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Osteocyte orientation in human lamellar bone and its relevance to the morphometry of periosteocytic lacunae
Metab.
Bone
Original
Dis.
8 Rel. Res.
article
Osteocyte Relevance GASTONE lstituto
Metabolic Bone Disease 8 Related Research @ by S.N.P.M.D. (Paris 19791
1, 325-333 (1979)
MAROTTl
di Anatomia
Address
Orientation in Human Lamellar Bone and Its to the Morphometry of Periosteocytic Lacunae Umana
for co/respondence
via Berengario,
Normale
I Cattedra
and reprints:
16, 41100 Modena,
UniversitB
Dr. G. Marotti,
The orientation of osteocyte lacunae in relation to the arrangement of collagen fibres has been analyzed in human lamellar osteonic bone. The results of microscopic observations under polarized and ordinary light and of measurements of the three main axes of the osteocyte lacunae show that the corresponding triaxial ellipsoid is oriented as follows: the major axis is always parallel to the length of the collagen flbres, in both longitudinally and transversely structured ,osteons; the intermediate and minor axes are respectively parallel and perpendicular to the surfaces of the lamellae which enclose the lacunae. The importance of these results is discussed in relatlon to the investigations of periosteocytic lacunae morphometry. - Osteons
lstituto
Italia.
di Anatomia
Umana Normale,
Universita
di Modena,
Italy.
Abstract
Key Words: Osteocyte
di Modena,
- Osteocyte
lacunae
osteocytes: small (inactive). large (lytic phase or secondary formation) and degenerated. The numbers of these three cell types found under normal, pathological and experimental conditions supply information about the changes in the metabolic activity of osteocytes (Baud and Auil, 1971; Krempien et al., 1976). It must be pointed out, however, that all the morphometric techniques for investigation of periosteocytic lacunae reported to date have overlooked two problems whose solution is a precondition to make reliable comparison of morphometric data between control and damaged tissue. These problems are: 1) the extent to which osteocytes lying in different fragments of compact and spongy bone differ in size under normal conditions, and 21 the orientation of the osteocyte cell body with respect to the plane of the histologic section.
- Morphometry.
Introduction While electron microscopic examination of bone has provided convincing evidence of the alternative osteolytic and osteoplastic activities of osteocytes (Baud 1962; Jande and Bblanger, 1969; Jande, 1971; Tonna, 1972), this technique allows examination of a limited number of cells. Consequently it is not the most suitable method for determining the extent of these two activities in various parts of the skeleton under normal, pathological or experimental conditions. For this reason periosteocytic osteolysis is usually investigated by microradiography (Jowsey. 1963; Duriez et al., 1965) or indirectly from the size of osteocyte lacunae measured in histologic sections, or on microradiographs, using a manual method or an image-analyzing computer (Meunier et al., 1971; 1973; Duriez. 1974). Another method widely utilized is based upon a differential count of three types of
With regard to the first problem, the maxima and minima for the physiological size of osteocytes must, obviously, be known to avoid mistaking what appears to be an [[originally large. osteocyte for an enlarged (and thus bone-dissolving) one, when the sclargenessn may fall within physiological limits. With regard to the second problem, it has been known since the last century (Ebner, 1875; Kijlliker, 1889) that osteocytes have an ellipsoid shape along three axes. As previously argued by Hennig and Elias (1963) and more recently by Boyde (19761 and by Boivin and Baud (1977), the principle on which stereology is based cannot be applied to such geometrical solids unless one knows their orientation with respect to the plane of the section. The results of our previous investigations carried out with the aim of solving these two problems have shown that under normal conditions the osteocyte population is made up of cells which cover a wide range of sizes. More specifically, it has been found that a relationship exists between the following three parameters: linear rate of bone deposition (appositional growth rate), size of osteoblasts, and size of
326
Gastone Marotti:
osteocytes. In other words, the size of the osteocytes and their lacunae, as found in individual bone fragments, depends on the size of the osteoblasts they originate from, and, therefore, ‘on the rate at which the fragments are themselves laid down (Marotti, 1976; Marotti et al.. 1976). I shall not discuss here the importance of this datum in morphometric analysis of periosteocytic lacunae; but say that the previously described differences in the size of osteocyte lacunae can be partly disregarded since measurements taken from several cells are analyzed using statistical methods.
Morphometry
of human osteocytes
1976; Marotti et al., 1976), the quantitative data reported in this study were obtained from osteocyte lacunae situated in the intermediate part of the wall of the various osteons examined, more precisely in a circular band of radium between 60 and 90 urn. In this way, it was possible to compare osteocyte lacunae having roughly the same dimensions in cross and longitudinal sections of the above-mentioned and two types of osteons - those with longitudinally those with transversely arranged fibres.
On the other hand, the error resulting from disregarding the orientation of the osteocyte cell body with respect to the plane of the section is inevitable. Therefore, I have attempted to find a method for establishing how osteocytes are oriented in bone tissue. I have assumed as a working hypothesis that the osteocyte is oriented in lamellar bone with its major axis parallel to that of the collagen fibres as the old German authors have pointed out (Kolliker, 1889). Fig. 1 shows schematically, above, an osteon with collagen fibres arranged longitudinally, and below, an osteon with fibres arranged transversely. If the afore-mentioned hypothesis is correct and the ellipsoid corresponding to the osteocyte cell body is coaxial to the collagen fibres, then its section areas should be as indicated in the two drawings.
