Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/56—Materials from animals other than mammals
  • A61K35/60—Fish, e.g. seahorses; Fish eggs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/01—Hydrolysed proteins; Derivatives thereof
  • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
  • A61K38/014—Hydrolysed proteins; Derivatives thereof from animals from connective tissue peptides, e.g. gelatin, collagen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/14—Macromolecular materials
  • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
  • A61L27/24—Collagen
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
  • A61L27/3843—Connective tissue
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0068—General culture methods using substrates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/50—Proteins
  • C12N2533/54—Collagen; Gelatin

Definitions

• the invention essentially relates to the use of collagen of aquatic origin for the production of supports intended for tissue engineering, as well as such supports and biomaterials.
• Collagen is a particularly favorable substrate for cell development. This is why this protein is widely used in several forms: matrices, gels, or films to produce reconstructed tissues comprising living cells.
• matrices, gels, or films to produce reconstructed tissues comprising living cells.
• collagen In the field of tissue engineering, a technique with a promising future, collagen has led in particular to the production of artificial skins or cartilages. To achieve a satisfactory result, collagen must be protected from enzymatic degradation originating from cell metabolism either by physical or chemical crosslinking processes or by the presence of natural macromolecules having strong interactions with the protein, or finally by a combination of the two. systems.
• Document EP-0 753 313 A1 describes a skin substitute using marine organisms based on a laminate comprising a layer or sheet of chitin extracted from mollusk or squid.
• the use of chitin is critical because when the chitin solution has been lyophilized, a chitin structure is obtained which constitutes an insoluble sponge which is not bio-degradable, the chitin not being degraded by the enzymes present in human skin.
• a fish skin collagen gel can be poured over the layer or sheet of chitin, which is left to dry in the refrigerator for about a week. As a result, the collagen layer is dense and is not porous. It cannot therefore be seeded with living cells.
• the laminate described in this document is an inert laminate and it is not intended that it can be seeded with living cells and keep the living character of these cells unlike the present invention described below.
• human cells develop very well on or inside certain supports made of fish collagen, preferably crosslinked.
• This marine collagen is preferably derived from the skin of fish of the teleost family, more particularly from fish having depigmented skin areas, even more particularly from flat fish, in particular those which are sinned industrially such as for example sole. , flounder, turbot, catfish and whose preparation of fillets involves butchering.
• the more preferred flatfish is sole, from which it is easy to cut the ventral part of non-pigmented skin.
• the inventors have been able to demonstrate that the human cells cultivated in these biomaterials maintain a normal metabolism.
• the bio-compatibility which allows conservation and proliferation capacity of living human cells with collagen extracted from the skin of fish of the teleost family was particularly not obvious to a person skilled in the art, and even more so when collagen is crosslinked and in particular chemically crosslinked, because in general the crosslinking of the collagen makes or risks making the crosslinked collagen non-bio-compatible, toxic with regard to living cells and in particular living human cells.
• biomaterials prepared according to the invention from fish collagen can be either films, or compressed sponges, or porous matrices which will be described as well as their methods of preparation in the examples given below.
• the present invention aims to solve the new technical problem consisting in the supply of new supports intended for tissue engineering capable of forming new biomaterials, that is to say capable of allowing a good proliferation of living, normal cells. or changed genetically, or malignantly to be cultured on said support and to be used in the context of these new biomaterials containing said living cells, for subsequent proliferation in vitro or in vivo.
• the present invention also aims to solve the new technical problem consisting in the supply of new supports intended for tissue engineering, at a low manufacturing cost and, moreover, at low risk of contamination, thus making them particularly suitable for provide new biomaterials.
• the main object of the present invention is also to solve the new technical problem consisting in the supply of new supports intended for tissue engineering, particularly well adapted to allow the cellular multiplication of living cells, normal or genetically modified, or malignant, in vitro. or in vivo, and whose structure is sufficiently compatible with an in vivo use in a mammal, in particular an animal or better a human being, while being different from the constitution of the tissues of such a mammal, such as an animal , or better and preferably a human being, in order to subsequently allow a differentiation between the newly synthesized tissues, and the old tissues of said mammal, preferably a human being.
