RU2638796C1 - Method for obtaining of two-component preparation for treatment of joints damage by low-invasive introduction into joint bag and preparation obtained by this method - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for obtaining of a two-component preparation for treatment of joint damage by low-invasive insertion of the said preparation into the joint bag, in the form of two components, the main and initiating, consisting in the fact that, in order to obtain the main component of the preparation, the blood plasma is transferred with a syringe and a transient filter with a pore size sufficient to ensure sterility, to one of the departments of the cryopack and kept until complete freezing, then the frozen blood plasma is partly defrosted, the first thawed plasma fraction is transferred to the empty compartment of the cryopack and removed from the cryopack by a syringe connected to the pack port, and the cryoprecipitate of the blood plasma remaining in the cryopack is thawed, then the cells which are chondrocyte precursors, are resuspended in the cryoprecipitate of the blood plasma in an amount sufficient to provide a therapeutic effect, and to make the initiating component of the preparation, solution of calcium chloride and thrombin is mixed in a balanced saline solution with addition of aminocaproic acid in an amount not exceeding the maximum recommended dose for a single administration in the joint bag, under certain conditions. A two-component preparation for treatment of joint damage.

EFFECT: method allows for cells localization at the site of administration, allowing the cells to migrate from the glue to the damage site and maintain functional activity.

8 cl, 5 dwg

Description

The invention relates to the field of cell biology and biotechnology, in particular to methods for producing cellular preparations for use in traumatology, orthopedics and maxillofacial surgery.

Local post-traumatic cartilage injuries, as well as degenerative-dystrophic cartilage injuries, such as Koenig’s disease, stage 1-2 osteoarthritis are common diseases. In some countries, up to 22% of the population suffers from this group of diseases, which makes the search for approaches to repair cartilage defects one of the most relevant areas of modern orthopedics and traumatology. Degenerative processes in the articular cartilage tissue lead to pain and loss of function of various joints in both older people due to age-related changes and in young people who subject joints to high and ultra-high loads during training and competition.

At present, there is no adequate way to regenerate the articular surface, which would ensure a complete and long-term restoration of the structure and functional activity of the damaged joint. Two main approaches to the treatment of damage to articular cartilage are described: conservative and operative. Conservative treatment of joints is carried out on the basis of chondroprotective drugs or non-steroidal anti-inflammatory drugs, and has a very limited duration, as well as a number of contraindications and complications with prolonged use of these drugs. Surgical treatment of joints is very invasive, accompanied by a number of complications, such as infections, aseptic loosening of the components of the endoprosthesis, liner wear, pain, stress-shielding and osteolysis, and also often requires repeated surgery, which is impossible due to the age of the patient and the high invasiveness of the procedure. Thus, at present, there is a need to improve existing or search for fundamentally new methods of treating joint injuries.

One of the promising approaches to the treatment of joint injuries is the minimally invasive administration of autologous chondrocytes, prechondrocytes, or autologous or allogeneic mesenchymal stromal cells immobilized in special support systems.

To immobilize cells at the injection site, as well as to increase the therapeutic effect, cells can be resuspended in non-toxic, biodegradable liquids or gels. Such liquids include, for example, fibrinogen-based liquids and gels (fibrin glue, its commercial analogues - Tissucol®, Tisseel®, Baxter, USA).

Ahmed TA, Giulivi A, Griffith M, Hincke M. Fibrin glues in combination with mesenchymal stem cells to develop a tissue-engineered cartilage substitute // Tissue Eng Part A. - 2011 - Vol. 17 - No. 3-4 - p. 323-335 describes a fibrin glue with mesenchymal stem cells (MSCs) and a method for its preparation. The method consists in obtaining cryoprecipitate from donated blood using a CryoSeal® FS device (Asahi Kasei Medical Co., Ltd) and thrombin using a Thrombin Processing Device ™ device (Thermogenesis corporation), the fibrinogen concentration in the cryoprecipitate being 20.1 mg / ml, and the concentration of thrombin is 38 U / ml. To stimulate chondrogenic differentiation, MSCs are cultured in DMEM containing 10 ^-7 M dexamethasone, 50 mg / ml ascorbic acid 2-phosphate, 100 mg / ml sodium pyruvate, 40 mg / ml L-proline, 1% insulin-transferrin-selenium mixture (BD Bioscience Pharmingen), 100 U / ml penicillin, 100 mg / ml streptomycin and 10 ng / ml TGF-β2 (R&D Systems). 85 ml of cryoprecipitate is mixed with 30 ml of human MSCs or chondrocytes at a concentration of 2 × 10 ⁷ cells / ml, then 85 ml of thrombin is added. Obtained as a result of the implementation of the described method, the drug allows to immobilize cells, the precursors of chondrocytes. However, the described method requires additional expensive equipment, in addition, the preparation obtained as a result of the described method has a high concentration of fibrinogen, and the glue (scaffold, frame) is so dense that it prevents the free migration of cells.

