Skull flap cleaning treatment method

Abstract

The invention provides a skull flap cleaning treatment method which comprises the following steps: S1, soaking a skull flap to be cleaned in normal saline, heating to normal temperature, and washing the skull flap with purified water; s2, the washed skull flap is put into ultrasonic cleaning equipment to be cleaned; s3, after the skull flap is soaked in 75% ethyl alcohol for a period of time, the soaked skull flap is cleaned with supercritical fluid; s4, the skull flap is cleaned through low-temperature carbon dioxide aerosol; s5, drying the skull flap treated in the step S4, putting the skull flap into a packaging bag, and vacuumizing; and S6, the cleaned skull flap is subjected to irradiation sterilization and then stored at low temperature. According to the skull flap cleaning treatment method provided by the invention, the loss of a large amount of active substances in the skull flap caused by the use of a large amount of chemical agents can be avoided, and the skull flap with a relatively complex structure and an internal structure of the skull flap can be cleaned.

Description

Technical Field

[0001] The present invention belongs to the medical field, specifically relates to the field of skull storage technology, and in particular relates to a skull flap cleaning and processing method. Background Art

[0002] The development of skull repair technology in the medical field is facing a key challenge: the preservation of autologous bone grafts. For patients with skull defects caused by accidents, autologous skull transplantation has become the preferred option over traditional methods such as titanium mesh and allogeneic bone transplantation due to its advantages such as perfect fit, zero rejection, and affordability. However, the key to this revolutionary technology lies in how to properly preserve the bone flap removed during surgery.

[0003] Currently, two methods of preservation exist in clinical practice: in vivo storage and in vitro storage. The former involves temporarily implanting the bone flap into the subcutaneous tissue of the patient's thigh or abdomen, which is then removed and repaired in a second surgery 3-6 months later. This traditional method carries numerous risks: repeated surgeries not only cause new trauma and scarring, but also increase the risk of infection. Even more problematic is the phenomenon of autologous bone resorption: over time, the bone tissue gradually atrophies, potentially leading to graft failure and inability to repair the original defect. In contrast, in vitro cryopreservation offers significant advantages. Using specialized refrigeration equipment to maintain a constant temperature, the bone flap avoids tissue resorption and the risk of a second surgery. This innovative method is particularly important in neurosurgery procedures at primary care hospitals, particularly during decompressive craniectomy for cerebral hemorrhage or severe head injury, where the intact bone flap provides an ideal material for subsequent repair. Clinical data demonstrate that cryopreserved autologous skull reimplantation not only perfectly restores the cranial structure but also outperforms traditional repair methods in postoperative healing and infection control. However, the in vitro storage of autologous skull bones faces multiple difficulties, especially the excessive use of chemical reagents leading to an imbalance in activity maintenance and the difficulty in completely removing the residual chemicals in the bone matrix. There is no clear consensus yet on whether these residues have long-term biological toxicity. Summary of the Invention

[0004] In view of the above shortcomings of the existing technology, the present application provides a skull flap cleaning and processing method, which not only avoids the use of a large amount of chemical agents that leads to a large loss of active substances in the skull flap, but also can clean the debris inside the skull flap, which is more conducive to subsequent use.

[0005] To achieve the above object, the present invention provides a method for cleaning and treating a skull flap, comprising the following steps:

[0006] S1. Soaking the skull flap to be cleaned in physiological saline and heating it to room temperature, and then rinsing the skull flap with purified water;

[0007] S2, placing the flushed skull flap into an ultrasonic cleaning device for cleaning;

[0008] S3, soaking the skull flap in 75% ethanol for a period of time, and then cleaning the skull flap using a supercritical fluid;

[0009] S4. Cleaning the skull flap using low-temperature carbon dioxide aerosol;

[0010] S5, drying the skull flap processed in step S4, placing it in a packaging bag and vacuuming it;

[0011] S6. After irradiation sterilization, the cleaned skull flap is stored at low temperature.

[0012] Furthermore, step S1 is specifically as follows:

[0013] Soak the skull flap in saline and place it in a -40°C refrigerator for 12-24 hours, then place it in a -20°C refrigerator for 2-4 hours.

[0014] The skull flap was immersed in physiological saline at room temperature for 30-60 minutes, and then the skull flap was taken out and rinsed with purified water.

[0015] In step S1, the skull flap is immersed in physiological saline and gradually heated from -80°C to room temperature, and then purified water at room temperature is used to remove the periosteum and blood on the surface of the skull flap.

[0016] Furthermore, in step S2, the skull flap is subjected to water bath ultrasound three times using 15° C. purified water in an ultrasonic cleaning device, each time for 10-15 minutes.

[0017] In step S2, tissue on the surface of the skull flap is removed by water bath ultrasonic cleaning.

[0018] Furthermore, step S3 is specifically as follows:

[0019] Soak the skull flap in 75% alcohol for 30-60 minutes;

[0020] The temperature is controlled at 30-37° C. and the pressure is controlled at 7-10 MPa, and the carbon dioxide that has reached a supercritical state is used to clean the immersed skull flap.

