CN111249529B - Bionic multilayer collagen scaffold for cartilage repair and preparation method thereof - Google Patents
Bionic multilayer collagen scaffold for cartilage repair and preparation method thereof Download PDFInfo
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- CN111249529B CN111249529B CN202010048585.XA CN202010048585A CN111249529B CN 111249529 B CN111249529 B CN 111249529B CN 202010048585 A CN202010048585 A CN 202010048585A CN 111249529 B CN111249529 B CN 111249529B
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—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
- 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
- A61L27/56—Porous materials, e.g. foams or sponges
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
- A61F2/02—Prostheses implantable into the body
- A61F2/28—Bones
- A61F2002/2835—Bone graft implants for filling a bony defect or an endoprosthesis cavity, e.g. by synthetic material or biological material
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
- A61F2310/00—Prostheses classified in A61F2/28 or A61F2/30 - A61F2/44 being constructed from or coated with a particular material
- A61F2310/00005—The prosthesis being constructed from a particular material
- A61F2310/00179—Ceramics or ceramic-like structures
- A61F2310/00293—Ceramics or ceramic-like structures containing a phosphorus-containing compound, e.g. apatite
-
- 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
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
Abstract
The invention relates to a medical material, in particular to the field of cartilage injury regeneration, and mainly discloses a bionic multilayer collagen scaffold for cartilage repair and a preparation method thereof. The scaffold product has a multilayer three-dimensional space structure, promotes in-vivo cell recruitment by reducing the immunogenicity of materials and adopting a multilayer overlapping design of a compact layer, a porous layer and a barrier layer, realizes in-vivo cartilage regeneration induced by a biomaterial without exogenous cells, optimizes a regeneration strategy, and is suitable for the treatment and regeneration of cartilage injury.
Description
Technical Field
The invention relates to a medical material, in particular to the field of cartilage injury regeneration, and mainly discloses a bionic multilayer collagen scaffold for cartilage repair and a preparation method thereof.
Background
Osteochondral defects at joint parts caused by trauma or bone diseases are common in clinic and seriously affect the life quality of patients, and become one of the main reasons of the existing limb disabilities. Knee disease can occur in people of all ages. Defects in the cartilage of the knee can result from trauma, sprains, excessive use of the knee, muscle weakness, or general wear. Due to the specific tissue structure and biological properties of articular cartilage, although many methods of treating articular cartilage defects are known, including but not limited to: drilling of subchondral bone, electrical stimulation, laser, drug and cell injection, gene therapy, etc., but the above methods have not achieved satisfactory repair effects.
In recent years, many research progresses in the field of articular cartilage injury regeneration, such as chondrocyte phenotype degeneration and maintenance, stem cell recruitment and differentiation, immune regulation, bioactive materials, and the like, and many clinical attempts have been made, but the overall effect is still unsatisfactory.
The tissue engineering technology which is developed rapidly provides a new strategy for the regeneration and repair of the cartilage. In tissue engineering, tissue cells are attached to a scaffold prepared from biological materials and implanted into a damaged part in vivo, so that the aims of repairing and reconstructing tissues or organs and recovering functions are finally fulfilled. With cell proliferation, the scaffold material is continuously degraded and replaced by extracellular matrix, and tissues with normal functions are gradually formed.
In current research, the scaffold materials for repairing articular cartilage are mainly Hyaluronic Acid (HA), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), collagen (collagen), chitosan (chitosan), etc., or the combination of several materials thereof is also common.
Among the above-mentioned natural active biomaterials, natural cartilage components such as collagen and hyaluronic acid are typically used, and biocompatibility can be improved by a purification process while maintaining activity. The artificially synthesized material has good biocompatibility and is convenient for precise control and in-situ forming, but generally lacks bioactivity and needs to be added with active molecules.
On one hand, the ideal construction strategy of the engineered cartilage composite tissue is an integrated three-dimensional scaffold mechanism with a functional interface; on the other hand, the ideal scaffold material has good biocompatibility, is beneficial to cell adhesion, proliferation and differentiation, can be degraded and absorbed, and can be adjusted and controlled manually to ensure that the degradation and absorption speed of the scaffold material is matched with the growth speed of in vivo new tissues; and good mechanical properties, and can meet the mechanical requirements of the implantation part.
