  various mechanical and chemical surface modifications of titanium dental implants , such as blasting , alkaline treatment , or hydroxyapatite coating , have been reported for improving the bone response during healing process [ 14 ] .
some reported the effectiveness of the surface coating or immobilization with cell adhesive protein , such as , fibronectin ( fn ) or laminin , for enhancing cell attachment , cell spreading , and cell activity [ 5 , 6 ] .
for biochemical surface treatment , several methods for covalent immobilization of cell adhesive proteins have been reported .
silane coupling reagents have been widely used for the immobilization of cell adhesive proteins [ 710 ] . on the contrary , hayakawa et al .
reported an easy and simple method for the immobilization of cell adhesive proteins onto a titanium surface , which was named the tresyl chloride - activated method [ 11 , 12 ] .
hayakawa et al . applied tresyl chloride directly on the titanium surface without the use of any solvent and found the basic hydroxyl groups of the titanium surface reacted with tresyl chloride [ 11 , 12 ] .
cell adhesive protein could be easily immobilized onto titanium surface through ionic interaction as shown in figure 1 , which was confirmed by quarts - crystal microbalance - dissipation measurements .
fibronectin- , collagen- , and fibronectin - derived peptides could be easily immobilized onto a titanium surface .
gene expression of osteoblast - like cells was monitored on titanium immobilized with fibronectin or fibronectin - derived grgdsp peptide and it was found that the expression levels of some genes related to the mineralization process , for example , bone sialoprotein and osteomodulin , were upregulated [ 1517 ] .  
many studies have shown that a rougher surface provided better bone formation or osteoconduction compared with a smooth surface
. however , the issues of optimal surface roughness and superficial morphology are still controversial and need to be clarified .
larsson et al . reported that differences of surface roughness between ra = 30.3  m ( machined ) and ra = 2.9  nm ( electropolishing ) varied the amount of bone after 6 weeks of implantation in rabbit cortical bone but not after 1 year of implantation .
they reported that the roughest plasma - sprayed surface ( ra = 33  nm ) exhibited the highest number of cell attachment cells and alp activity .
however , they also reported that the alp level of the polished surface with nanometer smoothness ( ra = 6  nm ) was much higher than that of the satin- ( ra = 0.83  m ) and grit - blasted ( ra = 11  m ) groups at 16 days of cell culture .  
previously we investigated the influence of two different surface roughness , namely , nanometer smoothing ( ra : approximately 2.0  nm ) and sandblasting ( ra : approximately 1.0  m ) , as well as biochemical treatment , namely , fibronectin immobilization using tresyl chloride - activated technique , of a titanium surface on osteoblast - like cell behavior . it was reported that the nanometer - smooth surface was beneficial for the differentiation of mc3t3-e1 cells and that fn immobilization provided better arrangement of attached cells . in the present study
, we aimed to evaluate cell viability of mc3t3-e1 and total protein content on the above - mentioned four different titanium surfaces , as a next series of our experiments .
  commercially pure titanium disk (  = 15  mm  1.0  mm , jis , japan industrial specification h 4600 , 99.9 mass% ti , furuuchi chemical corp . ,
nanometer - smooth titanium surface ( ti - smooth ) was prepared by polishing with diamond slurry with diamond particle diameters of 6  m , 3  m , and finally 1  m using a rapping machine ( rapping machine 12 in , tdc , corp . , miyagi , japan ) . after polishing ,
sandblasted surface ( ti sand ) was prepared by sandblasting with alumina powder ( 50  m ) at 5 atmospheric pressures for 5  s. after sandblasting , the specimen was cleaned ultrasonically with acetone - distilled water mixture and was then cleaned with 10  wt% hf and 5  wt% hno3 aqueous mixture for 20  s to remove any remaining alumina powder .
finally , the sandblasted specimen was cleaned ultrasonically again with acetone - distilled water mixture .
the surface roughness of nanometer - smooth titanium disk , which was determined with talysurf cci3000 ( 0.35  mm length and 12  nm pitch , ametek co. , ltd .
tokyo , japan ) , was 2.0  0.9  nm , and that of sandblasted titanium disk determined with surfcom-30a ( tokyo seimitsu , japan , 4  mm scale length and 0.8  mm pt . ) was 1.0  0.7  m .
fn immobilization on the titanium was performed in accordance with previous reports [ 11 , 12 ] ti smooth and ti sand disks were completely covered with tresyl chloride ( fluka , buchs , switzerland ) and then stored at 37c for two days .
afterwards , tresylated titanium disks were washed with double - distilled water followed by double - distilled water - acetone solution ( 50  :  50 ) and then dried and stored in a desiccator .
