CA1261290A

CA1261290A - Process for preparing biological mammalian implants

Info

Publication number: CA1261290A
Application number: CA000543031A
Authority: CA
Inventors: Petr Klement; Gregory J. Wilson; Herman Yeger
Original assignee: Hsc Research Development Corp
Current assignee: Hsc Research Development Corp
Priority date: 1986-07-28
Filing date: 1987-07-27
Publication date: 1989-09-26
Also published as: GB8618374D0; US4776853A

Abstract

ABSTRACT OF THE DISCLOSURE

A process for preparing biological material for implant in mammal's cardiovascular system, respiratory system or soft tissue is disclosed. The process comprises:
1) isolating from a suitable donor a desired tissue sample of the biological material;
2) extracting the tissue sample with an hypotonic buffer solution at a mild alkaline pH, the buffer solution including active amounts of proteolytic inhibitors and antibiotics;
3) extracting the tissue sample with a buffered solution having a high concentration of salt, the solution being at a mild alkaline pH and including a non-ionic detergent with protease inhibitors and antibiotics;
4) subjecting tissue sample to enzymatic digestion in a buffered saline solution, the enzymes consisting of purified protease-free deoxyribonuclease and ribonuclease;
5) extracting the tissue sample with an anionic detergent at a mild alkaline pH; and 6) storing the tissue sample in physiologic buffered solutions.

Description

~2612~) PROCESS FOR PREPARING

FIELD OF THE INVENTION
This invention relates to implantable prostheses for replacement of mammalian tissue including arteries, veins, valves and the like, and more particularly to prostheses made from biological material.
BACKGROUND OF TH~ INVENTION

Over the past three decades, numerous different types of vascular prostheses have been produced for the replacement of arteries and veins. Reasonable success has been achieved with large caliber vascular prostheses (greater than 6 mm internal diameter) using man-made polymers, notably Dacron and Teflon in both knitted and woven configurations. Expanded polytetrafluoroethylene grafts have, to date, been the most successful of commercially available vascular prostheses in smaller caliber (4-6 mm) configurations, but it is fair to say that further improvements are necessary to produce a small caliber vascular prosthesis which can really challenge autologous saphenous vein for overall efPicacy in both the peripheral vascular and coronary locations.
Biological va~cular grafts represent the most effective alternative de~ign pathway. With few exceptions, biological prostheses have been produced by methods which incorporated either proteolytic enzyme digestion followed by aldehyde fixation or aldehyde fixation alone. In some cases, ~urther ~urface modifications were performed with a variety of chemicals to modify surface charge. The objectives were to stabilize tissues by crosslinking the proteinaceous components and to alter favorably the thrombogenic properties of the vessels. In all instances where aldehyde fixation was used the resultant vessel was altered considerably with respect to its mechanical properties. Fixation, however, appeared to render these ve6sels less antigenic. In the case of heart valves, previously ~ixed with aldehydes, further treatment with an anionic detergent, sodium dodecyl sulfate, :~26~29~

substantially reduced the tendency of these devices to undergo calcification as disclosed in United States patent 4,323,358. Inhibition of calcification has also been attempted by incorporating a variety of inhibitors such as diphosphonates or chondroitan sulfates, as disclosed in United States patents 4,378,224 and 4,553,974. Both non-ionic and ionic detergents have been used in other ways, for example, to clear tissues of soluble materials prior to further treatment with aldehydes.
None of the treatments described above provided ~uperior performance to that which would be achieved in blood vessel replacements, with artificial entirely man-made materials (eg. polytetrafluoroethylene). In contrast, biological valve replacements have an appreciably better record of performance than prostheses from synthetic materials. A new approach is based upon the realization that the extracellular matrix (which is produced by connective tissue cells, the major cell component of vessels) provides the vessel with inherent mechanical properties, forms a highly integrated and dense network of croselinked fiber~ and iB essentially resistant to extraction by detergents and physiological solutions.
The advantages of retaining intact this extracellular matrix, composed primarily of a collagenous component but al80 including elastin and other tightly bound substance6, ha~ been explored by Klaus, B. and Duhamel, R. (W0 84/0488)) for the production of sterile body implants. In their method, a variety of tissues were extracted sequentially with non-ionic and ionic detergents to yield structures essentially free of cellular membranes, nucleic acids, lipids and cytoplasmic components. Dependent upon the particular application, these structures were further modified by fixation and/or sur~ace modi~ication. In the case of canine carotid arteries, treated with their protocol, they achieved acceptable results after 90 days post-implantation.

