Articles and methods for processing bioprosthetic tissue

Abstract

The present invention relates to a method of treating biological prosthesis tissue, said method comprising the use of cyclodextrin, preferably in combination with ethanol. The present invention also relates to an article for treating biological prosthesis tissue comprising cyclodextrin, and to a kit for treating biological prosthesis tissue comprising cyclodextrin.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201780089366.2, filed on November 30, 2017, entitled "Article and Method for Processing Biological Prosthetic Tissue", which was filed after PCT international application No. PCT / EP2017 / 080977 entered the Chinese national phase. Technical Field

[0002] This invention relates to the treatment of biological prosthetic tissues, particularly the treatment of biological tissues for cardiovascular prosthetics. Background Technology

[0003] Calcification is one of the leading causes of failure in prosthetic heart valves derived from glutaraldehyde pretreatment of bovine pericardium or porcine aortic valves.1-3 Such pretreatment is disclosed, for example, in U.S. Patent 5,931,969 (Baxter). The mechanism of this pathological calcification is not fully understood. In animal models, the initial sites of calcification have been shown to be the cell membrane, nucleus, and intracellular organelles, such as mitochondria in inactivated cells. The size and number of cell-associated calcification deposits increase with the duration of implantation. Subsequently, direct collagen calcification occurs in the cusp valve and elastin calcification occurs in the aortic wall. Various host factors (e.g., the young age of the recipient) and implantation factors (e.g., glutaraldehyde fixation) also exacerbate calcification events.

[0004] Biological tissues are currently primarily used to manufacture bioprosthetics, mainly for long-term implantation in the cardiovascular field. Typically, biological tissues used in cardiovascular bioprosthetics are represented by xenografts (such as natural valves, pericardial sacs, blood vessels, tendons, etc.) derived primarily from bovine or porcine tissues. Before being used to manufacture bioprosthetics and subsequently implanted, these biological tissues must undergo chemical treatment to avoid or mitigate any allogeneic or immune tissue reactions.

[0005] In this research field, biological tissues are typically chemically stabilized through a chemical reaction called "crosslinking," which aims to bind collagen and elastin fibers together and stabilize the extracellular matrix. Crosslinking also has the advantage of increasing the mechanical properties of the tissue to impart the necessary long-term durability. This last aspect is of particular interest for pericardial tissue or cusp valves of natural animal valves used to assemble and replace heart valve bioprosthetics for replacing valves in patients with aortic, mitral, tricuspid, or lung diseases.

[0006] Crosslinking chemistry has been a subject of much research for the past 50 years. Several methods using different molecules have been applied, but the one still in use today and primarily applied by heart valve manufacturers is glutaraldehyde.

[0007] However, due to the chemical processes between the free aldehyde groups of glutaraldehyde, phospholipids, fatty acids, and cholesterol, and the residual antigenicity of biological tissues... 1,2,3 Glutaraldehyde cross-linking or fixation promotes malnutrition-induced calcification. Extensive efforts in basic research over the years have focused on developing tissue processing methods to prevent calcification in glutaraldehyde-fixed xenograft tissues. The main anti-calcification strategies aim to extract lipids... 4 Or neutralize toxic aldehyde residues 5 Glutaraldehyde-fixed xenografts exhibit cellular / humoral rejection and undergo secondary calcification. 3 Tissue valve calcification also primarily begins within the already inactivated residual cells. 1 .

[0008] Cyclodextrin The structure of cyclodextrin Cyclodextrins are composed of 6, 7, or 8 monomers of D-(+) pyranose bound together by α,1-4 glucosidal bonds, forming a conical ring shape. Figure 1 Cyclic natural oligosaccharides. The three most common forms are α-CD (6 units), β-CD (7 units), and γ-CD (8 units).

[0009] Due to the formation of intramolecular hydrogen bonds, these macromolecules exhibit a three-dimensional rigid ring-shaped structure with an outer surface containing CH2OH groups and an internal cavity with hydrophobic properties. The internal cavity has a size that depends on the number of units constituting the cyclodextrin. 6 .

[0010] The presence of cavities and the solubility of hydrophilic alcohol functional groups in water endow cyclodextrins with the ability to complex in aqueous solutions.

[0011] At room temperature, cyclodextrin has the appearance of a crystalline white powder, is odorless, and has a mild sweetness.

[0012] The three-dimensional structure confines the hydroxyl groups at the outer boundaries, while only hydrogen and oxygen bonds exist within the cavity. This creates a hydrophobic central cavity, while the outer surface is hydrophilic. In this way, cyclodextrins gain the ability to accommodate hydrophobic molecules within the cavity while simultaneously being soluble in water. Conversely, the hydroxyl groups present on the outer surface can link with the aldehyde groups that ultimately exist in solution.

[0013] This explains the ability of cyclodextrins to increase the water solubility of hydrophobic substances. When molecules of appropriate polarity and size are accommodated within the cavity of a cyclodextrin, a supramolecular inclusion complex is formed. The impetus for inclusion involves various contributing factors, such as steric fitting, hydrophobic effects, van der Waals interactions, electrostatic interactions, and hydrogen bonding. The substance accommodated within the cavity of the cyclodextrin is called the "guest," while the cyclodextrin is called the "host."

[0014] The second advantage of inclusion complex formation lies in its ability to significantly modify the properties of molecules of interest (more accurately, "drugs" in our case) in numerous ways, such as improving drug stability, bioavailability, oral administration, and drug interactions with biological membranes or cells. This latter advantage readily explains why cyclodextrins have attracted so much attention and are already marketed in many industrial sectors worldwide, from food, cosmetics, and environmental engineering to chemical, pharmaceutical manufacturing and development.

