CN112042636A - Preservation method of autologous skull flap containing rhBMP-2 and provided with periosteum - Google Patents
Preservation method of autologous skull flap containing rhBMP-2 and provided with periosteum Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0294—Electromagnetic, i.e. using electromagnetic radiation or electromagnetic fields
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Abstract
The invention provides a method for preserving autologous skull flap containing rhBMP-2 and provided with a periosteum, which is a method for preserving the autologous skull flap at low temperature in vitro, has simple process and convenient operation, and can keep the activity and the sterility of the skull flap to the maximum extent, and meanwhile, the invention adds the recombinant human bone morphogenetic protein-2 (rhBMP-2) to improve the probability of successful healing and growth of the skull flap and reduce the occurrence of the absorption condition of the skull flap.
Description
Technical Field
The invention relates to the field of medicine and bioengineering, in particular to a method for preserving autologous skull flap containing rhBMP-2 and provided with a periosteum.
Background
The bone flap removing decompression is a commonly used treatment technology in neurosurgery, intracranial hypertension is caused by craniocerebral tumor, cerebral hemorrhage and craniocerebral trauma, and the bone flap removing decompression is needed when the medicine can not be controlled. The large scale skull defect patients have partial scalp subsidence caused by atmospheric pressure, which may cause intracranial pressure imbalance, brain tissue displacement, cerebral hemisphere blood flow reduction and cerebrospinal fluid circulation disorder, thereby causing a series of clinical manifestations, mainly including: headache, dizziness, irritability, epilepsy, no other interpretable discomfort and various mental disorders. Cranioplasty can not only repair skull defects, restore the appearance and protection function of the skull of a patient, but also effectively restore normal cerebrospinal fluid dynamics and cerebral cortex blood perfusion, is beneficial to reducing complications related to skull defects and is beneficial to the neurological function recovery of the patient.
The materials currently used for cranioplasty include autologous skull flap and artificial materials. Autologous skull preservation and transplantation is still used by many neurosurgeons because of the advantages of relative economy, small tissue reactivity, no need of shaping, physiological anatomical requirements, no rejection reaction and the like.
However, the safety and effectiveness of preservation of autologous skull remains controversial. The autogenous skull preservation can preserve the skull under the physiological state (such as the abdominal subcutaneous fat layer of a patient), but the pain of the patient can be increased, and the skull can be absorbed and thinned and the bone performance is reduced in the preservation process, so that the postoperative complications such as bone plate loosening and collapse can occur. The skull flap preserved in vitro at the deep low temperature can keep the activity of osteocytes, the bone guides in the frozen bone flap matrix are not inactivated, and the repaired bone flap can survive and be fused with the surrounding bone. However, the method requires special equipment for ultralow temperature preservation, which is not easy to be realized in ordinary hospitals. Meanwhile, the long-time deep low-temperature storage also has the risks of the performance reduction of the skull flap bone, the increase of the operation infection rate and the like.
Although the risk of infection, absorption and the like exists in the process of storing and replanting the autogenous skull, in 2016 (Chinese expert consensus for traumatic skull defect forming), the expert recommends that the storage and replanting of the autogenous skull is still advocated as the preferred scheme of the skull forming.
However, there are different ways how an autologous skull flap can be preserved, due to the different preservation mechanisms and neurosurgeons undertaking the surgery. Therefore, the quality of the preserved bone flap is different, and the incidence of infection and absorption after replantation is higher, thereby causing the secondary operation of the patient, increasing the pain of the patient and unnecessary dispute.
Disclosure of Invention
In order to solve the problems, the invention provides a method for preserving the autologous skull flap containing the rhBMP-2 and provided with the periosteum, which is a method for preserving the autologous skull flap in vitro at low temperature, has simple process and convenient operation, and can keep the activity and the sterility of the skull flap to the maximum extent, and meanwhile, the invention adds the recombinant human bone morphogenetic protein-2 (rhBMP-2) to improve the probability of successful healing and growth of the skull flap and reduce the occurrence of the absorption condition of the skull flap.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a preservation method of autologous skull flap containing rhBMP-2 and provided with periosteum comprises the following steps:
1) placing the skull flap taken out of the operation into a preservation solution C, and carrying out cold chain transportation to a hundred thousand grade aseptic operating room or an ultra-clean aseptic operating platform, wherein the skull flap with the periosteum comprises skull fragments bitten down during the operation, and the preservation solution C is a glycerin solution or plasma substitute colloidal solution containing antibiotics.
