WO2013093381A2

WO2013093381A2 - Method for the microscopic and macroscopic analysis of wound healing progress

Info

Publication number: WO2013093381A2
Application number: PCT/FR2012/053069
Authority: WO
Inventors: Christophe DARDENNE; Bernard Pipy; Agnès COSTE; Marielle BOUSCHBACHER; Christelle Laurensou
Original assignee: Laboratoires Urgo; Societe De Developpement Et De Recherche Industrielle; Universite Paul Sabatier Toulouse Iii; INSERM (Institut National de la Santé et de la Recherche Médicale)
Priority date: 2011-12-22
Filing date: 2012-12-21
Publication date: 2013-06-27
Also published as: WO2013093381A3; WO2013093381A9

Description

METHOD OF ANALYZING THE PROGRESSION OF WOUND HEALING AT MICROSCOPIC AND MACROSCOPIC LEVELS.

Application domain :

The present invention relates to a method of analyzing the progression of wound healing in real time by means of a medical device, non-invasive and adapted, which protects the wound and allows me to observe my croscopic progress healing of a wound as well as collecting wound exudates in order to carry out cell monitoring, but also biochemical and molecular analyzes of the metabolites and microorganisms contained in said exudates.

Previous Art:

Wound healing is a natural pathophysiological process, with human and animal tissue capable of repairing localized lesions through their own repair and regeneration processes.

The speed and quality of wound healing depend on the general condition of the affected organism, the etiology of the wound, the condition and location of the wound, and whether or not it occurs. infection, as well as genetic factors that may or may not predispose to healing disorders.

The natural healing of a wound occurs mainly in three successive phases, each of these phases being characterized by specific cellular activities that advance the repair process according to precise chronological sequences: the inflammatory phase, the granulation phase (or phase proliferative), and the ripening phase, giving the final appearance of the scar.

The first phase, the inflammatory phase, begins with the rupture of the blood vessels which triggers the formation of a clot (blood coagulation) mainly composed of fibrin and fibronectin, and which will constitute a temporary matrix. This matrix partially fills the lesion and will allow the migration within the injured area of the inflammatory cells recruited to ensure the debridement of the wound. The platelets present are going also release factors (eg cytokines, growth factors) allowing the recruitment of cells involved in the scar process. This phase is characterized by infiltration and activation on the site of the lesion, numerous inflammatory cells (such as neutrophils), but also cells ensuring the cleaning of the wound or debridement (such as macrophages).

The second phase corresponds to the development of the granulation tissue. We first observe a colonization of the wound by proliferation of fibroblasts. Then, cell migration from healthy vessels will allow neovascularization, or angiogenesis, of the injured tissue. In the granulation tissue, the fibroblasts are activated and will differentiate into myofibers with significant contractile properties, generated by the actin microfilaments, allowing contraction of the wound. These microfilaments are expressed by a protein: smooth muscle actin. These myofibroblasts therefore play a major role in the formation and maturation of the granulation tissue that will lead to the healing of the lesion. There is then migration of keratinocytes and reconstruction of the epidermis.

This phase is initiated by a decrease in the inflammatory state of the lesion accompanied by an apoptosis of the inflammatory cells.

The third phase of the process is a mainly maturation stage aimed at reconstructing a functional tissue that is as identical as possible to the original tissue. The previously formed granulation tissue is therefore remodeled. Part of the extracellular matrix is digested with proteases (essentially matrix metalloproteases and elastases), and progressive reorganization of the extracellular matrix is observed. Gradually, type III collagen, predominant in the granulation tissue, is replaced by type I collagen, the main matrix component of the dermis. At the end of the maturation phase, the fibroblasts, myofibroblasts and vascular cells see their proliferation and / or their activity reduced. Then, the excess cells die by apoptosis. Parallel to the remodeling of the extracellular matrix and to the apoptosis of the surplus cells.

The inflammatory phase is an essential phase of healing and must be transient. In some cases, it may be prolonged by the presence of certain infectious agents such as Staphylococcus aureus or Pseudomonas aeruginosa or by a pre-existing pathology such as diabetes, an immune deficiency or even venous insufficiency. The resolution of inflammation is a critical point that conditions the initiation of tissue repair. Disturbance of this phase will result in an abnormal prolongation of the inflammatory phase and result in the chronicity of the lesion by delaying tissue repair. The resolution of inflammation is a dynamic phenomenon that involves the apoptosis of inflammatory cells, their elimination and the appearance of anti-inflammatory, chemotactic and angiogenic mediators. One way to evaluate the success of this critical period, the resolution phase, is to analyze exudates from the wound (cells, mediators and metabolites). Indeed, inflammatory chronicity results in a larger influx of inflammatory cells, an excessive production of many cytokines and inflammatory chemokines and proteases that degrade the cell matrix. Conversely, the resolution phase involves the appearance of a certain type of macrophages responsible for the phagocytosis of apoptotic inflammatory cells. These macrophages are also involved in the production of mediators favoring the granulation phase as well as the tissue repair. Analysis of the exudate will allow the physician to evaluate the inflammatory phase and its resolution, and thus allow to choose to treat the wound with a therapeutic agent adapted to promote better healing.

