EP3972659A1 - Method for preventing the formation of calcified deposits and for inactivating xenoantigens in biological matrices - Google Patents

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

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Publication number
EP3972659A1
EP3972659A1 EP20728220.3A EP20728220A EP3972659A1 EP 3972659 A1 EP3972659 A1 EP 3972659A1 EP 20728220 A EP20728220 A EP 20728220A EP 3972659 A1 EP3972659 A1 EP 3972659A1
Authority
EP
European Patent Office
Prior art keywords
acid
solution
biological matrix
mixture
treated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20728220.3A
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German (de)
English (en)
French (fr)
Inventor
Filippo Naso
Alessandro Gandaglia
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Biocompatibility Innovation Srl
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Biocompatibility Innovation Srl
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Publication date
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Publication of EP3972659A1 publication Critical patent/EP3972659A1/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3625—Vascular tissue, e.g. heart valves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/216—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials with other specific functional groups, e.g. aldehydes, ketones, phenols, quaternary phosphonium groups
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/02—Treatment of implants to prevent calcification or mineralisation in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/20—Materials or treatment for tissue regeneration for reconstruction of the heart, e.g. heart valves
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking

Definitions

  • the present invention finds application in the medical, biomedical or veterinary field and, in particular, in the preparation of biological and biocompatible matrices to be implanted in the human or animal body.
  • bioprosthetic substitutes are currently undergoing a significant market expansion.
  • one sector that is representative is the cardiovascular sector, especially in terms of the social and health impact that the established practice of cardiac valve replacement can cause.
  • Biomedical technology is capable of developing and surgically applying valve prostheses for replacement that can imitate the opening and closing function of dysfunctional native valves.
  • a desirable bioprosthetic heart valve substitute (BHV) should be capable of allowing a trans-valve flow that can overlap that of the analogue original, healthy valve, to ensure a long lifetime and not generate haemolytic or thrombogenic effects.
  • valve substitutes that are most often used are biological prostheses derived from xenogeneic tissues, in particular from pig valves or valves produced with the bovine, equine or porcine pericardium.
  • the US patent application US 2014/018909 discloses a method for preserving tissues intended for applications in the cardiovascular field, from the products of reactions that accumulate in patients with diabetes and, in particular, resulting from the oxidation of lipid components. These catabolic products are named as AGEs and are responsible for accelerating the degeneration mechanisms of bioprosthetic substitutes, such as vessels and heart valves, in diabetic patients.
  • the method is disclosed in connection with Pentagalfoylglucose - PGG, only and it is performed on a collagen matrix obtained by decellularizing a porcine heart valve and an elastin matrix obtained by treating a previously decellularized porcine carotid artery with alkali.
  • the international patent application WO 01/21228 discloses the use of gallotanic acid to mitigate the calcification of biological or synthetic tissues that can be used for the production of heart valve substitutes.
  • the increased biocompatibility comprises the increase in one or more of: preventing the formation of calcified deposits, inactivating xenoantigens in biological matrices, preventing the thrombus formation in a biological matrix, preventing the lipid infiltration into a biological matrix, preventing the onset of inflammatory process mediated by the cellular component, prevents the biofouling processes on a biological matrix.
  • Figure 1 results of the assays on the phenolic compound release.
  • Figure 2 results of the in-vitro calcification assays.
  • Figure 3 the valve placed in the custom-fabricated silicone holder.
  • Asterisks indicate a significant difference between the untreated and treated groups for the specific parameter indicated.
  • Figure 10 some of the most significant commercial BHVs explanted from swine after 1 month of follow-up.
  • FACTATM treated BHVs D, E, and F
  • untreated BHVs B and C
  • non- implanted BHV A
  • Figure 11 macroscopic aspects of untreated commercial BHVs after 1 month of follow-up in a common swine model. It is evident the formation of blood clots (A) and of a fibrotic pannus affecting the cusps (A, C and D) and the suture ring of the prostheses (B).
  • Figure 12 Hematoxylin & Eosin staining of untreated commercial BHVs after 1 month of follow-up in swine.
  • the black arrows (box A and E) highlight the presence of a dense and homogeneous fibrotic layer on and in the ventricular surface of the leaflets.
  • a foreign body reaction is present in the other side of the leaflets (box C and G) with inflammatory cells penetration (box D and H).
