FR2809314A1 - Use of crosslinked collagen from teleostean fishes to prepare supports for artificial skin for in vitro testing of dermatological products - Google Patents

Abstract

Use of crosslinked collagen of aquatic origin is claimed, to prepare supports for tissue engineering. An Independent claim is included for biomaterial in the form of reconstituted conjunctive tissue prepared from collagen, containing cells obtained from young subjects or from aged subjects, suitable for use in the study of tissue ageing and the effectiveness of active ingredients on the ageing process.

Description

The invention essentially relates to the use of collagen of aquatic origin for the production of supports for tissue engineering, as well as such supports and biomaterials.

Collagen is a particularly favorable substrate for cell development. Therefore, this protein is widely used in several forms: matrices, gels, or films for producing reconstructed tissues comprising living cells.

In the field of tissue engineering, a technique that promises a great future, collagen has led in particular to the production of artificial skins or cartilages. To achieve a satisfactory result, the collagen must be protected from enzymatic degradation from cellular metabolism or physical or chemical crosslinking processes or by the presence of natural macromolecules with strong interactions with the protein or a combination of both systems.

Until now, for these tissue engineering applications, the collagen used in the supports intended to receive the cells was extracted from mammals and most often from bovine skin. The choice of this source was the good mechanical properties of the protein obtained after extraction, its resistance to enzymatic degradation and finally its amino acid composition very close to that of human collagen. For all these reasons it was legitimate to think that this collagen was the only one that could be suitable for culturing human cells.

The article by Yeh et al in Faseb Journal, Vol. 14, No. 4, March 15, 2000 entitled "a novel native matrix for tissue engineering." Analysis of cell-matrix interaction. Invokes the use of whale shark acellular collagen matrix for the study of its interaction with human skin keratinocytes and fibroblasts in order to study bio-compatibility. The shark belongs to the Selacian family different from the teleosts.

Derwent XP Abstract 002161598 refers to Japanese patent application JP 07/075566 of Marino Forum published March 20, 1995 relating to a method using fish-derived collagen as a culture base. However, this culture concerns fish cells.

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 mollusc or squid. Here, the use of chitin is critical because when chitin solution has been lyophilized, a chitin structure is obtained which constitutes an insoluble non-biodegradable sponge, the chitin not being degraded the enzymes present in the human skin. A fish skin collagen gel can be poured onto the chitin layer or sheet, which is allowed to dry in the refrigerator for about a week (see page 3, lines 5-8).

The procedure for extracting collagen from salmon skin is described on page 4, line 44, on page 5, line 3, in which the last step numbered 8 provides that the totally neutralized collagen solution is lyophilized ("freeze"). -dried ").

Further on page 5, line 28 and following, it is stated that the skin substitute of this prior invention comprises the shellfish chitin layer and a fish skin collagen layer, of which it is not further stated how it is applied on the chitin layer and it is understood that the collagen solution is reconstituted to be poured onto the previously prepared chitin sheet. The skin substitute is used by direct application of the chitin sheet on the surface of a wound (page 5, lines 30).

It is emphasized that salmon skin collagen has the advantage of a denaturation temperature considerably lower than that of bovine skin collagen (page 5, lines 5-6) and, on the other hand, it is emphasized in page 7, lines 35-36, in the tests that salmon skin collagen returns to the amorphous state at 37 C in a physiologically active environment, its superior efficacy compared to calf skin collagen for cell proliferation (page 7, lines 38-39).

This document thus demonstrates to those skilled in the art that the collagen must be used as it is and have not undergone any steps that can modify its structure, unlike the present invention which uses crosslinked collagen.

 Incidentally, it should be noted that the laminate described in this document is an inert laminate and it is not expected that it can be seeded with live cells keeping alive these cells in contrast to the present invention described hereinafter.

However, the inventors have unexpectedly found that human cells develop very well on or within certain supports consisting of crosslinked collagen of aquatic origin. This aquatic or marine collagen is preferably derived from the fish skin of the teleosts family, more particularly obtained from flatfish skins, in particular those that are fished industrially, for example sole, flounder and turbot. , the brill and the preparation of fillets involves a cutting. It may be advantageous in some applications to extract collagen depigmented skin. The most preferred flatfish is the sole which is easy to cut off the ventral portion of unpigmented skin from which collagen is produced.

In addition, the inventors have been able to demonstrate that the human cells cultured in these biomaterials maintained a normal metabolism. Biocompatibility, which allows the preservation and proliferation capacity of living human cells with cross-linked collagen extracted from fish skin, preferably from the teleost family, was particularly not obvious to one skilled in the art because the the art knows that the crosslinking of collagen makes or risks rendering crosslinked collagen non-bio-compatible, toxic towards living cells and in particular living human cells.

These biomaterials prepared according to the invention from cross-linked collagen of fish can be either films, compressed sponges or porous matrices which will be described as well as their methods of preparation in examples given below.

The object of the present invention is to solve the new technical problem consisting in the provision of new supports intended for tissue engineering capable of forming new biomaterials, that is to say able to allow a good proliferation of normal living cells. or genetically modified, or malignant to grow on said support and to use in the context of these new biomaterials containing said living cells, for subsequent proliferation in vitro or in vivo.

