a long segmental bone defects repair is one of the challenging problems in orthopaedic surgery .
although allogenic bone grafts are a current major option [ 13 ] , this technique is associated with problems of significant failure rates , poor mechanical properties , and immunological rejection .
porous materials are of significant importance for bone tissue engineering applications because of the good biological fixation to surrounding tissue through bone tissue .
porous titanium and titanium alloys have been investigated as they provide favourable mechanical properties with an elastic modulus closed to that of natural bone under a load bearing condition .  
surface characteristics of porous titanium are important determinants in its scaffold properties since the surface condition of titanium has been reported to play a critical role in bone formation associated with superior osteoblast adhesion and subsequent cell behaviors [ 610 ] . 
				
recent studies have raised a concern that degradation of biological ability with increase in adsorption of organic impurities on titanium - based biomaterials [ 6 , 7 ] .
this reduces hydrophilicity , adsorption of cell - binding proteins , and subsequent cell functions .
titanium - based biomaterials therefore need to be fabricated as clean surface without particular contamination such as titanium carbide .
there are a number of approaches in fabrication of porous titanium and titanium alloys such as sintering loose titanium powder or fibers , slurry sintering , and also rapid prototyping [ 3 , 4 , 11 , 12 ] .
initial surface contamination of sintered porous titanium would be unavoidable event as this is generally processed within the carbon graphite molds under a high thermal pressure [ 3 , 4 ] .
this study aimed to develop a contamination - free porous titanium scaffold by a plasma - activated sintering within an originally developed tin - coated graphite molds .
the surfaces of porous titanium sheets with or without a coated graphite mold were characterized .
the cell adhesion property of the porous titanium sheet was also evaluated in this study .
jis grade ii titanium ( ks-50 , kobe steel , tokyo , japan ) block was used as starting material .  
narrow titanium fibers were made by being turned with diameter approximetly 0.4 - 0.5  mm .
the surface of graphite molds were coated with thickness of 1.0  mm tin by presintering under vacuum with a condition of 1  mpa predisplacement and was processed by plasma - activated sintering under a vacuum at pressure of 20  mpa at 3800  a for 15  sec .
elemental titanium fibers were filled in the  30  mm  1.0  mm graphite mold with or without a tin coating .
the titanium fibers were subjected to the predisplacement with 1  mpa pressure in the graphite molds .
the porous titanium sheet  30  mm  0.5  mm was processed by plasma - activated sintering under a vacuum at pressure of 20  mpa , at 3800  a for 15  sec .
the surface topographies of the coatings on the specimens were then observed by sem ( s-2360n , hitachi , tokyo , japan ) .
the crystalline phases of the titanium samples before the tests were detected by tf - xrd ( xrd-6100 , shimadzu , kyoto , japan ) with cuk radiation .
the xrd was operated at 40  kv and 40  ma with a scanning speed of 0.02/4  s and a scanning range of 2060. 10  10  0.5  mm polished titanium plates and titanium sheets were subjected to cell culture .
an osteoblastic cell line , mc3t3-e1 , was obtained from the riken cell bank ( tsukuba , japan ) .
cells were cultured in  minimal essential medium ( gibco ) containing 10% fetal bovine serum ( gibco ) and 1% antibiotic ( penicillin , gibco ) under a 5% co2 atmosphere at 37c .
cells were suspended in the medium at 1  10  cells / ml and used for experiments .
a 1-ml quantity of floating cells was cultured onto titanium samples at 37c under 5% co2 for 1  d. a cell - counting kit ( dojindo , kumamoto , japan ) was used for the measurement of cell adhesion .
after incubation , each specimen was moved to another well and washed 3 times with pbs ( gibco ) to remove nonadherent cells .
adherent cells were mixed with 1  ml of medium and 100  l of reagent solution .
after 1  hr of incubation , the absorbance at 450  nm was measured .
the number of adherent cells was calculated from the activity of the original cell suspension .
adherent cells on each specimen after 1  hr of cultivation were dehydrated after being washed with pbs .
the cells were fixed with 3.7% formaldehyde in pbs and permeabilized by treatment with 0.1% triton x-100 ( sigma , tokyo , japan ) in pbs for 1  min .
