WO1996003093A1 - Bioprosthetic implants and method of making and using same - Google Patents
Bioprosthetic implants and method of making and using same Download PDFInfo
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- WO1996003093A1 WO1996003093A1 PCT/US1995/009590 US9509590W WO9603093A1 WO 1996003093 A1 WO1996003093 A1 WO 1996003093A1 US 9509590 W US9509590 W US 9509590W WO 9603093 A1 WO9603093 A1 WO 9603093A1
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- bioprosthetic
- valves
- lipids
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- 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
- A61L27/38—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 containing added animal cells
- A61L27/3804—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 containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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- 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
- A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
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- 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
Definitions
- the present invention relates to bioprosthetic implants from which the lipids have been removed by treatment with enzymes which degrade lipids, and methods of making and using same. Treatment with the lipid degrading enzymes, lipase and phospholipase, decreases the calcification of the bioprosthesis upon implantion in vivo.
- the bioprosthetic implant may preferably be recellularized prior to implantation.
- a bioprosthesis is a prosthesis made of biological as opposed to synthetic material.
- a bioprosthesis may be more effective than a synthetic prosthesis because it both physiologically and mechanically more closely resembles the body tissue which is to be replaced. This may be especially true in the case of a bioprosthesis serving both a structural and functional role within the recipient, such as a bioprosthesis implanted as a substitute blood vessel, heart valve, skin or other tissue.
- certain such bioprostheses may elicit an immune response in recipients due to the presence of antigenic components within the implant.
- such a bioprosthesis may calcify over time and induce thrombogenesis.
- tearing and cuspal calcification occur in well defined patterns. Whereas tearing generally occurs at the free edge and at the base of the cusps where they attach to the supporting stent (Ishihara et al., 1981; Pomar et al, 1984; Grabenwoger et al., 1992), calcification usually initiates within the valve cusps and spreads outward through the cusp surface.
- the disclosed methodology should result in reduction of some of the potentially damaging events that may occur in the processing of collagen-based tissues prior to transplantation (e.g., mechanical and biochemical events occurring during tissue procurement) and, in this sense, should reduce calcification resulting from structural damage to the bioprosthesis, the disclosed methodology is not specifically directed toward increased removal of lipids from the implant.
- German Patent 826,577 disclose ficin enzymatic treatment of collagen from mammals, which may then be fixed to aid in tissue handling, and implanted.
- the enzymes used in these approaches are proteolytic in nature, and thus are not expected to reduce the amount of lipid remaining in the bioprosthetic implant. Accordingly, these enzymatic approaches, like the approach employed in the European Patent Application 0 564 786, do not appear to remedy the ostensible failure of the methodology of United States Patent Nos. 4,801,299 and 4,776,853 to remove substantially all the lipids from the resultant bioprosthetic implant.
- the invention relates to a bioprosthetic implant which includes an extracellular matrix obtained by treatment of a collagen-based body-derived tissue with a 3093 PC ⁇ 7US95/09590
- the invention also relates to a method of implantation which comprises introducing the bioprosthetic implant into a recipient. Further, the invention relates to a method of preparing a bioprosthetic implant which comprises treating a collagen-based body-derived tissue with a lipid-degrading enzyme. The invention also relates to a method of recellularizing a bioprosthetic implant which comprises contacting the implant with cells, and maintaining the implant with the cells in tissue culture medium.
- FIGURE 1 is a photograph of porcine heart valve cusps stained with Oil-Red-o after the tissue has been extracted with a series of buffered solutions containing salts, a detergent, and enzymes that degrade nucleic acids. These valve cusps have been treated as set forth in Example 3.
- FIGURE 2 is a photograph of porcine heart valve cusps that were extracted with detergent and then subsequently treated with enzymes that degrade lipids, and stained with Oil-Red-0 as set forth in Example 3.
- the present invention relates to bioprosthetic implants from which the lipids have been removed by treatment with enzymes which degrade lipids, and methods of making and using same.
- the removal of the lipids from the bioprosthetic implants decreases the calcification of the bioprosthesis, resulting in increased durability of the bioprosthesis in vivo.
- the present invention further provides bioprosthesis derived from a mammalian body-derived tissue from which lipids have been removed through the use of a lipid-degrading enzyme, comprising a matrix of collagen and elastin, and lacking substantially all cellular and extracellular lipids, nucleic acids, cellular membranes and cytoplas ic components.
- the bioprosthesis of the present invention may be used to treat tissue failure, including heart disease due to the failure of a heart valve.
- a bioprosthetic implant according to the present invention comprises extracellular matrix obtained by treatment of a collagen-based body-derived tissue with a lipid-degrading enzyme.
- extracellular matrix comprises the intricate meshwork of interacting, extracellular acromolecules found in the extracellular space of most tissues.
- the extracellular matrix of the present invention is intact, i.e., the structure of collagen and elastin within the matrix is analogous to that found n vivo.
- the extracellular matrix may or may not further comprise cells, cell membranes, nucleic acids, lipids and cytoplasmic components.
- Body-derived tissue generally comprises any suitable collagen- containing tissue removed from the body and in the form of extracellular matrix, which may or may not further comprise elastin.
- the collagen-containing tissue used to derive the bioprostheses of the present invention may be selected from the group consisting of skin, blood vessels, heart valves, ligaments, tendons, bone, trachea, cartilage, dura mater, nerves and other such tissues.
