CN110237306A - Preparation method of bionic defect-filling material for repairing cartilage defect of knee joint - Google Patents
Preparation method of bionic defect-filling material for repairing cartilage defect of knee joint Download PDFInfo
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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
- A61L27/02—Inorganic materials
- A61L27/12—Phosphorus-containing materials, e.g. apatite
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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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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/56—Porous materials, e.g. foams or sponges
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y10/00—Processes of additive manufacturing
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y70/00—Materials specially adapted for additive manufacturing
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y80/00—Products made by additive manufacturing
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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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
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Abstract
The technical scheme of the preparation method of the bionic defect-filling material for repairing the cartilage defect of the knee joint comprises the following steps: step one, preparing a type I collagen solution and a type II collagen solution; step two, concentrating and measuring a type I collagen solution and a type II collagen solution, selecting the type I collagen solution and the type II collagen solution with different concentrations to prepare biological ink, and simulating components of different structural layers of the knee joint to add different materials as the biological ink; step three, 3D printing is carried out according to a pre-constructed computer model of the cartilage defect filling block of the knee joint with an internal anatomical structure and an external macroscopic structure, and biological ink is sprayed and printed to an ultralow-temperature copper plate to obtain a frozen block of the collagen scaffold; and step four, according to a thermal phase separation principle, carrying out vacuum freeze drying on the frozen block of the collagen scaffold, and finally preparing the collagen scaffold which is similar to the structure and components of the normal articular cartilage and has three structural layers (different holes and different components), wherein the collagen scaffold can be used for filling the articular cartilage defect of the knee joint.
Description
Technical field
Present invention relates particularly to a kind of for repairing the preparation method of the bionical material of filling a vacancy of defects of knee, passes through
Thermal inactive technical controlling 3d printed material hole configurations.
Background technique
Concentrated reflection of the 3D printing as digitizing technique is a kind of technology that manufacturing theory is successively accumulated based on material,
It is the various bone surgery individuations of realization, the effective means of precision, and the hot spot direction of current orthopaedics implant research.With
The development of 3D printing technique, can be patient " customized " high-precision operation plan and implant, make operation more rapidly, essence
Really, to improve the success rate of complicated operation.
3D printing technique have developed rapidly in recent years, especially obtain good development in orthopaedics and the department of stomatology, but now
Compare successful story all to concentrate in the product printing of single organization's structure.Compared with single bone tissue, articular cartilage at
Divide and structure is complex.Articular surface is wrapped by hyaline cartilage, is divided into hyaline cartilage layer, cartilaginous calcification layer from outside to inside
And subchondral bone, each interlayer are connect with close structure with distinctive form and are had different function.Therefore, qualification is printed
Bionic joint cartilage will also solve to mend defect three-dimensional in the technical foundation of the single bone material maturation of existing 3D printing
Problem is merged in the printing of the secondary Problems of Reconstruction of model and a variety of 3D printing materials.
3D printing can only control the structure of mm-cm, and current technology not can control the final μm-nm structure of printed material,
That is the hole configurations of material.And different pore sizes with hole configurations it is verified that can inducing mesenchymal stem cell it is different
Differentiation direction, and different cells is suitble to grow.The aperture of usual 200 μm of sizes is more suitable for the proliferation and life of cartilage cell
It is long, and the aperture of 50 μm of sizes is then more suitable for the proliferation and growth of osteoblast.
Thermally induced phase separation (Thermally InducedPhase Separation, TIPS) can be applied to it is many by
In the polymer that poor solubility cannot solve micro molding with wet, dry phase disengagement method.From substantially saying, TIPS process is just
It is that a kind of potential solvent is utilized, is non-solvent when being solvent at high temperature and low temperature, loses the immiscible of solvent power
Property be because lose thermal energy (i.e. hot as the driving force mutually separated).Because potential solvent be it is non-volatile, need with pair
It is solvent, is that a kind of liquid of non-solvent extracts it from finished product for polymer in potential solvent, to is formed micro-
Pore structure.This method is most blanket method in all phase disengagement methods, can be not only used for polar polymer, it can also be used to non-
Polar polymer；The parameter that this method needs to control in forming process is less, process stabilization easy to accomplish and continuous；And root
According to existing material, it is varied that TIPS process is formed by microstructure, it is easier to meet the particular/special requirement of poromerics；
In addition there is thermally induced phase separation that can generate respectively to identical microcellular structure on thick section, this characteristic makes TIPS method big in preparation
There is unique advantage in terms of volume of tissue engineering scaffold material.
