CN108159496A - A kind of bionical orientation cartilage frame of double factor program release and preparation method thereof - Google Patents
A kind of bionical orientation cartilage frame of double factor program release and preparation method thereof Download PDFInfo
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Abstract
The present invention discloses a kind of bionical orientation cartilage frame of double factor program release and preparation method thereof, the bionical orientation cartilage frame is combined by upper and lower materials at two layers, and upper surface layer composite material is made of the PLAG microballoons of collagen, chitosan, hyaluronic acid and load KGN；It is by the polylysine heparin sodium of collagen, chitosan, fibroin albumen and load TGF β 1 that lower floor, which transfers layer composite material,（PLL‑HS）Nanoparticle is made.Bionical orientation cartilage frame prepared by the present invention combines the good biocompatibility of collagen and biological degradability, the antibiotic property and biodegradable of chitosan, the excellent mechanical performances of fibroin albumen, and the lubrication and anti-inflammatory effect of hyaluronic acid, there is good promotion proliferation and differentiation to BMSCs, the alleviation to KOA symptoms and treatment can be reached while morning, mid-term KOA cartilage defects is repaired, be a kind of medical material of comparatively ideal novel osteoarthritis with cartilage defect reparation.
Description
Technical field
The invention belongs to bio-medical fields, and in particular to a kind of bionical orientation cartilage frame of double factor program release and
Preparation method.
Background technology
Knee osteoarthritis（KOA）It is that one kind the inflammation such as deformation, osteoproliferation and necrosis occurs with patient articular cartilage
The osteoarthritis disorders that venereal disease change is characterized, wherein with the regression of cartilage progressive and subchondral bone remodeling for main feature, it is clinical
It is mainly shown as kneecap joint rest pain, swelling, moving obstacle, deformity of knee etc., seriously affects the health of patient.
Clinic is broadly divided into the treatment of KOA two kinds of operative treatment and non-operative treatment.Operative treatment mainly have it is self or
Person's allograph bone cartilage transplantation, joint debridement art, Drilling and knee joint peripheral osteotomy, knee fusion, artificial total knee
Joint replacement etc.；Non-operative treatment mainly includes rehabilitation, drug therapy and acupuncture and tuina-massage treatment, wherein, supplemented with exogenous
Property hyaluronic acid have and repair destroyed barrier, improve synovia physiological function, alleviate arthralgia, bacterium and toxin etc. are invaded
Enter the effect of its protective barrier.
In articular cartilage in-situ regeneration, transforming growth factor（TGF-β1）Not only have and promote mesenchymal stem cell
（BMSCs）Be proliferated and be finally divided into the effect of cartilage cell, at the same also by inhibit the generation of matrix metalloproteinase and
It plays a significant role in KOA treatments.In addition, micromolecular compound Kartogenin in recent years（KGN）It is found to have extremely strong
Promote the effect that BMSCs promotes cartilage cell's lubricin protein secretions to cartilage differentiation, collaboration TGF-β 1 and BMP-7.Except this it
Outside, KGN may additionally facilitate tendon-bone junction and form chondroid tissue, and the cartilage energy that the KGN combined with chitosan is more unbonded
Power is stronger.Therefore, the treatment for being divided into disease defective tissue to target cell system using KGN directional inductions stem cell provides one kind
New effective ways.But TGF-β 1 and KGN there are action time it is relatively short, be easily degraded and act on the problems such as transient, such as
Fruit can be put by controlling sustained release, can preferably play its biological action.
In addition, normal cartilage is made of superficial layer, middle layer, lower floor, calcified layer, autologous bone pulp cavity BMSCs is participated in soft
Bone remoulding can reach middle layer or upper strata by calcified layer and lower floor.Bionic Design of the present invention is similar to natural cartilage structure
The cartilage frame with upper and lower double-layer structure, while in lower floor support introduce up/down perforation array micro-tubular structure（It is conducive to
BMSCs is transferred）, be incorporated in lower floor support contain promote BMSCs proliferation TGF-β 1, upper layer bracket contain promote BMSCs differentiation
KGN, and pass through material structure and factor embedding difference design realization TGF-β 1, the release of KGN bimoleculars program, it can reach effectively
Repair the purpose of cartilage defect.
