Ngf sustained-release based plga microsphere-pcl / gelma composite scaffold and preparation method and application thereof

Abstract

The application discloses a PLGA microsphere-PCL / GelMA composite scaffold based on nerve growth factor (NGF) sustained release and a preparation method and application thereof. The composite scaffold is constructed by embedding the NGF-loaded PLGA microsphere into the PCL framework and the GelMA hydrogel matrix, and a three-phase composite system with mechanical support, sustained release of biological factors and cell adhesion function is constructed. In vitro experiments show that the NGF-loaded composite scaffold significantly promotes the osteogenic differentiation of dental pulp stem cells, and the alkaline phosphatase activity is improved, the formation of mineralized nodules is increased, and the expression of osteogenesis-related genes RUNX2 , ALPL , COL1A1 and BGLAP is up-regulated. When applied to a rat skull critical defect model in vivo, the composite scaffold significantly promotes new bone formation and vascular nerve regeneration. Mechanism research shows that it realizes the regulation of osteogenesis and bone resorption by activating the PI3K / AKT signaling pathway. The composite scaffold is suitable for the repair of complex bone defects in the region rich in nerves and blood vessels, and has good clinical application prospect.

Description

TECHNICAL FIELD

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

[0002] The information disclosed in the background of the present application is only intended to increase the understanding of the overall background of the present application and should not necessarily be regarded as acknowledging or implying in any form that this information constitutes prior art known to those skilled in the art.

[0003] The repair of craniofacial bone defects faces great challenges, which mainly manifest in three aspects: first, the anatomical structure of this region 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, etc.; third, the repair process needs to take into account both morphological reconstruction and functional recovery. The current clinical repair strategies have limitations, and the traditional repair methods have obvious shortcomings: for example, 1. autologous bone transplantation is limited by the donor site, especially in large volume bone defect repair, and has the risk of large surgical trauma and multiple complications in the donor site; 2. allogeneic / xenogeneic bone materials have a wide range of sources, but face the risks of immune rejection and infection, and the long-term repair effect is not ideal; 3. metal / ceramic repair bodies can provide mechanical support, but lack biological activity and cannot achieve true functional regeneration.

[0004] The defects of existing tissue engineering technology are mainly reflected in: 1. one-sided design concept: most scaffolds only focus on the osteogenesis process, ignoring the core mechanism of "vascular-nerve synergistic regulation"; 2. technical bottlenecks in the application of biological factors: including poor factor stability (such as NGF (nerve growth factor) which is easily affected by enzyme and pH, degrades quickly in vivo and has a short biological activity maintenance time), uncontrollable release mode (burst release, which may cause side effects due to high concentration in a short time, and cannot maintain sustained action in the later period), and insufficient fusion of carrier system and scaffold; 3. low functional integration: there is a lack of comprehensive solutions that can simultaneously consider osteogenesis, promotion of vascularization, and nerve repair.

[0005] The key to 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), promote bone formation, and effectively induce the reconstruction of new blood vessels and nerve fibers, achieving a transition from "one-way osteogenesis" to "multi-dimensional regeneration", thereby meeting the clinical needs of complex bone defect repair. SUMMARY

[0006] Therefore, the application provides a PLGA microsphere-PCL / GelMA composite scaffold based on NGF slow release, a preparation method and application thereof, aims to realize controllable release of neurotrophic factors, reconstruct a "bone-vascular-nerve" network in a multidimensional bone defect area, and thus improve the repair effect.

[0007] In order to achieve the above-mentioned purpose, the application is realized by the following technical scheme:

[0008] In a first aspect, the application provides a PLGA microsphere-PCL / GelMA composite scaffold based on NGF slow release. The composite scaffold is composed of a PCL framework, a GelMA hydrogel matrix, NGF-loaded PLGA microspheres (P-NGF) and DPSCs (dental pulp stem cells). The PCL framework is prepared by premixing polycaprolactone and hydroxyapatite at a mass ratio of 2-5:1 and using a fused deposition modeling (FDM) three-dimensional printing technology. The NGF-loaded PLGA microspheres and the DPSCs are uniformly dispersed in the GelMA matrix, and the GelMA matrix is filled in the PCL framework.

[0009] The PCL framework is prepared by the FDM three-dimensional printing technology, forms a scaffold body with a regular porous structure, and provides mechanical support. The structure is usually arranged in an interlaced grid, and the printing path can include 0° / 90°, 0° / 60° / 120° or other orthogonal isometric angle designs. The pore size of the scaffold ranges from 200 to 800 μm, and is preferably 300-500 μm, so as to facilitate cell penetration and tissue ingrowth. The outer dimensions of the scaffold can be customized according to the defect site, and generally have a diameter ranging from 4 to 8 mm, preferably 5.0±0.5 mm, a height of 0.5-1.0 mm, preferably 0.6±0.1 mm, and a printing layer thickness controlled between 150-250 μm. The above structure parameters can be fine-tuned according to application requirements to meet the comprehensive requirements of porosity, mechanical properties and biological permeability for different tissue repair sites.

[0010] The GelMA hydrogel matrix uses gelatin methacrylate (GelMA) as the main body, and a photoinitiator to form a photosensitive crosslinking system to form a uniform gel structure for filling the pore area of the three-dimensional printed framework. The mass concentration of the GelMA prepolymer solution ranges from 3% to 15% (w / v), and is preferably 6%-10% (w / v), which can be adjusted according to the required scaffold mechanical strength and cell growth characteristics. The addition ratio of the photoinitiator LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate) ranges from 0.05% to 0.5% (w / v), and is preferably 0.2%-0.3% (w / v). After irradiation with a wavelength of 405 nm, the reaction time is controlled within 10-60 seconds to complete the crosslinking and curing.

[0011] The hydrogel can form a highly hydrophilic three-dimensional network structure in physical structure, and the pore size distribution is conducive to cell adhesion, nutrient exchange and waste discharge. As a biomimetic extracellular matrix, GelMA not only promotes stem cell adhesion and directional differentiation, but also has good compatibility with PLGA microspheres, which helps to embed the microspheres in situ and stabilize their spatial positioning during gel crosslinking, and realizes the sustained release of NGF. The above gel composition, crosslinking light intensity and time can be flexibly adjusted according to the actual repair site, factor release time and printing structure matching requirements, and has good adaptability and clinical transformation potential.

