Active maintenance process for long-term cryopreservation of autologous bone at deep low temperature

Abstract

The present invention discloses an active maintenance process for cryopreserving autologous bone at deep low temperature, belonging to the technical field of autologous bone preservation. The raw materials by weight include: 1-2 parts of L-proline, 5-8 parts of PVP / WS2 nanocomposite, 0.1-0.5 part of matrix metalloproteinase (MMPs) inhibitor, 1-2 parts of antifreeze protein (AFPs), 1-2 parts of non-permeating protective agent, 1-2 parts of trimethylammonium lauryl chloride, and 2-3 parts of oxygen carrier. This autologous bone cryopreservation medium 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 long-term deep low temperature cryopreservation, maintain the integrity of collagen fibers in bone tissue, and inhibit the decrease in the activity of growth factors.

Description

Technical Field

[0001] The present invention relates to the technical field of autologous bone preservation, and particularly to an active maintenance process for long-term cryopreservation of autologous bone at deep low temperature. Background Art

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

[0003] Deep low temperature preservation will affect the integrity of collagen fibers in bone tissue. In bone tissue preserved at -196°C, the structure of collagen fibers will change, showing phenomena such as thickening, separation, and formation of cavities, and this change is more obvious 24 hours after recovery.

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

[0005] As described in the above 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 long-term deep low temperature cryopreservation, maintain the integrity of collagen fibers in bone tissue, and inhibit the reduction of vascular endothelial growth factor activity.

[0006] Another purpose of the present invention is to provide an active maintenance process for long-term cryopreservation of autologous bone at deep low temperature, which can effectively preserve autologous bone for a long time.

[0007] One of the purposes of the present invention is achieved by adopting the following technical solution:

[0008] An autologous bone cryopreservation medium, the raw materials by weight include: 1 - 2 parts of L-proline, 5 - 8 parts of PVP / WS2 nanocomposite, 0.1 - 0.5 part of matrix metalloproteinase (MMPs) inhibitor, 1 - 2 parts of antifreeze protein (AFPs), 1 - 2 parts of non-permeating protectant, 1 - 2 parts of trimethyl lauryl ammonium chloride, and 2 - 3 parts of oxygen carrier.

[0009] Further, the raw materials by weight include: 2 parts of L-proline, 8 parts of PVP / WS2 nanocomposite, 0.3 part of matrix metalloproteinase (MMPs) inhibitor, 1 part of antifreeze protein (AFPs), 1 part of non-permeable protectant, 2 parts of trimethyl lauryl ammonium chloride, and 3 parts of oxygen carrier.

[0010] Further, the non-permeable protectant is one of sucrose and trehalose.

[0011] Further, the matrix metalloproteinase (MMPs) inhibitor is one of ilomastat and marimastat.

[0012] Further, the preparation method of the PVP / WS2 nanocomposite includes the following steps:

[0013] S1. Dissolve WS2 nanosheets and PVP in deionized water to form a mixed solution;

[0014] S2. Place the mixed solution in a hydrothermal reaction kettle and react at 150 - 200 °C for several hours;

[0015] S3. After the reaction is completed, cool to room temperature, and remove the unreacted substances by centrifugation and washing to obtain the PVP / WS2 nanocomposite.

[0016] Further, the oxygen carrier is one of cerium dioxide (CeO2) and vanadium pentoxide (V2O5).

[0017] The second object of the present invention is achieved by the following technical solution:

[0018] The process for maintaining the activity of autologous bone during long-term cryopreservation at deep low temperature includes the following steps:

[0019] S1. Obtain fresh autologous bone tissue, and use the pulsed flushing technique to wash the fresh autologous bone tissue to remove the blood, fat, and impurities on the surface to obtain the washed autologous bone tissue;

[0020] S2. Pre-freezing: Immerse the washed autologous bone tissue in a formaldehyde fixative containing methanol for 1 h. After fixation, put the fixed autologous bone tissue into a sterile plastic bag, add the autologous bone cryopreservation medium to the sterile plastic bag to cover the fixed autologous bone tissue, evacuate and seal the sterile plastic bag, put the sealed sterile plastic bag into a magnetic field generating device, and then freeze at 4 °C for 20 min together. During the pre-freezing process, apply a static magnetic field;

[0021] S3. Deep cryopreservation for a long term: After pre-freezing, the sealed sterile plastic bags are cryogenically frozen in two steps at -20°C and -80°C respectively, and finally the sealed sterile plastic bags are stored in liquid nitrogen at -196°C.

