AU4900900A - A method using potassium dihydrogen phosphate to reduce calcification of tissue - Google Patents

Description

WO 00/74692 PCT/AU00/00633 A METHOD USING POTASSIUM DIHYDROGEN PHOSPHATE TO REDUCE CALCIFICATION OF TISSUE FIELD OF THE INVENTION The present invention relates to a method for reducing calcification of tissue and 5 in particular use of such a method for reducing calcification of kangaroo tissue. BACKGROUND ART Bioprosthetic heart valves constructed from preserved porcine aortic valve leaflets or bovine pericardium have been widely used for over 30 years in valve replacement surgery. Although bioprosthetic heart valves and bioprosthetic 10 pericardial patches have reduced thrombogenicity and improved hemodynamic properties as compared to mechanical and synthetic substitutes, bioprostheses tend to calcify. In children, calcification occurs in 100% of recipients within 5 years of implantation. In adults this phenomenon occurs less frequently and 10 to 20% of 15 these valves tend to calcify within 10 years. Calcification of the implanted tissue, after an extended period of time, results in reduced flexibility, increased tissue stress, tissue ruptures and loss of function. The most important types of calcification deposits in porcine bioprostheses involve cuspal collagen and surface thrombi or vegetations. However, 20 calcification deposits also occur in valvular connective tissue cells, adipose tissue cells and the aortic wall. Although the mechanism of pathological calcification is not clearly understood, several factors have been identified as possible mediators, responsible for bioprosthetic heart valve calcification. These factors include deposition of blood 25 proteins in inter-collagen spaces (created by the leaching of fixed proteoglycans and lipids after transplantation), reaction of blood proteins with free aldehydes, immunological host reactions and the binding of free calcium to calcium binding sites in the collagen fibres and to free aldehydes.

WO 00/74692 PCT/AUO0/00633 -2 Attempts to overcome the problem of calcification have been, in the past, directed toward aspects of the fixation process such as fixation pressure, treatment of the tissue with biochemical blocking agents and glutaraldehyde-free fixation with dye-mediated photooxidation. 5 Early findings indicated that fixation of tissues by glutaraldehyde reduced the amount of calcification. However, there still existed a requirement to improve the efficiency of this method with the addition of blocking agents. Various anti calcification agents have been used, including 2-Amino oleic acid, surfactants, covalently bound aminohydroxypropane diphosphoric acid, aluminium and 10 toluidine blue. These agents require the tissue to be fixed and stabilised with a buffered glutaraldehyde solution prior to the addition of the blocking agent, thus creating a two step process of fixing and blocking. Thus there exists a need to improve the efficiency of methods of treatment of tissue to reduce calcification. 15 DISCLOSURE OF THE INVENTION The present invention provides a method of treating tissue to alleviate the occurrence of calcification comprising the step of: (i) Contacting the tissue with a solution of potassium dihydrogen phosphate or a salt thereof for sufficient time to allow the potassium 20 dihydrogen phosphate or salt thereof to impregnate the tissue, wherein potassium dihydrogen phosphate or salt thereof serves as a blocking agent and/or a buffer. At present most fixation methods are based on the principle of fixation of the tissue with a fixing solution of glutaraldehyde buffered with 25 hydroxyethylpiperazine - N-2-ethanesulphonic acid ("HEPES buffer") or sodium bicarbonate in order to stabilise the glutaraldehyde. To be effective in reducing the occurrence of calcification, this method needs to be enhanced with additional blocking agents. Surprisingly the present inventors have found that use of potassium dihydrogen phosphate in this method eliminates the requirement of an WO 00/74692 PCT/AUOO/00633 -3 additional buffer due to the fact that the potassium dihydrogen phosphate serves as a blocking agent and/or a buffer. In a preferred embodiment, the invention consists of the step comprising: fixing the tissue with a solution of glutaraldehyde and potassium dihydrogen 5 phosphate, wherein the potassium dihydrogen phosphate acts as both a buffer to stabilise the glutaraldehyde and/or a blocking agent to prevent or retard the onset of calcification. In a highly preferred embodiment of the invention, the tissue is selected from non-placental mammalian heart valve tissue or non-placental mammalian 10 pericardium. In an even more highly preferred embodiment, the tissue is selected from kangaroo heart valve tissue or kangaroo pericardium. BEST MODE(S) FOR CARRYING OUT THE INVENTION As used herein, the term "bioprostheses" is meant to include any device, which is 15 implanted in a mammal and is made from tissues derived from a mammal. Thus, the term includes heart valves and other heart components, vascular replacements or grafts, artificial hearts, urinary tract and bladder replacements, bowel and tissue resections in general, left ventricular devices, hip replacements, silastic breast implants, artificial tendons, electrodes, catheters and the like. 20 The present invention is not limited to treatment of tissue prior to insertion in a patient. It may also be used in relation to bioprostheses for which calcification after implantation is a clinical problem. In a preferred embodiment of the present method, the tissue is fixed with a solution comprising a fixing agent and potassium dihydrogen phosphate. 25 As used herein "fixing agent" includes any compound or formulation capable of stabilising the bioprostheses against in vivo enzymatic degradation. Such compounds include, but are not limited to, glutaraldehyde and formaldehyde. In one example, the fixing agent is glutaraldehyde.