[0012] The PLGA microspheres loaded with NGF (P-NGF) are composed of polylactic acid-glycolic acid copolymer (PLGA) and NGF, and are prepared into spherical particles by a double emulsion solvent evaporation method (W / O / W). The microspheres have a diameter range of 80-200 μm, preferably 100-150 μm, a smooth surface, a concentrated particle size distribution, and good structural stability and loading efficiency. The ratio of lactic acid (LA) to glycolic acid (GA) in the polymer raw material PLGA of the microspheres is 70:30 to 90:10, preferably 85:15, and the molecular weight range is 20-60 kDa, so as to regulate the in vivo degradation rate and drug release performance. The parameter setting can realize a sustained release period of NGF for 56-70 days, and the encapsulation efficiency can reach 70%-90%. The microspheres are uniformly dispersed in the GelMA hydrogel matrix after preparation, and are fixed in situ in the scaffold during the GelMA photo-crosslinking process, avoiding spatial drift after implantation or release efficiency decrease due to local exudation. This design not only ensures the targeted enrichment and continuous supply of NGF in the defect area, but also forms a microenvironment signal source to continuously activate the PI3K / AKT signaling pathway, inducing stem cell osteogenesis, angiogenesis and nerve repair.

[0013] The composite scaffold of the application realizes bone-vascular-nerve regeneration through the following synergistic mechanism: the components of the composite scaffold are physically embedded and photo-crosslinked to form a stable composite structure. The PCL skeleton provides mechanical support; the GelMA hydrogel supports cell adhesion, migration and differentiation; the PLGA microspheres realize sustained release of NGF (more than 56 days), activate the PI3K / AKT signaling pathway, and promote DPSC osteogenesis, angiogenesis and nerve repair; in vivo experiments show that it significantly up-regulates the expression of ALP, OCN, COL1, CD31, a-SMA, NF200 and other markers, and has high biological function integration.

[0014] In the second aspect, the application provides a preparation method of the PLGA microsphere-PCL / GelMA composite scaffold based on NGF sustained release of the first aspect, which specifically comprises the following steps:

[0015] (1) Inner phase preparation: NGF was dissolved in HEPES (4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid) buffer solution, and the concentration was controlled at 80-120 μg / mL;

[0016] (2) Oil phase preparation: PLGA was dissolved in dichloromethane at a concentration of 2-5% (w / v) to form an oil phase;

[0017] (3) Primary emulsification (W / O): under ultrasonic conditions, the inner water phase was emulsified into the oil phase to form a uniform W / O emulsion;

[0018] (4) Secondary emulsification (W / O / W): the above W / O emulsion was slowly dropped into a 0.2-1% (w / v) polyvinyl alcohol (PVA) aqueous solution, and a stable W / O / W double emulsion was formed under magnetic stirring;

[0019] (5) Solvent evaporation and particle solidification: continue stirring for 4-6 hours to make DCM volatilize completely, and the microspheres are solidified and formed;

[0020] (6) Microsphere collection and drying: after centrifugation and washing, freeze-drying for 24-48 hours to obtain dry P-NGF microspheres;

[0021] (7) Preparation of PCL framework: the PCL framework is prepared by pre-mixing polycaprolactone and hydroxyapatite (HA) at a mass ratio of 2-5:1 by melt deposition three-dimensional printing technology;

[0022] (8) Preparation of GelMA pre-polymer solution containing cells and microspheres: P-NGF microspheres and DPSCs (dental pulp stem cells) are mixed in a GelMA pre-polymer solution containing a photoinitiator to obtain a uniform suspension;

[0023] (9) Scaffold compounding and photocrosslinking: the suspension of step (8) is injected into the pores of the PCL framework, and then crosslinked under 405 nm light irradiation at 15 mW / cm 2 under light intensity conditions, and the crosslinking time is controlled at 30-50 seconds.

[0024] The final scaffold can be sterilized by ultraviolet light or aseptically packaged and stored, and is suitable for subsequent cell culture, animal implantation or in vivo tissue engineering research.

[0025] Preferably, in step (3), the ultrasonic treatment time is 30-60s;

[0026] Preferably, in step (4), the magnetic stirring is at 500-1000 rpm;

[0027] Preferably, in step (6), the centrifugal speed is 8000-12000 rpm;

[0028] Preferably, 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%; more preferably, the particle size is 100-150 μm.

[0029] Preferably, in step (7), the specific parameters of three-dimensional printing are as follows: nozzle diameter: 0.3 mm; nozzle temperature: 110℃; 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.

[0030] Preferably, in step (7), the scaffold prepared by three-dimensional printing has the following size (monomer): 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; scaffold mass control range: 15-30 mg.

[0031] Preferably, in step (8), the microsphere addition amount is 5-10 mg, corresponding to a total amount of NGF of 1-2 μg; the inoculation density of DPSCs is 1×10 6 cells / mL; the GelMA concentration is 6-10% (w / v), and the light initiator LAP concentration is 0.2-0.3% (w / v); the mass ratio of light initiator to GelMA is 1:20-1:40.

[0032] In a third aspect, the application provides an application of the NGF sustained-release based PLGA microsphere-PCL / GelMA composite scaffold prepared by the method of the second aspect or the NGF sustained-release based PLGA microsphere-PCL / GelMA composite scaffold prepared by the method of the first aspect in tissue regeneration and repair of bone defects.

[0033] Preferably, the composite scaffold provided by the application can be directly applied to tissue regeneration and repair of bone defects after preparation.

[0034] Preferably, the composite scaffold provided by the application can be applied to tissue regeneration and repair of bone defect areas with dense distribution of blood vessels and nerves and high complexity of repair, such as craniofacial bone defects, mandibular or zygomatic bone fracture reconstruction, nerve pathway related bone structure repair (such as spinal lateral process or lamina), and bone-cartilage transition areas with soft tissue interface.

[0035] The composite scaffold provided by the application can simultaneously induce regeneration of bone, blood vessels and nerves, has the comprehensive advantages of structural support, signal release and microenvironment simulation, and provides a new strategy for functional bone repair materials.

