Protective fluid and method for storing skull flap

Abstract

The present application relates to the technical field of protective liquid, and in particular to a protective liquid and a method for storing a skull flap. The protective liquid provided in the present application includes a mixture of dimethyl sulfoxide and serum, and the protective liquid is used to store the skull flap. Dimethyl sulfoxide is used as a solvent, and serum is dispersed in dimethyl sulfoxide. Dimethyl sulfoxide and serum have good mutual solubility, which can improve the dispersibility of serum. The protective liquid provided in the present application can improve the permeability of cell membranes to water through the synergistic effect of dimethyl sulfoxide and serum, reduce the water in the cells from leaking out of the cells, reduce the formation of ice crystals in the cells, and thus reduce the cell damage caused by the formation of ice crystals. At the same time, the use of a mixture of dimethyl sulfoxide and serum for tissue cryopreservation protection can further avoid the formation of ice crystals in the cells, so that the activity of the skull flap can be guaranteed as much as possible under the conditions of deep low temperature storage, so that the success rate of skull flap re-transplantation increases.

Description

Technical Field

[0001] The present application belongs to the technical field of protective fluid, and in particular, relates to a protective fluid and a method for storing a skull flap. Background Art

[0002] There is no widely recognized unified preservation method for the existing method of preserving the skull flap. Currently, the patents published do not have a preservation method or preservation conditions that can be widely recognized by the market.

[0003] The current medical method is to place the skull directly in a -80°C environment while waiting for the skull retransplantation operation. Therefore, the activity of the skull flap cannot be guaranteed, which in turn affects the recovery effect after surgery. Deep cryopreservation, as the most commonly used preservation method in biology and medicine, may cause changes in tissue morphology and cell death. Therefore, immersing the tissue in a preservation solution that can preserve tissue morphology and cell activity can be used as a suitable preservation method. Summary of the invention

[0004] The purpose of the present application is to provide a protective liquid and a method for storing a skull flap, aiming to solve the problem in the prior art that deep cryopreservation, as the most commonly used preservation method in biology and medicine, may cause morphological changes in tissues and cell death.

[0005] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0006] In a first aspect, the present application provides a protective solution, which includes a mixture of dimethyl sulfoxide and serum, and is used to store a skull flap.

[0007] The protective solution provided in the present application uses dimethyl sulfoxide as a solvent, and serum is dispersed in dimethyl sulfoxide. Dimethyl sulfoxide and serum have good mutual solubility and can improve the dispersibility of serum. The protective solution provided in the present application can improve the permeability of the cell membrane of the bone cells contained in the skull flap to water through the synergistic effect of dimethyl sulfoxide and serum, reduce the water in the bone cells from leaking out of the cells, reduce the formation of ice crystals in the bone cells, and thus reduce the damage to the bone cells caused by the formation of ice crystals. At the same time, the use of a mixture of dimethyl sulfoxide and serum for tissue cryopreservation protection can further avoid the formation of ice crystals in the cells, so that the activity of the skull flap can be guaranteed as much as possible under the conditions of deep low temperature storage, thereby increasing the success rate of skull flap re-transplantation.

[0008] In a second aspect, the present application provides a method for storing a skull flap, comprising the following steps:

[0009] The skull flap is immersed in the protective solution mentioned above for preservation.

[0010] In the method for storing skull flaps of the present application, the skull flap is immersed in the protective solution provided by the present application. The protective solution can increase the permeability of the cell membrane in the skull flap to water through the synergistic effect of dimethyl sulfoxide and serum, reduce the leakage of water from the cells, reduce the formation of ice crystals in the cells, and thus reduce the cell damage caused by the formation of ice crystals. Furthermore, the use of a mixture of dimethyl sulfoxide and serum for tissue cryopreservation protection can further avoid the formation of ice crystals in the cells, so that the activity of the skull flap can be maintained as much as possible under the conditions of deep low temperature storage, thereby increasing the success rate of skull flap re-transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained like these drawings without paying any creative work.

