titanium ( ti ) has been widely used in many types of biomedical applications . some of these applications are bone substitution materials : bone plates , bone screws , artificial joints , and dental implants . several major advantages of ti are its excellent biocompatibility , good osteoconductivity , superior corrosion resistivity , and sufficient strength . however , two major disadvantages exist : one is an elastic modulus higher than that of the cortical bone , and the other is a low bone bonding strength without surface modifications . an elastic modulus mismatch leads to  stress shielding  effects and causes bone resorption around the implant . one of the effective approaches to improving upon the above two problems is making the ti porous . the most popular and traditional process to produce porous ti produced by the above - mentioned processes shows a large plastic deformation under compressive stress ; however , it shows early fractures without showing sufficient deformation under tensile stress or bending stress . to improve the anisotropic plastic deformation property , a reduction of the porosity is the only solution , resulting in loss of the advantages of porous ti . materials commonly used in medical treatment today are calcium - phosphate cement paste , bio - absorbable polymer , and acrylic resin . the advantage of these materials is that they can be injected into the defect and fill it completely , and the material is geometrically stable in the defect . in contrast , metal materials can not be injected ; therefore , a metal piece for bone defect filling needs to be designed and shaped close to the defect . some studies reported on the use of porous ti granules that could fill defects . however , since close - packing of the granules into a defect is difficult , a large gap between the granules and bone may inhibit the osteoconduction . another type of porous ti that has gained interest is sintered ti fibre mesh [ 11 - 13 ] . sintered ti fibre mesh can exhibit high porosity , sufficient strength , and elastic deformation under bending stress , simultaneously however , the deformability of sintered ti fibre mesh is limited , and it needs to be designed and shaped to fill a bone defect . thus , in this study , a ti fibre knit block without sintering was developed . the tube was made from one ti fibre by knitting , and it was compressed into a block in a metal mould by cold pressing . because ti fibre does not bond mutually by sintering , the ti fibre knit block is flexible and can fit bone defects of any shape . the objective of this study is to evaluate the deformability , apparent elastic modulus , and bone ingrowth of a ti fibre knit block in vivo . fabrication of ti fibre knit block the ti fibre knit block in this study was produced from grade-1 pure ti fibre with a diameter of 150 m . prior to the knitting process , the ti fibre was soaked in a mixed solution of 10% hf and 40% hno3 for 1 min to remove its thick surface oxide layer . after soaking one ti fibre was knitted into a mesh tube of approximately 13 mm in diameter ( figure 1 ) . the knit pattern was a plain stitch with a mesh size of approximately 1.5 mm . the tube was inserted into a metal mould with a 3 mm inner diameter , and compressed to form a cylindrical block of 3 mm in height . in this study , the porosity was adjusted to approximately 90% ( group a ) , 70% ( group b ) , and 50% ( group c ) by controlling the length of the ti mesh tube . the ti fibre volume in a block was calculated from the weight of the block and ti density , and the porosity of the block was calculated from the ti fibre volume and apparent volume of the block with a 3 mm diameter and 3 mm height . image of ti mesh tube before compressing into a mould . 
  mechanical property evaluation to evaluate the elastic modulus and deformability , compressive stress was applied to the block using a universal testing instrument ( autograph ag-1knx , shimadzu , kyoto , japan ) . the loading direction was parallel to the cylinder axis , and the strain rate was 1  10s . because the block showed a large plastic deformation with loading , and the elastic modulus changed with increasing plastic deformation , the applied stress to the block started at 5 n and increased to 50 n in 5-n steps . to clarify the change in plastic deformation after loading , young s modulus of the block was calculated from the inclination of the linear unloading area of the stress - strain curve . to calibrate the displacement of the compressive test apparatus with loading , a copper rod of the same dimensions as the ti knit block was loaded , and the strain of the test apparatus was cancelled . the deformation of the ti knit block during the compressive test was observed and recorded with a digital camera . new bone formation was evaluated using 16 new zealand white rabbits , following the requirements of the ethics committee for animal research ( protocol no . 