Optically Monitoring Mineralization and Demineralization on

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Optically Monitoring Mineralization and Demineralization on Photoluminescent Bioactive Nanofibers Xiang Li,*,†,⊥ Yangyang Li,†,⊥ Xiaoyi Chen,‡ Binbin Li,† Bo Gao,§ Zhaohui Ren,† Gaorong Han,*,† and Chuanbin Mao*,†,∥ †

State Key Laboratory of Silicon Materials, School of Materials Science and Engineering, Zhejiang University, Hangzhou 310027, P.R. China ‡ Clinical Research Institute, Zhejiang Provincial People’s Hospital, Hangzhou 310014, P.R. China § Department of Prosthetic Dentistry, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, P.R. China ∥ Department of Chemistry & Biochemistry, Stephenson Life Sciences Research Center, University of Oklahoma, 101 Stephenson Parkway, Norman, Oklahoma 73019-5300, United States S Supporting Information *

ABSTRACT: Bone regeneration and scaffold degradation do not usually follow the same rate, representing a daunting challenge in bone repair. Toward this end, we propose to use an external field such as light (in particular, a tissue-penetrating near-infrared light) to precisely monitor the degradation of the mineralized scaffold (demineralization) and the formation of apatite mineral (mineralization). Herein, CaTiO 3 :Yb 3+ ,Er 3+ @bioactive glass (CaTiO3:Yb3+,Er3+@BG) nanofibers with upconversion (UC) photoluminescence (PL) were synthesized. Such nanofibers are biocompatible and can emit green and red light under 980 nm excitation. The UC PL intensity is quenched during the bone-like apatite formation on the surface of the nanofibers in simulated body fluid; more mineral formation on the nanofibers induces more rapid optical quenching of the UC PL. Furthermore, the quenched UC PL can recover back to its original magnitude when the apatite on the nanofibers is degraded. Our work suggests that it is possible to optically monitor the apatite mineralization and demineralization on the surface of nanofibers used in bone repair.

1. INTRODUCTION Bioactive glass (BG) materials have attracted worldwide attention due to their distinguished advantages, such as excellent biocompatibility, controllable degradation, and sound bioactivity.1 With an ever-growing understanding of the intricate interactions between bone cells and their microenvironments, more attention is now paid to the fabrication of scaffolds that better mimic the native extracellular matrix (ECM) in bone and can promote bone regeneration.2 One of such scaffolds is a nonwoven mat of BG nanofibers synthesized via electrospinning technique, which has been found to exhibit excellent bioactivity and osteogenic potential.3,4 When a BG scaffold is used to repair bone defect, the scaffold induces strong bonding to natural bone through rapid formation of bone−like hydroxycarbonate apatite (HCA) layer on the scaffold surface in the physiological environment.5 The HCA mineralization for bioactive glasses in body fluid is highly related to the formation of silanol (Si−OH) groups and the release of Ca2+ ions. Simulated body fluid (SBF) solution induces the nucleation of mineral on the silanol sites, leading to © XXXX American Chemical Society

the formation of an amorphous calcium phosphate (ACP) layer. As the (OH)− and (CO3)2− from the body fluid are incorporated into ACP, the ACP crystallizes into the HCA phase.6 It was also documented that the HCA layer formed could significantly improve cell activities such as cell attachment.7 Therefore, the mineralization ability of an implant material has been recognized as one iconic sign for its bone repairing ability. Thus, it has also been widely utilized as crucial evidence when evaluating the “bioactivity” properties of a biomaterial.8 During bone repairing, the scaffold material is expected to degrade in tune with the concurrent replacement by regenerated bone. Rapid degradation leads to burst release of ions from the scaffold, which may impede the expected cell response.9 Meanwhile, the slow ion release may induce insufficient bioactivity of the scaffold, and thus the delayed scaffold degradation can hinder the new bone regeneration. Received: January 26, 2016 Revised: March 10, 2016

