osteonecrosis of the femoral head ( onfh ) is a common disease in orthopedics throughout the world .
various accepted risks factors , including trauma , excessive steroid use , alcoholism , damage from radiation , sickle cell anemia , gaucher 's disease , and the exposure to high pressures , are reported [ 17 ] .
chronic groin pain and gait disturbance is one feature of this disease , which is considered to be caused by the secondary osteoarthritis followed by onfh .
furthermore , when the disease develops to the terminal stage with femoral head collapse and secondary osteoarthritis , a total hip replacement operation is required . to date , the pathogenesis process of onfh is still unclear .
it has been demonstrated that onfh is caused by the inadequate blood supply of subchondral bone [ 1015 ] .
the pathologic process involved in onfh starts with necrosis in femoral head bone tissue which is thought to be a result of the blood circulation disturbance to the femoral head . during the pathogenesis ,
the levels of various biomarkers in the femoral head bone , cartilage , synovium , and hip joint synovial fluid are changed , which make effects on the disease process .
it has been proved that , in the bone matrix of femoral head in patients with onfh , expression level of matrix metalloproteinase-2 ( mmp-2 ) is upregulated , which is related to the reduced repair capacity and altered bone remodeling . in the children 's onfh , an increase of proteoglycan fragments in the joint fluid was observed , which implied increased degradation proteoglycan and release of fragments from hip joint cartilage .
moreover , concentrations of proteoglycan fragments , c - propeptide of type - i1 collagen , mmp-3 , timp-1 , and the molar ratios of mmp-3/ttmp-1 are all increased in hip joint fluid of adult onfh patients even before any evident radiologic changes in the femoral head or hip joint were observed .
collectively , it appears that femoral head cartilage undergoes abnormal metabolic change before collapse in onfh .
the adamts ( a disintegrin and metalloproteinase with thrombospondin motifs ) family consists of 19 secreted zinc metalloproteinases with a precisely ordered modular organization including at least one thrombospondin type i repeat . to our knowledge , these enzymes have important effects in development , angiogenesis , and coagulation and have relationship with coagulation disorders , malignancy , lumbar disc herniation , rheumatoid arthritis , and osteoarthritis [ 2026 ] .
adamts-7 , a member of the adamts family , is widely expressed in various tissues .
adamts-7 has been reported to negatively regulate endplate cartilage differentiation , facilitate intimal hyperplasia , and mediate vascular remodeling as a novel locus for atherosclerosis .
furthermore , it has been found to degrade cartilage oligomeric matrix protein ( comp ) which is an important noncollagenous component of cartilage and may play a key role in osteoarthritis pathogenesis [ 2730 ] .
it is overexpressed in cartilage and synovium of arthritis patients , and the level of fragments of comp in patients ' serum is increased concurrently . in vitro ,
it could negatively mediate chondrocyte differentiation in cartilage differentiation by being a target of parathyroid hormone - related protein ( pthrp ) [ 29 , 31 ] .
moreover , adamts-7 is reported to be closely associated with tnf- and nf-b signaling pathway , as tnf- mediated nf-b activation facilitates expression of adamts-7 .
the aim of this study was to investigate the expression of adamts-7 in articular cartilage of onfh patients , to elucidate whether the expression was altered with cartilage degradation and radiological change , and to investigate the potential mechanisms involved , which may indicate new clinic therapeutic interventions for onfh .
ethical approval for this study was obtained from the medical ethics committee of qilu hospital , shandong university .
what is more , written informed consent from each patient included in this study was obtained .
patients and healthy controls with primary osteoarthritis , ankylosing spondylitis ( as ) , systemic lupus erythematosus ( sle ) , and acute or chronic inflammatory diseases were excluded .
all the patients had taken hip x - ray and magnetic resonance imaging ( mri ) examination .
human hip articular cartilage specimens were collected from patients with femoral neck fracture ( fnf ) without onfh and oa in x - ray as healthy control named group i ( n = 14 ; the average age was 64 years with a range of 48 to 85 years ) and patients with onfh (
n = 34 ; the average age was 51 years with a range of 41 to 70 years ) . according to arco classification standard
, articular cartilage specimens obtained from the onfh patients were categorized into two groups : group ii , 15 cases , stage ii - iii with no obvious collapse in x - ray ; group iii , 19 cases , stage iv with obvious femoral head collapse in x - ray .
