A method of cleaning a skull flap

Abstract

The application provides a skull flap cleaning method, comprising the following steps: S1, soaking the skull flap to be cleaned in normal saline and then washing the skull flap with purified water; S2, placing the washed 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 soaked skull flap with supercritical fluid; S4, cleaning the skull flap with low-temperature carbon dioxide gas aerosol; S5, drying the skull flap cleaned in step S4, placing the dried skull flap into a packaging bag and vacuumizing the packaging bag; and S6, irradiating and sterilizing the cleaned skull flap and then storing the sterilized skull flap at low temperature. The skull flap cleaning method provided by the application can not only avoid the loss of active substances in the skull flap caused by the use of a large amount of chemicals, but also clean the skull flap and the internal structure of the skull flap with a relatively complex structure.

Description

TECHNICAL FIELD

[0001] The application belongs to the medical field, and particularly relates to the field of skull storage, in particular to a skull flap cleaning and processing method. BACKGROUND

[0002] The 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 repair has become the preferred method over traditional methods such as titanium mesh and allogeneic bone transplantation due to its perfect fit, zero foreign body reaction, and economic advantages. However, the key to this revolutionary technology lies in how to properly preserve the bone flap removed during the operation.

[0003] There are two preservation methods in current clinical practice: in vivo storage and in vitro storage. The former involves temporarily implanting the bone flap into the patient's thigh or abdominal subcutaneous tissue, and then removing it for repair after 3-6 months. This traditional method, however, has many risks: repeated surgery not only causes new trauma and scars, but also increases the risk of infection. More problematic is the "autologous bone resorption phenomenon", which occurs as the bone tissue gradually atrophies over time, ultimately leading to graft failure and failure to repair the original defect. In contrast, in vitro cold storage technology has shown significant advantages by maintaining a constant temperature for the bone flap using specialized cold storage equipment, avoiding tissue absorption and the risk of secondary surgery. This innovative approach is particularly important in neurosurgery in primary hospitals, especially when dealing with bone flap decompression in cases of cerebral hemorrhage or severe craniocerebral injury, as the intact bone flap provides ideal material for subsequent repair. Clinical data shows that autologous skull replantation using cold storage preservation not only perfectly restores the physiological structure of the skull, but also has better postoperative healing and infection control indicators than traditional repair methods. However, autologous skull in vitro storage faces multiple challenges, particularly in maintaining the balance of active substances and the difficulty in completely removing residual chemicals from the bone matrix. Whether these residues have long-term biological toxicity is still not clear. SUMMARY

[0004] In view of the shortcomings of the prior art, the present application provides a skull flap cleaning and processing method which not only avoids the loss of active substances in the skull flap due to the use of a large amount of chemicals, but also cleans the debris inside the skull flap, which is more conducive to subsequent use.

[0005] To achieve the above purpose, the present application provides a skull flap cleaning and processing method, comprising the following steps:

[0006] S1, soaking the skull flap to be cleaned in physiological saline to room temperature, and then washing the skull flap with purified water;

[0007] S2, the skull flap after flushing into the ultrasonic cleaning equipment for cleaning;

[0008] S3, the skull flap soaked in 75% ethanol for a period of time, using supercritical fluid to clean the skull flap after soaking;

[0009] S4, by low carbon dioxide gas cleaning of the skull flap;

[0010] S5, the skull flap after drying step S4 treated into the packaging bag vacuum;

[0011] S6, the skull flap after cleaning irradiation sterilization and low temperature storage.

[0012] Further, step S1 is specifically:

[0013] The skull flap to be treated is soaked in physiological saline and placed in a-40℃ refrigeration equipment for pretreatment for 12-24h, and then placed in a-20℃ refrigeration equipment for pretreatment for 2-4h;

[0014] The skull flap is soaked in physiological saline at room temperature for 30-60min, and the skull flap is washed with purified water.

[0015] In step S1, the skull flap is soaked in physiological saline from-80℃ to room temperature, and then purified water at room temperature is used to remove the periosteum and bloodstains on the surface of the skull flap.

[0016] Further, in step S2, 15℃ purified water is used in the ultrasonic cleaning equipment to perform water bath ultrasonic for 3 times, each time for 10-15min.

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

[0018] Further, step S3 is specifically:

[0019] The skull flap is soaked in 75% alcohol for 30-60min;

[0020] The temperature is controlled at 30-37℃, the pressure is controlled at 7-10MPa, and the skull flap after soaking is cleaned by carbon dioxide reaching the supercritical state.

