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. 2023 Jan 5;15(2):2639–2655. doi: 10.1021/acsami.2c18545

Janus-Inspired Core–Shell Structure Hydrogel Programmatically Releases Melatonin for Reconstruction of Postoperative Bone Tumor

Wei Huang 1, Xiaoyue Wu 1, Yifan Zhao 1, Yanhua Liu 1, Bo Zhang 1, Mingxin Qiao 1, Zhou Zhu 1,*, Zhihe Zhao 1,*
PMCID: PMC9869893  PMID: 36603840

Abstract

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At present, surgery is one of the main treatments for bone tumor. However, the risk of recurrence and the large area of bone defects after surgery pose a great challenge. Therefore, a Janus-inspired core–shell structure bone scaffold was designed to achieve the self-programmed release of melatonin at different concentrations, clearing the residual tumor cells at early stage after resection and promoting bone repair later. The layered differential load designs inspired by Janus laid the foundation for the differential release of melatonin, where sufficient melatonin inhibited tumor growth as low dose promoted osteogenesis. Then, the automatically programmed delivery of melatonin is achieved by the gradient degradation of the core–shell structure. In the material characterization, scanning electron microscopy revealed the core–shell structure. The drug release experiment and in vivo degradation experiment reflected the programmed differential release of melatonin. In the biological experiment part, in vivo and in vitro experiments not only confirmed the significant inhibitory effect of the core–shell hydrogel scaffold on tumor but also confirmed its positive effect on osteogenesis. Our Janus-inspired core–shell hydrogel scaffold provides a safe and efficient means to inhibit tumor recurrence and bone repair after bone tumor, and it also develops a new and efficient tool for differential and programmed release of other drugs.

Keywords: bone regeneration, melatonin, Janus, core−shell hydrogel, antitumor

1. Introduction

Bone tumors are tumors that occur in the bone or its accessory tissues, including primary tumors and secondary tumors.1,2 Primary bone tumors may formulate localized lump, while secondary bone tumors may happen after the metastases from other parts of the body through blood or the lymphatic system.3 Comprehensive approaches based on surgical excision are the most used treatment for bone tumors.4 However, the surgical removal of the tumor inevitably results in some degree of bone loss, as well as residual tumor cells.5,6 How to eliminate the residual tumor cells in the early postoperative stage and promote the repair of bone defects later is an urgent issue after bone tumor surgery.

At present, common methods to inhibit tumor recurrence are radiotherapy and chemotherapy.7 Although the methods can effectively kill tumor cells, they have an inevitable negative impact on the whole body and are not conducive to the healing of bone defects after surgery. Common methods to promote bone repair, such as the use of growth factors, have little effect on inhibiting tumor recurrence.8 Previous reports showed that some studies used the controlled release of a variety of drugs to achieve the multi-effect of killing tumor cells and repairing bone defects, and some studies used the method of chemical synthesis to load drugs so as to achieve the delivery of drugs.2,9,10 Nevertheless, there are still some problems bothering us. When multiple drugs are used, the effects of each drug may interfere with each other, and the use of multiple drugs may also increase the burden of liver and kidney, while the chemical synthesis may put forward a higher requirement for synthetic conditions. In addition, usually chemically synthesized materials have limited drug loading. In this case, the multiple effects of melatonin on the antitumor property and osteogenesis promotion have attracted our attention.11,12 Melatonin is one of the hormones secreted by the pineal gland of the brain.13 The secretion of melatonin has a distinct circadian rhythm, and the secretion is suppressed during the day while active at night.14 Previous research showed that high concentrations of melatonin could significantly inhibit tumors, while low concentrations of melatonin could promote bone formation in vivo and in vitro.15−18 Thus, the key point to make use of the multifunction of melatonin is to achieve differential melatonin loading and release on time and demand so as to fit with the different requirements of the different stages in the postsurgery of bone tumors.

Tissue engineering has become one of the most commonly used approaches for bone tissue reconstruction and regeneration.19 Since there is a strong need to both inhibit tumor recurrence and promote bone repair after bone tumor surgery, the development of a bone tissue engineering scaffold with differential drug loading and delivery while with characteristic of programmed release of melatonin is a promising option. Based on the characteristics of melatonin, this paper designed a kind of Janus-inspired core–shell hydrogel bone scaffold Mel@Gel/Mel@HF. The core part of Mel@Gel/Mel@HF was composed of the composite of HAMA and F127DA (HF) loaded with a low concentration of melatonin (Mel@HF), which had high elasticity, relatively stable degradation performance, and excellent biocompatibility. Meanwhile, the shell part was made up of GelMA (Gel) loaded with a high concentration of melatonin (Mel@HF). The difference between the core layer and the shell layer including characteristics of materials, concentrations of melatonin, and release speed or time was inspired by the conformation difference of Janus material. Also, the core–shell structure controlled the melatonin release to guarantee that a high concentration of melatonin would be released fast in the early stage of postsurgery to eliminate residual tumor cells and a low concentration of melatonin could be gradually released to promote the osteogenesis process in the middle late stage. Mel@Gel/Mel@HF could not only differentially load melatonin but also automatically programmatically released melatonin in different stages according to the needs of treatment. From the perspective of functional difference, we had developed a conception of differential melatonin loading inspired by Janus conformation difference, which laid a material foundation for realizing the multifunction of melatonin. From the perspective of time difference, the automatically programmed release of different concentrations of melatonin in different treatment stages could be achieved by designing the core–shell structure hydrogel structures with different properties. In terms of the characteristics of bone repair, the shell hydrogel Gel not only had good biocompatibility but also could be degraded in a short time to release lots of melatonin so as to kill tumor cells and minimize the negative effect on osteoblasts. However, the inner core hydrogel material involved in bone repair not only possessed good stability and excellent mechanical properties but also had a long degradation time, which made it possible to provide an excellent scaffold for the bone repair process and long-term release of melatonin. Through these ingenious designs, the Janus-inspired core–shell hydrogel bone scaffold Mel@Gel/Mel@HF designed in this paper hopes to achieve the self-controlled release of melatonin at different concentrations to conform to the physiological process and realize the differential drug loading effect inspired by Janus to maximize the safety and effectiveness of melatonin, in which one drug has multiple effects.

