Joint Space Gap of Leptin-Def. ob/ob Mice in Response to Loading

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ORIGINAL! ARBEITEN

Joint Space Gap of Leptin-Def. ob/ob Mice in Response to Loading

GelenkspaLtweite Leptin-def. Mäuse unter biomechanischer Belastung

Das Hormon Leptin reguliert die Reifung und Entwicklung des Kno- chens. Unbekannt ist der Einfluß auf die Gelenke. Ziel der Studie war es, ohne die hormonelle Wirkung von Leptin die Auswirkung des Körpergewichtes auf den Gelenkspalt von Hüft- und Kniegelenk während des Wachstums zu untersuchen. Material und Methode:

Je 20 C57BL/6J-Lepob (ob/ob) weibliche leptindefiziente Mäuse wurden in eine Gruppe mit freier Futtermenge (Gruppe A) und ei- ne Gruppe mit einer kontrollierter Futtermenge (Guppe B) einge- teilt. Für die Analyse wurde ein Micro-CT (SkyScan 1072) verwen- det. Ergebnisse: Gruppe Α zeigte im Verlauf ab der 6. Lebenswoche ein signifikant höheres Körpergewicht als Gruppe B. Nach 20 Wo- chen betrug das Körpergewicht etwa das Doppelte. Statistisch sig- nifikante Unterschiede zeigten sich zwischen beiden Gruppen im Körpergewicht (A>B) und der Knochendichte (B>A); im Micro-Ct war die Anzahl der Trabekel (Tb.N.) nur im proximalen Femur der Gruppe Β erhöht. Einen signifikant weiteren Gelenkspalt des Hüft- gelenkes zeigte sich in coronarer, sagittaler und transversaler Ebe- ne der Gruppe Α und im Kniegelenk in der coronaren Ebene, im transversalen Querdurchmesser und in der subchondralen Slerose.

Signifikante Korrelationen konnten zwischen der Knochendichte und dem Querdurchmesser des Tibiaplateaus, zwischen der Anzahl der Trabekel des Hüft- und Kniegelenkes, dem Körpergewicht und zwischen der Anzahl der Trabekel des proximalen Femurs und dem Durchmesser des Hüftkopfes in coronarer, sagittaler und transver- saler Ebene gefunden werden. Zusammenfassung: Bei Leptindefi- zienz nimmt die Knochendichte bei zunehmendem Körpergewicht ab. Eine signifikante Änderung der Gelenkspaltweite lässt sich je- doch in beiden Gruppen im Micro-CT nicht nachweisen.

Autoren: Hansjoerg Heep1, Alexander Weg- ner1, Sebastian Hof- meister1, Christian We- demeyer1

Schlagworte: Leptin, Gelenkspalt, Mikro-CT, bomechanische Belas- tung

Keywords:leptin, joint space, microCT, biomechanical loading Zitierweise dieses Bei- trages: BlOmaterialien 2010; 11 (November) S. 31-36

Introduction

It is a known fact that body fat regulates bone metabolism by means of hormonal factors, and furthermore, that the effects of muscle and loading are s i g n a l i n g factors in m e c h a n o t r a n s - duction [12, 40]. Leptin, a peptide h o r m o n e produced predo- minantly by white fat cells, is one of these hormonal factors [1, 13, 14, 18, 30, 42], Leptin inhibits appetite; consequently, leptin deficiency induces obesity. With regard to bone metabolism it is not clear w h e t h e r leptin is a stimulator or an inhibitor of bone growth. Some study authors noted a positive relationship between serum leptin levels and bone mineral density [16, 28],

'Department of Orthopaedics, University of Duisburg-Essen, Pattbergstr. 1-3, 45329 Essen

whereas others observed a negative or no relationship [4, 33, 24, 29], which further c o n f o u n d s the interpretation of the effect of leptin on bone mass. Simha et al. concluded that the role played by leptin in b o n e metabolism in h u m a n s m a y de- pend on the stage of life [35]. This might explain its positive effect on b o n e f o r m a t i o n in the early stages of d e v e l o p m e n t . Leptin is also k n o w n to increase both proliferation and diffe- rentiation of the c h o n d r o c y t e population of skeletal growth centers in organ cultures [23, 26], All these findings strongly support the theory that leptin could stimulate b o n e growth in the early stages of life.

