Copyright 2007 Michal A. Ruchelsman. All rights reserved.
and electronic copies of this thesis and to grant others the right to do so.
MASSACHIUSETTS INST'ITT-E
OF TECHNOILOCY
12007
Author
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Department of Mechanical Engineering
June 2007
Certified by
U
Kimberly Hamad-Schifferli
Thesis Supervisor
Accepted by
-1
I
John H. Lienhard V
Chairman, Undergraduate Thesis Committee
Research conducted at Orthopedic Biomechanics Lab, Beth Israel Deaconess Medical Center and Harvard Medical School
Work closely with Julienne Brouwers, Ph.D. Candidate, Biomedical Engineering, EUT, The Netherlands
Under supervision of lab director: Mary Bouxsein mbouxsei@bidmc.harvard.edu

( i i=.
late T'hcV.is
The Effect of Zoledronate Treatment Timing on Lumbar and Caudal Vertebrae in
Ovariectomized Rats by
Michal Ruchelsman
Submitted to the
Department of Mechanical Engineering
June 2007
In Partial Fulfillment of the Requirements for the Degree of
Bachelor of Science in Engineering
bisphosphonates in rats as either a preventative treatment or recovering treatment for osteoporosis, few have directly compared the two treatment alternatives with respect to their effects on bone microstructure and strength. This paper, then, investigates the effects of treatment timing using Zoledronate [ZOL], a potent bisphosphonate, on the lumbar and caudal vertebrae in ovariectomized [OVX], female Wistar rats.
Methods: Twenty nine rats were divided into four groups according to their treatment:
OVX at week 0 (n=5), OVX+earlyZOL (20 gg/kg s.c. week 0, n=8), OVX+late ZOL (20 gg/kg s.c. week 8, n=7), and SHAM-OVX (n=9).
Results: Micro-computed tomography (gCT) evaluation of six parameters characterizing bone morphology [BV/TV, ConnD, SMI, TbTh, TbNr, and TbSp] showed slightly favorable effects with early ZOL treatment in the fourth lumbar [L4] vertebrae.
Compared to SHAM-OVX, OVX has a significantly (p<0.
0 5 ) lower BV/TV, higher SMI, and TbSp. OVX+earlyZOL had a significantly higher BV/TV than OVX and SHAM-
OVX and a lower TbSp than OVX. Decreasing trends but no statistically significant differences were reached in the cortical thickness with treatment, nor were there any differences in bone morphology between the groups in the sixth caudal vertebrae [CD6].
A two-way ANOVA revealed an interaction between the vertebral site and treatment group for BV/TV and TbSp. jCT and static compression tests on the L3 and L4 of rats in a secondary study revealed significant correlations in architectural parameters and biomechanical properties between the two vertebrae. L4 had a higher BV/TV, SMI, and minimum area [minA] and a lower TbNr and TbSp than L3, but L4 had lower values for stiffness, energy to failure [energy], and ultimate load. Regression analysis also showed statistically significant correlations between ultimate load [Uload] and total bone volume
[BV], energy and BV, Uload and minA, stiffness and minA, and energy and minA.
treatment and demonstrated the potential for clinical advantages using preventative therapy. Upon further research in understanding the vertebrae's response to ZOL at different time points after OVX, treatment for osteoporosis may be better directed.
Thesis Supervisor: Kimberly Hamad-Schifferli
Engineering
i
2 sSUr. i~l:` ?3

Abstract ..............
Table of Contents ......
List of Figures .........
List of Tables .........
Introduction ...........
Materials and Methods .
Results ............
Discussion ............
Conclusion ...........
Acknowledgements .....
Appendix ............
References .............
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MTi ch"l Rucel s:

