PLGA microsphere-PCL / GelMA composite scaffold based on NGF slow release as well as preparation method and application of PLGA microsphere-PCL / GelMA composite scaffold

Abstract

The invention discloses a PLGA microsphere-PCL / GelMA composite scaffold based on neurotrophic factor (NGF) slow release as well as a preparation method and application of the PLGA microsphere-PCL / GelMA composite scaffold. According to the composite scaffold, NGF-loaded PLGA microspheres are embedded into a PCL skeleton and a GelMA hydrogel matrix, and a three-phase composite system with mechanical support, biological factor sustained release and cell adhesion functions is constructed. In-vitro experiments show that the NGF-loaded composite scaffold can significantly promote osteogenic differentiation of dental pulp stem cells, which shows that the activity of alkaline phosphatase is improved, the formation of mineralized nodules is increased, and the expression of osteogenic related genes RUNX2, ALPL, COL1A1 and BGLAP is up-regulated. When the composite stent is applied to a rat skull critical defect model in vivo, new bone formation and vascular nerve regeneration are remarkably promoted. Mechanism research shows that regulation and control of osteogenesis and bone resorption are realized by activating a PI3K / AKT signal channel. The composite stent is suitable for repairing complex bone defects in areas with rich nerves and blood vessels, and has a good clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of porous biological scaffolds, and in particular to a PLGA microsphere-PCL / GelMA composite scaffold based on NGF sustained release, and a preparation method and application thereof. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] The repair of craniomaxillofacial bone defects faces major challenges, which are mainly reflected in three aspects: first, the anatomical structure of this area is complex, with dense distribution of nerves and blood vessels; second, severe bone tissue defects are often accompanied by soft tissue collapse, nerve damage and dysfunction; third, the repair process needs to take into account both morphological reconstruction and functional recovery. Currently, the commonly used repair strategies in clinical practice have limitations, and traditional repair methods have obvious shortcomings: for example, 1. Autologous bone transplantation faces donor site limitations, which is particularly prominent in the repair of large bone defects, and there are risks such as large surgical trauma to the donor site and multiple complications; 2. Although allogeneic / xenogeneic bone materials are widely available, they face the risk of immune rejection and infection, and the long-term repair effect is not ideal; 3. Although metal / ceramic restorations can provide mechanical support, they lack biological activity and cannot achieve true functional regeneration.

[0004] The defects of existing tissue engineering technology are mainly reflected in the following aspects: 1. One-sided design concept: most scaffolds only focus on the osteogenesis process and ignore the core mechanism of "vascular-neural coordinated regulation"; 2. There are technical bottlenecks in the application of biological factors: these include poor factor stability (such as NGF (neurotrophic factor), which is easily affected by enzymatic hydrolysis and pH, degrades rapidly in the body, and has a short biological activity duration), uncontrollable release mode (burst release, excessively high concentrations in a short time may cause side effects, and cannot maintain sustained effects in the later stage), and insufficient fusion between the carrier system and the scaffold; 3. Low functional integration: there is a lack of a comprehensive solution that can take into account the three functions of osteogenesis, vascularization promotion and nerve repair.

[0005] The key to a breakthrough in this field is to develop a new composite scaffold system with a multi-level structure that can achieve stable and sustained release of factors (such as NGF), and effectively induce neovascularization and nerve fiber reconstruction while promoting bone formation, thereby achieving a transition from "unidirectional osteogenesis" to "multidimensional regeneration", thereby meeting the clinical needs of complex bone defect repair. Summary of the Invention

[0006] In view of this, the present invention provides a PLGA microsphere-PCL / GelMA composite scaffold based on NGF sustained release and its preparation method and application, aiming to achieve the controlled release of neurotrophic factors and rebuild the "bone-blood vessel-nerve" network in the multi-dimensional bone defect area, thereby improving the repair effect.

[0007] In order to achieve the above object, the present invention is implemented through the following technical solutions: In its first aspect, the present invention provides a PLGA microsphere-PCL / GelMA composite scaffold for sustained release of NGF. The composite scaffold consists of a PCL framework, a GelMA hydrogel matrix, NGF-loaded PLGA microspheres (P-NGF), and DPSCs (dental pulp stem cells). The PCL framework is pre-mixed with polycaprolactone and hydroxyapatite in a mass ratio of 2-5:1 and produced using fused deposition modeling (FDM) three-dimensional printing technology. The NGF-loaded PLGA microspheres and DPSCs are uniformly dispersed within the GelMA matrix, which is then filled within the PCL framework.

[0008] PCL skeleton: It is prepared using FDM three-dimensional printing technology to form a scaffold body with a regular porous structure to provide mechanical support. The structure is usually arranged in a staggered grid, and its printing path may include 0° / 90°, 0° / 60° / 120° or other orthogonal angle designs. The pore size range of the scaffold is 200-800μm, preferably 300-500μm, to facilitate cell penetration and tissue ingrowth. The external dimensions of the scaffold can be customized according to the defect site. The general diameter range is 4-8 mm, preferably 5.0±0.5 mm, the height is 0.5-1.0 mm, preferably 0.6±0.1 mm, and the thickness of each printed layer is controlled between 150-250μm. The above structural parameters can be fine-tuned according to application requirements to meet the comprehensive requirements of porosity, mechanical properties and biopermeability of different tissue repair sites.

[0009] The GelMA hydrogel matrix utilizes gelatin methacryloyl (GelMA) as the main component, combined with a photoinitiator to form a photosensitive crosslinking system, forming a uniform gel structure that fills the pores of the 3D-printed scaffold. The mass concentration of the GelMA prepolymer solution ranges from 3%–15% (w / v), preferably 6%–10% (w / v), and can be adjusted based on the desired scaffold mechanical strength and cell growth characteristics. The photoinitiator LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate) is added in a ratio ranging from 0.05%–0.5% (w / v), preferably 0.2%–0.3% (w / v). Crosslinking and curing are completed by irradiation with 405 nm light, with a reaction time controlled within 10–60 seconds.

[0010] The hydrogel can form a highly hydrophilic three-dimensional network system in terms of physical structure, and the pore size distribution is conducive to cell attachment, nutrient exchange and waste discharge. As a biomimetic extracellular matrix, GelMA not only promotes stem cell adhesion and directional differentiation, but also has good composite compatibility with PLGA microspheres, which helps to embed microspheres in situ and stabilize their spatial positioning during the gel cross-linking process, thereby achieving sustained release support for NGF. The above-mentioned gel composition, cross-linking light intensity and time can be flexibly adjusted according to the actual repair site, factor release time and printing structure matching requirements, and have good adaptability and clinical transformation potential.

