Method and installation for treating tissue of human or animal origin, using dynamic circulation of supercritical fluid with additives

Abstract

Collagen-based tissue matrices (2) are treated in a reactor (1) with a supercritical carbon dioxide flow, including at least one chemical additive introduced via an introduction device (P2, V12) connected to a loop (20). The additive is injected into the liquid or supercritical carbon dioxide while the reactor is already pressurized. The process combines, in a dynamic treatment flow circulating in the loop (20) associated with the reactor, supercritical CO2 as a solvent and a chemical additive as a co-solvent to enhance the action on the treated tissue. The circulation of the additive, injected progressively and then recirculated with the CO2, forms part of a first treatment cycle. Several cycles, each with a different additive, can be carried out successively, separated by a step of separating the additive from the CO2, performed with or without maintaining pressurization and CO2 circulation. Abstract figure: Figure 1

Description

Title of the invention: Method and installation for treating tissue of human or animal origin, using dynamic circulation of an additive supercritical fluid. Technical field

[0001] This disclosure relates to the field of tissue processing, for example, tissues implanted during surgical procedures. More specifically, it concerns a process for cleaning, purifying, and / or decontaminating tissue of human or animal origin, using dynamic circulation of an additive-enhanced supercritical fluid. This process utilizes a supercritical fluid such as carbon dioxide combined with at least one reactive additive to help purify the tissue matrix. An installation equipped with a reactor and a circulation loop for the additive-enhanced supercritical fluid for implementing this process is also disclosed. Technological background

[0002] The use of supercritical carbon dioxide (CO2) as a decontaminant in the treatment of collagen-based tissue (i.e., bone, tendon, cartilage, or ligament tissue) is known from document FR 2735372 (or the equivalent US patent 5723012). This fluid in its supercritical state has excellent penetration capabilities by diffusion through the porous material of the tissue and helps to inactivate potentially present pathogens.

[0003] Treatments with chemically active agents and rinsing are carried out to obtain a product free of contaminants, impurities, or organic residues. In practice, the fabric can be immersed in the liquid solution containing the active agents. To be effective throughout the entire matrix, some of these agents, which are corrosive at atmospheric pressure, are used at sometimes high concentrations depending on the chemical treatment. This can result in the degradation of the matrix's mechanical and biological properties. More generally, it remains difficult to optimize the compromise between the effectiveness of the chemical treatment for decontamination and the effectiveness of removing undesirable residues at the end of the cleaning operations.

[0004] There is still room for improvement in obtaining a purified and decontaminated tissue or tissue matrix (for example, free of organic impurities and possible pathogens) and retaining good mechanical and biological properties. Summary

[0005] The present disclosure improves the situation, in particular by seeking to optimize the use of purifying and decontaminating products.

[0006] To this end, a process is proposed for treating tissue of human or animal origin by extracting organic matter residues, in order to purify and / or decontaminate said tissue, the process using carbon dioxide in a supercritical state and a reactor equipped with an inlet and an outlet, the process comprising the steps essentially consisting of: - place the fabric in an internal volume of the reactor; - pressurize the reactor, by introducing supercritical carbon dioxide into the internal volume via the inlet, so that the supercritical carbon dioxide reaches a region of contact with the tissue (typically at a determined pressure and temperature) and exits the reactor via the outlet; - maintain a flow of carbon dioxide in a supercritical state passing through the reactor, preferably by opening the reactor outlet using a valve regulating the pressure of the reactor's internal volume to a desired value; - add, for example upstream of the reactor, at least one chemical additive for purification and / or decontamination in the flow of carbon dioxide, liquid or supercritical, put into circulation to reach the contact region, in order to achieve in the internal volume a circulation of a treatment flow combining carbon dioxide in the supercritical state as an extraction solvent and at least one chemical additive as an extraction co-solvent, the concentration of the co-solvent being able to be determined according to its solubility in carbon dioxide in the supercritical state; - while maintaining pressurization, circulate the treatment flow, away from the contact area, in a loop that communicates with the reactor inlet, thus creating a recirculation that helps to make the treatment flow dynamic, allowing the co-solvent to penetrate the tissue matrix (therefore helping to purify and / or decontaminate the tissue) and, preferably, allowing the extraction and separation of some of the residues in the loop (such separation being feasible for example after the treatment phase with the additive).

[0007] The process may include one or more treatment cycles, each using a cosolvent. After each treatment cycle, at least one of the following measures may be taken: - the separation stage at the reactor outlet can be activated to allow purging of the chemical purification / decontamination additive and part of the residues from the internal volume, which are liquid and / or solid, with carbon dioxide recirculation maintained in the circuit. - after depressurization of the reactor, purging of the additive and residues remaining in the internal volume can be activated. - the reactor and the loop can be depressurized and compressed air can be injected into the reactor and the loop in order to evacuate, through an outlet placed downstream of the reactor, the chemical additive for purification / decontamination and part of the residues remaining in the internal volume, which are liquid and / or solid. - A rinse, for example with purified water, can be carried out in the same direction or in a different direction than the flow of the treatment stream in the reactor. This can facilitate the removal of any residues that may remain in the reactor's internal volume. The evacuation can be carried out via a separate channel from the inlet and outlet used for the supercritical carbon dioxide stream, or positioned at a bifurcation before the reactor inlet or after the outlet.

[0008] The process thus uses a residue extraction reactor, inside which a dynamic treatment flow circulates, joining / passing through a loop. This loop is a reactor maintained at a pressure and temperature that are respectively higher than the critical pressure and temperature of carbon dioxide, for example, above 73 bar and 31°C (the reactor is thus pressurized; pressures ranging from 100 to 400 bar can be used in certain configurations). The loop can contribute to the cleaning process by retaining, preferably by filtration, residues extracted from the matrix and preventing their recirculation. An advantage of this process is the combined effect of penetration into the tissue matrix, thanks to supercritical CO2, and chemical treatment with a co-solvent concentration that can be low / reduced, resulting in less degradation of the mechanical and biological properties of the tissue matrix.

[0009] In the process thus implemented, a cleaning / removal action is achieved, with some of the residue being flushed away with the dynamic treatment flow. The use of a loop and a dynamic flow tends to limit the accumulation of reagents or residues in the contact area, since with cycling, the volume of co-solvent introduced can be optimized (the total volume of co-solvent injected can be minimized). In some options, this volume is adjusted according to the amount of tissue matrix to be treated, the reactor volume, and the loop volume.

[0010] In advantageous configurations, a series of treatments, with a first additive followed by a second additive (each acting as a co-solvent of supercritical CO2), can be carried out without draining the reactor in which the treatment takes place. A separation stage accessible via a bypass in the loop can facilitate the removal / separation of the first additive. In other words, it is possible to avoid, in certain cases, a depressurization (during an intermediate phase, between two treatment cycles) that can be detrimental to tissue integrity.

[0011] In the process thus implemented, it is possible to control the effective concentration of reagent in the region of contact with the tissue matrix by adding additive directly into the supercritical carbon dioxide circulating to the reactor, for example by using an injection rate of the co-solvent proportional to the flow rate of supercritical carbon dioxide.

[0012] The process differs from solutions based on static treatment cycles, where the material is purified / decontaminated by immersion in a pure or diluted reagent bath. With a comparable / identical exposure time, it is possible to achieve cleaning efficiency with a much lower concentration and quantity of active product by using recirculation.

[0013] The process differs from solutions based on static treatment cycles, with material decontamination achieved by impregnation in supercritical carbon dioxide with an added co-solvent. With a comparable / identical exposure time, it is possible to use a larger quantity of co-solvent while maintaining the same co-solvent concentration at the fabric contact point. By also allowing the extraction of residues through the dynamic flow of supercritical carbon dioxide, superior purification / decontamination efficiency is achieved.

[0014] In some options, the additive is added to supercritical carbon dioxide at a volumetric flow rate ratio of 1 / 50 or at least a ratio between 1 / 5 and 1 / 100 (for example, between 1 / 10 and 1 / 100), which can be useful to avoid oversaturating the carbon dioxide with the co-solvent. The concentration of the chemical additive can be very low, compared to more conventional treatments, for good results. This promotes the preservation of the integrity of the tissue matrix, ensuring the preservation of biomechanical and biological properties. Thus, the process can facilitate adjusting the supply of the chemical decontamination additive as closely as possible to the need, for example, by avoiding exceeding a threshold concentration of the chemically active additive in the supercritical CO2.

[0015] In some embodiments, the process uses a reactor and a set of valves, as well as a filter and / or a separation stage, to separate residues, for example by including a solids separation filter and / or at least one collector. The use of a filter can allow the separation of solid residues that could hinder the maintenance of closed-loop circulation by blocking a pressure regulating valve. Such a filter, which prevents / limits the circulation of solid residues further downstream in the loop, can prevent the reintroduction of these residues into the contact region. The separation stage can be located in a part of the loop in which carbon dioxide circulates in gaseous or liquid form. The same applies to the filter. Unlike the filter, the separation stage allows the additive and the Carbon dioxide. In some options, the filter and separation stage are distributed on either side of a control valve (regulating the pressure in the reactor and located downstream of the reactor outlet). In this case, the separation stage involved in the collection / separation of the co-solvent can be placed downstream of the control valve and used only after a treatment cycle using a chemical additive (co-solvent).

[0016] According to a particular feature of the process, the carbon dioxide circulating in the loop successively passes into a gaseous state, downstream of a control valve (which may constitute the overflow, downstream of the reactor outlet), and then into a liquid state, by undergoing a cooling stage in a condenser. Thanks to pumping by a circulation pump located in the loop after the condenser (and upstream of the reactor inlet), it is possible to delimit a section of the loop in which the carbon dioxide circulates from the pump to the control valve (passing through the reactor), in a supercritical state.

[0017] The control valve, mounted on the loop at or shortly after / downstream of the reactor outlet, may consist of a back pressure regulator (BPR) that limits the gas flow exiting the reactor in order to maintain the desired pressurization level in the reactor. The back pressure regulator can be used both to circulate carbon dioxide as the sole solvent initially and then to circulate the treatment flow combining this solvent with a co-solvent.

[0018] The spillway may be located between the reactor outlet and a separation stage. A high-pressure pump is mounted in the loop, between the separation stage and the reactor inlet, and may be used to circulate carbon dioxide through the loop and compress it to pressurize the reactor to a given pressure. An additional high-pressure pump is mounted in parallel on the loop or on the reactor and may be used to introduce a chemical additive into the circuit at a predetermined flow rate and pressure.

