CN107537066A - A kind of bionical cartilage and its manufacture method based on 3D printing - Google Patents
A kind of bionical cartilage and its manufacture method based on 3D printing Download PDFInfo
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Abstract
The invention belongs to biomedical engineering technology field, particularly biomimetic material preparing technical field, a kind of bionical cartilage and its manufacture method based on 3D printing are disclosed, contains the manufacture method of a kind of bionical cartilage based on 3D printing and a kind of bionical cartilage of 3D printing.A kind of described bionical cartilage based on 3D printing is sandwich construction, as obtained by modified typeⅡ Collagen, modified hyaluronic acid, nanometer hydroxyapatite and cartilage cell by the bionical 3D printing of gradient.This kind of bionical cartilage has good biocompatibility, the mechanical property suitable with normal articular cartilage, is advantageous to the reparation of cartilage defect portion.
Description
Technical field
The invention belongs to biomedical engineering technology field, more particularly to a kind of bionical cartilage based on 3D printing and its
Manufacture method.
Background technology
Cartilage degeneration or damage are clinically relatively conventional as caused by wound or disease, but due to articular cartilage tissue
Special, inside have blood vessel, lymphatic vessel and a nerve, only has extremely low after traumatic damage or does not almost review one's lessons by oneself reactivation effectively
Power, if treatment is not in time or appropriately, serious dysfunction will be caused.It is identical with other process of tissue reparation, repair of cartilage
It is by normal cartilage, the marrow below defect or the synovial tissue around the formation of fibrin clot and subsequent defect damage
The cascade reaction of the chondroblast in source, ultimately form the repair tissue of damaged part.Existing several cartilage repair techniques
All it is tissue and marrow that defect has blood supply close under cartilage, generally connection as drilled under cartilage, clearing up Drilling or micro fractures
Close application and remove cartilage fragment, the operations such as wear joints are molded and joint is rinsed.But conventional art is by fibrocartilage sample group
Reparation cartilage defect is knitted, is formed, in terms of structure and mechanical property in composition, this repair tissue is different from normal cartilage（Normally
Articular cartilage is mainly made up of hyaline cartilage, and external stress is resisted as cushion pad）, thus unpredictable repair of cartilage is long-term
Clinical effectiveness.The patient treated by traditional approach, can there is an of short duration pain relief phase；But work as repair tissue
The symptom of progressivity occurs in regression, patient.Defect is repaired with appropriate cartilage packing material, is fast quick-recovery morbid state or wound
The effective ways of wound property cartilage defect tissue.The cartilage tissue engineered treatment for cartilage damage provides a kind of preferably treatment way
Footpath, but existing technology is difficult to prepare high quality similar in a kind of and normal articular cartilage physiological structure and physiological property
Cartilage repair material, there are many problems to have to be solved, it is immune after the fixation, homogeneous variant cell transplanting such as neocartilage tissue
The selection of rejection, seed cell and growth factor, how to solve the at a specified future date of cultured cartilage and degenerate, whether can endure for a long time
Load and friction etc..
And in recent years, design of the researcher to tissue engineering bracket proposes 4F criterions：Shape demand（Form）, performance tells
Ask（Function）, function demand（Formation）With it is implantable（Fixation）.Shape demand refers to that timbering material is necessary
The 3 D defects of complexity can be filled up completely with, and can be with inducing tissue regeneration；Performance demand refers to mechanical property of support etc.
The demand of missing tissues effect can be temporarily played before missing tissues reparation；It is corresponding raw that function demand refers to that material need to have
Thing activity, suitable environment can be provided for cell and is regenerated with promotion organization；It is implantable to refer to that support be implanted into by performing the operation
Human body, and play expected effect.Using bionics, external structure is adapted to the microstructure of tissue cell growth, analogue body inner ring
Border, coordinate propagation, differentiation, migration and apoptosis of different cells etc., be the developing direction for treating tissue defect.
The personalized support prepared using 3D printing techniques, it can accurately simulate the complicated three-dimensional microcosmic knot of natural tissues
Structure, support shape are identical with defective tissue, and can be by supporting the common printing imparting support of growth factor, cell biological
Activity, the method printed by gradient simulate the physiological structure of normal structure, so as to prepare a kind of and normal structure in structure
It is the inexorable trend that field of tissue engineering technology develops with the repair materials being no different in physiological function.
The content of the invention
The shortcomings that primary and foremost purpose of the present invention is to overcome prior art and deficiency, a kind of bionical cartilage based on 3D printing
Preparation method.
Another object of the present invention is to provide obtained by above-mentioned preparation method it is a kind of bionical soft based on 3D printing
Bone, it is characterised in that described bionical cartilage is counted by modified typeⅡ Collagen 20-80 parts, modified hyalomitome in parts by weight
Sour 20-80 parts, nanometer hydroxyapatite 10-20 parts and cartilage cell 0.1-0.5 parts pass through obtained by the bionical 3D printing of gradient.
