Cuttlebone grain mold and preparation method thereof

Abstract

The invention belongs to the technical field of medicine, and discloses a cuttlebone pattern mold and a preparation method thereof. The cuttlebone grain mold is prepared by a specific method comprising the following steps: filling a mixed rubber material of liquid silica gel mixed by a liquid A, a liquid B and the like according to a certain mass ratio into a culture dish of which the bottom is paved with an EDTA decalcified cuttlebone organic matter membrane, enabling S-shaped grains of the cuttlebone membrane to face upwards, removing bubbles by virtue of a vacuum bubble removing machine under the assistance of stirring, and performing vacuum drying to obtain the cuttlebone grain mold. Heating and curing temperature and time are controlled, cooling and molding are carried out, the mold is overturned for demolding, silica gel liquid of the same formula is poured for the second time, and the cuttlebone grain mold can be obtained after demolding. The cuttlebone texture mold has remarkable mechanical strength, hardness and high temperature resistance, remarkable osteogenesis performance and high transparency, can be used for preparing osteogenesis material textures, realizes accurate rubbing of cuttlebone textures, and has the advantages of low cost and good application performance.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and particularly relates to a cuttlefish bone texture mold and a preparation method thereof. Background Art

[0002] Bone tissue engineering is a promising strategy for treating bone defects. When the defect area exceeds the critical size of bone self-repair ability, bone repair materials made of various materials can serve as components of the extracellular matrix to provide an appropriate microenvironment for cell adhesion and bone ingrowth.

[0003] In bone tissue engineering, specific textures are designed on the surface of synthetic biomaterials to mimic the microstructure and physiological environment of natural bone tissue. This bionic texture structure can provide an in-vivo-like growth environment for osteoblasts, making it easier for cells to recognize and adapt to the material surface, thereby promoting the adhesion, proliferation, and differentiation of osteoblasts and inducing the formation of new bone tissue.

[0004] Research has found that the cuttlefish bone organic matrix membrane has a special S-shaped texture. The bionic periosteum with this texture can increase the contact area of osteoblasts, provide more adhesion sites for osteoblasts, and promote osteogenic differentiation. However, the simple extraction efficiency is low, and it is difficult to achieve large-scale production. Therefore, producing a texture mold for preparing cuttlefish bone bionic periosteum is crucial for improving production efficiency. Summary of the Invention

[0005] Object of the Invention: The object of the present invention is to provide a cuttlefish bone texture mold and a preparation method thereof. The bionic periosteum prepared by the cuttlefish bone texture mold provided by the present invention has good biocompatibility and bioactivity, and has the functions of promoting the adhesion, proliferation, and osteogenic differentiation of osteoblast-related cells, the angiogenesis of endothelial cells, and promoting bone defect repair.

[0006] Technical Solution: To achieve the above object, the technical steps adopted by the present invention are as follows: A preparation method of a cuttlefish bone texture mold, which comprises the following preparation steps: (1) Add liquid silicone A liquid and B liquid, etc. to a container according to a certain volume ratio, stir and mix evenly, and pour the mixed rubber material into a culture dish with a cuttlefish bone organic matter membrane decalcified with EDTA laid at the bottom, with the S-shaped texture of the cuttlefish membrane facing upwards; (2)After defoaming by a vacuum defoaming machine supplemented with stirring, control the heating and curing temperature and time, cool and mold. Flip the molded mold to achieve the demolding operation. Then place the demolded mold back into the petri dish. Take a brass block and accurately place it at the exact center of the molded mold. Then pour the silicone liquid with the same formula as in step (1) for the second time. After pouring, immediately place it in a vacuum environment for defoaming treatment. After sufficient defoaming, let it enter the curing stage. When the silicone is completely cured, carefully take out the brass block, and at this time, a spatial structure with cuttlebone patterns at the bottom can be obtained.

[0007] The cuttlebone pattern mold is obtained after demolding.

[0008] As a preferred solution, in the preparation method of a cuttlebone pattern mold described above, in step (1), the liquid silicone A liquid is the base rubber methyl vinyl silicone oil and the catalyst chloroplatinic acid, mixed in a mass ratio of 100:1 to 5, and the B liquid is the base rubber methyl vinyl silicone rubber (raw rubber) and the crosslinking agent hydrogen-containing silicone oil, mixed in a mass ratio of 100:0.6 to 100:12.

