Hierarchical periosteum-bone composite scaffold with staged release of dexamethasone and endothelial cell derivatives for efficient critical-sized bone defect repair

Abstract

Bone tissue engineering represents a promising approach for developing multifunctional biomaterials to facilitate bone regeneration. In this study, we designed a hierarchically composite scaffold that mimics both the periosteum and bone to investigate its effectiveness in repairing critical-sized bone defects. The periosteal layer of the bionic bilayered scaffold was created using a nonwoven mat of core-shell structured nanofibers. The fiber core consisted of a methylpropionylated gelatin (GelMA) hydrogel that encapsulated endothelial cell derivatives (ECd), while the shell was composed of a blend of poly(L-lactide-co-ε-caprolactone), bioactive glass (BG), and dexamethasone (DEX), enabling the staged release of the payloads. Then, a bulk of GelMA/BG/DEX hydrogel was integrated with the periosteal layer to construct a hierarchical periosteum-bone composite scaffold. Such a scaffold facilitated revascularization, anti-inflammation, and the promotion of mature bone formation through the biomimetic properties of organic-inorganic hybrid components and a three-dimensional porous structure, as well as the dual effects of staged release of DEX and ECd. In vivo, the scaffold significantly promoted repair of a 6-mm rat calvarial defect, accompanied by up-regulated expression of CD31, OPN, and type I collagen. Transcriptome sequencing analysis also revealed that the repair process is closely associated with the JAK2-STAT signaling pathway. Collectively, this bionic hierarchical scaffold enhanced critical-sized bone defect repair through synergistic multifunctional regulation, including enhanced angiogenesis, modulation of inflammation, and efficient osteogenic differentiation, demonstrating broad and promising clinical translation potential.

Graphical abstract

Schematic illustration revealing the fabrication of a hierarchical periosteum-bone composite scaffold with staged release of dexamethasone and endothelial cell derivatives for efficient critical-sized bone defect repair, owing to its outstanding performance in enhanced angiogenesis, inflammatory modulation, and efficient osteogenesis.

1. Introduction

The treatment of bone tissue defects is a big challenge in orthopedics, as various factors such as trauma, infection, tumors, osteonecrosis, and congenital malformations can lead to these problems [1]. To address these problems, autologous, allogeneic, and xenogeneic transplantation has evolved, but with separate advantages and disadvantages. The main disadvantages are infection in the donor area, pain, and the risk of rejection in allogeneic bone transplants [2]. At present, most studies on bone regeneration hydrogels and fibrous membranes focus on simulating bone composition and constructing the microenvironment required for bone healing [[3], [4], [5]]. However, simulating only bone components is not sufficient to achieve successful bone regeneration. In some studies, successful repair of critical-sized bone defects is closely associated with vascularization and immune regulation [[6], [7], [8]]. Specifically, the early inflammatory response driven by immune cells constructs a local microenvironment, which affects the behavior of stem cells, osteoblasts and osteoclasts, and most affects the speed of bone healing [9]. Macrophages are a key component of the immune system and play an important regulatory role in bone regeneration [10]. The classical process is that inflammation activates M1 macrophages to initiate the inflammatory response by secreting various pro-inflammatory cytokines such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS) [11]. Therefore, inhibiting macrophage polarization toward the M1 phenotype is very important for bone regeneration. In addition to the inflammatory response, local angiogenesis is also important for bone regeneration. Angiogenic factors can promote the proliferation and differentiation of vascular endothelial cells, thereby stimulating the growth of new blood vessels and providing more oxygen and nutrients to the regenerated tissue site [12]. In conclusion, regulating the local immune microenvironment and enhancing angiogenesis are essential for promoting bone tissue regeneration.

Natural bone tissue has a hierarchical structure, with the bone matrix and the periosteum on its surface. When a bone defect forms, the periosteum is also damaged [13]. The periosteum is super important for bone regeneration. It is mostly composed of collagen fibers, is densely vascularized, and provides essential growth factors and nutrients for bone growth [14]. The problem is that most scaffolds used in bone regeneration tissue engineering right now mimic the bone matrix and overlook the role of the periosteum [15]. Studies have shown that during fracture repair, the periosteum contributes more than 70% to early osteogenesis [16]. That is why, for tissue engineering technologies that promote bone regeneration, not only must the environment of the mineralized bone matrix be simulated, but the role of the periosteum must also be mimicked. Electrospinning technology has greatly advantages and can produce core-shell nanofiber membranes [17,18]. By loading different components into the shell and core layers, we achieve a graded release effect, thereby mimicking the role of the periosteum more effectively. Hydrogels are 3D scaffold with excellent biocompatibility that provides a growth environment similar to the natural extracellular matrix, promote tissue regeneration, and can be loaded with minerals and growth factors, which are crucial for bone tissue repair [19,20]. Methylpropionylated gelatin hydrogel (GelMA) is a modified gelatin with double bonds. It has great biocompatibility and cell activity, and its 3D structure is perfect for promoting cell growth and differentiation [21]. We can mimic the bone matrix environment by adding inorganic ions to the hydrogel, and GelMA can transition from a liquid state to a gel state under UV irradiation, making it suitable for use in irregular bone defect areas [22].

Bioactive glass (BG) is a biomaterial with excellent biocompatibility, osteoconductivity, and osteogenic properties, and it has attracted significant attention and research in bone regeneration [23,24]. It is mainly composed of oxides such as SiO₂, Na₂O, CaO, and P₂O₅, and the specific composition and ratios of these components directly determine its biological activity and mechanical properties [25]. The biological activity of BG primarily lies in its surface's ability to interact with body fluids and form a hydroxyapatite layer rich in calcium and phosphorus. This happens because the ions dissolved from BG react with those in body fluids [26]. As BG dissolves, it releases water-soluble ions, such as calcium, phosphorus, silicon, and sodium ions, which are directly derived from the corresponding oxides that compose the BG. Calcium and sodium ions dissolve and release first, forming a silica-rich region that serves as a nucleation site for hydroxyapatite deposition [27]. What makes this hydroxyapatite different is that it contains carbonate, distinguishing it from pure calcium phosphate bone grafts, such as hydroxyapatite and tricalcium phosphate, which lack carbonate [28]. This unique ion-release mechanism is what gives BG its specific properties and lets it boost bone regeneration [29].

Dexamethasone (DEX) is a synthetic glucocorticoid with anti-inflammatory, anti-allergic, immunosuppressive, and other effects. It is one of the earliest and most widely used inducers of osteogenic cell differentiation, and at an optimal concentration of 10 to 100 nM, it can strongly stimulate osteogenesis [[30], [31], [32], [33]]. During osteogenesis, DEX enhances bone formation by regulating immune cell activity. It can also be used alongside bone morphogenetic protein-2 (BMP-2), and by enhancing the biological activity of BMP-2, it promotes the differentiation and mineralization of osteoblasts [34]. This combined therapy significantly accelerates osteogenesis. Both ectopic osteogenesis and in vivo experiments have shown high alkaline phosphatase activity and strong mineralization with this pairing. Endothelial cells are single-layer cells lining the inner walls of blood vessels and are crucial for maintaining blood vessel health and function [35]. Endothelial cell derivatives (ECd) are basically the various bioactive substances that endothelial cells synthesize, secrete, or express. These substances play important roles in maintaining vascular function, regulating physiological activities, and participating in pathological processes. By loading BG, ECd, and DEX into core-shell nanofiber membranes, these components work synergistically to promote angiogenesis, which ultimately supports bone tissue formation.

