CN103893836B - A kind of screw of Absorbable rod compound interface and preparation method - Google Patents
A kind of screw of Absorbable rod compound interface and preparation method Download PDFInfo
- Publication number
- CN103893836B CN103893836B CN201410128124.8A CN201410128124A CN103893836B CN 103893836 B CN103893836 B CN 103893836B CN 201410128124 A CN201410128124 A CN 201410128124A CN 103893836 B CN103893836 B CN 103893836B
- Authority
- CN
- China
- Prior art keywords
- screw
- layer
- comp
- compound interface
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 150000001875 compounds Chemical class 0.000 title claims abstract description 31
- 238000002360 preparation method Methods 0.000 title claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 39
- 239000013612 plasmid Substances 0.000 claims abstract description 26
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims abstract description 18
- 229920001661 Chitosan Polymers 0.000 claims abstract description 18
- 229920002674 hyaluronan Polymers 0.000 claims abstract description 18
- 229960003160 hyaluronic acid Drugs 0.000 claims abstract description 18
- 239000002502 liposome Substances 0.000 claims abstract description 16
- 101150065984 Comp gene Proteins 0.000 claims abstract description 11
- 238000010079 rubber tapping Methods 0.000 claims abstract description 4
- 229920001432 poly(L-lactide) Polymers 0.000 claims description 27
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 claims description 22
- 239000012531 culture fluid Substances 0.000 claims description 13
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 229910052588 hydroxylapatite Inorganic materials 0.000 claims description 11
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000008367 deionised water Substances 0.000 claims description 8
- 229910021641 deionized water Inorganic materials 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000004593 Epoxy Substances 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 239000008187 granular material Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 229920006395 saturated elastomer Polymers 0.000 claims description 3
- 210000000845 cartilage Anatomy 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 10
- 210000003041 ligament Anatomy 0.000 abstract description 8
- 230000002308 calcification Effects 0.000 abstract description 7
- 238000001415 gene therapy Methods 0.000 abstract description 5
- 230000033228 biological regulation Effects 0.000 abstract description 3
- 230000006698 induction Effects 0.000 abstract description 3
- 208000015181 infectious disease Diseases 0.000 abstract description 3
- 230000002138 osteoinductive effect Effects 0.000 abstract description 3
- 230000009881 electrostatic interaction Effects 0.000 abstract description 2
- 239000010410 layer Substances 0.000 description 61
- 238000000034 method Methods 0.000 description 14
- 238000004519 manufacturing process Methods 0.000 description 7
- 210000000130 stem cell Anatomy 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 230000008569 process Effects 0.000 description 5
- 230000009977 dual effect Effects 0.000 description 4
- 239000005090 green fluorescent protein Substances 0.000 description 4
- 230000035876 healing Effects 0.000 description 4
- 101710176668 Cartilage oligomeric matrix protein Proteins 0.000 description 3
- 210000000988 bone and bone Anatomy 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000011164 ossification Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 102100027473 Cartilage oligomeric matrix protein Human genes 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000000432 density-gradient centrifugation Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 102000055007 Cartilage Oligomeric Matrix Human genes 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 206010058046 Post procedural complication Diseases 0.000 description 1
- 208000035965 Postoperative Complications Diseases 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 210000003559 hypertrophic chondrocyte Anatomy 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 238000009940 knitting Methods 0.000 description 1
- 229960000448 lactic acid Drugs 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 238000000707 layer-by-layer assembly Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000000399 orthopedic effect Effects 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 229920000447 polyanionic polymer Polymers 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000003746 surface roughness Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention provides a kind of screw of Absorbable rod compound interface, be made up of screw cap, screw rod, head of screw connector, gene thin layer, screw rod outer rim is provided with tapping screw thread, and gene thin layer covers thread surface.Gene thin layer by chitosan basal layer and from inside to outside successively interval be covered with the active layer that pEGFP-COMP plasmid layer that hyaluronic acid and liposome-2000 wrap up formed.The present invention utilizes the electrostatic interaction of chitosan, hyaluronic acid, liposome that plasmid pEGFP-COMP is deposited at interference screw surface layer-by-layer.The present invention has good biocompatibility and osteoinductive, the release of energy Effective Regulation COMP gene is to strengthen short calcification fibrous cartilage effect, improve host cell infection rate, induction ligament is implanted rear bone-ligament interface and is well healed, and can realize ligamentum intraarticulare reconstruction operations combined gene therapy.The present invention is reasonable in design, simple operation and with low cost, has extremely strong feasibility, is suitable for commercial application.
