A composite 3D printing scaffold with timing regulation of blood vessel and bone tissue regeneration and a preparation method and application thereof

Abstract

The application belongs to the field of biomedical materials and manufacturing thereof, and discloses a composite 3D printing scaffold with time sequence regulation of blood vessel and bone tissue regeneration and a preparation method and application thereof. The method loads osteogenic induction agents and vascular induction differentiation agents into two carrier materials with different degradation rates respectively, and carries out 3D printing and gel coating modification in the reverse order of the physiological repair process of vascularization first and then osteogenesis, to form a composite 3D printing scaffold with the inner layer loaded with osteogenic induction agents and the outer layer loaded with vascular induction differentiation agents. After the composite 3D printing scaffold is implanted into the body, the repair regeneration process of vascularization first and then osteogenesis is realized through the way of gel coating degradation first and then 3D printing scaffold degradation according to the different degradation rates of the composite materials, and the composite 3D printing scaffold has good life compatibility, biological activity and mechanical properties, and can be used as a biomedical material or self-repairing repair for repairing or treating bone defects, and has a good application prospect in the treatment and self-repair of bone defects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and their manufacturing, and more specifically, relates to a composite 3D printed scaffold with time-regulated vascular and bone tissue regeneration, its preparation method and application. Background Technology

[0002] Extensive bone defects caused by severe trauma, infection, resection of tumor-like lesions, osteoporosis, and congenital genetic diseases leading to skeletal developmental abnormalities are common in clinical practice, and their repair remains a challenge for orthopedic surgeons. Currently, common medical repair methods for bone defects include autologous bone grafting, allogeneic bone grafting, xenogeneic bone grafting, and metal implants. However, these traditional methods (such as autologous bone grafting, allogeneic bone grafting, and metal implants) have inherent limitations, including limited donor sources, immune rejection, mechanical mismatch, and the inability to personalize the procedure. Driven by urgent clinical needs, bone tissue engineering scaffold materials have emerged. A bone tissue engineering scaffold is a three-dimensional (3D) scaffold loaded with seed cells, cytokines, or bioactive factors. After implantation, it promotes cell adhesion, proliferation, and differentiation, providing a suitable environment for the growth of primitive mesenchymal stem cells, thereby promoting bone tissue regeneration. It can serve as a biomedical material or a self-repairing material. Currently, bone tissue engineering scaffolds mainly include polymer scaffolds, bioceramic scaffolds, and composite material scaffolds.

[0003] Polymers used in bone tissue engineering scaffolds include both natural and synthetic polymers. However, natural polymers suffer from insufficient mechanical strength and the inability to precisely control their degradation rate. Synthetic polymers also have drawbacks, such as poor cell affinity, poor water solubility, and the release of acidic degradation products like polylactic acid (PLA) after degradation, which can trigger inflammatory responses and swelling in local tissues. Commonly used bioceramic materials, such as hydroxyapatite (HA), are a major component of natural bone. Although they can bind to tissues through chemical bonds and form new bone tissue after implantation, the poor mechanical properties of HA mean that the new bone formed in porous HA scaffolds cannot withstand the mechanical loads required for remodeling.

[0004] Composite scaffolds are made of two or more different materials. While they can combine the advantages of different materials, the construction of bone tissue engineering scaffolds is very complex. Factors such as the scaffold's shape, mechanical properties, constituent materials, pore size, shape, and pore cross-linking characteristics all need to be considered during scaffold construction. Therefore, different composite scaffolds need to be researched for different purposes. For example, to improve the biocompatibility of scaffolds, patent CN 111097068 A uses hydroxyapatite particles, gelatin, and sodium alginate to create a biocompatible composite 3D-printed scaffold. The hydroxyapatite particles utilize osteogenic activity and osteoinductive properties, thereby promoting bone tissue regeneration and repair. To enhance the bone regeneration capacity of composite scaffolds, bone morphogenetic protein-2 (BMP-2) is loaded onto an HA / collagen / polylactic acid scaffold. New bone deposition is faster than with a pure scaffold. However, BMP-2 protein has secondary and tertiary structures and is prone to protein denaturation during transportation or storage. Moreover, an ideal bone tissue engineering scaffold should simulate the physiological repair process of bone defects, i.e., vascularization followed by osteoogenesis. However, existing bone tissue engineering scaffolds focus on promoting bone regeneration, neglecting the need to consider vascular reconstruction during bone tissue construction. Furthermore, achieving the physiological self-repair process of vascularization followed by osteoogenesis during bone defect repair remains a key technical challenge in the field of bone tissue engineering. Summary of the Invention

[0005] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a composite 3D-printed scaffold with time-regulated vascular and bone tissue regeneration, its preparation method, and its application. The aim is to discover that by loading osteogenic inducing agents and angiogenic differentiation agents onto two carrier materials with different degradation rates, and performing 3D printing in the reverse order of the physiological self-repair process of vascularization followed by osteoogenesis, a composite 3D-printed scaffold capable of time-released angiogenic differentiation agents and osteogenic inducing agents can be created. As a biomedical self-repairing material, it can simulate the physiological repair process of vascularization followed by osteoogenesis, significantly promoting bone regeneration. This solves the technical problem that existing 3D-printed scaffolds for bone defect repair cannot simulate the physiological repair process of vascularization followed by osteoogenesis.

[0006] To achieve the above objectives, according to one aspect of the present invention, a method for preparing a composite 3D-printed scaffold with time-regulated vascular and bone tissue regeneration is provided, comprising the following steps:

[0007] Osteogenic inducers and angiogenic differentiation inducers were loaded onto two carrier materials with different degradation rates and 3D printed in reverse order of the physiological repair process of vascularization first and then osteoogenesis. The slow-release carrier loaded with osteogenic inducers was first mixed with nano-hydroxyapatite and then 3D printed at a low temperature below 0°C to create a scaffold framework. The slow-release osteogenic inducers were loaded onto the 3D printed scaffold framework at the same time as the scaffold framework was formed.

[0008] Then, the fast-release carrier loaded with the angiogenic differentiation agent is coated onto the 3D printed scaffold skeleton to construct a composite 3D printed scaffold with different layers, so that the inner layer is loaded with the osteogenic inducing agent and the outer layer is loaded with the angiogenic differentiation agent.

[0009] The osteogenic inducing agent includes BMP-2 and OGP peptide; the angiogenic differentiation inducing agent includes VEGF and QK peptide.

[0010] Preferably, in the preparation method, the osteogenic inducer is an OGP peptide, the amino acid sequence of which is shown in SEQ ID NO:1.

[0011] Preferably, in the preparation method, the angiogenesis-inducing differentiation agent is QK peptide, whose amino acid sequence is shown in SEQ ID NO:5.

[0012] Preferably, in the preparation method, the low temperature is 0℃ to -20℃.

[0013] Preferably, the preparation method involves mixing the slow-release carrier, nano-hydroxyapatite, and OGP peptide in a mass ratio of 200:100:1 to 160:40:1, and co-melting them in a 1,4-dioxane solution; using a 3D printer for low-temperature printing with the following parameters: printing speed 20-55 mm / s, pneumatic pressure 80-150 kPa, receiving plate temperature between -10°C and -20°C, and a 27G printing needle; after 3D printing, the scaffold skeleton is freeze-dried at low temperature; the slow-release carrier is a hydrophobic polymer.

[0014] The freeze-dried scaffold framework is then immersed in a fast-release carrier solution containing QK peptides and photocured using ultraviolet light to obtain a composite 3D-printed scaffold with time-regulated vascular and bone tissue regeneration; the fast-release carrier is a hydrogel material or a hydrophilic polymer.

[0015] Preferably, in the preparation method, the slow-release carrier is selected from PLGA and PCL; the fast-release carrier is selected from gelatin, sodium alginate, collagen, and methacrylamide dextran.

[0016] Preferably, in the preparation method, the rapid-release carrier is methacrylamide dextran, and the mass ratio of QK peptide to methacrylamide dextran is 1:50 to 1:100.

[0017] According to another aspect of the present invention, a composite 3D-printed scaffold with time-regulated vascular and bone tissue regeneration is also provided, which is prepared according to the preparation method described in the present invention.

[0018] According to another aspect of the present invention, the application of a composite 3D-printed scaffold with time-regulated vascular and bone tissue regeneration in the preparation of self-repairing medical materials for bone defects is also provided.

[0019] Preferably, the composite 3D printed scaffold is used to prepare medical materials for self-repairing diabetic bone defects.

