CA1221641A

CA1221641A - Implantable biological tissue and process for preparation thereof

Info

Publication number: CA1221641A
Application number: CA000423664A
Authority: CA
Inventors: Aws S. Nashef; Alain F. Carpentier; Sophie Carpentier
Original assignee: American Hospital Supply Corp
Current assignee: American Hospital Supply Corp; Edwards Lifesciences Corp
Priority date: 1982-03-23
Filing date: 1983-03-15
Publication date: 1987-05-12
Also published as: IT1161803B; DE3367067D1; FR2523810B1; US4647283A; FR2523810A1; IT8320226A0; EP0105290A1; JPH0321521B2; JPH0430868B2; JPS62270163A; WO1983003335A1; EP0105290B1; US4648881A; JPS59500613A

Abstract

ABSTRACT OF THE INVENTION

A process for the preparation of implantable biological tissue and in particular bioprosthetic heart valves, which are prone to calcification after implantation. The process includes treatment of tissue with a hypophosphated isotonic solution prior to implantation which has been found effective in reducing calcification of the implanted tissue. The solution contains a physiologically acceptable salt of a divalent cation other than calcium.

Description

lZZ1641 IMPLANTABLE BIOLOGICAL TISSUE AND PROCESS FOR
PREPARATION THEREOF
With the ;ntroduction of glutaraldehyde preservation of biological tissue, and in particular porcine bioprosthetic heart valves, it has become possible to:- a) overcome the poor performance of early formaldehyde-preserved implanted tissue valvesi b) discontinue the use of homograft valves; and c) avoid the undesirable use of anticoagulants required to prevent thromboembolism associa-ted with the use of non-bioprosthetic (mechanical) heart valves, especially in children. Not unlike other similarly important discoveries, however, it appears that the glutaraldehyde-preserved bioprosthesis has created its own dilemma.
Although the relatively biologically inert glutaraldehyde-preserved valves of Carpentier and others have demonstrated excellent long-term dura-F bility in most instances, serious drawbacks suchh a~ tissue-fatigue and a propensity towards calcification have plagued i~ continued use. Moreover, it was initially contemplated that children and adolescents would be among those deriving the greatest benefit from the glutaraldehyde-preserved bloprosth~tic heart valves since the anticoagulants required with mechani~cal pros~ couldbe eliminated. Results from an increasing number of recent clinical studies indicate that severe calcification of these tissues with relatively short-term failure is prevalent among children and adolescents. Thus, despite their long-term durability and overall reduced incidence of complications, these glutaral-dehyde-preserved valves have been deemed by some to be unsuitable for use in children.
Calcification of tissue remains a mystery for the most part; however, it has previously been shown that various factors including calcium metabolism diseases, age, diet, degeneration of tissue components such as collagen, and turbulance are all involved to a certain extent. Recently, the occurrence of a

2 ~zz~641 specific calcium-binding amino acid, laid down after implantat;on of glutaralde-hyde-preserved porcine xenografts, has been demonstrated; and it has been postulated to play a role in calcification. While calcification has been accompanied by degradative changes in the glutaraldehyde-treated collagen fibers of the implanted tissue, it remains unclear whether the dystrophic calcification is a cause or the result of tissue degeneration. Nevertheless, there has been a continued effort to elucidate the source of the calcification problem with implanted tissue, with the hope that a remedy would be soon to follow. Heretofore, neither the sourceorcause of calcification in biological ;mplants nor the appropriate measures to prevent or reduce calcification in biolog;cal implants have been ascertained.
In accordance with the present invention, we have determined an underlying cause of calcification with biological implants, and in particular with glutaraldehyde-preserved valvular bioprostheses. Furthermore, we havecon-currently developed procedures which effectively reduce or mitigate calcifi-cation of implanted biological tissue.
One of the underlying causes of calcification in valvular bioprostheses, noted for the first time in our immediate studies, is the presence of phosphate in contact with the tissue prior to implantation in amounts which sustain calcification after implanation. The levels of phosphate normally found in shipping media, such as balanced salt solutions (Hank's, etc.), plasma, and 0.01to 0.10M phosphate-buffered-saline (PBS) conventionally used in glutaraldehyde-fixing solutions, all sustained calcification in tissue to varying degrees. Here-tofore, the deleteribus effects of phosphate in contact with biological implanttissue have not been appreciated, and accordingly researchers, clinicians and manufacturers alike have been unaware of the undesirable consequences caused by their treatment of these implants with phosphate-containing solutions ;
particularly because phosphate solutions such as Hank's, PBS, and glutaralde-hyde-PBS are so commonly used and even highly recommended. It would there-for be understandable why unwittingly the interchangeability ofPBS and bicarbonate buffers (having similar buffering capacities and pH ranges) might have been recommended for tissue storage resulting in sporadic substi-tution for phosphate-containing media. Moreover, in some instances, even bicarbonate-buffered tissue storage media contained high levels of phosphate.
Since the deleterious consequences of maintaining tissue implants in contact .