[0021] The skull flap is usually of various shapes, and the structure of part of the skull is not easy to clean. The use of supercritical fluid can clean the bloodstains or tissues in the skull flap that are not easy to clean. Moreover, the critical temperature of supercritical carbon dioxide is 31.04℃, which can clean the skull flap at room temperature.

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

[0023] Further, step S4 further comprises gradient cooling pre-cooling treatment of the skull flap, and the step is specifically:

[0024] After the skull flap is dried, the skull flap is placed in a bag, vacuumized, and placed in a-40℃ refrigeration device for 2-4 h;

[0025] Then, the skull flap is placed in a-80℃ refrigeration device for 12-24 h.

[0026] Further, in step S4, the carbon dioxide aerosol is directed sprayed to the surface of the skull flap by a controllable spraying device, and the spraying airflow of the carbon dioxide aerosol forms an angle of 0-90° with the horizontal plane.

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

[0028] Further, in step S4, the cleaning time is 5-15 min.

[0029] The cleaning of the skull flap by the low-temperature carbon dioxide aerosol can be performed at the storage temperature of the skull flap, which is not only conducive to the cleaning, but also facilitates the subsequent final cleaning and packaging and storage due to the stability of the environment.

[0030] Further, step S5 is specifically:

[0031] After the skull flap is dried, the skull flap is placed in a packaging bag;

[0032] The skull flap placed in the packaging bag is vacuumized for 30-60 s each time;

[0033] After every 3 times of vacuumization, the skull flap is soaked in purified water for 10-15 min until no blood and oil stains are exuded during vacuumization.

[0034] In step S5, the vacuumization can extract the blood and oil stains in the microporous structure of the skull. In the "vacuumization" in "the skull flap placed in the packaging bag is vacuumized for 30-60 s each time", it can be understood as achieving a vacuum state, and generally, the pressure is <-0.1 MPa. In addition, the "time" can be understood as the holding time in the vacuum state, and the purpose is to extract the blood and oil in the skull flap. It should be understood that the pressure of the vacuumization can be selected according to the actual situation or the experience of the person skilled in the art, as long as the subsequent operation of extracting the blood and oil can be realized.

[0035] The skull flap cleaning treatment method provided by the present application has the following beneficial technical effects:

[0036] 1. The skull flap cleaning method of the present application first removes the periosteum on the surface of the skull flap by soaking in physiological saline, then removes bloodstains on the surface of the skull flap by washing with purified water, then removes the tissue on the surface of the skull flap cleaned by ultrasonic waves, then further effectively cleans the complex structure of the skull or the microporous structure of the skull by supercritical fluid, then gradually cools and cleans by low-temperature carbon dioxide gas aerosol, then removes blood and oil stains in the microporous structure of the skull that are not easy to flush by vacuumizing the skull flap, and finally irradiates and sterilizes for low-temperature preservation. 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, facilitating the subsequent reuse of the skull.

[0037] 2. The skull flap cleaning method of the present application introduces supercritical fluid technology into the field of skull cleaning, and realizes efficient cleaning by precisely controlling the physical phase change of carbon dioxide. In the present application, supercritical carbon dioxide and aerosol carbon dioxide are used for collaborative cleaning. The supercritical fluid effectively removes blood and oil on the surface of the skull due to its high diffusivity and strong solubility, and the aerosol particles remove surface particulate matter through micro-jet impact. After cleaning, the carbon dioxide completely gasifies and escapes, achieving zero-residue cleaning effect. Compared with traditional processes, organic solvents are completely abandoned, avoiding the risk of chemical pollution; no ultrapure water flushing is needed, greatly improving water saving rate; the self-volatility of the cleaning medium eliminates the need for subsequent drying process, providing a green and sustainable new solution for the processing of biomedical materials.

[0038] 3. The skull flap cleaning method of the present application avoids the use of a large amount of chemical agents, which leads to a large loss of active substances in the skull flap, and even makes the skull flap completely lose activity and become dead bone; it also avoids the potential danger of long-term harm to the body caused by residual chemical substances in the skull flap.

[0039] 4. The skull flap cleaning method of the present application not only can clean irregularly shaped skull flaps, but also can clean the microporous structure in the skull flap.

