CN118055784A

CN118055784A - Method for producing a surface, in particular a heart prosthesis surface

Info

Publication number: CN118055784A
Application number: CN202280067773.4A
Authority: CN
Inventors: 菲利波·纳索; 亚历山德罗·甘达利亚; 乌戈·斯特凡内利
Original assignee: Biocompatibility Innovation Srl
Current assignee: Biocompatibility Innovation Srl
Priority date: 2021-10-06
Filing date: 2022-10-06
Publication date: 2024-05-17

Abstract

A method for preventing early active and passive degradation of a prosthesis to be contacted with a biological fluid is disclosed.

Description

Method for producing a surface, in particular a heart prosthesis surface

Background

The invention finds application in the medical field, in particular in the preparation of biological surfaces to be contacted with body fluids.

Glutaraldehyde-fixed Bioprosthetic Heart Valves (BHV) are reported to be prone to dystrophic calcification after medium/long term implantation in humans, a major limiting factor affecting their life. Calcification is a complex and multifactorial process that is not yet fully understood, including atherosclerotic tissue remodeling and prolonged exposure to mechanical stress. Glutaraldehyde (GLU) treatment should also be considered appropriately among the causes of calcified tissue malnutrition.

GLU is commonly used as a preferred fixative and sterilant for many commercial bioprosthetic products, particularly in surgical and transcatheter implantable heart valves (THV). THV implantation is considered a minimally invasive procedure involving the use of a guide catheter to position the prosthesis, avoiding open heart surgery. THV may be an option for persons with moderate or high risk of complications from surgical heart valve replacement. Unfortunately, GLU chemical instability is closely related to exposure of potential calcium binding sites (residual aldehydes, acids, schiff bases, etc.). Due to interactions between tissue amino acid residues and GLU, negatively charged carboxylic acid groups can be generated that can electrostatically interact with positively charged Ca ²⁺ ions, becoming a huge attractive site for calcium. Worse yet, even unreacted (free-to-reaction) aldehyde groups are readily oxidized to carboxyl residues by oxidation of air, blood and macrophages in the body.

To reduce the impact on the calcification process BHV manufacturers have proposed various modifications to the GLU fixation scheme, including the addition of new steps aimed at chemically stabilizing reactive aldehyde and carboxyl groups. GLU detoxification by urazole, diamine spacer extension, treatment by 2-amino oleic acid or incubation in ethanol are just some of the methods developed in the challenge of stabilizing GLU in hopes of delaying calcified tissue dystrophy.

Although calcification degradation of BHV is a long term event leading to eventual failure of such biomedical devices, it cannot be forgotten that, only within a few hours after implantation, a series of degenerative processes begin to affect the prosthesis, primarily damaging structural aspects of the device.

Early structural BHV degradation is a complex approach due to a variety of active and passive mechanisms (ACTIVE AND PASSIVE MECHANISM) that are closely related to each other. It has now been determined that degenerative active mechanisms are triggered by early host responses within hours after implantation, often associated with inflammation, subclinical small She Xieshuan formation, and/or bacterial infection. Passive deterioration, on the other hand, is closely related to graft fatigue, leading to leaflet formation holes, tears and wear.

Early structural degeneration-passive factor

It is well known that commercial BHV does not constitute viable tissue and, by definition, is unable to undergo extracellular matrix regeneration and remodeling, so any changes in the collagen network (delamination, structural rearrangements and destruction) caused by cyclic loading are considered irreversible lesions. The prolonged cyclic load during the accelerated wear test highlights a significant decrease in radial extensibility due to hardening of the effective collagen fiber network. Over time, the leaflets stiffen, resulting in abnormal mechanical stress distribution, thereby causing overload, particularly in the bending region and the suturing region around the stent. Histological evaluation of explanted BHV showed that small She Silie and collagen fiber bundle breaks are characteristic of high strain areas, even without associated calcification.

Early structural degradation-initiative factor

Subclinical small She Xieshuan formation frequently occurs in BHV replacement, more common in THV (frequency 13%) than in surgical BHV (4%). It relates to 30% BHV at 1 year post implantation. This pathology manifests clinical symptoms within the first 30 days after implantation, resulting in reduced leaflet motility. In patients with reduced leaflet motion (HALT-low attenuating leaflet thickening), the leaflet thickness changes significantly, with at least one valve tip being completely immobilized. The development of HALT represents a mild valve dysfunction associated with thrombosis, early calcification and/or valve leaflet degeneration.

Anticoagulant therapy (novel oral anticoagulants NOAC and warfarin) is effective in reducing HALT complications, but 50% of patients who cease anticoagulant therapy experience HALT recurrence. Notably, dual antiplatelet therapy (standard of care for transcatheter valve implantation) is not effective in preventing or treating subclinical leaflet thrombosis.

Bacterial infection is another alarming aspect that leads to early BHV regression, especially in the case of THV. Infectious 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 annually, with average hospitalization costs per patient exceeding $120,000. Despite improvements in diagnostic and surgical intervention, the 1 year mortality rate of IE has not changed over 20 years. If prolonged antibiotic treatment is inadequate, surgical valve replacement is required.

The ability of specific bacteria to colonize BHV is a key aspect of future heart valve replacement procedures, as THV has also shown good efficacy in low and medium risk patients, greatly increasing the number of people receiving such minimally invasive interventions. Replacement of degenerated surgical BHV is typically performed using the valve-in-valve method. In this case, THV is deployed within a dysfunctional surgical BHV without prior removal. This may lead to migration of bacteria from dysfunctional BHV to new THV, leading to bacterial growth and tissue colonization.

