PQQ-loaded porous ceramic support and preparation method and application thereof

Abstract

The invention provides a PQQ-loaded porous ceramic scaffold as well as a preparation method and application thereof, and belongs to the technical field of medical materials. The preparation method comprises the following steps: dispersing pyrroloquinoline quinone powder in a phosphate buffer solution to obtain a pyrroloquinoline quinone solution, putting a zinc-doped calcium phosphate porous ceramic scaffold into the pyrroloquinoline quinone solution, shaking, and drying to obtain the PQQ-loaded porous ceramic scaffold. Experimental verification shows that the zinc-doped stent can remarkably promote regeneration and repair of a bone defect area, and the bone repair efficiency of the stent can be further enhanced through the synergistic effect by introducing PQQ.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to a PQQ-loaded porous ceramic scaffold and a preparation method and application thereof. Background Art

[0002] Bone defects are a common problem in orthopedic clinics, primarily caused by factors such as fracture trauma, osteomyelitis, and tumors. Currently, treatments for bone defects primarily include autologous bone transplantation, allogeneic bone transplantation, and synthetic bone graft transplantation. However, autologous and allogeneic bone transplantation face numerous limitations in clinical application, such as limited donor sites, high costs, difficulty in tissue acquisition, and potential risks of infectious diseases. Therefore, the development of safe, effective, and economical bone repair materials has become a current research priority.

[0003] Synthetic bone substitutes have attracted widespread attention due to their advantages, including lack of immune rejection, no risk of disease transmission, and excellent osteoconductivity. β-tricalcium phosphate (β-TCP) has become one of the most commonly used and effective synthetic bone graft substitutes due to its excellent biocompatibility, osteoconductivity, and chemical composition similar to natural bone. However, traditional β-TCP materials have two major limitations in clinical application: first, the high risk of infection after implantation, which affects the therapeutic effect; second, the osteogenic activity needs to be further improved, especially in the repair of complex bone defects. These issues have restricted the widespread application of β-TCP materials in clinical practice.

[0004] In addition, pyrroloquinoline quinone (PQQ), a redox-cycling planar orthoquinone, was originally identified as a coenzyme of methanol dehydrogenase. Recent studies have found that PQQ can inhibit osteoclast differentiation in vitro and prevent osteoporosis by alleviating oxidative stress and osteoclast senescence.

[0005] Based on the above research, how to load PQQ onto a bone repair scaffold to obtain a new bone repair material with excellent mechanical properties, antibacterial activity and osteogenesis ability is a technical problem that needs to be solved at present. Summary of the Invention

[0006] The purpose of the present invention is to provide a PQQ-loaded porous ceramic scaffold and its preparation method and application, so as to solve the above technical problems.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing a PQQ-loaded porous ceramic scaffold, comprising the following steps:

[0009] 1) dissolving a calcium source and a zinc source in water to obtain a calcium-zinc mixed solution;

[0010] 2) drop the phosphorus source solution into the calcium-zinc mixed solution, add ammonia water to adjust the pH of the system, and obtain a suspension;

[0011] 3) hydrothermal reaction is performed on the suspension to obtain a zinc-doped calcium phosphate slurry;

[0012] 4) the zinc-doped calcium phosphate slurry is scraped onto a printing platform by using polyvinyl alcohol as a binder, and a zinc-doped calcium phosphate porous ceramic scaffold is obtained through photocuring 3D printing;

[0013] 5) pyrroloquinoline quinone powder is dispersed in a phosphate buffer to obtain a pyrroloquinoline quinone solution, and then the zinc-doped calcium phosphate porous ceramic scaffold is placed in the pyrroloquinoline quinone solution, shaken and dried, thereby obtaining a PQQ-loaded porous ceramic scaffold.

[0014] Further, the calcium source comprises calcium nitrate and / or calcium chloride; the zinc source comprises zinc nitrate and / or zinc chloride; the molar ratio of the calcium source and the zinc source is 8-9.5:0.5-2; the molar concentration of Ca 2+ + Zn 2+ in the calcium-zinc mixed solution is 1 mol / L.

[0015] Further, the molar ratio of the phosphorus source and the zinc source is 0.5-2:5-7.

[0016] Further, the ammonia water adjusts the pH of the system to 7-10.

[0017] Further, the temperature of the hydrothermal reaction is 100-200 DEG C, and the time of the hydrothermal reaction is 5-15 h.

[0018] Further, the thickness of the scraped zinc-doped calcium phosphate slurry is 150-200 mu m.

[0019] Further, the concentration of the pyrroloquinoline quinone solution is 100-200 mu g / mL.