[0040] The concept, specific structure and technical effects of the present application will be further described below with reference to the accompanying drawings, so as to fully understand the purpose, features and effects of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 is the decellularization effect diagram of the skull flap cleaning method embodiments 1-6 and comparative examples 1-2 of the present application. DETAILED DESCRIPTION

[0042] Following detailed description together with the examples will provide those skilled in the art with further instruction in the making, using and practicing the application. The general principles defined herein can be applied to other examples and applications without departing from the spirit of the application. The examples are described with specificity to meet statutory requirements, however the claims should not be construed as being limited to such examples.

[0043] It is to be understood that the figures shown in the following examples are merely schematic to illustrate the basic concept of the present application, and thus only the components related to the present application are shown in the figures, not the number, shape and size of the components as implemented in practice, the shape, number and ratio of the components in practice can be changed arbitrarily, and the layout of the components can be more complex. The terms such as "upper", "lower", "left", "right", "middle" and "one" used in the examples are only for the convenience of understanding the description, not to limit the scope of the application, and the change or adjustment of the relative relationship without substantial change of the technical content is also regarded as the scope of the application.

[0044] Some exemplary embodiments of the present application are described for illustrative purposes, it is to be understood that the present application can be implemented in other ways not specifically shown in the drawings.

[0045] The present application provides a skull flap cleaning method, comprising the following steps:

[0046] S1, the skull flap to be treated is taken out from the-80℃ freezing room, soaked in physiological saline, and then placed in a-40℃ freezing device for 12-24h, the skull flap soaked in physiological saline is placed in a-20℃ refrigeration device for 2-4h, and then soaked in physiological saline at room temperature for 30-60min; then the periosteum on the surface of the skull flap is removed, and the skull flap is washed with purified water to remove the bloodstains after the removal of the periosteum. During the placement at different temperatures, the physiological saline is replaced according to the specific conditions.

[0047] S2, the washed skull flap is placed in an ultrasonic cleaner, and 15℃ purified water is used for ultrasonic cleaning for 3 times, each time for 10-15min, and the tissue on the surface of the skull flap can be cleaned with scissors or a surgical knife before and after each ultrasonic cleaning.

[0048] S3. Immerse the skull flap in 75% ethanol for 30-60 minutes, controlling the temperature at 30-37℃ and the pressure at 7-10MPa. Then, clean the skull flap with supercritical carbon dioxide for 10-30 minutes at a flow rate of 0.1-10L / min. Next, place the skull flap in purified water and shake it in a 15℃ water bath for 30 minutes. This step can be repeated multiple times depending on the specific condition of the skull until no oil stains are produced.

[0049] S4. The skull flap undergoes gradient cooling pre-cooling: after drying, the skull flap is first placed in a bag, vacuum-sealed, and then placed in a -20℃ refrigerator for 2-4 hours, followed by a -40℃ refrigerator for 12-24 hours, and finally a -80℃ refrigerator for 12-24 hours. Then, the skull flap is removed from the packaging bag, and low-temperature carbon dioxide aerosol is sprayed onto the pre-cooled surface of the skull flap using a controlled spray device. During spraying, the carbon dioxide aerosol jet is at a 0-90° angle to the horizontal plane. The controlled spray device includes a multi-hole nozzle with orifice diameters of 10-200 μm, and the spray rate is controlled at 0.1-5 L / min; the cleaning time is 5-15 minutes. After cleaning, the carbon dioxide completely vaporizes and dissipates, further removing contaminants from the previous cleaning steps on the skull flap surface, leaving no chemical cleaning agent residue.

[0050] S5. Wrap the skull flap with sterile gauze and place it in a PA / PE packaging bag. Vacuum-dry the bag for 30-60 seconds each time. Do not completely seal the bag during the first vacuuming. After each vacuuming, wipe the skull flap dry with sterile gauze and replace the wrapping gauze. After every 3 vacuuming cycles, soak the skull flap in purified water for 10-15 minutes. This process can be repeated multiple times until no blood or oil seeps out during vacuuming. At this point, the skull flap is clean. The vacuuming time refers to the holding time under vacuum. A pressure of <-0.1 MPa is generally sufficient to remove blood and oil from the skull flap.

[0051] S6. After protecting the sharp edges of the cleaned skull flap with sterile gauze, vacuum-pack it in a packaging bag for irradiation sterilization and then store it at low temperature. The vacuum-packed skull flap is then sterilized by irradiation with 25 kGy of radioactive cobalt-60 and finally stored at low temperature for later use.