Invention content
Bionical orientation cartilage frame the purpose of the present invention is to provide a kind of release of double factor program and preparation method thereof,
It is on the basis of seed cell, growth factor and stock support three aspect factor has been considered, and a kind of of structure can allow
Endogenous BMSCs is easy to migrate, and the bionical orientation cartilage frame of proliferation and differentiation is successively completed during transferring, can be
Reach the alleviation to KOA symptoms and treatment while repairing morning, mid-term KOA cartilage defects.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of bionical orientation cartilage frame of claimed double factor program release is with collagen, chitosan, transparent
Matter is sour, the PLGA microballoons of load KGN prepare superficial layer composite material for raw material, with collagen, chitosan, fibroin albumen, load
The PLL-HS nanoparticles of TGF-β 1 transfer a layer composite material, then bond the two using fibroin albumen as adhesive for raw material preparation,
The bionical orientation cartilage frame with Dual-layer structure is made.
The collagenous source is in fish-skin, pigskin, ox-hide or beef tendon；The fibroin albumen is obtained from silk cocoon.
The preparation method of the bionical orientation cartilage frame of the double factor program release is also claimed in the present invention, including
Following steps：
First, the preparation of superficial layer composite material
（1）Collagen, chitosan are dissolved in glacial acetic acid respectively, the collagen solution of a concentration of 0.6 wt% and a concentration of 1 wt% is made
Chitosan solution；
（2）Hyaluronic acid is dissolved in the hyaluronic acid solution that a concentration of 1 wt% is made in distilled water or deionized water；
（3）By step（1）Gained collagen solution is uniformly mixed with chitosan solution whirlpool, is slow added into step（2）Institute
Hyaluronic acid solution is obtained, is uniformly mixed；Wherein, the volume ratio of collagen solution, chitosan solution and hyaluronic acid solution mixing is 1
~10:10:1~10；
（4）By step（3）Gained mixed solution magnetic stirrer over night adds crosslinking agent after freeze-dried and is crosslinked；It is described
Crosslinking agent be by 2- (N- morpholines) ethanesulfonic acid monohydrate, 1- ethyls-（3- dimethylaminopropyls）Carbodiimides and N- hydroxyls
Base succimide, which is dissolved in the ethanol solution that volumetric concentration is 40%, to be made, wherein 2- (N- morpholines) ethanesulfonic acid monohydrate, 1-
Ethyl-（3- dimethylaminopropyls）The molar ratio of carbodiimides and N- hydroxysuccinimides is 50:33:8；
（5）Product after crosslinking is rinsed with distilled water repeatedly to neutrality, answered after secondary freeze drying up to the superficial layer of freeze-drying
Condensation material；
（6）By patent（CN 106729987A）Disclosed in method prepare load KGN PLGA microballoons；
（7）By step（6）The PLGA microballoons of the load KGN of preparation are uniformly injected into the superficial layer composite material of freeze-drying with syringe
In, and be lyophilized again, superficial layer composite material is obtained, wherein the content of the PLGA microballoons of the load KGN factors is 0.03wt%；By institute
Obtain the cylinder that 5 mm of diameter, high 2 mm are made of card punch for superficial layer composite material；
2nd, the preparation of layer composite material is transferred
（8）Separately collagen, chitosan is taken to be dissolved in glacial acetic acid respectively, the collagen solution of a concentration of 0.6 wt% and a concentration of 1 wt% is made
Chitosan solution；
（9）Using patent（CN 106581776A）Disclosed in method the silk for obtaining a concentration of 1.343 wt% is extracted from silk cocoon
Fibroin；
（10）By step（8）Gained collagen solution is uniformly mixed with chitosan solution whirlpool, is subsequently placed in magnetic stirring apparatus
On be slowly stirred, be slow added into step（9）Gained fibroin albumen is uniformly mixed；Collagen solution, chitosan solution and fibroin egg
The mass ratio mixed in vain is 1:1:0~3；
（11）Prepare the PLL-HS nanoparticles of load TGF-β 1；
（12）By step（11）The PLL-HS nanoparticles of the load TGF-β 1 of preparation are added to step（10）In gained mixed liquor
Even mixing；
（13）By step（12）Gained mixed solution magnetic agitation 2 hours is then injected into mold and is slowly put into liquid vertically
Freeze overnight in nitrogen；Mould therefor construction such as patent（CN 106581776A）Described in；
（14）By step（13）Product after freeze-drying is crosslinked in crosslinking agent Geniposide；
（15）Cross-linking products are rinsed with distilled water repeatedly to neutrality, a layer composite material are transferred to obtain the final product after secondary freeze drying,
A concentration of 0.102 mg/cm of middle TGF-β 12；Then it is cut to the column of long 4 mm；
3rd, the preparation of cartilage frame
（16）By step（7）Gained cylindrical shape superficial layer composite material and step（15）Gained column transfers layer composite material and uses
Fibroin albumen is bonded, and is dried in 40 DEG C of baking oven, as the bionical orientation cartilage frame of double factor program release.
