ROS (reactive oxygen species) sensitive hydrogel composite polycaprolactone stent as well as preparation method and application thereof

Abstract

The invention discloses an ROS (reactive oxygen species) sensitive hydrogel composite polycaprolactone stent as well as a preparation method and application thereof, and belongs to the technical field of biological material preparation. The preparation method comprises the following steps: reacting 4-(bromomethyl) phenylboronic acid and TMPDA in a solvent, and collecting precipitate to prepare a cross-linking agent; blending and cross-linking the PVA solution and the cross-linking agent solution to prepare PVA / TPA hydrogel; taking a mixed solution containing PCL and nHA as printing slurry, and obtaining a PCL / nHA stent through 3D printing; and pouring the PVA / TPA hydrogel into the PCL / nHA scaffold, and freeze-drying to obtain the hydrogel. The ROS sensitive hydrogel composite polycaprolactone scaffold prepared by the invention is a composite material with soft and hard combination and communicated large and small pores, has excellent biocompatibility, ROS removal performance, anti-inflammatory performance and osteogenic differentiation performance, can enhance the bone regeneration capacity in an inflammatory microenvironment, and has wide application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of biocompatible material preparation, and particularly relates to a ROS-sensitive hydrogel composite polycaprolactone scaffold and its preparation method and application. Background Art

[0002] Scaffolds fabricated by 3D printing technology are widely used in the field of bone tissue engineering, with the advantages of customized size and shape and minimizing the consumption of excess volume. In addition, scaffolds with a porous structure and a large surface area can provide a specific three-dimensional space for cells, facilitating cell distribution and various cell interactions. However, due to the limitations of single materials and structures, traditional 3D printing technology cannot effectively simulate the extracellular matrix (ECM) structure, has insufficient ability to promote cell adhesion and proliferation, and cannot provide anti-inflammatory properties. Therefore, the structure and performance of 3D printed scaffolds need to be improved.

[0003] In recent years, researchers have prepared bone-mimicking scaffolds by introducing various modification methods such as bioactive nanomaterials or hydrophilic polymers to improve the physicochemical properties of traditional single 3D printed scaffolds. However, for the treatment of large-area bone defects of the inflammatory type, current research cannot prepare an adaptable functional scaffold according to the spatio-temporal relationship between early inflammation and mid- to late-stage bone loss, resulting in less than ideal effects of the implanted materials. Therefore, the present invention aims to construct a scaffold material that can coordinate the spatio-temporal relationship between early anti-inflammation and mid- to late-stage osteogenesis, by integrating an inflammation-triggered degradable ECM-like into a 3D printed framework to form a hierarchical structure scaffold with regional gradient degradation. Summary of the Invention

[0004] In order to solve the above technical problems, the object of the present invention is to provide a ROS-sensitive hydrogel composite polycaprolactone scaffold and its preparation method and application, so as to solve the problems that existing bone repair materials are not adaptable according to the spatio-temporal relationship between early inflammation and mid- to late-stage bone loss in the treatment of inflammatory bone defects, resulting in less than ideal effects of the implanted materials and defects.

[0005] The technical solution of the present invention to solve the above technical problems is as follows:

[0006] In the first aspect of the present invention, a preparation method of a ROS-sensitive hydrogel composite polycaprolactone scaffold is provided, comprising the following steps:

[0007] (1) React 4-(bromomethyl)phenylboronic acid and N,N,N’,N’-tetramethyl-1,3-propanediamine in a solvent and collect the precipitate to obtain a crosslinking agent;

[0008] (2) Blend and crosslink a polyvinyl alcohol solution and the solution of the crosslinking agent obtained in step (1) to obtain a PVA / TPA hydrogel;

[0009] (3) Using the mixed solution containing polycaprolactone and nano-hydroxyapatite as the printing paste, a PCL / nHA scaffold is obtained by 3D printing;

[0010] (4) Pouring the PVA / TPA hydrogel obtained in step (2) into the PCL / nHA scaffold, and freeze-drying to prepare a ROS-sensitive PVA / TPA-PCL / nHA hierarchical structure scaffold.

