Dual Photo‐Enhanced Interpenetrating Network Hydrogel with Biophysical and Biochemical Signals for Infected Bone Defect Healing

The healing of infected bone defects (IBD) is a complex physiological process involving a series of spatially and temporally overlapping events, including pathogen clearance, immunological modulation, vascularization, and osteogenesis. Based on the theory that bone healing is regulated by both biochemical and biophysical signals, in this study, a copper doped bioglass (CuBGs)/methacryloyl‐modified gelatin nanoparticle (MA‐GNPs)/methacrylated silk fibroin (SilMA) hybrid hydrogel is developed to promote IBD healing. This hybrid hydrogel demonstrates a dual‐photocrosslinked interpenetrating network mechanism, wherein the photocrosslinked SilMA as the main network ensures structural integrity, and the photocrosslinked MA‐GNPs colloidal network increases strength and dissipates loading forces. In an IBD model, the hydrogel exhibits excellent biophysical characteristics, such as adhesion, adaptation to irregular defect shapes, and in situ physical reinforcement. At the same time, by sequentially releasing bioactive ions such as Cu2+, Ca2+, and Si2+ ions from CuBGs on demand, the hydrogel spatiotemporally coordinates antibacterial, immunomodulatory and bone remodeling events, efficiently removing infection and accelerating bone repair without the use of antibiotics or exogenous recombinant proteins. Therefore, the hybrid hydrogel can be used as a simple and effective method for the treatment of IBD.


Introduction
Infected bone defects (IBDs), characterized by a protracted infection and bad prognosis, are frequently encountered during orthopedic, oral, and maxillofacial surgery.The healing of IBD is a coordinated regulation of the healing process of IBD through novel biomaterials is an important approach to address this problem. [9,10]Current spatiotemporal bone engineering strategies mainly focus on the development of a programmed delivery pattern to match physiological/pathological cascades. [11,12]One of the principal strategies to orchestrate tissue regeneration is based on the delivery of bioactive molecules.However, the effectiveness tends to decrease over time in vivo due to the suboptimal biodistribution of the released bioactive reagents and a reduction in their bioactivity due to enzymatic degradation. [13,14]At the same time, these strategies have the disadvantages of high cost and complex production.In comparison, bioactive ions (e.g., calcium, silicon, copper, magnesium, and cerium) have been found to exert long-term multifunctional effects through activating a series of biochemical signals that promote tissue regeneration, and they are relatively inexpensive and resistant to enzymatic hydrolysis, [13,[15][16][17][18][19][20][21] suggesting that these ions can be incorporated into the design of bioactive materials for regenerative purposes.
[24] The physiological effects of copper ions are dose-dependent.Specifically, high doses of copper ions exhibited antibacterial effects yet avoided the risk of drug resistance, [25] while low doses of copper ions promoted tissue repair responses. [26]Therefore, in the complex physiological cascade of IBD, a "multidimensional" regulation based on the staged release of Cu 2+ is a potential therapeutic approach.However, the single copper ion cannot be precisely regulated, and often cannot completely cover the above biological cascades.Bioactive glasses (BGs) are ideal carriers that can load multiple ions.Their own components (silicate and calcium) have been shown to be effective in stimulating osteogenesis and even immunomodulation, and other ions can also be added to increase function. [27,28]Therefore, we propose the use of copperdoped BGs(CuBGs) for the treatment of infected bone defects.Specifically, the early and rapid release of copper ions can play an antibacterial and angiogenic role, while the sustained release of calcium and silicon ions can continuously play an immunomodulatory and osteogenic role.This combined effect is consistent with the healing cascade of infected bone defects (copper ions for antibacterial and angiogenesis, calcium and silicon for osteogenesis and immunomodulation), the former antibacterial ensures a regeneration-friendly microenvironment, the latter participate in subsequent osteogenic repair events.
From a biophysical point of view, application of scaffolds with good physical characteristics (including mechanical adaptability and enabling viscotaxis) to the defect site is crucial for tissue regeneration. [4,29]Indeed, cells have been shown to sense the mechanical properties of their environment through a variety of mechanisms, including mechanoreceptors, local adhesions, and the actin cytoskeleton. [30]It has been reported that lower-stiffness scaffolds promoted adipose-derived stem/stromal cells adipogenesis while stiffer scaffolds favored their osteogenic commitment. [31]The use of stiffness-optimized hydrogels could also promote the polarization of immune cells toward a reparative phenotype. [32]In addition, the ability to adapt to the irregular shape of the defect site would be beneficial to the force conduction and local drug retention in bone repair, [4] and the correct viscosity would enhance the signal conduction between cells and biomaterials, which would be beneficial to tissue repair. [33]ydrogels have shown great potential to provide physiologically relevant microenvironments for cell proliferation and tissue regeneration, [34] while also serving as carriers for controlled drug release. [35]They are injectable, biocompatible, biodegradable, self-healing, and adaptable to various cavity sizes and shapes, which facilitate their application in tissue engineering. [34]Unfortunately, injectable hydrogels usually have weak mechanical properties and poor stability, making them unable to withstand external forces, thus limiting their application in bone engineering. [36]To overcome these limitations, photocrosslinked hydrogels, [37] which usually initiate chain polymerization via free radicals or bioorthogonal click reactions to achieve in situ curing, have become attractive biomaterials. [37,38]Such photocrosslinked hydrogels have better physical characteristics due to the balance between injectability and mechanical strength. [39]Therefore, combining photocrosslinked hydrogels and CuBGs to form a dual biophysical and biochemical signal regulation system may be a promising solution for the treatment of IBD.
Herein, based on the concept of "photo-controlled in situ hardening," we developed a dual photoenhanced interpenetrating network (IPN) by combining CuBGs, Methacryloyl-modified silk fibroin (SilMA), and Methacryloyl-modified gelatin nanoparticles (MA-GNPs) that enabled the bottom-up assembly of materials from the nanometer to macroscopic scale.The electrostatic interactions between the polymer matrix and CuBGs endow the composite hydrogel with improved interfacial compatibility between inorganic and organic matter.Before photocrosslinking, SilMA was combined as a liquid with a noncovalent crosslinked colloidal network with shear thinning and self-healing capabilities, generating a hybrid hydrogel with injectability, plasticity, and self-healing properties.After photocrosslinking, the network formed by SilMA was tightly combined with the colloidal network, and the mechanical strength increased by nearly 10 times and could resist cyclic stress.This dual-photocrosslinked interpenetrating network mechanism endowed the composite hydrogel with greater physical adaptability than a single network.A rat IBD model was used to study the ability of the hybrid hydrogel to provide biochemical and biophysical signals.The hybrid hydrogel showed ideal adaptability to the local complex environment of bone defects.Meanwhile, by sequentially releasing Cu 2+ , Ca 2+ , and Si 2+ bioactive ions from CuBGs on demand, the hybrid hydrogel spatiotemporally coordinated antibacterial, immunomodulatory and bone remodeling events.Overall, the dual photo-enhanced hydrogel could potentially break the current bottleneck in the treatment of IBD.

Preparation of the Hybrid Hydrogel
Figure 1A shows a schematic diagram of the two-step fabrication process for obtaining the hybrid interpenetrating network hydrogels with SilMA, MA-GNPs, and CuBGs.In a nutshell, the first network is formed when the three primary components are rapidly mixed, inducing self-assembly via electrostatic interactions.Next, in the presence a photo-initiator (LAP), ultraviolet (UV) light is used to further enhance the crosslinking between the MA-GNP gel and SilMA network in the hydrogel.The hydrogels containing SilMA, MA-GNPs, and CuBGs are referred to as SGC hydrogel, and the hydrogels containing only SilMA and MA-GNPs are referred to as SG hydrogel.
First, Fourier transform infrared (FT-IR) were used to confirm the modification of GMA on SF and MA on GNPs.Specifically, FT-IR spectra showed characteristic SilMA and MA-GNPS peaks of amide I, II and III at 1641, 1530, and 1240 cm −1 (Figure 1B). [40,41]CuBGs exhibited an inorganic amorphous structure.Furthermore, Zeta potential measurements revealed that SilMA and CuBGs were negatively charged, while MA-GNPs were positively charged (Figure 1C).The combination of the three induced an electrostatic attraction in SGC.Electrostatic interaction in SGC was supported by dynamic light scattering (DLS) analysis of the particle size.After mixing the three components, the mixture started to assemble, gradually forming larger clusters (Figure 1D).We observed, using transmisson electron microscope (TEM), that the CuBGs particle size was about ≈300-450 nm.After mixing CuBGs with MA-GNPs, we found that the MA-GNPs wrapped around the CuBGs (Figure 1E).In addition, the clusters gradually became smaller after incubation with NaCl (Figure 1F), which can disrupt ionic interactions, [42] suggesting electrostatic interactions is one of the stabilizing forces in the SGC hydrogel.Poor interfacial compatibility between the polymer matrix and inorganic particles has been reported, [43] which may lead to the disordered separation of CuBGs in hybrid hydrogels, thereby affecting their properties such as stability and mechanical properties.The electrostatic interactions in the SGC hybrid matrix would be beneficial to improve the interfacial compatibility between CuBGs and the polymer matrix.
For the hydrogel synthesis, we used gels with MA-GNP volume fractions higher than random close packing ( 11 RCP ≈ 0.64), corresponding to a MA-GNP concentration of 12% w/v, ensuring the formation of a dense colloidal network. [4]Since the change of SilMA concentration would have a large impact on the mechanical properties and injectability of the hydrogel, [33] we investigated changes in the rheological properties of hydrogels with different SilMA concentrations.Creep tests indicated that hydrogels with SilMA concentrations of 10% and 15% showed defects in their structure after applying certain stresses and some internal stresses could not be released, which may affect the hydrogel strength after deformation (Figure 1G).In contrast, the 20% and 25% SilMA systems exhibited better stress release.After structural damage, all hydrogels had good self-healing properties (Figure 1H).In the contact experiment after cutting, the same results were observed (Figure S1, Supporting Information).Most notably, hydrogels with 10%, 15%, 20% SilMA had the characteristic of shear thinning (Figure 1I).However, the 25% SilMA hydrogel had a decrease in viscosity at high shear rates due to its greater strength and viscosity, which drifts out of the test area during shear by centrifugal and frictional forces.This may be because SilMA, as a polymer, tends to aggregate thus reducing the performance of shear thinning.At the same time, we found that after photocrosslinking (Figure S2, Supporting Information) or increasing the concentration of SilMA (Figure 1J), there was both an increase in G′ and G″.However, when the SilMA content was increased, light transmission through the material decreased, resulting in slow light curing.It was also reported that an increase in SilMA concentration also delayed its degradation. [33]Therefore, considering the balance between the mechanical strength, curing time and injectability required for clinical treatment, 20% w/v SilMA was used in further experiments.
The microstructures of the hydrogels were observed using scanning electron microscopy (SEM, Figure 1K).SEM images showed that SilMA has a sparse porous structure and smooth surface, while MA-GNPs were closely packed spherical particles.The hybrid SGC hydrogel exhibited a porous network formed by the interconnection of SilMA and self-assembled MA-GNPs, suggesting a possible cohesive interaction between these two components.Meanwhile, The SEM-energy dispersive X-ray (SEM-EDX) elemental mapping of SGC hydrogel was recorded to identify the elemental composition, suggesting the uniform distribution of Cu Ca and Si elements in the SG hydrogel matrix (Figure 1L).In this construct, the SGC hydrogel forms a reinforced structure, in which SilMA is like a "rebar" that provides structural integrity and certain mechanical strength, while the MA-GNPs is like "concrete," to further enhance the mechanical strength while maintaining the dynamic properties.Compared with single-network gels formed by SilMA and MA-GNPs, the hybrid hydrogel had a significantly higher storage modulus (Figure S3, Supporting Information).The mechanism by which the dual network enhances the mechanical properties was demonstrated in our previous study. [4]Specifically, the gelatin colloid forms a continuous particle network concurrently with the polymer fibrous network, which provides improved injectability/formability of the resulting composite hydrogel, but also enhances toughness by enabling energy dissipation upon loading.Therefore, this SGC hybrid hydrogel with an interpenetrating network could exhibit excellent biophysical adaptability.

Characterization of the Hybrid Hydrogel
The injectability and malleability of hydrogels are important properties for clinical application.The SGC hybrid hydrogel exhibited good injectability and could be easily ejected from a syringe without causing needle clogging (Figure 2A).The shape of the gel can be controlled by injecting the hydrogel into molds with different shapes, such as circle, rectangle, and heart, which indicates that the hydrogel is adaptable and can meet any shape requirements for irregular bone defects caused by fractures, tumors, or other bone diseases.Furthermore, 3D printing, an advanced bio-fabrication technique that can generate patient-specific scaffolds with highly complex geometries, has been widely explored for the rapid design and fabrication of hydrogels. [44]Here, we printed a highly complex organ structure, the ear.This further illustrates the clinical application prospects of SGC hydrogels.
Generally, good adhesive properties of a hydrogel enable it to adhere to the defect's surface acting as a physical barrier, thereby achieving a physical hemostatic effect, and good adhesion is also conducive to subsequent osseointegration. [33]Therefore, the adhesive properties of the SGC hydrogel were investigated by lap shear strength experiments (Figure 2B,C).We found that the adhesive strength of the SGC hybrid hydrogel was significantly stronger than that of the single-network SilMA and MA-GNPs gels.
A hydrogel with suitable stiffness not only provides a stable environment for tissue regeneration, but also modulate cellular responses at the site of implantation. [45]As indicated above, the double network formed in the SGC hybrid increased the hydrogel's strength.The improvement in the mechanical strength of the hydrogel after photocrosslinking was investigated.As shown in Figure 2D, the UV-treated SGC hydrogel retained its original shape after being compressed (under a 500 g weight) without showing any signs of damage.Compression tests further showed that the Young's modulus of SGC hydrogel with UV light curing was significantly (≈10 times) higher than that of SGC hydrogel without UV treatment (Figure 2E).More notably, the SGC hydrogel without light curing showed increasing deformation of the structure after 5 and 10 cycles of compression or tensile tests, while after light curing, the Young's modulus of the SGC hydrogel hardly changed after 10 compression cycles (Figure 2F-I; and Figure S4, Supporting Information).Thus, the light-enhanced SGC hydrogel has enhanced mechanical properties and stability, which indicates that the hydrogel can resist external pressure well after implantation in vivo and withstand the mechanical microenvironment of the defect site.Meanwhile, compared with MA-GNP gels, SGC hydrogels exhibited a significantly higher swelling rate (Figure 2J), which would be beneficial for the movement of nutrients to promote cell infiltration into the scaffolds.In summary, dual-photocrosslinked SGC hydrogel exhibited excellent biophysical characteristics including injectability, local shape adaptability, 3D printability, and enhanced adhesive and mechanical properties.
In addition, we studied the release Cu 2+ ions from SGC as a source of biochemical signals.Unlike release from pure CuBGs, the release of Cu 2+ from CuBGs loaded in the SGC hydrogel was lower and slower (Figure 2K), which would avoid biotoxicity after application in vivo.Cu 2+ exhibited a biphasic release pattern, specifically, in the early stage, copper ions were released rapidly, while in the later stage, the ions begin were released at a steady lower rate.Cu 2+ is reported to have an antibacterial activity against methicillin-resistant S.aureus and E. coli at 1 ppm, [46] and the optimal dose of Cu 2+ to promote angiogenesis is about 0.7 ppm. [47]From our data, in the first 3 days, the concentration of copper ions released from the SGC hydrogel was > 1 ppm, and from the fifth day, the release of copper ions was maintained at about 0.7 ppm copper ions.The concentration of copper ions in this biphasic release mode is in the concentration range required for antibacterial activity and angiogenesis, which will be beneficial to realize the spatiotemporal control of pathogen clearance and repair in the IBD healing process.There have been many efforts to construct a drug delivery system that mimics the physiological healing process, but the shortcomings of existing strategies are the complex multidrug loading process and difficulty in maintaining drug activity.In comparison, strategies for single-drug concentration-dependent regulation could significantly simplify the preparation process and provide more controllable biological regulation functions. [48]

Antibacterial Activity of the SGC Hydrogel
Persistent bacterial infection of bone defects could have serious consequences, such as severe inflammation, local bone loss and destruction, and vascular injury.If bacteria could be killed and cleared in a timely fashion, it would help restore the disturbed bone immune microenvironment and greatly improve the treatment efficiency.The agar plate count method was used to assess the in vitro antibacterial activity of the SGC hydrogel against the pathogenic bacteria S. aureus and E. coli (Figure 3A,B; and Figure S5, Supporting Information).A large number of colonies on the gar plate demonstrated that the SG hydrogel possessed little bactericidal activity against S. aureus or E. coli.However, after culturing with the SGC hydrogel there was strong antibacterial activity against S. aureus and E. coli, with greater than 100-fold reduction in viable cells.Live/dead staining showed that the SGC hydrogel increased the number of damaged or dead bacteria, which supported the findings of plate count assays.In addition, one of the most important factors contributing to persistent drug resistant infections is the formation of bacterial biofilms, which prevent antimicrobials from encountering bacteria, delaying the healing of infected bone defects.Biofilms on microtiter plate wells were quantified with CV staining.The well coverage and stain quantification showed that the SGC hydrogel reduced the amount of biofilm by about 70% compared to the control, no hydrogel, group (Figure 3C).The results show that the SGC hydrogel can effectively inhibit and destroy bacterial biofilms.
To explore the antibacterial mechanism of the SGC hydrogel, we investigated the effect of SGC hydrogel treatment on reactive chemical species within the bacteria.It is known that Cu 2+ ions can increase intracellular ROS by catalyzing Fenton chemistry and generating hydroxyl radicals, and the elevated ROS levels lead to bacterial oxidative damage. [22]Use of the DCFH-DA probe showed that the bacteria treated with the SGC hydrogel produced a large amount of ROS, while the control group and SG hydrogel group had very low ROS levels (Figure 3D).Using dihydroethidium (DHE), a fluorescent probe for superoxide ions, and HKPerox-2, a green fluorescent probe with high selectivity and sensitivity for H 2 O 2 , we further confirmed the species of ROS (Figure 3E,F).Thus, the SGC hydrogel facilitated the formation of superoxide ions and H 2 O 2 inside the bacteria, suggesting that the Cu 2+ ions, through multiple ROS generation pathways, strengthened the antibacterial effect.At the same time, surprisingly, the SGC hydrogel also induced the production of NO (Figure S6, Supporting Information), which may contribute to bacterial killing. [49]In addition, Cu 2+ ions could reduce the potential difference across the membrane leading to membrane depolarization, and subsequently membrane leakage or even rupture. [50]From the SEM images, the bacterial cellular morphology in the control and SG groups were smooth, spherical (S. aureus), and rod-shaped (E.coli).Following SGC hydrogel treatment, there were fewer bacteria present, and the surface became indented or wrinkled, which was a sign that the bacteria were starting to die (Figure 3G).Therefore, according to these findings, the antibacterial mechanism of SGC hydrogels may be due to the induction of bacterial oxidative stress and membrane depolarization by the released Cu 2+ ions (Figure 3H).

