DE10196235B4 - Using collagen from aquatic animals as carriers for tissue engineering, useful e.g. as skin replacements and in screening for antiaging drugs - Google Patents

Abstract

Using collagen (A) of aquatic origin, particularly from the skin of teleost fishes, to prepare carriers (B) for tissue engineering, is new. Independent claims are also included for the following: (a) (B) prepared from (A); and (b) biological material (C), formed as reconstituted connective tissue or skin, prepared from (A).

Description

The This invention relates essentially to the use of fish collagen for the production of carriers for the Tissue formation and on these carriers.

collagen is a particularly advantageous substrate for cell development, which is why this protein in very big Scope in different forms - in Form of matrices, gels or films - for the production of reconstructed Tissues that contain living cells is used.

On the field of tissue formation, a process that has a great future promises collagen has in particular the formation of artificial Skin or artificial Cartilage allows. To achieve a satisfactory result, the collagen must against enzymatic degradation by cell metabolism either by physical or chemical crosslinking processes or by the Presence of natural macromolecules that interact with the protein, or finally through a combination of both systems are protected.

For this Tissue formation applications have been used in carriers for the uptake of cells Collagen so far from mammals and usually extracted from cowhide. The choice of this source was made on the basis of the good mechanical properties of the product obtained after extraction Protein, because of its persistence against enzymatic degradation and finally due to its amino acid composition, the very similar that of human collagen. For all these reasons was to legitimately assume that this collagen would be the only one that for the Cultivation of human cells is suitable.

In the article by Yeh et al. in "Faceb Journal, Vol. 14, No. 4, March 15th 2000, "A novel native matrix for tissue engineering. Analysis of cell-matrix interaction "will be the use a matrix from the acellular Collagen of a whale shark for the investigation of its interaction with keratinocytes and fibroblasts of human skin to study biocompatibility.

The "Derwent Abstract" XP 00 21 61 598 refers to Japanese Patent Application JP 07/075 566 of Marino Forum, published on March 20, 1995, in which a method is described in the fish collagen as a cultivation material is described. However, this cultivation is done with fish cells.

In the European Patent EP-A-0 753 313 describes a skin substitute made of a laminate, the one layer or a layer of a chitin extract from a Mollusca or a squid, i. using marine organisms, includes. Here the use of chitin is critical because then, if the chitin solution freeze-dried (lyophilized), a chitin structure is obtained, which is an insoluble, non-biodegradable sponge represents the chitin not by the enzymes contained in human skin is reduced. A collagen gel of fish skin may be on the layer or layer of chitin are infused, both in a refrigerator dried for about a week. Here is the collagen layer dense and non-porous. It can not be inoculated with living cells. That in this The laminate described in the document is an inert laminate and it is not to be expected that live cells can be inoculated and the living character of this cell can be maintained in contrast to the invention described below.

The Inventors are now surprising found that human cells on or inside of certain carriers, from fish collagen, preferably from cross-linked fish collagen, persist, develop very well. This marine collagen is preferably from from the skins of bony fish, in particular of fish, containing a non-pigmented Skin area, and especially from flatfish, in particular those which are industrially fished, e.g. Sole, plaice, Turbot, brill, cut in filet production become. A particularly preferred flatfish is the sole, at the non-pigmented abdominal skin section is easy to cut.

In addition, could the inventors show that human cells contained in these biomaterials be cultivated, maintain a normal metabolism. The Biokompatibität, which preserves and proliferates living cells with collagen extracted from bone fish skin was allowed, was particularly surprising for the One skilled in the art, when the collagen crosslinks and in particular chemically crosslinked because, in general, the crosslinking of collagen causes that cross-linked collagen is not biocompatible and toxic to living Cells, especially for living human cells, is.

These biomaterials can be either films or compressed (pressed) sponges or porous ones Matrices that are described together with their production methods in the examples below.

One The aim of the present invention is to provide the new technical Solve a problem, that consists of new vehicles for the To provide tissue formation, which are appro net for education of new biomaterials, i. which are suitable for the achievement a good proliferation of the normal, genetically modified (Modified) or malignant (malignant) living cells to be cultured on this carrier and within the framework of these new biomaterials that this contain living cells, for the subsequent one in vitro or in vivo proliferation.

One Another object of the present invention is to provide the new solve technical problem that consists of new vehicles for the Tissue formation at low cost and also with a To provide low risk of contamination, which is particularly are well suited for the production of new biomaterials.

