CN115177786A - Chitosan composite scaffold material loaded with montmorillonite/nano-hydroxyapatite in situ and preparation method thereof - Google Patents

Abstract

The invention discloses a chitosan composite scaffold material loaded with montmorillonite/nano hydroxyapatite in situ and a preparation method thereof, which can be used in the field of bone defect repair. The invention takes the in-situ regeneration thought and the self-assembly principle as guidance, so that the surface of the material is uniformly loaded with the polydopamine-coated montmorillonite and the nano-hydroxyapatite in situ, the support similar to a natural bone tissue structure is prepared by a bionic technology, organic materials and inorganic nano-particles are mutually doped, the binding property of the support material and the natural tissue interface is strong, and the support material has good mineralization capability and biocompatibility and is expected to become a novel bone repair material.

Description

A chitosan composite scaffold loaded with montmorillonite/nano-hydroxyapatite in situ material and method of making the same

technical field

The invention belongs to the field of composite materials, in particular to an in-situ loaded montmorillonite/nano hydroxyapatite chitosan composite scaffold material and a preparation method thereof, which can be used as a bone defect repair material.

Background technique

In situ tissue engineering uses external scaffold materials to induce self-repair of the injured site, and the final repaired tissue has good compatibility and adaptability with the body itself. In situ tissue engineering focuses on the design of acellular scaffolds, simplifying the fabrication process. Bioactive scaffolds with a similar bone tissue matrix microenvironment were implanted into the bone defect area to stimulate the influx of endogenous stem cells to the surface of the scaffold to promote the formation of new bone. This approach reduces the potential risk of immune rejection due to the implantation of exogenous stem cells and growth factors. The feature of in situ tissue repair strategy is that under the action of self-arousal and active repair of the body tissue, endogenous stem cells and growth factors in healthy tissues are migrated to the surface and interior of the scaffold for proliferation and osteogenic differentiation, so as to guide in situ. Repair bone defects and promote new tissue regeneration.

Chitosan scaffolds are widely used in bone tissue engineering. Chitosan scaffolds prepared by traditional methods have poor mechanical properties and cannot meet the needs of bone regeneration in defect sites. Based on this, the present invention provides an in-situ loaded montmorillonite/nano-hydroxyapatite chitosan composite scaffold material and a preparation method thereof. The acid-modified montmorillonite is added to effectively enhance the mechanical properties of the scaffold. In addition, the introduction of polydopamine can increase the adhesion of cells and growth factors on the scaffold surface, and at the same time provide nucleation sites for the directional growth of hydroxyapatite, which can effectively promote bone repair at the defect site.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an in-situ loaded montmorillonite/nano-hydroxyapatite chitosan composite scaffold material and a preparation method thereof, which take the concept of in-situ bone tissue engineering repair as the core, and perform biomimetic on the natural bone tissue structure. Natural montmorillonite is selected for modification and standby, soluble calcium salt and soluble phosphate are used as precursors of nano-hydroxyapatite, chitosan is used as the main material, combined with freeze-drying technology, the preparation has excellent mechanical properties and biocompatibility. The scaffold has a wide range of raw materials and is easy to operate.

To achieve the above object, the present invention adopts the following technical solutions:

An in-situ loaded montmorillonite/nano-hydroxyapatite chitosan composite scaffolding material uses natural polymer chitosan as the main material, modified montmorillonite as the reinforcing material, soluble calcium salt and soluble phosphate As the precursor of nano-hydroxyapatite, it was dissolved in an aqueous solution of acetic acid containing 1 % (V/V) and mixed uniformly, then injection-molded and freeze-dried to prepare in-situ supported montmorillonite/nano-hydroxyapatite. Chitosan composite scaffold material.

A preparation method of in-situ loaded montmorillonite/nano-hydroxyapatite chitosan composite scaffold material: acid-modified montmorillonite is coated with dopamine, soluble calcium salt and soluble phosphate are used as inorganic phase nano-hydroxyl The precursor of apatite can induce the self-polymerization of dopamine hydrochloride in an alkaline environment, while loading montmorillonite in situ, it also assists in inducing the nucleation of hydroxyapatite, and in situ biomimetic construction of montmorillonite/nano hydroxyapatite bone Repair the bracket.

