Understanding metaphysis biology:
Effects of training and age on bone parameters of
the third metatarsus in Thoroughbred horses
Research project of Drs. Anne Marks
Institute of Veterinary, Animal and Biomedical Sciences
Massey University, New Zealand
December 2009
Project Supervisors:
Prof. E.C. Firth / Dr. C.W. Rogers (NZ)
Prof. Dr. P.R. Van Weeren (NLD)
1
Table of Contents
Abstract ................................................................................................................................................... 3
Introduction ............................................................................................................................................ 4
How the long bones grow ................................................................................................................... 4
pQCT.................................................................................................................................................... 6
Bone parameters ................................................................................................................................ 7
Responses of the metatarsus to workload ......................................................................................... 8
Age related changes in the metaphysis .............................................................................................. 8
Research objectives ............................................................................................................................ 9
Material and methods .......................................................................................................................... 10
The MUGES population ..................................................................................................................... 10
Foals .................................................................................................................................................. 10
Scanning the bones ........................................................................................................................... 10
Statistical analysis ............................................................................................................................. 11
Results ................................................................................................................................................... 12
Trained versus Untrained Horses...................................................................................................... 12
High and Medium exercise intensity subgroups versus Untrained Horses ...................................... 14
Foals versus 2 year old Horses .......................................................................................................... 17
Discussion.............................................................................................................................................. 20
Trained versus Untrained Horses...................................................................................................... 20
Foals versus 2 year old Horses .......................................................................................................... 21
Use of the graphs and the metaphyseal profile ............................................................................... 22
Conclusions ........................................................................................................................................... 22
Acknowledgements............................................................................................................................... 23
References ............................................................................................................................................ 24
2
Abstract
Aim: to obtain more information about how the metaphyseal region in the horse develops during
early life and what influence initial training for racing has on the growth and development of the
metaphysis.
Methods: Graphs of the parameters Bone Mineral Content (BMC), Bone Area (BA) and Volumetric
Bone Mineral Density (BMDv) of the third metatarsal (Mt3)bone of trained and untrained 2-year old
Thoroughbred horses were compared. In these graphs, slices at increasing distances from the
metaphyseal growth plate were compared between the two groups. This way of imaging allowed to
compare the whole metaphyseal area between individuals instead of comparing only specific
measuring points.
To study the development of the physis near the time of birth, the metaphyseal profiles of still-born
foals were studied and compared with those of the 2 year old horses.
Results: The metaphyseal region of Mt3 responded in the medium exercise intensity subgroup by
making a denser cortex not a denser trabecular area, no response in BMC and BA was measured.
The high intensity exercise subgroup responded on the given exercise by increasing trabecular and
cortical BMC, trabecular and cortical area and cortical BMDv.
The metaphyseal region in the foals was different in all parameters measured compared to that of 2
year old horses.
Conclusions: it is possible to compare trained and untrained horses by comparing graphs that plot
BMC, BA or BMDv against distance from the growth plate. The metaphyseal profile was suitable in
comparing the different parameters of foals and 2 year old horses.
3
Introduction
In the equine racing industry, orthopaedic injury is one of the major reasons of horses being
incapable of finishing their race or continue with training at all. It is the most important reason for
euthanasia in race horses and a huge factor of economical loss in this industry. In other working
horses it is a common problem as well and options for treating bone fractures in horses can be
limited. (E. C. Firth, 2004). Improving how horses can be raised and trained could help to minimise
the chance of orthopaedic injury.
As exercise is known to affect bone development, an area of special interest has been the possibility
of increasing tissue resistance to deformation by training, with the aim of developing adequate
training schedules for young racehorses. Most work on the effects of exercise on bone development
in young racehorses has focused on either the diaphysis (mostly related to the occurrence of “sore”
or “bucked’ shins), or the epiphyseal area (because of the frequent condylar metacarpal or
metatarsal fractures) (E. Firth, 2005). The metaphyseal area and the area of the growth plate have
received little attention thus far.
In foals and the young of other species, fractures of the long bones occur most often in or close to
the growth plate. This is because the physis tissue is not as strong as the surrounding bone. Also,
trauma and other insults to the growth plate can lead to reduction or cessation of growth in part of
the physis, leading to oblique growth of the bone which can lead to deviation of the leg. ("Adams'
lameness in horses," 1987)
Despite the importance of this area, still very little is known about how exactly the growth plate
develops over time and what effect exercise has on this development.
This report describes a research project on the effects of training on the metaphysis in the third
metatarsal bone of Thoroughbred horses. Measurements with a pQCT scanner have been done on
bones of trained and untrained horses and on bones of stillborn foals to obtain more information
about the development of the metaphysis in the early life of the horse.
First information about the normal growth of the bone and the role of the physis is given as well as
some clarifications about bone parameters and bone strength. Then the population and measuring
methods used will be described where after the results will be presented. The last part of the report
evaluates the results and gives suggestions for future research.
