CN106730000A - A kind of chemical treatment method of homogeneous allogenic bone - Google Patents
A kind of chemical treatment method of homogeneous allogenic bone Download PDFInfo
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- CN106730000A CN106730000A CN201611103787.XA CN201611103787A CN106730000A CN 106730000 A CN106730000 A CN 106730000A CN 201611103787 A CN201611103787 A CN 201611103787A CN 106730000 A CN106730000 A CN 106730000A
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- A—HUMAN NECESSITIES
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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
- A61L27/3604—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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- A—HUMAN NECESSITIES
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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
- A61L27/365—Bones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61L27/3683—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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61L27/3683—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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61L27/3683—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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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- 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 provides a kind of chemical treatment method of homogeneous allogenic bone, including allograph bone processing cutting and freeze-drying.The method is will to be cleaned up with purified water after the processing cutting of allosome bone material, remove marrow, again successively with after NaOH, methyl alcohol, chloroform, dodecyl sodium sulfate, phosphate, ethanol postincubation, deep-frozen 16 25 hours in environment at 70 DEG C, then put it into freeze drier, freeze-drying is carried out according to the freeze-drying curve for setting.Homogeneous allogenic bone finished product is obtained after irradiation sterilization.The present invention uses NaOH, methyl alcohol, chloroform, dodecyl sodium sulfate, phosphate, alcohol Combined Treatment allograph bone raw material, marrow and wherein cell, albumen and fat constituent can effectively be removed, the immunogenicity of homogeneous allogenic bone can further be reduced by freeze-drying process simultaneously, while the three-dimensional structure and biomechanical property of bone tissue can be kept again.In addition, the chemical treatment method of present invention design is simple and easy to apply, large-scale production is facilitated to operate.
Description
Technical field
The present invention sets and is related to technical field of biological material, a kind of chemical treatment of allogeneic implantable biomaterial of specific design
Method.
Background technology
Fracture wound, tumour etc. can cause the defect of bone and structure to be destroyed.In existing technology usually using autologous bone or
Artificial bone etc. repairs the defect of bone.But, the materials of autologous bone are limited, while also having aggravated the wound of patient during materials
And increased the risk of complication.And artificial bone such as ceramics etc. for bone reparation when, its biomechanical property is poor, bone
Bioactivity also has much room for improvement.Homogeneous allogenic bone technology is using the qualified donor bone tissue of the mankind of legal donation, processed place
After reason, implanted as filler, be the substitute material of autologous bone transplanting.
Homogeneous allogenic bone is the bone alternate material being clinically most widely used at present.It has abundance, does not receive shape
State, size limit, have the advantages that preferable bone bioactivity clinically have preferable application prospect.Fresh is of the same race different
Body bone has stronger immunogenicity, and rejection can be induced after implanting.Although the host that homogeneous allogenic bone transplantation induces
Immune rejection will not typically cause the serious consequence of threat to life, but healing for bone graft is often disturbed in immune rejection
Close, and taken in excess or bone nonunion, deep infection of bone graft etc. can be caused, influence therapeutic effect.Bone tissue is by mineral matter, glue
Former, NCP and cell component composition.Mineral matter in bone does not have antigenicity, and collagen and NCP are only weak
Antigen, hematopoietic cell in its marrow of the antigenic stimulus of homogeneous allogenic bone transplantation, vascular endothelial cell, dendron are thin
Born of the same parents, macrophage and bone system cell.In order to reduce or eliminating the rejection of allograph bone, generally use in the world at present cold
Dry or cryogenic refrigeration processing method is freezed, sterilization is then carried out using gamma-rays according to line or oxirane.These processing methods
Have become international some authoritative institutions, such as the production standard in American. tissue storehouse and European organization storehouse.
The use of most homogeneous allogenic bone is clinically at present two categories below：Deep-frozen irradiation sterilization bone and freezing are dry
Dry irradiation sterilization bone.But it is simple that exempting from for homogeneous allogenic bone can not be completely eliminated using deep-frozen and the method for freeze-drying
Epidemic focus aspect is, it is necessary to further reduce the immunogenicity of bone using other methods.
The content of the invention
It is an object of the invention to provide a kind of immunogenic ingredient that can effectively remove in allosome bone material, while
Can be used for the chemical treatment method of the homogeneous allogenic bone of large-scale production.