[0036] Compared with the prior art, the application has the following beneficial effects:

[0037] (1) Construct a three-phase synergistic scaffold system with integrated structure and function to improve the overall performance and stability of the scaffold. The present application is based on 3D printing to construct a PCL skeleton, combined with GelMA hydrogel and P-NGF microspheres, to form a composite scaffold system with mechanical support-biological factor release-cell attachment triple function. Each component phase forms a stable chimeric interface through in-situ crosslinking, with good structural integrity and functional synergy.

[0038] (2) Achieve controllable release of non-model factor NGF and avoid burst release problems. The present application uses a double emulsion method to construct PLGA microspheres to encapsulate neurotrophic factor NGF, and by adjusting the ratio of lactic acid / hydroxyacetic acid of the polymer, the in-vivo degradation and release curve of NGF is regulated. The prepared microspheres can achieve a sustained release period of up to 56 days, effectively overcoming the problems of excessive early concentration and insufficient later release of traditional embedding systems.

[0039] (3) Induce "bone-vascular-nerve" synergistic regeneration to significantly improve the quality of functional repair. In in-vivo experiments, the composite scaffold can activate the PI3K / AKT signaling pathway through NGF release, and synergistically promote bone formation, neovascularization and nerve fiber construction. Studies have shown that it can significantly up-regulate functional markers such as ALP, OCN, COL1, CD31, α-SMA, NF200, etc., and the new bone volume fraction (BV / TV) is at least 2.5 times higher than the blank group, showing good multi-tissue repair ability.

[0040] (4) The mechanism is clear and has verifiability at the molecular level. RNA-seq and protein level analysis showed that the P-NGF microsphere scaffold can significantly activate the upstream factors of the PI3K / AKT pathway and inhibit the expression of osteoclast-related pathway genes, achieving bidirectional regulation of osteogenesis and bone resorption.

[0041] (5) The process is mild, the materials are safe, and it has good clinical convertibility. The whole scaffold uses biocompatible medical materials such as PLGA, PCL and GelMA, without using organic solvents or toxic ingredients. In-vitro cell viability and CCK-8 detection showed that there was no cytotoxicity to DPSCs, and the cell survival rate remained above 90%. The scaffold preparation process is stable and controllable, suitable for standardized production, and the structure and size are also suitable for multiple types of bone defect models (such as skull, mandible, spine, etc.), with good expandability and transformation prospects. BRIEF DESCRIPTION OF DRAWINGS

[0042] The drawings accompanying the specification of the present application form a part thereof, serve to provide further understanding of the present application, and together with the description, explain the present application. The description and illustrations of the exemplary embodiments of the present application serve to explain the present application, and do not constitute an improper limitation thereof.

[0043] Figure 1is the composite scaffold structure and physicochemical property analysis chart of the present application;

[0044] Figure 2 is the in vitro osteogenic induction experiment chart of the present application;

[0045] Figure 3 is the skull defect repair and bone regeneration analysis chart in vivo of the present application;

[0046] Figure 4 is the bone formation quantification and histological staining chart of the present application;

[0047] Figure 5 is the early and late osteogenic marker immunofluorescence analysis chart of the present application;

[0048] Figure 6 is the collagen marker expression and osteogenic related gene expression chart of the present application;

[0049] Figure 7 is the angiogenesis and nerve regeneration index analysis chart of the present application;

[0050] Figure 8 is the signal pathway activation mechanism verification chart of the present application;

[0051] Figure 9 is the composite scaffold preparation flow chart of the present application. DETAILED DESCRIPTION

[0052] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0053] Explanation of the terms of the invention

[0054] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0055] GelMA: 7.5% (w / v) pure GelMA hydrogel, without PCL, without microspheres, without cells, material control group (gel base);

[0056] gp: 3D printed PCL skeleton + GelMA hydrogel, without microspheres, without cells, scaffold structure blank control group;

[0057] Ro-gp: P-Ro 08-2750 microspheres are loaded in the gp scaffold (P-Ro 08-2750), without cells, drug release pathway inhibition group;

[0058] NGF-gp: P-NGF microspheres are loaded in the gp scaffold (P-NGF), without cells, drug release activation group;

[0059] Ro-GelMA: GelMA hydrogel system containing only Ro 08-2750 microspheres, no PCL scaffold, microsphere release / no scaffold system control;

[0060] NGF-GelMA: GelMA hydrogel system containing only NGF microspheres, no PCL scaffold, microsphere release / no scaffold system control;

[0061] GP: construct of loading DPSCs cells in gp scaffold, control group of animal experiment;

[0062] Ro-GP: construct of loading DPSCs cells in Ro-gp scaffold, inhibition group of animal experiment;

[0063] NGF-GP: construct of loading DPSCs cells in NGF-gp scaffold, treatment group of animal experiment.

[0064] The technical solutions of the present application will be further described below in combination with specific examples.

[0065] Example 1

[0066] (1) Preparation of P-NGF microspheres

[0067] Dissolve NGF in HEPES buffer to prepare an internal aqueous phase with a concentration of 100 μg / mL. Emulsify the above internal phase into 3% (w / v) PLGA (dichloromethane solution, LA:GA=85:15, Mw about 30-50 kDa) under ultrasonic conditions (power 200 W, 30 seconds) to form a W / O emulsion.

[0068] Subsequently, slowly drop the W / O emulsion into a 0.5% (w / v) PVA aqueous solution to form a stable W / O / W double emulsion under magnetic stirring (600 rpm). After continuous stirring for 6 hours to evaporate dichloromethane, precipitate the microspheres by centrifugation (8000 rpm, 5 min), wash them with deionized water for 3 times, and then freeze-dry to obtain dry P-NGF microspheres.

[0069] (2) Preparation of 3D printed PCL skeleton

[0070] Mix polycaprolactone (Mw≈80 kDa) and hydroxyapatite at a mass ratio of 4:1 uniformly. Use FDM technology and use a 3D BioArchitect printing platform for printing, with the following specific parameters:

[0071] Nozzle diameter: 0.3 mm;

[0072] Nozzle temperature: 110°C;

[0073] Printing pressure: 0.55 MPa;

[0074] Printing speed: 6 mm / s;

[0075] Layer height: 0.2 mm, using 0° / 90° interlaced printing mode;

[0076] Printing size: 30 mm x 30 mm x 0.6 mm (three layers).