How the long bones grow
The long bones of animals grow by a process called endochondral ossification, in which cartilage
serves as the precursor for the bone. Later, remodelling takes place and the initial bone is eventually
replaced. Endochondral ossification starts before birth, but it continues in all the growth plates
(metaphyseal and epiphyseal) in the young animal. (Getty, 1975) One of those growth plates, the
physis, is located between the metaphysis and the epiphysis and this is the area where the increase
in length of the bone takes place. Five different zones of the growth plate can be recognised: the
zone of reserve cartilage (the youngest), proliferation, hypertrophy, calcified matrix and the zone of
resorption (the oldest). (Figure 1)
4
Figure 1 zones of the growth plate (From Cormack DH. Essential Histology. 2nd Ed. Baltimore: Lippincott Williams &
Wilkins, 2001)
In the zone of proliferation, cartilage cells divide and produce matrix and a similar amount of
cartilage is resorbed in the zone of resorption. In this way the physis retains the same thickness. The
resorbed cartilage is replaced by cancellous bone. Due to the dividing of cells in the zone of
proliferation, the bone is getting longer as the epiphysis moves away from the diaphysis.
The width of the bone also increases during the early life of the foal, but by a different process called
appositional growth. In this process, new layers of bone are added to the surface of older bone or
cartilage. This occurs between the cortical lamellae and the periosteum.
As bone grows, constant remodelling occurs. Near the physis, bone from the outer surface of the
cortex is resorbed and bone on the inner surface of the cortex is added (Figure 2) The bone
produced at the metaphysis, which has a relatively large diameter and almost no cortex, is in this
way transformed into diaphyseal bone, which has a smaller diameter and a thick, distinct cortex.
New cartilage production slows and ceases at various stages after birth for the different physes. The
cartilage that is present in the physis is crossed by strands of bone, after which the whole physis is
gradually ossified, and is radiographically “closed”. The lengthening of the bone from the physis is
now complete. However, bone remains very active, and is constantly being resorbed and replaced in
response to hormonal and physical factors that act on bones. (Akers, 2008; Reece, 2009)
5
Figure 2 Sites of bone deposition and resorption in the process of lengthening and remodelling of long bones. Bone is
shown in gray, cartilage in colour. (From Cormack DH. Essential Histology. 2nd Ed. Baltimore: Lippincott Williams &
Wilkins, 2001)
pQCT
A way to study the strength of bones is to obtain the density of the material and its distribution
throughout the bone. Peripheral Quantitative Computed Tomography (pQCT) is the only method to
noninvasively and accurately measure dimensions and density, and predict bone strength.
Conventional CT-scanners can be used to obtain tissue densities. The CT-scanner “cuts” the bone in
serial adjacent slices. Software can than make three dimensional images which are now mainly used
for diagnostic purposes. Difficulties in using CT in veterinary practice include the need to
anaesthetise the animal, the costs, and the relative scarcity of such machines in clinics. (E. C. Firth,
2004). One of the benefits of using CT scanners in practice is their long-term precision, even in
portable scanners. (Wapniarz, et al., 1994)
Peripheral CT scanners were developed for imaging the lower arm and leg in people, in order to
identify higher fracture risk in people with osteoporosis. The software of these scanners generates a
three dimensional image, but the main use here has been quantifying density and dimensions of
various tissues. The density can be expressed in volumetric terms since the thickness of the slice is
known. This is one of the benefits of pQCT scanning; in this way it is possible to detect true bone
density independent of body height or bone size. Other techniques frequently used to obtain tissue
density, like DXA (Dual energy X-ray absorptiometry) are not able to do this. The pQCT software
defines Bone Mineral Content (BMC), Bone area (BA), and Volumetric Bone Mineral Density (BMDv)
of the cortex and the cancellous bone independently (E. C. Firth, 2004).
6
Bone parameters
BMC
The Bone Mineral Content (BMC) measures the mineral fraction that is apparent in the measured
bone slice. It is obtained by comparison of the absorption coefficient of the measured area to that of
a standardized phantom. (Ferretti, Cointry, & Capozza, 2003)
There is a linear relationship between bone strength and BMC in which the strength increases with
higher BMC. However, this relationship has an optimum where after breaking strength decreases
with increasing BMC, most likely due to increased stiffness of the bone. (Lawrence, et al., 1994)
Area
The surface area of the cross-section of the bone is an indication for its size and thus its strength.
Apart from the size, the shape of the bone is of importance and the distribution of the bone over
cortex or cancellous bone. Bone that is distributed further from the centre is more effective in
resisting bending and torsion forces than bone closer to the axis.
BMDv
When assessing bone strength and resistance of the bone to fractures, a third parameter that can be
used is the BMDv. This value corresponds to the apparent mineral density of the tissue, but includes
the pores, cells, vessels, etc. The mechanical properties of bone largely depend on the presence of
mineral in the matrix, and the modulus of elasticity is directly related to the BMDv of the bone tissue.