the articular cartilage tissues were dissected carefully from the weight - bearing region of femoral head and subsequently treated according to the corresponding downstream experiments .
the cartilage samples were dissected along the axial plane into pieces of 10  mm  5  mm  7  mm with a thin layer of subchondral bone and were fixed in 4% formaldehyde for over 24 hours in room temperature . after being decalcified in 10% ethylene diamine tetraacetic acid ( edta ) solution for over 2 weeks
the cartilage specimens were cut longitudinally into 4  m thick sections and stained with haematoxylin - eosin ( h&e ) and safranin - o .
we used the mankin score   ( table 1 ) to evaluate the cartilage degradation .
we use primary antibodies for adamts-7 ( 1  :  200 ; abcam biotechnology co. , ltd . ,
london , uk ) , tnf- ( 1  :  200 , beyotime institute of biotechnology , shanghai , china ) , and phospho - nf-b p65 ( 1  :  100 , bioss institute of biotechnology , beijing , china ) . a goat anti - rabbit immunoglobulin- ( igg- ) horseradish peroxidase ( hrp ) secondary antibody ( 1  :  200 ; beijing golden bridge biotechnology co. , ltd . ,
images were captured by a nikon eclipse 80i microscope ( nikon , tokyo , japan ) .
image - pro plus software ( media cybernetics ) was used to qualify the average optical density of the adamts-7 positive , tnf- positive , and phospho - nf-b p65 positive areas at 400x magnification .
2 - 3 grams of samples was put into liquid nitrogen and tripsised into powder .
dalian , china ) method according to the manufacturer 's instructions . the quantity and integrity of rna were verified .
total rna ( 1  g ) was reverse - transcribed in a total volume of 10  l containing 2  l 5x rt buffer ( toyobo co. , ltd . ,
japan ) , 0.5  l rt enzyme mix ( toyobo co. , ltd . ,
japan ) , and 0.5  l prime mix ( toyobo co. , ltd . ,
the reaction was performed at 37c for 15  min and 95c for 5  min .
rt - pcr reactions were carried out in triplicate with 100  ng cdna ( rna equivalent ) each with sybr green dye i master mix using a roche lightcycler ( roche , switzerland ) and the data were analyzed by lightcycler software 4.0.0.23 . to normalize adamts-7 gene expression ,
the genome - wide specificity of the primers was confirmed by blast searches ( genbank database ) .
a standard melting curve cycle was used to verify the quality of amplification and absence of primer dimer formation .
the relative expression levels of adamts-7 in each sample were analyzed using the 2 method .
tissue samples were conserved in liquid nitrogen ; total protein was extracted and determined using a bca protein assay kit according to the manufacturer 's instruction .
equal amounts of protein ( 10  g ) for each sample were resolved by 10% acrylamide - sds - page .
the samples were subsequently transferred to polyvinylidene difluoride membranes ( pvdf ; millipore , billerica , ma ) .
primary antibodies ( rabbit anti - adamts-7 , 1  :  3,000 , abcam biotechnology co. , ltd . , london , uk ; rabbit anti - comp , 1  :  3,000 , abcam biotechnology co. , ltd . ,
london , uk ) , and a goat anti - rabbit immunoglobulin- ( igg- ) horseradish peroxidase ( hrp ) secondary antibody ( 1  :  2,500 ; beijing golden bridge biotechnology co. , ltd . ,
equal amounts of protein loading were confirmed by reprobing the membranes with the rabbit anti - gapdh - hrp antibody ( 1  :  5,000 , abcam biotechnology co. ) .
protein bands were detected using a fluorchem e chemiluminescent western blot imaging system ( cell biosciences , santa clara , ca ) and quantified by densitometry analysis using imagej software ( national institutes of health , usa ) .
statistical analysis of the data was performed using unpaired student 's t - test and two - way analysis of variance ( anova ) with prism software followed by the student - newman - keuls post hoc test .
differences were considered to be statistically significant if the p value < 0.05 ( or ) or 0.01 ( or ) .