Finally, regulatory authorities require medical device biological evaluation-part 11 according to "ISO 10993-11:2017: systemic toxicity test (Biological evaluation of MEDICAL DEVICES-Part 11:Tests for systemic toxicity) "Material-mediated pyrogenicity test was performed on medical devices (MEDICAL DEVICE). The term pyrogen (Greek pyros: fire) defines the substance that causes fever. The pyrogenic reaction caused by the medical device may be caused by a number of reasons, depending on the presence of the so-called "material-mediated pyrogen". One well known and well characterized class of exogenous pyrogens is the endotoxins. Endotoxins are lipopolysaccharide components present on the cell wall of gram-negative bacteria. Another broad class of exogenous pyrogens are non-endotoxin pyrogens, which include substances such as lipoteichoic acid derived from gram-positive bacteria, and other compounds derived from fungi, yeasts, viruses, bacteria and parasites. The third class of non-endotoxin pyrogens is material-mediated pyrogens. Although there is no formal definition of material-mediated pyrogens, it is believed that they may leach from the medical device material or surface. Material-mediated pyrogens may also originate from contamination introduced during manufacture and packaging, such as residues from cutting fluids, mold release agents, cleaning agents and processing aids. Therefore, it is important to develop therapeutic methods that can improve BHV results without introducing chemicals or contaminants that lead to the pyrogenic reaction.

Disclosure of Invention

The inventors of the present patent application surprisingly found a method for preventing active and passive early degenerative changes in biological matrices (biological matrix, biological matrices), in particular medical devices, and even more particularly heart prostheses, achieving unprecedented GLU stability, resistance to surface platelet adhesion and fibrin release, and microbial surface colonization. Finally, this approach shows interesting improvements in biomechanics of the treated biomatrix.

Drawings

Figure 1-percentage of unreacted aldehyde groups and carboxyl groups reduced in the polyphenolic treated (polyphenolic-treated) GLU-immobilized pericardial patch (n=16 for each type of chemical group determination).

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

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

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

Fig. 5-correlation of young's modulus between F polyphenols treated and untreated samples (GLU, measured for each type of chemical group, n=36).

Object of the Invention

In a first object, the invention discloses a method for treating a surface to be contacted with a biological fluid.

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

According to a more preferred aspect, the surface is a surface of a bioprosthesis, which may be a heart prosthesis.

In a second object, the invention discloses a surface to be contacted with a biological fluid obtained with the method of the invention.

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

Medical devices, bioprostheses and cardiac prostheses comprising a surface according to the present invention do represent a further object of the present invention.

In a third object, the invention discloses a method of treating a disease comprising the use of a medical device, bioprosthesis or cardiac prosthesis of the invention.

According to a preferred aspect, the disease is heart disease.

According to one aspect, the disease occurs in humans, and according to another aspect, the disease occurs in animals.

Solutions comprising a phenolic compound or a mixture of phenolic compounds to be used in the process of the application represent a further object of the application.

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

In a still further object, the invention discloses a kit for performing the method of the invention.

Detailed Description

According to a first object, the present invention discloses a method for treating a surface to be contacted with a biological fluid.

Representative of biological fluids within the object of the present invention are blood, serum, plasma, vitreous gel, tears, urine, saliva, faeces; including synovial fluid, peritoneal fluid, pericardial fluid, pleural fluid and amniotic fluid.

Biological surfaces may be represented by surfaces of human or animal origin.

In particular, the surface of animal origin may be of equine, porcine or bovine origin, and preferably of porcine or bovine origin; such surfaces may be considered biological substrates.

In particular, the surface is a surface of a medical device.

The medical device according to the invention may be represented by: a heart valve; tendons; a ligament; pericardium; myofascial; dura mater; a tympanic membrane; intestinal submucosa; cartilage; fat and bone tissue; pelvis, abdomen, breast and dermis tissues.

According to another aspect of the invention, the surface is a surface of a bioprosthetic.

The bioprosthesis according to the present invention may be represented by: a blood vessel; a heart valve; tendons; a ligament; pericardium; myofascial; dura mater; a tympanic membrane; intestinal submucosa; cartilage; fat and bone tissue; pelvis, abdomen, breast and dermis tissues.

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

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

In an even more preferred embodiment, the heart valve that can be treated according to the invention is represented by a transcatheter implantable heart valve; the valve needs to be implanted through a catheter and folded to be accommodated within 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, phenolic compounds shall mean phenolic or polyphenolic compounds (phenolic or polyphenolic compound) (in some cases, both of which are referred to as "phenols (phenolic)" or "polyphenols (polyphenolic)", used herein as synonyms only), selected from the group comprising: phenols (phenols ), phenols (phenolic aldehydes), phenolic acids (phenolic acids), anilines (PHENYLAMINES, anilines), phenol compounds, flavonoids (flavonoids ), phenylpropanoids (phenylpropanoids) and tannins (tannins).

In particular, the phenolic compound is selected from the group comprising: vanillin, cinnamic acid, phenylalanine, coumarin, xanthones, catechins, flavanoids (flavonons), flavones (flavones ), chalcones, flavanols, leucoanthocyanidins, anthocyanidins, hydroxycinnamates.

More specifically, the phenolic compound may be selected from the group comprising: resveratrol, aloin, cynarate (cyanarin), epigallocatechin, tannic acid, caffeic acid, chlorogenic acid, hydroxytyrosol, rosmarinic acid, naringenin (narigenin), gallic acid, hesperetin, quinic acid, elemene (eleonolic acid), pinoresinol, luteolin, apigenin, isorhamnetin, kaempferol, myricetin, eriodictyol, hesperetin, naringin (naringenin), theaflavin, thearubigin, daidzein, genistein, glycitein, pterostilbene, delphinidin, malvidin, paeoniflorin, chicoric acid, ferulic acid, salicylic acid.

For the purposes of the present invention, derivatives of the above disclosed phenolic or polyphenolic compounds are also included; for example, salts or esters or isomers may 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 disclosed above.