The preparation method of the in-situ loaded montmorillonite/nano-hydroxyapatite chitosan composite scaffold material specifically includes the following steps:

(1) Weigh 5g of montmorillonite, add 100ml of 3M HCl solution, stir at a constant temperature in a water bath, centrifuge after the reaction, collect the lower solid precipitate, rinse it with deionized water for 5 to 6 times, dry it overnight, and grind the solid for later use to obtain the Acid-modified montmorillonite;

(2) Prepare 150ml of 10mmol/L tris(hydroxymethyl)aminomethane solution, adjust the pH to 8.5, add 1.5g of dopamine hydrochloride to it, stir at room temperature for 30min, add the acid-modified montmorillonite obtained in step (1) Soil, stirring at room temperature for 24h, monitoring pH; after the reaction, centrifuge, collect the lower solid precipitate, rinse with deionized water for 5 to 6 times, until the supernatant is clear and transparent, collect the solid and freeze-dried to obtain PDA-MMT, for subsequent use;

(3) Add 2g of chitosan powder to 100ml of acetic acid solution with a volume fraction of 1%, stir and dissolve in a constant temperature water bath at 65°C;

(4) The PDA-MMT obtained in step (2) is weighed and configured into a suspension, and then added to the chitosan solution prepared in step (3), stirred and mixed evenly;

(5) Add 2 ml each of 2 mol/L soluble calcium salt solution and 1.2 mol/L soluble phosphate solution to the mixed solution of chitosan and montmorillonite obtained in step (4) in turn, and stir well in a constant temperature water bath. , and evenly mixed to obtain the scaffold precursor solution;

(6) adding the scaffold precursor solution obtained in step (5) into the mold, and placing the mold at 4 °C overnight, then at -20 °C overnight, and finally at -80 °C for 48 h, to obtain the scaffold precursor;

(7) Immerse the scaffold precursor obtained in (6) in 500 ml of Tris/ethanol mixed solution with pH=10, and place it on a constant temperature shaker at 37°C with a rotating speed of 120 r/min. After soaking for 8 hours, wash with a large amount of deionized water. to neutral;

(8) Place the scaffold obtained in (7) at -20°C overnight, then place it in a -80°C refrigerator, and finally freeze-dry it in vacuum to obtain an in-situ loaded montmorillonite/nano-hydroxyapatite chitosan composite scaffold. .

In step (1), the temperature of the water bath is 45 °C and the time is 8 h; the drying temperature is 50 °C overnight.

In step (2), the stirring time at room temperature is 24 h, and the pH needs to be kept at 8.5. Dopamine-coated montmorillonite facilitates its attachment to and uniform dispersion in the scaffold. And dopamine has a certain induction effect on the formation of nano-hydroxyapatite.

In step (4), the PDA-MMT is weighed and configured so that the concentration of the suspension is 0.03-0.09 g/mL.

The soluble calcium salt used in step (5) is calcium nitrate or calcium chloride; the soluble phosphate used is dipotassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate or sodium dihydrogen phosphate; wherein the Ca/P molar ratio It is n(Ca ²⁺ ):n(PO ₄ ^3- )=1.67:1, and the stirring time is 3~4 h.

The Tris/ethanol mixed solution used in step (7) is to mix absolute ethanol and Tris solution according to a volume ratio of 1:1, and the concentration of Tris in the Tris/ethanol mixed solution is 10 mmol/L.

The freeze-drying temperature in step (8) was -80 °C, and the freeze-drying time was 48 h.

In the present invention, chitosan is used as the main material, modified montmorillonite, soluble calcium salt and phosphorus salt are added, and the porous scaffold is obtained by freeze-drying. The dopamine hydrochloride self-polymerizes under alkaline conditions to generate polydopamine, and at the same time, it can promote the nucleation of nano-hydroxyapatite, so that the dopamine-coated montmorillonite and nano-hydroxyapatite are uniformly distributed in the scaffold.

