CN111214704B - Method for preparing acellular nerve graft through supercritical fluid extraction reaction - Google Patents
Method for preparing acellular nerve graft through supercritical fluid extraction reaction Download PDFInfo
- Publication number
- CN111214704B CN111214704B CN202010114698.5A CN202010114698A CN111214704B CN 111214704 B CN111214704 B CN 111214704B CN 202010114698 A CN202010114698 A CN 202010114698A CN 111214704 B CN111214704 B CN 111214704B
- Authority
- CN
- China
- Prior art keywords
- reaction
- nerve
- peripheral nerves
- distilled water
- peripheral
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- A61L27/3604—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
-
- 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
- A61L27/3641—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
- A61L27/3675—Nerve tissue, e.g. brain, spinal cord, nerves, dura mater
-
- 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
-
- 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
-
- 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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- 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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
-
- 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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Dermatology (AREA)
- Transplantation (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Molecular Biology (AREA)
- Vascular Medicine (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Neurosurgery (AREA)
- General Chemical & Material Sciences (AREA)
- Neurology (AREA)
- Urology & Nephrology (AREA)
- Zoology (AREA)
- Peptides Or Proteins (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention relates to the field of biomedical engineering, and discloses a method for preparing a acellular nerve graft by supercritical fluid extraction reaction, which comprises the following steps: cutting peripheral nerve, removing external fat, blood vessel and connective tissue, and cleaning; immersing the cleaned peripheral nerves into a surfactant solution, placing the peripheral nerves into a high-temperature high-pressure reaction kettle, and introducing CO2Discharging air in the reaction kettle, then carrying out reaction, and taking out peripheral nerves after the reaction is finished; then, peripheral nerves were washed with a sodium lauryl sulfate solution and finally soaked with distilled water. The method greatly reduces the dosage and concentration of chemical reagents in the process of decellularization, has low reaction temperature, short action time and less reaction times, can effectively reduce immunogenicity, has little damage to nerve structures, not only keeps the integrity of the tubular structure of the Schwann cell basal lamina and the integrity and smoothness of the nerve basal lamina, but also keeps laminarin in axon regeneration, has good clinical use effect and is worthy of clinical popularization.
Description
Technical Field
The invention relates to the field of biomedical engineering, in particular to a method for preparing a acellular nerve graft by supercritical fluid extraction reaction.
Background
Peripheral nerve defect refers to the separation of peripheral nerve trunk caused by trauma or operation, and is a common clinical disease. Neurological defects are divided into four degrees, where defects of degrees I and II can be overcome by changing the position of the joint or by pathological methods such as free nerve trunk, anterior approach, diversion, lengthening or shortening of the bone joint, while defects above III (or defects at distances greater than 4 times the diameter of the defective nerve) must be repaired by nerve transplantation or various surrogate bridges. The nerve transplantation with the most definite curative effect is autologous nerve transplantation, namely, relatively unimportant nerves of a patient are transplanted to a defect part, and the nerve transplantation is still used as a 'gold standard' for evaluating the effectiveness of various nerve transplants at present, but the transplanted nerves have the defects of limited sources, difficult matching of tissue structures and sizes, long-term nerve failure in a transplantation supply area, difference in transplantation curative effect and the like, and the defects limit the wide development of autologous nerve transplantation. The allogeneic nerve transplantation needs to use the immunosuppressant for a long time, so the transplantation success rate is low and the application is limited. But the use of the allogeneic nerve (nerve) as a scaffold material has the advantages that: has an endoneurial tubular structure and further comprises a substance (e.g., laminin) that promotes nerve growth, facilitating the guidance and containment of nerve axon growth and passage. The application of the cell removing technology widens the application in the field of allogeneic nerve transplantation, removes living cells in allogeneic nerves, can greatly reduce the antigenicity of the living cells, and can be used as a scaffold material for nerve tissue engineering, and the basement membrane tube is well preserved.
