The effect of long-term methylprednisolone treatment on the femoral head in growing pigs

ELSEVIER

Journal of Orthopaedic Research

Journal of Orthopaedic Research 20 (2002) 661-668

www.elsevier.comllocate/orthres

The effect of long-term methylprednisolone treatment on the femoral head in growing pigs Wolf Drescher ',*, Haisheng Li a, Saren D. Jensen ', Jargen Ingerslev ', Ebbe S. Hansen ', Ellen M. Hauge ', Cody Bunger a "

Dcputnient

clf

Outhoptrrdit~r,Aurhus Lh~irersityFfo.~pitu/,8f)OO Aarhus, Denmark

' Ccntcr fi)r Hrtnopliiliu und Thruinbosis, Depurttiient of Clinic~irlhntnunolugy, Aurhux Uniwrsity Hospitul, 8000 Aurhus, Denimrk Depirtwwiii uf Clinical Iniinunology, .$urhus Uniwrsity Hospitul, HOOO Aurhus, Denmurk Received 14 September 2000; accepted 28 November 3,001

Abstract

The efTect of long term steroid treatment on coagulation, intraosseous pressure (IOP), femoral head (FH) blood flow, and histology in the normal organism was investigated in this study in growing pigs. From 24 growing female Danish Landrace pigs from 12 litters, 12 animals daily received 100 nig methylprednisolone orally for three months. Their 12 sister pigs served as controls without steroid treatment. Prothrombin time, activated partial thromboplastin time (aPTT), fibrinogen, and antithrombin 111 levels were recorded in jugular venous blood. Blood flow of the hip regions was measured by means of the radioactive microsphere technique. Metaphyseal and epiphyseal IOP of the left or right proximal femur were measured. Histomorphometry of the left or right FH epiphysis was performed. Major growth inhibition was found in the corticosteroid (CS) treated group. In CS pigs, aPTT was shortened to 500'0 compared to control pigs. Plasma fibrinogen was higher in the CS animals. Total F H blood flow was not digerent while regional blood flow in the cranial subregion of the F H epiphysis was higher in the CS group. Metaphyseal and epiphyseal 1OP of the proximal femur were not different in the CS animals. Histomorphometrically, cancellous bone volume (23 i ]'!,;I vs. 33 f ?'!,;I; p < 0.001) and bone turnover (10C! 2n.h vs. 55 f 8 ' ! 4 p < 0.001) of the F H epiphysis was lower in the CS than in the control group. The FH epiphysis of the CS animals invariably showed an irregular cartilage-bone interface with cartilaginous projections into the subchondral bone mainly in its cranial part. In normal growing pigs, long term high dose CS treatment has induced a hypercoagulable state of plasma via the intrinsic pathway of coagulation, cartilaginous projections into F H subchondral bone, FH osteopenia, and reduced bone turnover. Long-term steroid treatment did not produce F H necrosis or F H IOP alterations in the immature pig model. 0 3001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.

Introduction The effect of long term steroid treatment on the immature femoral head (FH)has not yet been investigated in a large animal model [40]. Nevertheless, children receiving methylprednisolone treatment have been reported to have disturbed growth and skeletal maturation [36]and osteonecrosis in 3.4% [lo] to 35% [34] of cases. Corticosteroids (CSs) have been suggested to cause changes in the coagulability of blood [37] and hypercoagulability has been related to skeletal lesions in children [39] and Legg-Perthes'-disease [ 151. Most

Corresponding author. Address: Department of Orthopaedics, University Hospital Eppendorf, D-20246 Hamburg, Germany. Tel.: +49-4192-90-25?6; fax: +49-4 192-90-3388. E-rwil U/~~I.C'.FS:wolt"[email protected] (W. Drescher). I

recent work has shown that short-term high dose methylprednisolone treatment caused selective bone blood flow reduction in growing pigs [7]. This study aims to elucidate the effect of chronic methylprednisolone treatment, as used after renal transplantation [ 101, on coagulation, FH hemodynamics, and bone mineralization in a porcine model.

