The effect of magnesium ions synergistic with mineralized collagen on osteogenesis/angiogenesis properties by modulating macrophage polarization in vitro and in vivo

In bone tissue engineering, the bone immunomodulatory properties of biomaterials are critical for bone regeneration, which is a synergistic process involving physiological activities like immune response, osteogenesis, and angiogenesis. The effect of the macrophage immune microenvironment on the osteogenesis and angiogenesis of various material extracts was examined in this experiment using Mg2+ and Nano-hydroxyapatite/collagen (nHAC) in both a single application and a combined form. This study in vitro revealed that the two compounds combined significantly inhibited the NF-κB signaling pathway and reduced the release of inflammatory factors from macrophages when compared with the extraction phase alone. Additionally, by contributing to the polarization of macrophages towards the M2 type, the combined effects of the two materials can significantly improve osteogenesis/angiogenesis. The results of in vivo experiments confirmed that Mg2+/nHAC significantly promoted bone regeneration and angiogenesis. This study offers a promising method for enhancing bone graft material osseointegration.


Introduction
Over the past, numerous biomaterials have been continuously created to improve osteogenic/angiogenic inductive capacity [1][2][3].Nano hydroxyapatite/ collagen (nHAC) is a biomimetic composite material that resembles natural materials in structure and composition, mainly composed of nanohydroxyapatite and type I collagen.NHAC has promising application potential in repairing defects and promoting new bone formation because of its excellent biocompatibility, biodegradability, osteoconductivity and osteoinductivity [4].However, Limitations in the biomechanical properties of nHAC make it difficult to maintain bone regeneration space in the load-bearing region [5].The repair of segmental bone defects can be achieved using medical metallic materials that possess high mechanical properties.In particular, its biological properties have made magnesium and its alloys an excellent material for orthopedic implants [6].
We have previously explored the introduction of magnesium-based metal materials to improve the mechanical strength of mineral collagen.Yu et al suggested that nHAC's mechanical properties are improved by combining it with Mg-Ca alloy and alleviates the degradation of the magnesium-based metal matrix to a certain extent [7,8].Subsequently, we also investigated the biological behavior and osteogenic mechanism of Mg 2+ /collagen type I, the main degradation product of Mg 2+ combined with nHAC, in promoting osteogenic precursor cell MC3T3-E1, and found that Mg 2+ and collagen type I could promote osteogenesis by regulating integrin α2β1-FAK-ERK1/2 signaling pathway [9].In the reconstruction of jaw defects, the interaction between bone graft materials and osteoblasts is not the only factor.During the process of bone defect repair, the crosstalk between chemokines, cytokines, and a variety of cells and biomaterials influence the bone tissue remodeling [4,10].A network of blood vessels that can carry vital nutrients and oxygen is also required for bone regeneration.Bone graft material interacts with immune cells and induces innate immune responses prior to interacting with bone and blood vessels [11].
Along with bone graft material being implanted, both innate and foreign microorganisms can subsequently colonize the implant surface and subsequently form microbial biofilms on the surface, which often cause complications associated with implantations, such as inflammation and other immune responses, failure of implants or disruptions of wound healing, and the adverse effects of scar tissue formation.Macrophages are an essential component of the immune cells [12].It initially participates in the local inflammatory response and tissue repair after trauma and then continues through the entire process of bone tissue formation, mineralization, and remodeling, making it a model cell [13].The inflammatory response to an implanted biomaterial is primarily mediated by macrophages.Macrophages are a highly heterogeneous group of immune cells that can polarize into a variety of phenotypes in response to the stimulation from the surrounding microenvironment, which are typically classified as M1 and M2 macrophages [14].M1 macrophages (classically activated macrophages) secrete interleukin-12 (IL-12) and interleukin-6 (IL-6), which exacerbate oxidative stress in bone defects, exacerbate local inflammation, and impair bone repair [15].M2 macrophages (alternatively activated macrophages) can secrete transforming growth factor (TGF-β) and interleukin-10 (IL-10), which can inhibit local inflammation and reduce osteogenic precursor cell differentiation [16,17].
Considering that the rapid explanation of magnesium-based metals in vivo is still the main reason limiting their use in clinical applications, their rapid degradation leads to a significant escape of magnesium ions.Nowadays several technological tools have proven to be excellent in improving the corrosion resistance of magnesium alloys, nonetheless the effect of magnesium ion concentration on immunomodulation should not be ignored when controlling the degradation rate of magnesium alloys.It has been shown that controlling magnesium ion concentration (∼100 mg l −1 Mg 2+ ) inhibits the TLR-NF-κB signaling pathway, induces an anti-inflammatory environment, and promotes osteogenic gene expression in mesenchymal stem cells [18].In addition, nHAC plays a critical role in inducing the transformation of macrophages into M2 type [19].However it is not known whether the combined application of magnesium ions and mineralized collagen can be more effective than a single material application.
Based on the above-mentioned reasons, Mg 2+ and nHAC were used in this study to explore whether Mg 2+ and nHAC can cooperatively regulate the immune response of macrophages and induce the polarization of macrophages to M2 phenotype.Furthermore, the effect of Mg 2+ combined with nHAC on osteogenic/angiogenic capacity through the modulation of the immune microenvironment was further investigated.Finally, the immune response and osseointegration capacity of Mg 2+ synergistic nHAC were investigated by in vivo experiments.

