Collagen Functionalized With Graphene Oxide Enhanced Biomimetic

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Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Collagen Functionalized With Graphene Oxide Enhanced Biomimetic Mineralization and in Situ Bone Defect Repair Chuchao Zhou,† Shaokai Liu,† Jialun Li, Ke Guo, Quan Yuan, Aimei Zhong, Jie Yang, Jiecong Wang,* Jiaming Sun,* and Zhenxing Wang*

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Department of Plastic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China S Supporting Information *

ABSTRACT: Biomimetic mineralization using simulated body fluid (SBF) can form a bonelike apatite (Ap) on the natural polymers and enhance osteoconductivity and biocompatibility, and reduce immunological rejection. Nevertheless, the coating efficiency of the bonelike apatite layer on natural polymers still needs to be improved. Graphene oxide (GO) is rich in functional groups, such as carbonyls (−COOH) and hydroxyls (−OH), which can provide more active sites for biomimetic mineralization and improve the proliferation of the rat bone marrow stromal cells (r-BMSCs). In this study, we introduced 0%, 0.05%, 0.1%, and 0.2% w/v concentrations of GO into collagen (Col) scaffolds and immersed the fabricated scaffolds into SBF for 1, 7, and 14 days. In vitro environment scanning electron microscopy (ESEM), energy-dispersive spectrometry (EDS), thermogravimetric analysis (TGA), micro-CT, calcium quantitative analysis, and cellular analysis were used to evaluate the formation of bonelike apatite on the scaffolds. In vivo implantation of the scaffolds into the rat cranial defect was used to analyze the bone regeneration ability. The resulting GO−Col−Ap scaffolds exhibited a porous and interconnected structure coated with a homogeneous distribution of bonelike apatite on their surfaces. The Ca/P ratio of 0.1% GO−Col−Ap group was equal to that of natural bone tissue on the basis of EDS analysis. More apatites were observed in the 0.1% GO−Col−Ap group through TGA analysis, micro-CT evaluation, and calcium quantitative analysis. Furthermore, the 0.1% GO−Col−Ap group showed significantly higher r-BMSCs adhesion and proliferation in vitro and more than 2-fold higher bone formation than the Col−Ap group in vivo. Our study provides a new approach of introducing graphene oxide into bone tissue engineering scaffolds to enhance biomimetic mineralization. KEYWORDS: graphene oxide, collagen, bonelike apatite, biomimetic mineralization, simulated body fluid

1. INTRODUCTION Large bone defects caused by traumatic injury, infection, or tumor resection pose a huge clinical burden worldwide.1 Current clinical treatments for large bone defects including autogenous grafts, allografts, and xenografts are generally limited by their inherent disadvantages such as donor site morbidity, disease or virus transmission, immunogenicity, and host−donor junction complications.2 Therefore, the use of bone graft substitutes as scaffolds to regenerate the bone tissue has attracted the interests of researchers. Type I collagen (Col) is one of the most prevalent collagen types found in the extracellular matrix (ECM), especially in tendon and bone tissues. It has been broadly used as a graft substitute due to its good biocompatibility and biodegradability.3,4 However, the mechanical strength and osteoconductivity of collagen scaffolds are still unsatisfactory.5 The presence of functional groups in collagen makes it convenient to modify, cross-link, or coat with other bioactive molecules to create collagen-based materials with tailored mechanical and biological properties.6 © XXXX American Chemical Society

Graft substitutes coated with CaP have been widely used because of their outstanding intrinsic bioactivity and osteoconductivity. The CaP coating was first introduced to treat the surface of titanium (Ti) metal implants using a plasma spraying method in the early 1950s. Since then, various types of methods aimed to prepare bioactive CaP coatings have been developed, including thermal spraying,7 sputter-coating,8 sol−gel deposition,9 dip-coating,10 and hot isostatic pressing.11 A common limitation of these methods is the high processing temperature which is unsuitable for polymers with low melting temperature, such as natural polymers and biological materials. Biomimetic mineralization using simulated body fluid (SBF) is a promising method that provides suitable conditions of temperature, ion concentration, and pH which are similar to human blood plasma. These biomimetic conditions could promote deposition of bonelike apatite (Ap) covered on graft substitutes.12 Received: October 9, 2018 Accepted: November 26, 2018 Published: November 26, 2018 A

DOI: 10.1021/acsami.8b17636 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

