Hydroxyapatite Nanowire@Magnesium Silicate CoreâShell

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Hydroxyapatite Nanowire@Magnesium Silicate Core−Shell Hierarchical Nanocomposite: Synthesis and Application in Bone Regeneration Tuan-Wei Sun,†,‡,⊥ Wei-Lin Yu,§,⊥ Ying-Jie Zhu,*,†,‡ Ri-Long Yang,†,‡ Yue-Qin Shen,†,‡ Dao-Yun Chen,§ Yao-Hua He,*,§,∥ and Feng Chen*,†,‡ †

State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China § Department of Orthopedics and ∥School of Biomedical Engineering, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, People’s Republic of China S Supporting Information *

ABSTRACT: Multifunctional biomaterials that simultaneously combine high biocompatibility, biodegradability, and bioactivity are promising for applications in various biomedical fields such as bone defect repair and drug delivery. Herein, the synthesis of hydroxyapatite nanowire@magnesium silicate nanosheets (HANW@MS) core−shell porous hierarchical nanocomposites (nanobrushes) is reported. The morphology of the magnesium silicate (MS) shell can be controlled by simply varying the solvothermal temperature and the amount of Mg2+ ions. Compared with hydroxyapatite nanowires (HANWs), the HANW@MS core−shell porous hierarchical nanobrushes exhibit remarkably increased specific surface area and pore volume, endowing the HANW@MS core−shell porous hierarchical nanobrushes with high-performance drug loading and sustained release. Moreover, the porous scaffold of HANW@MS/chitosan (HANW@MS/CS) is prepared by incorporating the HANW@MS core−shell porous hierarchical nanobrushes into the chitosan (CS) matrix. The HANW@MS/CS porous scaffold not only promotes the attachment and growth of rat bone marrow derived mesenchymal stem cells (rBMSCs), but also induces the expression of osteogenic differentiation related genes and the vascular endothelial growth factor (VEGF) gene of rBMSCs. Furthermore, the HANW@MS/CS porous scaffold can obviously stimulate in vivo bone regeneration, owing to its high bioactive performance on the osteogenic differentiation of rBMSCs and in vivo angiogenesis. Since Ca, Mg, Si, and P elements are essential in human bone tissue, HANW@MS core−shell porous hierarchical nanobrushes with multifunctional properties are expected to be promising for various biomedical applications such as bone defect repair and drug delivery. KEYWORDS: hydroxyapatite, nanowires, magnesium silicate, porous scaffolds, drug delivery, bone regeneration

1. INTRODUCTION

scaffolds were reported for successful bone integration and reconstruction in vivo.7,8 To achieve successful bone defect repair, the constituents and structures of the scaffolds need to be synergistically optimized.8 Natural bone is an inorganic−organic biocomposite that is composed of ∼70 wt % inorganic crystals (mainly hydroxyapatite (HAP, Ca10(PO4)6(OH)2)) and ∼30 wt % organic matrix (type I collagen). The structure of natural bone is hierarchically organized by HAP nanocrystals and collagen matrix at the macro-, micro-, and nanoscales.9,10 Inspired by the constituents and structure of natural bone, porous scaffolds based on

The reconstruction of large-sized bone defects caused by trauma, infection, and tumor resection is still a great challenge for orthopedic surgeons.1−3 Nowadays, autologous bone grafting and allogeneic bone grafting remain the mainstay of treatment for large-sized bone defects. However, these strategies are associated with high morbidity for the donor sites, limited supply for autografts, and serious risks of infection and immunological reaction.1,4 These drawbacks prevent their clinical applications and stimulate advanced techniques to overcome these limitations. Recently, bone tissue engineering has attracted considerable attention as a promising strategy for promoting bone regeneration.5,6 Various types of scaffolds were synthesized and investigated as alternatives to fill the missing bone segments and induce bone regeneration; however, only a small number of © XXXX American Chemical Society

Received: March 11, 2017 Accepted: April 28, 2017

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DOI: 10.1021/acsami.7b03532 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces inorganic−organic composites, such as HAP/collagen,11,12 HAP/chitosan,13−15 and HAP/collagen/chitosan,16,17 have been investigated for application in bone regeneration.18 Chitosan is a natural polymer which is similar to the polysaccharide in the extracelluar matrix, and has good biocompatibility, biodegradability, and low immunogenicity.19,20 Compared with collagen, chitosan has a rigid crystalline structure with higher stiffness which contributes to the osteogenic differentiation of the mesenchymal stem cells via the mechanical signaling pathways.21−23 Hydroxyapatite, as an important inorganic component of natural bone, has outstanding biocompatibility, good cell adhesiveness, and high osteoconductivity.24−26 Recent studies showed that HAP whiskers-based scaffolds exhibited enhanced bioactivity and biological responses due to the hierarchical structure and improved mechanical properties as compared with HAP particles-based scaffolds.12,27,28 Our previous study showed that HAP nanowires (HANWs) were highly flexible and could interweave with each other like fabric to construct hierarchical biomaterials with excellent mechanical properties and good biological properties.29−32 Thus, it is of great significance to develop hierarchical and porous scaffolds for bone tissue engineering using HANWs and chitosan as the building materials. The significant potential of Mg and Si elements has been demonstrated for promoting osteogenesis and angiogenesis.33,34 Zhai et al.35 reported that Mg/Si-containing bioceramics such as akermanite, bredigite, and diopside had a higher potential for osteogenesis and angiogenesis as compared with β-tricalcium phosphate (β-TCP) bioceramics. In addition, the akermanite and bredigite bioceramics exhibited more obvious stimulatory effects on osteogenesis and angiogenesis than diopside bioceramics owing to their larger released amounts of Si element. Chen et al.36 demonstrated that Ca−Si−Mg cement could stimulate cementogenesis and angiogenesis of human periodontal ligament cells. Wu et al.37 reported that MgSiO3 could improve osseointegration based on an immunomodulation induced by released Mg and Si ions, and promote de novo bone formation. It is thus significant to design multifunctional porous scaffolds with the hierarchical structure incorporating bioactive elements of Mg and Si to fulfill osteogenesis and angiogenesis. Our previous studies demonstrated that magnesium silicate nanostructured materials exhibited excellent biocompatibility, good biodegradability, high specific surface area, sustained release of Mg and Si ions, and high performance in drug loading and sustained delivery.38,39 Along with the high biocompatibility, good biodegradability, and bioactivity, when the scaffolds were endowed with the function of high-efficiency loading and sustainable release of various therapeutic molecules, their ability to control cellular responses and stimulate tissue reaction could be significantly increased.40,41 Hence the porous scaffolds containing magnesium silicate nanostructured materials exhibit a great potential to achieve high-performance bone regeneration. Herein, we report the synthesis of the HANW@MS core− shell porous hierarchical nanobrushes composed of hydroxyapatite nanowires as the core and magnesium silicate nanosheets (MS) as the shell. The performance of the HANW@MS core−shell porous hierarchical nanobrushes as the carrier for loading and releasing therapeutic drugs has been investigated. In addition, the hierarchical porous scaffold of HANW@MS/ chitosan (HANW@MS/CS) exhibits good bioactivity in inducing osteogenic differentiation of rat bone marrow derived mesenchymal stem cells (rBMSCs). Furthermore, the HANW@