Wherein, the mass concentration of glacial acetic acid used is 1%.
Step（11）The preparation method of the PLL-HS nanoparticles of the load TGF-β 1 is as follows：
1）By the polylysine that molecular weight is 7000-15000 Da（PLL）It is dissolved in deionized water, a concentration of 1 mg/mL is made
PLL solution；
2）0.4 mg TGF-β 1 is dissolved in 0.2 mL deionized waters, adds in the heparin sodium aqua of a concentration of 1 mg/mL of 5 mL,
Mixing is overnight；
3）By step 2）Gained mixed liquor is added to step 1）In the PLL solution of gained, 15 are shaken on vortex shaking mixer
S, then 30 min of incubation at room temperature is to get to the PLL-HS nanoparticles for containing TGF-β 1.
Preparation process of the present invention is simple, cheap and easy to get, and the bionical orientation cartilage frame of double factor program release prepared
In, superficial layer is in tridimensional network, transfers layer in micro-pipe shape, has good hygroscopicity and porosity, can allow endogenous
BMSCs is easy to migrate, and proliferation and differentiation are successively completed during transferring.
The present invention combines the good biocompatibility of collagen and biological degradability, the antibiotic property of chitosan and can biology drop
Xie Xing, the excellent mechanical performances of fibroin albumen and the lubrication of hyaluronic acid and anti-inflammatory effect have gained composite material good
The characteristics of biocompatibility well, nontoxicity, degradability and non-immunogenicity, and it has considered seed cell, growth
The factor and stock support three aspect factor, can reach pair while early, mid-term KOA cartilage defects are repaired to greatest extent
The alleviation and treatment of KOA symptoms are a kind of medical materials of comparatively ideal novel osteoarthritis with cartilage defect reparation.
Description of the drawings
Fig. 1 is the scanning electron microscope (SEM) photograph of the bionical orientation cartilage frame of double factor program release of the present invention.
Fig. 2 is rabbit knee osteoarthritis cartilage defect repair HE Coloration experiment result figures, and wherein A, B, C, D is respectively mould
Type control group superficial layer composite material group, transfers a layer composite material group, the bionical orientation cartilage frame that double factor program discharges
Group.
Specific embodiment
In order to which content of the present invention is made to easily facilitate understanding, With reference to embodiment to of the present invention
Technical solution is described further, but the present invention is not limited only to this.
Embodiment 1
1. the accurate 200 mg PLGA that weigh are dissolved in 4 mL dichloromethane, sonic oscillation forms oil phase；10 mg of precise again
KGN is placed in the ultra-pure water of 1 mL and the mixed liquor of methanol（1:1, v/v）In, it makes it completely dissolved to form inner aqueous phase at 37 DEG C.
Gained inner aqueous phase is slowly injected into gained oil phase, continues 10 min of stirring and emulsifying, it is uniformly dispersed and forms colostrum emulsion.It will
Colostrum emulsion is instilled in 50 mL, 1% PVA solutions containing 5 mL, 0.1% Tween-20s by 60 drops/min and forms outer aqueous phase, is continued
Stirring forms emulsion.Gained emulsion is fully centrifuged, abandons supernatant, then rinse centrifugation 3 times repeatedly through ultra-pure water, freeze-drying,
The PLGA microballoons of KGN must be loaded, are kept in dark place at -20 DEG C.
