The responses of osteoblasts to fluid shear stress depend on substrate chemistries

The responses of osteoblasts to fluid shear stress depend on substrate chemistries

Archives of Biochemistry and Biophysics 539 (2013) 38–50 Contents lists available at ScienceDirect Archives of Biochemistry and Biophysics journal h...

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Archives of Biochemistry and Biophysics 539 (2013) 38–50

Contents lists available at ScienceDirect

Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi

The responses of osteoblasts to fluid shear stress depend on substrate chemistries Yan Li a,b, Yanfeng Luo a,b,⇑, Ke Huang c, Juan Xing a,b, Zhao Xie c, Manping Lin a,b, Li Yang a,b, Yuanliang Wang a,b a

Key Laboratory of Biorheological Science and Technology (Chongqing University), Ministry of Education, Chongqing 400030, China Research Center of Bioinspired Materials Science and Engineering, College of Bioengineering, Chongqing University, Chongqing 400030, China National & Regional United Engineering Laboratory of Tissue Engineering, Department of Orthopaedics, Southwest Hospital, Third Military Medical University, Chongqing 400038, China b c

a r t i c l e

i n f o

Article history: Received 26 June 2013 and in revised form 21 August 2013 Available online 17 September 2013 Keywords: Self-assembled monolayer Material chemistry Fluid flow shear stress Osteoblasts Bone tissue engineering Synergistic responses

a b s t r a c t Natural bone tissue receives chemical and mechanical stimuli in physiological environment. The effects of material chemistry alone and mechanical stimuli alone on osteoblasts have been widely investigated. This study reports the synergistic influences of material chemistry and flow shear stress (FSS) on biological functions of osteoblasts. Self-assembled monolayers (SAMs) on glass slides with functional groups of OH, CH3, and NH2 were employed to provide various material chemistries, while FSS (12 dynes/cm2) was produced by a parallel-plate fluid flow system. Material chemistry alone had no obvious effects on the expressions of ATP, nitric oxide (NO), and prostaglandin E2 (PGE2), whereas FSS stimuli alone increased the production of those items. When both material chemistry and FSS were loaded, cell proliferation and the expressions of ATP, NO and PGE2 were highly dependent on the material chemistry. Examination of the focal adhesion (FA) formation and F-actin organization of osteoblasts before FSS exposure indicates that the FA formation and F-actin organization followed similar chemistry-dependence. The inhibition of FAs and/or disruption of F-actins eliminated the material dependence of FSS-induced ATP, PGE2 and NO release. A possible mechanism is proposed: material chemistry controls the F-actin organization and FA formation of osteoblasts, which further modulates FSS-induced cellular responses. Ó 2013 Elsevier Inc. All rights reserved.

Introduction Natural bone tissue receives both chemical cues and mechanical stimuli in physiological environments. Accordingly, in bone tissue engineering application, osteoblasts anchoring to a scaffold require appropriate chemical and mechanical stimuli to produce functional 3D bone tissue constructs. Generally, the chemical stimuli is provided by material chemistry of scaffolds, whereas mechanical stimuli is applied in vitro through scaffolds stretch, fluid flow shear, or hydrostatic compression [1,2]. Although the effects of material chemistry alone or mechanical loading alone on osteoblasts have been extensively studied [1,2,3–7], their synergistic contribution has not been investigated so far. Thus, it is essentially important to investigate the synergistic effects of material chemistry and mechanical loading on osteoblast responses and understand the potential mechanism. It is well known that material chemistry modulates cellular responses [3–5,8–13] and tissue formation [6,7] in vitro and in vivo. ⇑ Corresponding author at: College of Bioengineering, Chongqing University (Campus A), Chongqing 400030, China. Fax: +86 23 65102507. E-mail address: yfl[email protected] (Y. Luo). 0003-9861/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2013.09.005

For instance, Healy [3] demonstrated enhanced osteosarcoma cells attachment to NH2 surfaces compared to CH3 surfaces and guided spatial distribution of cells by controlling the distribution of NH2 and CH3. Similarly, Garcia [9] reported divergent osteoblasts adhesion and mineralization on well-defined NH2, COOH, CH3, and OH surfaces. In 3D scaffolds, Reis [7] found that sulfonic and phosphonic groups grafted on the starch and polycaprolactone (SPCL)1 blended scaffolds significantly promoted the proliferation of osteoblasts comparing with virgin SPCL scaffolds. Brodbeck et al. [6] employed a rat cage implant system with various surface chemistries to demonstrate increased in vivo apoptosis and reduce foreign body giant cell formation on hydrophilic and anionic implants compared to hydrophobic and cationic implants. In addition to material chemistry, appropriate mechanical loading is indispensable for the formation of normal bone tissue and maintenance of normal bone functions. For bone tissue in vivo, fluid shear stress (FSS) due to interstitial fluid flow through the canalicular spaces is regarded as the

1 Abbreviations used: SPCL, starch and polycaprolactone; Fss, fluid shear stress; ATP, adenosine triphosphate; NO, nitric oxide; PGE2, prostaglandin E2; SAMs, selfassembled monolayers; DIW, deionized water; PBS, phosphate-buffered saline; LDH, lactate dehydrogenase.