the cells were then incubated for 3  hrs in a rhodamine - conjugated phalloidin solution .
after the cells were washed with water , stress fiber formation and cell morphology were observed with the use of a fluorescence microscope ( e-600 , nikon , tokyo , japan ) .
the appropriateness of the hypothesis of homogeneous variances was investigated by means of bartlett 's test .
as shown in figure 1 , the porous titanium sheet was structured under the high thermal pressure . the titanium sheet processed with tin coating
was observed to be clean whereas a particular contamination was observed on the titanium sheet processed without tin coating .
the xrd analysis revealed that the titanium sheet prepared without tin coated graphite mold showed distinctive tic peaks while the sheet with coated graphite mold showed tic peak ( figure 2 ) .
the peaks attributable to rutile tio2 were detectable on the titanium sheet prepared with tin coated graphite mold .
the peaks attributable to tio2 were not detectable on titanium sheet processed without tin coating .
the number of adherent cells on titanium sheet processed with tin coating was significantly ( p = .002 ) higher than that without tin coating after 1  d ( figure 3 ) .
adherent cells on the titanium sheet with tin coating had begun to show stress fibers and widely extend , while the stress fiber formation and cell extension on titanium sheet without tin coating were not distinctive ( figure 4 ) .
the plasma - activated sintering is a rapid sintering method associated with self - heating phenomena within the powder .
this is capable of sintering metal or ceramic powders rapidly to its full density at a relatively lower temperature compared to the conventional furnace sintering methods .
the carbon graphite mold has been employed in the plasma - activated sintering due to its electroconductive property and thermostability [ 3 , 4 , 11 , 13 ] .
the direct heating of graphite mold and the large spark pulse current provide a very high thermal efficiency .  
the peak of tic was detected on the titanium sheet processed with graphite mold without tin coating . since the titanium fiber elements were directly in contact with the carbon graphite mold during processing ; a particular contamination such as tic is unavoidable event in this condition .   alternatively , the tic peak was not detectable on the titanium sheet processed within the tin - coated carbon graphite mold .
this modified plasma - activated sintering with the tin - coated graphite mold would be useful to fabricate the contamination - free titanium sheet .
recent study suggested that adsorption of organic impurities on titanium surface are responsible for reducing the initial cells adhesion , subsequent proliferation002c and differentiation [ 68 ] .
amount of carbon absorbed on the titanium sheet seems to be an important part in determining the initial affinity level for osteoblasts and new bone formation .
the present study demonstrated that number of adherent cells on the modified titanium sheet was much greater than that of bare titanium plate .
additionally , stress fiber formation and the extension of the cells were observed on the titanium sheets .
the initial adhesion of cells induces stress fiber formation , phosphorylation of focal adhesion kinase , and activation of other intracellular signal transduction molecules thereby affecting cell proliferation , differentiation and new bone formation .
thus , the contamination - free surface of modified titanium would be useful for new bone generation at a segmental bone defect in comparison with the unmodified sintered porous titanium . in conclusion ,
the tin - coated carbon graphite mold is a new method for processing a contamination - free porous titanium sheet .
this modified titanium sheet is expected to be a new tissue engineering material in orthopedic bone repair .
 this study aimed to develop a contamination - free porous titanium scaffold by a plasma - activated sintering within an originally developed tin - coated graphite mold . the surface of porous titanium sheet with or without a coated graphite mold was characterized . 
 the cell adhesion property of porous titanium sheet was also evaluated in this study . 
 the peak of tic was detected on the titanium sheet processed with the graphite mold without a tin coating . since the titanium fiber elements were directly in contact with the carbon graphite mold during processing , surface contamination was unavoidable event in this condition . 
 the tic peak was not detectable on the titanium sheet processed within the tin - coated carbon graphite mold . 
 this modified plasma - activated sintering with the tin - coated graphite mold would be useful to fabricate a contamination - free titanium sheet . 
 the number of adherent cells on the modified titanium sheet was greater than that of the bare titanium plate . 
 stress fiber formation and the extension of the cells were observed on the titanium sheets . 
 this modified titanium sheet is expected to be a new tissue engineering material in orthopedic bone repair . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion