decompressive craniectomy is very effective method of reducing intracranial pressure . in a recent prospective clinical trial , decompressive craniectomy was confirmed to have reduced the mortality by 50% in the patients with cerebral edema caused by cerebral infarction17,23,42,43 ) . in addition , it was effective for cerebral edema caused by traumatic brain damage2,19 ) , subarachnoid hemorrhage10,16 ) , intracerebral hemorrhage29 ) , and cranial venous and sinus thromboses7 ) . cranioplasty is performed after craniectomy when intracranial pressure is under control for functional and aesthetic restorations . during the cranioplasty procedure , autologous bone flaps are preferably used due to their advantages in storage , viability , cost , prevention of disease transmission , and aesthetics . however , cranioplasty that uses the autologous bone flap has a risk of developing complications such as infection , intracranial hemorrhage , seizure , and hydrocephalus . long - term complications , such as bone flap resorption and bone non - union , have incidence rates that reached 2 - 17%18,21,33 ) . according to the studies on human cranioplasty , the timing of performing cranioplasty is one of the critical factors that may develop autologous bone flap resorption and bone non - union15 ) . although generally accepted concept about timing of cranioplasty using autologous bone is that early cranioplasty has more risk of infection and delayed cranioplasty has risk of non - union or resoprtion of bone flap4,6,25,44 ) , the debates are still remained . another report showed non - union and/or resorption of bone flap occurred more frequently on early cranioplasty patients who had surgery - related infections35 ) . and the other said there were no relation of bone non - union to timing of cranioplasty32 ) . in this study , cranioplasty was performed on rabbits using frozen autologous bone flaps , while the levels of new bone formation and bone fusion after craniectomy were observed , according to the timing of cranioplasty to evaluate its efficacy . experiments were conducted in compliance with the animal experimentation ethics upon approval from the institutional animal care and use committee of our institute . eight adult ( 16 weeks or older ) male new zealand white rabbits with the body weights ranging from 3.2 kg to 4.2 kg were used in this study . four out of eight subjects were assigned to the delayed cranioplasty group ( dg ) , and the remaining four subjects were assigned to the early cranioplasty group ( eg ) . at day 0 , craniectomy was performed at the right frontoparietal bone of the dg rabbit , and the obtained bone flap was stored in a freezer at a temperature of -80. at day 28 or week 4 , bone defect was made in the right frontoparietal bone of the eg rabbit , and then , the bone flap was stored at the same condition . at day 56 or week 8 , the frozen bone flap was fixed on the bone defect area of the dg and eg rabbits . in order to obtain control data , the same procedures were made on the left cranial bone of the rabbits , and the bone flap was immediately fixed ( control group , cg ) . at day 112 or week 16 , the eg and dg rabbits were sacrificed to conduct radiological and histological tests on their cranial bones ( fig . the time span between craniectomy and cranioplasty was the healing time of bone defect and defined as " the primary healing time " , while the time between the cranioplasty and rabbit sacrifice was the bone formation time of the gap between bone flap and cranium and defined as " the secondary healing time " . in order to induce anesthesia on the rabbits , 0.5 ml / kg of ketamin hcl ( ketalar , yuhan corporation , seoul , korea ) and 0.25 mg / kg of xylazine hcl ( rompun , bayer , pittsburgh , pa , usa ) were mixed and injected intramuscularly . for local anesthesia and hemorrhage control , 2% lidocaine hcl ( yuhan corporation , seoul , korea ) containing 1 : 100000 epinephrine was injected subcutaneously . after a 4-cm long incision was made along the midline of the cranial skin , the muscles and periosteum were incised layer by layer to expose the cranial bone ( fig . in order to make the bone flap , a bone flap margin was formed 3 mm lateral to the sagittal suture , which was located at the right side for the experimental groups and the left side for the control group , using a trephine drill with a 10-mm outer diameter and 9-mm inner diameter . to avoid damaging the dura mater , using a 1-mm round burr , the space between the round bone - flap margin and the cranial bone was expanded out of the bone flap in order to create a 12-mm bone defect diameter ( fig . the reason why the size of the bone defect was decided at 12 mm was that the critical bone defect , which did not cause a spontaneous bone healing in rabbits , was reported to be 12 mm or larger37 ) . 