MS/CS porous scaffold can obviously stimulate in vivo bone regeneration by promoting osteogenesis and angiogenesis.

2. EXPERIMENTAL SECTION The information on the reagents used in the experiments and characterization of the as-prepared materials are described in the Supporting Information. 2.1. Preparation and Characterization of Hierarchical Porous Core−Shell-Structured Nanobrushes Composed of Hydroxyapatite Nanowires and Magnesium Silicate Nanosheets (HANW@MS). 2.1.1. Synthesis of Hydroxyapatite Nanowires (HANWs). The HANWs were synthesized according to the calcium oleate precursor solvothermal method reported previously by this research group.30 First, 200 mL of CaCl2 aqueous solution (0.15 M) and 200 mL of NaOH aqueous solution (1.25 M) were prepared and added separately into a mixture of ethanol (120 g) and oleic acid (120 g) under mechanical agitation. Then, 100 mL of NaH2PO4·2H2O aqueous solution (0.27 M) was added dropwise into the above mixture under continuing mechanical agitation. Finally, the resulting mixture was transferred into a Teflon-lined stainless steel autoclave with a volume of 1 L, sealed, and solvothermally treated at 180 °C for 36 h. The white product was collected by centrifugation and washed with ethanol and deionized water several times, and dispersed in ethanol for further use. 2.1.2. Synthesis of the HANW@SiO2. The classical Stöber method was adopted for the preparation of [email protected] In brief, 10 mL of deionized water and 1.2 mL of aqueous ammonia (28%) were separately added into 40 mL of an ethanol suspension containing HANWs with a concentration of 2 mg mL−1 under magnetic stirring, and the resulting suspension was treated by ultrasound for 1 h. A 0.4 mL volume of tetraethyl orthosilicate (TEOS) was added into the above suspension at room temperature under stirring. After 6 h of stirring, the product was collected by centrifugation, and washed three times using ethanol and deionized water, respectively, and dispersed in ethanol for further use. 2.1.3. Synthesis of Hierarchical Porous Nanobrushes of HANW@ MS. In a typical experiment, 10 mL of sodium acetate aqueous solution (1 M) and 10 mL of MgCl2·6H2O aqueous solution (0.04 M) were separately added into 10 mL of ethanol suspension containing HANW@SiO2 (5 mg mL−1) under magnetic stirring. Then, the resulting mixture was poured into a 50 mL Teflon-lined stainless steel autoclave, sealed, and solvothermally treated at 180 °C for 24 h.38 After the autoclave was cooled down, the product was obtained by centrifugation and washed with ethanol and deionized water three times, respectively, and dried at 60 °C overnight. Other samples were synthesized by similar procedures but varying experimental conditions such as solvothermal temperature or addition amount of MgCl2·6H2O in the reaction system. 2.2. In Vitro Drug Loading and Release. 2.2.1. In Vitro Vancomycin Loading. The as-prepared HANW@MS core−shell porous hierarchical nanobrushes were used as the drug carrier for vancomycin (VAN) loading. The powder of the HANW@MS core− shell porous hierarchical nanobrushes or HANWs (5 mg each) was dispersed in 2 mL of VAN aqueous solution (0.4 mg mL−1), which was treated with ultrasound for 15 min and constant shaking (120 rpm) at 37 °C for 24 h. Then, the suspension was centrifuged, and the concentration of VAN in the supernatant was determined by UV−vis absorption spectroscopy (UV-2300, Techcomp) at a wavelength of 280 nm. Hemoglobin (Hb) and a typical anticancer drug, doxorubicin hydrochloride (DOX), were used as the model protein and drug, respectively. For the typical loading procedure of Hb, the powder of the as-prepared HANW@MS core−shell porous hierarchical nanobrushes or HANWs (5 mg each) was dispersed in 2 mL of Hb aqueous solution with different concentrations (0.2−3 mg mL−1). Then, the suspensions were treated by ultrasound for 15 min and shaken constantly (120 rpm) at 37 °C for 24 h. The loading procedure of DOX was similar to that of Hb but using DOX aqueous solution instead of Hb aqueous solution. After the loading, the suspensions were centrifuged, and the concentrations of Hb or DOX in the supernatants were measured by the UV−vis absorption at 405 or 480 nm, respectively. B