[0019] At the outlet of the carbon dioxide circulation pump, the carbon dioxide can be brought to a supercritical state in a heat exchanger to raise the temperature of the recirculated stream (via the loop) re-entering the reactor. This achieves reactor pressurization exceeding a certain pressure threshold, for example, above the critical pressure of carbon dioxide, with the pressurization level possibly exceeding 100 bar, preferably between 140 and 180 bar, for example approximately 160 bar. The heat exchanger then raises the carbon dioxide stream to a certain temperature, for example above the critical temperature of carbon dioxide, preferably between 35 and 50 °C, for example 40 °C.

[0020] The reactor admits the fluid in a supercritical state, here CO2, in phases of dynamic circulation, as opposed to static impregnation. This allows not only penetration of the supercritical fluid into the entire tissue matrix, for example bone or cartilage tissue, a tendon, or a membrane, but also replenishment of the additive introduced as a co-solvent in the contact area. The outlet is optionally axially opposite the inlet when the reactor has an extensional direction or elongation axis, for example, a vertical or horizontal direction.

[0021] The temperature of the reactor, heated and above 31°C, can be maintained below a threshold, typically around 50°C, for example with an internal volume heated between 33 and 47°C, optionally between 37 and 43°C.

[0022] In embodiments of the process, one or more of the following provisions may be used: - Supercritical CO2 treatment with recirculation can start in the absence of chemical additives (and thus without any chemical additives present in the reactor, at least for the first treatment stage with recirculation). - A step to estimate a parameter representative of the total quantity of additive injected into the loop is carried out as a function of the total mass of the tissue to be treated present in the reactor, independently of the reactor's capacity and / or independently of the location / distribution of the tissue within the reactor's internal volume. - a flow rate of a co-solvent pump is set according to the result / parameter estimated in the estimation step and taking into account an additive concentration in a co-solvent solution containing said additive conveyed in the loop via the co-solvent pump. - The additive is added gradually, for example by taking into account the flow rate of the pump ensuring the circulation of supercritical CO2 in the loop, in order to allow its dissolution and distribution in the reactor and the loop. This can be achieved, for example, by choosing a total injection time that is greater than the transit time (residence time) in the reactor, and optionally also greater than the total time taken by the fluid to complete a full circuit (passage through the reactor and then the loop until returning to the reactor inlet). This time can be a characteristic parameter, calculated using a tracer. - the loop extends outside the reactor, between the reactor outlet and inlet. - at least one chemical purification / decontamination additive may be introduced into the loop upstream or downstream of a heat exchanger that heats the carbon dioxide stream from the pump provided in the loop before it enters the reactor. - the introduction of chemical additive is carried out between the outlet of the condenser / cooling means used to liquefy carbon dioxide and the inlet of the reactor, for example in a section where carbon dioxide is in liquid or supercritical state, whether or not there has been pressurization by the pump provided in the loop or heating by the exchanger. - each additive among the at least one additive is added by an additional pump separate from the pump provided in the loop, possibly using a fluid connection device connected to the loop upstream of the pump. - at least one of the following ratios is predetermined: a first ratio between the total volume of additive delivered by the additional pump and the mass of the tissue to be treated, the first ratio expressed in ml / g preferably being between 0.01 and 0.1 or 1, typically between 0.1 and 5, for example between 1 and 3; a second ratio between the volumetric flow rate delivered by the additional pump and the volumetric flow rate delivered by the pump provided in the loop, the second ratio preferably being between 1 / 5 or 1 / 10 and 1 / 100, typically between 1 / 30 and 1 / 70, for example between 1 / 45 and 1 / 55. - each of the at least one additive is preferably introduced in liquid form.

[0023] The process allows easy control of the injection conditions of the additive, which allows control of the respective concentration of carbon dioxide and cosolvent with respect to the tissue matrix to be treated.

[0024] In preferred embodiments, the supercritical carbon dioxide flow rate is between 1 and 100 L / h, for example, approximately 100 ml / min for a reactor with an internal volume of 1 liter. For a carbon dioxide flow rate of 100 ml / min, the co-solvent pump flow rate is, for example, between 0.1 and 30 ml / min, possibly between 1 and 10 ml / min depending on the additive.

[0025] The implementation of circulation in the loop, during the process, in order to make the processing flow dynamic may involve one and / or the other of the following arrangements: - a solvent and co-solvent circulation step is carried out in a closed circuit, in the circuit composed of the reactor and the loop, for example by using / opening valves allowing the reactor to remain pressurized and which allow recirculation in the loop. - the treatment flow can initially circulate without co-solvent in the reactor and in the loop, before an additive introduction step in the closed circuit, for example in the loop or in the reactor. - the reactor is functionally coupled, at the inlet, to a valve for the reintroduction, into the internal volume, of the flow recirculated via the loop. - at least one additive, for example a diluted or undiluted additive from a reservoir, is introduced into a section of the loop in which carbon dioxide flows in a supercritical state towards the reactor inlet. - the use of a spillway can make it possible to obtain a very large pressure difference between the reactor (high pressure) and the part of the loop located upstream of the carbon dioxide pump (which is a high pressure pump). - during addition, each chemical purification / decontamination additive is added into an injection line which joins, for example via a fluidic connection fitting, a communication channel in which carbon dioxide flows, so that each chemical additive flows as a co-solvent of the carbon dioxide in the supercritical state when it flows through the reactor to cross the tissue contact region. - the injection time for a given additive, several minutes, is optionally less than a period of circulation (dynamic circulation) of the closed-loop treatment flow without new additive input. - the communication route is part of the loop (loop segment). - the process includes: before said addition, a stabilization phase in which carbon dioxide circulates in a loop, in the circuit formed by the reactor and the loop, while the pressure in the reactor has reached or exceeded a predefined threshold (for example 73 bars) and the temperature has reached a target temperature range (for example above 31°C), so as to maintain the carbon dioxide in the supercritical state; and during said addition, maintaining the loop circulation of carbon dioxide in this circuit.

[0026] According to one particular feature, the process comprises carrying out, in the pressurized state of the reactor, at least one or two chemical treatments (in the contact area, to purify the tissue by extracting residues and to decontaminate by inactivating pathogens), each of which is carried out in a reactor operating mode with said recirculation, by using the loop. It is permissible to circulate the additive in the reactor, from the inlet to the outlet, and then through the rest of the circuit (the loop being connected to the inlet and outlet of the reactor) for a return to the reactor.

[0027] During each of these treatments which occur with this mode of operation, a chemical purification / decontamination additive (specific to each of said chemical treatments) may be successively: - introduced, by using a co-solvent pump (and for example in an open state of an additive introduction valve connected to the loop), into a section of the loop in which carbon dioxide circulates in the liquid or supercritical state (typically in the liquid state then in the supercritical state), at a volumetric flow rate, which is for example at least five or ten times lower, optionally one hundred times lower, for example fifty times smaller, compared to that of the carbon dioxide circulating in the loop; - during a supplementary recirculation step without addition, put to recirculate in the reactor and the loop (therefore following a loop circulation without co-solvent input), typically in the closed state of the additive introduction valve.

[0028] In some options, the volumetric flow rate of the additive can, for example, be greater than or equal to approximately two percent of the volumetric flow rate applied in the loop. An option with an additive introduction volumetric flow rate (via a co-solvent introduction valve) as low as 2% or less can also work for effective cleaning but with a longer processing time, particularly if the mass of tissue to be treated is large.

[0029] Generally, it may be preferable to inject a volume of additive, for example chosen from hydrogen peroxide and a weak acid (peracetic acid PPA for example as an additional additive added in a second treatment cycle following treatment with hydrogen peroxide), which represents a mass of additive much less than the total mass of tissue placed in the reactor, for example on the order of 25% or less of this mass of tissue.

[0030] In the case of a 35% hydrogen peroxide solution (35% being a percentage by weight), a ratio of approximately 1 ml + / - 0.5 ml of solution per 4 g of tissue to be treated can thus be used, as a non-limiting example. The same applies to a disinfectant acid solution, such as optionally an 18% PAA solution (peracetic acid diluted to 18% in 99% ethanol), where such a ratio can also be used. In what follows, the injected solution, which may thus have a certain level of dilution (for example, H₂O₂ diluted in H₂O, PAA diluted in ethanol), is referred to as the co-solvent.

[0031] In specific implementation details of the process, at least one of the following provisions may be applied: - each chemical additive is recirculated in the reactor and the loop before the end of the introduction of said additive in the open state of the inlet valve (the injection time of a given additive can be chosen longer than the transit time of the reactor, and possibly longer than the transit time of the reactor and the loop corresponding to a complete turn). - The reactor can operate with a variable pressure regime using a piston-type device - the reactor can operate with an agitator or an ultrasonic wave device in the internal volume. - after a period of operation in recirculation mode, for example for a sufficient duration corresponding to at least 3 or 4 times a transit time of the entire circuit, a purge of the reactor and the loop is carried out to allow the removal of residues. - and before a further treatment step using an additive, a recharging step with supercritical carbon dioxide is planned in the reactor, with an adjustment of the carbon dioxide flow rate for also predefined temperature and pressure conditions. - the parameter pair 'temperature and pressure' can be 40°C and 160 bars, with possibly a tolerance of 3% for each of these two values. - between two chemical treatments (treatments combining supercritical carbon dioxide and co-solvent), without moving the tissue and without stopping the carbon dioxide circulation pump in the reactor and in the loop, it is planned to: a / activate a separation stage connected in bypass of a section of the loop and accessible by opening a bypass valve (and typically by closing a valve (so-called primary valve) located downstream of a control valve); - during the intermediate phase between two chemical treatments, it may also be provided to: b / allow the co-solvent and the remaining residues to accumulate in the separation stage; c / purge the co-solvent and the remaining residues through valves in the separation stage; - it may also be planned to: d / deactivate the separation stage by closing the bypass valve and opening said primary valve, then restart a treatment with a second co-solvent. - between two chemical treatments (among the chemical treatments), and typically without displacement of the tissue (which remains in the internal volume of the reactor), it is planned successively to: a / depressurize the reactor and the loop (knowing that the carbon dioxide circulation pump, in the reactor and in the loop, has been stopped); b / drain the reactor using a suitable drain port (for example a lower port of the reactor which forms said outlet). - the loop is also drained after depressurization. - After draining, the following steps are also planned: c / purging the reactor and the loop with compressed air to remove co-solvent residues. Optionally, the following may also be planned: d / rinsing the reactor with purified water by introducing purified water upstream of the reactor and removing it downstream; the direction of purified water flow can be reversed. - When rinsing is planned, the compressed air purge can be carried out before and / or after step d / . In the absence of rinsing, the compressed air purge can follow (directly) the draining b / .

