A novel cryopreservation solution for skull materials

Abstract

This invention belongs to the field of biotechnology, specifically relating to a novel cryopreservation solution for skull materials. Specifically, the cryopreservation solution contains cardim iodine, microcrystalline cellulose, human serum albumin, phosphate buffer solution, calcium chloride, and potassium chloride, with a pH of 7.0-7.2. This cryopreservation solution, while improving sterilization effectiveness and maintaining a sterile environment for a long period, minimizes factors that negatively impact the viability and physical properties of bone tissue cells.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a novel cryopreservation solution for skull materials. Background Technology

[0002] For cerebral hemorrhage or traumatic brain injury, decompressive craniectomy is commonly performed clinically. Depending on the patient's condition and the location of the hemorrhage, the size and location of the cranial flap are determined before proceeding with the decompression surgery. my country has a consistently high incidence of hypertension globally, with cerebral hemorrhage and other diseases caused by hypertension accounting for 10%-15% of cases. Post-operative decompressive craniectomy often presents a serious challenge: how to handle the surgically removed cranial flap, and if reimplantation is necessary, how to preserve the skull in the short term. Currently, although various biomimetic materials have been approved for use as substitutes for natural bone tissue, they are inferior to autologous bone tissue in terms of biocompatibility, rejection, material cost, and patient acceptance. Therefore, considering postoperative healing outcomes, patient psychological well-being, and the economic burden on families, autologous cranial tissue that meets the surgical criteria is the most recommended and acceptable option for reimplantation by most clinicians and patients' families. Cranial cryopreservation technology is of great significance in the medical and health field for the efficient preservation of bone tissue removed during decompressive craniectomy due to stroke, tumors, or external injuries.

[0003] Short-term preservation of skull tissue requires adherence to aseptic techniques, minimal loss of physical properties, and ease of handling. The pretreatment, preservation conditions, and methods of skull preservation determine the long-term sterility of the sample. Currently, cryopreservation is the most common method for preserving biological materials, and they undergo rigorous aseptic treatment and testing before being stored in a storage facility. Therefore, the preservation method is a key factor in maintaining the long-term sterility of biological samples. Designing appropriate preservation containers and selecting suitable preservation methods are crucial and decisive factors in ensuring the sterility of biological samples.

[0004] Currently, most reported bone tissue preservation processes involve treating bone tissue with disinfectants such as alcohol and chlorhexidine, and using other organic solvents such as formalin for preservation. The components of these preservation solutions inevitably cause decalcification of the bone tissue, which directly leads to a decline in its physical properties. Furthermore, the preservation solutions reported at this stage lack components or conditions that maintain cell viability, easily causing metabolic disorders in bone tissue cells, and in severe cases, even loss of biological activity, thus negating the meaning and value of bone tissue preservation. Summary of the Invention

[0005] The preservation solution provided by this invention can reduce the problem of weakened bone cell vitality caused by decalcification during bone tissue preservation; compared with other cryopreservation solutions, it can maintain the physical properties of bone materials to the greatest extent. The preservation solution also contains components that maintain the pH balance, calcium ion balance, and osmotic pressure balance of the preservation solution, which can provide a stable acid-base microenvironment for skull tissue during long-term preservation, maintain calcium balance, stabilize osmotic pressure, and thus maintain the vitality of skull tissue cells.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a preservation solution (protective solution) for preserving bone tissue, the preservation solution containing cardim iodine, microcrystalline cellulose, human serum albumin, phosphate buffer solution, calcium chloride and potassium chloride.

[0008] Preferably, the pH value of the preservation solution is 7.0-7.2.

[0009] 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, and the pH value of the preservation solution is 7.0-7.2.

[0010] Preferably, the preservation solution contains 0.05% cardim iodine.

[0011] Preferably, the preservation solution contains 0.02% microcrystalline cellulose.

[0012] Preferably, the preservation solution contains 7% human serum albumin.

[0013] Preferably, the preservation solution contains 0.02% phosphate buffer solution.

[0014] Preferably, the preservation solution contains 0.04% calcium chloride.

[0015] Preferably, the preservation solution contains 0.01% potassium chloride.

[0016] Preferably, the pH value of the preservation solution is 7.0-7.2.

[0017] 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, and the pH value of the preservation solution is 7.0-7.2.

[0018] More specifically, the percentages of each component mentioned above are by mass, and each component is dissolved in water for use. More specifically, the water is sterile water or purified water.

[0019] On the other hand, the present invention provides a composition comprising the aforementioned preservation solution and cleaning solution, wherein the cleaning solution contains cardim iodine and microcrystalline cellulose.

[0020] Preferably, the concentration of cardim iodine in the cleaning solution is 0.01%-0.05%.

[0021] Preferably, the concentration of microcrystalline cellulose in the cleaning solution is 0.01%-0.02%.

[0022] Furthermore, the composition may also include a storage container (device). Specifically, the container is, for example, a polyimide (PI) container made of polystyrene. More specifically, the structure of the container is as shown in the attached figure. Figure 2 As shown.