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3 ~22~641 with phosphates were unknown, there was no deliberate intent on the part of clinicians or manufacturers of bioprostheses to avoid contacting the tissue with phosphate solutions.
In accordance with the present invention, we have developed processes which effectively reduce calcification of implanted biological tissue. These ~ccr~ advantageously reduce the tendency of bioprostheses towards calcifi-cation and overcome some of the problems associated with the durability of xenograft heart valves.
In accordance with the present invention, disclosed is an improved process for treating biological tissue prior to implantation which results in a mitigation or reduction of calcification thereof after implantationO In accor-dance with one embodiment,the process comprises contacting the tissue with a phosphate-deficient solution, the said solution having a level of phosphate de-creased to an amount effective in reducing calcification of said tissue after ;mplantation, said solution further being non-destructive or non-destabilizing to said tissue; and maintaining the tissue in contact with such a phosphate-deficient solution. In an alternate embodiment, the process comprises contac-ting the tissue with an amount of calcium-binding competing divalent cation effective in reducing calcification of tissue after implantation; and maintain-ing the tissue in contact with said cation.
In accordance with the present invention, it is contemplated that varioustypes of implantable biological tissue derived from numerous animal sources and parts of the anatomy can be made resistant to calcification. Thus, the tissue can be derived from, inter alia, bovine, porcine, horse, or rabbit; and can include tendons, ligaments, heart valves, or tissue used to construct heart valves such as dura mater and pericardium. It is further contemplated that tissue used for augme~tation such as skin patches, pericardial patches, aortic patches, and-tyYmpatlc membranes is suitable in the present invention. We have found, in accordance with the present invention, no significant difference in the rate of calcification between porcine pericardium and porcine aortic, tricuspid, and mitral valve tissue.
Although we are primarily concerned with tissue preparations which are fixed or tanned, such as glutaraldehyde-treated heart valves~ unfixed preserved tissue should benefit from our invention. In accordance with a preferred embod1rent o~ the present invention, ~*d6~ porcine heart valves or pericardi-. ~ .. . . ~ ~ .
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al tl~9ue fix~d ln glutaraldehyde, treated accordlng t~ th_ method Of the ptesent invention, and 8eparate1y implanted 8ubcutaneously ln rats and rabbit~, have unexpectedly ~nd beneficia11y demon8trated a 9u~talned mitigation or reduction of cnlci~ication of the biolo~ical ti~ue after implantatlon. Thi~
sustained mltigation of ca1clfication provide~ a method of 1ncreasing the durablllty of lmplanted ti9sue, partlcularly of ~eart valve b1Opro~theses.
In accordance with one embodiment of the present invention,.we have found that b;ological tissue maintained, prior to implantation, in a phosphate-deficient solution advantageously results in the sustained reduction or mitiga^
tion of calcification after implantation. In accordance with one embodiment of the present ~nvention, solut'ions characterized as phosphate-deficient are thosehaving levels of pi,osphate beiow a calcification-sustaining amount; that is, inan amount below the presently used level where no sign~ficant calcification reduction or mitigation is observed. Phosphate-deficient solutions are con-sidered to includethose~lav~llg substant~ally less p~lospl~ate t~lan the 0.01 to0.02M phosphate-buffered-saline solutions (PBS) ~onv~ntionally used for the preparation of tlssue prlor to lmplantatlon; and include solution~ whieh we have found effectlve ln reducing or mltlgating ca1c1f1cation after lmplantation.In a preferred enbodiment of the Pregent invention, these phosphate-deficient levels are below the phosp~late range normally found in plasma or balanced salt solutions such as Hank's, and Earle's which is from about 0.001 to about 0.00ZM.Furthermore, we find it most preferable to thoroughly rinse the blood from the host tissue after extraction in order to remove or substantially reduce any phosphate in the bloodfrom contact with the tissue.
Substantially phosphate-free solutions are those containing only trace amounts of phosphates; as in contaminat~ng amounts found in most chemicals used in the preparation of conventional tissue-treating solutions. We have found that HEPES buffer solutions, prepared in accordance with the present invention and further described below, contain in the`n'eighborhood of 2-4ppm phosphate-ion. Additionally, we ~ave found that some glutaraldehyde solutions prepared in accordance with the present ~nvention and further described below, contain in the neighborhood of 2-23ppm phosphate ion used as stabilizers by some manufacturers. We consider t~lese residual or trace amounts of phosphate to be inconsequential in accordance with the present ~nvention. Substànt~ally pllosphate-free solutions are the most preferred ~n accordance with the present invention.