[0077] After printing, natural cooling was performed, and ultraviolet irradiation sterilization was used, followed by cutting into disc-shaped implants with a diameter of 5 mm and a thickness of 0.6 mm under sterile conditions.

[0078] (3) Construction of composite scaffold

[0079] 5 mg of P-NGF microspheres (NGF loading capacity of 1 pg) were mixed with DPSCs (1 x 10 6 cells / mL) in a GelMA prepolymer solution (7.5% w / v GelMA + 0.25% w / v LAP) to prepare a homogeneous microsphere-cell-hydrogel mixture.

[0080] The mixture was injected into the pre-prepared PCL scaffold pores, and the light crosslinking was performed for 40 seconds under the irradiation of a 405 nm light source at 15 mW / cm 2 The intensity of the light. A complete composite scaffold was formed. This scaffold was named NGF-GP scaffold and was used for subsequent in vivo implantation experiments. All processes were performed under sterile conditions, and the obtained scaffold was stored at 4°C for standby.

[0081] Example 2

[0082] (1) Preparation of microspheres (P-Ro 08-2750)

[0083] 200 pg of Ro 08-2750 was dissolved in a 3% (w / v) PLGA solution in dichloromethane as an oil phase. The solution was dropped into a 0.5% PVA aqueous solution under high-speed emulsification conditions (high-speed homogenizer 12000 rpm, 30 seconds) to form an O / W emulsion.

[0084] DCM was volatilized for about 6 hours under magnetic stirring (600 rpm), and the microspheres were recovered by centrifugation (10000 rpm, 5 min), washed with deionized water for 3 times and freeze-dried to obtain dry P-Ro 08-2750 microspheres.

[0085] (2) Preparation of 3D printed PCL scaffold

[0086] The preparation method of the PCL / HA scaffold was the same as that in Example 1.

[0087] (3) Construction of composite scaffold

[0088] 5 mg of P-Ro 08-2750 microspheres (Ro 08-2750 loading amount is about 0.5 μg) were mixed with DPSCs (1 x 10 6 cells / mL) in GelMA prepolymer solution (7.5% w / v GelMA + 0.25% w / v LAP) to prepare a homogeneous microsphere-cell-hydrogel mixture.

[0089] The mixture was injected into the pre-prepared PCL scaffold pores, and the light crosslinking was performed under the irradiation of a 405 nm light source at 15 mW / cm 2 for 40 seconds to form a complete composite scaffold. The scaffold was named Ro-GP scaffold and was used for subsequent in vivo implantation experiments. All processes were carried out under sterile conditions, and the obtained scaffold was stored at 4°C for standby.

[0090] Comparative Example 1: Preparation method of composite scaffold without PLGA microspheres

[0091] The preparation process of the scaffold was the same as that of Example 1.

[0092] GelMA prepolymer solution (7.5% w / v GelMA + 0.25% w / v LAP) was only added with DPSCs (1 x 10 6 cells / mL), without the addition of PLGA microspheres. After uniform mixing, it was injected into the PCL scaffold pores, and the light crosslinking was completed under the irradiation of a 405 nm light source (15 mW / cm 2 ) for 40 seconds. The obtained composite structure was the GP scaffold.

[0093] The GP scaffold was used as a comparative scaffold without a drug delivery system for functional comparison and evaluation with Example 1 (NGF-GP) and Example 2 (Ro-GP).

[0094] Composite scaffold performance test:

[0095] (1) Cell viability test: DPSCs were inoculated in the GP and NGF-GP scaffolds and cultured for 1, 3, 5, and 7 days, respectively. Calcein-AM / PI double staining was used for live / dead cell staining to observe the effect of the scaffold on cell growth.

[0096] (2) Cell proliferation ability evaluation: CCK-8 kit was used to determine cell metabolic activity at each time point (1 / 3 / 5 / 7 days), and OD 450 values were used as indicators of cell proliferation ability.

[0097] (3) Osteogenic induction ability analysis:

[0098] Alkaline phosphatase (ALP) staining was used to detect early osteogenic activity at 7 days.

[0099] Mineralized nodule formation at day 21 was detected using Alizarin Red S staining;

[0100] and the expression levels of osteogenic genes were analyzed by quantitative PCR RUNX2, COL1A1, BGLAP

[0101] (4) Mechanical properties: Compression test was performed on both scaffolds to obtain stress-strain curve and Young's modulus, to evaluate the influence of microsphere loading on mechanical support.

[0102] (5) In vivo bone repair ability evaluation: GP and NGF-GP scaffolds were implanted into SD rat calvarial defect models, and Micro-CT scanning was performed at 4 and 12 weeks after surgery, respectively, 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 also 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 scaffolds on osteogenic differentiation and bone matrix formation.

[0103] (6) Vascular and neural generation evaluation: CD31, α-SMA, and NF200 immunofluorescence staining were used, and image analysis software was used to calculate the relative fluorescence intensity, to evaluate the influence of the scaffolds on vascular and neural regeneration.

[0104] The gene sequences of the osteogenic genes involved in the experiment are shown in Table 1 and Table 2:

[0105] Table 1 Gene sequences of osteogenic gene expression (human)

[0106]

[0107] Table 2 Gene sequences of osteogenic gene expression (rat)

[0108]

[0109] Composite scaffold performance:

[0110] Figure 1 ​​Figures showing the structure and physicochemical properties of the composite scaffolds of this invention. (A) Scanning electron microscope (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 particle 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 group of scaffolds 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. (GI) Swelling and degradation properties of the scaffolds: (G) Swelling rate change curves at different time points; (H) Statistical comparison of swelling rate over 24 hours; (I) Degradation curves of the scaffolds in collagenase solution. (J, K) Stress-strain curves and Young's modulus comparison. (L, M) Cell viability assessment using Calcein-AM / PI live / dead cell staining: (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) Cell proliferation assessment using the CCK-8 assay, with OD measured on days 1, 3, 5, and 7. 450 Value. 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.

