Isolation of MSCs
Investigations were conducted in accordance with institutional ethical standards, the Declaration of Helsinki and national and international guidelines. The study was approved by the China Medical University Animal Care and Use Committee. Sprague-Dawley (SD) rats, 4-6 weeks old, were euthanized by CO2. Rat femurs and tibias were dissected in a sterile hood, and bone marrow cells were collected in sterile phosphate buffered saline (PBS). BMSCs were separated by Histopaque-1083 (1.077g/ml, Sigma-Aldrich, USA) density gradient centrifugation at 400g for 20 min and placed in a flask at a density of 1×106 cells/ml. Cells were cultured at 37°C and 5% CO2. The cells will be passaged using 1x TrypLE express (Invitrogen, USA) when the confluency reached 70-80% after 5–7 days.
Isolation of EPCs
EPC preparation and culture were performed as previously described . Briefly, bone marrow mononuclear cells were washed twice in PBS, and suspended in EPC media (EGM-2 media supplemented with growth factor bullet kit (Lonza, Germany), at a density of 1x106 cells/ml. After 24 h incubation at 37°C and 5% CO2, non-adherent cells were removed and fresh medium was added. After 5–7 days, 90% confluence adhered cells were split for passage.
Flow cytometry analysis and immunofluorescence staining.
MSCs and EPCs were harvested at passage 4 for flow cytometry analysis and immunofluorescence staining. Briefly, 1x106 cells were washed with 10% FBS/PBS and centrifuged at 1000 rpm, 5 min to gather a pellet for flow cytometry analysis, MSCs were stained with FITC-conjugated rat anti-CD44, FITC-conjugated rat anti-CD90, FITC-conjugated rat anti-CD31 and FITC-conjugated rat anti-CD34 antibodies at a concentration of 2 mg/ml at 4°C. EPCs were stained with rat anti-CD31, rat anti-CD34, FITC-conjugated rat anti-CD11b and FITC-conjugated rat anti-CD133, Mouse IgG was served as negative controls. Cells were examined by flow cytometry with 10,000 events recorded for each condition. The results were analyzed by Flowjo software. For immunofluorescence staining, EPCs were co-stained with DPBS-E containing 10 µg/ml Dil-labeled acLDL and FITC-conjugated lectin (QiYue Technologies, China) for 1 h at 37°C. Preparations were then observed under fluorescence microscopy.
Experimental groups and induction culture conditions
Co-culture of MSCs and EPCs were established by using Transwells (Corning, USA). EPCs were incubated in the upper chamber, and MSCs were inoculated in the lower chamber at a density of 5×105 cells/cm2for both cell types (co-MSCs). Monolayer culture MSCs in a 6-well plate at a density of 5×105 cells/cm2 were used as control (MSCs). The cell culture media was osteogenic inducing media, with a final concentration of 10nM dexamethansone, 50ug/ml ascorbic acid and 10mM b-Glycerophosphate. Cells were incubated for 48 h at 37°C at 5% CO2. For specific inhibitors treatments, co-cultured MSCs were divided into four groups and cultured in osteogenic differentiation media. MSCs treated with 25uM SB203580(co-M+SB203580), 10uM FR180204(co-M+FR180204), or 10uM SP600125(co-M+SP600125). Inhibitor free group was used as control (co-M).
Microarray hybridization and data analysis
3 wells of co-MSCs were prepared for microarray analysis and MSCs alone were used as the control. The osteogenic differentiation will spend about 14 days, total RNA was extracted using TRIzol® Reagent according the manufacturer’s instructions (Invitrogen, USA) and genomic DNA was removed using DNase I (TaKara, Japan). An RNA-seq transcriptome library was prepared following instructions from the TruSeqTM RNA sample preparation Kit (Illumina, USA). Libraries were size selected for cDNA target fragments of 200–300 base pairs (bp) on 2% low range ultra agarose, followed by PCR amplification using Phusion DNA polymerase (NEB, USA) for 15 PCR cycles. After quantification by TBS380, the paired-end RNA-seq library was sequenced on the Illumina Novaseq 6000 (2 × 150 bp read length). The expression level of each transcript was calculated according to the FPKM method. RSEM (http://deweylab.biostat.wisc.edu/rsem/) was used to quantify gene abundance.Volcano plot were performed by R statistical package software, EdgeR was used for differential expression analysis . Gene ontology (GO) functional enrichment and KEGG pathway analyses were conducted on GOATOOLS (https://github.com/tanghaibao/Goatools) and KOBAS (http://kobas.cbi.pku.edu.cn/download.php) . Microarray data were analyzed on the online platform, Majorbio Cloud Platform (www.majorbio.com).
