Protein Corona of Magnetic Hydroxyapatite Scaffold Improves Cell

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Protein Corona of Magnetic Hydroxyapatite Scaffold Improves Cell Proliferation via Activation of MitogenActivated Protein Kinase Signaling Pathway Yue Zhu, Qi Yang, Minggang Yang, Xiaohui Zhan, Fang Lan, Jing He, Zhongwei Gu, and Yao Wu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.6b08193 • Publication Date (Web): 17 Mar 2017 Downloaded from http://pubs.acs.org on March 18, 2017

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Protein Corona of Magnetic Hydroxyapatite Scaffold Improves Cell Proliferation via Activation of Mitogen-Activated Protein Kinase Signaling Pathway Yue Zhu, Qi Yang, Minggang Yang, Xiaohui Zhan, Fang Lan, Jing He*, Zhongwei Gu and Yao Wu* National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, Sichuan 610064, P.R. China

Corresponding Author: *E-mail: [email protected] *E-mail: [email protected]

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ABSTRACT The beneficial effect of magnetic scaffolds on the improvement of cell proliferation has been well documented. Nevertheless, the underlying mechanisms about the magnetic scaffolds stimulating cell proliferation remain largely unknown. Once the scaffold enters into the biological fluids, a protein corona forms and directly influences the biological function of scaffold. This study aimed at investigating the formation of protein coronas on hydroxyapatite (HA) and magnetic hydroxyapatite (MHA) scaffolds in vitro and in vivo, and consequently its effect on regulating cell proliferation. The results demonstrated that magnetic nanoparticles (MNP)-infiltrated HA scaffolds altered the composition of protein coronas and ultimately contributed to increased concentration of proteins related to calcium ions, G-protein coupled receptors (GPCRs), and MAPK/ERK cascades as compared with pristine HA scaffolds. Noticeably, the enriched functional proteins on MHA samples could efficiently activate of the MAPK/ERK signaling pathway, resulting in promoting MC3T3-E1 cell proliferation, as evidenced by the higher expression levels of the key proteins in the MAPK/ERK signaling pathway, including mitogen-activated protein kinase kinases1/2 (MEK1/2) and extracellular signal regulated kinase 1/2 (ERK1/2). Artificial down-regulation of MEK expression can significantly down-regulate the MAPK/ERK signaling and consequently suppress the cell proliferation on MHA samples. These findings not only provide a critical insight into the molecular mechanism underlying cellular proliferation on magnetic scaffolds, but also have important implications in the design of magnetic scaffolds for bone tissue engineering.

KEYWORDS: protein corona, cell proliferation, MAPK signaling pathway, magnetic 2 ACS Paragon Plus Environment

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nanoparticles, magnetic hydroxyapatite scaffold, tissue engineering.

Due to their excellent biocompatibility and magnetic properties, magnetic nanoparticles (MNPs) have significantly advanced the development of magnetic scaffolds for bone tissue engineering in the past few decades.1-5 Currently, magnetic scaffolds are usually prepared by integrating MNPs with a matrix, including hydroxyapatite ceramics (HA)6,

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and biocompatible polymers, such as collagen,8,

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poly (L-lactide),10,

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poly-ε-caprolactone,12 and poly(lactic-co-glycolic acid)13, 14 etc. Studies have shown that when MNPs were conjugated with hydroxyapatite scaffolds, bone cell growth improved significantly.15 Nanofibrous scaffolds produced using needleless electrospinning from a mixture of poly-ε-caprolactone and magnetic particles accelerated the proliferation of mesenchymal stem cells (MSCs) in vitro.12 Furthermore, MNPs incorporated at small concentrations were found effective in increasing the rate of cell growth in gelatin-siloxanehybrid porous scaffolds.16 Although a variety of MNPs-added biomaterials have shown the ability to stimulate cell proliferation, the mechanisms by which magnetic scaffolds stimulate cell proliferation are still not well understood. One of the hypotheses is that each magnetic nanoparticle in the scaffold acts like a single magnetic domain at the nanoscale level to provide micro-motions at the interface between cells and scaffold, which might affect the ion channels on the cell membrane, and trigger the mechanotransduction pathway, leading to increased cell growth, proliferation and differentiation.9, 16, 17 Another study has demonstrated that a magnetic scaffold is supposed to attract and take up the growth factor 3 ACS Paragon Plus Environment