• the present invention relates to the use of collagen of aquatic origin for the production of supports intended for tissue engineering.
• collagen of aquatic origin means a collagen derived from tissues containing collagen of living beings of aquatic origin, these living beings are well known to those skilled in the art and include, for example, without limitation, aquatic mammals, especially marine mammals, jellyfish, and sea or freshwater fish. Those skilled in the art know, moreover, that these living beings have a skin which essentially contains collagen.
• the "collagen of aquatic origin” is extracted from the skin of fish from the teleost family, more particularly from fish having depigmented skin areas, even more particularly from flat fish and in particular fish mentioned above, the most preferred flat fish being sole.
• the collagen is obtained from the skin of fish, preferably in its native form.
• collagen has its reinforced mechanical resistance where its resistance to enzymatic digestion increased either by chemical and / or physical crosslinking, or by the addition of a natural macromolecule having strong interactions with collagen, either by a combination of the two processes.
• the collagen is used in the form of a porous matrix prepared from a collagen gel preferably subjected to a lyophilization step.
• the aforementioned porous matrix is crosslinked by physical crosslinking, preferably by hydrothermal dehydration or DHT.
• the above-mentioned porous matrix is crosslinked by chemical crosslinking, preferably with diphenylphosphorylazide or DPPA or with a carbodiimide and / or N-hydroxysuccinimide or with glutaraldehyde.
• the above-mentioned collagen can be in the form of a porous matrix prepared from preferably native marine collagen, mixed with chitosan and optionally at least one glycosaminoglycan, preferably chondroitin sulfate.
• the abovementioned collagen can be in the form of a porous matrix prepared from a collagen gel, said porous matrix being coated on at least one face with a membrane essentially compact collagen produced either by a collagen film prepared by drying, preferably in air or in a gaseous fluid, of a collagen gel, or by a very strongly compressed collagen sponge.
• the aforementioned compression of the very strongly compressed collagen sponge is carried out at a minimum pressure equal to approximately 50 bars (approximately 50.10 5 Pascal (Pa)), preferably between 50 bars (50.10 5 Pa ) and 200 bars (200.10 5 Pa), this compression having possibly been carried out at a temperature between 20 ° C and 80 ° C, even better between 40 ° C and 60 ° C.
• at least one of the two layers, respectively the porous layer and the essentially compact membrane comprises living cells, normal or genetically modified, or malignant, in particular from young or old subjects.
• the living cells are chosen from the group consisting of fibroblasts, keratinocytes, melanocytes, langerhan cells of blood origin, endothelial cells of blood origin, blood cells, in particular macrophages or lymphocytes, langerhan cells of blood origin, endotelial cells of blood, blood cells, adipocytes, sebocytes, chondrocytes, bone cells, osteoblasts, Merkel cells of blood origin, normal or genetically modified or malignant.
• the porous layer contains normal, genetically modified or malignant fibroblasts
• the essentially compact membrane contains normal or genetically modified or malignant living cells, in particular chosen from keratinocytes, melanocytes, cells of Merkel of blood origin, langerhans cells of blood origin, sebocytes, cells of blood origin, nerve cells.
• the above-mentioned essentially compact membrane is prepared before combining with the porous layer, preferably comprising a collagen sponge, in particular by preparing the membrane and depositing it on a collagen gel before the the whole is frozen and freeze-dried.
• the present invention also covers a support intended for tissue engineering, characterized in that it comprises collagen of aquatic origin as defined above or as it results from the following description taken as a whole , and integrating the examples which form an integral part of the present invention in their generality and with regard to any characteristic which appears to be new compared to a state of the art any technique, this characteristic being taken in its function and in its generality independently of the context of the example.
• the present invention also covers a biomaterial, for example in the form of a reconstituted connective tissue, or of reconstituted skin, characterized in that it has been prepared from collagen of aquatic origin as previously defined in all these aspects and as it also results from the following description as for the support of the second aspect above.
