, the injured bone possesses worse ability to heal and fight infection than normal bone does . in order to solve the problem associated with soft tissue and bone healing in such injuries the technique grafts autogenous cancellous bone matched in shape to the bone defect after debridement , and , while maintaining the wound open , the wound is covered with petroleum jelly gauze and drained . however , advancements in technology have led some researchers to combine vacuum - assisted closure ( vac ) with open bone grafting to treat patients with osteomyelitis or bone non - union , and a better curative effect has been attained . vac technology uses a medical - grade porous foam material ( aperture size 400600 m ) , called vacuseal , packed into a drainage tube with several holes . vacuseal is placed on the wound , and an adherent semipermeable membrane covering makes the wound airtight . this apparatus is then linked to a vacuum through a drainage bottle to create negative pressure . in this study , we propose to verify the impact of vac combined with open bone grafting in promoting rabbit bone graft vascularization ; we also seek to provide a theoretical basis for its clinical application . twenty - four male new zealand white rabbits weighing 2.8 to 3.2 kg aged four to five months ( from the wuhan wanqian jiaxing bio - technology co. , ltd , china ) were used in this study . the rabbits were fed with standard feed and housed in an ambient environment at constant temperature for at least one week prior to surgery . the rabbits were randomly divided into two groups : the experimental group and the control group . rabbits in the experimental group were grafted with allogeneic bone ( made from rabbit cancellous bone of the lower limbs provided by the second clinical college of wuhan university ) onto the proximal femur . then , the rabbits were singly placed in a cage and fed in such a way that they could not turn around to bite the tube . animals in the control group underwent a similar surgical procedure other than the fact that the surgical wound was closed by primary closure rather than with vacuseal . animals in the experimental group were anesthetized by injecting a 3% solution of sodium pentobarbital via the ear vein ( 30 mg / kg ) . following confirmation that the animals were anesthetized fur in the surgical area ( hind leg ) was removed and the skin was prepared using 8% na2s solution . after regular disinfection and draping , a 3 cm long incision was performed posterior to the midline on the thigh to expose the proximal femur . three allogeneic bones were grafted and banded with the proximal femur with a suture ( figure 1a ) . china ) was placed to cover the allogeneic bones ( figure 1b ) and was fixed to the wound with sutures . then , the semipermeable membrane ( smith & nephew co. , england ) was adhered to cover the vacuseal outside the wound ( figure 1c ) , and the dressing was linked to a negative pressure aspirator to maintain continuous vacuum aspiration at a pressure of about 60 mmhg to 75 mmhg ( figure 1d ) . the animals were housed in a special cage and an intramuscular injection of penicillin ( 400,000 u / ci ) was given for three days after surgery . the difference in the surgical procedure between the control and experimental groups was that the wound was sutured with vac in the experimental group but not in the control group . four rabbits from each group were sacrificed with air embolism separately at the 4 , 8 , and 12 week after surgery . then , we estimated the state of bone allograft vascularization in the callus based on the imaging . all the animals were injected with tetracycline hydrochloride ( aladdin reagent co. , ltd , china ) ( 35 mg / kg ) 15 d prior to sacrifice , and they were injected with calcein ( sinopharm chemical reagent co. , ltd , china ) ( 10 mg / kg ) 3 d prior to sacrifice . after sacrificing the animals , the bone allografts of the two groups the allografts were embedded with methyl methacrylate without decalcification over 24 h , and then the specimens were serial sectioned with slices of 5 micron thickness using a microtome ( jung type ) . the bone cortex and callus fluorescence staining for each slice was assessed by leica tcs sp2 confocal microscopy ( leica microsystems ir gmbh . , germany ) , and the distance between the two marking lines comprised of tetracycline hydrochloride and calcein was measured by aim image examiner ( carl zeiss microimaging gmbh . the bone allografts were accurately weighed after being rinsed , defatted , and dried off , then the specimens were decalcified with 0.1 g / ml trichloroacetic acid , and the calcium content of each specimen was determined using back titration . total protein was extracted from cancellous bone by hammer grinding and all protein samples were stored at 80c . the protein of each group was separated by 12% sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) and blotted to a polyvinylidene difluoride ( pvdf ) membrane . the membrane was blocked in tris - buffered saline with tween ( tbst ) with 5% skim milk for 2 h and incubated overnight at 4c with anti - fgf-2 antibody ( boster reagent co. , ltd , china ) ( 1:200 dilution ) . after washing in tbst for 5 times , the membrane was incubated with horseradish - peroxidase ( hrp)-coupled secondary antibody ( 1:50000 dilution ) at room temperature for 2 h. subsequently , the samples were washed with tbst again , and the membranes were incubated with an ecl detection kit ( beijing comwin biotech co. , ltd . , china ) for 5 min and exposed to medical x - ray film . bandscan was used to analyze the grey value of the film . to compare the difference in each test between the groups , spss17.0 for windows measurement data were presented as the mean sd and analyzed by student s t - test . enumeration data were presented as percentages and were analyzed by the chi - square test . in all cases , a significance level of p<0.05 was used . twenty - four male new zealand white rabbits weighing 2.8 to 3.2 kg aged four to five months ( from the wuhan wanqian jiaxing bio - technology co. , ltd , china ) were used in this study . the rabbits were fed with standard feed and housed in an ambient environment at constant temperature for at least one week prior to surgery . the rabbits were randomly divided into two groups : the experimental group and the control group . rabbits in the experimental group were grafted with allogeneic bone ( made from rabbit cancellous bone of the lower limbs provided by the second clinical college of wuhan university ) onto the proximal femur . then , the rabbits were singly placed in a cage and fed in such a way that they could not turn around to bite the tube . animals in the control group underwent a similar surgical procedure other than the fact that the surgical wound was closed by primary closure rather than with vacuseal . animals in the experimental group were anesthetized by injecting a 3% solution of sodium pentobarbital via the ear vein ( 30 mg / kg ) . following confirmation that the animals were anesthetized fur in the surgical area ( hind leg ) was removed and the skin was prepared using 8% na2s solution . after regular disinfection and draping , a 3 cm long incision was performed posterior to the midline on the thigh to expose the proximal femur . three allogeneic bones were grafted and banded with the proximal femur with a suture ( figure 1a ) . china ) was placed to cover the allogeneic bones ( figure 1b ) and was fixed to the wound with sutures . then , the semipermeable membrane ( smith & nephew co. , england ) was adhered to cover the vacuseal outside the wound ( figure 1c ) , and the dressing was linked to a negative pressure aspirator to maintain continuous vacuum aspiration at a pressure of about 60 mmhg to 75 mmhg ( figure 1d ) . the animals were housed in a special cage and an intramuscular injection of penicillin ( 400,000 u / ci ) was given for three days after surgery . the difference in the surgical procedure between the control and experimental groups was that the wound was sutured with vac in the experimental group but not in the control group . four rabbits from each group were sacrificed with air embolism separately at the 4 , 8 , and 12 week after surgery . the femurs of the two groups were assessed by lateral x - ray imaging . then , we estimated the state of bone allograft vascularization in the callus based on the imaging . all the animals were injected with tetracycline hydrochloride ( aladdin reagent co. , ltd , china ) ( 35 mg / kg ) 15 d prior to sacrifice , and they were injected with calcein ( sinopharm chemical reagent co. , ltd , china ) ( 10 mg / kg ) 3 d prior to sacrifice . after sacrificing the animals , the bone allografts of the two groups were obtained and were fixed in 10% paraformaldehyde , dehydrated , and defatted . the allografts were embedded with methyl methacrylate without decalcification over 24 h , and then the specimens were serial sectioned with slices of 5 micron thickness using a microtome ( jung type ) . the bone cortex and callus fluorescence staining for each slice was assessed by leica tcs sp2 confocal microscopy ( leica microsystems ir gmbh . , germany ) , and the distance between the two marking lines comprised of tetracycline hydrochloride and calcein was measured by aim image examiner ( carl zeiss microimaging gmbh . the bone allografts were accurately weighed after being rinsed , defatted , and dried off , then the specimens were decalcified with 0.1 g / ml trichloroacetic acid , and the calcium content of each specimen was determined using back titration . total protein was extracted from cancellous bone by hammer grinding and all protein samples were stored at 80c . the protein of each group was separated by 12% sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) and blotted to a polyvinylidene difluoride ( pvdf ) membrane . the membrane was blocked in tris - buffered saline with tween ( tbst ) with 5% skim milk for 2 h and incubated overnight at 4c with anti - fgf-2 antibody ( boster reagent co. , ltd , china ) ( 1:200 dilution ) . after washing in tbst for 5 times , the membrane was incubated with horseradish - peroxidase ( hrp)-coupled secondary antibody ( 1:50000 dilution ) at room temperature for 2 h. subsequently , the samples were washed with tbst again , and the membranes were incubated with an ecl detection kit ( beijing comwin biotech co. , ltd . , china ) for 5 min and exposed to medical x - ray film . to compare the difference in each test between the groups , spss17.0 for windows statistical software analysis was performed . measurement data were presented as the mean sd and analyzed by student s t - test . enumeration data were presented as percentages and were analyzed by the chi - square test . in all cases , a significance level of p<0.05 was used . by general observation and x - ray imaging ( figure 2a2f ) , some callus growths crawling from the femur to the bone graft were observed , and the dividing line could be seen at the 4 week after surgery . the femur connected a little more to the bone graft in the experimental group than in the control group . at the 8 week after surgery , more callus growth was observed around the bone graft , and the femur attached to the bone graft solidly in both groups . the dividing line was blurred in the experimental group while a part of the line was still visible in control group . at the 12 week after surgery , the bone graft connected to the femur entirely and a peripheral callus was beginning to form in both groups . fluorescent bone labeling demonstrated a discrete yellow fluorescent line ( tetracycline hydrochloride ) arranged around the bone graft along with scattered punctiform green fluorescence ( calcein ) present in the experimental group , while there was only scattered punctiform yellow fluorescence in the control group at the 4 week after surgery ( figure 3a , 3b ) . at the 8 week after surgery , two fluorescent ribbons with clear boundaries could be observed around the bone graft in the experimental group , while the corresponding ribbons were discontinuous and uneven in thickness in the control group ( figure 3c , 3d ) . at the 12 week after surgery , two annular - shaped fluorescent ribbons could be found in both groups , but the distance between the colored ribbons was significantly shorter in the control group than in the experimental group ( p<0.05 ) ( figure 3e , 3f , table 1 ) . the calcium content in the callus at the 4 , 8 , and 12 week after surgery was significantly different between the control and experimental group ( p<0.05 ) . the calcium content in the callus in the experimental group was significantly higher than in the control group at each time point after surgery ( p<0.05 ) . typical imaging of the expression of fgf-2 by western blot analysis in each group is shown in figure 5 . the grey value of each film in the experimental group was significantly higher than in the control group ( p<0.05 ) ( figure 6 ) . by general observation and x - ray imaging ( figure 2a2f ) , some callus growths crawling from the femur to the bone graft were observed , and the dividing line could be seen at the 4 week after surgery . the femur connected a little more to the bone graft in the experimental group than in the control group . at the 8 week after surgery , more callus growth was observed around the bone graft , and the femur attached to the bone graft solidly in both groups . the dividing line was blurred in the experimental group while a part of the line was still visible in control group . at the 12 week after surgery , the bone graft connected to the femur entirely and a peripheral callus was beginning to form in both groups . fluorescent bone labeling demonstrated a discrete yellow fluorescent line ( tetracycline hydrochloride ) arranged around the bone graft along with scattered punctiform green fluorescence ( calcein ) present in the experimental group , while there was only scattered punctiform yellow fluorescence in the control group at the 4 week after surgery ( figure 3a , 3b ) . at the 8 week after surgery , two fluorescent ribbons with clear boundaries could be observed around the bone graft in the experimental group , while the corresponding ribbons were discontinuous and uneven in thickness in the control group ( figure 3c , 3d ) . at the 12 week after surgery , two annular - shaped fluorescent ribbons could be found in both groups , but the distance between the colored ribbons was significantly shorter in the control group than in the experimental group ( p<0.05 ) ( figure 3e , 3f , table 1 ) . the calcium content in the callus at the 4 , 8 , and 12 week after surgery was significantly different between the control and experimental group ( p<0.05 ) . the calcium content in the callus in the experimental group was significantly higher than in the control group at each time point after surgery ( p<0.05 ) . typical imaging of the expression of fgf-2 by western blot analysis in each group is shown in figure 5 . the grey value of each film in the