Preparation method of skull for autologous replantation

Abstract

The invention provides a preparation method of a skull for autologous replantation, and relates to the technical field of skull treatment. The method comprises the following steps that the taken-out autologous skull is subjected to ultraviolet radiation and then conveyed into a clean room to be subjected to photographing treatment; sequentially carrying out iodophor soaking, alcohol soaking and normal saline flushing on the skull, and then carrying out surface tissue impurity removal and cleaning on the skull; putting the treated skull into a packaging bag, adding a cryopreservation solution to wrap the skull, and then performing vacuumizing and sealing treatment; and sterilizing and cooling the skull subjected to the sealing treatment, and finally feeding the skull into a bone library for cryopreservation. According to the method, the skull of a patient is taken out after an operation to serve as an autologous bone raw material, so that the skull has bone activity within a certain period of time, finally, the skull is replanted to the skull of the patient, the body burden and conflict psychology of the patient are reduced, the exclusive transportation, treatment and storage modes are adopted, the rejection of the patient is reduced, the risk of bone resorption is reduced, and the bone activity is guaranteed; the replanting rate is increased.

Description

Technical Field

[0001] The present application relates to the technical field of skull processing, and in particular to a method for preparing a skull for autologous replantation. Background Art

[0002] Skull treatment has always been a difficult problem in clinical practice, especially for neurosurgery. Decompressive craniectomy is an effective treatment for patients with craniocerebral trauma / cerebral hemorrhage. In order to achieve the purpose of surgical decompression, the excised bone flap cannot be restored. Therefore, most patients suffer from skull defects after the operation. Usually, in order to reduce the side effects of skull defects on patients, doctors recommend that patients return to the hospital for skull defect repair after about 3 months. In the past 10 years, the skull defect repair materials that clinicians have recognized are mostly medical titanium mesh, but titanium mesh also has shortcomings that make patients and doctors dissatisfied. For example, heat conduction can cause headaches or even epileptic symptoms, it is easy to deform and affect the appearance, and MRI examinations are also affected to varying degrees.

[0003] Many years ago, some scholars buried their own skulls in the patient's body (such as the inner thigh, abdomen, etc.), but when they were taken out and used a few months later, the autologous bone was absorbed, making it impossible to repair the original defect. Of course, burying the autologous skull in other parts of the body will cause new trauma and infection risks. In addition, due to different storage methods and temperatures, the activity of the autologous skull bone is not ideal. Summary of the invention

[0004] The purpose of the present application is to provide a method for preparing a skull for autologous replantation, which improves the activity of the skull and the transplantation rate of the skull replantation.

[0005] In order to solve the above technical problems, the technical solution adopted in this application is:

[0006] The present application provides a method for preparing a skull for autologous replantation, comprising the following steps:

[0007] S1. After the autologous skull is removed and exposed to ultraviolet light, it is brought into the clean room for photographing;

[0008] S2, sequentially soak the skull in iodine, alcohol, and saline, and then remove impurities and clean the skull surface tissue;

[0009] S3, placing the skull treated as above into a packaging bag, adding a cryopreservation solution to wrap it, and then vacuumizing and sealing it;

[0010] S4. Sterilize and cool the sealed skull, and finally send it to a bone bank for cryopreservation.

[0011] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0012] The present application obtains the patient's own skull as autologous bone material after surgery, so that the skull flap has bone activity for a certain period of time. When the patient recovers, the skull is re-implanted onto the patient's skull, thereby reducing the patient's physical burden and resistance. A unique transportation, processing, and storage method is used to reduce the patient's own rejection after surgery, reduce the risk of bone absorption, ensure bone activity, and increase the re-implantation rate. DETAILED DESCRIPTION

[0013] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0014] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to specific embodiments.

[0015] A method for preparing a skull for autologous replantation comprises the following steps:

[0016] S1. After the autologous skull is removed and exposed to ultraviolet light, it is brought into the clean room for photographing;

[0017] S2, sequentially soak the skull in iodine, alcohol, and saline, and then remove impurities and clean the skull surface tissue;

[0018] S3, placing the skull treated as above into a packaging bag, adding a cryopreservation solution to wrap it, and then vacuumizing and sealing it;

[0019] S4. Sterilize and cool the sealed skull, and finally send it to a bone bank for cryopreservation.

[0020] In some embodiments of the present application, the above-mentioned step S1 further includes removing the outer packaging of the autologous skull before it is introduced into the clean room, and the photographing process includes measuring and photographing the size and weight of the skull.

[0021] In some embodiments of the present application, the impurity removal in the above-mentioned step S2 specifically includes using a sterile surgical blade to remove soft tissue and impurities on the surface of the skull and remove sharp corners on the surface of the skull.

[0022] In some embodiments of the present application, in the above step S2, the iodine immersion time is 5 minutes, and the alcohol immersion time is 1 minute; and ultrasonic cleaning is performed using normal saline and gentamicin.

