research article enoxaparin prevents steroid-related...

Research ArticleEnoxaparin Prevents Steroid-Related Avascular Necrosis ofthe Femoral Head

Rainer Beckmann1 Hayfaa Shaheen1 Nisreen Kweider1 Alireza Ghassemi2

Athanassios Fragoulis1 Benita Hermanns-Sachweh3

Thomas Pufe1 Mamed Kadyrov1 and Wolf Drescher45

1 Department of Anatomy and Cell Biology RWTH Aachen University Wendlingweg 2 52074 Aachen Germany2Department of Oral and Maxillofacial Surgery RWTH Aachen University Pauwelsstraszlige 30 52074 Aachen Germany3Department of Pathology RWTH Aachen University Pauwelsstraszlige 30 52074 Aachen Germany4Department of Orthopedic and Trauma Surgery RWTH Aachen University Pauwelsstraszlige 30 52074 Aachen Germany5Department of Orthopaedic and Spine Surgery AGAPLESION EV BATHILDISKRANKENHAUS gemeinnutzige GmbHMaulbeerallee 4 31812 Bad Pyrmont Germany

Correspondence should be addressed toThomas Pufe tpufeukaachende

Received 19 February 2014 Revised 6 June 2014 Accepted 11 June 2014 Published 2 July 2014

Academic Editor Anastasios V Korompilias

Copyright copy 2014 Rainer Beckmann et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Nontraumatic osteonecrosis of the femoral head is still a challenging problem in orthopedic surgery It is responsible for 10of the 500000 hip replacement surgeries in the USA and affects relatively young active patients in particular Main reasons fornontraumatic osteonecrosis are glucocorticoid use alcoholism thrombophilia and hypofibrinolysis (Glueck et al 1997 Orth andAnagnostakos 2013) One pathomechanism of steroid-induced osteonecrosis is thought to be impaired blood flow to the femoralhead caused by increased thrombus formation and vasoconstriction To investigate the preventive effect of enoxaparin on steroid-related osteonecrosis we used male New Zealand white rabbits Osteonecrosis was induced by methylprednisolone-injection(1 times 20mgkg body weight) Control animals were treated with phosphate-buffered saline Treatment consisted of an injection of117mgkg body weight of enoxaparin per day (Clexane) in addition to methylprednisolone Four weeks after methylprednisolone-injection the animals were sacrificed Histology (hematoxylin-eosin and Ladewig staining) was performed and empty lacunae andhistological signs of osteonecrosis were quantifiedHistomorphometry revealed a significant increase in empty lacunae and necroticchanged osteocytes in glucocorticoid-treated animals as compared with the glucocorticoid- and Clexane-treated animals and withthe control group No significant difference was detected between the glucocorticoid and Clexane group and the control groupThis finding suggests that cotreatment with enoxaparin has the potential to prevent steroid-associated osteonecrosis

1 Introduction

Nontraumatic osteonecrosis of the femoral head remains achallenge to orthopedic surgeons If untreated it leads in80 of cases to complete collapse of the femoral head [1]The annual incidence of osteonecrosis in the USA is 50 000patients per year most commonly in young active patientswith an average age of 38 [2] The etiology of nontraumaticosteonecrosis is still not fully elucidated Osteonecrosis hasa multifactorial etiology Beside glucocorticoid therapy and

alcoholism hematological diseases like thrombophilia hy-pofibrinolysis and sickle-cell anemia and metabolic diseasessuch as Gaucherrsquos disease are associated with osteonecrosis[1ndash3] Glucocorticoids also directly affect osteocytes Theyinduce osteocyte apoptosis the first histological sign ofosteonecrosis [1 2] Osteocytes are important for the orches-tration of the bone cells The death of these cells leads toan impairment of bone remodeling and finally to a loss ofstructural integrity Another much-discussed theory of oste-onecrosis etiology is ischemia with subsequent damage to

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 347813 6 pageshttpdxdoiorg1011552014347813

2 The Scientific World Journal

the affected tissue Glucocorticoid therapy affects the bloodsupply Glucocorticoids induce fat-cell hypertrophy in thebone marrow and increase intraosseous pressure [4] Gluco-corticoids also may directly affect blood supply by increasingvasoconstriction This constellation is further enhanced byhyperlipidemia hypercoagulation and hypofibrinolysis ofthe circulatory system all of which are caused by glucocor-ticoid use [5]

