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Osteogenic commitment and differentiation of human mesenchymal stem cells by low‐intensity pulsed ultrasound stimulation

Low‐intensity pulsed ultrasound (LIPUS) as an adjuvant therapy in in vitro and in vivo bone engineering has proven to be extremely useful. The present study aimed at investigating the effect of 30 mW/cm2 LIPUS stimulation on commercially available human mesenchymal stem cells (hMSCs) cultured in basal or osteogenic medium at different experimental time points (7, 14, 21 days). The hypothesis was that LIPUS would improve the osteogenic differentiation of hMSC and guarantying the maintenance of osteogenic committed fraction, as demonstrated by cell vitality and proteomic analysis. LIPUS stimulation (a) regulated the balance between osteoblast commitment and differentiation by specific networks (activations of RhoA/ROCK signaling and upregulation of Ribosome constituent/Protein metabolic process, Glycolysis/Gluconeogenesis, RNA metabolic process/Splicing and Tubulins); (b) allowed the maintenance of a few percentage of osteoblast precursors (21 days CD73+/CD90+: 6%; OCT‐3/4+/NANOG+/SOX2+: 10%); (c) induced the activation of osteogenic specific pathways shown by gene expression (early: ALPL, COL1A1, late: RUNX2, BGLAP, MAPK1/6) and related protein release (COL1a1, OPN, OC), in particular in the presence of osteogenic soluble factors able to mimic bone microenvironment. To summarize, LIPUS might be able to improve the osteogenic commitment of hMSCs in vitro, and, at the same time, enhance their osteogenic differentiation.