2e ) , while the 9-mm bone flap was lifted in order to form a bone defect ( fig . the blood and soft tissues of the bone flap were removed , and the flap was wrapped up with gauze soaked sterilized saline . each bone flap was put in a sterilized and labeled plastic container before they were kept in a freezer ( cln-540u , nihon freezer , tokyo , japan ) at a temperature of -80. layer - to - layer suture was performed using absorbable sutures ( 3 - 0 vycryl ) and unabsorbable sutures ( 3 - 0 nylon ) . after anesthesia and skin incision that mentioned above , the fibrous tissues , which were formed during the primary healing time , were removed using a curette in order to expose the bone defect area ( fig . afterwards , the autologous bone flap , which has been kept in the freezer , was applied to each labeled rabbit in order to perform cranioplasty . the cranial bone and the bone flap were fixed using a titanium - alloy miniplate and 3-mm screws ( synthes inc . , , the cranial bone of the sacrificed rabbit was scanned by the micro - computed tomography ( micro - ct ) ( skyscan 1173 , skyscan , kontich , belgium ) . 4a , b ) and the three dimensional reconstructed images ( nrecon reconstruction program , skyscan , kontich , belgium ) ( fig . 4c , d ) were obtained . after confirming the round bone flap through the micro - ct images and 3d remodeling images , we defined the region of interest ( roi ) as the region within a 14-mm diameter from center point of bone flap . we measured the bone volume ( volroi ) and the bone surface ( surroi ) of roi ( fig . 5a ) . from volroi and surroi , the bone volume ( volbone flap ) and the bone surface ( surbone flap ) of the bone flap , and the volume afterwards , the ct scan images ( fig . 5b ) and the 3d reconstruction images ( fig . 5c ) of the new bone volume ( volnew ) and the new bone surface ( surnew ) were quantified and measured . volnew = volroi-(volbone flap+volmetal ) surnew = surroi-(surbone flap+surmetal ) after dehydration processing was completed , the graft was put in a methyl methacrylate solution in order to maintain a vacuum state . after the methyl methacrylate solution was permeated , the tissues were put in a 5 ml of methyl methacrylate solution and 50 l of jb-4 embedding kit component b was added in order to maintain a vacuum state for 10 minutes . nitrogen gas was injected to be activated at room temperature ( 24 - 28 ) for 24 hours and to prepare a plastic block . the tissues were consecutively cut in a 10-m thickness using a hard tissue cutter ( jung polycuts , leica biosystems , nussloch , germany ) . the sliced tissues were stained through the use of hematoxylin and eosin ( h - e ) , while goldner 's modified masson trichrome methods were used for optical microscopic observations . chicago , il , usa ) was used for statistical analyses . as a non - parametric test , kruskal - wallis test was used for the comparison among the dg , eg , and cg . as a post hoc test experiments were conducted in compliance with the animal experimentation ethics upon approval from the institutional animal care and use committee of our institute . eight adult ( 16 weeks or older ) male new zealand white rabbits with the body weights ranging from 3.2 kg to 4.2 kg were used in this study . four out of eight subjects were assigned to the delayed cranioplasty group ( dg ) , and the remaining four subjects were assigned to the early cranioplasty group ( eg ) . at day 0 , craniectomy was performed at the right frontoparietal bone of the dg rabbit , and the obtained bone flap was stored in a freezer at a temperature of -80. at day 28 or week 4 , bone defect was made in the right frontoparietal bone of the eg rabbit , and then , the bone flap was stored at the same condition . at day 56 or week 8 , the frozen bone flap was fixed on the bone defect area of the dg and eg rabbits . in order to obtain control data , the same procedures were made on the left cranial bone of the rabbits , and the bone flap was immediately fixed ( control group , cg ) . at day 112 or week 16 , the eg and dg rabbits were sacrificed to conduct radiological and histological tests on their cranial bones ( fig . the time span between craniectomy and cranioplasty was the healing time of bone defect and defined as " the primary healing time " , while the time between the cranioplasty