H2SO4, HCl, CaCl2, Na2SO4, KMnO4, H2O2, HAc, C2H6O, NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, and CNH2(HOCH2)3 were purchased from Sinopharm Chemical Reagent Co., Ltd. SBF solution was prepared according to Kokubo’s method.19 Fetal bovine serum (FBS) was purchased from Hyclone. Phosphatebuffered saline (PBS) and trypsin−EDTA were purchased from Servicebio technology Co., Ltd. Low-glucose Dulbecco’s modified Eagle’s medium (L-DMEM) and 1% penicillin and streptomycin were purchased from Hyclone (Logan, UT). Fluorescein diacetate (FDA), 3[4,5-dimehyl-2-thiazolyl]-2,5-diphenyl-2-H-tetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (Shanghai, China). 2.1. Fabrication of the GO−Col−Ap Scaffolds. 2.1.1. Synthesis of GO−Col Scaffolds. The GO−Col scaffolds were synthesized by chemical cross-linking as illustrated in Figure S1. Briefly, GO was prepared by the modified Hummer’s method.20 Solutions of 4% Col (4% w/v in 0.1 M HAc) and GO (0%, 0.1%, 0.2%, 0.4% w/v in 0.1 M HAc) were mixed at a 1:1 volume ratio (the final concentrations of GO were 0%, 0.05%, 0.1%, and 0.2% w/v) under sonication for 30 min. Then, the obtained solutions were cast in molds (5 mm in diameter and 1 mm in height). The molds were frozen overnight at −20 °C and subsequently lyophilized for 24 h at −50 °C under vacuum. Thereafter, the freeze-dried plates were taken out of the molds and immersed in 95% ethanol solution, followed by addition of EDC (10 mg/mL) and NHS (4 mg/mL). The scaffolds were kept for 24 h at room temperature to allow the collagen to cross-link with GO and form a stable GO−Col scaffold. The collagen scaffolds were formed using the same procedures described above. For removal of the byproducts of the chemical reactions, the scaffolds were washed with ddH2O three times, followed by freezing overnight at −20 °C and lyophilizing for 24 h at −50 °C under vacuum. 2.1.2. Biomimetic Apatite Deposition. The cross-linked scaffolds were immersed into CaCl2 and K2HPO4 solutions to accelerate biomimetic apatite deposition. Next, the scaffolds were immersed in 20 mL of 0.2 M CaCl2 solution for 3 min and then soaked in 20 mL of ddH2O for 10 s, followed by soaking in 20 mL of 0.2 M K2HPO4 solution for 3 min and then soaking in 20 mL of ddH2O for another 10 s. The entire experiment was repeated three times. These alternately soaked scaffolds were subsequently immersed in SBF for biomimetic apatite deposition (10 mL of SBF was poured into one well of a 6-welled culture plate containing one alternately soaked scaffold). After 5 min of vacuum treatment, the scaffolds were kept at 37 °C for 1 day, 7 days, and 14 days, and the SBF was renewed every 24 h to maintain a consistent ionic strength throughout the experiment. The SBF solution was prepared by dissolving NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2· 6H2O, CaCl2, and Na2SO4 in ddH2O and buffering to pH 7.40 at 36.5 °C with Tris (hydroxymethyl) aminomethane and aqueous 1 M HCl solution. The scaffolds were removed from the SBF, gently washed with ddH2O, and subsequently lyophilized at −50 °C for 24 h under vacuum. 2.2. Characterization of the Scaffolds. 2.2.1. Mass Increase of the Scaffolds after Biomimetic Apatite Deposition. The mass increase (MI) of the scaffolds after biomimetic apatite deposition in SBF was measured using an electronic analytical balance (accurate to 10−5 g). The mass increment was expressed as the difference between the M1 (mass of the scaffolds before alternately soaking in CaCl2 and K2HPO4 solution) and M2 (mass of the scaffolds after biomimetic immersion in SBF), described by eq 1.

However, there are still some improvements required to develop a uniform and thick enough coating on the natural polymers.13 Graphene oxide (GO), a single layer of sp2-bonded carbon atoms, has emerged as a promising substrate for constructing graft substitutes due to its superior mechanical and biological properties.14 The presence of epoxides, carbonyls, and hydroxyls on the surface of GO makes it easily dispersed in aqueous solutions and can provide anchor sites for binding with polymers or nanoparticles in a scaffold.15 Recent studies have shown that GO can serve as an effective reinforcement filler by enhancing the network structure of the scaffolds16,17 and as a biological activator in natural polymers such as collagen and chitosan through introducing a plethora of functional groups to more closely mimic the properties of native bone and coordinate ions to mineralize the scaffolds.18 Therefore, we hypothesized that GO nanosheets might significantly improve the biomineralization efficiency of collagen in SBF solutions. In this study, cross-linked and porous GO−Col scaffold was prepared by amidation and lyophilization of GO and Col. For an improvement of the osteoconductivity of the scaffold, the bonelike apatite was linked to GO and Col on the surface of the scaffold through soaking in SBF for 7 days (Figure 1). Then, environmental scanning electron microscopy

Figure 1. Schematic illustration of the fabrication of GO−Col−Ap scaffolds and their application in vivo. (1) The GO−Col scaffolds were fabricated by the cross-linking of collagen and graphene oxide. (2) Apatite crystals were deposited on the scaffolds after soaking in simulated body fluid (SBF) for 7 days. (3) The GO−Col−Ap scaffolds were implanted directly into rat cranial defects sites to evaluate orthotopic bone formation.

(ESEM), energy-dispersive spectrometry (EDS), Fourier transform infrared spectrometry (FTIR), thermogravimetric analysis (TGA), micro-CT, and calcium quantitative analysis were used to evaluate the formation of bonelike apatite on the scaffolds. Systematic in vitro experiments determining the cellular response (cell viability and cell proliferation) to the scaffolds were carried out to evaluate their cytocompatibility. Moreover, in vivo experiments evaluating the ability of scaffolds to repair critical-size bone defects in the rat skull were performed and further analyzed by CT scanning and histological examination.

