Thrombosis Research 131 (2013) 94–98

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Regular Article

Heapranase role in the treatment of avascular necrosis of femur head

Eli Peled a, Matthew Davis b, Elena Axelman c, Doron Norman a, Yona Nadir c,⁎a Department of Orthopedic B, Rambam Health Care Campus, Haifa, Israelb The Rappaport Faculty of Medicine, Technion, Haifa, Israelc Thrombosis and Hemostasis Unit, Department of Hematology, Rambam Health Care, Campus, Haifa, Israel

⁎ Corresponding author at: Thrombosis and Hemostatology and Bone, Marrow Transplantation, Rambam HeaIsrael. Tel.: +972 4 8543520; fax: +972 4 8543886.

E-mail address: y_nadir@rambam.health.gov.il (Y. N

0049-3848/$ – see front matter © 2012 Elsevier Ltd. Allhttp://dx.doi.org/10.1016/j.thromres.2012.09.018

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 22 August 2012Received in revised form 5 September 2012Accepted 21 September 2012Available online 9 October 2012

Keywords:Avascular necrosisHeparanaseTFPI

Background: Idiopathic avascular necrosis (AVN) of bone causes significant morbidity in adults although thepathophysiology is unknown. The present treatment options include systemic biphosphonate therapy andlocal bone drilling decompression, ameliorating the healing process and their by render the weight bearingfemur head less vulnerable to collapse. In the present study we demonstrate the involvement of heparanasein AVN and in the acceptable treatments.Methods: 56 female rats were studied. In 8 control rats AVN was induced by ligamentum teres ligation ofthe right femur while the left femur remained intact. In the rest of the rats, in addition to right femur AVN,treatment was added by subcutaneous biphosphonate therapy, femoral head drilling or combination of thetreatments. All rats were scarified after 6 weeks. Immunostaining of the femur heads were performed to

heparanase, tissue factor pathway inhibitor (TFPI), tissue factor (TF) and hematoxylin-eosin.Results: Staining of heparanase, TFPI and TF were most prominent in the bone-marrow tissue of the femurheads. Staining by hematoxylin-eosin revealed damaged femur heads with prominent heparanase and TFPIstaining in the femur with AVN compared to the contra lateral side of the same rat. No difference wasfound in the TF staining between the two sides. In the drilling and / or biphosphonate therapy groups, incontrast to the control group, femur heads were preserved with no significant difference in heparanaseand TFPI staining between the two sides.Conclusions: Heparanase and TFPI are locally elevated in the process of AVN and are normalized by theacceptable treatments. Inhibition of heparanase by heparins can potentially improve the nowadays therapymodalities.

© 2012 Elsevier Ltd. All rights reserved.

Introduction

Avascular necrosis (AVN) of bone, also known as osteonecrosis,is an idiopathic process of cellular death of bone tissue due to adisruption of blood supply, micro-infarctions and necrosis [1]. Thiscondition causes significant morbidity in adults and can lead to jointcollapse and arthritis. This pathology has the potential to affect anypart of the skeletal system; however, the most frequently affected siteis the femur head. Risk factors associated with the occurrence of AVNinclude prolonged corticosteroid use, alcoholism, hemoglobinopathies,Gaucher's disease, pregnancy, hyperbaric exposure, autoimmune dis-eases and hip trauma, although in many cases the etiology is unknown[2,3]. Activation of the coagulation system with local thrombosis waspreviously suggested [4,5] as a potentialmechanism. Treatment optionsinclude drilling of femur head and systemic biphosphonate therapy,supported by our previous publication in rate model, demonstrating

sis Unit, Department of Hema-lth Care Campus, Haifa 31096,

adir).

rights reserved.

significant improvement of femur head stabilization [6]. In the presentstudy we looked for coagulation markers to support the hypothesisthat drilling and biphosphonate treatment change the haemostaticbalance in the AVNprocess, giving a potential insight for themechanismof the treatment options.