Biocompatibility of the Hybrid Hydrogel
The biocompatibility of the hydrogels was tested by culturing different cell lines (MC3T3-E1, Raw264.7, and HUVECs) with the hydrogels.No significant difference in cell viability was observed between the hydrogel-treated and control groups using live/dead staining or the CCK-8 assay, indicating that the hybrid hydrogel is biocompatible (Figure S7, Supporting Information).This is because both SilMA and MA-GNPs are naturally derived and have good biocompatibility, and the loaded CuBGs had no obvious toxicity due to the slow-release effect of the hydrogel.In addition, although SilMA lacks specific sites for cell adhesion, MA-GNPs contains a large number of RGD sequences (tripeptide sequence of Arg-Gly-Asp), which would increase the adhesion ability of cells to SGC hydrogels and further promote cell proliferation. [51]

Immunomodulation Properties of the SGC Hydrogel
Macrophages are innate immune cells that play a key role in immune regulation and antimicrobial immune response in bone defect healing. [52]They are prime candidates for immune regulation due to their heterogeneity and plasticity.Specifically, macrophages are highly plastic cells that can polarize into various phenotypes and fulfill different roles.These roles are classified as "classical activation" (M1 phenotype), which promotes inflammatory responses and bacterial clearance, and "alternative activation" (M2 phenotype), which promotes immune regulation and tissue remodeling. [53]In IBD, the formation of a bacterial biofilm together with introduced biomaterial can induce a "frustrated" state in macrophages, [54] leading to a significantly reduced antibacterial capability, and a delayed M1-M2 transition which will cause chronic inflammation and delay the healing process.Therefore, rationally designing a biomaterial that can promote synergistic M1 antibacterial activity in the early stage of application and promote M1-M2 conversion in the later stage can be a good strategy for the treatment of IBD (Figure 4A).In this study, we observed the effect of SGC hydrogels on macrophages at different time points.polymerase chain reaction (PCR) analysis showed that at day 3, the expression of CD86, TNF-, and IL-6 in the SGC+LPS hydrogel group was higher than that in the LPS and SG+LPS groups.However, at day 7, CD206, TGF-, and ARG had the highest expression in the SGC+LPS group (Figure 4B).The results were confirmed by immunofluorescence staining, the expression of pro-inflammatory factors was high at day 3 day and decreased at day 7, while the expression of antiinflammatory factors showed opposite trend (Figure 4C).This phenomenon was encouraging as it fulfilled the afore-mentioned requirements for IBD healing.
Reports of the effect of CuBGs on macrophages are contradictory, [55,56] but this might be due to different doses of Cu 2+ ions being used. [28]High concentrations of Cu 2+ ions can promote the polarization of pro-inflammatory macrophages and enhance the immune response to clear pathogens by activating copper transporter 1 and copper transporter ATPase 1. [57] But, at the same time, some studies have found that low concentrations of Cu 2+ ions promote the anti-inflammatory phenotype of macrophages. [58,59]Therefore, the bimodal release pattern of Cu 2+ ions from the SGC hydrogel may achieve the spatiotemporal regulation of macrophages.In addition, BGs have been shown to promote the polarization of macrophages to the M2 phenotype by locally releasing active ions.For instance, Si ions released from BGs have been shown to inhibit the pro- inflammatory response activity of macrophages, and released Ca 2+ targets the Wnt/-catenin signaling pathway and IL-10 (M2 marker gene) transcription via calcium-sensing receptor activation in macrophages. [28]This would be beneficial to promote polarization to the macrophage M2 phenotype at later stages of IBD treatment.In summary, the SGC hydrogel may have spatiotemporal immunomodulatory capabilities through changing the ion concentrations in the local microenvironment.