[0011] NGF-loaded PLGA microspheres (P-NGF): Composed of poly(lactic-co-glycolic acid) (PLGA) and NGF, the microspheres are prepared using a double-emulsion solvent evaporation method (W / O / W) to form spherical particles. The microspheres have a diameter range of 80–200 μm, preferably 100–150 μm, and feature a smooth surface and a concentrated size distribution, exhibiting excellent structural stability and entrapment efficiency. The ratio of lactic acid (LA) to glycolic acid (GA) in the PLGA polymer is 70:30 to 90:10, preferably 85:15, and a molecular weight range of 20–60 kDa to control the in vivo degradation rate and drug release properties. This parameter setting enables sustained release of NGF for 56–70 days with an encapsulation efficiency of 70%–90%. After preparation, the microspheres are uniformly dispersed in the GelMA hydrogel matrix and are in situ fixed within the stent during the GelMA photocrosslinking process, preventing spatial drift or localized extravasation that could reduce release efficiency after implantation. This design not only ensures the targeted enrichment and continuous supply of NGF in the defect area, but also forms a microenvironment signal source, continuously activates the PI3K / AKT signaling pathway, and induces stem cell osteogenesis, angiogenesis and neural repair.

[0012] The composite scaffold described in this invention achieves bone-vascular-nerve regeneration through the following synergistic mechanism: The components of the composite scaffold are physically bonded and photocrosslinked to form a stable composite structure. The PCL skeleton provides mechanical support; the GelMA hydrogel supports cell adhesion, migration, and differentiation; and the PLGA microspheres achieve sustained release of NGF (for over 56 days), activating the PI3K / AKT signaling pathway and promoting DPSC osteogenesis, angiogenesis, and nerve repair. In vivo experiments have shown that the scaffold significantly upregulates the expression of markers such as ALP, OCN, COL1, CD31, α-SMA, and NF200, demonstrating a high degree of biofunctional integration.

[0013] In a second aspect, the present invention provides a method for preparing the PLGA microsphere-PCL / GelMA composite scaffold based on NGF sustained release according to the first aspect, which specifically comprises the following steps: (1) Preparation of internal phase: Dissolve NGF in HEPES (4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) buffer at a concentration of 80-120 μg / mL; (2) Oil phase preparation: PLGA was dissolved in dichloromethane at a concentration of 2-5% (w / v) to form an oil phase; (3) Primary emulsification (W / O): Under ultrasonic conditions, the inner water phase is emulsified into the oil phase to form a uniform W / O emulsion; (4) Secondary emulsification (W / O / W): The W / O emulsion was slowly dripped into a 0.2-1% (w / v) polyvinyl alcohol (PVA) aqueous solution to form a stable W / O / W double emulsion under magnetic stirring; (5) Solvent evaporation and particle solidification: Continue stirring for 4-6 hours to allow DCM to evaporate completely and the microspheres to solidify and form; (6) Microsphere collection and drying: After centrifugation and washing, freeze-dry for 24-48 hours to obtain dry P-NGF microspheres; (7) Preparation of PCL skeleton: The PCL skeleton is pre-mixed with polycaprolactone and hydroxyapatite (HA) at a mass ratio of 2-5:1 and prepared by fused deposition modeling 3D printing technology; (8) Preparation of GelMA prepolymer solution containing cells and microspheres: P-NGF microspheres and DPSCs (dental pulp stem cells) were mixed in GelMA prepolymer solution containing a photoinitiator to prepare a uniform suspension; (9) Scaffold composite and photocrosslinking: The suspension described in step (8) was injected into the pores of the PCL skeleton, and then irradiated with 405 nm light at 15 mW / cm 2 The cross-linking is carried out under light intensity conditions and the cross-linking time is controlled within 30-50 seconds.

[0014] The final scaffold can be UV sterilized or sterile packaged for storage, and is suitable for subsequent cell culture, animal implantation or in vivo tissue engineering research.

[0015] Preferably, in step (3), the ultrasonic treatment time is 30-60s; Preferably, in step (4), the magnetic stirring is performed at 500-1000 rpm; Preferably, in step (6), the centrifugal speed is 8000-12000 rpm; Preferably, the P-NGF microspheres prepared in step (6) are regular spherical, with a particle size of 80-200 μm and an encapsulation efficiency of 70-90%; more preferably, the particle size is 100-150 μm; Preferably, in step (7), the specific parameters of 3D printing are: nozzle diameter: 0.3 mm; nozzle temperature: 110°C; layer height: 0.2 mm; printing pressure: 0.55 MPa; printing speed: 6 mm / s; arrangement mode: 0° / 90° staggered grid structure; pore size range: 300-500 μm; Preferably, in step (7), the size of the scaffold prepared by three-dimensional printing (single body) is: diameter range: 4-8 mm, more preferably 5 mm±0.5 mm; height range: 0.5-1.0 mm, more preferably 0.6 mm±0.1 mm; skeleton quality control range: 15-30 mg.

[0016] Preferably, in step (8), the amount of microspheres added is 5-10 mg, corresponding to a total amount of NGF of 1-2 μg; the seeding density of DPSCs is 1×10 6 cells / mL; GelMA concentration is 6-10% (w / v), photoinitiator LAP concentration is 0.2-0.3% (w / v); the mass ratio of photoinitiator to GelMA is 1:20-1:40.

[0017] In the third aspect, the present invention provides the use of the NGF-sustaining PLGA microsphere-PCL / GelMA composite scaffold based on sustained release of NGF described in the first aspect of the present invention or the NGF-sustaining PLGA microsphere-PCL / GelMA composite scaffold prepared in the second aspect in tissue regeneration and repair of bone defects.

[0018] Preferably, the composite scaffold provided by the present invention can be directly used for tissue regeneration and repair of bone defects after preparation.

[0019] Preferably, the composite scaffold provided by the present invention can be applied to tissue regeneration and repair of bone defect areas with dense blood vessels and nerves and high repair complexity, such as: craniomaxillofacial bone defects, reconstruction of mandibular or zygomatic fractures, repair of bone structures related to neural pathways (such as scoliosis or vertebral laminae), and bone-cartilage transition zones with soft tissue interfaces.

[0020] The composite scaffold provided by the present invention can simultaneously induce the regeneration of three types of tissues: bone, blood vessels, and nerves. It has the comprehensive advantages of structural support, signal release, and microenvironment simulation, and provides a new strategy for functional bone repair materials.

[0021] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) Constructing a three-phase synergistic scaffold system with integrated structure and function to improve the overall performance and stability of the scaffold. This invention constructs a PCL skeleton based on 3D printing, combines GelMA hydrogel and P-NGF microspheres, and forms a composite scaffold system with the triple functions of mechanical support, biological factor release, and cell attachment. The constituent phases form a stable chimeric interface through in situ cross-linking, which has good structural integrity and functional synergy.

[0022] (2) Achieve controlled sustained release of the non-model factor NGF, avoiding the problem of explosive release. The present invention uses a double emulsion method to construct PLGA microspheres to encapsulate the neurotrophic factor NGF, and regulates its in vivo degradation and release curve by adjusting the polymer lactic acid / glycolic acid ratio. The prepared microspheres can achieve a sustained release period of up to 56 days, effectively overcoming the problems of high concentration in the early stage and insufficient release in the later stage of traditional encapsulation systems.