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principal mechanical stimuli responsible for bone adaption and remodeling [14–16]. The exact mechanisms by which osteoblasts sense and transform FSS into biological signals that are responsible for bone adaption and remodeling remain elusive. However, integrins and integrin-medicated cytoskeleton rearrangement are regarded as critical factors in this mechanotransduction [17–21]. Moreover, there are evidences that the initial formation of FAs is also a key factor in this mechanotransduction [22–28]. For example, the number and morphology of FAs considerably influence the FSS-induced short-term and mid-term responses, including the releases of adenosine triphosphate (ATP), nitric oxide (NO), and prostaglandin E2 (PGE2), which further regulate cell proliferation [14–15,22]. In addition, it has been reported that FSS-induced NO release requires complete cytoskeleton [28]. Previous studies have shown that both material chemistry and FSS are important regulators for osteoblasts behavior; however, their synergistic contributions have not been sufficiently studied. The aim of this study was to investigate the synergistic role of material chemistry and FSS in osteoblast responses and understand the possible mechanism of these responses. We chose self-assembled monolayers (SAMs) terminated with –OH, –CH3, and –NH2 functional groups to provide chemical stimuli and a parallel-plate fluid flow system to produce 12 dynes/cm2 FSS. The F-actin organization and FA formation of ROBs on various SAMs before FSS exposure were also investigated in order to understand the possible mechanism responsible for the synergistic responses. Materials and methods Preparation and characterization of SAMs on glass slides Blank glass slides were initially cleaned using acetone and ethanol, and subsequently rinsed with deionized water (DIW). All cleaned slides were stored in DIW before the introduction of chemical groups. To introduce –OH groups, the cleaned slides were dipped into freshly prepared Piranha solution (concentrated H2SO4:30% H2O2 = 7:3, v/v) at 80 °C for 1 h, and subsequently rinsed with excessive DIW and blown dry with nitrogen. The obtained slides were labeled as OH slides. To prepare NH2 slides, the OH slides were dipped into 1% solution of (3-aminopropyl) triethoxysilane (Alfa Aesar, USA) in acetone, and subsequently treated by refluxing 1 mL distilled water for 30 min. The obtained slides were rinsed with ethanol and DIW, and blown dry with nitrogen [29]. For CH3 slides, the OH slides were dipped into 5% solution of chloro(dimethyl)octadecylsilane (Sigma–Aldrich, USA) in hexane for 1 h, and subsequently rinsed with ethanol and DIW, and blown dry with nitrogen [30]. Finally, all the slides were placed in Petri dishes (Corning, USA), soaked in 75% alcohol overnight, and rinsed with sterile phosphate-buffered saline (PBS). To indicate that the preparation of the SAMs is successful, water contract angles of the slides were characterized by using a Model 200 video-based optical system (Future Scientific Ltd. Co., Taiwan, China). Six specimens were measured for each kind of SAMs, and three different points were selected for each specimen. All measurements were performed at room temperature by dropping 5 lL of ultra-pure water. In addition, the wettability has a significant effect on cell adhesion. Moderate wettability surface can make osteoblast adhesion be better than hydrophobic and hydrophilic surface [31].

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periosteum and surrounding connective tissue were removed by using tweezers. The calvaria bone was washed with PBS and cleaned with DMEM until the surface of bone is white and transparent, immersed into a small amount of fetal calf serum. The calvaria bone was cut into about 1  1  1 mm pieces, coated uniformly in 25 ml culture flask. Cultures were initiated in DMEM (Gibico, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS, Sijiqing, China), penicillin (100 U/mL), streptomycin (100 lg/mL), and 0.05% L-glutamine, and maintained in a humidified atmosphere of 5% CO2/95% air at 37 °C. The medium was changed every 2 days. After confluence, the cells were sub-cultured and identified by using the von Kossa staining method according to previously reported procedure [32]. The fourth to sixth passage of ROBs were used for experiments at a density of 2  105 cells/ slide. Fluid shear stress A parallel-plate flow chamber apparatus was employed to provide FSS [33]. The dimension of the flow chamber was 7.50 cm (length, L)  2.50 cm (width, W)  0.03 cm (height, H). FSS was produced by circulating 10 mL DMEM using a peristaltic pump (JieHeng, China). The produced FSS (s, dynes/cm2) was calculated according to equation s ¼ 6gQ =H2 W where g is the dynamic viscosity of the perfusate and Q is the flow rate. The employed FSS in this study was 12 dynes/cm2. All the components of the apparatus, except the pump, were maintained in a 37 °C incubator during the experiment and the medium was continuously saturated with 5% CO2/95% air. For FSS-loaded samples (denoted by X-FSS, where X represents surface chemistries), slides with attached ROBs were first mounted on the flow chamber and then exposed to FSS for a predetermined time. Other samples without FSS exposure (denoted by X, where X represents surface chemistries) were kept in Petri dishes at 37 °C in a humidified atmosphere of 5% CO2/95% air for the same predetermined time. Determination of ATP, NO, and PGE2 releases After seeded on SAMs for 48 h (reaching 80% confluence), ROBs were employed for the determination of ATP, NO, and PGE2 releases. After ROBs on slides were exposed to FSS for a predetermined time, 2 mL of medium was withdrawn and replenished with an equal volume of fresh culture medium to maintain a constant circulating fluid volume. The same procedure was performed for samples without FSS exposure.