In this study, we developed a hierarchical scaffold by combining core-shell fiber membranes with a photocrosslinked GelMA/BG/DEX hydrogel. We made the most of the complementary effects of the mineralized bone matrix and the periosteum to mimic the structure and function of natural bone tissue as closely as possible. This design promotes vascular regeneration, reduces inflammation, and ultimately supports effective bone regeneration.

2. Experimental section

2.1. Material

Type A gelatin and Triton X-100 were purchased from Sigma (Germany). BG was obtained from Hannotech Biosciences (China). Poly (L-lactide-co-ε-caprolactone) (PLCL) was purchased from Daigang (China). The photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphinate, LAP) and portable curing light source (405 nm, 3 W) were bought from EngineeringForLife (China). Hexafluoroisopropanol (HFIP) and MA were obtained from Macklin (China). Electrospinning machines were purchased from Yongkang (China). The BCIP/NBT alkaline phosphatase color development kit was purchased from Beyotime Biotechnology (China). Fetal bovine serum (FBS) was obtained from Gibco (USA). Phosphate-buffered saline (PBS), Dulbecco's modified Eagle medium (DMEM), penicillin-streptomycin solution (PS), Ethylenediaminetetraacetic acid (EDTA) solution, cell counting kit-8 (CCK-8), SP kit (Rabbit), H&E, Masson's trichome staining, Alizarin Red S, Calcein-AM/PI kit and radioimmunoprecipitation assay lysis buffer (RIPA lysates) were purchased from Solarbio (China). Anti-CD31 antibody, Anti-Osteopontin antibody, Anti-COL Ⅰ antibody, Anti-iNOS antibody, Anti-JAK2 antibody, Anti-p-JAK2 antibody, Anti-STAT antibody, Anti-p-STAT antibody, Anti-Actin antibody and Phalloidin-iFluor 488, 594 antibodies were purchased from Abcam (UK). FastPure Cell/Tissue Total RNA Isolation Kit V2, ChamQ Universal SYBR qPCR Master Mix, and HiScript Ⅲ RT SuperMix for qPCR were purchased from Vazyme (China). Gene primers of Actin, runt-related transcription factor 2 (RUNX2), and osteopontin (OPN) were purchased from Sangon Biotech (China). All reagents were used directly without further treatment.

2.2. Preparation and characterization of GelMA/ECd@PLCL/BG/DEX core-shell fiber

ECd was first extracted for core-shell nanofibers fabrication. Human umbilical vein endothelial cells (HUVECs) were cultured to 80% confluence, the complete medium was replaced, and cells were cultured for an additional three days. The complete medium was collected, frozen at −80 °C overnight, and lyophilized using a freeze dryer. The resulting freeze-dried powder was designated as ECd. The core-shell nanofiber membranes were fabricated via electrospinning equipment. The core solution was 10 wt% GelMA mixed with ECd, and the shell solution was 10 wt% PLCL mixed with 2% BG and 1% DEX. The distance from the coaxial needle to the collecting roller was 15 cm, and the applied voltage was 15 kV. Two independent injection pumps were used to deliver the two solutions at rates of 1 mL/h for the shell and 0.025 mL/h for the core. The coaxial needle had an inner diameter of 0.34 mm and an outer diameter of 0.84 mm. The electrospinning process was run for 2 h before termination. The core-shell fiber membrane was coated with Au/Pd and imaged by scanning electron microscopy (SEM). Transmission electron microscopy (TEM) imaging was used to confirm the core-shell structure. The average nanofiber diameter was measured by analyzing 100 nanofibers from SEM images using ImageJ. For further structural validation, ECd was replaced with FITC-BSA for the core-shell electrospinning process and the scaffold was observed and photographed under an inverted fluorescence microscope.

ECd is rich in bioactive factors that promote angiogenesis, thereby inducing neovascularization and accelerating tissue repair and healing. In order to screen the optimal concentration of ECd (0 %, 10 %, 20 %, 30 %, 40 %), the core-shell fibrous membranes loaded with different concentrations of ECd were immersed in DMEM medium to prepare the extract, and the function of HUVECs was detected by scratch test and CCK-8 test. The cells were treated with extracts containing different concentrations of ECd and were sampled at 24 and 48 h. The cells were stained with calcein-AM, and the images were collected by fluorescence microscope. The relative migration rate of cells was calculated by ImageJ software. In the CCK-8 cell viability assay, HUVECs were seeded in 24-well plates at a density of 5 × 10⁴ cells/well, and cultured for 1 d, 4 d, and 7 d using a fiber membrane extract containing ECd. Subsequently, the medium containing 10% CCK-8 reagent was added and incubated for 2 h. The absorbance of the supernatant at 450 nm was measured using a microplate reader to evaluate cell proliferation. In order to characterize the apatite formation ability of the core-shell fibrous membrane, the samples were immersed in simulated body fluid for 7 days. The samples were dried in a fume hood, and the surface morphology was observed by SEM, and the degree of mineralization was analyzed.

2.3. Fabrication and characterization of GelMA/BG/DEX hydrogel

GelMA hydrogel was fabricated as previously reported [36]. Briefly, 2 g of type A gelatin was dissolved in 20 mL of PBS at 60 °C, then 1.6 mL of methacrylic anhydride (MA) was added dropwise to the solution at 1.0 mL/h with vigorous stirring for 3 h. Next, 80 mL of preheated PBS was added to the mixture, and unreacted components were removed by dialysis against deionized water for 1 week using a 12-14 kDa dialysis membrane. The resulting solution was filtered through a 0.22 μm filter, frozen at −80 °C overnight, and lyophilized. To prepare the GelMA stock solution, 0.7 g of lyophilized GelMA was dissolved in 10 mL of PBS containing 0.5% (w/v) photoinitiator at 60 °C. GelMA/BG/DEX hydrogel was then prepared by adding DEX at 1 mg/mL and BG at 200 mg/mL to the GelMA stock solution and this formulation was used for all subsequent experiments.

