Osteopontin

Total cellular RNA was extracted from the cultures by using the RNeasy Protect Mini Kit with an on-column RNase-free DNase treatment (Qiagen, Hilden, Germany) [16 (link),37 (link),46 (link)]. RNA was eluted in 30 μl RNase-free water. Reverse transcription was carried out with 8 μl of eluate by using the 1^st Strand cDNA Synthesis kit for RT-PCR (AMV) (Roche Applied Science). An aliquot of the cDNA product (2 μl) was amplified with real-time PCR by using the Brilliant SYBR Green QPCR Master Mix (Stratagene, Agilent Technologies, Waldbronn, Germany) [16 (link)] on an Mx3000P QPCR operator system (Stratagene) as follows: (95°C, 10 minutes), amplification by 40 cycles (denaturation at 95°C, 30 seconds; annealing at 55°C, 1 minute; extension at 72°C, 30 seconds), denaturation (95°C, 1 minute), and final incubation (55°C, 30 seconds). The primers (Invitrogen GmbH) used were SOX9 (chondrogenic marker) (forward 5'-ACACACAGCTCACTCGACCTTG-3'; reverse 5'-GGGAATTCTGGTTGGTCCTCT-3'), type II collagen (COL2A1) (chondrogenic marker) (forward 5'-GGACTTTTCTCCCCTCTCT-3'; reverse 5'-GACCCGAAGGTCTTACAGGA-3'), type I collagen (COL1A1) (osteogenic marker) (forward 5'-ACGTCCTGGTGAAGTTGGTC-3'; reverse 5'-ACCAGGGAAGCCTCTCTCTC-3'), type X collagen (COL10A1) (marker of hypertrophy) (forward 5'-CCCTCTTGTTAGTGCCAACC-3'; reverse 5'-AGATTCCAGTCCTTGGGTCA-3'), alkaline phosphatase (ALP) (osteogenic marker) (forward 5'-TGGAGCTTCAGAAGCTCAACACCA-3'; reverse 5'-ATCTCGTTGTCTGAGTACCAGTCC-3'), matrix metalloproteinase 13 (MMP13) (marker of terminal differentiation) (forward 5'-AATTTTCACTTTTGGCAATGA-3'; reverse 5'-CAAATAATTTATGAAAAAGGGATGC-3'), osteopontin (OP) (osteogenic marker) (forward 5'-ACGCCGACCAAGGAAAACTC-3'; reverse 5'-GTCCATAAACCACACTATCACCTCG-3'), runt-related transcription factor 2 (RUNX2) (osteogenic marker) (forward 5'-GCAGTTCCCAAGCATTTCAT-3'; reverse 5'-CACTCTGGCTTTGGGAAGAG-3'), β-catenin (mediator of the Wnt signaling pathway for osteoblast lineage differentiation) (forward 5'-CAAGTGGGTGGTATAGAGG-3'; reverse 5'-GCGGGACAAAGGGCAAGA-3'), parathyroid hormone-related protein (PTHrP) (hypertrophy-associated gene) (forward 5'-CGACGACACACGCACTTGAAAC-3'; reverse 5'-CGACGCTCCACTGCTGAACC-3'), lipoprotein lipase (LPL) (adipogenic marker) (forward 5'-GAGATTTCTCTGTATGGCACC-3'; reverse 5'-CTGCAAATGAGACACTTTCTC-3'), peroxisome proliferator-activated receptor gamma 2 (PPARG2) (adipogenic marker) (forward 5'-GCTGTTATGGGTGAAACTCTG-3'; reverse 5'-ATAAGGTGGAGATGCAGGCTC-3'), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (housekeeping gene and internal control) (forward 5'-GAAGGTGAAGGTCGGAGTC-3'; reverse 5'-GAAGATGGTGATGGGATTTC-3') (all 150 nM final concentration) [13 (link),15 (link),16 (link),46 (link)-49 (link)]. Control conditions included reactions using water and non-reverse-transcribed mRNA. Specificity of the products was confirmed by melting curve analysis and agarose gel electrophoresis. The threshold cycle (Ct) value for each gene of interest was measured for each amplified sample by using the MxPro QPCR software (Stratagene), and values were normalized to GAPDH expression by using the 2^-ΔΔCt method, as previously described [16 (link)].