WO 00/74692 PCT/AUOO/00633 Glutaraldehyde stabilises the bioprostheses against in vivo enzymatic degradation. It does so by linking amino-containing proteins together during cross-linking reactions. Some of the cross-linked products are without free aldehyde groups and some still retain free aldehyde groups. During the cross 5 linking process, inter-collagen spaces are increased. In addition to its fixing properties, glutaraldehyde also acts as an excellent sterilising agent at low concentrations and reduces antigenicity of the tissue. Preferably, the tissue is treated with a concentration of glutaraldehyde of between 0.5% to 0.7%. Still more preferable the tissue is treated with a 10 glutaraldehyde concentration of between 0.6% to 0.7%. Even more preferable the tissue is treated with a glutaraldehyde concentration of between 0.6% and 0.65%. Most preferable the tissue is treated with a glutaraldehyde concentration of 0.625%. The inventors have found inter alia that monovalent cations such as potassium 15 and anions such as phosphate ions block carboxylic acid reactive groups on native collagen surfaces, which lowers calcification levels in tissues. Consequently, they have found that potassium dihydrogen phosphate is useful as a blocking agent. Moreover, it has a sufficiently low molecular weight such that it does not increase the osmolarity of the fixing solution. 20 Potassium dihydrogen phosphate is useful as a buffer to stabilise glutaraldehyde treated tissue. Previously an additional buffer such as HEPES buffer or sodium bicarbonate was required to be added to the glutaraldehyde solution to stabilise it. In the present method potassium dihydrogen phosphate acts as both a blocking agent and/or a buffer, thereby eliminating the need for an additional 25 buffer. As used herein the term "buffer" includes any substance which has the ability to bind or release hydrogen ions in solution, thus maintaining the pH of the solution relatively constant despite the addition of considerable quantities of acid or base. As used herein the term "blocking agent" is a chemical substance which 30 dissociates into electro-positive and electro-negative ions when dissolved in WO 00/74692 PCT/AUOO/00633 water and have the ability to bind to the respective opposite charged binding sites of a specific material or object. Preferably the concentration of potassium dihydrogen phosphate is between 0.05M and 0.07M. Preferably the concentration of potassium dihydrogen 5 phosphate is 0.067M. Preferably the pH of the fixing solution is between pH 7.3 and pH 7.5. Still more preferably the pH of the solution is between pH 7.35 and pH 7.45. Most preferably the pH of the fixing solution is pH 7.4. It is desirable that the fixing solution be brought to a pH of 7.4 by using a suitable 10 base. Preferably, the base is sodium hydroxide or potassium hydroxide. Preferably, the concentration of the base is between 0.05M and 0.15M. Still more preferable is a concentration of between 0.7M and 0.13M. Even more preferable is a concentration of between 0.9M and 0.11M. Most preferable is a concentration of 0.1M. 15 Preferably, the tissue is submerged in the fixing solution for a sufficient reaction time of 5 to 10 days. More preferably the tissue is submerged in the fixing solution for 6 to 9 days. Most preferably the tissue is submerged in the fixing solution for 7 days. The temperature at which the tissue is submerged in the fixing solution is 20 preferably 3 to 5 degrees. The most preferable temperature is 4 degrees Celsius. An advantage which results from treating tissue according to the invention is that the tissue does not oxidize as quickly, thereby increasing the period in which the tissue may be kept in storage. 25 In one embodiment of this invention, the tissue is selected from non-placental mammalian heart valve tissue or non-placental mammalian pericardium, which has less proteoglycan and a more densely arranged collagen structure than WO 00/74692 PCT/AUOO/00633 -6 porcine tissue. More preferable is tissue selected from kangaroo heart valve tissue or kangaroo pericardium. Kangaroo tissue has less proteoglycan and more densely arranged collagen structure than porcine tissue. Consequently, there are smaller inter-collagen 5 spaces. These features are in favour of lower calcium calcification potential because: (a) there are less available calcium binding sites; (b) there are less inter-collagen spaces for the invasion of host tissues by proteins, lipids, proteoglycan all of which have a potential to be 10 involved in the process of bioprosthetic calcification; and (c) there are less available amino groups. Thus, kangaroo tissue used as a bioprosthetic implant during cardiac repair procedures has a major advantage over porcine and bovine tissue because of its significantly low calcification rate without any anti-calcification treatment. 15 Kangaroo heart valve tissue treated according to the present method can be used to fabricate a stentless bioprosthetic valve, a stented bioprosthetic valve for and a bioprosthetic valve conduit. Kangaroo pericardium treated according to the present invention can be used as a bioprosthetic patch to repair intra-cardiac defects such as an atrio-septal 20 defect, a ventriculo-septal defect, ventricular reconstruction and reconstruction of defective in- and outflow tracts in children and adult human beings. Moreover, kangaroo pericardium can also be used as a bioprosthetic substitute for replacement of the human pericardium and a bioprosthetic substitute for the fabrication of bioprosthetic heart valves. 25 BRIEF DESCRIPTION OF THE DRAWINGS Further features of the invention are more fully described in the following Figures and Examples: Figure 1 shows the results of the Von Kossa Stain in Example 5.

WO 00/74692 PCT/AUOO/00633 -7 Figure 2 shows the calcification of kangaroo pericardial tissue versus bovine pericardial tissue after implantation into rat model. Figure 3 shows the results of the Von Kossa Stain in Example 6. Figure 4 shows the extractable calcium content of kangaroo and porcine 5 heart valve after 8 weeks of implantation in a rat model. Figure 5 shows the results of the Von Kossa Stain in Example 7. Figure 6 shows the extractable calcium content of kangaroo and porcine heart valve after 6, 8 and 12 months of implantation in a sheep model. Figures 7A and 7B are electron micrographs which revealed a significant 10 difference in inter-collagen spaces between the bioprosthetic bovine pericardium and the bioprosthetic kangaroo pericardium. Figures 8A and 8B are electron micrographs which revealed a significant difference in inter-collagen spaces between the bioprosthetic porcine aortic tissue and the bioprosthetic kangaroo aortic tissue. 15 Figure 9 are Von Kossa stains which revealed that heart valve tissue from kangaroo has a lower calcium content than heart valve tissue from porcine origin after eight weeks of implantation in a rat model. EXAMPLES Further features of the present invention are more fully described in the following 20 Examples. It is to be understood that the following Examples are included solely for the purposes of exemplifying the invention and should not be understood in any way as a restriction on the broad description set out above. Example 1 Harvesting of tissues 25 The pericardium (tissue sac surrounding the heart) or the aortic valve tissue (heart and main vessels) from adult Kangaroo species of body weight equal to or greater than 12 Kg, were harvested immediately after culling of the animal. Tissues were washed in cold (4 to 6 0C), sterile 0.9% saline solution for 2 to 3 WO 00/74692 PCT/AUOO/00633 minutes to remove excess blood. The aortic root, valve and 5 to 7 cm of the ascending aortic tube were removed using standard dissecting procedures. The left and right coronary arteries were separated from the heart muscle, leaving a 1 cm stump on each vessel. The valved aortic tube was washed with sterile, cold 5 saline solution for 1 to 2 minutes. Example 2 Fixing of tissues Tissues were fixed in a solution of 0.625% glutaraldehyde buffered with potassium dihydrogen phosphate to a final concentration of 0.067 M potassium 10 dihydrogen phosphate. The pH was adjusted to pH 7.4 with 0.1 M sodium hydroxide. Standard fixing procedures were used to fix the pericardium or aortic tissue and are briefly described below. The pericardium tissue was rinsed in 500 mL of cold (4 0C) balanced electrolyte solution Balsol (Baxter, Johannesburg, RSA) for 20 15 minutes. The tissue was then spread in a shallow, rectangular Perspex bowl, filled with the electrolyte solution at 4 *C. Any unwanted connective tissue and ligaments were carefully removed by standard dissection techniques. The pericardium was then stretched and fixed onto a square Perspex frame and submerged into the fixation solution at 4 0 C for 7 days. 20 The aortic tube was attached to a 10 cm long glass tube (with appropriate diameter) by slipping the distal end over the tube and tying with 1/10 black suture silk. The glass tube was then fixed over a 500 mL glass beaker. The outside of the aortic wall was wrapped in a gauze swab, soaked in cold 0.9% saline solution. The valve leaflets were fixed for 10 minutes under low pressure by 25 filling the aortic tube with the fixation solution, to a level approximately 2 to 3 cm above the closed valve leaflets. The resulting pressure was sufficient enough to keep the aortic valve in its closed configuration. During the filling period, the stumps of the coronary arteries were tied with 1/10 suture silk to prevent drainage of the fixation solution. After the 10 minute period, the valved tube was 30 submerged in the fixation solution at 4 0C for 7 days.

