WO1992018172A1 - Use of collagen which has been cross-linked by a cross-linking agent to produce collagen threads for forming an implant for filling soft tissue, and implant thereby obtained - Google Patents

Abstract

The use of collagen which has been cross-linked by a cross-linking agent to produce collagen threads for forming an implant for filling soft tissue is described. The cross-linking rate is preferably such that there occurs an increase of at least 15 C in the denaturation temperature of the cross-linked collagen relative to the native collagen. It is thereby possible to fill soft tissue, such as wrinkles, with reduced immunogenicity, good filling capacity and extended repair time.

Description

 Use of crosslinked collagen by a crosslinking agent for the manufacture of collagen threads intended to constitute an implant for the soft tissue furnishing, and implant so obtained.

We know that the average age of the population of developed countries is constantly increasing and that the concern for appearance in each individual becomes more and more strong.

This is why an increasing number of people are looking for treatments that generally reduce the effects of aging on the skin and in particular remove flaws from wrinkles.

The smoothing of wrinkled skin regions can be obtained by regular applications of cosmetic products. But the effects of such treatments only appear in the long term and are only effective on small wrinkles. For deep wrinkles, only the use of fillers allows immediate clear repairs.

Among the substances used, collagen is the one that provides the most satisfaction. In fact, this protein, even of animal origin, is generally well tolerated and its degradation rate is relatively slow.

However, in some individuals, immunogenicity reactions may appear and the filling obtained disappears between six months and a year after the injection of the intradermal collagen implant. In order to improve its biocompatibility, collagen can be rid of its telopeptides, non-helical ends of peptide chains. This operation is carried out by enzymatic treatment and results in an increase in the solubility of the protein, which makes it possible to obtain more concentrated solutions. The collagen thus transformed, thus called atelocollagen, can still cause immunogenicity reactions in 3% of cases.

The high viscosity of the collagen solutions makes it difficult to inject gels with concentrations greater than 6% in protein. As a result, the injected product contains

SUBSTITUTE SHEET Ci M i 94% water and maximum 6% collagen. As a result, the filling power is too low, in particular in the case of large wrinkles.

The object of the present invention is to solve the new technical problem consisting in providing a solution making it possible to provide a material for filling soft tissues which essentially does not have an immunogenicity reaction, and which has better filling power and a duration prolonged absorption.

The present invention makes it possible for the first time to resolve this new technical problem in a satisfactory, reliable, inexpensive manner which can be used on an industrial scale.

Thus, according to a first aspect, the present invention relates to the use of collagen crosslinked by a crosslinking agent, for the manufacture of collagen threads intended to constitute an implant for the filling of soft tissues.

According to a particularly preferred embodiment of the invention, crosslinking is carried out with a crosslinking rate such that there appears an increase of at least 15 ° C. in the denaturation temperature of the collagen relative to the native collagen, and even better at least 20 C.

The crosslinking agent can be chosen from any known collagen crosslinking agent. It may for example be an aldehyde such as a dialdehyde, in particular glutaraldehyde. However, preferably, the crosslinking agent is made up of diphenylphosphorylazide, or DPPA.

The crosslinking process in itself is well known to those skilled in the art. For a cross-linking process with DPPA, reference can be made to the specific previous request from the depositor published under the number FR-A-2645870, which is incorporated here by reference.

It is generally possible, in the context of the present invention to use a method of crosslinking of the bonding by introduction of azide groups on the carboxylic groups of the side chains of collagen such as that described in document FR-2617 855 also incorporated here by reference.

According to this process, the crosslinking is carried out by an esterification of the carboxylic groups of collagen, then the esters are successively transformed into hydrazides, then into acylazides. The acylazides finally react in basic medium with the amino functions of the side chains of collagen to give bonds of peptide type.

As the starting collagen, it is preferred to use a native collagen essentially of type I. It is also possible, in certain cases, to use atelocollagen although this is not a preferred embodiment.

According to a second aspect, the present invention also covers implants for the filling of soft tissues, characterized in that they comprise one or more collagen threads crosslinked by a crosslinking agent. Preferably, the crosslinking agent is diphenylphosphorylazide or DPPA.

As indicated above, the crosslinking rate is provided to obtain an increase of at least 15 ° C in the denaturation temperature of the crosslinked collagen compared to the native collagen, and even better of at least 20 ° C.

Cross-linking with diphenylphosphorylazide is preferred because it has the major technical advantage over other cross-linking treatments with a tanning agent such as an aldehyde, of not introducing foreign chemical agents into the biomaterial, while conferring with protein (here collagen) very good protection against enzymatic degradation as well as remarkable biocompatibility, an immunogenicity reaction being observed only in extremely exceptional cases.

The present invention will now be described in more detail using an example of preparation of a wire given simply by way of illustration and which therefore cannot in any way limit the scope of the invention. This example is given with reference to the single appended figure showing the thermograms obtained by Programmed Differential Calorimetry (CDP) on crosslinked collagen wires according to the invention and not crosslinked or

SUBSTITUTE SHEET native collagen. It is observed that the crosslinking according to the present invention makes it possible to obtain an increase in the collagen denaturation temperature of at least about 20 C.

Example of preparation of a crosslinked collagen wire intended to constitute an implant for the filling of soft tissues according to the present invention

A - Extraction of native collagen:

A gel is prepared from skins of calves previously washed and depilated with a mixture of lime-sulfide (lime: 4%, sodium sulfide: 3%).