One Another main object of the present invention is that solve new technical problem that consists of new vehicles for the To provide tissue formation, which are particularly suitable for propagation (Duplication) of normal, genetically modified or malignant living cells in vitro or in vivo and their structure with in vivo use is sufficiently compatible in a human while being simultaneously of is sufficiently different from the structure of human tissues, so that they are the subsequent differentiation between the newly synthesized Tissues and the ancient tissues of humans.

The present invention solves all of these technical problems for the first time in a satisfactory way at low cost with a low risk of contamination or without contamination while it is at the same time easily possible will identify the newly synthesized tissue, which for the skilled person particularly surprising in this field and was unexpected.

According to one first feature, the present invention therefore relates to a carrier Formation of human tissue by cell proliferation, the one porous Contains matrix, which is made of fish collagen, that of a lyophilization step was subjected, and wherein the porous matrix by applying a chemical process is crosslinked.

Of the As used herein, "fish collagen" means a collagen, derived from collagen-containing tissues of fish, i. seawater or freshwater fish. The person skilled in the art also knows that the skin of this Fish contains essentially collagen. The "fish collagen" is preferably made from fish from the Family of bony fish, in particular extracted from fish, have unpigmented areas, especially flatfish and in particular the above-mentioned fish, being the most preferred sole is the sole.

mechanical Strength of collagen or its resistance to enzymatic Degradation occurs either through chemical and / or physical crosslinking or by adding a natural one Macromolecule that interacts with the collagen in a strong interaction, or by a combination of both methods increased.

The Collagen is in the form of a porous Matrix made from a collagen gel, which has been subjected to a lyophilization step.

at an advantageous variant is the above-mentioned porous matrix by a chemical process, preferably with diphenylphosphorylazide or DPPA or with a carbodiimide and / or N-hydroxysuccinimide or crosslinked with glutaraldehyde.

at an advantageous embodiment may be the above-mentioned fish collagen of the porous matrix mixed with chitosan and optionally at least one glycosaminoglycan.

at Yet another advantageous embodiment of the invention the above porous Matrix on at least one side with a substantially compact, dense collagen membrane covered either by a collagen film, by drying a collagen gel, preferably in air or in a gas fluid, or from a compressed one Collagen sponge exists.

The above-mentioned compression of the compressed collagen sponge may be performed at a pressure of at least about 50 bar (50 x 10 ⁵ pascals (Pa)) and preferably at a pressure of between 50 and 200 bars (50 x 10 ⁵ - 200 x 10 ⁵ Pa) This compression has optionally been carried out at a temperature between 20 and 80 ° C and preferably between 40 and 60 ° C.

According to one further advantageous characteristic of the invention comprises the porous Matrix living cells consisting of normal, genetically modified or malignant living cells.

According to one advantageous embodiment the living cells are selected from the group that consists from fibroblasts, keratinocytes, melanocytes, Langerhans cells, the come from the blood, endothelial cells that come from the blood, Blood cells, in particular macrophages or lymphocytes, adipocytes, Sebocytes, chondrocytes, osteocytes, osteoblasts and Merkel cells, which originate from the blood, these cells being normal, genetically modified or malignant.

at a particularly advantageous embodiment contains the porous matrix normal, genetically modified or malignant live fibroblasts. In another advantageous embodiment, the essentially compact (dense) collagen membrane normal, genetic modified or malignant living cells, selected in particular from keratinocytes, Melanocytes, Merkel cells derived from the blood, Langerhans cells that derived from the blood, sebocytes, cells derived from the blood, and Neurons.

at Yet another advantageous embodiment of the invention It may be particularly valuable to use either "young" reconstructed skin of cells from young creatures, especially human ones Living things, are taken, or "aged (mature) "reconstructed Skin using cells derived from older animals, in particular human beings are taken to produce. These models make it possible, to improve our knowledge of the skin aging process and the influence of active ingredients to investigate this process.

According to one another particularly advantageous embodiment, the above-mentioned, essentially compact collagen membrane made before it with the porous one Matrix is combined, which preferably comprises a collagen sponge, in particular by production of the collagen membrane and application the same on a fish collagen gel, before the whole thing is frozen and is lyophilized.

According to one another embodiment The present invention also relates to a carrier, for example in the form of a biomaterial, a reconstructed connective tissue or a reconstructed skin is present.

According to one second feature, the present invention also relates to Use of fish collagen subjected to a lyophilization step is used to make a carrier formation of human tissue as defined above is, and the one porous Contains matrix, which is crosslinked by using a chemical process.

