CA1247007A - Treatment of collagenous tissue - Google Patents
Treatment of collagenous tissueInfo
- Publication number
- CA1247007A CA1247007A CA000488543A CA488543A CA1247007A CA 1247007 A CA1247007 A CA 1247007A CA 000488543 A CA000488543 A CA 000488543A CA 488543 A CA488543 A CA 488543A CA 1247007 A CA1247007 A CA 1247007A
- Authority
- CA
- Canada
- Prior art keywords
- tissue
- glutaraldehyde
- surfactant
- agent
- treatment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/44—Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/02—Treatment of implants to prevent calcification or mineralisation in vivo
Abstract
TREATMENT OF COLLAGENOUS TISSUE
Abstract 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 comprising treatment with at least one surfactant prior to fixation, treatment with agents which inhibit calcification, agents which resist attack by phagocytic cells and reducing agents.
Abstract 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 comprising treatment with at least one surfactant prior to fixation, treatment with agents which inhibit calcification, agents which resist attack by phagocytic cells and reducing agents.
Description
~2~
TREATMENT OF COLLAGENOUS TISSUE
This invention relates to a process for the treatment of collagenous tissue to render it suitable for use in prosthetic implants, and to the resulting tissue so treated. More particularly, the invention is concerned with a process for the treatment of collagenous tissue to adapt it to be used in a prosthetic implant and to promote the growth of endothelial cells thereon.
Prosthetic implants for use in humans have been known for some time and it also has been known to use natural tissue taken from animals including humans.
When natural tissue is used in an implant it is necessary to treat it to avoid problems after implant-ation, for example excessive mineralization or calcification and rejection bv the body's immune system.
Numerous treatments for improving the stability of prosthetic devices made from natural tissue have been proposed in the prior art.
Thus U.S. Patent No. 4,378,224 issued March 29, 1983 to Nimni et al discloses a process for improving the biophysical stability of bioprostheses for hetero-graft or allograft implantation made from animal tissue involving the formation of crosslinks in the protein structure of the tissue using the known cross-linking agent glutaraldeh,vde and soaking the tissue in an aqueous solution of a calcification inhibitor.
., .
Exampies of suitable calcification inhibitors mentioned by Nimmi et al are diphosphonates and 3-amino-1-hydro xypropane l,1-diphosphonic acid is mentioned as a typical diphosphonate, although no specific Example illustrating the use of this compound is given by Nimni et alO
Furthermore, although Nimni et al refer to harvesting and cleaning of the tissue prior to the glutaraldehyde treatment, there is no suggestion of any pretreatment with an appropriate surfactant to remove, substantially completely, deleterious material present in the tissue. Accordingly, the coating (column 2 line 23) provided by Nimni et al is essentially a surface phenomenon and crosslinking with glutaraldehyde and stabilization with the calcification inhibitor throughout the fibrous matrix of the tissue is not and can not be achieved by the Nimni procedure.
V.S. Patent No. 3988782 issued November 2, 1976 to Dardik et al discloses the preparation of prostheses in the form of tubes, patches and conduits from arteries and veins o~ umbilical cords using glutaraldehyde as a hardening agent.
U.S. Patent No. 3966401 issued June 29, 1976 to Hancock et al discloses the preparation o~ an implantable heart valve from porcine pericardial tissue in which the tissue is treated with glutaraldehyde as a tanning agent.
The inhibitory effect of various diphosphonates on aortic and kidney calcification in vivo is discussed in an article by M. Potokar and M. Schmidt-Dunker appearing in Atherosclerosis, 30 (1978) 313320.
7~
U.S. Patent No. 41206~9 issued Octo 17, 1978 to Schechter discloses the treatment of transplants with glutaraldehyde to enhance the retention time in the recipient. As in Nimni et al, supra, the treatment is essentially a surface treatment and no additional stabilization or like treatment is disclosed.
U.S. Patent No. 3562820 issued February 21, 1971 to ~raun, discloses the hardening with glutaraldehyde of tubular, strip and sheet form prostheses based on biological tissue.
U.S. Patent No. 4098571 issued July 4, 1978 to Miyata et al discloses a process for preparing a heteograft substitute blood vessel which comprises treating a pig blood vessel with a proteolytic emzyme to digest unwanted material and retain collagenous and elastic fiber constituents and then fixing the resulting blood vessel with, inter alia, a mixture of form-aldehyde and glutaraldehyde.
U.S. Patent No. 4323358 issued April 6, 1982 to 20 Lentz et al discloses treatment of a glutaraldehyde-fixed animal tissue with a solution of a watersoluble salt of a sulfated higher aliphatic alcohol, such as sodium dodecyl sulfate, allegedly to inhibit calcification of the tissue after implantation.
Although all of the above prior art proposals have some degree of success~ for example by inhibiting calcification to some extent and improving the bio-physical stability of prosthetic implants to some extent~
problems in these areas still remain. ~urthermore, 30 none of the aforesaid prior art disclosures express any recognition of the importance of promoting and enhancing the growth of endothelial cells on the surfaces of prosthetic implants.
7~
The endothelium is a layer of flat cells lining various cavities within the body, in particular blood vessels. The lining of endothelial cells provides a smooth surface so that blood cells and platelets can flow without being damaged. Endothelial cells are capable of producing and secreting substances with a variety of actions and the actions occuring at the blood endothelial interface contribute towards the well being of the organism as a whole; for example, the intact endothelium is nonthrombogenic because both circulating blood cells and the endoth~lial surface have a negative charge and thus repel each other. Each endothelial cell is closely linked to its ad~acent cells and the endothelial layer forms a selectively permeable membrane which resists the passive transfer of the fluid and cellular phases of blood.
While the intact endothelium acts as a primary barrier against the leakage of blood it also provides a prima facie indication to the body's immune system that foreign materials are not present, at least outside the blood vessels. However, if the endothelium is damaged, punctured or broken this automatically induces a response by the immune system which defends against foreign pathogens~ The immune system, which is generally capable of discriminating between self and foreign antigens, operates through a complex assortment of lymphocytes and phagocytic cells whose activities are adapted to produce a coordinated protective response to foreign pathogens. Thus, among the phagocytic cells involved in the immune system, white blood cells or leukocytes function primarily to defend the body against microorganisms. Another important group of phagocytes is the macrophages which are widely distributed throughout the body and act in concert with other phagocytes associated with the linings of blood vessels, i.e. the endothelium, in, for example, the bone marrow, liver, spleen and lymph nodes.
A more detailed description of the immune system is not considered necessary for a full understanding of the present invention, but recognition of the role played by endothelial cells is important for an appreciation of the improvement provided by the invention over the prior art.
Surprisingly, it has now been found that by performing the process of the present invention and, in particular, ensuring substantially complete removal of deleterious material from collagenous material by the essential initial step of said process, the ln vivo growth of endothelial cells upon prosthetic implants formed from tissue treated by the invention process is promoted. In addition to a marked improvement in the inhibition of mineralization or calcification.upon implantation, this permits the formation of implants which are less susceptible to rejection by the body's immune system than any produced by prior art procedures.
In accordance with the invention there is provided 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 L.~L~7~
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.
Collagen is a fibrous protein which occurs in - 15 vertebrates as the primary constituent of connective tissue fibrils. There are seven different types of collagen and type I is generally used for implants.
Fibrous animal tissue normally contains collagen in association with other proteinaceous material, particularly elastin. As used herein the term collagenous tissue is intended to mean collagen itself, particularly type I collagen, mixtures of collagen and elastin and animal tissues containing a significant proportion of collagen with or without elastin or other proteinaceous material. An essential requirement of the collagenous tissue to be used in the invention is that the protein molecules thereof contain free amino groups adapted to react with fixing or tanning reagents such as glutaraldehyde.
The preferred collagenous tissue is bovine pericardial tissue or porcine pericardial tissue. Such tissue is particularly suitable for the formation of the tissue leaflets in prosthetic heart valves~
particularly those made in accordance with the teachings of Ionescu et al U.S. Patent No. 4388735 issued ~une 21, lg83.
Other suitable forms of collagenous tissue which may be treated by the process of the invention are dura mater, fascia lata, valve tissue and vascular graft tissue.
Collagenous tissue, for example pericardial tissue, as it is initially removed from an animal requires cleaning to free it from unwanted contaminants. Usually the tissue is washed with sterile isotonic saline solution to remove excess blood and plasma proteins, and this conventional prewashing is a desirable initial step before performing the essential step according to the process of the invention of contacting tissue with at least one surfactant for a time sufficient to substantially completely remove deleterious material.
As used herein the term deleterious material is intended to mean material which blocks or clogs the fibrous matrix of the collagen or collagen/elastin tissue to be used as a prosthetic implant, which material, if not removed, would provide sites to initiate an immune response in the host organism with 25 consequential rejection of the implant or at least attack by host phagocytes. Deleterious material includes lipids, including lipoprotein and phospho-lipids, red blood cells, plasma protein, organelles and dead cell fragments, as well as free fatty acids, cholesterol, cholesterol esters and triglycerides.
The removal of deleterious material by the surfactant treating step of the invention opens up the fibrous structure of the collagenous tissue and this enables the glutaraldehyde used in the subsequent ~%~ 7 fixing step to substantially completely infiltrate the fibrous matrix of the collagenous tissue so that the reactive groups on the glutaraldehyde molecules bond to the free amino groups on the protein molecules of the collagenous tissue throughout the matrix.
Thus the surfactant treatment of the present invention serves the double purpose of, firstly, enabling the fibrous matrix of collagenous tissue to be thoroughly fixed throughout the matrix rather than 1~ merely on the surface; and, secondly, deleterious material is removed from the interstices of the fibrous matrix and is no longer present to be entrapped below the surface by the subsequent fixing step and to be available to present problems of rejection or attack by host phagocytes upon implantation.
Prior art procedures which have advocated treatment with surfactants after the fixing step, for example U.S.
Patent No. 4323358 supra, are substantially ineffective for removing deleterious material which is effectively bonded to the tissue by the fixing agent.
It has been found that the particular sequence of steps according to the present invention provides a significant improvement in terms of implant retention over the prior art.
The surfactant used in the surfactant treating step of the invention is a potent agent for removing deleterious material from animal tissue and care must be taken not to overdo the cleaning action and thereby damage the base tissue by using too strong a solution.
