CN110433342A - A kind of preparation method of compound VEGF and rhBMP-2 biphase coating articular prosthesis - Google Patents
A kind of preparation method of compound VEGF and rhBMP-2 biphase coating articular prosthesis Download PDFInfo
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/28—Materials for coating prostheses
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/606—Coatings
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Abstract
A kind of preparation method of compound VEGF and rhBMP-2 biphase coating articular prosthesis, it is related to nano coating field, the present invention is to solve current joint prosthesis coatings to promote interface angiogenesis, accelerates the formation of osteoid and reconstruction effect undesirable, causes stability poor；Especially, artificial joint handle controlled-release coating is made using ethyl cellulose and polyethylene glycol mixing as the compound rhBMP2 of slow-released system, it cannot promote interface angiogenesis, induced osteogenesis effect is poor, it is bad so as to cause joint prosthesis stability, cause risk and the revision procedure rate of the complication such as artificial prosthesis early stage loosening high, the problem of the long-term survival rate difference of prosthese, the present invention will be by VEGF165 and rhBMP-2, ethyl cellulose, Macrogol 4000, which combines, is prepared nano coating articular prosthesis, it can effectively improve the synostosis at joint prosthesis interface.The present invention is applied to articular prosthesis field.
Description
Technical field
The present invention relates to nano coatings, and in particular to a kind of compound VEGF and rhBMP-2 biphase coating articular prosthesis
Preparation method.
Background technique
Prosthetic replacement's treatment means common as orthopaedics, with the horizontal raising of surgical technic operation, prosthese with
The stability of bone interface is increasingly becoming the major issue of artificial joint replacement and revision procedure.Biotype artificial is closed
Section, the interface stability of joint and bone are the key that determine joint service life and improve patients ' life quality, wherein prosthese with
The determinant of bone interface stability depends primarily on synostosis degree.Increase vacation by improveing the coating of prosthetic surface
Body and the bone tissue at bone-contact interface form the direction for having become generally acknowledged articular prosthesis development.
Bone tissue, which is formed, is the complexity for being participated in and being adjusted by many factors and orderly process, bone morphogenetic protein
(BMP) it is to be currently known the uniquely energy growth factor that individually inducing bone mesenchymal stem cell breaks up to bone tissue direction, is bone
The regulatory factor of most critical during organizing the formation of, by acting synergistically with other cell factors in the tune of cell receptor level
Section, inducing cell are converted to bone and cartilage direction.Bone morphogenetic protein 2 (BMP-2) as a kind of cell factor for facilitating bone,
Effectively it can break up and be formed bone tissue to skeletonization direction by inducing bone mesenchymal stem cell, using the inducing action of BMP-2,
Increase prosthese and bone-contact interface into bone amount, to become the important application direction of prosthetic surface coating research.Many researchs
Have confirmed that BMP-2 promotes skeletonization definite effect effective.But BMP-2 needs because being easy to degrade loss by means of special
Slow-released system can obtain preferable biological effect.
Filling stem cell between many research trial VEGF inducing bone marrows all the time promotes revascularization to achieve certain effect
Fruit.Wherein the reports sodium alginate such as Janos M.Kanczler embedding VEGF being capable of significant inducing bone mesenchymal stem cell
Bone tissue is differentiated to form to skeletonization direction.But it is how cell factor and BMP-2 is compound and obtain good slow release effect
And keeping bioactivity is always the technology being concerned.
Ideal controlled-release coating should combine closely with articular surface, and thickness is uniform, not easy to lose, be easy to save and sterilize,
The process of the compound physiology skeletonization of slow-release time and it is easy to regulate and control, while requires biological safety height, histocompatbility is good, delays
The advantages that it is definite to release effect stability, is sustained process control, while chemical physical property also being required to stablize, it is not easy to be destroyed, be convenient for
The features such as disinfection.
The chemical structure of polyethylene glycol (PEG): HO (CH2CH2O) nH is formed by ethylene oxide polymerization, energy and multi-solvents
It is good compatible, be ideal solvent, property is stable, is unlikely to deteriorate, and has good compatibility with drug, can be convenient for medicine
The release and absorption of object, while medical surfaces being made smoothly to be not easy to be damaged.The chemical structure of ethyl cellulose (EC): [C6H7O2
(OC2H5)3] n plays a significant role, according to amount used in terms of being sustained release with good physical stability
Difference, can be with the rate of release of regulating medicine.The two is mainly used for the preparation of sustained-release dosage type drug, biofacies in pharmaceutical field
Capacitive safety is authenticated by U.S. FDA.
Present applicant selects the sustained-release matrix in pharmaceutical field, with above-mentioned ethyl cellulose (EC) and polyethylene glycol
Artificial joint handle controlled-release coating is made as the compound rhBMP2 of slow-released system (rhBMP-2) in mixing, can
By the effect of PEG make rhBMP-2 in conjunction with articular surface uniformly, closely, coating is smooth, loses small in art, is hardly damaged, again
Good slow release effect, the enhancing of joint prosthesis stability can be reached by the effect of EC.It is detailed in Publication No. CN
101721743B, patent name are as follows: the method for strengthening joint stability by using rhBMP-2 release coating on surface of artificial joint.But it should
Patent is unsatisfactory in terms of reinforcing joint prosthesis stability, that is, can not achieve and promote interface angiogenesis, accelerates osteoid
Formation and reconstruction, to improve work stability of joint.