[0020] Further, the shaking speed is 80-200 rpm, the temperature is 30-40 DEG C, and the shaking time is 6-8 d.

[0021] The drying temperature is 30-40 DEG C, and the drying time is 1-3 d.

[0022] The application also provides a PQQ-loaded porous ceramic scaffold prepared by the above preparation method.

[0023] The application provides an application of the PQQ-loaded porous ceramic scaffold in bone defect repair.

[0024] The application has the following beneficial effects:

[0025] 1、The present application successfully loads PQQ onto 10% Zn-β-TCPS by physical adsorption method, and the SEM and FTIR results all confirm the successful loading of PQQ, and the absorption peaks near 3450 cm -1 and 1650 cm -1 in the FTIR spectrum are further verified. Drug release experiments show that 10% Zn-β-TCPS has good sustained release capacity and is an ideal drug carrier.

[0026] 2、The protein adsorption experiment results show that zinc doping affects the protein adsorption behavior of β-TCPS. The 5% Zn-β-TCPS group has the lowest BSA adsorption amount due to the increase of negative potential, while the 10% and 20% Zn-β-TCPS improve the BSA adsorption through ion re-adsorption; at the same time, with the increase of zinc content, the adsorption capacity of the material for positively charged LSZ is significantly enhanced. In vitro degradation experiment shows that zinc doping slows down the degradation rate of the scaffold, and the degradation amount (about 10 wt%) of the zinc-doped scaffold in 12 weeks is significantly lower than that (30 wt%) of the undoped group. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 SEM diagram of PQQ / 10% Zn-β-TCPS prepared in the embodiment of the present application;

[0028] Figure 2 FTIR diagram of PQQ / 10% Zn-β-TCPS prepared in the embodiment of the present application;

[0029] Figure 3 Drug release curve diagram of PQQ / 10% Zn-β-TCPS prepared in the embodiment of the present application;

[0030] Figure 4 Protein adsorption column diagram of Zn-β-TCPS and PQQ / Zn-β-TCPS prepared in the embodiment of the present application, (A) adsorption of the scaffold to BSA; (B) adsorption of the scaffold to LSZ;

[0031] Figure 5 In vitro degradation of Zn-β-TCPS and PQQ / Zn-β-TCPS prepared in the embodiment of the present application (n=3);

[0032] Figure 6 Cell proliferation diagram of Zn-β-TCPS and PQQ / Zn-β-TCPS prepared in the embodiment of the present application, (A) proliferation of the cells co-cultured with the material extract for 1, 3, and 5 days; (B) proliferation of the cells cultured on the scaffold for 3 days;

[0033] Figure 7Figures of in vitro antibacterial experiment of different zinc-doped β-TCP prepared by the application, (A) OD value of material extract and E. coli co-cultured for 24 h; (B) OD value of material extract and S. aureus co-cultured for 24 h; (C) situation of material extract respectively co-coated with E. coli and S. aureus for 24 h (n = 3);

[0034] Figure 8 Figures of Micro-CT and quantitative analysis of new bone tissue of rabbit femur at 4 weeks after operation, (A) Micro-CT scanning image; (red marked in the figure is new bone) (B) quantitative analysis of bone volume fraction; (C) quantitative analysis of trabecular number; (D) quantitative analysis of trabecular separation / spacing; ("*" indicates that there is a significant difference between other groups and the control group, "*" p < 0.05, "**" p < 0.01, "***" p < 0.001; "#" indicates that there is a significant difference between other groups and the β-TCPS group, "#" p < 0.05, "##" p < 0.01, "###" p < 0.001);

[0035] Figure 9 Figures of Micro-CT and quantitative analysis of new bone tissue of rabbit femur at 12 weeks, (A) Micro-CT scanning image (red marked in the figure is new bone); (B) quantitative analysis of bone volume fraction; (C) quantitative analysis of trabecular number; (D) quantitative analysis of trabecular separation / spacing;

[0036] Figure 10 Figures of HE staining image of rabbit femur at 4 weeks after operation (under 20 times light microscope), wherein Defect is defect area, NB is newly formed bone tissue, and M is implanted scaffold material;

[0037] Figure 11 Figures of HE staining image of rabbit femur at 12 weeks after operation. DETAILED DESCRIPTION

[0038] The application provides a preparation method of a PQQ-loaded porous ceramic scaffold, which comprises the following steps:

[0039] 1) Dissolve a calcium source and a zinc source in water to obtain a calcium-zinc mixed solution;

[0040] 2) Drop a phosphorus source solution into the calcium-zinc mixed solution, add ammonia water to adjust the pH of the system, and obtain a suspension;

[0041] 3) Perform a hydrothermal reaction on the suspension to obtain a zinc-doped calcium phosphate slurry;

[0042] 4) Take polyvinyl alcohol as a binder, scrape the zinc-doped calcium phosphate slurry onto a printing platform, and perform light-curing 3D printing to obtain a zinc-doped calcium phosphate porous ceramic scaffold.