- the collagen-containing tissue may be obtained from any source, and preferably, will be obtained from a mammalian host. Even more preferably, the tissue will be obtained from a human, an ungulate (e.g., a caprine, bovine or porcine species), a canine or a feline.
- Human tissue including cadaver tissue is available through tissue banks and hospitals. Other mammal tissue can be obtained through suppliers of laboratory mammals, or through the meat processing industries.
- a lipid-degrading enzyme according to the present invention is any fat-splitting or lipolytic enzyme which cleaves a fatty acid residue from the glycerol residue in a neutral fat or a phospholipid.
- a preferred lipid- degrading enzyme is selected from the group consisting of Upases and phospholipases, and mixtures thereof. Even more preferred are the lipases triacylglycerol and diacylglycerol lipase, and the phospholipases phospholipase A,, A 2 , B, C and D.
- Preferred triacyglycerol lipases according to the present invention are triacylglycerol lipase from porcine pancreas (Boehringer Mannheim, Laval, Quebec) and triacylglycerol lipase Type XIII from Pseudomonas species (Sigma Chemical Co. , St. Louis, MO) .
- the lipases and/or phospholipases may be employed alone, or in appropriate combination to comprise the lipid degrading enzymes.
- triacylglycerol lipase from porcine pancrea and triacylglycerol lipase Type XIII from Pseudomonas species will be used in combination.
- These preferred triacylglycerol lipases may also be used in conjunction with other lipases and/or phospholipases.
- lipid-degrading enzyme may be employed at a concentration ranging from about 0.25 units/milliliter (ml) to about 50 units/ml. It is expected that the concentration of a particular lipid-degrading enzyme to be employed will differ according to its efficacy.
- porcine pancreatic triacylglycerol lipase may preferably be employed at a concentration ranging from about 0.5 units/ml to about 70 units/ml, more preferably, 1.0 units/ml to about 50 units/ml.
- the lipid- degrading enzyme will preferably be employed in a buffered solution. Even more preferably, the solution will be buffered at a pH of about 7.4. Suitable buffers to employ in the lipase solution, as well as the other tissue processing solutions of the present invention, include Tris-HCl, and other appropriate buffers such as are known in the art. preferably, however, the buffer will not result in the inactivation of the lipid- degrading enzyme.
- Processing of the collagen-based body-derived tissue in the solution comprised of the lipid-degrading enzyme can be carried out for any suitable length of time such that the amount of lipids remaining in the tissue is less than the amount of lipids which would be present were the tissue subjected to some other means of effecting lipid removal in the absence of processing using a lipid- degrading enzyme, such as, for example, detergent processing.
- a lipid- degrading enzyme such as, for example, detergent processing.
- processing with use of the lipid-degrading enzyme will be carried out for up to about two hours, more preferably for up to about eight (8) hours, and even more preferably for up to about 24 hours.
- a further increase in the processing time or amount of lipid-reducing enzyme employed will not result in a further decrease in the amount of lipid-reducing enzyme employed or processing time, respectively, based on such factors as the half-life of the lipid-degrading enzyme, the intrinsic ability of the lipid-degrading enzyme to hydrolyze lipid esters, etc. Accordingly, it is well within the means of the ordinary skilled artisan to optimize the precise manner in which tissue processing is to be carried out.
- processing with the lipid-degrading enzyme can be carried out at any suitable temperature.
- a low temperature such as a temperature less than about 20°C may be preferred.
- certain lipases may be more effective at an increased temperature, and in these cases, it may be desirable to carry out processing with the lipid-degrading enzyme at a temperature greater than about 20°C.
- processing with the lipid-degrading enzyme can be carried out at a temperature of between about 20°C and about 45°C, and even more preferably, at a temperature of about 37°C.
- a bioprosthetic implant may be suitable for implantation without further processing to reduce the immune potential of the implant.
- Glutaraldehyde has been used to stabilize collagen-based bioprosthetic implants and has demonstrated clinical utility for at least twenty (20) years for tissue heart valve implants and pericardial augmentation materials.
- Glutaraldehyde is a bifunctional agent and stabilizes collagen-based materials by covalently binding to free amino groups (i.e., crosslinking) , preventing recognition by the recipient of the implant of the collagen protein in the implant as a foreign material.
- any version of the commercial processes using glutaraldehyde or other acceptable crosslinking agents are suitable to prepare the implant of the present invention for implantation in cases where such further optional crosslinking is desired.
- Crosslinking agents and methods of using same are well known to those skilled in the art.
- a preferred process would mildly crosslink the material while maintaining native biomechanical properties Such a preferred process would still reduce the potential for the material to elicit an immune response.
- the bioprosthesis either may not require fixation at a conventional concentration of glutaraldehyde or may require fixation at a weaker concentration of glutaraldehyde than conventional concentrations, preferably in a solution of glutaraldehyde that is substantially less than 0.5% glutaraldehyde.
- the ideal bioprosthesis according to the present invention is biologically active tissue.
- the tissue prepared according to this invention preferably consists of a structurally sound collagen and elastin matrix which further may preferably be seeded with the recipient's fibroblasts or myofibroblasts and endothelium obtained from biopsy before the scheduled implantation surgery.
- the implant may be cultured in vitro before implantation as a viable graft.
- the implant preferably lacks substantially all lipids.
