Method of forming polysaccharide sponges for cell culture...

Details Method of forming polysaccharide sponges for cell culture... Method of forming polysaccharide sponges for cell culture...

1999-10-13

2002-01-01

Kuhns, Allan R. (Department: 1732)

Plastic and nonmetallic article shaping or treating: processes

With step of cooling to a temperature of zero degrees c. or...

C264S041000, C264S344000

Reexamination Certificate

active

06334968

ABSTRACT:

FIELD OF THE INVENTION
The present invention concerns new bioresorbable polysaccharide sponges, a method for their preparation and uses thereof for the cultivation of mammalian cells in vitro, as well as the use thereof as matrices, supports or scaffolds for implantation into a patient to replace damaged or removed tissue, the polysaccharide sponge implant serving as a substrate, matrix or scaffold for surrounding host tissue to invade it, proliferate thereon and eventually form an active part of the tissue or organ in which the implant was made, or the implant serving as an initial substrate for vascularization from the surrounding host tissue, and once vascularized, cells of choice grown in vitro or obtained from the host may be injected into the vascularized implant to enable a rapid acclimatization and proliferation of the cells which will subsequently form an active replacement for the organ of tissue that was damaged or removed. The polysaccharide sponge can also serve as a substrate, matrix or scaffold for the transplantation of cells initially grown thereon in vitro into a patient to replace damaged, removed or non-functioning tissue.
BACKGROUND OF THE INVENTION AND PRIOR ART
Porous, absorbable matrices fabricated from natural and synthetic polymers (see, for example, Yannas 1990; Natsumi et al., 1993; Grande, 1989; Vacanti, 1990; Mikos et al., 1993a; Mikos et al., 1993b Mikos et al. 1993c; and Langer and Vacanti, 1993), currently in use or under investigation as implants to facilitate regeneration of tissue in defects caused by disease, trauma or reconstructive surgical procedures. These matrices have been used alone or seeded with cells for the purpose of cell and tissue transplantation (Langer and Vacanti, 1993). Cell transplantation can provide an alternative treatment to whole organ transplantation for failing or malfunctioning organs such as liver and pancreas. As many isolated cell populations can be expanded in vitro using cell culture techniques, only a very small number of donor cells are needed to prepare a suitable implant. Consequently, when such cells are taken from a living donor, the living donor need not sacrifice an entire organ. Furthermore, for the purpose of gene therapy, gene transfer vectors can be introduced into various types of cells, such as, for example, hepatocytes, fibroblasts, keratinocytes, endothelial cells, and myoblasts, which are then transplanted back to the host for the production and local release of proteins and other therapeutic drugs or agents.
Another application of porous matrices has been as scaffolds to investigate the behavior of cells in a three-dimensional framework in vitro (Jain et al., 1990; Doane and Birk, 1991). In some applications, these porous matrices are designed to serve as analogues of the extracellular matrix in order to provide a suitable substrate for cell attachment to enable certain anchor-dependent processes such as migration, mitosis, and matrix synthesis (Folkman and Moscona, 1978). In this regard, it is considered that such analogues of the extracellular matrix may be able to modulate cell behavior in a similar fashion to the way in which the native extracellular matrix does so (see Madri and Basson, 1992), it being believed that the chemistry of these analogues, as well as their pore characteristics such as percentage porosity, pore size and orientation, may influence the density and distribution of the cells within the matrix and thereby affect the regeneration process when these analogues are used in transplantations.
Bioresorbable sponges can also provide a temporary scaffolding for transplanted cells, and thereby allow the cells to secrete extracellular matrix of their own to enable, in the long term, a complete and natural tissue replacement. The macromolecular structure of these sponges is selected so that they are completely degradable and are eliminated, once they have achieved their function of providing the initial artificial support for the newly transplanted cells. For these sponges to be useful in cell transplantations, they must be highly porous with large surface/volume ratios to accommodate a large number of cells, they must be biocompatible, i.e., non-toxic to the cells that they carry and to the host tissue into which they are transplanted, they must be capable of promoting cell adhesion and allowing the retention of the differentiated function of attached cells.
However, in most of the porous matrices described to date, the ones that have been successfully prepared and used in implants or transplants have been limited to those which carry a very thin layer of cells, being principally those which serve as skin substitutes or replacements (see, for example, Yannas, 1990). In view of this limited application, the matrices developed are ones in which the porosity and pore size thereof has been of the type that has been nearly sufficient to allow the dispersion of the thin layer of cells within the matrix. However, when such matrices are to be used with cells such as, for example, hepatocytes, which grow in aggregates of cells and with a thickness greater than the thickness which these earlier matrices are designed to support, a serious problem arises as regards the adequate diffusion of oxygen and nutrients to the inner cells within the matrix, with the result that these inner or lower layers of cells usually die. Thus, these earlier matrices may be useful for preparing skin equivalents, but are much less useful for preparing functional organ equivalents made up of multilayer cell aggregates, both in vitro and with subsequent transplantation use in vivo.
Most of the porous matrices developed to date, as noted above, are based on natural polymers such as collagen, or synthetic polymers from the lactic/glycolic acid family. The collagen-based matrices have several disadvantages, including: they degrade at relatively rapid rate; many disappearing as early as 4 weeks postimplantation (see Olde Damink et al., 1995; Ben-Yishay et al., 1995). Although the rate of degradation of the collagen matrix may be reduced by cross-linking with glutaraldehyde, the resulting cross-linked matrices, however, exhibited immunogenicity, calcification, and fibrous scarring when implanted for long periods (see Timple et al., 1980). Furthermore, collagen matrices are also not suitable for prolonged in vitro cultivation of cells due to a significant contraction of the collagen scaffold, which occurs after approximately one week of incubation, rendering this collagen scaffold less amenable to surgical handling when intended for use as a transplantation matrix (Ben-Yishay, 1995).
Other synthetic biodegradable foams based on poly(D, L-Lactic-co-glycolic acid) have been developed as scaffolds for tissue engineering, as noted above, but because these polymers are hydrophobic, when a cell suspension or culture media is placed on these foams or injected into their interior, the majority of their pores remain empty, resulting in he underutilization of the volume of these foams. In addition, studies have also shown that the degradation of these biodegradable foams results in the signifcant accumulation of acid products which significantly decreases the internal pH within the foam to less than pH 3.0 (see Park Lu and Crotts, 1995), which acidity is very harmful to the growing cells.
Alginates have also been used previously for the purpose of cell transplantation. Aiginates are natural polysaccharide polymers, the word “alginate” actually referring to a family of polyanionic polysaccharide copolymers derived from brown sea algae and comprising 1,4-Linked &bgr;-D-mannuronic (M) and &agr;-L-gluronic acid (G) residues in varying proportions. Alginate is soluble in aqueous solutions, at room temperature, and is capable of forming stable gels, particularly in the presence of certain divalent cations such as calcium, barium, and strontium. The unique properties of alginate, together with its biocompatibilty (see Sennerby et al., 1987 and Cohen et al., 1991), its relatively low cost and wide availability have made alginate an im

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