2.1 Preparation of CePO4 nanorods.
1.3539 g Ce (NO3)3·6H2O and 0.0990 g (NH4)2HPO4 were dissolved separately in 30 ml ultrapure water. The Ce (NO3)3 solution was added dropwise into the (NH4)2HPO4 solution under continuous stirring. During the dropping process, 1.0 M of HCl was utilized to keep the pH value at 0.5. After further stirring for 30 min, and the mixtures were transferred to the autoclave. The hydrothermal reaction was carried out at 180 °C for 24 h, and then was cooled to a room temperature. The final products were washed with distilled water and alcohol, and dried at 55 °C.
2.2 Preparation of CePO4/CS/GO scaffolds.
1.00 g CS was added into 25 ml CH3COOH solution, forming a CS solution after mechanical agitation for 2 h. 2.00 g CePO4 nanorods and 0.09 g GO nanosheets were added to the CS solution. The mixed solution was stirred for 2 h, and then was transfused to the mould of 24-well or 96-well plates. The samples were freeze-dried in a freezer at -60ºC for 48 h. The CePO4/CS/GO scaffolds were washed by deionized water for 6 days, and freez-dried again. Furthermore, CePO4/CS scaffolds were constructed by the same method without adding GO nanosheets.
2.3 Characterization.
The morphology and crystal structure of CePO4 nanorods was detected by transmission electron microscopy (TEM, JEOL2100, Japan) and selected area electron diffraction (SAED). Field-emission scanning electron microscopy (SEM, JSM-6380LV, Japan) and energy-dispersive spectrometry (EDS) were performed to observe the morphologies and element distributions of the nanorods and scaffolds. The phases of the samples were assayed by an X-ray diffractometer (XRD; D/max-III C, Japan) in a 2θ range of 10 ~ 70° with Cu Kα radiation. Functional groups in samples were detected by Fourier transform infrared spectroscopy (FTIR; PerkinElmer, USA) in the wavenumber range of 4000–500 cm-1. The binding energies of the Ce in the CePO4 nanorods were detected by an X-ray photoelectron spectroscopy (XPS, PHI5700 ESCA, USA). The light adsorption properties of the scaffolds were detected by a UV–vis spectrometer (UV3600, Shimadzu) in 400 ~ 1200 nm region. The Ce ion release performance from CePO4/CS porous scaffolds was investigated by soaking 0.175 g CePO4/CS/GO scaffolds in 4.5 ml ultrapure water. After released for different time, the concentrations of Ce3+ ions were determined by inductively coupled plasma/optical emission spectrometry (ICP/OES; Perkin Elmer, OPTIMA 3300 DV). To determine photothermal effects of the scaffolds, 7.80 mg samples were immersed in 100 µL ultrapure water. The temperatures were detected with time by a thermocouple thermometer upon the irradiation of NIR laser (λ = 808 nm, 4.6 W/cm2).
2.4 Cell Culture and toxicity.
MC3T3-E1, RAW264.7, and MDA-MB-231 cells were purchased from the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences (China). Human bone marrow mesenchymal stem cells (hBMSCs) were purchased from the Shanghai Bio-Chain Biological Technology Co., Ltd. (China). All processes were approved by the Animal Hospital of Shanghai Jiao Tong University. The α-minimum essential medium (α-MEM) and foetal bovine serum (FBS) were purchased from the Gibco line of Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Cells were cultured at 37 °C with 5% CO2. To examine the cell toxicity, 1 × 104 MC3T3-E1 cells/well were seeded in a 96-well plate and cultured for 24 hrs. The extraction solutions of CS, CePO4/CS, and CePO4/CS/GO scaffolds were used as the exchange solutions. The cell toxicity was examined with Cell Counting Kit-8 (CCK-8 Dojindo, Kumamoto, Japan) on days 1, 2, 3, and 4.
MDA-MB-21 cells (metastatic breast cancer cells) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS at 37 °C with 5% CO2. The cells were then mixed and cultured with the CS, CePO4/CS, and CePO4/CS/GO scaffolds (12 mm diameter, 2 mm height) in 24-well plates for 24 hrs under NIR radiation with a power density of 4.6 W cm − 2 for 3 min. The cell viability was examined with Cell Counting Kit-8 (CCK-8 Dojindo, Kumamoto, Japan) on days 1, 2, 3, and 4.
2.5 Alkaline Phosphatase Staining and Alizarin Red Staining.
MC3T3-E1 cells were cultured in a 24-well plate for 24 hrs. The CS, CePO4/CS, and CePO4/CS/GO extraction solutions were used as exchange solutions to test for cell osteogenesis. Cells were cultured in the extraction solutions for 7 and 21 days and fixed with paraformaldehyde (PFA), followed by two rinses with phosphate-buffered saline (PBS). The cells were then stained using an alkaline phosphatase (ALP) kit and alizarin red staining kit (Hongqiao, Shanghai, China) and observed under an optical microscope.