[0015] Among cyclodextrins, the β group is the most commonly used because the α group has too small a cavity, while the γ group, although very effective, is very expensive to manufacture.

[0016] Because it is non-toxic when taken orally, β-cyclodextrin ( Figure 2 They can be used in the pharmaceutical field. In this field, they are frequently used due to their main capabilities, which include: masking the unpleasant taste of certain drugs, converting liquid compounds into solid compounds, and further improving the bioavailability profile of many drugs due to increased water solubility.

[0017] Natural β-cyclodextrins cannot be used parenterally because they are nephrotoxic, but hydroxypropyl derivatives of these cyclodextrins (HP β-CD) (trade name Cavasol) are... ® ) and α-cyclodextrin can be used for parenteral administration because they do not exhibit any toxicity and allow for the formulation of drugs that are completely insoluble in water. Figure 2 ).

[0018] Conversely, methylated β-cyclodextrin (Mβ-cyclodextrin) is not suitable for parenteral administration. Even slightly lipophilic random Mβ-cyclodextrin does not readily penetrate lipophilic membranes, but it interacts with membranes more readily than hydrophilic cyclodextrin derivatives.

[0019] Sulfobutyl ether 7β-cyclodextrin (SBE7 β-CD) 7 It is another recently synthesized β-cyclodextrin. SBE7 β-CD is a highly water-soluble derivative of β-cyclodextrin, which can be expressed in Captisol. ®Commercially available. The water solubility of SBE7 β-CD (approximately 70 g / 100 ml at 25°C) is significantly higher than that of the parent β-cyclodextrin (1.85 g / 100 ml at 25°C). It has been approved for parenteral use and, due to its higher solubility, may be more effective than HP β-CD. Therefore, SBE7 β-cyclodextrin may be a promising alternative to HP β-cyclodextrin. (The following is a further description...) Figure 3 The structure represents the three isomers of SBE7β-cyclodextrin.

[0020] The toxicity characteristics of cyclodextrins and their derivatives have been extensively assessed. 9 Cyclodextrins are generally considered safe when administered orally because they do not cross the intestinal barrier. However, the route of administration can modify the toxicity of the same cyclodextrin. For example, natural β-cyclodextrin has been shown to exhibit limited toxicity after oral administration in animals, as the International Programme on Chemical Safety (IPCS; WHO Food Additives Series 32) limits the acceptable daily intake to 5 mg / kg body weight, while higher doses administered parenterally or subcutaneously can cause nephrotoxicity affecting the proximal tubules. The way cyclodextrins are cleared from the organism also depends on the route of administration. For instance, after intravenous injection in rats, HP β-cyclodextrin is primarily removed by glomerular filtration in the kidneys and excreted in the urine, while oral administration primarily results in excretion via feces in rats and dogs.

[0021] In summary, all toxicity studies indicate that oral cyclodextrin is practically non-toxic due to the absence of absorption from the gastrointestinal tract. Furthermore, numerous safety assessments suggest that even with parenteral administration, gamma-cyclodextrin, 2-hydroxypropyl β-cyclodextrin, sulfobutyl ether β-cyclodextrin, sulfated β-cyclodextrin, and maltose-based β-cyclodextrin appear to be safe. 8 .

[0022] Mode of action of cyclodextrin In the pharmaceutical field, novel cyclodextrin-based technologies of commercial interest are constantly being developed, which are beneficial to the biological properties of cyclodextrins, mainly involving drug delivery, biosafety, and therapeutic efficacy. 9 These novel cyclodextrins are mainly derived from natural β-cyclodextrins, and their properties depend primarily on their degree of substitution (…). Figure 4These involve methylated β-cyclodextrin derivatives, such as randomly methylated β-cyclodextrins (RAMEβ and KLEPTOSE® CRYSMEβ show 12, 6, and 4 methyl groups, respectively), HP-β-cyclodextrin with hydroxypropyl groups randomly substituted into the β-cyclodextrin molecule, and sulfobutyl ether-7-β-cyclodextrin (SBE7-β-cyclodextrin), which is currently being evaluated for the treatment of neurodegenerative diseases and atherosclerosis. Furthermore, γ-cyclodextrins have shown great therapeutic value because they do not exhibit any hypersensitivity reactions, unlike sugammadex. Modified cyclodextrins used in anesthesia to reverse the effects of neurovascular blocking drugs have been associated with allergic reactions in some patients. As therapeutic agents, cyclodextrins and their derivatives can act in two modes. The first mode implies a direct biological action of cyclodextrin on the cell membrane, while the second mode is more indirect, utilizing the encapsulation potential of cyclodextrin as a drug carrier.

[0023] The direct effect of cyclodextrins on cells lies in the extraction of lipids (cholesterol and phospholipids) and some proteins from the cell membrane, thereby modifying the molecular composition of the lipid bilayer and thus altering its properties. Figure 5 Alpha-cyclodextrin has been described as removing phospholipids, β-cyclodextrin as extracting phospholipids and cholesterol, while γ-cyclodextrin exhibits lower lipid selectivity than other cyclodextrins.

[0024] In the second mode, cyclodextrins are widely used as drug delivery carriers across the nasal mucosa, lung, eye, skin, intestine, and brain barriers because these molecules improve the delivery and bioavailability of hydrophilic, hydrophobic, and lipophilic drugs.