Within the scope of the present invention and the claims the "carrier of education of human tissue "for vehicles that should be used to carry out the cultivation and Proliferation (propagation) of normal, genetically modified or malignant living cells, whether in vitro or in vivo, wherein this proliferation is preferably applied in vivo to a People. It is clear that the invention is particularly preferably applicable is within the framework of tissue formation for the production of Biomaterials, from reconstructed connective tissues or reconstructed Skin. Within this framework, a first level includes general the cultivation of the carrier together with the living cells in vitro to form a biomaterial, a reconstructed connective tissue or a reconstructed skin, and then reconstructed a second stage at which this biomaterial Connective tissue or the reconstructed skin is applied in vivo to a person for the surgical reconstruction of a damaged or removed connective tissue or even for surgical reconstruction of skin as a replacement of a damaged one or remote area for any medical reason.

Appropriately includes the carrier for the Tissue formation, or preferably the biomaterial, reconstructed Connective tissue or the reconstructed skin cells, either in the essentially exclusively from young creatures or essentially exclusively from older creatures have been obtained, in particular to study the tissue aging process and especially the skin aging process and, where appropriate, for testing the efficiency of active ingredients or principles of action in this Process.

from that It can be seen that the invention provides a general solution to the above the above-mentioned new technical problems that are particularly is simple, low cost and low risk of Contamination and offers the possibility between Fish collagen and mammalian collagen or preferably human collagen, in the course of in vivo use has been synthesized to differentiate.

• – die allgemein als Ausgangsmaterial verwendete Fischhaut kann in großer Menge unter sehr sauberen Bedingungen erhalten werden;
• – die Gefahr einer infektiösen Kontamination ist sehr gering; insbesondere besteht keine bekannte Gefahr der Übertragung von Agentien vom Prionen-Typ;
• – schließlich kann, da die Aminosäure-Zusammensetzung von Fischkollagen relativ unähnlich derjenigen von Human-Kollagen ist, zwischen den beiden Proteinen mittels spezifischer Antikörper verhaltnismafüg leicht differenziert werden. Diese Methode ist sehr wertvoll insbesondere bei "in vitro"-Tests oder bei "in vivo"-Heilungs-Untersuchungen.

In fact, the use of fish collagen for the production of living artificial tissues has essentially three advantages compared to the use of mammalian collagen:

• The fish skin generally used as starting material can be obtained in large quantities under very clean conditions;
• - the risk of infectious contamination is very low; in particular, there is no known risk of transmission of prion-type agents;
• Finally, since the amino acid composition of fish collagen is relatively dissimilar to that of human collagen, it is possible to differentiate relatively between the two proteins by means of specific antibodies. This method is very valuable especially in "in vitro" tests or in "in vivo" healing studies.

Furthermore The use of fish collagen makes the immunoassay very much effective and allows a differentiation between fish collagen and newly synthesized collagen.

fish collagen has a native structure that it is resistant to enzymatic degradation Protects proteases, and the majority is responsible for its mechanical properties. It is therefore very important that the while applied to the extraction and cleaning operations Treatments affect the protein structure as little as possible (dismantle). This means that the helix structure and the intermolecular and intramolecular crosslinks should be maintained as much as possible. The inventors have achieved this in particular by using the in U.S. Patent No. 5,331,092 (published July 19, 1994) described method. Nevertheless, it is possible for certain applications that Use of partially uncrosslinked collagen, e.g. atelocollagen i.e. collagen that has lost part of its telopeptides, to consider.

For the majority The tissue building applications then become the mechanical properties of the Collagen improves and its resistance to enzymatic Dismantling is increased either by chemical and / or physical crosslinking methods or by Addition of natural Macromolecules which interact strongly with the protein or finally through a combination of both methods.

Of the Protecting fish collagen is all the more important as its natural stability is lower is that of mammalian collagen, the latter characteristic being at a lower level Hydroxyproline content is due.

The Biomaterials described above can be inoculated with living cells (inoculated) are used to produce living artificial tissues, either in the field of "in in vitro tests or in the pharmaceutical field for the repair of damaged tissues can be used.

Further Goals, characteristics and advantages of the invention will be apparent from the following explanatory description with reference to examples of the production of forms of fish collagen, which within the framework of the present invention for the production of carriers for tissue formation can be used and thus those carriers and biomaterials, which are given for illustration only and therefore, in no way limit the invention.

In The examples show the temperature in degrees Celsius, which Pressure is atmospheric pressure and the percentages are by weight unless otherwise specified.

The Examples 1 to 13 are examples of the preparation of fish collagen, as a carrier for the Tissue formation can be used. According to the invention used fish collagen subjected to a lyophilization step and the porous one Matrix is crosslinked using a chemical process.

