Method of cryopreserving bone tissue

Abstract

This invention belongs to the field of biotechnology, specifically relating to a method for cryopreserving bone tissue. Specifically, the method employs a combined vacuum-coated cryopreservation technique and a gradient cooling cryopreservation technique to maximize the preservation of the physical properties of the skull tissue during preservation, thereby improving preservation time and quality. The gradient cooling program involves uniformly lowering the temperature to -20°C at a rate of 1°C / min, then rapidly lowering it to -80°C at a rate of 10°C / min, and finally lowering it to -196°C at a rate of 25°C / min.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for cryopreserving bone tissue. Background Technology

[0002] While artificial bones can quickly resolve bone defects and prevent recurrence due to reimplantation caused by bone cancer or other factors, the price of artificial bone materials far exceeds the financial means of patients. Furthermore, the psychological anxiety and unpredictable stress caused by the "unnatural" nature of the transplanted bone cannot be avoided. From the perspective of improving patient recovery, patients still prefer to use autologous bone tissue that meets surgical criteria. Clinical treatment results show that using autologous bone tissue can effectively improve and maintain the healing speed of the bone window after surgery, effectively reduce rejection reactions caused by allogeneic or artificial bone, improve communication with patients during surgery, and thus increase the success rate of treatment. Therefore, most medical professionals and patients currently prefer to use autologous bone materials for postoperative reimplantation and repair of tissue defects.

[0003] However, current reports on bone tissue preservation techniques lack complete operational procedures and data on the impact of different disinfectants on sample quality during processing. Commonly used bone tissue preservation methods include wet preservation, cryopreservation, and room temperature preservation. As mentioned above, wet preservation commonly suffers from decalcification, making it difficult to maintain calcium balance and guarantee the physical properties of bone tissue after long-term preservation. Cryopreservation uses deep freezing at -80℃ or even -196℃, which can better maintain bone tissue viability; however, improper handling during the pre-implantation thawing and hydration processes can easily damage the physical properties of bone tissue, causing fractures and other phenomena. Furthermore, the thawing and hydration steps are cumbersome and prone to contamination. Room temperature preservation is suitable for bone tissue materials with slightly lower requirements for decalcification or deproteinization during preservation; however, decalcification and a decline in physical properties still occur during the preservation process. Summary of the Invention

[0004] The bone tissue method provided by this invention employs a combined vacuum-coated cryopreservation technique and a gradient cooling cryopreservation technique. By using vacuum and deep low temperature external conditions, the metabolic level of skull tissue cells is reduced to the lowest level that can maintain their viability. By using an appropriate gradient cooling program, the physical properties of skull tissue are preserved to the greatest extent during the preservation process, thereby improving preservation time and quality.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a method for cryopreserving bone tissue, the method comprising the following steps:

[0007] 1) Collect bone tissue and place it in a preservation device filled with preservation fluid;

[0008] 2) Eject air;

[0009] 3) Decrease to -20℃ at a constant rate of 1℃ / min, then rapidly decrease to -80℃ at 10℃ / min, and finally decrease to -196℃ at 25℃ / min.

[0010] Preferably, in step 1), it is necessary to ensure that the preservation solution can wet the surface of the bone tissue; more preferably, the amount of preservation solution added can be calculated according to V = h * S * 2 (V: volume of preservation solution; h: thickness of gel formed by preservation solution on the surface of skull, generally 2-3 mm; S: surface area of ​​skull bone flap, bone is taken according to the standard surface area for bone flap decompression surgery).

[0011] Furthermore, the bone tissue, after being processed by the above steps, is placed in liquid nitrogen for long-term preservation.

[0012] Preferably, the bone tissue has undergone cleaning, polishing, sterilization, and rinsing.

[0013] More preferably, the reagent used for cleaning is physiological saline and / or a cleaning solution containing cardim iodine and microcrystalline cellulose.

[0014] Preferably, the concentration of cardim iodine in the cleaning solution is 0.01%-0.05%.

[0015] Preferably, the concentration of microcrystalline cellulose in the cleaning solution is 0.01%-0.02%.