Currently, the loosening of prosthetic replacement's early stage prosthese is increasingly becoming the primary complication of artificial joint replacement.Surely
Qualitative is the key that determine joint service life and application quality, for the joint prosthesis that biomethanics is fixed, the bone at interface
Combination degree is the determinant of its stability.In the ongoing research for reinforcing joint prosthesis stability, on the one hand it is dedicated to
Prosthese geometric shape is improved, on the other hand reinforces stability of joint by improving prosthetic surface coating, passes through coating induced osteogenesis
The research of bone-prosthese interface Bone Ingrowth is promoted to become one of hot spot.Coating research is also showed by hydroxyapatite
(hydroxyapatites, HA) arrives the development trend of the coating modified research of prosthetic surface, how to improve to prosthese coating,
Promote interface angiogenesis, accelerate the formation and reconstruction of osteoid, further decreases the complication such as artificial prosthesis early stage loosening
Risk and revision procedure rate, make patient be easier to receive joint replacement surgery, improve the good results degree of operation, promote prosthese
Long-term survival rate.It is current urgent problem.
Summary of the invention
The purpose of the present invention is to solve current joint prosthesis coatings to promote interface angiogenesis, accelerates the shape of osteoid
At with reconstruction effect it is undesirable, cause stability poor；Especially, it is mixed using ethyl cellulose (EC) and polyethylene glycol as sustained release
Artificial joint handle controlled-release coating is made in the compound rhBMP2 of system (rhBMP-2), cannot promote interface blood vessel
It generating, induced osteogenesis effect is poor, and it is bad so as to cause joint prosthesis stability, cause the complication such as artificial prosthesis early stage loosening
Risk and revision procedure rate are high, the problem of the long-term survival rate difference of prosthese.And a kind of compound VEGF and rhBMP-2 two-phase is provided and is received
The preparation method of rice coating articular prosthesis.
A kind of preparation method of compound VEGF and rhBMP-2 biphase coating articular prosthesis of the invention, it be according to
What following steps carried out:
One, VEGF165 and rhBMP-2 are added separately to aqueous acetic acid, obtain VEGF165 mixed liquor, and
RhBMP-2 mixed liquor；VEGF165 mixed liquor is uniformly mixed with rhBMP-2 mixed liquor, 4h is freeze-dried, obtains rhBMP-2/
The mixed liquor of VEGF165；
Two, ethyl cellulose and Macrogol 4000 are added in chloroform, then 25 DEG C of ultrasonic dissolutions, add
The mixed liquor of rhBMP-2/VEGF165 obtains the mixed liquor of composite slow release rhBMP-2/VEGF165；
Three, the mixed liquor of composite slow release rhBMP-2/VEGF165 is sprayed on artificial femur handle, is subsequently placed in ultra-clean work
Make to dry in platform, be packaged in sealed bag, ethylene oxide cold sterilization is completed；Wherein, VEGF165, rhBMP-2, ethyl are fine
The mass ratio of dimension element and Macrogol 4000 is 0.005-0.015:1:23000~27000:4000~6000；RhBMP-2 and chlorine
Imitative mass volume ratio is 1mg:1.5~2.5mL.
The present invention include it is following the utility model has the advantages that
The present invention is for the first time by new sustained release system by vascular endothelial growth factor (VEGF165) and recombinant human bone form
Generation albumen 2 (rhBMP-2) is compound, while playing BMP-2 promotion skeletonization, promotes bone and prosthese interface using the VEGF same period
Vascularization, accelerate osteoid formation, reconstruction, can be comprehensive increase joint early stage stability.And ethyl fibre is added
Tie up the controlled-release coating that the slow-released system that plain (EC) and polyethylene glycol (PEG) construct is mixed and made into titanium plate surface.It not only can be by poly-
The effect of ethylene glycol (PEG) combines rhBMP-2 and VEGF drug uniformly with titanium plate surface and can be formed close smooth
Coating is easy to save, while can obtain good slow release effect according to the characteristics of ethyl cellulose (EC) again.
The present invention selects the VEGF factor to promote BMP-2 prosthese coating interface angiogenesis, accelerates the formation of osteoid and changes
It builds, proposes the new approaches of same period promotion bone interface vascularization, can effectively improve the synostosis at joint prosthesis interface, into one
Step reduces the risk and revision procedure rate of the complication such as artificial prosthesis early stage loosening, and patient can be made to be easier to receive joint replacement
Operation, improves the good results degree of operation, promotes the long-term survival rate of prosthese.
The design that controlled-release coating of the invention is established as bion articular prosthesis surface covering provides new thinking, solves
The high efficiency cell factor and the BMP-2 compound key technology to obtain good slow release effect inquire into VEGF to prosthese and bone circle
The facilitation of face vascularization provides foundation for clinical Transformation Application from now on.