Material and Methods

Study Group

The experiment was approved by the Technion Institute EthicCommittee for animal research. The blood circulation of right femurhead of 56 female Sprague–Dawley rats weighing 200–250 g wasinterrupted. The animals were housed in spacious cages to allowample ambulation and were operated after a minimum of a weekfor acclimation to the cage. They had free access to water and regularlaboratory chow at all times. The rats were anesthetized with anintramuscular injection of ketamine (120 mg/kg) and xylazine(17 mg/kg). They were placed on a heated operating table to preventhypothermia. After shaving of the skin, antisepsis, draping, and proxi-mally slitting of the cutis by a longitudinal incision over the greater

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trochanter, the gluteus maximus muscle was split in the direction ofits bundles and the anterior two-thirds of the gluteus medius musclewere detached from the bone. The anterolateral insertion of the articu-lar capsule was transected along the trochanteric ridge, the femur headwas dislocated, and the ligamentum teres was cut. With a number 11blade, the periosteum at the base of the neck of the femur head wasincised together with the reflected capsular fibers by circumferentiallysweeping the edge of the knife twice, at a 1- mm interval, aroundthe bone. The femur head was relocated. The articular capsule and thegluteal muscles were sutured with vicryl 3–0 stitches. The skin wasclosed with nylon 2–0 stitches.

Treatment groups 2–6 were treated by daily sub-cutenessAlendronate injections of 200 μg/kg/day and controls were treatedwith saline, both for a total of 42 days. Groups 2, 4, 6 had drilling ofthe AVN femur heads, as previously described [6]. For each rat, theright operated head was compared with the left, and the drillingand Alendronate treated groups were compared with the controlgroup.

All rats were sacrificed by CO2 inhalation on the 42nd postoperativeday, both femur heads were harvested. The specimens were fixed informalin for a week. Following decalcification in EDTA for two weeks,the femur heads were halved at the residue of the ligamentum teresinto anterior and posterior parts.

The experiment groups included:

1. AVN, n=8, Control group.2. AVN+drilling, n=8.3. AVN+Alendronate (after AVN for 6 weeks), n=11.4. AVN+drilling+Alendronate (after AVN for 6 weeks), n=9.5. AVN+Alendronate (beforeAVN for 1 week+after AVN for 6 weeks),

n=9.

A

B

Fig. 1. Histological damage of AVN is protected by treatments of drilling and / or biphosphonserve as control. Right figures. represent the right femur of the same rats following AVN. Nowith thick fibrous tissue in AVN (arrows). B. Representative figures. to the protective effebetween the AVN femur head (right) and the control (left). Images (x10 magnification) wercamera (Nikon, Tokyo, Japan).

6. AVN+drilling+Alendronate (before AVN for 1 week+after AVNfor 6 weeks), n=11.

Antibodies

Antibody 733was raised in rabbits against a 15 amino acid peptidethat maps at the N-terminus of the 50 kDa heparanase subunit. The733 antibody preferentially recognizes the 8+50 kDa heterodimervs. the 65 kDa latent proenzyme [7]. Polyclonal anti-rat tissue factor(TF) and polyclonal anti-rat tissue factor pathway inhibitor (TFPI)antibodies were purchased from Santa Cruz (Santa Cruz, CA).

Immunohistochemistry

Staining of formalin-fixed, paraffin-embedded 5-micron sectionswas performed. Slides were deparaffinized with xylene, rehydratedand endogenous peroxidase activity was quenched for 30 min by 3%hydrogen peroxide in methanol. Slides were then subjected to anti-gen retrieval by boiling (20 min) in 10 mM citrate buffer, pH 6. Slideswere incubated with 10% normal goat serum in PBS for 60 min toblock non-specific binding followed by incubation (20 h, 4 °C) withanti-TF, anti-TFPI, or anti-heparanase antibodies, diluted 1:100 inblocking solution. Slides were then extensively washed with PBScontaining 0.01% Triton X-100 and incubated with a secondaryreagent (Envision kit; Dako, Glostrup, Denmark) according to themanufacturer's instructions. Following additional washes, color wasdeveloped with the AEC reagent (Sigma, St. Louis, MO). The slideswere assessed blindly by two persons not knowing the allocatedtreatment group. Protein's immunostaining representative intensitywas rated as follows: none, weak, moderate and intense.

ate. Staining of femur heads by H&E. A. Left figures. represent the left femur of rats andt the damaged architecture including disappearance of chondrocytes and replacementct of drilling and / or biphosphonate treatments. No major difference was observede visualized through a 10x/0.82 MDC objective lens, captured with a Nikon E995 digital

96 E. Peled et al. / Thrombosis Research 131 (2013) 94–98

Results

The femur heads with AVN (RT side) showed a destructive pat-tern including disappearance of chondrocytes and replacement bythick fibrous tissue compared to the femur heads without AVN(LT side) (Fig. 1A). The femur heads with AVN and treatmentsof drilling and / or biphosphonate showed a preserved architecture(Fig. 1B).

