Collagen orientation
Samples:
All the slices mentioned below from left fibula and femur (first donor), and from tibia (second
donor) not subjected to the mechanical tests, were used for biological analysis.
The following regions of each section was obtained at different levels in the whole length of bones,
depending on the outcomes of the destructive testing described in point 2.3: into each level four
regions were analysed for the collagen orientation referring to anatomical landmarks.
FIBULA
8 sections that means
- 8 anterior regions
- 8 medial regions
- 8 lateral regions
- 8 posterior regions
TIBIA
8 sections that means
- 8 anterior regions
- 8 medial regions
- 8 lateral regions
- 8 posterior regions
FEMUR
9 sections that means
- 8 anterior regions
- 9 medial regions
- 9 lateral regions
- 8 posterior regions
(the missing regions were not detectable)
Toatal of samples: 82
Method:
The following description is extracted from: Beraudi A., et al., “Osteon classification in human fibular shaft by
circularly polarized light”, submitted to Cells Tissues Organs for publication.
The orientation of the collagen fibers was analysed by circular polarized light microscope
observation. The osteons were grouped using the modified Bigley et al., (2006) classification
[Bigley et al., 2006]
 Harvesting of the sample
Bones were harvested from the cadavers, fully embalmed by perfusion and immersion in
embalming solution [Van Sint Jan and Rooze, 1992]. All the tissues attached to the periosteum were
removed. Once retrieved, the bones were preserved at -20°C until testing.
Anatomical Landmarks were identified to determine the antero-posterior plane [Van Sint Jan,
2007], a suture thread was attached by acrylic glue on a reference scratch marked on the surface to
identify the anterior side.
Sequential cuts were performed to produce a series of bone cylinders of 8 mm in height. The
position of each cylinders was expressed as percentage of the total length of the bones (figure 1).
 Treatment of the samples
Each cylinder was embedded in poly-methylmethacrylate (Carlo Erba Reagenti SpA, Rodano,
Milano, Italy) with its distal surface supported on the container, according to the Boyde’s and
collaborators method (1984). Briefly, after accurate washing in water, the samples were dehydrated
and degreased in a vacuum with the following series of alcohol:
ethanol 80% overnight, 3 steps in ethanol 95% of 1h each, methanol 5 steps of 1h each at 37°C.
Infiltration occurred with 4 passages in poly-methylmethacrylate (Carlo Erba Reagenti SpA,
Rodano, Milan, Italy) first destabilized by chromatography with Aluminium oxide (Carlo Erba
Reagenti SpA, Rodano, Milan, Italy) and finally embedded in mixtures of poly-methylmethacrylate
and Di-n-butylphthalate (Carlo Erba Reagenti SpA, Rodano, Milan, Italy) at 3.5% w/v at 25°C. The
reaction mixture catalyst was benzoyl peroxide (Carlo Erba Reagenti SpA, Rodano, Milan, Italy) at
25% v/v.
 Slices preparation and analysis for CLP
From the embedded blocks, serial 100 ± 5 μm slices were obtained [Boyde et al., 1984; Boyde and
Riggs, 1990] with a 300 μm thick wafering blade (Leica 1600). The slices were compressed
between two slides while maintaining the correct orientation. On each slice, four regions, counting
the whole cortical wall thickness for a circumferential length of 3 mm, were identified at 90 degrees
each other, starting from the anterior side reference (figure 2). Therefore, the four regions were
located on the anterior (0°), lateral (90°), posterior (180°) and medial (270°) quadrant, respectively.
Each region was visualised at 4X objective lens magnification and 10X ocular lens magnification
by polarized circular light microscope (Leica DM EP). Since it was impossible the simultaneous
visualisation of the whole region, a series of photos was taken to map the whole region and acquired
by Leica DC 300 camera. Finally the photos were reassembled with Adobe Photoshop 7.0 in order
to obtain a digital image of each region on which the analysis was performed. Within each analyzed
region, the cortical wall thickness was determined as the mean value of three measurements
collected in a direction orthogonal to the external cross–section perimeter.
 Classification of the osteons
The collagen orientation within the osteons was determined on the basis of the osteon appearance.
The validated osteon classification proposed by Bigley et al. [Bigley et al., 2006] was used in this
study except for the two types of osteon classes (L and O) which were merged into a single class;
thus the classification used was interpreted according table 1.
This choice was done on the basis of a preliminary analysis on a bone cross section, performed in
double blind conditions, to assess the feasibility and the repeatability of the described procedure.
During this preliminary analysis it was established that:
o all the osteons considered to be "in a state of fusion", i.e. which had non-circular or partially
deformed geometry, had to be excluded from the reading since they cannot be classified
unambiguously;
o since osteons very often had a thin bright ring around the Haversian canal or around the
external perimeter, i.e. show forms between L and O that are not easy to attribute to one
class or the other, these two classes were merged into one.
Another cross section was treated as described except for staining (trichrome staining according to
Goldner, 1938 [Goldner, 1938]). This preliminary analysis was necessary to identify the large void
within the osteon structure. Since those were identified as blood vessels, it was decided that the
structure showing an internal canal of dimension of the same order of magnitude of the whole
structure dimension has to be excluded since they are non-capillary vessels.
All these criteria were used in analyzing the four regions of each cross section obtained from the
bones under investigation (figure 2). To be sure of the repeatability, half of the analyzed regions,
selected randomly, were analyzed twice by two operators (double blind condition).
Figure 1
Figure 2
A scheme representative of the serial cutting in bones.
The different levels from where the samples were obtained are
expressed in percentage of length along the whole bone length.
Fibula section in circularised polarized light
microscopy at 21% of length.
At 0°, 90°, 180° and 270° were selected the four regions
analyzed for osteons classification. In the red panel a
bigger view of a particular of the section in which were
pointed out the three types of osteons: T (bright
osteon), A (alternating osteon) and LO (dark-hooped
osteon).
Bar = 400 μm
%
lenght
19
%
lenght
20
22
29
21
37
31
47
37
29
%
lenght
37
44
55
63
64
70
73
69
78
85
82
75
58
81
osteon
A
M
A
L
P
FEMUR
M
A
L
P
L
M
P
TIBIA FIBULA
T: Bright osteons contain lamellae composed of collagen
fibers transverse to the osteon axis.
All lamella must appear bright, with no interspersed dark lamellae.
A: Alternating osteons contain lamellae composed of collagen fibers that
alternate between transverse and longitudinal to the osteon axis.
Two successive alternating pairs of lamellae are present away from the
peripheral boundary (hooped) or around the Haversian canal.
LO: Dark and hooped osteons contain lamellae prevalently longitudinal and
a portion of peripheral boundary composed of collagen fibers transverse
to the osteon axis with a bright appearance. A thick bright ring around
Haversian canal occurs.
Table 1
Classification of osteons type
for observation in circularly
polarized light microscopy and
for collagen fiber orientation
(Bigley et al., 2006 modified)
[Bigley et al., 2006].
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Res 5(6): 299-307.
Boyde, A., C.M. Riggs (1990) The quantitative study of the orientation of collagen in compact bone slices. Bone 11(1):
35-39.
Goldner, J. (1938) A modification of the Masson trichrome technique for routine laboratory purposes. Am J Path 14:
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