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Número de publicaciónEP0124596 A4
Tipo de publicaciónSolicitud
Número de solicitudEP19830903751
Número de PCTPCT/US1983/001702
Fecha de publicación26 Jun 1985
Fecha de presentación3 Nov 1983
Fecha de prioridad12 Nov 1982
También publicado comoCA1211716A, CA1211716A1, EP0124596A1, WO1984001894A1
Número de publicación1983903751, 83903751, 83903751.2, EP 0124596 A4, EP 0124596A4, EP-A4-0124596, EP0124596 A4, EP0124596A4, EP19830903751, EP83903751, PCT/1983/1702, PCT/US/1983/001702, PCT/US/1983/01702, PCT/US/83/001702, PCT/US/83/01702, PCT/US1983/001702, PCT/US1983/01702, PCT/US1983001702, PCT/US198301702, PCT/US83/001702, PCT/US83/01702, PCT/US83001702, PCT/US8301702
InventoresAws S Nashef, Guy Lowery
SolicitanteAmerican Hospital Supply Corp
Exportar citaBiBTeX, EndNote, RefMan
Enlaces externos:  Espacenet, Registro europeo de patentes
Chemical sterilization of implantable biological tissue.
EP 0124596 A4 (texto de WO1984001894A1) 
Resumen  
A process for treating biological tissue prior to implantation to effect sterilization thereof. In particular, the process includes the use of improved sterilant solutions containing conventional tissue sterilants such as glutaraldehyde or formaldehyde, alcohol, and surfactants to destroy resistant microorganisms.
Reclamaciones  (El texto procesado por OCR puede contener errores)
AMENDED CLAIMS
[received by the International Bureau on 28 February 1984(28.02.84); original claim 1 unchanged. Original claims 2 to 14 have been replaced by new claims 15 to 36; the text of the new claims follows]
15. A method for sterilizing biological tissue prior to implan¬ tation in an animal which comprises placing said tissue in an aqueous solution at a temperature of from about 30° to about 40°C for a time sufficient to destroy a substantial quantity of a microorganism, wherein said aqueous solution comprises: a sterilization effective amount of glutaraldehyde or formaldehyde; from about 0.1 to about 10 weight percent of a cationic, nonionic, or anionic surfactant; and from about 10 to about 30 percent of an aromatic or an aliphatic alcohol.
16. The method of Claim 15 wherein the solution comprises from about 0.2 to about 1 percent glutaraldehyde.
17. The method of Claim 15 wherein the solution comprises from about 3 to about 5 percent formaldehyde.
18. The method of Claim 15 comprising from about 20 to about 25 percent of an aliphatic alcohol.
19. The method of Claim 18 wherein said aliphatic alcohol has from 1 to about 5 carbon atoms.
20. The method of Claim 19 wherein the alcohol is ethanol or isopropanol.
21. The method of Claim 15 wherein the temperature is from about 32° to about 38°C.
22. The method of Claim 15 wherein the surfactant is from about 0.5 to about 1.5 weight percent.
23. The method of Claim 15 wherein the surfactant is cationic and is an alkyl quaternary amine or its halide salt.
24. The method of Claim 23 wherein the alkyl quaternary amine surfactant comprises a tertiary amine connected to either, an aromatic residue, an aliphatic residue, or a combination of both aromatic and aliphatic residues; said aromatic residue further com¬ prising benzene, pyridine, or napthylene, and said aliphatic residue is branched, unbranched, cyclic, saturated, or unsaturated.
25. The method of Claim 15 wherein the surfactant is nonionic and is a polyoxyalkylene ether, polyoxyalkylene alkylaryl ether, aliphatic ester, polyether, polyoxyalkylene ester, saccharide ester, or a combination thereof. 26. The method of Claim 15 wherein the surfactant is anionic and is a bile salt , salt of a fatty acid having from about 8 to about 20 carbon atoms , N-alkanoyl amino acid wherein the aliphatic portion has from about 8 to about 18 carbon atoms , N-acylated amino acid, sulfate of an aliphatic alcohol having from about 6 to about 18 carbon atoms, ethyleneoxide modified sulfate of an aliphatic alcohol, sulfated ethanol amide, sulfated alkyl phenol, alkane sulfonic acid, and alkylarylsulfonic acid.
27. The method of Claim 15 wherein the solution comprises from about 3 to about 5 percent formaldehyde; from about 20 to about 30 percent methanol, ethanol, or isopropanol; fyom about 0.5 to about 5 percent of a nonionic surfactant; and the temperature is from about 32 to about 38°C .
28. The method of Claim 15 wherein the solution comprises from about 3 to about 5 percent formaldehyde; from about 20 to about 25 percent aliphatic alcohol having from 1 to about 5 carbon atoms; from about 0.5 to about 5 percent of a nonionic surfactant; and the temperature is from about 32 to about 38°C.
29. The method of Claim 27 wherein the surfactant is sorbitan monooleate polyalkylene.
30. The method of Claim 15 wherein the solution comprises from about 0.2 to about 1 percent glutaraldehyde; from about 20 to about 25 percent methanol, ethanol, or isopropanol; from about 0.5 to about 5 percent of a nonionic surfactant; and the temperature is from about 32° to about 38°C.
31. The method of Claim 30 wherein the surfactant is sorbitan monooleate polyalkylene.
32. The method of Claim 28 wherein the biological tissue comprises tendon, ligament, heart valve, dura mater, pericardium, tympanic membranes and skin patches.
33. The method of Claim 28 wherein the biological tissue is a bioprosthetic heart valve; the microorganism is Microascus cinereus; the alcohol concentration is from about 20 to about 25 percent; the surfactant concentration is from about 0.5 to 1.5 percent; and the surfactant is sorbitan monooleate polyethylene.
34. A method for sterilizing biological tissue prior to implan¬ tation in an animal which comprises placing said tissue in an aqueous solution at a temperature of from about 32° to about 38 °C for a time
O PI IPO sufficient to destroy a substantial quantity of a formaldehyde- or glutaraldehyde-resistant microorganism; wherein said biological tissue comprises tendon, ligament, heart valve, dura mater, pericardium , tympanic membranes, and skin patches; and said aqueous solution comprises : from about 0.2 to about 1 percent glutaraldehyde or from about 3 to about 5 percent formaldehyde; from about 0.1 to about 1.5 weight percent of a cationic, nonionic, or anionic surfactant; and from about 20 to about 25 percent of an aromatic or an aliphatic alcohol, wherein said aliphatic alcohol has from 1 to about 5 carbon atoms.
35. The method of Claim 34 wherein said resistant micro¬ organism is Microascus cinereus.
36. The method of Claim 25 wherein said biological tissue is heart valve, and the surfactant is nonionic.
O H
Descripción  (El texto procesado por OCR puede contener errores)

CHEMICAL STERILIZATION OF IMPLANTABLE BIOLOGICAL TISSUE

Background of the Invention

The chemical processing of biological tissue such as that used in the preparation of heart valves , ligaments, tendons, tympanic membranes, and the like prior to implantation into humans involves a series of procedures to insure both the compatibility and the structural and functional integrity of the implanted tissue in its new host. Among these conventionally employed procedures are glutaraldehyde-fixation (tanning) and formaldehyde-sterilization. The glutaraldehyde-fixation of tissue prior to implantation renders it relatively biologically inert and has been widely accepted as a necessary step in the processing of the tissue. Likewise, the formaldehyde-sterilization of tissue prior to implantation has proven effective in destroying a large number of microorganisms either associated with the host-extraction of tissue or the subsequent handling of the tissue during processing. Unfortunately however, glutaraldehyde-fixation and subsequent formaldehyde-sterilization procedures presently employed have not effectively destroyed all microorganisms associated with implantable biological tissue; in particular that tissue used in the preparation of bioprosthetic heart valves. We have found for example that the mature spore form of Microascus cinereus exhibits resistance to conventional sterilization procedures. Moreover, the formaldehyde-sterilization procedures presently employed lack the ability to rapidly destroy many microorganisms associated with biological tissue , requiring long periods of exposure to sterilant solutions. Microascus cinereus is a fungus from the Ascomycete family which exhibits resistance to various concentrations of both glutaraldehyde and formaldehyde which are conventionally used in the processing of biological tissue prior to implantation. Microascus cinereus is a common environmental organism having worldwide geographic distribution, and has been clinically identified as being associated with minor dermal lesions.

