Materials Science and Engineering C 30 (2010) 1–7

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Materials Science and Engineering C

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Suppressed proliferation of mouse osteoblast-like cells by a rough-surfaced substrateleads to low differentiation and mineralization

T. Saito a,⁎, H. Hayashi b, T. Kameyama a,b, M. Hishida c, K. Nagai c, K. Teraoka a, K. Kato a

a Advanced Manufacturing Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2268-98 Anagahora, Shimoshidami, Moriyama-ku,Nagoya 463-8560, Japanb Department of Environmental Technology and Urban Planning, Nagoya Institute of Technology, Gokiso, Showa-ku, Nagoya 466-8555, Japanc Department of Biological Chemistry, Chubu University, 1200, Matsumoto-cho, Kasugai 487-8501, Japan

⁎ Corresponding author. Tel.: +81 52 762 7275; fax: +E-mail address: t-saito@aist.go.jp (T. Saito).

0928-4931/$ – see front matter © 2009 Published by Edoi:10.1016/j.msec.2009.06.010

a b s t r a c t

a r t i c l e i n f o

Article history:Received 28 November 2008Received in revised form 23 June 2009Accepted 29 June 2009Available online 6 July 2009

Keywords:OsteoblastSurface roughnessProliferationIntercellular communicationDifferentiationGene expression profiling

The cellular responses of mouse osteoblast-like MC3T3-E1 cells to the surface roughness were examined inthe sequential events of cell adhesion, proliferation, differentiation, and mineralization. The cells were platedand cultured on sandblasted borosilicate glass slideslips with different surface roughnesses. DNA synthesis atday 1 after plating and the cell number at day 5 significantly decreased as the surface roughness increased.The suppressed cell proliferation on the rough-surfaced substrates, closely related to the round cellmorphology, caused underdeveloped intercellular contacts via the gap junction due to the low population ofneighboring cells. Expressions of the representative osteoblastic genes at day 14, alkaline phosphataseactivity at day 21, and mineralization at day 28 were markedly reduced on the rough-surfaced substrates.These results clearly indicated that the reduced cell differentiation and mineralization resulted from the earlycellular responses of the suppressed cell proliferation depending on the surface roughness and theconsequent poor intercellular communication. The specific changes in the early gene expression profiles atday 1, depending on the surface roughness, were examined by a large-scale analysis of the gene expressionusing a mouse DNA chip. The ribosomal protein S6 kinase polypeptide 1 gene, which is a cell growth-relatedgene involved in the PI3-kinase/Akt pathway, was found to be the most down-regulated among the 4277screened genes.

© 2009 Published by Elsevier B.V.

1. Introduction

Implant surfaces play a major role in the successful biologicalfixation of an implant to its surrounding host tissue. Therefore, aprecise understanding of cell–implant interactions in the peri-implantregion is essential for designing biomaterials for clinical applications.After implantation, the implant surface is in contact with bodily fluidsand interacts with a number of proteins and different cell types [1]. Ithas been recognized that the behavior of cell adhesion, proliferationand differentiation on an implant mainly depends on its surfacephysico-chemical properties including hydrophilicity, roughness,texture, and morphology [2]. Moreover, implant surface-specificcellular responses are the result of a complex biological system thatincludes protein adsorption, receptor–ligand binding, and signaltransduction. Therefore, the relationships between the surfacephysico-chemical properties and cellular responses are quite compli-cated and far from being well understood. However, knowledge ofosteoblast responses to an implant is necessary in order to improve