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or other bio-agents via a driving magnetic force.6 Therefore, it is necessary to carry out the systematic studies of the cell proliferation promoted by MHA scaffolds. When a biomaterial is administered in vivo, its surface is immediately altered by endogenous proteins, leading to the formation of a macromolecular coating complex, usually called “protein corona”.18-20 The biological fate of biomaterials could be directly connected by the type and amount of the associated protein in the composition of the protein corona.19-22 Surface receptors could be activated via the adsorbed protein, resulting in the initiation of multiple signaling cascades that direct the cell response to biomaterial.23, 24 In addition, some special protein adsorptions typically involve kinases and phosphases, which alter the phosphorylation patterns of proteins to regulate downstream cell behaviors.24 For instance, the enhanced adsorption of serum fibronectin and vitronectin on PLLA/HAP scaffolds could protect MC3T3-E1 cells from apoptosis through the pathway of integrin-FAK-AKT (signal activated and phosphorylated FAK and Akt).25 The composition, formation and the quantity of the protein corona on magnetic scaffold would likely affect cell functions, including cell adhesion, migration, proliferation and differentiation. The protein corona strongly depends on the physicochemical properties of biomaterials including size,26-28 surface charge29,

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and

surface chemical functional groups,31-34 surface roughness,35 surface curvature,36 hydrophilic surfaces,37, 38 statistic magnetic field39 etc. For instance, gold nanoparticles with negatively charged can bind to fibrinogen and change its conformation, leading to promote interaction of nanoparticles with the integrin receptor.40 The magnetic field


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increased the adsorption of apolipoprotein in the protein corona of superparamagnetic iron oxides (SPIONs) to enhance the cellular uptake into 3T3 and HepG2 cell line.39 Nevertheless, these studies have primarily focused on the formation of protein corona at the surface of various nano biomaterials, especially for the nanoparticles.26-40 The development of such protein constructs onto magnetic scaffold and their physiological functions in bone regeneration have been rarely investigated, which may offer a greater understanding of the influence of protein corona on cell proliferation. Herein, we proposed a strategy to explore the mechanism about magnetic scaffolds promoting cell proliferation from the viewpoint of protein corona (Scheme 1). First, the magnetic hydroxyapatite ceramics (MHA) scaffold was prepared by embedding MNPs nanoparticles in hydroxyapatite ceramic (HA) scaffolds which is chemically similar to the inorganic component of bone matrix.41 The magnetic hydroxyapatite ceramics (MHA) demonstrate an excellent superparamagnetism and an enhanced effect on the MC3T3-E1 cell proliferation. Second, a systematic study of the formation of protein corona on the HA and MHA scaffolds was carried out both in vitro and in vivo. Third, the underlying mechanisms of the effects of protein corona on cell proliferation were thoroughly explored. The current study demonstrated that MHA scaffolds benefited the adsorption of the proteins related to cell proliferation. The higher concentration of the proteins related to MAPK/ERK cascade receptors and ion channels adsorbed on MHA scaffolds was closely correlated with the enrichment of the proteins related to cell proliferation on MHA scaffolds. Furthermore, these results showed that the accelerated adsorption of protein involved in upstream cascades receptor of the MAPK signaling pathway on MHA scaffolds might be a critical factor contributing to the enhancement of cell proliferation. 5 ACS Paragon Plus Environment

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This study provides a critical insight into the molecular mechanism underlying cellular proliferation on magnetic scaffolds.

Scheme 1. Schematic description of the experimental design. A) The preparation of MHA scaffolds (Φ 14 × 2 mm in vitro and Φ 2 × 6 mm in vivo). B) The study of cell proliferation on HA and MHA scaffolds by confocal laser scanning microscopy (CLSM). C) HA and MHA scaffolds (Φ 14 × 2 mm) were incubated in different biological environments including fetal bovine serum (100% FBS), extracellular secretion (ES) of MC3T3-E1 cells, the combination of 10% FBS and extracellular secretion (FBS+ES) in vitro and the femora of SD rats in vivo. 24 h post incubation under each set of conditions, scaffolds were removed and the protein coronas formed onto the scaffold were analyzed using BCA, SDS-PAGE, LC- MS/MS. D) The effect of MAPK/ERK signaling pathway on protein corona-mediated cell proliferation.


