RU2802139C1 - Method and an apparatus for sterilization of tendon transplants - Google Patents

Abstract

FIELD: medical biotechnology.

SUBSTANCE: invention can be used to obtain a sterile cryopreserved allogeneic tendon graft for subsequent use in traumatology, orthopedics, maxillofacial surgery, reconstructive microsurgery. The method of sterilization of tendon grafts is as follows: the tendons are impregnated with a cryopreservative solution, the graft is packed by placing the graft first in the first polyethylene cryopack with perforations perforated for gas and liquid, then in the second cryopack made with at least one hole permeable for gas and liquid, then the cryopacks with the graft are placed in a medical bag intended for sterilization in a gaseous medium, permeable in closed form for the corresponding sterilizing agent and impermeable to microorganisms, the medical bag with the graft is sealed, followed by sterilization in a supercritical carbon dioxide medium. During the sterilization process, the pressure is increased to 100 atm, the sterilization process is completed by lowering the pressure to the initial value at a rate of not more than 0.4 atm per minute. An apparatus for sterilizing tendon grafts is also disclosed.

EFFECT: invention is aimed at obtaining a sterile allogeneic tendon graft with the possibility of long-term storage at ultra-low temperatures, while maintaining the native tissue structure and physical and mechanical properties.

23 cl, 11 dwg, 3 tbl

Description

The field of technology to which the invention belongs

The invention relates to medicine, namely to medical biotechnology, and can be used to obtain a sterile cryopreserved allogeneic tendon graft for subsequent use in traumatology, orthopedics, maxillofacial surgery, reconstructive microsurgery.

State of the art

Ensuring biological safety is an important criterion in the production of transplants based on allogeneic tissues. An integral characteristic of biologically safe products is their sterility, the absence of infectious and immunological complications when using a transplant. Many grafts based on allogeneic tissues (bone, dura mater, pericardium) perform barrier and biomechanical functions without active functional load. On the contrary, the use of allogeneic tendons implies their active participation in the functioning of the musculoskeletal system, which requires the preservation of the structural and functional properties of the tendon after all transplant production procedures, including sterilization, which results in significant damage to the connective tissue structure of the transplant [G.C. Soaresa, D.A. Learmonth, M.C. Vallejo, S. P. Davila, P. González, R.A. Sousa, A.L. Oliveira, Supercritical CO2 technology: The next standard sterilization technique? // Materials Science and Engineering. 2019. 99: 520-540]. In this regard, it is relevant to develop an effective method for tendon sterilization, which ensures the preservation of biomechanical functions, including the preservation of the strength characteristics and integrity of the tendon connective tissue, the structure of the fibers in the graft. In addition, common methods of sterilization of medical devices, as a rule, irreversibly impair the ability of DNA to replicate and transcribe due to the influence of sterilizing factors that equally affect both pathogenic microorganisms and the transplant's own cells. In this regard, it is important to develop a method that ensures the preservation of the viability of tendon cells during sterilization to ensure normal engraftment and functioning of the graft.

The prior art method and apparatus for sterilizing tendon grafts using gamma radiation and an electron beam [E.Y. Elenes, S.A. Hunter Soft-tissue allografts terminally sterilized with an electron beam are biomechanically equivalent to aseptic, nonsterilized tendons //J Bone Joint Surg Am. 2014. Vol.96, No.16. R. 1321-1326]. The impact on the tendons is carried out by radiation of 17.1-21.0 KGy, followed by lyophilization of the obtained grafts. In this case, an industrial installation with a linear particle accelerator Electron Beam Accelerator (LAE-10; 10 MeV) is used for sterilization, containing an emitter, a linear accelerator, a microwave power supply system, a high-voltage power supply, an automated control system unit, a heat exchanger with water, forevacuum post TVP-K (mobile). The linear accelerator contains a working chamber with a movable belt for graft placement. For sterilization, the grafts are placed in individual impermeable bags. The sterilization process is carried out at a temperature of about -70°C, maintained using dry ice. The average duration of the sterilization procedure is 30 seconds. However, this method requires preliminary drying of the tendons, does not take into account the possible damage to the topography of collagen, the formation of protein crosslinks under the action of radiation during processing, and does not contain data on the histological picture of transplants after sterilization. In addition, the lyophilization of the tendon used in the manufacture of the graft causes fragility of the collagen fibers, which leads to a significant decrease in the biomechanical properties of the tendon grafts. It has been shown that after lyophilization in the tendons there is a breakdown and fragmentation of collagen fibers, the initial topography of collagen is disturbed, collagen is homogenized in large areas of the graft and the formation of numerous areas of destroyed tissue leads to cell death. Lyophilized tendons lose up to 40% of their original strength, while the diameter of the fibers changes by 3-4 times compared to the original - lyophilization and rehydration cause both thinning of the fibers and their sharp swelling. All this leads to a significant disruption of the original fiber architectonics and negatively affects the final clinical effect of allografts.

Promising for the sterilization of allografts is the use of supercritical carbon dioxide (SC CO ₂ ). Supercritical fluid (SCS) is a special aggregate form of matter at pressure and temperature above its critical point. SCS combine the properties of liquid and gas. In particular, SCS, due to their low viscosity, have a high penetrating ability, close to gases, as a result of which they can easily diffuse into tissues. At the same time, SCS have a density close to liquids, have a high dissolving potential, and can be considered as an environmentally friendly, non-toxic replacement for organic solvents. Sterilization in the medium of SC CO ₂ makes it possible to achieve the same effect of inactivation of viruses, bacteria, microscopic fungi and spores as with ionizing radiation.