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the implant composition or design to enhance the osseointegration[3]. Many in vitro studies have been carried out and provided someinsight into the response of specific cell types to biomaterial surfaceproperties. Mouse osteoblast-like MC3T3-E1 cells derived frommousecalvaria [4], human osteoblast-like MG63 [5], and Saos-2 [3] cellsderived from human osteosarcomas have often been used to probesurface influences in vitro. MG63 cells have been reported to exhibitroughness-dependent phenotypic characteristics and appear to bemore differentiated on rougher surfaces with respect to themorphology, extracellular matrix synthesis, alkaline phosphatase(ALP) specific activity, and osteocalcin (BGP) production [5]. In ourprevious study [6], we also showed that the surface roughnessenhances the osteoblastic differentiation of MC3T3-E1 cells. In orderto elucidate the direct effect of the surface roughness, the cells wereplated at a high cell density to form about a 100% confluent monolayeron the substrates, in which the DNA synthesis and cell growth werealmost arrested. From the results of the real-time reverse transcrip-tase-polymerase chain reaction (real-time RT-PCR) analysis of thegene expression of representative osteoblastic markers, ALP, Runx2,α1 chain collagen type I (Col1A1), and OPN, showed a higherexpression in the cells cultured on the rough-surfaced substrates. Inaddition, the intracellular ALP, and secreted BGP were also promi-nently expressed, and the matrix mineralization increased.

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In the present study, in order to obtain a better understanding ofthe regulatory mechanisms of osteoblast responses to the surfaceroughness, we focused on the early responses such as cell adhesion,morphology, and proliferation of MC3T3-E1 cells on the rough-surfaced substrates, and investigated how these early responsesinfluence the subsequent cellular development of osteoblasticdifferentiation and mineralization. Moreover, specific early changesin the gene expression profiles dependent on the surface roughnesswere analyzed using a mouse DNA chip in order to obtain a betterunderstanding of the regulatory mechanism of the early cellularresponses stimulated by the surface roughness.

2. Experimental

2.1. Substrates

Borosilicate glass slideslips (#S1112, Matsunami Glass Ind., Osaka,Japan)were sandblastedwith an ELP-1TR sandblaster (Elfo-tec, Nagoya,Japan). Half the area (38×26mm) of the sandblasted slideslips with Ravalues of 3.77, 1.99, 0.98, 0.48, and 0.04 µm was used as a culturesubstrate. The surface roughness was measured by a surface texture-measuring instrument (Handy Surf E-35A, Tokyo Seimitsu, Tokyo,Japan). Before the experimentswith cells, the substrateswere sonicatedin 70% ethanol for 15 min and sterilized at 160 °C for 2 h.

2.2. Cell culture

MC3T3-E1 cells were routinely maintained in a growth mediumconsisting of α-Minimum Essential Medium (α-MEM, Invitrogen,Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen)and antibiotics in a humidified 5% CO2 balanced-air incubator at 37 °C.To induce spontaneous differentiation into the mature osteoblasts, adifferentiation medium was prepared by adding 50 µg/ml ascorbicacid, 10 mM β-glycerophosphate, and 50 nM dexamethasone to thegrowth medium [7]. The cells were seeded at about 5×103 cells/cm2

onto 3 culture substrates in a 10-cm bacterial dish.

2.3. Cell morphology

MC3T3-E1 cells adhering to the substrate at day 1 after platingwere fixed by 2.5% glutaraldehyde and 1% OsO4 solutions for 1 h,then dried using a critical point drier after dehydration with ethanol(50–100%). Morphological estimation of the adhering cells wascarried out using a scanning electronic microscope (SEM, Hitachi S-3000, Hitachi, Tokyo, Japan) after being coated by Pt–Pd sputtering[8].

2.4. Dot blot analysis

Proteins (∼1.2 µg) were applied to a nitrocellulose membrane(Bio-Rad Laboratories, Hercules, CA) by dot-blotting, then blockedwith 3% gelatin in TBS buffer (20 mM Tris–HCl, 500 mM NaCl, pH 7.5)for 1 h. The membrane was washed with TTBS buffer (20 mM Tris–HCl, 500 mM NaCl, 0.05% Tween-20, pH 7.5) for 10 min, and thenexposed overnight to rabbit antisera reacting with mouse integrinsubunits α5 (Chemicon International, Temecula, CA), β1 (ChemiconInternational), talin (Chemicon International), and Cx43 (Sigma-Aldrich, St. Louis, MO). After removal of the unbound first antibodyby washing the membrane twice in TTBS for 5 min, the blots wereexposed to goat anti-rabbit IgG antiserum conjugated with ALP for2 h at room temperature and washed twice in TTBS for 5 min. Themembrane was washed in TBS for 5 min and color development wascarried out using a color development reagent (Bio-Rad Labora-tories) containing 5-bromo-4-chloro-3-indoyl phosphate and nitro-blue tetrazolium. The development was stopped by immersing themembrane in water for 10 min.

