Anticalcification Treatment For Impantable Biological Tissues Using Calcitonin

Abstract

This invention relates to the field of surgical implants, and in particular to a method of treating biomedical material, and more particularly bioprosthetic heart valves and tracheas, to mitigate calcification when implanted in a mammalian body.

Claims

1. A method for treating biological tissue, comprising exposing said tissue to a mixture of a fixative and Calcitonin in solution so as to create treated biological tissue.

2. The method of claim 1, wherein the fixative is glutaraldehyde.

3. The method of claim 1, wherein said Calcitonin is human Calcitonin.

4. The method of claim 1, wherein said Calcitonin is synthetic Calcitonin.

5. The method of claim 1, wherein said Calcitonin is salmon Calcitonin.

6. The method of claim 1, wherein said exposing is for a time period between 4 and 36 hours.

7. The method of claim 1, wherein said exposing is done at a temperature between 17 and 37 C.

8. The method of claim 1, wherein the solution is stirred during said exposing.

9. The method of claim 8, wherein said stirring is between 50 rpm and 100 rpm.

10. The method of claim 1, further comprising implanting said treated biological tissue in a human or animal so as to create an implanted biological tissue.

11. The method of claim 10, where said implanted biological tissue is selected from the group consisting of porcine aortic valves and pericardium, bovine pericardium, equine pericardium, seal aortic, pulmonary valve and pericardium, kangaroo aortic valve and pericardium and human seal, porcine and dog trachea.

12. The method of claim 10, wherein said implanted biological material comprises one or more arterial conduits of human or animal origin.

13. A method for treating biological tissue, comprising a) exposing said tissue to a mixture of a fixative and Calcitonin in solution so as to create treated biological tissue; and b) implanting said treated biological tissue in a human or animal so as to create implanted biological tissue.

14. The method of claim 13, wherein the fixative is glutaraldehyde.

15. The method of claim 13, wherein said Calcitonin is human Calcitonin.

16. The method of claim 13, wherein said Calcitonin is synthetic Calcitonin.

17. The method of claim 13, wherein said Calcitonin is salmon Calcitonin.

18. The method of claim 13, wherein said exposing is for a time period between 4 and 36 hours.

19. The method of claim 13, wherein said exposing is done at a temperature between 17 and 37 C.

20. The method of claim 13, wherein the solution is stirred during said exposing.

21. The method of claim 20, wherein said stirring is between 50 rpm and 100 rpm.

22. The method of claim 13, further comprising rinsing said treated biological tissue after step a) and before step b).

23. The method of claim 13, where said implanted biological tissue is selected from the group consisting of porcine aortic valves and pericardium, bovine pericardium, equine pericardium, seal aortic, pulmonary valve and pericardium, kangaroo aortic valve and pericardium and human, seal, porcine and dog trachea.

24. The method of claim 13, wherein said implanted biological material comprises one or more arterial conduits of human or animal origin.

25. The method of claim 13, wherein said implanted biological material comprises one or more venous conduits of human or animal origin.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The detailed description set forth below is intended as a description of the presently preferred embodiments of the invention, and is not intended to represent the only form in which the present invention may be constructed or utilized. The description sets forth the functions and sequence of steps for certain treatment embodiments of the invention.

[0024] In one embodiment, the method comprises placing the biological tissue for fixation and preservation in a buffered fixative solution (e.g. 0.5% at pH 7.4) and adding calcitonin. Thus, this embodiment involves the use of calcitonin at the time the fixative is used, i.e. together in a mixture. It is not intended that the present invention be limited to any particular source of calcitonin. However, salmon synthetic calcitonin (CT) is a convenient source.

[0025] As a pre-treatment step, the biomaterial can be first treated with a saline solution. For example, after harvesting, the biological tissue is rinsed and maintained in cold saline solution 0.9% NaCl (temperature between 2-12 C.), typically for a period of 1- 24 hours. Alternatively Hepa Saline Solution may be used. The Hepa saline solution can be made by the following formula: 20 liters of distilled water (Millipore-Direct-Q (18.2 M.cm) with 180.01g NaCl, 19.73 g NaHPO4, 121.72 g Na2HPO4, 20 ml HCL 1M (Ph 7.4).

[0026] In one embodiment, the method comprises placing the biological tissue for fixation and preservation in a buffered glutaraldehyde (Glut) solution 0.5% at pH 7.4, adding salmon synthetic calcitonin (CT), human calcitonin or of other origin, at various concentrations preferably between 0.1% up to 10%. In one embodiment, the first fixation process lasts for a first period (e.g. one to eight hours) and subsequently the biological tissues are embedded in or exposed to an accordingly fresh solution (of fixative and calcitonin) for a longer period (e.g. up to an including one week).

[0027] The reaction process can take place in a quiet environment. Alternatively, the reaction process can take place with stirring the solution between 50 rpm and 100 rpm at a temperature between 17 C. up to 37 C.

[0028] Following this novel anticalcification treatment of the present invention, the biological tissues can be sterilized (e.g. with a formaldehyde solution), as described in prior art, and then the tissues can be stored at buffered glutaraldehyde solution 0.25%-0.5% for a long period of time at room temperature, ready to use for mammal implantation. In another embodiment of the present invention, sterilization treatment can be achieved before the embedment of the biological tissue into this novel anticalcification treatment.

[0029] In another embodiment, calcitonin is used after the fixative treatment. As a pre-treatment step, the biomaterial can be first treated with a saline solution. For example, after harvesting, the biological tissue is rinsed and maintained in cold saline solution 0.9% NaCl (temperature between 2-12 C.), typically for a period of 1-24 hours. Alternatively, Hepa Saline Solution may be used. The Hepa saline solution can be made by the following formula: 20 liters of distilled water (Millipore-Direct-Q (18.2 M.cm) with 180.01 g NaCl, 19.73g NaHPO4, 121.72 g Na2HPO4, 20 ml HCL 1M (Ph 7.4).

[0030] In this particular embodiment, the tissue is next fixed using a fixative (e.g. 0.5% buffered glutaraldehyde) solution at room temperature for at least 1 hour. Then the buffered fixative (e.g. glutaraldehyde) solution is changed and the tissues are left for fixation for a longer period (e.g. 1 to 7 days). In this particular embodiment, the method further comprises placing the biological tissue, after being fixed with glutaraldehyde or other fixative compounds, in contact with Calcitonin solution for a time between 4 hours up to 36 hours at a temperature between 17 up to 34 C.

EXPERIMENTAL

[0031] In one embodiment, the method comprises placing the biological tissue for fixation and preservation in a buffered glutaraldehyde (Glut) solution 0.5% at pH 7.4, adding salmon synthetic calcitonin (CT), such as Miacalcic of Novartis Hellas A.B.E. In this experiment, two different concentrations of calcitonin were tested. One with 1 unit/100 ml (1%) and the other with 10 units/100 ml (10%). The first fixation process lasted one hour, at a pressure between 2-3 mmHg and subsequently the biological tissues were embedded in an accordingly new solution for one week.

[0032] Porcine aortic leaflets were selected as fresh tissue from a local slaughter house and were cut radially in three parts. Following harvesting, the biological tissue is rinsed and maintained in cold Hepa saline solution (temperature between 4-12 C.) between two to six hours.

[0033] Three groups of tissue were created. Group I (Glut only), Group II (Glut with 1% CT) and Group III (Glut with 10% CT). All tissues were then implanted subdermally in three sets of 8 (Group I), 9 (Group II) and 9 (Group III) male Wistar rats of 12 days old (Center for Experimental Surgery, Biomedical Research Foundation of the Academy of Athens). The rats were selected along with their mother and had a free alimentation regime. All tissues were rinsed three times in normal saline solution for 10 minutes each time before implantation. Each rat received four fragments of tissue at the dorsum, through four separate incisions (two at each side) each of 1 cm long, with a technique we have previously described elsewhere (Agathos E A et al: In vivo calcification of glutaraldehyde fixed cardiac valve and pericardium of Phoca Groenlandica: ASAIO 57(4):328-332, July/August 2011).

[0034] 21 days later the rats were euthanized by inhalation of CO.sub.2. All procedures were approved by the Animal Care Committee of the Academy of Athens and performed according to the Guide for the Care and Use of Laboratory Animals prepared by the Institutes of Laboratory Animal Resources, National Research Council and published by the National Academy Press, revised 1996 (NIH publication No. 85-23). The tissues were retrieved and after rinsing with distilled water 3 times, were lyophilized at 40 C. at high vacuum pressure of approximately 100 mmHg for 16 hours. The calcium content was then measured with flat atomic absorption technique.

[0035] For statistical analysis, the commercially available software package ANOVA Origin 8.0 for Windows (OriginLab Corporation, Northampton, Mass., USA) was used. P values of 0.05 or less were defined as a statistically significant difference.

RESULTS

[0036] The pre-implantation values for mg Ca/mg tissue of the various groups are listed in Table 1. Group I (control group) represents glutaraldehyde fixed tissues without anticalcification treatment, while Group II represents samples treated with buffered 1% CT solution and Group III represents samples treated with 10% CT solution.

[0037] The post implantation weight of the samples along with the values for mg Ca/mg tissue of the various groups are listed in Table 2, while Table 3 shows the cumulative results of Ca concentration in the various group and the statistical differences.

[0038] There was not significance difference between Groups II and III, even if Group II showed a less Ca concentration accumulation (5.16) than Group III (9.43) in the explanted tissues. All numeric data were expressed as meanstandard deviation (STDEV).

TABLE-US-00001 TABLE 1 Pre implantation Ca Content weight (gr) mg Ca/gr tissue Group I Mean 0.0039 1.79 STDEV() 0.0007 0.14 Group II Mean 0.0110 4.78 STDEV() 0.0007 0.079 Group III Mean 0.0120 2.88 STDEV() 0.0006 0.17 * no statistical difference between the various groups STDEV: Standard Deviation

TABLE-US-00002 TABLE 2 Post implantation Ca Content weight (gr) mg Ca/gr tissue Group I Mean 0.0086 126.95 STDEV() 0.0009 12.97 Group II Mean 0.0458 24.69 STDEV() 0.0212 2.71 Group III Mean 0.0453 27.16 STDEV() 0.0071 2.95 STDEV: Standard Deviation

TABLE-US-00003 TABLE 3 Cumulative results of post-implantation Ca concentration in the Various group and statistical difference Group I (Glut only) Group II (1% CT) Group III (10% CT) 8 rats 8 rats 9 rats 126.95 12.97 24.69 2.71 27.16 2.95 p < 0.05* p < 0.05** p = ns*** *The statistical difference between Group II and Group I **The statistical difference between Group III and Group I ***The statistical difference between Group II and Group III

[0039] It is understood that the analytical method for treating glutaraldehyde fixed biological tissue described herein represents only a presently preferred embodiment of the present invention. Various modifications and additions may be made to such embodiment without departing from the scope of the invention. For example, various fixing agents, such as aldehydes other than glutaraldehyde or other chemicals, may exhibit properties, similar to those of glutaraldehyde so as to make them suitable for use in the present invention and, thus, may likewise be utilized. Accordingly, these and other modifications and additions may be obvious to those skilled in the art and may be implemented to adapt the present invention for use in a variety of different applications.