and rabbit sacrifice was the bone formation time of the gap between bone flap and cranium and defined as " the secondary healing time " . in order to induce anesthesia on the rabbits , 0.5 ml / kg of ketamin hcl ( ketalar , yuhan corporation , seoul , korea ) and 0.25 mg / kg of xylazine hcl ( rompun , bayer , pittsburgh , pa , usa ) were mixed and injected intramuscularly . for local anesthesia and hemorrhage control , 2% lidocaine hcl ( yuhan corporation , seoul , korea ) containing 1 : 100000 epinephrine was injected subcutaneously . after a 4-cm long incision was made along the midline of the cranial skin , the muscles and periosteum were incised layer by layer to expose the cranial bone ( fig . in order to make the bone flap , a bone flap margin was formed 3 mm lateral to the sagittal suture , which was located at the right side for the experimental groups and the left side for the control group , using a trephine drill with a 10-mm outer diameter and 9-mm inner diameter . to avoid damaging the dura mater , the space between the round bone - flap margin and the cranial bone was expanded out of the bone flap in order to create a 12-mm bone defect diameter ( fig . 2d ) . the reason why the size of the bone defect was decided at 12 mm was that the critical bone defect , which did not cause a spontaneous bone healing in rabbits , was reported to be 12 mm or larger37 ) . 2e ) , while the 9-mm bone flap was lifted in order to form a bone defect ( fig . the blood and soft tissues of the bone flap were removed , and the flap was wrapped up with gauze soaked sterilized saline . each bone flap was put in a sterilized and labeled plastic container before they were kept in a freezer ( cln-540u , nihon freezer , tokyo , japan ) at a temperature of -80. layer - to - layer suture was performed using absorbable sutures ( 3 - 0 vycryl ) and unabsorbable sutures ( 3 - 0 nylon ) . after anesthesia and skin incision that mentioned above , the fibrous tissues , which were formed during the primary healing time , were removed using a curette in order to expose the bone defect area ( fig . afterwards , the autologous bone flap , which has been kept in the freezer , was applied to each labeled rabbit in order to perform cranioplasty . the cranial bone and the bone flap were fixed using a titanium - alloy miniplate and 3-mm screws ( synthes inc . , in order to induce anesthesia on the rabbits , 0.5 ml / kg of ketamin hcl ( ketalar , yuhan corporation , seoul , korea ) and 0.25 mg / kg of xylazine hcl ( rompun , bayer , pittsburgh , pa , usa ) were mixed and injected intramuscularly . for local anesthesia and hemorrhage control , 2% lidocaine hcl ( yuhan corporation , seoul , korea ) containing 1 : 100000 epinephrine was injected subcutaneously . after a 4-cm long incision was made along the midline of the cranial skin , the muscles and periosteum were incised layer by layer to expose the cranial bone ( fig . in order to make the bone flap , a bone flap margin was formed 3 mm lateral to the sagittal suture , which was located at the right side for the experimental groups and the left side for the control group , using a trephine drill with a 10-mm outer diameter and 9-mm inner diameter . to avoid damaging the dura mater , the space between the round bone - flap margin and the cranial bone was expanded out of the bone flap in order to create a 12-mm bone defect diameter ( fig . 2d ) . the reason why the size of the bone defect was decided at 12 mm was that the critical bone defect , which did not cause a spontaneous bone healing in rabbits , was reported to be 12 mm or larger37 ) . 2e ) , while the 9-mm bone flap was lifted in order to form a bone defect ( fig . the blood and soft tissues of the bone flap were removed , and the flap was wrapped up with gauze soaked sterilized saline . each bone flap was put in a sterilized and labeled plastic container before they were kept in a freezer ( cln-540u , nihon freezer , tokyo , japan ) at a temperature of -80. layer - to - layer suture was performed using absorbable sutures ( 3 - 0 vycryl ) and unabsorbable sutures ( 3 - 0 nylon ) . after anesthesia and skin incision that mentioned above , the fibrous tissues , which were formed during the primary healing time , were removed using a curette in order to expose the bone defect area ( fig . afterwards , the autologous bone flap , which has been kept in the freezer , was applied to each labeled rabbit in order to perform cranioplasty . the cranial bone and the bone flap were fixed using a titanium - alloy miniplate and 3-mm screws ( synthes inc . , west chester , pa , usa ) ( fig . at week 16 , the cranial bone of the sacrificed rabbit was scanned by the micro - computed tomography ( micro - ct ) ( skyscan 1173 , skyscan , kontich , belgium ) . 