[0052] Example 1: Cleaning and treatment of cranial flaps

[0053] The skull flap to be processed was removed from the -80℃ freezer, immersed in physiological saline, and then placed in a -40℃ freezer for 12 hours. It was then placed in a -20℃ refrigerator for 4 hours, followed by immersion in physiological saline at room temperature for 30 minutes. During this gradual temperature increase, the physiological saline was replaced as needed. The periosteum on the surface of the skull flap was then removed, and it was rinsed with purified water to remove any blood. The rinsed skull flap was then placed in an ultrasonic cleaner and ultrasonically cleaned three times with 15℃ purified water for 10 minutes each time. After each ultrasonic cleaning, the surface tissue of the skull flap was cleaned with scissors or a scalpel. At this point, the skull flap was still red, and there was still blood and oil in the skull that needed further cleaning. Although the surface tissue had been removed, further cleaning of the blood and oil on the skull flap was still necessary.

[0054] After immersing the skull flap in 75% ethanol for 30 minutes, the temperature was controlled at 31°C and the pressure at 7 MPa. Then, it was cleaned with supercritical carbon dioxide for 10 minutes at a flow rate of 10 L / min. The skull flap was then placed in purified water and agitated in a 15°C water bath for 30 minutes. This process could be repeated multiple times until no oil stains were produced. After this step, the skull flap was cleaned and appeared slightly yellow.

[0055] The skull flap was dried using sterile gauze, placed in a bag, vacuum-sealed, and then first 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 was then removed from the packaging bag, and low-temperature carbon dioxide aerosol was directionally sprayed onto the pre-cooled skull flap surface using a controlled spray device. During spraying, the carbon dioxide aerosol jet flow was at an angle of less than 90° to the horizontal plane. The controlled spray device included a multi-hole nozzle with a nozzle orifice diameter of 10 μm, a spray rate of 0.1 L / min, and a spraying time of 5 minutes. At this point, the skull flap appeared white, indicating it had been cleaned.

[0056] Wipe the treated skull flap dry with sterile gauze 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 completely seal it when vacuuming for the first time. Wipe the skull flap dry with gauze after each vacuuming. After every 3 vacuumings, soak the skull flap in purified water for 10 minutes. This process can be repeated multiple times until no blood or oil seeps out when vacuuming.

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

[0058] Example 2: Cleaning and treatment of cranial flaps (Part 2)

[0059] The skull flap to be processed was removed from the -80℃ freezer, immersed in physiological saline, and then placed in a -40℃ freezer for 18 hours. It was then placed in a -20℃ refrigerator for 2.5 hours, followed by immersion in physiological saline at room temperature for 35 minutes. During this gradual temperature increase, the physiological saline was replaced as needed. The periosteum on the surface of the skull flap was then removed, and it was rinsed with purified water to remove any blood. The rinsed skull flap was then placed in an ultrasonic cleaner and sonicated three times with 15℃ purified water for 11 minutes each time. After each ultrasonic cleaning, the surface tissue of the skull flap was cleaned with scissors or a scalpel. At this point, the skull flap was still red, and there was still blood and grease to be removed. While the surface tissue had been removed, further cleaning of the blood and grease was necessary.

[0060] After immersing the skull flap in 75% ethanol for 40 minutes, the temperature was controlled at 31.5℃ and the pressure at 7.4 MPa. It was then cleaned with supercritical carbon dioxide for 15 minutes at a flow rate of 7 L / min. The skull flap was then placed in purified water and agitated in a 15℃ water bath for 30 minutes. This process could be repeated multiple times until no oil stains were produced. After this step, the sutures and complex structures of the skull flap were cleaned and appeared slightly yellow.

[0061] The skull flap was dried using sterile gauze, placed in a bag, vacuum-sealed, and then first 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 was then removed from the packaging bag, and low-temperature carbon dioxide aerosol was directionally sprayed onto the pre-cooled surface of the skull flap using a controlled spray device. During spraying, the carbon dioxide aerosol jet flow maintained an angle of less than 90° with the horizontal plane. The controlled spray device included a multi-hole nozzle with a nozzle orifice diameter of 30 μm, a spray rate of 0.5 L / min, and a spraying time of 8 minutes. At this point, the skull flap appeared white, indicating it was clean, and the crevices and structurally complex parts of the skull flap were also cleaned.

[0062] Wipe the treated skull flap dry with sterile gauze 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 completely seal it when vacuuming for the first time. Wipe the skull flap dry with gauze after each vacuuming. After every 3 vacuumings, soak the skull flap in purified water for 11 minutes. This process can be repeated multiple times until no blood or oil seeps out when vacuuming.