Staining of heparanase, TFPI and TF were most prominent in thebone-marrow tissue of the femur head. There was no significant dif-ference in the pattern of staining of the analyzed proteins (Fig. 2A, B).

In the control group, staining of heparanase and TFPI were moreprominent in the RT femur with AVN compared to the LT femur(Fig. 2A, B). No difference was found in the TF staining between thetwo sides (data not shown).

In the groups with therapy (number 2–6), interestingly and incontrast to the control group, staining of heparanase and TFPI werenot different in the RT femur with AVN compared to the LT femur(Fig. 3A, B).

Discussion

Surgical osteonecrosis of the rat femur head is induced by detachingthe ligamentum teres and stripping the femoral neck periosteum [8].Hematopoietic necrosis of the bone marrow is seen from the secondday, and subchondral and epiphyseal bone necrosis are found on thefifth postoperative day [8]. Inflamed reaction around the joint, espe-cially from the joint capsule, promotes at first invasion of mesen-chymal cells into the femur head, which creates a healing processduring the second week and is replaced by well vascularized fibroustissue and elevated level of the proangiogenic protein vascular

B

A

Fig. 2. Heparanase and TFPI are elevated in AVN. Immunostaining of femur head to heparanafigs. represent the right femur of the same rat following AVN. Increased levels of heparanas(x20 magnification) were visualized through a 20x/0.82 MDC objective lens, captured with

endothelial growth factor (VEGF) [9]. This process involves macro-phages and osteoclast activity, which removes the necrotic bonethat is replaced by fibrous tissue and eventually by osteoblastdeposit and intramembranous bone. During the remodeling pro-cess, the spherical shape of the femoral head epiphysis and physisis distorted and becomes flattened [10,11]. The rapid repair processof the femoral necrosis by the newly formed bone brings earlyfemoral head architectural distortion and predispose to femurhead collapse. Several nitrogen-containing bisphosphonates werefound to inhibit osteoclast mediated bone absorption in vitro byinhibiting farnesyl diphosphate synthase, thereby preventing pro-tein post-translation processes in osteoclasts [12]. Alendronate is anitrogen-containing biphosphonate that inhibits osteoclasts activi-ty, resulting in inhibition of the remodeling and healing processesof the necrotic femur head bone [13]. Drilling was reported to bean effective treatment for osteonecrosis of femur head enabling de-compression of the intra-osseous pressure, relieve the compressiveeffects of interstitial edema, following reduced secondary ischemiaand attenuation of the healing process [14,15].

According to the results, treatment with drilling and / or thebisphosphonate Alendronate changed dramatically the expression ofheparanase and TFPI. Heparanase is a strong proangiogenic [16] andprothrombotic [17] factor that is involved in wound healing process.Heparanase was previously shown to up regulate TFPI and TF expres-sions [18,19] following release of TFPI from the endothelial cellsurface, resulting in increase endothelial cell surface coagulation.The increased level of heparanase 6 weeks after AVN, supports anongoing thrombotic and angiogenic process that unfortunatelymay end with femur head, a major weight bearing organ, distortion,weakening and disruption. Previously, Peled et al. [6,20,21] demon-strated that femur heads were preserved and remained intact with

se (A) and TFPI (B). Left figs. represent the left femur of a rat and serve as control. Righte (A) and TFPI (B) were observed in the right femur compared to the left side. Imagesa Nikon E995 digital camera (Nikon, Tokyo, Japan).

A

B

Fig. 3. Heparanase and TFPI are not elevated in AVN treated with drilling and / or Alendronare. Immunostaining of femur head to heparanase (A) and TFPI (B). Left figures. representthe left femur of a rat and serve as control. Right figures. represent the right femur of the same rat following AVN. No significant difference was found in heparanase (A) and TFPI (B)staining in the right femur compared to the left side. Images (x20 magnification) were visualized through a 20x/0.82 MDC objective lens, captured with a Nikon E995 digital camera(Nikon, Tokyo, Japan).

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the treatments of drilling and / or Alendronate. Now we have molec-ular explanation to these previous results, showing marked attenua-tion of the prothrombotic and proangiogenic heparanase proteinlevel, probably enabling more gradual healing and reducing thevulnerability of the femur head to fracture. As heparanse is stronglyinhibited by heparins, the present work gives a good rational to treatpatients with AVN with heparins for the weeks following the AVNpresentation, in accordance with previous publications supportingheparins use in osteonecrosis [22,23].