[0032] In an example of treatment involving several additives, it can be provided that: - at least one chemical additive for purification and / or decontamination is a first chemical additive, preferably oxygen peroxide, injected into the loop and introduced into the reactor as a co-solvent of carbon dioxide in the supercritical state, in order to carry out a chemical treatment in the contact region by circulating the first chemical additive in the reactor as well as in the loop which communicates with the reactor inlet. - in the pressurized state of the reactor and for example after a purging phase, the process includes the steps consisting essentially of: adding / applying a second chemical purification / decontamination additive, for example PAA, injected into the loop and introduced into the reactor as a co-solvent of carbon dioxide in the supercritical state. - The additives can be introduced without overlap or simultaneous presence of additives in the loop and the reactor, by carrying out the treatments with the first and second additives sequentially. A separation or purging of the circuit (which includes the reactor) is carried out in the interval between these two treatments. - Alternatively, we can plan to form a heterogeneous flow including simultaneously two types of compounds / co-solvents constituting chemically active additives to participate in the purification / decontamination of the tissue present in the reactor, during circulation in the loop.

[0033] According to one particular feature, the process includes performing a chemical treatment in the contact region by circulating the second chemical purification / decontamination additive through the reactor and the loop that connects to the reactor inlet. The second chemical additive is then reintroduced into the reactor via the loop as a co-solvent for supercritical carbon dioxide. Typically, the second additive can thus be circulated in a second treatment cycle using an additive flow, after the first additive has been removed from the reactor and the loop.

[0034] According to one option, the process may include, after a gradual depressurization following said chemical treatment using one or more chemical additives, one or more steps involving an additional agent as a co-solvent. For example, a third additive (ethanol) may be introduced into a third treatment cycle using an additive flow.

[0035] The process may include the steps essentially consisting of: - applying an additional decontamination and / or dehydration agent (in particular ethanol in one embodiment), injected into the loop and introduced into the reactor as a co-solvent of supercritical carbon dioxide; and - to carry out, in the pressurized state, a treatment in the contact region resulting from a combined circulation of the additional agent and carbon dioxide in the supercritical state, in the reactor and in the loop which communicates with said reactor inlet.

[0036] In some embodiments, the additional agent is introduced into the reactor via the loop as a co-solvent of supercritical carbon dioxide, for example, by being introduced through the same inlet valve used for the introduction of a first and / or second additive (one or more additional additives may be applied) circulated through the reactor before the selective addition of the additional agent. The additional agent may be a solvent suitable for neutralizing / solubilizing / carrying with it an acid used as a chemical additive in the process.

[0037] According to one option, a pressure regulating valve arranged in the loop downstream of the reactor following a direction of circulation of the treatment flow and upstream of the supercritical carbon dioxide circulation pump is actuated (for example by varying its degree of opening) to activate one or more pressure drops and rises in the reactor, during recirculation, so that the supercritical carbon dioxide and the co-solvent diffuse deeper into the tissue matrix, thus with the aim of making the action of the treatment more effective.

[0038] According to one aspect of this disclosure, a treatment facility is proposed that allows the purification and decontamination of a tissue (or tissue matrix) of human or animal origin suitable for optimizing the use of additive(s) involved in the effectiveness of the treatment.

[0039] More specifically, an installation is proposed for treating a tissue or tissue matrix, of human or animal origin and in particular collagen-based, by a supercritical carbon dioxide flow, for the implementation of a treatment process as described above, the installation comprising: - a reactor provided with an inlet and an outlet, the reactor delimiting an internal volume to receive the tissue, the reactor being able to be closed and pressurized to maintain the supercritical state of the carbon dioxide flow; - a pump and a heating device, designed and arranged to change carbon dioxide from a liquid state to a supercritical state, upstream of the reactor inlet in a direction of flow from the pump to the inlet; - a circuit comprising a loop passing through a point or region of introduction of liquid carbon dioxide, the loop extending between a first loop end connected to the reactor outlet and a second loop end connected to the reactor inlet, in order to allow recirculation of fluid from the reactor, from outlet to inlet, the pump being arranged in the loop between the first and second ends downstream of a condenser; - a fluidic connection device with the loop and associated with additive introduction means, to allow the addition of at least one chemical additive for tissue purification and / or decontamination in a section of the loop located downstream of the condenser and upstream of the reactor inlet, so that the fluidic connection device forms a mixer allowing a mixed flow combining supercritical carbon dioxide as a solvent and the additive as a co-solvent, to constitute a treatment flow (from the loop) reaching the tissue in the internal volume of the reactor.

[0040] Such an installation makes it possible to combine a pressurized reactor and a circulation passing through this reactor, to simultaneously admit a solvent and a co-solvent forming a dynamic flow for decontamination and residue extraction treatment, with the benefit of the penetrating properties of a supercritical fluid such as CO2. The supercritical fluid can come from a liquid carbon dioxide source connected to the loop.

[0041] For example, the installation may have an opening / closing assembly (equipped with valves arranged on the loop), which is capable of being configured in different states, including a first open state, in which a pressure regulating means or valve (which is, for example, part of the valves in the opening / closing assembly) allows the reactor pressurization to be maintained above a threshold (corresponding to a pressure exceeding the critical pressure of carbon dioxide), preferably greater than 100 bar, to actuate the recirculation of fluid passing through the loop, thereby contributing to making the treatment flow dynamic. In the reactor, the treatment flow may correspond to a flow that substantially follows an axial dispersion pattern in the reactor between the inlet and outlet (in the case of plug flow).The open state allows the reactor's inlet and outlet to be in fluidic communication with each end of the loop.

[0042] The opening / closing assembly can also be configured in at least one other state with the control valve open, for example, a state (second state) compatible with activation of a separation stage without depressurization or a state (third state) compatible with depressurization of the reactor and the loop. In this other state, some of the valves remain open, allowing the evacuation of co-solvent and residues formed during tissue processing and remaining in the internal volume and in the loop, which are liquid and / or solid residues. For this type of state, the loop can be modified locally and typically has / be connected to a bypass at which a co-solvent outlet / evacuation is formed. and residues (without the possibility of returning to the loop, the latter thus being ready for another chemical treatment afterwards).

[0043] Optionally, valves of the opening / closing assembly are configured, in a closed state (for example with a blocked / closed spillway or not supplied by stopping a valve provided at the outlet of the reactor), to allow the pressure increase in the internal volume of the reactor, under the effect of pumping by the pump, the closed state being maintained during a preliminary phase of pressure increase.

[0044] The separation stage is for example connected in bypass of a section of the loop and accessible by a determined setting of valve(s) of the opening / closing assembly, without being accessible in a recirculation phase via the loop (due to a different setting of the valve(s) considered), during the phases of treatment of the tissue with the flow of added carbon dioxide.

[0045] In options, the installation features a co-solvent pump, for example common to each type of additive, which can be activated during a loop circulation phase of carbon dioxide in and out of the reactor, passing to the supercritical state before circulating in the internal volume of the reactor.

[0046] The reactor geometry may be column-shaped. The height-to-diameter ratio can be on the order of 4 to 5 for the reactor in this configuration. A vertically elongated shape with a reduced cross-section, or a cross-section corresponding to the transverse size of the contact region corresponding to the position of the tissue(s), ensures that the supercritical carbon dioxide passes essentially through the material, minimizing peripheral flows that bypass the tissue being treated.

[0047] The ratio between the capacity of the loop and the internal volume of the reactor may be less than 2, for example on the order of 1.

[0048] According to one particular feature, the loop is equipped with a separation stage (such as a gravimetric or cyclone separator) to separate the co-solvent from residues discharged from the reactor by circulating in the loop. Such a separation stage can also separate residues discharged from the reactor by circulating in the loop. The separation stage is, for example, located in the loop downstream of the pressure control valve in the reactor. A filter may also be provided in the loop.

[0049] According to one particular feature, the loop is provided with a filtration stage (filter) typically designed to prevent extracted solid particles from clogging the control valve (which is, for example, a needle valve sensitive to obstruction, with a very small opening that maintains upstream pressure). The filter, or similar separator, thus constitutes a protective means for the control valve. In some embodiments, the filter is a component that can be disassembled and cleaned (reusable at least partially) at the end of the complete cycle. The filter can be placed at the reactor outlet and the pressure regulation device is, for example, interposed in the loop between the filter and two parallel sections of the loop, one of which includes the separation stage.

[0050] A filtration threshold of the filter can be set at one or several tens of micrometers, in order to trap impurities. This is, for example, a purely mechanical filtration.

[0051] The installation may include: - a source of liquid carbon dioxide connected to the loop upstream of the carbon dioxide circulation pump; - a source of chemical additive for purification and / or decontamination, connected via a fluidic communication fitting to a section of the loop extending between the carbon dioxide circulation pump and the reactor inlet; - an exhaust line, fluidly connected to the reactor when recirculation is activated, in order to evacuate at least some of the carbon dioxide and chemical decontamination additive, without depressurizing the reactor.

[0052] Optionally, the installation has one or more of the following features: - the discharge line includes a relief / control valve and forms a section of the loop. - the spillway is configured so that the discharge line circulates supercritical CO2 between the reactor outlet and the spillway. - the supercritical carbon dioxide source is possibly in the form of a bottle with a dip tube to deliver the carbon dioxide in liquid form into the loop. - The filter includes a filter medium. An area upstream of the filter medium, delimited within a filtration chamber, may contain a residue collection tank or base. It is understood that the filter is traversed by the treatment flow (flow that has already circulated within the reactor's internal volume) during an additive injection phase and, for example, during a closed-loop circulation phase without additive addition. Brief description of the drawings

[0053] Other features, details and advantages will become apparent from reading the detailed description below, and from analyzing the attached drawings, on which: - [Fig.1] is a diagram of an installation for implementing a tissue material purification process, equipped with a reactor, a recirculation loop equipped with a fluidic connection fitting device allowing the injection of one or more additives, each additive being able to be introduced during a treatment cycle which uses the recirculation loop associated with the reactor. - [Fig.2] is a flowchart of steps involved in the process of purifying tissue material, according to a non-limiting example of implementation. - Fig. 3 illustrates a liquid product supply route, associated here with several reservoirs, with a pump constituting the co-solvent pump in a treatment process using a fluid in a supercritical state. Description of the implementation methods

[0054] Several non-limiting examples of embodiments are set out in detail below. In the various figures, identical reference numerals indicate identical or similar elements.