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RESULTS AND DISCUSSION Physicochemical characterization of MHA scaffold The MHA scaffold was prepared via a simple immersion method (Scheme 1A).15 Briefly, MNPs 10 nm in diameter (Figure S1), were prepared using high-temperature decomposition42 and encapsulated in HA (Ф 14 × 2 mm or Ф 2 × 6 mm) scaffolds to produce MHA scaffolds through co-incubation for 24 h (Scheme 1A). Fourier transform infrared (FTIR) spectra revealed that the MNPs had successfully infiltrated the HA scaffold. Both -CH2 stretching of alkyl chains at 2923 and 2850 cm-1, which is related to the oleic acid14 on the surface of MNPs and the characteristic peaks of the HA scaffold were observed in MHA scaffolds (Figure 1A). However, the Fe-O characteristic band at 580 cm-1 was not detected on MHA scaffolds due to the superposition of phosphate group bands at 601 and 568 cm-1.43 A vibrating sample magnetometer (VSM) at 300 K was used to evaluate the magnetic properties of the MNPs and MHA scaffolds. Both MNPs and MHA scaffold exhibited superparamagnetism at room temperature because of the negligible coercivity and remanence44 in magnetic hysteresis curves (Figures S2 and 1B). The saturation magnetizations of MNPs and MHA scaffold were ≈ 56 emu/g and ≈ 2 emu/g, respectively (Figures S2 and 1B). Additionally, the stability of the MHA scaffold was measured by soaking it in water and measuring the mass of the iron released by atomic absorption spectrophotometers (AAS). As the soak time increased, the MHA scaffold gradually released iron. The local environment became saturated with iron over the course of 12 hours. Prolonging the soaking time further did not increase the amount of iron released, indicating the excellent stability of the MHA scaffold (Figure 1C).


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Finally, MHA released 0.32% of the adsorbed magnetite in H2O. Scanning electron microscopy (SEM) showed highly porous structures for both the HA and MHA scaffolds, indicating that the introduction of MNPs did not influence the porosity of the MHA scaffold (Figure 1D).

Figure 1. Physicochemical characterization of MHA. A) FTIR spectra of MHA scaffold, HA scaffold and MNPs; B) Magnetization curve of MHA scaffold; C) The mass of released iron of MHA scaffold soaking in water for different soak time; D) SEM micrographs of (a) HA and (b) MHA scaffolds.

The effects of MHA on osteoblast proliferation The effect of the HA and MHA scaffolds on osteoblast proliferation and adhesion were examined (Scheme 1B). The MC3T3-E1 cells cultured on MHA scaffold showed higher OD values than those on HA scaffolds for all culture times. Laser confocal 8 ACS Paragon Plus Environment

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scanning microscopy showed that the cell number on the MHA scaffold was larger than that on the HA scaffold on day 6, and the cells on MHA scaffold were spread better than those on the HA scaffold (Figure 2B). Overall, these results strongly suggested that the loaded MNPs in MHA scaffold were beneficial to the osteoblast cell proliferation, which is consistent with previous findings.15, 45

Figure 2. The effects of MHA and HA scaffolds on MC3T3-E1 cell proliferation: A) CCK-8 assay for proliferation of MC3T3-E1 cell cultured on HA and MHA scaffolds for 2, 4 and 6 days. Error bar represent means ± standard error of mean for n = 3 (*p < 0.05 as compared with HA); B) Confocal laser scanning microscopy images of MC3T3-E1 cells stained with FDA after cultivation on (a) HA and (b) MHA scaffolds for 6 days.