2.5. Cell proliferation

To monitor the DNA synthesis in proliferating cells, the 5-bromo-2'-deoxyuridine (BrdU) incorporation was analyzed using a CellProliferation Biotrak ELISA System kit (Amersham Biosciences,Uppsala, Sweden). This technique is based on the incorporation ofthe pyrimidine analogue BrdU instead of thymidine into the DNA ofthe S-phase cells. BrdU incorporation was detected according to themanufacturer's instructions. Briefly, after a 1-day culture, 10 µMBrdU labeling solution was added to the cultured cells on eachsubstrate for 2 h at 37 °C. After removing the labeling medium, thecells were fixed and then incubated with peroxidase-labeled anti-BrdU antibody for 90 min at room temperature. After washing 3times, the 3,3',5,5'-tetramethylbenzidine (TMB) substrate solutionwas added to the cultured cells on each substrate and incubated for30 min. The reaction was stopped by adding 1 M H2SO4 to each well.The optical density at 450 nm was measured to quantify the BrdUuptake.

Proliferated cells after a 5-day culture on the substrates with Ravalues of 3.77 and 0.04 µmwere visualized by staining the cells with aLive/DeadDouble StainingKit (Oncogene, SanDiego, CA). The cellswerestained with Cyto dye, a cell-permeable green fluorescent dye for livecells and propidium iodide (PI), a cell non-permeable red fluorescentdye for dead cells. Stained specimens were immediately observed by afluorescence microscope (Olympus) using a band-pass filter.

2.6. Real-time RT-PCR analysis

The total RNA was extracted from the 14-day cultured MC3T3-E1cells using Trizol reagent (Invitrogen). The first strand cDNA wassynthesized by the First-Strand Synthesis System (Invitrogen) using0.2 µg of the total RNA as a template for reverse transcription into cDNA.Quantitative real-time RT-PCR was carried out using a LightCyclerinstrument (Roche Molecular Biochemicals, Mannheim, Germany). Theoligonucleotide primers used in the amplification reaction were 5'-TGAACGGGAAGCTCACTGG-3' and 5'-TCCACCACCCTGTTGCTGTA-3' forGAPDH;5'-GTCCATGAGCAGAACTACATT-3' and5'-GGAGGATTCCAGATA-CAGG-3' for ALP; 5'-CCAAGCAGGAGGGCAATA-3' and 5'-AG-GGCAGCACAGGTCCTAA-3' for BGP; 5'-GTACCAGATGGGACTGTGGTT-3'and 5'-CTCCGGCCCACAAATCTC-3' for Runx2; 5'-TTGGGGCAAGACAG-TCAT-3' and 5'-GTTTGGGTTGTTCGTCTGTT-3' for Col1A1, 5'-GAG-GTGATAGCTTGGCTTA-3' and 5'-AGCTGACTTGACTCATGG-3' for OPN.Amplification was carried out in a 20 µl final volume containing 4 mMMgCl2, 0.5 µM of each sense and antisense primer, 2 µl of eachsynthesized cDNA, and 2 µl of the LightCycler-FastStart DNA MasterSYBR Green I (Roche). The amplification program included a 10 mininitial denaturation step at 95 °C for the Fast Start Taq DNA polymeraseactivation, followed by 40 cycles of 1 s annealing at 58 °C,15 s extensionat 72 °C, and 1 s denaturation at 95 °C. The specificity of the PCR productwas evaluated by determining the melt temperature (Tm) of the PCRproduct from the melt curve analysis. The cycle threshold (Ct) valueswere determined and the relative amounts of the mRNA wererepresented as the expression ratio using the Ct value obtained fromcells cultured on a substrate with Ra value of 0.04 µm.

2.7. ALP activity assay

The ALP activity of the supernatant, the same as those used in thedot blot analysis, was assayed at 37 °C using p-nitrophenylphosphate(PNP) as the substrate with ALP B-test kit (Wako Chemical Co., Osaka,Japan). The absorbance of p-nitrophenol formed from the enzymatichydrolysis was measured at 405 nm. The ALP activity was standar-dized as nmol of PNP per mg of protein per min. The proteinconcentration was determined by the method of Bradford [9] using aBio-Rad Laboratories protein assay kit with bovine serum albumin asthe standard.