The Examples 14 to 16 are in particular comparative tests within the framework of use of this fish collagen in the molds, which were prepared in some of Examples 1 to 13 within the Framework of the production of carriers for the Tissue formation. In the accompanying drawings show:

1 the proliferation of normal human fibroblasts in dermis equivalents, where the time expressed in days is plotted on the abscissa and the optical density x 1000 is plotted on the ordinate in units increasing by 100, the curve being diamonds that which is obtained when the carrier used is a porous matrix of fish collagen, and the squares curve is that obtained using bovine collagen; and

2 a similar curve for the proliferation of fibroblasts in dermis equivalents, in which the time, expressed in days, is plotted on the abscissa and the fluorescence, expressed in International Units (IU), is given on the ordinate, starting at 15,000 and increasing in each case 10,000 units, wherein the curve with the filled diamonds represents the fluorescence obtained in test 1, the curve with the filled squares represents the fluorescence obtained in test 2, the curve with the unfilled triangles represents the fluorescence obtained in test 3, and Finally, the curve with the crosses represents the fluorescence obtained in Test 4.

example 1

The Abbr. "M" stands for mol / l; "RPM" stands for RPM.

Production of a porous matrix of native fish collagen

The Collagen is obtained by the method of U.S. Patent No. 5,331 092, issued on July 19, 1994.

A. Preparation of the fish collagen

One Collagen gel is made from the abdominal skin of a sole that ground and then with a phosphate buffer having a pH of 7.8 with the following composition: 0.78 g / l potassium dihydrogen phosphate and 21.7 g / l disodium monohydrogen phosphate. The Washing is stirred for 1 h at a rate of 5 l of buffer per kg of ground material. Then the phosphate is washed through two consecutive washes softened Water removed, followed by a continuous centrifugation with 4000 rpm (Rousselet centrifuge) at a rate of 5 liters of water per kg of ground material connects. Then that will be ground Material with 0.25 M acetic acid solution in acidified to 10 liters of solution at a rate of 1 kg of ground material. The Gel is then centrifuged for 5 min at 4000 rpm.

The gel to be used consists of the obtained supernatant liquid, which has a collagen concentration between 0.5 and 2%.

B. Preparation of the porous matrix from the above obtained collagen gel

This gel is poured into a lyophilization trough at a rate of 20 g / cm ² . Then it is philospherized after freezing at -30 ° C and after heating to + 32 ° C lyo. The total lyophilization time is 16 h at a pressure of 400 microbar. The resulting matrix is then crosslinked by thermal dehydration (TDH), which consists in heating in an oven at 110 ° C under a vacuum of 400 microbar for 16 h.

Example 2

Preparation of a porous matrix containing diphenylphosphorylazide (DPPA) after in the European Crosslinked. Patent No. 4,666,829 issued July 24, 1996 is

The Collagen matrix of Example 1 is incubated for 24 hours in a solution the 5 to 250 μl DPPA / g collagen in 100 ml dimethylformamide (DMF). Then the collagen is rinsed in 100 ml of DMF to remove the DPPA. The DMF is then rinsed in 100 ml of a borate buffer solution with a pH of 8.9 (0.04M sodium tetraborate, 0.04M boric acid). After all the collagen is over Incubated overnight in the same borate buffer, then the borate buffer through 6-hour continuous do the washing up with softened Water removed.

Example 3

Preparation of a porous matrix containing carbodiimide and N-hydroxysuccinimide is crosslinked

The Fish collagen matrix of Example 1 is treated with EDC (ethyldimethylaminopropylcarbodiimide) at a concentration of 0.23 to 0.69 g / g collagen and with NHS (N-hydroxysuccinimide) at a level of 0.42 g / g collagen networked. After rinsing with softened Water, the collagen is lyophilized again.

Example 4

Preparation of a porous matrix containing glutaraldehyde is networked

The porous The matrix of the fish collagen of Example 1 becomes 24 to 96 hours long in a 0.6 to 1% GTA-containing solution of 20 ° C crosslinked. After rinsing with softened Water, the collagen is lyophilized again.

Example 5

porous Matrix made from the native fish collagen according to example 1 in combination with chitosan and a glycosaminoglycan as in European Patent No. 296,078 dated May 29, 1991

A solution of 2.5 g of chitosan in 356 ml of water and 1.9 ml of acetic acid and then a solution containing 1 g of chondroitin 4-sulfate in 400 ml of softened water becomes 600 g of 1.5% collagen gel. The mixture, which has a pH of about 4.0 will be followed touched and then lyophilized. The resulting sponge is crosslinked by TDH.

Example 6

porous Matrix as described in Example 1, covered with a collagen film

A. Production of the film

The collagen gel, which has a solids content 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 gel / cm ² trough. 10 to 40% glycerol can be added to the collagen gel. The collagen dried under these conditions forms a transparent film.