~26~29~) Healthy arteries or veins should provide material most suitable for the replacement of damaged vessels in terms of biological and mechanical properties.
An extraction protocol has been developed which provides substantially improved biological protheses which are highly biocompatible and long lasting replacements. The biological prosthesis is equivalent in compliance and mechanical strength to a healthy vessel through retention of elastic properties and highly resistant to calcification and thrombogenesis and hence in most situations, avoids the need to administer anti-thrombosis drugs.
~ccordingly, the invention removes soluble small and high molecular weight substances from natural tissue which will be used as the prosthesis while retaining the insoluble, collagenous and elastic "backbone" of the natural tissue. The tissue is extracted by a series of detergent and non-proteolytic enzymatic treatments.
SUMMARY OF THE INVENTION
According to an aspect of the invention, a process for preparing biological material for implant in a mammalian cardiovascular system, respiratory system or ~oft tissue by removing cellular membranes, nucleic acid~, lipids and cytoplasmic components and forms an extra cellular matrix having as major components collagens and elastins. The process comprises the following steps:
1) isolating from a suitable donor a desired tissue sample of the biological material;

2) extracting the tissue sample with a hypotonic buffer solution at a mild alkaline p~ for rupturing cells of the tissue sample, the hypotonic buffer solution including active amounts of proteolytic inhibitors and active amounts of antibiotic;

3) extracting the tissue sample with a buffered solution having a high concentration of salt, the solution being at a mild alkaline pH and including a non-ionic detergent and protease inhibitors and phenylmethyl-~1 sulfonylfluoride and active amounts of antibiotic;

4) subjecting the tissue sample to enzymatic digestion in a buffered saline solution, said enzymes consisting of purified protease-free deoxyribonuclease and ribonuclease;

5) extracting the tissue sample with an anionic detergent at a mild alkaline pH, and

6) storing the processed tissue sample in physiologic saline.
BRIEF DESCRIPTION OF THE D~ WINGS
Preferred embodiments of the invention are shown in the drawings, wherein:
Figure 1 is a view of unprocessed iliac artery examined with H and E staining;
Figure 2 is a view o~ the iliac artery of Figure 1 processed in accordance with this invention and observed with H and E staining; and Figure 3 is a graph comparing physical properties of fresh and processed artery.
~E~IT~ RTP~TON OF THE PREFERRED EMBO~IMENTS
This invention iB based upon the rationale that healthy arterie~ and veins and other relevant tissue should provide the ideal replacement vessel since they would be a structure most biologically equivalent to the damaged vessel in terms of size, shape, mechanical propertie~ and biochemistry. The components o~ the ve~sels that are nearly equivalent are the interstitial collagens, elastin and glycosaminoglycan6. All other components including cell membranes, cytoplasm, nuclear material and serum components could initiate an immunological rejection response and, therefore, necessitate complete removal. These latter components contribute only in a minor way to the mechanical properties of the vessel, 80 that, if the collagenous and elastic fraction can be retained intact and in its natural state, the mechanical properties would be essentially the same as the normal vessel. Because such ~L26~ 90 a processed vessel could be recellularized by host connective tissue cells, dlle to its biomatrix properties, it has the potential to provide optimal patency, healing characteristics and durability.
In the procedure, normal healthy vessels are surgically removed, cleaned to remove extraneous matter and immediately submersed in the first extraction solution which consists of a hypotonic buffer containing suitable protease inhibitors and antibiotics. The vessels are subsequently enclosed within a nylon or polyester mesh, so as to form a chamber, and extracted with the first solution, as above, for an additional 24 to 48 hours within a cylinder, and stirred at 2 to lO~C. The hypotonic condition allows the extraction to be initiated since cells are ruptured. In the second ~tep, the vessels are subjected to a buffered high salt ~olution containing a non-ionic detergent, protease inhibitors and antibiotics. This step is also carried out for 24 to 48 hours at 2 to 10C under stirring.
Both the high salt and detergent permit extraction of cytoplasmic components and soluble extracellular matrix components, but keep the rest of the extracellular matrix in an intact state. The carrying out of these steps at the lower temperatures inhibits auto-proteolysis. This high salt/detergent step is followedby several distilled water washes and equilibration with a buffered saline solution for the enzymatic portion o~
the procedure. The enzymes used are a mixture of purified, protease free, deoxyribonuclease and ribonuclease since both are required to remove nuclear material. It is also the advantage of such a combination that entrapped foreign agents such as bacteria and viruses would also be susceptible to these enzymes. This enzyme digestion is carried out with reciprocal shaking at pre~erably 37C (range 35~ to 38~C) for typically 2 to 12 hours, but more usually 4 to 6 hours, dependent on vessel size. In the last step of the extraction protocol, the vessels are extracted with an anionic detergent at alkaline pH. This anionic æ~2~