  • Box D and H magnification 40X Box
  • FIG. 13 Hematoxylin & Eosin staining of FACTATM treated commercial BHVs after 1 month follow-up in the swine. Where foreign body reaction occurs (box A), no cellular penetration is appreciable (box C). Box A, D and F magnification 4X. Box B, E and G magnification 10X. Box C magnification 40X.
  • Figure 14 Oil Red“O” staining for lipid identification.
  • Figure 15 Von Kossa staining of untreated commercial BHVs after 1 month follow-up in the swine.
  • Figure 16 Von Kossa staining of FACTATM treated commercial BHVs after 1 month followup in the swine. No calcium deposit was observable. Complete absence of micro-calcification. Box A, C and E magnification 4X. Box B, D and F magnification 10X;
  • Figure 17 Mallory Trichromic staining of untreated commercial BHVs after 1 month followup in the swine.
  • the collagenic matrix is recognizable in blue colour, the presence of structured thrombi in red/yellow.
  • Figure 18 Mallory Trichromic staining of untreated commercial BHVs after 1 month of follow-up in the swine. In red/yellow colour is clearly evident the presence of a fibrous pannus that culminates in a thrombus in the ventricular surface of the leaflet. Box A, magnification 4X, multiple panel magnification 10X.
  • FIG 19 Mallory Trichromic staining of FACTATM treated commercial BHVs after 1 month follow-up in the swine. No significant presence of abnormal fibrous formation, like pannus or structured thrombi. In some samples is clearly evident the preservation of the elastic component (box E and F, G and H). Box A, C, E and G magnification 4X. Box B, D, F and H magnification 10X.
  • Figure 20 staining of the reference commercial BHV (not implanted in the swine). H&E box A and B; Oil Red“O”, box C and D; Mallory Trichromic, box E and F; Von Kossa, box G and H. Box A, C, E and G magnification 4X. Box B, D, F and H magnification 10X.
  • Figure 21 multiple staining of FACTATM treated commercial BHVs after 1 month follow-up in the swine.
  • H&E box A and B Mallory Trichromic, box C and D; Oil Red“O”, box E; Von Kossa, box F.
  • Box B and D magnification 40X.
  • Figure 23 results of the Ca 2+ quantification comparison (by ICP analysis) in FACTATM treated (F) and untreated (C) leaflets from commercial bioprosthetic heart valve after 1 , 2 and 4 months follow-up in the WT mice.
  • Figure 24 results of the Ca 2+ quantification after Von Kossa histological staining on FACTATM treated (F) and not treated (C) samples analysis after 4 months follow-up in the WT mice.
  • Figure 25 results of the Ca 2+ quantification comparison (by ICP analysis) in FACTATM treated (F) and untreated (C) leaflets from commercial bioprosthetic heart valve leaflets after 1 and 2 months of follow-up in alpha-Gal KO mice.
  • the present invention discloses a method for preventing the formation of calcified deposits in a biological matrix.
  • said method comprises the step of contacting said biological matrix with a solution comprising a mixture of phenolic compounds for a period of fewer than two hours at the temperature of 35 ⁇ 2°C and in the dark.
  • the method disclosed also inactivates xenoantigens in biological matrices. According to another aspect of the invention, the method disclosed prevents the thrombus formation on a biological matrix.
  • the method disclosed prevents the lipid infiltration into a biological matrix.
  • the method disclosed prevents the onset of an inflammatory process mediated by the cellular component.
  • the method disclosed prevents the formation of the microorganism biofilm on a biological matrix (biofouling).
  • the method disclosed provides one or more of: preventing the formation of calcified deposits, inactivating xenoantigens in biological matrices, preventing the thrombus formation on a biological matrix, preventing the lipid infiltration into a biological matrix, preventing the onset of inflammatory process mediated by the cellular component, prevents the biofouling processes on a biological matrix.
  • the method disclosed provides for: preventing the formation of calcified deposits, inactivating xenoantigens in biological matrixes, preventing the thrombus formation on a biological matrix, preventing the lipid infiltration into a biological matrix and preventing the onset of inflammatory process mediated by the cellular component, prevents the biofouling processes on a biological matrix.
  • a biological matrix obtained with the method of the invention is another object of the present application.
  • a biological matrix obtained with the method of the invention for use in the treatment of heart diseases in the medical, biomedical and/or veterinary field represents a second object of the present application.
