Preparation method of collagen drug-loaded sponge for osteoporosis bone defect repair

Abstract

This invention provides a method for preparing a collagen-loaded drug-eluting sponge for osteoporotic bone defect repair. The method involves ultrasonically mixing a type I collagen solution, a graphene oxide solution, and an alendronate sodium solution at a volume ratio of 2:0.25 to 1:1, centrifuging to remove air bubbles, then adding the mixture to a container using a biopipette and freezing it once at -20 to -30°C, followed by freeze-drying to obtain product A. Solution B is then prepared by mixing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, an ethanol solution, and N-hydroxysuccinimide. Product A and solution B are crosslinked at 20–30°C, rinsed to remove impurities, and then freeze-dried twice to obtain the collagen-loaded drug-eluting sponge for osteoporotic bone defect repair. The collagen-loaded drug-eluting sponge prepared by this invention exhibits good biocompatibility, a suitable drug release time, and a bone regeneration-promoting effect, and can be used for osteoporotic bone defect repair.

Description

Technical Field

[0001] This invention relates to the fields of polymer composite materials and medical materials technology, specifically a method for preparing a collagen-loaded sponge for repairing osteoporotic bone defects. Background Technology

[0002] Large-segment bone defects caused by tumors, trauma, and other factors pose a significant clinical challenge. With an aging population, it's becoming increasingly common for patients with bone defects to also suffer from osteoporosis. Repairing bone defects in osteoporotic patients is more difficult, time-consuming, and consumes substantial social resources. Existing methods for treating bone defects, such as autologous and allogeneic bone transplantation, have drawbacks including donor site damage, limited availability, and high cost. Furthermore, osteoporosis increases the risk of complications such as fractures and nonunion. In recent years, biomaterials have emerged as a new approach for bone defect repair research and clinical application due to their wide availability, lack of donor site damage, and low cost.

[0003] Collagen (Col) is a common biomaterial and a major component of bone tissue matrix. Collagen sponges, prepared through further processing, have been applied in scientific research and clinical practice. Adding anti-osteoporosis drugs to collagen sponges is a promising method for treating osteoporotic bone defects. However, the loose, porous network structure formed inside the sponge hinders the adhesion of small drug molecules, leading to drug burst release, which not only negates the therapeutic effect but also introduces the risk of drug toxicity. Therefore, improving the drug-carrying capacity of collagen sponges and reducing drug burst release has become an urgent problem to be solved. Summary of the Invention

[0004] This invention addresses the problem of drug release in current biomaterials for treating osteoporosis and bone defects by providing a method for preparing a collagen drug-loaded sponge for osteoporosis bone defect repair. The collagen drug-loaded sponge has good biocompatibility, suitable drug release time, and the effect of promoting bone tissue regeneration.

[0005] To achieve the above-mentioned technical objectives, this invention provides a method for preparing a collagen-loaded sponge for repairing osteoporotic bone defects, the specific steps of which are as follows:

[0006] S1. Add type I collagen powder to an acetic acid solution with a concentration of 0.025–0.1 mol / L at a mass-to-volume ratio of 0.02–0.04 g / mL, and sonicate for 8–12 minutes in a water bath at 0–5°C to dissolve it into a homogeneous colloidal solution.

[0007] S2. Add graphene oxide powder to deionized water at a mass-volume ratio of 0.006-0.01 g / mL, and sonicate in a water bath at 20-30℃ until a homogeneous solution is obtained;

[0008] S3. Add sodium alendronate powder to deionized water at a mass-volume ratio of 0.008-0.015 g / mL, and sonicate until a homogeneous solution is obtained in a water bath at 20-30℃.

[0009] S4. Mix the type I collagen solution from step S1, the graphene oxide solution from step S2, and the sodium alendronate solution from step S3 at a volume ratio of 2:0.25 to 1:1, and then mix them evenly by ultrasonication in a water bath at 0 to 5°C.

