CN103893836B - A kind of screw of Absorbable rod compound interface and preparation method - Google Patents

Abstract

The invention provides a screw with an absorbable composite interface, which is composed of a screw cap, a screw rod, a screw head connector and a gene thin layer. The outer edge of the screw rod is provided with self-tapping threads, and the gene thin layer covers the surface of the thread. The gene thin layer is composed of a base layer of chitosan and an activation layer formed by a hyaluronic acid layer and a liposome-2000-wrapped pEGFP-COMP plasmid layer in sequence from the inside to the outside. The invention utilizes the electrostatic action of chitosan, hyaluronic acid and liposome to deposit the plasmid pEGFP-COMP layer by layer on the interface screw surface. The invention has good biocompatibility and osteoinductivity, can effectively regulate the release of the COMP gene to enhance the effect of promoting calcification of fibrocartilage, improve the transfection rate of host cells, induce good healing of the bone-ligament interface after ligament implantation, and realize Intra-articular ligament reconstruction surgery combined with gene therapy. The invention has reasonable design, convenient operation and low cost, has strong feasibility and is suitable for industrial application.

Description

A screw with absorbable composite interface and its preparation method

technical field

The invention belongs to the manufacture of interface screws in intra-articular ligament reconstruction, in particular to an absorbable composite interface screw assembled with COMP gene thin layer and a preparation method thereof.

Background technique

Arthroscopic ligament reconstruction is currently an effective surgical treatment for intra-articular ligament injuries and restoration of joint stability and function. According to statistics from the American Orthopedic Sports Medicine Society (AOSSM), at least 600,000 people worldwide undergo intra-articular ligament reconstruction every year. Accounted for 21.6% of joint surgeries. Although this technique has achieved great clinical efficacy, the problem of bone-ligament interface healing still plagues the majority of clinicians. Once the ligament-bone interface is poorly healed, it is likely to lead to the failure of the entire operation. Therefore, how to improve ligament-bone healing is a key factor in determining the long-term efficacy of surgery and preventing postoperative complications.

At present, the commonly used method of ligament fixation in clinic is fixation with absorbable interface screws through the bone marrow canal. Most interface screws are composed of polyL-lactic acid (PLLA). PLLA has good biocompatibility and suitable degradation speed. With the absorption of its own material, the bone tissue fills the entire tunnel synchronously, so that the implanted ligament and the tunnel form a good healing.

However, pure PLLA has no osteoinductive function, and the tunnel enlargement and unstable ligament fixation may be caused by the mismatch between the bone formation speed in the tunnel and the absorption degree of PLLA. Clinically, absorbable screws using composite hydroxyapatite (HAP) have improved to a certain extent in the induction of osteogenesis. However, due to the size difference between synthetic HAP and HAP in human bones, the osteogenesis effect is still not good. . Therefore, the preparation of absorbable interfacial screws by constructing 20nm HAP and PLLA similar to human HAP can improve its osteogenic activity.

At the same time, related studies have pointed out that the calcified fibrocartilage layer in the normal ligament-bone junction structure of the human body is the main transitional structure connecting ligaments and bone tissue. Calcified fibrocartilage is mainly composed of a small amount of hypertrophic chondrocytes surrounded by a large amount of extracellular matrix (ECM). Non-collagen protein is the main component of ECM, among which cartilage oligomeric matrix protein (COMP) can induce stromal stem cell adhesion, cartilage differentiation, participate in the regulation of the construction of ECM and bone tissue, and maintain the transitional structure of calcified cartilage.

Both chitosan and hyaluronic acid have good biocompatibility and degradability, and are widely used in the field of tissue engineering. Liposomes are often used as a tool for cell transfection because of their good fat solubility. By constructing pEGFP-COMP recombinant plasmids and encapsulating them with liposomes, the transfection efficiency of recombinant plasmids can be effectively improved, and the transfection can be monitored with the help of green fluorescent protein. Dyeing efficiency. With the help of layer-by-layer electrostatic self-assembly technology, pEGFP-COMP is assembled on the surface of the absorbable interface screw by using the positive and negative charges of chitosan, hyaluronic acid, and liposomes. Plasmid DNA is released for a long time, and gene therapy is used to promote the formation of calcified fibrocartilage to promote bone-ligament healing.