Detailed description of the invention
Fig. 1 is 500 × electron microscope before the degradation of sections observation HA coating in embodiment 1；
Fig. 2 is 8 days 300 × electron microscopes after the compound cells of sections observation HA coating in embodiment 1；
Fig. 3 is 1000 × electron microscope before the degradation of sections observation HA+VEGF coating in embodiment 1；
Fig. 4 is 8 days 1000 × electron microscopes after the compound cells of sections observation HA+VEGF coating in embodiment 1；
Fig. 5 is 1000 × electron microscope before the degradation of sections observation HA+BMP-2 group coating in embodiment 1；
Fig. 6 is 8 days 1000 × electron microscopes after the compound cells of sections observation HA+BMP-2 coating in embodiment 1；
Fig. 7 is 1000 × electron microscope before the degradation of sections observation HA+ controlled-release coating in embodiment 1；
Fig. 8 is 8 days 1000 × electron microscopes after the compound cells of sections observation HA+ controlled-release coating in embodiment 1；
Fig. 9 is 1000 × electron microscope before the degradation of sections observation HA+BMP-2+VEGF group coating in embodiment 1；
Figure 10 is 8 days 1000 × Electronic Speculum after the compound cells of sections observation HA+BMP-2+VEGF group coating in embodiment 1
Figure；
Figure 11 is that sections observation HA+ is sustained 1000 × electron microscope before the degradation of BMP-2 group coating in embodiment 1；
Figure 12 is 1000 × Electronic Speculum that sections observation HA+ is sustained 8 days after the compound cells of BMP-2 group coating in embodiment 1
Figure；
Figure 13 is that sections observation HA+ is sustained 1000 × electron microscope before the degradation of VEGF group coating in embodiment 1；
Figure 14 is 1000 × Electronic Speculum that sections observation HA+ is sustained 8 days after the compound cells of VEGF group coating in embodiment 1
Figure；
Figure 15 is that sections observation HA+ is sustained 1000 × electron microscope before the degradation of BMP-2/VEGF group coating in embodiment 1；
Figure 16 be embodiment 1 in sections observation HA+ be sustained BMP-2/VEGF group coating compound cells after 8 days 1000 ×
Electron microscope；
Figure 17 is 1000 × Electronic Speculum of the degradation 5 days that sections observation HA+ is sustained BMP-2/VEGF group coating in embodiment 1
Figure；
Figure 18 is 1000 × Electronic Speculum of the degradation 10 days that sections observation HA+ is sustained BMP-2/VEGF group coating in embodiment 1
Figure；
Figure 19 is 1000 × Electronic Speculum of the degradation 15 days that sections observation HA+ is sustained BMP-2/VEGF group coating in embodiment 1
Figure；
Figure 20 is 1000 × Electronic Speculum of the degradation 20 days that sections observation HA+ is sustained BMP-2/VEGF group coating in embodiment 1
Figure；
Figure 21 is the hip prosthesis photo for carrying different coating；
Figure 22 is Kunming dog hip replacement photo；
Figure 23 is Kunming dog HA group hip prosthesis postoperative X line piece；
Figure 24 is non-time-release group of hip prosthesis postoperative X line piece of Kunming dog；
Figure 25 is Kunming dog nano controlled-release group hip prosthesis postoperative X line piece；
Figure 26 is Kunming dog nano controlled-release group hip prosthesis postoperative X line piece；
Figure 27 is the postoperative CT scan piece of non-time-release group of hip prosthesis of Kunming dog；
Figure 28 is the postoperative CT scan piece of Kunming dog nano controlled-release group hip prosthesis；
Figure 29 is the postoperative CT scan piece of Kunming dog nano controlled-release group hip prosthesis；
Figure 30 is the compound VEGF and rhBMP-2 biphase coating electron microscope of the present invention.
Figure 31 is the technology of the present invention route map.
Specific embodiment
Specific embodiment 1: a kind of compound VEGF and rhBMP-2 biphase coating joint of this city embodiment is false
The preparation method of body, it is followed the steps below:
One, VEGF165 and rhBMP-2 are added separately to aqueous acetic acid, obtain VEGF165 mixed liquor, and
RhBMP-2 mixed liquor；VEGF165 mixed liquor is uniformly mixed with rhBMP-2 mixed liquor, 4h is freeze-dried, obtains rhBMP-2/
The mixed liquor of VEGF165；
Two, ethyl cellulose and Macrogol 4000 are added in chloroform, then 25 DEG C of ultrasonic dissolutions, add
The mixed liquor of rhBMP-2/VEGF165 obtains the mixed liquor of composite slow release rhBMP-2/VEGF165；
Three, the mixed liquor of composite slow release rhBMP-2/VEGF165 is sprayed on artificial femur handle, is subsequently placed in ultra-clean work
Make to dry in platform, be packaged in sealed bag, ethylene oxide cold sterilization is completed；Wherein, VEGF165, rhBMP-2, ethyl are fine
The mass ratio of dimension element and Macrogol 4000 is 0.005-0.015:1:23000~27000:4000~6000；RhBMP-2 and chlorine
Imitative mass volume ratio is 1mg:1.5~2.5mL.