[0010] S5. Centrifuge the mixture from step S4 to remove air bubbles, then add it to a container using a biological pipette and freeze it at -20 to -30°C for 10 to 15 hours. Then freeze-dry the frozen product in a vacuum freeze dryer to obtain product A.

[0011] S6. Prepare a solution by mixing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with an 85%-95% ethanol solution at a molar-volume ratio of 45-60 mmol / L; then add N-hydroxysuccinimide to the solution at a molar-volume ratio of 9-14 mmol / L, and shake to fully dissolve to form solution B.

[0012] S7. Take the product A prepared in step S5 and place it in a container. Quickly pour the solution B from step S6 into the container containing product A and completely immerse product A. Then shake and crosslink at 20-30°C.

[0013] S8. The cross-linking product from step S7 is repeatedly rinsed with deionized water to remove residual impurities, and then placed in an environment of -20 to -30°C for 10 to 15 hours to cool and freeze. Finally, the frozen product is vacuum freeze-dried in a vacuum freeze dryer to obtain the collagen drug-loaded sponge for osteoporosis bone defect repair.

[0014] The preferred technical solution of the present invention is as follows: the type I collagen powder in step S1 is selected from bovine Achilles tendon collagen powder.

[0015] The preferred technical solution of the present invention is as follows: In step S4, the mixture is ultrasonically mixed for 3 to 5 minutes in a water bath at 0 to 5°C until homogeneous.

[0016] The preferred technical solution of the present invention is as follows: In step S5, the mixture is centrifuged at 2500±200r / min for 3 to 5 min to remove air bubbles, and then added to a disc-shaped polycaprolactone container with a diameter of 5 to 10 mm and a height of 2 to 3 mm using a biological pipette and then frozen.

[0017] The preferred technical solution of the present invention is as follows: the vacuum freeze drying time in steps S5 and S8 is 20 to 24 hours, and the temperature is -25℃ to -15℃.

[0018] The preferred technical solution of the present invention is as follows: In step S7, the container containing solution B and product A is shaken at a speed of 10 to 15 times / minute for 6 to 8 hours to complete the crosslinking process of the product.

[0019] The preferred technical solution of the present invention is as follows: in step S8, the crosslinking product is rinsed with deionized water 2-4 times, each time for 3-5 minutes, to remove residual impurities.

[0020] The drug-loaded alendronate sodium in this invention is an anti-osteoporosis drug containing terminal amino groups. During the production of collagen sponges from type I collagen, the addition of graphene oxide increases the carboxyl group content. Subsequently, under the action of a cross-linking agent, the amino and carboxyl groups undergo a condensation reaction to form amide bonds, which increases the drug-loading capacity of the collagen sponge and prolongs the sustained-release effect. After cross-linking, a novel drug-loaded collagen sponge is obtained, exhibiting good biocompatibility, suitable drug release time, and the ability to promote bone tissue regeneration. Attached Figure Description

[0021] Figure 1 This is a photograph of the actual appearance of the collagen drug-loaded sponge prepared in the examples;

[0022] Figure 2 These are electron microscope scans at different magnifications of the collagen drug-loaded sponge prepared in the examples;

[0023] Figure 3 Fourier transform infrared spectra of collagen drug-loaded sponges containing different concentrations of graphene oxide and unloaded collagen sponges containing different concentrations of graphene oxide.

[0024] Figure 4 A comparison of Raman spectra of drug-loaded collagen sponges containing different concentrations of graphene oxide and unloaded collagen sponges containing different concentrations of graphene oxide.

[0025] Figure 5 This is a fluorescence micrograph of the collagen-loaded sponge from Experiment 1.

[0026] Figure 6 This is an electron microscope scan of the collagen-loaded sponge from Experiment 1;

[0027] Figure 7 This is the drug release curve of a collagen-loaded sponge containing different concentrations of graphene oxide in deionized water, as shown in Experiment 2.

[0028] Figure 8 This is a comparison of the bone tissue regeneration effects of the collagen-loaded sponge prepared in the examples. Detailed Implementation

[0029] The present invention will be further described below with reference to embodiments.