In the article by Kim YS, Choi YJ, Suh DS, Heo DB, Kim YI, Ryu JS, Koh YG. Mesenchymal stem cell implantation in osteoarthritic knees: is fibrin glue effective as a scaffold? // Am J Sports Med. - 2015 - No. 43 (1) - c. 176-85 describes a method for producing fibrin glue containing MSCs, which consists in using the commercially available Greenplast kit fibrin glue (Greencross), supplied in two syringes, in a syringe containing a lyophilized preparation of fibrinogen from human plasma with a concentration of 71.5-126 5 mg / ml add aprotinin solution with a concentration of 1100 kallikrein inactivating units in ml, and into another syringe containing thrombin with a concentration of 4.9-11 mg / ml, dissolved in a solution of calcium chloride with a concentration of 13.9-15.6 mg / ml, add cell suspension stromal vascular fraction of adipose tissue containing MSCs in a ratio of 1: 1 by volume. To obtain fibrin glue, a cell-thrombin suspension is mixed with a fibrinogen solution in a ratio of 1: 1 by volume using the Duploject system at the time of the introduction of cartilage damage to the surface. The authors were able to show the restoration of cartilaginous tissue of the knee joint after arthroscopic administration of the fibrin glue preparation with MSC. However, the fibrin glue used in the described method contains aprotinin as a catalyst for the formation of a fibrin clot. It has now been shown that aprotinin is highly toxic and can cause damage to the kidneys and the cardiovascular system. The FDA recommends replacing aprotinin with aminocaproic acid preparations. Also, the stromal vascular fraction of adipose tissue used as the cellular component of fibrin glue contains a relatively small amount of MSCs. In the absence of differentiation induction, not all MSCs contained in the fraction used can differentiate in the chondrocytic direction. The concentration of fibrinogen in the adhesive obtained by the described method is high enough to immobilize the cells, but too high to allow them to freely move to the damaged surface of the joint and differentiate in this place. In addition, the described method uses a commercial source of fibrinogen - the GreenPlast preparation, that is, the material is allogeneic for the patient, which increases the risk of complications, increases the cost of the procedure, and creates dependence on unique manufacturers. The described method is the closest to the claimed invention and is selected as a prototype.

An object of the present invention is to provide a method for preparing a preparation for treating joint damage by minimally invasive insertion into an articular bag and providing localization of cells at the injection site, allowing cells to migrate from the injection site to the site of injury and maintain functional activity. This method should satisfy the requirements of sterility and purity sufficient for the clinical use of the drug.

The problem is solved in that in the method of obtaining the drug for the treatment of joint damage, two separate components are prepared - the main and the initiating, while to obtain the main component of the drug, fresh blood plasma is transferred using a syringe and a transition filter with a pore size sufficient to ensure sterility, in one of the compartments of the cryopackage and kept at a temperature of minus 80 ° С until completely frozen, then the frozen blood plasma is partially thawed at 4 ° С, the first thawed fraction is moved blood plasma into an empty compartment of the cryopackage and remove it from the cryopackage with a syringe connected to the port of the packet. The cryoprecipitate of blood plasma remaining in the cryopackage is thawed. Chondrocyte precursors are obtained from the patient’s adipose or bone tissue, or from the umbilical cord using the standard method described in the article: Aizenshtadt A.A., Enukashvili N.I., Zolina T.L., Aleksandrova L.V., Smolyaninov A .B. Comparison of proliferative activity and phenotype of MSCs obtained from bone marrow, adipose tissue and umbilical cord // Bulletin of the North-West State Medical University. I.I. Mechnikov - 2015. - T. 7. - No. 2. - S. 14-22. In one embodiment of the method, the cells are incubated in the presence of factors that stimulate differentiation in the chondrogenic direction, for example, in a mixture of 2 × 10 ^-8 M dexamethasone, 0.5 μM ascorbic acid, 0.5% proline, 0.5% pyruvate and 5 nM TGF-β3. Before use, the cells are resuspended in cryoprecipitate of blood plasma. For the manufacture of the initiating component of the preparation, 0.1-1.0% calcium chloride, 32.5-500 U / ml thrombin is prepared in a balanced saline solution, for example, physiological saline, with the addition of aminocaproic acid in an amount not exceeding the maximum recommended dose for a single administration joint bag. Before administration, the main and initiating components of the drug are placed in different departments of the system for the simultaneous administration of drugs, for example, a Duploject syringe.

