Treatment of collagenous tissue with glutaraldehyde and aminodiphosphonate calcification inhibitor

Claim 2

A process for the treatment of collagenous tissue to adapt it for use in a prosthetic implant and to promote the growth of endothelial cells thereon after implantation, which comprises the steps of: (a) contacting said tissue with at least one surfactant for a time sufficient to substantially completely remove deleterious material and open up the fibrous structure of the collagenous tissue; (b) washing the resulting fibrous matrix to remove substantially all surfactant; (c) fixing the washed tissue with glutaraldehyde; (d) treating the glutaraldehyde-fixed tissue with a calcification-inhibiting agent, an agent which inhibits infiltration and attack by phagocytic cells upon implantation and/or an agent which inhibits infection; and (e) treating the resulting agent/matrix tissue with a reducing agent to stabilize the bonding of the glutaraldehyde of step (c) and the agent of step (d) to the tissue.

Claim 3

A process according to claim 1, in which the collagenous tissue is bovine or porcine pericardial tissue.

Claim 4

A process according to claim 1, in which the collagenous tissue is dura mater, fascia lata, valve tissue or vascular graft tissue.

Claim 5

A process according to claim 1, in which the surfactant is in the form of an aqueous solution containing 0.5 to 6% by weight of surfactant.

Claim 6

A process according to claim 4, in which said surfactant is a mixture of an anionic surfactant and a non-ionic surfactant.

Claim 7

A process according to claim 5, in which the anionic surfactant is 1% by weight sodium dodecyl sulfate and the non-ionic surfactant is 1% by weight octylphenoxy polyethoxy ethanol and/or 1% by weight polyoxyethylene (20) sorbitan monooleate.

Claim 8

A process according to claim 6, in which the collagenous tissue is contacted with said surfactant solution for about three hours at room temperature.

Claim 9

A process according to claim 4, in which the surfactant is an amphoteric surfactant.

Claim 10

A process according to claim 1, in which the collagenous tissue is pre-washed with saline solution to remove excess blood and plasma proteins, prior to treatment with surfactant.

Claim 11

A process according to claim 1, in which the fibrous matrix of tissue resulting from the surfactant treatment is soaked in aqueous glutaraldehyde solution for a time sufficient to fix the tissue by bonding the glutaraldehyde molecules to substantially all the reactive amino groups present in the protein molecules of the tissue.

Claim 12

A process according to claim 10, in which the concentration of glutaraldehyde is 0.25 to 1% by weight.

Claim 13

A process according to claim 11, in which the tissue is soaked in 0.5% by weight glutaraldehyde solution in the presence of 0.1M acetate buffer for a period of about three and a half hours, after which excess glutaraldehyde is washed from the tissue.

Claim 14

A process according to claim 10, in which the glutaraldehyde-fixed tissue is treated with a calcification-inhibiting agent containing reactive amino groups for a time sufficient to bond substantially all the free reactive groups of the bonded glutaraldehyde molecules to the reactive amino groups of the calcification-inhibiting agent.

Claim 15

The process according to claim 13, in which the calcification-inhibiting agent is an amino diphosphonate.

Claim 16

A process according to claim 14, in which the amino diphosphonate is selected from compounds of the formula: ##STR4##

Claim 17

A process according to claim 15, in which the amino diphosphonate is 3-amino-1-hydroxypropane-1,1-diphosphonic acid of formula (1) and the tissue is soaked in fresh saturated solutions of said amino diphosphonate in distilled water at a pH of 8.0 for three hours per day in each fresh solution over a period of three days.

Claim 18

A process according to claim 16, in which the tissue is soaked in glutaraldehyde between each fresh soaking with amino diphosphonate.

Claim 19

A process according to claim 14, in which the tissue is further treated with 5 to 10 mg/ml of sodium borohydride to stabilize the bonding of the amino diphosphonate and glutaraldehyde to the protein molecules of the tissue.

Claim 20

A process according to claim 10, in which the glutaraldehyde-fixed tissue is treated with (i) an agent which inhibits infiltration and attack by phagocytic cells or (ii) an agent which inhibits infection.

Claim 21

A process according to claim 19, in which said agent (i) is a sporin antibiotic having a free reactive amino group or methotrexate and said agent (ii) is cephalosporin C.

Claim 22

A process according to claim 19, in which the tissue is treated with sodium borohydride to stabilize the bonding of agent (i) or agent (ii) to the treated tissue.

