Bone, 13, 297-310, (1992) Printed in the USA. All rights reserved.

Copyright

8756-3282192 $5.00 + .OO 0 1992 Pergamon Press Ltd.

Age-Dependent Morphometric Alterations in the Distal Femora of Male and Female Rats W. SONTAG Kernforschungszentrum Karlsruhe, Institut fiir Genetik und fiir Toxicologic, Postjach 3640, W D-7500 Karlsruhe 1, Germany Address for correspondence and reprints: W. Sontag, Kemforschungszentmm Karlsruhe, Institut fiir Genetik und fur Toxikologie, Postfach 3640,

W D-7%0 Karl&he

1, Germany.

Abstract Morphologic parameters, bone area, bone-to-bone + marrow ratio, periosteal-to-periosteal + endocortical surface ratio, mean trabecular thickness, and surface-to-volume ratio were studied in the epiphysis and metaphysis of the distal femora of male and female rats (Heiligenberg strain) between birth and the end of the lifespan. With increasing age, bone area, bone-to-bone + marrow ratio, and mean trabecular thickness increases, whereas periosteal-to-periosteal + endocortical surface ratio and surface-to-volume ratio decreases in both parts of bone during the first 150 days. Afterwards, periosteal-to-per&teal + endocortical surface ratio, mean trabectdar thickness, and surface-to-volume ratio remain constant, whereas the bone area and the bone-tobone + marrow ratio decrease. Modeling dam were measured by use of the vital labeling technique with calcein. From the stained bone area, the bone formation, the bone resorption, and the periosteal mineral apposition rates have been calculated. The bone formation rate, about 13,OOO%/year in the metaphysis and 2,0OO%/year in the epiphysis, respectively, is greatest after birth and decreases continuously with increasing age to 3S%/year for both bone regions. During the first 150 days the bone resorption rate is lower than the bone formation rate, leading to an increase in bone area, but afterwards it is higher so that the area decreases. Likewise the periosteal mineral apposition rate is greater in the metaphysis (24 pm/day at day 50) than in the epiphysis (14 pm/day at day 50), but after 700 days it is comparable for both bone regions (0.07 @day). The absolute values of body weight, femur length, and bone area of epiphysis and metaphysis are greater in male rats; only the mean trabecular thickness and the periosteal mineral apposition rate are comparable in both sexes. The relative values of bone-to-bone + marrow ratio, periosteal-to-periosteal + endocortical surface ratio, bone formation rate, and bone resorption rate are comparable for both sexes. Key Words: Rat-Bone-Morphometry-Bone

turnover.

functions are controlled by hormones and vitamins; thus many investigations have been established to examine the role of vitamin D, estrogen, prostaglandin E,, thyroid or parathyroid hormones, or calcitonin on bone turnover and mineral metabolism (Baron & Vignery 1981; Marie & Travers 1983; Weinstein et al. 1984; Awbrey et al. 1985; Jee et al. 1985; Ueno et al. 1985; Harrison et al. 1986; Isaksson et al. 1987; Turner et al. 1987). Others examined the effects of castration, hypophysectomy, thymectomy, or orchidectomy on bone turnover (Thomgren et al. 1980; Gunther et al. 1980; Wronski et al. 1985; Giirkan et al. 1986; Verhas et al. 1986; Wronski et al. 1986; Turner et al. 1987); the influence of exercise (Steinberg & Trueta 1981; Forwood & Parker 1986; McDonald et al. 1986) and weightlessness (Jee et al. 1983; Eurell & Kazarian 1983; Cann & Adachi 1983) as well as the influence of fracture (Hansson et al. 1976; Simmons & Cohen 1980) on bone morphology have also been examined. All the experiments mentioned above were carried out under normal or pathological conditions for one or two defined ages of the animals. This was also true for most of the experiments studying the morphological structure of bone under normal conditions (Olerud & Lorenzi 1970; Tonna 1974; Doyle et al, 1977; Stenstrom et al. 1977; Whitson et al. 1978; Houghton & Dekel 1979; Simmons et al. 1979; Engesaeter et al. 1980; Indritz & Hegarty 1980; Sanchez et al. 1981; Kimmel & Jee 1982; Baron et al. 1984; Farley & Baylink 1986; Turner & Bell 1986). Only a few measured some morphological parameters over a wide range of age in rats (Aries 1941; Zucker & Zucker 1946; Acheson et al. 1959; Raman 1969; Hughes & Tanner 1970; Hansson et al. 1972; Vogel 1979; Nishimoto et al. 1985; MbuyiMuamba & Dequeker 1986), beagles (Anderson & Danylchuk 1979), or humans (Kragstrup et al. 1983). The aim of the present investigation was to determine some static and dynamic morphological parameters in the distal femora of male and female rats over the whole lifespan of the animals, and thereby to extend the work started in 1986 when the diaphysis of the rat femur was studied (Sontag 1986a,b).