In particular, the prior art method and apparatus for sterilizing tendon grafts [T. Baldini, K. Caperton, M. Hawkins, E. McCarty Effect of a novel sterilization method on biomechanical properties of soft tissue allografts // Knee Surgery, Sports Traumatology, Arthroscopy. 2016. Vol. 24. P. 3971-3975], including exposure of packed grafts to supercritical carbon dioxide. Sterilization is carried out on an installation containing a pneumatically controlled booster gas connected to a CO ₂ cylinder; taps, capillaries and pressure gauge for supplying CO ₂ to the reactor; 600 ml reactor with temperature control and pressure relief valve. However, this publication lacks information on sterilization conditions, including supercritical carbon dioxide treatment parameters, and does not show the effect of sterilization on the connective tissue structure.

The prior art method and apparatus for sterilizing tendons using supercritical carbon dioxide in a liquid medium [A. White, D. Burns, T. W. Christensen Effective terminal sterilization using supercritical carbon dioxide // Journal of Biotechnology. 2006. 123: 504-515]. Sterilization of allogeneic grafts based on soft tissues is carried out in the presence of a special reagent containing peracetic acid at a concentration of 13.5% to 18.5% and hydrogen peroxide at a concentration of 4.5% to 6%; before the sterilization procedure, the chamber with grafts is moistened within 2 hours. Sterilization of tendon grafts is carried out at a temperature of 35°C for 2 hours at a pressure of 97 atm. The method is carried out using a sterilization apparatus, which includes a standard carbon dioxide cylinder, an air compressor, a valve and filter system, a reactor, a system for removing carbon dioxide.

This method makes it possible to obtain biologically safe grafts, however, it requires the use of reagents that can disrupt the structure of collagen in the tendons. This publication also lacks information on the conditions of sterilization, including the duration and level of pressure during sterilization, does not show the effect of sterilization on the structure of the connective tissue, and there is no data on the histological picture of grafts after sterilization. In addition, the known method cannot be used for sterilization of grafts intended for cryopreservation, does not take into account the need for quarantine grafts.

Closest to the claimed invention is a method and apparatus for sterilizing allogeneic tissue grafts [ Perrut, M. (2012). Sterilization and virus inactivation by supercritical fluids (a review). The Journal of Supercritical Fluids, 66, 359-371. doi:10.1016/j.supflu.2011.07.007 ], based on the treatment of biomedical products inside a special container (basket) with supercritical carbon dioxide. For sterilization, gaseous carbon dioxide is compressed with a compressor to a certain pressure. After that, the gas under pressure enters the autoclave with a thermostat, which is preheated to the temperature of the transition of carbon dioxide to the supercritical state with the possible introduction of a sterilizing additive (ethanol, hydrogen peroxide, acetic or peracetic acid, water). The initial sample (biopolymers, allogeneic transplants) is placed in a reactor, which is a container equipped with a quick closing system and having a container (basket) for loading and unloading the material. The reactor is also equipped with pathogen-tight filters through which supercritical carbon dioxide fluid is passed at a given temperature (from 20 to 80°C) and pressure (from 150-300 atm), the sterilization time is from 10 to 170 minutes. At the end of sterilization, the pressure is released to atmospheric pressure. The results of the study showed that the material obtained after processing does not contain viable viral pathogens (HIV-1, Sindbis, Polio Sabin type 1, Pseudorabies), as well as a wide range of studied bacterial pathogens. This approach makes it possible to sterilize non-lyophilized grafts inside sterile containers and obtain biosafe products. The publication noted that the problems of using this method of sterilization of allogeneic transplants are the loss of the sterile properties of the material when it is removed from the reactor (container), which does not allow the preparation of sterile transplants suitable for long-term storage by cryopreservation, requires the use of special containers (baskets). In addition, the method does not specify decompression modes, there is no information about the obtained structural integrity of the graft tissues, the viability of cells in their composition, and the functional properties of soft tissue grafts.

The technical problem is the elimination of the above disadvantages of the known technical solutions and the development of a method and apparatus for sterilizing allogeneic tendon grafts suitable for cryopreservation, with the production of a safe allogeneic tendon graft having structural and mechanical properties similar to native tendon, preserving cell and tissue viability after sterilization and long-term (up to 1 year) storage at ultra-low temperatures.

Disclosure of invention

The technical result, to which the claimed invention is directed, is to obtain a sterile allogeneic tendon graft with the possibility of long-term storage at ultra-low temperatures, while maintaining native tissue structure and physical and mechanical properties.

The technical result is achieved by using a method for sterilizing tendon grafts, according to which the tendons are treated (impregnated) with a cryopreservative solution, packaged by placing the graft first in the first plastic cryopack with perforations permeable to gas and liquid, then in the second cryopack, made with at least one an opening located preferably in the upper part of the cryopackage, also made permeable to gas and liquid, then the cryopackets with the graft are placed in medical bags intended for sterilization in a gaseous medium, permeable in closed form for the corresponding sterilizing agent and impermeable to microorganisms; medical packages with a transplant are hermetically sealed, followed by sterilization of the packed transplant in a reactor in an environment of supercritical carbon dioxide for at least 1 hour, while during the sterilization process the pressure is increased to at least 100 atm. (10.13 MPa), complete the sterilization process by lowering the pressure to the initial value at a rate of not more than 0.4 atm (0.041 MPa) per minute.

