Collagen-Based

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Developing a Strontium-Releasing Graphene Oxide/CollagenBased Organic-Inorganic Nanobiocomposite for Large Bone Defect Regeneration via MAPK Signaling Pathway Yahong Chen, Zhiwei Zheng, Renpeng Zhou, Huizhong Zhang, Chuhsin Chen, Zhezhen Xiong, Kai Liu, and Xiansong Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b22606 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 4, 2019

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ACS Applied Materials & Interfaces

Developing a Strontium-Releasing Graphene Oxide/Collagen-Based Organic-Inorganic Nanobiocomposite for Large Bone Defect Regeneration via MAPK Signaling Pathway

Yahong Chen1, Zhiwei Zheng2.3*, Renpeng Zhou1, Huizhong Zhang1, Chuhsin Chen1, Zhezhen Xiong1, Kai Liu1*, Xiansong Wang1, 4 *

1 Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai 200011, P.R. China. 2 Department of Oral and Maxillofacial-Head and Neck Oncology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai, 200011, China. 3 Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology; National Clinical Research Center of Stomatology, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai, 200011, China. 4 Shanghai Key Laboratory of Tissue Engineering, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, National Tissue Engineering Center of China, 200011, P.R. China. * Corresponding authors [email protected] (Zhiwei Zheng) [email protected] (Kai Liu) [email protected] (Xiansong Wang)

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Abstract Significant effort has been dedicated to fabricating favorable biomaterial-based bone substitutes for the repair of large bone defects. However, the development of bone biomaterials with suitable physiochemical

and

osteoinductive

properties

remains

a

challenge.

Here,

novel

strontium-graphene oxide (Sr-GO) nanocomposites that allow long-term release of Sr ions are fabricated, which are used to reinforce collagen (Col) scaffolds through covalent crosslinking. The prepared Sr-GO-Col scaffold demonstrates significantly high water retention and mechanical properties compared with those of unmodified Col scaffolds. The Sr-GO-modified Col scaffolds display a strong effect on adipose-derived stem cells by facilitating cell adhesion and osteogenic differentiation and by promoting the secretion of angiogenic factors to stimulate the in vitro tube formation of endothelial cells. Additionally, the secretion of angiogenic VEGF and osteogenic BMP-2 proteins is increased, which may be attributed to the synergistic effects of GO and Sr on the activation of the MAPK signaling pathway. The Sr-GO-Col constructs were then transplanted into rat critical-size calvarial bone defects and showed the best bone regeneration and angiogenesis outcome at 12 weeks. Moreover, histological staining results show that the Sr-GO-Col group achieved complete defect bridging with newly formed bone tissue and the residual Sr-GO nanoparticles are phagocytosed and degraded by multinucleated giant cells. These findings reveal that the incorporation of inorganic Sr-GO nanocomposites into biocompatible Col scaffolds is a viable strategy for fabricating favorable substitutes that enhance the regeneration of large bone defects.

Keywords: Strontium; Graphene oxides; MAPK; Collagen; Bone substitute


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1. INTRODUCTION Large bone defects following infection, trauma and tumor resection typically require assistance to provide skeletal continuity.1-2 Bone autografts are the current “gold standard” but have some drawbacks including donor site morbidity and limited availability.3-4 Biomaterial-based bone graft substitute is considered to be a promising alternative to overcome this challenge in the treatment of large bone defect.5 Collagen (Col), the most abundant organic component in bone tissue, has been extensively studied as a bone substitute due to its biodegradability and favorable biocompatibility.6 However, the use of Col is hindered by its weak mechanical and bioactive properties.7-8 Considering that bone is a kind of organic-inorganic composite, inorganic bioactive materials such as bioactive glasses, β-tricalcium phosphate (β-TCP) and hydroxyapatite (HA) have been reported to be incorporated in composites to enhance the promotion of bone regeneration by Col.9-12 However, the therapeutic outcome of calcium phosphate-modified scaffolds is still limited due to poor osteoinductive and angiogenic properties.13-14 On the basis of their properties of excellent physical, mechanical and bioactive properties, graphene oxide (GO) have shown great potential in various biomedical applications.15-16 GO has been reported to enhance the cell adhesion and spreading and steer the osteogenic differentiation of stem cells.17-18 Furthermore, the rich oxygen-containing functional groups on GO, such as carboxyl, carbonyl, hydroxyl and epoxy groups, provide nucleation sites for the deposition of an HA layer, which is a prerequisite for bone formation.19 Hence, we utilized osteoconductive and osteoinductive GO as an ideal inorganic component to improve Col scaffolds. To further improve the bioactivity of GO, we previously tested a strategy incorporating an



