Preparation method of graded porous DEGDA / hydroxyapatite composite material and stent

Abstract

The invention provides a preparation method of a hierarchical porous DEGDA / hydroxyapatite composite material, which comprises the following steps: reacting HAP in a chitosan acid solution to obtain chitosan coated hydroxyapatite; the chitosan coated hydroxyapatite is put into a DMAC solution, acryloyl chloride and triethylamine are dropwise added, and modified chitosan coated hydroxyapatite is prepared; uniformly mixing the modified chitosan coated hydroxyapatite, DEGDA, a photoinitiator and a pore-foaming agent, and printing into a model by adopting photocuring; and cleaning and drying the model to obtain the DEGDA / hydroxyapatite composite material. The invention further discloses the stent obtained by adopting the preparation method. The chitosan wrapping the HAP is modified, agglomeration of the HAP is reduced, the dispersion uniformity of the HAP in DEGDA is improved, smooth connection is formed between holes, the uniformity of the hole channels is improved, and the holes in natural bone tissue are simulated to the maximum extent through large holes formed in a 3D printing mode and small holes formed by a pore-foaming agent.

Description

Technical Field

[0001] The present invention relates to a method for preparing a DEGDA / hydroxyapatite composite material with hierarchical pores and a scaffold prepared from the DEGDA / hydroxyapatite composite material. Background Art

[0002] A scaffold is a structure made of a specific material that can be artificially manufactured and implanted into the body to promote cell interaction in the body and help form new natural functional tissues. In recent years, scaffolds with hierarchical porous structures have occupied an important position in bone tissue engineering, which can provide a larger specific surface area and a larger volume for cell adhesion, proliferation, and growth. Scaffolds with interconnected and open pore structures are usually designed to support the migration of cultured cells into the interior of the scaffold, thus achieving a sufficient number of cells. Traditional methods for fabricating porous scaffolds include freeze-drying, phase separation, and electrospinning, etc., which can generate 3D porous structures with a tissue-like microenvironment. Currently, emerging technologies such as 3D printing emphasize precise control of porosity and gradient structures, and have been widely used in the field of bone tissue engineering by virtue of their design flexibility in the preparation of complex geometric structures.

[0003] However, low mechanical strength is a major challenge for porous structure scaffolds. When preparing porous scaffolds, various structural parameters such as pore size, pore size distribution, pore morphology, pore interconnectivity, and specific surface area need to be adjusted to match the bone simulation characteristics. The pore size range of scaffolds applied to tissue engineering is generally 20 - 1500 μm to simulate natural bone. The optimal pore size can provide a large number of cell sites and increase bone ingrowth. However, it is very difficult to achieve a uniform pore size. Therefore, a gradient porous structure has become the first choice, which has non-uniform pore sizes and can more effectively enhance cell migration, cell adhesion, and intracellular signal transduction compared to a uniform porous structure. At the same time, the mechanical properties of porous scaffolds with too large pore sizes also decrease significantly. However, few nanocomposites focus on preparing hierarchical porous nanostructures with adjustable mechanical properties. In addition, most of the early reported HAP / polymer composites for tissue engineering were prepared by traditional polymer processing techniques, which showed obvious disadvantages such as low flexibility in geometric design, long processing time, and dependence on expensive molds, making customization impractical. Summary of the Invention

[0004] To at least partially solve the above defects in the processing of HAP / polymer composites in the prior art, the present application first proposes a method for preparing a DEGDA / hydroxyapatite composite material with hierarchical pores, which includes the following steps:

[0005] (1) Put nano-HAP (hydroxyapatite) into the chitosan acidic solution, stir evenly and conduct the first reaction. After the first reaction is completed, filter the reactants to obtain the first filter residue. Then, after washing and drying the first filter residue, chitosan-coated hydroxyapatite is obtained;

[0006] (2) Put chitosan-coated hydroxyapatite into the DMAC (N,N-dimethylacetamide) solution, and then drop acryloyl chloride and triethylamine into the DMAC solution to form a reaction solution. Keep stirring and conduct the second reaction at room temperature. After the second reaction is completed, filter the reactants to obtain the second filter residue. Then, after washing and drying the second filter residue, modified chitosan-coated hydroxyapatite is prepared;

[0007] (3) Mix the modified chitosan-coated hydroxyapatite, DEGDA (diethyleneglycol diacrylate), photoinitiator and porogen evenly and use it as printing ink for photocuring printing to form a model;

[0008] Wash the model with absolute ethanol, then conduct post-curing, and then wash it with distilled water and freeze-dry to obtain the DEGDA / hydroxyapatite composite material.

[0009] In this application, the DEGDA / hydroxyapatite composite material is simply referred to as the composite material.

[0010] In step (1), to improve the dosage accuracy of nano-HAP, before putting nano-HAP into the chitosan acidic solution, it is best to heat and dry nano-HAP at a temperature of 60-90 °C for 5-10 h.

[0011] After the first reaction is completed, the washing of the first filter residue is preferably carried out with distilled water or deionized water. When drying the filtered filter residue, the drying temperature is controlled at a temperature of 70-100 °C and the drying time is controlled at 10-15 h.

[0012] The photoinitiator specifically uses the photoinitiator phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide.