Traditionally, wounds are covered with a bandage or compress to prevent contamination by infectious agents in the environment, but also to keep the wound in a moist environment that promotes healing. However, this protection system does not allow to analyze the exudates and to visualize the cicatrization in time real. In addition, the exudate accumulates in the dressing and saturates the absorbent medium of the dressing. The dressing must therefore be changed regularly to prevent maceration of the wound, which implies a risk of contamination of the wound that is temporarily exposed to the open air. In addition, the dressings may also adhere, even weakly to the wound, and thus induce lesions to the wound during their removal. These new aggressions cause a continuation of the inflammation, which has the consequence of prolonging the inflammatory phase and thus substantially modifying the kinetics of healing.

Implantable chambers have been developed to study the healing of dermal wounds in laboratory animals such as mice and pigs. However, these implantable chambers are inserted partially under the skin of the animal and are held in place by stitches. This method is therefore invasive, ie it has the major disadvantage of requiring a surgical procedure for implantation of the chamber partly under the skin of the animal and may therefore cause an inflammatory reaction in the subject in question. question, by creating tissue aggression during implantation. These tissue attacks will have harmful consequences on the good progress of the healing of the animal. In addition, these implantable chambers have essentially been developed to model the healing mechanisms in animals such as, for example, a mouse or pig. Thus, with such invasive chambers, it is impossible to perform reliable visual, cellular, biochemical and molecular monitoring of wounds, especially during the first days following the implantation of these chambers.

There is therefore a real need for a non-invasive method for taking pure exudates for real-time analysis using a non-invasive device that can visualize the progression of healing without necessarily having to open the device. or remove it. Invention

A first object of the present invention is a non-invasive method of analyzing the progression of wound healing which implements a device as described in patent application No. FR 11 62295 filed December 22, 2011. said method comprising the following steps:

fix the non-invasive device around the wound;

make a macroscopic observation of the wound and / or exudate; and or

withdrawing the exudate contained in said device and analyzing said exudate.

The device used in the method according to the invention comprises in particular: a chamber having a side wall, an open bottom end and an upper end, an access opening being disposed on the upper end and / or on the side wall of said chamber, said device being intended to be in contact with the skin and to circumscribe the wound during its use via an interface.

According to a particular embodiment of the invention, said device comprises a chamber having a side wall, an open bottom end and an upper end, an access opening being disposed on the upper end and / or on the side wall of said chamber, said device further comprising a removable closure means of the chamber which fits on the access opening of the chamber and a holding element, possibly of tubular shape, circumscribed or inscribed in said chamber, open to both ends, said device being intended to be in contact with the skin and circumscribing the wound during its use via an interface which is an adhesive disposed on a skirt of flexible material, said skirt being held between the side wall of the chamber and the holding member.

The device used in the method according to the invention can in particular be considered as a passive device for collecting exudates, as opposed to an active device. By active device, means a device where the exudates are harvested by the application of an effect or external stress, physical and / or mechanical, such as a negative pressure, such as for example vacuum devices or therapy devices by negative pressure, also called TPN. Active and passive devices can also be differentiated by the quality of exudates harvested. Indeed, by introducing a mechanical and / or physical stress in the active device, the exudates harvested are of a larger volume, and thus there is a dilution of the various cells, mediators, or cellular factors contained in said exudates. In addition, with the active devices, the cells found in the exudates can be dead and / or activated. By activated cells is meant cells extracted from their natural environment found in a situation of stress and which can then lose, among other things, their functional abilities, which is, for example, the case for macrophages and neutrophils. The advantage of the device used in the method of the present invention is therefore to allow the integrity and the functionalities of the different cell populations contained in the exudates resulting from the wound to be preserved. This gives a true "picture" of the wound.

In addition, the device used in the method of the present invention is a device for direct collection of exudates at the scar site. "Direct harvesting device" means a device in which the exudates are directly taken from their source of emission, in this case in the vicinity of the wound, the exudate therefore not having to pass through a chamber. or related reservoir and / or absorbent material prior to removal of the device. Direct harvesting thus preserves the integrity and physiology of the wound and its environment, all without disrupting wound healing. Thus, the device used in the method according to the invention is centered on the macroscopic and microscopic analysis of the exudates from the wound, but also the wound itself.

The device used in the method according to the invention is devoid of any absorbing means coming into contact with the exudate and the wound. This avoids maceration of the wound due to the saturation of said absorbent means by the exudate as may be the case with a dressing. In addition, the absence of absorbent means prevents any entrapment or degradation of proteins, metabolites and cells present in the exudate, all with the objective of recovering pure exudates and without loss of cells and metabolites. According to a particular embodiment, the device used in the method according to the invention is sealed at the level of the device / skin interaction zone of the subject. Thus, the exudate collected in the chamber can not escape through the edges of said device. The sealing of said device is preferably provided by the juxtaposition of the tubular chamber and the holding member, the components between which is found inserted the skirt of flexible material. The latter conferring in addition to the entire device conformability properties of good quality. Conformability of good quality means that the skirt of flexible material fits on the surface to which it is attached, taking a suitable form. The device once fixed on a flat surface or a non-flat surface makes it possible to retain the entire volume of the exudates collected at the wound level. It is the same for the volume of a solution which is injected into said device through a septum found on the closure means or on the chamber of the device. According to a preferred embodiment, it is also possible to add a lip made of a conformable semi-rigid material, possibly with shape memory, such as in particular PMMA (polymethyl methacrylate), POM-C (polyacetal copolymer), PEKK (polyether ketone ketone), silicone, polyurethane or polyamide, on the lower part of the holding member, which lip can be useful when the surface of the skin on which the device is attached is not flat , thus making it possible to ensure even further the sealing of said device. It should be noted that whatever the type of surface, for example a flat or non-planar surface, on which the device is fixed, the sealing of said device is demonstrated. Thus, the device used in the method of the present invention combines sealing and conformability by means of the juxtaposition of the tubular chamber, the holding element and the skirt of flexible material. In the first step of implementing this method, the sampling device is positioned in such a way as to circumscribe the wound without coming into contact with the wound, so that said device is fixed on the healthy skin around the wound, for example by virtue of an interface that is adhesive. The adhesive interface may consist of an adhesive which is applied either directly to the device just before positioning it around the wound, or on a skirt of conformable compliant material, or the device or skirt may be pre-coated by a adhesive during their manufacture. In this case, the adhesive interface is advantageously protected by a protective barrier which must be removed before positioning the device around the wound.

Once the device is positioned above the wound, sufficient pressure should be exerted on the upper part of the device until the adhesive is fully set and the device is completely fixed around the wound.

During the positioning step, and in order to improve the behavior of the device around the wound, it is possible to coat the periphery of the interface of the device as well as the skin in the vicinity of the wound and close to the device by the adhesive.

In order to facilitate the positioning step, it is best to visualize the wound in order to place the device on the healthy skin around the wound. For this, one can in particular remove the closure means of the device before positioning the latter. Indeed, the sampling device used in the method according to the invention comprises a closure means which allows easy access to the wound without removing the device. Said closing means is fixed on the upper part of the chamber of the device or on the side wall of the chamber of the device, in particular by a screw thread, a clip, a pin, a crimping means and / or a bayonet system . Once the device is attached to the skin around the wound by its adhesive interface, the closing means of the device is placed back on the chamber of said device, for example by screwing it or by means of a pin inserted into adapted orifices placed on the closure means and on the chamber of the device. The visualization of the wound allowing a good positioning of the device can also be performed without opening the device, looking through the closure means of said device, in particular when the closure means is made of transparent material and / or is provided with a transparent lens, possibly magnifying.

This positioning step of the device makes it possible to fix it around the wound temporarily and non-invasively. The device must be attached to the skin sufficiently for the duration of the healing.

The second step of the method according to the invention is the macroscopic observation of the wound and / or the exudate.

By macroscopic observation, the Applicant hears any analysis performed externally, that is to say non-invasively or without intervention on the site in question, the wound and / or exudate.

According to a preferred embodiment, the macroscopic observation of the wound and / or the exudate comprises the determination of the physico-chemical parameters of said wound and / or said exudate. By physico-chemical parameters is meant, in the sense of the present invention, different sensory characteristics of the wound and / or exudate, such as in particular the appearance of the wound (color, texture, depth, area, odor) and / or the appearance of the exudate (color, odor, clarity, viscosity).