[0021] Skull flaps often have various shapes, and some skull structures are difficult to clean. The use of supercritical fluid can remove blood and tissue from skull flaps that are difficult to clean. Furthermore, supercritical carbon dioxide has a critical temperature of 31.04°C, allowing skull flaps to be cleaned at room temperature.

[0022] Furthermore, in step S3, the flow rate of supercritical carbon dioxide is 0.1-10 L / min, and the cleaning time is 10-30 min.

[0023] Furthermore, step S4 also includes a gradient cooling pre-cooling treatment of the skull flap, the specific steps are:

[0024] After the skull flap is dried, it is placed in a bag, vacuumed, and placed in a -40°C refrigerator for 2-4 hours;

[0025] Then place it in a -80℃ freezer for 12-24 hours.

[0026] Furthermore, in step S4, carbon dioxide aerosol is directionally sprayed onto the surface of the skull flap for cleaning through a controllable spray device, and the carbon dioxide aerosol spray airflow forms an angle of 0-90° with the horizontal plane.

[0027] Furthermore, in step S4, the injection rate of the carbon dioxide aerosol is 0.1-5 L / min.

[0028] Furthermore, in step S4, the cleaning time is 5-15 minutes.

[0029] The cleaning of the skull flap with low-temperature carbon dioxide aerosol can be carried out at the storage temperature of the skull flap, which is not only conducive to cleaning, but the stable environment at this time also facilitates the subsequent final cleaning and packaging and storage.

[0030] Furthermore, step S5 is specifically as follows:

[0031] drying the skull flap and placing it in a packaging bag;

[0032] Vacuuming the skull flap in the packaging bag for 30-60 seconds each time;

[0033] After every three vacuuming operations, the skull flap was soaked in purified water for 10-15 minutes until no blood or oil seeped out during vacuuming.

[0034] The vacuuming in step S5 extracts the blood and grease from the microporous structure of the skull. The "vacuuming" in "vacuuming the skull flap in the packaging bag for 30-60 seconds at a time" can be understood as achieving a vacuum state, generally with a pressure of <-0.1 MPa, and the "time" can be understood as the duration of the vacuum state, in order to extract the blood and grease from the skull flap. It should be understood that the vacuuming pressure can be selected based on actual conditions or the experience of those skilled in the art, as long as it can achieve the subsequent blood and grease extraction operation.

[0035] The present application provides a method for cleaning and treating a skull flap, which has the following beneficial technical effects:

[0036] 1. The skull flap cleaning and processing method of the present invention first removes the periosteum on the surface of the skull flap by soaking it in physiological saline, then rinses the blood on the surface of the skull flap with purified water, and then cleans the tissue on the surface of the skull flap that has been ultrasonically cleaned. Then, the complex structure of the skull or the microporous structure of the skull is further effectively cleaned by supercritical fluid. Then, after gradually cooling, it is cleaned by low-temperature carbon dioxide aerosol. The skull flap is then vacuumed to remove blood and oil stains in the microporous structure of the skull that are difficult to rinse. Finally, it is irradiated and sterilized for low-temperature storage. The entire process gradually cleans the skull flap, not only avoiding the use of chemical agents, but also effectively cleaning the complex structure of the skull, making it easier to reuse the skull.

[0037] 2. The skull flap cleaning and processing method of the present invention introduces supercritical fluid technology into the field of skull cleaning, and realizes efficient cleaning by precisely controlling the physical phase transition of carbon dioxide. In this application, supercritical carbon dioxide and aerosol carbon dioxide are used for synergistic cleaning. Supercritical fluid effectively removes blood and grease from the skull surface by virtue of its high diffusivity and strong solubility, while aerosol particles remove surface particulate matter through micro-jet impact. After cleaning, carbon dioxide is completely vaporized and dissipated, achieving a zero-residue cleaning effect. Compared with traditional processes, organic solvents are completely abandoned to avoid the risk of chemical contamination; no ultrapure water is required for rinsing, which greatly improves the water saving rate; the self-volatile cleaning medium eliminates the subsequent drying process, providing a new green and sustainable solution for the processing of biomedical materials.

[0038] 3. The skull flap cleaning and treatment method of the present invention avoids the use of large amounts of chemical agents that may lead to a large loss of active substances in the skull flap, or even cause the skull flap to completely lose its activity and become dead bone; it also avoids the potential risk of long-term harm to the body caused by chemical substances remaining in the skull flap.

[0039] 4. The skull flap cleaning and processing method of the present invention can not only clean skull flaps of irregular shapes, but also clean the microporous structure in the skull flap.

[0040] The concept, specific structure and technical effects of this application will be further explained below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of this application. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 It is a diagram of the decellularization effect of Examples 1-6 and Comparative Examples 1-2 of the skull flap cleaning and treatment method of the present application. DETAILED DESCRIPTION

[0042] The following describes the embodiments of the present application through specific examples. Those skilled in the art can easily understand the other advantages and effects of the present application from the content disclosed in this specification. The present application can also be implemented or applied through other different specific embodiments. The details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that the following embodiments and features in the embodiments can be combined with each other unless they conflict.