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In addition to avoiding a calcification-sustaining phosphate erviron-ment for the tissue prior to implantation, it is preferable to employ solut-ions which are non destructive or non-destabilizing to the tissue. For example, we have found that heart valves fixed in 1% formaldehyde in water did not cal-cify after implantation in rats, whereas the same valves fixed in 1% formalde-hyde in PBS resulted in considerable calcification during the same period. The valves treated in the formaldehyde/water solution however, showed appreciable degenerationafterabout one month. The treatment of the valves in formalde-hyde/water,although in the absence of phosphate, adversely affect the stabili-zation of the tissue after implantation and should be avoided.
The phosphate-deficient solutions of the present invention include dis-t;lled water, buffer solutions, tissue-compatible compositions such as sodium chloride, saline, or combinations of these such as buffered-saline. In a preferred embodiment, the phosphate-deficient solution is unbuffered saline, and in a more preferred embodiment it is buffered saline. In all of these solutions, it is preferable to operate within a tissue-stabilizing pH range;
that is, within a pH range that ~ not deleterious to the tissue components. A
preferred pH range is from about 7.0 to about 7.6, and a more preferred pH
range is from about 7.1 to about 7.4. The most preferred pH in accordance with the present invention is 7.3.
Buffers used ir accordance with one embodiment of the present inven-tion are p~crfoctl~ s~able, non-interacting with the stabilization process, and have a buffering capacity sufficient to maintain an acceptable pH, particularly during fixation of the tissue. The choice of the appropriate buffer, and its concentration will depend upon specific tissue preparation conditions; varia-tions of which have been introduced by several manufacturers. Preferred buffers in accordance with the present invention include borate, carbonate, bic~ar~bonate, cacodylate (found to be non-toxic in animals) and other synthetic, artifical or organic buffers such as HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulph-onic acid; MOPS, morpholine propanesulphonic acid; and PIPES 1,4-piper-azinediethanesulphonic acid. We have found that tissue prepared in HEPES
buffer advantageously results in a significant reduction of calcification after implantation, and is therefore most preferred in the present invention.
In the present invent;on, the concentration of buffer in the phosphate-deficient solution is primarily chosen to maintain the tissue in a phosphate-:

