CN107007883A - A kind of repair of cartilage support and preparation method thereof - Google Patents
A kind of repair of cartilage support and preparation method thereof Download PDFInfo
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- CN107007883A CN107007883A CN201710084882.8A CN201710084882A CN107007883A CN 107007883 A CN107007883 A CN 107007883A CN 201710084882 A CN201710084882 A CN 201710084882A CN 107007883 A CN107007883 A CN 107007883A
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Abstract
The invention discloses a kind of preparation method of repair of cartilage support.This method includes：Utilize the mould of 3D printing technique print carriage；The bottom surface of mould has a plurality of the first fin being parallel to each other；A plurality of the second fin being parallel to each other being connected with the upper surface of the first fin；And a plurality of the 3rd fin being parallel to each other being connected with the upper surface of the second fin；The width of three is 100 500 μm；First fin intersects in predetermined angle with the second fin；Second fin intersects in predetermined angle with the 3rd fin；Prepare the mixed solution of fibroin and gelatin；Mixed solution is poured into mould, the liquid level of mixed solution was not had the 3rd fin；After solution solidification plastic to be mixed, mould is removed, the body of material of repair of cartilage support is obtained；Protein denaturation and chemical crosslinking processing, freeze-drying are carried out to it.Support prepared by this method provides more attachment sites and suitable growth microenvironment for BMSC enrichment, promotes damaged cartilage reparation.
Description
Technical field
The present invention relates to repair of cartilage support technology field, more particularly to a kind of repair of cartilage support and preparation method thereof.
Background technology
With the raising of sports level and sports participation, articular cartilage damage is increasingly common.Articular cartilage by
In its special histological anatorny feature, self-repairing capability is poor after damage.If such damage can not obtain timely and effective
Ground is treated, and may be in progress osteogenic arthritis, and white elephant is brought to patient and society.
The method of conventional articular cartilage damage reparation mainly has marrow stimulating technology (drilling and micro fractures), bone soft at present
Bone graft technique and chondrocyte cell transplantation technology etc..
Marrow stimulating technology (drilling and micro fractures) is the marrow contained in the blood clot using the marrow blood formation of release
Derived from Mesenchymal Stem Cells forms new cartilage, repair deficiency region.The technology has certain effect to cartilage damage, but bone
What bone marrow-drived mesenchymal stem differentiation was produced is fibrocartilage, and fibrocartilaginous biology performance and mechanical property are below normal soft
Bone, while the blood of release can not influence repairing effect in local fast storage, particularly with being repaired more for larger defect
Plus it is difficult.
Bone cartilage transplantation technology includes Cartilage transplantation and allogeneic cartilage is transplanted.This kind of technology can be to a certain degree
Upper mitigation patient pain, improvement function of joint, but there is research to show that bone and cartilage autotransplantation is possible to cause to supply area's cell
Death, and then cause for the corresponding symptom in area, while also existing and the agreeing with property of normal cartilage is undesirable, cytoactive during storage
The problems such as decline, limited donor graft source and transmission risk, limit its application.
Chondrocyte cell transplantation technology the disadvantage is that, the technology is still inevitably faced with during cultured chondrocytes
Dedifferente, the problems such as second operation wound and high cost, and compared with micro fractures are performed the operation, the technology is simultaneously without obvious
Clinical treatment advantage.
Therefore, difficult point and focus in clinical research are still to the research of cartilage damage reparation.
In recent years, tissue engineering technique is developed rapidly, is that articular cartilage damage reparation brings new thinking.Organizational project
Technology produces cambium mainly by the interaction and regulation and control of support, seed cell and biotic factor, reaches reparation because of wound
Wound, disease and tissue damage caused by aging and the purpose for recovering physiological function.Tissue engineering technique make it that articular cartilage is recognized
To be most hopeful by one of tissue and organ of tissue engineering technique successful regeneration.
The requirement of repair of cartilage support in design is in tissue engineering technique：1st, biodegradation does not produce noxious material；
2nd, good mechanics is provided for cambium to support；3rd, degradation speed matches with the newborn speed of tissue；4th, there can be hole
Performance, it is allowed to the disperse of nutriment and metabolite；5th, matching support and the anti-compression property of normal cartilage.
Current cartilage tissue engineered conventional support is divided into by making material：Support and biology that synthetic material is made
The support that natural material is made.Wherein, the support that artificial material is made be by it is artificial constructed prepare have specific physics and chemistry
The support of property, mechanical strength and material configuration, has some superiority, but the support that artificial material is made in terms of control of material
Biocompatibility and degradability are poor.The support being made up of natural material, is made especially by biological method, can promote just
The generation and remodeling of normal extracellular matrix, and the support that is made of natural material is in biocompatibility, biodegradable and into cartilage
There is incomparable advantage in terms of differentiation.
Biological raw material is divided into protein-based, such as fibroin, fibrin, collagen and gelatin, and carbohydrate,
Such as agarose, alginates, hyaluronic acid and chitosan.Wherein, fibroin is due to abundance, biocompatibility
Good, catabolite has no toxic side effect, good mechanical properties and it is cheap the advantages of, it is a kind of excellent as repair of cartilage
Timbering material；But its degradation speed is excessively slow, the Regeneration and Repair of cartilage is influenceed to a certain degree.Collagen be cartilage matrix it is main into
Point, it can adhere to the growth with sertoli cell very well, but the collagen of animal origin often has immunogenicity.Gelatin is used as glue
Former alternative materials, with immunogenicity it is low the characteristics of, obtain good effect in field of tissue engineering technology, but gelatin mechanical property
Can be poor, degradation speed is too fast to be limited it and is used separately as timbering material.
Find under study for action, in addition to the biological nature of timbering material in itself, the space structure of support, especially support
Three-dimensional structure and pore size, also repair significant to regenerating bone or cartilage.There are some researches show with three-dimensional stereochemical structure
Support be more beneficial for Chondrocyte Differentiation and propagation than simple planar structure.
Micro fractures technology is using the potential of the multi-direction differentiation of seed cell, the support being made with reference to biological raw material,
It is aided with cell factor again, repairs cartilage defect, because its is simple and easy to apply, low-cost, clinically have broad prospects.Micro- bone
Folding technology can escape mesenchymal stem cells MSCs (a kind of seed cell), be stimulated in the bracket by mechanics and biotic factor,
The purpose for repairing cartilage defect is reached to Chondrocyte Differentiation.But because of effective mesenchymal stem cells MSCs content after micro fractures
Less and easily from the outflow of cartilage defect area, it is difficult to be retained in part, causes neocartilage organization mechanicses weak, it is impossible to be subjected to joint
Activity, have impact on the effect of repair of cartilage.
During the present invention is realized, inventor has found that prior art at least has problems with：It is traditional by biology
Natural material directly lyophilized method can not realize minute design, and the support prepared is planar structure, without fine hole
The 3-D solid structure of gap, not only mechanical strength is poor, and is unfavorable for mesenchymal stem cells MSCs and is enriched with and retains on support
In defective region and differentiating cartilage-forming cell, new cartilage is formed.
