Effects of a Synbiotic Diet on Femur Bone
Structure in Aging Mice
Tina Wilson, Maureen Choman, Annette Gabaldón
Colorado State University-Pueblo, Department of Biology
Senior Honors Thesis
HONORS 481
Background:
Inside the gastrointestinal tract, live bacteria break down ingested substances that
the host cannot degrade any further. The bacteria receive food from the host, and
the host receives beneficial products from the bacteria. By breaking down what the
body cannot, both parties benefit. The term “prebiotics” refers to specific food for
the GI tract bacteria, and “probiotics” refer to the actual bacterial species. When
combined, the two terms are coined into what we know as “synbiotics”. The overall
concept with synbiotics involves finding the ideal bacteria with its ideal nutrients to
produce the best benefits for the host. It’s been discovered that nondigestible
oligosaccharides have beneficial effects on both bone structure as well as mineral
metabolism and absorption. The benefits include higher gut absorption levels of
calcium [3].
Regarding the mechanism of how it’s done, the bacterial probiotics produce a
short-chain fatty acid product in the gut of the host animal (Figure 1). This product
then promotes the increased level of mineral absorption by promoting the
production of enterocytes, which are intestinal absorption cells. With the increased
expression of enterocytes as well as calcium-binding proteins, more minerals are
absorbed, the health of the gut is improved, and the body is enabled to maintain
stronger bone structure with the supply of calcium [3]. The increase in calcium
absorption levels has been reported to coincide with an increase in bone density
values [4].
Synbiotic mechanism
Synbiotic mechanism
Probiotic bacteria
make short-fatty
acid chain
Probiotic
bacteria
products
make short-fatty
acid chain
products
Promotion of
enterocytes
Promotion of
enterocytes
Higher calcium
absorption in the
gut
Higher calcium
absorption in the
gut
Figure 1: The basic outline of the synbiotic mechanism, which involves: 1) probiotic bacteria producing
short-fatty acid chain products inside the host gut, 2) the probiotic products then promoting the
production of more enterocytes, and 3) the enterocytes helping to increase calcium absorption levels.
Diving further into the details of the mechanism, the bacterial probiotics produce a
phytase enzyme that is used to “free” up more minerals from phytate bondage for
absorption. The bacteria are also able to hydrolyse glycoside bonds in nutrients that
cannot be broken down by the intestines of the host. By breaking down the
nutrients that the host cannot, both the probiotic bacteria and the host animal
benefit with the increased availability of minerals [2].
The basic structure of the femur bone involves two parts, the cortical wall
and the medullary cavity (Figure 2). If the bone is compared to a metal pipe, the
cortical wall is the wall of the tube and the medullary cavity is the marrow-filled
space made by the shaft. With aging, it’s common for the cortical wall of long bones
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to be whittled away bit by bit. As the bones age, resorption occurs at the endosteal
(inner) surface of the bone and if not compensated for by bone deposition at the
periosteal (outer) surface, then cortical wall thinning may result.
Cortical bone
Periosteal
surface
Central Medullary
Cavity
Endosteal surface
Figure 2: Comparison of a cross sectional view of a long bone with that of a metal pipe. The outer surface
of the metal pipe would be the periosteal surface and the inner surface would be the endosteal surface.
The wall of the pipe would be the cortical bone and the space inside the pipe would be the central
medullary cavity. The circular figure to the right depicts what the long bone would look like if the central
medullary cavity expanded but no bone deposition occurs at the outer periosteal surface. This is
referred to as “cortical wall thinning”.
To determine which femur would be stronger, the cross sectional areas of the
cortical wall were measured to see which bone would have the thickest wall and the
smallest cavity. To examine the effects of age, a baseline group will be compared
directly with an aged control group. To examine the effects of synbiotics, a baseline
group will be compared with a test group given a synbiotic diet to see if the
probiotic bacteria will induce any reversing effects on the expanding central
medullary cavity to protect the thinning of the cortical bone wall.
Objectives:
The main objective of this subproject was to characterize the effects that both age
and synbiotics would have on the porosity of the cortical wall and central medullary
cross sectional area (CSA). The secondary objective is to see if the synbiotic diet
(SYN test groups) shows a reduction in any age-related porosity of the femur bone
wall. For this study, the 30% region of the femur bone shaft was examined due to
the greater presence of pores in that area when compared to the 50% and 70%
regions.