[0016] Preferably, the sterilization method is irradiation sterilization.

[0017] As described in this invention, the terms "irradiation sterilization" and "irradiation disinfection" refer to an effective method of killing microorganisms on most materials using electromagnetic waves generated by ionizing radiation. The types of radiation used for sterilization include alpha rays, beta rays, X-rays, and gamma rays; wherein alpha rays and beta rays are particle beams, and X-rays and gamma rays are electromagnetic waves.

[0018] Preferably, the irradiation sterilization described in this invention uses X-rays.

[0019] Preferably, the irradiation sterilization dose can be 20-50 Gy, specifically including 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, and 50 Gy.

[0020] Preferably, the irradiation sterilization dose is 25 Gy.

[0021] In this invention, Gy is an abbreviation for "Gray," which is the standard unit for the physical quantity "absorbed dose of ionizing radiation energy." Gray can be translated as "Gray," "Gray," or "Gray," and is sometimes simply called "Gray." In this invention, all of these concepts represent the same meaning and can be used interchangeably.

[0022] Preferably, the rinsing refers to cleaning the bone tissue with a preservation solution.

[0023] More preferably, the preservation solution used for rinsing is tested, including tests for fungi, bacteria, endotoxins, and mycoplasma. If the test is passed, the bone tissue can proceed to the next preservation step; if the test fails, the bone tissue can be subjected to irradiation sterilization again.

[0024] Specifically, the detection includes sterility testing using a culture method and quantitative endotoxin testing using a horseshoe crab reagent.

[0025] Most preferably, the bone tissue undergoes the following pretreatment: grinding to remove burrs from the surface of the bone tissue material, repeatedly cleaning residual bloodstains and bone powder with physiological saline and cleaning solution to remove residual blood clots and tissue debris as much as possible; cleaning the bone tissue with a cleaning solution containing 0.01%-0.5% cardim iodine and 0.01%-0.02% microcrystalline cellulose, followed by irradiation sterilization.

[0026] Preferably, the preservation solution contains cardim iodine, microcrystalline cellulose, human serum albumin, phosphate buffer solution, calcium chloride, and potassium chloride.

[0027] Preferably, the preservation solution contains 0.01%-0.05% cardim iodine, 0.01%-0.02% microcrystalline cellulose, 7% human serum albumin, 0.02% phosphate buffer solution, 0.02%-0.04% calcium chloride and 0.01%-0.02% potassium chloride.

[0028] Most preferably, the preservation solution contains 0.05% cardim iodine, 0.02% microcrystalline cellulose, 7% human serum albumin, 0.02% phosphate buffer solution, 0.04% calcium chloride and 0.01% potassium chloride.

[0029] Preferably, the pH value of the preservation solution is 7.0-7.2.

[0030] Preferably, the preservation device includes any type of container such as a preservation bag, preservation box, or preservation tube. Preferably, the material of the preservation device can be polyimide (PI). In a specific embodiment of the present invention, a preservation bag is used as the preservation device.

[0031] The "bone tissue" described in this invention can be derived from any animal, including but not limited to humans, chimpanzees, monkeys, horses, cattle, sheep, pigs, donkeys, camels, dogs, rabbits, cats, rats, mice, fish, birds, or insects. The bone tissue can originate from the skull, trunk bones, and limb bones.

[0032] Preferably, the bone tissue is derived from human tissue.

[0033] Most preferably, the bone tissue is skull tissue.

[0034] As described in this invention, the term "Cadexomer iodine" is a topical disinfectant that is a complex of iodine and cardemerol, containing an iodine concentration of 0.9%.

[0035] As described in this invention, "microcrystalline cellulose" (MCC) is a linear polysaccharide mainly composed of β-1,4-glucosidic bonds. It is a white, odorless, and tasteless crystalline powder composed of extremely fine, short rod-shaped or powdery porous particles that are free-flowing and formed by hydrolyzing natural cellulose to its ultimate degree of polymerization with dilute acid.