In accordance with the present invention, we have developed a process for treating biological tissue prior to implantation which effectively renders the mature spores of formaldehyde- and glutaraldehyde-resistant microorganisms such as Microascus cinereus susceptible to sterilization with conventional sterilants.

This process advantageously improves the efficacy of the tissue sterilization process while maintaining the biochemical, structural, and functional integrity of implantable tissue such as xenograft heart valves prosthesis. Moreover, we have unexpectedly found that many microorganisms already susceptible to formaldehyde or glutaraldehyde sterilization are destroyed considerably faster using the process and related compositions of the present invention.

Summary of the Invention

In accordance with the present invention, disclosed is an improved process for treating biological tissue prior to implantation in order to effect sterilization thereof. The process comprises contacting biological tissue under sterilizing conditions with a sterilization effective amount of a solution comprising formaldehyde or glutaraldehyde, alcohol, and a surfactant.

Detailed Description of the Invention

In accordance with the present invention, it is contemplated that various types of implantable biological tissue derived from numerous animal sources and parts of the anatomy can be made less resistant to conventional sterilization procedures. Thus, the

OMPI tissue can be derived from various sources including but not limited to bovine , porcine , horse, sheep , kangaroo, or rabbit; and can include tendons , ligaments, heart valves, or tissue used to construct heart valves such as dura mater and pericardium . It is further contemplated that tissue used for augmentation such as skin patches , pericardial patches , aortic patches , and tympanic membranes is suitable in the present invention. Moreover, it is contemplated that the surfaces of non-biological items such as surgical instruments, medical devices , valve conduits and the like made of plastic, cloth, or metal, can be effectively treated according to the present invention. In accordance with a preferred embodiment of the present invention, porcine tissue valves and bovine pericardial tissue used in the preparation of bioprosthetic heart valves was fixed in glutaraldehyde and treated with the improved sterilant solution of the present invention and was implanted subcutaneously in rabbits. The treated valve tissue which lost its resistance to conventional sterilants such as glutaraldehyde and formaldehyde showed no significant difference in the integrity of the valve tissue compared to the tissue treated in the conventional manner.

In accordance with one embodiment the present invention, formaldehyde-resistant organisms such as Microascus cinereus associated with implantable biological tissue have been made susceptible to sterilization, i.e. , destruction of the organism. It is contemplated from the results of our studies, however, that the present compositions are useful in destroying formaldehyde- resistant organisms on the surfaces of non-biological articles as well. We have unexpectedly found that the sterilant compositions in accordance with the present invention are more effective against these formaldehyde-resistant microorganisms on surfaces such as tissue than in solution. Although the reasons for this phenomena are not fully understood at present we have found that sterilant compositions containing formaldehyde and alcohol are effective in destroying Microascus cinereus in solution, but require a much longer time frame (days) to be effective on tissue. However, the presence of a surfactant renders the solution more effective in a shorter time frame (hours) .

We have found that additional clinically significant microorganisms which have previously been somewhat difficult to destroy by conventional formaldehyde or glutaraldehyde sterilization have been rendered more susceptible to destruction in accordance with the process of the present invention. We have advantageously found that the improved sterilant compositions of the present invention are effective against a large variety of organisms including but not limited to the following organisms: Aspergillus niger, Bacillus licheniformis , Bacillus pumilus, Bacillus subtilis var niger, Brucella suis, Candida albicans, Chaetomium globosum , Clostridium sporogenes , Enterobacter aerogenes, Escherichia coli, Klebsiella pneumoniae, Microascus cinereus, Mycobacterium chelonei-like organism , Mycobacterium fortuitum, Pseudomonas aeruginosa, Salmonella typhimurium , Serratia marcescens , Shigella sonnei, Staphylococcus aureus, Streptococcus faecalis .

In accordance with the present invention, the improved sterilant components may be used in admixture with the conventional sterilizing solutions containing glutaraldehyde or formaldehyde, or may be formulated into compositions as described hereinafter. Normally, from about a 0.2 to about 1% glutaraldehyde solution or from about 3 to about 5% formaldehyde solution is used to sterilize biological tissue prior to implantation. Preferable sterilant concentrations are 0.625% glutaraldehyde or from about 4 to about 5% formaldehyde.