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Hypothesis:
By comparing the control (CON) and the synbiotic (SYN) groups against the baseline
(BSL) group, the data will show the aftermath of two key factors: 1) what effects age
may/may not have on the cortical pore CSA and the central medullary cavity CSA,
and 2) what effects the synbiotic diet may/may not have on the cortical pore CSA
and the central cavity medullary CSA. I hypothesize that the data to support that the
synbiotic diet will have beneficial effects on the cortical bone wall of the femur bone
shaft.
Materials & Methods:
IP 1: Animal Care and Experimentation Protocol
Male albino mice at 10 months of age were separated into three test groups (Figure
3). The first group was the Baseline (BSL) group, where the mice would immediately
be euthanized. The second group was the Control (CON) group, where the mice
were fed a controlled diet of standard mouse food. The third group was the
Synbiotic (SYN) group, where the mice were being provided the controlled diet
enhanced with synbiotics. The synbiotic diet included fructooligosaccharides as a
prebiotic as well as Lactococcus lactic lactis and Lactobacillus acidophilus for the
probiotics. Both the controlled diet and synbiotic diets were equal in caloric quality.
The CON and SYN were fed daily over the course of 4 months; at the end of which,
the mice were euthanized. All experiments and housing of mice were conducted at
Idaho State University, Pocatello, Idaho. The protocol was reviewed and approved
by the Idaho State University IACUC (Institutional Animal Care and Use Committee).
Veterinary care and health monitory was provided on a regular basis. Euthanasia
was performed in accordance with American Veterinary Medical Association
(AVMA) guidelines. The research was in part by an Institutional Grant awarded to
Dr. Cynthia Blanton (Idaho State University) and by an Institutional SEED grant
awarded to Dr. Annette Gabaldón (CSU-Pueblo).
Baseline
Control
Synbiotic
10 months
n = 10 mice
14 months
n = 10 mice
14 months
n = 10 mice
Standard diet
Standard diet
Synbiotic diet
Figure 3: The Baseline (BSL) mice were labeled as BSL 1 through BSL 10, the Control (CON) mice were
labeled as CON 8 through CON 17, and the Synbiotic (SYN) mice were identified as SYN 1 through SYN 10.
At the end of the 4-month feeding period, the femur bones of the euthanized mice
were dissected, cleaned of adhering tissues, and scanned by a micro-computed
tomography system.
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7 scans at 30% region
Figure 4: The full vertical view of a baseline femur from the experiment. With the NHI ImageJ software
program, 7 scans at the 30% region were examined for each bone.
IP 2: micro-CT scanning and ImageJ Analysis
The NIH ImageJ software program was used to measure the total medullary cavity
cross sectional area of the sequential bone slices at the 30% proximal region of the
femur bones. To start, the ImageJ program was activated and a file containing the
bone sequence of a particular femur was imported. The brightness/contrast setting
was reset for clarity, and the zoom setting was set to 300% for measurement ease
(Figure 5).
Figure 5: Baseline Femur 1 before brightness/contrast adjustment is shown to the left. Baseline
Femur 1 after brightness/contrast adjustment is shown to the right, where the central medullary
cavity as well as the dim presence of pores can be seen in the cortical bone wall.
The 30% region was calculated by taking the last slide where bone was clearly
visible, multiplying that slide number by 0.3, then adding the slide number where
bone was first visible. Once the 30% region was calculated, 3 values of -5 intervals
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as well as 3 values of +5 intervals were included to provide a well-rounded average
for the 30% region. Back to the scan, the measurements were set to record the
perimeter and the area. The measurement scale units were set to be at 25.595
pixels/mm for every file. Once this was complete, the drawing tools of the ImageJ
program were used to outline the pores present and the total area of all pores were
recorded for each slide (Figures 6 and 7).
Figure 7: A close-up image of Baseline Femur 1. The red outline depicts the cortical pores and the blue
outline surrounds the central medullary cavity of the bone scan. The red outline of the pores shows how
the areas of the porous regions were profiled and measured.
By taking the total pore area for each slide, the average pore area of the 7 slides
provided a well-rounded average value of the pore area within that femur bone.
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Once the average pore areas were obtained, the values were subtracted from the
original cortical bone cross sectional area and added onto the medullary cavity cross
sectional areas for each respective bone.
To find more detail regarding the change in the femur bone’s structure due to both
age and diet, the cross sectional area for the main prominent medullary cavity was
determined by first, determining the CSA for the pores present in the cortical bone
wall, then subtracting that value from the total medullary cavity CSA values from the
original study conducted. Equation 1 below simplifies the concept.