[0009] As a more preferred solution, in the preparation method of a cuttlebone pattern mold described above, in step (1), the liquid silicone A liquid is the base rubber methyl vinyl silicone oil and the catalyst chloroplatinic acid, mixed in a mass ratio of 100:1, and the B liquid is the base rubber methyl vinyl silicone rubber (raw rubber) and the crosslinking agent hydrogen-containing silicone oil, mixed in a mass ratio of 100:5.

[0010] As a preferred solution, in the preparation method of a cuttlebone pattern mold described above, in step (1), the volume ratio of the liquid silicone A liquid to the B liquid added is 1:1 to 10:1.

[0011] As a more preferred solution, in step (1), the volume ratio of the A liquid to the B liquid added is 1:1.

[0012] As a preferred solution, the stirring time in step (1) is generally about 3 - 5 minutes.

[0013] As a preferred solution, the extraction method of the cuttlebone organic matter film in step (1) is: mix the cuttlebone block with an EDTA (disodium ethylenediaminetetraacetate) solution, reduce the air pressure to 0.08 MPa and keep the temperature constant at 30 °C. After all the calcium carbonate is etched, the cuttlebone organic matter film is obtained.

[0014] As a preferred solution, the operating temperature for defoaming by stirring with a vacuum defoaming machine in step (2) is 25 - 40 °C, and the operating time is 1.5 - 3 hours.

[0015] In step (2), the heating and curing temperature is 150 - 160 °C, and the time is about 1 - 2 minutes.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: In the present invention, the organic matter film of cuttlefish bone extracted from cuttlefish bone is used as a mold prototype, and a cuttlefish bone texture mold is prepared with the assistance of specific liquid silicone rubber A liquid and B liquid. Experiments have found that the cuttlefish bone texture mold can be used to prepare a biomimetic bone membrane with the effect of promoting osteogenic differentiation. The use of this mold greatly improves the preparation efficiency of the cuttlefish bone biomimetic bone membrane with texture, which is beneficial to the large-scale production of products. Moreover, the improvement of the cuttlefish bone biomimetic bone membrane preparation technology does not affect its biological activity and osteogenic ability. Description of the Drawings

[0017] Figure 1 Showing the physical picture of the cuttlefish bone texture mold and the microscopic texture picture on the mold surface; Figure 2 Showing the failure case picture of screening the addition amount of silicone rubber A liquid and B liquid; Figure 3 Showing the surface structure of the material prepared by the cuttlefish bone texture mold characterized by scanning electron microscopy; Figure 4 Showing the growth texture of rat bone marrow mesenchymal stem cells rBMSCs on the material surface, and the scale bar is 50 μm; Figure 5 Showing the survival state of mouse pre-osteoblast MC3T3-E1 on the material surface, and the scale bar is 200 μm; Figure 6 Showing the promoting proliferation effect of the material on different cells, MC3T3-E1 cells and rBMSCs cells; Figure 7 Showing the osteogenic activity of the material on MC3T3-E1 cells; Figure 8 Showing the influence of the material on early and late osteogenic differentiation. Detailed Embodiments

[0018] The following further describes the present invention in detail with reference to the embodiments, but the embodiments of the present invention are not limited thereto.

[0019] Embodiment 1 Preparation of the cuttlefish bone texture mold of the present invention Step (1) Use a weighing tool to accurately weigh the liquid silicone rubber A liquid and B liquid (the liquid silicone rubber A liquid is the base rubber methyl vinyl silicone oil and the catalyst chloroplatinic acid, mixed according to a mass ratio of 100:1, and the B liquid is the base rubber methyl vinyl silicone rubber (raw rubber) and the crosslinking agent hydrogen-containing silicone oil, mixed according to a mass ratio of 100:5), stir and mix them according to a volume ratio of 1:1 for 3 min, and pour 40 mL of the uniformly stirred rubber material into a culture dish lined with a cuttlefish bone membrane, with the S-shaped texture of the cuttlefish bone membrane facing upwards; Step (2): Turn on the vacuum pump to gradually reduce the pressure inside the container. The air bubbles in the silica gel will gradually escape. Control the operating temperature at 25°C. When there are no obvious air bubbles on the surface of the silica gel, turn off the vacuum pump. Control the heating and curing temperature at 150°C for 1 minute. After cooling and forming, flip the formed mold to achieve the demolding operation, and then place the demolded mold back into the petri dish. Take a brass block with dimensions of 50 mm × 40 mm × 5 mm and precisely place it at the center of the mold. Then, re-pour 30 mL of silica gel A and B liquids with the same formulation as in step (1). After pouring, immediately place it in a vacuum environment for defoaming treatment. After sufficient defoaming, let it enter the curing stage. When the silica gel is completely cured, carefully take out the brass block, and a spatial structure with cuttlebone-like patterns at the bottom can be obtained.