[0036] In a specific embodiment of the present invention, air is purged using a vacuum instrument, and the cooling process is controlled by a programmable cooling device. Specifically, the remaining gas is purged from the vacuum exhaust duct of the preservation device (preservation bag) using a vacuum instrument. The vacuum process is performed in small amounts and multiple times to ensure that the preservation device (preservation bag) fits tightly against the surface of the skull tissue. After the vacuum purging is completed, the exhaust duct is sealed, and the preservation device (preservation bag) is placed in the programmable cooling device, and then cooled according to the cooling program provided by the present invention.

[0037] On the other hand, the present invention provides a preservation solution containing cardim iodine, microcrystalline cellulose, human serum albumin, phosphate buffer solution, calcium chloride and potassium chloride.

[0038] Preferably, the preservation solution contains 0.01%-0.05% cardim iodine, 0.01%-0.02% microcrystalline cellulose, 7% human serum albumin, 0.02% phosphate buffer solution, 0.02%-0.04% calcium chloride and 0.01%-0.02% potassium chloride.

[0039] Most preferably, the preservation solution contains 0.05% cardim iodine, 0.02% microcrystalline cellulose, 7% human serum albumin, 0.02% phosphate buffer solution, 0.04% calcium chloride and 0.01% potassium chloride.

[0040] Preferably, the pH value of the preservation solution is 7.0-7.2.

[0041] On the other hand, the present invention also provides the application of the aforementioned preservation solution in the preservation of bone tissue.

[0042] The "bone tissue" described in this invention can be derived from any animal (e.g., mammals), including but not limited to humans, non-human primates, rodents, etc.

[0043] Preferably, the bone tissue is derived from sources including, but not limited to, the skull, trunk bones, and limb bones.

[0044] Preferably, the bone tissue is derived from human tissue.

[0045] Most preferably, the bone tissue is skull tissue.

[0046] In one specific embodiment, the bone tissue is autologous tissue, that is, bone tissue taken from the patient's own body is transplanted to the patient.

[0047] On the other hand, the present invention provides bone tissue preserved by the aforementioned method and its application in bone transplantation. It also provides a composition comprising bone tissue preserved by the aforementioned method and a preservation solution, the composition further comprising a preservation device.

[0048] Specifically, the bone tissue maintains high-quality physical properties and has a low decalcification rate even after long-term preservation.

[0049] Compared with the prior art, the present invention has at least the following beneficial effects:

[0050] The preservation method provided by this invention utilizes microcrystalline cellulose in the preservation solution to form an extremely thin covering gel on the surface of bone tissue under vacuum, replacing direct freeze-drying technology. Combined with the principle of programmed cooling, the cranial tissue is gradually adapted to the preservation conditions of a deep low-temperature environment, minimizing the adverse effects on the physical properties of the cranial tissue during cryopreservation. Attached Figure Description

[0051] Figure 1 This is the procedure for preserving skull tissue.

[0052] Figure 2 This is a schematic diagram of the planar structure of the skull preservation bag.

[0053] Figure 3 This is a scanning electron microscope image of the outer plate of the skull bone flap, preserved for 12 months, from sample group 7.

[0054] Figure 4 This is a scanning electron microscope image of the trabecular bone structure of the cancellous bone in the basilar region of the skull flap after 6 months of preservation in group 1.

[0055] Figure 5 This is a scanning electron microscope image of the trabecular and reticular structures of the cancellous bone in the large cranial flap barrier area of ​​sample group 4, preserved for 6 months.

[0056] Figure 6 This is a scanning electron microscope image of the outer table structure of the skull after 12 months of preservation of samples from group 4.

[0057] Figure 7 This is a scanning electron microscope image of the outer table structure of the skull after 12 months of preservation of samples from group 1. Detailed Implementation

[0058] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0059] Example 1: Preservation and Performance Testing of Skull Tissue

[0060] 1. Test materials

[0061] This technique requires the preparation of skull tissue cleaning solution, skull tissue preservation solution, and Ringer's solution. The solutions contain cardim iodine, microcrystalline cellulose, human serum albumin, phosphate buffer, calcium chloride, and potassium chloride. All reagents used must be pharmaceutical grade; reagents without pharmaceutical grade must at least meet analytical purity standards. After preparation, all reagents must be sterilized according to their physical properties and pass aseptic quality control before use. Reagents used for skull tissue sterility testing must conform to the testing standards of the Chinese Pharmacopoeia.