[0013] In step (3), when mixing the modified chitosan-coated hydroxyapatite, DEGDA (diethyleneglycol diacrylate), photoinitiator and porogen, use a high-speed stirrer with a stirring speed of 8000-10000 rpm.

[0014] In this application, in order to smoothly add HAP to the DEGDA material to form a composite material and improve the strength of DEGDA when used as an artificial bone scaffold material. In the scaffold material, in order to successfully complete the loading and transfer of cells to repair tissue defects or regenerate new tissues, it is necessary to form pores in the scaffold that mimic those in bone tissue to provide a larger specific surface area and larger volume for cell adhesion, proliferation, and growth. However, after forming the pores, the strength of the scaffold will inevitably be weakened. Therefore, it is necessary to improve the strength of the scaffold. HAP is a crystal chemical analogue of the natural bone mineral component and has long been used as the main inorganic component of bone tissue engineering synthetic materials. Adding it to DEGDA can improve the strength of DEGDA. However, when simply adding HAP, the strength of DEGDA cannot be effectively improved, and it will also have an adverse effect on the formation of pores in the scaffold.

[0015] In this application, chitosan is used to coat HAP, and chitosan is modified with acryloyl chloride and triethylamine. The acryloyl chloride reacts with the amino group of chitosan to reduce the agglomeration phenomenon of HAP, improve the dispersion uniformity of HAP in DEGDA, and make the connection between adjacent pores smoother. This not only improves the uniformity of the pores in the composite material but also enables the formation of the designed macropores in the composite material by means of photocuring 3D printing and the formation of the required micropores using porogens, so as to maximize the simulation of the pores in natural bone tissue and reduce the production cost of the composite material.

[0016] Due to the reduction of the agglomeration phenomenon of HAP and the formation of a smoother connection between adjacent pores, the composite material has a higher strength, and the porosity, specific surface area, and strength of the composite material are all improved, enabling the composite material to be used as a material for making artificial scaffolds.

[0017] Specifically, in step (1), the chitosan acidic solution is prepared from chitosan and an acetic acid aqueous solution, where the concentration of chitosan is 4 - 6 g / L and the concentration of acetic acid is 5 - 7 g / L; the mass ratio of HAP to chitosan is 1:(0.08 - 0.11). Under the above limitations, a uniform chitosan coating layer can be formed on the surface of HAP. The addition of chitosan can effectively improve the dispersion uniformity of HAP. When the proportion of chitosan is too low, the dispersion uniformity of HAP cannot be effectively improved, and after reaching a certain proportion, its dispersion effect remains unchanged.

[0018] Specifically, in step (1), the first reaction is carried out at room temperature, and the reaction time is 2 - 4 h. Under the above limitations, in an acidic environment, chitosan can successfully complete the cross-linking reaction and coat the HAP particles.

[0019] Specifically, in step (2), the concentration of chitosan-coated hydroxyapatite in the reaction solution is 100-130 g / L; the concentrations of acryloyl chloride and triethylamine are both 15-25 g / L.

[0020] Specifically, to ensure the smooth reaction of chitosan and its coating on HAP particles, in step (2), the reaction time of the second reaction is 5-8 h. To avoid too fast reaction, the second reaction is carried out at room temperature, so sufficient reaction time is required. If the reaction time is too short, part of the chitosan cannot be cross-linked and remains in a free state, unable to initiate the coating effect on HAP. However, too long reaction time is also unnecessary.

[0021] Specifically, to fully remove the unreacted acryloyl chloride and triethylamine, in step (2), the second filter residue is washed with absolute ethanol; vacuum drying is adopted, the drying time is 5-8 h, and the drying temperature is 30-60 °C.

[0022] Specifically, to form a porosity, specific surface area, and strength that all meet the corresponding requirements, in step (3), the weight ratio of modified chitosan-coated hydroxyapatite, DEGDA, photoinitiator, and porogen is (0.9-1.2):100:(0.8-1.2):(60-75).

[0023] Specifically, the porogen is a mixed solution of n-octanol, PVP, and absolute ethanol, and the mass ratio of n-octanol, PVP, and absolute ethanol is 70:(1.5-2.1):(27.9-28.5). In this application, a combined porogen is used to form the required small pores. The above absolute ethanol mainly serves as a solvent for PVP and plays an auxiliary role as a porogen. When preparing the porogen, first dissolve PVP in absolute ethanol to make a PVP ethanol solution, and then mix the PVP ethanol solution with n-octanol to form the porogen. The coordination of n-octanol, PVP, and absolute ethanol forms appropriate small pores. If only n-octanol or PVP ethanol solution is used, although a certain number of small pores can also be formed, the number of pores is insufficient, affecting the porosity and specific surface area of the DEGDA / hydroxyapatite composite material.

[0024] The combination of n-octanol, ethanol, and PVP can improve the uniformity of phase separation, and their synergistic effect optimizes the pore size, connectivity, and structural stability. More interconnected pore structures are formed instead of isolated holes.

[0025] n-Octanol is a long-chain alcohol with a certain lipophilicity, which can be well compatible with ink components such as DEGDA to form a stable mixed system. PVP is a hydrophilic polymer with good water solubility and biocompatibility. The combination of n-octanol and PVP ethanol solution can form a synergistic effect in the ink, improving the overall performance of the ink. The lipophilicity of n-octanol helps to interact with other components in the ink, enhancing the stability of the ink, while the hydrophilicity of PVP helps to improve the fluidity and film-forming property of the ink, making the printing process smoother.