[0020] After demolding, the cuttlebone-like pattern mold is obtained. The physical picture of the cuttlebone-like pattern mold is shown in Figure 1 。

[0021] Screening of the usage amounts of liquid silica gel A and B liquids: (1) Failure case one: In step (1), accurately measure according to the volume ratio of silica gel A liquid to silica gel B liquid of 2:1, stir and mix evenly for 3 minutes. Pour 40 mL of the evenly stirred rubber compound into the petri dish lined with cuttlebone membrane, with the S-shaped pattern of the cuttlebone membrane facing upwards; Step (2): Turn on the vacuum pump to gradually reduce the pressure inside the container. The air bubbles in the silica gel will gradually escape. Control the operating temperature at 25°C. When there are no obvious air bubbles on the surface of the silica gel, turn off the vacuum pump. Control the heating and curing temperature at 150°C for 1 minute. After cooling and forming, flip the formed mold to achieve the demolding operation, and then place the demolded mold back into the petri dish. Take a brass block with dimensions of 50 mm × 40 mm × 5 mm and precisely place it at the center of the formed mold. Then, re-pour 30 mL of the silica gel liquid from step (1). After pouring, immediately place it in a vacuum environment for defoaming treatment. After sufficient defoaming, let it enter the curing stage. When the silica gel is completely cured, carefully take out the brass block, and a spatial structure with cuttlebone-like patterns at the bottom can be obtained. The results are shown in Figure 2 -a.

[0022] (2) Failure case two: In step (1), accurately measure according to the volume ratio of silica gel A liquid to silica gel B liquid of 5:1, stir and mix evenly for 3 minutes. Pour 40 mL of the evenly stirred rubber compound into the petri dish lined with cuttlebone membrane, with the S-shaped pattern of the cuttlebone membrane facing upwards; Step (2): Turn on the vacuum pump to gradually reduce the pressure inside the container. The air bubbles in the silica gel will gradually escape. Control the operating temperature at 25°C. When no obvious air bubbles are seen on the surface of the silica gel, turn off the vacuum pump. Control the heating and curing temperature at 150°C for 1 minute. After cooling and forming, turn over the formed mold to achieve the demolding operation. Then place the demolded mold back into the petri dish. Take a brass block with dimensions of 50 mm × 40 mm × 5 mm and accurately place it at the exact center of the formed mold. Then pour 30 mL of silica gel liquid with the same formulation as in step (1) for secondary casting. After completion of casting, immediately place it in a vacuum environment for defoaming treatment. After sufficient defoaming, let it enter the curing stage. When the silica gel is completely cured, carefully take out the brass block, and a spatial structure with cuttlebone patterns at the bottom can be obtained. The results are shown in Figure 2 -b.

[0023] (3) Failure case three: Step (1): Accurately measure the silica gel A liquid and silica gel B liquid according to the volume ratio of 10:1, stir and mix evenly for 3 minutes. Pour 40 mL of the evenly stirred rubber compound into the petri dish lined with cuttlebone membrane, with the S-shaped pattern of the cuttlebone membrane facing upwards; Step (2): Turn on the vacuum pump to gradually reduce the pressure inside the container. The air bubbles in the silica gel will gradually escape. Control the operating temperature at 25°C. When no obvious air bubbles are seen on the surface of the silica gel, turn off the vacuum pump. Control the heating and curing temperature at 150°C for 1 minute. After cooling and forming, turn over the formed mold to achieve the demolding operation. Then place the demolded mold back into the petri dish. Take a brass block with dimensions of 50 mm × 40 mm × 5 mm and accurately place it at the exact center of the formed mold. Then pour 30 mL of silica gel liquid for secondary casting. After completion of casting, immediately place it in a vacuum environment for defoaming treatment. After sufficient defoaming, let it enter the curing stage. When the silica gel is completely cured, carefully take out the brass block, and a spatial structure with cuttlebone patterns at the bottom can be obtained. The results are shown in Figure 2 -c.