Dopamine is oxidized and self-polymerized to form polydopamine under alkaline conditions (pH>7.5). As a mussel-inspired polymer, polydopamine exhibits important properties such as good biocompatibility, low cytotoxicity, high reactivity, and high adhesion. There are a large number of catechol structures and protonated amino groups on the surface of polydopamine, which can serve as both the starting point for the covalent modification of the ideal molecule and the anchor point for the loading of transition metal ions. The catechol structure can enrich Ca ²⁺ on the surface of the material, promote the concentration of Ca ²⁺ to reach a supersaturated state, and provide nucleation sites for hydroxyapatite HAP to induce the mineralization of HAP on the surface of the material. Polydopamine can also hydrogen bond with chitosan, so polydopamine can be assembled on chitosan-based scaffolds.

Montmorillonite is a natural clay mineral with a layered structure, and the nanoscale of its lamellar structure is 1 nm. The crystal structure is a 2:1 type monoclinic system composed of two layers of silicon-oxygen tetrahedron sandwiching a layer of aluminum-oxygen octahedron. Its lamellar structure facilitates the insertion of polymer single chains to obtain intercalated nanocomposites. Montmorillonite has good biocompatibility and biological activity, and also has natural antibacterial properties. There are Na ⁺ , Ca ²⁺ , Mg ³⁺ and other cations between the montmorillonite structural layers. These cations can be exchanged and have the characteristics of ion exchange, water absorption, swelling, thixotropy, cohesion, and adsorption. The -OH contained on the surface and edge of montmorillonite sheets can be used as functionalized active sites. When the pH is lower than the isoelectric point of montmorillonite (PI=2), the montmorillonite is positively charged, and when the pH is greater than the isoelectric point, the montmorillonite is negatively charged. The addition of montmorillonite is beneficial to improve the mechanical properties and thermal stability of scaffold materials. However, due to the poor compatibility of montmorillonite with polymers, it is necessary to modify and reuse it. After acid modification, montmorillonite produces many small pores, which dissolve some Na ⁺ , K ⁺ , Al ³⁺ ions in the octahedron. The angle of the pores at both ends of the crystal increases, the specific surface area increases, and the diameter increases. The interlayer bonding force is greatly weakened, the interlayer lattice is cracked, the interlayer spacing is expanded, and after activation, the ion penetration is enhanced. After acid modification, the hydrogen bonds between the montmorillonite sheets are broken, and the interlayer spacing is expanded, which is helpful for polydopamine to insert into its sheet structure, and polydopamine can promote the in-situ growth of nano-hydroxyapatite inside it, In turn, the mechanical properties of the scaffold can be improved and bone regeneration at the injured site can be promoted.

Compared with other methods of bone repairing materials, the chitosan composite scaffold material loaded with montmorillonite/nano-hydroxyapatite in situ of the present invention has the following advantages:

(1) Under alkaline conditions, dopamine can self-polymerize to generate polydopamine, which connects the coated montmorillonite to the main body of the stent, and disperses evenly in the stent, effectively improving the mechanical properties of the stent;

(2) The in-situ polymerization of dopamine to generate PDA can effectively regulate the conversion of calcium phosphate to hydroxyapatite, accelerate mineralization, achieve uniform dispersion at the nanoscale, and improve the mechanical properties of the material;

(3) The surface of PDA contains a large number of active functional groups such as hydroxyl and amino groups, which can provide the required active groups for secondary grafting modification, and can also promote cell adhesion and proliferation and anchoring of active substances;

(4) In-situ cross-linking occurs between the in-situ regenerated PDA and chitosan molecules, which improves the porosity, hydrophilicity and mechanical strength of the material without other cross-linking agents;

(5) The composite scaffold has a wide range of materials, low price, and simple and easy manufacturing process.

Description of drawings

Fig. 1 is the macroscopic topography photograph of the composite scaffold obtained in Example 1, 2, 3, and 4;

Fig. 2 is the SEM image of the composite scaffold obtained in Examples 1, 2, 3, and 4;

Figure 3 is a schematic diagram of the intermolecular cross-linking reaction between chitosan and polydopamine;

Fig. 4 is the SEM images of the composite scaffolds obtained in Examples 1, 2, 3, and 4 after three days of mineralization;

FIG. 5 is the compressive force-displacement curve of the composite scaffolds obtained in Examples 1, 2, 3, and 4. FIG.