Protein Corona study in vitro Upon immersion in the biological media, the scaffolds interacted with the macromolecules, especially for proteins, resulting in the formation of protein corona on 9 ACS Paragon Plus Environment

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their surfaces.19 To test the hypothesis that the protein corona plays an important role in cellular responses to the scaffold, we examined the formation of the protein corona on the HA and MHA scaffolds that were placed in fetal bovine serum (100% FBS, the initial quantity was list in Table S1) at 37 °C for 24 h. The adsorbed proteins were separated using one-dimensional gel electrophoresis (SDS-PAGE) and visualized with Coomassie Brilliant Blue R250 staining. Significant differences in the composition of protein corona were assessed after adsorption onto the HA and MHA scaffold in FBS medium. High molecular weight proteins (Mw > 75 kDa) were found in the corona of HA samples, whereas MHA samples tended to adsorb small molecular weight proteins (Mw < 25 kDa) (Figure 3A). The results indicated that the presence of MNPs altered the pattern of protein distribution. During the cultivation process, cells continuously secreted extracellular secretions, which may have been unavoidably affected by the presence of biomaterial. In turn, the medicated extracellular secretions may affect the formation of the protein corona on the surface of the material or even change the composition of protein corona. For this reason, the formation of the protein corona was further examined on HA and MHA scaffolds in extracellular matrix environment. The HA and MHA scaffolds were placed in extracellular secretion (ES) medium and ES+FBS medium (method of preparation shown in the experimental section and Figure S3, the initial quantity was list in Table S1) at 37 °C for 24 h, respectively. Regardless of the sample used, the types and band intensities of proteins adsorbed in ES+FBS medium and ES medium were markedly lower than those in FBS medium (Figure 3A-C), which was consistent with the adsorption amounts of proteins determined by bicinchoninic acid assay (BCA, Figure S4). The phenomenon 10 ACS Paragon Plus Environment

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might have been caused by the different types of proteins and by the different affinities between the scaffolds and proteins in the three media.

Figure 3. SDS-PAGE gel (12%) of the protein corona obtained from HA and MHA in different incubation medium. A) FBS medium; B) ES medium; C) ES+FBS medium.

Nano liquid chromatography mass spectrometry (nano LC-MS/MS) was used to comprehensively identify the proteins. The amounts of proteins were calculated using spectral counting (SpC), which represents the total number of the MS/MS spectra for all peptides attributed to a detected protein. The SpC of each protein absorbed on the HA or MHA scaffold was normalized to the protein mass, expressed as the relative protein quantity using the following equation:46, 47 /

NpSpCk =∑

/

⨯ 100

Here, NpSpCk is the normalized percentage of the spectral count for protein k, SpC is the spectral count identified, and Mw is the molecular weight in kDa of protein k. The full list of the normalized SpC (NSpC) values for proteins identified in HA and MHA scaffolds after incubation in FBS, ES, and ES+FBS media were given in Tables S2-S7 in the 11 ACS Paragon Plus Environment

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supporting information section. The abundances of the adsorbed proteins with a value of above 1% in vitro were plotted as a heatmap (Figure 4). As expected, regardless of the medium used, the pattern of protein distribution changed with the addition of MNPs. The MHA scaffold was found to adsorb the number of proteins in the extracellular secretions same as the HA scaffold (Figure 5). In ES+FBS medium, the category and contents of the adsorbed proteins presented solely in ES were 13 and 16.65% for the HA scaffold, and 19 and 52.4% for the MHA scaffold. This high level of extracellular secretions on MHA scaffold indicated that MHA scaffold had a better affinity to the proteins from the extracellular secretions in ES+FBS medium.


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Figure 4. Heatmap data visualization of the abundance of the adsorbed proteins on HA and MHA scaffolds in the corona compositions under various in vitro conditions (FBS, ES and ES+FBS media) by liquid chromatography tandem mass spectrometry (LC-MS/MS). Values were quantified by spectral counting. Each row corresponds to a protein, and each column corresponds to different samples. The dark blue proteins are 13 ACS Paragon Plus Environment

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represented as “not detected” in the respective coronas. Only those proteins that constitute at least 1% of the protein corona on one of the scaffolds are shown.