[0011] The beneficial effects of the present invention are as follows: The preparation method of the present invention is simple, the conditions are mild, the equipment used for preparation is simple, the investment is small, it is conducive to mass production, and the application prospect is broad. By integrating the PVA / TPA hydrogel material with inflammation-triggered degradation into the PCL / nHA scaffold, a hierarchical structure scaffold with regional gradient degradation having biocompatibility, ROS scavenging ability, anti-inflammatory performance and osteogenic differentiation performance is obtained, which can effectively enhance the ability of bone regeneration in the inflammatory microenvironment.

[0012] Further, in step (1), the mass ratio of 4-(bromomethyl)phenylboronic acid to N,N,N',N'-tetramethyl-1,3-propanediamine is 3-7:1, and the solvent is DMF.

[0013] Further, the reaction time in step (1) is 24-36 h, and the reaction condition is room temperature.

[0014] Further, in step (2), the mass concentration of the polyvinyl alcohol solution is 3%-7%, and the mass concentration of the cross-linking agent solution is 3%-7%; the volume ratio of the polyvinyl alcohol solution to the cross-linking agent solution is (0.8-1.2):(0.8-1.2).

[0015] Preferably, in step (2), the mass concentration of the polyvinyl alcohol solution is 5%, the mass concentration of the cross-linking agent solution is 5%; the volume ratio of the polyvinyl alcohol solution to the cross-linking agent solution is 1:1.

[0016] Further, the cross-linking condition in step (2) is room temperature.

[0017] Further, in step (3), the mass ratio of polycaprolactone to nano-hydroxyapatite is (5-10):(2-4), the solvent is dichloromethane; the weight-average molecular weight of polyvinyl alcohol is 50000-100000.

[0018] Preferably, in step (3), the mass ratio of polycaprolactone to nano-hydroxyapatite is 7:3, the solvent is dichloromethane; the weight-average molecular weight of polyvinyl alcohol is 80000.

[0019] Further, the freeze-drying time in step (4) is 24-48 h.

[0020] Preferably, the freeze-drying time in step (4) is 48 h.

[0021] In the second aspect of the present invention, a ROS-sensitive hydrogel composite polycaprolactone scaffold prepared by the above preparation method is provided.

[0022] The beneficial effects of the present invention are as follows: The ROS-sensitive hydrogel composite polycaprolactone scaffold prepared by the present invention is a composite material combining soft and hard, with connected large and small pores, and has good biocompatibility, ROS scavenging performance, anti-inflammatory performance and osteogenic differentiation performance, which can enhance the bone regeneration ability under the inflammatory microenvironment.

[0023] In the third aspect of the present invention, an application of the above ROS-sensitive hydrogel composite polycaprolactone scaffold in the preparation of medical devices for the treatment of inflammatory bone defects is provided.

[0024] The beneficial effects of the present invention are as follows: By applying the prepared ROS-sensitive hydrogel composite polycaprolactone scaffold to medical devices for the treatment of inflammatory bone defects, the present invention has appropriate compressive stress. The bioactive scaffold combined with the ROS-sensitive hydrogel can effectively simulate the ECM structure while also having the ability to scavenge free radicals and ROS, and has a good therapeutic effect on inflammatory bone defect diseases, filling the gap in the design of implant scaffold materials for realizing the anti-inflammatory and osteogenic spatio-temporal characteristics in current bone tissue engineering.

[0025] The present invention has the following beneficial effects:

[0026] The equipment used in the preparation method of the present invention is simple and has a small investment. By using batch production, the prepared ROS-sensitive hydrogel composite polycaprolactone scaffold is a composite material combining soft and hard, with connected large and small pores, and has excellent biocompatibility, ROS scavenging performance, anti-inflammatory performance and osteogenic differentiation performance, which can enhance the bone regeneration ability under the inflammatory microenvironment, fill the gap in the design of implant scaffold materials for realizing the anti-inflammatory and osteogenic spatio-temporal characteristics in current bone tissue engineering, and has broad application prospects. Description of the Drawings

[0027] Figure 1 1H-NMR spectrum of the product obtained by incubating TPA and TPA with H 2 O 2 after incubation, where A is TPA and B is the product after incubating TPA with H 1 O 2 after incubation; 2

[0028] Figure 2 ​Macrographs and SEM images of the PCL / nHA scaffold and the PVA / TPA-PCL / nHA hierarchical structure scaffold in Test Example 1, where A and C are the macrograph and SEM image of the PCL / nHA scaffold respectively, and B and D are the macrograph and SEM image of the PVA / TPA-PCL / nHA hierarchical structure scaffold respectively;

