A directional multifunctional bone repair scaffold prepared by a pull-folding method and a preparation method and application thereof

Abstract

The present application relates to the technical field of bone repair scaffold, in particular to a directional multifunctional bone repair scaffold prepared by pulling and folding method and a preparation method and application thereof. The directional multifunctional bone repair scaffold comprises a polycaprolactone-collagen mixture with a honeycomb-shaped micropore structure, and a strontium phosphate mineralization layer covering the surface of the polycaprolactone-collagen mixture. The honeycomb forming process of "sugar stove" is imitated, that is, the repeated stretching and folding of the semi-solid mixture can induce structural orientation and porosity without solvent or special equipment. In-situ mineralization with strontium apatite further enhances the bioactivity, and Sr 2+ Dual role in angiogenesis and osteogenesis. Systemic characterization confirms SrAP mineralization uniformity and interface stability. In vitro studies show that 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. Rat calvarial defect model shows that the bone regeneration rate is 50.85% at 6 weeks, and micro-CT and histological examination shows mature vascularized bone formation.

Description

TECHNICAL FIELD

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

[0002] Accidental trauma and periosteal diseases can cause bone defects, and traditional treatment methods focus on autologous and allogeneic bone transplantation to repair defects; however, these methods have certain limitations, such as infection risk, scarcity of available grafts, and potential immune rejection problems. Tissue repair scaffolds are widely used as a highly promising strategy to solve bone defects that cannot be effectively recovered through the natural healing process.

[0003] The main function of the scaffold is to balance temporary mechanical support and efficient material transport, promote biological delivery and drive tissue regeneration. With further research, scientists have found that bone tissue is composed of highly ordered collagen fibers, which helps guide cell differentiation, migration and proliferation. Extensive research in recent years has shown that directional bone scaffolds not only promote the growth of new bone tissue, but also stimulate cells to produce extracellular matrix with ordered structure organization. For example, a variety of directional degradable polylactic acid scaffolds were prepared by electrospinning technology, and the directional scaffolds significantly enhanced in vivo bone regeneration and early vascularization. In addition, the application of directional scaffolds significantly improved the expression of key tissue development proteins compared with 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 customization of tissue-specific properties to ensure compatibility with host tissue needs. Over the past few decades, significant progress has been made in advanced manufacturing methods and related technologies, driving major innovations in the design and production of high-performance, directionally engineered scaffolds. Recent advances in the study of directional scaffold manufacturing have focused on four key technical methods, including particle leaching, electrospinning, freeze-drying, and 3D printing. Particle leaching provides a direct method for preparing scaffolds with tunable pore structures by controlling pore former size and morphology. However, this method typically relies on organic solvents and has limited pore interconnectivity. In addition, electrospinning technology can produce scaffolds with superior pore orientation and interconnectivity, effectively replicating the structure of the natural extracellular matrix. However, electrospun scaffolds typically exhibit limited mechanical performance, with 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 microstructures. However, it faces challenges in terms of limited mechanical strength and high production costs. Therefore, developing a simple, environmentally friendly, and cost-effective method to produce high-mechanical-performance, load-bearing-oriented porous scaffolds remains a major challenge in tissue engineering. SUMMARY

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

[0006] To achieve the above-mentioned purpose, the present application provides the following technical scheme: a directional multifunctional bone repair scaffold prepared by a pulling-folding method, comprising a polycaprolactone-collagen mixture with a honeycomb-like micropore structure inside, and a strontium phosphate mineralization layer covering the surface of the polycaprolactone-collagen mixture.

[0007] Further,

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

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

[0010] Further, the preparation steps include:

[0011] (1) Preparation of PCLSC scaffold

[0012] Preparation of PCL particles and collagen, uniformly mixed in a beaker, add an appropriate amount of anhydrous ethanol to cover the PCL and collagen, use a 70℃ oil bath to melt the PCL particle and collagen mixture, and frequently stir the material in the beaker during heating to accelerate dissolution, when the PCL solution is completely melted, use a clean wooden stick to repeatedly stretch in a 50℃, 75% ethanol solution for several hundred times, place the stretched PCL into deionized water for rapid solidification, cut the solidified solid into blocks or pieces to form a PCLSC scaffold;

[0013] (2) Weigh (NH4)2HPO4 powder and SrCl2·6H2O powder, respectively, dissolve in deionized water, under vigorous stirring, add (NH4)2HPO4 solution dropwise to strontium chloride solution, then add ammonia 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 with anhydrous ethanol and deionized water and freeze-dry to obtain a SrSC scaffold.

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

[0015] Further, 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 0.158g / ml, and the concentration of SrCl2·6H2O powder is 0.52g / ml.

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

[0017] Prepare 8.5g PCL particles with a molecular weight of 30,000 and 1.5g type I collagen, uniformly mix them in a beaker, add an appropriate amount of anhydrous ethanol to cover the PCL, use a 70℃ oil bath to melt the PCL mixture, and frequently stir the material in the beaker during heating to accelerate dissolution, prepare a solution to prevent rapid cooling in a 50℃, 75% ethanol solution in an incubator, when the PCL solution is completely melted, use a clean wooden stick to repeatedly stretch in a 75% ethanol solution for several hundred times, simulate the stretching process of sugar, place the stretched PCL into deionized water for rapid solidification, cut the solidified solid into blocks or pieces to form a PCLSC.

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

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

[0020] The SrSC scaffold prepared by the preparation method is applied to the preparation of a directional multifunctional bone repair scaffold.