Another object of the present invention is to solve the new technical problem of providing new supports for tissue engineering, low manufacturing cost and, in addition, low risk of contamination which are substantially entirely biodegradable. thus making it particularly suitable for providing new biomaterials.

The main object of the present invention is also to solve a new technical problem consisting in the provision of new supports intended for tissue engineering, particularly well adapted to allow cell multiplication of living cells, normal or genetically modified, or malignant cells, in vitro or in vitro. 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 and preferably a human being, in order to subsequently allow differentiation between the newly synthesized tissues and the ancient tissues of said mammal, a human being.

All of these technical problems are solved for the first time with the present invention in a satisfactory manner, low cost, low risk of contamination or without contamination, while allowing easy detection of newly synthesized tissues, this which is particularly unobvious and unexpected for a man of the art.

Thus, the present invention, according to a first aspect, relates to the use of crosslinked collagen of aquatic origin for the production of supports intended for tissue engineering.

By the term "collagen of aquatic origin" is meant 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 marine or freshwater fish. The person skilled in the art knows, elsewhere, that these living beings have a skin that essentially contains collagen. Preferably, the "collagen of aquatic origin" is extracted from the fish skin of the teleosts family, more particularly from the skin of flat fish, and in particular those which are fished industrially, for example the sole , dab, turbot, catfish, preferably sole, more particularly areas of depigmented fish skins.

According to an advantageous embodiment, the collagen is obtained from the skin of fish, preferably in its native form.

According to another advantageous embodiment of the invention, the collagen is crosslinked either by a chemical and / or physical crosslinking, or by addition of a natural macromolecule having strong interactions with the collagen, or by a combination of said methods. which also makes it possible to have its reinforced mechanical resistance or its resistance to enzymatic digestion increased.

According to yet another advantageous embodiment of the invention, the collagen is used in the form of a porous matrix prepared from a collagen which is preferably subjected to a freeze-drying step. According to yet another advantageous variant embodiment, the aforementioned porous matrix is crosslinked by physical crosslinking, preferably by hydrothermal dehydration or DHT.

According to yet another advantageous embodiment, the aforementioned porous matrix is crosslinked by chemical crosslinking, preferably with diphenylphosphorylazide or DPPA or with a carbodiimide and / or N-hydroxysuccinimide with glutaraldehyde.

According to an advantageous embodiment, the abovementioned collagen may be in the form of a porous matrix prepared from marine collagen, preferably native, chitosan-modified and optionally at least one glycosaminoglycan, preferably chondroitin sulfate.

According to yet another advantageous embodiment of the invention, the abovementioned collagen may be in the form of a porous matrix prepared from a collagen gel, said porous matrix being coated on at least one side of a membrane. essentially compact collagen made either by a collagen film prepared by drying, preferably in air or in a gaseous fluid, a collagen gel, or by a very highly compressed collagen sponge.

According to an equally advantageous variant embodiment, the above-mentioned compression of the highly compressed collagen sponge is carried out at a minimum pressure equal to about 50 bar (approximately 50 × 10 5 Pascal (Pa)), preferably between 50 bar (50 × 10 5 Pa) and 200 bars (200.105 Pa), this compression possibly being carried out at a temperature between 20 C and 80 C, more preferably between 40 C and 60 C.

According to yet another advantageous feature of the invention, the crosslinked collagen, in particular in the physical form of at least one layer, comprises living cells, normal or genetically modified, or malignant, in particular from young or old subjects.

According to an advantageous embodiment, the living cells are chosen from the group consisting of fibroblasts, keratinocytes, melanocytes, blood-born langerhan cells, endothelial cells of blood origin, blood cells, in particular macrophages or lymphocytes, langerhan cells. of blood origin, endotelial cells of blood origin, blood cells, adipocytes, sebocytes, chondrocytes, bone cells, osteoblasts, blood-derived Merkel cells, normal or genetically modified or malignant. According to another advantageous embodiment according to which the aforementioned cross-linked collagen is in the form of a porous matrix coated on at least one side of an essentially compact collagenous membrane, at least one of the two layers comprises said living, normal or modified living cells. genetically, or malignant, especially from young or old subjects. Preferably, the collagen of each layer is crosslinked.

According to a particularly advantageous embodiment, the porous layer contains normal fibroblasts, genetically modified or malignant, and the essentially compact membrane contains normal living cells or genetically modified or malignant, in particular selected from keratinocytes, melanocytes, Merkel of blood origin, cells of langerhans of blood origin, sebocytes, cells of blood origin, nerve cells.

According to yet another advantageous embodiment of the invention, it may be particularly advantageous to prepare either "young" reconstructed skin using cells taken from young subjects, or "aged" reconstructed skin obtained from cells taken from elderly subjects. Thanks to these models, it will be possible to improve knowledge on skin aging processes and to study the influence of active ingredients on this process.

According to another particularly advantageous embodiment, 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 whole be frozen and freeze-dried. The assembly is preferably crosslinked according to one of the three crosslinking techniques described chemical and / or physical crosslinking and / or addition of a natural macromolecule having strong interactions with collagen.

According to a second aspect, the present invention also covers a support intended for tissue engineering, characterized in that it comprises reticulated collagen of aquatic origin as defined above or as it results from the following description taken in its entirety. together, and integrating the examples are an integral part of the present invention in their generality and as regards any feature that appears to be new with respect to any state of the art, this feature being taken in its function and in its generality regardless of context of the example. According to a third aspect, the present invention also covers a biomaterial, for example in the form of reconstituted connective tissue, or reconstituted skin, characterized in that it has been prepared from crosslinked collagen of aquatic origin as previously defined in all these aspects and this also results from the following description as for the support of the second aspect above.