Osteoblasts culture Primary rat osteoblasts (ROBs) cultures were described in our previous paper [32]. Briefly, the calvaria bone of 1–5 d old SD rats was removed and placed in Petri dish containing PBS buffer. The

Fig. 1. Water contact angles of various SAMs with terminal NH2, OH, and CH3 compared to blank glass slides. The results were shown as mean ± SD. ‘‘⁄’’ represents that NH2, OH, and CH3 are significantly different from blank glass slides (P < 0.05).

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Fig. 2. (A–D) ATP release of ROBs receiving both material chemistry and FSS stimuli (X-FSS) compared to those receiving material chemistry stimuli alone (X) and FSS stimuli alone. (E) The differences of ATP releases of ROBs between X-FSS and X (X-FSS minus X). X represents material chemistry stimuli alone of NH2, OH, or CH3. Blank glass slides (Glass) were used as controls of various material chemistries. Glass-FSS was considered to provide FSS stimuli alone. FSS is 12 dynes/cm2. The results were shown as mean ± SD. Statistical analysis results are shown in Table 2. ‘‘⁄’’ represents that X-FSS is significantly different from X (P < 0.05).

ATP release The ATP release was detected by using an ATP Assay Kit (Beyotime, China) based on bioluminescence technology. Briefly, 500 lL of the withdrawn medium and 200 lL of luciferin-luciferase re-

agent were mixed for 5 s; thereafter, the fluorescence intensity was measured using the Multifunctional Microplate Reader (Biotek Synergy HT, USA). The ATP concentration was determined on the basis of the standard curve that was plotted by using the sup-

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Fig. 3. (A–D) NO production of ROBs receiving both material chemistry and FSS stimuli compared to those receiving material chemistry stimuli alone and FSS stimuli alone. (E) The differences of NO production of ROBs between X-FSS and X (X-FSS minus X). X represents material chemistry stimuli alone of NH2, OH, or CH3. Blank glass slides (Glass) were used as controls of various material chemistries. Glass-FSS was considered to provide FSS stimuli alone. FSS is 12 dynes/cm2. The results were shown as mean ± SD. Statistical analysis results are shown in Table 3. ‘‘⁄’’ represents that X-FSS is significantly different from X (P < 0.05).

plied ATP standards and normalized to the total cellular protein using a BCA Protein Assay Kit (Biotech, China).To ensure that the detected ATP was all extracellular ATP, the plasma membrane integrity was confirmed by surveying the lactate dehydrogenase (LDH) levels of the conditioned media by using a LDH Assay Kit (Jiangcheng, China).

NO release NO concentration was detected by using a Nitric Oxide Assay Kit (Beyotime, China) based on Griess Reagent. The withdrawn medium was permitted to react with Griess Reagent for 10 min, and then the absorbance was measured at 540 nm using a Microplate Reader (Bio-Rad, USA). The NO concentration was

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Fig. 4. (A–D) PGE2 production of ROBs receiving both material chemistry and FSS stimuli compared to those receiving material chemistry stimuli alone and FSS stimuli alone. (E) The differences of PGE2 releases of ROBs between X-FSS and X (X-FSS minus X). X represents material chemistry stimuli alone of NH2, OH, or CH3. Blank glass slides (Glass) were used as controls of various material chemistries. Glass-FSS was considered to provide FSS stimuli alone. FSS is 12 dynes/cm2. The results were shown as mean ± SD. Statistical analysis results are shown in Table 3. ‘‘⁄’’ represents that X-FSS is significantly different from X (P < 0.05).

determined according to the standard curve plotted by the supplied NaNO2 standards and normalized to the total cellular protein.