The macroscopic observation of the wound can thus consist of a visual or even olfactory follow-up of the state and progress of the healing of the wound. Thus, a simple observation of the wound can be performed using a camera, a video camera, or simply thanks to the human eye. A magnified observation can be performed using a magnifying glass, a dermoscope or in another register, via an application for "smartphone" as for example the application MOWA ("Mobile Wound Analyzer") . Finally, an observation offering even better visual resolutions can be implemented using a two-photon microscopy, a Raman scattering, a confocal microscopy, an MRI, a high-frequency ultrasound, or by capillaroscopy. This last type of technology allows a deep observation of the wound, of the order of a few hundred micrometers, which is impossible to the naked eye. It should be noted that standard monitoring means will be considered for each type of macroscopic visualization technique used. One of the preferred embodiments of the present invention resides in a photograph that can in particular be made without opening the device when it is made of transparent material and / or is provided with a transparent lens, possibly magnifying. This embodiment is particularly easy to implement. In the opposite case, the photographing is carried out after removing the closure means of the device, said closing means being returned to the closed position on the device once the photograph is taken. The frequency of taking photographs can be one photograph per day, which makes it possible to visualize the kinetics of the progression of the healing of the wound.

The different photographs are preferably taken with the same settings in order to be able to compare the photographs with each other. Thus, according to a particular embodiment, the camera is positioned at the same distance from the wound and with the same angle for each photograph, for example using a photography bench.

According to a preferred embodiment, the taking of the photograph can be followed or simultaneous with a comparison with one or more standard macroscopic tracking means. Thus, before taking the photograph, it is possible to position near the device calibration means such as in particular an object comprising graduations, and / or a color chart of well-defined hues. Once the various photographs have been taken, the calibration means makes it possible in particular to compare the photographs, even when the camera settings differ, and to make measurements of the wound on the photographs.

It should be noted that the method that is the subject of the present invention makes it possible, through the use of said non-invasive device, to observe the wound as a whole, an aspect that can not be found in methods of use. a device negative pressure therapy or vacuum creation. Indeed, these are methods in which the use of an occlusive dressing or an interface that is also occlusive is recurrent, and therefore the total visualization of the wound is impossible. The wound is best visualized only partially using these methods.

According to a preferred embodiment, biological analyzes are performed on the exudate to characterize and / or quantify the different cell populations contained. It is also possible to determine the presence but also to quantify the metabolites and / or microorganisms contained in said exudate.

In order to carry out these analyzes for monitoring the progress of the wound using the method according to the invention, the exudate contained in the device can be taken and said exudate can be analyzed. The analysis may in particular be a more specific evaluation of the physicochemical parameters of the exudate taken with respect to the same parameters observed without removal of the exudate. Thus it will be possible to determine more precisely the volume of exudate produced by the wound, but also all the parameters relating to the eigenvalues of said exudate (pH, T ° C, color, odor, clarity, viscosity).

Said removal of the exudate may in particular be carried out by means of a pipette, provided with a suction means such as a cone, and / or a syringe, either after opening of the closure means of the device, or through a septum positioned on the side wall of the device chamber or on the closure means of the device. Preferably, the removal of the exudate is carried out through a septum. The removal of the exudate through the septum makes it possible to preserve the sterility of the internal medium of the device vis-à-vis the external environment of said device. According to a preferred embodiment, the exudate is not absorbed by an absorbent material prior to sampling. According to another preferred embodiment, the exudate is not taken by a device applying a negative pressure to the wound, such as a vacuum pump.

When the exudate contained in the device is too viscous to be easily removed by syringe, it can be advantageously make the exudate less viscous by injecting into the device a physiological solution, such as for example physiological saline or a dilute solution of sodium chloride. According to a preferred embodiment, it is possible to aspirate and reinject several times the diluted exudate solution with physiological solution in order to wash the wound site and recover the maximum number of cells, metabolites and / or microorganisms in order to facilitate subsequent analyzes.

By metabolite, the Applicant hears all organic compounds likely to be found in a wound, such as for example, proteins (inflammatory or anti-inflammatory proteins, growth factors, extracellular matrix proteins such as collagen or fibrin). lipids (such as fatty acids, leukotrienes), amino acids, hormones, albumin, urea.

By microorganisms, the applicant hears all the microorganisms likely to be found on a wound, infected or not, such as, for example, bacteria (such as those composed of the genus Staphylococcus, for example, S.aureus, S. epidermidis, S.pyogenes, by the genus Enterococcus, for example E. faecalis, by the genus Pseudomonas, for example P. aeruginosa, by the Enterobacteriaceae family, of the E.coli type, Enterobacter sp., Proteus sp., Serratia sp., or by anaerobic bacteria such as Clostrium sp. or Bacteroides sp.), yeasts (Candida albicans), viruses (retroviruses, adenovirus), or parasites (ectoparasites, endoparasites, mesoparasites),

By cells, the Applicant hears all the cells likely to be found on a wound, such as in particular inflammatory cells (granulocytes), fibroblasts, or even keratinocytes.

The removed exudate is then transferred to a suitable container, such as in particular a test tube or a septum-filled bottle. According to a particular embodiment, when the removal of the exudate in the device is carried out through a septum and the exudate is transferred to a septum-filled flask, the exudate is not in contact with the open air which prevents its contamination by external pathogens.

