EP2498805A1

EP2498805A1 - Aqueous composition comprising gold nanoparticles, serum albumin and/or collagen for laser tissue welding

Info

Publication number: EP2498805A1
Application number: EP10763766A
Authority: EP
Inventors: Philippe Jenny; Marc Bernard; Franck Tricot
Original assignee: Franck Tricot; Marc Bernard
Current assignee: PRESTIGIACOMO, PHILIPPE VINCENT; CREASPINE
Priority date: 2009-11-09
Filing date: 2010-08-27
Publication date: 2012-09-19
Also published as: US20120220991A1; FR2952306A1; WO2011055047A1; JP2013510186A; FR2952306B1

Abstract

The invention relates to an aqueous composition used in the laser repair treatment of fibrous connective tissue, including: gold nanoparticles in a concentration of between 1.5 x 109 particles per ml and 1 x 1010 particles per ml; and albumin and/or collagen in a concentration of between 30 wt % and 45 wt %. The invention also relates to an assembly including such a composition.

Description

AQUEOUS COMPOSITION COMPRISING GOLD NANOPARTICLES, ALBUMIN SERUM AND / OR COLLAGEN FOR LASER TISSUE WELDING

The present invention relates to an aqueous composition used in the treatment of fibrous connective tissue repair.

The present invention also relates to a set used in the treatment of fibrous connective tissue repair.

Fibrous connective tissue is a relatively strong supporting tissue whose role is to protect the surrounding organs. Fibrous connective tissues may be predominantly collagen such as cartilage, dermis, ligaments, tendons, cornea.

Over time, fibrous connective tissues can become fragile to the point of cracking or tearing, causing tissue defects in their structure through which migration of the organic structure within the connective tissue can occur. or resulting in abnormal and painful mobility of an element or fragment.

Thus, in the case of distal hernias, the nucleus of the intervertebral disc (called "nucleus pulposis") escapes out of the ligamentous envelope ("annulus fibrosis") through a crack appeared in said envelope.

Usually, the orifice at the origin of this hernia is not repaired during the surgical procedure which consists in treating the protrusion of the disc material and the pain.

It is thus known to employ invasive surgical techniques consisting of suturing the tissue to be repaired. Such techniques traumatize the tissues surrounding the tissue to be repaired and only allow imperfect closure of the orifice because of their attachment in a damaged and mechanically fragile tissue.

There are other more specific methods for repairing said tissue defects using the application of polymeric staples on the lesion, mesh or metal barrier affixed to the injured part. These reported elements require a relatively invasive technique and generate a risk of a non-permanent mechanical fixation in a damaged tissue. However, all these techniques close in a superficial way the crack or the tear but do not make it possible to prevent or prevent imperfectly the painful post-operative operations and the recurrences of migration through the repaired tissues.

In the laboratory, it is known to use a biological or cyanoacrylic glue, alone or in combination with a standard suture. However, this technique called "standard gold" can not be used by a surgeon because the glue is not biocompatible.

It is known a repair technique used for certain types of tissues such as viscera and eyes based on the use of the laser. The laser can be adjusted to locally and accurately heat the targeted tissue. The heating generated is such that it destroys the fabric at the impact surface. Thus, it is possible to spray a tumor or to make an incision very accurately. The laser can also be used with less energy, capable of generating a smaller, localized heating, so as to bond or weld biological tissues.

Since the laser heats locally in a precise manner and at a chosen depth, the tissues traversed are less traumatized than in a conventional surgical technique where part of these surrounding tissues is incised. Moreover the laser beam can be brought by optical fibers allowing micro-invasive or even percutaneous techniques, much less traumatic than standard surgical techniques.

In the case of repair and welding of fibrous connective tissue, it is necessary to associate with the laser a biocompatible composition for repairing said tissues in the manner of a cement. This composition must also make it possible to obtain a waterproof repair that is long-lasting to avoid a new intervention.

In the treatment of fibrous connective tissue by laser, there is therefore a need for a composition allowing the closure of tissue defects, such as fissures or tears, while allowing the stabilization of the latter in order to avoid the reopening of these defects. .

An object of the present invention is therefore to provide a composition that can be used for the repair of fibrous connective tissue and that allows efficient and stable repair of tissue defects without the disadvantages mentioned above. For this purpose, according to a first aspect, the invention relates to an aqueous composition for use in the treatment of fibrous connective tissue repair by laser comprising:

gold nanoparticles in a concentration greater than 1, 5.10 ⁹ particles per ml and strictly less than 1 .10 ¹⁰ particles per ml, and

albumin and / or collagen in a mass concentration of between 30% and 45%.