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

[0064] The cranial 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 this application can clean the relatively complex cranial flap, minimizing the residual substances while preserving the complete cranial flap structure.

[0065] Example 3: Cleaning and treatment of cranial flaps (Part 3)

[0066] The skull flap to be processed was removed from the -80℃ freezer, immersed in physiological saline, and then placed in a -40℃ freezer for 20 hours. It was then placed in a -20℃ refrigerator for 3 hours, followed by immersion in physiological saline at room temperature for 40 minutes. During this gradual temperature increase, the physiological saline was replaced as needed. The periosteum on the surface of the skull flap was then removed, and it was rinsed with purified water to remove any blood. The rinsed skull flap was then placed in an ultrasonic cleaner and ultrasonically cleaned three times with 15℃ purified water for 12 minutes each time. After each ultrasonic cleaning, the surface tissue of the skull flap was cleaned with scissors or a scalpel. At this point, the skull flap was still red, and the surface tissue had been removed, but further cleaning of the blood and grease was still needed.

[0067] After immersing the skull flap in 75% ethanol for 45 minutes, the temperature was controlled at 32℃ and the pressure at 8 MPa. It was then cleaned with supercritical carbon dioxide for 20 minutes at a flow rate of 4 L / min. The skull flap was then placed in purified water and agitated in a 15℃ water bath for 30 minutes. This process could be repeated multiple times until no oil stains were produced. At this point, the skull flap was cleaned and appeared slightly yellow.

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

[0069] Wipe the treated skull flap dry with sterile gauze 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 completely seal it when vacuuming for the first time. Wipe the skull flap dry with gauze after each vacuuming. After every 3 vacuumings, soak the skull flap in purified water for 12 minutes. This process can be repeated multiple times until no blood or oil seeps out when vacuuming.

[0070] After the sharp edges of the cleaned skull flaps were protected with sterile gauze, they were vacuum-packed in packaging bags, irradiated and sterilized, and then stored at low temperature. An irradiation sterilization label was affixed to the second layer of packaging bags. The vacuum-packed skull flaps were then irradiated with cobalt-60 radioactive element with an irradiation dose of 25 kGy and finally stored in a refrigerator at -80°C.

[0071] Example 4: Cleaning and treatment of cranial flaps (Part 4)

[0072] The skull flap to be processed was removed from the -80℃ freezer, immersed in physiological saline, and then placed in a -40℃ freezer for 20 hours. It was then placed in a -20℃ refrigerator for 2 hours, followed by immersion in physiological saline at room temperature for 45 minutes. During this gradual temperature increase, the physiological saline was replaced as needed. The periosteum on the surface of the skull flap was then removed, and it was rinsed with purified water to remove any blood. The rinsed skull flap was then placed in an ultrasonic cleaner and ultrasonically cleaned three times with 15℃ purified water for 13 minutes each time. After each ultrasonic cleaning, the surface tissue of the skull flap was cleaned with scissors or a scalpel. At this point, the skull flap was still red, and the surface tissue had been removed, but further cleaning of the blood and grease was still needed.

[0073] After immersing the skull flap in 75% ethanol for 45 minutes, the temperature was controlled at 34℃ and the pressure at 8.7 MPa. It was then cleaned with supercritical carbon dioxide for 20 minutes at a flow rate of 2 L / min. The skull flap was then placed in purified water and agitated in a 15℃ water bath for 30 minutes. This process could be repeated multiple times until no oil stains were produced. At this point, the skull flap was cleaned and appeared slightly yellow.

[0074] The skull flap was dried using sterile gauze, placed in a bag, vacuum-sealed, and then first 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 low-temperature carbon dioxide aerosol was directionally sprayed onto the pre-cooled surface of the skull flap using a controlled spray device. During spraying, the carbon dioxide aerosol jet flow was at an angle of less than 90° to the horizontal plane. The controlled spray device included a multi-hole nozzle with a nozzle orifice diameter of 100 μm, a spray rate of 3.5 L / min, and a spraying time of 12 minutes. At this point, the skull flap had turned white, indicating that it had been cleaned.

[0075] Wipe the treated skull flap dry with sterile gauze 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 completely seal it when vacuuming for the first time. Wipe the skull flap dry with gauze after each vacuuming. After every 3 vacuumings, soak the skull flap in purified water for 11 minutes. This process can be repeated multiple times until no blood or oil seeps out when vacuuming.