Specific embodiment 2: the present embodiment is different from the first embodiment in that: composite slow release rhBMP-2/
The mixed liquor of VEGF165 is 50~150 microns in the coating thickness of artificial femur handle.It is other same as the specific embodiment one.
Specific embodiment 3: the present embodiment is different from the first embodiment in that: VEGF165, rhBMP-2, second
The mass ratio of base cellulose and Macrogol 4000 is 0.01:1:25000:5000.It is other same as the specific embodiment one.
Specific embodiment 4: the present embodiment is different from the first embodiment in that: the quality of rhBMP-2 and chloroform
Volume ratio is 1mg:2mL.It is other same as the specific embodiment one.
Specific embodiment 5: the present embodiment is different from the first embodiment in that: ultrasonic power 100-200W.
It is other same as the specific embodiment one.
Specific embodiment 6: the present embodiment is different from the first embodiment in that: the concentration of VEGF165 mixed liquor
For 8~12ug/mL, rhBMP-2 mixed liquid concentration is 0.5~1.5mg/mL.It is other same as the specific embodiment one.
Specific embodiment 7: the present embodiment is different from the first embodiment in that: the concentration of VEGF165 mixed liquor
For 10ug/mL, rhBMP-2 mixed liquid concentration is 1mg/mL.It is other same as the specific embodiment one.
Specific embodiment 8: the present embodiment is different from the first embodiment in that: the ethylene oxide low temperature
Disinfection is to be 50~60 DEG C in temperature to carry out disinfection.It is other same as the specific embodiment one.
The content of present invention is not limited only to the content of the respective embodiments described above, the group of one of them or several specific embodiments
The purpose of invention also may be implemented in contract sample.
Beneficial effects of the present invention are verified by following embodiment:
Embodiment 1
The present embodiment is coated on titanium sheet by preparing compound VEGF and rhBMP-2 biphase coating, then right
The mesenchymal stem cell obtained from experimental animal is tested:
A kind of preparation of the compound VEGF and rhBMP-2 biphase coating of the present embodiment, it be according to the following steps into
Capable:
1. the production of titanium sheet:
Diameter is 1cm, the titanium sheet for the titanium alloy material that thickness is about 2-3mm, and HA (hydroxyapatite) painting is coated in titanium sheet
Layer, about 100 ± 50 microns of coating layer thickness.The above titanium sheet and HA powder are provided and are made by system in Beijing Jing boat biomedical engineering center
Make, HA purity 95%, Ca/P 1.67, crystal rate 70%, porosity 40%.
2. the preparation of various non-time-release coatings:
RhBMP2 (rhBMP-2) uses Shanghai Ruibang Biological Material Co., Ltd.'s product, purity
95%.Using the recombinant human endothelial Porcine HGF (VEGF165) of company.RhBMP-2 and VEGF165 are separately added into acetic acid
Composite coating liquid is mixed and made into aqueous solution.Titanium plate surface after cold sterilization is distinguished to the mixed liquor of compound rhBMP-2,
The mixed liquor 0.05ml of the mixed liquor 0.05ml, compound rhBMP-2/VEGF165 of VEGF165 are freeze-dried 4h.
3. being sustained the preparation of rhBMP-2, VEGF coating
The polyethylene glycol (PEG) 4000 of 50mg ethyl cellulose (EC) Yu 10mg are weighed, 25 DEG C of ultrasounds of 2.0mL chloroform are added
Dissolution, respectively with the mixture of rhBMP-2,10ugVEGF165 of 1.0mg and the two, be made rhBMP-2, VEGF165,
RhBMP-2/VEGF165 mixed liquor.By the mixed liquor 0.05ml of the titanium plate surface difference composite slow release rhBMP-2 after cold sterilization
(containing about 0.05mg rhBMP-2,1.25mg ethyl cellulose, 0.25mg polyethylene glycol), is sustained the mixed liquor of VEGF165
0.05ml (containing about 0.0005mgVEGF165,1.25mg ethyl cellulose, 0.25mg polyethylene glycol), composite slow release rhBMP-2/
VEGF165 mixed liquor 0.05ml (containing about 0.05mg rhBMP-2 and 0.0005mgVEGF165,1.25mg ethyl cellulose,
0.25mg polyethylene glycol), it dries in superclean bench, is packaged in sealed bag, row ethylene oxide cold sterilization is spare.
4, sustained release rhBMP-2, VEGF coating titanium sheet of above-mentioned preparation is taken to carry out mesenchymal stem cell experiment:
1) experimental animal, Primary bone marrow mescenchymal stem cell isolation and culture:
Adult New Zealand large ear rabbit 2, weight about 1200g-2500g, by No.1 Hospital Attached to Harbin Medical Univ.
