Preservation solution for surgical transplantation and its preparation and use in transplant materials

Abstract

The application discloses a kind of surgical implantation with preservative and its preparation and application in transplant material, belong to biomedical engineering and surgical implantation material processing field, including BMSCs, recombinant collagen, isotonic solvent, cryoprotective agent, buffer system, the concentration of BMSCs is 1×10^4-1×10^7 Individual / mL, the concentration of the recombinant collagen is 0.5-50 μg / mL, the isotonic solvent is at least one in DMEM, DMEM / F12, alpha-MEM, RPMI 1640, 0.9% NaCl physiological saline, PBS, the buffer system makes the pH of preservative keep at 7.0-7.6.This application improves the cell activity maintenance ability of skull material in short-time preservation process by the synergistic effect of exogenous BMSCs and recombinant collagen, and forms cell-scaffold composite layer on the surface of bone piece, which is conducive to osteogenic repair, so as to improve the bone healing effect after transplantation.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering and surgical transplant material processing, and in particular to a preservation solution for surgical transplantation. Background Technology

[0002] Skull defects are a common problem in neurosurgery and craniofacial surgery, with causes including trauma, tumor resection, infection, and congenital malformations. Clinically, cranial reconstruction materials are often used for repair. Allogeneic cranial grafts have good mechanical compatibility and biocompatibility, but their preservation process must simultaneously meet the requirements of "thorough sterilization" and "preserving as much intracellular cellular and matrix activity as possible".

[0003] Low-temperature or cryopreservation of skull graft materials can preserve their tissue structure, cell and matrix activity to a certain extent. However, relying solely on cryopreservation makes it difficult to avoid microbial contamination of the preservation solution or bone tissue surface, increasing the risk of infection after long-term cryopreservation. Therefore, a combination of chemical, high-temperature or irradiation sterilization and cryopreservation is currently used to preserve skull graft materials for long-term preservation.

[0004] However, the conventional cryopreservation solutions currently used mainly serve to prevent freezing, moisturize, and maintain basic osmotic pressure. They are insufficient to provide additional osteoblast sources and microenvironment support for transplanted materials, resulting in bone healing after transplantation relying on the migration of cells from the recipient side and a longer healing cycle.

[0005] In addition, during short-term preservation or preoperative waiting, the surface cells and matrix microstructure of skull fragments may suffer stress damage; at the same time, the lack of scaffold components that can promote cell adhesion makes it difficult for exogenous cells to form a stable attachment on the surface of the bone fragment. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, one of the objectives of this invention is to provide a preservation solution for surgical transplantation that, through the synergistic effect of exogenous BMSCs and recombinant collagen, enhances the cell activity retention capacity of skull material during short-term preservation and forms a cell-scaffold composite layer on the surface of the bone fragment that is conducive to osteogenic repair, thereby improving the bone healing effect after transplantation.

[0007] To overcome the shortcomings of the prior art, a second objective of this invention is to provide a method for preparing a preservation solution for surgical transplantation. Through the synergistic effect of exogenous BMSCs and recombinant collagen, the cell activity retention capacity of the skull material during short-term preservation is improved, and a cell-scaffold composite layer conducive to osteogenic repair is formed on the surface of the bone fragment, thereby improving the bone healing effect after transplantation.

[0008] To overcome the shortcomings of the prior art, the third objective of this invention is to provide a method for preserving surgical transplant materials using a surgical transplant preservation solution, which improves the ability of skull materials to maintain cell activity during short-term preservation and forms a cell-scaffold composite layer on the surface of the bone fragment that is conducive to osteogenic repair, thereby improving the bone healing effect after transplantation.

[0009] To overcome the shortcomings of the prior art, the fourth objective of this invention is to provide a combination of transplant materials preserved using a surgical transplant material preservation method, which forms a cell-scaffold composite layer on the surface of the bone graft that is conducive to osteogenic repair, thereby improving the bone healing effect after transplantation.

[0010] To overcome the shortcomings of the prior art, the fifth objective of this invention is to provide a surgically prepared transplanted skull using a method for preserving transplanted materials, which forms a cell-scaffold composite layer on the surface of the bone fragment that is conducive to osteogenic repair, thereby improving the bone healing effect after transplantation.