Preparation of material extracts
MgSO 4 (Sigma-Aldrich, USA) was dissolved in deionized water and was filtered through a 0.22 lm filter (Corning, USA), and then diluted into cell culture medium formulated 100 mg l −1 .Nanohydroxyapatite/collagen (purchased from Beijing Aojing Medical Technology Co., Ltd) consists primarily of type I collagen (Col I) and nano-hydroxyapatite (nHA).The preparation of nHAC is exactly the same as the method previously studied [20] and cut into a disk-like shape (Φ5 mm × 2 mm).According to the ISO10993-1-2018 standard, prepare nHAC impregnation solution according to the ratio of material surface area and liquid volume to 3 cm 2 ml −1 .
The experimental groups were as follows, blank control group: DMEM complete medium; Mg 2+ group: 100 mg l −1 Mg 2+ solution; NHAC group: nHAC disc DMEM placed in complete medium; Mg 2+ + nHAC group: nHAC disk placed in 100 mg l −1 Mg 2+ solution.

Cell culture
Three cell types, MC3T3-E1, RAW 264.7 cells, and HUVEC cells, were involved in this work.All of the cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).In addition to 10% fetal bovine serum and 1% penicillin and streptomycin, all cells were cultured at 37 • C, 5% CO 2 , and 95% humid air in DMEM (HyClone, USA) medium.When the cells reached a confluence state of 80%-90%, the subculture was carried out.Cells of passages 3-8 were applied in this experiment.

Macrophage response to material extract 2.3.1. Cell proliferation
RAW 264.7 cells were plated at a concentration of 2 × 10 3 per well in 96-well plates.After 24 h, RAW 264.7 cells were cultured for 1, 3, and 5 d in the four experimental groups as described in section 2.1.At each time point, the medium was removed and replaced with 100 µl serum-free medium in order to prepare for the CCK8 assay.And then add 10 µl Cell Counting KIT-8 (CCK-8; APExBIO, USA) solution was in each well, after incubation at 37 • C for 2 h kept from light, while at 450 nm wavelength, the optical density (OD) values were analyzed using microplate reader (Bio-Tek, USA) (n = 5 in each group).

Flow cytometry
By measuring the expression of CCR7 and CD206 on the surface of M1 and M2 macrophages, phenotypic changes in RAW cells were determined using flow cytometry.RAW 264.7 were plated at 5 × 10 5 cells ml −1 in 6-well plates.After overnight growth, lipopolysaccharide (LPS, Beyotime) was applied for 2 h at a concentration of 1 µg ml −1 for polarizing M0 macrophages into M1 phenotypes.After being rinsed in PBS 3 times, the cell's medium was replaced by each group for 3 d.Flow cytometry was used to detect the expression of M1 and M2 surface markers on RAW after 3 d of culture in each of the four medium groups.Briefly, the cells were scraped off and centrifuged at 1000 rpm for 5 min.PBS with rat anti-mouse CCR7 antibody (1:100) (BD Pharmingen, USA) and rat anti-mouse CD206 antibody (1:100) (BioLegend, USA) at 4 • C for 30 min was used after the cells had been resuspended and blocked with 1% BSA/PBS.On a FACS flow cytometer, the cells were analyzed (BD).

Real-time quantitative PCR analysis
RAW cells were incubated in 6-well plates at a density of 5 × 10 5 cells ml −1 .After overnight incubation, the cells were treated with LPS at a concentration of 1 µg ml −1 for 2 h and then stimulated with the experimental groups described in section 2.1 for 3 d.The total RNA was extracted using the Trizol reagent (Ambion, USA).Here,1 mg RNA from each group was reversed transcribed into complementary DNA using the Prime Script™ RT reagent kit (Takara).For each gene, we used the same forward and reverse primers indicated in the literature [9, 18,].The expression of TNF-α IL-6, IL-1β, IL-10, IL-1ra, BMP-2, and PDGF-B at mRNA level was quantified by real-time PCR (Bio-Rad iQTM5 multicolor realtime PCR detection system) using SYBR Premix Ex Taq™ (Takara).The qPCR program was initiated at 95 • C for 30 s; 40 cycles of 95 • C for 15 s, and 60 • C for 30 s. β-actin was applied as the internal reference gene.Using version 2.0 of the iQTM5 optical system software for data analysis.In table 1, the primers for the target genes are listed.

