The freeze-dried prepolymers of GelMA and HAMA (Mw = 12–14 kDa, StemEasy, China) were fully dissolved in PBS (pH = 7.4) containing NHS and EDC under magnetic stirring at 50°C with the final concentrations of the polymers being 10% and 2% (w/v), respectively, to create the GelMA/HAMA double network hydrogels (O’Connell et al., 2016; Fan et al., 2020). Added photoinitiator 2-hydroxy-4’-(2-hydroxyethoxy)- 2-methylpropiophenone (1% w/v), in an addition reaction with photocrosslinkable OGP (MA-YGFGG, Qiangyao Company, China), GelMA/HAMA-OGP were photocrosslinked to create stable co-crosslinked hydrogels, with a final concentration of 10^–13 M. Finally, the solution is cured for 30 s to construct hybrid crosslinking network hydrogel by being exposed to UV light.

Gelation was shown using a tabletop technique. Simply put, the physical state of a hydrogel sample in a vial is tested by turning the vial, replicating the effect of injecting the hydrogel into the body in situ at physiological temperatures.

In order to evaluate the shape plasticity of the hydrogel, we observed whether the hydrogel could be solidified into the desired shape by using different shapes of molds.

To evaluate the injectability of the hydrogel, the double network hydrogel was added to a syringe and extruded through an 18-gauge needle (φ ≈ 1.20 mm) to observe the state of extrusion.

And, the gelation time was determined by the inversion method. The initial time point when the hydrogel was flowless in the inverted bottle at room temperature was recorded in triplicate.

The porosity of the hydrogels was determined by the ethanol displacement method. The initial weight W₀ and initial volume V₀ of the samples were recorded. The samples were soaked in anhydrous ethanol for 5 min. After removal, the residual ethanol was wiped from the sample surface and the weight W₁ was recorded. The density of ethanol was ρ. The porosity was calculated as: p o r o s i t y   % = W 1 − W 0   /   ρ V 0 × 100 %   n = 3 .

The scanning electron microscopy (SEM, Hitachi TM4000Plus, Japan) was used to examine the morphology of the GelMA/HAMA and GelMA/HAMA-OGP hydrogel scaffolds with a 15 kV acceleration voltage. The samples were lyophilized for SEM after being dehydrated in gradient ethanol and treated with 2.5% glutaraldehyde. Before SEM examination, platinum was sputter-coated onto the scaffold’s surface. There were three samples in each group. Five images were chosen from each sample at random.

Electrospray ionization mass spectrometry (ESI-MS) and high-performance liquid chromatography (HPLC) were used to analyze the OGP-MA.

GelMA, GelMA-OGP, HAMA, HAMA-OGP were characterized by Fourier transform infrared spectroscopy (FTIR, Nicolet iS10, United States). The hydrogels were first frozen at −80°C overnight, lyophilized, crushed and mixed with potassium bromide. The samples were then made into discs using a tablet press. Crushed samples ranging from 400 to 4,000 cm⁻¹ were used for analysis with a resolution of 4 cm⁻¹. 32 scans of the sample were performed for each FTIR measurement and the spectra obtained were the average of all scans.

A rheometer (MCR92, Anton Par) was used to investigate the hydrogels’ rheological characteristics to observe the change in viscosity with temperature, shear force and frequency respectively. Shear rates spanning 0.1–100 s⁻¹ were used to examine the viscosity properties. The temperature sweep test measured viscosity at temperatures ranging from 10 to 60°C. The fluctuation of storage modulus (G′) and loss modulus (G″) with frequency (ω; rad/s) allowed for the observation of frequency sweep.

To study the swelling rate of GelMA/HAMA and GelMA/HAMA-OGP hydrogels, they were weighed after freeze-drying and marked with an initial weight of W₀. The dried hydrogel samples were then immersed in PBS at room temperature for 0.5, 1, 2, 3, 4, 5, 6, 7 and 8 h. The mass of the wet hydrogel was recorded at each time point as W_t. Swelling Ratio (SR) was calculated as: SR=(W _t -W ₀ )/W ₀ × 100%.

To evaluate the degradation rate of GelMA/HAMA and GelMA/HAMA-OGP hydrogels, they were weighed after freeze-drying and labeled with an initial weight of W₀. The freeze-dried samples were immersed in PBS containing 2.6 U/ml collagenases at 37°C in a humidified 5% CO₂ atmosphere for 1, 2, 3, 5 and 7 days, then freeze-dried, weighed, and labeled with a weight of W_t. Degradation Ratio (DR) was calculated as: DR=(W ₀ -W _t )/W ₀ × 100%.