Figure 1: Chemical structure of Zoledronate
Figure 2: Representative lumbar vertebra
Figure 3: piCT manual selection of trabecular bone
Figure 4: ptCT evaluation of trabecular bone architecture
Figure 5: Schematic of sequence for specimen preparation and compression testing
Figure 6: Mean and standard deviation of parameters characterizing bone morphology in all groups in L4
Figure 7: Mean and standard deviation of cortical thickness in all groups in L4
Figure 8: Mean and standard deviation of parameters characterizing bone morphology in all groups in CD6
Figure 9: Correlations between L3 and L4 in biomechanical properties
Figure 10: Relationship between bone morphology and biomechanical properties
Table 1: FDA approved medications for treatment and prevention of osteoporosis
Table 2: Treatment group by site interaction ANOVA
Table 3: Correlations between L3 and L4 in bone morphology
Table 4: Correlations between L3 and L4 in biomechanical properties
Table 5: Correlations between bone morphology and biomechanical properties
I~glt~ I t~ .iii
*%:llhii;ii''ii-

1.1 Osteoporosis
According to the National Osteoporosis Foundation, osteoporosis is a skeletal disorder characterized by structural deterioration of bone tissue leading to decreased bone mass and increased bone fragility.
(26) Bone is a composite tissue that provides structural support for muscles and protection for organs while storing calcium and other minerals.
The skeleton consists of a cortical (dense) shell that serves primarily for protection and an inner trabecular (cancellous) core responsible for metabolic processes. Bone remodeling is a continuous process that consists of bone resorption and bone formation of both cortical and trabecular bone. Osteoclasts on the bone surface dissolve bone tissue during resorption while osteoblasts are responsible for synthesizing new bone matrix.
(l
8) In a healthy individual, the two stages are interdependent and balanced. However with aging and estrogen deficiency after menopause, an uncoupling of the two processes occurs, and bone resorption exceeds the rate of bone formation causing a net bone loss. This ultimately leads to osteoporosis and increased susceptibility to fracture.
1.2 Prevalence of Osteoporosis
An estimated ten million Americans are diagnosed with osteoporosis, and more than thirty million likely have low bone mass.(26) While both men and women are affected and significant risk has been reported in people of different ethnicities, of those already considered osteoporotic, eighty percent are women.
( 26) In women, bone loss especially accelerates after menopause when the ovaries halt estrogen production.
Women can lose up to twenty percent of their bone mass within as little as five years following menopause, increasing their susceptibility to osteoporosis.
If not prevented or treated, the disease can progress silently until a sudden fracture occurs. While all bones, loaded and non-loaded alike, can be affected, the most common and high-risk fracture sites associated with osteoporosis are the hip, spine and wrist. In fact, the disease is responsible for more than 1.5 million fractures annually, including an estimated 300,000 hip fractures, 700,000 vertebral fractures, 250,000 wrist fractures and
300,000 fractures at other sites.(26) In general, such fractures have serious implications by causing severe pain, limiting mobility, and even potentially causing prolonged, debilitating disabilities that require long-term care. Hip and vertebral fractures, in particular, often require hospitalization and surgery. In fact, twenty to twenty-five percent of hip fracture patients die within the first year due to complications. Consequently, it is imperative to establish both the best drug and regimen for treatment.
1.3 Prevention and Treatment with Zoledronate
While there is currently no cure for osteoporosis, one can still protect against the disease or at minimum, preserve current bone health by maintaining a diet rich in calcium and vitamin D and engaging in regular weight-bearing exercises.(26) There are several anabolic and anti-resorptive medications already approved by the FDA to treat osteoporosis (Table 1). Anabolic drugs stimulate new bone growth in excess of bone resorption, resulting in an increase in bone mass and a reduction in fracture risk.
Anti-resorptive agents, on the other hand, protect existing bone by preventing its natural breakdown. The most widely used among the anti-resorptive therapies are bisphosphonates that inhibit osteoclast bone resorption and have been shown clinically to cause modest increases in bone mineral density but greatly reduce the risk of fractures.
For patients with established osteoporosis, anti-resorptives can fail to fully restore bone mass and structure, and anabolic drugs may be required to stimulate bone formation.