[0012] Figure 1 is a flow chart of a method for storing a skull flap provided in an embodiment of the present application;

[0013] Figure 2 It is a micro-CT image of a skull flap provided in an embodiment of the present application;

[0014] Figure 3 This is an H-type vascular immunofluorescence staining image provided in an embodiment of the present application. DETAILED DESCRIPTION

[0015] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0016] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0017] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0018] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0019] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0020] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass in the embodiment description of the present application can be μg, mg, g, kg and other mass units known in the chemical industry.

[0021] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0022] In a first aspect, an embodiment of the present application provides a protective solution, which includes a mixture of dimethyl sulfoxide and serum, and the protective solution is used to store a skull flap.

[0023] In order to better preserve the skull flap, a protective solution is provided in an embodiment of the present application. Dimethyl sulfoxide (DMSO) is used as a solvent, and serum is dispersed in dimethyl sulfoxide. Dimethyl sulfoxide and serum have good mutual solubility and can improve the dispersibility of serum. The protective solution provided in the embodiment of the present application can improve the permeability of the cell membrane of the bone cells contained in the skull flap to water through the synergistic effect of dimethyl sulfoxide and serum, reduce the water in the bone cells from seeping out of the cells, reduce the formation of ice crystals in the bone cells, and thus reduce the damage to the bone cells caused by the formation of ice crystals. At the same time, the use of a mixture of dimethyl sulfoxide and serum for tissue cryopreservation protection can further avoid the formation of ice crystals in the cells, so that the activity of the skull flap can be guaranteed as much as possible under the conditions of deep low temperature storage, thereby increasing the success rate of skull flap re-transplantation.

[0024] In some embodiments, based on the mass percentage of the protective liquid being 100%, the protective liquid includes the following components:

[0025] Dimethyl sulfoxide 5~10%

[0026] Serum 90-95%

[0027] Hyaluronic acid 0-0.4%.

[0028] In some embodiments, the protective solution may contain hyaluronic acid (HA) or may not contain hyaluronic acid. The protective solution is compounded with the above-mentioned ingredients, and while fully exerting the respective effects of each component, there is a synergistic effect between the corresponding components, thereby giving the protective solution of the present application a protective effect on the skull flap.

[0029] In some embodiments, based on the mass percentage of the protective liquid being 100%, the protective liquid includes the following components:

[0030] Dimethyl sulfoxide greater than or equal to 5%, less than or equal to 10%

[0031] Serum >90%, <95%

[0032] The hyaluronic acid content is greater than 0% and less than or equal to 0.4%.

[0033] In order to better preserve the skull flap in the embodiment of the present application, a protective solution is provided. On the one hand, dimethyl sulfoxide is used as a solvent, and hyaluronic acid is dispersed in dimethyl sulfoxide. Dimethyl sulfoxide and hyaluronic acid have good mutual solubility, which can improve the dispersibility of hyaluronic acid, and hyaluronic acid has good biocompatibility and low immune response with the skull flap, so it is conducive to the storage of the skull flap. On the other hand, the protective solution provided in the embodiment of the present application can improve the permeability of the cell membrane of the bone cells contained in the skull flap to water through the synergistic effect of dimethyl sulfoxide, serum and hyaluronic acid, and slow freezing can make the water in the cells seep out of the bone cells, which can reduce the formation of ice crystals in the bone cells, thereby reducing the cell damage caused by ice crystal formation. At the same time, the application of dimethyl sulfoxide and serum mixed method for tissue cryopreservation protection can further avoid the formation of ice crystals in the cells, so that the skull flap can be kept as active as possible under the conditions of deep cryopreservation, so that the success rate of skull flap re-transplantation increases. Studies have shown that the skull flap preserved with a mixture of dimethyl sulfoxide and serum has a relatively high bone regeneration rate after replantation. Specifically, the bone regeneration rate of the protective solution provided in the embodiment of the present application is significantly higher than that of the skull flap to which no cryoprotection measures are applied. Specifically, through the results of micro-CT, it can be seen that the speed of connection between the skull flap in the group using hyaluronic acid and dimethyl sulfoxide and the surrounding bone tissue is significantly higher than that of the skull flap preserved only with dimethyl sulfoxide, and is significantly higher than that of the skull flap to which no cryoprotection measures are applied. At the same time, immunofluorescence staining was used to observe the H-type blood vessels, which are considered to be one of the signs closely related to bone regeneration. The number of H-type blood vessels can be considered to be a factor positively correlated with the rate of bone regeneration. The number of H-type blood vessels in the group protected by hyaluronic acid mixed with dimethyl sulfoxide is significantly higher than that in the group not preserved with hyaluronic acid.

