Synergistic Effects of Orbital Shear Stress on In Vitro Growth and Osteogenic Differentiation of Human Alveolar Bone-Derived Mesenchymal Stem Cells

Cellular behavior is dependent on a variety of physical cues required for normal tissue function. In order to mimic native tissue environments, human alveolar bone-derived mesenchymal stem cells (hABMSCs) were exposed to orbital shear stress (OSS) in a low-speed orbital shaker. The synergistic effects of OSS on proliferation and differentiation of hABMSCs were investigated. In particular, we induced the osteoblastic differentiation of hABMSCs cultured in the absence of OM by exposing hABMSCs to OSS (0.86–1.51 dyne/cm2). Activation of Cx43 was associated with exposure of hABMSCs to OSS. The viability of cells stimulated for 10, 30, 60, 120, and 180 min/day increased by approximately 10% compared with that of control. The OSS groups with stimulation of 10, 30, and 60 min/day had more intense mineralized nodules compared with the control group. In quantification of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) protein, VEGF protein levels under stimulation for 10, 60, and 180 min/day and BMP-2 levels under stimulation for 60, 120, and 180 min/day were significantly different compared with those of the control. In conclusion, the results indicated that exposing hABMSCs to OSS enhanced their differentiation and maturation.


Introduction
The stem cell is a complex microenvironment combining an extracellular matrix, cell-to-cell interactions, and other factors such as growth factors, physical factors, and various cytokines. Stem cells are exposed to high Ca 2+ concentrations and a variety of autocrine, paracrine, and endocrine signals (extrinsic factors) and they are attached to the ECM through integrin receptors [1][2][3][4][5][6][7][8][9][10]. Many researchers have already reported an influence of cell growth and differentiation with the use of physical stimulators. Also, we have previously reported the in vitro osteogenic effects of cell stimulation on human alveolar bone-derived mesenchymal stem cells (hABMSCs) using a simple rocking culture method [11].
Thereby, we ascertained that the shear stress on hABMSCs could significantly enhance cell migration, proliferation, and differentiation. Our previous study motivated us to identify other methods for simple cell stimulation.
Thus, we designed orbital shear stress (OSS), which considered another possible cell stimulation method with the concept that flow patterns within intraoral fluid in the mouth are circular. There have been several studies of the effects of OSS on cellular behaviors [2][3][4][5][6][7][8][9][10][11][12]. Steady laminar flow can induce the expression of many genes and proteins in stem cells. The physical forces have profound effects on the cytoskeleton and extracellular matrix. These cellular components are essential in maintaining the integrity of stem cells. In particular, gap junctions are membrane channels that mediate  the cell-to-cell movement of ions and small metabolites [5,6,13]. Some studies have reported that the Cx43 which is involved in gap junction channel activity in cells, including stem cells, might be induced by OSS to regulate cell growth and differentiation [6][7][8][9][10]. It has been suggested that the mode of cell-cell communication might be of particular importance in the skeleton, where various signals mediate gap junction communication and connexin biology in the bone [8-10, 14, 15]. Above all, one mechanism of cell-cell interaction is direct cell-cell communication via gap junctions, which are transmembrane channels that allow for the continuity of cytoplasm between communicating cells [13][14][15]. Cellular signaling occurs through distinct events: binding of stimuli secreted from neighboring cells or cell junctions and release in response to stimuli. Such signals affect cellular migration, growth, and differentiation [16][17][18]. The purpose of our study, therefore, was to investigate the synergistic effects of OSS on in vitro growth and osteogenic differentiation of hABMSCs for tissue engineering applications.  Figure 2: Cell metabolic viability as optical density of hABMSCs measured using WST-1 (a). DNA concentration as percent of initial hABMSCs measured using CyQUANT Cell Proliferation Assay Kit (b) ( = 3). Overhead brackets with asterisks indicate significant differences between groups.