[0025] When combined with active pharmaceutical ingredients to form complexes, cyclodextrins are also widely used to improve biocompatibility and enhance bioavailability, thereby increasing drug efficacy. Combining cyclodextrins with pharmaceutical compounds to form complexes has been applied in research for the treatment of atherosclerosis and neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Furthermore, cyclodextrins can be used as carriers, enabling selective binding to biomolecules of interest, as reported, for example, in the detection of cholesterol crystals in atherosclerosis.

[0026] Therefore, the direct mode of action of cyclodextrin has proven effective against cells, promoting the efficient extraction of cholesterol and phospholipids from lipid rafts in cell membranes. The indirect mode of action highlights the complexing ability of cyclodextrin, allowing for more efficient removal of lipids and aldehydes.

[0027] ethanol Ethanol has been used for many years to treat biological prosthetic tissues (such as aortic prosthetic valves, bovine or porcine pericardial tissue) with the aim of reducing the process of malnutrition calcification during long-term implantation.

[0028] Ethanol has been used alone or in combination with other substances, usually after cross-linking treatment with glutaraldehyde.

[0029] The following is a scientific review of various biological prosthetic tissue treatments based on ethanol (alone or in combination with other molecules). The possible mechanisms of action and efficacy of ethanol as an anti-calcification method have been analyzed.

[0030] Pretreatment of glutaraldehyde-crosslinked cusp valves with 80.0% ethanol extracted almost all cholesterol and phospholipids from the cusp valve samples. 10 It has been hypothesized that phospholipids present in the inactivated cells of bioprosthetics are the initial source of phosphorus in heart valve calcification caused by phosphoester hydrolysis. Other studies have also investigated the relationship between cholesterol and calcification in atherosclerotic plaques. Cholesterol levels have been shown to gradually increase with age and are directly associated with the risk of coronary artery disease. Cholesterol also alters calcium passage across cell membranes, intracellular calcium levels, and membrane fluidity in arterial smooth muscle cells. The mechanisms by which cholesterol content in cell membranes is associated with intracellular calcification are not yet fully understood.

[0031] These data strongly suggest that the conformational changes induced by ethanol pretreatment in collagen are stable and may be important in explaining the anti-calcification mechanism. This conformational change may be due to the observed reduction in cusp adsorption of lipids or proteins caused by ethanol pretreatment. This also necessitates further investigation into collagen and its role in calcification of prosthetic heart valves. 10 Further research is needed on protein-protein and protein-lipid interactions.

[0032] The conformational changes in cusp collagen induced by ethanol treatment are persistent. Furthermore, resistance to collagenase digestion is also observed. Therefore, it can be hypothesized that the anti-calcification effect and collagen conformational changes induced by ethanol pretreatment may lead to more durable biological prostheses. 10 .

[0033] Ethanol pre-incubation of glutaraldehyde-crosslinked porcine aortic valve prostheses is a highly effective pretreatment for preventing calcification of the porcine aortic apex valve in 60-day subcutaneous implantation in rats and 150-day mitral valve replacement in sheep. Ethanol was chosen as an anti-calcification agent because it is known to interfere with calcium metabolism in bone-line cells and fibroblasts. 11,12 The presence of ethanol has been shown to break down the acyl chains of phospholipids, which affect cell membranes and many cellular activities. 13 Furthermore, ethanol was shown to significantly inhibit calcium phosphate nucleation and phase transition due to its interaction with water. 14Previous publications on the inhibition of apical valve calcification in prosthetic heart valves by ethanol... 15 In this study, pretreatment with 80.0% ethanol extracted almost all the phospholipids and cholesterol from the glutaraldehyde-crosslinked cusps.

[0034] To understand the mechanism of action of ethanol in preventing calcification of prosthetic heart valves, lobular samples were analyzed for total lipid and cholesterol content before and after pretreatment.

[0035] Ethanol concentrations higher than 50.0% are very effective extractants for cholesterol and phospholipids, such as... Figure 6 The image shows that both were almost completely extracted. 15 Membrane-bound phospholipids are considered phosphorus donors in the initial stages of mineralization in prosthetic heart valves due to alkaline phosphatase hydrolysis. Complete removal of phospholipids from the initial calcification sites could partially explain the mechanism of action of ethanol. However, results from treatment with chloroform-methanol (2:1) indicate that this defatting protocol resulted in the complete extraction of total cholesterol and phospholipids (Table 1).

[0036] In a rat subcutaneous model, the implantation duration was extended to 60 days. Controls showed severe calcification (calcium level, 236 ± 6.1 μg / mg tissue). Pretreatment with 80.0% ethanol (pH 7.4, for 24 hours) was most effective, completely inhibiting calcification, with calcium levels comparable to those in unimplanted bioprosthetic tissue (calcium level, 1.87 ± 0.29 μg / mg tissue), while pretreatment with 60.0% ethanol was partially effective (calcium level, 28.5 ± 12.0 μg / mg tissue). Therefore, 80.0% ethanol pretreatment was found to be the optimal condition for preventing lobular calcification in 21-day and 60-day rat subcutaneous models.

[0037] Porcine aortic valve prostheses treated with ethanol and implanted in sheep models for 150 days showed a significant reduction in lobular calcium accumulation compared to the control (glutaraldehyde-fixed lobules), as described in Table 2.

[0038]

[0039] Table 1 Therefore, these data suggest that although lipid extraction may play a role in the mechanism of action of ethanol, lipid extraction alone cannot fully explain the anti-calcification efficacy of ethanol, and methanol may alter other factors affecting mineralization (Table 1).