[0034] In some embodiments, based on the mass percentage of the protective liquid being 100%, the protective liquid includes the following components:

[0035] Dimethyl sulfoxide greater than 5% and less than or equal to 10%

[0036] Serum greater than 90%, less than or equal to 95%

[0037] The hyaluronic acid content is greater than 0% and less than or equal to 0.4%.

[0038] In the examples of the present application, dimethyl sulfoxide, serum and hyaluronic acid are compounded to prepare the protective solution. The protective solution is compounded by the above-mentioned ingredients, and on the basis of giving full play to the respective effects of each component, there is a synergistic effect between the corresponding components, thereby giving the protective solution of the present application a protective effect on the skull flap.

[0039] In some embodiments, based on the mass percentage of the protective liquid being 100%, the protective liquid includes the following components:

[0040] Dimethyl sulfoxide greater than or equal to 5% and less than 10%

[0041] Serum greater than or equal to 90%, less than 95%

[0042] The hyaluronic acid content is greater than 0% and less than or equal to 0.4%.

[0043] In the examples of the present application, dimethyl sulfoxide, serum and hyaluronic acid are compounded to prepare the protective solution. The protective solution is compounded by the above-mentioned ingredients, and on the basis of giving full play to the respective effects of each component, there is a synergistic effect between the corresponding components, thereby giving the protective solution of the present application a protective effect on the skull flap.

[0044] In some embodiments, based on the mass percentage of the protective liquid being 100%, the protective liquid includes the following components:

[0045] Dimethyl sulfoxide greater than 5% and less than 10%

[0046] Serum greater than or equal to 90%, less than or equal to 95%

[0047] The hyaluronic acid content is greater than 0% and less than or equal to 0.4%.

[0048] In the examples of the present application, dimethyl sulfoxide, serum and hyaluronic acid are compounded to prepare the protective solution. The protective solution is compounded by the above-mentioned ingredients, and on the basis of giving full play to the respective effects of each component, there is a synergistic effect between the corresponding components, thereby giving the protective solution of the present application a protective effect on the skull flap.

[0049] In a second aspect, an embodiment of the present application provides a method for storing a skull flap, comprising the following steps:

[0050] Step S01. Soak the skull flap in the protective solution mentioned above for preservation.

[0051] In the method for storing a skull flap in an embodiment of the present application, the skull flap is immersed in a protective solution provided in an embodiment of the present application. The protective solution can increase the permeability of the cell membrane of the bone cells contained in the skull flap to water through the synergistic effect of dimethyl sulfoxide and serum, reduce the leakage of water from the cells, reduce the formation of ice crystals in the cells, and thus reduce the cell damage caused by the formation of ice crystals. Furthermore, the use of a mixture of dimethyl sulfoxide and serum for tissue cryopreservation protection can further avoid the formation of ice crystals in bone cells, so that the activity of the skull flap can be maintained as much as possible under the conditions of deep low temperature storage, thereby increasing the success rate of skull flap re-transplantation.

[0052] In some embodiments, the temperature of the preservation treatment can be -85°C to -75°C. In the method of storing the skull flap in the embodiment of the present application, these temperatures belong to deep cryopreservation temperatures, and the skull flap is kept as active as possible under the action of the protective liquid, thereby improving the success rate of skull flap re-transplantation. In the exemplary embodiment, the temperature of the preservation treatment may include but is not limited to -85°C, -83°C, -80°C, -78°C, and -75°C. Among them, -80°C is preferred.