[0029] Figure 3 Graphs of pressure-strain and bar graphs comparing the compressive moduli of the PCL / nHA scaffold and the PVA / TPA-PCL / nHA hierarchical structure scaffold in Test Example 1, where A is the pressure-strain graph and B is the bar graph comparing the compressive moduli;

[0030] Figure 4 Bar graph comparing the DPPH scavenging rates of the PCL / nHA scaffold and the PVA / TPA-PCL / nHA hierarchical structure scaffold in Test Example 1;

[0031] Figure 5 Graphs of the CCK8 test results after co-incubating bone marrow mesenchymal stem cells (BMSCs) with the PCL / nHA scaffold and the PVA / TPA-PCL / nHA hierarchical structure scaffold for 1 day, 4 days, and 7 days in Test Example 2, where Ctrl is the control group without treatment, PH is the PCL / nHA scaffold group, and PH-TP is the PVA / TPA-PCL / nHA hierarchical structure scaffold group;

[0032] Figure 6 Fluorescence microscopy images of intracellular ROS labeled with DCFH-DA fluorescent probe after co-incubating BMSCs with the PCL / nHA scaffold and the PVA / TPA-PCL / nHA hierarchical structure scaffold in a ROS environment in Test Example 2;

[0033] Figure 7 In vivo osteogenesis effect diagrams at 4W and 8W of the bone defect blank control group, the inflammatory bone defect model group, the inflammatory bone defect model + PCL / nHA scaffold group, and the inflammatory bone defect model + PVA / TPA-PCL / nHA hierarchical structure scaffold group in Test Example 2. Detailed implementation mode

[0034] The principles and features of the present invention will be described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention. Those not specified in the examples are carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not specified by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0035] Example 1:

[0036] A preparation method of a ROS-sensitive hydrogel composite polycaprolactone scaffold, comprising the following steps:

[0037] (1) Preparation of crosslinking agent TPA

[0038] Synthesized by the quaternary ammonium reaction between 4-(bromomethyl)phenylboronic acid and N,N,N’,N’-tetramethyl-1,3-propanediamine (TMPDA): 1 g of 4-(bromomethyl)phenylboronic acid and 0.2 g of TMPDA were mixed with 20 mL of DMF and stirred at room temperature for 24 h; the reaction solution was filtered by precipitation in 100 mL of TFH, and further washed three times with 20 mL of THF and a suction filtration device, and then freeze-dried in a freeze-vacuum dryer for 24 h to obtain pure TPA (N 1 -(4-bromobenzyl)-N 3 -(4-bromophenyl)-N 1 ,N 1 ,N 3 ,N 3 -tetramethylpropane-1,3-diamine), characterized by 1 H-NMR, and the results are shown in Figure 1 Figure A in it, proving that the corresponding compound was prepared. After incubation in 10 mmol / L H 2 O 2 at room temperature for 3 h, the crosslinking bonds of TPA were broken, generating new products, and the results are shown in Figure 1 Figure B in it, proving that TPA has ROS-sensitive chemical bonds.

[0039] (2) Preparation of PVA / TPA hydrogel

[0040] Prepare PVA / TPA hydrogel by room temperature crosslinking method: Add 0.5 g of polyvinyl alcohol (PVA) powder to 10 mL of deionized water, and stir in a 95 °C water bath until PVA is completely dissolved to make a 5 wt.% PVA solution; add 0.5 g of TPA powder obtained in step (1) to 10 mL of deionized water, and stir at room temperature until completely dissolved to make a 5 wt.% TPA solution; mix the 5 wt.% TPA solution and the 5 wt.% PVA solution evenly at a volume ratio of 1:1 at room temperature, and obtain PVA / TPA hydrogel after sufficient reaction.