MI (%) =

M 2 − M1 × 100 % M2

(1)

2.2.2. ESEM and EDS Evaluation. The surfaces of the scaffolds were characterized before and after the biomimetic coating process by environmental scanning electron microscopy (ESEM; Quanta 200, FEI company) at 10 kV. The ratio of the calcium and phosphate of the biomimetic coating was performed by energy-dispersive spectrometry (EDS). Prior to ESEM observation and EDS analysis, the scaffolds were sputter-coated with gold for 1 min. 2.2.3. XRD, FTIR, and TGA Evaluation. Crystalline phases were assayed using an X-ray diffractometer (XRD; Empyrean, PANalytical B.V.) at a scanning rate of 0.013°/s in a 2θ range from 5° to 40° with Cu

2. MATERIALS AND METHODS Graphene (500 meshes) was obtained from Acros Organic Company. Collagen, N-(3-(dimethylamino)propyl)-N-ethylcarbodiimide hydrochloride crystalline (EDC), and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (Shanghai, China). Reagents NaNO3, B


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Figure 2. Macro- and microimages of the scaffolds before and after biomimetic apatite deposition. (A) The colors of collagen and graphene oxide− collagen scaffolds were white and brown, respectively. (B) White crystals were deposited on the scaffolds after the biomimetic apatite deposition. As shown in the SEM images, (C) the scaffold surfaces before biomimetic apatite deposition were smooth, while (D) crystals were observed on the scaffolds after biomimetic apatite deposition at 1, 7, and 14 days. (E) The mass increases of the four groups were remarkable between 1 day and 7 days of incubation in SBF, but no significant difference was observed between 7 days and 14 days of incubation in SBF. (Scale bars in parts C and D, 10 μm; ***P < 0.001 vs Col−Ap at D1, &&&P < 0.001 vs 0.05% GO−Col−Ap at D1; ###P < 0.001 vs 0.1% GO−Col−Ap at D1; ^^^P < 0.001 vs 0.2% GO− Col−Ap at D1.) Kα radiation (k = 1.540 598 nm). Functional groups were confirmed using FTIR spectroscopy (VERTEX 70; Bruker company) from 4000 to 500 cm−1 at a resolution of 0.4 cm−1. The amount of apatite in the 3D scaffolds was determined using TGA (Diamond TG/DTA; PerkinElmer Instruments, Shanghai, China). The scaffolds were heated at a heating rate of 10 °C/min from 25 to 800 °C in N2 atmosphere. 2.2.4. Mechanical Evaluation. The elastic modulus was tested to evaluate the mechanical properties of the scaffold using an all-electric dynamic test instrument (ElectroPuls E1000, INSTRON) at a loading rate of 1 mm/min until the 80% compression of the samples was achieved. The slope of the initial linear portion of the stress−strain curves was calculated to obtain the elastic modulus. 2.2.5. Micro-CT Evaluation. For the observation of the biomimetic apatite deposition in the scaffolds, a micro-CT scanner (SkyScan 1176; Broker) was used with the following scanning parameters: 58 kV, 385 mA, and 18 μm slice thickness. Corresponding 3D images were reconstructed using VG studio software (Volume Graphics GmbH, Heidelberg, Germany). Apatite volume (AV) and the percentage of apatite volume relative to total volume (AV/TV) were determined by micro-CT assistant software (Scanco Medical, Zurich, Switzerland). 2.2.6. Calcium Quantitative Analysis. The calcium content assay was performed by dissolving the mineralized scaffolds in 0.4 mL of 0.5 M acetic acid overnight and quantifying them with a calcium assay kit (BioAssay Systems, Hayward, CA, http://www.bioassaysys.com) according to the manufacturer’s instruction. 2.3. In Vitro Cellular Evaluation. 2.3.1. Rat BMSC Isolation and in Vitro Culture. Rat bone marrow stromal cells (r-BMSCs) were isolated and harvested as described previously.21 In brief, newborn Sprague Dawley rats (3−5 days old) were euthanized by cervical dislocation, followed by soaking in 75% alcohol for 15 min. Femurs and tibias were separated from attached muscles and soft tissues. Then, cartilages at both end of the bones were cut off, followed by repeated washing of the cavities with the culture medium until the cavities appeared white. Finally, the fresh bone marrow tissues were seeded on

10 cm culture dishes with 7 mL of culture medium (L-DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin). The culture dishes were incubated (Thermo Scientific, Heracell 150i) at 5% CO2, 37 °C, and the culture medium was changed every 3 days. When the attached r-BMSCs became confluent, the BMSCs were passaged. 2.3.2. Cell Seeding on Scaffolds and Cell Adhesion Capability. For evaluation of the cytocompatibility of the scaffolds, each scaffold (n = 5) was seeded with 2 × 105 r-BMSCs in 20 μL of culture medium. The plates containing scaffolds were cultured in an incubator at 5% CO2, 37 °C, and the culture medium was changed every 3 days. After 2 h of the initial attachment period, the r-BMSCs seeded on scaffolds were transferred to a new 6-well plate. For assessment of cell adhesion capability, the number of r-BMSCs attached to the bottom of the plate was collected and counted (N). Cell adhesion capability (CAC) was calculated using the following equation: CAC (%) =

2 × 105 − N × 100% 2 × 105

(2)

where 2 × 10 in eq 2 was the total amount of cells loaded. 2.3.3. Evaluation of Cell Viability and Morphology. The cell viability and morphology of r-BMSCs seeded on the scaffolds were assessed by FDA cell imaging kit (Sigma-Aldrich) staining. In brief, the scaffolds were washed three times with phosphate-buffered saline (PBS) and subsequently immersed in 2 μM fluorescein diacetate (FDA) for 30 min at 37 °C. After 1, 7, and 14 days of incubation, scaffolds were examined by confocal fluorescence microscopy. For further evaluation of the cell morphology, the cell-seeded scaffolds were washed three times with PBS, fixed with 2.5% glutaraldehyde for 3 h, and washed again with PBS. Then, the scaffolds were dehydrated with a graded series of ethanol solutions, lyophilized at −50 °C under vacuum, and sputter-coated with gold for ESEM analysis. 2.3.4. Proliferation of r-BMSCs on the Scaffolds. The 2 × 105 rBMSCs were seeded on each scaffold in 6-well plates. After 1, 7, and 14 5

