Functionalized bone scaffold for bone defect repair and preparation method thereof

Abstract

The invention discloses a functional bone scaffold for bone defect repair as well as a preparation method and application thereof. The preparation method comprises the following steps: designing a three-dimensional structure of the scaffold according to a skull defect condition; preparing a PCL bracket by adopting a 3D printing technology; soaking the PCL stent in a mixed solution of icariin and dopamine, and enabling the mixed solution to be in contact with air; carrying out oscillation reaction on the reaction system for set time, and after the reaction is finished, taking out the stent, cleaning and drying to obtain the ICA-PPCL stent; and soaking the ICA-PPCL stent in a PBS (Phosphate Buffer Solution), soaking the ICA-PPCL stent in an exosome solution, and incubating for a set time to obtain the EXO-ICA-PPCL stent. A polyphenol structure of PDA and icariin can form a stable pi-pi accumulation effect, and the exosome is adsorbed through hydrophobic interaction, so that efficient loading and synchronous delivery of PDA and icariin are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of bone repair materials, and particularly relates to a functionalized bone scaffold for bone defect repair and a preparation method thereof. Background Art

[0002] The statements herein only provide background art related to the present invention and do not necessarily constitute prior art.

[0003] Skull defect is the local or full-thickness absence of the skull caused by pathological factors such as open craniocerebral trauma, tumor resection, surgical decompression, or infection.

[0004] Existing skull repair materials include bioactive ceramics, metals, synthetic polymers, etc. These materials have significant deficiencies in the coordinated regulation of mechanical adaptability, controllable degradation, and biological functionality.

[0005] PCL has broad application prospects in the field of biomedical engineering and can precisely construct complex three-dimensional structures through technologies such as fused deposition modeling and electrospinning, providing highly adaptable customized scaffolds for personalized skull defect repair. However, PCL-based scaffolds still face two major challenges in clinical translation: one is the biological functional defect, and its bio-inert and hydrophobic surface significantly inhibits the adhesion and differentiation of osteoblasts; the other is the imbalance between degradation and regeneration. The slow degradation of pure PCL does not match the bone regeneration cycle and may cause chronic inflammation.

[0006] Studies have shown that icariin (ICA) promotes the proliferation, differentiation, and mineralization of bone marrow mesenchymal stem cells (BMSCs) by activating classical osteogenic signaling pathways such as BMP / Smad and Wnt / β-catenin, while enhancing the expression of osteogenesis-related genes and inhibiting adipocyte differentiation, showing a strong osteogenic induction ability. These characteristics have enabled it to be widely used in the treatment of osteoporosis and have become a research hotspot in the field of bone defect repair.

[0007] Exosomes derived from mesenchymal stem cells (MSCs) can precisely regulate the proliferation of endothelial cells and the differentiation of osteoblasts and promote angiogenesis and bone regeneration due to the osteogenesis-related miRNAs and signaling proteins they carry. In addition, the function of exosomes can be further enhanced through pretreatment (such as magnetic nanoparticles, static magnetic fields, or hypoxia treatment), making them show stronger osteogenic and angiogenic abilities in bone defect repair. However, free exosomes are easily cleared rapidly in vivo and have problems such as poor targeting and low yield. Summary of the Invention

[0008] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a functionalized bone scaffold for bone defect repair, a preparation method thereof, and an application.

[0009] To achieve the above object, the present invention is realized by the following technical solutions:

[0010] In a first aspect, the present invention provides a method for preparing a functionalized bone scaffold for bone defect repair, comprising the following steps:

[0011] Design the three-dimensional structure of the scaffold according to the skull defect situation;

[0012] Prepare a PCL scaffold using 3D printing technology;

[0013] Immerse the PCL scaffold in a mixed solution of icariin and dopamine, and the mixed solution is in contact with air;

[0014] Shake the reaction system for a set time, after the reaction is completed, take out the scaffold, wash and dry it to obtain the ICA-PPCL scaffold;

[0015] After immersing the ICA-PPCL scaffold in PBS solution, immerse it in the exosome solution and incubate for a set time to obtain the EXO-ICA-PPCL scaffold.