Fig. 1. Scanning electron microscopic images of cell morphology (original magnification 1200×, bar=20 µm) of MC3T3-E1 cells cultured on the roughest substrate with Ra values of3.77 µm (A) and the smoothest substrate with Ra values of 0.04 µm (B) at day 1 after plating. Arrow heads in panels A and B indicate adhering cells on the substrates.

Fig. 2. Dot blot analysis of expressed focal adhesion components, integrin subunits α5(A), β1 (B), and talin (C) in MC3T3-E1 cells at days 1, 3, 5, and 7 after plating on theroughest substrate with Ra values of 3.77 µm (left lane) and the smoothest substratewith Ra values of 0.04 µm (right lane). Proteins (∼1.2 µg) from the cell lysates preparedfrom cultured cells were analyzed.with Ra values of 3.77 µm (left lane) and thesmoothest substrate with Ra values of 0.04 µm (right lane). Proteins (∼1.2 µg) from thecell lysates prepared from cultured cells were analyzed.

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2.8. Alizarin Red-S staining for mineralization

The mineralized matrix was stained for calcium by the AlizarinRed-S (AR-S, Sigma) staining method, as previously described [10].Briefly, at the end of the experimental period (at day 28), MC3T3-E1cells were washed with PBS, followed by fixation in ice-cold 70%ethanol for at least 1 h. Ethanol was then removed and the cells wererinsed with water and stained with 40 mmol/L AR-S (pH 4.2) for10 min at room temperature. The stained cells were further processedby 5 rinses with water, followed by a 15-minwash in PBSwith rotationto reduce the nonspecific AR-S staining. Stained cultures werephotographed followed by a quantitative destaining procedure using10% (w/v) cetylpyridinium chloride in 10 mmol/l sodium phosphate(pH 7.0) for 30 min at room temperature. The AR-S concentrationwasdetermined by absorbance measured at 562 nm using an AR-Sstandard curve in the same solution. Quantification of the mineralizedmatrix was expressed as the amount of bound AR-S per unit area ofthe cell culture surface (mg/cm2). The experimentswere performed intriplicate.

2.9. DNA chip assay

A Takara IntelliGene II mouse chip (Takara Bio, Inc., Kyoto, Japan),which was arrayed and immobilized with 4277 cDNA fragments ofmouse identified genes, was used for the DNA chip assay. The totalRNA was extracted at day 1 after plating with Trizol reagent fromMC3T3-E1 cells cultured on the substrates with Ra values of 3.77 and0.04 µm, then 3 µg of each RNA was labeled with Cy5 and Cy3,respectively, by the method of in vitro transcription (IVT) labeling[11]. The Cy3 and Cy5 labeled RNA were mixed together andhybridized with a Takara IntelliGene II mouse chip in hybridizationbuffer, 6× SSC containing 0.2% SDS, 5× Denhardt's solution, carrierDNA, and 50% formamide, at 65 °C for 14 h. After hybridization, theDNA chip was washed 3 times with 2× SSC containing 0.2% SDS at55 °C for 10 min and rinsed with 0.05× SSC. The hybridized DNA chipwas scanned using an Affymetrix 428 array scanner (Affymetrix, Inc.,Santa Clara, CA) and the signal intensity from each DNA spot wasanalyzed with BioDiscovery ImaGene Ver. 4.2 (BioDiscovery Corp., LosAngeles, CA).

3. Results

3.1. Cell morphology

The morphologies of the attached MC3T3-E1 cells at day 1 afterplating on the substrates with Ra values of 3.77, 1.99, 0.98, 0.48, and

0.04 µmwere observed by SEM. Representative pictures are shown inFig. 1 (A and B) to discern the substrate-dependent differences in theircell morphologies. Whereas cells on the smoothest substrate (control)exhibited a high degree of spreading and showed a highly flattenedmorphology (Fig. 1B), those on the roughest, of which the surface ishighly rugged and irregular, had a round shape (Fig. 1A).