The strongest staining in all groups was in the bone marrowtissue. In contrast to the bone and cartilage tissues, the bone marrowis a very vascular tissue containing numerous sinuses. As staining inright and left sides were different, we can conclude that the stainingwas of cells residing in the bone marrow and not circulating bloodcells or molecules in the plasma. These cells are potentially adiposetissue cells, macrophage, fibroblasts, endothelial cells, hematopoieticand endothelial progenitor stem cells.

Several studies and case reports have shown an increased fre-quency of thrombophilia, in patients suffering from both primaryosteonecrosis, as well as avascular necrosis secondary to steroid use[4,24]. Korompilias et al. reported that 50% of 204 patients suffering

AVN Damaged femur head Heparanas

AVN + Drilling and / or Alendronate Preser

Fig. 4. Schematic summarization of results. Heparanase and TFPI are locally increased in the dfemur head is preserved and heparanase and TFPI are normalized.

from AVN had resistance to activated protein C and that overall,83% of patients had an underlying coagulopathy [5]. Glueck et al.demonstrated that Factor VIII was more frequently high in 71 patientswith idiopathic osteonecrosis than in control subjects. High Factor VIIIand Factor V Leiden heterozygosity were more frequently present in62 patients with secondary osteonecrosis than in control subjects[25]. In view of the microcirculation thrombosis involved in AVN,inhibition of heparanase as a procoagulant protein can potentiallyimprove normal revascularization and improve prognosis.

The main limitation of the study is the fact that the local eleva-tion of heparanase and TFPI was demonstrated only by immuno-staining, although differences were very prominent (Fig. 2). Furtherhistological data and evaluation of hemostatic parameters in theplasma of patients with AVN, are an intriguing field for future re-search. In conclusion, by immunostaining, six weeks after AVNthere is a high level of heparanase and TFPI in the femur headcompared to unaffected femur head. Treatment with drilling and /or Alendronate significantly reduces their level in the AVN process,resulting in better preservation of the femur head structure. Hepa-rins are strong inhibitors of heparanase and may have additionalbeneficial effect.

e, TFPI

ved femur head Heparanase, TFPI

amaged femur head of AVN. Following treatment with drilling and / or Alendronate the

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Acknowledgments

The study was supported by the Israel Science Foundation (ISF),Legacy Heritage Clinical Research (2009-2012).

References

[1] Inoue A, Ono K. A histological study of idiopathic avascular necrosis of the head ofthe femur. J Bone Joint Surg Br 1979;61-B:138-43.

[2] Min BW, Song KS, Cho CH, Lee SM, Lee KJ. Untreated asymptomatic hips in pa-tients with osteonecrosis of the femoral head. Clin Orthop Relat Res 2008;466:1087-92.

[3] Kerachian MA, Cournoyer D, Harvey EJ, Chow TY, Begin LR, Nahal A, et al. New in-sights into the pathogenesis of glucocorticoid-induced avascular necrosis: micro-array analysis of gene expression in a rat model. Arthritis Res Ther 2010;12:R124.

[4] Cenni E, Fotia C, Rustemi E, Yuasa K, Caltavuturo G, Giunti A, et al. Idiopathic andsecondary osteonecrosis of the femoral head show different thrombophilicchanges and normal or higher levels of platelet growth factors. Acta Orthop2011;82:42-9.

[5] Korompilias AV, Ortel TL, Urbaniak JR. Coagulation abnormalities in patients withhip osteonecrosis. Orthop Clin North Am 2004;35:265-71 [vii].

[6] Peled E, Bejar J, Zinman C, Boss JH, Reis DN, Norman D. Prevention of distortion ofvascular deprivation-induced osteonecrosis of the rat femoral head by treatmentwith alendronate. Arch Orthop Trauma Surg 2009;129:275-9.

[7] Zetser A, Levy-Adam F, Kaplan V, Gingis-Velitski S, Bashenko Y, Schubert S, et al.Processing and activation of latent heparanase occurs in lysosomes. J Cell Sci2004;117:2249-58.

[8] Norman D, Reis D, Zinman C, Misselevich I, Boss JH. Vascular deprivation-inducednecrosis of the femoral head of the rat. An experimental model of avascularosteonecrosis in the skeletally immature individual or Legg-Perthes disease. IntJ Exp Pathol 1998;79:173-81.

