FIELD OF INVENTION

[001] The present invention relates to a method for anti-calcification of a tissue. More specifically, the present invention relates to a method for anti-calcification of a bioprosthetic tissue.

BACKGROUND

[001] Generally, a heart valve replacement may involve therapeutic excision of a native valve and its replacement with either a biological or an artificial valve. The heart valve may contain tissue-type or "bioprosthetic" valves and is to provide a viable and sustainable solution to the said problem. Bioprosthetic valves include flexible tissue leaflets (for example pericardium, bovine etc.) supported by a base structure (or a frame). The said tissue leaflet is processed by a series of steps such as flattening, washing etc. to finally deliver a tissue leaflet which is fixed on a frame and finally implanted inside a human body. The durability of tissue leaflet is limited by progressive structural deterioration primarily due to calcification. The presence of residual phospholipids and free aldehyde functional groups present due to glutaraldehyde fixation in tissue preparations causes tissue calcification. In fact, the existing tissue leaflets are calcified in relatively short period of time after implantation. Even though the current tissue leaflets are being treated with anti-calcification agent, the methods of treatment of tissue leaflet are ineffective in maintaining the anti- calcification properties of the tissue leaflet. There is another major problem with current method of treatment of calcification. The valves made of these leaflets have to be stored in hydrated condition to render it free from getting dry and this leads to more problem in logistics of these valves. Therefore, a method to manufacture heart valve tissues which overcomes the hurdles of conventional technology is needed.

SUMMARY [002] The present invention discloses a process for removal of free aldehydes from an aldehyde containing tissue is disclosed. The process involves quenching a tissue in the presence of buffer solution at a predefined temperature predefined time duration to obtain a quenched tissue. The quenching is followed by washing of the quenched tissue in the presence of distilled water and/or normal sterile saline and reduction of the quenched tissue in the presence of reducing agents to obtain a tissue which is less prone to calcification. Further, the tissue is subjected to dehydration process under high pressure and gentle fluid movement of glycerol.

BRIEF DESCRIPTION OF DRAWINGS [003] The summary above, as well as the following detailed description of illustrative embodiments, is better understood when read in conjunction with the appended drawings. For the purpose of illustrating the present disclosure, exemplary constructions of the disclosure are shown in the drawings. However, the disclosure is not limited to specific methods and instrumentalities disclosed herein. Moreover, those in the art will understand that the drawings are not to scale. [004] FIG.l depicts an exemplary flowchart of a process involved in dry conditioning of a bioprosthetic tissue in accordance with an embodiment of the present invention.

[005] FIG.2 depicts an exemplary flow chart of a process involved in anti-calcification of a bioprosthetic tissue in accordance with an embodiment of the present invention.

DETAILED DESCRIPTION OF DRAWINGS [006] Prior to describing the invention in detail, definitions of certain words or phrases used throughout this patent document will be defined: the terms "include" and "comprise", as well as derivatives thereof, mean inclusion without limitation; the term "or" is inclusive, meaning and/or; the phrases "coupled with" and "associated therewith", as well as derivatives thereof, may mean to include, be included within, interconnect with, contain, be contained within, connect to or with, couple to or with, be communicable with, cooperate with, interleave, juxtapose, be proximate to, be bound to or with, have, have a property of, or the like; Definitions of certain words and phrases are provided throughout this patent document, and those of ordinary skill in the art will understand that such definitions apply in many, if not most, instances to prior as well as future uses of such defined words and phrases. [007] Particular embodiments of the present disclosure are described herein below with reference to the accompanying drawings, however, it is to be understood that the disclosed embodiments are merely examples of the disclosure, which may be embodied in various forms. Well-known functions or constructions are not described in detail to avoid obscuring the present disclosure in unnecessary detail. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present disclosure in virtually any appropriately detailed structure.

