Preparation method and application of pre-vascularized bone repair scaffold

Abstract

The invention relates to the technical field of bone scaffold materials, and discloses a preparation method and application of a pre-vascularized bone repair scaffold, the preparation method of the pre-vascularized bone repair scaffold comprises the following steps: firstly, obtaining a porous scaffold and carrying out sulfonation treatment to obtain a sulfonated scaffold; then preparing a mixed solution containing gelatin and an extracellular matrix, and adding mesenchymal stem cells and / or endothelial cells to obtain a cell mixed preparation; and finally, contacting the cell mixed preparation with the sulfonated scaffold, and culturing to obtain the sulfonated scaffold. The pre-vascularized bone repair scaffold prepared by the preparation method disclosed by the invention simultaneously has excellent angiogenesis promoting capability and osteogenesis induction characteristic in vitro, and realizes vascularization-ossification synergistic interaction through a positive feedback loop mediated by a paracrine signal network; and a new thought and a new method are expected to be developed for clinical application of large-segment bone defect repair.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and in particular to a method for preparing and applying a prevascularized bone repair scaffold. Background Technology

[0002] Large-volume bone defects are often caused by traumatic injuries, congenital defects, and bone tumor resection. The damaged area exposes the medullary cavity, disrupting the original blood supply network and leading to local ischemia and hypoxia. This, in turn, induces osteoprogenitor cell apoptosis, ultimately resulting in necrotic tissue.

[0003] In related technologies, bone grafting is a common clinical intervention for achieving functional reconstruction of the damaged site. Traditional grafting methods typically use autologous or allogeneic bone with osteoconductive properties, but due to the lack of functional blood vessels, the implant is difficult to rapidly establish blood supply and is easily absorbed. Therefore, achieving effective coupling between functional vascular networks and bone regeneration, and developing biomimetic repair strategies to accelerate vascularized bone reconstruction, is key to treating large bone defects. Since vascularization is a slow process, with an average growth rate of only 5 μm per hour for newly formed capillaries, researchers have explored various methods to accelerate the vascularization process within implanted structures. For example, cytokines such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (FGF-2) are added to tissue-engineered scaffolds to induce angiogenesis; hollow channels are constructed using 3D printing and electrospinning techniques to promote host vascular infiltration. However, unidirectional ingrowth of peripheral vessels can easily cause necrosis in the central region of the implant, especially in critical-sized defects.

[0004] Therefore, there is an urgent need to develop a prevascularized bone repair scaffold with good mechanical properties, low hemolysis rate, and the ability to promote osteogenic differentiation and improve vascularization. Summary of the Invention

[0005] The first objective of this invention is to provide a method for preparing a prevascularized bone repair scaffold.

[0006] A second aspect of the present invention is to provide a prevascularized bone repair scaffold.

[0007] The third aspect of this invention aims to provide a method for preparing a prevascularized bone repair scaffold or the application of a prevascularized bone repair scaffold in the preparation of materials for repairing bone defects.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a method for preparing a prevascularized bone repair scaffold, comprising the following steps:

[0010] S1. Obtain a porous scaffold and perform sulfonation treatment to obtain a sulfonated scaffold;

[0011] S2. Prepare a mixed solution containing methacrylamide gelatin and extracellular matrix (ECM), and then add mesenchymal stem cells and / or endothelial cells to obtain a cell mixture preparation;

[0012] S3. The cell mixture is brought into contact with the sulfonated scaffold and cultured to obtain the final product.

[0013] According to some embodiments of the present invention, in step S1, the porous scaffold is a porous polyetheretherketone (PEEK) scaffold or a porous polyetherketone scaffold.

[0014] According to some embodiments of the present invention, the aperture of the porous support is 0.01 mm to 2.5 mm. For example, it can be 0.01, 0.1, 1, 1.2, 1.3, 1.4, 1.5, 1.8, 2.0, 2.2 or 2.5 mm, etc.

[0015] According to some embodiments of the present invention, the porosity of the porous support is 65% to 92%. Preferably, the porosity of the porous support is 80% to 92%, for example, it can be 80%, 82%, 85%, 88%, 90% or 92%, etc.

[0016] According to some embodiments of the present invention, in step S1, the specific method of the sulfonation treatment is as follows: the porous support is placed in concentrated sulfuric acid for treatment, followed by hydrothermal treatment, and then dried after cleaning.

[0017] According to some embodiments of the present invention, the temperature of the hydrothermal treatment is 95~110 °C.

[0018] According to some embodiments of the present invention, the hydrothermal treatment time is 3 to 5 hours.

[0019] According to some embodiments of the present invention, in step S2, the gelatin includes methacrylamide gelatin (GelMA) and / or methacrylamide hyaluronic acid gelatin.

[0020] According to some embodiments of the present invention, the final concentration of the methacrylamide gelatin in the mixed solution is 5% to 10%;

[0021] According to some embodiments of the present invention, the degree of substitution of the methacrylamide gelatin is 60% to 90%. For example, it can be 60%, 70%, 80%, or 90%, etc.

[0022] According to some embodiments of the present invention, in step S2, the final concentration of the extracellular matrix in the mixed solution is 1% to 5%.

[0023] According to some embodiments of the present invention, the extracellular matrix is ​​a bone-derived extracellular matrix.

[0024] Bone-derived extracellular matrix (ECM) can preserve the structure and function of the natural bone marrow microenvironment. For example, this matrix can well retain active components such as collagen, glycosaminoglycans, and growth factors, and can mimic the specific ecological niche in bone tissue, playing a role in regulating cell behavior and maintaining local microenvironment homeostasis. However, relying solely on ECM hydrogels cannot provide sufficient mechanical support in large bone defects.

[0025] According to some embodiments of the present invention, the content of glycosaminoglycans in the extracellular matrix is ​​2~8 μg / mg.

[0026] According to some embodiments of the present invention, the elastin content in the extracellular matrix is ​​5~8 μg / mg.

[0027] According to some embodiments of the present invention, in step S2, the mesenchymal stem cells include bone marrow mesenchymal stem cells (BMSCs).

[0028] According to some embodiments of the present invention, the endothelial cells include umbilical vein endothelial cells.

[0029] Endothelial cells are the main cells forming microvessels. Under normal two-dimensional culture conditions or without specific induction conditions, they typically do not spontaneously form budding structures. Previous studies have shown that endothelial cells and fibroblasts co-seeded in porous Cytopore 2 microcarriers and dynamically cultured in roller bottles for 48 hours before being embedded in fibrin gel can spontaneously form a microvessel-like network. Furthermore, this invention also reveals that BMSCs with higher differentiation capacity promote the vascularization of ECs, providing favorable conditions for constructing pre-vascularized bone tissue engineering scaffolds.

[0030] According to some embodiments of the present invention, in step S2, the mixed solution further comprises a free radical initiator.

[0031] According to some embodiments of the present invention, the free radical initiator includes LAP (Lithium phenyl-2,4,6-trimethylbenzoylphosphinate). Preferably, the final concentration of the LAP is 0.1% to 0.5%; more preferably, it is 0.2% to 0.3%.

[0032] According to some embodiments of the present invention, in step S2, the final concentration of the mesenchymal stem cells in the cell mixture is 0.5 × 10⁻⁶.6 ~2×10 7 per mL.

[0033] According to some embodiments of the present invention, the final concentration of the endothelial cells is 1 × 10⁻⁶. 6 ~4×10 7 per mL.

[0034] According to some embodiments of the present invention, the ratio of the mesenchymal stem cells to the endothelial cells is 1:1.8~2.2.

