As GelMA is a partially synthetic polymer derived from gelatin, the hydrolyzed form of type I collagen found in bone, we anticipate that utilizing this polymer to synthesize nanocomposite biomaterials for bone repair and regeneration could yield significant advantages. The synthesis of GelMA offers several benefits, such as enabling control over reproducibility, degree of methacryloyl substitution, and consequently, the initial mechanical properties of GelMA hydrogel [21]. Firstly, we observed the injectable properties of the synthesized GelMA and the composite hydrogel, which can be passed through a syringe needle and fill irregular defects (Fig. S1a-b). The rheometer results demonstrated that a range of composite hydrogels transitioned from liquid to gel in UV light irradiation (Fig. S1c-d). The storage modulus (G’) increased with incorporating BG (Fig. S1e). The successful synthesis of GelMA enabled observation of its porous structure using SEM imaging techniques (Fig. S2a). The average pore size of GelMA was 110 ± 27 μm (Fig. S2b). The pore sizes of different groups of hydrogels were also measured, and found that as the BG component content increased, the pore size of the hydrogel became smaller. Besides, the pore sizes of the hydrogel can be maintained by adding the PCL@GelMA coaxial fibers (Fig. S2c). Maintaining pore size provides an environmental basis for cell ingrowth and nutrient exchange, which is benefite for bone regeneration. Confirmation of gelatin modification with MA was achieved through NMR analysis (Fig. S2d) [22]. A comparative examination of gelatin and GelMA revealed a distinct double-bonded proton peak (= CH2) in the NMR spectrum of GelMA, which appeared at approximately 5.5 ppm. An additional minor peak at 5.85 ppm can be attributed to the acrylic protons originating from MA groups [23]. Meanwhile, the disappearance of the peak associated with lysine-NH2 (2.8 ppm) indicated the predominant grafting of MA onto lysine-NH2 groups within the gelatin backbone during the formation of GelMA [24]. Additionally, the new peaks at 5.4 and 5.6 ppm indicated the successful binding of the methacrylate groups to gelatin. The degree of methacrylation of GelMA was calculated to be ≈ 71.89 ± 0.33% as determined by the ratio of the integrated area of the lysine methylene signals (2.8-3.0 ppm) of GelMA and the phenylalanine signal (7.1–7.4 ppm) of unmodified gelatin [25].

PCL@GelMA coaxial nanofibers were fabricated successfully and observed by SEM and TEM (Fig. S3a). The inner layer (PCL) and outer layer (GelMA) can be seen in the TEM images. By measuring the SEM image, the diameter of the coaxial fiber was 879 ± 207 nm (Fig. S3b). The application of FT-IR revealed that the collagen peak (1645.12) remained after the ethanol production process, which contributed to the successful fabrication of coaxial fibers (Fig. S3c). Consequently, GelMA hydrogel was augmented with coaxial fibers to fabricate a fiber-reinforced hydrogel system. By employing GelMA as the outer layer of the coaxial fibers, it is anticipated that superior adhesion between the outer layer and the surrounding GelMA hydrogel will be achieved during the UV crosslinking process, which will increase the overall strength through the formation of an interpenetrating network.

The GelMA/BG-Fiber composite hydrogels, containing varying concentrations of BG (0, 1, 5, 10, 20% w/v), with or without PCL@GelMA coaxial nanofibers, were successfully synthesized and can be crosslinked using UV light. Figure 1 shows that the short coaxial nanofibers with varying concentrations of BG were successfully incorporated while preserving the porous structure of the hydrogel. The porous structure of hydrogels is essential for cell growth and angiogenesis [26]. The composite hydrogels exhibited robust swelling properties due to their highly interconnected porous structure. The swelling of hydrogels plays a pivotal role in the healing and regeneration of bone tissue as it facilitates the transportation of nutrients and the removal of waste products through diffusion [21]. The incorporation of BG significantly influences the swelling behavior of the hydrogels. The swelling characteristics of the hydrogels decreased with the increase in BG content, which may be attributed to the electrostatic interaction between the negatively charged silicon hydroxyl (–Si–OH) groups on the surface of BG and the positively charged amino (–NH2) groups in GelMA [9], whereas the incorporation of coaxial fibers had no significant effect (Fig. 2a-b). The hydration rate of the composite hydrogel with 20% BG-Fiber was 72%, which was lower than that of GelMA (89%) (Fig. 2c). The results demonstrate that the composite hydrogels exhibit favorable swelling ratios, which indicate their potential for efficient nutrient diffusion when transplanted into the body for bone regeneration.