[0043] 5) dispersing pyrroloquinoline quinone powder in phosphate buffer solution to obtain a pyrroloquinoline quinone solution, then placing the zinc-doped calcium phosphate porous ceramic scaffold into the pyrroloquinoline quinone solution, shaking and drying, to obtain a PQQ-loaded porous ceramic scaffold.

[0044] The calcium source comprises calcium nitrate and / or calcium chloride, preferably calcium nitrate; the zinc source comprises zinc nitrate and / or zinc chloride, preferably zinc nitrate; the molar ratio of the calcium source and the zinc source is 8-9.5:0.5-2, preferably 9.5:0.5, 9:1, or 8:2; the molar concentration of Ca 2+ + Zn 2+ in the calcium-zinc mixed solution is 1 mol / L.

[0045] In the present application, the molar ratio of the phosphorus source and the zinc source is 0.5-2:5-7, preferably 0.5-2:6.

[0046] In the present application, the ammonia water adjusting system adjusts the pH to 7-10, preferably pH=8.

[0047] In the present application, the temperature of the hydrothermal reaction is 100-200℃, preferably 120-180℃, further preferably 150℃; the time of the hydrothermal reaction is 5-15h, preferably 8-12h.

[0048] In the present application, the thickness of the zinc-doped calcium phosphate slurry is 150-200μm, preferably 175μm.

[0049] In the present application, the concentration of the pyrroloquinoline quinone solution is 100-200μg / mL, preferably 120-180μg / mL, further preferably 140-160μg / mL, more preferably 150μg / mL.

[0050] In the present application, the shaking speed is 80-200rpm, preferably 100rpm; the temperature is 30-40℃, preferably 37℃; the shaking time is 6-8d, preferably 7d.

[0051] The drying temperature is 30-40℃, preferably 37℃; the drying time is 1-3d, preferably 2d.

[0052] The present application also provides a PQQ-loaded porous ceramic scaffold prepared by the above preparation method.

[0053] The present application provides a PQQ-loaded porous ceramic scaffold for use in bone defect repair.

[0054] The technical solutions provided by the present application will be described in detail below in combination with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0055] Example 1

[0056] Ca(NO3)2-4H2O and Zn(NO3)2-6H2O were magnetically stirred until completely dissolved, (Ca 2+ + Zn 2+ ) concentration was 1 M, the molar ratio of Zn 2+ and Ca 2+ was 0.5:9.5. (NH4)2HPO4 was added to deionized water and magnetically stirred until completely dissolved, PO4 3- concentration was 0.6 M. 50 mL of (NH4)2HPO4 solution was added dropwise to an equal volume of Ca(NO3)2-4H2O and Zn(NO3)2-6H2O mixed solution using a pipette, (Ca 2+ + Zn 2+ ) / PO4 3- = 5 / 3. After dripping, stir for 5 min, then add 1.0 mL of ammonia water to adjust the system pH to 8, continue to stir for 25 min. The above suspension was moved into a reaction kettle, the reaction temperature was 150°C, and the reaction time was 8 h. The obtained sample was labeled as 5% Zn-β-TCP.

[0057] Example 2

[0058] The same as Example 1, except that the molar ratio of Zn 2+ and Ca 2+ was 1:9, and the sample was labeled as 10% Zn-β-TCP.

[0059] Example 3

[0060] The same as Example 1, except that the molar ratio of Zn 2+ and Ca 2+ was 2:8, and the sample was labeled as 20% Zn-β-TCP.

[0061] Example 4

[0062] The three-dimensional model was created and sliced using Materialise Magics software and 10dim slicing software, respectively. The thickness of each layer was 50 pm, and the sliced files were imported into a digital light processing (DLP) device. Polyvinyl alcohol (PVA) was used as a binder, and the prepared β-TCP, 5% Zn-β-TCP, 10% Zn-β-TCP, and 20% Zn-β-TCP slurries were poured into the blade slot of the DLP device, respectively. The blade level and height were adjusted, and then the material was laid, with a thickness of about 175 pm. After adjusting the printing parameters such as blade speed, first layer exposure time, remaining layer exposure time, and other layer exposure power, the "start" button was clicked to automatically print. After printing, the β-TCP scaffold (β-TCPS), 5% Zn-β-TCP scaffold (5% Zn-β-TCPS), 10% Zn-β-TCP scaffold (10% Zn-β-TCPS), and 20% Zn-β-TCP scaffold (20% Zn-β-TCPS) were gently scraped on the forming table, and the scaffolds were placed in an ultrasonic cleaner containing cleaning agent to wash off the residual slurry on the surface. The cleaned scaffolds were sintered after being placed at room temperature for 72 h, and the sintered scaffolds were sealed and stored for later use.