Claim 23

A process for the treatment of collagenous tissue to adapt it for use in a prosthetic implant and to promote the growth of endothelial cells thereon after implantation which comprises the sequential combination of the following steps: (1) contacting the tissue with at least one surfactant for a time sufficient to substantially completely remove deleterious material and open up the fibrous structure to form a matrix substantially free from lipids, red blood cells, plasma protein, organelles, and dead cell fragments; (2) rinsing the cleaned fibrous matrix resulting from step 1 with distilled water or saline solution to remove substantially all surfactant; (3) soaking said matrix in aqueous glutaraldehyde solution for a time sufficient to bond the glutaraldehyde molecules to substantially all the reactive amino groups present in the protein molecules of the tissue; (4) washing the glutaraldehyde-fixed tissue to remove excess glutaraldehyde; (5) treating the fixed tissue with an aqueous solution of amino diphosphonate containing reactive amino groups for a time sufficient to bond substantially all the free reactive groups of the bonded glutaraldehyde molecules to the reactive amino groups of the amino diphosphonate; (6) washing to remove excess amino diphosphonate; and (7) treating the diphosphonate-bonded tissue matrix with sodium borohydride to stabilize the bonding of the amino diphosphonate and glutaraldehyde to the protein molecules of the tissue; and (8) washing to remove excess sodium borohydride and, if desired, storing the resulting treated tissue in aqueous formaldehyde for subsequent use.

Claim 24

A process for the treatment of collagenous tissue to adapt it for use in a prosthetic implant and to promote the growth of endothelial cells thereon after implantation which comprises the sequential combination of the following steps: (1) contacting the tissue with at least one surfactant for a time sufficient to substantially completely remove deleterious material and open up the fibrous structure to form a matrix substantially free from lipids, red blood cells, plasma protein, organelles, and dead cell fragments; (2) rinsing the cleaned fibrous matrix resulting from step 1 with distilled water or saline solution to remove substantially all surfactant; (3) soaking said matrix in aqueous glutaraldehyde solution for a time sufficient to bond the glutaraldehyde molecules to substantially all the reactive amino groups present in the protein molecules of the tissue; (4) washing the glutaraldehyde-fixed tissue to remove excess glutaraldehyde; (5) treating the fixed tissue with an aqueous solution of amino diphosphonate containing reactive amino groups for a time sufficient to bond substantially all the free reactive groups of the bonded glutaraldehyde molecules to the reactive amino groups of the amino diphosphonate; (6) washing to remove excess amino diphosphonate; and (7) treating the diphosphonate-bonded tissue matrix with sodium borohydride to stabilize the bonding of the amino diphosphonate and glutaraldehyde to the protein molecules of the tissue; and (8) washing to remove excess sodium borohydride and, if desired, storing the resulting treated tissue in aqueous formaldehyde for subsequent use.

Claim 25

A process according to claim 22, in which the collogenous tissue is bovine or porcine pericardial tissue.

Claim 26

A process according to claim 22, in which the collagenous tissue is dura mater, fascia lata, valve tissue or vascular graft tissue.

Claim 27

A process according to claim 22, in which said collagenous tissue is pre-washed with isotonic saline solution to remove excess blood and plasma proteins prior to treatment with surfactant in step (1).

Claim 28

A process according to claim 22, in which step (1) is carried out with an aqueous solution containing 0.5 to 6% by weight of surfactant.

Claim 29

A process according to claim 26, in which said surfactant is a mixture of an anionic surfactant and a non-ionic surfactant.

Claim 30

A process according to claim 27, in which the anionic surfactant is 1% by weight sodium dodecyl sulfate and the non-ionic surfactant is 1% by weight octylphenoxy polyethoxy ethanol and/or 1% by weight polyoxyethylene (20) sorbitan monooleate.

Claim 31

A process according to claim 28, in which the collagenous tissue is contacted with said surfactant solution for three hours at room temperature.

Claim 32

A process according to claim 26, in which the surfactant is an amphoteric surfactant.

Claim 33

A process according to claim 22, in which step (3) is conducted in a solution having a glutaraldehyde concentration of 0.25 to 1% by weight.

Claim 34

A process according to claim 22, in which step (3) is conducted by soaking the collagenous tissue in 0.5% by weight glutaraldehyde in the presence of 0.1M acetate buffer for a period of about three and a half hours.

Claim 35

A process according to claim 22, in which the amino diphosphonate used in step (5) is selected from compounds of the formula: ##STR5##

Claim 36

A process according to claim 32, in which the amino diphosphonate is 3-amino-1-hydroxypropane-1,1-diphosphonic acid of formula (1) and the tissue is soaked in fresh saturated solutions of said amino diphosphonate in distilled water at a pH of 8.0 for three hours per day in each fresh solution over a period of three days.

Claim 37

A process according to claim 34, in which the tissue is soaked with glutaraldehyde between each fresh soaking with amino diphosphonate.

Claim 38

A process according to claim 22, in which step (7) is conducted with a solution having a concentration of sodium borohydride of 5 to 10 mg/ml.

Claim 39

A fixed and stabilized collagenous tissue adapted for use in a prosthetic implant produced by a process according to claim 22.

Claim 40

A storage-stable pack comprising a tissue according to claim 37 immersed in aqueous formaldehyde solution.