Non-contracting tissue equivalent

Details Non-contracting tissue equivalent Non-contracting tissue equivalent

2001-02-01

2002-10-29

Witz, Jean C. (Department: 1651)

Drug, bio-affecting and body treating compositions

Whole live micro-organism, cell, or virus containing

Animal or plant cell

C435S325000, C435S371000, C435S373000, C435S395000, C435S397000

Reexamination Certificate

active

06471958

ABSTRACT:

FIELD OF THE INVENTION
The invention relates generally to multicellular tissue-like compositions.
BACKGROUND OF THE INVENTION
Tissue equivalents are three-dimensional living multicellular tissue-like compositions. While these tissue equivalents have many uses, including tissue transplantation, screening and evaluation of new drugs, previous tissue equivalents have had limited utility because they contracted. For example, a tissue equivalent comprising a collagen matrix can exhibit as much as about 80% linear shrinkage, i.e., can contract to as little as twenty percent of the original diameter within a period of a few hours. This contraction produces a dense, opaque matrix which prevents the visualization of the contained cells by optical microscopy. The resulting tissue equivalent may resemble normal scar tissue more than the desired normal healthy tissue. The factors responsible for such contraction have not been systematically evaluated and studied, but may include collagen concentration and cell numbers.
In general, tissue equivalents are produced by combining at least one cellular component with at least one noncellular component. The design and construction of tissue equivalents is a branch of tissue engineering, which can be defined as the application of scientific principles to the design, construction modification, growth and maintenance of living tissues to form the desired composition.
Tissue-equivalents have numerous uses including: sources of tissue for transplantation; systems for screening and evaluating potential drugs, cosmetics and other consumer products; model systems for the study of multicellular processes such as wound healing; systems for establishing optimal conditions for trans-tissue delivery of hormones, cytokines or other biologically active materials and systems for introducing cells genetically engineered to produce a desired substance. It would be desirable to use such tissue equivalents to decrease dependency on cadaver tissue for grafts and transplants and to reduce dependency on animal testing in the development of new pharmaceuticals and consumer products.
“Tissue equivalent” as used herein includes, but is not limited to, artificially produced epithelial tissue, skin, cornea, connective tissue, cartilage, bone, and the like (see for example, U.S. Pat. Nos. 4,485,096; 4,485,097; 4,546,500; 4,539,716; 4,604,346; 4,835,102).
The cellular component of tissue equivalents may be derived from a number of sources. The cells comprising the cellular component may be autologous, that is, the donor and the recipient may be the same person. The cells are processed, incorporated into the non-contracting tissue equivalent, and transplanted back into the donor as part of the tissue equivalent. Alternatively, the cells may be allogenic, that is taken from a different donor than the recipient of the transplanted tissue equivalent, where both the donor and recipient are members of the same species. The cells also may be xenogeneic, i.e., derived from a donor of a different species from the recipient. In each of these cases, treatments are known in the art that reduce the likelihood of rejection or control the differentiation of the cellular component. Human cells, i.e., either autologous or allogenic cells, are preferred.
The noncellular component of tissue equivalents may comprise one or more of a group of compounds, including compounds normally secreted by cells to form a naturally occurring extracellular matrix. Suitable compounds include the collagens.
The collagens are a family of fibrous proteins that are secreted by connective tissue cells, as well as by a variety of other cell types. See generally, Alberts, B., et al.,
Molecular Biology of the Cell
, 3rd Ed., Garland Publishing, New York (1994) pp. 978-984. The characteristic feature of a typical collagen molecule is its long, stiff, triple-stranded helical structure, in which three collagen polypeptide chains, called &agr; chains, are wound around one another in a rope-like superhelix. About 25 distinct collagen &agr; chains have been identified, each encoded by a separate gene.
About fifteen different types of collagen have been described, which are characteristically composed of different combinations of specific &agr; chains. Type I collagen (collagen I) is the principal collagen of skin, tendon, ligaments, cornea, internal organs and bone. Collagen I is by far the most common, accounting for about 90% of body collagen. The &agr; chain composition of collagen I is [&agr;1(I)]
2
&agr;2(I).
Other fibrillar collagens are types II, III, V, VII and XI. Type II collagen (collagen II) cartilage, composed of [&agr;1(II)]
3
&agr; chains, is found in cartilage, the intervertebral discs of the spine and the vitreous humor of the eye. Type III collagen (collagen III), [&agr;1(III)]
3
, is found in skin, blood vessels and internal organs. Type V collagen (collagen V), [&agr;1(V)]
2
&agr;
2
(V), is found in the same tissues as type I collagen. Type XI collagen (collagen XI), &agr;1(XI)&agr;2(XI)&agr;3(XI), is found in the same tissues as collagen I. Alberts, et al., page 980.
In contrast to the above fibrillar collagens, network-forming collagens form a felt-like sheet or meshwork instead of rope-like fibers. An important network-forming collagen is collagen IV, [&agr;1(IV)]
2
&agr;2(IV), which forms the basal lamina. The basal lamina, sometimes called the basement membrane, is a thin mat of extracellular matrix that separates the epithelium from the underlying stroma/connective tissue. The basal lamina also separates many other types of cells, such as muscle cells and fat cells, from connective tissue.
In previous tissue equivalents, for example those described in Clark et al., J. Clin. Invest. 84: 1036-1040 (1989) and Montesano et al., Proc. Nat. Acad. Sci. U.S.A. 85: 4894-4897 (1988), the collagen matrix contracts after formation to a fraction of its original size (typically to about twenty percent of the original diameter) over a period of up to 48 hours. The contraction of the tissue equivalent as a whole follows the contraction of the collagen matrix. As a result, the matrix condenses forming a dense, opaque tissue which prevents visualization of the contained cells by transmitted light or fluorescence microscopy. The multiple factors responsible for contractions have not been studied systematically but it has been proposed that they include cell number and collagen concentration. In addition, unknown combinations of cytokines, such as presumably present in exogenously supplied serum such as fetal bovine serum (FBS) may be responsible for contraction.
Contraction of tissue equivalents may be desirable for some limited number of uses, for example, in wound closure or scar formation. However, extensive contraction produces an abnormally dense scar-like tissue that impedes normal tissue functions such as epithelialization, vascularization, pigmentation and hair growth. Contraction of tissue equivalents is thus a problem for which a solution has been sought for several years.
Previous non-contracting tissue equivalents have been constructed using pre-formed collagen sponge matrices. Collagen sponge matrices are composed of insoluble, covalently-linked, solid collagen fibrils. Covalent cross-links are formed between collagen fibrils by chemical reactions and thus cannot be readily reversed. The physical form of collagen sponges produced by chemical cross-linking can only be altered by digestion with collagenase, an enzyme which degrades collagen into its component amino acids. In addition, collagen sponges may retain the toxic chemical reagents used in cross-linking, such as aldehydes, which may leach into the host tissues, causing adverse reactions.
What is needed is a non-contracting tissue equivalent that provides dimensional stability and permits the monitoring of the functions of viability of the cellular component.
SUMMARY OF THE INVENTION
The present invention provides a method for producing a substantially non-contracting tissue equivalent comprising

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