GelMA/ECd@PLCL/BG/DEX core-shell fiber membranes were encapsulated in the GelMA/BG/DEX hydrogel, and the hydrogel was photocrosslinked under UV light for subsequent drug release studies. For DEX release profiling, a standard curve was established using DEX concentrations of 1, 5, 10, 25, and 50 μg/mL. The composite hydrogel scaffold was then weighed and immersed in 10 mL of PBS in centrifuge tube, placed in a constant temperature shaker and incubated at 37 °C with a shaking speed of 100 rpm. And 2 mL of PBS was collected at predetermined time points (0.5, 1, 2, 6, 18, 24, 36, 48, and 72 h). An equal volume of fresh PBS was added after each collection to maintain sink conditions. The absorbance of collected samples was measured at 242 nm using a UV-Vis spectrophotometer to calculate DEX concentration. For ECd sustained-release characterization, BSA was used as a substitute for ECd to prepare GelMA/BSA(30%)@PLCL/BG/DEX core-shell fibers, which were UV-crosslinked. Approximately 35 mg of the fiber membrane was immersed in 5 mL of PBS, and 1 mL of PBS was collected at designated time points (1 h, 12 h, 24 h, 2 d, 3 d, 7 d, 14 d, 21 d, 28 d, 35 d, 42 d, 49 d, 56 d, and 63 d), with fresh PBS added to replace the collected volume. BSA concentration in the collected PBS was measured using a protein quantification assay kit.

2.4. Evaluation of biocompatibility in vitro

The CCK-8 assay and live/dead cell staining were used to assess the effects of composite hydrogel scaffolds on cell viability. The experimental groups were designated as follows: Control, GelMA, GelMA/BG (GB), GelMA/BG/DEX (GBD), and GelMA/BG/DEX-GelMA/ECd@PLCL/BG/DEX (GBDE). Extracts from each group were prepared for subsequent cell experiments. MC3T3-E1 cells were cultured in extracts from different hydrogels for 1, 3, and 7 days, and the CCK-8 assay was conducted, with regular wells serving as controls. At each time point, the cells were washed three times with PBS, incubated in medium containing 10% CCK-8 reagent for 2 h, and the absorbance of the supernatant was measured at 450 nm using a microplate reader. For live/dead staining, MC3T3-E1 cells were cultured in scaffold extracts for 3 days, then stained with calcein-AM and propidium iodide (PI) according to the manufacturer's instructions, and incubated at 37 °C for 20 min. The cells were washed three times with PBS, imaged under an inverted microscope, and analyzed using ImageJ.

2.5. ALP staining

BMSCs were seeded in 24-well plates at 5 × 10⁴ cells/well, and cultured to 70% confluence, then co-cultured with hydrogel extracts and osteogenic induction medium at a 1:1 ratio for 14 days. Cells were washed twice with PBS and stained using the BCIP/NBT alkaline phosphatase color development kit. Images were acquired using an inverted microscope and analyzed with ImageJ software.

2.6. Alizarin Red S staining

BMSCs were seeded in 24-well plates at 5 × 10⁴ cells per well. cultured to 70% confluence, and co-cultured with hydrogel extracts and osteogenic induction medium at a 1:1 ratio for 21 days. Cells were washed twice with PBS and fixed with paraformaldehyde. Alizarin Red S staining was performed, and images were captured with an inverted microscope and analyzed with ImageJ.

2.7. Osteogenic-related gene expression

Quantitative real-time PCR (qRT-PCR) was employed to evaluate the expression of osteogenic-related genes, specifically ALP and RUNX2. BMSCs were seeded in 24-well plates at 5 × 10⁴ cells per well and co-cultured with hydrogel extracts along with osteogenic induction medium for 7 and 14 days. Total RNA was extracted using the FastPure Cell/Tissue Total RNA Isolation Kit, and equal amounts of RNA were reverse transcribed into cDNA using the HiScript III RT SuperMix for qPCR. Amplification was performed using SYBR qPCR Master Mix for 40 cycles, and relative gene expression was calculated using the −ΔΔCt method.

2.8. Anti-inflammatory assessment

RAW264.7 macrophages (CL-0190, Procell) were used to evaluate the anti-inflammatory properties of the scaffolds. The cells were cultured to an appropriate density and treated with scaffold extracts for 24 h. Immunofluorescence staining was performed to detect the expression of the pro-inflammatory marker, iNOS. Western blot (WB) analysis was conducted to quantify iNOS protein expression.

2.9. Immunofluorescence staining

Cells treated with scaffold extracts for 24 h were washed three times with PBS, fixed with 3% glutaraldehyde at room temperature for 10 min, and washed three times with PBS again. Cells were permeabilized with 0.1% Triton X-100 for 5 min at room temperature, followed by three PBS washes, then blocked with 1% BSA for 1 h and washed three times with PBS. Primary antibodies were added, and the cells were incubated overnight at 4 °C in the dark. After the incubation, the cells were washed once with 0.1% Tween 20 and twice with PBS (5 min each wash), then secondary antibodies were added and incubated for 1 h at room temperature in the dark. The secondary antibody was removed, and the cells were washed three times with PBS (5 min each wash). Finally, the cells were stained with a DAPI-containing solution for 5 min, washed three times with PBS, and observed under a fluorescence microscope.

2.10. Western blot

The cells were washed twice with PBS, and 500 μL of RIPA lysis buffer was added to lyse the cells on ice for 30 min. Lysed cells were scraped and transferred to 1.5 mL EP tubes, then centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was collected, and the protein concentration was measured using a microplate reader. Next, prepare the separation gel and stacking gel for SDS-PAGE, pouring each gel separately. Insert the comb into the gels and allow them to polymerize at room temperature for 40 min, then remove the comb. The sample volume of the protein sample was 5-10 μL, and the sample volume of the protein molecular weight standard was 2 μL. The sample was added to the gel sample hole. Electrophoresis was performed at 120 V for 90 min. When the protein band migrated to about 1 cm above the bottom of the gel, the electrophoresis was terminated. The protein was transferred to PVDF membrane by wet transfer system at 4 °C and 400 mA for 60 min. After the transfer was complete, the PVDF membrane was removed and washed 3 times with 1 × TBST buffer for 10 min each. Subsequently, the membrane was placed in a closed buffer and incubated at room temperature for 2 h. After blocking, the cells were washed 3 times with 1 × TBST buffer, 10 min each time. The membrane was incubated overnight at 4 °C with the diluted primary antibody. After the membrane was removed from the refrigerator and allowed to reach room temperature, it was washed three times with 1 × TBST buffer for 10 min each. Then they were incubated with diluted secondary antibody at room temperature for 1 h. After incubation, they were washed 3 times with 1 × TBST buffer, 10 min each time. Before use, the A and B solutions in the color development kit were mixed in equal volumes, evenly applied to the PVDF membrane, and incubated in the dark for 1 min. Finally, the membrane was placed in developer and imaged with ImageJ.