(Ferretti, Capozza, & Zanchetta, 1996)
Because the BMC and BA of the cross sectional slice are quantified and the thickness of the slice is
known, the volumetric density can be obtained, in either the slice or in a defined Region Of Interest
(ROI). This ROI can be only the cortex, only the trabecular bone or a manually defined area. If the ROI
comprises only trabecular bone, its BMDv will express the apparent density of that cancellous bone.
The BMDv of only cortical bone should be more closely proportional to the true mineral density of
the solid bone matrix, depending of the degree of porosity (Ferretti, et al., 2003), although even
here there is variation in BMDv within apparently (grossly and radiographically) homogeneous
cortical tissue.
Metaphyseal profile
In recent research on rodent bones, (Pitsillides, et al., 2009) have developed a way of describing the
change in the ratio of cross-sectional trabecular bone area on total area with increasing distance
from the growth plate. This so-called “metaphyseal profile” can be used to describe the morphology
of the metaphysis graphically and was used to compare the profiles of different animals treated with
bone-active agents. Other similar graphs where cortical bone area, trabecular and cortical BMC or
trabecular and cortical BMDv are plotted against distance from the growth plate can be used to focus
on growth and remodelling of the bone.
7
Responses of the metatarsus to workload
Diaphysis and epiphysis
Most of the research done thus far has focused on either the diaphysis or the epiphysis responses to
workload.
Some of the bone parameters in Mc3 of Thoroughbred horses had a significantly different response
in exercised and non-exercised groups. (E. C. Firth, Rogers, Doube, & Jopson, 2005)The bones of
horses exposed to initial two year old training for racing were larger in BMC, cortical BA and BMDv
than the bones of untrained horses. The architecture of the bones also differed: the bones of the
exercised group were less porous in the dorsal, dorso-medial and dorso-lateral aspects, in the nonexercised group the cortical porosity was evenly distributed. In the exercised group more new bone
formation was found at the dorsal aspect of the third metacarpal (Mc3) bone.
In the same Thoroughbred population, the density of the epiphysis was measured in the Mc3 of
trained and untrained horses. (E. C. Firth, et al., 2005) In the trained horses, the density of the
epiphysis was 37% greater than in the untrained group. The effect of training accounted for the
greatest amount of variation found in the density of the epiphysis.
Metaphysis
The proximal metaphysis of the metacarpus of the same population of thoroughbreds has been
studied (E.C. Firth, Rogers, & Van Weeren, Not published yet). These results might not be applicable
to the distal metacarpus because the proximal Mc3 closes before birth, and the distal metaphysis
closes around 15 months of age. (Ellenport, Hillmann, & Ghoshal, 1975)
In the study of (E.C. Firth, et al., Not published yet) the trabecular bone in the proximal metaphysis
of Mc3 had a significantly lower BMDv in the trained group. Most likely this was due to the relatively
large increase in bone area in comparison to the increase in BMC in the central part of the bone. In
the cortex the situation was reversed: the BMC was greater in the bones of the trained horses. The
BMDv in the cortex of the groups did not differ significantly, presumably because the BA of the
cortex was larger in the trained group, but BMC in this group was relatively more increased, leading
to no difference in BMDv. The greater stiffness of the bones due to the enlargement of the outer
cortical-subcortical bone most likely shielded the more centrally placed trabecular bone, resulting in
less strain, and thus a lower trabecular BMDv.
Age related changes in the metaphysis
As in the rest of the bone, the micro-architecture of the metaphysis changes with age. Not much
research has been done on this subject in horses but (Fürst, et al., 2008) studied the architecture of
the metaphysis of the radius and tibia of horses aged four to 21 years. The number of trabeculae
decreased significantly and the distance between trabeculae increased significantly with increasing
age. The mean cortical BMD of the metaphysis increased significantly with increasing age.
Based on these findings, it was assumed that the bone trabeculae are not replaced but rather
decrease in number with age. The trabecular thickness and volume also tended to decrease,
although the changes were not significant. The horses studied by (Fürst, et al., 2008) were older than
8
the ones used in our project. Still little is known about how these parameters develop from the just
born foal to the young horse.
Some former research on the relation between bone strength and age has been done without the
use of CT but with the use of three-point bending tests.(Lawrence, et al., 1994) The bone was tested
as the structure as a whole and a force was applied to the intact bone until failure. In this way a
breaking load was defined as the force required to break the bone. The BMC was also measured but
was obtained from bone cross-sections that were ether-extracted and ashed instead of using pQCT.
Findings here were that the breaking load increased with age and peaked at an age of 4.6 ± 1.8 years.
The BMC increased with age as well and reached its maximum at 6.0 ± 1.8 years.
Research objectives
The objectives of this project were to obtain more information about how the metaphyseal region in
the horse develops during early life and what influence initial training for racing has on the growth
and development of the metaphysis. This was obtained by comparing graphs of the parameters BMC,
BA and BMDv of the Mt3 of trained to untrained 2 year old Thoroughbred horses. In these graphs,
slices at increasing distances from the metaphyseal growth plate were compared between the two
groups. This way of imaging allowed to compare the whole metaphyseal area between individuals
instead of comparing only specific measuring points.