[0040]

[0041] Table 2 Stress-strain properties of ethanol-treated tissue were evaluated in aortic valves of porcine tissue apical valves. 16This study compared the uniaxial stress-strain properties of untreated porcine aortic cusp valves with those treated with glutaraldehyde, as well as the uniaxial stress-strain properties of valves incubated with glutaraldehyde followed by ethanol.

[0042] Untreated cusps provide a control (C), while glutaraldehyde-treated cusps (G) and glutaraldehyde-fixed ethanol-treated cusps (G+A) represent test samples.

[0043] Significant differences were found between groups (C) and (G + A) in the parametric maximum load (p = 0.002). Significant differences were also found between group (G + A) and the two groups (G) and (C) in the parametric maximum stress, with p values ​​of 0.047 and 0.007, respectively. Compared to the two groups (G) and (C), group (G + A) also showed increased stress elongation capacity (maximum displacement) {p = 0.025 and p = 0.049, respectively}. Compared to the two groups (G) and (C), group (G + A) also showed significantly higher maximum strain {p = 0.006 and p = 0.027, respectively}.

[0044] Ethanol treatment of glutaraldehyde-tanned tissue not only retains the increased tensile strength after glutaraldehyde tanning but also improves ductility in uniaxial tests in the circumferential direction. This alteration in physical properties could contribute to maintaining the durability of the aortic cusp valve. The reduced tendency for calcification and the valve tissue's ability to elongate under stress could help prevent structural and functional loss. However, it is appropriate to consider long-term in vivo durability studies of glutaraldehyde-tanned porcine aortic valves treated with ethanol in vivo to test this hypothesis.

[0045] In another study, the anti-calcification effect of ethanol on glutaraldehyde crosslinking was evaluated. The authors noted that low molecular weight alcohols (methanol, ethanol, and isopropanol) were effective in reducing calcification in porcine aortic valve apex. Following ethanol treatment, tissue storage in glutaraldehyde allowed for partial recovery of calcification, indicating the role of the ethanol-glutaraldehyde interaction in preventing tissue calcification. However, the anti-calcification effect of ethanol persisted when the porcine apical valve was stored in ethanol-glutaraldehyde. 17 .

[0046] In another study, Carpentier investigated in 2001 the effects of ethanol, ether, and surfactant treatments on pericardial samples pretreated with 0.6% glutaraldehyde. 18Lipid extraction was performed using ethanol, ether, or Tween 80 surfactant, or combinations thereof. The treated tissue was subcutaneously implanted into 50 young rats at 4 and 6 months of age. At 6 months post-implantation, calcium levels were significantly lower than in the control group, only in the ethanol and ether groups containing surfactants. Previous studies have shown that ethanol is quite effective in the extraction of phospholipids and that they play an important role in calcification. 10 Conversely, analysis showed that ethanol extraction did not completely eliminate triglycerides in the bovine pericardium, while ether extraction completely removed them. However, there was no significant difference in calcium content between the two groups 6 months after implantation, suggesting that triglycerides play a negligible role in calcium deposition. In summary, treatments using ethanol or ether alone, or surfactant alone, are less efficient than combinations of these treatments. The fact that combinations of treatments are more efficient than any single treatment suggests that the extraction mechanisms and the products extracted by each treatment are slightly different. As a practical conclusion of this work, it can be argued that increasing the ethanol concentration or its incubation time, or adding ether to surfactant treatments, may be beneficial in the preservation of currently used bioprosthetic valves.

[0047] Compared with surfactant alone (42.9±12.7 μg / mg dry tissue), the most effective pretreatment was the combination of ethanol and surfactant (calcium content 6 months after implantation: 15.5±6.8 μg / mg dry tissue), or the combination of ethanol, ether and surfactant (13.1±6.2 μg / mg dry tissue).

[0048] The anti-calcification effect of ethanol has been well-documented in numerous scientific publications. Although ethanol treatment was effective on porcine aortic apical valves and pericardial tissue, some slight residual calcification remained, suggesting that researchers explore additional combination treatments to further reduce malnutritional calcification deposits in tissues.

[0049] Connoly 19 The effects of ethanol-treated porcine aortic apical valves post-treated with sodium borohydride were evaluated. Ethanol pretreatment significantly inhibited calcification compared to control (13.3 ± 5.6 Ca μg / mg vs. 119.2 ± 6.6 Ca μg / mg tissue; p < 0.001). However, under optimized conditions, sodium borohydride reduction combined with ethanol pretreatment optimally reduced calcification (1.16 ± 0.1 Ca μg / mg; p < 0.05), while the level after sodium cyanoborohydride treatment (23.6 ± 10.4 Ca μg / mg) was not significantly different from the level after ethanol alone. Without ethanol pretreatment, neither reducing agent effectively inhibited calcification.

[0050] Other authors 20Ethanol was used as a phospholipid solvent in conjunction with amino acids to detoxify pericardial tissue. Bovine pericardial samples were fixed with 0.5% GA. Uriazole and glutamate were used to neutralize free aldehydes and some solvents (ethanol with octanol or octanediol) to reduce phospholipid content in bovine pericardial tissue. Uriazole and glutamate alone significantly reduced calcium... 2+ and inorganic phosphorus (IP) concentration (without any anti-calcification treatment, Ca 2+ (277.85±17.51 ​​μg / mg; IP: 147.07±8.32 μg / mg), but when used with organic solvents, Ca 2+ and the lowest concentration of inorganic phosphorus (Ca 2+ :0.05±0.04μg / mg; IP: 3.36±0.61μg / mg).

