin, naringenin, theaflavin, thearubigins, daidzein, genistein, glycitein, pterostilbene, delphinidin, malvidin, pelargonidin, peonidin, chicoric acid, ferulic acid, salicylic acid.

[0042] For the purposes of the present invention, derivatives of the above-disclosed phenolic or polyphenolic compounds are also encompassed; for instance, salts or esters or isomers may also be used.

[0043] In one embodiment of the invention, the solution of the invention comprises a mixture of two or more of the above-disclosed phenolic or polyphenolic compounds.

[0044] As per a preferred embodiment, the solution of the invention may comprise a mixture of two or more of the above-disclosed phenylpropanoids. Here below there are reported some components as well as some solutions according to the invention:

[0045] Table 1

[0046] According to the present invention, the disclosed fixation method does not comprise the use of aldehydes or aldehyde-like cross-linking agents.

[0047] For the present invention, said aldehyde-like cross-linking agents include glutaraldehyde, formaldehyde, or similar compounds or derivative compounds.

[0048] As per the preparation of the invention solution, a phenolic or polyphenolic compound is solubilized in an alcoholic solvent.

[0049] In case a mixture of the phenolic or polyphenolic compounds is prepared, then the solution of each compound is prepared separately and then admixed together.

[0050] According to a preferred embodiment of the invention, the solution comprises a mixture of phenolic compounds and more preferably a mixture of phenylpropanoid compounds.

[0051] For said purposes, a first component is solubilized in an alcoholic solution (Component A), preferably 10% of the final volume of the solution.

[0052] An alcoholic solvent according to the invention may comprise methanol, ethanol, isopropanol, butanol, etc., and preferably comprises or is represented by ethanol. Within the solution of the invention, a second component is solubilized in an isotonic buffered solution (Component B), preferably 90% of the final volume of the solution.

[0053] In one embodiment of the invention, the final solution is a hydroalcoholic solution.

[0054] According to the present invention, in the disclosed method the solution of a phenolic compound or a mixture of phenolic compounds preferably has a pH value of between 2 and 9.

[0055] Once it is prepared, the solution can optionally be filtered with a 0.22 pm filter.

[0056] As per the present invention, in the disclosed method the biological tissue is contacted with the solution of a phenolic compound or of a mixture of phenolic compounds for a period of at least 24 hours.

[0057] As per the present invention, the biological tissue is contacted with the solution of a phenolic compound or of a mixture of phenolic compounds for a period of at least 36, 48, 60 and up to 72 hours.

[0058] Preferably, the contact is continued for a period of about 48 hours.

[0059] In an even more preferred embodiment, the contacting step may comprise a first step with the use of a first solution and a second step with the use of a second solution.

[0060] In a preferred embodiment, the first contacting step is performed for 24 hours and the second contacting step is performed for 24 hours.

[0061] Each contacting step may be performed with the same solution or with a different solution; therefore, the first solution and the second solution may be equal or may be different.

[0062] According to a preferred embodiment of the invention, the method is performed in the dark and more preferably completely in the dark (i.e. avoiding any exposure to light).

[0063] According to a preferred embodiment, the method is performed while stirring the solution.

[0064] As per the temperature of the contacting step, it is preferably performed at a temperature between 0°C ± 40°C.

[0065] In a preferred embodiment of the invention, after the contacting step, the treated biological tissue, medical device, biological prosthesis, or cardiac prosthesis may be subjected to one or more washing steps.

[0066] Preferably, each of said washing steps is performed with a suitable buffer; for example, a suitable buffer may be represented by a phosphate buffer. In an embodiment, each washing step may be performed for a period of about 12-24 hours.

[0067] In another embodiment, each washing step may be performed for a period of about 6-12 hours.

[0068] According to an embodiment of the invention, the method for the fixation of a biological tissue may further comprise a step of treatment with a terpene compound or a mixture of terpene compounds.

[0069] The chemical formula of terpenes is (C5)n, wherein n represents the number of isoprenes present in the compound.

[0070] Examples of monoterpenes and monoterpenoids include geraniol, terpineol, limonene, myrcene, linalool, hinokitiol or pinene.

[0071] Iridoids derive from monoterpenes.

[0072] Examples of iridoids include aucubin and catalpol.

[0073] Examples of diterpenes and diterpenoids are cafestol, kahweol, cembrene and taxadiene. An example of a sesterterpenoid is geranylfarnesol.

[0074] Examples of triterpenes include squalene.

[0075] Examples of tetraterpenes include the acyclic lycopene, the monocyclic gamma-carotene, and the bicyclic alpha- and beta-carotenees.

[0076] Examples of sesquiterpenes and sesquiterpenoids include humulene, farnesenes, farnesol, and geosmin.