Further, the bionical cartilage is deep by bionical subchondral bone, bionical calcified layer, bionical cartilage successively from the bottom to top
As obtained by 3D printer printing structure, overall is in the inierpeneirating network structure of multilayer for layer, bionical intermediate layer and bionical superficial layer.
Described a kind of bionical cartilage and its manufacture method based on 3D printing, specifically comprises the following steps；
（1）The preparation of printed material：
1. the preparation of modified typeⅡ Collagen：TypeⅡ Collagen is added in 50 DEG C of PBS phosphate and is configured to quality
Concentration is 10% solution, then V typeⅡ Collagens PBS phosphate solutions by volume：V methacrylic anhydride=10:0.75
Add methacrylic anhydride to exist, stirring reaction 4 hours at 50 DEG C, after reaction terminates, resulting solution is placed in into molecular cut off is
Dialysed 7 days in 10000 bag filter, then obtained dialyzate be placed in -80 DEG C of freeze driers and is freeze-dried 48 hours,
Modified typeⅡ Collagen is produced, is placed in and stores at room temperature, it is standby；
2. the preparation of modified hyaluronic acid：Hyaluronic acid is dissolved in the hyalomitome for being configured to that mass concentration is 2% in distilled water
Acid solution, the pH to 8.5 for then adding the sodium hydroxide solution regulation solution that molar concentration is 1 mol/L obtain Sodium Hyaluronate
Solution, then m Sodium Hyaluronates in mass ratio：M dimethylaminoethyl methacrylate=1:3.5 add first methacrylic acid
Dimethylaminoethyl, after reacting 24h at 10 DEG C, resulting solution is placed in flowing water in the bag filter that molecular cut off is 14000
Dialyzate, is then placed in -80 DEG C of freeze driers and is freeze-dried 48 hours, produce modified hyaluronic acid, put by dialysis 7 days
Store at room temperature, it is standby；
3. after cartilage cell is digested, it is added in the cultured chondrocytes base containing the cow's serum that volume fraction is 10%, matches somebody with somebody
Cell concentration is made as 1 × 106—5×106mL-1Chondrocyte cell suspension, be placed in gas concentration lwevel as 5%, temperature 37
DEG C cell culture incubator in it is standby；The cartilage cell is the cartilage cell after 3 generations of normal culture；
（2）The preparation of each bionical layer presoma：
1. the preparation of bionical subchondral bone presoma：Light trigger Irgacure2959, step are sequentially added into distilled water（1）
The modification typeⅡ Collagen of preparation, the material added every time add a kind of lower material, then add nanometer after being completely dissolved
Hydroxyapatite, stirs dispersed in the solution to nanometer hydroxyapatite, obtains bionical subchondral bone precursor solution, standby
With；Wherein, light trigger Irgacure2959 concentration be 0.5wt%, the concentration of nanometer hydroxyapatite be 5-10wt%, it is modified
The concentration of typeⅡ Collagen is 5-10 wt%；
2. the preparation of bionical calcified layer presoma：Light trigger Irgacure2959, step are sequentially added into distilled water（1）System
Standby modified hyaluronic acid, the material added every time add a kind of lower material, then add nano hydroxyapatite after being completely dissolved
Lime stone, stirs dispersed in the solution to nanometer hydroxyapatite, obtains bionical calcified layer precursor solution, standby；Wherein,
Light trigger Irgacure2959 concentration is 0.5wt%, the concentration of nanometer hydroxyapatite is 1-5wt%, modified hyaluronic acid
Concentration be 1-3 wt %；
3. the preparation of bionical cartilage deep layer presoma：Light trigger Irgacure2959, step are sequentially added into distilled water（1）
The modified hyaluronic acid of preparation, step（1）The modification typeⅡ Collagen of preparation, the material added every time add again after being completely dissolved
Enter a kind of lower material, then add isometric step（1）The chondrocyte cell suspension prepared, gentle agitation are equal to mixing
It is even, bionical cartilage deep layer precursor solution is obtained, it is standby；Wherein, light trigger Irgacure2959 concentration is 0.5wt%, changed
Property hyaluronic acid concentration be 1-3 wt %, the concentration of modified typeⅡ Collagen be 10-15wt%, and the concentration of cartilage cell is 5
×105—2.5×106mL-1；
4. the preparation of bionical intermediate layer presoma：Light trigger Irgacure2959, step are sequentially added into distilled water（1）System