[0023] Furthermore, the composition may also include bone tissue preserved in the preservation solution.

[0024] Preferably, the bone tissue is derived from human tissue.

[0025] Preferably, the bone tissue is derived from the skull, trunk bones, and limb bones.

[0026] Most preferably, the bone tissue is skull tissue.

[0027] On the other hand, the present invention provides the application of the aforementioned preservation solution in the preservation of bone tissue.

[0028] Preferably, the bone tissue comprises bone tissue derived from any part of any animal, which can be a vertebrate or mammal. Specifically, the animal includes primates, rodents (e.g., mice, rats, guinea pigs), lagomorphs (e.g., domestic rabbits, hares), bovids (e.g., cattle), sheep (e.g., sheep), goats (e.g., goats), pigs (e.g., pigs), equines (e.g., horses), canids (e.g., dogs), felines (e.g., cats), birds (e.g., chickens; ducks; geese; companion birds such as canaries, budgerigars, etc.), marine mammals (e.g., dolphins, whales), reptiles (e.g., snakes, frogs, lizards, etc.), and fish.

[0029] Most preferably, the bone tissue is derived from human tissue.

[0030] Preferably, the bone tissue may be derived from the skull, trunk bones, and limb bones.

[0031] Most preferably, the bone tissue is skull tissue.

[0032] On the other hand, the present invention provides a method for preparing a bone tissue preservation solution, the method comprising weighing cardim iodine, microcrystalline cellulose, human serum albumin, phosphate buffer solution, calcium chloride and potassium chloride according to mass percentages, dissolving them in water, and adjusting the pH value to 7.0-7.2;

[0033] The components include: cardim iodine (0.01%-0.05% by mass), microcrystalline cellulose (0.01%-0.02% by mass), human serum albumin (7% by mass), phosphate buffer solution (0.02% by mass), calcium chloride (0.02%-0.04% by mass), and potassium chloride (0.01%-0.02% by mass).

[0034] Preferably, the cardihydric iodine has a mass percentage of 0.05%.

[0035] Preferably, the microcrystalline cellulose has a mass percentage of 0.02%.

[0036] Preferably, the human serum albumin has a mass percentage of 7%.

[0037] Preferably, the phosphate buffer solution has a mass percentage of 0.02%.

[0038] Preferably, the calcium chloride has a mass percentage of 0.04%.

[0039] Preferably, the potassium chloride has a mass percentage of 0.01%.

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

[0041] On the other hand, the present invention provides a method for preserving bone tissue, the method comprising rinsing the bone tissue twice with the aforementioned preservation solution, immersing the bone tissue in the aforementioned preservation solution, removing air, and then sealing and preserving it.

[0042] Specifically, the bone tissue needs to be immersed in a sufficient amount of preservation solution. More preferably, the appropriate volume of the preservation solution is calculated according to the following formula: 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 harvested according to standard surface area for bone flap decompression surgery).

[0043] Preferably, the method further includes the steps of cooling and freezing after the above-mentioned sealed storage.

[0044] Preferably, the cooling process is as follows: uniformly decreasing to -20°C at a rate of 1°C / min, then rapidly decreasing to -80°C at a rate of 10°C / min, and finally decreasing to -196°C at a rate of 25°C / min.

[0045] Preferably, the cryopreservation is carried out in an environment of -196°C, and more preferably, it is stored in liquid nitrogen.

[0046] Preferably, the bone tissue needs to be cleaned and sterilized by irradiation.

[0047] Specifically, the cleaning steps are as follows: grinding to remove burrs from the surface of the bone tissue material, repeatedly cleaning the residual bloodstains and bone powder with medical saline and cleaning solution to remove residual blood clots and tissue debris as much as possible, and finally cleaning with a skull tissue cleaning solution containing 0.01%-0.5% cardim iodine and 0.01%-0.02% microcrystalline cellulose.

[0048] Specifically, the irradiation sterilization uses X-rays, and 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. Bone tissue that fails the sterility test can be irradiated multiple times for sterilization.

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

[0050] 1. The bone tissue preservation solution provided by this invention contains a certain amount of components for maintaining calcium ion balance. The purpose is to take effective preventive measures from the source stage of bone tissue treatment to reduce the decalcification rate of bone tissue and improve the quality of bone tissue preservation.

[0051] 2. Appropriate components were added to maintain the pH, osmotic pressure, and metabolic balance of the preservation solution. The physical or chemical properties of each component in the preservation solution affect the bone tissue material, maintaining the acid-base balance, osmotic pressure balance, and metabolic reaction balance of the bone tissue preservation microenvironment, thereby providing favorable conditions for maintaining the vitality of bone tissue cells.

[0052] 3. The bone tissue preservation solution provided by this invention uses a combination of drugs that meet clinical standards and requirements. Compared with single-component disinfectants, it improves the disinfection effect and maintains a sterile environment for a long time while minimizing factors that negatively affect the vitality and physical properties of bone tissue cells.

