Quantification of osteocyte lacunar density and anisotropy of vascular canals in compact bone
Quantification of
osteocyte lacunar density and anisotropy of vascular canals in compact bone
-Introduction,
bone structure, remodelation, osteoporosis,
Remodelling of
Haversian bone may result in both spatial and temporal variability of osteocyte
lacunar density and arrangement of vascular canals.
-Parameters
and methods in bone histomorphometry, tetracycline labeling, disector
The most
commonly used histomorphometric parameters: [1]
1- Structural
parameters
Measures that
describe the biopsy specimen
-Core width (mm)
C.Wi Overall size
of the biopsy specimen, refers to the width of the entire biopsy specimen or
core. It is the distance between the inner and outer periosteal surfaces
-Cortical width
(μm)
Ct.Wi Distance between periosteal and
endocortical surfaces, he sum of the widths of inner and outer cortices of the
biopsy specimen.
-Bone volume /
tissue volume (%)
BV/TV Space taken up by mineralized and
unmineralized bone relative to the total size of a bone compartmen, is cancellous
bone volume as a percentage of tissue volume. The numerator includes matrix,
whether mineralized or not, and the denominator includes cancellous bone,
marrow, and associated soft tissue.
Measures that
describe the configuration of trabeculae in space:
Trabecular
thickness (μm)
Tb.Th Is the mean distance across individual
trabeculae
Trabecular number
(/mm)
Tb.N Number
of trabeculae that a line through a trabecular compartment would hit per
millimeter of its length, (BV/TV)/Tb.Th
- Trabecular
separation
Tb.Sp Is the mean distance between
individual trabeculae
Osteoid thickness
(μm)
O.Th Distance
between the surface of the osteoid seam and mineralized bone
Osteoid surface /
bone surface (%)
OS/BS Percentage
of bone surface covered by osteoid
Osteoid volume /
bone volume (%)
OV/BV Percentage
of bone volume consisting of unmineralized osteoid
Osteoblast
surface / bone surface (%)
Ob.S/BS Percentage
of bone surface covered by osteoblasts
Wall thickness
(μm)
W.Th Mean
thickness of bone tissue that has been deposited at a remodeling site, is the
mean distance between resting trabecular surfaces and cement lines
3- Dynamic
formation parameters
Mineralizing
surface / bone surface (%)
MS/BS Percentage
of bone surface showing mineralizing activity
Mineral
apposition rate (μm/d)
MAR Distance
between two tetracycline labels divided by the length of the labeling interval
Mineralization
lag time (d)
Mlt Time interval
between the deposition and mineralization of matrix
Bone formation
rate / bone surface (μm3×μm−2*y−1)
BFR/BS Amount of
bone formed per year on a given bone surface
4- Static
resorption parameters
ES/BS Percentage
of bone surface presenting a scalloped appearance
Osteoclast surface
/ bone surface (%)
Oc.S/BS Percentage
of bone surface covered by osteoclasts
-Aims
of the study
The aim of the
study was to assess locally specific numerical density of lacunes with a
three-dimensional unbiased counting method and to analyze anisotropy of
profiles of vascular canals in an embedded section of human tibia.
-Material and
methods
–
optical disector
–
Delaunay triangulation
-
bone biopsy
An
undecalcified 150 micrometers thick transversal section was sawed from the
diaphysis of tibia of a 70-year-old female, grinded to a 70-80 micrometers
thick section, polished, stained with basic fuchsin, and observed with an
optical microscope. We quantified numerical density of osteocyte lacunes in
compact bone underlying the lateral, medial, and posterior surface. Image
frames (n=42) were sampled in an systematic uniform random manner from cortical
and medullar layer round the tibial circumference. Each of the layers comprised
one half of the local thickness of the bone section. Series of seven optical
sections registered with respect to the Z-axis were photographed in each image
frame. The unbiased counting rule of optical disector was applied to these
series in order to assess the number of osteocyte lacunes per unit volume. Area
fraction of the profiles of vascular canals in the bone sections was estimated
with the point-counting method. Disector and point-grid counting was performed
with the Ellipse software (ViDiTo, Košice, Slovak Republic). The
two-dimensional anisotropy of profiles of vascular canals was assessed with the
Delaunay triangulation (Qhull software, The Geometry Center, Minneapolis MN,
USA). We carried out a 3-D reconstruction of vascular canal and surrounding
lacunes in a part of a randomly selected Haversian system (software Amira,
Mercury Computer Systems SAS, Merignac Cedex, France).
Results numerical density
of lacunes – anisotropy of vascular canals
Kruskal Wallis
ANOVA did not prove significant differences (p=0.051) in lacunar density among
the bone areas underlying the lateral (18088±2150 mm-3, mean±SD),
medial (16655±3772 mm-3) and posterior surfaces (20222±4109 mm-3).
Lacunar density was higher (p=0.032) in cortical (20356±3725 mm-3)
than in medullar layer (16287±2274 mm-3). Area fraction of profiles
of vascular canals was 10.5%. Delaunay triangulation of centres of gravity
revealed considerable anisotropy in arrangement of profiles of vascular canals
(n=1194). The 2-D arrangement of vascular canals was approximated by a network
of vertices forming isosceles triangles with average basis (perpendicular to
the bone surface) of 251±13 micrometers and height (parallel to the bone
surface) of 258±24 micrometers.
Osteocyte lacunar density in individual bone areas
underlying the lateral, medial and posterior surfaces of tibial shaft. (
mean±SD of 7 disectors) [mm-3]
|
layer:
|
facies lateralis
|
facies medialis
|
facies posterior
|
cortical
|
18958±2409
|
19119±3488
|
medulllar
|
17218±1566
|
14191±2122
|
17453±1603
|
Discussion
–
biological interpretation of quantitative parameters
–
časová naročnost
-
disektor- obecne v MM jedinná metoda umožnujici 3D
-
srovnání/přepočet 2D a 3D, vzorec
-Conclusion
We suggest the
combination of optical disector, stereological point-counting method, and
Delaunay triangulation to be a set of complementary methods useful for
description of local heterogeneities in lacunar density and arrangement of
vascular canals in microstructure of compact bone.
References:
[1] RAUCH F, Watching bone
cells at work: what we can see from bone biopsies, Pediatr Nephrol, 21: 457–462, 2006
[2] RAUCH F (2003)
Bone histomorphometry. In: Glorieux FH,Pettifor J, Jueppner H (eds) Pediatric
bone. Academic, San Diego, CA, pp 359–374
[3]RAUCH F, Travers R, Norman ME, Taylor A, Parfitt AM, Glorieux FH
(2002) The bone formation defect in idiopathic juvenile osteoporosis is
surface-specific. Bone 31:85–89
[3]GLORIEUX FH,
Travers R, Taylor A, Bowen JR, Rauch F, Norman M, Parfitt AM (2000) Normative
data for iliac bone histomorphometry in growing children. Bone 26:103–109
[4]PARFITT AM,
Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR
(1987) Bone histomorphometry: standardization of nomenclature, symbols, and
units.Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner
Res 2:595–610
Acknowledgments: Supported by the project
MSM0021620819 and by the grant GAAV 100110502.