Process for maintaining activity of autologous bone in deep hypothermia long-term cryopreservation

Abstract

The invention discloses an activity maintaining process for deep low-temperature long-term cryopreservation of autologous bones, and belongs to the technical field of autologous bone preservation, and the autologous bones comprise the following raw materials in parts by weight: 1-2 parts of L-proline, 5-8 parts of a PVP / WS2 nano composite material, 0.1-0.5 part of a matrix metalloproteinase (MMPs) inhibitor, 1-2 parts of antifreeze protein (AFPs), 1-2 parts of an impermeable protective agent, 1-2 parts of trilauryl methyl ammonium chloride and 2-3 parts of an oxygen carrier. According to the autologous bone cryopreservation culture medium, the use of a traditional cryoprotectant DMSO can be reduced, the toxicity of the traditional cryoprotectant DMSO to cells and tissues is reduced, the damage of ice crystals to the cells in the cryogenic long-term cryopreservation process is effectively relieved, the integrity of collagenous fibers in bone tissues is maintained, and the reduction of the activity of growth factors is inhibited.

Description

Technical Field

[0001] The invention relates to the technical field of autologous bone preservation, and in particular to a process for maintaining the activity of autologous bone during deep-low temperature long-term cryopreservation. Background Art

[0002] After removal, autologous bone tissue (such as skull flap) is cleaned and processed, and then gradually cooled to -20°C, -40°C, and -80°C, and stored in liquid nitrogen (-196°C) to maintain its biological activity and structural integrity. Dimethyl sulfoxide (DMSO) is usually used as a protective agent in cryopreservation medium to reduce damage to bone tissue during cryopreservation.

[0003] Deep cryopreservation will affect the integrity of collagen fibers in bone tissue. In bone tissue stored at -196°C, the structure of collagen fibers will change, manifesting as thickening, separation, and formation of cavities. This change is more obvious 24 hours after recovery.

[0004] Growth factors in bone tissue, such as vascular endothelial growth factor (VEGF), may experience a certain degree of activity reduction or structural changes during deep cryopreservation due to factors such as the toxicity of cryoprotectants and temperature changes. In bone tissue stored at -196°C, its ability to induce host cells to form new bone may be weakened due to cell death and reduced growth factor activity. Summary of the invention

[0005] As described in the above-mentioned prior art, one of the purposes of the present invention is to provide an autologous bone cryopreservation medium, which can reduce the use of the traditional cryoprotectant DMSO, reduce its toxicity to cells and tissues, effectively slow down the damage of ice crystals to cells during deep low temperature long-term freezing, maintain the integrity of collagen fibers in bone tissue, and inhibit the decrease of vascular endothelial growth factor activity.

[0006] The second purpose of the present invention is to provide a process for maintaining the activity of autologous bone during deep cryopreservation for a long time, which can effectively preserve autologous bone for a long time.

[0007] One of the purposes of the present invention is achieved by the following technical solution: An autologous bone cryopreservation medium, the raw materials according to weight parts include: 1-2 parts of L-proline, PVP / WS 2 5-8 parts of nanocomposite materials, 0.1-0.5 parts of matrix metalloproteinase (MMPs) inhibitor, 1-2 parts of antifreeze proteins (AFPs), 1-2 parts of non-permeable protective agents, 1-2 parts of trilauryl methylammonium chloride, and 2-3 parts of oxygen carriers.

[0008] Further, the raw materials in parts by weight include: 2 parts of L-proline, PVP / WS2 8 parts of nanocomposite materials, 0.3 parts of matrix metalloproteinase (MMPs) inhibitor, 1 part of antifreeze proteins (AFPs), 1 part of non-permeable protective agent, 2 parts of trilauryl methylammonium chloride, and 3 parts of oxygen carrier.

[0009] Furthermore, the non-permeable protective agent is one of sucrose and trehalose.

[0010] Furthermore, the matrix metalloproteinase (MMPs) inhibitor is one of ilomastat and marimastat.