Western blot
We performed a Western blot to assess the expression of NF-κB p65 and IκB-α.RAW cells were seeded in 6-well plates and grown to 80% confluence in 6well plates.After stimulating RAW cells for 2 h with 1 µg ml −1 LPS, the experimental group described in section 2.1 stimulated the cells for 3 d.Protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) from each group and then transferred to nitrocellulose membranes.After blockation in 5% non-fat dry milk in TBS-T buffer (25 mM Tris-HCl, 140 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h and incubated with primary antibodies against NF-κB (1:1000, Abcam), α-tubulin (1:5000, Abcam) and IκB-α (1:1000, Cell Signalling Technology) at 4 • C overnight.The membranes were then treated for 90 min at room temperature with a secondary antibody.Before applying chemiluminescent chemicals and exposing the TBST, it was cleaned.Image J was used to semi-quantitatively assess Western blot images.

Effects of RAW cell-conditioned culture medium on MC3T3-E1
2.4.1.Collection and preparation of RAW conditioned medium culture RAW 264.7 cells were plated in 6-well plates at a density of 5 × 10 5 cells per well.After 1 d, the complete culture-medium were removed and added with the DMEM complete medium, and each experimental group as described in section 2.1 respectively.After 2 d of culture, the medium supernatant of each group was extracted and mixed with DMEM medium in a ratio of 1:2.The obtained supernatants were RAW cell-conditioned medium.They were separated into four groups: RAW, Mg 2+ + RAW, nHAC + RAW, and Mg 2+ /nHAC + RAW.

Osteogenic-related gene expression
MC3T3-E1 cells were plated at 1 × 10 5 cells ml −1 in 6-well plates.Gene expression levels of osteogenicrelated genes including runt-related transcription factor 2 (Runx2), osteopontin (OPN), and osteocalcin (OCN) were determined after 7 d of culture in the RAW cell-conditioned media using the RT-PCR technology as mentioned in section 2.3.3.The sequence of the primers used is summarized in table 1.

Alkaline phosphatase activity
MC3T3-E1 cells were plated in a 6-well plate at a density of 5 × 10 4 cells ml −1 in a RAW cellconditioned medium.After culturing for 7 d, we used cell lysis buffer without inhibitors (Beyotime, China) to lyse cells and extract total proteins.According to the manufacturer's instructions, a bicinchoninic acid protein assay kit (Thermo Fisher, USA) was used to measure the total protein content, and an ALP kit (Beyotime, China) was utilized to detect the ALP activity.Results are reported in units per mg of total protein.

Extracellular matrix mineralization
MC3T3-E1 cells were plated in a 6-well plate at a density of 5 × 10 4 cells ml −1 after the cells were cultured for 7 d in RAW cell-conditioned medium.4% paraformaldehyde was applied to the cells for 30 min at room temperature, followed by 30 min of Alizarin Red S staining at 37 • C (Solarbio, China), then observed and captured for the formation of mineralized nodules under an inverted fluorescence microscope (Leica DM2500, Germany).Subsequently, the cetylpyridinium chloride (Solarbio, China) at a concentration of 10% was added to each well and incubated for 15 min at room temperature to dissolve the calcium nodules.Measuring the absorbance at 562 nm provides a semi-quantitative study of the mineralized extracellular matrix (n = 3 in each group).

Effects of RAW cell-conditioned culture medium on HUVECs 2.5.1. Gene expression of HUVECs
HUVECs were plated in a 24-well plate at a density of 1 × 10 4 cells per well.The expression levels of angiogenesis-related genes hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and VEGF receptor (KDR) were measured using RT-PCR technology as mentioned in section 2.3.3.After 7 d of culture in RAW conditioned medium.The primer sequences are summarized in table 1.

Tube formation assay
Growth factor reduced matrix gel (BD Matrigel 356234, USA) was thawed at 4

Subcutaneous experiment
Twenty-four male rats (about 250-300 g) and age of 5 weeks were used.Rats were intraperitoneal injected with pentobarbital sodium (40 mg kg −1 ) to anesthetize before surgery.The bilateral back of each rat was shaved and disinfected with 2% iodine prior to the procedure.One incisions were made on bilateral back of each rat, and the nHAC was implanted into the subcutaneous pocket of rats in the nHAC group.After that 0.1 ml magnesium ion-containing medium solution (concentration: 100 mg l −1 ) was injected surrounding the nHAC and the subcutaneous pocket of rats representing nHAC + Mg 2+ and Mg 2+ groups, respectively, while blank groups are not treated.The detailed implantation procedure is shown in figure 10(A).

Histological examination
After 4 weeks, the tissues harvested were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin and performed histological section.Acquired sections were stained with hematoxylin-eosin (H&E) for the observation of the angiogenesis.

Immunofluorescence analysis
Furthermore, to evaluate the polarization of macrophages surrounding the implants, the above acquired sections were stained with iNOS (Invitrogen, PA1-036, green, M1 marker) and CD206 (Abcam, ab64693, red, M2 marker) according to the manufacturer's instructions.The nuclei were stained with DAPI.The stained tissues were observed by using a fluorescence microscope.

