Directional multifunctional bone repair scaffold prepared by pulling-folding method and preparation method and application thereof

Abstract

The invention relates to the technical field of bone repair scaffolds, in particular to a directional multifunctional bone repair scaffold prepared through a pulling-folding method and a preparation method and application thereof. Comprising a polycaprolactone-collagen mixture and a strontium phosphate mineralization layer, wherein the polycaprolactone-collagen mixture is internally provided with a honeycomb micropore structure, and the strontium phosphate mineralization layer covers the surface of the polycaprolactone-collagen mixture. The honeycomb forming process of cooking sugar, namely repeated stretching and folding of a semi-solid mixture, is simulated, and structural orientation and porosity can be induced without a solvent or special equipment. In-situ mineralization is carried out by using strontium apatite, so that the biological activity is further enhanced, and the dual effects of Sr < 2 + > in angiogenesis and osteogenesis are utilized. The system characterization proves the mineralization uniformity and interface stability of SrAP. The in-vitro research shows that the bone cell migration is increased by 1.37 times, and the expression of VEGFA / RUNX2 is up-regulated by 1.15 times / 121.26 times. A rat skull defect model shows that the bone regeneration rate is 50.85% after 6 weeks, and micro-CT and histological examination shows that mature vascularized bones are formed.

Description

Technical Field

[0001] The present invention relates to the technical field of bone repair scaffolds, and in particular to a directional multifunctional bone repair scaffold prepared by a pulling-folding method, a preparation method and an application thereof. Background Art

[0002] Accidental trauma and periosteal diseases can cause bone defects. Traditional treatments focus on autologous and allogeneic bone grafts to repair the defects; however, these approaches have limitations, such as infection risk, scarcity of available grafts, and potential immune rejection. Tissue repair scaffolds have been widely adopted as a highly promising strategy to address bone defects that cannot be effectively restored through the natural healing process.

[0003] The main function of the scaffold is to balance temporary mechanical support with efficient material transport, promote biological delivery and drive tissue regeneration. As research deepens, scientists have discovered that bone tissue is composed of highly ordered collagen fibers, which helps guide cell differentiation, migration and proliferation. Extensive studies in recent years have shown that directional bone scaffolds not only promote the growth of new bone tissue, but also stimulate cells to produce extracellular matrix with ordered structural organization. For example, a variety of directional degradable polylactic acid scaffolds were prepared by electrospinning technology, and the centrally oriented scaffolds significantly enhanced bone regeneration and early vascularization in vivo. In addition, the application of directional scaffolds significantly increased the expression of key tissue development proteins compared to non-directional scaffolds. Therefore, the application of directional engineered scaffolds provides important advantages and is crucial for advancing bone regeneration strategies.

[0004] The development of tissue engineering scaffolds requires careful consideration of key parameters, including mechanical strength, biocompatibility, controlled degradation kinetics, and tailored tissue-specific properties to ensure compatibility with host tissue requirements. Significant progress has been made in the past few decades in the development of advanced manufacturing methods and related technologies, driving significant innovations in the design and production of high-performance, directed-engineering scaffolds. Recent advances in directed-engineering scaffold fabrication have focused on four key approaches: particle leaching, electrospinning, freeze-drying, and 3D printing. Particle leaching offers a straightforward method for preparing scaffolds with tunable pore structures by controlling the size and morphology of pore formers. However, this method typically relies on organic solvents and suffers from limited pore interconnectivity. Furthermore, electrospinning can produce scaffolds with superior pore orientation and interconnectivity, effectively replicating the structure of the native extracellular matrix. However, electrospun scaffolds often exhibit limited mechanical properties, manifested by low strength and stiffness. Freeze-drying forms porous scaffolds through solvent sublimation, providing highly interconnected pores. However, precise control of pore size and morphology remains challenging. 3D printing has gained significant attention in tissue engineering for creating scaffolds with precise microarchitecture. However, it faces the problems of limited mechanical strength and high production cost. Therefore, developing a simple, environmentally friendly and economical method to produce high mechanical properties, load-bearing oriented porous scaffolds remains a major challenge in tissue engineering. Summary of the Invention

[0005] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a directional multifunctional bone repair scaffold prepared by a pulling-folding method, and a preparation method and application thereof, so as to solve the problems involved in the background technology.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a directional multifunctional bone repair scaffold prepared by a pulling-folding method, comprising a polycaprolactone-collagen mixture with a honeycomb microporous structure inside, and a strontium phosphate mineralized layer covering the surface of the polycaprolactone-collagen mixture.

[0007] Further,

[0008] The honeycomb microporous structure is formed by repeatedly stretching, folding and re-stretching in a cyclic manner, and the pore size of the microporous structure is 80-120 μm;

[0009] The strontium phosphate mineralized layer is granular, and the particle diameter is less than 5 μm.

[0010] Furthermore, the preparation steps include:

[0011] (1) Preparation of PCLSC scaffolds

[0012] Prepare PCL particles and collagen, mix them evenly in a beaker, add an appropriate amount of anhydrous ethanol to cover the PCL and collagen, use a 70°C oil bath to melt the PCL particles and collagen mixture, and frequently stir the contents of the beaker during the heating process to accelerate dissolution. When the PCL solution is completely melted, use a clean wooden stick to repeatedly stretch it hundreds of times in a 50°C, 75% alcohol solution. Place the stretched PCL in deionized water to quickly solidify it. Cut the solidified solid into blocks or slices to form a PCLSC scaffold;

[0013] (2) Weigh (NH4)2HPO4 powder and SrCl2·6H2O powder and dissolve them in deionized water respectively. Under vigorous stirring, add the (NH4)2HPO4 solution dropwise to the strontium chloride solution. Then add ammonia water to adjust the pH to 10 and continue stirring for 1 hour. Place the PCLSC scaffold in the reaction solution for mineralization. After 7 days, thoroughly wash it with anhydrous ethanol and deionized water and freeze-dry it to obtain the SrSC scaffold.

[0014] Furthermore, the mass ratio of the PCL particles to collagen is 5.7:1.

[0015] Furthermore, the mass ratio of (NH4)2HPO4 powder to SrCl2·6H2O powder is 1:3.3; the concentration of (NH4)2HPO4 powder in deionized water is 0.158 g / ml, and the concentration of SrCl2·6H2O powder is 0.52 g / ml.