[0051] Kim 21 The aim of this published study was to evaluate, from the perspective of calcification and tissue elasticity, the synergistic effect of L-arginine and sodium borohydride (NaBH4) in glutaraldehyde-treated porcine pericardium using a simultaneous synergistic effect compared to ethanol and L-lysine. Porcine pericardium was fixed with 0.625% glutaraldehyde (2 days at 4°C followed by 7 days at room temperature). Glutaraldehyde fixation was completed after intermediate steps with either ethanol (80%; 1 day at room temperature) or L-lysine (0.1 M; 2 days at 37°C) or L-arginine (0.1 M; 2 days at 37°C). Compared with the control (175.5 ± 45.3 μg / mg), pretreatment with L-lysine and NaBH4 (183.8 ± 42.6 μg / mg, p = 0.804) and L-arginine and NaBH4 (163.3 ± 27.5 μg / mg, p = 0.621) did not significantly inhibit calcification, but pretreatment with ethanol and NaBH4 significantly inhibited calcification (38.5 ± 37.3 μg / mg, p = 0.003). Finally, NaBH4 pretreatment appeared to reduce calcification in porcine pericardium fixed with glutaraldehyde, but only with ethanol.

[0052] Another study 22 Enhanced to evaluate the efficiency of aluminum chloride, either isolated from or combined with ethanol, in preventing calcification and inflammation, in which fragments of porcine aortic wall were fixed in glutaraldehyde and subcutaneously implanted into young mice. Samples of porcine aortic wall were implanted into subcutaneous tissue. The samples were pretreated with three different methods: I (glutaraldehyde), I (glutaraldehyde + aluminum), and III (glutaraldehyde + ethanol + aluminum). Atomic absorption spectroscopy showed similar calcium levels in groups II and III, but significantly lower than in group I. Treatment with aluminum chloride inhibited calcification of the aortic wall samples after implantation and reduced inflammation. The combination of ethanol and aluminum chloride was even more effective in inhibiting calcification and also reduced inflammation.

[0053] The literature review above clearly demonstrates that ethanol treatment of bioprosthetic tissues is generally very effective in preventing dystrophic calcification of tissues after long-term implantation in animal models. When biomaterials are pretreated with ethanol and crosslinked with glutaraldehyde, the reduction in tissue calcium content relative to controls is always significant.

[0054] Ethanol is highly efficient in dissolving and extracting lipid rafts (cholesterol and phospholipids) from cell membranes, which are identified as a major cause of calcification. Optimal efficiency for lipid extraction is obtained at a concentration of 80%, but good reduction in tissue calcification is already visible at a concentration of 50%. 15 .

[0055] Alternative tissue cross-linking treatments (triglycidylamine, genipin, neomycin) or post-treatments (e.g., urazole, glutamate, sodium borohydride, aluminum chloride, etc.) only further reduce the tendency to calcify when combined with ethanol. Summary of the Invention

[0056] This invention covers the following items: 1. A method for processing biological prosthetic tissue, particularly biological tissue for cardiovascular prostheses; the method comprising using cyclodextrin, which is capable of removing phospholipids from the biological prosthetic tissue.

[0057] 2. The method according to Project 1, wherein the cyclodextrin is β-cyclodextrin.

[0058] 3. The method described in Project 1 or 2 is used to reduce calcification of biological prosthetic tissue.

[0059] 4. The method according to any one of the preceding items, used to reduce the residual toxicity of biological prosthesis tissue for storing the biological prosthesis in an aldehyde-free solution.

[0060] 5. The method according to any one of the preceding items, wherein it is combined with polyethylene glycol for achieving tissue dehydration and ethylene oxide for sterilization.

[0061] 6. The method according to any one of the preceding items further includes a rinsing step, wherein the cyclodextrin is used to remove residual free aldehyde molecules from the tissue.

[0062] 7. The method according to any one of the preceding items, wherein the cyclodextrin is selected from the group consisting of: γ-cyclodextrin, 2-hydroxypropylβ-cyclodextrin, sulfobutyl ether β-cyclodextrin, sulfated β-cyclodextrin, and maltodextrin.

[0063] 8. The method according to any one of the foregoing items further includes the use of ethanol.

[0064] 9. The method according to item 8, wherein ethanol and the cyclodextrin are used simultaneously.

[0065] 10. The method according to item 8 or 9, characterized in that ethanol is used first, followed by cyclodextrin.

[0066] 11. The method according to item 8 or 9, characterized in that cyclodextrin is used first, followed by ethanol.

[0067] 12. The method according to any one of the preceding items, including a crosslinking process.

[0068] 13. The method according to item 12, wherein the crosslinking process occurs after the use of cyclodextrin.

[0069] 14. The method according to item 13 includes a second use of cyclodextrin, which occurs after the crosslinking process.

[0070] 15. The method according to item 12, wherein the crosslinking process occurs before the use of cyclodextrin.

[0071] 16. An article for processing biological prosthetic tissue, particularly cardiovascular tissue; said article comprising cyclodextrin.

[0072] 17. The article according to item 16, wherein the cyclodextrin is β-cyclodextrin.

[0073] 18. The articles according to item 16 or 17 also include ethanol.

[0074] 19. A kit for treating biological prosthetic tissue, comprising two separate products: a first product containing cyclodextrin and a second product containing ethanol. Attached Figure Description

[0075] Figure 1 The structure of cyclodextrin is shown.

[0076] Figure 2 The structure of β-cyclodextrin is shown.

[0077] Figure 3 The structures of three isomers of SBE7β-cyclodextrin are shown.

[0078] Figure 4 This demonstrates a novel cyclodextrin.

[0079] Figure 5 This is a schematic diagram illustrating the direct effects of cyclodextrin on cells.

