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Research Article

Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro M.D. Aisha a, M.N.K. Nor-Ashikin a,c, A.B.R. Sharaniza c, H. Nawawi b,d, G.R.A. Froemming a,d,n a

Institute of Medical Molecular Biotechnology and Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh 47000, Selangor, Malaysia Center for Pathology Diagnostic and Research Laboratories, Clinical Training Center, Universiti Teknologi MARA, Sungai Buloh 47000, Selangor, Malaysia c DDH, Universiti Teknologi MARA, ShahAlam 40450, Selangor, Malaysia d I-PPerForM, Universiti Teknologi MARA, Selayang 47000 Selangor, Malaysia b

art ic l e i nf o

a b s t r a c t

Article history: Received 16 May 2015 Received in revised form 5 July 2015 Accepted 6 July 2015

Prolonged disuse of the musculoskeletal system is associated with reduced mechanical loading and lack of anabolic stimulus. As a form of mechanical signal, the multidirectional orbital fluid shear stress transmits anabolic signal to bone forming cells in promoting cell differentiation, metabolism and proliferation. Signals are channeled through the cytoskeleton framework, directly modifying gene and protein expression. For that reason, we aimed to study the organization of Normal Human Osteoblast (NHOst) cytoskeleton with regards to orbital fluid shear (OFS) stress. Of special interest were the consequences of cytoskeletal reorganization on NHOst metabolism, proliferation, and osteogenic functional markers. Cells stimulated at 250 RPM in a shaking incubator resulted in the rearrangement of actin and tubulin fibers after 72 h. Orbital shear stress increased NHOst mitochondrial metabolism and proliferation, simultaneously preventing apoptosis. The ratio of RANKL/OPG was reduced, suggesting that orbital shear stress has the potential to inhibit osteoclastogenesis and osteoclast activity. Increase in ALP activity and OCN protein production suggests that stimulation retained osteoblast function. Shear stress possibly generated through actin seemed to hold an anabolic response as osteoblast metabolism and functional markers were enhanced. We hypothesize that by applying orbital shear stress with suitable magnitude and duration as a non-drug anabolic treatment can help improve bone regeneration in prolonged disuse cases. & 2015 Elsevier Inc. All rights reserved.

Keywords: Normal Human Osteoblast cells Mechanical loading Orbital fluid shear stress Cytoskeleton Actin tubulin Anabolic stimulus Osteoblastogenesis Bone regeneration

1. Introduction Bone forming osteoblast and osteocyte cells are highly responsive to mechanical loading. This mechanosensing cells response by converting the stress into anabolic signal, promoting cell differentiation and bone formation. Alternately, lack in mechanical loading has been associated with rapid bone loss especially in prolonged disuse such as bed rest [1], cast immobilization due to fracture [2], in injury of the spinal cord [3] and astronauts under microgravity environment [4]. Under physiological condition, bone is continuously renewed through the balanced activity of bone forming osteoblasts (anabolic activity) and bone resorbing Abbreviations: ALP, alkaline phosphatase; NHOst, Normal Human Osteoblasts; OCN, osteocalcin; ECM, extracellular matrix; RANKL, receptor activator of nuclear factor kappa-B ligand; OPG, osteoprotegerin; CLSM, Confocal Laser Scanning Microscope; OBM, osteoblast basal medium n Corresponding author at: Institute of Medical Molecular Biotechnology (IMMB), Faculty of Medicine (Sungai Buloh Campus), Universiti Teknologi MARA, Jalan Hospital, 47000 Sungai Buloh, Selangor, Malaysia. Fax: þ 6 3 61265082. E-mail address: [email protected] (G.R.A. Froemming).