On the other hand, the concentration of surfactant and the period of treatment must be sufficient to achieve the desired result of substantially completely removing the deleterious material. Within these criteria it is preferred to use the surfactant in the form of an ~2~
aqueous solution containing 0~5 to 6% by weight of surfactant. A suitable treatment time is from two to six hours, preferably about three hours.
The surfactant may be an anionic surfactant, a non-ionic surfactant, an amphoteric surfactant or a mixture thereof.
Examples of suitable anionic surfactants are sodium dodecyl sulfate, sodium dodecyl sulfoacetate and sodium salt of alkaryl polyether sulfonate. Examples of suitable non-ionic surfactants are octylphenoxy polyethoxy ethanol (Triton X-100), polyoxyethylene (20) sorbitan monooleate (Tween 80), and polyoxyethylene (20) sorbitan monostearate (Tween 60). Examples of suitable amphoteric surfactants are sulfobetaines commonly known as Zwittergents.
It has been found that particularly advantageous results are obtained if the surfactant is mixture of an anionic surfactant and a nonionic surfactant; and a particularly preferred surfactant solution is one in which the anionic surfactant is 1% by weight sodium dodecyl sulfate and the nonionic surfactant is 1% by weight octylphenoxy polyethoxy ethanol and/or 1% by weight polyoxyethylene (20) sorbitan monooleate.
Preferably the collagenous tissue is contacted with said surfactant solution for about three hours at room temperature.
The surfactant is not only a potent cleansing agent but also a potential toxin and, accordingly, it is an important feature of the invention that, after the surfactant treatment, the fibrous matrix of collagenous tissue is thoroughly washed to remove sub-stantially all surfactant. This washing step may be conducted in any conventional manner, for example with 7~
saline solution or distilled water, and, to ensure sub-stantially complete removal of surfactant, the washing is continued until the formation of bubbles ceases.
After the above described treatment with surfactant and the washing step to remove substantially all trace of surfactant 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 ln the protein molecules of khe tissue. A suitable time for the fixation step is from two to twelve hours and substantially complete fixation is achieved preferably by the repeated soaking procedure described hereinafter. Preferably, the concentration of glutaraldehyde is 0.25 to 1~ by weight.
Fixation of animal tissue with glutaraldehyde to improve its characteristics and render it adaptable o for prosthetic implants is known in the art and this step, in and of itself, is not claimed to be inventive.
However, the special contribution provided by the invention with ~egard to this step is twofold:
Firstly, the soaking with glutaraldehyde is carried out only after deleterious material has been substantially completely removed from the collagenous tissue by the surfactant treatment; thus ensuring that the fibrous matrix is adapted to be fixed throughout, rather than merely on its surfaces.
Secondly, substantially complete fixation throughout the fibrous matrix is ensured by soaking the tissue in the glutaraldehyde for a time sufficient to bond the reactive groups on the glutar---11~
~2~7~
aldehyde molecules to substantially all the reactive amino groups present in the protein molecules of the tissue.
The described result is preferably achieved by repeated soakings in glutaraldehyde to effect multiple cross-linking in accordance with the procedure described hereinafter. The need for repeated soakings, not only in glutaraldehyde to effect multiple cross-linking, but also in calcification-inhibiting agents and anti-phagocytic agents, as described hereinafter, to achieve the cumulative saturation effect provided by the process of this invention has not been achieved in the prior art.
According to a preferred embodiment of the invention the tissue is soaked in 0.5~ by weight glutaraldehyde solution in the presence of 0.1 M
acetate buffer for a period of about three and a half hours. Subsequently, excess glutaraldehyde is washed from the tissue.
An important aspect of the invention is the inhibition of calcification on prostketic implants formed from tissue treated in accordance with the process of the invention.
It has been found that the presence of phosphate ions tends to increase the occurrence of calcification and accordingly the use of phosphate buffers in the steps of the inventive process is to be avoided, notwithstanding the efficiency of the intermediate washing steps.
Since control of pH is an important feature during the process, such control preferably is achieved with a nonphosphate buffer, preferably an acetate buffer.
The tissue fixed with glutaraldehyde is further treated in accordance with the invention firstly with a calcification inhibiting agent and/or an agent which inhibits infiltration and attack by phagocytic cells -12~
`` ~2~ 7 upon implantation and finally with a reducing agent which stabilizes the molecular bonds of the resulting agent/matrix ti~sue.
Mineralization, or more particularly calcification, on and around tissue implants after implantation results in reduced flexibility of the tissue and therefore decreased efficiency in the operation of the prosthesis in the host body. Various treatments have been proposed in the prior art to inhibit or reduce calcificat on and these have met with some degree of success. In particular, the use of specific compounds to inhibit calcification is known in the art. However, the special application of known calcification inhibitors, especially amino diphosphonates, in accordance with the proeess of the present invention results in a substantial improvement over prior art treatments and unexpected advantages in areas not investigated in prior art procedures.
Thus, prosthetic implants made from collagenous tissue treated in accordance with -the process of the present invention are found to be effective in resisting not only calcification but also thrombosis, infection and degeneration. These advantageous charaeteristics, which are exhibited to a degree substantially greater than that achieved in the prior art, are attributable to the fact that endothelial cell coverage on the implant is encouraged and the promotion of such coverage protects the implant from the reactions leading to thrombosis, calcification, infection and degeneration.
The stated improvement is attained by the particular combination of steps described above in which the agent used in the further treatment of the fixed tissue, as well as a calcification agent, may be an agent which inhibits infiltration and attack -13~
~2~
by phagocytic cells, for example, a sporin antibiotic having a free reactive amino group or methotrexate;
or an agent which inhibits infection, preferably cephalosporin C.
The said sporin antibiotic is derived from cyclosporin A, a known immunosuppresive drug having a molecular weight of 1202 and the structural formula illustrated in Figure 7 of the accompanying drawings.
For use as a treating agent in the process of the present invention the cyclopsorin A ring is opened at the position indicated by the arrow in the formula, to provide a free amino group for reaction with the free reactive groups on the glutaraldehyde molecules attached to the fixed tissue.
Methotrexate, or N-[4-[[(2,~-diamino-6-pteridinyl)methyl]methylamino]benzoylJ-L-glutamic acid is a known folic acid antagonist and antimetabolite having the formula:
N N NH
fOOH ~ / ~
HOOCCH2CH2CHNHC \ ~ H2 y > _ NCH 2 ~ I
This drug has two free amino groups available for reaction with the reactive groups on the glutaraldehyde-fixed tissue matrix.
Cephalosporin C i5 a known potent inhibitor of infection.
~.2~
For convenience ln terminology, the calcification-inhibiting agents and the immuno-suppresive agents and drugs used in the post-fixing step of the process according to the invention are referred to hereinafter by the generic term "drug" and, under this terminology, the desired effect produced by the process may be termed "drug immobilization" or "immunosuppression".
One of the advantageous results achieved by the drug immobilization provided by the process of the invention is an effective balance between:
(1) the encouragement or promotion of endothelial cell coverage;
t2) the enhancement of would healing; and (3) inhibition of rejection and attack by macrophage and other phagocytes.
A preferred application for tissue subjected to drug immobilization by the process of the invention is in the produc~ion of prosthetic heart valves whQrein the leaflets and sewing ring are formed from the treated 2n tissue. A particularly preferred embodiment of the tissue is produced when the drug is an amino diphosphonate calcium inhibitor and it has been found that prosthetic heart valves made from the preferred embodi-ment conform to the above balance in that examination of the prosthesis some two months after implantation shows:
1) Substantially complete endothelial cell coverage over the leaflets and sewing ring and no evidence of thrombosis, calcification, infection or degeneration;
TREATMENT OF COLLAGENOUS TISSUE
This invention relates to a process for the treatment of collagenous tissue to render it suitable for use in prosthetic implants, and to the resulting tissue so treated. More particularly, the invention is concerned with a process for the treatment of collagenous tissue to adapt it to be used in a prosthetic implant and to promote the growth of endothelial cells thereon.
Prosthetic implants for use in humans have been known for some time and it also has been known to use natural tissue taken from animals including humans.
When natural tissue is used in an implant it is necessary to treat it to avoid problems after implant-ation, for example excessive mineralization or calcification and rejection bv the body's immune system.
Numerous treatments for improving the stability of prosthetic devices made from natural tissue have been proposed in the prior art.
Thus U.S. Patent No. 4,378,224 issued March 29, 1983 to Nimni et al discloses a process for improving the biophysical stability of bioprostheses for hetero-graft or allograft implantation made from animal tissue involving the formation of crosslinks in the protein structure of the tissue using the known cross-linking agent glutaraldeh,vde and soaking the tissue in an aqueous solution of a calcification inhibitor.
., .
Exampies of suitable calcification inhibitors mentioned by Nimmi et al are diphosphonates and 3-amino-1-hydro xypropane l,1-diphosphonic acid is mentioned as a typical diphosphonate, although no specific Example illustrating the use of this compound is given by Nimni et alO
Furthermore, although Nimni et al refer to harvesting and cleaning of the tissue prior to the glutaraldehyde treatment, there is no suggestion of any pretreatment with an appropriate surfactant to remove, substantially completely, deleterious material present in the tissue. Accordingly, the coating (column 2 line 23) provided by Nimni et al is essentially a surface phenomenon and crosslinking with glutaraldehyde and stabilization with the calcification inhibitor throughout the fibrous matrix of the tissue is not and can not be achieved by the Nimni procedure.
V.S. Patent No. 3988782 issued November 2, 1976 to Dardik et al discloses the preparation of prostheses in the form of tubes, patches and conduits from arteries and veins o~ umbilical cords using glutaraldehyde as a hardening agent.
U.S. Patent No. 3966401 issued June 29, 1976 to Hancock et al discloses the preparation o~ an implantable heart valve from porcine pericardial tissue in which the tissue is treated with glutaraldehyde as a tanning agent.
The inhibitory effect of various diphosphonates on aortic and kidney calcification in vivo is discussed in an article by M. Potokar and M. Schmidt-Dunker appearing in Atherosclerosis, 30 (1978) 313320.
7~
U.S. Patent No. 41206~9 issued Octo 17, 1978 to Schechter discloses the treatment of transplants with glutaraldehyde to enhance the retention time in the recipient. As in Nimni et al, supra, the treatment is essentially a surface treatment and no additional stabilization or like treatment is disclosed.
U.S. Patent No. 3562820 issued February 21, 1971 to ~raun, discloses the hardening with glutaraldehyde of tubular, strip and sheet form prostheses based on biological tissue.
