Calcium density measurement in histological samples of trabecular bone of normal subjects: Relationship with aging

Arch. Gerontol. Geriatr. suppl. 3 (1992) 27-36 9 1992 ElsevierScience Publishers B.V." All rights reserved. 0167-4932/92/$05.00

27

CALCIUM DENSITY MEASUREMENT IN HISTOLOGICAL SAMPLES OF TRABECULAR BONE OF NORMAL SUBJECTS: RELATIONSHIP WITH AGING

P. B A L L A N T I , G. COEN a, G. M. BRAGA MARCAZZAN b, S. MAZZAFERRO a, E. CEREDA c and E. BONUCCI Department of Human Biopathology, Division ofaPathology., "La v e r s i t y , Viale Regina Elena, 324, 1-00161 Roma, Institute of 2nd DiOJl~I n R~ '~eSapienza" Uc~iVersity , Viale del I, "t e o al Ap~lie Physics, U n i v e r s i t y Festa del Perdono 7, 1-20122 Milano, CISE S . p . A . , P.O. Box Milano, Italy

Sapienza" UniMedical Clinic, Policlinico 155, of Milano, Via 12081; 1-20134

SUMMARY It is well-known that bone volume decreases with age both in normal subjects and p a r t i c u l a r l y in osteoporotic patients. It is not well demonstrated, however, whether bone loss is associated with changes in the composition of bone tissue and especially w i t h altered concentration of mineral elements. To v e r i f y whether calcium density changes with aging, autoptic specimens of iliac crest trabecular bone from 20 normal subjects between 21 and 66 years, 10 males and 10 females were analyzed by using a new method which allows the measurement of calcium density in a n o n - d e s t r u c t i v e way, on entire hist01ogic sections of the bone. Bone specimens were embedded in araldite and tissue sections, about 3 l~m thick and 4 x 4 mm size, were mounted onto p o l y v i n y l acetate films and analyzed by PIXE [proton induced X - r a y emission) using the CISE setup for calcium content determination. The same bone tissue sections were then mounted on glass slides, stained with the Von Kossa method and the volume of calcified bone was measured with a semiautomatic image analyzer (Videoplan). 3 to 4 sections from each subject were analy.zed and the values of calcium concentration were derived in ~ g / u l . Similar values of calcium density were found in males and females (535.6 + 77.1 and 539.2 + 74.1 u g l u l , r e s p e c t i v e l y ) . No s i g n i f i cant correlation be{-ween calcium dens~y and age was observed either in all cases ( r = 0.0925) or in males ( r = -0.0687) and in females ( r = 0.2676) separately. The unchanged calcium density d u r i n g aging obtained by combining PIXE and histomorphometric techniques demonstrates that the skeletal calcium reduction observed in old age and probably d u r i n g osteoporosis, is mainly due to the decrease of bone volume. Key'words: iliac crest trabecular bone, PIXE analy.sis, histomorphometry., calcium d e n s i t y , aging INTRODUCTION Histological

morphometric

results

demonstrate

that

iliac

crest

trabecular

bone volume declines 45-50 96 from 20 to 89 years of age in normal subjects. This reduction

in trabecular

volume occurs rather

regularly

in females and in

males, so that a v e r y severe osteopenia can be found (Ballanti et a l . , On the

basis of

its

histological

definition,

we speak

when bone tissue decreases below the physiological characteristic

for

sex,

age and

race (Avioli,

difficult

to distinguish

between

physiological

porosis.

Moreover,

the etiopathogenetic

1977).

about

1990}.

osteoporosis

rarefaction

threshold being

However,

it is sometimes

age-related

bone loss and osteo-

factors and the pathophysiological

lea-

28 t u r e s of this disease are still

largely

unknown and much remains to be disco-

v e r e d in this field. Recently, and p a r t i c u l a r l y

it has been supposed that the f r a g i l i t y of bone in e l d e r l y people in osteoporotic patients cannot be completely explained by the

reduced amount of bone. Accumulations of mechanical microdamages, t e c t u r a l alterations of t r a b e c u l a r t e x t u r e , may take place as well,

bone matrix and mineral abnormalities

in consequence to remodeling modifications,

cause of f u r t h e r weakening of bone ( F r o s t , The aim of this s t u d y was to v e r i f y aging

in bone of normal subjects,

bined

PIXE (Particle

content

in

microarchi-

Induced

1987).

w h e t h e r calcium d e n s i t y changes with

by" using a new method r e s u l t i n g

X-ray

bone histological

1985; Mosekilde et a l . ,

and can be

fluorescence Emission)

sections

and

histomorphometric

calcified bone volume in the same sections (Caruso et a l . ,

from com-

analysis of calcium evaluation

of the

1986).