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ACS Applied Materials & Interfaces 2.2.2. In Vitro DOX Drug Release. The DOX drug release behaviors of the as-prepared HANW@MS core−shell porous hierarchical nanobrushes or HANWs were investigated. For the preparation of the DOX-loaded HANW@MS core−shell porous hierarchical nanobrushes or DOX-loaded HANWs, the powder of HANW@MS core−shell porous hierarchical nanobrushes or HANWs (100 mg each) was added into the DOX aqueous solution (15 mL, 2 mg mL−1), followed by constant shaking (120 rpm) for 24 h at 37 °C, and the DOX-loaded HANW@MS core−shell porous hierarchical nanobrushes or DOXloaded HANWs were obtained by centrifugation and freeze-drying at −20 °C for 24 h. The DOX loading amount in HANW@MS core−shell porous hierarchical nanobrushes or HANWs was determined by measuring the DOX concentration in the supernatant with UV−vis absorption at a wavelength of 480 nm. For the drug release tests, the powder of DOX-loaded HANW@MS core−shell porous hierarchical nanobrushes or HANWs (8 mg) was immersed in 8 mL of phosphate buffered saline (PBS, pH 7.4). Then, the suspension was sealed and constantly shaken at 37 °C at a rate of 120 rpm. At different time periods, 4 mL of supernatant was collected to analyze the release amount of DOX by UV−vis absorption at a wavelength of 480 nm, and 4 mL of fresh PBS (pH 7.4) was added. 2.3. Preparation of Scaffolds and Characterization. 2.3.1. Fabrication of Scaffolds. The fabrication procedure of the HANW@MS/ chitosan (HANW@MS/CS) scaffold is as follows: first, a chitosan solution (30 mg g−1) was prepared by adding the powder of chitosan (3.000 g, medium viscosity) into 97.000 g of acetic acid aqueous solution (1 vol %) under stirring in water bath at 50 °C. Then, the HANW@MS aqueous suspension (30 mg g−1) was mixed uniformly with the asprepared chitosan solution at a weight ratio of 7:3 under stirring at room temperature. The homogeneous mixture was poured into the 24-well plate, frozen at −20 °C for 24 h, and followed by freeze-drying at −20 °C. Finally, the HANW@MS/CS scaffold was soaked in 1 M NaOH aqueous solution for 6 h, and washed with deionized water and ethanol to remove the residual NaOH, and dried at 37 °C. The HANWs/CS scaffold was prepared under the same conditions but using HANWs instead of HANW@MS. The chitosan (CS) scaffold was fabricated in the absence of HANWs or HANW@MS. The as-prepared scaffolds were observed by scanning electron microscopy (SEM; Hitachi S-3400, Japan). The CS, HANWs/CS, and HANW@MS/CS scaffolds were sterilized with 29 kGy of 60Co radiation before use. 2.3.2. Mechanical Properties of Scaffolds. Prior to testing, the CS, HANWs/CS, and HANW@MS/CS scaffolds (size 15 × 10 mm) were soaked in deionized water for 12 h. The mechanical properties of the rehydrated scaffolds were measured on a universal testing machine (Drick, China) at a displacement rate of 5 mm min−1, and the compressive modulus of the rehydrated scaffolds was determined based on the slope of the stress−strain curve in the strain ranging from 15 to 25%. 2.3.3. Ion Release from the HANW@MS/CS Scaffold. To investigate the release behaviors of Mg and Si elements from the HANW@MS/CS scaffold, the HANW@MS/CS scaffold (20 mg) was immersed in 15 mL of Dulbecco’s modified Eagle’s medium (DMEM; Sangon Biotech, Shanghai), sealed, and constantly shaken at a rate of 120 rpm at 37 °C in an oscillator (THI-92A). At different time periods, 3 mL of DMEM was withdrawn and measured using an inductively coupled plasma (ICP) optical emission spectrometer (JY 2000-2, Horiba) to analyze the concentrations of Mg and Si elements in the DMEM, and replaced with 3 mL of fresh DMEM. 2.3.4. Biomineralization. The apatite-forming ability of the scaffolds of the CS, HANWs/CS, and HANW@MS/CS was tested in 1.5 × simulated body fluid (SBF) solution.40 Each sample (10 mg) was soaked in 4 mL of 1.5 × SBF (pH 7.4) in an incubator at 37 °C. After 4 days of biomineralization, the scaffolds were washed three times with deionized water and dried. The apatite layer formed on the scaffolds was examined by SEM. 2.4. In Vitro Cellular Studies. 2.4.1. rBMSCs Culture. The rBMSCs were separated from the bilateral femurs of Sprague−Dawley (SD) rats (6 weeks old) by flushing out the bone marrow using the complete medium (Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1%

