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Article Cite This: Chem. Mater. 2018, 30, 4646−4657

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Assembly Preparation of Multilayered Biomaterials with High Mechanical Strength and Bone-Forming Bioactivity Jianmin Xue,†,‡ Chun Feng,†,‡ Lunguo Xia,§ Dong Zhai,† Bing Ma,† Xiaocheng Wang,†,‡ Bing Fang,§ Jiang Chang,† and Chengtie Wu*,†

Chem. Mater. 2018.30:4646-4657. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 10/05/18. For personal use only.

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State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, No. 1295 Dingxi Road, Shanghai 200050, People’s Republic of China ‡ University of Chinese Academy of Sciences, No. 19(A) Yuquan Road, Beijing 100049, People’s Republic of China § Center of Craniofacial Orthodontics, Department of Oral and Cranio-maxillofacial Science, School of Medicine, Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University, No. 639 Zhizaoju Road, Shanghai 200011, People’s Republic of China S Supporting Information *

ABSTRACT: The creation of a bone substitute with both excellent mechanical strength and bioactivity is still a challenge in bone tissue engineering biomaterials. To this end, inspired by the microstructure of nacre, multilayered graphene oxide/ chitosan/calcium silicate (GO/CTS/CS) biomaterials were successfully prepared via a bottom-up assembly approach. The GO/CTS/CS biomaterials emulated the “brick and mortar” layered microstructure via chemical assembly and the multilayered helical cylinder macrostructure. In addition, benefiting from the interface interactions in the layered microstructure as well as the multilayered helical cylinder macrostructure, the GO/CTS/CS biomaterials possessed high flexural strength (137.2 MPa), compressive strength (80.2 MPa), toughness (1.46 MJ/m3), and specific strength (124.7 MPa Mg−1 m−3), which are close to those of cortical bone. Furthermore, because of the bioactive chemical components of GO and CS, the multilayered GO/CTS/CS biomaterials significantly improved osteogenesis and angiogenesis in vitro. Moreover, the multilayered GO/CTS/CS biomaterials showed enhanced in vivo bone-forming ability due to the contributions of bioactive chemical components and the multilayered helical cylinder macrostructure. This work not only provides a biomaterial with excellent mechanical strength and bioactivity but also offers a strategy for fabrication of high-performance biomaterials based on bioinspired chemistry and engineering.

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INTRODUCTION Bone tissue regeneration remains a great challenge because of the complexity of tissue repair. In the past several decades, implanting a bone substitute has emerged as a common and effective strategy for repairing bone defects.1 The hierarchically ordered structure of human bone tissues endows them with excellent strength, high toughness, and the ability to deliver nutrition. However, traditional bone substitutes fail to provide both the desired mechanical strength and biological activity for bone regeneration. For instance, bioactive ceramics are brittle because of their poor fracture toughness, though they are biocompatible and biodegradable.2,3 Polymers, such as chitosan, polylactide, and polyglycolide, are too weak to fulfill the mechanical requirements of bone substitutes in the clinic despite their biocompatibility.4,5 Although metallic biomaterials, such as a titanium alloy, possess satisfactory mechanical strength, the insufficient bioactivity and degradability limit their further application in bone regeneration.6,7 Therefore, preparing a high-performance bone substitute with both excellent mechanical strength and bioactivity remains a challenge. © 2018 American Chemical Society

Intriguingly, over eons of evolution, many natural materials have developed superior mechanical properties because of their ordered structure, ranging from the nanoscale to the macroscale. Nacre, consisting of 95 vol % aragonite and 5 vol % organic (chitin and protein), has achieved an excellent combination of strength and toughness derived from its special “brick and mortar” layered structure and precise interface (mineral bridges, nanoasperities, etc.) between aragonite platelets and organic layers (Figure 1a).8−14 Although it is difficult to perfectly copy the multiscale features of nacre, many high-performance nacre−mimetic nanocomposites have been fabricated by constructing a “brick and mortar” layered structure and designing a combination of interface interactions.12,15−27 Therefore, it is reasonable to design a highperformance bone substitute by controlling its orderly layered structure at multiscales. However, most of the prepared nacre− mimetic composites were two-dimensional flexible films, which Received: March 27, 2018 Revised: June 21, 2018 Published: June 21, 2018 4646

DOI: 10.1021/acs.chemmater.8b01272 Chem. Mater. 2018, 30, 4646−4657

Article

Chemistry of Materials

Figure 1. Schematic illustration of the design and application of multilayered biomaterials. (a) Schemata of the “brick and mortar” layered microstructure of nacre. (b) Schemata of the GO/CTS/CS film inspired by the microstructure of nacre. (c) Schemata of scaffolds with the multilayered helical cylinder macrostructure. (d) Schemata of the application in tissue regeneration of multilayered scaffolds. The scaffolds possessed the “brick and mortar” layered microstructure and the multilayered helical cylinder macrostructure. (e) Different fabrication methods for three types of nacre−mimetic films: (e1) in situ and VSA method, (e2) ex situ and VSA method, and (e3) ex situ and ESA method. The prepared films were named IL-GO/CTS/CS, EL-GO/CTS/CS, and M-GO/CTS/CS, respectively.