In a few recent reports the animal model was a useful surrogate to investigate the mechanism of leptin. Mice with deficiency of leptin (obese; ob/ob) or its receptor (diabetic; db/db) are obe-

2 ] B l O m a t e r i a l i e n 11 (November), 2010

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Hansjoerg Heep: Joint Space Gap of Leptin-Def. ob/ob Mice in Response to Loading

se [1]. Both leptin and its receptors were found in murine fe- tal cartilage and b o n e template, as well as in the growth plate [21]. Takeda et al. and Karsenty described t h e l e p t i n - d e - p e n d e n t central control of b o n e remodeling via the s y m p a - thetic nervous system [18, 19, 36, 37]. The sterility of ob/ob mice should increase bone resorption and indeed, osteoclast numbers and the parameters of bone resorption did increase in ob/ob mice. Nevertheless, leptin-deficient mice h a v e a higher bone mass t h a n wild-type mice and completely correct their high bone mass by intracerebroventricular infusion of leptin at a rate that does not result in any detectable leak of leptin in ge- neral circulation [10].

In general, mechanical loading stress on bones causes tissue de- formation within the bone and stimulates the b o n e to adapt and remodel, ultimately improving resistance to osteoporosis.

While previous studies have s h o w n that bone parameters im- proved with weight-bearing exercise in normal subjects, we were interested in the co-influence of biomechanical loading on body weight and bone metabolism in subjects with leptin deficiency. There is so far no agreement regarding the positi- ve effect of leptin in the early stages of life.

The ability of three-dimensional micro-computed tomography to detect changes in a rat model was evaluated for b o n e mass [2, 9, 22, 27, 32, 34, 38]. It is a method to image, q u a n t i f y and study trabecular b o n e and the bone remodeling [32], A f t e r induction of osteoarthritis by drugs in the murine knee, Botter et al. showed that significant changes in subchondral bone architecture a n d could be detected and q u a n t i f i e d in 3D u s i n g micro-CT a n a l y s i s [5]. Ankle micro-CT a n a l y s e s de- monstrated b o n e destruction in a rat model [20].

The aim of this study was to investigate the differential effects of weight-bearing on j o i n t space in mice without the stimula- tion of leptin.

Materials and Methods

Animals

C57BL/6J-Lepo b (ob/ob) female mice were received f r o m the J a n v i e r Laboratory (Le Genest St Isle, France) at the age of fi- ve weeks. The animal experiment was approved by the local authorities according to the official guidelines.

The effects of dietaiy restriction in ob/ob and wild-type mice h a s been described in several studies [7, 15, 25]. The animals were housed one per cage with access to water ad libitum and a s t a n d a r d rodent diet (8640 Harlan Teklad 2 2 / 5 [ l ] ; Harlan Teklad, Madison, WI, USA) c o n t a i n i n g 1.13°/o c a l c i u m a n d 0.94% phosphorus. The animals were maintained u n d e r con- ditions of a t w e l v e - h o u r light and dark cycle w i t h the light switched on at 6.00 a.m.. Food intake and body weight were recorded daily. There were two experimental groups divided according to the provision of food. Group A included 20 ob/ob mice with access to food ad libitum, the other 20 ob/ob mice in Group Β received a limited amount of food (6 gr. each day).

All the animals were euthanized by mechanical procedure at the age of twenty weeks.

Micro-CT

A high resolution micro-CT (SkyScan 1072, Aartselaar, Bel- gium) was used to perform qualitative and quantitative analysis of the hip and knee. All samples were scanned by a c o m m e r - cially available m i c r o c o m p u t e d t o m o g r a p h i c s c a n n e r at the Department of Cardiology of the West German Heart Centre at the University of Duisburg-Essen.