The 3d of the interfacial structure of complex organization is printed, and is equally that the advantage of 3d printing technique is utilized to realize.Joint is soft
The calcified cartilage layer of bone structure between hyaline cartilage layer and subchondral bone, by the wavy damp cable architecture at interface thereon with
Hyaline cartilage layer closely connects, and bonds cable architecture by the rough and uneven in surface comb teeth-shaped at its lower interface and the mutual anchor of subchondral bone closes；
Special interface connection type is the connection area increased between articular cartilage interface in this, while it is strong to also increase connection
Degree.By traditional almost impossible this structure of reconstruction of organizational project, and the research of current 3D printing lays particular emphasis on bracket more
Apparent size, but the research for printing complicated internal structure is less.
The present invention addresses the above problem, and in conjunction with domestic and international present Research, the main component of articular cartilage tissue is made
It is constructed and articular cartilage tissue ingredient for bio-ink using 3D model reconstruction technology, 3D printing technique and bionics principle
Structure is identical, with host tissue integration frozen block, it is good to obtain a kind of repetitive rate, can industrialization repairing articular cartilage lack
The alternative material of damage.
Summary of the invention
The present invention provides a kind of preparation method of bionical material of filling a vacancy for repairing defects of knee, and can prepare has
Three-decker layer (three kinds of hole sizes and three kinds of components).
The technical solution of preparation method that the present invention mentions a kind of bionical material of filling a vacancy for repairing defects of knee includes:
Step 1: preparation Type I collagen solution and II Collagen Type VI solution；
Step 2: the concentration and measurement of Type I collagen solution and II Collagen Type VI solution, select the Type I collagen of various concentration molten
Liquid and II Collagen Type VI solution prepare bio-ink；
Step 3: select collagen as main bio-ink, knee joint hyaline cartilage layer, calcified cartilage layer and soft are simulated
The main component of osteoplaque adds different materials under bone, according to the computer of the defects of knee dutchman built in advance
Model (there is internal anatomy and external macrostructure) progress 3D printing, the layer-by-layer spray printing of bio-ink to ultralow temperature copper sheet,
Obtain consistent frozen block inside and outside a kind of and computer model；Since to build internal anatomy model in advance (transparent soft for computer
Osteoplaque, calcified cartilage layer and subchondral bone layer and interfacial structure), it can print out similar to three layers of different structure sheaf of knee joint；
Due to the different bio-ink of different structure layer choosing, the frozen block of three kinds of different components can be prepared；
Step 4: the frozen block printed is freeze-dried, due to having selected different collagen concentrations, can be obtained after freeze-drying
To different hole sizes；Finally, it is imitative that one kind with three-decker layer (three kinds of hole sizes and three kinds of components) can be prepared
Raw material of filling a vacancy, for filling knee joint endoprosthesis cartilage defect.
Preferably, in the above-mentioned methods,
In step 1, the preparation method of Type I collagen solution includes: separation pig tendon, and distilled water is cleaned, shreds and claim
Weight；Washing: being dipped in distilled water after the tendon shredded is washed 2-3 times with distillation, sets 4 DEG C of refrigerators 4 hours for use；Acid is molten:
Tendon moisture is blotted with filter paper, is dissolved in the 0.5M acetic acid of 100 times of bulking values, 4 DEG C of refrigerators is set and places 48 hours, and constantly shake
It swings；It saltouts: acid solution is centrifuged, take supernatant that 10% isometric sodium chloride solution is added after centrifugation, set 4 DEG C of refrigerators and saltout
Overnight, remaining sediment abandons it；Sour molten again: the liquid that will saltout is centrifuged, and takes precipitating that the 0.5M acetic acid of 6 times of volumes is added after centrifugation
It is molten to carry out again hypo acid, setting after 4 DEG C of refrigerators dissolve 4 hours becomes collagen extracting solution；Dialysis: by collagen extracting solution high speed
Low-temperature precipitation takes upper layer collagen solution, sets bag filter and dialyses to PH5.5 packing, -30 DEG C of preservations.