Materials and Methods
College of Dentistry, Seoul National University. hABMSCs were placed in 35 mm culture dishes at a density of 1.0 × 10 4 cells/cm 2 and cultured for 5 and 10 days. Cells were cultured in -minimum essential medium ( -MEM) containing 10% fetal bovine serum (FBS, Welgene Inc., Republic of Korea) and 10 mM ascorbic acid (L-ascorbic acid) and antibiotics (Antibiotic-Antimycotic solution, Gibco) at 37 ∘ C in a humidified atmosphere of 5% CO 2 (Steri-Cycle 370 Incubator, Thermo Fisher Scientific, USA). The cells were then incubated with osteogenic medium (100 nM dexamethasone, 50 g/mL of ascorbic acid, and 10 nM of -glycerophosphate; Sigma) for 10 days. The induction culture medium was  Healing rate (%) P > 0.05 (B) * P = 0.0189 * P < 0.05 * P = 0.0351 Figure 3: In vitro cell migration as representative optical microscopic images with OSS groups compared to static culture (A), indicating that stimulation groups exposed at 10, 30, and 60 min/day were significantly different ( * < 0.05) among groups (B) ( = 3).
changed every second or third day. The proliferation and osteogenic differentiation of the cells were examined after exposure to each OSS.

Stimulation Treatment of OSS and Experimental Device.
OSS was applied to confluent cell cultures using a low-speed orbital shaker (Benchmark Scientific, USA). The OSS was calculated using the following equation (1) [2]: where is shear stress, is the orbital radius of rotation of the shaker, is the density of the culture medium, is the viscosity of the medium, and is the frequency of rotation [2]. In this study, we calculated the values of shear stress at temporal points as shown in Figure 1 as 5, 10, 20, 30, and 40 rpm (revolutions per minute). The equation expresses constant magnitude of shear [17][18][19]. Figure 1 indicates temporal points for calculating values of OSS. The Reynolds number was calculated as 2 /V, where is the rotational speed of the orbital shaker, is the radius of rotation of the orbital shaker (17.5 mm), and V is the kinematic viscosity (1.012×10 −6 m 2 /s). hABMSCs were exposed to OSS (0.86-1.51 dyne/cm 2 ) with plate on the orbital shaker (Reynolds number of 121). There were six treatment groups, stimulated for 10, 30, 60, 120, and 180 min/day.

Cell Viability, DNA Analysis, and
In Vitro Cell Migration Assay. hABMSC proliferation was measured by WST-1 assay (EZ-Cytox Cell Viability Assay Kit, Daeillab Service Co., Ltd.). The formazan dye produced by viable cells was quantified by a multiwell spectrophotometer (Victor 3, Perkin Elmer, USA), measuring the absorbance of the dye solution at 460 nm. DNA concentration was quantified by fluorometry using the CyQUANT Cell Proliferation Assay Kit (Invitrogen), and the Fluorescence was measured using a Cytofluor II fluorescence multiwell plate reader with excitation of 485 nm and emission of 530 nm. In vitro cell migration was assessed by the CytoSelect Wound Healing Assay according to the manufacturer's protocols. Wound closure was measured by microscopy for up to 72 h, and photographs were taken. Cells were cultured with or without OSS, and cell morphology was observed by phase-contrast microscopy (Nikon TS100, Japan). hABMSCs were stimulated with exposure to OSS for 72 h, and the control was not exposed to OSS.

Measurement of Mineralized Nodule Formation.
All cells except control cells were exposed to OSS for 10 days. Nodule formation was checked routinely by phase contrast microscopy. The presence of mineralized nodules (calcium deposition) was determined by staining with Alizarin red, as described [20]. The ethanol-fixed cells and matrix were stained for 1 h with 40 mM Alizarin red-S (pH 4.2) and extensively rinsed with water. After photography, the bound stain was eluted with 10% (wt/vol) cetylpyridinium chloride, and the Alizarin red staining in the samples was quantified by measuring absorbance at 544 nm (Victor 3, Perkin Elmer, USA). Cells were fixed with 4% (wt/vol) formaldehyde in PBS for 15 min. And the cells were incubated in 5% (wt/vol) silver nitrate (Sigma-Aldrich, USA) for 1 h under ultraviolet light condition, followed by incubation in 5% (wt/vol) sodium thiosulfate (Sigma-Aldrich, USA) for 5 min. Last, the wells were rinsed with distilled water twice and air-dried, and mineralization images were captured using an optical microscope.