Material and Methods and bone preparation

Introduction

Animal keeping

The skeleton in humans and animals has two major functions. On the one hand it is a pool of mineral elements, such as phosphorus, calcium, sodium, potassium, zinc, or magnesium, and on the other hand it provides support for the whole body. Both

Male and female albino rats of the Heiligenberg strain (a substrain of Wistar rats) were used. The rats were kept in clear plastic cages with a metal grill top in an air conditioned room (23” to 25” C) with natural daylight illumination (12 h light and

298

W. Sontag: Morphometry in the femora of rats

12 h dark), and were fed on pellets (Altromin-R; 0.9% Ca and 0.75% P) and water ad libitum. For measurement of bone formation rates, the animals received multiple subcutaneous injections of 20 mg calcein per kg body weight at intervals of a few days (1.5% calcein in 240 mM NaHCO,). The interval between any two injections ranged from 2 days in young animals up to I4 days in the oldest animals, and were chosen so that the distance between adjacent fluorescence lines in bone would not be more than 8 pm. The animals were separated into groups of the same age but different body weight. At different time intervals after the first injection, two or more of the animals that had the same difference (one heavier and one lighter) in body weight from the mean body weight of the whole group were sacrificed. Six groups of male rats and five groups of female rats were used; the experimental protocol of the labeling is summarized in Table I. No measurements of the bone formation rates were made before 60 days, because at this early age most of the calcein lines appear to be too broad or diffuse to permit an accurate localization of the fluorescence lines and determination of the forming bone volume. Consequently, the static morphometric parameters were measured for both sexes at only the ages of 20 and 40 days. After sacrificing the rats by exsanguination under ether anaesthesia, the femora were taken out, and the soft tissue and muscles were carefully removed. The distal femora were fixed in formal saline, dehydrated, and embedded in methylmethacrylate. After embedding, the femora were cut longitudinally on a Leitz sawing microtome into sections about 80 pm thick. Before sawing, the embedded bone was positioned such that the cut area was parallel to the diaphysis. Two sections were sawed from the middle of the femora and mounted on glass slides. Measurements of samples The fluorescence of the bone samples was measured

in incident light with a Leitz microphotometer (MPVll) equipped with a fluorescence illuminator (Ploemopak 2. I with filter system G). After measurement of the fluorescence intensity, the samples were stained with alizarin red S (20 min in a 2% solution), and in a second measurement the structure of the mineralized bone was determined in digitized form by processing the photometer signal in transmitted green fight so that the hard tissue appeared black. The microscope stages were moved by stepping motors, which were controlled by a minicomputer (PDP I l/34). The measuring field was normally 20 pm’; but the field was reduced to 10 pm* if the fluorescence lines lay near the bone surfaces.

The scanning area was over the whole width of the epiphysis and metaphysis and in length for all bones 9 mm from the articular cartilage in the direction of the diaphysis (Fig. 1). The position of the x and y stepping motors and the digitized photometer signal are stored in a data file on a disc. The two data files containing the information on the fluorescence intensity and the bone structure, respectively, were combined to a new data file, which was the basis of calculating the various static and dynamic morphological parameters. The labeling protocol was chosen so that the distances between the single fluorescence lines are so close that they smelt during digitizing to a “continuum.” Thus the area of forming bone is equal to the calcein-labeled area. The calcein-labeled area extends from the bone surfaces deep into the bone. By determining the bone surface and the border of the fluorescence area in deep bone, and taking the shortest mean distance between both, the mineral apposition rate can be calculated. A detailed description of the vital labeling technique is given in previous publications (Sontag 1980, 1986a.b). In addition to the fully automatic measurements of bone parameters, some parameters on femora of selected ages were measured by use of a digitizer (MOP Digiplan I, Kontron). These parameters are the thickness of the trabeculae in the epiphysis, and the area, surface length, and thickness of the trabeculae in the metaphysis as a function of the distance from the epiphyseal plate. In the following, the nomenclature follows mostly the recommendations of the ASBMR committee (Parfitt et al. 1987). Calculation