[0023] (3) Inducing the coordinated regeneration of "bone-blood vessel-nerve" and significantly improving the quality of functional repair. In in vivo experiments, the composite scaffold can activate the PI3K / AKT signaling pathway through the sustained release of NGF, synergistically promoting bone formation, the construction of new blood vessels and nerve fibers. Studies have shown that it can significantly upregulate the expression of functional markers such as ALP, OCN, COL1, CD31, α-SMA, and NF200, and the new bone volume fraction (BV / TV) is at least 2.5 times higher than that of the blank group, showing good multi-tissue repair ability.

[0024] (4) The mechanism of action is clear and verifiable at the molecular level. RNA-seq and protein level analysis showed that P-NGF microsphere scaffolds can significantly activate upstream factors of the PI3K / AKT pathway and inhibit the expression of genes involved in the osteoclastogenesis-related pathway, thereby achieving bidirectional regulation of osteogenesis and bone resorption.

[0025] (5) The process is mild, the materials are safe, and it has good clinical translational applicability. The scaffold is made of medical materials such as PLGA, PCL, and GelMA with excellent biocompatibility, and no organic solvents or toxic components are used. In vitro cell viability and CCK-8 assays showed that it had no cytotoxicity to DPSCs, and the cell survival rate remained above 90%. The scaffold preparation process is stable and the parameters are controllable, making it suitable for standardized production. The structure and size are also suitable for various types of bone defect models (such as skull, mandible, spine, etc.), and it has good scalability and translational prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1This is an analysis diagram of the structure and physical and chemical properties of the composite stent of the present invention; Figure 2 This is a diagram of an in vitro osteogenic induction experiment of the present invention; Figure 3 This is an analysis diagram of skull defect repair and bone regeneration in vivo according to the present invention; Figure 4 It is the bone formation quantification and histological staining diagram of the present invention; Figure 5 This is an immunofluorescence analysis of early and late osteoblastic markers of the present invention; Figure 6 It is the collagen marker expression and osteogenesis-related gene expression graph of the present invention; Figure 7 This is an analysis chart of angiogenesis and nerve regeneration indicators of the present invention; Figure 8 This is a diagram verifying the activation mechanism of the signaling pathway of the present invention; Figure 9 This is a flow chart for preparing the composite stent of the present invention. DETAILED DESCRIPTION

[0028] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0029] Description of terms of the invention Unless stated otherwise, the terms used in the specification and claims have the following meanings.

[0030] GelMA: 7.5% (w / v) pure GelMA hydrogel without PCL, microspheres, or cells, material control group (gel base); gp: 3D printed PCL skeleton + GelMA hydrogel, without microspheres and cells, scaffold structure blank control group; Ro-gp: gp scaffold loaded with Ro 08-2750 microspheres (P-Ro 08-2750), no cells, drug release pathway inhibition group; NGF-gp: NGF microspheres loaded in gp scaffold (P-NGF), cell-free, drug release activation group; Ro-GelMA: GelMA hydrogel system containing only Ro 08-2750 microspheres, without PCL scaffold, and microsphere-release / no-scaffold system control; NGF-GelMA: GelMA hydrogel system containing only NGF microspheres, without PCL scaffold, and microsphere release / no scaffold system control; GP: construct of DPSCs cells loaded in gp scaffold, control group of animal experiments; Ro-GP: a construct in which DPSCs cells were loaded in a Ro-gp scaffold, used as the inhibitory group in animal experiments; NGF-GP: a construct in which DPSCs cells are loaded in an NGF-gp scaffold, used as the treatment group for animal experiments.

[0031] The technical solution of the present invention is further described below with reference to specific embodiments.

[0032] Example 1 (1) Preparation of P-NGF microspheres NGF was dissolved in HEPES buffer to prepare the internal aqueous phase at a concentration of 100 μg / mL. This internal phase was then emulsified into 3% (w / v) PLGA (dichloromethane solution, LA:GA = 85:15, Mw approximately 30-50 kDa) under ultrasonic conditions (power 200 W, 30 seconds) to form a W / O emulsion.

[0033] The W / O emulsion was then slowly dripped into a 0.5% (w / v) PVA aqueous solution under magnetic stirring (600 rpm) to form a stable W / O / W double emulsion. After stirring for 6 hours to evaporate the dichloromethane, the microspheres were precipitated by centrifugation (8000 rpm, 5 min), washed three times with deionized water, and freeze-dried to obtain dry P-NGF microspheres.

[0034] (2) Preparation of 3D printed PCL skeleton Polycaprolactone (Mw ≈ 80 kDa) and hydroxyapatite were mixed at a mass ratio of 4:1. Printing was performed using FDM technology and the 3D BioArchitect printing platform. The specific parameters are as follows: Nozzle diameter: 0.3 mm; Nozzle temperature: 110℃; Printing pressure: 0.55 MPa; Print speed: 6 mm / s; Layer height: 0.2 mm, using 0° / 90° staggered printing mode; Print size: 30 mm × 30 mm × 0.6 mm (three layers).

[0035] After printing, the implants were cooled naturally, sterilized by ultraviolet irradiation, and then cut into disc-shaped implants with a diameter of 5 mm and a thickness of 0.6 mm under sterile conditions.

[0036] (3) Construction of composite scaffold 5 mg of P-NGF microspheres (NGF loading 1 μg) were mixed with DPSCs (1×10 6cells / mL) were mixed in GelMA prepolymer solution (7.5% w / v GelMA + 0.25% w / v LAP) to prepare a homogeneous microsphere-cell-hydrogel mixture.

[0037] The mixed solution was injected into the pores of the prefabricated PCL skeleton and the pores were illuminated under a 405 nm light source and 15 mW / cm 2 Photocrosslinking was performed under high-intensity light for 40 seconds to form a complete composite scaffold. This scaffold, designated NGF-GP, was used for subsequent in vivo implantation experiments. All procedures were performed under sterile conditions, and the resulting scaffold was stored at 4°C until further use.

[0038] Example 2 (1) Microsphere preparation (P-Ro 08-2750) 200 μg of Ro 08-2750 was dissolved in a 3% (w / v) PLGA solution in dichloromethane to form the oil phase. This solution was then added dropwise to a 0.5% PVA aqueous solution under high-speed emulsification conditions (high-speed homogenizer at 12,000 rpm for 30 seconds) to form an O / W emulsion.

[0039] DCM was evaporated under magnetic stirring (600 rpm) for about 6 h, and the microspheres were recovered by centrifugation (10000 rpm, 5 min), washed three times with deionized water and freeze-dried to obtain dry P-Ro 08-2750 microspheres.

[0040] (2) Preparation of 3D printed PCL skeleton The preparation method of PCL / HA skeleton is the same as that in Example 1.

[0041] (3) Construction of composite scaffold 5 mg of P-Ro 08-2750 microspheres (Ro 08-2750 loading amount is about 0.5 μg) were mixed with DPSCs (1×10 6 cells / mL) were mixed in GelMA prepolymer solution (7.5% w / v GelMA + 0.25% w / v LAP) to prepare a homogeneous microsphere-cell-hydrogel mixture.