The pathogenesis of glucocorticoid-induced osteonecro-sis may involve intravascular thrombotic occlusion extravas-cular lipid deposition by fat emboli increased intraosseouslipocyte size or any combination of these This can increasebone-marrow pressure and exacerbate intraosseous circula-tory disturbances resulting in insufficient blood supply andfinally in necrosis [6] Enoxaparin is an established drugfor thrombosis prevention Glueck et al could arrest theprogression of Ficat I-II osteonecrosis in patients with pri-mary osteonecrosis by enoxaparin administration in a humanpilot study [7] Also in a rat model of mechanical inducedosteonecrosis by cutting the ligamentum teres and incessingthe periosteum showed enoxaparin treatment positive effectsThe treatment led to restoration of the necrotic area within4 weeks [8 9] Because of the positive effect of enoxaparinin inhibition of the progression of osteonecrosis and thereparative capacity in a mechanical induced osteonecrosismodel we investigated the preventive potential of enoxaparincotreatment during high dose glucocorticoid therapies

2 Materials and Methods

Osteonecrosis was induced in New Zealand White rabbits(male 3ndash45 kg bodyweight) by injecting once 20mgkg bodyweight methylprednisolone im (GC group 119899 = 6) andcontrol animals (119899 = 6) were treated with phosphate-bufferedsaline (PBS) The therapy group (GC + Clexane 119899 = 6)received 117mgkg body weight per day enoxaparin sodiumstarting with the methylprednisolone administration Theanimals were sacrificed four weeks after methylprednisoloneinjection For histology tissue samples were fixed in 3paraformaldehyde decalcified in 10 EDTA-Tris buffer andembedded in paraffin Hematoxylin and eosin and Ladewigstains were performed on 5 120583m thick slides This experimentcomplied with the German Law on Animal Experimentsand was approved by regional committee of animal experi-mentation ethics (Ministerium fur Energiewende Landwirt-schaft Umwelt und landliche Raume des Landes Schleswig-Holstein V742-72241121-39 (70-904))

21 Quantification of Empty Osteocyte Lacunae Measure-ments were performed on a stereological workstation (Olym-pus CAST grid Olympus Albertslund Denmark OlympusBX 50 microscope) and sections were visualized with aCCD color video camera (600 times 800 pixels JAI GlostrupDenmark) on a 1710158401015840 monitor The area of interest coveringthe whole femoral head was delineated on serial hematoxylinand eosin stained sections at low magnification (lens times125PlanApo) A computer program using systemic randomsampling selected 5 fields within the area of interest (in

the middle of the femoral head) Fields were evaluated persection (lens times40 UPlanApo NA = 070) by superimposinga grid of 16 crosses and counting the number of empty lacunaeand the area of necrotic changes per 1mm2 Two slides ofthe right femoral head per animal were evaluated Threeblinded investigators performed counting of empty osteocytelacunae and the number of necrotic changed osteocytes (cellsdegenerating pyknosis karyorrhexis karyolysis)

22 Statistical Analysis Numerical densities of empty lacu-nae and areas exhibiting necrotic changes were calculatedThe mean and SEM were calculated for every single sectionand for every group prior to calculation of the mean andSEM for each group and each investigated variable TheBartlett test was used to check for equal variances Box-Cox transformation was performed if necessary to achievehomoscedasticity Normal distribution was tested with theShapiro-Wilk test Parametric data were analyzed with one-way ANOVA followed by Tukey 119905-test Statistical significancewas established at119875 lt 005 Statistical analysis was performedusing Graph Pad Prism 5 and JMP 10