Preferably, in the above-mentioned methods,
In step 1, the preparation method of II Collagen Type VI solution includes: to cut hyaline cartilage, is thinly sliced, and degreasing is smash
Broken, system homogenate, then for 24 hours with 4M (pH7.5) the guanidine hydrochloride stirring of 10 times of volumes, centrifuge separation, precipitating is used after sufficiently washing
Pepsin digests 24~28h, centrifugation in acid condition, and supernatant is saltoutd with NaCL after EDTA terminates enzymatic hydrolysis, used again
Acetate dissolution, NaOH are neutralized, the collagen solution being collected into are adjusted to acidity, is saltoutd, and acid is molten, to water dialysis desalting, are prepared into II
Collagen Type VI solution.
Preferably, in the above-mentioned methods,
In step 2, the total protein content in Type I collagen solution and II Collagen Type VI solution is measured using bca method.
Preferably, in the above-mentioned methods,
In step 2, Type I collagen solution and II Collagen Type VI solution are concentrated through polyethylene glycol.
According to the method described in claim 5, it is characterized in that, through polyethylene glycol by Type I collagen solution and II Collagen Type VI
Solution concentration specifically includes: by the Type I collagen solution and II Collagen Type VI solution injection bag filter after extraction, sealing is placed on burning
In cup, pours into polyethylene glycol pulvis and cover entire bag filter, be concentrated at 4 DEG C.
Preferably, in the above-mentioned methods,
In step 3, hyaline cartilage layer choosing selects the II Collagen Type VI solution of concentration 5mg/ml as bio-ink；Calcification is soft
Osteoplaque select concentration 10mg/ml II Collagen Type VI solution and hydroxyapatite mixture as bio-ink；Subchondral bone layer
For the mixture of the Type I collagen solution and hydroxyapatite that select concentration 15mg/ml as bio-ink, each bio-ink collagen is dense
Degree is different.
Preferably, in the above-mentioned methods,
In step 3, knee cartilage computer model is converted into the executable mimeograph documents of 3D printer, it is different
Structure sheaf chooses corresponding spray head and bio-ink, by bio-ink spraying on -40 DEG C of low temperature copper sheet, due to the biology of ejection
Ink thickness be no more than 100 μm, can quick solidification, successively print and the consistent freezing of computer model appearance and internal structure
Block.
Preferably, in the above-mentioned methods,
In step 4, after the frozen block printed -80 DEG C of refrigerators of placement are continued freezing 24 hours, thermotropic phase is recycled
Separation principle is put into vacuum freeze drier and carries out freeze-drying 24 hours, the water sublimed in frozen block, due to bio-ink
Middle collagen concentration is different, can form different hole sizes internal；Finally, prepare similar to knee joint hyaline cartilage layer,
The bionical material of filling a vacancy of one kind of calcified cartilage layer and the knee cartilage of subchondral bone layer three-decker and three kinds of components.
In the above-mentioned technical solutions, different structure layer selects different bio-inks by simulating cartilaginous element, with building
Induction host's Derived from Mesenchymal Stem Cells direction and the microenvironment for being suitble to different cell growths；In addition, realizing group by 3D printing
The printing for knitting complicated interface structure between internal different structure layer is kept away by selecting different types of collagen as bio-ink
The additional bonds or chelating for exempting from different structure interlayer, bionical material size and the knee joint defect dutchman of filling a vacancy finally prepared
Computer model is consistent, and inside has three layers of different hole sizes and different component, and different interfacial structures.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
The present invention it is a kind of repair defects of knee bionical material of filling a vacancy preparation method specifically includes the following steps:
Step 1: the preparation of Type I collagen solution and II Collagen Type VI solution.
In view of mainly containing Type I collagen in tendon, II Collagen Type VI is mainly contained in cartilage, then respectively from pig tendon and thoroughly
I type and II Collagen Type VI are prepared in bright cartilage.