The content of the invention
In order to solve the above technical problems, the present invention provides a kind of repair of cartilage support and preparation method thereof.Technical scheme is such as
Under：
In a first aspect, the invention provides a kind of preparation method of repair of cartilage support, including：
The mould of repair of cartilage support is printed using 3D printing technique；The bottom surface of mould have it is a plurality of be parallel to each other first
Fin；A plurality of the second fin being parallel to each other being connected with the upper surface of first fin；And with second fin
A plurality of the 3rd fin being parallel to each other that is connected of upper surface；The width of first fin is 100-500 μm；Described second
The width of fin is 100-500 μm；The width of 3rd fin is 100-500 μm；First fin and described second convex
It is in predetermined angle that rib, which intersects,；Second fin intersects in predetermined angle with the 3rd fin；
Prepare the mixed solution of fibroin and gelatin；
The mixed solution is poured into mould, the liquid level of the mixed solution was not had the 3rd fin；
After after mixed solution solidification plastic, mould is removed, the body of material of repair of cartilage support is obtained；
Body of material to the repair of cartilage support carries out protein denaturation processing；
Body of material to the repair of cartilage support after protein denaturation processing carries out chemical crosslinking processing, freezes afterwards dry
It is dry, obtain repair of cartilage support.
Preferably, first fin is vertical with second fin；Second fin is vertical with the 3rd fin.
Preferably, the width of first fin is 300-500 μm；The width of second fin is 300-500 μm；Institute
The width for stating the 3rd fin is 300-500 μm.
It is highly preferred that the width of first fin, second fin and the 3rd fin is 400 μm.
Specifically, the gap between two neighboring first fin is 0.4-0.7mm；Between between two neighboring second fin
Gap is 0.4-0.7mm；Gap between two neighboring 3rd fin is 0.4-0.7mm.
Specifically, the liquid level of the mixed solution is than first fin, second fin and described 3rd convex
The high 0.1-0.3mm of height sum of rib.
Preferably, mass fraction of the fibroin in the mixed solution is 2.3%-4.6%；The gelatin exists
Mass fraction in the mixed solution is 2.3%-4.6%.
It is highly preferred that the mass fraction sum of the fibroin and the gelatin in the mixed solution is 6.9%.
Specifically, the protein denaturation processing is that the recovery support precursor is placed in into the ethanol that volume fraction is 95%
In solution overnight.
Specifically, the body of material of the repair of cartilage support after the processing to protein denaturation carries out chemical crosslinking processing
For the body of material of the repair of cartilage support after protein denaturation processing is placed in into the capital Buddhist nun that mass fraction is 0.3%-1%
Ambient temperature overnight in flat solution.
Specifically, the preparation method also includes：Carry out disinfection processing to the repair of cartilage support.
It is highly preferred that the preparation also includes：Glu-Pro-Leu-Gln-Leu-Lys-Met is coupled to the repair of cartilage branch
The surface of frame；The amino acid sequence of the Glu-Pro-Leu-Gln-Leu-Lys-Met such as SEQ ID NO：Shown in 1.
Specifically, the Glu-Pro-Leu-Gln-Leu-Lys-Met is coupled to the behaviour on the surface of the repair of cartilage support
It is as step：
Amination treatment is carried out to the repair of cartilage support first；
The repair of cartilage support after amination treatment is soaked in containing heterobifunctional crosslinking agent under conditions of lucifuge again
Solution in；
Then the repair of cartilage branch containing heterobifunctional crosslinking agent is placed on containing the mesenchymal stem cells MSCs
It is incubated in the solution of affine polypeptide；
The repair of cartilage support for finally having Glu-Pro-Leu-Gln-Leu-Lys-Met to surface modification is freeze-dried, system
Into the repair of cartilage support for coupling Glu-Pro-Leu-Gln-Leu-Lys-Met.
More specifically, described carry out amination treatment to the repair of cartilage support to be soaked in the repair of cartilage support
Volume fraction for 5%-15% ethylenediamine aqueous isopropanol in, temperature be 37 DEG C under conditions of, keep more than 1 hour.
More specifically, the solution containing heterobifunctional crosslinking agent is the 4- that quality volume fraction is 5%-15%
The dimethyl sulphoxide solution of (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group succinimide ester sodium；Amination treatment
Soak time of the repair of cartilage support afterwards in the solution of the heterobifunctional crosslinking agent is more than 1 hour.
More specifically, the solution containing Glu-Pro-Leu-Gln-Leu-Lys-Met is that mesenchymal stem cells MSCs is affine
The concentration of polypeptide is 0.05-0.15mg/mL phosphate buffer solution；The time of incubation is more than 1 hour.
Second aspect, the invention provides a kind of repair of cartilage support prepared using any of the above-described kind of preparation method.
The beneficial effect of technical scheme provided in an embodiment of the present invention is：
1st, a kind of preparation method for repair of cartilage support that the present invention is provided, is printed with three layers using 3D printing technique
The mould of the repair of cartilage support of fine fin structure, so as to prepare the repair of cartilage branch with fine space network
Frame, with more preferable mechanical strength.
2nd, by the design of the width of the fin to mould, the hole of the space network of repair of cartilage support is controlled
Size, and the liquid level of mixed solution did not had the 3rd fin, repair of cartilage support is had pedestal, formed fine and close membrane structure,
The mesenchymal stem cells MSCs for effectively preventing marrow from discharging flows into articular cavity, while avoiding joint fluid from being rushed to forming blood clot
Brush, is enriched with support for mesenchymal stem cells MSCs (BMSC) and provides more attachment sites and suitable growth microenvironment,
More mesenchymal stem cells MSCs is retained in defective region and differentiating cartilage-forming cell, form new cartilage, promote damage soft
Bone Defect Repari.
3rd, the material for the repair of cartilage support that preparation method of the invention is used is the mixed solution of fibroin and gelatin,
Noxious material is not produced with good biocompatibility and degradability, and after degrading.
Brief description of the drawings
Technical scheme in order to illustrate the embodiments of the present invention more clearly, makes required in being described below to embodiment
Accompanying drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the present invention, for
For those of ordinary skill in the art, on the premise of not paying creative work, other can also be obtained according to these accompanying drawings
Accompanying drawing.