In the context of the present description and the claims, the expression "supports intended for tissue engineering" means a support which will be used to carry out the cultivation and proliferation of living cells, normal or genetically modified, or malignant, whether in vitro. in vitro or in vivo, this proliferation being preferably applied in vivo to a mammal, comprising an animal, better and preferably a human.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 tissue or reconstituted skin In this context, 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 reconstituted connective tissue or reconstituted skin, then, in a second step, use of this biomaterial as reconstituted connective tissue or reconstituted skin in vivo on a mammal, e.g. animal and preferably and more preferably human, to reconstitute damaged or surgically abraded connective tissue, or likewise for reconstituted skin replacing an area of damaged skin or surgically removed for any medical reason.

Advantageously, the support intended for tissue engineering, or better the biomaterial, which is for example in the form of reconstituted connective tissue, of reconstituted skins, comprises cells either obtained almost exclusively from young subjects, or obtained It is mainly from elderly subjects, in particular to study the tissue aging process and in particular the skin, and possibly test the effectiveness of active ingredient ingredients on this process.

It will thus be understood that the invention makes it possible to solve all of the above-mentioned new technical problems of a particularly simple, low-cost, low-risk contamination and with a capacity for differentiation of aquatic collagen relative to collagen of the mammal. and preferably collagen of a newly synthesized human being when used in vivo.

Indeed, the implementation of fish collagen in the production of living artificial tissues provides three essential advantages over the mammalian source. The fish skin raw material generally used, can be obtained in abundance under conditions of great cleanliness.

. The danger of infectious contamination is very low. In particular, the risk of transmission of prion-type agents is unknown to date.

 Finally, since fish collagen has an amino acid composition that is relatively far apart from that of human collagen, the two proteins can be differentiated in a relatively easy manner by means of specific antibodies. This methodology will be valuable especially in in vitro tests or in vivo wound healing studies.

In addition, the use of marine collagen will make immunomarking very effective and allow the differentiation of marine collagen relative to newly synthesized collagen.

Fish collagen has a native structure that protects it from enzymatic degradation due to proteases and gives it much of its mechanical properties. It will therefore be very important during the operations of extraction and purification that the treatments used alter as little as possible the structure of the protein. This means that the helical structure as well as the inter and intramolecular crosslinks are preserved as much as possible. For this, the inventors have more particularly implemented the method described in US Patent No. 5331092 issued July 19, 1994. Nevertheless, for particular applications, the use of partially disintegrated collagen may be envisaged, for example atelocollagen that is, collagen having lost some of its lopeptides.

Then, for most tissue engineering applications the collagen mechanical properties will be enhanced and its resistance to enzymatic digestion increased either by chemical and / or physical crosslinking techniques, or by addition of natural macromolecules having strong interactions with the protein. or finally by a combination of the two processes.

The protection of fish collagen will be even more important that its natural stability is lower than that of mammalian collagen. This last characteristic is due to a lower level of hydroxyproline.

It is unexpected in the context of the invention that the cross-linked collagen can be used for the preparation of the previously described biomaterials which can be seeded with living cells and will thus give rise to living artificial tissues which can be used either in the field of testing. in vitro or in the pharmaceutical field for the repair of damaged tissues.

Other aims, characteristics and advantages of the invention will become clear in the light of the following explanatory description made with reference to the examples of embodiments of collagen form of aquatic origin that can be used in the context of the invention for the realization supports for tissue engineering, and thus constituting such supports as well as biomaterials, given merely by way of illustration and which therefore can not in any way limit the scope of the invention.

In the examples, the temperature is given in degrees Celsius, the pressure is atmospheric pressure, and the percentages are given by weight unless otherwise indicated.

Examples 1 to 13 are of course examples of collagen preparation usable as a tissue engineering support according to the invention.

examples 14 to 16 are particularly 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 for engineering tissue.

In the appended figures - FIG. 1 represents the proliferation of normal human fibroblasts in equivalent dermis, with the time expressed in days and abscissas and the optical density x 1000 as ordinates with units increasing by 100; the curve with the diamonds is that which is obtained by using as a support a porous matrix of aquatic collagen, here fish, and the curve with squares is obtained with bovine collagen <B>; </ B> and - FIG. 2 represents a similar proliferation curve of fibroblasts in equivalent dermis with the abscissa the time expressed in days and on the ordinate the fluorescence expressed in international unit, starting at 15,000 with units increasing by 10,000; the curve with the filled diamonds represents the fluorescence obtained in the context of test 1; the curve with square that obtained with test 2; the curve with the empty triangles being obtained with the test 3 and finally the curve with the crosses being that obtained with the test 4. EXAMPLE <U> 1 </ U> <U> Preparation of a porous matrix in native collagen </ U> The collagen is obtained according to the technique of US Patent 5331092 granted July 19, 1994. A - Obtaining the native aquatic collagen A collagen gel is prepared from ground ventral skin crushed and then washed with a phosphate buffer pH 7.8 whose composition is 0.78 g / l of potassium dihydrogenphosphate and 21.7 g / l of disodium monohydrogenphosphate. The washing is carried out with stirring for one hour at the rate of 5 of buffer per 1 kg of ground material. The phosphate is then removed by two successive washings with deionized water, followed by continuous centrifugation at 4000 rpm (Rousselet decanter), at a rate of 51 water per 1 kg of ground material.