[0011] Furthermore, the PVP / WS 2 The method for preparing the nanocomposite material comprises the following steps: S1. WS 2 Nanosheets and PVP were dissolved in deionized water to form a mixed solution; S2, placing the mixed solution in a hydrothermal reactor and reacting at 150-200° C. for several hours; S3. After the reaction is completed, the mixture is cooled to room temperature, and the unreacted substances are removed by centrifugation and washing to obtain the PVP / WS 2 Nanocomposite materials.

[0012] Furthermore, the oxygen carrier is one of cerium dioxide (CeO2) and vanadium oxide (V2O5).

[0013] The second object of the present invention is achieved by adopting the following technical solution: The process of maintaining the activity of autologous bone during long-term cryopreservation includes the following steps: S1. obtaining fresh autologous bone tissue, and washing the fresh autologous bone tissue using a pulse flushing technique to remove blood, fat and impurities on the surface, thereby obtaining washed autologous bone tissue; S2, pre-freezing: immersing the cleaned autologous bone tissue in a formaldehyde fixative containing methanol for 1 hour, after fixation, putting the fixed autologous bone tissue into a sterile plastic bag, adding the autologous bone cryopreservation medium into the sterile plastic bag, covering the fixed autologous bone tissue, evacuating the sterile plastic bag, sealing it, placing the sealed sterile plastic bag into a magnetic field generating device, and then freezing them together at 4° C. for 20 minutes, applying a static magnetic field during the pre-freezing process; S3. Deep-low temperature long-term freezing: After pre-freezing, the sealed sterile plastic bag is continuously cryopreserved at -20°C and -80°C in two steps, and finally the sealed sterile plastic bag is stored in liquid nitrogen at -196°C.

[0014] Furthermore, the magnetic field generating device is a Helmholtz coil or a permanent magnet. In the generated static magnetic field, it is ensured that the magnetic field uniformly covers the sample.

[0015] Furthermore, the magnetic field strength generated by the magnetic field generating device is 40-80 mT.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The invention provides an autologous bone cryopreservation culture medium, which selects L-proline and non-permeable protective agent with low toxicity cryoprotectant components to reduce chemical toxicity to bone cells. PVP / WS2 nanocomposite materials integrate the functions of inhibiting the formation, growth and rapid ablation of ice crystals, so they can significantly reduce ice crystal damage during cell cryopreservation, greatly improve cell preservation efficiency, and effectively slow down ice crystal damage to cells during deep-low temperature long-term freezing. During cryopreservation, adding MMPs inhibitors to cryopreservation medium can reduce the inhibitory effect of matrix metalloproteinases on collagen fiber degradation; using positively charged compounds (such as trilaurylmethylammonium chloride, TDMAC) to neutralize the negative charge on the surface of proteoglycans, thereby reducing its hydrophilicity and preventing collagen fibers from being affected by ice crystals during freezing, thereby maintaining the integrity of collagen fibers in bone tissue; at the same time, using oxygen carriers can use some oxygen carriers to maintain the hypoxic environment in cells, and vacuum encapsulation can reduce the oxygen content. The combination of the two can better maintain the hypoxic environment in cells, activate HIF, promote the expression of VEGF, and inhibit the reduction of growth factor activity. After autologous bone transplantation, it can improve bone conductivity and bone inductivity.

[0017] (2) The present invention provides a process for maintaining the activity of autologous bone in deep cryopreservation for a long time. The use of a magnetic field assisted freezing with a magnetic field strength of 40 to 80 mT can delay the fluidity of water in the sample. The use of a formaldehyde fixative containing methanol can reduce the shrinkage of the tissue to a certain extent. At the same time, formaldehyde can cross-link with amino acid residues such as lysine in collagen fibers to form a stable network structure, thereby reducing the structural damage of collagen fibers caused by physical factors such as ice crystal formation during the freezing process and enhancing the stability of collagen fibers to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0019] Figure 1The figure is a microscopic observation result of osteoblasts cultured for one week after cryopreservation of autologous bone by using the autologous bone cryopreservation medium provided in Example 1 through an activity maintenance process of deep low temperature long-term cryopreservation of autologous bone, taking tissue samples and culturing them in cell culture wells; Figure 2 The figure shows the microscopic observation results of osteoblasts cultured for three weeks after cryopreservation of autologous bone using the autologous bone cryopreservation medium provided in Example 1 through an activity maintenance process for long-term cryopreservation of autologous bone at deep low temperature, and then taking tissue samples and culturing them in cell culture wells. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with specific embodiments. It should be noted that, under the premise of no conflict, the embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0021] Matrix metalloproteinase (MMPs) inhibitors were ilomastat and marimastat, purchased from MedChemExpress.