In clinical practice, the cartilage injury is mainly caused by microfracture and autologous tissue transplantation, and is assisted by tissue engineering products such as cell therapy, biological materials and the like, for example, a collagen scaffold added with cells in vitro enhances autologous chondrocyte transplantation to be a collagen membrane, and the rough surface of the collagen membrane is used for inoculating chondrocytes separated, cultured and amplified in vitro. The autologous chondrocytes in this product are derived from the patient's biopsied knee cartilage tissue, which the investigator cultures and plants in a purified, resorbable, porcine-derived collagen membrane.
The collagen film product can be used for repairing adult cartilage defects, but has the defects of not obvious clinical effect, high cost, complex technology and the like. Meanwhile, according to research reports, a few patients have some side effects after treatment, mainly including joint pain, cold-like symptoms, headache or back pain.
Therefore, in order to simultaneously solve the problems of low biological material inductivity, high immunological rejection risk, low effectiveness and high treatment cost in the current cartilage repair practice, the design of the bionic space structure and the realization of the repair function of the bionic space structure must be considered in addition to the selection and preparation process of the scaffold bionic material.
Disclosure of Invention
The invention discloses a bionic multilayer collagen scaffold for cartilage repair and a preparation method thereof, which are used for researching interdisciplines of biomedical materials science, cell biology, joint surgery and the like.
The invention aims to create a brand-new articular cartilage functional repair material, promote the recruitment of in vivo cells through the design of a material space structure, and realize the in vivo cartilage functional regeneration independent of the induction of exogenous cells and biological materials.
In terms of the preparation method, the extraction and purification process of the pig tendon collagen is optimized and adjusted to ensure that the content of the collagen is more than 99 percent and the content of the polysaccharide is less than 0.1 percent, so as to improve the biocompatibility of the scaffold material and reduce the immunogenicity.
Then, by improving the prior product technology, the design of the collagen scaffold is optimized: the three-dimensional scaffold material is changed from a thin film, and the longitudinal porous main body structure is changed from a compact structure. The transverse laminated collagen porous scaffold with a longitudinal pore structure is prepared by adopting the processes of liquid nitrogen freezing and the like, so that the cell recruitment effect is improved, and the in-vivo cartilage induction regeneration activity is increased.
And finally, designing a multilayer composite collagen scaffold with a barrier layer, and compositing a hydroxyapatite barrier layer on the basal layer collagen layer to promote the regeneration of subchondral bone and the combination of the regenerated cartilage and the subchondral bone. The product is suitable for focal cartilage defect and partial combined subchondral bone defect.
In order to solve the technical problems, the technical scheme of the invention is as follows:
the bionic multilayer collagen scaffold is characterized by comprising an upper layer and a basal layer, wherein the layers are transversely and parallelly superposed, the upper layer is a collagen layer with a compact structure, and the basal layer is a collagen layer with a longitudinal cross-linked porous structure. Preferably, the collagen is extracted and purified from porcine tendon collagen, the content of collagen is more than 99%, the content of polysaccharide is less than 0.1%, and the percentages are mass percentages.
Preferably, the thickness of the upper dense layer is 0.1-0.5 mm, and the thickness of the substrate layer is 2.0-5.0 mm.
More preferably, the upper dense layer has a thickness of 0.3 mm and the base layer has a thickness of 2.5 mm.
Furthermore, a hydroxyapatite barrier layer is compounded on the other end, opposite to the dense layer, of the substrate layer to form a multilayer structure.