2) Under the aseptic operation environment, cleaning blood stain with isotonic saline, removing soft tissue except periosteum, soaking in 2% iodophor solution for 30 min after cleaning, cleaning with 75% alcohol for deiodination, washing with isotonic saline, and wiping with sterile gauze.
3) Under the aseptic operation environment, the hole is ground on the inner skull plate, and the hole is ground until reaching the outer skull plate without breaking the outer skull plate.
4) The ground and drilled skull fragments are mixed with recombinant human bone morphogenetic protein-2 (rhBMP-2) and carrier material, backfilled to the bone hole and waiting for solidification.
5) And after the curing is finished, coating and plugging the outer opening of each drilling hole by using bone wax, and irradiating the operated bone flap by using ultraviolet rays in a sterile operation environment.
6) And (3) placing the irradiated bone flap into an aseptic storage box A, simultaneously placing a small skull into another aseptic storage box B, and pouring a preservation solution D into the aseptic storage box A for immersion and sealing, wherein the preservation solution D is a culture medium solution added with antibiotics and cell antifreeze solution.
7) Cooling the skull flap in the storage box A to 0 ℃, transferring the skull flap into a cold box at the temperature of-20 ℃ for freezing for 6-8 hours, transferring the skull flap into a refrigerator at the temperature of-40 ℃ for freezing for 24-48 hours, then immersing the skull flap into liquid nitrogen for storage, carrying out bacterial culture examination on the small skull block and the storage solution, carrying out bone cell morphology detection on the small skull block, and if the detection result shows that the skull flap in the storage box A is sterile and the bone cell morphology meets the use requirement, indicating that the skull flap in the storage box A is also sterile and the bone cell morphology meets the use requirement, and being used for implantation.
Preferably, in step 1), the surgically removed skull flap with the periosteum is placed in a preservation box containing the preservation solution C for cold chain transportation.
Preferably, the antibiotic contained in the preservation solution C may be an antibiotic such as gentamicin, levofloxacin, vancomycin, penicillin, streptomycin, and the like. More preferably, the antibiotic is 500U-1000U/ml gentamicin.
Preferably, the glycerol solution contains 10 to 75 weight percent of glycerol, and can be glycerol fructose solution or glycerol fructose sodium chloride solution; the plasma substitute colloidal solution may be dextran or hydroxyethyl starch.
Preferably, in the step 3), the diameter of the grinding holes is 4 mm-6 mm, and the interval is 4 cm-6 cm.
Preferably, in step 4), each mg of skull fragment is mixed with 0.3mg to 0.5mg of recombinant human bone morphogenetic protein-2 (rhBMP-2), 1mg of carrier material.
More preferably, the support material is prepared from a mixture of 1: 1: 1, hydroxyapatite, lecithin and medicinal gelatin.
Preferably, in the step 5), the irradiation dose is 100000-500000 uW.s/CM2。
Preferably, the antibiotic added in the preservation solution D may be an antibiotic such as gentamicin, levofloxacin, vancomycin, penicillin, streptomycin, and the like. More preferably, the antibiotic is 500U-1000U/ml gentamicin.
Preferably, the preservation solution D includes M199 medium solution containing 10% to 20% (by weight) of dimethyl sulfoxide, DMEM medium solution containing 10% to 20% (by weight) of glycerol, and the like.
Preferably, the preservation box used in the method is made of low-temperature resistant medical polyethylene materials which are sterilized and disinfected, is provided with edge buckles, and achieves the sealing effect through double-layer sterile vacuum packaging.
The invention has the following beneficial effects:
and (I) the skull flap is cleaned by isotonic saline, so that the cell structure and the bioactivity protection of the periosteum tissue on the skull flap are facilitated, and the regeneration, the remodeling and the healing of the bone tissue after the skull flap is implanted are facilitated.
And (II) recombinant human bone morphogenetic protein-2 (rhBMP-2) is added, so that the success probability of the healing and growth of the skull flap is improved, and the absorption of the skull flap is reduced.
And (III) the disinfection and sterilization of the skull flap mainly adopts a medicinal biological method, avoids cell damage caused by high temperature or cobalt 60 radiation sterilization, furthest keeps the survival bone cells and prevents the denaturation of proteins, bone trabeculae, growth factors and the like.
And (IV) the process is simple, the operation is convenient, and the activity of the skull flap and the aseptic self-body skull flap can be kept to the maximum extent.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
In the following embodiments, the storage cases used are made of sterilized low-temperature resistant medical polyethylene material with edge buckles. The dimensions were 12X 16X 5 cm. The sealing is carried out by double-layer sterile vacuum packaging so as to meet the requirement that the medical material needs three-layer packaging.
