odontogenic keratocyst ( okc ) is a developmental odontogenic cyst with specific histopathological features and clinical behaviors , high recurrence rate , and aggressive behavior compared to other 
 developmental odontogenic cysts . due to its specific clinico - pathologic characteristics , its name has been changed to keratocystic 
 odontogenic tumor in the new classification of the world health organization ( who).[1 - 2 ] dentigerous ( follicular ) cyst is the most common odontogenic developmental cyst , which instigates from separation of dental follicle from crown of an unerupted tooth . the reactions between the odontogenic epithelium and its ectomesenchymal components have a role in the formation of odontogenic lesions , including odontogenic 
 cysts . blood vessels present in the connective stroma of odontogenic cysts help the growth of their epithelium by supplying oxygen and 
 nutrients.3 neoplastic tissues require angiogenesis in order to continue their growth , development and progression , which can also contribute to their invasion 
 and metastasis.[3 - 4 ] angiogenesis is defined as the formation of new blood vessels from the existing 
 blood vessels and has been evaluated in various lesions including breast cancer , melanoma , endometrial carcinoma , squamous cell carcinoma , lichen planus , ameloblastoma , 
 and some odontogenic cysts.[5 - 11 ] 
 one of the techniques used for the quantitative analysis of angiogenesis is mean vascular density ( mvd ) , which has been evaluated using various molecules , 
 including vascular endothelial growth factor ( vegf ) , von willebrand factor , factor viii , cd31 , cd34 , and cd105 ( endoglein).[12 - 13 ] 
 
 cd34 ( qbend 10 ) is a pan - endothelial marker and trans - membranous monomeric glycoprotein of the cell surface and is expressed in the normal and neoplastic endothelial cells of blood vessels.[14 - 18 ] 
 
 cd105 ( endoglein ) is a hypoxia - dependent membrane hemodimer disulfide glycoprotein , associated with cell proliferation , and expressed on activated vascular endothelial cells . since the expression of this protein increases in tumoral tissues compared with normal tissues , it can be inferred that expression of cd105 is related to tumor angiogenesis . several studies have shown that cd105 antibody has higher accuracy and specificity to bind to newly formed blood vessels in tumoral tissues compared to other pan - endothelial markers such as factor viii , von willebrand factor , cd31 and cd34 . [ 21 - 22 ] 
 only a limited number of studies have investigated stromal factors such as role of angiogenesis in odontogenic lesions reported by seifi et al 
 and de andrade santos et al . 
 