According to the original intention of the design, we confirmed the successful synthesis of the core–shell structure through scanning electron microscopy (SEM) and its related analysis. Fourier transform infrared (FTIR) and small angle scattering (SAXS) experiments confirmed that melatonin was evenly distributed in the inner core layer and outer shell layer, respectively. The differential drug release was verified by the in vivo degradation experiment and in vitro drug release experiment. In the biological experiment, in vitro and in vivo experiments confirmed that Mel@Gel/Mel@HF could not only inhibit tumor growth by inhibiting the expression of Yap1 of the Hippo pathway but also promote osteogenic differentiation and bone defect repair in vivo. This paper successfully developed the Janus-inspired core–shell hydrogel bone scaffold Mel@Gel/Mel@HF. Benefiting from the different design of core and shell layers inspired by Janus conformation difference, the multiple effects of different concentrations of melatonin on the antitumor property and osteogenesis are able to be achieved. Meanwhile, the core–shell structure can realize automatically programmed release of melatonin, where Mel@Gel/Mel@HF can release high concentrations of melatonin at the early stage after tumor surgery to effectively kill residual tumor cells and continuously and slowly release low concentrations of melatonin in the middle and late stages to promote osteogenesis in the process of bone repair. The shell and core layers of Mel@Gel/Mel@HF not only have good biocompatibility, but also the inner core hydrogel has good stability, excellent mechanical properties, and suitable degradability, which can avoid soft tissue invasion in the process of bone repair and provide a stable scaffold for bone repair. This study is expected to provide a new safe treatment method with simple synthesis and common raw materials for the full-course managing reconstruction of postoperative bone tumor, as well as a new effective tool for the differential loading and programmed loading of other drugs.

2. Materials and Methods

2.1. Cell Culture and Osteogenic Induction

Osteosarcoma cell lines U2OS and MG63 and melanoma cell line B16 were chosen as representatives of bone tumor. U2OS, MG63, and B16 cells were purchased from Procell (Wuhan, China). Bone marrow mesenchymal stem cells (BMSCs) were purchased from Cyagen Bioscience Company (Guangzhou, China). The raising conditions and osteogenic method are described in Supporting Information 1.1.

2.2. Synthesis of Mel@Gel/Mel@HF

Synthesis methods of GelMA, HAMA, and F127DA were referred to a previous study.20 The synthesis process of Gel/HF is shown in Figure 1a. Specifically, the solution containing 5 wt % HAMA and 5 wt % F127DA melted in advance was injected into the 3D-printed core–shell mold. After photocuring for 1 min, the solid Gel hydrogel was placed on the 3D-printed base, covered with the 3D-printed fence mold, and slowly injected with 10 wt % GelMA to wrap the HF core hydrogel before photocuring for 1 min. After removing the fence, the core–shell structure hydrogel missing the bottom was obtained, then 10 wt % Gel hydrogel was added to the missing part, and the Gel was cured for 1 min to successfully construct Gel/HF. The construction process of Mel@Gel/Mel@HF was similar to that of Gel/HF. It was only necessary to mix melatonin and core hydrogel solution by the proportion of 5 mg:4.3051 mL (5 mM) before injecting them into the mold. Similarly, the shell hydrogel only needed to mix melatonin and Gel according to 100 mg:4.3051 mL (100 mM) before injecting them into the mold. The leaching solution of the shell layer and core layer was acquired according to a previous report.21 The measurement method of the swelling ratio of Gel and HF is described in Supporting Information 1.2.

Figure 1.

Figure 1

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Synthesis and characterization of Gel/HF. (a) The diagram showed the synthesis process of the Gel/HF core–shell hydrogel scaffold. (b) The 1HNMR experiment showed the spectra of F127DA, HAMA, and Gel. The red box indicated the light curing group. (c) SEM observed the surfaces of the Gel shell hydrogel, HF core hydrogel, and Gel/HF core–shell structure. The white arrow pointed out the boundary between the core and shell layers. Scale bars, 250 μm. (d) The EDS spot scan showed the proportion of the elements C, N, and O where the content of the N element was higher in the shell hydrogel. (e) The EDS line scan showed the content change of C, N, and O elements, and the N element content increased in the Gel shell hydrogel. The black arrow showed the junction of core and shell layers. (f) The mapping experiment exhibited the distribution of C, N, and O elements. The white arrow labeled the boundary of core and shell layers. Scale bar, 100 μm. (g) The tensile strength test showed that the HF core hydrogel had higher tensile strength. (h) The compression strength test presented that the HF core hydrogel had the strongest compression strength, followed by the Gel/HF core–shell hydrogel scaffold and Gel shell hydrogel. *P < 0.05 and **P < 0.01.

2.3. Characteristic of Mel@Gel/Mel@HF

2.3.1. Scanning Electron Microscopy (SEM)

SEM (KYKY Technology Development Ltd., China) was applied to observe the surface of the hydrogel after gold spraying on a freeze-dried hydrogel. Energy-dispersive spectroscopy (EDS) analysis was operated with SEM under the condition of 10 kV and 45–55 s to analyze the element composition of core and shell layers. Mapping was performed to analyze the C, N, and O distribution with the help of SEM.

2.3.2. Fourier Transform Infrared (FTIR) Spectroscopy

An FTIR spectrometer (Thermo Fisher Scientific, USA) was used to analyze melatonin loading in Gel or HF. All samples were freeze-dried before testing, and the FTIR test was conducted under the conditions of a resolution of 4.000 cm–1, a moving mirror speed of 0.4747, and an aperture of 100.

2.3.3. Rheological Experiments

Rheological properties of hydrogels were measured by a Physica MCR 301 rheometer (Anton Paar, Austria). For the photocuring-related dependent elastic moduli (G′) and viscous moduli (G″), all samples were measured under the setting of 1% strain and 5 rad/s angular frequency. For the viscosity test, the setting was at a shear rate of 10.0 1/s. For detecting shear thinning, the shear rate varied from 0.01 to 1000 1/s.

2.3.4. Small-Angle X-ray Scattering (SAXS)

A CuKα X-ray beam with a wavelength of 1.54189 Å was applied to run the SAXS (D8 DISCOVER, Bruker, Germany) experiment to explore the distribution of melatonin in the core or shell hydrogel, and each sample was exposed for 5 s.

2.3.5. Tension and Compression Experiments

Tensile and compression experiments were conducted to assess the mechanical property of hydrogels. For the tensile experiment, the length of the model was 3.6 cm, the cross-sectional area was 2 mm2, and the tensile speed was 2 mm/min. For compression, the cross-sectional area of the compression model was 25 mm2, the height of the model was 5 mm, and the compression speed was 2 mm/min.

2.3.6. Release Curve of Melatonin

The melatonin-loaded hydrogel was located in 4 mL of PBS at 37 °C. For the initial 7 days, the drug release was measured every day, it was measured once every 3 days after 7 days, and the solution was measured by a UV–Vis spectrophotometer (Shimadzu, Japan). The wavelength of melatonin was 277 nm.