[0023] In some embodiments of the present application, the sealing process in the above-mentioned S3 step is specifically as follows: affix a patient information label to the vacuum-treated packaging bag, then put it into a layer of packaging bag and seal it, and finally put the double-layer packaged skull into a sterile bag to complete the three-layer sealing.

[0024] In some embodiments of the present application, the skull is sterilized by irradiation in the above-mentioned S4 step; the cooling is specifically to cool the skull to -120°C by programmed cooling, and the specific cooling process is: cooling from room temperature to -20°C at a rate of 1°C / min, then cooling from -20°C to -80°C at a rate of 25°C / min, and finally cooling from -80°C to -120°C at a rate of 10°C / min.

[0025] In some embodiments of the present application, the above-mentioned S2 step also includes an inspection of the cleaning after cleaning, and the specific steps are: use a membrane to filter the normal saline after ultrasonic cleaning of the skull, place the filtered filter membrane in tryptic soy peptone agar medium and culture it at 33°C for 3 days, and then culture it at 23°C for 5 days in Sabouraud dextrose agar medium, and observe whether the colony count meets the standard. If it does not meet the standard, the skull is repeatedly cleaned.

[0026] In some embodiments of the present application, the above-mentioned S4 step also includes a sterility test for the skull after the sterilization treatment. The specific steps are: the water after ultrasonic cleaning of the skull is used as the test solution, and it is sterilized under the same conditions as the skull. After sterilization, the direct inoculation method is used to add the test solution into the pancreatic casein soy peptone liquid culture medium and culture it at 23°C for 14 days and thioglycollate fluid culture medium at 33°C for 14 days, and observe whether the appearance of the culture medium is turbid. If there is no turbidity, it is negative and qualified; if it is turbid, it is positive and unqualified; if it is unqualified, the skull is sterilized again.

[0027] In some embodiments of the present application, the above-mentioned S4 step also includes a factory release step for the skull after the cryopreservation, and the factory release step includes a sterility test of the skull during cryopreservation and -196°C transportation before release.

[0028] In some embodiments of the present application, the above-mentioned sterility detection step of the skull during cryopreservation before leaving the factory is specifically as follows: the water after ultrasonic cleaning of the skull is used as the test solution, and it is sterilized and frozen under the same conditions as the skull. Using the direct inoculation method, the test solution is added to the trypsin soy liquid culture medium at 23°C and cultured for 14 days, the thioglycollate fluid culture medium at 33°C and cultured for 14 days, and the trypsin soy agar culture medium at 33°C and cultured for 3 days. The appearance of the culture medium is observed to be turbid. If there is no turbidity, it is negative and qualified; if it is turbid, it is positive and unqualified.

[0029] In some embodiments of the present application, the preparation methods of the above-mentioned tryptic soy agar medium (TSA), Sabouraud dextrose agar medium (SDA), thioglycollate fluid medium (FTM) and tryptic soy liquid medium (TSB) are as follows:

[0030] Prepare tryptic soy agar (TSA) according to the instructions of the medium, weigh 40 g, add 1000 ml of distilled water, and stir and heat until completely dissolved.

[0031] Sabouraud dextrose agar medium (SDA) was prepared according to the medium instructions. 65 g was weighed, 1000 ml of distilled water was added, stirred and heated until completely dissolved, and then boiled. After cooling, the pH values ​​of the two culture media were tested. If the pH values ​​met the requirements, they were sterilized at 121°C for 15 min and cooled to 45°C for distribution.

[0032] Weigh 29.29 g of thioglycollate fluid medium (FTM), add 1000 ml of distilled water, and stir and heat until completely dissolved.

[0033] Prepare TSB medium according to the medium instructions, weigh 30 g, add 1000 ml of distilled water, stir and heat until completely dissolved, boil, and test the pH values ​​of the two culture media after cooling. When the pH values ​​meet the requirements, package them and sterilize them at 121°C for 15 min for later use.

[0034] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0035] Example 1

[0036] This embodiment provides a method for preparing a skull for autologous replantation, which mainly includes the following steps:

[0037] 1. Skull treatment:

[0038] 1-1. After the autologous skull is removed and irradiated through the UV transmission window, the outer packaging is removed and transferred to the clean room.

[0039] 1-2. Sterilize the beaker, conical flask, waist tray, tweezers, surgical knife handle, scissors, brush, and square ruler by wet heat sterilization at 121°C for 30 minutes to ensure the sterility of the instruments.

[0040] 1-3. Open the inner packaging bags of the skull layer by layer on the clean workbench, take photos of the size and weight of the skull, soak the skull in iodine for 5 minutes, then use alcohol to deiodine, and then rinse the residual alcohol with sterile saline.