Standby modified hyaluronic acid, step（1）The modification typeⅡ Collagen of preparation, the material added every time add after being completely dissolved
A kind of lower material, bionical intermediate layer precursor solution is obtained, it is standby；Wherein, light trigger Irgacure2959 concentration is
0.5wt%, the concentration of modified hyaluronic acid are 2-5 wt %, and the concentration of modified typeⅡ Collagen is 10-15wt%；
5. the preparation of bionical superficial layer presoma：Light trigger Irgacure2959, step are sequentially added into distilled water（1）System
Standby modification typeⅡ Collagen, the material added every time add a kind of lower material after being completely dissolved, obtain bionical superficial layer
Precursor solution, it is standby；Wherein, light trigger Irgacure2959 concentration is 0.5wt%, is modified the dense of typeⅡ Collagen
Spend for 10-15wt%；
（3）The bionical printing of bionical cartilage：
By step（2）Each bionical layer presoma for preparing gained is pressed bionical subchondral bone presoma, bionical calcified layer presoma, imitated
Raw cartilage deep layer presoma, bionical intermediate layer presoma, the order of bionical superficial layer presoma
Printing is sequentially overlapped, wherein the print thickness of the bionical subchondral bone is arranged to the 5 ~ 10% of bionical cartilage gross thickness, institute
The print thickness for stating bionical calcified layer is arranged to the 2 ~ 5% of the bionical cartilage gross thickness, print thickness of the bionical cartilage deep layer and set
Be set to the 15 ~ 30% of the bionical cartilage gross thickness, print thickness in the bionical intermediate layer be arranged to bionical cartilage gross thickness 40 ~
55%th, the print thickness of the bionical superficial layer is arranged to the 10 ~ 20% of bionical cartilage gross thickness, produces a kind of based on 3D printing
Bionical cartilage；
Preferably, the molecular weight of described typeⅡ Collagen is 1 × 106kDa；
Preferably, the molecular weight of described hyaluronic acid is 1.2 ~ 1.5 × 106kDa；
Preferably, described nanometer hydroxyapatite, its particle size distribution range are 150-250 nm；
Preferably, the bionical printing of described described bionical cartilage, using 3D systems companies model 3D systems
ProX 800 stereolithography apparatus method 3D printer is printed, and the ultraviolet light time for setting every layer of bionical layer is 30
S, ultraviolet light intensity are 10 mW/CM2；
Beneficial effects of the present invention are：
（1）Bionical cartilage disclosed by the invention has that immunogenicity is low, abundant raw materials, has good biocompatibility, good
The advantages of mechanical property and vascularization ability for getting well.
（2）The present invention is to be modified typeⅡ Collagen, modified hyaluronic acid, nanometer hydroxyapatite and cartilage cell as base
Matter, the bionical printed materials of 3D are prepared, then carry out bionical printing using three-dimensional printing technology, prepare with multi-gradient knot
The bionical cartilage of structure, not only overcome the problem of traditional cartilage repair material phase structure causes repairing effect bad, Er Qietong
Crossing can effectively facilitate the bionical cartilage that cartilage cell and printed material are printed after compound the healing at damaged cartilage position.
（3）Bionical cartilage prepared by the present invention, it is that the bionical printing of gradient is carried out using 3D printing technique, compared to traditional
The preparation method of tissue engineering bone/cartilage material, preparation method disclosed in this invention have support micro-structural precise forming, favorably
It in sticking for growth factor or cell, and can be modeled according to patient's damaged part actual conditions, personalization can be really achieved and controlled
Treat.
Brief description of the drawings
The mechanics properties testing experimental result compares figure that Fig. 1 is done by embodiment 1,2,3 and control group；
The Cytotoxic evaluation experiments experiment result compares figure that Fig. 2 is done by embodiment 1,2,3 and control group；
The alkaline phosphatase that Fig. 3 is done by embodiment 1,2,3 and control group（ALP）Test experience result compares figure；
The glycosaminoglycan that Fig. 4 is done by embodiment 1,2,3 and control group（GAG）Secretory volume test experience result compares figure.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are unlimited
In this.