[0016] Furthermore, the specific steps of (1) include:

[0017] Prepare 8.5g of PCL particles with a molecular weight of 30,000 and 1.5g of type I collagen, mix them evenly in a beaker, add an appropriate amount of anhydrous ethanol to cover the PCL, use a 70°C oil bath to melt the PCL mixture, and frequently stir the contents of the beaker during the heating process to accelerate dissolution. Prepare a solution in an incubator with a 75% ethanol solution at 50°C to prevent rapid cooling. When the PCL solution is completely melted, use a clean wooden stick to repeatedly stretch it hundreds of times in the 75% alcohol solution to simulate the stretching process of sugar. The stretched PCL is placed in deionized water for rapid solidification. The solidified solid is cut into blocks or slices to form PCLSC.

[0018] Furthermore, the specific steps of (2) include:

[0019] 0.79 g of (NH4)2HPO4 powder and 2.6 g of SrCl2·6H2O powder were weighed and dissolved in 5 mL of deionized water respectively. Under vigorous stirring, the (NH4)2HPO4 solution was added dropwise to the strontium chloride solution, and then ammonia was added to adjust the pH to 10. The stirring was continued for 1 hour. The PCLSC scaffold was placed in the reaction system for mineralization. After 7 days, it was thoroughly washed with anhydrous ethanol and deionized water and freeze-dried to obtain the SrSC scaffold.

[0020] A preparation method is used to prepare an SrSC scaffold for use as a directional multifunctional bone repair scaffold.

[0021] Furthermore, the SrSC scaffold promotes cell migration, angiogenesis, and coordinates bone regeneration.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] Inspired by the traditional Chinese "stove sugar" process, the present invention has successfully developed a biomimetic strategy to construct a degradable bone repair scaffold with a hierarchical structure. By innovatively combining polycaprolactone with type I collagen and introducing strontium through biomineralization, a SrSC scaffold with uniaxially arranged microchannels was successfully prepared. Systematic in vitro and in vivo experiments have shown that the material exhibits significant advantages in bone tissue regeneration, angiogenesis and directional cell migration, providing a new solution to the problems of complex preparation process, insufficient bioactivity and low osteogenesis efficiency of traditional bone regeneration materials. Bone-vascular synergistic repair is achieved through structural bionics, element doping and interface modification, showing broad application prospects in the field of bone defect treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A is the SEM image of the stent formed when the stretching time is 100, 200 and 300 respectively according to the embodiment of the present invention.

[0025] Figure 1 B is the SEM image of the SrSC mineralization structure of the embodiment of the present invention.

[0026] Figure 1 C is a statistical diagram of the porosity of PCLSC-1, PCLSC-2 and PCLSC-3 according to the present invention.

[0027] Figure 1 D is a statistical diagram of the porosity of PCLSC, CaSC, and SrSC according to the embodiment of the present invention. Figure 1 E is the water contact angle diagram of PCLSC and SrSC in the embodiment of the present invention.

[0028] Figure 1F is a statistical graph of the in vitro degradation rates of PCLSC, CaSC and SrSC according to an embodiment of the present invention.

[0029] Figure 1 G is the FT-IR spectra of PCLSC, CaSC and SrSC according to the embodiment of the present invention.

[0030] Figure 1 H is the PCLSC, CaSC and SrSC of the present invention at 2000 cm -1 Up to 400cm -1 FT-

[0031] IR spectrum.

[0032] Figure 1 I is the element distribution diagram of SrSC according to the embodiment of the present invention.

[0033] Figure 1 J is the stress-strain curve when the directions of the stent channel are horizontal and vertical respectively.

[0034] Figure 1 K is the compression modulus when the directions of the stent channel are horizontal and vertical respectively.

[0035] Figure 2 A is a statistical graph of MC3T3-E1 cell viability cultured with extracts of different concentrations according to the examples of the present invention.

[0036] Figure 2 B is a live / dead cell staining image of MC3T3-E1 cells co-cultured with different groups in the example of the present invention.

[0037] Figure 2 C is an F-actin staining image of MC3T3-E1 cells co-cultured with different groups in the example of the present invention.

[0038] Figure 2 D is the image of the Transwell migration experiment of MC3T3-E1 cells in different groups in the examples of the present invention.

[0039] Figure 2 E is an image of the MC3T3-E1 cell scratch healing experiment in the PCLSC, CaSC and SrSC groups according to an embodiment of the present invention.

[0040] Figure 2 F is the statistical analysis of the Transwell migration experiment in the embodiment of the present invention.

[0041] Figure 2 G is the statistical analysis of the scratch healing experiment in the embodiment of the present invention.

[0042] Figure 2H is a SEM image of a scaffold with cells attached to it according to an embodiment of the present invention. Figure 2 I is a cell survival diagram after co-culture of cells and scaffolds according to an embodiment of the present invention.

[0043] Figure 3 A is a statistical graph of HUVEC cell viability cultured with extracts of different concentrations according to the present invention.

[0044] Figure 3 B is a live / dead cell staining image of HUVEC cells co-cultured in different groups according to an example of the present invention.

[0045] Figure 3 C is the transwell migration experiment image of HUVEC cells in the PCLSC, CaSC and SrSC groups in the example of the present invention.

[0046] Figure 3 D is an image of the scratch healing experiment of MC3T3-E1 cells in the PCLSC, CaSC and SrSC groups in the example of the present invention.

[0047] Figure 3 E is the statistical analysis of the Transwell migration experiment in the embodiment of the present invention.

[0048] Figure 3 F is the statistical analysis of the scratch healing experiment in the embodiment of the present invention.

[0049] Figure 3 G is a schematic diagram of angiogenesis experiments of PCLSC, CaSC and SrSC groups in an example of the present invention.

[0050] Figure 3 H is a statistical graph of the total tube length in the angiogenesis experiment of the embodiment of the present invention.

[0051] Figure 3 I is a statistical diagram of the number of master nodes in an embodiment of the present invention.

[0052] Figure 3 J is a statistical diagram of the number of nodes in the angiogenesis experiment of the embodiment of the present invention.

[0053] Figure 3 K is a statistical graph of the relative expression levels of the angiogenesis-related gene VEGFA in an embodiment of the present invention.

[0054] Figure 4 A is a schematic diagram of ALP staining of PCLSC, CaSC and SrSC groups in an example of the present invention.

[0055] Figure 4 B is a semi-quantitative analysis of ALP staining in an example of the present invention.