- the implant preferably lacks other solubilized cellular and extracellular components, and may preferably lack fixatives (such as cross-linking agents),and therefore has minimal antigenicity.
- the implant preferably is not chemically modified or crosslinked. Therefore, its mechanical behavior should substantially be the same as the natural biological tissue.
- the present invention provides an implant according to the present invention wherein all lipids are substantially removed.
- the invention also provides a method of preparing a bioprosthetic implant which comprises treating a collagen-based body-derived tissue with a lipid-degrading enzyme such that Figure 1 illustrates porcine heart valve cusps stained with Oil- Red-0 after the tissue has been extracted with a series of buffered solutions containing salts, a detergent, and enzymes that degrade nucleic acids, as in Example 3. These valve cusps have been treated in particular by the methods described in Kle ent et al.
- FIG. 1 is a photograph of porcine heart valve cusps that were extracted in accordance with the methods described in Klement et al . , then subsequently were treated with enzymes that degrade lipids in accordance with this invention, and stained with Oil-Red-O.
- stainable lipid clusters are dramatically reduced following lipase treatment, indicating that significant amounts of the lipid clusters have been removed.
- Table 1 presents quantitative calcification levels, measured by atomic absorption analysis, of various bioprosthetic implants prepared as in Example 5 implanted in young growing rats with an in vivo time of three weeks.
- the glutaraldehyde fixed specimens were prepared using a typical commercial process with a glutaraldehyde concentration of 0.5M.
- the "Cell Extracted” implants were prepared using a modified process as identified in the prior art by Klement, et al .
- the various Lipase treated specimens i.e., "Lipase A”, “Lipase B”, “Lipase C”, and “Lipase D" were prepared using extracted specimens that were further treated with varying concentrations of lipid-degrading enzymes, as set forth in Example 5.
- the Fresh specimen was prepared by washing tissue samples received directly from the abattoir. These samples were not further processed.
- the data in Table 1 confirms that there is an additional reduction in calcification in specimens treated with lipid-degrading enzymes as compared with those specimens prepared using only the extraction processes identified in the prior art. This data suggests that with the addition of a lipid-degrading enzyme treatment step following detergent extraction, reduced calcification levels, and subsequently longer clinical useful life of bioprosthetic implants may be achieved.
- the invention also provides a method of preparing a bioprosthetic implant which comprises treating a collagen-based body-derived tissue with a lipid-degrading enzyme. Accordingly, the present invention provides a method of treating a collagen based tissue sample of an animal, preferably a mammal, to remove cellular and extracellular lipids before implanting the material in a body of a person.
- the method includes extracting the tissue with detergents and lipid-degrading enzymes, including lipases or phospholipases, and mixtures thereof.
- the collagen-containing tissue may include skin, blood vessels, heart valves, ligaments, tendons, bone, cartilage, dura mater, nerves and other such tissues.
- the method of preparing a bioprosthetic implant which comprises a collagen-based body-derived tissue with a lipid-degrading enzyme may further preferably comprise extracting the implant with a solution comprising salt.
- this method may comprise extracting the implant with a solution comprising detergent. Moreover, preferably this method may further comprise extracting the implant with a solution comprising nuclease. These steps may be performed in any order, and optionally, any combination of steps may be combined with extraction with the lipid-degrading enzyme to obtain the bioprsothesis.
- the method of the present invention may comprise one or more steps, including extracting the implant tissue sample with one or more buffered salt solutions, extracting the tissue sample with one or more detergents, treating the tissue sample with one or more enzymes which degrade lipids, and storing the tissue sample in a physiologically buffered solution.
- the method may additionally include the steps of isolating a tissue sample of biological material from a suitable donor, extracting the tissue sample with one or more buffered salt solutions to rupture the cells of the tissue sample, extracting the tissue sample with buffer solutions containing one or more detergents, treating the tissue sample with buffer solutions containing one or more enzymes, such as nucleases, which degrade nucleic acids, treating the tissue sample with buffer solutions containing one or more enzymes which degrade lipids, i.e., a lipase, phospholipase, or mixture thereof, reextracting the tissue sample with one or more buffered salt solutions, re-extracting the tissue sample with one or more detergents, and storing the tissue sample in a physiologically buffered solution.
- enzymes such as nucleases, which degrade nucleic acids
- buffer solutions containing one or more enzymes which degrade lipids i.e., a lipase, phospholipase, or mixture thereof
- tissue may be decellularized by extraction in a first solution comprised of a hypotonic buffer, protease inhibitors and antibiotics, which results in cell lysis. Subsequently, the tissue may be extracted with a solution comprised of a high concentration of salt, a non-ionic detergent such as Tris-HCl, protease inhibitors and antibiotics. In this step, soluble components of the extracellular matrix, as well as cytoplasmic components, are extracted. The tissue may then be extracted with urea, which swells the tissue, making it more amenable to extraction.
- the tissue may be extracted with a suitable detergent, such as Chapso detergent.
- a suitable detergent such as Chapso detergent.
- Antibiotics, antifungal agents and protease inhibitors may be included in the extraction mixture as necessary or desired.
- Nucleases such as purified and protease-free ribonuclease and deoxyribonuclease may optionally be used to remove nuclear material (i.e., DNA and RNA) from the tissue, followed by detergent extraction and rinsing of the tissue.