[0077] Examples of sesquarterpenoids are ferrugicadiol and tetraprenylcurcumene.

[0078] Examples of norisoprenoids include the C13-norisoprenoid 3-oxo-a-ionol and 7,8- dihydroionone derivatives, such as megastigmane-3,9-diol and 3-oxo-7,8-dihydro-a-ionol.

[0079] According to an embodiment of the present invention, the disclosed method is performed on a biological tissue which may have been previously subjected to a pre-treatment step.

[0080] In particular, said pre-treatment step may be represented by a decellularization treatment.

[0081] For the purposes of the present invention, said pre-treatment step comprises the use of a decellularization solution comprising a decellularization agent or a mixture of decellularization agents. In particular, said decellularization agent may be represented by a cationic, anionic or nonionic detergent, hydroalcoholic solution, and hypo- or hypertonic solution, or a mixture thereof.

[0082] In particular, said detergents can be selected from non-ionic families such as Triton X-100, Tween 20, Tween 80, Igepal CA-630 or anionic families such as Sodium dodecyl sulfate, Sodium deoxycholate, or tauroursodeoxycholate, Dodecanoic acid sodium salt, Lauric acid sodium salt, Sodium laurate and bile salt derivatives or zwitterionic families such as Caprylyl sulfobetaine, 3-[(3- Cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), ASB, DDMAB, MAPCHO and LDAO.

[0083] For the purposes of the present invention, the pre-treatment with any one of the abovedisclosed agents may also provide any of the following effects: stabilizing or removing the cellular component, stabilizing or removing lipids, and lowering the antigenicity.

[0084] According to an embodiment of the present invention, the pre-treatment or the decellularization solution further comprises an anti-oxidant agent, or a mixture of anti-oxidant agents.

[0085] In particular, the anti-oxidant agent is selected from the group comprising: sodium ascorbate, glutathione, lipoic acid, uric acid, carotenes, alpha-tocopherol or ubiquinol.

[0086] Said anti-oxidant agent may have a concentration of 0.01 mM to 2 M.

[0087] According to an embodiment of the present invention, the pre-treatment or the decellularization solution further comprises a chelating agent, or a mixture of chelating agents.

[0088] In particular, the chelating agent is selected from the group comprising EDTA, EGTA, acetylacetonate, polyamines, oxalate, carbonate, crown ethers, glycinate, nitrilotriacetate or bipyridine.

[0089] Said chelating agent may have a concentration of 0.01 mM to 2 M.

[0090] According to an embodiment of the present invention, the pre-treatment solution may be flushed with an inert gas for the elimination of oxygen such as nitrogen for a time between 2 minutes and 8 hours.

[0091] As per the present invention, the method for the treatment of a biological tissue to be contacted with a biological fluid according to the invention is a method to preserve and maintain the proper structural biomechanical properties.

[0092] As per the present invention, the disclosed method is an anti-calcific method. A biological tissue obtained with the method of the invention represents another object of the present application.

[0093] A biological prosthesis, a medical device, and particularly a cardiac prosthesis comprising the biological tissue obtained according to the method of the present application represent further objects of the present invention.

[0094] A biological tissue obtained with the pre-treatment and the treatment method of the invention represents another object of the present application.

[0095] A biological prosthesis, a medical device, and particularly a cardiac prosthesis comprising the biological tissue or the surface obtained according to the pre-treatment and the treatment method of the present application represent further objects of the present invention.

[0096] As per the present invention, the method for the treatment of a biological tissue to be contacted with a biological fluid according to the invention is a protective method.

[0097] In particular, said protective method prevents thrombosis occurrences.

[0098] More in particular, the method of the invention is an anti-platelet adhesiveness method.

[0099] In particular, said protective method prevents calcification.

[0100] In particular, said protective method prevents microbial infection.

[0101] A per the present invention, the method ensure the immunological tolerance of the treated tissue in the case of homologous, autologous, allogeneic, isogenic and xenogenic transplants.

[0102] According to a third object of the invention, it is disclosed a method for the treatment of a disease comprising the use of the medical device, the biological prosthesis or the cardiac prosthesis above disclosed.

[0103] As per a preferred aspect, said disease is a heart disease.

[0104] As per an aspect, said disease is in a human, while per another aspect, said disease is in an animal.

[0105] In a particular embodiment, the method for the treatment of the disease according to the invention comprises a valve-in-valve approach, wherein a valve is deployed inside a dysfunctional valve without its preliminary removal.

[0106] As a further object of the present invention, it is disclosed the use of a solution comprising a phenolic compound or a mixture of phenolic compounds in the method of the present application. In particular, the preparation of said solution comprises a first step, wherein the phenolic compound is solubilized in an alcoholic solvent.