Description
Technical field
The invention belongs to the manufacture of ligamentum intraarticulare Reconstruction median surface screw, be specifically related to Absorbable rod compound interface screw of a kind of COMP gene thin layer assembling and preparation method thereof.
Background technology
Arthroscope inferior ligament Reconstruction treats ligamentum intraarticulare damage, recovery stability of joint and the effective surgical treatment of function at present, add up according to orthopedic sports medicine association of the U.S. (AOSSM), annual at least 60 ten thousand people in the whole world carry out ligamentum intraarticulare Reconstruction, account for 21.6% of operation on joint.Although this technology achieves huge curative effect clinically, bone-ligament interface healing difficult problem still annoyings numerous clinical workers, once ligament-bone interface poor healing, probably causes whole operative failure.Therefore how improving ligament-knitting is the key factor determining operation late result and prevention of postoperative complication.
At present, clinical conventional ligament fixing means fixes through bone marrow road absorbable interference screw fixation.Interference screw is many to be made up of Poly-L-lactic acid (polyL-lacticacid, PLLA).PLLA has good biocompatibility and suitable degradation speed, and along with the absorption of own material, the whole tunnel of filling that osseous tissue is synchronous, makes implantation ligament and tunnel form good healing.
But simple PLLA does not have self-bone grafting function, tunnel can be expanded because bone formation rate in tunnel is not mated with PLLA degree of absorption, ligament is fixing unstable.Current utilization composite hydroxylapatite (hydroxyapatite clinically, HAP) bioabsorbable interference screw has and to a certain degree improves in induced osteogenesis, but, because in synthetic HAP and skeleton, HAP exists size difference, cause skeletonization effect still not good.Therefore, prepare absorbable interference screw fixation with PLLA can improve its induced osteogenesis activity by building 20 nano-HAPs being similar to human body HAP.
Meanwhile, correlational study is pointed out, the calcification fibrous cartilage layer in human normal ligament-bone syndeton is the major shift structure connecting ligament and osseous tissue.Calcification fibrous cartilage holds a small amount of hypertrophic chondrocyte primarily of a large amount of extracellular matrix (extracellularmatrix, ECM) and forms.Noncollagen protein is as the key component in ECM, wherein cartilage oligo-substrate protein (cartilageoligomericmatrixprotein, COMP) there is the structure that the adhesion of inducer substance stem cell, cartilage differentiation, participation regulation and control ECM and osseous tissue are shown in, maintain the effect of calcified cartilage transition structure.
Chitosan, hyaluronic acid all have good biocompatibility and degradability, are widely used in field of tissue engineering technology.Liposome, because of the instrument of its good fat-soluble Chang Zuowei cell transfecting, by building pEGFP-COMP recombiant plasmid, and utilizes liposome to carry out wrapping up the transfection efficiency that effectively can improve recombiant plasmid, and convenient by green fluorescent protein monitoring transfection efficiency.By layer upon layer electrostatic self-assembling technique, utilize chitosan, hyaluronic acid, the charged positive negativity of liposome, pEGFP-COMP is assembled to absorbable interference screw fixation surface, impel gene thin layer degrade and discharge plasmid DNA for a long time in physiological conditions, calcification fibrous cartilage is formed to promote bone-Ligament healing to utilize gene therapy means to promote.
