Rom J Leg Med [22] 109-116 [2014]
DOI: 10.4323/rjlm.2014.109
© 2014 Romanian Society of Legal Medicine Histomorphometric evaluation of osteons inside the compacta of long bones
diaphysis. Implication in pathology
Gheorghe S. Dragoi1,*, Petru Razvan Melinte2, Ileana Dinca2, Mihaela Mesina Botoran2, Elena Patrascu2
_________________________________________________________________________________________
Abstract: The quantitative, micro-anatomic assessment of Havers systems within the structure of diaphysis compacta
of long bones imposes the acknowledgement and visualization of reference landmarks for morphometric. Authors consider that
the utilization of histolo-physics methods (examination in polarized light and/or interferential contrast) assures the visualization
of histotopographic landmarks necessary for performing these measurements. Histo-physics analysis of longitudinal sections
through tibia diaphysis compacta allowed authors to emphasize the spatial alternation of isotropic columns of osteons lamellas
with Havers central canals, with anisotropic columns and inter-osteon spaces. Authors consider that the measurement of osteon
diameters in the interval between two inter-osteon isotropic columns ensures the constancy of osteons’ geometry rigorous
evaluation.
Key Words: osteon, histomorphometry, histo-physic methods, osseous diaphysis compacta.
M
icro-anatomic qualitative and quantitative
analysis of osteons inside the diaphysis
of long bones can be useful in anthropology, forensics,
biomechanics and pathology to evaluate the age, physic
stress, histologic diagnosis of bone metabolic diseases,
and results of therapy on those diseases and to understand
the human skeleton ability to adapt to changes of life style.
A rigorous histomorphometric evaluation of osteon must
stand at the base of such an evaluation.
Studies on the dynamics of osteons dimensions
during ontogenesis are contradictory. Some researchers
signaled their decrease with aging in femur, ribs and
humerus (Currey, 1964; Burr, Ruff and Thompson, 1990;
Joshimo et al., 1994) and others noted an increase in
femur and tibia (Black, 1979).
The magnitude of phenotype changes
undergone by haversian systems raises many problems:
establishing micro-anatomic landmarks necessary for the
measurement of osteons’ diameters; geometric shape of
osteons; relations between osteons during remodeling
processes; anisotropic heterogeneity of osteons; relations
between Havers canals and Volkman canals and the system
of osteon lamella (Dragoi et al. 1998, 2001).The purpose
of the paper is to select and evaluate the micro-anatomic
landmarks used to perform osteon histomorphometry.
The objectives of the paper are imposed by
the structural heterogeneity of bone compacta, the
uncertainties to visualize the micro-anatomic landmarks
offered by standard micro-anatomic methods and lastly
by the use of histo-physics method of examination
in polarized and contrast interference light for
histomorphometric evaluation of osteons.
MATERIALS AND METHODS
The study was achieved on 6 adult tibia bones
(35-45 years old) from which we harvested 18 fragments
from the middle part of diaphysis; the fragments were
1) Romanian Academy of Medical Sciences, Bucharest, Romania
* Corresponding author: Prof.MD, PhD, E-mail [email protected]
2) University of Medicine and Pharmacy of Craiova, Department of Anatomy, Craiova, Romania
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Dragoi G.S. et al Histomorphometric evaluation of osteons inside the compacta of long bones diaphysis. Implication in pathology
3 mm thick in transverse and vertical plane sections.
Four fragments were processed by polishing and 10
fragments were prepared for paraffin embedding by
classical histologic methods. 5 microns serried sections
were stained using usual and special staining methids:
Hematoxiline-Eosine, Van Gieson, Gomori, PAS. For the
examination in polarized and contrast interference light,
the bone fragments were polished using a Ponce stone
and then fine glass-papier. When the fragments were thin
enough and transparent, they were grated with a knife
and then washed with water and alcohol to remove tissue
debris from their surface.
The fragments were then introduced in an
alcohol solution of blue anilin (anilin blue, 1g; alcohol
95%, 100 ml) for 3-4 hours; afterwards, the solution was
evaporated on a heated platinum. Lately, the fragments
were again polished on a fine rough surface using 2%
sodium chloride solution. Finally, the fragments were
rapidly washed in distilled water, dyed and mounted in
Canada balsam. Thus, we could visualize the Havers canals
in blue. Four fragments were prepared without staining
in anilin blue. The examination of fragments was carried
out using research microscope Nikon Eclipse E-600.
The images were captured by a digital camera through
Nis-Element Basic Research software. The images were
processed in Adobe Photoshop CS5 software.
RESULTS
The identification of landmarks needed for osteon
histomorphometry is possible through the knowledge of
the variable 3D shapes of oesteons and of the relations
between osteon lamella and Volkman and Havers canal
systems.
A. Micro-anatomic analysis of the variable
shape of osteons on transverse sections through diaphysis
compacta
The examination in polarized light of transverse
serried sections through fragments of tibia diaphysis
allowed the identification of three topographic sectors: a
sector limitrophe to the canal of diaphysis that contains
circumferential osseous lamella and a reduced number
of osteons; an intermediate sector with a great density
in osteons with variable relations – contiguity and/or
continuity to the system of interstitial osseous lamella,
and an external sector located near periosteum that
contains circumferential osseous lamella (Fig. 1 A, C).