[0080] Figure 6 Ethanol is shown to be an effective extractant.

[0081] Figure 7 The flowchart shows the degreasing process of the combination.

[0082] Figure 8 This demonstrates that phospholipid extraction can be performed in two separate stages after tissue cross-linking.

[0083] Figure 9 This demonstrates that β-cyclodextrin treatment can be performed directly on bioprosthetic tissues prior to the cross-linking process.

[0084] Figure 10 This demonstrates the further detoxification process based on cyclodextrin.

[0085] Figure 11 This shows a variant that is combined with the tissue dehydration process. Detailed Implementation

[0086] This invention relates in general to novel and original uses of cyclodextrin in the processing of biological prosthetic tissues.

[0087] The use of cyclodextrins in this treatment provides long-term mechanical and biological durability to the implanted bioprosthetic tissue. These properties are particularly important for heart valve bioprosthetics.

[0088] Cyclodextrins belong to a large family of molecules, but for the purposes of this invention, those β-group cyclodextrins are most favored. Functionalized β-cyclodextrins, particularly HP β-cyclodextrin and SBE β-cyclodextrin, have been approved for parenteral use. Therefore, they express all the desired chemical effects without causing any harm to the excretory organs, even when present in trace amounts.

[0089] Advantageously, cyclodextrin is used in combination with ethanol.

[0090] Tissue treatment with ethanol at concentrations above 50% water has proven highly effective in phospholipid extraction. The effectiveness of ethanol is closely related to glutaraldehyde cross-linking of bioprosthetic tissues. The mechanism is not fully understood, but ethanol treatment following a glutaraldehyde-based cross-linking process appears to be more effective.

[0091] Cyclodextrin action can be described as direct, primarily involving the extraction of lipid molecules; and indirect, involving the complexation of already extracted lipid molecules. In the second mode of action, cyclodextrin can complex phospholipids that have already been dissolved in ethanol.

[0092] Typically, when applied to biological tissues, the role of cyclodextrins can be explained by the spatial interaction or weak covalent bonding between their hydrophobic cavities and lipid molecules. In other words, it involves the weak covalent bonding between cyclodextrins and lipid molecules without any chemical reaction occurring.

[0093] US patent application US2002 / 137024 discloses the use of specific derivatives of cyclodextrin for treating biological tissues. This prior art teaches that sulfonated and sulfated polyanions can block calcium nucleation sites in biological tissues used in prosthetic devices. The mechanism of action of these chemicals is not described, but it can be clearly inferred from the teachings of this document that the sulfonated and / or sulfated functional groups are used to block calcium nucleation sites. In fact, the examples reported in US2002 / 137024 involve entirely different molecules that share only a sulfonate / sulfate group. One of the several examples of polyanions mentioned in US2002 / 137024 is sulfated cyclodextrin. It can be inferred that the sulfonate / sulfate group can block calcium nucleation sites, possibly due to the affinity between calcium and sulfate anions. In other words, there is a competitive effect of sulfate / sulfonate anions on the phosphate groups of phospholipids, although this is well known in the prior art, US2002 / 137024 clearly identifies the phosphate groups of phospholipids as calcium nucleation sites.

[0094] Typically, cyclodextrins do not contain functionalized sulfonate or sulfate groups. It should be emphasized that cyclodextrins are generally neutral molecules rather than ionic compounds. Sulfated cyclodextrins are merely one derivative of a large class of cyclodextrins, and functionalization aims to obtain more soluble or tolerable molecules for intravenous (iv) injection. Therefore, there is no recommendation in US2002 / 137024 to use cyclodextrins themselves as inhibitors of calcium nucleation sites. In fact, US2002 / 137024 even gives guidance contrary to the teaching of using cyclodextrins themselves as inhibitors of calcium nucleation sites.

[0095] In this invention, the selected cyclodextrin functions in a completely different manner from that described in US2002 / 137024. Due to its hydrophobic cavities, the cyclodextrin itself removes phospholipids from the tissue, thereby disrupting the phospholipid fatty chains.

[0096] Ethanol and cyclodextrin can be used simultaneously or separately in any order.

[0097] Select a biological prosthetic tissue (natural cusp valve, bovine or porcine pericardial tissue) that is free of defects and thickness. The selected patch or cusp valve undergoes a cross-linking process designed to stabilize the collagen and prevent any immune or allogeneic tissue reactions. The cross-linking process can be performed with different molecules, but typically uses glutaraldehyde at concentrations ranging from 0.1% to 1%, lasting 12 to 48 hours or longer.

[0098] Preferably, a combined degreasing treatment is performed ( Figure 7The combination of ethanol (35% to 80%) and β-cyclodextrin (10 mM to 200 mM) dissolved in a buffer solution at pH 7.4 was maintained at a temperature of 25°C to 40°C for 2 to 24 hours.

[0099] After degreasing, the patches are assembled into semi-finished or finished components, and then chemically sterilized with an aldehyde-based solution containing short-chain alcohol molecules.

[0100] The finished device is then stored in a solution consisting of 0.1% to 1% aldehydes and finally supplemented with 10% to 50% short-chain alcohol molecules.

[0101] To enhance the tissue detoxification process (which aims to remove aldehyde-free molecules from the bioprosthetic before implantation), a pre-implantation rinsing process is performed.

[0102] The pre-implantation rinse was performed using three equal 500 ml portions of a 10 mM to 200 mM β-cyclodextrin solution at a temperature of 15°C to 30°C.