- the tissue may be extracted with lipid-degrading enzymes to remove cellular and extracellular lipids and phospholipids, and washed in a solution comprised of heparin.
- the tissue may be further extracted using a detergent such as Chapso, and rinsed in an hypotonic solution.
- Antibiotics and antifungal agents may be employed in processing solutions and tissue culture media to maintain sterility of the bioprosthetic implant at various stages of processing.
- Antibiotics may be selected from the group consisting of gentamicin, kanamycin, penicillin, streptomycin, neomycin. vancomycin and mixtures thereof.
- a preferred antibiotic according to the present invention is gentamicin.
- Antifungal agents may be selected from the group consisting of amphotericin B, polymyxin B, fungizyme and nystatin. Other suitable antibiotics and antifungal agents such as are known in the art may also be employed in the present invention.
- protease inhibitors may also be employed in the present invention to inhibit proteolytic enzymes embedded in the collagen-based matrix which can cause degradation of various components of the matrix.
- Preferred protease inhibitors according to the present invention include phenylmethylsulfonyl fluoride (PMSF) , N-ethylmaleimide (NEM) , ethylene glycol-bis (2-aminoethyl ether)- N,N,N' ,N , -tetraacetic acid (EGTA) , leupeptin, aprotinin, pepstatin, and ethylenediaminetetraacetic acid (EDTA) .
- detergents may be used to remove cellular and antigenic components.
- removal can be accomplished using other chemical treatments (e.g. , using proteolytic enzymes such as chymotrypsin) , and that various approaches may be employed in combination (e.g., as in a preferred method set forth herein, wherein tissue is processed by incubation in solutions comprised of detergent, salts and enzymes) .
- proteolytic enzymes such as chymotrypsin
- Acceptable detergents for use in the present invention include those set forth in U.S. Patent No. 4,776,853, as well as polyoxyethylene (80) sorbitan nono- oleate (i.e., Tween 80), sodium deoxycholate, and 3-[3- cholamidopropyl)-dimethyla monio]-2-hydroxy-l- propansulfonate (Chapso; Boehringer Mannheim, Laval, Quebec) , DeoxyBigChap detergent, and Little Chap detergent.
- a preferred detergent according to this invention is Chapso detergent.
- other detergents such as are known and routinely used by those skilled in the art may also be employed.
- bioprosthetic implant preferably all solutions are processed, such as by filtering the solutions through a 0.22 uM filter prior to use, to ensure sterility of the resultant bioprosthetic implant.
- the bioprosthetic implant may also be sterilized by other means known to those skilled in the art (e.g., by exposure to ionizing radiation) .
- Hypotonic solutions which may be employed for rinsing and the high salt buffer solution which may be used to extract cytoplasmic and extracellular components may preferably be buffered, even more preferably at a pH of about 7.4.
- the present invention also provides a method of recellularizing a bioprosthetic implant obtained according to the present invention. This method comprises contacting an implant according to the present invention with cells, and maintaining the implant with the cells in suitable tissue medium.
- the endothelial cell (which typically lines the inner surface of this type of tissue) may be employed for recellularization.
- the fibroblasts or myofibroblasts are isolated from the same individual that will be the recipient for the bioprosthetic implant.
- the cells may be isolated from a mammal.
- the fibroblasts or myofibroblasts may be derived from organs or skin, as appropriate, which can be obtained by biopsy or other appropriate means such as are known to those skilled in the art. Alternately, the fibroblasts or myofibroblasts may be obtained from cadavers or fetal tissue.
- a preferred source of fibroblasts according to the present invention is gingiva or foreskin, and a preferred source of myofibroblasts is heart. Also preferred are cells taken from skin, gingiva or granulation tissue.
- Fibroblasts or myofibroblasts can be isolated by desegregating an organ or tissue, which can be done mechanically (e.g. , through use of blenders, grinders, homogenizers, pressure cells, etc.), through use of enzymes or chelating agents (e.g., trypsin, chymotrypsin, dispase, etc.) which allow single cell dispersions to be obtained, or by any means or combination of means which are known to those skilled in the art.
- the fibroblasts may be isolated using techniques which are standard such as cell separation, selective destruction of unwanted cells, cloning of specific cell types, filtration, fluorescence-activated cell sorting, etc.
- any commercially available medium can be used for the growth of cells on the bioprosthetic implant, such as RPMI 1640 and Dulbecco's Modified Minimal Medium.
- the medium may be supplemented as appropriate,for instance, with fetal calf serum, and may be further supplemented with cytokines, growth factors, and various interleukins as well as other growth-promoting agents, to maximize growth of cells in culture.
- Standard sterile tissue culture techniques should be employed, and the medium should be changed as appropriate.
- the invention also discloses a method of implantion which comprises introducing an implant according to the present invention into a recipient.
- the bioprosthetic implant has been recellularized prior to implantation.
- the invention also discloses a method of implanting in a body of a person a bioprosthesis in which substantially all of the cellular and extracellular lipids have been removed.
- the present invention further provides a kit for treating a tissue sample of a mammal to remove substantially all cellular and extracellular lipids before implanting the sample in a body of a person.
- the kit includes one or more receptacles containing, buffer solutions, and enzymes which degrade lipids, including lipases or phospholipases.
- the kit may include a receptacle containing enzymes which degrade nucleic acids and a receptacle containing detergents.