In accordance with the present invention, it is preferable to treat the tissue within a tissue-stabilizing pH range; that is, within a pH range that is not deleterious to the tissue components. Although the effectiveness of the present invention has been verified in a pH range from about 4.5 to about 7.5 , the preferred pH range is chosen for compatibility with the tissue. A preferred

OMPI pH range is from about 7.0 to about 7.6 , and a more preferred pH range is from about 7.1 to about 7.4. The most preferred pH in accordance with the present invention is 7.3.

Although we have found that the sterilant process of the present invention works well in phosphate buffer, HEPES buffer, or in non-buffered solutions, sterilant compositions used in accordance with one embodiment of the present invention are preferably stable, non-interacting with the sterilization process, and have a buffering capacity sufficient to maintain a tissue-acceptable pH. The choice of the appropriate buffer, and its concentration will depend upon specific tissue preparation conditions; variations of which have been introduced by several manufacturers. The buffers can be either conventional 0.01-0.02 M phosphate-buffered saline (PBS) or phosphate- deficient solutions such as those containing less phosphate than these 0.01 to 0.02 M PBS solutions, and preferably less than about 0.001 to about 0.002 M phosphate. Preferred buffers in accordance with the present invention include borate, carbonate, bicarbonate, cacodylate (found to be non-toxic in animals) , and other synthetic, artificial, or organic buffers such as HEPES N-2-hydroxyethylpiperazine-N,-2-ethanesulphonic acid; MOPS , 2-(N-morpholino) propanesulphonic acid; and PIPES ,

1 , 4-piperazinebis (ethanesulphonic acid) .

Preferably, the buffered or unbuffered solutions , used in accordance with the present invention should not interfere with the tissue sterilization process afforded by agents such as glutaraldehyde or formaldehyde. That is , they should not react with the agent or prevent the agent from achieving proper sterilization of the tissue. Illustrative of this are buffers containing primary and secondary amines such as tris(hydroxymethyl)aminomethane (Tris) , which are known to react with the aldehyde groups of glutaraldehyde or formaldehyde and thus interfere with the normal tissue sterilization process.

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OMPI In accordance with the present invention, the tissue may be stored and processed in conventional well-known conditions , and may be fixed (tanned) conventionally in 0.625% glutaraldehyde in either phosphate-buffered solutions , or phosphate-free buffers as described above. Tissue handling conditions outside of the sterilization process as conventionally known are not considered part of our present invention.

In accordance with the present invention, alcohols used to render the resistant microorganisms more susceptible to sterilization include aliphatic as well as aromatic alcohols and are preferably lower aliphatic alcohols containing from 1 to about 5 carbon atoms. Aliphatic alcohols include but are not limited to methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, t-butanol, cyclohexanol, n-octanol, allyl alcohol, and the like. Aromatic alcohols include benzyl alcohol, cresol, carbinol, and the like. Lower aliphatic alcohols in accordance with the present invention include but are not limited to methanol, ethanol, propanol, butanol, isopropanol, and pentanol. More preferably, alcohols in accordance with the present invention are ethanol and isopropanol. The concentration of alcohol to be used in the present invention falls in a range which will be limited at its lower end by the effectiveness in destroying resistant micro¬ organisms, and at its upper end by the impact or effect on the integrity of the tissue; and may depend somewhat on the type of alcohol used. We have found that ethanol concentrations falling within a range of from about 10% to about 30% are effective against these microorganisms , and thus describe a preferred range. A more preferred alcohol concentration is from about 20 to about 25% ethanol. The most preferred alcohol concentration is 22.5% ethanol.

