preparation of tetrapod - shaped alpha tricalcium phosphate granules ( tb ) : 
 the procedure used in the preparation of the tb was previously described , and the materials ( tetrabones ) were 
 obtained from next21 , k.k .
( tokyo , japan ) . preparation of bfgf - binding ion complex gel matrix ( f - ic gel ) : the 
 procedure used in the preparation of the f - ic gel was previously described [ 18 , 19 , 22 ] .
briefly , the ic gel formed spontaneously at room 
 temperature by mixing the same volume of alkali - treated collagen modified with citric acid 
 derivatives ( cads ) and atelocollagen derived from pig tissues ( nitta gelatin inc . ,
, recombinant human bfgf ( kaken pharmaceutical co. , ltd . , tokyo , 
 japan ) was added to the mixed solution at a final concentration of 100 
 ng / ml .
animals and surgical procedures : all the animal experiments in this study 
 were conducted in accordance with the guidelines of the animal care committee of the 
 graduate school of agricultural and life sciences at the university of tokyo .
five male 
 beagle dogs , aged 1319 months and weighing 10.511.3 kg , were used in the experiment .
intravenous propofol ( 8 mg / kg ) was used to induce anesthesia , which was maintained using 
 isoflurane ( 1.52.5% ) in oxygen .
fentanyl hydrate ( 1020 
 gkghr ) was administered as a constant - rate infusion 
 throughout the surgery to induce analgesia .
lactated ringer s solution ( 10 
 mlkghr ) was infused , and intravenous cefazolin ( 20 mg / kg ) 
 was administered before the skin incision was made and every 2 hr thereafter .
after 
 sterilization , the radius shaft was exposed through a craniolateral skin incision , and a 
 critically sized ( 20-mm long ) mid - diaphyseal segmental defect   was created using an oscillating saw .
a defect of the same size was 
 created in each side of the radius . a polypropylene mesh cage ( pmc ) , 9 mm in diameter and 28 
 mm in height , was fabricated to hold the tb granules in the defect , as previously described 
 ( fig .
both ends of the pmc were tucked between the 
 plate and intact bone ends .
then , the pmc containing the tb with or 
 without the bfgf or f - ic gel was inserted to the defect and fixed with an 8-hole stainless 
 bone plate 1.8 mm thick ( custom - made buttress plate , mizuho co. , ltd . ,
tokyo , japan ) and 8 
 screws ( 2.7-mm stainless cortical screw , synthes vet , tokyo , japan ) .
both ends of the pmc 
 were tucked between the plate and the intact bone ends .
cefazolin ( 20 mg / kg , subcutaneously twice a day ) and 
 buprenorphine ( 15 g / kg , intramuscularly 3 times a day ) were administered 
 for 3 days after surgery .
a robert - jones bandage was used in all the dogs 1 day after 
 surgery .
both ends of the pmc were tucked between the 
 plate and intact bone ends .
five dogs with 10 radial defects were included in this study and were randomly 
 divided into the following groups : tb group ( n=4 ) , implanted with pmc and tb ; 
 tb / f group ( n=3 ) , implanted with pmc , tb ( 1.2 g ) and bfgf solution ( 100 
 ng / ml , 1.0 ml ) ; and tb / f - ic group 
 ( n=3 ) , implanted with pmc , tb and f - ic gel ( 100 ng / ml , 1.0 
 ml ) . in the tb / f group , 100-ng / ml 
 bfgf / phosphate - buffered saline solution was poured into the tb during surgery .
fluorescent bone labeling : the dogs were intravenously injected with 
 oxytetracycline ( tc ; 20 mg / kg ; terramycin / la , pfizer japan , inc . , tokyo , japan ) 14 days 
 after surgery and calcein ( cl ; 20 mg / kg ; sigma - aldrich co. , tokyo , japan ) 21 days after 
 surgery .
radiography and micro - computed tomography : lateral radiographs were 
 obtained immediately after surgery and upon euthanasia . after removing the metallic 
 implants , with
the pmc retained , micro - computed tomographic ( micro - ct ) images ( smx-90ct , 
 shimadzu co. , kyoto , japan ) of the defects at the midsagittal sections were obtained .
histomorphometry : the harvested radius was fixed with 70% ethanol for 5 
 days , stained with villanueva bone stain for 7 days , dehydrated in ascending grades of 
 ethanol , defatted in an acetone - methyl methacrylate monomer mixture ( 1:2 ) and embedded in 
 methyl methacrylate ( wako chemicals , odawara , japan ) without decalcification .
histological 
 sections of 40-m thickness were cut along the midsagittal plane from the 
 intact proximal and distal ends of the radius and defect .
the specimens were examined 
 microscopically ( bx-53 , olympus co. , tokyo , japan ) under natural and polarized light and on 
 fluorescence microscopy .