The SEM images of composite hydrogels. (a) Different BG contents (0, 1, 5, 10, 20% v/w) were added to GelMA. (b) Different contents (0, 1, 5, 10, 20% v/w) of BG and PCL@GelMA coaxial nanofibers added in GelMA

The effect of BG and coaxial fibers on composite hydrogels’ hydration degree, mechanical, and physiological stability. (a-c) The swelling rate and hydration degree were weakened by adding BG and coaxial fibers. (d) The mechanical property was enhanced by adding BG and coaxial fibers. (e-f) Degradation behaviors of hydrogels in SBF showed slow weight loss after 28 days soaking. (g-h) Degradation behaviors of hydrogels in collagenase solution decreased the degradation rate by adding BG and coaxial fibers. (*p < 0.05, **p < 0.01, ***p < 0.001)

The mechanical properties of the hydrogel were significantly enhanced by incorporating PCL@GelMA coaxial nanofibers and BG. The GelMA shell of the coaxial fiber exhibited a remarkable affinity with the surrounding GelMA hydrogel, facilitating crosslinking under UV light irradiation and thereby augmenting the overall mechanical characteristics. During compression performance testing, the composite hydrogel containing PCL@GelMA coaxial nanofibers exhibited rupture at a compression level of 65%. Remarkably, upon completion of the test, the composite hydrogel fully recovered its initial appearance, whereas the absence of PCL@GelMA coaxial nanofibers in the hydrogel resulted in irreversible fragmentation (Fig. S4a). The 2% concentration of PCL@GelMA coaxial nanofibers of the composite hydrogel, solely supplemented with PCL@GelMA coaxial nanofibers, exhibited a significantly higher value compared to the content of 0% and 1%, while no significant difference was observed in comparison to the 3% content (Fig. S4b). This result was similar to that of Qiu [20], and therefore, 2% PCL@GelMA coaxial nanofibers were used in the subsequent experiments. The incorporation of PCL@GelMA coaxial nanofibers not only enhances the mechanical properties of the hydrogel but also maintains the hydrogel shape. The hydrogel exhibited a simultaneous increase in ultimate stress and compressive modulus with the addition of BG (Fig. 2d and Fig. S5a-b). The GelMA-containing BG hydrogels exhibited rupture at approximately 60% compression, whereas the composite hydrogel containing PCL@GelMA coaxial nanofibers exhibited rupture at a compression level of 65%. Notably, hydrogels with a BG content of 20% exhibited irrecoverable deformation when subjected to flat compression. Mechanical properties play a crucial role in the development of bone regeneration materials. Simple hydrogels possess limited mechanical strength and, therefore, necessitate the incorporation of PCL@GelMA coaxial nanofibers and BG to enhance the overall robustness. After incorporating these constituents, composite hydrogels retain their injectability and exhibit the potential for mending irregular bone defects.

The degradation properties of composite hydrogels are critical, and the optimal degradation scenario is the synchronization of hydrogel degradation and bone growth [27]. The degradation of composite hydrogels was assessed during a 28-day incubation period in SBF, approximating the duration required for bone healing and remodeling following fracture [21]. In SBF solution, the composite hydrogels containing 0%, 1%, and 5% BG exhibited significant degradation within two weeks and complete degradation by 4 weeks, while the hydrogels with 10% and 20% BG content demonstrated partial degradation (Fig. 2e-f). Collagenase-mediated degradation was rapid and complete within 24 h for hydrogels with 0%, 1%, and 5% BG content. Hydrogels containing 20% BG also exhibited significant degradation, reaching approximately 63% and 43% (fiber-containing) after 7 days (Fig. 2g-h). The addition of BG effectively slowed down the degradation process, possibly due to the strong interaction between BG and GelMA hindering the penetration of the collagenase solution. However, incorporating coaxial fibers did not significantly hinder the degradation, possibly due to the simultaneous degradation of the GelMA component in the shell and the hydrogel. The weight of the composite hydrogels exhibited fluctuations during the degradation measurements, which could be attributed to the hydrogel degradation along with the deposition of phosphates, which led to an increase in the weight of the sample. It can be inferred that the observed mass change is primarily attributed to the initial dissolution of the BG and the subsequent formation of mineralized crystals [28].