detergent extraction is carried out at ambient temperatures with stirring for 24 to 48 hours including a change to fresh solution appro~imately halfway through. After the anionic detergent step, the vessels are washed under stirring with large ~olumes of distilled water and finally stored in physiologic saline containing antibiotics.
It is appreciated that a variety of protease inhibitors and antibiotics may be used. Possible lo protease inhibitors include phenylmethylsulfonyl-floride, diisopropyl phosphofluoridate, ethylene-diaminetetraacetic acid, ethylene glycol-bis(~-amino-ethylether)N,N,N'N'-tetreaacetic acid and N-ethylmale-imide. The working range of these inhibitors is from 1 ~M to 25 mX dependent upon inhibitor used. The pre~erred inhibitors are ethylenediaminetetraacetic acid and phenylmethylsulfonyl~luoride. Useful gram (+ve) and gram (-ve) or broad spectrum antibiotics include ~ynthetic and semi-~ynthetic penicillins, streptomycin, gentamycin and cephalosporins. The preferred antibiotics are penicillins and streptomycin.
According to a preferred embodiment of this invention, the non-ionic detergent may be selected from the ~ollowing group TRITON X-100 (trademark), am octylphenoxy polyethoxyethanol, manu~actured by Rohm and Haa~; BRIJ-35 (trademark), a polyethoxyethanol lauryl ether, manu~actured by Atlas Chemical Co.; TWEEN 20 (trademark), a polyethoxyethanol sorbitan monolaureate, manu~actured by Rohm and Haas; and LUBROL-PX
(trademark), a polyethylene lauryl etller, manufactured by Rohm and Hass.
Suitable anionic detergents include those selected ~rom the group consisting of a salt of a sulfated higher aliphatic alcohol, sulfonated alkane and sul~onated alkylarene containing from 7 to 22 carbon atoms in a bran~hed or unbranched chain. The pre~erred anionic detergent is ~odium dodecyl sulphate.
It is appreciated that, when desired, suitable cro~slinking agents may be used to induce crosslinking ~6~2~

in the resulting collagen and elastin of the treated tissue. Suitable crosslinking agents include glutaraldehyde, carbodiimide and polyglycerol polyglycidyl ether.
It is appreciated that the treatment of this invention may be applied to a variety of sources of suitable tissue extracted from appropriate donors, including bovine, ovine, caprine, porcine and human sources. The tissue samples may include veins, arteries, pericardium, dura mater, ligaments, tendons, trachea and skin. Such components, when treated, may be u~ed for prostheses to replace arteries, veins, heart valves, ligaments and tendons, trachea and skin.
Ve~sel6 extracted by the above procedure were analyzed histologically, biochemically and mechanically prior to implantation. The morphology of the extracted vessel reveals the presence of a dense meshwork of collagen and elastin fibers, as seen in non-extracted vessels, but an absence of the cytoplasmic component and the soluble extracellular matrix component (ground substance). Biochemical analysis reveals the retention o~ collagen, ela~tin and glycosaminoglycans related to that ~ound in basement membranes while glycosaminoglycans of ground substance have been removed. The lumenal aspect o~ the vessel, as seen by scanning electron microscopy, shows a smooth membrane-like sur~ace, corresponding to a residual basement membrane, subtended in profile, by a prominent fibrillary component, corresponding to the internal lamina and underlying collagen fibers. The presence of the basal lamina components type IV collagen and laminin on the lumenal surface was confirmed immuno-histochemically. Mechanical te~ting showed that non-extracted and extracted ve~sels were virtually identical. The extracted sample had a slightly lower ~tif~ne~s at a given stre~s, and 81ightly increa8ed hysteresis losses. These differences were well below the expected experimental accuracy. Similar results ~2612~