Type I collagen preparation method generally includes: separation pig tendon, distilled water are cleaned, shred and weigh；Washing: it will cut
Broken tendon is dipped in distilled water after being washed 2-3 times with distillation, sets 4 DEG C of refrigerators 4 hours for use；Acid is molten: blotting tendon with filter paper
Moisture is dissolved in the 0.5M acetic acid of 100 times of bulking values, is set 4 DEG C of refrigerators and is placed 48 hours, and constantly shakes；It saltouts: acid is molten
Liquid is centrifuged, (- 8 DEG C of 10000rpm × 30min × 4 DEG C), takes supernatant that 10% isometric sodium chloride solution is added after centrifugation,
4 DEG C of refrigerators are set to saltout overnight (remaining sediment abandons it)；Sour molten again: the liquid that will saltout is centrifuged, (4000rpm × 10Min × 4
DEG C -8 DEG C), taking precipitating that the 0.5M acetic acid of 6 times of volumes is added after centrifugation, to carry out again hypo acid molten, set after 4 DEG C of refrigerators dissolve 4 hours at
For collagen extracting solution；Dialysis: collagen extracting solution high-speed low temperature being precipitated, upper layer collagen solution is taken, and sets bag filter dialysis
It is dispensed to PH5.5, -30 DEG C of preservations.
The preparation method of II Collagen Type VI generally includes: cutting hyaline cartilage, thinly slices, degreasing smashs to pieces, makes homogenate, so
Afterwards for 24 hours with 4M (pH7.5) the guanidine hydrochloride stirring of 10 times of volumes, centrifuge separation, precipitating is after sufficiently washing with pepsin in acid
Property under the conditions of digest 24~28h, centrifugation, supernatant is saltoutd after EDTA terminates enzymatic hydrolysis with NaCL, again with acetate dissolution, NaOH
It neutralizes, the collagen solution being collected into is adjusted to acidity, is saltoutd, acid is molten, to water dialysis desalting, is prepared into II Collagen Type VI solution.
Step 2: the concentration and measurement of Type I collagen solution and II Collagen Type VI solution.
Type I collagen solution and II Collagen Type VI solution are concentrated through polyethylene glycol.
Specifically: the collagen solution injection after extraction is about 12cm, diameter is about every bag of 10ml in the bag filter of 5cm.
Sealing is placed in beaker, is poured into polyethylene glycol (molecular weight 6000) pulvis and is covered entire bag filter, is concentrated at 4 DEG C, poly- second
Glycol collects the collagen solution after concentration since the part water in collagen solution can be sucked out for water imbibition, after 1.5h, measures albumen
After concentration, after adding weak acid to adjust PH, it is concentrated again using polyethylene glycol, until reaching required concentration.5mg/ml, 10mg/ml,
It is obtained after the multiple sour molten-concentration of the collagen solution of 15mg/ml.
Total protein content in Type I collagen solution and II Collagen Type VI solution is measured using bca method.
Step 3: the Type I collagen solution of various concentration and II Collagen Type VI solution is selected to prepare bio-ink, and simulate knee pass
The main component for saving hyaline cartilage layer, calcified cartilage layer and subchondral bone layer adds different material.
Since knee joint is divided into hyaline cartilage, calcified cartilage layer and subchondral bone three-decker towards deep from joint, respectively
Boundary clear between layer structure.
Since hyaline cartilage is mainly made of cartilage cell and the extracellular matrixs such as typeⅡ Collagen and proteoglycan, then
Hyaline cartilage selects II Collagen Type VI solution (concentration 5mg/ml) as bio-ink.
Calcified cartilage layer is mainly by a small amount of cartilage cell and the extracellular matrixs group such as typeⅡ Collagen and hydroxyapatite
At as biological ink after then calcified cartilage layer selects II Collagen Type VI solution (concentration 10mg/ml) and hydroxyapatite directly to mix
Water, II Collagen Type VI solution and hydroxyapatite mass ratio 3:1.
Subchondral bone layer is mainly made of osteoblast and the extracellular matrixs such as Type I collagen albumen and hydroxyapatite, then
Subchondral bone layer choosing is selected after Type I collagen solution (concentration 15mg/ml) and hydroxyapatite directly mix as bio-ink, I type
Collagen solution and hydroxyapatite mass ratio 1:1.
Since the main component of simulation knee joint hyaline cartilage layer, calcified cartilage layer and subchondral bone layer uses different lifes
Object ink, controllable frozen block inside different structure layer have different components.