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was isolated at 14 days after incubation. RNA quality was determined on 2100 Bioanalyzer (Agilent, USA) and quantified using the Nanodrop, ND-2000 (NanoDrop Technologies, USA). RNA reverse transcription was performed using the SuperScriptTM kit (Invitrogen, USA) and synthesized cDNA was used to perform qRT-PCR reactions . PCR amplifications were performed using specific primers(Table 1) for analyzing the expression of osteopontin(OPN), bone sialoprotein(BSP), Runt-related transcription factor 2(Runx2), TGF-beta activated kinase binding protein 1(TAB1), MKK6 and p38. Real time-PCR conditions were; 95°C for 1 min, followed by 95°C for 30 s, then 58°C for 40 s, over 35 cycles . The experiments are performed at least three times. GAPDH served as housekeeping gene. The Ct-method (2-ΔΔCT) was adopted for gene expression calculations.
Proteins were processed with the whole cell lysis assay kit (Keygen, China). Equal protein concentrations from each cell lysate were subjected to 10% SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA),Membranes were blocked in 5% bovine serum albumin at room temperture and probed with the following antibodies at 4°C overnight: OPN, BSP, RUNX2, JNK, phosphorylated-JNK, p38, phosphorylated-p38, ERK1/2 and phosphorylated-ERK1/2(Abcam, USA) at a dilution range of 1:500-1:1000. After this, membranes were washed 3 times with TBS-T and incubated with horseradish peroxide-conjugated secondary antibodies for 60 min at room temperature. Enhanced chemiluminescence reagents (Millipore, USA) were used to observe immunoreactive proteins, and blots were quantified using Image J software, measurements were performed in triplicate.
ALP activity assay
MSCs were harvested at 7, 14, and 21 days. ALP activity was determined using an ALP assay kit (Sigma, USA) according to manufacturer’s instructions. A BCA protein assay kit (Beyotime, China) was used to calculate total protein content for ALP activity normalization，measurements were performed in triplicate.
Calcium mineral deposition
Calcium deposits were determined by the Osteogenesis Quantitation Kit (Sigma-Aldrich, USA). Briefly, Cells were washed with PBS twice. Then cells were fixed with 10% formaldehyde and incubating at room temperature for 15 min. Subsequently, cells were washed with distilled water for 3 times and stained with 1x Alizarin Red at room temperature for 20 min. After acid extraction, extracted solution was measured at 405 nm. A serial dilution of ARS standards was prepared for quantitative analysis according manufacturer’s instructions, measurements were performed in triplicate.
p38 siRNA interference
For p38 silencing, siRNA against p38 (JTS, China) was transfected into MSCs using Lipofectamine 3000 transfection reagent (Thermo Fisher, USA) according to manufacturer’s instructions. An siRNA negative control sequence was used as a control. Cells were incubated at 37°C for 48 h, then treated with osteogenic inducing media. Monolayer MSCs and MSCs/EPCs co-culture groups treated with p38 siRNA or control siRNA were defined as; 1) MSCs+ control siRNA group (M+NC), 2) MSCs+p38 siRNA group (M+si), 3) MSCs/EPCs co-culture+control siRNA group (co-M+NC) and 4) MSCs/EPCs+p38 siRNA group (co-M+si). Alkaline phosphatase activity assay and alizarin red staining were performed on cells. Western blotting was performed to detect OPN, BSP and RUNX2 protein levels, and blots were quantified using Image J software in triplicate.
Statistical analysis was performed using SPSS-17.0 software. All data are expressed as the mean ±SD. Statistical analyses were performed with one-way analysis of variance or Student’s t-test. The probability level at which differences were considered statistically significant was P < 0.05.