According to a preferred embodiment, the solution of the invention may comprise a mixture of two or more of the phenylpropanoids disclosed above.

Some components and some solutions according to the invention are reported below:

Solution	Component A	Component B
			1	Resveratrol	Tannic acid
2	Resveratrol	Cynaro acid
			3	Aloin	Epigallocatechin (EGCG)
4	Aloin	Chlorogenic acid
			5	Caffeic acid	Tannic acid
6	Caffeic acid	Hydroxytyrosol
			7	Rosmarinic acid	Cynaro acid
8	Naringin	Gallic acid
			9	Hesperetin	Gallic acid

According to the preparation of the solution according to the invention, the phenolic or polyphenolic compound is dissolved in an alcoholic solvent.

In the case of preparing a mixture of phenolic or polyphenolic compounds, solutions of each compound are 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.

For this purpose, the first component is dissolved in an alcoholic solution (component a), preferably in an amount of 10% of the final volume of the solution.

The alcohol solvent according to the present invention may contain methanol, ethanol, isopropanol, butanol, etc., and preferably includes or is represented by ethanol.

In the solution of the invention, the second component is dissolved in an isotonic buffer solution (component B), preferably accounting for 90% of the final volume of the solution.

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

According to the invention, in the disclosed method, the solution of the phenolic compound or mixture of phenolic compounds preferably has a pH value between 5 and 7.

Once prepared, the solution may optionally be filtered using a 0.22 μm filter.

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

Preferably, the contacting is for a period of about one hour.

More preferably, the contacting is for a period of about 30 minutes.

In an even more preferred embodiment, the contacting comprises a first step and a second step.

In a preferred embodiment, the first contacting step is performed for 30 minutes and the second contacting step is performed for 30 minutes.

Optionally, between the two contacting steps, a rinsing step (also referred to as a washing step) may be performed.

According to a preferred embodiment of the invention, the process is carried out in the dark, and more preferably entirely in the dark (i.e. avoiding any light exposure).

According to a preferred embodiment, the process is carried out while stirring the solution.

Depending on the temperature of the contacting step, it is preferably carried out at a temperature of about +20℃.+ -. 10 ℃.

In a preferred embodiment of the invention, after the contacting step, one or more washing steps may be performed on the treated surface, medical device, bioprosthesis or cardiac prosthesis.

Preferably, each of said washing steps is carried out using a suitable buffer; for example, a suitable buffer may be represented by a phosphate buffer.

In one embodiment, each washing step may be performed for a period of time ranging from about 15 to 30 minutes.

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

According to one embodiment of the invention, the method disclosed therein is performed on a biological surface, which may be pre-subjected to a pretreatment step.

In particular, the pretreatment step may have one or more of the following effects:

the stability of the protein is achieved by the fact that,

-Stabilization or removal of the lipids,

-Stabilizing or removing the cellular structure of the cell,

-Reducing antigenicity.

For the purposes of the present invention, the pretreatment step may include a step of pretreatment with one or more of glutaraldehyde, formaldehyde, quercetin (quercetin) or genipin (genipin), and a treatment for removing phospholipids.

According to a particular embodiment of the invention, the biological surface may be subjected to a preparation step with a capping agent (CAPPING AGENT, covering agent) selected from the group comprising: glycerol, heparin, amines (i.e., alkylamines, amino alcohols, ethanolamine), amino acids (lysine, hydroxylysine, sulfamates, taurine, sulfamates, dextran sulfate, chondroitin sulfate), hydrophilic multifunctional polymers (i.e., polyvinyl alcohol, polyethylenimine), hydrophobic multifunctional polymers (i.e., α -dicarbonyl, methylglyoxal, 3-deoxyglucitol, glyoxal), hydrazides (i.e., adipohydrazide), N-disuccinimidocarbonate, carbodiimides (i.e., 1-ethyl-3-dimethylaminopropyl carbodiimide hydrochloride-EDC, N-cyclohexyl-N' - (2-morpholinoethyl) carbodiimide-CMC, 1, 3-dicyclohexylcarbodiimide-DCC, 2-chloro-1-methylpyridinium iodide-CMPI, 2-chloro-1-methylpyridinium iodide-CMPI), antibiotics, cell-collecting agents, blood compatibilizers, anti-inflammatory agents, antiproliferatives, reducing agents (i.e., sodium cyanoborohydride, sodium borohydride, sodium bisulfite+acetylacetone, sodium formate-formaldehyde, monoepoxy alkane, or polyalkylene oxide).

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

In particular, the stabilization method deactivates available reactive groups on the pretreated surface.

More particularly, the stabilization method deactivates available aldehyde groups and carboxyl groups on the pretreated surface.

According to the invention, the disclosed method is an anti-calcification method.

The surface obtained with the method of the application represents a further object of the application.

Bioprostheses, medical devices and in particular cardiac prostheses comprising surfaces obtained according to the method of the present application represent a further object of the present application.

The surface obtained using the pretreatment and treatment method of the present application represents another object of the present application.

Bioprostheses, medical devices and in particular cardiac prostheses comprising surfaces obtained according to the pretreatment and treatment method of the present application represent a further object of the present application.

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

In particular, the protection method prevents the occurrence of subclinical thrombosis.

More particularly, the method of the invention is an anti-platelet adhesion method.

Even more particularly, the method of the invention prevents the synthesis of fibrin.

Fibrin synthesis is associated with the activation of soluble fibrinogen into insoluble fibrin polymers. Such polymers aggregate laterally to form fibers, which then branch to create a three-dimensional network and interact with circulating platelets, resulting in the formation of fibrin clots that are critical for hemostasis and wound clotting. If the clot enters the blood stream, it is called a thrombus, it occludes medium/small blood vessels, leading to ischemia, stroke, and heart attack.