Angiogenesis and Osteogenesis Promoted by the SGC Hydrogel
Blood vessels supply bone tissue with essential nutrients, oxygen, growth factors, and hormones.Likewise, the osseointegration and bone defect repair processes may be enhanced by the vascular supply within tissue engineered implants. [60]Therefore, we investigated the effect of the SGC hydrogel on angiogenesis.By measuring cell migration with Transwell plates, we found that the SGC hydrogel induced increased cell migration of HU-VECs, which would support greater angiogenesis (Figure 5A).Meanwhile, qPCR results indicated that angiogenic genes (CD31, ANG-1, and VEGF) were more highly expressed in the SGC hydrogel group (Figure 5B).The tube formation assay was used to further explore the angiogenic ability of SGC hydrogels.More tubes were formed in the SGC hydrogel group than in the SG hydrogel group (Figure 5C).Semiquantitative analysis also revealed that the total length of tube branches as well as the number of tube nodes and branches were much larger in the SGC hydrogel group than in the other groups (Figure 5D).These results suggest that the SGC hydrogel has the potential to induce angiogenesis.
It has been previously reported that BGs could promote angiogenesis through dissolution and release of Si 2+ and Ca 2+ ions. [61]Also, Hypoxia-inducible factor-1a (HIF-1a), a major transcription factor regulating VEGF expression, can be accumulated and activated by Cu 2+ ions due to their ability to inhibit prolyl hydroxylases. [56]Therefore, the incorporation of Cu 2+ ions into BGs could further enhance the angiogenic ability.Indeed, according to our release results, the concentration of Cu 2+ ions released by the SGC hydrogel is maintained at about 0.7 ppm, the optimum angiogenesis-promoting concentration, which will be beneficial to the tissue repair process.Therefore, it is likely that the SGC hydrogel possesses pro-angiogenic ability due to the sustained release of pro-angiogenic bioactive ions.
In the early stages of osteogenic differentiation, alkaline phosphatase (ALP) is a useful marker.As shown in Figure 5E, the SGC hydrogel induced more ALP expression on day 7 than the SG hydrogel.In addition, mineralization in the late osteogenic differentiation stage was determined by Alizarin Red staining.The most calcium nodules were observed with the SGC hydrogel (Figure 5F).Gene expression assays for ALP, RUNX2, OSX, OPN, and COL-1 were subsequently performed to determine the molecular effects of the hydrogel on cell differentiation (Figure 5G).The highest ALP gene expression was seen in the SGC hydrogel group at day 7, OSX and RUNX2 were highly expressed at day 7 and day 14, OPN and COL-1 were highly expressed in the SGC group on day 14.These results indicate that the SGC hydrogel could enhance osteogenesis.It has been reported that during the process of osteogenic differentiation, osteogenesis-related genes are expressed in a certain order, [62] in which ALP is the gene expressed in early osteogenesis, OSX and RUNX2 are the genes expressed in the early and middle stages, and OPN and COL-1 are the genes expressed in late osteogenesis.The expression of osteogenic genes regulated by the SGC hydrogel is in line with this physiological expression sequence.Thus, the SGC hydrogel promoted the programmed expression of osteogenic genes through spatiotemporal regulation (Figure 5H).
Since their introduction, BGs have found widespread application in the field of bone regeneration and repair.As BGs degrade, a silicon-rich layer forms on their surface; this layer encourages the formation of hydroxyapatite (HA) and tightly combines with collagen fibers produced by osteoblasts. [63]Furthermore, leached ions such as Ca 2+ and Si stimulate osteoprogenitor cells at the genetic level and endow BGs with good osteoconductivity and osteoinductivity.Moreover, Cu 2+ ions, as divalent cations, have been shown to promote bone formation by activating skeleton interoception and downregulating sympathetic tone. [64]Therefore, the SGC hydrogel could release a variety of bioactive ions from CuBGs to promote bone formation.From a biophysical point of view, as mentioned before, mechanically enhanced hydrogels are more conducive to osteogenesis, compared to other hydrogels.Indeed, the SG hydrogel, without CuBGs, also induced cell migration and had higher ALP expression than the control in the early stage, which might indicate that the enhanced biophysical characteristics of the SC hydrogels could promote osteogenesis (Figure 5I).Therefore, the ability of SGC hydrogels to promote osteogenesis might be due to the synergistic effect of biochemical and biophysical signals.

In Vivo Therapeutic Performance of the SGC Hybrid Hydrogel
The in vivo therapeutic potential of the SGC hybrid hydrogel was examined in a bacteria-infected femoral critical sized bone defect model (Figure 6A).Following creation of the defect (2.8 mm diameter, 2 mm depth), saline (20 μL) was injected into the defects (Ctrl group).S. aureus (20 μL, 1×10 7 CFU), a major cause of clinical bone infection, was injected into bone defects.In S. aureus+SG and S. aureus+SGC groups, the SG and SGC hydrogels (100 μL) were injected into the defects simultaneously with the injection of S. aureus, and the defects were irradiated with UV for 30 s.The application of SGC hydrogel in the defect is shown in Figure S8 (Supporting Information).The hydrogel could well adapt to the shape of the defect after injection suggesting that the SGC hydrogel has a suitable physical adaptability.