 hence , the aim of this study was to determine mvd by using cd34 and cd105 markers in okcs and dentigerous cysts and evaluating their relationship with the biologic behaviors of these lesions . in this retrospective cross - sectional analytical study , the archive files of hamadan faculty of dentistry were evaluated from 1997 to 2010 . h&e slides were 
 evaluated and confirmed by two oral pathologists and 15 samples from each lesion with sufficient tissues and proper fixation were selected . inflammation was considered one of the factors affecting angiogenesis and therefore , as a confounding factor for the results . the files of patients with 
 the diagnosis of odontogenic lesions , including dentigerous cysts and okc were retrieved . their clinical and demographic data including age , gender , and locations of the lesions were recorded in tables . 
 slices with 4- thickness were prepared from paraffin blocks for immunohistochemical staining . the slides were deparaffinized in xylene and then hydrated in graded alcohol series . for cd34endogenous 
 peroxidase activity was blocked by incubating the slides in hydrogen peroxide ( 3% ) in phosphate buffer and for cd105 incubating the slides with 3% h2o2 in methanol for 30 minutes . antigen retrieval 
 for cd105 was performed by treating sections with proteinase - k for 5 minutes . antigen retrieval for cd34 was performed in a microwave oven for 10 minutes at 120 degree centigrade . to prevent nonspecic 
 reactions , sections were incubated with 10% serum for 10min.then monoclonal mouse anti - human cd105 antibody ( clone : sn6h ; product code : m3527 , dako , north america inc . ) and monoclonal mouse anti - human 
 cd34 antibody ( clone : qbend 10 , product code : m7165 a / s , glostrup , dako , denmark ) were applied as primary antibody . cd105 antibody was incubated at room temperature in a humidifying chamber for 90 minutes . 
 for cd34 , the sections were incubated with anti- cd34 antibody for 30 minutes at room temperature with a working dilution of 1:50 , followed by incubation with secondary biotinylated antibody and streptavidin 
 for 15 min each . diaminobenzidine was used to produce brown staining followed by counterstaining with mayer s hematoxylin . after each step the human 
 tonsillar tissue was used as the positive control and the primary antibody was eliminated and replaced with phosphate buffer saline for the negative control . 
  the technique described by weidner et al . the stained slides were evaluated under a light microscope ( olympus bx41 , japan ) 
 by two oral pathologists at 100400 magnification . in order to determine the mvd , three areas with the highest amount of vascularization , known as hot spots , were selected under low magnification ( 100 ) . microvessels were counted in each of the three elds at 400 magnication and the mean density was reported . positive staining for cd105 and cd34 was defined as each brown - colored endothelial cell separately ( or in aggregates ) was distinctively separated from the adjacent micro - vessels . the objects which seemed 
 to be originated from one blood vessel were counted if completely separated from it . blood vessels with muscular walls were excluded and the mean number 
 of blood vessels in the three selected was considered as mean vascular density ( mvd ) . the data were analyzed by pss16 statistical software with setting p value at 0.05 . descriptive statistics of clinical data in study groups 
  cd34 and cd105 were expressed in all samples . ( figure 1 ) the mean ( sd ) mvds with cd34 marker in dentigerous cysts and okcs were 8.482.8 and 12.245.8 respectively which shows mvd in 
 okc was higher than in dentigerous cysts . independent samples t - test revealed significant statistical difference between groups ( p= 0.032 ) . in addition , mean ( sd ) mvds with 
 cd105 marker in dentigerous cysts and okcs were 5.942.4 and 9.22.94 respectively , so mvd in okc was higher than that in dentigerous cysts . ( table 2 ) the evaluation of mvds in okc ( by cd105 and cd34 markers ) showed a statistical significant differences between these markers ( 9.22.94 and 12.245.8 respectively ) . the results showed mvd with the use of cd34 marker was significantly higher than that with the use of cd105 marker . independent samples t - test revealed significant difference between groups ( p= 0.017 ) . 
  a : cd 105 positive immunoreactivity in dentigerous cyst ( 400 ) b : cd 34 positive immunoreactivity in dentigerous cyst ( 400 ) c : cd 34 positive immunoreactivity in 
 odontogenic keratocyst ( 400 ) d : cd 105 positive immunoreactivity in odontogenic keratocyst ( 400 ) 
  cd34 and cd105 expression and mvd in studied groups 
  the mvds in dentigerous cysts with the use of cd105 and cd34 markers were 5.942.4 and 8.482.8 respectively . the mean number of stained vessels in dentigerous cysts with the use of cd34 
 marker was significantly higher than that with the use of cd105 marker . okc has aggressive behavior and a high recurrence rate compared to other developmental odontogenic cysts . in recent years , various factors such as p53 , mdm2 , ki67 , bcl2 , mmp , il-1 , 
 and tgf- markers which were supposed to be related to greater aggressive behavior of okc ( compared to other odontogenic cysts ) have been evaluated in various studies.[24 - 31 ] the growth mechanism of okc is different from dentigerous cyst . some investigators suggest the increase in some matrix metalloproteinase of connective tissue would affect the 
 okc growth . one of the effective factors that probably increase the angiogenesis in okc in comparison to dentigerous cysts would be the escalation of matrix metalloproteinase . browne was one of the pioneers to propose that the connective tissue wall of okc may have an important role in the pathogenesis of this lesion . angiogenesis has been previously studied in okc but the number of studies is limited . 
 among the many angiogenic markers , cd105 has higher accuracy and specificity to bind newly formed blood vessels and cd34 is a pan endothelial marker that shows stronger staining with endothelial cells with lower error rate in comparison to cd31 . 
 [ 15,21 - 22 ] 
 cd34 can not distinguish between the newly formed blood vessels and the old host ones but it has important role in evaluation of mvd . these points would justify why the current study evaluated the expression of these markers in odontogenic lesions . in this study cd34 and cd105 were expressed in all samples but mvd in okc was significantly higher than dentigerous cysts . several other studies , have also confirmed the role of angiogenesis as an important 
 factor in the aggressive behavior of okc compared to dentigerous cysts . alaeddini et al . determined mvd by using cd34 marker and reported that mvd was signicantly higher in okcs than in dentigerous cysts . they suggested that angiogenesis could 
 be one of the mechanisms possibly responsible for the different biological behaviors of these lesions . rubini et al . 
 observed a significant different expression of vascular endothelial 
 growth factor ( vegf ) in keratocysts compared to follicular cysts . they found that in all cases of okc , there was a statistically significantly higher expression of vegf whilst most follicular cysts were 
 negative for vegf expression . they concluded angiogenesis could be an active mechanism in the invasive growth of the okc . . 
 found cd105 to be strongly expressed in microvessels of okc compared with that in dentigerous cyst and normal oral mucosa . they suggested that tumor angiogenesis may be associated with locally aggressive 
 biologic behavior of odontogenic lesions and angiogenesis can show the prognosis and clinical behavior of odontogenic lesions . 
  unlike the first three studies that evaluated only 
 one marker of angiogenesis ( cd34 or cd105 ) , we studied two markers of angiogenesis and the marker used in the rubini et al . the results of all above mentioned studies and our study showed that angiogenesis was higher in okc compared with dentigerous cyst . angiogenesis is an important step in the majority of 
 physiologic and pathologic processes and is necessary for the cells to access to oxygen and nutrients in distances more than 12 mm from the blood vessels . therefore , more angiogenesis in okc compared to dentigerous cysts can indicate higher tissue metabolism . mvd which is considered as a quantitative analysis of angiogenesis in a lesion can predict the growth rate , 
 metastasis , patient survival , and the aggressive behavior . 
 