[0056] Figure 4 C is the immunofluorescence staining image of Bglap protein in the PCLSC, CaSC and SrSC groups according to the embodiment of the present invention.

[0057] Figure 4 D is the Runx2 protein immunofluorescence staining image of the PCLSC, CaSC and SrSC groups in the example of the present invention.

[0058] Figure 4 E is a semi-quantitative analysis of Bglap protein in an example of the present invention.

[0059] Figure 4 F is a semi-quantitative analysis of Runx2 protein in an example of the present invention.

[0060] Figure 4 G is a statistical graph of the relative expression levels of the ALP gene in an embodiment of the present invention.

[0061] Figure 4 H is a statistical diagram of the relative expression level of the Col1a1 gene in an example of the present invention.

[0062] Figure 4 I is a statistical diagram of the relative expression levels of the Runx2 gene in an embodiment of the present invention.

[0063] Figure 5 A is a schematic diagram of the SD rat skull defect experiment in an embodiment of the present invention.

[0064] Figure 5 B is the construction of the skull defect model in an embodiment of the present invention.

[0065] Figure 5 C is a three-dimensional reconstructed image of the skull of a SD rat at 4 weeks of age according to an embodiment of the present invention.

[0066] Figure 5 D is a three-dimensional reconstructed image of the skull of a 6-week-old SD rat in the example of the present invention.

[0067] Figure 5 E is the quantitative result of BV / TV obtained by micro-CT at 4 weeks in Example of the present invention.

[0068] Figure 5 F is the quantitative result of BV / TV obtained by micro-CT at 6 weeks in Example of the present invention.

[0069] Figure 5 G is the quantitative result of Tb.Th obtained by micro-CT at 6 weeks in the example of the present invention.

[0070] Figure 5 H is H&E staining of the example of the present invention at 6 weeks.

[0071] Figure 5I is Masson's trichrome staining at 6 weeks of the present invention.

[0072] Figure 5 J is the immunohistochemical staining of cyanate and OPN at the sixth week of the present invention.

[0073] Figure 5 K is the immunohistochemical staining of α-SMA and CD31 at the sixth week of the present invention.

[0074] Figure 6 SEM images of CaSC at different magnifications according to the embodiments of the present invention.

[0075] Figure 7 is the water contact angle of CaSC in the embodiment of the present invention.

[0076] Figure 8 This is an EDS image of CaSC according to an embodiment of the present invention.

[0077] Figure 9 Live / dead staining images of MC3T3-E1 cells treated with CaSC and SrSC extracts according to an embodiment of the present invention.

[0078] Figure 10 These are F-actin staining images of MC3T3-E1 cells treated with CaSC and SrSC extracts according to an embodiment of the present invention.

[0079] Figure 11 A is the CCK-8 test after the influence of SrSC on L929 cells in the embodiment of the present invention.

[0080] Figure 11 B is the live / dead cell staining of L929 cells after the effect of SrSC in the example of the present invention.

[0081] Figure 11 C is a Transwell migration experiment image of L929 cells after the influence of SrSC in an example of the present invention.

[0082] Figure 11 D is an image of a scratch healing experiment after the effect of SrSC on L929 cells in an example of the present invention.

[0083] Figure 11 E is the statistical analysis of the Transwell migration experiment after the influence of SrSC on L929 cells in the example of the present invention.

[0084] Figure 11 F is the statistical analysis of the scratch healing experiment after the influence of SrSC on L929 cells in the example of the present invention.

[0085] Figure 12Live / dead staining images of HUVEC cells treated with CaSC and SrSC extracts according to an embodiment of the present invention.

[0086] Figure 13 A is the expression levels of ALP, Col1a1 and Runx2 genes in MC3T3-E1 cells when the MC3T3-E1:HUVEC cell number ratio is 2:1 according to an example of the present invention.

[0087] Figure 13 B shows the expression levels of ALP, Col1a1 and Runx2 genes in MC3T3-E1 cells when the MC3T3-E1:HUVEC cell number ratio is 1:2 according to an example of the present invention.

[0088] Figure 14 This is the construction of the SD rat skull defect model in the examples of the present invention.

[0089] Figure 15 A is an H&E staining image of a SD rat skull pathological section stained 4 weeks after the embodiment of the present invention.

[0090] Figure 15 B is a Masson trichrome staining image of the SD rat skull pathological section stained 4 weeks after the embodiment of the present invention.

[0091] Figure 16 A is the quantitative CD31 fluorescence staining of the embodiment of the present invention.

[0092] Figure 16 B is the quantitative α-SMA fluorescence staining of an example of the present invention. DETAILED DESCRIPTION

[0093] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0094] All data in this application are expressed as mean ± standard error and analyzed using independent sample t-test and one-way analysis of variance. For CCK-8 assay, two-way analysis of variance was used. Statistical significance was defined as a P value < 0.05. Specifically, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns: no significant difference.

[0095] Example:

[0096] A directional multifunctional bone repair scaffold fabricated via a stretch-folding method comprises a polycaprolactone-collagen mixture with a honeycomb microporous structure, covered by a strontium phosphate mineralized layer. The honeycomb microporous structure is formed through repeated stretching, folding, and re-stretching, with a pore size of 80-120 μm. The strontium phosphate mineralized layer is granular, with a particle diameter of less than 5 μm.

[0097] A method for preparing a directional multifunctional bone repair scaffold prepared by a pulling-folding method comprises:

[0098] (1) Preparation of PCLSC scaffolds

[0099] Prepare 8.5g of PCL particles with a molecular weight of 30,000 and 1.5g of type I collagen, mix them evenly in a beaker, and add an appropriate amount of anhydrous ethanol to cover the PCL. Use a 70°C oil bath to melt the PCL mixture, and stir the contents of the beaker frequently during heating to accelerate dissolution. Prepare a solution in an incubator with a 75% ethanol solution at 50°C to prevent rapid cooling. When the PCL solution is completely melted, use a clean wooden stick to repeatedly stretch hundreds of times in a 75% alcohol solution to simulate the stretching process of sugar. The stretched PCL is placed in deionized water and quickly solidified. The solidified solid is cut into blocks or slices to form a PCL-collagen scaffold (PCLSC).

