Cell isolation, culture conditions, reagents, and antibodies
hBMSCs was provided by Cyagen Biosciences (Guangzhou, China), and it was confirmed as having the potential to induce the differentiation of osteoblasts, chondrocytes, and adipocytes. The cells were incubated in hBMSCs growth medium (Cyagen Biosciences, Guangzhou, China) at 37°C in a cell incubator containing 5% CO2 with the medium being replaced every three days. Cells were trypsinized and passaged at nearly 80–90% confluence, and only passages three to seven were cultured in the follow-up experiments.
Six to eight-week-old male C57BL/6 mice were used for the isolation of primary murine bone monocyte/macrophage precursors as described . Cells were differentiated into bone marrow-derived macrophages (BMMs) in complete Minimum essential medium Eagle Alpha modification (α-MEM, Gibco) containing 30 ng/ml of macrophage colony-stimulating factor (M-CSF) for 3–4 days at 37°C in a cell incubator with 5% CO2, and the medium was replaced every two days.
Recombinant human IL-34 (rhIL-34) was purchased from Novus Biologicals(CO, USA). Recombinant murine M-CSF and recombinant murine RANKL were purchased from Novoprotein Biotechnology (Shanghai，China). A phospho-p44/42 MAPK (ERK1/2) inhibitor (U0126; Selleck Chemicals) and a phospho-Akt inhibitor (MK-2206 2HCL; Selleck Chemicals) were used in this study. Specific antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), runt-related transcription factor 2 (RUNX2), collagen type I a 1 chain (COL1A1), extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-ERK1/2, Phospho-P38 MAPK, P38 MAPK, Phospho–NF-κB P65, NF-κB P65, Non-phospho (active) β-catenin, β-catenin, protein kinase B (AKT), Phospho-AKT, Phospho-IκBα, IκBα, Phospho-SAPK/JNK, SAPK/JNK, Nuclear factor of activated T-cells cytoplasmic 1 (NFATc1/NFAT2), C-Src were purchased from Cell Signaling Technology (Danvers, MA, USA). Specific antibodies against C-Fos and Cathepsin K were obtained from Abcam(Cambridge, United Kingdom). Specific antibodies against PGC1β and IL-34 were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).
To evaluate the effects of IL-34 on the proliferation of hBMSCs and mice bone marrow-derived macrophages (mBMMs), a 96-well plate was applied to culture the cells with a density of 5 × 103 cells/well and 8 × 103 cells/well in triplicate, individually. After a 24-h period for adhesion, different concentrations of IL-34 (0, 0.0001, 0.001, 0.01 ng/ml) were performed for 1, 3, 5, or 7 days. Afterward, the medium was changed, and 10 μl Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) buffer was added to each well, which was cultured for another four hours at 37°C. The optical density was measured on an ELX800 absorbance microplate reader (ELX808; BioTek, Winooski, VT, USA) at the wavelength of 450 nm (650 nm as reference).
For the determination of osteoblast differentiation in vitro, 12-well cell culture plates were applied to culture hBMSCs with a density of 3 × 104/cm2 at 37°C with 5% CO2. After three days, the cells were incubated in osteogenic differentiation medium (ODM; Dulbecco's modified eagle medium; 10% fetal bovine serum, 100 nM dexamethasone, 10 mM β-glycerophosphate, 1% penicillin-streptomycin and 0.05 mM L-ascorbic acid-2-phosphate) with different concentrations of IL-34 (0, 0.0001, 0.001, 0.01 ng/ml). Cells with ODM only were regarded as controls, and the medium of the treated cells was changed every three days.
Three days later, Alkaline Phosphatase (ALP) staining was performed. The cells were first fixed with 4% paraformaldehyde for 15 min, washed twice by phosphate buffer saline (PBS), and washed again but with double distilled water (ddH2O) every three minutes for three times. Then, the cells were stained with the BCIP/NBT ALP color development kit (Beyotime, Shanghai, China). In accordance with the manufacturer’s instructions, ALP activity was calculated by the ALP Assay Kit (Beyotime, Shanghai, China) after the cells were lysed by lysis buffer including with 1% Triton X-100, 20 mM Tris–HCl (pH 7.5), and 150 mM NaCl. Finally, the ALP activity was calculated at 405 nm by a microplate reader (ELX808; BioTek).
After inducting osteogenic differentiation for 12 days, Alizarin Red Staining (ARS) Kit (Cyagen, Guangzhou, China) was performed. The cells were fixed with 4% paraformaldehyde for 15 min after washed twice by PBS, and then washed with ddH2O every three minutes for three times before being stained with Alizarin Red S solution (Cyagen Biosciences, Guangzhou, China) for 30 min at room temperature. Afterward, the mineral deposition was observed and photographed using an inverted microscope with a digital camera. The stain was then absorbed by incubation with 10% cetylpyridinium chloride (MilliporeSigma, Billerica, MA, USA) for 1 h, and the solutions were plated on a 96-well plate with 200 μl/ well. The Optical density values at 560 nm of the microplate reader (ELX808; BioTek) determined the total protein concentration.
