Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Implant or insert
Reexamination Certificate
2000-07-14
2003-04-01
Russel, Jeffrey E. (Department: 1654)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Implant or insert
C424S423000, C424S424000, C424S425000, C424S443000, C424S484000, C435S397000, C435S398000, C435S401000, C435S402000, C530S356000, C623S015120
Reexamination Certificate
active
06541023
ABSTRACT:
SUBJECT OF THE INVENTION
The invention relates essentially to the use of collagen of aquatic origin for the production of supports for tissue engineering, and to such supports and biomaterials.
TECHNOLOGICAL BACKGROUND
Collagen is a particularly favorable substrate for cell development, which is why this protein is very widely used in several forms—matrices, gels or films—for the production of reconstructed tissues containing living cells.
In the field of tissue engineering, a technique which promises to have a great future, collagen has afforded the production of artificial skin or cartilage in particular. To achieve a satisfactory result, the collagen has to be protected from enzymatic degradation due to cell metabolism, either by physical or chemical crosslinking processes, or by the presence of natural macromolecules which interact strongly with the protein, or finally by a combination of both systems.
Hitherto, for these tissue engineering applications, the collagen used in supports for receiving cells was extracted from mammals and most frequently from bovine skin. The choice of this source was due to the good mechanical properties of the protein obtained after extraction, to its resistance to enzymatic degradation and finally to its amino acid composition, which is very similar to that of human collagen. For all these reasons, it was legitimate to think that this collagen was the only one suitable for the culture of human cells.
PURPOSES OF THE INVENTION
Now, the inventors have noticed, unexpectedly, that human cells develop very well on or inside certain supports consisting of preferably crosslinked fish collagen. In addition, the inventors have been able to demonstrate that human cells cultivated in these biomaterials preserve a normal metabolism. These biomaterials can be either films, or compressed sponges, or porous matrices, which will be described together with their methods of preparation in the Examples given below.
One object of the present invention is to solve the new technical problem which consists in providing novel supports for tissue engineering suitable for forming novel biomaterials, i.e. suitable for allowing a good proliferation of the normal, genetically modified or malignant living cells to be cultivated on said support and to be used within the framework of these novel biomaterials, containing said living cells, for subsequent proliferation in vitro or in vivo.
A further object of the present invention is to solve the new technical problem which consists in providing novel supports for tissue engineering at a low manufacturing cost and also with a low risk of contamination, thus making them particularly suitable for the provision of novel biomaterials.
A further main object of the present invention is to solve the new technical problem which consists in providing novel supports for tissue engineering which are particularly suitable for allowing the multiplication of normal, genetically modified or malignant living cells, in vitro or in vivo, and whose structure is sufficiently compatible with in vivo use in a mammal, particularly an animal or, preferably, a human being, while at the same time being different from the constitution of the tissues of said mammal, such as an animal or, preferably, a human being, so as to allow subsequent differentiation between the newly synthesized tissues and the old tissues of said mammal, preferably a human being.
The present invention solves all these technical problems for the first time in a satisfactory manner, at low cost, with a low risk of contamination or without contamination, while at the same time easily making it possible to identify the newly synthesized tissues, which is particularly non-obvious and unexpected for those skilled in the art.
SUMMARY OF THE INVENTION
Thus, according to a first feature, the present invention relates to the use of collagen of aquatic origin for the production of supports for tissue engineering, as well as the corresponding method of production thereof.
The expression “collagen of aquatic origin” is understood as meaning a collagen derived from collagen-containing tissues of living beings of aquatic origin; these living beings are well known to those skilled in the art and include for example, without implying a limitation, aquatic mammals, particularly marine mammals, jellyfish and saltwater or freshwater fish. Furthermore, those skilled in the art know that the skin of these living beings contains essentially collagen.
In one advantageous embodiment, the collagen is obtained from fish skin, preferably in its native form.
In another advantageous embodiment of the invention, the mechanical strength of the collagen or its resistance to enzymatic digestion is increased either by chemical and/or physical crosslinking, or by the addition of a natural macro-molecule which interacts strongly with collagen, or by a combination of both processes.
In yet another advantageous embodiment of the invention, the collagen is used in the form of a porous matrix prepared from a collagen gel which has preferably undergone a lyophilization step.
In yet another advantageous variant, the above-mentioned porous matrix is crosslinked by a physical method, preferably by thermal dehydration, or TDH.
In yet another advantageous variant, the above-mentioned porous matrix is crosslinked by a chemical method, preferably with diphenylphosphorylazide, or DPPA, or with a carbodiimide and/or N-hydroxysuccinimide, or with glutaraldehyde.
In one advantageous embodiment, the above-mentioned collagen can take the form of a porous matrix prepared from marine collagen (preferably native) mixed with chitosan and optionally at least one glycosaminoglycan, preferably chondroitin sulfate.
In yet another advantageous embodiment of the invention, the above-mentioned collagen can take the form of a porous matrix prepared from a collagen gel, said porous matrix being covered on at least one side with an essentially compact collagen membrane consisting either of a collagen film prepared by drying a collagen gel, preferably in air or a gaseous fluid, or of a very highly compressed collagen sponge.
In another advantageous variant, the above-mentioned compression of the very highly compressed collagen sponge is carried out at a pressure of at least about 50 bar (about 50.10
5
Pascal (Pa)) and preferably of between 50 bar (50.10
5
Pa) and 200 bar (200.10
5
Pa), this compression optionally having been carried out at a temperature of between 20° C. and 80° C. and preferably of between 40° C. and 60° C.
According to yet another advantageous characteristic of the invention, at least one of the two layers, i.e. the porous layer and the essentially compact membrane, comprises normal, genetically modified or malignant living cells originating particularly from young or elderly subjects.
In one advantageous embodiment, the living cells are selected from the group consisting of fibroblasts, keratinocytes, melanocytes, Langerhans' cells originating from the blood, endothelial cells originating from the blood, blood cells, particularly macrophages or lymphocytes, adipocytes, sebocytes, chondrocytes, osteocytes, osteoblasts and Merkel's cells originating from the blood, said cells being normal, genetically modified or malignant.
In one particularly advantageous embodiment, the porous layer contains normal, genetically modified or malignant fibroblasts and the essentially compact membrane contains normal, genetically modified or malignant living cells selected particularly from keratinocytes, melanocytes, Merkel's cells originating from the blood, Langerhans' cells originating from the blood, sebocytes, cells originating from the blood, and nerve cells.
In yet another advantageous embodiment of the invention, it may be of particular value to prepare either “young” reconstructed skin using cells taken from young subjects, or “aged” reconstructed skin obtained from cells taken from elderly subjects. These models will enable us to improve our knowledge of the skin ageing process and study the influence of active ag
Abdul Malak Nabil
Andre Valerie
Huc Alain
Coletica
Merchant & Gould P.C.
Russel Jeffrey E.
LandOfFree
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