Technical specifications of the micro-CT

The micro-CT system is based on a scanner developed for high-

resolution imaging [up to 4 p m cubic voxels] of sample sizes up to 2 cm'. The scanner uses a field x - r a y tube with an 8 pm spot-seize and expected lifetime of > 10,000 hours. The tube is operated at between 20 and 100 kiloelectron volts and a cur- rent of up to 100 micro amperes. For scanning, the samples are placed between the micro-focus X-ray source and a Char- ge-Coupled-Device (CCD) detector (matrix size: 1024 χ 1024 pi- xels, field of view: 25 mm2). In order to prevent samples from moving during scanning, the limbs are placed in a tightly fit- ting rigid plastic tube. In the scanner's chamber the specimens are placed on a stack of computer-controlled precision stages which are rotated in equiangular steps of 0.9° around a n a n - gle of 180°. When the object is placed between the X-ray source and the CCD-detector, the c o n e - b e a m of X-rays passes the object a n d t h e n hits the CCD-detector p r o d u c i n g 2 D - X - r a y images. A personal computer is used to control the s c a n n e r and store the CCD image data recorded at each angle of view during the s c a n n i n g process.

Tomographic image reconstruction

The X - r a y projection data of the s c a n n e d samples are then submitted to the resident reconstruction program (Cone-beam Reconstruction, Skyscan, Aartselaar, Belgium), which is based on a Feldkamp filtered back projection algorithm [43] resulting in a volume image of up to 10,243 voxels, each cubic voxel being 4 - 1 9 p m on one side, depending on how much of the specimen has to be imaged.

Cancellous bone assessment by micro-CT

The regions of interest (ROIs) from the proximal femur and tibia were selected for structural analysis of the cancellous bone. A cubic region of 0 . 5 x 0 . 5 x 0 . 5 m m 3 in the metaphysis of the tibia was adjacent to (1mm away from) the growth plate and femoral head. Trabecular thickness (Tb. Th) and trabecular number (Tb.N) were based directly on the 3-D data using medial axis transformation and distance transformation.

Joint space measurement by micro-CT

The center of the head of the hip was detected by "data viewer".

The m e a s u r e m e n t s were m a d e in micrometers. The smallest j o i n t space was measured from the longitudinal, sagittal and transversal slices of the center. The j o i n t space of the knee was measured from the transverse and sagittal slices between the medial condyle and the tibial plateau.

Statistical analysis

The data were analyzed and assessed using SPSS software (ver- sion 15.0; SPSS Institute Inc, Chicago, USA). Descriptive sta- tistics of all variables were determined including the mean and standard deviation of each group. The difference of all p a r a - meters between the two groups was assessed using the Stu- dent's t-Test because all parameters were normally distribu- ted [which was tested u s i n g the Kolmogorov Smirnov test].

Pearson's correlation coefficient was used to assess the relationship between all the trabecular b o n e parameters of the femur and tibia. A v a l u e of ρ <0.05 was considered to be statistically significant.

Results

No death or health deterioration occurred during this study.

The body weight at each time point in the two groups is shown in Figure 1.

Animals with an ad libitum diet (Group A) were found to increase body weight significantly at the age of six weeks in com- i parison with the lean mice (Group B). From this time point on,

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ORIGINAL· ARBEITEN

ο

Ξ 40 •

- GROUP A

- GROUP Β

9 11 13 15 17 A G E ( W E E K )

2,80

2

c Ü ä e CO

2,60

2,40

K-Qu»dra! = 0.1*

2900 3000 3100 Crossdiametcr of Tibial Plateau

Fig. 1: The curves show the change in body weight in the two groups. The animals in Group A had an ad libitum diet and reached a body weight of over 50 grams after 20 weeks, the animals in Group Β with a controlled diet reached a body weight of over 35 grams after 20 weeks. Inter-group difference was already significant at the age of six weeks (p<0.05).

Figure 2: In both groups, correlation of body weight and bone mineral density shows a decrease in bone mineral density as body weight increases.

the difference increased c o n s t a n t l y . A t t h e age of t w e n t y w e e k s t h e o b e s e mice w e r e a l m o s t t w i c e as h e a v y as t h e l e a n mice.

In b o t h G r o u p A a n d G r o u p Β t h e m i c e w i t h a h i g h b o d y w e i g h t h a d a l o w e r b o n e m i n e r a l d e n s i t y (BMD) (Figure 2} a n d a lower t r a b e c u l a r n u m b e r (Tb.N) in t h e p r o x i m a l f e m u r a n d ti- b i a (Table 1, F i g u r e 3).

The a n i m a l s w i t h a n ad l i b i t u m diet (Group A) w e r e f o u n d t o h a v e a s i g n i f i c a n t l y i n c r e a s e d j o i n t s p a c e in t h e h i p in t h e c o -

Table 1: Statistically significant differences between the ad libitum diet Group A and controlled diet Group Β for bone mineral density (BMD), body weight and trabecular number (TbN) in the ROI of the proximal femur (p < 0.05) and the statistical trend of the trabecular number (TbN) in the ROI of the proximal tibia (p = 0.058).

Figure 3: The correlation between bone mineral density and the cross-diameter of the tibial plateau shows that in both groups there was a significant decrease in bone mineral density as the cross-diameter of the tibial plateau increased (p < 0.01).

r o n a i y , s a g i t t a l a n d t r a n s v e r s e p l a n e s in c o m p a r i s o n with t h e l e a n m i c e ( G r o u p B) (Table 2).

The increase in t h e j o i n t s p a c e in t h e k n e e in t h e c o r o n a r y p l a - ne, f o r t h e t r a n s v e r s e d i a m e t e r of t h e tibial p l a t e a u a n d f o r t h e s u b c h o n d r a l sclerosis in t h e ad l i b i t u m diet G r o u p A w a s also s t a t i s t i c a l l y s i g n i f i c a n t .

Only o n e s i g n i f i c a n t c o r r e l a t i o n w a s f o u n d b e t w e e n b o n e m i - neral d e n s i t y ( m e a s u r e d b y micro-CT) a n d t h e c r o s s - d i a m e t e r of t h e tibial p l a t e a u (Figure 3).

C o r r e l a t i o n w a s f o u n d b e t w e e n t h e t r a b e c u l a r n u m b e r (Tb.N.) o f t h e k n e e a n d f e m u r ( m e a s u r e d b y m i c r o - C T ) a n d b o d y w e i g h t (Fig. 4).

C o r r e l a t i o n w a s a l s o f o u n d b e t w e e n t h e t r a b e c u l a r n u m b e r (Tb.N.) of t h e f e m u r ( m e a s u r e d b y micro-CT) a n d t h e d i a m e t e r of t h e f e m o r a l h e a d in t h e s a g i t t a l , c o r o n a r y a n d t r a n s v e r s e p l a n e s (Fig. 5).

Discussion

The a i m of t h e present s t u d y w a s to e x a m i n e c h a n g e s in t h e j o - i n t s p a c e i n d u c e d b y a r e s p o n s e t o b i o m e c h a n i c a l l o a d i n g in l e p t i n - d e f i c i e n t m i c e . C o r r e l a t i o n of b o d y w e i g h t a n d b o n e m i n e r a l d e n s i t y s h o w s t h a t in b o t h g r o u p s b o n e m i n e r a l d e n - sity d e c r e a s e d as b o d y w e i g h t increased. H o w e v e r , this did n o t i n d u c e a n y s i g n i f i c a n t c h a n g e s in t h e j o i n t s p a c e in e i t h e r o f t h e g r o u p s . O n l y o n e s i g n i f i c a n t c o r r e l a t i o n w a s f o u n d b e t - w e e n b o n e m i n e r a l d e n s i t y a n d t h e c r o s s - d i a m e t e r of t h e t i b i - al p l a t e a u .

A g r e a t d e a l of r e s e a r c h h a s c o n f i r m e d t h a t g r e a t e r b i o m e - chanical l o a d i n g d u e to high b o d y w e i g h t c o n t r i b u t e s to t h e increased b o n e d i m e n s i o n s o b s e r v e d n o t o n l y in o u r a n i m a l m o -

Group BMD

mean SD Weight (grams)

mean SD T b N femur

mean SD T b N tibia

mean SD

Group A

(ad libitum diet) 2.57 0.147 52.53 6.36 6.42 0.45 5.63 0.34

Group Β

(controlled diet) 2.68 0.138 35.65 3.50 6.76 0.55 5.90 0.52

Ρ value <0.05 < 0.001 0.038 0.058

BlOmaterialien 11 (November), 2010

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Figure 4: In both groups the correlation between the trabecular number (Tb.N.) and body weight shows a significant decrease in the trabecular number as body weight increases (p < 0.01).

J 700

1400 1450 1500 1550 Diameter of F e m o r a l Head SAG

1450 1475 1500 1525 1550 Diameter of Femoral Head COR

1400 1450 1500 1550 Diameter of F e m o r a l Head TRANS

del but also in obese h u m a n s [11]. At t h e s a m e time, it also contributes to an increased b o n e mass as a c o - i n f l u e n c e . In- creased loading of l o n g b o n e s p r o d u c e s t h e greatest m e c h a - nical stress on the subperiosteal s u r f a c e and stimulates b o n e f o r m a t i o n by subperiosteal e x p a n s i o n [41], A study of b o n e b i o m e c h a n i c s in adult rats with d i e t - i n d u c e d obesity s h o w e d s i g n i f i c a n t l y g r e a t e r b o n e strength in t h e obese rats t h a n in the controls [6], I w a m o t o et al. suggest that cancellous b o n e a d a p t a t i o n to treadmill exercise m a y be influenced by f a c t o r s such as mechanical l o a d i n g on t h e m e t a p h y s e a l b o n e a r c h i - tecture in the y o u n g g r o w i n g rat [17]. In a rat model of i m - mobilisation osteoporosis Laib at al. measured the p r o x i m a l part of the tibiae of t h e hind limbs in u n l o a d e d and control rats using 3D micro-CT. They show that most b o n e loss is c a u - sed by the t h i n n i n g of the trabeculae, a n d to a lesser e x t e n t by a decrease in their n u m b e r [5]. In the case of leptin-deficient mice o u r study shows in both g r o u p s a significant decrease in the t r a b e c u l a r n u m b e r as t h e d i a m e t e r s of t h e femoral h e a d increased.

With micro-CT it is possible to m o n i t o r p r o m i n e n t b o n y alterations such as o s t e o p h y t e f o r m a t i o n , t r a b e c u l a r remodeling, s u b c h o n d r a l b o n e plate thickening, a n d s u b c h o n d r a l sclerosis [39], Furthermore, micro-CT a r t h r o g r a p h y is a novel t e c h n i - que for indirect visualization of the distinct features and structural analysis of t h e rat knee j o i n t in s m a l l - a n i m a l research [31]. Botter et al. and Chappard at al. showed significant c h a n - ges in s u b c h o n d r a l b o n e architecture a f t e r induction of osteoarthritis or in advanced osteoarthritis [5, 8], Only Batiste et al.

d e m o n s t r a t e d p r o g r e s s i v e c a r t i l a g e d e g e n e r a t i o n a n d b o n e c h a n g e s using 4-T MRI and micro-CT correlated to m a c r o s c o - pic g r a d i n g [3]. However, n o n e of these a u t h o r s described t h e c h a n g e s which take place in the j o i n t space in hips and k n e e s affected by osteoarthritis.

In s u m m a r y , biomechanical loading leads to reduced b o n e mineral density due to a decrease in the number of trabeculae. De- creased body weight in leptin-deficient mice protects the mice against b o n e loss. This finding is consistent with the p r i n - ciple of t h e light-weight construction of bone. With micro-CT is it not possible to d e m o n s t r a t e c h a n g e s in j o i n t space in response to biomechanical loading. Differences in cartilage and t h e b o n e in j o i n t spaces will be e x a m i n e d in later studies correlated to h i s t o m o r p h o l o g y . It is not possible to conclude t h a t these results also apply to h u m a n beings.

Figure 5: The correlation between the trabecular number (Tb.N.) of the femur and the diameter of the femoral head in the sagittal, coronary and transverse planes shows in both groups a significant decrease in the trabecular number as the diameter of the femoral head increases (p < 0.01).

Abstract

Leptin w a s regulate b o n e d e v e l o p m e n t . However, there is so far no a g r e e m e n t c o n c e r n i n g the effect of leptin in t h e early stages of life. The aim of this study w a s to investigate t h e d i f - ferential effects of w e i g h t - b e a r i n g on j o i n t space in l e p t i n - d e - ficiency mice. Materials, Methods and Results: C57BL/6J-Lepob

(ob/ob) female mice with an a d - l i b i t u m - d i e t (Group A) w e r e f o u n d to increase b o d y weight significantly at t h e age of six weeks in c o m p a r i s o n with lean mice (Group B). At t h e age of twenty weeks the obese mice were almost twice as heavy as the lean mice. A high r e s o l u t i o n micro-CT (SkyScan 1072) w a s used to p e r f o r m q u a l i t a t i v e a n d q u a n t i t a t i v e analysis of t h e hip and knee. Significant statistical differences are shown between t h e two g r o u p s for b o d y weight and b o n e mineral d e n - sity, for the trabecular n u m b e r (Tb.N.) f o r t h e proximal femur, a increased j o i n t space in the hip in the coronary, sagittal a n d transverse planes for Group A, a increase in the j o i n t space in the knee in the c o r o n a r y plane, for t h e t r a n s v e r s e diameter of the tibial plateau and f o r the s u b c h o n d r a l sclerosis. Signifi- cant correlation was f o u n d between b o n e mineral density a n d the cross diameter of tibial plateau, between Tb.N. of k n e e / f e - Table 2: Statistically significant differences between the ad libitum diet Group A

and the controlled diet Group Β for joint space (JS) in the hip in the coronary, sagittal (p < 0.001) and transverse (p < 0,05) planes.

Group

JS hip sag

mean SD

JS hip cor

mean S D

JS hip trans

mean S D

Group A

(ad libitum diet) 85.99 19.44 103.56 38.14 142.85 91.28

Group Β

(controlled diet) 65.29 15.61 63.83 18.63 83.05 17.93

Ρ value < 0.001 < 0.001 <0.05

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mur and body weight, between Tb.N. of femur and diameter of the femora] head in the sagittal, coronary and transverse planes.

Conclusion

In both groups decreased bone mineral density as body weight increased. However, this did not induce any significant changes in the joint space in either of the groups in micro-CT.

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^ ^ m Dr. med.

^ ^ ^ ^ ^ ^ R B Hansjoerg Heep

^ ^ ^ P · " W Korrespondenzadresse:

^ H k i M u r Department of Orthopaedics, University of Duisburg-Essen Pattbergstr. 1-3

Essen

hansjoerg.heep@uni-due.de I f ^ ^ V ^ ^ · Phone.: 0201 /4089-2147

Fax: 0201/4089-2722 Akademischer Lebenslauf

Oberarzt, Orthopädische Klinik der Universität Duisburg- Essen im Evangel. Krankenhaus Essen-Werden

1986-1988 Studium der Chemie Universität Heidel- berg

1988-1994 Studium der Humanmedizin Heidelberg und München (LMU, Sommersemester

1988)

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