[0030] Example 1 provides a method for preparing a collagen-loaded sponge for repairing osteoporotic bone defects. The specific preparation steps are as follows:

[0031] S1. Take 40 mg of type I collagen powder and add it to 2 mL of 0.05 mol / L acetic acid solution. Sonicate in a water bath at 0-4℃ for 10 minutes until a homogeneous gel-like solution is obtained, which is the type I collagen solution. The type I collagen was purchased from Chengdu Kele Biotechnology Co., Ltd., or bovine Achilles tendon collagen extracted from bovine Achilles tendon can also be used.

[0032] S2. Take 8 mg of graphene oxide and add it to 1 mL of deionized water. Sonicate the solution in a 25°C water bath until it becomes a homogeneous solution, i.e., graphene oxide solution. The graphene oxide raw material was purchased from Suzhou CarbonFeng Technology Co., Ltd.

[0033] S3. Dissolve 10 mg of alendronate sodium in 1 ml of deionized water, and sonicate in a water bath at 20-30℃ until a homogeneous solution is obtained, which is the alendronate sodium solution; the alendronate sodium was purchased from Dalian Meilun Biotechnology Co., Ltd.

[0034] S4. Add 0.5 ml of the graphene oxide solution from step S2 to 1 ml of the type I collagen solution from step S1, and then add 0.5 ml of the sodium alendronate solution from step S3. Sonicate the solution in a water bath at 0-5°C to obtain a homogeneous mixed solution, wherein the graphene oxide concentration is 0.2%.

[0035] S5. Centrifuge at 2500 r / min for 5 min to remove air bubbles. The product is used to prepare a collagen drug-loaded sponge with 0.2% GO.

[0036] S6. Using a 100uL biological pipette, add the above product into a polycaprolactone disc with a diameter of 5mm and a height of 2mm. Place it in a freezer at -20 to -30℃ and slowly cool it for 10 hours. Remove the frozen product and place it in a vacuum freeze dryer to dry for 20 to 24 hours to obtain product A.

[0037] S7. Take 100 mL of ethanol solution with a concentration of 85%–95%, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to make the concentration 45–60 mmol / L, add N-hydroxysuccinimide to make the concentration 9–14 mmol / L; shake slightly to fully dissolve to obtain solution B;

[0038] S8. Quickly pour the solution B prepared in step S7 into the container along the wall of the polycaprolactone disk in step S6 and completely immerse the product A in the container. Soak the product A at 25°C for 6 to 8 hours to complete the crosslinking process.

[0039] S9. The crosslinked product was rinsed repeatedly with deionized water 4 times for 5 minutes each time to remove the remaining impurities; the product was then placed in a freezer at -20 to -30°C and slowly cooled and frozen for 10 hours to obtain a collagen drug-loaded sponge containing 0.2% graphene oxide.

[0040] The collagen drug-loaded sponge prepared in Example 1 is as follows: Figure 1 As shown, its microscopic state was observed under microscopes of different magnifications. Figure 2 As shown in the figure. Electron microscopy reveals that the microstructure of the drug-loaded sponge is a loose, porous structure with a rough surface and striped wrinkles; after implantation, it facilitates the exchange of tissue fluid components and cell migration and differentiation.

[0041] In Example 2, the homogeneous mixed solution prepared by repeating steps S1 to S3 in Example 1 was prepared. Then, 0.125 ml of the graphene oxide solution from step S2 and 0.375 ml of deionized water were added to the homogeneous mixed solution. The solution was then centrifuged at 2500 r / min for 5 min to remove air bubbles. Then, steps S6 to S9 in Example 1 were repeated to obtain a collagen drug-loaded sponge containing 0.05% graphene oxide.

[0042] Comparative Example 1: Alendronate sodium was removed, and the other steps were the same as in Example 1 to prepare a collagen sponge (unloaded with drug) containing 0.2% graphene oxide.

[0043] Comparative Example 2: Alendronate sodium was removed, and the other steps were the same as in Example 2 to prepare a collagen sponge (unloaded with drug) containing 0.05% graphene oxide.