[0022] Further, the magnetic field generating device is a Helmholtz coil or a permanent magnet. In the generated static magnetic field, ensure that the magnetic field uniformly covers the sample.

[0023] Further, the magnetic field intensity generated by the magnetic field generating device is 40 - 80 mT.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] The autologous bone cryopreservation culture medium provided by the present invention selects L-proline and a cryoprotectant component with low toxicity of a non-permeating protectant, reducing the chemical toxicity to bone cells. The PVP / WS2 nanocomposite integrates the functions of inhibiting the formation, growth and rapid ablation of ice crystals, so it can significantly reduce the ice crystal damage during the cryopreservation of cells, greatly improve the cell preservation efficiency, and effectively slow down the damage of ice crystals to cells during long-term deep cryopreservation. During the cryopreservation process, adding an MMPs inhibitor to the cryopreservation culture medium can reduce the degradation of collagen fibers by inhibiting matrix metalloproteinases; using a positively charged compound (such as trimethyl dodecyl ammonium chloride, TDMAC) to neutralize the negative charge on the surface of proteoglycans, thereby reducing its hydrophilicity and making the collagen fibers not affected by ice crystals during freezing, realizing the maintenance of the integrity of collagen fibers in bone tissue; at the same time, using an oxygen carrier can use some oxygen carriers to maintain a hypoxic environment inside the cells, and vacuum encapsulation can reduce the oxygen content. The combination of the two can better maintain the hypoxic environment inside the cells, activate HIF, promote the expression of VEGF, realize the inhibition of the reduction of growth factor activity, and improve the bone conductivity and osteoinductivity after autologous bone transplantation.

[0026] (2) The active maintenance process for long-term deep cryopreservation of autologous bone provided by the present invention. Using a magnetic field with a magnetic field intensity of 40 - 80 mT to assist freezing can delay the fluidity of water in the sample. Using a formaldehyde fixative containing methanol can reduce the shrinkage of tissues 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, reducing the structural damage of collagen fibers caused by physical factors such as ice crystal formation during freezing, and enhancing the stability of collagen fibers to a certain extent. Description of the Drawings

[0027] To more clearly illustrate the embodiments of the present invention, the accompanying drawings required for the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on the provided drawings.

[0028] Figure 1 It is a microscopic observation result diagram of osteoblasts cultured for one week after taking tissue samples and culturing them in cell culture wells after cryopreserving autologous bone by the active maintenance process of long-term deep cryopreservation of autologous bone using the autologous bone cryopreservation medium provided in Example 1.

[0029] Figure 2 It is a microscopic observation result diagram of osteoblasts cultured for three weeks after taking tissue samples and culturing them in cell culture wells after cryopreserving autologous bone by the active maintenance process of long-term deep cryopreservation of autologous bone using the autologous bone cryopreservation medium provided in Example 1. Detailed implementation manners

[0030] Next, in combination with specific embodiments, the present invention will be further described. It should be noted that, on the premise of no conflict, any combination of the following-described embodiments or technical features can form a new embodiment.

[0031] The matrix metalloproteinase (MMPs) inhibitors are ilomastat and marimastat, purchased from MedChemExpress.

[0032] Example 1

[0033] This example provides an autologous bone cryopreservation medium, and the raw materials by weight include: 2 parts of L-proline, 8 parts of PVP / WS2 nanocomposite, 0.3 part of the matrix metalloproteinase (MMPs) inhibitor ilomastat, 1 part of antifreeze protein (AFPs), 1 part of non-permeating cryoprotectant sucrose, 2 parts of trimethylammonium lauryl chloride, and 3 parts of oxygen carrier cerium dioxide.

[0034] In this example, the interaction between PVP and water molecules: PVP contains amide groups and can form hydrogen bonds with water molecules, thereby reducing the free movement of water molecules and reducing the formation of ice crystals.

[0035] In this example, the non-permeating cryoprotectant also has low cytotoxicity. By increasing the viscosity of the autologous bone cryopreservation medium, it can further reduce the free movement of water molecules. Acting synergistically with PVP, it can reduce the formation of ice crystals and enhance the ice crystal inhibition effect.