Detailed ways

In order to make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below with reference to specific embodiments, but the present invention is not limited thereto.

Example 1 (without both montmorillonite and dopamine)

(1) Add 2g of chitosan powder to 100ml of 1% acetic acid solution by volume, stir and dissolve in a constant temperature water bath at 65°C;

(2) Add 2 ml each of 2 mol/L calcium nitrate solution and 1.2 mol/L soluble dipotassium hydrogen phosphate solution to the chitosan solution obtained in step (1) in turn, wherein the Ca/P molar ratio is n(Ca ²⁺ ):n(PO ₄ ^3- )=1.67:1, fully stirred in a constant temperature water bath for 3 hours, and mixed evenly to obtain a scaffold precursor solution;

(3) Add the scaffold precursor solution obtained in step (2) into the mold, put the mold at 4 °C overnight, then at -20 °C overnight, and finally at -80 °C for 48 h;

(4) Immerse the scaffold precursor obtained in (3) in 500 ml of Tris/ethanol (volume ratio is 1:1, and the concentration of Tris is 10 mmol/L) mixed solution, and place it on a constant temperature shaker at 37 °C with a rotating speed of 120 r /min, after soaking for 8h, wash with plenty of deionized water until neutral.

(5) The scaffold obtained in (4) was placed at -20°C overnight, then placed in a -80°C refrigerator, and finally vacuum freeze-dried at -80°C for 24 hours to obtain a chitosan composite scaffold.

Example 2

(1) Weigh 5g of montmorillonite, add 100ml of 3M HCl solution, and stir at a constant temperature of 45°C in a water bath for 8h. After the reaction was completed, centrifugation was performed, and the lower solid precipitate was collected and washed with deionized water for 5 times. Dry at 50°C overnight, and grind the solid for later use to obtain acid-modified montmorillonite;

(2) Prepare 150ml of 10mmol/L tris(hydroxymethyl)aminomethane solution, and adjust the pH to 8.5. 1.5 g of dopamine hydrochloride was added thereto, and the mixture was stirred at room temperature for 30 min. The modified montmorillonite obtained in step (1) was added, stirred at room temperature for 24 hours, and the pH was monitored and kept at 8.5. After the reaction, centrifuge, collect the lower solid precipitate, rinse with deionized water for 5 times, until the supernatant is clear and transparent, collect the solid and freeze-dry it for subsequent use;

(3) Add 2g of chitosan powder to 100ml of 1% acetic acid solution by volume, stir and dissolve in a constant temperature water bath at 65°C;

(4) The PDA-MMT obtained in step (2) was weighed and configured into a suspension, the concentration of which was 0.03 g/mL. Add 2 ml of PDA-MMT suspension to the prepared chitosan solution. Stir to mix well.

(5) Add 2 ml of 2 mol/L calcium nitrate solution and 1.2 mol/L dipotassium hydrogen phosphate solution to the mixed solution of chitosan and montmorillonite obtained in step (4) in turn, wherein Ca/P moles The ratio is n(Ca ²⁺ ):n(PO ₄ ^3- )=1.67:1, the constant temperature water bath is fully stirred for 3h, and the mixture is uniform, that is, the scaffold precursor solution is obtained;

(6) Add the scaffold precursor solution obtained in step (5) into the mold, and place the mold at 4 °C overnight, then at -20 °C overnight, and finally at -80 °C for 48 h;

(7) Immerse the scaffold precursor obtained in (6) in 500ml Tris/ethanol (volume ratio is 1:1, Tris concentration is 10mmol/L) mixed solution, and place it on a constant temperature shaker at 37°C with a rotating speed of 120r /min, after soaking for 8h, wash with plenty of deionized water until neutral.

(8) The scaffold obtained in (7) was placed at -20°C overnight, then placed in a -80°C refrigerator, and finally freeze-dried in a vacuum at -80°C for 24 h, to obtain a PDA-modified modified montmorillonite uniformly doped montmorillonite. Composite bracket.