Bone implantation surgery
Twenty-four male rats (about 250-300 g) were randomly selected for the implantation experiments to evaluate the osseointegration effects of the Mineralized collagen combined with magnesium ions.The calvarium of the rat was disinfected by iodophor and alcohol.An incision was made and a cylindrical shaped defects with a diameter of 5 mm and a thickness of 2 mm of bone was created in the top of calvarium using a trephine drill.Continuous irrigation with saline solution was supplied at the same time to prevent tissue anhydration.
The nHAC was implanted into the cranial defect area of rats in the nHAC group.After that 0.1 ml magnesium ion-containing medium solution (concentration: 100 mg l −1 ) was injected surrounding the nHAC and the cranial defect area of rats representing nHAC + Mg 2+ and Mg 2+ groups, respectively, while blank groups are not treated.The detailed implantation procedure is shown in figure 11(A).
The surgical location was then closed and interrupted suture.The antibiotic and analgesics were administered for 3 d to avoid any infection and minimize pain.Animals were sacrificed by intravenous injection of air at 4 and 8 weeks after surgery.Defected calvarium containing implants were extracted for analysis.

Micro computerized tomography (micro-CT) assay
Rat calvarium were removed after 4 and 8 weeks of implantation.The skulls of model rat were harvested and fixed in neutral formalin for subsequent micro-computed tomography (micro-CT) to evaluate regenerated new bone.The bone trabeculae around the implant and the bone volume fraction (BV/TV) were analyzed.

Histological examination
Calvarium specimens were dehydrated with ethanol, then stained with hematoxylin & eosin (H&E) and Masson's trichrome staining.Subsequently, A methyl methacrylate solution was used for embedding the specimens.Specimens were sliced through a cutting and grinding system into hard tissue sections (EXAKT Apparatebau, Norderstedt, Hamburg, Germany), polished the femoral sections, and ground to 40-50 µm.Tissue sections were detected under a microscope (Olympus, Germany) after being stained with methylene blue/magenta.

Statistical analysis
SPSS software (version 21.0) was used for statistical analysis.All experiments were repeated three times and the experimental data were subjected to normal analysis and variance.The data measured were expressed as mean ± standard deviation, α = 0.05 as the test level, and the differences were considered statistically significant when P < 0.05.

Inflammatory response, osteogenesis, and angiogenesis potential of RAW cells 3.1.1. Results of cell proliferation assay
We cultured RAW 264.7 cells using four different culture conditions: blank control group, nHAC group, Mg 2+ group, and Mg 2+ + nHAC group.The CCK-8 kit was used to detect cell viability.In the Mg 2+ group, the OD values did not differ from the control group on the first day, whereas in the other material groups, the OD values increased.On the third and fifth days, the OD value of materials in each group increased relative to the control group, with the Mg 2+ + nHAC group having the highest OD value, as depicted in figure 1.

Flow cytometry results
Flow cytometry was used to determine the effects of different culture conditions on the phenotype switch of RAW 264.7 cells after stimulation with LPS.
Figures 2(a) and (b) depict, respectively, the expression of CCR7 (M1 surface marker) and CD206 (M2 surface marker).The CCR7-and CD206-positive fractions were statistically examined, as shown in figures 2(c) and (d).In comparison to the blank control group, the percentage of CCR7-positive cells decreased in the nHAC group, Mg 2+ group, and Mg 2+ + nHAC group, with the decrease in the Mg 2+ + nHAC group being particularly significant.
In contrast, compared to the blank control group, the percentage of CD206-positive cells was increased in the Mg 2+ , nHAC and Mg 2+ + nHAC groups, especially the Mg 2+ + nHAC group had a significant effect.

Results of real-time PCR
After 3 d, RT-PCR was used to quantify the expression of anti-inflammatory (IL-10 and IL-1ra) and pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6).In comparison to the control group, the Mg 2+ group, the nHAC group, and the Mg 2+ + nHAC group were able to stimulate the expression of IL-1ra and IL-10.In addition, the expression of IL-1ra and IL-10 reached the highest level in the Mg 2+ + nHAC group, as shown in figure 3(a).In contrast, the expression levels of pro-inflammatory cytokines TNF-α and IL-1β were reduced in the Mg 2+ , nHAC, and Mg 2+ + nHAC groups compared with the control group.The expression of the Mg 2+ + nHAC group was significantly decreased.However, the expression level of the IL-6 gene in the Mg 2+ group and nHAC group did not differ from that in the blank control group, and the expression of the IL-6 gene in the Mg 2+ + nHAC group was decreased, as shown in figure 3(b).Using RT-PCR, we also determined the expression of osteogenic and angiogenic-related factors at the mRNA level in RAW 264.7 cells.Compared to the blank control group, the osteogenesis-related gene BMP-2 and the angiogenesis-related gene PDGF-B were upregulated in the Mg 2+ , nHAC, and Mg 2+ + nHAC groups, with the effect on gene expression being statistically significant in the Mg 2+ + nHAC group.The expressions of BMP-2 and PDGF-B genes are presented in figures 3(c) and (d), respectively.