The technical scheme is that：A kind of chemical treatment method of homogeneous allogenic bone, including the processing of allograph bone is cut
And freeze-drying, wherein, the method is carried out according to the following steps：
（1）The allosome bone material processed after cutting is cleaned up with purified water, marrow is removed；
（2）Sodium hydroxide solution is configured with analytically pure NaOH, sodium hydroxide solution and allograph bone raw material are pressed into volume：
Quality is 5-2:Fat constituent is sloughed in 1 ratio mixing, constant temperature water bath concussion, untill in solution without substantially floating grease；
（3）By step（2）The allosome bone material methyl alcohol of gained, chloroform volume ratio are 3-1:1 mixed solution immersion 14-
18 hours；
（4）By step（3）The allosome bone material of gained is soaked 16-20 hours with sodium dodecyl sulfate solution；
（5）By step（4）The allosome bone material of gained is soaked 16-20 hours with phosphate buffer；
（6）By step（5）The allosome bone material of gained is with alcohol-pickled 16-20 hours；
（7）By step（6）It is 6.5- that the allosome bone material large-scale purification water of gained is cleaned to extreme trace cleaning fluid pH value repeatedly
7.5；
（8）By step（7）The allosome bone material of gained is put into the environment at -70 DEG C and freezes 16-25 hours；
（9）By step（8）The allosome bone material of gained carries out freeze-drying in being put into freeze drier；
（10）By step（9）The allosome bone material of gained carries out irradiation sterilization.
The chemical treatment method of homogeneous allogenic bone of the invention, it is preferred that the quality of the sodium hydroxide solution
Fraction is 2%.
The chemical treatment method of homogeneous allogenic bone of the invention, in a preferred embodiment, the water-bath
The temperature of isothermal vibration is 25-37 DEG C.
Step（4）Described in dodecyl sodium sulfate mass fraction be 0.25%.
The chemical treatment method of homogeneous allogenic bone of the invention, it is preferred that the concentration of the phosphate buffer
It is 0.1-0.2 mol/L, pH value is 7.0-7.4.
The chemical treatment method of homogeneous allogenic bone of the invention, it is preferred that the mass fraction of the alcohol is 75-
85%。
The chemical treatment method of homogeneous allogenic bone of the invention, in a preferred embodiment, the freezing
Drying time is 24 hours.
The chemical treatment method of homogeneous allogenic bone of the invention, it is preferred that the dosage of the irradiation sterilization is 20-
30kGy。
Know-why of the invention：By chemically treated method, allograph bone cell inactivation, cell surface protein can be made
Ingredient breakdown, reaches elimination or weakens antigenic purpose, so that the immunological rejection caused when weakening allogenic bone transplantation.This
Invention can effectively eliminate allosome bone material by removing the chemically treated methods such as marrow, de- cell, degreasing, de- albumen
It is middle remaining cell membrane, remove cellular matrix completely, eliminate immunogene, enable the homogeneous allogenic bone of implantation by host cell institute
Adapt to, and substantially reduce immunogenicity, immunological rejection disappears substantially.
The beneficial effects of the present invention are will be cleaned up with purified water after the processing cutting of allosome bone material, marrow is removed,
Use NaOH, methyl alcohol, chloroform, dodecyl sodium sulfate, phosphate, ethanol postincubation successively again, can effectively remove
The immunogenes such as cell membrane and cellular matrix in allosome bone material, greatly reduce the immunogenicity of homogeneous allogenic bone, while logical
Deep-frozen and freeze-drying process are crossed, the immunogenicity of homogeneous allogenic bone can be further reduced, while on the one hand protecting
The trailing edge slot structure of bone tissue is stayed, has been conducive to creeping for Gegenbaur's cell, on the one hand two also maintained preferable biomethanics
Performance.Additionally, the chemical treatment method step of present invention design is simply divided a word with a hyphen at the end of a line, large-scale production operation is advantageously implemented.
Specific embodiment
To make those skilled in the art more fully understand technical scheme, the present invention is made with reference to embodiment
Describe in further detail.
Embodiment 1
The processing of allogeneic bone material is cut into graininess, then clean with purified water and is removed marrow, then with analytically pure
NaOH configuration multiplies 2% sodium hydroxide solution, and sodium hydroxide solution and allograph bone raw material are pressed into volume：Quality is 2:1 ratio
Fat constituent is sloughed in example mixing, 37 DEG C of constant temperature water bath concussions, untill in solution without substantially floating grease.