The ground material is then acidified with a 0.25 M acetic acid solution at a rate of 1 kg of ground material per 10 1 of solution. The gel is then centrifuged at 4000 rpm for 5 minutes.

The gel that will be used is constituted by the obtained supernatant whose collagen concentration is between 0.5 and 2%.

B - Preparation of the porous matrix from the collagen gel obtained above This gel is cast in a freeze-drying tray at a rate of 20 g / cm 2. It is then freeze-dried after freezing -30 ° C. and heating to + 32 ° C. Freeze-drying lasts a total of 16 hours under a pressure of 400 microbars. The matrix obtained is then crosslinked by hydrothermal dehydration (DHT). This consists of heating in an oven at 110 C under a vacuum of 400 microbars for 16 hours. <U> EXAMPLE 2 </ U> <U> Preparation of a crosslinked porous matrix </ U> using <U> diphenylphosphorylazide (DPPA) <U> according to the technique described in European Patent No. 466,829 of the </ U> <U> July 24, 1996 </ U> The collagen matrix of Example 1 is incubated for 24 h in a solution containing 250 g of DPPA / g of collagen contained in 100 ml of dimethylformamide ( DMF). The collagen is then freed from DPPA by rinsing in <B> 100 ml DMF. The DMF is then removed by rinsing in 100 ml of borate buffer solution pH 8.9 (0.04 M sodium tetraborate, 0.04 M boric acid).

The collagen is finally incubated overnight in the same borate buffer. Finally, the borate buffer is removed by rinsing with deionized water continuously for 6 hours.

<U> EXAMPLE 3 </ U> <U> Preparation of a porous matrix crosslinked by the </ U> carbodiimide <U> and the </ U> N-hydroxysuccinimide The aquatic collagen matrix of Example 1 is crosslinked with PEDC (Ethyldimethylaminopropyl carbodiimide) at a concentration of 0.23 to 0.69 g / g of collagen, and with NHS (N-hydroxysuccinimide) at a concentration of 0.42 g / g of collagen.

After rinsing with deionized water, the collagen is freeze-dried again. <U> EXAMPLE 4 </ U> <U> Preparation of a porous matrix crosslinked by glutaraldeh The porous matrix of aquatic collagen of Example 1 is crosslinked for 24 to 96 hours in a solution containing 0.6 to 1% GTA at 20 C. After rinsing with deionized water, the collagen is again freeze-dried. <U> EXAMPLE 5 </ U> <U> Porous matrix prepared with the native aquatic <U> collagen of Example 1 in </ U> <U> association with </ U> chitosan <U > and a </ U> glycosaminoglycan <U> as described in the </ U> <U> European patent of 29 May 1991 N 296078 </ U> To 600 g of 1.5% collagen gel 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 having a pH of about 4.0 is then stirred and freeze-dried. The sponge obtained is crosslinked by DHT.

<U> EXAMPLE 6 </ U> <U> Porous matrix described in Example 1 surfaced with a collagen film </ U> A - Preparation of the film The collagen gel whose dry matter is between 0.3 and 0.8% is dried in an oven at 30 C or in a hood at a rate of 0.5 g of gel / cm 2 of tray.

In the collagen gel it is possible to add 10 to 40% glycerol. The collagen dried under these conditions forms a transparent film.

B-Association film with the porous matrix described above The native aquatic collagen gel of 0.5% to 2% dry matter, is deposited at a rate of 0.5g per cm 2 in a freeze-drying tray, then the collagen film is deposited on this gel and all is lyophilized.

The lyophilisate obtained is crosslinked by DHT. <B> <U> EXAMPLE 7 </ U> </ B> <U> <U> </ U> <U> Matrix Prepared <U> with a </ U> acid-soluble collagen gel <U> surfaced with a </ U> The process described in Example 6, the only difference being the nature of the gel cast on the film which consists of acid-soluble collagen prepared according to a well-known technique of the man of <U> EXAMPLE 8 </ U> <U> Porous matrix prepared with gel </ U> of atelocollagen <U> or surfaced with a film of </ U> <U> collagen </ U> The method is that indicated in Example 6, the only difference being the nature of the gel cast on the film which consists of atelocollagen ie collagen without telopeptide prepared according to a technique well known in the art. skilled in the art.

<U> EXAMPLE 9 </ U> <U> Porous matrix consisting of collagen associated with </ U> chitosan <U> and a </ U> glycosaminoglycan <U> surfaced with a film of </ U> collagen.

The method is that indicated in Example 6, but in this case the gel cast on collagen film consists of collagen, chitosan, a glycosaminoglycan. The preparation of this gel is described in Example 5. <U> EXAMPLE 10 </ U> All porous matrices surfaced with a collagen film described above can be crosslinked according to the techniques described in Examples 3 and 4.

<U> EXAMPLE 11 </ U> <U> Porous collagen matrix alone described in Example 1 surfaced with a compressed </ U> <U> sponge of collagen. </ U>

A - Preparation of the compressed sponge The collagen prepared as in Example 1 having a dry matter of between 0.3 and 1.5% is lyophilized to obtain a sponge having a weight of between 0.5 and 2 g / cm 2.