[0016] Although both icariin and exosomes have shown significant potential in bone regeneration, the synergistic delivery of the two has not been achieved. The polyphenol structure of PDA can form a stable π-π stacking interaction with icariin and adsorb exosomes through hydrophobic interactions, thus realizing the efficient loading and synchronous delivery of the two. Combining with the PDA-modified three-dimensional porous scaffold can further optimize the delivery efficiency, simulate the mechanical microenvironment of the bone matrix, and provide an ideal scaffold for cell proliferation and differentiation. This multifunctional composite material is expected to achieve the precise coupling of vascular network construction and bone regeneration by spatiotemporally coordinating exosome release and icariin sustained release, providing a new treatment paradigm for bone defect repair.

[0017] First, a bionic porous scaffold model is constructed based on SolidWorks software, the scaffold topology structure is optimized through hydrodynamic simulation, and the high-precision forming of a polycaprolactone (PCL) scaffold is realized using digital light processing (DLP) 3D printing technology. Subsequently, the surface of the scaffold is functionalized by polydopamine (PDA) in-situ deposition technology: on the one hand, the osteogenic active molecule icariin (ICA) is loaded using the strong adhesion property of PDA to construct an ICA-PPCL sustained release system with continuous osteogenic activity; on the other hand, pro-angiogenic exosomes (EXO) are fixed on the surface of the PDA coating through hydrophobic interactions, and finally an EXO-ICA-PPCL composite scaffold is formed.

[0018] In the initial stage of implantation, the scaffold improves the local hypoxic microenvironment through the rapid release of EXO, promotes angiogenesis and recruits endogenous stem cells; in the subsequent stage, it realizes the continuous delivery of ICA by virtue of the sustained-release characteristics of PDA, and directionally induces the osteogenic differentiation of stem cells by activating the osteogenic signaling pathway. This spatio-temporal delivery mode of "vascular first - osteogenesis second" can simulate the natural bone repair cascade process, and while achieving the spatial coupling of the vascular network and the newly formed bone tissue, significantly improve the repair quality of large bone defects.

[0019] When the mixed solution contacts with air, dopamine generates polydopamine through oxidative self-polymerization reaction; the reaction system is shaken to carry out the physical self-assembly and covalent polymerization of polydopamine.

[0020] Soaking the ICA-PPCL scaffold in PBS solution can wash away impurities.

[0021] In some embodiments, in the mixed solution of icariin and dopamine, the concentration of icariin is 0.1 - 1 mg / mL; the concentration of dopamine is 1 - 5 mg / mL.

[0022] Preferably, in the mixed solution of icariin and dopamine, the concentration of icariin is 0.3 - 0.6 mg / mL; the concentration of dopamine is 1 - 3 mg / mL.

[0023] In some embodiments, in the mixed solution of icariin and dopamine, the solvent is a mixture of Tris-HCl buffer and methanol, the pH value of the Tris-HCl buffer is 8 - 9, and the volume ratio of the Tris-HCl buffer to methanol is 6 - 10:1 - 2.

[0024] Tris-HCl maintains the weak alkalinity of the reaction system to make dopamine react; methanol is used to increase the solubility of icariin.

[0025] Preferably, in the mixed solution of icariin and dopamine, the solvent is a mixture of Tris-HCl buffer and methanol, the pH value of the Tris-HCl buffer is 8.5, and the volume ratio of the Tris-HCl buffer to methanol is 7 - 9:2.

[0026] Further preferably, the volume ratio of the Tris-HCl buffer to methanol is 8:2.

[0027] In some embodiments, the temperature of the shaking reaction is 35 - 40 °C, and the time of the shaking reaction is 20 - 30 h.

[0028] Preferably, the temperature of the shaking reaction is 36 - 38 °C, and the time of the shaking reaction is 20 - 25 h.

[0029] In some embodiments, the drying is vacuum freeze-drying.

[0030] In the following embodiments, the concentration of the exosome solution is 0.1-10 μg / μL.

[0031] Preferably, the concentration of the exosome solution is 0.5-5 μg / μL.

[0032] More preferably, the concentration of the exosome solution is 0.5-3 μg / μL.

[0033] In some embodiments, the incubation temperature is 3-10 °C and the incubation time is 1-10 h.