If the closure means has been removed to take the exudate sample, said closure means is returned to the closed position on the device after sampling.

The biological analyzes that can be performed on the exudate are multiple and varied. Thus, from the first day of the implementation of the method according to the invention, it is possible to carry out a phenotypic and functional monitoring of cell populations, such as, in particular, inflammatory cells or cutaneous cells. These analyzes, carried out in particular using flow cytometry, make it possible to follow the various stages of the cicatrization, particularly the inflammatory phase, although the follow-up of the second phase, called the granulation or epidermization phase (called also proliferation phase) can also be realized. These analyzes make it possible to verify that the granulation phase begins and that the inflammatory phase does not persist over time, which could hinder good healing. Thus, if flow cytometry is followed by the number of granulocytes present in the exudate, it will be seen at the very beginning of the inflammatory phase that the granulocytes are the majority. If the inflammatory phase proceeds correctly, a few days after the onset of the wound, the number of granulocytes will have greatly decreased, along with an increase in the number of monocytes and fibroblasts that allow the formation of granulation tissue. If it is found that the granulocytes are still very numerous three days after the onset of the wound, it is possible that the wound has a defect in the phase of resolution of the inflammation that can find its cause in a bacterial infection for example . If the type of bacteria is identified by the appropriate analysis, the doctor can then prescribe an antibiotic treatment, which will be advantageously injected into the device in order to be in contact with the wound and eliminate the infection before complications. 'appear.

Exudate analyzes can also be used to track mediators produced during the scarring, such as inflammatory cytokines (TNF-α, IL12, IL1-β, IL6, etc.) and anti-inflammatory cytokines (TGF-β and IL-10), lipid mediators such as leukotrienes, prostaglandins or even growth factors (VEGF, PDGF). These mediators can in particular be quantified by an ELISA assay. The analysis of metabolites, and more particularly of mediators contained in the exudate also makes it possible to follow the progress of the cicatrization. Thus, for example, if the exudate contains an abnormally high level of inflammatory cytokine content, this indicates that the inflammatory phase is not complete which may hinder the good healing of the wound. Indeed, the prolonged presence of inflammatory cytokines in the wound can cause degradation of the cell matrix and induce chronicity of the wound.

The method according to the invention may also comprise an additional step of introducing an aqueous solution or not into the device. These solutions may in particular make it possible to perform a washing of the wound, for example a physiological saline-type standard solution, sodium chloride, sterile water, soapy water or a solution of Ringer's lactate. It is also possible to envisage the introduction of a solution comprising an antibacterial agent and / or a therapeutic agent in order to treat the wound, in which case the bringing into contact of these solutions on the scar site will be prolonged, in comparison with the contacting a wash solution at the scar site without using a device. Thus, if it is desired to clean the wound, a conventional physiological solution may be brought into contact with the wound, for example after the step of positioning the device. The solution is allowed to act for a few minutes and removed from the device. In another way, if it is desired to treat the wound, a solution containing a therapeutically active agent may be brought into contact with the wound, for example every day after the macroscopic wound observation step and / or exudate or possibly after the exudate analysis step. The solution is left to act for several minutes to hours or days and then removed from the device if and only if the solution has not been fully absorbed into the wound. According to another embodiment of the present invention, the method comprises an additional step in which a solution comprising therapeutically active agents is brought into contact with the wound using said device. Thus, the device used in the method makes it possible to test new assets, in particular healing or antimicrobial active agents, directly in contact with the wound. Thus, it will be possible to use various galenic forms of assets, such as in particular asset solutions allowing a more localized action on the lesion, and several concentrations of active ingredients. This will test the effectiveness of certain assets and test the tolerance of the wound in relation to the concentration of each asset. The major advantage obviously resides in the possibility of testing a particular and atypical dosage form on the scar site, that is to say not possible in normal time without the aid of said device of the applicant, such as a solution liquid of active agents. In general, the assets are chosen from:

antibacterials such as polymyxin B, penicillins, clavulanic acid, tetracyclines, minocycline, chlorotetracycline, aminoglycosides, amikacin, gentamicin, neomycin, silver and its salts (sulfadi azi silver).

antiseptics such as sodium mercurothiolate, eosin, chlorhexidine, phenylmercury borate, hydrogen peroxide, Dakin liquor, triclosan, biguanide, hexamidine, thymol, lugol, povidone iodine, merbromine, benzalkonium and benzethonium chloride, ethanol and isopropanol.

antivirals such as acyclovir, famciclovir, ritonavir. antifungals such as polyenes, nystatin,

1 amphotericin B, natamycin, imidazoles (miconazole, ketoconazole, clotrimazole, econazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertoconazole, sulconazole, thiabendazole, tioconazole), triazoles, allylamines, terbinafine, 1 amorolfine, naftifine, butenafine. pain-relievers such as paracetamol, codeine, dextropropoxyphene, tramadol, morphine and its derivatives, corticosteroids and its derivatives.

the cicatrizants: KSOS, sucralfate, allantoin, collagen or hyaluronic acid.

Another object of the invention is a method for analyzing wound healing in an animal comprising the following steps:

create a wound;

- fix the sampling device around the wound;

make a macroscopic observation of the wound and / or exudate; and or

withdrawing the exudate contained in said device and analyzing said exudate.

The present method, as well as the method previously described above, are carried out on the animal. By animal is meant, in the sense of the present invention, an animal or the human being. The animal in which it is desired to study the progression of cicatrization may especially be a mouse, a pig and / or a rat. It may also be the human being, in particular people suffering from a metabolic disorder or pathology (such as diabetes, immunodeficiency, malnutrition, or venous insufficiency) which influences the phenomenon of healing.

In the case of murine, before creating the wound, said animal must be prepared for the operation. Thus, it proceeds first to the local or general anesthesia of the animal. In particular, anesthesia can be performed by injecting an anesthetic solution that can be a mixture of ketamine and xylazine. After anesthesia of the animal, we wait for the time necessary for the anesthesia to take effect. If necessary, the animal is then mowed on the area where it is desired to create the wound. This step is a step that can be overcome in the case of the human being. Nevertheless, depending on the case, it can be adapted, and this by means of local anesthesia of the wound creation zone. We then proceed to the creation of a wound on the animal. In particular, the wound can be superficial or deep, that is to say it goes as far as the hypodermis or to the muscle tissue.

Whether superficial or deep, the literature already describes several ways to achieve a wound. The wounds can be of different natures according to the fields of study which are privileged.

In the method according to the invention, the wound is created mechanically, chemically, thermally and / or by radiation. The wound can be created mechanically using tools such as scissors, a cookie cutter or "biopsy punch". In this case, we can trace the delineation of the future skin lesion, using for example a marker and a pattern. The skin of the animal can then be pinched to be cut with a chisel or with a scalpel, following the line delimiting the cutaneous lesion or the skin can be stretched to exert a pressure on the skin. using a cookie cutter or "biopsy punch". Superficial wounds can also be created mechanically. In particular, a "stripping" can be done by affixing an adhesive and then removing it from the future area of creation of the wound. By repeating the operation several times, it is thus possible to create a more or less superficial lesion on the surface of the skin. In a similar way, it is also possible to carry out a prolonged friction on the epidermis with the aid of tools such as a rasp. Other means of lesion creation can also be considered such as in particular chemicals. A caustic solution (sodium hydroxide, nitric acid ...) can be deposited on the part of the skin to be damaged. The concentration of the agent used and the duration of exposure will determine the depth of the wounds. This type of injury is similar to burns (hot and / or cold, electrical, radiation ...) that can also be reproduced. Thus, just like the use of chemicals, the depth and extent of the burn will depend essentially on the duration and physicochemical properties of the tools or solutions used. In particular, depression systems may be employed to produce detachments (suction bubbles) or phlyctenes. Crushing procedures can also be considered skin, but also ligation or vascular section procedures, to obtain ischemic wounds. It will also be possible to create a wound with a device emitting radiation, such as LASER or β or gamma ionizing rays.

The sampling device is then attached to the healthy skin around the wound by means of its adhesive interface as described above.

Macroscopic observation of the wound is preferentially performed by taking photographs of the wound as previously described. The biological monitoring of the wound by sampling the exudate contained in the device and its analysis is performed as described above. Thus, it is possible in particular to carry out the phenotypic and functional monitoring of various cell populations, such as, in particular, granulocytes, lymphocytes, monocytes, fibroblasts, and / or to carry out the monitoring of metabolites contained in the exudate such as that especially cytokines, lipid mediators, growth factors, or even the monitoring of microorganisms, as described above.

In addition, it may be interesting to study the influence of various pathological or physiological factors on the healing of the wound on the animal. Thus, one can compare the healing of an animal with a particular physiological state compared to that of a healthy animal. In addition, since the method according to the invention implements a non-invasive device, it is possible to reliably monitor the mediators and metabolites produced as soon as the wound is created in the animal, which was not possible with invasive devices implanted under the skin of the prior art.