The composition of the invention is particularly suitable for the repair of fibrous connective tissue with the aid of a laser. Indeed, the concentration of gold nanoparticles and albumin and / or collagen allows these two components to generate a more leakproof and durable seal than in the prior art. Surprisingly, outside of these concentration ranges, the weld is either unrealized or poorly sealed and not very durable.

Indeed, in these concentration ranges, the crosslinking of albumin and / or collagen is in a time of exposure compatible with surgical use, namely between 30 seconds and 40 seconds. This crosslinking time makes it possible to seal the weld.

In addition, by lowering the concentration of gold nanoparticles below 1, 5.10 ⁹ particles per ml, the exposure time necessary to obtain a weld is increased and therefore less tightness is achieved.

On the other hand, for a concentration greater than 1 .10 ¹⁰ particles per ml in gold nanoparticles, heating linked to the solder becomes more and more difficult to control. In this case, warming becomes more and more important which has the effect of reducing the adhesion between the albumin and / or the added collagen and the fibrous connective tissue, in particular the annulus fibrosis of a vertebral disc, which degrades under the effect of heat. As a result, the sealing of the weld decreases. If the exposure is continued, the heat induced destroys the cells of the fibrous connective tissue but also the surrounding cells.

By lowering the mass concentration of albumin and / or collagen below 30%, the crosslinking of the latter no longer forms a sufficiently strong film to seal the solder.

By increasing the mass concentration of albumin and / or collagen above 45%, the crosslinking is too strong and is no longer done with the fibrous connective tissue which decreases the seal. The gold nanoparticles of the composition of the invention are adapted to absorb the wavelength of the laser by restoring energy in the form of heat which causes the crosslinking of the albumin and / or collagen of the composition. By cross-linking, albumin and / or collagen form a biocompatible matrix filling the tissue defect, such as a crack or tear, and adhering to the fissure walls of the connective tissue. In other words, the composition of the invention used with the concentrations as indicated makes it possible to retain enough gold nanoparticles to allow the desired absorption of the wavelength of the laser in order to sufficiently heat the albumin and / or collagen in order to cross-link these which are in sufficient quantity to obtain a durable and waterproof weld. The particular composition of the invention makes it possible to stabilize the tissue defects in the depth and not only to close these defects superficially, as is the case in the prior art.

Thus, closeness and stability are achieved concomitantly in a single step and in a precise manner.

In addition, gold nanoparticles are chemically inert and do not produce a potentially toxic product to the surrounding tissues. The albumin and the collagen advantageously make it possible to create a resistant cement matrix over time and not to produce in their turn toxic by-product during their crosslinking.

According to other characteristics of the invention, the composition of the invention comprises one or more of the following optional characteristics considered alone or according to all the possible combinations:

the albumin and / or the collagen is of bovine or human origin, which makes it possible to ensure good biocompatibility with the tissue to be repaired;

the gold nanoparticles are of spherical, triangular or rod-like shape, which makes it possible to guarantee the desired absorption taking into account the "red shift";

the gold nanoparticles have their largest dimension of between 3 nm and 250 nm, which makes it possible to reduce the thermal diffusion and to concentrate the heat at the interface of the weld;

fibrous connective tissue is a cartilage, ligament or tendon. According to another aspect, the subject of the invention is a used assembly comprising:

an aqueous composition according to the invention;

a laser emitting at a wavelength between 0.78 μm and 1.4 μm.

The whole of the invention is particularly suitable for an effective and stable repair of fibrous connective tissue. Indeed, the laser used emits a beam of light at a wavelength that is able to pass without damaging the surrounding tissue while reaching the desired depth. The composition of the invention makes it possible to lower the wavelength thanks to the presence of chromophoric gold nanoparticles. In addition, the composition is biocompatible with the surrounding tissues even once this composition is denatured by contact with the laser. Therefore, the repair and consolidation performed by the whole of the invention are concentrated on the damaged tissues and do not affect the surrounding tissues.