C


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Figure 3. Characterization of the scaffolds. (A) EDS evaluation of the Ca/P ratios; the Ca/P ratio (1.67 ± 0.21) similar to that of the natural bone tissue (1.67) was observed in the 0.1% GO−Col−Ap group. EDS mapping of biomimetic apatite confirmed the homogeneous distribution of (B) calcium element and (C) phosphate element in the four scaffold groups. (D) Crystalline phases were evaluated in the XRD spectra; a gradually increasing intensity of the peak at 2θ = 31.8° was observed from the Col−Ap group to the 0.2% GO−Col−Ap group, and this change was attributed to the crystal growth at the (211) reflection of HA. (E) The phosphate group and hydroxyl group were characterized in the FTIR spectra at 1023, 961, 601, 561, and 3303 cm−1. (F) The TGA assay demonstrated that more apatite was deposited on 0.1% GO−Col−Ap and 0.2% GO−Col−Ap scaffolds. (G) Mechanical testing demonstrated that the elastic modulus in three groups of GO−Col−Ap scaffolds was higher than in Col−Ap scaffold, and the highest elastic modulus was observed in the 0.2% GO−Col−Ap group. days of incubation, the cell proliferation rate was evaluated using the MTT assay on the basis of the manufacturer’s instructions. 2.4. In Vivo Cranial Defect Study. 2.4.1. In Vivo Rat Cranial Defect Orthotopic Bone Formation. For an exploration of the ability of the GO−Col−Ap implants to form new bone in vivo, 24 male adult SD rats (age, 6 weeks; weight, 180−220 g; supplied by the animal center of Tongji medical college, Huazhong University of Science and Technology) were randomly divided into four groups: (1) Col−Ap; (2) 0.05% GO−Col−Ap; (3) 0.1% GO−Col−Ap; and (4) 0.2% GO− Col−Ap (n = 6 rats per group). The rats were anesthetized by inhaled isoflurane and subcutaneous buprenorphine injection, and a 2.0 cm sagittal incision was subsequently made on the middle of the scalp. Two parallel 5.0 mm critical cranial defects were drilled into each rat using a 5.0 mm diameter trephine (Nouvag AG, Goldach Switzerland). After the scaffolds were implanted into the defect areas, the incision was closed. 2.4.2. Micro-CT Evaluation. Animals were sacrificed at 4 and 12 weeks postoperation, and the skulls were collected for fixation with 10%

formalin (n = 3 rats per group). The bone tissue in the defect areas (diameter, 5 mm; height, 2 mm) was evaluated by micro-CT scanning. Three-dimensional images (mean threshold value = 226) of the samples were reconstructed by VG studio software, and the quantitative morphometric analyses were performed using micro-CT assistant software to determine the bone volume (BV), bone volume/tissue volume (BV/TV), and bone mineral density (BMD). The total soft tissue and bone tissue in the 5 mm rat cranial bone defect area are defined as tissue volume (TV). All surgical procedures in the experiments were approved by the Huazhong University of Science and Technology Animal Care and Use Committee. 2.4.3. Histological Observation. At the end of micro-CT evaluations, all samples were formalin-fixed for 7 days and subsequently decalcified in EDTA for 4 weeks. Thereafter, the samples were dehydrated through graded ethanol series and embedded in paraffin, followed by sectioning to 3 μm pieces and staining with hematoxylin and eosin (H&E, Sigma) and Masson’s Trichrome (Sigma) separately D


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Figure 4. Micro-CT analysis of the scaffolds. The (A) 3D reconstruction and (B) cross-section and coronal section images of scaffolds demonstrated that more apatite deposition was found in 0.1% GO−Col−Ap and 0.2% GO−Col−Ap groups. Quantitative analysis of the micro-CT data showed larger (C) apatite volume and (D) apatite volume/total volume ratio in 0.1% GO−Col−Ap and 0.2% GO−Col−Ap groups. (E) The calcium quantitative assay also confirmed these results. (*P < 0.05.) to examine the tissue morphology and new bone formation under the microscope (Eclipse Ni-E; Tokyo, Japan). For immunohistochemical analyses, sections were blocked by diluted ghost serum antibody and then incubated with OCN primary antibody (1:100 dilution, Abcam). Next, the sections were incubated with HRPlabeled goat antibody/mouse secondary antibody (Abcam) and subsequently colored by DAB reagent (DAKO). The sections were then stained with hematoxylin. The immunohistochemical staining samples were observed under a microscope. 2.5. Statistical Analysis. All data presented in this study are expressed as the mean ± SD. The means of two groups were compared using Student’s t test, and the means of multiple groups were compared using one-way analysis of variance (ANOVA) tests with post hoc contrasts by Student−Newman−Keuls test. P < 0.05 was considered to be a statistically significant difference.