The hypothesis was that the measured parameters BMC, BA and BMDv changed due to training.
Trained horses will have a higher trabecular and cortical BMC, BA and BMDv.
To study the development of the physis near the time of birth, the metaphyseal profiles of still-born
foals were studied and compared with those of the 2 year old horses.
The hypothesis for this comparison was that the foal metaphysis was different from the metaphysis
of 2-year old horses for the three parameters (BMC, BA and BMDv) measured. Foals will have a
greater trabecular BMC and trabecular area on total area and a smaller cortical BMC and cortical
area on total area in comparison to the 2-year-old horses. The BMDv will be smaller in both the
trabecular and the cortical bone of the foals compared to the 2-year-old horses.
9
Material and methods
The MUGES population
The material used in this study was part of a larger trial which had investigated the effect of early
race training on the musculoskeletal system of 2 year old Thoroughbred racehorses, described in
detail previously (E. C. Firth & Rogers, 2005; E. C. Firth, Rogers, Perkins, Anderson, & Grace, 2004).
A group of fourteen Thoroughbred fillies was studied, all of which except one were from the same
sire. They were identically raised at pasture on the same farm.
The study was non-randomised as the horses that underwent training were chosen by the trainer.
Seven of the horses were trained on grass and sand training tracks. This group was compared with
seven horses kept in yards without specific training. The horses were trained for 13 weeks, 6 days
per week, to a stage at which they were ready to participate in race trials. Distance and time worked
were recorded every day for each horse. Briefly, the exercise regimen consisted of 4 weeks slow
cantering, then 4 weeks fast cantering, followed by 5 weeks cantering with gallops superimposed
twice weekly. The amount of training was similar in all seven trained horses until galloping began.
Four of the seven horses (the high exercise intensity subgroup) completed the entire 13-week
programme; the other three trained horses formed the medium exercise intensity subgroup.
Horses were euthanised at least 13 weeks after training began. The bones were dissected free of
skin and tendons and were stored at –20oC.
Foals
The Mt3 of six foals born on several farms in New Zealand in the Palmerston North area were
studied. The foals were all still-born during September and October 2009, after a full-term gestation
length.
After post-mortem the bones of the foals were dissected free from bone and tendon. The bones of 5
foals were stored at -20oC, and those of one foal were stored in alcohol.
Scanning the bones
The Mt3 of each horse was scanned using a pQCT scanner (XCT2000 Stratec Medical, Pforzheim,
Germany). Twenty-five slices (thickness 2 mm, voxel size 0.4mm) with a 2mm distance between each
slice were taken, thus covering a distance along the metaphysis of 100mm.
To determine the site of the first slice a scout view was made so that the gross contour and features
of the bone could be viewed. The first slice was taken in the part of the epiphysis where the bone
was the widest. Because this was a very approximate way of defining were to start, the separate
slices were viewed after the scanning was finished. The slice then named as “MET 1“ was the first
slice with a purely elliptical shape and no presence of the formina of the blood vessels that supply
the growth plate.
Using the manufacturer’s software, the following parameters were determined in each slice: BMC,
BA and BMDv.
10
To distinguish cortex from trabecular bone “peel mode” approached every slice from the outside,
defined the outside of the bone as voxels having a density of > 280 mg/ cm3 and identified all the
bone it came across as cortex till it reached bone with a density of <710 mg/cm3. Voxels below this
threshold, in the metaphysis situated more centrally in the bone slice, were identified as trabecular
bone.
Statistical analysis
The parameters of interest (BMC, BA, BMDv) were obtained as an electronic extract using the
manufacturers software. After collation within Microsoft Excel the data were examined descriptively
within SPSS v 16 (SPSS Chicago, Il, USA).
To compare the graphs more specifically they were divided in four areas (MET1-6, MET7-12, MET1318 and MET19-23). The measured values of all the slices in one group were added and these derived
“area under curve” (AUC’s) were compared between treatment groups.
Differences in AUC’s between groups were examined using a general linear model. Within the model
AUC and treatment group were considered as fixed effects. Bodyweight at the time of tissue harvest
was included as a covariate in comparison of trained and untrained horses but not in the comparison
of foals to 2-year-old horses. For all statistical procedures the level of significance was set at p<0.05.
11
Results
Trained versus Untrained Horses
BMC
Trabecular BMC in μg
1200000
1000000
800000
600000
400000
200000
MET1
MET2
MET3
MET4
MET5
MET6
MET7
MET8
MET9
MET10
MET11
MET12
MET13
MET14
MET15
MET16
MET17
MET18
MET19
MET20
MET21
MET22
MET23
0
Metaphyseal slice
Figure 3 Trabecular BMC of Mt3 of Trained and Untrained Horses. = Untrained, =Trained
1200000
Cortical BMC in µg
1000000
trend
800000
600000
400000
200000
MET1
MET2
MET3
MET4
MET5
MET6
MET7
MET8
MET9
MET10
MET11
MET12
MET13
MET14
MET15
MET16
MET17
MET18
MET19
MET20
MET21
MET22
MET23
0
Figure 4 Cortical BMC of Mt3 of Trained and Untrained Horses. = Untrained, =Trained. Trend: 0,05<p<0,1
The trabecular and cortical BMC of the Mt3 of trained and untrained horses is shown in Figures 3
and 4. The slice MET1 represents the slice that was closest to the growth plate, but did not include
part of the growth plate. The shape of the BMC plots demonstrates the transition of the slices from
the metaphyseal to the diaphyseal region.