The antibiotics contained in the preservation solutions C and D may be vancomycin, penicillin, streptomycin, and other antibiotics except gentamicin and levofloxacin listed below, and may be added to an effective use concentration, for example, 500U to 1000U/ml gentamicin and 0.8mg to 2.0mg/ml levofloxacin.
The recombinant human bone morphogenetic protein-2 (rhBMP-2) and the carrier material used in the following examples may be commercially available products, or may be a composite product having similar functions, for example, Guoyou (Tokyo 2014 No. 3460233). The carrier material may also be self-formulated, for example, from a weight ratio of 1: 1: 1, hydroxyapatite, lecithin and medicinal gelatin.
The materials and methods used in the examples are conventional, unless otherwise specified.
Example 1
1. The skull flap with the periosteum (including the bitten skull fragment) taken down in the operation is put into a preservation box with preservation solution C, and is transported to a hundred thousand grade ultra-clean sterile operating platform through a cold chain.
The preservation solution C is a glycerol fructose solution containing 10% of glycerol and contains 500U/ml of gentamicin.
2. The blood stain was washed with isotonic saline on an ultra-clean sterile operating table and the soft tissue except periosteum was removed with a scalpel blade. Cleaning, soaking in 2% iodophor solution for 30 min, and cleaning with 75% alcohol to remove iodine. Finally, the mixture is washed clean by isotonic saline and wiped dry by sterile gauze.
3. On an ultra-clean sterile operating table, a grinding drill is used for grinding holes with the diameter of 5mm on the inner plate of the skull, and the hole is stopped when the hole reaches the outer plate of the skull without breaking the outer plate. At an interval of about 5cm, ground skull fragments (about 10mg) were mixed with 4mg of recombinant human bone morphogenetic protein-2 (rhBMP-2) and 10mg of carrier material (consisting of hydroxyapatite, lecithin and pharmaceutical gelatin in a weight ratio of 1: 1: 1), backfilled to bone holes and allowed to solidify.
4. And after the curing is finished, coating and plugging the outer openings of the drill holes by using bone wax.
5. Irradiating the operated skull flap with ultraviolet rays at dose of 100000uW.s/CM on ultra-clean sterile operating table2。
6. After irradiation, the skull flap is placed in a sterile storage box A, a small skull (about 0.5 cubic centimeter) is taken out at the same time, placed in another sterile storage box B, and simultaneously, the preservation solution D is poured in for immersion.
The preservation solution D is an M199 culture medium solution containing 10% of dimethyl sulfoxide, and 500U/ml of gentamicin is added.
7. And (5) fastening the storage box, and carrying out double-layer sterile vacuum packaging on the storage box.
8. And adhering the identity identification to the stored skull flap with the periosteum. Cooling the skull flap stored in the storage box A to 0 ℃, transferring the skull flap to a refrigerator with the temperature of minus 20 ℃ for freezing for 6 hours, transferring the skull flap to a refrigerator with the temperature of minus 40 ℃ for freezing for 24 hours, and then immersing the skull flap into liquid nitrogen for storage. And (4) carrying out bacterial culture examination on the small skull block and the preservation solution in the preservation box B, and carrying out bone cell morphology detection on the small skull block.
9. If the skull block in the preservation box B is detected to be sterile and the shape of the skull block meets the use requirement. Because the skull flap in the storage box A is processed under the same condition and in the same environment, the skull flap can be regarded as sterile and consistent in shape. When a patient carries out cranioplasty, the preserved skull flap is taken out from the liquid nitrogen bottle and is implanted after natural rewarming at room temperature.
Example 2
1. The skull flap with the periosteum (including the bitten skull fragment) taken down in the operation is put into a preservation box with preservation solution C, and is transported to a hundred thousand grade ultra-clean sterile operating platform through a cold chain.
The preservation solution C is a glycerol fructose sodium chloride solution containing 30% of glycerol and contains 0.8mg/ml of levofloxacin.
2. The blood stain was washed with isotonic saline on an ultra-clean sterile operating table and the soft tissue except periosteum was removed with a scalpel blade. Cleaning, soaking in 2% iodophor solution for 30 min, and cleaning with 75% alcohol to remove iodine. Finally, the mixture is washed clean by isotonic saline and wiped dry by sterile gauze.