[0011] One of the objectives of this invention is achieved through the following technical solution: A surgical transplant preservation solution includes bone marrow mesenchymal stem cells (BMSCs), recombinant collagen, an isotonic solvent, a cryoprotectant, and a buffer system. The concentration of BMSCs is 1×10^4-1×10^7 cells / mL, the concentration of recombinant collagen is 0.5-50 μg / mL, the isotonic solvent is at least one of DMEM, DMEM / F12, α-MEM, RPMI 1640, 0.9% NaCl saline, and PBS, and the buffer system maintains the pH of the preservation solution at 7.0-7.6.

[0012] Furthermore, the surgical transplant preservation solution also includes auxiliary components, which are at least one of protein protectants, energy substrates, antioxidants, antibiotics, antifungals, and osmotic pressure regulators.

[0013] Furthermore, the recombinant collagen is at least one of type I recombinant collagen and type III recombinant collagen.

[0014] The second objective of this invention is achieved by the following technical solution: A method for preparing the above-mentioned surgical transplant preservation solution includes the following steps: BMSCs were resuspended in isotonic solvent and cryopreservative was added to adjust the concentration of BMSCs to 1×10^4-1×10^7 cells / mL. The recombinant collagen was dissolved using a buffer system, and its concentration was adjusted to 0.5-50 μg / mL. The BMSCs solution and recombinant collagen solution are mixed at a volume ratio of 1:0.5-1.5 to prepare the preservation solution for surgical transplantation.

[0015] The third objective of this invention is achieved by the following technical solution: A method for preserving surgical transplant materials using the above-mentioned surgical transplant preservation solution includes the following steps: Skull harvesting: Fresh skull tissue is harvested, soft tissue and impurities are removed, and bone fragments are obtained by cutting. Preparation and precooling of surgical transplant preservation solution: Prepare surgical transplant preservation solution and precool to 0-10℃; the surgical transplant preservation solution includes BMSCs, recombinant collagen, isotonic solvent, and buffer system, wherein the concentration of BMSCs is 1×10^4-1×10^7 cells / mL, the concentration of recombinant collagen is 0.5-50 μg / mL, the isotonic solvent is at least one of DMEM, DMEM / F12, α-MEM, RPMI 1640, 0.9% NaCl saline, and PBS, and the buffer system maintains the pH of the preservation solution at 7.0-7.6; Chemical, high-temperature, or irradiation sterilization: Sterilizing bone fragments through chemical, high-temperature, or irradiation methods; Sealing of transplant materials: The sterilized bone fragments are immersed in a surgical transplant preservation solution for incubation and then placed together in a sterile, airtight container for sealing. Cryopreservation: Cool the sterile, sealed container containing bone fragments and preservation solution to a temperature not higher than -80°C, and then transfer it to an environment with a temperature not higher than -130°C for cryopreservation.

[0016] Furthermore, the method for preserving the surgical transplant material also includes a pre-reimplantation treatment step, which specifically involves: 1-2 hours before the reimplantation surgery, removing the sterile sealed container from the liquid nitrogen tank and allowing it to rewarm naturally at room temperature for 10-40 minutes, then transferring it to a 35-42℃ constant temperature water bath for 5-20 minutes. After rewarming, opening the sterile sealed container, removing the bone fragments, and rinsing them 3-6 times with sterile saline to obtain a finished product suitable for transplantation.

[0017] Furthermore, in the sealing step of the transplant material, the volume ratio of the bone fragment to the preservation fluid is 1:2 to 1:10.

[0018] Furthermore, in the cryopreservation step, the sterile sealed container is subjected to gradient cooling. The gradient cooling step specifically involves: first, cooling the cryopreservation solution to 4°C and holding it for 0.5-2 hours; then cooling it to -20°C and holding it for 1-4 hours; and finally cooling it to -80°C and holding it for 2-8 hours.

[0019] The fourth objective of this invention is achieved by the following technical solution: A method for preserving surgical transplant materials as described above, wherein the transplant material assembly includes bone fragments, a preservation solution, and a sterile sealed container, wherein the bone fragments and the preservation solution are both sealed within the sterile sealed container, the bone fragments are immersed in the preservation solution, and the sterile sealed container is kept in a low-temperature environment not exceeding -130°C.

[0020] The fifth objective of this invention is achieved by the following technical solution: A surgical transplant material preservation method described above provides a finished transplanted skull, comprising a bone fragment and a composite layer, wherein the composite layer is loaded on the surface of the bone fragment and is formed by combining BMSCs and recombinant collagen.