[0035] According to some embodiments of the present invention, in step S3, the contact includes injecting the cell mixture into the interior of the sulfonated scaffold.

[0036] According to some embodiments of the present invention, the culture medium used for the culture includes DMEM / F12 growth medium and ECM complete medium.

[0037] According to some embodiments of the present invention, the volume ratio of the DMEM / F12 growth medium to the ECM complete medium is 1:0.8~1.2.

[0038] In a second aspect, the present invention provides a prevascularized bone repair scaffold, which is prepared by the method for preparing the prevascularized bone repair scaffold described in the first aspect.

[0039] A third aspect of the present invention provides a method for preparing a prevascularized bone repair scaffold as described in the first aspect, or the application of a prevascularized bone repair scaffold as described in the second aspect in the preparation of materials for repairing bone defects.

[0040] The prevascularized bone repair scaffold of the present invention has at least the following beneficial effects:

[0041] (1) The prevascularized bone repair scaffold of the present invention has good hydrophilicity and excellent mechanical properties, which helps to promote cell attachment, tissue regeneration and biocompatibility, while supporting blood vessel sprouting and promoting cell migration.

[0042] (2) The prevascularized bone repair scaffold of the present invention has good cell compatibility and low hemolysis rate. In the presence of mesenchymal stem cells, the endothelial cells proliferate more significantly than those in single-cell culture.

[0043] (3) The prevascularized bone repair scaffold of the present invention has a high degree of vascularization.

[0044] (4) The prevascularized bone repair scaffold of the present invention has excellent early fixation and long-term stable osteogenic effects. By constructing a rabbit segmental bone defect model, the present invention found that the PVP-M and PVP-HM scaffolds have excellent early fixation and long-term stable osteogenic effects. In the PVP-M group, new bone grows from the defect edge to the inner region, but no obvious new bone tissue formation is observed in the central region of the scaffold. In the PVP-HM group, continuous callus is formed on both sides of the fracture end, and new trabecular bone structure is also visible in the central region of the scaffold. This indicates that the microvascular network formed in the PEKK-HM scaffold can provide a direct path for nutrient delivery and blood vessel ingrowth, and has significant advantages in promoting material exchange and bone tissue regeneration in the central region, and can significantly accelerate bone tissue regeneration.

[0045] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0046] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0047] Figure 1 The results show the acidic mucopolysaccharide content of natural bone and ECM, where a is H&E staining and b is AB&NFR staining.

[0048] Figure 2 The results show the main components of natural bone and ECM, where a is DNA content, b is collagen content, c is GAGs content, and d is elastin content.

[0049] Figure 3 The images show the CAD design top view and PLA printed physical images of the four types of brackets of this invention.

[0050] Figure 4 The present invention includes a design drawing and a physical photograph of the PEKK support structure.

[0051] Figure 5 The results of HUVEC skeleton staining and quantitative statistics after culturing PVP-H and PVP-HM for 1, 3, and 7 days are shown in the figure. Here, a is the skeleton staining result and b is the quantitative statistical result.

[0052] Figure 6 These are actual photographs and scanning electron microscope (SEM) images of the bracket of the present invention, where a is an actual photograph and b is a scanning electron microscope (SEM) image;

[0053] Figure 7 The images show the infrared spectra of the scaffold and its main components, with the left image showing the infrared spectrum of the scaffold and the right image showing the infrared spectra of the ECM, GelMA, and cross-linked hydrogel.

[0054] Figure 8 The surface roughness diagrams and quantitative analysis results of different supports of the present invention are shown below;

[0055] Figure 9 Surface hydrophilicity diagrams and quantitative analysis of different supports of the present invention;

[0056] Figure 10 The stress-strain curves of different supports in this invention are shown below;

[0057] Figure 11 Hemolytic performance diagrams and quantitative analysis of different stents of this invention;

[0058] Figure 12 This is a double staining image of live and dead cells on the PEKK composite scaffold of the present invention, where a represents the cell viability of BMSCs and b represents the cell viability of HUVECs.

[0059] Figure 13 This is a migration diagram of the HUVEC of the present invention;

[0060] Figure 14 This is a tubular diagram and quantitative analysis of the HUVEC of the present invention, where a is the tubular diagram and b is the quantitative analysis result;

[0061] Figure 15 The results of detecting the expression levels of angiogenesis-related genes in the stents of this invention are shown in Figure a, where a represents the expression levels of vascular-related genes in the two groups of stents after 7 days of culture, b represents VEGF in the culture medium on days 3 and 6, and c represents the change in the concentration of ANG-1.

[0062] Figure 16 The images show the expression levels of CD31 and EMCN genes in endothelial cells of two groups of scaffolds in this invention, as well as the results of vascular immunofluorescence analysis. In the images, a represents the expression levels of vascular-related genes in HUVECs cultured on the two groups of scaffolds for 7 days; and b represents the immunofluorescence staining image of CD31 and EMCN expression in HUVECs inside the scaffold.

[0063] Figure 17 The results of detecting ALP activity and osteogenic-related gene expression levels of BMSCs cultured on two scaffolds in this invention are shown in Figure a, where a represents ALP activity of BMSCs cultured on two scaffolds for 7 and 14 days, and b represents the expression level of osteogenic-related genes after 7 days of culture.

[0064] Figure 18 The results show the effect of HUVEC in the scaffold of this invention on osteogenic differentiation of BMSCs. In the figure, a is the change of BMP-2 concentration in the culture medium of the two scaffolds at 3 and 7 days, and b and c are the immunofluorescence staining images and quantitative analysis results of OCN in BMSCs in the scaffold at 14 days of culture; the cell nucleus is blue and the OCN is green.

[0065] Figure 19This is an immunofluorescence staining image of rabbit-derived primary umbilical vein endothelial cells and mesenchymal stem cells of the present invention, where a is endothelial cells and b is mesenchymal stem cells.

[0066] Figure 20 This is a surgical diagram of segmental bone defect in the rabbit femur according to the present invention;

[0067] Figure 21 The images shown are a photograph and b, taken 6 weeks after the invention, of the damaged area. Image a is the photograph, and image b is the CT scan. Detailed Implementation

[0068] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0069] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0070] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0071] Inventive concept:

[0072] This invention, for the first time, combines the mechanical advantages of PEKK with the biomimetic microenvironment of ECM to design a prevascularized PEKK-based porous scaffold (PVP). First, ECM was extracted from bovine cancellous bone, and its similarity to natural bone tissue components was verified. Second, the effect of ECM hydrogel on the budding behavior of encapsulated umbilical vein endothelial cells (HUVECs) was investigated. An in vitro co-culture system of HUVECs and BMSCs was constructed, and the ability of single-cell and two-cell cultures to form vascular networks and osteogenic differentiation on the scaffold was tested. Finally, the effect of the PVP scaffold on vascularized bone regeneration was evaluated using a rabbit segmental femoral defect model. This invention provides a new paradigm for developing bone repair materials that combine mechanical support and bioactivity, and has broad application potential.

[0073] The following will illustrate this with specific examples.

[0074] Example 1: PVP-M scaffold

[0075] This embodiment provides a method for preparing a prevascularized bone repair scaffold, which specifically includes the following contents.

[0076] 1. Extraction of bone-derived extracellular matrix

[0077] (1) Cut off the leg bone of the dairy cow, remove the hair and skin, cut the bone into pieces, soak it in 0.9% physiological saline, and boil it at 58°C for 3 hours.

[0078] (2) After draining the water, collect the cancellous bone part, add it to the mixer, add a small amount of liquid nitrogen, and quickly crush it.

[0079] Collect the bone fragments, wash them twice with pure water for 15 minutes each time, drain the water and weigh them.

