A composite hydrogel scaffold material with three-dimensional cell ingrowth and multiple osteogenesis-promoting properties and a preparation method thereof

Abstract

The application discloses a kind of composite hydrogel scaffold material with cell three-dimensional growth and multiple osteogenesis performance and preparation method thereof, method includes: ① using freeze-blasting method to prepare inorganic mineral / alkali-dissolved chitosan solution, after centrifugal defoaming, it is poured in mould and heated gelation and is aged to obtain inorganic mineral / alkali-dissolved chitosan gel;② using semi-freezing demolding technology to obtain the composite hydrogel scaffold with specific three-dimensional mesh structure;③ the scaffold is immersed in Tris-HCl buffer solution containing dopamine and carries out in-situ self-polymerization reaction;④ after dialysis of gel scaffold completely, on its surface, through chemical grafting reaction, introduce sodium alendronate, and further through ion capture process, obtain the composite hydrogel scaffold material loaded with osteogenesis ion.The hydrogel scaffold has excellent biocompatibility, degradability, cell three-dimensional growth capacity, cell proliferation and osteogenesis performance, and shows great application potential in bone repair field.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogel preparation, and in particular to a composite hydrogel scaffold material with three-dimensional cell ingrowth and multiple osteogenesis-promoting properties and a preparation method thereof. Background Art

[0002] Bone defects, defined as a disruption of bone structural integrity due to congenital or acquired causes, remain a highly prevalent clinical condition. Despite recent advances in tissue engineering and other medical fields, bone defects remain a challenging condition, with a high amputation rate. Currently, treatments for bone defects include bone transplantation, the Ilizarov technique, membrane-induced regeneration, gene therapy, and bone tissue engineering.

[0003] Tissue engineering technology primarily focuses on using scaffolds or hydrogels to create a cell growth environment similar to the extracellular matrix, thereby promoting cell proliferation and differentiation and inducing bone regeneration. Despite some progress, many challenges remain, such as poor bioactivity of tissue engineering materials, slow biomaterial degradation, difficulty in cellular incorporation, and limited osteogenesis, all leading to poor bone repair outcomes.

[0004] Therefore, the present invention designs and prepares a composite hydrogel scaffold material with three-dimensional cell ingrowth and multiple osteogenic properties. Through the material's excellent biocompatibility, degradability, three-dimensional cell ingrowth ability, cell proliferation promotion and multiple osteogenic properties, effective treatment of bone defects can be achieved. Summary of the Invention

[0005] The purpose of the present invention is to address the problem of slow bone defect repair and poor repair effect, and to provide a composite hydrogel scaffold material with three-dimensional cell ingrowth and multiple osteogenesis-promoting properties and a preparation method thereof.

[0006] The present invention is achieved by adopting the following technical solutions:

[0007] A composite hydrogel scaffold material with three-dimensional cell ingrowth and multiple osteogenic properties is designed to address the key points of bone defect treatment. It takes into account the three-dimensional ingrowth of cells within the scaffold, the promotion of cell proliferation after ingrowth, and the multiple promotion of osteogenic differentiation after proliferation. The material is made of chitosan, inorganic minerals, dopamine, and sodium alendronate as the main raw materials, and is produced using a combination of processes including freeze-blasting, nanocomposite, in-situ gelation, semi-freeze demolding, in-situ self-polymerization, and surface grafting. The preparation process includes: ① using the freeze-blasting method to prepare an inorganic mineral / alkali-soluble chitosan solution, centrifugally degassing it, pouring it into a mold, heating it for gelation, and aging it to obtain an inorganic mineral / alkali-soluble chitosan gel; ② using the semi-freeze demolding technology to obtain a composite hydrogel scaffold with a specific three-dimensional mesh structure; ③ immersing the scaffold in a Tris-HCl buffer containing dopamine for in situ self-polymerization reaction; ④ after the gel scaffold is completely dialyzed, sodium alendronate is introduced onto its surface through a chemical grafting reaction, and further an ion capture process is performed to obtain a composite hydrogel scaffold material loaded with osteogenic ions.

[0008] Specifically, the preparation method includes the following steps:

[0009] 1) using lithium hydroxide monohydrate and urea as a solvent system, adding chitosan as a solute, and adding a certain amount of inorganic minerals, with the remainder being water, stirring the solution at room temperature for 30-60 minutes, transferring it to a -80°C refrigerator and freezing it completely, then crushing and dissolving the ice cubes by external force at room temperature, repeating this process three times, and preparing a mixed solution by a freeze-blast method, and degassing the solution by low-speed centrifugation for later use;

[0010] 2) pouring the solution obtained in 1) into a customized mold, sealing it after self-leveling, transferring it to 60° C. for in-situ gelation, and aging it for 4-12 hours to obtain a composite physical hydrogel;

[0011] 3) Transfer the hydrogel to a refrigerator for freezing, and demould the sample in a semi-frozen state;

[0012] 4) After the demolded hydrogel scaffold is completely dialyzed, it is suspended and fixed in a dopamine DA / TrisHCl buffer solution with a pH of 8.5, and subjected to in situ self-polymerization reaction under stirring for 4-96 hours to obtain a composite hydrogel scaffold;

[0013] 5) reacting alendronate sodium with an excess of glutaraldehyde to introduce aldehyde groups into the alendronate sodium molecule, terminating the reaction with acetone, and drying to obtain alendronate sodium with aldehyde groups; after complete dialysis of the composite hydrogel scaffold, immersing the scaffold in an aqueous solution of alendronate sodium with aldehyde groups and reacting for 12-36 hours to obtain a composite hydrogel scaffold with alendronate sodium grafted on its surface;

[0014] 6) dialyzing a large amount of deionized water to remove unreacted alendronate sodium, and then transferring the resulting scaffold into an aqueous solution containing metal ions to achieve capture of the metal ions by the alendronate sodium. After dialysis with deionized water, a composite hydrogel scaffold material loaded with metal ions, having three-dimensional cell ingrowth and multiple osteogenic properties, is obtained.

[0015] In the above technical solution, further, in step 1), the concentration of lithium hydroxide monohydrate in the solution is 7-9wt%, the concentration of urea is 5-8wt%; the concentration of chitosan is 2-5wt%, and the molecular weight of chitosan is 500,000-1,200,000; the inorganic minerals include one or more of β-TCP, hydroxyapatite, etc., with a particle size of 200-800 nanometers, and the amount used is 1 / 3-2 times the mass of chitosan; the low-speed centrifugation speed is 3000-5000 rpm, and the centrifugation time is 5-15 minutes;

[0016] Furthermore, the mold customized in step 2) can generally be a mold with a columnar array, wherein one end of the mold with the array contains an array of raised columns, the height of the raised columns is 1-3 mm, the diameter or length and width of the raised columns is 200-1000 μm, and the spacing between the raised columns is 100-400 μm; for example, the array is composed of raised columns of specifications such as 400 μm*400 μm*3 mm or 600 μm*600 μm*3 mm or 800 μm*800 μm*3 mm, and the spacing between the raised columns can be 400 μm. For a specific schematic diagram, see the attached Figure 3 , and the mold parameters include but are not limited to the above three; the self-leveling process takes 5-15 minutes, and a customized mold box can be used for sealing and storage to prevent the loss of water in the hydrogel. The internal cross-sectional area of ​​the mold box is consistent with the cross-sectional area of ​​the mold, and the length is 0.5 cm larger than the mold; the solution gelation time is 4-8 hours;

[0017] Furthermore, the process of semi-frozen demoulding in step 3) is to freeze the sample in a -80°C refrigerator for 10 minutes, transfer the sample to a 25°C environment for thawing for 5 minutes, and demould the sample in a semi-frozen state;

[0018] Furthermore, in step 4), the concentration of DA in the DA / Tris HCl buffer is 1-4 mg / ml, and the magnetic stirring rate is 100-250 rpm; the gel scaffold is clamped and fixed using a needle-type clamp. The gel scaffold needs to be suspended and fixed in the DA / TrisHCl buffer. Otherwise, due to the large number of pores inside, the gel scaffold is very likely to break during the polymerization process. The needle-type clamp for suspension fixation covers a small area of ​​the gel scaffold surface and does not affect the deposition of DA on its surface; the gel scaffold needs to be turned over every 1 / 4 of the DA polymerization reaction time to ensure uniform PDA deposition;

[0019] Furthermore, in step 5), when glutaraldehyde reacts with alendronate sodium, the concentration of the alendronate sodium solution is 20-50 mg / ml, and the amount of glutaraldehyde used is greater than the molar equivalent of alendronic acid; the reaction temperature is 40-50° C., and the reaction time is 12-24 hours; the concentration of alendronate sodium with an aldehyde group in the alendronate sodium with an aldehyde group aqueous solution is 50-200 mg / ml, the amount of the hydrogel scaffold used is 1 / 10-1 / 5 of the mass of the solution, the reaction time is 12-36 hours, the reaction temperature is 37° C., and after the reaction, the gel is dialyzed for 3-7 days.

[0020] Furthermore, the metal ions in step 6) can be one or more of calcium ions, magnesium ions, zinc ions or copper ions, with a concentration of 0.05-0.15 g / ml, a reaction time of 12-24 hours, and a reaction temperature of 37°C.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] 1) The present invention utilizes chitosan, inorganic minerals, dopamine, and sodium alendronate as raw materials, and combines freeze-blasting, nanocompositing, in-situ gelation, semi-freeze demolding, in-situ autopolymerization, and surface grafting to produce a composite hydrogel scaffold material with three-dimensional cell ingrowth and multiple osteogenic properties. This raw material and combined process are unique to the present invention and have not been reported previously.