Materials
and Methods
The present investigation was carried out on secondary osteons in the mid-diaphyseal level of human tibiae from subjects of various age, all of whom died violent deaths and had no pathological lesions visible on autopsy. Measurements of the axes and of the section areas of osteocyte lacunae were performed on drawings made at an enlargement of 1,400 x of undecalcified ground sections (20-30 urn thick) perfectly polished and dried. The section areas of lacunae were calculated both mathematically from the axes of the ellipsoid and by planimeter directly from the drawings. The focal plane selected for each lacunae corresponded to its largest area. Cross, longitudino-radial and longitudino-tangential sections, both of osteons with longitudinally arranged collagen fibres and of osteons with transversely arranged fibres. were structured osteons examined (see Fig. 1). Alternately were not analyzed, because in these systems the collagen fibres run at an angle of nearly 90” in adjacent lamellae, and therefore it would be difficult to compare their orientation with that of the interposed osteocyte lacunae. The orientation of collagen fibres was established by examining decalcified sections, mounted in 10% NaCI, under polarized light.
Results As a representative example, I report data obtained from the mid-diaphysis of the left tibia of a 30-yearold man. Small variations of the size of osteocyte lacunae have been found in other subjects of the same and of different age (these data will be reported in a subsequent paper), but a close correlation between spatial orientation of osteocyte lacunae and the arrangement of collagen fibres was observed in all the subjects studied. Moreover, since the size of osteocytic lacunae decreases from the periphery towards the center of Haversian systems (Marotti,
_x ,-P’v
-
a3 = 5.5 al a3= 2.2 a2
\
I I I
I I
.
??
:__.
Fig. 1. Schematic representation of an osteon with longitudinally arranged fibres (above) and of an osteon, with transversal fibres (below]. The upper surface of each osteon represents its cross-section; the longitudinoradial and the longitudino-tangential sections, indicated on the left and on the right sides respectively, show the orientation of collagen fibres. x, y, z indicate in pm the three principal axes of the ellipsoid corresponding to the osteocyte cell body: a, is its cross section, a* its longitudinal section parallel to the plane subtended by the major and minor axes, as is its longitudinal section parallel to the plane subtended by the major and intermediate axes. The mean values in pm’, shown outside the brackets, were mathematically calculated from the axes of the lacunae: the mean values In brackets were obtained by planimeter from the drawings of the lacunae.
Gastone
Marotti:
Morphometry
of human osteocytes
327
Fig. 2. Osteons (a) with longitudinal fibres, (b) with transversal fibres. (c) with transversal fibres but partly tangentially sectioned; in the zone, marked off by arrows, the lacunae appear to be sectioned according to their flattened surface (a,) Microphotographs (1) under polarized light and (2) under ordinary light (a, b = x 150: c = x 180).
In close agreement with what was postulated in Fig. 1, the aspect of the osteocyte lacunae changes according to the orientation of collagen fibres with respect to the plane of the section. Independently of the type of osteon, the section areas of lacunae appear narrow and elongated (aJ where the fibres are longitudinally sectioned, i.e. birefringent under polarized light (Figs Sb, 31, whereas they appear more roundish [a,) where the fibres are cross-sectioned and show up
‘dark’ under polarized light [Fig. 2a). The flattened surface (as) of the osteocyte lacunae becomes visible in tangential sections of the osteons (Figs 2c, 4). The same variation in aspect may be observed in spongy bone (Fig. 5). Figs 6 and 7 show, at a higher magnification, the three aspects shown by osteocyte lacunae of the same size when viewed in cross, longitudino-radial and longitudino-tangential sections. fibres while The osteon in Figure 6 has longitudinal the osteon shown in Figure 7 has transverse fibres.
328
Gastone
Marotti:
Morphometry
of human osteocytes
Fig. 3. Longitudino-radial
section of an osteon with longiMicrophotographs (al under polarized tudinal fibres. light and [b) under ordinary light (x 110). A
Fig. 4. Longitudinal section of osteon with longitudinal fibres; the cementing lines are indicated by black segments. The peripheral part of the osteon appears sectioned radially, the central part tangentially. Note how the osteocyte lacunae appear correspondingly sectioned radially (a,) in the peripheral part and tangentially (a31 in the central part. Microphotograph under ordinary light (x 180). b
The above-mentioned observations appear to be confirmed by the morphometric data reported in Fig. 8. The intermediate axis x of the osteocyte lacunae was measured in cross and longitudino-tangential sections of longitudinally structured osteons; its mean value shows no significant differences in two types of sections. The major axis y was measured in both longitudino-radial and longitudino-tangential sections of longitudinally structured osteons and also in cross sections of transversely structured osteons; its mean value is lower in transversely structured osteons only. The minor axis z was measured both in cross and in longitudino-radial sections of longitudinally structured osteons and also in cross sections of transversely structured osteons; the mean value of this axis always measured 4 pm. probably because it corresponds to the ‘rotational’ axis of the cell (see the drawings of the triaxial ellipsoids reported on the right side in Fig. 1). The results indicate that the ellipsoid formed by the osteocyte cell body seems to be strictly oriented in lamellar bone according to the main arrangement of collagen fibres; i.e. it has the major axis parallel to the length of the fibres, and the intermediate
and minor axes respectively dicular to the bony lamellae osteocyte is situated.
parallel and perpenbetween which the
The upper drawing in Figure 1 shows the data of the section area of osteocyte lacunae. Note the striking differences between the values of this parameter; as indicated in the lower drawing, section a2 is 2.5 times greater than al (A% = 581 and section a3 is 5.5 times greater than a1 no/o = 84) and 2.3 times greater than a2 (no/o = 551.