• supports intended for tissue engineering means a support which will be used to carry out the culture and the proliferation of living cells, normal or genetically modified, or malignant, whether either in vitro or in vivo, this proliferation preferably being applied in vivo to a mammal, comprising an animal, better still and preferably a human being. It is understood that the invention finds a particularly preferred use in the context of tissue engineering for the manufacture of biomaterials, for example in the form of reconstituted connective tissues or reconstituted skins.
• a first step will generally be a culture of the support with said living cells in vitro to result in a biomaterial, for example in the form of a reconstituted connective tissue or of reconstituted skin, then, in a second step, the use of this biomaterial as reconstituted connective tissue or skin reconstituted in vivo on a mammal, for example animal and preferably and better still a human being, in order to reconstitute a connective tissue damaged or removed by surgery, or similarly for a skin reconstructed replacing a damaged or surgically removed area of skin for any medical reason.
• a mammal for example animal and preferably and better still a human being
• the support intended for tissue engineering or better still the biomaterial, which is for example in the form of a reconstituted connective tissue, or of reconstituted skins, comprises cells either obtained substantially exclusively from young subjects, or obtained substantially exclusively from elderly subjects, in particular to study the process of tissue aging and in particular skin aging and possibly test the effectiveness of active ingredients or principles on this process.
• the invention makes it possible to solve on the whole new technical problems stated above in a particularly simple manner, at low cost, with low risk of contamination and with a differentiation capacity of aquatic collagen relative to collagen of the mammal, and preferably of the collagen of a human being, newly synthesized during use in vivo.
• the raw material fish skin generally used can be obtained in abundance under very clean conditions.
• fish collagen has an amino acid composition relatively distant from that of human collagen, the two proteins can be relatively easily differentiated by means of specific antibodies.
• marine collagen will make the immunostaining very effective and allow the differentiation of marine collagen relative to the newly synthesized collagen.
• Fish collagen has a native structure which protects it from enzymatic degradation due to proteases and which gives it a large part of its mechanical properties. It will therefore be very important during the extraction and purification operations that the treatments used alter the structure of the protein as little as possible. This means that the helical structure as well as the inter and intramolecular crosslinks are preserved as much as possible.
• the protection of fish collagen will be all the more important as its natural stability is weaker than that of mammalian collagen. This last characteristic is due to a lower rate of hydroxyproline.
• biomaterials previously described can be seeded with living cells and will thus give rise to living artificial tissues which can be used either in the field of "in vitro" tests or in the pharmaceutical field for repairing damaged tissue.
• Other objects, characteristics and advantages of the invention will become clear in the light of the explanatory description which follows, given with reference to the embodiments of a form of collagen of aquatic origin which can be used in the context of the invention for the production of supports intended for tissue engineering, and thus constituting such supports as well as biomaterials, given simply by way of illustration and which therefore cannot in any way limit the scope of the invention.
• Examples 1 to 13 naturally constitute examples of collagen preparation which can be used as a tissue engineering support according to the invention.
• Examples 14 to 16 constitute in particular comparative tests, in the context of the use of this collagen of aquatic origin, in the forms prepared in some of Examples 1 to 13, in the context of the production of supports intended for tissue engineering.
• FIG. 1 represents the proliferation of normal human fibroblasts in equivalent dermis, with the time expressed in days and on the abscissa and the optical density x 1000 on the ordinate with units increasing by 100;
• the curve with the diamonds is that obtained using a porous matrix of aquatic collagen, in this case fish, as support, and the curve with squares is obtained with bovine collagen;
• FIG. 2 represents a similar proliferation curve of fibroblasts in equivalent dermis with the time expressed in days on the abscissa and the fluorescence expressed in international units on the ordinate, starting at 15,000 with units increasing by 10,000; the curve with the solid diamonds represents the fluorescence obtained in the context of test 1; the curve with the square that obtained with test 2; the curve with the empty triangles being obtained with test 3 and finally the curve with the crosses being that obtained with test 4.