Summary of the invention
An object of the present invention is to provide a kind of screw of Absorbable rod compound interface, it is the Absorbable rod compound interface screw of a kind of cartilage oligo-substrate protein (COMP) gene thin layer assembling, this interference screw has good biocompatibility and osteoinductive, the most important thing is that the release that effectively can regulate and control COMP gene is to strengthen short calcification fibrous cartilage effect, improve host cell infection rate, induction ligament is implanted rear bone-ligament interface and is well healed, for ligamentum intraarticulare reconstruction operations combined gene therapy provides new thought.
The screw of a kind of Absorbable rod compound interface of the present invention, be made up of screw cap 1, screw rod 2, head of screw 3, connector 4, gene thin layer 5, screw cap 1 is fixedly connected with by connector 4 with screw rod 2, head of screw 3 is arranged on screw rod 2 head end, screw cap 1 upper end is provided with the groove 6 corresponding with mounting screw torque spanner, screw rod 2 outer rim is provided with tapping screw thread 7, and gene thin layer 5 covers screw flight surface.
Described gene thin layer 5 by chitosan basal layer and from inside to outside successively interval be covered with the active layer that pEGFP-COMP plasmid layer that hyaluronic acid and liposome-2000 wrap up formed.Chitosan-based bottom is 1 layer, and in described active layer, the pEGFP-COMP plasmid layer of liposome and the total number of plies of hyaluronic acid are 4-24 layer; Described screw is made by hydroxyapatite and Poly-L-lactic acid.
Two of object of the present invention is to provide the preparation method of the screw of described Absorbable rod compound interface, passes through following steps:
(1) Poly-L-lactic acid (PLLA) is with 0.2gml
-1concentration be dissolved in Isosorbide-5-Nitrae-epoxy six ring solution, then add hydroxyapatite (HAP) powder that particle diameter is 20 nanometers, both mass ratioes above-mentioned are that 2:8 mixes;
(2) be that to add diameter be closely fill in the screw model of 4mm for the NaCl granule of 280-450um by diameter, be placed in the environment 1.5h of 70 DEG C of saturated steams, after room temperature cooling, the PLLA/20HAP mixture of step (1) gained is slowly added wherein, evacuation removing bubble under 0.07-0.08Mpa condition, in-40 DEG C of refrigerators, pre-freeze carried out freezing 24h after 3 hours, as sample;
(3) sample obtained in step (2) is immersed in 24h in distilled water, removing NaCl, lyophilization obtains PLLA/20HAP Absorbable rod compound interface screw again;
(4) PLLA/20HAP Absorbable rod compound interface screw obtained in step (3) is dipped in chitosan solution after 30 minutes, embathes 2-3 time with opti-DMEM culture fluid, each 1-2 minute;
(5) PLLA/20HAP Absorbable rod compound interface screw obtained in step (4) to be dipped in hyaluronic acid solution after 10-15 minute, to embathe 2-3 time with deionized water, each 1-2 minute;
(6) PLLA/20HAP Absorbable rod compound interface screw obtained in step (5) to be dipped in the opti-DMEM culture fluid of the pEGFP-COMP plasmid of liposome after 10-15 minute, to embathe 2-3 time with deionized water, each 1-2 minute;
(7) step (5) and step (6) is repeated successively, until obtain the Absorbable rod compound interface screw of required COMP gene thin layer assembling.
The concentration of described chitosan solution is 5mg/ml; Described hyaluronic acid solution concentration is 0.5mg/ml.
In the opti-DMEM culture fluid of the pEGFP-COMP plasmid of described liposome, mixed by 200ul liposome-2000 with 100ug plasmid, the pH value of culture fluid is 7.4.
The invention has the beneficial effects as follows:
1, the present invention has good biocompatibility and osteoinductive, the most important thing is that the release that effectively can regulate and control COMP gene is to strengthen short calcification fibrous cartilage effect, improve host cell infection rate, induction ligament is implanted rear bone-ligament interface and is well healed, and has wide clinical application prospect.