U.S. Patent No. 4098571 issued July 4, 1978 to Miyata et al discloses a process for preparing a heteograft substitute blood vessel which comprises treating a pig blood vessel with a proteolytic emzyme to digest unwanted material and retain collagenous and elastic fiber constituents and then fixing the resulting blood vessel with, inter alia, a mixture of form-aldehyde and glutaraldehyde.
U.S. Patent No. 4323358 issued April 6, 1982 to 20 Lentz et al discloses treatment of a glutaraldehyde-fixed animal tissue with a solution of a watersoluble salt of a sulfated higher aliphatic alcohol, such as sodium dodecyl sulfate, allegedly to inhibit calcification of the tissue after implantation.
Although all of the above prior art proposals have some degree of success~ for example by inhibiting calcification to some extent and improving the bio-physical stability of prosthetic implants to some extent~
problems in these areas still remain. ~urthermore, 30 none of the aforesaid prior art disclosures express any recognition of the importance of promoting and enhancing the growth of endothelial cells on the surfaces of prosthetic implants.
7~
The endothelium is a layer of flat cells lining various cavities within the body, in particular blood vessels. The lining of endothelial cells provides a smooth surface so that blood cells and platelets can flow without being damaged. Endothelial cells are capable of producing and secreting substances with a variety of actions and the actions occuring at the blood endothelial interface contribute towards the well being of the organism as a whole; for example, the intact endothelium is nonthrombogenic because both circulating blood cells and the endoth~lial surface have a negative charge and thus repel each other. Each endothelial cell is closely linked to its ad~acent cells and the endothelial layer forms a selectively permeable membrane which resists the passive transfer of the fluid and cellular phases of blood.
While the intact endothelium acts as a primary barrier against the leakage of blood it also provides a prima facie indication to the body's immune system that foreign materials are not present, at least outside the blood vessels. However, if the endothelium is damaged, punctured or broken this automatically induces a response by the immune system which defends against foreign pathogens~ The immune system, which is generally capable of discriminating between self and foreign antigens, operates through a complex assortment of lymphocytes and phagocytic cells whose activities are adapted to produce a coordinated protective response to foreign pathogens. Thus, among the phagocytic cells involved in the immune system, white blood cells or leukocytes function primarily to defend the body against microorganisms. Another important group of phagocytes is the macrophages which are widely distributed throughout the body and act in concert with other phagocytes associated with the linings of blood vessels, i.e. the endothelium, in, for example, the bone marrow, liver, spleen and lymph nodes.
A more detailed description of the immune system is not considered necessary for a full understanding of the present invention, but recognition of the role played by endothelial cells is important for an appreciation of the improvement provided by the invention over the prior art.
Surprisingly, it has now been found that by performing the process of the present invention and, in particular, ensuring substantially complete removal of deleterious material from collagenous material by the essential initial step of said process, the ln vivo growth of endothelial cells upon prosthetic implants formed from tissue treated by the invention process is promoted. In addition to a marked improvement in the inhibition of mineralization or calcification.upon implantation, this permits the formation of implants which are less susceptible to rejection by the body's immune system than any produced by prior art procedures.
In accordance with the invention there is provided 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 L.~L~7~
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.
Collagen is a fibrous protein which occurs in - 15 vertebrates as the primary constituent of connective tissue fibrils. There are seven different types of collagen and type I is generally used for implants.
Fibrous animal tissue normally contains collagen in association with other proteinaceous material, particularly elastin. As used herein the term collagenous tissue is intended to mean collagen itself, particularly type I collagen, mixtures of collagen and elastin and animal tissues containing a significant proportion of collagen with or without elastin or other proteinaceous material. An essential requirement of the collagenous tissue to be used in the invention is that the protein molecules thereof contain free amino groups adapted to react with fixing or tanning reagents such as glutaraldehyde.
The preferred collagenous tissue is bovine pericardial tissue or porcine pericardial tissue. Such tissue is particularly suitable for the formation of the tissue leaflets in prosthetic heart valves~
particularly those made in accordance with the teachings of Ionescu et al U.S. Patent No. 4388735 issued ~une 21, lg83.
Other suitable forms of collagenous tissue which may be treated by the process of the invention are dura mater, fascia lata, valve tissue and vascular graft tissue.
Collagenous tissue, for example pericardial tissue, as it is initially removed from an animal requires cleaning to free it from unwanted contaminants. Usually the tissue is washed with sterile isotonic saline solution to remove excess blood and plasma proteins, and this conventional prewashing is a desirable initial step before performing the essential step according to the process of the invention of contacting tissue with at least one surfactant for a time sufficient to substantially completely remove deleterious material.
As used herein the term deleterious material is intended to mean material which blocks or clogs the fibrous matrix of the collagen or collagen/elastin tissue to be used as a prosthetic implant, which material, if not removed, would provide sites to initiate an immune response in the host organism with 25 consequential rejection of the implant or at least attack by host phagocytes. Deleterious material includes lipids, including lipoprotein and phospho-lipids, red blood cells, plasma protein, organelles and dead cell fragments, as well as free fatty acids, cholesterol, cholesterol esters and triglycerides.
The removal of deleterious material by the surfactant treating step of the invention opens up the fibrous structure of the collagenous tissue and this enables the glutaraldehyde used in the subsequent ~%~ 7 fixing step to substantially completely infiltrate the fibrous matrix of the collagenous tissue so that the reactive groups on the glutaraldehyde molecules bond to the free amino groups on the protein molecules of the collagenous tissue throughout the matrix.
Thus the surfactant treatment of the present invention serves the double purpose of, firstly, enabling the fibrous matrix of collagenous tissue to be thoroughly fixed throughout the matrix rather than 1~ merely on the surface; and, secondly, deleterious material is removed from the interstices of the fibrous matrix and is no longer present to be entrapped below the surface by the subsequent fixing step and to be available to present problems of rejection or attack by host phagocytes upon implantation.
Prior art procedures which have advocated treatment with surfactants after the fixing step, for example U.S.
Patent No. 4323358 supra, are substantially ineffective for removing deleterious material which is effectively bonded to the tissue by the fixing agent.
It has been found that the particular sequence of steps according to the present invention provides a significant improvement in terms of implant retention over the prior art.
The surfactant used in the surfactant treating step of the invention is a potent agent for removing deleterious material from animal tissue and care must be taken not to overdo the cleaning action and thereby damage the base tissue by using too strong a solution.
On the other hand, the concentration of surfactant and the period of treatment must be sufficient to achieve the desired result of substantially completely removing the deleterious material. Within these criteria it is preferred to use the surfactant in the form of an ~2~
aqueous solution containing 0~5 to 6% by weight of surfactant. A suitable treatment time is from two to six hours, preferably about three hours.
The surfactant may be an anionic surfactant, a non-ionic surfactant, an amphoteric surfactant or a mixture thereof.
Examples of suitable anionic surfactants are sodium dodecyl sulfate, sodium dodecyl sulfoacetate and sodium salt of alkaryl polyether sulfonate. Examples of suitable non-ionic surfactants are octylphenoxy polyethoxy ethanol (Triton X-100), polyoxyethylene (20) sorbitan monooleate (Tween 80), and polyoxyethylene (20) sorbitan monostearate (Tween 60). Examples of suitable amphoteric surfactants are sulfobetaines commonly known as Zwittergents.
It has been found that particularly advantageous results are obtained if the surfactant is mixture of an anionic surfactant and a nonionic surfactant; and a particularly preferred surfactant solution is one in which the anionic surfactant is 1% by weight sodium dodecyl sulfate and the nonionic surfactant is 1% by weight octylphenoxy polyethoxy ethanol and/or 1% by weight polyoxyethylene (20) sorbitan monooleate.
Preferably the collagenous tissue is contacted with said surfactant solution for about three hours at room temperature.
The surfactant is not only a potent cleansing agent but also a potential toxin and, accordingly, it is an important feature of the invention that, after the surfactant treatment, the fibrous matrix of collagenous tissue is thoroughly washed to remove sub-stantially all surfactant. This washing step may be conducted in any conventional manner, for example with 7~
saline solution or distilled water, and, to ensure sub-stantially complete removal of surfactant, the washing is continued until the formation of bubbles ceases.
After the above described treatment with surfactant and the washing step to remove substantially all trace of surfactant 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 ln the protein molecules of khe tissue. A suitable time for the fixation step is from two to twelve hours and substantially complete fixation is achieved preferably by the repeated soaking procedure described hereinafter. Preferably, the concentration of glutaraldehyde is 0.25 to 1~ by weight.
Fixation of animal tissue with glutaraldehyde to improve its characteristics and render it adaptable o for prosthetic implants is known in the art and this step, in and of itself, is not claimed to be inventive.
However, the special contribution provided by the invention with ~egard to this step is twofold:
Firstly, the soaking with glutaraldehyde is carried out only after deleterious material has been substantially completely removed from the collagenous tissue by the surfactant treatment; thus ensuring that the fibrous matrix is adapted to be fixed throughout, rather than merely on its surfaces.
Secondly, substantially complete fixation throughout the fibrous matrix is ensured by soaking the tissue in the glutaraldehyde for a time sufficient to bond the reactive groups on the glutar---11~
~2~7~
aldehyde molecules to substantially all the reactive amino groups present in the protein molecules of the tissue.
The described result is preferably achieved by repeated soakings in glutaraldehyde to effect multiple cross-linking in accordance with the procedure described hereinafter. The need for repeated soakings, not only in glutaraldehyde to effect multiple cross-linking, but also in calcification-inhibiting agents and anti-phagocytic agents, as described hereinafter, to achieve the cumulative saturation effect provided by the process of this invention has not been achieved in the prior art.
According to a preferred embodiment of the invention the tissue is soaked in 0.5~ by weight glutaraldehyde solution in the presence of 0.1 M
acetate buffer for a period of about three and a half hours. Subsequently, excess glutaraldehyde is washed from the tissue.
An important aspect of the invention is the inhibition of calcification on prostketic implants formed from tissue treated in accordance with the process of the invention.
It has been found that the presence of phosphate ions tends to increase the occurrence of calcification and accordingly the use of phosphate buffers in the steps of the inventive process is to be avoided, notwithstanding the efficiency of the intermediate washing steps.
Since control of pH is an important feature during the process, such control preferably is achieved with a nonphosphate buffer, preferably an acetate buffer.
The tissue fixed with glutaraldehyde is further treated in accordance with the invention firstly with a calcification inhibiting agent and/or an agent which inhibits infiltration and attack by phagocytic cells -12~
`` ~2~ 7 upon implantation and finally with a reducing agent which stabilizes the molecular bonds of the resulting agent/matrix ti~sue.