(v/v) penicillin/streptomycin (Gibco, USA). Then the cells were cultured at 37 °C in a 5% CO2 incubator. The nonadherent cells were removed after 48 h of culture. Then, the attached rBMSCs were cultured and passaged at a confluence of 80−90%. The rBMSCs of passages 2−6 were used for the experiments. 2.4.2. Cell Adhesion and Proliferation. The rBMSCs were seeded on each scaffold (10 mm × 2 mm; 1 × 105 cells) and cultured for 3 days. For the cytoskeleton staining, the cells grown on the scaffolds were fixed in 4% paraformaldehyde, treated with Triton X-100 (0.5% v/v) for 5 min, stained in darkness for 30 and 5 min, respectively, with fluorescein isothiocyanate (FITC)−phalloidin (Sigma) and 4′,6-diamidino-2phenylindole (DAPI; Sigma), and then imaged using a confocal laser scanning microscope (CLSM; LSM 510, Zeiss). For SEM observation, the scaffolds with cells were fixed with 2.5% glutaraldehyde for 4 h, followed by dehydration using a series of ethanol solutions with gradient concentrations (50, 70, 80, 90, 95, and 100 vol %) for 10 min. After freeze-drying, the cells on the scaffolds were sputter-coated with platinum and observed using SEM. We evaluated the proliferation performance of rBMSCs on the scaffolds (Cell Counting Kit-8 assay, Dojindo, Japan). rBMSCs (1 ×105) were seeded on each scaffold and cultured for 1, 3, and 7 days. At each time point, the medium was replaced with 500 μL of fresh medium containing 10% CCK-8 solution in each well. After 2 h incubation, aliquots (100 μL) from each well were put in a 96-well plate for measurement. The absorbance of the samples was measured at a wavelength of 450 nm using a microplate reader (Bio-Rad 680, USA). 2.4.3. Gene Expression Analysis. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to study the effect of the scaffolds on the osteogenic gene expression level of rBMSCs. Briefly, the rBMSCs were seeded on the scaffolds at a density of 2 × 105 cells/ scaffold and cultured for 7 and 14 days. At each time point, total RNA was isolated by homogenizing the scaffolds with cells in 1 mL of Trizol reagent (Invitrogen). The complementary DNA (cDNA) was obtained using a PrimeScript first Strand cDNA Synthesis kit (Takara, Japan) following the manufacturer’s instructions. Quantification of cDNA of runt-related transcription factor 2 (Runx2), osteocalcin (OCN), osteopontin (OPN), and vascular endothelial growth factor (VEGF) was performed on an ABI7500 Thermal Cycler (Applied Biosystem, Australia) using a real-time PCR kit (SYBR Premix EX Taq, Takara). All assays were performed in triplicate. 2.5. In Vivo Bone Regeneration of the HANW@MS/CS Scaffold. 2.5.1. Animal Surgical Procedures. Twenty-four male SD rats (8 weeks old, 250−300 g) were used in the animal experiments and randomly divided into three groups: pure CS scaffold (n = 8), HANWs/ CS scaffold (n = 8), and HANW@MS/CS scaffold (n = 8). After anesthesia, a sagittal incision (1.0−1.5 cm) was prepared on the scalp and the calvarium was exposed by blunt dissection. Two defects with a diameter of 5 mm were created using an electric trephine (Nouvag AG, Goldach, Switzerland) under constant irrigation with normal saline. Then the defects were implanted with the scaffolds (size 5 × 2 mm). Finally, the incisions were closed using absorbable suture. Each rat received an intramuscular injection of penicillin postoperation. 2.5.2. Microfil Perfusion. To evaluate blood vessel formation in the defects, the rats were perfused with Microfil (Microfil MV-122; Flow Tech, Carver, MA) at 12 weeks postoperation. Briefly, after anesthesia, the rib cage of the rats was opened to expose the heart. After an angiocatheter penetrated the left ventricle, the right auricle was incised. Then, 50 mL of heparinized normal saline and 10 mL of Microfil working solution were perfused at 2 mL/min successively. Finally, the specimens were stored at 4 °C overnight to ensure complete polymerization of the contrast agent. 2.5.3. Microcomputed Tomography (Micro-CT) Assessment. Twelve weeks postoperation, the micro-CT (Skyscan 1176, Kontich, Belgium) was used to scan undecalcified calvaria at a resolution of 18 μm to evaluate the formation of new bone in the defects. After decalcification, the calvaria were scanned at a resolution of 9 μm to evaluate the formation of blood vessels in the defects. Threedimensional (3-D) images were reconstructed with CTVox program (Skyscan). Bone volume to total volume (BV/TV), blood vessel area, C


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Figure 1. Synthesis of porous hierarchical nanobrushes of hydroxyapatite nanowire@magnesium silicate nanosheets (HANW@MS) with core−shell nanostructure. (A) Schematic illustration of formation of HANW@MS nanobrushes. SEM micrographs (B, D, and F) and TEM micrographs (C, E, and G) of HANWs (B and C), HANW@SiO2 (D and E), and HANW@MS nanobrushes (F and G) synthesized using 0.4 mmol of MgCl2·6H2O by the solvothermal method at 180 °C for 24 h. and number in the defects were measured via the CTAn program (Skyscan). 2.5.4. Histological Observation. A graded series of ethanol solutions (50−100%) was used for dehydration; then, the undecalcified specimens were embedded in methyl methacrylate, and sectioned into 150 μm thick samples in the oriention of the coronary surface on a saw microtome (Leica SP1600, Germany). Then the coronary sections were polished to a thickness of ∼50 μm, and stained using van Gieson’s picrofuchsin method to identify the new bone, which was colored red. New bone area as a fraction of the total defect area was analyzed by Image pro (Media cybernetic, USA). 2.6. Statistical Analysis. One-way analysis of variance (ANOVA) and Student−Newman−Keuls post hoc tests were used for the statistical analysis. The data were given as the mean ± standard deviation (SD). The values of p < 0.05 were considered to be significant.

selected-area electron diffraction (SAED) (Figure S2). Then, the as-prepared HANWs are used as the cores for the preparation of core−shell nanowires of HANW@SiO2 by a classical Stöber method, and the thicknesses of the SiO2 shell are in the range 20−50 nm (Figure 1D,E). The X-ray powder diffraction (XRD) pattern of the asprepared core−shell nanowires of HANW@SiO2 (Figure 2D) exhibits the characteristic diffraction peaks of hydroxyapatite (Ca10(PO4)6(OH)2, JCPDS No. 09-0432) and a broad hump of amorphous silica around a 2θ value of ∼22°.39 Finally, the porous hierarchical nanobrushes of HANW@MS are obtained via a chemical-template etching process using MgCl2·6H2O as the magnesium source and sodium acetate as the alkali source under solvothermal conditions. In the reaction, the mild hydrolysis of acetate ions can generate hydroxide ions which can break the Si− O chains of silica to form silicate ions. Simultaneously, the released silicate ions can react with magnesium ions to generate magnesium silicate, which forms nanosheets and then the hierarchical structure on the surface of HANWs,38,39 as shown in Figure 1F,G. The HANW@MS nanobrushes are composed of the HANW as the core and a hierarchically nanostructured porous magnesium silicate shell (∼120 nm thick) self-assembled by the ultrathin magnesium silicate nanosheets (∼4.5 nm thick). The element mapping analysis of the HANW@MS nanobrushes indicates that the elements of Mg and Si are uniformly distributed in the shell region, while the elements of Ca and P are only observed in the core region (Figure 2C). The XRD patterns of HANWs and HANW@MS nanobrushes (Figure 2D) show