[0063] Example 5

[0064] A solution with a concentration of 150 pg / ml was prepared by adding PQQ powder to sterile phosphate buffered saline (PBS) as a solvent. The scaffolds obtained in Example 4 were placed in the above solution and shaken in a shaker (100 rpm, 37°C) for 7 days. The scaffolds were dried in an oven at 37°C for 2 days and stored in a sealed container for later use. The resulting scaffolds were labeled as PQQ / 5% Zn-β-TCP, PQQ / 10% Zn-β-TCP, and PQQ / 20% Zn-β-TCP, respectively.

[0065] Figure 1 The SEM image of PQQ / 10% Zn-β-TCP showed that PQQ was successfully loaded onto the 10% Zn-β-TCPS by physical adsorption. From the SEM image, it can be observed that PQQ molecules are uniformly distributed on the surface of the scaffold and do not change the original porous morphology of the scaffold. The high-magnification SEM image further shows that PQQ molecules form a good interfacial bond with the surface of the 10% Zn-β-TCPS, which is beneficial to the stable loading and sustained release of PQQ. It is worth noting that the surface of the scaffold still maintains appropriate roughness, and this microstructure is beneficial to cell adhesion and proliferation.

[0066] After loading PQQ onto the 3D-printed β-TCP scaffold by physical adsorption, Fourier transform infrared spectroscopy was used to characterize and analyze the material. Figure 2). The results show that no obvious PQQ characteristic peaks appear in the infrared spectrum of the PQQ-loaded β-TCP scaffolds compared to the non-PQQ-loaded β-TCP scaffolds. This phenomenon can be related to the way PQQ is loaded onto the surface of the scaffold and its content. It is worth noting that compared to 10% Zn-β-TCPS, the PQQ / Zn-β-TCPS (hereinafter PQQ / Zn-β-TCPS refers to PQQ / 10% Zn-β-TCPS) composite scaffold observed a significantly enhanced -OH and N-H stretching vibration absorption peak at about 3450 cm -1 . This can be due to the hydroxyl and amino groups in the PQQ molecule. In addition, the absorption peaks of -OH bending vibration and C=O stretching vibration were also observed to be enhanced near 1650 cm -1 . This further confirms the successful loading of PQQ. However, other characteristic peaks of PQQ (such as C=N stretching vibration at 1610 cm -1 and C=C aromatic ring vibration at 1450 cm -1 ) did not appear significantly.

[0067] To simulate the drug release performance of the scaffold in vivo, we conducted in vitro experiments. Ten PQQ / Zn-β-TCPS with a diameter of 5 mm and a thickness of 2 mm were immersed in 5 mL PBS. At predetermined time points, 1 mL of PBS solution was extracted and 1 mL of fresh PBS was added to maintain a constant medium volume. The released PQQ concentration was determined by high-performance liquid chromatography. All drug release tests were repeated 3 times under the same conditions. As shown in Figure 3 , the drug-loaded scaffold rapidly releases PQQ in the first 48 h and slows down only until 72 h. This is because PQQ is loaded onto Zn-β-TCPS by physical adsorption, and the drug is released quickly. The above results show that the scaffold is a good drug carrier with sustained-release ability.

[0068] Figure 4A is the adsorption behavior of BSA protein on Zn-β-TCPS with different zinc doping rates. The results show that the adsorption amount of BSA on β-TCPS decreases first and then increases with the increase of zinc doping amount. Specifically, the adsorption amount of 5% Zn-β-TCPS on BSA is the lowest, which may be due to the isoelectric point of BSA being 4.7, and being negatively charged in PBS solution with pH of 7.4. If electrostatic interaction dominates the adsorption process of BSA, theoretically, the increase of zinc doping amount should lead to the decrease of BSA adsorption amount. However, the adsorption of BSA is not only affected by electrostatic interaction, but also by the BSA binding sites on the surface of the material and its conformation. In addition, the scaffold material can adsorb calcium ions (positively charged) and phosphate ions (negatively charged) in the surrounding environment as binding sites. Therefore, the adsorption capacity of 5% Zn-β-TCPS on BSA is lower than that of β-TCPS, which may be due to the increase of negative charge on the surface of the material, which enhances the electrostatic repulsion between the negatively charged BSA molecules. In contrast, 10% Zn-β-TCPS and 20% Zn-β-TCPS show higher BSA adsorption capacity, and the mechanism may involve the recapture effect of free calcium ions and zinc ions in the solution by the material surface, thereby promoting the adsorption process of the protein.

[0069] Figure 4 The adsorption behavior of LSZ protein on Zn-β-TCPS with different zinc doping rates. Experimental data show that with the increase of zinc doping concentration, the adsorption capacity of β-TCPS on LSZ shows a significant enhancement trend, and the β-TCPS with 20% zinc doping shows the best adsorption performance. This result can be explained from the molecular properties of LSZ: in PBS buffer system with pH 7.4, due to the high isoelectric point (pI = 11.1) of LSZ, it carries a net positive charge, and its adsorption mechanism mainly depends on the electrostatic attraction between the material surface and the protein. Therefore, with the increase of zinc doping amount, the positive charge density on the material surface increases, further enhancing the adsorption capacity of the positively charged LSZ.

[0070] The present application selects acetic acid-sodium acetate buffer system with pH 4.5 as the degradation medium, which can effectively simulate the acidic environment mediated by osteoclast-mediated bone resorption. Figure 5 The mass loss of Zn-β-TCPS with different zinc doping ratios during the degradation process is shown. As can be clearly seen from the figure, the degradation mass loss of zinc-doped scaffolds at different time points is significantly lower than that of undoped β-TCPS. However, the degradation mass loss of Zn-β-TCPS with different zinc doping ratios is small, and the degradation amount of each group at 12 weeks is maintained at about 10wt%. In contrast, the degradation amount of undoped β-TCPS at 12 weeks is as high as 30wt%. This result shows that zinc doping significantly slows down the degradation rate of β-TCPS.

[0071] In vitro biological studies of scaffolds:

[0072] Material extract preparation: 0.5 g of sterile 5% Zn-β-TCP, 10% Zn-β-TCP, 20% Zn-β-TCP powder (autoclaved, 120°C, 2h) was added to centrifuge tubes containing 45 mL of α-MEM medium, respectively, and then placed in a shaker (100 rpm, 37°C) for 7 days. After the end of the release, the mixture was centrifuged (6000 rpm, 3 min) to collect the supernatant, which was then filtered to remove bacteria and 10% fetal bovine serum and 1% penicillin / streptomycin were added to the supernatant to form the ionized extract α-MEM medium, which was then stored in the refrigerator at 4°C until use. The PQQ / Zn-β-TCP extract was prepared by adding PQQ powder to the 10% Zn-β-TCP extract (30 μg / ml)

[0073] 0.4 g of 5% Zn-β-TCP, 10% Zn-β-TCP, 20% Zn-β-TCP powder was added to centrifuge tubes containing 40 mL of PBS, respectively, and then placed in a shaker (100 rpm, 37°C) for 7 days. After the end of the release, the mixture was centrifuged (6000 rpm, 3 min) to collect the supernatant, which was then filtered to remove bacteria and stored at room temperature until use.

[0074] Figure 6 A is the cell proliferation of the material extract and MC3T3-E1 cell culture for 1 day, 3 days, 5 days, with the increase of culture time, the activity of MC3T3-E1 cells in all experimental groups increases with time, indicating that the cells are in normal growth state. The results of 3 days show that there is no significant difference in the proliferation effect of MC3T3-E1 cells among the control group, β-TCP, 5% Zn-β-TCP and 20% Zn-β-TCP groups, indicating that these material extracts have no significant effect on cell proliferation in the short term. However, the 10% Zn-β-TCP and PQQ / Zn-β-TCP material extracts showed significant difference compared with the control group at 3 days, indicating that these two material extracts have a certain effect on cell proliferation in the early stage. The results of 5 days culture show that 5% Zn-β-TCP and 10% Zn-β-TCP material extracts show a certain effect on cell proliferation compared with the control group, and the effect of 10% Zn-β-TCP is more significant. It is worth noting that the PQQ / Zn-β-TCP group has the most obvious effect on cell proliferation, indicating that the introduction of PQQ significantly enhances the effect of the material on cell proliferation.