osteoclasts (catabolic activity). Tight coupling of this activity with the influence of mechanical loading is essential for the repair of micro-damages and fractures on bone. It is believed that mechanical loading in a form of orbital shear stress [5], fluid shear stress [6], vibration [7] and cell stretching [8] is a potent anabolic stimulus to enhance osteoblast metabolism [9]. More notably, the mechanosensing property in osteoblast accelerates osteogenesis process, fundamentally required to maintain bone strength. Nevertheless, the biological signals involved during bone mechanotransduction leading to enhanced osteogenesis are not fully understood. Therefore, it is necessary to investigate the underlying mechanisms of mechanotransduction in order to uncover targets for bone fracture healing. In vitro and in vivo models have shown that the mechanosensing properties of bone cells respond to loading by regulating cell differentiation and proliferation via biochemical activities. Cellular mechanosensing is initiated from the cell extracellular matrix (ECM) and into the cell interior. Mechanical load is transformed into biochemical signals which induce cell membrane protein conformational change. Alteration in the membrane protein structure modulates the cytoskeleton involved for cell metabolism,

http://dx.doi.org/10.1016/j.yexcr.2015.07.002 0014-4827/& 2015 Elsevier Inc. All rights reserved.

Please cite this article as: M.D. Aisha, et al., Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.07.002i

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differentiation, proliferation, protein synthesis, and apoptosis [10]. The integrin glycoprotein found on cell membrane acts as mechanoreceptors which spans the cell membrane under loading. Mechanical signal is then channeled into the cell through the cytoskeleton framework, subsequently modifying gene and protein expression. Signal reorganizes the cytoskeleton components namely actin, tubulin, and intermediate filaments as a mean of mechanical adaption. Tubulin fibers responsible for cell division was seen depolymerized under mechanical loading [11]. On the contrary, increase polymerization of actin fibers has been reported under mechanical stimulation. Moreover, actin polymerization was shown to accelerate osteoblast differentiation from the mesenchymal lineage [12]. During osteoblastogenesis, osteoblast cells derived from mesenchymal lineage express specific bone markers. Numerous studies have shown that mechanical loading has an anabolic effect on the regulation of osteoblastogenesis while catabolic effect on osteoclastogenesis process [13–15]. The anabolic and catabolic effect in bone occurs through the OPG/RANK/RANKL pathway. The membrane bound protein; receptor activator of nuclear factor kappa-B ligand (RANKL) produced by osteoblast is a vital factor for osteoclastogenesis. The process is initiated when the receptor activator of nuclear factor kappa-B (RANK) on the surface of osteoclast progenitor binds to RANKL, differentiating into functional osteoclast. However osteoclastogenesis can be inhibited when osteoprotegerin (OPG) a decoy receptor for RANKL binds to RANKL. Inhibiting RANK/RANKL pathway by OPG stimulates osteoblast formation through the RANKL/OPG pathway. A 48 h study of continuous mechanical stimulation on osteoblast cells showed an increase in the production of OPG, consequently decreasing the RANKL/OPG ratio [14]. It has been generally established that the ratio of RANKL to OPG manages the balance between bone resorption and formation. Although mechanical stimulation leads to reduced RANKL/OPG ratio, nevertheless the stimulation magnitude plays a role [15]. Studies from Kim et al. showed that low frequency 1 Hz stimulation increased OPG mRNA while at higher frequency 10 Hz, RANKL mRNA was reduced, in which both stimulation magnitudes resulted in decreased RANKL/OPG ratio [15]. Once osteoblast cells are fully differentiated from mesenchymal cells, multiple bone matrix proteins are being expressed. Specific bone proteins are used to monitor osteoblastic phenotype during stages in osteoblast differentiation and formation [16–19]. The expression of type I collagen (COL-1) protein and alkaline phosphatase (ALP) activity is usually observed at early stages of osteoblast differentiation particularly during in immature stage. As osteoblast mature, bone maturation proteins; osteopontin (OPN) and osteocalcin (OCN) are expressed at maximal levels [18]. Numerous studies have shown that mechanical stimulation increases bone matrix protein ALP, COL-1, OCN and OPN, required for matrix synthesis [19–21]. Enhanced expression of bone matrix protein was seen under low frequency mechanical stimulation (1–3 Hz), resulting in peak bone mass [22]. These proteins contribute to matrix synthesis and ECM production, which in turn increases bone mass. However, osteoblast response towards mechanical loading highly depends on the type of load, magnitude and duration [23]. Several in vitro studies have investigated the influence of mechanical loading on osteoblasts by applying load on the cell culture scaffold (collagen coated microcarrier) or directly onto the cells [24]. Kamkin could show that mechanical loading applied on osteoblast cells grown on collagen scaffolds increases osteoblast proliferation, ALP activity, OCN protein, and matrix production [25]. Given that osteoblast cells are highly responsive to mechanical signals, it is ultimately important to understand whether these anabolic signals are committed to the increase in