2, the present invention breaches the pattern of traditional operation treatment ligamentum intraarticulare damage, use operative treatment in conjunction with gene therapy, the COMP gene direct-assembling that calcification fibrous cartilage will be promoted to be formed is on screw surface and effectively control it and be discharged into local organization and play effect, for the treatment of ligamentum intraarticulare damage provides new thought.
3, utilize liposome pEGFP-COMP in manufacture method of the present invention, give full play to the fat-soluble of liposome and the affinity with cell membrane thereof, effectively can increase the cell transfecting efficiency of single plasmid layer, play the effect of gene therapy better.
4, manufacture method of the present invention utilizes the electrostatic interaction of chitosan, hyaluronic acid, liposome that plasmid pEGFP-COMP is deposited at interference screw surface layer-by-layer, not only simple operation but also with low cost, there is extremely strong feasibility, be suitable for commercial application.
Accompanying drawing explanation
Fig. 1 is the main TV structure schematic diagram of the present invention.
Fig. 2 is transverse section of the present invention gene thin layer assembling schematic diagram.
Fig. 3 is the stereoscan photograph of gene thin layer of the present invention surface complexion.
Fig. 4 is that the present invention increases surface contact angle variation tendency with the assembling gene thin layer number of plies.
Fig. 5 is that the present invention increases 260nm ultraviolet absorptivity variation tendency with the assembling gene thin layer number of plies.
Fig. 6 is the gene thin layer transfection mescenchymal stem cell Dual culture 48h fluorescent microscopy images of the different number of plies.
Fig. 7 is the gene thin layer transfection mescenchymal stem cell Dual culture 48hGFP fluorescence intensity change trend of the different number of plies.
Detailed description of the invention
The present invention is further described in conjunction with the accompanying drawings and embodiments.
embodiment 1
See Fig. 1, Fig. 2, the screw of a kind of Absorbable rod compound interface of the present invention, be made up of screw cap 1, screw rod 2, head of screw 3, connector 4, gene thin layer 5, screw cap 1 is fixedly connected with by connector 4 with screw rod 2, head of screw 3 is arranged on screw rod 2 head end, screw cap 1 upper end is provided with the groove 6 corresponding with mounting screw torque spanner, and screw rod 2 outer rim is provided with tapping screw thread 7, and gene thin layer 5 covers screw flight surface.
embodiment 2the Absorbable rod compound interface screw of COMP gene thin layer assembling manufactures and characterizes detection
(1) PLLA is with 0.2gml
-1concentration be dissolved in Isosorbide-5-Nitrae-epoxy six ring solution, then add the HAP powder that particle diameter is 20 nanometers, both mass ratioes above-mentioned are that 2:8 mixes;
(2) be that to add diameter be closely fill in the screw model of 4mm for the NaCl granule of 280-450um by diameter, be placed in the environment 1.5h of 70 DEG C of saturated steams, after room temperature cooling, the PLLA/20HAP mixture of step (1) gained is slowly added wherein, evacuation removing bubble under 0.07-0.08Mpa condition.In-40 DEG C of refrigerators, pre-freeze carried out freezing 24h after 3 hours;
(3) sample obtained in step (2) is immersed in 24h in distilled water, removing NaCl, lyophilization obtains PLLA/20HAP Absorbable rod compound interface screw again;
(4) PLLA/20HAP Absorbable rod compound interface screw obtained in step (3) is dipped in 5mg/ml chitosan solution after 30 minutes, embathes 2 times with opti-DMEM culture fluid, each 1 minute;
(5) PLLA/20HAP Absorbable rod compound interface screw obtained in step (4) is dipped in 0.5mg/ml hyaluronic acid solution after 10 minutes, embathes 2 times with deionized water, each 1 minute;
(6) pH PLLA/20HAP Absorbable rod compound interface screw obtained in step (5) being dipped in the pEGFP-COMP plasmid of liposome is in the opti-DMEM culture fluid of 7.4 after 10 minutes, embathes 2 times, each 1-2 minute with deionized water;
(7) step (5) and step (6) is repeated successively, until obtain the Absorbable rod compound interface screw of required COMP gene thin layer assembling.