The tendons are treated with a preservative solution outside the cryopackage under sterile conditions (in a laminar cabinet). As a preservative solution, a solution is used that provides cryoprotection of living cells from freezing at minus 80°C, for example, 10% DMSO solution, or 10% PEG-400 solution, or a combination thereof. For processing, the tendon graft is immersed in cryopreservation and incubated for approximately 30 minutes, shaking occasionally. After impregnation of the tendon with a preservative solution, excess cryoprotectant is removed from the tissue surface with a sterile napkin. The tendon grafts are then transferred into cryopacks. As cryopackages, packages are used that are resistant to ultra-low temperatures, providing transparency and elasticity, as well as resistance to mechanical stress, for example, containers of the CryoStore series (Origen Biomedical Inc., USA), articles CS50N, CS250N, CS500N, CS750N, CS1000N, CS2000N , https://www.delrus.ru/catalog/kletochnye-tekhnologii/raskhodnye-materialy-dlya-kletochnykh-tekhnologiy/2-raskhodnye-materialy-dlya-podgotovki-stvolovykh-kletok-k-zamorozke/41740-meshok-dlya -glubokoy-zamorozki-kletok-i-tkaney-196-s-obyem-750-ml/. As medical packaging bags for sterilization, high-density paper bags are used, for example, 60 g / m ² , with a double layer of polyester / polypropylene film, for example, SteriT® combined bags in rolls (Vinar, Russia), TU 9398- 083-11764404-2011, https://www.deznet.ru/catalog/upakovochnyy_material/rulony_ploskie/83688/, or https://www.sigma-med.ru/catalog/emkosti-i-pakety-dlia-sterilizatcii- i-dezinfektcii/rulon_kombinirovannyy_so_skladkoy_sterit_400mm_kh_80mm_kh_100m/. For effective sterilization of packaged grafts, cryopackets must contain perforations or holes for the free exit of moisture and gases during sterilization, as well as during subsequent defrosting after cryopreservation. In one of the embodiments of the invention, the openings of the first tendon pack are preferably between 0.5 and 1.2 mm, which are relatively evenly distributed along the length of the graft. The hole in the second cryopackage can be formed in the form of a non-sealed part of the package, 0.5 - 5.0 cm long.

For sterilization simultaneously with a graft, for example, up to 20 cm in size, at least two tendon samples up to 1 cm in size are formed for subsequent testing of samples for sterility and toxicity after freezing, while the samples are packaged and processed simultaneously with the graft under similar conditions.

For sterilization in an environment of supercritical carbon dioxide, the packed graft is immersed in a pressurized reactor, which is a stainless steel container with a sealed lid containing a valve for supplying CO ₂ . In one of the embodiments of the invention, for sterilization, the reactor is heated to a temperature of 35 ° C, after which carbon dioxide is fed into it through a valve in the lid using a compressor to a total pressure in the reactor of 100 atm., The sterilization time is at least 1 hour at the specified temperature , complete the sterilization process by lowering the pressure (decompression) to the initial value at a rate of 0.1 to 0.4 atm. per minute, for example, at a speed of 0.125 atm. in a minute.

After sterilization, the tendons are frozen and stored at minus 80°C.

The technical result is also achieved when using a device for sterilization of tendon grafts, including at least one reactor equipped with a lid, made with the possibility of placing a packed tendon graft in it and carrying out sterilization in an environment of supercritical carbon dioxide at a pressure of up to 100 atm .; a pressure generator or an electric pump, on the one hand connected to a source of CO ₂ , on the other hand, to the reactor through a valve, or fitting, or capillary, equipped with an actuator capable of supplying carbon dioxide to the reactor under pressure to ensure the sterilization process and pressure relief in the reactor at the end of the sterilization process at a rate of not more than 0.125 atm/min.

The reactor is made in the form of a vertically oriented cylindrical container made of stainless steel with the possibility of placing a packed graft having a length of at least 25 cm, a width of at least 8 cm, a thickness of at least 3 cm, while the graft is first placed in the first polyethylene cryobag with perforations, permeable to gas and liquid. The first package with the graft is placed in the second cryopackage, made with at least one hole permeable to gas and liquid. The graft packaged in cryobags is placed in a medical packaging bag intended for sterilization in a gaseous medium, permeable in closed form for an appropriate sterilizing agent and impervious to microorganisms.

The installation may contain a block of several reactors interconnected by a system of valves and capillaries (fittings).

The reactor is made with the possibility of heating to the temperature of the transition of carbon dioxide to the supercritical state, while it can be equipped with a means of its electric heating, or a water jacket, or a PID temperature controller, or a liquid thermostat. The reactor may also be provided with a pressure gauge to monitor the supercritical carbon dioxide pressure during sterilization.

The plant may be provided with an actuator for controlling the pressure in the compressor, such as a high pressure fine adjustment valve or a pressure regulator.

The installation can be equipped with an interface to enable remote control of the sterilization process from peripheral devices, a control unit for a high-pressure supply pump to the reactor.

Thus, the technical result of the invention is achieved through the use of certain conditions for sterilization in an environment of supercritical carbon dioxide, including the use of "slow" decompression, during which tissue damage and integrity of the proposed packaging system are not allowed. At the same time, the use of sequential "triple" packing of the graft in bags, two of which are made with holes, allows you to keep the impregnation of the graft with a cryoprojector before, after and during the entire sterilization process without violating the sterility regime for subsequent storage at low temperatures.

Brief description of the drawings

The invention is illustrated by drawings and photographs, where figure 1 shows a diagram of an industrial installation for sterilizing tendon grafts, figure 2 shows a portable version of the installation, which was made for research; Figure 3 is a photograph of a manufactured portable unit for tendon graft sterilization, FIG. 4-11 are photographs of a tendon graft showing the steps in its packaging prior to sterilization.