[0026] During the 3D printing process, the combination of n-octanol and PVP ethanol solution can promote the phase separation of the ink, forming a uniform pore structure. The chain structure of n-octanol helps to form dispersed holes in the ink, while the hydrophilicity of PVP helps to form channels between the holes, connecting the dispersed holes with each other.

[0027] The addition of PVP ethanol solution can, to a certain extent, improve the uniformity of phase separation, forming more interconnected pore structures rather than isolated holes. The holes formed by using n-octanol alone have poor continuity and smaller pore diameters. While the holes formed by using PVP ethanol solution alone are relatively large in size. The combined use of PVP ethanol solution and n-octanol, on the one hand, the macromolecular PVP can provide larger pore sizes in the scaffold, and at the same time, utilize the network structure of PVP to connect the dispersed n-octanol holes with each other, forming a pore channel network structure.

[0028] The holes in the composite material are formed after the porogen is removed. Ethanol, as a polar solvent, in addition to being a solvent for PVP, can also dissolve part of the DEGDA resin. However, neither ethanol nor n-octanol is soluble in the DEGDA polymer, thus promoting phase separation. The addition of PVP with polar groups can adjust the polarity of the mixed solvent, enhancing the driving force of phase separation, and the pyrrolidone group in PVP helps the good dispersion of HAP in the DEGDA matrix.

[0029] Preferably, the DEGDA / hydroxyapatite composite material has macropores with a size of 300 - 1000 μm and micropores with a size of 0.1 - 0.3 μm on the pore walls. Modern research shows that in addition to macropores with a size of 20 - 1500 μm, micropores smaller than 10 μm also help to improve the interaction between cells and the composite material and promote osteogenesis. Especially for micropores smaller than 1 μm, considering factors such as the limitations of photocuring printing and the strength of the composite material, the macropores in this application are limited between 300 - 1000 μm. To fully improve the interaction between cells and the composite material and enhance osteogenesis, the micropores in this application are limited between 0.1 - 0.3 μm as described above.

[0030] Secondly, the present application also discloses a scaffold prepared by using any one of the above DEGDA / hydroxyapatite composites with hierarchical pores. Description of the Drawings

[0031] Figure 1 is the Fourier transform infrared spectrum of HAP and the modified chitosan-coated hydroxyapatite prepared in Example 1.

[0032] Figure 2 are the SEM images of the scaffolds in Example 1, Comparative Examples 1-3.

[0033] Figure 3 is the pore size distribution diagram of the scaffolds in Example 1, Comparative Example 5 and Comparative Example 6.

[0034] Figure 4 are the SEM images of the surface morphologies of the scaffolds in Example 1, Comparative Example 1 and Comparative Example 3.

[0035] Figure 5 are the results of cytoskeleton staining of the scaffolds in Example 1, Comparative Examples 1-4. Detailed Embodiments

[0036] In the following examples and comparative examples, the macropores are all set to 500 μm and are directly formed during the photocuring printing process, and the micropores are formed by porogens.

[0037] In the following examples and comparative examples, the mass percentage of diethyleneglycol diacrylate (DEGDA) is 75%; the average particle size of hydroxyapatite particles (HAP) is 200 nm; the molecular weight of polyvinylpyrrolidone (PVP K30) is M w = 45000.

[0038] Example 1

[0039] Preparation of the 1# scaffold:

[0040] (1) Heat the nano-HAP at 80 °C for 6 hours for drying to remove the adsorbed water on the surface. Take 10 g of the dried nano-HAP and put it into the chitosan acidic solution, stir evenly and carry out the first reaction at room temperature for 3 h. After the first reaction is completed, filter the reactants to obtain the first filter residue. Then wash the first filter residue 3 times with distilled water and dry it at 80 °C for 12 h to obtain chitosan-coated hydroxyapatite.

[0041] The chitosan acidic solution is prepared from chitosan and acetic acid aqueous solution, where the concentration of chitosan is 5 g / L and the concentration of acetic acid is 6 g / L; the mass ratio of HAP to chitosan is 1:0.1.

[0042] (2) The chitosan-coated hydroxyapatite was put into the DMAC solution, and then acryloyl chloride and triethylamine were dropped into the DMAC solution to form a reaction solution. Stirring was maintained, and the second reaction was carried out at room temperature for 6 h. After the second reaction was completed, the reactants were filtered to obtain the second filter residue, which was then washed 3 times with anhydrous ethanol and then vacuum dried at 40 °C for 6 h to prepare the modified chitosan-coated hydroxyapatite. The modified chitosan-coated hydroxyapatite obtained in this example was labeled as m-CS@HAP.

[0043] The concentration of chitosan-coated hydroxyapatite in the reaction solution was 110 g / L; the concentrations of acryloyl chloride and triethylamine were both 20 g / L.