For the phosphate formation ability test, the composite hydrogels of each group were immersed in SBF for 7 days. Subsequently, the collected sample sections were examined using SEM. Particles were observed on the surface of the composite hydrogels, and EDS analysis revealed that these particles were enriched in calcium (Ca) and phosphorus (P) (Fig. S6a-b), which are the main components of bones. The composite hydrogel’s Ca/P ratio of the surface elements was 1.56 ± 0.03, which resembles that in artificial bone (ranging from 1.50 to 1.67) [29]. The deposition of phosphate crystals on the surface gradually increases with increasing BG content (Fig. S6c), which could also be seen on the surface of the fibers (Fig. 3). In the presence of SBF, the composite hydrogels exhibited rapid release of calcium and phosphorus ions, forming calcium phosphate crystals on their surface [30]. Incorporating coaxial fibers also facilitates the nucleation and growth of phosphate crystals, enhancing their crystallization capacity [31]. In conclusion, incorporating BG and coaxial fibers significantly mitigated swelling and hydration rate, decelerated degradation kinetics, and augmented mechanical properties and mineralization formation ability of the composite hydrogels.

The optical pictures and SEM images of composite hydrogels soaked in SBF for 7 days. (a) The optical pictures of different groups. (b) The formation of mineralized crystals on the surface increased with increased BG content. (c) Mineralized crystals form on the surface of the fibers

The inorganic components in bone biomaterials research include BG, β-tricalcium phosphate, and hydroxyapatite [32,33,34]. After implantation into damaged bones, BG is reabsorbed and integrated with the bone by forming a layer of apatite on its surface [35]. Although various polymers have been utilized in developing bone biomaterials, selecting polymers that closely emulate the natural bone microenvironment is imperative. Several studies have utilized both synthetic and natural polymers, such as chitosan, gelatin, and sodium alginate, to fabricate bone biomaterials containing BG [36,37,38,39]. The selection of gelatin, a natural polymer, is highly advantageous for establishing an environment that closely emulates the primary organic constituents of endogenous bones due to its hydrolyzed form derived from type I collagen present in the skeletal system. Collagen can enhance the metabolic activity of osteoblasts, thereby promoting osteogenesis, suppressing inflammation, inducing chondrogenesis, and improving bone mineral density [40]. Compared to gelatin, GelMA exhibited the primary advantage of a slower degradation rate and enhanced mechanical strength. In addition, the interaction between cells and the ECM is regulated by arginine-glycine-aspartic acid sequences within the GelMA structure, which serve as recognition sites for adhesive proteins that facilitate cell adhesion [41]. In summary, GelMA/BG-Fiber composite hydrogel is an excellent biomaterial that can effectively mimic the natural biological microenvironment of bone and has the potential to promote bone repair and regeneration.

The interaction between cells and hydrogels is essential. Upon examination of the cells adhered to the surface of the composite hydrogels, it was observed that they exhibited strong adhesion, with even crystalline formations emerging on their surfaces during the sustained release of ions from the BG (Fig. 4a). This phenomenon became more pronounced with increasing BG content. We initially investigated the effect of fiber supplementation on cell proliferation and observed no statistically significant differences (Fig. 4d). The biocompatibility of different composite hydrogels was assessed using Calcein-AM/PI (live/dead) staining. It was observed that an increase in the content of BG resulted in a higher number of dead cells, and 83% of the cells survived in the 20% BG of composite hydrogel in the extraction solution (Fig. 4b-c). We attribute these results to crystal growth. The incorporation of BG has been shown to release a significant quantity of ions, resulting in an initial alkaline pH in the solution, which subsequently affects cell proliferation [42]. The growth and proliferation of stem cells are also influenced by the content of BG in solution, with cell growth being stimulated only at an appropriate concentration [43]. Therefore, in this experiment, we hypothesize that the development of crystals, changes in solution pH, and BG content in the solution exert an inhibitory effect on cell proliferation.