[0024] In all failure cases, it was observed that the microscopic morphology of the mold could not truly reproduce the microscopic structure of the cuttlebone membrane surface, so it could not be applied to the preparation of bionic periosteum.

[0025] Example 2 Sample Preparation 1. Blank Sample Preparation PCL (polycaprolactone) was blended with 1,4-dioxane solution and heated in a 65 °C water bath until a 20% (w / v) polycaprolactone solution was formed. After shielding from light, 200 μL of a 3 mg / L nile red solution was added. After stirring evenly, it was poured into a petri dish and placed in a fume hood to evaporate the solution. After the solution was evaporated, a fluorescent polycaprolactone sheet was obtained. Cut 0.5 g and place it in a mold without the texture of cuttlebone. Heat and melt it in a 65 °C water bath, then place a brass block to assist in pressing into shape. After cooling and forming, cut it into a round sheet with a diameter of 20 mm and soak it in 75% alcohol for later use.

[0026] 2. Preparation of experimental samples PCL (polycaprolactone) was blended with 1,4-dioxane solution and heated in a 65 °C water bath until a 20% (w / v) polycaprolactone solution was formed. After shielding from light, 200 μL of a 3 mg / L nile red solution was added. After stirring evenly, it was poured into a petri dish and placed in a fume hood to evaporate the solution. After the solution was evaporated, a fluorescent polycaprolactone sheet was obtained. Cut 0.5 g and place it in the cuttlebone texture mold prepared in Example 1. Heat and melt it in a 65 °C water bath, then place a brass block to assist in pressing the texture into shape. After cooling and forming, cut it into a round sheet with a diameter of 20 mm and soak it in 75% alcohol for later use.

[0027] Example 3 Preparation of sample extract The sample prepared in Example 2 was extracted with serum-free α-MEM medium, and the extraction ratio (surface area / volume) was 6 cm 2 / mL, controlling the temperature at (37 ± 1) °C and the extraction time at (24 ± 1) h.

[0028] Example 4 Physicochemical property test 1. Test objects: The blank sample and experimental sample prepared in Example 2. The sample extract in Example 3.

[0029] 2. Scanning electron microscope: Two samples prepared in Example 2 were fixed on the conductive adhesive of the sample disk and subjected to sputter coating with a gold ion sputtering instrument. Adjust the scanning electron microscope instrument parameters, the high voltage (HV) is "15.00 kV", the detector (DET) is "high vacuum secondary electron imaging mode (ETD)", and the magnification is "500". Observe the surface and side morphologies of the samples, and the results are shown in Figure 3 .

[0030] As can be seen from Figure 3 , the surface of the sample prepared by the cuttlebone texture mold has a clear "S"-shaped groove structure, and the existence of this structure is beneficial to the adhesion and proliferation of osteoblasts and angioblasts.

[0031] In summary, the biomimetic periosteum prepared by the cuttlebone texture mold has a suitable surface morphology.

[0032] Example 5 Effect Evaluation 1. Cell Adhesion Experiment The sterilized test sample in Example 2 was evenly spread on the bottom of a six-well plate. Rat bone marrow mesenchymal stem cells (rBMSCs) were seeded at a density of 1×10 5 cells / well and cultured in a cell incubator. After the cells adhered to the wall, the culture medium was discarded, and the cells were washed twice with PBS for 10 min each time. Paraformaldehyde was added at room temperature for 20 min for fixation, followed by two washes with PBS. FITC-phalloidin at a concentration of 5 µg / mL was added at room temperature for 30 - 60 min for staining, and the cells were washed twice with PBS. Then, DAPI was added to stain the cell nuclei for 10 minutes, and the cells were washed twice with PBS again. After removing the excess moisture, a fluorescence mounting medium (neutral or slightly alkaline buffer plus an equal amount of glycerol) was added, a coverslip was placed on top, and the cells were observed under a fluorescence or confocal microscope. The results are shown in Figure 4 .