of’ the morphological

parameters

The surface-to-volume (S/V) ratio has been calculated by dividing the length of the bone surfaces (BS) through the area of mineralized tissue (BV) and multiplying it by the factor k = 4/n, assuming an isotropic orientation of the trabeculae. The change of the volume of BV between the two ages t, and t, is dependent on the bone formation rate (BFR) and the bone resorption rate (BRR). At age t, the BFR and the BRR are defined as CX,)and PO, respectively. Assuming that between the ages t,, and t, both rates decrease exponentially, we can describe the change of the bone volume dV during the time interval dt by the differential equation:

where At = t, - t,,. The solution of the differential eqn (1) with the initial condition V(0) = V, can be written as:

Table I. Experimental protocol of the multiple calcein labeling

Age” (days] 60 100 210 320 470 550 60 100 270 396 660

Sex 6 6 CT d d d P Z P P

Number of rats

Ar’ [days /

Age of killing

34 16 8 8 6 I4 20 20 6 32 6

2 3 3 5 IO I4 2 3 5 7 14

62, 70. 78. 85. 100, 110, 120, 130, 145 110. 120. 130. 150. 175. 200. 225, 2.50 230. 280. 300. 353 350. 410, 470. 530 500, 580,660 600, 650, 700, 750. 800. X60 62. 67, 75. 80, 90, 100, 115, 130 103. 110. 120. 130. 150. 170. 200, 220, 250 300, 350, 400 407. 425.450. 480. 510. 550, 590, 620. 660, 700, 740. 780, 810. 840 700. 760. 840

Idays

“Age means the age of the animals receiving the first injection of calcein. bag means the time interval between two successive injections of calcein until killing.

W. Sontag:

Morphometry

in the femora of rats

299

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Fig. 1. Body weight and length of the femur of male and female rats as a function of the age of the animals. Each symbol is the mean value between several measurements of body weight (2-30) and two measurements of the length of femora.

1/

=

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(2)

and k, = P&3. If both the bone formation and resorption rates are known, the bone volume V can be calculated. In the present investigation the bones had been labeled for many weeks; for each time point the fractional bone formation rate FBF was calculated as follows: with k, = q/cl

FBF = AVIV

(3)

where AV is the volume of new forming bone during the labeling time At from the beginning of treatment to the sacrifice of the animals, and V is the total bone volume at the age of sacrifice. From the fractional bone formation FBF the bone formation rate can be calculated with sufficient accuracy by the formula:

FBF = k,(l -

epmA’)

(4)

k,, a, and At have been explained for eqn (2). The bone resorption rate, which cannot be measured directly, is calculated using the difference equation (Frost 1969) AV(At) = AVF(At)

-

AVR(At)

(54

where AV is the change in the total bone volume during the short time interval At, and AVF and AVR are the changes of forming and resorbing bone volume, respectively. From eqn (5a), the bone resorption rate BRR at the age t can be determined with sufficient accuracy by knowing AV and AVF AVR(At) AVF(At) - AV(At) BRR(t) = 7 = At

(56)

W. Sontag: Morphometry in the femora of rats

300

Results Measurements

of’ the static parameters

Figure 1 shows the body weight and the length of the femora in male and female rats. Using a vernier, the length of the isolated femora were measured. In the older animals the variation between different animals was greater than the growth in length, so that in these cases it was calculated by determining the distance between the epiphyseal plate and the fluorescence lines in the distal metaphysis. In the younger animals it was found that about 75% of the growth in length occured in the distal and 25% in the proximal part of the femora. The development of the distal femora of female rats is demonstrated on eight computer printouts (Fig. 2). At the age of 20 days some parts of the spongy bone in the metaphysis near the epiphyseal plate are not fully calcified and cannot be detected; likewise, the cortical bone in epiphysis and metaphysis is not fully calcified. Between 20 and 40 days the remaining cartilage is transformed into calcified tissue. With increasing age the femora grow in length and width, and more and more spongy bone appears in the metaphysis. Up to 150 days, no qualitative change in length and width can be observed, but with increasing age a reduction of spongy bone in the metaphysis occurs; in addition. the width of the epiphyseal plate becomes smaller with increasing age.