Results of Western blot analysis
Figure 4 illustrates Western blot results.NF-κB p65 protein expression was reduced in the Mg 2+ group, the nHAC group, and the Mg 2+ + nHAC group compared to the control group.Specifically, the Mg 2+ + nHAC group had the greatest impact.In contrast, the protein expression of IκB-α was upregulated in the Mg 2+ group, the nHAC group, and the Mg 2+ + nHAC group, with the highest protein expression level in the Mg 2+ + nHAC group.

Effects of RAW cell-conditioned medium on osteogenic differentiation of MC3T3-E1
MC3T3-E1 cells were evaluated for ALP activity, mineralization and osteogenic gene expression of MC3T3-E1 stimulated by RAW conditioned medium, as a measure of the immune regulation of macrophages during osteogenic differentiation.

ALP activity analysis
Figures 5(a) and (b) demonstrated the ALP activity of MC3T3-E1 cells cultured in RAW cell conditioned medium of different groups for 7 d.Quantitative analysis showed that compared with the RAW group, the Mg 2+ + RAW group, nHAC + RAW group, and Mg 2+ /nHAC + RAW group significantly promoted the ALP activity of MC3T3-E1 cells.Especially, the Mg 2+ /nHAC + RAW group significantly increased cell ALP activity.According to both qualitative and quantitative findings, the Mg 2+ /nHAC + RAW group maintained the highest ALP activity.

Extracellular matrix mineralization assay results
Alizarin red staining was used to determine whether MC3T3-E1 mineralized in the extracellular matrix, as shown in figure 6(a).In the Mg 2+ /nHAC + RAW group, the red-stained area of the mineralization nodules in the extracellular matrix was significantly larger than that in the Mg 2+ + RAW group, nHAC + RAW group.In the RAW group, no mineralization nodules were observed.Figure 6(b) depicts the relative absorbance value of matrix mineralization, which confirms this result by indicating that the Mg 2+ /nHAC + RAW group has the highest mineralization ability of the extracellular matrix.

Results of real-time PCR
Real-time PCR was used to quantify the expression levels of Runx-2, OPN, and OCN at 7 d, and the results were presented in figure 7. Compared to the RAW group, the Mg 2+ + RAW group, the nHAC + RAW group, and the Mg 2+ /nHAC + RAW group all stimulated the expression of osteogenicrelated genes.Additionally, the osteogenic-related gene expression levels of a: Runx-2, b: OPN, and c: OCN had arrived at the highest gene expression in the Mg 2+ /nHAC + RAW group.

Tube formation assay
The endothelial tube formation assay is a classic in vitro angiogenesis test.Except for the RAW group, all groups exhibited a distinct capillary-like network.In addition, quantitative analysis outcomes are illustrated in figure 8(b).It displayed a significant increase in total capillary length in the Mg 2+ + RAW group, nHAC + RAW group, and Mg 2+ /nHAC + RAW group.In addition, the Mg 2+ /nHAC + RAW group is the most obvious.

Results of real-time PCR
Further exploration of representative endothelial marker gene expression of angiogenesis-related genes, such as KDR, HIF-α, and VEGF was carried out by real-time PCR at day 7 after HUVECs were cultured with the RAW cell conditioned medium.Compared with the RAW group, the gene expression levels of a: KDR, b: HIF-α, c: VEGF of HUVECs were significantly upregulated in the Mg 2+ + RAW group, nHAC + RAW group, and Mg 2+ /nHAC + RAW group, as shown in figure 9.In contrast, the Mg 2+ /nHAC + RAW group was able to induce greater KDR expression than the Mg 2+ + RAW and nHAC + RAW groups.

Subcutaneous implantation
To further estimate the capability of the magnesium ions to synergistically mineralize collagen to promote angiogenesis and healing, H&E was used to analyze the subcutaneous tissue of the implantation area, the results are shown in figure 10(B).It can be observed that all samples can promotes angiogenesis than the blank control group, and nHAC/Mg 2+ group provide more blood supply than the nHAC group and Mg 2+ group.
The results of immunofluorescence in vivo demonstrated that the fluorescence intensity of iNOS was observed to be comparable among the four groups, while CD206 expression exhibited significantly enhanced levels in the nHAC/Mg 2+ group as shown in figure 10(C).