[0023] 2) The hydrogel scaffold material prepared in the present invention has excellent biocompatibility, degradability, three-dimensional cell ingrowth ability, cell proliferation promotion and multiple osteogenic properties. The material first induces the three-dimensional ingrowth of cells in the gel scaffold through the three-dimensional ordered pore structure inside the scaffold, while the PDA coating on the surface of the scaffold promotes cell proliferation; then, the alendronate and its chelated metal ions on the surface of the scaffold promote cell osteogenic differentiation; and along with the degradation of the gel, the internal inorganic minerals are exposed, and calcium phosphate ions and calcium ions are dissolved, further exerting multiple osteogenic effects. The present invention targets the treatment points of bone defects, combines the three-dimensional ingrowth of cells inside the scaffold, promotes the proliferation of cell number after ingrowth, and promotes multiple osteogenic differentiation after proliferation. The careful design realizes the organic synergy between various functions and improves the efficacy of bone repair.

[0024] 3) The present invention uses a customized hydrogel gelation mold, which can achieve orderly regulation of the pore size of the three-dimensional holes in the hydrogel, and can provide a suitable growth environment and space for different cells; and the presence of the holes changes the previous cell growth on the surface of the hydrogel into three-dimensional body growth, greatly improving the rate of bone regeneration; at the same time, combined with the parameters of the mold and the mechanical properties of the hydrogel, an original semi-frozen demolding process was created. Since the mechanical properties of the hydrogel will decrease after the three-dimensional holes are introduced using the mold, direct demolding will cause the hydrogel to rupture. Therefore, in response to the above situation, the hydrogel sample is frozen together with the mold in a -80℃ refrigerator, and after exploring the melting time, it is determined that after 5 minutes of melting, that is, when the hydrogel is in a semi-frozen state, demolding can quickly obtain a complete sample;

[0025] 4) The present invention utilizes DA self-polymerization and Schiff base reaction to introduce a PDA coating and graft alendronate onto the surface of the hydrogel scaffold. This dual surface modification technology imparts the scaffold material with more prominent cell proliferation and osteogenesis-promoting properties, making it suitable for complex bone defect scenarios.

[0026] 5) The three-dimensional pore parameters of the hydrogel material used in this invention were determined by the inventors based on experimental results. Cells exhibit varying degrees of three-dimensional ingrowth within pores of varying sizes: smaller pores promote rapid lateral ingrowth but slower vertical ingrowth; larger pores exhibit the opposite behavior. Only when both lateral and vertical ingrowth are achieved can osteoblasts rapidly fill the scaffold, enhancing bone repair efficiency and effectiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Images of composite hydrogel scaffold materials with three-dimensional cell ingrowth and multiple osteogenic properties (a is the hydrogel material after in situ gelation and demolding, with samples prepared from molds having pore sizes of 400μm×400μm, 600μm×600μm, and 800μm×800μm from left to right; b is the corresponding final composite hydrogel scaffold material, with samples prepared from molds having pore sizes of 400μm×400μm, 600μm×600μm, and 800μm×800μm from left to right; the mold parameters include but are not limited to the three above);

[0028] Figure 2 Surface SEM images of composite hydrogel scaffolds with three-dimensional cell ingrowth and multiple osteogenic properties ((a), (b), and (c) are samples prepared using molds with pore sizes of 400μm×400μm, 600μm×600μm, and 800μm×800μm, respectively);

[0029] Figure 3The structural diagram of the composite hydrogel scaffold material mold with three-dimensional cell growth and multiple osteogenesis performance (a, b, c are 400 μm x 400 μm, 600 μm x 600 μm, 800 μm x 800 μm molds in sequence). DETAILED DESCRIPTION

[0030] The application is further illustrated below with specific examples.

[0031] Example 1:

[0032] 1) Using 8wt% lithium hydroxide monohydrate and 6.4wt% urea as a solvent system, adding 3wt% chitosan (molecular weight 800000) as a solute, and adding β-TCP of 1 / 2 mass of chitosan, and the rest is water. After stirring the solution at room temperature for 30 minutes, it is completely frozen in a-80℃ refrigerator, and then the ice is crushed and dissolved at room temperature. This process is repeated three times to obtain a 4wt% chitosan / 2wt% β-TCP (β-TCP particle size is 200nm) solution by freeze-thaw method, and the solution is centrifuged at 3000rpm for 10 minutes to remove bubbles;

[0033] 2) The above solution is poured into a mold with an array of 400 μm x 400 μm x 3 mm protruding columns, and after 15 minutes of self-leveling, a mold box is used for sealing to prevent water loss of the hydrogel, and the mold is transferred to a 60℃ temperature for 4 hours to realize in-situ gelation, and after 12 hours of aging, a chitosan / β-TCP composite physical hydrogel is obtained;