experimental group was significantly higher than in the control group ( p<0.05 ) ( figure 6 ) . for the high operation failure rates of former treatments of open , non - union bone fractures [ 410 ] , papineau introduced open bone graft technology to solve this problem . open bone grafting is also called the rhinelander - papineau or the papineau technique , and its characteristic benefits are conferred by the facts that the microporous structure of cancellous bone is advantageous to osteoblast agglomeration and growth , and that osteoblasts can obtain nutrition from surrounding tissue exudates for survival . new vessels grow easily and quickly to establish a blood supply because of the unique clearance of each bone graft and such vessels bring more nutrients including osteogenesis factors [ 1116 ] . draining wound exudates through gauze is better at preventing infection when applied with open bone grafts compared with traditional bone grafts [ 1719 ] . furthermore , the blood from the soft tissue bed or cancellous bone marrow cavity can seep out through the gap and solidify . afterwards , granulation tissue forms and covers the bone graft , and this is followed by capillary ingrowth , which provides the foundation for free skin grafting . all of these characteristics confirm that open bone grafting is applicable to patients with a poor state of local soft tissue and infectious non - union bone ; however , potential problems include needing treatment more frequently , and spending a longer period of time in hospital . fortunately , the emergence of vac greatly mitigates the said problems of open bone grafting . the basic working principle of vac is to use a vacuseal connected to a drainage tube between the wounds , which is collectively connected to negative pressure , enabling for the whole wound to drain and allow for clearance of exudate expeditiously . vac also contacts the wound closely and uses sustained active force provided by the negative pressure , enabling clearance of not only wound exudate , but also of pus and necrotic tissue in a complete and timely manner . vacuseal containing a porous structure has increased water permeability and ability to adsorb solutions better than petroleum jelly gauze . in addition , when exudate is eliminated , vac can inhibit bacterial growth and prevent the absorption of toxins and spread of infection . as the study of vac increases , more advantages to this technology have been discovered [ 3033 ] : ( 1 ) vac can promote vascular endothelial growth factor ( vegf ) generation in damaged tissue ; ( 2 ) the expression of cd34 significantly increases after vac treatment ; therefore , one can deduce that vac apparently promotes the proliferation of vascular endothelial cells and fibroblasts in the tissue around the wound and also increase microvascular density , thus promoting wound granulation tissue growth ; ( 3 ) the concentration of transforming growth factor - beta 1 ( tgf-1 ) and platelet - derived growth factor ( pdgf ) in wounds is significantly increased by vac ; ( 4 ) vac can decrease the expression of matrix metalloproteinase ( mmp)-1 , 2 , 9 , 13 mrna in wounds , and inhibit the degradation of gelatin and collagen by adjusting the balance of mmps to tissue inhibitors of metalloproteinase ( timp ) to promote wound healing . the role of active substances , including vegf and cd34 , tgf-1 , pdgf , and timp-1 in promoting bone revascularization has been confirmed [ 3438 ] . fgf2 is a member of the fibroblast growth factor family , which is widely distributed within human tissue , and it plays an important role in the repair of bone defects or fractures [ 3941 ] . in this experiment , we detected fgf2 in allografts by western blot analysis and found that the expression in the experimental group was significantly higher , which indicated that vac can promote the expression of fgf2 , and this may suggest that heightened fgf2 is probably one of the reasons behind vac promoting bone vascularization . vac in combination with open bone grafting is currently used in clinical settings mainly for complex open tibial bone non - union with soft tissue defects . because it can shorten fracture healing time , and the time for granulation tissue to cover the wound , it is recognized by a vast number of scholars . we had successfully treated 15 cases of complex tibial bone non - union using this technology since 2007 in our hospital , and the treatment efficacy has been satisfactory . in this experiment , the results of x - ray imaging , fluorescent bone labeling and calcium content in the callus indicated that vac plays a role in promoting bone allografts vascularization in rabbit , while the increased expression of fgf-2 in bone allografts may be the reason for this effect . we could not replace vacuseal every five to seven days strictly , but rather deciding on replacement time based on practical circumstances . in addition , this study only confirmed that vac could promote bone vascularization , but the effect was not quantified .