The present invention is prepared one kind and is based on the bionical cartilage of 3D printing as follows：
（1）The preparation of printed material：
1. the preparation of modified typeⅡ Collagen：Weigh a certain amount of typeⅡ Collagen（Molecular weight is：1×106kDa）Add
Enter into 50 DEG C of PBS phosphate to be configured to the solution that mass concentration is 10%, then V typeⅡ Collagens PBS phosphorus by volume
Acid salt solution：V methacrylic anhydride=10:0.75 adds methacrylic anhydride, and stirring reaction 4 hours at 50 DEG C, reaction terminates
Afterwards, the solution that reaction obtains is placed in the bag filter that molecular cut off is 10000 and dialysed 7 days, the dialyzate that then will be obtained
It is placed in -80 DEG C of freeze driers and is freeze-dried 48 hours, produces modified typeⅡ Collagen, be placed in and store at room temperature, it is standby；
2. the preparation of modified hyaluronic acid：Weigh a certain amount of hyaluronic acid（Molecular weight is 1.2 ~ 1.5 × 106kDa）It is dissolved in
The hyaluronic acid solution that mass concentration is 2% is configured in distilled water, then adds the sodium hydroxide that molar concentration is 1 mol/L
Solution adjusts the pH to 8.5 of solution, then m Sodium Hyaluronates in mass ratio：M dimethylaminoethyl methacrylate=1:3.5
Dimethylaminoethyl methacrylate is added, after reacting 24h at 10 DEG C, is added to the bag filter that molecular cut off is 14000
Middle flowing water is dialysed 7 days, then will obtain dialyzate and be placed in -80 DEG C of freeze driers be freeze-dried 48 hours, produce it is modified thoroughly
Bright matter acid, is placed in and stores at room temperature, standby；
3. after cartilage cell is digested, the cultured chondrocytes base containing the cow's serum of volume fraction 10% is added, is configured to cell
Concentration is 1 × 106—5×106mL-1Chondrocyte cell suspension, be placed in gas concentration lwevel as 5%, temperature is 37 DEG C of cell
It is standby in incubator；The cartilage cell is the cartilage cell after 3 generations of normal culture；
（2）The preparation of each bionical layer presoma：
1. the preparation of bionical subchondral bone presoma：Light trigger Irgacure2959, step are sequentially added into distilled water（1）
The modification typeⅡ Collagen of preparation, the material added every time adds a kind of lower material after being completely dissolved, then toward gained
Nanometer hydroxyapatite is added in solution（Particle size distribution range is 150-250 nm）, stir completely equal to nanometer hydroxyapatite
It is even to disperse in the solution, to obtain bionical subchondral bone precursor solution, it is standby；Wherein, light trigger Irgacure2959's is dense
The concentration spent for 0.5wt%, nanometer hydroxyapatite is 5-10wt%, and the concentration of modified typeⅡ Collagen is 5-10 wt%；
2. the preparation of bionical calcified layer presoma：Light trigger Irgacure2959, step are sequentially added into distilled water（1）System
Standby modified hyaluronic acid, the material added every time add a kind of lower material after being completely dissolved, then toward in the solution of gained
Add nanometer hydroxyapatite（Particle diameter distribution is 150-250 nm）, stir to nanometer hydroxyapatite and be thoroughly dispersed in solution
In, bionical calcified layer precursor solution is obtained, it is standby；Wherein, light trigger Irgacure2959 concentration is 0.5wt%, nanometer
The concentration of hydroxyapatite is 1-5wt%, and the concentration of modified hyaluronic acid is 1-3 wt %；
3. the preparation of bionical cartilage deep layer presoma：Light trigger Irgacure2959, step are sequentially added into distilled water（1）
The modified hyaluronic acid of preparation, step（1）The modification typeⅡ Collagen of preparation, the material added every time add again after being completely dissolved
Enter a kind of lower material, then add isometric step（1）The chondrocyte cell suspension prepared, gentle agitation are equal to mixing
It is even, bionical cartilage deep layer precursor solution is obtained, it is standby；Wherein, light trigger Irgacure2959 concentration is 0.5wt%, changed
Property hyaluronic acid concentration be 1-3 wt %, the concentration of modified typeⅡ Collagen be 10-15wt%, and the concentration of cartilage cell is 5
×105—2.5×106mL-1；
4. the preparation of bionical intermediate layer presoma：Light trigger Irgacure2959, step are sequentially added into distilled water（1）System
Standby modified hyaluronic acid, step（1）The modification typeⅡ Collagen of preparation, the material added every time add after being completely dissolved
A kind of lower material, bionical intermediate layer precursor solution is obtained, it is standby；Wherein, light trigger Irgacure2959 concentration is
0.5wt%, the concentration of modified hyaluronic acid are 2-5 wt %, and the concentration of modified typeⅡ Collagen is 10-15wt%；
5. the preparation of bionical superficial layer presoma：Light trigger Irgacure2959, step are sequentially added into distilled water（1）System
Standby modification typeⅡ Collagen, the material added every time add a kind of lower material after being completely dissolved, obtain bionical superficial layer
Precursor solution, it is standby；Wherein, light trigger Irgacure2959 concentration is 0.5wt%, is modified the dense of typeⅡ Collagen
Spend for 10-15wt%；
（3）The bionical printing of bionical cartilage：
By step（2）Each bionical layer presoma for preparing gained is pressed bionical subchondral bone presoma, bionical calcified layer presoma, imitated
Raw cartilage deep layer presoma, bionical intermediate layer presoma, the order of bionical superficial layer presoma
Be sequentially overlapped printing, wherein the print thickness of bionical subchondral bone layer be arranged to bionical cartilage gross thickness 5 ~ 10%, it is bionical
Calcified layer is 2 ~ 5%, bionical cartilage deep layer be 15 ~ 30%, bionical intermediate layer be 40 ~ 55%, bionical superficial layer be 10 ~ 20%, produce