Mineralization, or more particularly calcification, on and around tissue implants after implantation results in reduced flexibility of the tissue and therefore decreased efficiency in the operation of the prosthesis in the host body. Various treatments have been proposed in the prior art to inhibit or reduce calcificat on and these have met with some degree of success. In particular, the use of specific compounds to inhibit calcification is known in the art. However, the special application of known calcification inhibitors, especially amino diphosphonates, in accordance with the proeess of the present invention results in a substantial improvement over prior art treatments and unexpected advantages in areas not investigated in prior art procedures.
Thus, prosthetic implants made from collagenous tissue treated in accordance with -the process of the present invention are found to be effective in resisting not only calcification but also thrombosis, infection and degeneration. These advantageous charaeteristics, which are exhibited to a degree substantially greater than that achieved in the prior art, are attributable to the fact that endothelial cell coverage on the implant is encouraged and the promotion of such coverage protects the implant from the reactions leading to thrombosis, calcification, infection and degeneration.
The stated improvement is attained by the particular combination of steps described above in which the agent used in the further treatment of the fixed tissue, as well as a calcification agent, may be an agent which inhibits infiltration and attack -13~
~2~
by phagocytic cells, for example, a sporin antibiotic having a free reactive amino group or methotrexate;
or an agent which inhibits infection, preferably cephalosporin C.
The said sporin antibiotic is derived from cyclosporin A, a known immunosuppresive drug having a molecular weight of 1202 and the structural formula illustrated in Figure 7 of the accompanying drawings.
For use as a treating agent in the process of the present invention the cyclopsorin A ring is opened at the position indicated by the arrow in the formula, to provide a free amino group for reaction with the free reactive groups on the glutaraldehyde molecules attached to the fixed tissue.
Methotrexate, or N-[4-[[(2,~-diamino-6-pteridinyl)methyl]methylamino]benzoylJ-L-glutamic acid is a known folic acid antagonist and antimetabolite having the formula:
N N NH
fOOH ~ / ~
HOOCCH2CH2CHNHC \ ~ H2 y > _ NCH 2 ~ I
This drug has two free amino groups available for reaction with the reactive groups on the glutaraldehyde-fixed tissue matrix.
Cephalosporin C i5 a known potent inhibitor of infection.
~.2~
For convenience ln terminology, the calcification-inhibiting agents and the immuno-suppresive agents and drugs used in the post-fixing step of the process according to the invention are referred to hereinafter by the generic term "drug" and, under this terminology, the desired effect produced by the process may be termed "drug immobilization" or "immunosuppression".
One of the advantageous results achieved by the drug immobilization provided by the process of the invention is an effective balance between:
(1) the encouragement or promotion of endothelial cell coverage;
t2) the enhancement of would healing; and (3) inhibition of rejection and attack by macrophage and other phagocytes.
A preferred application for tissue subjected to drug immobilization by the process of the invention is in the produc~ion of prosthetic heart valves whQrein the leaflets and sewing ring are formed from the treated 2n tissue. A particularly preferred embodiment of the tissue is produced when the drug is an amino diphosphonate calcium inhibitor and it has been found that prosthetic heart valves made from the preferred embodi-ment conform to the above balance in that examination of the prosthesis some two months after implantation shows:
1) Substantially complete endothelial cell coverage over the leaflets and sewing ring and no evidence of thrombosis, calcification, infection or degeneration;
2) Substantially complete would healing with no evidence of macrophage debris or macrophage factor;
and
and
3) No evidence of rejection or inhibition of endothelial cell growth ~y macrophage (monocyte) attack.
The drug immobili~ation process is completed by stabilizing the fixed and drug-treated tissue with a reducing agent. Although in theory any reducing agent which will effectively reduce double bonds between carbon and nitrogen atoms may be used for this step including cyanoborohydride; to avoid any possible problems from toxic residues, the preferred reducing agent is sodium borohydride (NaBH4).
Thus, the preferred embodiment of the invention provides a process for the treatment of collagenous tissue to adapt it for use in a prosthetic implant and lS 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 glu~araldehyde solution for a time sufficient to bond the glutaralde-hyde molecules to substantially all the reactive amino groups present in the protein molecules of the tissue;
The drug immobili~ation process is completed by stabilizing the fixed and drug-treated tissue with a reducing agent. Although in theory any reducing agent which will effectively reduce double bonds between carbon and nitrogen atoms may be used for this step including cyanoborohydride; to avoid any possible problems from toxic residues, the preferred reducing agent is sodium borohydride (NaBH4).
Thus, the preferred embodiment of the invention provides a process for the treatment of collagenous tissue to adapt it for use in a prosthetic implant and lS 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 glu~araldehyde solution for a time sufficient to bond the glutaralde-hyde molecules to substantially all the reactive amino groups present in the protein molecules of the tissue;
(4) washing the glutaraldehydefixed tissue to remove excess glutaraldehyde;
'~2~7~
'~2~7~
(5) treating the fixed tissue with an aqueous solution of ~mino 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.
The preferred collagenous tissue is bovine or porcine pericardial tissue. Alternatively, the collagenous tissue may be dura mater, fascia lata, falve tissue or vascular graft tissue.
Preferably, the collagenous tissue is prewashed with isotonic saline solution to remove excess blood and plasma proteins prior to treatment with surfactant in step (1).
Preferably, step (1) is carred out with an aqueous solution containing 0.5 to 6~ by weight of surfactant;
the surfactant preferably being selected from those listed above. Particularly desirable resul s are obtained when said surfactant is a mixture of an anionic surfactant and a non-ionic surfactant.
A particularly preferred surfactant solution is one in which the anionic surfac~ant is 1% by weight sodium dodecyl sulfate and the nonionic surfactant is 1% by weight octylphenoxy polyethoxy ethanol and/or 1% by weight polyoxyethylene (20) sorbitan monooleate.
In carrying out step (1) it has been found that the sufficient time requirement is fulfilled when the collagenous tissue is contacted with said surfactant solution for two to six hours, preferably about three hours, at room temperature.
The fixing treatment of step (3) preferably is - conducted in a solution having a glutaraldehyde concentration of 0.25 to 1% by weight.
As described above, when treating collagenous tissue, particularly pericardial tissue, with a fixing solution this step is conducted by soaking the collagenous tissue in 0.5% by weight glutaraldehyde in the presence of 0.lM acetate bufLer for a period of about three and a half hours.
The glutaraldehyde-fixed tissue is then carefully washed, for example with deionized or acetate buffered water, to remove excess glutaraldehyde and then treated according to step (5).
Preferably, the amino diphosphonate used in step (5) is selected from compounds of the formula:
(1~ O OH ,O (2) o OH O
~\ I 1/ ~ I ;'/
HO - P - C - P - OHOH - P - C - P - OH
,' I \ J
HO I OH HO I OH
'F 2)2 (IH2)5 (3~ O OH O(4) O NH2 ~
~ ;J
HO - P ~ C P- OH HO-- P C-- P _ OH
HO j OH HO I OH
(I 2)10 (jCH2)2 NH2 N \
~2~
and (5) NH2 ~ ' /y HO P - C - ~ -OH
HO i OH
(IH2)2 A particularly preferred amino diphosphonate is 3-amino-hydroxypropane-l,1-diphosphonic acid of formula (1) and preferably the tissue is soaked in fresh saturated solutions of said amino diphosphonate in distilled water (16 mg/ml.) at a pH of 8.0 for three hours per day in each fresh solution over a period of three days.
Particularly advantageous results are obtained if the tissue is soaked in glutaraldehyde between each fresh soaking in amino diphosphonate. This in effect means repetition of steps (3), (4) and (5)O
The cumulative effect of multiple crosslinking on drug uptake ~i.e. amino diphosphonate uptake) by following this procedure i5 illustrated graphically in Figure 3 of the accompanying drawings; and this effect, as well as the effect of surfactant, temperature and fixation time on drug uptake is discussed hereinafter.
The next step in the treatment of the tissue after drug uptake is stabilization with sodium borohydride and this step (7) preferably is conducted with a solution having a concentration of sodium borohydride of 5 to 10 mgtml.
The process of the preferred embodiment is summarized in the following reaction scheme, wherein protein-NH2 representa a molecule of collagen or elastin in the collagenous tissue containing one free amino group and DP-NH2 represent a molecule of amino diphosphonate containing one free amino group.
~ ~ ~t~5~
Protein-NH2 ~ OC(CH2)3C0 ~ Protein- N=C(CH2)3Co H H H
~3) (53 DP - NH
Hydrolysis ~' ~'~ " ,~
Protein - N-C(CH2)3C=N - DP
(7) NaBH4 I Reduction Protein l ~ I(CH2)3C ~ ~ - DP
H H ~I H
In the above reaction scheme the numerals (3), tS) and (7) identify the relevant steps of the process and the final formula illustrates a fully saturated conjugate containing a terminal diphosphonate group.
The pr~sent invention will be more particularly described by reference to the accompanying drawings in which:
Figure 1 is a graph illustrating the effect of temperature and surfactant on drug uptake;
Figure 2 is a graph illustrating the effect of fixation time and temperature on drug uptake;
Figure 3 is a graph illustrating the effect of multiple cross-linking (fixation) on drug uptake;
Figure 4 is a graph illustrating the effect of pH on drug bonding;
Figure 5 is a graph illustrating the enhanced drug binding to pericardium achieved following the initial treatment with surfactant;
-20~
Figure 6 is a scanning electron micrograph, magnification X2000, showing endothelial cell coverage on a valve made from pericardial tissue treated in accordance with the invention after two months in a calf; and Figure 7 illustrates the structural formula of cyclosporin A.
In the graphs of Figures l to 5, the term "drug"
means 3-amino-1-hydroxypropane-l, l-diphosphonic acid.
Comparable results are obtainable with other amino diphosphonates in accordance with the invention.
The following Example illustrates in more detail the preferred embodiment of the invention.
EXAMPLE
The pericardium was removed from the heart of a calf. The pericardial tissue was then washed with 0~9% saline solution to remove excess blood and plasma proteins.
Fatty tissue and thick adherent tissue were removed.
The cleaned fat-free pericardial tissue was then cut into (5-lO cm x 5-10 cm) pieces and each piece of tissue (hereinafter referred to simply as "tissue") was treated according to the following procedure.