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

[0022] Compared with the prior art, the SrSC scaffold has the following beneficial effects:

[0023] The application is inspired by the traditional Chinese "stove sugar" process, and a biomimetic strategy is successfully developed to construct a hierarchical degradable bone repair scaffold. By innovatively combining polycaprolactone with type I collagen and introducing strontium elements through biomimetic mineralization, a SrSC scaffold with uniaxial aligned microchannels is successfully prepared. Systematic in vitro and in vivo experiments prove that the material has significant advantages in bone tissue regeneration, angiogenesis and cell directional migration, providing a new solution to the problems of complex preparation process, insufficient bioactivity and low osteogenesis efficiency of traditional bone regeneration materials. Through structural biomimicry, element doping and interface modification, bone-vascular coordinated repair is achieved, which has broad application prospects in the field of bone defect treatment. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 A is the SEM image of the scaffold formed when the stretching time of the embodiment of the application is 100, 200 and 300 respectively.

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

[0026] Figure 1 C is the porosity statistical diagram of PCLSC-1, PCLSC-2 and PCLSC-3 of the embodiment of the application.

[0027] Figure 1 D is the porosity statistical diagram of PCLSC, CaSC and SrSC of the embodiment of the application.

[0028] Figure 1 E is the water contact angle diagram of PCLSC and SrSC of the embodiment of the application.

[0029] Figure 1F is a statistical chart of the in vitro degradation rate of the PCLSC, CaSC and SrSC of the embodiment of the present application.

[0030] Figure 1 G is the FT-IR spectrum of the PCLSC, CaSC and SrSC of the embodiment of the present application.

[0031] Figure 1 H is the FT-IR spectrum of the PCLSC, CaSC and SrSC of the embodiment of the present application in the range of 2000 cm -1 to 400 cm -1 .

[0032]

[0033] Figure 1 I is the elemental distribution chart of the SrSC of the embodiment of the present application.

[0034] Figure 1 J is the stress-strain curve of the stent of the embodiment of the present application when the channel direction is horizontal and vertical, respectively.

[0035] Figure 1 K is the compression modulus of the stent of the embodiment of the present application when the channel direction is horizontal and vertical, respectively.

[0036] Figure 2 A is a statistical chart of the viability of MC3T3-E1 cells cultured by different concentrations of leaching solution of the embodiment of the present application.

[0037] Figure 2 B is a live / dead cell staining image of MC3T3-E1 cells co-cultured in different groups of the embodiment of the present application.

[0038] Figure 2 C is an F-actin staining image of MC3T3-E1 cells co-cultured in different groups of the embodiment of the present application.

[0039] Figure 2 D is a Transwell migration experiment image of MC3T3-E1 cells in different groups of the embodiment of the present application.

[0040] Figure 2 E is a scratch healing experiment image of MC3T3-E1 cells in PCLSC, CaSC and SrSC groups of the embodiment of the present application.

[0041] Figure 2 F is a statistical analysis of the Transwell migration experiment of the embodiment of the present application.

[0042] Figure 2 G is a statistical analysis of the scratch healing experiment of the embodiment of the present application.

[0043] Figure 2 ​H is the SEM image of the scaffold with cells attached in the embodiment of the application.

[0044] Figure 2 I is the cell survival graph after co-culturing the cells with the scaffold in the embodiment of the application.

[0045] Figure 3 A is the HUVEC cell viability statistical graph cultured by different concentrations of extractive solution in the embodiment of the application.

[0046] Figure 3 B is the HUVEC cell live / dead cell staining image co-cultured in different groups in the embodiment of the application.

[0047] Figure 3 C is the transwell migration experiment image of HUVEC cells in the PCLSC, CaSC and SrSC groups in the embodiment of the application.

[0048] Figure 3 D is the scratch healing experiment image of MC3T3-E1 cells in the PCLSC, CaSC and SrSC groups in the embodiment of the application.

[0049] Figure 3 E is the statistical analysis of the transwell migration experiment in the embodiment of the application.

[0050] Figure 3 F is the statistical analysis of the scratch healing experiment in the embodiment of the application.

[0051] Figure 3 G is the angiogenesis experiment schematic diagram of the PCLSC, CaSC and SrSC groups in the embodiment of the application.

[0052] Figure 3 H is the total tube length statistical graph in the angiogenesis experiment in the embodiment of the application.

[0053] Figure 3 I is the master node number statistical graph in the embodiment of the application.

[0054] Figure 3 J is the node number statistical graph in the angiogenesis experiment in the embodiment of the application.

[0055] Figure 3 K is the relative expression level statistical graph of the angiogenesis related gene VEGFA in the embodiment of the application.

[0056] Figure 4 A is the ALP staining schematic diagram of the PCLSC, CaSC and SrSC groups in the embodiment of the application.

[0057] Figure 4 B is the semi-quantitative analysis of the ALP staining in the embodiment of the application.

[0058] Figure 4 C is the immunofluorescence staining image of Bglap protein of the PCLSC, CaSC and SrSC groups in the embodiment of the application.

[0059] Figure 4 D is the immunofluorescence staining image of Runx2 protein of the PCLSC, CaSC and SrSC groups in the embodiment of the application.

[0060] Figure 4 E is the semi-quantitative analysis of Bglap protein in the embodiment of the application.

[0061] Figure 4 F is the semi-quantitative analysis of Runx2 protein in the embodiment of the application.

[0062] Figure 4 G is the statistical chart of the relative expression level of ALP gene in the embodiment of the application.

[0063] Figure 4 H is the statistical chart of the relative expression level of Col1a1 gene in the embodiment of the application.

[0064] Figure 4 I is the statistical chart of the relative expression level of Runx2 gene in the embodiment of the application.

[0065] Figure 5 A is the schematic diagram of the skull defect experiment of SD rats in the embodiment of the application.

[0066] Figure 5 B is the construction of the skull defect model in the embodiment of the application.

[0067] Figure 5 C is the three-dimensional reconstruction image of the skull of SD rats at 4 weeks in the embodiment of the application.