[0022] Example 1

[0023] This embodiment provides an autologous bone cryopreservation medium, the raw materials of which include, by weight: 2 parts of L-proline, PVP / WS 2 8 parts of nanocomposite materials, 0.3 parts of matrix metalloproteinase (MMPs) inhibitor ilomastat, 1 part of antifreeze proteins (AFPs), 1 part of non-permeable protective agent sucrose, 2 parts of trilaurylmethylammonium chloride, and 3 parts of oxygen carrier cerium dioxide.

[0024] In this embodiment, the interaction between PVP and water molecules: PVP contains amide groups, which can form hydrogen bonds with water molecules, thereby reducing the free movement of water molecules and reducing the formation of ice crystals.

[0025] In this embodiment, the non-permeable cryoprotectant also has low cytotoxicity, and can further reduce the free movement of water molecules by increasing the viscosity of the autologous bone cryopreservation medium, and synergize with PVP to reduce the formation of ice crystals and enhance the ice crystal inhibition effect.

[0026] In this embodiment, L-proline is a permeable cryoprotectant that can quickly penetrate the cell membrane, enter the cell, bind to the water molecules in the cell, inhibit the formation of ice crystals at low temperatures, reduce the loss of water in the cell, and protect the structure and function of the protein in the cell.

[0027] In this embodiment, the application of antifreeze protein is as follows: antifreeze protein is extracted from cold-resistant organisms and added to autologous bone cryopreservation medium to inhibit the formation and growth of ice crystals, and can reduce the damage of ice crystals to chondrocytes and proteoglycan structures, and reduce the damage of ice crystals to collagen fibers.

[0028] In this embodiment, the inhibition of matrix metalloproteinases (MMPs): adding MMPs inhibitors to the cryopreservation medium can reduce the degradation effect of matrix metalloproteinases on collagen fibers.

[0029] In this embodiment, the negative charge is neutralized by using a positively charged compound (such as trilauryl methyl ammonium chloride, TDMAC) to neutralize the negative charge on the surface of proteoglycan, thereby reducing its hydrophilicity and preventing the collagen fibers from being affected by ice crystals during the freezing process.

[0030] In this embodiment, during the cryopreservation process, oxygen carriers can be used to maintain the hypoxic environment in the cells, and vacuum encapsulation can reduce the oxygen content. The combination of the two can better maintain the hypoxic environment in the cells, activate HIF, and promote the expression of VEGF.

[0031] In this embodiment, the preparation method of the PVP / WS2 nanocomposite material comprises the following steps: S1. WS 2 Nanosheets and PVP were dissolved in deionized water to form a mixed solution; S2, placing the mixed solution in a hydrothermal reactor and reacting at 150-200°C for several hours; S3. After the reaction is completed, cool to room temperature, remove unreacted substances by centrifugation and washing to obtain PVP / WS 2 Nanocomposite materials.

[0032] In this embodiment, the PVP / WS2 nanocomposite material integrates the functions of inhibiting the formation, growth and rapid melting of ice crystals, thus significantly reducing ice crystal damage during cell cryopreservation and greatly improving cell preservation efficiency. Specifically, the research results show that: 1) During the cooling process, the PVP / WS2 nanocomposite material can regulate ice crystal nucleation, significantly reducing the supercooling degree of the autologous bone cryopreservation medium, thereby significantly reducing the ice crystal damage suffered by the sample during the cooling process.

[0033] 2) Based on the adsorption-inhibition effect, it was found that PVP / WS2 nanocomposites can selectively adsorb to the ice crystal interface, thereby inhibiting the recrystallization of ice crystals during the rewarming process; 3) Based on the photothermal conversion effect of nanomaterials, the rewarming process: During the rewarming process, the frozen bone tissue is placed under a light irradiation device and the WS2 The photothermal conversion effect quickly melts ice crystals, rapidly warms up bone tissue, and reduces damage during the rewarming process.