The lyophilizate is compressed for 5 to 60 seconds at a temperature between 20 and 60 C and a pressure between 200 and 200 bar (50 to 200 Pa).

B - Association of the compressed sponge with the porous matrix The collagen gel described in Example 1 is deposited at a rate of 0.5 g per cm 2 in a freeze-drying tray. The compressed sponge is then deposited on this gel and the whole is lyophilized. A porous collagen sponge surfaced with a compressed collagen sponge is thus obtained. The whole is crosslinked by DHT as described in Example 1. <U> EXAMPLE 12 </ U> <U> Porous matrix consisting of collagen, </ U> chitosan <U> and </ U> glycosaminoglycan < U> as </ U> described <U> in Example 5 and surfaced with the compressed sponge. </ U>

The collagen, chitosan and glycosaminoglycan gel, prepared according to the method of Example 5, is deposited at the rate of 0.5 g per unit in a freeze-drying tray, then the compressed sponge is deposited in this gel and the whole is lyophilized. The lyophilizate is then crosslinked as described in Example 1.

<U> EXAMPLE 13 </ U> All porous matrices surfaced with a compressed collagen sponge described above can be crosslinked according to the techniques described in Examples 2, 3 and 4.

<U> EXAMPLES 14 TO 16: Comparative assays for cell metabolism between </ U> <U> <U> bovine <U> and </ U> collagenous matrices <U> EXAMPLE 14: TEST OF </ U> <FIBROBLAST CELLULAR VIABILITY <1> <U> Preparation of Equivalent Derms </ U> For this comparison test, a porous aquatic matrix is crosslinked with DPPA, according to Example 2. .

By way of comparison, one manufactures a comparative porous matrix with collagen of bovine origin, called the bovine matrix, also reticulated with DPPA under the same conditions as those of Example 2.

Normal human fibroblasts from the 7th seeded pool of young donors are seeded into each of the aquatic and bovine matrices at 250,000 cells per cm 2 for the Proliferation and Proliferation Study. protein synthesis, which is seeded at 300 000 cells per cm2 for the aquatic and bovine matrices for histology studies.

The cultures of these aquatic and bovine matrices are respectively carried out in medium composed of DMEM / HAM F 12 in a 50/50 (v / v) ratio supplemented with 10% fetal calf serum, 100 IU / ml penicillin, g / ml of gentamycin, 1 g / ml of amphotericin B, 50 g / ml of vitamin C.

This culture is carried out for 1 month by changing the culture medium 3 times a week. II - <U> Analyzes carried out </ U> 1) <U> Measurement of the cell viability by reaction with MTT 1% by weight of MTT (that is to say the 3- (4) - (dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide) in the culture medium.

Incubation is carried out for 2.5 hours at 37 ° C.

After incubation for this time, 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 salt MTT into blue crystals of formazan.

Cell viability was achieved after 1, 5, 7, 22 days and one month of culture.

To determine the average values, 6 samples were made for each matrix.

<B><U> TABLE <SEP> I </ U></B>
<tb><U> DE <SEP> RESULTS </ U>
<tb> Days <SEP> Matrix <SEP> Deviation <SEP> at <SEP> Matrix <SEP> Deflection <SEP> Standard
<tb><U> aquatic </ U><SEP> standard <SEP><U> average </ U><SEP> bovine <SEP><U> average </ U>
<tb> 1 <SEP> 487 <SEP> 24 <SEP> 403 <SEP> 40
<tb> 5 <SEP> 604 <SEP> 19 <SEP> 393 <SEP> 59
<tb> 7 <SEP> 520 <SEP> 56 <SEP> 398 <SEP> 64
<tb> 22 <SEP> 608 <SEP> 30 <SEP> 680 <SEP> 40 These results are also the subject of the curves of the appended FIG. note that the curve with the diamonds is that obtained with the aquatic matrix and the curve with the squares is that obtained with the bovine matrix.

observes from the results that, quite surprisingly, the aquatic matrix not only provides a support for the survival of normal human fibroblasts but also the cell proliferation of these normal human fibroblasts, while at the same time constituting a much better culture support during the three first weeks. It can therefore be concluded from these tests that aquatic collagen is surprisingly particularly suitable for carrying out a tissue engineering support, in particular for in vitro applications and even more importantly in vivo for constituting biomaterials containing living cells in particular and preferably living cells of humans.

2) <U> Measurement of </ U> protein syntheses </ U> The synthesis of proteins secreted for 3 days in culture medium without serum of fetal calf was evaluated after one month of mature dermes equivalents, such as obtained after the month of culture under the conditions reported above in the preparation of equivalent dermes.

The assay is performed by Pierce's microBCA method. In parallel, the cell density was evaluated by a test under the conditions described above.

The relative protein content corresponds to the protein content reduced to 1 unit of cell density expressed in optical density or OD, in order to reason at equivalent cell concentration. The results obtained are listed in Table II below.