To plot the release profile of OGP in the hydrogel materials, we collected soaked supernatants of GelMA/HAMA-OGP hydrogels on 1, 3, 5, 7, and 14 days. The BCA protein concentration assay kit (Solarbio, China) was used to determine total protein concentrations.

The commercially available pre-osteoblast cell line MC3T3-E1 (National Infrastructure of Cell-line Resource, China) was cultured in MEM Alpha basic medium (α-MEM, Gibco, United States) containing 10% FBS (Gibco, United States) and 1% penicillin and streptomycin (Gibco, United States) at 37°C and 5% CO₂.

The Cell Counting Kit-8 (CCK-8, Solarbio, China) assays was applied for cell viability. The hydrogels were cured in 48-well plates, sterilized with 75% ethanol for 30 min, then UV irradiated for 1 h, and then washed with PBS for 5 min with three changes of PBS. Then the MC3T3-E1s cells were seeded onto hydrogel surface with a density of 2×10³ cells/200 μL per well. After culture for 1, 3, 5, and 7 days, a 200 μL media containing 20 μL of the CCK-8 staining solution was added to each well. The plates were incubated for 2 hours at 37°C in an incubator with 5% CO₂ humidity (Thermo Fisher Scientific, United States). Then, a microplate spectrometer (Tecan, Switzerland) was used to detect the absorbance intensity at 450 nm.

Live/Dead staining was carried out using Live/Dead Cell test kit (Beyotime, China). Firstly, MC3T3-E1s suspension with a density of 1×10³ was inoculated into 24-well tissue culture plates and incubated for 24 h. Cells were put to each well 72 h and stained with caledrin AM/PI. To distinguish between living and dead cells, add 250 μL of the cell detection reagent working solution to each well. 30 min incubation at 37°C protected from light was performed. Images were then taken with an inverted fluorescence microscope (Nikon, Japan).

In addition, cellular adhesion to the hydrogel composites was also evaluated. 1 × 10⁴ cells per well were inoculated on GelMA/HAMA and GelMA/HAMA-OGP hydrogels in 24-well plates. The actin protein was stained with FITC-Phalloidin (Beyotime, China) for 20 min and the nuclei were re-stained with 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, China) for 3 min. The morphology and adhesion of attached cells were examined under fluorescence microscopy.

The MC3T3-E1s cells were seeded into 6-well plates with a density of 1×10⁵ cells per well. When cell confluence reached 70%–80%, the complete medium was replaced with osteogenic differentiation medium. Osteogenic induction medium contained α-MEM, 10% FBS, 1% penicillin/streptomycin, 10 nM dexamethasone (Sigma-Aldrich, United States), 10 mM β-glycerophosphate (Sigma-Aldrich, United States), and 0.2 mM ascorbic acid (Sigma-Aldrich, United States). The GelMA/HAMA and GelMA/HAMA-OGP hydrogel scaffolds were soaked in osteogenic medium for 48 h to obtain extracts, which was then used to further incubate the cells for an additional 48 h.

For the ALP activity assay, cells were washed with PBS after 7 days of osteogenic induction, followed by lysis of cells and collection of cell lysates. The micro-BCA protein assay kit (Beyotime, China) was used to measure the total intracellular protein content before adjusting for ALP activity. The next step was to determine the ALP activity using an alkaline phosphatase test kit (Beyotime, China) in accordance with the manufacturer’s instructions. The absorbance readings were calculated using a microplate spectrometer and measured at 405 nm.

BCIP/NBT alkaline phosphatase staining kit (Beyotime, China) was used in accordance with the manufacturer’s instructions to carry out the ALP staining. Briefly, 6-well plates of MC3T3-E1 induced for 7 days were fixed with 95% ethanol, flushed with PBS buffer, and treated with 500 μL dye. The cells were then washed with PBS and imaged using an Olympus microscope and an EPSON scanner.

For Alizarin Red S (ARS) staining, cells were preserved with 95% ethanol for 15 min after 14 days of osteogenic induction. Cells were then gently rinsed three times with PBS buffer and stained for 5 min with 1% Alizarin Red S (pH = 4.2, Solarbio, China). Finally, the unbound dye was gently washed off the cell surface with PBS buffer. Under an inverted microscope, the cells were examined for orange-red mineralized nodules and documented. Subsequently, 10% cetylpyridinium chloride (CPC, Solarbio, China), which dissolves alizarin red in the presence of calcium deposition, was added to the alizarin red-stained culture plates. The optical density was measured at 562 nm after the supernatant had been collected.