It is 1 by the allosome bone material methyl alcohol of above-mentioned gained, chloroform volume ratio:1 mixed solution soaks 16 hours；
Then allosome bone material obtained in the previous step is soaked with the phosphate buffer of 0.1mol/L pH7.2, the time is 16 hours；So
Allosome bone material obtained in the previous step is soaked with the sodium dodecyl sulfate solution that mass fraction is 0.15% afterwards, the time is 16 small
When；Again with the allosome bone material alcohol-pickled obtained in the previous step that mass fraction is 75%, after dehydration 16 hours, with substantial amounts of pure
Change water to clean to extreme trace cleaning fluid pH value repeatedly is 7.0；Allosome bone material after cleaning is put into the environment at -70 DEG C and is freezed
After 20 hours, be put into freeze drier carries out freeze-drying 24 hours according to the curve for setting.It is finally that freeze-drying is complete
Into allograph bone under the Co-60 of 25kGy dosage irradiation sterilization.
Embodiment 2
Allogeneic bone material is processed into cut growth strip, then clean with purified water and is removed marrow, then with analytically pure
NaOH configuration multiplies 2% sodium hydroxide solution, and sodium hydroxide solution and allograph bone raw material are pressed into volume：Quality is 2.5:1
Ratio mixes, and fat constituent is sloughed in 37 DEG C of constant temperature water bath concussions, untill in solution without substantially floating grease.
It is 1 by the allosome bone material methyl alcohol of above-mentioned gained, chloroform volume ratio:1 mixed solution soaks 16 hours；
Then allosome bone material obtained in the previous step is soaked with the phosphate buffer of 0.2mol/L pH7.2, the time is 18 hours；So
Allosome bone material obtained in the previous step is soaked with the sodium dodecyl sulfate solution that mass fraction is 0.25% afterwards, the time is 18 small
When；Again with the allosome bone material alcohol-pickled obtained in the previous step that mass fraction is 75%, after dehydration 19 hours, with substantial amounts of pure
Change water to clean to extreme trace cleaning fluid pH value repeatedly is 7.0；Allosome bone material after cleaning is put into the environment at -70 DEG C and is freezed
After 20 hours, be put into freeze drier carries out freeze-drying 24 hours according to the curve for setting.It is finally that freeze-drying is complete
Into allograph bone under the Co-60 of 25kGy dosage irradiation sterilization.
Embodiment 3
Allogeneic bone material is processed into cut growth strip, then clean with purified water and is removed marrow, then with analytically pure
NaOH configuration multiplies 3% sodium hydroxide solution, and sodium hydroxide solution and allograph bone raw material are pressed into volume：Quality is 3:1 ratio
Fat constituent is sloughed in example mixing, 37 DEG C of constant temperature water bath concussions, untill in solution without substantially floating grease.
It is 1 by the allosome bone material methyl alcohol of above-mentioned gained, chloroform volume ratio:1 mixed solution soaks 16 hours；
Then allosome bone material obtained in the previous step is soaked with the phosphate buffer of 0.2mol/L pH7.2, the time is 18 hours；So
Allosome bone material obtained in the previous step is soaked with the sodium dodecyl sulfate solution that mass fraction is 0.3% afterwards, the time is 18 small
When；Again with the allosome bone material alcohol-pickled obtained in the previous step that mass fraction is 75%, after dehydration 19 hours, with substantial amounts of pure
Change water to clean to extreme trace cleaning fluid pH value repeatedly is 7.0；Allosome bone material after cleaning is put into the environment at -70 DEG C and is freezed
After 20 hours, be put into freeze drier carries out freeze-drying 24 hours according to the curve for setting.It is finally that freeze-drying is complete
Into allograph bone under the Co-60 of 25kGy dosage irradiation sterilization.