were obtained for carotid and iliac artery samples (canine).
Our processed biological vascular grafts implanted as grafts in the femoral and carotid artery sites (15 implants in 8 dogs) as well as the abdominal aGrta (1 dog) were evaluated for follow-up periods ranging from 3 to 1361 days, with further follow-up continuing on three of the grafts. All implants were processed canine carotid or iliac arteries with the exception of the aortic interposition graft which was processed aorta.
The implants at femoral and common carotid positions were performed with end-to-side anastomoses using grafts o~ 5 to 8 cm in length and 5 to 6 mm in diameter with the host artery ligated near each anastomosis and divided at the end of the procedure. The dogs used were screened with a range of biochemical and hematological tests so that normal, healthy subjects were chosen. No anticoagulation was employed at any time nor were anti-platelet medications given before, during or after implantation. Graft patency was assessed by palpation and/or angiography. Explantation was scheduled to provide graft~ for examination over a range of follow-up intervals, balancing the need for long term follow-up to assess continued patency and the biological fate of the implants with the need for sequential morphological assessment of these implants. Each graft was opened longitudinally, photographed and submitted ~or svaluation by microscopy, using the same techniques as applied to samples of the biografts prior to implantation.
The following exemplifies preferred embodiments of the invention in preparing vascular grafts and their biological properties. It is appreciated that, although the following examples are directed to vascular grafts, the processes of this invention are equally useful in preparing variou~ type o~ mammalian implants derived from natural sources, which include arteries, veins, heart valves and the like.

~26~1~

Example 1 - Details of Extraction Process as Used with Arteries 1. A variety of arteries have been treated: femoral, iliac, carotid, aortic.
5 2. The vessels are resected and cleaned of adhering connective tissues and debris prior to extraction.
3. Cleaned vessels are immediately placed into the first extraction solution called Solution A, which consists of: 10 mM Tris.HCL, 5 mM EDTA at pH 8.0 supplemented with 50 U/ml penicillin/streptomycin combination tstock of 10,000 U/ml penicillin and lo,OOo ~g/ml streptomycin GIBC0) and 1 ~M PMSF
(phenylmethylsulfonyl fluoride - an antiproteolytic agent). Extraction is carried out with vessels enclosed in Nitex envelopes in a cylinder at 5C, with stirring for 24 hours (24 hours to 48 hours).
4. The vessels are placed into the second extraction solution, Solution B consisting of: 50 mM Tris.HCL, 1.5 M KCL, 1% Triton X-100 (a non-ionic detergent), 5 mM
20 EDTA at pH 8.0 supplemented with 1 ~M PMSF and 50 U/ml penicillin/streptomycin as in Solution A. Extraction is carried out with vessels in Nitex envelopes in a cylinder at 5C with stirring for 24 hours (range of 24 to 72 hours). The volume ratios of solutions A or B to tissue are a minimum of 100:1.
5. The vessels are washed three times in 100:1 volumes of either purified (Milli Q~system 0.2 u filtered water or same water after autoclaving) water and then for 30 minutes to 1 hour in Hanks buffered salt solution 30 (GIBC0) containing 10 mM Hepes buffer and 50 U/ml penicillin/streptomycin at 37 with rocking.
6. The vessels are treated enzymatically as follows:
The vessels are transferred to solutions containing .75 mg/15 ml DNase I ~Type III - Sigma) and RNase (Type lA -35 Sigma) 1.25 mg/15 ml and roc~ed for 4 to 6 hours at 37C.