Positions in the figures indicate: 1 - source of carbon dioxide (for example, a cylinder with CO ₂ ), 2 - valve for supplying carbon dioxide to the distribution unit or pump; 3 - a pressure generator or a high pressure electric pump, which can be made with a control unit, 4 - an actuator configured to supply carbon dioxide to the pressurized reactor to ensure the sterilization process, which in a particular case can be a high pressure control valve, or a distribution unit with a pressure gauge for supplying CO ₂ to the reactor, 5 - a reactor that can be equipped with valves for pressurizing and releasing it, as well as a temperature and pressure monitoring and control system, 6 - a laminar flow cabinet for placing reactors, 7 - an actuator , made with the possibility of releasing pressure in the reactor at the end of the sterilization process (automated pressure relief system), 8 - data exchange interface for remote control of the installation from peripheral devices, 9 - peripheral device with a software environment for monitoring the parameters of the installation (for example, a computer or tablet, or smartphone), 10 - means of heating the reactor (thermostat).

Implementation of the invention

The method can be implemented using an installation for sterilization with supercritical carbon dioxide, as shown in figures 1 and 2. Figure 1 shows an embodiment of an industrial installation with a set of reactors and a remote control system for the sterilization process, figure 2 shows a portable (or laboratory) embodiment installation.

The installation contains at least one reactor 5 with a lid, a pressure generator or an electric pump 3, connected to a source of carbon dioxide 2, on the one hand, on the other hand, connected through an actuator 4 to the reactor with the possibility of supplying carbon dioxide under pressure to ensure sterilization process at a pressure of at least 100 atm with the ability to control the pressure and rate of carbon dioxide supply to the reactor.

In one embodiment, the plant may use a pressure generator, such as a HiP high pressure generator (https://www.highpressure.com/products/pump-products-and-systems/specialty-pumps-and-controllers/manual -pressure-pump-generators/), which is a manually operated piston pump.

In another embodiment of the invention, the unit may use an electric, high-performance, two-piston pump, such as the SFT-25 pump, equipped with pressure controls (https://www.supercriticalfluids.com/products/supercritical-fluid-extraction-products/sft-25 / )

A 40-liter cylinder with a membrane valve can serve as a source of CO ₂ in a portable version of the installation. In the industrial embodiment of the installation, other containers and reservoirs equipped with means of controlled gas supply to the generator can be used as CO ₂ sources.

The reactor 5 used in the installation is made with the possibility of placing a packed tendon graft in it and carrying out sterilization in an environment of supercritical carbon dioxide at a pressure of at least 100 atm. (10.13 MPa). In one of the embodiments of the invention, the reactor is made in the form of a vertically oriented cylindrical container made of stainless steel with the possibility of heating to a temperature not lower than 35°C. The reactor is equipped with a dial gauge to control the pressure of supercritical carbon dioxide during sterilization. If it is necessary to increase the productivity of the installation, a block of several reactors connected to each other using a system of valves and capillaries can be used (for example, https://www.highpressure.com/products/valves-fittings-tubing/taper-seal-valves-fittings -and-tubing/low-pressure-tubing/). To heat the reactor to the supercritical CO ₂ transition temperature and maintain the required temperature for a given time, the reactor is equipped with appropriate means 10, which can have a different design solution known from the prior art, providing its prescribed function. In one of the embodiments of the invention, metal, ceramic, or nozzle electric heaters or heating elements, for example, clamp or ring heaters, placed on the side of the outer surface of the reactor, can be used as such a means, ensuring a snug fit of the heater to the heated surface and contact of the heating elements with the housing reactor. The heating element in these devices is a high resistance wire. Clamp electric heaters are fixed to the reactor by tie-downs of two types (tie and “lugs”). Electric heaters are usually equipped with a thermocouple to control the temperature of the heated surface. Possible options for electric heaters that can be used in the claimed invention are presented on the websites of their manufacturers (see, for example, https://electro-nagrev.ru/catalog/promyshlenennye_nagrevateli/homutovye-nagrevateli/). The heating power in one of the embodiments of the installation can be controlled using the TRM10 PID controller with RS-485 (see, for example, https://owen.ru/product/trm10). The reactor can be heated by other means, for example, by immersing it in a water bath equipped with a liquid thermostat (see, for example, https://www.lauda.de/ru/termostatirujushchee-oborudovanie/termostaty/nagrevajushchie-termostaty/eco -s-prozrachnoi-vannoi/product/ECO-ET-15-S) to ensure that the water in the bathroom is heated to the required temperature (not lower than 35°C).

The reactor is configured to accommodate packaged grafts of at least 20x8x3 cm as shown in FIG. In this case, the package is essentially triple - it consists of three packages placed one inside the other: the first, a polyethylene cryopackage with perforations permeable to gas and liquid, in which the graft is placed; the second cryopackage, provided with at least one hole permeable to gas and liquid, and made with the possibility of placing the first package with the transplant; the third package - a medical packaging bag, made with the possibility of placing the second package in it. The stages of graft packaging are shown in Fig. 4 - 11.

The plant is equipped with actuators 4 and 7 to control the pressure and rate of carbon dioxide supply from the compressor to the reactor and pressure relief. In one of the embodiments of the invention, the installation is equipped with an actuator for controlling pressure (not lower than 100 atm) and pressure release rate (not coarser than 0.125 atm / min or 0.041 MPa) during the sterilization process, which is a high pressure fine adjustment valve (for example, https ://www.highpressure.com/products/valves-fittings-tubing/taper-seal-valves-fittings-and-tubing/taper-seal-needle-valves/), alone or in combination with a pressure regulator (e.g. https: //mvif.ru/regulyatory-davleniya-go-serii-b-pr50), or an automatic pressure regulator (for example, https://bimedis.com/thar-abpr-200-m506058) equipped with a pressure sensor, a fine stepper motor driven adjustments with PID based control system and remote control and monitoring capability from peripheral devices via communication interface. The actuator can be installed directly on the reactor lid, or connected to the reactor using a system of valves and high pressure capillaries.