DISTAL I

Figures 3 and 4 show some static morphological parameters, such as the area of calcified tissue, the bone-to-bone + marrow ratio, the periosteal-to-periosteal + endocortical surface ratio, the mean trabecular thickness, and the surface-to-volume ratio, in the epiphysis and metaphysis of male and female rats as a function of the age of the animals. As can be seen from Figure 2. it is not possible to obtain identical cuts through the middle of the distal femora. Therefore, for some animals serially cut sections are analyzed. The results indicate that such absolute parameters as the area of bone and marrow and area of labeled bone. length of the surfaces, and width of the epiphysis, show a variation between sections, whereas the relative parameters, such as bone-to-bone + marrow ratio, periosteal-to-periosteal + endocortical surface ratio, and surface-to-volume ratio are independent of the position of the cuts over a wide range. Therefore. in Figs. 3 and 4, up to 200 days the area of bone has been corrected to a mean width of the epiphysis, assuming no growth in width in adult animals. With increasing age, the mean trabecular thickness in the epiphysis and metaphysis increases to a constant level (Figs. 3 and 4). whereas the surface-to-volume ratio decreases to a constant level. But up to an age of 150 days, no change in the two parameters can be observed, with no significant differences appearing between male and female rats (r-test, p < 0.95). The mean trabecular thickness in the epiphysis (80 pm) is larger than in the metaphysis (64 pm), whereas the surface-to-volume ratio

FEMORA OF FEMALE RATS

I

Fig. 2. Eight computer printouts showing the development

of the distal femora of female rats from age 20 days fo 800 days.

W. Sontag:

Morphometry in the femora of rats

301

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Age (days) Fig. 3. Bone area, bone-to-bone to-volume ratio in the epiphysis

+ marrow ratio, periosteal-to-periosteal + endocortical bone surface ratio, mean trabecular of male and female rats as a function of the age of the rats.

in the epiphysis is about 17% lower than in the metaphysis. The area of calcified tissue grows with increasing age to a maximum lying between 130 days and 180 days, and decreases nearly linearly after this age. During the first 80 days of age, the bone area of epiphysis and metaphysis are comparable for both sexes, but after that the bone area in epiphysis and metaphysis is greater in male rats. In the metaphysis the bone area, surface, and mean thickness of trabeculae have been measured as a function of the distance from the epiphyseal plate by use of a digitizer. Whereas the mean

of 25 mm2/mm3

thickness,

and surface-

thickness of all trabeculae as a function of age is shown in Fig. 4, Fig. 5 presents trabecular thicknesses, bone areas, lengths of surfaces, and S/V ratios for five selected ages of female rats as a function of the distance from the epiphyseal plate. The four data sets presented are values collected in bands of OS-mm widths parallel to the epiphyseal plate. As can be seen from Fig. 5, area and surface show similar behavior; in 20-day-old rats most of the bone is near the epiphyseal plate, but with increasing age the maximum shifts to larger values of about 0.75 mm, and decreases afterwards. The maximum values show behavior similar

W. Sontag:

302

Morphometry

in the femora of rats

METAPHYSIS 7

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Age (days) Fig. 4. Bone area, bone-to-bone + marrow ratio, periosteal-to-periosteal + endocortical bone surface ratio, mean trabecular to-volume ratio in the metaphysis of male and female rats as a function of the age of the rats.

to the whole bone area (Fig. 4); with increasing age the values also increase, but after 200 days they are decreasing again. The trabecular thickness is lowest near the epiphyseal plate and increases to a maximum at about one millimeter (100 d and 200 d) or to a constant level (20 d, 450 d, and 800 d). The S/V ratios show a similar pattern; with increasing distance they increase to a maximum and decrease again later. At a distance of 4 mm the values are independent of age. The trabecular area and surface as a function of the distance from the epiphyseal plate can be de-

thickness,

and surface-

scribed by a lognormal distribution. In Fig. 6 the relationship between trabecular area, age, and the distance from the epiphyseal plate in female rats are shown in a 3-D plot. The peak in this figure is not significance; it comes only from the fact that the trabecular area as a function of the distance from the epiphyseal plate is described by a lognormal distribution (Fig. 5, top), and with decreasing age the mean value p, shifts to zero, so that in young rats the maximum area is near the epiphyseal plate. But below the age of 20 days, no measurements have been made; on

W. Sontag:

Morphometry

in the femora of rats

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Distance from the epiphyseal plate (mm) Fig. 5. Trabecular area, trabecular surface, trabecular thickness, and surface-to-volume ratio as a function of the distance from the epiphyseal in the metaphysis. The values of female rats of five ages (20 d, 100 d, 200 d, 450 d, 800 d) are presented.

the other side in the young animals the epiphyseal plate is very broad with pure calcification (Fig. 2), so that it is difficult to define a neutral point. Measurements of the dynamic parameters With the vital labeling technique, the new forming bone area is marked with the calcein-calcium complex, which can be seen in a fluorescence microscope. Therefore with this technique it is

plate

possible to determine the new bone area qualitatively (where?) and quantitatively (how much?). Normally, humans or animals receive two single injections of a fluorochrome, and from the length and the distance of the two labels the growing bone area canbe calculated (Frost 1969). But the forming and modeling of soonav bone is so complex, even in fast growing animals, that the a new -I forming bone area cannot be determined-with our computer-controlled automatic method (Sontag 1980). Therefore we stained the forming bone area at more than two different times.

W. Sontag:

Morphometry

in the femora of rats

Fig. 6. 3-D plot of the trabecular area of female rats as a function of the age of the animals and the distance from the epiphyseal Four examples of calcein-labeled distal femora are given in Fig. 7. The first row shows bones of 60-day-old female rats which were labeled for 4 days (picture A) or 60 days (picture B). Whereas 4 days after the first label, bone is labeled only in the metaphysis near the epiphyseal plate, 60 days later most of the trabeculae in the metaphysis are newly formed. In the epiphysis the newly forming bone area is much smaller, only on the periosteal sides, the articular cartilage, and some trabeculae bone is forming. The lower row shows the situation in older bones: 260-day-old male rats are labeled for 60 days (picture C), whereas picture D shows 400-day-old female rats labeled for six months. In picture C the growth in length (labeled area near the epiphyseal plate) and the modeling of trabeculae (partially labeled trabeculae in the middle and the end of the metaphysis) can be seen, whereas the comparison between the two pictures shows the reduced growth in length and the reduction of spongy bone. From the fact that only one periosteal side is forming (picture C right and picture D left) it can be concluded that the whole distal femur has drifted to one side. In Fig. 8 the fractional bone formation is shown for the I I groups of male and female rats. The fractional bone formation is defined as the ratio of labeled bone to total bone area. The values are separated for epiphysis and metaphysis, whereas the lines are calculated by use of eqn (4) and regression analysis. In the young animals, the forming rate is so large in the metaphysis that after 40 days most of the hard tissue is replaced by new bone. As can be seen from Fig. 8, with bone formation rates greater than 70% to 80%, the agreement between calculated and experimental values is not very good, for which there may be two reasons. Firstly, eqn (4) is only an approximation of the general eqn (2). so that with increasing fractional bone formation the variation

plate

increases; but the second and main reason is that in eqns (2) and (4) the assumption has been made that the sides of formation and resorption are separated. This is surely correct for short times of labeling, but with increasing time of labeling more and more of the bone is stained, so that the possibility of resorption of stained bone increases proportionally. tn the same group of animals the slope of the fractional bone formation is greater in the metaphysis than in the epiphysis (Fig. 8). but in the same region of bone the slope becomes smaller with increasing age at the start of labeling. Figure 9 presents the calculated values of the bone formation rate from Fig. 8 by using eqn (4) from day 60 up to day 800. Before 60 days, the bone formation rates are calculated from the increase of the bone volume (Figs. 3 and 4). The bone formation rates decrease continuously with increasing age in male and female rats in the epiphysis and the metaphysis. In male rats the bone formation rate is greater in the metaphysis up to 100 days, whereas in the epiphysis this only holds up to 200 days. After 800 days no difference between male and female rats, and metaphysis and epiphysis, can be detected (t-test, p < 0.95). With the method of vital labeling, only bone-forming areas can be measured, but with the information on the forming bone area and the total bone volume, the bone resorption rate can be determined by using eqn (5). However, it is evident that in the calculation of the resorption rate, the variation of the new boneforming area and the total bone area is included, so that the variation of the resorption rate is much greater than the variation of the formation rate. The resorption rate (Fig. 10) is calculated by using eqn (5b) and an age difference At of 10 days. By comparison of Figs. 9 and 10, a similar behavior of formation and resorption rates in the metaphysis can be seen; but in the first

W. Sontag:

Morphometry

in the femora of rats

305

Fig. 7. Four computer printouts of the distal femur. The animals of 60 days are labeled between 60 and 64 days (A), between 60 and 120 days (B), between 260 and 320 days (C) and between 400 and 520 days (D). The pictures A, B, and D are from female rats and picture C is from a male rat. The grey area is unlabeled bone and the darker area is calcein-labeled bone; the field of measurement was 20 pm*. 200 days the formation rate is greater, and thereafter the resorp tion rate. During the first 200 days the formation rate in the epiphysis decreases, whereas the resorption rate increases to a maximum. After 200 days in the epiphyses, both formation and resorption rates decrease, whereby the resorption rate is greater than the formation rate.