3. RESULTS AND DISCUSSION 3.1. Formation and Characterization of Porous Hierarchical Nanobrushes of HANW@MS. The formation procedure of porous hierarchical nanobrushes of HANW@MS is demonstrated in Figure 1A. First, hydroxyapatite nanowires (HANWs) are synthesized using CaCl2, NaOH, and NaH2PO4· 2H2O in a mixture of oleic acid, ethanol, and deionized water by the calcium oleate precursor solvothermal method at 180 °C for 36 h. SEM and transmission electron microscopy (TEM) analyses reveal that the lengths of the as-prepared HANWs range from several tens of micrometers up to more than 100 μm, while the diameters of HANWs are tens of nanometers (Figure 1B,C, and Figure S1 in the Supporting Information). The as-prepared HANWs have a single-crystalline structure which is supported by D


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Figure 2. SEM micrograph (A), TEM micrograph (B), and element mapping (C) of porous hierarchical HANW@MS nanobrushes synthesized using 0.4 mmol of MgCl2·6H2O by the solvothermal method at 180 °C for 24 h. (D) XRD patterns of as-prepared HANWs, HANW@SiO2, and HANW@MS nanobrushes. (E) Nitrogen adsorption−desorption isotherm curves of HANWs and HANW@MS nanobrushes.

that the HANWs are well-crystallized and belong to a single crystal phase of hydroxyapatite with a hexagonal structure (Ca10(PO4)6(OH)2, JCPDS No. 09-0432). The chemical composition of the shell can be indexed to magnesium silicate with a talc structure (Mg3Si4O10(OH)2·H2O, JCPDS No. 030174). Furthermore, we investigated the effects of the solvothermal temperature and the amount of MgCl2·6H2O on the morphology of HANW@MS nanobrushes. The magnesium silicate shells of HANW@MS nanobrushes synthesized at 200 °C are slightly denser than those synthesized at 140 °C (Figure S3). Moreover, the HANW@MS nanobrushes prepared with 0.8 mmol of MgCl2·6H2O (∼140 nm thick) exhibit much denser and thicker magnesium silicate shells than those prepared with 0.2 mmol of MgCl2·6H2O (∼55 nm thick) (Figure 3). These experimental results indicate that the thickness and denseness of the magnesium silicate shells of HANW@MS nanobrushes can be simply controlled by varying the solvothermal temperature and the amount of MgCl2·6H2O. The nitrogen adsorption−desorption isotherm curves of HANWs and HANW@MS nanobrushes synthesized using 0.4 mmol of MgCl2·6H2O are shown in Figure 2E. After coating with the shell of magnesium silicate nanosheets, the HANW@MS nanobrushes display remarkably higher Brunauer−Emmett− Teller (BET) specific surface area (SBET) and pore volume (VP) than those of HANWs. The SBET of HANW@MS nanobrushes

Figure 3. TEM micrographs of porous hierarchical HANW@MS nanobrushes prepared by the solvothermal method at 180 °C for 24 h using 0.2 mmol of MgCl2·6H2O (a, b) and 0.8 mmol of MgCl2·6H2O (c, d).

(295.6 m2 g−1) is about 6 times that of HANWs (52.6 m2 g−1). In addition, the VP of HANW@MS nanobrushes (0.71 cm3 g−1) is about 3.5 times that of HANWs (0.20 cm3 g−1). Moreover, the E


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Figure 4. Drug loading and release performance of HANWs and porous hierarchical HANW@MS nanobrushes as drug carriers. (A) Loaded amount of vancomycin (VAN) by 5 mg of HANWs or HANW@MS in 2 mL VAN aqueous solution (0.4 mg mL−1). (B) Protein loading curve of 5 mg of HANWs or HANW@MS in 2 mL of Hb aqueous solution with different initial concentrations (0.2−3 mg mL−1). (C) Drug loading curve of 5 mg of HANWs or HANW@MS in 2 mL of DOX aqueous solution with different initial concentrations (0.2−3 mg mL−1). (D) DOX drug release profiles of DOX-loaded HANWs and HANW@MS in PBS (pH 7.4) for different times.

range from 0.2 to 0.8 mg mL−1, the DOX molecules are almost completely loaded by the HANW@MS nanobrushes (Figure 4C). At the DOX concentration of 3.0 mg mL−1, the loaded amount of DOX in HANW@MS nanobrushes (2568.8 ± 39.3 μg) is about 9.5 times that of HANWs (269.0 ± 31.9 μg). The experimental results clearly indicate the high drug loading capacity of the HANW@MS nanobrushes. The drug release profile of the HANW@MS nanobrushes was investigated using the DOX-loaded HANW@MS drug delivery system as a representative example. As shown in Figure 4D, the DOX-loaded HANWs drug delivery system shows a relatively rapid drug release in the first 0.5 day (65.3 ± 5.4 μg of DOX released) and an almost complete release within 6 days (105.4 ± 7.6 μg of DOX released). However, the DOX-loaded HANW@ MS drug delivery system exhibits a sustainable drug release performance. The DOX release from the HANW@MS nanobrushes is relatively fast in the first 0.5 day (97.2 ± 4.4 μg of DOX released), and then slows down and still continues over 4 weeks (354.8 ± 8.7 μg of DOX released). These experimental results suggest that the porous hierarchical nanobrushes of HANW@ MS exhibit great potential as the high-efficiency platform for loading and long-term release of various therapeutic drug molecules. Mg and Si are essential elements in the human body. Our previous studies demonstrated that magnesium silicate nanostructured materials have high biocompatibility and good biodegradability.38,39 By choosing suitable drug molecules, the biomaterials/scaffolds may have the ability to control or stimulate cellular responses and tissue reaction.40,41 Therefore, along with the intrinsic chemical and biological properties, the as-