[0075] As Figure 6As shown in Fig. 5A and 5B, the proliferation of MC3T3-E1 cells after 3 days of direct culture with different scaffold materials showed that there was no significant difference in cell proliferation between 5% Zn-β-TCPS and β-TCPS, indicating that low concentration of zinc doping had little effect on cell proliferation. 10% Zn-β-TCPS and PQQ / Zn-β-TCPS groups showed obvious proliferation-promoting effect, indicating that appropriate amount of zinc doping (10%) and the introduction of PQQ could significantly promote the proliferation of MC3T3-E1 cells. However, unlike the results of the extract experiment, the 20% Zn-β-TCPS group showed a certain inhibitory effect on cell proliferation, indicating that high concentration of zinc doping may have a negative impact on cell growth under direct contact conditions.

[0076] In vitro antibacterial effect:

[0077] To evaluate the in vitro antibacterial performance of zinc-doped β-TCP materials, the extract of the materials was co-cultured with E. coli and S. aureus for 24 h, and analyzed by OD value determination and colony counting method. As shown in Fig. 6A and 6B, the OD values of the Zn-β-TCP group were significantly lower than those of the control group, indicating that the introduction of zinc ions significantly enhanced the antibacterial effect of β-TCP. To further verify this result, the extract was co-coated with bacterial solution and cultured for 24 h, and the results are shown in Fig. 6C. The co-coating experiment showed that the number of colonies in the Zn-β-TCP group was significantly less than that in the control group, and the number of colonies in the 20% Zn-β-TCP group was the least, which was consistent with the in vitro co-culture results, indicating that high concentration of zinc doping could significantly inhibit the growth of bacteria. Figure 7 Figure 7 C. The co-coating experiment showed that the number of colonies in the Zn-β-TCP group was significantly less than that in the control group, and the number of colonies in the 20% Zn-β-TCP group was the least, which was consistent with the in vitro co-culture results, indicating that high concentration of zinc doping could significantly inhibit the growth of bacteria.

[0078] The above studies can be seen that the extract and scaffold proliferation experiments of MC3T3-E1 cells and live and dead staining results after rBMSCs cell culture showed that all material extracts did not show cytotoxicity, and appropriate amount of zinc doping (10%) and PQQ modification could promote cell proliferation by direct contact. The results of in vitro antibacterial experiment confirmed that zinc-doped β-TCP materials had good antibacterial performance, indicating that the introduction of zinc ions could improve the antibacterial effect of β-TCP, and the antibacterial effect was enhanced with the increase of zinc doping amount.

[0079] In vivo osteogenesis-promoting effect of scaffold:

[0080] ​Male New Zealand white rabbits were selected to establish a femoral defect model: 30 healthy New Zealand white rabbits weighing about 2.5 kg (8 weeks) were randomly divided into 4 groups. After the rabbits were anesthetized by intravenous injection of pentobarbital sodium (30 mg / kg) through the ear margin, the right hind leg femur lateral ankle area was selected for surgical preparation (shaving, disinfection, and towel laying), and the rabbit was kept in a supine position. A 6 cm longitudinal incision was made in the surgical area, and the soft tissue was separated layer by layer to expose the lateral malleolus of the femur. A 2 mm diameter and 5 mm deep bone defect was made at the lateral malleolus using an electric drill, and physiological saline was dripped to dissipate the heat generated by the electric drill. β-TCPS, 10% Zn-β-TCPS, and PQQ / 10% Zn-β-TCPS were implanted into the defect, respectively, and the group without implantation of the scaffold served as the control group. After implantation, the wound was sutured and disinfected with iodophor, and 400,000 IU of penicillin was injected into the muscle of the rat daily for 3 days to prevent wound infection. The animals were sacrificed at 4 weeks and 12 weeks, respectively, and the femur was fixed in 4% paraformaldehyde solution for subsequent experiments. All animal experiments in this study strictly followed the requirements of the Medical Committee of Southwest Jiaotong University (Approval Number: SWTJU-2503-NSFC(146)).

[0081] Figure 8 The Micro-CT scan images of the scaffold implanted into the rabbit femoral condyle defect site after 4 weeks, with the red area marked as new bone tissue. The results show that the control group (without implantation of the scaffold) has very little new bone; the β-TCPS group (without zinc doping) failed to completely scan the scaffold cross-section due to the implantation angle of the scaffold, but obvious new bone formation can still be observed; a large amount of new bone can be seen at the edge of the scaffold pores in the 10% Zn-β-TCPS group and the PQQ / Zn-β-TCPS group, indicating that the scaffold has good bone guiding effect. Quantitative analysis results Figure 8 B-D) show that at 4 weeks after surgery, the BV / TV and Tb.N of the 10% Zn-β-TCPS group and the PQQ / Zn-β-TCPS group were significantly higher than those of the β-TCPS group; at the same time, the Tb.Sp of the two groups was significantly lower than that of the β-TCPS group. These data indicate that zinc-doped scaffolds (10% Zn-β-TCPS and PQQ / Zn-β-TCPS) can significantly promote new bone formation and increase the density of bone trabeculae, and have better bone repair ability.