Example 3

(1) Weigh 5g of montmorillonite, add 100ml of 3M HCl solution, and stir at a constant temperature of 45°C in a water bath for 8h. After the reaction was completed, centrifugation was performed, and the lower solid precipitate was collected and washed with deionized water for 5 times. Dry at 50°C overnight, and grind the solid for later use to obtain acid-modified montmorillonite;

(2) Prepare 150ml of 10mmol/L tris(hydroxymethyl)aminomethane solution, and adjust the pH to 8.5. 1.5 g of dopamine hydrochloride was added thereto, and the mixture was stirred at room temperature for 30 min. The modified montmorillonite obtained in step (1) was added, stirred at room temperature for 24 hours, and the pH was monitored and kept at 8.5. After the reaction, centrifuge, collect the lower solid precipitate, rinse with deionized water for 5 times, until the supernatant is clear and transparent, collect the solid and freeze-dry it for later use;

(3) Add 2g of chitosan powder to 100ml of 1% acetic acid solution by volume, stir and dissolve in a constant temperature water bath at 65°C;

(4) The PDA-MMT obtained in step (2) was weighed and configured into a suspension, the concentration of which was 0.06 g/mL. Add 2 ml of PDA-MMT suspension to the prepared chitosan solution. Stir to mix well.

(5) Add 2 ml of 2 mol/L calcium nitrate solution and 1.2 mol/L dipotassium hydrogen phosphate solution to the mixed solution of chitosan and montmorillonite obtained in step (4) in turn, wherein Ca/P moles The ratio is n(Ca ²⁺ ):n(PO ₄ ^3- )=1.67:1, the constant temperature water bath is fully stirred for 3h, and the mixture is uniform, that is, the scaffold precursor solution is obtained;

(6) Add the scaffold precursor solution obtained in step (5) into the mold, and place the mold at 4 °C overnight, then at -20 °C overnight, and finally at -80 °C for 48 h;

(7) Immerse the scaffold precursor obtained in (6) in a mixed solution of 500ml Tris/ethanol (volume ratio is 1:1, Tris concentration is 10mmol/L), and place it on a constant temperature shaker at 37°C with a rotating speed of 120r /min, after soaking for 8h, wash with plenty of deionized water until neutral.

(8) The scaffold obtained in (7) was placed at -20°C overnight, then placed in a -80°C refrigerator, and finally freeze-dried in a vacuum at -80°C for 24 h, to obtain a PDA-modified modified montmorillonite uniformly doped montmorillonite. Composite bracket.

Example 4

(1) Weigh 5g of montmorillonite, add 100ml of 3M HCl solution, and stir at a constant temperature of 45°C in a water bath for 8h. After the reaction was completed, centrifugation was performed, and the lower solid precipitate was collected and washed with deionized water for 5 times. Dry at 50°C overnight, and grind the solid for later use to obtain acid-modified montmorillonite;

(2) Prepare 150ml of 10mmol/L tris(hydroxymethyl)aminomethane solution, and adjust the pH to 8.5. 1.5 g of dopamine hydrochloride was added thereto, and the mixture was stirred at room temperature for 30 min. The modified montmorillonite obtained in step (1) was added, stirred at room temperature for 24 hours, and the pH was monitored and kept at 8.5. After the reaction, centrifuge, collect the lower solid precipitate, rinse with deionized water for 5 times, until the supernatant is clear and transparent, collect the solid and freeze-dry it for later use;

(3) Add 2g of chitosan powder to 100ml of 1% acetic acid solution by volume, stir and dissolve in a constant temperature water bath at 65°C;

(4) The PDA-MMT obtained in step (2) was weighed and configured into a suspension, the concentration of which was 0.09 g/mL. Add 2 ml of PDA-MMT suspension to the prepared chitosan solution. Stir to mix well.