3 Results

Ladewig staining revealed normal bone-tissue structure inthe control group (Figures 1(a) and 1(b)) and a strong ab-normal increase in empty osteocyte lacunae in the gluco-corticoid-treated group (Figure 1(c)) In the group treatedwith enoxaparin the number of empty osteocyte lacunaereturned to control level (Figure 1(d)) The bone-liningcells appeared to have less number of pyknotic changednuclei in the enoxaparin-treated group compared to theGC group (Figure 1(d)) Also the number of osteonecroticchanged osteocytes increases after glucocorticoid treatment(Figure 2) The cotreatment with enoxaparin reduced thenecrotic signs in osteocytes (Figure 2) Histomorphometryrevealed a significant increase in empty lacunae and necroticchanged osteocytes in glucocorticoid-treated (GC-treated)animals as compared both with controls and with animalstreated with glucocorticoid and enoxaparin (control versusGC control versus GC + Clexane and GC versus GC + Clex-ane 119875 lt 00001) No significant differences in the number ofempty lacunae nor necrotic changed osteocytes were detectedbetween the glucocorticoid and enoxaparin cotreated group(GC + Clexane) and the control group (Figures 2 3 and 4)

4 Discussion

The literature contains several rabbit models of steroid-induced osteonecrosis [6 10] We chose from among thesethe osteonecrosis model established by Yamamoto et al[11] We used this model to address the question whetherthe anticoagulant enoxaparin could be used as a preventivetherapy against glucocorticoid-induced osteonecrosis [5 7]In the current study administration of the glucocorticoidmethylprednisolone leads to osteonecrosis in the femoralhead as demonstrated by the increased number of emptyosteocyte lacunae and osteonecrotic changed osteocytes

The Scientific World Journal 3

(a) (b)

(c) (d)

Figure 1 Ladewig stained sagittal sections of the femoral head Methylprednisolone treatment induces osteonecrosis demonstrated by anincrease of empty osteocyte lacunae (c) in comparison to PBS treated control animals (a and b) Enoxaparin cotreatment leads to a decreaseof empty osteocyte lacunae and adipocytes (d) Bar represents 100 120583m

400

300

200

100

0

Control GC GC + Clexane

lowastlowastlowast lowastlowastlowast

Empt

y la

cuna

e per

mm

2

Figure 2 Enoxaparin treatment decreases glucocorticoid-inducedosteonecrosis The application of enoxaparin with methylprednisol-one reduces significantly the amount of empty osteocyte lacunaeper mm2 in the femoral head of rabbits due to methylprednisolonetreatment to control levels The graph represents the mean scorewith SEM of empty lacunae Statistical analysis was performed withone-way ANOVA followed by Bonferronirsquos multiple comparison test(control versus GC control versus GC+Clexane andGC versus GC+ Clexane) ( lowastlowastlowast119875 lt 00001)

which are pathological features of osteonecrosis The addi-tional treatment with enoxaparin results in a subsequent

decrease in the number of necrotic changed osteocytes andempty lacunae as compared with the GC animals Thissuggests that cotreatment with enoxaparin may have a pre-ventive effect against steroid-associated osteonecrosis Theassociation between corticosteroid therapy and osteonecrosishas been well established since 1957 when the first case reportof vascular lesions from glucocorticoid therapy in patientswith rheumatoid arthritis was published [12] The femoralhead because of its nonredundant blood supply is vulnerableto ischemic damage caused by capillary occlusions Onemuch-discussed pathomechanism of osteonecrosis of thefemoral head is ischemia Glucocorticoids lead to vasocon-striction Drescher and colleagues [9 13] were previouslyable to demonstrate increased vasoconstriction and coagu-lability following high-dose glucocorticoid application in aswine modelmdashpossibly leading to reduced blood supply andenhanced capillary occlusion causing ischemia Glucocorti-coids are known to suppress the production of vasodilatorssuch as prostacyclin and nitric oxide [14] But glucocorticoidsalso modulate the vascular response to vasoregulators Inmany species such as rats rabbits and humans an enhancedvasoconstrictive response to catecholamines and endothelinhas been observed while the response to bradykinin avasodilator was reduced [12 15] Beside the general reducedoxygen and nutrient supply caused by vasoconstriction theliterature also discusses heritable or acquired risk factors


(a) (b)

(c) (d)

Figure 3 Enoxaparin treatment prevents necrotic changes of osteocytes Methylprednisolone treatment induced osteonecrosis demonstratedby necrotic changes of osteocytes (c + d) in comparison to PBS treated control animals (a) Enoxaparin cotreatment leads to a decrease ofnecrotic changed osteocytes (b) Bar represents 50 120583m