In particular, the protection method of the present invention has been demonstrated to prevent and avoid the anchoring of circulating platelets on biological surfaces treated and/or pretreated according to the present invention.

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

Bioprostheses, medical devices and cardiac prostheses comprising surfaces obtained according to the pretreatment and treatment methods of the present application represent a further object of the present application.

According to the invention, the method according to the invention for treating a surface to be contacted with a biological fluid is a method of preserving and maintaining (PRESERVE AND MAINTAIN) the appropriate structural biomechanical properties.

In particular, the methods of the present invention have been demonstrated to increase the elongation properties of biologically treated tissue.

In particular, the method preserves the collagenous structure of BHV leaflets.

Thus, the method according to the invention for treating a surface to be contacted with a biological fluid is a protective method for maintaining the proper physiological hemodynamic and hydrodynamic properties of the treated BHV.

Bioprostheses, medical devices and cardiac prostheses comprising surfaces obtained according to the method of the present application represent a further object of the present application.

According to the invention, the method according to the invention for treating a surface to be contacted with a biological fluid is an antimicrobial and antiviral method, since it is a method of disinfection of the treated surface.

In particular, the antimicrobial methods of the present invention have bactericidal effects.

More particularly, the antimicrobial methods of the present invention are active against microorganisms that cause the onset of endocarditis.

In one embodiment, the microorganism is Gram ⁺ bacteria (Gram ⁺ bacteria), gram ^- bacteria (Gram ^- bacteria), yeast and mold.

In another embodiment, the microorganism is a mycobacterium, such as mycobacterium chelonii (Mycobacterium chelonae).

Thus, the method according to the invention for treating a surface to be contacted with a biological fluid is an anti-gram ⁺ method, an anti-gram ^- method, an anti-yeast method, an anti-mould method, an anti-mycobacterial method.

In particular, the antiviral methods of the present invention have virucidal effects on viruses.

More particularly, the virucidal effect is according to standard EN 14476.

In particular, the viruses belong to the families picornaviridae, adenoviridae and caliciviridae.

More particularly, the microorganisms are poliovirus type 1 LSc-2ab (RVB-1260), adenovirus type 5 (ATCC VR-5) and murine norovirus strain S-99 (RVB-651).

Bioprostheses, medical devices and cardiac prostheses comprising surfaces obtained according to the pretreatment and optional treatment methods of the present application represent a further object of the present application.

More particularly, the bioprosthesis may be represented by a cardiovascular prosthesis obtained with the pretreatment and optional treatment methods of the present invention.

According to a third object of the present invention, a method of treating a disease is disclosed, comprising the use of the medical device, bioprosthesis or cardiac prosthesis disclosed above.

According to a preferred aspect, the disease is heart disease.

According to one aspect, the disease is a disease in humans, and according to another aspect, the disease is a disease in animals.

In a particular embodiment, the method for treating a disease according to the present invention comprises a valve-in-valve approach (valve-in-valve approach) wherein the valve is deployed within a dysfunctional valve without requiring prior removal thereof.

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

In particular, the preparation of the solution comprises a first step in which the phenolic compound is dissolved in an alcoholic solvent.

For example, phenolic compounds from table a above may be dissolved.

If desired, further phenolic compounds may be dissolved in an isotonic buffer solution.

For example, phenolic compounds from table B above may be dissolved.

According to a preferred embodiment, the solution of compound a comprises 10% by volume of the final solution and the solution of compound B comprises 90% by 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.

The invention is further described in connection with the experimental section below.

The following experimental part shows the results of the measurements performed on the surface treated according to the present invention.

Polyphenol solution

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

Solution	Component A	Component B
			1	Resveratrol 3 + -2 mg/ml	Tannic acid 4+ -3.5 mg/ml
2	Resveratrol 3 + -2 mg/ml	Cynarate 2+ -1.5 mg/ml
			3	Aloin 1.5+ -1 mg/ml	Epigallocatechin 2+ -1 mg/ml
4	Aloin 1.5+ -1 mg/ml	Chlorogenic acid 4+ -3 mg/ml
			5	Caffeic acid 2+ -1.5 mg/ml	Tannic acid 4+ -3.5 mg/ml
6	Caffeic acid 2+ -1.5 mg/ml	Hydroxytyrosol 4+ -2.5 mg/ml
			7	Rosmarinic acid 2.5+ -2 mg/ml	Cynarate 2+ -1 mg/ml
8	Naringin 1+ -0.5 mg/ml	Gallic acid 1.5+ -1 mg/ml
			9	Hesperetin 2+/-1.5 mg/ml	Gallic acid 1.5+ -1 mg/ml

In particular, the surface has been treated with a solution 5 according to the disclosure above.

Preparation of polyphenols solution 5

Caffeic acid as component a was weighed according to the indicated concentration and dissolved in ethanol to 10% of the final volume of the polyphenol mixture. Tannic acid as component B column was weighed according to the indicated concentration and dissolved in modified phosphate buffer to 90% of the final volume of the polyphenol mixture. Both steps are performed in the dark. When dissolution is complete, the two solutions are mixed. The pH was adjusted to 5-7. The solution was filtered through a 0.22 μm filter. This solution is referred to as solution 5 or the polyphenol solution.

Other solutions according to the present invention may be prepared similarly to the above disclosure.

Glutaraldehyde stabilization

Tissue processing

Multiple bovine pericardium were carefully selected to obtain rectangular patches (n=32). All patches were subjected to a preliminary GLU crosslinking treatment. Briefly, pericardial tissue was incubated in buffered GLU solution in a dark room for three steps, each step for 24 hours. For this example, the GLU solution was 0.6% + -0.5% v/v for the first and second steps, followed by 0.2% + -0.15% v/v for the third step. The GLU-treated pericardial patch was subjected to two washing steps in phosphate buffer for 15 minutes each.