The tissue was immersed in a surfactant solution comprising 1% by weight sodium dodecyl sulfate and 1~
by weight octylphenoxy polyethoxy ethanol, commercially available under the Trade Mark Triton XlO0. The tissue was soaked in the surfactant solution at room temperature (23 to 25C.) for a period of three hours.
The tissue was removed from the surfactant solution and thoroughly rinsed with saline solution in a strainer until no more bubbles were seen coming from the tissue and vesicles were removed by suction and washings. It is to be understood that the importance of this washing step is to ensure substantially complete removal of surfactant from the tissue and the nature of the washing solution is not critical, for example, distilled water, deionized water or 0.05M
acetate buffer solution having a pH of 5.5 may be used instead of saline solution.
After the aforesaid washing step, the tissue was soaked in 0.5% by weight glutaraldehyde in O.lM acetate buffer solution for about three and a half hours.
The fixed tissue was rinsed in 0.05M acetate buffer (or deionized water) to remove excess glutaraldehyde and immersed in a saturated drug solution comprising 16 mg/mlO of 3-amino-1-hydrox~propane-1,1-diphosphonic acid in 0.05M acetate buffer. The tissue was soaked in the drug solution for a period of two to three hours.
After the initial drug bonding step the tissue was reimmersed in 0.05M acetate buffer/glutaraldehyde solution and soaked therein for twelve hours.
The tissue was then again rinsed and soaked in drug solution for a period of two to three hours.
The fixation and drug-bonding steps were then repeated two more times.
The cumulative effect of repeating the glutaralde-hyde and amino diphosphonate treatments is illustrated graphically in Figure 3 of the accompanying drawings and these steps may be repeated as long as there is any significant increase in drug uptake. In practice, the performance of each step four times, i.e. three repetitions of each, is normally sufficient to obtain substantially maximum uptake of drug. The overall process is dependent upon the amino group conjugation of amino diphosphonate via glutaraldehyde to the amino acid (lysine) of the collagen or elastin in the tissue.
After the final repetition of the drug treatment the tissue was soaked in a solution containing 5 mg/ml of sodium borohydride for thirty minutes at 25C.
Finally the tissue was removed from the sodium borohydride solution, rinsed to remove excess borohydride and placed in storage under 0.5~ glutaraldehyde or formaldehyde solution until required for use.
Tissue valves prepared from tissue treated according to the above procedure are normally ~ept in 4% formaldehyde solution until required for implantation.
It is important to note that, before implantation, the valve may be rinsed with sodium borohydride solution or glycine (lOmg/ml) to remove formaldehyde.
The removal of formaldehyde reduces tissue necrosis and enhances wound healing.
A number of tissue valves were made from tissue treated in accordance with the procedure illustrated in the above Example and these valves were implanted in calves. The calves were slaughtered after two ?5 months and the valves examined. There were no signs of calcification and endothelial cell coverage was substantial as indicated in Figure 6 of the accompanying drawings. These results demonstrate the substantial improvement achieved by the process of the present invention.
The improved effects achieved by the process of the present invention are demonstrated by the results illustrated graphically in Figures 1 to 5 of the drawings and shown photographically in Figure 6 of the drawings.
Referring to Figure 1 of the drawings, it will be seen that uptake of drug increases steadily for about 30 minutes at room temperature (25C) reaching a maximun of about 2.9~ without a surfactant pre-treatment. Further soaking time and increase oftemperature, up to 37C., has very little effect on the drug uptake.
Treatment of the tissue with surfactant according to the process of the invention, indicated ~y curves (S), not only increases the uptake time, up to ~0 minutes, but also increases the amount of drug taken up by the tissue.
Furthermore, the drug uptake is more temperature dependent and treatment at 37C. increases the drug uptake by 2% over that achieved at 25C.
Figure 2 illustrates the effect of fixation time and temperature on drug uptake. It is to be noted that increased fixation time does not necessarily increase the drug uptake. A fixation time of 24 hours appears to be optimum for maximum drug uptake up to a drug treatment time of two hours. ~owever, increasing the temperature of fixation, from 25C to 37C., provides a significant increase in drug uptake.
Figure 3 illustrates the effect of multiple cross-linking, i.e. repeated fixation treatme~ts, on drug uptake. Starting at time O with a tissue initially fixed in glutaraldehyde according to the procedure described hereinabo~e, this graph illustrates the -~4-effect of repeated drug treatments, D, of periods of 120 minutes each intersperced with repeated treatments with glutaraldehyde, G, represented by the arbitrary time periods ab, cd and ef. These fixation periods may vary from two or three up to about twelve hours, indicated by broken lines in the drawing, and naturally there is no drug uptake during these fixation periods.
However, by following this repetitive procedure the effect is cumulative and a considerable increase in drug uptake is achieved. It will be noted that the curve for the drug uptake at the end of the first treatment period has almost flattened out and the subsequent increases achieved by following the stated repetitive procedure is totally unexpected and certainly not attained or attainable by the procedures disclosed in the prior art.
Figure ~ illustrates the effect of pH on drug (DP) bonding followed by reduction with sodium borohydride. It will be seen that bonding trends to increase with more alkaline solutions.
The effect of the initial surfactant treatment in enhancing the binding of the drug to pericardial tissue is illustrated in Figure 5. The percentage of drug conjugated to the tissue was determined by use of radioactive tracers and the tests were conducted to compare the drug uptake achieved using (1) fresh pericardial tissue with no surfactant or fixation as a control; (2) pericardial tissue fixed with glutaralde-hyde (G.A.) after treatment with Triton X100 (Tx);
(3) pericardial tissue fixed with glutaraldehyde after treatment with polyoxyethylene (20) sorbitan monooleate, commercially available under the Trade Mark Tween (Tw); and (~) paricardial tissue ~ixed with glutaralde-hyde after treatment with sodium dodecyl sulfate tSDS).
It will be seen that a significant increase in drug uptake is achieved following the pretreatment with sodium dodec~l sulfate, an anionic surfactant. This effect is even further enhanced by using a mixture of anionic and nonionic surfactants according to the preferred embodiment of the invention.
Figure 6 shows the substantially complete coverage of the tissue surface with endothelial cells achieved after an implantation time of only two months. This effect serves to demonstrate the success of the implant and, in particular, the achievement of the advantageous results discussed herein with regard to the substantial absence of thrombosis, calcification, infection ox degeneration to a degree not achieved in the prior art.
(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.
The preferred collagenous tissue is bovine or porcine pericardial tissue. Alternatively, the collagenous tissue may be dura mater, fascia lata, falve tissue or vascular graft tissue.
Preferably, the collagenous tissue is prewashed with isotonic saline solution to remove excess blood and plasma proteins prior to treatment with surfactant in step (1).
Preferably, step (1) is carred out with an aqueous solution containing 0.5 to 6~ by weight of surfactant;
the surfactant preferably being selected from those listed above. Particularly desirable resul s are obtained when said surfactant is a mixture of an anionic surfactant and a non-ionic surfactant.
A particularly preferred surfactant solution is one in which the anionic surfac~ant is 1% by weight sodium dodecyl sulfate and the nonionic surfactant is 1% by weight octylphenoxy polyethoxy ethanol and/or 1% by weight polyoxyethylene (20) sorbitan monooleate.
In carrying out step (1) it has been found that the sufficient time requirement is fulfilled when the collagenous tissue is contacted with said surfactant solution for two to six hours, preferably about three hours, at room temperature.
The fixing treatment of step (3) preferably is - conducted in a solution having a glutaraldehyde concentration of 0.25 to 1% by weight.
As described above, when treating collagenous tissue, particularly pericardial tissue, with a fixing solution this step is conducted by soaking the collagenous tissue in 0.5% by weight glutaraldehyde in the presence of 0.lM acetate bufLer for a period of about three and a half hours.
The glutaraldehyde-fixed tissue is then carefully washed, for example with deionized or acetate buffered water, to remove excess glutaraldehyde and then treated according to step (5).
Preferably, the amino diphosphonate used in step (5) is selected from compounds of the formula:
(1~ O OH ,O (2) o OH O
~\ I 1/ ~ I ;'/
HO - P - C - P - OHOH - P - C - P - OH
,' I \ J
HO I OH HO I OH
'F 2)2 (IH2)5 (3~ O OH O(4) O NH2 ~
~ ;J
HO - P ~ C P- OH HO-- P C-- P _ OH
HO j OH HO I OH
(I 2)10 (jCH2)2 NH2 N \
~2~
and (5) NH2 ~ ' /y HO P - C - ~ -OH
HO i OH
(IH2)2 A particularly preferred amino diphosphonate is 3-amino-hydroxypropane-l,1-diphosphonic acid of formula (1) and preferably the tissue is soaked in fresh saturated solutions of said amino diphosphonate in distilled water (16 mg/ml.) at a pH of 8.0 for three hours per day in each fresh solution over a period of three days.
Particularly advantageous results are obtained if the tissue is soaked in glutaraldehyde between each fresh soaking in amino diphosphonate. This in effect means repetition of steps (3), (4) and (5)O
The cumulative effect of multiple crosslinking on drug uptake ~i.e. amino diphosphonate uptake) by following this procedure i5 illustrated graphically in Figure 3 of the accompanying drawings; and this effect, as well as the effect of surfactant, temperature and fixation time on drug uptake is discussed hereinafter.
The next step in the treatment of the tissue after drug uptake is stabilization with sodium borohydride and this step (7) preferably is conducted with a solution having a concentration of sodium borohydride of 5 to 10 mgtml.
The process of the preferred embodiment is summarized in the following reaction scheme, wherein protein-NH2 representa a molecule of collagen or elastin in the collagenous tissue containing one free amino group and DP-NH2 represent a molecule of amino diphosphonate containing one free amino group.
~ ~ ~t~5~
Protein-NH2 ~ OC(CH2)3C0 ~ Protein- N=C(CH2)3Co H H H
~3) (53 DP - NH
Hydrolysis ~' ~'~ " ,~
Protein - N-C(CH2)3C=N - DP
(7) NaBH4 I Reduction Protein l ~ I(CH2)3C ~ ~ - DP
H H ~I H
In the above reaction scheme the numerals (3), tS) and (7) identify the relevant steps of the process and the final formula illustrates a fully saturated conjugate containing a terminal diphosphonate group.