The preparation method of the bionic multilayer collagen scaffold for cartilage repair comprises the following steps:
1. compact layer-longitudinal cross-linked porous layer collagen scaffold
Step 1, crushing the stripped tendon, and adding 10-fold volume of 4M guanidine hydrochloride, wherein the guanidine hydrochloride is prepared from 0.05M Tris-HCl with the pH value of 7.5;
suspending, crushing and homogenizing, stirring at 4 ℃ for 24h, centrifuging at 12000g for 20min, and separating to obtain a supernatant and a precipitate;
step 4, dissolving the tendon by using a glacial acetic acid solution containing pepsin, continuously treating for a period of time until the mixture becomes colorless, transparent and viscous liquid, centrifuging for 20min at 4 ℃ under the centrifugal force of 5000g, and collecting supernatant, namely crude tendon collagen stock solution;
step 5, adding the sodium chloride solution into the solution, continuously stirring until white flocculent precipitate is separated out from the solution, and continuously adding the sodium chloride solution until the precipitate is not separated out;
step 6, centrifuging for 20min at the temperature of 4 ℃ and under the centrifugal force of 5000g to obtain white precipitate;
step 7, adding an acid solution with a certain concentration into the precipitate for dissolving, and continuously and repeatedly carrying out salting-out treatment on the dissolved collagen solution for 2-4 times;
step 8, continuously dialyzing in ultrapure water for 3 days, and changing the liquid for 3 times every day to remove inorganic salts in the collagen solution;
step 9, freezing and molding the tiled 4mg/mL collagen solution, soaking and dehydrating the collagen solution by using a hypertonic buffer solution, and extruding the collagen solution to form a film with the thickness of 0.1-0.5 mm;
step 10, paving a layer of collagen solution on the surface of the membrane again, wherein the thickness of the collagen solution is 2.0-5.0 mm, moving the membrane into a refrigerator with the temperature of-80 ℃, pre-cooling the membrane for 2 hours at low temperature, freezing the membrane in liquid nitrogen for 2 hours, and performing vacuum freeze drying to obtain the collagen scaffold which has a compact layer and a longitudinal porous structure.
2. Porous collagen-hydroxyapatite multilayer collagen scaffold: and (2) suspending the nano hydroxyapatite powder in distilled water, controlling the solid-to-liquid ratio to be about 20/80, performing ultrasonic dispersion for 15min at low temperature, quickly adding the nano hydroxyapatite powder to the collagen surface precooled at low temperature of a refrigerator at minus 80 ℃ in the step 10, standing overnight at 4 ℃, transferring the collagen surface to liquid nitrogen for freezing for 2h, and performing vacuum freeze drying to obtain the porous collagen-hydroxyapatite multilayer scaffold composite material.
Further, the application of the bionic multilayer collagen scaffold material in the aspect of preparing a cartilage repair device is also included.
At this stage, physicochemical characterization of materials and safety evaluation:
1) chemical property detection
a) The purity of the hydroxyapatite is determined according to GB 23101.3;
b) determining the total protein content of the collagen by a Kjeldahl method;
c) hydroxyproline content of collagen was determined by hydroxyproline assay;
d) the content of heavy metal is measured according to 0821-heavy metal detection method in the fourth part of Chinese pharmacopoeia 2015 edition;
e) the pH value is measured according to GB9724
f) The requirement of sodium hyaluronate is determined according to YY/T0606.9-2007.5;
g) the content of glucurone is determined according to YY/T0606.9-2007 appendix A;
2) physical property detection
a) The size is measured with a vernier caliper with a precision of 0.02 mm, and the particles are measured with reference to GB/T1480;
b) the compressive strength is detected according to GB/T1964;
c) detecting porosity, density and volume weight according to GB/T1966;
d) the aperture is detected according to GB/T1967 electron microscope scanning method.
3) Evaluation of biosafety
a) Genotoxicity, carcinogenicity and reproductive toxicity tests were tested according to GB/T16886.3;
b) the interaction with blood test was tested according to GB/T16886.4;
c) the in vitro cytotoxicity test was tested according to GB/T16886.5;
d) the post-implantation local response test was tested according to GB/T16886.6;
e) the qualitative and quantitative tests of the degradation products are tested according to GB/T16886.9 and GB/T16886.13;
f) the stimulation and sensitization test is tested according to GB/T16886.10;
g) systemic acute, subacute, subchronic and chronic toxicity tests were tested according to GB/T16886.11;
h) the pharmacokinetic test of the degradation products is tested according to GB/T16886.16;
i) the pyrogen reaction test is checked according to YY/T1500-;
j) the sterility test is carried out according to GB/T19973.2;
k) the bacterial endotoxin assay was tested by YY/T1295-2015.
(2) Evaluation of the effectiveness of the material:
1) evaluation of in vitro efficacy
a) Cell proliferation: and (3) taking the material or the material leaching liquor, inoculating bone marrow mesenchymal stem cells/chondrocytes (primary isolated culture cells), measuring the number of the cells at different time points after inoculation by using a CCK-8 kit, and drawing a cell proliferation curve. Meanwhile, a PBS treatment group is used as a blank control, an untreated group is used as a negative control, and SPSS software is used for counting and analyzing the experimental results. For materials with significant proliferative activity, the pro-proliferative activity was further confirmed using BrdU labeling.
b) Cell chemotaxis: the bottom of the well plate was coated with material and 1 × 105 bone marrow mesenchymal stem cells/chondrocytes were added.