800

600

400

200

0


lowast lowast

Nec

rotic

chan

ged

oste

ocyt

es p

erm

m2

Figure 4 Enoxaparin treatment decreases glucocorticoid-inducednecrotic changes of osteocytes The application of enoxaparinwith methylprednisolone reduces significantly the amount necroticchanged osteocytes per mm2 in the femoral head of rabbits due tomethylprednisolone treatment to control levels The graph repre-sents the mean score with SEM of empty lacunae Statistical analysiswas performed with one-way ANOVA followed by Bonferronirsquosmultiple comparison test (control versus GC control versus GC +Clexane and GC versus GC + Clexane) ( lowast119875 lt 005)

for femoral head osteonecrosis related to hypercoagulabilityhemoglobinopathies steroids angiogenesis and oxidative

stress [16] Dysregulated coagulation parameters play a sub-stantial role in the etiology of osteonecrosis reviewed byOrth and Anagnostakos [3] Glueck et al could demonstratethrombophilia and hypofibrinolysis in patients with idio-pathic and GC induced secondary osteonecrosis [17] Muta-tions of the factor V Leiden and prothrombin genes couldbe observed in patients with idiopathic osteonecrosis andsecondary osteonecrosis [18 19] Several studies have demon-strated the involvement of plasminogen activator inhibitor-1(PIA-1) PIA-1 suppresses the generation of plasmin leadingto hypofibrinolysis Mutation in the PIA-1 gene was identifiedas a risk factor of glucocorticoid-induced osteonecrosis [1420] Kerachian et al could observe an increased PIA-1expression in prednisone-induced osteonecrosis model inWistar rats by Affymetrix analysis [21]

The pathomechanism of osteonecrosis also involves otherfactors of coagulation regulation Wu et al [22] in a pro-teomic study were able to show a lower level of antithrombinIII an inhibitor of the blood coagulation in the serum ofpatients with osteonecrosis M L te Winkel et al [23] werealso able to show a decrease in antithrombin serum level aswell as a decrease in the level of protein S a cofactor of proteinC which acts as an anticoagulative The expression of theseanticoagulative proteins differs significantly in patients whodeveloped osteonecrosis during dexamethasone treatment ascompared with patients who did not develop osteonecrosisM L teWinkel et al [23] also showed increased expression of


factor X after 3 weeks of dexamethasone treatment contribut-ing to development of symptomatic osteonecrosis Cenniet al [24] showed also reduced inhibition of coagulationdue to reduced protein C activity in combinationwith hyper-coagulation due to increasedD-dimer formation in glucocor-ticoid-induced osteonecrosis in a group of 18 patients Plateletaggregation is also modulated by glucocorticoids Sebaldtet al [25] found that patients with osteonecrosis showedinhibited synthesis of prostacyclin (PGI

2) a potent inhibitor

of platelet aggregationIntravascular coagulation is also caused by fat emboli a

complication after glucocorticoid therapy Fat emboli depos-its in vessels and sinusoids may indirectly induce coagulationby activating the complement pathway and causing deposi-tion of immune complex [26]

Glucocorticoid treatment influences the coagulation andthrombus formation at several points The increased coagu-lation in combination with the induced vasoconstriction byglucocorticoids enhances the risk of capillary occlusion fol-lowed by necrosis

In this study we investigate the effect of heparin derivateenoxaparin treatment as a preventive therapy option for usein conjunction with glucocorticoid therapies Enoxaparin isa commonly used drug for thrombosis prevention The mainaction of heparin is to increase inhibition of the serineprotease factor Xa the critical component connecting theintrinsic and extrinsic activations of the coagulation pathwayHeparin binds to antithrombin III and induces a conforma-tion change The ability of this complex to render factor Xainactive thereby undergoes a 300-fold increase as comparedto antithrombin III by itself A secondmechanism is the bind-ing of the heparin-antithrombin III-complex to thrombinwhich leads to the inactivation of thrombin Thrombin isrequired to convert soluble fibrinogen into an insoluble fibrinclot (reviewed in [27])