[0044] (3) Using a high-speed stirrer, at a stirring speed of 9000 rpm, the modified chitosan-coated hydroxyapatite, DEGDA, photoinitiator, and porogen were mixed evenly to form printing ink, and then photocuring printing was carried out to form a model. The weight ratio of the modified chitosan-coated hydroxyapatite, DEGDA, photoinitiator, and porogen was 1:100:1:66. The porogen was composed of n-octanol, PVP, and anhydrous ethanol mixed in a mass ratio of 70:1.8:28.2. When preparing the porogen, PVP was first dissolved in anhydrous ethanol, and then mixed with n-octanol and stirred evenly.

[0045] The model was washed with anhydrous ethanol to remove the unreacted materials, and then irradiated with ultraviolet light with a wavelength of 365 nm for 30 minutes for post-curing. Then, it was ultrasonically washed 3 times in anhydrous ethanol for 30 minutes each time, washed with distilled water for 30 minutes, and finally freeze-dried to obtain the 1# scaffold.

[0046] HAP and the modified chitosan-coated hydroxyapatite in this example were detected, and the Fourier transform infrared spectrum shown as follows was obtained. In the infrared spectrum of m-CS@HAP, the absorption peaks at 2917 cm Figure 1 and 2844 cm -1 were clearly visible. These two peaks corresponded to the symmetric and asymmetric stretching vibrations of the C-H of the alkyl chain and chitosan, respectively. In addition, after grafting, a new absorption peak, namely, the stretching vibration peak of the C=O bond at 1701 cm -1 , appeared in the FTIR spectrum. This feature indicated that the grafting reaction was successful and confirmed the modification effect on the material surface. -1

[0047] Example 2

[0048] Preparation of the 2# scaffold:

[0049] (1) Heat the nano-HAP at 50 °C for 10 hours to dry it and remove the water adsorbed on the surface. Take 10 g of the dried nano-HAP and put it into the chitosan acidic solution. Stir evenly and conduct the first reaction at room temperature for 2 h. After the first reaction is completed, filter the reactants to obtain the first filter residue. Then wash the first filter residue 3 times with distilled water and dry it at 70 °C for 15 h to obtain chitosan-coated hydroxyapatite.

[0050] The chitosan acidic solution is prepared from chitosan and acetic acid aqueous solution. The concentration of chitosan is 6 g / L, and the concentration of acetic acid is 5 g / L; the mass ratio of HAP to chitosan is 1:0.11.

[0051] (2) Put the chitosan-coated hydroxyapatite into the DMAC solution, and then drop acryloyl chloride and triethylamine into the DMAC solution to form a reaction solution. Keep stirring and conduct the second reaction at room temperature for 5 h. After the second reaction is completed, filter the reactants to obtain the second filter residue. Then wash the second filter residue 3 times with anhydrous ethanol and vacuum dry it at 30 °C for 8 h to prepare modified chitosan-coated hydroxyapatite.

[0052] The concentration of chitosan-coated hydroxyapatite in the reaction solution is 100 g / L; the concentrations of acryloyl chloride and triethylamine are both 15 g / L.

[0053] (3) Using a high-speed stirrer, at a stirring speed of 8000 rpm, mix the modified chitosan-coated hydroxyapatite, DEGDA, photoinitiator, and porogen evenly as printing ink, and conduct photocuring printing to form a model. The weight ratio of the modified chitosan-coated hydroxyapatite, DEGDA, photoinitiator, and porogen is 1.1:100:1.2:61. The porogen is composed of n-octanol, PVP, and anhydrous ethanol with a mass ratio of 70:1.5:28.5. When preparing the porogen, first dissolve PVP in anhydrous ethanol, and then mix it with n-octanol and stir evenly.

[0054] Wash the model with anhydrous ethanol to remove the unreacted materials, then irradiate it with ultraviolet light with a wavelength of 365 nm for 30 minutes for post-curing. Then conduct ultrasonic washing in anhydrous ethanol 3 times, 30 minutes each time, wash it with distilled water for 30 minutes, and finally conduct freeze-drying to obtain the 2# scaffold.

[0055] Example 3

[0056] Preparation of the 3# scaffold:

[0057] (1) Heat the nano-HAP at 90 °C for 5 hours to dry it and remove the surface-adsorbed water. Take 10 g of the dried nano-HAP and put it into the chitosan acidic solution. Stir evenly and conduct the first reaction at room temperature for 4 h. After the first reaction is completed, filter the reactants to obtain the first filter residue. Then wash the first filter residue 3 times with distilled water and dry it at 80 °C for 12 h to obtain chitosan-coated hydroxyapatite.

[0058] The chitosan acidic solution is prepared from chitosan and acetic acid aqueous solution, where the concentration of chitosan is 4 g / L and the concentration of acetic acid is 6 g / L; the mass ratio of HAP to chitosan is 1:0.08.

[0059] (2) Put the chitosan-coated hydroxyapatite into the DMAC solution, and then drop acryloyl chloride and triethylamine into the DMAC solution to form a reaction solution. Keep stirring and conduct the second reaction at room temperature for 8 h. After the second reaction is completed, filter the reactants to obtain the second filter residue. Then wash the second filter residue 5 times with absolute ethanol and vacuum dry it at 60 °C for 5 h to obtain modified chitosan-coated hydroxyapatite.

[0060] The concentration of chitosan-coated hydroxyapatite in the reaction solution is 130 g / L; the concentrations of acryloyl chloride and triethylamine are both 25 g / L.