In the opti-DMEM culture fluid of the pEGFP-COMP plasmid of described liposome, mixed by 200ul liposome-2000 with 100ug plasmid, the pH value of culture fluid is 7.4.
Gene thin layer of the present invention surface complexion is detected by field emission scanning electron microscope.The surface contact angle assembling each layer in gene thin layer assembling process is successively detected by quiet contact angle measurement.By assembling the 260nm ultraviolet absorptivity of one group of hyaluronic acid+pEGFP-COMP plasmid layer in UV spectrophotometer measuring gene thin layer assembling process successively.
In this example, hyaluronic acid and the total number of plies of pEGFP-COMP plasmid layer are not less than 4 layers can maintain the more stable gene thin layer structure in interference screw surface, by increasing the gene thin layer number of plies to reach the object that regulation and control interference screw reprints gene dosage.Total number of plies is best with 4-24 layer, thus realizes plasmid DNA guarantee finite concentration sustained release in special time, avoids the too much number of plies to cause the plasmid DNA short time to discharge simultaneously and is difficult to control or excessive.
In this example, interference screw can soak 30 minutes in chitosan solution, but do not represent the present invention and be confined to 30 minutes, the object of soaking be to enable chitosan layer by its with positive charge fully adsorb interference screw surface, the abundant formation of soak time long chitosan polycation layer, for polycation thin layer basis is laid in gene thin layer assembling subsequently.Therefore soak time is fully formed as suitable with chitosan layer.
In this example, interference screw can soak 10-15 minute in hyaluronic acid and opti-DMEM culture fluid, but do not represent the present invention and be confined to 10-15 minute, the object of soaking is to enable hyaluronic acid and liposome plasmid layer to invest interference screw surface in interval successively by the principle of layer upon layer electrostatic self assembly, the long abundant formation being more conducive to polyanion or polycation layer of soak time, make it cover more complete at interference screw, monolayer is more smooth.Therefore soak time reaches Optimal coverage effect with each layer and is advisable.
In this example, after interference screw soaks and terminates in each solution, all need to carry out washing by soaking with deionized water, in each process, deionized water embathes number of times is 2-3 time, and each 1-2 minute is advisable.Reason is that the main purpose of embathing removes the bilayer that may be formed within the same layer or multilamellar gathers anions and canons, in order to avoid it is stable to cause package assembly subsequently to lose, embathes overlong time simultaneously and easily causes the multiple structure assembled to dissociate.
See Fig. 3, result shows, and increasing surface roughness with the assembling gene thin layer number of plies increases gradually, illustrates that assembling process median surface screw surfactant continues to increase.
See Fig. 4, result shows, and increasing surface contact angle with the assembling gene thin layer number of plies is undulatory property change.Illustrate that screw surface, assembling process median surface is in two kinds of metaboly assemblings.
See Fig. 5, result shows, and increasing 260nm ultraviolet absorptivity with the assembling gene thin layer plasmid number of plies increases.Increase with the assembling number of plies is described, gene dosage increases gradually.
embodiment 3the efficiency of gene thin layer to mescenchymal stem cell transfection of the different number of plies is probed into.
Adopt the manufacture method of example 1, manufacture the Absorbable rod compound interface screw of the COMP gene thin layer assembling of 0,4,8 and 12 layer respectively.When adopting density-gradient centrifuga-tion method and difference, adherent method cultivates SD Rat Mesenchymal Stem Cells, and mescenchymal stem cell is inoculated into the interference screw Dual culture of six orifice plates and the different number of plies, cell-seeding-density is 40000cells/cm
2.Add 10% hyclone by DMEM culture medium to cultivate.After 48h with GFP fluorescence in each group of the different number of plies of fluorescence microscope to detect cell transfecting situation, by GFP fluorescence intensity in each group of IPP6.0 computed in software.