WO 00/74692 PCT/AUOO/00633 Both pericardium and aortic tissue may be stored at 4 *C in fixation solution until further use. The maximum time that the tissue may be stored in fixation solution is 18 months. Example 3 5 Electron microscopic evaluation of bioprosthetic pericardium Three randomised samples of each bovine and kangaroo fixed pericardium were prepared for scanning electron microscopic to investigate the ultrastructural arrangement of the collagenous matrix, consisting of collagen fibres and elastin. Results (electron micrograph 7A and 7B) revealed a significant difference in 10 inter-collagen spaces between the bioprosthetic bovine pericardium (Figure 7A) and the bioprosthetic kangaroo pericardium (Figure 7B). Inter-collagen spaces in Figure 7A were visibly less densely arranged than in Figure 7B. Example 4 Electron microscopic evaluation of bioprosthetic aortic tissue 15 Three randomised samples of each porcine and kangaroo fixed aortic tissue were prepared for scanning electron microscopic to investigate the ultrastructural arrangement of the collagenous matrix, consisting of collagen fibres and elastin. Results (electron micrograph 8A and 8B) revealed a significant difference in inter-collagen spaces between the bioprosthetic porcine aortic tissue (Figure 8A) 20 and the bioprosthetic kangaroo aortic tissue (Figure 8B). Inter-collagen spaces in Figure 8A were visibly less densely arranged than in Figure 8B. Example 5 Pericardial tissue implanted in a rat model The effectiveness of non-placental mammalian pericardial tissue (kangaroo) 25 compared with placental mammalian pericardial tissue (bovine) was determined by animal implant studies in an internationally recognised rat model. Implants were analysed for calcification using Von Kossa stain and extractable calcium content.