  • a method for the treatment of heart diseases in humans or animals comprising the use of a biological matrix obtained according to the method of the invention.
  • a solution comprising a phenolic compound or a mixture of phenolic compounds to be used in the methods of the present patent application represents a further object of the invention.
  • the present invention discloses a kit for performing the invention method.
  • the present invention discloses a method for preventing the formation of calcified deposits in a biological matrix.
  • a calcification may occur on the surface of a biological matrix or inside a biological matrix.
  • FACTATM a method according to the invention.
  • said method comprises the step of contacting the said biological matrix with a solution comprising a mixture of phenolic compounds.
  • biological matrices are xenomatrices, i.e. they are not of human origin.
  • they can have equine, porcine or bovine origin.
  • biological matrices shall be intended as: vessels, cardiac valves, tendons, ligaments, pericardium, muscular fasciae, dura mater, tympanic membrane, intestinal submucosa, cartilages, adipose tissue and bone tissue, pelvic, abdominal and breast tissue.
  • biological matrices are selected in the group comprising cardiovascular prostheses (cardiac valves) and pericardial tissue patches.
  • cardiac valve An example of such cardiac valve is represented by TrifectaTM Valve with GlideTM Technology and EpicTM Mitral Valve (Abbott/St. Jude Medical, St. Paul, MN, USA).
  • Sapien 3, Sapien 3 Ultra and Sapien XT transcatheter heart valve (Edwards Lifesciences, Irvine, CA, USA), Inspiris Resilia, Intuity Valve System, Magna Easy, Perimount RSR and Perimount Valve (Edwards Lifesciences, Irvine, CA, USA), AvalusTM, Contegra Valved Conduit, FreestyleTM, Hancock II TM, MosaicTM and MosaicTM Ultra, MelodyTM and CoreValve transcatheter valve replacement platform (Medtronic, Minneapolis, MN, USA), Acurate NeoTM and Lotu EdgeTM Aortic Valve System (Boston Scientific, Marlborough, MA, USA), Accufit® Transapical Mitral Valve (Sinomed, Tianjin, China), Xinli® (KingstronBio, Jiangsu, China), BioconduitTM, BiomitralTM, BiopulmonicTM Conduit and Injectable Biopulm
  • the cardiac valves which can be treated according to the present invention are represented by trans-catheter implantable heart valves (TAVI); said valves require to be implanted through a catheter and, accordingly, need to be pliable to be housed within the catheter.
  • TAVI trans-catheter implantable heart valves
  • a phenolic compound within the phenolic compound mixture above referred shall be intended as a phenolic or polyphenolic compound (in some instances used here as synonyms) selected from the group comprising: simple phenols, phenolic aldehydes, phenolic acids, phenylamines, phenol compounds, flavonoids, phenylpropanoids and tannins.
  • a phenolic compound is selected in the group comprising: vanillin, cinnamic acids, phenylalanine, coumarins, xanthones, catechins, flavononids, flavones, chalcones, flavanonols, flavanols, leucoanthocyanidin, anthocyanidin, hydroxycinnamic acids, phenylpropanoids.
  • a phenolic compound can be selected in the group comprising: resveratrol, aloin, cyanarin, epigallocatechin, tannic acid, caffeic acid, chlorogenic acid, hydroxytyrosol, rosmarinic acid, narigenin, gallic acid, hesperidin, quinic acid, eleonolic acid, pinoresinol, luteolin, apigenin, tangeritin, isorhamnetin, kaempferol, myricetin, eriodictyol, hesperetin, naringenin, theaflavin, thearubigins, daidzein, genistein, glycitein, pterostilbene, delphinidin, malvidin, pelargonidin, peonidin, chicoric acid, ferulic acid, salicylic acid.
  • curcumin can also be used.
  • derivatives of the above disclosed phenolic or polyphenolic compound are also encompassed; for instance, salts or esters may also be used.
  • the solution comprises a mixture of two or more of the above disclosed phenylpropanoids.
  • each phenylpropanoid may be present in a concentration comprised between about 0.2-5 mg/ml ⁇ 0.5 mg/ml (w/total volume of the solution).
  • the step of contacting is performed for a period of time of less than 2 hours.
  • the step of contacting is performed for a period of about one hour.
  • the contacting step is continued for a period of about 30 minutes.
  • a first contacting step is repeated for one additional 30- minute period.