Fluorescence Microscopy and Confocal Laser Scanning
Analysis. Cells were washed in phosphate-buffered saline (PBS, Sigma-Aldrich, Milwaukee, WI, USA), fixed in a 4% paraformaldehyde solution (Sigma-Aldrich, Milwaukee, WI, USA) for 20 min, and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, WI, Milwaukee, USA) for 15 min. Cells were incubated with TRITC-conjugated phalloidin, antivinculin, its secondary antibody (Millipore Cat. no. AP124F), and DAPI (Millipore, Billerica, MA, USA) for 1 h to stain actin filaments, focal contacts, and nuclei, respectively. Cytoskeleton organization was visualized using an actin cytoskeleton and focal adhesion staining kit (FAK100; Millipore, Billerica, MA) according to the manufacturer's instruction. Cells were mounted in glycerol/buffer on a glass slide after extensive washing with PBS. Images of labeled cells were obtained using a fluorescence image restoration microscope (Applied Precision, USA).
To investigate specific proteins, cells were incubated with TRITC-conjugated phalloidin, antiosteocalcin, its secondary antibody (Cat. no. AB10911, Millipore), and DAPI (Millipore, Billerica, MA, USA) for 1 h to stain actin filaments, focal contracts, and nuclei, respectively. In addition, the major intermediate filament protein of the cells was visualized using an anti-Cx43 antibody (Cat. no. AB1728, Millipore) according to ALP activity (ng/hour/protein) * P < 0.05 Figure 6: ALP activity cultured in different types of hABMSCs exposed with OSS for 7 days. The groups exposed at 10, 30, 60, and 120 min/day were significantly different among groups ( = 3).
the manufacturer's protocol. Immunostaining with primary antibodies was used as a control, and at least two independent stainings were performed. Cells were mounted in glycerol/buffer on a glass slide after extensive washing with PBS. Images of labeled cells were obtained by a Confocal Laser Scanning Microscope (Carl Zeiss, LSM710).

Statistical Analysis.
Statistical analysis was carried out using the SAS Statistical Analysis System for Windows v9.3 (SAS Institute, Inc., Cary, NC, USA). Statistical significance between control and treatment groups was compared withtest, two-way ANOVA, and Duncan's multiple range tests at * < 0.05. The data are reported as the mean ± standard deviation.

Cell Viability and Growth Are Enhanced by OSS.
Cell metabolic viability of hABMSCs was measured using optical density and WST-1 according to Figure 1. The cell viability of the 40 rpm group when exposed at 10 min/day increased more than 10% over those of 10 and 20 rpm groups (Figure 2(a)). DNA concentration (Figure 2(b)) as a percentage of initial hABMSC measured using the CyQuant cell proliferation with OSS stimulation (40 rpm). Specifically, we observed that 40 rpm and OSS stimulation of 10, 30, and 60 min/day induced greater cell metabolic activity. OSS groups had higher cell metabolic viability than control group. We have indicated that OSS in short term stimulated the cell growth and proliferation in vitro whereas hABMSCs proliferation was associated with decrease with exposure to laminar shear stress for long term. The in vitro hABMSCs migration result was shown in Figure 3. The in vitro cell migration shown in the optical microscopic images (A) showed that the difference between OSS and static culture groups was significant, and the OSS groups exposed at 10, 30, and 60 min/day also showed significant differences ( * < 0.05) (B). Based on the cell growth, migration assay, and DNA proliferation, hABMSCs proliferated significantly (about 20%) under OSS condition of 10 and 30 min/day when compared with that of control. We could consider that OSS does produce laminar shear stress on the cell-seeded culture dish, which is related to increased proliferation. Figure 4 showed representative confocal images of hABMSCs cultured for 5 days in static conditions (a1-d1) or at 10 min/day (a2-d2), 30 min/day (a3-d3), 60 min/day (a4-d4), 120 min/day (a5-d5), and 180 min/day (a6-d6) by OSS in the absence of OM; cell nuclei (a1-a6), actin filaments (b1-b6), gap junctions (Cx43, c1-c6), and merged images (d1-d6) of the fluorescence stains. The Cx43 indicated more intense staining in OSS groups in the absence of OM compared with the control. Gap junction communication is important in bone cells [21], where the channels are involved in mechanical transmission [22][23][24], induction of cytokines in osteoblasts [25], and coordination of hormonal responses [26,27]. In osteoblast-like cells in vitro, Cx43 is the dominant connexin subtype and likely plays an important role in normal skeletal development [28][29][30]. Many studies have demonstrated a mutual relationship between cell growth and the expression of tissue-specific genes during mineralization [31][32][33]. In this respect, we could assure that gap junction (Cx43) was accelerated by OSS compared with that of control in the absence of OM. Figure 5 presented representative optical fluorescence microscopy images of hABMSCs cultured for 5 days in static conditions (a1-d1) or  Mineralized nodule (absorbance of 562 nm) * P < 0.05 P > 0.05 Figure 9: Representative optical microscopic images of hABMSCs after Alizarin red staining treatment with static condition (a1, b1) or at 10 min/day (a2, b2), 30 min/day (a3, b3), 60 min/day (a4, b4), 120 min/day (a5, b5), and 180 min/day (a6, b6) by OSS treatment with OM on 5 days and 10 days, respectively. OSS groups of 10, 30, and 60 min/day were intense compared to those of control. Representative microscopic images of hABMSCs after Von-Kossa staining with static condition (c1, d1) or at 10 min/day (c2, d2), 30 min/day (c3, d3), 60 min/day (c4, d4), 120 min/day (c5, d5), and 180 min/day (c6, d6) by OSS treatment with OM. Mineralized nodule as optical density measured after destaining treatment (C). OSS induction exposed at 10, 30, and 120 min/day groups was significantly different ( * < 0.05) among groups ( = 3, bar = 1 mm).