[0041] (3) Preparation of PCL / nHA scaffold (PH)

[0042] Fabrication of 3D printed PCL / nHA scaffolds in a 3D printer by room temperature extrusion method: Dissolve 7 g of polycaprolactone (PCL, Mw = 80,000) in 28 mL of dichloromethane at room temperature for 1 h under closed conditions. Take 3 g of nano-hydroxyapatite (nHA) raw materials, sieve them through an 800-mesh sieve, and dissolve them in 10 mL of dichloromethane. Uniformly mix the nHA solution into the PCL solution to obtain a PCL / nHA slurry in a diluted flowing state. Filter it through an injection needle (diameter: 0.21 mm). Stir the filtered slurry to volatilize dichloromethane until the slurry presents a wall-hanging creamy state to obtain a PCL / nHA slurry in a printing state. Print it through an injection needle (diameter: 0.26 mm) in a 90° cross and double-wire overlapping design pattern. The printing parameters are as follows: The designed size of the 3D grid structure is 10 mm × 10 mm × 6 mm, the line spacing is 0.8 mm, the layer height is 0.25 mm, the number of layers is 24 layers, the printing speed is 8 mm / sec, and the extrusion air pressure is 0.25 MPa. After printing, place the scaffold in a vacuum freeze dryer and dry it for 7 days to remove the residual dichloromethane.

[0043] (4) Preparation of ROS-sensitive hydrogel composite polycaprolactone scaffold (PVA / TPA-PCL / nHA hierarchical structure scaffold)

[0044] Perfuse the PVA / TPA hydrogel prepared in step (2) into the PCL / nHA scaffold obtained in step (3) by vacuum impregnation method: Immerse the PCL / nHA scaffold into the PVA / TPA hydrogel, place it in a vacuum environment, wait for the gel to completely immerse the scaffold, and freeze-dry it for 48 h to obtain a ROS-sensitive PVA / TPA-PCL / nHA hierarchical structure scaffold (PH-TP).

[0045] Example 2:

[0046] A preparation method of a ROS-sensitive hydrogel composite polycaprolactone scaffold, comprising the following steps:

[0047] (1) Preparation of cross-linking agent TPA

[0048] Synthesize by quaternary ammonium reaction between 4-(bromomethyl)phenylboronic acid and N,N,N',N'-tetramethyl-1,3-propanediamine (TMPDA): Mix 1 g of 4-(bromomethyl)phenylboronic acid and 0.2 g of TMPDA with 20 mL of DMF and stir the reaction at room temperature for 24 h; Filter the reaction solution by precipitation in 100 mL of TFH, and further wash it three times with 20 mL of THF and a suction filtration device, and place it in a freeze vacuum dryer and freeze-dry it for 24 h to obtain pure TPA (N 1 -(4-bromobenzyl)-N 3 -(4-bromophenyl)-N 1 ,N 1 ,N3 , N 3 -tetramethylpropane-1,3-diamine).

[0049] (2) Preparation of PVA / TPA hydrogel

[0050] Prepare PVA / TPA hydrogel by room-temperature crosslinking method: Add 0.6 g of polyvinyl alcohol (PVA) powder to 10 mL of deionized water, stir in a 95 °C water bath until PVA is completely dissolved to make a 6 wt.% PVA solution; add 0.4 g of TPA powder obtained in step (1) to 10 mL of deionized water, stir at room temperature until completely dissolved to make a 4 wt.% TPA solution; mix the 4 wt.% TPA solution and the 6 wt.% PVA solution evenly at a volume ratio of 1:1 at room temperature, and prepare PVA / TPA hydrogel after full reaction.

[0051] (3) Preparation of PCL / nHA scaffold

[0052] Manufacture a 3D-printed PCL / nHA scaffold in a 3D printer by room-temperature extrusion method: Dissolve 8 g of polycaprolactone (PCL, Mw = 80000) in 28 mL of dichloromethane at room temperature for 1 h under closed conditions, take 3.5 g of nano-hydroxyapatite (nHA) raw material, sieve it through an 800-mesh sieve, and dissolve it in 10 mL of dichloromethane; evenly mix the nHA solution into the PCL solution to obtain a diluted and flowing PCL / nHA slurry; filter through an injection needle (diameter: 0.21 mm); stir the filtered slurry to volatilize dichloromethane until the slurry shows a wall-hanging creamy state to obtain a printable PCL / nHA slurry; print through an injection needle (diameter: 0.26 mm) in a 90° cross and double-wire overlapping design pattern, and the printing parameters are as follows: the designed size of the 3D grid structure is 10 mm × 10 mm × 6 mm, the line spacing is 0.8 mm, the layer height is 0.25 mm, the number of layers is 24 layers, the printing speed is 8 mm / sec, and the extrusion air pressure is 0.25 MPa. After printing, place the scaffold in a vacuum freeze dryer and dry for 7 days to remove the residual dichloromethane.