[9] Vadasz Z, Misselevich I, Norman D, Peled E, Boss JH. Localization of vascular endo-thelial growth factor during the early reparative phase of the rats' vesselsdeprivation-induced osteonecrosis of the femoral heads. Exp Mol Pathol2004;77:145-8.

[10] Peskin B, Shupak A, Levin D, Norman D, Jacob Z, Boss JF, et al. Effects ofnon-weight bearing and hyperbaric oxygen therapy in vasculardeprivation-induced osteonecrosis of the rat femoral head. Undersea HyperbMed 2001;28:187-94.

[11] Levin D, Norman D, Zinman C, Rubinstein L, Sabo E, Misselevich I, et al. Treatmentof experimental avascular necrosis of the femoral head with hyperbaric oxygen inrats: histological evaluation of the femoral heads during the early phase of the re-parative process. Exp Mol Pathol 1999;67:99–108.

[12] Dunford JE, Thompson K, Coxon FP, Luckman SP, Hahn FM, Poulter CD, et al.Structure-activity relationships for inhibition of farnesyl diphosphate synthasein vitro and inhibition of bone resorption in vivo by nitrogen-containingbisphosphonates. J Pharmacol Exp Ther 2001;296:235-42.

[13] Reszka AA, Rodan GA. Nitrogen-containing bisphosphonate mechanism of action.Mini Rev Med Chem 2004;4:711-9.

[14] Lee MS, Hsieh PH, Chang YH, Chan YS, Agrawal S, Ueng SW. Elevated intraosseouspressure in the intertrochanteric region is associated with poorer results inosteonecrosis of the femoral head treated by multiple drilling. J Bone Joint SurgBr 2008;90:852-7.

[15] Marker DR, Seyler TM, Ulrich SD, Srivastava S, Mont MA. Do modern techniquesimprove core decompression outcomes for hip osteonecrosis? Clin Orthop RelatRes 2008;466:1093-103.

[16] Vlodavsky I, Abboud-Jarrous G, Elkin M, Naggi A, Casu B, Sasisekharan R, et al. Theimpact of heparanese and heparin on cancer metastasis and angiogenesis.Pathophysiol Haemost Thromb 2006;35:116-27.

[17] Nadir Y, Brenner B, Fux L, Shafat I, Attias J, Vlodavsky I. Heparanase enhances thegeneration of activated factor X in the presence of tissue factor and activated fac-tor VII. Haematologica 2010;95:1927-34.

[18] Nadir Y, Brenner B, Gingis-Velitski S, Levy-Adam F, Ilan N, Zcharia E, et al.Heparanase induces tissue factor pathway inhibitor expression and extracellularaccumulation in endothelial and tumor cells. Thromb Haemost 2008;99:133-41.

[19] Nadir Y, Brenner B, Zetser A, Ilan N, Shafat I, Zcharia E, et al. Heparanase inducestissue factor expression in vascular endothelial and cancer cells. J ThrombHaemost 2006;4:2443-51.

[20] Peled E, Bejar J, Zinman C, Reis DN, Boss JH, Ben-Noon H, et al. Alendronate preservesfemoral head shape and height/length ratios in an experimental rat model: Acomputer-assisted analysis. Indian J Orthop 2009;43:22-6.

[21] Bejar J, Peled E, Boss JH. Vasculature deprivation–induced osteonecrosis ofthe rat femoral head as a model for therapeutic trials. Theor Biol Med Model2005;2:24.

[22] Glueck CJ, Freiberg RA, Sieve L, Wang P. Enoxaparin prevents progression ofstages I and II osteonecrosis of the hip. Clin Orthop Relat Res 2005;435:164-70.

[23] Norman D, Miller Y, Sabo E, Misselevich I, Peskin B, Zinman C, et al. The effectsof enoxaparin on the reparative processes in experimental osteonecrosis of thefemoral head of the rat. APMIS 2002;110:221-8.

[24] Omeroglu H, Inan U. Inherited thrombophilia may be a causative factor forosteonecrosis of femoral head in male patients with developmental dysplasia ofthe hip: a case series. Arch Orthop Trauma Surg 2012;132:1281-5.

[25] Glueck CJ, Freiberg RA, Wang P. Heritable thrombophilia-hypofibrinolysis andosteonecrosis of the femoral head. Clin Orthop Relat Res 2008;466:1034-40.