[0034] Preferably, the incubation temperature is 3-5 °C and the incubation time is 1-5 h.

[0035] In a second aspect, the present invention provides a functionalized bone scaffold for bone defect repair, which is prepared by the preparation method.

[0036] The beneficial effects obtained by one or more of the above embodiments of the present invention are as follows:

[0037] A. Aiming at the deficiencies of existing bone scaffolds in functional modification, the present invention aims to design and optimize the topological structure of the scaffold through SolidWorks software modeling combined with hydrodynamic simulation analysis to achieve a high permeability and an appropriate wall shear stress (WSS) adaptation rate. A bone scaffold with excellent mechanical properties and biocompatibility is prepared by 3D printing using the biodegradable material polycaprolactone (PCL). The ica-ppcL scaffold modified with polydopamine is successfully prepared by the "one-pot method". The scaffold combined with hypoxic exosomes exhibits a significant angiogenesis-promoting function. The constructed EXO-ICA-PPCL functionalized scaffold can effectively promote angiogenesis and osteogenic induction, thereby improving the bone repair effect.

[0038] B. Aiming at the problem that it is difficult to balance the material degradation rate and the new bone regeneration rate of the functionalized bone scaffold, exosomes are rapidly released in the initial stage of bone repair to promote the rapid formation of the vascular network, while icariin is slowly and continuously released. Along with the gradual degradation of the PCL scaffold, the drug continuously exerts its pharmacological effect. This design not only optimizes the degradation behavior of the scaffold but also ensures the quality of new bone regeneration, ultimately achieving high-quality mature bone regeneration and solving the problem of poor osteogenic quality in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0040] Figure 1 It is a three-dimensional model diagram of the bone scaffold according to an embodiment of the present invention. Among them, a is arranged in a circular pattern; b is arranged in a linear pattern;

[0041] Figure 2 It is a wall shear stress distribution diagram according to an embodiment of the present invention. Among them, a is homogeneous scaffold A; b is homogeneous scaffold B;

[0042] Figure 3 It is a flow velocity distribution diagram of the scaffold according to an embodiment of the present invention. Among them, a is homogeneous scaffold A; b is homogeneous scaffold B;

[0043] Figure 4 It is a PPCL loading map of ICA under different solvent ratios according to an embodiment of the present invention. Among them, a is the loading efficiency; b is the loading amount;

[0044] Figure 5 It is an X-ray photoelectron spectroscopy diagram of three groups of scaffolds of PCL, PPCL and ICA-PPCL in an embodiment of the present invention;

[0045] Figure 6 It is a Fourier transform infrared spectrometer of three groups of scaffolds of PCL, PPCL and ICA-PPCL in an embodiment of the present invention;

[0046] Figure 7 Among them, a is the ultraviolet absorption spectrum scanning curve diagram of ICA, and b is the standard curve made from a;

[0047] Figure 8 It is an in vitro release kinetics diagram of ICA in an embodiment of the present invention. Among them, a is the standard curve, and b is the cumulative release curve;

[0048] Figure 9 It is a diagram after exosomes treat HUVECs cells in an embodiment of the present invention. Among them, a is the Western blot result of VEGF protein expression, and b is the quantitative analysis result;

[0049] Figure 10 It is a scanning electron microscope diagram of PCL, PPCL, and EXO-ICA-PPCL in an embodiment of the present invention;

[0050] Figure 11 It is a fluorescence diagram of the surface of the scaffold after different treatments in an embodiment of the present invention;

[0051] Figure 12 It is a diagram of the effect of low-concentration ICA on the activity of two types of cells in an embodiment of the present invention. Among them, a is BMSCs, and b is HUVECs;

[0052] Figure 13It is the blood safety investigation diagram of PPCL, ICA-PPCL, EXO-PPCL and EXO-ICA-PPCL in the embodiments of the present invention. Among them, a is the appearance diagram of hemolysis phenomenon: (+) positive control, (-) negative control, and b is the quantitative result diagram of hemolysis experiment.