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122i64~
deficient environment or effectively replace phosphate already present in the F tissue; while at the same time controlling the pH of the solution. It is important that ~ amount of this buffer, either alone or in combinatifon w;th solutions such as saline, be used that will effectively reduce~or maintain the immersed tissue in a phosphate-deficient environment. In a preferred embodi-ment, a buffer having a concentration of from about O.OO1 to about O.10M
HEPES is used. In a more preferred embodiment, a buffer having a concentra-tion from about 0.002 to about 0.050M HEPES is used. The most preferred buffer for glutaraldehyde-fixation is one having a concentration of about 0.02M HEPES.
Preferably, the buffered or unbuffered solutions, used in accordancewith the present invention should not interfere with the tissue stabilizing process afforded by fixing agents such as glutaraldehyde. That is, they should notreact with the fixing agent or preventthe fixing agent from achieving proper fixation of the tissue. Illustrative of this are buffers containing primary and second-ary amines such as tris(hydroxymethyl)aminomethane (Tris), which are known to react with the aldehyde groups of gluteraldehyde and thus interfere with the normal tissue stabilization process. Buffers such as Tris, although substanti-ally free of phosphate, adversely affect the stabilizati-on of the tissue afterimplantation and should therefore be avoided.
In accordance with one embodiment of the present invention, we have found that transient exposure of biological tissue to a phosphate-deficient or substantially phosphate-freesolution is ineffective in reducing calcification.
Unexpectedly, wehave found that, in one embodiment, the biological tissue must be maintained in a substantially phosphate-deficient solution from the time the phosphate is removed from contact with the tissue in any one of the various steps to a time immediately prior to implantation. For example, we have found that porcine tissue treated with either a phosphate-deficient ~ ; solution or a substantially phosphate-free solution after extraction and during 3 ~ shipping exhibits Jcvcrc calcification if calcification-sustaining phosphate solutions are used in the fixation and post-fixation stages of preparation. On the other hand, we have found that tissue treated with PBS, a calcification-sustaining solution, after extraction and during shipping will effect reduced calcification if substantially phosphate-free solutions are used in the fixationand post-fixation stages of preparation. Tissue treated with substantially :, :

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phosphate-free solutions after extract;on, dur~ng shipp~ng, during fixatlon, and post-fixation storage and sterili~ation are maximally effective in sus-taining reduced calcification.
~ In accordance with one embodiment of the present ~nvention, it is `` 5 preferred to mainta~n the biological tissue in a phosphate-deficient solution during the post-fixation period; that is from a time subsequent to fixation through storage and sterilization such as in formaldehyde, to a time immediate-ly ~rlrto implantation. It-~is more preferred to contact the biolog~cal tissue with a substantially p~losp~ate-deficient solution during fixation and maintain the tissue in contact wi.t~h;such a phosphate-deficient solution to a time in~ediately prior to implantatlan. ~refer~ly the phosphat~de~iclent solutlon 18 a component of the fixation solution. It is most preferred to contact the tissue. immediately after extraction with a phosphate-deficient solution; and maintain the tissue in a phosphate-deficient solution during shipping, fixation, and post-fixation storage and sterilization to a time immediately prior to implantation.
In a preferred embodiment, the tissue is contacted with a substantially phosphate-deficient solution during fixation wherein said solution is a compo-nent of the fixation solution. Buffered and unbuffered glutaraldehyde-saline solutions, preferably from about 0.2 to about 6.0 weight percent glutaraldehyde and most preferably about 0.5 to about 0.7 weight percent glutaraldehyde, are pre~erred in accordance with the present invention. A more preferred fixati~n solution comprises buffered-saline having from about 0.5 to about 07 wt% glutaral-dehyde. We have .f~und that a substantially phosphate-free fixation solution COIIIpriSing ~.02N ~IEPES-buffered-saline containing about 0.625 weight per-cent glutaraldehyde at a pH of from about 7.1 to about 7.5 is effective in reducing calcification after implantation and is therefore a most preferred embodiment of the present invention.
It is further contemplated that biological tissue, shipped and/or fixed in the presence of calcification-sustaining amounts of phosphate such as about 0.01-0.02M PBS, can be thoroughly rinsed or otherwise treated with a phosphate-deficient solution prior to implantation in order to remove phosphate there-from and thus effect a reduction or mitigation of calcification in the .
implanted tissue in accordance with the present invention. Rinsing or treating : implantable tissue containing a calcification-sustaining amount of phosphate prior to ~mplantat~on ~s n~st pre~erably accompl~shed witI~ a substantially .