nitrogen adsorption−desorption isotherms of HANWs and HANW@MS nanobrushes belong to the typical type IV isotherm with a type H3 hysteresis loop, indicating the presence of mesopores.43 The high SBET and VP can be explained by the hierarchically porous shell of HANW@MS nanobrushes constructed with ultrathin magnesium silicate nanosheets. 3.2. In Vitro Drug Loading and Release. Magnesium silicate nanostructured materials have high specific surface areas, and are favorable for drug delivery. In this work, we further investigated the performance of HANWs and HANW@MS nanobrushes as the drug carriers in loading and release of the drug and protein, and a typical antibiotic drug of vancomycin (VAN), a typical anticancer drug of doxorubicin hydrochloride (DOX), and a typical protein of hemoglobin (Hb) were chosen for investigation. First, the VAN loading performance in HANWs and HANW@MS nanobrushes was investigated at a fixed VAN concentration of 0.4 mg mL−1. As shown in Figure 4A, the loaded amount of VAN in HANW@MS nanobrushes (227 ± 6.4 μg) is about 22 times of that of HANWs (10.5 ± 4.7 μg). Then, the loading behaviors of Hb or DOX in HANWs and HANW@MS nanobrushes were investigated by varying the initial concentration of Hb or DOX. As shown in Figure 4B, the loaded amount of Hb in HANWs and HANW@MS nanobrushes increases with increasing Hb initial concentration from 0.2 to 0.8 mg mL−1 and then reaches a plateau with the Hb initial concentration ranging from 1.2 to 3.0 mg mL−1. At the concentration of 3.0 mg mL−1, the loaded amount of Hb in HANWs and HANW@MS nanobrushes is 573.1 ± 48.6 and 1286.4 ± 24.7 μg, respectively. Moreover, the HANW@MS nanobrushes exhibit a high capacity of loading DOX drug molecules. When the DOX concentrations F


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Figure 5. Characterization of chitosan (CS) scaffold, HANWs/CS scaffold and HANW@MS/CS scaffold. (A) SEM micrographs of CS scaffold (a−c), HANWs/CS scaffold (d−f), and HANW@MS/CS scaffold (g−i). (B) SEM micrographs of CS scaffold (a), HANWs/CS scaffold (b), and HANW@ MS/CS scaffold (c) after soaking in ×1.5 SBF for 4 days. The scale bars in each column are the same in (A) and (B). (C) Stress−strain curves and (D) compressive modulus of scaffolds of CS, HANWs/CS, and HANW@MS/CS under wet condition. (E) Mg and Si element release profiles from HANW@MS/CS scaffold in DMEM for different times.

HANWs/CS and HANW@MS/CS scaffolds (Figure 5A(e,f,h,i)). In contrast, the surface of pore walls of the pure CS scaffold is smooth (Figure 5A(b,c)). Compared with the pure CS scaffold, the HANWs/CS and HANW@MS/CS scaffolds exhibit significantly enhanced apatite-forming abilities. As shown in Figure 5B, after soaking in ×1.5 SBF for 4 days, a large amount of apatite nanosheets is observed on the surface of HANWs/CS and HANW@MS/CS scaffolds, whereas no obvious apatite is found on the surface of the pure CS scaffold. The mechanical properties of the as-prepared HANWs/CS and HANW@MS/CS scaffolds together with the pure chitosan scaffold as the control sample are shown in Figure 5C. The compressive modulus of HANW@MS/CS and HANWs/CS scaffolds under wet condition in the strain ranging from 15 to 25% are 6.18 ± 0.52 and 7.84 ± 0.77 kPa, respectively, which are higher than that of the pure CS scaffold (5.38 ± 0.66 kPa) (Figure 5D). Moreover, the HANW@MS/CS scaffold exhibits a sustainable release performance of Mg and Si elements for a relatively long period of time (21 days) in DMEM (Figure 5E), which is consistent with the good degradability of magnesium

prepared HANW@MS nanobrushes with high drug loading capacities and sustainable drug release properties exhibit great potential as smart platforms to achieve high performance in various biomedical applications. 3.3. Preparation and Characterization of HANW@MS/ CS Scaffold. To examine the properties of HANW@MS nanobrushes on the in vitro bioactivity and in vivo bone regeneration, the porous HANW@MS/CS scaffold with 70 wt % HANW@MS was fabricated by hybridizing HANW@MS nanobrushes with the biocompatible chitosan (CS) based on a facile freeze-drying method. The pure CS scaffold and HANWs/ CS scaffold were also prepared and used as the control samples. The as-prepared scaffolds of pure CS, HANWs/CS, and HANW@MS/CS are highly porous and exhibit a hierarchical interconnective structure with controllable pore sizes ranging from 200 to 300 μm (Figure 5A(a,d,g)). Higher magnification SEM images further reveal that the HANWs or HANW@MS nanobrushes are uniformly incorporated in the chitosan matrix and interweave with each other to form a porous fabric-like structure, leading to the rough surface of pore walls of the G


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Figure 6. In vitro studies of scaffolds of CS, HANWs/CS, and HANW@MS/CS. (A) Cytoskeleton observation of rBMSCs cultured on scaffolds of CS (a), HANWs/CS (b), and HANW@MS/CS (c) for 3 days. (B) SEM micrographs of rBMSCs on scaffolds of CS (a), HANWs/CS (b), and HANW@ MS/CS (c) after culture for 3 days. The scale bars in (A) or (B) are the same. (C) RT-qPCR analysis of gene expression (Runx2, OCN, OPN, and VEGF) of rBMSCs cultured on scaffolds of CS, HANWs/CS, and HANW@MS/CS for 7 and 14 days; (a) Runx2, (b) OCN, (c) OPN and (d) VEGF. ∗, p < 0.05 compared to CS scaffold; #, p < 0.05 compared to HANWs/CS scaffold.