osteogenic cytokine, bone morphogenetic protein (BMP)-2, and demonstrated that this promising hybrid strategy significantly promoted bone regeneration after in vivo implantation.17 However, due to their intricate tertiary structures and short biological half-lives, a high dose of bioactive factors is often required and limit their clinical applications.20 Bioactive ions, such as copper (Cu), magnesium (Mg) and strontium (Sr), have emerged as potential therapeutic agents that can enhance bone formation due to their osteogenic and angiogenic effects.21-22 Therapeutic inorganic ions have certain properties that are superior to those of cytokines in certain aspects; for example, inorganic ions are not at risk of decomposition, are less specific or sensitive to microenvironmental conditions, and incur lower expenses. Among the many bioactive elements, Sr2+ is an essential trace element that has been reported to play an important role in maintaining human tissue functions, especially in bone.23 Because of its dual effects, namely, the ability to promote osteoblasts and inhibit osteoclasts, Sr preparations have been used as a clinical pharmaceutical agent for the treatment of osteoporosis patients.24-25 Given the individual beneficial properties of Sr, GO and Col, we fabricated a Sr-GO-Col-based organic-inorganic nanobiocomposite for large bone defect regeneration. A Sr-GO nanocomposite was first fabricated by anchoring Sr2+ nanoparticles on a GO substrate. Due to the abundant amino functional groups on Col, a hybrid Sr-GO-Col-based scaffold was readily formed by amidation. In this study, we hypothesized that the original angiogenic and osteogenic properties of the Sr-GO-Col nanobiocomposite would be further enhanced via the slow release of Sr ions. We evaluated the angiogenic and osteogenic potential of the nanobiocomposite in a rat cranial defect model and probed the potential mechanisms of regeneration (Scheme 1).


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Scheme 1. A brief schematic of the Sr-GO-Col nanobiocomposite fabrication and experimental design of this study.

2. MATERIALS AND METHODS Preparation of Sr-GO Nanocomposites: The Sr-GO nanocomposites were in situ synthesized and characterized as described elsewhere.17 Briefly, GO was fabricated according to modified Hummers method,26 and 20 mL of an aqueous SrCl2 solution (10 mM) was gradually added to 20 mL of a GO solution (0.5 mg mL-1) under stirring conditions and the mixed solution was then kept static at room temperature 72 h. The resulting nanocomposite was then washed in deionized water twice and separated by centrifugation (10000 rpm, 15min). The morphology and thickness of the prepared Sr-GO were evaluated using a Cypher atomic force microscope (AFM, Multi-Mode Nanoscope V, Veeco) operating in tapping mode under ambient conditions. Scanning electron microscopy (SEM) images were obtained on a ZEISS Ultra 55 field emission. Fabrication and Characterization of the Sr-GO-Col construct: The hybrid Sr-GO-Col construct



was fabricated via chemical crosslinking. A Col solution (2 wt% dissolved in 0.1 M acetic acid) and a Sr-GO solution with the same volume were mixed under magnetic stirring for 30 min to obtain a uniform mixture. To develop a series of Sr-GO-Col, Sr-GO was homogeneously mixed with different contents of Col (0-1.25 wt%). Then, N-hydroxysuccinimide (NHS, 0.025 M) and N-(3-(dimethylamino) propyl)-N-ethylcarbodiimide hydrochloride crystalline (EDC, 0.1 M) were added to the mixture to allow the crosslinking of Col and Sr-GO. Porous Sr-GO-Col scaffolds were obtained by freeze-drying the hydrogel. The samples with 0.25, 0.5, and 1.25 wt% Sr-GO were named 0.25 Sr-GO-Col, 0.5 Sr-GO-Col and 1.25 Sr-GO-Col, respectively. The morphology of the lyophilized Sr-GO-Col scaffolds was characterized by SEM at an accelerating voltage of 10 kV, and the chemical compositions were analyzed via Fourier transform infrared (FTIR) spectroscopy, and Raman spectra and X-ray diffraction (XRD) patterns. The release property of Sr ions from the Sr-GO-Col scaffolds were measured as previously reported.23 The scaffolds were placed in physiological saline at 37 ℃ under slow stirring. After a given time interval, the amount of released Sr ions in the saline was analyzed using inductively coupled plasma optical emission spectrometry (ICP-OES, Varian, USA). Physiochemical and Mechanical Characterization of Sr-GO-Col Scaffolds: Water contact angle (WCA) measurements was performed as previously reported.27 At RT, a 2 μL sessile droplet of deionized water was dropwise added onto the scaffold surface. The images of the droplet were obtained by an image analysis microscope (OCA 40 contact angle system, Data Physics Instruments GmbH, Germany). Water uptake and water retention rate measurements were carried out as previously described.28 Briefly, dry cylindrical scaffolds with a diameter of 15 mm and a length of 10 mm were weighed