[0034] 3) The above hydrogel is placed in a-80℃ refrigerator for 10 minutes, and then taken out and placed in a 25℃ environment for 5 minutes to make the sample in a semi-frozen state for demolding, and a three-dimensional gel scaffold with a 400 μm x 400 μm mesh array is obtained;

[0035] 4) After the above hydrogel scaffold is completely dialyzed with deionized water, it is suspended and fixed in a 2mg / ml DA / Tris HCl buffer solution with a pH of 8.5 using a needle clamp, and after 24 hours of in-situ polymerization under the condition of magnetic stirring at 150rpm (turning over every 6 hours), a composite hydrogel scaffold is obtained;

[0036] 5) An excess amount of glutaraldehyde is reacted with 50mg / ml alendronate sodium at 40℃ for 12 hours to introduce an aldehyde group on the alendronate sodium molecule, and after the reaction is terminated using acetone, the alendronate sodium with an aldehyde group is dried. After the above hydrogel scaffold is completely dialyzed, it is immersed in a 100mg / ml alendronate sodium solution with an aldehyde group, and after 24 hours of reaction, a composite gel scaffold with alendronate sodium grafted on the surface is obtained;

[0037] 6) The unreacted alendronate sodium was dialyzed against a large amount of deionized water and then transferred to a 0.15 g / ml magnesium chloride aqueous solution for reaction at 37° C. for 24 hours to allow the alendronate sodium to capture magnesium ions on the scaffold surface. After dialysis against deionized water, a composite hydrogel scaffold material loaded with metal ions, exhibiting three-dimensional cell ingrowth, and multiple osteogenic properties was obtained.

[0038] The resulting hydrogel scaffold material is black, with pores measuring approximately 380 μm x 380 μm (the gel volume shrinks slightly after PDA polymerization). Live-dead cell fluorescence staining of the hydrogel after seven days of co-culture with bone marrow mesenchymal stem cells demonstrated excellent biocompatibility. A 28-day in vitro degradation experiment showed that the material degraded by approximately 59.4%. The material was applied to a rat skull defect model, and the skull was repaired well after one month. Example 2:

[0039] 1) Using 8 wt% lithium hydroxide monohydrate and 6.4 wt% urea as the solvent system, 3 wt% chitosan (molecular weight 2,000,000) was added as the solute, and 1 / 2 the mass of β-TCP of chitosan was added at the same time, with the remainder being water. The solution was stirred at room temperature for 30 minutes, then transferred to a -80°C refrigerator to freeze completely, and then the ice cubes were crushed and dissolved at room temperature. This was repeated three times to prepare a 4 wt% chitosan / 2 wt% β-TCP (β-TCP particle size of 200 nm) solution by freeze-blasting, and then degassed by low-speed centrifugation at 3000 rpm for 10 minutes for later use;

[0040] 2) The above solution was cast into a mold having an array of 400 μm × 400 μm × 3 mm raised columns. After 15 minutes of self-leveling, the mold was sealed in a mold box to prevent water loss in the hydrogel. The solution was then transferred to 60°C and kept at this temperature for 4 hours to achieve in situ gelation. After 12 hours of aging, the chitosan / β-TCP composite physical hydrogel was obtained.

[0041] 3) The hydrogel and the mold were placed in a -80°C freezer and frozen for 10 minutes. After removal, the sample was thawed at 25°C for 5 minutes. The sample was demolded in a semi-frozen state to obtain a three-dimensional gel scaffold with a 400 μm × 400 μm mesh array.

[0042] 4) After the hydrogel scaffold was completely dialyzed with deionized water, it was suspended and fixed in 2 mg / ml DA / Tris HCl buffer at pH 8.5 using a needle clamp. After in situ autopolymerization under magnetic stirring at 150 rpm for 24 hours (turning over every 6 hours), a composite hydrogel scaffold was obtained;

[0043] 5) reacting an excess of glutaraldehyde with 50 mg / ml alendronate sodium at 40° C. for 12 hours to introduce aldehyde groups into the alendronate sodium molecule. The reaction was terminated with acetone and dried to obtain alendronate sodium with aldehyde groups. After complete dialysis, the hydrogel scaffold was immersed in a 100 mg / ml aqueous solution of alendronate sodium with aldehyde groups and reacted for 24 hours to obtain a composite gel scaffold with alendronate sodium grafted on its surface.

[0044] 6) The unreacted alendronate sodium was dialyzed against a large amount of deionized water and then transferred to a 0.15 g / ml magnesium chloride aqueous solution for reaction at 37° C. for 24 hours to allow the alendronate sodium to capture magnesium ions on the scaffold surface. After dialysis against deionized water, a composite hydrogel scaffold material loaded with metal ions, exhibiting three-dimensional cell ingrowth, and multiple osteogenic properties was obtained.