flexural strength (137.2 MPa), compressive strength (80.2 MPa), toughness (1.46 MJ/m3), and specific strength (124.7 MPa Mg−1 m−3), nearly the same as those of cortical bone. In addition, the mechanical properties of multilayered biomaterials could be easily adjusted by regulating the fabrication method, chemical component content, and intermediate channel diameter of scaffolds, thus satisfying the different needs of human bone in mechanical strength for required applications. Furthermore, the multilayered biomaterials possessed good cytocompatibility and excellent in vitro osteogenesis and angiogenesis bioactivities. During the repair of rat femoral defects, the multilayered GO/CTS/CS biomaterials showed good in vivo bone-forming ability. Considering the high mechanical strength and bioactivity,

were difficult to use in repairing bone defects.18,20,21,27 Furthermore, besides their mechanical properties, the previous studies about nacre−mimetic composites mainly focused on their electronic conductivity or optoelectronic properties rather than their biological properties.21,24 Thus, it is of great significance to explore a nacre−mimetic strategy for preparing three-dimensional biomaterials for further application. Herein, we successfully fabricated multilayered graphene oxide/chitosan/calcium silicate (GO/CTS/CS) biomaterials by using a simple bottom-up assembly approach (Figure 1). GO and CTS were selected as the “bricks” and “mortar”, respectively, while CS was utilized to improve bioactivity. The GO/CTS/CS biomaterials possessed the “brick and mortar” layered microstructure and the multilayered helical cylinder macrostructure. The multilayered biomaterials exhibited high 4647


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Figure 2. Surface and cross-section morphology of CTS, CTS/CS, and three types of GO/CTS/CS films. (a) Overview morphology of five films. (b and c) Cross-section morphologies of five films at different magnifications, indicating that GO/CTS/CS films clearly exhibited a layered microstructure. IL-GO/CTS/CS and EL-GO/CTS/CS films exhibited a flat layered microstructure, while the layered microstructure of the MGO/CTS/CS film was tortuous. (d) EDS mapping images of five films, suggesting the presence of Ca and Si elements in GO/CTS/CS films. The scale bar is 10 μm.

solution to achieve the in situ synthesis of CS. With respect to the ex situ and VSA and ex situ and ESA methods, the prepared amorphous CS was added to the mixture. The pure GO and CTS films were prepared though vacuum filtration of the GO suspension and CTS solution, respectively. Subsequently, the two-dimensional nacre−mimetic GO/CTS/CS films were coaxially folded into three-dimensional multilayered GO/ CTS/CS scaffolds (Figure 1c). Therefore, the scaffolds possessed an orderly layered structure at multiscales, including the “brick and mortar” layered microstructure and the multilayered helical cylinder macrostructure. Characterization of Nacre−Mimetic GO/CTS/CS Films. The cross-section morphology of GO/CTS/CS films was observed to confirm the existence of the “brick and mortar” layered structure in films. Compared with CTS and CTS/CS films, three types of GO/CTS/CS films showed obviously layered microstructure with thicknesses of 70−500 nm, which were induced by self-assembly of GO in the films (Figure 2b,c).16 IL-GO/CTS/CS and EL-GO/CTS/CS films exhibited a flat layered microstructure, while the layered microstructure of the M-GO/CTS/CS film was tortuous, suggesting that vacuum filtration provided stronger force in assisting the selfassembly of GO nanosheets in the films (Figure 2b and Figure S3). However, for the cross-section morphology of the ELGO/CTS/CS film, CS particles originating from ex situ synthesis clearly existed (red arrow in Figure 2c), which partly disturbed the laminated microstructure. From scanning

the prepared multilayered biomaterial is believed to be a promising candidate for repairing bone tissue defects.

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RESULTS AND DISCUSSION Design and Preparation Strategy. GO was an ideal candidate for the “bricks” because of its outstanding mechanical properties and the abundant functional groups on its surface.16,20,28 It had been reported that GO exhibited good biocompatibility and could stimulate bone formation.29−31 CTS was widely used as a biomedical material and had many functional groups, which may lead to the interaction with GO nanosheets, thus being reasonably selected as the adhesive “mortar”.32 CS, as an “intensifying factor”, possessed distinct bone-forming ability by releasing Ca and Si ions to promote osteogenic differentiation of stem cells.33,34 The few-layer GO was previously fabricated through liquid ultrasonic exfoliation from bulk graphite oxide before incorporation into the films (Figure S1). Three different nacre−mimetic GO/CTS/CS films, i.e., IL-GO/CTS/CS, ELGO/CTS/CS, and M-GO/CTS/CS, were first prepared by three different methods, namely, in situ synthesis CS and vacuum filtration-assisted self-assembly (in situ and VSA), ex situ synthesis CS and vacuum filtration-assisted self-assembly (ex situ and VSA), and ex situ synthesis CS and evaporationinduced self-assembly (ex situ and ESA), respectively (Figure 1e). For the in situ and VSA method, Ca2+ and SiO32− were added to the mixture of the GO suspension and the CTS 4648