[008] The present invention discloses a method for dry conditioning of a tissue. In various embodiments, the present invention pertains to improved fixation, anti-calcification followed by the dehydrating the tissue. Such tissue may be an animal tissue utilized for medical purposes such as in artificial heart valves. Various animal tissues that can be used can be of bovine, porcine, ovine, canine origin. In an embodiment, the tissue is bovine pericardium tissue. The method of the present invention involves a series of steps (an exemplary method is elaborated below). [009] The said method overcomes various drawbacks of the conventional technology for example to deliver a resultant tissue which can be stored in dry conditions. The storage in the dry conditions renders the tissue less prone to calcification thereby reducing potential toxicity associated with the glutaraldehyde liquid storage medium and removes the step of rinsing the implant prior to implantation. Such tissue may be utilized for medical purposes such as in artificial heart valves. In an exemplary embodiment, the method involves a process of anti-calcification by means of without limitation, treatment of the tissue with preheated solution, quenching of the tissue and alcohol washing etc. The anti-calcification process may involve chemical alteration of the tissue for example, removal of free aldehydes groups, extraction of phospholipids etc. to make it less prone to calcification. [0010] The tissue obtained from the said process may then be utilized for a predefined application. In an embodiment, the tissue is sutured/stitched to a frame to form a bioprosthetic heart valve.

[0011] Now referring specifically to the drawings, FIG. 1 represents a flow chart depicting a process involved in dry conditioning of a bioprosthetic tissue. In an embodiment of the present invention, the tissue is a bovine pericardial tissue. In an embodiment of the present invention, the dry conditioning of the bioprosthetic tissue may involve without limitation, the following steps: flattening, fixation, anti-calcification, dehydration and storage of the tissue.

[0012] The raw material required for the said process includes one or more tissue sheets. The tissue may be obtained from any animal derived tissue known in the art depending upon the nature of application. For example, for heart valve applications, the selected animal tissues must be strong, flexible and compatible for being utilized as a xenograft. The shape, size and dimensions of the tissue sheet may be unrestrictive. The tissue is trimmed and is washed with deionized water, salt solution, saline and/or other suitable washing solutions. In an embodiment, the tissue is placed in phosphate-buffered saline (PBS, 0.1 M, pH 7.4±0.2) and immediately transported to laboratory.

[0013] In accordance with an embodiment of the present invention, the process of dry conditioning of tissue is initiated at step 101. In the said step, the thickness of the tissue is trimmed and maintained by the process of flattening. The thickness of the tissue is flattened to make the tissue uniform throughout its length and breadth. In an embodiment, the tissue is flattened to a uniform thickness of 0.30-0.50 mm. In an embodiment, the said process is performed by feeding the tissue to a multi-roll mill, for example a two roll mill. The two roll mill includes two adjacently placed cylinders (for example metal cylinders). The two cylinders may be disposed in a vertical or horizontal plane. The advantage of using the multi-roll mill is obtainment of a more uniform tissue in lesser time as compared to conventional flattening process. Alternately, the flattening process may be performed via a known process applicable to the teachings of the present invention.

[0014] The tissue having uniform thickness is then engaged in the process of fixation at step 103. The process of fixation of the tissue is done for fixing fats and proteins in the tissue so that they become non-reactive. The fixation process renders the tissue non-antigenic, mechanically strong and minimizes tissue degradation. Moreover, the fixation of the tissue may change the collagen properties and renders the tissue acceptable to human host.

[0015] The tissue may be fixed by treatment with any aldehyde compounds (fixative agents) known in the art. The fixative agents may include without limitation Glutaraldehyde (GA),

Formaldehyde, Glutaraldehyde acetals, Epoxy compound, Acyl azide, Dye-mediated photo- oxidation Cyanimide, and Carbodiimides etc. The treatment of the tissue with aldehyde compound causes the tissue to retain some content of aldehyde during the process of fixation thereby forming an aldehyde containing tissue. In an embodiment, the process of fixation en hances the strength and stability of the tissue.

[0016] In an embodiment, the tissue is treated with glutaraldehyde solution for the process of fixation. In an exemplary embodiment of the fixation, the tissue is fixed with low concentration glutaraldehyde solution in the range of 0.20% to 0.80%, preferably 0.50%, and more preferably 0.625% at a temperature ranging between 20°C-80°C for time duration of 06 days to 02 months.

[0017] In another embodiment, the tissue is rinsed with saline solution followed by placement on the fixative tray. The tissue placed in fixative tray is exposed to 0.625% glutaraldehyde solution at room temperature for 06-08 days with gentle fluid movement for better adherence to the tissue.