[0042] The mixed solution was injected into the pores of the prefabricated PCL skeleton and the pores were illuminated under a 405 nm light source and 15 mW / cm 2 Photocrosslinking was performed under high-intensity light for 40 seconds to form a complete composite scaffold. This scaffold, designated Ro-GP, was used for subsequent in vivo implantation experiments. All procedures were performed under sterile conditions, and the resulting scaffold was stored at 4°C until further use.

[0043] Comparative Example 1: Preparation method of composite scaffold without PLGA microspheres The preparation process of the scaffold is the same as that in Example 1.

[0044] Only DPSCs (1×10 6 cells / mL), without adding PLGA microspheres, mixed evenly and injected into the pores of PCL scaffolds under a 405 nm light source (15 mW / cm 2 ) was irradiated for 40 seconds to complete the photocrosslinking, and the resulting composite structure was the GP scaffold.

[0045] The GP stent was used as a comparative example stent without a drug delivery system for functional comparison evaluation with Example 1 (NGF-GP) and Example 2 (Ro-GP).

[0046] Composite bracket performance test: (1) Cell viability detection: DPSCs were inoculated into GP and NGF-GP scaffolds and cultured for 1, 3, 5, and 7 days, respectively. Live / dead cell staining was performed using the Calcein-AM / PI double staining method to observe the effect of the scaffold on cell growth.

[0047] (2) Evaluation of cell proliferation ability: CCK-8 kit was used to measure cell metabolic activity at different time points (1 / 3 / 5 / 7 days), OD 450 The value was used as an indicator of cell proliferation ability.

[0048] (3) Analysis of osteogenic induction ability: Alkaline phosphatase (ALP) staining was used to detect early osteogenic activity on day 7; Alizarin red S staining was used to detect the formation of mineralized nodules on day 21; And quantitative PCR analysis RUNX2, COL1A1, BGLAP The expression levels of osteogenic genes.

[0049] (4) Mechanical properties: Compression tests were performed on the two scaffolds to obtain stress-strain curves and Young's modulus to evaluate whether microsphere loading affects the mechanical support.

[0050] (5) Evaluation of in vivo bone repair ability: GP and NGF-GP scaffolds were implanted into the SD rat skull defect model, and Micro-CT scans were performed at 4 weeks and 12 weeks after surgery to analyze parameters such as bone volume fraction (BV / TV), trabecular thickness (Tb.Th), and bone porosity (Po.V / TV). HE and Masson staining were performed to observe new bone formation, and multiple immunofluorescence labeling was used to detect the expression levels of osteogenic markers, including ALP (alkaline phosphatase), OCN (osteocalcin), and COL1 (type I collagen), to further evaluate the promoting effect of the scaffold on osteogenic differentiation and bone matrix formation.

[0051] (6) Angiogenesis and neurogenesis assessment: CD31, α-SMA, and NF200 immunofluorescence staining were performed, and the relative fluorescence intensity was calculated using image analysis software to evaluate the effect of the stent on angiogenesis and neurogenesis.

[0052] The gene sequences of osteogenic genes expressed in the experiment are shown in Table 1 and Table 2: Table 1 Gene sequences of osteogenic genes expressed (human)

[0053] Table 2 Gene sequences of osteogenic genes expressed (rat)

[0054] Composite bracket performance: Figure 1 Figures illustrating the structure and physicochemical properties of the composite scaffolds of the present invention. (A) Scanning electron microscopy (SEM) images of the scaffolds, showing the overall morphology and surface microstructure of the gp, Ro-gp, and NGF-gp scaffolds (Scale bar = 100 μm). (B, C) Microsphere size distribution: (B) P-Ro 08-2750 microspheres; (C) P-NGF microspheres. (D, E) Mechanical and rheological properties: (D) Final storage modulus (G′) of each scaffold group after photocrosslinking. (E) Time-dependent evolution of G′ during UV irradiation. (F) In vitro cumulative release curves of P-Ro 08-2750 and P-NGF microspheres. (G1) Scaffold swelling and degradation properties: (G) Swelling rate curves at different time points; (H) Statistical comparison of swelling rate after 24 hours; (I) Scaffold degradation curves in collagenase solution. (J, K) Comparison of stress-strain curves and Young's modulus. (L, M) Calcein-AM / PI live / dead cell staining to assess cell viability: (L) Fluorescence micrographs on days 1, 3, 5, and 7 (green: live cells; red: dead cells; scale bar = 500 μm); (M) Quantitative analysis of cell viability. (N) CCK-8 assay to assess cell proliferation; OD values ​​were measured on days 1, 3, 5, and 7. 450 Value. ns means no significant difference ( P >0.05); “*” indicates P <0.05, “**” indicates P <0.01, “***” indicates P <0.001, “****” indicates P <0.0001.

[0055] SEM ( Figure 1 A) shows that all three scaffolds present a uniform 3D porous structure, the PCL skeleton forms a stable support structure, the GelMA hydrogel fills the gaps, and the microspheres are clearly embedded in the matrix. Particle size distribution analysis ( Figure 1B, C) show that the average diameter of Ro 08-2750 microspheres is about 90 μm, and the average diameter of NGF microspheres is about 120 μm, both of which are suitable for subsequent sustained-release applications. Figure 1 F) shows that both types of microspheres achieved sustained drug release. NGF microspheres showed an initial burst release, releasing nearly 70% of the encapsulated factor within 21 days, followed by a plateau phase, showing a biphasic release pattern. In contrast, Ro08-2750 microspheres showed a slower and more sustained release profile, with sustained release for more than 56 days, indicating a more sustained release capacity. Rheological analysis of GelMA-based hydrogels ( Figure 1 D, E) show that GelMA, Ro-GelMA and NGF-GelMA were illuminated at 405 nm (15 mW / cm 2 ), G′ increased rapidly, indicating effective photocrosslinking behavior. The final G′ values ​​of each group were slightly different, with pure GelMA showing the highest modulus, indicating that the incorporation of drug-loaded microspheres did not significantly impair the gelation performance. Swelling experiment ( Figure 1 G, H) show that all four scaffold groups exhibited considerable swelling capacity in phosphate-buffered saline (PBS). Pure GelMA scaffolds showed the highest swelling rate within 24 hours, reflecting high water absorption. The introduction of PCL slightly reduced the swelling capacity of the GP group. Further reductions were observed in the NGF-GP and Ro-GP groups, both of which showed statistically significant differences compared to pure GelMA ( P <0.05). These results indicate that the addition of drug-loaded microspheres may change the network structure and water absorption rate of the hydrogel to some extent, but the scaffold still exhibits satisfactory swelling properties, which is beneficial to maintaining the stability of the cell microenvironment and nutrient exchange. Figure 1 I) showed that the degradation trend of PBS containing collagenase was stable in all groups. Mechanical testing ( Figure 1 J, K) showed that pure GelMA had low mechanical strength, while the addition of PCL scaffolds significantly improved the compressive modulus and load-bearing capacity of the gp, Ro-gp, and NGF-gp groups. Notably, the Young's modulus of the NGF-gp scaffold was slightly higher compared to the other groups, indicating its excellent comprehensive mechanical properties. In addition, the presence of DPSCs was confirmed by phenotypic characterization. For biocompatibility assessment, DPSCs cultured with scaffold extracts were then subjected to live / dead staining ( Figure 1 L). On days 1, 3, and 7, all groups showed high cell viability, abundant live cells (green), minimal dead cells (red), and cells maintained a good distribution and healthy morphology, indicating good cell compatibility. Quantitative fluorescence analysis ( Figure 1 M) showed that there was no statistically significant difference in cell survival rate among the groups ( P>0.05), indicating negligible cytotoxicity. Further evaluation using CCK-8 assay ( Figure 1 N) confirmed that none of the scaffold extracts (gp, Ro-gp, and NGF-gp) inhibited DPSC proliferation. Absorbance at 450 nm continued to increase after 4 hours and on days 1, 3, and 7, indicating continued cell growth. Overall, these results confirm that the developed scaffold has excellent biocompatibility and can effectively support the survival and proliferation of DPSCs, providing a reliable foundation for establishing a pro-regenerative microenvironment conducive to osteogenesis and tissue repair.