2.11. Effectiveness of double-layer scaffolds on bone regeneration in vivo

All experimental procedures were approved by the Qingdao University Laboratory Animal Welfare Ethics Committee (202301SD65202401089) and performed in strict compliance with institutional laboratory animal care and use guidelines. The bone regeneration efficacy of the composite hydrogel bilayer scaffold was evaluated using a critical-sized cranial defect model in 8-week-old male Sprague-Dawley (SD) rats. A total of 60 rats were randomly divided into five groups: Control, GelMA, BG, GBD, and GBDE. Rats were anesthetized using isoflurane administered through an anesthesia machine at a flow rate of 1 L/min. The surgical area was shaved and disinfected, a 2-cm sagittal incision was made on the skull to expose the calvaria and skin retractor was used to retract the soft tissue. A 6-mm diameter circular defect was created on one side of the sagittal suture using a dental trephine drill, with careful preservation of the dura mater and underlying brain tissue. Hematomas were rinsed with sterile saline, a matching core-shell fiber membrane was placed over the defect, and the defect was filled with the corresponding hydrogel, which was UV-crosslinked for 30 s. The incision was closed with 4-0 absorbable sutures. Postoperatively, the rats were housed individually in standard cages without any activity restrictions. At 4 weeks and 8 weeks after surgery, the rat skulls were collected for Micro-CT imaging and histological analysis to assess the extent of bone regeneration.

2.12. Micro-CT analysis

Cranial bone samples collected at 4 weeks and 8 weeks post-surgery were scanned by Micro-CT, and the scanned data were reconstructed into 3D images. Image analysis was performed using Analyzer software, and bone regeneration was assessed by the ratio of new bone volume to total bone volume (BV/TV).

2.13. Histological sample preparation

After Micro-CT scanning, the samples were fixed in paraformaldehyde for 24 h, then decalcified in EDTA decalcification solution for 8 weeks (solution changed twice weekly). Decalcified samples were dehydrated in a dehydrator, embedded in paraffin, and sectioned at 5 μm slices. H&E, Masson, and immunohistochemical staining were performed on the sections, which were then observed under an upright microscope, and images were collected.

2.14. Transcriptome sequencing

Tissue samples (n = 3) from the defect repair sites of the GelMA and GBDE groups were collected at 8 weeks post-operation for transcriptome sequencing. Following sequencing, RAW264.7 macrophages were cultured for 24 h with extracts from the two composite hydrogel scaffold groups, and qPCR was performed to validate the transcriptome sequencing findings. The JAK2-STAT signaling pathway was identified as a key pathway for regulating bone regeneration in sequencing data analysis. In this study, the key role of this pathway was further verified by WB analysis of the protein extracts of two groups of macrophages.

2.15. Statistical analysis

Differences between groups were compared using one-way analysis of variance (one-way ANOVA), followed by Student's t-test for all pairwise combinations. All data were derived from at least three independent repeated experiments, and the results were expressed as mean ± standard deviation (Mean ± SD). Significance levels are indicated as follows: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

3. Results and discussion

3.1. Preparation and characterization of GelMA/ECd@PLCL/BG/DEX core-shell fiber

Intramembranous ossification is the core link of bone regeneration. Natural periosteum is rich in capillaries and osteogenic growth factors, which can not only provide oxygen and nutrients for the process of bone remodeling, but also significantly promote bone tissue repair and osteogenesis by activating local osteogenesis-related activities [[37], [38], [39]]. However, the clinical application of autologous periosteum has obvious limitations: on the one hand, due to the scarcity of tissue sources, it is difficult to meet the high demand for repair. On the other hand, the collection process may lead to complications such as donor-site infection [40]. Allogeneic periosteal transplantation also faces thorny problems, the immune rejection is difficult to avoid, and there is a potential risk of disease transmission. These factors have restricted its wide application in clinical practice [41]. Tissue-engineered bionic periosteum, designed to mimic the structure and function of natural periosteum, addresses these limitations of traditional methods and holds great therapeutic potential [42]. Such synthetic scaffold-periosteum materials are easily fabricated and effectively reduce immune response and disease risks associated with acellular periosteum matrices.

Coaxial electrospinning is a widely used technique for fabricating core-shell structured biomaterials, in which core and shell phases are injected into coaxial needles of different inner diameters. After applying an electric field, the solution will be stretched and solidified to form a nanofiber membrane [43,44]. Nanofiber structure has a significant regulatory effect on cell proliferation and differentiation [[45], [46], [47]]. In this study, we prepared a core-shell composite membrane with nano-scale random structure by electrospinning. To simulate the composition and function of natural bone matrix, BG was added to the shell phase of the membrane, and DEX was added to leverage its anti-inflammatory activity and osteogenic induction potential. The inner core of the membrane is loaded with ECd to exert its pro-angiogenic effect. Unlike a single angiogenic factor (such as VEGF) or osteogenic factor, ECd, as a complex bioactive factor library, can not only directly promote angiogenesis, but also indirectly enhance osteogenic efficiency through paracrine effects, and form an irreplaceable synergy with BG and DEX, providing multi-dimensional regulatory support for bone regeneration [48]. ECd is a complex library of bioactive factors, including VEGF, bFGF and Ang-1. Among them, VEGF can effectively promote the proliferation of vascular endothelial cells and the formation of lumen structure, bFGF mainly regulates the growth of vascular branches, and Ang-1 can significantly enhance the stability and integrity of the vascular wall. The synergy of these three factors completely covers the whole process of angiogenesis, branch extension and functional maturation, and successfully overcomes the inherent limitations of single use of VEGF-only induction of angiogenesis, but cannot guarantee the stable physiological function of blood vessels [49]. In order to screen out the optimal loading concentration of ECd, the core-shell fibrous membranes with ECd loading of 0 %, 10 %, 20 %, 30 % and 40 % were prepared in this study, and the extracts of each membrane were prepared. The migration and proliferation of HUVECs were evaluated using the scratch test and CCK-8 assay. The results of the scratch test showed no significant difference in cell migration rate between the 10 %, 20 %, 30 %, and 40 % ECd groups at 24 h. After 48 h of culture, cell migration in the 30 % ECd fiber membrane extract treatment group was significantly improved, with a statistical difference compared with the other concentration groups (Fig. 1a and b). The CCK-8 proliferation assay further confirmed that cell proliferation in the 30 % ECd extract group was significantly higher than in the other experimental groups at 4 d and 7 d (Fig. 1c). Based on the above experimental results, 30% was selected as the optimal ECd loading concentration for all subsequent experiments.

Fig. 1.