Fig. 1 is the stereogram of the mould of the repair of cartilage support shown in an exemplary embodiment of the invention；
Fig. 2 is the sectional view of the mould of the repair of cartilage support shown in an exemplary embodiment of the invention；
Fig. 3 is the top view of the mould of the repair of cartilage support shown in an exemplary embodiment of the invention；
Fig. 4 be an exemplary embodiment of the invention shown in cut out after repair of cartilage support upward view；
Fig. 5 is Fig. 4 electron microscope；
Fig. 6 be an exemplary embodiment of the invention shown in cut out after repair of cartilage support top view；
Fig. 7 is Fig. 6 electron microscope；
Fig. 8 is the result figure that the repair of cartilage support that the embodiment of the present invention 3 is provided carries out mechanical strength measure；
Fig. 9 is the result figure that the repair of cartilage support that the embodiment of the present invention 3 is provided carries out external degradation measure；
Figure 10 is the result figure that the repair of cartilage support that the embodiment of the present invention 3 and embodiment 5 are provided carries out CCK-8 measure；
Figure 11 is the result figure that the repair of cartilage support that the embodiment of the present invention 3 and embodiment 5 are provided carries out DNA measure；
Figure 12 is that the repair of cartilage support that the embodiment of the present invention 3 and embodiment 5 are provided carries out the knot that mucopolysaccharide (GAG) is determined
Fruit is schemed；
Figure 13 is the laser confocal microscope observed result figure that the embodiment of the present invention 7 is provided；
Figure 14 is the result figure of the repair of cartilage for the zoopery that the embodiment of the present invention 8 is provided.
Embodiment
To make technical scheme and advantage clearer, below in conjunction with accompanying drawing embodiment of the present invention is made into
One step it is described in detail.
In a first aspect, the invention provides a kind of preparation method of repair of cartilage support, including：
The mould of repair of cartilage support is printed using 3D printing technique；As shown in Figures 1 to 3, mould is a groove, the bottom of groove
Face has a plurality of the first fin 1 being parallel to each other；Be connected with the upper surface of the first fin 1 it is a plurality of be parallel to each other it is second convex
Rib 2；And a plurality of the 3rd fin 3 being parallel to each other being connected with the upper surface of the second fin 2；The width of first fin 1 can
Think 100-500 μm；The width of second fin 2 can be 100-500 μm；The width of 3rd fin 3 can be 100-500 μm；
First fin 1 intersects in predetermined angle with the second fin 2；Second fin 2 intersects in predetermined angle with the 3rd fin 3；
Prepare the mixed solution of fibroin and gelatin；
Mixed solution is poured into mould, the liquid level of mixed solution was not had the 3rd fin；
After solution solidification plastic to be mixed, mould is removed, the body of material of repair of cartilage support is obtained；
Body of material to repair of cartilage support carries out protein denaturation processing；
Body of material to the repair of cartilage support after protein denaturation processing carries out chemical crosslinking processing, freezes afterwards dry
It is dry, obtain repair of cartilage support.
A kind of beneficial effect of the preparation method for repair of cartilage support that the present invention is provided：
1st, a kind of preparation method for repair of cartilage support that the present invention is provided, is printed with three layers using 3D printing technique
The mould of the repair of cartilage support of fine fin structure, so as to prepare the repair of cartilage branch with fine space network
Frame, with more preferable mechanical strength.
2nd, by the design of the width of the fin to mould, the hole of the space network of repair of cartilage support is controlled
Size, and the liquid level of mixed solution did not had the 3rd fin, repair of cartilage support is had pedestal (fine and close membrane structure), effectively
The mesenchymal stem cells MSCs that ground prevents marrow from discharging flows into articular cavity, while avoid joint fluid from being washed away to forming blood clot,
It is enriched with for mesenchymal stem cells MSCs (BMSC) on support and more attachment sites and suitable growth microenvironment is provided, is made more
Many mesenchymal stem cells MSCs are retained in defective region and differentiating cartilage-forming cell, form new cartilage, promote damaged cartilage to repair
It is multiple.
3rd, the material for the repair of cartilage support that preparation method of the invention is used is the mixed solution of fibroin and gelatin,
Noxious material is not produced with good biocompatibility and degradability, and after degrading.
Those skilled in the art can use 3D drawing techniques draw out repair of cartilage support mould shape, recycle
3D printing technique prints the mould of repair of cartilage support.The mould can be groove as shown in Figure 1.The bottom surface of the groove has many
The parallel width of bar is 100-500 μm of the first fin 1, is 100-500 μm with a plurality of parallel width that the first fin 1 intersects
The second fin 2, a plurality of parallel width intersected with the second fin 2 is 100-500 μm of the 3rd fin 3.Because cartilage is repaiied
The mould of multiple support has the network structure of such crossings on different level so that the repair of cartilage support of preparation has crossings on different level
Network structure.The mixed solution of fibroin and gelatin is poured into mould, and the liquid level of mixed solution did not had the 3rd fin,
The solid netted knot with pedestal (fine and close membrane structure) and the hole with multiple 100-500 μm of 100-500 μ ms can be formed
Structure.Not only mechanical strength is high for the repair of cartilage support of 3 D stereo network structure with such hole, and can be effectively
The mesenchymal stem cells MSCs for preventing marrow from discharging flows into articular cavity, while avoiding joint fluid from being washed away to forming blood clot, is
Mesenchymal stem cells MSCs (BMSC) is enriched with support provides more attachment sites and suitable growth microenvironment, makes more
Mesenchymal stem cells MSCs be retained in defective region and differentiating cartilage-forming cell, form new cartilage, promote damaged cartilage reparation.
First fin intersects in predetermined angle, 0 ° with the second fin<Predetermined angle≤90 °, preferably 45 °~90 °, more
Preferably 60 °~90 °, more preferably 75 °~90 °, in order to easy to process etc., most preferably 90 °.Second fin and the 3rd
It is in predetermined angle, 0 ° that fin, which intersects,<Predetermined angle≤90 °, preferably 45 °~90 °, more preferably 60 °~90 °, further
Preferably 75 °~90 °, in order to easy to process etc., most preferably 90 °.
As shown in Figure 1 to Figure 3, the first fin 1 can be that antarafacial is vertical with the second fin 2, shape angle in 90 °；Second is convex
Rib 2 can be that antarafacial is vertical with the 3rd fin 3, shape angle in 90 °；First fin 1 is parallel with the 3rd fin 3；As shown in figure 3,
3rd fin 3 is not located at the surface of the first fin 1, i.e. plane where the center line of the two and horizontal plane out of plumb.Certainly,
It is also conceivable for a person skilled in the art that the first fin and the angle of the second fin formation are less than 90 °, the second fin and the 3rd convex
Prismatic into angle be less than 90 °, and formed the two angles be able to can also be differed with identical；3rd fin can be located at
The surface of first fin, i.e., plane and horizontal plane where the center line of the two, certainly, the first fin and the 3rd fin
Can be with not parallel.The position relationship of foregoing the first fin, the second fin and the 3rd fin can be formed required for the present invention
Fine crossings on different level network structure, according to the width of the first fin, the second fin and the 3rd fin so that according to the mould
Repair of cartilage support prepared by tool has the hole of pedestal (fine and close membrane structure) and multiple 100-500 μm of 100-500 μ ms, promotees
Enter damaged cartilage reparation.
It should be noted that the step of utilization 3D printing technique of the present invention prints the mould of repair of cartilage support is with preparing
The step of mixed solution of fibroin and gelatin, first operates the two to walk without obvious priority logical order, those skilled in the art
Which of rapid step can.