[0021] Compared with existing technologies, the surgical transplant preservation solution of this invention improves the cell activity retention capacity of skull materials during short-term preservation through the synergistic effect of exogenous BMSCs and recombinant collagen, and forms a cell-scaffold composite layer on the surface of the bone fragment that is conducive to osteogenic repair, thereby improving the bone healing effect after transplantation.

[0022] Compared with existing technologies, the surgical transplant material preservation method of the present invention can improve the ability of skull materials to maintain cell activity during short-term preservation and form a cell-scaffold composite layer on the surface of the bone fragment that is conducive to osteogenic repair, thereby improving the bone healing effect after transplantation. Attached Figure Description

[0023] Figure 1 This is a flowchart of the preparation method of the preservation solution for surgical transplantation according to the present invention; Figure 2 This is a flowchart of the surgical transplant material preservation method of the present invention; Figure 3 A more detailed flowchart of the surgical transplant material preservation method of the present invention; Figure 4 According to Figure 2 Surgical transplant material preservation methods for preparing transplant material assemblies; Figure 5 According to Figure 3 Finished transplanted skull prepared using a method for preserving surgical transplant materials; Figure 6 The staining result of product a prepared according to the preservation solution and preservation method for surgical transplantation in Example 1 is shown in the figure. Figure 7 The staining result of finished product b prepared according to the preservation solution and preservation method for surgical transplantation in Example 2 is shown in the figure. Figure 8 The staining results of control c, prepared according to the conventional preservation solution and preservation method of control example 1, are shown in the figure. Detailed Implementation

[0024] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicating orientation or positional relationships, are based on the orientation or positional relationships shown in the accompanying drawings and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. In the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0025] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Example

[0027] The preservation solution for surgical transplantation includes BMSCs (bone marrow mesenchymal stem cells), recombinant collagen, isotonic solvent, cryoprotectant, and buffer system. The concentration of BMSCs is 1×10^4-1×10^7 cells / mL, more preferably 5×10^5-2×10^6 cells / mL; the concentration of recombinant collagen is 0.5-50 μg / mL, more preferably 2-10 μg / mL; the isotonic solvent is selected from at least one of DMEM, DMEM / F12, α-MEM, RPMI 1640, 0.9% NaCl saline, and PBS; the cryoprotectant is selected from at least one of 1%-15% v / v DMSO and 10-200 mM trehalose; and the buffer system maintains the pH of the preservation solution at 7.0-7.6.

[0028] like Figure 1 As shown, the preparation of surgical transplant preservation solution A1: Third-generation BMSCs were collected and identified by flow cytometry as CD29+, CD44+, CD34-, and CD45-, with a cell purity ≥95%. BMSCs were resuspended in sterile physiological saline (containing 25 mM HEPES, pH 7.2-7.4), and 10% v / v DMSO and 100 mM trehalose were added to adjust the BMSC concentration to 1×10^6 cells / mL. Type I recombinant collagen was dissolved in a buffer system and its concentration was adjusted to 5 μg / mL. BMSCs and type I recombinant collagen were mixed at a volume ratio of 1:1 to obtain surgical transplant preservation solution A1.

[0029] The surgical transplant preservation solution of this application can be used for short-term preservation or direct transplantation at 2-25℃. The short-term preservation time shall not exceed 12 hours, preferably not more than 6 hours.

[0030] BenShen Surgical Transplant Preservation Solution enhances the cell viability retention of skull material during short-term preservation through the synergistic effect of exogenous BMSCs and recombinant collagen, and forms a cell-scaffold composite layer on the bone fragment surface that is conducive to osteogenic repair, thereby improving the bone healing effect after transplantation.

[0031] The BMSCs in the surgical transplant preservation solution of this application can participate in osteogenic processes after transplantation, provide a cell source for bone healing, and form a biocompatible scaffold on the bone fragment surface with recombinant collagen, thereby improving the adhesion and survival of BMSCs and improving the microenvironment on the bone fragment surface.

[0032] This application adopts a ready-to-use or kitted component approach, which can be quickly prepared and used in clinical short-turnover scenarios, reducing reliance on complex equipment and having strong process adaptability.

[0033] The recombinant collagen in this application has a clear source and low immunogenicity; by setting the range of cell concentration and collagen concentration, it is easy to carry out large-scale production and batch consistency control.