PGE2 release PGE2 release was measured by using a Rat PGE2 ELISA Assay Kit (Yuan-Ye Chemical reagents, China). The absorbance was detected

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Fig. 5. (A) PI values of ROBs receiving both material chemistry and FSS stimuli (X-FSS) compared to those receiving material chemistry stimuli alone (X) and FSS stimuli alone. (B) Representative histograms of the PI cell-cycle profiles. X represents material chemistry stimuli alone of NH2, OH, or CH3. Blank glass slides (Glass) were used as controls of various material chemistries. Glass-FSS was considered to provide FSS stimuli alone. FSS is 12 dynes/cm2. ROBs on various SAMs were exposed to FSS for 1 h and then cultured for 24 h before PI detection. The results were shown as mean ± SD. ‘‘⁄’’ represents that NH2, OH and CH3 are significantly different from Glass (P < 0.05). (C) The differences of PI values between X-FSS and X (X-FSS minus X).

at 450 nm and the PGE2 concentration was determined on the basis of the standard curve plotted by the supplied PGE2 standards and normalized to the total cellular protein.

following formula: PI% ¼ ½ðS þ G2=MÞ=ðG0=G1 þ S þ G2=MÞ% , where S, G2/M, and G0/G1 represent the cell numbers in phase S, phase G2 and M, and phase G0 and G1, respectively, in cell cycle.

Determination of the proliferative index (PI)

Focal adhesion formation and F-actin organization

After being exposed to FSS for 1 h, ROBs were cultured for another 24 h without FSS exposure. Subsequently, the cells were trypsinized, washed with PBS, and fixed in 70% ethanol at 4 °C. The cell suspension was centrifuged at 2000 r/min for 10 min. The collected ROBs were washed with PBS and then 1 mL of PBS was added to obtain the cell suspension again. Following this, 20 lL of RNAse (10 mg/mL) was added to the obtained cell suspension and it was then incubated at 37 °C for at least 30 min. Then, 50 lL of pyridine iodide was added to the cell suspension and it was incubated in the dark at 37 °C for another 30 min. Finally, the cell cycle stages were detected using a Flow Cytometry (BD FACS Vantage SE, USA) and analyzed using Cell quest software (Becton Dickinson). The cell proliferative index (PI%) was calculated using the

After seeded on SAMs for 2, 12 h and 48 h (80% confluence, equivalent to osteoblasts before FSS), ROBs were stained for FA and cytoskeleton visualization. Before staining, ROBs were fixed through incubation in 4% (w/v) paraformaldehyde in PBS for 30 min and permeabilized for 10 min with 0.25% (v/v) Triton X100 in PBS. Subsequently, 1% BSA in PBS was added to block the non-specific binding sites by incubating with ROBs for 1 h. To observe FA formation, vinculin was stained by initially incubating with a mouse monoclonal anti-vinculin antibody (Abcam, UK) at room temperature for 60 min, and subsequently incubating with a FITC-conjugated anti-mouse immunoglobulin secondary antibody (ZSGB-Bio, China) at room temperature for another 60 min. F-actin was stained using Rhodamine-Phalloidin (Molecular

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Fig. 6. Focal adhesion and F-actin formation of ROBs on various SAMs without FSS exposure after incubation for 2, 12, and 48 h. The cells were fixed, permeabilized, and double labeled for vinculin (green), F-actin (red), and nuclei (blue). The yellow areas are vinculin, partially colocalized with F-actin in the focal adhesion plaques. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

Table 1 The number of vinculin (vinculin number/cell) and the area of F-actin (lm2/cell). Time

Glass NH2 CH3 OH

2h

12 h

48 h

Vinculin

F-actin

Vinculin

F-actin

Vinculin

F-actin

96 ± 13 153 ± 21 23 ± 5 18 ± 9

61 ± 8 98 ± 12 35 ± 14 33 ± 9

143 ± 26 227 ± 34 78 ± 19 75 ± 10

87 ± 12 110 ± 27 42 ± 15 52 ± 24

263 ± 29 372 ± 32 150 ± 23 144 ± 29

132 ± 23 168 ± 25 79 ± 21 86 ± 19

The results were shown as mean ± SD.

Probes, Invitrogen, USA) at room temperature for 60 min. Finally, the nuclei were stained by incubation with bisBenzimide H 33258 (Sigma–Aldrich, USA) at room temperature for 10 min. The stained specimens were washed, mounted in glycerol, and exam-

ined by a confocal laser scanning microscope (CLSM; TCS SP5, Leica, Germany). The number and total area of vinculin and F-actin was counted using Image J.

Inhibition of focal adhesions and disruption of F-actin To inhibit the FA formation of ROBs, suspended ROBs were first incubated under rotation for 1 h at 37 °C in DMEM containing 0% fetal calf serum and 500 lg/ml RGDS peptide (Glbiochem, China). Thereafter, ROBs were permitted to seed on the various SAMs for 48 h in the continued presence of RGDS. To disrupt F-actin organization, ROBs were seeded on SAMs for 48 h and then cultured in DMEM containing 0% fetal calf serum and 1 lM cytochalasin B (Sigma–Aldrich, USA) for another 1 h. The FAs and F-actin organization before FSS exposure and the ATP, NO, and PGE2 releases

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Table 2 The statistical analysis results of Fig. 2(E and F), Fig. 8A and Fig. 9A. The ‘‘⁄’’ represents that it has significant difference at the confidence of 0.05. The ‘‘NS’’ represents that it has no significant difference at the confidence of 0.05. Time(min)

NH2-FSS

CH3-FSS

NH2-FSS minus NH2 1 CH3-FSS (CH3-FSS minus CH3)

OH-FSS (OH-FSS minus OH)

1 2 3 4 5 10 15 1 2 3 4 5 10 15

2

CH3-FSS minus CH3

3

4

5

10

15

1

2

3

4

5

10

15

⁄ ⁄ ⁄ ⁄ ⁄ ⁄ ⁄ NS



NS



NS



NS



NS



NS



NS



Table 3 The statistical analysis results of Fig. 3(E and F), Fig. 4(E and F), Fig. 8B and Fig. 9C. The ‘‘⁄’’ represents that it has significant difference at the confidence of 0.05. The ‘‘NS’’ represents that it has no significant difference at the confidence of 0.05. Time(min)

NH2-FSS NH2-FSS minus NH2 5

CH3-FSS (CH3-FSS minus CH3)

OH-FSS (OH-FSS minus OH)

5 10 15 30 45 60 5 10 15 30 45 60

10

15

CH3-FSS CH3-FSS minus CH3 30

45

60

5

10

15

30

45

60

⁄ ⁄ ⁄ ⁄ ⁄ ⁄ NS



NS



NS



after FSS exposure were determined according to the methods described in Sections of 2.6 and 2.4, respectively. Statistical analysis Data were expressed as means ± SD (n P 6 for all experiments). Single factor analysis of variance (ANOVA) technique with OriginPro (version 8.0) was used to assess the statistical significance of results. The confidence interval was set to 0.05. Results Water contact angle measurements of SAMs Various SAMs with terminal –NH2, –CH3, and –OH groups were prepared on glass slides. Fig. 1 shows the static water contact angles of different substrates. The contact angle of CH3 substrate was the highest (97.9 ± 1.8°), while that of OH substrate had the lowest (11.4 ± 1.2°). The water contact angles of NH2 and blank glass was 61.7 ± 2.4° and 28.6 ± 1.6°, respectively. The hydrophilicity decreased following OH > Glass > NH2 > CH3. According to the Berg limit of 65° [34], only CH3 surfaces are hydrophobic. ATP release responding to FSS depends on material chemistry First, we examined the effects of material chemistry alone and FSS alone on the ATP releases of ROBs. To ensure that the detected

NS



NS

⁄ ⁄

NS

ATP is actively released by ROBs, the plasma membrane integrity was monitored by detecting and comparing the LDH levels in the conditioned media with FSS and without FSS. The LDH from ROBs on glass without FSS exposure was employed as the control. Compared to the control LDH, the LDH levels from ROBs on various SAMs both with and without FSS exposure demonstrated no significant difference, suggesting that the ROBs plasma membrane was not disrupted by the loaded FSS or surface chemistries and the detected ATP was actively released by ROBs. When ROBs received chemical stimuli alone, that is, when ROBs were cultured on various SAMs without FSS exposure, the concentrations of released ATP on NH2, CH3, and OH were almost identical to those on Glass (the blank sample) and demonstrated no observable change with incubation time (Fig. 2A–D). This implies that material chemistry has negligible effects on ATP releases of ROBs. On the other hand, when ROBs received FSS exposure alone (labeled as Glass-FSS), the corresponding ATP release was significantly higher than that without FSS exposure (Fig. 2A); the ATP release reached its maximum after ROBs were exposed to FSS for 1 min. This is consistent with previously reported results [35,36]. When ROBs received both material chemistry and FSS stimuli (labeled as CH3-FSS, OH-FSS, and NH2-FSS), the corresponding ATP releases were significantly higher than those of ROBs that were exposed to material chemistry alone, and they displayed a similar trend to Glass-FSS (Fig. 2B–D). Despite the common trend, differentiated ATP releases were observed for the same exposure time to FSS when the material chemistry was different (Fig. 2E).

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Fig. 7. Inhibition of focal adhesions and disruption of F-actin of ROBs on various SAMs after incubation for 48 h without FSS exposure. The cells were fixed, permeabilised, and double labeled for vinculin (green), F-actin (red), and nuclei (blue). The yellow areas are vinculin, partially colocalized with F-actin in the focal adhesion plaques. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

NH2-FSS led to largest ATP releases, while CH3-FSS and OH-FSS led to similarly lower ATP releases. Fig. 2F indicates the differences between the ATP releases of ROBs that are exposed to both material chemistry and FSS, and those that are exposed to material chemistry alone. If material chemistry did not interfere with the role of FSS in ATP releases, the differences should have been identical for all samples because the loaded FSS was the same (12 dynes/cm2). However, the results indicate that the differences in ATP releases demonstrated clear material chemistry-dependence. This result suggests that material chemistry influences the ATP releases of ROBs responding to FSS exposure, although it alone has no direct influence on the ATP releases of ROBs. NO and PGE2 releases responding to FSS depend on material chemistry The releases of NO and PGE2 of ROBs that were exposed to material chemistry alone and FSS alone was further detected.