B. Combination of the film with the above described porous matrix

The native fish collagen gel having a solids content of 0.5 to 2% is deposited in a lyophilization trough at a rate of 0.5 g / cm ² , then the collagen film is applied to this gel and the whole is lyophilized. The resulting lyophilizate is crosslinked with TDH.

Example 7

porous Matrix made with an acid-soluble Collagen gel and covered with a collagen film

The Procedure is the same as in Example 6, with the only difference exists with respect to the type of gel cast on the film, the from acid soluble Collagen produced by a process as is well known to those skilled in the art.

Example 8

porous Matrix, made with a atelocollagen gel and covered with a collagen film

The Procedure is the same as in Example 6, with the only difference which is related to the type of gel cast on the film, the consists of atelocollagen, that is of telopeptide-free collagen, prepared by a method well known to those skilled in the art has been.

Example 9

porous Matrix consisting of collagen in combination with chitosan and one Glycosaminoglycan and covered with a collagen film

The The procedure is the same as in Example 6 except that that in this case the gel that is poured onto the collagen film consists of collagen, chitosan and a glycosaminoglycan. The production This gel is described in Example 5.

Example 10

All porous as described above Matrices covered with a collagen film may relax be crosslinked, as in Examples 2, 3 and 4 are described.

Example 11

Porous matrix only from collagen, as described in Example 1, with a compressed one Collagen sponge is covered

A. Preparation of the compressed sponge

The collagen gel with a solids content between 0.3 and 1.5%, prepared as in Example 1, is lyophilized to give a foam weighing between 0.5 and 2 g / cm ² .

The lyophilizate is pressed for 5 to 60 seconds at a temperature between 20 and 60 ° C and a pressure between 50 and 200 bar (50-200 x 10 ⁵ Pa).

B. Combination of the compressed sponge with the porous one matrix

The collagen gel described in Example 1 is deposited in a lyophilization trough at a rate of 0.5 g / cm ² . The compressed sponge is then applied to this gel and the whole is lyophilized to obtain a porous collagen sponge covered with a compressed collagen sponge. The whole is crosslinked by TDH as described in Example 1.

Example 12

porous Matrix consisting of collagen, chitosan and glycosaminoglycan, as described in Example 5, covered with a compressed sponge

The collagen, chitosan and glycosaminoglycan gel prepared by the method of Example 5 is deposited in a lyophilization trough at a rate of 0.5 g / cm ² , the compressed sponge is then applied to this gel and the whole is lyophilized. The lyophilizate is then crosslinked by TDH as described in Example 1.

Example 13

All porous as described above Matrices covered with a compressed collagen sponge, can be crosslinked according to the methods as described in Examples 2, 3 and 4 are described.

Examples 14 to 16: Tests for comparison cell metabolism of bovine and fish collagen matrices

Example 14: Test for cell viability of fibroblasts.

I. Preparation of Dermis Equivalents

For this A comparison test is first a porous fish matrix crosslinked with DPPA prepared according to Example 2.

For comparison, a comparative porous matrix, referred to as a bovine matrix, which is also crosslinked with DPPA, is prepared from bovine collagen under the same conditions as in Example 2 posed.

Normal human fibroblasts taken from a young donor pool used in the 7th passage are inoculated to each of the fish and bovine matrices at a rate of 250,000 cells / cm ² in the case of proliferation. and protein synthesis studies and inoculated at a rate of 300,000 cells / cm ² in the case of the fish and bovine matrices destined for histological studies.

These Fish and bovine matrices are cultured in a medium containing consists of DMEM / HAM F 12 in a vol./vol. ratio of 50/50, supplemented with 10% fetal calf serum, 100 IU / ml penicillin, 25 μg / ml gentamicin, 1 μg / ml Amphotericin B and 50 μg / ml Vitamin C.

These Cultivation is carried out for one month using the culture medium exchanged three times a week becomes.

II. Analyzes performed

1) Determination of cell viability by implementation with MTT

the Culture medium is 1 wt% MTT (i.e., 3- (4- (dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) added. The incubation is carried out at 37 ° C. for 2.5 hours.

To this incubation period, the conversion product (formazan blue) solubilized in DMSO and its optical density becomes 550 nm read.

The the optical density obtained is proportional to the activity of succinate dehydrogenases, the light yellow tetrazolium salt MTT in blue formazan crystals can convert.

The Cell Viability became after 1, 5, 7 and 22 days Cultivation and determined after 1-month cultivation. For determination the averages were for each matrix produced 6 samples.