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initially and at 24 h postimmersion in water. The water uptake and retention rates were estimated by blotting with filter paper and by centrifuging the wetted scaffolds (500 rpm, 3 min), respectively. Cytotoxicity Assays: Cytotoxicity Assays were performed to determine the suitable concentration of Sr-GO for fabricating the construct in this study. Human adipose-derived stem cells (hADSCs), one of the most practical clinically applied autologous stem cells,29 were selected for subsequent studies. hADSCs and HUVECs were seeded into the Sr-GO-Col constructs fabricated with different concentrations of Sr-GO (0-1.25 wt%) for 1 day and 3 days and then assessed by using a CCK8 assay. In addition, the proliferation rates of the hADSCs were also evaluated by a 5-ethynyl-2’-deoxyuridine (EdU) DNA Cell Proliferation kit (detailed protocol: Supporting information). Cell Viability, Morphology and Adhesion on Sr-GO-Col Scaffolds: The biocompatibility of the resulting scaffold was visualized using a live/dead staining assay (Invitrogen, USA). The cell-seeded constructs were incubated with the staining kit for 10 min at RT and imaged with a confocal laser scanning microscope (CLSM, Leica, Germany). The morphologies of the hADSCs and HUVECs on the scaffolds were also observed by SEM at 24 h after seeding (detailed protocol: Supporting information). To analyze cell adhesion activity, hADSC adhesion on different scaffolds was analyzed by the expression of integrin β1 after 12 h of culture. The cells were incubated with integrin β1 (Abcam, Cambridge, UK) overnight at 4 ℃. Nuclei and cytoskeletal structures were stained with DAPI (Invitrogen) and phalloidin (Sigma), respectively. Fluorescence images were visualized by CLSM. Detection of hADSC Osteogenic Differentiation on Sr-GO-Col Scaffolds: The hADSCs



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incubated on Sr-GO-Col scaffolds were cultured with osteoinductive medium, and after 7 days the cells were then stained with an alkaline phosphatase (ALP) kit (Beyotime, China).30 Alizarin red S (ARS) staining was performed after incubation for 14 days. Immunofluorescence staining was used to explore the expression of the osteogenic marker osteocalcin (OCN). Microcomputed tomography (micro-CT) was used to evaluate apatite deposition after 14 days of incubation. The samples were fixed in 4% PFA for 20 min; afterwards, the samples were scanned and reconstructed 3D images using the micro-CT system software (μCT-80, Scanco Medical, Switzerland). Quantitative Real-time Polymerase Chain Reaction (qRT-PCR) and Western Blot: Total RNA and proteins were isolated from the hADSCs after osteoinductive culture. The genes encoding ocn, osteopontin (opn), Osterix (Osx), and alp were analyzed and gene-specific primers with GADPH was selected as a reference gene (primers and detailed protocol: Supporting information Table S1). The following antibodies were used for western blot analyses: runt-related transcription factor (Runx2), extracellular signal-related kinases (ERK), phosphorylated (p-)ERK, protein kinase 38 (P38), p-P38, vascular endothelial growth factor (VEGF) and BMP-2 antibodies (detailed protocol: Supporting information). Angiogenic Effects of the Sr-GO-Col Nanobiocomposite: After culturing on different substrates for 2 days, the hADSCs were digested, and their gene expression levels of vegf and platelet-derived growth factor (pdgf-bb) were analyzed. For the in vitro recruitment assay, different culture media (CMs) were placed in the lower chamber of Transwell inserts according to the

group

designation:

Negative

control

[NC]-DMEM

with

1%

FBS;

Col

group

[CM(Col)]-DMEM with 1% FBS that was previously conditioned with hADSCs-seeded Col for 2


