A nanoparticle / polymer composite scaffold with good dispersibility and antioxidant activity, and its preparation method and application

Abstract

The present invention is applicable to the field of biotechnology, and provides a nanoparticle / polymer composite scaffold with good dispersibility and antioxidant activity, and a preparation method and application thereof. The method includes: adding an aqueous solution of calcium chloride, magnesium chloride, manganese chloride and sodium phosphate into a mixed solution of oleic acid and absolute ethanol, forming manganese-substituted whitlockite nanoparticles coated with oleic acid by a hydrothermal method, and forming a composite scaffold by utilizing the high interfacial compatibility between oleic acid and the polymer. The present invention utilizes the manganese-substituted whitlockite nanoparticles coated with oleic acid to exert antioxidant and osteogenic biological effects in the oxidative stress bone defect microenvironment, and at the same time realizes the uniform dispersion of the nanoparticles in the polymer through the constructed nanoparticle / polymer composite scaffold. These two strategies synergistically regulate the repair of the oxidative stress bone defect region, and solve problems such as nanoparticle aggregation, mechanical property decline, limited cell interaction, and inability to relieve the oxidative stress state.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a nanoparticle / polymer composite scaffold with good dispersibility and antioxidant activity, and a preparation method and application thereof. Background Art

[0002] Bone defect is one of the major challenges in the global health field. Traditional treatment methods such as autologous bone grafting and allogeneic bone grafting, although having certain effects, face problems such as donor site complications, risk of immune rejection, and scarcity of donor sources. In addition, bone defects are often accompanied by local microvascular rupture and insufficient oxygen supply, forming a hypoxic microenvironment, which leads to an increase in the levels of reactive oxygen species and reactive nitrogen species, triggering oxidative stress. Excessive reactive oxygen species not only induce apoptosis of osteoprogenitor cells and mature osteoblasts, but also inhibit the expression of key osteogenic markers, thereby disrupting the osteogenic differentiation process and ultimately hindering bone regeneration. In view of these challenges, there is an urgent need to develop new treatment strategies to effectively address bone defects and achieve their repair and regeneration.

[0003] Tissue engineering technology provides a new idea for developing scaffold materials with both mechanical support and osteogenic induction functions. Among them, polycaprolactone (PCL) has become a matrix material widely used in 3D printing due to its excellent biocompatibility, mechanical properties and easy processability. However, polycaprolactone itself has problems of insufficient mechanical strength and lack of bioactivity, and it is difficult to effectively induce osteogenic differentiation. Traditional methods introduce functional inorganic nanoparticles into the polymer matrix to provide bioactive ions, but due to the poor interfacial compatibility between the functional inorganic nanoparticles and the polymer matrix, problems such as nanoparticle aggregation, mechanical property degradation and limited cell interaction often occur. Therefore, the present invention proposes a nanoparticle / polymer composite scaffold with good dispersibility and antioxidant activity, and a preparation method and application thereof. Summary of the Invention

[0004] The purpose of the present invention is to provide a nanoparticle / polymer composite scaffold with good dispersibility and antioxidant activity, and a preparation method and application thereof, aiming to solve the problems raised in the above background art.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A manganese-substituted whitlockite nanoparticle coated with oleic acid is prepared by the following method:

[0007] An aqueous mixed solution of calcium chloride, magnesium chloride, manganese chloride and sodium phosphate is added to a container containing oleic acid and absolute ethanol. After stirring evenly, the reaction mixture reacts at 150 - 200 °C for 10 - 12 h to obtain a mixture of manganese-substituted whitlockite nanoparticles immersed in a mixed solution of oleic acid and ethanol. After centrifugation and washing, oleic acid-coated manganese-substituted whitlockite nanoparticles are obtained;

[0008] Among them, the molar ratio of calcium chloride, magnesium chloride, manganese chloride, and sodium phosphate is 9:0 - 1:0 - 1:6; the volume ratio of oleic acid and absolute ethanol is 4 - 5:16.

[0009] Furthermore, the total molar amount of calcium chloride, magnesium chloride and manganese chloride, and the molar ratio of sodium phosphate is 9:1:6.

[0010] An application of the above-mentioned oleic acid-coated manganese-substituted whitlockite nanoparticles is used in the preparation of a nanoparticle / polymer composite scaffold with good dispersibility and antioxidant activity.