[0043] It should be noted that the diagrams provided in the following embodiments are only schematic illustrations of the basic concept of the present application. Therefore, the diagrams only show the components related to the present application and are not drawn according to the number, shape, and size of the components in actual implementation. In actual implementation, the type, quantity, and proportion of each component can be changed at will, and the component layout type may also be more complex. Terms such as "upper", "lower", "left", "right", "middle", and "one" cited in the embodiments are only for the convenience of description and are not intended to limit the scope of the implementation of the present application. Changes or adjustments in their relative relationships should also be considered as the scope of the implementation of the present application without substantially changing the technical content.

[0044] Some exemplary embodiments of the present application are described for the purpose of illustration. It should be understood that the present application may be implemented in other ways not specifically shown in the drawings.

[0045] The present invention provides a method for cleaning and treating a skull flap, comprising the following steps:

[0046] S1. Remove the skull flap from the -80°C freezer, soak it in saline, and place it in a -40°C freezer for 12-24 hours. Then, place the skull flap, also soaked in saline, in a -20°C refrigerator for 2-4 hours. Then, soak it in saline at room temperature for 30-60 minutes. Remove the periosteum from the skull flap and rinse it with purified water to remove any blood. When storing the skull flap at different temperatures, change the saline solution accordingly.

[0047] S2. Place the flushed skull flap in an ultrasonic cleaning machine and use 15°C purified water for three times of ultrasonic cleaning, each time for 10-15 minutes. Use scissors or a scalpel to clean the tissue on the surface of the skull flap before and after each ultrasonic cleaning.

[0048] S3. Soak the skull flap in 75% ethanol for 30-60 minutes, control the temperature to 30-37°C and the pressure to 7-10 MPa, and then clean the skull flap with supercritical carbon dioxide for 10-30 minutes at a flow rate of 0.1-10 L / min. Then place the skull flap in purified water and oscillate in a 15°C water bath for 30 minutes. This step can be repeated multiple times according to the specific condition of the skull until no oil stains are produced.

[0049] S4. Pre-cool the skull flap using a gradient cooling method. After drying the skull flap, place it in a bag, evacuate it, and place it in a -20°C refrigerator for 2-4 hours. Then, place it in a -40°C refrigerator for 12-24 hours, and then place it in a -80°C refrigerator for 12-24 hours. Then, remove the skull flap from the packaging bag and spray low-temperature carbon dioxide aerosol onto the surface of the pre-cooled skull flap through a controllable injection device. During injection, the carbon dioxide aerosol jet is at an angle of 0-90° to the horizontal plane. The controllable injection device includes a multi-hole nozzle with a nozzle aperture of 10-200μm and a controlled injection rate of 0.1-5L / min. The cleaning time is 5-15 minutes. After cleaning, the carbon dioxide is completely vaporized and dissipated, further cleaning away the contaminants on the skull flap surface after the previous steps, and no chemical cleaning agent remains.

[0050] S5. Wrap the skull flap with sterile gauze and place it in a PA / PE packaging bag to vacuum. Each time, the vacuuming time is 30-60 seconds. Do not completely seal the bag during the first vacuuming. After each vacuuming, wipe the skull flap dry with sterile gauze and replace the sterile gauze. Soak the skull flap in purified water for 10-15 minutes after every three vacuuming. Repeat this process until no blood or oil seeps out during vacuuming. The skull flap is now clean. The vacuuming time is the time the vacuum is maintained. When the vacuum is reached, a pressure of <-0.1MPa is usually sufficient to extract the blood and oil from the skull flap.

[0051] S6. Protect the sharp edges of the cleaned skull flap with sterile gauze, vacuum-pack it in a packaging bag, sterilize it with irradiation, and then store it at low temperature. Irradiate the vacuum-packed skull flap with 25 kGy of radioactive cobalt-60 and store it at low temperature until ready for use.

[0052] Example 1 Cleaning of skull flap

[0053] The skull flap to be processed is removed from the -80°C freezer, soaked in saline, and then placed in a -40°C freezer for 12 hours. The skull flap, also soaked in saline, is then placed in a -20°C refrigerator for 4 hours and then soaked in saline at room temperature for 30 minutes. During this gradual warming process, the saline solution is changed as needed. The periosteum on the skull flap is then removed and rinsed with purified water to remove any blood. The rinsed skull flap is then placed in an ultrasonic cleaner and sonicated three times in a 15°C purified water bath for 10 minutes each. After each ultrasonic cleaning, the skull flap's surface tissue is cleaned with scissors or a scalpel. At this point, the skull flap still appears red, and blood and grease remain in the skull, requiring further cleaning. Although the surface tissue has been removed, further cleaning of the blood and grease is necessary.

