Interleukin‐4‐loaded hydrogel scaffold regulates macrophages polarization to promote bone mesenchymal stem cells osteogenic differentiation via TGF‐β1/Smad pathway for repair of bone defect

Abstract Objective Tissue engineering is a promising strategy for repair of large bone defect. However, the immune system reactions to biological scaffold are increasingly being recognized as a crucial factor influencing regeneration efficacy. In this study, a bone‐bioactive hydrogel bead loaded with interleukin‐4 (IL‐4) was used to regulate macrophages polarization and accelerate bone regeneration. Methods IL‐4‐loaded calcium‐enriched gellan gum (Ca‐GG + IL‐4) hydrogel beads were synthesised. And the effect on cell behaviour was detected. Furthermore, the effect of the Ca‐GG + IL‐4 hydrogel bead on macrophage polarization and the effect of macrophage polarization on bone mesenchymal stem cells (BMSCs) apoptosis and osteogenic differentiation were evaluated in vitro and in vivo. Results BMSCs were able to survive in the hydrogel regardless of whether IL‐4 was incorporated. Immunofluorescence staining and qPCR results revealed that Ca‐GG + IL‐4 hydrogel bead could promote M2 macrophage polarization and increase transforming growth factor (TGF)‐β1 expression level, which activates the TGF‐β1/Smad signalling pathway in BMSCs and promotes osteogenic differentiation. Moreover, immunohistochemical analysis demonstrated Ca‐GG + IL‐4 hydrogel bead could promote M2 macrophage polarization and reduce cell apoptosis in vivo. In addition, micro‐CT and immunohistochemical analysis at 12 weeks post‐surgery showed that Ca‐GG + IL‐4 hydrogel bead could achieve superior bone defect repair efficacy in vivo. Conclusions The Ca‐GG + IL‐4 hydrogel bead effectively promoted bone defect regeneration via regulating macrophage polarization, reducing cell apoptosis and promoting BMSCs osteogenesis through TGF‐β1/Smad pathway. Therefore, it is a promising strategy for repair of bone defect.


| INTRODUC TI ON
Large bone defect remains a major clinical problem worldwide, which could be caused by tumour resection, congenital abnormality and trauma. Bone graft therapy is currently the most common clinical treatment. 1 However, there are several factors that limit the use of this clinical therapy, including bone-related disorders and limited donor supplies. 2 Recently, tissue engineering has become a promising approach for large bone defects regeneration. 3,4 An important issue regarding bone tissue engineering is the construction of biological scaffold with biofunction to modulate cellular behaviour such as proliferation, migration and differentiation. 5 Because of the capacity of imitating many matrix parameters of natural extracellular matrix (ECM), hydrogel-based biomaterials have become promising candidates for a large number of tissue engineering applications. 6 The hydrophilic polymeric networks of hydrogels can retain water without losing their inner structure, and they can also be used as carriers for drug and protein delivery. [7][8][9] Gellan gum (GG) hydrogel is a biodegradable polysaccharide with high cytocompatibility and has been approved by the United States Food and Drug Administration as a biomaterial. [10][11][12] Numerous studies on the application of GG hydrogels have been carried out in tissue engineering, including studies on brain tissue engineering and cartilage and intervertebral disc applications. 13,14 GG hydrogel-based matrices have great potential for the controlled delivery of stem cells, different drugs and growth factors in situ. 15 Biomineralization mediated by ionic exchange with the surrounding microenvironment has recently gained substantial attention and has been reported to promote bone mesenchymal stem cells (BMSC) osteogenic differentiation. 16,17 A previous study showed that Ca-GG hydrogels exhibited strong mineralization capacity because the incorporated calcium ions facilitate phosphate ion deposition and further mineralization. Furthermore, Ca-GG hydrogel beads are compatible with a wide range of molecular drug delivery applications. 18 Macrophages are myeloid precursor cells that derived from bone marrow and are an important part of the body's innate immune system. Macrophages play an important role in inflammation, host defence, tissue repair and metabolism. 19 Mononuclear macrophages possess the characteristics of diversity and plasticity, and they can differentiate into different phenotypes and play different roles in different microenvironments; this differentiation is called macrophage polarization, and it plays an important role in many physiological and pathological processes. 20 Studies show that the IRF1/STAT1 signalling pathway, which is activated by interferon (IFN)-γ and TLRs, can mediate M1 type polarization, while the IRF4/STAT6 signalling pathway, which is activated by IL-4 and IL-13, can mediate M2 type polarization. 21 M1 macrophages are characterized by the high expression of proinflammatory factors and nitrogen oxides, which have a killing effect on microorganisms. M2 macrophages are characterized by the high expression of scavenger molecules and exert highly efficient phagocytic and immunomodulatory functions; M2 macrophages can promote tissue repair. [22][23][24] The aggregation and differentiation of BMSCs at the site of bone defect and the subsequent formation of bone and cartilage are regulated by multiple factors of the local microenvironment. 25 The interaction and precise interregulation between macrophages and cells related to bone formation play increasingly important roles in both endochondral and membranous osteogenesis. 26 Some inflammatory cytokines secreted by M1 macrophages, such as tumour necrosis factor-α (TNF-α), could result in BMSCs apoptosis. And some inflammatory cytokines secreted by M2 macrophages could promote the osteogenic differentiation of BMSCs. 27 In this study, bone-bioactive Ca-GG hydrogel beads were successfully synthesized. Interleukin-4 (IL-4), one of the cytokines that can regulate the transformation of macrophages from the proinflammatory M1 phenotype into the anti-inflammatory M2 phenotype, 28 was incorporated into the hydrogel beads to regulate macrophage polarization and promote bone regeneration. The aqueous and ECMlike microenvironment in the GG hydrogel bead makes it a suitable IL-4 delivery vehicle. Then the effects of the Ca-GG + IL-4 hydrogel beads on macrophage immunomodulation, BMSCs apoptosis and osteogenic differentiation were evaluated both in vitro and in vivo.