[0068] Figure 5 D is the three-dimensional reconstruction image of the skull of SD rats at 6 weeks in the embodiment of the application.

[0069] Figure 5 E is the quantitative result of BV / TV obtained by micro-CT at 4 weeks in the embodiment of the application.

[0070] Figure 5 F is the quantitative result of BV / TV obtained by micro-CT at 6 weeks in the embodiment of the application.

[0071] Figure 5 G is the quantitative result of Tb.Th obtained by micro-CT at 6 weeks in the embodiment of the application.

[0072] Figure 5 H is the H&E staining at 6 weeks in the embodiment of the application.

[0073] Figure 5 I is Masson's trichrome staining of the inventive example at 6 weeks.

[0074] Figure 5 J is immunohistochemical staining of cyanide and OPN at 6 weeks of the inventive example.

[0075] Figure 5 K is immunohistochemical staining of a-SMA and CD31 at 6 weeks of the inventive example.

[0076] Figure 6 SEM images of CaSC at different magnifications of the inventive example.

[0077] Figure 7 Water contact angle of CaSC of the inventive example.

[0078] Figure 8 EDS image of CaSC of the inventive example.

[0079] Figure 9 Live / dead staining image of MC3T3-E1 cells treated with CaSC and SrSC extract of the inventive example.

[0080] Figure 10 F-actin staining image of MC3T3-E1 cells treated with CaSC and SrSC extract of the inventive example.

[0081] Figure 11 A is CCK-8 detection of the effect of SrSC on L929 cells of the inventive example.

[0082] Figure 11 B is live / dead cell staining of the effect of SrSC on L929 cells of the inventive example.

[0083] Figure 11 C is Transwell migration experiment image of the effect of SrSC on L929 cells of the inventive example.

[0084] Figure 11 D is scratch healing experiment image of the effect of SrSC on L929 cells of the inventive example.

[0085] Figure 11 E is statistical analysis of Transwell migration experiment of the effect of SrSC on L929 cells of the inventive example.

[0086] Figure 11 F is statistical analysis of scratch healing experiment of the effect of SrSC on L929 cells of the inventive example.

[0087] Figure 12Figure 1 shows the live / dead staining images of HUVEC cells treated with CaSC and SrSC leaching solution according to the embodiments of the present application.

[0088] Figure 13 Figure 2 shows the expression levels of ALP, Col1a1 and Runx2 genes in MC3T3-E1 cells when the MC3T3-E1:HUVEC cell ratio is 2:1 according to the embodiments of the present application.

[0089] Figure 13 Figure 3 shows the expression levels of ALP, Col1a1 and Runx2 genes in MC3T3-E1 cells when the MC3T3-E1:HUVEC cell ratio is 1:2 according to the embodiments of the present application.

[0090] Figure 14 Figure 4 shows the construction of SD rat skull defect model according to the embodiments of the present application.

[0091] Figure 15 Figure 5 shows the H&E staining images of SD rat skull pathological section after 4 weeks of staining according to the embodiments of the present application.

[0092] Figure 15 Figure 6 shows the Masson trichrome staining images of SD rat skull pathological section after 4 weeks of staining according to the embodiments of the present application.

[0093] Figure 16 Figure 7 shows the quantitative CD31 fluorescent staining according to the embodiments of the present application.

[0094] Figure 16 Figure 8 shows the quantitative a-SMA fluorescent staining according to the embodiments of the present application. DETAILED DESCRIPTION

[0095] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0096] All data in the present application are expressed as mean ± standard error, and independent sample t test and one-way analysis of variance are used for analysis. For CCK-8 determination, two-way analysis of variance is used. Statistical significance is defined as P value < 0.05. Specifically, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns: no significant difference.

[0097] EMBODIMENT

[0098] A directional multifunctional bone repair scaffold prepared by a pulling-folding method, comprising a polycaprolactone-collagen mixture with a honeycomb-like micropore structure inside, and a strontium phosphate mineralization layer covering the surface of the polycaprolactone-collagen mixture. The honeycomb-like micropore structure is formed by repeated stretching, folding and re-stretching in a cyclic manner, and the pore size of the micropore structure is 80-120 μm; the strontium phosphate mineralization layer is granular, and the particle diameter is less than 5 μm.

[0099] A preparation method of a directional multifunctional bone repair scaffold prepared by a pulling-folding method, comprising:

[0100] (1) Preparation of PCLSC scaffold

[0101] Prepare 8.5 g of PCL particles with a molecular weight of 30,000 and 1.5 g of type I collagen, and uniformly mix them 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 frequently stir the material in the beaker during heating to accelerate dissolution. Prepare a solution to prevent rapid cooling in a 50°C 75% ethanol solution in an incubator. After the PCL solution is completely melted, use a clean wooden stick to repeatedly stretch it in the 75% alcohol solution for several hundred times, simulating the stretching process of sugar. Place the stretched PCL in deionized water for rapid solidification. Cut the solidified solid into blocks or pieces to form a PCL-collagen scaffold (PCLSC).

[0102] (2) In-situ mineralization of PCLSC

[0103] Weigh 0.79 g of (NH4)2HPO4 powder and 2.6 g of SrCl2·6H2O powder, and dissolve them in 5 mL of 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 system for mineralization. After 7 days, wash thoroughly with anhydrous ethanol and deionized water and freeze-dry to obtain a SrSC scaffold.