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days; GO-Col group [CM(GO-Col)]-DMEM with 1% FBS that was previously conditioned with hADSCs-seeded GO-Col for 2 days; and Sr-GO-Col group [CM(Sr-GO-Col)]-DMEM with 1% FBS that was previously conditioned with hADSCs-seeded GO-Col for 2 days. The HUVECs were cultured in the upper chamber of Transwell inserts. After 24 h, the cells migrated into the bottom side of the Transwell were stained and counted. An in vitro tubular formation assay was performed using Matrigel (BD Bioscience) according to the manufacturer's specifications. The HUVECs were seeded into 24-well plates coated with Matrigel, and different CM were added. After 6 h, the cells were imaged. Animal Study: The bone regeneration potential of the Sr-GO-Col nanobiocomposite was evaluated using a critical size cranial bone defect. After anesthetized by intraperitoneal injection of pentobarbital (Nembutal 3.5 mg/100 g), a 2 cm midsagittal incision was made, and two 5 mm size defects were created symmetrically on both sides of the calvaria bone using a diameter trephine. Afterwards, the scaffolds were transplanted into the bone defects. Twenty-four male rats (8-week-old male) with 48 defects were created and were randomly allocated into four groups: defect without treatment, Col group, GO-Col group and Sr-GO-Col group with a sample size of 12 defects. Micro-CT: The calvaria were harvested at 4 and 12 weeks post-implantation for micro-CT analysis (n = 4 defects for each time point). A three-dimensional reconstruction of the images was performed for the region of interest containing the scaffold, and the bone volume density (BV/TV) and the bone mineral density (BMD) were evaluated. Micro-CT Angiography: For identification of neovascularization at 12 weeks postoperation, micro-CT angiography was performed (n = 4 defects).31 Local vascular were observed by



micrangium perfusion with Microfil (Flowtech, Carver, MA, USA). After complete decalcification in 10% EDTA, micro-CT images of the vascular morphology were obtained. Calvarial undecalcified sections: The calvaria were collected, fixed in 4% PFA, and embedded in polymethylmethacrylate (PMMA) and then sectioned in the middle plane to produce 50-mm-thick undecalcified sections. For fluorescent double-labeling, alizarin red (30 mg/kg) and calcein (20 mg/kg) were subcutaneously injected into the rats at 6 and 9 weeks postoperation, respectively.32 The sections were imaged using a CLSM, and the integrated optical density (IOD) was used to determinate the distribution of alizarin red and calcein green for analyzing the new bone formation. The sections were then stained with Van Gieson and toluidine blue to quantify bone regeneration. Calvarial decalcified sections: The calvaria complete decalcification in 10% EDTA and embedded in paraffin blocks. Histological sections (5 μm) were prepared for subsequent H&E staining and Masson’s trichrome. To analyze the expression of OCN, OPN, Runx2 of the newly formed tissue, immunofluorescence staining was performed on the paraffin sections. Briefly, immunohistochemical staining was performed with primary anti-Runx2 antibody (1:200, Abcam), anti-OCN antibody (1:200, Abcam) and anti-OPN antibody (1:1000, Abcam), followed by 488 or 594 Alexa Fluor® secondary antibodies (Invitrogen) and DAPI before mounting and viewing the sections by CLSM. Different regions of the samples were imaged, and the average immunofluorescence from triplicate constructs was calculated. Statistical Analysis: All measurements are presented as the mean ± standard deviation. Statistical analyses were performed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test using GraphPad Prism software (GraphPad Software Inc., USA). The level


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of significance was set at p < 0.05. 3. RESULTS 3.1. Fabrication and Characterization of the Sr-GO-Col Scaffolds The as-prepared GO sheet by the modified Hummers method26 with a size of up to approximately 20 μm and a uniform thickness of approximately 1 nm (Figure 1a).17, 33-35 When the Sr2+ solution was gradually dropwise into the GO solution, the Sr2+ ions were anchored by the oxygen-containing functional groups on GO and began to grow into clusters. The Sr-GO complex formed flocculent structures, and the solution gradually separated. Figure 1b and c shows an SEM and TEM images of the Sr-incorporated GO sheet. The GO sheets have a smooth surface, and Sr nanoparticles formed during incubation are represented by white dots, ranges from 20 to 50 nm.

Figure 1. Characterization of the Sr-GO-Col scaffolds. a) Typical AFM image of a GO sheet.



b, c) SEM and TEM images of Sr nanoparticles deposited on the GO surface (Sr-GO). d) Optical images before lyophilization and SEM images of the lyophilized Col and Sr-GO-Col scaffolds. e) Raman spectra of the Sr-GO nanocomposites, Col scaffolds and Sr-GO scaffolds. f) Release of the Sr ions from the Sr-GO-Col scaffolds.