the histomorphometric measurements were made using a semiautomatic 
 image analyzing system ( system supply , ina , japan ) .
the standard bone histomorphometric 
 nomenclature , symbols and units described in the report of the american society for bone and 
 mineral research histomorphometry nomenclature committee   were used . the ratios ( 100% ) of bone distance - tissue distance ( bd / td ) , bone 
 volume - tissue volume ( bv / tv ) and lamellar bone volume / bone volume ( lbv / bv ) ; the number of 
 vessels / analysis area ( n.ve/mm ) and mineral apposition rate ( mar , 
 m / day ) were calculated for each sample .
the bd / td was calculated for 
 each sample in the midline of the defect .
lamellar bone was defined as the regular parallel alignment of collagen as distinguished 
 from woven bone , which was defined as a haphazard organization of collagen fibers . 

n.ve/mm was calculated in 3 longitudinal areas ( cranial , medial and caudal ) of 
 the defect . a 20-mm rectangle ( 1  20 mm ) in each area was analyzed .
vessels 
 were defined as luminal structures containing red blood cells . using fluorescence 
 microscopy ,
the mar was calculated by dividing the bone thicknesses between the pairs of 
 lines marking the administration of the fluorescent bone markers tc and cl by the time 
 intervals between the doses ( tc - cl , days 14 and 21 ; cl - osteoid , days 21 and 28 ) . statistical analysis : results were expressed as mean  standard deviation . 

statistical analysis was performed using the statview version 5.01 software ( sas institute 
 inc . ,
statistical differences were analyzed using one - way analysis of 
 variance followed by the tukey - kramer test for multiple comparisons .
surgical outcome and clinical findings : macroscopically , the defect and 
 pmc were stabilized well using a plate and screws without tb leakage during the surgery . all 
 the dogs tolerated the surgical procedures and had a complete , uneventful recovery .
c ) radiographs and ( a  c ) micro - computed tomographic images of the radial segmental 
 bone defects in each treatment group 4 weeks after surgery .
( a , a ) tb group ; ( b , b ) 
 tb / f group ; and ( c , c ) tb / f - ic group .
the arrows indicate the osseous callus formation 
 extending from the intact proximal bone cortex over the defect .
dr : distal radius , pr : 
 proximal radius . ) and micro - ct images ( fig .
2a2c ) showed 
 that the tb granules were placed in the defects without leakage but with slight caudal 
 displacement of the middle portion of the tb .
although it was difficult to distinguish the 
 newly formed bone in the intergranular pores of the tb , osseous callus formation extending 
 from the intact bone cortex over the implant was observed 4 weeks after surgery in each 
 group .
( a  c ) radiographs and ( a  c ) micro - computed tomographic images of the radial segmental 
 bone defects in each treatment group 4 weeks after surgery .
( a , a ) tb group ; ( b , b ) 
 tb / f group ; and ( c , c ) tb / f - ic group .
the arrows indicate the osseous callus formation 
 extending from the intact proximal bone cortex over the defect .
j ) histological sections ( villanueva bone stain ) at the midsagittal plane of the 
 tb ( a ) , tb / f ( b ) and tb / f - ic groups ( c ) 4 weeks after surgery .
dr : distal radius , pr : 
 proximal radius , pmc : polypropylene mesh cage .
the arrows indicate the distance of the 
 distal and proximal new bone tissues from the midline of the defect .
scale bar=5 
 mm . shows the histological sections for each group . in the tb / f - ic group ,
more newly 
 formed bone was observed , especially from the distal radius , compared with that in the tb 
 and tb / f groups .
4.histological findings of newly formed bone ( a  c : natural light , d  f : polarized light ) 
 and neovascularization ( g  i : natural light ) in the tb ( a , d and g ) , tb / f ( b , e and h ) 
 and tb / f - ic groups ( c , f and i ) 4 weeks after surgery .
. shows the histological findings from the defect in each group . as shown in fig .
4a4c , the histological sections under natural 
 light revealed more extensive bone formation in the tb / f - ic group than in the tb and tb / f 
 groups
. detailed analysis of the histological sections under polarized light revealed that 
 the newly formed bone in each group mainly consisted of woven bone ( fig .
furthermore , more neovascularization was observed in the 
 tb / f - ic group than in the tb and tb / f groups ( fig . 

the yellow and 
 green lines indicate the tetracycline and calcein signals , respectively , in the tb 
 ( a ) , tb / f ( b ) and tb / f - ic groups ( c ) 4 weeks after surgery : the thicknesses ( i ) from 
 day 14 to 21 and ( ii ) from day 21 to 28 . scale bar=50 m . shows the histological sections of the fluorescence - labeled newly formed bone with tc 
 ( yellow lines ) and cl ( green lines ) signals in each group .
the mineral apposition during 
 certain periods after implantation was analyzed by examining the incorporation of the 
 fluorescence labels , tc and cl , into the bones .
the bone thicknesses of the 2 pairs of 
 lines , tc - cl and cl - osteoid , did not differ grossly among the groups ( fig .