The pr~sent invention will be more particularly described by reference to the accompanying drawings in which:
Figure 1 is a graph illustrating the effect of temperature and surfactant on drug uptake;
Figure 2 is a graph illustrating the effect of fixation time and temperature on drug uptake;
Figure 3 is a graph illustrating the effect of multiple cross-linking (fixation) on drug uptake;
Figure 4 is a graph illustrating the effect of pH on drug bonding;
Figure 5 is a graph illustrating the enhanced drug binding to pericardium achieved following the initial treatment with surfactant;
-20~
Figure 6 is a scanning electron micrograph, magnification X2000, showing endothelial cell coverage on a valve made from pericardial tissue treated in accordance with the invention after two months in a calf; and Figure 7 illustrates the structural formula of cyclosporin A.
In the graphs of Figures l to 5, the term "drug"
means 3-amino-1-hydroxypropane-l, l-diphosphonic acid.
Comparable results are obtainable with other amino diphosphonates in accordance with the invention.
The following Example illustrates in more detail the preferred embodiment of the invention.
EXAMPLE
The pericardium was removed from the heart of a calf. The pericardial tissue was then washed with 0~9% saline solution to remove excess blood and plasma proteins.
Fatty tissue and thick adherent tissue were removed.
The cleaned fat-free pericardial tissue was then cut into (5-lO cm x 5-10 cm) pieces and each piece of tissue (hereinafter referred to simply as "tissue") was treated according to the following procedure.
The tissue was immersed in a surfactant solution comprising 1% by weight sodium dodecyl sulfate and 1~
by weight octylphenoxy polyethoxy ethanol, commercially available under the Trade Mark Triton XlO0. The tissue was soaked in the surfactant solution at room temperature (23 to 25C.) for a period of three hours.
The tissue was removed from the surfactant solution and thoroughly rinsed with saline solution in a strainer until no more bubbles were seen coming from the tissue and vesicles were removed by suction and washings. It is to be understood that the importance of this washing step is to ensure substantially complete removal of surfactant from the tissue and the nature of the washing solution is not critical, for example, distilled water, deionized water or 0.05M
acetate buffer solution having a pH of 5.5 may be used instead of saline solution.
After the aforesaid washing step, the tissue was soaked in 0.5% by weight glutaraldehyde in O.lM acetate buffer solution for about three and a half hours.
The fixed tissue was rinsed in 0.05M acetate buffer (or deionized water) to remove excess glutaraldehyde and immersed in a saturated drug solution comprising 16 mg/mlO of 3-amino-1-hydrox~propane-1,1-diphosphonic acid in 0.05M acetate buffer. The tissue was soaked in the drug solution for a period of two to three hours.
After the initial drug bonding step the tissue was reimmersed in 0.05M acetate buffer/glutaraldehyde solution and soaked therein for twelve hours.
The tissue was then again rinsed and soaked in drug solution for a period of two to three hours.
The fixation and drug-bonding steps were then repeated two more times.
The cumulative effect of repeating the glutaralde-hyde and amino diphosphonate treatments is illustrated graphically in Figure 3 of the accompanying drawings and these steps may be repeated as long as there is any significant increase in drug uptake. In practice, the performance of each step four times, i.e. three repetitions of each, is normally sufficient to obtain substantially maximum uptake of drug. The overall process is dependent upon the amino group conjugation of amino diphosphonate via glutaraldehyde to the amino acid (lysine) of the collagen or elastin in the tissue.
After the final repetition of the drug treatment the tissue was soaked in a solution containing 5 mg/ml of sodium borohydride for thirty minutes at 25C.
Finally the tissue was removed from the sodium borohydride solution, rinsed to remove excess borohydride and placed in storage under 0.5~ glutaraldehyde or formaldehyde solution until required for use.
Tissue valves prepared from tissue treated according to the above procedure are normally ~ept in 4% formaldehyde solution until required for implantation.
It is important to note that, before implantation, the valve may be rinsed with sodium borohydride solution or glycine (lOmg/ml) to remove formaldehyde.
The removal of formaldehyde reduces tissue necrosis and enhances wound healing.
A number of tissue valves were made from tissue treated in accordance with the procedure illustrated in the above Example and these valves were implanted in calves. The calves were slaughtered after two ?5 months and the valves examined. There were no signs of calcification and endothelial cell coverage was substantial as indicated in Figure 6 of the accompanying drawings. These results demonstrate the substantial improvement achieved by the process of the present invention.
The improved effects achieved by the process of the present invention are demonstrated by the results illustrated graphically in Figures 1 to 5 of the drawings and shown photographically in Figure 6 of the drawings.
Referring to Figure 1 of the drawings, it will be seen that uptake of drug increases steadily for about 30 minutes at room temperature (25C) reaching a maximun of about 2.9~ without a surfactant pre-treatment. Further soaking time and increase oftemperature, up to 37C., has very little effect on the drug uptake.
Treatment of the tissue with surfactant according to the process of the invention, indicated ~y curves (S), not only increases the uptake time, up to ~0 minutes, but also increases the amount of drug taken up by the tissue.
Furthermore, the drug uptake is more temperature dependent and treatment at 37C. increases the drug uptake by 2% over that achieved at 25C.
Figure 2 illustrates the effect of fixation time and temperature on drug uptake. It is to be noted that increased fixation time does not necessarily increase the drug uptake. A fixation time of 24 hours appears to be optimum for maximum drug uptake up to a drug treatment time of two hours. ~owever, increasing the temperature of fixation, from 25C to 37C., provides a significant increase in drug uptake.
Figure 3 illustrates the effect of multiple cross-linking, i.e. repeated fixation treatme~ts, on drug uptake. Starting at time O with a tissue initially fixed in glutaraldehyde according to the procedure described hereinabo~e, this graph illustrates the -~4-effect of repeated drug treatments, D, of periods of 120 minutes each intersperced with repeated treatments with glutaraldehyde, G, represented by the arbitrary time periods ab, cd and ef. These fixation periods may vary from two or three up to about twelve hours, indicated by broken lines in the drawing, and naturally there is no drug uptake during these fixation periods.
However, by following this repetitive procedure the effect is cumulative and a considerable increase in drug uptake is achieved. It will be noted that the curve for the drug uptake at the end of the first treatment period has almost flattened out and the subsequent increases achieved by following the stated repetitive procedure is totally unexpected and certainly not attained or attainable by the procedures disclosed in the prior art.
Figure ~ illustrates the effect of pH on drug (DP) bonding followed by reduction with sodium borohydride. It will be seen that bonding trends to increase with more alkaline solutions.
The effect of the initial surfactant treatment in enhancing the binding of the drug to pericardial tissue is illustrated in Figure 5. The percentage of drug conjugated to the tissue was determined by use of radioactive tracers and the tests were conducted to compare the drug uptake achieved using (1) fresh pericardial tissue with no surfactant or fixation as a control; (2) pericardial tissue fixed with glutaralde-hyde (G.A.) after treatment with Triton X100 (Tx);
(3) pericardial tissue fixed with glutaraldehyde after treatment with polyoxyethylene (20) sorbitan monooleate, commercially available under the Trade Mark Tween (Tw); and (~) paricardial tissue ~ixed with glutaralde-hyde after treatment with sodium dodecyl sulfate tSDS).
It will be seen that a significant increase in drug uptake is achieved following the pretreatment with sodium dodec~l sulfate, an anionic surfactant. This effect is even further enhanced by using a mixture of anionic and nonionic surfactants according to the preferred embodiment of the invention.
Figure 6 shows the substantially complete coverage of the tissue surface with endothelial cells achieved after an implantation time of only two months. This effect serves to demonstrate the success of the implant and, in particular, the achievement of the advantageous results discussed herein with regard to the substantial absence of thrombosis, calcification, infection ox degeneration to a degree not achieved in the prior art.
Claims (10)
1. 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, characterized in that it 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.
(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.
2. A process according to claim 1, characterized in that the surfactant is a mixture of 1% by weight sodium dodecyl sulfate and 1% by weight octylphenoxy polyethoxy ethanol and/or 1% by weight polyoxyethylene (20) sorbitan monooleate and the collagenous tissue is contacted with said surfactant solution for about three hours at room temperature.
3. A process according to claim 1, or claim 2 characterized in that the collagenous tissue is prewashed with saline solution to remove excess blood and plasma proteins, prior to treatment with surfactant.
4. A process according to claim 1, characterized in that the fibrous matrix of tissue resulting from the surfactant treat-ment is soaked in 0.5% by weight glutaraldehyde solution in the presence of 0.1 M acetate buffer for a period of about three and a half hours, after which excess glutaraldehyde is washed from the tissue.
5. A process according to claim 4, characterized in that the glutaraldehyde-fixed tissue is treated with an agent which inhibits calcification 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 and said calcification-inhibiting agent is an amino diphosphonate selected from compounds of the formula:-(1) (2) (3) (4) and (5)
6. A process according to claim 4, characterized in that the glutaraldehyde-fixed tissue is treated with (i) a sporin antibiotic having a free reactive amino group or methotrexate or (ii) cephalosporin C.
7. A process according to claim 5 or 6, characterized in that tissue is further treated with sodium borohydride to stabilize the treated tissue.
8. 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 characterized in that it 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.
(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.
9. A process according to claim 1, 2 or 3, characterized in that the collagenous tissue is bovine or porcine pericardial tissue; dura mater; fascia lata; valve tissue or vascular graft tissue.