According to other characteristics of the invention, the assembly of the invention comprises one or more of the following optional characteristics considered alone or according to all the possible combinations:

the laser emits in a wavelength equal to approximately 808 nm, which makes it possible to avoid damaging the surrounding tissues;

the laser is pulsed or continuous;

the laser is configured to present a beam with a diameter of between 3 mm ² and 7 mm ² , which makes it possible to accurately repair small areas of fabric;

the laser is associated with a collimator and with an optical fiber system which allows easy manipulation of the light beam;

the laser is configured to present an angle of incidence with respect to the normal of the impact surface on the fibrous tissue of between 45 ° and 60 °, which makes it possible to have an optimal angle of incidence in the composition of the 'invention.

The invention will be better understood on reading the nonlimiting description which follows, with reference to the appended figures.

- Figure 1 is a schematic cross section of a healthy vertebral disc; - Figure 2 is a schematic cross section of a vertebral disc with a herniated disc;

FIG. 3 is a schematic perspective view of the assembly of the invention used for the repair of an incision made in a vertebral disc;

FIG. 4 is a schematic cross section of a vertebral disc presenting an open incision;

FIG. 5 is a schematic cross section of a vertebral disc having a repaired incision by the laser technique employing the assembly of the invention;

FIG. 6 is a schematic transverse section of a vertebral disc having an incision repaired by the laser technique using the assembly of the invention, comprising an annular passage in which is inserted a pipe connected to a syringe under pressure allowing inject saline with methylene blue into the nucleus.

The present invention makes it possible to repair the tissue defects of fibrous connective tissue such as cartilage, such as the meniscus of the knee and the facets, the ligaments, such as the annulus fibrosis of the vertebral disc, the dermis, the tendons, the cornea.

Tissue defects are typically in the form of tears or cracks that induce tissue damage in the thickness of the tissue.

The aqueous composition of the invention is thus used in the treatment of fibrous connective tissue repair by laser and comprises:

- gold nanoparticles in a concentration between

1, 5.10 ⁹ particles per ml and 1 .10 ¹⁰ particles per ml, and

albumin and / or collagen in a mass concentration of between 30% and 45%.

The composition of the invention is particularly suitable for the repair of fibrous connective tissue using a laser. Indeed, a fibrous connective tissue may have tissue defects such as tears or cracks.

The gold nanoparticles of the composition of the invention are particularly suitable for absorbing the wavelength of the laser and for restoring energy in the form of heat which causes the crosslinking of the albumin and / or collagen of the composition of the invention. By crosslinking, albumin and / or collagen form a biocompatible matrix filling the tissue defect, such as a crack or tear. Thus, the composition of the invention used with the laser makes it possible to stabilize the tissue defects in the thickness and not only to close these defects superficially, as is the case in the prior art.

Thus, in a single step, it is possible to permanently stabilize the tissue defect by a composition comprising non-toxic constituents and not producing hazardous by-products for the surrounding tissues.

The concentration of gold nanoparticles and albumin and / or collagen allow these two components to produce a more leakproof and durable seal than in the prior art. Surprisingly, outside the ranges of concentrations, the weld is either unrealized or not or tight and durable.

Indeed, in these concentration ranges, the crosslinking of albumin and / or collagen is done in a time of exposure compatible with surgical use, namely between 30 seconds and 40 seconds. This crosslinking time makes it possible to seal the weld.

On the other hand, for a concentration greater than 1 .10 ¹⁰ particles per ml in gold nanoparticles, heating linked to the solder becomes more and more difficult to control. In this case, heating becomes more and more important which has the effect of reducing the adhesion between the albumin and / or the added collagen and the fibrous connective tissue, in particular the annulus fibrosis of a vertebral disc, which degenerate under the effect of the heat r. In this way, the sealing of the weld decreases. If the exposure is continued, the heat induced destroys the cells of the fibrous connective tissue but also the surrounding cells.

By increasing the mass concentration of albumin and / or collagen above 45%, the crosslinking is too strong and is no longer done with the fibrous connective tissue which decreases the seal.

Albumin and / or collagen have the advantage of improving the strength of the weld performed in the tissue defect and to obtain a reliable weld. Indeed, after crosslinking, albumin and collagen act as an adhesive which forms a binding matrix among the collagen fibers already present in the fibrous connective tissue.

In addition, using collagen, the structural support is improved while increasing the post-welding repair speed because collagen is one of the endogenous materials of connective tissue.

The albumin and / or collagen may be of bovine or human origin which ensures good biocompatibility with the tissue to be repaired.