[0034] This embodiment also provides a process for maintaining the activity of autologous bone during long-term cryopreservation at a deep temperature, including the following steps: S1. obtaining fresh autologous bone tissue, and using pulse flushing technology to clean the fresh autologous bone tissue to remove blood, fat and impurities on the surface, thereby obtaining cleaned autologous bone tissue; S2, pre-freezing: the cleaned autologous bone tissue is immersed in a formaldehyde fixative containing methanol for 1 hour. After fixation, the fixed autologous bone tissue is placed in a sterile plastic bag, an autologous bone cryopreservation culture medium of this embodiment is added to the sterile plastic bag to cover the fixed autologous bone tissue, the sterile plastic bag is evacuated and sealed, and the sealed sterile plastic bag is placed in a Helmholtz coil of a magnetic field generating device, and then frozen together at 4°C for 20 minutes. During the pre-freezing process, a static magnetic field is applied with a magnetic field strength of 60mT to ensure that the magnetic field evenly covers the sample. Experiments show that the application of a magnetic field assisted freezing with a magnetic field strength of 40 to 80mT can delay the fluidity of water in the sample, and the best effect is achieved when the magnetic field strength is 60mT.

[0035] S3. Deep-low temperature long-term freezing: After pre-freezing, the sealed sterile plastic bags are continuously cryopreserved at -20℃ and -80℃ in two steps, and finally the sealed sterile plastic bags are stored in liquid nitrogen at -196℃.

[0036] In this embodiment, formaldehyde is a commonly used tissue fixative, which mainly forms stable chemical bonds by cross-linking with amino groups in proteins, thereby enhancing the stability of tissue structure. In bone tissue, collagen fibers are one of the main protein components. Formaldehyde can cross-link with amino acid residues such as lysine in collagen fibers to form a stable network structure, reduce the structural damage of collagen fibers caused by physical factors such as ice crystal formation during freezing, and enhance the stability of collagen fibers to a certain extent. To reduce problems such as tissue hardening and deformation. For example, the use of formaldehyde fixatives containing methanol (such as Roti®-Histofix) can reduce tissue shrinkage to a certain extent.

[0037] In this embodiment, although the sterile plastic bag can provide a sterile environment, there is still air and moisture in the package, which may lead to the formation of ice crystals. The effect of vacuum sealing in this embodiment is as follows: Reduce moisture content: Vacuum sealing removes air from the packaging bag, which also reduces the moisture content in the package. During the freezing process, moisture is the main source of ice crystal formation. By reducing moisture, the formation of ice crystals can be significantly reduced.

[0038] Rapid freezing: The vacuum environment can accelerate the freezing process and cool the sample quickly. Rapid freezing helps to form smaller ice crystals, which are less damaging to cells and tissue structures. This is because small ice crystals grow more slowly and will not cause severe mechanical damage to cell membranes and organelles like large ice crystals.

[0039] Less space for ice crystals to grow: Vacuum sealing reduces the air space around the sample, leaving ice crystals with less room to grow during their formation. This helps limit the size and number of ice crystals, which reduces damage to the sample structure.

[0040] Reduced mechanical damage: The formation and growth of ice crystals can cause mechanical damage to cells and tissue structures. Small ice crystals cause less damage to cell membranes and organelles, while large ice crystals may cause cell rupture and damage to tissue structure. Vacuum sealing indirectly protects the structural integrity of the sample by reducing the formation of ice crystals.

[0041] Maintaining the integrity of cell membranes: Vacuum sealing can reduce the mechanical pressure of ice crystals on cell membranes, thereby maintaining the integrity of cell membranes.

[0042] Reduce solute effect: During the freezing process, as the water freezes, the solute concentration in the unfrozen portion increases, which can cause cell dehydration and protein denaturation. Vacuum sealing can reduce the freezing of water, thereby alleviating this solute effect and protecting the structure of cells and tissues.