  • a rinsing step may be performed between the two contacting steps.
  • the method is performed completely in the dark, i.e. avoiding any exposure to light.
  • the temperature of the contacting step it is preferably performed at the temperature of about +35°C ⁇ 2°C.
  • the matrix after the contacting step, may be subjected to one or more washing steps.
  • washing step is performed with a suitable buffer; for example, a suitable buffer may be represented by phosphate buffer.
  • Each washing step may be performed for a period of about 15 minutes.
  • the method can be performed on both native biological matrix and biological matrix that had previously been treated for other purposes, for instance for stabilizing the protein, lipid and cell structures as well as lowering the potential antigenic action of the host.
  • biological matrices may comprise: decellularized extracellular matrix, partially digested matrix and gelatins from animal origin.
  • the biological matrix before treating the biological matrix according to the method of the invention, it can be subjected to a treatment with glutaraldehyde, formaldehyde and quercetin.
  • the method disclosed also inactivates xenoantigens in biological matrices.
  • xenoantigen it is intended of animal origin that can be recognized by the immune system and can induce an antibody/immune-mediated/inflammatory response in the human host organism; in the present description the terms “xenoantigen”, “antigen”, “xenogeneic antigen”, “epitope” and “crucial antigen” can have the same meaning, and can be used together or to substitute for each other.
  • “xenoantigen” refers to the alpha-Gal epitope.
  • the method disclosed prevents the thrombus formation and clots on and in a biological matrix. According to a further aspect of the invention, the method disclosed prevents the lipid infiltration into a biological matrix.
  • the method disclosed prevents the onset of an inflammatory process mediated by the cellular component.
  • the method disclosed prevents the formation of the microorganism biofilm on a biological matrix (biofouling).
  • the method of the invention has proved to prevent the formation of a biofilm formed by Staphylococcus aureus by decreasing the adhesive capacity on the prostheses surface.
  • the method of the invention is effective on both Gram- and Gram + bacteria.
  • a biological matrix obtained with the method of the invention is another object of the present application.
  • said biological matrix is represented by a cardiovascular bio-prostheses obtained with the method disclosed, which has a significant decrease in the formation of calcific deposits, structured thrombus, lipoproteins infiltration and significant resistance to bacterial adhesion and consequent tissue colonization.
  • the present invention discloses the biological matrix obtained with the method above described for use in the treatment of heart diseases in the medical or in the veterinary field.
  • said solution is prepared by mixing two or more phenolic compounds as above disclosed.
  • a sodium phosphate buffer can be used.
  • the mixture of phenolic compounds may be prepared in a solution of NaOH.
  • the solution of the invention may be added with a suitable enzyme endowed with oxidase activity; for instance, there can be used tyrosinase, L-gulonolactone oxidase, laccase, etc.
  • the solution is preferably prepared in the dark, i.e. avoiding any exposure to light.
  • said kit comprises:
  • the phenolic compounds may be present in powder form to be dissolved in the buffer.
  • knockout animal for the alpha-Gal antigen refers to an animal in which the gene that encodes for the alpha-galactosyltransferase enzyme has been silenced. Such enzyme is responsible for attacking the membrane glycoproteins and lipoproteins of the alpha-Gal epitope. Its absence causes the production of tissues that lack the epitope in question entirely and which in this respect are entirely comparable to the tissue of the human body. In the present invention, knockout animal vascular tissues for the alpha-Gal antigen were used as absolute negative control.
  • Trifecta GTTM bioprosthetic aortic heart valves were extracted from their packaging and subjected to two washing steps in phosphate buffer for 15 minutes each. Different solutions were prepared based on the phenolic mixtures reported above and filtered with a 0.2 pm sterile membrane. The Trifecta GTTM valves were incubated with each single solution under moderate but constant stirring in the dark, for two step of 25 ⁇ 5 minutes each, at room temperature.
  • tissue samples from the treated and untreated TrifectaTM valves were placed in test tubes to which a phosphate buffer is added up to a final volume comprised between 1000 pl_ and 1500 mI-.
  • a monoclonal mouse antibody, directed against the alpha-Gal epitope, is added (in the present example this is an IgM clone called M86), at the preferable concentration of [1 :50] v/v and the whole is incubated for 120 ⁇ 10 minutes at 37 ⁇ 2°C under constant but moderate stirring.
