IT202100025796A1 - METHOD FOR THE PREPARATION OF SURFACES, IN PARTICULAR OF HEART PROSTHESES - Google Patents

Description

Title: ?A method for the preparation of surfaces, in particular of cardiac prostheses?

Background of the invention

The present invention finds application in the medical field and, in particular, in the preparation of biological surfaces in contact with body fluids.

? Bioprosthetic heart valves fixed with glutaraldehyde (BHV) have been reported to be subject to dystrophic calcification after medium/long-term implantation in humans, this being the main limiting factor influencing their longevity. Calcification? a complex and multifactorial process not yet fully understood, which includes atherosclerosis-like tissue remodeling and prolonged exposure to mechanical stress. Among the causes responsible for calcific tissue dystrophy, treatment with glutaraldehyde (GLU) must also be taken into due consideration.

GLU? often used as the fixative and sterilizing agent of choice for many commercial bioprosthetic products, especially in surgical and transcatheter implantable heart valves (THVs). The THV system? considered a minimally invasive procedure that involves the use of a guiding catheter for positioning the prosthesis, avoiding surgery to open the heart. THVs may be an option for people at intermediate or high risk of complications from surgical heart valve replacement. Unfortunately, the instability? GLU chemistry? closely involved in the exposure of potential calcium binding sites (residual aldehydes, acids, Schiff bases, etc.). As a result of the interaction between tissue amino acid residues and GLU, negatively charged carboxylic acid groups can be created that can interact electrostatically with positively charged Ca2+ ions, becoming a huge attraction site for calcium. To make matters worse, even free-reacting aldehyde groups can be easily oxidized to carboxyl residues via air, in vivo blood, and macrophage oxidation.

To reduce the influence on the calcification process, BHV manufacturers have proposed several changes in GLU fixation protocols, including the addition of new steps aimed at chemical stabilization of the reactive aldehyde and carboxyl groups. Detoxification of GLU by urazole, extension of the diamine spacer, treatment with 2-amino oleic acid or incubation in ethanol are just some of the processes developed in the challenge of stabilizing GLU, with the hope of delaying calcific tissue dystrophy .

Although calcified degeneration of BHVs is the long-term event generally responsible for the ultimate failure of such biomedical devices, it should not be forgotten that there are a series of degenerative processes that begin to affect the prosthesis already? a few hours after implantation, mainly compromising the structural aspect of the device.

The early structural degeneration of BHV? a complex path due to multiple active and passive mechanisms closely related to each other. ? It is now well established that the active degenerative mechanisms are triggered by the early response of the host within a few hours after implantation, often related to inflammation, subclinical thrombosis of the flaps and/or bacterial infection. On the other hand, passive deterioration? strictly related to graft fatigue with consequent formation of holes, laceration and abrasion of the flaps.

Early structural degeneration - passive factors

As ? known, commercial BHVs do not constitute viable tissue and, by definition, are not capable of regenerating and remodeling the extracellular matrix in such a way as to account for any changes in the collagen network (delamination, structural rearrangements and destruction) resulting from cyclic damage loading irreversible. Prolonged cyclic loading during accelerated wear tests showed a marked decrease in extensibility. radial due to the stiffening of the effective collagen fiber network. The stiffening of the flaps over time, causing anomalies in the distribution of mechanical stresses, leads to overloads especially in the folding areas and in the suture areas around the stent. Histological evaluation of explanted BHVs demonstrated that leaflet tears and ruptured collagen fiber bundles are characteristic of a high-strain area, even in the absence of associated calcifications.

Early structural degeneration - active factors

Is subclinical leaflet thrombosis possible? frequently verified in the replacement of BHVs, more? commonly in THVs (13% frequency) compared to surgical BHVs (4%). It involves 30% of BHVs 1 year after implantation. This pathology shows clinical signs within the first 30 days of implantation, resulting in a reduction in mobility. of the flap. In patients with reduced mobility? of the flaps (HALT - hypoattenuating thickening of the flaps), the thickness of the flaps was significantly altered with complete immobility? of at least one valve cusp. HALT development represents a mild form of valve dysfunction related to a thrombus, early calcification, and/or degeneration of the valve leaflets.

Anticoagulant therapy (both new NOAC oral anticoagulants and warfarin)? effective in reducing the complications of HALT, but HALT is recurred in 50% of patients in whom anticoagulation was been interrupted. ? It is important to note that dual antiplatelet therapy, the standard of care for transcatheter valve implantation, is not found to be effective in the prevention or treatment of subclinical leaflet thrombosis.

The bacterial infection? another worrying aspect that contributes to the early degeneration of BHVs, especially in the case of THVs. Infective endocarditis (IE) has a significant impact on both population and patient management. In the United States there are 40,000 to 50,000 new cases/year, with average hospital costs exceeding $120,000/patient. Despite improvements in diagnosis and surgery, mortality? 1 year after EI? remained unchanged in over 2 decades. In cases where prolonged antibiotic treatment is not sufficient, ? surgical valve replacement is required.

The capacity? of specific bacteria to colonize BHVs? a crucial aspect for the future of heart valve replacements because? THVs have also shown good results in medium and low risk patients, enormously expanding the number of people subjected to this type of minimally invasive intervention. Replacement of a degenerated surgical BHV is often performed using a valve-invalve approach. In this case, a THV is deployed within the dysfunctional surgical BHV without its preliminary removal. There? could potentially lead to bacterial migration from the dysfunctional BHV to the new THV resulting in bacterial growth and tissue colonization.