[0043] Example 2

[0044] This embodiment provides an autologous bone cryopreservation medium, the raw materials of which include, by weight: 1 part of L-proline, PVP / WS 2 5 parts of nanocomposite materials, 0.1 parts of matrix metalloproteinase (MMPs) inhibitor Marimastat, 1.5 parts of antifreeze proteins (AFPs), 2 parts of non-permeable protective agent trehalose, 1 part of trilaurylmethylammonium chloride, and 2 parts of oxygen carrier vanadium oxide.

[0045] In this embodiment, PVP / WS 2 The method for preparing the nanocomposite material comprises the following steps: S1, dissolving WS2 nanosheets and PVP in deionized water to form a mixed solution; S2, placing the mixed solution in a hydrothermal reactor and reacting at 150-200°C for several hours; S3. After the reaction is completed, cool to room temperature, remove unreacted substances by centrifugation and washing to obtain PVP / WS 2 Nanocomposite materials.

[0046] This embodiment also provides a process for maintaining the activity of autologous bone during long-term cryopreservation at a deep temperature, including the following steps: S1. obtaining fresh autologous bone tissue, and using pulse flushing technology to clean the fresh autologous bone tissue to remove blood, fat and impurities on the surface, thereby obtaining cleaned autologous bone tissue; S2, pre-freezing: immerse the cleaned autologous bone tissue in a formaldehyde fixative containing methanol for 1 hour, and after fixation, put the fixed autologous bone tissue into a sterile plastic bag, add an autologous bone cryopreservation medium of this embodiment into the sterile plastic bag, cover the fixed autologous bone tissue, evacuate the sterile plastic bag, seal it, put the sealed sterile plastic bag into a Helmholtz coil of a magnetic field generating device, and then freeze them together at 4° C. for 20 minutes. During the pre-freezing process, apply a static magnetic field with a magnetic field strength of 40 mT to ensure that the magnetic field evenly covers the sample; S3. Deep-low temperature long-term freezing: After pre-freezing, the sealed sterile plastic bags are continuously cryopreserved at -20℃ and -80℃ in two steps, and finally the sealed sterile plastic bags are stored in liquid nitrogen at -196℃.

[0047] Example 3

[0048] This embodiment provides an autologous bone cryopreservation medium, the raw materials of which include, by weight: 1.5 parts of L-proline, PVP / WS 2 7 parts of nanocomposite materials, 0.5 parts of matrix metalloproteinase (MMPs) inhibitor Marimastat, 2 parts of antifreeze proteins (AFPs), 1.5 parts of non-permeable protective agent trehalose, 1.5 parts of trilaurylmethylammonium chloride, and 2.5 parts of oxygen carrier vanadium oxide.

[0049] In this embodiment, PVP / WS 2 The method for preparing the nanocomposite material comprises the following steps: S1. WS 2 Nanosheets and PVP were dissolved in deionized water to form a mixed solution; S2, placing the mixed solution in a hydrothermal reactor and reacting at 150-200°C for several hours; S3. After the reaction is completed, cool to room temperature, remove unreacted substances by centrifugation and washing to obtain PVP / WS 2 Nanocomposite materials.

[0050] This embodiment also provides a process for maintaining the activity of autologous bone during long-term cryopreservation at a deep temperature, including the following steps: S1. obtaining fresh autologous bone tissue, and using pulse flushing technology to clean the fresh autologous bone tissue to remove blood, fat and impurities on the surface, thereby obtaining cleaned autologous bone tissue; S2, pre-freezing: immerse the cleaned autologous bone tissue in a formaldehyde fixative containing methanol for 1 hour, and after fixation, put the fixed autologous bone tissue into a sterile plastic bag, add an autologous bone cryopreservation medium of the present embodiment into the sterile plastic bag, cover the fixed autologous bone tissue, evacuate the sterile plastic bag, seal it, put the sealed sterile plastic bag into a permanent magnet of a magnetic field generating device, and then freeze them together at 4°C for 20 minutes. During the pre-freezing process, apply a static magnetic field with a magnetic field strength of 80mT to ensure that the magnetic field evenly covers the sample; S3. Deep-low temperature long-term freezing: After pre-freezing, the sealed sterile plastic bags are continuously cryopreserved at -20℃ and -80℃ in two steps, and finally the sealed sterile plastic bags are stored in liquid nitrogen at -196℃.