human plasma fibronectin ( harbor bio - products , ma , usa ) was dissolved in phosphate - buffered saline ( pbs ) solution ( ph 7.4 ) at a concentration of 100  g / ml for cell attachment assay .
tresylated titanium disks were immersed in the fibronectin - pbs solution for 24 hours at 37c and then rinsed with double - distilled water . finally , the titanium disks were dried with a gentle stream of dry air and stored in a desiccator .
thus , fibronectin - immobilized ti smooth ( ti smooth / fn ) and ti sand ( ti - sand / fn ) disks were prepared .
the fn immobilization was confirmed by an x - ray photoelectron spectroscope ( xps ; axis ultra , kratos analytical , uk ) , which was equipped with a monochromatized alk x - ray source operated at 15  kv and 15  ma .
the binding energy scale for each spectrum was calibrated against the c1s peak at 284.8  ev .
an n1s peak of amide bonds in fibronectin was detected at 399.9  ev .  
the cells were cultured in mem ( invitrogen , md , usa ) supplemented with 10% heat - inactivated fetal bovine serum , 66.7  g / ml kanamycin sulfate , and 284  m l - ascorbic acid 2-phosphate at 37c in a humidified atmosphere of 95% air and 5% co2 .
subsequently , the cells were cultured to subconfluence in 100  mm standard dishes ( falcon , becton - dickinson labware , franklin lakes , nj , usa ) and transferred to bioflex collagen i - coated 24-well plates ( falcon ) after treatment with 0.25% trypsin / edta ( invitrogen , md , usa ) .
classical medium without any bone differentiation components was employed in the preset study for evaluating the influence of fibronectin immobilization and surface roughness on the cell behavior [ 22 , 23 ] .
four different surfaces , that are , ti smooth , ti smooth / fn , ti sand , and ti sand / fn were evaluated by cell assay .
mc3t3-e1 cells were seeded on each titanium disc in 24-well plates at a density of 5  10 cells / cm and incubated for 11 days .
cell viability was determined by ( 3-(4,5-di - methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) assay by using vialight plus kit ( lonza group ltd , basel , switzerland ) . at predetermined time intervals ( 1 and 11 days ) ,
attached cells were rinsed with pbs and 200  l of culture medium was added .
then , 200  l of the cell lysis reagent was added , and cells were incubated in the orbital shaker for 10  min at 600  rpm , and 100  l of cell medium was transferred to a white plate .
afterwards , 100  l of atp monitoring reagent plus was added , and the plate was incubated for 2  min in the dark condition ( inside the luminometer ) before the measurement .
measurement was taken with the plate reader ( wallac 1420 arvosx , perkinelmer , inc .
four runs were performed and four specimens were analyzed for each substrate and each period .  
mc3t3-e1 cells were seeded on each titanium disc in 24-well plates at a density of 5  10 cells / cm and incubated for 11 days . to determine the total cellar protein , bca protein assay kit ( thermo fisher scientific k.k .
200  l of the cell lysis reagent was added to each well , and then 10  l of each specimen was pipette and 90  l of cell lysis buffer was added into a 96-well plate . 100  l of the reagent ( micro bca kit ; thermo fisher scientific inc . ,
the enzyme reactions were conducted for 30  min at 37c , and absorbency at 570  nm was immediately measured using a plate reader ( bio rad model 550 , bio - rad laboratories , inc .
after 1 day of cell culture , titanium disks were rinsed thoroughly in pbs , mounted on a piece of cork , and fixed in 2.5% glutaraldehyde in phosphate buffer ( ph 7.4 ) . following fixation ,
disks were dehydrated through a graded series of ethanol ( 50 , 60 , 70 , 80 , 90 , 95 , and 100% ) and then dried with tetramethylsilane . after ion coating with gold , the morphology of the cells
was observed using a scanning electron microscope ( s4000 , hitachi , tokyo , japan ) at an accelerating voltage of 5  kv .  
significant differences were determined by one - way analysis of variance ( anova ) using graphpad software ( graphpad prism , graphpad software inc .
the results of mtt assay are shown in figure 2 . at 1 day of cell culture
, there were no significant differences among four different titanium surfaces . at 11 days ,
ti sand / fn showed the significantly highest cell viability than ti sand , no significant differences were detected between ti smooth / fn and ti smooth . between 1 day and 11 days of cell culture , there were no significant differences for each titanium surface . 