The drug obtained by the implementation of the proposed method is presented in the form of two components - the main and initiating, while the main component of the drug is cryoprecipitate obtained from blood plasma, with a fibrinogen concentration of 2-18 mg / ml, into which cells, chondrocyte precursors are added, and the initiating component of the drug contains 32.5-500 U / ml of thrombin, 0.1-1.0% calcium chloride and aminocaproic acid in an amount not exceeding the maximum recommended dose for a single injection into the joint bag, in the SBA ansirovannom saline, such as saline. Both components of the drug are intended to be introduced into the joint bag simultaneously and independently, for example, using a “Duploject” syringe with two separate exits or a combination of “Duploject” with a long double-lumen needle, while mixing the components of the drug and the formation of glue occurs immediately at the time of introduction into the articular bag.

Unlike the prototype, the inventive method is characterized by the use of conventional equipment such as a freezer and a refrigerator for the preparation of cryoprecipitate of blood plasma, and the use of a cryopackage, a vacuum tube and a transition filter with a pore diameter sufficient to ensure sterility, for example 0.22 microns, allows the whole cycle preparation of cryoprecipitate of blood plasma in a closed system, providing the sterility necessary for the drug intended for administration to the patient. The inventive method of obtaining cryoprecipitate of blood plasma allows you to prepare a drug with a fibrinogen content sufficient to localize the cells at the injection site, and allowing the cells to migrate to the surface of the damaged joint. The resulting preparation of the proposed method contains a lower concentration of fibrinogen, 2-18 mg / ml instead of 71.5-126.5 mg / ml. This allows you to create the necessary density of fibrin glue, sufficient to localize the cells at the injection site, while allowing the cells to migrate to the surface of the damaged joint. The number of cells introduced into the main component of the drug is not limited by the claimed method, and depends on the treatment method and the material used to obtain the cells. In the treatment of various joint injuries, a different number of cells, the precursors of chondrocytes, is required, therefore, so many of these cells are introduced into the drug that can produce a therapeutic effect. The use of MSCs induced in the chondrogenic direction by keeping differentiation inducers in solution, for example, in a mixture of 2 × 10 ^-8 M dexamethasone, 0.5 μM ascorbic acid, 0.5% proline, 0.5% pyruvate, and 5 nM TGF-β3 , allows to increase the number of active cells and their functional activity in relation to differentiation into chondrocytes and further repair of cartilage damage, and also accelerates the regeneration processes. In addition, unlike the prototype, the cells are part of the main component of the drug containing fibrinogen, since calcium chloride in the initiating component of the drug significantly reduces the functional activity of the cells. Calcium chloride in the initiating component of the drug provides polymerization of fibrinogen in a concentration of not less than 0.1%, however, in a concentration above 1.0% it causes pronounced apoptosis of chondrocytes [Amin A.K., Huntley JS, Bush PG, Simpson AHRW, Hall AC Chondrocyte death in mechanically injured articular cartilage - the influence of extracellular calcium. Journal of Orthopedic Research. 2009. - Vol. 27. - P. 778-784]. The inventive method does not limit the amount of aminocaproic acid introduced into the initiating component of the drug. Aminocaproic acid inhibits fibrinolysis and, thus, improves the characteristics of fibrin glue. The amount of aminocaproic acid administered can be limited only by medical indications, which will determine the maximum dose for administration into the joint bag without pronounced side effects on the patient's body.

