due to an aging   population together with a lifestyle that requires less movement , many elderly today have weak bone structure . one complication with a weaker bone structure is an increased fracture risk , especially in the vertebras . however there are complications associated with the use of pmma , such as cement leakage and increased risk of adjacent fractures     [ 2 , 3 ] , so there is increased interest in finding alternative materials . calcium phosphate cements ( cpcs ) , which were first presented in the beginning of the 80s     [ 4 , 5 ] , are alternatives to pmma . thanks to the chemical resemblance to bone the cpcs have good biocompatibility   and are resorbed in vivo     [ 6 , 7 ] . cpcs consist of calcium phosphate salts , which upon mixing with water dissolve and then precipitate to form a solid body of either brushite or apatite depending on the ph of the solution . there are already numerous commercial cpcs used in the clinical practice as bone void fillers ; however very few are intended for use in vertebroplasty . there are two major obstacles for the cpcs when to be used in spinal applications : handling and radiopacity . to obtain a cpc with sufficient working time , which can be injected through a g11 or g13 needle without filter pressing , is not an easy task , and various publications on injectability and setting regulators can be found     [ 913 ] . recently an innovative approach was taken where water was exchanged for glycerol as mixing liquid . this gives the surgeon unlimited time for the injection , and the viscosity remains constant until the premixed calcium phosphate cement ( pcpc ) is in place and the cement comes in contact with water . the premixed concept has been shown to work on both brushite and apatite cements     [ 1416 ] . during injection into the vertebra it is important to be able to follow the cement with x - ray to avoid leakage . in the case of pmma this is solved by adding either zro2 or baso4     [ 17 , 18 ] . a number of additives have been suggested to add radiopacity to cpcs , such as bismuth      and strontium . however very little has been published describing cpc with zro2 and baso4 , which are well documented for use in pmma cements . the aim of this paper is to evaluate a premixed calcium phosphate cement containing zro2 , regarding its setting time , compressive strength , and radiopacity as well as its in vivo behaviour in a vertebral rat model . the powder part of the cement consisted of -tricalciumphosphate ( -tcp , fluka ) , monocalcium phosphate monohydrate ( mcpm , scharlau ) , and zirconia ( zro2 , sigma - aldrich ) . mcpm and -tcp were mixed in equimolar amounts . to this 0 , 10 , 20 , 30 , or 40% ( w / w ) zro2 was added . the powder was then mixed with glycerol ( 99% , sigma - aldrich ) . in all evaluated compositions the powder / liquid ratio was 4.2  g / ml . before mixing , the -tcp and zro2 were sterilized using heat ( 200c during 2 hours ) , and the mcpm was sterilized by soaking in 70% ethanol for   > 24 hours . after mixing , the paste was injected into cylindrical moulds  6  mm , height 12  mm open at both ends for compression strength testing and then immersed in 37c phosphate buffered saline ( pbs , ph 7.4 , sigma - aldrich ) to simulate physiological ph and start the setting reaction . after 24 hours the samples were removed from the mould , and a universal testing machine ( shimadzu ags - h ) was used with a cross - head speed of 1  mm / min ; the maximum compressive stress was recorded for each sample . a thin plastic film was placed between the sample and the crosshead in order to reduce the effect of defects deriving from the mould . was injected into 4 cylindrical moulds  6  mm , height 3  mm for setting time . to initiate the setting of the cement the moulds the cement was considered to have set when the sample could support the 453.5  g gillmore needle with a tip diameter of 1.06  mm without breaking . the mean between the time when the sample supported the weight and the previous time where the sample broke under the load was regarded as the setting time . the x - ray opacity was measured at 1  mas , with 40 and 80  kv . a 1  mm sample of a pmma - based cement ( vertebroplastic , depuy , usa ) , which has barium sulphate as radiopacifier , was used as control along with an aluminium wedge 0.52.5  mm in 0.5  mm steps . the cement used in the in vivo studies 1  ml syringes were prefilled with the premixed cement under aseptic conditions 4 days before the in vivo experiments . three - month old , 480600  g male wistar rats were anesthetized with halothane . the surgical area was washed with betadine three times , and a ligature was applied at the root of the tail to prevent bleeding . after removing the distal part of the tail a 2.0  mm diameter and 3.5  mm deep hole was drilled axially into the 4th or 5th tail vertebra . in order to stall the regeneration of bone a stainless steel wire