[0080] (3) Add 25 mL of hydrochloric acid (0.5 mol / L) to each gram of bone fragments and stir at 1000 rpm for 24 hours at room temperature. Discard the hydrochloric acid and wash with plenty of pure water 3 times, 5 minutes each time.

[0081] (4) Prepare a chloroform / methanol mixed solution in a 1:1 ratio and stir at 600 rpm for 1 hour at room temperature.

[0082] (5) Wash with methanol 3 times and pure water 3 times, each time for 5 minutes. Then collect bone fragments by centrifugation and freeze-dry overnight.

[0083] (6) Dilute the pancreatic enzyme at a ratio of 1:4, add 40 mL of pancreatic enzyme dilution to each gram of bone fragments, seal the container and shake it in a shaker at 37°C for 24 hours.

[0084] (7) Wash twice with PBS, each time for 15 minutes.

[0085] (8) Prepare a 1% penicillin-streptomycin solution with PBS, stir at 4°C for 24 hours, and freeze dry overnight.

[0086] (9) Prepare a 1 mg / mL pepsin solution using 0.01 M hydrochloric acid. Add 0.1 mL of pepsin solution to each milligram of bone fragments and digest at room temperature for 96 hours.

[0087] (10) Transfer the digested bone fragments to a 15% NaCl solution, stir for 20 minutes at room temperature, and then transfer to 4°C and continue stirring for 2 hours.

[0088] (11) Collect bone fragments, add pure water and stir well, then dialyze using a 3500 Da dialysis bag at 4°C until the solution is colorless. Finally, freeze-dry and store at -20°C for later use. The obtained sample is named ECM.

[0089] 2. Characterization of bone-derived extracellular matrix

[0090] The ECM obtained above was characterized in comparison with natural bone tissue, and the specific methods are as follows:

[0091] (1) Detection of acidic mucopolysaccharide content:

[0092] First, the ECM and natural bone tissue were fixed with 4% paraformaldehyde. The natural bone tissue was then immersed in EDTA decalcification solution and decalcified in a constant-temperature shaker until softened. The softened bone tissue was then subjected to ethanol gradient dehydration. Afterward, the natural bone tissue and ECM were embedded in paraffin wax, cut into 10 μm sections using a microtome, and the residual cellular components were observed by hematoxylin and eosin (H&E) staining. The distribution of acidic mucopolysaccharides was analyzed by Alcian blue and nuclear solid red (AB&NFR) staining.

[0093] (2) Hydroxyproline content detection:

[0094] To determine the hydroxyproline content in bone tissue and ECM, 10 mg of sample was added to 1.0 mL of 6.0 M HCl solution and heated at 100°C for 5 hours until no solid residue remained. The hydrolyzed sample was rinsed with running water and cooled to room temperature. The hydroxyproline content of each sample was determined using a hydroxyproline assay kit. Collagen content was calculated based on approximately 13.4% hydroxyproline content in collagen.

[0095] (3) Detection of glycosaminoglycan content:

[0096] To determine the glycosaminoglycan content, 5.0 mg of natural bone tissue and ECM were added to 5.0 mL of papain digestion solution and digested in a 60°C water bath for 16 hours. After digestion, the sample was centrifuged at 7000 rpm for 10 minutes to remove cell debris. 1.0 mL of the supernatant was used to determine the glycosaminoglycan content using the dimethyl methylene blue staining method, with chondroitin sulfate as a standard sample.

[0097] (4) DNA concentration detection:

[0098] To determine the DNA concentration in the sample, 10 mg of natural bone tissue and ECM were added to 5.0 mL of papain digestion solution and digested in a 60°C water bath in the dark for 16 hours. After digestion, the supernatant was separated by centrifugation, and 1.0 mL was taken and the DNA concentration in the sample was determined using the Quant-iT™ PicoGreen™ dsDNA detection kit.

[0099] (5) Elastin content detection:

[0100] Take 10 mg of natural bone tissue and ECM, add 10 mL of PBS, and homogenize the sample thoroughly using a tissue homogenizer. After homogenization, incubate the sample overnight at 4°C to allow it to fully dissolve. Centrifuge at 5000 rpm for 10 minutes, collect the supernatant, dilute to 100 mL, and determine the elastin content in the sample using an elastin ELISA kit.

[0101] Staining results of natural bone and ECM as follows: Figure 1 As shown, compared to natural bone tissue, cell nuclei in ECM are almost entirely removed, while glycosaminoglycans and collagen fibers are largely retained.

[0102] The main component test results are as follows Figure 2 As shown, the collagen content in ECM was 524.75±35.49 μg / mg, higher than that in natural bone tissue; the glycosaminoglycan content was 4.57±2.08 μg / mg, and the elastin content was 6.00±0.75 μg / mg, similar to that in natural bone tissue. This indicates that the ECM extraction process successfully removed the cellular components of bone tissue while retaining the main active ingredients, which is beneficial for mimicking the microenvironment of natural bone tissue. Furthermore, the DNA content in ECM was 10.82 ng / mg, a 93.2% reduction compared to natural bone tissue, meeting the requirement of less than 50 ng / mg in the national standard GB-T16886.

[0103] 3. Structural Design of PEKK Support

[0104] Different support structures were designed using CAD software, and the porosity (ф) of the support was derived: ф = (Vtotal - Vmaterial) / Vtotal × 100%. In this embodiment, four three-dimensional models were first constructed using computer-aided design (CAD) software, with structures as follows: Figure 3 As shown in the diagram. All scaffolds have a wire diameter of 400 μm. The first scaffold has a uniform porous structure with a pore size of 500 μm and a porosity of 67.1%. The second scaffold simulates the hollow structure of a long bone, with a dense outer layer and a sparse inner layer, and a maximum pore size of 1.4 mm × 1.3 mm in the middle, with a porosity of 89.2%. The third scaffold has uniform triangular pores with a base diameter of 3 mm × a height of 2.2 mm and a porosity of 85.5%. The fourth scaffold also has a biomimetic structure with a maximum pore size of 1.8 mm × 1.8 mm in the middle and a porosity of 85.8%. The printability of different structures was verified using PLA filament. The results showed that only the fourth structure could not be printed completely. Based on a comprehensive consideration of the scaffold structure in terms of porosity and biomimetic properties, the second scaffold structure was ultimately selected.

[0105] A porous PEKK scaffold with a diameter of 7 mm, a height of 15 mm, a maximum central aperture of 1.4 mm × 1.3 mm, and a porosity of 89.2% was printed using a 3D medical printer and matching PLA filament. The PEKK scaffold structural design drawing and actual product image are shown below. Figure 4 As shown.

[0106] 4. Fabrication of composite scaffolds

[0107] The composite scaffold sample was prepared using the PEKK scaffold printed by the above-mentioned fused deposition modeling technology. The preparation process includes the following steps:

[0108] S1. Immerse the PEKK stent in concentrated sulfuric acid for 1 minute, then place it in pure water. Place the stent in a reaction vessel filled with 2 / 3 pure water and heat at 100°C for 4 hours. After cooling to room temperature, wash with water and alcohol, then dry. This sample is labeled SPEKK. Then, sterilize the SPEKK by immersing it in 75% ethanol, followed by washing in sterile PBS and air-drying.

[0109] S2. Weigh the ECM using a balance, then place the ECM in a petri dish. Sterilize it by irradiating it with a 254 nm UV lamp for 20 minutes, turning it off for 5 minutes, and then irradiating it for another 20 minutes. After that, seal the dish and place it in a laminar flow hood overnight for later use.