4a , b ) and the three dimensional reconstructed images ( nrecon reconstruction program , skyscan , kontich , belgium ) ( fig . 4c , d ) were obtained . after confirming the round bone flap through the micro - ct images and 3d remodeling images , we defined the region of interest ( roi ) as the region within a 14-mm diameter from center point of bone flap . we measured the bone volume ( volroi ) and the bone surface ( surroi ) of roi ( fig . 5a ) . from volroi and surroi , the bone volume ( volbone flap ) and the bone surface ( surbone flap ) of the bone flap , and the volume afterwards , the ct scan images ( fig . 5b ) and the 3d reconstruction images ( fig . 5c ) of the new bone volume ( volnew ) and the new bone surface ( surnew ) were quantified and measured . after dehydration processing was completed , the graft was put in a methyl methacrylate solution in order to maintain a vacuum state . the tissues were put in a 5 ml of methyl methacrylate solution and 50 l of jb-4 embedding kit component b was added in order to maintain a vacuum state for 10 minutes . nitrogen gas was injected to be activated at room temperature ( 24 - 28 ) for 24 hours and to prepare a plastic block . the tissues were consecutively cut in a 10-m thickness using a hard tissue cutter ( jung polycuts , leica biosystems , nussloch , germany ) . the sliced tissues were stained through the use of hematoxylin and eosin ( h - e ) , while goldner 's modified masson trichrome methods were used for optical microscopic observations . chicago , il , usa ) was used for statistical analyses . as a non - parametric test , kruskal - wallis test was used for the comparison among the dg , eg , and cg . as a post hoc test no subject died or experienced complications such as infection , and none of them dropped out of the experiments conducted in this study . the new bone formation observed in the micro - ct images was represented as bone volume ( volnew ) and bone surface ( surnew ) ( table 1 ) . the mean new bone volume of the dg was 53.7810.86 mm , while the mean bone surface was 726.60170.99 mm . the mean new bone volume and mean bone surface of the eg were 50.137.18 mm and 706.2377.26 mm , respectively . the mean new bone volume of the cg was 31.5112.84 mm , while the mean bone surface was 436.65132.24 mm . statistically significant differences were observed in the mean new bone volume ( p=0.024 ) and bone surface ( p=0.007 ) among the three groups ( table 2 ) . in the post hoc test , no statistically significant difference in bone volume ( p=0.886 ) and bone surface ( p=0.886 ) was observed between the dg and eg . when the cg was compared with the eg and dg , significant differences in bone volume ( p=0.028 ) and bone surface ( p=0.008 ) similar to the findings in the micro - ct images , new bone formation was observed in the microscopic examination of the h - e stain and goldner 's stain in all of the rabbit tissues . the new bone formation was observed as a form of 1 ) a bony islet between the bone flap and the edge of the cranial bone ( fig . 6a ) , 2 ) a bony islet at the lower part of the bone flap ( fig . 6b ) , 3 ) a new bone formation at the edge of the cranial bone ( fig . 6c ) , and 4 ) a bone flap incorporation between the bone flap and the edge of the cranial bone ( fig . cranioplasty , which is performed after craniectomy , has aesthetic and functional advantages , and it is used in a wide variety of diseases . however , various complications such as infection , intracranial hemorrhage , bone flap resorption , depressed bone flap , cerebral vasospasm , and hydrocephalus can develop after the cranioplasty procedure , and their incidence rate has been reported to be 15 - 36.5%4,5,8,14,20,27,32,36 ) . the materials that are most frequently used in cranioplasty include autologous bone flap , poly - methyl - methacrylate ( pmma ) , and hydroxy apatite . the pmma is known to show a similar frequency of complication development to that of the autologous bone flap , but its osteogenesis capability is much less than that of the autologous bone flap22,24 ) . hydroxy apatite can supplement the low osteogenesis capability of pmma ; however , it is expensive38 ) . the autologous bone flap is the most widely used cranioplasty material due to its superiority in the viability , cost , prevention of disease tranmission , and aesthetics to artificial bones . nevertheless , long - term complications