[0061] (3) Using a high-speed stirrer, at a stirring speed of 10,000 rpm, mix the modified chitosan-coated hydroxyapatite, DEGDA, photoinitiator and porogen evenly to form printing ink, and conduct photocuring printing to form a model. The weight ratio of the modified chitosan-coated hydroxyapatite, DEGDA, photoinitiator and porogen is 0.9:100:1.1:75. The porogen is composed of a mixture of n-octanol, PVP and absolute ethanol with a mass ratio of 70:2.1:27.9. When preparing the porogen, first dissolve PVP in absolute ethanol, and then mix it with n-octanol and stir evenly.

[0062] Wash the model with absolute ethanol to remove the unreacted materials, and then irradiate it with ultraviolet light with a wavelength of 365 nm for 30 minutes for post-curing. Then conduct ultrasonic washing in absolute ethanol 3 times, 30 minutes each time, wash it with distilled water for 30 minutes, and finally conduct freeze-drying to obtain the 3# scaffold.

[0063] Example 4

[0064] Preparation of the 4# scaffold:

[0065] (1) Heat the nano-HAP at 70 °C for 6 hours to dry it and remove the surface-adsorbed water. Take 10 g of the dried nano-HAP and put it into the chitosan acidic solution. Stir evenly and carry out the first reaction at room temperature for 3 h. After the first reaction is completed, filter the reactants to obtain the first filter residue. Then wash the first filter residue 3 times with distilled water and dry it at 100 °C for 10 h to obtain chitosan-coated hydroxyapatite.

[0066] The chitosan acidic solution is prepared from chitosan and acetic acid aqueous solution, where the concentration of chitosan is 5 g / L and the concentration of acetic acid is 7 g / L; the mass ratio of HAP to chitosan is 1:0.09.

[0067] (2) Put the chitosan-coated hydroxyapatite into the DMAC solution, and then drop acryloyl chloride and triethylamine into the DMAC solution to form a reaction solution. Keep stirring and carry out the second reaction at room temperature for 7 h. After the second reaction is completed, filter the reactants to obtain the second filter residue. Then wash the second filter residue 4 times with absolute ethanol and vacuum-dry it at 50 °C for 6 h to obtain modified chitosan-coated hydroxyapatite.

[0068] The concentration of chitosan-coated hydroxyapatite in the reaction solution is 120 g / L; the concentrations of acryloyl chloride and triethylamine are both 22 g / L.

[0069] (3) Using a high-speed stirrer, mix the modified chitosan-coated hydroxyapatite, DEGDA, photoinitiator and porogen evenly at a stirring speed of 9000 rpm as the printing ink, and carry out photocuring printing to form a model. The weight ratio of the modified chitosan-coated hydroxyapatite, DEGDA, photoinitiator and porogen is 1.2:100:0.8:70. The porogen is composed of n-octanol, PVP and absolute ethanol mixed in a mass ratio of 70:2.0:28.0. When preparing the porogen, first dissolve PVP in absolute ethanol, and then mix it with n-octanol and stir evenly.

[0070] Wash the model with absolute ethanol to remove the unreacted materials, then irradiate it with ultraviolet light with a wavelength of 365 nm for 30 minutes for post-curing. Then carry out ultrasonic washing in absolute ethanol 3 times, 30 minutes each time, wash it with distilled water for 30 minutes, and finally carry out freeze-drying to obtain the 4# scaffold.

[0071] Comparative Example 1

[0072] Preparation of the 5# scaffold:

[0073] This comparative example is basically the same as Example 1, except that step 2 is cancelled and the scaffold is directly prepared using chitosan-coated hydroxyapatite.

[0074] Comparative Example 2

[0075] Preparation of the 6# scaffold:

[0076] (1) Heat the nano-HAP at 80 °C for 6 hours to dry it and remove the surface-adsorbed water.

[0077] (2) Using a high-speed stirrer, mix nano-HAP, DEGDA, photoinitiator, and porogen evenly at a stirring speed of 9000 rpm to form printing ink, and perform photocuring printing to form a model. The weight ratio of nano-HAP, DEGDA, photoinitiator, and porogen is 1:100:1:66. The porogen is composed of a mixture of n-octanol, PVP, and absolute ethanol with a mass ratio of 70:1.8:28.2. When preparing the porogen, first dissolve PVP in absolute ethanol, then mix it with n-octanol and stir evenly.

[0078] Wash the model with absolute ethanol to remove the unreacted materials, then irradiate it with ultraviolet light with a wavelength of 365 nm for 30 minutes for post-curing. Then perform ultrasonic washing in absolute ethanol 3 times, 30 minutes each time, wash with distilled water for 30 minutes, and finally perform freeze-drying to obtain the 1# scaffold.

[0079] In this comparative example, nano-HAP was directly used to prepare the scaffold.

[0080] Comparative Example 3

[0081] Preparation of the 7# scaffold:

[0082] (1) Heat the nano-HAP at 80 °C for 6 hours to dry it and remove the surface-adsorbed water.

[0083] (2) Using a high-speed stirrer, mix nano-HAP, DEGDA, photoinitiator, and porogen evenly at a stirring speed of 9000 rpm to form printing ink, and perform photocuring printing to form a model. The weight ratio of nano-HAP, DEGDA, photoinitiator, and porogen is 2:100:1:66. The porogen is composed of a mixture of n-octanol, PVP, and absolute ethanol with a mass ratio of 70:1.8:28.2. When preparing the porogen, first dissolve PVP in absolute ethanol, then mix it with n-octanol and stir evenly.