7. The vessels are washed briefly one time in purified water for 30 minutes or transferred directly to solution C.
~ r~ade,"l~rl~

10 ~90

8. The vessels are mounted in Nitex~envelopes and extracted with Solution C consisting of 50 mM TRIS.HCl at pH s.o with 1% SDS (sodium dodecyl sulphate) for 24 hours (range 24 to 96 hours) at ambient temperature.
5 9. The vessels are washed in >loo:l volumes of water or saline at least three times over 24 hours (range 24 to 96 hours).
lo. The vessels are stored in either Han~s buffered salt solutlon with Hepes (lo - 25 mM) and penicillin and streptomycin or in phosphate buffered saline with the same antibody at 4C. The penicillin and streptomycin concentration is raised to 100 U/ml and 100 ~g/ml respectively.
The extraction procèdure is initiated by hypotonic 15 ly6i8 of the tissue cells. Antibiotics are included from the onset of the process. No cell poisons, such as azide are used in this process which is initiated by the hypotonic lysis of the tissue cells. A high salt, non-ionic detergent combination is used to extract a substantial proportion of the cytoplasmic components.
The high salt solution generally includes a salt concentration in the range of 1 to 2 Molar of the de~ired salt. In accordance with this Example, the preferred salt is potassium chloride at 1 to 2 Molar, usually 1.5 M. This type of salt will not precipitate in colder solutions at the higher concentrations. It is known from cultured cell work that this combination i6 gentle and leaves behind a cell cytoskeleton but completely permeabolizes the cell. A combined use of DNase and RNase is used under physiologic conditions to remove nuclear material, both enzymes being used together to provide an effective removal.
Exam~le 2 ~orpholoqv: To assess the cellularity of the graft and the presence of other extractable components, #tandard histological methods were employed.
Unprocessed and processed vessels were fixed in ~ormaldehyde, paraffin embedded and samples stained with hematoxylin and eosin (H and E) for general k ll histology and with the van Gieson stain for the discrimination of collagen and elastin. To assess the topography of the endothelial side of the graft, grafts were examined with scanning electron microscopy (SEM) using a standard method of preparation. Briefly, tissues were fixed in glutaraldehyde, dehydrated, critical point dried and gold coated and examined in a JEOL 35S~scanning electron microscope.
A photomicrograph of a processed vessel such as a lo processed aorta revealed a highly uniform intimal sur~ace. H and E staining of a carotid artery ~unproce~ed) revealed the presence of a normal media rich in smooth muscle cells a6 shown schematically in Figure 1. The tiesue sample 10 consists of predominantly smooth muscle cells 12, each haing a nucleus 14. The cells are surrounded by collagen bundle6 and elastic laminae 18. Alternatively, a Van Gieson stain has revealed a prominent array of elastic laminae and a moderate amount of collagen interspersed between the elastic laminae. After processing and staining with H and E stain, Figure 2 shows ~chematically that the treated sample 20 has the cellular material removed, leaving behind the collagen 16 and elastin 18. Figure 2 shows the intact organization o~ these ~tructures and the appearance of a more open architecture. Scanning electron microscopy of the intima of a processed artery has revealed that the endothelial covering ha~ been removed, leaving behind a substratum which represents the basement membrane. The presence o~ two basement membrane components, type ~V
collagen and laminin, was demonstrated by immunofluorescent labelling with specific antibodies.
The SEM also revealed the high degree o~ fibrillar complexity which is present in natural vessel~, ¢onsisting o~ fine and cour~e collagen fibers interwoven with ela~tin.
~Xp~ple 3 BiochemistrY: In order to determine the extent to which collagen, elastin and inter~titial ground I ~dc,~a~