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Figure 3. Characterization of nacre−mimetic GO/CTS/CS films. (a) XRD pattern of different films, indicating the orderly laminated microstructure of GO/CTS/CS films. (b) FTIR spectra and (c) Raman spectra of different films, suggesting the presence of hydrogen bonding and covalent cross-linking between GO and CTS and the coordination interactions between Ca2+ and functional groups in GO/CTS/CS films. (d) Possible interactions in GO/CTS/CS films, including hydrogen bonding (blue circles), covalent cross-linking (yellow circles), and coordination interactions between Ca2+ and functional groups (brown arrows).

The broad peaks in the range of 3000−3700 cm−1 in FIIR spectra of three types of GO/CTS/CS films might be ascribed to the hydrogen bonding interaction between the functional groups of GO and CTS (blue circles in Figure 3d).22,35−38 After the GO had been mixed with CTS and CS, the intensity of the characteristic peak for the carboxyl group (CO, 1718 cm−1) in GO decreased while the intensity of the peak of the N−H deformation vibration (1575 cm−1) appeared, as a result of the covalent cross-linking between the amino group of CTS and the carboxyl group of GO (yellow circles in Figure 3d).39 Raman spectral analysis showed that the ID/IG ratios of both IL-GO/CTS/CS and EL-GO/CTS/CS films were obviously lower than that of the GO/CTS film, suggesting that Mn+ ions enhanced the interaction between GO and CTS.15 Park et al.40 reported that chemical interactions existed between the functional groups of GO and divalent metals ions (like Mg2+ and Ca2+). Xing et al.41 demonstrated that Ca2+ ions could produce coordination interactions with oxygen-containing functional groups of GO and sodium alginate. Therefore, it is reasonable to speculate that there are interactions between Ca2+ and oxygen-containing functional groups of GO and CTS in GO/CTS/CS films (brown arrows in Figure 3d). Therefore, three types of interactions might exist in GO/CTS/CS films, including hydrogen bonding and covalent cross-linking between GO and CTS and the coordination interactions between Ca2+ and oxygen-containing functional groups. Three types of GO/CTS/CS films might possess similar molecular structure, but the interactions between various components of the IL-GO/CTS/CS film were much stronger than that of the

electron microscopy (SEM) analysis, IL-GO/CTS/CS films prepared by the in situ and VSA method had a uniformly layered microstructure in films because of the in situ addition of Ca and Si ions and the vacuum filtration method. The molecular structure and interface interactions of GO/ CTS/CS films were further explored by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and Raman spectral analysis. The XRD patterns of three types of GO/CTS/CS films clearly showed a sharp diffraction peak, indicating the orderly laminated structure in films (Figure 3a).15 The 2θ value of the diffraction peak of the GO/CTS film was smaller than that of the pure GO film, which means the d spacing of GO/CTS increased after the addition of CTS, suggesting that CTS successfully entered GO nanosheets and was distributed well in the interlayers of the GO/CTS film.35 In addition, after in situ incorporation of CS, the 2θ value of the diffraction peak of the IL-GO/CTS/CS film was still smaller than that of the GO/CTS film, implying that Ca and Si ions entered the GO nanosheets. However, for the EL-GO/ CTS/CS and M-GO/CTS/CS films, the amounts of CTS and CS particles being inserted into GO nanosheets might be smaller than that of the IL-GO/CTS/CS film, thus resulting in the 2θ values of diffraction peaks being smaller than that of the pure GO film but larger than that of the IL-GO/CTS/CS film. Energy-dispersive spectroscopy (EDS) mapping and X-ray photoelectron spectroscopy (XPS) also further confirmed the presence of Ca and Si elements and CTS in three different GO/CTS/CS films (Figure 2d and Figure S4). 4649


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Figure 4. Mechanical properties of nacre−mimetic GO/CTS/CS films. (a) Overview of GO/CTS/CS films being folded into different shapes (scale bar of 1 cm). Tensile strengths of (b) three types of GO/CTS/CS films with different GO:CTS mass ratios and (c) IL-GO/CTS/CS films with different GO, CTS, and CS contents. (d) Toughness values of different films. These results demonstrated that IL-GO/CTS/CS films possessed a tensile strength and a toughness significantly higher than those of other films.