2.6 Cell Adhesion.
The hBMSCs were cultured in 24-well plates at a density of 1 × 104 cells/well with CS, CePO4/CS, and CePO4/CS/GO for 12 hrs. The cells were fixed with 2.5% glutaraldehyde for 20 min and then dehydrated in ethanol with concentration gradients of 75%, 85%, 95%, and 100% before being observed under scanning electron microscopy (SEM, Siriaon 200, FEI, Hillsboro, OR, USA).
2.7 Flow Cytometry.
The CS, CePO4/CS, and CePO4/CS/GO scaffolds were added to MDA-MB-231 cell culture medium, and the cells were exposed to radiation from a near-infrared (IR) spectrometer with a power density of 4.6 W cm-2 for 3 min. The apoptotic cells were detected using flow cytometry (annexin V/PI staining) according to the protocol provided by the manufacturer (BD Bioscience, USA). The RAW264.7 cells were then added to the CS, CePO4/CS, and CePO4/CS/GO scaffolds. The cells with the CePO4/CS/GO scaffolds were divided into two groups; one group was exposed to NIR radiation, and other was not. According to the protocol provided by the manufacturer (BD Bioscience, USA), the polarization of the macrophages was examined by flow cytometry using anti-mouse CD16/32-PE (cat. No. 553145) and anti-mouse CD206-Alexa 647 (cat. No. 565250).
2.8 Immunofluorescence.
The CS, CePO4/CS, and CePO4/CS/GO scaffold materials were added to other batches of RAW264.7 cell culture medium. The cells with CePO4/CS/GO were divided into two groups; one group was exposed to NIR radiation, and the other was not. The cells were fixed with 4% PFA fixation solution for 20 min. The CD206 (1:200) antibody and CD16/32 (1:200) antibody were employed for the immunofluorescence analysis. All antibodies were purchased from Cell Signaling Technology.
2.9 Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) and western blot.
The MC3T3-E1 cells were seeded at a concentration of 4 × 106 in extraction solutions of CS, CePO4/CS, and CePO4/CS/GO scaffolds. The total RNAs were collected with an RNeasy Mini kit (Qiagen: Valencia, CA, USA) after 5 days of culture. Reverse transcriptase (TaKaRa) was employed to transcribe the RNA to complementary DNA (cDNA). SYBR1 Premix ExTaqTM II (TaKaRa) was added to the Real-Time PCR and ABI 7500 Sequencing Detection System (AppliedBiosystems, Foster City, CA). The PCR primer was designed as follows:
GAPDH forward 5'-CACCACCATGGAGAAGGCCG-3'
and reverse 5'-ATGATGTTCTGGGCAGCCCC-3'
Runx2 forward 5'-GACTGTGGTTACCGTCATGGC-3'
and reverse 5'-ACTTGGTTTTTCATAACAGCGGA-3'
ALP forward 5'-AGAAGTTCGCTATCTGCCTTGCCT − 3'
and reverse 5'-TGGCCAAAGGGCAATAACTAGGGA − 3'
BMP-2 forward 5′-CCGCTCCACAAACGAGAAAA-3′
and reverse 5′-CAGCAAGGGGAAAAGGACAC-3′
OCN forward 5'-TAGCAGACACCATGAGGACCATCT-3'
and reverse 5'-CCTGCTTGGACATGAAGGCTTTGT-3'
The expression levels of COL1, bone morphogenetic protein (BMP-2), P-Smad1/5, RUNX2, and GAPDH were examined with CS, CePO4/CS, and CePO4/CS/GO scaffold extraction solutions. After 3 days of culture, radioimmunoprecipitation assay (RIPA) lysate containing protease inhibitor was added to the MC3T3-E1 cells, and they were left for 20 min. The cells were centrifuged at 12000 rpm for 20 min, and the supernatant was collected. The protein content of the supernatant was analysed by a bicinchoninic acid assay (BCA). The supernatant was transferred to gel pores for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a poly (vinylidene fluoride) (PVDF) membrane after electrophoresis. The membrane was stored in 5% low-fat milk for 1 hr. Primary antibody was added, and the samples were incubated at room temperature for 4 hrs. GAPDH(1:1000), P-Smad1/5 (1:1000), RUNX2 anti-bodies (1:1000), BMP-2 anti-bodies (1:1000), and COL1 anti-bodies (1:1000) were purchased from Cell Signaling Technology (Shanghai, China). Finally, horseradish peroxide (HRP) secondary antibody was added to the sample and incubated for 1 hr. Analysis was then performed with the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE).