[0110] S3. Dissolve 5% methacrylamide gelatin (GelMA, degree of substitution 90%) in PBS solution containing 0.25% LAP (Lithium phenyl-2,4,6-trimethylbenzoylphosphinate). Heat to 50 °C in the dark and filter the solution through a 0.22 μm membrane. After it returns to room temperature, add the sterilized ECM to the solution and stir in the dark for 2 hours to obtain a GelMA / ECM mixed solution (containing 5% GelMA and 2% ECM).

[0111] S4. Mesenchymal stem cells (BMSCs) were digested with trypsin, and the digestion was terminated in DMEM / F12 complete medium. Cell counts were then performed. The cell pellet was collected by centrifugation at 1500 rpm for 2 minutes and added to the prepared GelMA / ECM mixture to achieve a BMSC concentration of 1.5 × 10⁻⁶. 6 The sample was collected at a concentration of 1 / mL and then injected into the SPEKK scaffold prepared above. The sample was then irradiated with 405 nm UV light for 3 minutes. The sample was collected and placed in a well plate with mixed culture medium (DMEM / F12 growth medium: ECM complete medium = 1:1) for later use. This sample was designated as PVP-M.

[0112] Example 2: PVP-H support

[0113] This embodiment provides a method for preparing a prevascularized bone repair scaffold, which differs from Embodiment 1 in that: in the preparation process of the composite scaffold (step S4), mesenchymal stem cells (BMSCs) are replaced with human umbilical vein endothelial cells (HUVECs), and the concentration of HUVECs in the injection solution reaches 3.0 × 10⁻⁶. 6 Quantity / mL. The remaining steps are the same.

[0114] The sample prepared using the above method is designated as PVP-H.

[0115] Example 3: PVP-HM scaffold

[0116] This embodiment provides a method for preparing a prevascularized bone repair scaffold, which differs from Embodiment 1 in that step S4 differs in the preparation process of the composite scaffold, as follows:

[0117] Mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) were digested separately with trypsin. Cell counts were performed after digestion was terminated with culture medium. The cell pellet was collected by centrifugation at 1500 rpm for 2 minutes and added to the prepared GelMA / ECM mixed solution to achieve a BMSC concentration of 1.5 × 10⁻⁶. 6 The concentration of HUVEC reached 3.0 × 10⁶ cells / mL. 6 Samples / mL. After mixing thoroughly, the solution is injected into the SPEKK scaffold and irradiated with 405 nm UV light for 3 minutes. The samples are then collected and placed in well plates with mixed culture medium (DMEM / F12 growth medium: ECM medium = 1:1) for later use. This sample is designated as PVP-HM.

[0118] Comparative Example 1: PEKK stent

[0119] This comparative example provides a PEKK stent, which is obtained using the preparation method of the PEKK stent in Example 1.

[0120] Comparative Example 2: SPEKK Bracket

[0121] This comparative example provides an SPEKK stent, which is obtained using the preparation method of the SPEKK stent in Example 1.

[0122] Comparative Example 3: PVP Bracket

[0123] This comparative example provides a PVP scaffold, which differs from Example 1 in that step S4 of the composite scaffold preparation process is different, namely, mesenchymal stem cells (BMSCs) and / or human umbilical vein endothelial cells (HUVECs) are not added, as detailed below:

[0124] After thoroughly mixing the GelMA / ECM solution, inject it into the SPEKK scaffold and irradiate it with 405 nm UV light for 3 minutes. Collect the sample and place it in a well plate with mixed culture medium (DMEM / F12 growth medium: ECM medium = 1:1) for later use. This sample is designated as PVP.

[0125] Example 1: The effect of cells on angiogenesis

[0126] To enhance the angiogenesis potential of prevascularized scaffolds, HUVECs and BMSCs were simultaneously added to the hydrogel at a cell density of 2:1. The group without BMSCs was designated the PVP-H group, and the group with BMSCs was designated the PVP-HM group. After addition, the capillary network morphology was observed during culture.

[0127] HUVEC skeleton staining results are as follows Figure 5 As shown in Figure a, after 1 day of culture, HUVECs in both groups were not fully expanded and the cells were spherical. After 3 days of culture, HUVECs began to differentiate. The PVP-H group had fewer budding endothelial cells, while the PVP-HM group had more budding endothelial cells, and the formation of three-dimensional capillary-like tubular structures could be preliminarily observed. After 7 days of culture, only a few interconnected capillary networks formed in the PVP-H group, while the PVP-HM group formed extensive vascular-like structures through vascular budding.

[0128] Furthermore, quantitative analysis of the vascular-like structures was performed, and the results were as follows: Figure 5 As shown in b, on day 3, the number of crosspoints in the PVP-H group was similar to that in the PVP-HM group, but the total vessel length in the PVP-HM group increased by 32.1%. After 7 days of culture, the number of crosspoints in the PVP-HM group was 79.2% higher than that in the PVP-H group, and the total vessel length increased by 36.0% compared to the PVP-H group. This indicates that BMSCs accelerated the angiogenesis process of HUVECs and promoted the formation of a stable microvascular system by the PVP scaffold after 7 days of in vitro culture.

[0129] Example 2: Characterization of Composite Stents

[0130] This embodiment characterizes the PEKK, SPEKK, and PVP scaffold materials obtained in the above embodiments, specifically including the following contents.

[0131] 1. Scanning electron microscopy observation

[0132] The stent was washed with pure water and then freeze-dried. The surface morphology of the stent was observed using a scanning electron microscope. Optical photographs and surface morphology of the stent are shown below. Figure 6As shown, the scaffold has a dense outer layer and a sparse inner layer, with the internal pores filled with hydrogel. Scanning electron microscopy revealed that the smooth surface of PEKK makes it difficult for cells to attach and grow, affecting initial cell spreading and subsequent growth and differentiation. During the sulfonation reaction, the introduced sulfonic acid groups (-SO3H) increase the distance between PEKK molecular chains, leading to internal stress and the formation of a microporous structure, which significantly increases the specific surface area of ​​PEKK. The hydrogel in the central part of the scaffold forms a three-dimensional network structure through cross-linking, providing ample three-dimensional growth space for cells and allowing them to migrate and interact through the pores, promoting cell regeneration and repair.

[0133] 2. FTIR spectral analysis

[0134] Fourier transform infrared spectroscopy was used at 4000-400 cm⁻¹. -1 Detecting functional group information of hydrogels. FTIR spectroscopy analysis, such as... Figure 7 As shown, PEKK, SPEKK, and PVP are displayed at 1652 cm. -1 1493 cm -1 and 918 cm -1 A C=O stretching vibration peak caused by the stretching vibration of the aromatic group in PEKK was observed at position 1452 cm⁻¹. SPEKK showed a peak at 1452 cm⁻¹. -1 An asymmetric stretching peak with O=S=O appeared at 1082 cm⁻¹. -1 A symmetrical stretching peak with O=S=O appeared at 3272 cm⁻¹, and at the same time, a peak was observed at 3272 cm⁻¹. -1 A new peak appeared at this position, which may be the OH bond in the SO3H group. Three characteristic peaks of amide bonds appeared in the infrared spectra of ECM, GelMA, and the cross-linked hydrogel, namely amide I (1630 cm⁻¹). -1 ), Amide II (1540 cm) -1 ) and amide III (1236 cm) -1 It is worth noting that the ECM is 3299 cm. -1 The peak shifted to 3283 cm⁻¹ at Hydrogel. -1 At this location, a significant redshift is observed, indicating that hydrogen bonds have formed between ECM and GelMA, significantly enhancing the intermolecular interaction forces and providing strong evidence for the formation of ECM-GelMA hydrogels.