Cranial bone implantation
To visually display the influences of nHAC/Mg 2+ on new bone formation in vivo, the 3D reconstructed micro-CT images allowed visualization of bone defect healing process at the defect site.Figure 11(B) showed that an increase in bone regeneration area in the nHAC/Mg 2+ group as compared to nHAC group and Mg 2+ group.Figure 11(C) shows the calculated values of BV/TV.In both week 4 and week 8, the BV/TV values were significantly higher in the nHAC/Mg 2+ group compared with the other three groups, indicating that the combined action of nHAC and Mg 2+ synergistically enhances the growth of calvarial bone.
Meanwhile, at week 4, the H&E staining of the calvarial bone showed that there was a visible improvement in the healing of nHAC/Mg 2+ group compared with the other three groups.After 8 weeks, the calvarium in the nHAC/Mg 2+ group increased bone mineralization and formation around the defect than nHAC group and Mg 2+ group.Moreover, as shown in the figure 11(D), there was substantial fibrous tissue in the defect area of the calvarium in the blank control group.

Discussion
The repair of bone defects is a replicable process involving three successive but interdependent stages: inflammation, repair and remodeling [20].Soon after bone graft material implantation, M1-type macrophages recruit other immune cells and mesenchymal stem cells to trigger a cascade of inflammatory responses [21,22].However, the long-term chronic inflammatory response may lead to implant failure.During normal fracture healing, proinflammatory M1 macrophages gradually change to antiinflammatory M2 macrophages, a process that represents the resolution of inflammation and consequent osteogenic differentiation [23].Therefore, timely regulation of macrophage transformation to the M2 phenotype and appropriate regulation of the inflammatory response is essential for the success of bone regeneration.Macrophages can transform into M1 type in response to LPS stimulation and release proinflammatory factors, such as TNF-α, IL-6, and IL-1β [24].In this experiment, it was found that the expression effect of the TNF-α, IL-6, and IL-1β gene in macrophages stimulated by LPS was decreased due to the presence of the material, especially in the Mg 2+ + nHAC group.In contrast, the presence of biomaterials increased the expression of anti-inflammatory factors IL-10 and IL-1ra produced by anti-inflammatory M2 macrophages, again significantly more so in the Mg 2+ + nHAC group.This phenomenon suggests that Mg 2+ and nHAC have a synergistic role in inducing macrophage conversion to the M2 phenotype.The CCK-8 results showed that Mg 2+ and nHAC promoted the proliferative activity of macrophages and exhibited good cytocompatibility, which is consistent with the previously reported results [25,26].The combined effect of the two substances in regulating the conversion of M1 macrophages to the M2 phenotype was stronger than that of the materials alone, further suggesting that the two substances can synergistically promote macrophage proliferation while inducing the polarization of macrophages from the M1 to the M2 phenotype, thereby increasing the secretion of anti-inflammatory factors by macrophages and inhibiting the inflammatory response of macrophages.Besides, flow cytometry results showed the Mg 2+ + nHAC group was efficient in reducing the proportion of M1 macrophage surface marker CCR7-positive cells, whereas the percentage of M2 macrophage surface marker CD206-positive cells increased compared to applying Mg 2+ and nHAC alone, validating these results.
Studies have found that magnesium sulfate can reduce inflammation by inhibiting the TLR-NF-κB signaling pathway [27], which is one of the pathways involved in inflammation.Under LPS stimulation, Toll-like receptor 4 (TLR4) in the Toll family can induce activation the NF-κB signaling pathway and mitogen-activated protein kinases (MAPK) [28].After stimulation, NF-κB can induce the transcription of inflammatory genes such as TNF-α, IL-1, and IL-6.When cells are unstimulated, NF-κB binds to its inhibitor I-κB, which exists in the cytoplasm as an inactive NF-κB/I-κB α complex and prevents NF-κB from binding to DNA.However, when cells are exposed to pro-inflammatory signals (e.g.cytokines, pathogens, and risk-associated molecules), the NF-κB pathway is activated.With the less expression of I-κB α gene, NF-κB dimers migrate into the nucleus, resulting in inflammatory factor gene expression [29].The most abundant form of NF-κB family members is the heterodimer of p65/p50 [30], and some studies have shown that p65 inhibition reduces the secretion of pro-inflammatory factors [31].In this study, we found that all material groups could attenuate the expression of NF-κB p65 protein by inhibiting the degradation of IκBα and thus reducing the expression of proinflammatory cytokines, which was more significant in the Mg 2+ + nHAC group.In conclusion, in addition to the anti-inflammatory effect of Mg, nHAC also affects the inhibition of inflammation, which may be closely related to nHAC nanomorphology [20].Ni et al demonstrated that bone-mimicking nanoparticles (BMnP) possess potent immunomodulatory properties and can promote M2 macrophage polarization in vitro and in vivo [32].Lv et al also successfully constructed nano-convex microarray morphology on the surface of 316LSS and induced M1-type macrophages to polarize to M2, effectively inhibiting the release of inflammatory factors [33].Moreover, surface hydrophilicity also has a significant effect on the immune response.It is generally accepted that hydrophilic surfaces inhibit inflammation more than hydrophobic surfaces, and nHAC is a biograft material with good hydrophilic properties.Lv et al showed that hydrophilic surfaces can promote integrin β1 gene expression, and integrin β1 is closely related to PI3K/Akt activation, which inhibits NF-κB activation through Akt signaling pathway [34].NHAC could release Ca 2+ during degradation [35], and some researchers have found that a certain concentration of Ca 2+ also inhibits the activation of the NF-κB signaling pathway [36].Mg 2+ and nHAC can thereby reduce the inflammatory response and stimulate the transformation of macrophages to the M2 phenotype, which may be related to the inhibition of the NF-κB signaling pathway.The combined effect is larger than that of either material alone, demonstrating that nHAC and Mg have a synergistic effect when used together.