Methods Study desigii The experiment complied with the Danish Law 011 Animal Experiments and was approved by the Danish Ministry of Justice. J. nr. 1998-561-67. Twentyfour immature female Danish Landrace pigs 10 weeks of age from I2 litters were randomly assigned to one of two groups of 13-a glucocorticoid treated experimental group (CS) and an untreated control group (NCS) of 12 sister pigs receiving no steroid

0736-0266/02/$ - see front matter 0 2002 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 7 3 6 - 0 2 6 6 ( 0 1 ) 0 0 1 8 3 - 8

treatment. Following three months of treatment. coagulation parameters were determined. Central hemodynamics were examined and regional blood flow (RBF) was recorded by means of the radioactive microsphere ( M S ) technique in the F H epiphysis. metaphysis, hip joint capsule, ligamentum teres, acetabular bone, and reference regions. Metaphyseal and epiphyseal intraosscons pressure (IOP) of the left or right proximal femur were measured and histomorphometry of the contralateral side was performed. i Z l ~ ~ ~ l ~ ~ ~ rudniini,s/rution c ~ ~ i ~ i s ~ ~ l o i ~ ~ ~

The CS group of animals received oral methylprednisolone 100 mg per day (Medrol; Pharmacia & Upjohn, Copenhagen, Denmark) mixed into the morning feed. The dosage of methylprednisolone in this study was approximated to doses used in pediatric renal transplant patients [22]. All pigs were hept in separate boxes at the Danish Institute of Agricultural Sciences, Research Center Foulum, the controls for 98.2 (91-107) days, and the CS animals for 96.9 (91-106) days (mean and range). During that time period, the CS animals received a cumulative dose of 9.7 g (9.1 ~10.6g ) methylprednisolone. By giving 100 mg methylprednisolone per day. the initial dosage was 3.6 (0.1) nig(kg bodyweight) (mean, SEM). The mean dosage during the first month was 3.0 (0.1) mg(kg bodyweight)-', 2.3 (0.1) mg(kg bodyweight)-' during the second, and 1.9 (0.1) mg(kg bodyweight) [ during the third month.

'

After three months, the animals were premedicated with 25 mg midazolam (Dormicum; Hoffman La Roche, Basel, Switzerland) and 200 mg azaperon (Stresnil; Janssen Pharmaceutica, Bcerse, Belgium) intramuscularly. Intravenous anesthesia was induced by 20 mg etomidate (Hypnomidate; Janssen Pharmaceutica, Beerse, Belgium), and after orotracheal intubation maintained by a combination of 30 nil ketamin (Ketaminol Vet; Veterinaria, Switzerland) 50 mgml I , 4 ml pethidinhydrochloride (Petidin Amino; Amino AG, Switzerland), 6 ml midazolam (Dormicum; Hoffman-La Roche, Basel, Switzerland) 5 mgm-'], 6 ml pancuron (Pavulon; Organon Teknika, Turnhout, Belgium) 2 mgml-I, and 4 ml isotonic saline at a rate of 20 ml h I . The pig was positioned supine and ventilated o n a Servo Ventilator 900 (Siemens-Elema; Sweden) with hips in neutral position during blood flow measurements.