Table 1: FDA approved medications for treatment and prevention of osteoporosis
Anti-resorptive Agents
Bisphosphonates
Anabolic Agents
Parathyroid Hormone Therapy
Alendronate
Risedronate
Ibandronate
Calcitonin
Estrogen Therapy
Raloxifene [Selective Estrogen Receptor Molecule]
Zoledronate (zoledronic acid, ZOL, Fig. 1), the focus of this research, is a bisphosphonate that has been developed for osteoporosis treatment but has not yet been approved by the FDA. ZOL is a heterocyclic imidazole bisphosphonate that in
N pharmacologically active doses suppresses osteoclast bone resorption and turnover in rats _ without interfering with osteoblast activity, thereby maintaining bone mass.
( 26) ZOL has been shown to be more potent than previously developed bisphosphonates and can thus achieve 0-
similar effects with lower dosages and less frequent therapy or better results with the same Figure 1: Chemical structure of ZOL 2 ' 8) treatment regimen.
( 6
,7, 16 ) Approximately fifty percent of the administered drug binds to the bone
Chemical Formula: CsH
1 0
N
2
0
7
P
2
IUPAC name: (1-hydroxy-2-imidazol-1-ylmatrix and is slowly released into systemic
1-phosphono-ethyl)phosphonic acid circulation, with the rest being excreted in the urine within 24 hours.
( 27 ) Animal studies have shown that ZOL inhibits bone loss and increases bone strength in mature, estrogendeficient rats.
1.4 Experimental Osteoporosis and Rat Model
The ovariectomized [OVX] rat is an established animal model for studying postmenopausal osteoporosis.(4-9,14,21-25) While mice, sheep and other nonhuman primates have been used in experimental osteoporosis studies, ovariectomized rats are most widely used, and various manipulations have been implemented to induce sustained bone loss.
OVX is commonly performed and has been widely accepted as a suitable model, for there are no significant differences in bone behavior between surgical and natural menopause.(4,
24 ) OVX in rats mimics the estrogen depletion that accompanies menopause in women, stimulating an increase in bone turnover and an acceleration of bone loss.(9,25)
In particular, OVX has been shown to have a decreasing effect on bone volume fraction, connectivity, and trabecular number and an increasing effect on the trabecular separation and structural model index. OVX and menopause are both associated with changes in bone remodeling such that bone is synthesized more slowly than the bone matrix is degraded, ultimately leading to osteoporosis.
In general, there are two models for evaluating the efficacy of new therapies for osteoporosis: 1. Begin treatment immediately after OVX to prevent bone loss 2. Initiate drug therapy after established bone loss to recover and stabilize bone mass. These two models reflect the two clinical treatment alternatives. Continuous, anti-resorptive therapy with ZOL has been studied in the OVX rat using both preventative and recovering treatment regimens and has been shown to mitigate the effects of decreasing bone mass and increasing bone fragility that accompany aging. (5 -7
,17) More specifically, Glatt determined the protective effect of ZOL treatment on bone mass, structure, and strength

in estrogen-deficient rats.
(6 )
Horby et. al. studied the effects of a one-year administration to mature, OVX rats, and confirmed it as a suitable model for postmenopausal osteoporosis.
7) In addition, Rhee et. al. determined that upon withdrawing PTH, sequential therapy with ZOL effectively maintained the PTH-acquired bone quantity and quality.
( 7)
But in addition to preventing bone loss in rats, ZOL has shown to be a promising therapeutic for patients suffering from bone metastases and osteoporosis.(7,19)
1.5 Objectives of the Current Study
A quantitative study on the effects of Zoledronate therapy on bone morphology and strength may improve the treatment of osteoporosis. While there have been numerous studies demonstrating the effect of bisphosphonates in rats as either a preventative treatment or recovering therapy with the onset of osteoporosis, few have directly compared the two treatment alternatives with respect to their effects on bone microstructure and biomechanical properties. Specifically, uncertain are the effects of
ZOL when administered at OVX versus at a period of established bone loss. Previously in an in vivo study in rats, ZOL was shown to fully inhibit the effects of OVX in the proximal tibia using preventative treatment and partially alleviate symptoms with recovering treatment.
(
To further determine the clinical relevance of preventative therapy and ZOL's fracture efficacy this study has the following objectives:
Primary Objectives:
1. Characterize the effect of ZOL on vertebral microstructure in OVX rats i. Determine whether the effect of ZOL on vertebral microstructure depends on the time-point the therapy is administered after OVX.
ii. Determine whether the effect of OVX and ZOL on vertebral microstructure differs in loaded [fourth lumbar, L4] and unloaded
[sixth caudal, CD6] vertebrae.
Secondary Objectives:
2. Determine whether the bone microarchitecture and biomechanical properties in L4 can be predicted based on those values for L3.
3. Determine relationships between vertebral morphology and compressive biomechanical properties.
Michal Ruchelsman
Januarsy 20)07