The formation of trabecular bone in the epiphysis and metaphysis is very complex, so that the calculation of the apposition rate is not possible with our method. Therefore, Fig. 11 shows only the periosteal apposition rate. The apposition rate, was calculated from the distance between the periosteal surface and the first calcein injection line for the first two time points of sacrifice

W. Sontag:

306

Morphometry

in the femora of rats

Epiphysis

6CI-

Epiphysis

Age

(days)

Fig. 8. Fractional bone formation rate in the epiphysis and metaphysis of male and female rats as a function of the time of labeling. The different symbols indicate different groups of rats; each symbol is the mean value of four measurements, whereas the lines represents regression analysis using

eqn 4.

of each group. Because the sacrifice times are different in each group and the distance of the first label from the surface depends on the age of the animals, the calculated values have a large variation. However, the behavior of the apposition rates in Fig. I1 shows a pattern similar to the formation rates in Fig. 9; the values in the metaphysis are initially greater than in the epiphysis, decrease for both with increasing age, and at about 600 days have similar values.

The last figure (Fig. 12) qualitatively shows the sides of formation and resorption in the distal femora. The development of the distal femur is very complex, the bone grows from the epiphyseal plate in length, the diameter increases over the whole bone, and simultaneously the whole bone drifts transversely in one direction. This three-dimensional movement of bone results in different behavior in young and old rats. In young animals the epiphysis over the whole periosteal

W. Sontag: Morphometry

in the femora of rats

Bone formatton

Perlosteol miner01 opposhon rote

rote

10'

I

&

’

zoo

LOO

@lo

600

z+

Age(days)

Fig. 11. Periosteal mineral apposition rates of the epiphysis and the metaphysis of male and female rats as a function of the age of the animals.

Fig. 9. Bone formation rates of the epiphysis and the metaphysis of male and female rats as a function of the age of the animals.

surface is growing, whereas the opposite endosteal surface is resorbing, but because of the transversal drift, the formation and resorption rates are greater at one side than on the opposite side. In the metaphysis the perimeter grows, too, but the diameter, which is greatest near the epiphyseal plate, decreases in the direction of the diaphysis. Therefore, the whole periosteal surface on the compacta is resorbing and the opposite endosteal surface is growing; the adjacent trabeculae are included into the new forming bone areas. The transversal drift has the effect that resorption and formation is greater on one side than on the opposite side. The spongy bone formed near the epiphyseal plate moves through the metaphysis and is resorbed near the diaphysis. In the older rats the femur is growing in length as well as in width, and also the transversal drift is considerably reduced. Little bone-formation activity can be observed at one periosteal surface side in epiphysis and metaphysis, on the articular cartilage, and the epiphyseal plate. In the metaphysis, the formation of new bone near the epiphyseal plate is much lower than the resorption near the diaphysis. In the spongy bone of epiphysis and metaphysis in the older animals, some trabecular bone formation can be observed, and from the fact that overall trabecular thickness is constant (Fig. 4), it can be concluded that on the opposite side resorption has occurred. Discussion Bone mass and shape result from four main determinants: genetic endowment, mechanical loading, and the endocrine and nutr-

Bone resorption

‘O’_

rote

400

600

d

Ageldoysl Fig. 10. Bone resorption rates of the epiphysis and the metaphysis male and female rats as a function of the age ‘of the animals.