[0053] Figure 14 Among them, (a) is the Micro-CT image after implanting different composite scaffolds at the skull defect of rats for 10 weeks; (b) is the analysis result of BV / TV; (c) is the analysis result of BS / BV; (d) is the analysis result of Tb.Th; (e) is the analysis result of BMD.

[0054] Figure 15 Among them, a is the comparison diagram of stress-strain curves of PCL, PPCL, and ICA-PPCL; b is the comparison diagram of compressive strengths of PCL, PPCL, and ICA-PPCL. Detailed implementation manners

[0055] It should be noted that the following detailed description is illustrative and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0056] The present invention will be further described below in conjunction with embodiments.

[0057] Embodiment 1

[0058] 1) Establishment of the stent structure model

[0059] Two three-dimensional models of bone scaffolds with different micropore arrangements were designed using SolidWorks software. The stent structure was constructed based on a cylindrical unit cell geometric model, and the overall geometric size was a cylinder with a diameter of 5 mm and a height of 2 mm. The interior of the stent was designed as a regular porous structure, with the pore size ranging from 450 to 500 μm and the radial spacing between pores being 200 μm.

[0060] The main difference between the two homogeneous scaffolds lies in the spatial arrangement of the micropores: both adopt a circumferential arrangement in the axial direction, while in the radial direction, homogeneous scaffold a is designed with a circumferential arrangement, and homogeneous scaffold b is designed with a linear arrangement. The two configurations are as shown in Figure 1 a and b in.

[0061] 2) Preparation and characterization of ICA-PPCL scaffolds

[0062] When preparing a PCL scaffold using Digital Light Processing (DLP) 3D printing technology, first optimize the three-dimensional structure of the scaffold through SolidWorks software, export the final model as an STL format file, and import it into the DLP printing system. By precisely regulating the key process parameters, the preparation of a PCL scaffold with micron-level accuracy can be achieved. Wash the PCL scaffold with dimensions of 5×5×2 mm thoroughly with deionized water and dry it for later use.

[0063] Prepare a solvent by mixing Tris-HCl buffer with a concentration of 10 mmol / L and a pH of 8.5 and methanol in a volume ratio of 8:2. Then, add dopamine and icariin in sequence, so that the concentration of dopamine is 2 mg / mL and the concentration of icariin is 0.5 mg / mL to obtain a mixed solution of icariin and dopamine.

[0064] Immerse the PCL scaffold in the mixed solution of icariin and dopamine, allow the mixed solution to contact the air, and shake the reaction system at 37°C for 24 h.

[0065] After the shaking ends, take out the scaffold, wash it three times with deionized water to remove the unreacted substances, and obtain the ICA-PPCL scaffold after vacuum freeze-drying for 12 h.

[0066] PPCL refers to a polydopamine-coated scaffold (PDA / PCL), and the preparation method is the same as above, with the concentration of icariin being 0.

[0067] Successfully prepared an icariin-functionalized scaffold modified with polydopamine (ICA-PPCL) by the "one-pot" reaction.

[0068] The characterization results show that a uniform black coating layer is formed on the surface of the scaffold. The N1s peak (399.9 eV) in the XPS spectrum confirms the successful coating of polydopamine; FTIR spectral analysis indicates the existence of intermolecular hydrogen bond interactions between icariin and the polydopamine coating, confirming the successful loading of ICA. The mechanical property test shows that the compressive strength of the scaffold meets the mechanical requirements for skull defect repair, laying an important foundation for subsequent in vivo implantation research.

[0069] The prepared PPCL scaffold shows excellent drug-loading performance for ICA, with the highest loading rate reaching 85.55±1.25% and the loading amount being 87.57±1.67 mg / g, meeting the effective drug dosage. Based on the interfacial adhesion characteristics of the PDA coating, study the in vitro release kinetic behavior of ICA and achieve a sustained release effect for up to one month. This slow-release characteristic is expected to maintain a long-term effective therapeutic drug concentration locally in bone defects and provide a favorable microenvironment for promoting bone tissue regeneration.

[0070] 3) Preparation and Characterization of EXO-ICA-PPCL Scaffolds

[0071] After soaking the ICA-PPCL scaffolds in PBS solution, place them in a 96-well plate and add 150 μL of exosome solution with a concentration of 1 μg / μL to each well. Incubate the samples in a shaker at 4°C for 4 h to prepare the EXO-ICA-PPCL scaffolds.