osteogenesis function and can it be applied clinically. Therefore, we aimed to study continuous mechanical stimulation through orbital fluid shear stress on Normal Human Osteoblast (NHOst) cells. Of special interest were the effects of orbital fluid shear stress on the reorganization of the cytoskeleton towards NHOst metabolism, proliferation, and on osteogenic phenotypic markers.

2. Methods 2.1. Orbital fluid shear stimulation A total of 2.5  105 NHOst cells/cm2 were seeded into a T-25 Flask with full growth medium (OBM™). After pre-incubation for 24 h, OBM medium was replaced and 20 mM of HyQ HEPES was added. Culture flask was sealed with parafilm and transferred into a shaking incubator. NHOst cells were continuously shaken for 72 h at 250 RPM at 37 °C to induce orbital fluid shear stress [6]. Unstimulated NHOst cells grown at 37 °C with 5% CO2 in a waterjacketed incubator with full growth medium for 3 days served as a control. 2.2. Change in NHOst cytoskeleton NHOst cells were cultured on the 0.025 g Cytodex™ 3 microcarrier collagen beads (GE Healthcare) in a petri dish at density of 500,000 cells/ml. After 24 h of pre-incubation, NHOst cells attached onto Cytodex™ 3 were transferred to Lab-Tek II chamber slides (Thermo Fisher Scientific Inc., USA). Cells were exposed to orbital fluid shear stress at 24, 48 and 72 h. Unstimulated cells served as control. After each time points, cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature and stained according to our optimized method [26]. Fixed cells were incubated with DAPI (300 nM in PBS) for 20 min to stain the nucleus and then permeabilised with 0.1% Triton X-100 in PBS for 10 min. Alexa Fluors 635 Phalloidin (6.6 mM in PBS) together with anti-α-tubulin conjugated with FITC diluted (1:50) in blocking solution (10% BSA, and 1% Triton X-100 in PBS) was added to the permeabilised cells and incubated for 1 h to stain actin and tubulin fibers. After 1 h, cells were washed twice with PBS and counterstained with DAPI (300 nM in PBS) for 30 min. Chambers were removed from the slides and cover slip was mounted with Prolongs Gold Antifade reagent to minimize photobleaching. Slides were stored at 4 °C in the dark until scanned with a Confocal Laser Scanning Microscope (CLSM) (Leica TCS SP5). Fluorescence intensity of nucleus (Excitation: 358 nm, Emission: 461 nm), actin (Excitation: 633 nm, Emission: 647 nm) and tubulin (Excitation: 494 nm, Emission: 518 nm) fibers was measured according to their wavelength. 2.3. Mitochondrial activity NHOst mitochondrial activity was measured via MTS-based assay after 72 h of orbital fluid shear stress. A volume of 20 ml CellTiter 96s Aqueous One Solution (317 μg/ml) was added to the medium contained in each well (1:5). After incubation for 2 h, production of solubilised formazan was measured by absorbance at 490 nm. Mean optical density (OD) readings were converted into percentage (%) relative to control. 2.4. NHOst proliferation Trypan Blue stain was used to measure the rate of NHOst cell proliferation. Both stimulated and control NHOst cells were harvested and resuspended in OBM. A volume of 20 ml of Trypan Blue

Please cite this article as: M.D. Aisha, et al., Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.07.002i

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was added to the cell suspension to make up a dilution factor of 1. A total of 12 ml of cell-dye mixture was transferred to hemocytometer chambers. NHOst cells were viewed under the microscope. Live cells were counted to obtain the cell proliferation rate.

each well consisting of both control and stimulated cells was read at 450 nm wavelength. Protein concentration of OPG, RANKL and OCN was determined from the standard curve constructed using the O.D. value against each standard concentration.