  • the samples were subjected to centrifugation at 14,750 x g for 30 ⁇ 5 minutes at ambient temperature.
  • a 96-well plate was prepared, in which the bottom of the wells is lined with 100 pL per cell of alpha-Gal/serum albumin at 5 pg/ml in phosphate buffer.
  • the plate thus prepared was incubated for 60 ⁇ 10 minutes at a temperature comprised between 30°C and 40°C, although it is preferable to stabilize everything at 37°C.
  • the blocking was done with 300 pL per well with serum albumin, followed by incubation for 60 ⁇ 10 minutes at ambient temperature, in darkness. Subsequently, 3 washes as above were performed. For each well, 100 pL of supernatant, taken after centrifugation from each treated sample, was added. The samples were loaded into the plate, each type of sample occupying the wells of an entire column. There follows incubation of the plate for 120 ⁇ 10 minutes at a temperature comprised between 30°C and 40°C, although it is preferable to stabilize everything at 37°C.
  • the plate is then incubated again for 60 ⁇ 10 minutes at a temperature comprised between 30°C and 40°C, although it is preferable to stabilize everything at 37°C.
  • valve treated and non-treated has been subjected to the following in-vitro and in-vivo analysis.
  • the FACTATM treatment is based on the action of a mixture containing specific polyphenols according to the present invention.
  • polyphenols at certain dosages can be toxic (LD50 5 g/Kg), accumulating in the liver and kidneys.
  • LD50 5 g/Kg toxic
  • To confirm the stability of the FACTATM treatment we evaluated the release of phenolic residues over time.
  • a set of untreated commercial BHV leaflets were analyzed at same time-point.
  • the total polyphenol content present in the solutions was evaluated using a protocol based on the reaction, under conditions of alkaline pH, between the phenolic compounds eventually present in the storage solution and diazonium salts.
  • the reaction product is a stable chromophore that can be detected and quantified by absorbance analysis at a wavelength of 480 nm.
  • the concentration of phenols present in the sample is quantified by an external calibration method with a catechin standard and is therefore expressed as a millimolar concentration of catechin equivalent (mM).
  • Leaflets from untreated (B) and FACTATM treated (BT) commercial BHVs, native (WT) and FACTATM treated native aortic porcine leaflets (WT T) and alpha-Gal knockout porcine aortic leaflets (KO) were incubated in pooled normal human serum (Innovative Research, Peary Court Novi, Ml) with 2% of penicillin and streptomycin for 14 days at 37°C.
  • a set of samples was incubated in PBS in the same condition. After incubation, samples were washed twice in PBS for 10 min and subjected to acid hydrolysis in HNO 3 at 110°C for 12 h.
  • Calcium evaluation was performed in hydrolyzed samples by inductively coupled plasma according to the directives of the EPA6010D method and expressed as pg Ca 2+/ mg of dry defatted weight (d.d.w.).
  • FACTATM treated BHV shows a decrease in the calcific propensity of 90% (B vs BT p ⁇ 0.05) and of 44.4% (BT vs KO, p ⁇ 0.05) when compared with untreated BHVs (B) and alpha-Gal knockout tissues (KO) respectively.
  • Knockout tissue is considered the least calcifying biological support and is used as an absolute negative control in xenotransplant-induced calcification studies.
  • FACTATM treated BHV has been shown to calcify almost 50% less than the KO standard.
  • the hydrodynamic performance of the valves was assessed under simulated pulsatile flow in a Vivitro® Pulse Duplicator System (Vivitro Labs Inc., Victoria, BC, Canada).
  • the flow simulator was used to model the left side of the heart.
  • the test valves were fitted into a custom-fabricated silicon holder (Figure3) and placed in the aortic position of the pulsatile flow system.
  • a 25 mm Bjork- Shiley tilting disc valve was used in the mitral position as a reference valve.
  • the testing was performed in 0.9% (w/v) NaCI at room temperature.
  • the valves were tested under five pulsatile flow conditions (Table 1), according to ISO 5840-3. The flow conditions corresponded to cardiac outputs between 2.5-9 l/min.
  • the diastolic/systolic pressure was set to 80/120 mm Hg, whereas the systolic duration occupied 35% of the cardiac cycle.
  • the hydrodynamic performance of the valves was assessed in terms of the mean pressure drop (MPD) and peak pressure drop (PPD) across the valve, root mean square forward flow through the valve (QRMS), peak forward flow through the valve (QPEAK), valve effective orifice area (EOA), reverse flow through the valve during valve closure and while the valve was fully closed, and valve energy losses.