2.4. Evaluation of the In Vitro Assay of Mel@Gel/Mel@HF

2.4.1. Transwell Assay

Transwell assay was performed to evaluate the migration ability of tumor cells. Approximately 5 × 104 cells were added into the top chamber (24-well; the pore size was 8 μm; Corning, USA), and the lower chamber was immersed into the leaching solution. Twenty-four hours later, the migrating cells on the lower surface of the membrane were fixed with 4% paraformaldehyde for 20 min and stained with crystal violet. The images were collected by a microscope. The quantitative analysis was conducted after treating the chamber with 33% glacial acetic acid for 10 min, and the result was read at 570 nm.

2.4.2. EDU Assay

Cell proliferation was evaluated by EDU assay. U2OS cells were seeded into a 96-well plate with the density of 5 × 103 per well. A Cell-Light EdU Apollo In Vitro Kit was bought from RiboBio (Guangzhou, China), and EDU assay was conducted under the instruction of the manufacturer. Detailed experimental methods are shown in Supporting Information 1.3.

2.4.3. Colony Assay

Tumor cells were inoculated into six-well plates with the density of 1000 per well, and then 2.5 mL of complete culture medium of tumor cells, GelMA leaching solution, and Mel@Gel leaching solution was added. After 10 days, the well plates were fixed with 4% paraformaldehyde for 30 min before being washed three times with PBS and stained with 0.1% crystal violet. After washing with PBS, a stereomicroscope was used to take pictures and count the number of colonies.

2.4.4. Cell Cycle Experiment

A total of 1 × 106 cells were collected after being washed three times with PBS. One milliliter of DNA holding solution and 10 μL of permeabilization solution were added and underwent vortex oscillation for 5–10 s to mix evenly. The cells were incubated for 30 min at room temperature away from light. The lowest loading speed was selected, and detection was performed on a flow cytometer (Beckman Coulter, USA).

2.4.5. Cell Apoptosis Experiment

The cell apoptosis experiment was conducted under the instruction of MultiSciences Biotech (Hangzhou, China). The details are depicted in Supporting Information 1.4.

2.4.6. Alkaline Phosphatase (ALP) and Alizarin Red S (ARS) Staining

The ALP staining reagent was bought from Beyotime (Shanghai, China). ALP staining was conducted under the direction of the manufacturer. ARS staining was conducted with 0.2% Alizarin Red S solution of Solarbio (Beijing, China). The details are depicted in Supporting Information 1.5.

2.4.7. Real-Time PCR (RT-PCR) and Western Blotting (WB)

RT-PCR was applied to detect mRNA relative expression, while WB was applied to detect protein expression. Detailed experimental methods are shown in Supporting Information 1.6. All primers are available in the Supporting Information, Table S1.

2.5. Evaluation of the In Vivo Assay of Mel@Gel/Mel@HF

2.5.1. Micro-CT

Rat skull samples were analyzed by micro-CT (SCANCO Medical AG, Fabrikweg 2, CH-8306 Bruettisellen, Switzerland) to evaluate the bone formation around the skull defect. Micro-CT adopted the scanning parameters of 70 kVp, 200 μA, AL 0.5 mm, 1 × 300 ms, and voxel size of 10.0 μm, and the reconstruction of the skull defect model was conducted with the help of SCANCO Medical visualizer software.

2.5.2. In Vivo Assay

Animal experiments in the study were carried out in accordance with the ISO 10993 standard. All procedures for animal experiments followed all ethical guidelines for laboratory animals and were approved by the Ethics Committee of West China Hospital of Stomatology, Sichuan University (WCHSIRB-D-2022-634). For the in vivo degradation experiment of materials, 6 week-old male SD rats were purchased from Dossy. Mel@Gel/Mel@HF and Gel/HF were placed subcutaneously into the rat’s back and then sutured, and the surgery process is shown in Figure S1. At the 1st, 6th, and 10th weeks, the materials were taken out and weighed, every time point contained three rats, and the measured weights are recorded in Tables S2 and S3. For biocompatibility evaluation, the blood of rats was collected for the routine blood test and blood biochemistry test. Meanwhile, the heart, liver, spleen, lung, kidney, and skin were also collected for HE staining at week 10, and the inflammation score evaluation was conducted in the way described in a previous study.22

For melanoma homotransplantation, female C57BL/6 mice aged 6 weeks were purchased from Dossy Experimental Animals Co., Ltd. (Chengdu, China). A total of 1 × 106 cells suspended in 200 μL of PBS were injected subcutaneously into the right flank of the C57BL/6 mice. Seven days later, the C57BL/6 mice were randomly divided into three groups, with each group containing five mice. Also, Gel/HF and Mel@Gel/Mel@HF were put beneath the melanoma at day 7, while the control group was sutured without putting anything. The volume of melanoma was measured every 2 days. C57BL/6 mice were sacrificed at day 15 after homotransplantation, and the melanoma was collected.

HE staining and TUNEL staining as well as immunohistochemistry staining were performed after the melanoma was collected. HE staining was conducted to observe the morphology of tumor overall, TUNEL staining was performed to assess the apoptosis in tumor, and immunohistochemistry staining detected the expression of YAP1. HE staining, TUNEL staining, and immunohistochemistry staining were performed under the instruction of the manufacturer.

For skull defects, 6 week-old male SD rats were purchased from Dossy (Chengdu, China). SD rats were divided into three groups randomly, which were the Control group, HF group, and Mel@HF group, and each group contained five rats. A 5 mm-diameter skull defect was created with a drill after the SD rats were anesthetized. Two months later, the skull defects of rats were collected for micro-CT analysis, HE staining, Masson staining, Sirius staining, and immunofluorescence staining. HE staining was applied to observe the overall situation around the skull defect. Masson staining was designed to detect new bone and new fibers. Sirius staining was used to stain new fibers, and immunofluorescence staining detected the expression of OPN. HE staining, Masson staining, Sirius staining, and immunofluorescence staining were conducted according to the instruction of the manufacturer.

2.6. Statistical Analysis

Statistical analysis was performed with SPSS 22.0 (SPSS, Inc., Chicago, IL, USA). The data was described by the form of the means ± standard deviation (SD). The statistical analysis between two groups was unpaired t test, and the statistical analysis for more than two groups was one-way analysis of variance (ANOVA). P < 0.05 was regarded as statistically significant.