The evaluation of biocompatibility of composite hydrogels. (a) SEM images of MC3T3-E1 on composite hydrogels at day 3. (b) Calcein-AM/PI (live/dead) staining of different composite hydrogels. (c) Results of Calcein-AM/PI (live/dead) staining by counting the number of live cells. (d) The CCK8 assay showed that the cell growth was inhibited with the increase in BG. (*p < 0.05, ***p < 0.001)

Angiogenesis and osteogenesis are essential for bone regeneration in tissue engineering scaffolds. The impact of hydrogels with varying BG contents on HUVEC tube formation assays was assessed. The composite hydrogel containing 20% BG exhibited the most extensive tube network formation, continuous tube walls, and the most significant number of meeting points, segments, and branches (Fig. 5a-d). Figure 5e shows cell migration images of HUVECs after culturing with the extraction solution of the composite hydrogels for 24 and 48 h. The corresponding quantitative migration areas filled by HUVECs are shown in Fig. 5f-g. The composite hydrogel containing 20% BG exhibited larger migration areas filled by HUVECs than the other groups. To assess the differentiating effect of hydrogels on cells, we isolated and characterized BMSCs based on their multipotent differentiation potential and cell surface-specific markers (Fig. S7) and then cultured BMSCs in the extraction solution of composite hydrogels. By evaluating the alizarin red staining of BMSCs cultured for 14 days, it was observed that the area of positive staining was significantly increased in the composite hydrogels containing 10% (9.7%) and 20% BG (12.8%) (Fig. 6a-b). Quantitative analysis of ALP showed that at 7 days, ALP expression was higher in the composite hydrogel containing 1% BG (0.24 U/L), while at 14 days, ALP expression was significantly increased in the composite hydrogel containing 20% BG (0.27 U/L) (Fig. 6c). Regarding gene expression analyzed by qPCR, the inclusion of 5%, 10%, and 20% BG hydrogels significantly enhanced the expression of OPN when BMSCs were cultured for 7 days. Additionally, after 14 days, incorporating a 20% BG composite hydrogel increased the expression levels of ALP (9.1 times), RUNX2 (2.7 times), and OPN (2.3 times) in BMSCs (Fig. 6e-f). ALP is an early indicator of osteogenesis and closely correlates with the biomineralization process. RUNX2 and OPN are crucial ECM proteins that serve dual functions, playing a pivotal role in the survival of osteoclasts as well as bone mineralization [44]. The hydrogels containing 20% BG exhibited the highest capacity for mineralized crystal formation, which may transiently impede cell proliferation but ultimately promote osteogenic differentiation. Consequently, this particular group was chosen for in vivo experimentation. Therefore, adding bioglass releases various ions, such as calcium, phosphorus, and silicon ions, and these can produce phosphate crystals when in contact with body fluids. Such crystals have a high hardness, which can constitute both the bone mechanism and the signal for osteogenic differentiation that can be generated for stem cells, promoting the expression of osteogenesis-related genes such as ALP, RUNX2, OPN, which is conducive to the differentiation of cells to osteoblasts.

Effect of scaffolds on tube formation of angiogenesis. (a) Tube formation images of HUVECs. (b) Number of junctions, (c) total branching length, and (d) total segment length per field of view. (e) The images of HUVECs cultures for 24 and 48 h and (f, g) quantitative statistics of cell migration rates in cell scratch assay (n = 3). (*p < 0.05, **p < 0.01, ***p < 0.001)

The osteogenic differentiation detected by alizarin red staining, ALP quantification, and qPCR. (a-b) Alizarin red staining after cultured BMSCs with the composite hydrogel extraction solution for 14 days. The 10% and 20% BG composite hydrogels can significantly increase the positive staining area. (c) Quantitative analysis of ALP shows that the expression of ALP in the composite hydrogel with 1% BG content was higher at 7 days, while the composite hydrogel with 20% BG content significantly promoted ALP expression at 14 days. (d-e) qPCR results detecting the osteogenic gene (ALP, RUNX2, and OPN) expression of BMSCs cultured with different composite hydrogels. (*p < 0.05, ***p < 0.001)

To evaluate the osteogenic potential of the composite hydrogels in vivo, we initially implanted the lyophilized hydrogels into the subcutaneous tissue of rats. Subsequently, we obtained samples for Micro-CT analysis after 2 and 4 weeks. Compared to hydrogels without fibers, composite hydrogels containing fibers showed significantly enhanced mineralization after 2 and 4 weeks in vivo (Fig. S8). The fibers may offer additional attachment sites to form mineralized crystals, promoting crystal nucleation and subsequent crystal growth. Subsequently, a rat skull defect model was established, and the composite hydrogel was injected into the defect site, followed by UV light curing for 10 s. The samples were collected and analyzed by Micro-CT at 4, 8, and 12 weeks. Subsequently, the samples were decalcified, sectioned, and stained with H&E, Masson’s trichrome stain, and immunohistochemical staining (Fig. 7a). The 20% BG composite hydrogel demonstrated significant osteogenesis in Micro-CT detection compared to the other groups at all three-time points (Fig. 7b). Moreover, although the presence of coaxial fibers did not exert a significant effect on bone regeneration, it was observed that the bone tended to be located more centrally within the skull defect. The quantitative bone volume/tissue volume (BV/TV) data was computed based on the 3D Micro-CT data. At 12 weeks, the 20% BG composite hydrogels repaired approximately 25% and 23% of the bone defects, which was much higher than the control group (10%) (Fig. 7c). This finding suggests that fibers may serve as nucleation sites for crystal formation, facilitating and enhancing bone regeneration. Currently, the prominence of 3D printing technology is driving advancements in bone formation, but it is challenging to apply to repair irregular defects [45, 46]. The strength of our study lies in its ability to cater to diverse application scenarios involving irregular defects while being easily implementable.