which led to a remarkable increase of mass at each time-point in the quantitative assay (Figure 2E). Significant differences were observed (p < 0.001) at day 7 (Col−Ap, 174.2% ± 13.8%; 0.05% GO−Col−Ap, 170.8% ± 21.3%; 0.1% GO−Col−Ap, 220.0% ± 18.3%; 0.2% GO−Col−Ap, 225.8% ± 7.9%) relative to day 1 (Col−Ap, 82.9% ± 8.2%; 0.05% GO−Col−Ap, 79.3% ± 7.0%; 0.1% GO−Col−Ap, 95.3% ± 7.1%; 0.2% GO−Col−Ap, 101.8% ± 9.1%), but no significant differences were observed between day 7 and day 14. Given these findings, the 7 day period of SBF treatment was chosen for the following in vitro and in vivo experiments. 3.2. Characterization of the Scaffolds. The EDS analysis demonstrated that the crystals on the scaffolds were mainly composed of Ca element and P element, and the Ca/P ratios were calculated (Figure 3A). A Ca/P ratio (1.67 ± 0.21) was observed in the 0.1% GO−Col−Ap group, which was closest to that of the natural bone tissue (1.67). EDS mapping of calcium element (Figure 3B) and phosphate element (Figure 3C) distributions confirmed the uniform and homogeneous apatite deposition in the scaffolds. The color bar on the left side represents an increasing element content from black to pink. The color of the Col−Ap group and 0.05% GO−Col−Ap group tended to blue, while that of the 0.1% GO−Col−Ap group and 0.2% GO−Col−Ap group tended to green. These color differences indicated a greater crystal content in 0.1% GO− Col−Ap and 0.2% GO−Col−Ap groups. X-ray diffraction measurements were carried out to analyze the crystalline pattern of the scaffolds (Figure 3D). The broad diffuse peak observed at about 20° corresponded to collagen and was recorded in Col, Col−Ap, and the three groups of GO−Col−Ap scaffolds. The peaks at 26.1° and 31.8° were detected in the biomimetic

3. RESULTS 3.1. Macro- and Microimages of Scaffolds before and after Biomimetic Apatite Deposition. The colors of the collagen scaffold and three groups of GO−Col scaffolds were white and brown, respectively. The brown color of GO−Col scaffolds increased with the GO concentration from 0.05% to 0.2% w/v (Figure 2A). After 7 days of soaking in SBF, white crystals were deposited on the scaffolds, and the color of GO− Col scaffolds changed to white (Figure 2B). The SEM images revealed the surface morphologies of scaffolds and crystals at the microscale level. Porous and interconnected structures were observed in the four groups of scaffolds. The surface of Col scaffold was smooth while folded and uneven surfaces were observed in three groups of GO−Col scaffolds (Figure 2C). After biomimetic apatite deposition in SBF, homogeneous crystals were deposited on the surface of scaffolds (Figure 2D), E


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Figure 5. In vitro cellular evaluation of the scaffolds. (A) Confocal laser microscopic imaging of r-BMSCs cultured on the scaffolds with FDA staining at day 1, day 7, and day 14 (green: live cells). (D) Quantitative analysis revealed more cells in the 0.1% GO−Col−Ap group at day 1, day 7, and day 14. (B) The cells attached to the surfaces of the scaffolds at day 7 were evaluated using SEM, and clustered morphologies were observed in 0.1% GO− Col−Ap and 0.2% GO−Col−Ap groups. Higher adhesion ability was observed in 0.1% GO−Col−Ap group on the basis of (C) the cell adhesion assay, corresponding to (D) more living cell numbers at day 1. (E) All groups showed time-dependent proliferation rates, and the 0.1% GO−Col−Ap group showed the highest proliferation rates at day 7 and day 14. (Scale bar in part B, 10 μm; *p < 0.05; **p < 0.01; ***p < 0.001.)

61.2%, 62.7%, 65.2%, and 65.6%, respectively, which indicated that more apatites were deposited in 0.1% GO−Col−Ap and 0.2% GO−Col−Ap groups. A compressing test was performed to evaluate the mechanical properties of the scaffolds (Figure 3G). The results suggested that the elastic modulus of Col−Ap scaffolds (0.20 ± 0.17 MPa) was significantly lower than the elastic modulus of the scaffolds incorporated with graphene oxide (0.05% GO−Col−Ap, 0.27 ± 0.04 MPa; 0.1% GO−Col−Ap, 0.34 ± 0.04 MPa; and 0.2% GO−Col−Ap, 0.51 ± 0.06 MPa). The highest elastic modulus was observed in the 0.2% GO−Col−Ap group. 3.3. Micro-CT Analysis of the Scaffolds. The apatite deposition in the scaffolds was visualized and analyzed using the micro-CT method. Cylinder-shaped scaffolds were observed in the 3D reconstruction images (Figure 4A), and a uniform distribution of the apatite layer was observed (Videos S1−S4, Supporting Information). Cross-section and coronal section images demonstrated that many apatites attached to the surfaces

mineralized scaffolds, which corresponded to the (002) and (211) diffraction peaks of HA,22 respectively. An additional peak at 32.2° was detected in the GO−Col−Ap scaffolds. As the concentration of graphene oxide in the scaffolds increased, a raising peak intensity was observed at 2θ = 31.8°, and this change was attributed to the crystal growth at the (211) reflection of HA. FTIR spectra were performed to analyze the chemical composition of the scaffolds (Figure 3E). In the biomimetic mineralized scaffolds, the peaks at 1023, 961, 601, and 561 cm−1 corresponded to the vibration of the PO43− groups of apatite.15 The peak at 1634 cm−1 corresponded to the CO stretch of amide I, and NH bending of amide II band was observed at 1547 cm−1 while the peak around 1200−1300 cm−1 corresponded to NH bending of amide III bands.13 TGA was carried out to analyze the apatite contents in the scaffolds after biomimetic mineralization (Figure 3F). The remaining weights of Col, Col−Ap, 0.05% GO−Col−Ap, 0.1% GO−Col−Ap, and 0.2% GO−Col−Ap scaffolds were 27.9%, F