The operation of the installation can be controlled remotely, for which it can be equipped with appropriate means of receiving and transmitting the corresponding signals using the Internet. In particular, the installation can be equipped with a data exchange interface 8 with peripheral devices 9 to enable remote control of the sterilization process from peripheral devices, the introduction of control parameters for all devices and actuators included in the installation, namely, a pressure generator (pump), a reactor and a mechanism pressure control and depressurization rate, in one software environment.

The unit can operate from mains or autonomous power supply.

Prior to sterilization, tendon specimens are mechanically cleaned of subcutaneous adipose tissue and muscle tissue. The blood of the tissue donor is sent to the study of the presence of bloodborne infections. Until the results of the studies are obtained, the allografts selected for further processing are placed in a physiological solution of an antibiotic, for example, vancomycin in an amount of 1 g per 100 ml of saline. solution - 0.9% sodium chloride. In the absence of bloodborne infections, the tendons are treated with a cryoprotectant, sterilized, frozen and stored at -80°C.

The preparation of the preservative solution is carried out in compliance with the rules of asepsis and antisepsis in a laminar flow cabinet. The cryopreservative 99.8% DMSO is diluted with DMEM to a final concentration of 10%, 100% PEG-400 is also diluted with DMEM to a concentration of 10% or 15%. A mixture of DMSO and PEG-400 solutions can be used as a cryopreservative. To prepare a mixture, for example, 10 ml of 100% PEG-400 is mixed with 5 ml or 10 ml of 99.8% DMSO and adjusted to the required concentration (10% PEG-400 + 5% or 10% DMSO) with DMEM medium to obtain 100 ml . final solution of cryopreservative. Impregnation of tendons with a cryoconservative solution can be carried out in separate containers. For better impregnation, containers with grafts can be placed on a shaker for about 20-30 minutes.

After 30 minutes under sterile conditions of the box, the grafts are removed from the cryopreservative solution, dried with napkins, removing excess moisture, and placed in perforated plastic cryopackets (Fig.5 and Fig.8), for example, a CryoStore container (Origen Biomedical Inc., USA), articles CS750N meshok-dlya-glubokoy-zamorozki-kletok-i-tkaney-196-s-obyem-750-ml/). After that, the cryopackages with the graft are placed in medical packages for sterilization, for example, the combined package 250 mm "SteriT®" (Vinar, Russia), TU 9398-083-11764404-2011 (https://www.deznet.ru/catalog/upakovochnyy_material /rulony_ploskie/83688/, or https://www.sigma-med.ru/catalog/emkosti-i-pakety-dlia-sterilizatcii-i-dezinfektcii/rulon_kombinirovannyy_so_skladkoy_sterit_400mm_kh_80mm_kh_100m/), and hermetically sealed (Fig.11). At the same time, for sterilization, at least two tendon samples up to 1 cm in size are formed simultaneously with a graft up to 20 cm in size for subsequent testing of samples for sterility and toxicity after freezing, the samples are packaged and processed simultaneously with the graft (Fig. 6-7, 9-10) under similar conditions.

After packaging, the grafts are sent for sterilization. Why grafts packed in bags are loaded into a stainless steel reactor, closed with a sealed lid equipped with a valve for supplying pressure. The reactor is heated to a temperature of 35°C, after which carbon dioxide is supplied to the reactor using a compressor to a final pressure in the reactor of 100 atm. The duration of sterilization is at least 1 hour.

The sterilization process is completed using slow decompression of the reactor to the initial pressure at a rate of 0.1 - 0.4 atm. per minute, preferably 0.125 atm. per minute, which can take from 12 to 24 hours. Upon completion of depressurization, the reactor in a laminar flow hood is opened, the tendon packs are removed and checked for visible damage. In the absence of damage, the grafts are labeled and sent to a freezer at a temperature of -80°C for further storage.

The claimed method using manufactured portable sterilization unit (see figure 2, 3) was obtained a number of grafts under various processing conditions in supercritical carbon dioxide.

The portable unit contained a carbon dioxide cylinder 1, a valve for supplying carbon dioxide 2 (High Pressure Equipment Co. (USA), part number 15-11AF1) to a distribution unit 4 (High Pressure Equipment Co. (USA) part number 15-15AF1) with a pressure gauge (MP-2U, NPO "Manometr" (Russia)), as well as a high-pressure pump 3 connected to it (High Pressure Equipment Co. (USA), article 68.5.75-15), installed in a laminar flow cabinet 6 (Holten Maxi Safe manufactured by Thermo Fisher Scientific (USA)), thermostat 10 (Lauda ECO ET 15S, article L001098, manufactured by Lauda Dr. R. Wobser Gmbh & Co (Germany)) with immersed reactors 5 (High Pressure Equipment Co. (USA) article OC-5) equipped with pressure relief valves 7 (High Pressure Equipment Co. (USA), item 15-12AF1), _CO2 flow meter (LZTM-15 (PRC)) and a clamp (vice) for opening the reactors ( Master 32721, Zubr (Russia)). The pressurized carbon dioxide cylinder was connected with a carbon dioxide valve using a stainless steel capillary to a distribution unit, which was connected to the pressurization pump and reactors by the same stainless steel tubes and valves. The reactors were placed in an autoclave with a thermostat, which, in turn, was placed in a laminar flow cabinet. After cooling the high pressure pump, the carbon dioxide supply valve was opened and the cylindrical capacity of the compressor was filled, after which carbon dioxide was fed into the reactors and compressed to a pressure of 100 atm. The valves on the distribution unit and on the valve for supplying carbon dioxide to the reactor were closed and the sterilization process began. After 1 hour, the valve on the carbon dioxide supply valve with a pressure gauge was opened to provide a controlled release of pressure. As soon as the pressure in the reactor was equal to atmospheric pressure, the supply and release valves were closed, and the treated graft was removed from the reactor.

