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
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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.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410128124.8A CN103893836B (en) | 2014-04-01 | 2014-04-01 | A kind of screw of Absorbable rod compound interface and preparation method |
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| CN201410128124.8A CN103893836B (en) | 2014-04-01 | 2014-04-01 | A kind of screw of Absorbable rod compound interface and preparation method |
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| WO2010117389A1 (en) * | 2009-04-07 | 2010-10-14 | University Of Arkansas | Advanced bio-compatible nanocomposite surface coatings for implants and tissue engineering scaffolds |
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| 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 |
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| 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 |
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| CN103893836A (en) | 2014-07-02 |
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