Contents of the invention

One of the objectives of the present invention is to provide a screw with an absorbable composite interface, which is a kind of absorbable composite interface screw assembled with a thin layer of cartilage oligomeric matrix protein (COMP) gene. The interface screw has good biocompatibility and Osteoinductive, the most important thing is that it can effectively regulate the release of the COMP gene to enhance the effect of promoting calcification of fibrocartilage, increase the transfection rate of host cells, induce good healing of the bone-ligament interface after ligament implantation, and provide a basis for intra-articular ligament reconstruction surgery. Gene therapy provides a new way of thinking.

A screw with an absorbable composite interface according to the present invention is composed of a screw cap 1, a screw rod 2, a screw head 3, a connector 4, and a gene thin layer 5. The screw cap 1 and the screw rod 2 are fixedly connected by a connector 4, and the screw The head 3 is arranged on the head end of the screw 2, the upper end of the screw cap 1 is provided with a groove 6 corresponding to the installation screw torque wrench, the outer edge of the screw 2 is provided with a self-tapping thread 7, and the gene thin layer 5 covers the surface of the screw thread.

The gene thin layer 5 is an activation layer formed by a base layer of chitosan and a pEGFP-COMP plasmid layer covered with a hyaluronic acid layer and a liposome-2000-wrapped layer sequentially from the inside to the outside. The chitosan base layer is 1 layer, and the total number of pEGFP-COMP plasmid layers and hyaluronic acid layers wrapped by liposomes in the activation layer is 4-24 layers; Made with lactic acid.

The second object of the present invention is to provide the preparation method of the screw with absorbable composite interface, through the following steps:

(1) L-polylactic acid (PLLA) was dissolved in 1,4-epoxyhexacyclic solution at a concentration of 0.2 gml ^-1 , and then hydroxyapatite (HAP) powder with a particle size of 20 nm was added, and the mass of the above two The ratio is 2:8 for mixing;

(2) Add NaCl particles with a diameter of 280-450um into a screw model with a diameter of 4mm to fill it tightly, place it in an environment saturated with water vapor at 70°C for 1.5h, and after cooling at room temperature, put the PLLA/20HAP obtained in step (1) Add the mixture slowly, vacuumize under the condition of 0.07-0.08Mpa to remove air bubbles, pre-freeze in -40°C refrigerator for 3 hours and then freeze for 24h, as a sample;

(3) Soak the sample prepared in step (2) in distilled water for 24 hours, remove NaCl, and freeze-dry again to obtain PLLA/20HAP absorbable composite interface screws;

(4) Soak the PLLA/20HAP absorbable composite interface screw prepared in step (3) in the chitosan solution for 30 minutes, then soak it with opti-DMEM culture solution for 2-3 times, each time for 1-2 minutes ;

(5) Soak the PLLA/20HAP absorbable composite interface screw prepared in step (4) in the hyaluronic acid solution for 10-15 minutes, then soak it with deionized water 2-3 times, 1-2 minutes each time ;

(6) Soak the PLLA/20HAP absorbable composite interface screw prepared in step (5) in the opti-DMEM culture medium of the liposome-encapsulated pEGFP-COMP plasmid for 10-15 minutes, then rinse with deionized water 2-3 times, 1-2 minutes each time;

(7) Steps (5) and (6) were repeated in sequence until the required absorbable composite interface screw assembled with the COMP gene thin layer was obtained.

The concentration of the chitosan solution is 5mg/ml; the concentration of the hyaluronic acid solution is 0.5mg/ml.

In the opti-DMEM culture fluid of the liposome-encapsulated pEGFP-COMP plasmid, 200ul liposome-2000 and 100ug plasmid were mixed, and the pH value of the culture fluid was 7.4.

The beneficial effects of the present invention are:

1. The present invention has good biocompatibility and osteoinductivity, and the most important thing is that it can effectively regulate the release of the COMP gene to enhance the effect of promoting calcification of fibrocartilage, improve the transfection rate of host cells, and induce bone- The ligament interface is well healed and has broad clinical application prospects.

2. The present invention breaks through the traditional mode of surgical treatment of intra-articular ligament injury, uses surgical treatment combined with gene therapy to directly assemble the COMP gene that promotes the formation of calcified fibrocartilage on the surface of the screw and effectively controls its release to the local tissue to exert its effect, providing joint The treatment of internal ligament injury provides a new idea.

3. Utilize liposome to wrap pEGFP-COMP in the manufacturing method of the present invention, give full play to the fat solubility of liposome and its affinity with cell membrane, can effectively increase the cell transfection efficiency of single plasmid layer, better exert the efficacy of gene therapy.