[0107] For instance, a phenolic compound from the above List A may be solved.

[0108] If necessary, a further phenolic compound can be solubilized in an isotonic buffered solution.

[0109] For instance, a phenolic compound from the above List B may be solved.

[0110] According to a preferred embodiment, the solution of compound A represents 10% (volume) of the final solution and the solution of compound B represents 90% (volume) of the final solution.

[0111] According to a preferred aspect, the preparation of the solution is carried out in the dark and preferably in complete darkness, i.e. avoiding any exposure to light.

[0112] The present invention will be further described in connection with the following experimental section.

[0113] The following experimental session shows the results of assays carried out according to the present invention.

[0114] Bovine pericardial patch preparation

[0115] Bovine pericardium was obtained from a certified abattoir (Inalca Spa, Castelvetro di Modena, Italy) and transported to the laboratory under controlled conditions. The pericardial samples were harvested from the anterior region of the heart after washing thoroughly in sterile phosphate-buffered saline (PBS) at 4°C. The most homogeneous portions of pericardial tissue were selected through the use of an optical viewer and the thickness measurement (MTG, Digital Material Thickness Gauge, ELECTROMATIC Equipment Co., New York). The selected pericardium area was mechanically cleaned from adipose tissue debris and dissected. The samples were placed into 12cmx10cm bespoke frames (Figure 1).

[0116] Direct crosslinking treatment with polyphenols

[0117] The frames containing the samples are subjected, for a time between 1 and 72 hours (in the example described 24 hours), to treatment in an isotonic phosphate buffer at a pH between 2 and 9 (in the example pH 5) containing a total concentration of polyphenols, among the various proposals in table 1 , between 0.001 mg / ml and 25 mg / ml (in the example 1mg / ml was used). The treatment is carried out at a temperature between 0°C and 40°C (in the example it was set at 20°C) under moderate but constant stirring in the dark. Optionally, the treatment can be carried out in a single step or in multiple steps up to a maximum of 5 (in the example a single treatment step was conducted).

[0118] After the crosslink with the polyphenolic mixture, washings in isotonic solution (in the example phosphate buffer was used) followed for a time between 5 minutes and 5 days (in the example they were washed for 8 hours). Optionally washes can be carried out in a single step or multiple steps (in the example two steps of 4 hours each were performed).

[0119] The samples thus prepared were labeled as “Example 1”.

[0120] Pre-treatment before polyphenol-crosslink

[0121] Pre-treatment

[0122] The frames containing the samples are subjected for a time between 1 and 24 hours (in the example described 8 hours) to a treatment in a hypotonic solution (in the example deionized water) containing an anionic detergent (in the example sodium dodecanoate) at a concentration between 0.01% and 5% v / v (in the example a concentration of 0.4% was used). The treatment is carried out at a temperature between 0°C and 40°C (in the example it was set at 4°C) under moderate but constant stirring.

[0123] Optionally, a series of washes with a hypotonic hydroalcoholic solution (with an alcohol concentration between 0.5% and 95% v / v) can follow. In the example, two washes lasting 4 hours each were carried out with a solution of deionized water with a percentage of 10% v / v of isopropanol (ethanol, butanol, isopropyl, isobutyl, n-butyl, cetyl, allyl can also be used).

[0124] The frames containing the samples move forwards for a time between 1 and 24 hours (in the example described 8 hours) to treatment in an isotonic solution (in the example phosphate buffer) containing a non-anionic detergent (in the example sodium Tween 80) at a concentration between 0.01% and 15% v / v (in the example a concentration of 1% was used). The treatment is carried out at a temperature between 0°C and 40°C (in the example it was set at 4°C) under moderate but constant stirring.

[0125] Optionally, a series of washes with an isotonic solution can follow. In the example, two washes lasting 4 hours each were carried out with a solution of phosphate buffer. The frames containing the samples move forwards for a time between 1 and 24 hours (in the example described 8 hours) to treatment in an isotonic hydroalcoholic solution with an alcohol concentration between 1 % and 95% v / v (in the example 60% of ethanol plus 10% isopropanol). The treatment is carried out at a temperature between 0°C and 40°C (in the example it was set at 4°C) under moderate but constant stirring.

[0126] A series of washes with an isotonic solution can follow. In the example, two washes lasting 4 hours each were carried out with a solution of phosphate buffer.