[0033] It can be seen from Figure 4 that compared with the free and disordered growth state of cells in the blank group, the material prepared from the cuttlebone texture mold has a tendency to grow along the texture direction and then hook and extend between the grooves.

[0034] 2. Cell Viability and Cytotoxicity Staining The test sample prepared in Example 2 was pre-incubated with α-MEM complete medium for 1 h and evenly attached to the bottom of a glass-bottom cell culture dish (diameter 20 mm). Mouse pre-osteoblasts MC3T3-E1 were seeded at a density of 1×10 5 cells / well, cultured, and stained. The results are shown in Figure 5 .

[0035] It can be seen from Figure 5 that there are only a very small number of red-stained cells in the field of view, the overall cell growth condition is good, and the number of apoptotic cells is very small, indicating good biocompatibility. At the same time, it can be seen that the cells grow along the direction of the S-shaped texture.

[0036] 3. Cell Proliferation Experiment Rat bone marrow mesenchymal stem cells (rBMSCs) and mouse pre-osteoblasts MC3T3-E1 were respectively seeded in 96-well plates at a density of 1×10 4 cells / mL. After the cells adhered to the wall, the culture medium was replaced with the sample extract prepared in Example 3. After culturing for 1, 3, and 5 days respectively, the extract in the culture plate was aspirated, and the prepared CCK-8 reaction solution was added to the wells under light protection. After incubating at 37 o °C for 30 min, the OD value was measured at 450 nm. The results are shown in Figure 6 .

[0037] It can be seen fromFigure 6 It can be seen that, compared with the blank control, the experimental group has a proliferative effect on MC3T3-E1 cells and rBMSCs cells to varying degrees.

[0038] 4. Osteogenic differentiation promotion effect experiment Alkaline phosphatase (ALP) activity detection: Seed mouse pre-osteoblast MC3T3-E1 cells in a 12-well plate at a density of 1×10 5 cells / well, add 1000 μL / well of α-MEM complete medium. After the cell confluence reaches 60%, replace the complete medium with the sample extract prepared in Example 3, and then add osteogenic induction medium. Observe the cell growth status daily. Replace all the medium on the first day, and then replace half of the medium every two or three days. Induce for 7 d and 14 d respectively. After the induction is completed, aspirate the medium in the wells, wash twice with PBS, add 150 μL of Western and IP cell lysis buffer to each well, lyse on ice for 30 min, scrape and pipette with a pipette tip to detach the cells, centrifuge after collecting the lysate, take the supernatant, and use a BCA kit to detect the protein concentration. When detecting, dilute the sample by half, add 25 μL to each well, and detect the protein concentration according to the instructions. The results are shown in Figure 7 .

[0039] It can be seen from Figure 7 that, compared with the blank control, the presence of S-shaped patterns is beneficial to enhancing the alkaline phosphatase activity of MC3T3-E1 cells.

[0040] 5. Alkaline phosphatase (ALP) color development experiment Seed mouse pre-osteoblast MC3T3-E1 cells in a 12-well plate at a density of 1×10 5 cells / well, add 1000 μL / well of α-MEM complete medium. After the cell confluence reaches 60%, replace the complete medium with the sample extract prepared in Example 3, and then add osteogenic induction medium. Observe the cell growth status daily. Replace all the medium on the first day, and then replace half of the medium every two or three days. Induce for 7 d and 14 d respectively. After the induction is completed, aspirate the medium in the wells, wash twice with PBS, fix with paraformaldehyde at room temperature for 10 min, wash 5 times with PBS, 3 min each time. Add 200 μL / well of BCIP / NBT staining working solution to fully cover, incubate at room temperature in the dark for 30 min, remove the working solution, wash twice with ultrapure water, counterstain the cell nuclei with neutral red staining solution for 5 minutes for easy observation, wash thoroughly with distilled water, and then observe the staining situation and under the microscope. The results are shown in Figure 8 .

[0041] Von Kossa staining experiment: Seed mouse pre-osteoblast MC3T3-E1 cells in a 12-well plate at a density of 1×10 5The density of seeds per well was seeded into a 12-well plate with the sterilized sample of Example 2 already paved. After the cells adhered to the wall, the medium was replaced with osteogenic induction medium. After 21 days of osteogenic induction, 4% paraformaldehyde was added for fixation for 10 minutes, and then washed 3 times with PBS. 500 μL of Von Kossa silver solution was added to each well and irradiated under strong light for 20 minutes. The supernatant was discarded. After washing 3 times with PBS, it was observed and photographed under a microscope. The results are shown in Figure 8 .