(5) Add 2 ml of 2 mol/L calcium nitrate solution and 1.2 mol/L dipotassium hydrogen phosphate solution to the mixed solution of chitosan and montmorillonite obtained in step (4) in turn, wherein Ca/P moles The ratio is n(Ca ²⁺ ):n(PO ₄ ^3- )=1.67:1, the constant temperature water bath is fully stirred for 3h, and the mixture is uniform, that is, the scaffold precursor solution is obtained;

(6) Add the scaffold precursor solution obtained in step (5) into the mold, and place the mold at 4 °C overnight, then at -20 °C overnight, and finally at -80 °C for 48 h;

(7) Immerse the scaffold precursor obtained in (6) in 500ml Tris/ethanol (volume ratio is 1:1, Tris concentration is 10mmol/L) mixed solution, and place it on a constant temperature shaker at 37°C with a rotating speed of 120r /min, after soaking for 8h, wash with plenty of deionized water until neutral.

(8) The scaffold obtained in (7) was placed at -20°C overnight, then placed in a -80°C refrigerator, and finally freeze-dried in a vacuum at -80°C for 24 h, to obtain a PDA-modified modified montmorillonite uniformly doped montmorillonite. Composite bracket.

It can be seen from FIG. 1 that the macroscopic topography of the prepared scaffold shows that the scaffold has a cylindrical three-dimensional structure. Its diameter is 12.5mm, and the composite scaffold after lyophilization is gray-white. With the increase of PDA-MMT content, the color of the scaffold gradually deepened and showed grayish white.

It can be seen from Figure 2 that the four composite scaffolds all have high pore structures, and the scaffolds have interconnected microporous structures, which are highly similar to the multi-level hierarchical structure of natural bone. In addition, under the magnification of 200X, it can be clearly observed that the pore diameter of Example 1 is between 50 and 200 μm, while the pore diameter of Examples 2, 3 and 4 is between 100 and 300 μm. Both meet the requirement of 200~400 μm pore size of natural bone. At the same time, it can be seen that the surface of the stent is relatively rough under high magnification. There are a large number of crystals evenly distributed on the inner wall of the stent, which are nHAP crystals formed by the in situ conversion of calcium phosphate crystals generated by the interaction of soluble calcium salts and phosphates.

Figure 3 is a schematic diagram of the mechanism of the reaction between polydopamine and chitosan molecules. Dopamine can self-polymerize to form PDA under alkaline conditions. The PDA molecule contains many phenolic hydroxyl groups, which can be further oxidized to quinone groups under alkaline conditions. The CS molecular chain contains many -NH ₂ , which can form new chemical bonds with the quinone group in PDA through Schiff base reaction or Michael addition reaction.

Figure 4 shows the SEM images of the material after 1 day and 3 days of mineralization in SBF. After 1 day of mineralization, nHAP was generated on the surface of the material, and with the increase of the amount of PDA-MMT added, the amount of nHAP generated on the surface of the scaffold gradually increased. In addition, the nHAP generated on the surface of the scaffolds in each group increased with time. A fully covered mineralized layer is gradually formed. The results indicated that the addition of PDA-MMT facilitated the aggregation and ion exchange of Ca ²⁺ and PO ₄ ^3- on the scaffold surface. PDA can promote the nucleation of nHAP and the transformation of the crystal form of nHAP.

Figure 5 is the force and displacement images during the secondary compression process measured by the texture analyzer after the material has absorbed water. With the increase of the loading force, the stent is gradually compressed, and the stent can return to its original state when the loading force is gradually withdrawn. This result indicates that the stent can maintain a stable state in a humid environment in vivo. And with the increase of the amount of PDA-MMT added, the maximum load force of the scaffolds increased to 2.5N, 2.53N, 2.97N and 3.03N, respectively. It shows that the addition of PDA-MMT helps to enhance the mechanical properties of the scaffold, which is sufficient to maintain the support function of the defect site.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims (9)