Finally, do regulatory agencies require medical devices to be tested for pyrogenicity? mediated by the material following "ISO 10993-11:2017 Biological evaluation of medical devices - Part 11: Testing for systemic toxicity". The term pyrogen (from the Greek pyros: fire) defines the substances that cause fever. A pyrogenic response induced by a medical device can be due to different causes depending on the presence of the so-called "material-mediated pyrogens". A class of well-known and well-characterized exogenous pyrogens? the class of endotoxins. Endotoxins are lipopolysaccharide components present on the cell walls of Gram-negative bacteria. Another broad class of exogenous pyrogens are nonendotoxic pyrogens, which include substances such as lipoteichoic acid from Grampositive bacteria and other compounds from fungi, yeasts, viruses, bacteria, and parasites. The third class of non-endotoxic pyrogens? that of material-mediated pyrogens. While there is no formal definition of material-mediated pyrogens, it is thought that they may leach from materials or surfaces of medical devices. Material-mediated pyrogens can also result from contamination introduced during manufacturing and packaging, such as cutting fluid residues, mold releases, detergents, and processing aids. Therefore, ? It is of fundamental importance to develop treatments capable of improving the outcomes of BHVs without introducing chemicals or contaminants responsible for increasing pyrogenic reactions.

Summary of the invention

The inventors of the present patent application have surprisingly found a method to prevent both active and passive early degenerative events in a biological matrix (in particular of medical devices and even more particularly of cardiac prostheses) obtaining GLU stabilization, resistance and superficial Without precedents. platelet adhesion and release of fibrin and surface colonization of microorganisms. Finally, this method showed an interesting improving effect on the properties biomechanics of the biological matrix treated.

Brief description of the figures

Figure 1 ? Percentage decrease in free-to-react aldehyde and carboxyl groups in GLU-fixed pericardial patches treated with polyphenols (n=16 for each type of chemical group determination).

Figure 2 ? Percentage of thrombus accumulation in polyphenol-treated and untreated pericardial samples (GLU) (n=6 for each type of chemical group determination).

Figure 3 ? Comparison of tensile strength between polyphenol-treated and untreated samples (GLU, n=36 for each type of chemical group determination).

Figure 4 ? Percent elongation comparison between polyphenol-treated and untreated samples (GLU, n=36 for each type of chemical group determination).

Figure 5 ? Correlation of Young's modulus between polyphenol-treated and untreated F samples (GLU, n=36 for each type of chemical group determination).

Object of the invention

In a first subject matter, the present invention describes a method for treating a surface to be contacted with biological fluids.

According to a preferred aspect, said surface? the surface of a medical device.

According to a more favorite, said surface ? the surface of a biological prosthesis, which can? be a cardiac prosthesis.

In a second object, the present invention describes a surface to be contacted with biological fluids obtained by the method of the invention.

According to a preferred aspect, said surface? the surface of a medical device.

According to a more favorite, said surface ? the surface of a biological prosthesis, which can? be a cardiac prosthesis.

The medical device, the biological prosthesis and the cardiac prosthesis comprising the surface according to the invention represent further objects of the invention.

In a fourth subject matter, the present invention discloses a method for treating a disease comprising the use of the medical device, biological prosthesis, or cardiac prosthesis of the invention.

According to a preferred aspect, said disease? a heart disease.

In one respect, called disease? in a human being, while for another aspect, called disease? in an animal.

A solution comprising a phenolic compound or a mixture of phenolic compounds to be used in the method of the present application represents a further object of the invention.

According to another object of the invention, a method for preparing the solution of the invention comprising a phenolic compound or a mixture of phenolic compounds is disclosed.

In yet another subject matter, the present invention discloses a kit for carrying out the method of the invention. Detailed description of the invention

According to a first aim, the present invention describes a method for treating a surface to be contacted with biological fluids.

A biological fluid within the scope of the present invention? represented by blood, serum, plasma, vitreous gel, tears, urine, saliva, feces; including synovial, peritoneal, pericardial, pleural, and amniotic fluid. A biological surface can be represented by a surface of human or animal origin.

In particular, the surfaces of animal origin can be of equine, porcine or bovine origin and preferably of porcine or bovine origin; such surfaces can be considered as biological matrices. According to a particular aspect of the invention, said surface? an artificial surface.

In particular, this artificial surface? a surface of a medical device.

The medical devices according to the present invention can be represented by: artificial vessel, heart valve, tendon, ligament, pericardium, muscle fascia, dura mater, tympanic membrane, intestinal submucosa, cartilage, adipose and bone tissue, pelvic, abdominal, breast and tissue dermal.

According to another aspect of the invention, said surface? the surface of a biological prosthesis.

A biological prosthesis according to the invention can? be represented by: a vessel, a heart valve, a tendon, a ligament, pericardium, muscle fascia, dura mater, tympanic membrane, intestinal submucosa, cartilage, adipose and bone tissue, pelvic, abdominal, breast and dermal tissue.

In a preferred embodiment, the biological prosthesis according to the invention is represented by a cardiovascular prosthesis, such as a heart valve, or a patch of pericardial tissue.