3. Results and Discussion

3.1. Preparation and Characterization of Mel@Gel/Mel@HF

The Janus-inspired core–shell hydrogel scaffold Mel@Gel/Mel@HF was synthesized by loading melatonin into the core–shell structure Gel/HF. Therefore, the construction of the core–shell structure Gel/HF acted as the key barrier to realize the differential loading of melatonin. Figure 1a shows the synthesis process of Gel/HF. Figure S2 shows the images of Gel/HF. To construct Gel/HF, GelMA, HAMA, and F127DA materials were first synthesized, and 1HNMR results showed that the measured spectra of GelMA, HAMA, and F127DA were consistent with previous reports (Figure 1b).23−25 SEM was used to observe Gel, HF, and Gel/HF where a clear boundary between the core and shell layers was spotted, and the left side, which was the core part of the boundary, was similar to HF and the right side, which was the shell part, was like Gel (Figure 1c). Since F127DA contained no N element, the EDS spot scan analysis showed that the nitrogen content of the core layer was 9.99%, while that of the shell layer was 19.54%, which was in line with the fact that the nitrogen content of the Gel shell hydrogel was higher than that of the HF core hydrogel (Figure 1d), and not surprisingly, it was also observed that the nitrogen element of the shell layer increased during the EDS line scan from the core hydrogel to shell hydrogel (Figure 1e). In the mapping analysis, the distribution of C, N, and O elements between the core and shell layers was also obviously different where clear boundaries could also be observed (Figure 1f). In terms of physical properties, the swelling ratio of the inner core HF was significantly lower than that of Gel (Figure S3). The mechanical strength test showed that the tensile strength of HF is significantly higher than that of Gel, while the compressive strength of Gel/HF ranked between Gel and HF (Figure 1g,h). More interestingly, if the material is damaged, it can be repaired by dropping the same material, which is similar to the self-healing ability of hydrogels (Figures S4–S6).26

Mel@Gel/Mel@HF was designed to kill residual tumor cells and promote bone healing, so it was the key point to find the balance between eliminating tumor cells and protecting bone marrow stem cells from the excessive harm of high concentrations of melatonin. Combining the insight into eliminating tumor cells and promoting the osteogenesis of BMSCs, a concentration of 100 mM loaded in the Gel shell hydrogel was chosen for further study, which was named as the Mel@Gel shell hydrogel group according to the results of CCK8 assay (Figures S7–S9). To figure out the most suitable concentration of melatonin in the HF core hydrogel, the quantitative analysis of ALP was applied, which showed that the concentration of melatonin at 5 mM in the inner core layer promoted the osteogenesis process best, and the group of the HF core hydrogel containing 5 mM melatonin was named as the Mel@HF core hydrogel group (Figure S10). After loading melatonin into Gel/HF, FTIR experiments were used to detect the drug loading. The results showed that when melatonin was loaded into the Gel shell hydrogel, some wavenumbers attributed to melatonin like 1172 and 925 cm–1 occurred in the measured spectra compared to the spectra of Gel. Similarly, a wavenumber like 1537 cm–1 that belonged to melatonin was also detected after melatonin was loaded into the HF core hydrogel in comparison to HF (Figure 2a). Melatonin was evenly distributed in the Gel and HF hydrogels according to the results of the small angle scattering (Figure 2c). In the drug release experiment, the release rate of melatonin in the Gel hydrogel exceeded 80% within 7 days, while the release of melatonin in the HF hydrogel was relatively slower with about 60% of melatonin released within 30 days (Figure 2b). The porosity of Gel was bigger than that of HF, which also supported the finding that the release of melatonin was faster in Gel (Figure S11). The rheology experiment of core and shell layers was divided into groups that either loaded melatonin or not. In the study of shell layer rheology, the results of shear rate-dependent viscosity indicated that the viscosity of the Gel group remained stable around 0.07 Pa·s, while that of the Mel@Gel group remained stable around 0.14 Pa·s (Figure 2d). In the analysis of the dynamic time-sweep rheological experiment, G′ was initially lower than G″ of both Gel and Mel@Gel groups, and G′ would surpass G″ at about 35th second after light curing was activated at the time of 30th second. Notably, either G′ or G″ of the Mel@Gel group was always higher than its counterpart in the Gel group (Figure 2e). After photocuring, frequency-dependent G′ and G″ were detected where G′ of both groups was stable around 10–3 MPa, while G″ hovered around 10–5 MPa. Similarly, the G′ and G″ of the Mel@Gel group dominated those of the Gel group (Figure 2f). The rheology experiment of the HF core hydrogel showed the similar tendency with the results of the Gel shell hydrogel. To put it simply, both HF and Mel@HF groups had stable viscosity where Mel@HF was higher in shear rate-dependent viscosity (Figure 2g). G′ would exceed G″ after light curing quickly where melatonin loading slightly increased G′ and G″ compared to those of the HF hydrogel after light curing in the dynamic time-sweep rheological experiment (Figure 2h). Also, after light curing, G′ was an order of magnitude greater than G″, and G′ and G″ essentially remained steady (Figure 2i).

Figure 2.

Figure 2

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Detection of melatonin loading and release as well as rheological properties of Mel@Gel/Mel@HF. (a) FTIR spectroscopy detected the spectra of Mel, Mel@Gel, Gel, Mel@HF, and HF. The red circle indicated some peaks belonging to Mel while occurring in Mel@Gel or Mel@HF. (b) Release curve of Mel from Mel@Gel and Mel@HF. The red circle labeled the degraded time point of Mel@Gel in vivo. (c) The small angle scattering assay showed that melatonin was evenly distributed in Mel@Gel or Mel@HF. (d) The shear rate-dependent viscosity showed that Gel and Mel@Gel had stable viscosity as the shear rate increased. (e) The dynamic time-sweep rheological analysis of Gel and Mel@Gel exhibited the change process of G′ and G″ during the process of light curing. (f) Frequency-dependent G′ and G″ were tested after Gel and Mel@Gel were lightly cured. (g) The shear rate-dependent viscosity showed that HF and Mel@HF exhibited stable viscosity as the shear rate went up. (h) The dynamic time-sweep rheological analysis of HF and Mel@HF showed the change process of G′ and G″ during the process of light curing. (i) Frequency-dependent G′ and G″ were applied on the light curing product of HF and Mel@HF.

To apply the multifunction of melatonin at different concentrations, a conception of differential melatonin loading based on Janus differential conformation came into mind.27 Furthermore, a core–shell structure hydrogel scaffold was taken into consideration to achieve the automatically programmed release of melatonin so as to meet the needs of different treatment stages. In this study, we directly observed the double-layer hydrogels and their boundary through SEM. EDS and mapping analysis also supported the existence of the core–shell structure through the detection of the content of the nitrogen element. As a result, the hydrogel with a core–shell structure was effectively produced. Moreover, based on the good stability and excellent mechanical properties of the core hydrogel, it might offer stable support for the bone repair process and prevent soft tissue from encroaching on the location of the bone defect, creating a favorable environment for bone repair.