[0053] (4) Preparation of ROS-sensitive hydrogel composite polycaprolactone scaffold (PVA / TPA-PCL / nHA hierarchical structure scaffold)

[0054] Perfuse the PVA / TPA hydrogel prepared in step (2) into the PCL / nHA scaffold obtained in step (3) by vacuum impregnation method: Immerse the PCL / nHA scaffold into the PVA / TPA hydrogel, place it in a vacuum environment, wait for the gel to completely immerse the scaffold, and freeze-dry for 48 h to prepare a ROS-sensitive PVA / TPA-PCL / nHA hierarchical structure scaffold.

[0055] Example 3:

[0056] A preparation method of a ROS-sensitive hydrogel composite polycaprolactone scaffold, comprising the following steps:

[0057] (1) Preparation of crosslinking agent TPA

[0058] Synthesized by the quaternary ammonium reaction between 4-(bromomethyl)phenylboronic acid and N,N,N',N'-tetramethyl-1,3-propanediamine (TMPDA): Mix 1 g of 4-(bromomethyl)phenylboronic acid and 0.2 g of TMPDA with 20 mL of DMF, and stir the reaction at room temperature for 24 h; precipitate the reaction solution in 100 mL of TFH and filter, and further wash three times with 20 mL of THF and a suction filtration device, and place it in a freeze-drying vacuum dryer for freeze-drying for 24 h to obtain pure TPA (N 1 -(4-bromobenzyl)-N 3 -(4-bromophenyl)-N 1 ,N 1 ,N 3 ,N 3 -tetramethylpropane-1,3-diamine), characterized by 1 H-NMR, and the results are as shown in Figure 1 Figure A in, proving that the corresponding compound was prepared. After incubation in 10 mmol / L H 2 O 2 at room temperature for 3 h, the crosslinking bonds of TPA were broken, generating new products, and the results are as shown in Figure 1 Figure B in, proving that TPA has ROS-sensitive chemical bonds.

[0059] (2) Preparation of PVA / TPA hydrogel

[0060] Prepare PVA / TPA hydrogel by the room temperature crosslinking method: Add 0.4 g of polyvinyl alcohol (PVA) powder to 10 mL of deionized water, and stir in a 95 °C water bath until the PVA is completely dissolved to make a 4 wt.% PVA solution; add 0.6 g of TPA powder obtained in step (1) to 10 mL of deionized water, and stir at room temperature until completely dissolved to make a 6 wt.% TPA solution; mix the 6 wt.% TPA solution and the 4 wt.% PVA solution evenly at a volume ratio of 1:1 at room temperature, and obtain PVA / TPA hydrogel after sufficient reaction.

[0061] (3) Preparation of PCL / nHA scaffold

[0062] Fabrication of 3D-printed PCL / nHA scaffolds in a 3D printer by room-temperature extrusion: Under closed conditions, 6 g of polycaprolactone (PCL, Mw = 80,000) was dissolved in 28 mL of dichloromethane at room temperature for 1 h. 2.5 g of nano-hydroxyapatite (nHA) raw material was sieved through an 800-mesh sieve and then dissolved in 10 mL of dichloromethane. The nHA solution was uniformly mixed into the PCL solution to obtain a PCL / nHA slurry in a diluted flowing state. The slurry was filtered through an injection needle (diameter: 0.21 mm). The filtered slurry was stirred to volatilize dichloromethane until the slurry showed a wall-hanging creamy state, obtaining a PCL / nHA slurry in a printable state. The PCL / nHA slurry was printed through an injection needle (diameter: 0.26 mm) in a 90° cross and double-filament overlapping design pattern. The printing parameters were as follows: the designed size of the 3D grid structure was 10 mm × 10 mm × 6 mm, the line spacing was 0.8 mm, the layer height was 0.25 mm, the number of layers was 24, the printing speed was 8 mm / sec, and the extrusion air pressure was 0.25 MPa. After printing, the scaffolds were placed in a vacuum freeze dryer and dried for 7 days to remove the residual dichloromethane.