substance (hyaluronic acid, etc.) has been removed during the extraction, vessels were analyzed with biochemical methods. Unprocessed and processed vessels were treated as follows:
A) Collagen - the acid pepsin soluble fraction was analyzed.
B) Elastin - the fraction remaining after alkali extraction was analyzed.
C) Matrix (soluble connective tissue) - no further extraction required, the entire samples were analyzed.
For all three analyses, samples were hydrolyzed in hydrochloric acid and the content of hydroxyproline for (A ~ B~ and N-acetylglucosamine indicative of hyaluronic acid of ground substance and N-acetylgalactosamine indicative of tightly bound glycosaminoglycans of basement membranefor lÇL determined. Analyses were performed with a Beckman Amino Acid Analyzer.
E-Aminocaproic acid was used as the internal standard.
The results are expressed per mg dry weight of vessel.
Arteries before and after processing are compared for their content of collagen, elastin and ground substance in Table 1. The results show: 1) no change in the elastin content; 2) a minimal and insignificant difference in collagen content: and 3) a significant loss of ground ~ubstance in processed vessels as indicated by the decrease of N-acetylglucosamine ~GlcNac) content. GlcNac is a major component of hyaluronic acid. Loss of glycosaminoglycans would be expected as they are highly soluble. This data demonstrates that processed vessels are essentially free of physiologically soluble substances and cells but retain their network of crosslinked collagen and elastin fiberc.

~2~

Vessel Before After 5 com~onent Processing Processing Collagen 303 ~g/mg 329 ~g/mg Elastin 559 ~g/mg 647 ~g/mg Glyco6aminoglycans - GlcNac 38.16 nM/mg 0.046 nN/mg - GalNac 17.86 nM/mg 15.24 nM/mg Units Nanomolar (nM) per mg dry weight vessel sample.
Microgram (~g) per mg dry weight vessel sample.
It is noted that at least 10% of the dry weight of ve6sels prior to processing reflects the cellular component which is missing from the processed vessels.
Examle 4 Mech~nical: In this test, stress-strain curves were obtained for unprocessed and processed vessels as follows: Five mm wide circumferential strips were washed three times in Hanks physiological solution and mounted on an Instron TT-~ Universal Testing Machine such that each sample could be stretched between the two grips and the resulting load and extension measured.
The information was digitized on a Tektronix~computer and converted to stress-strain data. Sample dimensions for this analysis were obtained by photographic recording o~ sample gauge length and mean width and by thickness measurement with a Mitutayo non-rotating thickness gauge. Strip volume was assumed to be constant throughout the test. Final stress-strain curves were plotted by computer~
Figure 3 illustrates a typical stress-strain curve for comparison of fresh and extracted aorta. The stress-strain curve~ obtained for the fresh and extracted ~amples were virtually identical. The extracted sample ~howed very slightly lower stiffnes~ at a given stress, and slightly increased hystere~is losses. These differences were well below the expected ~ Tr~ er~ k 3~

experimental accuracy. Therefore, in low strain testing, the extraction procedure did not significantly affect mechanical performance of the vessel wall.
Similar results were obtained for carotid and iliac artery samples.
Exam~le 5 In-Vivo Evaluation A summary of the status at explanation or on continued follow-up of the 16 grafts is presented in Table 2. Only one of the grafts was occluded with thrombus and this occlusion occurred during the first week, as assessed by palpation, although the graft was not explanted until 185 days post-implantation because of the presence of a patent graft in the contralateral femoral artery.
Of the 15 grafts patent on angiographic evaluation, 13 which were explanted at follow-up intervals of 3 to 484 days and confirmed by direct observation to be widely patent with minimal mural thrombus.
The aortic interposition graft was mainly of interest in regard to possible aneurysm formation.
Angiography at three years follow-up revealed no aneurysm formation. All explanted grafts were free of aneurysm formation up to 434 days ~ollow-up and two femoral position grafts evaluated angiographically at 125 day~ and still being followed up after 856 days are free of aneurysm formation.
Preliminary histological evaluation of the explanted grafts revealed no in~lammatGry response of the new host to the graft and no calcification.