Screening of ECd concentration encapsulated in the core-shell fibers. (a) Cell migration test of core-shell fiber membranes with different content of ECd. (b) Migration quantitative results of the cell migration assay. (c) CCK-8 assay shows that the core-shell fiber membranes with 30% ECd content have the best ability to promote cell proliferation. (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

We successfully prepared the GelMA/ECd@PLCL/BG/DEX core-shell nanofiber membrane. SEM images revealed a random nanofiber morphology (Fig. 2a), and TEM images confirmed the core-shell structure of individual nanofibers (Fig. 2b), which was further verified by fluorescence microscopy (Fig. 2c and d). The core-shell nanofiber membrane was cut into circular discs (Fig. 2e) and embedded in the GelMA/BG/DEX hydrogel matrix, yielding the composite hydrogel scaffold (Fig. 2f). SEM characterization of the scaffold cross-section revealed a distinct bilayer structure consisting of a nanofiber membrane layer and a porous hydrogel layer (Fig. 2g). To evaluate surface mineralization capacity, the composite hydrogel scaffold was immersed in FBS solution for 7 days. SEM observations showed hydroxyapatite coatings on the surface of each nanofiber (Fig. 2h), indicating that the fibers provided nucleation sites for mineralization within the nanofiber membrane. Meanwhile, hydroxyapatite aggregates were observed in the hydrogel phase (Fig. 2i), confirming the composite hydrogel scaffold's excellent mineralization capability. Besides, the average diameter of core-shell fibers measured 803.49 ± 217.55 nm (Fig. 2j). This core-shell structure allows the incorporation of various factors and demonstrates sustained release.

Fig. 2.

Characterizations of periosteum-bone bilayer scaffold. (a) SEM image of core-shell fiber membranes. (b) TEM image of the core-shell fiber. (c, d) Fluorescence images of a single core-shell fiber. (e) Digital photograph of core-shell fiber membrane. (f) Digital photograph of periosteum-bone bilayer scaffold. (g) SEM image of the interface of the periosteum-bone bilayer scaffold. (h) SEM image of the core-shell fiber membrane after mineralization. (i) SEM image of the composite hydrogel scaffold after mineralization. (j) Fiber diameter of core-shell fibers. The average diameter of core-shell fibers is 803.49 ± 217.55 nm. (k) Results of the sustained release curve of DEX. (l) Results of the sustained release curve of ECd. (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

The inorganic components commonly used in bone biomaterials include BG, β-tricalcium phosphate and hydroxyapatite [50,51]. After these materials are implanted into the bone defect site, a hydroxyapatite coating forms on the bone surface, which can be reabsorbed by the body and integrated with the bone tissue [52]. The formation of bone-induced hydroxyapatite is mainly regulated by the local ion microenvironment. Although pH plays a secondary role, it can still affect this process through a synergistic effect. Firstly, the osteoinductive components are mainly composed of SiO₂, Na₂O, CaO, and P₂O₅. After implantation, ion exchange occurs between the material surface and tissue fluid, significantly increasing local calcium and phosphorus ion concentrations, creating a microenvironment with high calcium and phosphorus ion concentrations, and providing the necessary material basis for the nucleation of hydroxyapatite. At the same time, the Si ions released by the bone-inducing component will form silanol groups upon adsorbing calcium ions, which can serve as nucleation sites for hydroxyapatite deposition, thereby accelerating crystal growth and mineralization. Secondly, in the early stage of bone defect repair, products such as lactic acid produced by inflammatory cells can lead to a slight decrease in local pH. This weakly acidic microenvironment can promote the dissolution of osteoinductive components and further increase local ion concentrations. However, it should be noted that too low pH value will inhibit the nucleation of hydroxyapatite. The DEX loaded in the scaffold can exert anti-inflammatory activity, effectively regulate the intensity of the local inflammatory response, maintain pH stability, prevent interference from excessive accumulation of acidic substances in the mineralization process, and ensure the normal formation of hydroxyapatite.

A variety of polymers have been used in the research and development of bone tissue engineering biomaterials. The core of material screening is to construct a highly bionic functional system with natural bone microenvironment. Existing studies often combine synthetic polymers with natural polymers such as chitosan, gelatin, and sodium alginate to prepare bone repair biomaterials loaded with BG [[53], [54], [55]]. Among them, gelatin, as a natural polymer, has a significant advantage in simulating the microenvironment of the main organic components of endogenous bone. Collagen, the core component of gelatin, can effectively enhance the metabolic activity of osteoblasts, promote osteogenic process, reduce local inflammatory response, help cartilage formation and increase bone density [56]. Compared with pure gelatin, GelMA has a slower degradation rate and better mechanical strength. At the same time, the arginine-glycine-aspartic acid adhesion sequence in its molecular structure can serve as an extracellular matrix recognition site to regulate cell-matrix interactions and effectively promote cell adhesion and colonization [57]. Bioactive factors play a key role in regulating osteogenic differentiation. DEX, as a classic osteogenic inducer, can significantly promote the osteogenic differentiation of human bone marrow mesenchymal stem cells and enhance the osteogenic activity of biomaterials [58]. A large number of studies have confirmed that DEX-loaded scaffold materials have good biocompatibility and efficient osteogenic induction ability [59,60]. Based on this, this study introduced DEX into the composite scaffold system to provide strong support for bone tissue regeneration with its dual effects of anti-inflammation and osteogenesis. In summary, GelMA/BG/DEX composite hydrogel scaffold has become a promising bone tissue engineering repair material due to its advantages of bionic microenvironment construction, excellent mechanical properties and dual biological activity regulation. In summary, GelMA/BG/DEX composite hydrogel scaffold has become a promising bone tissue engineering repair material due to its advantages of bionic microenvironment construction, excellent mechanical properties and dual biological activity regulation.

In order to systematically evaluate the comprehensive performance of the scaffold, five groups of experiments were set up in this study: the control group (Control), the simple GelMA group, the GelMA/BG group (GB), the GelMA/BG/DEX group (GBD), and the GelMA/BG/DEX-GelMA/ECd@PLCL/BG/DEX group (GBDE). The primary focus was on investigating the sustained-release characteristics of DEX and ECd in the GBDE group. The results showed that DEX release followed a typical two-phase mode: the first 48 h were a rapid release stage, followed by a stable, sustained release period (Fig. 2k). The release curve is due to the unique structural design of the composite hydrogel scaffold: the porous structure of GelMA hydrogel has excellent hydration and degradability, which can realize the early rapid release of encapsulated DEX and exert immediate anti-inflammatory effect. The moderate degradation rate of PLCL shell not only ensures the smooth connection between the early rapid release of DEX and the later sustained release, but also achieves the medium-term stable controlled release of ECd. In addition, the PLCL shell can serve as a physical barrier to prevent the sudden release of ECd before scaffold degradation. With the gradual degradation of the shell, ECd was continuously released with the slow decomposition of the GelMA core layer, which was synergistic with the late sustained release of DEX and the continuous ion effect of BG, and finally achieved the integrated regulation of long-term anti-inflammatory, angiogenesis and osteogenesis. This sustained release mode can not only maintain a stable anti-inflammatory effect, but also continuously drive osteogenic cell differentiation. Studies have confirmed that DEX can induce osteogenic differentiation by up-regulating the expression of osteogenic related proteins such as RUNX2 and Osterix [61,62]. These dual regulatory mechanisms of anti-inflammation and osteogenesis further highlight the important application value of DEX in bone tissue engineering.