Animal Lab. provides.It is spare that 1ml heparin is extracted using 5ml syringe.10% amobarbital ear edge is chosen with 30mg/kg standard
Intravenous injection anesthesia White Rabbit.White Rabbit bilateral posterior superior iliac spine cropping preserved skin, using iodophor disinfection 3 times, centered on point of puncture
Sterilize radius about 10cm or so.It is intracavitary that iliac bone marrow is passed to using medullo-puncture needle, extracts medullo-puncture needle core out, medullo-puncture needle tail portion connects containing liver
The 5ml syringe marrow aspiration 1ml of element.Marrow heparin mixed liquor about 2ml is injected in 25ml Tissue Culture Flask, is added in culture bottle
Enter 3ml α-MEM culture medium.32 healthy adult dogs are selected in zoopery, and male and female are unlimited, weight about 15-20kg, real by breathing out medical university
Animal center observation, raising are tested, is tested after determining animal health, free from infection.
2) cell culture, passage and identification:
Liquid is changed according to 1 time/3 days frequencies.Culture bottle culture solution at the middle and upper levels is outwelled, PBS liquid 2ml is added and shakes 6-10 times,
Culture solution is discarded, fresh medium 3ml is instilled.Using inverted phase contrast microscope observation cell be in shuttle shape, can be formed cell mass and
Twist it is distributed.When cell confluent cultures bottom of bottle area about 95%, or while forming intensive cladding cell mass, passes on.Cell
The culture medium in culture bottle is outwelled when passage first, PBS liquid rinses 2 times, each 1min, and pancreatin 1ml is added.Microscopically observation
To cell curling it is agglomerating after, be added DMEM culture medium 3ml terminate digestion, blow and beat bottom of bottle repeatedly using pipette.Use pipette
Cell suspension is added in 15ml centrifuge tube and is centrifuged, 1000rp/min is centrifuged 3min, discards supernatant liquid, and DMEM culture medium is added
6ml piping and druming is suspended, and is separately added into two 25ml Tissue Culture Flasks.After mesenchymal stem cell chooses the progress of the 3rd generation cell
It is continuous to test and identify whether be mesenchymal stem cell using flow cytometer progress CD34 (-), CD44 (+).Fluidic cell
Instrument detection provides help by Harbin Medical University's nervous physiology laboratory.
Experiment detection is grouped to the mesenchymal stem cell after above-mentioned culture, specific as follows:
One, it is grouped situation:
According to this requirement of experiment be divided into HA group, BMP group, VEGF group, slow releasing pharmaceutical group, BMP+VEGF group, sustained release BMP group,
It is sustained VEGF group, is sustained BMP+VEGF group, totally 8 groups, and the titanium sheet of respective coatings is prepared by the standby of ethylene oxide cold sterilization
With.Using 24 orifice plates according to random group forming criterion by above-mentioned steps 4) mesenchymal stem cell that obtains is added and applies with corresponding
It is co-cultured in the culture plate of layer titanium sheet.Every group of 9 titanium sheet, totally 3 culture plates.The healthy adult dog of zoopery part 32, it is female
Male unlimited, weight about 15-20kg is randomly divided into 8 groups.
Two, Testing index:
2.1 alkaline phosphatases:
According to this requirement of experiment be divided into HA group, BMP group, VEGF group, slow releasing pharmaceutical group, BMP+VEGF group, sustained release BMP group,
It is sustained VEGF group, is sustained BMP+VEGF group, totally 8 groups, and the titanium sheet of respective coatings is prepared by the standby of ethylene oxide cold sterilization
With.Mesenchymal stem cell is added in the culture plate with respective coatings titanium sheet according to random group forming criterion using 24 orifice plates
It co-cultures.Every group of 9 titanium sheet, totally 3 culture plates.Cellular alkaline phosphatase assay is carried out respectively, is observed various coatings and is lured
The ability that mesenchymal stem cell breaks up to skeletonization direction is led (to build up company by Nanjing and AKP microplate reader method alkaline phosphatase is provided
Enzyme reagent kit).
The measurement of 2.2 Electronic Speculum:
Electronic Speculum observes the degradation situation of controlled-release coating, pore size and how many.Cellular morphology University material scientific and engineering
It assists to complete in institute's scanning electron microscope room).Whether break up in shuttle shape to skeletonization direction, the attaching of cell and titanium plate surface coating porosity
Situation.(assisting to complete by Northeast Forestry University's college of materials science and engineering scanning electron microscope room).
The measurement of 2.3PCR:
Expression, that is, Runt-related factor-2 (Runx2), Osteocalcin of PCR measurement osteogenesis gene
Genes, CXC chemokine cell-surface receptor-4 (Cxcr4) the mRNA transcriptional level of totally three genes
Situation of change (is assisted to complete) by Harbin longitude and latitude encyclopaedia Bioisystech Co., Ltd.Experiment is divided into HA group (1), sustained release VEGF group
(2), BMP group (3), sustained release VEGF+BMP-2 group (4), slow releasing pharmaceutical group (5) are sustained.
Target gene Real Time PCR detection primer.Following primer is designed according to objective gene sequence:
2.4 continuous modes:
2.4.1 extracting sample total serum IgE
Total serum IgE in tissue samples is extracted with ultrapure RNA extracts kit (Simple P Extract RNA kit).