( a  j ) histological sections ( villanueva bone stain ) at the midsagittal plane of the 
 tb ( a ) , tb / f ( b ) and tb / f - ic groups ( c ) 4 weeks after surgery .
dr : distal radius , pr : 
 proximal radius , pmc : polypropylene mesh cage .
the arrows indicate the distance of the 
 distal and proximal new bone tissues from the midline of the defect .
histological findings of newly formed bone ( a  c : natural light , d  f : polarized light ) 
 and neovascularization ( g  i : natural light ) in the tb ( a , d and g ) , tb / f ( b , e and h ) 
 and tb / f - ic groups ( c , f and i ) 4 weeks after surgery .
the yellow and 
 green lines indicate the tetracycline and calcein signals , respectively , in the tb 
 ( a ) , tb / f ( b ) and tb / f - ic groups ( c ) 4 weeks after surgery : the thicknesses ( i ) from 
 day 14 to 21 and ( ii ) from day 21 to 28 .
6.results of the histomorphometric analyses of the newly formed bone 4 weeks after 
 surgery .
( a ) the distances of the proximal and distal new bone tissues per total 
 tissue distance ( bd / td ) , ( b ) the volume of bone tissue per volume of total tissue 
 ( bv / tv ) , ( c ) the volume of lamellar bone tissue per volume of bone tissue ( lbv / bv ) , 
 ( d ) the number of vessels per square millimeter ( n.ve/mm ) and ( e ) the 
 mineral apposition rate ( mar ) .
the 
 asterisks indicate statistically significant differences between the groups ( one - way 
 analysis of variance followed by the tukey - kramer test for multiple comparison ; 
 p<0.05 ) . shows the results of the histomorphometry using the histological findings in figs . 4 and 
 5 . as shown in fig .
6 ( a , b and d ) , 
 bd / td , bv / tv and n.ve/mm were significantly higher in the tb / f - ic group than in 
 the other groups 4 weeks after surgery .
the values of bd / td , bv / tv and n.ve/mm 
 in the tb / f - ic group were 61.6  7.0% , 17.9  2.0% and 7.3  0.4 , respectively ; however , no 
 significant differences in these 3 parameters were found between the tb / f and tb groups .
the 
 values of bd / td , bv / tv and n.ve/mm in the tb versus tb / f group were 36.9  2.6% 
 versus 44.1  2.9% , 10.1  0.3% versus 9.5  1.3% and 3.7  0.3 versus 4.5  0.8 , 
 respectively .
as shown in fig . 6 ( c and 
 e ) , no significant differences in lbv / bv and mar values were observed 
 between the groups .
results of the histomorphometric analyses of the newly formed bone 4 weeks after 
 surgery .
( a ) the distances of the proximal and distal new bone tissues per total 
 tissue distance ( bd / td ) , ( b ) the volume of bone tissue per volume of total tissue 
 ( bv / tv ) , ( c ) the volume of lamellar bone tissue per volume of bone tissue ( lbv / bv ) , 
 ( d ) the number of vessels per square millimeter ( n.ve/mm ) and ( e ) the 
 mineral apposition rate ( mar ) .
the 
 asterisks indicate statistically significant differences between the groups ( one - way 
 analysis of variance followed by the tukey - kramer test for multiple comparison ; 
 p<0.05 ) .
in our recent study , the combination of tb and f - ic gel facilitated neovascularization and 
 new bone formation in a rabbit segmental femoral defect model .
the results also demonstrated that the combination successfully 
 facilitated neovascularization and new bone formation within 4 weeks after surgery in 
 critically sized ( 20 mm ) segmental radial defects in dogs . in the present study ,
the number of vessels in the defects in the tb / f - ic group was 
 significantly higher than that in the tb and tb / f groups , whereas no significant difference 
 was found between the tb and tb / f groups .
these results suggest that the f - ic gel induced 
 angiogenic effects in the dogs .
our previous studies demonstrated that the ic gel alone 
 markedly induced vessel growth and that its combined use with bfgf significantly enhanced 
 the vascularization ability of the ic gel in rats   and rabbits .
furthermore , the enhanced 
 neovascularization in the tb / f - ic group was accompanied by a significantly greater ingrowth 
 of newly formed bone than that in the tb and tb / f groups , suggesting that the increased 
 formation of new bone might be because of the promotion of neovascularization by the f - ic 
 gel .
vessels produced in the defect were essentially 
 within the ecm , which functioned as a natural scaffold for the vessel structure .
the ic gel 
 has a stable three - dimensional matrix structure and is degradable to develop vascular 
 network by a variety of proteases generally released from vascular cells and related cells . 