The way stem cells participate in tissue generation, maintenance, and repair, saving themselves from depletion and protecting the host from over-exuberant stem-cell proliferation, is regulated by a specific anatomic entity defined by Schofield in 1978 and described by Scadden in 2006 as stem-cell niche (Scadden, 2006;Schofield, 1978). Stem-cells receive specific signals from physical and biochemical clues of this niche environment by direct contact with basal membrane proteins and by binding with soluble mediators like growth factors and cytokines, or insoluble extracellular matrix proteins, acting like paracrine regulators of stem-cell function (Griffin, Butler, Seifalian, & Kalaskar, 2015).
Alternatively, adult stem or progenitor cells, such as MSCs, represent a promising resource for new bone formation via cellbased tissue engineering. These cells can be isolated from various adult tissues and are able to promote tissue repair through trophic effects on other cell types and immune-regulatory activities (Griffin et al., 2015). Until now, tissue engineering approaches were characterized by the use of hMSCs isolated by different tissues, alone or in combination with a biomimetic scaffold and/or a physical approach (such as ultrasound, pulsed electromagnetic fields or others), in order to accelerate bone restoration (Baker, Sohn, & Tuan, 2015).
Therefore, it is fundamental to understand how to control and guide the fate of stem cells in culture by providing external stimuli, that is using specific surface topography and chemistry with the help of biomimetic biomaterials, or delivering chemical, and genetic clues in the form of growth factors and cytokines (Griffin et al., 2015;Papadimitropoulos et al., 2014). The loss of MSC properties in vitro, probably due to the absence of the niche environment, might limit the utility of MSCs in cell-based regenerative medicine (Wang, Cheung, et al., 2014). To avoid this phenomenon, threedimensional environments (i.e., bioreactors) have been proposed to expand MSC, but these approaches require an initial phase of MSC growth on plastic, which is inevitably associated with the selection of adherent cellular fractions, already depleted of their earlier progenitors properties (Di Maggio et al., 2012).
Recently, it has been showed that low intensity pulsed ultrasound (LIPUS) is able to positively influence the maintenance of MSC stemness (Kusuyama et al., 2015). LIPUS stimulation at 30 mW/cm 2 is an established, widely used and FDA-approved therapeutic treatment for accelerating bone healing in fractures and in delayed or established nonunions (Angle, Sena, Sumner, & Virdi, 2011;Augat, Simon, Liedert, & Claes, 2005;El-Mowafi & Mohsen, 2005). LIPUS is a form of mechanical energy that is transmitted through and into living tissues as an acoustic pressure wave with compressions. It is able to induce micromotion on the extra cellular matrix (ECM), produce stable cell cavitation and increase cell permeability with pathways activation (mechanotransduction) (Azuma et al., 2001;Claes & Willie, 2007;Rawool, Goldberg, Forsberg, Winder, & Hume, 2003). These changes lead to downstream alterations in gene expression, resulting in an acceleration of bone repair by up-regulating bone specific genes and signaling pathways such as the mitogen-activated protein kinase (MAPK) cascade (Angle et al., 2011;Appleford, Oh, Cole, Protivínský, & Ong, 2007;Claes & Willie, 2007;Fung, Cheung, Pounder, Harrison, & Leung, 2014;Hu et al., 2014;Uddin & Qin, 2013). Additional reports suggest that ultrasound alters cellular membrane properties (cellular adhesion, membrane permeability, calcium flux, and proliferation), activating signal-transduction pathways that lead to proteomes regulation (Fischell, Abbas, Grant, & Siegel, 1991;Maxwell et al., 1994). Importantly, exposure to ultrasound caused an increase in intracellular calcium in many cells types, such as fibroblast, monocyte, and mesenchymal stem cells, suggesting that the mechanical effects disrupt the normal function of the membrane, permitting leaking of calcium into the cell. Cells employ calcium as a cofactor in regulating the activity of enzymes, many of which are associated with signal-transduction pathways and relative proteomes modulation (Johns, 2002). Regarding these aspects, through mass spectrometry (MS) based proteomic methods, it was attempted to define the expression profiling of membrane proteins in MSCs undergoing osteoblast differentiation, illustrating the effects of many growth factors on this process (Foster et al., 2005;Kratchmarova, Blagoev, Haack-Sorensen, Kassem, & Mann, 2005;Salasznyk et al., 2005). A comparison of the protein expression profiles in undifferentiated and fully differentiated hMSC might reveal expressional differences that provide important clues into the mechanism underlying the process from stem cells to osteoblasts.
The overall purpose of this study was to verify whether LIPUS stimulation was able to allow the osteoblast lineage commitment of hMSCs and differentiation. To this end, LIPUS with spatially averaged and temporally averaged (SATA) intensity of 30 mW/cm 2 was applied to hMSCs cultured in basal or osteogenic medium at different experimental time points. The MSC differentiation response to shortand long-term LIPUS stimulation was analyzed in terms of (a) changes in the expression of protein markers, through MS-based quantitative proteomic technologies, in order to identify protein networks; and (b) the modulation of different stem cell markers (CD73, CD90, CD105, OCT-3/4, NANOG, SOX2) and osteogenic pathways; both activated by LIPUS (Kuhn & Tuan, 2010;Liu & Lee, 2014;Pricola, Kuhn, Haleem-Smith, Song, & Tuan, 2009;Scherzed et al., 2016;Yoon et al., 2014 Untreated OM group: cells were cultured in OM and not exposed to LIPUS treatment; (d) LIPUS OM group: cells were cultured in OM and treated with LIPUS. Culture plates were then placed on the ultrasound transducer array with a thin layer of standard ultrasound gel and exposed to LIPUS for 20 min/day for five consecutive days/week. The untreated groups were handled in the same way, but the ultrasound generator was switched off. At the end of LIPUS stimulation time, a culture plate for each group was cultured for further 7 days at the same condition in the incubator but without LIPUS exposure (indicated as "off").

| hMSC viability (WST-1 test)
WST-l colorimetric reagent (Roche Diagnostics GmbH, Manheim, Germany) was used to evaluate cell viability. Briefly, WST-1 reagent (10% vol/vol) was added to the cell monolayer in each well. After 4 hr of incubation, the formazan dye produced by viable cells was quantified spectrophotometrically at 450 nm by Bio-Rad Microplate Reader (Bio-Rad Laboratories, Hercules, CA) and results were reported as percentage of viable cells compared to Untreated MSCGM group.