[0018] The tissue may be placed on any fixative tray having an even surface in contact with the fixative in order to achieve uniform fixation. Alternately, the fixation process may be performed via a known process applicable to the teachings of the present invention. [0019] Optionally and additionally the fixed tissue may be stored in a low concentrated glutaraldehyde solution ranging 0.25% to 0.50% to keep the tissue moist and viable to use for a longer time. In an embodiment, the fixed tissue is stored at room temperature under vacuum sealed jar with 80ml of 0.625% glutaraldehyde solution for the storage purpose. However, GA has been reported to accelerate the calcification process, which considerably limits its application. Calcification is thus the main cause of long-term failure of GA-fixed pericardial valves. Therefore, the anti-calcification treatment is required to reduce in-vivo calcification of the tissue.

[0020] Following fixation at the previous step, the tissue is subjected to anti-calcification at step 105. Various chemical alteration strategies may be adopted to mitigate calcification such as treatment of the tissue with pre-heated solution for removal of free aldehyde groups from the tissue, removal of phospholipids of the cell membrane etc.

[0021] The free aldehyde groups on the tissue make the potential binding site for the calcium. Phosphorus which is present inside the phospholipids of the cell membrane is also a binding site for the calcium as it is known as the substrate for the calcium. Therefore, blocking of such groups is necessary to minimize calcification. [0022] At step 107, the aldehyde free tissue is dehydrated. The process of dehydration may be performed by any process known in the art, such as, treating the reduced tissue with a dehydrating agent(s). The dehydrating agents utilized in the said process may include organic solvents such as, without limitation, Glycerol: Isopropyl alcohol (IPA), Methanol, IPA, IPA: Polyethylene glycol (PEG), Acetone, IPA: Acetone etc. The ratio of aforementioned dehydrating agents may be varied in the range of 90:10 to 10:90. The glycerol used for dehydration may minimize the GA toxicity by avoiding storage in GA solution.

[0023] At step 109, the dehydrated tissue is subjected to sterilization by means of without limitation ethylene oxide gas treatment. In an embodiment, the dehydrated tissue is used to fabricate heart valve. The fabricated heart valve may be further sterilized by means of without limitation ethylene oxide gas.

[0024] At step 111, the dehydrated tissue is temporarily stored for subsequent application. The tissue obtained from the aforesaid steps can be stored in dry conditions that is, it does not require storage in solvents. In an embodiment, the dehydrated tissue is stored at a temperature from 10°C to 15°C.

[0025] In accordance with an embodiment of the present invention, FIG.2 represents a flow chart depicting a process involved in anti-calcification of the tissue. The process of anti-calcification of the tissue commences at the step 201, in which the tissue is treated with preheated GA solution. The chemical compounds employed for treatment of the tissue may include without limitation, formaldehyde, glutaraldehyde, di-aldehyde starch, hexamethylene di-isocyanate and polypoxy compounds etc. In an embodiment, the treatment with the pre heated GA solution causes its free aldehyde groups concentration to decrease upto 70-80% to the original concentration.

[0026] In an embodiment, The GA solution constitutes of the GA in a concentration ranging from 0.1% to 5.0%, preferably 1.5% to 2.5% and more preferably 1.8% is used. In an embodiment, the GA solution is heated to a temperature ranging between 30°C to 80°C, preferably 65 °C to 75 °C for 06-14 days in oil bath with reflux assembly. In an embodiment, following heat treatment of GA solution, the pH of solution is adjusted between 6.0 to 10.0 or more preferably 7.4.

[0027] In an embodiment, the preheated and pH adjusted GA solution is used for the heat treatment of the fixed tissue. The tissue may be rinsed with the normal saline solution 03-05 times to remove traces of GA groups. In an embodiment, the rinsed tissue is immersed with the GA solution in the fixative tray at a temperature ranging between 40°C to 70°C, more preferable at 48 °C to 54 °C for the time period of 08 days to 60 days, more preferably for the 08-14 days in a vacuum oven or in an orbital shaker. The orbital shaker may enable gentle fluid movement to give better surface contact of the tissue with the GA solution. In an embodiment, after heat treatment the tissue color turns brownish which is an indicator of completion of heat treatment. This tissue may be further rinsed with deionized water or more preferably with saline solution.

[0028] The treatment with pre-heated GA solution may facilitate removal of aldehyde groups and acid groups which are coupled to the tissue thereby making the tissue less prone to

calcification.