Figure 5. Venn diagrams report the number of unique proteins identified in ES and ES+FBS media formed coronas and their respective overlap: the number of proteins identified solely in ES medium (red color), the number identified solely in ES+FBS medium (dark blue color), and the number identified both in ES medium and in ES+FBS medium (light blue color). In the pie charts, the percentage composition of proteins identified solely in ES+FBS medium is shown as in dark blue color, and the percentage composition of proteins identified both in ES medium and in ES+FBS medium is shown as in light blue color. The category of protein in ES medium same as in ES+FBS medium is listed: the proteins found in single scaffold (black color), and the proteins found both in HA and MHA scaffolds (red color). A) HA scaffold; B) MHA scaffold.

Several bioanalytical tools, such as Uniprot and David, were used to further classify the bound proteins according to biological processes and molecular function (Figure 6A and Figure S5). Considering that the proliferation of MC3T3-E1 cells was influenced by the scaffolds, the proteins were divided into five groups including proteins related to cell 14 ACS Paragon Plus Environment

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proliferation, calcium ions, iron ions, G-protein coupled receptors (GPCRs), and cascade receptors of the MAPK/ERK signaling pathway. As anticipated, the concentrations of these proteins of five groups were much higher in MHA scaffolds than HA scaffolds. Particularly, G-protein coupled receptors constitute the large family of cell-surface molecules involved in signal transmission and these receptors play key physiological roles in stimulating intracellular signaling pathways to influence the cell proliferation.48, 49

In this way, the adsorbed proteins related to G-protein coupled receptors onto the MHA

scaffold may facilitate the MHA-cell interaction. As expected, more G-protein formed on MHA scaffolds in the ES and ES+FBS media were greater than those on HA scaffolds (Figure 6E), indicating clearly positive effects of MHA scaffold on adsorbing the proteins related to G-protein coupled receptor. Furthermore, heterotrimeric G proteins were found to stimulate effector molecules that include phosphodiesterases, phospholipase (PLC), and phosphoinositide 3-kinases (PI3K), thereby activating the production of second messengers and increasing the intracellular concentration of Ca2+ and the opening or closing of various ion channels.49 Based on these findings, it is here assumed that MHA scaffold enriched with proteins related to calcium ions and iron ions can enhance cell proliferation. Notably, high adsorption of annexin (ANXA2), desmoglein-1-beta (DSG1B), cholinergic receptor, nicotinic, alpha polypeptide 10 (CHRNA10), and desmoplakin (DSP) were observed on the MHA scaffold in the ES and ES+FBS media (Figure 6C). Results also showed the MHA scaffolds bind more proteins related to iron ions, including cytochrome P450 2U1 (CYP2U1) and beta-globin (HBBT1) than HA scaffolds (Figure 6D). However, these phenomena were no observed in FBS medium, and the amounts of the proteins related to calcium ions and iron ions absorbed on the MHA 15 ACS Paragon Plus Environment

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and HA scaffolds were similar (Figure 6C-D). This might be partially due to the effects of the cellular microenvironment. Most biological responses mediated by GPCRs are not dependent on a single biochemical route but rather result from the integration of the functional activity of an intricate network of intracellular signaling pathways.49 It has been reported that GPCRs modulate downstream Ras signaling, particularly MAPK activation.50 For this reason, we further analyzed the proteins related to MAPK/ERK signaling pathway on both MHA and HA scaffolds (Figure 6F). The amounts of proteins related to MAPK signaling pathway absorbed on MHA scaffolds were 0.5 fold, 1 fold, and 10 fold of those found on HA in FBS, ES, and ES+FBS media, respectively (Figure 6F). Interestingly, the protein coronas formed on MHA scaffolds comprised considerably greater amounts of complement C3 and PRDX2 compared to protein coronas formed on HA scaffolds in the three media. These proteins were distributed in upstream cascades of the MAPK signaling pathway. Complement C3 plays a central role in the activation of MAKP signaling pathway via GPCRs protein.51 Peroxiredoxins (PRDX2) can increase and sustain ERK and MEK activation.52 In this way, it appeared that the composition of protein corona might be the critical factors to affect the cell proliferation. The MHA scaffold was found to facilitate the adsorption of the proteins related to calcium ions, iron ions, G-protein coupled receptors, and the MAPK/ERK signaling pathway, which may ultimately affect the cell function.