[0036] In this embodiment, L-proline is a permeating cryoprotectant that can quickly penetrate the cell membrane, enter the cell, bind to the water molecules inside the cell, inhibit the formation of ice crystals at low temperatures, and at the same time reduce the loss of water inside the cell, protecting the structure and function of the proteins inside the cell.

[0037] In this embodiment, the application of antifreeze proteins: extracting antifreeze proteins from cold-tolerant organisms and adding them to the autologous bone cryopreservation medium to inhibit the formation and growth of ice crystals, and being able to reduce the damage of ice crystals to the structure of chondrocytes and proteoglycans, and reduce the damage of ice crystals to collagen fibers.

[0038] In this embodiment, inhibiting matrix metalloproteinases (MMPs): adding MMPs inhibitors to the cryopreservation medium can reduce the degradation of collagen fibers by matrix metalloproteinases.

[0039] In this embodiment, neutralizing negative charges: using a positively charged compound (such as trimethyl dodecyl ammonium chloride, TDMAC) to neutralize the negative charges on the surface of proteoglycans, thereby reducing their hydrophilicity and making the collagen fibers not affected by ice crystals during freezing.

[0040] In this embodiment, during the cryopreservation process, some oxygen carriers can be used to maintain a low-oxygen environment inside the cells, and vacuum encapsulation can reduce the oxygen content. The combination of the two can better maintain the low-oxygen environment inside the cells, activate HIF, and promote the expression of VEGF.

[0041] In this embodiment, the preparation method of the PVP / WS2 nanocomposite includes the following steps:

[0042] S1. Dissolve WS2 nanosheets and PVP in deionized water to form a mixed solution;

[0043] S2. Place the mixed solution in a hydrothermal reaction kettle and react at 150 - 200 °C for several hours;

[0044] S3. After the reaction is completed, cool to room temperature, and remove the unreacted substances by centrifugation and washing to obtain the PVP / WS2 nanocomposite.

[0045] In this embodiment, the PVP / WS2 nanocomposite integrates the functions of inhibiting the formation, growth, and rapid ablation of ice crystals, so it can significantly reduce the ice crystal damage during cell cryopreservation and greatly improve the cell preservation efficiency. Specifically, the research results show that:

[0046] 1) During the cooling process, the PVP / WS2 nanocomposite has the effect of regulating ice crystal nucleation, significantly reducing the supercooling degree of the autologous bone cryopreservation medium, thereby being able to significantly reduce the ice crystal damage suffered by the sample during the cooling process.

[0047] 2) Based on the adsorption-inhibition effect, it was found that the PVP / WS2 nanocomposite could selectively adsorb to the ice crystal interface, thereby inhibiting ice crystal recrystallization during rewarming;

[0048] 3) At the same time, based on the photothermal conversion effect of the nanomaterial, during the rewarming process: when rewarming, the frozen bone tissue is placed under a light irradiation device, and the photothermal conversion effect of WS2 is used to rapidly melt the ice crystals, enabling the bone tissue to rapidly warm up and reducing the damage during the rewarming process.

[0049] This embodiment also provides an active maintenance process for cryopreserving autologous bone at ultra-low temperature for a long time, including the following steps:

[0050] S1. Obtain fresh autologous bone tissue, and use the pulsed flushing technique to clean the fresh autologous bone tissue to remove blood, fat, and impurities on the surface, obtaining the cleaned autologous bone tissue;

[0051] S2. Pre-freezing: Immerse the cleaned autologous bone tissue in a formaldehyde fixative containing methanol for 1 h. After fixation, put the fixed autologous bone tissue into a sterile plastic bag, add a kind of autologous bone cryopreservation medium of this embodiment to the sterile plastic bag to cover the fixed autologous bone tissue, evacuate and seal the sterile plastic bag, and then put the sealed sterile plastic bag into the Helmholtz coil of the magnetic field generating device, and then freeze together at 4 °C for 20 min. During the pre-freezing process, apply a static magnetic field with a magnetic field intensity of 60 mT to ensure that the magnetic field evenly covers the sample; experiments show that applying a magnetic field with a magnetic field intensity of 40 - 80 mT to assist freezing can delay the fluidity of water in the sample, and the effect is the best when the magnetic field intensity is 60 mT.