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maintained at 90 °C for 3 h. The particles formed were collected by centrifugation, washed for three times with ethanol, and dried at 80 °C. CaTiO3:Yb3+,Er3+ nanoparticles were further annealed in air at 700 °C for 3 h. 2.2. Fabrication of CaTiO3:Yb3+,Er3+@BG Nanofibers. The electrospinning precursor solution was obtained by a two-solution method. First, 1.67 g of tetraethyl orthosilicate (TEOS, Sigma-Aldrich Inc.), 0.21 g of triethylphosphate (TEP, Sigma-Aldrich), 0.675 g of calcium nitrate (Ca(NO3)2·4H2O, Sigma-Aldrich), 0.2 mL of acetic acid (A.R., Sinopharm Chemical, China), and 10 mL of pure ethanol (99.9 vol %, Sinopharm Chemical, China) were mixed under stirring at room temperature. Subsequently, deionized water was added with different water/TEOS ratios (2, 4, and 6) to control the BG precursor hydrolysis, which was crucial to the bioactivity properties of BG materials. After 1.5 h of stirring, 3 mL of N,N-dimethylformamide (DMF) was added. Finally, 0.9 g of polyvinylpyrrolidone (PVP, Mw = 1 300 000, Sigma−Aldrich Inc.) was added to the BG precursor solution under constant stirring to obtain solution A. CaTiO3:Yb3+,Er3+ nanoparticles were dispersed in the ethanol with a concentration of 2 mg/mL, then ultrasonicated for 2 h to obtain solution B. Five ml B solution was added dropwise into solution A using micropipette under stirring to obtain the electrospinning precursor. Subsequently, the precursors were transferred into a single nozzle electrospinning setup, as shown in Figure 2. The distance and the applied voltage between the needle tips and the collector were set at 15 cm and 7−10 kV, respectively. The precursor flow rate was set at 0.8 mL h−1. The as-spun BG composite fibers were collected on stainless steel grids. The electrospun fibers were then dried at 80 °C in oven for 12 h, and sintered in air at 600 °C to eliminate organic additives. 2.3. An in Vitro Study of CaTiO3:Yb3+,Er3+@BG Nanofibers. To study the mineralization process, 20 mg of composite fibers was immersed in 20 mL of simulated body fluid (SBF) at 37 °C for different periods (3, 6, 12, 24, 48, 72, and 120 h). All types of mineralized nanofibers were collected, rinsed in deionized water, and air-dried. Furthermore, the reversible UC PL effect of the nanofibers during apatite dissolving process was examined. An amount of 20 mg of CaTiO3:Yb3+, Er3+@BG nanofibers, which were mineralized in SBF for 2 days, was immersed in an acidic aqueous solution (pH = 3) for different periods (0, 6, 12, 24, 48, and 72 h) at 37 °C. The structure evolution and PL properties of the nanofibers were examined at each time point during the formation and dissolving of apatite using scanning electron microscopy (FESEM, Hitachi SU-70, Japan) and a fluorescence spectrophotometer under excitation with a wavelength of 980 nm (PL, FLSP920, Edinburgh). Confocal fluorescence microscopy (BX61WI_FV1000, Olympus, Japan) was used to observe the PL quenching phenomenon of nanofibers during the apatite formation. Bone marrow-derived mesenchymal stem cells (BMSCs) were cultured in 96-well plates using H-DMEM medium with 10% FBS (heat-inactivated fetal bovine serum) at 37 °C in a humidified atmosphere containing 5% CO2, and allowed to attach to the bottom of the plates, then the cells were incubated with fresh media containing CaTiO3:Yb3+,Er3+@BG fibers. The fibers with different hydrolysis degrees for 1, 3, and 5 days were cut into mesh plates with the same diameter (similar mass) and fixed to the well edge of 96-well culture plates. After the incubation, MTT (20 μL, 10 mg/mL) solution was added to each well of the plate, and the cells were incubated for 4 h further. Subsequently, the cells were lysed using DMSO (150 μL). The absorbance was measured at 490 nm using a microplate reader (Tecan, Durham, NC). Tests were performed in sextuplicate. The cell morphology was observed with SEM after the cells were fixed with 2.5% glutaraldehyde, dehydrated with a graded series of ethanol (70, 90, and 100%), and coated with gold. 2.4. Characterization. The X-ray diffraction (XRD) analysis of the as-prepared nanofibers was conducted on a Philips X pert PRO X-ray diffractometer with Cu (Kα) radiation. The morphology and microstructure of nanofibers were examined via field emission scanning electron microscopy (FESEM, Hitachi SU-70, Japan) and high-resolution transmission electron microscopy (HRTEM, Tecnai F20, FEI). The UC emission spectrum measurements were carried out