Sixteen patches were incubated with the inventive solution 5 prepared as disclosed above at Room Temperature (RT) under moderate but constant stirring in the dark for two steps of 25±10 minutes each. At the end of the incubation, the treated patches were washed five times in phosphate buffer for 15 to 30 minutes each. The sample is referred to as "treated".

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

Determination of the free carboxyl content

The pericardial patch was incorporated into OCT (optimal cutting temperature) and frozen by immersion in isopentane pre-chilled in liquid nitrogen. A MirrIR slide suitable for infrared reflectance studies was then used as support to make frozen sections of 7 μm thickness and each selected area was analysed by 64 scans with an FT-IR microscope in reflectance and mosaic mode. The detector used was a 4cm ^-1 resolution FPA. The treated tissue showed a lower concentration of carboxyl groups at wavenumber 1233cm ^-1 (corresponding to C-O bond stretching of carboxyl groups) than the sample fixed in GLU alone. Considering that the total number of quantified free carboxyl groups in the GLU-fixed samples is 100% of the available groups, the treated samples reported a reduction equal to 76% of the total.

Determination of the free aldehyde group content

150Ml of solution A was prepared: in ultrapure water, 0.2M citric acid, 0.5M sodium hydroxide and 8mM tin (II) chloride. 25ml of solution B was prepared: in a dark screw cap bottle, 0.22M ninhydrin was dissolved in 25ml ethylene glycol monomethyl ether (cellosolve). One volume of solution a was mixed with an equal volume of solution B and mixed in the dark for 45 minutes to obtain solution C. Note that 2ml of solution C is required for each sample to be analyzed.

The tissue samples should be prepared such that the wet weight of each sample is about 20mg. Each sample was incubated in 2ml of solution C at 100 ℃ for 20 minutes in the dark, cooled with water and diluted with 15ml of 50% isopropanol. The color development can be read at 570nm within 30 minutes. The nanomoles of aldehyde groups were determined relative to glycine standards.

Considering that the total amount of free aldehyde quantified in the GLU-fixed sample is 100% of the available groups, the treated sample reported a reduction equal to 56.3% of the total amount.

Fig. 1 shows the percentage reduction of unreacted aldehyde and carboxyl groups in the treated pericardial tissue patches (n=16 for each type of chemical group determination).

Platelet adhesion evaluation

Tissue processing

To evaluate the propensity for platelet adhesion and fibrin release under flow conditions, an in vitro blood flow model was used. Multiple bovine pericardium were carefully selected to obtain rectangular patches (n=12). All patches were subjected to a preliminary GLU crosslinking treatment. Briefly, pericardial tissue was incubated in buffered GLU solution in a dark room for three steps, each step for 24 hours. For this example, the GLU solution was 0.6% + -0.5% v/v for the first and second steps, followed by 0.2% + -0.15% v/v for the third step. The GLU-treated pericardial patch was subjected to two washing steps in phosphate buffer for 15 minutes each.

Six patches were incubated with the inventive solution 5 prepared as disclosed above at Room Temperature (RT) for two steps of 25±10 minutes each with moderate but constant stirring in the dark. At the end of the incubation, the treated patches were washed five times in phosphate buffer for 15 to 30 minutes each. The sample is referred to as "treated".

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

Platelet adhesion quantification

Heparinized bovine blood was collected from 3 different animals and thrombus quantified by adding a radioisotope. The pericardial tissue strips were deployed in 25.4mm catheters and a blood flow of 2.5L/min was achieved using peristaltic pumps for 1 hour. The strips were washed with saline and placed in a gamma counter to quantify the radiation (reflecting the relative thrombosis). Glu has an average emission value of 73.133 counts per minute (cpm), and the treated sample has an average emission value of 35.165cpm. In fig. 2, the results are expressed as a percentage of decrease in platelet propensity in polyphenol treated tissue, where the GLU sample is considered to be 100%.

Mechanical properties of the treated tissue

Tissue processing

Seventy-two rectangular strips (about 10mm long and 8mm wide) of bovine pericardium were treated with the GLU solution as disclosed above ("tissue processing"). The GLU-treated pericardial bands were subjected to two washing steps in phosphate buffer for 15 minutes each. Thirty-six patches were incubated with solution 5 disclosed above in the dark at room temperature for two steps, 25±10 minutes each, with moderate but constant agitation. At the end of the incubation, the treated patches were washed five times in phosphate buffer for 15 to 30 minutes each. The sample is referred to as "treated".

The remaining GLU-fixed pericardial band was used as a control (GLU, n=36).

Evaluation of mechanical Properties

Each strip was mounted on a tensile apparatus for uniaxial tensile testing (Thumler Z-X500 equipped with a 100N/500N load cell) with a rubber clamp. The initial length of the sample (distance between clamps) was set to 50mm. The tensile test was performed at 50 mm/min.

Each strip was dimensioned in terms of length (useful length 50 mm), width and thickness (measured average) and the cross-sectional area (x thickness) was calculated. The following parameters were obtained from each stretch curve:

breaking strength [ N ], expressed as the maximum strength before failure;

ultimate tensile strength [ MPa ], expressed as maximum strength divided by cross-sectional area (UTS);

failure strain [% ], expressed as strain at maximum strength;

Young's modulus [ MPa ] expressed as the slope of the linear region of the stress-strain curve (elastic phase).

For each parameter, an average value is calculated from the samples, one value being obtained for each patch. Again, an average value was calculated from the patches, one value being obtained for each test group.

Fig. 3 shows the ultimate tensile strength comparison between treated and untreated samples (GLU). Ultimate Tensile Strength (UTS), commonly referred to simply as Tensile Strength (TS), is the maximum stress that a material can withstand when stretched or pulled before breaking. No statistically significant differences from the control samples (GLU) were reported for polyphenol treatment.