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Figure 6. In vivo evaluation of the scaffolds using the critical-sized defect. (A, C) Three-dimensional reconstruction and (B, D) coronal section analysis of the defect areas at 4 weeks and 12 weeks. (A, B) New bone was formed in the four groups at 4 weeks, and (C, D) almost complete healing of the bone defects was observed in 0.1% GO−Col−Ap and 0.2% GO−Col−Ap groups at 12 weeks. Dotted red circle: defect area. Quantitative analysis of the micro-CT data of (E) bone volume and (F) bone volume/total volume ratio showed more than 2-fold higher BV, BV/TV, and BMD in 0.1% GO− Col−Ap and 0.2% GO−Col−Ap groups than in the other two groups (**P < 0.01; ns, no significance).

days. We found that proliferation rate was higher in the 0.1% GO−Col−Ap group at 7 days and 14 days (Figure 5E). 3.5. In Vivo Evaluation of Scaffolds. 3.5.1. Micro-CT Evaluation of Ability of the Scaffolds to Repair Critical-Sized Bone Defect. Micro-CT was used to evaluate the therapeutic efficacy of the scaffolds in a large bone defect at 4 weeks and 12 weeks postimplantation. A small amount of newly formed bone was observed in 3D reconstruction images (Figure 6A) and coronal images (Figure 6B) after 4 weeks of treatment. When the treatment period was prolonged to 12 weeks, 0.1% GO− Col−Ap and 0.2% GO−Col−Ap groups exhibited more newly formed bone tissue than the other two groups both in 3D reconstruction images (Figure 6C) and in coronal images (Figure 6D). Similarly, quantitative analysis revealed that 1% GO−Col−Ap and 0.2% GO−Col−Ap groups had more than 2fold higher bone volume (Figure 6E), bone volume/total volume ratio (Figure 6F), and bone mineral density (Figure 6G) than the other two groups. 3.5.2. Histological Analysis of Bone Regeneration. H&E staining (Figure 7A−D) and Masson’s Trichrome staining (Figure 7E−H) of the scaffolds after implantation for 4 weeks revealed that the new bone tissue with bone lacunas (yellow arrow in Figure 7L,P,Q−X) and central canals (red arrow in Figure 7Q−X) were observed in the four groups, and significantly more bone tissue was observed in the 0.1% GO− Col−Ap group. After 12 weeks of implantation, dense and

of the scaffolds, and only a small number of apatites were located within the scaffolds (Figure 4B). Quantitative analysis of apatite volume (AV) (Figure 4C) and apatite volume relative to the total volume (AV/TV) (Figure 4D) confirmed that more apatite was deposited in 0.1% GO−Col−Ap and 0.2% GO−Col−Ap groups. These results were further confirmed by the calcium quantitative evaluation (Figure 4E) after 7 days of soaking in SBF. 3.4. In Vitro Cellular Evaluation of the Scaffolds. The viability and distribution of r-BMSCs cultured on the scaffolds were evaluated by confocal laser microscopy at 1 day, 7 days, and 14 days after cell seeding (Figure 5A). More cells survived in the 0.1% GO−Col−Ap group at day 1, which was in accordance with the cell adhesion capability results (Figure 5C), suggesting that higher cell adhesion capability contributed to the survival of more cells in the scaffolds at day 1. When the culture period was prolonged to 7 days, a significant higher growth rate was observed in the 0.1% GO−Col−Ap group as shown by the calculated number of living cells in the scaffolds (Figure 5D). For further evaluation of the cell morphologies in the scaffolds after 7 days of incubation, SEM was used. Fewer and single cells were attached to the surface of the scaffolds in Col−Ap and 0.05% GO−Col−Ap groups. However, clustered and more cells were observed in 0.1% GO−Col−Ap and 0.2% GO−Col−Ap groups (Figure 5B). An MTT assay was used to assess the proliferation of the cells in the scaffolds at 1 day, 7 days, and 14 G


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Figure 7. Histological analysis of the scaffolds after implantation for 4 weeks. (A−D) H&E staining, (E−H) Masson’s Trichrome staining, and highmagnification images of (red rectangle and I−P) the center region and (yellow rectangle and Q−X) the edge region in the four groups. New bone formation with (yellow arrow in L, P, Q−X) bone lacunas and (red arrow in Q−X) central canals was observed in all groups. (yellow rectangle in C and G) Much more bone tissue was formed and distributed at the edge of the defect area in the 0.1% GO−Col−Ap group. Some of the new bone tissues with (yellow arrow in L and P) bone lacunas were formed at the (red rectangle in D and H) center of the defect area in the 0.2% GO−Col−Ap group. Dotted red line: initial defect edge. (Scale bar in A−H, 500 μm; in I−X, 100 μm.)