2. 1 Experimental Design
2.1.1 Ovariectomy and ZOL treatment [performed by Julienne Brouwers]
(2)
Twenty-nine female Wistar rats (retired breeder, 35 weeks-old) were kept under standard laboratory conditions, stratified by weight and assigned to four groups. All rats underwent OVX or SHAM-OVX at week 0. Success of OVX was confirmed by analyzing ovarian tissue size and the atrophy of the uterine horns. Osteoporosis was induced by ovariectomy in rats but no drug treatment was given [OVX, n=5]. Rats in the early treatment group were administered Zoledronate (donated by Novartis
Pharmaceutical, Basel, Switzerland) at a single dose of 20 jg/kg s.c. immediately following ovariectomy [OVX+early ZOL, n=8], corresponding to preventative treatment.
The late treatment group was given the same single dose at week 8 [OVX+late ZOL, n=7], representing treatment once the disease has already been established. To subject the rats in the fourth group to the same stress as the other three treatment groups, surgery but no ovariectomy was performed [SHAM-OVX, n=9].
2.1.2 Secondary Study
The secondary study consisted of ten rats, including five Wistar rats that had unsuccessful ovariectomies and five Sprague Dawley rats that had undergone a variety of treatments including parathyroid hormone and bone morphogenetic protein-6 therapy.
spinous process
2.2 Microcomputed Tomography
At sixteen weeks post OVX the rats were sacrificed, and the vertebrae (Fig. 2) were carefully excised with a scalpel. Vertebrae were wrapped in saline-soaked gauze and stored at -20'C. The vertebrae were thawed at room temperature in a water bath, and giCT [uCT40, Scanco Medical AG] was used to determine six parameters that characterize bone morphology of the fourth lumbar [L4] and sixth caudal [CD6] vertebrae in each rat: bone volume fraction [BV/TV], connectivity density [ConnD], structure model index [SMI], and trabecular number [TbNr], thickness [TbTh] and spacing [TbSp]. The L4 were scanned at a resolution of 16jim and CD6 at 12pm. Cortical thickness of the L4 was also analyzed for endosteal expansion. Cortical and trabecular bone was manually selected (Fig. 3), and after the pCT images were subjected to
Gaussian filtration and segmentation, bone architecture was automatically evaluated (Fig.
4). jtCT was similarly performed on the L3 and L4 of five of the secondary rats. In addition to the above six parameters, minimum cross-sectional area [minA] and total bone volume [BV] was evaluated for the vertebrae in the secondary study.
/ vertebral body Superio
tranverse process pedicle spinous process
Axial View Lateral View
Inferior
Figure 2: Representative lumbar vertebra
29
Figure 3: pICT manual selection of Figure 4: pCT evaluation of trabecular