• a collagen gel is prepared from ground ventral sole skins, then washed with a phosphate buffer pH 7.8, the composition of which is: 0.78 g / 1 of potassium dihydrogen phosphate and 21.7 g 1 of disodium monohydrogen phosphate.
• the washing is carried out with stirring for one hour at a rate of 5 l of buffer for 1 kg of ground material.
• the phosphate is then removed by two successive washes with deionized water, then by continuous centrifugation at 4000 rpm (Rousselet decanter), at the rate of 5 l of water per 1 kg of ground material.
• the ground product is then acidified with a 0.25 M acetic acid solution at a rate of 1 kg of ground material for 10 1 of solution.
• the gel is then centrifuged at
• the gel which will be used consists of the supernatant obtained whose collagen concentration is between 0.5 and 2%.
• This gel is poured into a lyophilization tray at a rate of 20 g / cm 2 . It is then lyophilized after freezing at -30 ° C and heating to + 32 ° C. The lyophilization lasts a total of 16 hours under a pressure of 400 microbars. The matrix obtained is then crosslinked by hydrothermal dehydration
• Example 1 The collagen matrix of Example 1 is incubated for 24 h in a solution containing 5 to 250 ⁇ l of DPPA / g of collagen contained in. 100 ml of dimethylformamide (DMF). The collagen is then freed from DPPA by rinsing in 100 ml of DMF. The DMF is then eliminated by rinsing in 100 ml of a borate buffer solution pH 8.9 (sodium tetraborate
• the collagen is finally incubated overnight in the same borate buffer. Finally, the borate buffer is removed by rinsing with continuous permuted water for 6 h.
• the aquatic collagen matrix of Example 1 is crosslinked with EDC (Ethyldimethylaminopropyl carbodiimide) at the concentration of 0.23 to 0.69 g / g of collagen, and with NHS (N-hydroxysuccinimide) at the concentration of 0.42 g / g of collagen.
• EDC Ethyldimethylaminopropyl carbodiimide
• NHS N-hydroxysuccinimide
• the collagen After rinsing with deionized water, the collagen is again lyophilized.
• the porous aquatic collagen matrix of Example 1 is crosslinked for 24 to 96 h in a solution containing 0.6 to 1% of GTA at 20 ° C. After rinsing with deionized water, the collagen is again lyophilized.
• Porous matrix prepared with the native aquatic collagen of example 1 in combination with chitosan and a glycosaminoglycan as described in the European patent of May 29, 1991 N ° 296078 To 600 g of gel with 1.5% of collagen, are added 2.5 g of chitosan dissolved in 356 ml of water and 1.9 ml of acetic acid, then a solution containing 1 g of chondroitin 4 sulfate contained in 400 ml of deionized water. The mixture, the pH of which is approximately 4.0, is then stirred and then lyophilized. The sponge obtained is crosslinked by DHT.
• Porous matrix described in Example 1 surfaced with a collagen film
• Collagen gel has a dry matter is between 0.3 and '0.8% is dried in an oven at 30 ° C or under a hood at a rate of 0.5 g of gel / cm tray.
• the collagen gel it is possible to add 10 to 40% glycerol.
• the collagen dried under these conditions forms a transparent film.
• the native aquatic collagen gel of 0.5% to 2% in dry matter is deposited at the rate of 0.5 g per cm 2 in a lyophilization tray, then the collagen film is deposited on this gel and the whole is lyophilized.
• the lyophilisate obtained is crosslinked with DHT.
• Porous matrix prepared with an acid-soluble collagen gel surfaced with a collagen film
• Example 6 The process is that indicated in Example 6, the only difference being the nature of the gel cast on the film which is made of acid-soluble collagen prepared according to a technique well known to those skilled in the art.