[0127] Polyphenolic crosslink

[0128] The frames containing the pre-treated samples are subjected, for a time between 1 and 72 hours (in the example described 48 hours), to treatment in an isotonic phosphate buffer at a pH between 2 and 9 (in the example pH 5) containing a total concentration of polyphenols, among the various proposals in Table 1 , between 0.001 mg / ml and 25 mg / ml (in the example 0.5mg / ml was used). The treatment is carried out at a temperature between 0°C and 40°C (in the example it was set at 20°C) under moderate but constant stirring in the dark. Optionally, the treatment can be carried out in a single step or in multiple steps up to a maximum of 5 (in the example two steps of 24 hours each were done).

[0129] After the crosslink with the polyphenolic mixture, washings in isotonic solution (in the example phosphate buffer was used) followed for a time between 5 minutes and 5 days (in the example they were washed for 8 hours). Optionally washes can be carried out in a single step or multiple steps (in the example two steps of 4 hours each were performed).

[0130] The samples thus prepared were labeled as “Example 2”.

[0131] Biomechanical assessment

[0132] Strips were obtained from each GA, Examplel , and Example2 pericardial patch (about 10 mm long and 8 mm wide). Each stripe was mounted on the tensile equipment to undergo a uniaxial tensile test fixed with rubber grips. The sample's initial length (distance between the grips) was set as 50 mm (Figure 2).

[0133] Each stripe was dimensionally characterized in terms of length (useful length 50 mm), width, and thickness (average value of the measurements), and the cross-sectional area was calculated (with x thickness). The following parameters were obtained from each tensile curve:

[0134] • Breaking strength [N], in terms of maximum strength before failure;

[0135] • Ultimate tensile strength [MPa], in terms of maximum strength divided by the cross-sectional area (UTS);

[0136] • Failure strain [%], in terms of strain at the maximum strength;

[0137] • Young’s Modulus [MPa], in terms of the slope of the linear region of the stress-strain curve (elastic phase)

[0138] For each parameter, the average value was calculated from the samples, obtaining one value for each patch. The average value was calculated again from the patches, obtaining one value for each test group.

[0139] The results of Example 1 are shown in Figure 3.

[0140] The samples subjected to direct crosslinking with the polyphenolic mixture (Example 1 , n=10), exhibited the same ultimate tensile strength as the samples fixed in glutaraldehyde (GA, n=10), but showed a surprisingly significative increase in elongation (p<0.05). Consequently, the increase in elasticity translates into a decrease in Young’s modulus.

[0141] The results of Example 2 are shown in Figure 4.

[0142] The samples subjected to pre-treatment before the crosslinking with the polyphenolic mixture (Example 2, n=10), exhibited the same elongation as the samples fixed in glutaraldehyde (GA, n=10), but showed a surprisingly significative increase in braking force (p<0.05).

[0143] Shrinking temperature assessment

[0144] Tinius Olsen oven with temperature controller was fitted in a Tinius Olsen uniaxial tensile testing rig, which was fitted with a 1 kN load cell. The top grip of a bespoke holder was mounted to the load cell, which was kept outside the oven, through a hole in the ceiling of the oven, whilst the bottom grip was securely mounted on the floor of the oven. An RS-Pro K-type thermocouple was placed next to the grips of the holder and connected to an Omega UTC-USB data logger to monitor the oven-enclosure temperature. The pericardial strips were clamped between the grips of the holder that allowing for a gauge length of the samples of 30mm. For each sample, testing was initiated at room temperature (RT; 21°C-25°C). After mounting, the samples were stretched at a rate of 10mm / min until a load of 0.6 N was registered. Once the 0.6 N preloading was achieved, the length of the samples was maintained constant while the temperature was increased to 90°C at a rate of 0.3°C / s.

[0145] During testing, the time in seconds (s) and the temperature in °C were recorded by the datalogger that was connected to the thermocouple were recorded, whilst the time in seconds and force in Newtons (N) were recorded by the Horizon software of the uniaxial tensile testing rig. The temperature that corresponded to the first increase of the force during the testing phase of maintaining the sample length constant was taken as the shrinkage temperature of the sample.

[0146] Table 2 - Comparison of shrinking temperature expressed as °C between standard glutaraldehyde-fixed bovine pericardial strip (n=10, GA), direct polyphenol crosslinking (n=10, Example 1), and pre-treated and polyphenol crosslinked (n=10, Example 2) samples. (Mean ± SD).

[0147] The shrinkage temperature is a parameter adopted to determine the effectiveness of a fixation treatment. In this specific case, the samples cross-linked directly with the polyphenols (Example"!) showed a slightly lower temperature than the reference ones fixed with glutaraldehyde (GA), but the difference is very limited and does not necessarily lead to negative consequences (a native unfixed bovine pericardium generally has a shrinkage temperature around 60°C). As regards the samples pre-treated and then crosslinked with polyphenols (Example2) the temperatures recorded are not statistically different from the GA reference samples.