[0055] With reference to [Fig. 1], which shows an installation for implementing the process, a reactor 1 is provided to achieve a desired pressurization level. In the installation, reactor 1 allows the tissue 2 to be treated / purified to be placed in its internal volume VR. Tissue 2 is a biological tissue (of human or animal origin), for example, based on a bone matrix, which may be in several pieces or as a monolithic block. In the examples described below, the treatment process aims to purify and / or decontaminate collagen-based tissue matrices. The purging and / or rinsing operations of reactor 1 will be described briefly, insofar as these steps do not specifically relate to the extraction action carried out by a solvent (a fluid in a supercritical state such as CO2) or the combined action carried out by this solvent and a co-solvent. Example of installation

[0056] Reactor 1 of the installation has an inlet 5 and an outlet 6 to allow the circulation of a stream of supercritical carbon dioxide (CO2) through reactor 1. A valve VI can be connected in a manner known per se to a carbon dioxide source 3, optionally by forming a fluid communication connection with a loop 20, which will be described in detail later. The pressurization device includes a pump 8, for example, a pump for regulating the flow rate of the carbon dioxide. The pump 8 is capable of compressing the liquid carbon dioxide to a pressure that allows it to reach a supercritical state. A valve V2 is located downstream of the pump 8, which can allow reactor 1 to be isolated for loading or unloading.

[0057] The installation can take the form of a circuit, having a loop 20 passing through a point or region of introduction of liquid carbon dioxide. Such a loop 20 extends, for example, between a first loop end 21 connected to the outlet 6 of reactor 1 and a second loop end 22 connected to the inlet 5 of reactor 1, as in the non-limiting case illustrated in [Fig. 1]. The loop 20 thus associated with reactor 1 makes it possible to constitute a circuit compatible with circulation in a closed loop. More generally, loop 20 can allow recirculation of fluid from reactor 1, from outlet 6 to inlet 5.

[0058] As can be clearly seen in [Fig.1], a CO2 supply line L5 can be formed for the reactor, on which the following are arranged successively (in series): - the refrigeration unit 7; - the pressurization device with pump 8, which here forms a high-pressure pump; - a heater 9, for example in the form of a heat exchanger which uses a circulation of a heat transfer fluid or an electrical resistance.

[0059] Before being introduced into reactor 1, the carbon dioxide is thus heated by the heater 9 so that at the outlet of this heater, the carbon dioxide is in a supercritical state. In some embodiments, the supercritical state can be obtained upstream of this heater outlet 9, for example at the outlet of the pump 8. The heating can be carried out to obtain a temperature between 31° and 60°C.

[0060] Reactor 1 is suitable for being closed and pressurized to maintain the supercritical state of the carbon dioxide stream. However, it is permissible to release a stream, typically through outlet 6 (here with valve V4 open), which circulates in a loop 20 and then returns to reactor 1. The carbon dioxide stream can be gaseous, for example at a temperature of approximately 50°C, in a section L2 or L3 of loop 20, the pressure being lower than that in reactor 1, for example on the order of 50 bar. Cooling can be provided in loop 20, in an intermediate section L4, to allow liquefaction of the carbon dioxide, facilitating its circulation and compression by pump 8.

[0061] During this type of circulation (loop) to pass through reactor 1 and loop 20 back to the inlet 5 of reactor 1, a branch or section L3, on which optional separator means SI, S2 are located to separate residues from the gaseous CO2 (for example, by performing a liquid-gas and / or solid-gas separation), is inaccessible. The inlet valve V8 in this gaseous CO2 purification branch is kept closed, while the valve V7, associated with the line L2 not passing through the separator means SI, S2, is open. The separator means SI, S2 may be of the cyclonic or gravimetric type.

[0062] Loop 20 can then successively include, starting from outlet 6 of reactor 1: - an L1 evacuation line as the first section of loop 20, which forms an end 21 of loop 20 (here connected to the outlet 6 of reactor 1) and joins a valve 10 forming a spillway; - a second section L2 of gaseous CO2 circulation which joins the open recirculation valve VI1 and allows the flow of CO2 to continue (as seen in [Fig. 1], a vent EV2 can be provided just upstream of the valve VI1); - a third section L3 constituting a bypass from the line L2, allowing gaseous CO2 to circulate in a separation stage SI, S2 before reaching the recirculation valve VI1; - a fourth CO2 circulation section L4, extending from valve V1 to pump 8, which cools and liquefies the circulating carbon dioxide; and - the L5 feed line, as the fifth section of loop 20, with a circulation of CO2 in liquid and then supercritical state, forming a second end 22 of loop 20 for connection to an inlet 5 of reactor 1.

[0063] More generally, the loop 20 can take many forms. Although the loop 20 is shown in Figures 1 and 3 as linear, such a loop 20 can have different branches in parallel, for example upstream of pump 8 and / or downstream of pump 8. A reactor inlet 5 corresponding to one end 22 of the loop 21 has been described. However, it may also be possible to distribute the flow using several inlets of reactor 1.

[0064] In alternative embodiments, all or part of the loop 20 can also be used to contribute to the processing of tissues 2 distributed / distributed in two reactors in parallel. In this case, it suffices, for example, to duplicate the respective ends 21 and 22 of the loop 20, to correspond to outputs 6 and corresponding inlets 5 of reactors 1.

[0065] As illustrated in the non-limiting example in [Fig. 1], the reactor 1 of the installation can be interposed between the pump 8 and the overflow valve 10. The pump 8 can be designed and arranged to: - to change carbon dioxide from a liquid state to a supercritical state, upstream of the inlet 5 of reactor 1 following the direction of circulation (going from pump 8 towards inlet 5); - in a mode with recirculation (as illustrated for example in [Fig.1]) which uses an additive injected as a co-solvent downstream of pump 8, perform pressurization in the presence of recirculated co-solvent with carbon dioxide as the solvent.

[0066] The installation features a fluidic connection RC device with loop 20, which is, for example, associated with additive introduction means V12. In the non-limiting embodiment of [Fig. 1], such an RC device is shown upstream of heater 9 but an arrangement upstream of pump 8 or closer to reactor 1 (near inlet 5, for example downstream of heater 9) is of course possible.

[0067] The means V12, which include, for example, an inlet valve coupled, where appropriate, to several respective reservoirs RI, R2, R3, R4, can deliver a solution in liquid form directly into a supercritical CO2 stream, i.e., into the line stream L5 subjected to high pressure, for example, between 100 and 200 bar. Alternatively, the solution is delivered further upstream, into a CO2 stream that is still liquid. In the non-limiting case of [Fig. 1], the means V12 can allow the addition of at least one chemical additive 11, 12, 13 for tissue purification / decontamination in a section of the loop 20 located downstream of the pump 8 and upstream of the inlet 5, such that the connecting RC device forms a mixer. Thus, a mixed / heterogeneous flow combining supercritical carbon dioxide as the treatment solvent and the additive as a treatment co-solvent can be conveyed together into the internal volume VR of the reactor 1 to reach the tissue 2. The means V12 can be parameterized so that the liquid flow rate is significantly lower than the CO2 flow rate in the L5 section of the loop 20 (and more generally throughout the entire circuit). Regardless of the introduction point (chosen just upstream or downstream of pump 8), a pump P2, referred to hereafter as the co-solvent pump, is included as part of the introduction means V12. This pump P2 is configured with a flow rate that depends on the flow rate of pump 8, in order to ensure the dissolution of the co-solvent in the solvent (supercritical carbon dioxide). For example, the ratio of additive (co-solvent) flow rate to solvent flow rate can be between 1 / 5 and 1 / 100. In some embodiments, this ratio is 1 / 50. At the inlet of reactor 5, a treatment flow containing the additive can thus be admitted.As described later, the chemically active additive can be introduced in a diluted form in another liquid, preferably with the possibility of controlling the concentration of the chemical component involved in the treatment of the tissue to remove impurities / residues, in particular by controlling a ratio between a parameter representative of the mass or total concentration of additive introduced into reactor 1 and the total mass of tissue to be treated.

[0068] Reactor 1 is designed and arranged to admit an additive as a co-solvent when the internal volume VR is already pressurized. To allow activation of loop 20 while maintaining this pressurized state, the installation may include an opening / closing assembly equipped with valves (V2, V4, V10, V7, V11); such a valve assembly may have an opening configuration compatible with series circulation between the reactor and the loop. This assembly may allow recirculation of fluid, in a first open state, the pressure regulating valve 10 allowing a maintenance of a pressurization of reactor 1 beyond a threshold, preferably greater than 100 bars.

[0069] In the case illustrated in [Fig. 1], numerous valves or check / vent systems are shown. Of course, the loop 20 can be simplified in the number of valves, for example by integrating a different valve system at least in certain parts of the circuit, valves directly into the separator means SI, S2 or other comparable arrangement.

[0070] At the end of a treatment cycle in the pressurized reactor 1, the valve arrangement V7, V8 located downstream of the spillway 10 can be modified / configured differently to use the bypass line L3, in order to access separator means SI, S2. More generally, purging steps can use all or part of the loop 20 as well as one or more separators SI, S2, which are typically separate from the filter 4 located upstream of the valve 10. Here, the co-solvent and the residual materials carried by / dissolved in the carbon dioxide that remained in reactor 1 are discharged via the reactor outlet 6, circulated through the valve V8, with the bypass valve V7 closed, and recovered at the separator means SI, S2, here in this example at the outlet of a first separator SI, via a purge valve V9.The installation may also include a second separator S2, this second separator S2 having a V10 valve for purging organic matter. More than two separate separators may be provided, depending on the requirements.

[0071] The step of recovering residues and co-solvent by the separation stage / means SI, S2 is carried out while carbon dioxide is circulated in section L3, so that this solvent is recycled in the circuit via a valve 11 joining section L4, valve V7 being closed. Steps in the process

[0072] After the fabric 2, possibly placed on a suitable container or support, is positioned within the internal volume VR of the reactor 1, this fabric 2 can remain in a fixed position, and a contact region is defined, located intermediately between the inlet 5 and the outlet 6. When the reactor 1 is arranged vertically, for example, forming a column, the fabric 2 can thus be positioned at a height intermediate between the inlet 5, located at the upper end of the reactor 1, and the outlet 6, formed at the lower end of the reactor 5. A lid including the inlet 5 can be used to hermetically seal the internal volume VR, which is delimited by the reactor body. The access ports to the reactor 1 can be limited, for example, by primarily including the inlet 5 and the outlet 6, and possibly an additional port that can serve as a vent.

[0073] In order to purify / decontaminate the tissue 2, the process can begin with a first treatment cycle involving the initial introduction of supercritical carbon dioxide into the internal volume VR, for example by configuring the opening / closing assembly to allow communication: - exit 6 with section Ll, as seen in [Fig.l] for example; - entrance 5 with section L5. Carbon dioxide can circulate in a loop while under pressure in reactor 1, in a supercritical state.