- Lipase Type XIII from Pseudomonas species.
- the following reagents were obtained from Gibco (Ontario, Canada): BRI, Heparin, and Phenylmethyl Sulfonylfluoride (PMSF) .
- Urea was obtained from Fisher Scientific (Ontario, Canada) .
- Other reagents used are commercially available from commercial suppliers.
- the cusps were placed on the stage of a dissecting stereo microscope with the ventricularis side up. By gently grasping and carefully pulling on the ventricularis with forceps, the two layers could be separated along their natural cleavage plane, which appeared to be the midline of the spongiosa. Dissection progressed from the base, where the lipids were most abundant, through the body of the cusp towards the coaptation region. Since the spongiosa did not continue into the Nodulus Arantius and into the coaptation region, it was not possible to separate the layers and examine these areas for lipids. Saline solution was applied repeatedly to keep the tissue moist.
- lipid clusters could be easily visualized and their distribution could be quantified using a point counting technique.
- the dissected valve cusp was first placed on a piece of celluloid divided into 144 quadrants (12 x 12) and each quadrant was examined at high magnification for the presence of lipid clusters.
- the presence of lipids was quantified using a conventional stereological point counting method.
- An estimate of the spatial density of lipids was obtained by counting the number of grid points in the eyepiece that overlay lipid clusters in each quadrant.
- the distribution of lipids in the cusp was visualized by expressing the lipid count in each quadrant as a "density". Since each quadrant had a total of 100 grid points, the values ranged from 0 to a maximum of 100. This density or darkness of each quadrant was therefore proportional to the number of occurrences of lipid clusters in that particular quadrant. A density of "zero" or white, meant that no lipids were found in a given quadrant.
- the density mapimages of all cusps were spatially averaged together. Spatial averaging was done by summing up the pixel values of all the images at a particular x,y pixel location and dividing by the number of images.
- the target image was a simplified, idealized cusp shape, to which all the real cusp density plot images were warped.
- the same grid used for the source image was applied to the ideal cusp shape, the "target image”, and the nodes of the "target mesh” were positioned to correspond to lie over the same landmarks as they did on the source image.
- the source mesh was warped to align the source mesh nodes with the target mesh nodes.
- the underlying source image was therefore warped along with the mesh, so that its outline corresponded to the ideal cusp shape.
- the lipid clusters were excised from the aortic valve cusps, and the lipids were extracted and identified by thin layer chromatography (TLC), and gas/liquid chromatography (GLC) , as per Huff et al., 1993.
- TLC thin layer chromatography
- LLC gas/liquid chromatography
- 2 cusps from each extraction were placed in a 3:2 mixture of hexane/isopropanol, were dried under nitrogen, resuspended in 2:1 chloroform/methanol, and were separated by TLC with petroleum ether/diethyl ether/acetic acid (84:15:1).
- the free cholesterol, esterified cholesterol, and triglyceride spots were identified by exposure to iodine. The spots were then scraped from the TLC plate and extracted, and the mass for each constituent was determined by GLC. (4) Light Microscopy
- FIGURES 1 and 2 were taken through the dissecting microscope, showing the lipid clusters under white light illumination and following staining with Oil-Red-O.
- Lipids generally did not extend into the belly of the valve cusp, and no lipids were observed in the coaptation region or near the free edge.
- the amount of lipid found in a given cusp was highly variable, ranging from zero occurrences up to 3% coverage of cusp area.
- the distribution of lipids in each of the three cusps was similar, with a greater occurrence in the left coronary cusp.
- Lipids were found in 19 of 20 valves in the left coronary cusp, 15 of 20 in the non coronary cusp, and 11 of 20 in the right coronary cusp. More importantly, 20 out of 20 valves had lipids in at least two of the three cusps.
- lipid clusters occur with surprising abundance in the aortic valve cusps of juvenile pigs.
- a sampling of valves from three abattoirs in the Ontario region was positive in all cases, suggesting that the presence of lipids may be universal and not dependent upon a particular strain of pig or method of farming. While the reason for the presence of lipid droplets in aortic valve cusps is presently unclear, it may be related to the high protein, high fat diet that farm pigs are fed to encourage rapid growth.
- Thubrikar has shown that calcification occurs at the base of the valve cusps (Thubrikar et al. , 1983), in the same areas that we found large amounts of lipids. Less calcification has generally been found in the belly of the cusps and towards the Nodulus Arantius, areas where we found no lipids.
- the correlation between the distribution of lipids in porcine aortic valve cusps, and clinically significant calcification of the prosthetic valves is particularly evident from images of x-rayed valves, (Cipriano et al . , 1984) .
- the tissue of porcine aortic valve cusps was extracted with a series of buffered solutions containing salts, a detergent, and enzymes that degrade nucleic acids. These valve cusps were treated using methods described in the prior art (ie, particularly as set forth in US Patent No. 4,776,853). Lipids present in valve cusps prepared according to the prior art were compared to the present example, and as set forth in Example 4. We dissected the fibrosa and the ventricularis of such extracted valve cusps using a icrodissection technique (Vesely et al . , 1992). Following such dissections, we discovered clusters of spherically shaped Objects or droplets, roughly 50 micrometers in size. Staining with Oil-Red-0 indicated that these droplets were lipids (Knuth, 1984; Boyan et al . , 1989).