Fig. 4 shows the percent elongation comparison between treated and untreated samples (GLU).

Fig. 5 shows the young's modulus correlation between treated and untreated samples (GLU). The percent elongation is closely related to Young's modulus. Young's modulus or modulus of elasticity is a characteristic, property, of a material that expresses the relationship between stretching and deformation under uniaxial loading conditions and under elastic (reversible) behavior of the material. It is defined as the ratio between the applied stress and the resulting deformation. As the young's modulus increases, the stiffness of the material increases. An increase in the elastic level of the treated tissue results in a concomitant decrease in its young's modulus compared to untreated pericardium (GLU).

The increased elasticity allows for better distribution of mechanical loads, particularly in the BHV region where greater pressures are favored. This can avoid the formation of tears and preserve the collagenous structure of the valve leaflet.

Sterilization potential of polyphenols solutions

The Bactericidal Activity (BA) of the polyphenol solutions of the present invention was evaluated against the following different microorganisms: staphylococcus aureus (Staphyloccoccus aureus) ATCC 6538, pseudomonas aeruginosa (Pseudomonas aeruginosa) ATCC 9027, enterococcus faecalis (Enterococcus faecalis) ATCC 29212, listeria monocytogenes (Listeria monocytogenes) ATCC 19111, salmonella typhimurium (Salmonella enterica typhimurium) ATCC 14028, streptococcus viridis (Streptococcus viridans) ATCC 6249, mycobacterium tuberculosis (Mycobacterium chelonae) ATCC 35752, candida albicans (Candida albicans) ATCC 10231 and aspergillus brasiliensis (Aspergillus brasiliensis) ATCC 16404.

The BA assay consisted of a suspension method in which bacteria were incubated with a known concentration of the solution 5 of the invention (inoculum) for 24 hours in a single pass. At the end of the incubation time, the contents of each tube were inoculated into a specific agar medium in a 90mm sterile petri dish, and after dilution in tryptic salt broth (MRD broth) depending on the microorganism tested, a pour plate or spread plate technique was used. The plates obtained are then incubated under specific conditions and temperatures, according to the growth requirements of each microorganism.

Preparation of inoculum

For each type of microorganism, the microbial suspension in the MRD broth was quantified by spectrophotometry at 620nm wavelength in a disposable 10mm optical path cuvette. Measuring absorbance of an aliquot of the suspension: a range between 0.150 and 0.460 corresponds to a cell concentration between 1x 10A 8CFU/ml and 3x 10A 8CFU/ml (where Candida albicans is between 1x 10A 7CFU/ml and 3x 10A 7 CFU/ml). For streptococcus stomatus (Streptococcus oralis), quantification was performed by cell counting under a microscope, since there was no correlation between absorbance measurements and bacterial concentration.

The data are elaborated by comparing the growth on the samples with the bacterial concentration at t0 to observe the effect of growth promotion or inhibition and expressed as a percentage of bactericidal activity. The results were compared to control samples (antibiotic and ethanol solutions).

The following table reports the percent bactericidal activity compared to standard antibodies and ethanol solutions.

Virucidal potential of polyphenol solutions

The virucidal activity of polyphenol solution 5 was evaluated according to the following guidelines: test methods and requirements, european Standard EN 14476:2013+A2:2019/UNI EN 14476:2019-chemical disinfectants and preservatives. Quantitative suspension tests are used to evaluate virucidal activity in the medical field. Test methods and requirements (stage 2/step 1). The following strains were tested for virucidal activity: poliovirus type 1 LSc-2ab (RVB-1260), adenovirus type 5 (ATCC VR-5) and murine norovirus S99 (RVB-651). The following table reports the percent virucidal activity of polyphenol solution 5 diluted to 80% (corresponding to the highest possible concentration that can be assessed according to the method).

	Percent viral inactivation
		Poliovirus LSc-2ab-RVB1260 of type 1	99.00±3.20
Adenovirus 5-ATCC VR5	99.70±1.02
		Murine norovirus S99-RVB651	99.48±1.54

Anti-adhesion effect of polyphenol solution on different strains of microorganisms

Anti-adhesion activity on the treated surface was evaluated with reference to the following different microorganisms: staphylococcus aureus ATCC 6538, pseudomonas aeruginosa ATCC 9027, enterococcus faecalis ATCC 29212, listeria monocytogenes ATCC 19111, salmonella typhimurium ATCC 14028, streptococcus grass ATCC 6249, mycobacterium tuberculosis ATCC 35752, candida albicans ATCC 10231, and aspergillus brasiliensis ATCC 16404.

Each microbial strain was grown overnight in dedicated broth at 37 ℃. At the end of the incubation, colony forming Units (UFCs) were counted to determine the effective concentration of microorganisms.

Tissue processing

90 Samples of about 2cm ² bovine pericardium each have been treated with the GLU solution as disclosed above ("tissue processing"). The 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 present invention according to the disclosed method, followed by 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 used as controls (GLU).

Evaluation of anti-adhesion Effect

The polyphenol treated and untreated patches were washed with PBS and incubated overnight at room temperature in PBS+antibiotics (300. Mu.g/mL). Different types of antibiotics specific for each type of microbial strain (neomycin, penicillin, cephalosporin, polymyxin, rifamycin, leap-years-mycin, quinolone, sulfonamide, macrolide, lincosamide, tetracycline, aminoglycoside, doxycycline, minocycline, ampicillin, amoxicillin/clavulanic acid, azithromycin, carbapenems, piperacillin/tazobactam, quinolones, chloramphenicol, ticarcillin, trimethoprim/sulfamethoxazole) are used. After overnight incubation, the tissue patches were thoroughly washed in PBS to remove any trace amounts of unbound antibiotic. Subsequently, the treated and untreated samples were individually exposed to different microbial strains (microbial load 1x 10 ⁷ CFU/mL) for 90 minutes at room temperature under moderate but constant agitation. At the end of the incubation, the tissue samples were subjected to three moderately vortexing channels to promote detachment of loosely bound bacteria. Finally, by400 Homogenizes the sample and serial dilutions of the homogenate obtained are spread in a petri dish containing a suitable selective growth medium. After incubation at 37 ℃ for 24 hours, colony forming units for each type of sample were counted.