[0056] Figure 2 :In vitro osteogenic induction experiment. The ability of the scaffold extract to induce DPSCs osteogenic differentiation was evaluated by ALP and ARS staining, combined with the quantitative analysis of mineralized nodules and osteogenic genes ( RUNX2, ALPL, COL1A1, BGLAP ) qRT-PCR results, reflecting the osteogenesis-promoting effect of the scaffold. (A) Representative images of ALP and ARS staining. The upper row shows ALP staining on day 7, and the darkened staining area indicates enhanced intracellular phosphatase activity; the lower row shows ARS staining on day 21, and the orange-red nodules represent the formation of mineralized nodules. Scale bar = 100 μm. (B) Quantitative analysis of ALP activity (King unit / g protein). (C) After ARS staining and CPC treatment, the absorbance was measured at 560 nm to quantify the mineralized nodules. (DG) qRT-PCR expression analysis of osteogenesis-related genes in DPSCs on day 7, including RUNX2 (D) ALPL (E) COL1A1 (F) and BGLAP (OCN) (G). ns indicates no significant difference ( P >0.05); “*” indicates P <0.05, “**” indicates P <0.01, “***” indicates P <0.001, “****” indicates P <0.0001.

[0057] At the early stage of induction (day 7), ALP staining ( Figure 2 A) The NGF-gp group showed the strongest enzyme activity, followed by the NGF group, while the control group showed only weak staining. Quantitative analysis of ALP activity ( Figure 2 B) confirmed these findings, showing that ALP levels in the NGF-gp group were significantly higher than those in the control group ( P <0.05, while the activity of the Ro-gp group was significantly decreased ( P<0.01). These results indicate that NGF effectively promotes the early osteogenic differentiation of DPSCs, while Ro 08-2750 has a significant inhibitory effect at this stage. At the late stage of osteogenesis (day 21), ARS staining ( Figure 2 A) shows that both the NGF-gp and free NGF groups had numerous orange-red mineralized nodules, indicating a significantly higher degree of calcification compared with the control group. In contrast, negligible staining was observed in the Ro 08-2750 and Ro-gp groups. Quantitative ARS assessment ( Figure 2 C) Further, mineralization was enhanced in the NGF-gp group, whereas mineralization was significantly inhibited in the Ro-gp group. Collectively, these findings suggest that sustained NGF release significantly enhances late-stage mineralization, whereas Ro 08-2750 inhibits late-stage osteogenesis.

[0058] To further elucidate the molecular mechanisms of these effects, key osteogenic genes RUNX2, ALPL, COL1A1 and BGLAP The expression of osteocalcin (OCN) was analyzed by qRT-PCR on day 7. Figure 2 DG) showed that NGF-gp significantly upregulated RUNX2, ALPL, COL1A1 and BGLAP These findings suggest that Ro 08-2750 not only broadly inhibits the transcriptional activation of key osteogenic factors and extracellular matrix genes but also may impair the overall osteogenic potential of cells.

[0059] Figure 3 Figure 1 shows in vivo analysis of skull defect repair and bone regeneration. The figure shows the rat skull defect model establishment process, scaffold implantation images, Micro-CT 3D reconstructions, and transverse, coronal, and sagittal slices. Bone remodeling indicators (BV / TV, Tb.Th, Tb.N, Tb.Sp, and Po.V / TV) were quantitatively analyzed at 4 and 12 weeks. (A) Schematic diagram of the animal experiment process. (B) Micro-CT analysis at 4 and 12 weeks: 3D reconstruction images on the left, transverse, coronal, and sagittal images on the right. The defect area is indicated by a yellow circle or box. Scale bar = 2 mm. (C) Surgical implantation site. The upper image shows a 5 mm skull slice and the exposed defect area; the lower image shows the scaffold and its implantation process. (D) Quantitative analysis of bone regeneration parameters at weeks 4 and 12, including bone volume fraction (BV / TV, %), trabecular thickness (Tb.Th, μm), trabecular number (Tb.N, 1 / mm), trabecular spacing (Tb.Sp, μm), and porosity (Po.V / TV, %). ns indicates no significant difference ( P >0.05); “*” indicates P <0.05, “**” indicates P<0.01, “***” indicates P <0.001, “****” indicates P <0.0001.

[0060] To evaluate the in vivo bone regeneration ability of the prepared scaffolds, a critical-sized calvarial defect model (5 mm in diameter) was established in Sprague-Dawley rats ( Figure 3 A). GP, Ro-GP, and NGF-GP scaffolds were implanted into the defect site, conforming to the defect geometry ( Figure 3 C). New bone formation was assessed using micro-computed tomography (micro-CT) at 4 and 12 weeks after implantation ( Figure 3 B, D). 3D reconstruction and multiplanar slice images showed that the control group did not exhibit significant bone bridging at both time points, and the defects were essentially unhealed. In contrast, all scaffold-treated groups (GP, Ro-GP, and NGF-GP) showed new bone growth extending from the defect edge toward the center as early as week 4, with progressive thickening observed at week 12. Notably, the NGF-GP group demonstrated a more continuous and densely mineralized bone structure at both time points. Quantitative micro-CT analysis showed that BV / TV, Tb-Th, and Tb-N increased significantly in all scaffold-treated groups compared with the control group. Among them, the NGF-GP group showed the most significant improvement. In addition, the NGF-GP group had the lowest Tb Sp and Po V / TV values, indicating improved bone quality and defect closure.

[0061] Radar chart based on micro-CT parameters ( Figure 4 A) Shows that the NGF-GP group exhibited the most favorable bone regeneration characteristics at 4 and 12 weeks, while the Ro-GP group showed minimal improvement. The control group exhibited high trabecular spacing and porosity, indicating insufficient bone repair. These results are consistent with 3D imaging data and quantitative analysis, confirming that the NGF-GP scaffold promoted superior bone regeneration in vivo.