The method according to the invention may also comprise an additional step of washing the wound with a physiological solution comprising an antibacterial agent and / or a therapeutic agent, as described above. Thus, if it is desired to clean the wound, a solution comprising an antibacterial agent may be placed in contact with the wound, for example after the step of positioning the device. The solution is allowed to act for a few minutes and removed from the device. On the contrary, if we wish to treat the wound, a solution containing a therapeutically active agent may be placed in contact with the wound, for example several minutes or even hours or days and this every day after the photographing step and / or after the stage of analysis of the exudate. The solution is allowed to act on the site and removed from the device if and only if it has not been fully absorbed into the wound. This additional step can in particular make it possible to study the influence and the effectiveness of a therapeutically active agent on the healing of the wound of the animal.

It will thus be possible, for example, to apply the method according to the invention to two groups of animals, an animal control group which is not treated with a therapeutically active agent and a test group of animals which receives treatment with a therapeutically active agent every day. By comparing the photographs taken between the two groups of animals, and / or by comparing the biological analyzes of the exudates, it will be possible to check whether the therapeutically active agent improves wound healing and to adapt the dosage or the frequency of administration of the treatment. .

The invention will be better understood in the light of the following figures which are only illustrative and can not in any way limit the invention.

Figure 1 is a cross sectional view of a device for implementing the invention.

Figure 2 is a photograph of a mouse on which a dorsal wound has been created.

Figure 3 is a photograph of the mouse of Figure 2 on which the chamber of a sampling device was placed on the healthy skin around the wound by an adhesive.

Figure 4 is a photograph of the mouse of Figure 3 on which the closure means was fixed to the device chamber by means of a pin.

Figure 5 shows different photographs of a wound on a healthy mouse, the photographs being taken at different time intervals from the day of wound creation (D0) to 14 days after wound creation (D14).

FIG. 6 represents the different results of the flow cytometric analysis of the cell populations contained in the exudate resulting from a wound on a healthy mouse, the analyzes being carried out at different time intervals starting from the first day after the creation. from the wound (J1) until the 7th day after the creation of the wound (J7).

Figure 7 is a graph showing the amount of an anti-inflammatory cytokine (TGF-β), measured by ELISA, contained in exudates from dorsal wounds in healthy mice.

The invention will be described in more detail with the aid of the following examples which are given for purely illustrative and non-limiting purposes.

Examples:

Example 1: Creation of a wound and placement of the device on the back wound of the mouse.

In order to create a wound on a mouse as photographed in FIG. 2, the mouse is first anesthetized by injecting a mixture of ketamine and xylazine intraperitoneally. Then, cut the skin of the mouse with a chisel, on its dorsal part.

The device (1) of FIG. 1 is placed on the mouse. The device (1) consists of a tubular chamber (2) comprising a lateral wall (3), and two extremities, lower (4) and upper ( 5), open. Said lower end (4) of the tubular chamber (2) has an outer collar (6) of flexible material. The interface (7) of the device (1) consists of a thin layer of surgical glue applied to the lower surface of the collar (6) of the tubular chamber (2). A closing means (8) of the device (1) is fixed to the upper part of the tubular chamber (2) by means of a pin (9).

The closure means is removed from the device by unhooking it. The pin (9) is removed in order to remove the means of closure (8) of the device (1). The lower surface of the flange (6) of the tubular chamber (2) is coated with a thin layer of surgical glue to form the interface (7) of the device. The device (1) is positioned above the wound so as to circumscribe it. Sufficient pressure is applied on the upper part of the device (1) until the adhesive is completely taken on the skin of the mouse according to FIG. 2. The outer surface and the periphery of the collar (6) are coated. device (1) as well as the skin near the surgical glue device until the adhesive is fully set as shown in FIG. 3. The closure means (8) are placed on the device by inserting a pin (9). ) and inserting it into orifices placed on the chamber (2) of the device (1) and on the closing means (8) in order to hold these two elements together. The device (1) is then fixed on the back of the mouse as photographed in FIG.

EXAMPLE 2 Photographic follow-up of the progress of wound healing of a dorsal wound in mice.

The analysis of the progression of the cicatrization of a wound is carried out in a healthy mouse on which a wound has been created according to example 1 and positioned a device (1) still according to example 1. The mouse is anesthetized with a gas and arranged on a photography bench with regular graduations to perform a calibration. The pin (9) is removed in order to remove the closure means (8) from the device (1). We take photography with a conventional camera, which can be found on the market. The closure means (8) is replaced on the device by heating a pin (9) and inserting it into orifices placed on the chamber (2) of the device (1) and on the closing means (8) in order to maintain these two elements together. One photograph per day is taken, from the day of the creation of the wound and for 14 days. The different photographs taken are presented in Figure 4. As seen in the photographs, from OJ to D5, corresponding to the inflammatory phase and the start of the proliferative phase, the size of the wound does not vary, but attend training a granulation tissue gradually covering the muscular fascia exposed during the excision of the cutaneous tissue. From day 5 to day 14 the wound size decreases thanks to the development of the granulation tissue containing the myofibroblasts allowing the contraction of the wound. At day 14, the wound is almost closed.