In clinical trials enoxaparin has proved more effectivethan other heparins in managing acute coronary syndromes(ACS) [22 23] Low-molecular-weight heparins (LMWHs)are used as anticoagulant and antithrombotic drugsThey aremore effective against both venous and arterial thrombosisthan unfractionated heparin [28] By comparison LMWHshave a lower affinity to plasma proteins and endothelial cellsa higher affinity to factor Xa a greater capacity to releasetissue factor pathway inhibitor from endothelial cells a morepredictable dose-response relationship and a longer plasmahalf-life with dose-independent clearance kinetics They arealso relatively resistant to neutralization by platelet factor 4[28] It is now widely accepted that LMWHs have individualbiochemical and pharmacological profiles [25 26]

Several clinical studies were performed to access enoxa-parin as a therapy option to stop the progression of oste-onecrosis Glueck et al could arrest the progression of oste-onecrosis of Ficat stages 1 and 2 by enoxaparin therapy in 19of 20 patients with thrombophilic-hypofibrinolytic disordersover an observation period of 24-month follow-up andonly 20 of Ficat stages 1 and 2 hips of patients withsecondary osteonecrosis did not progress further [7] Thepatients of this study have already amanifested osteonecrosisOur study demonstrates a preventive effect of enoxaparin

treatmentwhen simultaneously administratedwith high doseglucocorticoid therapiesWe were able to demonstrate signif-icant reduced signs of early osteonecrosis The simultaneousadministration of Enoxaparin to high dose GC reduces thenumber of necrotic changed osteocytes and empty osteocytelacunae The use of enoxaparin in a chirurgical inducedosteonecrosis rat model demonstrated an improvement ofquantities of necrotic and newly formed bone compared tocontrol group after 1 month [7]

Future clinical studies are needed to clarify the extentto which patients receiving high doses of GC can benefitfrom cotreatment with enoxaparin to prevent osteonecrosisOne salient area of relevance for such study will be in organtransplantation where due to progress in terms of immuno-suppressive treatment and surgical techniques the survivalrate of transplant patients has increased as well approaching90 of patients 5 years or more after kidney transplant [29]In spite of these advances a number of complications maystill affect the success of organ substitution Of these bonedisease is one of the most frequently reported [29] Patientsundergoing immunosuppressive therapy with steroids mayspare damage to the femoral head simply by cotreatment withenoxaparin

5 Conclusion

Enoxaparin cotreatment has the potential to inhibit the osteo-cyte necrosis a severe side effect of high dose glucocorticoidtherapies

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Rainer Beckmann Hayfaa Shaheen Mamed Kadyrov andWolf Drescher equally contributed to this work

Acknowledgments

The study was in part supported by the German ResearchFoundation (Deutsche Forschungsgemeinschaft) GrantsDR4492-1 PU2145-2 VA 2202-1 PU2144-2 and PU2143-2 in part by a grant from the Medical Faculty Kiel(FoFo) in part by the Forschungsschwerpunkt Muskel- undSkelettsystem (Musculo-Skeletal Science Kiel MSS-Kiel) ofthe University Hospital of Schleswig-Holstein Kiel in partby a grant from the Interdisciplinary Centre for ClinicalResearch (IZKF) within the Faculty of Medicine at theRWTH Aachen University (T9-3 T9-5) and in part bythe Excellence Initiative of the German Federal and StateGovernments

References

[1] M A Mont L C Jones and D S Hungerford ldquoCurrentconcepts reviewmdashnontraumatic osteonecrosis of the femoral


head ten years laterrdquo Journal of Bone and Joint Surgery A vol88 no 5 pp 1117ndash1132 2006

[2] M A Mont and D S Hungerford ldquoNon-traumatic avascularnecrosis of the femoral headrdquo Journal of Bone and Joint SurgeryA vol 77 no 3 pp 459ndash474 1995

[3] P Orth and K Anagnostakos ldquoCoagulation abnormalities inosteonecrosis and bonemarrow edema syndromerdquoOrthopedicsvol 36 no 4 pp 290ndash300 2013

[4] G Motomura T Yamamoto K Miyanishi A Yamashita KSueishi and Y Iwamoto ldquoBone marrow fat-cell enlargementin early steroid-induced osteonecrosismdasha histomorphometricstudy of autopsy casesrdquo Pathology Research and Practice vol200 no 11-12 pp 807ndash811 2005