Therefore, an ideal bone scaffold should be degraded at a similar rate, or even the same rate, with the new bone formation. A variety of studies have been carried out to synthesize BG scaffold materials with tunable degradation rate in the past decades.10 However, patients, with different age, gender, and health conditions, have remarkably different bone regeneration ability.11 It is unlikely to use one BG scaffold material to repair the bone defect of different patients with an expected outcome. Although many techniques, such as atomic force microscopy (AFM) and nuclear magnetic resonance (NMR), have been previously used to quantify mineral crystal growth rates,12−14 the current approaches are usually timeconsuming, needing sophisticated instrument. To tackle this challenge, there needs to be a simply method for externally monitoring the behavior of the BG scaffold (e.g., the structural and chemical composition changes), which will instruct us to apply the external stimulation strategies to accelerate or postpone the bone repairing process. Recently, the upconversion (UC) photoluminescence (PL) biomaterials have been widely investigated for drug delivery, cell imaging, and tumor diagnosis/therapy.15,16 The UC PL lanthanide materials emit high-energy visible photons after being excited by near-infrared (NIR) photons.17 Meanwhile, the NIR excitation results in weak autofluorescence background because the UV-excitable biological tissues and fluorescent drug molecules that interfere with normal phosphor luminescence cannot be excited by NIR radiation.18 In addition, NIR light with strong penetration ability will be safe to the human body and less harmful to cells.19 Recently, Er3+ doped and Er3+/Yb3+ codoped nanomaterials were successfully developed for the targeted drug delivery with expected PL efficiency.20 To avoid the possible toxicity of lanthanide ions in the physiological environment, a host material is needed to prevent its release.21 Calcium titanate (CaTiO3) is an established bioceramic, which has been widely used as a coating material for orthopedic implants.22 When used for biomedical applications, comparing to the current NaYF4 UC nanomaterials reported,23 CaTiO3 is of superior biocompatibility and contains Ca and Ti that are beneficial for bone formation.24 These properties make CaTiO3 a very promising host material for the UC photonic crystal for tissue engineering applications. More importantly, Er3+ ions can be doped and “caged” within the lattice of CaTiO3, which can avoid possible free release of lanthanide element due to its perovskite crystal structure and the dimensional matching between Er and Ca atoms.25 Herein, Er3+/Yb3+ doped CaTiO3 (CaTiO3:Yb3+, Er3+) nanoparticles are proposed to serve as an optical reporter for monitoring the HCA mineralization and demineralization on the 70SiO2−25CaO−5P2O5 BG nanofibers decorated with the nanoparticles.