[0020] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects:

[0021] This method loads osteogenic inducing agents and angiogenic differentiation inducing agents onto two carrier materials with different degradation rates. Following the reverse order of the physiological repair process—vascularization followed by osteoogenesis—3D printing and gel coating modification are performed to prepare a composite 3D-printed scaffold with an inner layer loaded with osteogenic inducing agents and an outer layer loaded with angiogenic differentiation inducing agents. This scaffold can release the angiogenic differentiation inducing agents and osteogenic inducing agents sequentially. When implanted in the body as a medical material, this composite 3D-printed scaffold can achieve a repair and regeneration process—vascularization followed by osteoogenesis—by degrading the gel coating first, then the 3D-printed scaffold, according to the different degradation rates of the composite materials. This effectively matches the physiological regeneration needs of diabetic bone defects. The composite 3D-printed scaffold provided by this invention has good biocompatibility, bioactivity, and mechanical properties, and shows great promise as a biomedical material for the treatment and self-repair of bone defects. Attached Figure Description

[0022] Figure 1 In the figure, A represents the appearance of different 3D printed scaffolds, B to D represent the scanning electron microscope images of different 3D printed scaffolds, and E represents the Ca and P elemental analysis results of different 3D printed scaffolds.

[0023] Figure 2 In the figure, A represents the Fourier transform infrared spectral scans of different composite 3D printed scaffolds, B represents the X-ray photoelectron spectra of different composite 3D printed scaffolds, and C represents the mechanical property tests of different composite 3D printed scaffolds.

[0024] Figure 3 In the figure, A represents the calcium release from different 3D printed scaffolds, B represents the phosphorus release from different 3D printed scaffolds, C represents the static hydrophilic angle of different 3D printed scaffolds, and D represents the release rate of OGP and QK on the PHOQ scaffold.

[0025] Figure 4 The effects of different 3D-printed scaffolds on the proliferation activity of MC-3T3-E1 cells.

[0026] Figure 5 In the middle section, A represents the Calcein / Propidium iodide (PI) staining of different 3D printed scaffolds, and B is a bar chart showing the live-to-dead ratio of different 3D printed scaffolds.

[0027] Figure 6The results show the scaffold staining of MC-3T3-E1 cells on different composite 3D printed scaffolds.

[0028] Figure 7 In Figure A, HUVECs migration experiments were conducted outside the scaffolds of different composite 3D printed scaffolds. In Figure B, a bar chart showing the migration rate of HUVECs in different composite 3D printed scaffolds was presented.

[0029] Figure 8 In the diagram, A represents the tube formation assay of HUVECs, B is a bar chart showing the number of nodules, C is a bar chart showing the total length of tubes formed, D represents the mRNA expression level of the VEGF gene, and E represents the mRNA expression level of the NOTCH-1 gene.

[0030] Figure 9 In the chart, A represents alkaline phosphatase (ALP) staining, B represents alizarin red (ARS) staining, C represents a bar chart showing the percentage of ALP-positive areas, and D represents a bar chart showing the percentage of ARS-positive areas.

[0031] Figure 10 In the diagram, A represents Micro-CT scan reconstruction, B represents bone mineral density (BMD), C represents BV / TV, and D represents regenerated bone volume.

[0032] Figure 11 In the diagram, A represents the HE staining result, B represents the Masson staining result, C represents the NB / TB statistical bar chart, and D represents the LB / TB statistical bar chart.

[0033] Figure 12 In the diagram, A represents alkaline phosphatase (ALP) staining, B represents a statistical bar chart of alkaline phosphatase (ALP) staining, C represents osteocalcin (OCN) staining, and D represents a statistical bar chart of osteocalcin (OCN) staining.

[0034] Figure 13 In the diagram, A represents CD206 immunofluorescence staining, B represents CD31 staining, C represents a CD206 statistical bar chart, D represents a CD31 statistical bar chart, and E represents a blood vessel diameter statistical bar chart. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0036] Unlike other tissues, the normal bone tissue surrounding bone defects can provide seed cells for bone regeneration at the defect site. Although defect repair can be achieved simply by filling the defect with scaffold material, the main considerations of this invention are how to recruit more seed cells and achieve the timed release of different bioactive factors to simulate the physiological repair process of vascularization followed by osteoogenesis.

[0037] This invention discovers that osteogenic inducers and angiogenic differentiation agents are loaded onto two carrier materials with different degradation rates, and 3D printed in the reverse order of the physiological repair process of vascularization followed by osteoogenesis. First, a slow-release carrier loaded with the osteogenic inducer is mixed with nano-hydroxyapatite and then 3D printed to create a scaffold framework loaded with the slow-release osteogenic inducer. Then, a fast-release carrier loaded with the angiogenic differentiation agent is coated onto the 3D-printed scaffold framework, constructing a composite 3D-printed scaffold with an inner layer loaded with the osteogenic inducer and an outer layer loaded with the angiogenic differentiation agent. This composite 3D-printed scaffold, as a pharmaceutical material, can achieve rapid release of the angiogenic differentiation agent in vitro or in vivo to promote early vascularization. Subsequently, as the slow-release carrier gradually degrades, the osteogenic inducer is continuously and slowly released, dominating the mid-to-late-stage osteogenic process. In particular, the osteogenic inducer is an osteogenic growth peptide (OGP peptide), and the angiogenic differentiation agent is a short peptide mimicking vascular endothelial growth factor (QK peptide). It was found that the two have a synergistic effect, jointly promoting bone regeneration in bone defects, with significantly better results than either OGP peptide or QK peptide alone.

[0038] Based on this, the present invention provides a method for preparing a composite 3D printed scaffold with time-regulated vascular and bone tissue regeneration, comprising the following steps:

[0039] Osteogenic inducers and angiogenic differentiation inducers were loaded onto two carrier materials with different degradation rates. 3D printing was performed in reverse order of the physiological repair process, which involves vascularization followed by osteoogenesis. First, the slow-release carrier loaded with the osteogenic inducer was mixed with nano-hydroxyapatite and then 3D printed at low temperatures to create a scaffold framework. Simultaneously, the slow-release osteogenic inducer was loaded onto the 3D-printed scaffold framework. The low temperature range was 0℃ to -20℃. Printing at low temperatures helps maintain the bioactivity of the osteogenic inducer and avoids the degradation of its activity by high temperatures.

[0040] Then, a fast-release carrier loaded with angiogenic differentiation agent is coated onto a 3D-printed scaffold skeleton to construct a composite 3D-printed scaffold with different layers, so that the inner layer is loaded with osteogenic inducing agent and the outer layer is loaded with angiogenic differentiation agent.

[0041] The osteogenic inducer includes bone morphogenetic protein-2 (BMP-2) and osteogenic growth peptide (OGP). BMP-2 is a pleiotropic growth factor belonging to the TGF-β superfamily, primarily involved in bone formation, tissue repair, and developmental regulation. The OGP peptide mainly participates in bone formation, bone marrow repair, and osteoblast function regulation. The amino acid sequence of the OGP peptide is selected from ALKRQGRTLYGFGG (SEQ ID NO:1). OGP is a potent osteogenic peptide that stimulates osteoblast proliferation, differentiation, and mineralization, promoting bone matrix synthesis. Although the amino acid sequence of the OGP peptide varies in different studies, it consistently promotes bone formation and repair. In some embodiments, the osteogenic inducer is an OGP peptide with the amino acid sequence shown in SEQ ID NO:1 (ALKRQGRTLYGFGG).

[0042] The angiogenesis-inducing differentiation agent includes vascular endothelial growth factor (VEGF) and a short peptide (QK peptide) that mimics VEGF. The QK peptide can mimic the activity of VEGF, specifically binding to and activating VEGF receptors, promoting the proliferation, migration, and lumen formation of vascular endothelial cells, thereby inducing angiogenesis. The amino acid sequence of the QK peptide is selected from KLTWQELYQLKYKGI (SEQ ID NO:5).

[0043] In some embodiments, the angiogenic differentiation agent is a QK peptide with the amino acid sequence KLTWQELYQLKYKGI as shown in SEQ ID NO:5.

[0044] Hydroxyapatite (HA) has a chemical composition similar to that of natural bone tissue and exhibits good biocompatibility and bone regeneration-promoting activity.

[0045] The slow-release carrier is selected from hydrophobic polymers with slow degradation (such as PLGA and PCL). In some embodiments, the slow-release carrier is PLGA. Mixing the slow-release carrier loaded with osteogenic inducers with nano-hydroxyapatite before printing allows for the loading of osteogenic inducers into the inner layer of the scaffold, enabling long-term sustained release and avoiding the side effects of high doses, while also enhancing the mechanical properties of the scaffold. Furthermore, the degradation products of PLGA do not negatively impact fresh bone tissue and can even promote cell growth and angiogenesis through these degradation products.

[0046] The rapid-release carrier is selected from hydrogel materials that degrade quickly (such as gelatin, sodium alginate, and collagen) or hydrophilic polymers. In some embodiments, the rapid-release carrier is methacrylamide dextran (DexMA). After QK peptides are co-melted with DexMA, they are applied to the surface of the scaffold or its macroporous structure through physical adsorption, simple soaking, or coating. DexMA hydrogel has excellent biocompatibility and biodegradability, facilitating the rapid diffusion and release of QK peptides after scaffold implantation, promoting early vascularization. In addition, the DexMA coating fills and connects the pores within the 3D-printed scaffold, further improving the mechanical properties of the scaffold.