[0042] It can be seen from Figure 8 that compared with the blank control, for MC3T3-E1 cells, there is an osteogenic differentiation-promoting ability to varying degrees in both the early and late stages of osteogenesis.

[0043] In summary, the material prepared by the cuttlebone texture mold has better biocompatibility and in vitro osteogenic differentiation-promoting effect.

Claims

1. A preparation method of a cuttlebone texture mold, characterized in that It includes the following preparation steps: (1) Add liquid silicone rubber A liquid and B liquid into a container according to a certain mass ratio, stir and mix evenly, and then pour the mixed silicone rubber material into a culture dish with an EDTA decalcified cuttlebone organic matter film laid at the bottom, with the S-shaped texture of the cuttlebone organic matter film facing upwards; (2) Then, after defoaming with a vacuum defoaming machine assisted by stirring, control the heating and curing temperature and time, cool and form, turn over the formed mold to perform the demolding operation, then place the demolded mold back into the culture dish, take a brass block and place it at the center position of the mold, then pour the silicone rubber A liquid and B liquid with the same formula as in step (1) for the second time. After pouring, immediately place it in a vacuum defoaming machine for defoaming treatment. After sufficient defoaming, let it enter the curing stage. When the silicone rubber is completely cured, carefully take out the brass block. At this time, a spatial structure with cuttlebone texture at the bottom can be obtained. Finally, perform the demolding process again to successfully prepare a mold with cuttlebone texture.

2. The preparation method of a cuttlebone texture mold according to claim 1, characterized in that, In step (1), the liquid silicone rubber A liquid is methyl vinyl silicone oil and the catalyst chloroplatinic acid, mixed according to a mass ratio of 100:1 to 100:5, and the B liquid is methyl vinyl silicone rubber and the crosslinking agent hydrogen-containing silicone oil, mixed according to a mass ratio of 100:0.6 to 100:

12.

3. The preparation method of a cuttlebone texture mold according to claim 1 or 2, characterized in that, In step (1), the volume ratio of the added liquid silicone rubber A liquid and B liquid is 1:1 to 10:

1.

4. The preparation method of a cuttlebone texture mold according to claim 3, characterized in that, In step (1), the volume ratio of the liquid silicone rubber A liquid and B liquid is 1:

1.

5. The preparation method of a cuttlefish bone texture mold according to claim 1, wherein, The extraction method of the EDTA decalcified cuttlebone organic matter film in step (1) is: mix the cuttlebone block with disodium ethylenediaminetetraacetate solution, reduce the air pressure to 0.08 MPa and keep the temperature constant at 30 °C. After all calcium carbonate is etched, the cuttlebone organic matter film can be obtained.

6. The preparation method of a cuttlebone texture mold according to claim 1, wherein, The stirring time in step (1) is 3 - 5 min.

7. The preparation method of a cuttlebone texture mold according to claim 1, characterized in that, In step (2), the operating temperature of the vacuum defoaming machine for defoaming is 25 - 40 °C, and the operating time is 1.5 - 3 h.

8. The preparation method of a cuttlebone texture mold according to claim 1, characterized in that, Pour 40 mL of the mixed solution of liquid silicone rubber A liquid and B liquid for the first time onto an EDTA decalcified cuttlebone organic matter film with a specification of 60 mm × 60 mm.

9. The preparation method of a cuttlebone texture mold according to claim 1, characterized in that, In step (2), the heating and curing temperature is 150 - 160 °C, and the time is 1 - 2 min.

10. A method for preparing a bionic periosteum, characterized in that, The preparation method includes the following steps: Blend polycaprolactone with a 1,4-dioxane solution, obtain a polycaprolactone solution with a mass concentration of 10 - 30% by water bath heating, stir evenly, pour it into a culture dish, and place it in a fume hood to volatilize the solution. After the solution is volatilized, a polycaprolactone sheet is obtained. Cut a part and place it in a cuttlebone texture mold, heat and melt it in a water bath, and then place a brass block to assist in forming the texture by pressing the sheet.

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