Western blotting was performed as previously described [24] (link), [25] (link), [27] (link), [28] (link), [29] (link), [30] (link), [31] (link), [55] (link), [61] (link). Briefly, cells were collected and lysed in Laemmli buffer. Cleared total cell lysate was denatured by boiling and loaded onto a 4–20% gradient SDS–PAGE. After electrophoretic separation, proteins were transferred to an Immobilon-P membrane. Membrane was blocked with SuperBlock Blocking Buffer, and probed with the primary antibody, anti-osteopontin, anti-osteocalcin, and anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with a secondary antibody conjugated with horseradish peroxidase. The proteins of interest were detected by using SuperSignal West Pico Chemiluminescent Substrate kit.

The osteoblastic marker osteopontin (Opn) gene expression was evaluated on day 10 by RT-qPCR. The total RNA was extracted using the SV Total RNA Isolation System kit (Promega, Fitchburg, WI, USA) and reverse transcription reaction was carried out to synthesize the complementary DNA (cDNA) using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). For RT-qPCR, the SYBR Green system, and primers (Table 1) were used in the QuantStudio™ 7 Flex System device (Applied Biosystems, Waltham, MA, USA). The reactions were done (n = 4) and the data analysed using the cycle threshold value (Ct). The expression of the constitutive gene eukaryotic translation initiation factor 2B, subunit 1 alpha (Eif2b1) was evaluated, and the 2^-ddCt method was used to compare the gene expression of the experimental groups [49 (link),50 (link)].

The hWJ-MSC ability to differentiate toward the osteogenic fate was evaluated in the presence of CB for the entire period of the induction. Cells were seeded at 4000 cells/cm² in 24-well or in 6-well plates for Alizarin Red S staining and gene expression analysis, respectively. For both evaluations, when cells reached 70% confluence, they were cultured in technical duplicate with standard (not induced) or StemPro Osteogenesis Differentiation medium (Thermo-Fisher Scientific, Waltham, MA, USA). For Alizarin Red S staining experiments, hWJ-MSCs were treated with DMSO or CB at 0.1, 1, and 3 μM and untreated cells were used as control (CTR); for Alizarin Red S staining and gene expression analysis, hWJ-MSCs were treated only with CB 1 μM and results were compared to those of DMSO-treated cells; both analyses were performed at 3, 7, 14, and 21 days from the beginning of the differentiation protocol. In all experiments, media were changed twice a week for 21 days.
The quantitative and qualitative assessment of osteogenic differentiation was performed by staining calcium deposits with Alizarin Red S solution after 21 days from the beginning of the differentiation protocol. Briefly, cells were fixed with 4% formaldehyde solution in PBS for 15 min and stained with Alizarin Red S solution 1% w/v (Sigma-Aldrich Co., St. Louis, MO, USA) in deionized water solution (pH = 4.1) for 15 min. The exceeding dye was removed by washing wells with deionized water. Images were acquired using the Leica Labovert FS Inverted Microscope (Wetzlar, Germany) with a Leica MC170 HD Imaging System Camera (Wetzlar, Germany). To quantify the amount of calcium deposits, the Alizarin Red S dye was solubilized with 10% acetic acid (Sigma-Aldrich Co., St. Louis, MO, USA); the solution was loaded in a 96-well plate in technical triplicate to measure the absorbance at 405 nm using a spectrophotometer plate reader (Wallac 1420 Victor2 Multilabel Counter; Perkin Elmer, Waltham, MA, USA). In parallel, for the gene expression analysis, cells were lysed at the chosen experimental time points for RNA extraction (see Section 4.7). Both Alizarin Red S staining and gene expression analysis were performed in biological triplicate (n = 3).
In addition, to confirm the relation between CB, the cytoskeleton changes, and osteogenesis process, the expression and distribution of F-Actin and osteopontin, were analyzed at 7 days from the beginning of the osteogenic protocol. hWJ-MSCs were cultured in osteogenic differentiation medium and were treated with CB 1 μM for the entire induction period or for only 24 h followed by CB washout. DMSO-treated cells were used as control. Immunofluorescence analysis was conducted as reported in Section 4.11, and specific primary AB anti-osteopontin (69498; AbCam, Cambridge, UK) was diluted at 1:200 in a solution containing 2% BSA (Sigma-Aldrich Co., St. Louis, MO, USA) and 0.15% Triton X-100 in PBS.