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Figure 6. Classification of identified protein coronas found on HA and MHA scaffolds according to their biological processes and molecular functions in FBS, ES, and ES+FBS media, respectively. Each bar represents the percentage of adsorbed proteins in each category for HA and MHA scaffolds. A: Biological process and molecular function; B: Proteins related to cell proliferation; C: Proteins related to calcium ion; D: Proteins


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related to iron ion; E: Proteins related to G-protein-coupled receptors; F: Proteins related to MAPK/ERK signaling pathway.

Protein Corona study in vivo Protein profiles of HA and MHA scaffolds were also evaluated in vivo. HA and MHA cylinders were implanted into the femora of sprague dawdley (SD) rats and allowed to remain for 24 h. The BCA assay results illustrated that the infiltration of MNPs in MHA scaffold did not affect the saturated adsorption amount of proteins in vivo (Figure 7A). However, protein adsorption profiles in vivo differed considerably from in vitro profiles (Figure 7B and Figure 4). Significantly more protein categories were observed for the protein coronas formed on both HA and MHA scaffolds in vivo, which might be partly attributable to the complexity of the environment in vivo. About 400 different proteins were detected and identified by nano liquid chromatography mass spectroscopy. Individual protein spectral counts are listed in Tables S8-9, Supporting information. The common serum protein (e.g. serum albumin and serotransferrin), abundant blood cell proteins (e.g. hemoglobin subunit beta-1, protein Hba-a2, hemoglobin subunit beta-2, and hemoglobin subunit alpha-1/2, are all related to iron ions) accounted for the majority of the detected proteins in HA and MHA scaffolds (Figure S6).


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Figure 7. Analysis of protein corona adsorbed on HA and MHA scaffolds in vivo for 24 h. A) The amount of adsorbed proteins on HA and MHA scaffolds by the BCA assay; B) Gel electrophoresis to visualize proteins associated with HA and MHA scaffolds in vivo; C) Venn diagram reports on the number of unique proteins identified in HA and MHA scaffolds in vivo and their respective overlap.

The in-vivo results further underscored the in-vitro results that MHA scaffold enhanced the protein binding ability of the five groups (Figure 8). As the MHA scaffold was exposed to the in vivo environment, the proportion of proteins related to cell proliferation increased (Figure 8B). Particularly, the proteins related to calcium ion 19 ACS Paragon Plus Environment

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(Figure 8C), GPCRs (Figure 8E), and MAPK/ERK cascade receptor (Figure 8F) were enriched on the MHA scaffolds, which was in agreement with the in vitro results. The higher concentration of these functional proteins related to calcium ion and MAPK/ERK cascade receptors were detected on the surface of MHA scaffolds, indicating the essential roles played by the calcium ion and MAPK signaling pathway in the stimulation of cell proliferation. Overall, the preliminary in-vivo study further supported in vitro results that the protein corona of MHA scaffold was markedly influenced by MNPs. In particular, a strong correlation was observed between cell proliferation and protein corona, as evidenced by the enrichment of proteins related to cell proliferation in MHA scaffold than HA scaffold both in vivo and in vitro. Furthermore, the greater content of the proteins related to MAPK/ERK cascade receptors and ion channels adsorbed on MHA scaffolds was closely correlated with the enrichment of the proteins related to cell proliferation on MHA scaffold, suggesting that cell proliferation had a correlation with MAPK/ERK signaling pathway or calcium ions. These two correlations were further explored to determine which one played a more important role in cell proliferation.


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Figure 8. Classification of identified protein corona found on HA and MHA scaffolds according to their biological processes and molecular functions in vivo. Each bar shows the percentage of adsorbed proteins in each category for HA and MHA scaffolds. A: Biological process and molecular function; B: Proteins related to cell proliferation; C: Proteins related to calcium ion; D: Proteins related to iron ion; E: Proteins related to G-protein-coupled receptors; F: Proteins related to MAPK/ERK signaling pathway. 21 ACS Paragon Plus Environment