[0076] After the sharp edges of the cleaned skull flap were protected with sterile gauze, it was vacuum-packed in a packaging bag, irradiated and sterilized, and then stored at low temperature. An irradiation sterilization label was affixed to the second packaging bag. The vacuum-packed skull flap was then irradiated with 25 kGy of radioactive cobalt-60 and finally stored in a -80°C refrigerator.

[0077] Example 5: Cleaning and Treatment of the Skull Flap

[0078] The skull flap to be processed was removed from the -80℃ freezer, immersed in physiological saline, and then placed in a -40℃ freezer for 22 hours. It was then placed in a -20℃ refrigerator for 2 hours, followed by immersion in physiological saline at room temperature for 50 minutes. During this gradual temperature increase, the physiological saline was replaced as needed. The periosteum on the surface of the skull flap was then removed, and it was rinsed with purified water to remove any blood. The rinsed skull flap was then placed in an ultrasonic cleaner and ultrasonically cleaned three times with 15℃ purified water for 14 minutes each time. After each ultrasonic cleaning, the surface tissue of the skull flap was cleaned with scissors or a scalpel. At this point, the skull flap was still red, and the surface tissue had been removed, but further cleaning of the blood and grease was still needed.

[0079] After immersing the skull flap in 75% ethanol for 50 minutes, the temperature was controlled at 35.8℃ and the pressure at 9.4 MPa. It was then cleaned with supercritical carbon dioxide for 25 minutes at a flow rate of 0.8 L / min. The skull flap was then placed in purified water and agitated in a 15℃ water bath for 30 minutes. This process could be repeated multiple times until no oil stains were produced. At this point, the skull flap had been cleaned and appeared slightly yellow.

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

[0081] Wipe the treated skull flap dry with sterile gauze 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 completely seal it when vacuuming for the first time. Wipe the skull flap dry with gauze after each vacuuming. After every 3 vacuumings, soak the skull flap in purified water for 13 minutes. This process can be repeated multiple times until no blood or oil seeps out when vacuuming.

[0082] After the sharp edges of the cleaned skull flap were protected with sterile gauze, it was vacuum-packed in a packaging bag, irradiated and sterilized, and then stored at low temperature. An irradiation sterilization label was affixed to the second packaging bag. The vacuum-packed skull flap was then irradiated with 25 kGy of radioactive cobalt-60 and finally stored in a -80°C refrigerator.

[0083] Example 6: Cleaning and Treatment of Skull Flaps

[0084] The skull flap to be processed was removed from the -80℃ freezer, immersed in physiological saline, and then placed in a -40℃ freezer for 24 hours. It was then placed in a -20℃ refrigerator for 2 hours, followed by immersion in physiological saline at room temperature for 60 minutes. During this gradual temperature increase, the physiological saline was replaced as needed. The periosteum on the surface of the skull flap was then removed, and it was rinsed with purified water to remove any blood. The rinsed skull flap was then placed in an ultrasonic cleaner and ultrasonically cleaned three times with 15℃ purified water for 15 minutes each time. After each ultrasonic cleaning, the surface tissue of the skull flap was cleaned with scissors or a scalpel. At this point, the skull flap was still red, and the surface tissue had been removed, but further cleaning of the blood and grease was still needed.

[0085] After immersing the skull flap in 75% ethanol for 60 minutes, the temperature was controlled at 37°C and the pressure at 10 MPa. Then, it was cleaned with supercritical carbon dioxide for 30 minutes at a flow rate of 0.1 L / min. The skull flap was then placed in purified water and agitated in a 15°C water bath for 30 minutes. This process could be repeated multiple times until no oil stains were produced. At this point, the skull flap was cleaned and appeared slightly yellow.

[0086] The skull flap was dried using sterile gauze, placed in a bag, vacuum-sealed, and then refrigerated at -20°C for 4 hours, followed by -40°C for 12 hours, and finally -80°C for 24 hours. The skull flap was then removed from the packaging bag, and low-temperature carbon dioxide aerosol was directionally sprayed onto its surface using a controlled spray device. During spraying, the carbon dioxide aerosol jet was held at an angle of less than 90° to the horizontal plane. The controlled spray device included a multi-hole nozzle with a 200μm orifice diameter, a spray rate of 5L / min, and a cleaning time of 15 minutes. At this point, the skull flap appeared white, indicating it had been cleaned.