twelve weeks later the animals were anesthetized again , and the implanted wire was removed . the hole was filled either with pmma ( heraeus palacos r ) ( n = 5 ) , pcpc ( n = 5 ) , or with impacted human lyophyilized bone chips ( n = 5 ) , and the wound was closed . the last two vertebrae ( last operated vertebra plus one healthy vertebra ) were fixed in 4% formaldehyde and analyzed with micro - ct and histology . single isotope nano - spect / ct ( bioscan ) imaging acquisitions were performed on 2 - 3 rats from each group in order to follow the integration of the bone substitutes and bone regeneration . nano - spect / ct was carried out weekly for 6 weeks and once again on the 12th week . the rats were inoculated with 0.5  ml , 150  mbq of tc - methyl diphosphonate ( tc - mdp ) through the tail vein under halothane anesthesia . metastable technetium ( tc ) is tagged onto a phosphonate compound such as mdp to generate tc - mdp , which selectively concentrates in osteoblasts . the selective accumulation of tc - mdp is ensured by both chemical adsorption onto the surface of the hydroxyapatite in bone and incorporation into the crystalline structure of hydroxyapatite . two hours later the animals were anesthetized with euthasol intraperitoneally , and 30-minute image acquisitions were performed . regeneration activity was expressed as a percentage compared to the proximal healthy vertebra depending on isotope density . the operated vertebrae were examined with   microtomography ( skyscan 1172 x - ray microtomography skyscan , kontich , belgium ) . scans were carried out applying a 60  kv voltage and an al - filter . microtomographical reconstruction was obtained by rotating the view through 180 degrees ( rotation step 0.5 degrees ) . the formaldehyde - fixed 4th and 5th vertebrae were decalcified by immersing the samples in biodec - r solution for 1 week . five micron longitudinal sections were cut from the paraffin blocks and mounted on glass slides . conventional hematoxylin - eosin ( merck & co ) staining was used to confirm the results of the micro - ct measurements . the compressive strength is reduced with an increasing content of zro2 as seen in figure 1 . when there is no zro2 in the cement the compressive strength is 13.5 ( 0.6 )  mpa , and with 40% zro2 the compressive strength is lowered to 8.0 ( 1.2 ) mpa . an increasing amount of zro2 also increased the setting time as seen in figure 2 . 0% zro2 gives a setting time of 20 ( 4 )  min , 20% zro2 has a setting time of 26 ( 2 ) and the longest setting time was measured with 40% zro2 , 37 ( 2 )  min .   from the x - ray images in figure 3 it could be concluded that 20% zro2 gave a sufficient radiopacity , comparable to 11.5  mm aluminium and vertebroplastic at 40  kv and 80  kv . a good radio contrast was obtained in vivo with the pcpc , which can be observed in the micro - ct images in figure 6 . the animals tolerated all the implants well , there was no sign of wound infection or rejection . the ct graph ( blue line in figure 4 ) follows the structure of the two vertebrae , while the pixel intensity is much higher in the operated vertebra compared to the healthy control vertebra when looking at the red line in figure 4 . the same trend was observed in all animals . in all three groups the values are higher than those of the normal bone as seen in figure 5 . the osteoblast activity in this cavitary defect is higher than the normal bone and remains high during the first 5 weeks after implantation . the overall activity is comparable among the groups , with a slight trend of pcpc for an even higher activity , especially at week 2 , while a slight decrease can be observed in the bone chips and the pmma groups . the osteoblast activity did not return to normal levels ( unit 100 in the graph ) in any animals in any group during the study . in figure 6 as seen in the figure , in the vertebrae filled with   pmma ( upper ) , the defect was completely filled with pmma and no bone formation was observed . this result was consistent in all animals in the pmma group .    in the vertebrae filled with bone chips there was no bony connection between the bone chips and the new bone as seen in the cross - section ct image in figure 6 ( bottom ) . there was some bone formation ; however the bone chips are still demarcated from the bone . in the histology image in figure 7(b ) bone chips are seen to have direct contact with newly formed bone .   in the case of pcpc in the vertebrae ( middle image in figure 6 ) however the zro2 present in the pcpc for radiopacity is not resorbed , and thus residues of the cement can be observed in the newly formed bone . histology results , ( figure 7(a ) ) confirmed the nanospect and ct results for the pcpc . an increasing amount of zro2 in the cement pastes will decrease the amount of reactive material ( mcpm and -tcp ) . when the cement comes in contact with water the reactive material will dissolve and monetite crystals start to form giving the paste its strength . accordingly the compressive strength was reduced from 13.5 ( 0.6 ) to 8.0 ( 1.2 )  mpa with an increase of zro2 from 0 to 40% zro2 ( figure 1 ) . in relation to this 0% zro2 gave a setting time of 20 ( 4 ) minutes compared to 37 ( 2 ) minutes with 40% zro2 ( figure 2 ) . the same trend was seen in a previous publication on premixed monetite cement   where high strength samples also showed shorter setting time .   