[0029] In another embodiment, the preheated formaldehyde solution is used for the heat treatment of the fixed tissue. The fixed tissue may be Immersed in the pre-heated formaldehyde solution at a temperature ranging between 40°C to 80°C, more preferable at 45°C to 50°C for the time period of 01 day to 15 days, or more preferably for the 04-08 days. After heat treatment tissue may be further rinsed with deionized water or more preferably with saline solution or Phosphate buffer solution to remove traces of salt.

[0030] Followed by heat treatment at the step 201, the tissue is subjected to FEP treatment at step 203. However, other sterilants may also be used. The alternate embodiment of FEP includes Glutaraldehyde Carbodiimides, epoxides and/or other combinations of aldehydes instead of formaldehyde (F), category of alcohols such as without limitation Isopropyl alcohol, octanol, octandiol instead of Ethanol (E) and category of non-ionic surfactants such as without limitation, octylphenoxy polyethoxy ethanol, polyoxyethylene, polysorbate 60 instead of polysorbate 80 (P).

[0031] In an embodiment, the FEP treatment acts as an anti-calcification and a bio-burden reduction step. The heat treated tissue may be immersed in the FEP solution for time duration of approximately 01 hour to 48 hours at a temperature of between 25-45 °C. The formaldehyde present in the FEP may fix tissue by cross-linking of primary amino groups in proteins with nearby nitrogen atoms in protein or DNA through a -CH2- linkage, ethanol may remove residual phospholipid content which is binding substrate for the calcium and polysorbte 80 has tendency to adsorb at surface thereby blocking calcium binding sites in the tissue. [0032] In an embodiment, the tissue is placed in a vacuum sealed jar containing approximately 50-90 ml of the FET solution, more preferably 80ml in the orbital shaker for gentle fluid movement. This jar is placed in orbital shaker at temperature of 25 °C to 45 °C, more preferably 37 °C to 40 °C with gentle fluid movement for time duration of approximately 03 - 06 hours. Due to gentle shaking, the process may be completed within short span of period. After completion of treatment, the tissue may be washed with ethanol, deionized water, saline solution or phosphate buffer saline and stored in 0.625% glutaraldehyde solution.

[0033] At step 205, the tissue is again processed for removal of free aldehydes

(untreated/unconjugated aldehydes). The said process may involve, without limitation, quenching of tissue, washing and/or reduction of the tissue. The quenching of the tissue may be performed with the help of buffer solutions in a predefined concentration. The quenching agents may include without limitation tris-glycine, isopropyl alcohol (IPA), water or mixture thereof.

[0034] In an exemplary embodiment, the process of quenching is performed with the help of buffer solution without limitation, tris-glycine buffer (say 5x- lOx). The tris-glycine buffer may be diluted with water and isopropyl alcohol (IPA). The constituents of the buffer may include tris, glycine, and isopropyl alcohol (I PA). The percentage compositions of said constituents in the tris- glycine buffer may be tabulated as follows.

[0035] The process of quenching may be continued for one or more days until all aldehydes are removed from the tissue. In an embodiment, the tissue is quenched for predefined time duration of around 02 days at a predefined temperature of around 10-20°C.

[0036] In an embodiment, the absence of free aldehydes in the tissue post quenching may be validated by subjecting the filtrate to laboratory tests. In an embodiment, the filtrate is subjected to Benedict's test, Fehling test and/or GC analysis.

[0037] Optionally, the tissue may be subjected to (GA) extraction process post quenching with the help of buffers known in the art. The extracting buffer may include without limitation acetic acid buffer, Tris-Glycine buffer etc. In an embodiment, the process of (GA) extraction is performed following the process of quenching and washing at the previous step. I n an embodiment, the process of (GA) extraction is performed at around pH of 4, at a temperature of around 37°C for time duration of approximately one week. [0038] At step 207, the tissue is subjected to a process of reduction in order to remove residual free aldehydes from the tissue. The reduction may be performed by treating the tissue with reducing agents. The reducing agent may include without limitation, Lithium aluminium hydride (LiAIH4), UBHET3, Lithium borohydride (LiBH4), Magnesium borohydride (Mg(BH4)2), Calcium borohydride (Ca(BH4)2), Aluminum borohydride (AI(BH4)3), Zinc borohydride (Zn(BH4)2), etc. in presence of solvents without limitation, methanol, ethanol, etc.