2. MATERIALS AND METHODS 2.1. Synthesis of CaTiO3:Yb3+,Er3+ Nanoparticle. CaTiO3:(18 mol %)Yb3+,(2 mol %)Er3+ nanoparticles were synthesized by a coprecipitation method. Stock solution of ethylenediaminetetraacetic acid disodium salt (EDTA, Aladdin, China) was prepared by dissolving 1.00 g of EDTA into 100 mL of deionized water. The pH value was adjusted at 11 using ammonia−water. Ca(NO)2·4H2O (SigmaAldrich, Germany), Yb(NO3)3·5H2O, and Er(NO3)3·5H2O (SigmaAldrich, Germany) were dissolved into 10 mL of ethanol under stirring for 30 min at room temperature, and subsequently 5 mmol of Ti(OC4H9)4 (Aladdin, China) was added to prepare a transparent precursor solution. Ca/Ti molar ratio was set at 1.5. The precursor solution was then added into the EDTA stock solution via a syringe pump with a flow rate of 60 mL/h. The reactant solution was B

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Figure 1. (a) X-ray diffraction patterns, (b) SEM image (inset shows the particle size distribution), and (c) UC PL spectrum of CaTiO3:Yb3+,Er3+ nanoparticles. via an F-7000 spectrophotometer under 980 nm excitation, and the PL images was recorded using a confocal laser scanning microscopey (BX61WI_FV1000, Olympus, Japan) equipped a 980 nm laser diode.

3. RESULTS AND DISCUSSION 3.1. The Synthesis of CaTiO3:Yb3+,Er3+@BG Nanofibers. The orthorhombic crystal structure of the CaTiO3:Yb3+,Er3+ nanoparticles synthesized was confirmed by XRD analysis (Figure 1a). The nanoparticles are spherical and well-dispersed with a mean diameter of ∼60 nm(Figure 1b). Under the excitation of 980 nm NIR laser, the nanoparticles present strong anti-Stokes emission: green and red emission (Figure 1c). The red emission (∼660 nm) presents a much higher intensity than the green emission (∼550 nm). The doping of Yb3+ ions populates directly the 4 F9/2 (Er3+) level, which induces the enhanced red(4F9/2 → 4 I15/2) emission.26 The CaTiO3:Yb3+,Er3+@BG nanofibers were synthesized by particles-electrospinning (Figure 2). When the water/TEOS ratio used during electrospinning was set at 2, the nanofibers were of ∼280 nm in average diameter. With the increased water/TEOS ratio to 4 and 6, the average fiber diameter was found to increase to ∼330 and ∼440 nm, respectively (Figure 2c a n d d ) . T h e n a n o fi b e r s w i t h o u t t h e C a T i O3:Yb3+,Er3+nanoparticles were amorphous, as reflected by a broad band characteristic of amorphous SiO2 network structure.27 The examination using energy dispersive spectroscopy (EDS) shows the coexistence of Si, Ca, P, and O elements in the BG fibers, and the Si/Ca/P ratios (atom %) of BG fibers with water/TEOS ratio of 2, 4, and 6 are calculated to be 72.6/ 24.3/4.1, 73.9/23.5/2.6, and 73.09/22.8/4.2, respectively, reflecting that the chemical composition of BG contents in all three fibers is close to 70SiO2-25CaO-5P2O5 (Figure S1), as expected. In comparison, for CaTiO3:Yb3+,Er3+@BG nanofibers, the peaks at 33° and 47°coincide with (112) and (220) planes of CaTiO3:Yb3+,Er3+ nanoparticles(Figure 3a).28 The

Figure 2. (a) Schematic diagram of electrospinning setup (insets show the high-speed camera image of jetting). (b−d) SEM images of the CaTiO3:Yb3+,Er3+@BG nanofibers, with water/TEOS ratio of 2, 4 and 6, after calcination.

microstructure of both nanofibers was examined using TEM. It was found that the CaTiO3:Yb3+,Er3+nanoparticles were successfully incorporated with BG fibers (Figures 3b,c and S2). Furthermore, the lattice fringes presented in the HRTEM image confirm the presence of crystalline CaTiO3 nanoparticles within the BG fibers after annealing (Figure 3d). The distance between the adjacent lattice fringes of CaTiO3:Yb3+,Er3+@BG nanofibers is ∼0.269 nm, which is well consistent with the interplanar spacing of (112) of CaTiO3crystal (JCPDS Card No. 82-0228). C