we used h&e and safranin - o staining to determine the cartilage degradation in onfh patients ; mankin score was also evaluated .
the cartilages of fnf patients had mild moderated perichondrium , and the cells organizations were normal or mildly irregular with little chondrocyte clusters and necrosis ; the safranin - o staining was normal or mildly reduced with intact tidemark .
groups ii and iii exhibit moderate - marked irregularity and more chondrocyte clusters and necrosis ; the safranin - o staining of group ii showed moderate - marked reduction while group iii was almost not stained ; their tidemark integrity was destroyed ( figure 1 ) .
we measured the mankin score , and groups ii and iii exhibited significantly higher score than group i , in which group iii got the highest ( figure 1(d ) ) .
the results indicated that , during the pathogenesis of onfh , the cartilage has a progressive degradation .
the immunohistochemistry results revealed that adamts-7 and tnf- were expressed in all three groups , but the levels of adamts-7 and tnf- were significantly increased in degraded cartilage of groups ii and iii .
furthermore , we also found a statistically significant increase in the expression of adamts-7 and tnf- in group iii compared to in group ii ( figure 2 ) .
the results suggested that the level of adamts-7 was markedly upregulated with the degradation of cartilage .
mrna is expressed in both degraded cartilage from onfh patients and relatively normal cartilage from fnf patients . when we compared the expression levels of the target gene , a statistically significant increase in the gene expression of adamts-7
was obtained in degraded cartilage compared with the relatively normal cartilage ( figure 3 ) .
what is more , in different groups of the degraded cartilage , there was also a significant increase in the adamts-7 expression in cartilage of hip joint at stage 4 compared with that at stage 2 - 3 .
adamts-7 has been proved to degrade comp in oa and some circulation diseases [ 3436 ] .
so we examined the adamts-7 and comp protein level in fnf and onfh patients ' cartilage samples .
the comp degradation was also observed in onfh cartilage . with the increase of adamts-7 ,
the result indicates a positive association between increased adamts-7 and comp degradation in onfh patients . in order to investigate the mechanisms
involved in the increased expression of adamts-7 in cartilage of onfh patients , we stained phospho - nf-b p65 in the cartilage of both onfh and fnf patients .
the results showed that the onfh groups exhibited significantly higher expression of phospho - nf-b p65 compared with the fnf group , while phospho - nf-b p65 was rarely expressed in relatively normal cartilage ( figure 5 ) .
moreover , the adamts-7 and phospho - nf-b p65 are expressed synchronously in degraded and relatively normal cartilage .
it has been proved that adamts-7 plays an important role in the pathogenesis of oa ; it directly associates with and degrades comp in vitro , and the level of adamts-7 is elevated in cartilage patients with arthritis .
the cartilage of patients with onfh exhibits a degraded condition with a relatively femoral head bone formative condition concurrently , which is different from patients with other hip diseases . in the present study ,
we first evaluated the cartilage degradation levels of onfh and fnf patients by mankin score and examined the expression pattern of adamts-7 .
our results suggested that adamts-7 expression level and mankin score had comparable trend , which prompted us to speculate whether the adamts-7 expression was significantly increased with the degradation of cartilage ( figures 1 and 2 ) .
one possible explanation is that , in onfh patients , the cartilage degradation is also related to adamts-7 . in all three groups
, the onfh groups demonstrated an elevated level of adamts-7 compared to the fnf group . in the comparison between the different stages of onfh ,
the levels of adamts-7 expression in the patients with onfh changes ( stage 4 ) were significantly increased compared with those observed in stage 2 and stage 3 patients ( figure 2 ) . in the stage
4 onfh group , which performed secondary osteoarthritis caused by onfh , level of adamts-7 was the highest among the stages .
similarly , rt - pcr with specific primers for human adamts-7 showed that adamts-7 mrna level exhibits a parallel trend as the immunohistochemistry results , which is in line with adamts-7 expression pattern in primary osteoarthritis . on the other hand ,
the adamts-7 expression level was enhanced just before the secondary osteoarthritis was formed or the femoral head collapsed , which indicates cartilage degradation throughout the whole process of onfh pathogenesis and that adamts-7 participated in cartilage degradation even in the early stage of onfh .