Tendon sterilization experiments were carried out in several stages.

At the first stage, an effective cryoprotectant was selected for joint use during sterilization using supercritical carbon dioxide, which allows tendon grafts to be stored at ultralow temperatures without losing their structural and functional characteristics. We used tendon grafts m. tibialis anterior taken from tissue donors. For storage at ultralow temperatures, endocellular/penetrating cryoprotectants (dimethyl sulfoxide (DMSO), polyethylene glycol (PEG-400), glycerol) and exocellular/nonpenetrating cryoprotectants (glucose solution, albumin solution) were used. To assess the safety of tendons after thawing, the properties of grafts for rupture and stretching-shear were determined, the general morphology of the tendons, topography, compaction and integrity of collagen fibers, and the safety of cellular elements were assessed microscopically. In the presence of 10% DMSO, 10% or 15% PEG-400, a combination of 5% or 10% DMSO and 10% PEG-400, the structure of collagen fibers and cells did not undergo visible changes compared to the control, while in all experiments with non-penetrating cryopreservatives the topography and orientation of the fibers were clearly disturbed, and deformation of many cells in the tendons was observed. Thus, penetrating cryopreservatives based on 10% DMSO, 10% or 15% PEG-400, combinations (as a mixture) of 5% or 10% DMSO with 10% PEG-400 are the most effective for cryopreservation of tendon grafts.

Packaging of grafts before sterilization

When selecting packaging for tendon sterilization with supercritical carbon dioxide, non-perforated cryopackages were initially used. At the same time, it was noted that the supercritical fluid of carbon dioxide did not enter the cryopackages, and the grafts, following the results of bacterial seeding, were not sterile. In this regard, experiments were carried out using a perforated cryopackage as shown in FIG. 4., and a high-density paper medical bag. During decompression, the cryoprotectant came out of the perforations as a result of a relatively rapid (>1 atm/min) decrease in carbon dioxide pressure and the paper bag got wet. Such packages were sterilized or torn when removed from the reactor. In this connection, additional packaging was used, namely, another cryopackage with a hole. Such a three-layer package ensured complete sterilization of the biological material without violating the tightness of the outer package after decompression. Thus, high-quality sterilization of the tendon in supercritical carbon dioxide is possible when it is placed in a triple package - first in a cryopack with perforations, then in a cryopack with a hole of 0.5-5.0 cm, and then in a medical bag made of high-density paper, for example , 60 g/m2, with a double layer of polyester/polypropylene film.

At the next stage, the conditions for sterilization of tendon grafts using supercritical carbon dioxide were determined. Sterilization technology with supercritical carbon dioxide is characterized by the absence of toxicity, high penetrating power, pathogen inactivation, as well as minimal damaging effects on the structure of tissues and cells. [A. Bernhardt, M.Wehrl, B. Paul, T. Hochmuth, M. Schumacher, K. Schütz, M. Gelinsky Improved Sterilization of Sensitive Biomaterials with Supercritical Carbon Dioxide at Low Temperature // PLoS One. 2015; 10(6): e0129205]. At the same time, there is conflicting information in the publications regarding the effect of sterilization conditions on the biomechanical characteristics of the resulting graft.

To identify significant parameters of tendon sterilization in combination with selected cryopreservatives, bacteriological, histological and animal studies were carried out.

The tendon grafts were divided into 2 groups depending on the decompression conditions: one half of the samples were sterilized under slow decompression conditions (pressure was released in the mode from 0.1 to 0.4 atm per minute), the other half - under fast decompression conditions ( pressure relief was carried out in the mode of 10 atm per minute or more). Within each group, additional subgroups were identified to evaluate the effect of the duration of sterilization (1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 72 hours). Prior to sterilization, the tendon samples were immersed for 30 minutes in a cryopreservative pre-prepared in the required concentrations as described previously. After sterilization, the tendons were frozen and stored at -80°C for 14 days. Subsequently, the grafts were thawed and evaluated: sterility, tissue appearance, physical and mechanical properties, as well as the structural integrity and toxicity of the obtained tendons. The research results are presented in table 1.

Table 1. Quality assessment of tendon grafts after different sterilization regimens Treatment time with supercritical carbon dioxide fluid 1 hour 3 hours 6 hours 12 hours 24 hours 72 hours Evaluation parameters Slow pressure release Sterility + + + + + + Tissue toxicity - - - - - - Structural integrity on histological preparations + + + + + + Physical and mechanical properties (tensile strength) + + + + + + Preservation of tendon cells + + + + + + Rapid pressure relief Sterility - - - - - - Tissue toxicity Not conducted Structural integrity on histological preparations - - - - - - Physical and mechanical properties (tensile strength) - - - - - - Preservation of tendon cells - - - - - -

Testing tendon grafts for sterility

Grafts ready for sterility testing were divided into 3 groups: grafts after sterilization with slow (group 1) and fast (group 2) pressure release, and the control group - tendons that were not sterilized. After opening the packages with transplants in a laminar flow cabinet in compliance with the rules of asepsis and antisepsis, the samples were immersed in 2 bacteriological media - Sabouraud broth, thioglycol medium.

It should be noted that in group 2, the graft packages were either microdamaged or, due to the cavitation process, with a wet paper backing. Tissue sterility was assessed on days 7 and 14.