| Preparation of Ca-GG + IL-4 hydrogel beads and conditioned media
GG powder (0.5 g, Sigma-Aldrich) and an appropriate volume of glycidyl methacrylate (Sigma-Aldrich) were added to 50 mL distilled water with continuous stirring at room temperature for 8 hours. Then, cold acetone was used to precipitate the reaction products. Next, the solution was further purified by dialysis and freeze-dried. Then, dry methacrylated GG was dissolved in distilled water to form a 1% (w/v) GG solution. Then, 100, 200 or 300 ng recombinant mouse IL-4 was added to 1 mL GG solution, respectively. The 0.1 mol/L calcium chloride aqueous solution was prepared using CaCl 2 anhydrous powder (Merck-Millipore).

| Cell culture
Mouse BMSCs and the murine-derived macrophage cell line RAW 264.7 (RAW) were used in this study. The BMSCs were isolated and cultured according to previous studies. 29,30 The RAW cells were cultured in DMEM (HyClone) supplemented with 10% FBS, 100 units/mL penicillin and 100 mg/mL streptomycin in a cell incubator.

| Proliferation capacity assay
The proliferation capacity of RAW 264.7 cells and BMSCs was detected using a Cell Counting Kit-8 (CCK-8, Dojindo). We seeded 2000 RAW 264.7 cells in a 96-well tissue culture plate. After 24 hours of incubation, we replaced the culture medium with 200 μL of conditioned media. Regular DMEM with 10% FBS was used as a control. At the designed time points (days 1, 2 and 3), the proliferation capacity of each well was tested using a CCK-8 assay according to the manufacturer's protocol. Briefly, 100 μL DMEM including 10 μL CCK-8 solution was added to each well at each time point and then incubated at 37°C for 1 hour. Then, the absorbance was measured at 450 nm.
The same assay was used to examine the proliferation capacity of BMSCs.

| Co-culture of BMSCs and macrophages
To determine the effects of macrophages treated with different conditioned media on the osteogenic differentiation of BMSCs, a transwell co-culture system was used. Macrophages were seeded on Hanging Cell Culture Inserts containing 0.4-μm pores in each well (Millipore) at a density of 5 × 10 4 per well in 6-well plates. The macrophages were firstly treated using 100 ng/mL LPS and 10 ng/mL IFN-γ for 24 hours and then incubated with different conditioned media ( Figure 3A). BMSCs were seeded in flat-bottom 24-well transwell plates at a density of 5 × 10 3 cells per well.