We employed RT-PCR to measure the expression of genes linked to osteogenesis, including RUNX family transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteoblast-specific genes (Osterix), as well as osteocalcin (OCN) and osteopontin (OPN). Total RNA from the cells was extracted using the Mini BEST universal RNA Extraction Kit (TaKaRa, Japan) after 7 and 14 days of osteogenic induction, and concentrations were determined. The Prime Script RT kit (TaKaRa, Japan) was used for reverse transcription. By using TB Green Premix Ex Taq IIRT-PCR (TaKaRa, Japan), the cDNA template was amplified. As an internal reference control, GAPDH was utilized. Table 1 displays the primer sequences utilized for PCR amplification. A Light Cycler/Light Cycler 480 system from Roche (Switzerland) was used to perform RT-PCR.

To evaluate the expression of osteogenic proteins, RUNX2 antibodies (Proteintech, China), Osterix (Abcam, United States) and OPN antibodies (Proteintech, China) were used to label target proteins. During the inoculation process, 500 cells per well were inoculated into 6-well plates for 7 days. The cells were immobilized with 4% paraformaldehyde for 15 min, and then penetrated with 0.5% Triton X-100 solution for 10 min. After blocking non-specific antigens incubated at 4°C overnight with primary staining buffer (Beyotime, China), the cells were then immersed in secondary staining buffer at room temperature for 45 min. The nucleus is labeled with DAPI to locate the cell. Fluorescent images are captured under an inverted fluorescence microscope in a dark environment.

Statistical analysis was conducted by using GraphPad Prism 5.0 software (San Diego, United States). The mean and standard error of the mean were used to represent all data. To ascertain the statistical difference between the two groups, a two-tailed Student’s t-test was applied. Data from more than two groups were compared using the one-way ANOVA method. Statistics were deemed significant at p < 0.05.

Figure 1 depicts the steps in the production of GelMA/HAMA-OGP hydrogels. After exposure to UV light, the liquid GelMA/HAMA-OGP could transform into a gel-like solid at fast rate (Figure 2A). The hydrogel can be made into different shapes of hydrogel materials by using different shapes of molds (Figure 2B). The shear thinning behavior of the hydrogel demonstrated its excellent injectability and its ability to pass through the 18-gauge needle smoothly, facilitating the clinical implementation of the injection procedure (Figure 2C). Gelation time was not significantly different between GelMA/HAMA and GelMA/HAMA-OGP hydrogels, both of which could be rapidly light-cured and molded in a short time, demonstrating that the cross-linking of OGP did not affect the curing of the hydrogels (Figure 2D). The porosities of GelMA/HAMA and GelMA/HAMA-OGP hydrogels were 68.2% and 66.7%, respectively. The two groups did not significantly differ from one another (Figure 2E). A suitable porosity facilitates material exchange and is a necessary property for an ideal biomaterial (Loh and Choong, 2013; N’Diaye et al., 2013). The morphology of the hydrogels was observed by SEM technique (Figure 2F), and it is clear that there are no appreciable differences in the internal structures of GelMA/HAMA and GelMA/HAMA-OGP. This indicates that our encapsulation of OGP proteins in GelMA/HAMA dual network hydrogels did not change their structures. These hydrogel materials have appropriate pore size and provide favorable physical conditions for three-dimensional cell growth and material exchange (Lee and Shin, 2007). In contrast to conventional single-component hydrogels (Gu et al., 2018), small and large pores of dual-network hydrogels exist simultaneously and are dotted and connected to each other. This structure is crucial for biological behavior, as previous studies have demonstrated that smaller pores facilitate cell anchoring and adhesion, and larger pore sizes facilitate cell proliferation and spreading (Tang et al., 2016). Thus, it appears that GelMA/HAMA-OGP hydrogels allow for cell entry, internal and external circulation of nutrients, and metabolic waste efflux.