[0103] In another embodiment, phospholipid extraction can be performed in two separate stages after tissue cross-linking. Figure 8 First, ethanol treatment is performed, followed by β-cyclodextrin treatment. Both treatments are based on... Figure 7 Perform at the same concentrations and under the same conditions as described above.

[0104] like Figure 8 The phospholipid extraction can be performed in reverse order, with the intention of exposing the patient to β-cyclodextrin followed by ethanol treatment at the same concentration and under the same conditions.

[0105] A combined treatment with ethanol and β-cyclodextrin can be performed, with the expectation of directly treating the bioprosthetic tissue with β-cyclodextrin prior to the cross-linking process. Figure 9 Then, it is treated with an ethanol solution. The concentrations of ethanol and β-cyclodextrin can be adjusted according to... Figure 7 The same applies as described above. If necessary, the separation process can be applied in reverse order.

[0106] In the crosslinking process ( Figure 9 The previously anticipated principle behind β-cyclodextrin treatment was based on the direct activity of cyclodextrin in defatting biological prosthetic tissue. This is consistent with the same extractive activity observed in cyclodextrin expression in experiments, where these molecules are able to extract cholesterol and other lipids from atherosclerotic plaques in arterial vessels (the FDA has approved cyclodextrin as an orphan drug for rare pediatric diseases in which infants at 2–3 years of age exhibit abnormally high plasma cholesterol concentrations with atherosclerotic plaques).

[0107] In another embodiment, following the phospholipid extraction described in the previous treatment with ethanol and cyclodextrin, the process may include a further detoxification process based on cyclodextrin, aimed at effectively removing residual aldehyde molecules ( Figure 10 To store biological prostheses in an antibacterial, aldehyde-free storage solution, this chemical treatment modification is necessary. As previously mentioned, removing aldehydes from the storage solution is quite important because storing biological prostheses in glutaraldehyde has been shown to partially overturn the positive anti-calcification effect given by ethanol treatment. This is why, in the preceding embodiments, the storage medium, such as... Figures 7 to 9 The reason for adding a certain amount of short-chain alcohol to the glutaraldehyde-based solution is as follows.

[0108] Tissue dehydration combined with ethylene oxide sterilization represents a significant step forward in the processing of bioprosthetic tissues. This is done to facilitate the storage of bioprosthetics and avoid chemical sterilization and its associated processing, especially when they must collapse and be used in transcatheter procedures.

[0109] The previous treatment embodiments, presented as possible variations, can be combined with tissue dehydration processes. For example, in Figure 11 In this process, after defatting, a detoxification process is performed using β-cyclodextrin to remove aldehyde molecules from the tissue. Once detoxification is complete, the tissue dehydration process can begin. This treatment is based on the gradual removal of water from biological prosthetic tissue using 80% to 90% polyethylene glycol (e.g., MW 100 to 800) in an aqueous solution. The treatment is performed at a temperature of 20°C to 50°C for 12 to 48 hours. For more effective dehydration, short-chain alcohols can be added at a concentration of 10% to 20%.

[0110] The tissue is dried for several hours in a clean environment to complete the dehydration process. This allows for the final storage of the biological prosthesis in a dry package, which is sterilized with ethylene oxide.

[0111] All of the above processes can be performed on the semi-finished components or directly on the final assembled biological prosthesis. In this case, the process can be applied as a separate prosthesis treatment.

[0112] Bibliography 1.Schoen FJ, Levy RJ. Calcification of tissue heart valvesubstitutes: progress toward understanding and prevention. Ann Thorac Surg2005;79:1072-80.

[0113] 2.Konakci KZ, All B, Blumer R, Hoetzenecker W, Roth G, Moser B,Boltz-Nitulescu G, Gorlitzer M, Klepetko W, Wolner E, Ankersmit HJ. Alpha-GALon bioprostheses: xenograft immune response in cardiac surgery. Eur J ClinInvest 2005:35:17-23.

[0114] 3.Manji RA, Zhu LF, Nijjar NK, Rayner DC, Korbutt GS, Churchill TA,Rajotte RV, Koshal A, Ross DB. Glutaraldehyde-fixed bioprosthetic heart valve conduits calcify and fail from xenograft rejection. Circulation 2006:114:318–27.

[0115] 4.Vyavahare N, Hirsch D, Lerner E, Baskin JZ, Schoen FJ, Bianco R,Kruth HS, Zand R, Levy RJ. Prevention of bioprosthetic heart valvecalcification by ethanol preincubation. Efficacy and mechanisms. Circulation1997:95:479–88.

[0116] 5.Jorge-Herrero E, Fernandez P, Escudero C, Garcl ́a-Pa ́ez JM,Castilo-Olivares JL. Calcification of pericardial tissue pretreated withdifferent amino acids. Biomaterials 1996:17:571-5.

[0117] 6.Bina Gidwani, Amber Vyas. A comprehensive review on cyclodextrin-based carriers for celivery of chemotherapeutic cytotoxic anticancer drugs.BioMed Research International Volume 2015, Article ID 198268, 15 pageshttp: / / dx.doi.org / 10.1155 / 2015 / 198268.

[0118] 7.Ankitkumar S. Jain, Abhijit A. Date, Raghuvir R. S. Pissurlenkar,Evans C. Coutinho, Mangal S. Nagarsenker. Sulfobutyl Ether7 β-Cyclodextrin(SBE7 β-CD) Carbamazepine complex: preparation, characterization, molecularmodeling, and evaluation of in vivo anti-epileptic activity. AAPSPharmSciTech, Vol. 12, No. 4, December 2011;1163-75.

[0119] 8.E.M. Martin Del Valle. Cyclodextrins and their uses: a review.Process Biochemistry 39 (2004) 1033–1046.