Claims (8)
1. the processing cutting and freeze-drying of a kind of chemical treatment method of homogeneous allogenic bone, including allograph bone, it is characterised in that
The method is carried out according to the following steps：
（1）The allosome bone material processed after cutting is cleaned up with purified water, marrow is removed；
（2）Sodium hydroxide solution is configured with analytically pure NaOH, sodium hydroxide solution and allograph bone raw material are pressed into volume：
Quality is 5-2:Fat constituent is sloughed in 1 ratio mixing, constant temperature water bath concussion, untill in solution without substantially floating grease；
（3）By step（2）The allosome bone material methyl alcohol of gained, chloroform volume ratio are 3-1:1 mixed solution immersion 14-
18 hours；
（4）By step（3）The allosome bone material of gained is soaked 16-20 hours with sodium dodecyl sulfate solution；
（5）By step（4）The allosome bone material of gained is soaked 16-20 hours with phosphate buffer；
（6）By step（5）The allosome bone material of gained is with alcohol-pickled 16-20 hours；
（7）By step（6）It is 6.5- that the allosome bone material large-scale purification water of gained is cleaned to extreme trace cleaning fluid pH value repeatedly
7.5；
（8）By step（7）The allosome bone material of gained is put into the environment at -70 DEG C and freezes 16-25 hours；
（9）By step（8）The allosome bone material of gained carries out freeze-drying in being put into freeze drier；
（10）By step（9）The allosome bone material of gained carries out irradiation sterilization.
2. the chemical treatment method of the homogeneous allogenic bone according to claim, it is characterised in that the sodium hydroxide solution
Mass fraction be 2%-5%.
3. the chemical treatment method of the homogeneous allogenic bone according to claim, it is characterised in that the constant temperature water bath concussion
Temperature be 25-37 DEG C.
4. the chemical treatment method of the homogeneous allogenic bone according to claim, it is characterised in that the dodecyl sodium sulfonate
The mass fraction of sodium is 0.1%-0.3%.
5. the chemical treatment method of the homogeneous allogenic bone according to claim, it is characterised in that the phosphate buffer
Concentration be 0.1-0.2 mol/L, pH value is 7.0-7.4.
6. the chemical treatment method of the homogeneous allogenic bone according to claim, it is characterised in that the quality of the alcohol point
Number is 75-85%.
7. the chemical treatment method of the homogeneous allogenic bone according to claim, it is characterised in that the sublimation drying
It is 24 hours.
8. the chemical treatment method of the homogeneous allogenic bone according to claim, it is characterised in that the agent of the irradiation sterilization
It is 20-30kGy to measure.
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| CN201611103787.XA CN106730000A (en) | 2016-12-05 | 2016-12-05 | A kind of chemical treatment method of homogeneous allogenic bone |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611103787.XA CN106730000A (en) | 2016-12-05 | 2016-12-05 | A kind of chemical treatment method of homogeneous allogenic bone |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107753159A (en) * | 2017-11-10 | 2018-03-06 | 湖北联结生物材料有限公司 | The reinforced homogeneous allogenic bone cervical vertebral fusion cage of the compound new bone formation of multi-disc |
| CN111069149A (en) * | 2019-12-20 | 2020-04-28 | 河北鑫康辰生物技术有限公司 | Medical method for cleaning allogeneic bone |
| CN115414531A (en) * | 2022-09-20 | 2022-12-02 | 上海亚朋生物技术有限公司 | Method for removing fat and endotoxin of allogeneic bone |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102335458A (en) * | 2010-07-20 | 2012-02-01 | 上海安久生物科技有限公司 | Chemical treatment method for bone allograft |
| CN104288839A (en) * | 2014-09-22 | 2015-01-21 | 中国人民解放军第四军医大学 | Method for preparing dual-factor carrying type hybrid bionic bone scaffold and application of dual-factor carrying type hybrid bionic bone scaffold |
-
2016
- 2016-12-05 CN CN201611103787.XA patent/CN106730000A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102335458A (en) * | 2010-07-20 | 2012-02-01 | 上海安久生物科技有限公司 | Chemical treatment method for bone allograft |
| CN104288839A (en) * | 2014-09-22 | 2015-01-21 | 中国人民解放军第四军医大学 | Method for preparing dual-factor carrying type hybrid bionic bone scaffold and application of dual-factor carrying type hybrid bionic bone scaffold |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107753159A (en) * | 2017-11-10 | 2018-03-06 | 湖北联结生物材料有限公司 | The reinforced homogeneous allogenic bone cervical vertebral fusion cage of the compound new bone formation of multi-disc |
| CN111069149A (en) * | 2019-12-20 | 2020-04-28 | 河北鑫康辰生物技术有限公司 | Medical method for cleaning allogeneic bone |
| CN115414531A (en) * | 2022-09-20 | 2022-12-02 | 上海亚朋生物技术有限公司 | Method for removing fat and endotoxin of allogeneic bone |
| CN115414531B (en) * | 2022-09-20 | 2024-06-04 | 上海亚朋生物技术有限公司 | Method for removing fat and endotoxin from allogeneic bone |
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Application publication date: 20170531 |