[0041] 1-4. Use a sterile surgical blade to remove the soft tissue on the surface of the skull to ensure that the skull is clean. Use a grinder to remove the sharp corners of the skull to get the skull to prevent the storage package from being scratched during storage.

[0042] 1-5. Use 500ml of sterile saline and one vial of 80,000 units of gentamicin to clean the skull with ultrasound for 15 minutes to inhibit bacteria. The water after cleaning should be kept for future use.

[0043] 1-6. Place the processed skull in an EVA bag, add an appropriate amount of cryopreservative solution (enough to fully cover the skull), and vacuum seal it. Then attach the prepared patient information label to the skull package, and then seal the skull in a layer of EVA packaging bag. Finally, place the double-wrapped skull in a pre-prepared sterile bag to complete the three-layer sealed packaging for storage.

[0044] 2. Bioburden detection:

[0045] 2-1 First sterilize the tweezers, scissors, conical flask, and disposable filter cup.

[0046] 2-2. Weigh 40g of tryptic soy peptone agar medium, add 1000ml of distilled water, stir and heat until completely dissolved, and dispense. Autoclave at 121℃ for 15min for later use. Weigh 65g of Sabouraud dextrose agar medium, heat to one liter of distilled water until completely dissolved, and autoclave at 121℃ for 15min for later use. Use membrane filtration method to detect whether the bioburden of the culture medium is qualified: put the filter membrane after filtering the liquid into tryptic soy peptone agar medium at 33℃ for 3 days, and Sabouraud dextrose agar medium at 23℃ for 5 days.

[0047] 2-3. Irradiation sterilization: The packaged skull is sterilized by irradiation sterilization process with an irradiation dose of 25kGy.

[0048] 3. Sterility testing:

[0049] 3-1. Sterilize instruments at 121°C with moist heat.

[0050] 3-2. Weigh 40g of tryptic soy peptone agar medium, add 1000ml of distilled water, stir and heat until completely dissolved, and dispense. Autoclave at 121℃ for 15min for use. Dissolve 29.25g of thioglycollate fluid medium in 1L of purified water, dispense into appropriate containers, heat to boil until completely dissolved, and autoclave at 121℃ for 15min. Weigh 30g of tryptic soy peptone agar in 1L of purified water, heat to boil until completely dissolved, and autoclave at 121℃ for 15min.

[0051] 3-3. The water used for ultrasonic treatment of skull bones was sterilized under the same conditions as skull bones, and then the culture medium was tested by direct inoculation method. Trypticase soytone liquid culture medium was cultured at 23℃ for 14 days, thioglycollate fluid culture medium was cultured at 33℃ for 14 days, and trypticase soytone agar culture medium was cultured at 33℃ for 3 days (testing the operating table environment).

[0052] 4. Cryopreservation of skull:

[0053] 4-1. Use a programmed cooling device to program the temperature of the sterilized skull to -120°C. Quickly move the skull after program cooling to a liquid nitrogen tank at -196°C for 24-hour monitoring and storage.

[0054] 5. Skull reimplantation:

[0055] 5-1. 121℃ moist heat sterilization, sterilization of instruments.

[0056] 5-2. Tryptic soy peptone liquid culture medium: Weigh 40g of tryptic soy peptone agar culture medium, add 1000ml of distilled water, stir and heat until completely dissolved, and divide into portions. Autoclave at 121℃ for 15min for use. Thioglycollate fluid culture medium: Dissolve 29.25g in 1L of purified water, divide into appropriate containers, heat to boil until completely dissolved, and autoclave at 121℃ for 15min. Weigh 30g of tryptic soy peptone agar and dissolve in 1L of purified water, heat to boil until completely dissolved, and autoclave at 121℃ for 15min.

[0057] 5-3. Use direct inoculation method to take out the spare water used for skull ultrasonic treatment and use direct inoculation method to perform sterility test on the culture machine. Culture the culture medium with tryptic soytone liquid at 23℃ for 14 days, culture the culture medium with thioglycollate liquid at 33℃ for 14 days, and culture the culture medium with tryptic soytone agar at 33℃ for 3 days.

[0058] 5-4. Bioburden test records, receipt, factory inspection report, factory inspection records, pre-shipment sterility inspection records, pre-freezing sterility inspection records, sterilization certificate, replantation process, and factory labels.

[0059] 5-5. The skull is shipped out of the warehouse at -196℃.

[0060] In this example, rats were used for the experiment. After being subjected to the process of this example and frozen in liquid nitrogen, the activity of bone cells was detected. The results are shown in Table 1.

[0061] Table 1

[0062]

[0063] As can be seen from Table 1, after the rat skull bones were removed from the body, they were sterilized with iodine after simulated cold chain transportation and frozen in liquid nitrogen at -196°C for 5 days. After 1.5 hours of resuscitation and digestion, the viability of rat skull bone cells was detected to be 57.60%.

