Cellular matrix

Details Cellular matrix Cellular matrix

1999-12-20

2003-09-23

Lankford, Jr., Leon B. (Department: 1651)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Solid support and method of culturing cells on said solid...

C435S401000, C424S426000, C530S326000, C623S023720

Reexamination Certificate

active

06623963

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a matrix suitable, inter alia, for the support, retention, and/or growth of living cells in a form suitable, for example, for cell transplantation. More particularly, it pertains to a reconstituted collagen matrix suitable for cell growth, such as chondrocyte cell growth for use in chondrocyte cell transplantation.
BACKGROUND OF THE INVENTION
Injuries to the cartilage of the knee or other joints often result from abnormal mechanical loads which deform the tissue matrix. The loads applied to the joint can rupture the collagen network in the matrix and decrease the stiffness of the tissue.
Cartilage injuries are difficult to treat because articular cartilage has a limited capacity for regeneration once it has been damaged. Type II collagen is the main structural protein of the extracellular matrix in articular cartilage. Type II collagen, similar to other types of collagen, is comprised of three collagen polypeptides which form a triple helix configuration. The polypeptides are intertwined with each other and possess at each end telopeptide regions which provide the crosslinking between the collagen polypeptides. Collagen matrices in their natural state contain numerous crosslinked triple helices and the individual molecules have a molecular weight of about 300,000 daltons. Type II collagen is found almost exclusively in animal cartilage, while other types of collagen are found in animal hides, membranes, and bones.
Excessive degradation of Type II collagen in the outer layers of articular surfaces of joints is also caused by osteoarthritis. The collagen network is accordingly weakened and subsequently develops fibrillation whereby matrix substances, such as proteoglycans, are lost and eventually displaced entirely. Such fibrillation of weakened osteoarthritic cartilage can reach down to the calcified cartilage and into the subchondral bone (Kempson, G. E. et al., Biochim. Biophys. Acta 1976, 428, 741; Roth, V. and Mow, V. C., J. Bone Joint Surgery, 1980, 62A, 1102; Woo, S. L. -Y. et al., in
Handbook of Bioengineering
(R. Skalak and S. Chien Eds), McGraw-Hill, New York, 1987, pp. 4.1-4.44).
A method for regeneration-treatment of cartilage would be useful for treating arthritis and other joint conditions and could be performed at an earlier stage of joint damage, thus reducing the number of patients needing more extensive procedures, such as artificial joint replacement surgery. With such preventive methods of treatment, the number of patients developing osteoarthritis would also decrease.
Methods for growing and using chondrocyte cells are described by Brittberg, M. et al. (New Engl. J. Med. 1994, 331, 889). Autologous transplants using cells grown with these methods are also disclosed. Additionally, Kolettas et al. examined the expression of cartilage-specific molecules, such as collagens and proteoglycans, under prolonged cell culturing (J. Cell Science 1995, 108, 1991). They found that, despite morphological changes during culturing in monolayer cultures (Aulthouse, A. et al., In Vitro Cell Dev. Biol., 1989, 25, 659; Archer, C. et al., J. Cell Sci. 1990, 97, 361; Hänselmann, H. et al., J. Cell Sci. 1994, 107, 17; Bonaventure, J. et al., Exp. Cell Res. 1994, 212, 97), when compared to suspension cultures grown over agarose gels, alginate beads or as spinner cultures (retaining a round cell morphology) tested by various scientists, such morphologies did not change the chondrocyte—expressed markers such as types II and IX collagens and the large aggregating proteoglycans, aggrecan, versican and link protein did not change (Kolettas, E. et al., J. Cell Science 1995, 108, 1991).
In addition, chondrocyte cells from donors have been grown in vitro to form neocartilage which has been implanted into animals (Adkisson et al., “A Novel Scaffold-Independent Neocartilage Graft for Articular Cartilage Repair,” ICRS 2
nd
Symposium International Cartilage Repair Society, Nov. 16-18, 1998). Further, chondrocyte cells have been seeded onto the cartilage surface of osteochondral cores to attempt cartilage regeneration (Albrecht et al., “Circumferential Seeding of Chondrocytes: Towards Enhancement of Integrative Cartilage Repair,” ICRS 2
nd
Symposium International Cartilage Repair Society, Nov. 16-18, 1998). Articular surface defects in knee joints have been treated with various cultured chondrocytes (Stone et al., Operative Techniques in Orthopaedics 7(4), pp. 305-311, October 1997 and Minas et al., Operative Techniques in Orthopaedics 7(4), pp. 323-333, October 1997).
Membranes and certain of their general properties are described in the following references:
U.S. Pat. No. 5,837,278—Geistlich et al. describe a collagen-containing membrane which is resorbable and is used in guided tissue regeneration. The membrane has a fibrous face which allows cell growth thereon and a smooth face opposite the fibrous face which inhibits cell adhesion thereon. The membrane product is derived from a natural collagen membrane (from the hide or tendons of calves or piglets) and, although treated, it is described as maintaining its natural structural features. The collagen is purified with alkaline agents to defat the collagen and degrade substances, and then the purified collagen is acidified, washed, dried, degreased, and optionally crosslinked. The fats are saponified. The membrane is described as containing about 95% by weight native collagen.
PCT WO 96/25961—Geistlich et al. describe a matrix for reconstructing cartilage tissue which consists of Type II collagen, optionally including crosslinking. In producing the matrix, cartilage is taken from an animal and frozen, subjected to size reduction, dewatered, defatted, washed, and treated with alkaline materials. Non-collagen alkaline soluble proteins are denatured, destroyed, dissolved, and eliminated. Dialysis and freeze drying are mentioned as possible treatment steps. The matrix material is stamped to form a required shape and then it is sterilized.
U.S. Pat. No. 4,424,208—Wallace et al. describe an injectable collagen implant material comprising particulate crosslinked atelopeptide collagen and reconstituted atelopeptide collagen fibers dispersed in an aqueous carrier. The atelopeptide form of collagen lacks the native telopeptide crosslinking. In the method described in the '208 patent, collagen obtained from bovine or porcine corium (sub-epithelial skin layer) is softened by soaking in a mild acid; depiliated; comminuted by physical treatment, such as grinding; solubilized by treatment with acid and a proteolytic enzyme; treated with an alkaline solution; and freed of enzyme. The crosslinked gel form of collagen is formed by radiation-induced or chemical-induced crosslinking, such as by addition of glutaraldehyde. Meanwhile, the fibrous form of collagen is produced by neutralizing the solution with a buffer, such as Na
2
HPO
4
. The collagen content of the injectable implant comprises 5-30% fibrous collagen and 70-98% of the crosslinked gel form of collagen.
U.S. Pat. No. 4,488,911—Luck et al. describe the formation of collagen fibers free of the immunogenic, telopeptide portion of native collagen. The telopeptide region provides points of crosslinking in native collagen. The fibers, which may be crosslinked, are described for use as sponges, prosthetic devices, films, membranes, and sutures. In the method described in the '911 patent, (non-Type II; Type I and others) collagen obtained from tendons, skin, and connective tissue of animals, such as a cow, is dispersed in an acetic acid solution, passed through a meat chopper, treated with pepsin to cleave the telopeptides and solubilize the collagen, precipitated, dialyzed, crosslinked by addition of formaldehyde, sterilized, and lyophilized. The '911 patent indicates that its disclosed method obtains the atelocollagen form of collagen, free from noncollagen proteins, such as glycosaminoglycans and lipids. Further, it describes that the collagen may be used as a gel to make, for example, a membrane, film, or sponge and that the deg

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