[0034] like Figure 2-3 As shown, this embodiment 1 also provides a method for preserving surgical transplant materials using the above-mentioned surgical transplant preservation solution A1, including the following steps: Skull harvesting: Fresh skull tissue is harvested, soft tissue and impurities are removed, and bone fragments are obtained by cutting. Preparation and precooling of preservation solution for surgical transplantation: Prepare low-temperature preservation solution and precool to 0-10℃; Chemical, high-temperature, or irradiation sterilization: Sterilizing bone fragments through chemical, high-temperature, or irradiation methods; Sealing of transplant materials: After sterilization, the bone fragments are immersed in the preservation solution for incubation and then placed together in a sterile, airtight container for sealing. Cryopreservation: The sterile, sealed container containing bone fragments and preservation solution is cooled first to no higher than -80°C, and then transferred to an environment with a temperature no higher than -130°C for cryopreservation. Pre-implantation treatment: 1-2 hours before the implantation surgery, remove the sterile sealed container from the liquid nitrogen tank and allow it to rewarm naturally at room temperature for 10-40 minutes. Then, transfer it to a constant temperature water bath at 35-42℃ for 5-20 minutes. After rewarming, open the sterile sealed container, remove the bone fragments, and rinse them with sterile saline 3-6 times to obtain a finished product that can be used for transplantation.

[0035] In existing technologies, irradiation sterilization can kill potential bacteria, fungi, and viral spores, and is one of the recognized "gold standards" for terminal sterilization in the tissue bank industry. The current mainstream method for preserving skull transplant materials is to combine irradiation sterilization with cryopreservation. Therefore, this application mainly adopts low-temperature irradiation sterilization. The specific steps of low-temperature irradiation sterilization are as follows: placing the bone slices in an ice bath or cold room and monitoring the temperature with a temperature probe to maintain the temperature of the bone slices at 0-10℃ throughout the low-temperature irradiation sterilization process; preferably 2-6℃.

[0036] The irradiation device uses any one of gamma rays, electron beams, or X-rays, with an absorbed dose of 10-35 kGy and an irradiation time of 10-120 min. In this embodiment, the irradiation device uses gamma rays, with an absorbed dose of 20 kGy and an irradiation time of 45 min.

[0037] In the sealing step of the transplant material, BMSCs are adsorbed onto the surface of the bone graft under the action of recombinant collagen. When the bone graft is immersed in surgical transplant preservation solution A1 for incubation, the volume ratio of bone graft to surgical transplant preservation solution A1 is 1:2-1:10, preferably 1:3-1:5. In this embodiment, the volume ratio of bone graft to surgical transplant preservation solution A1 is 1:4. The incubation time is 10-120 min, preferably 20-60 min.

[0038] In the pretreatment steps before replanting, the water bath temperature is preferably 37-40℃; the water bath rewarming time is preferably 8-12 min; and the water bath is preferably rinsed 4-5 times with sterile physiological saline.

[0039] This application involves irradiating and sterilizing bone fragments, and then freezing them using a newly prepared surgical transplant preservation solution. This achieves effective sterilization while reducing the damage to tissue activity caused by irradiation and temperature fluctuations, thereby reducing the risk of transplant infection and improving post-transplant healing.

[0040] like Figure 4As shown, this application also provides a method for preserving surgical transplant materials, which includes a combination of transplant materials, comprising bone fragments, a preservation solution, and a sterile sealed container. Both the bone fragments and the preservation solution are sealed in the sterile sealed container, with the bone fragments immersed in the preservation solution. The sterile sealed container is kept in a low-temperature environment not exceeding -130°C.

[0041] like Figure 5 As shown, this application also provides a surgical transplant bone product prepared according to the above-described method for preserving surgical transplant materials after reimplantation treatment. The surgical transplant bone product includes a bone fragment and a composite layer. The composite layer is loaded on the surface of the bone fragment and is formed by combining BMSCs and recombinant collagen. Example

[0042] The difference between Example 2 and Example 1 is that, based on Example 1, the surgical transplant preservation solution A2 also includes auxiliary components, which include at least one of protein protectants, energy substrates, antioxidants, antibiotics, antifungal agents and osmotic pressure regulators, and the recombinant collagen is a combination of type I recombinant collagen and type III recombinant collagen.