[5] C J Glueck R Freiberg T Tracy D Stroop and P WangldquoThrombophilia and hypofibrinolysis Pathophysiologies ofosteonecrosisrdquo Clinical Orthopaedics and Related Research no334 pp 43ndash56 1997

[6] L Yang K Boyd S C Kaste L Kamdem R J Rahija andM V Relling ldquoA mouse model for glucocorticoid-inducedosteonecrosis effect of a steroid holidayrdquo Journal of OrthopaedicResearch vol 27 no 2 pp 169ndash175 2009

[7] C J Glueck R A Freiberg L Sieve and P Wang ldquoEnoxaparinprevents progression of stages I and II osteonecrosis of the hiprdquoClinical Orthopaedics andRelated Research no 435 pp 164ndash1702005

[8] J Bejar E Peled and J H Boss ldquoVasculature deprivationmdashinduced osteonecrosis of the rat femoral head as a model fortherapeutic trialsrdquo Theoretical Biology and Medical Modellingvol 2 2005

[9] J H Boss and IMisselevich ldquoOsteonecrosis of the femoral headof laboratory animals the lessons learned from a comparativestudy of osteonecrosis in man and experimental animalsrdquoVeterinary Pathology vol 40 no 4 pp 345ndash354 2003

[10] J Jia W Yao M Guan et al ldquoGlucocorticoid dose determinesosteocyte cell faterdquoThe FASEB Journal vol 25 no 10 pp 3366ndash3376 2011

[11] T Yamamoto T Irisa Y Sugioka and K Sueishi ldquoEffectsof pulse methylprednisolone on bone and marrow tissuescorticosteroid-induced osteonecrosis in rabbitsrdquo Arthritis andRheumatism vol 40 no 11 pp 2055ndash2064 1997

[12] C H Kemper J W Baggenstoss and A H Slocumb ldquoTherelationship of therapy with cortisone to the incidence ofvascular lesions in rheumatoid arthritisrdquo Annals of InternalMedicine vol 46 no 5 pp 831ndash851 1957

[13] T Masada K Iwakiri Y Oda et al ldquoIncreased hepaticcytochrome P4503A activity decreases the risk of developingsteroid-induced osteonecrosis in a rabbit modelrdquo Journal ofOrthopaedic Research vol 26 no 1 pp 91ndash95 2008

[14] S Yang and L Zhang ldquoGlucocorticoids and vascular reactivityrdquoCurrent Vascular Pharmacology vol 2 no 1 pp 1ndash12 2004

[15] T Yamamoto E F DiCarlo and P G Bullough ldquoThe prevalenceand clinicopathological appearance of extension of osteonecro-sis in the femoral headrdquo The Journal of Bone and Joint SurgeryB vol 81 no 2 pp 328ndash332 1999

[16] J Seamon T Keller J Saleh and Q Cui ldquoThe pathogenesisof nontraumatic osteonecrosisrdquo Arthritis vol 2012 Article ID601763 11 pages 2012

[17] C J Glueck R Freiberg T Tracy D Stroop and P WangldquoThrombophilia and hypofibrinolysis pathophysiologies ofosteonecrosisrdquo Clinical Orthopaedics and Related Research no334 pp 43ndash56 1997

[18] C Glueck R Freiberg and G Boriel ldquoThe role of the factorV leiden mutation in osteonecrosis of the hiprdquo Clinical andAppliedThrombosisHemostasis vol 19 no 5 pp 499ndash503 2013

[19] M G Lykissas I D Gelalis I P Kostas-Agnantis G Vozonelosand A V Korompilias ldquoThe role of hypercoagulability in thedevelopment of osteonecrosis of the femoral headrdquo OrthopedicReviews vol 4 no 2 article e17 2012

[20] WDrescher K PWeigertMH Bunger J Ingerslev C Bungerand E S Hansen ldquoFemoral head blood flow reduction andhypercoagulability under 24 h megadose steroid treatment inpigsrdquo Journal of Orthopaedic Research vol 22 no 3 pp 501ndash508 2004

[21] M A Kerachian D Cournoyer E J Harvey et al ldquoNewinsights into the pathogenesis of glucocorticoid-induced avas-cular necrosis microarray analysis of gene expression in a ratmodelrdquo Arthritis Research and Therapy vol 12 no 3 articleR124 2010

[22] R W Wu F S Wang J Y Ko C J Wang and S WuldquoComparative serum proteome expression of osteonecrosis ofthe femoral head in adultsrdquo Bone vol 43 no 3 pp 561ndash5662008

[23] M L te Winkel I M Appel R Pieters and M M vanden Heuvel-Eibrink ldquoImpaired dexamethasone-related in-crease of anticoagulants is associated with the developmentof osteonecrosis in childhood acute lymphoblastic leukemiardquoHaematologica vol 93 no 10 pp 1570ndash1574 2008

[24] E Cenni C Fotia E Rustemi et al ldquoIdiopathic and secondaryosteonecrosis of the femoral head show different thrombophilicchanges and normal or higher levels of platelet growth factorsrdquoActa Orthopaedica vol 82 no 1 pp 42ndash49 2011

[25] R J Sebaldt J R Sheller J A Oates L J Roberts II and GA Fitzgerald ldquoInhibition of eicosanoid biosynthesis by gluco-corticoids in humansrdquo Proceedings of the National Academy ofSciences of the United States of America vol 87 no 18 pp 6974ndash6978 1990

[26] J P Jones Jr ldquoFat embolism and osteonecrosisrdquo OrthopedicClinics of North America vol 16 no 4 pp 595ndash633 1985

[27] SMasuko andR J Linhardt ldquoChemoenzymatic synthesis of thenext generation of ultralow MW heparin therapeuticsrdquo FutureMedicinal Chemistry vol 4 no 3 pp 289ndash296 2012

[28] I Manduteanu M Voinea M Capraru E Dragomir and MSimionescu ldquoA novel attribute of enoxaparin inhibition ofmonocyte adhesion to endothelial cells by a mechanism involv-ing cell adhesion moleculesrdquo Pharmacology vol 65 no 1 pp32ndash37 2002

[29] L Giannini S Nobile and M Sartori ldquoOrgan transplantationand glucocorticoid-induced osteoporosisrdquo in Glucocorticoid-Induced Osteoporosis A Giustina A Angeli and E CanalisEds Karger Basel Switzerland 2002

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


the affected tissue Glucocorticoid therapy affects the bloodsupply Glucocorticoids induce fat-cell hypertrophy in thebone marrow and increase intraosseous pressure [4] Gluco-corticoids also may directly affect blood supply by increasingvasoconstriction This constellation is further enhanced byhyperlipidemia hypercoagulation and hypofibrinolysis ofthe circulatory system all of which are caused by glucocor-ticoid use [5]

The pathogenesis of glucocorticoid-induced osteonecro-sis may involve intravascular thrombotic occlusion extravas-cular lipid deposition by fat emboli increased intraosseouslipocyte size or any combination of these This can increasebone-marrow pressure and exacerbate intraosseous circula-tory disturbances resulting in insufficient blood supply andfinally in necrosis [6] Enoxaparin is an established drugfor thrombosis prevention Glueck et al could arrest theprogression of Ficat I-II osteonecrosis in patients with pri-mary osteonecrosis by enoxaparin administration in a humanpilot study [7] Also in a rat model of mechanical inducedosteonecrosis by cutting the ligamentum teres and incessingthe periosteum showed enoxaparin treatment positive effectsThe treatment led to restoration of the necrotic area within4 weeks [8 9] Because of the positive effect of enoxaparinin inhibition of the progression of osteonecrosis and thereparative capacity in a mechanical induced osteonecrosismodel we investigated the preventive potential of enoxaparincotreatment during high dose glucocorticoid therapies