since the method used for testing the setting time measures how fast the cement obtains a certain strength this relation as the zro2 content increases it was noted during the experiments that the viscosity of the cement decreases . since the zro2 has higher density than the -tcp and mcpm more zro2 implies that the total powder volume will be smaller when using the same weight of powder . with less powder volume the glycerol will more easily encase the powder particles thus reducing the viscosity . with 20% zro2 the compressive strength was 11.8 ( 1.2 )  mpa and the setting time 26 ( 2 ) minutes , which are acceptable values for certain vertebral fractures , and this also gave a good radiopacity . therefore this formulation was chosen for the in vivo studies . as seen in the results from the nano - spect ( figure 5 ) the osteoblast activity in this cavitary defect is higher than the normal bone and remains high during the 12-week duration of the study . although there were too few animals in the study to do statistical analysis on the results from the nano - spect a slight trend could be observed indicating that the pcpc has a positive effect on the osteoblast activity compared to the other groups . the monetite formed when pcpc sets is chemically less stable than apatite under in vivo conditions where the ph is ~7.4 , thus it can be resorbed faster , than , for example , apatite forming cpcs and bone grafts . when the pcpc is resorbed , calcium and phosphate ions are released which stimulate the osteoblasts     . this could explain why the osteoblast activity seems to be slightly higher in the pcpc animals . the pmma fills the defect , and it can not be resorbed , thus any new bone formation into the cavity is blocked . the results from the bone chips on the other hand show new bone formation into the cavity ; however the bone chips do not show signs of bony reconsolidation , and it is clearly demarcated from the host bone . this is probably due to the highly compacted structure of the bone chips , so their osteoinductive potential can only be seen on the contact surface , and penetration of the graft by the host tissue is limited .   in the pcpc group new bone formation was observed and there is connection between the new bone and the material . this indicates that the pcpc has osteoconductive properties , which has been shown for monetite in previous publications     [ 25 , 26 ] . the resorption of pcpc and new bone formation , that is , the remodelling , seems to be in balance in this model . the zro2 is not resorbed ; however the ct results indicate that good bone integration was achieved around the zro2 . previous studies where zro2 was used in contact with bone also report good bone - implant contact . in future studies the bone integration of zro2 will be evaluated more thoroughly . the premixed calcium phosphate cement containing 20% zro2 was found to have excellent handling , good radiopacity , and sufficient strength and setting time . all materials evaluated in the study filled the bone cavity , and in each case there was a strong osteoblast activity at the injury site . however , in spite of the osteoblast activity , pmma blocked bone healing , and even bone chips seemed to be less effective than pcpc .
in   this work a radiopaque premixed calcium phosphate cement ( pcpc ) has been   developed and evaluated in vivo . 
 radiopacity was obtained by adding 040 % zirconia to the cement paste . 
 the effects of zirconia on setting time , strength and radiopacity were evaluated . in the in vivo study a 2 by 3.5  mm cylindrical defect in a rat vertebrae was filled with either the pcpc , pmma or bone chips . 
 nano - spect ct analysis was used to monitor osteoblast activity during bone regeneration . 
 the study showed that by adding zirconia to the cement the setting time becomes longer and the compressive strength is reduced . 
 all materials evaluated in the in vivo study filled the bone defect and there was a strong osteoblast activity at the injury site . in spite of the osteoblast activity , pmma blocked bone healing and the bone chips group showed minimal new bone formation . at 12 weeks the pcpc was partially resorbed and replaced by new bone with good bone ingrowth . 
 the radiopaque pcpc may be considered to be used for minimal invasive treatment of vertebral fractures since it has good handling , radiopacity and allows healing of cancellous bone in parallel with the resorption of the cement .