- Example 4 We determined that there was little difference in the amount of lipids present in the valves dissected and stained with Oil-Red-0 in accordance with Example 2 and in the valves extracted, dissected and stained with Oil-Red-0 in accordance with Example 3. Thus, as described in Example 2, it would be helpful if endogenous lipids could be more effectively removed from tissue samples before implantation. Such a novel methodology is set out in Example 4.
- biological material is removed from a suitable donor.
- heart valves were removed from slaughtered pigs and subjected to a multistep process designed to lyse the cells and extract the cellular constituents. Namely, the valves were placed in large beakers and were subjected to a series of processing steps, constantly agitating the solutions with mechanized stirrers.
- valves were first soaked in buffers (0.05M Tris- HC1, pH 7.4) containing 0.05M NaCl and enzyme inhibitors (lmM PMSF, lmM EDTA, 0. luM Aprotinin, luM Leupeptin, luM Pepstatin) for 24 hours at 4°C.
- the valves were then soaked in buffers (0.05M Tris-HCl, pH 7.4) containing 1.5M NaCl and enzyme inhibitors (lmM PMSF, lmM EDTA, O.luM Aprotinin, luM Leupeptin, and luM Pepstatin) for 24 hours at 4°C.
- valves were rinsed in 0.05 M Tris buffer (pH 7.4) with enzyme inhibitors (lmM PMSF, lmM EDTA, O.luM Aprotinin, luM Leupeptin, luM Pepstatin) at 4°C numerous times.
- enzyme inhibitors lmM PMSF, lmM EDTA, O.luM Aprotinin, luM Leupeptin, luM Pepstatin
- the valves were then soaked in buffer (0.05M Tris-HCl, pH 7.4) containing 8M Urea for 1 hour at 4°C.
- the valves were rinsed six (6) times in Tris buffer pH 7.4 with enzyme inhibitor (lmM PMSF, lmM EDTA, O.luM Aprotinin, luM Leupeptin, luM Pepstatin) for one hour at 4°C.
- valves were soaked in buffer (0.05M Tris-Hcl, pH 7.4) containing 8 mM Chapso detergent and enzyme inhibitors (lmM PMSF, lmM EDTA, O.luM Aprotinin, luM Leupeptin, luM Pepstatin) for 24 hours at 4°C.
- the valves were rinsed six times in Tris buffer pH 7.4 with enzyme inhibitors (lmM PMSF, lmM EDTA, O.luM Aprotinin, luM Leupeptin, luM Pepstatin) for one hour each rinse at 4°C.
- Gentamicin (lmL) was added as necessary.
- valves were then soaked in 200 mL buffer (0.05 M Tris-HCl, pH 7.4) containing 4 mg DNase and 20 mg RNase for 24 hours at 37°C.
- the valves were exhaustively rinsed six times in 0.05M Tris buffer pH 7.4 with enzyme inhibitors (lmM PMSF, lmM EDTA, O.luM Aprotinin, luM Leupeptin, luM Pepstatin) for one hour with each rinse at 4°C.
- enzyme inhibitors lmM PMSF, lmM EDTA, O.luM Aprotinin, luM Leupeptin, luM Pepstatin
- Gentamicin was added as necessary.
- the valves were soaked again in buffer (0.05M Tris-HCl, pH 7.4) containing 8mM Chapso detergent for 24 hours at 4°C.
- the valves were subjected to six washes in 0.05M Tris-HCl pH 7.4 for one hour each rinse at
- valves were soaked in suitable buffers (0.05M Tris-HCl pH 7.4) containing varying concentrations of lipases and phospholipases for 24 hours at 37°C. Then the valves are soaked in buffer (0.05M Tris-Hcl) pH 7.4) containing 10 mg/ml heparin at 4°C. The valves are soaked in buffer ).05M Tris-HCl pH 7.4) containing 8mM Chapso detergent for 24 hours at 4°C. Then the valves were rinsed six times in 0.05M Tris buffer (pH 7.4) at 4°C.
- valves were then soaked in sterile culture medium (DMEM) with antibiotic (Gentamicin) with six (6) changes of antibiotic-containing medium over 1 hour and then stored in the same medium.
- DMEM sterile culture medium
- antibiotic Genetamicin
- the valves may then be implanted or cultured as described below. This process was found to remove substantially all of the cellular and extracellular lipids.
- the urea, DNase and RNase break up the cellular components.
- the detergent solubilizes and removes these components from the biological tissues through multiple extractions and rinses.”
- the urea also solubilizes and removes various connective tissue components.
- Variations may be made to the above process of extraction including using different detergents, different concentrations of lipases or phospholipases to extract lipids or phospholipids and performing the extraction steps at different pHs or at different temperatures.
- the process of extraction could be used on any biological material and the steps of extraction could be repeated or lengthened or several steps could be combined or one or more steps could be eliminated in an effort to remove substantially all of the lipids and phospholipids.
- porcine pancreatic triacylglycerol lipase and Pseudomonas triacylglycerol lipase Type XIII were used concurrently in the same lipid-degrading solution as follows:
- Triacylglycerol Lipase (units/ml) Pseudomonas Type XIII Porcine Pancreatic
- the amount of lipids present in the various valve tissues was determined by a combination of TLC and GLC as set forth in Example l. Whereas the amounts of free cholesterol, esterified cholesterol and triglyceride were greatest in the fresh tissue which was not subjected to any extraction protocol, these components were reduced in the tissue valves prepared according to U.S. Patent No. 4,776,853, and were further reduced in the tissue valves prepared according to the present invention.