A kind of bionical cartilage based on 3D printing；
Embodiment 1
A kind of bionical cartilage based on 3D printing is prepared as stated above by the present invention, wherein：In the preparation of step printed material
The molecular weight of typeⅡ Collagen used is 1 × 106KDa, the molecular weight of hyaluronic acid used is 1.2 × 106kDa；Step
Used nanometer hydroxyapatite particle size distribution range is 150-250 nm in the preparation of each bionical layer presoma；Bionical soft
Dosage part of nanometer hydroxyapatite is 5 mass parts, the use of modified typeⅡ Collagen in the preparation process of bone sending down the fishbone presoma
The dosage measured as 5 mass parts, light trigger Irgacure2959 is 0.5 mass parts；The preparation process of bionical calcified layer presoma
The dosage of middle modified hyaluronic acid be 30 mass parts, nanometer hydroxyapatite with for 5 mass parts, light trigger
Irgacure2959 dosage is 0.5 mass parts；The use of modified hyaluronic acid in the preparation process of bionical cartilage deep layer presoma
The dosage measured as 25 mass parts, modified typeⅡ Collagen is 5 mass parts, the dosage of cartilage cell is 0.1 mass parts, light-initiated
Agent Irgacure2959 dosage is 0.5 mass parts；The use of modified hyaluronic acid in the preparation process of bionical intermediate layer presoma
The dosage measured as 25 mass parts, modified typeⅡ Collagen is 5 mass parts, light trigger Irgacure2959 dosage is 0.5
Mass parts；The dosage of modified typeⅡ Collagen is 5 mass parts, light trigger in the preparation process of bionical superficial layer presoma
Irgacure2959 dosage is 0.5 mass parts；The wherein bionical subchondral bone layer in the bionical printing step of bionical cartilage
Print thickness is arranged to that the 10% of bionical cartilage gross thickness, bionical calcified layer is 2%, bionical cartilage deep layer is 15%, bionical intermediate layer
It is 18% for 55%, bionical superficial layer；
Embodiment 2
A kind of bionical cartilage based on 3D printing is prepared as stated above by the present invention, wherein：In the preparation of step printed material
The molecular weight of typeⅡ Collagen used is 1 × 106KDa, the molecular weight of hyaluronic acid used is 1.2 × 106kDa；Step
Used nanometer hydroxyapatite particle diameter distribution is 150-250 nm in the preparation of each bionical layer presoma；Under bionical cartilage
In the preparation process of bone presoma dosage part of nanometer hydroxyapatite be 10 mass parts, the dosage of modified typeⅡ Collagen be
10 mass parts, light trigger Irgacure2959 dosage are 0.5 mass parts；Change in the preparation process of bionical calcified layer presoma
The dosage of property hyaluronic acid be 10 mass parts, nanometer hydroxyapatite with for 5 mass parts, light trigger Irgacure2959
Dosage is 0.5 mass parts；The dosage of modified hyaluronic acid is 15 mass parts, changed in the preparation process of bionical cartilage deep layer presoma
The dosage of property typeⅡ Collagen is 10 mass parts, the dosage of cartilage cell is 0.25 mass parts, light trigger
Irgacure2959 dosage is 0.5 mass parts；The dosage of modified hyaluronic acid in the preparation process of bionical intermediate layer presoma
Dosage for 15 mass parts, modified typeⅡ Collagen is 10 mass parts, light trigger Irgacure2959 dosage is 0.5 matter
Measure part；The dosage of modified typeⅡ Collagen is 10 mass parts, light trigger in the preparation process of bionical superficial layer presoma
Irgacure2959 dosage is 0.5 mass parts；The wherein bionical subchondral bone layer in the bionical printing step of bionical cartilage
Print thickness is arranged to that the 5% of bionical cartilage gross thickness, bionical calcified layer is 5%, bionical cartilage deep layer is 30%, bionical intermediate layer
It is 20% for 40%, bionical superficial layer；
Embodiment 3
A kind of bionical cartilage based on 3D printing is prepared as stated above by the present invention, wherein：In the preparation of step printed material
The molecular weight of typeⅡ Collagen used is 1 × 106KDa, the molecular weight of hyaluronic acid used is 1.2 × 106kDa；Step
Used nanometer hydroxyapatite particle diameter distribution is 150-250 nm in the preparation of each bionical layer presoma；Under bionical cartilage
In the preparation process of bone presoma dosage part of nanometer hydroxyapatite be 10 mass parts, the dosage of modified typeⅡ Collagen be
20 mass parts, light trigger Irgacure2959 dosage are 0.5 mass parts；Change in the preparation process of bionical calcified layer presoma
The dosage of property hyaluronic acid be 10 mass parts, nanometer hydroxyapatite with for 10 mass parts, light trigger Irgacure2959
Dosage be 0.5 mass parts；In the preparation process of bionical cartilage deep layer presoma the dosage of modified hyaluronic acid be 5 mass parts,
The dosage of modified typeⅡ Collagen is 20 mass parts, the dosage of cartilage cell is 0.5 mass parts, light trigger
Irgacure2959 dosage is 0.5 mass parts；The dosage of modified hyaluronic acid in the preparation process of bionical intermediate layer presoma
Dosage for 5 mass parts, modified typeⅡ Collagen is 20 mass parts, light trigger Irgacure2959 dosage is 0.5 matter
Measure part；The dosage of modified typeⅡ Collagen is 20 mass parts, light trigger in the preparation process of bionical superficial layer presoma
Irgacure2959 dosage is 0.5 mass parts；The wherein bionical subchondral bone layer in the bionical printing step of bionical cartilage
Print thickness is arranged to that the 5% of bionical cartilage gross thickness, bionical calcified layer is 5%, bionical cartilage deep layer is 30%, bionical intermediate layer
It is 10% for 50%, bionical superficial layer；
Embodiment 4
Comparative example：（A kind of multi-gradient bionic joint cartilage material preparation process based on 3D printing（Application reference number is
CN201510752927.5 embodiments 1 are prepared）.