[0047] In some embodiments, the preparation method is as follows:

[0048] A copolymer of glycolide and lactide (PLGA: 75:25), nano-hydroxyapatite (HA, particle size 50nm), and OGP peptide (ALKRQGRTLYGFGG) were mixed at a mass ratio of 200:100:1 to 160:40:1 and co-melted in a 1,4-dioxane solution. The mixture was then cryogenically printed using a 3D printer with the following parameters: printing speed 20-55mm / s, pneumatic pressure 80-150kPa, receiving plate temperature between -10℃ and -20℃, and a 27G printing nozzle. After printing, the 3D-printed scaffold was transferred to a cryogenic freeze dryer for freeze-drying, cleaning, and freezing to obtain an OGP-functionalized 3D-printed PLGA / HA scaffold.

[0049] The PLGA / HA scaffold was immersed in a composite DexMA hydrogel containing QK peptide (KLTWQELYQLKYKGI), and the composite 3D printed scaffold was photocured using 405nm UV light for 3 minutes (25mW / cm²). 2 This process yields a composite 3D-printed scaffold with time-regulated vascular and bone tissue regeneration. The mass ratio of QK peptide to DexMA in the composite DexMA hydrogel containing QK peptide (KLTWQELYQLKYKGI) is 1:50 to 1:100.

[0050] The composite 3D-printed scaffold prepared according to this method can sequentially release angiogenesis-inducing and osteogenic-inducing agents in vivo after implantation. First, QK is rapidly released to promote early vascularization, followed by sustained release of OGP to dominate subsequent osteoogenesis, thus achieving vascularization before osteoogenesis. This avoids the antagonistic or competitive effects that might occur with the simultaneous release of the two factors. Sequential release refers to the release of two or more active substances (such as OGP and QK) from the scaffold according to a pre-designed sequence and time points, which mimics the biosignals required at different stages of natural tissue healing.

[0051] This invention also provides a composite 3D-printed scaffold with time-regulated vascular and bone tissue regeneration, which is prepared according to the method described in this invention. The composite 3D-printed scaffold has a fence-like structure with a distinct 3D-printed grid pattern and irregularly shaped cavities.

[0052] This invention also provides the application of a composite 3D-printed scaffold with time-regulated vascular and bone tissue regeneration in the preparation of self-repairing medical materials for bone defects. The composite 3D-printed scaffold is prepared according to the method described in this invention. It is particularly suitable for the preparation of medical materials for repairing diabetic bone defects.

[0053] The following are examples.

[0054] OGP usually refers to osteogenic growth peptide. It is a potent bone-forming peptide that stimulates osteoblast proliferation, differentiation, and mineralization, and promotes bone matrix synthesis.

[0055] QK typically refers to a short peptide that mimics vascular endothelial growth factor (VEGF). It specifically binds to and activates VEGF receptors, promoting the proliferation, migration, and lumen formation of vascular endothelial cells, thereby inducing angiogenesis.

[0056] HA refers to hydroxyapatite, whose chemical composition is similar to the mineral composition of natural bone tissue, and it has good biocompatibility and bone regeneration-promoting activity.

[0057] PLGA (poly(glycolic acid-lactide) copolymer) is a biodegradable synthetic polymer, also known as polylactic-glycolic acid copolymer, polylactic-co-glycolic acid copolymer, or poly(glycolic acid-lactide). PLGA is produced by copolymerizing glycolide and lactide using ring-opening polymerization and direct melt polymerization processes. It exhibits good biocompatibility and adjustable degradability. Its degradation products do not negatively impact fresh bone tissue and can even promote cell growth and angiogenesis through these degradation products.

[0058] DexMA (methacryloyldextrin) is a water-soluble polymer prepared by grafting methacrylate onto dextran molecules. It exhibits excellent biocompatibility and biodegradability, and DexMA hydrogels can be used as 3D printing scaffold materials to support cell growth and tissue regeneration.

[0059] In the following examples, HA was derived from Aladdin (H485315), PLGA from Shenzhen Maiqi Biotechnology (Mqsw221101), and DexMA from Suzhou Yongqinquan (EFL-DexMA-200k). The amino acid sequence of the OGP peptide was ALKRQGRTLYGFGG, and the amino acid sequence of the QK peptide was KLTWQELYQLKYKGI, all synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. Experimental data were analyzed using SPSS software (version 22.0, IBM, USA), and results are expressed as mean ± standard deviation. One-way ANOVA and post-hoc Tukey's test were used to determine statistical significance between groups, with a p-value < 0.05 considered statistically significant.

[0060] Example 1: Preparation of different medical material-composite 3D printed scaffolds

[0061] 1. Fabrication of a time-released OGP and QK composite 3D-printed scaffold

[0062] Ideally, bone regeneration typically involves vascularization followed by osteoogenesis. To mimic the physiological healing process, this embodiment prepares a time-released OGP and QK composite 3D-printed multilayer scaffold in reverse order of the physiological bone injury healing process. This allows for the rapid release of QK (outer layer bioactive component) to promote early vascularization, followed by the sustained release of OGP (inner layer bioactive component) to dominate subsequent osteoogenesis. The specific fabrication process is as follows:

[0063] 1.1 Fabrication of OGP-functionalized 3D-printed PLGA / HA scaffolds

[0064] (1) Preparation of 3D printing ink: 3.2g of glycolide-lactide copolymer (PLGA) (75:25, molecular weight 100k), 0.8g of nano-hydroxyapatite (HA, particle size 50nm) and 20mg of OGP peptide (ALKRQGRTLYGFGG) were dissolved in 20mL of 1,4-dioxane solution and stirred on a magnetic stirrer for 24h to complete the preparation of 3D printing ink.

[0065] (2) 3D printing of OGP-functionalized PLGA / HA bracket: The printing ink in (1) was printed at low temperature using a 3D printer (Regenovo, Hangzhou). The printing parameters were: printing speed 50mm / s, pneumatic pressure 0.1MPa, receiving plate temperature -15℃, and printing needle 27G. After printing, the 3D printed bracket was transferred to a low-temperature freeze dryer for freeze drying. Then, the bracket was washed three times with double-distilled water and freeze-dried again for later use.

[0066] 1.2 Preparation of a time-controlled release OGP and QK composite 3D printed scaffold

[0067] (1) Preparation of methacrylated dextran (DexMA) loaded with QK peptide: 0.5 g of methacrylated dextran (molecular weight 500 k, EFL) and 5 mg of QK peptide (KLTWQELYQLKYKGI) were dissolved together in PBS solution containing 0.25% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) for later use.

[0068] (2) Preparation of time-released OGP and QK composite 3D printing scaffold: The composite DexMA gel prepared in (1) above was coated onto the OGP-functionalized 3D printing PLGA / HA scaffold prepared in 1.1, so that it completely immersed the 3D printing scaffold. Then, the composite 3D printing scaffold was photocured with 405nm ultraviolet light for 3 minutes (25mW / cm). 2 The composite 3D printed scaffold of time-released OGP and QK was prepared by freeze-drying at low temperature and then stored at -20℃ for later use. The composite 3D printed scaffold of time-released OGP and QK is referred to as PHOQ scaffold.

[0069] 2. Preparation of pH

[0070] (1) Preparation of 3D printing ink: 3.2g of glycolide-lactide copolymer (PLGA) (75:25, molecular weight 100k) and 0.8g of nano-hydroxyapatite (HA, particle size 50nm) were dissolved together in 20mL of 1,4-dioxane solution and stirred on a magnetic stirrer for 24h to complete the preparation of 3D printing ink.

[0071] (2) 3D printing of PLGA / HA bracket: The printing ink in (1) was printed at low temperature using a 3D printer (Regenovo, Hangzhou Genuofei). The printing parameters were: printing speed 50mm / s, pneumatic pressure 0.1MPa, receiving plate temperature -15℃, and printing needle 27G. After printing, the 3D printed bracket was transferred to a low temperature freeze dryer for freeze drying. Then, the bracket was washed three times with double distilled water and freeze-dried again at -20℃ for storage. It is referred to as PH bracket.

[0072] 3. Preparation of PHQ

[0073] (1) Preparation of 3D printing ink: 3.2g of glycolide-lactide copolymer (PLGA) (75:25, molecular weight 100k) and 0.8g of nano-hydroxyapatite (HA, particle size 50nm) were dissolved together in 20mL of 1,4-dioxane solution and stirred on a magnetic stirrer for 24h to complete the preparation of 3D printing ink.

[0074] (2) 3D printing of PLGA / HA bracket: The printing ink in (1) was printed at low temperature using a 3D printer (Regenovo, Hangzhou Genuofei). The printing parameters were: printing speed 50mm / s, pneumatic pressure 0.1MPa, receiving plate temperature -15℃, and printing needle 27G. After printing, the 3D printed bracket was transferred to a low temperature freeze dryer for freeze drying. Then the bracket was washed with double distilled water 3 times and freeze dried again for later use.