mature bone tissue was observed at the margins of the four groups through H&E staining (Figure 8A−D) and Masson’s Trichrome staining (Figure 8E−H). The defects in 0.1% GO− Col−Ap and 0.2% GO−Col−Ap groups were almost filled with dense bone tissues, while the defect healing of the other two groups was only partial. For further evaluation of the osteogenic potential of the scaffolds, the expression of OCN was immunohistochemically determined and analyzed after 4 weeks and 12 weeks of implantation. The brown areas indicate a positive expression of OCN (Figure 9) in the implants. After 4 weeks of implantation, the Col−Ap group exhibited a lower positive area than other groups (Figure 9A−H). At the end of 12 weeks of implantation, new bone tissues were observed in 0.1% GO−Col−Ap and 0.2% GO−Col−Ap groups, and the positive brown stainings were distributed in the bone lacunas (red arrow in Figure 9O,P). The positive brown stainings confirmed that new bone tissues were

regenerated in 0.1% GO−Col−Ap and 0.2% GO−Col−Ap groups.

4. DISCUSSION Biomimetic mineralization using simulated body fluids to coat bonelike apatite onto the surfaces of scaffolds has been widely used to improve the osteoconductivity of the implanted graft substitutes.12 However, developing a homogeneous and thick enough bonelike apatite coating on the surfaces of natural polymers is still unsatisfactory.13 In this study, we developed a biomimetic GO−Col−Ap scaffold and explored both the optimal GO concentration (0.1% w/v) in the scaffold and the optimal immersion time (7 days) in SBFs to obtain the best outcomes. We successfully healed the critical-size bone defect by implanting the biomimetic GO−Col−Ap scaffolds and confirmed the osteoinduction activity of the scaffolds in vivo. In addition, we proved that conjugation of GO into Col scaffolds H


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Figure 8. Histological analysis of the scaffolds after implantation for 12 weeks. (A−D) H&E staining, (E−H) Masson’s Trichrome staining, and highmagnification images of (red rectangle and I−P) the center region and (yellow rectangle and Q−X) the edge region in the four groups. Dense and mature bone tissue with more (yellow arrow in Q−X) bone lacunas and (red arrow in Q−X) central canals was observed in the four groups at 12 weeks relative to 4 weeks. The defects in 0.1% GO−Col−Ap and 0.2% GO−Col−Ap groups were almost filled with dense bone tissue, but the defects in the other two groups were partially filled. Some of the new bone tissues with (yellow arrow in L and P) bone lacunas and (red arrow in L and P) central canals were formed at the (red rectangle in D and H) center of the defect area in the 0.1% GO−Col−Ap group. Dotted red line: initial defect edge. (Scale bar in A−H, 500 μm; in I−X, 100 μm.).

ment of an apatite coating on the substrate in SBF, a bioactive surface containing functional groups is needed to induce the apatite deposition, and the presence of sufficient functional groups could accelerate the deposition process.26 Another factor contributing to apatite deposition is the enhanced surface roughness.27 A rough surface has a large surface area which provides more contact with the clusters in SBF, thereby accelerating the apatite deposition. For the collagen scaffold, a smooth surface was observed in ESEM images (Figure 2C), and a limited number of functional groups were reported.28 These limitations reduce the efficacy of its biomimetic mineralization in SBF. In addition, in this study, the complex 3D architecture of the Col scaffold prevented the crystal from growing into the inner part of the scaffolds (Figure S2). On the basis of the crystal forming process and the limitation of the collagen scaffolds, several methods were proposed to accelerate the efficacy of the biomimetic mineralization, such as

enhanced the efficiency of the biomimetic mineralization process in SBF. Biomimetic mineralization using SBF provides biological conditions in terms of pressure, temperature, and pH, which facilitates the formation of a thick and continuous bonelike apatite layer on the surface of the substrate.3 Compared with several other coating techniques, biomimetic mineralization in SBF mimics the natural crystallization process.23 SBF offers a suitable supersaturated environment around the substrates, and hence provides the necessary clusters for the nucleation process and formation of the amorphous calcium phosphate (ACP) which is a component of the prenucleation clusters.24 The negatively charged functional groups on the substrates facilitate the binding of ACP and reorganize the clusters into orderly crystals. Furthermore, the electrostatic interactions between clusters and functional groups promote the crystal growth, morphological, and aggregation processes.25 For the developI


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Figure 9. OCN staining images of the four scaffold groups after implantation for (A−H) 4 weeks and (I−P) 12 weeks. The positive brown staining in bone lacunas was observed in 0.1% GO−Col−Ap and 0.2% GO−Col−Ap groups after 12 weeks of implantation (red arrow in O and P). (Scale bar in A−D and I−L, 100 μm; in E−H and M−P, 25 μm.)