The chemical structure of the Sr-GO composites was investigated by UV-vis spectroscopy (Figure S1). The UV-vis spectrum of GO displays two characteristic peaks, 232 nm and 304 nm, which corresponds to the aromatic C–C bonds and C=O bonds, respectively. After the incorporation Sr2+, the characteristic peak of GO at 304 nm exhibited a redshift, which is likely because of the interactions between Sr ions and carboxylic groups. In contrast to the absorption spectrum of SrCl2, that of the Sr nanoparticles shows a broad peak, indicating that the Sr nanoparticles was amorphous.

After the crosslinking treatment by EDC, the white Col scaffold and the black Sr-GO-Col hybrid scaffold both exhibited porous structures (Figure 1d, S2-3). The SEM images revealed that the two scaffolds were both highly porous with interconnected structures, while the Sr-GO-Col scaffold exhibited a rougher surface than that of the Col scaffold. FTIR spectra were used to analyze the chemical structure of the Sr-GO-Col scaffolds (Figure S4a), which confirmed the amido bonds formed in the scaffolds. XRD patterns of the Sr-GO-Col scaffolds were also analyzed, and the structure was determined to be amorphous (Figure S4b), which is consistent with the results of UV-vis spectroscopy.

The Raman spectra of the Sr-GO powder revealed two

peaks, at 1351 cm−1and 1590 cm−1, which corresponded to the D and G bands, respectively (Figure 1e). After the crosslinking of Sr-GO and collagen to form the Sr-GO-Col scaffold, the D and G bands were still obvious, which confirmed that the scaffolds contained crosslinks between


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Sr-GO and collagen. The releasing profiles of Sr2+ from SrCl2 solution and Sr-GO-Col scaffolds were analyzed by ICP-OES. As shown in Figure 1f, the diffusion of Sr2+ was very fast from SrCl2 solution. However, for Sr-GO-Col scaffolds, the Sr particles anchored by the GO and the release of Sr2+ was much slower. This is preferable for bone regeneration, since the scaffold can maintain the moderate concentration of Sr2+ ions at a long time. 3.2. Evaluation of Physiochemical and Mechanical Properties of the Sr-GO-Col hybrid Scaffolds As shown in Figure 2a, the WCA of the scaffolds in the Sr-GO-Col group decreased gradually as the content of Sr-GO nanoparticles increased from 0.25 wt% to 1.25 wt%, indicating that the presence of Sr-GO nanoparticles enhanced the hydrophilicity of the Col scaffold. We then tested the water uptake and water retention properties of the Sr-GO-Col scaffolds (Figure 2b). After soaking overnight in water, the Sr-GO-Col scaffolds had higher water uptake rates (4778%-6739%) and water retention rates (1684%-2814%) than those of the Col scaffold (4526% uptake and 1364% retention rates). Furthermore, the overall mechanical properties of the scaffolds were measured by compression testing (Figure 2c). The stress-strain curves of the Sr-GO-Col scaffolds revealed enhanced mechanical properties of the hybrid scaffold compared with those of the bare Col scaffold. Specifically, the compressive modulus of the Sr-GO-Col scaffolds (0.5 wt%) was 74.8 ± 8.1 kPa, which was higher than that of the Col scaffolds under the same conditions (p< 0.05). When the content of Sr-GO increased to 1.25 wt%, the compression modulus of the Sr-GO-Col scaffolds further increased to 96.5 ± 7.2 kPa. These results revealed that the hybrid Sr-GO-Col scaffolds would be more suitable for the application of bone substitute.



Figure 2. The physiochemical and mechanical properties and cytotoxicity of the Sr-GO-Col scaffolds. a) Optical images and hydrophilicity analysis of the Col and Sr-GO-Col (0.25, 0.5, and 1.25 wt%) scaffolds. WCA decreased with the increasing ratio of Sr-GO nanocomposites. b) Water uptake and water retention analysis. c) Stress-strain curves and compressive modulus analysis. d) CCK-8 assay of cell viability for Col and Sr-GO-Col (0.25, 0.5, 0.75, 1 and 1.25 wt%) substrates at 1 and 3 days. e) EdU proliferation assay. The EdU results showed that hADSC proliferation was not impaired on the 0.25 and 0.5 Sr-GO-Col scaffolds. (*, p < 0.05 compared with the Col control.)