We examined the actin cytoskeletal formation at day 1 andrevealed that the actin filaments were organized in stress fibers on thesmoothest substrate; however, those on the roughest substrateshowed more diffused actin cytoskeletons with less stress fiberformation (data not shown), which means that the surface roughnesscauses significant alterations in the actin cytoskeleton formation.

3.2. Cell adhesion

Next, in order to examine whether the morphology of MC3T3-E1cells is related to focal adhesion or not, we investigated the influence

Fig. 3. Assessment of DNA synthesis in MC3T3-E1 cells at day1 after plating on thesubstrates with Ra values of 3.77, 1.99, 0.98, 0.48, and 0.04 µmmeasured by 5-bromo-2'-deoxyuridine (BrdU) incorporation. The incorporated BrdU labeling of the newlysynthesized DNA was detected by immunoassay at an absorbance of 405 nm. The cellnumber for each substrate was about the same (∼5×103 cells/cm2). The error barsindicate the standard deviation (n=3). ⁎Pb0.05; ⁎⁎Pb0.01; compared to the smoothestsubstrate.

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of the surface roughness on the focal adhesion organization. Within afocal adhesion, the cytoplasmic domain of the integrin is linked to theactin cytoskeleton via a complex array of protein–protein interactions.One of these intracellular cytoskeleton associated proteins is talin. Theexpression of the focal adhesion components, e.g., the integrinsubunits α5, β1 (fibronectin receptor), and talin, was analyzed by adot blot analysis. As shown in Fig. 2, the integrin and talin expression

Fig. 4. Fluorescence microscopic images of MC3T3-E1 cells at days 1 and 5 after plating on thwith Ra values of 0.04 µmwith Ra values of 3.77 µm (left lane) and the smoothest substratfluorescent dye for live cells and propidium iodide, a cell non-permeable red fluorescent dy

varied with time in the culture and were also surface dependent [12].At day 1, the expression of these constituents was very weak, and hadalmost the same expression level on both surfaces with Ra values of3.77 and 0.04 µm. However, more than 3 days after plating, theexpression of the constituents on the roughest substrate was muchlower than those on the smoothest one. These results suggest thatthe round cell morphology formed on the roughest substrate at day 1(Fig. 1A) is initially determined by the surface morphology of thesubstrate, and then the intracellular cytoskeletal arrangement andfocal adhesion components follow and support the cell morphology.

3.3. Cell proliferation

To investigate the effect of the surface roughness on the DNAsynthesis at day 1, MC3T3-E1 cells of a low cell density cultured oneach substrate were incubated with BrdU. The incorporated BrdUlabeling newly synthesized DNA was detected by immunoassay at anabsorbance of 405 nm. As shown in Fig. 3, cells seeded onto therougher substrates with Ra values of 3.77–0.98 µm showed much lessBrdU incorporation than those seeded onto the smoother substrateswith Ra values of 0.48 and 0.04 µm, suggesting that the surfaceroughness significantly inhibits the cells from entering the S-phase.

The effect of the surface roughness on the cell growth was alsoconfirmed by fluorescence microscopic observations of the live anddead cells cultured on the roughest and smoothest substrates. Theinitial number of cells attached on both substrates at day 1 wasevaluated bymeasuring the L-lactate dehydrogenase (LDH) activity ofthe cell lysate and were confirmed to be almost the same (data notshown), which is also evident in Fig. 4A and B. After a 5-day culture,the proliferated cells covered all of the smoothest substrate (Fig. 4D);

e roughest substrate with Ra values of 3.77 µm (left lane) and the smoothest substratee with Ra values of 0.04 µm (right lane) stained with Cyto dye, a cell-permeable greene for dead cells.

Fig. 5. Dot blot analysis of expression of a gap junction protein, a gap junction protein,connexin (Cx) 43, in MC3T3-E1 cells cultured on the substrates with Ra values of 3.77,1.99, 0.98, 0.48, and 0.04 µm at days 7, 14, 21, and 28 after plating. Proteins (∼1.2 µg) ofthe cell lysates were analyzed.