[0064] In summary, the method for preparing a skull for autologous reimplantation in an embodiment of the present application has the following advantages: the present application removes the patient's own skull as autologous bone raw material after surgery, so that the skull flap has bone activity for a certain period of time. When the patient recovers, the skull is reimplanted onto the patient's skull, thereby reducing the patient's physical burden and resistance. A unique transportation, processing, and storage method is used to reduce the patient's own rejection after surgery, reduce the risk of bone absorption, ensure bone activity, and increase the reimplantation rate.

[0065] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present application.

Claims

1. A method for preparing a skull for autologous replantation, characterized in that: The following steps are involved: S1. After the autologous skull is removed and exposed to ultraviolet light, it is brought into the clean room for photographing; S2, sequentially soak the skull in iodine, alcohol, and saline, and then remove impurities and clean the skull surface tissue; S3, placing the skull treated as above into a packaging bag, adding a cryopreservation solution to wrap it, and then vacuumizing and sealing it; S4. Sterilize and cool the sealed skull, and finally send it to a bone bank for cryopreservation.

2. The method for preparing a skull for autologous replantation according to claim 1, characterized in that: The step S1 includes removing the outer packaging of the autologous skull before it is introduced into the clean room, and the photographing process includes measuring and photographing the size and weight of the skull.

3. The method for preparing a skull for autologous replantation according to claim 1, characterized in that: The impurity removal in step S2 specifically includes using a sterile surgical blade to remove soft tissue and impurities on the surface of the skull and remove sharp corners on the surface of the skull.

4. The method for preparing a skull for autologous replantation according to claim 1, characterized in that: In the step S2, the iodine immersion time is 5 minutes, and the alcohol immersion time is 1 minute; the cleaning adopts ultrasonic cleaning with a mixture of physiological saline and gentamicin, the amount of gentamicin added is 160,000 units / L, and the ultrasonic cleaning time is 15 minutes.

5. The method for preparing a skull for autologous replantation according to claim 1, characterized in that: The sealing process in step S3 is specifically as follows: affix a patient information label to the vacuum-treated packaging bag, then place the bag in a first-layer packaging bag and seal it, and finally place the double-layer packaged skull in a sterile bag to complete the three-layer sealing.

6. The method for preparing a skull for autologous replantation according to claim 1, characterized in that: In the S4 step, the skull is sterilized by irradiation sterilization with an irradiation dose of 25 kGy; the cooling is specifically to cool the skull to -120°C by programmed cooling; the cooling process is: cooling from room temperature to -20°C at a rate of 1°C / min, then cooling from -20°C to -80°C at a rate of 25°C / min, and finally cooling from -80°C to -120°C at a rate of 10°C / min.

7. The method for preparing a skull for autologous replantation according to claim 4, characterized in that: The S2 step also includes a cleaning inspection after cleaning, and the specific steps are: use a membrane to filter the physiological saline after ultrasonic cleaning of the skull, place the filtered filter membrane in a tryptic soy peptone agar medium at 33°C for 3 days, and culture it at 23°C for 5 days in a Sabouraud dextrose agar medium, and observe whether the colony count meets the standard. If it does not meet the standard, the skull is repeatedly cleaned.

8. The method for preparing a skull for autologous replantation according to claim 4, characterized in that: The S4 step also includes a sterility test for the skull after the sterilization treatment, and the specific steps are: the water after ultrasonic cleaning of the skull is used as the test solution, and it is sterilized under the same conditions as the skull. After sterilization, the direct inoculation method is used to add the test solution to the tryptic soy casein liquid culture medium at 23°C and the thioglycollate fluid culture medium at 33°C for 14 days, and observe whether the appearance of the culture medium is turbid. If there is no turbidity, it is negative and qualified; if it is turbid, it is positive and unqualified; if it is unqualified, the skull is sterilized again.

9. The method for preparing a skull for autologous replantation according to claim 1, characterized in that: After the cryopreservation in step S4, the skull is also shipped out of the factory. The shipping step includes sterility testing of the skull during cryopreservation and -196°C transportation before shipping.

10. The method for preparing a skull for autologous replantation according to claim 4, characterized in that: The sterility test steps for the skull during cryopreservation before shipment are as follows: the water after ultrasonic cleaning of the skull is used as the test solution, and it is sterilized and cryopreserved under the same conditions as the skull. The test solution is added to the tryptic soy peptone liquid culture medium at 23°C and cultured for 14 days, the thioglycollate fluid culture medium at 33°C and cultured for 14 days, and the tryptic soy peptone agar culture medium at 33°C and cultured for 3 days. The appearance of the tryptic soy peptone liquid culture medium and the thioglycollate fluid culture medium is observed to see if they are turbid. If there is no turbidity, it is negative and qualified; if there is turbidity, it is positive and unqualified.

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