Results are expressed as the percentage reduction of adherent microorganisms (each microbial strain, n=5) assessed in the treated pericardial patch compared to the untreated GLU-immobilized pericardial patch.

The following table reports the percentage reduction of adherent microorganisms assessed in the treated pericardial patch. The percentage value (n=5 for each microbial strain) was determined by comparison between treated and untreated GLU-immobilized pericardial patches.

Non-pyrogenicity of polyphenol-treated tissue

Monocyte activation assays (MATs) have been qualified and validated by the European alternate method validation center (ECVAM) in 2005 and the coordination Committee (ICCVAM) between alternate method validation institutions in 2008, and can be used to detect pyrogens. Since 2010, it has become one of the pharmacopoeia methods of european pharmacopoeia pyrogen detection (chapter 2.6.30) and was tested by the FDA "industrial guidelines-pyrogen and endotoxin: questions and solutions. Monocyte activation assay (MAT) is a human in vitro replacement for rabbit pyrogen assay (RPT) and can detect a full range of pyrogens, including endotoxin and non-endotoxin pyrogens (NEP).

Tissue processing

Eighteen samples of bovine pericardium, each about 2cm ², have been treated with the GLU solution as disclosed above ("tissue processing"). The GLU-treated pericardial samples were subjected to two washing steps in phosphate buffer for 15 minutes each. Nine patches were incubated with solution 5 at room temperature in the dark for two steps, 25.+ -. 10 minutes each, with moderate but constant agitation. At the end of the incubation, the treated patches were washed five times in phosphate buffer for 15 to 30 minutes each. The sample is referred to as "treated".

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

Pyrogenicity assessment

The sample was placed in 40ml endotoxin free water at 37 ℃ under moderate shaking for 1 hour. Water was analyzed using the MAT test. Briefly, contacting water with human monocytes mimics what happens in humans: monocytes are activated and produce various types of cytokines including interleukin-6 (IL 6) in the presence of pyrogens. Cytokines were then detected using an immunological assay (ELISA) involving specific antibodies and enzymatic chromogenic reactions.

The following table reports endotoxin unit assessments in treated and untreated (GLU) pericardial patches. Reference value to be considered as pyrogen: NMT 20 EU/appliance.

The advantages of the method of the present invention will be apparent in view of the disclosure reported above.

In particular, it has been demonstrated that the method of the present invention does not alter the surface treated and optionally pretreated according to the disclosure above and other properties of medical devices, bioprostheses and in particular cardiac prostheses comprising said surface.

As another advantage, the disclosed methods have been shown to deactivate available aldehyde and carboxyl reactive groups on treated or pretreated surfaces.

Furthermore, the method of the present invention prevents impairment of BHV functionality, as platelet deposition on the fibrin network results in the occurrence of subclinical small She Xieshuan formation (SLT), which in turn results in altered valve leaflet motility.

It has been found that the protective effect of the method of the invention results in a better distribution of mechanical loads, which avoids tearing, wear and hole formation.

As a further advantage, the method of the present invention prevents microbial adhesion and biofilm formation on the treated and optionally pretreated surface.

Again, the disclosed method avoids bacterial and viral contamination of the surface treated and optionally pretreated according to the present invention.

As a still further advantage, the disclosed methods preserve the non-pyrogenic nature of the treated and optionally pretreated surfaces.

Claims

1. A method for treating a surface to be contacted with a biological fluid, the method comprising the step of contacting the surface with a solution comprising a phenolic compound or a mixture of phenolic compounds.

2. The method of claim 1, wherein the surface is a surface of a medical device.

3. The method of claim 1 or 2, wherein the surface is a surface of a bioprosthesis.

4. A method according to any of the preceding claims 1 to 3, wherein the surface is the surface of a heart prosthesis or heart valve or pericardial tissue patch or surgical heart valve.

5. The method of any one of the preceding claims, wherein the biological fluid is selected from the group comprising: blood, serum, plasma, vitreous gel, tears, urine, saliva, stool; including synovial fluid, peritoneal fluid, pericardial fluid, pleural fluid and amniotic fluid.

6. The method according to the preceding claim, wherein the phenolic compound or mixture of phenolic compounds is selected from the group comprising: phenols, phenolic acids, anilines, phenolic compounds, flavonoids, phenylpropanoids and tannins.

7. The method according to the preceding claim, wherein the phenolic compound or mixture of phenolic compounds is selected from the group comprising: vanillin, cinnamic acid, phenylalanine, coumarin, xanthone, catechin, flavanone, flavone, chalcone, flavanol, leuco anthocyanidin, anthocyanin, and hydroxycinnamic acid.

8. The method according to the preceding claim, wherein the phenolic compound or mixture of phenolic compounds is selected from the group comprising: resveratrol, aloin, cynarate, epigallocatechin, tannic acid, caffeic acid, chlorogenic acid, hydroxytyrosol, rosmarinic acid, naringenin, gallic acid, hesperetin, quinic acid, elemene, pinoresinol, luteolin, citrus flavones, isorhamnetin, kaempferol, myricetin, eriodictyol, hesperetin, naringin, theaflavin, thearubigin, daidzein, genistein, glycitein, pterostilbene, delphinidin, dimethyl delphinidin, malvidin, paeoniflorin, chicoric acid, ferulic acid, salicylic acid.