2.5. Apoptosis

2.8. Statistical analysis

Type of cell death induced by orbital fluid shear stress was detected using flow cytometry through double staining with Annexin V-FITC and PI. NHOst cells were harvested after stimulation and staining was performed according to the manufacture's protocol (Beckman Coulter). Cells were analyzed using a Flow Cytometer (Coulters Epicss XL-MCL™Beckman Coulter, USA) within 30 min. Untreated and unstained NHOst cells were used as both negative and positive controls. 2.6. Alkaline phosphatase activity The p-nitrophenol (pNP) produced by alkaline phosphatase due to dephosphorylation of nitrophenyl phosphate (pNPP) was measured using a colorimetric assay kit ((BioVision). ALP activity (U/mL) measured from cell supernatant after orbital fluid shear stimulation was performed according to manufacturer's protocol. Absorbance was measured at 405 nm to measure the amount of pNP produced. Production of ALP from stimulated cells was compared to the standard curve and applied to the equation A/V/T [pNP produced (mmol)/volume of sample (mL)/reaction time (mins)] to generate ALP activity. 2.7. Enzyme-linked immunosorbent assay Human osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), and osteocalcin (OCN) protein was measured using cell supernatant. ELISA (R&D Systems) was performed according to the manufacture's protocol. Absorbance for

3. Results 3.1. Orbital fluid shear stress reorganizes NHOst cytoskeleton NHOst cells grown on collagen coated microcarrier showed significant changes in the arrangement of actin and tubulin fibers. Orbital fluid shear (OFS) stress appeared to facilitate actin polymerization as the fibers looked much more condensed and brighter in stain compared to control after 24, 48 and 72 h (Fig. 1). Moreover, the projection of actin fibers from the perinuclear to the lamellipodium region of the cell in shear stressed cells were similar to controls. Conversely, shear stress resulted in the disassembly of the microtubular network as the fibers were less condensed compared to control after 48 h and 72 h. At these time points, tubulin fibers were seen fragmented after shear stress. Moreover, tubulin fibers appeared to be accumulated in the perinuclear region of the cell. However, by looking at the images, OFS stress showed no morphological signs of apoptosis or necrosis of the osteoblasts.

48 h

72 h

Orbital shear stress

Control

24 h

Statistical analysis using Student's t-test was used from the statistical package SPSS (version 17.0) software. Level of significance between control and stimulated group was determined. Statistical significant level was set at *po 0.05 and **po0.01. All results were calculated and presented as mean 7standard deviation (SD).

Fig. 1. OFS stress rearranged NHOst cytoskeleton components. Actin was polymerized while tubulin was seen to be depolymerized after 48 h and 72 h of shear stress. Fixed NHOst cells were stained with Anti-α-Tubulin-FITC for tubulin fibers (green), Alexa Fluors635 Phalloidin for actin fibers (red), and DAPI for nucleus (blue) after 24 h, 48 h, and 72 h for both shear stressed and control cells. Cells were viewed with CLSM under 40x magnification (Scale bar:75 mm).

Please cite this article as: M.D. Aisha, et al., Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.07.002i

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Metabolism Proliferation

**

% of NHOst metabolism & proliferation

120 100 80 60 40 20 0 Control

Orbital shear stress

Fig. 2. OFS stress increased NHOst mitochondrial activity and proliferation after 72 h. NHOst cells were incubated with MTS-tetrazolium compound at 37 °C for 2 h. The result represents the means of 3 biological replicates and 3 technical replicates (n¼9) 7 SD. Statistical analysis by Student's t-test showed significant differences between shear stressed and control (100%). **: p o0.01.