In a more embodiment? preferred, a heart valve treatable according to the present invention? represented by a surgical heart valve.

In an even more embodiment? preferred, the heart valve treatable according to the present invention? represented by a transcatheter implantable heart valve; said valve needs to be implanted via a catheter and are folded to be housed inside the catheter.

According to the method of the invention, the disclosed surface is contacted with a solution comprising a phenolic compound or a mixture of phenolic compounds.

For the purposes of the present invention, a phenolic compound is to be understood as a phenolic or polyphenolic compound (in some cases both are referred to only as "phenolic" or "polyphenolic") used here as synonyms) selected from the group comprising: phenols, phenolic aldehydes, phenolic acids, phenylamines, phenolic compounds, flavonoids, phenylpropanoids and tannins.

In particular, a phenolic compound? chosen from the group including: vanillin, cinnamic acids, phenylalanine, coumarins, xanthones, catechins, flavonones, flavones, chalcones, flavanonols, flavanols, leucoanthocyanidins, anthocyanidins, hydroxycinnamic acids.

More, in particular, a phenolic compound can be selected from the group including: resveratrol, aloin, cyanrin, 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, eriodictiol, esperetin, naringenin, theaflavin, tearubigins, daidzein, genistein, glycitein, pterostilbene, delphinidin, malvidin, pelargonidin, peonidin, chicoric acid, ferulic acid, salicylic acid.

For the purposes of the present invention, the derivatives of the phenolic or polyphenolic compound described above are also included; for example, salts or esters or isomers can also be used.

In one embodiment of the invention, the solution of the invention comprises a mixture of two or more? of the phenolic or polyphenolic compounds described above.

According to a preferred embodiment, the solution of the invention can include a mixture of two or more? of the phenylpropanoids described above.

Below are some components and some solutions according to the invention:

As for the preparation of the solution of the invention, a phenolic or polyphenolic compound is solubilized in an alcoholic solvent.

In case a mixture of the phenolic or polyphenolic compound is prepared, the solution of each compound is prepared separately and then mixed together.

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

To this end, a first component is solubilized in an alcoholic solution (Component A), preferably 10% of the final volume of the solution.

An alcoholic solvent according to the invention can include methanol, ethanol, isopropanol, butanol, etc. and preferably includes or ? 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.

In one embodiment of the invention, the final solution is? a hydroalcoholic solution.

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 between 5 and 7.

Once prepared, the solution can? be optionally filtered with a 0.22 ?m filter.

According to the present invention, in the described method the surface is placed in contact with the solution of a phenolic compound or a mixture of phenolic compounds for a period of time less than 2 hours.

Preferably, the contact is continued for a period of approximately one hour.

More? preferably, the contact is continued for a period of approximately 30 minutes.

In an even more embodiment? preferred, the contact includes a first phase and a second phase. In a preferred embodiment, the first contact step is performed for 30 minutes and the second contact step is performed for 30 minutes.

Optionally, between the two contact phases, can? a rinsing phase (also called washing phase) must be carried out.

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).

According to a preferred embodiment, the method is performed by stirring the solution.

As regards the temperature of the contact phase, it is preferably carried out at a temperature of approximately 20°C? 10?C.

In a preferred embodiment of the invention, after the contact phase, the treated surface, medical device, biological prosthesis or cardiac prosthesis can be subjected to one or more? washing phases. Preferably, each of said washing steps is carried out with a suitable buffer; for example, a suitable swab can? be represented by a phosphate buffer.

In one embodiment, each washing step can be performed for a period of approximately 15-30 minutes.

In another embodiment, each washing step can be performed for a period of approximately 12-48 hours.

According to an embodiment of the present invention in which the described method is performed on a biological surface, said biological surface can be previously subjected to a pre-treatment phase.

In particular, this pre-treatment phase can have one or more? of the following effects:

- stabilizing proteins,

- stabilize or remove lipids,

- stabilize or remove cellular structures,

- lowering antigenicity.

For the purposes of the present invention, said pretreatment phase can? include a pre-treatment phase with one or more? between glutaraldehyde, formaldehyde, quercetin or genipin as well as? a treatment for the removal of phospholipids.

According to a particular embodiment of the invention, before subjecting the biological surface to the pre-treatment phase, it can be subjected to a preparation step with a capping agent selected from the group comprising: glycerol, heparin, amines (i.e. alkylamines, amino alcohols, ethanolamine), amino acids (lysine, hydroxylysine, amino sulfonates, taurine, amino sulfates, dextran sulfate, chondroitin sulfate), hydrophilic multifunctional polymers (e.g. -deoxyglucosone, glyoxal), hydrazides (e.g. adipic hydrazide), N,N-disuccinimidyl carbonate, carbodiimide (e.g. 1-ethyl-3-3-dimethylaminopropyl carbodiimide hydrochloride ? EDC, N-cyclohexyl-N'-(2-morpholino ethyl )carbodiimide ? CMC, 1,3-di-cyclohexyl carbodiimide ? DCC, 2-chloro-1-methyl pyridinium iodide - CMPI, 2-chloro-1-methyl pyridinium iodide - CMPI), antibiotics, cell recruitment agents, hemocompatibility agents, anti-inflammatory agents, antiproliferative agents, reducing agents (e.g. sodium cyanoborohydride, sodium borohydride, sodium bisulfite acetylacetone, formic acid-formaldehyde, mono-, di- or polyepoxy alkanes) .