Further, FTIR experiments and small angle scattering confirmed the uniform distribution of melatonin in Mel@Gel/Mel@HF. The drug release test showed that the shell hydrogel of Mel@Gel/Mel@HF could rapidly release a large amount of melatonin within 7 days, addressing the issues with removing remaining tumor cells of the surgical area at the initial stage after surgery. On the other hand, the core part of Mel@Gel/Mel@HF continuously provided positive factors for bone repair by releasing melatonin in low concentration for a long time, which coincided with the long period of bone repair. In addition, the results of shear rate-dependent viscosity pointed out that both the core and shell hydrogels with or without melatonin were homogeneous, which made it possible to develop a uniform drug loading system. The experiment in dynamic time-sweep rheology then showed that all hydrogels mentioned in this study could be cured by light by detecting the change of G′ and G″ during the light curing process. Logically, the final test of the light-cured product demonstrated that the hydrogel could continue to exist steadily after light curing.

3.2. Biodegradation and Biocompatibility of Mel@Gel/Mel@HF

In terms of biodegradation of Gel/HF and Mel@Gel/Mel@HF, in vivo experiments were conducted. Targeting at exploring the in vivo degradation experiment of Gel/HF and Mel@Gel/Mel@HF, Gel/HF or Mel@Gel/Mel@HF was put subcutaneously into the rat’s back. At the time points of weeks 1, 6, and 10, the materials were acquired from the rat’s back to analyze the degradation ratio by the method of measuring weight loss. At week 1, it was observed that the shell hydrogel was completely degraded, while the core hydrogel still remained. By the end of the 6th week, the proportion of degradation reached almost 80% for both Gel/HF and Mel@Gel/Mel@HF, and Gel/HF as well as Mel@Gel/Mel@HF would completely degrade at week 10 (Figure 3a). For the biocompatibility of Gel/HF and Mel@Gel/Mel@HF, the blood of rats and the heart, liver, spleen, lung, kidney, and local skin of rats were collected for further analysis. Through the results of blood routine and blood biochemical indicators, it was implied that implantation of Mel@Gel/Mel@HF did not cause systemic inflammation and metabolic function damage significantly (Figure 3b,d). Through HE staining (Figure 3c) and its score evaluation (Figure S12), no significant damage was observed in the slices of organs or skin. In addition, the results of live dead staining, staining experiment of cytoskeletons, and CCK8 assay of the core layer hydrogel, which was mainly involved in bone repair, are shown in the Supporting Information, Figures S13–S15. One of the most important properties of materials used in medicine is biocompatibility, and in vivo studies demonstrated that the novel Janus-inspired Mel@Gel/Mel@HF exhibited excellent compatibility, making Mel@Gel/Mel@HF a candidate in bone tumor-related bone repair. After surgical resection of the bone tumor, tumor cells needed to be eliminated quickly in the early stage. Later, it was necessary to provide some long-term promoting factors and a good scaffold for the attachment of osteoblasts during the repair of bone defects. The Janus-inspired core–shell hydrogel Mel@Gel/Mel@HF perfectly matched the phased requirements of the bone tumor postoperation. Due to the fast degradation of the shell layer of Mel@Gel, lots of melatonin carried by the Gel hydrogel would release like flood to drown bone tumor cells. However, the core hydrogel in the inner core layer was much more stable, and the HF hydrogel in vivo took more than a month to decompose entirely, giving the core layer of Mel@HF the capacity to release melatonin over an extended period of time and to act as a space-occupying support without interfering with soft tissue. The differential release of melatonin verified by the drug release experiment, the core–shell structure confirmed by SEM, and the difference of degradation speed made up the most important characters that allowed the automatically programmed release of different concentrations of melatonin to satisfy the requirements for different stages.

Figure 3.

Figure 3

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Evaluation of material degradability and biosafety. (a) Degradation curve of Gel/HF and Mel@Gel/Mel@HF in vivo. The blue circle labeled the degradation time point of the shell hydrogel. (b) Hematological examination of rat blood was carried out after subcutaneous implantation of Gel/HF and Mel@Gel/Mel@HF in the backs of SD rats at week 10. (c) The heart, liver, spleen, lung, kidney, and skin of control, Gel/HF, and Mel@Gel/Mel@HF groups were collected for HE staining. Scale bars, 100 μm. (d) Blood biochemistry examination of rat blood was carried out after subcutaneous implantation of Gel/HF and Mel@Gel/Mel@HF in the backs of SD rats at week 10.

3.3. Mel@Gel Shell Hydrogel Suppresses Osteosarcoma and Melanoma In Vitro

To explore the antitumor effect of Mel@Gel/Mel@HF, the shell layer of Mel@Gel leaching solution was obtained to treat melanoma cell line B16 and osteosarcoma cell lines U2OS and MG63. Figure 4a exhibits the experimental principle of the antitumor effect of the leaching solution from Mel@Gel in vitro. CCK8 assay showed the effect of 100 mM melatonin loaded in GelMA on U2OS, B16, and BMSCs, and the results indicated that the concentration of melatonin at 100 mM began to influence the proliferation of BMSCs significantly, while the concentration of melatonin at 100 mM exposed a significant inhibitory effect on the proliferation of B16 and U2OS (Figure 4b). Transwell assay and its corresponding quantitative analysis indicated that migration of B16 and U2OS cells was impeded by the leaching solution from Mel@Gel compared with control and Gel groups (Figure 4c,f). In colony formation assay, control and Gel groups formulated more colonies than the Mel@Gel group after 10 days of treatment of leaching solution (Figure 4d), and the semiquantitative analysis of colony assay presented the same evidence as colony assay (Figure 4g). Meanwhile, it was found that the Mel@Gel group decreased the fluorescence intensity of EDU staining of B16 in contrast with control and Gel groups (Figure 4e,h). To figure out the effect of Mel@Gel on the cell cycle and cell apoptosis, flow cytometry assay was applied to detect the cell cycle and cell apoptosis. The results of cytometry showed that the Mel@Gel group prolonged the G0/G1 period of both B16 and U2OS while reducing the period of G2/M of B16 and U2OS, which hindered the mitotic process of cells (Figure 4i and Figure S16). In cell apoptosis assay, Mel@Gel induced more apoptosis of B16 and U2OS obviously compared to control and Gel groups (Figure 4j and Figure S17). The results of Transwell assay, colony assay, and cell cycle of MG63 presented in the Supporting Information also supported the inhibitory role of Mel@Gel in MG63 (Figures S18–S20).

Figure 4.

Figure 4

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The leaching solution of the Mel@Gel shell hydrogel inhibits tumor cells. (a) Effect of leaching solution from the Mel@Gel shell hydrogel on tumor cells. (b) CCK8 assay revealed the influence of leaching solution from Mel@Gel whose Mel concentration was 100 mM. (c) The leaching solution of the Mel@Gel group suppressed cell migration of B16 and U2OS detected by Transwell assays. Scale bars, 50 μm. (d) Colony formation was inhibited by the Mel@Gel group compared to Gel and control groups in B16 and U2OS. (e) Fluorescence intensity in the Mel@Gel group reduced, while control and Gel groups shared similar intensity in EDU staining. Scale bars, 100 μm. (f) Semiquantitative analysis of Transwell formation assay. (g) Semiquantitative analysis of colony formation assay. (h) Semiquantitative analysis of EDU staining. (i) The Mel@Gel group shortens the period of G2/M compared to Gel and control groups in the cell cycle experiment. (j) The cell apoptosis test showed that the Mel@Gel group increased the apoptosis of B16 and U2OS. *P < 0.05, **P < 0.01, and ***P < 0.001.

In a previous report, it was reported that a high concentration of melatonin could inhibit the migration, proliferation, invasion, and other activities of tumor cells, while a low concentration of melatonin was good for osteogenesis.28 Melatonin based on different concentrations can play different roles in killing tumor cells and promoting bone repair, which is just in line with the need for killing tumor cells in the early stage after tumor resection and promoting bone repair in the middle and late stages. Thus, from the perspective of effect, it was necessary to make use of a high concentration of melatonin to kill tumor cells and utilize a low concentration of melatonin to promote bone repair. At the same time, from the time dimension, to produce the therapeutic effect of melatonin at various treatment stages, it was necessary to achieve the programmed release and staging release of melatonin of varying concentrations. Therefore, the Janus-inspired core–shell hydrogel scaffold came into mind. The Janus-inspired core–shell hydrogel scaffold Mel@Gel/Mel@HF was designed to achieve the goal of differential melatonin loading and automatically programmed release of melatonin so as to apply the multifunction of melatonin to the antitumor property and promotion of bone repair. The differential melatonin concentrations inspired by Janus conformation provided the material foundation of realizing the multifunction of melatonin, and the core–shell structure made it possible for the automatically programmed release of melatonin in different stages. Here, the antitumor effect of Mel@Gel/Mel@HF was not only preliminarily verified through experiments such as CCK8 assay, Transwell assay, colony assay, and EDU staining, but the optimal concentration for both the antitumor effect and avoidance of excessive osteogenic impair was also selected through in vitro experiments. However, the mechanism of the inhibitory role of Mel@Gel in tumors still remained unclear.

3.4. Mel@Gel Shell Hydrogel Plays an Inhibitory Role in U2OS and B16 through the Hippo Signaling Pathway

To explore the underlying mechanism of Mel@Gel on the antitumor effect, U2OS was chosen for the RNA-sequence experiment. Compared with the Mel@Gel group, 3138 genes were upregulated while 2936 genes were downregulated in the control group. When comparing the Gel group with the Mel@Gel group, the Gel group had 2724 genes upregulated and 2638 genes downregulated (Figure 5a). Gene ontology (GO) analysis presented the most enriched biological processes (BPs), cellular components (CCs), and molecular functions (MFs), and some of them were involved in cell proliferation (Figure 5b). Taking the Mel@Gel group as the comparison standard, control and Gel groups had some common terms related to cell proliferation in BP that were upregulated, such as sister chromatid segregation, nuclear chromosome segregation, chromosome segregation, and sister chromatid cohesion, which indicated that cell proliferation in the Mel@Gel group was inhibited. As for CC and MF, it was also found that cell proliferation items like the chromosomal region, chromosome, centromeric region, ATPase activity, and ATPase activity were also downregulated in the Mel@Gel group. In addition, some other terms related to excessive movement and migration of cells (such as hyperactivity of animals) were also suppressed by Mel@Gel. In Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, it was found that some cancer-involved pathways were enriched the most (Figure 5c). For example, microRNA was widely studied in the regulation of tumor via interaction with upstream long noncoding RNA or downstream target genes.29 mTOR and Hippo pathways were hotspots in the field of tumor regulation.30,31 In addition, some other pathways like the cell cycle were also closely related to tumor cell behavior. In the heatmap analysis, the key downstream gene of the Hippo pathway, Yap1, was found to be expressed low in the Mel@Gel group (Figure 5d). Therefore, the Hippo pathway was selected for further study in this research. The Hippo pathway is consisted of a set of highly conserved kinases, including the kinase cascade, MST, lats, the downstream effector unphosphorylated yes1 associated transcriptional regulator (YAP1) and its homologous transcriptional cofactor TAZ, and the TEAD family of transcription factors,32 which play a significant role in carcinogenesis, tissue regeneration, and the functional regulation of stem cells.30,33,34 When the Hippo pathway is inhibited, unphosphorylated Yap/TAZ enters the nucleus to bind to TEADS and other transcription factors (TFs), which further affects gene transcription and participates in the regulation of cell proliferation, migration, and apoptosis, but when the Hippo pathway is activated, phosphorylated Yap/TAZ will bind to 14-3-3 and remain in the cytoplasm rather than entering the nucleus, thereby inhibiting cell proliferation and tissue growth.35−38 WB experiment was performed to detect YAP1 protein expression in B16 and U2OS cells. WB and its corresponding quantitative analysis results indicated that the Mel@Gel group reduced the protein level of YAP1 significantly, implying that Mel@Gel played the inhibitory role in B16 and U2OS by suppressing the expression of YAP1 of the Hippo pathway (Figure 5e,f). The illustration demonstrated the mechanism by which Mel@Gel inhibited tumor cells (Figure 5g).

Figure 5.

Figure 5

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The leaching solution of the Mel@Gel shell hydrogel downregulates Yap1 to suppress the activity of tumor cells. (a) The volcano map showed the distribution of differentially expressed genes comparing the control or Gel group with the Mel@Gel group. (b) GO analysis exhibited the most enriched items related to cancer activity upregulated in the control or Gel group. The red box labeled some items related to cancer activity. (c) KEGG analysis uncovered the most enriched pathways after U2OS was treated with a complete medium, leaching solution of Gel, or leaching solution of Mel@Gel. The red box selected pathways involved in cancer regulation. (d) The heatmap presented the differentially expressed genes among control, Gel, and Mel@Gel groups. The red box selected the downregulated gene Yap1 in the Mel@Gel group. (e, f) WB and its quantitative analysis detected the protein expression of YAP1 in control, Gel, and Mel@Gel groups of B16 and U2OS. (g) The schematic diagram illustrated the mechanism of Mel@Gel leaching solution on inhibiting tumor. ***P < 0.001.