Experimental group 1~3：For a kind of bionical cartilage based on 3D printing of the gained of embodiment 1~3.
（1）The detection of mechanical property：By the anti-bacterial hydrogel recovery support of the 3D printing prepared by above-described embodiment 1~3
Mechanics evaluation experiment is carried out with comparative example, using the mechanics machine test comparison examples of Instron 5575 and the compressibility of experimental group
Can, compression speed is arranged to 0.5mm/min, for a diameter of 6 mm of the sample of test, a height of 4.5 mm.Instrument is pressed before test
Specification is returned to zero, and test is tested using auto-reset function every time afterwards, and every group of sample sets 5 Duplicate Samples, experiment knot
Fruit is as shown in Figure 1.
It is it can be seen that bright by the compressive strength of the bionical cartilage embodiment 1~3 obtained by the method disclosed in the present
It is aobvious compared with comparative example will height, the compressive strength of embodiment 3 is 2 times of comparative example, and compressive strength is up to 5.16 MPa.
（2）Cytotoxic evaluation is tested：By a kind of bionical cartilage based on 3D printing prepared by above-described embodiment 1~3
Cytotoxic evaluation experiment is carried out with comparative example（Tested by national standard GB/T 16886.5-2003）, comparative example 1~3
It is as shown in Figure 2 with the experimental result of comparative example pair.
Cytotoxicity testing result shows embodiment 1~3, and its is right after being co-cultured 1 day and 7 days with medulla mesenchyma cell
For the cell answered with respect to proliferation rate more than 90%, cytotoxicity is rated 0 grade, it was demonstrated that and it has good cell compatibility, and
After comparative example and medulla mesenchyma cell co-culture 1 day and 7 days its corresponding cell proliferation rate relatively be respectively 89.3% and
87.5%, cytotoxicity is rated 1 grade, has slight cytotoxicity.In addition, co-culture the extension embodiment 1~3 of time
Relative to be significantly improved compared to proliferation rate, its cell is more negative group high with respect to proliferation rate after 7 days for embodiment 2 and 3（
Higher than 100%）, it was demonstrated that medulla mesenchyma cell can be promoted using the bionical cartilage obtained by preparation method disclosed in this invention
Growth, have good biocompatibility.
（3）Alkaline phosphatase（ALP）Detection
By a kind of bionical cartilage based on 3D printing prepared by above-described embodiment 1~3 and comparative example respectively with medulla mesenchyma
Cell carries out using its Alkaline Phosphatase Kit after co-culturing 7 days（ALP assay KIT, purchased from Bio protocol reagents
Box company）Its alkaline phosphatase enzyme values is detected, the self-bone grafting ability of contrast experiment's group and comparative example.Experimental result such as Fig. 3
It is shown.
Alkaline phosphatase（alkaline phosphate ALP）It is the mark for breaking up Gegenbaur's cell, bone matrix can be promoted
Mineralising.It was found from experimental result, compared with comparative example, the ALP values of embodiment 1 ~ 3 substantially will height compared with comparative example.As can be seen here,
The prepared bionical cartilage based on 3D printing of this hair has higher self-bone grafting ability, can promote luring for damaged cartilage tissue
Lead regeneration.
（4）Glycosaminoglycan（GAG）Secretory volume detection
By a kind of bionical cartilage based on 3D printing prepared by above-described embodiment 1~3 and comparative example respectively with medulla mesenchyma
Cell carries out using Dimethylmethylene blue test kit after co-culturing 7 days and 14 days（DMMB assay KIT, purchased from Bio
Protocol kits company）The secretory volume of glycosaminoglycan is detected, concrete operations are operated by kit specification.Experiment knot
Fruit such as Fig. 4 shows.