Discussion On the basis of the results reported in this paper, the following conclusions may be drawn: (al in order for morphometric investigations on osteocytes and/ or on their lacunae to be reliable, they must take into account the orientation of the triaxial ellipsoid, corresponding to the osteocyte cell body, with respect to the plane of the histologic section; (bl the orientation of the osteocytes in lamellar bone can be established by examination under polarized light of the arrangement of collagen fibres. The importance of the former conclusion has already been
Gastone Marotti:
Fig. 5.
taphyseal
Morphometry
of
human osteocytes
Trabecula of mespongy bone.
Microphotographs [al under polarized light and [k) u;ler ordinary light The osteocyte longitudilacunae ’appear nally sectioned in birefringent (bright) areas, cross sectioned in ‘dark’
areas light.
under
polarized
emphasized by various authors (Henning and Elias, 1963; Boyde, 1976; Boivin and Baud, 19771: however no technical procedures have been developed to avoid the errors which arise from disregarding the orientation of the osteocyte lacunae in the bony fragments examined. By means of the scanning electron microscope it has been shown that human secondary osteons which appear ‘dark’ under polarized light contain fibres with orientation having little or no transverse component, whereas birefringent osteons possess fibre orientation with transverse and longitudinal components (Frasca et al.. 1977). This means that
polarized light microscopy can only be used to obtain a statistical mean orientation of the collagen. However, as shown by the data reported in the present investigation, polarized light seems to be a good tool to establish, from the practical viewpoint, the main orientation of osteocyte lacunae in lamellar bone. The lower value of the major axis y of the osteocyte lacunae recorded in cross-sections of transversely structured osteons, in comparison with the values obtained from longitudinal sections of longitudinally structured osteons (Fig. 81, probably reflects the fact that in the former osteons the collagen fibres do not
Gastone
330
Marotti:
Morphometry
of human osteocytes
Shows the three aspects - al, a2, a3 of osteocyte lacunae of the same dimensions when respectively observed in cross, longitudino-radial and longitudino-tangential sections of longitudinai;de;tructured osteons. ordinary light Microphotographs [x 1 400). Fig. 6.
all run in a horizontal direction, as has been shown by Frasca et al. (1977). Thus in cross-sections of transversely structured osteons a certain number of osteocyte lacunae are probably not intersected accorFor this reason we suggest ding to their major axis. that periosteocytic lacunae morphometry be carried out on bone structures that appear ‘dark’ under the fibres are crosspolarized light viz. where sectioned. Several investigations on the morphometry normal and in various
have already been published of periosteocytic lacunae in pathological and experimental
conditions. However, all the data reported fail to take into account the orientation of the osteocyte ellipsoids. It is worth noting, in this connection, that increments in size of osteocyte lacunae by 1923%, with respect to controls, have been considered indicative of an increased osteolytic activity (Meunier et al., 1971; Meunier and Bernard, 1976; Lok and Jaworski, 1976). On the other hand, a decrement of about 16% recorded after calcitonin treatment (Duriez. 1974) or parathyroidectomy (Lok and Jaworski, 1976) has been ascribed to increased osteoplastic activity. Meunier and Bernard (1976) have also shown that in hyperparathyroidism the incre-
Gastone
Marotti:
Morphometry
of
human
osteocytes
- a,. a2, a, - of Fig. 7. The three aspects osteocyte lacunae of the same dimensions when respectively observed in longitudino-radial, cross and longitudino-tangential sections of transversely structured osteons. Microphotographs under ordinary light (x 1 400). Compared to fig. 6, note how the section areas of lacunae a,, ai, a, are practically the two types of osteons.
ment in size of osteocyte lacunae, with respect to controls, is greater in decalcified than in undecalcified samples: this datum indicates that the morphometric method applied by these authors seems to be correct and that the bone dissolving activity of osteocytes appears to be enhanced in primary hyperparathyroidism. Without in any way detracting from the value of these studies, one should point out that, in light of the results reported in this paper, the increase of 1923% or the decrease of 16%. with respect to controls, of the size of osteocyte lacunae does not
equal
in
prove conclusively that the osteolytic or the osteoplastic activities of osteocytes are respectively enhanced. In my opinion, percentage differences of this order could surely be significant if the morphometric analyses had taken the orientation of the osteocytes into consideration. The fact that the same osteocyte lacuna may show differences of 55-84% between its section areas, depending on how it is sectioned (Fig. l), offers grounds for suspecting that the percentage increments (1923%) or decrement (16%) in size of the osteocyte lacunae with respect to controls, as reported in the abovementioned articles, does not exclusively reflect a
1
4
30-
M=9
7
30-
D.S.2 3 E.St0.2
M=lO
r.iS.~ 2
1
E.S:01
X
IO-
IOi
M=22
50
OS.? 5 E.S.f 0.5
P
14
14
50-
Y 14
28
1'4
’ is’
14
70
I .: m D.S.il E.S.tO.1
50
50
30
.. . . . .
.I:
28
m=4 D.S.t 1 ES to.1
P
Gastone
Marotti:
Morphometry
of
human
osteocytes
333
Fig. 8. Frequency histograms of values in pm of the three principal axes (x, y, L) of the osteocyte lacunae. In the osteon drawn below each histogram, the arrangement of collagen fibres and the plane of the section, where the measurements were recorded, are indicated. M = mean: D.S. = standard deviation: ES. = standard error (see text).
