Claims:1. A process for anti-calcification of a tissue, the process comprising:
a. treating a tissue with a pre-heated solution of a fixative agent;
b. treating the tissue with a sterilant;
c. refluxing one or more times a tissue in a distillation apparatus in the presence of one or more refluxing agents at a predefined temperature; and
d. washing the reduced tissue with alcohol to yield an anti-calcified tissue.
2. The process as claimed in claim 1 wherein the tissue is a bioprosthetic tissue.
3. The process as claimed in claim 1 wherein the pre-heated solution of the fixative agent comprises a solution pre-heated at 70°C temperature for 6-8 days.
4. The process as claimed in claim 1 wherein the pre-heated solution of the fixative agent is a pre-heated solution of glutaraldehyde.
5. The process as claimed in claim 1 wherein the sterilant is a solution of formaldehyde, ethanol and polysorbate 80.
6. The process as claimed in claim 1 wherein the refluxing agents comprise a mixture of one or more of isopropyl alcohol (IPA), ethanol and methanol.
7. The process as claimed in claim 1 wherein the predefined temperature is 60-100oC.
8. The process as claimed in claim 1 wherein the distillation apparatus is a soxhelt apparatus.
9. The process as claimed in claim 1 wherein the alcohol is ethanol with 99.9% purity.
10. A process for dry conditioning of a tissue, the process comprising:
a. providing a tissue;
b. flattening the tissue in a multi-roll mill to yield a uniform tissue;
c. treating the uniform tissue with a fixative agent to yield a fixed tissue;
d. anti-calcifying the fixed tissue by the process of claim 1 to yield an anti-calcified tissue; and
e. dehydrating the anti-calcified tissue with one or more dehydrating agents for subsequent storage in dry conditions.
11. The process as claimed in claim 10 wherein the multi-roll mill is a two-roll mill.
12. The process as claimed in claim 10 wherein the fixative agent is an aldehyde solution.
13. The process as claimed in claim 10 wherein the one or more of dehydrating agents comprise glycerol, glycerol derivatives, isopropyl alcohol, polyethylene glycol, acetone and methanol.
14. The process as claimed in claim 10 wherein the dehydrated tissue heart valve is sterilized by using ethylene oxide gas.
, Description:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(Section 10 and Rule 13)

1. TITLE OF THE INVENTION:
ANTI-CALCIFICATION OF A TISSUE
2. APPLICANTS:
Meril Life Sciences Pvt. Ltd., an Indian company, of the address of the address Bilakhia House Muktanand Marg, Chala, Vapi, Gujarat - 396191

The following specification particularly describes the invention:

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 human body.
[002] 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 leaflet is 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 anti-calcification. The valves made of these tissue 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 existing conventional technology is needed.

SUMMARY
[003] The present invention discloses a process for anti-calcification of a tissue. The process comprises treating a tissue with a pre-heated solution of fixative agent, treating the tissue with a sterilant, refluxing one or more times a tissue in a distillation apparatus in the presence of one or more refluxing agents at a predefined temperature and washing the reduced tissue with an alcohol to yield an anti-calcified tissue. Further, the tissue is subjected to dehydration process under high pressure and gentle fluid movement of glycerol.

BRIEF DESCRIPTION OF DRAWINGS
[004] 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.
[005] FIG.1 depicts an exemplary flowchart of a process involved in dry conditioning of a bioprosthetic tissue in accordance with an embodiment of the present invention.
[006] FIG.2 depicts an exemplary flowchart of a process involved in anti-calcification of a tissue in accordance with an embodiment of the present invention.

DETAILED DESCRIPTION OF DRAWINGS
[007] 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.
[008] 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.
[009] The present invention discloses a method for dry conditioning of a tissue. Such tissue may be an animal tissue utilized for medical purposes such as in artificial heart valves. In an embodiment, the tissue is a bovine pericardium tissue. The method of the present invention involves a series of steps (an exemplary method is elaborated below). The said method overcomes the drawbacks of the conventional technology to deliver a resultant tissue which can be stored in dry conditions. Such tissue may be utilized for medical purposes such as in artificial heart valves. In an exemplary embodiment, the method involves the removal of free aldehydes groups. Moreover, the removal of the free aldehyde groups may be achieved by a process of refluxing which renders the tissue less susceptible 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 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. 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 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 fixed tissue. In an embodiment, the process of fixation enhances 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 20oC-80oC 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] 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.
[0021] 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 1:1, 90:10 and 10:90. The glycerol used for dehydration may minimize the GA toxicity by avoiding storage in GA solution.
[0022] At step 109, the dehydrated tissue is subjected to sterilization. In an embodiment, the dehydrated tissue is used to fabricate heart valve. The sterilization of the tissue is performed by means of without limitation ethylene oxide gas.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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).
[0030] 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
[0031] In an embodiment, the tissue is placed in a vacuum sealed jar containing approximately 50-90 ml of the Formaldehyde ethanol polysorbate (FEP) 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.
Constituent Concentration (v/v)%
Polysorbate 80 1-10
Ethanol 24-37
Formaldehyde 10-25