Printed material is known in the art general knowledge used in 3D printing technique.In order to obtain the intact present invention's
Repair of cartilage support, the material of preferred mold is polystyrene or polystyrene of high intensity etc., and polystyrene and polyphenyl
Propylene etc. is soluble in D- limonenes, so that remove it, and D- limonenes do not destroy the knot of repair of cartilage support
Structure, and generation environment does not pollute.It will be appreciated by persons skilled in the art that remove mould process, it is necessary to less than
Carried out at a temperature of gelatin fusing point, to prevent gelatin to be melted into liquid, destroy the structure of repair of cartilage support, reduce repair of cartilage
The mechanical performance of support.
In practical situations both, after mould is removed, the ethanol solution of volume fraction 95% can also be used soft to what is obtained
The body of material of bone repairing support is cleaned repeatedly, generally 3 to 5 i.e. removable D- limonenes being built-up in thereon of cleaning.
The hole of the repair of cartilage support of the present invention be enriched with for mesenchymal stem cells MSCs on repair of cartilage support with
Growth is very important.In order to obtain the hole for being more beneficial for mesenchymal stem cells MSCs enrichment and growth, the present invention is to soft
The fin of the mould of bone repairing support has carried out series of optimum：
In a kind of specific embodiment of the mould of repair of cartilage support, as depicted in figs. 1 and 2, the first fin 1 with
The antarafacial of second fin 2 is vertical；Second fin 2 is vertical with the antarafacial of the 3rd fin 3.3rd fin 3 be located at the first fin 1 just on
Side, the i.e. center line of the center line of the first fin and the 3rd fin is in the same plane.
In a kind of preferably embodiment of the mould of repair of cartilage support, the width of the first fin can be 300-
500μm；The width of second fin can be 300-500 μm；The width of 3rd fin can be 300-500 μm.Art technology
Personnel are it is understood that the width of the first fin, the second fin and the 3rd fin can be the same or different.
Length, width and height mentioned by the present invention are length, width and the height understood on people's ordinary meaning.With
Exemplified by first fin, as shown in Fig. 2 the length of the first fin 1 is the distance from front end to rear end of the first fin 1, first is convex
The width of rib 1 refers to the distance of the first fin from left to right, the height of the first fin 1 refer to the first fin 1 from upper end to
The distance of lower end.
Can be 0.4-0.7mm for the gap between two neighboring first fin of the mould of repair of cartilage support；Phase
Gap between adjacent two the second fins can be 0.4-0.7mm；Gap between two neighboring 3rd fin can be 0.4-
0.7mm, three can be the same or different.Gap between two neighboring first fin mentioned herein refers to, such as Fig. 2
It is shown, the rightmost of the first fin 1 positioned at left side of two neighboring first fin 1 to the first fin 1 positioned at right side most
The distance on the left side.The definition in the gap between the definition in the gap between two neighboring 3rd fin and two neighboring second fin
The definition in the gap between two neighboring first fin is identical, refers to determining for the gap between two neighboring first fin
Justice.
In a kind of embodiment of the mould of repair of cartilage support, mold height can be 0.5-1cm groove, the
The width of one fin, the second fin and the 3rd fin can be 400 μm；Gap between two neighboring first fin can be
0.6mm；Gap between two neighboring second fin can be 0.6mm；Gap between two neighboring 3rd fin can be
0.6mm；The thickness of the side wall of mould can be 1mm, and the thickness of bottom surface can be 1mm.
The step of below by being related in the preparation method of repair of cartilage support disclosed by the invention, is described in detail.
Prepare the mixed solution of fibroin and gelatin, be technological means commonly used in the art, i.e., by fibroin with it is bright
Glue is dissolved in water, and is formed the respective aqueous solution, is matched according to required concentration.
Because in actual applications, the good mechanical properties of fibroin, degradation rate is slow, although and gelatin immunogene
Property very well, but mechanical property is slightly worse, and degradation rate is fast, in order that repair of cartilage support has good mechanical property, and
And the regeneration rate of its degradation rate and cartilage matches, mass fraction of the fibroin in mixed solution is 2.3%-
4.6%, preferably 2.3%；Mass fraction of the gelatin in mixed solution be 2.3%-4.6%, preferably 4.6%, further
The mass fraction sum of ground, fibroin and gelatin in mixed solution is 6.9%.
Those skilled in the art can judge when mixed solution solidifies plastic according to practical experience.Mix under normal circumstances
Solution static more than 5 minutes in a mold, you can realize that mixed solution solidifies plastic, that is to say, that form repair of cartilage support
Body of material.
Because fibroin can be with soluble in water, it is necessary to carry out denaturation treatment to it, and because gelatin melts at high temperature,
Therefore it is difficult to make protein denaturation using high temperature, and is contemplated that toxicity problem, therefore, the present invention is selected recovery support
Body of material be placed in volume fraction be 95% ethanol solution in overnight, overnight typically 8-12 hours time, make
Fibroin is denatured, and after protein denaturation is handled, the body of material of repair of cartilage support is provided with certain intensity, and
Dissolution phenomena will not occur for fibroin in subsequent processes.By giving full play to the respective excellent of fibroin and gelatin
Gesture, realizes the performance optimization of repair of cartilage support mechanics and degraded.
In actual applications, before to the body of material progress chemical crosslinking processing of repair of cartilage support, first cleaned with water
Fall to adhere to the ethanol in the body of material of repair of cartilage support.Without specified otherwise, water used in biological field is usually to steam
Distilled water, distilled water, deionized water or ultra-pure water etc..
For the repair of cartilage support after protein denaturation processing body of material carry out chemical crosslinking processing generally can be with
Genipin solution that carbodiimides solution that use quality fraction is 0.01%-0.3%, mass fraction are 0.3%-1% or
Mass fraction is 0.1%-2% glutaraldehyde etc., the material master of the repair of cartilage support after such as protein denaturation can be handled
Body is placed in ambient temperature overnight in the genipin solution that mass fraction is 0.5%.Time overnight is generally understood that 8-12 hours.
Room temperature is generally understood that 18-25 DEG C.
The body of material of repair of cartilage support after handling chemical crosslinking is freeze-dried, and can specifically use freezing
Drier machine carries out vacuum freeze-drying processing, and this is this area conventional technology, and those skilled in the art can be according to reality
Situation is grasped, and therefore not to repeat here by the present invention.
, will also be to the repair of cartilage support before being repaired using the repair of cartilage support of the present invention to damaged cartilage
Carry out disinfection processing or the processing that carried out disinfection when preparing and completing to repair of cartilage support, and finished product is made in packaging.
