CN110743039A - Preparation method of autologous skull used for replanting material - Google Patents
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
- A61L27/3645—Connective tissue
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
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- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
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Abstract
The invention relates to a preparation method of an autologous skull used for a replantation material, which comprises the following steps: extracting an autologous skull sample and carrying out pretreatment; repeatedly freezing and thawing the pretreated autologous skull sample, and removing cells through protease solution treatment; carrying out decalcification operation on the autologous skull sample subjected to cell removal in an acid solution; carrying out degreasing and inactivation on the autologous skull sample subjected to decalcification operation; freeze-drying and sterilizing the inactivated autologous skull sample; and adding growth factors into the sterilized autologous skull sample to obtain the replant material. The replanting material prepared by the method provided by the invention has good biocompatibility, and no inflammatory reaction and no self-absorption after operation.
Description
Technical Field
The invention relates to the field of biological materials, in particular to a preparation method of an autologous skull used for a replantation material.
Background
The skull-removing decompression is an effective treatment method for patients with increased intracranial pressure, and in order to achieve the aim of intracranial decompression, the removed bone flap cannot be transplanted back immediately, so that the patients need to perform skull defect repair after the condition of the disease is stable. At present, the skull defect repairing materials can be divided into metal materials, high polymer materials, allogeneic bones, xenogeneic bones and other materials, but no material can completely replace the autogenous skull in the aspects of rejection reaction, plasticity, pressure resistance, shock resistance, cold resistance, thermal insulation and the like.
The autogenous skull has the advantages of good tissue biocompatibility and mechanical properties, good bone induction and bone conduction, complete accordance of the skull flap with the anatomical and physiological requirements of human bodies, and the like, and is undoubtedly considered as the gold standard of clinical bone grafting materials. However, the preparation technology of the self skull flap which can meet the clinical application has larger obstacles, and the self skull preservation preparation method is divided into an in-vivo preservation method and an in-vitro preservation method. In vivo preservation is disadvantageous in that it requires the patient to bear the risk of additional surgical trauma and infection and the phenomenon of autologous bone resorption during in vivo preservation, resulting in the inability to repair the original defect. The method for preserving in vitro includes deep low temperature preservation, high temperature disinfection, chemical reagent disinfection and the like. The skull bone repairing liquid is easy to pollute in deep low temperature storage, the mechanical performance is obviously reduced after high temperature disinfection, the biocompatibility is poor only by chemical reagent disinfection, and the phenomenon of body bone absorption is generated in the skull bone replanting process, so that the original defect can not be repaired. At present, the problems of good biocompatibility, no inflammatory reaction after operation and self absorption which can meet the clinical application are solved.
Disclosure of Invention
In view of the above, the invention provides a preparation method of an autologous skull used for a replantation material, which has good biocompatibility and no inflammatory reaction and no self-absorption after operation.
In order to solve the technical problems, the technical scheme of the invention is a preparation method for a replantation material by adopting an autologous skull, which is characterized by comprising the following steps:
extracting an autologous skull sample and carrying out pretreatment;
repeatedly freezing and thawing the pretreated autologous skull sample, and removing cells through protease solution treatment;
carrying out decalcification operation on the autologous skull sample subjected to cell removal in an acid solution;
carrying out degreasing and inactivation on the autologous skull sample subjected to decalcification operation;
freeze-drying and sterilizing the inactivated autologous skull sample;
and adding growth factors into the sterilized autologous skull sample to obtain the replant material.
Preferably, the pretreatment specifically comprises:
removing soft tissue on the surface of the skull, and drilling holes on the skull by using a drill bit, wherein the hole diameter is about 0.5cm-0.7cm, and the hole spacing is 2-3 cm.
Preferably, the cell removal method specifically comprises:
repeatedly freezing and thawing an autologous skull sample for 2-5 times, and carrying out freeze thawing on the autologous skull sample by using a mixed solution of 0.05 wt% of trypsin and 0.02 wt% of EDTA at 37 ℃ and 5% of CO in volume concentration2Shaking is continued for 24h under the condition, and the solution is washed by ultrapure water.