Organic surfactants within the scope of the present invention include anionic, cationic, and nonionic surfactants and their salts. Preferred salts of the surfactants in the present invention include sodium and potassium. Anionic surfactants of the present invention are those having a relatively large hydrophobic region of hydrocarbon residues including both aliphatic groups , aromatic groups and combinations thereof bonded to a negatively charged ionic group. The aliphatic residues may be branched chains, straight chains , cyclic , heterocyclic , saturated or unsaturated. These hydrophobic residues may either be connected directly to an anionic group such as carboxylate, sulfate, or sulfonate; or connected thereto through an intermediate linkage such as an ester, amide, sulfonamide , ether, or aryl group . Anionic surfactants in one embodiment of the present invention are those having carboxylates bonded to the alkyl side chain of a steroid or through amino acids in the side chain; such as in the bile acids. Illustrative bile acids in accordance with the present invention include but are not limited to deoxycholic acid, cholic acid, lithocholic acid, taurocholic acid, and glycocholic acid, and their salts. Anionic surfactants in accordance with the present invention further include those having a carboxylate group bonded to a straight-chained aliphatic group preferably having from about 8 to about 20 carbon atoms; such as the sodium salts of fatty acids. Anionic surfactants containing carboxylate groups in accordance with the present invention further include those having the carboxylate group coupled to a hydrophobic portion through an amide, sulfonamide, or ester linkage such as in the N-alkanoyl amino acids and N-acylated amino acids. Illustrative of N-alkanoyl amino acids are those including but not limited to surfactants having the formula R., CONR2CHR3CO2- where R- is an aliphatic residue preferably having from about 8 to about 18 carbon atoms, R„ is hydrogen or methyl, and R„ is a conventional amino acid side chain. Illustrative side chains include the non-polar aliphatic side chains of alanine, leucine, isoleucine, valine, and proline; the aromatic rings of phenylalanine and tryptophan; the polar side chains of glycine, serine, threonine, cystine, and the like; and the charged polar groups of aspartic acid, glutamic acid, lysine, and the like. Preferred carboxlate containing surfactants in accordance with this embodiment of the present invention are those containing an amide linkage such as N-lauroylsarcosine. Anionic surfactants in accordance with an alternate embodiment of the present invention include sulphates of aliphatic alcohols such as alkyl sulfates having from about 6 to 18 carbon atoms , ethylene oxide modified sulfates of aliphatic alcohols , sulfated ethanol amides, or alkyl phenols such as the sulfonated alkylphenyl ethers. Further anionic surfactants include alkane sulfonic acids and alkylaryl sulfonic acids. Illustrative of the sulphates of aliphatic alcohols is sodium dodecyl sulfate. Alkane sulfonic acids in accordance with the present invention include those having the sulfur directly attached to the hydrophobic residue, such as 1-decane sulfonic acid; or coupled through an ester, amide, or ether; such as N-methyltaurine. Alkylaryl sulfonates are those having the sulfur directly attached to an aromatic ring such as phenyl or napthyl which is, in turn, coupled to the hydrophobic residue preferably having from about 8 to about 18 carbon atoms. Illustrative of this latter type of surfactant is dodecylbenzene sulfonic acid.

Cationic surfactants in accordance with the present invention include alkyl quaternary amines and their halide salts. Preferable surfactants in the present invention include the chlorine and bromine salts of tertiary amines connected directly to a hydrophobic residue or connected thereto through an amide linkage. Preferably the amines are directly connected to a relatively large hydrophobic portion having an aromatic residue such as benzene, pyridine or napthylene; aliphatic chain which is branched, unb^anched, cyclic, saturated, or unsaturated; or a combination of both aromatic and aliphatic residues. Illustrative alkyl quaternary ammonium surfactants include but are not limited to cetylpyridinium chloride, cetyltrimethyl ammonium bromide, trimethylphenyl ammonium chloride, decyltrimethyl ammonium bromide, hexdecyltrimethyl ammonium bromide, and the like.

Nonionic surfactants in accordacne with the present invention include polyoxyalkylene ethers, polyoxyalkylene alkylaryl ethers , aliphatic esters, polyethers, polyoxyalkylene ester derivatives ,

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OMPI saccharide ester derivatives, and combinations thereof. Nonionic polyoxyalkylene , and preferably polyoxyethylene , ethers are those having a relatively long hydrophobic residue and a hydroxyl end connected by one or more alkylene oxide residues. Examples of polyoxyalkylene ethers are polyoxyethylene lauryl ether (Brij) , polyoxyethylene oleyl ether, polyoxyethylene cetyl ether, and the like. Nonionic polyoxyalkylene, and preferably polyoxyethylene, alkylaryl ethers are those having a relatively large hydrophobic residue and a hydroxyl end connected thereto by an aryl, such as benzene or napthaline and one or more alkylene oxide residues. Examples of polyoxyalkylene alkylaryl ethers include polyethylene Glycol p-Isooctyl phenyl ethers such as Triton X-100 and the like. Nonionic polyethers are those having the formula CH3(CH2)-O-(C2H4O M where N is about 11 , and M is about 23.