[0045] Compared with Example 1, the molecular weight of chitosan is increased, and the prepared hydrogel scaffold material is black, with a pore size of approximately 390 μm × 390 μm inside the scaffold (the gel volume shrinks slightly after PDA polymerization, but due to the increase in the molecular weight of chitosan, the degree of shrinkage is less than that in Example 1). After the hydrogel was co-cultured with bone marrow mesenchymal stem cells for 7 days, live and dead cell fluorescence staining results demonstrated excellent biocompatibility of the material, and the results of a 28-day in vitro degradation experiment demonstrated that the material degraded by approximately 47.2%. It was applied to a rat skull defect model, and the skull was repaired well after one month.

[0046] Example 3:

[0047] 1) Using 8 wt% lithium hydroxide monohydrate and 6.4 wt% urea as the solvent system, 3 wt% chitosan (molecular weight 800,000) was added as the solute, and 1 / 2 the mass of β-TCP of chitosan was added at the same time, with the remainder being water. The solution was stirred at room temperature for 30 minutes, then transferred to a -80°C refrigerator to freeze completely, and then the ice cubes were crushed and dissolved at room temperature. This was repeated three times to prepare a 4 wt% chitosan / 2 wt% β-TCP (β-TCP particle size of 200 nm) solution by freeze-blasting, and then degassed by low-speed centrifugation at 3000 rpm for 10 minutes for later use;

[0048] 2) The above solution was cast into a mold having an array of 600 μm × 600 μm × 3 mm raised columns. After 15 minutes of self-leveling, the mold was sealed in a mold box to prevent water loss in the hydrogel. The solution was then transferred to 60°C and kept at this temperature for 4 hours to achieve in situ gelation. After 12 hours of aging, the chitosan / β-TCP composite physical hydrogel was obtained.

[0049] 3) The hydrogel and the mold were placed in a -80°C freezer and frozen for 10 minutes. After removal, the sample was thawed at 25°C for 5 minutes. The sample was demolded in a semi-frozen state to obtain a three-dimensional gel scaffold with a 400 μm × 400 μm mesh array.

[0050] 4) After the hydrogel scaffold was completely dialyzed with deionized water, it was suspended and fixed in 2 mg / ml DA / Tris HCl buffer at pH 8.5 using a needle clamp. After in situ autopolymerization under magnetic stirring at 150 rpm for 24 hours (turning over every 6 hours), a composite hydrogel scaffold was obtained;

[0051] 5) reacting an excess of glutaraldehyde with 50 mg / ml alendronate sodium at 40° C. for 12 hours to introduce aldehyde groups into the alendronate sodium molecule. The reaction was terminated with acetone and dried to obtain alendronate sodium with aldehyde groups. After complete dialysis, the hydrogel scaffold was immersed in a 100 mg / ml aqueous solution of alendronate sodium with aldehyde groups and reacted for 24 hours to obtain a composite gel scaffold with alendronate sodium grafted on its surface.

[0052] 6) The unreacted alendronate sodium was dialyzed against a large amount of deionized water and then transferred to a 0.15 g / ml magnesium chloride aqueous solution for reaction at 37° C. for 24 hours to allow the alendronate sodium to capture magnesium ions on the scaffold surface. After dialysis against deionized water, a composite hydrogel scaffold material loaded with metal ions, exhibiting three-dimensional cell ingrowth, and multiple osteogenic properties was obtained.

[0053] Compared with Example 1, the mold parameters were adjusted, and the resulting hydrogel scaffold material was black, with a pore size of approximately 580 μm × 580 μm inside the scaffold (the gel volume shrank slightly after PDA polymerization). The results of live and dead cell fluorescence staining after co-culture of the hydrogel with bone marrow mesenchymal stem cells for 7 days demonstrated excellent biocompatibility of the material, and the longitudinal growth distance of bone marrow mesenchymal stem cells in the hydrogel scaffold after 3 days was increased by 16.8% compared with the scaffold in Example 1, and the lateral coverage was 9.3% lower. The results of a 28-day in vitro degradation experiment demonstrated that the material degraded by approximately 68.1%. The material was applied to a rat skull defect model, and the skull was repaired well after one month.

[0054] Example 4:

[0055] 1) Using 8 wt% lithium hydroxide monohydrate and 6.4 wt% urea as the solvent system, 3 wt% chitosan (molecular weight 800,000) was added as the solute, and 3 / 2 of the mass of chitosan β-TCP was added at the same time, with the remainder being water. The solution was stirred at room temperature for 30 minutes, then transferred to a -80°C refrigerator to freeze completely, and then the ice cubes were crushed and dissolved at room temperature. This was repeated three times to prepare a 4 wt% chitosan / 2 wt% β-TCP (β-TCP particle size of 200 nm) solution by freeze-blasting, and then degassed by low-speed centrifugation at 3000 rpm for 10 minutes for later use;

[0056] 2) The above solution was cast into a mold having an array of 400 μm × 400 μm × 3 mm raised columns. After 15 minutes of self-leveling, the mold was sealed in a mold box to prevent water loss in the hydrogel. The solution was then transferred to 60°C and kept at this temperature for 4 hours to achieve in situ gelation. After 12 hours of aging, the chitosan / β-TCP composite physical hydrogel was obtained.