In one embodiment of the method, autologous blood plasma is used to obtain the main component of the drug, for this, blood is taken from a patient in a vacuum tube with an anticoagulant, such as heparin, EDTA or any other, blood plasma and blood cells are separated by centrifugation. The use of autologous blood plasma avoids the risks associated with the use of allogeneic materials and obtain a completely non-immunogenic drug.

In another embodiment of the method, umbilical cord plasma is used to obtain the main component of the drug, for this, umbilical cord blood is collected in a vacuum tube with an anticoagulant, such as heparin, EDTA or any other, blood plasma and blood cells are separated by centrifugation. The use of cord blood plasma can reduce the risk of donor infection, as Long-term stored, pre-tested material is used. In addition, umbilical cord blood plasma is significantly richer in biologically active substances that improve cell survival and graft formation compared with the blood of an adult donor.

The invention is illustrated by the following graphic materials.

In FIG. Figure 1 shows the fibrinogen content in platelet-free plasma (PPP), cryoprecipitate and residual plasma (A) and the dependence of its concentration in cryoprecipitate on the number of freeze-thaw cycles (B). The data are presented in the form of mean, error of the mean, and values of all elements of the sample are also given. * - significant differences, p <0.05.

In FIG. 2 shows the polymerization time of fibrin glue depending on the concentration of thrombin.

In FIG. Figure 3 presents microphotographs of MSCs in fibrin glue on the 4th day of cultivation at a concentration of fibrinogen in cryoprecipitate of blood plasma 18 g / l (A) and on the 2nd (B) and 4th (C) day of cultivation at a concentration of fibrinogen in cryoprecipitate plasma blood 2.4 g / l, and the control culture of MSCs on the 4th day of cultivation (G). The scale is 100 microns.

In FIG. 4 presents the results of the determination of the immunophenotype of MSCs migrated from fibrin glue, before differentiation (MSC) and on the 7th day (Prechondrocytes). (A) A table with the results of determining the percentage of cells positive for the presence of surface markers of MSCs. (B) A histogram of the intensity of staining of cells with antibodies to the surface marker CD44. An increase in CD44 expression, characteristic of the onset of differentiation in the chondrocytic direction, was shown.

In FIG. 5. presents micrographs of MSC stained with antibodies to type II collagen (I) before differentiation (A) and on the 7th day after the start of differentiation in the chondrocytic direction (B). The nuclei are stained with DAPI (II). The combined image is shown in FIG. 5 (III).

The method was carried out as follows.

An example embodiment of the invention.

Preparation of thrombin solution.

40 μl of 5% aminocaproic acid, 20 μl of 10% calcium chloride, and 940 μl of phosphate-buffered saline or saline (0.9% sodium chloride) were mixed. Thrombin (Baxter or Technology-Standard, Novosibirsk) was dissolved to a concentration of 500 U / ml or 32.5 U / ml in the resulting mixture. The polymerization time of fibrin glue with the introduction of the drug depends on the concentration of thrombin (Fig. 2).

Preparation of cryoprecipitate of blood plasma.

Venous blood was collected in a standard 9 ml blood collection tube containing an anticoagulant such as lithium heparin, then the tube was transported to a laboratory. Blood was centrifuged, for example, in a centrifuge СМ-6М (Elmi, Latvia), 15 min at 2200 g. Without opening the tube, the supernatant, or plasma, was taken using a hypodermic needle connected to a syringe. A hypodermic needle was removed, a filter with a pore diameter of 0.22 μm with the corresponding side, luer female, was connected to a syringe with plasma. A needle with a caliber of no more than 21-23G was connected to the second output of the filter, luer male. A cryopackage port, for example, Macopharma, was introduced to this needle, after which plasma was squeezed out of the syringe into one of the cryopackage compartments. The collected plasma was frozen at -80 ° C, for example, in a Sanyo MDF-U74V Ultra-low Temperature Laboratory Freezer freezer. A cryopack with frozen plasma was placed in a container with a temperature of + 2-4 ° C, for example, in a pharmaceutical refrigerator HF-250-3 "POZIS". As soon as the contents of the bag reached the consistency of “gruel” with a small amount of liquid, squeezed “gruel” with chilled tweezers, squeezing the liquid fraction into the empty compartment of the cryopackage, then quickly removed it through the cryopackage port. After that, the remaining cryopackage was thawed. Fibrinogen levels were determined using the Klaus coagulometric method.