[0062] Figure 4Quantification of bone formation and histological staining. Radar charts were used to integrate various structural parameters to comprehensively compare the regeneration effects of different groups; histological staining was used to display new bone formation under HE staining and Masson trichrome staining, and bone area analysis based on Masson images was used to evaluate the quality and maturity of new bone tissue. (A) Radar charts of bone parameters (BV / TV, Tb.Th, Tb.N, Tb.Sp, Po.V / TV) at weeks 4 and 12. (B, C) HE staining (left) and Masson trichrome staining (right) of bone tissue sections at weeks 4 and 12. Images include low-power (scale bar = 1 mm) and high-power views (scale bar = 200 μm). (D) Quantitative analysis of new bone area at weeks 4 and 12 based on Masson staining. ns indicates no significant difference ( P >0.05); “*” indicates P <0.05, “**” indicates P <0.01, “***” indicates P <0.001, “****” indicates P <0.0001.

[0063] To further evaluate the histological characteristics of scaffold-induced bone regeneration, tissue samples were collected from the calvarial defect site at 4 and 12 weeks after surgery for HE and Masson's trichrome staining ( Figure 4 B, C). H&E staining showed that at both time points, the defect area in the control group was primarily filled with fibrous tissue, with minimal evidence of new bone formation. In contrast, the GP, Ro-GP, and NGF-GP groups exhibited nascent bone plate-like structures at 4 weeks, which matured into more organized bone tissue by 12 weeks. Masson's trichrome staining revealed dark blue to bluish-purple bone tissue, with a clear boundary between the newly formed bone and surrounding tissue. The NGF-GP group exhibited continuous bone plate formation as early as 4 weeks, and by 12 weeks, osteoid lamellar structures were observed, with the most extensive and intense staining. The GP group showed moderate bone formation, while the Ro-GP group showed limited new bone formation, which was greater than that of the control group but significantly lower in quantity and maturity than that of the NGF-GP group.

[0064] Quantitative analysis of new bone area ( Figure 4 D) Further confirmed the histological findings. The percentage of new bone in the NGF-GP group was significantly higher than that in all other groups at 4 and 12 weeks ( P <0.001). The bone area of ​​the GP group was also significantly greater than that of the control group ( P <0.0001), while the Ro-GP group increased slightly, but was still significantly lower than the GP group ( P<0.05), indicating limited osteogenic potential. Taken together, these results suggest that NGF microsphere-loaded scaffolds not only promote early bone formation but also support long-term bone maturation and remodeling. In contrast, sustained release of Ro 08-2750 may impair osteoblast recruitment, bone matrix deposition, and tissue remodeling during bone regeneration.

[0065] Figure 5 Immunofluorescence analysis of early and late osteogenic markers. Immunofluorescence staining images of ALP and OCN at 4 weeks and 12 weeks and their three-dimensional intensity distribution diagrams (Z axis represents fluorescence intensity), and provide quantitative fluorescence intensity analysis results at each time point. (A, B) Immunofluorescence staining images of ALP and OCN expression at 4 weeks and 12 weeks and the corresponding three-dimensional peak diagrams. ALP and OCN are marked in red, and DAPI is marked in blue. The scale bars are 500μm, 200μm, and 50μm, respectively. In the three-dimensional diagram, the X and Y axes represent the spatial coordinates, and the Z axis represents the fluorescence intensity. (C, D) Quantitative analysis of the fluorescence intensity of ALP and OCN at 4 weeks and 12 weeks. ns indicates that the difference is not significant ( P >0.05); “*” indicates P <0.05, “**” indicates P <0.01, “***” indicates P <0.001, “****” indicates P <0.0001.

[0066] Figure 6 This is a graph showing the expression of collagen markers and osteogenesis-related genes. It is used to display the expression of COL1 at different time points and three-dimensional fluorescence images, combined with qRT-PCR analysis. Alpl 、 Col1a1 and Bglap Gene expression levels in tissue samples. (A) Immunofluorescence images and three-dimensional peak intensity images of COL1 expression at weeks 4 and 12; COL1 is labeled in red, DAPI is labeled in blue; scale bars are 500 μm, 200 μm, and 50 μm, respectively; the X and Y axes represent spatial position, and the Z axis represents fluorescence intensity. (B) Quantitative analysis of fluorescence intensity of COL1 at weeks 4 and 12. (CE) Expression of COL1 in bone tissue at weeks 4 and 12 Alpl 、 Col1a1 and Bglap qRT-PCR expression analysis of genes; ns indicates no significant difference ( P >0.05); “*” indicates P <0.05, “**” indicates P <0.01, “***” indicates P <0.001, “****” indicates P <0.0001.

[0067] To further investigate the regulatory effects of the scaffold on osteogenesis, immunofluorescence staining was performed to evaluate the expression of ALP, OCN, and type I collagen (COL1) in the calvarial defect area at 4 and 12 weeks after implantation. In addition, 3D surface maps were generated using FIJI software to enhance visualization of fluorescence intensity and spatial distribution ( Figure 5 AB, Figure 6 A).

[0068] At week 4, the GP group showed obvious ALP-related red fluorescence in the osteogenic area. The control group showed a weak signal, and the Ro-GP group showed even lower fluorescence. In contrast, the NGF-GP group showed the strongest ALP signal, characterized by a clear high-intensity peak on the 3D surface map ( Figure 5 A). By week 12 ( Figure 5 A) ALP expression increased in all groups, with the NGF-GP group maintaining the highest intensity, followed by the GP group, while the Ro-GP group remained relatively weak. Quantitative analysis of fluorescence intensity ( Figure 5 C) confirmed that ALP expression in the NGF-GP group was significantly higher than that in all other groups at both time points ( P <0.05). Although the ALP expression in the Ro-GP group exceeded that in the control group, it was still significantly lower than that in the NGF-GP group. OCN staining results ( Figure 5 B) Following a similar trend, OCN, a marker of late osteogenic differentiation, was most widely distributed and expressed most intensely in the NGF-GP group. At week 4, distinct areas of red fluorescence were already evident, and by week 12, these areas displayed characteristics of mature mineralized tissue. Quantitative analysis ( Figure 5 D) showed that at both time points, the OCN expression in the NGF-GP group was significantly higher than that in the control group, while the Ro-GP group showed moderate improvement but still significantly lower than that in the NGF-GP group. Figure 6 A) The expression of COL1 was weak in the control group, but significantly increased in the GP, Ro-GP, and NGF-GP groups, with the NGF-GP group showing the strongest signal. Figure 6 A) COL1 expression continued to increase in all groups, with the NGF-GP group maintaining the highest intensity. Quantitative fluorescence analysis ( Figure 6 B) showed that at both time points, the expression of COL1 in the NGF-GP group was significantly higher than that in the control group and the Ro-GP group ( P <0.001).

[0069] To further validate these findings at the transcriptional level, total RNA was extracted from bone defect tissues and analyzed. Alpl 、 Col1a1 and Bglap qRT-PCR analysis was performed on the gene encoding OCN ( Figure 6 CE). The mRNA expression levels of the three genes in the NGF-GP group were the highest at both time points, followed by the GP group, and the control group had the lowest expression. It is worth noting that compared with the control group, the expression of Bglap in the NGF-GP group was significantly upregulated as early as week 4 ( P <0.001), and this difference further increased by week 12. These results suggest that NGF delivery plays a sustained and powerful role in promoting bone mineralization and extracellular matrix maturation during the later stages of bone regeneration.