And after the cells grow to be full, making a slight scratch line along the bottom of the dish by using a 10-microliter gun head, cleaning the cells at the scratch by using PBS (phosphate buffer solution), marking, culturing the cells by using a serum-free culture medium, observing and collecting pictures every 12 hours, and counting the migration number of the cells.
c) Cell differentiation: qRT-PCR experiments: putting equal parts of materials into a 6-well plate, inoculating 1 × 105 bone marrow mesenchymal stem cells/on each piece of material, removing culture medium at different time points after inoculation, adding Trizol reagent, transferring the solution into a non-RNA enzyme centrifuge tube, extracting RNA, and performing reverse transcription to obtain cDNA. Relative expression levels of mRNA of the dry gene and the cartilage marker gene at different time points are detected by qRT-PCR by taking the GAPDH gene as an internal reference. ② Western blot experiment: putting the porous material into a 6-hole plate in equal parts, inoculating 1 × 105 bone marrow mesenchymal stem cells on each material, digesting and collecting the cells at different time points after inoculation, and adding a lysis solution to extract the total protein of the cells. And (3) quantifying the total protein by using a BCA kit, determining the loading volume, and then carrying out electrophoresis, membrane transfer, antigen-antibody reaction, color development, determination of band gray values of internal reference GAPDH and cell specific protein, and analyzing the relative expression level of the cell specific protein at different time points.
2) Study of in vivo (animal) Experimental effectiveness (histology, imaging, biomechanics)
The miniature pig is used as an animal model, models are manufactured on bilateral knee joints of the miniature pig, a cartilage punching device is used for manufacturing a full-layer cartilage defect with the knee joint diameter of 16 mm at a femoral load part of the knee joint of the miniature pig, a collagen bracket or gel is also used for filling the cartilage defect part, an operation incision is sewed layer by layer, and the experimental animal is allowed to move freely in a cage. Extracting peripheral blood of the experimental animal for biochemical index detection at 3 months, 6 months and 12 months after the operation; performing nuclear magnetic resonance scanning on knee joints of experimental animals to detect the filling condition of cartilage defects, the combination of repair tissues and host tissues and the combination condition of repair cartilage and subchondral bone; the experimental animal is sacrificed to take a femur specimen, and a micro-nano comprehensive mechanical testing system is used for analyzing the forward vertical load, the shear modulus, the elastic modulus, the friction coefficient and the boundary state of the repaired tissue of the cartilage defect area and the normal cartilage area respectively so as to detect the biomechanical characteristics of the repaired tissue; detecting the remodeling condition of subchondral bone by using Micro CT; performing HE staining, toluidine blue staining and safranin O staining on histological sections, evaluating the repair condition by adopting an ICRS histological scoring system, performing immunohistochemical staining on the sections by using type I collagen, type II collagen, type X collagen, osteocalcin and Lubricin, and observing the degeneration of the new tissues by using an apoptosis detection kit; obtaining the degradation product residues of important organ detection materials of the liver, the kidney, the brain, the heart and the like of the experimental animal.
The multilayer collagen scaffold disclosed by the invention shows physicochemical properties and biocompatibility meeting design purposes and requirements, the in vitro cell survival rate of the multilayer collagen scaffold is greater than 80% of that of a control, peripheral tissue necrosis, obvious inflammatory reaction and infection are not seen in vivo research, cartilage regeneration is effectively promoted for three months, the cartilage regeneration is maintained for 12 months, and the cartilage is not degenerated.
The material is implanted in the articular cartilage defect of the miniature pig through arthroscopy and open surgery, the articular cartilage is induced and maintained to be phenotypic after 3 months of surgery, and regenerated cartilage does not degenerate under the load and mechanical stimulation after 6 months and one year of surgery.
The filling rate of cartilage defect is more than 95%, and the repair tissues are transparent cartilage tissues; the repaired cartilage tissue can bear the pressure 6 times of the body weight, the dynamic friction coefficient is less than 0.005, and the extension strength reaches 5-25 Mpa; the repair tissue is in seamless connection with the host tissue, the cartilage layer in the repair tissue is promoted to be in seamless connection with the subchondral bone layer, and the regeneration of the subchondral bone and the combination of the new cartilage and the subchondral bone are realized.