[0053] 4. The bone tissue preservation solution 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, it allows the skull tissue to gradually adapt to the preservation conditions of a deep low-temperature environment, minimizing the adverse effects on the physical properties of the skull tissue during cryopreservation. Attached Figure Description

[0054] Figure 1 This is the procedure for preserving skull tissue.

[0055] Figure 2 This is a schematic diagram of the planar structure of the skull preservation bag.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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

[0061] 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.

[0062] Example 1: Preservation and Performance Testing of Skull Tissue

[0063] 1. Test materials

[0064] 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.

[0065] 2. Skull tissue preservation process

[0066] (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.

[0067] 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.

[0068] Table 1. Grouping of Autologous Cranial Bone Cleansing Solution

[0069] 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

[0070] (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.

[0071] (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 ).

[0072] 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.

[0073] 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.

[0074] Table 2. Grouping of Autologous Skull Preservation Solution

[0075] 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

[0076] (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.

[0077] 3. Quantitative detection and results of endotoxins

[0078] 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.

[0079] 1) Results of sterility test before freezing

[0080] 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.

[0081] Table 3. Results of sterility testing of skull before cryopreservation

[0082]

[0083] 2) Post-resuscitation sterility test results

[0084] 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.

[0085] Table 4. Results of sterility testing of the skull after 6 months of storage

[0086]

[0087] Table 5. Results of skull sterility test after 12 months of storage

[0088]

[0089]

[0090] 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.

[0091] 4. Observe the microstructure of skull tissue

[0092] 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.

[0093] 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.

[0094] 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.

[0095] Figure 5The 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.

[0096] 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.

[0097] Figure 7 The 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.

[0098] 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.

[0099] 5. Elemental analysis of skull tissue

[0100] 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.

[0101] Table 6. Elemental analysis results of cranial bone in preservation fluid of group 1

[0102]

[0103] Table 7. Elemental analysis results of cranial bone in preservation fluid of group 6

[0104]

[0105] Table 8. Elemental analysis results of cranial bone in preservation fluid of group 7

[0106]

[0107] 6. Test of the compressive strength of skull tissue

[0108] 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.

[0109] Table 9. Results of cranial pressure testing in preservation fluid of group 1

[0110] 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

[0111] Table 10. Results of cranial pressure testing in group 6 preservation fluid

[0112] 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

[0113] 7. Impact resistance test of autologous skull tissue

[0114] 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.

[0115] Table 11. Results of skull toughness test in group 1 preservation solution

[0116] serial number Sample information / test results <![CDATA[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

[0117] Table 12. Results of skull toughness test in group 6 preservation solution

[0118] serial number Sample information / test results <![CDATA[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

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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 composition comprising a preservation solution and a cleaning solution; The preservation solution contains cardim iodine, microcrystalline cellulose, human serum albumin, phosphate buffer solution, calcium chloride, and potassium chloride; 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, and the pH value of the preservation solution is 7.0-7.2; The cleaning solution contains cardim iodine and microcrystalline cellulose.

2. The composition of claim 1, wherein the concentration of cardim iodine in the cleaning solution is 0.01%-0.05%.

3. The composition of claim 1, wherein the concentration of microcrystalline cellulose in the cleaning solution is 0.01%-0.02%.

4. The composition of claim 1, wherein the composition further comprises bone tissue preserved in a preservation solution.

5. The composition of claim 4, wherein the bone tissue is derived from human tissue.

6. The composition of claim 4, wherein the bone tissue is derived from the skull, trunk bones, and limb bones.

7. The composition of claim 4, wherein the bone tissue is cranial bone tissue.

8. A method for preparing a bone tissue preservation solution, the method comprising weighing cardim iodine, microcrystalline cellulose, human serum albumin, phosphate buffer solution, calcium chloride and potassium chloride according to mass percentages, dissolving them in water, and adjusting the pH value to 7.0-7.

2. 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, and the pH value of the preservation solution is 7.0-7.

2.

9. A method for preserving bone tissue, the method comprising immersing the bone tissue in the preservation solution of claim 1, removing air, and then sealing for preservation.

10. The method of claim 9, wherein the formula for calculating the bone tissue volume is V=h S 2, h is the thickness of the gel formed by the preservation solution on the surface of the skull, and S is the surface area of ​​the skull flap.

11. The method of claim 9, further comprising the steps of cooling and freezing after the above-mentioned sealed storage.

12. The method of claim 11, wherein the cooling procedure is to uniformly reduce the temperature to -20°C at a rate of 1°C / min, then rapidly reduce it to -80°C at a rate of 10°C / min, and finally reduce it to -196°C at a rate of 25°C / min.

13. The method of claim 11, wherein the cryopreservation is performed at -196°C.

14. The method of claim 13, wherein the contents are stored in liquid nitrogen for preservation.

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