3.2. Orbital fluid shear stress enhances NHOst mitochondrial activity and proliferation Continues shear stress resulted in an increase in osteoblast mitochondrial activity and proliferation rate. Compared to control, orbital shear stressed NHOst increased mitochondrial enzyme activity by 14.3 70.03% (Fig. 2), however, changes were not significant. Nevertheless, shear stress significantly enhance NHOst proliferation rate by 17.97 0.02% (p o0.01) relative to control cells (Fig. 2). The consequent increase in NHOst proliferation rate after shear stress corresponded to a decrease in the number of apoptotic cells by 0.09% compared to control (Fig. 3). 3.3. Orbital fluid shear stress promotes osteoblastogenesis Shear stress decreased RANKL protein concentration by 2.8 folds (130.4 70.003 pg/mL) but increased OPG concentration by 5.3 folds (800 70.070 pg/mL) compared to control NHOst cells (Fig. 4a). Changes in the concentration of osteoblastogenesis protein markers lead to the reduction of RANKL to OPG ratio from 1 to 0.16 after 72 h (Fig. 4b). Reduced RANKL/OPG ratio in response to orbital fluid shear indicates increase in osteoblastic activity. 3.4. Shear stress retained osteoblasts functionality markers We demonstrated that the increase in the NHOst proliferation rate supported the expression of bone matrix markers. Continues shear stress boosted ALP activity by 0.0034 70.001 U/mL/mmol compared to control (Fig. 5a). Meanwhile, stimulation showed no significant changes in OCN protein concentration compared to control after 72 h (Fig. 5b).

Live Cells Apoptosis Necrosis

Control

Orbital shear stress

p

99.33 ± 0.05 0.31 ± 0.02 0.15 ± 0.06

99.46 ± 0.15 0.22 ± 0.14 0.22 ± 0.06

0.230 0.162 0.005

Fig. 3. Percentage of apoptotic and necrotic cell death induced in response to orbital fluid shear stress.

4. Discussion 4.1. Orbital fluid shear stress reorganized NHOst cytoskeleton components Bone forming osteoblast cells sense shear stimulation and respond through an anabolic way (Fig. 6). Initial response to shear stress induces deformation and changes to cells that can be sensed by the cytoskeleton cellular network, consisting of actin and tubulin fibers which mediate cellular functions for cell motility, attachment, molecular transport, cell division, protein synthesis and others [27]. Intact cytoskeleton components facilitate tighter adhesion between the cell and the ECM, important for cellular communication. The family of integrin glycoproteins located on the cell membrane senses mechanical signals through the interaction between cell-ECM for intracellular signaling. The transmembrane receptor integrins, are mechanoreceptors that bridges between the cytoskeleton and the ECM for transmission of mechanical signals. More specifically, focal adhesions serving as linker proteins, attaches the integrins to actin fibers. Hence, mechanosignals are then converted into intracellular biochemical signals required during mechanical adaption. Conversion into biochemical signals drive changes in the intracellular protein confirmation, leading to rearrangement in cytoskeleton components. Moreover, protein confirmation releases mechanosomes which are focal adhesion-associated protein. Our unpublished data showed that CD44, a membrane glycoprotein functioning as adhesion protein for cell to ECM attachment was increased after 72 h of stimulation. The increase in CD44 protein expression seemed to correspond to the polymerization and condensation of actin fibers. Similarly, Meazzini et al., showed that 2 h of mechanical stimulation resulted in condensation of actin fibers [11]. However in our study, polymerization of actin fibers was not solely dependent on shear stress per se but also due to the type of culture substratum. Our previous study have showed that Cytodex 3 microcarrier coated with collagen represented as an excellent scaffold to support osteoblast growth microenvironment [28]. Collagen coated scaffolds has been shown to improve osteoblast attachment, spreading, proliferation [29] and mineralization [30]. Actin fibers were reported to be thicker and much condensed when osteoblast were grown on collagen scaffolds [31]. Meanwhile, Maezzini showed that polymerization of actin fibers not only facilitated tighter adhesion between osteoblast to the ECM for cellular communication, but also promoted osteoblast differentiation [11]. Actin were seen condensed during osteogenic differentiation [11]. Similarly, in our study, the polymerization of actin fibers could promote the anabolic activity of pre-osteoblast differentiation via osteoblastogenesis, as the expression of OPG protein was significantly increased. Balance between RANKL and OPG determines the net outcome of bone resorption and bone formation activity. High RANKL/OPG ratio represents bone catabolic activity where cells differentiate into osteoclasts, meanwhile, low ratio represents the anabolic bone activity as cells differentiate into osteoblasts (Fig. 6). OPG is a decoy receptor for RANKL and are both produced by osteoblast cells [32]. Binding of OPG to RANKL promotes osteoblastogenesis and blocks osteoclastogeneis. Our results showed that OPG protein was increased significantly while RANKL was reduced after orbital shear stress. This shows that orbital fluid shear stress was responsible in reducing RANKL/OPG ratio. Shear stress radiated anabolic signal and may promote preosteoblast differentiation, which in return reduces bone turnover in prolonged disuse. Shear stressed NHOst demonstrated mechanoadaptation properties by retaining normal osteoblast formation process. Previously, a study showed that stimulation increased RANKL/OPG ratio, consequently promoting osteoclastogenesis [33]. However, a recent study could show that stimulated