𝑻𝒐𝒕𝒂𝒍 𝑴𝒆𝒅𝒖𝒍𝒍𝒂𝒓𝒚 𝑪𝑺𝑨 − 𝑪𝒐𝒓𝒕𝒊𝒄𝒂𝒍 𝑷𝒐𝒓𝒆 𝑪𝑺𝑨 = 𝑪𝒆𝒏𝒕𝒓𝒂𝒍 𝑴𝒆𝒅𝒖𝒍𝒍𝒂𝒓𝒚 𝑪𝒂𝒗𝒊𝒕𝒚 𝑪𝑺𝑨
Equation 1
Results:
The central medullary cavity extended from the proximal to the distal ends of the
femur shaft for the three test groups. With age, it’s common for the cortical wall of
the bone to be whittled away bit by bit. So by comparing the CON and the SYN
groups against the BSL group, the data would show the aftermath of two key factors:
1) what effects age may/may not have on cortical bone density and central
medullary cavity CSA, and 2) what effects the synbiotic diet may/may not have on
the cortical bone density and the central cavity medullary CSA. The following bones
are currently being studied and therefore the respective data have not been
included in this paper: BSL 4, BSL 9, CON 8, CON 9, CON 10, CON 17, and SYN 3. On a
side note, SYN 3 was purposefully excluded due to the extensive damage of the
femur bone condyle. The breaking of the SYN 3 femur bone was after the study and
was unrelated to the experiment.
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Table 1 The total medullary CSA values for 30% femur bone regions of BSL, CON, and SYN test groups.
Total Medullary CSA
Femur
Med CSA (mm2)
Med SD
BSL 1
0.892
0.022
BSL 2
0.668
0.071
BSL 3
0.887
0.043
BSL 5
0.49
0.027
BSL 6
0.88
0.033
BSL 7
0.646
0.056
BSL 8
0.635
0.092
BSL 10
1.302
0.031
Average
0.8
0.047
Femur
Med CSA (mm2)
Med SD
CON 11
1.116
0.067
CON 12
0.926
0.061
CON 13
0.593
0.065
CON 14
0.699
0.031
CON 15
1.011
0.05
CON 16
0.923
0.05
Average
0.878
0.054
Femur
Med CSA (mm2)
Med SD
SYN 1
1.034
0.059
SYN 2
0.928
0.104
SYN 4
1.185
0.054
SYN 5
1.084
0.064
SYN 6
0.989
0.046
SYN 7
0.617
0.063
SYN 8
0.656
0.064
SYN 9
0.989
0.102
SYN 10
0.591
0.124
Average
0.897
0.076
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Table 2: The cortical pore CSA values at 30% femur bone regions for BSL, CON, and SYN test groups.
Cortical Pore CSA
Femur
BSL 1
BSL 2
BSL 3
BSL 5
BSL 6
BSL 7
BSL 8
BSL 10
Average
Average pore area (mm2)
0.023
0.012
0.066
0.039
0.049
0.023
0.022
0.015
0.031125
Femur
CON 11
CON 12
CON 13
CON 14
CON 15
CON 16
Average
Average pore area (mm2)
0.03
0.115
0.011
0.029
0.061
0.117
0.0605
SD
0.022216
0.059522
0.015435
0.022572
0.0205
0.018898
0.027
Femur
SYN 1
SYN 2
SYN 4
SYN 5
SYN 6
SYN 7
SYN 8
SYN 9
SYN 10
Average
Average pore area (mm2)
0.016
0.019
0.017
0.1
0.122
0.076
0.048
0.118
0.103
0.068
SD
0.012542
0.015951
0.01618
0.016682
0.030427
0.013734
0.045329
0.045091
0.018017
0.024
SD
0.725
0.007159
0.023365
0.02264
0.018662
0.010384
0.017791
0.001549
0.013
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Table 3: The central cavity medullary CSA values at 30% femur bone regions for BSL, CON, and SYN test
groups. The values shown were determined by taking the total medullary cavity CSA values from Table 1
and subtracting the cortical pore CSA values from Table 2.
Central Cavity Medullary CSA (mm2)
Femur
BSL 1
BSL 2
BSL 3
BSL 5
BSL 6
BSL 7
BSL 8
BSL 10
Average
.