Table I Results

These results are also used for the curves in the attached 1 ,

It it should be noted that the curve with the diamonds the one is that was obtained with the fish matrix, and that the curve with the squares is the one that received with the bovine matrix becomes.

The Results show completely surprising that the fish matrix is a carrier not just survival of normal human fibroblasts, but also the proliferation (Propagation) of these normal human fibroblasts allowed simultaneously even a much better culture carrier during the first three weeks represents.

It can therefore be concluded from these tests that fish collagen is surprisingly particularly suitable for the production of a tissue-forming carrier, in particular for in vitro applications and even, in particular, in vivo applications for the production of biomaterials containing living cells, in particular and preferably those of humans.

2. Determination of protein synthesis

The Synthesis of proteins within 3 days in a culture medium which was free of fetal calf serum was reduced 1-month maturation of dermis equivalents, as stated after 1 month of cultivation under the above in the preparation the dermis equivalents conditions were obtained. The determination was performed by the micro BCA method of Pierce.

The Cell density was measured in parallel by an MTT test among the above determined conditions.

• * mittlere Standardabweichung

The relative protein content corresponds to the protein content per unit of the cell density, expressed by the optical density or OD, so that the cell concentration in question is equivalent. The results obtained are shown in Table II below. Table II Results of protein synthesis

• * mean standard deviation

As in Table I the mean is based on 6 samples.

3. Histology

The after 21 days Cultivation of the fish and bovine collagen matrices obtained Dermis equivalents are fixed in a 2% paraformaldehyde solution and then in a Osmium tetroxide solution postfixed, dehydrated, included in Epon, it becomes one Mean preparation prepared and in a transmission electron microscope (Jeol 1200) at CMEAGB (Lyon, France).

Conclusions

These Results indicate a very good colonization of the three-dimensional Matrices, whether they come from fish or beef. After 3 weeks of cultivation the cell density is equivalent in both matrix types. The fish matrix However, better cell adhesion appears at the beginning of the experiment to allow, what by the proliferation investigation in the first Week of cultivation is displayed, and thus a better colonization to allow for short cultivation times.

What As far as protein synthesis is concerned, these are the fibroblast synthesis capabilities (relative protein levels) also equivalent after 1 month of culture.

These Results show that the developed fish collagen matrices the production of dermis equivalents enable a good quality, the results obtained with these matrices being comparable are with those obtained with bovine collagen matrices.

Fibroblasts were detected in bovine and fish matrices in a transmission electron microscope. In both types of matrix, the presence of a large amount of newly synthesized extracellular matrix was noted. The newly synthesized extracellular matrix can open Because of the periodic streaking of fibers from deposited collagen, they are differentiated in comparison with the collagen clusters that form the three-dimensional matrix of the original sponge.

Example 15

Influence of the different ones Types of cross-linking of fish collagen matrices on viability the cell

It The following tests are done to determine the influence of different Types of cross-linking of fish collagen matrices on viability to examine the cell.

I) Preparation of dermis equivalents

a) used carrier or matrix

It become different collagen carriers or matrices made using different proportions collagen in the collagen gel to make the porous matrix and optionally using a different cross-linking agent, as indicated below:

1) test 1

For this Test becomes a porous one Matrix in the form of a porous Sponge made from a fish collagen gel, which consists of 1.3% by weight Fish collagen, which was frozen at -80 ° C after standard lyophilization Example 2 and then with DPPA in a proportion of 250 μl / g Sponge was crosslinked in the dry state.

2) test 2

For this Test becomes a porous one carrier made in the form of a fish sponge from a fish collagen gel, comprising 0.7% by weight fish collagen frozen at -80 ° C and then one Subjected to standard lyophilization and with DPPA in a proportion of 250 μl / g dry sponge as in test 1 is crosslinked.

3) Test 3

For this Test, the operation is the same as in Test 1, but with with the exception that the crosslinking with EDC according to Example 2 in an amount 0.46 g / g dry sponge.

4) Test 4

It becomes a porous one carrier prepared comprising a sponge of fish collagen that was prepared from a fish collagen gel containing 1.1% by weight fish collagen, frozen at -80 ° C and then subjected to standard lyophilization and DPPA in a proportion of 250 μl / g dry sponge as in test 2 was crosslinked, the difference in the proportion of 1.1 wt .-% fish collagen.

In In all these tests the fish collagen originates from the abdominal skin of the Sole, as indicated in Example 2.

b) Cultivation of fibroblasts on these matrices

As Example 14 uses normal human fibroblasts, these but come from the eighth passage.

The inoculation is performed at a rate of 275,000 cells per cm ² .