[0135] 3. Surface roughness

[0136] Surface roughness is an important factor affecting the performance of tissue-engineered scaffolds in bone regeneration, and is usually quantified by the Ra value; the lower the Ra value, the smoother the surface. In this test example, the surface roughness Ra of the scaffold was determined using a material friction and wear testing machine.

[0137] Test results as follows Figure 8 As shown, PEKK has a smooth surface and the lowest roughness; the micropores on the surface of SPEKK allow the scaffold to achieve an optimal surface roughness range (0.5-2 μm), which provides sufficient adhesion sites for cells without causing excessive mechanical stress to them. Since the PVP test uses a freeze-dried composite scaffold, it exhibits the highest roughness.

[0138] 4. Hydrophilicity test

[0139] The hydrophilicity of scaffolds has a significant impact on cell adhesion, tissue regeneration, and biocompatibility. More hydrophilic surfaces can significantly enhance cell adhesion by reducing the interfacial tension between the material and cells. The surface hydrophilicity of a material is usually measured by its contact angle. In this test example, a contact angle meter was used to determine the water contact angle of the scaffold surface to characterize its hydrophilic properties.

[0140] The results are as follows Figure 9 As shown, droplet morphology observation reveals that the droplets on the PEKK sample exhibit obtuse angles at the interface, while their interfaces with the other two samples show acute angles. Quantitative analysis indicates that the water contact angle of PEKK is 112.46°±4.01°, classifying it as a hydrophobic surface, while the water contact angle of SPEKK is 70.83°±2.43°, classifying it as a hydrophilic surface. This is likely due to the introduction of hydrophilic sulfonic acid groups during the sulfonation reaction. The water contact angle of PVP is 13.29°±2.02°, primarily because the ECM filling the scaffold is a complex network structure composed of biomacromolecules such as proteins and polysaccharides, containing various hydrophilic components, thus making the scaffold as a whole hydrophilic.

[0141] 5. Mechanical property testing

[0142] The mechanical properties of scaffolds are closely related to the bone regeneration process. Softer matrix materials can support blood vessel budding and promote cell migration, making them more suitable for angiogenesis. However, the differentiation of osteoprogenitor cells and the maintenance of bone tissue usually depend on harder matrix materials. In this test, the compressive modulus and stress-strain curves of the scaffold were determined using a universal testing machine.

[0143] The results are as follows Figure 10 As shown, the PVP composite scaffold has a compressive strength of 5.1 MPa and an elastic modulus of 31.3 MPa. The similarity of this composite scaffold to the mechanical properties of cancellous bone indicates its potential application in the field of bone tissue regeneration.

[0144] Test Example 3: Hemolytic Test

[0145] This test example examines the hemolytic activity of the PEKK, SPEKK, and PVP stent materials prepared in the above embodiments. The specific test method is as follows:

[0146] One mL of anticoagulated bovine whole blood was incubated at 3000 rpm for 5 min, and the supernatant was discarded. 50 mL of physiological saline was added to prepare a 2% diluted blood solution. The stents were then immersed in 1 mL of this diluted blood and incubated at 37°C for 1 h. The physiological saline group served as the negative control group, and the group receiving 0.1% Triton X-100 served as the positive control group. After incubation, the samples were centrifuged at 3000 rpm for 5 min, and the samples were photographed. 100 μL of the supernatant from each sample was added to a 96-well plate, and the absorbance was measured at 540 nm. The hemolysis rate (HI) was calculated as (AB) / (CB) × 100%, where A represents the absorbance of the sample group; B represents the absorbance of the negative control group; and C represents the absorbance of the positive control group.

[0147] The results are as follows Figure 11 As shown, 0.1% Triton X-100 was used as the positive control group, and 0.9% NaCl was used as the negative control group. The experimental results showed that the hemolysis rates of PEKK, SPEKK, and PVP were 0.45%, 1.58%, and 0.90%, respectively, all below 5%. Therefore, the PEKK composite scaffold exhibits good blood compatibility. This excellent performance is mainly attributed to the porous structure design of PEKK, which avoids mechanical hemolysis; simultaneously, the hydrophilicity of the PEKK / ECM scaffold effectively reduces erythrocyte membrane damage by lowering interfacial tension, thereby further enhancing blood compatibility.

[0148] Example 4: Cell compatibility test

[0149] This test example describes the cell compatibility testing of the PVP-M, PVP-H, and PVP-HM scaffold materials prepared in the above embodiments. The specific testing methods are as follows:

[0150] 1. Cell extraction and culture

[0151] In vitro cell experiments used human umbilical vein endothelial cells (HUVECs) and rat bone marrow mesenchymal stem cells (BMSCs). HUVECs are immortalized cell lines, and the growth medium used was 89% high-glucose DMEM medium, 10% fetal bovine serum, and 1% penicillin-streptomycin solution. In vivo animal experiments used rabbit umbilical vein endothelial cells (P3) and rabbit bone marrow mesenchymal stem cells (P3).

[0152] Primary rabbit umbilical vein endothelial cells were purchased from iCell. The culture medium used was ECM medium (500 ml basal medium, 25 ml fetal bovine serum, 5 ml endothelial cell growth supplement) and 5 ml penicillin / streptomycin solution (P / S). Primary rabbit bone marrow mesenchymal stem cells were extracted from 1-2 month old male New Zealand rabbits. After euthanasia, both hind limbs were collected and disinfected by immersing in 75% ethanol solution for 10 minutes. They were then transferred to a laminar flow hood, the skin of the legs was peeled off, and the femur and tibia were carefully removed. The femur and tibia were immersed in 10% penicillin / streptomycin solution, and the surrounding muscles and connective tissue were carefully removed. The femur and tibia were separated, ensuring the integrity of the medullary cavity. The ends of the long bones were then cut open, and ECM medium was used to flush the medullary cavity. The flushing fluid was collected into cell culture dishes. The rinsing fluid from one femur and tibia was cultured in a 10 cm culture dish and placed in an incubator at 37°C with 5% CO2. The medium was changed every 2 days. When the cell confluence reached more than 85%, the cells were passaged.

[0153] 2. Fluorescent staining

[0154] The purity of the two primary cell lines was identified by immunofluorescence staining for von Willebrand factor (vWF) and CD44, respectively: cell spreaders were placed in 24-well plates, and 2.0 × 10⁶ cells were seeded per well. 4 Cells were cultured in an incubator for 2 hours or overnight. The culture medium was removed from the wells, and the cells were washed once with PBS. Then, 4% paraformaldehyde solution was added and the cells were fixed at 4°C for 30 minutes. The cells were then washed three times with PBS for 5 minutes each time. After this procedure, the slides were incubated overnight at 4°C.

[0155] Preparation of blocking solution for glass slides: Mix 0.5% Triton X-100 solution with PBS at a 1:1 volume ratio, and then add 10% serum. After aspirating any residual liquid from the surface of the slides, place them in a culture dish, add 50 μL of blocking solution to each slide, and perform permeabilization treatment at room temperature for 2 hours.

[0156] After permeation and blocking, the vWF antibody was diluted with PBS at a ratio of 1:100, and the CD44 antibody was diluted with PHEBS at a ratio of 1:100. 50 μL of each diluted solution was dropped onto a glass slide and incubated overnight at 4°C.

[0157] Under room temperature and light-protected conditions, goat anti-rabbit IgG (H+L) secondary antibody was diluted with PBS at a ratio of 1:500 and incubated with rabbit umbilical vein endothelial cells and rabbit primary bone marrow mesenchymal stem cells, respectively. After incubation for 2 hours, the cells were washed three times with PBS for 5 minutes each time, and then stained with DAPI (DAPI:PBS=1:1000) for 5 minutes. The washing was repeated three times. After incubation for 2 hours, the cells were washed three times with PBS (5 minutes each time), and the same DAPI staining and washing procedures were performed.