[0054] After soaking the skull flap in 75% ethanol for 30 minutes, the skull flap was cleaned with supercritical carbon dioxide at a flow rate of 10 L / min at a controlled temperature of 31°C and a pressure of 7 MPa for 10 minutes. The skull flap was then placed in purified water and shaken in a 15°C waterbath for 30 minutes. This process can be repeated several times until no oil stains are present. After this step, the skull flap is clean and appears slightly yellow.

[0055] The skull flap is dried using sterile gauze, placed in a vacuum bag, and then placed in a -20°C refrigerator for 2 hours, then in a -40°C refrigerator for 24 hours, and finally in a -80°C refrigerator for 12 hours. The skull flap is then removed from the packaging bag and a controlled spray device is used to spray low-temperature carbon dioxide aerosol onto the pre-cooled surface of the skull flap. During spraying, the carbon dioxide aerosol jet is directed at an angle of less than 90° to the horizontal plane for cleaning. The controlled spray device includes a multi-hole nozzle with a nozzle aperture of 10 μm, a spray rate of 0.1 L / min, and a spray cleaning time of 5 minutes. At this point, the skull flap appears white and has been cleaned.

[0056] Use sterile gauze to dry the skull flap treated above and wrap it with sterile gauze. Place the wrapped skull flap in a PA / PE packaging bag and vacuum it for 30 seconds each time. Do not seal it completely during the first vacuuming. Wipe the skull flap dry with gauze after each vacuuming. Soak the skull flap in purified water for 10 minutes after every three vacuumings. Repeat this process until no blood or oil seeps out during vacuuming.

[0057] After protecting the sharp edges of the cleaned skull flap with sterile gauze, the skull flap was vacuum-packed in three layers of PA / PE bags, sterilized by irradiation, and then stored at low temperatures. An irradiation sterilization label was affixed to the second layer of PA / PE bags, and the vacuum-packed skull flap was irradiated and sterilized with radioactive element cobalt-60 at a dose of 25 kGy, and finally stored in a -80°C refrigerator.

[0058] Example 2: Cleaning of skull flap

[0059] The skull flap to be processed was removed from a -80°C freezer, soaked in saline, and then placed in a -40°C freezer for 18 hours. The skull flap, also soaked in saline, was then placed in a -20°C refrigerator for 2.5 hours and then soaked in saline at room temperature for 35 minutes. During this gradual warming process, the saline was changed as needed. The periosteum on the skull flap was then removed and rinsed with purified water to remove any blood. The rinsed skull flap was then placed in an ultrasonic cleaner and sonicated three times in a 15°C purified water bath for 11 minutes each. After each ultrasonic cleaning, the skull flap's surface tissue was cleaned with scissors or a scalpel. At this point, the skull flap still appeared red, and blood and grease remained within the skull flap. Although the surface tissue had been cleaned, further cleaning of the blood and grease was required.

[0060] After soaking the skull flap in 75% ethanol for 40 minutes, the skull flap was cleaned with supercritical carbon dioxide at a flow rate of 7 L / min for 15 minutes at a controlled temperature of 31.5°C and a pressure of 7.4 MPa. The skull flap was then placed in purified water and shaken in a 15°C waterbath for 30 minutes. This process was repeated several times until no oil stains were present. After this step, the cranial flap's cracks or complex structures were cleaned, leaving it with a slightly yellowish appearance.

[0061] The skull flap is dried with sterile gauze, placed in a vacuum bag, and then placed in a -20°C refrigerator for 2 hours, then in a -40°C refrigerator for 20 hours, and finally in a -80°C refrigerator for 14 hours. The skull flap is then removed from the packaging bag and a controlled spray device directs a low-temperature carbon dioxide aerosol onto the pre-cooled surface of the skull flap. The carbon dioxide aerosol jet is sprayed at an angle of less than 90° to the horizontal plane for cleaning. The controlled spray device includes a multi-hole nozzle with a nozzle diameter of 30 μm. The spray rate is 0.5 L / min, and the spray cleaning time is 8 minutes. At this point, the skull flap appears white, indicating that it has been cleaned, and any gaps or complex structures in the skull flap have also been cleaned.

[0062] Use sterile gauze to dry the skull flap treated above and wrap it with sterile gauze. Place the wrapped skull flap in a PA / PE packaging bag and vacuum it for 40 seconds each time. Do not seal it completely during the first vacuuming. Wipe the skull flap dry with gauze after each vacuuming. Soak the skull flap in purified water for 11 minutes after every three vacuumings. Repeat this process until no blood or oil seeps out during vacuuming.

[0063] After protecting the sharp edges of the cleaned skull flap with sterile gauze, the skull flap is vacuum-packed in a packaging bag, irradiated and sterilized, and then stored at low temperature. An irradiation sterilization label is affixed to the second layer of packaging bag, and the vacuum-packed skull flap is irradiated and sterilized with the radioactive element cobalt 60 with a radiation dose of 25kGy, and finally stored in a -80°C refrigerator.