Analysis of Gap Junction (Cx43) and OCN with OM.
Gap junction intercellular communication is the most direct way of achieving such signaling, and gap junction communication through connexin-mediated junctions, in particular connexin 43 (Cx43), plays a major role bone development [45]. Given the important role of Cx43 in controlling development and differentiation, especially in bone cells, controlling the expression of Cx43 may provide control over cell-to-cell communication and may help overcome some of the challenges in craniofacial tissue engineering [45][46][47].
Connexins play a major role in response to many mechanical, electrical, chemical, and hormonal stimuli and help regulate cell homeostasis as well as calcium signaling and differentiation [26,48,49]. Therefore, controlling fluid flow like OSS can also potentially induce the opening of Cx43 hemichannels in osteocytes and other bone cells allowing for enhanced cell-cell communication and bone formation [50][51][52]. The major premise of functional tissue engineering is to provide physical cues to cells as a means of enhancing proliferation, differentiation, and tissue formation. Physical stimulation of cells in monolayer enhances gap junction function [23,49,50,53,54]. Thus, the mechanisms with relation to the enhanced tissue regeneration that are subjected to physical stimulation may be gap junction mediated [55].

Quantitative Analysis of BMP-2 and VEGF Proteins.
Quantitative analysis of VEGF and BMP-2 proteins was performed with conditioned medium. VEGF protein of cells in the OSS induction group showed significant differences, as shown in Figure 12 (30 and 120 min/day; * < 0.05, 10, 60, and 180 min/day; * * < 0.001). BMP-2 protein in the OSS induction group also indicated significant differences (60, 120, and 180 min/day; * < 0.05). The interaction between VEGF and BMP-2 is dependent on the ratios of angiogenic and osteogenic factors. Osteogenic factors such as BMP-2 can stimulate osteoblasts, and VEGF can modulate vascularization [56][57][58].

Conclusions
In this study, we investigated the synergistic effects of OSS on in vitro growth and osteogenic differentiation of hABMSCs. The results indicated that OSS stimulation treatment has an important effect on the activation of mechanotransduction. Cell viability stimulated for 10, 30, and 60 min/day increased by about 10% compared with that of the control. We also found an effect of OSS on the osteogenic differentiation of hABMSCs with OM and without OM, respectively. The OSS groups with OM and without OM that underwent stimulation for 10, 30, and 60 min/day showed more intense staining compared with the control. We also quantified VEGF and BMP-2 protein expression levels after stimulation for 10, 60, and 180 min/day and found that VEGF protein levels and BMP-2 protein levels after 60, 120, and 180 min/day were significantly different from levels measured in the control. In conclusion, this study showed that exposing hABMSCs to OSS stimulation enhanced cell differentiation and maturation.