such as bone non - union and resorption , which develop after the cranioplasty procedure using the autologous bone flap , are the major causes that require re - operation . in a cranioplasty using autologous bone flap , bone flap incorporation occurs in the process of revascularization , osteoconduction , resorption , and osteogenesis12 ) . in a bone union process , bone remodeling is completed through the repetition of bone resorption and new bone formation . bone resorption is known to happen when problems arise during the bone reunion process39 ) . the patient - side risk factors of bone resorption are known to include young age , traumatic cranial damage , multiple cranial fractures , and the size of the cranial defect . meanwhile , iatrogenic factors , which include the timing of cranioplasty , have been reported13,15,21,40 ) . according to previous studies conducted on the timing of cranioplasty , its early performance resulted in an increase in the incidence of infection , while delayed performance resulted in an increase in the resorption of the autologous bone flap4,6,25,35,44 ) . however , piedra et al.32 ) reported that there is no difference in the frequency of the development of complications , such as bone non - union and resorption , and in the infection between the early and delayed cranioplasty performance groups . in the case of cranioplasty performed within two months after craniectomy , schuss et al.35 ) reported a high frequency of infection , and resultant bone non - union and resorption . in this study , the timing of cranioplasty was set at 4 and 8 weeks with the eg and dg , and no significant difference in new bone formation was observed between the groups . furthermore , when the frozen autologous bone flap was used on rabbits within 8 weeks , an active new bone formation was radiologically and histologically confirmed , and no significant difference in new bone formation was observed according to the freezing period . there are several methods of storing the autologous cranial bone during the period between the procedures of craniectomy and cranioplasty . the method of freezing the autologous bone flap was introduced in the 1950s1,11,30 ) , and since then , it has been the most widely used method . the most important factor to consider for a successful bone flap incorporation after cranioplasty is the viability of the bone flap9,28,34 ) . furthermore , the frozen autologous bone flap is known to have a comparatively favorable viability . when the frozen autologous bone flap was histologically analyzed at temperatures between -17 and -80 , the harvesian system of the bone tissues and structural proteins were confirmed to have remained the same regardless of the freezing period3,31,34 ) , and the osteocytes were observed even until the 35th month after freezing34 ) . moreover , the freezing storage method is superior to the other methods in terms of preserving the cellular architecture30,34 ) . an example of this is the cytoplasm of the lacunar cells , which was well preserved41 ) . in the frozen cranial bone , new blood vessels are remodeled , and they play the role of an architectural frame that assists the growth of the osteoprogenitor cells . in addition , revascularization and infiltration of the osteoblasts occur from the edge of the cranial bone toward the bone flap34 ) . a new bone formation was observed at the edge of the cranial bone close to the bone flaps of all the dg and eg rabbits not only in the micro - ct , but also in the histological results . a bony islet formation was also observed . according to sultan et al.41 ) , the bony islet formation contributed to the promotion of bone flap incorporation and the reduction of bone flap resorption . when cranioplasty is performed immediately after craniectomy , the best bone flap viability and a favorable new bone formation are usually observed41 ) . in this study , however , the cg 's new bone formation was significantly the worst when compared with the eg and dg . this may be due to the difference in the total healing time - no primary healing time in the cg , 8 weeks in the dg and 4 weeks in the eg - even though the three groups had the same secondary healing time of 8 weeks . according to sohn et al.37 ) , osteogenesis is a consistent process , but the time of initiating bone marrow maturity and bony islet formation in rabbits after the craniectomy procedure was 8 weeks after the surgery . this means that it takes 8 to 12 weeks to clearly observe and assess bone flap incorporation , bone remodeling , and osteogenesis . in this study , early