[0074] This corresponds to a first circulation, which is, for example, a circulation without addition. With reference to the example in [Fig. 1], valve V4 shown in [Fig. 1] can then be opened (reactor drain valve V14 closed), while the weir 10 allows fluid circulation towards the recirculation valve VI1. Sections L4 and L5 are involved during cycle start-up to allow the reactor to be filled with CO2, and are also used in a similar way during such a first circulation.

[0075] With reference now to [Fig. 2], the successive steps of placing 50 the material to be treated in reactor 1, and of supplying / distributing 51 CO2, can constitute process steps that precede step 52 of introducing additive(s) via a suitable valve V12, connected to the supply line L5. Filling reactor 1 with CO2 under pressurization, for example to 160 bar, and filling loop 20 can correspond to a limited duration, shorter in any case than the duration of the treatment steps.

[0076] During step 51, pump 8 operates to ensure that reactor 1 is traversed by a dynamic flow of supercritical CO2. An example of the flow rate delivered by pump 8 is a continuous flow of 5 kg / h under stable temperature and pressure conditions. After step 51 and for steps 52a, 52b, or 52c, which involve the insertion of a co-solvent (with additive), pump 8 remains running. While carbon dioxide circulates in a loop within the circuit (reactor 1 + loop 20), and a desired pressure and temperature are reached, for example, 160 bar and 40°C, in reactor 1, and the pressure / flow rate / temperature parameters are stable, the insertion of at least one co-solvent can begin.The injections carried out in steps 52a, 52b or 52c can be parameterized with a predefined dosage, taking into account a parameter representative of the total quantity of additive injected which is calculated / dosed according to the total mass of the tissue 2 to be treated present in reactor 1, independently of the capacity of reactor 1 or the location / distribution of tissue 2 in the internal volume VR. .

[0077] While carbon dioxide in a supercritical state circulates dynamically in reactor 1, beginning its cleaning action (in particular degreasing) In the region of contact with tissue 2, the introduction means V12, P2 deliver the co-solvent, for example using a co-solvent pump P2 which delivers the latter in liquid form into a co-solvent supply line LO. A chemical additive 11, 12 or 13 can thus be added for a complementary action, for example chemical / enzymatic, carried out in conjunction with the action of supercritical carbon dioxide, and following the flow of this solvent.

[0078] Once the additive 11, 12, or 13 has been injected into reactor 1 as a co-solvent, typically at a comparatively low volumetric flow rate compared to that of CO2, a different flow than that circulating during the first circulation with CO2 is recirculated in loop 20: this results in a second type of circulation corresponding to a dynamic treatment flow with additive. The co-solvent insertion valve V12 is opened and the pump P2 is started without interrupting the circulation, so as to inject a volume of liquid into the pressurized CO2, thus typically without stopping pump 8. In some options, heating the heterogeneous flow (CO2 in a supercritical or near-supercritical state and additive) by the heater 9 can maintain stable parameters in reactor 1, without the temperature, pressure, or flow rate at inlet 5 varying significantly.

[0079] The installation allows for dosing the quantity of additive, by controlling: - on the one hand, the volumetric flow rate of the co-solvent (which can be a solution with the chemical agent diluted in the liquid solution), this flow rate being able to be substantially constant by adjusting the pump P2; - and on the other hand, the duration of injection. In some variations, the quantity of additive can also be predetermined without resorting to constant volumetric flow rate injection, for example by using a gradient or controlled variation in the flow rate, or by alternating injection phases with pauses, while controlling / fixing in advance the total quantity injected in sequences.

[0080] The installation can enable a treatment process to be carried out with a sequence of treatment phases using a particular additive. Here, using the same additive introduction device or means (P2, V12), the same introduction point formed on section L5 of the loop 20 can be used. [Fig. 1] shows, for example, the use of an RC connection fitting, which can optionally be included in the co-solvent inlet valve V12. The liquid additive can be introduced into a section of the loop where the carbon dioxide is itself either liquid or supercritical.For example, this additive can be introduced before the reactor inlet 5, possibly upstream of the pump 8, right at the outlet of the condenser 7.

[0081] With reference to [Fig. 3], the pump P2 can receive liquid supplied from a co-solvent reservoir R, which can be filled with various additives 11, 12, or 13, optionally mixed with a substance 14 such as water or another solvent (aqueous or non-aqueous). The term co-solvent can thus refer to a solution injected into the CO2 stream (supercritical or possibly still liquid) and which will be brought into contact with the material (fabric 2) to be treated. The co-solvent can contain at least one active ingredient and an associated diluent.

[0082] For example, a first reservoir RI is provided for a first additive 11, for example, hydrogen peroxide. Other reservoirs R2 and R3 can be used to store a second additive, for example, an acid such as PAA, and a third additive or component, for example, ethanol. Valves 41, 42, 43, and 44, associated with each of these reservoirs RI, R2, R3, and R4, are shown here. By opening valves 41 and 44, simultaneously or not, to fill the co-solvent supply reservoir R, with flow control, it is possible, for example, to produce a 35% hydrogen peroxide solution (or another desired percentage). Similarly, using valves 42 and 43, it is possible to prepare a solution of peracetic acid diluted in ethanol with a defined percentage, for example 18-20% peracetic acid (or another desired percentage as needed). The case of H2O2 and peracetic acid as active ingredients has been cited.Of course, other molecules or substances can be used, provided that such a substance, combined with supercritical carbon dioxide, has a purifying or decontaminating effect on the material to be treated.

[0083] Of course, in other options, the reservoir R can be eliminated and different co-solvent supply lines can be used. If necessary, this can be achieved by means of introducing additives arranged differently, for example in parallel with each other up to a junction point with the loop 20, downstream of the pump 8.

[0084] The treatment of matrix / tissue 2 in reactor 1 is carried out, in each treatment cycle, by a flow of supercritical carbon dioxide, supplemented by the introduction (for example, during the circulation of CO2 in reactor 1 and loop 20) of at least one chemical additive injected by the introduction means P2, V12 in the form of a liquid solution while reactor 1 is already pressurized. The combination of supercritical CO2 as a solvent and the chemical additive as a co-solvent can enhance the cleaning / disinfecting action on the treated tissue. At the outlet 6 of the pressurized reactor 1, the flow circulates in the end 21 of loop 20 and then rejoins the rest of the loop via the overflow 10. The circulation of a first additive, injected gradually and then recirculated with the CO2, forms part of a first treatment cycle. Several cycles, each with an additive, can follow one another, separated by a separation of the co-solvent and residues at constant pressure or a depressurization and purging, as detailed a little later.

[0085] In some options, the valve forming the weir 10 is controlled and regulates the upstream pressure by allowing a trickle of fluid to flow through, in this case a carbon dioxide-based fluid. By way of non-limiting example, between the valve 10 and the pump 8, the pressure in the loop 20 (in sections L2, L3, and L4) can be approximately 50 bar. Example of operating parameters

[0086] Carbon dioxide CO2 is pumped here in liquid form by pump 8. This liquid is preheated upstream of the extraction reactor 1 in order to be introduced into it in a supercritical state.

[0087] It is worth recalling that fluids in a supercritical state can be defined as gases placed under temperature and pressure conditions such that their properties are intermediate between those of gases and those of liquids. They are also called "dense gases" or "expanded liquids." For a given chemical substance, the precise point on the temperature-pressure diagram at which the two phases, liquid and vapor, merge into one is called the critical point. Above this critical temperature (Te) and critical pressure (Pc), the fluid is in the so-called "supercritical" state.

[0088] The installation uses supercritical carbon dioxide at least in a portion of the loop corresponding to section L5 and in the internal volume VR of the pressurized reactor. As it passes through reactor 1, the supercritical carbon dioxide solubilizes a large portion of the organic matter, primarily lipids, in tissue 2. In particular, it can dissolve the fats in bone marrow tissue contained within bone tissue.

[0089] To achieve this action, the implementation conditions may vary. To illustrate, the following conditions are given as a non-limiting example: - the pressures prevailing in reactor 1 range from 100 to 200 bar, so a pressure of 160 bar can be chosen. Such pressure conditions are compatible with temperatures that remain well below 90 or 100°C, for example, temperatures between 35 and 50°C: a temperature of 40°C can optionally be chosen. - For a given quantity of material to be treated (total mass of tissue 2 in reactor 1 in the non-limiting example of [Fig. 1]), a total volume of co-solvent to be injected can be determined by the operator according to the nature of the co-solvent, its concentration, and the desired effect: one usable parameter is the co-solvent / mass of material (tissue 2) ratio, expressed in ml / g. This is referred to as the first ratio in what follows. A first ratio of 0.25 ml / g is chosen in some of the experimental cases that follow, but, of course, a completely different ratio could be suitable (particularly depending on the principle The active ingredient is more or less diluted in the co-solvent solution, this second ratio necessarily changes). - It is permissible to adjust the flow rate of the co-solvent pump P2 according to the flow rate of the supercritical CO2 pump 8, so as to ensure the dissolution of the co-solvent in the solvent. The co-solvent flow rate / supercritical CO2 flow rate ratio, hereinafter referred to as the second ratio, can vary depending on the co-solvent delivered via the introduction means P2, V12. With the solutions considered, the second ratio is, for example, between 1 / 5 and 1 / 100, with a ratio of 1 / 50 being a possible choice.

[0090] The installation, particularly depending on the reactor(s) 1 and the type of associated loop 20, may vary in its structure and capacity, which may depend on the total mass of tissue 2 disposed of in a given internal volume VR. The volume VR may be greater than the volume of the loop 20 that completes the circuit (the loop volume here does not include the separator means SI, S2, which are not used in closed-circuit recirculation). Depending on the volumes of reactor 1 and loop 20, a flow rate can be applied to the pump 8 adapted to the desired effect on the tissue 2.

[0091] At each stage of the treatment, secondary parameters (variables) of the process can be deduced from primary parameters as indicated above. Thus, with a known pump flow rate (supercritical CO2 flow rate), the co-solvent flow rate can be deduced based on the second ratio. Similarly, the volume of co-solvent added can depend on the quantity of material to be treated according to the first ratio mentioned above.

[0092] The injection time can be calculated based on the co-solvent flow rate and the volume (total volume) to be injected via the introduction means P2, V12. The transit time through reactor 1 is calculated based on the supercritical carbon dioxide flow rate and the internal volume VR of reactor 1. Furthermore, the initial contact time of the co-solvent with the material / tissue 2 (corresponding to the first-passage time of the co-solvent over the material) is typically equal to the sum of the injection time and the transit time through reactor 1.