See Fig. 6,7, result shows, and increases with the interference screw gene thin layer number of plies, and GFP fluorescence intensity increases.Illustrate that the gene thin layer on interference screw effectively discharges gene, and improve gradually with gene dosage increase transfection efficiency.
embodiment 4the gene thin layer of the different number of plies becomes fibrous cartilage to affect on mescenchymal stem cell
Adopt the manufacture method of example 1, manufacture the Absorbable rod compound interface screw of the COMP gene thin layer assembling of 0,4,8 and 12 layer respectively.When adopting density-gradient centrifuga-tion method and difference, adherent method cultivates SD Rat Mesenchymal Stem Cells, and mescenchymal stem cell is inoculated into the interference screw Dual culture of six orifice plates and the different number of plies, cell-seeding-density is 20000cells/cm
2.Add 10% hyclone by DMEM culture medium to cultivate, change liquid once every 2 days.With 0.25% trypsinization after 5d, collecting cell, uses TRIzol cell lysis, carries row RT-PCR after RNA, detects the expression of II type collagen fiber in each group of the different number of plies.
Without the need to elaborating further, believe content disclosed before employing, those skilled in the art can apply the present invention to greatest extent.Therefore, embodiment is above interpreted as only illustrating, and not limits range of application of the present invention by any way.So substantial scope of the present invention will be determined by appended claims and equivalents thereof.
Claims (4)
1. the screw of an Absorbable rod compound interface, it is characterized in that, by screw cap (1), screw rod (2), head of screw (3), connector (4), gene thin layer (5) is formed, screw cap (1) is fixedly connected with by connector (4) with screw rod (2), head of screw (3) is arranged on screw rod (2) head end, screw cap (1) upper end is provided with the groove (6) corresponding with mounting screw torque spanner, screw rod (2) outer rim is provided with tapping screw thread (7), gene thin layer (5) covers screw flight surface, described gene thin layer (5) by chitosan basal layer and from inside to outside successively interval be covered with the active layer that pEGFP-COMP plasmid layer that hyaluronic acid and liposome-2000 wrap up formed, chitosan-based bottom is 1 layer, in described active layer, the pEGFP-COMP plasmid layer of liposome and the total number of plies of hyaluronic acid are 4-24 layer, described screw is made by hydroxyapatite and Poly-L-lactic acid.
2. the preparation method of the screw of a kind of Absorbable rod compound interface according to claim 1, is characterized in that, pass through following steps:
(1) Poly-L-lactic acid is with 0.2gml
-1concentration be dissolved in Isosorbide-5-Nitrae-epoxy six ring solution, then add the HAP powder that particle diameter is 20 nanometers, both mass ratioes above-mentioned are that 2:8 mixes;
(2) be that to add diameter be closely fill in the screw model of 4mm for the NaCl granule of 280-450um by diameter, be placed in the environment 1.5h of 70 DEG C of saturated steams, after room temperature cooling, the PLLA/20HAP mixture of step (1) gained is slowly added wherein, evacuation removing bubble under 0.07-0.08Mpa condition, in-40 DEG C of refrigerators, pre-freeze carried out freezing 24h after 3 hours, as sample;
(3) sample obtained in step (2) is immersed in 24h in distilled water, removing NaCl, lyophilization obtains PLLA/20HAP Absorbable rod compound interface screw again;
(4) PLLA/20HAP Absorbable rod compound interface screw obtained in step (3) is dipped in chitosan solution after 30 minutes, embathes 2-3 time with opti-DMEM culture fluid, each 1-2 minute;
(5) PLLA/20HAP Absorbable rod compound interface screw obtained in step (4) to be dipped in hyaluronic acid solution after 10-15 minute, to embathe 2-3 time with deionized water, each 1-2 minute;
(6) PLLA/20HAP Absorbable rod compound interface screw obtained in step (5) to be dipped in the opti-DMEM culture fluid of the pEGFP-COMP plasmid of liposome after 10-15 minute, to embathe 2-3 time with deionized water, each 1-2 minute;
(7) step (5) and step (6) is repeated successively, until obtain the Absorbable rod compound interface screw of required COMP gene thin layer assembling.