[0100] (2) In situ mineralization of PCLSC

[0101] Weigh 0.79 g of (NH₄)₂HPO₄ powder and 2.6 g of SrCl₂·6H₂O powder and dissolve each in 5 mL of deionized water. Under vigorous stirring, the (NH₄)₂HPO₄ solution was added dropwise to the strontium chloride solution. Ammonia was then added to adjust the pH to 10, and stirring was continued for 1 hour. The PCLSC scaffold was placed in the reaction system for mineralization. After 7 days, the scaffold was thoroughly washed with anhydrous ethanol and deionized water and freeze-dried to obtain the SrSC scaffold.

[0102] Preparation of comparative CaSC scaffolds:

[0103] Weigh 0.79 g of (NH4)2HPO4 powder and dissolve it in 5 mL of deionized water. Separately, dissolve 1.1 g of CaCl2 solid in 5 mL of deionized water. At room temperature, slowly add the (NH4)2HPO4 solution dropwise to the calcium chloride solution while rapidly stirring with a magnetic stirrer. The (NH4)2HPO4 solution should be added within 40 minutes. After adding the (NH4)2HPO4 solution, adjust the pH of the reaction system to 10 by adding ammonia water and continue stirring vigorously for 1 hour. Place the PCLSC scaffold in the stirred reaction system for mineralization deposition. After 7 days of deposition, remove the scaffold and wash it multiple times with anhydrous ethanol and deionized water to remove any undeposited solids. It is then frozen in a -20°C freezer and transferred to a freeze dryer for freeze-drying to obtain the CaSC scaffold.

[0104] Preparation of comparative HAP:

[0105] Dissolve 1.2M (NH4)2HPO4 and 2M CaCl2 in 10mL of deionized water, respectively. Stir rapidly at 90°C, then add the (NH4)2HPO4 solution dropwise to the calcium chloride solution. The addition should be complete within 40 minutes. Aqueous ammonia is added to the reaction system to adjust the pH to 10, and stirring is continued at 90°C for 1 hour. The reaction product is then incubated at 90°C for 48 hours. The precipitated product is washed with anhydrous ethanol and deionized water multiple times to obtain hydroxyapatite (HAP).

[0106] Preparation of comparative sample SrAP:

[0107] Dissolve 1.2M (NH₄)₂HPO₄ and 2M SrCl₂·6H₂O in 10mL of deionized water. Add the (NH₄)₂HPO₄ solution dropwise to the strontium chloride solution at 90°C. Adjust the pH of the reaction system to 10 with aqueous ammonia and continue stirring for 1 hour. After 48 hours, wash the product with deionized water and anhydrous ethanol to obtain strontium apatite (SrAP).

[0108] 1. Structural features:

[0109] Experimental methods

[0110] (1) Characteristic detection of HAP and SrAP

[0111] The dried HAP and SrAP powders were placed on the sample stage of a Fourier transform infrared spectrometer to detect the infrared absorption spectra of the samples.

[0112] (2) Characterization of PCLSCs, CaSCs, and SrSCs

[0113] PCLSCs, CaSCs, and SrSCs were immersed in 75% ethanol, sterilized with ultraviolet light for 48 h, and then washed twice with phosphate-buffered saline (PBS).

[0114] PCLSC, CaSC, and SrSC scaffolds were adhered to conductive adhesive and then gold-sprayed for 240 seconds. The gold-sprayed samples were placed on the stage of a scanning electron microscope to observe the porous structure of both the unmineralized and mineralized scaffolds. Mineralized CaSC and SrSC scaffolds were fixed with conductive adhesive and gold-sprayed. Field-emission scanning electron microscopy was used to observe the mineralization and elemental distribution near the scaffold's pore structure.

[0115] Select smooth PCLSC, CaSC and SrSC scaffolds and measure their static water contact angles using an optical contact angle meter. Fill the calorimetric cup with absolute ethanol and measure its mass w1 using an electronic balance. s Place the stent in the calorimetric cup, expel all bubbles from the stent voids, and then fill it with absolute ethanol. The measured mass at this point is w2. Remove the sample filled with absolute ethanol and measure the total mass of the calorimetric cup and the remaining absolute ethanol as w3. Let the density of ethanol at the test temperature be ρ. The volume of the stent pore wall is Volume of the inner hole of the stent The porosity is calculated as follows:

[0116]

[0117] In vitro degradation experiments were conducted using a lysozyme solution that simulated body fluids. The samples were completely immersed in a 0.1 mg / mL lysozyme solution, and the solution was replaced once a week. The degradation temperature was 37°C.

[0118] The experimental procedure is as follows: First, the PCLSC, CaSC and SrSC scaffolds were processed into standard cubes (5mm long × 5mm wide × 5mm high). A universal testing machine equipped with a 500N compression fixture was used, the loading rate was 1mm / min, and zero calibration was performed. Each sample was placed vertically in the center of the fixture platform, and then the compression program was started until the 50% strain termination point was reached. The stress-strain curve was recorded in real time, and the slope of the linear elastic region was selected to calculate the compression modulus. Each group of scaffolds was tested in the transverse and longitudinal channel directions, and each measurement was repeated three times. The final data are expressed as the mean ± standard error and are obtained after normality verification.

[0119] Experimental results:

[0120] Figure 1Figure A shows the morphological and structural characteristics of PCLSC scaffolds subjected to different stretching and folding cycles (labeled as PCLSC-1, PCLSC-2, and PCLSC-3, respectively). Scanning electron microscopy (SEM) analysis revealed that increasing the number of stretching and folding cycles significantly improved the pore structure, including pore size uniformity and interconnectivity.