[0104] Preparation of a comparative sample CaSC scaffold:

[0105] Take 0.79 g of (NH4)2HPO4 powder and dissolve in 5 mL of deionized water. Take 1.1 g of CaCl2 solid and dissolve in 5 mL of deionized water. Slowly add the (NH4)2HPO4 solution to the calcium chloride solution at room temperature with rapid stirring using a magnetic stirrer. The (NH4)2HPO4 solution should be added within 40 minutes. After the (NH4)2HPO4 solution is added, adjust the pH of the reaction system to 10 by adding ammonia water and continue to stir 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 with anhydrous ethanol and deionized water several times to remove the un-deposited solids. Then freeze it in a -20 °C refrigerator and transfer it to a freeze dryer for freeze-drying to obtain the CaSC scaffold.

[0106] Preparation of comparative sample HAP:

[0107] Dissolve 1.2 M (NH4)2HPO4 and 2 M CaCl2 in 10 mL of deionized water, respectively. Stir rapidly at 90 °C, then add the (NH4)2HPO4 solution dropwise to the calcium chloride solution. The dropwise addition process should be completed within 40 minutes. Add ammonia water to the reaction system to adjust the pH to 10, and continue to stir at 90 °C for 1 hour. Then, place the reaction product at 90 °C for 48 hours. Wash the deposition product with anhydrous ethanol and deionized water several times to obtain the reaction product hydroxyapatite HAP.

[0108] Preparation of comparative sample SrAP:

[0109] Dissolve 1.2 M (NH4)2HPO4 and 2 M SrCl2·6H2O in 10 mL of deionized water, respectively. At 90 °C, add the (NH4)2HPO4 solution dropwise to the strontium chloride solution, and adjust the pH of the reaction system to 10 with ammonia water, and continue to stir for 1 hour. After 48 hours, wash the product with deionized water and anhydrous ethanol to obtain the reaction product strontium apatite SrAP.

[0110] 1. Structural characteristics:

[0111] Experimental methods

[0112] (1) Characteristic detection of HAP and SrAP

[0113] Place the dried HAP and SrAP powders on the sample table of the Fourier transform infrared spectrometer and detect the infrared absorption spectrum of the sample.

[0114] (2) Characteristic detection of PCLSC, CaSC and SrSC

[0115] PCLSC, CaSC and SrSC were immersed in 75% ethanol and sterilized with UV light for 48 hours, then washed twice with phosphate buffered saline (PBS).

[0116] PCLSC, CaSC and SrSC scaffolds were adhered to conductive glue and then gold-sprayed for 240 seconds. The samples after gold-spraying were placed on the sample stage of a scanning electron microscope to observe the porous structure of unmineralized scaffolds and mineralized scaffolds. Mineralized CaSC and SrSC scaffolds were fixed with conductive glue and sprayed with gold. Field emission scanning electron microscopy was used to observe the mineralization and element distribution near the scaffold pore structure.

[0117] Smooth and flat PCLSC, CaSC and SrSC scaffold pieces were selected, and the static water contact angle of the scaffolds was measured using an optical contact angle measuring instrument. The absolute ethanol was filled into the calorimetric cup, and the mass w1 was measured using an electronic balance. The mass w s of the scaffold was placed into the calorimetric cup, all the air bubbles in the scaffold interstices were discharged, and then it was filled with absolute ethanol. At this time, the mass w2 was measured. The sample filled with absolute ethanol was taken out, and the total mass w3 of the calorimetric cup and the remaining absolute ethanol was measured. Let the density of ethanol at the test temperature be p. The volume of the scaffold pore wall The volume of the scaffold inner hole The calculation formula of porosity is as follows:

[0118]

[0119] In vitro degradation experiments were carried out by using lysozyme solution of simulated body fluid, and the sample was completely immersed in 0.1 mg / mL lysozyme solution, the solution was replaced every week, and the degradation temperature was 37°C.

[0120] The experimental procedure is as follows: first, the PCLSC, CaSC and SrSC scaffolds were processed into standard cubes (5 mm long x 5 mm wide x 5 mm high). A universal testing machine equipped with a 500N compression clamp was used, with a loading rate of 1 mm / min, and zero calibration was performed. Each sample was placed vertically in the center of the clamp 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. The transverse and longitudinal channel directions of each group of scaffolds were tested, and each measurement was repeated three times. The final data is represented as mean ± standard error, and is obtained after normality verification.

[0121] Experimental results:

[0122] Figure 1A demonstrates the morphological and structural characteristics of PCLSC scaffolds (100, 200, 300 cycles are marked as PCLSC-1, PCLSC-2, PCLSC-3, respectively). Scanning electron microscope (SEM) analysis shows that increasing the number of stretching-folding cycles significantly improves the pore structure, including pore size uniformity and interconnectivity.