Please cite this article as: M.D. Aisha, et al., Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.07.002i

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OPG RANKL

900

1.1 1

**

800

0.9

700

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600

RANKL/OPG Ratio

Concentration (pg/mL)

5

500 400 300

0.7 0.6 0.5 0.4 0.3

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**

100

0.1 0

0 Control

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Fig. 4. OFS stress promotes osteoblastogenesis. (a) Level of OPG protein expression was elevated after 72 h under stress compared to control. Osteoblastogenesis is regulated when the (b) ratio of RANKL/OPG was reduced from 1 to 0.16. Optical density readings for RANKL and OPG from control and stressed cells were converted into pg/mL obtained from the standard curve. Data presented as mean from 3 biological replicates (n¼ 3) 7SD. Statistical analysis by Student's t-test showed significant differences between shear stress and control. **: po 0.01.

MC3T3-E1 osteoblast cells reduced the ratio between RANKL/OPG in vitro [34]. Differences in RANKL/OPG ratio were demonstrated to be selectively dependent on the magnitude and duration of stimulation [33,34]. 4.2. Cytoskeleton reorganization improved NHOst metabolism and proliferation under shear stress Cytoskeletal rearrangement responds to the activation of a secondary messenger cascade, promoting active metabolism and proliferation response [35–37]. Low mechanical stimulation at 1 Hz applied in human osteoblasts culture significantly increased osteoblast proliferation by 16.4–100% [7]. Conversely, optimum

proliferation rate was attained in a study stimulated with higher magnitude and duration; 20 Hz for 24 h [38]. Similarly, our study showed that orbital shear stress increased the proliferation rate along with NHOst mitochondrial metabolism. Meanwhile, in a much higher stimulation magnitude, 60 Hz for 24 h, increase in osteoblasts mitochondrial activity was observed [38]. Substantial increase in NHOst cell proliferation under stimulation was concurrent to a reduction in NHOst apoptosis. Our results are in agreement with studies done by Li Xuan on osteoblast cells isolated from rat calvarial bones [39]. They showed that continuous stimulation at 0.5 Hz for 72 h on rat osteoblast cells reduced apoptosis. At the same time, expression of apoptotic executioner marker, Caspase 3 activity, was deregulated after 72 h indicating

0.006

5 4.5

0.005 OCN Concentration (ng/mL)

ALP activity (U/mL/µmol)

4 0.004

0.003

0.002

3.5 3 2.5 2 1.5 1

0.001

0.5 0 Control

Orbital shear stress

0 Control

Orbital shear stress

Fig. 5. Bone matrix markers; ALP activity (a) was enhanced while OCN protein (b) was reduced after 72 h stimulation. Optical density readings from shear stressed and control cells were converted into respective units and obtained from the standard curve. Data presented as mean from 3 biological replicates (n ¼3) 7 SD. Statistical analysis by Student's t-test showed no significant differences.