• Porous matrix prepared with an atelocoUagene gel or surfaced with a collagen film
• Example 6 The process is that indicated in Example 6, the only difference being the nature of the gel cast on the film which is made atelocoUagen, that is to say collagen without telopeptide prepared according to a technique well known to those skilled in the art.
• Porous matrix consisting of collagen associated with chitosan and a glycosaminoglycan surfaced with a collagen film.
• Example 6 The process is that indicated in Example 6, but in this case the gel poured onto the collagen film consists of collagen, chitosan, a glycosaminoglycan. The preparation of this gel is described in Example 5.
• porous matrices surfaced with a collagen film described above can be crosslinked according to the techniques described in Examples 2, 3 and 4.
• Porous collagen alone matrix described in Example 1 surfaced with a compressed collagen sponge.
• the collagen gel prepared as in Example 1 and having a dry matter of between 0.3 and 1.5% is lyophilized so as to obtain a sponge having a weight of between 0.5 and 2 g / cm 2 .
• the lyophilisate is compressed for 5 to 60 seconds, at a temperature between 20 and 60 ° C and a pressure between 50 and 200 bars (50 to 200.10 5 Pa).
• the collagen gel described in Example 1 is deposited at a rate of 0.5 g per
• Porous matrix consisting of collagen, chitosan and glycosaminoglycan as described in Example 5 and surfaced with the compressed sponge.
• the collagen, chitosan and glycosaminoglycan gel prepared according to the method of Example 5, is deposited at a rate of 0.5 g per cm in a freeze-drying tray, then the compressed sponge is deposited on this gel and the assembly is lyophilized. The lyophilisate is then crosslinked by DHT as described in Example 1.
• bovine matrix a comparative porous matrix with collagen of bovine origin, known as bovine matrix, also crosslinked with DPPA, under the same conditions as those of Example 2.
• bovine matrix collagen of bovine origin
• DPPA DPPA
• the culture of these aquatic and bovine matrices is carried out respectively in medium composed of DMEM / HAM F 12 in a 50/50 (v / v) ratio supplemented with 10% fetal calf serum, 100 IU / ml of penicillin, 25 ⁇ g / ml gentamycin, 1 ⁇ g / ml amphotericin B, 50 ⁇ g / ml vitamin C.
• This culture is carried out for 1 month by changing the culture medium 3 times a week.
• MTT that is to say 3- (4- (dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide) is added to the culture medium.
• Incubation is carried out for 2.5 hours at 37 ° C.
• the optical density of the transformation product (formazan blue) is read at 550 nm after solubilization in DMSO.
• the optical density obtained is proportional to the activity of the succinate dehydrogenases which are capable of carrying out the transformation of the light yellow tetrazolium MTT salt into blue crystals of formazan.
• the aquatic matrix not only constitutes a support allowing the survival of normal human fibroblasts but also the cell proliferation of these normal human fibroblasts, while even constituting a much better culture support during first three weeks. It can therefore be concluded from these tests that the aquatic collagen is, surprisingly, particularly suitable for producing a tissue engineering support in particular for applications in vitro and even and especially in vivo to constitute biomaterials containing living cells and in particular and preferably living cells of human beings.
• the assay is carried out by the Pierce microBCA method.
• fibroblasts could be observed in matrices of bovine and aquatic origin. In both types of matrices, we note the presence of an abundant neosynthesized extracellular matrix.
• the neosynthesized extracellular matrix can be differentiated by the periodic striation of the deposited collagen fibers compared to the collagen clusters forming the three-dimensional matrix of the starting sponge.
• Various collagen supports or matrices are prepared by using various proportions of collagen in the collagen gel used to make the porous layer or matrix and optionally using a different crosslinking agent, as follows:
• a porous matrix is produced in the form of a porous sponge from an aquatic collagen gel prepared from 1.3% by weight of aquatic collagen, which is frozen at -80 ° C. and that it is subjected to a standard lyophilization in accordance with Example 2 and which is then subjected to crosslinking with DPPA in a proportion of 250 ⁇ l / g of sponge in the dry state.