content, because CTS could fill up the interlayer gap and this could result in a strong combination between GO and CTS (Figure S5). Furthermore, the toughness of films was calculated according to the previous report.21,42 It was found that CTS played an important role in the improvement in the toughness of GO/CTS/CS films, considering CTS exhibited a toughness higher than that of GO as shown in Figure 4d. Compared with those of the pure GO and CTS films, the toughness value of the IL-GO/CTS/CS film was improved to 1.46 MJ/m3, which was lower than that of natural nacre (1.8 MJ/m3)15 but higher than that of bone (1.2 MJ/m3).15 The toughness values of EL-GO/CTS/CS and M-GO/CTS/CS films were improved to 1.06 and 0.92 MJ/m3, respectively, which were also close to that of bone. In brief, the IL-GO/ CTS/CS film made by the in situ and VSA method exhibited a high tensile strength and toughness because of the uniform layered microstructure and strong intermolecular interactions in films. Considering the optimized tensile strength and toughness, films with theoretical components of 20 wt % CS, 26.7 wt % GO, and 53.3 wt % CTS (0.5:1 GO:CTS by weight) were further selected for the preparation of multilayered scaffolds. Subsequently, the two-dimensional films were further transformed to three-dimensional (3D) scaffolds with multilayered helical macrostructure to repair bone defects (Figure 5a). The macrostructure endowed the multilayered scaffolds with excellent compressive strength and flexural strength. The compressive strength of the IL-GO/CTS/CS scaffold reached 80.2 MPa, which was 8.8 and 2.4 times higher than those of the GO (9.1 MPa) and CTS (33.2 MPa) scaffolds, respectively. In

latter two materials, indicating in situ synthesis of the GO/ CTS/CS film displayed distinct advantages for both uniform microstructure and possible mechanical strength. Investigation of Mechanical Properties. The mechanical properties of GO/CTS/CS films were evaluated by a series of mechanical tests. As shown in Figure 4a, GO/CTS/CS films were very flexible in being transformed into desired shapes, which was convenient for later fabricating three-dimensional multilayered scaffolds. Compared with GO/CTS/CS films prepared by ex situ and VSA and ex situ and ESA methods, the IL-GO/CTS/CS film made by the in situ and VSA method possessed a higher tensile strength with the same chemical components, which could be contributed to the uniform layered microstructure and strong intermolecular interactions in films. Then, IL-GO/CTS/CS films with different chemical components were prepared by the in situ and VSA method to investigate the effect of chemical components on tensile strength. After CTS and GO had been mixed, the tensile strength of binary GO/CTS films (32.0−40.3 MPa) was much higher than that of pure CTS films (13.3 MPa) and pure GO films (20.9 MPa), which verified our speculation about the existence of hydrogen bonds and covalent cross-linking between GO and CTS. Compared with that of pure GO, the addition of CS obviously improved the tensile strength of GO/ CS films, indicating the possible chemical interactions between the functional groups of GO and Ca ions. The highest tensile strength of IL-GO/CTS/CS films was measured as 64.3 MPa, which was 4.8 and 3.1 times higher than that of the pure CTS and pure GO films, respectively. In addition, it was observed that the tensile strength increased with an increase in CTS 4650


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Figure 5. Mechanical properties of multilayered GO/CTS/CS scaffolds. (a) Overview of multilayered scaffolds. (b) Compressive strength and (c) flexural strength of multilayered scaffolds. The results suggested that IL-GO/CTS/CS scaffolds exhibited excellent compressive strength and flexural strength. (*p < 0.05; **p < 0.01; ***p < 0.001).

the mechanical strength of our GO/CTS/CS biomaterials. The different interactions among GO, CTS, and CS in the layered microstructure efficiently reinforced the tensile strength and toughness of our biomaterials, while the multilayered helical cylinder structure at the macroscale endowed the biomaterials with superior flexural strength and compressive strength. Taken together, the multilayered GO/CTS/CS biomaterials possessed excellent mechanical properties and could satisfy different demands for bone regeneration, especially in loadbearing application. In Vitro Biological Performance. To evaluate in vitro osteogenesis and angiogenesis bioactivities, cell proliferation, cell attachment, and gene expression assays were investigated. The rabbit bone marrow stromal cells (rBMSCs) were used to explore the in vitro osteogenesis of GO/CTS/CS films. The level of proliferation of rBMSCs in all groups increased with culture time, while the cellular amount of the M-GO/CTS/CS group was clearly smaller than that of IL-GO/CTS/CS and EL-GO/CTS/CS groups (Figure 6a). For cell attachment and morphology, the rBMSCs spread well and closely adhered to the surface of all GO/CTS/CS films with numerous pseudopodia after 7 days (Figure 6c). It is reported that vascularization plays a vital role in new bone formation.45 Therefore, human umbilical vein endothelial cells (HUVECs) were further applied to investigate in vitro angiogenesis. The number of HUVECs cultured on three types of GO/CTS/CS films significantly increased after 7 days (Figure 6b). The spread morphology of cells suggested that all GO/CTS/CS films supported the adhesion of HUVECs (Figure 6d). Therefore, both IL-GO/CTS/CS and EL-GO/CTS/CS films