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phosphate-free solution.
In accordance with an alternate embodiment of the present invention, we have unexpectedly found that biological tissue treated with divalent cations prior to implantation, and maintained in contact with said ions advantageously reduces or mitigates calcification of the tissue after implantation. The divalent ions added to the tissue are believed to effectively compete for the calcium-binding sites in the tissue; particularly after implantation when, ~n at least some instances, additional calcium-binding sites are generated. We have found that by increasing the amount of divalent ions such as magnesium ions in tissue after fixation in glutaraldehyde-PBS, we have effected a sustained reduction or mitigation of calcification; whereas ti6sue fixed in glutaraldehyde-PBS with increased amounts of magnesium ions and subsequently exposed to additional amounts of calcification-sustaining phosphate did not exhibit a reduction or mitigation of calcification. Thus, in accordance with the present invention, it is not considered necessary to maintain the divalent cation treated tissue in contact with a phosphate-deficient solution prior to or during treatment with the ion;however, it is preferred that the tissue be maintained in a ; phosphate-deficient solution after divalent ion treatment to a time prior to implantation.
; In accordance with the present invention, it is contemplated that any divalent ion which effectively competes for the calcium-binding sites in the tissue will reduce calcifica-tion. Accordingly, preferred divalent cations include barium, magnesium, strontium, copper, zinc, silver and mercury. We have found that tissue treated with barium, strontium and 30 magnesium ions prior to implantation effectively reduce calcifi-cation after implantation in accordance with the present invention.
~` In a preferred embodiment of the present invention the magneisum ion is derived from magnesium salt solutions;
35 most preferably water-soluble salt solutions such as magnesium chloride (MgC12), magnesium sulfate (MgSO4), and magnesium `- carbonate ~MgCO3). In the most preferred embodiment, the magnesium salt comprises magnesium ion in an amount effective in reducing or mitigating calcification of tissue after implant-40 ation. Although the concentration of the magnesium ion and the ,:~ - - , . - , ' ' - , .- , ' .

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~ZZ1641 g time it is in contact with the tissue can vary, we find it preferable that the tissue is substantially saturated with solutions containing effective amounts of this ion.
In accordance with the present invention, effective amounts of divalent cations such as magnesium ion in contact with the tissue are considered to be those amounts in excess of the quantities found in certain PBS, balanced salt solu-tions, and plasma. Conventional PBS solutions usually contain on the order of 0.001 weight percent magnesium ion; balanced salt solutions on the order of 0.002 weight percent; and the upper limit in plasma is in the order of about 0.003 weight percent. A preferred amount of magnesium ion is one that exceeds from about 0.003 to about 0.004 weight percent. The maximal amount of magnesium ion contemplated to be useful in the present invention is that level required to maintain an isotonic solution. We have found that saturating tissue with solutions containing about 0.03 percent magnesium ion has effected a reduction of calcification in tissue, and represents the preferred process. This was accomplished by immersing tissue in a 0.26 weight percent solution o-f magnesium chloride which affords a 0.03 weight percent solution of magnesium ions.
Solutions such as balanced salt solutions, having more than about 0.03 percent magnesium ion normally found in plasma would have a similar beneficial effect and would be preferred in accordance with the present invention.
In accordance with one embodiment of the present invention, we have found that extracted porcine valvular tissue shipped in HEPES-buffered-saline fixed in HEPES-buffered-saline containing 0.25 weight percent magnesium chloride and 0.625 weight percent glutaraldehyde, rinsed in HEPES-buffered-saline containing 0.26 weight percent magnesium chloride, sterilized in HEPES-buffered-saline containing 0.26 weight percent magnesium chloride and about 4.0 weight percent formaldehyde, rinsed and stored in HEPES-buffered-saline containing 0.26 weight percent magnesium chloride and 0.625 weight percent glutaraldehyde, until immediately prior to implantation advantageously ~nd significantly reduces or mitigates calcification of the tissue and implantation. Thus, in accordance with one embodiment of the present invention, it is most preferred to glutaraldehyde-fix the biological tissue in a - ~:
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- 9a -HEPES-buffered-saline solution containing an effective amount of magnesium, and maintain the solution in contact with said HEPES-buffered-saline solution and magnesium ion to a time immediately prior to implantation.
It is further contemplated, in accordance with the present invention, that biological tissue processed in the absence of effective amounts of divalent cations such as magnesium can be rinsed in sterile solutions, such as magnesium chloride, immediately prior to implantation to effect a reduction of calcification.
The present invention is further illustrated by the following examples which are not intended to be limiting.