[0052] S3. Ultra-low temperature long-term cryopreservation: After pre-freezing, the sealed sterile plastic bags are continuously cryo-frozen in two steps at -20 °C and -80 °C respectively, and finally the sealed sterile plastic bags are stored in liquid nitrogen at -196 °C.

[0053] In this embodiment, formaldehyde is a commonly used tissue fixative. It mainly forms stable chemical bonds by cross-linking with groups such as 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, reducing the structural damage of collagen fibers caused by physical factors such as ice crystal formation during freezing, and enhancing the stability of collagen fibers to a certain extent to reduce problems such as tissue hardening and deformation. For example, using a formaldehyde fixative containing methanol (such as Roti®-Histofix) can reduce tissue shrinkage to a certain extent.

[0054] 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 effects of vacuum sealing in this embodiment are as follows:

[0055] Reduce moisture content: Vacuum sealing reduces the air in the packaging bag and 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.

[0056] Rapid freezing: The vacuum environment can accelerate the freezing process and quickly cool the sample. Rapid freezing helps to form smaller ice crystals, and smaller ice crystals cause less damage to cell and tissue structures. This is because the growth rate of small ice crystals is slower and they do not cause serious mechanical damage to cell membranes and organelles like large ice crystals.

[0057] Reduce the space for ice crystal growth: Vacuum sealing reduces the air space around the sample, making it lack sufficient space for ice crystals to grow during the formation process. This helps to limit the size and quantity of ice crystals, thereby reducing the damage to the sample structure.

[0058] Reduce mechanical damage: The formation and growth of ice crystals cause mechanical damage to cell and tissue structures. Small ice crystals cause less damage to cell membranes and organelles, while large ice crystals may lead to cell rupture and damage to tissue structures. Vacuum sealing indirectly protects the structural integrity of the sample by reducing the formation of ice crystals.

[0059] Maintain the integrity of the cell membrane: Vacuum sealing can reduce the mechanical pressure of ice crystals on the cell membrane, thereby maintaining the integrity of the cell membrane.

[0060] Reduce solute effects: During the freezing process, as water freezes, the solute concentration in the unfrozen part increases, which may lead to cell dehydration and protein denaturation. Vacuum sealing can reduce the freezing of water, thereby alleviating this solute effect and protecting the structures of cells and tissues.

[0061] Example 2

[0062] This embodiment provides an autologous bone cryopreservation medium, and the raw materials by weight include: 1 part of L-proline, 5 parts of PVP / WS2 nanocomposite, 0.1 part of marimastat, an inhibitor of matrix metalloproteinases (MMPs), 1.5 parts of antifreeze protein (AFPs), 2 parts of non-permeable protective agent trehalose, 1 part of trimethylammonium lauryl chloride, and 2 parts of oxygen carrier vanadium oxide.

[0063] In this embodiment, the preparation method of the PVP / WS2 nanocomposite includes the following steps:

[0064] S1. Dissolve WS2 nanosheets and PVP in deionized water to form a mixed solution;

[0065] S2. Place the mixed solution in a hydrothermal reactor and react at 150 - 200 °C for several hours;

[0066] S3. After the reaction is completed, cool to room temperature, and remove the unreacted substances by centrifugation and washing to obtain the PVP / WS2 nanocomposite.

[0067] This example also provides an active maintenance process for long-term cryopreservation of autologous bone, including the following steps:

[0068] S1. Obtain fresh autologous bone tissue, and use the pulsed flushing technique to wash the fresh autologous bone tissue to remove the blood, fat, and impurities on the surface to obtain the washed autologous bone tissue;

[0069] S2. Pre-freezing: Immerse the washed autologous bone tissue in a formaldehyde fixative containing methanol for 1 h. After fixation, put the fixed autologous bone tissue into a sterile plastic bag, add a kind of autologous bone cryopreservation medium of this example to the sterile plastic bag to cover the fixed autologous bone tissue, evacuate and seal the sterile plastic bag, put the sealed sterile plastic bag into the Helmholtz coil of the magnetic field generating device, and then freeze at 4 °C for 20 min together. During the pre-freezing process, apply a static magnetic field with a magnetic field strength of 40 mT to ensure that the magnetic field uniformly covers the sample;

[0070] S3. Long-term cryopreservation at deep low temperature: After pre-freezing, continuously freeze the sealed sterile plastic bags at -20 °C and -80 °C in two steps, and finally store the sealed sterile plastic bags in liquid nitrogen at -196 °C.