[0093] Once the planned volume of co-solvent has been injected, the co-solvent (with the corresponding additive 11, 12, or 13) circulates through the circuit until it completes a full cycle of the installation. The cycle time is defined by the flow rate of pump 8 and the sum of the volume VR of reactor 1 and the volume of the rest of the circuit (here, loop 20). If a longer contact time between the co-solvent and the material is required, the mixture (supercritical solvent / co-solvent) can simply be recycled. Optionally, a certain number N of revolutions can be set once all the co-solvent has been introduced into the circuit and has reached section L5. Practical experimental example

[0094] In this example, a total mass of 320 g of bone tissue is placed in reactor 1 and the envisaged process involves several types of treatments, one after the other: - first with hydrogen peroxide, for example with this active ingredient added in a 35% liquid solution (possibly an undiluted commercial solution), - then with PAA (peracetic acid) diluted 2.7 times in 99% ethanol, i.e. a PAA concentration of 18% in the co-solvent solution, - then with 99% ethanol.

[0095] The volume of each co-solvent to be applied is predetermined; in this non-limiting example, it is 0.25 ml of co-solvent to treat 1 g of material (e.g., 1 g of bone). The installation may have the following characteristics: - a circuit with a volume of approximately 2 L, of which 1 L corresponds to the internal volume VR of reactor 1. - Pump 8 is set to deliver a flow rate of 5 kg / h, or approximately 104 ml / min (for a supercritical CO2 density of approximately 0.8 at 160 bar). The second flow rate ratio (volumetric flow rate) of pumps P2 and P8 is 1 / 50.

[0096] The treatment steps are carried out at 160 bar and 40°C in reactor 1. The deduced values ​​are as follows: - The co-solvent flow rate is 2.08 ml / min (104 / 50). - The volume of co-solvent to be injected is 80 ml (0.25*320). - The injection time is 38 minutes 27 seconds (80 / 2.08); this time can obviously be rounded to 38 or 39 minutes. - The reactor transit time is 9 minutes 37 seconds (1000 / 104). - The time of one cycle within the circuit for a complete lap is approximately 20 minutes (2000 / 104).

[0097] The experimental example is carried out with recirculation for 100 minutes, i.e. 5 treatment cycles, to obtain a total contact time (in the contact region) of 138 min, and this at each stage using a given additive / active ingredient. The complete sequence of treatment process phases, here based on the specified (but not exhaustive) values, corresponds to the following: a. Insertion of the material to be treated (fabric 2) into reactor 1 b. Continuous flow of supercritical carbon dioxide at 5 kg / h at 160 bar and 40 °C, using pump 8 c. Insertion of 35% hydrogen peroxide for 38 minutes (80 ml), upstream of reactor 1 in section L5, using the introduction means P2, V12 (including pump P2), this active substance forming a first additive 11 d. Maintaining a (closed) recirculation loop for 100 minutes (5 revolutions in the closed circuit) e. Purging of reactor 1 and the rest of the circuit, then rinsing with purified water to remove any residual material extracted from tissue 2 f. Carbon dioxide recharging followed by restarting the supercritical carbon dioxide flow at 5 kg / h at 160 bar and 40°C g. Insertion of 18% PAA for 38 minutes (80 ml) upstream of reactor 1, using the introduction means P2, V12 (including pump P2), this active substance forming a second additive 12 h. Maintaining recirculation for 100 minutes (5 cycles) i. Purge, for example resulting from the activation of the SI separation stage, S2 to recover and purge the PAA for 60 minutes j. Insertion of 99% ethanol for 38 minutes (80 ml) upstream of reactor 1, using the introduction means P2, V12 (including pump P2), this active substance forming a third additive 13 k. Maintaining a (closed) recirculation loop for 50 minutes (2.5 turns) 1. Purge, for example resulting from the activation of the SI separation stage, S2 to recover and purge the ethanol for 60 minutes m. Insertion of 99% ethanol for 38 minutes (80 ml) upstream of reactor 1, using the introduction means P2, V12 (including pump P2) n. Maintaining a (closed) recirculation loop for 50 minutes (2.5 turns) o. Stopping pump 8 and purging reactor 1 and the rest of the circuit (loop 20) to remove ethanol p. Outlet of reactor 1 of the cleaned fabric (processed material).

[0098] Of course, the main treatment steps (ae / or fi / , j-1 and / or mp / ) can be adjusted as needed, if necessary by substituting one active ingredient for another (i.e., by changing the co-solvent). With reference to [Fig. 2], after step 50 of placing the tissue 2 in the reactor 1 (see phase a / indicated above), once the reactor 1 is closed and fitted with the airtight communication connections to the ends 21, 22 of the loop 20, and after step 51 of filling and dynamic circulation of supercritical CO2 (which can correspond to phase b / , possibly repeated in f / , j / or m / ), it is understood that step 52 of additive introduction is carried out progressively and in a controlled manner to achieve a relatively low ratio between the total volume of co-solvent injected and the mass of tissue 2 to be treated in the reactor 1.

[0099] The detailed experimental example above corresponds to a special case. Optionally, the ethanol treatment step or other final treatment / rinsing step may include fewer substeps or may not require two rinsing cycles. The number of treatments can be higher or lower than that of ethanol. In other examples, to treat more tissue (larger mass), whether the reactor volume is different (larger) or not, a similar injected volume of co-solvent can be used, but with a higher concentration of active ingredient. Multiple variations are applicable regarding the precise injection method and the duration of each recirculation, while pre-determining the quantity of active ingredient (co-solvent) based on the mass of tissue to be treated.

[0100] Limiting this total quantity can prove beneficial for preserving the collagenous tissue matrix and maintaining the mechanical properties of the material being treated. Phases c and d correspond respectively to step 52a, the injection of the first additive, and step 53, the recirculation of a (mixed) treatment stream. Phases g and h correspond respectively to step 52b, the injection of the second additive, and the repetition of step 53, the recirculation of the resulting (mixed) treatment stream. Similarly, step 52c corresponds to the injection of a third chemical agent, for example, ethanol.Arrow 19 after outlet 6 of reactor 1 reflects the residues and illustrates that treatment residues are carried into loop 20, with the possibility of extracting / separating some of them so as not to return these residues to reactor 1, knowing that the treatment options may require one or more full turns during recirculation step 53.

[0101] Step 54, evacuation / cleaning, corresponding to the end of a cycle, involves depressurizing reactor 1 using vent EV1, which may include a valve. Such vent EV1 may be a column-type vent at the top of the reactor, possibly formed at inlet 5. Pump 8 is stopped in order to slowly depressurize (approximately 10 bar / min up to a first threshold, for example, 100 bar, then a slower decrease, here 4 bar / min down to 73 bar or a comparable second threshold, and 2 bar / min until atmospheric pressure is reached) by opening vent EV1. Step 54 may include purging and / or rinsing substeps. For example, reactor 1 can be drained by opening its valve V14 coupled to outlet 6 (here a low drain valve) and closing the valve forming the vent EV1 and opening the regulating valve 10, forming a spillway.This is followed by purging of the co-solvent residues, carried out for example with compressed air for a defined period, shorter than the duration of a treatment step with recirculation loop, for example on the order of 10 to 60 minutes. After 5 minutes of purging, the recirculation valve Vil can be closed.

[0102] It should be noted that, in options allowing for the cleaning of the discharged carbon dioxide, line L2 is not used during step 54, with valve V8 open (and valve V7 closed). Thus, the separator means SI, S2 separate the residues discharged during step 54 via line / section L3.

[0103] Although not illustrated in [Fig.2], step 54 with purging may include or be followed by a rinsing step using purified water, for example when the chemical additive used is H2O2 or a comparable oxidizing and disinfecting active substance, typically diluted in water.

[0104] As an example for rinsing with purified water, the following sequence of operations can be envisaged, starting from a purged reactor 1 (having undergone the basic purging phases of step 54): - carry out a first injection of purified water (if necessary from tank R4 or other available tank), which reaches the contact region, through a V2 inlet valve of the reactor, for example using a pneumatic or electric pump. - open the V14 drain valve (low drain of reactor 1 here in the example of [Fig.l]), with the V4 valve (reactor outlet and forming an access valve to loop 20) which is closed, which allows not to interfere with loop 20. - once the water has been drained (via outlet 6) and is clean, close valve V14. - close valve V2 and open valve V3 purified water inlet connected to outlet 6, here corresponding to lower port of reactor 1. - open the V5 valve associated with the inlet 5 of reactor 1, so as to allow a high purge, i.e. to evacuate the rinsing water. - Optionally, measure the peroxide level at the outlet of valve V5 (which is a high-pressure purge valve), then dilute the solution 1 / 100th before measuring the remaining peroxide level. Verify that the level is below a threshold, for example 2 mg / l, then stop the water injection via outlet 6. - To purge the water from reactor 1, open the inlet valve V2 of reactor 1, open valve V14 and inject compressed air. Close valve V5 as soon as no more water is flowing out of valve V5. - purge residues, in particular co-solvent residues, with compressed air for 60 minutes, by injection with valve V2 held open, while leaving valve V4 open so that the purge applies to loop 20. After 5 min, close recirculation valve VI1. - stop the compressed air supply.

[0105] Compressed air may originate from a region or point or inlet separated from reactor 1 by at least one loop section equipped with one or more valves (valve V2 on the inlet side 5; valve V1 and / or valve V4 on the outlet side 6). For example, this inlet point may correspond to a connection upstream of the chiller unit 7, with a compressed air supply valve V6. Optionally, compressed air may be injected through a connection common to that used for the liquid CO2 inlet, as illustrated in [Fig. 1].

[0106] Of course, the rinsing process can vary in its implementation, for example by modifying the installation, the duration, or certain control parameters that allow the transition between rinsing with purified water and purging with compressed air. Each purge constitutes a way of stopping the action of the co-solvent and at the same time removing the residues extracted from the material (fabric 2) by the combined action of the solvent (supercritical fluid) and the co-solvent (chemical additive).

[0107] After step 54, whether or not completed by rinsing with purified water, the installation can be prepared again for another dynamic flow treatment step with an additive solvent. For this, CO2 can fill loop 20 and reactor 1 (i.e., the circuit), the recirculation mode can be activated, and pump 8 is started to circulate supercritical CO2 in reactor 1, before introducing a new additive.