addition, the compressive strength of the IL-GO/CTS/CS scaffold was close to the bottom level of human cortical bone (100−230 MPa), showing promising potential for application in load-bearing bone regeneration.2,43 For the EL-GO/CTS/ CS scaffold, its compressive strength reached 69.2 MPa, which was also much higher than that of traditional 3D printing bioceramic scaffolds (6−46 MPa) used in bone regeneration.44−46 Although the compressive strength of the M-GO/ CTS/CS scaffold (55.9 MPa) was not as good as those of the two former groups, it was still much higher than that of human cancellous bone (2−12 MPa) and thus could fully satisfy the requirement for non-load-bearing bone regeneration.43,47 The flexural strengths of IL-GO/CTS/CS and EL-GO/CTS/CS scaffolds were measured to be 137.2 and 112.1 MPa, respectively, which met the requirement for that of human cortical bone (50−150 MPa).43 Furthermore, the flexural strength of multilayered scaffolds could be adjusted by changing the intermediate channel diameter (Di) of scaffolds (Figure S7c,d). The high specific strength is a typical characteristic of lightweight and high-strength materials. Therefore, the specific flexural strength of multilayered scaffolds was further compared with that of other related materials (Table S1).17,22,23,43,48 The specific strengths of ILGO/CTS/CS (124.7 MPa Mg−1 m−3) and EL-GO/CTS/CS (93.4 MPa Mg−1 m−3) scaffolds were similar to that of cortical bone (42−125 MPa Mg−1 m−3)43,48 and significantly higher than those of CaCO3/CTS artificial nacre (26−33 MPa Mg−1 m−3)23 and HA/poly(methyl methacrylate) (PMMA) artificial nacre (27−36 MPa Mg−1 m−3).17 The measurements of mechanical properties showed that the orderly layered structure at multiscales significantly increased 4651


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Figure 6. In vitro cell proliferation and adhesion. Proliferation of (a) rBMSCs and (b) HUVECs cultured on different films. Morphology of (c) rBMSCs and (d) HUVECSs after seeding on different films for 7 days. IL-GO/CTS/CS and EL-GO/CTS/CS films improved the attachment and proliferation of rBMSCs and HUVECs, showing good cytocompatibility.

can improve in vitro osteogenic and angiogenic differentiation of tissue cells.33,34,53,54 Unlike the GO/CTS film, three types of GO/CTS/CS films could continuously release Ca and Si ions (Figure S8). Therefore, besides the function of GO, it is reasonable to speculate that the released Ca and Si ions from GO/CTS/CS films also contribute to the improved in vitro osteogenesis and angiogenesis bioactivities. In Vivo Osteogenic Activity. Encouraged by excellent in vitro bioactivity, we further sought to confirm the in vivo boneforming bioactivity of multilayered GO/CTS/CS biomaterials. The rat femoral defect model was applied to investigate the in vivo bone regeneration of scaffolds. The morphology and structure of samples were analyzed by micro-CT after implantation for 8 weeks. Previous studies reported that CTS could efficiently accelerate the bone regeneration process in rat bone defection.32,55 In this work, both multilayered GO/CTS/ CS scaffolds and the CTS scaffold could induce new bone formation in the femoral defects (Figure 8a,b), suggesting that the GO/CTS/CS scaffolds exhibited good in vivo boneforming ability. In addition, histological analysis performed by Van Gieson’s picrofuchsin stain showed that the newly formed bone had successfully grown into the interlamination gap of scaffolds (green arrows in Figure 8c). These results demonstrated that the multilayered helical cylinder structure

strongly supported the attachment and proliferation of both rBMSCs and HUVECs, showing good cytocompatibility. Subsequently, the expression of important markers (OCN, BSP, RunX2, and BMP2) for osteoblast differentiation was determined (Figure 7a−d). It is reported that GO could influence osteogenic differentiation of stem cells and enhance osteogenic gene expression in vitro.49,50 Compared with the pure CTS film, the GO/CTS film could improve the expression of some osteogenic genes because of the function of GO, which was consistent with previous reports.29,30,51,52 Additionally, three groups of GO/CTS/CS films showed osteogenic gene expression levels significantly higher than those of blank and pure CTS groups after 3 and 7 days, demonstrating the substantially improved in vitro osteogenic activity of GO/CTS/CS films. Similarly, the angiogenic gene expression assays showed that the GO/CTS film and three types of GO/CTS/CS films also significantly improved the expression of angiogenic genes (HIF-1α, KDR, VEGF, and eNOS) of HUVECs after 3 and 7 days, suggesting their good in vitro angiogenesis bioactivity (Figure 7e−h). It was also found that three groups of GO/CTS/CS films possessed levels of expression of some osteogenic and angiogenic genes (OCN, BSP, HIF-1α, and KDR) that were higher than those of the GO/CTS film. Previous studies reported that Ca and Si ions 4652