[0072] The extracted hypoxic exosomes exhibited significant pro-angiogenic functions. WB confirmed that hypoxic exosomes could significantly upregulate the expression level of VEGF in human umbilical vein endothelial cells (HUVECs). Based on this, the microstructure surface morphology of the EXO-ICA-PPCL functionalized scaffolds after adsorbing exosomes was observed by SEM, and at the same time, fluorescence labeling was used to confirm the uniform distribution of exosomes on the scaffold surface.

[0073] Through hydrodynamic simulation, it was determined that the topological structure of homogeneous scaffold a had the highest permeability of 9.08411E-09 (m 2 ), as shown in Figure 2 The wall shear stress accounted for 94.89%, and the best flow velocity distribution was as shown in Figure 3 .

[0074] Permeability Calculation

[0075] According to Darcy's law, substitute the measured pressure gradient value into the porous medium permeability calculation formula (1-1) to obtain the permeability of A and B type homogeneous scaffolds, and compare it with the permeability of natural cancellous bone.

[0076]

[0077] In the formula, k is the permeability; Q is the volume flow rate inside the scaffold; v is the viscosity of the fluid; L is the total height of the scaffold; Δp is the pressure gradient; A is the area of the upper and lower cross-sections of the scaffold.

[0078] Table 1

[0079]

[0080] The proportion of wall shear stress (5×10 -5 -2.5×10 -2 Pa) was calculated to be 94.89%.

[0081] As shown in Figure 4As shown in the figure, by optimizing the reaction solvent system, the effects of different volume ratios of Tris-HCl buffer to methanol (4:6, 5:5, 6:4, 7:3, 8:2, 9:1) on the loading effect of icariin (ICA) were investigated. The experimental results showed that when the volume ratio of Tris-HCl to methanol was 8:2, the PPCL scaffold had the best loading effect on ICA, with a loading rate of 85.55 ± 1.25% and a loading amount of 87.57 ± 1.67 mg / g.

[0082] As Figure 5 The physicochemical properties of the three groups of scaffolds, PCL, PPCL, and ICA-PPCL, were characterized by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). The results showed that the polydopamine coating was successfully deposited on the surface of the scaffolds, and its characteristic N element was significantly detected. As Figure 6 Infrared spectroscopy analysis showed that obvious changes occurred in the characteristic peaks of the ICA-PPCL sample: the stretching vibration characteristic peaks of N-H and O-H shifted from 3200 cm -1 to 3333 cm -1 , accompanied by the phenomenon of peak broadening. These changes in spectral characteristics confirmed the formation of a stable hydrogen bond interaction between icariin and polydopamine, providing strong evidence for their effective combination.

[0083] The stress-strain curves obtained from the compression test showed that the three groups of specimens, PCL, PPCL, and ICA-PPCL, all reached the peak yield strength at a compression strain of 8.73% ± 0.2%, and then showed typical plastic deformation characteristics, manifested as a stress plateau accompanied by densification deformation, as Figure 15 shown in a of Figure 15 . The quantitative results showed that the average compressive strength of each group of scaffolds was within the range of the mechanical parameters of human cancellous bone (compressive strength 0.6 - 15 MPa), as

[0084] shown in b of Figure 7 The experiment used PDA as the solvent system to systematically investigate the absorption characteristics of different concentrations of ICA solutions (10 - 50 μg / mL) at a wavelength of 360 nm. As 2 The results showed that with the gradient increase of the ICA concentration, its absorbance showed a linear increase (R

[0085] = 0.9999), confirming that this wavelength can be used for the quantitative detection of ICA. Figure 8The results showed that during the 28-day release period, ICA presented a continuous and stable release characteristic, and the final cumulative release rate reached 81.09±0.76%.

[0086] This release characteristic was attributed to the effective binding of ICA to the scaffold material through intermolecular forces such as π-π stacking and hydrogen bonding, thereby achieving precise regulation of the drug release rate. This release characteristic can ensure that after the scaffold is implanted into the rat cranial defect model, it can maintain a long-term effective local drug concentration, provide a continuous pharmacological effect for bone tissue repair, avoid the phenomenon of sudden drug release, and significantly improve the therapeutic effect and safety.