The metabolic activity of hADSCs and HUVECs on a series of Sr-GO-Col scaffolds (0.25-1.25 wt%) was examined at 1 and 3 days using the CCK-8 assay (Figure 2d). The Sr-GO-Col scaffolds did not elicit significant cytotoxicity in hADSCs and HUVECs at a Sr-GO content below 0.75


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wt%. EdU analysis further verified that hADSC proliferation was not impaired on the 0.25 and 0.5 Sr-GO-Col scaffolds (Figure 2e). Considering various results, such as hydrophilicity and mechanical properties, and in vitro cell proliferation, we chose 0.5 Sr-GO-Col as a suitable scaffold for further study.

Figure 3. The viability activity and adhesion of cells cultured on Sr-GO-Col scaffolds. a) Live/dead staining of HUVECs and hADSCs at 1 and 3 days. b, c) Cell viability analysis. d) SEM images of HUVECs and hADSCs growth on Col, GO-Col, and Sr-GO-Col scaffolds. e, f) Immunofluorescence staining of integrin β1 expression in the hADSCs after seeding on the scaffolds for 12 h and semiquantitative analysis. In these images, F-actin was stained with phalloidin, and the nuclei were stained with DAPI (*, p < 0.05, compared with the Col group; #, p < 0.05, compared with the GO-Col group).



3.3. Evaluation of the In Vitro Cytocompatibility of the Sr-GO-Col Nanobiocomposite To verify the hypothesis that the Sr-GO-Col scaffolds can provide a suitable microenvironment for bone regeneration, we first evaluated the cell viability and adhesion on the Sr-GO-Col scaffolds. A live/dead analysis of hADSCs and HUVECs was initially performed to visualize the cytotoxicity of the Sr-GO-Col materials in the hADSCs and HUVECs (Figure 3a, b, c). The results showed that more than 78% of the hADSCs seeded on the Sr-GO-Col scaffolds remained viable after 1 day of culture and greater than 88% remained viable after 3 days of culture; these results were comparable to those for the Col group (p > 0.05). HUVECs on the Sr-GO-Col scaffolds showed similar trends, and most of the cells remained viable. Interestingly, the hADSCs on the Sr-GO-Col scaffolds exhibited greater spreading and migrated deeper into the scaffolds than the hADSCs cultured on the Col and GO-Col scaffolds (Figure 3a). SEM observations of cell morphology indicated that most of the hADSCs and HUVECs exhibited a spindle-like morphology on the Col, GO-Col and Sr-GO-Col scaffolds (Figure 3d). Notably, the spreading of hADSCs and HUVECs was more obvious in the Sr-GO-Col group. Similarly, the expression of the cell adhesion-related protein integrin β1 was higher in the GO-Col group than in the Col group, and this difference was more prominent after the incorporation of Sr2+ (Figure 3e, f). 3.4. Sr-GO-Col Nanobiocomposite Promoted Osteogenesis of hADSCs In Vitro To further elucidate whether the Sr-GO nanocomposite could promote bone regeneration, the effects of the Sr-GO-Col scaffolds on the potential osteogenic differentiation of hADSCs were evaluated. Micro-CT was used to visualize and analyze the extent and 3D distributions of mineralization among the different scaffolds (Figure 4a). Evidently, GO-functionalized Col led to


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marked mineralization of the constructs, and mineralization was further increased by functionalization with Sr-GO nanocomposites. In addition, the early osteogenic differentiation of hADSCs was measured by ALP staining at day 7. As shown in Figure 4b, the ALP activity of both GO-Col and Sr-GO-Col scaffolds was enhanced compared with that of Col alone and was significantly higher in the Sr-GO-Col group than in the GO-Col group. On day 14, ARS confirmed that the osteogenic differentiation of hADSCs was promoted in the GO-Col and Sr-GO-Col groups (Figure 4b), and ARS decreased among the groups in the following order: Sr-GO-Col > GO-Col > Col. Furthermore, the expression level of the osteogenic marker OCN significantly increased in the Sr-GO-Col group after 14 days of incubation. The stimulating effects of the Sr-GO-Col nanobiocomposite on the osteogenic differentiation of hADSCs were further investigated by examining osteogenic-related gene and protein expression. The expression levels of osteogenic genes in hADSCs, including ocn, opn, osx, and alp, were highest in the Sr-GO-Col group (p GO-Col group > Col control group. Furthermore, residual Sr-GO was observed in the multinucleated giant cells, which mainly located between the regenerated bone tissues (Figure S5).