In the preferred embodiment of the present invention, the preparation method also includes：Mesenchymal stem cells MSCs is affine
Polypeptide is coupled to the surface of repair of cartilage support；The amino acid sequence of Glu-Pro-Leu-Gln-Leu-Lys-Met such as SEQ ID NO：
It is EPLQLKM shown in 1, with 7 amino acid, abbreviation E7.The Glu-Pro-Leu-Gln-Leu-Lys-Met can be raised effectively
BMSC, and without species specificity.The Glu-Pro-Leu-Gln-Leu-Lys-Met property is stable, can functionally more added with
The mesenchymal stem cells MSCs of micro fractures operation release is raised on effect ground, and cell is retained in defective region, is cartilage defect area
More repair cells are provided.Property for Glu-Pro-Leu-Gln-Leu-Lys-Met is special in Publication No. CN102229646A
Existing play-by-play in sharp document, therefore not to repeat here by the present invention.
The Glu-Pro-Leu-Gln-Leu-Lys-Met of the present invention is artificial synthesized sequence, by the biological section of BeiJing ZhongKe's matt
Skill Co., Ltd synthesizes.Mesenchymal stem cells MSCs is synthesized on polyethylene glycol (PEG-PS) resin using solid phase method affine many
Peptide, the substantially step of synthesis is：
A. in a nitrogen environment, 3- maleimidoproprionic acids are activated with dicyclohexylcarbodiimide；
B. the amino acid residue 3- maleimidoproprionic acids of activation and peptide molecule being connected on resin reacts, and makes
Maleimide maleimide is coupled in peptide termini；With 95% trifluoroacetic acid by maleimide maleoyl-
The peptide molecule of imines activation is cut from PEG-PS resins, is dispensed after freezing.
The operating procedure that Glu-Pro-Leu-Gln-Leu-Lys-Met is coupled to the surface of repair of cartilage support is：
Amination treatment is carried out to repair of cartilage support first；
The repair of cartilage support after amination treatment is soaked in containing heterobifunctional crosslinking agent under conditions of lucifuge again
Solution in；
Then the repair of cartilage branch containing heterobifunctional crosslinking agent is placed on affine containing mesenchymal stem cells MSCs
It is incubated in the solution of polypeptide；
The repair of cartilage support for finally having Glu-Pro-Leu-Gln-Leu-Lys-Met to surface modification is freeze-dried, system
Into the repair of cartilage support for coupling Glu-Pro-Leu-Gln-Leu-Lys-Met.
The operating procedure that the surface of repair of cartilage support will be coupled to Glu-Pro-Leu-Gln-Leu-Lys-Met below is entered
Row is described in detail.
It can be that repair of cartilage support is soaked in into volume fraction is 5%- to carry out amination treatment to repair of cartilage support
In 15% ethylenediamine aqueous isopropanol, under conditions of temperature is 37 DEG C, kept for more than 1 hour.
Solution containing heterobifunctional crosslinking agent can be 4- (the N- maleimides that mass body fraction is 5%-15%
Amine methyl) hexamethylene -1- carboxylic acid sulfonic group succinimide ester sodium dimethyl sulphoxide solution；Repair of cartilage after amination treatment
Soak time of the support in the solution of heterobifunctional crosslinking agent is more than 1 hour.
Solution containing Glu-Pro-Leu-Gln-Leu-Lys-Met is that the concentration of Glu-Pro-Leu-Gln-Leu-Lys-Met is
(0.01M, pH value is 7.2) preferably Glu-Pro-Leu-Gln-Leu-Lys-Met to 0.05-0.15mg/mL phosphate buffer solution
Concentration be 0.1mg/mL；The time of incubation is more than 1 hour.
In second aspect, present invention also offers the repair of cartilage support prepared using any of the above-described kind of preparation method.
It was found from from the statement of the above-mentioned mould to repair of cartilage support, the mould of the invention by limiting repair of cartilage support
The structure of tool is come the structure of the repair of cartilage support prepared by determining.By controlling the liquid level of mixed solution not have the 3rd fin,
I.e. the liquid level of mixed solution is higher than the height sum of the first fin, the second fin and the 3rd fin so that the repair of cartilage support
With pedestal (fine and close membrane structure).The present invention repair of cartilage support mould structure and the structure of repair of cartilage support be
Opposite, i.e. the groove of the corresponding repair of cartilage support of fin of mould.
In a kind of embodiment of repair of cartilage support repair of cartilage support have pedestal (as shown in Figure 4) and
Formed between multiple grooves on pedestal, groove and intersect to form hole (as shown in Figure 6), pedestal is the liquid level by mixed solution
The fine and close membrane structure that the 3rd fin is formed is not crossed；The shape of the groove is corresponding with the fin shape of above-mentioned mould.Such as Fig. 6
Shown, the upper surface of repair of cartilage support is to pour into mixed solution after mould, the face fitted with the bottom surface of mould.With this
The mesenchymal stem cells MSCs that planting the repair of cartilage support of structure can effectively prevent marrow from discharging flows into articular cavity, keeps away simultaneously
Exempt from joint fluid and wash away the blood clot to be formed, be enriched with for mesenchymal stem cells MSCs (BMSC) on support and more attachment positions are provided
Point and suitable growth microenvironment, make more mesenchymal stem cells MSCs be retained in defective region and differentiating cartilage-forming cell, shape
Cheng Xin cartilage, promotes damaged cartilage reparation.
As shown in figure 5 and figure 7, the repair of cartilage support has the 3 D stereo net of pedestal to the electron microscope of repair of cartilage support
The space structure of shape, 3 D stereo network structure designs to be formed via 3D printing, is made up of rib and middle pore structure, can be with
Mesenchymal stem cells MSCs in being repaired for articular cartilage damage provides more suitable existence microenvironment；Pedestal and 3 D stereo
Network structure is connected, and its structure is one layer of fine and close fibroin-gelatin, can effectively be hindered during repairing compound articulation cartilage damage
The marrow blood that gear micro fractures are discharged enters articular cavity, and the mesenchymal stem cells MSCs required for repair of cartilage is effectively protected
Stay in 3 D stereo network structure and top surface membrane structure, so that the reparation of significantly more efficient promotion damaged cartilage.
The high 0.1- of height sum according to the liquid level of mixed solution than the first fin, the second fin and the 3rd fin
0.3mm, it is known that the thickness of the pedestal of repair of cartilage support of the invention is about 0.1-0.3mm.
In actual applications, the repair of cartilage support of the present invention can be designed according to the thickness of the cartilage of required reparation
Height.
The repair of cartilage support is made up of the mixed solution of fibroin and gelatin, may be simply referred to as SFG supports.Fibroin
All it is that natural biologic material has good biocompatibility, degradability and nontoxicity with gelatin.Preferably, fibroin exists
Mass fraction in mixed solution is 2.3%-4.6%；Mass fraction of the gelatin in mixed solution is 2.3%-4.6%.
The speed of the degradation rate and regenerating bone or cartilage that obtain repair of cartilage support in the range of this mass component matches.It is highly preferred that silkworm
The mass fraction sum of silk-fibroin and gelatin in mixed solution is 6.9%.