• Test 2 For this test, a porous support is prepared in the form of an aquatic sponge from an aquatic collagen gel comprising 0.7% by weight of aquatic collagen, which is subjected to freezing at -80 ° C, then standard lyophilization and crosslinking with DPPA at 250 ⁇ l / g dry as in test 1.
• a porous support comprising an aquatic collagen sponge obtained from an aquatic collagen gel comprising 1.1% by weight of aquatic collagen, which is subjected to freezing at -80 ° C., then to lyophilization standard and crosslinking with DPPA at 250 ⁇ l / g dry as in test 2, the difference residing in a proportion of 1.1% by weight of aquatic collagen.
• the aquatic collagen is derived, as in Example 2, from the ventral sole skin. b) Culture of fibroblasts on these matrices
• Example 14 Is used as in Example 14, normal human fibroblasts but which are taken at 8 th passage.
• the culture medium is composed of DMEM / HAM F12 50/50 (v / v) added with 10% by weight of fetal calf serum, 100 IU 7 ml of penicillin, 25 ⁇ g / ml of gentamycin, 1 ⁇ g / ml of amphotericin B , 50 ⁇ g / ml of vitamin C.
• the culture is carried out for 1 month by changing the medium 3 times a week.
• 4 matrices are used in order to carry out an average for each type of test and to measure the mean standard deviation.
• Alamar Blue is added at a rate of 2% by weight of a culture medium used, when it is desired to measure the cell viability on a sample taken from the culture medium.
• the fluorescence is read, on the basis of an excitation at 530 nm and an emission at 590 nm).
• the intensity of the fluorescence obtained is proportional to the metabolic activity of the cells.
• the cell viability measurement is carried out on 10 samples after 1, 4 6, 11 17 days of culture. The results are expressed in the table or below.
• Table 3 show the fibroblastic proliferation curves in equivalent dermis.
• the solid diamond curve is that performed with test 1; the curve in full square is that carried out with test 2; the triangle curve is that carried out with test 3 and the curve with the crosses is that carried out with test 4.
• the different matrices prepared can allow good fibroblastic growth after 17 days of culture.
• the fibroblasts adhere well to their three-dimensional support and divide very quickly in order to colonize the matrix.
• the proliferation profile is very slightly variable from one type of matrix to another, but after 17 days of culture, the fibroblastic density is comparable regardless of the manufacturing process.
• This test is similar to that of Example 14, except that a histology with immunostaining is carried out.
• Example 14 These are equivalent dermes from Example 14, the culture being carried out under the conditions of Example 14.
• This culture is therefore carried out for three weeks by changing the medium three times a week, the seeding of normal human fibroblasts having taken place at 300,000 cells per cm 2 as was indicated in Example 14.
• the fixation is carried out with paraformaldehyde at a content of 4% by weight, then a dehydration and a paraffin inclusion are carried out.
• Tissue Tek OCT compound is carried out, that is to say an inclusion liquid supplied by Miles, Elkhart, Indiana, USA, and a cold cut at 7 ⁇ m.
• Immunostaining is performed as follows: i. With a first rabbit human anti-collagen type I antibody (dilution 1/40) ii. A second anti-rabbit antibody FITC (Fluorescein Iso ThioCyanate) (dilution 1/160)
• the supports constituted respectively by an aquatic matrix and a bovine matrix form more or less loose pores in which the fibroblasts adhere.
• On the surface there is a higher proportion of fibroblasts forming a favorable surface treatment of the equivalent dermis for producing reconstructed skin.
• the distribution of fibroblasts is homogeneous in aquatic and bovine sponges.
• the matrix of aquatic origin is only very weakly marked by the human anti-collagen antibody.

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• 2001
  • 2001-05-25 WO PCT/FR2001/001629 patent/WO2001092322A1/fr active Application Filing
  • 2001-05-25 JP JP2002500933A patent/JP3747412B2/ja not_active Expired - Fee Related
  • 2001-05-25 DE DE10196235T patent/DE10196235B4/de not_active Expired - Fee Related

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