[0111] SEM ( Figure 1 A) shows that all three scaffolds exhibit a uniform 3D porous structure, with the PCL framework forming a stable support structure, GelMA hydrogel filling the gaps, and microspheres clearly embedded in the matrix. Particle size distribution analysis ( Figure 1 (B, C) indicates that the average diameter of the Ro 08-2750 microspheres is approximately 90 μm, and the average diameter of the NGF microspheres is approximately 120 μm, both suitable for subsequent sustained-release applications. In vitro release profile ( Figure 1 (F) indicates that both types of microspheres achieved sustained drug release. NGF microspheres exhibited an initial burst release, releasing nearly 70% of the encapsulated factor within 21 days, followed by a plateau phase, demonstrating a biphasic release pattern. In contrast, Ro08-2750 microspheres exhibited a slower and more sustained release characteristic, with sustained release exceeding 56 days, indicating a more durable release capability. Rheological analysis of the GelMA-based hydrogels ( Figure 1 (D, E) shows that GelMA, Ro-GelMA, and NGF-GelMA perform well under 405nm illumination (15mW / cm²). 2) Next, G' rapidly increased, indicating that it has effective photo-crosslinking 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 experiments ( Figure 1 G, H) showed that all four scaffold groups exhibited considerable swelling capacity in phosphate buffered saline (PBS). The pure GelMA scaffold 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 reduction was 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 of the hydrogel to some extent, but the scaffolds still exhibit satisfactory swelling performance, which is beneficial to maintaining the stability of the cell microenvironment and nutrient exchange. In vitro degradation curves ( Figure 1 I) showed that the degradation trend of collagenase-containing PBS was stable in all groups. Mechanical tests ( Figure 1 J, K) showed that the mechanical strength of pure GelMA was low, while the addition of PCL scaffolds significantly improved the compression modulus and load-bearing capacity of the gp, Ro-gp, and NGF-gp groups. Notably, the NGF-gp scaffold had a slightly higher Young's modulus compared to the other groups, indicating excellent overall mechanical performance. In addition, the presence of DPSCs was confirmed by phenotypic characteristics. To assess biocompatibility, DPSCs cultured with scaffold extracts were then subjected to live / dead staining ( Figure 1 L). At 1, 3, and 7 days, all groups showed high cell viability, with abundant live cells (green) and minimal dead cells (red), and cells maintained 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 between groups ( P >0.05), indicating that cell toxicity was negligible. Further evaluation using the CCK-8 assay ( Figure 1 N) confirmed that the scaffold extracts (gp, Ro-gp, NGF-gp) did not inhibit DPSC proliferation. The absorbance at 450 nm continued to increase at 4 hours and at 1, 3, and 7 days, indicating continuous cell growth. Overall, these results confirm that the developed scaffolds have excellent biocompatibility and can effectively support the survival and proliferation of DPSCs, providing a reliable foundation for constructing a pro-regenerative microenvironment that is conducive to osteogenesis and tissue repair.

[0112] Figure 2Figure. The in vitro osteogenic induction experiment. The ability of the scaffold extract to induce osteogenic differentiation of DPSCs was evaluated by ALP and ARS staining, combined with mineralized nodule quantification analysis and osteogenic genes (ALP, OCN, BSP, and RUNX2) qRT-PCR results, reflecting the osteogenesis promotion of the scaffolds. (A) Representative images of ALP and ARS staining. The upper row is ALP staining at day 7, and the darkened areas indicate enhanced intracellular phosphatase activity; the lower row is ARS staining at day 21, and the orange-red nodules represent the formation of mineralized nodules. Scale bar = 100 pm. (B) Quantitative analysis of ALP activity (King unit / g protein). (C) After CPC treatment following ARS staining, the absorbance was measured at 560 nm to quantify mineralized nodules. (D-G) qRT-PCR expression analysis of DPSCs osteogenic-related genes at day 7, including (D), (E), (F), and (G) OCN. ns indicates not significant (p > 0.05); "*" indicates p < 0.05, "**" indicates p < 0.01, "***" indicates p < 0.001, and "****" indicates p < 0.0001. RUNX2, ALPL, COL1A1, BGLAP RUNX2 ALPL COL1A1 BGLAP P P P P P

[0113] At the early stage of induction (day 7), ALP staining (A) showed that the NGF-gp group had the strongest enzymatic activity, followed by the NGF group, while the control group only showed weak staining. Quantitative analysis of ALP activity (B) confirmed these findings, with the ALP level of the NGF-gp group being significantly higher than that of the control group (p < 0.05), while the activity of the Ro-gp group was significantly reduced (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 (A) showed that both the NGF-gp group and the free NGF group had a large number of orange-red mineralized nodules, indicating a significantly higher degree of calcification compared to the control group. In contrast, the staining observed in the Ro 08-2750 and Ro-gp groups was negligible. Quantitative ARS assessment (C) further demonstrated that the NGF-gp group had enhanced mineralization, while the Ro-gp group had significantly inhibited mineralization. Overall, these findings indicate that sustained NGF release significantly enhances late-stage mineralization, while Ro 08-2750 inhibits the late-stage osteogenic process. Figure 2 Figure 2 P P Figure 2 Figure 2

[0114] ​​​​​​​​​​​​​​​​To further elucidate the molecular mechanisms of these effects, key osteogenic genes RUNX2, ALPL, COL1A1 and BGLAP Expression of (encoding osteocalcin, OCN) was analyzed by qRT-PCR on day 7. Figure 2 DG) showed that NGF-gp significantly upregulated RUNX2, ALPL, COL1A1 and BGLAP Ro 08-2750 significantly inhibited the expression of key osteogenic factors and extracellular matrix genes. These findings suggest that Ro 08-2750 not only broadly inhibits the transcriptional activation of key osteogenic factors and extracellular matrix genes, but may also impair the overall osteogenic potential of cells.

[0115] Figure 3 This section presents an analysis of in vivo cranial defect repair and bone regeneration. It showcases the establishment process of a rat cranial defect model, scaffold implantation images, Micro-CT 3D reconstruction images, and transverse / coronal / sagittal section images. Quantitative analysis of bone regeneration indices (BV / TV, Tb.Th, Tb.N, Tb.Sp, Po.V / TV) was performed at 4 and 12 weeks. (A) Schematic diagram of the animal experiment process. (B) Micro-CT analysis at weeks 4 and 12: The left side shows the 3D reconstruction image, and the right side shows the transverse, coronal, and sagittal section images. The defect area is marked with a yellow circle or square. Scale bar = 2 mm. (C) Surgical implantation scene. The upper image shows a 5 mm cranial section 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.