Gold nanoparticles can reduce the power of the laser by selecting a low wavelength, usually less than 820nm. Gold nanoparticles are capable of selectively absorbing wavelengths of the light spectrum, transforming into heat and transferring heat to collagen and / or surrounding albumin. The gold nanoparticles reduce the wavelength of the laser beam so as to limit the interaction between the light beam and the tissues. This is particularly important for a heterogeneous medium in which tissues surrounding the tissue to be repaired are sensitive to wavelengths lower than that tolerated by the tissue to be repaired. For example, there is the case of the repair of an annulus fibrosis of a vertebral disc in the case of a herniated disc.

In addition, gold nanoparticles have the advantage of being chemically inert and non-toxic. In addition, they do not produce harmful or toxic tissue degradation by-products.

The term "nanoparticles" means nanoparticles having their largest dimension between 3 nm and 1 000 nm, or even between 3 nm and 250 nm. The small size of these nanoparticles reduces the thermal diffusion that occurs from the treatment site and concentrates the heat at the interface of the weld which minimizes the damage to the tissues surrounding the tissue to be repaired.

Gold nanoparticles are typically gold nanoparticles formed by agglomerating gold atoms.

The gold nanoparticles may be hollow or encasing a silica or silicate core.

The gold nanoparticles can be functionalized by a coating of polyethylene glycol (PEG). Thus, advantageously, the nanoparticles are stabilized in the aqueous composition, avoiding the flocculation of these nanoparticles which facilitates the manipulation of nanoparticles.

The gold nanoparticles can be of any suitable form to guarantee the desired absorption by limiting the "red shift" corresponding to the absorption spectral shift in the wavelengths of the red, namely less than 800 nm. Thus, these particles may be spherical, triangular or stick-type.

The composition of the invention is carried out by diluting the gold nanoparticles in deionized water so as to obtain a concentration of between 1.5 × 10 ⁹ particles per ml and 1 × 10 ¹⁰ particles per ml and albumin and / or collagen so as to obtain a concentration by mass relative to the final composition of between 30% and 45%.

The composition of the invention can be more or less viscous, so that it can be used as a foam. In this case, it is advantageously possible to inject the composition of the invention at the desired location and that said composition remains at the injected location without flowing or leaking which allows a more thorough repair of the fabric by compared to a liquid composition. The gel advantageously makes it possible to treat a tissue defect in vivo by preventing the body fluid from carrying away the composition before the welding is performed. Thus, the surgical procedure is simplified by avoiding keeping the area to be treated "dry".

According to another aspect, the invention also relates to an assembly used in the treatment of fibrous connective tissue repair by laser comprising:

the aqueous composition of the invention; and

a laser emitting at a wavelength in the near infrared, namely between 0.78μηη and 1.4μηη.

The whole of the invention is particularly suitable for an effective and stable repair of fibrous connective tissue. Indeed, the laser and the composition used in the context of the invention advantageously have a safety vis-vis the surrounding tissues. Indeed, the laser beam passes through the different tissues before reaching the tissue to be repaired without causing harmful interaction for the tissues passed through, such as a heating. In addition, the composition is adapted to be biocompatible and not generated by toxic byproducts. The repair and consolidation performed by the whole of the invention are thus concentrated on the damaged fabric. More precisely, the laser is focused on the composition of the invention at the place where the repair is to take place, namely in the tissue defect. The gold nanoparticles absorb the wavelength emitted by the laser beam and emit heat captured by the albumin and / or collagen molecules surrounding the gold nanoparticles. Under the effect of heat, macromolecules of albumin and / or collagen polymerize to become a hardening matrix adhesive, filling the cracks or tears in the fibrous connective tissue. Thus, in the repaired portion of the fibrous connective tissue, a durable structural cohesion of said tissue that fills the tissue defect is provided.

The laser emits in a wavelength equal to about 808 nm which is an optimal wavelength that gold nanoparticles are capable of absorbing to restore heat to albumin and / or collagen. In addition, at this wavelength, there is a weaker interaction with the tissues surrounding the tissue to be repaired.

The laser may be for example a laser diode.

The laser can be pulsed or continuous. A pulsed laser makes it possible, if necessary, to send more powerful pulses of light in energy and to concentrate the energy over a very short time while not inducing a higher temperature rise than the continuous laser.

The laser power is between 10 kW and 12 kW.

The laser may be configured to have a beam diameter of between 3 mm ² and 7 mm ² or diameter adapted to the size of the area to be treated by keeping a power flux (per cm ²⁾ identical thereby fix accurately small areas of fabric.

The laser can be associated with a collimator that allows focusing at a given surface point.