Magnesium and mineralized collagen potentiate not only osteogenic and angiogenic effects but also modulate the secretion of more pro-angiogenic/osteogenic related factors by macrophages [37].Different macrophage phenotypes produce distinct cytokines and play distinct roles.M2 macrophages are involved in the final phase of tissue repair, and moderate polarization of macrophages towards M2 can promote osteogenesis.Bone morphogenetic protein BMP-2 can indirectly stimulate osteogenic differentiation via the secretion of M2 macrophages, and macrophages play an essential role in bone regeneration via the BMP signaling pathway [38].In addition, M2 macrophages secrete cytokines involved in later angiogenesis, particularly PDGF-B, which stabilizes angiogenesis [39].In our study, we examined the gene expression levels of BMP-2 and PDGF-B, both of which were significantly increased in the Mg 2+ + nHAC group, suggesting that Mg 2+ , together with nHAC, promotes the polarization of macrophages towards the M2 type and may have a beneficial effect on osteogenic differentiation and angiogenesis.Mg 2+ synergetic nHAC produced a favorable osteoimmun microenvironment, which was associated with the induction of macrophage conversion to M2 type and the synergistic promotion of macrophage proliferation, as well as an increase in cytokine secretion, according to the present study.Increased BMP-2 and PDGF-B gene expression may contribute indirectly to osteogenic differentiation and angiogenesis via a paracrine mechanism.To further investigate, macrophage-conditioned media was applied to cultivate MC3T3-E1 and HUVEC cells in order to assess the potential impact of Mg 2+ combined with nHAC on regulating the immune milieu for osteogenic differentiation and angiogenic characteristics.
ALP activity is usually used as a marker of early osteogenic differentiation, while extracellular matrix mineralization is thought to play an important role in late osteogenic differentiation.During mineralization, osteoblasts secrete alkaline phosphatase, which subsequently forms calcium deposits, usually calcium phosphate [38].In this experiment, alkaline phosphatase activity and alizarin red staining were used to test the osteogenic activity of MC3T3-E1 cultured in RAW cell conditioned medium from different material groups.The ALP activity results showed a similar trend to the cell mineralization results, that is the osteogenic differentiation and mineralization of MC3T3-E1 cells was promoted in the Mg 2+ + RAW, nHAC + RAW, and Mg 2+ /nHAC + RAW groups, with the osteogenic differentiation effect being particularly significant in the Mg 2+ /nHAC + RAW group.To further validate the effect of RAW cell-conditioned medium on the osteogenic activity of MC3T3-E1, we also assessed the expression of osteogenic genes such as RUNX2, OPN, and OCN by RT-PCR, a pivotal transcription factor for osteoblast differentiation.OPN and OCN are important genes in mineral deposition [40].The present analysis showed that the expression of the osteogenic genes RUNX-2, OPN, and OCN was significantly enhanced in MC3T3-E1 cells cultured in the Mg 2+ /nHAC + RAW group.The results of bone regeneration were highly dependent on the phenotypic polarization of macrophages and imply that BMP-2 produced by macrophages influences osteogenesis.Through a more favorable bone immunological environment of macrophages, it was revealed that the combination of Mg and nHAC might boost the osteogenic differentiation potential of MC3T3-E1.Based on the above, we further verified the bone integration performance of magnesium ion combined mineralized collagen material through in vivo experiments.The results showed that compared with other groups, the combination of magnesium ions and mineralized collagen significantly promotes bone formation in the defect area of rat cranial bones.This is consistent with the in vitro osteogenesis results.
Providing oxygen and nutrients for bone regeneration, as well as transporting osteogenic precursor cells and secreting numerous growth factors required by osteoblasts, the vascular network is vital to the bone repair process [41,42].For example, VEGF can activate VEGF receptors on osteoblasts to promote bone regeneration [43] and can also promote bone regeneration by promoting the secretion of hypoxia-inducible factor 1α (HIF-1α) [44].The control of vasogenesis by biological substances has been explored extensively.In prior research, we discovered that the CS-nHAC/Mg-Ca composite material not only promoted osteogenic differentiation and mineralization of MC3T3-E1 cells but also had angiogenic properties [45].However, angiogenesis and immune responses are interdependent.To investigate the effect of RAW macrophage-conditioned medium on angiogenesis in each material group, we used qRT-PCR to assess the expression levels of HIF-1α, KDR, and VEGF genes.The results demonstrated that HUVEC cells in the Mg 2+ /nHAC + RAW group significantly promoted the expression levels of HIF-1α, KDR, and VEGF genes, which suggests that the combination of Mg and nHAC may not only enhance angiogenesis but also indirectly affect osteogenesis by stimulating the secretion of growth factors using endothelial cells.While it has been shown that hypoxic conditions can upregulate HIF-1α expression and enhance inflammatory damage in EC [46], it has also been demonstrated that both NF-κB and HIF pathways would be mutually exclusive if activated simultaneously in the inflammatory response [47].It is the inhibition of the NF-κB pathway through the HIF activation pathway that is important to attenuate the overly intense inflammatory response as well as to reduce the inflammatory response in chronic hypoxia.In addition, the tube formation assay is the traditional method for determining the in vitro differentiation trend of HUVEC cells.These results also demonstrated that the Mg 2+ /nHAC + RAW group had a pro-angiogenic effect on HUVEC cells.Meanwhile, in vivo experiments confirmed that the combination of Mg and nHAC synergistically promoted angiogenesis.These findings indicate that the angiogenic effects is largely a consequence of high levels of PDGF-B gene expression in M2 macrophages.
In conclusion, the combined application of low concentrations of Mg 2+ and nHAC in this study discovered that Mg 2+ combined with nHAC regulated the inflammatory response of macrophages by inhibiting the NF-κB pathway and promoted the polarization of macrophages to the M2 phenotype, which was significantly more effective than the application of either material alone.It also displays that the combination of multiple components in composite materials can strengthen the complementary advantages and obtain more perfect biological properties than single components.In addition, it can also exhibit good osteogenic and angiogenic activities.