2. the PLL that molecular weight is 7000-15000 Da is dissolved in deionized water, the PLL of a concentration of 1 mg/mL is made
Solution.0.4 mg TGF-β 1 is dissolved in 0.2 mL deionized waters again, adds in the heparin sodium aqua of a concentration of 1 mg/mL of 5 mL,
Mixing is overnight.The mixed liquor of gained heparin sodium and TGF-β 1 is added in PLL solution, is shaken on vortex shaking mixer
15 s, then 30 min of incubation at room temperature is to get to the PLL-HS nanoparticles for containing TGF-β 1.
3. weighing 45.1127 g beef tendon collagens to be dissolved in the glacial acetic acid solution of 100 mL a concentration of 0.1%, it is placed on stirring
The beef tendon collagen solution that mass fraction is 0.6 % is made in stirring and dissolving on device.
4. weighing 1.0 g chitosans to be dissolved in the glacial acetic acid solution of 100 mL a concentration of 0.1%, it is placed on blender and stirs
Dissolving is mixed, the chitosan solution that mass fraction is 1% is made.
5. weighing 0.5 g hyaluronic acids to be dissolved in 50 mL distilled water or deionized water solution, it is placed on blender and stirs
Dissolving is mixed, the hyaluronic acid solution that mass fraction is 1 % is made.
6. the silk cocoon shredded is put into the Na that mass fraction is 0.5%2CO3In solution, 30 are placed in 98 ± 2 DEG C of water-baths
Min removes the colloid on silk cocoon, is dried after operating 3 times repeatedly, obtains fibroin；At 78 DEG C, fibroin is weighed, with mass ratio 1:
25 in CaCl2/H2O/C2H5OH mixed solutions（CaCl2、H2O、C2H5The molar ratio of OH is 1:8:2）Middle dissolving, filtered through gauze obtain
To coarse filtration liquid；Coarse filtration liquid is poured into the bag filter that molecular cut off is 8000-14000 3 d that dialyse, removes calcium chloride and second
Alcohol；It refilters, obtains silk fibroin protein solution；5 clean glass dishes are taken, are weighed（M0）, suitable silk is added in each culture dish
Fibroin is weighed（M1）, weigh after being placed in 80 DEG C of 8 h of baking oven, then weigh every 30 min, until weight is not changing（M2）.
Use formula：The concentration of fibroin albumen（%）=[(M2-M1)/(M1-M0)] × 100% calculates concentration, is averaged.The fibroin of extraction
A concentration of the 1.343% of albumen.
7. the beef tendon collagen solution of 0.6 % is added in the centrifuge tube of 50 mL, whirlpool on whirlpool device is placed on, is then pressed
Volume ratio 10:10:1 adds in 1% chitosan solution mixing in whirlpool, is slow added into the hyaluronic acid solution of 1 %, is made
Collagen/chitosan/hyaluronic acid solution of mixing.The PLGA microballoons of making are added to collagen/shell by the amount of 500 μ g/mL again
It in glycan/hyaluronic acid mixed solution, by it, is placed on magnetic stirring apparatus and is stirred overnight, be then placed in cold in freeze drier
The composite material dry, cross-linking agents is recycled to be lyophilized is lyophilized, crosslinking agent used is by 2- (N- morpholines) ethanesulfonic acid monohydrate
（MES）, 1- ethyls -3（3- dimethyl aminopropyls）Carbodiimides (EDC) and N- maloyls (NHS) are dissolved in volumetric concentration
To be made in 40% ethanol solution, the mass ratio of MES/EDC/NHS is 50：33：8.Product after crosslinking is rushed repeatedly with distilled water
Neutrality is washed till, secondary freeze drying simultaneously disinfects to load the superficial layer composite material of the KGN factors.Then superficial layer is answered
Condensation material is fabricated to the cylinder of a diameter of 5 mm, a height of 2 mm with the card punch of a diameter of 5 mm.