MATERIALS AND METHODS Sample processing.

I liac c r e s t bone specimens were taken at the autopsy

from 20 subjects who died s u d d e n l y or v i o l e n t l y eases which

could a f f e c t

bone metabolism.

and have had no clinical d i s -

Subjects were selected

cover uniformly the age range between 20 to 69 y e a r s ,

in o r d e r

to

t h e y were equally d i v i -

ded between males and females (Table I ) . Table I AGE (years) AND SEX D I S T R I B T U I O N OF 20 NORMAL SUBJECTS STUDIED Decades of age (years) Sex

20 - 29

30 - 39

40 - 49

50 - 59

60 - 69

Males

21, 26

32, 38

40, 49

54, 57

63, 65

Females

26, 27

31, 35

42, 47

54, 56

63, 66

Vertical specimens of bone, about 8 mm t h i c k and about 2.5 cm in l e n g t h , were obtained approximately 2 cm behind the a n t e r i o r s u p e r i o r iliac spine, corr e s p o n d i n g to the usual site selected for the iliac crest biopsy in patients with metabolic bone diseases. T h e y were c u t t r a n s v e r s a l l y fragments of t r a b e c u l a r buffered

to pH 7.2

with

bone, 0.1

so as to obtain 6 - 8 thin

which were then fixed M phosphate

buffer,

in 4 ~o paraformaldehyde

dehydrated

in alcohol

and

embedded in araldite w i t h o u t decalcification. Sections of 3 um t h i c k n e s s with an area of about 4 x 4 mm were c u t by an ultramicrotome

(Porter-Blum

MT1,

Sorwall

Inc.,

Norwalk,

Connecticut,

USA)

29 using glass k n i v e s .

For each s u b j e c t ,

one randomly

of 3 - 4 embedded fragments was collected.

selected section from each

The sections were placed on home-

made p o l y v i n y l acetate s u p p o r t films with a thickness of 50 ]Jg/cm 2, which was circumscribed

by a plastic

duce both the X - r a y

ring.

Such a v e r y thin s u b s t r a t e film allows to r e -

background

due to proton

and electron

Bremsstrahlung,

and sample heating u n d e r PIXE analysis carried out in vacuum. PIXE analysis.

The

sections

underwent

calcium

content

determination

by

means of PIXE technique using a 2.8 MeV proton beam p r o v i d e d by the 3.5 MV Tandem Van de Graaff accelerator of the CISE (Milan). mg/cm 2 t h i c k

AI

foil

and

was collimated

by

The beam crossed a 1.5

means of

Ta

phragms in o r d e r to obtain a uniform charge d i s t r i b u t i o n

and

graphite

dia-

and a spot with a dia-

meter as wide as to cover the whole section. The c u r r e n t was settled down to a few nA in o r d e r to reduce damage of the sample and to avoid and dead time c o r r e c t i o n s .

The sections were mounted in a s c a t t e r i n g

which could be p r e s s u r i z e d at 50 T o r r heating.

Fluorescence

mounted at 90 ~

strong

X-rays

were

of p u r e

detected

He in o r d e r

by

means

of

pile up chamber

to reduce sample a SI{Li)

detector

and separated from the scattering chamber by a 50 um t h i c k

Mylar window ( s u p p o r t i n g

the d i f f e r e n c e in p r e s s u r e ) .

The absolute value of Ca

content was estimated by means of a calibration c u r v e obtained by irradiation of many r e f e r e n c e samples. Morphometric analysis. A f t e r PIXE i r r a d i a t i o n , the same sections were mounted on glass slides w i t h o u t removing the p o l y v i n y l acetate film. P a r t i c u larly,

the film-coated

assure

the

side of the specimen was placed on the glass surface to

histochemical

staining

reaction.

In

some cases where

the

sections

became damaged because of the p r e v i o u s handling processes, adjacent and nonirradiated

sections were stained.

(1901) histochemical

The sections were reacted with the Von Kossa

method which allowed to detect the sites of localization

the anions of calcium salts by development of a typical black stain. tic evaluation of the calcified semiautomatic

image a n a l y z e r

bone volume was c a r r i e d (Kontron,

Munich,

out with

Germany)

the e n t i r e section.

In detail,

the two-dimensional

results

Morphomet-

the Videoplan

by evaluating

microscopic fields with an objective x4 and total magnification

of

serial

x260, t h r o u g h o u t

( p e r ~m 2) of the cal-

cified matrix area of bone trabeculaeo

p r o v i d e d by the a n a l y z e r ,

transformed

~m 3) values on the basis of stereologic

in three-dimensional

{per

were d i r e c t l y

p r i n c i p l e s and, then multiplied by 3, since the section t h i c k n e s s was 3 ~m. Calcium (~gl~l)

density

measurement.

wa's obtained by d i v i d i n g

by the calcified bone volume ( ~ I ) , Statistical analysis.