another crucial feature of the ic gel is that it serves as a reservoir of the bfgf as an 
 angiogenic factor .
given that bfgf is positively charged and the cad part of the ic gel is 
 negatively charged , the bfgf can bind to the cad with ionic bonds , fixing itself inside the 
 ic gel .
therefore , the bfgf stimulates neovascularization and accelerates the development of 
 a vascular network in the gel .
the concentration of bfgf ( 100 
 ng / ml ) in the ic gel used in this study was the minimum 
 level required to achieve the full effect on neovascularization .
bfgf is known as an osteoinductive factor [ 2 , 17 ] and has been demonstrated at a single local 
 application ( 100200 g / site ) to produce osteoinduction in animal bone 
 defects or fracture models in several species including rats , rabbits , dogs and nonhuman 
 primates [ 9 , 11 , 12 , 15 ] .
kawaguchi et al . 
 reported that the bfgf at high concentrations acted on osteoblastic cells and stimulated not 
 only bone formation but also bone resorption .
recent studies demonstrated that using a low 
 dose of the bfgf ( rabbits , 1.4 g / site ; dogs , 0.15 
 g / site ) combined with collagen minipellets as a drug delivery system 
 successfully facilitated bone regeneration in femoral segmental defects in rabbits and 
 guided bone regeneration ( gbr ) sites in dogs [ 7 , 8 ] .
although the bfgf dose ( 100 ng / site ) 
 in this study was much lower than that used in previous studies , the f - ic gel in this study 
 successfully facilitated bone regeneration via the binding of the bfgf to the ic gel with 
 ionic bonds .
in addition , the f - ic gel was compatible with various granular artificial bones , in 
 contrast to the collagen minipellets .
the tb used in this study consisted of 1-mm 
 tetrapod - shaped granules and formed intergranular pores of an appropriate size ( 100400 
 m ) , interconnecting to facilitate cellular and vascular invasions when 
 packed together .
therefore , the combination of the 
 tb and f - ic gel may provide an ideal scaffold for bone regeneration in large bone defects 
 that require an osteoconductive scaffold and vascular network . in this study
, we calculated the lbv / bv and mar to precisely evaluate the osteoinductive 
 effect of the low - dose bfgf in the ic gel .
the lbv / bv value indicates the percentage of 
 remodeling area occupied by newly formed bone and is supposed to reflect an aspect of the 
 bone healing process in which initially formed bone , woven bone , is gradually replaced by 
 lamellar bone through cooperative actions of osteoblasts and osteoclasts .
meanwhile , the mar 
 value indicates the linear rate of production of calcified bone matrix by the osteoblasts . 

the present study demonstrated that neither the lbv / bv nor mar values differed significantly 
 among the groups , suggesting that the ic gel in combination with the bfgf facilitated 
 increased bone regeneration through neovascularization rather than by enhancing bone 
 turnover , although the bfgf within the ic gel might have also contributed to bone 
 regeneration .
however , longer observation periods than 4 weeks are needed to get more 
 precisely evaluation of bone turnover , because lamellar bone appears relatively late phase 
 of the bone healing process . in conclusion , the present study revealed that the combination of tetrapod - shaped alpha 
 tricalcium phosphate granules and the f - ic gel facilitated bone regeneration by inducing 
 neovascularization in a canine segmental radial defect model .
this combination may be a 
 clinically suitable scaffold for the treatment of segmental long bone defects . however , 
 additional long - term studies are necessary to predict its clinical efficacy .
 abstractthe effect of tetrapod - shaped 
 alpha tricalcium phosphate granules ( tetrabones [ tb ] ) in combination with 
 basic fibroblast growth factor ( bfgf)-binding ion complex gel ( f - ic gel ) on bone defect 
 repair was examined . 
 bilateral segmental defects 20-mm long were created in the radius of 
 5 dogs , stabilized with a plate and screws and implanted with 1 of the following : tb ( tb 
 group ) , tb and bfgf solution ( tb / f group ) , and tb and f - ic gel ( tb / f - ic group ) . 
 dogs were 
 euthanized 4 weeks after surgery . 
 radiographs showed well - placed tb granules in the 
 defects and equal osseous callus formation in all the groups . 
 histomorphometry revealed 
 that the number of vessels and volume of new bone in the tb / f - ic group were significantly 
 higher than those in the other groups . 
 however , no significant differences in 
 neovascularization and new bone formation were observed between the tb / f and tb groups . 
 
 furthermore , no significant difference in the lamellar bone volume or rate of mineral 
 apposition was observed among groups . 
 these results suggest that increased bone formation 
 might have been because of the promotion of neovascularization by the f - ic gel . 
 therefore , 
 the combinatorial method may provide a suitable scaffold for bone regeneration in large 
 segmental long bone defects . 
MATERIALS AND METHODS
RESULTS
DISCUSSION