  • MPD mean pressure drop
  • PPD peak pressure drop
  • QRMS root mean square forward flow through the valve
  • QPEAK peak forward flow through the valve
  • EOA valve effective orifice area
  • the data was analyzed in Microsoft Excel® and Prism® 7 for Windows (v7.03, GraphPad Software Inc., California) and expressed as mean ⁇ standard deviation (SD). Two-sided unpaired I- test was used to assess significant differences between the treated and untreated groups, at the
  • the mean dynamic and static regurgitation volumes of the untreated and treated groups at each flow conditions are illustrated in Figure 7.
  • the dynamic regurgitation (closing volume) and static regurgitation (closed volume) for both groups were negative representing back-flow through the valves.
  • the regurgitation volumes were more significant for the lower flow conditions and increased with increasing flow rates. No statistically significant differences were found in the regurgitation volumes between the untreated and treated groups.
  • the mean energy losses for the treated and untreated groups are illustrated in Figure 8. With increasing flow condition, an opposite pattern was observed for the energy losses in the forward flow and closed phase. The lowest energy loss during the forward flow phase was observed for the low-flow condition (60/60) and, increased with increasing flow rates. In contrast, the highest energy loss during the closed phase was observed for the low-flow condition and decreased with increasing flow rates. There was no significant difference in the energy losses between the untreated and treated groups, in any of the flow conditions tested.
  • the stress-strain curves were used to calculate the elastic and collagen phase slopes, transition stress and strain, failure strain and ultimate tensile strength.
  • the data was analysed in Excel and expressed as mean ⁇ SD. Two-sided unpaired t-test was used to assess significant differences between the treated and untreated groups at the 0.05 confidence level.
  • Tissues were embedded in OCT compound (Tissue Tek; Sakura Finetek, Tokyo, Japan), cryo- cooled in liquid nitrogen, and cut into 6-mm cryo-sections. Histological analyses were performed by the use of commercial kit from Bio-Optica (Milan, Italy) according to the indications provided by the manufacturer. The histo-kits used are listed in Table 2.
  • leaflets from untreated valves report the presence of a homogeneous fibrotic pannus on the ventricular surface as highlighted by the black arrows in Figure 12 (Box A and E).
  • a foreign body reaction is clearly visible in all the explanted cusps, both untreated ( Figure 12) and FACTATM treated commercial BHVs ( Figure 13). It should be noted that in untreated commercial valves the cellular component is able to penetrate the matrix (Fig. 12 box D and H).
  • the FACTATM treatment seems to be able to exert a barrier effect that counteracts the penetration into the inner stroma of the inflammatory cellular component (Fig. 13, box C) responsible for the subsequent degeneration of the extracellular matrix.
  • the FACTATM treated commercial BHVs are optimally preserved from calcium deposition, as can be seen in Figure 16.
  • Figure 18 is reported a panel showing the presence of a fibrous pannus that culminates in a structured thrombus formation.
  • FIG 21 shows a panel of different histological staining of a FACTATM treated commercial BHVs. This panel summarizes all the improvements that the FACTATM treatment provides to the bioprosthetic tissue. It is clearly evident the barrier effect exerted by the treatment; in fact, the host cellular population is unable to penetrate within the matrix (box B and D). This barrier effect is further confirmed by the absence of any lipid infiltration as showed in the box E. Finally, the tissue is free from calcific deposition (box F).
  • the anti-adhesive bacterial activity on FACTATM treated and untreated commercial valves was evaluated with the bacterial species of Staphylococcus aureus ATCC 6538 (gram-positive).
  • the bacteria were grown overnight in Tryptic Soy Broth (TSB) at of 37°C.
  • TTB Tryptic Soy Broth
  • the total bacterial load was assessed by 10-factor serial dilutions in TSB (10-1 to 10-7), sown in Petri dishes with appropriate selective medium (MSA-Mannitol Selective Agar) and kept in overnight incubator. At the end of the incubation, the units forming colony (UFC) were counted to determine the effective concentration of the microorganism.
  • optical density at 600 nm was determined from each tiled dilution, in order to verify the linearity between the latter and the effective microbial load of the broth.