Production and cultivation of MSCs

MSCs were isolated from adipose tissue or umbilical cord, as described in: Aizenshtadt A.A., Enukashvili N.I., Zolina T.L., Alexandrova L.V., Smolyaninov A.B. Comparison of proliferative activity and phenotype of MSCs obtained from bone marrow, adipose tissue and umbilical cord // Bulletin of the North-West State Medical University. I.I. Mechnikov - 2015. - T. 7. - No. 2. - S. 14-22. Samples of subcutaneous adipose tissue were crushed, then kept at 37 ° C in a 0.2% solution of collagenase in phosphate-buffered saline, and then centrifuged, for example, in a SM-6M centrifuge (Elmi, Latvia), 10 min at 400 g. The supernatant was removed, the pellet was placed in a 75 cm ² culture vial, into which 10-15 ml of the complete culture medium was previously added, for example, MEM-α / Advanced Stem Cell Media (HyClone, Germany) supplemented with 20% fetal bovine serum for MSCs or Advanced Supplement for Stem Cells Serum Replacement (HyClone, Germany). The vials were placed in a CO ₂ incubator, for example BBD 6220 (Thermo, Germany).

The umbilical cord vessels were washed with a balanced saline solution, such as Versen's solution, using a syringe, filled with a 0.2% collagenase solution in the same buffer, washed and re-filled with a collagenase solution in PBS. Then, the umbilical cord was clamped on both sides, placed in a Petri dish and kept in a shaker-incubator, for example, ES-20 (Biosan, Latvia), for 1 h at a temperature of 37 ° C and gentle stirring. After this, the collagenase solution was collected and the umbilical vein was thoroughly washed with physiological saline, for example, PBS. The collagenase solution and saline after washing were collected and centrifuged, for example, in a centrifuge СМ-6М (Elmi, Latvia), 10 minutes at 400 g. The supernatant was removed, the pellet was placed in a 75 cm2 culture vial, into which 10-15 ml of the complete culture medium was previously added, for example, MEM-α or Advanced Stem Cell Media (HyClone, Germany) supplemented with 20% fetal bovine serum for MSCs or Advanced Supplement for Stem Cells Serum Replacement (HyClone, Germany). The vials were placed in a CO2 incubator, for example, BBD 6220 (Thermo, Germany).

When the cells reached 50% confluency, factors stimulating chondrogenic differentiation were added to the cell culture vial, for example, a mixture of 2 × 10 ^-8 M dexamethasone, 0.5 μM ascorbic acid, 0.5% proline, 0.5% pyruvate and 5 nM TGF-β3. The minimum incubation time was 1 hour, the maximum - 5-7 days.

Preparation of fibrin glue with cells localized in it.

Sterilized 1 ml of cryoprecipitate of blood plasma, for example, using a syringe attached to the port of the cryopackage. The culture fluid was removed from the cell culture vials of the chondrocyte precursors intended for transplantation, then a 0.25% solution of trypsin pre-heated to 37 ° C was added to the vials, and immediately the cell fluid from the vial was collected in a test tube, centrifuged, for example , in a centrifuge СМ-6М (Elmi, Latvia), 7 minutes at 300-500 g. The supernatant was removed, a 2.5% sterile solution of albumin in saline was added to the tube, and the pellet was resuspended. Then the liquid from the vial was collected in a test tube, centrifuged, for example, in a centrifuge СМ-6М (Elmi, Latvia), 7 minutes at 300-500 g. The supernatant was removed, 1 ml of blood plasma cryoprecipitate was added to the tube, and the pellet was resuspended.

Selected 1 ml of cryoprecipitate of blood plasma with cells in a syringe. In another syringe, 1 ml of the initiating solution was taken.

0.5 ml of blood plasma cryoprecipitate was mixed with the cells and 0.5 ml of the initial solution using the Duploject application kit (Baxter), a system of two syringes with a common needle, or using two standard 2 ml medical syringes, directly introducing the components into the wells 6 well plate.