The peptide was characterized by ESI-MS and its molecular structure was determined (Figure 3A). The [M + H]⁺ of OGP was found at 568.76 Da, consistent with its theoretical molecular weight at 567.61 Da. HPLC analysis showed a high purity of 99.23% for the peptide (Figure 3B). These results demonstrate the successful synthesis of OGP-MA. In the FTIR spectra of GelMA-OGP, compared to GelMA, they all produced absorption peaks at the same position with only minor absorption peak migration, indicating that the peptides have the same groups after modification. However, compared to GelMA spectra, its absorption peaks at 1,631, 1,542 and 1,236 cm⁻¹ were enhanced, where the absorption peaks were mainly caused by C=O, C-N and N-H groups. These indicate that the peptide OGP grafted to GelMA hydrogel caused the enhancement of absorption peaks at the same position, so the peptide successfully modified GelMA material (Figure 3C). In the FTIR spectrogram of HAMA-OGP, its absorption peak at 1,298 cm⁻¹ becomes faint compared to HAMA, while a new absorption peak appears at 1,235 cm⁻¹, where the absorption peak is related to the peptide N-H bending vibration. Meanwhile, the absorption peaks of C=O stretching vibration near 1,750∼1,400 cm⁻¹ were all shifted to some extent, which were related to the peptide C=O structure. All these indicate that OGP interacts with HAMA correlatively, generating covalent bonding and grafting on HAMA successfully (Figure 3D). The rheological performance of hydrogel materials was characterized for the effects of shear, temperature and frequency on the viscosity. The viscosity profile of this hydrogel varied linearly at room temperature, independent of temperature, with more stable production conditions. By increasing the temperature, the fluidity of the hydrogel increases significantly, facilitating the injection operation (Figure 3E). These hydrogel solutions exhibit a similar diminishing trend with increasing shear rate and are viscous in the low shear rate range. When the shear rate approached 10 s⁻¹ or greater, the viscosity substantially falled, indicating that they are injectable and malleable, which facilitates bone repair operations during treatment (Figure 3F). The storage modulus (G′) and loss modulus (G″) of GelMA/HAMA and GelMA/HAMA-OGP hydrogel tended to be stable in the scan range, and over the frequency range, the energy storage modulus was above the loss modulus, indicating that the mesh structure of hydrogel maintains a stable gel state throughout the test range (Figure 3G). The dilatability of hydrogels is strongly related to its network structure, mechanical strength, and capacity to govern the transport of cellular nutrients and metabolites, in addition to its capacity to absorb bodily fluids. The swelling curves showed that GelMA/HAMA and GelMA/HAMA-OGP hydrogels almost reached the equilibrium swelling state after 5 h of immersion in PBS, with the swelling ratios of 22.5% ± 1.9%, 21.6% ± 0.5%, respectively (Figure 3H). The rate at which the osteogenic scaffold material degrades is another crucial characteristic since it should coincide with the rate at which new bone tissue is forming (Koons et al., 2020; Wei et al., 2020). We employed collagenases to examine the biodegradation behavior of this hydrogel because they are abundant in the physiological milieu of humans and are crucial for biomaterials to in vivo breakdown (Wu et al., 2021). The degradation experiments showed that the degradation rates of the hydrogels were 60.8% ± 0.7%, 59.2% ± 1.6% after 7 days of incubation in PBS containing collagenases, with very similar degradation trends (Figure 3I). In addition, we also examined the in vitro release of OGP loaded in GelMA/HAMA-OGP hydrogels. As shown in Figure 3J, the cumulative release profile showed that the initial burst of OGP release occurred within 24 h, accounting for approximately 18% of the overall amount. The initial abrupt release was able to effectively stimulate cell proliferation and differentiation in the bone defect area (Fanaee et al., 2021). OGP protein release was observed for up to 14 days, which is due to the synergistic effect of physical encapsulation and chemical interlocking of the dual network hydrogel to ensure sustained drug release. The release rate of OGP gradually slowed down with time, and the cumulative release of OGP at 14 days was about 70%, which indicates that GelMA/HAMA-OGP hydrogel has sustained release properties and provides a stable drug delivery system. The slow release of OGP is beneficial to maintain the effective concentration of extracellular drugs in the scaffold to promote bone tissue regeneration and repair sufficiently and durably (Jayaraman et al., 2015).

The cytocompatibility of the hydrogels was assessed by cell viability and cell adhesion. We firstly demonstrated the biosafety of GelMA/HAMA-OGP hydrogels by CCK-8 and live-dead staining assays. The three groups did not significantly differ from one another, and all three groups of cells increased with time (Figure 4A). The experimental results showed that GelMA/HAMA-OGP composite hydrogel has great biocompatibility and biosafety. Calcein AM/PI staining was used to distinguish live and dead cells. Most of the stained cells were alive and green in color, and only a few red fluorescent spots appeared (Figure 4B). In addition, most of the cells showed polyhedral morphology, which indicated that MC3T3-E1 could survive, spread, and adhere well on the gel surface, and the hydrogel composite provided a suitable biological microenvironment.

Early cell adhesion is critical for subsequent cell differentiation and directly affects subsequent biological behavior (Shuai et al., 2018). To investigate cell expansion and morphology, the cytoskeleton was stained and photographed after MC3T3-E1 was cultured for 24 h on various hydrogels. MC3T3-E1 adhered to the hydrogel surface in a polygonal structure, exhibiting a well-stretched morphology and extending more contact pseudopods (Figure 4C). These findings imply that the hydrogel serves as a useful matrix for promoting cell adhesion and proliferation, and that integrin-mediated binding can improve the cell-matrix interaction between MC3T3-E1 and hydrogels by binding to the RGD motif in the gel matrix (Xiao et al., 2019). Moreover, the hydrogel’s cross-linked structure, appropriate viscoelasticity, and rough surface promote cell attachment (Li et al., 2018).