[0057] 3) The hydrogel and the mold were placed in a -80°C freezer and frozen for 10 minutes. After removal, the sample was thawed at 25°C for 5 minutes. The sample was demolded in a semi-frozen state to obtain a three-dimensional gel scaffold with a 400 μm × 400 μm mesh array.

[0058] 4) After the hydrogel scaffold was completely dialyzed with deionized water, it was suspended and fixed in 2 mg / ml DA / Tris HCl buffer at pH 8.5 using a needle clamp. After in situ autopolymerization under magnetic stirring at 150 rpm for 24 hours (turning over every 6 hours), a composite hydrogel scaffold was obtained;

[0059] 5) reacting an excess of glutaraldehyde with 50 mg / ml alendronate sodium at 40° C. for 12 hours to introduce aldehyde groups into the alendronate sodium molecule. The reaction was terminated with acetone and dried to obtain alendronate sodium with aldehyde groups. After complete dialysis, the hydrogel scaffold was immersed in a 100 mg / ml aqueous solution of alendronate sodium with aldehyde groups and reacted for 24 hours to obtain a composite gel scaffold with alendronate sodium grafted on its surface.

[0060] 6) The unreacted alendronate sodium was dialyzed against a large amount of deionized water and then transferred to a 0.15 g / ml magnesium chloride aqueous solution for reaction at 37° C. for 24 hours to allow the alendronate sodium to capture magnesium ions on the scaffold surface. After dialysis against deionized water, a composite hydrogel scaffold material loaded with metal ions, exhibiting three-dimensional cell ingrowth, and multiple osteogenic properties was obtained.

[0061] Compared with Example 1, the amount of β-TCP was increased, and the resulting hydrogel scaffold material was black, with a pore size of approximately 390 μm × 390 μm inside the scaffold (the gel volume shrank slightly after PDA polymerization, but the degree of volume shrinkage decreased due to the increase in the amount of β-TCP). After the hydrogel was co-cultured with bone marrow mesenchymal stem cells for 7 days, live and dead cell fluorescence staining results demonstrated excellent biocompatibility of the material, and 28-day in vitro degradation test results demonstrated that the material degraded by approximately 51.3%. It was applied to a rat skull defect model, and the skull was repaired well after one month.

[0062] Example 5:

[0063] 1) Using 8 wt% lithium hydroxide monohydrate and 6.4 wt% urea as the solvent system, 3 wt% chitosan (molecular weight 800,000) was added as the solute, and 1 / 2 the mass of β-TCP of chitosan was added at the same time, with the remainder being water. The solution was stirred at room temperature for 30 minutes, then transferred to a -80°C refrigerator to freeze completely, and then the ice cubes were crushed and dissolved at room temperature. This was repeated three times to prepare a 4 wt% chitosan / 2 wt% β-TCP (β-TCP particle size of 200 nm) solution by freeze-blasting, and then degassed by low-speed centrifugation at 3000 rpm for 10 minutes for later use;

[0064] 2) The above solution was cast into a mold having an array of 400 μm × 400 μm × 3 mm raised columns. After 15 minutes of self-leveling, the mold was sealed in a mold box to prevent water loss in the hydrogel. The solution was then transferred to 60°C and kept at this temperature for 4 hours to achieve in situ gelation. After 12 hours of aging, the chitosan / β-TCP composite physical hydrogel was obtained.

[0065] 3) The hydrogel and the mold were placed in a -80°C freezer and frozen for 10 minutes. After removal, the sample was thawed at 25°C for 5 minutes. The sample was demolded in a semi-frozen state to obtain a three-dimensional gel scaffold with a 400 μm × 400 μm mesh array.

[0066] 4) After the hydrogel scaffold was completely dialyzed with deionized water, it was suspended and fixed in 2 mg / ml DA / Tris HCl buffer at pH 8.5 using a needle clamp. After in situ autopolymerization under magnetic stirring at 150 rpm for 24 hours (turning over every 6 hours), a composite hydrogel scaffold was obtained;

[0067] 5) reacting an excess of glutaraldehyde with 50 mg / ml alendronate sodium at 40° C. for 12 hours to introduce aldehyde groups into the alendronate sodium molecule. Terminate the reaction with acetone and dry the resulting alendronate sodium with aldehyde groups. After complete dialyzation, the hydrogel scaffold was immersed in a 200 mg / ml aqueous solution of alendronate sodium with aldehyde groups and allowed to react for 24 hours to obtain a composite gel scaffold with alendronate sodium grafted on its surface.