The presence of bacterial and fungal flora was revealed in groups 2, 3, regardless of the time of sterilization, both on the 7th and 14th days. This indicates that the method of tissue sterilization with rapid release of pressure does not ensure the sterility of the final graft. On the contrary, in the 1st group on days 7 and 14, no flora growth was detected, which proves the effectiveness of the method of sterilizing allogeneic tendons with a supercritical carbon dioxide fluid with slow decompression. It has also been proven that the time of sterilization does not affect the final result. However, taking into account the conditions for creating a supercritical medium (the temperature in the reactor is 35°C) and the toxic effect of cryopreservatives on the tissue, a minimum graft processing time in the reactor of 1 hour is sufficient.

Study of graft toxicity in cell culture

The study of the toxicity of tendon grafts was carried out in a culture of multipotent mesenchymal stromal cells (MMSCs) from the bone marrow of tissue donors of passages 3-9. Only grafts with proven sterility (slow decompression) were used to assess toxicity. Fragments of tendon grafts 1.0-1.5 cm long were placed in experimental wells of a 6-well plate and a suspension containing 50,000 MMSCs was added. In parallel, 50,000 MMSCs were injected into wells without tendon grafts (control). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, USA) at 37°C and 5% CO ₂ for 3 days. For microscopic analysis, the cells were stained with a vital fluorochrome dye based on tripaflavin and rhodamine. The total number of cells (thousands/cm ² ), their morphology, and the integrity of cell membranes (CCM, in points) were assessed.

An in vitro study showed that all types of transplants studied did not have a toxic effect on cells. In the control and in all experimental wells, after 3 days of cultivation, a subconfluent monolayer was formed with a cell density of 17-18 thousand/cm². MMSC had a characteristic fusiform appearance, the level of CCM in all the experimental ones was 34-36 points and did not significantly differ from that in the control.

Study of sterility and toxicity of allogeneic tendons in an animal model (rats)

The study was carried out on 4 laboratory animals (rats) with the permission of the local ethical committee of the GBUZ NII SP im. N.V. Sklifosovsky DZM. Pre-prepared allogeneic calcaneal tendons of rat paws were sterilized with supercritical carbon dioxide and cryopreserved. Sterilization mode - 100 atm., 35°C 1 hour, slow pressure release 0, 125 atm. in a minute. After thawing, allogeneic tendons were transplanted to rats in the region of the biceps femoris muscle, in a specially formed cavity on the right paw - the experimental paw. The left paw was not subjected to any influences - the control paw. Before transplantation, the tendon was washed for 15 minutes in a physiological solution of 0.9% NaCl from the cryopreservative. Sterility and toxicity were assessed on the 14th day. On the 14th day the animals were active, nutrition was not disturbed, physiological functions were normal. When examining the area of intervention: postoperative wounds without signs of inflammation, normal color.

This study showed that the grafted tendon has no toxicity and does not cause suppuration or necrosis of nearby tissues.

Macro- and microscopic analysis of graft tissue after sterilization

The macroscopic analysis of the grafts and their appearance differed from native tissue only in the group where rapid pressure release was used. Tendon samples, regardless of the time of sterilization, were edematous, enlarged, and in some cases, damage to the fibers was visible.

Histological analysis showed that in the group with slow decompression, collagen fibers retained their integrity and topography, were parallel oriented, and contained only local minor tears. The intensity of collagen autofluorescence along the entire length of the fibers corresponded to the norm. On the contrary, in the group with rapid decompression, severe edema was observed in the grafts, many fibers were decondensed with disintegration into separate fibrils and the formation of extensive ruptures. The intensity of collagen autofluorescence along the entire length of the fibers was 2.1-3.5 times lower than in native samples.

The preservation of tendon cells was also assessed in histological preparations.

Histological examination showed that after sterilization and cryopreservation procedures, tendon grafts were found to contain tendinocyte cells with normal structural organization. In most cells, the structure of cell nuclei and chromatin in their composition, the structure of the cytoplasm does not undergo visible changes compared to native tissue. The number of cells with a pycnotized (nonfunctional) nucleus, with deformations of the cytoplasm does not exceed 10-15% of their total number in the graft. Thus, the proposed methods of tendon treatment do not cause pronounced cell death in the composition of finished grafts.

Study of the physical and mechanical properties of allogeneic transplants

The mechanical properties of the samples were evaluated on a LLOYD Instruments LR5K Plus testing machine with a tensile rate of 5 mm/min. Because the rapid decompression group grafts did not meet the study parameters for sterility and structural integrity, it was decided not to include them in the strength study. In the slow decompression group, the grafts were thawed and washed in physiological sodium chloride solution for half an hour. During the study, the following parameters were determined: load at break, N; ultimate strain, %; rigidity, N/mm, ultimate stress, MPa. It was found that at different times of treatment of tendons with supercritical fluid in all experimental samples, the stiffness and ultimate stress did not differ significantly from similar values in the control (native tendons). The level of ultimate strain in all experimental samples was significantly lower than in the control (Table 2). The load at break in the samples processed for 1 hour did not significantly differ from the control, while in all other experimental groups (treatment for 3-10 hours), the load at break was 1.6-1.8 times lower than in the control and in samples treated for 1 hour (p<0.05). Thus, sterilization with supercritical carbon dioxide fluid for at least 1 hour is optimal.

Table 2. Effect of the duration of sterilization with supercritical carbon dioxide fluid on the mechanical characteristics of tendon grafts Processing time, h Breaking load, N Limit deformation, % Rigidity, N/mm Ultimate stress, MPa control 601±100 63±20 65±10 80±10 1 623±100 34±20* 70±10 85±10 3 427±60* 28±15* 80±10 82±10 6 363±50* 38±15* 78±10 69±10 10 332±50* 31±10* 83±10 75±10 *p<0.05 relative to control

When comparing the mechanical properties of the tendons, sterilized by the method specified in the prototype, and the method proposed by us, it was found that sterilization with a supercritical fluid of carbon dioxide for 1 hour according to the developed method allows you to better maintain the original mechanical parameters of the tendons (Table 3), while simultaneously allowing them to be carried out for a long time. storage.