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on the other hand, the number of cells on the roughest substrate wasmuch lower (Fig. 4C). No dead cells were observed by staining withpropidium iodide on either substrate (Fig. 4C and D). These resultsindicate that the difference in the cell number after the 5-day cultureresulted not from the initial cell number differences nor from celldeath, but from inhibited proliferation by the surface roughness.

3.4. Gap junction formation

Gap junctions are membrane-spanning channels that facilitateintercellular communication by allowing small signaling molecules topass from cell to cell [13]. Each gap junction is comprised of twohexamers termed connexons that, in turn, are comprised of sixsubunits termed connexins (Cx) [14]. A previous study has demon-strated that the expression of Cx43, a specific gap junction proteinpredominant in bone, is increased in the more highly differentiatedosteoblasts [15]. One role for Cx43 as a channel used by bone cells isconsidered to be the passage of differentiation signals [16]. Given theexpression of Cx43 in MC3T3-E1 cells cultured on each substrate(Fig. 5), gap junctional intracellular communication via Cx43 seems to

Fig. 6. Gene expression ratio of osteogenic proteins in MC3T3-E1 cells cultured on thesubstrates with Ra values of 3.77, 1.99, 0.98, 0.48, and 0.04 µm at day14 after plating. Thecycle threshold (Ct) values were determined by quantitative real-time RT-PCR and theratio was determined by defining the Ct value=1 for the smoothest substrate (n=3).⁎Pb0.05; compared to the smoothest substrate.

be much lower in cells cultured on the rougher substrates with Ravalues of 3.77–0.98 µm because the low cell density preventscommunication among neighbors.

3.5. Osteoblast-specific gene expression

The expression of osteoblast-specific genes in MC3T3-E1 cellsgrown on each culture substrate for 14 days was semi-quantitativelyanalyzed using real-time RT-PCR. The osteoblast-specific genesinclude ALP, BGP, Runx2, ColIA1, and OPN. For each sample, thehouse-keeping gene GAPDHwas expressed at a similar level (data notshown). The gene expression results (Fig. 6) showed that the ALP, BGP,and Runx2 genes were expressed at much lower levels in cellscultured on the rougher substrates with Ra values of 3.77–0.98 µmthan on the smoother ones with Ra values of 0.48 and 0.04 µm, whilethe expression of the Col1A1 and OPN genes was less changed withthe surface roughness.

3.6. Alkaline phosphatase activity

ALP is a representative enzyme of osteoblastic differentiation, andthe ALP activity was determined as an indicator of osteoblasticdifferentiation in the MC3T3-E1 cells. A different ALP activity wasfound depending on the surface roughness and on the duration of theculture (data not shown). The maximum ALP activities were observedfor the cells at day 21. As shown in Fig. 7, the ALP activities of cells onthe rougher substrates with Ra values of 3.77–0.98 µmwere much lessthan of those on the smoother substrates with Ra values of 0.48 and0.04 µm. This tendency of the ALP activity was consistent with the ALPpositive cell staining (data not shown). These results indicate thatosteoblasts cultured on smooth surfaces maintain their ability toexpress ALP, while a rough surface seems to significantly reduce it.

3.7. Mineralization

AR-S staining has been used for decades to evaluate calcium-richdeposits in cell cultures. At the end of the experimental period (atday 28), MC3T3-E1 cells on each substrate were stained with AR-Scalcium stain. The AR-S concentration (mg/cm2 substrate) wasdetermined as shown in Fig. 8. Similar to the results obtained fromthe experiments of the osteoblast-specific gene expression and ALPactivity, reduced matrix mineralization was also observed in cellscultured on the rougher substrates with Ra values of 3.77–0.98 µm.

Fig. 7. Intracellular ALP activity of MC3T3-E1 cells at day 21 after plating on thesubstrates with Ra values of 3.77, 1.99, 0.98, 0.48, and 0.04 µm. The error bars indicatethe standard deviation (n=3). ⁎Pb0.05; ⁎⁎Pb0.01; compared to the smoothestsubstrate.

