Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Treating animal or plant material or micro-organism
Reexamination Certificate
2000-07-03
2003-03-11
Witz, Jean C. (Department: 1651)
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Treating animal or plant material or micro-organism
C435S265000, C435S268000
Reexamination Certificate
active
06531310
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to a process for treating tissues using viable or nonviable cells, preferably microorganisms. More particularly, the invention relates to the use of microorganisms a an in vitro source of compositions for the processing of tissue to produce biomaterials useful in the production of bioprostheses.
Bioprostheses, i.e., bioprosthetic devices, are used to repair of replace damaged or diseased organs, tissues and other structures in humans and animals. Bioprostheses must be generally biocompatible with the recipient patient since they are typically implanted for extended periods of time. Bioprostheses can include artificial hearts, artificial heart valves, ligament repair material, vessels repair material, surgical patches constructed of mammalian tissue and the like. Considerable effort has been invested in the development of bioprosthetic heart valves, and the following discussion, for simplicity, will focus on these devices. It is to be understood, however, that the subject matter of this application is not limited to heart valves.
Currently available prostheses for the replacement of defective heart valves and other vascular structures may be classified as mechanical or bioprosthetic. Although mechanical values have the advantage of proven durability through decades of use, they frequently are associated with a high incidence of blood clotting on or around the valve, necessitating continuous treatment with anticoagulants. Bioprosthetic heart valves constructed from materials derived from biological tissues were introduced in the early 1960's. Bioprosthetic heart valves are typically derived from porcine aortic valves or are manufactured from other biological materials such as bovine pericardium. Bioprostheses can include a combination of tissue-derived materials and synthetic materials.
A major rationale for the use of biological material for heart valves is that the profile and surface characteristics of biological material are optimal for laminar, nonturbulent flow. The result is that intravascular clotting is less likely to occur than with mechanical valves. This reduction in thrombogenicity has been well documented in clinical use of glutaraldehyde-fixed bioprosthetic valves. Glutaraldehyde fixes tissue by reacting to form covalent bonds with free amino groups in proteins, thereby chemically crosslinking nearby proteins.
Generally, bioprosthetic heart valves begin failing after about seven years following implantation, and few bioprosthetic valves remain functional after 20 years. Replacement of a degenerating valve prosthesis subjects the patient to additional surgical risk, especially in the elderly and in situations of emergency replacement. While failure of bioprostheses is a problem for patients of all ages, it is particularly pronounced in younger patients. Over fifty percent of bioprosthetic valve implants in patients under the age of 15 fail in less than five years, due to calcification.
Mineralization, e.g. calcification, appears to be the primary process leading to degeneration of bioprostheses. Efforts to address the calcification problem have included treating glutaraldehyde-fixed valves with compounds such as toluidine blue, sodium dodecyl sulfate and diphosphonate to reduce calcium nucleation. Other approaches include removal of reactive glutaraldehyde moieties from the tissue by a chemical process. Xenograft tissue, i.e., tissue from a species other than the species of the recipient patient, typically is fixed with glutaraldehyde prior to implantation to reduce the possibility of immunological rejection.
Still other approaches include development of alternative fixation techniques, since evidence suggests that the glutaraldehyde fixation process itself may contribute to calcification and mechanical deterioration. In addition, since nonviable cells present in transplanted tissue are sites for calcium deposition, investigators have developed various processes (“decellularization” processes) to remove cellular structure from the valve matrix. For example, detergents and nucleases have been used to obtain an extracellular matrix from tissue for use as graft material.
Fixation and treatment to reduce calcification generally involve harsh conditions that tend to sterilize the tissue. Absence of living microorganisms, primarily bacteria and fungi, is an important consideration for any bioprosthetic material intended for implantation into a patient since implantation of a nonsterile implant may be catastrophic for the patient. Thus, previous approaches to preparation of medical implants, including bioprostheses, generally have avoided introduction of microorganisms or, at least, have not encouraged the growth of microorganisms around and within the implant material.
SUMMARY OF THE INVENTION
In a first aspect, the invention involves a method for processing tissue including the steps of:
a) inoculating the tissue with a solution having microorganisms that are selected to produce one or more compounds effective to process the tissue; and
b) incubating the tissue in the solution under conditions effective for processing the tissue by the compounds produced by the selected microorganisms. The process can include the step of treating the tissue to substantially remove or inactivate the microorganisms. Preferred tissue, prior to processing, includes cells and an extracellular matrix. The one or more compounds can include a compound effective at decellularizing the tissue while leaving the extracellular matrix substantially intact. The selected microorganisms can carry one or more exogenous genes encoding enzymes effective for decellularizing the tissue. The solution used during incubation can include an additive effective at reducing enzyme activities detrimental to the matrix.
The one or more compounds can include a replacement extracellular matrix constituent effective to reinforce an extracellular matrix within the tissue. A preferred replacement extracellular matrix constituent can be selected from the group consisting of proteoglycans, glucosamioglycans, collagen, elastin, glycoproteins and lipoproteins. The one or more compounds can also include an antioxidant. The one or more compounds can include a surface modification chemical effective to treat the tissue. The surface modification chemical can be selected from the group consisting of heparin, RGD sequence containing peptides, fibroblast growth factors, transforming growth factors and other chemotactants. The one or more compounds can include a compound effective to crosslink proteins.
The method of the invention can further include the step of adding a growth medium effective for selecting for proliferation of desired microorganisms. The method can also further include the step of monitoring the solution to estimate numbers of the microorganisms or to determine metabolic activity of the microorganisms.
The inoculated microorganisms preferably are selected to have low levels of endotoxin. The inoculated microorganisms can be selected for susceptibility to one or more inactivating mechanisms selected from the group consisting of ionizing radiation, ultraviolet irradiation, antibiotics and chemical exposure. The selected inoculated microorganisms can carry an exogenous, inducible suicide gene, such that the processing includes inducing the suicide gene.
The selected inoculated microorganisms can produce anti-microbial agents effective for inhibiting growth of undesired microorganisms in the solution. The incubation can be performed in the presence of a media supplement effective to enhance or to inhibit specific cellular activity. The selected inoculated microorganisms preferably would be resistant to antimicrobial agents effective for inhibiting growth of undesired microorganisms.
The method of the invention can further include the step of forming a bioprosthetic article from the tissue. Preferred microorganisms include
Micrococcus luteus
bacteria.
In another aspect, the invention involves a method for decellularizing a tissue, the tissue including cells and a
Borner William H.
Mirsch, II M. William
Montoya Susan I.
Schroeder Richard F.
Altera Law Group LLC
St. Jude Medical Inc.
Witz Jean C.
LandOfFree
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