Calcium

density

of

calcified

the calcium content (]Jg),

bone

matrix

measured by PIXE,

measured by morphometry.

Linear regression analysis was p e r f o r m e d .

30 RESULTS The variations of calcium density determinations,

obtained within

the 3-4

sections measured for each subject ranged from 3.15 to 28.62 ~ (mean + S.D. = 14.56 + 6.90). Taking the mean values of 3 - 4 determinations for each subject, a good correlation

was found

between calcium content of bone,

measured by

PIXE, and calcified bone volume, determined by morphometry (Figure I ) .

8-

Calcium 7 content (~) s

~

9

9

9

o"

5

4

ee/~=

9

r :0.9008 p
9 9

1

9

4

8

6

10

12

14

16

Calcified Bone Volume ( . m 3 x l ( ) 6) Figure 1. The correlation between calcium content of bone determined by PIXE, and calcified bone volume measured by morphometry, in the 20 normal subjects. -,-,-,-,-,-,-,-,-,-,-,-

The results of calcium density obtained in the total pool of both male and female 20 normal subjects are shown in Figure 2.

Linear regression analysis

showed no significant correlation ( r = 0.0925) between calcium density and age. Average calcium density was 537.4 + 73.6 ~g/~l bone (mean + S . D . ) . tion of the results picture:

regarding

males and females did

Separa-

not change this overall

the average values were 535.6 + 77.1 and 539.2 + 74.1

ug/~l

bone,

B

respectively.

Coefficients of age correlation

of calcium density

remained also

below significance when analyzed for males (Figure 3) or females (Figure 4).

DISCUSSION Our study wanted to contribute to the knowledge concerning the composition of trabecular bone, with special reference to calcium density,

and to its

31

800-

Cslclum density

(.o~i)

n=20 r-0.0925 p=NS

700

600 500 9

i 9

400

300

. .I . . . I . .

20

I

30

I

40

I

I

I

50 age (years)

!

I

60

I

70

Figure 2. The age correlation of calcium density obtained in the total pool of the 20 normal subjects {black squares and circles mean females and males, respectively). The correlation coefficient { r ) is very low and does not reach significance criteria.

800-

Calcium density (.O~0

n=10 r--0.0687 p.NS

700

600 e___..__ 500

9

400 300 20

I

30

I

I

40

w

I

!

50 age (years)

I"

60

I

I

70

Figure 3, The age correlation of calcium density obtained in 10 normal male subjects. The correlation coefficient ( r ) is very low and does not reach signiFicance criteria.

32

800"

Calcium density

n-10 r.0.2676 p.NS

700 600 9

IL

9

9

500 400, 300

,

i

20

i

i

30

i

40

i

I

50 age (years)

i

i

i

60

70

F i g u r e 4. The age correlation of calcium d e n s i t y obtained in 10 normal female subjects. The correlation coefficient ( r ) is v e r y low and does not reach s i g n i ficance c r i t e r i a . -O-O-O-O-O-O-O-O-O-

possible

modifications

during

aging,

by

applying

a new combined

method

of

PIXE and morphometric analysis. The variation longing

of calcium d e n s i t y

to identical

subjects,

found

in the 3-4 d i f f e r e n t

sections

was in some cases h i g h e r than 20 96. T h i s

cates that the method used by us may r e q u i r e some f u r t h e r

beindi-

improvements.

In

this respect, evaluation of mean values obtained from several measurements can contribute

to

improve

the

reliability

of

the

results.

Moreover,

it

should

be

emphasized that the values of calcium d e n s i t y of t r a b e c u l a r calcified bone tissue obtained with our technique in normal subjects are comparable to those r e p o r t e d by

using

other

techniques

1960; Ascenzi et a l . , al.,

such

as q u a n t i t a t i v e

microradiography

1960) and atomic absorption

spectrometry

(Rowland,

(Pugliarello

et

1973) in normal human and animal osteons of cortical bone, and e x p r e s s e d

in identical units. The good correlation

found between the calcium c o n t e n t of bone and calci-

fied bone volume s u p p o r t s the assumption that more than 98 ~ of the total body calcium

is

localized

in

the calcified

matrix

of bone

reasonable to assume on the basis of this correlation in osteoid tissue, are also irradiated

bone marrow cells, by

the proton

(Raisz,

i.e.,

It

is also

that the calcium contained

araldite and p o l y v i n y l

beam,

1977).