<U> TABLE <SEP> II </ U>
<tb><U> RESULTS <SEP> FROM <SEP> SYNTHESIS <SEP> PROTEIOUE </ U>
<tb> Collagen <SEP> of <SEP> support <SEP> Matrix <SEP> Matrix <SEP> bovine
<tb> aquatic
<tb><U> Average <SEP> Average </ U>
<tb> Density <SEP> cell <SEP> 2.12 <SEP> 0.09 <SEP> 1.91 <SEP> 0.13
<tb><B><U> (DO) </ U></B></B>
<tb> Proteins <SEP> ml <SEP> 494 <SEP> 48 <SEP> 499 <SEP> 32
<tb> Content <SEP> in <SEP> proteins <SEP> 233 <SEP> 18 <SEP> 262 <SEP> 23
<tb> relative
<tb> * <SEP>: <SEP> Standard <SEP> Deflection <SEP> As for Table I, the average results from an average of 6 samples. <U> Histology </ U> The equivalent dermas obtained after culturing for 21 days of culture from the aquatic and bovine collagen matrices are fixed in a 2% paraformaldehyde solution and then post-fixed in a solution of osmium tetroxide , dehydrated, included in Epon, cut and observed by transmission electron microscopy (Jeol 1200) at CMEABG (Lyon, France). <U> Conclusions </ U> These results indicate a very good colonization of three-dimensional matrices whether aquatic or bovine. After three weeks of culture, the cell density is equivalent in both types of matrices. However, it seems that the aquatic matrix allows a better cellular adhesion at the beginning of the experimentation as indicated by the proliferation study during the first week of culture and thus a better colonization of the short culture times.

With regard to protein synthesis, after one month, the fibroblast synthesis capacities (relative protein content) are also equivalent.

These results indicate that the aquatic collagen matrices developed have allowed the preparation of equivalent dermes of good quality; the results obtained with these matrices being comparable to those obtained with bovine collagen matrices.

In transmission electron microscopy, fibroblasts could be observed in matrices of bovine and aquatic origin. In both types matrices, there is the presence of an abundant extracellular matrix neosynthesized. The neosynthesized extracellular matrix can be distinguished by the periodic striation of deposited collagen fibers compared to the collagenous clusters forming the three-dimensional matrix of the starting sponge.

<U> EXAMPLE 15 </ U> <U> Influence of the different <U> types <U> of </ U> crosslinking <U> of aquatic collagenic matrices </ U> <U> on cell viability </ To study the influence of the different types of crosslinking of aquatic collagen matrices on cell viability, the following tests are carried out: 1) <U> Preparation of equivalent dermes </ U> a) Support <U> or matrix used </ U> prepares various collagen supports or matrices using various proportions of collagen in the collagen gel used to make the porous layer or matrix and possibly using a different crosslinking agent, as follows <Test 1 </ U>. test, a porous sponge matrix is made from an aqueous collagen gel prepared from 3% by weight of aquatic collagen, which is frozen at -80 C and subjected to standard lyophilization according to Example 2 and which is then at a crosslinking with DPPA at a proportion of 250 g / g of sponge in the dry state.

2) <U> Test 2 </ U> For this test, a porous support in the form of an aquatic sponge is prepared from an aqueous collagen gel comprising 0.7% by weight of aquatic collagen, which is subjected to freezing at -80 ° C, followed by standard freeze-drying and crosslinking with DPPA at 250 μl / g as in Run 1.

3) <U> Test 3 </ U> For this test, the procedure is as for Test 1, if only the crosslinking with fEDC, according to Example 3, at a proportion of 0.46 g / g of dry sponge.

<U> Assay 4 </ U> prepares 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 to - 80 C, followed by standard freeze-drying and crosslinking with DPPA <I> at 250 μg / g dry, as in the test, the difference being in a proportion of 1.1% by weight of aquatic collagen.

In all of these tests, the aquatic collagen is derived, as in Example 2, the ventral skin sole. b) <U> Culture of fibroblasts on these matrices </ U> As in Example 14, normal human fibroblasts which are taken at the 8th passage are used.

Seeding is carried out at 275,000 cells per cm 2.

The culture medium is composed of DMEM / HAM F12 50/50 (v / v) supplemented with 10% by weight of fetal calf serum, 100 IU / ml of penicillin, 25 μg / ml of gentamycin, Fg / ml of amphotericin B, 50 μg / ml of vitamin C.

The culture is carried out for 1 month by changing the medium 3 times a week.

For each test, 4 matrices are used to average each test type and measure the average standard deviation.

II) <U> Assays performed </ U> <U> Measurement of cell viability by reaction to </ U> Alamar <U> Blue (redox marker </ U> <U>) </ U> UAlamar Blue is added at a rate of 2% by weight of a culture medium used, at the moment when it is desired to measure the cell viability on a sample taken on the culture medium.

After incubation at 2 h at 37 ° C., the fluorescence is read, on the basis of excitation at 530 nm emission at 590 nm).

The fluorescence intensity obtained is proportional to the metabolic activity of the cells.

The cell viability is measured on 10 samples after 1, 46, and 11 days of culture.

The results are shown in Table III below.

The results are given in international unit of fluorescence as a function of time.