When analyzing with 10x and 20x objectives, we
noticed the variable shape and dimensions of osteons.
The central canal Havers is ellipsoid in shape and is
limited by intense bi-refringent osseous lamella (Fig. 1 B,
D, F). In some places, central canal Havers is interrupted
by a communication to a Volkman canal (Fig. 1 A, F). The
distance between osteons is variable and can be evaluated
by the space interval between central canals Havers (Fig.
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1 A, B). When examining with 4x and 10x objectives,
we observed that osteons group in two, three of four
formations, circumscribed by bi-refringent osseous
lamella (Fig. 1 B, C, E).
B. Micro-anatomic analysis of osteon structural
heterogeneity on transverse sections through diaphysis
compacta
The structure of osteons is variable during
ontogenesis. On transverse sections through new born
tibia diaphysis, stained with Hematoxiline Eosine, we
identified osteons centered by Havers canals, surrounded
by osteoblast arranged in circumferential trajectories,
osteoblasts that present cytoplasm expansions with radial
distribution (Fig. 2 A).
When examining the transverse serried sections
through adult tibia diaphysis, stained with Hematoxiline
Eosine, PAS and argentic impregnation Gomori, we
noted the structural heterogeneity of osteons by the
presence of phenotype changes generated by remodeling
processes (Fig. 2 A-H). The examination with 20x and 40x
objectives of osteons in direct and contrast interferential
light, allowed us to identify two borderline structures:
“linea cementalis” at the periphery of Havers system and
“resorption line”, as a clear, circumferential space that
corresponds to the future “linea cementalis” of the osteon
that is about to differentiate (Fig. 2 C-E).
On the sections of Gomori argentic impregnation,
linea cementalis appears like a sinusoid path (Fig. 2 G).
The anisotropy of collagen fiber fascicles decreases from
the center to the periphery of osteons (Fig. 2 F). When
examining the sections using differential interference
contrast microscope, the resorption line appears very
well contoured. At the intersection between three osteon
Havers canals and a Volkman canal, we visualized the
primordial structures of newly formed osteons (Fig. 2 H).
C. Micro-anatomic analysis of space relations
between osteon lamella and the system of Havers and
Volkman canals on vertical sections
When examining the vertical, sagittal and
transverse sections, osteons appear as columns centrally
crossed by Havers canals and sometimes by Volkman
canals (Fig. 3). One can easily notice the striate aspect of
the microscope field due to the quasiparallel alternation of
columns made of osseous lamella and Havers canals (Fig.
3A). When examining with 10x and 20x objectives, the
central canals Havers are crossed under variable angles
creating images resembling to letters M, X, Y (Fig. 3).
The comparative analysis of transverse and longitudinal
sections proves the heterogeneous relations between
osteons and the variability of landmarks that can be taken
into consideration to achieve a rigorous morphometric
analysis (Fig. 4 J-L). When examining the longitudinal
sections in polarized light, after rotating the polarizer
with 90 degrees, the columns of osseous lamella appear
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Figure 1. Transverse sections through human tibia diaphysis compacta. The structural heterogeneity of compacta is determined
by the variability of geometrical shape and spatial distribution of osteons. 1.Osteonum secundarium; 2.Canalis osteoni
(Centralis); 3.Linea cementalis; 4.Canalis transversus; 5.Lamella interstitialis; 6.Canalis ossis; 7. Lamella circumferentialis externa;
8.Heterogeneous distribution of osteons in 2/3 external of diaphysis compacta; 9.Lamella osteoni; 10.Osteons in reshuffle. Ground
sections, unstained. Examination in direct light (A) and in polarized light (B-F). Microphotos by Nikon Digital Sight DS-Fi 1
High-Definition Color Camera Head; x 28 (C); x 70 (A,F), x 140 (B), x 280 (D,E).
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Dragoi G.S. et al Histomorphometric evaluation of osteons inside the compacta of long bones diaphysis. Implication in pathology
Figure 2. Reshuffle and reshaping of tibia diaphysis compacta. 1.Canalis osteoni (Centralis); 2. Linea calcificationis; 3. Lacuna
osteocyti; 4. Processus osteocyti; 5. Osteoblasts line; 6. Linea cementalis of a secondary osteon; 7.Linea cementalis of an osteon
about differention; 8. Lamella interstitialis; 9. Newly-formed Lamella osteoni; 10. Lacuna osteocyti; 11. “Cone funnel” formed by
the spatial orientation of osteon’s lamellas during the reshuffling process. Parafin sections. Hematoxylin-Eosin coloration (A-D),
Van Gieson coloration (B), Mac Mannus- PAS (E) coloration; Reduced silver nitrate Gomori (G, H);Ground sections, unstained
(F). Examination in direct light (A, B, C, E, G); Examination in polarized light (F); Interferential contrast (D, H).Microphotos by
Nikon Digital Sight DS-Fi 1 High-Definition Color Camera Head; x 70 (C, D, E, G, H), x 140 (A, B, F).