3.5. Mel@HF Core Hydrogel Stimulates the Osteogenesis of BMSCs

To evaluate the osteogenic effect of Mel@Gel/Mel@HF, the leaching solution of Mel@HF was acquired to show its effect on BMSCs. Above all, a diagram has exhibited the experiment process of using Mel@HF leaching solution to promote BMSC osteogenesis (Figure 6a). First, surface antigen flow cytometry showed that CD29, CD44, and CD90 had high expression and CD45 had low expression, which conformed to the character of BMSCs (Figure 6b). Then, 5 mM melatonin was chosen for loading into the HF hydrogel. ALP and ARS staining showed that the Mel@HF group presented a stronger ALP intensity and more calcium nodules compared with control and HF groups (Figure 6e). Quantitative ALP analysis and semiquantitative analysis of ARS suggested that the Mel@HF group had the strongest ALP activity and formulated the most calcium nodules among three groups as well (Figure 6c,d). In the process of osteogenic differentiation of BMSCs, the ALP enzyme is active in the early stage.39 With the passage of time, the activity of ALP decreases, while the calcium nodules gradually come into formation.40,41 Therefore, in the late stage of the BMSC osteogenesis process, the osteogenic activity of BMSCs can be better reflected by detecting the formation of calcium nodules. Moreover, judging the osteogenic ability of BMSCs can also be achieved by detecting osteogenesis-related genes and osteogenesis-related proteins by PCR and WB experiments, respectively. In the PCR experiment, the mRNA expressions of Runx2, Osx, and Opn were enhanced in the Mel@HF group, while the HF group did not change significantly in comparison with the control group (Figure 6f). The WB experiment also detected the protein levels of RUNX2, OSX, and OPN, demonstrating the positive effect of the Mel@HF group on osteogenic differentiation of BMSCs (Figure 6g,h). As a member of the RUNX family, Runx2 plays a crucial role in the proliferation, development, and differentiation of osteoblasts.42 The expression of Runx2 varies at different stages of the preosteoblast maturation process, peaking in immature osteoblasts and gradually decreasing in mature osteoblasts.43 In addition, Runx2 also regulates the expression of Osx, an osteoblast-specific transcription factor encoded by the Sp7 gene, which plays an important role in osteoblast differentiation.44,45 OPN is a glycosylated protein widely found in the extracellular matrix and is widely expressed in both humans and animals, secreted by osteoblasts and osteoclasts, and involved in the homeostasis of bone formation.46,47 Through in vitro experiments of BMSC osteogenesis, we have preliminarily proved that the core hydrogels loaded with low concentrations of melatonin could effectively promote the osteogenic differentiation of BMSCs. Combined with the results of antitumor in vitro experiments, we could preliminarily believe that it was hopeful to achieve the goal of tumor inhibition in the early stage after tumor surgery and subsequent bone repair promotion by realizing the automatically programmed release of melatonin from the core and shell hydrogels. Therefore, the in vivo experiment would further be operated to verify the in vitro results.

Figure 6.

Figure 6

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The leaching solution of the Mel@HF core hydrogel promotes the osteogenic differentiation of BMSCs. (a) The diagrammatic sketch showed the effect of leaching solution from the Mel@HF core hydrogel on the osteogenesis of BMSCs. (b) Flow cytometry detected the expression of the surface characteristic antigens of CD29, CD44, CD90, and CD45 on BMSCs. (c) Quantitative ALP analysis showed the effect of the leaching solution from HF or Mel@HF on BMSC osteogenic differentiation. The concentration of Mel in Mel@HF was 5 mM. (d) The semiquantitative analysis of ARS was performed after ARS staining to assess calcium nodule formation. (e) ALP staining and ARS staining were applied to evaluate the osteogenesis activity of control, HF, and Mel@HF groups. Scale bars, 1 mm. (f) RT-PCR detected the gene expression of Runx2, Osx, and Opn to clarify the osteogenesis-promoting effect of Mel@HF. (g, h) The expression of RUNX2, OSX, and OPN proteins was figured out by the WB experiment and its corresponding quantitative analysis. **P < 0.01 and ***P < 0.001.

3.6. Mel@Gel/Mel@HF Inhibits the Melanoma In Vivo

For clarifying the antitumor effect of Mel@Gel/Mel@HF, B16 cells were injected subcutaneously into the C57BL/6 mouse, and pictures of the melanoma were taken according to groups 15 days later (Figure 7a). The growing curve of melatonin was recorded every 2 days to show the growing situation of melanoma. From the curve, it was indicated that Mel@Gel/Mel@HF did slow down the growth of melanoma, while Gel and control groups shared similar growing speed (Figure 7b). After the melanoma was harvested, HE staining, TUNEL staining, and immunohistochemical staining were applied to further evaluate the effect of Mel@Gel/Mel@HF on melanoma and its underlying mechanism. HE staining showed the overall morphology of B16 melanoma (Figure 7c). In the TUNEL assay, it was observed that Mel@Gel/Mel@HF increased the apoptosis of B16 cells obviously compared with Gel and control groups (Figure 7d). Therefore, it could be concluded that Mel@Gel/Mel@HF did play an inhibitory role in B16 in vitro and in vivo. To further explore the expression of YAP1 on the growth of melatonin, the immunohistochemical staining experiment was conducted to analyze the expression of YAP1 protein. Immunohistochemical staining results illustrated that YAP1-positively stained B16 cells in Mel@Gel/Mel@HF were significantly lower than those in Gel and control groups, which confirmed the results of the in vitro experiment (Figure 7e). Combining the results of in vivo and in vitro experiments, Mel@Gel/Mel@HF actively contributed to preventing tumor growth by suppressing the expression of YAP1.

Figure 7.

Figure 7

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Mel@Gel/Mel@HF inhibits the growth of B16 melanoma in vivo. (a) B16 melanoma was taken out and photographed 15 days after the subcutaneous injection of B16 in control, Gel/HF, and Mel@Gel/Mel@HF groups. (b) The volume of B16 melanoma recorded every 2 days was drawn into a line chart to show the growing situation of B16 melanoma. (c) HE staining showed the overall view of melanoma. Scale bars, 50 μm. (d) TUNEL staining showed that Mel@Gel/Mel@HF did increase apoptosis cells in vivo. The cell nucleus exhibited blue fluorescence, and green fluorescence was displayed by apoptosis cells. Scale bars, 50 μm. (e) Immunohistochemical assays evaluated the expression of YAP1 protein. Scale bars, 50 μm. ***P < 0.001.

Cell death is mainly divided into necrosis and apoptosis.48 Between them, cell apoptosis is a programmed death process playing an important role in the treatment of tumor cells. Many tumor treatment methods have the effect of promoting tumor cell apoptosis.49 For example, chemotherapy can often cause tumor cell apoptosis. In addition, the apoptosis of tumor cells can be also regulated by many genes. In a previous report, the low expression of Yap1 could promote the apoptosis of tumor cells and achieve the inhibitory effect on tumors.50 In this study, both in vivo and in vitro detection methods found that Yap1 expression under the treatment of Mel@Gel declined, which showed that the large amount of melatonin released from Mel@Gel in a short time postsurgery could work as an inhibitory factor for tumor. Taken together, it was concluded that Mel@Gel/Mel@HF was distinguished with suppressing tumors.

3.7. Mel@Gel/Mel@HF Promotes the Healing of Bone Defects

Mel@Gel/Mel@HF was not only accountable for suppressing the relapse of tumor but also was expected for improving bone formation in bone defect areas. Therefore, we conducted a skull defect model to assess the bone repair ability of the Mel@Gel/Mel@HF bone scaffold. At first, Mel@Gel/Mel@HF was translocated onto the skull defect, and 2 months later, the skull of the rat was harvested for micro-CT reconstruction. The results implied that the Mel@Gel/Mel@HF group had the best skull defect repair effect, while the Mel/HF group slightly accelerated the healing of the skull defect compared to the control group (Figure 8a). BV/TV, Tb.N, Tb.Th, and Tb.Sp based on micro-CT supported the conclusion of micro-CT (Figure 8b). HE staining and its inflammation score as well as bone healing score, Sirius red staining, and tissue immunofluorescence staining were further operated to analyze the skull defect area. HE staining showed the overall morphology of the skull defect area (Figure 8c). The inflammation score evaluating the inflammation situation and the bone healing score evaluating the healing of the skull defect are shown in Figure S21. Also, Sirius red staining presented that more fibers were produced in the Mel@Gel/Mel@HF group than the other two groups, while Gel/HF formulated more fibers than the control group (Figure S22). In Masson staining, it was observed that greater new bone formation occurred around the skull defect of the Mel@Gel/Mel@HF group, but the control group had the least new bone formation (Figure 8d). In addition, tissue immunofluorescence staining of the bone defect showed that OPN-positively stained cells in Gel/HF and control groups were obviously less than those in the Mel@Gel/Mel@HF group (Figure 8e,f). The core–shell hydrogel scaffold Mel@Gel/Mel@HF, which was inspired by Janus, was developed with the intention of achieving differential melatonin loading and autonomously timed release of melatonin by taking advantage of using melatonin’s antitumor and bone-healing properties; meanwhile, Mel@Gel/Mel@HF was expected to function as a bone scaffold. The differential melatonin concentrations inspired by Janus conformation were the foundation of realizing the multifunction of melatonin, and the core–shell structure guaranteed the automatically programmed release of melatonin. When Mel@Gel/Mel@HF is implanted into the bone defect site with tumor cells, the residual tumor cells around the bone defect should be eliminated in a short time first to clear the operation area for later bone repair. Thus, GelMA, a widely studied hydrogel that possessed good biocompatibility and could carry lots of drugs, was chosen for the shell hydrogel. Within 1 week, GelMA would degrade completely to release a large amount of melatonin to kill tumor cells. At the same time, excessive damages to the cells involved in the later bone repair stage should be avoided as far as possible. Naturally, it was crucial to strike a balance between removing tumor cells and minimizing excessive harm to the stem cells surrounding the bone defect. In this study, the balance between killing tumor cells and promoting bone defect was initially explored in the in vitro experiment. Then, in the in vivo study, Mel@Gel/Mel@HF showed the best bone healing effect, followed by the Gel/HF group and, last, the control group, which proved the successful seeking of the balance between tumor cell killing and bone repair promoting. As for the Gel/HF group, the better outcome compared to the control group may originate from its scaffold effect. Although the shell hydrogel degraded fast to release a large amount of melatonin in a short time, which may damage osteogenesis-related cells to a certain extent, thanks to the good biosafety, the scaffolding effect in the osteogenesis process, and the long-term release of the low concentration of melatonin, the Mel@Gel/Mel@HF group formed the most amount of new bone around the skull defect.

Figure 8.

Figure 8

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Mel@Gel/Mel@HF accelerates the healing of bone defects in vivo. (a) Micro-CT reconstruction of the skull defect of rat 2 months after the transplantation of control, Gel/HF, and Mel@Gel/Mel@HF groups. (b) BV/TV, Tb.N, Tb.Th, and Tb.Sp required by micro-CT analysis showed the bone repair of control, Gel/HF, and Mel@Gel/Mel@HF groups. (c) HE staining showed the overall view of the skull defect. The red arrow pointed to the new bone. Scale bars, 1 mm and 500 μm. (d) Masson staining showed the fiber formation and new bone formation in control, Gel/HF, and Mel@Gel/Mel@HF groups. The red arrow showed the new bone, while the orange arrow showed the new fibers. Scale bars, 1 mm and 500 μm. (e) Tissue immunofluorescence staining and (f) its semiquantitative analysis of OPN were enhanced in the Mel@Gel/Mel@HF group compared to Gel/HF and control groups. Scale bars, 500 μm. *P < 0.05 and **P < 0.01.

4. Conclusions

To sum up, the successful realization of multiple effects of melatonin by the Janus-inspired Mel@Gel/Mel@HF scaffold mainly relied on the design physiology of achieving the automatically programmed release of different concentrations of melatonin and the effect of the bone scaffold. The raw materials for Mel@Gel/Mel@HF are accessible and easy to synthesize, and the synthesis process is straightforward. Mel@Gel/Mel@HF provides a fresh treatment approach to preventing tumor recurrence after tumor resection and encouraging bone healing. At the same time, the Janus-inspired core–shell hydrogel scaffold Mel@Gel/Mel@HF designed in this study also provides a new and safe tool for differential drug loading and automatically programmed release of other drugs.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (grant no. 82101076), the Postdoctoral Research Foundation of China (no. 2020M683334), the Research Funding from West China School/Hospital of Stomatology Sichuan University (no. RCDWJS2021-7), the Research and Develop Program from West China School/Hospital of Stomatology Sichuan University (no. RD-02-202109), and the Construction of Chengdu AI Application Development Industrial Technology Foundation Public Service Platform (2021-0166-1-2).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.2c18545.

  • Methods and materials containing the detailed description of cell culture and osteogenic induction, EDU staining, equilibrium swelling experiment, cell apoptosis experiment, ALP staining and ARS staining, real-time PCR (RT-PCR), and western blotting (WB); tables containing primer sequences used for quantitative real-time PCR analysis and degradation data of Gel/HF in vivo and Mel@Gel/Mel@HF in vivo; figures including the image of Gel and Gel/HF hydrogels, swelling ratio experiment, CCK8 assay, quantitative ALP analysis, statistical analysis of the cell cycle, statistical analysis of apoptosis, Transwell assay and colony assay and cell cycle experiment of MG63, operation process of in vivo degradation and in vivo safety evaluation, Sirius staining of the skull defect, and so on (PDF)

Author Contributions

Z. Zhu and Z. Zhao designed the study and reviewed the manuscript. W.H. finished the majority of the manuscript. W.H., X.W., and Y.Z. provided assistance in painting figures. Y.L., B.Z., and M.Q. reviewed the manuscript. All authors read and approved the final manuscript.

The authors declare no competing financial interest.

Supplementary Material

am2c18545_si_001.pdf (2MB, pdf)

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