[0148] Bending test

[0149] To characterize furtherly the polyphenolic crosslinking effect, a “bending test” has been performed. Briefly, pericardial strips belonging to the GA, Example"! , and Example2 groups (n=10 for each group) were clamped by a grip on one of their shorter sides. They were then placed in front of graph paper, taking a specific point as a reference. The opposite sample ending was then left free, and the gravity effect on the free extremity was analyzed. The difference (in degrees) formed between the axis of the specimen in its undeformed and deformed configurations was measured and reported as an “angle of bend” (Figure 5). A high self-sustenance is directly related to a higher tissue crosslinking effect. The lower the specimen self-maintenance, the greater the angle of bend. Angles of bend were simply evaluated through Imaged software (National Institute of Mental Health, USA).

[0150] Table 3 - Bending angle assessment (mean ± SD).

[0151] Together with the angle detection, it was decided to conduct a simple specimen thickness investigation. From the pre-recorded images, sample thickness was therefore determined using Imaged software.

[0152] Table 4 - Thickness samples measurement expressed in millimeters (mean ± SD).

[0153] The results showed that there were no statistically significant differences regarding the passive flexion capacity between the samples (Table 3). Crosslinking with polyphenols, whether direct or preceded by a pre-treatment, does not lead to changes in the thickness of the pericardium, but it makes the samples more homogeneous with each other by lowering the standard deviations (Table 4).

[0154] Xenoantigens inactivation

[0155] The xenoantigens inactivation was carried out considering as reference the number of the aGal epitopes. Data were expressed as a percentage of reduction compared to untreated pericardial tissue. Generally, to quantify the presence of aGal, each strip was divided into three parts of approximately 1cm2each. The weight of each part was recorded after gently blotting it on filter paper. The specimens were then incubated with primary anti-Gal IgG antibody aGaloMab [1 :40.000] for two hours at 37°C with gentle stirring. Finally, they were centrifuged at 14,340 g for 30 minutes at 4°C.

[0156] To coat a Polysorp 96-well plate, 50 pl of aGal / HSA (Human serum albumin; Dextra Laboratories, Berkshire, UK), 5 pg / ml, was added and incubated for 2 hours at 37°C. After washing three times with PBS, 300 pl per well of 1 % HSA (Human serum albumin; Sigma, St. Louis, MO, USA) in PBS was used for blocking and incubated for 2 hours at room temperature in darkness. The wells were rewashed three times. A set of wells was loaded with 100 l of supernatant from untreated (NT), glutaraldehyde fixed (GA), direct polyphenol crosslinked (Examplel) and pre-treated plus polyphenol crosslinked (Example2) samples and incubated overnight at 4°C in darkness. After washing, a secondary HRP- conjugate antibody [1 :500] (Dako Cytomation, Glostrup, Denmark) was loaded. Finally, 100 pl of horseradish peroxidase substrate buffer was added to each well for 30 minutes at room temperature in darkness. A plate reader measured the plate absorbance at 450 nm (Skyscan, Thermo Scientific, Waltham, Massachusetts, USA).

[0157] Table 5 - Percentage of alpha-Gal antigen inactivation following different treatments (n=12 for each treatment) obtained through comparison with the number of epitopes originally present in an original bovine pericardial tissue (NT, untreated). (Mean ± SD)

[0158] Protective effects from thrombosis

[0159] GA, Examplel and Example2 pericardial samples (n=10 for each) underwent a Thrombin Generation Assay Test (TGA, Haemoscan, Groningen, Netherlands) which is based on a special plasma product that enables the determination of thrombin activity in an incubation medium after this has been exposed to a biomaterial. This method is suited to evaluate the haemocompatibility of biomaterials and medical devices according to the international standard ISO 10993-4.

[0160] Specimens were processed by following the instructions provided by the manufacturer. Briefly, tissues were incubated in modified human plasma (plasma was provided by the manufacturer) with subsequent withdrawals at different time points. The thrombin concentration of the samples was determined from a calibration curve of optical density at 405 nm. The thrombin generation curve for each specimen was constructed by plotting the thrombin concentration versus the time points at which the samples were taken. The curve is used to determine the speed of thrombin generation, expressed as per cm2of the sample. Reference materials were provided by the manufacturer, in particular: Polydimethylsiloxane (REF 1) — a low propensity to thrombin generation, and Medical steel (REF 2) — a high propensity to thrombin generation. The results are expressed as a percentage of the decrease / increase in thrombogenic activity compared to the reference material REF2 (Medical steel, high thrombogenic activity) and REF 1 (Polydimthylsoloxane, low propensity to thrombin generation).