Therefore, the beneficial effects of the invention are as follows: the cell recruitment can be promoted due to the three-dimensional multilayer three-dimensional structure; the scaffold material subjected to the purification process has good biocompatibility and low immunogenicity; the cartilage defect is repaired by the acellular biomaterial, so that the cost can be greatly reduced, the operation process is simplified, the treatment time is shortened, and the complications are reduced.
Drawings
FIG. 1 is a schematic structural view of a multilayered collagen scaffold.
1-dense collagen layer
2-longitudinal cross-linked porous collagen layer
3-hydroxyapatite barrier layer
Fig. 2 is a schematic view of the shape of a porous collagen scaffold.
Fig. 3 is an articular cartilage repair situation.
Detailed Description
Example 1
Crushing the stripped tendon, and adding 10-fold volume of 4M guanidine hydrochloride, wherein the pH value of the guanidine hydrochloride is 7.5, and the guanidine hydrochloride is prepared in 0.05M Tris-HCl;
suspending, pulverizing, homogenizing, stirring at 4 deg.C for 24 hr, centrifuging at 12000g for 20min, and separating to obtain supernatant and precipitate;
washing the precipitate with Tris-HCl buffer and 0.5mM acetic acid, and adding acetic acid overnight to remove proteoglycan;
dissolving tendon with glacial acetic acid solution containing pepsin, continuously treating for a while until the mixture turns into colorless, transparent and viscous liquid, centrifuging at 4 deg.C under 5000g centrifugal force for 20min, and collecting supernatant as crude tendon collagen stock solution;
adding sodium chloride solution into the solution, continuously stirring until white flocculent precipitate is separated out from the solution, and continuously adding the sodium chloride solution until the precipitate is not separated out;
centrifuging at 4 deg.C and 5000g centrifugal force for 20min to obtain white precipitate;
adding an acid solution with a certain concentration into the precipitate for dissolving, and continuously and repeatedly carrying out salting-out treatment on the dissolved collagen solution for 2-4 times;
continuously dialyzing in ultrapure water for 3 days, and changing the solution 3 times per day to remove inorganic salts in the collagen solution;
freezing and molding the tiled 4mg/mL collagen solution, soaking and dehydrating the collagen solution by using a hypertonic buffer solution, and extruding the collagen solution to form a film with the thickness of 0.5 mm;
and (3) paving a layer of collagen solution on the surface of the membrane, wherein the thickness of the collagen solution is 2.0 mm, moving the membrane into a refrigerator with the temperature of-80 ℃, pre-cooling the membrane for 2 hours at low temperature, freezing the membrane in liquid nitrogen for 2 hours, and performing vacuum freeze drying to obtain the collagen scaffold which has a compact layer and a longitudinal porous structure.
Example 2
Crushing the stripped tendon, and adding 10-fold volume of 4M guanidine hydrochloride, wherein the pH value of the guanidine hydrochloride is 7.5, and the guanidine hydrochloride is prepared in 0.05M Tris-HCl;
suspending, pulverizing, homogenizing, stirring at 4 deg.C for 24 hr, centrifuging at 12000g for 20min, and separating to obtain supernatant and precipitate;
washing the precipitate with Tris-HCl buffer and 0.5mM acetic acid, and adding acetic acid overnight to remove proteoglycan;
dissolving tendon with glacial acetic acid solution containing pepsin, continuously treating for a while until the mixture turns into colorless, transparent and viscous liquid, centrifuging at 4 deg.C under 5000g centrifugal force for 20min, and collecting supernatant as crude tendon collagen stock solution;
adding sodium chloride solution into the solution, continuously stirring until white flocculent precipitate is separated out from the solution, and continuously adding the sodium chloride solution until the precipitate is not separated out;
centrifuging at 4 deg.C and 5000g centrifugal force for 20min to obtain white precipitate;
adding an acid solution with a certain concentration into the precipitate for dissolving, and continuously and repeatedly carrying out salting-out treatment on the dissolved collagen solution for 2-4 times;
continuously dialyzing in ultrapure water for 3 days, and changing the solution 3 times per day to remove inorganic salts in the collagen solution;
freezing and molding the tiled 4mg/mL collagen solution, soaking and dehydrating the collagen solution by using a hypertonic buffer solution, and extruding the collagen solution to form a film with the thickness of 0.2 mm;
and (3) paving a layer of collagen solution on the surface of the membrane, wherein the thickness of the collagen solution is 3.0 mm, moving the membrane into a refrigerator with the temperature of-80 ℃, pre-cooling the membrane for 2 hours at low temperature, freezing the membrane in liquid nitrogen for 2 hours, and performing vacuum freeze drying to obtain the collagen scaffold which has a compact layer and a longitudinal porous structure.