silicate.39 The release rate of Si element from the HANW@MS/ CS scaffold is faster than that of Mg element, which may be caused by the factor that Mg ions in the DMEM inhibit the release of Mg ions from the HANW@MS/CS scaffold. 3.4. Effects of Different Scaffolds on Cell Adhesion and Proliferation. The biocompatibility of the as-prepared scaffolds was assessed in terms of cell adhesion and proliferation. As shown in Figure 6A, the analysis of cytoskeleton staining reveals that the rBMSCs spread well and maintain their phenotype on the HANWs/CS and HANW@MS/CS scaffolds, whereas the number of attached cells is significantly reduced and the morphology of the attached cells is irregular on the CS scaffold. In addition, the SEM micrographs confirm that the rBMSCs well spread with clear and prominent filopodia on the HANWs/CS and HANW@MS/CS scaffolds, whereas the cells have a spherical shape on the CS scaffold (Figure 6B). These experimental results demonstrate that the HANWs/CS and HANW@MS/CS scaffolds can significantly enhance the adhesion and spreading of rBMSCs, which can be explained by the rough surfaces of the HANWs/CS and HANW@MS/CS scaffolds. As determined by the CCK-8 assay (Figure S4), both HANWs/CS and HANW@MS/CS scaffolds are favorable for the proliferation of rBMSCs, and the proliferation rate on the HANW@MS/CS scaffold is higher than that on the HANWs/ CS scaffold on days 3 and 7. On the contrary, there is no

significant proliferation for the cells on the CS scaffold in 7 days. The experimental results confirm that the HANW@MS/CS scaffold is highly biocompatible and favorable for cell spreading and proliferation, which is consistent with previous studies on the good biocompatibility of HANWs and magnesium silicate.29,31,32,44 3.5. Effect of HANW@MS/CS Scaffold on Gene Expression of rBMSCs. The abilities of the scaffolds to stimulate osteogenic and angiogenic responses were evaluated by directly culturing rBMSCs on various scaffolds. After culturing for 7 and 14 days, the expression of osteogenic and angiogenic genes including Runx2, OCN, OPN, and VEGF was evaluated by RT-qPCR analysis (Figure 6C). Compared with the CS scaffold, the rBMSCs cultured on the HANWs/CS and HANW@MS/CS scaffolds exhibit remarkably higher expression of Runx2, OCN, and OPN on days 7 and 14, and the HANW@MS/CS scaffold exhibits the highest expression of Runx2, OCN, and OPN. In addition, the HANW@MS/CS scaffold also enhances the expression of VEGF compared with the scaffolds of HANWs/ CS and CS on days 7 and 14. These experimenal results indicate that the abilities of these scaffolds in stimulating osteogenic responses of rBMSCs follow the trend HANW@MS/CS scaffold > HANWs/CS scaffold > CS scaffold (Figure 7). The HANWs/CS and HANW@MS/CS scaffolds display a critical role in the osteogenic differentiation of rBMSCs. On the one hand, the mechanical properties of the scaffold can H


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Figure 7. Schematic illustration of potential applications of HANW@MS/CS scaffold for enhanced osteogenic differentiation of rBMSCs and angiogenesis for bone regeneration. The HANW@MS/CS scaffold can release functional elements of Mg and Si to promote the osteogenic differentiation of hBMSCs into osteoblasts in vitro and subsequently simulate the formation of new bone and neovascularization to enhance bone regeneration in vivo.

and the stimulatory effects were improved with increasing Si concentration in the silicate extracts.35 Thus, the experimental results suggest that the HANW@MS/CS scaffold is promising for application in bone regeneration. 3.6. In Vivo Bone Regeneration. The efficacies of the HANW@MS/CS scaffold for bone defect repair were evaluated by implanting the porous HANW@MS/CS scaffold into a critical-sized rat calvarial defect model. After 12 weeks postimplantation, the formation of bone and blood vessels in the calvarial defect region was investigated by micro-CT and histological analyses. As shown in Figure 8A (interior and coronal views), there is almost no healing in the defects implanted with the CS scaffold, and much more new bone formation is observed in the HANW@MS/CS group than in the HANWs/CS group. The quantitative analysis demonstrates that the percentage of newly formed bone volume (BV/TV) in the HANW@MS/CS group (40.15 ± 6.11%) is significantly higher than that in the HANWs/CS group (25.06 ± 3.74%) or the CS group (4.92 ± 1.24%) (Figure 8C). To evaluate the neovascularization effect of the as-prepared scaffolds, the newly formed blood vessels in the defect regions were assessed using Microfil perfusion and micro-CT scanning. The 3-D images (Figure 8B) reveal that more new blood vessels have been formed in the defect regions implanted with the HANW@MS/CS scaffold than those implanted with the HANWs/CS and CS scaffolds. Quantification analysis of the newly formed blood vessels further demonstrates that the HANW@MS/CS group exhibits a significantly greater blood vessel number and larger blood vessel area in the calvarial defect

significantly influence the fate of stem cells, and the scaffold with high stiffness is favorable for the bone-related gene expression of rBMSCs.21,22 Thus, the enhanced mechanical properties of the HANWs/CS and HANW@MS/CS scaffolds may promote osteogenic differentiation of rBMSCs. On the other hand, the hierarchical and porous structures of the scaffolds of HANWs/ CS and HANW@MS/CS mimic the natural structure of human trabecular bone, which is beneficial to the osteogenic differentiation of rBMSCs.45 The Ca and P elements released from the scaffolds of HANWs/CS and HANW@MS/CS can provide a positive chemical environment for the stem cell niche and further influence the stem cell behavior and induce their osteogenic differentiation.46 In addition, the more obvious effect of the HANW@MS/CS scaffold on the osteogenic differentiation of rBMSCs compared with the HANWs/CS scaffold can be explained by the additionally released Mg and Si elements. The sustained release of Mg and Si elements could significantly stimulate the osteogenic differentiation of mesenchymal stem cells. Previous studies showed that Mg element is crucial to bone formation and metabolism, and the doping of Mg could improve the differentiation of MSCs into osteoblasts.47,48 Chen et al. reported that Mg ions could stimulate the cementogenesis and angiogenesis of human periodontal ligament cells, and the stimulatory effect was enhanced by the Mg ion concentration of the medium.36 Si element could also significantly enhance osteogenic differentiation and stimulate Wnt- and Shh-related signaling pathways of BMSCs.49 Moreover, Si element could stimulate the osteogenic and angiogenic differentiation of cells, I