[0121] Porosity analysis showed that the porosity of the scaffold gradually increased with the increase of the number of stretching and folding cycles ( Figure 1 B). The pore size of PCLSC-3 is mainly distributed in the optimal range of 80-120 μm, a critical size that is beneficial for accelerated osteogenesis in bone regeneration applications. After 7 days of in vitro mineralization, SrSC and CaSC scaffold derivatives were successfully prepared from PCLSC precursors ( Figure 1 C, Figure 6 ). SrSC scaffolds show a consistent strontium phosphate mineralization layer along the pore wall, such as Figure 1 As shown in Figure C, the particle diameter is less than 5 μm and the distribution is uniform. In contrast, the CaSC scaffold exhibits an uneven calcium phosphate deposition pattern with irregular particle morphology, as shown in Figure 5. Figure 6 Quantitative analysis showed that the porosity of the scaffold decreased by 12.40% after mineralization. Figure 1 D. An ideal osteoconductive scaffold needs to meet dual structural criteria: (1) a macroporous network (>100 μm) to facilitate cell migration; and (2) sufficient porosity (>30%) to maintain nutrient supply. The PCLSC matrix exhibited an interconnected porosity of over 69.5%—exceeding the 60% threshold required for osteoblast infiltration. Although mineralization reduced the void volume, both CaSCs and SrSCs maintained a functional porosity above 50%, which enabled remodeling of the extracellular matrix during bone formation ( Figure 1 D). In addition, the inherent hydrophobicity of PCL limits its cell adhesion ability, as evidenced by a water contact angle of 82.7° ( Figure 1 E). After the surface was treated with phosphate coating, the hydrophilicity was significantly enhanced, and the contact angles of CaSC and SrSC were reduced to 50.2° and 53.9°, respectively ( Figure 7 This transition from a hydrophobic to a hydrophilic state improves fibroblast adhesion. As a degradable polymer, PCL exhibits gradual degradation in vitro. Figure 1 Figure 5 shows that PCLSC, CaSC, and SrSC scaffolds maintain stable degradation over time. This slow degradation provides ample time for new cell growth, supporting tissue regeneration. To characterize the mineralized components on the SrSC scaffold, we performed FT-IR spectroscopy and elemental mapping analysis. The characteristic PO43- absorption band of SrAP was observed in the SrSC scaffold at 1017 cm -1 ( Figure 1GH), confirming the successful incorporation of phosphate. Elemental mapping showed the uniform distribution of P, O, N, and Sr throughout the scaffold structure ( Figure 1 I). These results confirmed that SrAP mineralized particles were successfully deposited on the polymer matrix, and calcium phosphate mineralization was also observed on CaSC ( Figure 8 ). In addition, the engineered channel-aligned scaffolds exhibited significant structural anisotropy, a key property that mimics natural bone structure. To systematically evaluate their mechanical properties, we tested their strength through uniaxial compression tests (transverse / longitudinal axis). The stress-strain curves showed that the mineralized SrSC scaffolds maintained excellent compressive strength under biaxial loading, demonstrating good structural stability ( Figure 1 J). Notably, the compressive modulus of SrSC (25.35 MPa in transverse direction and 37.10 MPa in longitudinal direction) was not statistically significantly different from that of the control and CaSC groups, indicating that strontium doping did not alter the intrinsic mechanical properties of the scaffolds ( Figure 1 K). Comprehensive physicochemical characterization confirmed that SrSC meets the requirements of an ideal bone regeneration material in terms of mechanical properties, structural integrity, and compositional controllability.

[0122] 2. In vitro cell experiments on scaffolds

[0123] Experimental methods:

[0124] Preparation of scaffold extract: Extraction was performed using complete culture medium according to the method in GB_T 16886.12-2017. Sterile scaffolds were placed in complete culture medium and extracted at a concentration of 0.1 g / mL at 37°C for 72 hours. The culture medium was then filtered through a sterile 0.22 μm pore size filter to obtain the usable scaffold extract.

[0125] Preparation of osteogenesis medium: Add 10mM sodium β-glycerophosphate, 50μg / mL vitamin C and 10mM dexamethasone to complete culture medium. Set up four concentration gradients of scaffold extract: 0.1, 0.05, 0.025 and 0.0125g / mL. Use these extract concentrations to detect the cytotoxicity of MC3T3-E1, HUVEC and L929 cells. Cells were cultured in extracts of different concentrations, and the extracts were removed at predetermined times. 10% CCK-8 working solution was added, and the cells were cultured in a cell culture incubator for 1-4 hours. The absorbance at 450nm was detected using an enzyme detector. The cell viability after co-culture with the extract was then calculated. The formula for calculating cell viability is as follows:

[0126]

[0127] In the formula, OD s Indicates the absorbance of the experimental well (cultured with the extract), ODc represents the absorbance of the control well (cultured with normal culture medium), while OD b = represents the absorbance of blank wells (incubated with standard culture medium but without cells). MC3T3-E1, HUVEC, and L929 cells were cultured with CaSC and SrSC scaffold extracts, respectively, and stained at predetermined time points. A working dye solution was prepared using the Calcein-AM / PI double staining kit according to the ratio. The working solution was added to the cell wells, incubated at 37°C for 15 minutes, and then washed with PBS. Finally, the cells were observed and photographed under a confocal microscope.

[0128] MC3T3-E1 cells were co-cultured with the scaffold extract at a concentration of 0.0125 g / mL. Changes in cell morphology were observed on the 3rd and 7th days of culture. The cells were fixed with 4% paraformaldehyde and treated with 0.05% Triton X-100. Actin-Tracker Red-555 antibody (1:200) diluted in 5% bovine serum albumin (BSA) was added and incubated in the dark for 30 minutes. The cells were washed with PBS containing 0.05% Triton X-100. Then, the slides were stained with DAPI Fluoromount-G TM Seal with anti-fade mounting medium. Finally, observe the cells using a confocal microscope.

[0129] The cells were seeded in six-well plates and cultured until confluence. Subsequently, lines were drawn on the cell surface and the cells were washed with PBS. The cells continued to be cultured in 0.0125 g / mL extract (serum-free). At the predetermined time points, the differences in cell migration in the blank areas of the lines at the same position were observed to verify the effect of the scaffold extract on cell migration. The closed area (%) of the experimental group = blank area / blank area of ​​the control group × 100%. 2×10^4 cells were seeded in the Transwell chamber, and 500 μL of 0.0125 g / mL scaffold extract was added to the lower chamber. The cells were cultured normally, and the Transwell chamber was removed at the predetermined time points and fixed with 4% paraformaldehyde. The cells were stained with 0.1% crystal violet solution, and the cells in the upper chamber were wiped off. The cells that migrated to the lower chamber were observed under an optical microscope, and the number of migrated cells was analyzed using ImageJ software.

[0130] Select a sterile scaffold of appropriate size and place it in a 24-well plate. Add 1 mL of cell suspension to the scaffold to verify its cell adhesion and changes in cell morphology. Add the suspension slowly multiple times to ensure that as many cells as possible remain on the scaffold. Then place it in a cell culture incubator for 24 hours. Subsequently, live / dead cell staining was performed, and the bioactivity of the cells on the scaffold was observed using a confocal microscope. The cell-laden scaffold was fixed with 2.5% glutaraldehyde. It was sequentially immersed in 30% ethanol for 90 minutes, 50% ethanol for 60 minutes, 70% ethanol for 30 minutes, 90% ethanol for 5 minutes, and 100% ethanol for 30 minutes for gradual dehydration. Then freeze-dried. Gold was sprayed on the dried scaffold under vacuum conditions and observed and photographed using a scanning electron microscope.