Extracted porcine aortic heart valve tissue was thoroughly rinsed, shipped, fixed with 0.625 weight percent glutaraldehyde, sterilized in about 4~ formaldehyde, stored at about 4 to 25C, all in the presence of a phosphate-deficient isotonic (285+15 milliosmols) solution containing 0.885 weight percent sodium chloride at pH 7.3, rinsed in sterile saline to remove '' ~

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lO ~2Z1641 residual glutaraldehyde at a time immediately prior to implantation, and implanted in growing rabbits. The valve tissue was retrieved up to six weeks later at regular one-week intervalsO After retrieval, the extent of tissue calcification was assessed by quantitatively monitoring the weight percent calcium in dried tissue using atomic absorption analysis; and histologically by visually monitoring the degree of calcification in Von Kossa-stained tissue sections. The extent of calcification was simultaneously and identically assessed for heart valve tissue which was rinsed, shipped, fixed with 0.625 weight percent glutaraldehyde, sterilized in about 4% formaldehyde, stored at about 4 to 25C all in the presence of an isotonic solution containing 0.02M
phosphate, 0.885 weight percent sodium chloride at pH 7.3 (0.02M PBS), rinsed in sterile saline to remove residual glutaraldehyde at a time immediately prior to implantation, and implanted in growing rabbits.
Both the histologic and quantitative results indicate that the implanted valve tissue treated with the phosphate-deficient solution effected a significant reduction in calcification compared to the valvue tissue treated with 0.02M PBS.EXAMPLE II
Experiments identical to those of example I were conducted with the exception that the tissue was implanted in mature rabbits and retrieved up to six months later at regular one-month intervals. Likewise, bothfhi~stologic and quantitative results indicate that implanted valve tissue~with the '~ phosphate-deficient solution effected a significant reduction in calcification compared to the valve tissue treated with 0.02M PBS.
EXAMPLE III
Experiments identical to those of Example II were conducted with the exception that the phosphate-deficient solution further contained 0.54/grams/liter of the sodium salt of HEPES during rinsing and shippingi and 5.39 grams/liter of the sodium salt of HEPES during fixation, storage, and sterilization. Again, both histologic and quantitative results indicate that theimplanted valve tissue treated with the phosphate~deficient solution effected a reduction in calcification compared to the valve tissue treated with 0.02M PBS.
EXAMPLE IV
` Experiments identical to those of Example I were conducted with the exceptionthat the phosphate-deficient solutions further contained 0.54 grams/liter of the sodiuln salts of HEPES during rinsing and shipping; 5.39 :