[0053] In some embodiments, after the preservation treatment, the step of detecting the bone regeneration after the skull flap is replanted by immunofluorescence staining method is also included to further confirm the activity of the skull flap. Further, the immunofluorescence staining method includes at least one of DAPI, Sp7 (OSX), Emcn / CD31, and MicroCT. Specifically, DAPI: is a fluorescent dye that can strongly bind to DNA. Because DAPI can penetrate the intact cell membrane, it can be used for staining cells and fixed cells. Sp7 (OSX): acts as a major regulator of bone formation during embryonic development and maintenance of adult bone homeostasis. It is a marker of osteoblasts, and the observation of OSX can be used as a marker of osteogenic effect, that is, the more OSX is expressed, the better the osteogenic effect. Emcn / CD31: In the mammalian skeletal system, osteogenesis and angiogenesis are closely related in the bone growth and regeneration process in bone modeling and the bone homeostasis process in bone remodeling. Recent studies have expanded our understanding of the molecular and cellular mechanisms responsible for coupling angiogenesis and bone formation. H-type vessels, named for their high expression of endomucin (Emcn) and CD31, have recently been identified and have the ability to induce bone formation. MicroCT: Microcomputed tomography (microCT or μCT) is a non-destructive imaging tool used to generate high-resolution three-dimensional (3D) images consisting of two-dimensional (2D) transaxial projections or "slices" of the target specimen. The results of its imaging can demonstrate changes in bone mass, that is, the more white locations, the better the osteogenesis.

[0054] In order to make the implementation details and operations of the present application clearly understood by those skilled in the art, and to demonstrate the significant improvement of the protective solution and the method for storing the cranial flap in the embodiments of the present application, the above technical solutions are illustrated by multiple embodiments below. The raw materials or reagents used in the following embodiments are all commercially available or homemade.

[0055] Example 1

[0056] This embodiment provides a protective liquid.

[0057] Taking the mass percentage of the protective liquid as 100%, the protective liquid includes the following components:

[0058] Dimethyl sulfoxide 5%

[0059] Serum 95%.

[0060] Example 2

[0061] This embodiment provides a protective liquid.

[0062] Taking the mass percentage of the protective liquid as 100%, the protective liquid includes the following components:

[0063] Dimethyl sulfoxide 10%

[0064] Serum 90%.

[0065] Example 3

[0066] This embodiment provides a protective liquid.

[0067] Taking the mass percentage of the protective liquid as 100%, the protective liquid includes the following components:

[0068] Dimethyl sulfoxide 9.9%

[0069] Serum 90%

[0070] Hyaluronic acid 0.1%.

[0071] Example 4

[0072] This embodiment provides a method for storing a skull flap. Select 8-12 week old C57 / B6J female mice to perform skull defect and re-transplantation surgery. Please refer to Figure 1 As shown, it specifically includes the following steps:

[0073] Step S01. A 3.5 mm defect is artificially created in the middle of the sagittal suture by using a cranial drill with an outer diameter of 3.5 mm and an inner diameter of 3 mm, and physiological saline is used for cooling during the process of creating the defect;

[0074] Step S02. After taking out the skull flap fragment with a diameter of 3 mm, rinse the blood cells on its surface with physiological saline, place it in an EP tube containing 500 μl of the protective solution in Example 1, and directly place it in a -80°C refrigerator for deep cryopreservation for one week;

[0075] Step S03. After one week, the skull flap fragments are replanted in situ and allowed to recover naturally in 4 weeks. After 4 weeks, the replanted skull flap together with the surrounding bone tissue is removed for subsequent analysis.

[0076] Example 5

[0077] The experiment was carried out according to steps S01 to S03 in Example 4, except that the protective solution in step S02 was the protective solution of Example 2.

[0078] Example 6

[0079] The experiment was carried out according to steps S01 to S03 in Example 4, except that the protective solution in step S02 was the protective solution of Example 3.

[0080] Comparative Example 1

[0081] Taking the mass percentage of the protective liquid as 100%, the protective liquid includes the following components:

[0082] Serum 100%.