1. a kind of preparation method of the chitosan composite scaffold material of in-situ load montmorillonite/nano-hydroxyapatite, it is characterized in that: the montmorillonite after coating acid modification with dopamine, soluble calcium salt and soluble phosphoric acid As the precursor of inorganic phase nano-hydroxyapatite, the salt induces the self-polymerization of dopamine hydrochloride in an alkaline environment, and at the same time supports montmorillonite in situ, it assists in inducing the nucleation of hydroxyapatite, and in situ biomimetic construction of montmorillonite/nano Bone repair scaffold with hydroxyapatite. 2. the preparation method of the chitosan composite scaffold material of in-situ loaded montmorillonite/nano-hydroxyapatite according to claim 1, is characterized in that, comprises the following steps: (1) Weigh 5g of montmorillonite, add 100ml of 3M HCl solution, stir at a constant temperature in a water bath, centrifuge after the reaction, collect the lower solid precipitate, rinse it with deionized water for 5 to 6 times, dry it overnight, and grind the solid for later use to obtain the Acid-modified montmorillonite; (2) Prepare 150ml 10mmol/L tris(hydroxymethyl)aminomethane solution, adjust the pH to 8.5, add 1.5g dopamine hydrochloride to it, stir at room temperature for 30min, add the acid-modified montmorillonite obtained in step (1) Soil, stirring at room temperature for 24h, monitoring pH; after the reaction, centrifuge, collect the lower solid precipitate, rinse with deionized water for 5 to 6 times, until the supernatant is clear and transparent, collect the solid and freeze-dried to obtain PDA-MMT, which is for subsequent use; (3) Add 2g of chitosan powder to 100ml of acetic acid solution with a volume fraction of 1%, stir and dissolve in a constant temperature water bath at 65°C; (4) The PDA-MMT obtained in step (2) is weighed and configured into a suspension, and then added to the chitosan solution prepared in step (3), stirred and mixed evenly; (5) Add 2 ml each of 2 mol/L soluble calcium salt solution and 1.2 mol/L soluble phosphate solution to the mixed solution of chitosan and montmorillonite obtained in step (4) in turn, and stir well in a constant temperature water bath. , and evenly mixed to obtain the scaffold precursor solution; (6) adding the scaffold precursor solution obtained in step (5) into the mold, placing the mold at 4 °C overnight, then at -20 °C overnight, and finally freezing at -80 °C for 48 h to obtain the scaffold precursor; (7) Immerse the scaffold precursor obtained in (6) in 500 ml of Tris/ethanol mixed solution with pH=10, and place it on a constant temperature shaker at 37°C with a rotating speed of 120 r/min. After soaking for 8 hours, wash with a large amount of deionized water. to neutral; (8) Place the scaffold obtained in (7) at -20°C overnight, then place it in a -80°C refrigerator, and finally freeze-dry it in vacuum to obtain an in-situ loaded montmorillonite/nano-hydroxyapatite chitosan composite scaffold. . 3. The preparation method according to claim 2, characterized in that: the temperature of the water bath in step (1) is 45°C and the time is 8 h; the overnight drying temperature is 50°C. 4 . The preparation method according to claim 2 , wherein the monitoring pH in step (2) needs to be kept at 8.5. 5 . 5. The preparation method according to claim 2, characterized in that: in step (4), the PDA-MMT is weighed and configured so that the concentration of the suspension is 0.03-0.09 g/mL. 6. preparation method according to claim 2, is characterized in that: the soluble calcium salt used in step (5) is calcium nitrate or calcium chloride; The soluble phosphate used is dipotassium hydrogen phosphate, potassium dihydrogen phosphate, hydrogen phosphate Disodium or sodium dihydrogen phosphate; wherein the Ca/P molar ratio is n(Ca ²⁺ ):n(PO ₄ ^3- )=1.67:1, and the stirring time is 3-4 h. 7. preparation method according to claim 2 is characterized in that: the Tris/ethanol mixed solution used in step (7) is to mix dehydrated alcohol and Tris solution according to volume ratio 1:1, and Tris/ethanol mixes The concentration of Tris in the solution was 10 mmol/L. 8. The preparation method according to claim 2, wherein the temperature of freeze-drying in step (8) is -80°C, and the freeze-drying time is 48 h. 9. An in-situ loaded montmorillonite/nano-hydroxyapatite chitosan composite scaffold prepared by the method of any one of claims 1-8.

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