[0051] Experimental example Autologous bone was cryopreserved using the autologous bone cryopreservation medium provided in Example 1 through the process of maintaining the activity of autologous bone cryopreservation at a deep low temperature for a long time. After 5 human tibiae that had been cryopreserved in liquid nitrogen at -196°C for more than 6 months in sterile plastic bags were revived, fine forceps were used to sample tissue samples with a diameter of 3 mm to 5 mm, and the tissue samples were cultured in cell culture wells. The osteoblasts were directly observed under a microscope after one and three weeks of culture to evaluate the activity of the osteoblasts. The results are as follows: Figure 1 and Figure 2 shown.

[0052] from Figure 1 It can be seen that after one week of culture, the growth of osteoblasts has been observed in Example 1; Figure 2 It can be seen that after three weeks of culture, a large number of osteoblasts were observed in Example 1, indicating that it has good osteogenic ability when used in autologous transplantation.

[0053] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. An autologous bone cryopreservation medium, characterized in that The raw materials include, by weight: 1-2 parts of L-proline, 5-8 parts of PVP / WS2 nanocomposite material, 0.1-0.5 parts of matrix metalloproteinase inhibitor, 1-2 parts of antifreeze protein, 1-2 parts of non-permeable protective agent, 1-2 parts of trilauryl methyl ammonium chloride, and 2-3 parts of oxygen carrier.

2. The autologous bone cryopreservation medium according to claim 1, characterized in that: The raw materials include, by weight: 2 parts of L-proline, 8 parts of PVP / WS2 nanocomposite materials, 0.3 parts of matrix metalloproteinase inhibitors, 1 part of antifreeze protein, 1 part of non-permeable protective agent, 2 parts of trilauryl methylammonium chloride, and 3 parts of oxygen carriers.

3. The autologous bone cryopreservation medium according to claim 1, characterized in that: The non-permeable protective agent is one of sucrose and trehalose.

4. The autologous bone cryopreservation medium according to claim 1, characterized in that: The matrix metalloproteinase inhibitor is one of ilomastat and marimastat.

5. The autologous bone cryopreservation medium according to claim 1, characterized in that: The preparation method of the PVP / WS2 nanocomposite material comprises the following steps: S1, dissolving WS2 nanosheets and PVP in deionized water to form a mixed solution; S2, placing the mixed solution in a hydrothermal reactor and reacting at 150-200° C. for several hours; S3. After the reaction is completed, the mixture is cooled to room temperature, and unreacted substances are removed by centrifugation and washing to obtain the PVP / WS2 nanocomposite material.

6. The autologous bone cryopreservation medium according to claim 1, characterized in that: The oxygen carrier is one of cerium dioxide and vanadium oxide.

7. A process for maintaining the activity of autologous bone during long-term cryopreservation at low temperatures, characterized in that: The following steps are involved: S1. obtaining fresh autologous bone tissue, and washing the fresh autologous bone tissue using a pulse flushing technique to remove blood, fat and impurities on the surface, thereby obtaining washed autologous bone tissue; S2, pre-freezing: immersing the cleaned autologous bone tissue in a formaldehyde fixative containing methanol for 1 hour, after fixation, putting the fixed autologous bone tissue into a sterile plastic bag, adding an autologous bone cryopreservation medium according to any one of claims 1 to 6 into the sterile plastic bag, covering the fixed autologous bone tissue, evacuating the sterile plastic bag, sealing it, placing the sealed sterile plastic bag into a magnetic field generating device, and then freezing them together at 4° C. for 20 minutes, and applying a static magnetic field during the pre-freezing process; S3. Deep-low temperature long-term freezing: After pre-freezing, the sealed sterile plastic bag is continuously cryopreserved at -20°C and -80°C in two steps, and finally the sealed sterile plastic bag is stored in liquid nitrogen at -196°C.

8. The process for maintaining the activity of autologous bone during long-term cryopreservation at a deep temperature as claimed in claim 7, characterized in that: The magnetic field generating device is a Helmholtz coil or a permanent magnet.

9. The process for maintaining the activity of autologous bone during long-term cryopreservation at a deep temperature as claimed in claim 8, characterized in that: The magnetic field strength generated by the magnetic field generating device is 40-80 mT.

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