Preferably, the repeated freezing and thawing is specifically:
freezing at-80 deg.C in sterile sealed box containing autogenous bone and ultrapure water 1:1, taking out after 10-18 hr, melting at 37 deg.C, and repeating the above steps for 3 times.
Preferably, the decalcification operation is specifically as follows:
and (3) continuously shaking the autologous skull sample subjected to the decellularization treatment in a HCL solution with the concentration of 0.4-0.6mol/L for 2-3 days until the calcium content is 10-40 wt%, and cleaning the autologous skull sample to be neutral by using ultrapure water.
Preferably, the degreasing is specifically as follows:
shaking with chloroform/methanol mixed solution at a ratio of 1:1 for 4-6 hr, washing with PBS twice, and washing with ultrapure water.
Preferably, the inactivation specifically comprises:
placing the degreased autologous bone into peroxyacetic acid/ethanol mixed solution, and continuously shaking the autologous bone matrix for 4-6 hours;
then 0.25 wt% Triton X-100+0.25 wt% sodium deoxycholate solution is used for treatment for 24h, and the solution is washed for a plurality of times by ultrapure water.
Preferably, the lyophilization is specifically:
placing the inactivated autologous bone material into a freeze dryer for sublimation and dehydration until the residual water amount in the sample is 2% -6%.
Preferably, the sterilization is specifically: packaging in a super clean bench, and sterilizing with gamma-ray at a dose of 20-25 kGy.
Preferably, the adding of the growth factors is specifically that the autologous bones are soaked in physiological saline for rehydration for 30 minutes, and then the growth factors are added into the autologous bone boreholes.
The invention has the advantages that the method of combining physics, chemistry and enzyme preparation is adopted to effectively remove the cell immunogenicity and keep the inorganic components and collagen of the bone matrix, and the autologous bone material has no obvious inflammation and immunologic rejection after being implanted. The chemical reagent and the method for removing part of the bone matrix are adopted, so that the decalcification is uniform, a good crawling growth microenvironment can be effectively provided for host cells, the bone conduction and the bone induction are good, the bone growth is promoted, and the bone healing is accelerated. Growth factors (BMPs) are added to promote the generation of new bones in the autologous bone graft material, solve the problem that the degradation speed of calcium in the transplanted bone is too high, and completely meet various performance requirements required by the bone filling material.
Drawings
Fig. 1 is a schematic diagram of an autologous skull sample provided by an embodiment of the invention.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present invention, the present invention will be further described in detail with reference to the following embodiments.
Enriching the description after the completion of the embodiment
In the invention, the immunogen removing process adopts a method of combined treatment of physical, chemical and enzyme preparations to basically or completely remove the immunogen in the autologous bone. The cell removing process comprises the steps of breaking and dissolving the cell membrane of the bone tissue by repeated freezing and thawing under the condition of keeping better biomechanical property, digesting the residual cell membrane and cell matrix by trypsin to separate the cell components from the bone matrix, further removing cytoplasm and nucleus by using an anion detergent Triton X-100 and sodium deoxycholate, and simultaneously accelerating washing and completely removing the cell matrix by combining with shaking.
Hair brushIn the method, a three-dimensional porous sponge-like structure in a bone matrix is preserved by using HCl solution decalcification, a good microenvironment is provided for the creeping growth of host blood vessels and cells, and meanwhile, holes are drilled in bone flaps to increase the areas of transplanted bones and decalcification solution, so that the problem of uneven decalcification caused by large bone flap areas is effectively solved, and proper Ca is ensured to be reserved in the bone matrix+As a calcified core of new bone, provides nuclei for the deposition of calcium phosphate. Bone formation-producing proteins (BMPs) are highly potent osteoinductive growth factors that allow mesenchymal cells on a substrate to differentiate into osteoblastic chondrocytes and osteocytes. The Decalcified Bone Matrix (DBM) is used as an osteoinductive scaffold and is combined with added bones to form mesenchymal cells in tissues to grow and differentiate into osteoblasts after being implanted with generating proteins (BMPs), and finally, new osteogenesis is formed to completely replace own tissues.