To assess the effect of the ZOL treatment timing in OVX rats on the biomechanical properties of the lumbar spine, L3 was subjected to compression testing and L4 to fatigue loading (performed by Julienne Brouwers; results not included). To assist with determining fatigue loading parameters [i.e. magnitude of load] and other future experimental procedures, compression tests were performed on the L3 and L4 of ten secondary rats. Specimens were thawed in a water bath prior to preparation and testing and were kept hydrated with saline throughout. A low speed IsoMet saw was used to cut plano-parallel ends, and a custom jig was used for fixation. The vertebral body was cut to a height of 4.5mm such that the growth plates were removed (Fig. 5A) and was separated from the posterior elements (spinous, transverse, articular processes) by cutting through pedicles using a single diamond saw (Fig. 5B). The anterior processes were clipped off, and all remaining soft tissue was removed. Samples were placed on a lower platen of Synergie 200 (MTS Systems, MN, USA). A preload of 3N was applied, and the vertebrae were compressed along the longitudinal axis at a displacement rate of
3mm/min until failure (Fig. 5C and Fig. 5D). Load-displacement curves were recorded in
TestWorks 4 Control and Acquisition Software and analyzed for ultimate load, ultimate displacement and strain, energy to failure, and stiffness, (Fig. 5E).
D Load cell
L
1
platen
F1 vertebral body
F
5
2so
2Wo
0
0 oUS 0.1 0.15 02
Displacement (mm) Du
Fig. 5: Schematic of sequence for specimen preparation and compression testing
The vertebral body was cut to length with piano-parallel ends, and the growth plates were removed using parallel diamond blades (A).
The body was then isolated from posterior elements (B).
Compression testing was performed using Synergie 200 Electro-
Mechanical Materials Testing System (MTS Systems, MN, USA)
(C). The vertebrae were compressed between platens along its longitudinal axis at a displacement rate of 3mm/min (D). A representative load-displacement curve for L3 and L4 with marked values of ultimate load (Fu), ultimate displacement (D,), energy to failure (area under curve) and stiffness is shown (E).
All statistical analyses were performed using StatView. For each tCT structural parameter, the mean and standard deviation were determined for each group for the L4 and CD6. An Analysis of Variance [ANOVA] plus Bonferroni test was used to determine significant differences (p<0.05) between treatment groups. A two-factor ANOVA was used to determine whether the effect of ZOL on vertebral microstructure differs in loaded
[L4] and unloaded [CD6] vertebrae. Simple regression analysis was used to determine relationships for bone morphology and compressive biomechanical properties between
L3 and L4A in the secondary study. Finally, regression analysis was also performed to determine whether significant correlations exist between bone morphology and biomechanical properties.
e ...
,,..-

3.1 Effect of ZOL Treatment Timing on L4
3.1.1 Effect of OVX on Vertebral Trabecular Bone
Compared to the SHAM-OVX group, the OVX group resulted in a significantly lower BV/TV, higher SMI and TbSp (p<0.05, Fig. 6, Appendix Table 1). While ConnD and TbNr tended to be substantially deteriorated in the OVX group, these differences were not statistically significant.
3.1.2 Effect of ZOL on Vertebral Trabecular Bone
In the early ZOL group, BV/TV was significantly higher and TbSp lower than the
OVX group (Fig. 6, Appendix Table 1). Late ZOL treatment tended to have favorable effects on all structural parameters compared to the OVX group, though the differences were not significant. Both ZOL groups were not significantly different from the SHAM-
OVX except for TbTh, which was significantly higher in the OVX+earlyZOL group than in SHAM-OVX. No significant differences were found between the OVX+earlyZOL and
OVX+lateZOL groups. However, a trend showed a slightly favorable bone microstructure for early treatment over late ZOL therapy.
BV/TV Conn.D
SMI
100
80
60
1 2 3 4
'TbNr TbTh TbSp
0.1
0.08
0.06
0.04
0.02
1 2 3 4
0
--
1 2 3 4 1 2 3 4
Figure 6: Mean and standard deviation of parameters characterizing bone morphology in all groups for L4 I=OVX, 2=OVX+lateZOL, 3=OVX+EarlyZOL, 4=SHAM-OVX. Symbols
$"' indicate statistically significant differences at p<0.
0 5
.

