Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-09-15
2003-01-07
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C424S409000, C424S489000, C435S091100, C435S177000, C435S182000, C435S239000, C435S395000, C435S403000, C530S382000, C530S383000, C530S402000
Reexamination Certificate
active
06503731
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to fibrin microbeads, methods for preparing the fibrin microbeads, and their use as vehicles for culturing, separating and transporting cells, as vehicles for transplanting cells involved in wound healing, and as vehicles for tissue engineering.
Fibrin clots are formed in vivo based upon the reaction of fibrinogen and thrombin in the presence of calcium ions. The initial phase of wound healing starts after the formation of fibrin clot, and involves the mobilization of cells from surrounding undamaged tissue. Normally, the earliest cells mobilized to the wound are inflammatory where they are active for a period of at least 1-3 days following injury. Subsequently, they are displaced by cells of the mesenchyme lineage which are immobilized into, navigate through and digest fibrin and replace fibrin with extracellular matrix (ECM) of different collagen types, fibronectin and hyaloron. Endothelial cells also infiltrate the fibrin and generate microcapillary structures. Ultimately, these cells replace the provisional fibrin matrix with granulation tissue populated by parenchymal cells and vasculature in secreted ECM.
Human fibroblasts are the major cellular entities responsible for the regeneration of the extracellular matrix (ECM) within the wound bed. Human fibroblasts also express specific membrane receptors to fibrinogen and thrombin. In the case of skin wounds, human fibroblasts reform the matrix of the dermis. For example, during the course of healing of an incisional skin wound, human fibroblasts are mobilized from the surrounding tissue and enter into the fibrin clot, help dissolve it and generate as well as reform the collagens (i.e. type I and type III) in the extracellular matrix. Based upon these properties of human fibroblasts, fibroblast implants have been suggested as a means for supplementing the body's natural wound healing regime (Gorodetsky, R., et al.
Radiat. Res.
125:181-186 (1991)).
Benzoylated hyaluronic acid (HA) sheets containing holes or pores have been used as a carrier for fibroblasts and keratinocytes for wound healing (Andreassi, L., et al.
Wounds
3 (3): 116-126 (1991)). Specifically, HA sheets are cultured with these cells and then affixed to the site of the burn injury, where the cells migrate out of the sheet and accelerate the rate of wound re-granulation. A major problem with implanted HA sheets, however, is that they are not metabolized by tissue, are cumbersome to administer, and may result in long-term immunological problems.
Purified fibrin(ogen) (which is known in the art as a mixture of fibrin and fibrinogen) and several of its lytic fragments (i.e. FPA, FPB, D and E) have been shown to be chemotactic to a variety of cells including macrophages, human fibroblasts (HF) and endothelial cells (Gorodetsky, R., et al.
J. Lab. Clin. Med.
, in press (1997); Brown, L. F., et al.
Amer. J. Pathol.
142:273-283 (1993); Clark, R. A. F., et al.
J. Invest. Dermatol.
79:624-629 (1982); Ciano, P. S., et al.
Lab. Invest.
54:62-69 (1986); Dejana, E., et al.
J. Clin. Invest.
75:11-18 (1985)). Thrombin also has been shown to exert proliferative effect on various cells including fibroblasts, endothelial cells, and to enhance wound healing in rat skin (Kang, Y. H., et al.
J. Histochem. Cytochem.
39:413-423 (1991); Shuman, F.,
NY Acad. Sci.
408:228-235 (1986); Biedermann, B., et al.
J. Lab. Clin. Med.
124:339-347 (1994)).
Fibrin microbeads have been described in the prior art for use as drug delivery systems ((Ho, et al.
Drug Dev. and Ind. Pharm.
20(4):535-546 (1994); Senderoff, et al.
J. Parenteral Sci. & Tech.
45(1):2-6 (1991)). However, it has not been suggested or taught in the prior art that such fibrin microbeads have chemotactic and/or proliferative effects on any cells. Furthermore, the fibrin microbeads of Ho, et al. and Senderoff, et al. would not be particularly useful or desirable as vehicles for culturing cells. In this regard, the Ho, et al. microbeads contain glutaraldehyde which cross-links proteins and destroys certain biologically active sites, thereby interfering with the binding of the microbeads to cells. Glutaraldehyde treatment may also render the microbeads immunogenic. The Senderoff, et al. microbeads contain essentially the same relatively low degree of cross-linking as fibrin. Thus, the Senderoff, et al. microbeads are not stable in aqueous solutions and therefore would not be useful as vehicles for culturing cells which require matrices that do not readily dissolve in aqueous solutions.
SUMMARY OF THE INVENTION
The present invention provides fibrin microbeads that, unlike the fibrin microbeads of the prior art, do not contain any exogenous cross-linking agents such as glutaraldehyde that can damage certain biologically active sites that permit the microbeads to react with various types of cells. In addition, the fibrin microbeads of the present invention, unlike the prior art fibrin microbeads, contain extensive cross-linking of fibrin(ogen) which renders the fibrin microbeads stable for prolonged periods in aqueous solution, a property which is particularly desirable for use as vehicles for culturing cells, and for other uses.
Accordingly, it is an object of the present invention to provide fibrin microbeads that are biologically active and comprise extensively cross-linked fibrin(ogen).
It also is an object of the present invention to provide a method for preparing the fibrin microbeads of the present invention.
It is a further object of the present invention to provide a composition comprising cells bound to the fibrin microbeads of the present invention.
It is a still further object of the present invention to provide methods for culturing and separating one cell type from another using the fibrin microbeads of the present invention.
In addition, it is an object of the present invention to provide a method for transplanting cells using the fibrin microbeads of the present invention.
It also is an object of the present invention to provide a method for promoting healing of a wound using cells bound to the fibrin microbeads of the present invention.
Finally, it is an object of the present invention to provide a method for engineering tissue using the fibrin microbeads of the present invention.
Additional objects of the present invention will be apparent from the description which follows.
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Ho et al. “Fibrin-based drug delivery systems. II. The preparation and char
Gorodetsky Raphael
Marx Gerard
Amster Rothstein & Ebenstein
Hadasit Medical Research & Development Ltd.
Naff David M.
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