[0087] Such as Figure 9 The significant pro-angiogenic function of hypoxic exosomes was verified by Western blot (WB) experiments. The expression of the internal reference protein β-tubulin in the normoxic and hypoxic groups was almost the same, and the color of the VEGF protein band in the hypoxic group was significantly darker than that in the normoxic group. Normalized with β-tubulin as the internal reference protein, hypoxic exosomes activated the angiogenic function of endothelial cells by significantly increasing the VEGF protein expression by 3.7-fold, providing a molecular mechanism basis for its application in vascularized bone regeneration. Subsequently, the ICA-PPCL scaffold was co-incubated with exosomes to successfully prepare the functionalized EXO-ICA-PPCL scaffold.

[0088] Figure 10 The microscopic surface morphology of the scaffold after adsorbing exosomes was observed by scanning electron microscopy (SEM). The results showed that exosomes were evenly distributed on the surface of the scaffold, and the typical "cup-shaped" or refractive morphological characteristics of exosomes could be seen.

[0089] Meanwhile, Figure 11 The uniform distribution of exosomes on the surface of the scaffold was further confirmed by fluorescence labeling technology.

[0090] EXO-ICA-PPCL Fluorescence Labeling

[0091] Exosomes were labeled with the green fluorescent dye DiO. The DiO fluorescent dye was prepared as a 10 mg / mL DMSO stock solution and then diluted to a 100 μM dye working solution with PBS. 100 μg of exosome solution was added to the dye working solution, vortexed and mixed for 1 min, and incubated at 37°C in the dark for 30 min. After incubation, the free dye was removed, and the labeled exosomes were co-incubated with the ICA-PPCL scaffold for 4 h. The distribution of exosomes on the scaffold was observed using a fluorescence inverted microscope.

[0092] The experimental results showed that the EXO-ICA-PPCL scaffold not only retained the excellent properties of the ICA-PPCL scaffold, but also further enhanced its angiogenic ability through the loading of exosomes, providing dual-functional support for bone defect repair and significantly improving the biological performance and therapeutic effect of the scaffold.

[0093] Biocompatibility and in vivo bone repair effect of the EXO-ICA-PPCL scaffold

[0094] Figure 12 The effect of ICA on the viability of bone marrow mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) was investigated by CCK8. BMSCs and HUVECs were seeded into 96-well plates at a density of 5.0×10 3 cells / well, respectively. After the cells adhered overnight, different concentrations of ICA extracts were added and co-cultured with the cells for 24 and 48 hours, with blank medium as the control. Subsequently, CCK-8 reagent was added, and the cells were incubated at 37°C in the dark for 1 hour, and the OD value was measured at a wavelength of 450 nm to detect the relative cell viability. Each group was subjected to five independent experiments and statistical analysis.

[0095] The results showed that the low concentration of ICA released from the ICA@PDA scaffold after 48-hour immersion did not affect cell viability, demonstrating excellent cytocompatibility and providing a safe and effective delivery system for cranial defect repair.

[0096] As Figure 13 The blood safety of four groups of scaffold materials was investigated by hemolysis experiments. The results fully demonstrated that the scaffold to be implanted had excellent blood compatibility and could be safely used for the repair and treatment of cranial defects. Hemolysis experiments were used to evaluate the blood safety of the delivery system. The PPCL, ICA-PPCL, EXO-PPCL, and EXO-ICA-PPCL scaffolds were mixed with 2% rabbit red blood cell suspension, and a negative control group (normal saline, 0% hemolysis) and a positive control group (water, 100% hemolysis) were set. After incubation at 37°C for 2 hours, centrifugation (1000 g, 5 minutes) was performed, and the hemolysis phenomenon of each group was observed and photographed. Subsequently, the supernatant was taken and its OD value was measured at a wavelength of 540 nm for further analysis of the hemolysis situation.