2.10 Construction of Animal Model.
All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. All mice were provided by the Research Council of the Animal Center of the Shanghai Ninth People's Hospital (China). All mice were kept at room temperature of 20–26 °C, with a relative humidity of 70% and light intensity ≥ 200 lux (12 h light/dark) and provided with sufficient food and water. MDA-MB-231 cells (5 × 107 metastatic breast cancer cells) were subcutaneously injected at the underarm of the mice. Mice with a tumour size of 6 mm in diameter were randomly divided into 4 groups (n = 4): control, CS, CePO4/CS, CePO4/CS/GO groups. An incision was performed on the edge of the tumour, and the scaffold was installed. All mice were exposed to NIR radiation (0.55 W/cm) for 10 min. The tumour tissue temperature was monitored by a LIRTM A320 camera. All mice were exposed to NIR radiation once a day for 14 days. After 14 days, the mice were given full anaesthesia, and the immunofluorescence intensity of the metastatic breast cancer cells was measured with IVIS scanning (PerkinElmer, USA). At the end of the process, all mice were euthanized, and the tumour tissues were removed, followed by the measurement of the volume. After the quantitative measurement, the tumour tissues were fixed in 4% PFA, embedded in paraffin, and sliced, and the sliced tissues from each group were subjected to caspase-3 immunofluorescence staining.
Another animal model was initiated using Sprague-Dawley rats with defects in the skull. Female Sprague-Dawley rats of 200–250 g were selected as the model animal. An animal model with bilateral bone defects in the skull was used to measure the polarization of macrophages and the regeneration of bone. The diameter and height of the bone defects were 5 mm and 2 mm, respectively. CS, CePO4/CS, and CePO3/CS/GO scaffolds (n-5) were filled into the defects, and the scalp was sutured. The new bone formation and osteogenesis were examined by multicolour immunofluorescence. Intraperitoneal injections of the immunofluorescence markers alizarin red (30 mg/kg, Sigma-Aldrich) and calcein (30 mg/kg, Sigma-Aldrich) were given 3 and 21 days before sacrificing the rats, respectively. All rats were euthanized after 12 weeks, and the skulls with the scaffolds in the defects were dissociated. The skulls were stored in 4% phosphate formaldehyde solution for 7 days and subjected to micro-CT (mCT-80, Scanco Medical AG, Switzerland) examination. The parameters were as follows: a voltage of 90 kV, a current of 88 µA, and a pixel size of 28 µm. The 3D model was reconstructed after the scanning. The bone mineralization density (BMD) and new bone volume/tissue volume (BV/TV) were analysed by software. The undecalcified samples were fixed by bone cement, and the skulls were sectioned along the sagittal plane using a microtome (Leica, Hamburg, Germany). The immunofluorescence was measured by scanning confocal microscopy (Leica, Heidelberg, Germany; Alizarin red: 543/580–670 nm, Calcein: 488/500–550 nm). The bone mineralization rate was measured by a PC-based analysis system. The skulls were stored in 10% ethylene diamine tetraacetic acid (EDTA) for 30 days after the removal of the soft tissues. Finally, we dewaxed the sample and placed it in: xylene for 5 minutes, xylene for 5 minutes, xylene for 5 minutes, 100% ethanol for 2 minutes, 100% ethanol for 2 minutes, 100% ethanol for 2 minutes, and 90% ethanol for 2 minutes. 80% ethanol for 2 minutes; deionized water for 2 minutes. Put in the hematoxylin dye solution for 5 minutes. Eosin stained for 5 minutes. Subsequently, it was taken out and placed in 80% ethanol for 2 minutes; 90% ethanol for 2 minutes; 100% ethanol for 2 minutes; 100% ethanol for 2 minutes; xylene for 5 minutes; and 100% ethanol for 2 minutes. Hematoxylin-eosin (HE) staining and Masson staining were performed for a morphology analysis. The above samples were washed with TBST, and the dewaxed sections were taken out and placed in a blocking solution for 1 hour on a shaker. After the end of the time, the blocked sections were washed 3 times with TBST for 10 minutes each time. Then carry out a primary antibody reaction. The antibody of the target protein was prepared in advance, and the primary antibody (iNOS, 1:200; CD206, 1:200) was diluted with the primary antibody dilution according to the instructions, and dropped on the surface of the slice. The primary antibody was applied and allowed to stand overnight at 4 °C. After the end of the primary antibody, the primary antibody was collected and the membrane was washed with TBST solution for 10 minutes each time. Finally, the secondary antibody was incubated, protected from light, and allowed to stand at room temperature for 2 hours. The polarization index of the sample was detected by fluorescence microscopy.
2.11 Data Analysis.
SPSS 13.0 software (Statistical Package for the Social Science, USA) was used to analyze the data. It presented as the Means ± SD. One-way analysis of variance was used to determine statistically significant differences. P ≤ 0.05 as statistically significant.