[0070] Figure 7 The following images are an analysis of angiogenesis and neural regeneration indicators. Double immunofluorescence staining and 3D intensity maps of CD31, α-SMA (a vascular marker), and NF200 (a neural marker) were used to assess the efficacy of bone-vascular-neural coordinated reconstruction. Quantitative fluorescence analysis and integrated radar images of six markers (ALP, OCN, COL1, CD31, α-SMA, and NF200) were combined to assess the efficacy of bone-vascular-neural coordinated reconstruction. (A) Double immunofluorescence staining of CD31 (red) and α-SMA (green) at weeks 4 and 12, along with 3D peak fluorescence intensity maps. Scale bars are 500 μm, 200 μm, and 50 μm, respectively. The X and Y axes represent spatial position, and the Z axis represents fluorescence intensity. (B) Immunofluorescence staining of NF200 (red) at weeks 4 and 12, along with 3D peak fluorescence intensity maps. Scale bars are 500 μm, 200 μm, and 50 μm, respectively. The X and Y axes represent spatial position, and the Z axis represents fluorescence intensity. (C) Quantitative analysis of relative fluorescence intensity of CD31, α-SMA, and NF200. (D) Radar chart of the expression levels of six markers (ALP, OCN, COL1, CD31, α-SMA, and NF200) at week 4 and week 12; ns indicates no significant difference ( P >0.05); “*” indicates P <0.05, “**” indicates P <0.01, “***” indicates P <0.001, “****” indicates P <0.0001.

[0071] During bone tissue repair, the coordinated regeneration of blood vessels and nerves is crucial for achieving functional recovery. To systematically evaluate the effect of each scaffold on angiogenesis, double immunofluorescence staining of CD31 (red) and α-SMA (green) was performed on the defect area at 4 and 12 weeks after surgery to assess the formation of mature vascular structures. Simultaneously, the neuronal marker NF200 (red) was used to assess nerve fiber regeneration. The results were visualized using 3D fluorescence surface maps generated by FIJI software ( Figure 7 AB). In terms of angiogenesis ( Figure 7A) At week 4, there was almost no detectable vascular signal in the control group. The GP group showed limited CD31- and α-SMA-positive areas, while the Ro-GP group showed weak and discontinuous signals. In contrast, the NGF-GP group showed dense, co-localized vascular structures with significantly enhanced fluorescence intensity, indicating that early angiogenesis was effectively promoted. By week 12, all groups showed increased blood vessels, and the CD31 / α-SMA co-expression in the NGF-GP group was further expanded, and the vascular morphology was more mature and continuous. In terms of nerve regeneration ( Figure 7 B) At week 4, no obvious NF200 signal was observed in the control group, while the GP and Ro-GP groups showed red fluorescence. In contrast, the NGF-GP group showed uniform distribution of NF200-positive nerve fibers, indicating the beginning of nerve regeneration. By week 12, NF200 expression in the NGF-GP group was further elevated, showing higher fluorescence intensity and wider spatial distribution. Although the GP and Ro-GP groups showed some improvement, their expression levels were still significantly lower than that of the NGF-GP group. Quantitative analysis ( Figure 7 C) further supports these observations. At weeks 4 and 12, the expression levels of CD31, α-SMA, and NF200 in the NGF-GP group were significantly higher than those in the control group ( P <0.001), and were significantly higher than those in the GP and Ro-GP groups. It is worth noting that although the Ro-GP group had a slight increase compared with the control group, its expression levels of all markers were still lower than those in the GP group, indicating that blocking the NGF signaling pathway may impair vascular and neural regeneration to some extent. In order to comprehensively evaluate the overall regenerative performance of each scaffold in terms of osteogenesis, angiogenesis, and neurogenesis, radar charts were constructed using six representative markers: ALP, OCN, COL1 (osteogenesis); CD31, α-SMA (angiogenesis); NF200 (neurogenesis) ( Figure 7 D). At week 4, the NGF-GP group demonstrated superior performance across all three functional domains, with radar plots displaying a characteristic "hexagonal expansion" pattern, indicating balanced and robust regeneration. In contrast, the Ro-GP group exhibited significant contraction across multiple dimensions, particularly in vascular and neural markers. By week 12, the NGF-GP group maintained its superior performance across all parameters. Improvements in the Ro-GP group were limited but remained significantly lower than those in the GP group, while the control group consistently maintained the lowest values, reflecting its limited spontaneous repair capacity. Overall, these results highlight the combined regenerative advantages of NGF-based scaffolds in promoting coordinated bone-vascular-neural regeneration.

[0072] Figure 8This is a diagram to verify the activation mechanism of the signal pathway. It shows the principal component analysis of RNA-seq PCA clustering, the volcano plot of differentially expressed genes, the GO and KEGG pathway enrichment analysis diagram, the osteogenesis / osteoclast related gene heat map, the GSEA enrichment curve, and the Western blot detection diagram and grayscale quantitative diagram of the PI3K / AKT pathway, revealing the chain of evidence for the molecular mechanism of scaffold activation. (A) Principal component analysis (PCA), the distance between points reflects the difference and clustering trend of the overall gene expression profile between samples. (B) Volcano plot of differentially expressed genes (DEGs) between the NGF-GP group and the Ro-GP group; up-regulated genes are shown in red (right), and down-regulated genes are shown in green (left) (|log2FC|≥1, P <0.05). (C) Gene ontology (GO) enrichment analysis results (including biological processes, cellular components, and molecular functions). The bar chart shows the significantly enriched functional categories. (D, E) Heat map of osteogenesis-related and osteoclast-related gene expression, red indicates high expression, and blue indicates low expression. (F, G) GSEA enrichment analysis diagram: (F) Gene sets positively enriched in the NGF-GP group (such as oxidative phosphorylation, cytoskeletal proteins, and actin binding) and their NES values, p value, q (G) Negatively enriched gene sets in the Ro-GP group (e.g., extracellular matrix, osteogenesis, collagen trimers) and statistical results. (H) KEGG pathway enrichment analysis scatter plot. Circle size represents the number of enriched genes, and color represents significance; red boxes indicate significant enrichment of the PI3K / AKT pathway. (I, J) Western blot analysis of PI3K and AKT phosphorylation levels and quantitative analysis of their grayscale values. ns indicates no significant difference ( P >0.05); “*” indicates P <0.05, “**” indicates P <0.01, “***” indicates P <0.001, “****” indicates P <0.0001.