[0011] An application of the above-mentioned oleic acid-coated manganese-substituted whitlockite nanoparticles is used in the preparation of a drug for antioxidant and bone defect repair.

[0012] A nanoparticle / polymer composite scaffold with good dispersibility and antioxidant activity is prepared by the following method:

[0013] Take the above-mentioned oleic acid-coated manganese-substituted whitlockite nanoparticles, add them to polycaprolactone dissolved in dichloromethane, continuously stir until dichloromethane volatilizes, and use a 3D printer through fused deposition modeling technology to prepare a composite scaffold with uniform pore size and specific shape, obtaining a nanoparticle / polymer composite scaffold with good dispersibility and antioxidant activity.

[0014] Furthermore, the mass ratio of the oleic acid-coated manganese-substituted whitlockite nanoparticles to polycaprolactone is 1:1 - 10.

[0015] Furthermore, the mass fraction of the oleic acid-coated manganese-substituted whitlockite nanoparticles is 30%.

[0016] An application of the above-mentioned nanoparticle / polymer composite scaffold with good dispersibility and antioxidant activity is used in the preparation of a composite scaffold for promoting bone defect repair under oxidative stress.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] The present invention proposes a method with a simple synthesis process and low cost, having two significant advantages and technical contributions. On the one hand, manganese-substituted whitlockite nanoparticles coated with oleic acid are utilized to exert antioxidant and osteogenic biological effects in the oxidative stress bone defect microenvironment. On the other hand, through the constructed nanoparticle / polymer composite scaffold, the uniform dispersion of nanoparticles in the polymer is achieved. These two strategies synergistically regulate the repair of the oxidative stress bone defect region, solving problems such as nanoparticle aggregation, decline in mechanical properties, limited cell interaction, and inability to relieve the oxidative stress state. Description of the Drawings

[0019] Figure 1 Particle size distribution diagrams, TEM images, XRD patterns, and EDS spectra of 0% Mn-OAWH, 20% Mn-OAWH, and 50% Mn-OAWH nanoparticles in Example 1; where A is the particle size distribution diagram and TEM image of 0% Mn-OAWH nanoparticles; B is the particle size distribution diagram and TEM image of 20% Mn-OAWH nanoparticles; C is the particle size distribution diagram and TEM image of 50% Mn-OAWH nanoparticles; D is the XRD pattern of each nanoparticle; E is the EDS spectrum of 20% Mn-OAWH nanoparticles; F is the EDS spectrum of 50% Mn-OAWH nanoparticles.

[0020] Figure 2 FTIR characterization, dispersion experiment results, and EDS Mapping images in Example 2; where A is the FTIR spectra of 0% Mn-OAWH, 20% Mn-OAWH, 50% Mn-OAWH, and APWH nanoparticles; B is the difference in the dispersion of each nanoparticle in dichloromethane; C is the difference in the dispersion of each nanoparticle in polycaprolactone dissolved in dichloromethane; D is the EDS Mapping image of the 20% Mn-OAWH / PCL composite scaffold.

[0021] Figure 3 Investigation of the effect of the composite scaffold on the proliferation of rat bone marrow mesenchymal stem cells and its ability to scavenge reactive nitrogen and reactive oxygen species under normal and oxidative stress conditions in Example 3; where A is the proliferation of rBMSCs cells cultured in the leaching solution medium of each group of composite scaffolds under normal conditions; B is the proliferation of rBMSCs cells cultured in the leaching solution medium of each group of composite scaffolds under oxidative stress conditions; C is the scavenging rates of DPPH and superoxide anion by PCL, 0% Mn-OAWH / PCL, and 20% Mn-OAWH / PCL.

[0022] Figure 4 ALP staining of rBMSC cells treated with the osteogenic induction medium of the composite scaffold leaching solution for 14 days in Example 4.

[0023] Figure 5 For the establishment of the critical bone defect model in rats in Example 5, the implantation of the scaffold, and the bone regeneration situation after 8 weeks; where A is the establishment of the critical bone defect model in rats and the implantation of the scaffold; B is the Micro-CT three-dimensional reconstruction image after 8 weeks. Detailed implementation manners

[0024] For a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solutions of the present invention will be described in detail below, but it should not be construed as a limitation on the scope of implementation of the present invention.

