autologous bone grafts remain the gold standard for the treatment of congenital craniofacial bone disorders , such as alveolar cleft [ 18 ] . however , autologous bone grafts have potential problems , which include donor site morbidity and limitations to the amount of bone that can be harvested [ 913 ] . porous beta - tricalcium phosphate ( -tcp ) , which is now commercially available , is known for its osteoconductive and biodegradable properties . however , its use as a replacement for autologous bone grafts remains controversial [ 1418 ] . according to recent studies , various growth factors exhibit osteogenic properties [ 8 , 1923 ] , such as bone morphogenetic protein 2 ( bmp-2 ) [ 2436 ] , basic fibroblast growth factor ( b - fgf ) [ 3742 ] , platelet derived growth factor ( pdgf ) [ 4346 ] , transforming growth factor - beta 1 ( tgf-1 ) [ 4750 ] , and vascular endothelial growth factor ( vegf ) [ 5154 ] . in general , growth factors administered in solution form are readily diffused or degraded in vivo [ 26 , 50 , 55 , 56 ] . thus , their enhanced and prolonged bioactivity at the target site is necessary to reduce bolus dosage , especially in pediatric patients . recent studies suggest that granulocyte colony - stimulating factor ( g - csf ) promotes fracture healing or osteogenesis [ 5760 ] . because g - csf is an essential drug most frequently used to treat neutropenia secondary to chemotherapy , it is widely administered not only to adults but also to pediatric patients [ 6169 ] . accordingly , its biosafety is well established through extensive use in clinical contexts compared to other growth factors . commercially available -tcp ( superpore , pentax , tokyo , japan ) was used in the present study as an osteoconductive scaffold and space - maintaining material . to investigate the bone regenerative properties of g - csf , topical supplementation either in solution or in sustained release form with a gelatin hydrogel system the purpose of this study was to investigate whether g - csf with or without a controlled release system stimulates bone regeneration in combination with -tcp using a rat calvarial defect model [ 7073 ] . the present study was approved by the institutional committee of animal experiments at hokkaido university ( institutional animal care and use committee protocol number 12 - 0017 ) . fourteen wistar rats ( male , 13 weeks old ; weight , 250350  g ) were purchased from sankyo labo service corporation ( tokyo , japan ) . a total of 27 calvarial defects were randomly divided into nine treatment groups , with a total of three defects per treatment group . in solution - based treatment groups , defects were filled with a -tcp disc containing normal saline alone ( group a , control ) or 1 ( group b ) , 5 ( group c ) , or 20  g of g - csf ( group d ) . in controlled release groups , defects were filled with a -tcp disk with an overlaid gelatin hydrogel sheet incorporating normal saline alone ( group e ) or , 1 ( group f ) , 5 ( group g ) , or 20  g g - csf ( group h ) . the remaining defects were left empty to measure spontaneous healing ( group i ) ( table 1 ) . commercially available porous -tcp blocks ( superpore ) blocks were cut into discs 5  mm in diameter and 1  mm thick using a fine surgical saw and round bur . briefly , a mixed acidic gelatin - glutaraldehyde aqueous solution was cast into a polypropylene dish ( 80  80  mm ) and maintained at 4c for 12 hours . the water content of gelatin hydrogels ( weight ratio of water present in hydrogel to wet hydrogel ) was 95  wt% . gelatin hydrogels were designed so that degradation would be complete in approximately two weeks under in vivo conditions [ 29 , 39 , 49 , 74 ] . hydrogel sheets were cut into discs 5  mm in diameter and 1  mm thick . human recombinant g - csf was kindly supplied by kyowa kirin co. ( tokyo , japan ) . to prepare gelatin hydrogels incorporating g - csf , 20  l of normal saline solution containing 1 , 5 , or 20  g g - csf similarly , 20  l of g - csf - free normal saline was dropped onto a freeze - dried hydrogel to obtain g - csf empty hydrogels . animals were anesthetized by intraperitoneal administration of pentobarbital sodium ( 50  mg / kg ) . subsequently , a skin incision was made and subperiosteal dissection was performed under a surgical microscope to raise the periosteal flaps . a bone defect 5  mm in diameter was then prepared on each side lateral to the sagittal suture using a fine surgical bur under copious sterile saline irrigation . defects were filled with bone substitutes according to the groups described above ( table 1 and figure 1 ) . periosteal flaps were repositioned using a 4 - 0 nylon suture , and the skin was closed with a running 4 - 0 nylon suture . specimens were prepared for decalcified sectioning by immersing them in 10% ethylenediaminetetraacetic acid ( edta ) for four weeks . embedded samples were then sectioned into 3  m slices parallel to the sagittal suture across the center of each calvarial defect using a microtome ( leica , sm2000r ) . hematoxylin and eosin ( he ) staining was used for histological analysis and aniline blue staining was used for histomorphometric analysis . each specimen was examined under a light microscope and digital photographs were obtained for histological evaluation of a region corresponding to the center of the calvarial defect ( figure 1(b ) ) . high magnification images with aniline blue staining ( 1.001  mm or 1360  1024 pixels ) of the most - central area of the defect were quantified to measure the percentage of newly formed bone and remaining bioceramics using imaging software ( adobe photoshop cs5 ) . statistical analysis was performed using kruskal - wallis one - way analysis of variance ( anova ) . figure 2 shows low magnification images of decalcified specimens stained with he along the midline of each calvarial defect . no remaining gelatin hydrogel or surgical site infections were observed . in group a ( control group ) , newly formed trabecular bone was observed focally but failed to occupy the entire defect . in groups b  d ( solution - based treatment groups ) and in group e ( g - csf - free gelatin hydrogel group ) , newly formed trabecular bone was observable but failed to fill the defect . in group f ( 1  g g - csf gelatin hydrogel group ) , newly formed bone tissue nearly bridged the calvarial gap , whereas residual -tcp was also present . in group g ( 5  g g - csf gelatin hydrogel group ) , most of the defect was occupied with newly formed bone tissue ; moreover , sparse residual -tcp was observed . in contrast , group h ( 20  g g - csf gelatin hydrogel group ) showed focal formation of new bone surrounded by fibrous connective tissue at the superficial area of the defect with the presence of remaining biomaterials . in group i ( untreated defect group ) , the defect was filled with fibrous connective tissue with hardly any newly formed bone . figure 3 shows higher magnification images of groups e ( e ) and g ( g ) . in group g , newly formed bone was observed immediately below the periosteal flap and multinuclear giant cells were detected around the newly formed bone . in contrast , in group e , the formation of fibrous tissue and blood vessels was significant compared with newly formed bone in the subperiosteal region . figure 4 shows high magnification images of aniline blue staining in which matured bone tissue exhibits homogeneous dark blue and entrapped osteocytes . figure 5 shows the percentage of newly formed bone and remaining -tcp per high - powered field . in groups a , b , c , d , e , and h , defects had a tendency to be occupied by more remaining -tcp compared to newly formed bone tissue . in group a ( control ) , the percentages of newly formed bone and remaining -tcp were 20.77%  25.44% and 35.01%  2.01% , respectively . in contrast , in groups f and g ( 1  g and 5  g g - csf gelatin hydrogel groups ) , the percentage of newly formed bone ( 54.84%  9.46% and 69.53%  5.35% for groups f and g , resp . ) conspicuously exceeded values of remaining -tcp ( 20.47%  2.89% and 14.76%  7.36% for groups f and g , resp . ) . the values were significantly higher in groups f and g compared to the control group ( p < 0.01 ) . there was no significant difference between groups a ( control ) and i ( empty defect ) . values corresponding to groups b , c , d , e , and h showed no significant difference compared to that of group a. 
 figure 7 shows the percentage of remaining -tcp , which was used to evaluate biodegradability in vivo . there was no significant difference between groups a , b , c , d , e , f , and h. in contrast , only in group g ( 5  g g - csf gelatin hydrogel group ) the percentage was significantly lower compared to group a ( 14.76%  7.36% versus 33.53%  0.80% , p < 0.05 ) . this result indicated a prominent enhancement of the biodegradable properties of -tcp , which was further accelerated by 5  g g - csf in sustained release form . in the present study , we demonstrated that the controlled release of low - dose ( 1  g and 5  g ) g - csf significantly enhanced bone regeneration when combined with a -tcp disc . moreover , administration of 5  g g - csf using a controlled release system significantly promoted the biodegradable properties of -tcp . according to our results , this tissue - engineering approach combining -tcp and the sustained release of g - csf is potentially feasible and promising for clinical use . to our knowledge , this is the first report which demonstrates the bone regeneration properties of g - csf at membranous ossification sites . because systemic administration of 510  g g - csf / kg / day is commonly used for pediatric malignancies [ 64 , 6668 ] , the results shown here indicate that notably low doses of g - csf ( 15  g / defect/2 weeks ) with controlled release can promote osteogenesis . in this study , we used -tcp as an osteoconductive scaffold and space - maintaining material . in the present study , although the control group ( -tcp alone ) showed a greater tendency for bone formation compared to the untreated defect group , there were no significant differences between the groups . furthermore , the defect in the control group had more residual -tcp than newly formed bone tissue . these results suggest that -tcp alone implantation is not sufficient to fill the defect with regenerated bone in the craniofacial region . some experimental studies have confirmed the osteoconductive properties of -tcp , which were comparable to autologous bone grafts [ 15 , 17 , 70 ] . however , other groups have emphasized versatility by combining -tcp with autologous bone fragments [ 16 , 7779 ] , growth factors [ 45 , 46 , 8084 ] , simvastatin , or stem cells [ 18 , 85 , 86 ] in both experimental and clinical studies . interestingly , ishida et al . reported that topical application of g - csf had bone regenerative properties via neovascularization and osteogenesis . that study revealed a significant increase in cd34 cells  an endothelial and hematopoietic progenitor - enriched cell population  in capillaries corresponding to the bone defect site . the study also showed that g - csf was responsible for mobilizing osteoblasts to the bone defect site . in addition , recent studies demonstrated the promotion of fracture healing by cd34 cells [ 58 , 8789 ] . kuroda et al . reported the first successful clinical case of a tibial