In contrast, the ECd release curve for the scaffold core layer load was more stable, with release remaining relatively constant after about 3 weeks (Fig. 2l). The three-week sustained release phase is highly consistent with the key window of angiogenesis during bone repair, which can continuously provide bioactive factors and effectively avoid the problems of rapid release and depletion of a single pro-angiogenic factor. Although ECd lacks endogenous osteogenic induction activity, its potent pro-angiogenic effect can provide sufficient oxygen and nutrients for osteoblasts and significantly reduce osteoblast apoptosis. The mature vascular network can accelerate the removal of local metabolic waste, reduce the accumulation of inflammatory factors, and synergize with the anti-inflammatory effect of DEX to further optimize the osteogenic microenvironment during bone repair. At the same time, the enhanced vascular network can promote the local diffusion of BG active ions and DEX, and further improve its osteogenic induction efficiency. The above differentiated sequential release characteristics endow the composite hydrogel scaffold with strong early anti-inflammatory function, and can provide long-term and all-round biological support for bone tissue repair, so as to realize the synergistic regulation of anti-inflammatory, osteogenesis and angiogenesis.

3.2. The impact of composite hydrogel scaffold on cells in vitro

The interaction between hydrogels and cells is a key indicator for evaluating the biocompatibility and functionality of biomaterials. To systematically explore the regulation of different scaffold materials on cell biological behavior, this study extracted extracts from each experimental group and co-cultured them with cells to evaluate cell survival and proliferation. The results of live/dead cell staining showed that cell viability in all experimental groups remained high and was not significantly different between groups (Fig. 3a and b). The CCK-8 assay further verified this conclusion: after 1, 4, and 7 days of culture, there was no statistical difference in cell proliferation between groups (Fig. 3c). The above results confirm that the composite hydrogel scaffold constructed in this study has excellent biocompatibility, which can provide an ideal microenvironment for cell adhesion, growth and proliferation, and lay a solid biological foundation for its subsequent application in the field of bone tissue engineering.

Fig. 3.

Biocompatibility of composite hydrogel scaffolds (a) Calcein-AM/PI (live/dead) staining. (b) Quantitative results of Calcein-AM/PI (live/dead) staining. There are no statistical differences between the groups. (c) CCK-8 results of different composite hydrogel scaffold groups.

To further evaluate the osteogenic efficiency of the composite hydrogel scaffold, a series of osteogenic differentiation characterization experiments was conducted. Alkaline phosphatase (ALP) is a core extracellular enzyme secreted by osteoblasts and a key marker of osteoblast activity. It plays an important role in matrix mineralization and is recognized as a landmark marker of early osteogenic differentiation [63]. The results of ALP staining showed that ALP activity in the GBD and GBDE groups was significantly higher than in the other groups (Fig. 4a and b), confirming that both materials significantly enhance early osteogenic differentiation of cells. Biomineralization is a core evaluation index of late osteogenic differentiation and directly reflects the mineralization and deposition abilities of fully differentiated stem cells. In this study, alizarin red S (ARS) staining was used to evaluate the deposition level of extracellular calcium nodules. The results showed that calcium deposition in the GBDE group was significantly better than in the other groups on the 21st day of culture, indicating superior performance in regulating late osteogenic cell differentiation (Fig. 4a and c). The molecular mechanism underlying the scaffold's osteogenic induction was further explored using qRT-PCR. At 7 days of culture, RUNX2 expression in the composite hydrogel scaffold group was significantly up-regulated. At 14 days of culture, OPN gene expression in this group also reached a peak (Fig. 4d and e). Among them, RUNX2 is the core transcription factor that regulates the expression of key proteins involved in bone and cartilage development. OPN, a highly abundant phosphorylated glycoprotein in bone matrix, is an important marker of late-stage osteogenic differentiation and plays a key role in accelerating bone regeneration and remodeling. The above results fully confirm that the composite hydrogel scaffold effectively promotes osteogenic differentiation of BMSCs at both early and late stages, providing solid experimental support for its clinical application in bone tissue engineering.

Fig. 4.

Detection of osteogenic properties of the cells. (a) ALP and ARS staining pictures. (b) Semi-quantitative analysis of ALP staining. (c) Semi-quantitative analysis of ARS staining. (d, e) Results of qRT-PCR at days 7 and 14. The GBDE group showed the greatest osteogenic effect among the BMSC groups. (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

The local inflammatory response and the immune microenvironment are key regulators of fracture healing and bone regeneration [64]. Macrophages, as core immunoregulatory cells, exhibit significant phenotypic plasticity and can be divided into pro-inflammatory (M1) and anti-inflammatory (M2) subtypes based on functional differences [65]. To explore the regulatory effect of the composite hydrogel scaffold on macrophage polarization, RAW264.7 macrophages were co-cultured with extracts from each material in this study, and the phenotypic transformation of cells was systematically analyzed. Immunofluorescence staining showed that the expression level of iNOS in the GBD and GBDE groups was significantly lower than in other groups (Fig. 5a and Fig. S1). As a characteristic marker of M1 macrophages, the expression level of iNOS is directly related to the pro-inflammatory phenotype of cells. The results were further verified by WB. The expression of iNOS protein in the GBD group and the GBDE group was significantly down-regulated (Fig. 5b and c). The above data confirmed that the DEX loaded into the scaffold can effectively inhibit macrophage polarization from the resting state (M0) to the pro-inflammatory state (M1) and exhibit a significant anti-inflammatory effect. This regulatory mechanism has important biological significance: inhibiting an excessive inflammatory response helps construct an immune microenvironment suitable for bone regeneration. By targeting macrophage polarization, the composite hydrogel scaffold can effectively alleviate local inflammation and further promote tissue repair and regeneration, providing a new strategy to optimize fracture healing.

Fig. 5.

Anti-inflammatory assay of composite hydrogel scaffold. (a) iNOS immunofluorescence staining. (b) WB assay of iNOS. (c) Semi-quantitative analysis of WB results. The anti-inflammatory effects of GBD and GBDE were not statistically different. (∗P < 0.05).

3.3. Composite hydrogel scaffold promotes bone regeneration in vivo

In order to comprehensively evaluate the osteogenic efficacy of the composite hydrogel scaffold in vivo, a rat critical-size skull defect model was constructed in this study. The core-shell fibrous membrane was cut to a size that matched the defect and implanted. The hydrogel was injected into the periphery of the fibrous membrane to fill the bone defect, and cross-linked and solidified by ultraviolet light irradiation for 30 s. The new bone formation was evaluated by micro-CT at 4 and 8 weeks after the operation. Subsequently, the specimens were fixed, decalcified, paraffin-embedded and sectioned. H&E staining, Masson trichrome staining and immunohistochemical detection were used to further analyze the histological morphology and osteogenic-related protein expression, which confirmed that the composite hydrogel scaffold had excellent ability to promote bone regeneration in vivo (Fig. 6a and b). The results of H&E and Masson trichrome staining showed that a thin layer of new bone tissue was formed on the surface of the fibrous membrane in the GBDE group at 4 weeks after the operation. Compared with the GB group, lymphocyte infiltration in the GBDE group was significantly reduced, suggesting that its anti-inflammatory activity was significantly enhanced. At 8 weeks after operation, mature bone tissue was closely attached to the surface of the fiber membrane in the GBDE group, and some new bone tissue grew into the fiber membrane (Fig. 6c and d). The above results show that the core-shell fibrous membrane not only has no bone growth inhibition but also significantly enhances bone regeneration and repair by providing mineralization nucleation sites, releasing osteogenic-related active factors, and promoting the formation of a suitable osteogenic matrix. In summary, the composite hydrogel scaffold exhibits excellent osteogenic performance and good biocompatibility in vivo, and has broad clinical transformation and application prospects in bone defect repair.