[0062] 2. Skull tissue preservation process

[0063] (1) Cleaning of skull tissue materials: The skull tissue was obtained from the decompression surgery of the Department of Neurosurgery of the First Affiliated Hospital of Xinjiang Medical University. The skull tissue obtained by the operation was polished to remove the burrs on the surface of the skull tissue material. It was repeatedly cleaned with medical saline and cleaning solution to remove residual blood stains and bone powder, and to remove residual blood clots and tissue debris as much as possible. The last cleaning was done with the skull tissue cleaning solution described in Table 1, the composition of which is: Cardim iodine: 0.01%-0.5%; microcrystalline cellulose: 0.01%-0.02%; the balance is pure water.

[0064] The skull tissue cleaning solution was prepared by weight percentage and a preservation solution was prepared. The solution was weighed using a 0.01 g balance and dissolved or diluted with sterile water for injection (cardim iodine must be dissolved in a small amount of 95% ethanol to prepare the stock solution). All operations were performed at room temperature.

[0065] Table 1. Grouping of Autologous Cranial Bone Cleansing Solution

[0066] Group 1 Group 2 Group 3 Kadim Iodine 0.01％ 0.25％ 0.5％ microcrystalline cellulose 0.01％ 0.01％ 0.02％ pure water margin margin margin

[0067] (2) Skull tissue weighing and irradiation sterilization: a medical balance was used to weigh the skull tissue in a clean area, and an irradiation instrument (VitalBeam linear accelerator, USA, X-ray) was used to set 25Gy for irradiation sterilization.

[0068] (3) Rinsing and soaking of skull tissue: Rinse the skull tissue twice with sufficient amount of the preservation solution shown in Table 2. Take the preservation solution from the second rinsing for sterility testing. After collecting the preservation solution used for sterility testing, remove as much residual preservation solution as possible. The rinsing is then complete. Place the skull tissue in a clean, homogenized preservation bag and heat-seal the bag opening. Based on the weight of the skull tissue, inject an appropriate amount of preservation solution through the preservation solution filling port of the preservation bag. Calculate the injection volume according to V = h * S * 2 (V: volume of preservation solution; h: thickness of the gel formed by the preservation solution on the skull surface, generally 2-3 mm; S: surface area of ​​the skull bone flap; for bone flap decompression surgery, bone is harvested according to the standard surface area). Ensure that the tissue surface is thoroughly infiltrated (see schematic diagram of the skull preservation bag planar structure as shown in Table 2). Figure 2 ).

[0069] The preservation solution consists of: cardim iodine: 0.01%-0.5%; microcrystalline cellulose: 0.01%-0.02%; human serum albumin: 4%-7%; phosphate buffer: 0.01%-0.03%; calcium chloride: 0.02%-0.04%; potassium chloride: 0.1%-0.02%; and the balance is pure water.

[0070] The skull preservation solution was prepared according to the laboratory solution preparation and dilution methods. The required amount was planned, and the weight of the drug was calculated according to the mass percentage. The weight was measured using a balance of 0.01 g / L. The drug was dissolved or the stock solution was diluted with sterile water for injection. All solution preparation was carried out at room temperature.