Example 3
Crushing the stripped tendon, and adding 10-fold volume of 4M guanidine hydrochloride, wherein the pH value of the guanidine hydrochloride is 7.5, and the guanidine hydrochloride is prepared in 0.05M Tris-HCl;
suspending, pulverizing, homogenizing, stirring at 4 deg.C for 24 hr, centrifuging at 12000g for 20min, and separating to obtain supernatant and precipitate;
washing the precipitate with Tris-HCl buffer and 0.5mM acetic acid, and adding acetic acid overnight to remove proteoglycan;
dissolving tendon with glacial acetic acid solution containing pepsin, continuously treating for a while until the mixture turns into colorless, transparent and viscous liquid, centrifuging at 4 deg.C under 5000g centrifugal force for 20min, and collecting supernatant as crude tendon collagen stock solution;
adding sodium chloride solution into the solution, continuously stirring until white flocculent precipitate is separated out from the solution, and continuously adding the sodium chloride solution until the precipitate is not separated out;
centrifuging at 4 deg.C and 5000g centrifugal force for 20min to obtain white precipitate;
adding an acid solution with a certain concentration into the precipitate for dissolving, and continuously and repeatedly carrying out salting-out treatment on the dissolved collagen solution for 2-4 times;
continuously dialyzing in ultrapure water for 3 days, and changing the solution 3 times per day to remove inorganic salts in the collagen solution;
freezing and molding the tiled 4mg/mL collagen solution, soaking and dehydrating the collagen solution by using a hypertonic buffer solution, and extruding the collagen solution to form a film with the thickness of 0.3 mm;
spreading a layer of collagen solution with the thickness of 2.5 mm on the surface of the membrane, and transferring into a refrigerator with the temperature of 80 ℃ below zero for precooling for 2 hours at low temperature;
suspending the nano hydroxyapatite powder in distilled water, controlling the solid-to-liquid ratio to be about 20/80, performing ultrasonic dispersion for 15min at low temperature, rapidly adding the nano hydroxyapatite powder to the surface of the collagen pre-cooled at low temperature in a refrigerator at-80 ℃, standing overnight at 4 ℃, transferring the collagen to liquid nitrogen for freezing for 2h, and performing vacuum freeze drying to obtain the porous collagen-hydroxyapatite multilayer scaffold composite material.
Example 4
Crushing the stripped tendon, and adding 10-fold volume of 4M guanidine hydrochloride, wherein the pH value of the guanidine hydrochloride is 7.5, and the guanidine hydrochloride is prepared in 0.05M Tris-HCl;
suspending, pulverizing, homogenizing, stirring at 4 deg.C for 24 hr, centrifuging at 12000g for 20min, and separating to obtain supernatant and precipitate;
washing the precipitate with Tris-HCl buffer and 0.5mM acetic acid, and adding acetic acid overnight to remove proteoglycan;
dissolving tendon with glacial acetic acid solution containing pepsin, continuously treating for a while until the mixture turns into colorless, transparent and viscous liquid, centrifuging at 4 deg.C under 5000g centrifugal force for 20min, and collecting supernatant as crude tendon collagen stock solution;
adding sodium chloride solution into the solution, continuously stirring until white flocculent precipitate is separated out from the solution, and continuously adding the sodium chloride solution until the precipitate is not separated out;
centrifuging at 4 deg.C and 5000g centrifugal force for 20min to obtain white precipitate;
adding an acid solution with a certain concentration into the precipitate for dissolving, and continuously and repeatedly carrying out salting-out treatment on the dissolved collagen solution for 2-4 times;
continuously dialyzing in ultrapure water for 3 days, and changing the solution 3 times per day to remove inorganic salts in the collagen solution;
freezing and molding the tiled 4mg/mL collagen solution, soaking and dehydrating the collagen solution by using a hypertonic buffer solution, and extruding the collagen solution to form a film with the thickness of 0.1 mm;
spreading a layer of collagen solution on the surface of the membrane with the thickness of 5.0 mm, and transferring into a refrigerator with the temperature of 80 ℃ below zero for precooling for 2 hours at low temperature;
suspending the nano hydroxyapatite powder in distilled water, controlling the solid-to-liquid ratio to be about 20/80, performing ultrasonic dispersion for 15min at low temperature, rapidly adding the nano hydroxyapatite powder to the surface of the collagen pre-cooled at low temperature in a refrigerator at-80 ℃, standing overnight at 4 ℃, transferring the collagen to liquid nitrogen for freezing for 2h, and performing vacuum freeze drying to obtain the porous collagen-hydroxyapatite scaffold multilayer composite material.