[0116] To evaluate the in vivo bone regeneration capacity of the prepared scaffold, a critical-sized skull defect model (5 mm in diameter) was established in Sprague-Dawley rats. Figure 3 A). GP, Ro-GP, and NGF-GP stents are implanted at 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 post-implantation. Figure 3B, D). 3D reconstruction and multiplanar reformation images showed that the control group did not exhibit significant bone bridging at both time points and the defect was essentially non-healing. In contrast, all scaffold-treated groups (GP, Ro-GP, and NGF-GP) showed new bone growth extending from the defect margins towards the center as early as 4 weeks and progressive thickening was observed at 12 weeks. Notably, the NGF-GP group exhibited more continuous and denser mineralized bone structures at both time points. Quantitative micro-CT analysis showed that BV / TV, Tb-Th, and Tb-N were significantly increased in all scaffold-treated groups compared to the control group. Among them, the NGF-GP group showed the most significant improvements. Moreover, the NGF-GP group had the lowest Tb Sp and Po V / TV values, indicating improved bone quality and defect closure.

[0117] Radar plots based on micro-CT parameters Figure 4 A) indicate that the NGF-GP group exhibited the most favorable bone regeneration characteristics at 4 and 12 weeks, while the Ro-GP group showed the least improvement. The control group exhibited high trabecular spacing and porosity, indicating insufficient bone repair. These results are consistent with the 3D imaging data and quantitative analysis, confirming that the NGF-GP scaffold promoted superior bone regeneration in vivo.

[0118] Figure 4 Quantification of bone formation and histological staining images. Radar plots were used to integrate the structural parameters for comprehensive comparison of the regeneration effects of different groups; histological staining was used to demonstrate new bone formation under HE staining and Masson trichrome staining, and bone area analysis based on Masson images was used to assess the quality and maturity of the newly formed bone tissue. (A) Radar plots of bone parameters (BV / TV, Tb.Th, Tb.N, Tb.Sp, Po.V / TV) at 4 and 12 weeks. (B, C) HE staining (left) and Masson trichrome staining (right) of bone tissue sections at 4 and 12 weeks. Images include low magnification (scale bar = 1 mm) and high magnification views (scale bar = 200 pm). (D) Quantitative analysis of the new bone area based on Masson staining at 4 and 12 weeks. ns indicates not significant (p > 0.05); "*" indicates p < 0.05, "**" indicates p < 0.01, "***" indicates p < 0.001, and "****" indicates p < 0.0001. P >0.05); "*" indicates P <0.05, "**" indicates P <0.01, "***" indicates P <0.001, and "****" indicates P <0.0001.

[0119] To further evaluate the histological characteristics of scaffold-induced bone regeneration, tissue samples were collected from the calvarial defect sites at 4 and 12 weeks postoperatively for HE and Masson trichrome staining Figure 4B, C). H&E staining showed that the defect area of the control group was mainly filled with fibrous tissue at both time points, with little evidence of new bone formation. In contrast, the GP, Ro-GP, and NGF-GP groups exhibited newly formed bone plate-like structures at 4 weeks, which matured into more organized bone tissue by 12 weeks. For Masson's trichrome staining, the bone tissue appeared dark blue to blue-purple with a clear boundary between newly formed bone and surrounding tissue. The NGF-GP group exhibited continuous bone plate formation as early as 4 weeks, and by 12 weeks, a bone-like lamellar structure was 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 the control group but significantly lower in quantity and maturity than the NGF-GP group.

[0120] Quantitative analysis of new bone area Figure 4 D) further confirmed the histological findings. The percentage of new bone at 4 and 12 weeks was significantly higher in the NGF-GP group than in all other groups 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 showed a slight increase but was still significantly lower than the GP group P <0.05), indicating limited osteogenic potential. In summary, these results indicate that scaffolds loaded with NGF microspheres not only promote early bone formation but also support long-term bone maturation and remodeling. In contrast, the sustained release of Ro 08-2750 can disrupt osteoblast recruitment, bone matrix deposition, and tissue reconstruction during the bone regeneration process.

[0121] Figure 5 immunofluorescence analysis of early and late osteogenic markers. Immunofluorescence staining images of ALP and OCN at 4 and 12 weeks and their three-dimensional intensity distribution graphs (Z-axis represents fluorescence intensity) are shown, and the results of quantitative fluorescence intensity analysis at each time point are provided. (A, B) Immunofluorescence staining images and corresponding three-dimensional peak graphs of ALP and OCN expression at 4 and 12 weeks. ALP and OCN are labeled in red, and DAPI is labeled in blue. The scale bars are 500 μm, 200 μm, and 50 μm, respectively. In the three-dimensional graphs, the X and Y axes represent spatial coordinates, and the Z axis represents fluorescence intensity. (C, D) Quantitative analysis of ALP and OCN fluorescence intensity at 4 and 12 weeks. ns indicates no significant difference P >0.05); "*" indicates P <0.05, "**" indicates P <0.01, "***" indicates P <0.001, "****" indicates P <0.0001.

[0122] Figure 6This image shows the expression of collagen markers and osteogenic-related genes. It illustrates the expression of COL1 at different time points and displays a three-dimensional fluorescence image, combined with qRT-PCR analysis. Alpl , Col1a1 and Bglap Gene expression levels in tissue samples. (A) Immunofluorescence and three-dimensional peak intensity maps of COL1 expression at weeks 4 and 12; COL1 is marked in red, and DAPI in blue; scale bars are 500 μm, 200 μm, and 50 μm; X and Y axes represent spatial location, and Z axis represents fluorescence intensity. (B) Quantitative analysis of COL1 fluorescence intensity at weeks 4 and 12. (CE) Expression levels 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.

[0123] To further investigate the regulatory role of the scaffold in osteogenesis, immunofluorescence staining was performed to assess the expression of ALP, OCN, and type I collagen (COL1) in the skull defect area at 4 and 12 weeks post-implantation. Furthermore, 3D surface maps were generated using FIJI software to enhance the visualization of fluorescence intensity and spatial distribution. Figure 5 AB Figure 6 A).