[0044] The collagen-loaded sponges containing different concentrations of graphene oxide prepared in Examples 1 and 2, and the unloaded collagen sponges containing different concentrations of graphene oxide prepared in Comparative Example 1 and Comparative Example 2 were subjected to Fourier transform infrared spectroscopy and Raman spectroscopy to analyze their composition and molecular structure changes. The specific analytical results are as follows: Figure 3 and Figure 4 As shown, Figure 3 Fourier transform infrared spectra of collagen drug-loaded sponges containing different concentrations of graphene oxide and unloaded collagen sponges containing different concentrations of graphene oxide. Figure 3 The black dashed line in the middle shows the characteristic peak of the amide bond formed after preparation, and the black triangle indicates the characteristic peak of sodium alendronate. The infrared spectroscopy results show that amide bonds were formed in the collagen drug-loaded sponge and connected to the loaded sodium alendronate. Figure 4 A comparison of Raman spectra of drug-loaded collagen sponges containing different concentrations of graphene oxide and unloaded collagen sponges containing different concentrations of graphene oxide. Figure 4 The D and G bands shown by the black dashed lines are characteristic bands of graphene oxide. Raman spectroscopy results confirm that the collagen drug-loaded sponge has been modified with graphene oxide.

[0045] The following tests were conducted on the collagen drug-loaded sponge prepared in Example 1;

[0046] Experiment 1: Detection of the biocompatibility of collagen-loaded sponge cells. The specific experimental steps are as follows:

[0047] Experiment 1: Detection of the biocompatibility of collagen-loaded sponge cells. The specific experimental steps are as follows:

[0048] (1) Sacrifice the 1-3 day old SD rats by cervical dislocation and disinfect them by soaking them in 75% alcohol for 10 minutes; remove the long bones of the limbs under sterile conditions and carefully remove the attached muscles and other tissues.

[0049] (2) Remove the cartilage at both ends of the long bone with ophthalmic scissors after disinfection to expose the bone marrow cavity. Use a 1mL syringe to draw low-glucose culture medium containing 10% fetal bovine serum and insert it into the bone marrow cavity to rinse the bone marrow cavity. Repeat 3-5 times until the long bone turns white. Collect the rinsing solution in a culture dish and shake it evenly.

[0050] (3) Transfer the culture dish to a regular incubator and let it stand for five days without changing the medium. After the primary adherent cells have grown to a confluence, change the medium and passage the cells.

[0051] (4) The mesenchymal stem cells passed down to the third generation were added together with the culture medium to the collagen drug-loaded sponge prepared in Example 1, and an appropriate amount of culture medium was added before placing it in an incubator and changing the medium every day.

[0052] (5) On day 14 of the culture process, the cells were stained with FDA / PI staining solution. The cell-collagen sponge complex was then placed under a laser confocal microscope to observe cell viability, as detailed below. Figure 5 As shown, Figure 5 The morphology under a fluorescence microscope shows that a large number of mesenchymal stem cells survive on the drug-loaded sponge and are evenly distributed along the main structure of the sponge, with few dead cells (green fluorescence indicates FDA-labeled live cells, and red fluorescence indicates PI-labeled dead cells).

[0053] (6) After fixing the cell-collagen sponge complex with glutaraldehyde, it was dehydrated by gradient ethanol solution, sputter-coated with gold, and then placed under a scanning electron microscope to observe the cell morphology on the surface of the collagen sponge, as shown in the following figures. Figure 6 As shown, Figure 6The gold area in the middle shows the morphology of mesenchymal stem cells surviving on the collagen-loaded sponge.

[0054] Experiment 2: Determination of sustained-release drug curve of collagen-loaded sponge. The specific steps are as follows:

[0055] (1) Take equal mass of collagen drug-loaded sponge containing 0.2% graphene oxide prepared in Example 1 and collagen drug-loaded sponge containing 0.05% graphene oxide prepared in Example 2 and immerse them in 2 mL of distilled water as experimental group 1 and experimental group 2. At the same time, weigh equal mass of collagen sponge without drug loading in comparative example 1 and comparative example 2 as control group 1 and control group 2. Place both experimental groups and two control groups in an environment of 37°C.