bone flap incorporation was assessed through the early assessment on the healing process of the defect area . the primary healing time of the eg and dg was 4 weeks and 8 weeks , respectively , and their total healing time was 12 weeks and 16 weeks , respectively , while the total healing time of the cg was 8 weeks . this implies that the time of initiating new bone formation and bone flap incorporation is affected not only by the healing time after cranioplasty ( secondary healing time ) , but also by the total period after craniectomy ( primary and secondary healing time ) . accordingly , the primary healing time given prior to bone transplantation may positively affect the bone healing process . the bone remodeling period after fracture on rabbits is widely known as 6 - 8 weeks of period-1 week of resoprtion , 0.5 - 1 week of reversal and 4.5 - 6 weeks of bone formation ( on human , total 17 weeks-2 weeks of resorption , 2 weeks of reversal and 13 weeks of bone formation)26 ) . at this point of view , the cranioplasty performing before end of bone remodeling period would have some advantages to obtain bone fusion . thus , we think that cranioplasty performed within 8 weeks of primary healing time could result in good bone fusion . small sample size and the lack of power analysis for estimating appropriate sample are major concerns of this study . despite of small sample size , the experimental result using animal models that the period between craniectomy and cranioplasty could affect the bone fusion is a meaningful result of this study . another limitation is the different periods of total healing time and primary healing time among groups , because the duration of healing time after craniectomy has been known as one of important factors of bone fusion and union . we suggested more precisely designed study that focused on variations of healing duration among the groups should be mandatory . considering the preliminary characteristic of this study , these limitations are expected to be surmounted on well - designed large study on future . in this study , the cranioplasty procedure conducted on rabbits using the autologous bone flap frozen for less than 8 weeks resulted in favorable new bone formation and bone flap incorporation . furthermore , not only the post - cranioplasty healing time ( secondary healing time ) , but also the primary healing time , which was the time between the procedures of craniectomy and cranioplasty , were considered to positively affect the new bone formation and bone flap incorporation processes . based on the confirmation in this study regarding the positive healing process of rabbits even during the delayed period , the delayed period between the craniectomy and cranioplasty procedures in humans may positively affect the bone flap incorporation process .
objectivethe timing of cranioplasty and method of bone flap storage are known risk factors of non - union and resorption of bone flaps . in this animal experimental study 
 , we evaluated the efficacy of cranioplasty using frozen autologous bone flap , and examined whether the timing of cranioplasty after craniectomy affects bone fusion and new bone formation.methodstotal 8 rabbits ( male , older than 16 weeks ) were divided into two groups of early cranioplasty group ( eg , 4 rabbits ) and delayed cranioplasty group ( dg , 4 rabbits ) . 
 the rabbits of each group were performed cranioplasty via frozen autologous bone flaps 4 weeks ( eg ) and 8 weeks ( dg ) after craniectomy . in order to obtain control data , 
 the cranioplasty immediate after craniectomy were made on the contralateral cranial bone of the rabbits ( control group , cg).the bone fusion and new bone formation were evaluated by micro - ct scan and histological examination 8 weeks after cranioplasty on both groups.resultsin the micro - ct scans , the mean values of the volume and the surface of new bone were 50.137.18 mm3 and 706.2377.26 mm2 in eg , 53.7810.86 mm3 and 726.60170.99 mm2 in dg , and 31.5112.84 mm3 and 436.65132.24 mm2 in cg . in the statistical results , significant differences were shown between eg and cg and between dg and cg ( volume : p=0.028 and surface : p=0.008 ) . 
 the histological results confirmed new bone formation in all rabbits.conclusionwe observed new bone formation on all the frozen autologous bone flaps that was stored within 8 weeks . 
 the timing of cranioplasty may showed no difference of degree of new bone formation . 
 not only the healing period after cranioplasty but the time interval from craniectomy to cranioplasty could affect the new bone formation .