When exposed to material chemistry alone, ROBs on various SAMs produced small amounts of NO (Fig. 3A–D) and PGE2 (Fig. 4A–D). The NO and PGE2 release for the ROBs that were exposed to FSS alone (Glass-FSS), were significantly higher than those for ROBs without FSS exposure (Glass) (Figs. 3A and 4A). The releases of NO and PGE2 gradually increased and reached a plateau at a FSS exposure time of 15 min. These results are similar to the previously reported ones [37,38]. When both material chemistry and FSS were loaded, the productions of NO and PGE2 were also significantly increased compared to those of ROBs that were exposed to material chemistry alone (Fig. 3B–D) and Fig. 4B–D). Furthermore, similar material chemistry-dependence of NO and PGE2 releases responding to FSS was observed following a pattern NH2-FSS > OH-FSS  CH3FSS (Figs. 3E and 4E). The differences between the NO and PGE2 releases for ROBs exposed to both material chemistry and FSS and those exposed to

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Fig. 8. FSS-induced responses of ROBs on various SAMs after focal adhesions inhibition: (A) ATP releases; (B) NO releases; (C) PGE2 releases. The results were shown as mean ± SD. There is no significant difference among various SAMs for PGE2 releases at a confidence level of 0.05. Statistical analysis results of A and B are shown in Tables 2 and 3, respectively. ‘‘⁄’’ represents that X-FSS is significantly different from X (P < 0.05). There is no significant difference among various SAMs for PGE2releases at a confidence level of 0.05.

material chemistry alone are shown in Figs. 3F and 4F, respectively. The results indicate that both the NO and PGE2 differences also demonstrated obvious material chemistry-dependence, as exhibited by ATP releases, suggesting that material chemistry exerts influence on the NO and PGE2 releases of ROBs responding to FSS exposure, although it alone has no direct influence on the NO and PGE2 releases of ROBs. Cell proliferation responding to FSS depends on material chemistry The cell proliferation potential can be indicated by cell proliferative index (PI), which is the ratio of cell number in G2/M phase and S phase to the total cell number. The PI values of ROBs on various SAMs without and with FSS exposure were determined by using flow cytometry and the result is illustrated in Fig. 5A. The typical histograms of the PI cell-cycle profiles are shown in Fig. 5B. When ROBs were cultured on SAMs without FSS, the PI values varied from material chemistries, decreasing in the order of NH2 > CH3  OH. When FSS was added and ROBs received both material chemistry and FSS stimuli, the corresponding PI values increased and demonstrated material chemistry-dependence as well. The differences between the PI values for ROBs exposed to both material chemistry and FSS and those exposed to material chemistry alone are shown in Fig. 5C and these differences followed the pattern

NH2 > CH3  OH. Compared with Glass, NH2 magnified while CH3 and OH diminished the contribution of FSS to cell proliferation. Focal adhesion formation and F-actin organization of ROBs on various SAMs before FSS exposure To elucidate the possible mechanism by which material chemistry differentially influences the releases of ATP, NO and PGE2, and cell proliferation, the FA formation and F-actin organization in ROBs receiving material chemistry stimuli alone were examined using a CLSM. The pictures of ROBs after cultured on SAMs for 2, 12, and 48 h are shown in Fig. 6, where the blue, green, and red represent nuclei, vinculin, and F-actin, respectively. After ROBs were cultured for 2 h (Fig. 6), some FAs formed and F-actin filaments appeared on NH2 slides, whereas no FAs or F-actin filaments were observed on CH3 and OH slides. On blank glass slides, only a few fuzzy F-actin filaments formed without any FAs. After culturing for 12 h (Fig. 6), short linear focal adhesion plaques and longitudinal F-actin stress fibers were observed on both NH2 and glass slides, whereas only very small or punctate FAs started to develop and no F-actin stress fibers formed on OH slides. On CH3 slides, neither FAs nor F-actin stress fibers formed (Fig. 6). After ROBs were cultured for 48 h, all ROBs on NH2 contained a large number of FAs and clear F-actin stress fibers, whereas ROBs on OH and CH3 displayed few FAs and