[0075] (3) Preparation of methacrylamide dextran (DexMA) loaded with QK peptide: 0.5 g of methacrylamide dextran (molecular weight 500 kJ, EFL) and 5 mg of QK peptide (KLTWQELYQLKYKGI) were dissolved together in PBS solution containing 0.25% phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP) for later use. The composite DexMA gel prepared above was coated onto the 3D printed PLGA / HA scaffold prepared in (2), so that it completely immersed the 3D printed scaffold, and then the composite 3D printed scaffold was photocured with 405 nm ultraviolet light for 3 minutes (25 mW / cm). 2 The composite 3D printed scaffold of time-released OGP and QK was prepared, and after being freeze-dried again at low temperature, it was stored at -20℃ for later use. It is referred to as PHQ scaffold.

[0076] 4. Preparation of PHO

[0077] (1) Preparation of 3D printing ink: 3.2g of glycolide-lactide copolymer (PLGA) (75:25, molecular weight 100k), 0.8g of nano-hydroxyapatite (HA, particle size 50nm) and 20mg of OGP peptide (ALKRQGRTLYGFGG) were dissolved in 20mL of 1,4-dioxane solution and stirred on a magnetic stirrer for 24h to complete the preparation of 3D printing ink.

[0078] (2) 3D printing of OGP functionalized PLGA / HA bracket: The printing ink in (1) was printed at low temperature using a 3D printer (Regenovo, Hangzhou Genuofei). The printing parameters were: printing speed 50mm / s, pneumatic pressure 0.1MPa, receiving plate temperature -15℃, and printing needle 27G. After printing, the 3D printed bracket was transferred to a low temperature freeze dryer for freeze drying. Then, the bracket was washed with double distilled water 3 times and freeze dried again for later use. It is referred to as PHO bracket.

[0079] Example 2 Characterization of different composite 3D printed scaffolds

[0080] Composite 3D printed scaffolds PHOQ, PH, PHQ and PHO were prepared according to the different 3D printed scaffold preparation methods in Example 1, and were characterized.

[0081] (1) Microtubule morphology

[0082] The geometry and structure of different 3D-printed scaffolds were observed using scanning electron microscopy (SEM, ZEISS, Germany). The surface morphology of the samples was characterized using SEM at a working voltage of 3.0 kV. The elemental concentrations and distributions of Ca and P on the surface of the 3D-printed scaffolds were determined using energy dispersive spectroscopy (EDS). The results are as follows: Figure 1 As shown. Figure 1 In the figure, A represents the appearance of different 3D printed scaffolds, B to D represent the scanning electron microscope images of different 3D printed scaffolds, and E represents the Ca and P elemental analysis results of different 3D printed scaffolds.

[0083] Figure 1 As shown in Figure A, the four 3D-printed scaffolds, PHOQ, PH, PHQ, and PHO, all exhibit a clear fence-like structure with a distinct 3D-printed mesh pattern. Scanning electron microscopy (SEM) analysis reveals highly interconnected pores throughout the scaffold structure. Figure 1 (B to C). Notably, the pore morphology of the different 3D printed scaffolds differs: the PHO and PHOQ scaffolds exhibit irregularly shaped cavities, while the PH and PHQ groups primarily have circular pores. The pore size of the PHOQ group is smaller than that of the PH group; this difference may be attributed to the DexMA surface coating covering and filling some of the pores. Energy dispersive spectroscopy (EDS) analysis shows that C, O, Ca, and P are clearly detected on the surface of all different 3D printed scaffolds. Figure 1 (E). It is worth noting that the Ca and P contents of the PHQ and PHOQ groups were lower than those of the PH and PHO groups, which may be due to the DexMA coating on the surface of the 3D printed scaffold.

[0084] (2) Physicochemical characterization

[0085] Different composite 3D printing scaffold materials were mixed and ground with KBr, and the results were analyzed using Fourier transform infrared spectroscopy (FTIR, Thermo, USA) in the range of 400–4000 cm⁻¹. -1Variations in surface functional groups within the wavenumber range were investigated; the hydrophilicity of the scaffold was tested using a contact angle meter (Dataphysics, Germany); and the elemental composition and chemical state of the scaffold were analyzed using X-ray photoelectron spectroscopy (XPS, Thermo, USA). Mechanical properties of the composite 3D-printed scaffold (8 mm in diameter and 8 mm in height) were tested using a mechanical testing apparatus (CARE, China) at a compression rate of 1 mm / min. The stress-strain curves of the composite 3D-printed scaffold were calculated using the measured loads and displacements. Results are as follows: Figure 2 As shown, Figure 2 In the figure, A represents the Fourier transform infrared spectral scans of different composite 3D printed scaffolds, B represents the X-ray photoelectron spectra of different composite 3D printed scaffolds, and C represents the mechanical property tests of different composite 3D printed scaffolds.

[0086] like Figure 2 As shown in Figure A, Fourier transform infrared (FTIR) spectroscopy analysis results indicate that the four 3D printed scaffolds, PHOQ, PH, PHQ, and PHO, show significant differences in optical density at 1025, 1453, and 1749 cm⁻¹. -1 Characteristic absorption bands are present at all locations, corresponding to COC stretching, CH bending, and C=O stretching vibrations, respectively. Notably, the PHQ and PHOQ groups show absorption bands at 2917 cm⁻¹. -1 (CH vibration) and 3100-3650cm -1 An additional peak appeared at (OH vibration), confirming the successful DexMA coating on both PHQ and PHOQ 3D-printed scaffolds. XPS analysis detected characteristic peaks at 133.08 eV (P 2p), 286.08 eV (C 1s), 348.08 eV (Ca 2p), and 530.08 eV (O 1s), indicating that nano-hydroxyapatite was successfully integrated into the 3D-printed scaffold. Figure 2 (Middle B). Although Figure 2 The results in C indicate that modifying QK or OGP can reduce the mechanical properties of the scaffold to some extent, but the mechanical properties of the PHQ and PHOQ scaffolds are better than those of the PHO group. This may be because the DexMA coating fills and connects the pores in the 3D printed scaffold, thereby improving its mechanical properties.

[0087] (3) Detection of calcium, phosphorus, OGP and QK peptide release

[0088] 3D-printed scaffolds (PHOQ, PH, PHQ, and PHO) with dimensions of 4 × 10 × 10 mm³ were prepared and placed in 10 mL of PBS solution. Supernatants were collected on days 1, 3, 5, 7, 10, and 15. Calcium and phosphorus content in the 3D-printed scaffolds was detected using inductively coupled plasma optical emission spectrometry (ICP-OES, PerkinElmer, USA), and release curves were plotted. Release curves of QK and OGP in the 3D-printed scaffolds were detected using BSA simulations. Supernatants were collected at the specified time points (days 1, 3, 5, 7, 10, 15, 20, 25, and 30), and release simulations were performed using a BCA protein assay kit. Results are as follows: Figure 3 As shown, Figure 3 In the figure, A represents calcium release from different 3D printed scaffolds, B represents phosphorus release from different 3D printed scaffolds, C represents the static hydrophilic angle of different 3D printed scaffolds, and D represents the release rates of OGP and QK on the PHOQ scaffold. In the figure, ‡ represents PHO vs. PH, p < 0.05; ❉ represents PHO vs. PHOQ, p < 0.05; ♟ represents PHOQ vs. PH, p < 0.05; § represents PHOQ vs. PHQ, p < 0.05; D is a comparison of OGP and QK release from the PHOQ scaffold, and * represents OGP vs. QK, p < 0.05.

[0089] Figure 3 The calcium and phosphorus release results showed no statistically significant difference in the release rates of Ca and P between the PHQ and PH scaffold groups. However, the PHO scaffold group released Ca and P more rapidly than the other groups, indicating that OGP functionalization can promote the release of Ca and P from the 3D printed scaffold. Although the PHOQ scaffold group, after being coated with QK-functionalized DexMA, exhibited slightly slower Ca and P release compared to the PHO scaffold, its Ca and P release rate was significantly better than that of the PH and PHQ scaffold groups. Figure 3 (A, B). Static water contact angle testing showed that after adding QK and OGP, the contact angle of the 3D printed scaffold gradually decreased, with the PHOQ scaffold group exhibiting the smallest hydrophilic angle. Figure 3 The results (C) indicate that these 3D printed scaffolds, after functionalization by QK and OGP, have stronger hydrophilicity, with the PHOQ scaffold exhibiting the best hydrophilicity. Figure 3 The results showed that QK was released faster in the PHOQ scaffold than in the OGP scaffold. This result is consistent with the healing process (blood vessel formation followed by bone re-formation) involved in bone healing, suggesting that the PHOQ scaffold could serve as a biomimetic material to promote bone defect repair.