[0064] The skull flap in Example 2 has gaps and uneven structures. It is often difficult to clean the residual substances in these structures using conventional cleaning methods. However, the cleaning method of the present application can clean skull flaps with more complex structures, while retaining the complete skull flap structure and minimizing the residual substances therein.

[0065] Example 3: Cleaning and Treatment of Skull Flap

[0066] The skull flap to be processed is removed from a -80°C freezer, soaked in saline, and then placed in a -40°C freezer for 20 hours. The saline-soaked skull flap is then placed in a -20°C refrigerator for 3 hours and then soaked in saline at room temperature for 40 minutes. During this gradual warming process, the saline solution is changed as needed. The periosteum on the skull flap is then removed and rinsed with purified water to remove any blood. The rinsed skull flap is then placed in an ultrasonic cleaner and sonicated three times in a 15°C purified water bath for 12 minutes each. After each ultrasonic cleaning, the skull flap's surface tissue is cleaned with scissors or a scalpel. At this point, the skull flap still appears red, and the surface tissue has been cleaned, but further cleaning of the skull flap to remove blood and grease is required.

[0067] After soaking the skull flap in 75% ethanol for 45 minutes, the skull flap was cleaned with supercritical carbon dioxide at a flow rate of 4 L / min at a controlled temperature of 32°C and a pressure of 8 MPa for 20 minutes. The skull flap was then placed in purified water and shaken in a 15°C waterbath for 30 minutes. This process was repeated several times until no oil stains were present. At this point, the skull flap was clean and had a slightly yellowish appearance.

[0068] The skull flap was dried using sterile gauze, placed in a vacuum bag, and then placed in a -20°C refrigerator for 3 hours, then in a -40°C refrigerator for 18 hours, and finally in a -80°C refrigerator for 17 hours. The skull flap was then removed from the packaging bag and a controlled spray device directed a low-temperature carbon dioxide aerosol onto the pre-cooled surface of the skull flap. The carbon dioxide aerosol jet was sprayed at an angle of less than 90° to the horizontal plane for cleaning. The controlled spray device included a multi-hole nozzle with a nozzle aperture of 60 μm, a spray rate of 1 L / min, and a spray cleaning time of 10 minutes. At this point, the skull flap had taken on a white color, indicating that it had been cleaned.

[0069] Use sterile gauze to dry the skull flap treated above and wrap it with sterile gauze. Place the wrapped skull flap in a PA / PE packaging bag and vacuum it for 45 seconds each time. Do not seal it completely during the first vacuuming. Wipe the skull flap dry with gauze after each vacuuming. Soak the skull flap in purified water for 12 minutes after every three vacuumings. Repeat this process until no blood or oil seeps out during vacuuming.

[0070] After protecting the sharp edges of the cleaned skull flap with sterile gauze, the packaging bag is vacuum-packed and irradiated for sterilization and then stored at low temperature. An irradiation sterilization label is affixed to the second layer of packaging bag. The vacuum-packed skull flap is irradiated and sterilized with the radioactive element cobalt 60 with a radiation dose of 25kGy and finally stored in a -80°C refrigerator.

[0071] Example 4: Cleaning of skull flap

[0072] The skull flap to be processed is removed from a -80°C freezer, soaked in saline, and then placed in a -40°C freezer for 20 hours. The saline-soaked skull flap is then placed in a -20°C refrigerator for 2 hours and then soaked in saline at room temperature for 45 minutes. During this gradual warming process, the saline solution is changed as needed. The periosteum on the skull flap is then removed and rinsed with purified water to remove any blood. The rinsed skull flap is then placed in an ultrasonic cleaner and sonicated three times in a 15°C purified water bath for 13 minutes each. After each ultrasonic cleaning, the skull flap's surface tissue is cleaned with scissors or a scalpel. At this point, the skull flap still appears red, and the surface tissue has been removed. However, further cleaning of the skull flap to remove blood and grease is required.

[0073] After soaking the skull flap in 75% ethanol for 45 minutes, the skull flap was cleaned with supercritical carbon dioxide at a flow rate of 2 L / min at a controlled temperature of 34°C and a pressure of 8.7 MPa for 20 minutes. The skull flap was then placed in purified water and shaken in a 15°C waterbath for 30 minutes. This process was repeated several times until no oil stains were present. At this point, the skull flap was clean and had a slightly yellowish appearance.

[0074] The skull flap was dried using sterile gauze, placed in a vacuum bag, and then placed in a -20°C refrigerator for 3 hours, then in a -40°C refrigerator for 16 hours, and finally in a -80°C refrigerator for 20 hours. The skull flap was then removed from the packaging bag and a controlled spray device directed a low-temperature carbon dioxide aerosol onto the pre-cooled surface of the skull flap. The carbon dioxide aerosol jet was sprayed at an angle of less than 90° to the horizontal plane for cleaning. The controlled spray device included a multi-hole nozzle with an aperture of 100 μm, a spray rate of 3.5 L / min, and a spray cleaning time of 12 minutes. At this point, the skull flap had taken on a white color, indicating that it had been cleaned.