There were no significant differences between the trained and untrained horses for either cortical or
trabecular BMC, with both treatment groups having similar profiles. In reflection of the anatomy of
the region examined, the BMC of the trabecular area was highest in the slices closest to the growth
12
plate and declines towards the diaphysis. The BMC of the cortical area increased towards the
diaphysis.
At the AUC closest to the growth plate there was a trend of a higher cortical BMC in the trained
horses.
Area
Trabecular area in mm3
1400
1200
1000
800
600
400
200
MET1
MET2
MET3
MET4
MET5
MET6
MET7
MET8
MET9
MET10
MET11
MET12
MET13
MET14
MET15
MET16
MET17
MET18
MET19
MET20
MET21
MET22
MET23
0
Metaphyseal slice
Figure 5 Trabecular area of Mt3 of Trained and Untrained Horses. = Untrained, =Trained.
1400
Cortical area in mm3
1200
1000
800
600
400
200
MET1
MET2
MET3
MET4
MET5
MET6
MET7
MET8
MET9
MET10
MET11
MET12
MET13
MET14
MET15
MET16
MET17
MET18
MET19
MET20
MET21
MET22
MET23
0
Figure 6 Cortical area of Mt3 of Trained and Untrained Horses. = Untrained, =Trained
The area of the cortex and the cancellous bone is presented in Figures 5 and 6. The shape of the
graphs of both treatment groups was similar. The trabecular area values decrease as the slices
progress towards the diaphyseal region. In contrast the cortical area increases with the progression
towards the diaphyseal region. There were no significant differences in cortical or trabecular area
between the two treatment groups.
13
BMDv
Trabecular BMDv in mg/cm3
600.0
500.0
400.0
300.0
200.0
100.0
MET1
MET2
MET3
MET4
MET5
MET6
MET7
MET8
MET9
MET10
MET11
MET12
MET13
MET14
MET15
MET16
MET17
MET18
MET19
MET20
MET21
MET22
MET23
0.0
Figure 7 Trabecular Density of Mt 3 of Trained and Untrained Horses. = Untrained, =Trained
Cortical BMDv in mg/cm3
1200.0
*
1100.0
1000.0
900.0
*
800.0
700.0
MET1
MET2
MET3
MET4
MET5
MET6
MET7
MET8
MET9
MET10
MET11
MET12
MET13
MET14
MET15
MET16
MET17
MET18
MET19
MET20
MET21
MET22
MET23
600.0
Figure 8 Cortical Density of Mt3 of Trained and Untrained Horses. = Untrained, =Trained. *: p<0,05
The trabecular and cortical density of the various slices of the Mt3 are presented in Figure 7 and 8.
The plots of the trained and the untrained horses were similar for both the trabecular and cortical
density. There was no significant difference between the trained and untrained horses at any AUC
for trabecular density. In contrast the plots of the cortical bone differed significantly at all AUC’s.
High and Medium exercise intensity subgroups versus Untrained Horses
In the graphs shown above it was prominent that variation between horses was much larger in the
group trained animals than in the group untrained animals, indicated by the larger error bars in the
graphs of the trained horses. This was most likely due to the different amount of exercise the horses
in the trained group did because of lameness in three of them at various stages of the training
programme. (E. C. Firth, et al., 2004). To examine the effect of the amount of training on the
different parameters in more detail, the trained horses were divided in a “high exercise intensity
subgroup” which finished the whole training programme and the “medium exercise intensity
14
subgroup” which consisted of horses who became lame somewhere during the programme. For
more details see (E. C. Firth, et al., 2004).
BMC
Trabecular BMC in µg
1200000
1000000
800000
600000
400000
200000
MET1
MET2
MET3
MET4
MET5
MET6
MET7
MET8
MET9
MET10
MET11
MET12
MET13
MET14
MET15
MET16
MET17
MET18
MET19
MET20
MET21
MET22
MET23
0
Figure 9 Trabecular BMC of Mt3 of Trained and Untrained Horses. = Untrained, =Medium exercise intensity
subgroup =High exercise intensity subgroup. *: p<0,05 Between High exercise intensity subgroup and Untrained
horses.