Figure 8. Evaluation of bone tissue regeneration at 12 weeks using Van Gieson and toluidine blue staining and fluorescence labeling. a) Van Gieson and toluidine blue staining of undecalcified calvarial sections. In the Sr-GO-Col group, the newly formed bone exhibited a cortical bone-like tissue structure. b) Summary of the Van Gieson staining results. c) Fluorescent images of newly formed bone with different scaffolds, which were labeled with calcein and alizarin red. d, e) Summary of fluorescence labeling results. (*, p < 0.05, compared with the Col group; #, p < 0.05, compared with the GO-Col group).


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Immunohistochemical analysis was carried out to further evaluate the early neovascularization and osteogenic activity of different scaffolds at 4 weeks (Figure 9). The Sr-GO-Col group had the higher expression of osteogenic transcription factor Runx2 higher than those of the other groups (Figure 9a, b). Immunofluorescence staining for the late-stage bone markers OCN and OPN illustrated that the defects in the Col groups were primarily filled with loose tissues without OCN accumulation. The expression levels of OCN and OPN in the defective sites of the Sr-GO-Col group were significantly higher than those of the other groups (Figure 9a, b). Furthermore, as shown by the immunohistochemistry staining results in Figure 9c and d, the Sr-GO-Col group had significantly more CD31-positive vessels than did the other groups, indicating that incorporation of the Sr-GO nanocomposite effectively enhanced the osteoinductive and angiogenic activities of the Col scaffolds.

Figure 9. Immunohistochemical analysis. a) Immunostaining of RUNX2, OCN, and OPN of in vivo implants at 4 weeks. b) Positive expression ratios of RUNX2, OCN, and OPN. c, d)



Immunohistochemistry of CD31 and semiquantitative analysis. e) CD68 (red) and CD206 (green) coimmunolabeling to assess the profiles of M2 macrophages. f, g) Summary analysis of CD68+ and CD206+CD68+ cell ratios. (*, p < 0.05, compared with the Col group; #, p < 0.05, compared with the GO-Col group)

Recent studies have shown that anti-inflammatory M2 macrophages play an important role in the tissue repair process.36-37 Hence, the M2 macrophage phenotypes around the scaffold were examined at 4 weeks (Figure 9e). In particular, the presence of CD68 (a common marker for pan-macrophages) and CD206 (a marker for M2 macrophages) was investigated. The total number of macrophages (CD68+ cells) was decreased in the Sr-GO-Col group. In addition, the total number of M2 macrophages (CD68+CD206+cells) was increased in the Sr-GO-Col group (Figure 9f, g). These data suggested that Sr-GO effectively modulates macrophage polarization toward the M2 profile, which is beneficial for tissue regeneration.

4. DISCUSSION In this study, we developed an inorganic-organic Sr-GO-Col nanobiocomposite and demonstrated its potential to successfully repair large bone defects and regenerate bone-like tissue through the release of Sr2+ ions. Sr-GO nanocomposite materials were fabricated by depositing strontium nanoparticles on the surface of GO, and a prolonged and sustained release of Sr ions was observed. Then, a biocompatible scaffold was fabricated via the chemical crosslinking of GO and Col. The efficacy of the Sr-GO-Col scaffolds in regenerating bone defects and the underlying mechanism were systematically investigated. The resulting Sr-GO-Col scaffolds achieved the


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required physiochemical, biocompatible and bioactive properties for in vivo bone substitutes. The resulting Sr-GO-Col scaffold demonstrated a modulus that was 3.1-fold higher than that of the Col scaffold and showed highly porous and interconnected structures. In addition, our data also showed higher hydrophilicity and water retention rates after the incorporation of Sr-GO, which are beneficial properties for biomedical applications. These results also imply that the incorporation of Sr nanoparticles would not compromise the superior physiochemical properties of GO, which are comparable to previous studies showing that the incorporation of GO could enhance the water uptake and mechanical properties of the construct. This may be accomplished by the geometric constraint of GO that imposed a higher degree of constraint on the mobility of polymer chains, and increased the degree of crosslinking between the of the abundant functional groups on the surface of GO and Col.17,28 Furthermore, cytotoxicity assays revealed that the Sr-GO-Col scaffolds did not exhibit cytotoxicity at a Sr-GO content below 0.5 wt%, where the difference in cell viability between Sr-GO-Col and Col scaffold is neglectable. This is consistent with our previous data showing that GO is cytotoxic when its concentration was greater than 50 µg mL−1,17 corresponding to 0.5 wt% in the present study. Consequently, we chose 0.5 Sr-GO-Col as a suitable scaffold for further biological evaluation in vitro and in vivo. Notably, the incorporation of 0.5 wt% GO has also been shown to affect cell spreading and morphology and to promote the growth of hADSCs deep in the scaffolds. These findings may be attributed to the rich oxygen-containing functional groups in GO, which provide an abundance of binding sites for serum proteins and, resulting in bettter cell adhesion performance.17 Importantly, the incorporation of Sr2+ further enhanced cell adhesion and spreading. This phenomenon was also reported in previous studies in which cell adhesion and