[0161] Table 6 - Percentage of the decrease / increase in thrombogenic activity compared to the reference material REF2 (Medical steel, high thrombogenic activity) and REF 1 (Polydimthylsoloxane, low propensity to thrombin generation). (Mean ± SD).

[0162] The fixation with glutaraldehyde (GA) guarantees a good protective effect against the formation of thrombi, lowering the risk of thrombosis by up to 30% compared to medical steel (reference material considered to be highly thrombogenic). However, the treatment with polyphenols has shown surprising results, confirming how it is almost possible to obtain a behavior similar to a polymeric material used in the medical field REF 1 (Polydimethylsiloxane) considered non- thrombogenic. On average, the samples treated with polyphenols (directly or after pre-treatment) showed a risk of thrombosis similar to that of a REF 1 material.

[0163] In-vitro calcification propensity assessment

[0164] GA, Example"! , and Example2 samples (n=10 for each) were gently blotted on filter paper, and their weight was recorded. Samples were subsequently incubated in pooled normal human serum with 2% penicillin and streptomycin for 14 days at 37°C. As a control, a set of samples was incubated just in PBS in the same condition.

[0165] After incubation, samples were washed twice in isotonic phosphate buffer for 10 minutes and subsequently subjected to acid hydrolysis in HCI 6N at 110°C for 12 hours. Calcium evaluation was performed in hydrolyzed samples by inductively coupled plasma according to the directives of the EPA6010D method and expressed as ig Ca2+ / mg of dry defatted weight (ddw).

[0166] Table 7 - Calcium evaluation performed in hydrolyzed samples by inductively coupled plasma expressed as pg Ca27mg of dry defatted weight (ddw). (Mean ± SD)

[0167] Compared to the samples in bovine pericardium fixed in glutaraldehyde (GA), the direct treatment with the polyphenols (Example"!) showed a decrease in the calcific propensity of 95.11 %, while the pre-treatment followed by the crosslink with the polyphenols (Example2) was able to reduce the calcific propensity up to 98.75%.

[0168] Resistance to Tissue Bacterial Adhesion

[0169] The anti-adhesive bacterial activity on GA, Examplel , and Example2 (n=5 for each) pericardial sample was evaluated regarding the Staphylococcus aureus (S. aureus) ATCC 6538 (gram positive). The bacteria were grown overnight in Tryptic Soy Broth (TSB) at 37°C. The total bacterial load was assessed by 10-factor serial dilutions in TSB (10-1 to 10-7), sown in Petri dishes with appropriate selective medium (MSA-Mannitol Selective Agar), and kept in an overnight incubator. Following incubation, the CFU were counted to determine the effective concentration of the microorganism. Furthermore, the optical density at 600 nm was determined from each tiled dilution, to verify the linearity between the latter and the effective microbial load of the broth.

[0170] Samples were isolated using a biopsy punch (3 mm in diameter), to obtain the same effective surface for bacterial adhesion. To eliminate any bacterial load before the adhesiveness test, the punched leaflets samples were washed with isotonic buffer and incubated overnight at RT in phosphate buffer (PBS), supplemented with gentamicin (300 pg / mL) under moderate but constant agitation. Following overnight incubation, the samples were washed extensively in PBS to remove any remaining antibiotics that could skew the test results.

[0171] Subsequently, the samples were exposed to S. aureus bacterial suspensions (bacterial load 1 x107CFU / mL) for 90 min at RT under moderate but constant agitation. The samples were then subjected to three moderate vortexing passages to facilitate the detachment of the loosely bound bacteria. Finally, the samples were homogenized (Ultra-Turrax, IKA, Germany), and serial dilutions of the obtained homogenates were plated in Petri dishes containing the appropriate selective growth media. Finally, after 24 hs of incubation at 37°C, the CFU were counted for each type of sample. The results are expressed as a percentage by calculating the reduction in bacterial adhesion of the samples treated with polyphenols compared to the number of bacteria attached in the samples treated with glutaraldehyde (therefore considered as 100% of the bacteria capable of adhering).

[0172] Table 8 - Percentage of S. aureus adhesiveness in polyphenol-treated samples compared to glutaraldehyde fixed ones (mean ± SD).

[0173] From the above disclosure, there will be evident advantages of the method of the present invention. In particular, the method of the invention has proved not to alter the other properties of the treated and optionally pre-treated according to the above disclosure, and of the medical devices, biological prosthesis, and particularly cardiac prosthesis comprising said surfaces.

[0174] As another advantage, the disclosed method has been shown to ensure an improvement in the biomechanical features of the treated or pre-treated surface.

[0175] In addition, the method of the invention prevents the impairment of the functionality of the treated or pre-treated surface avoiding thrombosis, calcification, and infection.