8. the beef tendon collagen solution of 0.6 %, 1% chitosan solution and 1.343% fibroin albumen are in addition weighed, by matter
Measure ratio 1：1：0.5 first by beef tendon collagen solution, the whirlpool under the action of whirlpool blender mixes 2~3 minutes with chitosan solution,
It is then placed on magnetic stirring apparatus and is slowly stirred, be slowly added to fibroin albumen, and continue stirring 2 hours, it is outstanding to obtain mixing
Turbid.Again by the PLL-HS nanoparticles for containing TGF-β 1 prepared by 1 a concentration of 0.102 mg/cm of TGF-β2It is added to mixing
In suspension, continue stirring 10 minutes, then carry out vacuumize process, stand 10~15 h again later to get to 1/ glue of TGF-β
Original/chitosan/Silk fibroin gel shape suspension.
9. by 1/ collagen/chitosan of gained TGF-β/Silk fibroin gel shape suspension injection conduit molding die, with
The both ends of iron staff confined bed pipe shaping mould.By conduit molding die direct-axis to slowly and evenly immersing liquid in a manner of fishing
In nitrogen, it is solid that cold leaching is carried out to 1/ collagen/chitosan of TGF-β/Silk fibroin gel shape suspension in injection conduit molding die
Type processing, after conduit molding die is completely immersed in liquid nitrogen, continues to retain 10 h in liquid nitrogen.By conduit molding die together with interior
1/ collagen/chitosan of TGF-β/Silk fibroin gel shape suspension of portion's injection is transferred to cold in -90 DEG C~-70 DEG C of refrigerator together
It hides.Conduit molding die is taken out from -90 DEG C~-70 DEG C of refrigerator, the rapid iron staff for removing conduit molding die both ends.It will
Conduit molding die is positioned over the freeze drier being pre-chilled and is lyophilized 3 days.Conduit molding die after freeze-drying is positioned over vacuum
It under state and is warmed to room temperature, keeps 6 h, release vacuum state.Composite material from catheter mold is taken out, that is, is prepared more
1/ collagen/chitosan of micropore TGF-β/fibroin albumen conduit composite material.
10. weighing Geniposide and absolute ethyl alcohol respectively, it is mixed the capital Buddhist nun for being configured to that mass concentration is 0.5%~1.5%
Flat alcohol mixed solution.1/ collagen/chitosan of more micropore TGF-β prepared/fibroin albumen conduit composite material is put into capital
Crosslinking Treatment is carried out in the flat alcohol mixed solution of Buddhist nun, crosslinking time is 48 h；After crosslinked processing, by more 1/ glue of micropore TGF-β
Original/chitosan/fibroin albumen composite material deionized water and the ethyl alcohol that mass concentration is 95% clean repeatedly.It is more after cleaning
1/ collagen/chitosan of micropore TGF-β/fibroin albumen composite material is again placed in being lyophilized in freeze drier immediately, finally uses Co60
Irradiate 1/ collagen/chitosan of more micropore TGF-β/fibroin albumen composite material carry out disinfection processing to get to load TGF-β 1
PLL-HS nanoparticles transfer a layer composite material, are then cut to the column of long 4 mm.
11. by the columnar layer composite material that transfer of the superficial layer composite material and gained of gained cylindrical shape with a small amount of silk
Fibroin is bonded, and the bionical orientation cartilage frame discharged to get double factor program is dried in 40 DEG C of baking oven.
Embodiment 2
（1）The preparation of knee osteoarthritis cartilage defect models：20 Male New Zealand rabbits are bought, are randomly divided by weight
4 groups, every group 5, it is respectively：A groups, model control group；B groups, superficial layer composite material group；C groups transfer a layer composite material group；D
Group, the bionical orientation cartilage frame group of double factor program release.The intramuscular injection Su-Mian-Xin Ⅱ 0.3mL/ after adaptability is fed 1 week
Kg establishes osteoarthritis using preceding right-angled intersection ligament amputation.Postoperative continuous 1 week intramuscular injection Benzylpenicillin sodium salt（150000
U/ pcs/day）, continue to feed January and daily appropriate exercise.Continue 0.3 mL/kg Su-Mian-Xin Ⅱs of intramuscular injection after January to carry out
It anaesthetizes, then longitudinally slit knee joint epidermis and musculature under gnotobasis, with manual corneal trephine in kneecap joint table
Face drills, and the animal model of gained is knee osteoarthritis cartilage defect models.Injury tissue size is 5 mm of diameter,
High 4 mm.