3. On an ultra-clean sterile operating table, a grinding drill is used for grinding a hole with the diameter of 4mm on the inner plate of the skull, and the hole is stopped when the hole reaches the outer plate of the skull without breaking the outer plate. At about 6cm intervals, ground burr bone fragments (about 10mg) were mixed with bone powder (guoyuan (national food and drug administration) (2014 No. 3460233)) 10mg, backfilled to the bone hole and allowed to cure.
4. And after the curing is finished, coating and plugging the outer openings of the drill holes by using bone wax.
5. Irradiating the operated skull flap with ultraviolet rays at a dose of 250000uW.s/CM on an ultra-clean sterile operating table2。
6. After irradiation, the skull flap is placed in a sterile storage box A, a small skull (about 0.5 cubic centimeter) is taken out at the same time, placed in another sterile storage box B, and simultaneously, the preservation solution D is poured in for immersion.
The preservation solution D is a DMEM medium solution containing 15% of glycerol, and 0.8mg/ml levofloxacin is added.
7. And (5) fastening the storage box, and carrying out double-layer sterile vacuum packaging on the storage box.
8. And adhering the identity identification to the stored skull flap with the periosteum. Cooling the skull flap stored in the storage box A to 0 ℃, transferring the skull flap to a refrigerator with the temperature of minus 20 ℃ for freezing for 7 hours, transferring the skull flap to a refrigerator with the temperature of minus 40 ℃ for freezing for 36 hours, and then immersing the skull flap in liquid nitrogen for storage. And (4) carrying out bacterial culture examination on the small skull block and the preservation solution in the preservation box B, and carrying out bone cell morphology detection on the small skull block.
9. If the skull block in the preservation box B is detected to be sterile and the shape of the skull block meets the use requirement. Because the skull flap in the storage box A is processed under the same condition and in the same environment, the skull flap can be regarded as sterile and consistent in shape. When a patient carries out cranioplasty, the preserved skull flap is taken out from the liquid nitrogen bottle and is implanted after natural rewarming at room temperature.
Example 3
1. The skull flap with the periosteum (including the bitten skull fragment) taken down in the operation is put into a preservation box with preservation solution C, and is transported to a hundred thousand grade ultra-clean sterile operating platform through a cold chain.
The preservation solution C is hydroxyethyl starch solution containing 75% of glycerol and contains 1000U/ml gentamicin.
2. The blood stain was washed with isotonic saline on an ultra-clean sterile operating table and the soft tissue except periosteum was removed with a scalpel blade. Cleaning, soaking in 2% iodophor solution for 30 min, and cleaning with 75% alcohol to remove iodine. Finally, the mixture is washed clean by isotonic saline and wiped dry by sterile gauze.
3. On an ultra-clean sterile operating table, a grinding drill is used for grinding holes with the diameter of 6mm on the inner plate of the skull, and the hole is stopped when the hole reaches the outer plate of the skull without breaking the outer plate. At an interval of about 4cm, ground skull fragments (about 10mg) were mixed with 5mg of recombinant human bone morphogenetic protein-2 (rhBMP-2) and 10mg of carrier material (consisting of hydroxyapatite, lecithin and pharmaceutical gelatin in a weight ratio of 1: 1: 1), backfilled to bone holes and allowed to solidify.
4. And after the curing is finished, coating and plugging the outer openings of the drill holes by using bone wax.
5. Irradiating the operated skull flap with ultraviolet rays at 500000uW.s/CM on an ultra-clean sterile operating platform2。
6. After irradiation, the skull flap is placed in a sterile storage box A, a small skull (about 0.5 cubic centimeter) is taken out at the same time, placed in another sterile storage box B, and simultaneously, the preservation solution D is poured in for immersion.
The preservation solution D is a DMEM medium solution containing 20% glycerol and added with 1000U/ml gentamicin.
7. And (5) fastening the storage box, and performing double-layer sterile vacuum two-layer packaging on the storage box.
8. And adhering the identity identification to the stored skull flap with the periosteum. Cooling the skull flap preserved in the preservation box A to 0 ℃, transferring to a refrigerator with the temperature of minus 20 ℃ for freezing for 8 hours, transferring to a refrigerator with the temperature of minus 40 ℃ for freezing for 48 hours, and then immersing into liquid nitrogen for preservation. And (4) carrying out bacterial culture examination on the small skull block and the preservation solution in the preservation box B, and carrying out bone cell morphology detection on the small skull block.
9. If the skull block in the preservation box B is detected to be sterile and the shape of the skull block meets the use requirement. Because the skull flap in the storage box A is processed under the same condition and in the same environment, the skull flap can be regarded as sterile and consistent in shape. When a patient carries out cranioplasty, the preserved skull flap is taken out from the liquid nitrogen bottle and is implanted after natural rewarming at room temperature.