[0071] Table 2. Grouping of Autologous Skull Preservation Solution

[0072] Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Group 7 Kadim Iodine 0.50％ 0.25％ 0.01％ 0.50％ 0.50％ 0.50％ 0.50％ microcrystalline cellulose 0.02％ 0.02％ 0.02％ 0.01％ 0.02％ 0.02％ 0.02％ Human serum albumin 7％ 7％ 7％ 7％ 4％ 7％ 7％ Phosphate buffer solution 0.02％ 0.02％ 0.02％ 0.02％ 0.02％ 0.02％ 0.02％ Calcium chloride 0.04％ 0.04％ 0.04％ 0.04％ 0.04％ 0.02％ 0.04％ Potassium chloride 0.02％ 0.02％ 0.02％ 0.02％ 0.02％ 0.02％ 0.01％ pure water margin margin margin margin margin margin margin solution pH value 7.0-7.2 7.0-7.2 7.0-7.2 7.0-7.2 7.0-7.2 7.0-7.2 7.0-7.2

[0073] (4) Cryopreservation of skull tissue: A vacuum instrument is used to remove residual gas from the vacuum exhaust tube of the preservation bag. The vacuum process is performed in small, repeated steps to ensure the preservation bag adheres tightly to the skull tissue surface. After vacuum exhaust is complete, the exhaust tube opening is sealed, the preservation bag is labeled, and it is placed in a preservation box and then placed in a programmable cooling device. Following the cooling procedure described in this technology, after completing the programmed cooling, it is moved to -196℃ for deep cryopreservation. The cooling procedure described in this technology is as follows: Taking a room temperature of 20℃ as an example, the temperature is uniformly reduced to -20℃ at a rate of 1℃ / min in the programmable cooling device, then rapidly reduced to -80℃ at a rate of 10℃ / min, and finally reduced to -196℃ at a rate of 25℃ / min.

[0074] 3. Quantitative detection and results of endotoxins

[0075] Sterility testing includes pre-freezing sterility testing and post-resuscitation sterility testing, including tests for fungi, bacteria, endotoxins, and mycoplasma. Pre-freezing sterility testing: The preservation solution used to rinse the skull tissue is collected and subjected to qualitative sterility testing using a culture method, while endotoxins are quantitatively detected using Limulus Amebocyte Lysate (LAL) reagent. Post-resuscitation sterility testing: The skull tissue is removed from the preservation bag and repeatedly rinsed twice with Ringer's solution and medical saline to thoroughly remove the preservation solution. The skull tissue is then rinsed twice with medical saline, the first rinse using Ringer's solution, followed by a second rinse. After rinsing, sterility testing is performed. The rinsing solution from the second saline rinse is collected and subjected to sterility testing using a culture method, while endotoxins are quantitatively detected using LAL reagent.

[0076] 1) Results of sterility test before freezing

[0077] Microorganisms (including fungi and bacteria) and mycoplasma were detected by culture method, and endotoxins were quantitatively detected by Limulus amebocyte lysate (LAL) reagent. Table 3 shows the sterility test results of the four groups of samples before freezing.

[0078] Table 3. Results of sterility testing of skull before cryopreservation

[0079]

[0080] 2) Post-resuscitation sterility test results

[0081] Tables 4 and 5 show the sterility test results of the skull tissue after the four groups of samples were frozen in the group 7 preservation solution for 6 months and 12 months, respectively, using the same method and technique.

[0082] Table 4. Results of sterility testing of the skull after 6 months of storage

[0083]

[0084] Table 5. Results of skull sterility test after 12 months of storage

[0085]

[0086] In summary, based on the biological sample testing methods in the Chinese Pharmacopoeia (2020), qualitative test results showed that different concentrations of cardim iodine preservation solution could effectively prevent bacterial, fungal, and mycoplasma infections. Quantitative endotoxin testing showed that the endotoxin levels in 0.01% (group 3), 0.25% (group 2), and 0.5% (group 1) cardim iodine preservation solutions fluctuated upwards at 6 and 12 months, but still met the requirements for biological sample testing in the Chinese Pharmacopoeia (2020). However, the 0.5% (group 1) cardim iodine preservation solution showed... Compared to other concentration groups, the cardim iodine preservation solution at this concentration maintained a low endotoxin content in skull tissue preserved for 12 months with significant differences. This indicates that this concentration of cardim iodine preservation solution can more effectively inhibit sample infection, reduce Gram-negative bacterial contamination rate, and effectively maintain cell viability. Furthermore, according to literature reports, no adverse effects on cells have been found at this concentration. Therefore, this concentration (0.5% cardim iodine) can maintain a sterile state for a longer period during skull cryopreservation and is a safe concentration for biological sample preservation.