Nonionic aliphatic esters include aliphatic fatty acid esters , polypropyleneglycol fatty acid esters such as propyleneglycol monostearate , and glycerol fatty acid esters such as glycerol monostearate. Aliphatic fatty acid esters are those having the formula R.COORg where R. is an alkyl preferably having from about 8 to about 20 carbon atoms, and R_ is an aliphatic residue having from about 1 to about 5 carbon atoms. Saccharide and polyoxyalkylene ester derivatives are those having either a 5 or 6 carbon sugar in the former or a polyoxyalkylene chain in the latter coupled to a relatively long hydrophobic residue through an ester bond. Illustrative saccharide derivatives include sorbitol coupled to fatty acids to form surfactants such as sorbitan trioleate, sorbitan strearate, sorbitan monooleate, and the like.

Polyoxyalkylene ester derivatives include polyoxyethylene monooleate, polyoxyethylene monostearate, and the like.

Combinations of polyoxyalkylene ether derivatives and sorbitol ester derivatives found to be useful in the present invention include polyoxyethylene sorbitan fatty acid derivatives such as polyoxyethylene (20) sorbitan monooleate ( Poly sorb ate-80 , Tween-80 manufactured by DIFCO) . In accordance with the preferred embodiment of the present invention, the concentration of surfactant is from about 0.1 to about 10 percent (w/v) and more preferably from about 0.5 to about 5 percent. Most preferably, the surfactant concentration is from about 0.5 to about 1.5 percent.

In accordance with a preferred embodiment of the present invention, the tissue, surgical instruments, and medical devices are treated with the improved sterilant composition at temperatures ranging from about 20°C to about 100°C. More preferably the temperature is maintained from about 20°C to about 40°C. We have unexpectedly found that sterilization is enhanced at temperatures above room temperature (20°C) to a range of from about 30 to about 40°C. A most preferred range is from about 32°C to about 38° C. Temperatures above this physiological temperature range might be harmful to the biological tissue, but would not be harmful to other surgical instruments or medical devices in accordance with the present invention.

In accordance with the present invention, the biological tissue is exposed to the improved sterilant composition for a period of time related to the quantity of microorganism present and the level of sterility assurance desired; and consequently the time can be varied according to needs. In accordance with a preferred embodiment of the present invention, and by way of example, tissue requiring a decrease in microorganisms (Microascus

0 — 6 cinereus) from about 10 to 10 requires an exposure time of at least 7 hours at 35+3° C. The interplay between time and temperature would dictate that if the temperature is increased, the time of exposure would necessarily decrease. Accordingly, if the temperature is decreased, then the exposure time would have to be increased dramatically.

In accordance with the present invention, a sterilant composition is considered potentially effective if it shows about a

90% reduction of the organisms being tested. Preferably, the

OMPI

, wipo . effective solution shows a complete destruction of a large quantity

(10 5 to 106 ) of test organisms within an acceptable time frame

(from about 7 to about 8 hours) . Moreover, sterilant compositions are considered particularly effective if the organism destroyed is on a surface or on tissue (substrate) . As discussed above, we have found that solutions containing formaldehyde and ethanol without surfactant are effective against mircoorganisms in solution, but are not as effective against Microascus cinereus on tissue. We have further found that biological tissue (bioprosthetic heart valves) exposed to surfactant , rinsed free of surfactant, and then exposed to sterilants such as glutaraldehyde and alcohol are less effective against Microascus cinereus. These observations unexpectedly show that the tissue must be exposed to the glutaralde¬ hyde or formaldehyde, alcohol, and surfactant simultaneously for optimal effect. In accordance with the present invention, the tissue may be exposed to these three components in separate steps or in a single step . A single-step exposure of tissue to the improved sterilant compositions of the present invention is preferred. c .