The method aims to solve the side effects and the defects of the skull repairing material at the present stage, adopts a method of combining physical reagents, chemical reagents and enzyme preparations to remove immunogenicity in skull tissues, simultaneously keeps inorganic components of bones of the skull and a collagen structure, and ensures that autologous bone materials are sterile to avoid postoperative infection; decalcification techniques using chemical agents to treat and remove portions of the bone matrix provide a suitable microenvironment for host cell growth and osteoconductive scaffolds for Bone Morphogenetic Proteins (BMPs). On the basis of the above, growth factors (BMPs) are added to promote the growth and proliferation of osteocytes and the bony healing, and the replacement of transplanted bone materials by new bones is accelerated.
The first embodiment of the invention is manufactured by the following steps in combination with the attached drawings and the detailed implementation mode:
(1) decellularization and skull drilling: as shown in fig. 1, a fresh skull flap is taken out in a clean environment, placed in a sterile sealed box and injected with ultrapure water, placed in a refrigerator and frozen for 10 hours in an environment of-80 ℃, and rapidly rewarmed for about 0.5 hour in a water bath at 37 ℃; the tissue cells are fully broken in the processes of temperature reduction and rewarming, and the cells are autolyzed. Removing soft tissues on the surface of the skull by using a surgical tool; the skull is drilled by a drill, the diameter of the holes is about 0.5cm, the distance between the holes is 2cm, and the bone edge is about 3cm away from the drilled hole and is not drilled. Freezing the autologous bone material in the environment of 80 ℃ and rapidly rewarming in a water bath of 37 ℃, repeatedly freezing and thawing for 2 times, continuously oscillating for 24 hours at 37 ℃ and 5% CO2 by using a mixed solution of 0.05% trypsin and 0.02% EDTA, cleaning for 2 times by using a PBS (pH 7.4) solution after oscillation is finished, and cleaning for 2 times by using ultrapure water;
(3) decalcification (DBM): placing the autologous bone material subjected to the cell removal treatment in 0.5mol/l HCL solution, continuously shaking for 3 days, carrying out surface decalcification until the calcium content is 13.4 wt%, and washing with ultrapure water for several times until the autologous bone material is neutral;
the detection of the calcium content after the surface decalcification specifically comprises the following steps:
TABLE 1 example 1 calcium content determination after decalcification
| No. | Test items | NCL/wt％ | Test results/wt% | Test method |
| 1 | Ca | 0.001 | 13.4 | (1) |
The test method comprises the following steps:
sample treatment and detection references USEPA30521996& USEPA6010D: 2014; and analyzed by ICP-OES.
Remarking:
n.d Not Detected (< MDL);
MDL ═ Method selection Limit (Method detection Limit);
the samples were heterogeneous.
(4) Degreasing: shaking with chloroform/methanol 1:1 mixed solution for 5 hr, washing with PBS (pH 7.4) for 2 times, and washing with ultrapure water for 4 times.
(5) Inactivation of microorganisms: placing autologous bone material into peroxyacetic acid/ethanol (10g/L peroxyacetic acid and 24% ethanol) mixed solution, continuously shaking autologous bone matrix for 4 hours, and cleaning with ultrapure water for 3 times; treated with 0.25% Triton X-100+ 0.25% sodium deoxycholate solution (10 mM Tris HCl, 5mM EDTA, pH 7.4) for 24h, and rinsed with ultra pure water until no foam is formed.
(6) And (3) freeze drying: placing the inactivated autologous bone material into a freeze dryer for sublimation and dehydration until the residual water amount in the sample is 2-6%.