Central Cavity Medullary CSA (mm2)
0.878
0.656
0.821
0.451
0.831
0.623
0.613
1.287
0.77
CON 11
CON 12
CON 13
CON 14
CON 15
CON 16
Average
1.086
0.811
0.582
0.67
0.95
0.806
0.818
SYN 1
SYN 2
SYN 4
SYN 5
SYN 6
SYN 7
SYN 8
SYN 9
SYN 10
Average
1.018
0.909
1.168
0.984
0.867
0.541
0.608
0.871
0.488
0.828
The values determined for the Central Cavity Medullary CSA, as shown in Chart 3,
represent the cross sectional area of the main cavity that is prominently seen as the
“main shaft” of the femur.
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Figure 8: The total medullary CSA values for the BSL, CON, and SYN test groups from the original study.
This chart depicts the average CSA from Table 1. The image to the right depicts the total medullary CSA,
which include both cortical pores (in orange) and central medullary cavity (in purple).
Figure 9: The cortical pore CSA values determined in this subproject study for the BSL, CON, and SYN test
groups. This chart depicts the average CSA from Table 2. The image to the right depicts the cortical pore
CSA, which is filled in with orange.
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Figure 10: The central cavity medullary CSA values determined by taking the total medullary CSA values
from Table 1 and subtracting the cortical pore CSA values shown in Table 2. The image to the right shows
the central cavity medullary CSA, which is filled in with purple.
Discussion:
Seeing as this is an ongoing subproject, the currently compiled data can only be used
to see if there are any general trends. The main study measured the total medullary
cavity CSA, but this means that when just the cavity space was being measured for
the ImageJ program would have included the pores in the cortical bone. The
subproject focused just on those cortical pores. So by taking both into account, we
can determine the central medullary cavity and what changes may or may not have
occurred to it due to age or diet.
Looking at the total medullary cavity CSA data on Figure 8, there is quite a
difference between the baseline and aged control group femurs. This suggests that
the cavities inside the bone expand with the course of age. By comparing the aged
control with the aged synbiotic femurs, there is great overlap of standard deviation.
This suggests that there is no big difference between the two. Preliminary data from
the main study shows that there are 2 general trends: 1) Age seems to have a big
impact on the size of the cavities, and 2) There appears to be no influence of the
synbiotic diet on the total medullary cavity CSA.
According to Figure 9, the cortical pores also appeared to follow the same
general trends. By comparing the baseline to the aged control, the cortical pore CSA
was doubled in just a 4-month experimental period. By comparing the aged control
to the aged synbiotic, the standard deviation overlap showed no influence of the diet
on the cortical pore CSA.
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And finally to the main focus of the study, Figure 10 shows the data of just
the central medullary cavity. By comparing the baseline to the aged control group, it
was found that age has a severe impact on the expanding size of the central
medullary cavity CSA. Within a 4-month period, the central medullary cavity CSA
had increased by 0.048-mm2. (Keeping in mind that these are femurs from mice, this
is a big increase.) By comparing the aged control with the aged synbiotic group,
there was barely a 0.010-mm2 difference, which indicates no influence of the diet on
the size of the cavity.
In conclusion, it was found that age plays a major part in the expansion of the
central medullary cavity. And while this preliminary data shows the effects of age,
without more information about the actual strength of the femurs, nothing is set in
stone. This subproject brought forth more questions than answers found. It’s rather
exciting because with just this preliminary data, more questions are leading more
subjects of future research. We aim to study the 50% and 70% regions of the bones
to see if this general trend continues down the shaft of the femur. We’re also
currently in the works of conducting a 3-point bend test to test the mechanical
strength of each femur.
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References:
1. Choman M, Blanton C, Gabaldon A. 2015. The Effect of a Synbiotic Diet on Bone
Structure in Aging Male Mice.
2. Parvaneh K, Jamaluddin R, Karimi G, Erfani R. 2014. Effect of probiotics
supplementation on bone mineral content and bone mass density. Scientific World
Journal. 2014;2014:595962. doi:10.1155/2014/595962.
3. Scholz-Ahrens K.E., Ade P, Marten B, Weber P, Timm W, Acil Y, Gluer C.C.,
Schrezenmeir J. 2007. Prebiotics, Probiotics, and Synbiotics Affect Mineral
Absorption, Bone Mineral Content, and Bone Structure. American Society for
Nutrition. 137(3): 8385-8465.
4. Weaver CM. 2015. Diet, gut microbiome, and bone health. Current Osteoporosis
Reports. 13(2): 125-130.
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