‘lytic’ or a ‘plastic’ action by osteocytes respectively. It may derive, at least partly, from measurements performed on osteocyte lacunae of unknown orientation. Additionally these studies were carried out on fragments taken bioptically from the iliac crest viz. on spongy bone where the orientation of collagen
fibres, and therefore of the osteocytes, varies considerably not only between different trabeculae but also even in nearby portions of the same trabeculae (see Fig. 5).
Acknowledgements: Research aided Italian National Research Council.
by a grant
from
the
Baud, C.A.: Morphology and inframicroscopic structure osteocytes. Acta Anat. (Easel), 51: 209-225, 1962.
of
References
Baud, C.A., Auil, E.: Osteocyte differential count in normal human alveolar bone. Acta Anat. (Base/j, 78: 321-327. 1971. Boivin, G., Baud, periosteocytaires.
C.A.: La morphometrie Acta Anat. (Base/),
des lacunes 99: 356, 1977.
Boyde, A.: Resolution, sampling and the determination lacunar size. In: Bone histomorphometry (Meunier. ed.), pp. 399-412. Paris: Armour Montagu, 1976.
of P.J..
Duriez. J.: Les modifications calciques peri-osteocytaires. Etude microradiographique a I’analyseur automatique d’images. Noun. Presse med., 3: 2007-2010, 1974. Duriez. J.. Ghosez. J.P., Flautre. B.: La resorption ou lose periosteocytaire et son role possible da& la destruction du tissu osseux. Press m&d.. 73: 2581-2585, 1965. Ebner, V. von: Uber den feineren Bau der Knochensubstanz. Akad. Wiss. Wien, 72: 49-138, 1875. Frasca, P.. Harper, R.A., Katz, J.L.: Collagen tations in human secondary osteons. (Basef), 98: l-13. 1977.
fibre Acta
orienAnat.
Hennig, A., Elias, H.: Sections through tri-axial ellipsoids. Proc. 1st Intern. Congress of Stereology. 43/l - 43/12 (1963). Jande, S.S.: Fine structural study of osteocytes and their surrounding bone matrix with respect to their age in young chiks. J. Ultrastruct. Res.. 37: 279-300, 1971.
Jowsey, J.: Microradiography of bone resorption. In: Mechanisms of hard tissue destruction (Sognnaes, R.F., ed.). pp. 447-469. Washington, D.C.: Amer. A. Advanc. Sci., 1963. Kolliker, A.: Handbuch der 6 Anflage 1. W. Engelman.
Gewebelhere des Leipzig, 1889.
Menschen.
Krempien, B., Ritz, E., Geiger, G.: Behaviour of osteocytes in various ages and chronic uremia. Morphological studies in human cortical bone. Proc. 1st Workshop on Bone Morphometry [Z.F.G. Jaworski ed.), pp. 288--296. Ottawa: University of Ottawa Press, 1976. Lok, E.. Jaworski, Z.F.G.: Changes in the periosteocytic lacunae size observed under experimental conditions in adult dog. Proc. 1st Workshop on Bone Morphometry (Z.F.G. Jaworski ed.), pp. 297-300. Ottawa: University of Ottawa Press, 1976. Marotti. G.: Decrement in volume of osteoblasts during osteon formation and its effect on the size of the corresponding osteocytes. In: Bone histomorphometry (Meunier. P.J., ed.), pp. 385-397. Paris: Armour Montagu, 1977. Marotti. G.. Ledda, M., Delrio, N.. Fadda. M.: Distribution and size of osteocytic lacunae in secondary osteons of the dog. In: Exploration morphologique et fonctionnelle du sqielette, I Symposium CEMO-(Courvoisier, B.. Donath, A., eds.), pp. 225-230. Geneve: Editions MBdecine et HygiBne. 1976. Meunier. P.J., Bernard, J.: Morphometric analysis of pe1st Workshop on Bone riosteocytic osteolysis. Proc. Morphometry (Z.F.G. Jaworski ed.), pp. 279-287. Ottawa: University of Ottawa Press, 1976. Meunier. P.J., Bernard, J., Courpron, P., Vignon, G.: Use of an imaqe analvzino computer for the study of osteocytic behaviour -in -bone ‘diseases. Proc. . IX Europ. Symp. on Calc. Tiss., pp. 203-208. Wien: Facta-Publication, 1973. Meunier, P.J., Bernard, J., Vignon, G.: The measurement of periosteocytic enlargement in primary and secondary hyperparathyroidism. Israel J. Med. Sci., 3: 482-485, 1971. Tonna, E.A.: Electron microscopic evidence of alternating osteocytic-osteoclastic and osteoplastic activity in the perilacunar walls of aging mice. ‘Connective T&s. Res., 1: 221-230. 1972.
Jande, S.S., Belanger, L.F.: Ultrastructural changes associated with osteocytic osteolysis in normal trabecular bone. Anat. Rec., 183: 204, 1969.
Received: June 28, 1978 Revised: October 12, 1978 Accepted: December 20, 1978
RESUME
L’ORIENTATION OSTEOCYTAIRE LACUNES PERIOSTEOCYTAIRES
DANS
1’0s
LAMELLAIRE
HUMAIN
ET SON INFLUENCE
SUR LA MORPHOMETRIE
DES
L’orientation des lacunes osteocytaires par rapport a I’organisation des fibres collagenes a 6th Ctudiee dans’ 1’0s haversien humain. Les resultats de I’observation microscopique en lumiire normale et polarisee et les mesures des trois axes principaux des lacunes ostdocytaires ont montre que I’ellipsoide triaxiale est orientee comme suit : I’axe principal est toujours parallele aux fibres collag&res. tant dans les osteones longitudinaux que transversaux ; les axes intermediaire et mineur sont respectivement paralkle et perpendiculaire i la surface de la lamelle qui inclut la lacune de I’ostbocyte. L’importance de ces rtfsultats est discutCe par rapport aux etudes sur la morpho. metric de la lacune periosteocytaire.