[0097] A rat critical-sized cranial defect model was constructed and randomly divided into five groups: the PPCL scaffold group, the ICA-PPCL scaffold group, the EXO-PPCL scaffold group, the EXO-ICA-PPCL scaffold group, and the blank control group. After ten weeks, the rats were sacrificed, the skulls were taken for micro-CT scanning, and the defect sites were analyzed to obtain bone morphometric parameters. As Figure 14Ten weeks after stent implantation, the difference in new bone mass among groups was further significant. Only a small amount of new bone tissue grew outside the defect in the control group, indicating its limited bone healing ability, which was in line with the characteristics of the critical-sized bone defect model. The combined treatment group EXO-ICA-PPCL achieved the best bone regeneration effect (BV / TV 60.3±4.0%), and the defect area was quickly completely covered by new bone. Its BS / BV (4.9±1.2%) value was significantly reduced, reflecting a denser new bone structure; the Tb.Th (0.61±0.02mm) value increased, indicating enhanced three-dimensional bone trabecular structure; and the BMD (976.9±18.9mg / cm 3 ) value was the highest, confirming the highest degree of bone mineralization. These data demonstrated from multiple dimensions that EXO-ICA-PPCL achieved a regenerative effect closest to natural bone tissue by coordinately regulating the bone structure remodeling and mineralization processes.

[0098] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a functionalized bone scaffold for bone defect repair, characterized in that: It includes the following steps: Design the three-dimensional structure of the scaffold according to the skull defect situation; Prepare the PCL scaffold by 3D printing technology; Immerse the PCL scaffold in a mixed solution of icariin and dopamine, and the mixed solution is in contact with air; Oscillate the reaction system for a set time. After the reaction is completed, take out the scaffold, wash and dry it to obtain the ICA-PPCL scaffold; After soaking the ICA-PPCL scaffold in PBS solution, immerse it in the exosome solution and incubate for a set time to obtain the EXO-ICA-PPCL scaffold.

2. The preparation method of the functionalized bone scaffold for bone defect repair according to claim 1, wherein: In the mixed solution of icariin and dopamine, the concentration of icariin is 0.1-1 mg / mL; the concentration of dopamine is 1-5 mg / mL.

3. The preparation method of the functionalized bone scaffold for bone defect repair according to claim 1, wherein: In the mixed solution of icariin and dopamine, the concentration of icariin is 0.3-0.6 mg / mL; the concentration of dopamine is 1-3 mg / mL.

4. The preparation method of the functionalized bone scaffold for bone defect repair according to claim 1, wherein: In the mixed solution of icariin and dopamine, the solvent is a mixture of Tris-HCl buffer and methanol. The pH value of the Tris-HCl buffer is 8-9, and the volume ratio of the Tris-HCl buffer to methanol is 6-10:1-2; Preferably, in the mixed solution of icariin and dopamine, the solvent is a mixture of Tris-HCl buffer and methanol. The pH value of the Tris-HCl buffer is 8.5, and the volume ratio of the Tris-HCl buffer to methanol is 7-9:2; Preferably, the volume ratio of the Tris-HCl buffer to methanol is 8:

2.

5. The preparation method of the functionalized bone scaffold for bone defect repair according to claim 1, characterized in that: The temperature of the oscillating reaction is 35-40 °C, and the time of the oscillating reaction is 20-30 h; Preferably, the temperature of the oscillating reaction is 36-38 °C, and the time of the oscillating reaction is 20-25 h.

6. The preparation method of the functionalized bone scaffold for bone defect repair according to claim 1, characterized in that: The concentration of the exosome solution is 0.1-10 μg / μL.

7. The preparation method of the functionalized bone scaffold for bone defect repair according to claim 1, characterized in that: The concentration of the exosome solution is 0.5-5 μg / μL; Preferably, the concentration of the exosome solution is 0.5-3 μg / μL.

8. The preparation method of the functionalized bone scaffold for bone defect repair according to claim 1, wherein: The temperature of the incubation is 3-10 °C, and the time of the incubation is 1-10 h.

9. The preparation method of the functionalized bone scaffold for bone defect repair according to claim 1, characterized in that: The temperature of the incubation is 3-5 °C, and the time of the incubation is 1-5 h.

10. A functionalized bone scaffold for bone defect repair, characterized in that: Prepared by the preparation method according to any one of claims 1-9.

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