In a kind of preferred embodiment of repair of cartilage support, the surface of repair of cartilage support has coupled medulla mesenchyma
Stem cell is affine polypeptide, the amino acid sequence such as SEQ ID NO of the Glu-Pro-Leu-Gln-Leu-Lys-Met：Shown in 1, it is
EPLQLKM, referred to as E7 polypeptides.Repair of cartilage support with E7 polypeptides can specifically raise the bone of micro fractures operation release
Bone marrow-drived mesenchymal stem, and cell is retained in defective region, provide more repair cells for cartilage defect area.
Embodiment 1 extracts fibroin
Load 2L ultra-pure water in beaker, boil, and weigh 4.24g Na2CO3It is poured slowly into wherein；By 5g silk cocoon
After (Guangdong silkworm base) is shredded with scissors, pour into boiling water, soak 30min and be sufficiently stirred for, make the sericin in silk cocoon
All it is dissolved in water, to remove the sericin in silk cocoon；
Boil after 30min, take out the fibroin albumen of flocculence, use ultrapure water cooling, unnecessary moisture is extruded, afterwards by it
It is put into 1L beaker, adds ultra-pure water stirring 30min, squeeze the water out, repeat 2-3 times afterwards；
Pure fibroin albumen as fibroin is taken out afterwards, is spread out on aluminium foil, and ventilation, which is stayed overnight, dries；
According to fibroin:LiBr (lithium bromide)=1:4 weight ratio, weighs the LiBr of respective quality, and be made into water
The 9.3M LiBr aqueous solution.The LiBr aqueous solution is poured on fibroin, fibroin is completely covered by solution；
Mixed solution is heated to 60 DEG C, 4h is kept, is completely dissolved it, it is transparent amber.It is then saturating with ultra-pure water
Analysis 3 days, and by the solution low-temperature centrifugation 20min (4 DEG C, 9000rpm) after dialysis；
Pipette supernatant and centrifuge again, the fibroin of acquisition is then placed in sealing preserve in 4 DEG C.
Acquisition for fibroin is not related to the inventive point of the present invention, and the present invention, which only provides one kind, can obtain higher
The acquisition methods of the fibroin of purity, those skilled in the art can be obtained by other conventional meanses or from purchasing on the market
Buy.
The design and preparation of the 3D moulds of embodiment 2
Utilize the mould of AutoCAD Software on Drawing repair of cartilage supports.Wherein, the height of mould is 1cm；The side wall of mould
Thickness with bottom surface is 1mm.The bottom surface of mould has 28 the first fins being parallel to each other；It is vertical with the first fin antarafacial,
And the second fin that 28 be connected with its upper surface are parallel to each other；And it is vertical with the second fin antarafacial, and with its upper table
The 3rd fin that be connected 28 articles of face are parallel to each other；Form 3 layers of fin altogether, the first fin, the second fin and the 3rd fin
Width is 0.4mm with height, and length is 3cm；
Then the mould of repair of cartilage support is printed, institute using Table top type 3D printer (first facing three-dimensional, China)
The wire rod of selection is HIPS (high intensity polystyrene, 1.75mm) (dodging casting science and technology, China).
The preparation of the repair of cartilage support of embodiment 3
By fibroin and gelatin in mixed solution respective mass fraction 0% and 6.9% (first group), 2.3% and
4.6% (second group), 4.6% and 2.3% (the 3rd group) are mixed, and after being sufficiently stirred for, are poured into before gelatin plastic
In mould (mixed solution should not have the 3rd fin, it is ensured that form fine and close top surface membrane structure), 60 DEG C of vacuum drying chamber is placed in
Middle 10min so that mould is filled up completely with by composite solution, then takes out；
Solution to be mixed solidifies after plastic (5min) at room temperature, and mould is put into D- limonenes (nine orange industry, China)
In 3 days, mould is removed；
Keeping temperature is needed to be less than gelatin melt temperature during removing mould, and liquid is changed in timing.After mould is removed,
Lyophilized obtained porous support, and it is placed again into the mould that remnants are removed in limonene.
D- limonenes are washed away using 95% ethanol afterwards, support is put into 95% ethanol overnight after cleaning up, makes silkworm
Silk-fibroin denaturation solidification；
After the completion of processing, support is put into the Geniposide (the rich biotechnology in day, China) that mass fraction is 0.5wt% water-soluble
In liquid, ambient temperature overnight makes gelatin and Geniposide complete crosslinking.The support after crosslinking is taken out, after being cleaned with ultra-pure water, ring is used
Bore (Φ 4.00mm) and the support after the crosslinking of bulk is drilled to columned support, be freeze-dried, obtain columned soft
Bone repairing support, Co 60 illumination-based disinfection is being carried out using preceding to it.
Because the material of repair of cartilage support is fibroin and gelatin, therefore, repair of cartilage support of the invention also may be used
To be referred to as fibroin-gelatin support (SFG supports).
The fibroin that embodiment 4 is prepared to embodiment 3-gelatin support carries out mechanical strength and external degradation is determined
Three kinds of fibroins-gelatin support prepared by Example 3, apparatus measures compressive deformation is compressed and strong using mechanics
Degree, calculates the mechanical strength of three kinds of fibroin-gelatin supports；As shown in figure 8, with the increase silk egg of fibroin concentration
The mechanical strength enhancing of in vain-gelatin support.
Above-mentioned three kinds of fibroins-gelatin support is weighed respectively, is then immersed in PBS (0.01M, pH=7.2),
Take out SFG within 7 days, the 14th day and the 21st day, after freeze-drying and weigh, calculate degradation in vitro.As shown in figure 9, using merely
Repair of cartilage support prepared by gelatin is degraded rapidly, and mass fraction of the fibroin in mixed solution is brought up into 4.6%
When then degradation rate substantially slow down, for match cartilage regeneration rate, selection second group be used as final fibroin-gelatin branch
Frame concentration ratio, carries out follow-up test.
Wherein, first group, second group and third component do not represent what fibroin and gelatin were each accounted in mixed solution
Mass fraction is respectively 0% and 6.9%, 2.3% and 4.6%, 4.6% and 2.3%.
E7 polypeptides are coupled on fibroin-gelatin support that concentration ratio is second group by embodiment 5
It is 10% ethylenediamine solution with isopropanol dose volume fraction, takes fibroin and gelatin that concentration ratio is second group
Support is immersed in solution (37 DEG C, 1 hour), carries out amination treatment.
Distilled water is rinsed 3 times；By 10mg heterobifunctionals crosslinking agent (4- (N- maleimidomehyls) hexamethylene -1- carboxylics
Sour sulfonic group succinimide ester sodium, Sulfo-smcc) dissolve into 100 μ l dimethyl sulfoxide (DMSO)s (Dimethyl sulfoxide,
DMSO it is now with the current) in solution.Fibroin-gelatin support 1 hour after amination is soaked in light protected environment.