<U> TABLE <SEP> III </ U>
<tb><U> VIABILITY <SEP> CELL <SEP> (u <SEP> I <SEP> Fluorescence) </ U>
<tb> Time <SEP> TEST <SEP> 1 <SEP> TEST <SEP> TEST <SEP> 3 <SEP> TEST4
<tb>'bear)
<tb><U> Average </ U><SEP> SD * <SEP><U> Average <SEP> Average </ U><SEP> SD * <SEP> Average <SEP> SD *
<tb> 1 <SEP> 21734 <SEP> 1184 <SEP> 30535 <SEP> 1888 <SEP> 25528 <SEP> 6820 <SEP> 28461 <SEP> 3805
<tb> 4 <SEP> 3161 <B> 1 </ B><SEP> 920 <SEP> 35623 <SEP> 3544 <SEP> 36404 <SEP> 3570 <SEP> 45126 <SEP> 2930
<tb> 6 <SEP> 43144 <SEP> 2500 <SEP> 35244 <SEP> 2095 <SEP> 37819 <SEQ> 4170 <SEQ> 41254 <SEQ> 3396
<tb> 11 <SEP> 42808 <SEQ> 1481 <SEP> 38532 <SEP> 2537 <SEP> 42442 <SEP> 3112 <SEP> 44508 <SEP> 2329
<tb> 17 <SEP> 45484 <SEQ> 2426 <SEQ> 45094 <SEQ> 1470 <SEQ> 43963 <SEQ> 8285 <SEQ> 43939 <SEQ> 4521
<tb> Order <SEP> 1 <SEP> 2 <SEP> 3 <SEP> 4
<tb> * <SEP>: <SEP> Standard <SEP> Diversion The results in Table 3 are also shown in Figure 2 appended.

They show the fibroblastic proliferation curves in equivalent dermis.

The diamond-shaped curve is complete with test 1; the solid square curve is that carried out with the test 2; the triangle curve is the one carried out with the test 3 and the curve with cross is that carried out with the test 4.

The time is expressed in days on the abscissa and the fluorescence in Ul with a scale starting at 15 000 and increasing per unit from 10 000 to 55 000. The results lead to the following conclusions. <U> Conclusions </ U> The results indicate that the different matrices prepared can allow good fibroblastic growth after 17 days of culture. Whatever the preparation of the aquatic collagen matrices, the fibroblasts adhere well to their three-dimensional support and divide very rapidly 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.

The different types of crosslinking used either with DPPA or with fEDC do not seem to influence cell renewal. After almost 3 weeks of culture, the stability of the matrices is excellent, namely little digestion, little contraction.

 EXAMPLE <U> Test Demonstrating the Advantages of Aquatic Collagen for Demonstration </ U> <U> of Neosynthesized Human Collagen Assay </ U> This assay is similar to that of Example 14, except that that performs a histology with immuno-labeling.

The test is carried out as follows: 1) <U> Preparation of equivalent dermes </ U> These are equivalent dermas of 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 indicated in Example 2) <U> Histology </ U> a) <U> Classical Histology </ U> carries out the fixation with paraformaldehyde at a content of 4 wt., followed by dehydration and paraffin embedding.

then performs 7 gin sections and Mallory Haidenhain staining after dewaxing and rehydration.

b) Immunolabeling also fixed with 4% by weight paraformaldehyde, achieved an inclusion in Tissue Tek OCT compound, ie an inclusion liquid supplied by Miles, Elkhart, Indiana, USA, and a cold cut at 7 will read.

 Immunostaining is performed as follows i. With a first rabbit human anti-collagen type 1 antibody (1/40 dilution) ii. A second FITC conjugated anti-rabbit antibody (Fluorescein ThioCyanate) (1I160 dilution) against staining with DAPI (4 ', 6-diamidino-2-phenylindole, dilactate). 3) <U> Results </ U> It is found that 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 surfacing of the equivalent dermis for the production of a reconstructed skin. The distribution of fibroblasts is homogeneous in aquatic and bovine sponges.

In immunolabeling, it is found that the bovine collagen matrix is labeled with the human type I anti-collagen antibody (crossing). On the other hand, the matrix of aquatic origin is only very slightly marked by the human anti-collagen antibody.

The use of sponges composed of aquatic collagen is thus favorable to the demonstration of the neosynthesized extracellular matrix.

These results are explained by the work of Professor Hartmann reactions of the various antigens with respect to the different antibodies determined by the measurements of the optical densities after immunostaining data Table IV says Hartmann hereinafter