figures 4 and 5 show the sem views of attached cells after 1 day of cell culture .
fn immobilization did not have any influence on the morphologies of attached cells but did have some influence on the number of attached cells .  
cells attached on nanometer smooth titanium ( ti smooth , ti smooth / fn ) showed the flat shape with a large and thin cytoplasmic layer and with numerous filopodia .
the filopodia was extending from the cell body to the titanium surface and higher magnification indicated the presence of short - fiber - like structure , which is presumed to be microvilli . comparing ti smooth and ti smooth / fn , ti smooth / fn surface showed more attached cells and attached cells showed better arrangement on ti smooth / fn surface rather than on ti smooth ( figure 3 ) .  
attached cells on sandblasted surface ( ti sand and ti sand / fn ) were slightly less spread than on nanometer smooth surface ( ti smooth , ti smooth / fn ) .
some of them had a globular appearance , especially cell on ti sand / fn .
more globular structure was observed on ti sand / fn and higher magnification clearly showed the globular structure of attached cell ( figure 4 ) .
in this study , we evaluated cell viability and total protein contents of mc3t3-e1 cells on four different titanium surfaces , ti smooth , ti smooth / fn , ti sand , and ti sand / fn .
the influences of mechanical treatment ( nanometer smoothing and sandblasting ) and biochemical treatment ( with and without fibronectin immobilization ) of a titanium surface were investigated .  
previously , we monitored dna amounts , alp activity , octeocalcin production and mineralization behavior to four different surfaces same as the present study .
it revealed that surface roughness enhanced the differentiation of mc3t3-e1 but not fn immobilization .  
the cell viability for ti sand / fn showed greater cell viability than that for ti sand .
although the reason is not clear , the combination of sandblasting and fn immobilization provided better cell viability .
pugdee et al . reported that significantly more mc3t3-e1 cells attached to fn - immobilized titanium disk than to untreated titanium disk at 30  min after cell seeding .
however , the present study showed no remarkable enhancement of cell attachment by fn immobilization , which was determined by total protein contents .
it is presumed that the controversial results were caused by the difference of cell assay time , 30  min versus 11 days and the difference of number of seeded cells , 1  10 versus 5  10 cells / cm .
fn is a major extracellular matrix protein , and cultured cells produce fn by themselves during cell assay .
it concluded that fn immobilization only enhanced the initial cell attachment , not proliferation .  
cell on ti sand / fn showed the highest cell viability , but not the highest total protein contents .
it is suggested that the activities and/or morphologies of cells on ti sand / fn were influenced by the combination of surface roughness and fn immobilization .
sem observation appeared that more attached cells were present on the ti smooth / fn surface than on ti smooth .
therefore , the arrangement of attached cells is controlled by the presence of immobilized fn .  
classical medium without any bone differentiation components was employed in the preset study for evaluating the influence of fibronectin immobilization and surface roughness on the cell behavior [ 22 , 23 ] .  
fn could be easily immobilized on both nanometersmooth and sandblasted titanium surfaces by our originally developed method , namely , the tresyl chloride - activation technique [ 11 , 12 ] .
first they treated titanium surface with a mixture of sulfuric acid and hydrogen peroxide to reproduce titanium oxide surface layer and then applied - silane - coupling agents to oxide titanium layer for the immobilization of proteins .
another advantage of tresyl chloride - activation technique is that any types of proteins and cytokine such as bmp or tgf- can be immobilized on titanium .
the influence of the immobilization of other types of proteins or cytokines , for example , laminin or bmp , on the cell behaviors should be further investigated .
 the present study was aimed to evaluate the viability and total protein contents of osteoblast - like cells on the titanium surface with different surface mechanical treatment , namely , nanometer smoothing ( ra : approximately 2.0  nm ) and sandblasting ( ra : approximately 1.0  m ) , and biochemical treatment , namely , with or without fibronectin immobilization . 
 fibronectin could be easily immobilized by tresyl chloride - activation technique . 
 mc3t3-e1 cells were seeded on the different titanium surfaces . 
 cell viability was determined by mtt assay . at 1 day of cell culture 
 , there were no significant differences in cell viability among four different titanium surfaces . at 11 days , 
 sandblasted titanium surface with fibronectin immobilization showed the significantly highest cell viability than other titanium surface . 
 no significant differences existed for total protein contents among four different titanium surfaces at 11 days of cell culture . 
 scanning electron microscopy observation revealed that smoothness of titanium surface produced more spread cell morphologies , but that fibronectin immobilization did not cause any changes of the morphologies of attached cells . 
 fibronectin immobilization provided greater amount of the number of attached cells and better arrangement of attached cells . in conclusion , 
 the combination of sandblasting and fibronectin immobilization enhanced the cell viability and fibronectin immobilization providing better arrangements of attached cells . 