According to the present invention, the method for treating a surface to be contacted with a biological fluid according to the invention is? a stabilization method.

In particular, this stabilization method inactivates the reagent groups available on the pre-treated surface. More? in particular, this stabilization method inactivates the aldehyde and carboxyl groups available on the pre-treated surface.

According to the present invention, the described method is an anti-limescale method.

A surface obtained with the method of the invention represents another object of the present application. A biological prosthesis, a medical device and in particular a cardiac prosthesis comprising the surface obtained according to the method of the present application represent further objects of the present invention.

A surface obtained with the pretreatment and treatment method of the invention represents another object of the present application.

A biological prosthesis, a medical device and in particular a cardiac prosthesis comprising the surface obtained according to the pretreatment and the treatment method of the present application represent further objects of the present invention.

According to the present invention, the method for treating a surface to be contacted with a biological fluid according to the invention is? a protective method.

In particular, this protection method prevents the onset of subclinical thrombosis.

More? in particular, the method of the invention ? an adhesive method? antiplatelet.

Even more? in particular, the method of the invention prevents the release of fibrin.

The release of fibrin? linked to the activation of soluble fibrinogen into insoluble fibrin polymers. These polymers aggregate laterally to form fibers, which then branch to form a three-dimensional network and, by interacting with circulating platelets, lead to the formation of the fibrin clot essential for hemostasis and wound coagulation. If the clot enters the bloodstream it is called a thrombus and can obstruct small/medium sized vessels causing ischemia, stroke and heart attack.

In particular, the protective method of the invention has been shown to prevent and avoid the anchoring of circulating platelets on biological surfaces treated and/or pre-treated according to the invention.

A surface obtained with the method of the invention represents another object of the present application.

A biological prosthesis, a medical device and in particular a cardiac prosthesis comprising the surface obtained according to the method of the present application represent further objects of the present invention.

A surface obtained with the pretreatment and treatment method of the invention represents another object of the present application.

A biological prosthesis, a medical device and a cardiac prosthesis comprising the surface obtained according to the pretreatment and treatment method of the present application represent further objects of the present invention.

According to the present invention, the method for treating a contact surface with a biological

fluid according to the invention ? a method to preserve and maintain the correct properties? structural biomechanics.

In particular, the method of the invention has been shown to increase the properties of elongation of biologically treated tissues.

In particular, this method preserves the collagen structure of the BHVs flaps.

Consequently, the method for treating a surface to be contacted with a biological fluid according to the invention is ? a protective method to maintain the correct properties? physiological hemo and fluid dynamics of the treated BHVs.

A surface obtained with the method of the invention represents another object of the present application.

A biological prosthesis, a medical device and a cardiac prosthesis comprising the surface obtained according to the method of the present application represent further objects of the present invention.

A surface obtained with the pretreatment and treatment method of the invention represents another object of the present application.

A biological prosthesis, a medical device and a cardiac prosthesis comprising the surface obtained according to the pretreatment and treatment method of the present application represent further objects of the present invention.

According to the present invention, the method for treating a contact surface with a biological

fluid according to the invention ? an antimicrobial and antiviral method, as it is? a disinfectant method of the treated surface.

In particular, the antimicrobial method of the invention has a bactericidal action.

More? in particular, the antimicrobial method of the invention has a bactericidal action against microorganisms responsible for the onset of endocarditis.

In one embodiment, said microorganisms are Gram+ bacteria, Gram- bacteria, yeasts and molds.

In another embodiment, said microorganisms are mycobacteria, such as Mycobacterium chelonae.

Consequently, the method for treating a surface to be contacted with a biological fluid according to the invention is ? an anti-Gram+ method, an anti-Gram- method, an anti-yeast method, an anti-mold method, an anti-mycobacteria method.

In particular, the antiviral method of the invention has a virucidal action against viruses.

More? in particular, this virucidal action? according to EN 14476.

In particular, these viruses belong to the Picornaviridae, Adenoviridae and Caliciviridae families.

More? in particular, said microorganisms are Poliovirus Type 1 LSc-2ab (RVB-1260), Adenovirus Type 5 (ATCC VR-5) and Murine norovirus strain S-99 (RVB-651).

A surface obtained with the method of the invention represents another object of the present application. A biological prosthesis, a medical device and a cardiac prosthesis comprising the surface obtained according to the method of the present application represent further objects of the present invention. A surface obtained with the pretreatment and treatment method of the invention represents another object of the present application.

A biological prosthesis, a medical device and a cardiac prosthesis comprising the surface obtained according to the pretreatment and possibly the treatment method of the present application represent further objects of the present invention.

More, in particular, this biological prosthesis can be represented by a cardiovascular prosthesis obtained with the pretreatment and possibly the treatment method of the invention.

According to a third object of the invention, a method for treating a disease comprising the use of the above-described medical device, biological prosthesis or cardiac prosthesis is disclosed. According to a preferred aspect, said disease? a heart disease.

In one respect, called disease? in a human being, while for another aspect, called disease? in an animal.

In a particular embodiment, the method for treating the disease according to the invention comprises a valve-in-valve approach, in which a valve is deployed inside a dysfunctional valve without its preliminary removal.

As a further object of the present invention, a solution comprising a phenolic compound or a mixture of phenolic compounds for use in the method of the present application is disclosed.