[0025] The following describes the specific implementation of the present invention in detail in combination with specific embodiments.

[0026] Example 1: Add 4 mL of oleic acid and 16 mL of absolute ethanol into a 50 mL beaker, and stir at a speed of 600 rpm for 10 min at room temperature to form a homogeneous solution. Subsequently, add 4.5 mL of 0.25 M CaCl2 aqueous solution, 0.40 mL of 0.25 M MgCl2 aqueous solution, 0.10 mL of 0.25 M MnCl2 aqueous solution, and 5 mL of 0.15 M Na3PO4 aqueous solution to the mixed solution in sequence, and continue to stir at a speed of 600 rpm for 20 min at room temperature. Transfer the obtained mixed solution to a 50 mL polytetrafluoroethylene-lined reaction kettle, and heat it at 200 °C for 12 h. After cooling to room temperature, collect the precipitate by centrifugation, and wash it three times with ethanol to remove impurities. Finally, dry the product in an oven at 40 °C for 24 h, and grind it into fine powder to obtain Mn-OAWH nanoparticles with a manganese content of 20% (denoted as 20%Mn-OAWH).

[0027] To synthesize Mn-OAWH nanoparticles with different manganese contents, use the same steps as those for preparing 20%Mn-OAWH, but adjust the dosages of MgCl2 and MnCl2 aqueous solutions: use 0.25 mL of MgCl2 and 0.25 mL of MnCl2 aqueous solutions respectively to obtain Mn-OAWH nanoparticles with a manganese content of 50% (50%Mn-OAWH).

[0028] For the synthesis of oleic acid-functionalized whitlockite nanoparticles without manganese substitution (0%Mn-OAWH), adjust the dosage of MgCl2 aqueous solution to 0.5 mL and omit MnCl2.

[0029] As Figure 1 shown in A-C above, the above-mentioned nanoparticles are spherical with a diameter of about 17 nm; XRD analysis proves that the synthesized nanoparticles are whitlockite ( Figure 1In D); The presence of Mn in the synthesized 20% Mn-OAWH and 50% Mn-OAWH was observed by EDS energy spectrum characterization, proving that manganese ions successfully replaced some magnesium ions ( Figure 1 In E and F).

[0030] Example 2: After preparing 0% Mn-OAWH, 20% Mn-OAWH, and 50% Mn-OAWH nanoparticles according to the steps of Example 1, they were added to polycaprolactone dissolved in dichloromethane. The mass ratio of the nanoparticles to polycaprolactone was 3:7. Stirring was continued until the dichloromethane evaporated. A composite scaffold with uniform pore size and specific shape was prepared by using a 3D printer through fused deposition modeling technology, that is, a nanoparticle / polymer composite scaffold with good dispersibility and antioxidant activity (0% Mn-OAWH / PCL, 20% Mn-OAWH / PCL, 50% Mn-OAWH / PCL composite scaffolds) was obtained.

[0031] The FTIR characterization results show that compared with the traditional aqueous phase synthesis method (APWH), the surface of the whitlockite nanoparticles synthesized with oleic acid and absolute ethanol as the mixed solvent is coated with oleic acid ( Figure 2 In A). The results of the dispersibility experiment show that the dispersibility of the manganese-substituted whitlockite nanoparticles coated with oleic acid in dichloromethane and in polycaprolactone dissolved in dichloromethane is better than that of the traditional aqueous phase whitlockite ( Figure 2 In B and C). The uniform distribution of Ca, Mg, Mn, and P elements in the 20% Mn-OAWH / PCL composite scaffold was observed by EDS energy spectrum characterization, indicating that the manganese-substituted whitlockite nanoparticles coated with oleic acid are uniformly dispersed in polycaprolactone ( Figure 2 In D).