taken together , the data implied that adamts-7 might be involved in the cartilage pathogenesis of onfh . in the current study , we examined the comp and adamts-7 levels in fnf and onfh patients .
comp is a prominent component of cartilage extracellular matrix ( ecm ) ; loss of comp may lead to susceptibility of other cartilage matrix proteins , including col ii and aggrecan , and cause the degradation of cartilage .
we revealed a parallel alternation between comp degradation and increased adamts-7 ( figure 4 ) .
it is reasonable to assume that cartilage degradation in onfh depends , at least in part , on comp degradation ; what is more , adamts-7 upregulation greatly enhanced comp degradation in onfh patients , which may strongly contribute to cartilage degradation in onfh .
collectively , in the pathogenesis of onfh , cartilage degradation may stem from comp degradation induced by adamts-7 .
tnf- is a well - described mediator of the inflammatory response and is synthesized and released in many diseases including oa . in the previous studies ,
tnf- has been reported to play a crucial role in cartilage destruction in arthritis [ 39 , 40 ] .
the increased expression of adamts-7 in the joint tissues of degenerative diseases , such as oa and rheumatoid arthritis ( ra ) , patients is due to proinflammatory cytokines , including tnf- [ 31 , 41 ] .
tnf- enhances mmps and adamts expressions which could in turn cause exaggerated cartilage matrix degradation , while adamts-7 also upregulated tnf- expression in vitro and in vivo ; it is indicated that adamts-7 and tnf- form a positive feedback regulatory loop in cartilage .
our immunohistochemistry results showed that the tnf- expression exhibited a concurrent increase with the increased expression of adamts-7 in the radiographic groups ( figure 2 ) . on the basis of the present study and the literature ,
our results suggest that at a late stage both primary oa and onfh process a comparable inflammation status for the expression profile of tnf- , major inflammatory cytokine , is similar .
it also proved the concept that tnf- can enhance adamts-7 expression in onfh patients and indicated that tnf- also played an important role in the pathogenesis of onfh .
it has been reported that the hip labrum and synovium of the hip joint of onfh patients exhibited less synovitis , less tnf expression than that of oa .
our results showed that the expression of tnf- was enhanced in cartilage of onfh patients .
in contrast , the traumatic injury of femoral neck fracture has a relatively short time process that simply does not facilitate the synthesis of tnf-. nf-b is a critical transcriptional regulatory factor which has been extensively investigated .
nf-b is activated and enters the cell nucleus and then it combines with specific dna motifs and stimulates the expression of corresponding gene if the cells are stimulated by various cytokines .
tnf--mediated activation of the nf-b signaling pathway plays an important role in the pathogenesis of oa .
what is more , the upregulation of adamts-7 mediated by tnf- is primarily through the nf-b signaling pathway .
our results showed that the level of phospho - nf-b p65 expression in onfh patients was much higher than in femoral neck fracture patients ( figure 5 ) .
it is interesting to surmise that tnf- upregulates adamts-7 expression throughout nf-b pathway in cartilage degradation of onfh patients . in conclusion
, this study suggests that adamts-7 is associated with and might be involved in cartilage degradation during the pathogenesis of onfh , and its expression appears to interact with tnf- mediated by nf-b .
the detailed underlying mechanisms of these functions are still unknown , and subsequent studies are required to explore the related molecular interactions and signaling pathways .
level of adamts-7 is elevated in articular cartilage of osteonecrosis of femoral head ; admats-7 is positively associated with tnf- and nf-b .
 adamts-7 has been reported to exaggerate cartilage degeneration and to be associated with tnf- and nf-b signaling pathway . in this study 
 we compared the expression of adamts-7 , tnf- , and phospho - nf-b in patients with femoral neck fracture ( fnf ) and osteonecrosis of femoral head ( onfh ) at different stages . 
 we found that expression of adamts-7 , tnf- , and phospho - nf-b was significantly upregulated in onfh patients ' articular cartilage and related to the pathogenesis of onfh . 
 thus we conclude that adamts-7 level appears to be positively associated with expression of tnf- and phospho - nf-b p65 in cartilage , which may imply its association with cartilage destruction of onfh . 