[0124] At week 4, the GP group showed significant ALP-related red fluorescence in the osteogenic region. The control group showed weak signal, and the Ro-GP group showed even lower fluorescence. In contrast, the NGF-GP group exhibited the strongest ALP signal, characterized by clear high-intensity peaks 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 at both time points, ALP expression in the NGF-GP group was significantly higher than in all other groups. P <0.05). Although 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 5B) followed a similar trend. As a marker of late osteogenic differentiation, OCN was most widely distributed and expressed most strongly in the NGF-GP group. By week 4, distinct areas of intense red fluorescence were evident, and by week 12, these areas exhibited the characteristics of mature mineralized tissue. Quantitative analysis ( Figure 5 D) demonstrated that OCN expression was significantly higher in the NGF-GP group than in the control group at both time points, while the Ro-GP group showed moderate improvement but remained significantly lower than the NGF-GP. At week 4 ( Figure 6 A), COL1 expression was weak in the control group but significantly enhanced in the GP, Ro-GP, and NGF-GP groups, with the NGF-GP group exhibiting the strongest signal. By week 12 ( 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 COL1 expression was significantly higher in the NGF-GP group than in the control and Ro-GP groups at both time points ( P <0.001).

[0125] To further validate these findings at the transcriptional level, total RNA was extracted from the bone defect tissues and subjected to qRT-PCR analysis of Alpl , Col1a1 and Bglap (OCN-encoding gene) ( Figure 6 C-E). The NGF-GP group had the highest mRNA expression levels of the three genes at both time points, followed by the GP group, and the control group had the lowest expression. Notably, Bglap expression was significantly upregulated in the NGF-GP group as early as week 4 ( P <0.001), and this difference was further increased by week 12. These results indicate that the delivery of NGF plays a sustained and robust role in promoting bone mineralization and extracellular matrix maturation during the later stages of bone regeneration.

[0126] Figure 7This image shows the analysis of angiogenesis and nerve regeneration indicators. It includes dual immunofluorescence staining and three-dimensional intensity maps of CD31, α-SMA (vascular marker), and NF200 (neural marker). Quantitative fluorescence analysis and integrated radar maps of six markers (ALP, OCN, COL1, CD31, α-SMA, NF200) were used to assess the synergistic bone-vascular-nerve regeneration effect. (A) Dual immunofluorescence staining images of CD31 (red) and α-SMA (green) at weeks 4 and 12, and their three-dimensional fluorescence intensity peak maps; scale bars are 500 μm, 200 μm, and 50 μm, respectively; X and Y axes represent spatial location, and Z axis represents fluorescence intensity. (B) Immunofluorescence staining images of NF200 (red) at weeks 4 and 12, and their three-dimensional intensity maps; scale bars are 500 μm, 200 μm, and 50 μm, respectively; X and Y axes represent spatial location, and Z axis represents fluorescence intensity. (C) Quantitative analysis of relative fluorescence intensity of CD31, α-SMA, and NF200. (D) Radar plots of expression levels of six biomarkers (ALP, OCN, COL1, CD31, α-SMA, 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.

[0127] During bone tissue repair, coordinated regeneration of blood vessels and nerves is crucial for functional recovery. To systematically evaluate the impact of each scaffold on angiogenesis, dual immunofluorescence staining of CD31 (red) and α-SMA (green) was performed on the defect area at 4 and 12 weeks postoperatively 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 fluorescent surface maps generated using FIJI software. Figure 7 AB). In terms of angiogenesis ( Figure 7 (A) At week 4, the control group showed almost no undetectable vascular signals. 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 exhibited dense, co-localized vascular structures with significantly enhanced fluorescence intensity, indicating effective promotion of early angiogenesis. By week 12, all groups showed increased angiogenesis, with further expansion of CD31 / α-SMA co-expression in the NGF-GP group, and more mature and continuous vascular morphology. Regarding nerve regeneration ( Figure 7(B) At week 4, no significant 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 onset of neurogenesis. By week 12, NF200 expression in the NGF-GP group was further increased, showing higher fluorescence intensity and wider spatial distribution. Although the GP and Ro-GP groups showed some improvement, their expression levels remained significantly lower than those in 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 significantly higher than the GP and Ro-GP groups. Notably, although the Ro-GP group showed a slight increase compared to the control group, its expression levels on all markers were still lower than the GP group, suggesting that blocking the NGF signaling pathway may impair angiogenesis and neurogenesis to some extent. To comprehensively evaluate the overall regenerative performance of each scaffold in terms of osteogenic, angiogenesis, and neurogenesis, radar maps were constructed using six representative markers: ALP, OCN, COL1 (osteogenic); CD31, α-SMA (angiogenesis); NF200 (neurogenesis) ( Figure 7 (D) At week 4, the NGF-GP group demonstrated superior performance in all three functional domains, with radar plots showing a characteristic "hexagonal expansion" pattern, indicating balanced and robust regeneration. In contrast, the Ro-GP group showed significant contraction across multiple dimensions, particularly in vascular and neural markers. By week 12, the NGF-GP group maintained superior performance across all parameters. The Ro-GP group showed limited improvement but remained significantly lower than the GP group, while the control group consistently had the lowest values, reflecting its limited spontaneous repair capacity. Overall, these results highlight the comprehensive regenerative advantages of NGF-based scaffolds in promoting coordinated bone-vascular-neural regeneration.

[0128] Figure 8 This is a validation diagram of the signaling pathway activation mechanism. It shows principal component analysis (PCA) of RNA-seq clusters, differentially expressed gene volcano plots, GO and KEGG pathway enrichment analysis plots, osteogenic / osteoclast-related gene heatmaps, GSEA enrichment curves, and Western blot detection and grayscale quantitative plots of the PI3K / AKT pathway, revealing the evidence chain of the scaffold activation molecular mechanism. (A) Principal component analysis (PCA), the distance between points reflects the differences in the overall gene expression profiles and clustering trends among samples. (B) Volcano plot of differentially expressed genes (DEGs) between the NGF-GP group and the Ro-GP group; upregulated genes are shown in red (right), and downregulated 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), with bar charts showing significantly enriched functional categories. (D, E) Heatmaps of osteogenic and osteoclast-related gene expression, with red indicating high expression and blue indicating low expression. (F, G) GSEA enrichment analysis plots: (F) Gene sets positively enriched in the NGF-GP group (e.g., oxidative phosphorylation, cytoskeletal proteins, actin binding) and their NES values. p value, q Values; (G) Gene sets negatively enriched in the Ro-GP group (e.g., extracellular matrix, osteogenic, collagen trimer) and statistical results. (H) Scatter plot of KEGG pathway enrichment analysis. The size of the circle represents the number of enriched genes, and the color represents significance; the red box indicates significant enrichment of the PI3K / AKT pathway. (I, J) Western blot detection of PI3K and AKT phosphorylation levels and their gray values ​​for quantitative analysis. ns indicates no significant difference ( P >0.05); "*" indicates P <0.05, "**" indicates P <0.01, "***" indicates P <0.001, "****" indicates P <0.0001.