To determine the osteoclast differentiation in vitro, mBMMs were seeded into 96-well plates (8 × 103 cells/well) in triplicate with osteoclastogenic medium (complete α-MEM with 30 ng/ml M-CSF and 100 ng/ml RANKL) and various concentrations of IL-34 (0, 0.0001, 0.001, 0.01 ng/ml). Cells without treatment were regarded as controls. Cells with complete α-MEM (30 ng/ml of IL-34 and 100 ng/ml RANKL) were used to check our IL-34 worked. In the process of cultivation, the medium was changed every two days. Afterwards, cells were washed twice with PBS, fixed in 4% paraformaldehyde for 15 min, washed twice with PBS again, and stained for tartrateresistant acid phosphatase (TRAP) staining (Sigma-Aldrich, Hannover, Germany), according to the manufacturer’s instructions. TRAP-positive cells with no less than two nuclei were considered as mature osteoclasts. The number and spread area of mature osteoclasts were measured by ImageJ software (National Institutes of Health, Bethesda, MD, USA).
RNA extraction and quantitative RT-PCR
Gene expression levels of osteoclast and osteoblast formation were measured by quantitative RT-PCR (qRT-PCR). hBMSCs (3 × 104 cells/cm2) and mBMMs (1 × 105 cells/well) were seeded in six-well plates and cultured in the medium. The isolation and measurement of total cellular RNA was performed using the RNAiso reagent (Takara Bio, Kusatsu, Japan) and NanoDrop 2000. The absorbance of the samples at 260 nm was calculated in accordance with the manufacturer’s instructions (Thermo Fisher Scientific, MA, USA). Total RNA (#1 μg) was reverse transcribed into complementary DNA (cDNA) in a 20 μl reaction volume (Takara). Two μl cDNA was used as the template with Power SYBR® Green PCR Master Mix (Takara), qRT-PCR was performed in triplicate by the ABI StepOnePlus System (Thermo Fisher Scientific). As housekeeping genes, 18S or β-actin was used, and all of the reactions were repeated three times independently. Sangon Biotech (Shanghai, China) synthesized all of the primers used in this work. Primer sequences are listed in Table 1. The qRT-PCR reaction was: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, and 60°C for 30 s. The expression levels of all of the genes were evaluated by 2-△△Ct method.
Western blotting analyses
To investigate the effects of IL-34 on multiple signaling pathways, hBMSCs (3 × 104 cells /cm2) and mBMMs (5 × 105 cells/well) were seeded in six-well plates and cultured in the medium. Total lysates of cells were extracted by lysis in for 30 min on ice in a ripa buffer containing with phosphatase and protease inhibitor cocktails (Beyotime). The centrifugation to clear the lysates and collect the supernatants was set at 14,000 rpm for 10 min at 4°C. After electrophoresis, the SDS polyacrylamide gel was transferred to a polyvinylidene fluoride (PVDF) membrane (MilliporeSigma), which was then probed with the primary antibodies. Then, membranes were blocked with 10% non-fat milk and 0.1% Tween in tris-buffered saline for 1 h at room temperature. Afterward, the membranes were incubated at 4°C overnight with primary antibodies. After washing with 0.1% Tween in tris-buffered saline for three times and incubation with horseradish peroxidase-conjugated secondary antibodies (anti-mouse or anti-rabbit; Beyotime) for 1 h at room temperature, proteins were visualized using an enhanced chemiluminescent detection reagent (MilliporeSigma) and an XRS chemiluminescence detection system (Bio-Rad Laboratories, Hercules, CA, USA).
A 12-well plate was used to place hBMSCs in induction medium, and a fluorescence microscope (EU5888; Leica Camera, Wetzlar, Germany) was used for the evaluation of RUNX2 and COL1A1. At room temperature, the cells were treated with 4% paraformaldehyde. After 15 min, hBMSCs were permeabilized for five min with 0.1% Triton X-100 in PBS, and blocked in 2% bovine serum albumin for 30 min. Fixed cells were washed and incubated overnight with anti-RUNX2 (1:500; CST) and anti-COL1A1 (1:500; CST). Then the fluorescence-conjugated secondary antibody (Beyotime) was added to the cells for 60 min, and DAPI (Nanjing KeyGen Biotech, Nanjing, China) was used to stain the nuclei for five min. The images were captured by a fluorescence microscope (Leica Camera) and the fields were selected randomly.