[0176] Advantageously, the disclosed method can replace the fixation treatment with aldehydes or other fixative compounds, such as glutaraldehyde, formalin, and formaldehyde, through a direct crosslink treatment based on polyphenols.

[0177] Optionally, such a fixation can be preceded by a pre-treatment for stabilizing the tissue before the polyphenolic crosslink.

[0178] References

[0179] 1. Boer II, Buettner FFR, Schridde A, Klingenberg M, Sarikouch S, Haverich A, et al. Antibody formation towards porcine tissue in patients implanted with crosslinked heart valves is directed to antigenic tissue proteins and aGal epitopes and is reduced in healthy vegetarian subjects. Xenotransplantation 2017;24(2). doi: 10.1111 / xen.12288.

[0180] 2. Huma P, Bezuidenhout D, Torrianni M, Hendriks M, Zilla P. Optimization of diamine bridges in glutaraldehyde treated bioprosthetic aortic wall tissue. Biomaterials 2002;23:2099-2103. doi: 10.1016 / s0142-9612(01)00302-7.

[0181] 3.Yokoyama Y, Sakurai Y, Kuno T, Takagi H, Fukuhara S. Externally mounted versus internally mounted leaflet aortic bovine pericardial bioprosthesis: meta-analysis. Gen Thorac Cardiovasc Surg 2023;71 :207-215. doi: 10.1007 / s11748-022-01904-5.

[0182] 4. Konakci KZ, Bohle B, Blumer R, Hoetzenecker W, Roth G, Moser B, et al. Alpha-Gal on bioprostheses: xenograft immune response in cardiac surgery. EurJ Clin Invest 2005;35:17-23. doi: 10.1111 / j.1365-2362.2005.01441. x.

[0183] 5. Mangold A, Szerafin T, Hoetzenecker K, Hacker S, Lichtenauer M, Niederpold T, et al. Alpha-Gal specific IgG immune response after implantation of bioprostheses. Thorac Cardiovasc Surg 2009;57:191-5. doi: 10.1055 / S-0029- 1185395.

[0184] 6. Park CS, Oh SS, Kim YE, Choi SY, Lim HG, Ahn H, et al. Anti-alpha-Gal antibody response following xenogeneic heart valve implantation in adults. J Heart Valve Dis 2013;22:222-229.

[0185] 7. llchino G, Murakami H, Mukohara N, Tanaka H, Nomura Y, Miyahara S, et al. Modes of the bioprosthetic valve failure of the porcine and pericardial valves in the mitral position. Eur J Cardiothorac Surg 2022;62:ezab506. doi: 10.1093 / ejcts / ezab506.

[0186] 8. Stacchino C, Bona G, Bonetti F, Rinaldi S, Della CL, Grignani A. Detoxification process for glutaraldehyde treated bovine pericardium: biological, chemical and mechanical characterization. J Heart Valve Dis 1998;7:190-194.

[0187] 9. Copic D, Bormann D, Direder M, Ankersmit HJ. Alpha-Gal-specific humoral immune response and reported clinical consequence for cardiac valve replacement in patients below 65 years: moving beyond conjecture. Eur J Cardiothorac Surg 2022;62:ezac227. doi: 10.1093 / ejcts / ezac227.

Claims

CLAIMS:

1. A method for the fixation of a biological tissue comprising the step of subjecting said biological tissue to a contacting step with a solution comprising a phenolic compound or a mixture of phenolic compounds for a period of at least 24 hours.

2. The method for the fixation of a biological tissue according to claim 1 , wherein said period is of at least 36, 48, 60 and up to 72 hours.

3. The method for the fixation of a biological tissue according to the preceding claim 1 or 2, which does not comprise the use of aldehydes or aldehyde like cross-linking agents.

4. The method for the fixation of a biological tissue according to the preceding claim, wherein said aldehydes or said aldehyde like cross-linking agent are selected from glutaraldehyde or formaldehyde, or similar compounds or derivative compounds.

5. The method for the fixation of a biological tissue according to any one of the preceding claims, wherein said contacting step is performed in the dark.

6. The method for the fixation of a biological tissue according to any one of the preceding claims, wherein said contacting step is performed at a temperature up to 40°C.

7. The method for the fixation of a biological tissue according to any one of the preceding claims, wherein said biological tissue is part of a medical device.

8. The method for the fixation of a biological tissue according to any one of the preceding claims 1 to 6, wherein biological tissue is a biological surface.

9. The method for the fixation of a biological tissue according to the preceding claim, wherein said biological surface is part of a medical device.

10. The method for the fixation of a biological tissue according to any one of the preceding claims 1 to 8, wherein said biological surface is the surface of a biological prosthesis.