nonunion treated with topically applied g - csf - mobilized cd34 cells . some reports have shown that cd34 cells play an important role in releasing angiogenic factors , including vascular endothelial growth factor ( vegf ) , hepatocyte growth factor ( hgf ) , and fibroblast growth factor 2 ( fgf2 ) [ 58 , 90 , 91 ] . moreover , the differentiation capacity of cd34 cells into osteoblasts has been shown in previous reports [ 59 , 92 ] . in the present study , the controlled release g - csf groups showed more newly formed bone immediately below the periosteum compared to the other groups . on the other hand , rojbani et al . reported that osteoprogenitor cells differentiate from the dura mater . presumably , the sustained release of g - csf may stimulate periosteal cells along an osteogenic lineage , resulting in enhanced bone formation . in the present study , we used gelatin hydrogel as a sustained release carrier of g - csf . various growth factors have been shown to have bone regenerative properties , such as bone morphogenetic proteins ( bmps ) [ 2436 ] , b - fgf [ 3742 , 93 ] , pdgf [ 4346 ] , tgf-1 [ 4750 ] , and vegf [ 5154 ] . bmp-2 has the strongest osteoinductive activity in promoting ectopic bone regeneration [ 26 , 29 ] and has been approved by the food and drug administration for use in orthopedics and oral surgery [ 30 , 34 , 35 ] . in general , growth factors administered in solution form are easily diffused or degraded prior to achieving full bioactivity [ 26 , 50 , 55 , 56 ] . therefore , commercially available bmp-2 in combination with a collagen sponge kit must contain milligram amounts of the growth factor ( 1.5  mg / ml ) [ 30 , 34 , 35 ] . potential risk for local inflammatory responses should be taken into consideration after topical application . in order to reduce bolus dosage , enhanced and one of the practical ways to control the in vivo release of growth factors is to use gelatin hydrogel , in which the growth factor is physicochemically immobilized and subsequently released in proportion to hydrogel degradation [ 74 , 94 ] . in the present study , the water content of gelatin hydrogels ( weight ratio of water present in hydrogel to wet hydrogel ) was 95  wt% . the hydrogels were designed so that degradation would be complete in approximately 2 weeks under in vivo conditions [ 29 , 39 , 49 , 50 , 74 ] . gelatin is commercially available and its biosafety is well established through its long clinical use as a plasma expander and drug ingredient . in the present study , the controlled release of 5  g g - csf ( group g ) significantly promoted the osteoconductive properties and biodegradability of -tcp . improved biodegradability compared to hydroxyapatite is a major characteristic of porous -tcp [ 71 , 9597 ] . biodegradability is generally thought to occur in harmony with bone remodeling , in which -tcp allows tissue fluid dissolution and absorption by osteoclasts in vivo [ 72 , 95 ] . reported that g - csf increased both osteoclast activity and bone resorption in the bone marrow , triggering an increase in the number of mesenchymal precursor cells in the bone marrow using a mouse model . in another study , pdgf modified -tcp resorption , although the underlying mechanism was not provided . some studies have shown that bmp-2 does not facilitate -tcp resorption [ 70 , 99 ] . in group g of the present study , we can speculate from the results that the controlled release of 5  g g - csf may stimulate the mobilization and differentiation of mesenchymal precursor cells in the periosteum as well as osteoclast activation . in contrast , group h ( 20  g g - csf gelatin hydrogel group ) showed less new bone formation and -tcp resorption . this might be explained by the multidifferentiation potential of g - csf - mobilized progenitor cells , which is consistent with previously published reports [ 92 , 100102 ] . interestingly , ishida et al . stated that topical application of 50  g g - csf did not induce bone regeneration according to preliminary data . moreover , some reports have shown that sustained release of g - csf enhances tendon - bone integration with significantly more formation of sharpey 's fibers and microvessels . these results led us to speculate that a prolonged high concentration of topical g - csf drives progenitor cells toward fibrous tissue formation rather than osteogenesis . therefore , sustaining relatively low concentrations of topical g - csf can play an important role in inducing balanced bone regeneration and -tcp resorption . our findings suggest an optimal dose of 5  g per defect for controlled release of g - csf , which is consistent with previously published reports [ 59 , 103 ] . first , the study was designed using small animals and a limited number per experimental group . second , although some reports accept the calvarial defect rat model [ 7073 ] , the decortication procedure may not fully reflect clinical situations of congenital craniofacial anomalies , since some evidence suggests that fractures mobilize cd34 cells from the bone marrow into the peripheral blood [ 88 , 89 ] . third , we used histomorphometric analysis to characterize newly formed bone and biodegradation of -tcp ; however , we did not identify cd34 cells or evaluate the activity of osteogenic cells at the bone defect site . future studies should incorporate experimental models without decortication , larger animals , and immunohistochemical analysis . in conclusion , controlled release of 5  g g - csf using a gelatin hydrogel system the present results indicate that the combination of g - csf slow - release and -tcp is feasible and promising for the treatment of congenital craniofacial bone defects .