10. A fixed and stabilized collagenous tissue adapted for use in a prosthetic implant produced by a process according to claim 1, 2 or 8.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US640,725 | 1984-08-14 | ||
US06/640,725 US4553974A (en) | 1984-08-14 | 1984-08-14 | Treatment of collagenous tissue with glutaraldehyde and aminodiphosphonate calcification inhibitor |
Publications (1)
Publication Number | Publication Date |
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CA1247007A true CA1247007A (en) | 1988-12-20 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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CA000488543A Expired CA1247007A (en) | 1984-08-14 | 1985-08-12 | Treatment of collagenous tissue |
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US (1) | US4553974A (en) |
EP (1) | EP0174737A3 (en) |
JP (1) | JPS61137825A (en) |
AU (1) | AU558688B2 (en) |
CA (1) | CA1247007A (en) |
DK (1) | DK366785A (en) |
MX (1) | MX161342A (en) |
ZA (1) | ZA856106B (en) |
Families Citing this family (108)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4883864A (en) * | 1985-09-06 | 1989-11-28 | Minnesota Mining And Manufacturing Company | Modified collagen compound and method of preparation |
US5108923A (en) * | 1986-04-25 | 1992-04-28 | Collaborative Research, Inc. | Bioadhesives for cell and tissue adhesion |
GB8618374D0 (en) * | 1986-07-28 | 1986-09-03 | Hsc Res Dev Corp | Biological vascular prostheses |
WO1988001155A1 (en) * | 1986-08-20 | 1988-02-25 | The Children's Medical Center Corporation | Biomaterial implant with a net positively charged surface |
US4786287A (en) * | 1986-10-10 | 1988-11-22 | Baxter Travenol Laboratories | Process for decreasing residual aldehyde levels in implantable bioprosthetic tissue |
JPS63117121A (en) * | 1986-11-05 | 1988-05-21 | Honda Motor Co Ltd | Controlling method for composite intake system of internal combustion engine |
US4865602A (en) * | 1986-11-06 | 1989-09-12 | Collagen Corporation | Gamma irradiation of collagen/mineral mixtures |
US4838888A (en) * | 1987-04-17 | 1989-06-13 | Baxter Travenol Laboratories, Inc. | Calcification mitigation of implantable bioprostheses |
US5139527A (en) * | 1987-12-17 | 1992-08-18 | Immuno Aktiengesellschaft | Biologic absorbable implant material for filling and closing soft tissue cavities and method of its preparation |
US4976733A (en) * | 1988-02-03 | 1990-12-11 | Biomedical Design, Inc. | Prevention of prosthesis calcification |
US4969912A (en) * | 1988-02-18 | 1990-11-13 | Kelman Charles D | Human collagen processing and autoimplant use |
US5746775A (en) * | 1988-04-01 | 1998-05-05 | The Board Of Regent6S Of The University Of Michigan | Method of making calcification-resistant bioprosthetic tissue |
US5024841A (en) * | 1988-06-30 | 1991-06-18 | Collagen Corporation | Collagen wound healing matrices and process for their production |
US4950483A (en) * | 1988-06-30 | 1990-08-21 | Collagen Corporation | Collagen wound healing matrices and process for their production |
US5110604A (en) * | 1988-06-30 | 1992-05-05 | Collagen Corporation | Processes for producing collagen matrixes and methods of using same |
US5002566A (en) * | 1989-02-17 | 1991-03-26 | Baxter International Inc. | Calcification mitigation of bioprosthetic implants |
US4996193A (en) * | 1989-03-03 | 1991-02-26 | The Regents Of The University Of California | Combined topical and systemic method of administration of cyclosporine |
US5540931A (en) * | 1989-03-03 | 1996-07-30 | Charles W. Hewitt | Methods for inducing site-specific immunosuppression and compositions of site specific immunosuppressants |
US5100429A (en) * | 1989-04-28 | 1992-03-31 | C. R. Bard, Inc. | Endovascular stent and delivery system |
US6004261A (en) * | 1989-04-28 | 1999-12-21 | C. R. Bard, Inc. | Formed-in-place endovascular stent and delivery system |
AT398276B (en) * | 1989-05-31 | 1994-11-25 | Sorin Biomedica Spa | METHOD FOR PREPARING BIOLOGICAL IMPLANTATION MATERIAL |
US5609626A (en) * | 1989-05-31 | 1997-03-11 | Baxter International Inc. | Stent devices and support/restrictor assemblies for use in conjunction with prosthetic vascular grafts |
DE4028088A1 (en) * | 1990-09-05 | 1992-04-16 | Berg Ernes Elme Dipl Ing | Resorbable implant for antibody concn. - used in immunological diagnostics, testing and antibody prodn. |
DE69210225T2 (en) * | 1991-02-14 | 1996-12-05 | Baxter Int | Manufacturing process for flexible biological tissue transplant materials |
US5192312A (en) * | 1991-03-05 | 1993-03-09 | Colorado State University Research Foundation | Treated tissue for implantation and methods of treatment and use |
US5513662A (en) * | 1991-12-31 | 1996-05-07 | Osteotech, Inc. | Preparation of bone for transplantation |
US5476516A (en) * | 1992-03-13 | 1995-12-19 | Albert Einstein College Of Medicine Of Yeshiva University | Anticalcification treatment for aldehyde-tanned biological tissue |
US5509932A (en) * | 1993-04-08 | 1996-04-23 | Keogh; James R. | Fixed tissue medical devices comprising albumin-binding dyes |
US6203755B1 (en) * | 1994-03-04 | 2001-03-20 | St. Jude Medical, Inc. | Electron beam sterilization of biological tissues |
EP1452153A1 (en) * | 1994-03-14 | 2004-09-01 | Cryolife, Inc | Treated tissue for implantation and preparation methods |
US5595571A (en) * | 1994-04-18 | 1997-01-21 | Hancock Jaffe Laboratories | Biological material pre-fixation treatment |
CA2186374A1 (en) * | 1994-04-29 | 1995-11-09 | William Carl Bruchman | Improved blood contact surfaces employing natural subendothelial matrix and method for making and using the same |
JPH09512184A (en) * | 1994-04-29 | 1997-12-09 | ダブリュ.エル.ゴア アンド アソシエイツ,インコーポレイティド | Improved blood contact surface utilizing endothelium on subendothelial extracellular matrix |
US5976104A (en) * | 1994-08-19 | 1999-11-02 | Lifenet Research Foundation | Recirculation method for cleaning essentially intact bone grafts using pressure mediated flow of solutions and bone grafts produced thereby |
US5556379A (en) * | 1994-08-19 | 1996-09-17 | Lifenet Research Foundation | Process for cleaning large bone grafts and bone grafts produced thereby |
US5797871A (en) * | 1994-08-19 | 1998-08-25 | Lifenet Research Foundation | Ultrasonic cleaning of allograft bone |
US5977034A (en) * | 1994-08-19 | 1999-11-02 | Lifenet Research Foundation | Composition for cleaning bones |
AUPN174495A0 (en) | 1995-03-15 | 1995-04-06 | Ketharanathan, Vettivetpillai | Surgical prostheses |
WO1996032905A1 (en) * | 1995-04-19 | 1996-10-24 | St. Jude Medical, Inc. | Matrix substrate for a viable body tissue-derived prosthesis and method for making the same |
DK2111876T3 (en) * | 1995-12-18 | 2011-12-12 | Angiodevice Internat Gmbh | Crosslinked polymer preparations and methods for their use |
US6458889B1 (en) | 1995-12-18 | 2002-10-01 | Cohesion Technologies, Inc. | Compositions and systems for forming crosslinked biomaterials and associated methods of preparation and use |
US6833408B2 (en) | 1995-12-18 | 2004-12-21 | Cohesion Technologies, Inc. | Methods for tissue repair using adhesive materials |
US7883693B2 (en) | 1995-12-18 | 2011-02-08 | Angiodevice International Gmbh | Compositions and systems for forming crosslinked biomaterials and methods of preparation of use |
US5782931A (en) * | 1996-07-30 | 1998-07-21 | Baxter International Inc. | Methods for mitigating calcification and improving durability in glutaraldehyde-fixed bioprostheses and articles manufactured by such methods |
US6121041A (en) | 1996-07-31 | 2000-09-19 | St. Jude Medical, Inc. | Use of microorganisms for decellularizing bioprosthetic tissue |
US5782914A (en) * | 1996-11-29 | 1998-07-21 | Bio-Vascular, Inc. | Method for preparing heterogeneous tissue grafts |
US5862806A (en) * | 1997-10-30 | 1999-01-26 | Mitroflow International, Inc. | Borohydride reduction of biological tissues |
US20080077251A1 (en) * | 1999-06-07 | 2008-03-27 | Chen Silvia S | Cleaning and devitalization of cartilage |
US8563232B2 (en) | 2000-09-12 | 2013-10-22 | Lifenet Health | Process for devitalizing soft-tissue engineered medical implants, and devitalized soft-tissue medical implants produced |
US6214054B1 (en) | 1998-09-21 | 2001-04-10 | Edwards Lifesciences Corporation | Method for fixation of biological tissues having mitigated propensity for post-implantation calcification and thrombosis and bioprosthetic devices prepared thereby |
US6509145B1 (en) * | 1998-09-30 | 2003-01-21 | Medtronic, Inc. | Process for reducing mineralization of tissue used in transplantation |
US6214055B1 (en) * | 1998-10-30 | 2001-04-10 | Mures Cardiovascular Research, Inc. | Method and kit for rapid preparation of autologous tissue medical devices |
US20020081732A1 (en) * | 2000-10-18 | 2002-06-27 | Bowlin Gary L. | Electroprocessing in drug delivery and cell encapsulation |
US7615373B2 (en) * | 1999-02-25 | 2009-11-10 | Virginia Commonwealth University Intellectual Property Foundation | Electroprocessed collagen and tissue engineering |
WO2002018441A2 (en) * | 2000-09-01 | 2002-03-07 | Virginia Commonwealth University Intellectual Property Foundation | Electroprocessed fibrin-based matrices and tissues |
US6903244B1 (en) | 1999-02-26 | 2005-06-07 | University Of Utah Research Foundation | Mice which are +/− or −/− for the elastin gene as models for vascular disease |
US6471723B1 (en) | 2000-01-10 | 2002-10-29 | St. Jude Medical, Inc. | Biocompatible prosthetic tissue |
US20070055367A1 (en) * | 2000-03-15 | 2007-03-08 | Orbus Medical Technologies, Inc. | Medical device with coating that promotes endothelial cell adherence and differentiation |
US9522217B2 (en) | 2000-03-15 | 2016-12-20 | Orbusneich Medical, Inc. | Medical device with coating for capturing genetically-altered cells and methods for using same |