[0090] Example 3: Biocompatibility Evaluation of Different Composite 3D Printed Scaffolds

[0091] MC3T3-E1 cells are a mouse embryonic osteoblast precursor cell line capable of differentiating into osteoblasts and osteocytes. This study aims to investigate the application of different composite 3D-printed scaffolds as medical materials in bone defect repair. This example uses MC3T3-E1 cells to evaluate the biocompatibility of 3D-printed scaffolds (PHOQ, PH, PHQ, PHO), as detailed below:

[0092] (1) Cell proliferation

[0093] MC3T3-E1 cells were seeded into 96-well plates mounted on a 3D-printed scaffold, with a density of 5 × 10⁶ cells per well. 3 Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), with the medium changed every other day. Cell proliferation was assessed using a CCK-8 assay kit (Dojindo, Japan) after 1, 3, 5, and 7 days of incubation. At each time point, serum-free culture medium containing 10% CCK-8 was added. After incubation at 37°C in the dark for 1 hour, the culture medium was collected, and absorbance was measured at 450 nm using a microplate reader (Thermo Scientific, USA). The effects of different 3D-printed scaffolds on the proliferation activity of MC-3T3-E1 cells are shown in the figure below. Figure 4 As shown in the figure. * indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001, and so on.

[0094] Figure 4 The results showed that as the number of days progressed, MC3T3-E1 cells cultured on the four 3D-printed scaffolds (PHOQ, PH, PHQ, and PHO) all exhibited a growth trend, with the PHOQ scaffold showing the best growth. After the fifth day, the growth of MC3T3-E1 cells cultured on the PH, PHQ, and PHO scaffolds became more consistent. However, compared to the PH, PHQ, and PHO scaffolds, the cell proliferation rate of the PHOQ scaffold group was significantly increased (p<0.05), which is likely due to the synergistic effect of the internal OGP and the surface QK during the release process. Figure 4 The results showed that the PHOQ scaffold had good cell compatibility and cell proliferation promotion, which may be due to the synergistic effect of the internal OGP and the surface QK during the release process.

[0095] (2) Live and dead staining

[0096] Cytotoxicity of 3D-printed scaffolds was assessed using a live / dead assay kit containing calcein-AM and propidium iodide (PI) (Beyotime, China). 2 × 10⁻⁶ scaffolds were used. 4 / MC3T3-E1 cells were seeded on the above-mentioned composite 3D-printed scaffold and incubated for 1 day. After washing with PBS, the cells were stained with calcein / PI solution, and then the cell viability (Live / Dead) was observed using a confocal laser scanning microscope (CLSM, Nikon, Japan). The effects of different 3D-printed scaffolds on the viability of MC-3T3-E1 cells are shown in the figure. Figure 5 As shown, Figure 5 In the figure, A represents the Calcein / Propidium iodide (PI) staining of different 3D printed scaffolds, and B is a bar chart of the live-to-dead ratio of different 3D printed scaffolds, with a scale bar of 200 μm.

[0097] Figure 5 Live / dead staining showed that MC3T3-E1 cells attached strongly to all four 3D-printed scaffolds: PHOQ, PH, PHQ, and PHO. There was no significant difference in cell density among the PH, PHO, and PHQ scaffold groups. Figure 5 (A, B) Compared to the PH scaffold group, PHO scaffold group and PHQ scaffold group, we observed that the PHOQ scaffold group had higher cell density, better cell compatibility and the fewest dead cells detected. This indicates that the 3D printed scaffold of the PHOQ scaffold group, after combining OGP and QK peptides, further enhanced the biological performance of the scaffold and promoted cell survival and adhesion.

[0098] (3) Cell morphology

[0099] 2×10 4 / MC3T3-E1 cells were seeded on different composite 3D-printed scaffolds and cultured for 2 days to observe cell morphology. After washing three times with PBS, the cells were fixed with 4% paraformaldehyde solution for 15 minutes, soaked in 0.1% Triton X-100 (Sigma-Aldrich, Burlington, MA, USA) for 10 minutes, and washed twice with PBS. Phalloidin and 4,6-diamino-2-phenylindole dihydrochloride (DAPI, Sigma, MA, USA) were stained overnight and for 2 minutes, respectively, to label actin and cell nuclei. Cell morphology on the scaffolds was then observed using a confocal laser scanning microscope (CLSM, Nikon, Japan). The scaffold staining results of MC-3T3-E1 cells on different composite 3D-printed scaffolds are shown in the figure. Figure 6 As shown, the scale bar is 200 μm.

[0100] Figure 6Fluorescent staining of the cytoskeleton showed that MC3T3-E1 cells adhered and spread well on the PHQ and PHOQ scaffolds, and the tentacles of the cells in the PHQ and PHOQ groups were more densely distributed and extended more widely than those in the PH and PHO groups.

[0101] The above experimental results show that the PHOQ scaffold surface has better biocompatibility after being coated with a composite hydrogel coating of OGP and QK functionalization.

[0102] Example 4 Evaluation of in vitro migration, angiogenesis, and osteogenic properties of different composite 3D printed scaffolds

[0103] (1) In vitro migration and angiogenesis properties of different composite 3D printed scaffolds

[0104] Using human umbilical vein endothelial cells (HUVECs) as the experimental subject, the angiogenic effect of the scaffold extract was evaluated through scratch assays and tube formation assays, as detailed below:

[0105] HUVECs were planted at 5.0 × 10⁻⁶ per well. 5 HUVECs were seeded at a density of 1000 g / well in 12-well plates. After reaching 90% confluence, the cells were starved for 24 hours. Scratches were then created using the tip of a 200 μL pipette, followed by rinsing to remove unattached cells and cell debris. The culture medium was then replaced with an extract of the 3D-printed scaffold (the 3D-printed scaffold extract was prepared according to ISO 10993-12:2021). HUVEC migration was observed under a light microscope at 0, 12, and 24 hours, and the acquired images were analyzed using ImageJ 1.8.0 software. The migration rate was calculated using the formula: Migration rate (%) = (S0 - S...) / (S0 - S0 ... t ) / S0×100%; where S0 is the initial area of ​​the scratch, S t This represents the area at each time point. Results of the in vitro cell migration performance of different composite 3D-printed scaffolds are as follows: Figure 7 As shown; Figure 7 In Figure A, HUVECs migration experiments outside the scaffolds of different composite 3D printed scaffolds are shown. In Figure B, HUVECs migration rate statistics are shown in the bar chart. * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0106] Matrigel (Sigma, USA) was used for tube formation experiments. 200 μL of Matrigel was added to each well of a 24-well plate and incubated at 37°C for gelation. The extract from the 3D-printed scaffold was then used as the culture medium at a concentration of 2.0 × 10⁻⁶. 4HUVECs were seeded into each well at a density of 100 cells / well. After 6 hours of culture, they were observed and photographed under a light microscope. For quantitative analysis of the tubular structures, the Angiogenesis Analyzer included with ImageJ 1.8.0 software was used to analyze the length, number of branches, connections, and nodes of the tubular structures. The in vitro tubular formation effect results of different composite 3D-printed scaffolds are shown below. Figure 8 As shown in A to C; Figure 8 In the diagram, A represents the tube formation test of HUVECs, B is a bar chart of the number of nodules, and C is a bar chart of the total tube length.

[0107] The expression levels of angiogenesis-related mRNAs were detected by quantitative polymerase chain reaction (qPCR). In brief, total RNA was extracted from human umbilical vein endothelial cells (HUVECs) and reverse transcribed, followed by qPCR analysis using a real-time quantitative thermal cycler (LC 96, Roche, Switzerland). The primer sequences used in this embodiment are detailed in Table 1.

[0108] Table 1 Primer sequences for angiogenesis genes

[0109]

[0110] Through 2 -△△Ct The relative expression levels of angiogenesis-related genes were calculated, with β-actin used as an internal reference gene for normalization. The mRNA expression results of VEGF (vascular endothelial growth factor) and NOTCH1 genes are shown below. Figure 8 As shown in D and E, Figure 8 In the figure, D represents the mRNA expression level of the VEGF gene, and E represents the mRNA expression level of the NOTCH1 gene.

[0111] In this embodiment, angiogenesis assays and HUVEC migration assays were used to determine the angiogenesis induction performance of different 3D printed scaffolds. Figure 7 HUVEC migration experiments showed that, compared with the PH group scaffold, the PHO, PHQ, and PHOQ group scaffolds significantly enhanced HUVEC migration ability after 12 hours of co-culture. The PHOQ group exhibited the highest migration rate among the four groups, with a significantly faster cell migration speed than the PH group. However, after 24 hours of incubation, there was no statistically significant difference between the PHO and PHQ group scaffolds. Compared to the PHO and PHQ groups, the PHOQ group scaffold further enhanced HUVEC migration (p < 0.05), indicating that OGP and QK on the PHOQ group scaffold synergistically promoted and accelerated HUVEC migration.