(7) Irradiation sterilization: packaged in a clean room and sterilized by gamma-ray at a dose of 25 kGy.
(8) Addition of growth factor (BMP): before use, autologous bones are soaked in physiological saline for rehydration for 30 minutes, and then long factors (BMPs) are added into the autologous bone boreholes.
(9) The bacteria in the steps (1) to (5) are operated in a sterile operation table, and the oscillation is performed at a low-temperature oscillation temperature of below 10 ℃.
The second embodiment is manufactured by the following steps in combination with the attached drawings and the detailed implementation mode:
(1) decellularization and skull drilling: taking out a fresh skull bone flap in a clean environment, putting the fresh skull bone flap into a sterile sealing box, injecting ultrapure water, placing the sterile sealing box in a refrigerator, freezing for 18 hours in an environment of-80 ℃, and rapidly rewarming for about 0.5 hour in a water bath at 37 ℃; the tissue cells are fully broken in the processes of temperature reduction and rewarming, and the cells are autolyzed. Removing soft tissues on the surface of the skull by using a surgical tool; the skull is drilled by a drill, the diameter of the holes is about 0.7cm, the distance between the holes is 2.5cm, and the bone edge is about 2.5cm away from the drilled holes without drilling. Freezing the autologous bone material in the environment of 80 ℃ and rapidly rewarming in a water bath of 37 ℃, repeatedly freezing and thawing for 2 times, continuously oscillating for 24 hours at 37 ℃ and 5% CO2 by using a mixed solution of 0.05% trypsin and 0.02% EDTA, cleaning for 2 times by using a PBS (pH 7.4) solution after oscillation is finished, and cleaning for 2 times by using ultrapure water;
(2) decalcification (DBM): placing the autologous bone material subjected to the cell removal treatment in 0.5mol/l HCL solution, continuously shaking for 2 days, performing surface decalcification to ensure that the calcium content is 25.7 wt%, and washing with ultrapure water for several times to be neutral; calcium content was measured as in example 1.
(3) Degreasing: shaking with chloroform/methanol 1:1 mixed solution for 4 hr, washing with PBS (pH 7.4) for 2 times, and washing with ultrapure water for 4 times.
(4) Inactivation of microorganisms: placing autologous bone material into peroxyacetic acid/ethanol (10g/L peroxyacetic acid and 24% ethanol) mixed solution, continuously shaking autologous bone matrix for 4 hours, and cleaning with ultrapure water for 3 times; shaking with 0.25% Triton X-100+ 0.25% sodium deoxycholate solution (solvent 10mM Tris HCl, 5mM EDTA, pH 7.4) at 4 deg.C for 24 hr, and washing with ultrapure water several times.
(5) And (3) freeze drying: placing the inactivated autologous bone material into a freeze dryer for sublimation and dehydration until the residual water amount in the sample is 2% -6%.
(6) Irradiation sterilization: packaged in a clean bench and sterilized by gamma-ray at a dose of 20 kGy.
(7) Addition of growth factor (BMP): before use, autologous bones are soaked in physiological saline for rehydration for 30 minutes, and then long factors (BMPs) are added into the autologous bone boreholes.
(8) The bacteria in the steps (1) to (5) are operated in a sterile operation table, and the oscillation is performed at a low-temperature oscillation temperature of below 10 ℃.