After completion of the polymerization of fibrin glue with chondrocyte precursor cells, 2 ml of the complete culture medium was added to the wells of the plate, the plates were closed and kept at 37 ° C for 7 days. Change of environment was carried out every 2 days. The cells were studied both growing in the gel and those who left it. Using light microscopy, we evaluated the appearance of cells, the number of cells growing outside the gel, the number of single cells and colonies in the gel. After 7 days, the fibrin clot was removed, and the cells that left it (approximately 20% of the total number of cells in the well) were used to study the ability of cells from fibrin glue to differentiate in the chondrocytic direction.

Cells that left the gel were cultured until they reached 60-70% confluency and then transferred to a chondrogenic culture medium containing 10% chondrogenic serum substitute (hMSC Mesenchymal Stem Cell Chondrocyte Differentiation Medium, Lonza) and cultured according to the manufacturer's protocol. Using light microscopy methods, cell morphology was evaluated on days 2 and 4 from transfer to a chondrogenic medium (Fig. 3). At a low concentration of fibrinogen (B), part of the cells leaves the fibrin clot within 48 hours after the start of cultivation. Further, such cells are spread on a substrate and proliferate. On day 4, the culture of cells that left the fibrin glue (B) is morphologically indistinguishable from the control (MSCs isolated and cultured according to the standard protocol) (G). Immunophenotype cells were evaluated by flow cytometry methods before and after the start of differentiation (Fig. 4). In the process of differentiation, an increase in the expression of the marker CD44, a receptor for hyaluronic acid, is observed, which is characteristic of chondrocytes.

The success of differentiation was also evaluated by the appearance of cells stained with antibodies to collagen II (Fig. 5). The onset of expression in type II collagen cells is an early sign of chondrogenic differentiation.

The resulting preparation can be introduced into the articular bag by arthroscopy or arthrotomy to treat congenital or acquired degenerative cartilage damage. The list of diseases to which this method can be applied includes (but is not limited to) the following diseases: acute or chronic osteoarthritis, cartilage tissue formation during endoprosthetics, arthrosis, Koenig's disease, ankylosis.

The applicant asks to consider the submitted application materials "A method for producing a two-component preparation for the treatment of joint damage by minimally invasive insertion into the joint bag and a preparation obtained by this method" for the grant of a patent of the Russian Federation for an invention.

Claims (8)

1. A method of obtaining a two-component drug for the treatment of joint damage, carried out by minimally invasive injection into the joint bag, the drug in the form of two components - the main and the initiating one, which consists in transferring blood plasma using a syringe and a transition filter with pore size sufficient to ensure sterility, in one of the compartments of the cryopackage and kept at a temperature of minus 80 ° C until completely frozen, then frozen plasma VI is partially thawed at 4 ° С, the first thawed blood plasma fraction is transferred to the empty compartment of the cryopackage and it is removed from the cryopackage by a syringe connected to the packet port, and the cryoprecipitate of the blood plasma remaining in the cryopackage is thawed, then the precursor cells are resuspended in the cryoprecipitate of blood plasma , in an amount sufficient to provide a therapeutic effect, and for the manufacture of the initiating component of the drug, a solution of 0.1-1.0% calcium chloride, 32.5-500 U / ml thrombin is mixed saline solution with the addition of aminocaproic acid in an amount not exceeding the maximum recommended dose for a single injection into the joint bag. 2. The method according to claim 1, wherein the autologous blood plasma of the patient is used as the blood plasma. 3. The method according to claim 1, wherein the cord blood plasma is used. 4. The method according to claim 1, wherein mesenchymal stem cells obtained from the adipose or bone tissue of a patient are used as cells precursors of chondrocytes. 5. The method according to claim 4, wherein the cells, the precursors of chondrocytes, are kept in the presence of factors stimulating differentiation in the chondrogenic direction before use. 6. The method of claim 1, wherein mesenchymal stem cells obtained from the umbilical cord are used as cells precursors of chondrocytes. 7. The method according to claim 6, wherein the cells, the precursors of chondrocytes, are kept in the presence of factors stimulating differentiation in the chondrogenic direction before use. 8. A two-component preparation for the treatment of joint damage, carried out by minimally invasive introduction into the joint bag, obtained by the method according to any one of paragraphs. 1-7.

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