[0084] Wash the model with absolute ethanol to remove the unreacted materials, then irradiate it with ultraviolet light with a wavelength of 365 nm for 30 minutes for post-curing. Then perform ultrasonic washing in absolute ethanol 3 times, 30 minutes each time, wash with distilled water for 30 minutes, and finally perform freeze-drying to obtain the 1# scaffold.

[0085] In this comparative example, nano-HAP was directly used to prepare the scaffold.

[0086] Comparative Example 4

[0087] Preparation of the 8# scaffold:

[0088] This comparative example is basically the same as Example 1, except that the modification of chitosan-coated hydroxyapatite was cancelled, and the scaffold was directly prepared with DEGDA.

[0089] Comparative Example 5

[0090] Preparation of the 9# scaffold:

[0091] This comparative example is basically the same as Example 1, except that the porogen consists only of n-octanol.

[0092] Comparative Example 6

[0093] Preparation of the 10# scaffold:

[0094] This comparative example is basically the same as Example 1, except that the porogen consists only of PVP and absolute ethanol, and the mass ratio of PVP to absolute ethanol is 6:94.

[0095] The SEM images of the scaffolds prepared in Example 1 and Comparative Examples 1-3 are as Figure 2 shown, where Figure 2 (a) is the scaffold in Example 1, Figure 2 (b) is the scaffold in Comparative Example 1, Figure 2 (c) is the scaffold in Comparative Example 2, Figure 2 (d) is the scaffold in Comparative Example 3.

[0096] It can be seen from Figure 2 that when the nano-HAP content reaches 2 wt%, obvious agglomeration of nano-HAP particles occurs, filling the larger voids between the pores, thus reducing the number of open pores. Compared with the surfaces of the scaffolds in Comparative Examples 1 and 2, the connections between the pores in the scaffold of Example 1 are smoother. This is because triethylamine, as a catalyst, promoted the reaction between acryloyl chloride and the amino group of chitosan, improving the uniform dispersion of the modified hydroxyapatite in the 3D printing ink, reducing the agglomeration phenomenon, resulting in smoother and stronger connections between the pores, thereby improving the overall mechanical properties of the scaffold.

[0097] When the content of nano-HAP is low, a large amount of particle agglomeration does not occur, the pores are not blocked, and the surface of the scaffold is more conducive to cell growth; while when the HAP content is high, the whole scaffold can better promote bone mineralization and integration. A larger specific surface area helps for more sufficient material exchange and energy transfer between cells and the external environment. Therefore, an appropriate porosity can effectively achieve the balance between cell growth and bone mineralization. The porosities of the scaffolds in Examples 1-4 and Comparative Examples 1-6 are 45.4%, 44.9%, 45.7%, 44.4%, 44.5%, 43.75%, 34.1%, 47.0%, 28.5%, and 32.6% respectively. It can be seen that, under the same addition amount, the porosity of the scaffold in Example 1 is slightly increased. The porosity of Comparative Example 4 without adding modified chitosan-coated hydroxyapatite is the highest, indicating that as long as other substances are added, the porosity in the scaffold will be reduced, but modified chitosan-coated hydroxyapatite is beneficial to reducing the reduction range of porosity.

[0098] As the content of nano-HAP increases, more HAP particles are filled inside the scaffold, occupying more space, resulting in a reduction in the contact area, a decrease in porosity, and ultimately a decrease in the specific surface area. However, the modified nano-HAP makes the surface of the scaffold smoother, and the specific surface area is increased compared with other components.

[0099] The porosities of Comparative Examples 6 and 7 are low, indicating that only using PVP ethanol solution or n-octanol cannot form a large number of pores in the scaffold.

[0100] The specific surface areas of the scaffolds in Examples 1-4 and Comparative Examples 1-6 are 21.8 m 2 / g, 21.4 m 2 / g, 22.3 m 2 / g, 21.2 m 2 / g, 19.9 m 2 / g, 19.2 m 2 / g, 15.7 m 2 / g, 28.1 m 2 / g, 16.8 m 2 / g, 19.5 m 2 / g. It can be seen that, under the same addition amount, the specific surface area of the scaffold in Example 1 is slightly increased, and the specific surface area roughly corresponds to the porosity.

[0101] In addition to porosity and specific surface area, pore size distribution is also an important parameter for evaluating bone scaffold materials. Modern research shows that in addition to macropores of 20 - 1500 μm, micropores less than 10 μm also help to improve the interaction between cells and the scaffold and promote osteogenesis, especially micro-pores below 1 μm. The pore size ranges of the micro-pores in the scaffolds of Example 1, Comparative Example 5, and Comparative Example 6 are as Figure 3As shown, from Figure 3 It can be seen that the pore size ranges of the micropores of the scaffolds in Example 1, Comparative Example 5, and Comparative Example 6 are roughly the same. However, the number of micropores in Comparative Example 5 and Comparative Example 6 is significantly reduced compared to Example 1, which also leads to a corresponding decrease in the porosity and specific surface area of Comparative Example 5 and Comparative Example 6. A new characteristic peak appears at about 8 μm in the scaffold of Example 1, indicating that the modified HAP generates more uniform pores by enhancing the interaction with the surface.