[0087] 4. Observe the microstructure of skull tissue

[0088] The skull tissue samples that were frozen and then revived under the above preservation conditions were observed using scanning electron microscopy to examine the microstructure of the bone tissue. For the compact bone portion of the outer plate, the structure of the outer ring bone lamellar layer and the integrity of the Haver tubular system were observed. For the trabecular bone portion of the cancellous bone, the regularity and density of the bone tissue cells and the destruction of the Haver tubular system were observed.

[0089] Figure 3 The image shows a scanning electron microscope (SEM) image (100 μm) of the outer plate of the skull bone flap, preserved for 12 months, from sample group 7. The image shows that the outer plate of the bone flap has a firm texture and the surrounding structure of the Haver tubular system is densely arranged. No structural collapse or breakage was observed.

[0090] Figure 4 The scanning electron microscope (SEM) image (100 μm) of the trabecular bone structure of the cancellous bone in the diploic area of ​​the skull flap, preserved for 6 months, shows that the diploic bone tissue is regular and dense, and no damage to the Haver canal system is observed.

[0091] Figure 5 The image shows a scanning electron microscope (SEM) image (100 μm) of the trabecular and mesh structures of the cancellous bone in the large cranial flap barrier area of ​​sample group 4 after 6 months of preservation. The image shows that the structure around the mesh is smooth, dense, and without any cracking or collapse.

[0092] Figure 6 The image shows a scanning electron microscope (SEM) image (100 μm) of the outer plate of the skull after 12 months of preservation of sample 4. The image shows that the outer plate structure is flat, but some areas show signs of disintegration and cracking.

[0093] Figure 7The image shows a scanning electron microscope (SEM) image (100 μm) of the outer plate structure of the skull after 12 months of preservation of the sample from group 1. The image shows that the outer plate structure is flat and there are no signs of disintegration or cracking.

[0094] The high polymer properties of microcrystalline cellulose can serve as an osmotic pressure protectant and binder, maintaining osmotic pressure balance and necessary hydration during long-term cryopreservation of bone tissue cells. Skull tissues preserved for 6 months using group 1 and group 4 microcrystalline cellulose preservation solutions showed no significant differences in the ultrastructure of the outer plate and Haver canal system. Figure 5 Ultrastructural observation of skull tissue preserved for 12 months revealed that the outer plate of the skull tissue in the 0.01% microcrystalline cellulose group (group 4) showed a small amount of fracture. Figure 6 In contrast, the above-mentioned phenomena were not observed in the skull tissue preserved for 12 months in the 0.01% microcrystalline cellulose group. Figure 7 This indicates that a high concentration of microcrystalline cellulose is more conducive to maintaining the hydration environment per unit area of ​​the surface of the skull tissue in contact with it, and is less prone to disintegration. Considering the outer plate disintegration phenomenon that occurred in group 4, and excluding human damage factors such as surgery and pretreatment, the microcrystalline cellulose preservation solution with this content may not be suitable for long-term skull tissue preservation. It is believed that the microcrystalline cellulose content of group 1 (0.02%) can improve the quality of skull preservation and is conducive to maintaining the physical properties of skull tissue.

[0095] 5. Elemental analysis of skull tissue

[0096] Scanning electron microscopy was used to perform multi-point continuous elemental analysis on resuscitated skull bone flap tissue, detecting elements such as O, Ga, P, Na, and Cl, calculating their relative contents, and evaluating the effect of this technique on the chemical element content of bone tissue after statistical analysis with the control group. Table 6 shows the elemental analysis results of four samples stored for 12 months in group 1, Table 7 shows the results of group 6, and Table 8 shows the results of group 7.

