The application of additive manufacture (3D printing technology) has revolutionized bone tissue engineering due to its remarkable ability in simulating tissues by producing precise layer-by-layer deposition of bioactive and biocompatible materials (Murphy and Atala, 2014; Markstedt et al., 2015; Daly et al., 2016a; Groll et al., 2016). This advanced technology has helped to establish models of tumor, drug-releasing, and organ grafts since it was first introduced in 1998 (Kim et al., 1998). Key to the successful application of 3D-printing technology in bone tissue engineering is an appropriate selection of ink based on the working mode of the 3D printing machine such as drops inkjet (Saunders and Derby, 2014), micro-valve dispensing (Gudapati et al., 2016), laser-assisted techniques (Sorkio et al., 2018), and continuous micro-extrusion (Ozbolat and Hospodiuk, 2016). As the most frequently used mode used in bone tissue engineering, extrusion-based printing products show excellent performance on versatility, multiple modes of solidification, and towel adaptation in printing complex structures (Ozbolat and Hospodiuk, 2016; Placone and Engler, 2018). An applicable bioactive ink should be well extrudable to prevent blockage and able to produce 3D structures with high shape fidelity and mechanical stability. An appropriate bioactive ink should also behave like the extracellular matrix: it can protect cells, provide a stable environment, and subsequently promote the functional expression of cells (Parak et al., 2019). Although many investigations have been conducted by other researchers, an applicable single-component hydrogel with multiple features to greatly fit extrusion-based 3D printing machines has not been achieved (Zhang et al., 2014; Pei et al., 2016; Du et al., 2019).

During the process of optimizing cellular function and tissue integration, the bioactivity of bioink is a critical factor as it is crucial to the creation of a microenvironment similar to extracellular matrix (ECM) in bone. The bioactivity of current hydrogel-based bioinks needs to be improved in cell biocompatibility and bioactivity (adhesion and proliferation), hydrophilicity, osteoconductivity, and osteoinductivity (Ma et al., 2018). One promising approach is to blend inorganic bioactive components with organic matrices, which has been widely used in tissue engineering in recent years (Laurenti et al., 2016).

Silk fibroin (SF), natural polymeric fibrin consisting of 18 amino acids (Palcone et al., 2018; Parak et al., 2019) derived from Bombyx mori cocoons, has excellent mechanical properties, biocompatibility, and biodegradability (Du et al., 2019). The applications of SF in biomedicine and 3D printing have been extensively reported in recent years (Wang et al., 2008; Cao and Wang, 2009; Jiang et al., 2009; Shi et al., 2017; Zhong et al., 2019). The featured gelation process of the SF solution would take place under specified conditions on account of the conformational transitions during which the SF molecule chain transfers from random coil to β-sheet (Wang et al., 2008). Among those conditions, Polyethylene glycol (PEG) gelated SF hydrogel was successfully applied as an injectable tissue repair material in a recent study (Wang et al., 2015). However, pure SF gelation was shown to lack osteogenic activity in vivo (Zhang et al., 2011), and the natural fibroin is difficult to degrade because of its special crystallization and orientation, as well as compact structure (Cao and Wang, 2009).

Sodium alginate, which was extracted from native brown seaweed (Phaeophyceae) and composed of two uronic acids, β (1–4) linked D-mannuronic acid (M) and α (1–4) linked L-guluronic acid (G) (Lee and Mooney, 2012; Venkatesan et al., 2015), was shown to be another prominent biopolymer for the fabrication of hydrogels (de Moraes et al., 2014; Gong et al., 2016). SA has been widely used as a kind of biomaterial for biomedical applications not only because SA regulates the biodegradation of SF (Zheng et al., 2018; Wang et al., 2020) but also because it helps stabilize organization on the surface to maintain inner stability when exposed to calcium chloride (Smrdel et al., 2006).

In this study, we novelty prepared multiple crosslinking hydrogels with inorganic material magnesium phosphate-based gels. Magnesium phosphate is considered a potential solution to promoting the osteogenic activity function of the hydrogel. Indeed, in recent years, magnesium phosphate (Klammert et al., 2011; Mestres and Ginebra, 2011; Babaie et al., 2016; Babaie et al., 2017) has captured more attention as an alternative for bone replacement. As highlighted by Ostrowski et al. (2016) and Nabiyouni et al. (2018), many MgP phases, such as struvite (MgNH₄PO_4˙6H₂O) or newberyite (MgHPO_4˙3H₂O) have advantages of better solubility and smaller tendency while transforming into lower soluble phases in vivo (Tamimi et al., 2011), since Mg²⁺ ions suppress Hydroxyapatite formation by stabilizing gel-like amorphous calcium phosphate phases to inhibit crystal growth (Yang et al., 2011). This inhibition leads to faster resorption and is likely to pronounce bone regeneration capacity, which has already been demonstrated in a couple of studies carried out in small animal models (Kanter et al., 2018). However, a notable weakness of magnesium phosphate as a blended material applied in extrusion-based 3D printing machines is the clogging of particles that may lead to blockage during extrusion. Previous research has proposed a potential solution to overcoming this—the novel thixotropic magnesium phosphate-based gel (TMP-BG) for 3D bioprinting (Laurenti et al., 2016; Chen et al., 2020). TMP-BG was shown to have excellent printability (Chen et al., 2018).