[0056] (2) Collect the soaking solution at 12, 24, 36, 48 hours, and 4, 6, 8, 12, 16, 20, 24, 31 days, and replace it with 2 mL of distilled water;

[0057] (3) Place the soaking solution in different 25mL volumetric flasks, add 1mL of 1% ammonium persulfate solution, heat in a boiling water bath for 10 minutes, and cool to room temperature; add 2mL of vanadium molybdate reagent, and dilute to the mark with distilled water; shake well.

[0058] (4) Measure the absorbance using a spectrophotometer, compare it with the standard curve to obtain the concentration, calculate the total amount of drug released, and plot the drug release curve. This experiment was repeated three times, and the plotted curves are shown below. Figure 7 As shown, through Figure 7 It can be seen that the drug release cycle can be as long as 30 days. The results also verify that graphene oxide plays a role in sustained drug release in the entire collagen drug-loaded sponge. The higher the concentration of graphene oxide, the slower the drug release rate.

[0059] Experiment 3: Detection of the bone regeneration activity of collagen-loaded sponge in osteoporosis patients. The specific steps are as follows:

[0060] (1) Ten-week-old female SD rats were anesthetized with sodium pentobarbital, their abdomens were shaved, disinfected three times with povidone-iodine, and deiodinated with 75% alcohol. Sterile gloves were put on and sterile surgical drapes were laid. Then, a midline abdominal incision was made 2 cm above the pubic symphysis. The skin was cut layer by layer to the abdominal cavity. The left adipose tissue was gently turned outward to expose the left fallopian tube, and the mulberry-shaped ovary could be seen at the top.

[0061] (2) Separate the fat fascia tissue around the ovary, ligate the blood vessels and cut off the ovary. Check for bleeding. The sham surgery group removed a fat tissue of the same volume as the ovary, while the sham surgery group removed a fat tissue of the same volume as the ovary.

[0062] (3) Remove the right ovary using the same procedure.

[0063] (4) After confirming that there is no intra-abdominal bleeding, the peritoneum, abdominal muscles and skin are sutured layer by layer with silk thread; after disinfecting the incision with active iodine, the rat is placed on clean bedding and kept warm in a warm place.

[0064] (5) Three days after the operation, 80,000 U of penicillin was injected into the leg muscles of the rats to prevent infection. Then, the female ovariectomized SD rats were raised to 22 weeks of age and anesthetized with sodium pentobarbital. The top of the head was shaved, the rats were disinfected with iodine three times, and the iodine was removed with 75% alcohol. The rats were then covered with sterile gloves and sterile surgical drapes.

[0065] (6) Make a midline incision in the skull, cut the skin and muscles layer by layer, cut the periosteum along the midline, carefully separate the periosteum completely and turn it to one side, use a small animal electric skull drill to drill a 5mm diameter full-thickness defect between the two parietal bones of the rat, fill it with the unloaded collagen sponge in Comparative Example 1 (control group) and the collagen drug-loaded sponge prepared in Example 1 (experimental group), respectively, cover the defect and collagen sponge with the periosteum of the skull, and carefully suture the periosteum with silk thread, and suture the muscles and scalp layer by layer.

[0066] (7) After disinfecting the incision with active iodine, place the rat on clean bedding and keep it warm in a warm place. Inject 80,000 U of penicillin into the thigh muscle of the rat for 3 consecutive days after the operation to prevent infection.

[0067] (8) After 12 weeks of normal feeding, rats were euthanized by cervical dislocation. The rat skull was removed, and the attached muscle and fascia tissue was shaved off. The skull was scanned using a small animal Micro-CT scanner, and the skull image was reconstructed using a computer. (Specific details are as follows...) Figure 8 As shown, the top image is a 3D reconstruction of the skull defect, and the bottom image is a cross-sectional image of the largest bone defect; the left side is a typical image of the control group, and the right side is a typical image of the experimental group.