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Figure 7. In vitro osteogenic gene and angiogenic gene expression. (a−d) In vitro osteogenic differentiation of rBMSCs on different films: osteogenic gene expression of (a) OCN, (b) BSP, (c) RunX2, and (d) BMP2. (e−h) In vitro angiogenic differentiation of HUVECs on different films: angiogenic gene expression of (e) HIF-1α, (f) KDR, (g) VEGF, and (h) eNOS. GO/CTS/CS films significantly improved the expression of osteogenic genes and angiogenic genes compared to the blank, CTS, and GO/CTS groups, indicating excellent in vitro osteogenesis and angiogenesis (*p < 0.05; **p < 0.01; ***p < 0.001).

promotion of the in vivo bioactivity of multilayered GO/ CTS/CS scaffolds might originate from the functions of GO and CS, indicating that these bioactive components not only improved in vitro bioactivity but also induced bone formation in vivo. Therefore, the multilayered helical cylinder macrostructure could successfully induce the growth of new bone tissues in the inner section of the biomaterials and bioactive

at the macroscale of scaffolds could induce the ingrowth of new bone tissues in the inner section of multilayered materials. Furthermore, the three multilayered GO/CTS/CS scaffolds firmly integrated with the growing peripheral bone, while there was an obvious gap between CTS scaffolds and peripheral bone (Figure 8d), suggesting the improved in vivo boneforming bioactivity of the GO/CTS/CS scaffolds. The 4653


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Figure 8. In vivo bone formation of multilayered GO/CTS/CS scaffolds after their implantation in rat femoral defects. (a) Transverse and (b) cross-section views of femoral defects reconstructed by micro-CT [OB, old bone tissues; NB, new bone tissues (red zones); materials, green zones]. (c and d) Histological images achieved using Van Gieson’s picrofuchsin stain of different scaffolds 8 weeks after implantation (red zones, new bone tissues). Newly formed bone had successfully grown into the interlamination gap of the multilayered scaffolds, and the growing peripheral bone was firmly integrated with multilayered scaffolds, suggesting multilayered scaffolds had good in vivo bone-forming ability.

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components stimulated the formation of new bone, which led to good the in vivo bone-forming ability of the multilayered GO/CTS/CS scaffolds.

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MATERIALS AND METHODS

Materials. Chitosan (CTS), calcium nitrate tetrahydrate [Ca(NO3)2·4H2O], sodium metasilicate nonahydrate (Na2SiO3·9H2O), acetic acid, and NH3·H2O were purchased from Sinopharm Chemical Reagent Co., Ltd. Graphite oxide powders were obtained from Shanghai Haoye Electronic Technology Co., Ltd. Amorphous calcium silicate (CS) particles were synthesized by chemical precipitation of Ca(NO3)2·4H2O and Na2SiO3·9H2O without sintering. Fabrication of Nacre−Mimetic GO/CTS/CS Films. Graphite oxide was dispersed into deionized water and ultrasonicated for 4 h to obtain a 1 wt % graphene oxide (GO) suspension. A 5 wt % CTS solution was prepared by dissolving CTS into a 3 wt % acetic acid solution. Three types of nacre−mimetic GO/CTS/CS films, i.e., ILGO/CTS/CS, EL-GO/CTS/CS, and M-GO/CTS/CS, were prepared by three different methods, namely, (1) in situ synthesis CS and vacuum filtration-assisted self-assembly (in situ and VSA), (2) ex situ synthesis CS and vacuum filtration-assisted self-assembly (ex situ and VSA), and (3) ex situ synthesis CS and evaporation-induced selfassembly (ex situ and ESA). For the in situ and VSA method, Ca(NO3)2·4H2O and Na2SiO3·9H2O were added to the mixture of the GO suspension and CTS solution to achieve the in situ synthesis of CS. After being stirred 4 h, the mixture was filtered with a vacuum filter to obtain composite films. With respect to the ex situ and VSA method, the prepared amorphous CS was added to the mixture and stirred for 4 h, and then composite films were prepared via vacuum filtration. For the ex situ and ESA method, the prepared amorphous CS was added to the mixture and stirred for 4 h, and then composite films were prepared via evaporation. The pure GO and CTS films were prepared via vacuum filtration of the GO suspension and CTS solution, respectively. Fabrication of Multilayered GO/CTS/CS Scaffolds. The threedimensional multilayered GO/CTS/CS scaffolds were fabricated by curling up the two-dimensional GO/CTS/CS films into spirals, and these shapes were maintained with molds. The curled films were placed in a draft cupboard for ∼24 h to hold their shape. Finally, the molds were removed, and the scaffolds were prepared. Therefore, the scaffolds possessed an orderly layered structure at multiscales, including the “brick and mortar” microstructure and the multilayered helical cylinder macrostructure.