3. the preparation method of the screw of a kind of Absorbable rod compound interface according to claim 2, is characterized in that, the concentration of chitosan solution is 5mg/ml, and hyaluronic acid solution concentration is 0.5mg/ml.
4. the preparation method of the screw of a kind of Absorbable rod compound interface according to claim 2; it is characterized in that; in the opti-DMEM culture fluid of the pEGFP-COMP plasmid of liposome, mixed by 200ul liposome-2000 with 100ug plasmid, the pH value of culture fluid is 7.4.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201410128124.8A CN103893836B (en) | 2014-04-01 | 2014-04-01 | A kind of screw of Absorbable rod compound interface and preparation method |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201410128124.8A CN103893836B (en) | 2014-04-01 | 2014-04-01 | A kind of screw of Absorbable rod compound interface and preparation method |
Publications (2)
Publication Number | Publication Date |
---|---|
CN103893836A CN103893836A (en) | 2014-07-02 |
CN103893836B true CN103893836B (en) | 2016-01-27 |
Family
ID=50985613
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201410128124.8A Expired - Fee Related CN103893836B (en) | 2014-04-01 | 2014-04-01 | A kind of screw of Absorbable rod compound interface and preparation method |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN103893836B (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110916737B (en) * | 2019-11-26 | 2020-10-27 | 杭州电子科技大学 | Degradable screw and manufacturing method thereof |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1656223A (en) * | 2002-03-28 | 2005-08-17 | 组织基因股份公司 | Bone generation by gene therapy |
CN101052427A (en) * | 2004-10-06 | 2007-10-10 | 拜克技术有限公司 | Transparent hyaluronic acid coated bone implanting appliance |
CN201404282Y (en) * | 2009-02-25 | 2010-02-17 | 中国人民解放军第三军医大学 | Antibacterial peptide carrying slow release bone screw |
CN101690676A (en) * | 2009-10-26 | 2010-04-07 | 上海交通大学 | Absorbable metal intramedullary nail and preparation method thereof |
CN102085397A (en) * | 2011-01-19 | 2011-06-08 | 浙江普洛家园生物医学材料有限公司 | Absorbable implant material in bone and preparation method thereof |
CN102387825A (en) * | 2009-04-10 | 2012-03-21 | 麦德托尼克瓦斯科尔勒公司 | Implantable medical devices having bioabsorbable primer polymer coatings |
CN203201952U (en) * | 2013-04-03 | 2013-09-18 | 宁波金鹏高强度紧固件有限公司 | Durable screw |
CN203201939U (en) * | 2013-04-01 | 2013-09-18 | 宁波金鹏高强度紧固件有限公司 | Semicircular self-tapping screw |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010117389A1 (en) * | 2009-04-07 | 2010-10-14 | University Of Arkansas | Advanced bio-compatible nanocomposite surface coatings for implants and tissue engineering scaffolds |
-
2014
- 2014-04-01 CN CN201410128124.8A patent/CN103893836B/en not_active Expired - Fee Related
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1656223A (en) * | 2002-03-28 | 2005-08-17 | 组织基因股份公司 | Bone generation by gene therapy |
CN101052427A (en) * | 2004-10-06 | 2007-10-10 | 拜克技术有限公司 | Transparent hyaluronic acid coated bone implanting appliance |
CN201404282Y (en) * | 2009-02-25 | 2010-02-17 | 中国人民解放军第三军医大学 | Antibacterial peptide carrying slow release bone screw |
CN102387825A (en) * | 2009-04-10 | 2012-03-21 | 麦德托尼克瓦斯科尔勒公司 | Implantable medical devices having bioabsorbable primer polymer coatings |
CN101690676A (en) * | 2009-10-26 | 2010-04-07 | 上海交通大学 | Absorbable metal intramedullary nail and preparation method thereof |