9. A method according to any one of the preceding claims, wherein the method comprises the step of contacting the surface with a solution of the phenolic compound or a solution of a mixture of phenolic compounds for a period of time less than 2 hours.

10. A method according to any one of the preceding claims, wherein the method comprises a first contact step and a second contact step, wherein a rinsing step is performed between the first contact step and the second contact step.

11. A method according to any one of the preceding claims, wherein the method comprises a pretreatment step with one or more compounds selected from the group comprising: glutaraldehyde, formaldehyde, quercetin or genipin.

12. The method of any one of the preceding claims, wherein the pre-treatment step further comprises removing phospholipids.

13. The method of any of the preceding claims, wherein the pretreatment step further comprises a preparation step comprising the use of a capping agent.

14. The method of any one of the preceding claims, which is one or more of the following: methods of preserving and maintaining the proper structural biomechanical properties, methods of protecting for maintaining the proper physiological hemodynamic and hydrodynamic properties of the treated surface, antimicrobial and antiviral methods for disinfecting the treated surface.

15. A surface to be contacted with a biological fluid obtained according to the method of any one of claims 1 to 14.

16. A medical device or bioprosthesis comprising the surface of claim 15.

17. Medical device according to the preceding claim, selected from the group comprising: a heart valve; tendons; a ligament; pericardium; myofascial; dura mater; a tympanic membrane; intestinal submucosa; cartilage; fat and bone tissue; pelvis, abdomen, breast and dermis tissues.

18. A method for treating a disease comprising using the medical device or bioprosthesis of claim 16.

19. The method for treating a disease according to claim 18, wherein the disease is heart disease.

20. A method for treating a disease according to claim 18 or 19 wherein the disease is a disease in a human or animal.

21. A method for treating a disease according to any one of claims 18 to 20 wherein the method comprises a valve-in-valve method.

22. A biocidal method for treating a surface to be contacted with a biological fluid, the method comprising the step of contacting the surface with a solution comprising a phenolic compound or a mixture of phenolic compounds.

23. The biocidal method according to claim 22 wherein the surface is a surface of a medical device.

24. The biocidal method according to claim 22 or 23 wherein the surface is a surface of a bioprosthetic.

25. The biocidal method according to any one of the preceding claims 22-24 wherein the surface is a surface of a heart prosthesis or heart valve or pericardial tissue patch or surgical heart valve.

26. A biocidal method according to any one of the preceding claims 22-2 wherein the biological fluid is selected from the group comprising: blood, serum, plasma, vitreous gel, tears, urine, saliva, stool; including synovial fluid, peritoneal fluid, pericardial fluid, pleural fluid and amniotic fluid.

27. The biocidal method according to the preceding claim, wherein the phenolic compound or mixture of phenolic compounds is selected from the group comprising: phenols, phenolic acids, anilines, phenolic compounds, flavonoids, phenylpropanoids and tannins.

28. The biocidal method according to the preceding claim, wherein the phenolic compound or mixture of phenolic compounds is selected from the group comprising: vanillin, cinnamic acid, phenylalanine, coumarin, xanthone, catechin, flavanone, flavone, chalcone, flavanol, leuco anthocyanidin, anthocyanin, and hydroxycinnamic acid.

29. The biocidal method according to the preceding claim, wherein the phenolic compound or mixture of phenolic compounds is selected from the group comprising: resveratrol, aloin, cynarate, epigallocatechin, tannic acid, caffeic acid, chlorogenic acid, hydroxytyrosol, rosmarinic acid, naringenin, gallic acid, hesperetin, quinic acid, elemene, pinoresinol, luteolin, citrus flavones, isorhamnetin, kaempferol, myricetin, eriodictyol, hesperetin, naringin, theaflavin, thearubigin, daidzein, genistein, glycitein, pterostilbene, delphinidin, dimethyl delphinidin, malvidin, paeoniflorin, chicoric acid, ferulic acid, salicylic acid.

30. A biocidal method according to any one of the preceding claims 22-29 wherein the method comprises the step of contacting the surface with a solution of the phenolic compound or a solution of a mixture of phenolic compounds for a period of time less than 2 hours.

31. A biocidal method according to any one of the preceding claims 22-30 wherein the method comprises a first contact step and a second contact step, wherein a rinsing step is performed between the first contact step and the second contact step.

32. A biocidal method according to any one of the preceding claims 22-31 wherein the method comprises a pretreatment step with one or more compounds selected from the group comprising: glutaraldehyde, formaldehyde, quercetin or genipin.

33. A biocidal method according to any one of the preceding claims 22-32 wherein the pretreatment step further includes removing phospholipids.

34. A biocidal method according to any one of the preceding claims 22-33 wherein the pretreatment step further comprises a preparation step comprising the use of a capping agent.

35. A biocidal method according to any one of the preceding claims 22-34 wherein the biocidal activity is against microorganisms causing the onset of endocarditis.

36. A biocidal method according to any one of the preceding claims 22-35 wherein the microorganism is gram ⁺ bacteria, gram ^- bacteria, yeast, mould or virus.

37. The biocidal method according to any one of the preceding claims 22-36 wherein the microorganism is a mycobacterium (mycobacteria).

38. A biocidal method according to any one of the preceding claims 22 to 37 wherein the virus belongs to the families picornaviridae, adenoviridae and caliciviridae.

39. A surface obtained with the biocidal method according to any one of the preceding claims 22 to 38.

40. Bioprostheses, medical devices and cardiac prostheses comprising a surface according to the preceding claim.

41. The bioprosthesis according to the preceding claim, represented by a cardiovascular prosthesis.