[0102] Although a higher porosity and larger pore size are helpful for supporting cell migration, an overly large pore size will reduce the mechanical strength of the scaffold, thereby limiting its application in tissue engineering. Therefore, while ensuring that the scaffold has a high porosity and large pore size, it is also necessary to ensure that it has sufficient mechanical strength.

[0103] The coating of chitosan can not only enhance the mechanical strength of the material but also improve its biocompatibility. Therefore, the porous scaffold containing modified chitosan-coated hydroxyapatite can not only maintain a high porosity and specific surface area to provide a good growth surface for cells but also, due to its high HAP content, is more conducive to promoting bone mineralization and integration.

[0104] The compressive strengths of the scaffolds in Example 1 - 4 and Comparative Example 1 - 6 are 9.96 MPa, 10.31 MPa, 9.85 MPa, 10.52 MPa, 7.21 MPa, 5.80 MPa, 3.96 MPa, 2.93 MPa, 9.21 MPa, and 9.54 MPa respectively. It can be seen that, under the same addition amount, the strength of the scaffold in Example 1 has a significant increase compared to Comparative Example 1 and 2, indicating that the modified hydroxyapatite can effectively improve the strength of the scaffold.

[0105] This is because hydroxyapatite, as an inorganic filler, is difficult to disperse uniformly in the printing ink. However, the modified nano-HAP can be more uniformly dispersed in the 3D printing ink, and this effect is reflected in the SEM images of the porous scaffolds. Figure 4 SEM images of the surface morphologies of the scaffolds in Example 1, Comparative Example 1, and Comparative Example 3 are respectively shown, where Figure 4 (a) is the SEM image of the surface morphology of the scaffold in Example 1, Figure 4 (b) is the SEM image of the surface morphology of the scaffold in Comparative Example 1, Figure 4 (c) is the SEM image of the surface morphology of the scaffold in Comparative Example 3. From Figure 4It can be seen that, compared with Comparative Example 1, the HAP particles in the scaffold of Comparative Example 3 are significantly agglomerated, with a relatively high degree of agglomeration and a large number of particles. This leads to a weakening of the interfacial interaction between the polymer matrix and the HAP particles, and a lack of sufficient adhesion force between the particles to maintain the structural strength, resulting in a significant decrease in the compressive strength. In contrast, compared with Comparative Example 1, in Example 1, the vast majority of HAP particles adhere to the joints between the pores, enhancing the mechanical strength of the porous scaffold. In Comparative Example 1, most of the pores are blocked by HAP particles, resulting in a lower compressive strength than that of Example 1.

[0106] The swelling ratios of the scaffolds in each example and comparative example were measured. Within 24 hours, the swelling ratios of all scaffolds remained at about 120%, indicating that HAP has little effect on the water absorption capacity of the scaffolds. This is mainly because the hydrophilicity of the DEGDA matrix dominates the water absorption process, so the swelling ratio remains relatively stable.

[0107] Cell compatibility of the scaffold:

[0108] The specific steps are as follows:

[0109] (1.1) Preparation of the extract of the porous scaffold

[0110] Prepared according to the protocol established by the International Organization for Standardization (ISO 10993-12). The scaffold was added to the culture medium to prepare a stock solution of 200 mg / mL. After incubation at 37 °C for 24 hours, it was centrifuged at 2000 rpm for 5 minutes, and the supernatant was collected. The supernatant was filtered and sterilized through a 0.22 μm sterile filter membrane and stored.

[0111] (1.2) Cell culture

[0112] RAW 264.7 cells were used in the in vitro study. RAW 264.7 cells were purchased from ScienCell Research Laboratories. RAW 264.7 cells were cultured in a medium supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin (P / S), and incubated in a humidified environment at 37 °C, 95% air and 5% CO 2 2. Once the cells reached approximately 90% confluence, they were passaged using 0.25% (w / v) trypsin-EDTA. Only cells from the 3rd to 6th passages were used in the experiment.

[0113] (1.3) Cell seeding and attachment

[0114] To seed RAW 264.7 cells onto the scaffold materials, the scaffolds were first printed to fit 24-well plates or culture dishes. To ensure sterility, before cell seeding, each scaffold was soaked in 75% ethanol and exposed to ultraviolet (UV) light for disinfection. RAW 264.7 cells were seeded onto 24-well plates (3×10 4 cells per scaffold) to study the effect of each scaffold on cell adhesion. RAW 264.7 cells were cultured for 24 hours. The cells on the scaffolds were stained with F-actin to show the cytoskeleton (Fluor TM 488 labeling kit, abbkine, China), and the cell nuclei were stained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, Beyotime, China). Finally, the stained samples were observed using a confocal laser scanning microscope (CLSM, Nikon, Japan).

[0115] (1.4) Cell compatibility assay

[0116] According to the manufacturer's instructions, Cell Counting Kit-8 (CCK-8, Dojindo, Japan) was used to evaluate the proliferation of RAW 264.7 cells. Specifically, 3×10 5 cells per well were seeded onto the scaffold samples in 96-well plates. After incubation with 10% (v / v) CCK-8 solution at 37 °C for 1 hour, the absorbance was measured at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, USA). The results were analyzed using GraphPad Prism 9 software.