In particular, the preparation of said solution includes a first phase, in which the phenolic compound is solubilized in an alcoholic solvent.

For example, can be resolved a phenolic compound from list A above.

If necessary, an additional phenolic compound can be added. be solubilized in an isotonic buffered solution. For example, can be resolved a phenolic compound from list B above.

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

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.

Will this invention come? further described in connection with the following experimental section. The following experimental session shows the results of tests carried out on surfaces treated according to the present invention.

Polyphenolic solutions

The following table reports some examples of polyphenolic solutions according to the invention.

In particular, the surfaces were treated with Solution 5 as described above.

Preparation of the Polyphenolic Solution 5

Caffeic acid as Component A? was weighed according to the indicated concentrations and solubilized in ethanol to a final volume of 10% of the polyphenolic mixture. Tannic acid as column Component B? was weighed according to the indicated concentrations and solubilized in modified phosphate buffer at 90% of the final volume of the polyphenolic mixture. Both steps are performed in the dark. When is solubilization? complete, the two solutions are mixed. The pH? adjusted to 5-7. The solution is filtered with a 0.22 ?m filter. This solution ? referred to as Solution 5 or Polyphenolic Solution. Other solutions according to the present invention can be prepared in analogy with what has been described. Stabilization with glutaraldehyde

Fabric treatment

Several bovine pericardias were carefully selected to obtain rectangular patches (n=32). All the patches were subjected to a preliminary GLU cross-linking treatment. In short, the pericardial tissue is was incubated in a buffered GLU solution for three passages of 24 hours each in a dark room. For this example, a GLU solution ? a 0.6%? 0.5% v/v for the first and second passes followed by 0.2% ? 0.15% v/v for the third. The GLU-treated pericardial patches were subjected to two washing steps in phosphate buffer for 15 minutes each.

Sixteen patches were incubated with Solution 5 of the invention prepared as described above under moderate but constant stirring in the dark, for two phases of 25 ? 10 minutes each, at room temperature (RT).

At the end of the incubation, the treated patches were subjected to five washes in phosphate buffer for 15-30 minutes each. Samples are indicated as TREATED.

The remaining GLU fixed pericardial patches were adopted as controls (GLU, n=16).

Determination of the content of free carboxyl groups

Pericardial patches were embedded in Optimal Cutting Temperature (OCT) and frozen by immersion in isopentane precooled in liquid nitrogen. Cryosections with a thickness of 7 ?m were then produced using MirrIR slides suitable for infrared reflectance studies as a support and analyzed by FT-IR microscopy in reflectance and mode? mosaic with 64 scans for each selected area. The detector used? FPA with 4 cm<-1> resolution. The treated tissues showed a lower concentration of carboxyl groups at the wave number 1233 cm<-1> (corresponding to the stretching of the C-O bonds of the carboxyl groups) compared to the samples fixed only in GLU.

Considering the total free carboxyl groups quantified in the GLU fixed samples as 100% of the available groups, the treated samples reported a decrease equal to 76% of the total.

Determination of the content of free aldehyde groups

Prepare 150 ml of solution A: 0.2 M citric acid, 0.5 M sodium hydroxide, and 8 mM tin(II) chloride in ultrapure water. Prepare 25 ml of Solution B: 0.22 M ninhydrin dissolved in 25 ml of Ethylene Glycol Monomethyl Ether (Cellosolve) in a dark bottle with screw cap. Combine one volume of Solution A with an equal volume of Solution B and mix in the dark for 45 minutes to obtain Solution C. Please note that 2 ml of Solution C is required for each sample to be analyzed.

Tissue samples should be prepared to have a wet weight of approximately 20 mg each. Each sample is incubated at 100?C in 2 ml of Solution C for 20 minutes in the dark, cooled in water and diluted with 15 ml of 50% Isopropanol. The developed color is read at 570 nm within 30 minutes. The nmoles of the aldehyde groups are determined relative to a glycine standard.

Considering the total free aldehyde quantified in the GLU fixed samples as 100% of the available groups, the treated samples reported a decrease equal to 56.3% of the total.

Figure 1 shows the percentage decrease in free-reacting aldehyde and carboxyl groups in treated pericardial tissue patches (n=16 for each type of chemical group determination).

Evaluation of adhesiveness? platelet

Fabric treatment

To evaluate the propensity for adhesiveness? platelet and fibrin release under flow conditions, ? An in vitro blood flow model was adopted. Several bovine pericardiums were carefully selected to obtain rectangular patches (n=12). All the patches were subjected to a preliminary GLU cross-linking treatment. In short, the pericardial tissue is was incubated in a buffered GLU solution for three passages of 24 hours each in a dark room. For this example, a GLU solution ? a 0.6%? 0.5% v/v for the first and second passes followed by 0.2% ? 0.15% v/v for the third. The GLU-treated pericardial patches were subjected to two washing steps in phosphate buffer for 15 minutes each.

Six patches were incubated with Solution 5 of the invention prepared as described above under moderate but constant stirring in the dark, for two phases of 25 ? 10 minutes each, at room temperature (RT).

At the end of the incubation, the treated patches were subjected to five washes in phosphate buffer for 15-30 minutes each. Samples are indicated as TREATED.

The remaining GLU fixed pericardial patches were adopted as controls (GLU, n=6).