In 1998, Sondell et al, Lund university in Sweden, applied sodium deoxycholate and polyethylene glycol tert-octyl phenyl ether (Triton X-100) to prepare rat acellular nerves, not only removed all cell components such as Schwann cells, but also kept the tubular structure of the Schwann cell substrate layer intact, the preparation steps were: (1) oscillating and dipping in distilled water for 12 h; (2) oscillating and extracting for 12 hours in a 3 percent TritonX-100 solution; (3) oscillating and extracting for 24 hours in a 4 percent sodium deoxycholate solution; (4) repeating the above steps for 2-4 times according to the diameter of the optic nerve; (5) washing with distilled water for 30 min. The extractant used in the method is TritonX-100 and sodium deoxycholate. TritonX-100 is a commonly used nonionic surfactant and emulsifier, which is considered a relatively mild detergent that is not denatured and is often used in biochemical applications to solubilize proteins. His role is to lyse cells to extract proteins and organelles. But its decellularization is not complete for thick and dense tissues. Sodium deoxycholate has the main functions: cell lysis, protein solubilization, especially the solubilization of some water insoluble proteins, such as membrane proteins; therefore, deoxycholate sodium is decellularized completely, but the damage to the nerve structure is also large. In addition, the method has long reaction time: a single reaction generally takes 24 hours, and 3-5 times are needed to remove cells. In summary, the currently widely used chemical decellularization systems have the following disadvantages: 1. the concentration and the reaction capacity of the chemical decellularization reaction solution are strong; 2. the chemical decellularization reaction time is too long, and the reaction times are too many; 3. because the water system has poor penetration force, the protein is not completely removed, and the residual value is high.
Disclosure of Invention
In view of the above, the present invention provides a method for preparing a acellular nerve graft by supercritical fluid extraction reaction, which greatly reduces the dosage and concentration of acellular chemical reagents, and has the advantages of low reaction temperature, short action time, less reaction times and little damage to nerve structures.
In order to solve the technical problems, the invention provides a method for preparing a acellular nerve graft by supercritical fluid extraction reaction, which comprises the following steps:
1) cutting peripheral nerves, removing external fat, blood vessels and connective tissues of the peripheral nerves, and performing shaking bath in distilled water for 12 h;
2) immersing peripheral nerves obtained by the treatment of the step 1) into a solution containing a surfactant, placing the solution into a high-pressure reaction kettle, and introducing CO2Discharging air in the high-pressure reaction kettle, then carrying out reaction under the conditions that the temperature is 25-35 ℃ and the pressure is 15-25 Mpa so as to remove the protein of the peripheral nerves, opening the high-pressure reaction kettle after the reaction is finished, and taking out the peripheral nerves from which the protein is removed, wherein the surfactant is TritonX-100 or TritonX-200;
3) washing the protein-removed peripheral nerve obtained in the step 2) with a sodium dodecyl sulfate solution for 3 hours, and then soaking the peripheral nerve with distilled water to obtain the acellular nerve graft.
Preferably, in the method provided by the invention, the surfactant in the step 2) is triton x-200.
Preferably, in the method provided by the invention, the content of the surfactant in the solution containing the surfactant in the step 2) is 1-3 wt%.
Preferably, in the method provided by the invention, the reaction time in the step 2) is 0.5-3 h.
Preferably, in the method provided by the present invention, the mass concentration of the sodium dodecyl sulfate solution in step 3) is 1%.
Preferably, in the method provided by the present invention, the soaking in step 3) is specifically: soaking the peripheral nerves cleaned with sodium dodecyl sulfate solution with distilled water, and replacing distilled water every 20min for three times.