[0117] The CCK-8 assay was used to evaluate the cytotoxicity of the scaffolds in Example 1 and Comparative Examples 1-4 on RAW 264.7 cells. Compared with the control group, the cell viability in Comparative Example 3 decreased significantly because there was agglomeration of nano-HAP inside the scaffold in Comparative Example 3, and higher concentrations of HAP particles directly contacted the cell surface, which might damage the cell membrane structure, leading to cell injury and triggering cell death. While the cell viability in Example 1 was relatively high, comparable to that in Comparative Examples 1 and 2, indicating that the scaffold with modified hydroxyapatite not only had a hierarchical porous structure but also could maintain high mechanical properties and certain cell compatibility.

[0118] In addition, RAW 264.7 cells cultured for 1 day in the scaffolds in Example 1 and Comparative Examples 1-4 were evaluated by cytoskeleton staining. Figure 5 The fluorescence staining images of cells cultured for 24 hours in each scaffold are shown Figure 5 (a), Figure 5 (b), Figure 5 (c), Figure 5 (d).Figure 5 (e) Fluorescence staining images of cells corresponding to the scaffolds of Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4, respectively. Figure 5 In the figure, the cytoskeleton (green) and the cell nucleus (blue) are labeled, and the differences in cell morphology among different samples can be observed. The cells corresponding to Example 1, Comparative Example 1, 2, and 4 showed strong adhesion and cluster morphology, with a large number of cells, normal morphology, and the HAP in the scaffold had no significant negative impact on the cells. The cytoskeleton (green) showed a relatively uniform and dense distribution, indicating that the cells could grow and divide normally. After modification, the HAP reduced the aggregation effect and decreased the particle size, thus improving the cell adhesion and proliferation ability on the scaffold. The cytoskeleton staining in the fluorescence image was relatively clear, indicating that Example 1, Comparative Example 1, 2, and 4 were safe and had good biocompatibility. In contrast, the cell morphology corresponding to Comparative Example 3 changed significantly. There were fewer cell clusters in the fluorescence image, poor cell adhesion, uneven cytoskeleton distribution, and a significant reduction in the number of cells. It may be due to the adverse effect of the aggregation effect of high-concentration HAP on the cells. The large particles of HAP changed the cell adhesion force, resulting in abnormal cell growth or division.

Claims

1. A method for preparing a DEGDA / hydroxyapatite composite material having hierarchical porosity, characterized in that: The steps include: (1) adding nano-HAP into a chitosan acid solution, stirring evenly and performing a first reaction, filtering the reactant after the first reaction is completed to obtain a first filter residue, and then washing and drying the first filter residue to obtain chitosan-coated hydroxyapatite; (2) putting chitosan-coated hydroxyapatite into a DMAC solution, then dropping acryloyl chloride and triethylamine into the DMAC solution to form a reaction solution, keeping stirring, and performing a second reaction at room temperature. After the second reaction is completed, filtering the reactant to obtain a second filter residue, and then washing and drying the second filter residue to obtain modified chitosan-coated hydroxyapatite; (3) mixing the modified chitosan-coated hydroxyapatite, DEGDA, a photoinitiator and a porogen evenly as printing ink, and performing photocuring printing to form a model; The model was cleaned with anhydrous ethanol, then post-cured, washed with distilled water, and freeze-dried to obtain the DEGDA / hydroxyapatite composite material.

2. The preparation method according to claim 1, characterized in that: In step (1), the chitosan acid solution is prepared from chitosan and acetic acid aqueous solution, wherein the concentration of chitosan is 4-6 g / L, the concentration of acetic acid is 5-7 g / L; and the mass ratio of HAP to chitosan is 1:(0.08-0.11).

3. The preparation method according to claim 1, characterized in that: In step (1), the first reaction is carried out at room temperature and the reaction time is 2-4 hours.

4. The preparation method according to claim 1, characterized in that: In step (2), the concentration of chitosan-coated hydroxyapatite in the reaction solution is 100-130 g / L; the concentrations of acryloyl chloride and triethylamine are both 15-25 g / L.

5. The preparation method according to claim 1, characterized in that: In step (2), the reaction time of the second reaction is 5-8h.

6. The preparation method according to claim 1, characterized in that: In step (2), the second filter residue is washed with anhydrous ethanol; vacuum drying is adopted, the drying time is 5-8 hours, and the drying temperature is 30-60°C.

7. The preparation method according to claim 1, characterized in that: In step (3), the weight ratio of modified chitosan-coated hydroxyapatite, DEGDA, photoinitiator and porogen is (0.9-1.2):100:(0.8-1.2):(60-75).

8. The preparation method according to claim 1, characterized in that: The porogen is a mixed solution of n-octanol, PVP and anhydrous ethanol, and the mass ratio of n-octanol, PVP and anhydrous ethanol is 70:(1.5-2.1):(27.9-28.5).

9. The preparation method according to claim 1, characterized in that: The DEGDA / hydroxyapatite composite material has macropores of 300-1000 μm and small pores of 0.1-0.3 μm on the pore wall.

10. A stent prepared using the DEGDA / hydroxyapatite composite material with hierarchical porosity according to any one of claims 1 to 9.

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