Quantification of adhesiveness? of platelets

Heparinized bovine blood? was collected from 3 different animals and is the radioisotope for the quantification of thrombi was added. Strips of pericardial tissue were deployed in a 25.4 mm conduit and blood flow was at 2.5 L/min ? been enabled with a peristaltic pump for 1 hour. The strips were rinsed with saline and placed in a gamma counter for radiation quantification (reflective of relative thrombosis). The average radiation values are 73.133 counts per minute (cpm) for Glu and 35.165 cpm for the treated samples. In Figure 2 the results are expressed as a percentage of reduction in platelet propensity in tissues treated with polyphenols, taking into account GLU samples as 100%.

Property? mechanics of the treated fabric

Fabric treatment

Seventy-two rectangular strips (approximately 10 mm long and 8 mm wide) of bovine pericardia were processed with GLU solution as described above (“Tissue processing”). The GLU-treated pericardial strips were subjected to two washing steps in phosphate buffer for 15 minutes each. Thirty-six patches were incubated with Solution 5 described above under moderate but constant shaking in the dark, for two phases of 25 ? 10 minutes each, at room temperature. At the end of the incubation, the treated patches were subjected to five washes in phosphate buffer for 15-30 minutes each. Samples are indicated as TREATED.

The remaining fixed pericardial GLU strips were adopted as controls (GLU, n=36).

Property valuation? mechanical

Every strip? was mounted on the tensile equipment to be subjected to a uniaxial tensile test secured with rubber grips (Thumler Z3-X500 equipped with 100 N / 500 N load cell). The initial length of the sample (distance between the handles) ? was set at 50 mm. The tensile test? was performed at 50 mm/min.

Every strip? been characterized dimensionally in terms of length (useful length 50 mm), width and thickness (average value of the measurements) and? the cross-sectional area (with x thickness) was calculated. From each traction curve the following parameters were obtained:

? Breaking strength [N], in terms of maximum strength before breaking;

? Ultimate strength [MPa], in terms of ultimate strength divided by cross-sectional area (UTS); ? Deformation at break [%], in terms of deformation at maximum strength;

? Young's modulus [MPa], in terms of the slope of the linear region of the stress-strain curve (elastic phase);

For each parameter? The average value was calculated from the samples, obtaining a value for each patch. The average value? was calculated again from the patches, resulting in a value for each test group.

Figure 3 shows the comparison of the tensile strength between treated and untreated samples (GLU). Ultimate Tensile Strength (UTS), often abbreviated to tensile strength (TS), is ? the maximum stress that a material can? endure while being stretched or pulled before breaking. The polyphenolic treatment does not show a statistically significant difference compared to the control sample (GLU).

Figure 4 shows the percentage elongation comparison between treated and untreated samples (GLU).

Figure 5 shows the correlation of Young's modulus between treated and untreated samples (GLU). The percentage of elongation? closely related to the Young's Modulus. Young's Modulus or Modulus of Elasticity? ? a characteristic, characteristic of a material, which expresses the relationship between tension and deformation in the case of uniaxial loading conditions and in the case of elastic (reversible) behavior of the material. ? defined as the ratio of the applied stress to the resulting strain. As the Young's Modulus increases, the stiffness also increases. of the material. The increase in the level of elasticity? of the treated tissue leads to a consequent decrease in its Young's Modulus compared to the untreated pericardium (GLU).

The gain in elasticity? it allows a better distribution of the mechanical load, especially to the advantage of the BHV areas subject to greater pressure. This avoids the formation of tears and preserves the collagen structure of the valve leaflets.

The bactericidal potential of the polyphenolic solution

The activity? bactericidal (BA) of the polyphenolic solutions of the invention? been evaluated for the following different microorganisms: Staphyloccoccus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Enterococcus faecalis ATCC 29212, Listeria monocytogenes ATCC 19111, Salmonella enterica typhimurium ATCC 14028, Streptococcus viridans ATCC 6249, a non-tuberculous mycobacterium Myco bacterium chelonae ATCC 35752, a yeast Candida albicans ATCC 10231 and a fungus Aspergillus brasiliensis ATCC 16404.

The BA assay consists of a suspension method with a single incubation for 24 hours of the bacteria at a known concentration of Solution 5 of the invention (inoculum). At the end of the incubation time, the contents of each test tube are sown in sterile 90 mm Petri dishes in a specific agar medium, with a pour plate or spread plate technique depending on the microorganisms tested, after dilution in Tryptone Salt Broth (MRD broth) . Then the plates obtained are incubated at specific conditions and temperatures based on the growth needs of each microorganism.

Preparation of the inoculum

For each type of microorganism, ? a microbial suspension in MRD Broth was quantified through the spectrophotometer at a wavelength of 620 nm in a disposable cuvette with a 10 mm path. The absorbance of an aliquot part of the suspension is measured: the range between 0.150 and 0.460 corresponds to a cell concentration between 1x10^8 CFU/ml and 3x10^8 CFU/ml (with Candida albicans between 1x10^7 CFU /ml and 3x10^7 CFU/ml). For Streptococcus oralis, since? there was no correlation between the absorbance measurement and the concentration of bacteria, the quantification ? was performed by cell counting under a microscope.