TABLE <SEP> IV
<tb><U> Cross Reaction <SEP> cross <SEP> human, <SEP> bovine, <SEP> fish <SEP> (test <SEP> Elisa) </ U>
<tb> Antigens <SEP> Collagen <SEP> Collagen <SEP> Collagen
<tb><U> Antibody </ U><SEP> I <SEP> sole <SEP> I <SEP> human <SEP> I <SEP> bovine
<tb> 201 <SEP> 1 (225)
<tb> 1/25 <SEP> 190 <SEP>><SEP><B> 815 </ B>
<tb> 1/50 <SEP> 210 <SEP>><SEP> 548
<tb> 00 <SEP> 73 <SEP><B> 1233 </ B><SEP> 234
<tb> 1/200 <SEP> 43 <SEP> 605 <SEP> 136
<tb> 1/400 <SEP> 56 <SEP> 326 <SEP> 165
<tb> 50121 <SEP> (03)
<tb><B> 1 </ B> / 25 <SEP> 180 <SEP> 1550 <SEP>>
<tb> 1/50 <SEP> 130 <SEP> 1094 <SEP>>
<tb> 1/100 <SEP> 158 <SEP> 536 <SEP>>
<tb> 1/200 <SEP> 96 <SEP> 305 <SEP> 967
<tb> 1/400 <SEP> 109 <SEP> 215 <SEP> 728
<tb><B> 71 (01) </ b>
<tb> 1/25 <SEP> 1880 <SE> 64 <SEP> 73
<tb> 1/50 <SEP> 1043 <SEP> 193 <SEP> 32
<tb> 00 <SEP> 571 <SEP> 51 <SEP> 33
<tb> 1/200 <SEP> 523 <SEP> 51 <SEP> 87 (>: optical density greater than 2000) The results are expressed in OD x 103 (optical density at 450 nm) <U> Legends </ U> : 20111 (225): human anti-collagen I 50121 (03): anti-collagen I bovine <B> 50171 </ B>): anti-collagen I fish (sole) From this table of results, it appears that whatever either the antibody (anti-type 1 human collagen, anti-collagen type 1 bovine, anti-collagen type I sole), in immunostaining, the difference is much greater between the human collagen the sole collagen than between the collagen human and bovine collagen. As a result, in a fish collagen matrix, the collagen synthesized by human fibroblasts can be highlighted much more easily. This confirms the results described above obtained by immunolabeling with human type I anti-collagen collagen synthesized in the fish collagen matrix, which is a particularly unexpected and advantageous result of the invention.

Claims (2)

<U> CLAIMS </ U> 1. Use of crosslinked collagen of aquatic origin, preferably derived from fish skin of the teleost family, for the production of supports for tissue engineering. 2. Use according to claim 1, characterized in that the collagen is obtained from the fish skin of the family of teleosts more particularly from the skin of flat fish, in particular those are caught industrially as for example the sole, the dab, the turbot the catfish, preferably the sole, even more particularly depigmented fish skin areas. Use according to claim 1 or 2, characterized in that the collagen is crosslinked either by chemical and / or physical crosslinking, or by addition of a natural macromolecule having strong interactions with the collagen, or by a combination of said methods, which also allows to have its reinforced mechanical strength or its resistance to enzymatic digestion increased. 4. Use according to one of claims 1 to 3, characterized in that the collagen is used in the form of a porous matrix prepared from collagen gel preferably subjected to a freeze-drying step. 5. Use according to claim 4, characterized in that said porous matrix is crosslinked by physical crosslinking, preferably by hydrothermal dehydration or DHT. Use according to claim 4 or 5, characterized in that the aforementioned porous matrix is crosslinked by chemical crosslinking, preferably with diphenylphosphorylazide or DPPA or with a carbodiimide or hydroxysuccinimide or with glutaraldehyde. Use according to one of the preceding claims, characterized in that the abovementioned collagen is in the form of a porous matrix prepared from preferably native aquatic collagen, mixed with chitosan optionally at least one glycosaminoglycan, preferably chondroitin sulfate. Use according to one of the preceding claims, characterized in that the abovementioned collagen is in the form of a porous matrix prepared from a collagen gel, said porous matrix being coated on at least one side of a membrane essentially compact collagen made either by a collagen film prepared by drying, preferably in air or in a gaseous fluid, a collagen gel or by a very highly compressed collagen sponge. 9. Use according to one of the preceding claims, characterized in that crosslinked collagen, in particular in the physical form of at least one layer, comprises living cells, normal or genetically modified, or malignant, in particular from young subjects or elderly subjects. Use according to one of claims 6, 7 to 9, characterized in that the aforementioned cross-linked collagen is in the form of a porous matrix coated at least one side of a substantially compact collagenous membrane, at least one of the two layers comprises genetically modified or malignant normal living cells, especially from young or elderly subjects. 11. Use according to claim 9 or 10, characterized in that 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, blood-born langerhan cells, blood-born endotelial cells, blood cells, adipocytes, sebocytes, chondrocytes, bone cells, osteoblasts, blood-derived Merkel cells, normal or genetically modified cells or malignant cells. 12. Use according to claim 10 or 11, characterized in that the porous layer contains normal fibroblasts, genetically modified or malignant, and the substantially compact membrane contains normal living cells or genetically modified or malignant, in particular selected from keratinocytes, melanocytes, blood-derived Merkel cells, blood-born langerhan cells, sebocytes, cells of blood origin, nerve cells. Use according to one of Claims 8 to 12, characterized in that the abovementioned compression of the highly compressed collagen sponge is carried out at a minimum pressure of approximately 50 bar (50 × 10 5 Pa), preferably of between 50 bar (50 × 10 5 Pa). Pa) and 200 bar (200.105 Pa), this compression possibly being carried out at a temperature between 20 C and 80 C, more preferably between 40 C and 60 C. 14. Use according to one of claims 8 to 13, characterized in that the substantially compact membrane is prepared prior to combining with the porous layer, preferably comprising a collagen sponge, in particular by preparing the membrane and depositing it on a collagenic gel before the assembly is frozen and lyophilized. 15. Support for tissue engineering characterized in that it comprises crosslinked collagen as defined in any one of the preceding claims. 16. Biomaterial, for example in the form of a reconstituted connective tissue, or reconstituted skin, characterized in that it has been prepared from the cross-linked collagen as defined in any one of the preceding claims, advantageously this biomaterial comprising cells is 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 and possibly test the effectiveness of active ingredients on this process.