At day 3 and day 7, we extracted the surrounding tissue from the defects for bacterial culture (Figure 6B; and Figure S9, Supporting Information).Up to 7 days, compared with the S. aureus group, SG hydrogel had no obvious antibacterial effect, while after treatment with SGC hydrogel, the bacterial clearance rate was 98.6%±0.6%.Therefore, the in vivo results showed that the SGC hydrogel has a long-lasting antibacterial activity.Meanwhile, we collected the serum for ELISA (Figure 6C).At 3 days, compared with the Ctrl group, the expressions of inflammatory factors IL-6 and TNF- in the serum of the S. aureus, S. aureus+SG, and S. aureus+SGC groups were all up-regulated, and the expression of TNF- in the S. aureus+SGC group was the highest.At 7 days, after treatment with SGC hydrogel, the expressions of IL-6 and TNF- were all down-regulated, while that in S. aureus and S. aureus+SG groups the levels of inflammatory factors were still high, indicating that SGC hydrogel regulated the inflammatory response in vivo through antibiosis and immune regulation.
Overall photographs of femur samples were collected at 2 and 4 weeks (Figure The S. aureus group without any treatment showed delayed fracture healing.On the contrary, the bone repair was accelerated after treatment with SGC hydrogel, exceeding that of the Ctrl group.To assess the information about newly formed bone tissue within defects more precisely, micro-CT was performed.As shown in the CT images and 3D reconstructed images (Figure 6E,F), at 2 weeks, large bone defects existed in the S. aureus group and S. aureus+SG group, which may due to the persistent bacterial infection affecting the bone healing process, resulting in the destruction of cortical bone and cancellous bone.In comparison, there was more bone repair in S. aureus+SGC and Ctrl group.At the end of 4 weeks, cortical bone regenerated significantly in S. aureus+SGC group, and the newly formed bone bridged well.Besides, the BV/TV of S. aureus+SGC group was up   to 62%±2.0%, which was significantly higher than that in other groups as measured by quantitative analysis of micro-CT scans.Bone microarchitecture, in addition to bone volume, is thought to be a significant factor in determining bone mechanical strength.The increase of Tb.N, Tb.Th, and the decrease of Tb.Sp in the S. aureus+SGC group compared to other groups all suggested that SGC hydrogel could repair bone microarchitecture under infection (Figure 6G).Van Gieson's (VG) staining were further performed to further verify the regulation of various biological events in the process of IBD healing by hydrogels from a microscopic aspect, whereby osteoid was stained blue and mature bone tissue was stained red (Figure 7A).At week 2, the S. aureus and S. aureus+SG groups showed mainly bone destruction, with a small amount of bone tissue.Whereas in the control and SGC+S.aureus group, new bone had grown into the defect and a bridge had been established.Until 4 weeks, the S. aureus group still had not completely bridged.In the S. aureus+SG group, although there was a certain amount of bridging, the trabecular bone was still relatively loose, and the bone height did not recover.As we expected, there was no obvious interface between the newly formed bone and the host bone in the control and the SGC+S.aureus group, and the morphology was completely restored, which indicated that SGC hydrogel promoted osteogenesis and osseointegration.
To further understand the spatiotemporal behavior of newly formed bone, a series of fluorescently labeled mineral dyes were subcutaneously injected at predetermined intervals (Figure 7B).As a result, the red fluorescence (labeled on newly formed bone at 1st week) were barely observed in S. aureus and S. aureus+SG groups, while some blue and green fluorescence (labeled on newly formed bone at 2nd and 3rd week) could be seen, indicating the bacterial infection delayed new bone formation at early osteogenic stage.In comparison, all of the three fluorescence signals were strong in S. aureus+SGC group (Figure 7C), suggesting that SGC hydrogel helped build regenerative microenvironment at the early stage of osteogenesis and could maintain the effect to middle and late osteogenic stages, resulting in excellent new bone formation.Additionally, we further verified this result by establishing an infected osteochondral defect model and SGC hydrogel also exhibited ideal therapeutic effect (Figure S10, S11, Supporting Information).Overall, SGC hydrogel could exert an antibacterial effect at early stage, promote a regenerationfriendly microenvironment [65] and then initiate a series of prorepair events (Figure 7D).In comparison, the autologous immune regulation system takes a longer time to eliminate bacterial stimulation, thus delaying bone repair.
Combining the in vitro and in vivo results, we confirmed that the SGC hydrogel owns the ability to spatiotemporally meet the needs of biochemical and biophysical signals at different stages of IBD healing (Figure 8).At the time of implantation, due to the dual-photo crosslinking IPN of the SGC hydrogel, it could be cured in situ after injection and molding, and achieve physical hemostasis.Due to the excellent mechanical properties and stability, the SGC hydrogel could resist external pressure well after implantation in vivo and match the mechanical microenvironment of the defect site.This physical adaptability of the implanted biomaterial is thought to be critical for tissue regeneration.In addition, the rapid release of copper ions in the early stage of implantation is beneficial for the killing of bacteria, and for increasing the clearance of the pathogens by activating M1 macrophages.As changes in the ionic microenvironment increased the proportion of M2 macrophages, this multifunctional hydrogel could help suppress the inflammatory response and promote angiogenesis and tissue remodeling.Therefore, the above results demonstrated that SGC hydrogels have great promise for hastening the recovery from IBD.

Conclusion
In this study, a promising hybrid hydrogel with excellent biophysical adaptability was developed to promote rapid healing of IBD by spatiotemporally regulating the healing process.The hydrogel was composed of CuBGs, photo-crosslinked SilMA and MA-GNPs.To provide biophysical signals from the dualphotocrosslinked interpenetrating network formed by SilMA and MA-GNPs, the hydrogel could be reinforced in situ by UV irradiation.The cured hydrogel exhibited excellent biophysical properties, such as adhesion, adaptation to the irregular shape and desirable mechanical properties.To overcome the biological cascade disorder in the healing process of IBD, this hydrogel achieved spatiotemporal regulation of the healing process of IBD by releasing different biochemical signals from CuBGs at different time points.It was demonstrated in treating IBD in rats that the SGC hydrogel could effectively kill bacteria and remove pathogens in the early stage of implantation and promote the polarization of macrophages to the M2 phenotype later, and finally promote angiogenesis and osteogenesis.Therefore, the SGC hydrogel is expected to provide a simple, minimally invasive and effective treatment for the repair of infected bone defects without exogenous addition of expensive biological factors or resistance-inducing antibacterial drugs.In summary, the novel SGC hydrogel is expected to become a new strategy for the clinical treatment of IBD.

Experimental Section
Synthesis and Characterization of the Hybrid Hydrogel: MA-GNPs were prepared using the procedure described by Diba et al., [40] SilMA solution was prepared according to the method of Kim et al. and CuBGs (95SiO 2 /2.5CaO/2.5CuO(in mol%)) was synthesized by a modified Stöber method. [41,66]The detailed steps are shown in the Supporting Information.For the hybrid hydrogel, following its loading into a luer-lock medical syringe, 1 mL of SilMA solution (either 10%, 15%, 20%, or 25% w/v) containing the photoinitiator lithium phenyl(2,4,6-trimethylbenzoyl) phosphinate (LAP) (0.25% w/v) was combined with 120 mg freeze-dried MA-GNPs powder and 10 mg CuBGs powder in a separate syringe, and the resulting mixture was repeatedly extruded to produce hybrid hydrogels.The mixed hybrid hydrogel was then exposed to UV light (30 mW cm −2 ) at the distance of 5 cm for 30 s in order to cause photo-crosslinking.The morphology of the hybrid hydrogel was observed using scanning electron microscopy (FEI, Quanta 450, USA), and the elements C, Ca, Si, and Cu were quantified and their distributions obtained by energy-dispersive x-ray spectroscopy (EDX) mapping.Specifically, the hydrogels were frozen and broken up at −80 °C, and then the water was taken out by freeze-drying them.Then, gold was put on the cross sections of the hydrogel samples to make them more conductive.
FT-IR, Zeta Potential, DLS, and TEM: To study graft copolymerization reactions, FT-IR spectroscopy was used to confirm the structure of SilMA, MA-GNPs, and CuBGs (Frontier, PerkinElmer, Rodgau, UK).The Zeta potential was measured using a Zetasize Nano-S instrument (Malvern Instruments Ltd.).SilMA, MA-GNPs, and CuBGs were dispersed in HPEPS (5 mm) and mixed in the ratio of 20:12:1.DLS (Zetasizer Nano-S, NanoBrook 90 Plus PLAS) was used to measure the average particle size.Different concentrations of NaCl (0, 1, 5, or 10 mm) were added to the solution to observe the particle size change.For TEM, the CuBGs and CuBGs/MA-GNPs were dispersed in ethanol and after sonication for 5 min, the dispersed suspensions were examined Rheological and Mechanical Tests: The rheological properties were assessed using a AR2000ex rheometer (DHR, TA Instruments, USA)) with a parallel plate (diameter 20 mm).The operating gap was 1 mm, and the temperature was set to 25 °C.Creep tests were conducted at 5 Pa for 1 min to measure gel deformation, and the constant stress was removed to detect the recovery.The shear failure test was carried out with 1 Hz shear frequency and strain ranging from 0.1% to 100% to assess the self-healing properties of the samples.The light curing process adopted time sweep, and the testing process used ultraviolet light curing with a frequency of 1 Hz.Hydrogels' shear-thinning behavior was measured by altering the shear rate from 0.1 to 100 s-1 and recording the resulting changes in viscosity.The universal testing machine was used for all mechanical tests.Supporting Information displays the detailed procedures.
Tensile tests were also used to examine the hydrogels' self-healing ability after being damaged.The hydrogel samples were cut into two pieces and then brought into direct contact.Five minutes after contacting, samples were observed.
Injecting, Molding, and Printing Tests: Rhodamine was added to the hydrogel to give it a red color, and the hydrogel was subsequently extruded for writing and extruded into molds with different shapes before UV crosslinking.The hydrogel was put into a syringe and printed using a 3D printer with a needle tip movement speed of 10 mm s −1 and an extrusion pressure of 500 kPa.
Equilibrium Swelling and Release Behavior: Before being weighed, the hydrogels were lyophilized.After 24 h in 3 mL of phosphate buffered solution (PBS; pH = 7.4), the hydrogels were weighed after they had swollen.Following this formula, it was able to determine the final swelling ratio of the hydrogel where W s and W d represent the weight of the swollen and the lyophilized gel, respectively.
To determine the amount of Cu 2+ released from the hybrid hydrogel, 1 g of SGC hydrogel was soaked in 5 mL of PBS at room temperature for 10 days.Two and a half milliliters of PBS were removed daily for analysis using an inductively coupled plasma-atomic emission spectrometer (ICP-AES, Vista AX, Varian, USA), and 2.5 mL of fresh PBS was added back into the immersion solution.
Agar Assay, Bacterial Live, and Dead Staining: 1 g hydrogels were incubated with Staphylococcus aureus or Escherichia coli (1 × 10 6 CFU mL −1 ) in 5 mL of tryptic soy broth (TSB, Sigma, USA) at 37 °C for 12 h with shaking to test their in vitro antibacterial activity.Then,100 μL of the TSB was placed on an agar plate after dilution, spread evenly and incubated at 37 °C.Images of the plates were taken after 24 h, and the total number of colonies was counted using image J software.Bacterial viability after incubation with hydrogels was assessed using the Live/Dead BacLight Bacterial Viability Kit (Thermo Fisher, USA).Detailed methods are described in the Supporting Information.
Biofilm Assay of Antibacterial Activity: Wells in a 48-well microtiter plate were inoculated with 500 μL of an overnight TSB culture of E. coli or S. aureus cells (1 × 10 6 CFU mL −1 ) and incubated at 37 °C for 48 h to develop a static biofilm.After a further incubation with hydrogels for 24 h, crystal violet (CV, 0.2% w/v) staining was used to quantify the bacteria in the biofilms.Detailed methods are described in the Supporting Information.
Exploration of Antibacterial Mechanism of SGC Hydrogel: After being incubated with E. coli or S. aureus cells (1 × 10 6 CFU mL −1 ) at 37 °C for 12 h, the hydrogel samples were fixed in 2.5% glutaraldehyde overnight for SEM analysis.The ROS level of the bacteria incubated with the hydrogel was measured with the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) assay (Reactive Oxygen Species Assay Kit, cat#S0033; Beyotime) (see details in the Supporting Information, Section 1.7).The special type of ROS generated within the bacteria treated by SGC hydrogel was evaluated by DHE (as probe to O 2 -), HKPerox-2 (as probe to H 2 O 2 ).S. aureus hydrogel (n = 3).A critical sized defect (2.8 mm in diameter, 2 mm in depth) was drilled at the long bone mid-shaft of femur under isoflurane gas and local anesthesia with lidocaine.S. aureus cells (20 μL 1 × 10 7 CFU mL −1 ) in the PBS were injected into the defect to simulate an infected bone defect.After injecting the hydrogel into the defect, UV (30 s) was used to crosslink the hydrogel.The knee joint capsule and skin were stitched together layer by layer after the ligaments have been repositioned.The rats were euthanized at 3 and 7 days, 2 and 4 weeks after implantation to collect samples from their knees.At 3 and 7 days, the tissue around the defect was excised and cultured in TSB for bacteria enumeration using the agar assay.At the same time, serum was collected assessed with TNF- and IL-6 ELISA kits.
Micro-CT Analysis of Bone Defects: Extracted tissues were rinsed in PBS after being fixed in 4% PFA at 4 °C for 48 h.Before CT scanning at 70 kV and 112 A (vivaCT40, SCANCO Medical AG, Switzerland), the fixed tissue samples were soaked in ethanol for 30 min.Following the acquisition of CT scans, 3D images were reconstructed and assessed for bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N) at the site of the defect.
Hard Tissue Section Preparation and Histological Staining: After micro-CT examination, the dehydrated specimens were embedded in methyl methacrylate, and 30 μm thick sections were prepared using a hard tissue sawing system (E200CP, EXAKT Verteriebs, Germany).Sections were stained with VG according to the previous method. [67]Olympus VS200 system was used for observation.
Sequential Fluorescent Labeling: Based on previous studies, a multicolor sequential fluorescent labeling approach was used to elucidate the process of new bone formation and mineralization.Specifically, at 1st, 2nd, and 3rd week after surgery, alizarin red, tetracycline hydrochloride, and calcein were, respectively, injected in the neck of rats subcutaneously (30 mg kg −1 ) and confocal microscope was used for fluorescence observation.
Statistical Analysis: Data are presented as means ± standard deviation.All experiments were repeated in triplicates.One-way or two-way analysis of variance (ANOVA) were used to assess the multiple comparisons by GraphPad Prism 7.0.Statistical significance level was set at p < 0.05, where ns means not significant.* p<0.05, ** p<0.01, *** p<0.001.