They are then released in a bath containing ammonium chloride (2%) and sodium metabisulfite (0.5%). They are then neutralized, then the salts are removed by two washes with water. They are then ground, then washed with phosphate buffer pH 7.8 (potassium dihydrogen phosphate 0.78 g / 1 and disodium hydrogen phosphate 21.7 g / 1). The phosphate is then removed by two successive washes with deionized water.

The ground material is then acidified with a 10% acetic acid solution, the amount of actic acid being 5% relative to the collagen. The ground material is then kneaded in order to obtain a homogeneous paste. This paste is then diluted to obtain a gel having a concentration of 1.5% in native collagen.

B - Preparation of the wires

The gel obtained is degassed under vacuum and then poured (flow rate = 7 ml / min) under pressure through a die in an ammonia acetone solution (V / V 70/30) which is renewed every 250 ml of gel.

The wires obtained are air dried. After drying, they can have a diameter of between 0.15 mm and 2 mm depending on the die (a die of 1 mm in diameter gives a dry thread of about 0.15 mm in diameter).

REPLACEMENT SHEET C - Chemical crosslinking

The wires are incubated for 24 h at 4 ° C. in a solution of dimethylfor amide (DMF) containing 0.5% of diphenylphosphoryl azide (DPPA) (concentration expressed in volume / volume).

05 The wires are then freed from DPPA by rinsing with DMF. The DMF is then eliminated by rinsing with a borate buffer solution pH 8.9 (sodium tetraborate 0.04 M, boric acid 0.04 M). The strands are finally incubated overnight in a borate buffer ^" pH 8.9, then are rinsed several times with water.

10 permuted before being put in an aqueous solution containing 10% glycerol.

The wires are then air dried and sterilized by gamma irradiation at a dose of 25 kGy.

15 D - Stability-thermal

The influence of the crosslinking of the wire by tanning was demonstrated by a differential scanning calorimeter. The latter allows on the one hand to measure the amount of heat involved in the thermal denaturation of collagen and on the other

20 part to determine the temperatures of beginning and end of the phenomenon of transformation of the structure. The higher these temperatures, the greater the stability of the protein structure. This will lead to better resistance to the enzymatic degradation of the biomaterial. ^ 5 In the example, it is possible to see that tanning with azides leads to an increase of 20 C in the denaturation temperature (Fig. 1). As a result, the implanted wire will have an extended resorption period when it is implanted in an intradermal situation. 30

E - Method of using the crosslinked wire as a soft tissue filling implant (example: filling of wrinkles)

The technique is simple, it consists in implanting the tanned thread in the dermis. The wire used has a diameter of 0.8 mm. ^ 5 To do this, two ways are essentially used:

REPLACEMENT SHEET - Introduction of thread to thread puller

The thread is introduced into the dermis at the level of the wrinkle using a special instrument, the thread puller. This instrument consists of a stainless steel rod, mounted on a handle and terminated by an eye. The thread puller is introduced under the skin through a small incision made perpendicular to the end of the wrinkle; it is then led by the surgeon, on the subcutaneous path of the wrinkle until the eye comes out at the other end.

At this time, the thread is introduced into the eye, the thread puller is brought back on the reverse path so that the thread is implanted all along the wrinkle.

Finally, the wire is cut using surgical scissors so that each of its ends remains buried under the skin.

At the end of the intervention, the depression of the wrinkle is filled by the volume of the wire, it is only very difficult to detect with the eye.

F - Results-of implantation

A series of interventions carried out on 5 patients at the level of the wrinkles of the nasolabial fold, by implantation of collagen wire crosslinked by DPPA with a diameter of 0.8 mm with the thread puller method gave the following results:

- Immediately after implantation, tolerance is excellent; no abnormal pain or inflammation could be detected in the treated patients. The aesthetic result is very satisfactory and the wrinkles have disappeared or are considerably reduced.

- At four months, the thread is still perfectly tolerated and no aesthetic difference can be detected in comparison with the immediate result.

REPLACEMENT SHEET

Claims

1. Use of collagen crosslinked by a crosslinking agent for the production of collagen threads intended to constitute an implant for filling soft tissues. 2. Use according to claim 1, characterized in that the crosslinking rate is such that it appears an increase of at least 15 ° C in the denaturation temperature of the crosslinked colla¬ gene compared to the native collagen, and better at least 20 ° C. 3. Use according to claim 1 or 2, caracté¬ ized in that the crosslinking agent is chosen from an aldehyde such as glutaraldehyde, or preferably diphenyl¬ phosphorylazide or DPPA. 4. Use according to one of claims 1 to 3, characterized in that the collagen is essentially type I native collagen or atelocollagen. 5. Implant for the filling of soft tissues, characterized in that it comprises at least one collagen wire crosslinked by a crosslinking agent. 6. Implant according to claim 5, characterized in that the rate of crosslinking of the collagen shows an increase of at least 15 ° C in the denaturation temperature of the crosslinked collagen relative to the native collagen, and better still of at least 20 ° C. 7. Implant according to claim 5 or 6, characterized in that the crosslinking agent is chosen from an aldehyde, in particular glutaraldehyde, or preferably diphenyl¬ phosphorylazide or DPPA. 8. Implant according to one of claims 5 to 7, charac¬ terized in that the collagen is native collagen essentially of type I or atelocollagen. REPLACEMENT SHEET