| BMSC apoptosis assay
Bone mesenchymal stem cell apoptosis was detected using an apoptosis detection kit (KeyGEN) according to the manufacturer's protocol. Briefly, BMSCs were harvested after 1 and 3 days of co-culture with macrophages. Then, the BMSCs were resuspended using 1× annexin-binding buffer (100 μL). After 30 minutes of incubation with 1 μL propidium iodide (PI, 100 μg/mL) and 5 μL Annexin V in the dark, the apoptosis rate of the BMSCs was analysed using flow cytometry. In addition, the total protein was harvested on days 1 and 3, and cleaved caspase 3 protein expression levels were detected using Western blotting.

| Osteogenic differentiation assay
Bone mesenchymal stem cells and macrophages were seeded in a Transwell co-culture system. After 24 hours of incubation, the BMSC culture media were replaced with osteogenic medium (10 mmol/L β-glycerophosphate, 50 μmol/L l-ascorbic acid 2-phosphate and 100 nmol/L dexamethasone). The alkaline phosphatase (ALP) activity was detected at day 7. After 2 weeks of induction, calcium deposition was assessed using Alizarin Red S (ARS) staining.
The total RNA and protein were harvested after 7 days of co-culture to determine the expression of osteogenesis-related genes and proteins using qPCR and Western blot.

| Activation of TGF-β1/Smad signalling pathway
The total protein was harvested from the BMSCs after 5 days of co-culture. To investigate the activation of the TGF/Smad signalling pathway in the BMSCs, the related proteins, including TGF-β1 receptor (TGF-β1R), phosphorylated Smad2(p-Smad2) and phosphorylated Smad3(p-Smad3), were analysed using Western blot. To further confirm the participation of the TGF/Smad pathway, we inhibited TGF-β1R expression of BMSCs by siRNA. Two siRNA sequences were used in this study and the sequences were: 5′UUUCCCAGAGUACCAGAGCT-T3′. The TGF-β1/Smad signalling pathway-related proteins, including the TGF-β1 receptor, p-Smad2 and p-Smad3, were analysed using Western blot.

| In vivo animal experiment and histological evaluation
All Ca-GG + IL-4 (10 ng) hydrogel beads were implanted. 27 We implanted 3 Ca-GG hydrogel beads into the defects of the rats in Ca-GG hydrogel bead group. And 3 Ca-GG hydrogel beads loading 10 ng of IL-4 were implanted into the defects of the rats in Ca-GG + IL-4 group.

| Evaluation of osteogenesis in vivo
To evaluate the bone regeneration capacity of Ca-GG + IL-4 hydrogel bead in vivo, a critical-sized bone defect was created in the rat mandible using a 5-mm diameter trephine burr. 31

| Statistical analysis
All data are expressed as mean values ± SD. We used GraphPad Prism 8.0 to compute statistical significance between 2 groups with 2-tailed Student's t test. And the statistical significance between more than 2 groups was compared by one-way ANOVA followed by Dunnett's multiple comparisons test. P < .05 was considered statistically significant.

| Structure characteristic, cell viability, cell proliferation and IL-4 release profile
SEM analysis showed that the micro-structure of Ca-GG was porous ( Figure 1B), which facilitates the loaded IL-4 to enter the surrounding tissues in vivo. The diameter of Ca-GG hydrogel bead is (2.03 ± 0.11) mm ( Figure 1C), and the porosity of Ca-GG hydrogel is 74.14 ± 2.27%. The cumulative release profiles are plotted in Figure 1D. The conditioned media with or without IL-4 did not significantly change the proliferation rates compared to the control media ( Figure 1E). Live and dead staining results demonstrated that Ca-GG hydrogel with or without IL-4 had no significant effect on the ratio of live cells in the gels ( Figure 1F).

| Effect of conditioned media on macrophage polarization in vitro
Immunofluorescence staining results demonstrated that LPS + IFN-γ treatment resulted in higher expression of CCR7, and macrophages in the Ca-GG + IL-4 hydrogel bead groups was significantly higher than that of LPS + IFN-γ alone group ( Figure 2F).  Figure 4D,E). The qPCR data were consistent with the following Western blotting results ( Figure 4F).