[0129] To further investigate the molecular mechanism of osteogenic action of NGF-loaded scaffolds, bone tissue samples were collected 12 weeks post-implantation for RNA-seq transcriptome analysis, followed by Western blot analysis to verify the activation status of key signaling pathways. Principal component analysis (PCA) Figure 8 A) revealed a clear separation between the NGF-GP and Ro-GP groups, indicating different clustering patterns in their global gene expression profiles. Volcano plot analysis ( Figure 8 B) It was determined that 264 genes in the NGF-GP group were significantly upregulated and 796 genes were significantly downregulated (|log2FC|≥1, P <0.05). Gene Ontology (GO) enrichment analysis ( Figure 8 C) indicates that differentially expressed genes (DEGs) are primarily associated with biological processes such as "response to stimuli," "phylogeny," and the cellular component "extracellular region," suggesting that NGF may promote bone regeneration by regulating cellular stress responses and the extracellular microenvironment. A heatmap of genes related to osteoblast and osteoclastogenesis (…) Figure 8D, E) further indicated that osteogenic transcription factors such as Smad1, Smad4, Smad5, Runx2 and Bmpr2 were upregulated in the NGF-GP group, while the Ro-GP group exhibited elevated expression of osteoclast-related genes, including Ctsk, Acp5 and Tnfrsf11b. These findings suggested that NGF mediates a dual regulation 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, which are 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 trimer". KEGG pathway analysis ( Figure 8 H) identified multiple classical signaling cascades related to bone remodeling, 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 evaluated ( Figure 8 I). Western blot results showed that the expression of phosphorylated PI3K (p-PI3K) and AKT (p-AKT) was significantly increased in the NGF-GP group. Gray scale 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). Overall, 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 coordinating bone repair at the transcriptional and protein levels.

[0130] Figure 9 A flowchart for preparing the composite scaffold of the present application. It is used to show the preparation process of the neurotrophic factor NGF or Ro 08-2750 loaded PLGA microspheres, and the overall process of embedding them into a three-dimensional printed PCL scaffold and photo-crosslinking GelMA to form a composite scaffold, as well as its application scenario in a SD rat calvarial defect model.

[0131] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. PLGA microspheres- PCL / GelMA composite scaffolds based on NGF sustained release, characterized in that, The composite scaffold is composed of a PCL framework, a GelMA hydrogel matrix, NGF-loaded PLGA microspheres, and dental pulp stem cells DPSCs; The PCL framework is prepared by premixing polycaprolactone and hydroxyapatite at a mass ratio of 2-5:1 and using a fused deposition modeling three-dimensional printing technology; The NGF-loaded PLGA microspheres and the DPSCs are uniformly dispersed in the GelMA matrix, which is filled in the PCL framework. 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 PLGA has a lactic acid to hydroxyacetic acid ratio of 70:30 to 90:

10. The PLGA has a molecular weight range of 20-60 kDa.

2. The composite stent of claim 1, wherein, The GelMA hydrogel matrix uses gelatin methacrylate as the main body and cooperates with a photoinitiator to form a photosensitive crosslinking system, thereby forming a uniform gel structure. The mass concentration of the GelMA prepolymer solution ranges from 3% to 15% (w / v), and the proportion of the photoinitiator LAP added ranges from 0.05% to 0.5% (w / v).

3. The method of claim 1, wherein the NGF sustained-release based PLGA microsphere-PCL / GelMA composite scaffold is prepared by the steps of: The steps include: (1) Inner phase preparation: NGF is dissolved in 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid buffer, with a concentration controlled at 80-120 μg / mL; (2) Oil phase preparation: PLGA is 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 above W / O emulsion is slowly dropped into a 0.2-1% (w / v) polyvinyl alcohol aqueous solution, and a stable W / O / W double emulsion is formed under magnetic stirring; (5) Solvent evaporation and particle solidification: continuous stirring is performed for 4-6 hours to completely volatilize dichloromethane, and the microspheres are solidified and formed; (6) Microsphere collection and drying: after centrifugation and washing, the P-NGF microspheres are obtained by freeze-drying for 24-48 hours; (7) Preparation of the PCL framework: the PCL framework is prepared by premixing polycaprolactone and hydroxyapatite at a mass ratio of 2-5:1 and using a fused deposition modeling three-dimensional printing technology; (8) Preparation of a GelMA prepolymer solution containing cells and microspheres: P-NGF microspheres and dental pulp stem cells DPSCs are blended in a GelMA prepolymer solution containing a photoinitiator to prepare a uniform suspension; (9) Scaffold composite and photo-crosslinking: the suspension described in step (8) is injected into the PCL scaffold pores, and then crosslinked under 405 nm light irradiation at 15 mW / cm 2 intensity conditions, and the crosslinking time is controlled at 30-50 seconds, and it is obtained.

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

5. The production method according to claim 3, wherein In step (4), the magnetic stirring speed is 500-1000 rpm.

6. The production method according to claim 3, wherein The P-NGF microspheres prepared in step (6) are regular spherical, have a particle size of 80-200 μm, an encapsulation rate of 70-90%, and a centrifugation speed of 8000-12000 rpm.

7. The production method according to claim 3, wherein The microspheres are added in an amount of 5-10 mg, corresponding to a total amount of NGF of 1-2 μg; the DPSCs are seeded at a density of 1×10 6 cells / mL; the GelMA concentration is 6-10% (w / v), and the light initiator LAP concentration is 0.2-0.3% (w / v); the mass ratio of light initiator to GelMA is 1:20-1:

40.

8. Use of the NGF sustained-release based PLGA microsphere-PCL / GelMA composite scaffold according to any one of claims 1-2 or prepared by the method according to any one of claims 3-7 in the preparation of a tissue regeneration and repair material for bone defects.

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