acetate film (which

could c o n t r i b u t e

to the overall

33 calcium amount revealed) is i r r e l e v a n t , as compared with the amount of this element contained in the calcified matrix of bone trabeculae. Our results demonstrate that calcium concentration

in the bone tissue does

not v a r y with e i t h e r the sex or the age in normal subjects. in agreement with

those of B u r n e l l

sorption

spectrophotometry,

position

and

specifically

did

in

et al.

not find

calcium

These results are

(1982) who by means of atomic a b significant

concentration

differences (mEqlg

in bone com-

bone

mineral

and

mEqlg bone) according to age or sex in iliac crest bone of 48 normal subjects between 30 to 80 years of age. These a u t h o r s ,

h o w e v e r , showed an overall r e -

duction of percentual mineral components of bone and skeletal calcium deficiency in a s u b g r o u p of 56 postmenopausal osteoporotic patients. Moreover, our f i n d i n g s are in agreement with those of O b r a n t and Odselius (1986) who did not find a g e - d i f f e r e n c e s

in calcium concentration

( w e i g h t %) of

the iliac c r e s t from six men aged 70-80 and from six men aged 20-30 y e a r s . The results were obtained by examining the central p a r t of 5 bone trabeculae selected in 100 IJm g r o u n d subject

by

sections (the size of each field was I

means of an electron

probe e n e r g y

dispersive

~m 2 ) from each

X-ray

microanalysis

(EDX) t e c h n i q u e . Significant (glkg),

age-dependent

P (glkg),

(1990)

studying

variations

and of o t h e r elements,

normal

control

becular

iliac c r e s t

bone.

thods,

EDX

inductively

and

in

the

coupled

(ICPOES), to compare the r e s u l t s .

of

were n e i t h e r found by

and osteoporotic

These authors

concentration

plasma

optical

Particularly,

Basl~ et al.

s u b j e c t s in cortical

used two fundamentally

calcium and t r a -

different

emission

me-

spectrometry

EDX analysis was performed on

500 um t h i c k sections, and f o u r measurement windows, 25 x 25 ~m, were selected on cortical and on t r a b e c u l a r bone. Studies

on the composition

of normal

and of osteoporotic

bone are few.

With specific r e f e r e n c e to osteoporosis, the results are often c o n f l i c t i n g , normal

calcium

density

(Manicourt

et a l . ,

creased one ( B i r k e n h ~ g e r - F r e n k e l ,

1981;

Basl~ et a l . ,

1966; B u r n e l l et a l . ,

1982).

cies between these studies could be due to the d i f f e r e n t ment,

since

chemical

all

of

analysis

them

are g e n e r a l l y

spectrophotometry,

which

t i v e chemical analysis, methods,

some

which

technical

based on methods,

imply d e s t r u c t i o n

performed

1990) or a d e The d i s c r e p a n -

methods of measure-

disadvantage. such

Particularly,

as atomic

of the sample.

Other

the most t r a d i t i o n a l exclude microscopic

little method,

spots of histological

absorption

non-destruc-

by electron probe and by n e u t r o n

allow to measure only v e r y

roradiography, sections,

present

having

activation

sections.

Mic-

implies examination of quite t h i c k

resoJution

of s t r u c t u r a l

tails. All these limitations can be avoided with our method.

and cytologic

de-

34 In conclusion, the combined use of the PIXE and the morphometric techniques allow us to perform the non d e s t r u c t i v e analysis of calcium concentration in entire histological sections of trabecular bone, offering a potentially advantageous use for the s t u d y of metabolic bone diseases. The measurement of calcium density obtained with this method demonstrates that no consistent modifications of this parameter occur in bone matrix with aging, and the skeletal calcium deficiency usually found in old age and p a r t i c u l a r l y in osteoporotic patients is essentially due to the reduction of calcified bone volume. ACKNOWLEDGEMENTS This investigation was p a r t l y

supported by g r a n t s from the Ministry of

U n i v e r s i t y and Scientific and Technologic Research and the National Research Council of Italy (INV 92 3 091).

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35 Pugliarello, M . C . , Vitturo F., de Bernard, B . , Bonuccio E. and Ascenzi, A. (1973): Analysis of bone composition at the microscopic level. Calcif. Tissue Res., 12, 209-216. Raisz, L.G. (1977): Bone metabolism and calcium regulation. In: Avioli, L . V . and Kraneo S.M. (Eds.) Metabolic Bone Disease. Vol. I. pp. 1-48. Academic Press, New York, San Francisco, London Rowland, R.E. (1960): Quantitative microradiography of bone. Meal. Phys., 3, 525-528, Von Kossa, J. (1901): 0bet die im Organismus k~nstlich erzeugbaren Verkalkungen. Beitr. Path. A n a t . , 29, 163-202 (in German).