（2）In above-mentioned knee osteoarthritis cartilage defect models, in addition to A groups, planted respectively at B, C, D group defect
Enter superficial layer composite material, transfer layer composite material and embodiment 1 prepare double factor program release bionical orientation cartilage branch
Frame（Superficial layer composite material transfers layer composite material respectively by embodiment 1 step 1-7 and step 8-10 preparation）.It is implanted into material
Kneecap is resetted afterwards and is successively tightly sewed up a wound, is raised with Iodophor to wound disinfection and individually.Postoperative continuous intramuscular injection in 1 week
Benzylpenicillin sodium salt（150000 U/ pcs/day）, during which normally give diet.Animal is put to death in the excessive Su-Mian-Xin Ⅱ of injection in the 16th week, is taken out
Sample carries out pathological section, as a result Fig. 2 is shown in HE staining analysis.
Fig. 2 the result shows that, the repair of cartilage of the bionical orientation cartilage frame of double factor program release is with obvious effects better than single
The superficial layer and transfer a layer composite material that the factor discharges, are a kind of the medical of comparatively ideal knee osteoarthritis cartilage defect repair
Material.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification should all belong to the covering scope of the present invention.
Claims (10)
1. a kind of bionical orientation cartilage frame of double factor program release, it is characterised in that：Using collagen, chitosan as main original
Material coordinates with hyaluronic acid, the PLGA microballoons of load KGN and fibroin albumen, the PLL-HS nanoparticles of load TGF-β 1 make respectively
For superficial layer composite material and a layer composite material is transferred, then the two is bonded using fibroin albumen as adhesive, being made has up and down
The bionical orientation cartilage frame of double-layer structure.
2. the bionical orientation cartilage frame of double factor program release according to claim 1, it is characterised in that：The collagen
From fish-skin, pigskin, ox-hide or beef tendon；The fibroin albumen is obtained from silk cocoon.
3. a kind of preparation method of the bionical orientation cartilage frame of double factor program release as described in claim 1, feature
It is：Include the following steps：
First, the preparation of superficial layer composite material
（1）Collagen, chitosan are dissolved in glacial acetic acid respectively, collagen solution and chitosan solution is made；
（2）Hyaluronic acid solution is made by hyaluronic acid is soluble in water；
（3）By step（1）Gained collagen solution is uniformly mixed with chitosan solution whirlpool；
（4）By step（2）Gained hyaluronic acid solution is slowly added to step（3）In gained mixed liquor, it is uniformly mixed；
（5）By step（4）Gained mixed solution magnetic stirrer over night adds crosslinking agent after freeze-dried and is crosslinked；
（6）Product after crosslinking is rinsed with distilled water repeatedly to neutrality, answered after secondary freeze drying up to the superficial layer of freeze-drying
Condensation material；
（7）Prepare the PLGA microballoons of load KGN；
（8）By step（7）The PLGA microballoons of the load KGN of preparation are uniformly injected into the superficial layer composite material of freeze-drying with syringe
In, and be lyophilized again, obtain superficial layer composite material；
（9）By step（8）The cylinder of 5 mm of diameter, high 2 mm are made of card punch for gained superficial layer composite material；
2nd, the preparation of layer composite material is transferred
（10）Separately collagen, chitosan is taken to be dissolved in glacial acetic acid respectively, collagen solution and chitosan solution is made；
（11）Prepare fibroin albumen；
（12）By step（10）Gained collagen solution is uniformly mixed with chitosan solution whirlpool, is subsequently placed in magnetic stirring apparatus
On be slowly stirred；
（13）By step（11）Gained fibroin albumen is slowly added into step（12）In gained mixed liquor, it is uniformly mixed；
（14）Prepare the PLL-HS nanoparticles of load TGF-β 1；
（15）By step（14）The PLL-HS nanoparticles of the load TGF-β 1 of preparation are added to step（13）In gained mixed liquor
Even mixing；
（16）By step（15）Gained mixed solution magnetic agitation 2 hours is then injected into mold and is slowly put into liquid vertically
Freeze overnight in nitrogen；
（17）By step（16）Product after freeze-drying is crosslinked in crosslinking agent；
（18）Cross-linking products are rinsed with distilled water repeatedly to neutrality, transfer a layer composite material to obtain the final product after secondary freeze drying, so
It is cut to the column of long 4 mm afterwards；
3rd, the preparation of cartilage frame
（19）By step（9）Gained cylindrical shape superficial layer composite material and step（18）Gained column transfers layer composite material and uses
Fibroin albumen is bonded, the bionical orientation cartilage frame of 40 DEG C of drying, the as release of double factor program.