[0131] Matrigel was added to a 48-well plate and placed in a cell culture incubator until solidified. After counting the HUVEC cells, 2 × 10⁴ cells were added to each well. After culturing in a scaffold extract solution at a concentration of 0.0125 g / mL for 6 hours, HUVEC angiogenesis was observed and photographed under a microscope. Images were analyzed using ImageJ software.

[0132] MC3T3-E1 cells were cultured in a 24-well plate containing 0.0125 g / mL scaffold extract solution. After a certain period of time, alkaline phosphatase (ALP) staining was performed at predetermined time points to directly evaluate the osteogenic effect of the scaffold. After culturing for a period of time, the cells were washed with PBS and fixed with 4% paraformaldehyde. The BCIP solution and NBT solution were diluted into the ALP staining buffer to form the working solution. The working solution was added to the fixed cells for staining. After staining, the cells were washed with deionized water and observed under a microscope. The ALP staining area was semi-quantitatively analyzed using ImageJ software.

[0133] MC3T3-E1 cells were cultured in a scaffold extract at a concentration of 0.0125 g / mL. After a certain period of culture, the cells were fixed with 4% paraformaldehyde. The cells were first treated with 0.5% Triton X-100 and then blocked with 3% BSA for 1 hour. Diluted primary antibodies were added and incubated at room temperature for 3 hours. The cells were washed with PBST (0.05% Tween-20, 99.95% PBS). The cells were incubated with Alexa Fluor 594-labeled goat anti-rabbit IgG (H+L) antibody for 1 hour in the dark. The cells were washed again with PBST. Finally, DAPI Fluoromount-G was used. TM Cells were blocked with anti-fluorescence quenching mounting medium and observed under a confocal microscope. Quantitative analysis was performed using ImageJ software.

[0134] MC3T3-E1 cells and HUVEC cells were co-cultured with the scaffold extract at a concentration of 0.0125 g / mL. Cells were harvested at the designated time points. After washing with PBS, the cells were transferred to a centrifuge tube and lysed with the appropriate amount of RNA-easy. Two-fifths volume of RNase-free ddH2O was added, mixed, and allowed to stand for 5 minutes. The supernatant was centrifuged at 12,000 × g for 15 minutes, and the supernatant was retained. Two-fifths volume of isopropanol was added, mixed, and allowed to stand for 15 minutes. The supernatant was centrifuged again at 12,000 × g for 10 minutes to produce a white precipitate. The precipitate was washed three times with 75% ethanol and dried. The appropriate amount of RNase-free ddH2O was added for thorough dissolution, and RNA concentration was determined using a Nanodrop micro-spectrometer. Reverse transcription was performed using 5 μg of RNA, incubated at 25°C for 10 minutes, 45°C for 15 minutes, and finally at 85°C for 5 seconds. The reverse transcription product was stored at -20°C. Using 10 ng of cDNA as template, add 10 μL of a 2x concentrated universal SYBR Green Fast qPCR mix, 0.4 μL of a 10 μM forward primer, and 0.4 μL of a 10 μM reverse primer (see Table 1). Add ddH₂O to a total volume of 20 μL to prepare the qPCR reaction system. A qPCR reaction protocol was established. β-actin was used as a housekeeping gene, and the relative expression level of the target gene was determined using the 2-ΔΔCt method.

[0135] Table 1. Primers used in qPT-PCR experiments.

[0136]

[0137] Experimental results:

[0138] (1) SrSC scaffolds promote cell migration

[0139] Mouse calvarial osteoblasts (MC3T3-E1) were cultured using scaffold extracts at different concentrations (0.1, 0.05, 0.025, and 0.0125 mg / mL) to verify the biocompatibility of the scaffold. CCK-8 assay results showed that the extracts at concentrations of 0.025 and 0.0125 mg / mL had no cytotoxicity to MC3T3-E1 cells. Figure 2 A). Live / dead staining of cells cultured with 0.025 mg / mL extract revealed almost no dead cells ( Figure 2 B and Figure 9 ), confirming that this concentration is non-toxic to cells. MC3T3-E1 cells, as an osteoblast precursor cell line, will form osteoblasts similar to fibroblasts after induction and differentiation. After three days of co-culture, it was clearly observed that the cells in the CaSC and SrSC groups had significantly formed a fibroblast-like and oriented morphology ( Figure 2 C and Figure 10 This demonstrates that CaSCs and SrSCs have the ability to induce osteoblastic precursor cell differentiation. Sr has a favorable effect on cell migration. To verify the ability of the mineralized scaffold to stimulate MC3T3-E1 cell migration, a Transwell migration assay and a cell scratch healing assay were performed. Figure 2 D clearly shows the difference in the number of migrated cells. Figure 2 The statistical data in F demonstrated that SrSCs significantly promoted the migration of MC3T3-E1 cells compared with the control group and CaSC group. The scratch healing assay also confirmed that SrSCs significantly promoted the migration of MC3T3-E1 cells ( Figure 2 E and 2G). After co-culture, scanning electron microscopy images showed that the cells were well attached to the scaffolds of SrSC and CaSC ( Figure 2 H) To verify the unique directional porous structure of the scaffold, we co-cultured the scaffold with cells and observed the cell survival within the pores. Figure 2 I shows that MC3T3-E1 cells can grow along the pore walls of the scaffold. The number of cells in the pores of the PCLSC scaffold is the least, followed by the CaSC scaffold.

[0140] To evaluate biocompatibility, L929 fibroblasts were co-cultured with CaSC and SrSC extracts, and CCK-8 assay was performed ( Figure 11 A). The results showed that both scaffolds had good cytocompatibility. In addition, live / dead staining showed that no cytotoxic effect was detected and the surviving cells maintained normal morphology ( Figure 11 B). Notably, both material variants exhibited bioactive effects on cellular function, as evidenced by enhanced cell migration in the SrSC and CaSC groups compared to the control group ( Figure 11 Importantly, SrSCs outperformed CaSCs in terms of osteoprogenitor cell adhesion and directional guidance, indicating their greater suitability as osteogenic implant materials.