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Figure 3. (a) X-ray diffraction patterns of BG fibers and CaTiO3:Yb3+,Er3+@BG nanofibers. (b,c) TEM images of (b) BG fibers and (c) CaTiO3:Yb3+,Er3+@BG nanofibers (insets show the corresponding SAED patterns). (d) HRTEM images of CaTiO3:Yb3+,Er3+@BG nanofibers. (e) UC PL spectra of BG fibers and CaTiO3:Yb3+,Er3+@BG nanofibers (insets show the optical images of both nanofibers under 980 nm excitation, captured via confocal laser scanning microscopy).

With 980 nm NIR laser excitation, pure BG nanofibers without the nanoparticles do not emit light. In contrast, the UC PL spectra of CaTiO3:Yb3+,Er3+@BG nanofibers are strongly dominated by the strong red emission centered at ∼660 nm as well as weak green emission at ∼550 nm (Figure 3e), which is due to the presence of OH groups in the CaTiO3:Yb3+,Er3+@ BG nanofibers, resulting in the quenched green emission and the enhanced red emission.29 These emission peaks agree well with the earlier studies on the doped Er3+ ions.30 The CaTiO3:Yb3+,Er3+@BG nanofibers under 980 nm excitation were further confirmed using confocal microscopy (Figures 3e and S3). 3.2. The Cytotoxicity of CaTiO3:Yb3+,Er3+@BG Nanofibers. The cytotoxicity has been always a crucial factor when one biomaterial is expected to be used as a bone scaffold. All types of CaTiO3:Yb3+,Er3+@BG nanofibers with different hydrolysis degrees (X ratio = 2, 4, and 6) were cultured with BMSCs, and MTT assay was used for the cytotoxicity assessment. An increasing trend of cell growth in a time dependent manner was observed in each group (X ratio = 2, 4, and 6) (Figure 4). No statistically significant difference was found in the proliferation rate of BMSCs among three BG nanocomposite scaffolds with different degrees of hydrolysis. The cell proliferation on all CaTiO3:Yb3+,Er3+@BG nanofibers shows a similar or gently faster growth rate than the positive control after being cultured for 1, 3, and 5 days, implying that the nanofibers do not present negative effect on the proliferation of the BMCSs. Similar results were also confirmed by using CCK8 assay (FigureS4). SEM images show that BMSCs were spread on all types of CaTiO3:Yb3+,Er3+@BG nanofibers after being cultured for 3 days (Figure S5). All cells maintained the normal morphology and were spread actively on the nanofibrous surface with numerous cytoplasmic extensions, confirming the typical osteoblast cellular growth. Such phenomenon has also been reported in previous literature,

Figure 4. Proliferation of bone marrow-derived mesenchymal stem cells (BMSCs) on CaTiO3:Yb3+,Er3+@BG nanofibers with water/ TEOS ratio (X) of 2, 4, and 6.

which has proved that the lanthanide based UC PL materials exhibit no or low cytotoxicity in vivo.31 This in vitro study suggests that the CaTiO3:Yb3+,Er3+@BG nanofibers are of good cytocompatibility and can be a promising candidate for bone scaffolds. 3.3. UC Photoluminescence Responses during Simulated Body Fluid (SBF) Mineralization. The CaTiO3:Yb3+,Er3+@BG nanofibers synthesized at different water/ TEOS ratio values were immersed in SBF to induce the mineralization for different times. The apatite mineral is formed and remarkably enhanced on the surface of all nanofibers from 3 to 72 h. At 72 h, the nanofibers (water/TEOS ratio = 2) are heavily covered by flocculent-like bone-like apatite. When water/TEOS ratio is increased from 2 to 6, the apatite coverage on the fiber surface becomes significantly weakened (Figure S6). The TEM examination was carried out to uncover the apatite layer formed at the fiber surface after being immersed in SBF for 72 h. With the increased X from 2 to 6, the apatite layer formed on BG fibers remains its flocculent-like structure D

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Figure 5. UC PL emission spectra of CaTiO3:Yb3+,Er3+@BG nanofibers with water/TEOS ratio (X) = (a) 2, (b) 4, and (c) 6 immersed in SBF solution for 0−120 h. Insets show the confocal microscopy images of the nanofibers after soaking for 3, 24, and 72 h. (d) Variation of UC PL emission intensity of all nanofibers during soaking in SBF.