Step 4: having internal anatomy (under hyaline cartilage layer, calcified cartilage layer and cartilage according to what is built in advance
Osteoplaque and interfacial structure) and the computer model of defects of knee dutchman of external macrostructure carry out 3D printing, it is raw
The layer-by-layer spray printing of object ink obtains and the consistent frozen block of computer model appearance and internal structure to ultralow temperature copper sheet.
Using more nozzle printings, the bio-ink of different structure layer is added in print cartridge, syringe needle precision is 100 μm, different
Structure sheaf can select different bio-inks according to the difference of spray head.
In print procedure, according to computer instruction, bio-ink is squeezed out on -40 DEG C of temperature of ultralow temperature copper sheet,
Since copper plate temperature is extremely low, spray head is every to spray one layer of bio-ink thickness no more than 100 μm, can freeze immediately.
If there is different bio-inks in same layer, is sprayed by different syringe needles, print next layer after the completion, pass through
3D printing controls the interfacial structure between the freezing block structure of mm-um and different structure layer, until completing, finally prints one kind
With frozen block consistent inside and outside computer model.
Since computer builds internal anatomy model in advance, it can print out three layers of different structure sheaf；Due to different knots
The different bio-ink of structure layer choosing, the frozen block printed have different structure layer, and different structure layer has different component.
Step 5: the frozen block printed is freeze-dried, due to having selected different collagen concentrations, can be obtained after freeze-drying
To different hole sizes, collagen concentration is higher, and the aperture formed after vacuum freeze drying is smaller, and vice versa；Finally, it can make
Standby different structure layer out has the bionical material of filling a vacancy of different three kinds of hole sizes and different component, to close for filling knee joint
Save cartilage defect.
By Thermal inactive technology, there are different apertures, the aperture knot of control μm-nm under various concentration using collagen
Structure.
Printed frozen block is placed -80 DEG C of refrigerators to continue after freezing 24 hours, Thermal inactive principle is recycled to put
Enter vacuum freeze drier and carry out freeze-drying 24 hours, the water sublimed in frozen block is directly sublimed into freeze-drying process
Gas, due to the difference of collagen concentration, the difference of moisture content forms the hole configurations in different size aperture, finally prepares
The bionical material of filling a vacancy for being used to repair defects of knee with different hole sizes.
Bionical material of filling a vacancy has the hole configurations of internal mutual Communicating, and collagen concentration is higher, the inside aperture after freeze-drying
It is smaller, there is the defects of knee of external macroscopic view to fill a vacancy block structure, also there is Normal Knee interior laminate layer structure and not
With the interfacial structure between structure sheaf.
Due to knee joint include three layers of different structure, each layer of structure have different ingredients, different hole sizes and
Similar to the interfacial structure between normal configuration layer, breaks up and be conducive to different directions to be conducive to host's mescenchymal stem cell
Cartilage cell, fibrocartilage cells and the osteoblast of host is to the migration of freezing block of material and adheres to.
The above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although with reference to the foregoing embodiments
Invention is explained in detail, those skilled in the art should understand that: it still can be to aforementioned each implementation
Technical solution documented by example is modified or equivalent replacement of some of the technical features；And these modification or
Replacement, the spirit and scope for technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution.
Claims (9)
1. a kind of for repairing the preparation method of the bionical material of filling a vacancy of defects of knee, which is characterized in that including following
Step:
Step 1: preparation Type I collagen solution and II Collagen Type VI solution；
Step 2: the concentration and measurement of Type I collagen solution and II Collagen Type VI solution, select various concentration Type I collagen solution and
II Collagen Type VI solution prepares bio-ink, and simulates the component of knee joint hyaline cartilage layer, calcified cartilage layer and subchondral bone layer
Add different materials；
Step 3: filling a vacancy according to the defects of knee with internal anatomy and external macrostructure built in advance
The computer model of block carries out 3D printing, and the layer-by-layer spray printing of bio-ink to ultralow temperature copper sheet obtains and computer model appearance one
The frozen block of cause；
Step 4: the frozen block printed is freeze-dried to obtain different hole sizes, preparing different structure layer has not
Bionic knee joint cartilage defect with three kinds of hole sizes and different component is filled a vacancy material, for filling knee joint endoprosthesis cartilage
Defect.