How much the secretory volume of glycosaminoglycan has with medulla mesenchyma cell differentiation and the speed of cartilage cell that forms maturation
Close.Being shown from experimental result, the secretory volume of the glycosaminoglycan of embodiment 1 ~ 3 is substantially higher at 7 days and 14 days compared with comparative example,
And the secretory volume for increasing the glycosaminoglycan of experimental group 1 ~ 3 with incubation time is in rising trend, the osamine of embodiment 3 at 14 days
Glycan secretory volume is 2 times of the secretory volume of the glycosaminoglycan of comparative example up to 430.15.As can be seen here, this hair it is prepared based on
The bionical cartilage of 3D printing has higher glycosaminoglycan secretory volume, and medulla mesenchyma cell differentiation can be promoted thin for cartilage
Born of the same parents, be advantageous to the reparation of damaged cartilage tissue.
Obviously, the above embodiment of the present invention is only intended to clearly illustrate example of the present invention, and is not pair
The restriction of embodiments of the present invention；For those of ordinary skill in the field, may be used also on the basis of the above description
To make other changes in different forms, there is no necessity and possibility to exhaust all the enbodiments；It is all this
All any modification, equivalent and improvement made within the spirit and principle of invention etc., should be included in the claims in the present invention
Protection domain within.
Claims (8)
1. a kind of bionical cartilage based on 3D printing, it is characterised in that by modified typeⅡ Collagen, modified hyaluronic acid, receive
Rice hydroxyapatite and cartilage cell are made by the bionical 3D printing of gradient.
2. a kind of bionical cartilage based on 3D printing according to claim 1, it is characterised in that count in parts by weight by changing
Property typeⅡ Collagen 20-80 parts, modified hyaluronic acid 20-80 parts, nanometer hydroxyapatite 10-20 parts and cartilage cell
0.1-0.5 parts form.
3. a kind of bionical cartilage based on 3D printing according to claim 1, it is characterised in that the bionical cartilage is under
It is supreme to be beaten successively by bionical subchondral bone, bionical calcified layer, bionical cartilage deep layer, bionical intermediate layer and bionical superficial layer by 3D
Print machine printing structure gained, overall is in the inierpeneirating network structure of multilayer.
4. a kind of manufacture method of bionical cartilage based on 3D printing according to claim any one of 1-3, its feature exist
In described manufacture method comprises the following steps that：
（1）The preparation of printed material：
1. the preparation of the modified typeⅡ Collagen：TypeⅡ Collagen is added in 50 DEG C of PBS phosphate and be configured to
Mass concentration is 10% typeⅡ Collagen PBS phosphate solutions, and then V typeⅡ Collagens PBS phosphate is molten by volume
Liquid：V methacrylic anhydride=10:0.75 adds methacrylic anhydride, and stirring reaction 4 hours at 50 DEG C, will after reaction terminates
Resulting solution be placed in molecular cut off be 10000 bag filter in dialyse 7 days, then by obtained dialyzate be placed in -80 DEG C it is cold
It is freeze-dried 48 hours in lyophilizer, produces modified typeⅡ Collagen, be placed in and store at room temperature, it is standby；
2. the preparation of modified hyaluronic acid：Hyaluronic acid is dissolved in the hyalomitome for being configured to that mass concentration is 2% in distilled water
Acid solution, the pH to 8.5 for then adding the sodium hydroxide solution regulation solution that molar concentration is 1 mol/L obtain Sodium Hyaluronate
Solution, then m Sodium Hyaluronates in mass ratio：M dimethylaminoethyl methacrylate=1:3.5 add methacrylic acid two
Methylamino ethyl ester, after reacting 24h at 10 DEG C, it is saturating that resulting solution is placed in flowing water in the bag filter that molecular cut off is 14000
Dialyzate, is then placed in -80 DEG C of freeze driers and is freeze-dried 48 hours, produce modified hyaluronic acid, be placed in by analysis 7 days
Store at room temperature, it is standby；
3. after cartilage cell is digested, it is added in the cultured chondrocytes base containing the cow's serum that volume fraction is 10%, matches somebody with somebody
Cell concentration is made as 1 × 106—5×106mL-1Chondrocyte cell suspension, be placed in gas concentration lwevel be 5%, temperature 37
DEG C cell culture incubator in it is standby；The cartilage cell is the cartilage cell after 3 generations of normal culture；
（2）The preparation of each bionical layer presoma：
1. the preparation of bionical subchondral bone presoma：Light trigger Irgacure2959, step are sequentially added into distilled water（1）
The modification typeⅡ Collagen of gained is prepared, the material added every time adds a kind of lower material, then added after being completely dissolved
Nanometer hydroxyapatite, stirs dispersed in the solution to nanometer hydroxyapatite, and it is molten to obtain bionical subchondral bone presoma