[0039] Lastly at step 209, the tissue is subjected to ethanol washing. This step may remove the staining caused by the heat treatment step and also extract phospholipids from the tissue cellular matrix. Phosphorus, which is present inside the phospholipids are the potential binding site for calcium. Therefore extraction of phospholipids will mitigate the in-vivo calcification after implantation.

[0040] A specific embodiment for quenching of glutaraldehyde fixed tissue is elaborated with the help of following example.

[0041] A fresh bovine pericardium is obtained from the bovine animal donor whose age is below 24 months. The bovine pericardium is rinsed with saline solution. The fresh bovine pericardium is trimmed in a specified size i.e. 10X12cm and rinsed with normal saline solution and subjected to the fixation process with 0.625% GA solution for chemical modification of the tissue. The fresh bovine pericardium tissue is placed in a fixative tray with 0.625% GA solution for time duration of 08-14 days at room temperature. The fixed bovine pericardium tissue is subjected to heat treatment process. The preheated GA solution having GA concentration of 1.8% and pH of approximately 7.4 is used. The heating is performed with the help of oil bath attached with reflux condenser (for negligible loss of solution during pre-heating step). The bovine pericardium tissue turns pale yellow to brownish in color which indicate the completion of heat treatment step.

[0042] The heat treated tissue is washed with saline solution with gentle shaking to remove traces. This heat treated tissue is further subjected to FET based bio-burden reduction process. At this step, the tissue is in contact with surfactant, alcohol and cross-linking agent. The alcohol aids in extraction the phospholipids from the tissue which is the potential binding side for the calcium.

[0043] The glutaraldehyde fixed tissue is quenched with quenching agent such as Tris-Glycine buffer. The Tris-glycine buffer is diluted with Isopropyl alcohol (IPA) and water to make a buffer solution containing 80mM Tris, 180mM Glycine and 20% Isopropyl alcohol (IPA). The tissue is quenched with 9x Tris-glycine buffer for two days on a mechanical shaker at a temperature between 10-20°C until all aldehydes are removed. The results of quenching are analyzed by performing gas chromatography (GC) of the quenched filtrate. Analysis of gas chromatography shows increased glutaraldehyde concentration in the filtrate up to 10 hours. After 10 hours, the glutaraldehyde concentration is constant which shows all glutaraldehydes are quenched from the tissue. The tissue is washed repeatedly with distilled water to remove excess quenching agents. The filtrate is tested for Benedict's test and Fehling test. It is observed that the reaction does not give Cu ₂ 0 precipitate which stands for negative test results. The negative result confirms the absence of aldehydes in the reaction mixture which validate removal of free aldehydes from the tissue.

[0044] Post the quenching process, the tissue was subjected to reduction with 0.8% LiAIH ₄ in Methanol for 18 hours. The reduction process confirms total removal of residual glutaraldehyde in the quenching filtrate. The presence of any residual glutaraldehyde in the solution is verified by performing gas chromatography. The gas chromatography result shows no peak for glutaraldehyde which confirms the complete removal of excess glutaraldehyde from the tissue. The final filtrate is subjected to Benedict's test and Fehling test. It is observed that reaction does not give Cu ₂ 0 precipitate which stands for negative test results. The negative result confirms the absence of free aldehydes in the reaction mixture which validate removal of free aldehydes from the tissue. [0045] Followed quenching, the tissue is subjected to several ethanol washes. The tissue is immersed in a vacuum jar containing 80ml of 99.9% purity ethanol on the orbital shaker at 50rpm at a temperature of around 37°C±02 ^o C for time duration of 12 hours.

[0046] Further, the tissue is subjected to dehydration process under high pressure and gentle fluid movement of glycerol. The dehydration step may replace oxidane molecules by glycerol and may penetrate high amount of glycerol inside the tissue by applying an adequate pressure. Thus, the tissue will become dehydrated and can be stored in a "solid" state rather than "liquid" state. The dehydrated tissue is further used for the heart valve fabrication and suitable for the EtO sterilization. [0047] While the invention has been described in its preferred embodiments, it is to be understood that the words which have been used are words of description and not of limitation. Therefore, changes may be made within the appended claims without departing from the true scope of the invention.