Bone
Original
Dis.
8 Rel. Res.
article
Osteocyte Relevance GASTONE lstituto
Metabolic Bone Disease 8 Related Research @ by S.N.P.M.D. (Paris 19791
1, 325-333 (1979)
MAROTTl
di Anatomia
Address
Orientation in Human Lamellar Bone and Its to the Morphometry of Periosteocytic Lacunae Umana
for co/respondence
via Berengario,
Normale
I Cattedra
and reprints:
16, 41100 Modena,
UniversitB
Dr. G. Marotti,
The orientation of osteocyte lacunae in relation to the arrangement of collagen fibres has been analyzed in human lamellar osteonic bone. The results of microscopic observations under polarized and ordinary light and of measurements of the three main axes of the osteocyte lacunae show that the corresponding triaxial ellipsoid is oriented as follows: the major axis is always parallel to the length of the collagen flbres, in both longitudinally and transversely structured ,osteons; the intermediate and minor axes are respectively parallel and perpendicular to the surfaces of the lamellae which enclose the lacunae. The importance of these results is discussed in relatlon to the investigations of periosteocytic lacunae morphometry. - Osteons
lstituto
Italia.
di Anatomia
Umana Normale,
Universita
di Modena,
Italy.
Abstract
Key Words: Osteocyte
di Modena,
- Osteocyte
lacunae
osteocytes: small (inactive). large (lytic phase or secondary formation) and degenerated. The numbers of these three cell types found under normal, pathological and experimental conditions supply information about the changes in the metabolic activity of osteocytes (Baud and Auil, 1971; Krempien et al., 1976). It must be pointed out, however, that all the morphometric techniques for investigation of periosteocytic lacunae reported to date have overlooked two problems whose solution is a precondition to make reliable comparison of morphometric data between control and damaged tissue. These problems are: 1) the extent to which osteocytes lying in different fragments of compact and spongy bone differ in size under normal conditions, and 21 the orientation of the osteocyte cell body with respect to the plane of the histologic section.
- Morphometry.
Introduction While electron microscopic examination of bone has provided convincing evidence of the alternative osteolytic and osteoplastic activities of osteocytes (Baud 1962; Jande and Bblanger, 1969; Jande, 1971; Tonna, 1972), this technique allows examination of a limited number of cells. Consequently it is not the most suitable method for determining the extent of these two activities in various parts of the skeleton under normal, pathological or experimental conditions. For this reason periosteocytic osteolysis is usually investigated by microradiography (Jowsey. 1963; Duriez et al., 1965) or indirectly from the size of osteocyte lacunae measured in histologic sections, or on microradiographs, using a manual method or an image-analyzing computer (Meunier et al., 1971; 1973; Duriez. 1974). Another method widely utilized is based upon a differential count of three types of
With regard to the first problem, the maxima and minima for the physiological size of osteocytes must, obviously, be known to avoid mistaking what appears to be an [[originally large. osteocyte for an enlarged (and thus bone-dissolving) one, when the sclargenessn may fall within physiological limits. With regard to the second problem, it has been known since the last century (Ebner, 1875; Kijlliker, 1889) that osteocytes have an ellipsoid shape along three axes. As previously argued by Hennig and Elias (1963) and more recently by Boyde (19761 and by Boivin and Baud (1977), the principle on which stereology is based cannot be applied to such geometrical solids unless one knows their orientation with respect to the plane of the section. The results of our previous investigations carried out with the aim of solving these two problems have shown that under normal conditions the osteocyte population is made up of cells which cover a wide range of sizes. More specifically, it has been found that a relationship exists between the following three parameters: linear rate of bone deposition (appositional growth rate), size of osteoblasts, and size of
326
Gastone Marotti:
osteocytes. In other words, the size of the osteocytes and their lacunae, as found in individual bone fragments, depends on the size of the osteoblasts they originate from, and, therefore, ‘on the rate at which the fragments are themselves laid down (Marotti, 1976; Marotti et al.. 1976). I shall not discuss here the importance of this datum in morphometric analysis of periosteocytic lacunae; but say that the previously described differences in the size of osteocyte lacunae can be partly disregarded since measurements taken from several cells are analyzed using statistical methods.
Morphometry
of human osteocytes
1976; Marotti et al., 1976), the quantitative data reported in this study were obtained from osteocyte lacunae situated in the intermediate part of the wall of the various osteons examined, more precisely in a circular band of radium between 60 and 90 urn. In this way, it was possible to compare osteocyte lacunae having roughly the same dimensions in cross and longitudinal sections of the above-mentioned and two types of osteons - those with longitudinally those with transversely arranged fibres.
On the other hand, the error resulting from disregarding the orientation of the osteocyte cell body with respect to the plane of the section is inevitable. Therefore, I have attempted to find a method for establishing how osteocytes are oriented in bone tissue. I have assumed as a working hypothesis that the osteocyte is oriented in lamellar bone with its major axis parallel to that of the collagen fibres as the old German authors have pointed out (Kolliker, 1889). Fig. 1 shows schematically, above, an osteon with collagen fibres arranged longitudinally, and below, an osteon with fibres arranged transversely. If the afore-mentioned hypothesis is correct and the ellipsoid corresponding to the osteocyte cell body is coaxial to the collagen fibres, then its section areas should be as indicated in the two drawings.