Fig. 8.Mineralizedmatrix in MC3T3-E1 cells cultured for 28 days on the substrates withRa values of 3.77, 1.99, 0.98, 0.48, and 0.04 µm stained for calcium by the Alizarin Red-S.The bound Alizarin Red-S was solubilized with 10% (w/v) cetylpyridinium chloridesolution and the concentration was determined by absorbance measured at 562 nm.Quantification of the mineralized matrix is expressed as the amount of bound AR-S perunit area of the cell culture surface (mg/cm2). The error bars indicate the standarddeviation (n=3). ⁎Pb0.05; ⁎⁎Pb0.01; compared to the smoothest substrate.

Table 1Expression of specific genes on the roughest surface (Ra=3.77 µm) relative to thesmoothest one (Ra=0.04 µm).

Gene name Code GeneBankaccession

Expressionratio

Down-regulationRibosomal protein S6 kinasepolypeptide 1

Rps6ka1 NM_009097 0.05

Son cell proliferation protein Son NM_019973 0.09RAB, member of RAS oncogenefamily-like 3

Rabl3 NM_026297 0.14

CD3 antigen, epsilonpolypeptide

Cd3e NM_007648 0.16

Transmembrane protease,serine 2

Tmprss2 NM_015775 0.18

Early growth response 1 Egr1 NM_007913 0.18Ras homolog gene family,member E

Rnd3 NM_028810 0.19

Monooxygenase, DBH-like 1 Moxd1 NM_021509 0.20Peptidyl arginine deiminase,type III

Padi3 NM_011060 0.21

Annexin A11 Anxa11 NM_013469 0.21Complement factor H-related 1 Cfhr1 NM_015780 0.22Chloride channel K1 Clcnka NM_024412 0.23Leucine-rich repeat kinase 2 Lrrk2 NM_025730 0.23Granzyme A Gzma NM_010370 0.23Adenylate cyclase activatingpolypeptide 1 receptor 1

Adcyap1r1 NM_007407 0.23

Ets variant gene 5 Etv5 NM_023794 0.24Small proline-rich protein 1A Sprr1a NM_009264 0.24Myosin Vb Myo5b NM_008661 0.26Killer cell lectin-like receptor,subfamily D, member 1

Klrd1 NM_010654 0.26

ELAV-like 2 Elavl2 NM_010486 0.27Estrogen receptor-bindingfragment-associated gene 9

Ebag9 NM_019480 0.27

Lymphocyte antigen 9 Ly9 NM_008534 0.28Inhibitor of DNA binding 2 Id2 NM_010496 0.29Myelin protein zero Mpz NM_008623 0.31Natriuretic peptide receptor 3 Npr3 NM_008728 0.33Chemokine (C-X-C motif)ligand 12

Cxcl12 NM_013655 0.33

Up-regulationHeme oxygenase (decycling) 1 Hmox1 NM_010442 3.67Olfactomedin 1 Olfm1 NM_019498 3.81Tubulin polymerization-promotingprotein family member 3

Tppp3 NM_026481 4.10

Down-regulation: ratio b1:3, up-regulation ratio N3:1.

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3.8. Gene expression profiling of MC3T3-E1 cells responding to thesurface roughness

Gene expression profiling of MC3T3-E1 cells at day 1 after platingon the roughest surface compared with that on the smoothest surfacewas obtained by DNA chip assay. Of the total 4277 genes on theIntelliGene II mouse chip, we discovered that 26 genes weresignificantly down-regulated and that 3 genes were up-regulated inresponse to the surface roughness at a level more than threefold less(1:3) or greater (3:1) (Table 1). Among them, the Rps6ka1 gene,which is a cell growth-related gene and involved in the PI3-kinase/Aktsignal transduction pathway [17], was the most down-regulated (lessthan a 1:20 relative expression).

4. Discussion

The influence of the surface roughness on the cellular responses ofosteoblast-like MC3T3-E1 cells was investigated in the sequentialevents of cell adhesion, proliferation, differentiation, and mineraliza-tion. In this study, we focused on early events, such as cell adhesionand proliferation, and investigated how these early responsesinfluence later cell fates, such as differentiation and mineralization.