2.4.2 reverse transcription
The first chain of cDNA (PrimeScript is synthesized with reverse transcription reagent boxTMRT reagent kit) reverse transcription is carried out,
RNA template, primer, 5xRT mix and RNase-freeWater dissolution are placed in spare on ice.
The first part of 20ul reaction system is added into reaction tube for according to the form below, mixes.
65 DEG C are incubated for 5 minutes, and rapid ice bath 2-10 minutes, of short duration centrifugation made the solution on tube wall be collected into tube bottom.
Continue that following reagent is added into the above reaction solution:
It gently inhales and plays mixing, 37 DEG C are incubated for 40 minutes.Keep the temperature 10 minutes for 70 ° after reaction.
2.4.3RealTimePCR
Instrument and analysis method
With ABI 7500 (U.S.) fluorescence quantitative PCR instrument, using 2-△△CTThe relative quantitative assay of method progress data.
1 target gene standard curve RealTimePCR of table design
2 sample RealTimePCR detection design of table
3, mesenchymal stem cell is grouped experiment detection, interpretation of result:
3.1 coating monitoring indexes and Electronic Speculum result alkaline phosphatase data are as shown in table 3.
3 coating monitoring index of table and Electronic Speculum result alkaline phosphatase data
The results are shown in Table 4 for 3.2 grouping real-time quantitative PCR measurements osteogenesis gene expression quantity (using HA group as control group).
4 real-time quantitative PCR of table measures osteogenesis gene expression quantity data
3.3 slice Electronic Speculum measurement results are as shown in Fig. 1 to 20.
By the above results result it is found that indicating that the alkaline phosphatase of sustained release and non-time-release BMP-2/VEGF165 have obviously
Increase, PCR measure expression, that is, Runt-related factor-2 (Runx2) of osteogenesis gene, Osteocalcin genes,
The variation feelings of CXC chemokine cell-surface receptor-4 (Cxcr4) the mRNA transcriptional level of totally three genes
Condition shows, sustained release BMP-2/VEGF165 is significantly raised, and the hole that the degradation of Electronic Speculum controlled-release coating as the result is shown is formed is similar to
Honeycomb structure, pore size is uniform, rough surface, is conducive to cell attachment.Sustained release and non-time-release BMP-2, sustained release and non-time-release
BMP-2/VEGF165 cell, cellular morphology are in that shuttle shape shows that bone marrow mesenchymal stem cells break up to osteoblast direction more, sustained release
VEGF165 coating surface cell is presented flat area and becomes larger, and prompts mesenchymal stem cell to endothelial cell differentiation.
Embodiment 2
A kind of preparation of compound VEGF and rhBMP-2 biphase coating articular prosthesis of the present embodiment, it is according to following
What step carried out:
The concentration of VEGF165 mixed liquor is 10ug/mL, and rhBMP-2 mixed liquid concentration is 1mg/mL.
One, VEGF165 and rhBMP-2 are added separately to aqueous acetic acid, obtain the VEGF165 that concentration is 10ug/mL
The rhBMP-2 mixed liquor that mixed liquor and concentration are 1mg/mL；VEGF165 mixed liquor is mixed with rhBMP-2 mixed liquor
It is even, it is freeze-dried 4h, obtains the mixed liquor of rhBMP-2/VEGF165；
Two, the polyethylene glycol (PEG) 4000 of 50mg ethyl cellulose (EC) Yu 10mg are weighed, is added 25 DEG C of 2.0mL chloroform
Ultrasonic dissolution；Be added composite slow release rhBMP-2/VEGF165 mixed liquor 0.05ml (containing about 0.05mg rhBMP-2 and
0.0005mgVEGF165,1.25mg ethyl cellulose, 0.25mg polyethylene glycol)；It is dried in superclean bench, is packaged in sealing
In bag, row ethylene oxide cold sterilization is spare to get the nano coating.
Wherein, rhBMP2 (rhBMP-2) uses Shanghai Ruibang Biological Material Co., Ltd.'s product,
Purity 95%.Using the recombinant human endothelial Porcine HGF (VEGF165) of company.
Three, hollow metal type femoral bone end prosthesis (hollow prosthesis, HP) makes:
The hollow femur handle prosthese for being suitble to dog femoral proximal end is designed, Cr-Al alloy material, the long 4cm of prosthese, proximal end prosthese are bored
Shank diameter lcm.Proximal end hollow chamber internal diameter 0.6cm, deep lcm, there are six the aperture that diameter is 3mm, prostheses on chamber surrounding wall
At handle proximal end 1/2 with sintering process prepare HA coating, about 100 ± 50 microns of coating layer thickness.Separately spray the preparation of non-time-release and step 2
Controlled-release coating, all prosthese row oxirane disinfections are spare.
Prosthese manufactured in the present embodiment is subjected to zoopery:
1, operation method:
1.1, experimental model: the excision of right hindlimb femoral head is chosen in this experiment, hollow prosthesis none-cement fixes implantation
Model.
1.2, anesthesia: 10% yellow Jackets 25mg/kg, intravenous injection.Maintain venous access simultaneously conventional defeated in art
Liquid gives intravenous infusion of penicillin.