1200000
Cortical BMC in µg
1000000
800000
600000
400000
200000
MET1
MET2
MET3
MET4
MET5
MET6
MET7
MET8
MET9
MET10
MET11
MET12
MET13
MET14
MET15
MET16
MET17
MET18
MET19
MET20
MET21
MET22
MET23
0
Figure 10 Cortical BMC of Mt3 of Trained and Untrained Horses. = Untrained, =Medium exercise intensity subgroup
=High exercise intensity subgroup. *: p<0,05 Between High exercise intensity subgroup and Untrained horses. Trend:
0,05<p<0,1 Between Medium exercise intensity subgroup and Untrained horses.
The trabecular and cortical BMC of untrained horses and horses of the medium and high exercise
intensity subgroup are shown in Figure 9 and 10. In the trabecular area there were no significant
differences between the medium exercise intensity subgroup and the untrained horses but in the
cortex there was a trend towards a higher BMC for the medium exercise intensity subgroup in all
AUC’s. When comparing the bones of the horses in the high exercise intensity subgroup with the
untrained horses the BMC was significantly higher in almost all AUC’s. Only in the trabecular area
closest to the diaphysis no difference in BMC was found.
15
Area
Trabeucalr area in mm3
1600
1400
1200
1000
800
600
400
200
MET1
MET2
MET3
MET4
MET5
MET6
MET7
MET8
MET9
MET10
MET11
MET12
MET13
MET14
MET15
MET16
MET17
MET18
MET19
MET20
MET21
MET22
MET23
0
Figure 11 Trabecular area of Mt3 of Trained and Untrained Horses. = Untrained, =Medium exercise intensity
subgroup =High exercise intensity subgroup. *: p<0,05 Between High exercise intensity subgroup and Untrained
horses. Trend: 0,05<p<0,1 Between High exercise intensity subgroup and Untrained horses.
1600
1400
1200
800
600
400
200
0
MET1
MET2
MET3
MET4
MET5
MET6
MET7
MET8
MET9
MET10
MET11
MET12
MET13
MET14
MET15
MET16
MET17
MET18
MET19
MET20
MET21
MET22
MET23
Cortical area in mm3
1000
Figure 12 Cortical area of Mt3 of Trained and Untrained Horses. = Untrained, =Medium exercise intensity subgroup
=High exercise intensity subgroup. *: p<0,05 Between High exercise intensity subgroup and Untrained horses. Trend:
0,05<p<0,1 Between High exercise intensity subgroup and Untrained horses.
The area of the cortex and the cancellous bone of untrained horses and horses of the high and
medium exercise intensity subgroup is presented in Figures 11 and 12. Again there were no
differences between the medium exercise intensity subgroup and the untrained horses, but in all
areas of the metaphysis a difference between the high exercise intensity subgroup and the
untrained horses was found. In the trabecular area closest to the diaphysis and in the cortical area
consisting of the slices MET7-12 this difference was only a trend.
16
BMDv
No differences were found when comparing the trabecular density of horses of the medium and
high exercise intensity subgroup and untrained horses. In the cortex there were no significant
differences between the high and medium exercise intensity subgroup apart from the area that
consisted of slices MET13-18 where a significant lower cortical density was found in the medium
exercise subgroup horses. Both groups of trained horses had a higher cortical density than the
untrained horses. In the AUC closest to the growth plate this difference was only a trend for both
trained groups.
Foals versus 2 year old Horses
500
450
400
350
300
250
200
150
100
50
0
MET1
MET2
MET3
MET4
MET5
MET6
MET7
MET8
MET9
MET10
MET11
MET12
MET13
MET14
MET15
MET16
MET17
MET18
MET19
MET20
MET21
MET22
MET23
Trab. BMC/Total area in μg/mm3
BMC
1000
900
800
700
600
500
400
300
200
100
0
MET1
MET2
MET3
MET4
MET5
MET6
MET7
MET8
MET9
MET10
MET11
MET12
MET13
MET14
MET15
MET16
MET17
MET18
MET19
MET20
MET21
MET22
MET23
Cortical BMC / Total area in
μg/mm3
Figure 13 Trabecular BMC/Total area of Foals and 2 year old Horses. = 2 year old Horses, =Foals *: p<0,05
Figure 14 Cortical BMC/Total area of Mt3 of Foals and 2 year old Horses. = 2 year old Horses, =Foals *: p<0,05
The trabecular and cortical BMC on total area of foals and 2 year old untrained horses is shown in
Figures 13 and 14. In the trabecular area closest to the growth plate the graph of the foals had a
different shape than that of the 2 year old horses but no significant difference between treatment
groups was found in this AUC. In the AUC’s more towards the diaphysis the trabecular BMC on total
area of the foals was significantly greater than that of the 2 year old horses.
17
The shape of the plots of cortical BMC on total area were similar for foals and 2 year old horses. The
cortical BMC on total area was significantly smaller for all AUC’s in the foals.