[0108] With reference to [Fig. 1], the carbon dioxide extracted via one end 21 of the loop 20 passes successively into a gaseous state, downstream of a regulating valve called a weir 10, and then into a liquid state to continue circulating in the loop 20. The filter 4 allows the retention of residues, for example, solid residues. This filtration is carried out here on line L1 in which the carbon dioxide circulates in a supercritical state. The recirculation, downstream of the valve V1, allows the pump 8 to bring back, to reactor 1, a treatment flow free of solid residues with the advantages of a solvent fluid in a supercritical state, which forms a dynamic flow.

[0109] To promote contact, an additional stirring action may be introduced, for example, by using an ultrasonic device, optionally with a movable bar. Such a device can dislodge impurities from the material and facilitate their removal, with circulation in reactor 1 towards outlet 6 and loop 20 allowing such impurities to leave the contact / treatment region. In alternative embodiments, all or part of loop 20 may be formed in or on reactor 1. Optionally, an internal partition provided in the reactor can allow some of the residues to be separated into a sub-region of the internal volume VR, interposed (depending on the direction of carbon dioxide flow) between the contact region and outlet 6.More generally, it is understood that the pressurization of reactor 1 is compatible with a dynamic effect, allowing the removal of residues (via a discharge line, here corresponding to section L1, which is fluidly connected to reactor 1 when recirculation is activated) to a second region offset / distinct from the contact region. By removing at least some of the carbon dioxide and the chemical additive 11, 12 or 13 for purification and / or decontamination, without depressurizing reactor 1, the discharge line or section L1. (including the weir 10) helps to limit / reduce the concentration of additive in the contact area, also reducing the presence of residues in this area.

[0110] An option with a loop 20, for example external to reactor 1, ensures that co-solvent is removed from the contact region le, thus limiting the concentration of active ingredient / additive 11, 12, 13 in this contact region le. With an external loop 20, reactor 1 can remain simple in design.

[0111] At the end of the process, the fabric 2 placed in reactor 1 can be retrieved after opening reactor 1. A drying phase can then be carried out, for example in a ventilated oven, at a temperature between 30 and 50 °C, for example at 40 °C. The drying time can be, for example, between 6 and 12 hours. The duration can be adjusted according to the efficiency of the drying device used.

[0112] More generally, the duration of many steps of the process can vary, depending on the weight of the tissue to be treated and the flow rate of supercritical carbon dioxide introduced into reactor 1. Processing steps 51, 52 may last just long enough for a mass of carbon dioxide and a quantity of additive 11, 12, 13 passing through the tissue 2 (the mass of which is itself determined) to have reacted in the contact region. With the use of a loop 20, it is possible to interrupt the CO2 supply from the cylinder 3 or similar source quite early, well in advance of the end of the treatment, when the mass of carbon dioxide used is sufficient to fill the volume of the circuit.

[0113] The treatment process, with a dynamic circulation of supercritical CO2, in which the additive is added in the form of a liquid co-solvent, allows effective cleaning without alteration thanks to a concentration of active product which remains moderate, in particular low compared to more conventional processes by simple soaking and impregnation of the fabric in a chemical agent.

[0114] In some examples, the number of co-solvents may exceed two or three. A particular case has been described with three co-solvents used, in the following order: hydrogen peroxide, PAA, and ethanol, as additives in the loop through which carbon dioxide circulates in a supercritical state. However, in variations, a short preliminary treatment, for example shorter in duration, may be used, during which the tissue is exposed to an active substance.

[0115] For example, a water-diluted additive, such as hydrogen peroxide, can optionally be introduced at an earlier stage, for example as a first dose already present in the reactor (before any recirculation loop, if applicable) or injected at the same time as the initial CO2 filling to reach the desired pressurization state. In this case as well, contact with the active substance, hydrogen peroxide for example, can be achieved at a low concentration. In some embodiments, the first dose can be taken into account when calculating Next, the additional additive (hydrogen peroxide or similar) is introduced as a co-solvent, preferably gradually. The first dose may represent only 1 to 10%, or in any case a small fraction compared to the total quantity of additive injected (first dose < second dose injected gradually).

[0116] Loop 20 offers the advantage of being able to optimize treatment efficiency, not necessarily by increasing the number of reagent molecules present in reactor 1 at a given time, but by creating a dynamic and for example by increasing the frequency of passage of these molecules in a reactive zone (contact zone), by the return of the molecules (extracted out of reactor 1 by loop 20 via an outlet 6) to the inlet 5 of reactor 1 following a circulation in a flow directed towards / passing through the tissue 2 to be treated.

[0117] The total amount of material used is better utilized, with improved yield, and can be reduced, if necessary, by slightly increasing the duration of the treatment in order to increase the number of passes and therefore the contact between an active component and the tissue to be cleaned.

[0118] Circulation can be carried out in a reactor 1 having a general column shape, in order to create mixing within the internal volume VR, following a plug flow regime (similar to the piston of a syringe). The combination of a filter 4 and a separation stage contributes to purification under dynamic treatment conditions. The filter 4 allows the purification of the output stream from reactor 1 by separating solid particles through purely mechanical separation (without chemical treatment) and with a submillimeter filtration threshold, for example, greater than or equal to 10 µm. It is understood that the active substances can pass through such a filter 4: therefore, there is no reduction in the co-solvent concentration (the additive circulates) due to this separation stage.

[0119] One or more separation stages may be provided to recover the co-solvent or active product at the end of a treatment phase / step 54. A bypass valve may allow access to this or these separation stages only at the end of a treatment cycle or phase. Experimental case study, not exhaustive

[0120] As an experiment, an animal bone matrix weighing approximately 320 or 330 grams was cleaned, with a cycle time of approximately 100 minutes for each co-solvent. The purging steps are the same between each cleaning cycle with the action of a chemically active agent. Satisfactory cleaning quality corresponds to the removal of sufficient impurities while maintaining conventional properties compatible with implantation (here, test). adhesion of MC3T3 cells to the bone matrix tested after 48 hours and implantation test on a rat calvaria bone defect model).

[0121] In this example, approximately 320 g of bone tissue is treated with undiluted 35% hydrogen peroxide and PAA (peracetic acid) diluted 2.7 times in 99% ethanol, resulting in a PAA concentration of 18% in the co-solvent solution, followed by treatment with 99% ethanol. Reactor 1 may have a capacity of approximately 1 liter. More generally, it is understood that the capacity of reactor 1 can vary depending on the quantity of material / tissue to be treated.

[0122] The bone tissue 2 can be distributed, in the zone / region of contact with the gas flow circulating in the reactor 1, in several blocks, for example, staggered vertically or in the principal direction of flow and / or arranged side by side transversely with respect to the principal direction of flow. Alternatively, the tissue 2 can be in a single block (a whole femur being a non-limiting example). The method can also be applied to several tendons.

[0123] According to this treatment, the volume of each co-solvent (corresponding to an additive 11, 12, 13) to be applied is predetermined at 0.25 ml of co-solvent to treat 1 g of bone. This represents 80 ml of added volume, which is similar to the experimental conditions described above. The concentrations of the co-solvents are measured in ml of active ingredient per gram of material to be treated, particularly reduced by circulating them in a supercritical carbon dioxide stream and using a circulation loop 20.

[0124] The process shows an effectiveness of purification / decontamination despite the saving / reduction in active agent(s), which is transposable to any type of bone matrix or tissue of human or animal origin to be treated and with a different number of cycles and / or a choice (quantitative or qualitative) of different cleaning agents.

[0125] A comparison concerning bone generation was established for two respective types of blocks, each consisting of a block of spongy bone of porcine origin. The tests were carried out on rat calvaria for both types of blocks compared in order to regenerate a critical size defect. The blocks of the first type were treated (Test A) according to a process as described above, with three successive additives introduced in three treatment cycles with added carbon dioxide. The blocks of the second type were treated (Test B) according to a process that also involves three additives, each with a treatment of comparable duration, by a chemical action obtained in a different form, namely: after degreasing with supercritical CO2 under the same conditions, the blocks were successively soaked in a 35% hydrogen peroxide solution, a 1 mol / L sodium hydroxide solution, and a 99% ethanol solution.

[0126] The analysis of the results uses a parameter representative of the amount of bone generated in the region of interest measured by micro-computed tomography. This parameter is here the BV / TV parameter (bone volume to total volume), expressed as a percentage.

[0127] The results are as follows: % BV / TV 0 days 30 days 60 days Number of defects 12 12 8 Test A 15.8 22.3 25.9 Test B 16.7 16.5 18.3

[0128] A faster growth in the percentage of bone material occupied by the blocks from Test A, obtained according to the process, is observed. Thus, it is found that the action obtained with the process is more effective for bone regeneration than for bone chemically treated with a liquid-phase soaking process (Test B). Furthermore, the action of the treatment process (Test A) makes the material regenerate more rapidly. Regarding the results, statistically significant differences are obtained: - for the increase in bone regeneration with Test A, with p < 0.05 between J0 and J30 and p < 0.001 between J0 and J60; - for the comparison between Test A and Test B of bone regeneration with p < 0.05 at J60.

[0129] This disclosure is not limited to the embodiments described above, only by way of example, but encompasses all the variants that a person skilled in the art may consider in the context of the protection sought.

[0130] For example, although a reaction chamber in the VR volume has been illustrated in [Fig.1], a multi-chamber arrangement, each chamber housing at least one tissue 2, can be provided using one or more reactors 1. In addition, controlled flow pulsations, for example during step 51 and / or one of steps 52a, 52b, 52c, can be envisaged.

[0131] Also, it is quite clear that the number of chemical additive(s) can vary depending on the treatment sought.

Claims

1. Demands A process for treating a tissue (2) of human or animal origin by extracting organic matter residues, in order to purify and / or decontaminate said tissue (2), the process using carbon dioxide in a supercritical state and a reactor (1) having an inlet (5) and an outlet (6), characterized in that the process comprises the steps essentially consisting of: - dispose (50) the tissue in an internal volume (VR) of the reactor (1); - pressurize (51) the reactor (1), by introducing into the internal volume (VR) carbon dioxide in the supercritical state via the inlet (5), so that the carbon dioxide in the supercritical state reaches a region (le) of contact with the tissue (2) and exits the reactor (1) via the outlet (6); - add at least one chemical additive (11, 12, 13) for purification and / or decontamination to the stream of liquid or supercritical carbon dioxide, circulated to reach the contact region (l), in order to achieve, within the internal volume (VR), the circulation of an additive-enhanced CO2 stream forming a treatment stream combining supercritical carbon dioxide as a solvent and at least one chemical additive (11, 12, 13) as a co-solvent; and - while maintaining said pressurization, circulating said additive-enhanced CO2 flow, away from the contact region (the), in a loop (20) which communicates with said inlet (5) of the reactor, thereby creating a recirculation contributing to making the treatment flow dynamic and to purifying and / or decontaminating the tissue (2), preferably by allowing the extraction and separation of a portion of the residues in the loop (20), said loop (20) in which the additive-enhanced CO2 flow circulates extending between a first end (21) of the loop connected to the outlet (6) of the reactor (1) and a second end (22) of the loop connected to the inlet (5) of the reactor (1), including a condenser (7) or cooling means for cooling the fluid to be recirculated and liquefying the carbon dioxide, the addition of at least one chemical additive being carried out between the outlet of the condenser or cooling means and the inlet (5) of the reactor (1),the process also allows the carbon dioxide circulating in the loop (20) to be successively converted to a gaseous state, downstream of a, regulating valve (10), then in liquid state, by being subjected to a cooling stage in the condenser (7).