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Fig. 9. FSS-induced responses of ROBs on various SAMs after F-actin disruption: (A) ATP releases; (B) NO releases; (C) PGE2 releases. The results were shown as mean ± SD. The results were shown as mean ± SD. Statistical analysis results of A and C are shown in Tables 2 and 3, respectively. There is no significant difference among various SAMs for NO releases at a confidence level of 0.05.

vague F-actin stress fibers (Fig. 6). With respect to ROBs on glass slides, the number of FAs was fewer and the formation of F-actin stress fibers was not significantly different as compared to those on NH2 slides (Fig. 6). The statistical results of FAs and F-actin using Image J were shown in Table. 1. In a brief summary, FA formation and F-actin organization in ROBs evidently depended on material chemistry following the pattern NH2 > Glass > OH  CH3, which is consistent with the reported results [9,30].

without F-actin disruption (Fig. 4). However, the disruption of Factin resulted in a considerable reduction in the release of NO on all SAMs (Fig. 9B).When both FA was inhibited and F-actin was disrupted (Fig. 7), all FSS-induced responses of ROBs including the releases of ATP, NO, and PGE2 nearly disappeared on various SAMs and demonstrated no material chemistry-dependence (Fig. 10).

Discussion Focal adhesion inhibition and F-actin disruption of ROBs To further elucidate whether the different FA formation and Factin organization on various SAMs are responsible for the differentiated cellular responses of ROBs to FSS on various SAMs, the FA formation was inhibited and/or F-actin organization was disrupted before FSS exposure and then FSS-induced ATP, NO, and PGE2 releases were examined. When FA was inhibited (Fig. 7), both ATP and NO releases decreased to some extent on all SAMs and maintained a similar pattern of FSS-NH2 > FSS-CH3  FSS-OH (Fig. 8A and B) compared to that without FA inhibition (Figs. 2 and 3). On the other hand, PGE2 release declined dramatically and demonstrated no statistical difference between various SAMs (Fig. 8C). When F-actin was disrupted (Fig. 7), the releases of ATP and PGE2 slightly decreased and maintained a similar pattern of FSS-NH2 > FSS-CH3  FSS-OH (Fig. 9A and C) compared to that

In this study, we found that the responses of ROBs to FSS are highly dependent on material chemistry and the possible mechanism is that material chemistry regulates the responses of ROBs to FSS by controlling the F-actin organization and FA formation. Our findings provide valuable guidance for the design and selection of scaffold chemistry and the application of FSS to induce appropriate osteoblasts responses and produce normal bone tissues. The material chemistry was represented by SAMs on glass slides with terminal NH2, CH3, and OH. Blank glass slides (Glass) were used as the control of SAMs. FSS was provided by a parallel plate flow chamber and an FSS of 12 dynes/cm2 was employed. Since the preparation of SAMs on glass slides is well established, only the water contact angles were determined to indicate the formation of NH2, CH3, and OH SAMs. The detected water contact angles as shown in Fig. 1 are similar to those reported in previous studies [29,39], indicating that the preparation of the SAMs is successful.

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Fig. 10. FSS-induced response of ROBs on various SAMs after simultaneous FA inhibition and F-actin disruption: (A) ATP releases; (B) NO releases; (C) PGE2 releases. The results were shown as mean ± SD. The results were shown as mean ± SD. There is no significant difference among various SAMs for ATP, NO, and PGE2 releases, respectively, at a confidence level of 0.05.

The releases of NO and PGE2 are known to play an important role in bone formation and remodeling, and they are essential early responses of osteoblasts to FSS [40–42]. In this light, NO and PGE2 releases were selected as parameters to indicate the early responses of ROBs. Moreover, ATP, as a signaling molecule, is the precondition for other follow-up cellular responses including NO and PGE2 production, and cell proliferation. ATP release is one of the fastest responses of ROBs to FSS and can be employed to indicate the sensitiveness of ROBs to FSS [36]. Therefore, we also considered ATP releases in this study. Cell proliferation was examined to indicate the long-term responses of ROBs. We first examined the short-term responses of ROBs to FSS alone, including ATP releases as well as NO and PGE2 releases. Blank glass slides (Glass) were selected as a substrate for ROB cultures. As expected, FSS induced quick changes in the release of ATP, NO, and PGE2 (Fig. 2A, Fig. 3A, and Fig. 4A). To examine the synergistic effects of material chemistry and FSS on the releases of ATP, NO, and PGE2, the blank glass slides were replaced by NH2, CH3, and OH slides and the same FSS was applied. As compared with Glass-FSS, the sensitiveness of ROBs to FSS, characterized by ATP releases, was enhanced by NH2 whereas weakened by CH3 and OH (Fig. 2E). As a result, the differentiated releases of NO and PGE2 followed the pattern: NH2-FSS > CH3FSS  OH-FSS (Figs. 3E, and 4E). To further elucidate the possible contributions of material chemistry to the releases of ATP, NO, and PGE2, the effect of mate-