[0082] Figure 9is the Micro-CT scan image of the scaffold implanted in the femoral condyle defect site of rabbits after 12 weeks. The red marked part is the newly formed bone. Compared with the Micro-CT scan result at 4 weeks, it can be seen that the newly formed bone tissue gradually increases with the extension of the implantation time. The undoped zinc β-TCPS appears cracks and broken, while 10% Zn-β-TCPS and PQQ / Zn-β-TCPS do not appear this situation, which also proves that the 10% Zn-doped Zn-β-TCPS has good mechanical properties. At 4 weeks of implantation, the new bone tissue mainly appears at the edge of the scaffold pores, and with the extension of the implantation time to 12 weeks, the new bone tissue gradually fills the internal pores of the scaffold. At the same time, PQQ / Zn-β-TCPS shows more new bone than 10% Zn-β-TCPS, so the introduction of PQQ can indeed further promote osteogenesis. At 4 weeks of implantation, the material basically does not degrade, and the complete material can be clearly seen. After 12 weeks of implantation, the material at the interface between the scaffold and the host bone begins to degrade, but the overall scaffold degradation is less, and still maintains the integrity of its structure. The new bone tissue of 3D reconstruction was quantitatively analyzed, which was consistent with the four-week result. The new bone amount of 10% Zn-β-TCPS and PQQ / Zn-β-TCPS groups was high and dense; compared with the 10% Zn-β-TCPS group, the BV / TV value of the PQQ / Zn-β-TCPS group at 4 weeks had no significant difference, but was higher than that of the 10% Zn-β-TCPS group at 12 weeks, indicating that the PQQ / Zn-β-TCPS group grew faster after 4 weeks, indicating that the introduction of PQQ can continuously promote the bone regeneration in the later stage. These results confirm that 10% zinc doping can enhance the mechanical properties and osteogenic activity of the scaffold, and PQQ modification can further optimize the bone repair effect.

[0083] Figure 10 is the HE staining image of the scaffold implanted in the femoral condyle defect of rabbits after 4 weeks. The defect area of the control group is mainly filled with fibrous tissue, and little new bone is formed; a small amount of new bone formation can be seen around the material in the β-TCPS group, and there is a slight inflammatory reaction; there is part of new bone tissue around the material in the 10% Zn-β-TCPS group and the PQQ / Zn-β-TCPS group, and the new bone tissue in the scaffold of each group is not connected in large pieces because the material has not been degraded, which is not conducive to the connection between the new bone tissues.

[0084] As Figure 11As shown, at 12 weeks postoperatively, the control group still had obvious incomplete repair phenomenon in the defect area, and the newly formed bone (NB) was limited; the β-TCPS group could see the formation of new bone, but the material (M) and host bone interface integration was poor; the 10% Zn-β-TCPS group showed significantly improved bone regeneration effect, and the newly formed bone not only surrounded the surface of the material, but also penetrated into the interior of the material pores, and the material-bone interface integrated well; the PQQ / Zn-β-TCPS group showed the best repair effect, and the newly formed bone tissue fully filled the defect area, and the material and the host bone formed a tight integration. It is particularly noteworthy that compared with 4 weeks, the maturity of the newly formed bone of all experimental groups at 12 weeks was significantly improved, and the bone trabecula arrangement was more ordered, and the bone tissue maturity of the PQQ / Zn-β-TCPS group was the highest, and the newly formed bone and the host bone were almost indistinguishable, forming a complete bony connection. These results further confirmed that zinc doping not only enhanced the stability of the scaffold, but also significantly promoted the bone regeneration process, and the introduction of PQQ further enhanced this promoting effect, and showed the best bone repair performance in long-term implantation.

[0085] From the above examples, the present application provides a PQQ-loaded porous ceramic scaffold and a preparation method and application thereof. The present application uses 3D printing technology to prepare β-tricalcium phosphate with different zinc doping rates synthesized by hydrothermal method into a scaffold with a regular porous structure, and loads a small molecule drug PQQ on the scaffold, and carries out material characterization, in vitro cell compatibility and antibacterial effect, in vivo bone formation effect research, and the conclusions are as follows:

[0086] (1) Regular porous β-TCP scaffolds with different zinc doping rates were prepared by using light-cured 3D printing technology, and it was found that 10% Zn-β-TCPS had the best mechanical properties (compressive strength 0.55 MPa). PQQ was successfully loaded on 10% Zn-β-TCPS by physical adsorption method, and it was confirmed that it had good drug release ability. In addition, zinc doping significantly promotes the adsorption of proteins on the scaffold and slows down the degradation rate of the scaffold, and PQQ loading reduces the amount of protein adsorption.