The data were processed by comparing the growth on the sample with the concentration of bacteria at t0, to see an effect in promoting or inhibiting growth, and expressed as a percentage of activity? bactericidal. The results were compared with control samples (antibiotics and ethanol solutions).

The table below shows the percentage of activity? bactericidal compared to standard antibodies and ethanolic solution.

The virucidal potential of the polyphenolic solution

The activity? virucidal of Polyphenolic Solution 5? been assessed according to the guidelines: Test method and requirements European Standard EN 14476:2013+A2:2019/UNI EN 14476:2019? Chemical disinfectants and antiseptics. Quantitative test in suspension for the evaluation of the activity? virucidal in the medical area. Test method and requirements (Phase 2/Phase 1). The virucidal test? was performed regarding the following virus strain: Poliovirus Type 1 LSc-2ab (RVB-1260), Adenovirus Type 5 (ATCC VR-5) and Murine norovirus S99 (RVB-651). The table below shows the percentage of activity? virucide of Polyphenolic Solution 5 diluted to 80% (corresponding to the maximum possible concentration that can be evaluated according to the method).

Anti-adhesive effect of the polyphenolic solution on different strains of microorganisms

The activity? anti-adhesive on treated surfaces? was evaluated with reference to the following different microorganisms: Staphyloccoccus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Enterococcus faecalis ATCC 29212, Listeria monocytogenes ATCC 19111, Salmonella enterica typhimurium ATCC 14028, Streptococcus viridans 6249, a non-tuberculous mycobacterium Mycobacterium which lonae ATCC 35752, a yeast Candida albicans ATCC 10231 and a fungus Aspergillus brasiliensis ATCC 16404.

Each strain of microorganism? was grown overnight in a dedicated broth at 37?C. At the end of the incubation, were the units counted? colony forming (CFU) to determine the actual concentration of the microorganism.

Fabric treatment

Ninety samples of approximately 2 cm<2 > each of bovine pericardia were processed with the GLU solution as described above (?Tissue processing?). GLU-treated pericardial samples were subjected to two washing steps in phosphate buffer for 15 minutes each. At the end of the incubation, 45 patches were treated with Solution 5 of the invention according to the method described and then subjected to five washes in phosphate buffer for 15 to 30 minutes each. These samples are referred to as TREATED.

The remaining GLU-fixed pericardial samples were adopted as control (GLU).

Evaluation of the anti-adhesive effect

Polyphenol-treated and untreated patches were washed with PBS and incubated overnight at room temperature in PBS antibiotics (300 ?g/mL). Different types of antibiotics specific for each type of strain of microorganisms have been used (neomycin, penicillin, cephalosporin, polymyxin, rifamycin, lipiarmicin, quinolone, sulfonamide, macrolide, lincosamide, tetracycline, aminoglycoside, doxycycline, minocycline, ampicillin, amoxicillin/clavulanic acid , azithromycin, carbapenems, piperacillin/tazobactam, quinolones, chloramphenicol, ticarcillin, trimethoprim/sulfamethoxazole). After overnight incubation, tissue patches were washed extensively in PBS to remove any traces of unbound antibiotic. Subsequently, the treated and untreated samples were individually exposed to the different strains of microorganisms (microorganism load 1 X 10<7 >CFU/mL) for 90 minutes at room temperature under moderate but constant stirring. At the end of the incubation, tissue samples were subjected to three moderate vortexing steps to facilitate detachment of loosely bound bacteria. Finally, were the samples homogenized with Stomacher? 400 and serial dilutions of the obtained homogenates, plated in Petri dishes containing the appropriate selective growth media. After 24 hours of incubation at 37?C, the units? formed by colonies were counted for each sample type.

The results were expressed as a percentage decrease in attached microorganisms assessed in the treated pericardial patches compared to the untreated fixed GLU pericardial patches (n=5 for each strain of microorganisms).

The table below shows the percentage decrease in attached microorganisms evaluated in the treated pericardial patches. The percentage value? was determined by comparing fixed and untreated GLU pericardial patches (n=5 for each strain of microorganisms).

Non-pyrogenic? of the fabric treated with polyphenols

The monocyte activation test (MAT)? was qualified and validated for the detection of pyrogens by the European Center for the Validation of Alternative Methods (ECVAM) in 2005 and by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) in 2008. ? has been among the compendium methods for the detection of pyrogens in the European Pharmacopoeia since 2010 (Chapter 2.6.30) and mentioned by the FDA "Guidance For Industry? Pyrogen and Endotoxins testing: Questions and Answers". The monocyte activation test (MAT)? the in vitro human alternative to the rabbit pyrogen test (RPT) and allows

detection of the full range of pyrogens, including endotoxins and non-endotoxic pyrogens (NEPs). Fabric treatment

Eighteen bovine pericardial samples of approximately 2 cm2 each were processed with the GLU solution as described above ("Tissue processing"). GLU-treated pericardial samples were subjected to two washing steps in phosphate buffer for 15 minutes each. Nine patches were incubated with Solution 5 under moderate but constant shaking in the dark, for two phases of 25 ? 10 minutes each, at room temperature. At the end of the incubation, the treated patches were subjected to five washes in phosphate buffer for 15-30 minutes each. Samples are indicated as TREATED.

The remaining nine fixed pericardial GLU samples were adopted as controls (GLU, n=9).

Evaluation of pyrogenicity?