CN102085397A (en) * | 2011-01-19 | 2011-06-08 | 浙江普洛家园生物医学材料有限公司 | Absorbable implant material in bone and preparation method thereof |
CN203201939U (en) * | 2013-04-01 | 2013-09-18 | 宁波金鹏高强度紧固件有限公司 | Semicircular self-tapping screw |
CN203201952U (en) * | 2013-04-03 | 2013-09-18 | 宁波金鹏高强度紧固件有限公司 | Durable screw |
Also Published As
Publication number | Publication date |
---|---|
CN103893836A (en) | 2014-07-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Zhao et al. | Promoting in vivo early angiogenesis with sub-micrometer strontium-contained bioactive microspheres through modulating macrophage phenotypes | |
CN102657893B (en) | Medical nano-fiber sponge material and preparation method and application thereof | |
CN101584885B (en) | Preparation method of three-layer lead tissue regenerating velum with gradient | |
CN101736430B (en) | Method for preparing silk fibroin nano-fibre with skin-care effect | |
CN101184450B (en) | Cell-free graft composed of substrate and serum | |
CN102302804B (en) | Hydroxyapatite-based biological composite scaffold and tissue engineered bone | |
EP2682135B1 (en) | Non-woven fabric containing bone prosthetic material | |
CN107456607A (en) | Guide Periodontal Tissue Regeneration film of new " sandwich " structure a kind of of difunctionalization and its preparation method and application | |
CN106362216B (en) | A kind of calcium and magnesium silicate porous ceramic ball orbital implant and preparation method thereof | |
JP2013536246A5 (en) | ||
CN104645414B (en) | Titanium-based surface antibacterial and bone tissue regeneration inductivity functional coating and its preparation method and application | |
Xu et al. | Effects of strontium-incorporated micro/nano rough titanium surfaces on osseointegration via modulating polarization of macrophages | |
CN107261213A (en) | A kind of active hydrogel and preparation method thereof and the application in operation wound reparation | |
CN103751847A (en) | Preparation method of tissue-regeneration-promoting controlled-release multiple-growth-factor self-assembled coating | |
GB2573092A (en) | Porous scaffold for the delivery of therapeutic agents | |
CN101934095A (en) | Injectable strengthened phosphate lime/hydrogel microcapsule tissue engineering bone as well as preparation method and application thereof | |
CN103480042A (en) | Artificial dura mater spinalis, and preparation method and use method thereof | |
Wu et al. | Electrospun fibers immobilized with BMP-2 mediated by polydopamine combined with autogenous tendon to repair developmental dysplasia of the hip in a porcine model | |
CN104250655A (en) | BMP(bone morphogenetic protein)-2/VEGF(vascular endothelial growth factor)165 double gene modified bone marrow mesenchymal stem cells and preparation method thereof | |
CN105169480A (en) | Scaffold material applied to vertebral fusion | |
CN103893836B (en) | A kind of screw of Absorbable rod compound interface and preparation method | |
CN102139125A (en) | Growth-factor-containing nanofibre porous composite material capable of repairing bone and preparation method thereof | |
CN101411893A (en) | Titanium or titanium alloy material with modified surface as well as preparation method and use thereof | |
CN105797217A (en) | Porous micro-sphere bone repair material and preparing method thereof | |
Gao et al. | Strategy of a cell-derived extracellular matrix for the construction of an osteochondral interlayer |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160127 Termination date: 20160401 |