[0112] and Figure 8The results of the tube formation experiment showed that the PHOQ group was significantly superior to the PH, PHO, and PHQ groups in both the total length of the tubes formed and the number of nodes. Specifically, the mRNA expression levels of VEGF and NOTCH1 genes in the cells of the PHQ and PHOQ scaffold groups were significantly higher than those in the PH and PHO scaffold groups. The high expression of VEGF and Notch1 promotes angiogenesis, confirming that QK is gradually released from the scaffold surface, significantly enhancing the scaffold's angiogenesis induction ability. These findings indicate that the PHOQ scaffold, after being coated with QK hydrogel, possesses enhanced angiogenesis properties. This composite material retains the bioactivity of QK, meeting the requirements for angiogenesis induction in bone regeneration and addressing the challenge of insufficient angiogenesis in bone healing.

[0113] (2) Evaluation of the in vitro osteogenic properties of different composite 3D printed scaffolds

[0114] MC3T3-E1 cells were loaded at 5.0 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells per well in 12-well plates. The culture medium consisted of Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), mixed 1:1 with the scaffold extract, and the medium was changed every other day. After 14 days of culture, cells in each group were fixed with 4% paraformaldehyde solution and then stained with alkaline phosphatase (ALP) (Beyotime, China). The stained cells were observed under a light microscope and analyzed using ImageJ 1.8.0 software. After 21 days of culture, the degree of extracellular matrix mineralization was determined using Alizarin Red S (ARS, Beyotime, China) staining, and the resulting images were analyzed using ImageJ 1.8.0 software. The in vitro osteogenic induction performance evaluation results of different composite 3D-printed scaffolds are shown below. Figure 9 As shown, Figure 9 In the chart, A represents alkaline phosphatase (ALP) staining, B represents alizarin red (ARS) staining, C represents a bar chart showing the percentage of ALP-positive areas, and D represents a bar chart showing the percentage of ARS-positive areas.

[0115] like Figure 9 As shown in Figure A, a small amount of ALP staining (blue-purple) areas were observed in the PH and PHQ groups, while significantly more ALP-positive areas were found in the PHO and PHOQ groups, with the deepest staining depth in the PHOQ group. ALP activity assays also showed that the PHOQ group exhibited the most significant expression among all groups (mean positive staining area percentage was 55.16±4.48%), followed by the PHO group, with a staining area of ​​47.24±3.65%. Figure 9(C). These findings suggest that the presence of HA within the scaffold combined with sustained OGP release can improve osteogenic differentiation of MC3T3-E1 cells.

[0116] Figure 9 The B-mode image shows the calcium deposition effect in MC3T3-E1 cells cultured in different scaffold extracts for 21 days. ARS staining showed a similar trend to ALP staining. The PHO and PHOQ groups induced more calcium nodule formation than the PH and PHQ groups. The PHOQ group showed the highest mineralization level (mean positive area 30.90±2.49%), and the positive area in the PHOQ group was significantly increased compared to the PH, PHO, and PHQ groups (p<0.05). Figure 9 (D). The superior osteogenic induction capacity exhibited by the PHOQ group can be attributed to the synergistic effect of the dual release of QK and OGP within the scaffold on the mineralization of MC3T3-E1 cells.

[0117] The above experimental results show that the PHOQ scaffold has excellent ability to promote angiogenesis and osteogenic induction. It can release QK first to promote angiogenesis in a time sequence, and then continuously release OGP to synergistically promote bone regeneration. This is consistent with the physiological self-healing process of bone injury. The PHOQ scaffold can be used as a bone repair biomaterial to promote the repair of bone defects.

[0118] Example 5: In vivo verification in animal experiments

[0119] Diabetes mellitus, through hyperglycemia, accumulation of advanced glycation end products (AGEs), increased reactive oxygen species (ROS), and inflammatory responses, inhibits osteoblast differentiation and proliferation, promotes osteoclast activity, leading to reduced bone formation, decreased bone density, and decreased bone regeneration capacity. To verify that the PHOQ scaffold can serve as a medical material for bone repair and promote bone defect repair, this embodiment first constructs a diabetic animal model and then artificially induces bone injury to simulate an application scenario where the body's own bone regeneration and healing capacity is low.

[0120] 1. Modeling and Grouping

[0121] (1) Animal feeding: Male Sprague-Dawley (SD) rats weighing approximately 300g were purchased from the Guangdong Provincial Experimental Animal Center and housed at Shenzhen Lingfu Top Biotechnology Co., Ltd., with free access to water and 12-hour light-dark alternation. The animal experimental protocol was approved by the Animal Ethics Committee of Shenzhen Lingfu Top Biotechnology Co., Ltd. (Project No.: IACUCNO:2023-0302).

[0122] (2) Establishment of a diabetic animal model: After a 7-day adaptation period, rats were placed in a fasting state for 24 hours, and then injected intraperitoneally with streptozotocin (STZ) at a dose of 75 mg / kg. Blood glucose was measured 3 days later. If the blood glucose level was higher than 16.1 mmol / L, the diabetic rat model was considered to have been successfully established.

[0123] (3) Anesthesia and surgical preparation: SD rats were anesthetized with isoflurane gas and anesthesia was maintained with a breathing mask. After anesthesia, the rats' heads were prepared, the hair on the heads was shaved with a razor, and the limbs were fixed in a prone position. After disinfection with povidone-iodine, a 1.5 cm long incision was made along the midline of the head, and then a circular critical-size bone defect with a diameter of 5 μm was drilled with a dental drill.

[0124] (4) Experimental intervention and postoperative management: The skull defect area of ​​SD rats was filled with different composite 3D printed scaffolds, which were designated as PH, PHO, PHQ and PHOQ groups, respectively, and the incision was closed by suturing layer by layer. After the operation, the rats were housed separately. All experimental rats were given 30 mg / kg of ampicillin and 2 mg / kg of meloxicam to prevent infection and relieve pain.

[0125] 2. Micro-CT scan

[0126] Rats were euthanized at 4 and 8 weeks post-surgery by intraperitoneal injection of an overdose of chloral hydrate, and their skull tissues were harvested. Micro-CT (NEMO, China) was used to scan and reconstruct skull regeneration and bone mineral density, with parameters of 80 kV voltage, 100 μA current, and 18 μm voxel size. The acquired skull tomographic sections were reconstructed into 3D images using the accompanying reconstruction software, with a grayscale setting of 220. Three-dimensional bone morphology measurements were used to determine the volume of newly regenerated bone, bone volume / total volume (BV / TV), and bone mineral density (BMD) in diabetic rats. The micro-CT scan analysis results of different composite 3D-printed scaffolds for skull defect repair in diabetic rats are shown below. Figure 10 As shown, Figure 10 In the diagram, A represents Micro-CT scan reconstruction, B represents bone mineral density (BMD), C represents BV / TV, and D represents regenerated bone volume.

[0127] 3. Animal sampling and embedding sections

[0128] (1) Decalcification: After Micro-CT scan, fix with 4% paraformaldehyde at room temperature for 24 hours, rinse with tap water to remove residual formaldehyde, and then immerse the skull tissue in 20% EDTA decalcification solution for decalcification, changing the solution twice a week.

[0129] (2) Tissue trimming: After decalcification, rinse with running water again, trim excess tissue from the skull sample, cut along the midline, place it in the embedding cassette, rinse with running water again for 4 hours to remove residual fixative.

[0130] (3) Dehydration and clearing: The above samples were placed in 50%, 75%, 85%, 95%, 100% and 100% alcohol solutions for dehydration, 2 hours for each gradient. The dehydrated samples were then placed in xylene for clearing twice, 30 minutes each time.

[0131] (4) Paraffin impregnation and embedding: The transparent skull sample was then immersed in the melted paraffin solution three times, each time for 2 hours. After the skull sample was fully impregnated with paraffin, it was placed in an embedding machine for embedding.

[0132] (5) Sectioning: The embedded skull sample was fixed on a microtome and sectioned to a thickness of 4 μm. After sectioning, the sections were placed in a 65°C oven for 6 hours for later use.

[0133] To further evaluate the quality of skull bone regeneration in rats, this example also employed HE and Masson staining for histological analysis, as detailed below:

[0134] 4. HE staining

[0135] (1) Dewaxing and rehydration: The above-mentioned skull tissue sections were baked at 65°C for 30 minutes and then dewaxed and rehydrated. They were immersed in xylene twice for dewaxing, each time for 15 minutes. Then, they were rehydrated by passing through 100%, 95%, 85%, 75% and 50% alcohol solutions in stages, each gradient treatment for 2 minutes. Finally, they were placed in tap water for the next step of processing.

[0136] (2) Hematoxylin staining: Immerse the sections in hematoxylin dye for 5 minutes and wash thoroughly with water.