In the context of a surgical operation, the surgeon can not always bring the beam to the location of the lesion, but the latter must position the laser beam towards the lesion via organs or bones, such as the spine in the case of a herniated disc. To do this, it is possible to pass the light beam through an endoscope or a handpiece closer to the area to be repaired.

The laser can also be associated with an optical fiber system making it easier to manipulate the beam and adjust the point of light from the light beam at the location of the fabric to be repaired.

The laser can be configured to have an angle of incidence with respect to the normal of the impact surface on the fibrous tissue between 45 ° and 90 °, preferably between 45 ° and 60 ° which allows to have a optimal incidence through the composition of the invention.

Alternatively, it is possible to stick a patch comprising collagen and / or albumin directly on the tear or crack. The use of such a patch makes it possible to further increase the mechanical strength of the solder by up to 50% and to avoid the loss of the aqueous composition when the latter is hydrated.

Comparative example of the resistance of a repair of a herniated disc lesion.

Presentation of a herniated disc

As shown in FIG. 1, a healthy or normal vertebral disc 5 has a center 3 ("nucleus pulposis") surrounded by a vertebral ligament 7 ("annulus fibrosis"). The vertebral disc is also surrounded by vertebrae 9 which house the spinal cord from which the nerve roots emerge. The annulus fibrosis 7 is surrounded by muscle and tissue. As illustrated in FIG. 2, a disc herniation corresponds to a protrusion 13 of the center 3 through the vertebral ligament 7.

Preparation of an example of an aqueous composition of the invention

The composition used in this example is prepared as follows.

The albumin solution is prepared by diluting about 2 g of a bovine serum albumin in about 5 ml of deionized water. The final concentration obtained is about 40% by weight, namely 40 g per 100 ml.

The solution of gold nanoparticles is prepared by centrifuging at

10,000 rpm for 30 minutes a solution composed of about 11 ml of deionized water with a concentration of about 2.47 × 10 ⁹ particles. A deposit of gold nanoparticles is obtained on which the deionized water floats. About 10.61 ml of the supernatant water is removed and the deposit is dissolved in the remaining water. An aqueous solution comprising a concentration of gold nanoparticles of about 7 × ^{10 10} particles per ml is then obtained. Approximately 142.9 μΙ of the solution of gold nanoparticles is taken and introduced into a 1.5 ml tube, and about 857.1 μΙ of the final albumin solution are added to obtain a final solution of approximately 1 ml with a concentration of about 1 .10 ¹⁰ nanoparticles per ml and a mass concentration of about 34% albumin.

Laser employed.

A medical laser diode emitting at about 808nm with a power of about 7kW connected to a laser diode source 15, a laser driver and a temperature controller is used. In the embodiment shown in FIG. 3, the laser diode, the laser diode source, the laser driver and the temperature controller are contained in the same receptacle 15. The laser is coupled to a collimator 16 forming a luminous task of approximately 3.1 mm diameter and fiber optic cabling 17.

The luminous exit point of the light beam may be attached to a metal rod positioned at an angle of about 50 ° to the perpendicular of the impact surface of the annulus fibrosis to be repaired, said surface being disposed at about 10cm from the output of the light beam of the optical fibers.

Two thermocouples are connected to a temperature data logger. The thermocouple about 0.4mm in diameter can be inserted into the annulus in a depth of about 55mm from the area to be repaired and on both sides of the tissue defect. The temperature is read in real time and reaches a maximum value of about 45 ° C.

Preparation of porcine vertebral discs

The vertebral discs are taken from 40kg pigs

80kg within 4 hours of their death.

The discs thus collected are cleaned of all the meat, in particular using a scalpel.

The vertebral discs are frozen at about -18 ° C to be kept until manipulations

Sample preparation

The repair of herniated discs on porcine vertebral discs is simulated by incision in annulus fibrosis 7 (see Figure 4). The incision is either unrepaired or repaired 21 (see Figure 5) following two different methods. As illustrated in FIG. 4, the incision is made by a scalpel 31 radially (arrow 33) with respect to the center 3.

A first control disk is prepared with an open incision, ie not repaired.

A second disc is prepared by repeating the incision by the "standard gold" technique, namely the direct injection into the incision formed by two drops of a cyanoacrylate glue injected into the incision and on which it has been closed. the entrance of the incision by stitches.

As shown in Fig. 5, a third disc 5 is prepared by repairing the incision 21 with the assembly of the invention as follows. After making the incision, the composition 41 of the invention is injected appropriately into the incision 21 by an appropriate means 43.