of

tional environments (Heaney 1986). Although interactions among the four are complex, in laboratory rats the most important factor seems to be the genetic determination, because nutrition is equal and movement is similar for all rats. The influence of the genetic endowment can be seen in the distribution of the body weight; for each age it follows a Gaussian distribution, but the deviations in both directions are sometimes more than 50%. After 300 days the mean body weight of the female rats is nearly constant, whereas the male rats grow over the whole life span (Fig. 1). For one-year-old Heiligenberg rats the body weight is 260 g (female) and 460 g (male). Thus, the body weight of the Heiligenberg rats is about 20% lower than that of Sprague-Dawley rats (Sontag 1983), but comparable to the body weight of most of the Norwegian rat strains used for laboratory studies (Altman & Dittmer 1964). With increasing age, body and skeletal weights increase, but there is no linear correlation; the body weight increases more than skeletal weight (Sontag 1984; O’Flaherty 1988). For one-year-old Sprague-Dawley rats, skeletal weight is about 6% of the body weight (Sontag 1983), which can be assumed to be the proportion for most of the rat strains (O’Flaherty 1988). The whole femur grows in length from birth up to about 250 days; afterwards the variation between the different animals is greater than the change in length so that the length seems to be constant (Fig. 1). But from the vital labeling experiments it can be seen that over the whole lifespan of the animals, trabecular bone is stained with calcein near the epiphyseal plate, which is never closed in our rat strain. This indicates a growth in length over the whole lifespan (Fig. 7). The growth in length of the whole femur is the sum of the growth on the distal epiphyseal plate (about 66%) and the proximal epiphyseal plate (about 34%), so that bone turnover is considerably greater in the distal than in the proximal femur. With increasing age the femora grow in length, until at 300 days they reach a plateau of 48 mm (male) and 34 mm (female), respectively (Fig. 1). The same has been observed in Wistar rats (Mbuyi-Muamba & Dequeker 1986; Kalu 1989) and albino rats (Aries 1941); in the adult rats the femora have a maximum length of 39 mm (albino rats), 38 mm (female Wistar rats), or 44 mm (male Wistar rats). The growth in length (endochondral ossification) in the metaphysis occurs in zones along the epiphyseal plate (growth plate). The growth plate is composed of hyaline cartilage with mesenchymal cells (zone l), which differentiate into chondrocytes (zone 2) that synthesize cartilage templates (zone 3). The chondrocytes become larger (hypertrophic chondrocytes) and OSsification centers are formed in the hyaline matrix (zone 4). Outgoing from the ossification centers, more and more of the cartilage templates are calcified, forming the early trabecular

W. Sontag: Morphometry

308

Young rats

Old

in the femora of rats

rats

Fig. 12. Schematic description of the sides of bone formation (arrows) and bone resorption (triangles) in the distal femora of young (left) and old (right) rats. The size of the arrows and triangles is an indication of the size of formation and resorption rates.

structure of the metaphysis (zone 5). By the modeling processes of synthesizing new bone and resorbing bone, this primary spongiosa is transformed into the secondary spongiosa (zone 6). Finally, at the end of the metaphysis, the trabecular structure is resorbed and .the metaphysis is turned into the relatively bonefree area of the diaphysis (zone 7) (Kahn et al. 1984; Jee 1988). With alizarin red S, only the calcified tissue is stained so that the zones 1 to 4 are invisible (Figs. 2 and 7). The processes of forming and resorbing the spongiosa are quantified in Fig. 5. With increasing distance from the epiphyseal plate, both the trabecular area and surfaces+ and the mean thickness of the trabeculae increase to a maximum lying about one millimeter away. For larger distances, area and surfaces decrease again, whereas the trabecular thickness seems to be constant, and only between ages 100 and 200 days is there a diminution. The rapid decrease of the trabecular area beyond one millimeter from the epiphysial plate is the result of two different mechanisms. On the one hand, with increasing distance from the epiphyseal plate the diameter of the femur decreases (Figs. 2, 7, and I I), and therefore the bone atea also decreases; on the other hand, with increase in distance more and more of the spongiosa is resorbed. The length of the spongiosa increases from 3 mm (20 days) to a maximum of about 8 mm (200 days), and is reduced for older ages to about 5.5 mm (Fig. 5).

The development of the femur can be separated into two age groups, young animals from birth up to about 200 days and adult rats over 200 days. In young animals the femur is growing in all directions, with a rapid increase of the bone and marrow volume. Both the areas of calcified bone in the epiphysis and in the metaphysis increase to a maximum (Figs. 3 and 4, above). The linear correlation between bone and the total area indicates that the same is true for marrow. Not only do the number of trabeculae increase, but also their thicknesses (Figs. 3 and 4) to a maximum of about 80 p,m in the epiphysis and 62 p,rn in the metaphysis. Although the distribution of the trabecular thickness in the epiphysis is relatively homogeneous, in the metaphysis it is dependent on the distance from the epiphyseal plate (Fig. 5). The maximum values of the trabecular thickness in the middle of the metaphysis are comparable with those in the epiphysis. The gain of spongy bone in the epiphysis and metaphysis has the consequence of decreasing the S/V ratio and the periosteal-tototal surface ratio. In this age group the differences in the morphological parameters between male and female rats can be neglected. After 200 days the growth and development of the femur is mostly complete and the external form does not change further, but there are modeling processes over the whole lifespan. A steady state of the bone area cannot be observed because the

W. Sontag:

Morphometry

in the femora of rats

variation among various animals is too large. After reaching the maximum, the bone area decreases linearly and is replaced by marrow; therefore the bone-to-bone + marrow ratio decreases and the periosteal-to-total surface ratio increases (Figs. 3 and 4). In female rats the bone area is only 83% (epiphysis) and 70% (metaphysis) of the corresponding values of male rats. No differences between male and female rats have been found in the mean trabecular thickness and in the relative parameters boneto-bone + marrow ratio and periosteal-to-total surface ratio. Only in the metaphysis is the surface-to-volume ratio larger in female than in male rats (Fig. 4). Qualitatively it can be said that with increasing age the femur is not only growing in length and diameter, but also a transversal drift of the whole distal part can be observed (Figs. 7B and 12). From the periosteal apposition rate in the metaphysis and epiphysis (Fig. 11) and the corresponding values in the diaphysis (Sontag 1986a,b), it can be concluded that this drift is greatest in the middle of the femur and decreases in the direction of the distal end. The diameter of the distal femur is greatest near the growth plate and decreases continuously in the direction of the diaphysis (Figs. 2 and 7). This has the consequence that during growth in length the compacta of the metaphysis must be resorbed on the periosteal surfaces, whereas new bone is formed on the opposite endosteal surfaces; but this general behavior becomes overlayed by the transversal drift. These complex age-dependent processes are quantitatively summarized by two dynamic parameters, bone formation rate and bone resorption rate. The bone formation and bone resorption rates of the diaphysis (Sontag 1986a,b), metaphysis, and epiphysis (Figs. 9 and 10) have a similar course. At birth they are greatest and decrease continuously with increasing age. The only exception of the general behavior is the resorption. rate of the epiphysis (Fig. 10). which shows an increase between birth and about 60 days and decreases thereafter. No measurements with the vital labeling technique have been made before 60 days; therefore we can only speculate as to why these morphological alterations occur. The structure of the epiphysis is not yet fully mineralized, in contrast to the diaphysis and metaphysis. Therefore, in the first weeks after birth the bone forming rate is indeed normal, but the resorption rate seems to be. reduced, and only normal values resume when the epiphysis is fully mineralized. Gamer (1987) measured the bone-forming rate in the epiphysis of young male Sprague-Dawley rats and found values of 1244 (47 days), 720 (63 days), 206 (91 days), and 16O%/year (147 days). These values are two-fold larger at 47 days in our data and comparable at 147 days. One reason for these age-dependent differences could be the fact that Gamer (1987) measured the bone formation rate only in selected areas, whereas in this study it was measured in the whole epiphysis. In contrast, Wronski et al. (1988) measured the bone-formation rate in the metaphysis of the proximal tibiae of 90-day-old female Sprague-Dawley rats (1 #O%/year) using the tetracycline method. This value is only 30% greater than the values measured in the metaphysis of the distal femora of our Heiligenberg rats. Comparing formation and resorption rates of one sex and one part of the bone in the first weeks after birth, formation rate is always larger than the corresponding resorption rate, causing growth of the bone area. After 150 days, the resorption rate becomes larger than the formation rate and the bone area decreases. Both formation and resorption rates, are greatest in the metaphysis and lowest in the epiphysis (Figs. 9 and lo), whereas the diaphysis lies between both during the first 200 days; afterwards the diaphysis has the lowest values (Sontag 1986a,b). Only in the case of the formation rate the diminution in the diaphysis is not so great as in the epiphysis and metaphysis, so that after 700 days the formation rate in the diaphysis is greater

309

than the corresponding values for epiphysis and metaphysis. During the first 100 days in epiphysis and metaphysis, the values for female rats are lower than for male rats, but after that no differences between the two sexes can be detected.

I appreciate the patient and skillful assistance of B. and I. Person, and thank Dr. E. Drosselmeyer for critically

Acknowledgment:

Braunstein

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12. I992 12. I992