left ventricle under fluoroscopic control through the sheath in the carotid artery. Another pigtail catheter (6.0 F ) wab advanced into the thoracic aorta via the other sheath within the Same carotid artery. Each vial of MS contained 5.0 = 10" spheres suspended in 5 ml lo"/;, Dextran. The vial was agitated for 5 min on a Whirlimixer" (Fisons; Loughborough, England) before injection. The spheres were injected through the pigtail catheter into the left ventricle over a period of 30 s followed by flushing with 5 ml 37 "C heparin-saline. Reference blood sampling from the aorta was started 30 s before sphere injection and continued until 4 min after the injection. For cardiac output (CO) determination, the total activity in each vial was measured in an Amersham Calibrator (ARC 120; Capintec Inc.. NJ, LJSA)before injection. After the experiment, all the remnants were measured and subtracted to obtain the injected dose in MegaBecquerel ( M Bq). A predetermined MBq quantity of MS was suspended in tap water and vortexed, and I0 I-ml aliquots were withdrawn for determination of counting elticiency (counts per minute/MBq) of the gamma counter for the isotope used. After killing the pig with intravenous iiijection of pentobarbital 0.4 in1 kg-' bodyweight the FH epiphysis, hip joint capsule. and Iigamentum teres were each harvested in toto. The F H epiphysis was carefully separated from the epiphyseal plate and cut into 16 rectangular columns perpendicular to the growth plate. Samples from proximal femoral metaphyseal cortical and cancellous. and acetabular bone were removed. All samples were distributed into preweighed counting vials. Non-osseous reference samples were taken from heart muscle at the apex, from gluteal skeletal muscle, and from gluteal subcutaneous fat. The reference blood samples and tissue samples were counted for gamma activity (Packard Cobra, Packard Instrument Company, Meriden, CT, USA) using correction for background and decay during counting. The R B F of each predefined region (RBFh,t,p,i, ml min 100g ) was determined according to Hales [IS] as

'

where Culups~denotes the count rate o f a predefined region (counts per minute, cpm), C K t b the count rate of the reference blood sample from the thoracic aorta (cpm), SR the sampling rate of the reference blood sample (mlmin-'), and W ~ y B I othe p ~ weight ~ of the biopsy (gj. C O was calculated from MS injectates (MS,,l,)and total MS count in the reference blood (MS,,f) using the equation

Coqulurion t~ic'usurcviiwt

In general anesthesia a 6 F sheath (Fast-CathiM; Daig Corp., Minnetonka, USA) was inxrted unilaterally into the jugular vein from which three 5 ml citrate blood samples were taken immediately. The samples were centrifuged for 10 min at 4000 cycles per minute at room temperature. Using platelet free plasma, the prothrombin time (PT: thromboplastin reagent Excel S, Organon Teknika, Turnhout, Belgium) the activated partial thromboplastin time ( a m , reagent Platelin LS, Organon Teknika, Turnhout, Belgium) functional fibrinogen as derived from the amplitude of the PT test, and antithrombin 111 (AT-111; Coaniatic Antithrombin, Chromogenix, Molndal. Sweden) levels were recorded on an ACL-3000 CoagLrhtion Analyser- (Instrnmentation Laboratory, Milan, Italy) employing routine reagents as described. For these parameters, reference intervals for iiorinal pig plasma have not been established.

Blood flow

I~I~FUO.S.S~YJU.S pw.ssurcj

i?ii'ir~suI'L'iiic'tit

One F H of each animal was randomized for IOP measurement in the FH metaphysis and epiphysis [5].From a lateral open approach, an intraosseous cannula (Radner" biopsy cannula j with an inner diameter of 1.5 mm was inserted first into the proximal femoral metaphysis and then further advanced into the F H epiphysis. The position of the tip of the cannula was secured by fluoroscopy, and the stylet removed. The cannula was flushed with heparin-saline solution (5000 U 1 I ) and filled with saline by means of a spinal needle. The pressure transducer ( h i f l o w ; Baxter Healthcar-e Corporation. Santa Ana, C A ) was zeroadjusted and via saline-filled manometer tube (Portexm;". length of 200 cm, inner diameter 1.5 nim) connected to the cannula. The 1OP was monitored on a CardioMed CM-4008 Physiological Trace System (Medi-Stim AS, Oslo, Norway).