[0087] Wipe the treated skull flap dry with sterile gauze 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 completely seal it when vacuuming for the first time. Wipe the skull flap dry with gauze after each vacuuming. After every 3 vacuumings, soak the skull flap in purified water for 15 minutes. This process can be repeated multiple times until no blood or oil seeps out when vacuuming.

[0088] After the sharp edges of the cleaned skull flap were protected with sterile gauze, it was vacuum-packed in a packaging bag, irradiated and sterilized, and then stored at low temperature. An irradiation sterilization label was affixed to the second packaging bag. The vacuum-packed skull flap was then irradiated with 25 kGy of radioactive cobalt-60 and finally stored in a -80°C refrigerator.

[0089] Comparative Example 1

[0090] The skull flap to be processed was removed from the -80℃ freezer and placed in a -40℃ freezer for 12 hours, then placed in a -20℃ refrigerator for 4 hours. After that, it was soaked in physiological saline at room temperature for 30 minutes, and then rinsed with purified water. The rinsed skull flap was then placed in an ultrasound device and sonicated three times with purified water at 15℃, each time for 10 minutes. After each ultrasound cleaning, the tissue on the surface of the skull flap was cleaned with scissors or a scalpel.

[0091] After soaking the skull flap in 75% ethanol for 30 minutes, place it in purified water and shake in a 15°C water bath for 30 minutes. This process can be repeated multiple times until no oil stains are produced.

[0092] After drying the skull flaps treated above, place them in a PA / PE packaging bag and vacuum-seal them for 30 seconds each time. Do not completely seal the bag during the first vacuuming. After each vacuuming, wipe the skull flaps dry with gauze. After every 3 vacuuming cycles, soak the skull flaps in purified water for 15 minutes. This process can be repeated multiple times until no blood or oil seeps out during vacuuming.

[0093] After protecting the sharp edges of the cleaned skull flap with sterile gauze, it was vacuum-packed in three layers of PA / PE bags for irradiation sterilization and then stored at low temperature. An irradiation sterilization label was affixed to the second layer of PA / PE bag. The vacuuming process must be completed while the skull is still frozen, and the packaging time should be controlled within 5 minutes. It was then placed in a -80°C freezer to await irradiation sterilization. The vacuum-packed skull flap was then sterilized by irradiation with 25 kGy of radioactive cobalt-60.

[0094] Comparative Example 2

[0095] The skull flap to be processed was removed from the -80℃ freezer and placed in a -40℃ freezer for 24 hours, then placed in a -20℃ refrigerator for 2 hours. After that, it was soaked in physiological saline at room temperature for 60 minutes, and then rinsed with purified water. The rinsed skull flap was then placed in an ultrasound device and sonicated three times with purified water at 15℃, each time for 15 minutes. After each ultrasound cleaning, the tissue on the surface of the skull flap was cleaned with scissors or a scalpel.

[0096] After soaking the skull flap in 75% ethanol for 60 minutes, place it in purified water and shake in a 15°C water bath for 30 minutes. This process can be repeated multiple times until no oil stains are produced.

[0097] After drying the skull flaps treated above, place them in a PA / PE packaging bag and vacuum-dry them for 60 seconds each time. Do not completely seal the bag during the first vacuuming. After each vacuuming, wipe the skull flaps dry with gauze. After every 3 vacuuming cycles, soak the skull flaps in purified water for 10 minutes. This process can be repeated multiple times until no blood or oil seeps out during vacuuming.

[0098] After protecting the sharp edges of the cleaned skull flap with sterile gauze, it was vacuum-packed in three layers of PA / PE bags for irradiation sterilization and then stored at low temperature. An irradiation sterilization label was affixed to the second layer of PA / PE bag. The vacuuming process must be completed while the skull is still frozen, and the packaging time should be controlled within 5 minutes. It was then placed in a -80°C freezer to await irradiation sterilization. The vacuum-packed skull flap was then sterilized by irradiation with 25 kGy of radioactive cobalt-60.

[0099] The fat content and water content of the cleaned cranial flaps of Examples 1-6 and Comparative Examples 1-2 were measured, 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. Lower water content is more conducive to subsequent irradiation sterilization and storage of the skull. The skull flap cleaning treatment method provided by this 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 reabsorption process, and also reducing the water content of the skull, which is conducive to the penetration of irradiation sterilization and the storage of the skull during the process.