[0071] Example 3

[0072] This example provides an autologous bone cryopreservation medium, and the raw materials by weight include: 1.5 parts of L-proline, 7 parts of PVP / WS2 nanocomposite, 0.5 part of marimastat, an inhibitor of matrix metalloproteinases (MMPs), 2 parts of antifreeze protein (AFPs), 1.5 parts of non-permeable cryoprotectant trehalose, 1.5 parts of trimethylammonium lauryl chloride, and 2.5 parts of oxygen carrier vanadium oxide.

[0073] In this example, the preparation method of the PVP / WS2 nanocomposite includes the following steps:

[0074] S1. Dissolve WS2 nanosheets and PVP in deionized water to form a mixed solution;

[0075] S2. Place the mixed solution in a hydrothermal reactor and react at 150 - 200 °C for several hours;

[0076] S3. After the reaction is completed, cool to room temperature, and remove the unreacted substances by centrifugation and washing to obtain the PVP / WS2 nanocomposite.

[0077] This embodiment also provides an active maintenance process for cryopreserving autologous bone in the long term at deep low temperature, including the following steps:

[0078] S1. Obtain fresh autologous bone tissue, and use the pulsed irrigation technique to wash the fresh autologous bone tissue to remove the blood, fat and impurities on the surface to obtain the washed autologous bone tissue;

[0079] S2. Pre-freezing: Immerse the washed autologous bone tissue in a formaldehyde fixative containing methanol for 1 h. After fixation, put the fixed autologous bone tissue into a sterile plastic bag, add a kind of autologous bone cryopreservation medium of this embodiment to the sterile plastic bag to cover the fixed autologous bone tissue, evacuate and seal the sterile plastic bag, put the sealed sterile plastic bag into the permanent magnet of the magnetic field generating device, and then freeze it at 4 °C for 20 min together. During the pre-freezing process, apply a static magnetic field with a magnetic field strength of 80 mT to ensure that the magnetic field evenly covers the sample;

[0080] S3. Deep low temperature long-term cryopreservation: After pre-freezing, continuously freeze the sealed sterile plastic bags at -20 °C and -80 °C in two steps, and finally store the sealed sterile plastic bags in liquid nitrogen at -196 °C.

[0081] Experimental example

[0082] Use the autologous bone cryopreservation medium provided in Example 1 to cryopreserve autologous bone through the active maintenance process of cryopreserving autologous bone in the long term at deep low temperature. After 5 cases of human tibia cryopreserved in liquid nitrogen at -196 °C for more than 6 months are resuscitated, use fine forceps to sample tissue samples with a diameter of 3 mm to 5 mm, take the tissue samples and culture them in cell culture wells, and directly observe the osteoblasts after culturing for one week and three weeks to evaluate the viability of the osteoblasts. The results are as Figure 1 and Figure 2 shown.

[0083] From Figure 1 it can be seen that after culturing for one week, the growth of osteoblasts has been observed in Example 1; from Figure 2 it can be seen that after culturing for three weeks, a large number of osteoblasts are observed in Example 1, indicating good osteogenic ability for autologous transplantation.

[0084] The above embodiments are only the preferred embodiments of the present invention, and the scope of protection of the present invention cannot be limited by this. Any non-substantial changes and substitutions made by those skilled in the art on the basis of the present invention belong to the scope of protection required by the present invention.

Claims

1. An autologous bone cryopreservation culture 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; 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.

2. The autologous bone cryopreservation culture medium according to claim 1, wherein 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 culture medium according to claim 1, wherein The non-permeable protective agent is one of sucrose and trehalose.

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

5. The autologous bone cryopreservation culture medium according to claim 1, wherein The oxygen carrier is one of cerium dioxide and vanadium oxide.

6. The process for maintaining the activity of autogenous bone during long-term cryopreservation at deep low temperature, 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 5 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.

7. The active maintenance process for long-term cryopreservation of autologous bone at deep low temperature according to claim 6, characterized in that, The magnetic field generating device is a Helmholtz coil or a permanent magnet.

8. The active maintenance process for long-term cryopreservation of autologous bone at deep low temperature according to claim 7, characterized in that, The magnetic field strength generated by the magnetic field generating device is 40-80 mT.

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