The present invention is further illustrated by the following examples which are not intended to be limiting.

Example I Extracted porcine aortic heart valve tissue was thoroughly rinsed and shipped in an isotonic (285+15 milliosmols) solution of 0.02M phosphate-buffered saline (0.885 weight percent sodium chloride) at pH 7.3 and at about 4°C , fixed in 0.625 weight percent glutaraldehyde in an isotonic solution at pH 7.4 and at room temperature.

Example II A suspension of Microascus cinereus in a phosphate-buffered solution having a spore count of 10 5" to 106 spores /ml was injected into various portions of a bioprosthetic heart valve prepared from the extracted tissue of Example I to give a final inoculum level of 7.1x10 spores per valve. The valve tissue was further exposed to 100ml of a 0.02M phosphate-buffered

OivIPI solution containing 4+0.4 percent formaldehyde, 20 percent ethanol (100% anhydrous ethanol) , pH 7.4 at 20-22°C for 48 hours . The effect of the sterilant solution on the growth of Microascus cinereus was assessed at regular intervals by removing the valves from the solution, placing them in a fluid fungal support nutrient medium and visibly determining the viable spores remaining on the valve after treatment for 48 hours. At the end of 48 hours, no reduction in spore count was evident in any of 5 samples of the valve tissue tested.

_ E _x—amp _le — HI A suspension of Microascus cinereus in a phosphate-buffered solution having a spore count of 10 to 10 spores /ml was injected into various portions of a bioprosthetic heart valve prepared from the extracted tissue of Example I to q give a final inoculum level of 5.6x10 spores per valve. The valve tissue was further exposed to 100ml of a 0.02M phosphate-buffered solution containing 4+0.4 percent formaldehyde, 20 percent ethanol, 1 percent Tween-80 (sorbitan monooleate polyoxyethylene) , pH 7.4 at 20-22 °C for 48 hours. The effect of the sterilant solution on the growth of Microascus cinereus was assessed at regular intervals by removing valve from the solution, placing them in a fluid fungal support nutrient medium and visibly determining the viable spores remaining on the tissue after treatment for 48 hours. From this analysis, we found that 3 of 5 samples tested had growth of the organism after 48 hours, and that the average time required to reduce the spore count by a factor of 10 (a 1 log reduction) is about 578.9 minutes (9 hours and 39 minutes) .

Example IV A _ suspension of — M—i—c—r—o—a—s—c—u—s——— c—i—n—e—r—e—u—s— having a spore count of 10 to 10 spores /ml was injected into various portions of a bioprosthetic heart valve prepared from the extracted tissue of Example I to give a final inoculum level of 1.6x10 spores per valve was further treated as in Example III. At the end of 24 hours, no logarithmic reduction in spore count was evident in any of 5 samples of the valve tissue tested.

s VIPO Example V A suspension of Microascus cinereus having a spore count of 10 5 to 106 spores/ml was injected into various portions of a bioprosthetic heart valve prepared from the extracted

3 tissue of Example I to give a final inoculum level of 7.7x10 spores per valve, after which the valve tissue was further treated as in

Example III with the exception that the tissue was treated at 32°C for 24 hours. At the end of 4 hours only 2 of 5 samples tested

• showed organism growth; and after 6 hours , none of 5 samples tested showed organism growth. Moreover, the time required to reduce spore count by a factor of 10 is about 47 minutes.

Example VI A suspension of Bacillus pu _milas in a phosphate- buffered solution having a spore count of 10 to 10 spores per ml was injected into various portions of a bioprosthetic heart valve prepared from the extracted tissue of Example I to give a final

4 inoculum level of 1.6x10 spores /valve, after which the valve tissue was further treated as in Example III with the exception that the tissue was treated at 32°C. At the end of 30 minutes, there was no growth of Bacillus pumilas detected in any of 5 samples of the valve tissue tested.