[0032] At step 205, the tissue is further processed for removal of free aldehydes (untreated/unconjugated aldehydes) by means of reflux. In an embodiment, the tissue is subjected to reflux via refluxing in a soxhlet extractor using solvents such as Methanol, Ethanol and IPA by varying the temperature in a range of 45-650C. The concentration of free glutaraldehyde extracted in the solvents is checked at regular time intervals of 1 hour for up to 24 hours by means of without limitation gas Chromatography. The reflux action is stopped once concentration of the free aldehydes is constant after a particular time interval. Alternatively the reflux action can also be performed using vacuum in the soxhlet extractor.
[0033] Additionally, the removal of aldehydes in the tissue may be further validated by subjecting the filtrate to laboratory tests. In an embodiment, the filtrate present in the soxhlet apparatus is subjected to Benedict’s test and Fehling test.
[0034] Lastly at step 207, 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.
Anti-calcification of the tissue is elaborated in the following examples:
EXAMPLE 1
[0035] 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.
[0036] 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.
[0037] The glutaraldehyde fixed tissue is washed with a IPA:Saline (20:80) mixture for 05 wash cycles. The cross-linked glutaraldehyde-fixed tissue is kept in a soxhlet apparatus maintained at a temperature of around 800 C for a process of reflux. The tissue is subjected to reflux in the presence of solvent ethanol until all the free aldehydes are extracted from the tissue. The concentration of extracted glutaraldehyde at different time is checked by Gas chromatography (GC) analysis and it is found that, maximum glutaraldehyde concentration is achieved after 12 hours of refluxing of the tissue. After 12 hours of treatment, the concentration remains constant which shows that all free glutaraldehyde is extracted from the tissue. The tissue is washed repeatedly with distilled water to remove excess cross-linking and refluxing agents. The filtrate is tested for Benedict’s test and Fehling’s test. It is observed that the reaction does not give Cu2O 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.
[0038] Followed refluxing, 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°C for time duration of 12 hours.
[0039] Further, the tissue is subjected to dehydration process under high pressure and gentle fluid movement of glycerol in a ratio of Glycerol:IPA (1:1), IPA:PEG (1:1), IPA:Acetone (1:1). 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.
EXAMPLE 2
[0040] In the present example, the steps involved in anti-calcification of a tissue for example fixation of a tissue, heat treatment of the tissue, ethanol washing, dehydration and storage is identical as given in previous example. The glutaraldehyde fixed tissue is washed with a IPA:Saline (20:80) mixture for 5 wash cycles. The cross-linked glutaraldehyde-fixed tissue is kept in a soxhlet apparatus maintained at a temperature of around 65 °C for a process of reflux. The tissue is subjected to reflux in the presence of solvent methanol until all the free aldehydes are extracted from the tissue. The concentration of extracted glutaraldehyde at different time is checked by Gas chromatography (GC) analysis and it is found that, maximum glutaraldehyde concentration is achieved after 16 hours of refluxing of the tissue. After 16 hrs of treatment, the concentration remains constant which shows that all free glutaraldehydes are extracted from the tissue. The tissue is washed repeatedly with distilled water to remove excess cross-linking and refluxing agents. The filtrate is tested for Benedict’s test and Fehling’s test. It is observed that the reaction does not give Cu2O 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.
EXAMPLE 3
[0041] In the present example, the steps involved in anti-calcification of a tissue for example fixation of a tissue, heat treatment of the tissue, ethanol washing, dehydration and storage is identical as given in the example 1. The glutaraldehyde fixed tissue is washed with a IPA:Saline (20:80) mixture for 05 wash cycles. The cross-linked glutaraldehyde-fixed tissue is kept in a soxhlet apparatus maintained at a temperature of around 85 °C for a process of reflux. The tissue is subjected to reflux in the presence of solvent Isopropyl alcohol (IPA) until all the free aldehydes are extracted from the tissue. The concentration of extracted glutaraldehyde at different time is checked by Gas chromatography (GC) analysis and it is found that, maximum glutaraldehyde concentration is achieved after 16 hours of refluxing of the tissue. After 16 hours of treatment, the concentration remains constant which shows that all free glutaraldehydes are extracted from the tissue. The tissue is washed repeatedly with distilled water to remove excess cross-linking and refluxing agents. The filtrate is tested for Benedict’s test and Fehling’s test. It is observed that the reaction does not give Cu2O 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.
[0042] 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.