[0097] Table 6. Elemental analysis results of cranial bone in preservation fluid of group 1

[0098]

[0099] Table 7. Elemental analysis results of cranial bone in preservation fluid of group 6

[0100]

[0101] Table 8. Elemental analysis results of cranial bone in preservation fluid of group 7

[0102]

[0103]

[0104] 6. Test of the compressive strength of skull tissue

[0105] The samples were revived after preservation, and pressure resistance tests were performed to measure the pressure values ​​after the bone flap tissue was damaged. The results were statistically analyzed and compared with the control to evaluate the viscoelasticity and robustness of the skull tissue. Table 9 shows the pressure resistance test results of four samples preserved in group 1 for 12 months, and Table 10 shows the test results of group 6.

[0106] Table 9. Results of cranial pressure testing in group 1 preservation fluid

[0107] serial number Sample information / test results Crushing pressure (N) Crushing kilogram force (KGf) 1 Sample 1 5013 511.53 2 Sample 2 4415 450.51 3 Sample 3 3375 344.39 4 Sample 4 2993 305.41

[0108] Table 10. Results of cranial pressure testing in group 6 preservation fluid

[0109] serial number Sample information / test results Crushing pressure (N) Crushing kilogram force (KGf) 1 Sample 1 4893 499.29 2 Sample 2 4115 419.90 3 Sample 3 2833 289.08 4 Sample 4 2758 281.43

[0110] 7. Impact resistance test of autologous skull tissue

[0111] The impact toughness characteristics of the skull were measured in the preserved and revived samples, and the load-bearing capacity and elastic properties of the skull tissue were evaluated compared with the control. Table 11 shows the impact resistance test results of four samples preserved in group 1 for 12 months, and Table 12 shows the test results of group 6.

[0112] Table 11. Results of skull toughness test in group 1 preservation solution

[0113] serial number Sample information / test results <![CDATA[Charpy impact toughness characteristic value (J / cm 2 )]]> 1 Sample 1 5.56 2 Sample 2 4.79 3 Sample 3 4.38 4 Sample 4 4.11

[0114] Table 12. Results of skull toughness test in group 6 preservation solution

[0115] serial number Sample information / test results <![CDATA[Charpy impact toughness characteristic value (J / cm 2 )]]> 1 Sample 1 4.88 2 Sample 2 4.11 3 Sample 3 3.97 4 Sample 4 3.44

[0116] Calcium ions can replenish the calcium lost during wet storage, reduce decalcification, and maintain calcium balance, compared to low-Ga... 2+ The elemental content of skull tissue preserved in the preservation solution group (group 6) for 12 months showed that all four skull tissue samples preserved showed significant Ga content. 2+ Loss phenomenon (Table 4), Ga 2+ Loss can easily lead to Ga 2+ Imbalance can directly lead to a decline in the physical properties of skull tissue (Tables 7 and 9), therefore low Ga... 2+ The content is not conducive to the long-term preservation of skull tissue.

[0117] K + Excessive K content may activate cell metabolism, accelerate the cell metabolic rate, and cause cellular energy loss, which is detrimental to long-term tissue preservation. During the cryopreservation of skull tissue cells, it is essential to maintain a low metabolic rate; an excessively high metabolic rate will be detrimental to the long-term preservation of skull tissue. A comparison of the two groups of K... +The results of elemental content analysis of skull tissue preserved for 12 months in preservation solutions (groups 1 and 7) showed high K content. + The concentration of K+ did not significantly affect the elemental content of the preserved skull tissue (Tables 3 and 5). Considering that high concentrations of K+ in the preservation solution may promote cell metabolic rate, thereby increasing cellular energy consumption, and that excessively high metabolic rate may trigger a chain reaction of other metabolic factors, when the components in the preservation solution used to maintain energy metabolism (such as human serum albumin) are rapidly depleted, it will ultimately lead to a decline in the quality and physical properties of the preserved skull, which is not conducive to the long-term preservation of skull tissue. Therefore, K+ in group 7 was selected. + The concentration is the recommended concentration for preparing skull preservation solution.