1.3, surgical operation step: experiment lateral position, right hindlimb routine preserved skin, 2% tincture of iodine, 75% alcohol disinfecting visual area
Skin, paving aseptic operation towel.Straight cut is about 12cm on the outside of the right near end of thighbone of row, successively cuts skin, subcutaneous tissue and deep muscle
Film appears femoral head through anterolateral muscle gap blunt separation；Femoral head is cut off, marrow is expanded, salt water rinses Using prosthesis behind visual area
Femur, A group are implanted into common prosthese；B group 10, it is implanted into ten collagen protein sponge of hollow metal prosthese ++ 0.5 milligram of rhBMP-2, C
Group 10/implantation hollow metal prosthese and slow-release rhBMP-2；C group 10: after bone marrow stromal cells in vitro osteogenic induction
Plant back Periprosthetic；D group 10: for sustained release BMP and plant group is returned in bone marrow stroma stem cell culture.
1.4, zoopery sample is taken and is prepared:
Two experiments of A group were put to death using depletion method respectively at postoperative 3,6 months；Using depletion method point at 3,6,12 months
Each two experimental dogs of remaining dead each group of other places, completely take out right hindlimb femur along former operative approach.Completely remove surface attachment
Soft tissue and periosteum, taking the photograph the near end of thighbone of experimental dog after X-ray film includes under prosthesis handle about 5cm is completely sawed.With WK-150 type
Low-speed diamond cutting machine is cut (revolving speed is 10 revs/min), is accomplished and retains metal prosthesis handle and surrounding 0.5cm range
Cortex bone about 2cm × 2cm × l cm sample.All histological specimens are put immediately into 10% formalin fixed.
1.5, the preparation of undecalcified bone embedding and grinding: the sample flowing water after fixation is rinsed overnight, then with 70%,
80%, 90%, 95%, 100% graded ethanol is dehydrated step by step, xylene soak is transparent, polymethyl methacrylate and adjacent benzene two
Formic acid dibutyl ester 3:1 mixing is gathered and is embedded under catalyzed room temperature.Bone specimen after embedding is produced with Germany
Leica1600 diamond dust undecalcified slicer carries out serial section, about 500 microns of thickness, then successively uses 100#, 400#,
The grinding of 500# waterproof abrasive paper first cleans the polishing of the one side of slice, surface, adhered on glass slide with epoxy resin continue to grind it is another
Face, fine grinding and flowing water rinse cooling after first roughly grinding when grinding, and grinding is to being no more than 100 microns, and last surface is cleaned, row
Giemsa-eosin stains.Under the colouring method, bone tissue dyes pink, and osteocyte core is blue, mature bone tissue
Color is deeper, and freshman bone tissue's color is shallower.
1.6, statistical procedures:
It is for statistical analysis using Spss17.0 software package, ALP enumeration data data with the variance analysis of duplicate measurements into
Row compares.Gene difference analysis uses the variance analysis of completely randomized design data between PCR group.P < 0.05 indicates that difference is aobvious
It writes, P < 0.01 indicates difference highly significant.
2. result is observed
2.1, gross examination of skeletal muscle: the firm situation of prosthese and prosthese week when the ordinary circumstance of animal, art area wound are reacted, drawn materials
Enclose the general condition of tissue.
2.2, x ray photograph: whether there is or not lucent areas, eburnation degree, the Periprosthetic of Periprosthetic between observation prosthese and bone bed
Whether there is or not absorptions for sclerotin.
2.3, undecalcified bone tissue is ground: observe under an optical microscope: 1) fibr tissue is long in interface and prosthese hole
Enter, calcification, new bone growth situations such as；2) Periprosthetic new bone formation situation；3) associativity of prosthese chamber and outer boundary in bone
Matter and bonding state.
2.4, bone histomophormetry: 1) the synostosis rate of three groups of prosthese outer boundaries is measured；2) B, C group prosthese
Bone Ingrowth percentage in hole.
2.5,3D row operative site x ray photograph before putting to death: intramuscular injection quadracycline (30mg/kg) carries out fluorescence mark
Note, the femur upper segment that cutting machine (ISOMET Germany) is cut with implantation prosthese at a slow speed carry out histological observation, and sample is anhydrous third
2 weeks are fixed in ketone, organic glass embedding, German LEICA1600 saws formula slicer hard tissue slicing, thickness 170-200UM, VAN
GIESON picric acid-moral training.Common light microscopic and fluorescence microscopy microscopic observation, from the hard tissue slicing of three groups of every animal
In randomly select 2！Appliance computer image analysis system (German ZEISS AXIOVERT100) is random at implantation prosthese interface
4 visuals field are chosen, the length and interface total length of visual field inner boundary synosteosis are measured, calculate interface bone contact ratio.Bone contact ratio
=visual field median surface combines length/visual field median surface total length * 100%.
3. Biomechanics test:
Interface shear strength is measured using push-out method.