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11 1;~21641 grams/liter of the sodium salt of HEPES during fixation, storage and sterili-zation; and 2.6 grams/liter of MgCl2 6H20 in the fixation, storage, and steri-lization solutions. Again, both histologic and quantitative results indicate that implanted valve tissue treated with the phosphate-deficient solution effec-ted a significant reduction in calcification compared to the valve tissuetreated with 0.02M PBS.
EXAMPLE V
Experiments identical to those of Example V were conducted with the exception that the tissue was implanted in mature rabbits and retrieved up to six months later at regular one-month intervals. Both histologic and quanti-tative results indicate that implanted valve tissue treated with the phosphate-deficient solution effected a significant and sustained reduction in calci-fication compared to the valve tisue treated with 0.02 M PBS.
EXAMPLE VI
Experiments identical to those of Example I were conducted with the exception that the phosphate-deficient solution further contained 0.05M
cacodylate buffer, and the valvewas retrieved up to only two weeks later at weekly intervals. Furthermore, the extent of calcification was simultaneously and identically assessed usin~ 0.012M phosphate, 0.885 weight % sodium 20F chloride at pH 7.3 (0.012M ~ ) prior to implantation. The histologic resultsindicate that the implanted valve tissue treated with phosphate-deficient solution effected a slight reduction in calcification after one week compared to the valve tissue treated with 0.012M PBS. Thereafter, mild calcification was observed.
EXAMPLE VII
r Experiments identical to those of Example VI were conducted with the exception that 0.1M borate buffer was substituted for cacodylate. The histo-logic results were similar to those in Example VI.
XAMPLE VIII
Experiments identical to those of Example IV were conducted with the exception that neither solution for comparison was phosphate-deficient; that is, the fixation, storage and stèrilization solutions wcrc su~^~alÇnqcd 0,02M PBS
instead of HEPES buffer. Both histologic and quantitative results indicate that the implanted valve tissue treated with the phosphate-containing magnesium chloride solution effected a reduction in calcification compared i ' ..

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to the valve tissue treated without magnesium chloride.
EXAMPLE IX
Experiments identical to those of Example III were conducted in order to assess the integrity of the tissue after inplantation. The results of our analysis indicate that there was no significant difference in: shrinkage temperature;
Ninhydrin values; leachable uronic acid in an acid mucopoly-saccharide leach; the stability of the tissue subjected to pronase digestion; histological staining; amino acid analysis;
ultrastructure as assessed by transmission electron microscopy;
or moisture content.
EXAMPLE X - XXII
Extracted procine heart valve tissue was interchange-ably treated with phosphate-deficient solutions and phosphate-containing solutions at various stages of processing (pre-fixing glutaraldehyde-fixation, and post-fixation) prior to implantation in rats in order to assess the degree of calcification mitigation afforded by each stage of treatment. The results in Table I
summarize our findings after up to two months in some cases after implantation. In the table, Gp represents glutaraldehyde-treated tissues in 0.02M PBS; FH O represents formaldehyde-treated tissue in water; GWater represe~ts glutaraldehyde-treated tissue in water; Fp represents formaldehyde-treated tissue in 0.02M PBS:
GHEpES represents glutaraldehyde-treated tissue in 0.002M HEPES;
GCarbonate represents glutaraldehyde-treated tissue in carbonate buffer; saline/HEPES represents 0.002M HEPES-buffered-saline; and BSS represents balanced salt solution.
EXAMPLE XXIII
Experiments identical to those of Example IV were conducted with the exception that MgC12 6H2O was replaced with BaC12-2H2O at a concentration of 2.2 grams/litre. The results indicate that barium had an effect on calcification similar to magnesium.
EXAMPLE XXIV
Experiments identical to those of Example IV were conducted with the exception that MgC12 6H2O was replaced with SrC12-2H2O at a concentration of 2.3 grams/litre. The results indicate that strontium had an effect on calcification similar to magnesium.
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CALCIFICATION STUDIES IN nA'l'S

Treatment Degree of Calcification Pre-Tanning Tanning Post-Tannlng PBS Gp phosphate severe PBS F phosphate-def icient reduced H20 (degeneration) PBS F phosphate severe P (degeneration) PBS water phosphate-def icient reduced PBS GHEPES phosphate-def icient reduced PBS Gcarbonate phosphate-deficient reduced Hank's G phosphate severe (BSS) P
Saline/HEPES Gp phosphate severe Hank's Gsaline/HEPES phosphate-def icient reduced (BSS) PBS Gsaline/HEPES phosphate-def icient reduced Saline/HEPEs GS~ine/HEPES phosphate-deficient (sustained) PBS G MgCl-phosphate reduced P de~lcient ~sustained) PBS Gp MgCl2-Gp severe The present invention has been described in specific detail and in reference to its preferred embodimentsi however, it is to be understood by those skilled in the art that modifications and changes can be made thereto without departing from the spirit and scope thereof.