[0087] (2) It is confirmed by cell experiments that appropriate zinc doping (10%) and PQQ modification not only do not show cytotoxicity, but also can promote the proliferation of MC3T3-E1 cells and the osteogenic differentiation of rBMSCs, and PQQ and zinc ions have a synergistic enhancement effect. In addition, zinc doping significantly improves the antibacterial performance of β-TCP material, and the antibacterial effect is enhanced with the increase of zinc doping amount. The results show that the zinc-doped and PQQ-modified β-TCP material has good biocompatibility, osteoinductive property and antibacterial property.

[0088] (3) Through the rabbit femoral condyle defect model evaluation confirmed that 10% Zn-β-TCPS and PQQ / Zn-β-TCPS showed good biological safety in vivo application, no toxic effects on major organs were observed. Micro-CT three-dimensional reconstruction and histological staining analysis showed that compared with the blank control group and β-TCPS, the zinc-doped scaffold can significantly promote the regeneration and repair of bone defect area. Notably, PQQ / Zn-β-TCPS showed the most excellent osteogenic performance, the number and quality of new bone tissue were significantly better than that of simple zinc-doped scaffold, which confirmed that the introduction of PQQ can further enhance the bone repair efficiency of the scaffold through synergistic effect.

[0089] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for preparing a PQQ-loaded porous ceramic scaffold, characterized in that: The following steps are involved: 1) dissolving a calcium source and a zinc source in water to obtain a calcium-zinc mixed solution; 2) adding the phosphorus source solution dropwise to the calcium-zinc mixture, and adding ammonia water to adjust the pH of the system to obtain a suspension; 3) subjecting the suspension to a hydrothermal reaction to obtain a zinc-doped calcium phosphate slurry; 4) Using polyvinyl alcohol as a binder, the zinc-doped calcium phosphate slurry is scraped onto a printing platform and then subjected to light-curing 3D printing to obtain a zinc-doped calcium phosphate porous ceramic scaffold; 5) Pyrroloquinoline quinone powder is dispersed in a phosphate buffer solution to obtain a pyrroloquinoline quinone solution, and then the zinc-doped calcium phosphate porous ceramic scaffold is placed in the pyrroloquinoline quinone solution, shaken, and then dried to obtain a PQQ-loaded porous ceramic scaffold.

2. The method for preparing a PQQ-loaded porous ceramic scaffold according to claim 1, characterized in that: The calcium source comprises calcium nitrate and / or calcium chloride; the zinc source comprises zinc nitrate and / or zinc chloride; the molar ratio of the calcium source to the zinc source is 8-9.5:0.5-2; the calcium zinc mixed solution contains calcium nitrate and / or calcium ... 2+ +Zn 2+ The molar concentration is 1 mol / L.

3. The method for preparing a PQQ-loaded porous ceramic scaffold according to claim 1 or 2, characterized in that: The molar ratio of the phosphorus source to the zinc source is 0.5-2:5-7.

4. The method for preparing a PQQ-loaded porous ceramic scaffold according to claim 3, characterized in that: The ammonia water is used to adjust the pH of the system to 7-10.

5. The method for preparing a PQQ-loaded porous ceramic scaffold according to claim 1, 2 or 4, wherein: The temperature of the hydrothermal reaction is 100-200° C., and the time of the hydrothermal reaction is 5-15 hours.

6. The method for preparing a PQQ-loaded porous ceramic scaffold according to claim 5, characterized in that: The thickness of the zinc-doped calcium phosphate slurry applied by scraping is 150-200 μm.

7. The method for preparing a PQQ-loaded porous ceramic scaffold according to claim 6, characterized in that: The concentration of the pyrroloquinoline quinone solution is 100-200 μg / mL.

8. The method for preparing a PQQ-loaded porous ceramic scaffold according to claim 1, 2 or 7, wherein: The shaking speed is 80-200 rpm, the temperature is 30-40°C, and the shaking time is 6-8 days; The drying temperature is 30-40°C and the drying time is 1-3 days.

9. The PQQ-loaded porous ceramic scaffold prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the PQQ-loaded porous ceramic scaffold according to claim 9 in repairing bone defects.

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