Lentiviral packaging and cell infection
A lentiviral package was applied by Cyagen Biosciences (Guangzhou, China), including lentiviral particles to overexpress IL-34 (IL-34 overexpressed (OE) group) and lentiviral GFP particles (the negative control (NC) group). hBMSCs (3 × 104 cells /cm2) were seeded in six-well plates and cultured in the medium. When hBMSCs reached at 30%–50% confluence, cells were cultured in lentiviral particles together with 5 μg/ml polybrene in the growth medium, in accordance with the manufacturer’s instructions. The GFP fluorescence was regarded as the efficiency of transduction and the culture medium was replaced 12 h later. When at a confluence of 80%–90%, the cells were passaged and used the experiments here described. The qRT-PCR and Western blotting analyses were performed to determine the difference in osteo-specific genes and proteins.
An ELISA (Mskbio, Wuhan, China) was used to evaluate the concentrations of IL-34 in the culture medium of both OE and NC. After the cells were infected with the lentiviral package and then cultured for 24 h, the medium was collected to measure the concentrations of IL-34, according to the manufacturer’s instructions.
In vivo evaluation in animals
This study was approved by the Institutional Animal Care and Use Committee of the Second Affiliated Hospital, School of Medicine, Zhejiang University (approval number: 2018-078). In accordance with the Animal Care and Use Committee guidelines of Zhejiang province together with the laboratory animals’ care and use guidelines, we performed the animal experiments. Thirty-six male Sprague Dawley rats (eight-week-old, 200 g) from the Academy of Medical Sciences of Zhejiang Province were used as tibial bone defect or ovariectomized (OVX) models . Eighteen rats were separated equally and haphazardly into three groups (n = 6 per group): (1) Blank group: defects without treatment; (2) PBS group: defects treated with PBS (negative control group treated with PBS); and (3) IL-34 group: defects treated with local injection of IL-34 (20 μl IL-34 at 0.01 ng/ml). Briefly, rats were anesthetized by intraperitoneal injection with 0.3% pentobarbital sodium (Sigma) at 30 mg/kg body weight. After wiping the knee joint with alcohol, the closed reduction and internal fixation were performed by a 1.3-mm intramedullary fixation pin set into the tibial cavity. A 5 × 2 mm2 tibial defect was formed in all the rats nearly 7 mm from the proximal tibial growth plate by a grinding drill and penetrated through the cortex of the bone. The operation was performed on the same leg for each group. The incision was then sewed up with 4–0 absorbable sutures. Local injection with 20 μl IL-34 (0.01 ng/ml) in the tibial defect sites of the rats from IL-34 group every two days after operation, and the rats from rest groups were treated with (PBS group) or without (BLANK group) 20 μl PBS. The remaining 18 rats were randomized into three groups of six rats each: sham-surgery rats treated with PBS (BLANK group), OVX rats treated with PBS (OVX group), and OVX rats treated with IL-34 (OVX + IL-34 group). One wk after ovariectomy, IL-34 (0.1 ug/kg) or PBS was injected intraperitoneally into each OVX rat every two days. After two wk and eight wk, the rat tibial bone defects model and the OVX rat model were euthanized using excess anesthesia, respectively. No deaths or side effects occurred during the intervention. The tibia from each rat was collected and scanned by microcomputed tomography (Micro-CT). Specimens from the rat tibial bone defects model were fixed in 4% paraformaldehyde. One day later, they were decalcified by 10% ethylene diaminetetra acetic acid (EDTA, Sigma) with 0.1 M PBS (pH 7.4) for eight wk, with a mixture change per wk. After decalcification, specimens were embedded in paraffin, and sections of the proximal tibias were obtained for H&E, SO/FG, and Masson’s trichrome analysis.
Micro-CT and bone histomorphometric analyses
The tibias were analyzed by a Micro-CT (Scanco Medical, Brüttisellen, Switzerland) instrument with scanning method set at an isometric resolution of 14.8 μm with an exposure time of 300 ms. The X-ray energy settings were 70 kV and 80 μA. Trabecular bone volume per total volume (BV/TV), trabecular bone surface per bone volume (BS/BV), mean trabecular thickness (Tb.Th), mean trabecular number (Tb.N), mean trabecular separation (Tb. Sp), and mean connectivity density (Conn-Dens) were quantified to evaluate the microstructure of the tibias.
Results are expressed as means ± SD. SPSS software (v.22.0; IBM, Armonk, NY, USA) was used to perform the statistical analyses. All of the experiments were independently accomplished no less than three times. Statistical differences were evaluated by two-tailed Student’s t-test or one-way ANOVA with Bonferroni’s post hoc test. A two-way ANOVA with Bonferroni multiple comparisons post hoc test was used in the comparison of the treated groups at different time points. A P ≤ 0.05 was regarded as being significantly different.