  • Commercial FACTATM treated and untreated BHVs leaflets were cut with a punch for biopsies (3 mm in diameter), in order to obtain the same useful surface for bacterial adhesion.
  • the tissue punches thus obtained were washed with PBS and incubated overnight at room temperature in PBS + gentamicin (300 pg/mL) under moderate but constant agitation.
  • the commercial BHVs tissue punches were washed extensively in PBS to remove any remaining antibiotics. Subsequently, the FACTATM treated and untreated samples were exposed to S. aureus bacterial suspensions (bacterial load 1 X 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 subject to three moderate vortexing mix to facilitate the detachment of the loosely bound bacteria.
  • tissue punches were homogenized by Ultraturrax and serial dilutions of the obtained homogenates, plated in Petri dishes containing the appropriate selective growth media. Finally, after 24 hours of incubation at 37°C, the colony-formed units were counted for each type of samples.
  • the bacterial anti-adhesive activity was calculated using the following formula:
  • CT is the bacterial charge of the tissue sample obtained from a commercial BHV treated with the FACTATM technology and CNT is the one achieved in tissue pinches from commercial BHVs (untreated).
  • the FACTATM treatment significantly limits the adhesive capacity of S. aureus (reduction of 96 ⁇ 4%) on the tissue surface.
  • ICP - Inductive Coupled Plasma
  • the untreated original samples (C) showed a considerable tendency to calcify just after the first month of implantation. The trend continues significantly during the follow-up resulting in a calcium deposition of 7.17 pg/mg of d.d.w. as quantified at the end of the fourth month of the implant (Table 1 ).
  • the FACTATM treated samples (F) report a negligible amount of calcium even after 4 months follow-up. Noteworthy, the calcium content in the F samples does not exceed significantly the total calcium detected in the UN, confirming no calcium uptake over time (Table 1).
  • Table 1 Average of calcium content expressed as pg/mg of d.d.w. in FACTATM treated (F) and untreated (C) samples after different follow-up.
  • the purpose of the test was the evaluation of the calcium uptake in leaflets excised from a commercially available Trifecta GTTM BHV and its FACTATM treated counterparts, once implanted in an alpha-Gal knockout (KO) murine animal model.
  • the KO mouse is a specifically developed BCI-owned murine model genetically modified to silence the expression of the alpha-Gal xenoantigen.
  • Alpha-Gal KO mice are characterized by having an immunological response mechanism similar to the human where the alpha-Gal antigen stimulates the production of specific anti-Gal antibodies.
  • Leaflets were implanted sub-cutis in the back of C57BI/6 alpha-Gal KO mice.
  • ICP - Inductive Coupled Plasma
  • the amount of calcium determined in the F samples at 1 -month as well as 2-months of follow-up was negligible as it is lower than the ICP quantification threshold (0.048 pg/mg).
  • the calcium content in the F samples does not exceed the total calcium detected in the UN, confirming no calcium uptake at land 2 months follow-up.
  • the C specimens implanted in KO show a pro-calcific effect by reporting a significant and homogenous presence of calcium.
  • the results demonstrate a clear role of the alpha-Gal in promoting calcium deposition in the KO mice as it happens into humans.
  • FACTATM treatment proves to be effective in counteracting the calcification even in a critical physiological system like the alpha-Gal KO model.
  • the invention method is capable of limiting the deposition of calcium salts, therefore preventing the formation of episodes of calcific dystrophy of the valve.
  • the invention method has shown to protect the treated cardiovascular bio-prostheses against the formation of blood clots and structured thrombi.
  • the method of the invention allows to avoid the lipoproteins infiltration from blood circulation and the consequent onset of cell-mediated inflammatory tissue response in the treated cardiovascular bio-prostheses.
  • the method disclosed in the present invention proved to be very stable and safe.
  • the method proved to be able to inhibit the formation of calcified deposits both at the in-vitro and in-vivo investigations.
  • the BHVs treated with FACTATM were clearly non-thrombogenic and protected from cellular and lipid infiltration.
  • the invention kit it is aimed at the autonomous treatment of bioprosthetic substitutes that are already prepared, with a method according to the invention as described above, useful for health facilities like clinics and hospitals.
  • the invention is susceptible of numerous modifications and variations, all of which are within the scope of the appended claims.

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CN118105544A (zh) * 2022-11-30 2024-05-31 上海微创心通医疗科技有限公司 生物组织材料及其制备方法和应用
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