Ideal osteogenic biomaterials should be effective at promoting cellular osteogenic differentiation together with strong biocompatibility. The osteogenic induction ability of GelMA/HAMA-OGP hydrogel scaffolds was evaluated by ALP staining, ARS staining, immunofluorescence staining and RT-PCR. ALP staining and ALP assay kits allow for the qualitative and quantitative analysis of ALP activity, which can be employed as an early marker of cellular osteogenic differentiation. Positive staining outcomes for the ALP assay were seen in all groups (Figure 5A). Notably, the GelMA/HAMA-OGP hydrogel group showed more positive staining results at day 7 compared to the control and GelMA/HAMA hydrogel groups. ALP activity was also measured further to assess the osteogenic development. The ALP activity survey showed that the ALP activity of both GelMA/HAMA-OGP group was significantly higher than that of the control group (****p < 0.0001) and GelMA/HAMA group (***p < 0.001) (Figure 5C). This result suggests that GelMA/HAMA hydrogel promotes MC3T3-E1 differentiation toward osteoblasts and the presence of OGP contributes to the upregulation of ALP. After 14 days of co-culture with hydrogel, MC3T3-E1 cells were stained with alizarin red to demonstrate calcium nodule development, a phenotypic hallmark of late osteogenic differentiation, which demonstrated the capacity for late osteogenic differentiation. The findings revealed that the blank group had tiny crimson nodules. The GelMA/HAMA group in the control group displayed an increase in red nodules, however the overall number was not significant. Numerous red mineralized nodules and robust positive red reaction were seen in the GelMA/HAMA-OGP hydrogel group (Figure 5B). Similar results were obtained when the OD values of mineralized nodules that were solubilized in CPC were quantified (Figure 5D). According to the aforementioned findings, MC3T3-E1 cells undergoing late osteogenic differentiation can considerably increase calcium deposition and mineralization in GelMA/HAMA-OGP hydrogel.

To further explore the osteogenic differentiation of MC3T3-E1 cells on different samples, we use RT-PCR to detect the gene expression of osteogenic-related proteins after 7 and 14 days of induced osteogenesis, including ALP (early marker of osteogenic differentiation) (Manolagas, 2020; Vimalraj, 2020), RUNX2 (central regulator of osteoblast differentiation (Wang et al., 2022)), OCN (regulates Ca²⁺ homeostasis and bone mineralization as a bone mineralization stage marker (Manolagas, 2020; Wei and Karsenty, 2015)), OPN (major bone non-collagen component (Rodan, 1995) and Osterix (transcriptional regulator involved in osteoblast development and bone regeneration (Sinha and Zhou, 2013)). As shown in Figures 6A–E, the expression of five genes associated with osteogenic differentiation was upregulated in the GelMA/HAMA-OGP group compared with other groups, confirming their osteogenic activity. In conclusion, the GelMA/HAMA-OGP group enhances the osteogenic ability of MC3T3-E1 cells in vitro and is a promising scaffold material to promote osteogenic differentiation.

We used immunostaining assays to measure the synthesis of several osteogenic-related proteins after 7 days. Among the early indicators of osteoblast differentiation, RUNX2 regulates the transcription of genes encoding osteogenic-specific matrix proteins and modulates the expression of osteogenic-specific matrix protein genes in the MC3T3-E1 cell line (Yan et al., 2022). Osterix is a downstream gene of RUNX-2 and plays an important role in the induction of osteoblast differentiation and bone formation (Liu et al., 2020). As shown in Figures 7A, B, the expression of RUNX2 was evaluated by immunofluorescence staining on day 7. The levels of RUNX2 were higher in the GelMA/HAMA-OGP hydrogel group than in the control and GelMA/HAMA hydrogel groups, with an increased number of positive cells and enhanced immunofluorescence signal. In addition, OPN is a marker of middle to late osteogenic differentiationis and considered to be an important factor in bone remodeling (Depalle et al., 2021). Similarly, the GelMA/HAMA- OGP hydrogel scaffolds showed the greatest staining intensity and proportion of positive areas compared to the other two groups (Figure 7C). Among these three groups, the fluorescence intensity of various proteins (e.g., Runx2, Osterix and OPN) all in the GelMA/HAMA-OGP group showed the highest intensity (Figure 7D).