Example 3: Cellular monitoring of the advanced healing of a dorsal wound in a healthy mouse.

The analysis of the progression of the healing of a wound is carried out on a healthy mouse on which a wound has been created according to example 1 and positioned a sampling device according to example 1. Before taking the exudate , the mouse is anesthetized with a gas. A physiological solution is injected into the syringe through a septum placed on the closure means of the device. The exudate diluted with physiological saline is aspirated and reinjected three times with the syringe so as to thoroughly wash the site of the wound and recover the maximum number of cells for analysis.

The diluted exudate is pipetted and transferred to a septum vial.

In order to determine the infiltration kinetics of the granulocytes, characterized by the expression of the Ly6G and 7/4 antigens on their surface, the cells are incubated for 15 minutes with Ly6g and 7/4 anti-mouse antibodies each coupled to fluorochromes. . Finally the cells of the exudate are washed again. The cell populations of the exudate are analyzed by flow cytometry. The data is acquired by a FACScalibur (BD Biosciences) cytometer using CellQuest Pro software.

Exudate sampling and flow cytometry analysis were performed 24 hours after wound creation and repeated daily for one week. The results, showing the different fluorescence intensities for each antibody, are grouped together in FIG. 6. At post-lesion, an exudate composed essentially of granulocytes, represented by a strong Ly6G and 7/4 marking, is observed. From D2 post-lesion, a gradual decrease in Ly6G labeling is observed until its total disappearance, while the 7/4 labeling is preserved. This analysis of The cell populations composing the exudate can be used to monitor the progression of the inflammatory phase, characterized by the infiltration of the granulocytes, then by their disappearance at the start of the proliferative phase. EXAMPLE 4 Comparison of the number of anti-inflammatory cytokines contained in the exudate resulting from a dorsal wound of a healthy mouse

The progress of wound healing is monitored on a group of 2 healthy mice. On each of the two mice, a wound was created according to Example 1 and a sampling device was always positioned according to Example 1. Before taking the exudate, each mouse was anesthetized with a gas. A physiological solution of known volume is injected into the syringe through a septum placed on the chamber of the device. The exudate diluted with physiological saline is aspirated and reinjected three times with the syringe so as to wash the site of the wound well, to homogenize the exudate. The diluted exudate is taken by syringe and transferred to a septum vial and centrifuged to recover only the supernatant containing the cytokines. Inflammatory and anti-inflammatory cytokines contained in the exudate are analyzed by ELISA and are performed 48 hours after the creation of the wound and repeated daily for one week. The cytokine TGF-β assay is shown in FIG. 6. A strong progression of the level of anti-inflammatory cytokines of the TGF-β type is observed as early as D2 post-lesion. This overproduction of this anti-inflammatory cytokine reaches its peak at 5 days after the end of the inflammatory phase, before returning to a normal stage at the end of the healing process.

Claims

A non-invasive method of analyzing the progress of wound healing, comprising the steps of:

attach a non-invasive collection device around the wound;

make a macroscopic observation of the wound and / or exudate and / or

withdrawing the exudate contained in said device and analyzing said exudate.

2. Method according to claim 1 characterized in that the device comprises a chamber having a side wall, an open bottom end and an upper end, an access opening being disposed on the upper end and / or the side wall of said chamber, said device further comprising a removable closure means of the chamber which fits on the access opening of the chamber and a holding element, possibly of tubular shape, circumscribed or inscribed in said chamber, open to both ends, said device being intended to be in contact with the skin and circumscribing the wound during its use via an interface which is an adhesive disposed on a skirt of flexible material, said skirt being held between the side wall of the chamber and the holding member.

3. Method according to claim 1 or 2, characterized in that the macroscopic observation of the wound and / or exudate comprises the determination of physico-chemical parameters of said wound or said exudate.

4. Method according to any one of claims 1 to 3, characterized in that the macroscopic observation is followed or simultaneous to a comparison with one or more standard macroscopic monitoring means.

5. Method according to any one of claims 1 to 4, characterized in that the exudate is taken through a septum positioned on the sampling device.

6. Method according to any one of claims 1 to 5, characterized in that the analysis of the exudate makes it possible to determine the metabolites, the cellular components, and / or the microorganisms contained in said exudate and comprises the determination of the parameters physico-chemical said exudate.

7. Method according to any one of claims 1 to 6, characterized in that it comprises a step of washing the wound.

8. Method according to any one of claims 1 to 7, characterized in that it comprises an additional step in which a washing solution or a solution comprising one or more therapeutically active agents is brought into contact with the wound to the wound. using said device.