Compared with the prior art, the invention uses supercritical carbon dioxide as a reaction system, only uses mild polyethylene glycol tert-octyl phenyl ether (TritonX-100) or TritonX-200 which is milder and more suitable for nervous tissues to dissolve protein, and completely removes the protein with immunogenicity. The used supercritical fluid has stronger penetrating power similar to gas and larger density and solubility similar to liquid, greatly reduces the dosage and concentration of chemical reagents in the process of decellularization, simultaneously has low reaction temperature, short action time and less reaction times, can effectively reduce immunogenicity, has small damage to the structure of nerves, not only keeps the integrity of the tubular structure of the Schwann cell basal lamina and the integrity and smoothness of the nerve basal lamina membrana tube, but also keeps the lamina essence which is vital in the regeneration of axon. Has good clinical use effect and is worthy of clinical popularization.
Detailed Description
For a further understanding of the invention, reference will now be made to the preferred embodiments of the present invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the present invention and is not intended to limit the scope of the claims which follow.
All of the starting materials of the present invention, without particular limitation as to their source, may be purchased commercially or prepared according to conventional methods well known to those skilled in the art.
The invention provides a method for preparing a acellular nerve graft by supercritical fluid extraction reaction, which comprises the following steps:
1) cutting peripheral nerves, removing external fat, blood vessels and connective tissues of the peripheral nerves, and performing shaking bath in distilled water for 12 h;
2) immersing peripheral nerves obtained by the treatment of the step 1) into a solution containing a surfactant, placing the solution into a high-pressure reaction kettle, and introducing CO2Discharging air in the high-pressure reaction kettle, then carrying out reaction under the conditions that the temperature is 25-35 ℃ and the pressure is 15-25 Mpa so as to remove the protein of the peripheral nerves, opening the high-pressure reaction kettle after the reaction is finished, taking out the peripheral nerves from which the protein is removed, and arranging a plurality of holes on the surface of the high-pressure reaction kettleThe active agent is TritonX-100 or TritonX-200;
3) washing the protein-removed peripheral nerve obtained in the step 2) with a sodium dodecyl sulfate solution for 3 hours, and then soaking the peripheral nerve with distilled water to obtain the acellular nerve graft.
Specifically, the present invention includes cutting peripheral nerve (such as sciatic nerve) of mammal (such as mouse and dog), cutting to certain length, eliminating peripheral nerve fat, blood vessel and connective tissue, and soaking in distilled water for 12 hr. After the cleaning is finished, the cleaned peripheral nerves are immersed in a surfactant solution and placed in a high-pressure reaction kettle, and CO is introduced into the high-pressure reaction kettle2And (3) discharging air in the reaction kettle, then carrying out reaction under the conditions that the temperature is 25-35 ℃ and the pressure is 15-25 Mpa so as to remove the protein of the peripheral nerves, and opening the high-pressure reaction kettle after the reaction is finished, and taking out the peripheral nerves from which the protein is removed. Supercritical fluid extraction is the most advanced physical extraction technology at present, abbreviated as sfe (supercritical fluid extraction). At lower temperatures, the gas is converted to a liquid with increasing pressure, and the volume of the liquid increases with increasing pressure, there always being a critical temperature (Tc) and critical pressure (Pc) for a particular substance, above which the substance does not become liquid or gas, which is the critical point. In the range above the critical point, the state of matter is between gas and liquid, and the fluid in this range becomes a Supercritical Fluid (SF). The supercritical fluid has stronger penetrating power similar to gas, larger density and solubility similar to liquid, has good solvent property, and can be used as a solvent for extracting and separating monomers. CO 22Is safe, nontoxic and cheap liquid, and is supercritical CO2Has the advantages of similar gas diffusion coefficient, liquid dissolving power and zero surface tension, can quickly permeate into solid substances to extract essence, and has the characteristics of high efficiency, difficult oxidation, pure nature, no chemical pollution and the like. Because the nerve tissue is smaller, the tissue wall is thin, and the nerve tissue structure can be damaged if the pressure is too high, therefore, the invention introduces CO into the high-pressure reaction kettle2In the course of the subsequent reaction, the reaction is selectedThe temperature is 25-35 ℃, the reaction pressure is 15-25 Mpa, and CO is filled in the reaction kettle under the reaction condition2Change of gas state, CO2Conversion of gas to supercritical CO2Fluid or semi-fluid and extracts immunogenic proteins. In the invention, the reaction time is preferably 0.5-3 h, and more preferably 1-3 h. The surfactant selected in the invention is TritonX-100 or TritonX-200; TritonX-100 is a commonly used nonionic surfactant and emulsifier in CO2Has good solubility, and simultaneously utilizes supercritical CO2Has gas-like diffusion coefficient and liquid dissolving power, can quickly permeate into nerves to dissolve protein, thereby completely removing the protein, and greatly reducing the damage to nerve structures because sodium deoxycholate with strong destructive power is not used and the reaction time and times are greatly shortened. TritonX-200 is a milder surfactant, and therefore TritonX-200 is more preferred for the decellularization of neural tissue.