Example VII A suspension of Chaetomium globosum in a e a phosphate-buffered solution having a spore count of 10 to 10 spores per ml was injected into various portions of a bioprosthetic heart valve prepared from the extracted tissue of Example I to

4 give a final inoculum level of 2.2x10 spores /valve, after which the valve tissue was further treated as in Example III with the exception that the tissue was treated at 32°C. At the end of 30 minutes, there was no growth of Chaetomium globosum detected in any of 5 samples of the valve tissue tested.

Example VIII The extracted tissue of Example I was exposed to 100ml of a 0.02M phosphate-buffered saline solution containing

22.5 percent ethanol, 4.0+0.4 percent formaldehyde , and 1.2 percent sorbitan monooleate polyoxyethylene (Tween-80) , pH 7.3 at

35°C and resulted in complete sterilization after 8 hours. The treated tissue was further analyzed to assess the integrity of the tissue after exposure to the sterilization. The results of our analysis show that there was no significant difference in the cross-link stability as indicated by shrinkage temperature, tissue stability as indicated by pronase digestion; amino acid analysis, ninhydrin analysis; uronic acid content, histologic examination as indicated by staining with Hematoxylin-Eosin, aldehyde fuschin, PAS/alcian bluue, and Trichrome; surface morphology as determined by scanning electron microscopy and transmission electron microscopy. Subcutaneous implantation of tissue in growing and mature rabbits was performed, and the degree of collagen fiber degeneration, host cellular infiltration, and tissue calcification (a significant concern for tissue valves) were determined for the tissue exposed to the current sterilant and tissue exposed to the improved sterilant. Host cell infiltration was comparable for both groups, while collagen fiber degeneration was less for the improved process. Tissue calcification was also less for the tissue exposed to the improved sterilant.

Example IX A suspension of Microascus cinereus in a phosphate-buffered solution having a spore count of 10 to 10 spores per ml was inoculated into 100 ml of 0.02M phosphate-buffered sterilant solution containing 4+0.4 percent formaldehyde, 20 percent ethanol, and 1 percent Tween-80 , pH 7.51 at 20-22°C to give a final inoculum level of 3.3xl03 spores /ml. The inoculated sterilant solution was incubated with stirring; and 1.10 ml aliquots were taken at intervals , filtered through 0.45μ filter to collect spores, and placed in solid media containing a fungal support nutrient where it was treated at 33° C for 10 days to determine the number of spores remaining after treatment by visual counting. After 8 hours of exposure to the sterilant solution, no more spores were detected; and the average time required to reduce the spore count by a factor of 10 is about 114 minutes. Example X The experiment of Example IX was repeated in all essential details with the exception that the spore-inoculate sterilant solution was treated at 33°C rather than 20-22°C . After 4 hours of exposure to the sterilant solution, no more spores were detected, and the average time required to reduce the spore count by a . factor of 10 is about 61 minutes.

The present invention has been described in specific detail and in reference to its preferred embodiments; however, it is to be understood by those skilled in the art that modifications and changes can be made thereto without departing from the spirit and scope thereof.

Citas de patentes
Patente citada Fecha de presentación Fecha de publicación Solicitante Título
US4315919 *6 Oct 198016 Feb 1982Edward ShanbromDepyrogenation process
US4323358 *30 Abr 19816 Abr 1982Vascor, Inc.Method for inhibiting mineralization of natural tissue during implantation
Otras citas
Referencia
1 *See also references of WO8401894A1
Clasificaciones
Clasificación internacionalA61L27/36, A61B17/00, A61L2/16, A61L2/00, A61B18/00, A01N1/02
Clasificación cooperativaA61L2/0088, A61L27/3687, A61L27/507
Clasificación europeaA61L27/36H2, A61L27/50E, A61L2/00P4A
Eventos legales
FechaCódigoEventoDescripción
14 Nov 1984AKDesignated contracting states:
Designated state(s): CH DE FR GB LI
16 Ene 198517PRequest for examination filed
Effective date: 19841102
29 Oct 198617QFirst examination report
Effective date: 19860908
14 Oct 1987RAP1Transfer of rights of an ep published application
Owner name: BAXTER TRAVENOL LABORATORIES, INC.
27 Dic 198918DDeemed to be withdrawn
Effective date: 19890601
8 Ago 2007RIN1Inventor (correction)
Inventor name: LOWERY, GUY
Inventor name: NASHEF, AWS, S.