[0141] (2) SrSC scaffold promotes angiogenesis

[0142] Effective bone defect repair requires robust vascularization to ensure nutrient supply and removal of metabolic waste. To evaluate this critical aspect, we first evaluated the biocompatibility of the scaffold using umbilical vein endothelial cells (HUVECs) cultured with different concentrations (0.1, 0.05, 0.025, and 0.0125 mg / mL) of CaSC / SrSC extract. While CaSCs remained cytocompatible at 0.1 mg / mL, SrSCs exhibited optimal biocompatibility at 0.05 mg / mL ( Figure 3 A), Live / dead staining confirmed that there were few dead cells even at the lowest concentration ( Figure 3B and Figure 12 Given the key role of endothelial cells in osteogenesis, we next investigated the directional migration of cells into the bone defect area. Transwell migration assays and cell scratch healing assays showed that the migration effect of the SrSC scaffold was more significant than that of the control group, with both the number of migrated cells and the closure area being higher than those of the CaSC scaffold ( Figure 3 CF). In addition, the superiority of SrSC in angiogenesis was further confirmed by the tube formation test. At a concentration of 0.0125 mg / mL, the vascular network formation time induced by SrSC extract was 1.35 times longer than that of CaSC, with 1.76 times more main connection points and 1.69 times more connection points ( Figure 3 Mechanistically, the expression level of angiogenesis-related genes (VEGFA) in HUVEC cells in the SrSC group was significantly higher than that in the CaSC group, demonstrating that SrSC can effectively stimulate endothelial cells to express VEGFA and activate angiogenesis, thereby providing a favorable microenvironment for the growth of bone cells.

[0143] (3) SrSC scaffold promotes bone regeneration in vitro

[0144] The osteogenic differentiation potential of SrSCs was systematically verified by in vitro evaluation. Preliminary alkaline phosphatase (ALP) staining showed that at a concentration of 0.0125 mg / mL, SrSCs had a better potential to promote the differentiation of MC3T3-E1 pre-osteoblasts than CaSCs, with ALP activity 1.51 times higher than that of CaSCs on day 7 ( Figure 4 A, B). To further verify the osteogenic potential of SrSCs, we used immunofluorescence to detect the osteogenesis-related proteins Bglap and Runx2. Supplementary immunofluorescence analysis showed that the osteogenic markers Bglap and Runx2 were significantly upregulated in SrSC-treated cells compared with CaSCs ( Figure 4 CF). In addition, molecular profiling confirmed this trend, with the expression levels of ALP, Runx2, and Col1a1 genes in the SrSC group significantly higher than those in the CaSC group, which also indicated that SrSC had a more pronounced osteogenic potential ( Figure 4 To recreate the in vivo osteogenic microenvironment, we established a MC3T3-E1:HUVEC co-culture model (ratios of 2:1 and 1:2). Under these physiologically relevant conditions, SrSCs maintained their osteoinductive advantage, inducing 1.29- to 1.89-fold higher expression of osteogenic genes compared to CaSCs ( Figure 13 This consistent enhancement in both mono- and co-culture systems strongly suggests that SrSCs have an intrinsic capacity to orchestrate bone regeneration through multicellular regulatory mechanisms, providing strong preclinical evidence for their translational potential.

[0145] 3. In vivo stent experiments

[0146] Test method:

[0147] All animal experiments were performed in accordance with the procedures approved by the Experimental Animal Welfare and Ethics Committee of Qingdao University (Laboratory Approval No. 20241122sd4420250120176176). Twenty 8-week-old female Sprague-Dawley (SD) rats were randomly divided into four groups: blank control group, PCLSC group, CaSC group, and SrSC group. After anesthesia, the skull was exposed by hair removal and medical iodine disinfection, and two critical full-thickness bone defects with a diameter of 6 mm were created using a dental drill.

[0148] Continuously flush with saline to cool. The scaffolds of the three experimental groups were cut longitudinally into 6 mm thick slices, weighed and sterilized. The blank control group was not implanted with any material, while the PCLSC, CaSC and SrSC groups were implanted with sterile prepared scaffolds. The incision was closed with sutures and disinfected with iodophor. Postoperatively, each animal received 40,000 units of penicillin per day for 3 days. At 4 and 6 weeks after surgery, the rats were killed by cervical dislocation, and their skulls were removed and fixed in 4% paraformaldehyde.

[0149] Each specimen was imaged using a Quantum GX2 high-resolution micro-CT scanner set at 90 kV scanning voltage and 88 μA current. Subsequently, three-dimensional reconstruction of the specimen images was performed to evaluate the repair efficacy of rat calvarial defects. Bone volume fraction (BV / TV) and trabecular thickness (Tb / Th) were quantitatively analyzed (n = 4).

[0150] Each sample was decalcified using a commercially available ethylenediaminetetraacetic acid (EDTA) solution, which was changed every 48 hours for four weeks. After decalcification, the samples were rinsed overnight in running water. Subsequently, the samples were dehydrated through a gradient of alcohol, paraffin-infiltrated, and embedded in 4-μm sections. Finally, the samples were stained with hematoxylin and eosin (H&E), Masson's trichrome, immunohistochemistry (IHC), and immunofluorescence.

[0151] Experimental results:

[0152] Based on the positive results of the in vitro study, a rat full-thickness skull defect model (6 mm in diameter) was established to verify the osteogenic effect of SrSCs in vivo. Micro-CT analysis was used to systematically evaluate the osteogenic changes of the scaffold-implanted groups (SrSCs, CaSCs, and PCLSCs) and the blank control group ( Figure 5 A, B and Figure 14). 3D reconstruction images obtained by Micro-CT showed that at 4 weeks, the SrSC-implanted group had significantly more skull bone formation than the other groups, with the bone volume fraction (BV / TV) reaching 19.1%, while the control group had only 3.74%. At this time, neither PCLSC (10.48%) nor CaSC (14.18%) showed significant osteogenic activity ( Figure 5 C, E). By week 6, the SrSC-treated defect area had almost completely formed bone bridges, while CaSCs showed slow, measurable regeneration of bone. Notably, SrSCs maintained their architectural superiority, with trabecular bone thickness (Tb.Th) of 0.60 mm at week 6 compared to 0.38 mm for CaSCs ( Figure 5 D, F, G). In contrast, little new bone formation was observed in the PCLSC-implanted area, with almost no new bone growth observed.