[0032] Example 3: 0% Mn-OAWH / PCL, 20% Mn-OAWH / PCL, and 50% Mn-OAWH / PCL composite scaffolds were prepared according to the steps of Example 1 and Example 2. First, the detection of cell proliferation was carried out, indicating that when the molar concentration of manganese ions is 20% or less, there is no obvious effect on the proliferation of rat bone marrow mesenchymal cells (rBMSCs) ( Figure 3In A). The complete medium was replaced with a composite scaffold extract medium, and 200 μmol / L H2O2 was added to establish an oxidative stress cell model. CCK-8 assays were performed on the 1st and 3rd days respectively. The results showed that the OD values of the Control+H2O2, PCL+H2O2, and 0% Mn-OAWH / PCL+H2O2 groups were lower than those of the Control group on the 1st and 3rd days, indicating the successful establishment of the oxidative stress cell model. However, the OD value of the 20% Mn-OAWH / PCL+H2O2 group was comparable to that of the Control group without H2O2, indicating that the 20% Mn-OAWH / PCL composite scaffold 2+ effectively scavenged reactive oxygen species, protected rBMSCs from oxidative stress, and promoted subsequent bone defect repair ( Figure 3 In B). DPPH detection kits and superoxide anion detection kits were used to evaluate the ability of the 20% Mn-OAWH / PCL scaffold to scavenge reactive nitrogen and reactive oxygen species. The results showed that the PCL group and the 0% Mn-OAWH / PCL group had almost no antioxidant ability. In contrast, the 20% Mn-OAWH / PCL group showed strong antioxidant activity ( Figure 3 In C).

[0033] Example 4: 0% Mn-OAWH / PCL and 20% Mn-OAWH / PCL composite scaffolds were prepared according to the steps of Examples 1 and 2. Alkaline phosphatase (ALP) staining was used to determine the effect of the composite scaffold extract on rBMSCs. After treating the cells for 14 days, compared with the Control group, the number of ALP-positive cells in the group treated with the composite scaffold extract was significantly increased, the staining became deeper, and it showed a manganese ion concentration dependence ( Figure 4 ).

[0034] Example 5: 0% Mn-OAWH / PCL and 20% Mn-OAWH / PCL composite scaffolds were prepared according to the steps of Examples 1 and 2. Each group of composite scaffolds was implanted into the critical bone defect of the rat skull ( Figure 5 In A). Compared with the Control group, PCL group, and 0% Mn-OAWH / PCL group, the 20% Mn-OAWH / PCL group had the best effect in promoting bone tissue regeneration. ( Figure 5 In B).

[0035] The above are only the preferred embodiments of the present invention. It should be noted that for those skilled in the art, without departing from the concept of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent.

Claims

1. A nanoparticle / polymer composite scaffold with good dispersibility and antioxidant activity, characterized in that, It is prepared by the following method: Take oleic acid-coated manganese-substituted whitlockite nanoparticles, add them to polycaprolactone dissolved in dichloromethane, continuously stir until dichloromethane volatilizes, and use a 3D printer through fused deposition modeling technology to prepare a composite scaffold with uniform pore size and specific shape, obtaining a nanoparticle / polymer composite scaffold with good dispersibility and antioxidant activity; The oleic acid-coated manganese-substituted whitlockite nanoparticles are prepared by the following method: Add a mixed aqueous solution of calcium chloride, magnesium chloride, manganese chloride and sodium phosphate to a container containing oleic acid and absolute ethanol. After stirring evenly, the reaction mixture reacts at 150-200 °C for 10-12 h to obtain a mixture of manganese-substituted whitlockite nanoparticles immersed in a mixed solution of oleic acid and ethanol. After centrifugation and washing, oleic acid-coated manganese-substituted whitlockite nanoparticles are obtained; Among them, the total molar amount of calcium chloride, magnesium chloride and manganese chloride, and the molar ratio of sodium phosphate is 9:1:6; the volume ratio of oleic acid and absolute ethanol is 4-5:

16.

2. The nanoparticle / polymer composite scaffold with good dispersibility and antioxidant activity according to claim 1, wherein The mass ratio of the oleic acid-coated manganese-substituted whitlockite nanoparticles to polycaprolactone is 1:1-10.

3. The nanoparticle / polymer composite scaffold with good dispersibility and antioxidant activity according to claim 1, wherein The mass fraction of the oleic acid-coated manganese-substituted whitlockite nanoparticles is 30%.

4. Use of a nanoparticle / polymer composite scaffold having good dispersibility and antioxidant activity according to any one of claims 1-3, characterized in that, It is applied to the preparation of a composite scaffold for promoting bone defect repair under oxidative stress.

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