[0123] Pore size analysis shows that the scaffold porosity gradually increases with the number of stretching-folding cycles Figure 1 B). The pore size of PCLSC-3 is mainly distributed in the optimal range of 80-120 pm, which is beneficial for accelerated osteogenesis in bone regeneration applications. After 7 days of in vitro mineralization, SrSC and CaSC scaffold derivatives Figure 1 C, Figure 6 ) are successfully prepared from PCLSC precursors. The SrSC scaffold shows a consistent strontium phosphate mineralization layer along the pore wall, as shown in Figure 1 C, with a particle diameter of less than 5 pm and uniform distribution. In contrast, the CaSC scaffold exhibits an uneven calcium phosphate deposition pattern with irregular particle morphology, as shown in Figure 6 . Quantitative analysis shows that the porosity of the scaffold after mineralization decreases by 12.40%, as shown in Figure 1 D. An ideal bone conduction scaffold needs to meet dual structural criteria: (1) a large pore network (>100 pm) to facilitate cell migration; (2) sufficient porosity (>30%) to maintain nutrient supply. The PCLSC matrix exhibits an interconnected porosity of more than 69.5% - exceeding the 60% threshold required for osteoblast infiltration. Although mineralization reduces the void volume, both CaSC and SrSC maintain a functional porosity of more than 50%, which enables remodeling of the extracellular matrix during bone formation Figure 1 D). In addition, the inherent hydrophobicity of PCL limits its cell adhesion ability, which is confirmed by a water contact angle of 82.7° Figure 1 E). After surface treatment with phosphate coating, the hydrophilicity is significantly enhanced, with contact angles of CaSC and SrSC decreasing to 50.2° and 53.9°, respectively Figure 7 . This transition from hydrophobic to hydrophilic state improves the adhesion of fibroblasts. As a degradable polymer, PCL exhibits gradual degradation characteristics in vitro. Figure 1 F shows that PCLSC, CaSC, and SrSC scaffolds maintain stable degradation over time. This slow degradation provides sufficient 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 at 1017 cm -1 ( Figure 1G-H), confirming the successful incorporation of phosphate. Elemental mapping showed uniform distribution of P, O, N and Sr throughout the scaffold structure Figure 1 I). These results confirmed the successful deposition of SrAP mineralized particles on the polymer matrix, while calcium-phosphorus mineralization was also observed on CaSC Figure 8 ). In addition, the engineered channel-aligned scaffold exhibited remarkable structural anisotropy, which is a key property to mimic the natural bone structure. To systematically evaluate its mechanical performance, we tested its strength by uniaxial compression test (transverse / longitudinal axis). The stress-strain curves indicated that the SrSC scaffold after mineralization still maintained excellent compressive strength under bidirectional loading, showing good structural stability Figure 1 J). Notably, the compressive modulus of SrSC (transverse 25.35 MPa, longitudinal 37.10 MPa) showed no statistically significant difference compared with the control and CaSC groups, indicating that strontium doping did not change the inherent mechanical properties of the scaffold Figure 1 K). Comprehensive physicochemical characterization confirmed that SrSC met the requirements of ideal bone regeneration materials in terms of mechanical properties, structural integrity, and controllable composition.

[0124] 2. In vitro cell experiments of the scaffold

[0125] Experimental methods:

[0126] Preparation of scaffold extract: According to the method of GB_T 16886.12-2017, extraction was performed using complete culture medium. Sterile scaffolds were placed in complete culture medium at a ratio of 0.1 g / mL under constant temperature conditions at 37°C for 72 hours. Subsequently, the culture medium was filtered through a sterile 0.22 μm pore size filter to immediately obtain the available scaffold extract.

[0127] Preparation of osteogenic medium: 10 mM sodium β-glycerophosphate, 50 μg / mL vitamin C and 10 mM dexamethasone were added to the complete culture medium. Four concentration gradients of scaffold extract were set: 0.1, 0.05, 0.025 and 0.0125 g / mL. These extract concentrations were used to detect the cytotoxicity of MC3T3-E1, HUVEC and L929 cells. Cells were cultured in different concentrations of extract, and the extract was removed at the predetermined time. 10% CCK-8 working solution was added, and the cells were further incubated in the cell incubator for 1-4 hours. The absorbance at 450 nm was detected using an enzyme detector. Then the cell viability after co-culture with the extract was calculated. The formula for calculating cell viability is as follows:

[0128]

[0129] In the formula, OD s represents the absorbance of the experimental well (cultured with extract), ODc OD represents the absorbance of the control well (cultured with normal medium), while OD b blank well (cultured with normal medium but without cells). MC3T3-E1, HUVEC and L929 cells were cultured with CaSC and SrSC scaffold leachate, respectively, and stained at predetermined time points. The dye working solution was prepared according to the proportion of Calcein-AM / PI double staining kit. The working solution was added to the cell well plate, incubated at 37°C for 15 min, and then washed with PBS. Finally, it was observed and photographed under a confocal microscope.

[0130] MC3T3-E1 cells were co-cultured with scaffold leachate at a concentration of 0.0125 g / mL. The changes in cell morphology were observed on the 3rd and 7th day 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 for 30 minutes in the dark. The cells were washed with PBS containing 0.05% Triton X-100. Then, the glass slides were sealed with DAPI Fluoromount-G TM antifading mounting medium. Finally, the cells were observed using a confocal microscope.

[0131] Cells were seeded in a six-well plate and cultured to confluence. Subsequently, the cells were scratched on the surface and washed with PBS. The cells were cultured in 0.0125 g / mL leachate (serum-free). At predetermined time points, the differences in cell migration in the blank area of the same position were observed to verify the effect of scaffold leachate on cell migration. Experimental group closed area (%) = blank area / control group blank area x 100%. 2 x 10^4 cells were seeded in a Transwell chamber, and 500 μL of 0.0125 g / mL scaffold leachate was added to the lower chamber. The cells were normally cultured, and the Transwell chamber was removed at the predetermined time point 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 a light microscope, and the number of migrated cells was analyzed using ImageJ software.

[0132] A sterile scaffold of appropriate size is selected and placed in a 24-well plate. 1 mL of cell suspension is added to the scaffold to verify its adhesion to the cells and changes in cell morphology. The suspension is added slowly and multiple times to ensure as many cells as possible remain on the scaffold. It is then placed in a cell culture incubator for 24 hours. Subsequently, live / dead cell staining is performed and the biological activity of the cells on the scaffold is observed using a confocal microscope. The cell-loaded scaffold is fixed with 2.5% glutaraldehyde. It is 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, gradually dehydrated. It is then freeze-dried. Gold is sprayed on the dried scaffold under vacuum conditions, and observed and photographed using a scanning electron microscope.

[0133] Matrigel is added to a 48-well plate and placed in a cell culture incubator until it solidifies. After counting the HUVEC cells, 2 x 10^4 cells are added per well. After 6 hours of culture in a scaffold extract solution at a concentration of 0.0125 g / mL, the HUVEC cells are observed and photographed under a microscope to observe their angiogenesis. Analysis is performed using ImageJ software.