11. The method for the fixation of a biological tissue according to anyone of the preceding claims 7 to 10, wherein said medical device or said biological prosthesis may be represented by cardiac valve, tendon, ligament, pericardium, muscular fasciae, cornea and crystalline intraocular lens, dura mater, tympanic membrane, intestinal submucosa, cartilage, adipose and bone tissue, pelvic, abdominal, breast, and dermal tissue.

12. The method for the fixation of a biological tissue according to any one of the preceding claims 8 to 11 , wherein said biological surface is the surface of a cardiac prosthesis or a cardiac valve or a pericardial tissue patch or a surgical heart valve.

13. The method for the fixation of a biological tissue according to any one of the preceding claims, wherein said biological tissue is to be in contact with a biological fluid selected in the group comprising: blood, serum, plasma, vitreous gel, tears, urine, saliva, faeces; including synovial, peritoneal, pericardial, pleural and amniotic fluid.

14. The method for the fixation of a biological tissue according to any one of the preceding claims, wherein said phenolic compound or said mixture of phenolic compounds are selected from the group comprising: phenols, phenolic aldehydes, phenolic acids, phenylamines, phenol compounds, flavonoids, phenylpropanoids and tannins.

15. The method for the fixation of a biological tissue according to the preceding claim, wherein said phenolic compound or said mixture of phenolic compounds are selected from the group comprising: vanillin, cinnamic acids, phenylalanine, coumarins, xanthones, catechins, flavonons, flavones, chaicones, flavanonols, flavanols, leucoanthocyanidin, anthocyanidin, hydroxycinnamic acids.

16. The method for the fixation of a biological tissue according to the preceding claim, wherein said phenolic compound or a mixture of phenolic compounds is selected from the group comprising: resveratrol, aloin, cynarin, epigallocatechin, tannic acid, caffeic acid, chlorogenic acid, hydroxytyrosol, rosmarinic acid, narigenin, gallic acid, hesperitin, quinic acid, eleonolic acid, pinoresinol, luteolin, apigenin, tangeritin, isorhamnetin, kaempferol, myricetin, eriodictyol, hesperetin, naringenin, theaflavin, thearubigins, daidzein, genistein, glycitein, pterostilbene, delphinidin, malvidin, pelargonidin, peonidin, chicoric acid, ferulic acid, salicylic acid.

17. The method for the fixation of a biological tissue according to any one of the preceding claims, wherein said method comprises a first step of contacting said biological tissue with a first solution of said phenolic compound or of a mixture of said phenolic compounds and a second step of contacting said biological tissue with a second solution of said phenolic compound or a mixture of said phenolic compounds.

18. The method for the fixation of a biological tissue according to the preceding claim, wherein said method comprises a first and a second contacting step, wherein between said first and said contacting second step a washing step is performed.

19. The method for the fixation of a biological tissue according to any one of the preceding claims, further comprising a step of treatment with a terpene compound or a mixture of terpene compounds.

20. The method for the fixation of a biological tissue according to any one of the preceding claims, wherein said method comprises a pretreatment step of decellularization.

21. The method for the fixation of a biological tissue according to the preceding claim, wherein said decellularization is performed with a decellularization solution comprising a decellularization agent selected from anionic, cationic, non-ionic detergents, hydroalcoholic solution, hypotonic or hypertonic solutions.

22. The method for the fixation of a biological tissue according to the preceding claim, wherein said decellularization step comprises one or more steps of contacting with said decellularization solution, wherein said decellularization solution may comprise the same decellularization agent or not.

23. The method for the fixation of a biological tissue according to the preceding claim, wherein said decellularization solution further comprises an anti-oxidant agent or a mixture of anti-oxidant agents.

24. The method for the fixation of a biological tissue according to the preceding claim, wherein said anti-oxidant agent is selected from the group comprising: sodium ascorbate, glutathione, lipoic acid, uric acid, carotenes, alpha-tocopherol or ubiquinol.

25. The method for the fixation of a biological tissue according to any one of the preceding claims 21 to 24, wherein said decellularization solution further comprises a chelating agent or a mixture of chelating agents.

26. The method for the fixation of a biological tissue according to the preceding claim, wherein said chelating agent is selected from the group comprising EDTA, EGTA, acetylacetonate, polyamines, oxalate, carbonate, crown ethers, glycinate, nitrilotriacetate or bipyridine.

27. The method for the fixation of a biological tissue according to any one of the preceding claims, which ensure the immunological tolerance of the treated tissue in the case of homologous, autologous, allogeneic, isogenic and xenogenic transplants.

28. The method for the fixation of a biological tissue according to any one of the preceding claims, which prevents thrombosis occurrences, calcification, microbial infection.

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