[0075] Use sterile gauze to dry the skull flap treated above and wrap it with sterile gauze. Place the wrapped skull flap in a PA / PE packaging bag and vacuum it for 45 seconds each time. Do not seal it completely during the first vacuuming. Wipe the skull flap dry with gauze after each vacuuming. Soak the skull flap in purified water for 11 minutes after every three vacuumings. Repeat this process until no blood or oil seeps out during vacuuming.

[0076] After protecting the sharp edges of the cleaned skull flap with sterile gauze, the skull flap is vacuum-packed in a packaging bag, irradiated and sterilized, and then stored at low temperature. An irradiation sterilization label is affixed to the second layer of packaging bag. The vacuum-packed skull flap is irradiated and sterilized with the radioactive element cobalt 60 with a radiation dose of 25kGy, and finally stored in a -80℃ refrigerator.

[0077] Example 5 Cleaning and Treatment of Skull Flap

[0078] The skull flap to be processed is removed from a -80°C freezer, soaked in saline, and then placed in a -40°C freezer for 22 hours. The saline-soaked skull flap is then placed in a -20°C refrigerator for 2 hours and then soaked in saline at room temperature for 50 minutes. During this gradual warming process, the saline solution is changed as needed. The periosteum on the skull flap is then removed and rinsed with purified water to remove any blood. The rinsed skull flap is then placed in an ultrasonic cleaner and sonicated three times in a 15°C purified water bath for 14 minutes each. After each ultrasonic cleaning, the skull flap's surface tissue is cleaned with scissors or a scalpel. At this point, the skull flap still appears red, and the surface tissue has been removed, but further cleaning of the skull flap to remove blood and grease is required.

[0079] After soaking the skull flap in 75% ethanol for 50 minutes, the skull flap was cleaned with supercritical carbon dioxide at a controlled temperature of 35.8°C and a pressure of 9.4 MPa for 25 minutes at a flow rate of 0.8 L / min. The skull flap was then placed in purified water and shaken in a 15°C waterbath for 30 minutes. This process was repeated several times until no oil stains were present. At this point, the skull flap was clean and had a slightly yellowish appearance.

[0080] The skull flap was dried with sterile gauze, placed in a vacuum bag, and then placed in a -20°C refrigerator for 4 hours, then in a -40°C refrigerator for 14 hours, and finally in a -80°C refrigerator for 22 hours. The skull flap was then removed from the packaging bag and a controlled spray device directed a low-temperature carbon dioxide aerosol onto the pre-cooled surface of the skull flap. The carbon dioxide aerosol jet was sprayed at an angle of less than 90° to the horizontal plane for cleaning. The controlled spray device included a multi-hole nozzle with a nozzle aperture of 150 μm, a spray rate of 4 L / min, and a spray cleaning time of 14 minutes. At this point, the skull flap had taken on a white color, indicating that it had been cleaned.

[0081] Use sterile gauze to dry the skull flap treated above and wrap it with sterile gauze. Place the wrapped skull flap in a PA / PE packaging bag and vacuum it for 50 seconds each time. Do not seal it completely during the first vacuuming. Wipe the skull flap dry with gauze after each vacuuming. Soak the skull flap in purified water for 13 minutes after every three vacuumings. Repeat this process until no blood or oil seeps out during vacuuming.

[0082] After protecting the sharp edges of the cleaned skull flap with sterile gauze, the skull flap is vacuum-packed in a packaging bag, irradiated and sterilized, and then stored at low temperature. An irradiation sterilization label is affixed to the second layer of packaging bag. The vacuum-packed skull flap is irradiated and sterilized with the radioactive element cobalt 60 with a radiation dose of 25kGy, and finally stored in a -80℃ refrigerator.

[0083] Example 6 Cleaning of skull flap

[0084] The skull flap to be processed is removed from a -80°C freezer, soaked in saline, and then placed in a -40°C freezer for 24 hours. The saline-soaked skull flap is then placed in a -20°C refrigerator for 2 hours and then soaked in saline at room temperature for 60 minutes. During this gradual warming process, the saline solution is changed as needed. The periosteum on the skull flap is then removed and rinsed with purified water to remove any blood. The rinsed skull flap is then placed in an ultrasonic cleaner and sonicated three times in a 15°C purified water bath for 15 minutes each. After each ultrasonic cleaning, the skull flap's surface tissue is cleaned with scissors or a scalpel. At this point, the skull flap still appears red, and the surface tissue has been removed. However, further cleaning of the skull flap to remove blood and grease is required.

[0085] After soaking the skull flap in 75% ethanol for 60 minutes, the skull flap was cleaned with supercritical carbon dioxide at a flow rate of 0.1 L / min for 30 minutes at a controlled temperature of 37°C and a pressure of 10 MPa. The skull flap was then placed in purified water and shaken in a 15°C waterbath for 30 minutes. This process was repeated several times until no oil stains were present. At this point, the skull flap was clean and had a slightly yellowish appearance.