[0134] MC3T3-E1 cells are cultured in a 24-well plate containing a 0.0125 g / mL scaffold extract solution. After a certain period of time, alkaline phosphatase (ALP) staining is performed at predetermined time points to directly evaluate the osteogenic effect of the scaffold. After a certain period of time, the cells are washed with PBS and fixed with 4% paraformaldehyde. BCIP solution and NBT solution are diluted in ALP staining buffer to working solution. The working solution is added to the fixed cells for staining. After staining, the cells are washed with deionized water and observed under a microscope. Semi-quantitative analysis of the ALP-stained area is performed using ImageJ software.

[0135] MC3T3-E1 cells are cultured in a scaffold extract solution at a concentration of 0.0125 g / mL. After a certain period of time, the cells are fixed with 4% paraformaldehyde. The cells are first treated with 0.5% Triton X-100 and then blocked with 3% BSA for 1 hour. Diluted primary antibody is added and incubated at room temperature for 3 hours. The cells are washed with PBST (0.05% Tween-20, 99.95% PBS). The cells are incubated with Alexa Fluor 594-labeled goat anti-rabbit IgG (H+L) antibody for 1 hour in the dark. The cells are washed again with PBST. Finally, the cells are mounted with DAPI Fluoromount-G TM The cells are blocked with anti-fluorescence quenching mounting medium. The cells are observed under a confocal microscope. Quantitative analysis is performed using ImageJ software.

[0136] MC3T3-E1 cells and HUVEC cells were co-cultured with the scaffold extract at a concentration of 0.0125 g / mL. Cells were collected at predetermined time. After washing the cells with PBS, they were transferred to centrifuge tubes and an appropriate amount of RNA-easy was added for lysis. Two-fifths volume of RNase-free ddH2O was added, mixed well and left for 5 minutes. Centrifugation was performed at 12000 x g for 15 minutes and the supernatant was retained. Two-fifths volume of isopropanol was added, mixed well and left for 15 minutes. Centrifugation was performed again at 12000 x g for 10 minutes and the white precipitate was retained. The white precipitate was washed with 75% ethanol three times and dried. An appropriate amount of RNase-free ddH2O was added to completely dissolve the precipitate and the RNA concentration was determined using a Nanodrop ultramicro spectrometer. Reverse transcription was performed using 5 μg of RNA, which was incubated at 25 °C for 10 minutes, 45 °C for 15 minutes and finally 85 °C for 5 seconds. The reverse transcription product was stored in a refrigerator at -20 °C. 10 ng of cDNA was used as a template, 10 μL of 2x concentrated universal SYBR Green Fast qPCR mixture, 0.4 μL of forward primer (10 μM) and 0.4 μL of reverse primer (10 μM) (see Table 1) were added. ddH2O was added to a total volume of 20 μL as a qPCR reaction system. The qPCR reaction program was established. β-actin was used as a housekeeping gene and the relative expression level of the target gene was detected by the 2^-△△Ct method.

[0137] Table 1. Primers in qPT-PCR experiments.

[0138]

[0139] Experimental results:

[0140] (1) SrSC scaffolds promote cell migration

[0141] Mouse calvarial osteoblasts (MC3T3-E1) were cultured with different concentrations (0.1, 0.05, 0.025 and 0.0125 mg / mL) of scaffold extract to verify the biocompatibility of the scaffold. CCK-8 results showed that the extract at concentrations of 0.025 and 0.0125 mg / mL had no cytotoxicity to MC3T3-E1 cells Figure 2 A). After live / dead staining of cells cultured with 0.025 mg / mL extract, almost no dead cells were observed Figure 2 B and Figure 9 ), confirming that this concentration was not toxic to the cells. MC3T3-E1 cells are a preosteoblast cell line that forms osteoblast-like fibroblasts after induction and differentiation. After co-culturing for three days, it was observed that the cells in the CaSC and SrSC groups had formed fibroblast-like and oriented morphologies Figure 2 C andFigure 10 This demonstrates that CaSC and SrSC have the ability to induce osteoblast progenitor cell differentiation. Sr has a good effect on cell migration. To verify the migration ability of mineralized scaffolds for MC3T3-E1 cells, Transwell migration assays and cell scratch healing assays were performed. Figure 2 D clearly shows the difference in the number of migrating cells. Figure 2 Statistical data from F demonstrate that SrSC significantly promoted the migration of MC3T3-E1 cells compared to the control group and the CaSC group. The scratch healing assay also confirmed that SrSC significantly promoted the migration of MC3T3-E1 cells. Figure 2 E and 2G). After co-culture, scanning electron microscopy images showed that the cells adhered well to the scaffolds of SrSC and CaSC (E and 2G). Figure 2 H). To verify the unique directional porous structure of the scaffold, we co-cultured the scaffold with cells and observed cell survival within the pores. Figure 2 The results showed that MC3T3-E1 cells could grow inward along the scaffold pore walls. The PCLSC scaffold pores contained the fewest cells, followed by the CaSC scaffold.

[0142] To assess biocompatibility, L929 fibroblasts were co-cultured with CaSC and SrSC extracts, followed by CCK-8 analysis. Figure 11 A). The results showed that both scaffolds had good cell compatibility. Furthermore, live / dead staining revealed no detectable cytotoxic effects, and surviving cells maintained normal morphology. Figure 11 B). Notably, both material variants exhibited bioactivity effects on cell function, as evidenced by enhanced cell migration in the SrSC and CaSC groups compared to the control group. Figure 11 Importantly, SrSC outperforms CaSC in osteoprogenitor cell adhesion and directional guidance, indicating its greater suitability as an osteogenic implant material.