functionalizing the substrate surfaces by soaking into acid/base solution prior to immersion in SBF, increasing ion concentration of SBF solution, and adjusting concentration of selected ions.12 However, these methods focus on preprocessing of the templates or changing composition of the SBF solution; few attempts have been tried on the scaffolds themselves. Constructing composite biomaterials could improve the mechanical and physiochemical properties of the scaffolds, and therefore may enhance the efficacy of the biomimetic mineralization. For the GO−Col scaffolds, GO exhibited a homogeneous distribution in the collagen scaffolds, resulting in even functional groups on the surface (Figures S3 and S4). The large number of carboxyl (−COOH) and hydroxyl (−OH) groups in the graphene oxide increase its capacity to interact with clusters in SBF.29 Upon the consideration of the consumption of functional groups in GO by the cross-linking reactions between the carboxyl groups of GO and the amide groups of collagen molecules, a low concentration of GO (0.05% w/v) might not provide additional functional groups for nucleation. A high concentration of GO (0.1% w/v) provided a suitable match between GO and Col groups, which improved the efficacy of the biomimetic mineral coating process (Figure 3) and formed bonelike apatite (Ca/P = 1.67 ± 0.21) with similar properties to natural bone tissue. Interestingly, through the quantitative experiments of micro-CT analysis, calcium quantitative assay, and TGA evaluation, we found that the group with the highest concentration of GO (0.2% w/v) had an equal amount of apatite compared to that with 0.1% w/v (Figure 3). Thus, the fabricated apatite-coated GO−Col scaffolds demonstrated enhanced efficacy in terms of the biomimetic mineralization in SBF after the introduction of GO in the scaffolds. Furthermore, we found no significant difference in the amount of apatites in the highGO-concentration groups (0.1% and 0.2% w/v) at 7 days and 14 days of incubation in SBF. After 7 days of incubation in SBF, the

functional groups on surfaces of the scaffolds may already have full interaction with clusters to form the mature crystals. The 0.1% GO−Col−Ap group showed high in vitro bioactivity, including cell adhesion and cell proliferation (Figure 5). This improvement of in vitro bioactivity in this group may be explained by the incorporation of GO and presence of the bonelike apatite coating. First, incorporation of GO into collagen scaffolds increased the roughness of the scaffold surfaces (Figure 2C), which provided a suitable environment for r-BMSCs to adhere.30 Second, the large surface area, π−π stacking, and hydrophilic functional groups of GO enhanced the adsorption capacity for surrounding proteins, which provided a favorable microenvironment for r-BMSC adhesion and proliferation.31 Third, the bonelike apatite coating in the 0.1% GO−Col−Ap group (Ca/P ratio: 1.67 ± 0.21) also provided a biomimetic microenvironment for r-BMSC adhesion and proliferation.32 The cytotoxicity of graphene oxide for tissue engineering is highly controversial. Many reports have demonstrated the timeand concentration-dependent cytotoxicity of graphene oxide in the form of suspensions.33 However, some researchers demonstrated that the biological response and cytotoxicity of graphene-oxide-incorporated scaffolds were markedly different from those of suspended graphene-oxide-based particles.34 Graphene oxide in the scaffolds provides sites for adsorption of biomolecules and cells, resulting in enhanced cell attachment and proliferation in vitro rather than blocking the supply of nutrients.35 Once the graphene-oxide-based scaffolds are implanted into the bone defect site, the scaffolds may be partially degraded by the peroxidases, taken up by macrophages of the mononuclear phagocytic defense system, and finally transferred to other organ systems, such as renal, lungs, liver, and spleen.36 Functionalizing the graphene oxide with biocompatible molecules and reducing the concentration (less than 100 mg/mL) and size (nanosize) of graphene oxide were reported to J



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ACKNOWLEDGMENTS This work was supported in part by the Industry of National Public Welfare Scientific Research (201502029), National Natural Science Foundation of China (81501688, 81601701, 81701922, 81873941), the Natural Science Foundation of Hubei Province (2017CFB263), and the Science Foundation of Wuhan Union Hospital (2016ZYCX034) to Z.W.

reduce the cytotoxicity of graphene-oxide-based scaffolds in vitro and in vivo.14,37−39 The mechanical properties of the bone graft substitutes have been reported to play an essential role in determining the fate of cells. Many reports have confirmed the notion that a softer matrix is better for chondrogenic differentiation, while stiffer matrices are better for osteogenic differentiation.40−42 In this study, the elastic moduli of the four scaffold groups were higher than 100 kPa, which was found to be suitable for osteogenic differentiation.43,44 The graphene-oxide-incorporated scaffolds exhibited higher elastic modulus than Col−Ap scaffolds, being highest in 0.2% GO−Col−Ap scaffolds. The enhanced mechanical properties may be due to the EDC treatment in the cross-linking procedure.43 Additionally, incorporation of GO increased the degree of cross-linking, developing a higher degree of geometric constraint on the mobility of scaffold polymer chains.16

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b17636. Schematic illustration of the preparation of GO−Col−Ap scaffolds and their applications in vivo, SEM images of the coronal section of the scaffolds, characteristics of the GO−Col solutions and scaffolds, and SEM analysis and EDS evaluation of the GO−Col scaffolds (PDF) Video S1: In Videos S1−S4, apatite deposition in the scaffolds was visualized and analyzed using the micro-CT method, and a uniform distribution of the apatite layer was observed (AVI) Video S2 (AVI) Video S3 (AVI)

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REFERENCES

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5. CONCLUSION We demonstrated that, by adding GO, the biomineralization efficiency of collagen was improved. The suitable GO concentration was found to be 0.1% w/v, and immersion time in SBF was 7 days. The 0.1% GO−Col−Ap 3D porous scaffold showed high therapeutic effects for repairing critical-sized rat cranial defects.

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Video S4 (AVI)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Zhenxing Wang: 0000-0002-2436-0372 Author Contributions †

These authors contributed equally to this work. C.Z. and S.L. performed experiments. C.Z., J.L., K.G., Q.Y., A.Z., and J.Y. analyzed the data. C.Z., S.L., and Z.W. wrote the manuscript. J.W., Z.W., and J.S. designed the study. All authors reviewed the manuscript. Notes

The authors declare no competing financial interest. K


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L