[0083] Comparative Example 2

[0084] The experiment was carried out according to steps S01 to S03 in Example 4, except that no protective liquid was used in step S02.

[0085] Comparative Example 3

[0086] The experiment was carried out according to steps S01 to S03 in Example 4, except that the protective solution in step S02 was the protective solution of comparative example 1.

[0087] Performance Testing

[0088] At the same time, they were divided into four groups: no protection measures were taken (Control group, recorded as Control), 5% dimethyl sulfoxide + 95% serum was used for cryoprotection (5% dimethyl sulfoxide group, recorded as 5% DMSO), 10% dimethyl sulfoxide + 90% serum was used for cryoprotection (10% dimethyl sulfoxide group, recorded as 1% DMSO), and 0.1% hyaluronic acid + 9.9% dimethyl sulfoxide + 90% serum (0.1% hyaluronic acid group, recorded as 1% HA) was used for storage.

[0089] Please refer to Figure 2 As shown, 1wks is 1 week, 2wks, 3wks, and 4wks are 2 weeks, 3 weeks, and 4 weeks respectively. After the skull flaps of Examples 4 to 6 and Comparative Example 2 were preserved for one week and then replanted in situ, it was observed that more and more bone tissue connections were found in the skull flaps during the four weeks. The results of micro-CT show that the speed of connection between the skull flaps in the hyaluronic acid + dimethyl sulfoxide + serum group and the surrounding bone tissue was significantly higher than that of the skull flaps preserved only with dimethyl sulfoxide + serum, and was even significantly higher than that of the skull flaps without any cryoprotection measures.

[0090] Please refer to Figure 3 As shown, wks has the same meaning as Figure 2In the same way, DAPI, OSX, Emcn, and CD31 are the corresponding detection substances and methods. At the same time, immunofluorescence staining is used to observe H-type blood vessels. H-type blood vessels are considered to be one of the signs closely related to bone regeneration. The number of H-type blood vessels can be considered a factor positively correlated with the rate of bone regeneration. Figure 3 The number of H-type blood vessels in the group protected by hyaluronic acid and serum mixed with dimethyl sulfoxide was higher than that in the group not preserved by hyaluronic acid.

[0091] In summary, in Examples 1 and 2, the protective liquid dimethyl sulfoxide is used as a solvent, and the serum is dispersed in dimethyl sulfoxide. Dimethyl sulfoxide and serum have good miscibility and can improve the dispersibility of serum. Therefore, it is beneficial to store skull flaps. The skull flap preserved using a mixture of dimethyl sulfoxide and serum has a relatively high bone regeneration rate after replantation.

[0092] In Example 3, the protective solution dimethyl sulfoxide is used as a solvent, and serum and hyaluronic acid are dispersed in dimethyl sulfoxide. Dimethyl sulfoxide, serum and hyaluronic acid have good mutual solubility, which can improve the dispersibility of serum, and hyaluronic acid has good biocompatibility and low immune response with the skull flap, so it is conducive to the storage of the skull flap. The skull flap preserved with a mixture of dimethyl sulfoxide and serum has the highest bone regeneration rate after replantation. In addition, the protective solution in Example 3 is the best embodiment.

[0093] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A use of a protective liquid in storing a skull flap, wherein: The protective solution includes a mixture of dimethyl sulfoxide and serum, and the protective solution also includes hyaluronic acid. Taking the mass percentage of the protective solution as 100%, the protective solution includes the following components: The dimethyl sulfoxide 5-10% The serum is 90-95% The hyaluronic acid is 0.1% to 0.4%.

2. A method for storing a skull flap, characterized in that: The steps include: The skull flap is immersed in the protective solution as described in claim 1 for preservation.

3. The method for storing a skull flap according to claim 2, characterized in that: The storage temperature is -85°C to -75°C; and / or The preservation treatment also includes a step of detecting the bone regeneration after the skull flap is replanted using an immunofluorescence staining method.

4. The method for storing a skull flap according to claim 3, characterized in that: The immunofluorescence staining method includes at least one of DAPI, Sp7, Emcn / CD31, and MicroCT.

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