[0143] (2) SrSC stents promote angiogenesis

[0144] Effective bone defect repair requires robust vascularization to ensure nutrient supply and removal of metabolic waste. To assess this crucial aspect, we first evaluated the biocompatibility of scaffolds using umbilical vein endothelial cells (HUVECs) cultured in CaSC / SrSC extract at different concentrations (0.1, 0.05, 0.025, and 0.0125 mg / mL). While CaSC maintained cell compatibility at 0.1 mg / mL, SrSC exhibited the best biocompatibility at 0.05 mg / mL. Figure 3 A), live / dead staining confirmed that there were almost no dead cells even at the lowest concentration. Figure 3B and Figure 12 ) Given the key role of endothelial cells in the osteogenic process, we next investigated the directional migration of cells to the bone defect area. Transwell migration experiments and cell scratch healing experiments showed that the migration effect of SrSC scaffolds was more significant than that of the control group, and the number of cell migration and the closed area were higher than those of CaSC scaffolds Figure 3 C-F). 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 SrSC extract induced the formation of blood vessel networks 1.35 times longer than CaSC, 1.76 times more main connection points, and 1.69 times more connection points Figure 3 G-J). Mechanistically, the expression level of the angiogenesis-related gene (VEGFA) in HUVEC cells in the SrSC group was significantly higher than that in the CaSC group, proving that SrSC can effectively stimulate endothelial cells to express VEGFA and activate angiogenesis ability, thus providing a favorable microenvironment for the growth of bone cells.

[0145] (3), SrSC scaffolds promote bone regeneration in vitro

[0146] The osteogenic differentiation potential of SrSC was systematically verified by in vitro evaluation. Preliminary alkaline phosphatase (ALP) staining showed that at a concentration of 0.0125 mg / mL extract, SrSC had a better potential than CaSC to promote the differentiation of MC3T3-E1 preosteoblasts, with 1.51 times higher ALP activity than CaSC at day 7 Figure 4 A, B). To further verify the osteogenic potential of SrSC, we used immunofluorescence to detect the osteogenesis-related proteins Bglap and Runx2. Supplementary immunofluorescence analysis showed that the osteogenesis markers Bglap and Runx2 were significantly upregulated in SrSC-treated cells compared to CaSC Figure 4 C-F). In addition, the molecular profile confirmed this trend, with significantly higher expression levels of ALP, Runx2, and Col1a1 genes in the SrSC group than in the CaSC group, which also indicated that SrSC had a more obvious osteogenic potential Figure 4 G-I). To reproduce the in vivo osteogenic microenvironment, we established a MC3T3-E1:HUVEC co-culture model (ratio of 2:1 and 1:2). Under these physiologically relevant conditions, SrSC maintained its bone induction advantage, with 1.29 to 1.89 times higher expression of induced osteogenic genes than CaSC Figure 13 ) This consistent enhancement in single culture and co-culture systems strongly suggests that SrSC has an inherent ability to coordinate bone regeneration through multi-cell regulatory mechanisms, providing strong preclinical evidence for its transformation potential.

[0147] 3, in vivo experiments of scaffolds

[0148] Test method:

[0149] All animal experiments were performed in accordance with the approved procedures of the Experimental Animal Welfare Ethics Committee of Qingdao University (Laboratory Approval Number: NO.20241122sd4420250120176176). A total of twenty 8-week-old female Sprague-Dawley (SD) rats were selected and randomly divided into four groups: blank control group, PCLSC group, CaSC group, and SrSC group. After anesthesia, the skull was exposed by depilation and medical iodine disinfection, and two key full-thickness bone defects with a diameter of 6 mm were manufactured using a dental drill bit,

[0150] Continuous flushing with normal 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, respectively. The incision was closed with suture and disinfected with iodophor. After operation, each animal received 40000 units of penicillin treatment per day for 3 days. At 4 and 6 weeks after operation, the rats were sacrificed by cervical dislocation, and their skulls were removed and fixed in 4% paraformaldehyde.

[0151] Each sample was imaged using a Quantum GX2 high-resolution micro-CT scanner, set to a scan voltage of 90 kV and a current of 88 μA. Subsequently, the sample images were reconstructed in three dimensions to evaluate the repair effect of rat skull defects. The bone volume fraction (BV / TV) and trabecular thickness (Tb.Th) were quantitatively analyzed (n = 4).

[0152] Each sample was decalcified using a commercially available ethylenediaminetetraacetic acid (EDTA) solution, and the decalcification solution was replaced every 48 hours for four weeks. After decalcification, the samples were rinsed in flowing water for a long time (overnight). Subsequently, the samples were dehydrated in an alcohol gradient, followed by paraffin infiltration and embedding, with a slice thickness of 4 μm. Finally, the samples were subjected to hematoxylin-eosin (H&E) staining, Masson's trichrome staining, immunohistochemical (IHC) staining, and immunofluorescence staining.

[0153] Experimental results:

[0154] Based on the positive results of in vitro studies, a rat full-thickness skull defect model (6 mm in diameter) was established to verify the osteogenic effect of SrSC in vivo. Through micro-CT analysis, the scaffold-implanted groups (SrSC, CaSC and PCLSC) and the blank control group ( Figure 5 A, B and Figure 14Micro-CT obtained three-dimensional reconstructed images showed that the skull of the SrSC implant group formed significantly more than the other groups at 4 weeks, with a bone volume fraction (BV / TV) of 19.1%, while the control group was only 3.74%. At this time, PCLSC (10.48%) and CaSC (14.18%) did not show significant osteogenic activity Figure 5 C, E) By the 6th week, the SrSC-treated defect area almost completely formed a bone bridge, while the CaSC showed slow, measurable regenerative bone. Notably, SrSC remained structurally superior, with a trabecular thickness (Tb.Th) of 0.60 mm at 6 weeks, while CaSC was 0.38 mm Figure 5 D, F, G) In contrast, the PCLSC implant area showed less new bone formation, with almost no new bone growth.