2. the method according to claim 1, wherein in step 1, the preparation method packet of Type I collagen solution
Include: separation pig tendon, distilled water are cleaned, shred and weigh；Washing: it is dipped in after the tendon shredded is washed 2-3 times with distillation
In distilled water, 4 DEG C of refrigerators are set 4 hours for use；Acid is molten: blotting tendon moisture with filter paper, is dissolved in the 0.5M vinegar of 100 times of bulking values
In acid, sets 4 DEG C of refrigerators and place 48 hours, and constantly shake；It saltouts: acid solution is centrifuged, supernatant addition etc. is taken after centrifugation
10% sodium chloride solution of volume sets 4 DEG C of refrigerators and saltouts overnight, and remaining sediment abandons it；It is sour molten again: the liquid that will saltout carry out from
The heart, taking the 0.5M acetic acid of precipitating 6 times of volumes of addition to carry out again after centrifugation, hypo acid is molten, and setting after 4 DEG C of refrigerators dissolve 4 hours becomes collagen
Protein extract；Dialysis: collagen extracting solution high-speed low temperature is precipitated, upper layer collagen solution is taken, sets bag filter and dialyse to PH5.5
Packing, -30 DEG C of preservations.
3. the method according to claim 1, wherein in step 1, the preparation method packet of II Collagen Type VI solution
It includes: cutting hyaline cartilage, thinly slice, degreasing smashs to pieces, makes homogenate, is then stirred with 4M (pH7.5) guanidine hydrochloride of 10 times of volumes
For 24 hours, it is centrifugated, precipitating digests 24~28h, centrifugation, supernatant warp in acid condition after sufficiently washing with pepsin
EDTA is saltoutd after terminating enzymatic hydrolysis with NaCL, uses acetate dissolution again, and NaOH is neutralized, and the collagen solution being collected into is adjusted to acidity,
It saltouts, acid is molten, to water dialysis desalting, is prepared into II Collagen Type VI solution.
4. the method according to claim 1, wherein measuring Type I collagen solution using BCA method in step 2
With the total protein content in II Collagen Type VI solution.
5. the method according to claim 1, wherein in step 2, through polyethylene glycol by Type I collagen solution and
The concentration of II Collagen Type VI solution.
6. according to the method described in claim 5, it is characterized in that, through polyethylene glycol that Type I collagen solution and II Collagen Type VI is molten
Liquid concentration specifically includes: by the Type I collagen solution and II Collagen Type VI solution injection bag filter after extraction, sealing is placed on beaker
In, it pours into polyethylene glycol pulvis and covers entire bag filter, be concentrated at 4 DEG C.
7. the method according to claim 1, wherein hyaline cartilage layer choosing selects concentration 5mg/ml in step 3
II Collagen Type VI solution as bio-ink；The II Collagen Type VI solution and hydroxy-apatite of calcified cartilage layer selection concentration 10mg/ml
The mixture of stone is as bio-ink；Subchondral bone layer choosing selects the Type I collagen solution of concentration 15mg/ml and mixing for hydroxyapatite
Object is closed as bio-ink.
8. the method according to claim 1, wherein in step 3, by defects of knee dutchman
Computer model is converted to the executable mimeograph documents of 3D printer, and different structure layer choosing takes corresponding spray head and bio-ink, will
Bio-ink sprays on -40 DEG C of low temperature copper sheet, and every layer of bio-ink thickness of every layer of ejection is no more than 100 μm, successively beats
It prints off and the consistent frozen block of computer model appearance and internal structure.
9. according to the method described in claim 8, it is characterized in that, the frozen block printed is placed -80 DEG C in step 4
After refrigerator continues freezing 24 hours, recycling Thermal inactive principle to be put into vacuum freeze drier, to carry out freeze-drying 24 small
When, the water sublimed in frozen block that prints simultaneously forms different hole sizes inside frozen block；Finally, preparing has
Similar to the bionical benefit of defects of knee of knee joint hyaline cartilage layer, calcified cartilage layer and subchondral bone layer structure and component
Lack material.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910378353.8A CN110237306A (en) | 2019-05-08 | 2019-05-08 | Preparation method of bionic defect-filling material for repairing cartilage defect of knee joint |
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