The proliferation of MC3T3-E1 cells assessed by both BrdUincorporation into DNA and fluorescence microscopic observationwas obviously suppressed as the surface roughness increased.Negatively impacted proliferation by the surface roughness has alsobeen reported in cultures of fetal rat calvarial cells [18], MG-63 cells[19], and MC3T3-E1 cells [20]. Considering that the cells on the roughsurfaces had a round shape and diffused actin filaments, thesuppressed proliferation could be closely related to the cell morphol-ogy determined by the surface topography of the substrates. In theprevious studies, the epithelial cells, fibroblasts, and bone cells werereported to use the topography of the substrates for orientation andmigration, a process known as contact guidance [21]. The endothelialcells attached on the planar substrates micropatterned with adhesiveand nonadhesive regions were shown to take on the size and shape ofthe underlying adhesive island [22]. Miura et al. [23] reported that thecells attached on a closely packed particle monolayer showed astretched narrow stalk-like shape and the cell growth was suppressedcompared with a flat surface. They concluded that cell extension andshape could be related to the cell growth and the pitch is a key factorfor determining later cell fates. These results clearly indicate that cellmorphology is significantly influenced by the degree of cell spreading,

which is governed by its 2-D or 3-D surface geometry of a substrate.Therefore, the round cell morphology with less stress fiber formationand decreased proliferation observed on the rough-surfaced sub-strates in this study could be similarly explained with geometricalrestriction of cell spreading caused by 3-D surface of the sandblastedsubstrates.

As a result of the suppressed proliferation, the cell population onthe rough-surfaced substrates remained low and the development ofintracellular communication via gap junction failed. Differentiationwas examined on the basis of the expression of representativeosteoblastic genes at day 14, and ALP activity at day 21. Runx2 isknown to play essential role in osteoblastic differentiation and boneformation after initiation by bone morphogenic proteins [24]. Runx2has been shown to induce the expression of osteoblastic genes, such asthe BGP, collagen α1 and OPN genes, by inducing their promoteractivities [25]. In our present study, the expression of Runx2 gene wassignificantly suppressed on the rougher substrates with Ra values of3.77–0.98 µm and those of ALP and BGP genes were also suppressed,while those of col1A1 and OPN were less changed with the surfaceroughness. As a marker of osteoblastic enzyme activity, ALP activitywas quantified. With accordancewith its gene expression, ALP activitywas also decreased on the rougher substrates. Mineralization of the

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cells was also shown to be reduced on the rougher substrates. Thepoor osteoblastic differentiation and mineralization on the roughsubstrates probably resulted from the sequential events of therestriction of the cell spreading, the round cell morphology withpoor stress fiber organization, the suppressed cell proliferation andsubsequent poor intercellular communication.

We investigated the specific changes in the early gene expressionprofiles of MC3T3-E1 cells dependent upon the surface roughness. Forthis purpose, we carried out a large-scale analysis of the geneexpression using a mouse DNA chip (total of 4277 genes). As shownin Table 1, we identified 26 genes down-regulated and 3 genes up-regulated in response to the surface roughness at a level of more thanthreefold less (1:3) or greater (3:1); notably, 8 geneswere significantlydown-regulated (less than a 1:5 relative expression). The most down-regulated gene among the 4277 genes was the ribosomal protein S6kinase polypeptide 1 gene (Rps6ka1), which is known to be a cellgrowth-related gene and involved in the PI3-kinase/Akt signaltransduction pathway. Keeping this pathway active is important forcell growth and prevents the cells from undergoing apoptosis [17]. Itwas previously reported that the expression of the Rps6ka1 gene issuppressed in the all-trans retinoic acid (ATRA) treated HL-60 cells(human promyelocytic leukemia cell line), indicating that this gene'sproducts play a role in the HL-60 cell growth [17]. Currently, little isknown about the molecular mechanism whereby the surface rough-ness exerts its effect on the cell development. Based on the results ofthe present study, it is suggested that the surface roughnesssignificantly down-regulates the expression of the Rps6ka1 geneduring the very early developmental stage of MC3T3-E1 cells andsignificantly suppresses cell proliferation by affecting modularphosphorylation in the signal transduction pathways.

5. Conclusions

The present study indicated that the surface roughness of animplant plays a major role in affecting cell morphology and specificchanges in early gene expressions, including modular phosphoryla-

tion in the signal transduction pathways and control of early events ofproliferation and cell–cell communication of the osteoblasts, whichconsequently affects later differentiation and mineralization.

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