00391.01 ) at the cole nationale vtrinaire dalfort ( maisons - alfort , val - de - marne , france ) . all surgical procedures were performed under general anaesthesia using an intramuscular injection of ketamine chlorate ( 15 mg / kg ) . the tibia of the rabbit was shaved and disinfected with 10% povidone - iodine solution . after anaesthetic and disinfection procedures , a flap was raised , and three bone defects of 3 mm in depth and 3 mm in diameter were drilled in each tibia in imitation of the similar study on the bone scaffolds fabricated by ti fibre . the bone defects were randomly designated to the specimen from the three porosities ( figure 2 ) . blocks of groups a , b , and c were implanted from left to right . micro computed tomography four weeks or 12 weeks after implantation , the animals were euthanized with an anaesthesia overdose , and the bone around the defects was cut out . after fixation , the bone formation within the defect was examined using micro - ct ( skyscan 1176 , bruker microct , kontich , belgium ) . all scans were obtained at 80 kv and 300 ma , using a cu + al filter . three - dimensional images were created using reconstruction software ( nrecon , bruker microct , kontich , belgium ) , and analysed using viewing software ( data viewer , bruker microct , kontich , belgium ) . the cylindrical volume of interest ( voi ) was 3 mm in diameter and 3 mm in height , and 200 slices in height were created . the volume of the newly formed bone in the defect was evaluated using analysis software ( ct - analyser , bruker micro ct , kontich , belgium ) . the upper threshold of the signal intensity was set to 85/256 to eliminate existing bone in the image , and the lower threshold of the signal intensity was set to 25/256 to eliminate ti fibre and pores in the image ( figure 3 ) . the volume fraction of the newly formed bone in each defect area was calculated using the following equation : 
 vnb = volume of newly formed bonevolume of bone defect 
  where vnd is the volume fraction of the newly formed bone . histological analysis after ct scanning , the bone specimens were dehydrated and infiltrated in a series of mixed liquid of ethanol and a light curing resin ( technovit 7200 vlc , heraeus kulzer gmbh , hanau , germany ) with an ethanol mixing ratio of 100% , 70% , 50% , 30% , 15% , and 0% under constant vacuuming . after complete infiltration , the specimens were embedded in light curing resin ( technovit 7200 vlc , heraeus kulzer gmbh , hanau , germany ) . the resin blocks after curing were subjected to a non - decalcified cut using a low - speed diamond band saw ( bs-300cp , meiwafosis co. , ltd , tokyo , japan ) , and then grind sectioning using waterproof silicon carbide grinding paper of p800 , p1000 , and p1200 with a final thickness of approximately 30 m at the central section . the specimen was then stained with toluidine blue according to staining protocol for plastic sections . histological analyses were performed using a light microscope ( bz-9000e ; keyence , osaka , japan ) . the images were analysed using image analysing software ( image j ver . 1.43u , national institute of health , maryland , u.s.a . ) , and the amount of newly formed bone stained with toluidine blue in the bone defect area was calculated . the area fraction of the newly formed bone in the defect area was calculated using the following equation : 
 snb = area of newly formed bonearea of bone defect 
  where snb is the area fraction of the newly formed bone . all data were statistically analysed using a student s t - test ( microsoft excel 2010 , microsoft , redmond , wa , usa ) , and p value of less than 0.01 was considered for significance . morphology and porosity of ti fibre knit block the fabricated ti fibre knit blocks are shown in figure 4 . the group a specimen with the highest porosity showed loosely compacted ti fibre , while the group c specimen with the lowest porosity showed densely compacted fibre . no fibre folding or fractures were observed in the sem images of each block ( figure 5 ) . the sem images also showed a rough surface of ti fibre after the surface oxide layer was removed ; however , no cracks were observed . the porosities of the blocks of groups a , b , and c were 88% , 69% , and 50% , respectively . although pore size distribution was not estimated in this research , many large pores were observed in the group a specimen , while few large pores were observed in the group c specimen . mechanical properties of ti fibre knit block figure 6 shows the stress - strain curves of the compressive test for the ti fibre knit block . the group a specimen showed a large plastic deformation with the first loading of 5 n , 10 n , and 15 n. this specimen showed less plastic deformation with the second loading of the forces ; however , it showed considerable hysteresis between loading and unloading . the plastic deformation decreased with a loading of more than 20 n , and the difference in the stress - strain curve between the first loading and second loading also decreased . a unique feature of the stress - strain curves was that a linear area was barely observed in the loading curve ; however , the initial area of the unloading curve was linear and appropriate for young s modulus calculation . the group b specimen showed a smaller plastic deformation than the group a specimen ; however , the reduction in the plastic deformation with increasing applied stress was smaller than that of the group b specimen . the deformation of groups a , b , and c with a 50 n load was 2.49 mm , 1.95 mm , and 0.63 mm , respectively . the plastic deformation after unloading of a 50 n load was 2.26 mm , 1.68 mm , and 0.38 mm , respectively . figure 7 shows the ti fibre knit blocks after a loading of 50 n. the group a specimen showed a large deformation in the directions perpendicular and parallel to the specimen axis . this buckling was slightly observed in the group b specimen , and barely observed in the group c specimen . ti fiber knit block after loading of 50 n. 