spreading were found to increase with increasing strontium content.38 The osteoinductive properties of the Sr-GO-Col construct was also investigated. Similar to previous studies, inorganic GO had favorable osteoinductive properties and was beneficial for osteogenesis. In this study, the GO-Col scaffolds promoted the osteogenic differentiation of hADSCs and accelerated the mineralization deposition process when compared with that of the Col scaffolds. This is consistent with previous findings showing that GO could steer stem cells toward osteogenic differentiation.39-40 Importantly, the incorporation of Sr ions further improved osteogenesis, and the osteogenic gene and protein expression levels of the hADSCs in the Sr-GO-Col group were much higher than those in the other groups. These observations are consistent with data showing that Sr ions are beneficial for cell proliferation and osteogenic differentiation.24-25 To further explain the molecular mechanism by which the Sr-GO nanocomposite enhanced osteogenic differentiation, we detected the expression of Runx2 and the phosphorylation of ERK 1/2 and P38, which have been recognized to be necessary for the activation of Runx2. As confirmed in previous studies, GO could promote VEGF and BMP-2 secretion of stem cells by phosphorylating ERK 1/2 via activation of the integrin β1/FAK signaling pathway.22,41 This phenomenon is consistent with our studies, which showed that the expression of integrin β1 was enhanced after cell culture on the GO-Col or Sr-GO-Col scaffolds. Moreover, western blot analysis revealed that the incorporation of Sr ions further facilitated the phosphorylation of ERK 1/2 and P38 and the activation of Runx2, this was also confirmed in previous research.23 Notably, the enhanced expression of VEGF and BMP-2 was revealed to be coupled with the activation of MAPK, which subsequently promoted the tube formation of HUVECs and facilitated the


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osteogenesis of hADSCs. These results inferred that Sr-GO nanocomposites could synergistically promote angiogenesis and osteogenesis via the MAPK signaling pathways. Further investigation is required to fully understand the signaling mechanism. The vascularized bone regeneration processes of Sr-GO-Col scaffolds without stem cells were carefully investigated in this study. We showed that the Sr-GO-Col group exhibited both bone-like regenerated tissue that bridged the defects from the periphery and in situ bone formation island tissue from the center region of the defect. This finding is probably attributed to the incorporation of Sr-GO, where Sr and GO act synergistically to enhance osteogenesis and angiogenesis,38,42 achieving the best defect healing outcome. Because the degradation of graphene is on the time scale of months, residual Sr-GO materials was observed between the regenerated bone tissue after 12 weeks postsurgery. This is consistent with previous research which indicated that the resident GO were probably phagocytosed and subsequently degraded by the multinucleated giant cells.22 Local host inflammatory responses also play a crucial role in determining the outcome of the biomaterial implanted. Recently, local tissue M2 macrophages have been reported to be key regulators of tissue repair and regeneration.36-37,43 Previous studies have revealed that Sr2+ ions have anti-inflammatory and immunomodulatory effects.36 Notably, Sr-GO-Col significantly induced macrophage polarization toward the M2 phenotype, indicating that the Sr-GO-Col scaffold was favorable for bone defect repair. 5. CONCLUSIONS In summary, the Sr-GO nanocomposites with a prolonged release of Sr2+ ions were successfully fabricated. Incorporating Sr-GO nanoparticles into the Col construct significantly increased the physiochemical and mechanical properties of the constructs. Notably, the Sr-GO-Col hybrid



scaffolds could provide preferable microenvironments for in vitro osteogenesis, and significantly promoted in vivo vascularized bone regeneration in a rat calvarial defect model. These results suggest that the Sr-GO-Col scaffold could serve as a promising bone substitute for the treatment of large bone defect.

Acknowledgements This research was supported by the National Natural Science Foundation of China (81671839, 81471878 and 81272128).

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