Fig. 6.

Micro-CT, H&E, and Masson staining of samples at 4 and 8 weeks. (a, b) Micro-CT imaging and quantitative assessments showed that the GBDE group demonstrated the most effective ability to promote bone regeneration in vivo. (c) H&E staining results. (d) Masson staining result. Mature bone formation was observed in the GBDE group at 8 weeks, and the regenerated bone grew into the core-shell fibrous membrane. (∗P < 0.05, ∗∗∗P < 0.001).

Angiogenesis is the core link of bone regeneration, which can transport nutrients, bioactive factors and osteoblasts for local tissues, promote new bone formation, accelerate tissue repair, and effectively remove metabolic waste and toxic substances [66,67]. CD31 is a specific marker of newly formed endothelial cells and is widely used for quantitative analysis of new microvessels. It is a classic index to evaluate the ability of implant materials to promote angiogenesis [[68], [69], [70]]. The results of immunohistochemical staining for CD31 in this study showed that CD31 expression was significantly increased at 4 and 8 weeks after operation in the GBDE group, and the amount of neovascularization tissue was significantly higher than in other groups (Fig. 7a and b). This pro-angiogenic effect is due to the synergistic effect of GelMA hydrogel matrix, BG and ECd: BG can promote angiogenesis by releasing calcium ions to up-regulate the secretion of vascular-related cytokines and enhance endothelial cell adhesion [71]. Additionally, its silicic acid component can further enhance the pro-angiogenic effect by up-regulating nitric oxide synthase expression [72]. ECd is derived from endothelial cell culture medium and is rich in a variety of pro-angiogenic factors, creating an ideal microenvironment for angiogenesis. In summary, the composite hydrogel scaffold significantly promotes angiogenesis through multi-channel synergy, thereby accelerating bone regeneration. In addition, the expression level of OPN in the GBDE group was the highest, suggesting that the introduction of DEX and the loading of core-shell fibrous membranes could significantly up-regulate the expression of osteogenesis-related proteins. This result was highly consistent with the conclusion that the amount of new bone formation in the GBDE group was the most significant in the micro-CT test (Fig. 7c and d). Collagen type I, as a core component of bone matrix, plays a key role in bone regeneration, and its expression is also significantly up-regulated in the GBDE group (Fig. 7e and f). The above results confirm that the composite hydrogel scaffold can not only efficiently promote angiogenesis but also significantly enhance bone regeneration by up-regulating the expression of osteogenesis-related proteins, providing an integrated, multifunctional solution for bone defect repair.

Fig. 7.

Immunohistochemical staining of the samples. (a, b) CD31 immunohistochemical staining and semi-quantitative analysis. The GBDE group had the highest CD31 expression. (c, d) OPN immunohistochemical staining and semi-quantitative analysis. The GBDE group had the highest OPN expression. (e, f) COL I immunohistochemical staining and semi-quantitative analysis. The GBDE group had the highest COL I expression. (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001).

In order to comprehensively evaluate the biocompatibility of the composite hydrogel scaffold in vivo, the main organs, such as the heart, liver, spleen, lung, and kidney of rats in each group, were taken for histological staining analysis at 8 weeks after operation. The results of H&E staining showed that there was no obvious inflammatory infiltration, tissue degeneration or pathological damage in the main organs of rats in each group, which confirmed that the composite hydrogel scaffold constructed in this study had excellent in vivo biocompatibility and no significant systemic toxicity and organ damage risk (Fig. S2). Good biocompatibility is the core prerequisite for clinical transformation and application of biomaterials. The composite hydrogel scaffold can effectively promote bone regeneration without adverse effects on key organs, further verifying its clinical application potential as a bone defect repair material and laying a key safety foundation for subsequent pre-clinical research and transformation applications.

3.4. RNA transcriptome sequencing

To elucidate the molecular mechanism of bone defect repair in the GBDE group, RNA transcriptome sequencing was performed on bone defect tissue samples from the GBDE and control groups, and differences in gene expression between the two groups were systematically analyzed. To elucidate the molecular mechanism of bone defect repair in the GBDE group, RNA transcriptome sequencing was performed on bone defect tissue samples from the GBDE and control groups, and differences in gene expression between the two groups were systematically analyzed (Fig. 8a). The above DEGs are widely involved in the core signaling pathways of bone regeneration regulation, including: JAK-STAT signaling pathway (Prl6a1, Csf3, Cxcl6 and other genes are up-regulated), BMP signaling pathway (Vstm2a, Sox11 and other genes are up-regulated), WNT signaling pathway (Drd2, Vax2, Hnf1b and other genes are up-regulated) and ERK signaling pathway (Chrna10, Ccl20, Psca and other genes are up-regulated). At the same time, pro-angiogenic genes (Nox1, Drd2, Pla2g2f) were significantly activated, while inflammatory response-related genes (Nr1h4, Lep, Serpinb1b) were significantly inhibited. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis further confirmed that the HIF-1 signaling pathway, PPAR signaling pathway, CAMP signaling pathway, and JAK-STAT signaling pathway were significantly up-regulated in the GBDE group (Fig. 8b). In summary, the GBDE composite hydrogel scaffold can initiate a synergistic biological effect that promotes bone regeneration by simultaneously regulating immune responses, angiogenesis, and osteogenic differentiation-related genes and signaling pathways (Fig. 8d). Among them, the JAK2-STAT pathway forms a precise cross-regulatory network with the BMP and Wnt pathways during bone regeneration: p-STAT can directly up-regulate the transcription of genes such as Vstm2a and Sox11 downstream of the BMP pathway and positively drive the osteogenic process. The BMP-2 secreted upon activation of the BMP pathway can, in turn, enhance JAK2 phosphorylation, forming a positive feedback loop. In addition, the activation of the JAK2-STAT pathway can also up-regulate the expression of Wnt pathway key genes Drd2 and Vax2, strengthen Wnt/β-catenin signaling, and ultimately promote osteoblast proliferation and bone matrix synthesis. To verify the reliability of transcriptome sequencing results, five differentially expressed genes were randomly selected for qPCR validation, and the results were highly consistent with the sequencing data (Fig. S3).

Fig. 8.