US20030229393A1 (en) * | 2001-03-15 | 2003-12-11 | Kutryk Michael J. B. | Medical device with coating that promotes cell adherence and differentiation |
US8088060B2 (en) * | 2000-03-15 | 2012-01-03 | Orbusneich Medical, Inc. | Progenitor endothelial cell capturing with a drug eluting implantable medical device |
EP1263484B1 (en) | 2000-03-15 | 2007-05-16 | OrbusNeich Medical, Inc. | Coating which promotes endothelial cell adherence |
US8460367B2 (en) * | 2000-03-15 | 2013-06-11 | Orbusneich Medical, Inc. | Progenitor endothelial cell capturing with a drug eluting implantable medical device |
US7510572B2 (en) * | 2000-09-12 | 2009-03-31 | Shlomo Gabbay | Implantation system for delivery of a heart valve prosthesis |
AUPR217300A0 (en) * | 2000-12-20 | 2001-01-25 | Ketharanathan, Vettivetpillai | Method of creating biological and biosynthetic material for implantation |
US20020177223A1 (en) * | 2001-03-12 | 2002-11-28 | Ogle Mathew F. | Methods and compositions for crosslinking tissue |
US7078163B2 (en) * | 2001-03-30 | 2006-07-18 | Medtronic, Inc. | Process for reducing mineralization of tissue used in transplantation |
CN1556719A (en) * | 2001-09-24 | 2004-12-22 | 诺沃斯特公司 | Methods and apparatus employing ionizing radiation for treatment of cardiac arrhythmia |
US7008591B2 (en) * | 2001-10-17 | 2006-03-07 | Edwards Lifesciences Corporation | Supercritical fluid extraction process for tissue preparation |
US6878168B2 (en) | 2002-01-03 | 2005-04-12 | Edwards Lifesciences Corporation | Treatment of bioprosthetic tissues to mitigate post implantation calcification |
US8308797B2 (en) * | 2002-01-04 | 2012-11-13 | Colibri Heart Valve, LLC | Percutaneously implantable replacement heart valve device and method of making same |
US20060079439A1 (en) * | 2002-03-27 | 2006-04-13 | University Of Utah Research Foundation | Elastin prevents occlusion of body vessels by vascular smooth muscle cells |
EP1594459B1 (en) | 2002-12-30 | 2010-02-17 | Angiotech International Ag | Drug delivery from rapid gelling polymer composition |
EP1508343B1 (en) * | 2003-08-21 | 2015-11-04 | AddBIO AB | Bisphosponate coated implant device and method therefor |
US7955788B2 (en) * | 2003-10-30 | 2011-06-07 | Medtronic, Inc. | Bioprosthetic tissue preparation with synthetic hydrogels |
WO2005086831A2 (en) * | 2004-03-10 | 2005-09-22 | Orbus Medical Technologies, Inc. | Endothelial ligand binding coated medical device |
US7648676B2 (en) * | 2004-04-20 | 2010-01-19 | Rti Biologics, Inc. | Process and apparatus for treating implants comprising soft tissue |
US20060228252A1 (en) * | 2004-04-20 | 2006-10-12 | Mills C R | Process and apparatus for treating implants comprising soft tissue |
WO2006083260A2 (en) | 2004-04-28 | 2006-08-10 | Angiotech Biomaterials Corporation | Compositions and systems for forming crosslinked biomaterials and associated methods of preparation and use |
EP2946666B1 (en) * | 2004-04-30 | 2017-11-15 | OrbusNeich Medical, Inc. | Medical device with coating for capturing genetically-altered cells and methods of using same |
US20050266390A1 (en) * | 2004-06-01 | 2005-12-01 | Yuichiro Ueda | Processes for removing cells and cell debris from tissue and tissue constructs used in transplantation and tissue reconstruction |
WO2005118014A2 (en) * | 2004-06-01 | 2005-12-15 | Medtronic, Inc. | Decellurisation processes for making bioprotheses |
EP1796602A4 (en) | 2004-09-17 | 2016-10-19 | Angiotech Pharm Inc | Multifunctional compounds for forming crosslinked biomaterials and methods of preparation and use |
DE102004047247B3 (en) * | 2004-09-22 | 2006-03-16 | Auto Tissue Gmbh | Sterilization process for the production of implantable or transplantable biological material |
US7989157B2 (en) * | 2005-01-11 | 2011-08-02 | Medtronic, Inc. | Solution for storing bioprosthetic tissue used in a biological prosthesis |
US8445278B2 (en) * | 2005-03-01 | 2013-05-21 | Medtronic, Inc. | Process for producing decellularized biological tissues |
US8764820B2 (en) * | 2005-11-16 | 2014-07-01 | Edwards Lifesciences Corporation | Transapical heart valve delivery system and method |
EP2077718B2 (en) | 2006-10-27 | 2022-03-09 | Edwards Lifesciences Corporation | Biological tissue for surgical implantation |
US9101691B2 (en) | 2007-06-11 | 2015-08-11 | Edwards Lifesciences Corporation | Methods for pre-stressing and capping bioprosthetic tissue |
FR2917625B1 (en) * | 2007-06-20 | 2009-09-18 | Perouse Soc Par Actions Simpli | TREATMENT OF IMPLANTABLE MEDICAL DEVICES RESISTANT TO CALCIFICATION |
US9744043B2 (en) | 2007-07-16 | 2017-08-29 | Lifenet Health | Crafting of cartilage |
US8357387B2 (en) | 2007-12-21 | 2013-01-22 | Edwards Lifesciences Corporation | Capping bioprosthetic tissue to reduce calcification |
US8211165B1 (en) | 2008-01-08 | 2012-07-03 | Cook Medical Technologies Llc | Implantable device for placement in a vessel having a variable size |
US20100040593A1 (en) * | 2008-08-15 | 2010-02-18 | Orthopeutics, Lp | Formulations for nonsurgical exogenous crosslink therapy |
US8439970B2 (en) | 2009-07-14 | 2013-05-14 | Edwards Lifesciences Corporation | Transapical delivery system for heart valves |
EP2656863B1 (en) | 2010-03-23 | 2019-09-18 | Edwards Lifesciences Corporation | Methods of conditioning sheet bioprosthetic tissue |
US8906601B2 (en) | 2010-06-17 | 2014-12-09 | Edwardss Lifesciences Corporation | Methods for stabilizing a bioprosthetic tissue by chemical modification of antigenic carbohydrates |
AU2015202239B2 (en) * | 2010-08-24 | 2016-11-17 | Collagen Solutions Nz Limited | Biomaterials with enhanced properties and devices made therefrom |
US9095430B2 (en) | 2010-08-24 | 2015-08-04 | Southern Lights Ventures (2002) Limited | Biomaterials with enhanced properties and devices made therefrom |
US9351829B2 (en) | 2010-11-17 | 2016-05-31 | Edwards Lifesciences Corporation | Double cross-linkage process to enhance post-implantation bioprosthetic tissue durability |
WO2012082952A2 (en) | 2010-12-14 | 2012-06-21 | Colibri Heart Valve Llc | Percutaneously deliverable heart valve including folded membrane cusps with integral leaflets |
US9381082B2 (en) | 2011-04-22 | 2016-07-05 | Edwards Lifesciences Corporation | Devices, systems and methods for accurate positioning of a prosthetic valve |
US10238771B2 (en) | 2012-11-08 | 2019-03-26 | Edwards Lifesciences Corporation | Methods for treating bioprosthetic tissue using a nucleophile/electrophile in a catalytic system |
US10149757B2 (en) | 2013-03-15 | 2018-12-11 | Edwards Lifesciences Corporation | System and method for transaortic delivery of a prosthetic heart valve |
US9615922B2 (en) | 2013-09-30 | 2017-04-11 | Edwards Lifesciences Corporation | Method and apparatus for preparing a contoured biological tissue |
US10959839B2 (en) | 2013-10-08 | 2021-03-30 | Edwards Lifesciences Corporation | Method for directing cellular migration patterns on a biological tissue |
EP3134033B1 (en) | 2014-05-29 | 2018-04-04 | Edwards Lifesciences CardiAQ LLC | Prosthesis and delivery device |
CN113164258A (en) | 2018-11-01 | 2021-07-23 | 爱德华兹生命科学公司 | Transcatheter regeneration pulmonary valve |
Family Cites Families (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR1317584A (en) * | 1961-03-15 | 1963-02-08 | Olin Mathieson | Method of treatment of bone and other tissues |
AT261800B (en) * | 1966-08-22 | 1968-05-10 | Braun Internat Gmbh B | Process for the manufacture of tubular, smooth or threaded tissue-blood vessel prostheses |
US3988782A (en) * | 1973-07-06 | 1976-11-02 | Dardik Irving I | Non-antigenic, non-thrombogenic infection-resistant grafts from umbilical cord vessels and process for preparing and using same |
DE2360797C2 (en) * | 1973-12-06 | 1985-05-23 | Henkel KGaA, 4000 Düsseldorf | Pharmaceutical preparations |
US3966401A (en) * | 1974-07-01 | 1976-06-29 | Hancock Laboratories Incorporated | Preparing natural tissue for implantation so as to provide improved flexibility |
IL47062A (en) * | 1975-04-10 | 1979-07-25 | Yeda Res & Dev | Process for diminishing antigenicity of tissues to be usedas transplants by treatment with glutaraldehyde |
JPS5230097A (en) * | 1975-09-02 | 1977-03-07 | Kaneyasu Miyata | Method of mounting different substitute blood vessel |
US4378224A (en) * | 1980-09-19 | 1983-03-29 | Nimni Marcel E | Coating for bioprosthetic device and method of making same |
US4323358A (en) * | 1981-04-30 | 1982-04-06 | Vascor, Inc. | Method for inhibiting mineralization of natural tissue during implantation |
US4402697A (en) * | 1982-08-25 | 1983-09-06 | Extracorporeal Medical Specialties, Inc. | Method for inhibiting mineralization of natural tissue during implantation |
WO1984001879A1 (en) * | 1982-11-12 | 1984-05-24 | American Hospital Supply Corp | Surfactant treatment of implantable biological tissue to inhibit calcification |
-
1984
- 1984-08-14 US US06/640,725 patent/US4553974A/en not_active Expired - Fee Related
-
1985
- 1985-08-09 EP EP85305681A patent/EP0174737A3/en not_active Withdrawn
- 1985-08-12 CA CA000488543A patent/CA1247007A/en not_active Expired
- 1985-08-13 AU AU46130/85A patent/AU558688B2/en not_active Ceased
- 1985-08-13 MX MX206273A patent/MX161342A/en unknown
- 1985-08-13 DK DK366785A patent/DK366785A/en not_active Application Discontinuation
- 1985-08-13 ZA ZA856106A patent/ZA856106B/en unknown
- 1985-08-14 JP JP60179218A patent/JPS61137825A/en active Pending
Also Published As
Publication number | Publication date |
---|---|
ZA856106B (en) | 1987-03-25 |
DK366785D0 (en) | 1985-08-13 |
AU4613085A (en) | 1986-03-27 |
EP0174737A3 (en) | 1987-03-11 |
AU558688B2 (en) | 1987-02-05 |
EP0174737A2 (en) | 1986-03-19 |
MX161342A (en) | 1990-09-10 |
US4553974A (en) | 1985-11-19 |
JPS61137825A (en) | 1986-06-25 |
DK366785A (en) | 1986-02-15 |
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