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Claims

1. A process for treating biological tissue prior to implantation of said tissue in a human or animal, comprising contacting said tissue, during a step in the preparation thereof, with a phosphate-deficient solution which is effective in reducing calcification of said tissue after implantation, and maintaining said tissue in a phosphate-deficient solution until immediately prior to implantation of said tissue.

2. The process of claim 1, wherein said solution is substantially phosphate-free.

3. The process of claim 1, wherein said tissue is maintained in said solution during the post-fixation period.

4. The process of claim 1, wherein said tissue is contacted with said solution during fixing of said tissue and is maintained in said solution during the post-fixation period.

5. The process of claim 1, wherein said tissue is contacted with said solution immediately after extraction from an animal source, and is maintained in said solution until immediately prior to implantation.

6. The process of claim 1, wherein said tissue is derived from an animal source selected form the group consisting of bovine, porcine, horse, or rabbit source.

7. The process of claim 1 or 6, wherein said tissue is selected from the group consisting of tendons, ligaments, heart valves, tissue used to construct heart valves, dura mater, pericardium, skin patches, aortic patches, and tympanic membranes.

8. The process of claim 1, wherein said solution comprises a buffer.

9. The process of claim 8, wherein said buffer is selected from the group consisting of borate, carbonate, bicarbonate, cacodylate, HEPES, MOPES, or PIPES.

10. The process of claim 9, wherein said solution comprises from about 0.001 M to about 0.10M HEPES.

11. The process of claim 10, wherein said solution comprises from about 0.002M to about 0.050M HEPES.

12. The process of claim 1, wherein said solution comprises saline.

13. The process of claim 1, wherein the pH of said solution is within a tissue-stablizing pH range.

14. The process of claim 11, wherein said pH is between about 7.0 and 7.6.

15. The process of claim 12, wherein said pH is between about 7.1 and about 7.4.

16. The process of claim 1, wherein said tissue is fixed by contact with said phosphate-deficient solution, said solution additionally comprising glutaraldehyde as a fixing agent.

17. The process of claim 16, wherein said solution comprises saline and from about 0.2 to about 6.0 weight percent glutaraldehyde.

18. The process of claim 17, wherein said solution comprises from about 0.5 to about 0.7 weight percent glutaraldehyde.

19. The process of claim 17 or 10, wherein said solution additionally comprises a buffer, wherein said buffer is substantially non-reactive with said fixing agent.

20. The process of claim 1, wherein said tissue additionally is contacted with an amount of a divalent cation effective in reducing calcification of said tissue after implantation, and is maintained in said phosphate-deficient solution from the time of contact with said divalent cation until immediately prior to implantation of said tissue.

21. The process of claim 20, wherein said divalent cation is a magnesium ion.

22. The process of claim 21, wherein said tissue is contacted with an amount of magnesium ions that exceeds about 0.004 weight percent, up to a concentration required to maintain an isotonic solution.

23. The process of claim 21, wherein said tissue is contacted with magnesium ions derived from a solution containing a magnesium salt.

24. The process of claim 23, wherein said magnesium salt is selected from magnesium chloride, magnesium sulfate, or magnesium carbonate.

25. The process of claim 24, wherein said tissue is substantially saturated with the solution containing said magnesium salt.

26. The process of claim 25, wherein the solution containing said magnesium salt comprises about 0.03 weight percent of magnesium ions.

27. The process of claim 20, wherein said tissue is maintained in contact with said divalent cation from the time of initial contact with said cation until immediately prior to implantation of said tissue.

28. Implantable biological tissue treated according to the process of claim 1.

29. The tissue of claim 28, wherein said tissue is derived from an animal source selected from the group consisting of bovine, porcine, horse, and rabbit sources.

30. The tissue of claim 29, wherein said tissue is selected from the group consisting of tendons, ligaments, heart valves, tissue used to construct heart valves, dura mater, pericardium, skin patches, aortic patches, and tympanic membranes.