SUMMARY OF 16 HSC-PBVG IMPL~NTED IN g pOGS
5 Side of Graft Follow-Up Patency Implantation Days RFA 3 Patent 10 LCCA 19 Patent RFA 19 Patent RFA 72 Patent RFA 104 Patent LFA lo~ Patent 15 RCCA 121 Patent RFA 186 Occluded*
LFA 186 Patent LFA 196 Patent RFA 196 Patent 20 LCCA 301 Patent LFA 434 Patent LF~ 856+ +Patent RFA 856+ +Patent Aorta 1361+ +Patent RFA - right ~emoral artery LFA - le~t ~emoral artery RCCA - right common carotid artery LCCA - left common carotid artery *Occlusion during the first week post implantation + animal i8 alive - long term follow-up According to this invention, a procedure i~
provided for the manu~acture o~ processed biolcgical vascular and other biological material gra~ts in which a segment of artery or the like is rendered ~ree of soluble macromolecular components which would be expected to induce an immune reaction in the new host yet the normal mechanical properties of the tissue segment are retained through preservation of the elastin and collagen framework.
Accordingly, the invention provides, vascular grafts of acceptable characteristics as determined by morphological, biochemical and mechanical testlng techniques and preliminary in vivo testing performed through the implantation o~ 16 vascular biografts in 9 dogs, 15 o~ which have been 5 to 6 mm in diameter implanted in the carotid or ~emoral locations. The results are conclusive regarding the invention's utility 3~

with only one early thrombotic occlusion, no aneurysm formation with follow-up of up to three years and no graft calcification or inflammation detected on histological examination at follow-up to 434 days.
Although preferred embodiments of the invention have been described herein in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OF PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for preparing biological material for implant in mammal's cardiovascular system, respiratory system or soft tissue by removing cellular membranes, nucleic acids, lipids and cytoplasmic components and forming an extracellular matrix having as major components collagens and elastins, said process comprising the following sequence of steps:
1) isolating from a suitable donor a desired tissue sample of said biological material;
2) extracting said tissue sample with a hypotonic buffer solution at a mild alkaline pH for rupturing cells of said tissue sample, said hypotonic buffer solution including active amounts of proteolytic inhibitors and active amounts of antibiotic;
3) extracting said tissue sample with a buffered solution having a high concentration of salt, said solution being at a mild alkaline pH and including a non-ionic detergent and the proteolytic inhibitors and active amounts of antibiotic;
4) subjecting said tissue sample to enzymatic digestion in a buffered saline solution, said enzymes consisting of purified protease-free deoxyribonuclease and ribonuclease;
5) extracting said tissue sample with an anionic detergent at a mild alkaline pH, and 6) storing said tissue sample in physiologic buffered solutions.

2. The process of claim 1, wherein said extraction solution of steps 2) and 3) include the antibiotics penicillin and streptomycin.

3. A process of claim 1, wherein said extraction solution of steps 2), 3) and 5) are each at a mild alkaline pH in the range of 7.5 to 9.5.

4. A process of claim 1, wherein said suitable donor is human, bovine, ovine, porcine or caprine.

5. A process of claim 4, wherein said tissue sample is selected from the group consisting of veins, arteries, pericardium, dura mater, ligaments, tendons, trachea and skin.

6. A process of claim 5, wherein said biological material for implant is used for prostheses in vascular grafts, heart valves, replacement ligaments and tendons, trachea and skin.

7. A process of claim 1, wherein said non-ionic detergent of step 3) is selected from the group consisting of octylphenoxy polyethoxyethanol, polyethoxyethanol lauryl ether, polyethoxyethanol sorbitan monolaureate and polyoxyethylene lauryl ether.

8. A process of claim 7, wherein said selected non-ionic detergent is octylphenoxy polyethoxyethanol.

9. A process of claim 1, wherein said anionic detergent is selected from the group consisting of a salt of a sulfated higher aliphatic alcohol, sulfonated alkane and sulfonated alkylarene containing from 7 to 22 carbon atoms in a branched or unbranched chain.

10. A process of claim 9, wherein said anionic detergent is sodium dodecyl sulfate.

11. A process of claim 1, wherein said high salt concentration in said solution of step 3) is in the range of 1 to 2 M of a desired physiological acceptable salt.

12. A process of claim 11, wherein said desired salt is KCl.

13. A process of claim 1, wherein said extraction solution of steps 2) and 3) include the protease inhibitors ethylenediaminetetraacetic acid and phenylmethylsulfonylfluoride.

14. A process of claim 3, wherein said extraction solution of steps 2) and 3) include the protease inhibitors ethylenediaminetetraacetic acid and phenylmethylsulfonylfluoride.

15. A process of claim 1, wherein steps 2) and 3) are carried out at a temperature in the range of 2° to 10°
C.