[0063] (4) Preparation of ROS-sensitive hydrogel composite polycaprolactone scaffolds (PVA / TPA-PCL / nHA hierarchical structure scaffolds)

[0064] The PVA / TPA hydrogel prepared in step (2) was infused into the PCL / nHA scaffolds obtained in step (3) by vacuum impregnation: The PCL / nHA scaffolds were immersed in the PVA / TPA hydrogel and placed in a vacuum environment. After the gel was completely immersed in the scaffolds, they were freeze-dried for 48 h to obtain ROS-sensitive PVA / TPA-PCL / nHA hierarchical structure scaffolds.

[0065] Experimental example 1: Morphological and physicochemical property characterization

[0066] (1) The PVA / TPA-PCL / nHA hierarchical structure scaffolds and CL / nHA scaffolds prepared in Example 1 were taken for macroscopic appearance and SEM characterization. The results are as Figure 2 shown. It can be seen from the figure that the hydrogel was uniformly filled in the pores of the scaffolds, forming a composite material with a combination of hard and soft and connected large and small pores.

[0067] (2) Mechanical property characterization

[0068] Experimental method: Referring to the preparation steps in Example 1, rectangular parallelepiped PVA / TPA-PCL / nHA hierarchical structure scaffolds and PCL / nHA scaffolds with dimensions of 10 mm × 10 mm × 6 mm were prepared for characterization respectively. They were placed on the compression plate, and at room temperature, a universal mechanical testing machine was used to compress the rectangular parallelepiped scaffolds to 30% of the total height at a compression strain rate of 0.5 mm / min.

[0069] Experimental results: As Figure 3 shown, compared with the PCL / nHA scaffold used for comparison, the PVA / TPA-PCL / nHA hierarchical structure scaffold prepared in Example 1 of the present invention has an increased compressive stress by adding hydrogel to the scaffold.

[0070] (3) DPPH scavenging rate of PVA / TPA-PCL / nHA hierarchical structure scaffold

[0071] Experimental method: Referring to the preparation steps of Example 1, cylindrical PVA / TPA-PCL / nHA hierarchical structure scaffolds and PCL / nHA scaffolds with a diameter of 14 mm and a height of 1 mm were respectively prepared for characterization. They were incubated in the dark at room temperature for 30 min in 500 μL of DPPH working solution. The incubation solution of each group was placed in a 96-well plate, and the absorbance was measured at 517 nm with an enzyme-labeling instrument.

[0072] Experimental results: As Figure 4 shown, compared with the PCL / nHA scaffold used for comparison, the PVA / TPA-PCL / nHA hierarchical structure scaffold prepared in Example 1 of the present invention significantly improves the free radical scavenging rate of the scaffold by adding PVA / TPA hydrogel, indicating that the PVA / TPA-PCL / nHA hierarchical structure scaffold has excellent ROS scavenging ability.

[0073] Test Example 2: Application of PVA / TPA-PCL / nHA hierarchical structure scaffold in the treatment of inflammatory bone defects

[0074] (1) Biocompatibility

[0075] Experimental method: Referring to the preparation steps in Example 1, cylindrical PCL / nHA scaffolds and PVA / TPA-PCL / nHA hierarchical structure scaffolds with a diameter of 14 mm and a height of 0.5 mm were respectively prepared, and they were co-incubated with bone marrow mesenchymal stem cells (BMSC) for 1 d, 4 d, and 7 d, and their in vitro biocompatibility was evaluated by CCK8 detection.

[0076] Experimental results: As Figure 5 shown, after BMSC was incubated with different scaffolds, with the increase of the incubation days, the cell volume also showed an increasing trend, and there was no significant difference compared with the control group (without treatment), indicating that the scaffolds prepared in the present invention have excellent cell compatibility.

[0077] (2) ROS scavenging ability

[0078] Experimental method: Referring to the preparation steps in Example 1, cylindrical PCL / nHA scaffolds and PVA / TPA-PCL / nHA hierarchical structure scaffolds with a diameter of 14 mm and a height of 0.5 mm were respectively prepared. In H 2 O2 BMSCs treated with different scaffolds were incubated in the presence of

[0079] Experimental results: As Figure 6 shown, H 2 O 2 2 will induce the production of intracellular ROS in BMSCs, and this ROS can be captured by the DCFH-DA probe, showing green fluorescence under a fluorescence microscope. When the ROS-sensitive hydrogel composite scaffold of the present invention is used, the production of these ROS is reduced, which means that the PVA / TPA-PCL / nHA hierarchical structure scaffold prepared by the present invention helps to reduce the damage of ROS to cells.