As a result, the autologous skull flap containing the rhBMP-2 and the periosteum obtained in the above embodiment can improve the success probability of the healing growth of the skull flap and reduce the occurrence of skull flap absorption.
Claims (10)
1. A preservation method of autologous skull flap containing rhBMP-2 and provided with periosteum comprises the following steps:
1) placing the skull flap taken out of the operation into a preservation solution C, and carrying out cold chain transportation to a hundred thousand grade aseptic operating room or an ultra-clean aseptic operating platform, wherein the skull flap with the periosteum comprises skull fragments bitten down during the operation, and the preservation solution C is a glycerin solution or plasma substitute colloidal solution containing antibiotics;
2) under the aseptic operation environment, cleaning blood stain with isotonic saline, removing soft tissues except periosteum, soaking in 2% iodophor solution for 30 min after cleaning, cleaning with 75% alcohol for deiodination, washing with isotonic saline, and wiping with sterile gauze;
3) under the aseptic operation environment, grinding holes on the inner skull plate, stopping when the outer skull plate is reached, and not breaking the outer skull plate;
4) mixing the ground and drilled skull fragments with recombinant human bone morphogenetic protein-2 and a carrier material, backfilling to bone holes and waiting for solidification;
5) after the curing is finished, coating and plugging the outer openings of the drill holes with bone wax, and irradiating the operated bone flap with ultraviolet rays in a sterile operation environment;
6) placing the irradiated bone flap into an aseptic storage box A, simultaneously placing a small skull into another aseptic storage box B, pouring a preservation solution D into the aseptic storage box A for immersion and sealing, wherein the preservation solution D is a culture medium solution added with antibiotics and cell antifreeze solution;
7) cooling the bone flap in the storage box A to 0 ℃, transferring the bone flap into a cold box at the temperature of-20 ℃ for freezing for 6-8 hours, transferring the bone flap into a refrigerator at the temperature of-40 ℃ for freezing for 24-48 hours, then immersing the bone flap into liquid nitrogen for storage, performing bacterial culture examination on the small skull block and the storage solution in the storage box B, performing bone cell morphology detection on the small skull block, and if the bone cell morphology is detected to be sterile and meets the use requirement, indicating that the bone flap in the storage box A is also sterile and the bone cell morphology meets the use requirement, and can be used for implantation.
2. The method for preserving the autologous skull flap with the periosteum according to claim 1, wherein the skull flap with the periosteum removed in the operation in the step 1) is placed in a preservation box containing preservation solution C for cold chain transportation.
3. The method for preserving the autologous skull flap with periosteum of rhBMP-2 according to claim 1, wherein the antibiotics contained in the preservation solution C comprise gentamicin, levofloxacin, vancomycin, penicillin and streptomycin.
4. The method for preserving the autologous skull flap with the periosteum of the rhBMP-2 according to any one of claims 1-3, wherein the glycerol solution contains 10-75% by weight of glycerol, including glycerol fructose and glycerol fructose sodium chloride; the plasma substitute colloidal solution comprises dextran and hydroxyethyl starch.
5. The method for preserving the autologous skull flap with the periosteum according to claim 1, wherein the diameter of the milled hole is 4mm to 6mm and the interval is 4cm to 6cm in step 3).
6. The method for preserving the autologous skull flap with periosteum containing rhBMP-2 of claim 1, wherein in the step 4), 0.3mg to 0.5mg of the recombinant human bone morphogenetic protein-2, 1mg of the carrier material is mixed with each mg of the skull fragment.
7. The method for preserving the autologous skull flap with the periosteum according to claim 1 or 6, wherein the carrier material is prepared from the following components in a weight ratio of 1: 1: 1, hydroxyapatite, lecithin and medicinal gelatin.
8. The method for preserving the autogenous skull flap with periosteum according to claim 1, wherein the irradiation dose in step 5) is 100000-500000 uW.s/CM2。
9. The method for preserving the autologous skull flap with periosteum of rhBMP-2 as claimed in claim 1, wherein the antibiotics added in the preserving fluid D comprise gentamicin, levofloxacin, vancomycin, penicillin and streptomycin.
10. The method for preserving the rhBMP-2-containing autologous skull flap with the periosteum according to claim 1 or 9, wherein the preservation solution D comprises an M199 culture medium solution containing 10-20% of dimethyl sulfoxide and a DMEM culture medium solution containing 10-20% of glycerol.
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