The Culture medium consists of DMEM / HAM F 12 (Vol./Vol.) 50/50, supplemented with 10% fetal calf serum, 100 IU / ml penicillin, 25 μg / ml gentamicin, 1 μg / ml Amphotericin B and 50 μg / ml Vitamin C.

The Cultivation is performed for one month, with the medium three times is exchanged per week.

For each Test 4 matrices are used to give an average for each Test type form, and it will be the mean standard deviation certainly.

II) Analyzes performed

Determination of viability of the cell, implementation with Alamar blue (redox marker

Alamar Blue is added in an amount of 2% by weight to the culture medium used at the moment, if desired is, the viability to determine the cell of a sample taken from the culture medium.

To incubation for 2 h at 37 ° C for 20 min, the fluorescence on the Based on excitation at 530 nm and emission at 590 nm read. The intensity the fluorescence obtained is proportional to the metabolic activity of the cells.

The viability the cells are harvested at 10 samples after 1, 4, 6, 11 and 17 days of culture certainly. The results are shown in Table III below.

• * Standard-Abweichung

The results show the fluorescence in International Units (IU) as a function of time. Table III Cell viability (IU fluorescence)

• * Standard deviation

The results in Table III are also included in the attached 2 used. They show the curves of fibroblast proliferation in dermis equivalents.

The Curve with filled Lozenge corresponds to Test 1, the curve to solid squares corresponds to the test 2, the curve with triangles corresponds to the test 3 and the curve with crosses corresponds to the test 4.

The Time is indicated in days on the abscissa and the fluorescence is given in IU (International Units) on the ordinate on a scale starting at 15,000 and in levels of 10,000 Unit increases to 55,000. The results allow the to draw the following conclusions.

Conclusions

The Results show that the different matrices produced allow good growth of fibroblasts after 17 days of culture. Independently the preparation of the fish collagen matrices adhere to the fibroblasts good at her three-dimensional carrier and divide very quickly, whereby they colonize the matrix.

The Proliferation profile varies very little from a matrix type however, the fibroblast density is after 17 days of culture comparable, independent from the manufacturing process.

The applied different types of networking, either with DPPA or performed with EDC were not appear to affect the cell renewal. To practically 3 weeks Cultivation is the stability excellent matrices, there is a low digestion and a slight contraction.

Example 16

Test to demonstrate the Benefits of fish collagen for the identification and determination of newly synthesized human collagen

This Test is similar that of Example 14, except that one Histology is performed with an immunolabeling. The test is carried out as follows:

1) Preparation of dermis equivalents

there are the dermis equivalents of Example 14, wherein the cultivation under the conditions of Example 14 is performed.

This culture is therefore carried out for three weeks with the medium being changed three times a week after inoculating normal human fibroblasts at a rate of 300,000 cells / cm ² as indicated in Example 14.

2) histology

a) Conventional histology

The Fixation is done with paraformaldehyde in a concentration of 4 % By weight, after the material is dehydrated and enclosed in paraffin has been.

Thereon follow the production of 7 μm sections and the Mallory Haidenhain stain after removal of the paraffin and rehydration.

b) immunolabeling

A Fixation is carried out again with 4 wt .-% paraformaldehyde, the Material is in a tissue Tek OCT compound, i. in an inclusion liquid supplied by Miles, Elkhart, Indiana, USA will, and in the cold a 7 μm section is prepared.

• i. ein erster Kaninchen-Antihuman-Kollagen vom Typ I-Antikörper (Verdünnung 1/40) und
• ii. ein zweiter Antikaninchen-Antikörper werden gekuppelt mit FITC (Fluorescein Isothiocyanat) (Verdünnung 1/160).

The immunolabeling is carried out as follows:

• i. a first rabbit anti-human collagen type I antibody (dilution 1/40) and
• ii. a second anti-rabbit antibody is coupled with FITC (fluorescein isothiocyanate) (1/160 dilution).

When counterstain becomes DAPI (4 ', 6-diamidino-2-phenylindoldilactate) used.

3) Results

It it was found that carrier, which consist of a fish matrix and a cattle matrix, more or form less loose pores to which the fibroblasts adhere.

A greater proportion of fibroblasts are found on the surface, giving them a favorable Form coating on the dermis equivalent for the production of reconstructed skin. The distribution of fibroblasts is homogeneous in fish and bovine sponges.

at The immunolabeling was found to be from bovine collagen prepared matrix by the anti-human collagen type I antibody (cross) is marked.

on the other hand the fish matrix is only very weakly marked by the anti-human collagen antibody.

The Use of sponges, which consist of fish collagen, therefore favors the identification the newly synthesized extracellular matrix.