WO 00/74692 PCT/AUO0/00633 - 10 Implantation of pericardial tissue A total of 18 male Albino Wistar rats, weighing 200 to 250 g, were anaesthetised with an intra-peritoneal injection of Nembutal (45 mg/kg) (Abbott Laboratories, AUS). Rats were prepared for abdominal surgery using standard techniques. 5 For example, the abdominal muscle wall was shaved and disinfected with 5% diluted chlorohexidine gluconate (ICI Pharmaceuticals, Perth AUS) in 70% ethanol (Merck Chemicals, Perth AUS) and a lengthwise midline skin incision of approximately 3 to 4 cm in length was made in the ventral surface. The skin and subcutaneous layers were separated from the underlying muscle and one small 10 pouch was formed in the muscle wall on either side of the midline incision by a small incision followed by blunt dissection of the muscle wall. One piece each of kangaroo or bovine pericardium of size 1.5 cm x 1.5 cm, was subjected to a standard rinsing protocol in sterile 0.9% saline solution to remove residual and excess glutaraldehyde and inserted into each muscle pouch. From 15 an anterior perspective, the bovine pericardium was implanted into the left muscle pouch and the kangaroo pericardium was inserted into the right pouch. A small metal ligaclip was attached to the top end of the left muscle pouch to serve as a marker for the bovine pericardium. The skin was closed with 5/0 prolene sutures and the animal returned to its cage. 20 From the total of 18 implants from both kangaroo and bovine origin, five implants were explanted from animals at either 4, 6 or 8 weeks after implantation. Implanted pericardial tissues were removed from animals euthenased with an overdose of Barbiturates (Abbott Laboratories, AUS). Analysis of calcification of implants by Von Kossa Stain 25 The abdominal wall was removed from the rat before removal of the implants. Samples of three implants in each group (4, 6 or 8 weeks) were used for qualitative calcium analysis by means of the Von Kossa staining technique for calcium (Schoen FJ et al1985) WO 00/74692 PCT/AUOO/00633 - 11 Determination of calcium content in implants The remaining implants (n= 12) were dissected free of host tissue and dried in a Biotherm incubator (Selby Scientific, WA) at 70 0C for 48 hours. The dried samples were weighed and the calcium content extracted in a 5.0 ml volume of 5 6N Hydrochloric acid (Merck, Perth AUS) (Tsao JW et al, 1988). Extracted calcium content was measured by atomic absorption spectrophotometry and expressed as mg of calcium per g of tissue (dry weight). Results of Von Kossa Stain The presence of calcium deposits were reported according to the following visual 10 grading system during light microscopic investigation: 0 = no calcification + = mild calcification ++ = moderate calcification +++ = severe calcification 15 Results of this experiment are presented in Figure 1. Extractable calcium content of implants The pericardial tissue from kangaroo has a significantly lower calcium content than pericardial tissue from bovine origin after 4, 6 and 8 weeks of implantation into the rat model. The concentration of calcium in bovine tissue was 20 significantly greater (P<0.05) at 0.533 ± 0.09 mg/g dry weight (mean ± SD) after 4 weeks of implantation compared to 0.025 ± 0.001 mg/g dry weight (mean + SD) in the kangaroo pericardial tissue. Furthermore, the concentration of calcium in bovine tissue increased to 0.963 ± 0.43 mg/g dry weight (mean ± SD) after 6 weeks of implantation compared to no significant increase in the 25 concentration of calcium in kangaroo tissue at the same time point [0.039 ± 0.02 mg/g dry weight (mean ± SD). By 8 weeks after implantation, there was a slight but insignificant increase (P>0.05) in the concentration of calcium in kangaroo tissue [0.070 ± 0.04 mg/g dry weight (mean ± SD)]. No further increase in the concentration of calcium in bovine tissue was observed after 8 weeks of WO 00/74692 PCT/AUOO/00633 - 12 implantation [0.936 ± 0.37 mg/g dry weight (mean ± SD)]. Individual results are presented in Figure 2. Example 6 Aortic tissue implantation in a rat model 5 The effectiveness of non-placental mammalian (Kangaroo) heart valve tissue as an alternative to placental mammalian (porcine) heart valve tissue was compared in a rat model. Implants were analysed for calcification using Von Kossa stain and extractable calcium content. Implantation of aortic tissue 10 Male Albino Wistar rats (n=10), weighing 200 to 250 g were anaesthetised with an intra-peritoneal injection of Nebutal (45 mg/kg) and prepared for abdominal surgery using standard techniques. Briefly, the abdominal muscle wall was shaved and disinfected with 5% diluted chlorhexidine gluconate in ethanol and a lengthwise midline skin incision of approximately 3 to 4 cm in length was made in 15 the ventral surface. The skin and subcutaneous layers were separated from the underlying muscle and one small pouch was formed in the muscle wall on either side of the midline incision by a small incision followed by blunt dissection of the muscle wall. One piece each of kangaroo or porcine valve leaflets were subjected to a 20 standard rinsing protocol whereby the tissue was rinsed in two separate rinsing bowls filled with 250 mL of sterile 0.9% saline solution for 15 minutes. This removed residual and excess glutaraldehyde. The leaflets were then inserted into each muscle pouch. From an anterior perspective, the porcine leaflet was implanted into the left muscle pouch and the kangaroo leaflet was inserted into 25 the right pouch. A small metal ligaclip was attached to the top end of the left muscle pouch to serve as a marker for the porcine valve tissue. The skin was closed with 5/0 prolene sutures and the animal returned to its cage. Rats were euthenased with Barbitrates, 8 weeks after implantation of the heart valve leaflets.