Area
1
Trabecular area/ Total area
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
MET23
MET22
MET21
MET20
MET19
MET18
MET17
MET16
MET15
MET14
MET13
MET12
MET11
MET10
MET9
MET8
MET7
MET6
MET5
MET4
MET3
MET2
MET1
0
Figure 15 Trabecular area/Total area of Foals and 2 year old Horses.= 2 year old Horses, =Foals *: p<0,05
1
Cortical area/Total area
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
MET23
MET22
MET21
MET20
MET19
MET18
MET17
MET16
MET15
MET14
MET13
MET12
MET11
MET10
MET9
MET8
MET7
MET6
MET5
MET4
MET3
MET2
MET1
0
Figure 16 Cortical area/ Total area of Foals and 2-year-old Horses. = 2-year-old Horses, =Foals *: p<0,05
The cross-sectional area of the cortical and trabecular bone divided by the total cross-sectional area
is presented in Figures 15 and 16. The shape of the graphs of both treatment groups were similar but
their position on the y-axis differed. The ratio of trabecular area and total area of the foals was
significantly greater in all AUC’s. On the other hand, in the cortical bone, the ratio of cortical area
and total area of the foals was significantly smaller in all AUC’s.
18
BMDv
Trabecular BMDv in mg/cm3
1200.0
1000.0
800.0
600.0
400.0
200.0
MET1
MET2
MET3
MET4
MET5
MET6
MET7
MET8
MET9
MET10
MET11
MET12
MET13
MET14
MET15
MET16
MET17
MET18
MET19
MET20
MET21
MET22
MET23
0.0
Figure 17 Trabecular density of Foals and 2 year old Horses = 2 year old Horses, =Foals *: p<0,05
Cortical BMDv in mg/cm3
1200.0
1000.0
800.0
600.0
400.0
200.0
MET1
MET2
MET3
MET4
MET5
MET6
MET7
MET8
MET9
MET10
MET11
MET12
MET13
MET14
MET15
MET16
MET17
MET18
MET19
MET20
MET21
MET22
MET23
0.0
Figure 18 Cortical Density of Foals and 2 year old Horses. = 2 year old Horses, =Foals *: p<0,05
The trabecular and cortical density of the Mt3 of foals and untrained 2 year old horses are presented
in Figure 17 and 18. The shapes of the graphs differed in the area closest to the growth plate. In the
trabecular bone, the measured density here was significantly higher in the 2 year old horses than in
the foals. The cortical bone of the foals was significantly less dense in all AUC’s. In the region closest
to the growth plate, the density increased more from slice to slice in the foals, represented by a
steeper graph.
19
Discussion
Trained versus Untrained Horses
BMC
The BMC of the trabecular bone decreased with the progression towards the diaphyseal region and
became almost zero in the slices furthest away from the growth plate. In this region the cortical BMC
was the greatest, indicating that the cortex gives the Mt3 its strength in the more diaphyseal part of
the metaphysis while the trabecular bone does so in the area closest to the growth plate.
There was no significant difference in BMC between treatment groups when comparing all the
trained horses with the untrained horses. However, when comparing only the high exercise intensity
subgroup with the untrained horses, a significant difference in all areas apart from the trabecular
area closest to the growth plate could be seen. This indicates that only in the horses that completed
the training programme, the stimulus was strong enough to increase the BMC of the metaphysis.
Area
The plots (Figures 5, 6, 11 and 12) of the trabecular and cortical bone area had the same shape in
both treatment groups and presented nicely how the shape and constitution of the bone changed
from the metaphyseal to the diaphyseal region.
There was no difference in size of the trabecular or cortical area between the medium exercise
intensity subgroup and untrained horses. On the other hand, there was a difference between the
high exercise intensity subgroup and the untrained horses in all areas of the cortical and trabecular
bone. This indicates that in the medium exercise subgroup the stimulus was not high enough to
trigger the bone to increase in cross-sectional area but that this was the case in the high exercise
subgroup. From previous research on the same Mt3 by (E. C. Firth, et al., 2005) it is known that the
diaphysis responded to exercise in the same way.
BMDv
There was a clear response of the cortical bone on the forces experienced by the bone during
exercise. The density of the cortex of the Mt3 was higher in all AUC’s when comparing both the
medium and high exercise intensity subgroups to the untrained horses. In contrast, in the trabecular
part of the very same bones no significant effect was observed. This illustrates the strategy of the
bone in dealing with the higher physical stress applied by the training. Instead of strengthening the
whole bone, effectively only the cortex is strengthened and in this way takes over the extra load.
Dividing the horses in medium and high exercise intensity subgroups gave more insight in how the
actual amount of exercise influences the reaction of the bone. It appears that galloping, which hardly
happened in the medium exercise group, but twice a week for five weeks in the high exercise
subgroup is an important factor in the response of the bone to exercise. However, the groups in this
study were very small (n=3 for the medium exercise intensity subgroup and n=3 for the high exercise
intensity subgroup) which might be one of the reasons why no response to training was seen in the
medium exercise intensity subgroup.