[0104] Furthermore, the cleaned skull flaps from Examples 1-6 and Comparative Examples 1-2 were decalcified, dehydrated, paraffin-embedded, sectioned, stained with hematoxylin and eosin, dehydrated, and mounted. The flaps were then examined under a microscope, and images were acquired and analyzed to observe any residual cells. Figure 1 a-1f correspond to the decellularization detection results of the skull flap surface after cleaning in Examples 1-6, and Figure 1 g-1h correspond to the decellularization detection results on the surface of the skull flaps after washing in Comparative Examples 1-2, respectively. Figure 1 a-1b corresponds to Examples 1-2, and it can be seen that there are fewer cell nuclei and cell debris remaining after decellularization treatment, indicating that the decellularization effect is better; Figure 1 c-1d corresponds to Examples 3-4, and it can be seen that a small number of cell nuclei and cell fragments remain after decellularization treatment, indicating that the decellularization effect is good; Figure 1e-1f corresponds to Examples 5-6, and it can be seen that no cell nuclei or cell debris remain after decellularization treatment, indicating that the decellularization effect is good. Figure 1 Comparative examples 1-2 (g-1h) show a significant amount of residual cell nuclei and cell debris after decellularization, indicating a generally poor decellularization effect. This demonstrates that the cranial flap cleaning method provided by this invention can more effectively clean the skull, effectively remove lipids from the skull, achieve good decellularization, 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. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this application should still be covered by the claims of this application.

Claims

1. A method for cleaning and treating cranial bone flaps, characterized in that, Includes the following steps: S1. Immerse the skull flap to be cleaned in physiological saline and heat it to room temperature, then rinse the skull flap with purified water. S2. Place the rinsed skull flap into an ultrasonic cleaning device for cleaning. S3. After immersing the skull flap in 75% ethanol for a period of time, the skull flap after immersion is cleaned with supercritical fluid. S4. First, the skull flap is pre-cooled by gradient cooling, and then the skull flap is cleaned by low-temperature carbon dioxide aerosol. The gradient cooling pre-cooling step specifically involves drying the skull flap, first placing it in a bag, vacuuming it, and then placing it in a -20℃ refrigerator for 2-4 hours, then placing it in a -40℃ refrigerator for 12-24 hours; and finally placing it in a -80℃ refrigerator for 12-24 hours. S5. After drying the skull flap processed in step S4, place it in a packaging bag and vacuum it. S6. After cleaning, the skull flaps are sterilized by irradiation and then stored at low temperature.

2. The method for cleaning and treating cranial flaps according to claim 1, characterized in that, Step S1 is as follows: The skull flaps to be treated were immersed in physiological saline and then placed in a -40℃ refrigeration unit for pretreatment for 12-24 hours, and then placed in a -20℃ refrigeration unit for pretreatment for 2-4 hours. Immerse the skull flap in physiological saline at room temperature for 30-60 minutes, then remove the skull flap and rinse it with purified water.

3. The method for cleaning and treating cranial flaps according to claim 1, characterized in that, In step S2, the skull flap is subjected to water bath ultrasound three times in an ultrasonic cleaning device using purified water at 15°C, with each session lasting 10-15 minutes.

4. The method for cleaning and treating cranial flaps according to claim 1, characterized in that, Step S3 is as follows: Soak the skull flap in 75% alcohol for 30-60 minutes; The skull flap was cleaned by supercritical carbon dioxide at a controlled temperature of 30-37℃ and a pressure of 7-10 MPa.

5. The method for cleaning and treating cranial flaps 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 method for cleaning and treating cranial flaps according to claim 1, characterized in that, In step S4, carbon dioxide aerosol is directionally sprayed onto the surface of the skull flap using a controllable spraying device for cleaning. The airflow of the carbon dioxide aerosol is at an angle of 0-90° to the horizontal plane.

7. The method for cleaning and treating cranial flaps according to claim 6, characterized in that, In step S4, the injection rate of carbon dioxide aerosol is 0.1-5 L / min.

8. The method for cleaning and treating cranial flaps according to claim 6, characterized in that, In step S4, the cleaning time is 5-15 minutes.

9. The method for cleaning and treating cranial flaps according to claim 1, characterized in that, Step S5 is as follows: After the skull flap is dried, it is placed in a packaging bag; The cranial flap, once placed in the packaging bag, is vacuum-sealed for 30-60 seconds each time. After each 3 vacuuming cycles, soak the skull flap in purified water for 10-15 minutes until no blood or oil seepage occurs during vacuuming.

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