[0118] This invention compares the differences in elemental composition and physical properties between currently reported wet preservation solutions for skull tissue and four samples preserved using Group 7 preservation solution. Excluding factors affecting bone tissue characteristics and condition such as age, daily contact environment, and living conditions, the skull material preserved using Group 7 solution showed higher levels of key elements influencing its properties, such as calcium, oxygen, and phosphorus—critical chemical elements for maintaining bone mass—than those in the previously reported preservation solutions. This indicates that using this preservation solution can maintain a lower decalcification rate and chemical element loss rate. Test results for compressive strength and impact toughness show that skull tissue preserved using Group 7 solution has higher impact resistance and impact toughness characteristics, demonstrating that using Group 7 preservation solution can effectively maintain skull characteristics and improve the quality of reimplantation.

[0119] In addition, human serum albumin is used to maintain cell viability for long-term preservation of biological samples. Considering the long-term nature of biological sample preservation and the fact that the preservation solution will not be changed or the preserved tissue will not be replenished with energy during the preservation process, and based on the fact that high concentrations of human serum albumin have not been reported to have adverse effects on cells, 7% human serum albumin is recommended as the concentration of the preservation solution.

Claims

1. A method for cryopreserving bone tissue, the method comprising the following steps: 1) Collect bone tissue and place it in a preservation device filled with preservation fluid; 2) Eject air; 3) Decrease to -20℃ at a constant rate of 1℃ / min, then rapidly decrease to -80℃ at 10℃ / min, and finally decrease to -196℃ at 25℃ / min; After being processed in steps 1)-3), the bone tissue was placed in liquid nitrogen for long-term preservation. The preservation solution contains 0.05% cardim iodine, 0.02% microcrystalline cellulose, 7% human serum albumin, 0.02% phosphate buffer solution, 0.04% calcium chloride and 0.01% potassium chloride; The pH value of the preservation solution is 7.0-7.

2.

2. The method of claim 1, wherein the bone tissue has been treated by cleaning, grinding, sterilization and / or rinsing.

3. The method of claim 2, wherein the reagent used for cleaning is physiological saline and / or a cleaning solution, wherein the cleaning solution contains cardim iodine and microcrystalline cellulose.

4. The method as described in claim 3, wherein the concentration of cardim iodine in the cleaning solution is 0.01%-0.05%.

5. The method according to claim 3, wherein the concentration of microcrystalline cellulose in the cleaning solution is 0.01%-0.02%.

6. The method of claim 2, wherein the sterilization method is irradiation sterilization.

7. The method of claim 6, wherein the irradiation sterilization uses X-rays.

8. The method of claim 6, wherein the irradiation sterilization dose is 20-50 Gy.

9. The method of claim 6, wherein the irradiation sterilization dose is 25 Gy.

10. The method of claim 2, wherein the rinsing refers to cleaning bone tissue with a preservation solution.

11. The method of claim 1, wherein the preservation device comprises a preservation bag, a preservation box, and a preservation tube.

12. The method of claim 11, wherein the material of the storage device is polyimide.

13. The method of claim 1, wherein the bone tissue is derived from the skull, trunk bones, and limb bones.

14. The method of claim 1, wherein the bone tissue is derived from a human.

15. The method of claim 1, wherein the bone tissue is cranial tissue.

16. A preservation solution for long-term preservation of bone tissue, said preservation solution containing 0.05% cardim iodine, 0.02% microcrystalline cellulose, 7% human serum albumin, 0.02% phosphate buffer solution, 0.04% calcium chloride and 0.01% potassium chloride; The pH value of the preservation solution is 7.0-7.

2.

17. The use of the preservation solution of claim 16 in the preservation of bone tissue.

18. The application as described in claim 17, wherein the bone tissue is derived from the skull, trunk bones, and limb bones.

19. The application as described in claim 17, wherein the bone tissue is derived from a human.

20. The application as described in claim 17, wherein the bone tissue is autologous tissue.

21. The application as described in claim 17, wherein the bone tissue is cranial tissue.

22. Bone tissue preserved by the method of claim 1.

23. The bone tissue as described in claim 22, wherein the bone tissue maintains high-quality physical properties and has a low decalcification rate even after long-term preservation.

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