The samples were placed for 1 hour in 40 ml of endotoxin-free water under moderate stirring at 37?C. The water ? was analyzed with the MAT test. In short, water is been brought into contact with human monocytic cells, imitater? There? that happens in the human body: in the presence of pyrogens, monocytes are activated and produce different types of cytokines including interleukin-6 (IL6). Cytokines are then detected using an immunoassay (ELISA) involving specific antibodies and an enzymatic color reaction.

The following table shows the evaluation of the Unit? of endotoxins in treated and untreated pericardial patches (GLU). Reference value to be considered pyrogenic: NMT 20 EU/device.

Given the above description, the advantages of the method of the present invention will be evident. In particular, the method of the invention has proven not to alter the other properties? of the surfaces treated and possibly pre-treated as described, and of the medical devices, biological prostheses and in particular cardiac prostheses comprising said surfaces.

As an additional advantage, the described method has been shown to inactivate available aldehyde and carboxyl reagent groups on the treated or pretreated surface.

Furthermore, since? platelet deposition on the fibrin network? responsible for the onset of subclinical leaflet thrombosis (SLT), which in turn is responsible for the alteration of mobility? of the valve leaflets, the method of the invention prevents the impairment of the functionality? of BHVs.

Yes ? found that the protective action of the method of the invention is responsible for a better distribution of the mechanical load, which avoids the formation of tears, abrasions and holes.

As a further advantage, the method of the invention prevents the adhesion of microorganisms and the formation of biofilms on the treated and possibly pre-treated surfaces.

Furthermore, the disclosed method avoids bacterial and viral contamination of a surface treated and possibly pre-treated according to the present invention.

As a further advantage, the method described maintains the pyrogen-free characteristic of the treated and possibly pre-treated surfaces.

Claims (21)

Claims: 1. A method for treating a surface to be contacted with biological fluids, said method comprising the step of contacting said surface with a solution comprising a phenolic compound or a mixture of phenolic compounds. 2. Method according to claim 1, wherein said surface is? the surface of a medical device. 3. Method according to claim 1 or 2, wherein said surface is? the surface of a biological prosthesis. 4. Method according to any of the preceding claims 1 to 3, wherein said surface is? the surface of a cardiac prosthesis or heart valve or a patch of pericardial tissue or a surgical heart valve. 5. Method according to any of the preceding claims, wherein said biological fluid is? chosen from the group including: blood, serum, plasma, vitreous gel, tears, urine, saliva, feces; including synovial, peritoneal, pericardial, pleural, and amniotic fluid. 6. Method according to the previous claim, wherein said phenolic compound or said mixture of phenolic compounds are selected from the group comprising: phenols, phenolic aldehydes, phenolic acids, phenylamines, phenolic compounds, flavonoids, phenylpropanoids and tannins. 7. Method according to the previous claim, wherein said phenolic compound or said mixture of phenolic compounds are selected from the group comprising: vanillin, cinnamic acids, phenylalanine, coumarins, xanthones, catechins, flavonones, flavones, chalcones, flavanonols, flavanols, leucoanthocyanidins, anthocyanidins, hydroxycinnamic acids. 8. Method according to the previous claim, wherein said phenolic compound or a mixture of phenolic compounds is? chosen from the group including: resveratrol, aloin, cyanarin, 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, eriodictiol, hesperetin, naringenin, theaflavin, thearubigins, daidzein, genistein, glycitein, pterostilbene, delphinidin, malvidin, pelargonidin, peonidin, chicoric acid, chicoric acid, ferulic acid, salicylic acid. 9. A method according to any one of the preceding claims, wherein said method comprises the step of contacting said surface with the solution of said phenolic compound or a mixture of said phenolic compounds for a period of time less than 2 hours. 10. A method according to any one of the preceding claims, wherein said method comprises a first and a second contact step, wherein a rinsing step is performed between said first and said second contact step. 11. Method according to any of the preceding claims, wherein said method comprises a pretreatment step with one or more? of the compounds selected from the group comprising; glutaraldehyde, formaldehyde, quercetin or genipin. 12. Method according to any one of the preceding claims, wherein said pretreatment step further comprises the removal of phospholipids. 13. A method according to any one of the preceding claims, wherein said pretreatment step further comprises a preparation step comprising the use of a capping agent. 14. Method according to any of the previous claims, which is? one or more? between: a method to preserve and maintain properties? adequate structural biomechanics, a protective method to maintain the properties? hemodynamics and physiological fluid dynamics of the treated surface, an antimicrobial and antiviral method for disinfection of the treated surface. 15. Surface in contact with biological fluids obtained according to the method of any one of claims 1 to 14. 16. A medical device or biological prosthesis comprising the surface of claim 15. 17. Medical device according to the previous claim, which is? selected from the group including: artificial vessel, heart valve, tendon, ligament, pericardium, muscle fascia, dura mater, tympanic membrane, intestinal submucosa, cartilage, adipose and bone tissue, pelvic, abdominal, breast and dermal tissue. 18. A method for treating a disease comprising the use of the medical device or biological prosthesis according to claim 16. 19. A method for treating a disease according to claim 18, wherein said disease is? a heart disease. 20. A method for treating a disease according to claim 18 or 19, wherein said disease is? in humans or animals. 21. A method for treating a disease according to any one of claims 18 to 20, wherein said method comprises a valve-in-valve approach.