[0043] Preparation of surgical transplant preservation solution A2: Third-generation BMSCs were collected and identified by flow cytometry as CD29+, CD44+, CD34-, and CD45-, with a cell purity ≥95%. BMSCs were resuspended in sterile physiological saline (containing 25 mM HEPES, pH 7.2-7.4), and 10% v / v DMSO and 100 mM trehalose were added to adjust the BMSC concentration to 1×10^6 cells / mL. Type I and Type III recombinant collagen were dissolved in a buffer system, and the concentrations of both types of recombinant collagen were adjusted to 5 μg / mL and 5 μg / mL, respectively. BMSCs, Type I recombinant collagen, and Type III recombinant collagen were then mixed at a volume ratio of 1:1:1 to obtain surgical transplant preservation solution A2.

[0044] Regarding the aforementioned surgical transplant preservation solution A2, this Example 2 provides a method for preserving surgical transplant materials using surgical transplant preservation solution A2. The general steps are the same as in Example 1, with the difference being: In the cryopreservation process, the sterile sealed container undergoes segmented gradient cooling. Specifically, the segmented gradient cooling process involves first cooling the cryopreservation solution and bone fragments to 4°C and holding them at that temperature for 0.5-2 hours, then cooling them to -20°C and holding them for 1-4 hours, followed by cooling them to -80°C and holding them for 2-8 hours. Finally, the samples are stored in liquid nitrogen gas or liquid phases. The storage temperature is preferably no higher than -150°C, and the storage period can reach 12 months, preferably no more than 6 months.

[0045] The segmented gradient cooling can be achieved using a programmed cooling device. In the prior art, after irradiation sterilization, the bone matrix is ​​directly placed in a -80°C freezer or -196°C liquid nitrogen for freezing and preservation. This is a sudden cooling process with an uncontrollable cooling rate. The segmented gradient cooling of this application allows the protective agent to play a better role and prevents ice crystals from damaging the bone matrix that has been "weakened" by irradiation.

[0046] The surgical transplant material preservation method of this application differs from a simple combination of irradiation sterilization and cryopreservation. Instead, it involves segmented gradient cooling of a sterile, sealed container after irradiation, which allows the protective agent to function better and prevents ice crystals from damaging the bone matrix that has been "weakened" by irradiation, while achieving all the beneficial effects of this invention.

[0047] Compare with Example 1 Conventional cryopreservation solutions are used, which include isotonic solvents, cryoprotectants, osmotic pressure regulators, and buffer systems. These solutions primarily serve to prevent freezing, maintain humidity, and preserve basic osmotic pressure.

[0048] The method for preserving surgical transplant materials using conventional preservation solutions is roughly the same as above: first, the bone fragments are irradiated and sterilized, and then directly placed in a -80°C freezer or -196°C liquid nitrogen for cryopreservation.

[0049] Experimental Example Experimental materials: A rabbit weighing 2-3 kg was taken, and the rabbit skull was divided into 3 pieces. Since rabbit skulls and human skulls are highly similar in bone tissue structure, collagen composition, cell type and physicochemical properties, they can objectively reflect the effects of preservation solution on bone tissue activity, structural integrity and transplantation performance. Therefore, rabbit skull models can effectively replace human skulls for the efficacy verification of this invention, which meets the norms and ethical requirements of preclinical research.

[0050] Three skull fragments were prepared according to the preservation solutions and methods corresponding to Examples 1, 2, and 1 (Control Example 1), respectively, to obtain product a, product b, and control c. These were then subjected to microbial culture detection (37℃, 24h) and TUNEL staining detection using a biological microscope. Figure 6-8 The table below shows the staining results for product a, product b, and control c, with red indicating apoptotic cells. Table 1 Experimental Results Analysis of experimental results: In the above experimental results, microbial detection is achieved by directly observing the samples under a microscope, which allows for a more intuitive observation of the bacteria on the skull surface. This data shows that only control c showed tiny rod-shaped bacteria, while no bacteria grew on finished products a and b. This indicates that the surgical transplant preservation solution and preservation method of this application can avoid microbial contamination of the preservation solution or bone tissue surface, reducing the risk of infection after long-term cryopreservation.

[0051] Regarding the staining, control c showed a large number of apoptotic cells, while finished products a and b showed only a small number of apoptotic cells. This indicates that the surgical transplant preservation solution and preservation method of this application can improve the cell activity retention of skull materials during short-term preservation and form a cell-scaffold composite layer on the bone surface that is conducive to osteogenic repair, thereby improving the bone healing effect after transplantation.