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MAPK/ERK signaling pathway critical for the protein-corona-mediated cell proliferation The MAPK/ERK signaling pathway was examined first due to its function in controlling cell proliferation through the regulation of related genes.53 The key markers, in the MAPK/ERK signaling pathway, MEK1/2 and ERK1/2, play important roles in magnetically-mediated cell proliferation.54 Figure 9 showed a dramatic regulation effect of MHA scaffold on the activation of the MAPK/ERK signaling pathway, in contrast to a weaker dependence for the HA scaffold. Throughout the culture time, the MEK1/2 expression on the MHA scaffold increased continuously while the MEK1/2 expression on the HA scaffold increased only for the first 4 days, followed by a drop. Overall, there was considerably more MEK1/2 expression in MHA samples than in HA samples (Figure 9A). A similar trend was observed for ERK1/2 expression. Significantly higher expression of ERK1/2 was observed on the MHA samples than those in HA samples (Figure 9B). The western blot results also showed that the higher expression of MEK1/2 and ERK1/2 were produced in the MC3T3-E1 cells grown on the MHA scaffolds (Supporting information, Figure S7). Next, the expression of C-fos, which might be considered as a bridge between ERK1/2 and cell proliferation, was assessed.55 The analysis of C-fos further corroborated this trend. At each point in time, the expression of C-fos from the MC3T3-E1 cells grown on the MHA scaffold was significantly more pronounced than on the HA scaffold (Figure 9C). The expression levels of C-fos, MEK1/2 and ERK1/2 were found to be interconnected. A strong correlation was also observed between the level of expression of


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C-fos and the OD values, which were significantly up-regulated in cells grown on the MHA scaffolds.

Figure 9. MAPK signaling pathway-related protein expression levels of MC3T3-E1 cells on HA and MHA scaffolds for 2, 4 and 6 days. A) MEK1/2; B) ERK1/2; C) C-fos. Error bar represents means ± standard error of mean for n = 3 (*p < 0.05, **p < 0.01 and ***p < 0.001, relative to HA).

For further validation of the roles of MAPK pathway in protein-corona mediated cell proliferation, cells were pretreated with a specific inhibitor PD98059 of MEK1/2, which inhibited MEK-1-medicated activation of MAPK.56 As shown in figure 10A and 10B, the 23 ACS Paragon Plus Environment

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levels of expression of MEK1/2 and ERK1/2 on the MHA scaffold were all visibly decreased while the expression levels on the HA scaffold were not significantly different (compared to Figure 9A-B). A similar trend was observed regarding the level of C-fos expression, which was considerably downregulated (Figure 10C and 9C). CCK-8 assay and CLSM were performed to further confirm the role of classical MAPK/ERK signaling pathway and to determine whether the level of cell proliferation could be changed by the inhibition of this pathway (Figure 10D-E). Results showed that the MHA-promoted cell proliferation was significantly suppressed (Figure 10D and 10E-a), whereas the presence of an inhibitor did not change the results of cell proliferation on HA samples (Figure 10D and 10E-b). On the basis of these results, it was concluded that classical MAPK/ERK signaling pathway of MC3T3-E1 cells on MHA scaffold was activated and subsequent downstream proteins associated with cell proliferation (C-fos) were up-regulated. However, HA had a much less pronounced effect on the activation of the MAPK/ERK signaling pathway.


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Figure 10. Evaluation of the key proteins in MAPK/ERK signaling pathway and cell proliferation after treated by PD98059 (specific inhibitor MEK1/2). MC3T3-E1 cells were planted on HA and MHA scaffolds and treated with 10 µmol PD98059 for 2, 4 and 6 days. A) MEK1/2; B) ERK1/2; C) C-fos; D) cell proliferation; E) confocal laser scanning microscopy images of MC3T3-E1 cells stained with FDA after cultivation on (a) 25 ACS Paragon Plus Environment

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HA and (b) MHA for 6 days. Error bar represents means ± standard error of mean for n = 3 (*p < 0.05, **p < 0.01 and ***p < 0.001, relative to HA).