The above detailed description of the multilayered collagen scaffold material and the method for preparing the same and the use thereof with reference to the specific embodiments is illustrative and not restrictive, and several examples may be cited within the scope of the present invention, so that variations and modifications thereof may be made without departing from the general concept of the present invention and the scope thereof is intended to be covered by the appended claims.
Claims (3)
1. A bionic multilayer collagen scaffold for cartilage repair is characterized by having a bionic multilayer three-dimensional space structure and comprising an upper layer and a basal layer; wherein, the layers are transversely and parallelly superposed, the upper layer is a collagen layer with a compact structure, and the basal layer is a collagen layer with a longitudinal cross-linked porous structure; the collagen is extracted and purified from pig tendon collagen, wherein the content of collagen is more than 99 percent, the content of polysaccharide is less than 0.1 percent, and the percentages are mass percentages; a hydroxyapatite barrier layer is compounded on the other end of the substrate layer relative to the compact layer;
the preparation method of the bionic multilayer collagen scaffold comprises the following steps:
step 1, crushing the stripped tendon, and adding 10-fold volume of 4M guanidine hydrochloride, wherein the guanidine hydrochloride is prepared from 0.05M Tris-HCl with the pH value of 7.5;
suspending, crushing and homogenizing, stirring at 4 ℃ for 24h, centrifuging at 12000g for 20min, and separating to obtain a supernatant and a precipitate;
step 3, fully washing the precipitate obtained in the step 2 with Tris-HCl buffer solution and 0.5mM acetic acid, and then adding acetic acid for overnight to remove proteoglycan;
step 4, dissolving the mixture by using a glacial acetic acid solution containing pepsin, continuously treating the mixture for a period of time until the mixture becomes colorless, transparent and viscous liquid, centrifuging the mixture for 20min at the temperature of 4 ℃ under the centrifugal force of 5000g, and collecting supernatant to obtain crude tendon collagen stock solution;
step 5, adding the sodium chloride solution into the solution, continuously stirring until white flocculent precipitate is separated out from the solution, and continuously adding the sodium chloride solution until the precipitate is not separated out;
step 6, centrifuging for 20min at the temperature of 4 ℃ and under the centrifugal force of 5000g to obtain white precipitate;
step 7, adding an acid solution into the precipitate obtained in the step 6 for dissolving, and continuously and repeatedly carrying out salting-out treatment on the dissolved collagen solution for 2-4 times;
step 8, continuously dialyzing in ultrapure water for 3 days, and changing the liquid for 3 times every day to remove inorganic salts in the collagen solution;
step 9, freezing and molding the tiled 4mg/mL collagen solution, soaking and dehydrating the collagen solution by using a hypertonic buffer solution, and extruding the collagen solution to form a film with the thickness of 0.1-0.5 mm;
step 10, spreading a layer of collagen solution on the surface of the collagen obtained in the step 9, wherein the thickness of the collagen solution is 2.0-5.0 mm, and moving the collagen solution into a refrigerator with the temperature of minus 80 ℃ for precooling for 2 hours at low temperature;
and 11, suspending the nano hydroxyapatite powder in distilled water, controlling the solid-to-liquid ratio at 20/80, performing ultrasonic dispersion for 15min at low temperature, quickly adding the nano hydroxyapatite powder to the surface of the longitudinal porous structure collagen precooled at low temperature in a refrigerator at-80 ℃ in the step 10, standing overnight at 4 ℃, then transferring the collagen surface to liquid nitrogen for freezing for 2h, and finally performing vacuum freeze drying to obtain the porous collagen-hydroxyapatite scaffold composite material.
2. A biomimetic multilayer collagen scaffold according to claim 1, wherein the thickness of the upper dense layer is 0.3 mm and the thickness of the base layer is 2.5 mm.
3. Use of a biomimetic multilayer collagen scaffold according to claims 1-2 for the preparation of a cartilage repair device.
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