The phosphate buffer solution (PBS) (0.01M, PH=7.2) of 0.1M ethylenediamine tetra-acetic acids (EDTA) is prepared, is buffered with this
Liquid rinses fibroin-gelatin support 3 times；Glu-Pro-Leu-Gln-Leu-Lys-Met (E7 polypeptides) is dissolved in PBS (concentration
For：0.1mg/ml), it is added in support, is incubated at room temperature 2 hours, E7 polypeptides is fully coupled on support, realize E7 polypeptides pair
The surface modification of fibroin-gelatin support, it is standby after being freeze-dried 24 hours.
Before use, carrying out Co 60 illumination-based disinfection to the fibroin-gelatin support for coupling E7 polypeptides.
The CCK-8 of embodiment 6 is determined and DNA and mucopolysaccharide (GAG) measure
By 1.5 × 105Individual/ml mesenchymal stem cells MSCs is inoculated in fibroin-gelatin support (SFG supports) respectively
With on the fibroin-gelatin support (SFG-E7 supports) for coupling E7 polypeptides, complete medium culture is added, respectively the
1st, 3,5 and 7 days when discard culture medium, after PBS is rinsed 2 times, add the new culture medium containing 10 microlitres of CCK-8 working solutions
200 microlitres.37 DEG C, 5%CO2Under the conditions of hatch 2 hours.Take 100 microlitres of above-mentioned solution to be added in 96 orifice plates, determine OD values,
As a result as shown in Figure 10, fibroin-gelatin support (SFG supports) and the fibroin-gelatin support for coupling E7 polypeptides
(SFG-E7 supports) can be good support mesenchymal stem cells MSCs propagation, and couple the silk egg of E7 polypeptides
In vain-gelatin support is better than fibroin-gelatin support.
By 1.5 × 105Individual/ml mesenchymal stem cells MSCs is inoculated on SFG and SFG-E7 respectively, adds training completely
Support base culture, the 7th, 14 and 21 days when discard culture medium, after PBS is rinsed 2 times, add 200 microlitres of papains, 60 DEG C of bars
Under part overnight, support and cell are completely dissolved；
Take 20 microlitres of above-mentioned lysates to instill after 96 orifice plates, add 200 microlitres of the solution of Hoechst 33258 or 200 micro-
Rise DMMB solution, 37 DEG C hatching 30 minutes after determine OD values.Determine DNA conditions：Exciting light 360nm, transmitting light 460nm.Survey
Determine GAG conditions：OD values are determined at 520nm.Actual value is corrected according to DNA and GAG standard curves, as a result respectively such as Figure 11 and Figure 12
It is shown, the support dermoskeleton bone marrow-drived mesenchymal stem that two kinds of supports can be good into cartilage differentiation, and coupled E7 polypeptides
Fibroin-gelatin support it is more preferable to the effect into cartilage differentiation for promoting external mesenchymal stem cells MSCs.
Growth of the confocal laser scanning microscope mesenchymal stem cells MSCs of embodiment 7 on repair of cartilage support is with dividing
Cloth
It is 1.5 × 10 by density5The culture medium drop of individual/ml mesenchymal stem cells MSCs is planted on SFG and SFG-E7,
37 DEG C are incubated 1 hour；
1ml MEM- α culture mediums (without nucleotides and deoxynucleotide), 37 DEG C of incubator cultures are added in 6 orifice plates；
After culture 3 days, support is taken out, PBS is rinsed 2 times；
Quality volume fraction is fixed for 4% paraformaldehyde, rhodamine-phalloidine (160nM, 2ml) lucifuge dye cell
Skeleton 4 hours；
PBS is rinsed 2 times, Hoechst33258 (1:800,2ml) nucleus is redyed 1 hour；
PBS is rinsed 2 times, laser confocal microscope observation mesenchymal stem cells MSCs distribution situation.
Figure 13 is laser confocal microscope observed result figure.Figure 13 shows mesenchymal stem cells MSCs in two kinds of supports
On growth and distribution situation.Two kinds of supports can support the adhesion and growth of stem cell well, couple E7 polypeptides
Fibroin-gelatin support can adhere to more mesenchymal stem cells MSCs, illustrate to couple the fibroin of E7 polypeptides-bright
Glue support is more conducive to mesenchymal stem cells MSCs enrichment.
Cartilage defect repair in 8 zooperies of embodiment-rabbit body
Take adult male New Zealand White Rabbit, 2.9 ± 0.3kg of body weight.It is randomly divided into 3 groups：MF groups (simple micro fractures),
SFG groups (SFG supports+micro fractures), SFG-E7 groups (the SFG supports+micro fractures for coupling E7 polypeptides).
3 ﹪ amobarbitals are injected to 3 groups of new zealand white rabbits by auricular vein to anaesthetize, dosage is 1mL/
kg.After rabbit is successfully anaesthetized, dorsal position, fixing head and four limbs, the right patella of exposure, by field of operation preserved skin, Iodophor routine are taken
Sterilization, alcohol takes off iodine, spreads aseptic operation list；Flexing knee joint, approach is hit exactly before knee and cuts skin, continuation enters by the kneecap of inner side
Open articular cavity in road；Stretch knee-turn up kneecap-to go down on one's knees, exposure femoral bone pulley.With 4mm Diameter Corneals ring by femoral bone pulley surface
Vertically pierce (depth is 1mm), and take out the cylindrical cartilage bolt under boring, the full-thickness cartilage defects area of femoral bone pulley is made.Hand
Art operation must be carefully, it is to avoid the tissue such as damage synovial membrane, muscle, meniscus.
MF groups：Micro fractures processing is only done in cartilage defect area, and (aperture 0.5mm, pitch of holes 1mm, depth is to have blood and fat drips
Ooze out for standard), the filling of any support is not done.
SFG groups：First in cartilage defect area row microfrature, then by the cartilage defect of SFG stenter to implant femoral bone pulleys
Place, fills up defect, layer-by-layer suture articular cavity.
SFG-E7 groups：First in cartilage defect area row microfrature, the then cartilage of SFG-E7 stenter to implant femoral bone pulley
At defect, defect, layer-by-layer suture articular cavity are filled up.
The knee joint of Post operation each group rabbit does not do any outer fixing process；U/, intramuscular injection penicillin 800,000, continuous 3
My god；Standard animal forage feed, room temperature keeps 15-20 DEG C, cleaning drinking-water, sub-cage rearing, freely activity.
The healing state of the cartilage damage of above-mentioned 3 groups of rabbits is detected after microfrature June.As a result it is as shown in figure 14：
Micro fractures group (MF)：Gross examination of skeletal muscle still fails to fill up cartilage defect area, defect periphery for 6 months after finding microfrature
There is osteoproliferation performance；Magnetic resonance imaging (MRI) and Hematoxylin-eosin (HE) dyeing show that cartilage defect is not filled by, toluene
Amine indigo plant dyeing shows that the content of proteoglycan in cartilage defect area repair tissue is relatively low, is fibrocartilage reparation, II Collagen Type VI is exempted from
Epidemic disease histochemical staining shows that II Collagen Type VI content is low in tissue.