2. A method according to claim 1, wherein carbon dioxide is pumped by a pump (8) provided in the loop, the loop (20) extending outside the reactor (1), between the outlet (6) and the inlet (5) of the reactor, and wherein at least one chemical additive (11, 12, 13) is introduced into the loop (20) upstream or downstream of a heat exchanger (9) which heats a carbon dioxide stream from the pump (8) provided in the loop to reach or exceed the supercritical temperature of carbon dioxide, the method comprising connecting a separation stage (S1, S2) so that the latter, provided in bypass with respect to the loop (20), is accessible by a determined setting of one or more valve(s), without being accessible in the recirculation phase via the loop (20) to obtain said dynamic treatment stream.

3. A method according to claim 2, wherein each additive among the at least one chemical additive (11, 12, 13) is added, preferably in liquid form, by an additional pump (P2) separate from the pump (8) provided in the loop, and wherein at least one of the following ratios is predetermined: - a first ratio between the total volume of additive delivered by the additional pump (P2) and the mass of the tissue (2) to be treated, the first ratio expressed in ml / g preferably being between 0.01 and 1; - a second ratio between the volumetric flow rate delivered by the additional pump (P2) and the volumetric flow rate delivered by the pump (8) provided in the loop, the second ratio preferably being between 1 / 5 and 1 / 100 and chosen taking into account the solubility of the chemical additive in carbon dioxide in the supercritical state, for example between 1 / 45 and 1 / 55.

4. A method according to any one of the preceding claims, comprising a step (52a, 52b, 52c) of circulating the solvent and co-solvent in a closed circuit within the circuit composed of the reactor (1) and the loop (20), by opening valves (V2, V4, V10, V7, V11) allowing recirculation in the loop (20) and keeping the reactor (1) pressurized, said reactor (1) being functionally coupled, at the inlet (5), to a valve (V2) for the reintroduction, into the internal volume (VR), of the CO2 additized flow recirculated via the loop (20); and in which at least one chemical additive (11, 12, 13), from a reservoir (RI, R2, R3), is introduced into a section of the loop (20) in which carbon dioxide in the supercritical state flows towards the inlet (5).

5. A method according to any one of the preceding claims, wherein during the addition, each chemical additive (11, 12, 13) is added into an injection line (LO) which joins a communication channel (L5) with the tissue contact region (1e) (2), such that each chemical additive (11, 12, 13) flows in the communication channel (L5) as a co-solvent of the supercritical carbon dioxide flowing in the reactor (1) to cross the contact region (1e), and wherein said communication channel (L5) forms a segment of the loop (20).

6. A method according to any one of the preceding claims, comprising: - before said addition, a stabilization phase in which carbon dioxide circulates in a loop in the circuit formed by the reactor (1) and the loop (20), while the pressure in the reactor has reached or exceeded a predefined threshold and the temperature has reached a target temperature range, so as to maintain the carbon dioxide in the supercritical state; and - during said addition, maintaining the loop circulation of carbon dioxide in said circuit.

7. A method according to any one of the preceding claims, comprising carrying out, in the pressurized state of the reactor (1), at least one chemical treatment in the contact region (1e), which is carried out in a reactor operating mode with said recirculation, by using the loop (20) which is connected to the inlet (5) and outlet (6) of the reactor (1), whereby a chemical additive (11, 12, 13) is: - introduced, by using a co-solvent pump and in an open state of an additive introduction valve (V12) connected to the loop (20), into a section of the loop in which carbon dioxide circulates in the liquid or supercritical state, at a flow rate volume at least ten times smaller, for example fifty times smaller, compared to that of the carbon dioxide circulating in the loop (20); - during a complementary recirculation step without addition, put to recirculate in the reactor (1) and the loop (20), in the closed state of the valve (VI2) for the introduction of additive.

8. A method according to claim 7 when it depends on claim 2, wherein, between two chemical treatments combining supercritical carbon dioxide and co-solvent, without moving the fabric (2) and without stopping a carbon dioxide circulation pump (8) in the reactor (1) and in the loop (20), it is provided that: a / said separation stage (SI, S2) connected in bypass of a section (L2) of the loop (20), this stage being accessible by opening a bypass valve (V8) and closing a primary valve (V7) located downstream of the control valve (10); b / allow the co-solvent and the remaining residues to accumulate in the separation stage (SI, S2); c / purge the co-solvent and the remaining residues by valves (V9, V10) of the separation stage (SI, S2);d / deactivate the separation stage (SI, S2) by closing the bypass valve (V8) and opening said primary valve (V7) then restart a treatment with a second co-solvent.;

9. A method according to claim 7, wherein between two chemical treatments combining supercritical carbon dioxide and co-solvent, without moving the fabric (2), it is provided to: a / stop a carbon dioxide circulation pump (8) in the reactor (1) and in the loop (20), depressurize the reactor (1) and the loop (20); b / drain the reactor (1), using a lower drain port of the reactor which forms said outlet (6), and drain the loop (20); c / purge the reactor (1) and the loop (20) with compressed air; and d / optionally, rinse the reactor (1) with purified water by introducing purified water upstream of the reactor (1) and removing it downstream of the reactor (1), the direction of circulation of the purified water being able to be reversed.

10. A method according to any one of the preceding claims, wherein at least one chemical purification additive and / or decontamination is a first chemical additive, preferably oxygen peroxide, injected into the loop (20) during an addition step (52a) and introduced into the reactor (1) as a co-solvent of carbon dioxide in the supercritical state, in order to: - carry out a chemical treatment in the contact region (le) by circulating the first chemical additive in the reactor (1) as well as in the loop (20) which communicates with said reactor inlet (5), the process further comprising, in the pressurized state of the reactor (1), the steps consisting essentially of: - adding (52b) a second chemical purification / decontamination additive, preferably peracetic acid, into the loop (20) in order to introduce the second chemical additive into the reactor (1) as a co-solvent of carbon dioxide in the supercritical state;and - carry out a chemical treatment in the contact area (the) by circulating the second chemical purification / decontamination additive in the reactor as well as in the loop (20) which communicates with said reactor inlet, the second chemical additive being reintroduced into the reactor (1) via the loop (20) as a co-solvent of carbon dioxide in the supercritical state, during a recirculation step (53) with the second additive.;

11. A method according to any one of the preceding claims, wherein the pressure regulating valve (10), arranged in the loop (20) downstream of the reactor (1) in a direction of flow of the treatment stream and upstream of a supercritical carbon dioxide circulation pump (8), is actuated to activate one or more pressure drops and rises in the reactor (1), during recirculation.

12. Installation for treating a tissue or tissue matrix, of human or animal origin and in particular based on collagen, by a flow of carbon dioxide in a supercritical state, for carrying out the process according to any one of the preceding claims, the installation comprising: - a reactor (1) having an inlet (5) and an outlet (6), delimiting an internal volume (VR) to receive the tissue (2), the reactor (1) being able to be closed, pressurized and heated to maintain the supercritical state of the carbon dioxide flow; - a pump (8) and a heating device, designed and arranged to change carbon dioxide from a liquid state to a supercritical state, upstream of the inlet (5) of the reactor (1) in a direction of flow from the pump (8) to the inlet (5); - a condenser (7); - a circuit comprising a loop (20) passing through a point or region of introduction of carbon dioxide in liquid form, the loop (20) extending between a first end (21) of the loop connected to the outlet (6) of the reactor (1) and a second end (22) of the loop connected to the inlet (5) of the reactor (1), in order to allow a recirculation of fluid from the reactor, from the outlet (6) to the inlet (5), the pump (8) being disposed in the loop (20) between the first and second ends (21, 22) downstream of the condenser (7) which allows the carbon dioxide of the fluid from the reactor (1) to be recirculated to be liquefied; - a fluidic connection device (RC) with the loop (20) and associated with additive introduction means (P2, V12), to allow the addition of at least one chemical additive (11, 12, 13) for tissue purification and / or decontamination in a section of the loop (20) located downstream of the condenser (7) and upstream of the inlet (5), so that the connection device (RC) forms a mixer allowing a mixed flow combining supercritical carbon dioxide as a solvent and the additive as a co-solvent, to constitute a treatment flow from the loop and reaching the tissue (2) in the internal volume (VR); - a separation stage (SI, S2) connected to the reactor outlet and accessible via a bypass of the loop (20) when a bypass valve (V8) is open, said stage being able to be activated to allow the purging of the chemical additive (11, 12, 13) and a portion of the residues from the internal volume (VR), which are liquid and / or solid; and - an opening / closing assembly provided with valves (V2, V4, 10, V7, V11) arranged on the loop (20), said assembly being capable of being configured in a first open state, in which a pressure regulating valve (10) which is part of said valves allows the reactor (1) to be pressurized above a threshold exceeding the critical pressure of carbon dioxide, possibly exceeding 100 bar, to actuate said recirculation of fluid passing through the loop (20) which is a recirculation without passing through the separation stage (SI, S2), contributing to making the processing flow dynamic; the opening / closing assembly also being able to be configured: - in a second state compatible with an activation of said separation stage (SI, S2) without depressurization, - or in a third state compatible with a depressurization of the reactor (1) and of the loop (20), to evacuate the co-solvent and residues formed during the treatment of the tissue (2) and remaining in the internal volume (VR) and in the loop (20), which are liquid and / or solid.

13. Installation according to claim 12, wherein the loop (20) is provided with a filter (4) disposed at the outlet (6) of the reactor to retain solid residues, while the separation stage (SI, S2) disposed downstream of the filter (4) allows the co-solvent and residues evacuated from the reactor (1) to be separated by circulating in the loop (20), the pressure regulating valve (10) being disposed in the loop (20) between the filter (4) and two parallel sections (L2, L3) of the loop (20), one of which includes the separation stage (SI, S2).