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Figure 3. Longitudinal sections through tibia diaphysis compacta. The relations between Havers central canals, Volkman
transverse canals and osteons lamellar columns. 1.Canalis transversus; 2. Canalis osteoni (Canalis centralis); 3. Longitudinal
columns of osteons lamellas. Ground sections, unstained. Examination in direct light.Microphotos by Nikon Digital Sight DS-Fi
1 High-Definition Color Camera Head. x 28 (A); x 70 (B, D, E), x 140 (C), x 280 (F).
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Dragoi G.S. et al Histomorphometric evaluation of osteons inside the compacta of long bones diaphysis. Implication in pathology
Figure 4. Location and comparative relations of Havers canals, Volkman canals and osteons lamellas on transverse (A-I) and
longitudinal (J-L) sections. 1. Canalis osteoni (Canalis centralis); 2. Canalis transversus; 3. Longitudinal columns with osteons
lamellas; 4. Transverse diameter of osteons; 5. Interosteon space. Ground sections, unstained. Examination in direct light (A-K)
and in polarized light (L). Microphotos by Nikon Digital Sight DS-Fi 1 High-Definition Color Camera Head; x 70 (A-L).
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Romanian Journal of Legal Medicine anisotropic and Havers canals and osteons boundaries
appear isotropic (Fig. 4 L). The lateral limits of osteons
are marked by isotropic bands of similar intensity and
trajectory as the ones determined by Havers canals.
The belonging of different structures to
isotropic bands is achievable either by comparative
analysis of images visualized in direct or polarized light,
or by moving the polarizer by 45 degrees (Fig. 4). The
comaparative histomorphometric analysis of osteons
proves a great difference between the values obtained
when measuring landmarks on transverse sections
(linea cementalis) than when measuring on longitudinal
sections (isotropic spaces between osteons). The first
values are 25% greater than the last ones.
DISCUSSIONS
Microscopic anatomy of bones compacta
still presents many unsolved problems regarding the
geometry and structural heterogeneity of osteons,
space and time relations between osseous lamella and
Volkman and Havers canal systems from one hand, and
between osteons and inter-osteon spaces on the other
hand, and regarding the morphometric data as function
of landmarks choosing. The osteon systems inside tibia
diaphysis compacta were differently studied using
methods that allowed the micro-anatomic-topographic
visualization of bone tissue (Hematoxiline Eosine
stain), collagen fibers (Van Gieson stain), argirofil
collagen fibers (Gomori argentic impregnation) and
neutral mucous-polysaccharide (PAS). We examined
comparatively the serried sections in direct, polarized
and interference contrast light.
By analyzing our observations on Havers
systems we proved the important role played by some
micro-anatomic research methods in the qualitative
analysis of osteon geometry, osteon relations and in
describing the trajectory of osseous lamella around
central and inter-osteon canals.
The variable shape and diameter of osteons
and chaotic space distribution of Havers systems on
transverse serried sections disoriented us during the
selection of microanatomic landmarks that can serve
for histomorphometric measurements. Nevertheless,
the analysis of transverse sections, offers important
information on osteon boundaries represented by
linia cementalis that borders secondary osteons and
by resorption line that states the beginning of a new
genesis and remodeling cycle. The examination of
Vol. XXII, No 2(2014)
those sections brings data on location of landmarks for
histomorphometric studies.
The analysis of longitudinal serried sections
in direct light reveals important knowledge about the
relations between Havers and Volkman canals and
columns of osseous lamella. Although one can easily
notice the parallelism of columns inside Havers system,
we were not able to establish with certainty the boundary
between osseous lamella of two adjacent osteons.
Nevertheless, on those sections, we could measure
the distances between Havers canals of two neighbor
osteons but we could not manage to morphometric
evaluate the osteon as a micro-anatomic system.
The examination in polarized light of
longitudinal sections allowed the visualization of interosteon boundaries as an isotropic longitudinal band at
that level, that we consider an important landmark for
osteon histomorphometric studies.
CONCLUSIONS
1. Establishment of qualitative and quantitative
evaluation criteria of bone tissue depends on the time
and space evolution of Havers system during bone turnover, regenesis and remodeling.
2. The heterogeneity of shape, structure and
relations between Havers system elements (canals and
lamella) represent an important factor in the qualitative
and quantitative analysis of bone compacta.
3. Permanent remodeling of osteons by the
turn over of cells that form “multicellular bone unit”
determines the instability in time and space that
impends osseous histomorphometric studies.
4. The variable relations between osteons and
between osteons and the structures inside inter-osteon
spaces on transverse sections, increases the difficulty
of choosing stable landmarks for histomorphometric
analysis.
5. The examinations of sections through
diaphysis compacta, in polarized or interference
contrast light offers supplemental information on
space distribution of osteon osseous lamella and on the
relations between them and Havers and Volkman canal
systems.
6. The space alternation of isotropic and
anisotropic columns offers valid landmarks for
measuring the osteon diameter, represented by the
isotropic columns that form the lateral margins of
osteons.
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