Moderate influence of Ca2+ signals on the protein-corona-mediated cell proliferation Additionally, the results of the current MS assay demonstrated that the MHA scaffold adsorbed more proteins related to calcium ions than HA scaffold regardless of the protein medium used. This showed the calcium ion channel to be another possible mechanism by which cell proliferation was affected. Ca2+ signals can be mediated by Ca2+/calmodulin (CaM) protein kinase II (CaMKII), a ubiquitous serine/threonine protein kinase that is activated by Ca2+ and CaM to phosphorylate diverse substrates involved in cell cycle control.57-59 The activities of calmodulin (CaM) in MC3TC-E1 cells grown on HA and MHA scaffolds were examined to determine whether MHA stimulates cell proliferation through the calcium signaling pathway or not. ELISA assay of CaM in figure 11A demonstrated that the levels of total CaM protein expression on MHA scaffold were only slightly higher than those on HA scaffold at 4 and 6 days. Here, 10 µmol of W7, an inhibitor of CaM, was used to treat the cells.60 Then the cell proliferations with or without the presence of W7 were examined. The results also suggested that W7 inhibitor did not influence the overall trend but somehow suppressed the cell proliferation on the MHA scaffold at later stage, as evidenced by the observed significant down-regulation of OD values on MHA samples at 6 days (Figure 11B). Collectively, the results indicated that the calcium channels might be correlated with the cell proliferation, but only to a limited degree.


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However, several papers have reported a link between ERK activation and Ca2+ signal.61, 62 MAPK may function as a converging point of signaling cascades induced by a variety of upstream signal. CaM may act on one particular upstream signal that directly target MAPK modules or not. It raises the question of whether calcium channels activate the MAPK/ERK signaling pathway and subsequently influence cell proliferation or not. W7 was used to inhibit the CaM activities and then the levels of MEK1/2 and ERK1/2 expression were examined (Figure 11 C-D). The presence of W7 inhibitor did not change the overall trends of MEK1/2 expression (Figure 11C), except for the observed significant down-regulation of OD values on MHA samples at 4 days as compared with the MHA before the inhibition (Figure 11B). As observed in MEK1/2 expression, less influence was found on the ERK1/2 marker, despite the observed decreased in ERK1/2 level on the MHA sample at 6 days (Figure 11D). To sum up, blockade of CaM activities slightly inhibited the levels of MEK1/2 and ERK1/2 expression, suggesting that MAPK-induced osteoblast growth was weakly dependent on the calcium channel. Collectively, these results suggested that adsorption of protein corona during cell cultivation is critical to stimulate cell proliferation through the activation of MAPK signaling pathway, where the MHA scaffold plays an essential role in formation of protein corona. To the best of our knowledge, this finding has not been previously reported. Adding MNPs to the HA scaffold would produce magnetic fields which is from the magnetic domain provided by the MNPs in a nanoscale field. Those tiny magnetic fields strongly affected the formation of the protein corona. Magnetism has already been shown


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to play a critical role in the formation of protein corona.39 Moreover, the different compositions of protein coronas affect cellular responses to the biomaterial. Particularly, the adsorption of specific proteins may activate various transcription factors or receptor, resulting in highly orchestrated signal and cellular events. The current experiments indicated that proteins, upstream receptor of the MAPK signaling pathway, were adsorbed in large concentrations on the MHA scaffolds. These proteins act as upstream promoters to stimulate the signaling cascade of MAPK/ERK signaling pathway, resulting in accelerating cell proliferation (Figure 12). Although there is still no clear understanding of the regulatory mechanism, the current findings may provide further understanding of the orchestrated signaling cascades and cell proliferation during bone regeneration, and have significant implications in the design of magnetic scaffolds for orthopedic applications.


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Figure 11. The effect of CaM on the MAPK/ERK signaling pathway: A) the CaM expression of MC3T3-E1 cells cultured on HA and MHA for 2, 4 and 6 days; B) Proliferation of cells cultured on HA and MHA with and without W7; C) Effects of CaM on MEK1/2 activation of MAPK signaling activity. MC3T3-E1 cells were incubated with W7 at 10 µmol; D) Effects of CaM on ERK1/2 activation of MAPK signaling activity. MC3T3-E1 cells were incubated with W7 at 10 µmol; Error bar represents means ± standard error of mean for n = 3. **p < 0.01 versus HA. #p < 0.05, ##p < 0.01, ###p < 0.001 versus MHA (non-inhibitor W7). +p

Protein Corona of Magnetic Hydroxyapatite Scaffold Improves Cell

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