4. the preparation method of the bionical orientation cartilage frame of double factor program release according to claim 3, feature exist
In：In the preparation of superficial layer composite material, a concentration of 0.6 wt% of collagen solution used, a concentration of the 1 of chitosan solution used
Wt%, a concentration of 1 wt% of hyaluronic acid solution used；The volume ratio of three's mixing is 1 ~ 10:10:1~10.
5. the preparation method of the bionical orientation cartilage frame of double factor program release according to claim 3, feature exist
In：Step（5）Described in crosslinking agent be by 2- (N- morpholines) ethanesulfonic acid monohydrate, 1- ethyls-（3- dimethylaminopropyls）
Carbodiimides and N- hydroxysuccinimides, which are dissolved in the ethanol solution that volumetric concentration is 40%, to be made；
Wherein 2- (N- morpholines) ethanesulfonic acid monohydrate, 1- ethyls-（3- dimethylaminopropyls）Carbodiimides and N- hydroxyls
The molar ratio of succimide is 50:33:8.
6. the preparation method of the bionical orientation cartilage frame of double factor program release according to claim 3, feature exist
In：The content of the PLGA microballoons of load KGN is 0.03 wt% in superficial layer composite material obtained by step 1.
7. the preparation method of the bionical orientation cartilage frame of double factor program release according to claim 3, feature exist
In：It transfers in the preparation of layer composite material, a concentration of 0.6 wt% of collagen solution used, a concentration of the 1 of chitosan solution used
A concentration of 1.343 wt% of wt%, fibroin albumen used；The mass ratio of three's mixing is 1:1:0~3.
8. the preparation method of the bionical orientation cartilage frame of double factor program release according to claim 3, feature exist
In：The preparation method of the PLL-HS nanoparticles of the load TGF-β 1 is as follows：
1）The PLL that molecular weight is 7000-15000 Da is dissolved in deionized water, the PLL solution of a concentration of 1 mg/mL is made；
2）0.4 mg TGF-β 1 is dissolved in 0.2 mL deionized waters, adds in the heparin sodium aqua of a concentration of 1 mg/mL of 5 mL,
Mixing is overnight；
3）By step 2）Gained mixed liquor is added to step 1）In the PLL solution of gained, 15 are shaken on vortex shaking mixer
S, then 30 min of incubation at room temperature is to get to the PLL-HS nanoparticles for containing TGF-β 1.
9. the preparation method of the bionical orientation cartilage frame of double factor program release according to claim 3, feature exist
In：Step（17）Described in crosslinking agent be Geniposide.
10. the preparation method of the bionical orientation cartilage frame of double factor program release according to claim 3, feature exist
In：A concentration of 0.102 mg/cm of TGF-β 1 in layer composite material is transferred obtained by step 22。
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| CN113877000A (en) * | 2021-10-13 | 2022-01-04 | 科笛生物医药(无锡)有限公司 | Microsphere composition for injection and application thereof |
| CN115957377A (en) * | 2023-01-11 | 2023-04-14 | 四川大学 | Injectable functionalized collagen-based hydrogel for cartilage repair |
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