The third embodiment of the invention is manufactured by the following steps in combination with the attached drawings and the detailed implementation mode:
(1) decellularization and skull drilling: taking out a fresh skull bone flap in a clean environment, putting the fresh skull bone flap into a sterile sealing box, injecting ultrapure water, placing the sterile sealing box in a refrigerator, freezing for 14 hours in an environment of-80 ℃, and rapidly rewarming for about 0.5 hour in a water bath at 37 ℃; the tissue cells are fully broken in the processes of temperature reduction and rewarming, and the cells are autolyzed. Removing soft tissues on the surface of the skull by using a surgical tool; the skull is drilled by a drill, the diameter of the holes is about 0.5cm, the distance between the holes is 2cm, and the bone edge is about 3cm away from the drilled hole and is not drilled. Freezing the autologous bone material in the environment of 80 ℃ and rapidly rewarming in a water bath of 37 ℃, repeatedly freezing and thawing for 2 times, continuously oscillating for 24 hours at 37 ℃ and 5% CO2 by using a mixed solution of 0.05% trypsin and 0.02% EDTA, cleaning for 2 times by using a PBS (pH 7.4) solution after oscillation is finished, and cleaning for 2 times by using ultrapure water;
(3) decalcification (DBM): placing the autologous bone material subjected to the cell removal treatment in 0.5mol/l HCL solution, continuously shaking for 1 day, performing surface decalcification until the calcium content is 36.9 wt%, and washing with ultrapure water for several times until the autologous bone material is neutral; calcium content was measured as in example 1.
(4) Degreasing: shaking with chloroform/methanol 1:1 mixed solution for 5 hr, washing with PBS (pH 7.4) for 2 times, and washing with ultrapure water for 4 times.
(5) Inactivation of microorganisms: placing autologous bone material into peroxyacetic acid/ethanol (10g/L peroxyacetic acid and 24% ethanol) mixed solution, continuously shaking autologous bone matrix for 4 hours, and cleaning with ultrapure water for 3 times; treated with 0.25% Triton X-100+ 0.25% sodium deoxycholate solution (10 mM Tris HCl, 5mM EDTA, pH 7.4) for 24h, and rinsed with ultra pure water until no foam is formed.
(6) And (3) freeze drying: placing the inactivated autologous bone material into a freeze dryer for sublimation and dehydration until the residual water amount in the sample is 2-6%.
(7) Irradiation sterilization: packaged in a clean room and sterilized by gamma-ray at a dose of 25 kGy.
(8) Addition of growth factor (BMP): before use, autologous bones are soaked in physiological saline for rehydration for 30 minutes, and then long factors (BMPs) are added into the autologous bone boreholes.
(9) The bacteria in the steps (1) to (5) are operated in a sterile operation table, and the oscillation is performed at a low-temperature oscillation temperature of below 10 ℃.
The fourth embodiment of the invention is manufactured by the following steps in combination with the attached drawings and the detailed implementation mode:
(1) decellularization and skull drilling: taking out a fresh skull bone flap in a clean environment, putting the fresh skull bone flap into a sterile sealing box, injecting ultrapure water, placing the sterile sealing box in a refrigerator, freezing for 10-18 hours in an environment of-80 ℃, and rapidly rewarming for about 0.5 hour in a water bath at 37 ℃; the tissue cells are fully broken in the processes of temperature reduction and rewarming, and the cells are autolyzed. Removing soft tissues on the surface of the skull by using a surgical tool; the skull is drilled by a drill, the diameter of the holes is about 0.5cm, the distance between the holes is 2cm, and the bone edge is about 3cm away from the drilled hole and is not drilled. Freezing the autologous bone material in the environment of 80 ℃ and rapidly rewarming in a water bath of 37 ℃, repeatedly freezing and thawing for 2 times, continuously oscillating for 24 hours at 37 ℃ and 5% CO2 by using a mixed solution of 0.05% trypsin and 0.02% EDTA, cleaning for 2 times by using a PBS (pH 7.4) solution after oscillation is finished, and cleaning for 2 times by using ultrapure water;
(3) decalcification (DBM): placing the autologous bone material subjected to the cell removal treatment in 0.5mol/l formic acid solution, continuously shaking for 3 days, carrying out surface decalcification until the calcium content is 6.3 wt%, and washing with ultrapure water for several times until the autologous bone material is neutral;
(4) degreasing: shaking with chloroform/methanol 1:1 mixed solution for 5 hr, washing with PBS (pH 7.4) for 2 times, and washing with ultrapure water for 4 times.