CONCLUSION

In summary, inspired by the orderly layered structure of nacre, we successfully fabricated multilayered GO/CTS/CS biomaterials by combining the GO self-assembly method and the vacuum filtration-assisted assembly strategy. The multilayered biomaterials possessed the “brick and mortar” layered microstructure and the multilayered helical cylinder macrostructure. Among them, the IL-GO/CTS/CS scaffold had excellent mechanical properties, which matched the strength of cortical bone. The mechanical properties of multilayered biomaterials could be effectively controlled by regulating the fabrication method, chemical component content, and intermediate channel diameter of scaffolds, thus satisfying the requirements of different bone defects with different strengths. Furthermore, the multilayered biomaterials could significantly promote the in vitro osteogenesis of rBMSCs and the angiogenesis of HUVECs. Moreover, the multilayered biomaterials also possessed good in vivo bone-forming ability. Our studies suggested that the interface interactions in the layered microstructure as well as the multilayered helical cylinder macrostructure of the scaffolds may play a key role in their excellent mechanical strength, and the bioactive chemical components of GO and CS together with the macrostructure were greatly important for improving the bioactivity of the scaffolds. Such a multilayered biomaterial is believed to be a promising candidate for load-bearing bone regeneration. This work may also provide a strategy for the design and fabrication of high-performance tissue engineering biomaterials based on bioinspired engineering. 4654


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Chemistry of Materials Characterization of Materials. The microstructure of different films was characterized by SEM (SU8220, Hitachi). The elemental distribution was observed by SEM with EDS. The XRD patterns of different films were characterized by XRD (Rigaku D/Max-2550 V, Geigerflex). FIIR spectra of different films were recorded with a Fourier transform infrared spectrometer (Nicolet 380, Thermo Scientific). Raman spectra of different films were recorded with a laser micro-Raman spectrometer (Thermo Nicolet). XPS (ESCAlab250, Thermo Fisher Scientific) was used to aid in the elemental analysis of materials. The tensile strength and compressive strength of materials were determined by using a computer-controlled universal testing machine (AG-I, Shimadzu). The static mechanical test machine (INSTRON 5566) was used to test the flexural strength of scaffolds. The toughness of different films was calculated according to eq 1:21,42

U=

∫0

σf

σ dε

for 30 min before SEM analysis. The morphology of HUVECs was observed by SEM (SU8220, Hitachi). The proliferation of HUVECs was evaluated by the MTT assay. HUVECs were cultured on different films in ECM for 1, 3, and 7 days (37 °C, 5% CO2) and added with 300 μL of the MTT solution (0.5 mg/mL) at each time point. After the culture medium had been removed, the formazan product was solubilized in DMSO. Afterward, a 100 μL aliquot was taken from each well and transferred to a fresh plate. The absorbance value at 590 nm was measured with a microplate spectrophotometer (EpochTM, Bio-Tek Instruments). The HUVECs were seeded on the films and cultured in ECM (Sciencell) at 37 °C and 5% CO2. After 3 and 7 days, the total RNA was harvested by using Trizol reagent (Invitrogen). The mRNA expression of angiogenesis-related genes (VEGF, KDR, eNOS, and HIF-1α) of HUVECs was measured by real-time quantitative RTPCR (RT-qPCR) on StepOnePlus Realtime PCR systems (Applied Biosystems). The relative expression level of each gene was calculated by using the 2−ΔΔCt method. In Vivo Osteogenesis of Multilayered GO/CTS/CS Scaffolds. All animal procedures were approved by the Animal Research Committee of the Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University. To investigate the osteogenesis of multilayered GO/CTS/CS scaffolds, 18 white rats were selected as femoral defect models, and cylindrical defects (Φ 3.5 mm × 4 mm) were made in their femoral condyle of hind limbs by surgery. Eight weeks after implantation, all of the rats were sacrificed and the samples were removed. The morphology and structure of samples were analyzed by micro-CT (SKYSCAN1172, SKYSCAN). After that, the samples were further dehydrated and embedded in PMMA. Three longitudinal sections for each sample were prepared, and the histological images were acquired with Van Gieson’s picrofuchsin stain to observe the newly formed bone tissues. Statistical Analysis. All the data were expressed as means ± the standard deviation and analyzed using one-way analysis of variance with a post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001.