20
If more of the parameters measured could be analysed this would probably give a better
understanding of how exactly the bone responds. Interesting parameters would be the Strength
Strain Index (SSI) to measure real bone strength as well as Periosteal Circumference (PeriC) and
Endosteal Circumference (EndoC) to see if the measured BA increased by increase of the size of the
whole bone or by increasing the number or the thickness of the trabeculae and narrowing of the
pores in the cortical bone.
The chosen way to compare the slices (by defining “MET 1” by looking at the shape of the slices and
the position of the blood vessels) has given a satisfactory result. However, this method is not
absolutely accurate as the slices have a thickness of 2mm and the space between slices was 2mm as
well. In this way the actual areas that were compared could be several millimetres apart.
Foals versus 2 year old Horses
BMC
The trabecular BMC on total area of the foals was higher while the cortical BMC on total area was
lower in the foals compared to the 2 year old horses. In the foal there was thus less difference
between cortex and trabecular bone which made the bone a more plastic structure which is able to
accomplish rapid growth. However, in adult bones, the thicker, denser cortex is designed to deal
efficiently with the large forces that affect the third metatarsal bone.
In the area closest to the growth plate, the graphs of trabecular BMC had different shapes for the
foals and the 2 year old horses. The BMC on total area started lower in the foals than in the control
horses, then increased and decreased again towards MET 7. Most likely, the low values in the slices
adjacent to the growth plate were measured because parts of the growth plate cartilage were
included and measured as trabecular bone here. The growth plate of foals is very uneven and parts
of it will have been part of the slices closest to the growth plate.
Area
The foal bones had a larger trabecular area on total area and a smaller cortex on total area than the
2 year old horses. The cortex of foals was also less dense than that of the 2 year old horses. This
illustrates how the growing long bone differs from bone where the growth plate has already closed.
In the metaphyseal area of the foal bone there is constant formation of new trabecular bone.
Towards the diaphysis the diameter of the bone has to become smaller. As the bone grows in length,
bone is added on the inside of the cortex and resorbed on the outside. In this way, the diameter of
the bone decreases towards the diaphysis. If the cortex of the foal bone was thicker and denser,
more bone would have to be added on the inside and resorbed at the outside and rapid growth
would not be possible.
BMDv
The trabecular density was similar in the slices MET7 to MET23 for foals and 2 year old horses. In
contrast, in the slices closest to the growth plate, the density was significantly lower in the Mt3 of
the foals. When assessing the function of this area in the foals this is logical: in the area of the
growth plate the trabecular bone is just formed and not yet ossified while in 2 year old horses the
growth plate is closed and ossified to strengthen the bone.
21
In the cortical region closest to the growth plate a density of < 710 mg/cm3 was measured. This was
unexpected because it is below the threshold used for defining the cortex. Most likely these low
values were due to the way the scanner software differentiated between cortex and trabecular bone.
In the foal bones were there is almost no cortex close to the growth plate, the software still defines
a cortex, even if the density is <710 mg/cm3.
Use of the graphs and the metaphyseal profile
In the graphs used, the relationship between the values BMC, BA, and BMDv, on the y-axis and the
metaphyseal slices on the x-axis was shown. These graphs give at a glance a graphic record of the
composition of the metaphysis. Because the metaphysis in young animals is a dynamic structure
with a different composition in every location, this graphic presentation is very suitable for
examining this part of the bone. With control graphs of normal horses, the influence of internal and
external factors on the metaphysis can be examined by comparing the different plots.
For the metaphyseal profiles made from the foals and 2-year-old horses (thus the ratio Trabecular
area/Total area to distance from the growth plate) it would be useful to study more age groups and
a higher number of bones in future research. In this way a “normal” metaphyseal profile could be
obtained for different ages, and individual horses or groups could be compared with this control
profile.
Conclusions
It was possible to compare trained and untrained horses by comparing graphs that plot BMC, BA or
BMDv against distance from the growth plate.
The metaphysis of the Mt3 of the medium exercise subgroup did only respond significantly on the
given exercise by increasing the cortical density. There was a trend towards a greater cortical BMC in
this group. On the other hand, the Mt3 of the high exercise intensity subgroup responded not only
by increasing the cortical density, but also by increasing the cortical and trabecular area and cortical
and trabecular BMC.
The metaphyseal profile was suitable in comparing the different parameters between foals and 2
year old horses. In the foals, the trabecular BMC and area on total area were greater and the cortical
BMC and area on total area were smaller than in the two year old horses. The BMDv in the cortex
was higher in the 2 year old horses. No difference in trabecular density was measured, apart from in
the area closest to the growth plate, were the trabecular density of the foals was smaller.
22
Acknowledgements
Special thanks go to Prof. Dr. P.R. Van Weeren (Utrecht University, The Netherlands) for his efforts
which made it possible for me to do this research project at Massey University (New Zealand). I also
want to thank my supervisors Prof. Elwyn Firth and Dr. Chris Rogers from Massey University for all
their help, support and good ideas and for the great time I had at Massey.
Furthermore I want to thank my fellow student Nynke Wester for helping me with the dissections of
the foal legs and Daan Sprünken for all the usefull criticism and reading my report.
23
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