Materials
and Methods
The present investigation was carried out on secondary osteons in the mid-diaphyseal level of human tibiae from subjects of various age, all of whom died violent deaths and had no pathological lesions visible on autopsy. Measurements of the axes and of the section areas of osteocyte lacunae were performed on drawings made at an enlargement of 1,400 x of undecalcified ground sections (20-30 urn thick) perfectly polished and dried. The section areas of lacunae were calculated both mathematically from the axes of the ellipsoid and by planimeter directly from the drawings. The focal plane selected for each lacunae corresponded to its largest area. Cross, longitudino-radial and longitudino-tangential sections, both of osteons with longitudinally arranged collagen fibres and of osteons with transversely arranged fibres. were structured osteons examined (see Fig. 1). Alternately were not analyzed, because in these systems the collagen fibres run at an angle of nearly 90” in adjacent lamellae, and therefore it would be difficult to compare their orientation with that of the interposed osteocyte lacunae. The orientation of collagen fibres was established by examining decalcified sections, mounted in 10% NaCI, under polarized light.
Results As a representative example, I report data obtained from the mid-diaphysis of the left tibia of a 30-yearold man. Small variations of the size of osteocyte lacunae have been found in other subjects of the same and of different age (these data will be reported in a subsequent paper), but a close correlation between spatial orientation of osteocyte lacunae and the arrangement of collagen fibres was observed in all the subjects studied. Moreover, since the size of osteocytic lacunae decreases from the periphery towards the center of Haversian systems (Marotti,
_x ,-P’v
-
a3 = 5.5 al a3= 2.2 a2
\
I I I
I I
.
??
:__.
Fig. 1. Schematic representation of an osteon with longitudinally arranged fibres (above) and of an osteon, with transversal fibres (below]. The upper surface of each osteon represents its cross-section; the longitudinoradial and the longitudino-tangential sections, indicated on the left and on the right sides respectively, show the orientation of collagen fibres. x, y, z indicate in pm the three principal axes of the ellipsoid corresponding to the osteocyte cell body: a, is its cross section, a* its longitudinal section parallel to the plane subtended by the major and minor axes, as is its longitudinal section parallel to the plane subtended by the major and intermediate axes. The mean values in pm’, shown outside the brackets, were mathematically calculated from the axes of the lacunae: the mean values In brackets were obtained by planimeter from the drawings of the lacunae.
Gastone
Marotti:
Morphometry
of human osteocytes
327
Fig. 2. Osteons (a) with longitudinal fibres, (b) with transversal fibres. (c) with transversal fibres but partly tangentially sectioned; in the zone, marked off by arrows, the lacunae appear to be sectioned according to their flattened surface (a,) Microphotographs (1) under polarized light and (2) under ordinary light (a, b = x 150: c = x 180).
In close agreement with what was postulated in Fig. 1, the aspect of the osteocyte lacunae changes according to the orientation of collagen fibres with respect to the plane of the section. Independently of the type of osteon, the section areas of lacunae appear narrow and elongated (aJ where the fibres are longitudinally sectioned, i.e. birefringent under polarized light (Figs Sb, 31, whereas they appear more roundish [a,) where the fibres are cross-sectioned and show up
‘dark’ under polarized light [Fig. 2a). The flattened surface (as) of the osteocyte lacunae becomes visible in tangential sections of the osteons (Figs 2c, 4). The same variation in aspect may be observed in spongy bone (Fig. 5). Figs 6 and 7 show, at a higher magnification, the three aspects shown by osteocyte lacunae of the same size when viewed in cross, longitudino-radial and longitudino-tangential sections. fibres while The osteon in Figure 6 has longitudinal the osteon shown in Figure 7 has transverse fibres.
328
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Morphometry
of human osteocytes
Fig. 3. Longitudino-radial
section of an osteon with longiMicrophotographs (al under polarized tudinal fibres. light and [b) under ordinary light (x 110). A
Fig. 4. Longitudinal section of osteon with longitudinal fibres; the cementing lines are indicated by black segments. The peripheral part of the osteon appears sectioned radially, the central part tangentially. Note how the osteocyte lacunae appear correspondingly sectioned radially (a,) in the peripheral part and tangentially (a31 in the central part. Microphotograph under ordinary light (x 180). b
The above-mentioned observations appear to be confirmed by the morphometric data reported in Fig. 8. The intermediate axis x of the osteocyte lacunae was measured in cross and longitudino-tangential sections of longitudinally structured osteons; its mean value shows no significant differences in two types of sections. The major axis y was measured in both longitudino-radial and longitudino-tangential sections of longitudinally structured osteons and also in cross sections of transversely structured osteons; its mean value is lower in transversely structured osteons only. The minor axis z was measured both in cross and in longitudino-radial sections of longitudinally structured osteons and also in cross sections of transversely structured osteons; the mean value of this axis always measured 4 pm. probably because it corresponds to the ‘rotational’ axis of the cell (see the drawings of the triaxial ellipsoids reported on the right side in Fig. 1). The results indicate that the ellipsoid formed by the osteocyte cell body seems to be strictly oriented in lamellar bone according to the main arrangement of collagen fibres; i.e. it has the major axis parallel to the length of the fibres, and the intermediate
and minor axes respectively dicular to the bony lamellae osteocyte is situated.
parallel and perpenbetween which the
The upper drawing in Figure 1 shows the data of the section area of osteocyte lacunae. Note the striking differences between the values of this parameter; as indicated in the lower drawing, section a2 is 2.5 times greater than al (A% = 581 and section a3 is 5.5 times greater than a1 no/o = 84) and 2.3 times greater than a2 (no/o = 551.