[0068] 6) The unreacted alendronate sodium was dialyzed against a large amount of deionized water and then transferred to a 0.15 g / ml magnesium chloride aqueous solution for reaction at 37° C. for 24 hours to allow the alendronate sodium to capture magnesium ions on the scaffold surface. After dialysis against deionized water, a composite hydrogel scaffold material loaded with metal ions, exhibiting three-dimensional cell ingrowth, and multiple osteogenic properties was obtained.

[0069] Compared with Example 1, the amount of sodium alendronate grafted on the surface of the scaffold increased, the prepared hydrogel scaffold material was black, and the pore size inside the scaffold was approximately 380 μm × 380 μm (the gel volume shrank slightly after PDA polymerization). After the hydrogel was co-cultured with bone marrow mesenchymal stem cells for 7 days, the live and dead cell fluorescence staining results demonstrated that the material had excellent biocompatibility. The results of the 28-day in vitro degradation experiment demonstrated that the material degraded by approximately 55.2%. It was applied to a rat skull defect model, and the skull repair condition was better than that in Example 1 after one month.

Claims

1. A composite hydrogel scaffold material with three-dimensional cell ingrowth and multiple osteogenic properties, characterized by: The hydrogel scaffold material is prepared using chitosan, inorganic minerals, dopamine, and sodium alendronate as main raw materials, and is produced using a combination of processes including freeze-blasting, nanocomposite, in-situ gelation, semi-freeze demoulding, in-situ self-polymerization, and surface grafting. The preparation method comprises the following steps: 1) Using lithium hydroxide monohydrate and urea as a solvent system, chitosan is added as a solute, along with a certain amount of inorganic minerals, and the remainder is water. After stirring the solution at room temperature for 30-60 minutes, transfer it to a -80°C freezer and freeze it completely. Then, crush the ice cubes at room temperature by external force to dissolve them. Repeat this process at least three times to prepare a mixed solution using the freeze-blast method. The solution is then degassed by low-speed centrifugation and set aside for use. 2) The solution obtained in 1) is poured into a customized mold, sealed after self-leveling, transferred to 60°C for in-situ gelation, and aged for 4-12 hours to obtain a composite physical hydrogel; the customized mold is a mold with a columnar array, and one end of the mold with the array contains an array of raised columns, the height of the raised columns is 1-3 mm, the diameter or length and width of the raised columns is 200-1000 μm, and the spacing between the raised columns is 100-400 μm; the self-leveling process lasts 5-15 minutes, and the solution gelation time is 4-8 hours; 3) Transfer the hydrogel to a -80°C freezer and freeze for 10 minutes. Then transfer the sample to a 25°C environment and thaw for 5 minutes. Demold the sample in a semi-frozen state. After demolding, the hydrogel scaffold forms a three-dimensional ordered pore structure. 4) After the hydrogel scaffold is completely dialyzed, it is suspended and fixed in a dopamine DA / Tris HCl buffer solution with a pH of 8.5, and subjected to in situ self-polymerization reaction under stirring for 4-96 hours to obtain a composite hydrogel scaffold; 5) reacting alendronate sodium with an excess of glutaraldehyde to introduce aldehyde groups into the alendronate sodium molecule, terminating the reaction with acetone, and drying to obtain alendronate sodium with aldehyde groups; after complete dialysis of the composite hydrogel scaffold, immersing the composite hydrogel scaffold in an aqueous solution of alendronate sodium with aldehyde groups and reacting for 12-36 hours to obtain a composite hydrogel scaffold with alendronate sodium grafted on its surface; 6) A large amount of deionized water is dialyzed to remove unreacted alendronate sodium, and the dialyzed scaffold is transferred to an aqueous solution containing metal ions to achieve the capture of metal ions by alendronate sodium. After deionized water dialysis, a composite hydrogel scaffold material loaded with metal ions with three-dimensional cell ingrowth and multiple osteogenic properties is obtained.

2. The composite hydrogel scaffold material with three-dimensional cell ingrowth and multiple osteogenic properties according to claim 1, characterized in that: The hydrogel also has excellent biocompatibility, degradability, three-dimensional cell ingrowth ability, cell proliferation promotion and multiple osteogenic properties; the material induces three-dimensional cell ingrowth in the gel scaffold through the three-dimensional ordered pore structure inside the scaffold, while the PDA coating on the scaffold surface promotes cell proliferation; alendronate and its chelated metal ions on the scaffold surface promote cell osteogenic differentiation; and as the gel degrades, the inorganic minerals inside the scaffold are exposed, dissolving calcium phosphate ions and calcium ions, which can further exert multiple osteogenic effects.