 therefore , more angiogenesis and higher mvd in okc compared to dentigerous cysts can indicate more aggressive biologic behavior , higher recurrence rate and more growth and proliferation . it might be possible to control the aggressive behavior of this cyst with anti - angiogenic treatments . the result of this study was different from result of the study performed by de andrade santos et al . . 
 studied the expression of cd105 in okcs , dentigerous cysts , and radicular cysts . they showed that micro vessel count was higher in dentigerous cyst than in okcs and the differences in the biologic behavior of these lesions do not appear to be associated with the angiogenic index . our results based on cd105 are not in agreement with the results of the study of de andrade santos et al . this difference might be attributed to the differences in methodology such as counting method and number of samples . 
 
 in the present study , mvds based on cd105 marker in okc and dentigerous cysts were significantly less than those with cd 34 . , 
 miyata et al . , kumagai et al.,[39 
 and czekierdowski which compared the expression of cd105 and cd34 antibodies in different lesions have shown a lower mvd based on cd105 marker compared to cd34 marker . the results of the current study were consistent with their results since the expression of cd105 is a prominent feature of newly formed vessels ; moreover , its expression in previously formed vessels and also in the endothelium of the vessels of normal tissues is little or even negative . nevertheless , cd34 is expressed in the vascular endothelial cells of normal and neoplastic tissues and can not distinguish the newly formed blood vessels from the old blood vessels of host . compared to cd34 , these findings suggest that cd105 is a more specific and sensitive marker for angiogenesis . 
 
 the results of this study would probably contribute in better understanding of the role of angiogenesis in the biological behavior of okc and dentigerous cyst . further studies with larger number of cases are required to elucidate the role of angiogenesis in the aggressive behavior of okc . in the present study , mvd in okc was higher than that in dentigerous cysts , which can indicate that angiogenesis is one of the possible mechanisms involved in the greater 
 aggressive biologic behavior and higher recurrence rate of okc compared to dentigerous cysts .