Figure 6. (a−c) UC luminscence emission spectra variation and (d−f) intensity variation of 660 nm emission (the intensity ratio between after and before demineralization) of mineralized CaTiO3:Yb3+,Er3+@BG nanofibers when immersed in an acidic aqueous solution at 37 °C (X is the water/ TEOS ratio).

mineralization, and nearly completely quenched when mineralized for 120 h. When the nanofibers with water/TEOS ratio of 2 were soaked in SBF, the intensity of the emission spectra was rapidly decreased (Figure 5). The confocal microscopy images show that, when immersed in SBF for 3 h, the red emission pattern of the nanofibers becomes dark. With soaking prolonged to 24 h, the red emission is hardly observed. It is completely shielded when the soaking time is 72 h. At the increased water/TEOS ratio (4 and 6), the quenching of red emission is slower. Namely, during the mineralization process, the UC PL quenching from the nanofibers shows a quite direct relationship with the bone-like apatite formation on the

characteristics, and becomes thinner dramatically. The selected area electron diffraction (SAED) patterns of the three samples present clear diffraction rings, confirming the polycrystalline nature of apatite coverage formed (Figure S7). The HCA mineralization for bioactive glasses in SBF is highly related to ion exchange ability. TEOS hydrolysis process is vitally important to the integrity of Si−O−Si network for BG materials. With increased water/TEOS ratio, the integrity of Si−O−Si network is improved, and consequently the mineralization ability of BG materials in SBF is weakened.32 More interestingly, the UC PL emission of CaTiO3:Yb3+,Er3+@ BG nanofibers at ∼660 nm is significantly quenched during E

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the nanofibers were weakened with marginal degree (Figure 7d). The relative UC PL intensity variation at ∼660 nm indicates there is no distinguishable variation of UC PL intensity with the decreased fiber diameter due to the degradation (Figure 7e). However, as demonstrated above, the UC PL emission of CaTiO3:Yb3+,Er3+@BG nanofibers at ∼660 nm was significantly quenched during mineralization in SBF, and quenched completely when mineralized for 120 h. Therefore, the loss of emission was mainly as the result of mineralization on the surface of the fibers. It should be noted is that the degradation effect of the fibers does assist the optical quenching phenomenon. 3.6. The Mechanism Analysis. The UC PL quenching and recovery during mineralization and demineralization may be understood based on different mechanisms (Scheme 1).When CaTiO3:Yb3+,Er3+@BG nanofibers are immersed in SBF, a large quantity of flocculent-like apatite, consisting of Ca2+, PO43−, CO32−, and OH− ions, is formed on the surface of the nanofibers. The PL emission of rare earth ions can be quenched in the environments where high phonon frequencies are present, such as a vibration frequency near 3450 cm−1 of −OH groups.26 Furthermore, with the growth of mineral layer, a screening effect may also be induced by absorbing (or scattering) the excitation light and the UC emission. Therefore, the PL emission becomes invisible when the quenching effect is enhanced to reach a certain degree due to continuous mineralization. Photoluminescent properties of Eu3+ ions were used to monitor the apatite formation on CaB2O4 powder previously.33 It was found that the relationship between the luminescence intensity and the soaking time was difficult to determine because the excitation light usually only penetrates to a very thin depth on the surface. In contrast, in our study, the apatite formation process on the CaTiO3:Yb3+,Er3+@BG nanofibers can be qualitatively monitored via the photoluminescence evolution. The nanofibers with X = 2 present the strongest ion exchange ability and thus the apatite forming ability. Hence, this type of nanofibers shows the most rapid quenching of PL emission. With enhanced hydrolysis degree, the nanofibers at water/TEOS ratio of 4 and 6 shows relatively weakened optical quenching effect. In contrast, during the demineralization process in an acidic aqueous solution, the apatite formed on the fiber surface is degraded, and the UC PL intensity is remarkably enhanced. When the apatite layer is fully dissolved, the PL emission of all nanofibers recovers back to their original spectral intensity. One notable fact is that no distinguishable difference between the UC PL recovery rates for all types of CaTiO3:Yb3+,Er3+@BG nanofibers is observed. Under the same acidic condition, the apatite layer formed on all of the nanofibers is dissolved at a similar rate. In comparison, during the immersion in SBF, the mineralization rate is remarkably varied for the fibers with different apatite forming ability. Therefore, CaTiO3:Yb3+,Er3+@ BG nanofibers present dramatically different PL quenching phenomena during mineralization, but show a similar PL recovery trend during the demineralization process.