Table 3. Comparison of tendon sterilization techniques with supercritical carbon dioxide fluid Processing time, h Breaking load, N Limit deformation, % Rigidity, N/mm Ultimate stress, MPa control 601±100 63±20 65±10 80±10 Prototype method 330±79* 40±10* 77±32* 90±15 Proposed-method 623±100** 34±10* 70±10 85±10 *p<0.05 relative to control
**p<0.05 relative to the prototype method

Thus, the proposed method makes it possible to obtain sterile and functionally effective allogeneic tendon grafts.

Claims (25)

1. A method for sterilizing tendon grafts, characterized in that the tendons are impregnated with a cryopreservative solution, packaged by placing the graft first in the first polyethylene cryopack with perforations perforated for gas and liquid, then in the second cryopack made with at least one permeable hole for gas and liquid, then the cryopackages with the graft are placed in a medical bag designed for sterilization in a gaseous medium, permeable in the closed form for the corresponding sterilizing agent and impermeable to microorganisms, the medical bag with the graft is sealed with subsequent sterilization in a supercritical carbon dioxide medium, while during the sterilization process, the pressure is increased to 100 atm, the sterilization process is completed by lowering the pressure to the initial value at a rate of not more than 0.4 atm per minute. 2. The method according to claim 1, characterized in that the tendons in cryopackages with a preservative solution are kept for impregnation for up to 30 minutes, preferably with periodic shaking. 3. The method according to claim 1, characterized in that a solution is used as a preservative solution that provides cryoprotection of living cells at minus 80°C. 4. The method according to claim 3, characterized in that a 10% DMSO solution, or a 10% PEG-400 solution, or a 15% PEG-400 solution, or a mixture of the mentioned solutions of DMSO and PEG-400, or a mixture containing solutions of 10% PEG-400 and 5% DMSO. 5. The method according to claim 1, characterized in that after impregnation of the tendons with a preservative solution, moisture is removed from the surface before packaging. 6. The method according to claim 1, characterized in that transparent elastic bags are used as the first and second cryopackets, resistant to mechanical stress, characterized by resistance to ultra-low temperatures. 7. The method according to claim 1, characterized in that paper bags with a double layer of polyester or polypropylene film are used as medical bags for sterilization. 8. The method according to claim 1, characterized in that for sterilization, simultaneously with a transplant up to 25 cm in size, at least two tendon samples up to 1 cm in size are formed for subsequent testing of samples for sterility and toxicity after freezing, while the samples are packed and processed simultaneously with the graft under similar conditions. 9. The method according to claim 1, characterized in that for sterilization in an environment of supercritical carbon dioxide, the packed transplant is immersed in a pressurized reactor, which is a stainless steel container with a sealed lid containing a valve, or fitting, or capillary for supplying CO ₂ gas . 10. The method according to claim 9, characterized in that for sterilization the reactor is heated to a temperature of 35 ° C, after which gas is supplied to it through a valve in the lid using a compressor to a total pressure in the reactor of 100 atm, the sterilization time is at least , 1 hour at the specified temperature, complete the sterilization process by lowering the pressure to the initial value at a rate of 0.125 atm per minute. 11. The method according to claim 1, characterized in that the openings of the first cryopackage for packing tendons have dimensions of 0.5-1.2 mm, which are evenly distributed along the length of the graft. 12. The method according to claim 1, characterized in that the hole in the second cryopackage is a non-sealed part of the package up to 5.0 cm long. 13. The method according to claim 1, characterized in that after sterilization, the tendon is stored at a temperature of minus 80 °C. 14. Installation for sterilization of tendon grafts, including at least one reactor, equipped with a lid, configured to accommodate a packed graft placed first in the first polyethylene cryopack with perforations permeable to gas and liquid, then in the second cryopack made with at least one hole permeable to gas and liquids, then into a medical packaging bag, and sterilization in an environment of supercritical carbon dioxide at a pressure of up to 100 atm, a pressure generator or an electric pump, on the one hand connected to a source of carbon dioxide (CO ₂ ), on the other hand, to the reactor through an actuator with the possibility of supplying carbon dioxide to the reactor under pressure to ensure the sterilization process and depressurize the reactor at the end of the sterilization process with speed not more than 0.4 atm/min. 15. Installation according to claim 14, characterized in that the reactor is made in the form of a vertically oriented cylindrical container made of stainless steel. 16. Installation according to claim 14, characterized in that it contains more than one reactor, interconnected by a system of valves and capillaries. 17. The installation according to claim 14, characterized in that the reactor is made with the possibility of heating to the temperature of the transition of carbon dioxide to the supercritical state. 18. Installation according to claim 17, characterized in that the reactor is equipped with means for its electrical heating, or a water jacket, or a PID temperature controller, or a liquid thermostat. 19. Installation according to claim 14, characterized in that the actuator is a high pressure control valve or pressure regulator. 20. Installation according to claim 14, characterized in that the reactor is equipped with a pressure gauge to control the pressure of supercritical carbon dioxide during sterilization. 21. Installation according to claim 14, characterized in that it is equipped with a data exchange interface to enable remote control of the sterilization process from peripheral devices. 22. The installation according to claim 14, characterized in that it is equipped with a control unit for a high pressure supply pump to the reactor. 23. Installation according to claim 14, characterized in that the reactor is configured to accommodate a packed graft having a length of at least 25 cm, a width of at least 8 cm, and a thickness of at least 3 cm.