- levels of triglycerides were about two to three times less than levels observed for tissue valves prepared according to U.S. Patent No. 4,776,853.
- extraction according to U.S. Patent No. 4,776,853 resulted in levels of cholesterol ester which were only about seven times less than levels of cholesterol ester observed for fresh tissue, cholesterol ester was not detectable in tissue valves prepared using the Lipase B and Lipase C conditions.
- a bioprosthetic implant prepared according to the method of the present invention is substantially free of lipids.
- use of a lipid-degrading enzyme according to this invention is effective at reducing lipid levels below those remaining after conventional detergent extraction, e.g., below about 50 ug triglycerides per gram of wet tissue, and/or about 8 ug cholesterol ester per gram of wet tissue, especially with respect to pig heart valves.
- the glutaraldehyde fixed tissue valve exhibited substantially greater calcification than the fresh tissue, which was not processed in any fashion.
- calcification was substantially reduced in the tissue valves prepared according to the present invention, and in the tissue valves prepared according to the Lipase C and Lipase D conditions in particular. Calcification in these tissues was further substantially reduced over the calcification observed for the tissue valve prepared according to U.S. Patent No. 4,776,853.
- the biological material may be cultured with the recipient's cells to partially repopulate the valve.
- the cells may be fibroblasts or myofibroblasts or both. The cells are
- suitable, non-neoplastic connective tissue source taken from the recipient. Examples include cells taken from skin, gingiva, or granulation tissue.
- tissue culture medium typically Dulbecco's modified Eagle's medium - DMEM
- tissue pieces were cut in a containment cabinet with a sterile scalpel into 1 X 1 mm pieces and transferred to a sterile tissue culture dish where they were covered with a sterile glass coverslip and incubated at 37ac in 5% C0 2 in culture medium containing 10% fetal calf serum, 50 ⁇ g/ l gentamicin.
- fibroblasts or myofibroblasts reach confluency from the explanted gingival tissue, the material was removed, the cells washed with sterile balanced salt solution, two times, and the cells were treated with buffered trypsin solutions by standard techniques to passage the cells. Following several passages, selected dishes of cells were washed and fixed in situ with formalin, then immunohistochemically stained with antibodies to vimentin, cytokeratin, muscle specific actin, and human fibroblasts to verify that the cells were fibroblasts or myofibroblasts.
- the biological material was washed with tissue culture medium, then placed in a sterile culture dish or flask, and once adherent to the dish, covered with medium containing 5 X 10 4 fibroblasts or myofibroblasts and incubated at 37sc with 5% C0 2 in culture medium supplemented with 10% fetal calf serum (FCS) , cytokines and antibiotics.
- FCS fetal calf serum
- the cells were cultured in the presence of cytokines, growth factors, transforming growth factors, various interleukins and other generally anabolic cytokines.
- porcine aortic valve cusps fibroblasts or myofibroblasts were typically present on the surface and superficially within the extracted cusp connective tissue matrix by the tenth day of culture, as determined by histological studies.
- Hufnagel CA Basic concepts in the development of cardiovascular prostheses. Am.J.Surg. 137:285-300, 1979.
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP95927528A EP0804127A1 (en) | 1994-07-28 | 1995-07-27 | Bioprosthetic implants and method of making and using same |
AU31531/95A AU3153195A (en) | 1994-07-28 | 1995-07-27 | Bioprosthetic implants and method of making and using same |
MXPA/A/1997/000727A MXPA97000727A (en) | 1994-07-28 | 1997-01-28 | Bioprotesic implants and method to produce and break my |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US28205994A | 1994-07-28 | 1994-07-28 | |
US08/282,059 | 1994-07-28 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1996003093A1 true WO1996003093A1 (en) | 1996-02-08 |
Family
ID=23079930
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1995/009590 WO1996003093A1 (en) | 1994-07-28 | 1995-07-27 | Bioprosthetic implants and method of making and using same |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0804127A1 (en) |
AU (1) | AU3153195A (en) |
CA (1) | CA2196213A1 (en) |
WO (1) | WO1996003093A1 (en) |
ZA (1) | ZA956249B (en) |
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998049972A2 (en) | 1997-05-02 | 1998-11-12 | St. Jude Medical, Inc. | Differential treatment of prosthetic devices |
US5855620A (en) * | 1995-04-19 | 1999-01-05 | St. Jude Medical, Inc. | Matrix substrate for a viable body tissue-derived prosthesis and method for making the same |
WO2000064381A2 (en) | 1999-04-28 | 2000-11-02 | St. Jude Medical, Inc. | Heart valve prostheses |