Please cite this article as: M.D. Aisha, et al., Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.07.002i

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Fig. 6. Schematic diagram of anabolic signals transmitted by orbital fluid shear stress as a form of mechanical stimulation. Adhesion protein complexes located on cell membrane operate as mechanoreceptors to transduce shear signal into biochemical activity via the actin network. Biochemical signals can respond in an anabolic manner through (i) promoting the polymerization of actin fibers, (ii) stimulates osteoblastogenesis and (iii) enhance the expression of bone mineralizing markers.

apoptosis was inhibited [39]. 4.3. Cytoskeleton reorganization retained bone functional proteins Increase in actin polymerization not only promoted osteoblast metabolism, differentiation, and proliferation but also facilitated tighter adhesion between osteoblast cells to the culture substratum. We found that longer duration was required to detach NHOst cells cultured on Cytodex 3 microcarrier under stimulation in comparison to unstimulated cells. Increase rigidity of osteoblast adhering to ECM has been shown to promote bone mineralization [30], meanwhile reduced rigidity activates apoptosis [40]. In our study, improved NHOst rigidity under orbital fluid shear could be one of the factors involved in apoptosis inhibition. Rearragement of the cytoskeleton directs the autocrine signals to regulate cytokines involved in cell differentiation and proliferation [19]. Cytoskeletal rearrangment transfers mechanical signals into the cell through biological reactions. Signal is then channeled into the cell and throughout the cytoskeleton framework, directly modifying gene and protein expression in a specified manner (Fig. 6). Studies have shown that under shear stress, proteins reponsible for ECM assembly such as ALP, BMP-2, and Collagen type 1 and OCN [7,11] are expressed. Numerous studies have shown that when osteoblast cells exposed to shear stress over 24 h, expression of ALP activity and mRNA was increased [20,41,42]. When MC3T3-E1 osteogenic cells exposed to mechanical stimulation at 0.5 Hz for 72 h, level of ALP activity was significanlty increased [21]. ALP activity remained increased up to day 7 under continues mechanical stress [21]. In aggrement to previous findings, we could show that orbital fulid shear stress increased ALP activity after 72 h. However, no changes to bone mineralization marker OCN was observed. Nevertheless studies have shown that after prolonged (1–2 weeks) shear stimulation, OCN protein was significanlty increased [43] followed by ECM mineralization [44]. Our study expressed both early (ALP activity) and late (OCN protein) bone protein markers which are necessary for matrix synthesis and mineralization. Via polymerized actin fibers, anabolic signals generated from orbital fluid shear enhanced NHOst metabolism, proliferation and improved osteogenic phenotype markers. Production of ALP and OCN proteins suggests that osteoblast function was retained in

response to shear stress. Our findings showed that signals from orbital shear stress responded in an anabolic manner and are committed to increasing osteogenesis function. For that reason, with suitable orbital shear stimulation magnitude and duration, we suggest that applying orbital shear stress as a non-drug anabolic treatment may improve bone regeneration in prolonged disuse or even to slow down osteoporosis progression.

Conflict of interest All authors have no conflicts of interest.

Acknowledgment We would also like to thank the staff of the Institute of Medical Molecular Biotechnology (IMMB), particularly Ms. Salina Othman for her excellent operating skills of the Leica Confocal Laser Scanning Microscope and Mrs. Norita Salim for her skillful molecular technical assistance. This study was funded by the Research Excellent Fund (DANA), University Teknologi MARA (UiTM). Grant code: 600-RMI/ST/DANA 5/3 Dst (71/2011).

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Please cite this article as: M.D. Aisha, et al., Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.07.002i

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Please cite this article as: M.D. Aisha, et al., Orbital fluid shear stress promotes osteoblast metabolism, proliferation and alkaline phosphates activity in vitro, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.07.002i