[0080] (3) Anti-inflammatory osteogenic performance

[0081] Experimental method: Referring to the preparation steps in Example 1, cylindrical PCL / nHA scaffolds and PVA / TPA-PCL / nHA hierarchical structure scaffolds with a diameter of 3 mm and a height of 2 mm were respectively prepared and implanted into a rat inflammatory alveolar bone defect model for evaluating the in vivo treatment effect.

[0082] Experimental results: As Figure 7 shown, the figures are the osteogenesis effect diagrams at 4W and 8W in vivo of the bone defect blank control group, the inflammatory bone defect model group, the inflammatory bone defect model + PCL / nHA scaffold group, and the inflammatory bone defect model + PVA / TPA-PCL / nHA hierarchical structure scaffold group. BV represents the volume of bone tissue, and TV represents the total tissue volume. It can be seen from the figure that the PVA / TPA-PCL / nHA hierarchical structure scaffold prepared in Example 1 of the present invention has significantly stronger osteogenic ability compared with the PCL / nHA scaffold group and other control groups, indicating that the PVA / TPA-PCL / nHA hierarchical structure scaffold prepared by the present invention can more effectively promote bone tissue regeneration in an inflammatory microenvironment.

[0083] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a ROS-sensitive hydrogel composite polycaprolactone scaffold, characterized in that: The following steps are involved: (1) reacting 4-(bromomethyl)phenylboronic acid and N,N,N',N'-tetramethyl-1,3-propylenediamine in a solvent and collecting the precipitate to prepare a crosslinking agent; (2) blending and cross-linking the polyvinyl alcohol solution and the solution of the cross-linking agent obtained in step (1) to prepare a PVA / TPA hydrogel; (3) using a mixed solution containing polycaprolactone and nanohydroxyapatite as a printing slurry to obtain a PCL / nHA scaffold by 3D printing; (4) The PVA / TPA hydrogel obtained in step (2) is poured into the PCL / nHA scaffold and freeze-dried to obtain a scaffold.

2. The method for preparing the ROS-sensitive hydrogel composite polycaprolactone scaffold according to claim 1, characterized in that: In the step (1), the mass ratio of 4-(bromomethyl)phenylboronic acid to N,N,N',N'-tetramethyl-1,3-propylenediamine is 3-7:1, and the solvent is DMF.

3. The method for preparing the ROS-sensitive hydrogel composite polycaprolactone scaffold according to claim 1, characterized in that: The reaction time in step (1) is 24-36 hours, and the reaction condition is room temperature.

4. The method for preparing the ROS-sensitive hydrogel composite polycaprolactone scaffold according to claim 1, characterized in that: In the step (2), the mass concentration of the polyvinyl alcohol solution is 3%-7%, and the mass concentration of the crosslinking agent solution is 3%-7%; the volume ratio of the polyvinyl alcohol solution to the crosslinking agent solution is (0.8-1.2): (0.8-1.2).

5. The method for preparing the ROS-sensitive hydrogel composite polycaprolactone scaffold according to claim 1, characterized in that: The cross-linking condition in step (2) is room temperature.

6. The method for preparing the ROS-sensitive hydrogel composite polycaprolactone scaffold according to claim 1, characterized in that: In the step (3), the mass ratio of polycaprolactone to nano-hydroxyapatite is (5-10):(2-4), the solvent is dichloromethane; and the weight average molecular weight of polyvinyl alcohol is 50,000-100,000.

7. The method for preparing the ROS-sensitive hydrogel composite polycaprolactone scaffold according to claim 1, characterized in that: The freeze-drying time in step (4) is 24-48 hours.

8. A ROS-sensitive hydrogel composite polycaprolactone scaffold, characterized in that: The method is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the ROS-sensitive hydrogel composite polycaprolactone scaffold according to claim 8 in the preparation of medical devices for the treatment of inflammatory bone defects.

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