US6283980B1 (en) | 1996-08-30 | 2001-09-04 | Verigen Transplantation Services Internt'l | Method, instruments, and kit for autologous transplantation |
GB2375771A (en) * | 2001-05-24 | 2002-11-27 | Univ Leeds | Decellularisation of tissue implant material |
US6652583B2 (en) | 2000-04-07 | 2003-11-25 | Rhode Island Hospital | Cardiac valve replacement |
EP1390017A1 (en) * | 2001-05-08 | 2004-02-25 | Verigen Transplantation Service International (VTSI) AG | Reinforced matrices |
US6866668B2 (en) | 1998-08-14 | 2005-03-15 | Verigen Transplantation Service International (“VTSL”) AG | Methods, instruments and materials for chondrocyte cell transplantation |
WO2005042043A2 (en) * | 2003-10-28 | 2005-05-12 | Medtronic, Inc. | Methods of preparing crosslinked materials and bioprosthetic devices |
WO2009025398A1 (en) * | 2007-08-23 | 2009-02-26 | National University Corporation, Tokyo Medical And Dental University | Decellularizing solution, method of preparing decellularized tissue, graft and culture member |
WO2011099007A1 (en) | 2010-02-10 | 2011-08-18 | Nayacure Therapeutics Ltd. | Pharmaceutical compositions and methods for the treatment and prevention of cancer |
WO2016132357A1 (en) | 2015-02-16 | 2016-08-25 | Nayacure Therapeutics Ltd. | Modified blood clots |
EP3095470A1 (en) | 2008-02-07 | 2016-11-23 | Shahar Cohen | Compartmental extract compositions for tissue engineering |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1985005274A1 (en) * | 1984-05-24 | 1985-12-05 | Oliver Roy Frederick | Implant tissue |
US4976733A (en) * | 1988-02-03 | 1990-12-11 | Biomedical Design, Inc. | Prevention of prosthesis calcification |
-
1995
- 1995-07-27 CA CA002196213A patent/CA2196213A1/en not_active Abandoned
- 1995-07-27 ZA ZA956249A patent/ZA956249B/en unknown
- 1995-07-27 WO PCT/US1995/009590 patent/WO1996003093A1/en not_active Application Discontinuation
- 1995-07-27 AU AU31531/95A patent/AU3153195A/en not_active Abandoned
- 1995-07-27 EP EP95927528A patent/EP0804127A1/en not_active Withdrawn
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1985005274A1 (en) * | 1984-05-24 | 1985-12-05 | Oliver Roy Frederick | Implant tissue |
US4976733A (en) * | 1988-02-03 | 1990-12-11 | Biomedical Design, Inc. | Prevention of prosthesis calcification |
Cited By (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5855620A (en) * | 1995-04-19 | 1999-01-05 | St. Jude Medical, Inc. | Matrix substrate for a viable body tissue-derived prosthesis and method for making the same |
US6379367B1 (en) | 1996-08-30 | 2002-04-30 | Verigen Transplantation Service International (Vtsi) Ag | Method instruments and kit for autologous transplantation |
US6283980B1 (en) | 1996-08-30 | 2001-09-04 | Verigen Transplantation Services Internt'l | Method, instruments, and kit for autologous transplantation |
WO1998049972A2 (en) | 1997-05-02 | 1998-11-12 | St. Jude Medical, Inc. | Differential treatment of prosthetic devices |
US6866668B2 (en) | 1998-08-14 | 2005-03-15 | Verigen Transplantation Service International (“VTSL”) AG | Methods, instruments and materials for chondrocyte cell transplantation |
WO2000064381A2 (en) | 1999-04-28 | 2000-11-02 | St. Jude Medical, Inc. | Heart valve prostheses |
US6652583B2 (en) | 2000-04-07 | 2003-11-25 | Rhode Island Hospital | Cardiac valve replacement |
EP1390017A1 (en) * | 2001-05-08 | 2004-02-25 | Verigen Transplantation Service International (VTSI) AG | Reinforced matrices |
EP1390017A4 (en) * | 2001-05-08 | 2008-10-01 | Verigen Ag | Reinforced matrices |
GB2375771A (en) * | 2001-05-24 | 2002-11-27 | Univ Leeds | Decellularisation of tissue implant material |
US7053051B2 (en) | 2003-10-28 | 2006-05-30 | Medtronic, Inc. | Methods of preparing crosslinked materials and bioprosthetic devices |
WO2005042043A3 (en) * | 2003-10-28 | 2005-07-21 | Medtronic Inc | Methods of preparing crosslinked materials and bioprosthetic devices |
WO2005042043A2 (en) * | 2003-10-28 | 2005-05-12 | Medtronic, Inc. | Methods of preparing crosslinked materials and bioprosthetic devices |
WO2009025398A1 (en) * | 2007-08-23 | 2009-02-26 | National University Corporation, Tokyo Medical And Dental University | Decellularizing solution, method of preparing decellularized tissue, graft and culture member |
EP3095470A1 (en) | 2008-02-07 | 2016-11-23 | Shahar Cohen | Compartmental extract compositions for tissue engineering |
US10071185B2 (en) | 2008-02-07 | 2018-09-11 | Nayacure Therapeutics Ltd. | Compartmental extract compositions for tissue engineering |
WO2011099007A1 (en) | 2010-02-10 | 2011-08-18 | Nayacure Therapeutics Ltd. | Pharmaceutical compositions and methods for the treatment and prevention of cancer |
WO2016132357A1 (en) | 2015-02-16 | 2016-08-25 | Nayacure Therapeutics Ltd. | Modified blood clots |
Also Published As
Publication number | Publication date |
---|---|
CA2196213A1 (en) | 1996-02-08 |
AU3153195A (en) | 1996-02-22 |
ZA956249B (en) | 1996-05-10 |
EP0804127A1 (en) | 1997-11-05 |
MX9700727A (en) | 1997-11-29 |
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