nanofibers synthesized at different water/TEOS ratios. The nanofibers with water/TEOS ratio of 2 present the fastest optical quenching effect and strongest HCA forming ability (Figures 5 and S5). One notable fact is that, although the UC PL quenching phenomenon and mineralization trend are well corresponding to each other, the relationship between such two processes remains at a semiquantitative level. 3.4. UC Photoluminescence Responses during Apatite Degradation. To further uncover the PL evolution during demineralization, CaTiO3:Yb3+,Er3+@BG nanofibers, mineralized in SBF for 2 days, were immersed in an acidic aqueous solution (pH = 3) at 37 °C. After soaking for 24 and 72 h, the apatite formed at the surface of composite nanofiber dissolves rapidly due to the reaction between hydroxyl groups of apatite and hydrogen ions. The surface of all three nanofibers become smooth and the apatite layer formed is degraded in a completed fashion after 72 h in the acidic aqueous media (Figure S8). The PL spectra of the nanofibers soaked in the acidic aqueous solution for different periods are generally enhanced (Figure 6a−c). The relative PL intensity variation of the nanofibers, which is the ratio between the PL emission at 660 nm after and before immersion (Figure 6d−f), confirms the gradual recovering of UC PL intensity during demineralization. Further, the recovering of UC PL intensity presents a similar trend. No distinguishable difference between the PL enhancement rates of three types of CaTiO3:Yb3+,Er3+@BG nanofibers is observed, which is a significantly different scenario comparing to the UC PL quenching process during apatite formation. 3.5. UC Photoluminescence Responses during Fiber Degradation. To verify the PL quenching effect during mineralization, CaTiO3:Yb3+,Er3+@BG nanofibers (X = 6) were immersed in deionized water and degrade for different periods (0 h-24 h) at 37 °C. After soaking, the mean diameter of the composite nanofiber decreased from ∼440 nm to ∼230 nm (Figures 7 and S9) due to the breaking-down of Si−O−Si network by water molecules. In consequence, the PL spectra of

4. CONCLUSIONS In summary, to realize a “smart” bone scaffold, BG nanofibers decorated with CaTiO3:Yb3+,Er3+UC PL nanoparticles were used to show that it is possible to optically monitor the mineralization and demineralization of apatite on the BG nanofibers, a well-established biomaterial for repairing bone defects. Both mineralization and demineralization of CaTi-

Figure 7. (a−c) SEM images of the CaTiO3:Yb3+,Er3+@BG nanofibers (X ratio = 6) soaked in deionized water for (a) 0, (b) 6, and (c) 24 h. (d) UC luminescence emission spectra variation during degradation in deionized water for 0−24 h. (e) Fiber mean diameter and the intensity variation of 660 nm emission (the intensity ratio between after and before degradation) of mineralized nanofibers when immersed in deionized water solution at 37 °C (X is the water/TEOS ratio). F