4. The manufacturing method of the present invention utilizes the electrostatic action of chitosan, hyaluronic acid, and liposomes to deposit the plasmid pEGFP-COMP on the surface of the interface screw layer by layer, which is not only convenient to operate but also low in cost, and has strong feasibility , suitable for industrial applications.

Description of drawings

Fig. 1 is a schematic diagram of the front view of the structure of the present invention.

Fig. 2 is a schematic diagram of cross-sectional gene thin-layer assembly of the present invention.

Fig. 3 is a scanning electron micrograph of the surface appearance of the gene thin layer of the present invention.

Fig. 4 shows the change trend of surface contact angle with the increase of the number of assembled gene thin layer layers in the present invention.

Fig. 5 is the change trend of 260nm ultraviolet absorbance with the increase of the number of assembled gene thin layer layers of the present invention.

Fig. 6 is a fluorescent micrograph of co-cultured 48h of gene thin-layer transfected mesenchymal stem cells with different numbers of layers.

Figure 7 shows the change trend of fluorescence intensity of 48hGFP co-cultured with gene thin layer transfected mesenchymal stem cells with different layers.

detailed description

The present invention will be further described in conjunction with drawings and embodiments.

Example 1

Referring to Fig. 1 and Fig. 2, a screw with an absorbable composite interface according to the present invention is composed of a screw cap 1, a screw rod 2, a screw head 3, a connector 4, and a gene thin layer 5, and the screw cap 1 and the screw rod 2 pass through The connector 4 is fixedly connected, the screw head 3 is set on the head end of the screw rod 2, the upper end of the screw cap 1 is provided with a groove 6 corresponding to the torque wrench for the installation screw, the outer edge of the screw rod 2 is provided with a self-tapping thread 7, and the gene thin layer 5 covers on the screw thread surface.

Example 2 Manufacture and characterization detection of absorbable composite interface screw assembled by COMP gene thin layer

(1) PLLA was dissolved in 1,4-epoxyhexacycline solution at a concentration of 0.2gml ^-1 , and then HAP powder with a particle size of 20 nm was added, and the mass ratio of the above two was 2:8 for mixing;

(2) Add NaCl particles with a diameter of 280-450um into a screw model with a diameter of 4mm to fill it tightly, place it in an environment saturated with water vapor at 70°C for 1.5h, and after cooling at room temperature, put the PLLA/20HAP obtained in step (1) The mixture is slowly added therein, and the air bubbles are removed by vacuuming under the condition of 0.07-0.08Mpa. Pre-freeze in -40°C refrigerator for 3 hours and then freeze for 24 hours;

(3) Soak the sample prepared in step (2) in distilled water for 24 hours, remove NaCl, and freeze-dry again to obtain PLLA/20HAP absorbable composite interface screws;

(4) Soak the PLLA/20HAP absorbable composite interface screw prepared in step (3) in a 5 mg/ml chitosan solution for 30 minutes, then soak it twice with opti-DMEM culture solution, 1 minute each time;

(5) Immerse the PLLA/20HAP absorbable composite interface screw prepared in step (4) in 0.5 mg/ml hyaluronic acid solution for 10 minutes, then soak it with deionized water twice, each time for 1 minute;

(6) Soak the PLLA/20HAP absorbable composite interface screw prepared in step (5) in the opti-DMEM culture medium with pH 7.4 of the liposome-encapsulated pEGFP-COMP plasmid for 10 minutes, and rinse with deionized water Soak twice, 1-2 minutes each time;

(7) Steps (5) and (6) were repeated in sequence until the required absorbable composite interface screw assembled with the COMP gene thin layer was obtained.

In the opti-DMEM culture fluid of the liposome-encapsulated pEGFP-COMP plasmid, 200ul liposome-2000 and 100ug plasmid were mixed, and the pH value of the culture fluid was 7.4.

The surface appearance of the gene thin layer of the present invention is detected by a field emission scanning electron microscope. The surface contact angles of each layer assembled sequentially during the gene thin layer assembly process were detected by static drop contact angle measuring instrument. The 260nm ultraviolet absorbance of a group of hyaluronic acid+pEGFP-COMP plasmid layers sequentially assembled during the gene thin layer assembly process was detected by an ultraviolet spectrophotometer.