[0137] (3) Color separation: Immerse the slices in 1% (v / v) hydrochloric acid alcohol solution for 5-7 seconds, then quickly remove and wash with water.

[0138] (4) Blue inversion: Immerse the slices in ammonia water (1:400) for 1 minute to invert blue, then remove and wash with water again.

[0139] (5) Eosin staining: Immerse the section in 75% ethanol solution for 30 seconds, remove and spin dry, then immerse in eosin dye for 40 seconds, and quickly remove and wash with water.

[0140] (6) Mounting: Immerse the slides in 100% ethanol for 1 minute, remove and air dry, then mount with neutral quick-drying gel. HE staining results are as follows. Figure 11 As shown in A, Figure 11The "▲" in the middle represents a 3D-printed scaffold, the same below; NB represents newly formed bone, LB represents lamellar bone; TB represents total regenerated bone. Figure 11 In the middle, C is a bar chart of NB / TB statistics, and D is a bar chart of LB / TB statistics.

[0141] 5. Masson staining

[0142] (1) Dewaxing and rehydration: Same as above.

[0143] (2) Iron hematoxylin staining: outline the tissue area with an immunohistochemical pen, add iron hematoxylin dye and stain for 5 minutes, then wash thoroughly with water.

[0144] (3) Color separation: Same as above.

[0145] (4) Spring Red Staining: Add Spring Red dye and stain for 2 hours, then wash thoroughly with water.

[0146] (5) Phosphomolybdic acid color separation: Add 1% phosphomolybdic acid solution for color separation treatment for 1 minute.

[0147] (6) Aniline blue staining: Add aniline blue dye for 2 minutes and then spin dry.

[0148] (7) Wash with glacial acetic acid: Immerse the slices in 1% glacial acetic acid solution for 10 seconds.

[0149] (8) Mounting: Same as above. Masson staining results are as follows. Figure 11 As shown in B.

[0150] 6. Immunohistochemistry

[0151] (1) Dewaxing and rehydration: Same as above.

[0152] (2) Antigen retrieval: Immerse the slides in antigen retrieval solution, microwave for 8 minutes, add pure water to make up the evaporation volume, heat again for 8 minutes, and then rinse thoroughly with water after returning to room temperature. See Table 4 for the types of antigen retrieval solutions.

[0153] (3) Blocking and inactivation: Immerse the slices in peroxidase blocking solution for 15 minutes and wash thoroughly with water.

[0154] (4) Blocking: Outline the tissue area with an immunohistochemical pen, add blocking goat serum and incubate for 40 minutes.

[0155] (5) Primary antibody incubation: Spin dry the sections, add primary antibody to fully cover the tissue, and incubate overnight at 4°C in a humidified chamber. Remove the sections the next day, warm them again, and wash thoroughly with water. The primary antibody dilution ratios are ALP (1:500 dilution, ET1601-21, HUABIO) and OCN (1:250 dilution, ER1919-20, HUABIO).

[0156] (6) Secondary antibody incubation: Add secondary antibody (1:250 dilution) and incubate at room temperature for 40 minutes, then wash thoroughly with water.

[0157] (7) Color development: Prepare the working solution by adding 1 drop of DAB concentrate to 1 mL of diaminobenzidine (DAB) diluent. Add the DAB working solution to cover the tissue. Observe under a microscope until a distinct brown color appears and forms a sharp contrast with the surrounding tissue, then stop the color development and wash thoroughly with water.

[0158] (8) Hematoxylin staining: Immerse the slices in hematoxylin dye for 1 minute and wash thoroughly with water.

[0159] (9) Color separation: Same as above.

[0160] (10) Sealing: Same as above. Evaluation results of the osteogenic properties of different composite 3D printed scaffolds for repairing skull defects in diabetic rats are as follows: Figure 12 As shown, Figure 12 In the diagram, A represents alkaline phosphatase (ALP) staining, B represents a statistical bar chart of alkaline phosphatase (ALP) staining, C represents osteocalcin (OCN) staining, and D represents a statistical bar chart of osteocalcin (OCN) staining. "▲" represents a 3D printed scaffold.

[0161] 7. Immunofluorescence

[0162] (1) Dewaxing, rehydration, antigen retrieval, peroxidase inactivation and blocking are the same as above.

[0163] (2) Primary antibody incubation: Spin-dry the sections, add primary antibody to fully cover the tissue, and incubate overnight at 4°C in a humidified chamber. Remove the sections the next day, warm them again, and wash thoroughly with water. The primary antibody dilution ratios are CD206 (1:1000 dilution, ab300621, Abcam) and CD31 (1:1000 dilution, ab281583, ab281583).

[0164] (3) Secondary antibody incubation: Add Alexa Fluor 594 and Alexa Fluor 488 fluorescent secondary antibodies (1:250 dilution) respectively, incubate at room temperature for 40 minutes, and then wash thoroughly with water.

[0165] (4) The mixture was sealed by adding a glycerol mounting medium containing DAPI and observed under a laser microscope. The results are as follows: Figure 13 As shown in the figure. The evaluation results of the immunogenic and angiogenic properties of different composite 3D-printed scaffolds for the repair of skull defects in diabetic rats are as follows. Figure 13 As shown, Figure 13 In the diagram, A represents CD206 immunofluorescence staining, B represents CD31 staining, C represents a CD206 statistical bar chart, D represents a CD31 statistical bar chart, E represents a blood vessel diameter statistical bar chart, and "▲" represents a 3D printed stent.

[0166] Animal experiment results:

[0167] (1) Micro-CT scan

[0168] like Figure 10 As shown, at 4 and 8 weeks after scaffold implantation, bone regeneration was only observed in the peripheral portion of the skull defect in the PH and PHQ groups, with cavities still visible between the scaffold struts, indicating insufficient new bone formation. Although the cavities in the PHO scaffold were filled with new bone tissue after 8 weeks, gaps remained between the scaffold and the defect boundary. In contrast, the PHOQ scaffold significantly improved bone regeneration in the skull defect, with the gap between the defect boundary and the scaffold cavity essentially filled by newly regenerated bone tissue. Figure 10 As shown in Figure A, at 4 and 8 weeks post-implantation, Micro-CT scans revealed that the PHOQ group dominated in all measured parameters of bone regeneration. The BV / TV ratios were calculated to be 7.87±0.61% and 15.93±0.55%, respectively (p<0.05, vs. PH, PHO, and PHQ groups). Figure 10 (B) Bone volume / total volume (BV / TV) and bone mineral density (BMD) were significantly increased (p<0.05 compared with the PH, PHO, and PHQ groups). Bone mineral density (BMD) was significantly increased; at 4 and 8 weeks after implantation, the BMD in the PHOQ group was 1.15±0.01 g / cm³. 3 and 1.24±0.01g / cm 3 (Compared with the PH, PHO, and PHQ groups, p<0.05) Figure 10 (C). Furthermore, compared to the PH and PHQ groups, although the PHO group also showed significantly enhanced bone regeneration indices, with marked increases in new bone mass, BV / TV, and BMD (p<0.05), the new bone mass in the PHOQ group was only 3.93±0.33 mm. 3 and 7.13±0.22mm 3 The value was significantly higher than the other three groups (p<0.05). Figure 10 (Middle D). The enhanced osteogenic activity observed in these results, particularly in scaffolds containing OGP (PHO and PHOQ groups), confirms that OGP is an effective active ingredient for stimulating bone regeneration. The PHOQ scaffold induced the highest levels of bone regeneration volume and density, which can be attributed to the synergistic effect of angiogenesis and osteogenic activity induced by QK and OGP release.

[0169] (2) HE and Masson staining

[0170] Depend on Figure 11HE staining results showed that, 4 weeks after scaffold implantation, significant new cell migration could be observed near the scaffolds in the PHO, PHQ, and PHOQ groups, with deeper staining and more pronounced cell infiltration observed in the PHOQ group. Figure 11 (A). Furthermore, new bone formation was observed above the scaffold in the PHOQ group at 4 and 8 weeks after implantation, and new bone formation also appeared around the scaffold in the PHO group at 8 weeks.

[0171] Depend on Figure 11 Masson staining results showed that at 4 and 8 weeks after scaffold implantation, only limited new bone tissue and blood vessels were observed around the PH group scaffold; while the PHQ group showed significantly more blood vessels, but new bone formation was still insufficient, and cell migration around the scaffold was inadequate. This indicates that the release of pro-angiogenic biomolecules can effectively improve vascularization, but in the process of tissue regeneration, the presence of pro-angiogenic biomolecules alone in the scaffold cannot effectively enhance bone regeneration. In contrast, the PHO group scaffold showed significantly more new bone formation and cell infiltration, and at 8 weeks, red calcified lamellar bone structures appeared on the scaffold surface, including some blood vessels. The PHOQ group scaffold not only induced new bone formation but also showed significant cell infiltration, and the diameter of the newly formed blood vessels around it was also larger. Figure 11 (B) It is worth noting that the upper layer of the scaffold in the PHOQ group showed a significantly thicker calcified lamellar bone structure, a feature that was more prominent than that in the PHO group.