| hMSC characterization
To demonstrate hMSC phenotype maintenance, CD73, CD90, and  Tables 1 and 2). The mean threshold cycle was used for the calculation of relative expression using the 2 −ΔΔCt method, against GAPDH as housekeeping gene (Livak & Schmittgen, 2001  Digested samples were then centrifuged at 14,000 g for 10 min at 4°C. The resulting supernatant, containing the peptide mixture, was extracted using the 100 µl Bond Elute OMIX C18 pipette tips (Agilent Technologies Italia SpA, Cernusco sul Naviglio-Milan, Italy). Eluted peptide mixtures were vacuum dried and reconstituted in 5% acetonitrile 0.1% formic acid for mass spectrometry analyses. Duplicates of each sample were used for the following proteomic analysis.

| Generation of the reference spectral library
Approximately, 2 µg of tryptic peptides of each sample were run for Data-Dependent Acquisition (DDA) analysis. The resulting list of protein/peptides was used for construction of the Sequential Window and solvent B (2% water and 0.1% formic acid in acetonitrile). The sample was loaded in the trap column at a flow rate of 5 µl/min for 10 min using a solvent, from loading pump, containing 2% acetonitrile and 0.1% v/v TFA in water and eluted at a flow rate of 300 nl/min using a gradient method according to which solvent B is linearly increased from 2% to 10% within 10 min, from 10% to 30% within 110 min, and then to 60% within 15 min; afterwards, phase B is further increased to 95% within 2 min. Phase B is maintained at 95% for 10 min to rinse the column. Finally, B is lowered to 2% over 2 min and the column re-     Table S1, sheet "Protein quantification").
The comparison "Untreated MSCGM versus Untreated OM" was considered as the "reference comparison" since cells in MSCGM maintained their sternness, while in OM were induced  Table S2 and Figure S1).

In the LIPUS MSCGM versus Untreated OM comparison it was
interesting to note that about 58% of modulated proteins (45 up-regulated and 30 down-regulated out of 128 differentially expressed) showed the same trend in the reference comparison.

Moreover, STRING analysis of the modulated protein dataset,
showing only high confidence functional connections (confidence score ≥0.7), highlighted two interesting aspects.
The first one concerned the down regulation in LIPUS MSCMG cells, of proteins associated to Ras homolog gene family, member A (RhoA) protein and its activity as a positive regulator of hMSCs osteogenic differentiations (Figure 4a) (Chen et al., 2011

| DISCUSSION
Different studies have been performed on the effects of LIPUS on cells attempting to describe the cellular response to biomechanical stimuli (Frairia & Berta, 2011) or transient membrane permeability (Engler, Sen, Sweeney, & Discher, 2006;Huang, Holfeld, Schaden, Orgill, & Ogawa, 2013;McBeath, Pirone, Nelson, Bhadriraju, & Chen, 2004). In the present study, the role of LIPUS stimulation (SATA intensity at 30 mW/cm 2 ) in hMSC osteogenic commitment and differentiation was investigated using an in vitro model, where LIPUS stimulation was evaluated in terms of proteome, gene expression, and proteins release modulation.
PCA and heat map clustering analyses of the obtained proteomic data, suggested that the most significant differences in protein expression profiles of MSCs are due to the specific growth medium (MSCGM or OM) rather than the exposure to LIPUS. Very few studies were focused indeed on osteogenic differentiation of hMSC (Gao, Walmsley, Cooper, & Scheven, 2016;Hu et al., 2014;Yue et al., 2013) mediated by LIPUS treatment. In this sense, our proteomics analysis revealed the existence of differences in osteoblast commitment and differentiation potential between all experimental groups. Thus, deeper proteomic data analysis showed the peculiar ability of LIPUS to positively regulate several proteins and pathways associated with stemness, such as the autophagosomes proteins SQSTM1, SRSF2, TMEM109, or Glycolysis/Gluconeogenesis proteins (CKAP4,COX4I, ALDOA) (Folmes & Terzic, 2016;Folmes et al., 2011), and to negatively regulate signaling networks associated to the differentiation of MSCs into osteoblasts, such the RhoA/ROCK pathway (Chen et al., 2011;Fang et al., 2008;Yun et al., 2016). In particular, our quantitative proteomic analysis indicated that, regardless the grown medium, inhibited cell proliferation, thus resulting in significantly lower dsDNA content at each experimental time point, which was likely due to the start of the differentiation process (Wang, Peng, et al., 2014).