• (>: die optische Dichte beträgt mehr als 2000)

These results are illustrated by the studies of Professor Hartmann on the responses of various antigens to different antibodies as determined by optical density measurements after immunolabeling given in Table IV below or the Hartmann Table: , Bovine and fish collagen (Elisa)

• (>: the optical density is more than 2000)

The results are expressed by the OD × 10 ³ (optical density at λ = 450 nm).
Key:
20111 (225): Type I anti-human collagen
50121 (03): Type I anti-bovine collagen
50171 (01): Type I anti-fish (sole) collagen

These Results table shows that regardless of the antibody (type anti-human collagen I), anti-bovine type I collagen, anti-sole collagen from Type I) in immunolabeling the difference between human collagen and sole collagen is much larger As a result, between human collagen and bovine collagen. As a result, can in a fish collagen matrix synthesized by human fibroblasts Collagen can be identified much easier. This confirms the abovementioned results obtained by immunolabelling Collagen obtained in the fish collagen matrix with the Anti-human collagen was synthesized by type I antibody a particularly unexpected and advantageous result of the invention represents.

Claims (21)

carrier a formation of human tissue by cell proliferation, the a porous one Contains matrix that from fish collagen, that is a lyophilization step was subjected, and wherein the porous matrix by applying a chemical process is crosslinked. carrier according to claim 1, wherein a crosslinking agent is used, the selected is made from diphenylphosphoryl azide or DPPA, a carbodiimide, N-hydroxysuccinimide and glutaraldehyde. carrier according to claim 1 or 2, wherein the fish collagen is from the skin of a Bone fish is native. carrier according to one of the claims 1 to 3, wherein the fish collagen comes from the skin of a flatfish. carrier according to one of the claims 1 to 4, wherein the fish collagen from the skin of an industrial fished fish selected from sole, plaice, Turbot and brill. carrier according to one of the claims 1 to 5, wherein the porous Matrix contains a fish collagen, mixed with chitosan and optionally at least one Glycosaminoglycan. carrier according to one of the claims 1 to 6, wherein the porous Matrix on at least one side with a substantially compact, dense collagen membrane is selected, which is selected from a collagen film, which is prepared by drying a collagen film in air or in a gas fluid, and from a compressed collagen sponge. carrier according to one of the claims 1 to 7, wherein the porous Matrix comprises living cells selected from the group consisting of normal living cells, genetically modified living cells and malignant living cells. carrier according to claim 8, wherein the living cells are selected from the group consisting of fibroblasts, keratinocytes, melanocytes, Langerhans Cells that are derived from the blood, endothelial cells that are from the blood blood cells, macrophages, lymphocytes, adipocytes, sebocytes, Chondrocytes, osteocytes, osteoblasts and Merkel cells resulting from come from the blood. carrier according to claim 8 or 9, wherein the porous matrix is living fibroblasts contains which are selected from normal, genetically modified or malignant fibroblasts. carrier according to claim 7, 8, 9 or 10, wherein the substantially compact Collagen membrane normal, genetically modified or malignant living Contains cells, the selected are made up of keratinocytes, melanocytes, Merkel cells, from the Blood, Langerhans cells, that come from the blood, sebocytes, cells that come from the blood, and nerve cells. carrier according to one of the claims 8 to 11, wherein the living cells are derived from human beings. carrier according to one of the claims 8 to 12, wherein the living cells are from young animals. carrier according to one of the claims 8 to 12, wherein the living cells are from old animals. A carrier according to any one of claims 7 to 14, wherein the compression of the compressed collagen sponge was carried out at a pressure of at least 50 bar (50 × 10 ⁵ Pa) at a temperature of between 20 ° C and 80 ° C. carrier according to one of the claims 7 to 15, wherein the substantially compact collagen membrane is produced is prior to combination with the porous matrix by preparation the compact collagen membrane and then by deposition of the same on the fish collagen gel, before the combination of collagen gel and the compact collagen membrane is frozen and lyophilized. carrier according to one of the claims 7 to 16 in the form of a biomaterial, a reconstructed connective tissue or a reconstructed skin. carrier according to one of the claims 7 to 17, which contains cells, the practically exclusively come from young creatures, or virtually exclusively old creatures come to investigate the tissue aging process and for testing the efficiency of drugs in the skin aging process. Use of fish collagen, that of a lyophilization stage is subjected to for producing a carrier of formation of human A fabric as defined in any one of claims 1 to 18 and the one porous Contains matrix, which is crosslinked by using a chemical process. Use of a carrier according to one of claims 1 to 18 for implementation a cultivation and proliferation of living cells in vitro or in vivo. Use according to claim 20 for in vitro tests or for repair of injured Tissues.