WO 00/74692 PCT/AUOO/00633 - 13 Analysis of calcification of implants by Von Kossa Stain The abdominal wall was removed from the rat before removal of the implants. Samples of three implants from both kangaroo and porcine origin were analysed for qualitative calcium by means of the Von Kossa staining technique for 5 detecting calcium (Schoen FJ, et al, 1985). Determination of calcium content in implants The remaining implants (n= 7) were dissected free of host tissue and dried in a Biotherm incubator (Selby Scientific, WA) at 70 CC for 48 hours. The dried samples were weighed and the calcium content extracted in a 5.0 mL volume of 10 6N Hydrochloric acid (Merck, Perth AUS) (Tsao JW et al, 1988). Extracted calcium content was measured by atomic absorption spectrophotometry and expressed as mg of calcium per g of tissue (dry weight). Results of Von Kossa Stain The presence of calcium deposits were reported according to the following visual 15 grading system during light microscopic investigation: 0 = no calcification + = mild calcification ++ = moderate calcification +++ = severe calcification 20 Individual results of this experiment are presented in Figure 3. Extractable calcium content of implants The heart valve tissue from non-placental mammal tissue (kangaroo) has a significantly lower (P<0.0001) calcium content than heart valve tissue from porcine origin after 8 weeks of implantation in the rat model. The mean 25 concentration of extractable calcium in porcine tissue was 52.96 ± 4.71 (SD) mg/g dry weight compared to the mean concentration of 6.77 ± 2.72 (SD) mg/g dry weight in kangaroo heart tissue. Individual results are shown in Figure 4.

WO 00/74692 PCT/AUOO/00633 Example 7 Aortic tissue implant in sheep model The effectiveness of non-placental mammalian (Kangaroo) heart valve tissue as a prosthesis was investigated in the sheep model. Implants were analysed for 5 calcification using Von Kossa stain and extractable calcium content. Implantation of aortic tissue Juvenile sheep (n=12), weighing 16 to 22 kg were anaesthetised with sodium pentothal (Abbott Laboratories, AUS) and 2% halothane prior to surgery. A left lateral thoracotomy was performed and the descending aorta exploited through 10 the 4 th and 5 th intercostal space. The ductus ateriosus was ligated with 1/0 black suture silk. A segment of the descendics aorta, about 5 to 7 cm, in length was dissected free from surrounding fatty tissue and mobilised. The sheep was cooled down to 32 'C by means of external ice packs as well as with topical cooling inside the thoracic cavity. A segment (4cm) was isolated by cross 15 clamping of the aorta and the segment excised. The bioprosthetic kangaroo aortic valve, with a piece of its aortic tube, was sewn end-to-end into the sheep's aorta. The clamps were removed, the sheep warmed and the thoracic cavity closed. The sheep was allowed to recover from anaesthesia and returned to its paddock. Porcine aortic tissue was implanted into a different sheep using the 20 same procedure. Implant valves were explanted at 6, 8 and 12 months after implantation. Analysis of calcification of implants by Von Kossa Stain The aortic implants were excised from the sheep, after being sacrificed. Samples of three implants from both porcine and kangaroo origin were analysed 25 for qualitative calcium by means of the Von Kossa staining technique for detecting calcium (Schoen FJ et al, 1985).

WO 00/74692 PCT/AUOO/00633 - 15 Determination of calcium content in implants The implants from the remaining sheep (n= 9) were dissected free of host tissue and dried in a Biotherm incubator (Selby Scientific, WA) at 70 0C for 48 hours. The dried samples were weighed and the calcium content extracted in a 5.0 mL 5 volume of 6N Hydrochloric acid (Merck, Perth AUS) (Tsao JW et al, 1988) Extracted calcium content was measured by atomic absorption spectrophotometry and expressed as mg of calcium per g of tissue (dry weight). Results after Von Kossa staining procedure The presence of calcium deposits were reported according to the following visual 10 grading system during light microscopic investigation: 0 = no calcification + = mild calcification ++ = moderate calcification +++ = severe calcification 15 Individual results of this experiment are presented in Figure 5. Extractable calcium content of implants The heart valve tissue from non-placental mammal tissue (kangaroo) has a significantly lower (P<0.01) calcium content than heart valve tissue from porcine origin 6, 8 and 12 months after implantation in the sheep model. The mean 20 concentration of calcium in porcine tissue 6 months after implantation was 53.79 ± 6.42 (SD) mg/g dry weight, compared to a concentration of calcium of 0.452 + 0.38 mg/g dry weight in the kangaroo tissue implants. By 8 months, the concentration of calcium in porcine tissue implants had increased to a mean of 173.8 ± 75.5 (SD) mg/g dry weight, which was significantly higher than the mean 25 concentration of calcium in kangaroo tissue implants [3.05 ± 1.30 (SD) mg/g dry weight] (P<0.01). Furthermore, the concentration of calcium in kangaroo implant tissues after 12 months remained low [10.63 ± 5.53 (SD) mg/g dry weight]. Individual results are presented in Figure 6.