[0172] Further analysis was conducted to quantify the evaluation of regenerated bone by analyzing the ratio of newly formed bone area to total bone area (NB / TB) and the ratio of lamellar bone area to total bone area (LB / TB). The results were consistent with the observed phenomena; both the PHO and PHOQ groups showed higher NB / TB and LB / TB ratios at 4 and 8 weeks, indicating greater collagen fiber deposition in the bone defect area. Figure 11(C and D) Among them, the PHOQ group showed better bone regeneration. Eight weeks after implantation, the NB / TB and LB / TB ratios of all groups increased significantly. The NB / TB ratio of the PHOQ group was 48.57±0.89%, and the LB / TB ratio was 25.54±0.79%, both significantly higher than those of the PH and PHQ groups. These experimental results confirm the beneficial effects of integrating OGP osteogenic growth peptides into 3D-printed scaffolds. Furthermore, coating the OGP-loaded scaffold with the angiogenic factor QK further endows it with excellent angiogenic capacity and effectively induces the regeneration of large-diameter vessels, thereby compensating for the insufficient bone regeneration of QK alone and further enhancing the ability of OGP to promote new bone formation and calcification in patients with bone defects. This also indicates that the PHOQ scaffold is a composite bone tissue engineering scaffold with dual QK and OGP delivery functions, serving as an ideal platform for enhancing bone tissue regeneration and achieving the desired vascularization, and can be used to prepare bone repair biomaterials to promote the regeneration and repair of defective or damaged bone tissue.

[0173] (3) Immunohistochemistry

[0174] Depend on Figure 12 Results A and B show that 4 weeks after stent implantation, ALP staining revealed significantly higher ALP expression levels in the PHO and PHOQ groups compared to the PH and PHQ groups (p<0.05). Specifically, the ALP expression area ratio in the PHO group was 4.75±0.59%, while it was 5.67±0.43% in the PHOQ group. Notably, 8 weeks after implantation, the ALP area ratio in the PHO group increased to 5.67±0.54%, while it increased to 7.08±0.70% in the PHOQ group. Compared to the PHO group, the PHOQ group showed significantly higher ALP levels (5.67±0.54% vs 7.08±0.70%, p<0.05), and also significantly higher than the PHQ group (p<0.001), indicating a significant increase in ALP osteogenic expression over time after PHOQ stent implantation.

[0175] Figure 12 The staining results of OCN in the middle are consistent with the trend observed in ALP staining results. Figure 12 (C and D). Notably, at 4 and 8 weeks post-implantation, the OCN expression levels in the PHO and PHOQ groups were significantly higher than those in the PH and PHQ groups (p<0.05). Specifically, the OCN expression level in the PHO group was 5.92±0.23% at 4 weeks and 7.04±0.09% at 8 weeks; while the OCN expression level in the PHOQ group was 6.84±0.44% at 4 weeks and 8.07±0.34% at 8 weeks (p<0.05). Figure 12(C, D) This indicates that OCN, as a late-stage osteogenic marker, showed significantly higher expression levels than ALP at 8 weeks, and the OCN-positive rate in the PHOQ group was significantly higher than that in the PHO group at 8 weeks (p<0.05). Furthermore, at 8 weeks, the expression levels in the PHOQ group were significantly higher than the other three groups in both ALP and OCN staining (p<0.05). These results demonstrate that the PHOQ scaffold possesses optimal osteogenic capacity, promoting osteoogenesis not only in the early stages but also enhancing bone mineralization in the later stages. This proves that the time-released QK and OGP in this 3D-printed scaffold (PHOQ scaffold) synergistically promote better bone formation.

[0176] The host's innate immune response also plays a crucial role in bone regeneration. CD206 (also known as the mannose receptor) is a transmembrane glycoprotein that participates in tissue repair, anti-inflammatory responses, and immune regulation. It is a marker of M2 macrophages and is closely related to their anti-inflammatory and tissue repair functions. In this embodiment, the immunofluorescence of CD206 was also used to evaluate the immunomodulatory effect of the 3D-printed scaffold in vivo. Figure 13 The results from the study showed that, 14 days after stent implantation, CD206 expression gradually increased across the PH, PHO, PHQ, and PHOQ groups. The CD206 expression level in the PHQ group was 1.37±0.14%, significantly higher than that in the PH and PHO groups (p<0.05). The PHOQ group showed the strongest expression, at 1.98±0.15% (significantly higher than the PH, PHO, and PHQ groups, p<0.05). Figure 13 (C). In this experiment, the enhanced expression of these CD206 cells may be related to the combined anti-inflammatory effects of QK and OGP. QK has been reported to indirectly induce M2 macrophage polarization by regulating the VEGF signaling pathway. OGP has also been shown to have osteogenic and immunomodulatory effects, possibly by promoting the secretion of anti-inflammatory factors and increasing the positive proportion of M2 macrophages, thereby reducing local inflammatory response and improving the bone repair environment.

[0177] To further elucidate the angiogenesis potential of 3D-printed scaffolds in vivo, we conducted a detailed study on their implantation efficacy. Fourteen days after implantation, the expression of CD31, a typical vascular biomarker around the scaffold, showed an increasing trend across groups. Figure 13(B) Statistical analysis of CD31-positive areas showed that the positive area in the PHQ group was 4.39±0.49%, significantly larger than that in the PH and PHO groups (p<0.05). The CD31-positive area in the PHOQ group was 5.81±0.59%, significantly larger than the other three groups (p<0.05). Notably, on day 14, the diameter of neovascularization around the stent in the PHOQ group was significantly increased compared to the PH and PHO groups, reaching 17.65±2.17 μm (p<0.05). In contrast, the vessel diameters in the PH, PHO, and PHQ groups were all less than 15 μm, indicating that QK and OGP on the PHOQ stent can also effectively synergistically promote angiogenesis in vivo. Figure 13 D, E).

[0178] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a composite 3D-printed scaffold with time-regulated vascular and bone tissue regeneration, characterized in that, Includes the following steps: Osteogenic inducer and angiogenic differentiation inducer were loaded onto two carrier materials with different degradation rates. The slow-release carrier loaded with osteogenic inducer was first mixed with nano-hydroxyapatite and then 3D printed at a low temperature below 0°C to create a scaffold skeleton. While forming the 3D printed scaffold skeleton, the slow-release osteogenic inducer was loaded onto it. Then, the fast-release carrier loaded with the angiogenic differentiation agent is coated onto the 3D printed scaffold skeleton to construct a composite 3D printed scaffold with different layers, so that the inner layer is loaded with the osteogenic inducing agent and the outer layer is loaded with the angiogenic differentiation agent. The osteogenic inducing agent includes BMP-2 and / or OGP peptide; the angiogenic differentiation inducing agent includes VEGF and / or QK peptide; The slow-release carrier is selected from PLGA or PCL; the fast-release carrier is selected from gelatin, sodium alginate, collagen or methacrylamide dextran.

2. The preparation method according to claim 1, characterized in that, The osteogenic inducer is an OGP peptide, the amino acid sequence of which is shown in SEQ ID NO:

1.

3. The preparation method according to claim 2, characterized in that, The angiogenic differentiation agent is QK peptide, whose amino acid sequence is shown in SEQ ID NO:

5.

4. The preparation method according to any one of claims 1 to 3, characterized in that, The low temperature range is 0℃ to -20℃.

5. The preparation method according to claim 4, characterized in that, The slow-release carrier, nano-hydroxyapatite, and OGP peptide were mixed in a mass ratio of 200:100:1 to 160:40:1 and co-melted in a 1,4-dioxane solution. Low-temperature printing was performed using a 3D printer with the following parameters: printing speed 20-55mm / s, pneumatic pressure 80-150kPa, receiving plate temperature between -10℃ and -20℃, and 27G printing needle. After the 3D printing was completed, the support frame was freeze-dried at low temperature. The freeze-dried scaffold framework was then immersed in a fast-release carrier solution containing QK peptides and photocured using ultraviolet light to obtain a composite 3D-printed scaffold with time-regulated vascular and bone tissue regeneration.

6. The preparation method according to claim 5, characterized in that, The mass ratio of the QK peptide to the methacrylamide dextran is 1:50 to 1:

100.

7. A composite 3D-printed scaffold with time-regulated vascular and bone tissue regeneration, characterized in that, Prepared according to the preparation method described in any one of claims 1 to 6.

8. The application of a composite 3D-printed scaffold with time-regulated vascular and bone tissue regeneration prepared by the method according to any one of claims 1 to 6 in the preparation of medical materials for self-repairing bone defects.

9. The application as described in claim 8, characterized in that, The composite 3D printed scaffold is used to prepare self-repairing medical materials for diabetic bone defects.

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