Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Solid support and method of culturing cells on said solid...
Reexamination Certificate
2000-09-26
2004-02-10
Naff, David M. (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...
C424S426000, C424S093700, C435S180000
Reexamination Certificate
active
06689608
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention is generally in the field of polymeric materials, and in particular in the area of biocompatible artificial matrices for implantation of cells.
Loss of organ function can result from congenital defects, injury or disease. Many times treatment with drugs or surgery is not in itself sufficient and the patient dies or is severely disabled. One approach for treatment has been to transplant donor organs or tissue into the patient. Drugs such as cyclosporin can be used to prevent tissue rejection. However, there is a tremendous shortage of donor organs, most of which must come from a recently deceased individual.
There have been a number of attempts to culture dissociated tissue and implant the cells directly into the body. One of the problems with implanting dissociated cells into the body is that they do not form three dimensional structures and the cells are lost by phagocytosis and attrition. One approach to overcome this problem is described by U.S. Pat. No. 4,352,883 to Lim, wherein cells are encapsulated within alginate microspheres, then implanted. While this method can sometimes maintain viable functioning cells, the cells do not form organs or structures and rarely result in long term survival and replication of the encapsulated cells. Most cells have a requirement for attachment to a surface in order to replicate and to function.
The first attempts to culture cells on a matrix for use as artificial skin, which requires formation of a thin three dimensional structure, were described by Yannas and Bell in a series of publications. They used collagen type structures which were seeded with cells, then placed over the denuded area. A problem with the use of the collagen matrices was that the rate of degradation is not well controlled. Another problem was that cells implanted into the interior of thick pieces of the collagen matrix failed to survive.
One method for forming artificial skin by seeding a fibrous lattice with epidermal cells is described in U.S. Pat. No. 4,485,097 to Bell, which discloses a hydrated collagen lattice that, in combination with contractile agents such as platelets and fibroblasts and cells such as keratinocytes, is used to produce a skin-equivalent. U.S. Pat. No. 4,060,081, to Yannas et al. discloses a multilayer membrane useful as synthetic skin which is formed from an insoluble non-immunogenic material which is nondegradable in the presence of body fluids and enzymes, such as cross-linked composites of collagen and a mucopolysaccharide, overlaid with a non-toxic material such as a synthetic polymer for controlling the moisture flux of the overall membrane. U.S. Pat. No. 4,458,678 to Yannas et al. discloses a process for making a skin-equivalent material wherein a fibrous lattice formed from collagen cross-linked with glycosaminoglycan is seeded with epidermal cells. A disadvantage to the first two materials is that the matrix is formed of a “permanent” synthetic polymer. In the third case, the matrix can be biodegradable but, since it is formed primarily of collagen, only by enzymatic action, which occurs in an uncontrolled manner.
U.S. Pat. No. 4,520,821 to Schmidt describes a similar approach that was used to make linings to repair defects in the urinary tract. Epithelial cells were implanted onto synthetic non-woven biodegradable polymeric matrices, where they formed a new tubular lining as the matrix degraded. The matrix served a two fold purpose—to retain liquid while the cells replicated, and to hold and guide the cells as they replicated. However, this approach is clearly limited to repair or replacement of very thin linings.
Vacanti, et al.,
Arch. Surg
. 123, 545-549 (1988), describes a method of culturing dissociated cells on biocompatible, biodegradable matrices for subsequent implantation into the body was described. This method was designed to overcome a major problem with previous attempts to culture cells to form three dimensional structures having a diameter of greater than that of skin. Vacanti and Langer recognized that there was a need to have two elements in any matrix used to form organs: adequate structure and surface area to implant a large volume of cells into the body to replace lost function and a matrix formed in a way that allowed adequate diffusion of gases and nutrients throughout the matrix as the cells attached and grew to maintain viability in the absence of vascularization. Once implanted and vascularized, the porosity required for diffusion of the nutrients and gases was no longer critical.
However, even with the method described by Vacanti, the implant was initially constructed in vitro, then implanted. It is clearly desirable to be able to avoid the in vitro step. It is also desirable to have better ways that can be used to form synthetic, biodegradable matrices that can be implanted and sustain cell growth in vivo, degrading in a controlled manner to leave functional, viable cells organized to form an organ equivalent.
It is therefore an object of the present invention to provide a polymeric material which can be implanted into the body, vascularized and used as a means to achieve a high survival rate for dissociated cells injected into the matrix.
It is a further object of the present invention to provide a biocompatible, polymeric implant which can be implanted with cells without prior in vitro culturing and then degrades at a controlled rate over a period of time as the implanted cells replicate and form an organ structure.
SUMMARY OF THE INVENTION
Polymeric materials are used to make a pliable, non-toxic, implantable porous template for vascular ingrowth and into which cells can be injected. The pore size, usually between approximately 100 and 300 microns, allows vascular and connective tissue ingrowth throughout approximately 10 to 90% of the matrix following implantation, and the injection of cells uniformly throughout the implanted matrix without damage to the cells or patient. The introduced cells attach to the connective tissue within the matrix and are fed by the blood vessels. The preferred material for forming the matrix or support structure is a biocompatible synthetic polymer which degrades in a controlled manner by hydrolysis into harmless metabolites, for example, polyglycolic acid, polylactic acid, polyorthoester, polyanhydride, or copolymers thereof. The elements of these materials can be overlaid with a second material to enhance cell attachment. The polymer matrix is configured to provide access to ingrowing tissues to form adequate sites for attachment of the required number of cells for viability and function and to allow vascularization and diffusion of nutrients to maintain the cells initially implanted.
As described in the examples, highly-porous biocompatible and biodegradable polymers forms were prepared and implanted in the mesentery of rats for a period of 35 days to study the dynamics of tissue ingrowth and the extent of tissue vascularity, and to explore their potential use as substrates for cell transplantation. The advancing fibrovascular tissue was characterized from histological sections of harvested devices by image analysis techniques. The rate of tissue ingrowth increased as the porosity and/or the pore size of the implanted devices increased. The time required for the tissue to fill the device depended on the polymer crystallinity and was less for amorphous polymers versus semicrystalline polymers. The vascularity of the advancing tissue was consistent with time and independent of the biomaterial composition and morphology.
REFERENCES:
patent: 4060081 (1977-11-01), Yannas et al.
patent: 4186448 (1980-02-01), Brekke
patent: 4352883 (1982-10-01), Lim
patent: 4391909 (1983-07-01), Lim
patent: 4458678 (1984-07-01), Yannas et al.
patent: 4485097 (1984-11-01), Bell
patent: 4520821 (1985-06-01), Schmidt et al.
patent: 4563489 (1986-01-01), Urist
patent: 4846835 (1989-07-01), Grande
patent: 4897267 (1990-01-01), Bontempts et al.
patent: 5041138 (1991-08-01), Vacanti et al.
patent: 5064866 (1991-11-01), Toyomoto et al.
patent: 5096814 (1
Griffith Linda G.
Langer Robert S.
Mikos Antonios G.
Sarakinos Georgios
Vacanti Joseph P.
Bieker-Brady Kristina
Clark & Elbing LLP
Massachusetts Institute of Technology
Naff David M.
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