(5) Inactivation of microorganisms: placing autologous bone material into peroxyacetic acid/ethanol (10g/L peroxyacetic acid and 24% ethanol) mixed solution, continuously shaking autologous bone matrix for 4 hours, and cleaning with ultrapure water for 3 times; treated with 0.25% Triton X-100+ 0.25% sodium deoxycholate solution (10 mM Tris HCl, 5mM EDTA, pH 7.4) for 24h, and rinsed with ultra pure water until no foam is formed.
(6) And (3) freeze drying: placing the inactivated autologous bone material into a freeze dryer for sublimation and dehydration until the residual water amount in the sample is 2% -6%.
(7) Irradiation sterilization: packaged in a clean room and sterilized by gamma-ray at a dose of 25 kGy.
(8) Addition of growth factor (BMP): before use, autologous bones are soaked in physiological saline for rehydration for 30 minutes, and then long factors (BMPs) are added into the autologous bone boreholes.
(9) The bacteria in the steps (1) to (5) are operated in a sterile operation table, and the oscillation is performed at a low-temperature oscillation temperature of below 10 ℃.
The fifth embodiment of the invention is manufactured by the following steps in combination with the attached drawings and the detailed implementation mode:
(1) decellularization and skull drilling: taking out a fresh skull bone flap in a clean environment, putting the fresh skull bone flap into a sterile sealing box, injecting ultrapure water, placing the sterile sealing box in a refrigerator, freezing for 10-18 hours in an environment of-80 ℃, and rapidly rewarming for about 0.5 hour in a water bath at 37 ℃; the tissue cells are fully broken in the processes of temperature reduction and rewarming, and the cells are autolyzed. Removing soft tissues on the surface of the skull by using a surgical tool; the skull is drilled by a drill, the diameter of the holes is about 0.5cm, the distance between the holes is 2cm, and the bone edge is about 3cm away from the drilled hole and is not drilled. Freezing the autologous bone material in the environment of 80 ℃ and rapidly rewarming in a water bath of 37 ℃, repeatedly freezing and thawing for 2 times, continuously oscillating for 24 hours at 37 ℃ and 5% CO2 by using a mixed solution of 0.05% trypsin and 0.02% EDTA, cleaning for 2 times by using a PBS (pH 7.4) solution after oscillation is finished, and cleaning for 2 times by using ultrapure water;
(3) decalcification (DBM): placing the autologous bone material subjected to the cell removal treatment in 0.5mol/l formic acid solution, continuously shaking for 7 days, carrying out surface decalcification until the calcium content is 0.039 wt%, and washing with ultrapure water for several times until the autologous bone material is neutral;
(4) degreasing: shaking with chloroform/methanol 1:1 mixed solution for 5 hr, washing with PBS (pH 7.4) for 2 times, and washing with ultrapure water for 4 times.
(5) Inactivation of microorganisms: placing autologous bone material into peroxyacetic acid/ethanol (10g/L peroxyacetic acid and 24% ethanol) mixed solution, continuously shaking autologous bone matrix for 4 hours, and cleaning with ultrapure water for 3 times; treated with 0.25% Triton X-100+ 0.25% sodium deoxycholate solution (10 mM Tris HCl, 5mM EDTA, pH 7.4) for 24h, and rinsed with ultra pure water until no foam is formed.
(6) And (3) freeze drying: placing the inactivated autologous bone material into a freeze dryer for sublimation and dehydration until the residual water amount in the sample is 2% -6%.
(7) Irradiation sterilization: packaged in a clean room and sterilized by gamma-ray at a dose of 25 kGy.
(8) Addition of growth factor (BMP): before use, autologous bones are soaked in physiological saline for rehydration for 30 minutes, and then long factors (BMPs) are added into the autologous bone boreholes.
(9) The bacteria in the steps (1) to (5) are operated in a sterile operation table, and the oscillation is performed at a low-temperature oscillation temperature of below 10 ℃.