[0158] One drop of fluorescent mounting medium was placed on the surface of the glass slide, and the image was acquired using a fluorescent inverted microscope.

[0159] 3. Cell compatibility testing of composite scaffolds

[0160] The scaffolds to be tested were placed in well plates and cultured for 1, 3, and 7 days. The viability and proliferation of cells in the scaffolds were assessed by observing the fluorescence inside the scaffolds using a Live / Dead cell viability assay kit.

[0161] Results of double staining of live and dead cells as follows Figure 12 As shown, the two cell types are uniformly distributed in the hydrogel. While cell aggregation may be observed in the two-dimensional image, it is clear that HUVECs proliferate more significantly in the presence of BMSCs than in single-cell culture, and BMSCs show a similar trend. Fluorescence analysis revealed that the cell viability of BMSCs after 1, 4, and 7 days of culture in the PVP-M scaffold was 89.49%, 92.66%, and 96.65%, respectively, while in the PVP-HM scaffold, the viability was 89.09%, 95.80%, and 92.06%. Similarly, the cell viability of HUVECs after 1, 4, and 7 days of culture in the PVP-H scaffold was 89.81%, 85.43%, and 78.70%, respectively, while in the PVP-HM scaffold, the viability was 80.82%, 87.44%, and 85.37%.

[0162] The above results indicate that ECM provides a favorable microenvironment for cell survival, UV irradiation does not affect the survival of cells encapsulated in the PEKK composite scaffold, and the designed structure provides ample space for nutrient exchange.

[0163] Example 5: Characterization of the microvascular network in a composite stent

[0164] This test example characterizes the microvascular network of the PVP-M, PVP-H, and PVP-HM stent materials prepared in the above embodiments. The specific methods are as follows:

[0165] 1. Scratch test

[0166] In the migration experiment of HUVECs, HUVECs were transported at a rate of 5.0 × 10⁻⁶. 4Cells were evenly seeded at a density of cells / well in 24-well plates until a monolayer of cells covered the wells. Then, using a sterile 10 μL pipette tip, a straight, uniform-width scratch was made vertically along a pre-marked reference line at a constant speed from the bottom of the plate. The cells were gently rinsed three times with pre-warmed PBS to remove detached cells. The medium was then replaced with high-glucose medium, and a support was placed on top of the Transwell chamber. The culture plate was continuously incubated at 37°C in a 5% CO2 incubator. At 0, 4, 8, 12, and 24 hours, the same scratched area was continuously observed and imaged using an inverted microscope. Finally, quantitative evaluation was performed using ImageJ software.

[0167] The results of the scratch test are as follows Figure 13 As shown, after 4 hours of culture, the scratch area in the PVP and PVP-M groups was significantly reduced compared to the control group. After 24 hours, the control group still had a large scratch area, the scratch area in the PVP group decreased further, while the PVP-M group almost completely healed. Quantitative analysis of the scratch healing rate was consistent with the observation results. After 24 hours of in vitro culture, the healing rate was 61.95±1.84% in the control group, 74.42±4.58% in the PVP group, and 96.81±1.95% in the PVP-M group. This indicates that ECM retains pro-angiogenic cytokines, which can enhance the migration ability of HUVECs, and the pro-angiogenic cytokines secreted by BMSCs can further promote the migration of HUVECs, which is a key condition for pre-angiogenesis.

[0168] 2. Tube forming experiment

[0169] In the tube formation experiment of HUVEC, the substrate gel was first thawed at 4°C. Using a pre-cooled pipette tip, 150 μL of substrate gel was added to each well of a 24-well plate, ensuring it completely and evenly covered the bottom surface. The plate was then placed in a 37°C incubator for 30-60 minutes to allow the substrate gel to completely gel. 1.0 × 10⁶ μL of substrate gel was seeded per well. 5 HUVECs were placed in the upper chamber of a Transwell incubator at 37°C with 5% CO2. Tubular structure formation was observed using an inverted microscope at 2, 4, and 6 hours, and vessel length and branch number were calculated in three random regions using ImageJ software.

[0170] The results are as follows Figure 14As shown, in the blank group, only a small number of effective lumens could be observed after 6 hours of culture; in the PVP group, no significant changes were observed after 2 hours of culture, multiple small tubes could be observed after 4 hours of culture, and a partially integrated network of small tubes could be observed after 6 hours of culture; while in the PVP-M group, scattered punctate nodules or dendritic protrusions could be observed after 2 hours of culture, lumen formation could be observed after 4 hours of culture, and the lumen was completely closed after 6 hours of culture. This indicates that the degree of vascularization in the PVP-M group was significantly higher than that in the other groups.

[0171] Furthermore, calculations of tubule length and branch number under high-power fields revealed that the PVP group accelerated tubule formation after 4 hours, while the PVP-M group maintained the strongest tubule formation capacity from 2 hours onwards. These results indicate that cytokines retained within the ECM promote HUVEC differentiation, leading to tubule formation, while the paracrine function of BMSCs accelerates lumen formation, enabling early vascularization and osteogenic processes. Through the synergistic effect of microenvironment regulation and paracrine signaling, both facilitate the transition from single-cell behavior to a capillary-like network.

[0172] 3. Detection of angiogenesis-related gene expression levels

[0173] This experiment assessed the relative levels of the angiogenesis-related genes VEGF and ANG-1 in endothelial cells using qPCR. Figure 15 Results a showed that after 7 days of culture, the expression level of VEGF in the PVP-HM group was higher than that in the PVP-H group. EILSA was used to test the concentrations of VEGF and ANG-1 secreted by the PVP-H and PVP-HM groups. Figure 15 (b and c in the original text). The results showed that after 3 days of in vitro culture, the concentration of VEGF in the PVP-H group was 156.82 pg / mL and the concentration of ANG-1 was 12.37 ng / mL, while the concentration of VEGF in the PVP-HM group was 292.75 pg / mL and the concentration of ANG-1 was 17.89 ng / mL. After 6 days of in vitro culture, the concentration of VEGF in the PVP-H group was 120.55 pg / mL and the concentration of ANG-1 was 10.05 ng / mL, while the concentration of VEGF in the PVP-HM group was 316.13 pg / mL and the concentration of ANG-1 was 19.49 ng / mL.

[0174] The above results indicate that MSCs added to the stent can stimulate angiogenesis by secreting VEGF-A, increase microvascular density, increase the secretion of Ang-1, stabilize blood vessels, and improve the maturity of the lumen.

[0175] 4. Capillary subtype detection

[0176] Capillary subtypes in bone tissue exhibit high heterogeneity, and can be classified into H-type, R-type, S-type, and traditional sinusoidal capillaries (possibly corresponding to L-type). These subtypes show significant differences in anatomical location, molecular markers, and function. To further investigate the capillary subtypes formed in pre-vascularized scaffolds, this experiment used qPCR to detect the expression levels of CD31 and EMCN genes in endothelial cells of two scaffold groups, and also performed vascularization immunofluorescence analysis. The specific methods are as follows:

[0177] The scaffolds were cultured in a mixed medium at 37°C for 7 days. After washing the scaffolds three times with PBS, 4% paraformaldehyde was added and incubated at room temperature for 30 minutes. Then, 1 mL of 0.1% Triton X-100 was added for permeation for 10 minutes, followed by blocking with 1% BSA-PBS solution for 30 minutes. The scaffolds were washed three times before each addition of new solution. The scaffolds were then immersed in a mixed primary antibody solution of CD31 (1:300) and EMCN (1:200) and incubated overnight at 4°C. After washing three times with PBS, the corresponding secondary antibody mixture (1:500) was added and incubated at room temperature for 1 hour. After washing, DAPI solution was added at a 1:1000 concentration and incubated at room temperature for 10 minutes. A 3D image (736.57 μm × 736.57 μm × 69 μm) was captured using a confocal microscope.