2 Materials and Methods

Osteonecrosis was induced in New Zealand White rabbits(male 3ndash45 kg bodyweight) by injecting once 20mgkg bodyweight methylprednisolone im (GC group 119899 = 6) andcontrol animals (119899 = 6) were treated with phosphate-bufferedsaline (PBS) The therapy group (GC + Clexane 119899 = 6)received 117mgkg body weight per day enoxaparin sodiumstarting with the methylprednisolone administration Theanimals were sacrificed four weeks after methylprednisoloneinjection For histology tissue samples were fixed in 3paraformaldehyde decalcified in 10 EDTA-Tris buffer andembedded in paraffin Hematoxylin and eosin and Ladewigstains were performed on 5 120583m thick slides This experimentcomplied with the German Law on Animal Experimentsand was approved by regional committee of animal experi-mentation ethics (Ministerium fur Energiewende Landwirt-schaft Umwelt und landliche Raume des Landes Schleswig-Holstein V742-72241121-39 (70-904))

21 Quantification of Empty Osteocyte Lacunae Measure-ments were performed on a stereological workstation (Olym-pus CAST grid Olympus Albertslund Denmark OlympusBX 50 microscope) and sections were visualized with aCCD color video camera (600 times 800 pixels JAI GlostrupDenmark) on a 1710158401015840 monitor The area of interest coveringthe whole femoral head was delineated on serial hematoxylinand eosin stained sections at low magnification (lens times125PlanApo) A computer program using systemic randomsampling selected 5 fields within the area of interest (in

the middle of the femoral head) Fields were evaluated persection (lens times40 UPlanApo NA = 070) by superimposinga grid of 16 crosses and counting the number of empty lacunaeand the area of necrotic changes per 1mm2 Two slides ofthe right femoral head per animal were evaluated Threeblinded investigators performed counting of empty osteocytelacunae and the number of necrotic changed osteocytes (cellsdegenerating pyknosis karyorrhexis karyolysis)

22 Statistical Analysis Numerical densities of empty lacu-nae and areas exhibiting necrotic changes were calculatedThe mean and SEM were calculated for every single sectionand for every group prior to calculation of the mean andSEM for each group and each investigated variable TheBartlett test was used to check for equal variances Box-Cox transformation was performed if necessary to achievehomoscedasticity Normal distribution was tested with theShapiro-Wilk test Parametric data were analyzed with one-way ANOVA followed by Tukey 119905-test Statistical significancewas established at119875 lt 005 Statistical analysis was performedusing Graph Pad Prism 5 and JMP 10

3 Results

Ladewig staining revealed normal bone-tissue structure inthe control group (Figures 1(a) and 1(b)) and a strong ab-normal increase in empty osteocyte lacunae in the gluco-corticoid-treated group (Figure 1(c)) In the group treatedwith enoxaparin the number of empty osteocyte lacunaereturned to control level (Figure 1(d)) The bone-liningcells appeared to have less number of pyknotic changednuclei in the enoxaparin-treated group compared to theGC group (Figure 1(d)) Also the number of osteonecroticchanged osteocytes increases after glucocorticoid treatment(Figure 2) The cotreatment with enoxaparin reduced thenecrotic signs in osteocytes (Figure 2) Histomorphometryrevealed a significant increase in empty lacunae and necroticchanged osteocytes in glucocorticoid-treated (GC-treated)animals as compared both with controls and with animalstreated with glucocorticoid and enoxaparin (control versusGC control versus GC + Clexane and GC versus GC + Clex-ane 119875 lt 00001) No significant differences in the number ofempty lacunae nor necrotic changed osteocytes were detectedbetween the glucocorticoid and enoxaparin cotreated group(GC + Clexane) and the control group (Figures 2 3 and 4)

4 Discussion

The literature contains several rabbit models of steroid-induced osteonecrosis [6 10] We chose from among thesethe osteonecrosis model established by Yamamoto et al[11] We used this model to address the question whetherthe anticoagulant enoxaparin could be used as a preventivetherapy against glucocorticoid-induced osteonecrosis [5 7]In the current study administration of the glucocorticoidmethylprednisolone leads to osteonecrosis in the femoralhead as demonstrated by the increased number of emptyosteocyte lacunae and osteonecrotic changed osteocytes


(a) (b)

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5 Conclusion






Acknowledgments


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Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















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5 Conclusion






Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)


800

600

400

200

0


lowast lowast

Nec

rotic

chan

ged

oste

ocyt

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m2
















5 Conclusion






Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























5 Conclusion






Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article enoxaparin prevents steroid-related...

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