Transcriptome sequencing of the samples. (a) The volcano plot depicts the transcriptome results for differentially expressed genes in the GBDE group compared to the control group. (b) KEGG enrichment analysis. (c) Heatmap of clustering between the GBDE group and the control group. (d) Schematic representation of immunogenesis, angiogenesis, and osteogenesis. (e-i) WB verification of the JKA2-STAT pathway and semi-quantitative analysis of the results. (∗P < 0.05).

Further analysis confirmed that the JAK2-STAT signaling pathway plays a central regulatory role in bone regeneration. WB results showed that, compared with the control group, JAK2 protein expression in the GBDE group was significantly up-regulated, and its phosphorylation (p-JAK2) was significantly increased. The total STAT protein expression showed a downward trend, whereas its phosphorylation level (p-STAT) was significantly up-regulated (Fig. 8e–i). The above results indicate that GBDE composite scaffolds can mediate bone regeneration by activating the JAK2-STAT signaling pathway. The molecular mechanism is that the bioactive factors released by the scaffold can up-regulate JAK2 expression and induce its phosphorylation and activation. The activated p-JAK2 further catalyzes the phosphorylation of STAT, initiates the downstream signaling cascade, and finally promotes bone regeneration. The up-regulated expression of CD31, OPN and type I collagen is directly regulated by the upstream and downstream molecular mechanisms of the JAK2-STAT pathway. When the JAK2-STAT pathway is activated, p-STAT translocates into the nucleus as a key transcription factor, specifically binds to the STAT binding elements in the promoter regions of CD31, OPN and type I collagen genes, initiates target gene transcription, and ultimately mediates the up-regulation of the expression of the above osteogenesis and angiogenesis-related proteins.

Compared with the single component scaffold, the periosteum-bone composite scaffold constructed in this study accurately mimics the natural layered structure of periosteum and bone tissue. Among them, the periosteal biomimetic layer can provide osteogenic active factors and induce the formation of functional vascular networks, while the bone matrix biomimetic layer provides stable mechanical support and suitable mineralized microenvironment for bone regeneration. The bionic design effectively overcomes the functional limitations of a single structural scaffold, and the synergistic effect among multiple components can compensate for the inherent defects of a single component. For example, scaffolds loaded with BG alone lack anti-inflammatory and pro-angiogenic functions, while scaffolds loaded with ECd alone are difficult to achieve synergistic regulation of the inflammatory response and osteogenic process. Based on the sequential degradation characteristics of core-shell structure and materials, this scaffold realizes the sequential synergistic effect of DEX, ECd and BG, which can accurately release the corresponding active ingredients according to the different stages of bone repair, and provide continuous and targeted biological support for the whole cycle of bone regeneration.

The periosteum-bone composite scaffold exhibits sequential and staged precise regulation function in the process of bone repair, and its mechanism runs through the whole cycle of bone regeneration. In the early stage of inflammation regulation, the rapidly degrading GelMA hydrogel was the first to release DEX, which effectively inhibited macrophage polarization to the M1 pro-inflammatory phenotype and reduced the secretion of pro-inflammatory factors. At the same time, calcium and silicon ions released by BG synergistically exert anti-inflammatory effects and jointly construct an immune microenvironment with low inflammatory levels, laying an ideal foundation for subsequent angiogenesis and osteogenic differentiation. During the angiogenesis stage, the progressive degradation of the PLCL shell enables long-term, sustained release of ECd. The active factors, such as VEGF and bFGF, are enriched in ECd and can activate the PI3K-AKT signaling pathway in vascular endothelial cells, thereby initiating endothelial cell migration and lumen formation. Among them, calcium ions released by BG enhance endothelial cell adhesion, and silicon ions promote vasodilation by up-regulating nitric oxide synthase expression. The three cooperate to construct a mature, stable vascular network that delivers sufficient oxygen and nutrients to osteoblasts. During osteogenic differentiation and matrix mineralization, sustained DEX release activates the JAK2-STAT signaling pathway, strongly drives osteogenic differentiation, and promotes the directional differentiation of bone marrow mesenchymal stem cells into osteoblasts. The calcium and phosphorus ions continuously released by BG serve as the core raw materials for hydroxyapatite deposition and accelerate the mineralization and maturation of the bone matrix. At the same time, the periosteal biomimetic layer of the scaffold provides specific adhesion sites for osteoblasts to guide the orderly growth of new bone from the defect surface to the inside. The scaffold realizes the significant up-regulation of angiogenesis and osteogenesis-related genes and the effective down-regulation of inflammation-related genes through the multi-link synergistic integration of immune regulation, angiogenesis and osteogenic differentiation, and completes the sequential precise regulation of the whole cycle of bone repair, and finally realizes the efficient repair of bone defects. In summary, the GBDE group significantly accelerated bone defect regeneration by targeting angiogenesis and osteogenesis-related gene expression and by inhibiting inflammatory genes. The above research results provide a key experimental basis for elucidating the molecular regulation mechanism of composite hydrogel scaffolds in bone tissue engineering.

4. Conclusions

In this study, a composite bone repair scaffold material of GelMA/ECd@PLCL/BG/DEX core-shell fiber membranes combined with GelMA/BG/DEX hydrogel was successfully constructed. Relying on the synergistic regulation effect of multiple components, the scaffold exhibits excellent anti-inflammatory, osteogenic and pro-angiogenic biological properties. Among them, BG can continuously release active ions such as calcium, phosphorus and silicon in the physiological microenvironment, effectively induce the formation of mineralized crystals and hydroxyapatite deposition on the bone surface and lay a core material foundation for bone mineralization and bone regeneration. DEX can accurately optimize the local microenvironment for bone repair by inhibiting excessive inflammation and positively regulating osteogenic differentiation. ECd efficiently mediates angiogenesis, provides sufficient nutrition for bone regeneration, and accelerates the repair process of bone defects. The results of in vivo animal experiments confirmed that the composite hydrogel scaffold could significantly promote the formation of new bone and vascular tissue, and up-regulate the expression of key functional factors such as CD31, OPN, and type I collagen. Transcriptome sequencing analysis further revealed that the molecular mechanism of the above-mentioned bone regeneration effect was closely related to the activation of the JAK2-STAT signaling pathway. In summary, the multifunctional composite hydrogel scaffold developed in this study shows great application potential in the repair and treatment of bone defects due to its excellent biocompatibility, sequential biological activity release and coordinated regulation of full-cycle bone regeneration. It provides a new strategy for bone tissue engineering and complex tissue regeneration and repair, and has good clinical transformation and application prospects.

CRediT authorship contribution statement

Chenghao Yu: Data curation, Formal analysis, Investigation, Methodology, Software, Writing – original draft. Yuanfei Wang: Formal analysis, Funding acquisition, Investigation, Writing – review & editing. Lei Xie: Investigation, Methodology. Qiuping He: Formal analysis, Methodology. Tengbo Yu: Conceptualization, Resources, Supervision. Tong Wu: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.