Liquid, it is standby；Wherein, light trigger Irgacure2959 concentration be 0.5wt%, the concentration of nanometer hydroxyapatite be 5-
10wt%, the concentration of modified typeⅡ Collagen is 5-10 wt%；
2. the preparation of bionical calcified layer presoma：Light trigger Irgacure2959, step are sequentially added into distilled water（1）System
Standby modified hyaluronic acid, the material added every time add a kind of lower material, then add nano hydroxyapatite after being completely dissolved
Lime stone, stirs dispersed in the solution to nanometer hydroxyapatite, obtains bionical calcified layer precursor solution, standby；Wherein,
Light trigger Irgacure2959 concentration is 0.5wt%, the concentration of nanometer hydroxyapatite is 1-5wt%, modified hyaluronic acid
Concentration be 1-3 wt %；
3. the preparation of bionical cartilage deep layer presoma：Light trigger Irgacure2959, step are sequentially added into distilled water（1）
The modified hyaluronic acid of preparation, step（1）The modification typeⅡ Collagen of preparation, the material added every time add again after being completely dissolved
Enter a kind of lower material, then add isometric step（1）The chondrocyte cell suspension prepared, gentle agitation are equal to mixing
It is even, bionical cartilage deep layer precursor solution is obtained, it is standby；Wherein, light trigger Irgacure2959 concentration is 0.5wt%, changed
Property hyaluronic acid concentration be 1-3 wt %, the concentration of modified typeⅡ Collagen be 10-15wt%, and the concentration of cartilage cell is 5
×105—2.5×106mL-1；
4. the preparation of bionical intermediate layer presoma：Light trigger Irgacure2959, step are sequentially added into distilled water（1）System
Standby modified hyaluronic acid, step（1）The modification typeⅡ Collagen of preparation, the material added every time add after being completely dissolved
A kind of lower material, bionical intermediate layer precursor solution is obtained, it is standby；Wherein, light trigger Irgacure2959 concentration is
0.5wt%, the concentration of modified hyaluronic acid are 2-5 wt %, and the concentration of modified typeⅡ Collagen is 10-15wt%；
5. the preparation of bionical superficial layer presoma：Light trigger Irgacure2959, step are sequentially added into distilled water（1）System
Standby modification typeⅡ Collagen, the material added every time add a kind of lower material after being completely dissolved, obtain bionical superficial layer
Precursor solution, it is standby；Wherein, light trigger Irgacure2959 concentration is 0.5wt%, is modified the dense of typeⅡ Collagen
Spend for 10-15wt%；
（3）The bionical printing of bionical cartilage：
By step（2）Each bionical layer presoma for preparing gained is pressed bionical subchondral bone presoma, bionical calcified layer presoma, imitated
Raw cartilage deep layer presoma, bionical intermediate layer presoma, the order of bionical superficial layer presoma are sequentially overlapped printing, produce one kind
Bionical cartilage based on 3D printing；The print thickness of wherein described bionical subchondral bone be arranged to bionical cartilage gross thickness 5 ~
10%th, the print thickness of the bionical calcified layer is arranged to the 2 ~ 5% of the bionical cartilage gross thickness, printing of the bionical cartilage deep layer
Thickness is arranged to the 15 ~ 30% of the bionical cartilage gross thickness, print thickness in the bionical intermediate layer and is arranged to bionical cartilage gross thickness
40 ~ 55%, the print thickness of the bionical superficial layer be arranged to the 10 ~ 20% of bionical cartilage gross thickness.
5. the manufacture method of a kind of bionical cartilage based on 3D printing according to claim 4, it is characterised in that described II
The molecular weight of collagen type is 1 × 106kDa。
6. the manufacture method of a kind of bionical cartilage based on 3D printing according to claim 4, it is characterised in that described
The molecular weight of bright matter acid is 1.2 ~ 1.5 × 106kDa。
7. the manufacture method of a kind of bionical cartilage based on 3D printing according to claim 4, it is characterised in that described to receive
The particle size distribution range of rice hydroxyapatite is 150-250 nm.
8. the manufacture method of a kind of bionical cartilage based on 3D printing according to claim 4, it is characterised in that using type
Number printed for 3D systems ProX 800 stereolithography apparatus method 3D printer, the purple of every layer of bionical layer is set
The outer light irradiation time is 30 s, and ultraviolet light intensity is 10 mW/CM2。
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| CN113440651A (en) * | 2020-08-06 | 2021-09-28 | 华夏司印(上海)生物技术有限公司 | Hydrogel composition, hydrogel, bionic cartilage and preparation method and application thereof |
| CN112354016A (en) * | 2020-11-24 | 2021-02-12 | 重庆工程职业技术学院 | Bionic artificial bone material and production method thereof |
| CN115161268A (en) * | 2021-03-19 | 2022-10-11 | 高雄医学大学 | Method for preparing cartilage cell lamina with regional layering and application thereof |
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