rial chemistry alone was investigated. ROBs on NH2, CH3, and OH produced only a small amount of ATP, NO, and PGE2 (Fig. 2B–D, Fig. 3B–D, and Fig. 4B–D). This implies that material chemistry alone exerts negligible effects on the releases of ATP, NO, and PGE2. Therefore, if material chemistry functions independently and does not interfere with the role played by FSS, the differences of ATP, NO, and PGE2 releases between the samples receiving both material chemistry and FSS stimuli and the corresponding samples receiving material chemistry stimuli alone should be identical because the loaded FSS for all samples is constant (12 dynes/cm2). However, the observation is on the contrary. These differences were obviously dependent on material chemistry with NH2-FSS the highest, followed by CH3-FSS and OH-FSS (Figs. 2F, 3F, and 4F). Therefore, we speculated that material chemistry interferes with the role of FSS in the releases of ATP, NO, and PGE2 and this interference occur even before FSS loading. Initial adhesion and spreading of osteoblasts, including integrin-medicated cytoskeleton rearrangement [17–21] and initial formation of FA [22–28], have been regarded as critical factors that influence FSS-related mechanotransduction. On the other hand, adhesion formation and cytoskeleton rearrangement at the early stage of matrix–cell interactions are regulated by material chemistry [9]. Therefore, FA and cytoskeleton are the common components by which both material chemistry and FSS influence osteoblasts behavior. Accordingly, it can also be hypothesized that FA and cytoskeleton are possible early coupling points where

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material chemistry contribute to the divergent responses of ROBs to FSS on various SAMs. To verify this hypothesis, the development of F-actin organization and FAs on various SAMs before FSS exposure were monitored (Fig. 6). It was observed that material chemistry differentially altered the F-actin organization of ROBs following the pattern NH2 > OH  CH3, and FAs following NH2 > CH3  OH. These patterns are consistent with previously reported cases and different hydrophilicity and electrostatic effects are believed to be responsible for these patterns [9,30]. By comparing with the patterns of FSS-induced releases of ATP, NO and PGE2 (Fig. 2E and F, Fig. 33E and F, Fig. 44E and F), it is observed that the releases of ATP, NO and PGE2 followed the same pattern of focal adhesions and F-actin organization. On the other hand, inhibition of FAs alone and disruption of F-actin alone eliminated the material dependence of FSS-induced PGE2 release (Fig. 8C) and NO release (Fig. 9B), respectively, while simultaneous FAs inhibition and F-actin disruption erased all the material chemistry dependence of ATP, NO, and PGE2 releases (Fig. 10). These results verified our hypothesis that material chemistry regulates the responses of ROBs to FSS by controlling FA formation and F-actin organization. It should be noted that FA formation and F-actin organization do not equally contribute to the FSS-induced ATP, NO, and PGE2 releases. FSS-induced NO release requires intact actin while PGE2 release does not [28]. Accordingly, the FSS-induced PGE2 release of ROBs was mainly determined by the FA formation on various SAMs and followed the pattern of FAs (see Figs. 4 and 6 without FA inhibition; see Figs. 7 and 8C with FA inhibition), whereas the NO release was mainly modulated by F-actin organization and followed the pattern of F-actin (see Figs. 3 and 6 without F-actin disruption; see Figs. 7 and 9B with F-actin disruption). Cell proliferation as a long-term response of ROBs was further investigated to evaluate the synergistic effect of material chemistry and FSS by using PI. ROBs were exposed to FSS for 1 h on various SAMs and then cultured for another 24 h before being examined for cell proliferation. As expected, better FA formation and F-actin organization positively contribute to cell proliferation [30]. The cell proliferation of ROBs on various SAMs without FSS exposure followed the pattern NH2 > Glass > CH3  OH (Fig. 5A). Moreover, better FA formation and F-actin organization on NH2 slides led to higher ATP, NO, and PGE2 releases of ROBs when exposed to FSS (Figs. 2E, 3E, and 4E), which further improved cell proliferation [43–45]. As a result, compared to Glass, NH2 magnified while CH3 and OH diminished the cell proliferation of ROBs responding to FSS (Fig. 5C). Conclusions In summary, this study demonstrated for the first time that the responses of osteoblasts to FSS including the releases of ATP, NO and PGE2 and the cell proliferation are significantly dependent on materials chemistry, following the pattern of NH2 > CH3  OH. The material chemistry differentially regulates the FSS role through controlling the F-actin organization and focal adhesion formation of osteoblasts which further mediate the short-term and long-term cellular behavior responding to FSS. NH2 magnifies while CH3 and OH diminish the osteoblasts responses to FSS. These findings provide a possible mechanism by which material chemistry influences osteoblasts responses to FSS and may provide guidance for the design of scaffold chemistry and selection of mechanical loading in order to in vitro produce functional bone tissues. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (Nos. 30970700, 11032012, and 30973065).

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