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Scheme 1. Mechanism of PL Responses of CaTiO3:Yb3+,Er3+@BG Nanofibers during Mineralization and Demineralization

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O3:Yb3+,Er3+@BG nanofibers have been successfully tracked in a visualized and qualitative manner by monitoring their UC PL emission. The UC PL intensity is quenched during the mineralization, and the quenching rate reflects the mineralization rate. Furthermore, the UC PL of such nanofibers can gradually recover back to its original magnitude due to the demineralization. This study may open up a new avenue to the future investigations on monitoring scaffold degradation and bone formation in vivo.

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(1) Hench, L. L.; Paschall, H. Direct chemical bond of bioactive glassceramic materials to bone and muscle. J. Biomed. Mater. Res. 1973, 7, 25−42. (2) Liu, W.; Thomopoulos, S.; Xia, Y. Electrospun nanofibers for regenerative medicine. Adv. Healthcare Mater. 2012, 1, 10−25. (3) Allo, B. A.; Lin, S.; Mequanint, K.; Rizkalla, A. S. Role of bioactive 3D hybrid fibrous scaffolds on mechanical behavior and spatiotemporal osteoblast gene expression. ACS Appl. Mater. Interfaces 2013, 5, 7574−7583. (4) Beachley, V.; Katsanevakis, E.; Zhang, N.; Wen, X. A novel method to precisely assemble loose nanofiber structures for regenerative medicine applications. Adv. Healthcare Mater. 2013, 2, 343−351. (5) Jones, J. R.; Tsigkou, O.; Coates, E. E.; Stevens, M. M.; Polak, J. M.; Hench, L. L. Extracellular matrix formation and mineralization on a phosphate-free porous bioactive glass scaffold using primary human osteoblast (HOB) cells. Biomaterials 2007, 28, 1653−1663. (6) Hench, L. L. The story of Bioglass®. J. Mater. Sci.: Mater. Med. 2006, 17, 967−978. (7) Gil-Albarova, J.; Salinas, A. J.; Bueno-Lozano, A. L.; Roman, J.; Aldini-Nicolo, N.; García-Barea, A.; Giavaresi, G.; Fini, M.; Giardino, R.; Vallet-Regí, M. The in vivo behaviour of a sol−gel glass and a glassceramic during critical diaphyseal bone defects healing. Biomaterials 2005, 26, 4374−4382. (8) Kokubo, T.; Takadama, H. How useful is SBF in predicting in vivo bone bioactivity? Biomaterials 2006, 27, 2907−2915. (9) Castaño, O.; Sachot, N.; Xuriguera, E.; Engel, E.; Planell, J. A.; Park, J.-H.; Jin, G.-Z.; Kim, T.-H.; Kim, J.-H.; Kim, H.-W. Angiogenesis in bone regeneration: tailored calcium release in hybrid fibrous scaffolds. ACS Appl. Mater. Interfaces 2014, 6, 7512−7522. (10) Lin, S.; Ionescu, C.; Pike, K. J.; Smith, M. E.; Jones, J. R. Nanostructure evolution and calcium distribution in sol−gel derived bioactive glass. J. Mater. Chem. 2009, 19, 1276−1282. (11) Jones, J. R. Review of bioactive glass: from Hench to hybrids. Acta Biomater. 2013, 9, 4457−4486.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b00290. EDS data; SEM and TEM images; confocal microscopy images; cell proliferation data (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Author Contributions ⊥

X.L. and Y.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (51103128), Provincial Natural Science Foundation of Zhejiang Province (LY15E020005 and LZ16E030001), and “Qianjiang Talent” program of Zhejiang province (2013R10037). G

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DOI: 10.1021/acs.langmuir.6b00290 Langmuir XXXX, XXX, XXX−XXX