[0120] 9.Caroline Coisne, Sébastien Tilloy, Eric Monflier, Daniel Wils,Laurence Fenart, Fabien Gosselet. Cyclodextrins as emerging therapeutic toolsin the treatment of cholesterol-associated vascular and neurodegenerativediseases. Molecules 2016, 21, 1748.

[0121] 10.Narendra R. Vyavahare, Danielle Hirsch, Eyal Lerner, Jonathan Z.Baskin, Robert Zand, Frederick J. Schoen, Robert J. Levy. Prevention ofcalcification of glutaraldehyde-crosslinked porcine aortic cusps by ethanolpreincubation: Mechanistic studies of protein structure and water–biomaterialrelationships. J Biomed Mater Res, 40, 577–585, 1998.

[0122] 11.W. K. Ramp and D. N. Demaree, “Inhibition of net calcium effluxfrom bone by ethanol in vitro,” Am. J. Physiol., 246, C30– C36 (1984).

[0123] 12.K. E. Friday and G. A. Howard, “Ethanol inhibits human bone cellproliferation and function in vitro,”Metabolism, 40, 562–565 (1991).

[0124] 13.E. Rubin and R. Hagai, “Ethanol-induced injury and adaptation inbiological membranes,”Fed. Proc., 41, 2465–2471 (1982).

[0125] 14.M. S. Tung and T. J. O’Farrell. The effect of ethanol on thesolubility of dicalcium phosphate dihydrate in the system Ca(OH)2-H3PO4-H2Oat 37°C. J. Mol. Liq., 56, 237–243 (1993).

[0126] 15.Narendra Vyavahare, Danielle Hirsch, Eyal Lerner, Jonathan Z.Baskin, Frederick J. Schoen, Richard Bianco, Howard S. Kruth, Robert Zand,Robert J. Levy. Prevention of bioprosthetic heart valve calcification byethanol preincubation. Circulation. 1997;95:479-488.

[0127] 16.Anil Madhav Patwardhan, M.Ch., Pradeep Vaideeswar. Stress straincharacteristics of glutaraldehyde treated porcine aortic valve tissuefollowing ethanol treatment. IJTCVS 2004; 20: 67–71.

[0128] 17.Vyavahare NR, Jones PL, Hirsch D, Levy RJ. Prevention ofglutaraldehyde-fixed bioprosthetic heart valve calcificatio by alcoholpretreatment: further mechanistic studies. J Heart Valve Dis. 2000 Jul; 9(4):561-6.

[0129] 18.Ming Shen, Ali Kara-Mostefa, Lin Chen, Michel Daudon, MarcThevenin, Bernard Lacour, and Alain Carpentier. Effect of ethanol and etherin the prevention of calcification of bioprostheses. Ann Thorac Surg 2001;71:S413–6.

[0130] 19.Connolly JM, Alferiev I, Kronsteiner A, Lu Z, Levy RJ. Ethanolinhibition of porcine bioprosthetic heart valve cusp calcification isenhanced by reduction with sodium borohydride. J Heart Valve Dis. 2004 May;13(3):487-93.

[0131] 20.Hyoung Woo Changa, Soo Hwan Kimb, Kyung-Hwan Kima, Yong Jin Kima.Combined anti-calcification treatment of bovine pericardium with aminocompounds and solvents. Interactive CardioVascular and Thoracic Surgery 12(2011) 903–907.

[0132] 21.Kwan-Chang Kim, Soo-Hwan Kim, Yong-Jin Kim. Detoxification of glutaraldehyde treated porcine pericardium using L-Arginine & NABH4. Korean JThorac Cardiovasc Surg 2011;44:99-107.

[0133] 22. Evandro Antonio Sardeto, Francisco Diniz Affonso da Costa, Iseu do Santo Elias Affonso da Costa, João Gabriel Roderjan, Eduardo Discher, Ricardo Alexandre Schneider, Carlos Henrique Gori Gomes, Claudinei Colattusso, Daniel Précoma, Andrea Dumsch, Sergio Veiga Lopes, Jairo Leal. Efficacy of AlCl3 and ethanol in the prevention of calcification of fragments of porcine aortic wall fixed in GDA.

Claims

1. A method for processing biological prosthetic tissue, particularly biological tissue for cardiovascular prostheses; the method comprising using cyclodextrin, which is capable of removing phospholipids from the biological prosthetic tissue.

2. The method according to claim 1, wherein, The cyclodextrin is β-cyclodextrin.

3. The method according to claim 1 or 2, used to reduce calcification of biological prosthetic tissue.

4. The method according to any one of the preceding claims, used to reduce the residual toxicity of biological prosthesis tissue for storing the biological prosthesis in an aldehyde-free solution.

5. The method according to any one of the preceding claims, wherein it is combined with polyethylene glycol for achieving tissue dehydration and ethylene oxide for sterilization.

6. The method according to any one of the preceding claims further includes a rinsing step, wherein, Cyclodextrins are used to remove residual free aldehyde molecules from tissues.

7. The method according to any one of the preceding claims, wherein, The cyclodextrin is selected from the group consisting of: γ-cyclodextrin, 2-hydroxypropylβ-cyclodextrin, sulfobutyl ether β-cyclodextrin, sulfated β-cyclodextrin, and maltodextrin.

8. The method according to any one of the preceding claims further includes the use of ethanol.

9. The method according to claim 8, wherein, Ethanol and the cyclodextrin are used simultaneously.

10. The method according to claim 8 or 9, characterized in that, Ethanol was used first, followed by cyclodextrin.

Images