Method: one is randomly selected in the undecalcified slice of each sample, using computer color Pathologic image analysis system
System (Image-process system) is observed, is measured.This system include camera system, imaging system (fluorescent screen) and with
Electronic computer connected image plate and cursor.Its working principle is that the image that light microscopic undertissue is sliced is shown by camera system
Show on fluorescent screen, the electronic computer being connected from fluorescent screen is different or shown with cursor to be measured according to tissue gray scale
Image carries out automatic identification and measurement, and shows measured data.2 visual field ((A groups are randomly selected in prosthese outer boundary
4 visuals field of specimen selection), the synosteotic length of prosthese outer boundary and interface total length in the visual field are measured, Interface Bone knot is calculated
Conjunction rate takes synostosis rate of its average value as the sample.Measurement method: by undecalcified histotomy in low power microscopic observation
The combination situation of bone tissue and prosthese interface marks the total length that bone is contacted with prosthese along interface with cursor under high power lens,
The part that bone tissue is directly contacted with prosthese is marked with cursor according to Interface Bone-prosthese combination situation with same method,
It is handled through computer, synostosis rate: synostosis rate (%)=B1/AIX 1009b is calculated according to the following formula.Wherein Al is bone
The total length contacted with prosthese, B1 are the physical length that bone is directly contacted with prosthese.In addition, in B, in two groups of C of prosthese hole,
2 visuals field are randomly selected, measurement hole site tissue grows into area (A2) and bone tissue grows into area ((B2), by following public affairs
Formula calculates the Bone Ingrowth percentage in hollow prosthesis hole: Bone Ingrowth percentage (%)=B2/A2X100%.Its average value is taken to make
For the Bone Ingrowth percentage of the sample hole.
4, interpretation of result:
All coatings of the present embodiment and the plant of Chinese Academy of Sciences's medicine assist to complete, and zoopery prosthese and HA powder are by capital boat biology
It sets up the project by our design requirement production, the research and development of prosthese initial stage with the high joint Medical Group cooperation of prestige at engineering in medicine center.
The experimental section of big animal (Kunming dog) hip replacement is completed as depicted in figures 22 and 23.Kunming dog hip joint art
X-ray result is as shown in Figure 24 to 26 afterwards.Postoperative CT scan HA group, non-time-release group, nano controlled-release group (note as shown in Figure 27 to 29
Numerical portion has computer to automatically generate in figure, and numerical portion content does not influence the expressed meaning of picture).
Claims (7)
1. a kind of preparation method of compound VEGF and rhBMP-2 biphase coating articular prosthesis, it is characterised in that it be according to
What following steps carried out:
One, VEGF165 and rhBMP-2 are added separately to aqueous acetic acid, obtain VEGF165 mixed liquor and rhBMP-2 is mixed
Close liquid；VEGF165 mixed liquor is uniformly mixed with rhBMP-2 mixed liquor, is freeze-dried 4h, obtains the mixed of rhBMP-2/VEGF165
Close liquid；
Two, ethyl cellulose and Macrogol 4000 are added in chloroform, then 25 DEG C of ultrasonic dissolutions, add rhBMP-2/
The mixed liquor of VEGF165 obtains the mixed liquor of composite slow release rhBMP-2/VEGF165；
Three, the mixed liquor of composite slow release rhBMP-2/VEGF165 is sprayed on artificial femur handle, is subsequently placed in superclean bench
In dry, be packaged in sealed bag, ethylene oxide cold sterilization is completed；Wherein, VEGF165, rhBMP-2, ethyl cellulose
Mass ratio with Macrogol 4000 is 0.005~0.015:1:23000~27000:4000~6000；RhBMP-2 and chloroform
Mass volume ratio be 1mg:1.5~2.5mL.
2. a kind of preparation method of compound VEGF and rhBMP-2 biphase coating articular prosthesis according to claim 1,
It is characterized in that the mixed liquor of composite slow release rhBMP-2/VEGF165 is 50~150 microns in the coating thickness of artificial femur handle.
3. a kind of preparation method of compound VEGF and rhBMP-2 biphase coating articular prosthesis according to claim 1,
It is characterized in that the mass ratio of VEGF165, rhBMP-2, ethyl cellulose and Macrogol 4000 are 0.01:1:25000:
5000。
4. a kind of preparation method of compound VEGF and rhBMP-2 biphase coating articular prosthesis according to claim 1,
It is characterized in that the mass volume ratio of rhBMP-2 and chloroform is 1mg:2mL.
5. a kind of preparation method of compound VEGF and rhBMP-2 biphase coating articular prosthesis according to claim 1,
It is characterized in that ultrasonic power is 100-200W.
6. a kind of preparation method of compound VEGF and rhBMP-2 biphase coating articular prosthesis according to claim 1,
It is characterized in that the concentration of VEGF165 mixed liquor is 8~12ug/mL, rhBMP-2 mixed liquid concentration is 0.5~1.5mg/mL.
7. a kind of preparation method of compound VEGF and rhBMP-2 biphase coating articular prosthesis according to claim 1,
It is characterized in that the ethylene oxide cold sterilization, is to be 50~60 DEG C in temperature to carry out disinfection.
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