3. A method for preparing a composite hydrogel scaffold material having three-dimensional cell ingrowth and multiple osteogenic properties as claimed in claim 1 or 2, characterized in that: The steps are as follows: 1) Using lithium hydroxide monohydrate and urea as a solvent system, chitosan is added as a solute, along with a certain amount of inorganic minerals, and the remainder is water. After stirring the solution at room temperature for 30-60 minutes, transfer it to a -80°C freezer and freeze it completely. Then, crush the ice cubes at room temperature by external force to dissolve them. Repeat this process at least three times to prepare a mixed solution using the freeze-blast method. The solution is then degassed by low-speed centrifugation and set aside for use. 2) The solution obtained in 1) is poured into a customized mold, sealed after self-leveling, transferred to 60°C for in-situ gelation, and aged for 4-12 hours to obtain a composite physical hydrogel; the customized mold is a mold with a columnar array, and one end of the mold with the array contains an array of raised columns, the height of the raised columns is 1-3 mm, the diameter or length and width of the raised columns is 200-1000 μm, and the spacing between the raised columns is 100-400 μm; the self-leveling process lasts 5-15 minutes, and the solution gelation time is 4-8 hours; 3) Transfer the hydrogel to a -80°C freezer and freeze for 10 minutes. Then transfer the sample to a 25°C environment and thaw for 5 minutes. Demold the sample in a semi-frozen state. After demolding, the hydrogel scaffold forms a three-dimensional ordered pore structure. 4) After the hydrogel scaffold is completely dialyzed, it is suspended and fixed in a dopamine DA / Tris HCl buffer solution with a pH of 8.5, and subjected to in situ self-polymerization reaction under stirring for 4-96 hours to obtain a composite hydrogel scaffold; 5) reacting alendronate sodium with an excess of glutaraldehyde to introduce aldehyde groups into the alendronate sodium molecule, terminating the reaction with acetone, and drying to obtain alendronate sodium with aldehyde groups; after complete dialysis of the composite hydrogel scaffold, immersing the composite hydrogel scaffold in an aqueous solution of alendronate sodium with aldehyde groups and reacting for 12-36 hours to obtain a composite hydrogel scaffold with alendronate sodium grafted on its surface; 6) A large amount of deionized water is dialyzed to remove unreacted alendronate sodium, and the dialyzed scaffold is transferred to an aqueous solution containing metal ions to achieve the capture of metal ions by alendronate sodium. After deionized water dialysis, a composite hydrogel scaffold material loaded with metal ions with three-dimensional cell ingrowth and multiple osteogenic properties is obtained.

4. The method for preparing a composite hydrogel scaffold material with three-dimensional cell ingrowth and multiple osteogenic properties according to claim 3, characterized in that: Step 1) The solution comprises a lithium hydroxide monohydrate concentration of 7-9 wt%, a urea concentration of 5-8 wt%, a chitosan concentration of 2-5 wt%, and a chitosan molecular weight of 500,000-1,200,000. The inorganic minerals include one or more of β-TCP and hydroxyapatite, each having a particle size of 200-800 nm and an amount of 1 / 3-2 times the mass of the chitosan. The low-speed centrifugation speed is 3,000-5,000 rpm, and the centrifugation time is 5-15 minutes.

5. The method for preparing a composite hydrogel scaffold material with three-dimensional cell ingrowth and multiple osteogenic properties according to claim 3, characterized in that: Step 4) The DA concentration in the DA / Tris HCl buffer is 1-4 mg / ml, and the magnetic stirring rate is 100-250 rpm. The gel scaffold is clamped with a needle-type clamp to suspend and fix in the DA / Tris HCl buffer. The gel scaffold needs to be turned over every 1 / 4 of the DA polymerization reaction time to ensure uniform PDA deposition.

6. The method for preparing a composite hydrogel scaffold material with three-dimensional cell ingrowth and multiple osteogenic properties according to claim 3, wherein: In step 5), when glutaraldehyde reacts with alendronate sodium, the concentration of the alendronate sodium solution is 20-50 mg / ml, and the amount of glutaraldehyde used is greater than the molar equivalent of alendronic acid; the reaction temperature is 40-50° C., and the reaction time is 12-24 hours; The concentration of sodium alendronate with aldehyde groups in the aqueous solution of sodium alendronate with aldehyde groups is 50-200 mg / ml, the amount of the hydrogel scaffold used is 1 / 10-1 / 5 of the mass of the solution, the reaction time is 12-36 hours, the reaction temperature is 37° C., and the gel is dialyzed for 3-7 days after the reaction is completed.

7. The method for preparing a composite hydrogel scaffold material with three-dimensional cell ingrowth and multiple osteogenic properties according to claim 3, characterized in that: The metal ions in step 6) are one or more of calcium ions, magnesium ions, zinc ions or copper ions, with a concentration of 0.05-0.15 g / ml, the reaction time is 12-24 hours, and the reaction temperature is 37°C.

8. Use of a composite hydrogel scaffold material having three-dimensional cell ingrowth and multiple osteogenic properties prepared by the method of any one of claims 3 to 7, characterized in that: The material is used as a bone defect repair gel scaffold material in the field of bone tissue engineering.

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