Through the test results of the first to fifth examples, when the calcium residual quantity is 12% of the decalcified bone matrix, the newly born femur is induced to the maximum, and when the calcium residual quantity is less than 12%, the osteogenesis quantity is induced to be reduced, but when the decalcification is serious, the size and the mechanical properties of the bone matrix are affected, so that the autogenous bone ingrowth and the freeze-dried Surface Decalcified Bone (SDBM) can be induced, and the calcium content is generally 9-15% and has partial bone induction and bone conduction functions.
ICP detection results according to examples 1-3 show that the 0.6M HCl solution is removed for 3 days to substantially meet the requirements, and the residual amount of calcium element (calcium content) is 13.4%. The results shown in examples 4-5 reached 6.3 wt% in three days, which was less than 12%, so the treatment with formic acid mixed solution resulted in unstable data and severe yellowing of the bone matrix after decalcification. The invention is proved to have better effect by using HCl.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (10)
1. A preparation method of an autologous skull for a replantation material is characterized by comprising the following steps:
extracting an autologous skull sample and carrying out pretreatment;
repeatedly freezing and thawing the pretreated autologous skull sample, and removing cells through protease solution treatment;
carrying out decalcification operation on the autologous skull sample subjected to cell removal in an acid solution;
carrying out degreasing and inactivation on the autologous skull sample subjected to decalcification operation;
freeze-drying and sterilizing the inactivated autologous skull sample;
and adding growth factors into the sterilized autologous skull sample to obtain the replant material.
2. The method according to claim 1, characterized in that the pre-treatment is in particular:
removing soft tissue on the surface of the skull, and drilling holes on the skull by using a drill bit, wherein the hole diameter is about 0.5cm-0.7cm, and the hole spacing is 2-3 cm.
3. The method according to claim 1, wherein the decellularization is in particular:
repeatedly freezing and thawing an autologous skull sample for 2-5 times, and carrying out freeze thawing on the autologous skull sample by using a mixed solution of 0.05 wt% of trypsin and 0.02 wt% of EDTA at 37 ℃ and 5% of CO in volume concentration2Shaking is continued for 24h under the condition, and the solution is washed by ultrapure water.
4. The method according to claim 3, wherein said repeated freezing and thawing is in particular:
freezing at-80 deg.C in sterile sealed box containing autogenous bone and ultrapure water 1:1, taking out after 10-18 hr, melting at 37 deg.C, and repeating the above steps for 3 times.
5. The method according to claim 1, characterized in that the decalcification operation is in particular:
and (3) continuously shaking the autologous skull sample subjected to the decellularization treatment in a HCL solution with the concentration of 0.4-0.6mol/L for 2-3 days until the calcium content is 10-40 wt%, and cleaning the autologous skull sample to be neutral by using ultrapure water.
6. The method according to claim 1, wherein the degreasing is in particular:
shaking with chloroform/methanol mixed solution at a ratio of 1:1 for 4-6 hr, washing with PBS twice, and washing with ultrapure water.
7. The method according to claim 1, characterized in that the inactivation is in particular:
placing the degreased autologous bone into peroxyacetic acid/ethanol mixed solution, and continuously shaking the autologous bone matrix for 4-6 hours;
then 0.25 wt% Triton X-100+0.25 wt% sodium deoxycholate solution is used for treatment for 24h, and the solution is washed for a plurality of times by ultrapure water.
8. The method according to claim 1, characterized in that the lyophilization is in particular:
placing the inactivated autologous bone material into a freeze dryer for sublimation and dehydration until the residual water amount in the sample is 2% -6%.
9. The method according to claim 1, characterized in that the sterilization is in particular: packaging in a super clean bench, and sterilizing with gamma-ray at a dose of 20-25 kGy.
10. The method according to claim 1 or 2, wherein the adding of the growth factors is specifically that the autologous bones are soaked in physiological saline for 30 minutes for rehydration, and then the growth factors are added into the autologous bone boreholes.
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