The animal experiment patterns and analysis of Micro-CT results. (a) The skull defect model of 6 mm diameter was established, and the samples were analyzed at 4, 8, and 12 weeks. (b-c) Micro-CT results showed that adding BG and coaxial fiber induced more bone regeneration. (*p < 0.05, **p < 0.01, ***p < 0.001)

The results of H&E and Masson’s trichrome staining of each group are presented in Fig. 8. The GelMA group was still not completely degraded at 4 weeks. However, upon the addition of BG, a substantial formation of blood tissue and bone-like matrix was observed, accompanied by an enhanced infiltration of lymphocytes. At 8 weeks, the BG group exhibited more multinucleated osteoclasts, while lymphocytes remained abundant. By 12 weeks, lymphocyte levels started to decline, accompanied by an increase in the quantity and size of the osteoid matrix and a significant reduction in the osteoclast population. As shown in Fig. 9a-b, hydrogels (including BG and coaxial fibers) dramatically increased the expression of OPN in immunohistochemical staining at all periods. Especially at 12 weeks, OPN expression in the GelMA-PCL@GelMA-BG group was 5 times higher than in the control group. CD31, a marker of nascent endothelial cells, is commonly employed for neonatal microvessel quantification to assess the angiogenesis of implanted materials [47, 48]. In immunohistochemical staining for CD31, the GelMA-PCL@GelMA-BG group had more CD31 expression and more vascular tissue production at 8 and 12 weeks (Fig. 9c-d). The observed results may be attributed to the synergistic interaction between the hydrogel matrix and the particles released from BG. In the GelMA-BG group, a substantial amount of CD31-labeled cavity formation was observed, which was infrequent in the GelMA-PCL@GelMA-BG group. Consequently, these findings suggest that including coaxial fibers effectively restricts cavity formation. It has been demonstrated that silicates can upregulate nitric oxide synthase, thereby inducing angiogenesis [49]. Calcium ions also facilitate angiogenesis, thereby augmenting the secretion of vascular-related cytokines and promoting endothelial cell adhesion [50]. BG, which releases calcium ions, may facilitate this process. Moreover, blood vessels can deliver nutrients, bioactive factors, and osteogenesis-related cells to promote new bone formation and transport metabolic waste or toxic products for accelerated repair. In conclusion, the novel advantages of the GelMA/BG-Fiber composite hydrogel enhanced biocompatibility and improved osteogenic and angiogenic marker expression, which are crucial for promoting bone regeneration.

H&E and Masson’s trichrome staining of the samples. (a-b) Adding BG and coaxial fibers promoted the early increase of osteoclasts and the formation of a bone-like matrix. The yellow arrows indicate the formation of a bone-like matrix and bone

Immunohistochemical staining of the samples. (a-b) Immunohistochemical staining of osteoblastic marker (OPN) shows that the addition of BG and coaxial fibers promotes the expression of OPN. (c-d) Immunohistochemical staining of angiogenic marker (CD 31) shows that adding BG and coaxial fibers promotes the expression of CD 31 at 8 and 12 weeks. (*p < 0.05, **p < 0.01, ***p < 0.001)

Notwithstanding the preliminary findings of the present study, certain limitations were identified. For instance, the composite hydrogel displays inadequate strength, rendering it incapable of providing adequate support for the bone defect at the weight-bearing site. Consequently, it can only be employed as an auxiliary measure subsequent to internal fixation. Secondly, the hydrogel is of low viscosity, which carries the risk of detachment from the bone defect site. It is our intention to further improve the hydrogel composite in order to facilitate osteogenesis and vascularisation, which will be more pertinent to the context of bulk bone defect surgery.