To optimize the performance of the composites, a new type of 3D-print assisted hybrid SF-SA/MgP hydrogel scaffold was prepared in this study. To adequately utilize the advantages of the composites in hydrogel and minimize their shortages simultaneously, formulations were adjusted and MgP concentration was made in different ratios. Next, the mechanical properties, rheological, bioactivity, in vitro behaviors, and printability were characterized and optimized. Then living cells were co-cultured with the hybrid hydrogel scaffolds. Their viability and osteogenic differentiation ability in printed constructs were evaluated. The hybrid mechanisms of the hydrogel scaffolds were also explored in this step.

The objective was to find an alternative scaffold with the assistance of 3D printing technology that promotes osteodifferentation in the early stage and matches degradation with new bone deposition. To achieve the goal, a new prototype of hybrid hydrogel consisting of sodium alginate and MgP-based gel in PEG-gelated silk fibroin hydrogel was created to strengthen the mechanical properties of hydrogel and balance the degradation rate with the newly formed tissues. Previous studies have demonstrated that pore size and the 3D spatial structure of the scaffolds are key factors for cell proliferation, differentiation, and extracellular matrix (ECM) production during bone regeneration (Gudapati et al., 2016; Ma et al., 2018). It has been shown that in bone repair, cells prefer scaffolds with pore sizes between 100 and 300 µm for better proliferation, differentiation, and ECM production ability (Ma et al., 2018). Hence, a 3D-printing hydrogel scaffold with macropores around 200 µm was highly preferred in our study.

In our previous study, we designed an injectable silk-MgP hydrogel for the repair of the defect in bone tissues. However, in follow-up experiments we found that this kind of injectable hydrogel was seriously stuck with the loading plate of the 3D-print machine, making it impossible for a pre-designed scaffold to be fabricated and defect repaired. To overcome this issue, we made improvements by adding a low concentration of sodium alginate, with which we were able to fabricate a hydrogel bioink that can be applied for an extrusion 3D-print system.

In this newly hydrogel bioink, SF was the main component for the fabrication of the scaffold as SF is a natural polymer with excellent biocompatibility that can mimic the function of extracellular materials. There have been many reports on the successful application of SF-made 3D-printed scaffolds (Zhong et al., 2019; Kim et al., 2021a; Kim et al., 2021b). Theoretically, the silk protein can be catalytically hydrolyzed into amino acids under the action of proteolytic enzymes, then those amino acids would be metabolized by the body (Zheng et al., 2018). A recent study also revealed that the addition of sodium alginate can regulate the degradation rate of SF (Wang et al., 2020). The intermolecular covalent bonds and hydrogen bonds of SA macromolecule help SA form an interactive polymer network structure with SF which can affect the amount of silk II in SF hydrogel (Wang et al., 2020). In the following stage, the printed scaffold would be soaked into 0.2 M CaCl₂ to further reinforce the scaffold and better maintain its stability (Hua et al., 2010; Hong et al., 2016). The selection of 0.5%wt of SA is based on the fact that the transition of silk fibroin molecular in the follow-up experiments would not be affected while the viscosity of the SF solution was improved in this concentration, which is beneficial for the dispersion of MgP nanoparticles.

A thixotropic magnesium phosphate was also applied in this study in the form of nanoparticles instead of the traditional inorganic MgP particles. Laurenti, et al. were the first research group to study the fabrication and application of magnesium phosphate-based gel in bone repair (Laurenti et al., 2016). The MgP nanoparticles can evenly spread in the solution as a result of the ionization; the positive and negative charges simultaneously present lead to electrostatic and van der Waals interactions (Roth and Lenhoff, 1995). The particles dispersed in the solution, forming a 3D network known as the “house of cards” structure. (Barnes, 1997; Mewis and Wagner, 2009). By applying mechanical forces, thixotropic materials can be liquefied to flow; when the mechanical stress stops, the particles were driven into contact by Brownian motion to reform the 3D network again and the liquid-like dispersion is converted to solid-like gel. (Mewis and Wagner, 2009; Bergaya and Lagaly, 2013).