Fibroin-gelatin support (SFG) group：Generally, defect has filling, but not exclusively；The visible soft bone nonunions of MRI
Continuous, subchondral bone has capsule table；HE and Toluidine blue staining are shown in that defect is filled, and GAG and II Collagen Type VI content are lower slightly.
Couple fibroin-gelatin support (SFG-E7) group of E7 polypeptides：General form is similar to normal cartilage, defect
Filling is complete, and cartilage is glossy；The visible cartilage of MRI results is continuous, and subchondral bone is abnormal without capsule change etc.；HE, toluidine blue and exempt from
Epidemic disease groupization shows that defect filling is good, is tightly combined with perienchyma, and GAG and II Collagen Type VI content are close to normal cartilage.
Cartilage defect repair description of test in rabbit body, it is affine many that what the present invention was provided has coupled mesenchymal stem cells MSCs
The repair of cartilage support of peptide and the repair of cartilage support of Glu-Pro-Leu-Gln-Leu-Lys-Met is not coupled can promote cartilage
Repair, wherein the repair of cartilage support for coupling Glu-Pro-Leu-Gln-Leu-Lys-Met promotes the effect of repair of cartilage more preferable.
It the above is only for the ease of it will be understood by those skilled in the art that technical scheme, not to limit this hair
It is bright.Within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., should be included in the present invention
Protection domain within.
SEQUENCE LISTING
<110>The Third Affiliated Hospital of Peking University
<120>A kind of repair of cartilage support and preparation method thereof
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 7
<212> PRT
<213> Artificial
<220>
<223>Artificial synthesized amino acid sequence
<400> 1
Glu Pro Leu Gln Leu Lys Met
1 5
Claims (17)
1. a kind of preparation method of repair of cartilage support, it is characterised in that including：
The mould of repair of cartilage support is printed using 3D printing technique；The bottom surface of mould have it is a plurality of be parallel to each other it is first convex
Rib；A plurality of the second fin being parallel to each other being connected with the upper surface of first fin；And with second fin
A plurality of the 3rd fin being parallel to each other that upper surface is connected；The width of first fin is 100-500 μm；Described second is convex
The width of rib is 100-500 μm；The width of 3rd fin is 100-500 μm；First fin and second fin
It is in predetermined angle to intersect；Second fin intersects in predetermined angle with the 3rd fin；
Prepare the mixed solution of fibroin and gelatin；
The mixed solution is poured into mould, the liquid level of the mixed solution was not had the 3rd fin；
After after mixed solution solidification plastic, mould is removed, the body of material of repair of cartilage support is obtained；
Body of material to the repair of cartilage support carries out protein denaturation processing；
Body of material to the repair of cartilage support after protein denaturation processing carries out chemical crosslinking processing, is freeze-dried afterwards,
Obtain repair of cartilage support.
2. preparation method according to claim 1, it is characterised in that first fin is vertical with second fin；It is described
Second fin is vertical with the 3rd fin.
3. preparation method according to claim 1, it is characterised in that the width of first fin is 300-500 μm；Described
The width of two fins is 300-500 μm；The width of 3rd fin is 300-500 μm.
4. preparation method according to claim 3, it is characterised in that first fin, second fin and the described 3rd
The width of fin is 400 μm.
5. preparation method according to claim 1, it is characterised in that the gap between two neighboring first fin is 0.4-
0.7mm；Gap between two neighboring second fin is 0.4-0.7mm；Gap between two neighboring 3rd fin is 0.4-
0.7mm。
6. preparation method according to claim 1, it is characterised in that the liquid level of the mixed solution is more convex than described first
The high 0.1-0.3mm of height sum of rib, second fin and the 3rd fin.
7. preparation method according to claim 1, it is characterised in that the fibroin quality in the mixed solution point
Number is 2.3%-4.6%；Mass fraction of the gelatin in the mixed solution is 2.3%-4.6%.
8. preparation method according to claim 7, it is characterised in that the fibroin and the gelatin are in the mixed solution
In mass fraction sum be 6.9%.
9. preparation method according to claim 1, it is characterised in that the protein denaturation processing is by the recovery support
Body of material is placed in the ethanol solution that volume fraction is 95% overnight.
10. preparation method according to claim 1, it is characterised in that the repair of cartilage branch after the processing to protein denaturation
The body of material of frame carries out chemical crosslinking and is processed as the body of material of the repair of cartilage support after protein denaturation processing
It is placed in ambient temperature overnight in the genipin solution that mass fraction is 0.3%-1%.
11. preparation method according to claim 1, it is characterised in that also include：Carry out disinfection place to the repair of cartilage support
Reason.
12. according to the preparation method of any one of claim 1 to 11, it is characterised in that also include：By mesenchymal stem cells MSCs
Affine polypeptide is coupled to the surface of the repair of cartilage support；The amino acid sequence of the Glu-Pro-Leu-Gln-Leu-Lys-Met
Such as SEQ ID NO：Shown in 1.
13. preparation method according to claim 12, it is characterised in that couple the Glu-Pro-Leu-Gln-Leu-Lys-Met
Operating procedure to the surface of the repair of cartilage support is：
Amination treatment is carried out to the repair of cartilage support first；
The repair of cartilage support after amination treatment is soaked in containing the molten of heterobifunctional crosslinking agent under conditions of lucifuge again
In liquid；
Then the repair of cartilage branch containing heterobifunctional crosslinking agent is placed on affine containing the mesenchymal stem cells MSCs
It is incubated in the solution of polypeptide；
The repair of cartilage support for finally having Glu-Pro-Leu-Gln-Leu-Lys-Met to surface modification is freeze-dried, and coupling is made
It is associated with the repair of cartilage support of Glu-Pro-Leu-Gln-Leu-Lys-Met.
14. preparation method according to claim 13, it is characterised in that described that amination treatment is carried out to the repair of cartilage support
It it is 37 DEG C in temperature for the repair of cartilage support is soaked in the ethylenediamine aqueous isopropanol that volume fraction is 5%-15%
Under conditions of, kept for more than 1 hour.
15. preparation method according to claim 13, it is characterised in that the solution containing heterobifunctional crosslinking agent is matter
It is 5%-15% 4- (N- maleimidomehyls) hexamethylene -1- carboxylic acid sulfonic group succinimide ester sodium to measure volume fraction
Dimethyl sulphoxide solution；During the immersion in the solution of the heterobifunctional crosslinking agent of repair of cartilage support after amination treatment
Between be more than 1 hour.
16. preparation method according to claim 13, it is characterised in that described containing Glu-Pro-Leu-Gln-Leu-Lys-Met
Solution be Glu-Pro-Leu-Gln-Leu-Lys-Met concentration be 0.05-0.15mg/mL phosphate buffer solution；The time of incubation
For more than 1 hour.
17. repair of cartilage support prepared by a kind of preparation method of any one of utilization claim 1 to 16.
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