If the peripheral nerve selected in the invention is thicker (the diameter of the nerve is more than 3mm), the process can be repeated once, specifically, the peripheral nerve taken out after one-time reaction is immersed in a new surfactant solution and placed in a high-pressure reaction kettle, and CO is introduced2And discharging the air in the high-pressure reaction kettle, and then reacting at the temperature of 25-35 ℃ and under the pressure of 15-25 Mpa, wherein the reaction time is preferably 0.5-3 h. At this point, the peripheral nerves after the re-reaction have completely removed the immunogenic proteins. The mass concentration of the surfactant solution in the invention is preferably 1-3%.
After the peripheral nerves of the protein with completely removed immunogenicity are obtained, because the used supercritical fluid has stronger penetrating power similar to gas and larger density and solubility similar to liquid, and the dosage and concentration of the late-stage decellularization chemical reagent are greatly reduced, the invention can adopt a small amount of sodium dodecyl sulfate solution with the mass concentration of 1 percent to clean the peripheral nerves for 3 hours, and then soak the peripheral nerves with distilled water to finally obtain the decellularized nerve graft of the invention, and in the process, the specific soaking steps preferably comprise: the peripheral nerves cleaned with the sodium dodecyl sulfate solution were soaked with distilled water, and the distilled water was changed every 20 min.
In order to further illustrate the present invention, the following will describe the method for preparing a acellular nerve graft by supercritical fluid extraction reaction provided by the present invention in detail with reference to the examples.
Example 1
(1) Cutting 5 rat sciatic nerves, removing external nerve fat, blood vessel and connective tissue, cutting into 20mm length, and placing in distilled water for shaking bath for 12 hr;
(2) immersing 5 sciatic nerves after oscillation bath in 100mL TritonX-200 solution with mass concentration of 3%, placing in 1000mL high-temperature high-pressure reaction kettle, introducing CO2The air in the kettle is discharged, and then the pressure is increased to the set pressure of 15Mpa at room temperature (25 ℃) for reaction for 1 h. After the reaction, the reaction vessel was opened, and 5 sciatic nerves from which the immunogenic protein was completely removed were taken out.
(3) The 5 sciatic nerves from which the immunogenic protein was completely removed were washed with 200mL of 1% sodium dodecyl sulfate solution for 3 hours, then soaked in 200mL of distilled water, and the distilled water was replaced every 20 minutes, thereby obtaining the acellular nerve graft product of this example.
Example 2
(1) Cutting 5 rat sciatic nerves, removing external nerve fat, blood vessel and connective tissue, cutting into 20mm length, and placing in distilled water for shaking bath for 12 hr;
(2) immersing 5 sciatic nerves after oscillation bath in 100mL TritonX-200 solution with mass concentration of 1%, placing in 1000mL high-temperature high-pressure reaction kettle, introducing CO2The air in the kettle is discharged, and then the pressure is increased to the set pressure of 20Mpa at room temperature (35 ℃) for reaction for 3 hours. After the reaction, the reaction vessel was opened, and 5 sciatic nerves from which the immunogenic protein was completely removed were taken out.