WO 00/74692 -16- PCT/AUOO/00633 Throughout the specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. 5 REFERENCES 1. Schoen FJ, Levy RJ, Nelson AC, Bernhard WF, Nashef A and Hawley M, 1985. 'Onset and progression of experimental bioprosthetic heart valve 10 calcification'. Laboratory Investigation, 52(5): 523-532. 2. Tsao JW, Schoen FJ, Shankar R, Sallis JD, Levy RJ, 1985. 'Retardation of calcification of bovine pericardium used in bioprosthetic heart valves by phosphocitrate and a synthetic analogue' Biomat 9: 393-397

Claims (22)

1. Potassium dihydrogen phosphate or a salt thereof when used as a blocking agent and/or a buffer.

2. A method of treating tissue to alleviate the occurrence of calcification 5 comprising the step of : (i) Contacting the tissue with a solution of potassium dihydrogen phosphate or a salt thereof for sufficient time to allow the potassium dihydrogen phosphate or salt thereof to impregnate the tissue, wherein the potassium dihydrogen phosphate or salt thereof serves as 10 a blocking agent and/or a buffer.

3. A method of treating tissue to alleviate the occurrence of calcification comprising the step of fixing the tissue with a solution comprising a fixing agent and potassium dihydrogen phosphate, wherein the potassium dihydrogen phosphate acts as a buffer and/or a blocking agent. 15

4. A method of treating tissue according to claim 3 wherein the fixing agent is glutaraldehyde.

5. A method of treating tissue according to claim 4 wherein the concentration of glutaraldehyde is between 0.5% to 0.7%.

6. A method of treating tissue according to claim 5 wherein the concentration of 20 glutaraldehyde is between 0.6% to 0.7%.

7. A method of treating tissue according to claim 6 wherein the concentration of glutaraldehyde is between 0.6% to 0.65%.

8. A method of treating tissue according to claim 7 wherein the concentration of glutaraldehyde is 0.625%. WO 00/74692 -18- PCT/AUO0/00633

9. A method of treating tissue according to claim 3 wherein the fixing agent is formaldehyde.

10. A method of treating tissue according to any one of the preceding claims wherein the concentration of potassium dihydrogen phosphate is between 5 about 0.05M and 0.07M.

11. A method of treating tissue according to claim 10 wherein the concentration of potassium dehydrogen phosphate is 0.067M.

12. A method of treating tissue according to any one of claims 3 to 11 wherein the pH of the fixing solution is between about 7.3 and 7.5. 10

13. A method of treating tissue according to claim 12 wherein the pH of the fixing solution is between 7.35 and 7.45.

14. A method of treating tissue according to claim 13 wherein the pH of the fixing solution is 7.4.

15. A method of treating tissue according to any one of claims 3 to 14, wherein 15 the tissue is fixed for 5 to 10 days.

16. A method of treating tissue according to claim 15 wherein the tissue is fixed for 6 to 9 days.

17. A method of treating tissue according to claim 16 wherein the tissue is fixed for 7 days. 20

18. A method of treating tissue according to any one of claims 3 to 17 wherein the temperature at which the tissue is fixed is about 3 to 5 degrees Celsius.

19. A method of treating tissue according to claim 18 wherein the temperature at which the tissue is fixed is 4 degrees Celsius. WO 00/74692 -19- PCT/AUOO/00633

20. A method of treating tissue to alleviate the occurrence of calcification comprising the step of fixing the tissue with a solution comprising glutaraldehyde and potassium dihydrogen phosphate wherein: (a) the concentration of glutaraldehyde is 0.625%; 5 (b) the concentration of potassium dihydrogen phosphate is 0.067M; (c) the pH of the fixing solution is 7.4; (d) the tissue is fixed for 7 days; and (e) the temperature at which the tissue is fixed is 4 degrees 10 Celsius.

21. A method of treating tissue according to any one of the preceding claims wherein the tissue is selected from non-placental mammalian heart valve tissue and non-placental mammalian pericardium.

22. A method of treating tissue according to any one of the preceding claims 15 wherein the tissue is selected from kangaroo heart valve tissue and kangaroo pericardium.