 
 dotted line indicates the original height . although the porosities of the specimens were different , young s modulus of the specimens of groups a , b , and c were not significantly different . micro computed tomography analysis the effects of specimen porosity and time after implantation on a volume fraction of new bone ( vnd ) are shown in figure 9 . a dotted line indicates the porosity , namely , the maximum vnd at the porosity . four weeks after implantation , the group c specimen with 50% porosity showed the highest vnd ( 46.7% ) , and new bone filled nearly all pores . with increasing porosity , vnd decreased . twelve weeks after implantation , vnd of the group c specimen ( 45.2% ) did not increase because new bone already filled nearly all of the pores at four weeks after implantation . vnd of the group b specimen with a 69% porosity ( 35.5% ) increased from that at four weeks after implantation , and had a vnd similar to that of group c ; however , new bone did not fill all pores . vnd of the group a specimen with an 88% porosity ( 21.7% ) did not increase from that at four weeks after implantation . histological evaluation typical histological cross - section images of the blocks at four weeks and 12 weeks after implantation are shown in figure 10 . area stained with light blue shows an existing bone , and area stained with dark blue shows a new bone . the histological observation showed healing of the bone around the ti fibre knit block , and did not show an inflammatory response in any specimens . new bone formation in the group a specimen of 88% porosity was limited to the area near the tibia bone surface . new bone formation in the group b specimen and group c specimen were observed in the entire ti fibre knit block . typical histological cross section images of the blocks ( toluidine blue stain , original magnification x10 ) . four weeks after implantation , snb of the group a specimen ( 17.8% ) was significantly lower than that of the group c specimen ( 28.4% ) . twelve weeks after implantation , snb of the group a ( 19.1% ) , group b ( 27.6% ) , and group c specimens ( 28.9% ) was not significantly different . interestingly , although the dependence of porosity on snb as histologically evaluated was similar to that of vnd evaluated using ct data , snb was clearly lower than that evaluated using ct data . area fraction of new bone 4 weeks and 12 weeks after implantation . 
 
 * significant different ( p < 0.01 ) . morphology and mechanical properties of ti fibre knit block the ti fibre knit block developed in this research is made of a single fibre , and no fibre fracture was observed in the blocks . because the diameter of the fibre is small , the fibre ends can potentially penetrate tissue and cause inflammation . the risk of such trouble in this block is , however , minimum , because there are minimum numbers of fibre ends . because the ti mesh tube was compressed to the cylindrical block , nearly all parts of the fibre in the block are not straight , and the directions of each part are not uniform , as observed in figure 4 and figure 5 . when parts of the fibre are oriented nearly perpendicular to the block axis , the deformation in the loading direction should be small with initial loading . by contrast , when parts of the fibre are oriented nearly parallel to the block axis , the deformation should be large with initial loading . because the fibre of the group a specimen was lightly compressed , the directions of the parts of the fibre were potentially close to random . therefore , many parts of the fibre were oriented close to parallel to the block axis , and showed large deformations with initial loading . by contrast , because the fibre of the group c specimen was heavily compressed , many parts of the fibre were potentially oriented perpendicular to the block axis , and showed small deformations with initial loading . although the initial loading curves of the block were not linear , the initial unloading curves were linear . macroscopically , linear elastic recovery is a typical behaviour ; however , it is not certain that the recovery of each part of the fibre is also linear . the mechanism of linearity mismatches between loading and unloading was not clarified in this research . however , the pairing of nonlinear loading curves and linear unloading curves reminds us of the loading and unloading curves of a dynamic hardness test result , and suggests this is not necessarily unusual behaviour . it is reported that the elastic modulus of the porous ti decreased as the porosity increased . however , the elastic modulus of the block in this research did not change when the porosity changed . the reason for this decreased effect of porosity is also the orientation of the parts of the fibre in the block . the parts of the fibre close to perpendicular to the block axis show less deformation with loading ; therefore , the number density of parts of the fibre parallel to the block axis mainly affects the elastic modulus . as discussed above , major parts of the shorter fibre of the group a specimen , and minor parts of the longer fibre of the group c specimen , were potentially parallel to the axis . for the three types of blocks in this research , the number densities of the parts of the fibre parallel to the block axis were potentially similar , and the elastic modulus were similar . after the plastic deformation of the compressive test , each specimen indicated compressive plastic deformation along the block axis . the compressive deformation induced a porosity reduction , and potentially led to an increase in elastic modulus . it is noted that the compressive deformation during block production did not change the elastic modulus of each type of