[0178] The expression levels of CD31 and EMCN genes are as follows: Figure 16 As shown in Figure a, after 7 days of culture, the expression levels of CD31 and EMCN genes in the PVP-HM group were higher than those in the PVP-H group. Subsequently, immunofluorescence staining of CD31 and EMCN was performed on the PVP-HM and PVP-H scaffolds after one week of in vitro culture. The results are shown in Figure a. Figure 16 As shown in b, HUVECs in both groups of stents exhibited budding, forming luminal structures. The positive expression levels of CD31 and EMCN in the PVP-HM group were significantly higher than those in the PVP-H group. This indicates that the paracrine function of MSCs can promote the formation of HUVECs into an H-type vascular network.

[0179] Example 6: Osteogenic Differentiation Capacity Detection of BMSCs

[0180] To assess the osteogenic differentiation potential of BMSCs within the prevascular scaffold, this test case involved culturing a PEKK / ECM scaffold (PVP-M prepared in Example 1) containing only BMSCs and a PEKK / ECM scaffold (PVP-HM) containing BMSCs / HUVECs in an osteogenic induction environment (osteogenic medium: ECM medium = 1:1) and performing relevant tests.

[0181] 1. Alkaline phosphatase activity detection

[0182] The early osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) was observed by detecting the activity of alkaline phosphatase (ALP) on days 7 and 14. The specific method is as follows:

[0183] After culturing the encapsulated cells in hydrogels in osteogenic induction medium (osteogenic medium: ECM medium = 1:1) for 7 and 14 days, the cells were washed three times with low-temperature PBS, and then 0.2% Triton-X100 was added. The samples were then homogenized using a sterile, enzyme-free tissue homogenizer and placed on ice for 30 minutes. After centrifugation at 7000 rpm for 5 minutes, the supernatant was collected for ALP activity assay.

[0184] Test results as follows Figure 17 As shown in Figure a, ALP activity in the PVP-HM group was higher than that in the PVP-M group on day 7. On day 14, ALP activity in both groups decreased slightly. Although the ALP activity in the PVP-HM group was higher, it was not significantly different from that in the PVP-M group. Further co-culturing BMVs and RUVEC-BMVs with BMSCs revealed that the RUVEC-BMV group showed greater ALP expression and calcium nodule formation, as well as higher osteogenic gene and protein expression than other groups. These results indicate that the presence of HUVECs contributes to osteogenic differentiation of BMSCs.

[0185] 2. Detection of expression levels of osteogenic-related genes

[0186] To further investigate the effects of HUVECs encapsulated in the scaffold on osteogenic differentiation-related genes in bone marrow mesenchymal stem cells (BMSCs), this experiment used RT-qPCR to detect the expression of four osteogenic-related genes in BMSCs: RUNX2, ALP, COLⅠ, and OCN. The specific detection methods are as follows:

[0187] The scaffold was cultured in culture medium (osteogenic medium: ECM medium = 1:1) for 7 days before PCR testing. The results are as follows: Figure 17 As shown in b, after 7 days of scaffold culture in osteogenic induction medium, the gene expression of BMSCs in the PVP-HM group was significantly higher than that in the PVP-M group, indicating that the paracrine function of HUVECs can promote the differentiation of BMSCs towards osteogenic direction.

[0188] 3. BMP-2 enzyme-linked immunosorbent assay

[0189] To further investigate the mechanism of action of HUVEC on BMSCs, this experiment used ELISA to test the concentration of BMP-2 in the culture medium of the two scaffolds after 3 and 6 days of culture. The specific method is as follows:

[0190] The scaffold was placed in a 24-well plate, 1 mL of growth medium was added, and the plates were incubated for 3 days and 7 days, respectively. The culture medium was then collected, the supernatant was collected by centrifugation, and the relevant cytokines released by the cells in the scaffold were detected using the BMP-2 enzyme-linked immunosorbent assay kit.

[0191] Test results as follows Figure 18 As shown in Figure a, after 3 days of in vitro culture, the concentration of BMP-2 in the culture medium of the PVP-M group was 1042.00 pg / mL, and that of the PVP-HM group was 1970.67 pg / mL. After 7 days of in vitro culture, the concentration of BMP-2 in the culture medium of the PVP-M group was 1567.33 pg / mL, and that of the PVP-HM group was 2481.34 pg / mL. The concentrations in both groups were significantly lower in the PVP-M group than in the PVP-HM group, indicating that HUVECs can promote osteogenic differentiation of BMSCs by secreting BMP-2.

[0192] 4. Immunofluorescence analysis

[0193] OCN expression typically increases significantly in the later stages of osteogenic differentiation and is a characteristic marker protein secreted by mature osteoblasts. To further verify the promoting effect of HUVECs on osteogenic differentiation of BMSCs, this experiment observed the protein expression of OCN in BMSCs within two scaffolds after 14 days of in vitro culture using immunofluorescence staining. The specific methods are as follows:

[0194] The scaffolds were cultured in osteogenic induction medium at 37°C for 14 days. After fixation, permeabilization, and blocking, the scaffolds were immersed in OCN (1:300) primary antibody solution and incubated overnight at 4°C. After washing three times with PBS, the corresponding secondary antibody solution (1:500) was added, and the scaffolds were incubated at room temperature for 1 hour. After washing, DAPI solution was added at 1:1000, and the scaffolds were incubated at room temperature for 10 minutes. Three-dimensional images were then captured using a confocal microscope.

[0195] Test results as follows Figure 18 As shown in Figures b and c, the green fluorescence area in the PVP-HM group is higher than that in the PVP-M group. Semi-quantitative analysis shows that the fluorescence intensity of OCN in the PVP-HM group is approximately twice that of the PVP-M group, indicating that the PVP-HM group has more significant OCN protein expression, and the paracrine function of HUVECs can enhance the osteogenic differentiation effect of BMSCs.

[0196] Example 7: In vivo experiment

[0197] Based on the results of the above in vitro studies, this study further investigated the angiogenesis and osteogenic function of the prevascularized PEKK composite scaffold in a rabbit segmental bone defect model. Specifically, the following content was included.

[0198] 1. Obtaining rabbit primary cells

[0199] To avoid immune rejection of xenogeneic cells by the host tissue, primary rabbit cells of two seed cell types were first extracted in this experiment. To ensure the purity of the extracted umbilical vein endothelial cells (UVECs) and BMSCs, immunofluorescence analysis was used; UVECs were stained with vWF antibody, and BMSCs were stained with CD44 antibody.

[0200] Test results as follows Figure 19 As shown, the purity of both cell types reached over 90%, laying a solid foundation for subsequent animal experiments.

[0201] 2. Obtain PVP, PVP-M, or PVP-HM brackets.

[0202] Referring to the methods in Examples 1 and 3, mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs) were replaced with the rabbit-derived primary cells (UVECs and BMSCs) prepared above to prepare the corresponding scaffolds.

[0203] 3. Establishment of a rabbit segmental bone defect model

[0204] A segmental femoral defect model was established using 18 ordinary New Zealand rabbits aged 4-6 months and weighing 2-4 kg. The animals were obtained from the Shenzhen Medical Device Testing Center. All experimental methods were reviewed and approved by the Experimental Animal Ethics Committee of the Shenzhen Medical Device Testing Center.

[0205] First, the rabbit's preoperative weight was measured. Anesthesia was induced with an injection of 0.1 mL / kg of sulfadiazine, followed by maintenance anesthesia with 2% isoflurane. The surgery was performed under sterile conditions. The rabbit was placed in a lateral recumbent position, and the fur on one limb was shaved. The area was disinfected sequentially with iodine and alcohol, and draped. A longitudinal skin incision was made along the femoral surface projection on the posterolateral thigh, exposing the thigh muscles layer by layer. The thigh muscles and periosteum were bluntly dissected along the intermuscular spaces to fully expose the femur. A 15 mm section of bone was then harvested from the mid-shaft of the femur using a miniature bone drill, creating a large bone defect. After hemostasis and irrigation of the surgical area with saline, a 7 mm diameter, 15 mm high scaffold (three sets of porous scaffolds PVP, PVP-M, or PVP-HM pre-cultured for 7 days) was implanted into the bone defect. The scaffold was fixed to the defect with self-tapping screws, two screws above and two below. Finally, the muscle layer, subcutaneous tissue, and skin incision were sutured intermittently layer by layer. The surgical procedure is as follows: Figure 20 As shown. Three days post-surgery, rabbits were injected with penicillin at a dose of 60,000 units per kilogram. At 6 and 12 weeks post-implantation, the rabbits were euthanized using CO2, and the femur from the operated side was harvested, fixed with 4% paraformaldehyde, and further analysis was performed to evaluate the repair effect.

[0206] Following stent implantation, the rabbits survived well for one week post-surgery, with no infection or death. The PEKK stent resulted in severe fractures in the second week, leading to a high mortality rate in this group. Six weeks post-surgery, the animals' leg condition was as follows... Figure 21 As shown in a, the surgical area in the PEKK group showed significant swelling, while the surgical sites in the other two groups appeared normal with no redness or swelling.

[0207] 4. Micro-computed tomography (CT) scan

[0208] The original image was obtained by placing the fixed sample on the scanning table of the MicroCT instrument and scanning it. The scanning accuracy was 70 μm. The original image was reconstructed using the 3D reconstruction software Recon. The ROI of the target region was analyzed using the data analysis software Avatar.

[0209] Micro-CT scan analysis results are as follows Figure 21 As shown in b, after 6 weeks, the PVP group exhibited severe fractures and scaffold displacement, with significant misalignment at both ends of the femur and no new bone regeneration. In contrast, the two scaffolds encapsulated with cells showed shrinkage of the defect area and ingrowth of new bone as healing time progressed. This indicates that the early fixation and long-term stable osteogenic effects of the PVP-M and PVP-HM scaffolds were superior to those of the PVP group. In the PVP-M group, new bone grew from the defect edge inwards, but no significant new bone tissue formation was observed in the central area of ​​the scaffold. In contrast, the PVP-HM group not only formed continuous callus on both sides of the fracture ends, but also showed newly formed trabecular bone structures in the central area of ​​the scaffold. This suggests that the microvascular network formed in the PEKK-HM scaffold provides a direct pathway for nutrient delivery and vascular ingrowth, demonstrating a significant advantage in promoting material exchange and bone tissue regeneration in the central area, and significantly accelerating bone regeneration.

[0210] In summary, this invention provides a method for preparing and applying a prevascularized bone repair scaffold. This invention successfully constructed a prevascularized PEKK bone repair scaffold (PVP). The PEKK scaffold prepared by fused deposition modeling (FDM) has a porous structure, providing ample space for angiogenesis while offering suitable mechanical support for bone defect repair. The ECM retains active components such as collagen, glycosaminoglycans, and elastin from the natural bone matrix, better mimicking the specific ecological niche in bone tissue. In vitro experiments showed that a 2% ECM three-dimensional matrix could induce HUVEC germination. After co-culturing with BMSCs for 7 days, HUVECs could form a capillary-like three-dimensional network structure within the scaffold, forming a prevascularized scaffold. Animal experiments established a rabbit critical femoral defect model, demonstrating good in-situ tissue repair effects. This study innovatively prepared a prevascularized PEKK scaffold, achieving a synergistic effect of vascularization and osteogenic formation through a positive feedback loop mediated by paracrine signaling networks, which is expected to open up new ideas and methods for the clinical application of large-segment bone defect repair.

[0211] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for preparing a prevascularized bone repair scaffold, characterized in that, Includes the following steps: S1. Obtain a porous scaffold and perform sulfonation treatment to obtain a sulfonated scaffold; S2. Prepare a mixed solution containing gelatin and extracellular matrix, and then add mesenchymal stem cells and / or endothelial cells to obtain a cell mixture preparation; S3. The cell mixture is brought into contact with the sulfonated scaffold and cultured to obtain the final product.

2. The preparation method according to claim 1, characterized in that, In step S1, the porous support is a porous polyetheretherketone support or a porous polyetherketoneketone support. Preferably, the aperture of the porous support is 0.01 mm to 2.5 mm; Preferably, the porosity of the porous support is 65% to 92%.

3. The preparation method according to claim 1, characterized in that, In step S1, the specific method of the sulfonation treatment is as follows: the porous support is placed in concentrated sulfuric acid for treatment, followed by hydrothermal treatment, and then dried after cleaning. Preferably, the temperature of the hydrothermal treatment is 95~110 ℃; Preferably, the hydrothermal treatment time is 3-5 hours.

4. The preparation method according to claim 1, characterized in that, In step S2, the gelatin includes methacrylamide gelatin and / or methacrylamide hyaluronic acid gelatin; Preferably, the final concentration of gelatin in the mixed solution is 5% to 10%; Preferably, the degree of substitution of the methacrylamide gelatin is 60% to 90%.

5. The preparation method according to claim 4, characterized in that, In step S2, the final concentration of the extracellular matrix in the mixed solution is 1% to 5%; Preferably, the extracellular matrix is ​​a bone-derived extracellular matrix; Preferably, the content of glycosaminoglycans in the extracellular matrix is ​​2~8 μg / mg; Preferably, the elastin content in the extracellular matrix is ​​5~8 μg / mg.

6. The preparation method according to claim 1, characterized in that In step S2, the mesenchymal stem cells include bone marrow mesenchymal stem cells; Preferably, the endothelial cells include umbilical vein endothelial cells.

7. The preparation method according to claim 6, characterized in that, In step S2, the final concentration of the mesenchymal stem cells in the cell mixture is 0.5 × 10⁻⁶. 6 ~2×10 7 cells / mL; Preferably, the final concentration of the endothelial cells is 1×10⁻⁶. 6 ~4×10 7 cells / mL; Preferably, the ratio of mesenchymal stem cells to endothelial cells is 1:1.8~2.

2.

8. The preparation method according to any one of claims 1 to 7, characterized in that, In step S3, the contact includes injecting the cell mixture into the sulfonated scaffold; Preferably, the culture medium used for the culture includes DMEM / F12 growth medium and ECM complete medium; Preferably, the volume ratio of the DMEM / F12 growth medium to the ECM complete medium is 1:0.8~1.

2.

9. A prevascularized bone repair scaffold, characterized in that, The prevascularized bone repair scaffold was prepared using the preparation method described in any one of claims 1 to 8.

10. The method for preparing the prevascularized bone repair scaffold as described in any one of claims 1 to 8, or the application of the prevascularized bone repair scaffold as described in claim 9 in the preparation of materials for repairing bone defects.

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