[0052] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A preservation solution for surgical transplantation, characterized in that: The solution includes bone marrow mesenchymal stem cells (BMSCs), recombinant collagen, an isotonic solvent, a cryoprotectant, and a buffer system. The concentration of BMSCs is 1×10^4-1×10^7 cells / mL, the concentration of recombinant collagen is 0.5-50 μg / mL, the isotonic solvent is at least one of DMEM, DMEM / F12, α-MEM, RPMI 1640, 0.9% NaCl saline, and PBS, and the buffer system maintains the pH of the preservation solution at 7.0-7.

6.

2. The surgical transplant preservation solution according to claim 1, characterized in that: The surgical transplant preservation solution also includes auxiliary components, which are at least one of protein protectants, energy substrates, antioxidants, antibiotics, antifungals, and osmotic pressure regulators.

3. The surgical transplant preservation solution according to claim 1, characterized in that: The recombinant collagen is at least one of type I recombinant collagen and type III recombinant collagen.

4. A method for preparing a surgical transplant preservation solution as described in any one of claims 1-3, characterized in that, Includes the following steps: BMSCs were resuspended in isotonic solvent and cryoprotectant was added to adjust the concentration of BMSCs to 1×10^4-1×10^7 cells / mL. The recombinant collagen was dissolved using a buffer system, and its concentration was adjusted to 0.5-50 μg / mL. The BMSCs solution and recombinant collagen solution are mixed at a volume ratio of 1:0.5-1.5 to prepare the preservation solution for surgical transplantation.

5. A method for preserving surgical transplant materials using the surgical transplant preservation solution as described in any one of claims 1-3, characterized in that, Includes the following steps: Skull harvesting: Fresh skull tissue is harvested, soft tissue and impurities are removed, and bone fragments are obtained by cutting. Preparation and precooling of surgical transplant preservation solution: Prepare surgical transplant preservation solution and precool to 0-10℃; the surgical transplant preservation solution includes BMSCs, recombinant collagen, isotonic solvent, and buffer system, wherein the concentration of BMSCs is 1×10^4-1×10^7 cells / mL, the concentration of recombinant collagen is 0.5-50 μg / mL, the isotonic solvent is at least one of DMEM, DMEM / F12, α-MEM, RPMI 1640, 0.9% NaCl saline, and PBS, and the buffer system maintains the pH of the preservation solution at 7.0-7.6; Chemical, high-temperature, or irradiation sterilization: Sterilizing bone fragments through chemical, high-temperature, or irradiation methods; Sealing of transplant materials: The sterilized bone fragments are immersed in a surgical transplant preservation solution for incubation and then placed together in a sterile, airtight container for sealing. Cryopreservation: Cool the sterile, sealed container containing bone fragments and preservation solution to a temperature not higher than -80°C, and then transfer it to an environment with a temperature not higher than -130°C for cryopreservation.

6. The method for preserving surgical transplant materials according to claim 5, characterized in that: The method for preserving surgical transplant materials also includes a pre-reimplantation treatment step, which specifically involves: 1-2 hours before the reimplantation surgery, removing the sterile sealed container from the liquid nitrogen tank and allowing it to rewarm naturally at room temperature for 10-40 minutes, then transferring it to a 35-42℃ constant temperature water bath for 5-20 minutes. After rewarming, opening the sterile sealed container, removing the bone fragments, and rinsing them 3-6 times with sterile saline to obtain a finished product suitable for transplantation.

7. The method for preserving surgical transplant materials according to claim 5, characterized in that: In the sealing step of the transplant material, the volume ratio of the bone fragments to the preservation solution is 1:2 to 1:

10.

8. The method for preserving surgical transplant materials according to claim 5, characterized in that: In the cryopreservation step, the sterile sealed container is subjected to a gradient cooling process. Specifically, the gradient cooling process is as follows: first, the cryopreservation solution is cooled to 4°C and held for 0.5-2 hours; then, it is cooled to -20°C and held for 1-4 hours; and finally, it is cooled to -80°C and held for 2-8 hours.

9. A combination of transplant materials preserved by the surgical transplant material preservation method according to claim 5, characterized in that: The transplant material assembly includes bone fragments, preservation solution, and a sterile sealed container. The bone fragments and preservation solution are both sealed in the sterile sealed container. The bone fragments are immersed in the preservation solution. The sterile sealed container is kept in a low temperature environment not higher than -130°C.

10. A finished transplanted skull prepared according to the method for preserving surgical transplant materials according to claim 6, characterized in that: The transplanted skull product includes a bone fragment and a composite layer. The composite layer is loaded on the surface of the bone fragment and is formed by combining BMSCs and recombinant collagen.

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