In this example, the total number of layers of hyaluronic acid layer and pEGFP-COMP plasmid layer is not less than 4 layers, which can maintain a relatively stable gene thin layer structure on the surface of the interface screw, and increase the number of gene thin layer layers to achieve the regulation of the amount of genes transferred by the interface screw the goal of. The total number of layers is 4-24 layers, so as to ensure the continuous release of plasmid DNA at a certain concentration within a certain period of time, while avoiding the short-term release of plasmid DNA that is difficult to control or excessive due to too many layers.

In this example, the interface screw can be soaked in the chitosan solution for 30 minutes, but it does not mean that the present invention is limited to 30 minutes. On the surface of the screw, the chitosan polycation layer was fully formed after soaking for a long time, which laid the foundation of the polycation thin layer for the subsequent gene thin layer assembly. Therefore soaking time is advisable with fully forming chitosan layer.

In this example, the interface screw can be soaked in hyaluronic acid and opti-DMEM culture solution for 10-15 minutes, but it does not mean that the present invention is limited to 10-15 minutes. The purpose of soaking is to make the hyaluronic acid layer and lipid The plastid-encapsulated plasmid layer can be attached to the surface of the interface screw at intervals by means of the principle of layer-by-layer electrostatic self-assembly. Longer soaking time is more conducive to the full formation of the polyanion or polycation layer, making it more complete in the interface screw coverage and a smoother single layer. . Therefore, the best soaking time is for each layer to achieve the best coverage effect.

In this example, after the interface screws are soaked in each solution, they all need to be soaked and washed with deionized water, and the number of times of soaking with deionized water in each process is 2-3 times, and each time is preferably 1-2 minutes. The reason is that the main purpose of rinsing is to remove the double or multi-layer polyanions and cations that may be formed in the same layer, so as not to cause the subsequent assembly structure to lose stability. Dissociate.

Referring to Figure 3, the results show that the surface roughness gradually increases with the increase of the number of assembled gene thin layers, indicating that the interface screw surface active substances continue to increase during the assembly process.

Referring to Figure 4, the results show that the surface contact angle fluctuates as the number of assembled gene thin layers increases. It shows that the surface of the interface screw is assembled alternately with two substances during the assembly process.

Referring to Fig. 5, the results show that the 260nm ultraviolet absorbance increases with the increase of the number of plasmid layers of the assembled gene thin layer. It shows that as the number of assembly layers increases, the number of genes increases gradually.

Example 3 Exploration of transfection efficiency of mesenchymal stem cells with gene thin layers of different layers.

Using the manufacturing method of Example 1, absorbable composite interface screws assembled with 0, 4, 8 and 12 layers of COMP gene thin layers were manufactured respectively. SD rat mesenchymal stem cells were cultured by density gradient centrifugation and time-difference adherence method. The mesenchymal stem cells were seeded into six-well plates and co-cultured with interface screws of different layers. The seeding density of the cells was 40000cells/cm ² . Cultured in DMEM medium plus 10% fetal bovine serum. After 48 hours, the GFP fluorescence in each group with different layers was observed with a fluorescence microscope to detect the cell transfection, and the GFP fluorescence intensity in each group was calculated by IPP6.0 software.

Referring to Figures 6 and 7, the results show that the fluorescence intensity of GFP increases with the increase of the number of interfacial screw gene thin layers. It shows that the gene thin layer on the interface screw releases genes effectively, and the transfection efficiency gradually increases with the increase of the number of genes.

Example 4 Effects of Gene Thin Layers with Different Numbers of Layers on Mesenchymal Stem Cell Fibrocartilage

Using the manufacturing method of Example 1, absorbable composite interface screws assembled with 0, 4, 8 and 12 layers of COMP gene thin layers were manufactured respectively. SD rat mesenchymal stem cells were cultured by density gradient centrifugation and time-difference adherence method. The mesenchymal stem cells were inoculated into six-well plates and co-cultured with different layers of interface screws. The seeding density of the cells was 20000cells/cm ² . Cultured with DMEM medium plus 10% fetal bovine serum, the medium was changed every 2 days. After 5 days, digest with 0.25% trypsin, collect cells, lyse cells with TRIzol, extract RNA and perform RT-PCR to detect the expression of type II collagen fibers in each group with different layers.

Without further elaboration, it is believed that one skilled in the art can, using the preceding disclosure, utilize the present invention to its fullest extent. Therefore, the foregoing embodiments should be understood as illustrative only and not limiting the scope of application of the present invention in any way. Therefore, the main scope of the present invention is to be determined by the appended claims and their equivalents.

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.