(3) The 5 sciatic nerves from which the immunogenic protein was completely removed were washed with 200mL of 1% sodium dodecyl sulfate solution for 3 hours, then soaked in 200mL of distilled water, and the distilled water was replaced every 20 minutes, thereby obtaining the acellular nerve graft product of this example.
Example 3
(1) Cutting 5 dog sciatic nerves, removing external nerve fat, blood vessel and connective tissue, cutting into 30mm length, and placing in distilled water for shaking bath for 12 hr;
(2) immersing 5 dog sciatic nerves after shaking bath into 300mL of TritonX-100 solution with the mass concentration of 3%, placing the solution into a 1000mL high-temperature high-pressure reaction kettle, and introducing CO2The air in the kettle is discharged, and then the pressure is increased to 25Mpa at room temperature (35 ℃), and the reaction is carried out for 2 hours. After the reaction is finished, opening the reaction kettle, taking out 5 sciatic nerves of the dogs, pouring out residual liquid in the high-temperature high-pressure reaction kettle, adding 300mL of TritonX-100 solution with the mass concentration of 3%, immersing the 5 sciatic nerves of the dogs which have undergone the primary reaction, and introducing CO2The reaction was carried out once more after the air in the reaction vessel was vented, and 5 canine sciatic nerves from which the immunogenic protein was completely removed were finally obtained.
(3) 5 dog sciatic nerves of which the immunogenicity is completely removed are cleaned by 300mL of 1% sodium dodecyl sulfate solution for 3 hours, then soaked by 300mL of distilled water, and the distilled water is replaced every 20 minutes, so that the acellular nerve graft product is obtained.
While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (3)
1. A method for preparing a acellular nerve graft by supercritical fluid extraction reaction is characterized by comprising the following steps:
1) cutting peripheral nerves, removing external fat, blood vessels and connective tissues of the peripheral nerves, and performing shaking bath in distilled water for 12 h;
2) immersing peripheral nerves obtained by the treatment of the step 1) into a solution containing a surfactant, placing the solution into a high-pressure reaction kettle, and introducing CO2Discharging air in the high-pressure reaction kettle, then carrying out reaction under the conditions that the temperature is 25-35 ℃ and the pressure is 15-25 Mpa so as to remove the protein of the peripheral nerves, opening the high-pressure reaction kettle after the reaction is finished, and taking out the peripheral nerves from which the protein is removed, wherein the surfactant is TritonX-100 or TritonX-200; the content of the surfactant in the solution containing the surfactant is 1-3 wt%; the reaction time is 0.5-3 h;
3) cleaning the peripheral nerve from which the protein is removed obtained in the step 2) with a sodium dodecyl sulfate solution for 3 hours, and then soaking the peripheral nerve with distilled water to obtain a decellularized nerve graft; the mass concentration of the sodium dodecyl sulfate solution is 1 percent.