specimen . the difference in the above two compressive deformations was that the block was produced in a mould and there was a wall beside it , while the block was deformed freely perpendicular to the block axis during the compressive test . however , the effect of this difference was not clarified in this research . to discuss the mechanical behaviour of the blocks quantitatively , a three - dimensional analysis of the fibre shape in the block is required . however , a numerical analysis is still difficult because the shape of the fibre is complicated , and the shape should be different in each block . increasing the group a specimen showed buckling with plastic deformation , in other words , it showed deformation parallel to and perpendicular to the loading direction , simultaneously . however , the group c specimen showed only plastic deformation parallel to the block axis . flexible plastic deformation of a high - porosity block suggests that these blocks should fill a non - uniform bone defect effectively with compression into the defect . by contrast , limited plastic deformation of a low - porosity block suggests that these blocks might be appropriate to fill a cylindrical defect but are not appropriate for non - uniform defects , especially a defect whose entrance size is smaller than the inner size . bone formation of ti fibre knit block both the x - ray micro - ct analysis and histological analysis indicated new bone formation in the blocks ; however , snb based on a histological analysis was lower than vnd based on an x - ray micro - ct analysis . one possible reason was that the histological sections in this study were sliced parallel to the block axis . because the new bone grew into the block from the outside , vnd in the outer area of the block is higher than or similar to that of the inner area . therefore , when the sections were sliced perpendicular to the axis , snb should have been close to vnd of the entire specimen . when the sections were sliced parallel to the block axis , snb changes with the slice position the mismatch between vnd and snb suggests a limitation of the histological analysis in this research , and the efficacy of x - ray micro - ct analysis . the ti fibre knit block with higher porosity showed less bone formation in this research . there are many research studies that indicate the effect of the pore size of porous material on bone formation [ 16 - 25 ] , and they suggest there is an appropriate range of pore sizes . , the pore size distribution could not be analysed from x - ray micro - ct data . however , the results potentially indicated that the group c specimen consisted of pores of appropriate size , and the pore size of the group a specimen was larger than that . this leads to a difference in new bone formation speed between the types of specimen . appropriate porosity of ti fibre knit block the deformation of the specimen after the compressive test suggested the porosity should decrease after compression . therefore , the group a specimen , which had the lowest bone formation in this research , could increase its bone formation after compression into a bone defect . concerning the other important clinical property , deformability with compression , the group a specimen indicated promising data . therefore , the group a specimen , a specimen with high porosity and high deformability , is appropriate for bone defect filling . after filling into the defect , the effect of in situ porosity changes with bone defect filling will be evaluated in the future . the titanium fibre knit block in this research exhibited excellent properties with controlling porosity of the block . the specimen of higher porosity indicated excellent deformability , demonstrating potential appropriateness for bone defect filling material . the titanium fibre knit block with 88% porosity is potentially appropriate for bone defect filling , potentially indicating good bone ingrowth after porosity reduction with compression into it . osaka , japan , for the ti fibre knit block production , which was supported in part by a manufacturing grant from the small and medium enterprise agency , japan .
abstractobjectivesthe objective of this study is to develop a ti fibre knit block without sintering , and to evaluate its deformability and new bone formation in vivo.material and methodsa ti fibre with a diameter of 150 m was knitted to fabricate a ti mesh tube . 
 the mesh tube was compressed in a metal mould to fabricate porous ti fibre knit blocks with three different porosities of 88% , 69% , and 50% . 
 the elastic modulus and deformability were evaluated using a compression test . 
 the knit block was implanted into bone defects of a rabbit s hind limb , and new bone formation was evaluated using micro computed tomography ( micro - ct ) analysis and histological analysis.resultsthe knit blocks with 88% porosity showed excellent deformability , indicating potential appropriateness for bone defect filling . 
 although the porosities of the knit block were different , they indicated similar elastic modulus smaller than 1 gpa . 
 the elastic modulus after deformation increased linearly as the applied compression stress increased . 
 the micro - ct analysis indicated that in the block with 50% porosity new bone filled nearly all of the pore volume four weeks after implantation . 
 in contrast , in the block with 88% porosity , new bone filled less than half of the pore volume even 12 weeks after implantation . 
 the histological analysis also indicated new bone formation in the block.conclusionsthe titanium fibre knit block with high porosity is potentially appropriate for bone defect filling , indicating good bone ingrowth after porosity reduction with applied compression .