Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Implant or insert
Reexamination Certificate
2001-08-28
2004-06-01
Naff, David M. (Department: 1651)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Implant or insert
C435S001100, C530S353000, C530S356000, C530S402000
Reexamination Certificate
active
06743435
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for processing animal tissue, particularly methods for dispersing decellularized human skin to produce injectable tissue matrix compositions.
BACKGROUND
Collagen is one of the most abundant proteins in the animal kingdom and is the primary structural component of connective tissues, such as skin, ligaments, tendons, and bones. Collagen possesses many properties, including high tensile strength, low immunogenicity, semipermeability, and solubility, that make it particularly suitable for use in the preparation of various biomaterials and other medical products, including medical prostheses, implantable compositions, cellular and acellular grafts, and other tissue replacement materials. Recently, injectable collagen formulations have been widely used in a variety of applications, including as tissue bulking compositions and as ophthalmic implants.
Collagen compositions are often prepared from skin, ligaments, or tendons by dispersion, digestion and/or dissolution. Dispersion typically involves mechanically shearing the tissue to produce a crude homogenate tissue matrix. Tissue digestion and dissolution generally entails enzyme degradation of a portion of the non-helical telopeptide portions of the collagen molecules, followed by purification steps, to produce a solution of telopeptide-poor collagen. The solubilized collagen can be reconstituted into molecular aggregates and occasional fibrils by neutralizing the enzyme digested, purified collagen solution.
Although it is generally desirable to use autologous tissues, i.e., those derived from the recipient of the implant, in the preparation of injectable collagen-containing compositions, there is seldom an ample supply of transplantable autologous tissues. Thus, allograft tissues and xenograft tissues are often used. Unfortunately, allogeneic and xenogenic tissues may be rejected by the recipient organism and may be associated with a greater risk of disease transmission. However, approximately 450,000 allograft tissues are transplanted each year in the U.S., and during the past decade, improvements in donor screening and serological testing have greatly reduced the risk of infectious disease transmission. For example, there have been no confirmed transmissions of AIDS through tissue transplants distributed by the Musculoskeletal Transplant Foundation, Edison, N.J., (MTF) in more than 10 years following the development of sensitive and accurate test methods. The estimated odds of contracting the AIDS virus from a transplant are less than 1 in 1.67 million (annual), without the inclusion of viral inactivation and sterilization steps. However, allograft implants may still be rejected following implantation. Allograft skin, used to treat burns, eventually becomes rejected, primarily due to allorecognition of Class II MHC antigens associated with Langerhans cells present in the epidermal layer of skin.
Thus, there is a need for improved methods for preparing injectable collagen compositions, particularly methods that are able to make use of allogeneic and xenogenic tissue sources while avoiding the complications that often accompany implantation of such materials.
SUMMARY OF THE INVENTION
The present invention features a method for processing dermal tissue for implantation into a subject. The method includes the steps of: (a) removing the epidermal layer of the dermal tissue to produce de-epidermalized tissue; (b) incubating the de-epidermalized tissue in at least one processing solution to remove cells from the de-epidermalized tissue, thereby producing a decellularized tissue matrix; and (c) exposing the decellularized tissue matrix to an acylating agent, wherein the ratio of acylating agent to wet tissue weight is about 0.003:1 or less. In a particularly preferred embodiment, the decellularized tissue matrix is treated, e.g., by cryomilling, to increase its surface area prior to acylation. This processing using low levels of acylating agent combined with cryomilling consistently results in relatively high yields of dispersed tissue matrix having a high resistance to trypsin.
In one embodiment of the invention, the dermal tissue is de-epidermalized by exposing the dermal tissue to a hypertonic salt solution, which allows for separation of the dermis and epidermis. The de-epidermalized tissue may then be processed by incubation in a variety of processing solutions. For example, the de-epidermalized tissue is preferably incubated in a series of decellularization solutions, including a high pH (e.g., sodium hydroxide) solution, a low pH (e.g., hydrochloric acid or phosphoric acid) solution, and a solvent (e.g., reagent alcohol). Incubation in such solutions may be in any order. For example, the de-epidermalized tissue may be incubated in low pH solutions first and then in high pH solutions and solvent solutions (or vice versa). In addition to removing cells from the tissue, this process also leads to inactivation of viruses and other contaminants in the tissue. Optionally, the tissue may be exposed to any of a number of viral inactivating agents before, during, or after the decellularization process.
In another related aspect, the invention provides a method for dispersing decellularized animal tissue, which method involves contacting any type of decellularized animal connective tissue with a solution comprising an acylating agent, wherein the ratio of acylating agent to wet tissue weight is about 0.003:1 or less. As discussed above, the decellularized tissue may be treated, for example by cryomilling, to increase the surface area of the tissue prior to decellularization.
The tissues processed according to the methods of the invention may come from autogenic, allogeneic, or xenogenic sources. In various preferred embodiments, the tissue is mammalian, preferably human; the acylating agent is an anhydride such as glutaric anhydride or succinic anhydride; and the ratio of acylating agent to wet tissue weight is within the range of about 0.002:1 to about 0.001:1.
The methods of the invention can be used to produce injectable, dispersed collagen compositions that have a trypin resistance greater than about 40%, preferably greater than about 50%, more preferably greater than about 70%, and most preferably than 90%. Preferably, the dispersed collagen matrix compositions of the invention are injectable through needles as small as 30 gauge.
In another aspect, the invention features a method of using the compositions of the invention for altering the condition of in situ tissue of a mammalian subject. The method involves placing an effective amount of the composition, e.g., as an injectable flowable mass, or formed into a putty like spreadable mass or finely divided distributable particles, at the in situ tissue site to be altered.
Other advantages and features of the present invention will be apparent from the following detailed description thereof and from the claims.
DEFINITIONS
By “acylating agent” is meant an agent that transfers an acyl group to another nucleophile. Examples of acylating agents include anhydrides, acid chlorides, sulfonyl chlorides, and sulfonic acids.
The terms “autologous” and “autogenic” refer to tissues or cells which originate with or are derived from the recipient, whereas the terms “allogeneic” and “allograft” refer to tissues or cells which originate with or are derived from a donor of the same species as the recipient. The terms “xenogenic” and “xenograft” refer to tissues or cells that originate with or are derived from a species other than that of the recipient.
By “cryomilling” is meant a reduction in size by homogenizing or pulverizing the tissue in the presence of liquid nitrogen or other such solutions that cause the tissue to remain in a frozen state during the homogenizing or pulverizing process.
By “decellularized tissue” is meant tissue that is substantially free of cells or cellular debris (i.e. a substantially acellular tissue matrix) as determined by light microscopy or by biochemical methods capable of identifying cells or cellular debri
Ciarametaro Peter D.
DeVore Dale P.
Barlow Josephs & Holmes, Ltd.
Collagen Matrix Technologies, Inc.
Naff David M.
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