[0153] To evaluate the microstructural characteristics of the newly formed bone tissue, hematoxylin-eosin (H&E) staining and Masson's trichrome staining were systematically performed. Four weeks after surgery, H&E and Masson's trichrome staining images showed that new bone was formed in the defect area of ​​the SrSC group, indicating that this group has unique osteogenic capacity, whereas no detectable mineralization was observed in the control group, PCLSC group, or CaSC group. Figure 15 At 6 weeks after implantation, H&E staining images showed gradual changes: the defects in the control group were completely occupied by fibrous tissue (marked as "FT"), while the PCLSC group showed partial fibrous infiltration. New bone appeared in the CaSC group (marked as "NB"), while the SrSC group showed significantly expanded mineralized bone deposition ( Figure 5 H). Masson trichrome staining confirmed these findings, showing SrSC-induced collagen maturation and vascular infiltration (Figure I).

[0154] Osteocalcin (OCN) and osteopontin (OPN) were selected as specific osteogenic markers and systematic immunohistochemical analysis was performed on skull tissue sections from the four groups. Although the SrSC group showed extensive new bone formation, these areas showed limited positive staining, which was attributed to the steric exclusion effect of rapid matrix mineralization, which hindered the penetration of dye ( Figure 5 Undifferentiated mesenchymal cells surrounding the scaffold exhibited distinct staining patterns. This spatial variation in staining patterns suggests that the microenvironment in which SrSCs were implanted not only effectively promoted the differentiation of undifferentiated mesenchymal cells into the osteoblast lineage, but also that the rapid deposition of mineralized components within the new bone matrix may have sterically hindered dye penetration. Taken together, these results demonstrate the superior osteoinductive capacity of the microenvironment created by SrSCs.

[0155] In vivo experiments showed that the fluorescence intensity of angiogenesis markers α-SMA and CD31 was the highest in the SrSC group by immunofluorescence staining, while that in other groups was relatively low ( Figure 5 K, Figure 16 Consistent with the in vitro results, in vivo experiments further confirmed SrSC's effective cell-guiding effect and its promotion of cranial bone regeneration. This not only demonstrates the beneficial effects of strontium on bone regeneration but also provides new insights into the development of directional porous scaffolds.

[0156] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A directional multifunctional bone repair scaffold prepared by a pulling-folding method, characterized in that: The invention comprises a polycaprolactone-collagen mixture with a honeycomb microporous structure inside and a strontium phosphate mineralized layer covering the surface of the polycaprolactone-collagen mixture.

2. The directional multifunctional bone repair scaffold prepared by the pulling-folding method according to claim 1, characterized in that: The honeycomb microporous structure is formed by repeatedly stretching, folding and re-stretching in a cyclic manner, and the pore size of the microporous structure is 80-120 μm; The strontium phosphate mineralized layer is granular, and the particle diameter is less than 5 μm.

3. The method for preparing a directional multifunctional bone repair scaffold prepared by a pulling-folding method according to claim 1, characterized in that: The preparation steps include: (1) Preparation of PCLSC scaffolds Prepare PCL particles and collagen, mix them evenly in a beaker, add an appropriate amount of anhydrous ethanol to cover the PCL and collagen, use a 70°C oil bath to melt the PCL particles and collagen mixture, and frequently stir the contents of the beaker during the heating process to accelerate dissolution. When the PCL solution is completely melted, use a clean wooden stick to repeatedly stretch it hundreds of times in a 50°C, 75% alcohol solution. Place the stretched PCL in deionized water to quickly solidify it. Cut the solidified solid into blocks or slices to form a PCLSC scaffold; (2) In situ mineralization of PCLSC (NH4)2HPO4 powder and SrCl2·6H2O powder were weighed and dissolved in deionized water respectively. Under vigorous stirring, the (NH4)2HPO4 solution was added dropwise to the strontium chloride solution, and then ammonia was added to adjust the pH to 10. The stirring was continued for 1 hour. The PCLSC scaffold was placed in the reaction solution for mineralization. After 7 days, it was thoroughly washed with anhydrous ethanol and deionized water and freeze-dried to obtain the SrSC scaffold.

4. The method for preparing a directional multifunctional bone repair scaffold prepared by a pulling-folding method according to claim 1, characterized in that: The mass ratio of the PCL particles to collagen is 5.7:

1.

5. The method for preparing a directional multifunctional bone repair scaffold prepared by a pulling-folding method according to claim 1, characterized in that: The mass ratio of (NH4)2HPO4 powder and SrCl2·6H2O powder is 1:3.3; the concentration of (NH4)2HPO4 powder in deionized water is 0.158 g / ml, and the concentration of SrCl2·6H2O powder is 0.52 g / ml.

6. The method for preparing a directional multifunctional bone repair scaffold prepared by a pulling-folding method according to claim 1, characterized in that: The specific steps of (1) include: Prepare 8.5g of PCL particles with a molecular weight of 30,000 and 1.5g of type I collagen, mix them evenly in a beaker, add an appropriate amount of anhydrous ethanol to cover the PCL, use a 70°C oil bath to melt the PCL mixture, and frequently stir the contents of the beaker during the heating process to accelerate dissolution. Prepare a solution in an incubator with a 75% ethanol solution at 50°C to prevent rapid cooling. When the PCL solution is completely melted, use a clean wooden stick to repeatedly stretch it hundreds of times in the 75% alcohol solution to simulate the stretching process of sugar. The stretched PCL is placed in deionized water for rapid solidification. The solidified solid is cut into blocks or slices to form PCLSC.

7. The method for preparing a directional multifunctional bone repair scaffold prepared by a pulling-folding method according to claim 1, characterized in that: The specific steps of (2) include: 0.79 g of (NH4)2HPO4 powder and 2.6 g of SrCl2·6H2O powder were weighed and dissolved in 5 mL of deionized water respectively. Under vigorous stirring, the (NH4)2HPO4 solution was added dropwise to the strontium chloride solution, and then ammonia was added to adjust the pH to 10. The stirring was continued for 1 hour. The PCLSC scaffold was placed in the reaction system for mineralization. After 7 days, it was thoroughly washed with anhydrous ethanol and deionized water and freeze-dried to obtain the SrSC scaffold.

8. Use of the SrSC scaffold prepared by the preparation method according to any one of claims 2 to 7 as a directional multifunctional bone repair scaffold.

9. The use according to claim 8, characterized in that: The SrSC scaffold promotes cell migration, angiogenesis, and coordinates bone regeneration.

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