2. The method according to claim 1, wherein the surfactant in step 2) is TritonX-200.
3. The method according to claim 1, wherein the soaking in step 3) is specifically: soaking the peripheral nerves cleaned with sodium dodecyl sulfate solution with distilled water, and replacing distilled water every 20min for three times.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010114698.5A CN111214704B (en) | 2020-02-25 | 2020-02-25 | Method for preparing acellular nerve graft through supercritical fluid extraction reaction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010114698.5A CN111214704B (en) | 2020-02-25 | 2020-02-25 | Method for preparing acellular nerve graft through supercritical fluid extraction reaction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111214704A CN111214704A (en) | 2020-06-02 |
| CN111214704B true CN111214704B (en) | 2022-02-25 |
Family
ID=70811934
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010114698.5A Active CN111214704B (en) | 2020-02-25 | 2020-02-25 | Method for preparing acellular nerve graft through supercritical fluid extraction reaction |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111214704B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111686303A (en) * | 2020-06-09 | 2020-09-22 | 上海亚朋生物技术有限公司 | Preparation method of allogeneic or xenogeneic tendon and bone tendon complex |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6562605B1 (en) * | 1995-11-13 | 2003-05-13 | Genencor International, Inc. | Extraction of water soluble biomaterials from fluids using a carbon dioxide/surfactant mixture |
| CN104288838A (en) * | 2014-10-22 | 2015-01-21 | 林贤丰 | Kit for obtaining multiple-organ and tissue extracellular matrix and using method of kit |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017025053A1 (en) * | 2015-08-11 | 2017-02-16 | Dar-Jen Hsieh | Preparation of acellular cartilage graft and uses thereof |
| GB2543540B (en) * | 2015-10-21 | 2020-10-14 | Videregen Ltd | Decellularising tissue |
-
2020
- 2020-02-25 CN CN202010114698.5A patent/CN111214704B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6562605B1 (en) * | 1995-11-13 | 2003-05-13 | Genencor International, Inc. | Extraction of water soluble biomaterials from fluids using a carbon dioxide/surfactant mixture |
| CN104288838A (en) * | 2014-10-22 | 2015-01-21 | 林贤丰 | Kit for obtaining multiple-organ and tissue extracellular matrix and using method of kit |
Non-Patent Citations (1)
| Title |
|---|
| "化学萃取法制备人类同种异体神经支架的实验研究";江长青等;《实用手外科杂志》;20080310;第22卷(第1期);第27-29页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111214704A (en) | 2020-06-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3571362B2 (en) | Method of treating bone tissue and related implantable biomaterial | |
| BR9912180B1 (en) | Soft, foldable fabric sample and process for its preparation. | |
| EP3365035B1 (en) | Decellularising tissue | |
| KR101982760B1 (en) | Preparation method of hydrogel based on decellularized tissue using supercritical fluid-organic solvent system | |
| Ackbar et al. | Decellularized ovine esophageal mucosa for esophageal tissue engineering | |
| KR101410533B1 (en) | A method for treating material derived from biological tissue | |
| CN107164298A (en) | Supercritical fluid technique prepares the method that soft tissue removes cellular matrix | |
| WO2006122533A2 (en) | Bioartificial heart tissue graft and method for the production thereof | |
| CN111214704B (en) | Method for preparing acellular nerve graft through supercritical fluid extraction reaction | |
| CN110612128A (en) | Method for producing decellularized tissue, and apparatus for producing decellularized tissue | |
| CN104288838A (en) | Kit for obtaining multiple-organ and tissue extracellular matrix and using method of kit | |
| Shahabipour et al. | Scaffolds derived from cancellous bovine bone support mesenchymal stem cells' maintenance and growth | |
| CN109621008B (en) | Acellular nerve graft and preparation method thereof | |
| Chaschin et al. | Decellularization of the human donor aortic conduit by a new hybrid treatment in a multicomponent system with supercritical CO2 and Tween 80 | |
| CN103436489A (en) | Reagent for acellular processing of animal skin tissue, and processing method thereof | |
| CN105920671B (en) | Preparation method of nerve graft and product thereof | |
| CN111518744A (en) | Liver acellular scaffold construction method based on irreversible electroporation technology | |
| CN107137769A (en) | A kind of preparation method of the full organ acellular matrix of heart | |
| CN107029298A (en) | A kind of medical acellular dermal matrix and preparation method thereof | |
| Sokol et al. | Biocompatibility analysis of the decellularized bovine pericardium | |
| Say et al. | Sonication-assisted perfusion decellularization of whole porcine kidney | |
| CESUR et al. | Decellularization of cow aorta via supercritical CO2 | |
| JP2020006130A (en) | Method for producing decellularized tissue | |
| Tokpayev et al. | Decellularization of bone tissue in a supercritical carbon dioxide environment | |
| JP2020005622A (en) | Method for producing decellularized tissues, and apparatus for producing decellularized tissues |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |