CN116836265A - Sheep spine fiber annulus collagen and extraction method and application thereof - Google Patents
Sheep spine fiber annulus collagen and extraction method and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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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
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
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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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
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Abstract
The application relates to collagen, in particular to a collagen extraction method and application, wherein the extraction method comprises the following steps of (1) removing upper and lower back films from a cut sheep spine and then extracting fibrous rings; (2) Crushing the fiber ring, and then carrying out enzymolysis by trypsin to obtain a cell-removed fiber ring; (3) Washing the cell-free fiber ring, and then carrying out enzymolysis by using nuclease to obtain the cell-free fiber ring; (4) And (3) cleaning the fiber ring with the nucleic acid removed, extracting by an acid enzyme combination method, balancing ions, and dialyzing to obtain the collagen of the fiber ring of the sheep backbone. The method can greatly reduce the influence of cell fragments and nucleic acid fragments on the extraction efficiency of the collagen, effectively solves the problems of more impurities, insufficient purity and extraction efficiency of the collagen in the traditional extraction process of the collagen in the fibroring, and provides a good biological resource regeneration method for the sheep spinal column.
Description
Technical Field
The application relates to collagen, in particular to a collagen extraction method and application.
Background
The annulus is a ring composed of a variety of proteins and biological fibers that are widely present in the human and animal body and perform important physiological functions.
Collagen is one of the most important components in the annulus fibrosus, and plays a vital role in structural support of the annulus fibrosus, mechanical enhancement and activity maintenance. In addition, collagen is also receiving a great deal of attention as a glycoprotein that is most widely distributed in nature, and also by virtue of its own special triple helix structure.
In the food field, collagen is considered as a natural thickener, tackifier with extremely high nutritional value; in the medical field, collagen is widely used in skin filling, moisturizing and anti-aging products due to its high biocompatibility; in the scientific field, collagen is used as a main component of extracellular matrix in the development of biomaterials such as cell 3D culture, wound healing, drug delivery, and vascular prostheses.
Sheep, one of the earliest domestic animals domesticated by humans, has its own demand increasing year by year with the progress of human society and the improvement of the living standard. However, sheep spines, which account for 10% -15% of the weight of the fleshy material, are often regarded as non-fleshy offcuts (except for special uses) and discarded.
The extraction of the collagen in the fiber ring of the sheep backbone provides a channel for the treatment of the sheep backbone leftover materials, so that not only is the regeneration of biological resources realized, but also the production of medical products related to bone health promotion and bone tissue growth enhancement can be realized by utilizing the unique characteristics of the collagen from the fiber ring.
At present, the traditional method for extracting the collagen comprises a hot water extraction method, an acid extraction method, an enzyme extraction method, an alkali extraction method and the like, but the method is easy to cause serious corrosion to an extraction container and equipment and secondary pollution to raw materials due to long extraction time (the time in industrialization is usually as long as a plurality of weeks).
Meanwhile, as the sheep spine fiber ring contains various nerve cells, cell fragments, nucleic acid fragments and various intracellular impurity proteins can not be removed after the traditional method is adopted, so that the extracted sheep spine fiber ring collagen protein has more impurities, low purity and extraction rate and other industrial production problems.
Disclosure of Invention
The application provides sheep spine fiber ring collagen and an extraction method thereof, aiming at the problem of extracting collagen in sheep spine fiber ring in the prior art.
In order to solve the technical problems, the application is solved by the following technical scheme:
the method for extracting the collagen from the fiber ring of the sheep spine comprises the following steps:
s1, removing fascia and adipose tissue from a cut sheep spine, and extracting a fibrous ring;
s2, crushing the fiber ring obtained in the step S1, and then carrying out enzymolysis by trypsin to remove various cells so as to obtain a cell-removed fiber ring;
by carrying out trypsin treatment on the freshly extracted annulus fibrosus, various cells in the annulus fibrosus, including neuron cells or glial cells, can be effectively removed, the influence of intracellular hybrid proteins on the extraction of target collagen can be effectively reduced, and the collagen extraction efficiency is improved.
Step S3, cleaning the cell-free fiber ring obtained in the step S2, and then carrying out enzymolysis by using nuclease to remove internal miscellaneous DNA and RNA so as to obtain the cell-free fiber ring;
by carrying out nuclease treatment on the fiber ring, the influence of broken cell leakage nucleic acid on the extraction of target collagen can be effectively reduced, the content of impurities in the collagen can be effectively reduced, the extraction time of an acid enzyme method is shortened, and the extraction efficiency of the collagen is improved.
Step S4, cleaning the fiber ring with the nucleic acid removed in the step S3, and extracting by an acid enzyme combination method to obtain fiber ring collagen supernatant;
step S5, regulating pH balance and ion balance of the supernatant of the fibrous ring collagen in the step S4 to obtain a fibrous ring collagen solution;
step S6, dialyzing the annular collagen solution in the step S5;
and S7, performing freeze-drying and drying after dialysis to obtain the sheep spine fiber ring collagen.
Simultaneously, by carrying out trypsin and nuclease treatment on the fiber ring, the nucleic acid pollution caused by singly using trypsin or the impurity protein pollution caused by insufficient cell removal caused by singly using nuclease can be effectively avoided.
Preferably, the step S1 specifically includes the steps of:
s11, removing membranes and adipose tissues from the cut sheep spinal column;
step S12, cleaning for 3 times by using a cleaning solution, and extracting the fiber ring after cleaning, wherein the cleaning solution is sterilized water, phosphate buffer solution or cell culture medium solution.
Preferably, the step S2 specifically includes the steps of:
s21, cutting or cutting the fiber ring into small pieces;
s22, soaking small pieces of fiber rings in trypsin solution for enzymolysis for 24-48 hours at the temperature of 30-37 ℃ to obtain cell-removed fiber rings, wherein trypsin comprises one or more of acid trypsin, neutral trypsin and alkaline trypsin; the mass volume ratio of the fiber ring to the trypsin solution is 0.1 g-2 g:40-80 mL, and the concentration of the trypsin solution is 0.1% -0.6%.
Preferably, the step S3 is to soak the cell-free fiber ring in nuclease solution with the concentration of 3% -8% for enzymolysis to obtain the cell-free fiber ring, wherein the nuclease comprises ribonuclease, deoxyribonuclease, demethylase or nucleotide shearing enzyme, the volume ratio of the fiber ring to the nuclease solution is 0.5 g-3 g:50-200 mL, the soaking time is 8-16 h, and the soaking temperature is 30-37 ℃.
Preferably, the step S4 specifically includes the steps of:
s41, soaking the fiber ring with the removed nucleic acid in a cleaning solution for 24-48 hours to obtain the fiber ring with the removed impurities; wherein the cleaning solution comprises phosphate buffer solution, polyethylene glycol octyl phenyl ether solution or sterilized water;
step S42, extracting the impurity-removed fiber ring by an acid enzyme combination method to obtain fiber ring collagen supernatant, wherein the acid enzyme combination method is to add the impurity-removed fiber ring into an acid solution, wherein the acid solution used in the acid enzyme combination method is acetic acid or hydrochloric acid solution with the concentration of 0.3-0.8 mol/L, the enzyme used in the acid enzyme combination method is pepsase, and the ratio of the pepsase consumption to the enzymolysis fiber ring mass is 1-3: 10-15, wherein the extraction temperature of the acid enzyme combination method is 1-8 ℃ and the extraction time is 24-48 h;
and step S43, after the extraction by an acid enzyme combination method is finished, performing ultracentrifugation by using 2000 g-8000 g to obtain the supernatant of the fibrous ring collagen.
Preferably, the step S5 specifically includes the steps of:
s51, standing the supernatant of the fibrous ring collagen for 24-48 hours;
s52, performing pH balance adjustment on the supernatant of the fibrous ring collagen by using 2-8 mol/L NaOH solution;
and step S53, performing ion balance adjustment on the fiber ring collagen supernatant adjusted in the step S52 by using a phosphate buffer solution to obtain a fiber ring collagen solution.
Preferably, in step S6, the solution of the collagen is dialyzed by a dialysis bag, the molecular weight cut-off of the dialysis bag is 5000-10000, and the dialysis time is 24-48 hours.
Preferably, the freeze-drying temperature in the step S7 is-40 ℃ to-80 ℃; the freeze-drying time is 24-72 h.
The sheep spine fiber ring collagen is extracted by adopting a sheep spine fiber ring collagen extraction method, and the extracted sheep spine fiber ring collagen has the characteristics of type I collagen and contains at least two different alpha chains and one beta chain segment.
The application of the sheep spine fiber ring collagen is applied to hydrogel.
The application has the remarkable technical effects due to the adoption of the technical scheme:
the method is simple in operation and easy to industrialize, cell fragments, nucleic acid fragments and impurity proteins in the collagen from the annulus fibrosis can be effectively removed, the collagen with high purity can be prepared, the extraction rate of the collagen can be effectively improved, and the time for extraction is shortened. The extracted collagen is white spongy solid after dynamic, has good water solubility and extremely high similarity with the human collagen in composition, and can be widely applied to the preparation and production of medical skin moisturizing and filling products, implantable biomedical materials, bone repair and regeneration promoting materials and drug delivery materials.
Drawings
FIG. 1 is a diagram showing the comparison between the extracted and the extracted results of the collagen from the annulus fibrosus in example 1.
FIG. 2 is a SDS-PAGE electrophoresis of the fibrous ring collagen according to example 1.
FIG. 3 is a cell adhesion and cell compatibility test of the collagen of the annulus fibrosis in example 1.
FIG. 4 is a nuclear magnetic resonance spectrum of double bond modified annular fiber collagen in example 1.
Fig. 5 is a photograph of a real object of the collagen hydrogel of the annulus fibrosus in example 1 and a photo-curing rheological test.
FIG. 6 is a cell adhesion to cell compatibility test of the collagen hydrogel of example 1.
Fig. 7 shows the extraction yield of the collagen from the annulus fibrosus in examples 1 to 5.
Detailed Description
The present application will be described in further detail with reference to the accompanying drawings and examples.
Example 1
As shown in fig. 1 to 7, the method for extracting the collagen from the fiber ring of the sheep spine comprises the following steps:
step S1, taking sheep spines from which connective tissues and elastic fibers are removed,
step S2, washing for 3 times by using PBS, cutting the fiber ring into small pieces of 1cm X1 cm to obtain the crushed fiber ring, soaking the fiber ring in a trypsin solution with the concentration of 0.4% for enzymolysis for 36 hours at the temperature of 37 ℃, replacing the trypsin solution every 4 hours in the soaking process, and removing various cells in the fiber ring, such as myocardial cells, fiber cells and the like to obtain the cell-free fiber ring.
Step S3, soaking the cell-removed fiber ring in a 5% concentration deoxyriboendonuclease nuclease solution for enzymolysis for 12 hours at 37 ℃ to remove various nucleic acids in the fiber ring, such as deoxyribonucleic acid and the like, so as to obtain the nucleic acid-removed fiber ring.
And S4, soaking the fiber ring with the nucleic acid removed in a polyethylene glycol octyl phenyl ether solution (1%) and cleaning for 48 hours. Weighing the cleaned fiber ring, adding the fiber ring into 0.5mol/L acetic acid solution, then adding pepsin with the weight of 1/8 of the fiber ring into the mixed solution, carrying out enzymolysis for 24 hours at the temperature of 4 ℃, and carrying out ultracentrifugation with 5000g after the enzymolysis is finished to obtain fiber ring collagen supernatant.
And S5, standing the fiber ring collagen supernatant for 24 hours, then adjusting the pH of the supernatant to 7.0 by using a 5mol/L NaOH solution, and adjusting the ion balance of the supernatant by using a phosphate buffer solution added with 5 times of the volume of the supernatant to obtain the fiber ring collagen solution.
And S6, after the fiber ring collagen solution is obtained, dialyzing the solution for 24 hours by using a dialysis bag with the molecular weight cut-off of 7500.
Step S7, freeze-drying at-80 ℃ for 48 hours to obtain the sheep spine fiber ring collagen, as shown in figure 1.
As shown in FIG. 2, in the present embodiment, SDS-PAGE electrophoresis test is performed on the obtained sheep spinal fibrous ring collagen, and the test result proves that the obtained protein is collagen, has no impurity band and has higher purity;
as shown in figure 3, the experimental results of cell adhesion and cell compatibility of the obtained sheep spinal fibrous ring collagen are shown to be good in biocompatibility and can promote cell adhesion by using human embryonic lung fibroblasts (NHFL) and Human Umbilical Vein Endothelial Cells (HUVEC) human non-small cell lung cancer cells (A549).
Example 2
The difference is that in step S2 of this example, the annulus fibrosus is digested with 0.4% trypsin solution at 37deg.C for 24h.
Example 3
The difference is that in step S2 of the annulus fibrosis of this example, the annulus fibrosis is immersed in a 0.2% trypsin solution as in example 1.
Example 4
The procedure is as in example 1, except that the decellularized fiber ring in step S3 of this example is digested in 5% strength DNase solution at 37℃for 16 hours.
Example 5
The procedure is as in example 1, except that the decellularized fiber ring in step S3 of this example is immersed in a 3% strength solution of deoxyriboendonuclease.
Comparative example 1:
the difference from embodiment 1 is that this embodiment specifically includes the following steps:
step S1, taking sheep spines from which connective tissues and elastic fibers are removed,
step S2, washing for 3 times by using PBS, cutting the fiber ring into small pieces with the length of 1cm X1 cm, obtaining the crushed fiber ring, soaking the fiber ring in a trypsin solution with the concentration of 0.4% for enzymolysis for 36 hours at the temperature of 37 ℃, and replacing the trypsin solution every 4 hours in the soaking process to obtain the cell-free fiber ring.
Step S3, soaking the cell-free fiber ring in polyethylene glycol octyl phenyl ether solution (1%) and cleaning for 48 hours. Weighing the cleaned fiber ring, adding the fiber ring into 0.5mol/L acetic acid solution, then adding pepsin with the weight of 1/8 of the fiber ring into the mixed solution, carrying out enzymolysis for 24 hours at the temperature of 4 ℃, and carrying out ultracentrifugation with 5000g after the enzymolysis is finished to obtain fiber ring collagen supernatant.
And S4, standing the fiber ring collagen supernatant for 24 hours, then adjusting the pH of the supernatant to 7.0 by using a 5mol/L NaOH solution, and adjusting the ion balance of the supernatant by using a phosphate buffer solution added with 5 times of the volume of the supernatant to obtain the fiber ring collagen solution.
And S5, after the fiber ring collagen solution is obtained, dialyzing the solution for 24 hours by using a dialysis bag with the molecular weight cut-off of 7500.
And step S6, freeze-drying at-80 ℃ for 48 hours to obtain the sheep spine fiber ring collagen.
Comparative example 2:
the difference from embodiment 1 is that this embodiment specifically includes the following steps:
step S1, taking sheep spines from which connective tissues and elastic fibers are removed,
s2, washing for 3 times by using PBS, and then cutting the fiber ring into small pieces of 1cm X1 cm to obtain the crushed fiber ring;
and S3, soaking the fragmented fiber ring in a 5% concentration deoxyribose endonuclease solution for enzymolysis, and carrying out enzymolysis for 12 hours at 37 ℃ to obtain the nucleic acid-removed fiber ring.
And S4, soaking the fiber ring with the nucleic acid removed in a polyethylene glycol octyl phenyl ether solution (1%) and cleaning for 48 hours. Weighing the cleaned fiber ring, adding the fiber ring into 0.5mol/L acetic acid solution, then adding pepsin with the weight of 1/8 of the fiber ring into the mixed solution, carrying out enzymolysis for 24 hours at the temperature of 4 ℃, and carrying out ultracentrifugation with 5000g after the enzymolysis is finished to obtain fiber ring collagen supernatant.
And S5, standing the fiber ring collagen supernatant for 24 hours, then adjusting the pH of the supernatant to 7.0 by using a 5mol/L NaOH solution, and adjusting the ion balance of the supernatant by using a phosphate buffer solution added with 5 times of the volume of the supernatant to obtain the fiber ring collagen solution.
And S6, after the fiber ring collagen solution is obtained, dialyzing the solution for 24 hours by using a dialysis bag with the molecular weight cut-off of 7500.
And step S7, freeze-drying at-80 ℃ for 48 hours to obtain the sheep spine fiber ring collagen.
Comparative example 3:
the method for extracting the sheep spine fiber ring collagen by adopting the traditional collagen extraction mode comprises the following specific operation steps:
step S1, taking sheep spines from which connective tissues and elastic fibers are removed;
step S2, washing 3 times by using PBS, and then cutting the fiber ring into small pieces of 1cm multiplied by 1 cm. After the crushed fiber ring is obtained, the cleaned fiber ring is weighed and added into 0.5mol/L acetic acid solution, then pepsin with the weight of 1/8 of that of the fiber ring is added into the mixed solution, and enzymolysis is carried out for 24 hours at the temperature of 4 ℃.
And S3, after enzymolysis is finished, using 5000g of ultracentrifugation, and standing supernatant for 24h.
And S4, adding NaCl into the annular fiber collagen supernatant until the final concentration of NaCl in the mixed solution is 5mol/L, standing for 24 hours, centrifuging at 5000g, and taking out precipitate to obtain the salted-out annular fiber collagen.
And S5, re-dissolving the salted-out annulus fibrosus collagen by using sterilized water, dialyzing the solution for 24 hours by using a dialysis bag with the molecular weight cut-off of 7500, and then freeze-drying at the temperature of minus 80 ℃ for 48 hours to obtain the sheep spinal column annulus fibrosus collagen.
As shown in FIG. 7, the comparison of the extraction rates of the collagens in example 1 and example 2 and example 3 shows that the optimal concentration of trypsin used in the extraction process of the sheep spinal fibrous ring collagen of the present application is 0.4%, and the enzymolysis time is 36h.
By comparing the extraction rates of the collagens in example 1 with those in example 4 and example 5, the optimal use concentration of nuclease in the extraction process of the sheep spine fiber ring collagen of the application is 5%, and the enzymolysis time is 12h
By comparing the extraction rates of the collagens in examples 1 to 5 and comparative example 1 with that in comparative example 2, it is demonstrated that in the extraction process of the collagen of the sheep spine fiber annulus according to the present application, the combination treatment of trypsin and nuclease can significantly increase the extraction rate of collagen because the simultaneous treatment of trypsin and nuclease can effectively avoid nucleic acid contamination caused by the use of trypsin alone or impurity protein contamination caused by insufficient cell removal caused by the use of nuclease alone.
By comparing the extraction rates of the collagens in examples 1 to 5 and comparative example 3, the extraction method of the collagen in the fiber annulus of the sheep backbone is obviously superior to the traditional extraction method of the collagen in the fiber annulus of the sheep backbone.
Example 6
The sheep spine fiber ring collagen is extracted by the method for extracting the sheep spine fiber ring collagen in any one of embodiments 1-5, and the extracted sheep spine fiber ring collagen has the characteristic of I-type collagen and contains at least two different alpha chains and one beta chain segment.
The sheep spine fiber annulus collagen obtained by the embodiment has good solubility, can be dissolved in various solvents (such as sterilized water, acetic acid, hydrochloric acid and the like) with high concentration (more than 30 mg/mL), effectively solves the waste of sheep spine leftover materials, and effectively realizes the regeneration of biological resources.
Meanwhile, the extracted sheep spine fiber ring collagen can meet the requirements of engineering medical materials, and fills the blank of fiber ring collagen in the domestic market.
Example 7
The application of the sheep spine fiber ring collagen applies the sheep spine fiber ring collagen in the embodiment 6 to the hydrogel, and specifically comprises the following steps:
after the sheep spine fiber ring collagen is obtained in the step (1), double bonds are modified on the surface of the sheep spine fiber ring collagen by using active molecules, wherein the active molecules can be one or more of methacrylic anhydride, allylamine hydrochloride, norbornene dianhydride and acrylic anhydride, and in the embodiment, double bonds are modified on the surface of the collagen by using methacrylic anhydride to obtain the double bond modified sheep spine fiber ring collagen.
As shown in fig. 4, the double bond modified sheep spinal fiber ring collagen is characterized by using a nuclear magnetic hydrogen spectrogram, and the test result proves that the double bond modified sheep spinal fiber ring collagen is successfully synthesized.
Step (2), after the double bond modified sheep spine fiber ring collagen is obtained, dissolving the collagen by using a neutral solution, wherein the neutral solution is preferably PBS or sterilized water, and dissolving the double bond modified sheep spine fiber ring collagen into a solution with the concentration of 5% -20% by using the sterilized water to obtain a double bond modified sheep spine fiber ring collagen solution;
step (3), after the double bond modified sheep spine fiber ring collagen solution is obtained, adding a photoinitiator with the concentration of 0.01% -0.1%, and irradiating for 20-40 s by ultraviolet light to obtain the sheep spine fiber ring collagen hydrogel, wherein the photoinitiator is LAP, DPB or 2959, and the concentration after adding the solution is preferably 0.01% -0.1%, and is preferably 0.05%.
And an appropriate amount of LAP was added thereto to give a final LAP concentration of 0.05%.
As shown in fig. 5, the photo-curing process test is performed on the double-bond modified sheep spinal fiber ring collagen by using a rheometer, and the test result proves that the double-bond modified sheep spinal fiber ring collagen can be cured within 20 seconds to form hydrogel, and the hydrogel modulus is in the common commercial biomedical hydrogel modulus range.
As shown in fig. 6, the obtained sheep spinal fibrous ring collagen hydrogel was subjected to cell adhesion and cell compatibility experiments by using human embryonic lung fibroblasts (NHFL) and Human Umbilical Vein Endothelial Cells (HUVEC) human non-small cell lung cancer cells (a 549), and the test results prove that the obtained fibrous ring collagen has good biocompatibility and good cell adhesion.
It is to be understood that, based on one or several embodiments provided in the present application, those skilled in the art may combine, split, reorganize, etc. the embodiments of the present application to obtain other embodiments, which do not exceed the protection scope of the present application.
In summary, the foregoing description is only of the preferred embodiments of the present application, and all equivalent changes and modifications made in accordance with the claims should be construed to fall within the scope of the application.
Claims (10)
1. The extraction method of the sheep spine annulus fibrosus collagen is characterized by comprising the following steps of:
s1, removing fascia and adipose tissue from a cut sheep spine, and extracting a fibrous ring;
s2, crushing the fiber ring obtained in the step S1, and then carrying out enzymolysis by trypsin to obtain a cell-removed fiber ring;
step S3, cleaning the cell-free fiber ring obtained in the step S2, and then carrying out enzymolysis by using nuclease to obtain the cell-free fiber ring;
step S4, cleaning the fiber ring with the nucleic acid removed in the step S3, and extracting by an acid enzyme combination method to obtain fiber ring collagen supernatant;
step S5, regulating pH balance and ion balance of the supernatant of the fibrous ring collagen in the step S4 to obtain a fibrous ring collagen solution;
step S6, dialyzing the annular collagen solution in the step S5;
and S7, performing freeze-drying and drying after dialysis to obtain the sheep spine fiber ring collagen.
2. The method for extracting the collagen from the fibrous annulus of the sheep spine according to claim 1, wherein the method comprises the following steps:
the step S1 specifically comprises the following steps:
s11, removing membranes and adipose tissues from the cut sheep spinal column;
step S12, cleaning for 3 times by using a cleaning solution, and extracting the fiber ring after cleaning, wherein the cleaning solution is sterilized water, phosphate buffer solution or cell culture medium solution.
3. The method for extracting the collagen from the fibrous annulus of the sheep spine according to claim 1, wherein the method comprises the following steps:
the step S2 specifically comprises the following steps:
s21, cutting or cutting the fiber ring into small pieces;
s22, soaking small pieces of fiber rings in trypsin solution for enzymolysis for 24-48 hours at the temperature of 30-37 ℃ to obtain cell-removed fiber rings, wherein trypsin comprises one or more of acid trypsin, neutral trypsin and alkaline trypsin; the mass volume ratio of the fiber ring to the trypsin solution is 0.1 g-2 g:40-80 mL, and the concentration of the trypsin solution is 0.1% -0.6%.
4. The method for extracting the collagen from the fibrous annulus of the sheep spine according to claim 1, wherein the method comprises the following steps:
step S3 is to soak the cell-free fiber ring in nuclease solution with the concentration of 3-8% for enzymolysis to obtain the cell-free fiber ring, wherein the mass volume ratio of the fiber ring to the nuclease solution is 0.5-3 g:50-200 mL, the soaking time is 8-16 h, and the soaking temperature is 30-37 ℃.
5. The method for extracting the collagen from the fibrous annulus of the sheep spine according to claim 1, wherein the method comprises the following steps:
the step S4 specifically comprises the following steps:
s41, soaking the fiber ring with the removed nucleic acid in a cleaning solution for 24-48 hours to obtain the fiber ring with the removed impurities; wherein the cleaning solution comprises phosphate buffer solution, polyethylene glycol octyl phenyl ether solution or sterilized water;
step S42, extracting the impurity-removed fiber ring by an acid enzyme combination method to obtain fiber ring collagen supernatant, wherein the acid enzyme combination method is to add the impurity-removed fiber ring into an acid solution, wherein the acid solution used in the acid enzyme combination method is acetic acid or hydrochloric acid solution with the concentration of 0.3-0.8 mol/L, the enzyme used in the acid enzyme combination method is pepsase, and the ratio of the pepsase consumption to the enzymolysis fiber ring mass is 1-3: 10-15, wherein the extraction temperature of the acid enzyme combination method is 1-8 ℃ and the extraction time is 24-48 h;
and step S43, after the extraction by an acid enzyme combination method is finished, performing ultracentrifugation by using 2000 g-8000 g to obtain the supernatant of the fibrous ring collagen.
6. The method for extracting the collagen from the fibrous annulus of the sheep spine according to claim 1, wherein the method comprises the following steps:
the step S5 specifically comprises the following steps:
s51, standing the supernatant of the fibrous ring collagen for 24-48 hours;
s52, performing pH balance adjustment on the supernatant of the fibrous ring collagen by using 2-8 mol/L NaOH solution;
and step S53, performing ion balance adjustment on the fiber ring collagen supernatant adjusted in the step S52 by using a phosphate buffer solution to obtain a fiber ring collagen solution.
7. The method for extracting the collagen from the fibrous annulus of the sheep spine according to claim 1, wherein the method comprises the following steps:
in the step S6, the annular collagen solution is dialyzed by a dialysis bag, the molecular weight cut-off of the dialysis bag is 5000-10000, and the dialysis time is 24-48 h.
8. The method for extracting the collagen from the fibrous annulus of the sheep spine according to claim 1, wherein the method comprises the following steps:
the freeze-drying temperature in the step S7 is-40 ℃ to-80 ℃; the freeze-drying time is 24-72 h.
9. A sheep backbone fibre ring collagen, which is characterized in that: the method for extracting the sheep spine fiber ring collagen is characterized in that the sheep spine fiber ring collagen obtained by extraction through the method for extracting the sheep spine fiber ring collagen according to any one of claims 1-9 has the characteristics of type I collagen and contains at least two different alpha chains and one beta chain segment.
10. An application of sheep backbone fibre ring collagen is characterized in that: use of a sheep spine annulus fibrosus collagen according to any one of claims 1-9 in a hydrogel.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4789663A (en) * | 1984-07-06 | 1988-12-06 | Collagen Corporation | Methods of bone repair using collagen |
| KR20180028229A (en) * | 2016-09-08 | 2018-03-16 | 주식회사 엘앤씨바이오 | The Method of High-yield and High-purity Manufacturing of Allo-collagen Composition Extracted From Human origin |
| US20190160109A1 (en) * | 2016-01-11 | 2019-05-30 | Technische Universiteit Eindhoven | Notochordal cell matrix as a bioactive lubricant for the osteoarthritic joint |
| CN109939262A (en) * | 2019-03-19 | 2019-06-28 | 皖南医学院第一附属医院（皖南医学院弋矶山医院） | A kind of preparation method and applications of bFGF hydrogel |
| CN110655568A (en) * | 2019-09-27 | 2020-01-07 | 成都维德医疗器械有限责任公司 | Method for extracting collagen by acid enzymolysis method |
| CN110652611A (en) * | 2019-11-08 | 2020-01-07 | 华中科技大学同济医学院附属协和医院 | Genipin cross-linked fiber ring acellular matrix hydrogel and preparation method thereof |
| CN110935067A (en) * | 2019-12-09 | 2020-03-31 | 皖南医学院第一附属医院（皖南医学院弋矶山医院） | Polyurethane/acellular fiber ring matrix fiber scaffold and preparation and application thereof |
| WO2021118379A1 (en) * | 2019-12-12 | 2021-06-17 | Ineb - Instituto De Engenharia Biomédica | Fetal decellularized nucleus pulposus material and methods for obtaining pharmaceutic compositions to be used in the treatment of intervertebral disc degeneration and back pain |
-
2023
- 2023-08-07 CN CN202310985391.6A patent/CN116836265B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4789663A (en) * | 1984-07-06 | 1988-12-06 | Collagen Corporation | Methods of bone repair using collagen |
| US20190160109A1 (en) * | 2016-01-11 | 2019-05-30 | Technische Universiteit Eindhoven | Notochordal cell matrix as a bioactive lubricant for the osteoarthritic joint |
| KR20180028229A (en) * | 2016-09-08 | 2018-03-16 | 주식회사 엘앤씨바이오 | The Method of High-yield and High-purity Manufacturing of Allo-collagen Composition Extracted From Human origin |
| CN109939262A (en) * | 2019-03-19 | 2019-06-28 | 皖南医学院第一附属医院（皖南医学院弋矶山医院） | A kind of preparation method and applications of bFGF hydrogel |
| CN110655568A (en) * | 2019-09-27 | 2020-01-07 | 成都维德医疗器械有限责任公司 | Method for extracting collagen by acid enzymolysis method |
| CN110652611A (en) * | 2019-11-08 | 2020-01-07 | 华中科技大学同济医学院附属协和医院 | Genipin cross-linked fiber ring acellular matrix hydrogel and preparation method thereof |
| CN110935067A (en) * | 2019-12-09 | 2020-03-31 | 皖南医学院第一附属医院（皖南医学院弋矶山医院） | Polyurethane/acellular fiber ring matrix fiber scaffold and preparation and application thereof |
| WO2021118379A1 (en) * | 2019-12-12 | 2021-06-17 | Ineb - Instituto De Engenharia Biomédica | Fetal decellularized nucleus pulposus material and methods for obtaining pharmaceutic compositions to be used in the treatment of intervertebral disc degeneration and back pain |
Non-Patent Citations (3)
| Title |
|---|
| LIYUN GUO等: "Preparation and Characterization of a Novel Decellularized Fibrocartilage "Book" Scaffold for Use in Tissue Engineering", PLOS ONE, vol. 10, no. 12, 4 December 2015 (2015-12-04), pages 1 - 11 * |
| 刘晨等: "京尼平交联脱细胞纤维环基质/壳聚糖水凝胶与兔纤维环源干细胞的生物相容性", 中国组织工程研究, vol. 20, no. 21, 20 May 2016 (2016-05-20), pages 3143 - 3149 * |
| 石坤等: "水凝胶再生修复退变椎间盘的研究进展", 中国修复重建外科杂志, vol. 34, no. 3, 15 March 2020 (2020-03-15), pages 275 - 284 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN118557798A (en) * | 2024-06-20 | 2024-08-30 | 苏州先觉新材料科技有限公司 | Hydrogel-electrostatic spinning film double-layer mixed bracket and preparation method and application thereof |
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