Discussion On the basis of the results reported in this paper, the following conclusions may be drawn: (al in order for morphometric investigations on osteocytes and/ or on their lacunae to be reliable, they must take into account the orientation of the triaxial ellipsoid, corresponding to the osteocyte cell body, with respect to the plane of the histologic section; (bl the orientation of the osteocytes in lamellar bone can be established by examination under polarized light of the arrangement of collagen fibres. The importance of the former conclusion has already been
Gastone Marotti:
Fig. 5.
taphyseal
Morphometry
of
human osteocytes
Trabecula of mespongy bone.
Microphotographs [al under polarized light and [k) u;ler ordinary light The osteocyte longitudilacunae ’appear nally sectioned in birefringent (bright) areas, cross sectioned in ‘dark’
areas light.
under
polarized
emphasized by various authors (Henning and Elias, 1963; Boyde, 1976; Boivin and Baud, 19771: however no technical procedures have been developed to avoid the errors which arise from disregarding the orientation of the osteocyte lacunae in the bony fragments examined. By means of the scanning electron microscope it has been shown that human secondary osteons which appear ‘dark’ under polarized light contain fibres with orientation having little or no transverse component, whereas birefringent osteons possess fibre orientation with transverse and longitudinal components (Frasca et al.. 1977). This means that
polarized light microscopy can only be used to obtain a statistical mean orientation of the collagen. However, as shown by the data reported in the present investigation, polarized light seems to be a good tool to establish, from the practical viewpoint, the main orientation of osteocyte lacunae in lamellar bone. The lower value of the major axis y of the osteocyte lacunae recorded in cross-sections of transversely structured osteons, in comparison with the values obtained from longitudinal sections of longitudinally structured osteons (Fig. 81, probably reflects the fact that in the former osteons the collagen fibres do not
Gastone
330
Marotti:
Morphometry
of human osteocytes
Shows the three aspects - al, a2, a3 of osteocyte lacunae of the same dimensions when respectively observed in cross, longitudino-radial and longitudino-tangential sections of longitudinai;de;tructured osteons. ordinary light Microphotographs [x 1 400). Fig. 6.
all run in a horizontal direction, as has been shown by Frasca et al. (1977). Thus in cross-sections of transversely structured osteons a certain number of osteocyte lacunae are probably not intersected accorFor this reason we suggest ding to their major axis. that periosteocytic lacunae morphometry be carried out on bone structures that appear ‘dark’ under the fibres are crosspolarized light viz. where sectioned. Several investigations on the morphometry normal and in various
have already been published of periosteocytic lacunae in pathological and experimental
conditions. However, all the data reported fail to take into account the orientation of the osteocyte ellipsoids. It is worth noting, in this connection, that increments in size of osteocyte lacunae by 1923%, with respect to controls, have been considered indicative of an increased osteolytic activity (Meunier et al., 1971; Meunier and Bernard, 1976; Lok and Jaworski, 1976). On the other hand, a decrement of about 16% recorded after calcitonin treatment (Duriez. 1974) or parathyroidectomy (Lok and Jaworski, 1976) has been ascribed to increased osteoplastic activity. Meunier and Bernard (1976) have also shown that in hyperparathyroidism the incre-
Gastone
Marotti:
Morphometry
of
human
osteocytes
- a,. a2, a, - of Fig. 7. The three aspects osteocyte lacunae of the same dimensions when respectively observed in longitudino-radial, cross and longitudino-tangential sections of transversely structured osteons. Microphotographs under ordinary light (x 1 400). Compared to fig. 6, note how the section areas of lacunae a,, ai, a, are practically the two types of osteons.
ment in size of osteocyte lacunae, with respect to controls, is greater in decalcified than in undecalcified samples: this datum indicates that the morphometric method applied by these authors seems to be correct and that the bone dissolving activity of osteocytes appears to be enhanced in primary hyperparathyroidism. Without in any way detracting from the value of these studies, one should point out that, in light of the results reported in this paper, the increase of 1923% or the decrease of 16%. with respect to controls, of the size of osteocyte lacunae does not
equal
in
prove conclusively that the osteolytic or the osteoplastic activities of osteocytes are respectively enhanced. In my opinion, percentage differences of this order could surely be significant if the morphometric analyses had taken the orientation of the osteocytes into consideration. The fact that the same osteocyte lacuna may show differences of 55-84% between its section areas, depending on how it is sectioned (Fig. l), offers grounds for suspecting that the percentage increments (1923%) or decrement (16%) in size of the osteocyte lacunae with respect to controls, as reported in the abovementioned articles, does not exclusively reflect a
1
4
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M=9
7
30-
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1
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IOi
M=22
50
OS.? 5 E.S.f 0.5
P
14
14
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30
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28
m=4 D.S.t 1 ES to.1
P
Gastone
Marotti:
Morphometry
of
human
osteocytes
333
Fig. 8. Frequency histograms of values in pm of the three principal axes (x, y, L) of the osteocyte lacunae. In the osteon drawn below each histogram, the arrangement of collagen fibres and the plane of the section, where the measurements were recorded, are indicated. M = mean: D.S. = standard deviation: ES. = standard error (see text).