(1)

where U is the energy per volume absorbed, σ is the stress, ε is the strain, and σf is the failure strain. In Vitro Osteogenesis of Nacre−Mimetic GO/CTS/CS Films. The rabbit bone marrow stromal cells (rBMSCs) were used to explore the in vitro osteogenesis of GO/CTS/CS films. The BMSCs were isolated from the femurs of rabbits (1 month old). The cells were cultured in α-MEM (HyClone) containing 10% fetal calf serum (Invitrogen), 1% (v/v) penicillin, and 1% (v/v) streptomycin (Invitrogen) at 37 °C and 5% CO2. The films were cut into small rounds (Φ = 10 mm) and sterilized with ultraviolet radiation. The rBMSCs were seeded on CTS, GO/CTS, IL-GO/CTS/CS, EL-GO/ CTS/CS, and M-GO/CTS/CS films in 48-well culture plates (1 × 104 cells/film). To observe the adhesion and morphology of rBMSCs, the cellseeded films were rinsed with a phosphate-buffered saline (PBS) solution and anchored with 2.5% glutaraldehyde for 20 min. After that, cellular samples were sequentially dehydrated in graded ethanol [30, 40, 50, 60, 70, 80, 90, 95, and 100% (v/v)] and dried in hexamethyldisilazane (HMDS) for 30 min before SEM analysis. The morphology of BMSCs was observed by SEM (SU8220, Hitachi). The proliferation of rBMSCs on films was evaluated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The rBMSCs were cultured on different films for 1, 3, and 7 days and added with 300 μL of the MTT solution (0.5 mg/mL) at each time point. After that, the culture medium was removed and the formazan product was solubilized in dimethyl sulfoxide (DMSO). Afterward, a 100 μL aliquot was taken from each well and transferred to a fresh plate. The absorbance value at 590 nm was measured with a microplate spectrophotometer (EpochTM, Bio-Tek Instruments). The rBMSCs were seeded on films and incubated in differentiation medium (DMEM supplemented with 10 mM β-glycerol phosphate, 0.2 mM ascorbic acid, and 10% FBS) in 48-well culture plates (37 °C, 5% CO2). After 3 and 7 days, the total RNA was isolated using Trizol reagent (Invitrogen). The mRNA expression of osteogenesis-related genes (RUNX2, BMP2, OCN, and BSP) was measured by real-time quantitative RT-PCR (RT-qPCR) on StepOnePlus Realtime PCR systems (Applied Biosystems). The relative expression level of each gene was calculated by using the 2−ΔΔCt method.46 In Vitro Angiogenesis of Nacre−Mimetic GO/CTS/CS Films. Human umbilical vein endothelial cells (HUVECs) were applied to investigate in vitro angiogenesis in this work. HUVECs were cultured in endothelial cell medium (ECM, Sciencell, Carlsbad, CA) at 37 °C and 5% CO2. As described above, the GO/CTS/CS films were also cut into small rounds with a diameter of 10 mm and sterilized with ultraviolet radiation. After that, the HUVECs were cultured on different films in 48-well culture plates (1 × 104 cells/film). To observe the adhesion and morphology of HUVECs, the cellseeded films were rinsed with a PBS solution and anchored with 2.5% glutaraldehyde for 20 min. After that, cellular samples were sequentially dehydrated in graded ethanol [30, 40, 50, 60, 70, 80, 90, 95, and 100% (v/v)] and dried in hexamethyldisilazane (HMDS)

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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/acs.chemmater.8b01272. Transmission electron microscopy images of graphene oxide after ultrasonication (Figure S1), surface morphologies of different films (Figure S2), additional SEM images of the cross-section morphologies of different films (Figure S3), XPS data of three types of GO/CTS/ CS films (Figure S4), cross-section morphology of the IL-GO/CTS/CS film with different GO:CTS mass ratios (Figure S5), toughness of three types of GO/ CTS/CS films (Figure S6), additional mechanical properties of multilayered scaffolds (Figure S7), water contact angles and ion release profiles of five different films (Figure S8), cell morphologies after seeding on different films (Figure S9), thermogravimetric analysis of different films (Figure S10), and a comparison of the mechanical performance of multilayered GO/CTS/CS scaffolds with natural materials and other bulk artificial nacre (Table S1) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Jiang Chang: 0000-0003-1462-6541 Chengtie Wu: 0000-0002-5986-591X 4655


Article

Chemistry of Materials Author Contributions

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J.X. designed, planned, and performed the experiments, analyzed data, and drafted the manuscript. C.F. and X.W. helped in the fabrication of materials and discussed the data. L.X. and B.F. performed animal experiments. D.Z. performed in vitro biological experiments. B.M. helped in animal experiments. J.C. provided reagents and revised the paper. C.W. initiated, designed, and supervised the study and revised the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Key Research and Development Program of China (2016YFB0700803), the National Natural Science Foundation of China (51761135103, 31370963, and 81430012), the Key Research Program of Frontier Sciences, CAS (QYZDB-SSW-SYS027), and the Science and Technology Commission of Shanghai Municipali ty (1 74 419 03 700 , 1 6DZ2 260 60 3, and 15XD1503900).

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