Conclusions
In summary, the findings of this study in vitro suggest that the combination of low concentrations of Mg 2+ and nHAC may reduce the inflammatory response of macrophages by inhibiting the NF-κB signaling pathway, and also play a synergistic effect to promote the transformation of macrophages into M2-type, generating an appropriate bone immune microenvironment to enhance bone tissue reconstruction and osseointegration.

Figure 2 .
Figure 2. Results of flow cytometry.The phenotype of RAW264.7 cells stimulated by LPS was changed under different culture conditions, a, and b represent differences in expression levels of M1 surface marker CCR-7 and M2 surface marker CD206, c, d represent the statistical results for CCR7-and CD206-positive macrophages from three repeated experiments.(* = P < 0.05).

Figure 4 .
Figure 4. Results of Western blot analysis.Expression of NF-κB signaling pathway protein in RAW264.7 cells stimulated with LPS under different culture conditions.In RAW264.7 cells, a and b respectively represent the difference in NF-κB p65 protein expression level, and a and c respectively represent the difference in IκB-α protein expression level.The differences among the groups were statistically significant.( * = P < 0.05).

Figure 5 .
Figure 5. ALP activity analysis.(a) Corresponding stained areas of MC3T3-E1 cells after 7 d of culture in different groups of RAW cell conditioned medium, and (b) quantitative results of ALP activity.( * = P < 0.05).

Figure 6 .
Figure 6.Extracellular matrix mineralization assay results.MC3T3-E1 cells were cultured in different groups of RAW cell-conditioned media for 14 d.Matrix mineralization image (a) and semi-quantitative analysis results of cell mineralized nodules after lysis (b).( * = P < 0.05).

Figure 7 .
Figure 7. Quantification of the expression levels of osteogenesis-related genes (a) RUNX2, (b) OPN, and (c) OCN in MC3T3-E1 cells after 7 d of culture in different groups of RAW cell conditioned media.(* = P < 0.05).

Figure 8 .
Figure 8.In vitro angiogenic images of HUVEC cells after 7 d of culture on stromal gels with different groups of RAW cell conditioned medium (a) and corresponding total vessel length (b).( * = P < 0.05).

Figure 10 .
Figure 10.(A) Scheme of subcutaneous implantation process.(B) Histological evaluation of the different samples for 4 weeks after subcutaneous implantation in rats, the black arrows indicate angiogenesis.(C) Immunofluorescent staining of the tissue surrounding the different samples after subcutaneous implantation for 4 weeks.Scale bars, 50 µm.

Figure 11 .
Figure 11.(A) Scheme of the implantation process.(B) Micro-CT 3D reconstruction of cranial defect sites.(C) Calculated bone volume/tissue volume (BV/TV).(D) Histological evaluation of the different samples for 4 and 8 weeks after calvarium implantation in rats.The black arrows indicate new bone formation.

Table 1 .
The sequence of primers used for RT-PCR analysis.
• C, 50 µl matrix gel was first coated on a 96-well plate and incubated for 30 min at 37 • C to set matrix gel.