Generally, the hybrid hydrogel was confirmed to be a satisfying bioink for 3D scaffold production. The FT-IR spectrum of four hydrogel films shared similar structures with no obvious deviation, illustrating that the amount of MgP nanoparticles would not affect the transformation to the β-sheet in the SF solution. The absorption bands at a frequency ranging from 1,620 to 1,640 cm⁻¹ and 1,695 to 1700 cm⁻¹ corresponded to the functional groups in silk II form (Hu et al., 2006). As Figure 2A showed, the peaks at 1,634 cm⁻¹ represented the existence of β-sheet structure (Kweon et al., 2001), but the value of transmittances decreased as the amount of the MgP increased.

For rheological analysis, an ideal substance for extrusion-based 3D printing should be shear-thinning, thixotropic, easily extruded, and with fine formability (Chung et al., 2013; Schuurman et al., 2013; Ozbolat and Hospodiuk, 2016). The four hydrogels in our study all showed well shear-thinning properties. The G′ value was higher than that of G″ in frequency sweep mode in all samples, showing that the G′ and G″ were independent of the change in frequency. This result indicated the gel-like behavior and a stable decentralized system of the bio-ink (Malkin et al., 2004).

To study the biocompatibility and bioactivity of the components in the hydrogel, MSCs were first seeded on 2D hydrogel films for cell viability and then incubated with extractions of biomaterials for ALP activity because the hydrogel films would collapse after 5 days due to the swelling of hydrogel. As shown in Figure 3, the CCK-8 analysis was in contradiction with the fluorescent staining. The decreased OD value in M3 on day 7 can be explained by the crack of hydrogel and washing with PBS. The extractions of hydrogels contained the releasing of Mg²⁺ with different concentrations; the results of CCK-8 and ALP activity exquisitely reflected the proper concentration of MgP which optimally promoted the osteogenic activity. The results of proliferation analysis and ALP activity were in accordance with a previous study (Wang et al., 2017), which revealed that low Mg²⁺ concentration (i.e., 0.1 × 10⁻³ M) had an inhibitory effect on the differentiation of osteoblasts while an overdosed Mg²⁺ concentration (i.e., 18 × 10^–3 M) was harmful to osseous metabolism (Basdra and Komposch, 1997; Wang et al., 2017).

In the process of the 3D scaffold fabrication, the air pressure was set at 100kPa, far less than the 0.45 MPa as described by Andrew C Daly, et al (Daly et al., 2016b). Figures 5A,B showed the shape fidelity and accuracy of the scaffold, reflected by the bar width and smoothness after the 4-layer printing. The SF-SA scaffold had a smoother surface but wider line than the SF-SA/MgP scaffold since SF-SA/MgP bio-ink had better recovery. The diameter of the syringe was finally set at 510 µm because the MgP nanoparticles would clog and block the finer syringe. This may be explained by the uneven gelation of the hydrogel. To overcome this problem, several improvements were proposed, such as the extension of stirring, ultrasonic, and syringe replacement with a conical nozzle. However, no feasible and sustainable solution had been found. Micro morphology of the scaffolds under SEM (Figure 5C,D) showed that the micropores of SF-SA/MgP were better for the medium penetration and absorption, as well as cell migration and adhesion. The results also explained why the performance of cell proliferation on M2.5 suppressed the performance on M0 because the M0 film had a smoother surface and more unevenly distributed micropores, making it difficult for cell attachment especially when the cell confluency reached over 80%.

The combination of MgP-based gel with silk fibroin and a low concentration of sodium alginate has been shown in previous research to be a highly effective hybrid hydrogel for 3D printing. We constructed an SF-SA/MgP hydrogel scaffold for bone tissue engineering with a content of 2.5% MgP. This hydrogel scaffold had good porosity with macro-pores around 200 µm between bars and outstanding biochemical properties that are beneficial to the growth of BMSCs. The mean diameter of micro-pores was 57.885 ± 5.341 µm. Moreover, as a bioactive hydrogel, it positively stimulated the osteogenic activity of cells due to the release of Mg²⁺. Although the softness of SF-SA/MgP hydrogel can provide a microenvironment similar to ECM to use as cell-loaded bioink, it cannot be applied in force-loading structures like mandibular. The absence of experiments in vivo cannot explain the relationship between osteogenesis and degradation in detail. Overall, this 3D-printing SF-SA/MgP hydrogel provides a potential solution to the bone defect issue and has the potential to be put into practice in future bone tissue engineering.