[0068] The comparison between the left and right sides reveals that collagen-loaded sponges can significantly promote bone tissue regeneration in osteoporosis and improve the repair effect of bone defects.

[0069] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Those skilled in the art will understand that various modifications, alterations, substitutions, combinations, and simplifications can be made to the above embodiments without departing from the overall spirit and concept of the present invention. All such modifications, alterations, substitutions, combinations, and simplifications are equivalent substitutions and are included within the protection scope of the present invention. The protection scheme of the present invention is defined by the appended claims.

Claims

1. A method for preparing a collagen-loaded drug-eluting sponge for repairing osteoporotic bone defects, characterized in that, The specific steps are as follows: S1. Add type I collagen powder to an acetic acid solution with a concentration of 0.025–0.1 mol / L at a mass-to-volume ratio of 0.02–0.04 g / mL, and sonicate for 8–12 minutes in a water bath at 0–5°C to dissolve it into a homogeneous colloidal solution. S2. Add graphene oxide powder to deionized water at a mass-volume ratio of 0.006-0.01 g / mL, and sonicate in a water bath at 20-30℃ until a homogeneous solution is obtained; S3. Add sodium alendronate powder to deionized water at a mass-volume ratio of 0.008-0.015 g / mL, and sonicate until a homogeneous solution is obtained in a water bath at 20-30℃. S4. Mix the type I collagen solution from step S1, the graphene oxide solution from step S2, and the sodium alendronate solution from step S3 at a volume ratio of 2:0.25 to 1:1, and then mix them evenly by ultrasonication in a water bath at 0 to 5°C. S5. Centrifuge the mixture from step S4 at 2500±200r / min for 3-5min to remove air bubbles. Then, add it to a disc-shaped polycaprolactone container with a diameter of 5-10mm and a height of 2-3mm using a biological pipette. Freeze the product at -20 to -30℃ for 10-15 hours. Then, freeze-dry the frozen product under vacuum in a vacuum freeze dryer to obtain product A. S6. Prepare a solution by mixing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride with an 85%-95% ethanol solution at a molar-volume ratio of 45-60 mmol / L; then add N-hydroxysuccinimide to the solution at a molar-volume ratio of 9-14 mmol / L, and shake to fully dissolve to form solution B. S7. Take the product A prepared in step S5 and place it in a container. Quickly pour the solution B from step S6 into the container containing product A and completely immerse product A. Then shake and crosslink at 20-30°C. S8. The cross-linking product from step S7 is repeatedly rinsed with deionized water to remove residual impurities, and then placed in an environment of -20 to -30°C for 10 to 15 hours to cool and freeze. Finally, the frozen product is vacuum freeze-dried in a vacuum freeze dryer to obtain the collagen drug-loaded sponge for osteoporosis bone defect repair.

2. The method for preparing a collagen-loaded sponge for repairing osteoporotic bone defects according to claim 1, characterized in that: The type I collagen powder used in step S1 is bovine Achilles tendon collagen powder.

3. The method for preparing a collagen-loaded sponge for repairing osteoporotic bone defects according to claim 1, characterized in that: In step S4, the mixture is ultrasonically mixed for 3-5 minutes in a water bath at 0-5°C until homogeneous.

4. The method for preparing a collagen-loaded sponge for repairing osteoporotic bone defects according to claim 1, characterized in that: The vacuum freeze-drying time in steps S5 and S8 is 20 to 24 hours, and the temperature is -25°C to -15°C.

5. The method for preparing a collagen-loaded sponge for repairing osteoporotic bone defects according to claim 1, characterized in that: In step S7, the container containing solution B and product A is shaken at a speed of 10-15 times / minute for 6-8 hours to complete the product crosslinking process.

6. The method for preparing a collagen-loaded sponge for repairing osteoporotic bone defects according to claim 1, characterized in that: In step S8, the crosslinked product is rinsed with deionized water 2-4 times, each time for 3-5 minutes, to remove residual impurities.

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