wi~ti~~~ri~in~nt

Histology RBF measurement was undertaken in general anesthesia by means of 15 itni Cerium 141 labeled MS (New England Nuclear, Boston, M A ) in the hip and reference regions [19,25]. Sheaths (7.0 F: Fast-Cath, Daig Corporation, Minnetonka, USA) were placed in one common carotid artery. Systolic, diastolic, and mean arterial blood pressure were monitored i n a carotid artery by a pressure transducer (Uniflow; Baxter Healthcare Corporation, Santa Ana, CA) on a CardioMed CM-4008 Physiological Trace System (Medi-Stim AS, Oslo, Norway). on which also ECG and rectal temperature were monitored continuously. Blood gas analysis was performed half-hourly on an ABL 510 (Radiometer AIS, Copenhagen, Denmark). Fur administration of the MS, a pigtail catheter (6.0 F; Cook", Denmark) was advanced into the

In order to estimate mineralizing activity in the F H epiphysis. tetracycline labeling was done by gluteal intramuscular tetracycline injection (Supramycin: Griinenthal, Aachen, Germany) 10 and 3 days at a dose of 20 mglkglday before killing the animal [9]. Both FHs were harvested immediately after sacrifice. The FH not randomized for IOP and RBF measurement was harvested for histology. FH epiphyseal slices were sampled in the frontal plane in order to allow for comparison with RBF in the corresponding subregions of the contralateral FH. The slices 0.5-1 cm of thickness were fixated in 70";) alcohol, cmbedded undecalcified in niethylmetacrylate, cut into 7 pm slices p ~ d l eto l the frontal plane. and stained with Goldner Trichrome. Two

12: Drrsc,her et 01. / Journal of Orthopnrdic Resrurrh 20 (2002)662-668

664

sections from each animal were mounted unstained for epifluorescent microscopy. Stereologically unbiased estimates of FH cancellous bone volume were obtained by point counting using an Olympus BX light microscope equipped with an eyepiece-integrated reticle at a magnification of 60. The area of interest for trabecular bone volume and mineralizing bone surface was the cancellous bone tissue demarcated by the subchondral bone and the growth plate. A total of 2000 hits on the tissue (bone marrow) were sampled systematically and randomly [17]. Bone volume was estimated as the number of hits on cancellous bone and osteoid divided by the number of hits on the tissue (BV/TV, Bone volume/tissue volume). In a randomly selected subpopulation ( n = 10) the coeficient of variation for BV/TV was 7'!,0 as estimated by double counting. The surface extent of tetracycline deposits in mineralizing bone was evaluated in the frontal FH epiphyseal slices [9]. Tetracycline deposits where active mineralization is ongoing. In remodeling bone, administering two doses of tetracycline separated by an interval results in two tetracycline lines separated by a distance. The labelled surface is an estimate of bone turnover. A total of 400 hits on the bone surface were sampled systematically and randomly in the frontal FH epiphyseal slices. The stereological requirement of isotropic uniform random sampling was thereby only fulfilled in part [I]. Mineralizing surface MS was estimated as the sum of the number of hits on double-labelled bone surface and half the number of hits on singlelabelled bone surface divided by the total number of hits on bone surface (MS/BS, Mineralizing surface/bone surface). The FH epiphysis was examined qualitatively for signs of osteonecrosis. Bone tissue was considered necrotic when at least one of the following signs were present: ( I ) bone marrow cell necrosis, (2) osteocyte pycnosis, ( 3 ) empty osteocyte lacunae. (4) blurred and swollen lamellae, or (5) sequestered bone tissue surrounded by mesenchymal reaction [4?] o r late signs of osteonecrosis like vascular ingrowth, creeping substitntion, marrow fibrosis. decreased subchondral bone tissue, or chondral collapse [31]. The sections were examined for signs of microthrombosis or fat deposition.

+

Stat i.c.tics

+

Results are presented as mean SEM. The coagulation, blood flow, IOP, and BV/TV data were log10 transformed and normal distribution was documented by Q-Q-plotting. Homogeneity of variances in the transformed data was checked by Levene's test. Comparison of the two experimental groups was done by the unpaired samples t-test at p < 0.05 significance level.

Table 1 Coagulation in steroid-(CS) and non-treated (NCS) animals

NCS (n = 10) Prothrombin time PT (s) Activated partial thromboplastin time aPTT (s) Plasma fibrinogen (gl-') Antithrombin Ill AT-111 (U/I) NS = not significant

CS ( n = 8)

Mean

SEM

Mean

SEM

p <

10.2

0.2

10.0

0.1

NS

34.5

3.6

16.1

3.7

0.001

6.0

0.4 0.0

9.5 1.2

1.8 0.2

0.05 NS

0.9

pigs showed macroscopically visible clotting before analysis. These four animals were excluded from the analysis. In CS pigs ( n = 8), the aPTT was shortened to 50% compared to control pigs ( n = 10, Table I ) plasma fibrinogen displayed a significant tendency to increase in C'S animals (Table I ) . Regional bloodJon.

Complete RBF data were obtained from 10 control and 9 CS animals (Table 2). RBF of the total FH epiphysis was not different in the NCS and the CS group (Table 3 ) . RBF in the cranial subregion of the FH epiphysis was 14.3 =k 2.0 mlmin-' 1OOg-' in the NCS group (n = 10 hips), (Fig. l ) , significantly lower than 22.5 3.3 mlmin-' l0Og-' in the CS group of animals ( n = 9), (p < 0.05). In the remaining hip regions, no RBF difference was found between the control and CS group of animals. Gluteal subcutaneous fat RBF was higher in the CS treated animals (Table 3 ) .

*

Results Total bodyweight (TBW)at the start of the study was 27.9 (0.7) kg in the CS animals and 27.2 (0.7) kg in the controls. TBW at sacrification was 59.2 (2.3) kg in the CS pigs ( n = 12), and 109.9 (0.7) kg in the control group ( n = 12; p < 0.001). Mean daily weight gain was 0.326 (0.03) kg in the CS animals and 0.846 (0.02) kg in the controls. The CS treated pigs appeared shorter in axial length, lowet- in shoulder height, had more subcutaneous fat, and thinner extremities. One of the control animals was lost related to surgery, and one was lost related to anesthesia. In two of the CS animals, the pigtail catheter for reference blood sampling from the aorta was clotted, and one of the CS animals was lost related to anesthesia. Coagulation

Blood samples from the first NCS pig and the first CS pig were not analyzed because of delay in equipment supply. Samples from 1 NCS pig showed hemolysis, 1 CS pig died during anesthesia, and samples from 2 CS

Iritriiosseous pressure A total of 19 animals was available for IOP measurement as for the reasons mentioned above. An adequate, central position of the cannula in the FH metaphysis and epiphysis was obtained in eight animals in the control group and eight animals in the CS group. Metaphyseal and epiphyseal IOP of the proximal femur were not increased in the CS animals (Table 4).

Table 2 Central hemodynamics in steroid-(CS)and non-treated (NCS)animals

Specific C O (mlmin (kg BW)..') Mean arterial pressure (mmHg) Heart rate (beatsmin-')

'

NS = not significant.

NCS ( n = 10)

CS

Mean

SEM

Mean

SEM

59

3

50

4

NS

94

3

88

4

NS

84

6

86

4

NS

(fl

= 9)

It: Drescher et al. I Journal Table 3 RBF of the hip (ml min

'

Region

100 g

of

665

Orthopardic, Rcmirch 20 (2'002)66,7468

Table 4 IOPs of the proximal femur (mmHg)

')

NCS ( n = 10)

CS ( n = 9 )

Mean

SEM

Mean

SEM p <

13.2 33.8

2.0 7.5

18.4 32.3

2.7 4.7

NS NS

13.1

1.8

11.0

1.3

NS

19.2

2.8

20.3

1.4

NS

27.0 3.6 3.4 1.1

3.8 1.1 0.6 0.2

18.6 3.0 3.3 2.4

1.1 0.7 0.4 0.7

NS NS NS 0.05

NCS ( n = 8 )

CS ( n = 8)

Mean

SEM

Mean

SEM

12.9 15.3

2.4 3.1

16.3 12.8

4.3

~~

Femoral head epiphysis Femoral head growth plate Proximal femoral corticalis Proximal femoral metaphyseal cancellous bone Acetabular bone Capsule Ligamenturn teres Gluteal subcutaneous fat NS = not significant

The macroscopic size of the FH was smaller in all CS animals (Fig. 3). Histology

The FH epiphysis was found to be osteopenic in the CS animals compared to that of the NCS animals (BV/ TV 33 z!= 1% vs. 34 4~ 2%; p < 0.001; Fig. 2). FH epiphyseal bone turnover was lower in the CS animals as the mineralizing activity was much less in steroid treated animals compared to control animals. The extent of double-labelled surface as well as the extent of singleplus double-labelled surface was lower in the CS group (MS/BS 10 5 2%)VS. 55 5 8%; p < 0.001). All the FH epiphyses of the CS animals showed a scalloped interface between the articular cartilage and the underlying bone mainly in the cranial angle between articular cartilage and the epiphysis, (Fig. 3). These cartilaginous projections extended into the subchondral

Femoral head metaphysis Femoral head epiphysis NS = not significant.

2.2

NS NS

Fig. 2. Gross overview of the femoral head in the frontal plain of the CS treated animals to the left and non-treated (NCS) animals to the right (Goldner Trichrome stain).

plate (Fig. 4). These finger- and tongue-like shaped cartilaginous projections had a morphology different from the overlying articular cartilage. The chondrocytes were still in columnar arrangement, but were disturbed in their zonal organization. Besides the columns of chondrocytes, empty tubes of cartilaginous matrix were found. The cartilaginous projections showed tetracycline labelling indicating ongoing enchondral ossification. This was not seen in any of the control animals. The chondrocytes localized above the projections had lost their zonal organization, thus being disturbed in their maturation. No signs of necrosis were encountered in any of the groups. In the CS group, the hematopoetic marrow was predominant in the subchondral region being gradually replaced by adipocytes near the FH epiphysis, thus forming a clear cellular gradient (Fig. 3 ) .

Fig. I. The perfusion pattern of the FH with and without long-term methylprednisolone treatment in a view from superomedial. The cranial subregion is marked.

666

Fig. 3. ( a ) Subchondral lacunae in the cranial part of the femoral head epiphysis of the CS animals. ( b ) Regularly delimited subchondral plate in the femoral head epiphysis of the control animals.

Fig. 4.(a) Tongue- and ( b ) finger-like lacuna projecting into the subchondral bone of' the fernordl head (Goldner trichrome stain). Chondrocytes without clear zonal qtructure along with empty tubes of cartilaginous matrix are found.

No signs of microthrombosis or fat deposition were found.

Furthermore, an increased plasma fibrinogen concentration was found in the methylprednisolone treated animals. Also in clinical studies, increased fibrinogen plasma levels have been reported under steroid treatDiscussion ment [37].Increased plasma fibrinogen is associated with enhanced hyperviscosity and hypercoagulability [30]. Hypercoagulability has previously been suggested The porcine coagulation system essentially employs the same set of coagulation factors as found in all as a possible cause of osteonecrosis [17-14,23,43] and Perthes' disease [l 11. In otherwise healthy pigs, steroid other mammals, including human [31]. Our results in the treatment caused a hypercoagulable state in the present untreated NCS pigs for PT, aPTT, AT-111. and the study. The activated partial thromboplastin time that fibrinogen levels are approximately equal to the correwas significantly shortened in CS pigs displays the glosponding human values [4,21]. IOP of the FH metaphbal function of the intrinsic pathway of coagulation [24]. ysis and epiphysis in this study was not increased in the Since the global coagulation measure of the extrinsic steroid treated animals. Likewise, total FH RBF was pathway of coagulation, the PT, was unchanged in CS unchanged by CS treatment in the present study while being increased in the cranial subregion of the FH (Fig. trcated pigs, suspicion is raised that one or more coagulation factors in the early intrinsic coagulation phase 1 ). Long-term steroid treatment has shown to cause inare affected by methylprednisolone. Patients receiving creased IOP of the F H [39] and decreased FH blood high dose methylprednisolone for Idiopathic Thromboflow in mature rabbits [38]. These studies suggested that cytopenic Purpura have shown to develop hypergrowth of fat cells causes a rise in IOP, and thereby to coagulability due to increased activity of the intrinsic compress the thin-walled sinusoids, with subsequent coagulation factors VIII and von Willebrand factor [B]. decrease in bone blood flow [41]. The lower trabecular

bone volume found in the CS treated pigs in the present study might counteract an increase in IOP. In the present study, growth of marrow fat cells was not found, and subcutaneous fat blood flow was significantly higher in the steroid treated animals. In a study in steroidtreated systemic lupus erythematosus patients, FH perfusion was also greater than in controls [3]. In contrast to the above studies, the present study was performed in growing pigs at an age where weight gain is enormous, and the animals received higher doses of cortisone. Steroid treatment in this study was started at the age of 10 weeks. At this age, the ossific nucleus still occupies <100% of the FH epiphysis [30]. Histopathology in the present study revealed an irregularly shaped subchondral bone plate with chondrous lacunae mainly in the cranial area exclusively in the CS treated animals. Although no signs of microvascular occlusion could be found, the chondrous lacunae might represent local zones of microcirculation disturbed by hypercoagulability and hyperviscosity [30]. Compatible histologic findings have been reported in meningococcemia and disseminated intravascular coagulation in children [ 16, 291. These findings may also be a direct effect of steroid treatment as the glucocorticoid receptor has been demonstrated in chondrocytes and osteoblasts [33]. In the present study, total weight gain stunted by a mean 520 grams per day in the methylprednisolone treated group. Glucocorticoid therapy in children has been shown to suppress somatomedin generation and thereby to cause growth retardation [8]. Cessation of growth, narrowing and premature closure of the growth plates have been documented in young rabbits under methylprednisolone treatment [32]. It has been shown that glucocorticoids act locally on the growth plate to inhibit longitudinal bone growth in rabbits [2]. Trueta has reported compatible histopathologic findings under ischemia of the F H epiphysis in growing rabbits [35,36]. He introduced local ischemia by filling a channel drilled into the F H epiphysis with polyethylene. Ossification stopped where local blood supply was impaired by the drill channel. This lead to chondrous ossification defects histopathologically compatible to our findings. F H necrosis was found after a single high-dose methylprednisolone injection in adult rabbits [42]. In contrast to the present findings, previous work has shown a 2-3 fold decrease of FH blood flow after 2 weeks of high dose intramuscular methylprednisolone treatment in immature pigs [7].No histologic signs of F H N were found in the present material. The steroid dosage in the present study was lower and maintained longer than in the previous study and than in studies in rabbits in which osteonecrosis was found [42]. Using the same methylprednisolone protocol as in the present study, the authors have found decreased vertebral endplate and cancellous bone blood flow in growing pigs [6] which may be due to the different blood supply [37].

In conclusion, this study has shown hypercoagulability and changes in the metabolic function of FH bone after long-term steroid treatment. The effect of steroid treatment in immature pigs was growth inhibition, lower bone turnover, and osteoporosis as found in humans. Three months of steroid administration induced a hypercoagulable state via the intrinsic pathway of coagulation in the growing pig. Long-term steroid treatment did not produce F H necrosis or FH IOP alterations in the immature pig model.

Acknowledgements This work was supported by The Institute of Experimental Clinical Research, Aarhus University Hospital, Aarhus, Denmark and The Danish Rheumatism Association. The first author had a PhD-grant from Aarhus University, Aarhus, Denmark.

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