[0086] The skull flap is dried using sterile gauze, placed in a vacuum bag, and then placed in a -20°C refrigerator for 4 hours, then in a -40°C refrigerator for 12 hours, and finally in a -80°C refrigerator for 24 hours. The skull flap is then removed from the packaging bag and a controlled spray device directs a low-temperature carbon dioxide aerosol onto the pre-cooled surface of the skull flap. The carbon dioxide aerosol jet is sprayed at an angle of less than 90° to the horizontal plane for cleaning. The controlled spray device includes a multi-hole nozzle with an aperture of 200 μm, a spray rate of 5 L / min, and a spray cleaning time of 15 minutes. At this point, the skull flap has turned white, indicating that it has been cleaned.

[0087] Use sterile gauze to dry the skull flap treated above and wrap it with sterile gauze. Place the wrapped skull flap in a PA / PE packaging bag and vacuum it for 60 seconds each time. Do not seal it completely during the first vacuuming. Wipe the skull flap dry with gauze after each vacuuming. Soak the skull flap in purified water for 15 minutes after every three vacuumings. Repeat this process until no blood or oil seeps out during vacuuming.

[0088] After protecting the sharp edges of the cleaned skull flap with sterile gauze, the skull flap is vacuum-packed in a packaging bag, irradiated and sterilized, and then stored at low temperature. An irradiation sterilization label is affixed to the second layer of packaging bag. The vacuum-packed skull flap is irradiated and sterilized with the radioactive element cobalt 60 with a radiation dose of 25kGy, and finally stored in a -80℃ refrigerator.

[0089] Comparative Example 1

[0090] Remove the skull flap from the -80°C freezer, place it in a -40°C freezer for 12 hours, then place it in a -20°C refrigerator for 4 hours. Then, soak it in saline at room temperature for 30 minutes, and then rinse it with purified water. Place the rinsed skull flap in an ultrasonic device and sonicate it three times in a 15°C purified water bath, each time for 10 minutes. After each ultrasonic cleaning, clean the surface tissue of the skull flap with scissors or a scalpel.

[0091] Soak the skull flap in 75% ethanol for 30 minutes, then place it in purified water and shake it in a 15°C water bath for 30 minutes. Repeat this process several times until no oil stains are left.

[0092] After drying the skull flap treated as above, place it in a PA / PE packaging bag and vacuum it for 30 seconds each time. Do not seal it completely during the first vacuuming. Dry the skull flap with gauze after each vacuuming. Soak the skull flap in purified water for 15 minutes after every three vacuumings. Repeat this process until no blood or oil seeps out during vacuuming.

[0093] After protecting the sharp edges of the cleaned skull flap with sterile gauze, the skull flap is vacuum-packed in three layers of PA / PE bags, sterilized by irradiation, and stored at low temperatures. The second layer of PA / PE bag is labeled "irradiation sterilization." The vacuum-packing process must be completed while the skull is still frozen, and the packaging time must be controlled within 5 minutes. The skull flap is then stored in a -80°C refrigerator pending irradiation sterilization. The vacuum-packed skull flap is sterilized by irradiation with 25 kGy of radioactive cobalt-60.

[0094] Comparative Example 2

[0095] Remove the skull flap from the -80°C freezer, place it in a -40°C freezer for 24 hours, then place it in a -20°C refrigerator for 2 hours. Then, soak it in saline at room temperature for 60 minutes, and rinse it with purified water. Place the rinsed skull flap in an ultrasonic device and sonicate it three times in a 15°C purified water bath, each time for 15 minutes. After each ultrasonic cleaning, clean the surface tissue of the skull flap with scissors or a scalpel.

[0096] Soak the skull flap in 75% ethanol for 60 minutes, then place it in purified water and shake it in a 15°C water bath for 30 minutes. Repeat this process several times until no oil stains are left.

[0097] After drying the skull flap treated as above, place it in a PA / PE packaging bag and vacuum it for 60 seconds each time. Do not seal it completely during the first vacuuming. Dry the skull flap with gauze after each vacuuming. Soak the skull flap in purified water for 10 minutes after every three vacuumings. Repeat this process until no blood or oil seeps out during vacuuming.

[0098] After protecting the sharp edges of the cleaned skull flap with sterile gauze, the skull flap is vacuum-packed in three layers of PA / PE bags, sterilized by irradiation, and stored at low temperatures. The second layer of PA / PE bag is labeled "irradiation sterilization." The vacuum-packing process must be completed while the skull is still frozen, and the packaging time must be controlled within 5 minutes. The skull flap is then stored in a -80°C refrigerator pending irradiation sterilization. The vacuum-packed skull flap is sterilized by irradiation with 25 kGy of radioactive cobalt-60.

[0099] The fat content and water content of the cleaned skull flaps of Examples 1-6 and Comparative Examples 1-2 were tested, and the results are shown in Table 1.