‘lytic’ or a ‘plastic’ action by osteocytes respectively. It may derive, at least partly, from measurements performed on osteocyte lacunae of unknown orientation. Additionally these studies were carried out on fragments taken bioptically from the iliac crest viz. on spongy bone where the orientation of collagen
fibres, and therefore of the osteocytes, varies considerably not only between different trabeculae but also even in nearby portions of the same trabeculae (see Fig. 5).
Acknowledgements: Research aided Italian National Research Council.
by a grant
from
the
Baud, C.A.: Morphology and inframicroscopic structure osteocytes. Acta Anat. (Easel), 51: 209-225, 1962.
of
References
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C.A.: La morphometrie Acta Anat. (Base/),
des lacunes 99: 356, 1977.
Boyde, A.: Resolution, sampling and the determination lacunar size. In: Bone histomorphometry (Meunier. ed.), pp. 399-412. Paris: Armour Montagu, 1976.
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Duriez. J.: Les modifications calciques peri-osteocytaires. Etude microradiographique a I’analyseur automatique d’images. Noun. Presse med., 3: 2007-2010, 1974. Duriez. J.. Ghosez. J.P., Flautre. B.: La resorption ou lose periosteocytaire et son role possible da& la destruction du tissu osseux. Press m&d.. 73: 2581-2585, 1965. Ebner, V. von: Uber den feineren Bau der Knochensubstanz. Akad. Wiss. Wien, 72: 49-138, 1875. Frasca, P.. Harper, R.A., Katz, J.L.: Collagen tations in human secondary osteons. (Basef), 98: l-13. 1977.
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Hennig, A., Elias, H.: Sections through tri-axial ellipsoids. Proc. 1st Intern. Congress of Stereology. 43/l - 43/12 (1963). Jande, S.S.: Fine structural study of osteocytes and their surrounding bone matrix with respect to their age in young chiks. J. Ultrastruct. Res.. 37: 279-300, 1971.
Jowsey, J.: Microradiography of bone resorption. In: Mechanisms of hard tissue destruction (Sognnaes, R.F., ed.). pp. 447-469. Washington, D.C.: Amer. A. Advanc. Sci., 1963. Kolliker, A.: Handbuch der 6 Anflage 1. W. Engelman.
Gewebelhere des Leipzig, 1889.
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Krempien, B., Ritz, E., Geiger, G.: Behaviour of osteocytes in various ages and chronic uremia. Morphological studies in human cortical bone. Proc. 1st Workshop on Bone Morphometry [Z.F.G. Jaworski ed.), pp. 288--296. Ottawa: University of Ottawa Press, 1976. Lok, E.. Jaworski, Z.F.G.: Changes in the periosteocytic lacunae size observed under experimental conditions in adult dog. Proc. 1st Workshop on Bone Morphometry (Z.F.G. Jaworski ed.), pp. 297-300. Ottawa: University of Ottawa Press, 1976. Marotti. G.: Decrement in volume of osteoblasts during osteon formation and its effect on the size of the corresponding osteocytes. In: Bone histomorphometry (Meunier. P.J., ed.), pp. 385-397. Paris: Armour Montagu, 1977. Marotti. G.. Ledda, M., Delrio, N.. Fadda. M.: Distribution and size of osteocytic lacunae in secondary osteons of the dog. In: Exploration morphologique et fonctionnelle du sqielette, I Symposium CEMO-(Courvoisier, B.. Donath, A., eds.), pp. 225-230. Geneve: Editions MBdecine et HygiBne. 1976. Meunier. P.J., Bernard, J.: Morphometric analysis of pe1st Workshop on Bone riosteocytic osteolysis. Proc. Morphometry (Z.F.G. Jaworski ed.), pp. 279-287. Ottawa: University of Ottawa Press, 1976. Meunier. P.J., Bernard, J., Courpron, P., Vignon, G.: Use of an imaqe analvzino computer for the study of osteocytic behaviour -in -bone ‘diseases. Proc. . IX Europ. Symp. on Calc. Tiss., pp. 203-208. Wien: Facta-Publication, 1973. Meunier, P.J., Bernard, J., Vignon, G.: The measurement of periosteocytic enlargement in primary and secondary hyperparathyroidism. Israel J. Med. Sci., 3: 482-485, 1971. Tonna, E.A.: Electron microscopic evidence of alternating osteocytic-osteoclastic and osteoplastic activity in the perilacunar walls of aging mice. ‘Connective T&s. Res., 1: 221-230. 1972.
Jande, S.S., Belanger, L.F.: Ultrastructural changes associated with osteocytic osteolysis in normal trabecular bone. Anat. Rec., 183: 204, 1969.
Received: June 28, 1978 Revised: October 12, 1978 Accepted: December 20, 1978
RESUME
L’ORIENTATION OSTEOCYTAIRE LACUNES PERIOSTEOCYTAIRES
DANS
1’0s
LAMELLAIRE
HUMAIN
ET SON INFLUENCE
SUR LA MORPHOMETRIE
DES
L’orientation des lacunes osteocytaires par rapport a I’organisation des fibres collagenes a 6th Ctudiee dans’ 1’0s haversien humain. Les resultats de I’observation microscopique en lumiire normale et polarisee et les mesures des trois axes principaux des lacunes ostdocytaires ont montre que I’ellipsoide triaxiale est orientee comme suit : I’axe principal est toujours parallele aux fibres collag&res. tant dans les osteones longitudinaux que transversaux ; les axes intermediaire et mineur sont respectivement paralkle et perpendiculaire i la surface de la lamelle qui inclut la lacune de I’ostbocyte. L’importance de ces rtfsultats est discutCe par rapport aux etudes sur la morpho. metric de la lacune periosteocytaire.