[0073] To further investigate the molecular mechanism of the osteogenesis-promoting effect of NGF-loaded scaffolds, bone tissue samples were collected 12 weeks after implantation for RNA-seq transcriptome analysis, followed by Western blot analysis to verify the activation status of key signaling pathways. Figure 8 A) Revealed a clear separation between NGF-GP and Ro-GP groups, indicating different clustering patterns in their global gene expression profiles. Volcano plot analysis ( Figure 8 B) 264 genes were significantly up-regulated and 796 genes were significantly down-regulated in the NGF-GP group (|log2FC| ≥ 1, P <0.05). Gene ontology (GO) enrichment analysis ( Figure 8 C) shows that the differentially expressed genes (DEGs) are mainly related to biological processes such as "response to stimulus", "system development" and "extracellular region", which indicates that NGF may promote bone regeneration by regulating cellular stress response and extracellular microenvironment. Heat map of genes related to osteogenesis and osteoclastogenesis ( Figure 8 D, E) further showed that osteogenic transcription factors such as Smad1, Smad4, Smad5, Runx2, and Bmpr2 were upregulated in the NGF-GP group, while the Ro-GP group showed increased expression of osteoclast-related genes, including Ctsk, Acp5, and Tnfrsf11b. These findings suggest that NGF plays a dual regulatory role by promoting osteogenesis and inhibiting osteoclast-mediated bone resorption. GSEA analysis ( Figure 8 F, G) revealed significant positive enrichment of pathways such as "oxidative phosphorylation," "cytoskeletal proteins," and "actin binding" in the NGF-GP group, processes closely related to cellular energy metabolism, migration, and structural remodeling of bone tissue. In contrast, the Ro-GP group showed negative enrichment in pathways related to extracellular matrix organization and bone matrix formation, such as "extracellular matrix," "ossification," and "collagen trimers." KEGG pathway analysis ( Figure 8 H) Multiple canonical signaling cascades associated with bone remodeling were identified, among which the PI3K / AKT pathway showed the most significant enrichment. To verify the activation of this pathway, the phosphorylation levels of PI3K and AKT were assessed ( Figure 8 I). Western blot results showed that the expression of phosphorylated PI3K (p-PI3K) and AKT (p-AKT) increased significantly in the NGF-GP group. Grayscale analysis ( Figure 8 J) confirmed that the p-PI3K / PI3K and p-AKT / AKT ratios in the NGF-GP group were significantly higher than those in the Ro-GP group and the control group ( P <0.05). Collectively, these findings suggest that NGF-loaded scaffolds promote bone regeneration by activating the PI3K / AKT signaling pathway, upregulating osteogenic transcription factors, and inhibiting osteoclast gene expression, thereby orchestrating bone repair at the transcriptional and protein levels.

[0074] Figure 9 This is a flow chart for preparing the composite scaffold of the present invention. It demonstrates the preparation of neurotrophic factor NGF or Ro 08-2750-loaded PLGA microspheres, their embedding into a 3D-printed PCL framework, and the overall process of photocrosslinking GelMA to form the composite scaffold. It also demonstrates its application in a SD rat skull defect model.

[0075] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. PLGA microsphere-PCL / GelMA composite scaffold based on NGF sustained release, characterized in that: The composite scaffold is composed of a PCL skeleton, a GelMA hydrogel matrix, NGF-loaded PLGA microspheres, and dental pulp stem cells (DPSCs); The PCL skeleton is pre-mixed with polycaprolactone and hydroxyapatite in a mass ratio of 2-5:1 and prepared by fused deposition modeling 3D printing technology; The NGF-loaded PLGA microspheres and DPSCs are uniformly dispersed in the GelMA matrix, and the GelMA matrix is ​​filled in the PCL skeleton.

2. The composite stent according to claim 1, wherein: The GelMA hydrogel matrix uses gelatin methacryloyl as the main component and is combined with a photoinitiator to form a photosensitive cross-linking system to form a uniform gel structure. The mass concentration of GelMA prepolymer solution ranged from 3% to 15% (w / v), and the addition ratio of photoinitiator LAP ranged from 0.05% to 0.5% (w / v).

3. The composite stent according to claim 1, wherein: The NGF-loaded PLGA microspheres have a particle size of 100-150 μm, an NGF encapsulation rate of 70-90%, and are dispersed in the GelMA hydrogel; the ratio of lactic acid to glycolic acid in the PLGA is 70:30 to 90:

10.

4. The method for preparing the PLGA microsphere-PCL / GelMA composite scaffold based on NGF sustained release according to claim 1, characterized in that: The following steps are involved: (1) Preparation of internal phase: Dissolve NGF in 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid buffer at a concentration of 80-120 μg / mL; (2) Preparation of oil phase: PLGA was dissolved in dichloromethane at a concentration of 2-5% (w / v) to form an oil phase; (3) Primary emulsification: Under ultrasonic conditions, the inner water phase is emulsified into the oil phase to form a uniform W / O emulsion; (4) Secondary emulsification: The W / O emulsion was slowly dripped into a 0.2-1% (w / v) polyvinyl alcohol aqueous solution to form a stable W / O / W double emulsion under magnetic stirring; (5) Solvent evaporation and particle solidification: Continue stirring for 4-6 hours to allow DCM to evaporate completely and the microspheres to solidify and form; (6) Microsphere collection and drying: After centrifugation and washing, freeze-dry for 24-48 hours to obtain dry P-NGF microspheres; (7) Preparation of PCL skeleton: The PCL skeleton is pre-mixed with polycaprolactone and hydroxyapatite in a mass ratio of 2-5:1 and prepared by fused deposition modeling 3D printing technology; (8) Preparation of GelMA prepolymer solution containing cells and microspheres: P-NGF microspheres and dental pulp stem cells (DPSCs) were mixed in GelMA prepolymer solution containing a photoinitiator to prepare a uniform suspension; (9) Scaffold composite and photocrosslinking: The suspension described in step (8) was injected into the pores of the PCL skeleton, and then irradiated with 405 nm light at 15 mW / cm 2 The cross-linking is carried out under light intensity conditions and the cross-linking time is controlled within 30-50 seconds.

5. The preparation method according to claim 4, wherein In the step (3), the ultrasonic treatment time is 30-60s.

6. The preparation method according to claim 4, wherein In the step (4), the magnetic stirring speed is 500-1000 rpm.

7. The preparation method according to claim 4, wherein The P-NGF microspheres prepared in step (6) are regular spherical, with a particle size of 80-200 μm and an encapsulation rate of 70-90%; the centrifugal speed is 8000-12000 rpm.

8. The preparation method according to claim 4, wherein In step (8), the amount of microspheres added is 5-10 mg, corresponding to a total amount of NGF of 1-2 μg; the seeding density of DPSCs is 1×10 6 cells / mL; GelMA concentration was 6-10% (w / v), photoinitiator LAP concentration was 0.2-0.3% (w / v); the mass ratio of photoinitiator to GelMA was 1:20–1:

40.

9. Use of the NGF-sustaining PLGA microsphere-PCL / GelMA composite scaffold according to any one of claims 1 to 3 or the NGF-sustaining PLGA microsphere-PCL / GelMA composite scaffold prepared by the preparation method according to any one of claims 4 to 8 in tissue regeneration and repair of bone defects.

10. The use according to claim 9, characterized in that The composite scaffold can be applied to tissue regeneration and repair of bone defect areas where blood vessels and nerves are densely distributed and the repair is relatively complex.

Images