Then, the laser is used as indicated above whose beam is focused on the incision in order to perform the welding.

Annular passages 51 are also made with the scalpel for receiving pipes or sensors 53 (see FIG. 6).

The sample is fixed and prepared in one of the annular passages 51 made in the annulus fibrosis.

1 cm laterally on each side of the incision and about 0.75 cm deep. The hose 55 is connected to a syringe 57 under pressure for injecting through the hose 55 a salt solution with methylene blue into the nucleus. Syringe 55 is set at about 3 ml. min ^"1 .

The saline solution is prepared by diluting about 2.16 g of sodium chloride in about 250 ml and adding methylene blue to color. The coloring of the saline solution intended to be injected into the heart of the vertebral disc makes it possible to identify visually when the tissue defect leaks liquid.

During the injection, the intradiscal pressure is measured by a pressure sensor 59 which can be connected to a computer-based computer unit 60. In order to use only one annular passage 51, it is possible to use Y type bypass device 61 in order to choose the injection or the pressure measurement.

A healthy disc 5, namely without lesion, has an annulus fibrosis 7 able to deform elastically to contain the saline solution. On the other hand, a disc having undergone a herniated disc and being repaired presents at the level of the incision a fragility which is translated by the leak the solution saline through the incision repaired if the repair is not effective enough. In the context of the comparative example, the pressure is measured in order to detect the maximum pressure. This maximum pressure corresponds to the amount of maximum injected saline solution retained by the annulus fibrosis. Beyond the maximum pressure, the saline solution leaks out of said annulus. Thus, the measurement of the maximum pressure makes it possible to detect the robustness and the tightness of the repair of the incision.

The maximum intra-discal pressure of a healthy vertebral disc supported is generally greater than 32 bars, which corresponds to the maximum pressure used in the context of this example. Typically, the injected volume of the saline solution before being expelled by a healthy annulus is substantially equal to 5 ml.

Results of the pressure measurement

For each type of disk repaired or not, about fifteen attempts were made. The value given below corresponds to the average of the results obtained on the said fifteen tests.

The maximum pressure measured corresponds to the pressure of the saline solution beyond which said solution leaks out of the annulus fibrosis of the vertebral disc.

The vertebral disc comprising the unrepaired incision has a maximum supported pressure of about 4.4 bar to 1, 5 bars.

The vertebral disc comprising the incision repaired according to the Gold Standard method has a maximum pressure supported from about 10 bar to 5 bars.

The vertebral disc comprising the incision repaired with the assembly of the invention has a maximum pressure supported of about 25 bar to 2 bars.

As indicated above, a healthy vertebral disc supports a pressure greater than 32 bars, in particular substantially equal to 34.5 bars.

Thus, the vertebral disc repaired by all of the assembly of the invention has a weld that supports a pressure close to that supported by a healthy vertebral dice, namely with an undamaged year. Therefore, the weld made through the assembly of the invention is particularly effective. This invention is not limited to the repair of a herniated disc but may also be used in connection with cartilage repair or other fibrous connective tissue.

Claims

1. An aqueous composition for use in the treatment of fibrous connective tissue repair by laser comprising:

albumin and / or collagen in a mass concentration of between 30% and 45%.

2. Composition according to the preceding claim, wherein the albumin and / or collagen is of bovine or human origin.

3. Composition according to one of claims 1 and 2, wherein the gold nanoparticles are spherical, triangular or rod-type.

4. Composition according to any one of the preceding ications, wherein the gold nanoparticles have their largest dimension of between 3 nm and 250 nm.

5. The method according to any one of the preceding claims, wherein the fibrous connective tissue is a cartilage, a ligament or a tendon.

6. Set comprising:

an aqueous composition according to one of the preceding claims;

a laser emitting at a wavelength of between 0.78 μm and 1.4 μm.

7. An assembly according to the preceding claim, wherein the laser emits in a wavelength equal to about 808 nm.

8. The assembly of claim 6 or 7, wherein the laser is pulsed or continuous.

9. An assembly according to any one of claims 6 to 8, wherein the laser is configured to have a beam with a diameter of between 3 mm ² and 7 mm ² .

10. An assembly according to the preceding claim, wherein the laser is associated with a collimator and an optical fiber system.

1 1. An assembly according to any one of claims 6 to 10, wherein the laser is configured to have an angle of incidence with respect to the normal of the impact surface on the fibrous tissue between 45 ° and 60 °.