Figure 1 .
Figure 1.Synthesis and the structure of the SGC hydrogel.A) Illustration of the formation of the interpenetrating polymer network within the hybrid hydrogel.B) FT-IR spectra for freeze-dried SilMA, MA-GNPs, and CuBGs.C) The Zeta potential of CuBGs, SilMA, MA-GNPs, and a mixture of the three.D) The size of CuBGs, SilMA, MA-GNPs, and a mixture of the three.E) TEM image of CuBGs and a mixture of CuBGs and MA-GNPs.F) The size of the SGC particles after incubation with different concentrations of NaCl.G) Creep tests of hybrid hydrogels with different concentrations of SilMA.H) Selfhealing behavior of hydrogels with different concentrations of SilMA during destructive shearing and recovery.I) Shear-thinning property of hydrogels with different concentrations of SilMA.J) Time-dependent rheological studies of hybrid hydrogels with different concentrations of SilMA.K) SEM images of SilMA, MA-GNPs, and SCG hydrogel.L) EDX mapping of C, Si, Ca, and Cu in SGC hydrogels.

Figure 2 .
Figure 2. Physical properties of the hybrid hydrogels.A) Photographs of the injectability, moldability, and printability (image of an ear 3D printed using DLP printer) of the SGC hybrid hydrogel.B) Schematic of the procedure for the measurement of lap shear strength.C) Representative stress/strain curves of SGC, MA-GNP, and SilMA hydrogels.D) The SGC hydrogel with and without light-curing was compressed by a 500 g weight on the hydrogel for 30 s and the dimensions of the recovered hydrogels were measured.E) Representative stress-strain curves for compression of the SGC hydrogels with and without light-curing.F-I) The hysteresis curves of the SGC hydrogels with and without light-curing upon cyclic compressive loading and unloading.J) Equilibrium swelling study of hydrogels.K) Concentration of Cu 2+ released from pure CuBGs and SGC hydrogels over 10 days.

Figure 3 .
Figure 3. Antibacterial activity of the SGC hydrogel.A) Agar plate assay of S. aureus and E. coli showing the antibacterial potential of SGC hydrogel.B) Live/dead fluorescence images of S. aureus and E. coli after being incubated with the SG or the SGC hydrogel (Live/dead cells are stained green/red, respectively).C) Crystal violet staining of bacterial biofilms incubated with the SG or the SGC hydrogel and percentage quantitative biofilm formation under different conditions (n = 3).D) DCFH staining of bacteria showing the generation of ROS.E) DHE staining of bacteria showing the formation of superoxide ions.F) HKPerox-1 staining of bacteria showing the formation of H 2 O 2 .G) Color enhanced SEM imaging of S. aureus and E. coli cultured on the surface of SG and SGC hydrogels.H) Schematic diagram of the antibacterial mechanisms of the SGC hydrogel.

Figure 4 .
Figure 4. Macrophage polarization in vitro in response to the SGC hydrogels.A) Schematic illustration of the effect of the SGC hydrogel on macrophage polarization and subsequent tissue repair.B) Relative gene expression of M1 phenotype markers (CD86, TNF-, and IL-6) and M2 phenotype markers (CD206, ARG, and TGF-) in macrophages at day 3 and 7. C) Fluorescence images of iNOS (red) and CD206 (green) staining of Raw264.7 cells after culture with hydrogels at day 3 and 7.

Figure 5 .
Figure 5. SGC hydrogel promoted angiogenesis and osteogenesis.A) Analysis of HUVECs migration behavior using the Transwell assay with SG or SGC hydrogels.B) Expression of angiogenesis-related genes CD31, ANG, and VEGF determined by qRT-PCR on day 3. C) HUVEC tube formation when cultured with SG or SGC hydrogels for 12 h.D) Semiquantitative analysis of vascular tube formation (n = 3).E) ALP staining on day 7 and F) Alizarin red staining on day 28.The SGC hydrogels stimulated better osteogenesis.G) qRT-PCR analysis of expression of osteogenesis-related genes (ALP, Runx2, OSX, OPN, COL-1) in cells cultured with the SG or SGC hydrogel.H) The programmed expression of osteogenesis-related genes at different times induced by SGC hydrogel.I) Schematic diagram of the angiogenesis and osteogenesis mechanisms induced by the SGC hydrogel.

Figure 6 .
Figure 6.In vivo evaluation of bone regeneration after SGC hydrogel treatment on infected critical size bone defects of SD rats.A) Schematic diagram illustrating the timeframe of the in vivo study.B) Bacterial culture at day 3 and day 7, colony plate counts for the different groups.C) ELISA of TNF- and IL-6 in serum.D) Gross observations of defect repair at 2 and 4 weeks postsurgery.E) 2D micro-CT coronal, transverse and sagittal views of femur defect area.F) 3D images reconstructed from micro-CT analysis of hydrogels implanted in rat femurs for 2 and 4 weeks.G) Quantification of bone volume/total volume (BV/TV), trabecular bone number (Tb.N), trabecular thickness (Tb.Th), and trabecular space (Tb.Sp) of the defects.

Figure 7 .
Figure 7. A) VG staining of hard tissue sections of infected femoral critical sized bone defects with SGC application.B) Scheme showing the timeframe for subcutaneous injection of mineral dyes.C) Sequential fluorescent labeling observation showing new mineral deposition at IBD (red: Alizarin red, 1st week; blue: Tetracycline hydrochloride, 2nd week; green: Calcein, 3rd week).D) Schematic diagrams showing regenerative effect of SGC at early, middle, and late osteogenic stages.

Figure 8 .
Figure 8. Schematic diagram showing the design principle of the hybrid hydrogel and the mechanism of the hybrid hydrogel induced infected bone defect regeneration through spatiotemporally regulating the healing process.