| The activation of TGF-β1/Smad pathway in BMSCs
The protein expression of TGF-β1R, p-Smad2 and p-Smad3 in Ca-GG + IL-4 groups was significantly increased compared with that of LPS + IFN-γ alone group after 5 days of co-culture ( Figure 5A,B).
And the expression levels of TGF-β1R mRNA were similar to protein expression ( Figure 5C). To further confirm the participation of the TGF/Smad pathway, we inhibited TGF-β1R expression by siRNA and the knockout rate was 73.86 ± 7.01% ( Figure 5D-F). ALP staining showed that inhibition of the TGF/Smad pathway reduced the osteogenic capacity of BMSCs compared to that observed in the Ca-GG + IL-4 group ( Figure 5G). The increased expression of osteogenic-related genes in BMSCs induced by Ca-GG + IL-4 hydrogel bead-conditioned media was reversed by the inhibition of the TGF-β1/Smad pathway ( Figure 5H,I).

| Early-stage host response and macrophage polarization in vivo
The tissue surrounding the implanted material presented sig- post-implantation in vivo ( Figure 6C).

| Cell apoptosis assay in vivo
Immunohistochemical analysis showed that a lower level of TNF-α expression was observed in Ca-GG + IL-4 group at day 7 post-implantation compared to that of other 2 groups ( Figure 7A). However, no significant difference was observed in the expression of TNF-α at day 3 post-implantation. TUNEL assay results demonstrated that a significantly low level of cell apoptosis was observed at 7 days after implantation in Ca-GG + IL-4 group. Similarly, no significant difference was observed at day 3 post-implantation ( Figure 7B).

| Bone regeneration capacity analysis
Micro-CT examination showed that newly formed bone in Ca-GG + IL-4 group expanded to most of the bone defect area. The ratio of new BV to total BV (BV/TV, %) was 8.56 ± 2.16% in NC group, 18.44 ± 3.40% in GG group, and 29.63 ± 4.32% in Ca-GG + IL-4 group. The quantification analysis of the newly formed bone showed that the Ca-GG + IL-4 group had up to 3.46-fold more newly formed bone volume than that of NC group ( Figure 8A). And the Quantification analysis of bone mineral density, trabecular number and trabecular separation showed that Ca-GG + IL-4 hydrogel beads achieve superior bone defect repair efficacy ( Figure 8B). Immunohistochemical analysis of Runx-2 and OCN showed that the osteogenetic activity of the Ca-GG + IL-4 group was higher than that of other 2 groups ( Figure 8C,D).

| D ISCUSS I ON
Host immune reaction to tissue engineering scaffold has been recognized as a crucial factor that determines therapeutic efficacy. 32,33 This study presented evidence that immunomodulation patterns play a crucial role in osteogenesis during bone defect regeneration. Bone regeneration involves a series of complex and continuous physiological processes. 34 Several kinds of cells, including macrophages, stem cells, osteoblasts, osteoclasts and endothelial progenitor cells, are recruited from their local niches and then develop into mature bone tissues and vessels. 35,36 The reaction between tissue engineering scaffold and surrounding cells may determine the therapeutic efficacy; therefore, the scaffold material always delivers a drug to manage the host response, such as immunomodulatory drugs. 37,38 M1 phenotype macrophages secrete factors such as SDF-1, which is involved in BMSC migration, while M2 phenotype macrophages produce factors such as TGF-β1, BMP-2 and VEGF, which promote bone regeneration. 39 Increasing studies have shown that the precise and sequential polarization of M1 and M2 macrophages accelerates the tissue regeneration process. 40,41 Here, the data in this study showed that the M1 phenotype rapidly infiltrated into the bone defect area at the early stage of injury, while the M2 phenotype showed a gradually increasing trend. Hydrogel is an ideal material that can effectively mimic the ECM microenvironment and consequently promote cell migration and regeneration outcomes. 42 Some studies have shown that Ca-GG hydrogels are functional bone-bioactive materials that are not only able to mineralize but also compatible with efficient drug delivery applications in a wide range of molecular weights. 18,43 Previous study showed that proper delivery of IL-4 can generate the most preferable M1/M2 macrophage profile, resulting in a pro-healing microenvironment coupled with enhanced downstream osteogenesis. 27 Therefore, we developed a strategy that using Ca-GG hydrogel bead to delivers IL-4 for repair of jaw defect.
The therapeutic outcome of mandibular defects in this study showed that the sustained local release of IL-4 from the bone-bio-