[0100] Table 1

[0101]

[0102]

[0103] As can be seen from Table 1, the lower the fat content, the lower the immunogenicity, and the lower the water content is more conducive to the subsequent irradiation sterilization and storage of the skull. The skull flap cleaning and treatment method provided by the present invention can more effectively remove the fat on the surface of the skull flap, thereby reducing immunogenicity, reducing the absorption and rejection reaction of the skull during autologous return, and can also reduce the water content of the skull, which is beneficial to the penetration of irradiation sterilization and the storage of the skull.

[0104] Furthermore, the cleaned skull flaps of Examples 1-6 and Comparative Examples 1-2 were decalcified, dehydrated, wax-soaked, embedded, sliced, stained with hematoxylin and eosin, dehydrated, and mounted. The skull flaps were examined under a microscope, and image acquisition and analysis were performed to observe the residual cells. Figure 1 a-1f correspond to the decellularization test results of the skull flap surface after cleaning in Examples 1-6, and Figure 1 g-1h correspond to the decellularization test results of the skull flap surface after cleaning in comparative examples 1-2, respectively. Figure 1 a-1b corresponds to Example 1-2, and it can be seen that fewer cell nuclei and cell fragments remain after decellularization, indicating that the decellularization effect is better; Figure 1 c-1d corresponds to Example 3-4, where a small amount of cell nuclei and cell debris remaining after decellularization can be seen, indicating a good decellularization effect; Figure 1e-1f corresponds to Example 5-6. It can be seen that no cell nuclei and cell debris remain after decellularization, indicating that the decellularization effect is good. Figure 1 g-1h Comparative Examples 1-2 correspond to the presence of a relatively large number of cell nuclei and cell fragments remaining after decellularization, indicating that the decellularization effect is average. This demonstrates that the skull flap cleaning method provided by the present invention can more effectively clean the skull, effectively remove the oil content in the skull, achieve a relatively good decellularization effect, and control the water content of the skull, facilitating subsequent reuse of the skull.

[0105] The above embodiments are merely illustrative of the principles and effects of this application and are not intended to limit this application. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical concepts disclosed in this application shall be covered by the claims of this application.

Claims

1. A method for cleaning and treating a skull flap, characterized in that: The following steps are involved: S1. Soaking the skull flap to be cleaned in physiological saline and heating it to room temperature, and then rinsing the skull flap with purified water; S2, placing the flushed skull flap into an ultrasonic cleaning device for cleaning; S3, soaking the skull flap in 75% ethanol for a period of time, and then cleaning the skull flap using a supercritical fluid; S4. Cleaning the skull flap using low-temperature carbon dioxide aerosol; S5, drying the skull flap processed in step S4, placing it in a packaging bag and vacuuming it; S6. After irradiation sterilization, the cleaned skull flap is stored at low temperature.

2. The skull flap cleaning method according to claim 1, characterized in that: Step S1 is specifically as follows: Soak the skull flap in saline and place it in a -40°C refrigerator for 12-24 hours, then place it in a -20°C refrigerator for 2-4 hours. The skull flap was immersed in physiological saline at room temperature for 30-60 minutes, and then the skull flap was taken out and rinsed with purified water.

3. The skull flap cleaning method according to claim 1, characterized in that: In step S2, the skull flap is subjected to water bath ultrasound three times using 15° C. purified water in an ultrasonic cleaning device, each time for 10-15 minutes.

4. The skull flap cleaning method according to claim 1, characterized in that: Step S3 is specifically as follows: Soak the skull flap in 75% alcohol for 30-60 minutes; The temperature is controlled at 30-37° C. and the pressure is controlled at 7-10 MPa, and the carbon dioxide that has reached a supercritical state is used to clean the immersed skull flap.

5. The skull flap cleaning method according to claim 4, characterized in that: In step S3, the flow rate of supercritical carbon dioxide is 0.1-10 L / min, and the cleaning time is 10-30 min.

6. The skull flap cleaning method according to claim 1, characterized in that: Step S4 also includes a gradient cooling pre-cooling treatment of the skull flap, and the specific steps are as follows: After the skull flap is dried, it is placed in a bag, vacuumed, and placed in a -40°C refrigerator for 2-4 hours; Then place it in a -80℃ freezer for 12-24 hours.

7. The skull flap cleaning method according to claim 1, characterized in that: In step S4, a controllable injection device is used to spray carbon dioxide aerosol onto the surface of the skull flap for cleaning, and the carbon dioxide aerosol injection airflow forms an angle of 0-90 degrees with the horizontal plane.

8. The skull flap cleaning method according to claim 7, characterized in that: In step S4, the injection rate of the carbon dioxide aerosol is 0.1-5 L / min.

9. The skull flap cleaning method according to claim 7, characterized in that: In step S4, the cleaning time is 5-15 minutes.

10. The skull flap cleaning method according to claim 1, characterized in that: Step S5 is specifically as follows: drying the skull flap and placing it in a packaging bag; Vacuuming the skull flap in the packaging bag for 30-60 seconds each time; After every three vacuuming operations, the skull flap was soaked in purified water for 10-15 minutes until no blood or oil seeped out during vacuuming.

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