[0155] To evaluate the microstructural features of the newly formed bone tissue, hematoxylin-eosin staining (H&E staining) and Masson's trichrome staining techniques were systematically employed. At 4 weeks postoperatively, H&E staining images and Masson's trichrome staining images showed new bone formation in the SrSC group defect area, indicating that this group had a unique osteogenic ability, while no detectable mineralization was observed in the control group, PCLSC group, or CaSC group Figure 15 At 6 weeks post-implantation, H&E staining images showed gradual changes: the control group defect was completely occupied by fibrous tissue (labeled "FT"), while the PCLSC group showed partial fibrous infiltration. The CaSC group showed new bone (labeled "NB"), while the SrSC group exhibited significantly expanded mineralized bone deposition Figure 5 H) Masson's trichrome staining results confirmed these findings, showing collagen maturation and vascular infiltration induced by SrSC (Figure I).

[0156] Bone osteocalcin (OCN) and osteopontin (OPN) were chosen as specific osteogenic markers for systematic immunohistochemical analysis of the four groups of skull tissue sections. Although the SrSC group showed extensive new bone formation, these areas showed limited positive staining, which was attributed to the rapid matrix mineralization creating a spatial exclusion effect that hindered dye penetration Figure 5 J) Undifferentiated mesenchymal cells surrounding the scaffold showed significant staining characteristics. The spatial distribution of the staining pattern indicated that the microenvironment of the SrSC implant not only effectively promoted the differentiation of undifferentiated mesenchymal cells to the osteoblast lineage, but the rapid deposition of mineralized components in the new bone matrix may have also created a spatial hindering effect on dye penetration. In summary, these results demonstrate that the microenvironment formed by SrSC has superior osteogenic induction ability.

[0157] In vivo experiments showed that the fluorescence intensity of angiogenesis markers α-SMA and CD31 was the highest in the SrSC group, and the other groups were relatively low Figure 5 K, Figure 16 ) which was consistent with the results of in vitro experiments. In vivo experiments more powerfully confirmed the effective guiding effect of SrSC on cells and its promoting effect on skull regeneration. This not only proved the beneficial effect of strontium element on bone regeneration, but also provided a new idea for the development of directional porous scaffolds.

[0158] Although embodiments of the present application have been shown and described, it is to be understood that various modifications, substitutions, replacements and changes can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an oriented multifunctional bone repair scaffold by a pull-folding method, characterized in that, The preparation method of the oriented multifunctional bone repair scaffold prepared by the pulling-folding method comprises the following steps: The preparation steps comprise: (1) Preparation of PCLSC scaffold Prepare PCL particles and collagen, uniformly mix them in a beaker, add an appropriate amount of anhydrous ethanol to cover the PCL and collagen, melt the PCL particle and collagen mixture using a 70 ℃ oil bath, and frequently stir the substances in the beaker during heating to accelerate dissolution, after the PCL solution is completely melted, repeatedly stretch a clean wooden stick in a 50 ℃ 75% ethanol solution for several hundred times, quickly solidify the stretched PCL in deionized water, cut the solidified solid into blocks or pieces to form a PCLSC scaffold; (2) In-situ mineralization of PCLSC Weigh (NH4)2HPO4 powder and SrCl2·6H2O powder, respectively dissolve them in deionized water, under vigorous stirring, add the (NH4)2HPO4 solution dropwise into 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 clean and freeze-dry the scaffold to obtain a SrSC scaffold.

2. The preparation method of the oriented multifunctional bone repair scaffold prepared by the pulling-folding method according to claim 1, characterized in that: The mass ratio of the PCL particles and collagen is 5.7:

1.

3. The preparation method of the oriented multifunctional bone repair scaffold prepared by the 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.

4. The method of claim 1, wherein the oriented multifunctional bone repair scaffold prepared by the pull-folding method is characterized by, The specific steps of (1) comprise: Prepare 8.5 g PCL particles with a molecular weight of 30,000 and 1.5 g type I collagen, uniformly mix them in a beaker, add an appropriate amount of anhydrous ethanol to cover the PCL, melt the PCL mixture using a 70 ℃ oil bath, and frequently stir the substances in the beaker during heating to accelerate dissolution, prepare a solution to prevent rapid cooling in a 50 ℃ 75% ethanol solution in an incubator, after the PCL solution is completely melted, repeatedly stretch a clean wooden stick in a 75% ethanol solution for several hundred times to simulate the stretching process of sugar, quickly solidify the stretched PCL in deionized water, cut the solidified solid into blocks or pieces to form a PCLSC.

5. The method of claim 1, wherein the oriented multifunctional bone repair scaffold prepared by the pull-folding method is characterized by, The specific steps of (2) comprise: Take 0.79 g (NH4)2HPO4 powder and 2.6 g SrCl2·6H2O powder, respectively dissolved in 5 mL deionized water, under vigorous stirring, the (NH4)2HPO4 solution is added dropwise into the strontium chloride solution, then ammonia water is added to adjust the pH to 10, and continue to stir for 1 hour, the PCLSC scaffold is placed in the reaction system for mineralization, after 7 days, washed thoroughly with anhydrous ethanol and deionized water and freeze-dried to obtain the SrSC scaffold.

6. The SrSC scaffold prepared by the preparation method of any one of claims 1-5 as a directed multifunctional bone repair scaffold.

7. Use according to claim 6, characterized in that: The SrSC scaffold promotes cell migration, promotes angiogenesis, and coordinates bone regeneration.

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