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Figure 8. Micro-CT assessment of newly formed bone and blood vessels in rat calvarial defect regions after implantation of scaffolds of CS, HANWs/CS, and HANW@MS/CS for 12 weeks. (A) Three-dimensional (3-D) and coronal views of reconstructed calvarial images. (B) Newly formed blood vessels presented by 3-D reconstructed images. Morphometric analysis was completed for the percentage of newly formed bone volume (BV/TV) (C), blood vessel number (D), and blood vessel area (E) in the defects. ∗, p < 0.05 compared to CS scaffold; #, p < 0.05 compared to HANWs/CS scaffold.

regions (vessel number 50 ± 7; vessel area 13.26 ± 0.98%) as compared to the HANWs/CS group (vessel number 29 ± 8; vessel area 9.66 ± 1.91%) or the CS group (vessel number 13 ± 5; vessel area 5.38 ± 2.21%) (Figure 8D,E). The histological analysis of bone regeneration is shown in Figure 9. The experimental results in van Gieson’s picrofuchsin staining of representative undecalcified sections from each group show that a greater amount of new bone has been formed in the HANW@MS/CS group than in the HANWs/CS group, and typical bone structure is scarcely observed in the CS group (Figure 9A). The histomorphometric analysis reveals that the percentage of new bone area in HANW@MS/CS, HANWs/CS, and CS groups is 39.41 ± 4.25, 22.99 ± 4.39, and 3.15 ± 0.84%, respectively, with a significant difference (p < 0.05) (Figure 9B). Consistent with the in vitro experimental results, the HANW@MS/CS scaffold can significantly facilitate the formation of new bone and blood vessels as compared to the scaffolds of CS and HANWs/CS. The combination of the nanostructured magnesium silicate with HANWs can significantly improve both in vitro and in vivo bioactivity of the HANW@MS/CS scaffold. The experimental results of in vitro biomimetic mineralization reveal that the HANW@MS/CS

scaffold has an excellent apatite-forming ability. The formed apatite layer can endow the scaffold with high osteoconductivity and enhance the adhesion of osteogenic cells in surrounding tissues.50 The porous structure of the HANW@MS/CS scaffold promotes cell attachment and proliferation, and simultaneously induces the osteogenic differentiation of rBMSC.45,51,52 The sustained release of Mg and Si elements contributes to osteogenic and angiogenic induction, bone formation, osseointegration, and blood vessel formation (Figure 7).35,48 The experimental results in this study demonstrate that the HANW@MS/CS scaffold can significantly promote bone regeneration by simulating osteogenesis and angiogenesis, suggesting that the porous HANW@MS/CS scaffold is promising for application in bone regeneration.

4. CONCLUSIONS The porous hierarchical nanobrushes of HANW@MS composed of hydroxyapatite nanowire as the core and magnesium silicate nanosheets as the shell have been synthesized through a solvothermal approach based on a chemical-template etching strategy. The as-prepared HANW@MS nanobrushes possess J


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Figure 9. Histological analysis of bone regeneration. (A) Van Gieson’s picrofuchsin stained sections of rat calvarial defect regions after implantation with scaffolds of CS (a−c), HANWs/CS (d−f), and HANW@MS/CS (g−i) for 12 weeks. (B) Percentage of new bone area in the defects. ∗, p < 0.05 compared to CS scaffold; #, p < 0.05 compared to HANWs/CS scaffold.

Notes

high specific surface area and pore volume, resulting in high performance in loading and sustained release of various drugs, proteins, and biomolecules. Moreover, the as-prepared HANW@MS/CS scaffold with a porous and hierarchical structure exhibits a sustainable release of Mg and Si elements, and can significantly promote the adhesion and spreading of rat bone marrow derived mesenchymal stem cells (rBMSCs). Compared with the CS and HANWs/CS scaffolds, the HANW@MS/CS scaffold can significantly enhance the osteogenic and angiogenic differentiation of rBMSCs in vitro and facilitate the formation of new bone and blood vessels in vivo. Thus, the as-prepared HANW@MS nanobrushes and the HANW@MS/CS scaffold are promising for applications in various biomedical fields such as drug delivery and bone regeneration.

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The authors declare no competing financial interest.

ACKNOWLEDGMENTS

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REFERENCES

Financial support from the Science and Technology Commission of Shanghai (15JC1491001), the National Natural Science Foundation of China (51472259), the Youth Innovation Promotion Association of CAS (2015203), and the National Key Research and Development Program of China (2016YFA0203700) is gratefully acknowledged.

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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.7b03532. Reagents used in the experiments and characterization of the as-prepared materials; SEM micrograph and selectedarea electron diffraction pattern of the as-prepared hydroxyapatite nanowires (HANWs); TEM micrographs of porous hierarchical HANW@MS nanobrushes; CCK-8 assay of the proliferation of rBMSCs on the scaffolds of the CS, HANWs/CS, and HANW@MS/CS (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Y.-J.Z.). *E-mail: [email protected] (Y.-H.H.). *E-mail: [email protected] (F.C.). ORCID

Ying-Jie Zhu: 0000-0002-5044-5046 Yao-Hua He: 0000-0002-2829-8908 Author Contributions

⊥ T.-W.S. and W.-L.Y.: These authors contributed equally to this work.

K


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