3.1.3 Effect of OVX and ZOL on Vertebral Cortical Bone
There were no significant differences in cortical thickness between the groups for
L4 (Fig. 8, Appendix Table 1). However, cortical thickness tended to increase after OVX and decrease with ZOL treatment.
0.25
0.2
0.15r
0.1
0.05r
1 2 3 4
Figure 7: Mean and standard deviation of cortical thickness in all groups for L4
3.2 Effect of ZOL Treatment Timing on CD6
Zoledronate treatment did not influence vertebral microstructure in CD6. There were no statistically significant differences between any of the groups, nor were there any substantial trends in the bone morphology of CD6 (Fig. 9, Appendix Table 1).
BV/TV Conn.D SMI
0.4
1
0.3
0.5
0.2
0
0.1
0
12
TbNr
3
-0.5
TbTh
1 2 3
TbSp
0.1
0.8
2
0.08
0.06
0.04
0.6
0.4
[ r I
1
0.02
0.2
1 v
0
I!
1 2 3 4 1 2 3 4 1 2 3 4
Figure 8: Mean and standard deviation of parameters characterizing bone morphology in all groups in CD6

3.3 Comparison of the Effect of OVX and ZOL in CD6 and L4
The effect of ZOL on vertebral bone microstructure differed in lumbar and caudal vertebrae, as evidenced by a significant treatment group by site interaction in the twofactor ANOVA. Significant differences were found for BV/TV (p<0.0578) and TbSp
(p<0.0535) in the group*site analysis, but there were no significant differences for the remaining parameters characterizing bone morphology.
Table 2: Treatment group by site interaction ANOVA
P-value Group P-value Site P-value Group*Site
BV/TV
ConnD
SMI
TbNr
TbTh
TbSp
0.0984
0.2429
0.0089
0.2975
0.0896
0.4772
0.0007
<0.0001
0.0252
0.0002
0.4251
0.0009
0.0578
0.3119
0.3745
0.1301
0.2337
0.0535
3.4 Secondary Study
3.4.1 Correlations Between L3 and L4 in Bone Morphology
Regression Analysis revealed significant correlations (p<0.05, Table 3) when comparing the bone morphology between a rat's L3 and L4. L4 showed a higher BV/TV,
SMI, and minimum cross-sectional area [minA] and a lower TbNr and TbSp than L3.
Good but statistically insignificant correlations were found for total bone volume [BV],
ConnD and TbTh between the L3 and L4.
Table 3: Correlation
BV/TV
ConnD
SMI
TbNr
TbTh
TbSp minA
BV
Regression Equation y = -0.16+1.462x
y 6.478+0.912x
y = 0.043+1.687x
y = 0.366+0.901x
y 0.061+0.327x
y 0.111+0.585x
y = -0.041+1.10x
y = -12.64+1.90x
R
... . .. . . r ... . . •
2
L4 v. L3 y
P-value
0.882
0.0179
0.631
0.1083
0.863
0.790
0.580
0.774
0.912
0.633
0.0225
0.0437
0.1345
0.0491
0.0114
0.1075
in
.

3.4.2 Correlations Between L3 and L4 in Biomechanical Properties
Regression Analysis revealed significant correlations (p<0.05, Fig. 11, Table 4) when comparing the biomechanical properties between a rat's L3 and L4. L4 showed lower values ultimate load [Uload], energy to failure [energy], and stiffness than L3.
Ultimate strain [Ustrain] and ultimate displacement [Udisp] yielded neither good nor significant correlations between L3 and L4.
400
350
300 c 250
200
150
100
150 200 250
Uload L3
Y = 17.286 + .872 * X; R^2 = .762
300
4000
3500
3000
L 2500
2000
350 400
28
26
24
22
> 20
2 18
16
14
12
10
12 14 16 18 20 22 24 26 28 30 32
Energy L3
Y = 1.392 +.859 * X; RA2 =.723
.28
S .26
.24 -
.22 -
-J
C.
.A .2
.18-
.16-
.14-
*
0
0
.
.0....
0
.12-
" "" I "
12 .14 .16 .18 .2
Udisp L3
Y = .081 + .502 * X; RA2 = .256
' ' I '." ' I '
.22 .24 .26
1500
1000
I
5( )0 1000 1500 2000 2500 3000 3500 4000 4500
Stiffness L3
Y = +.597 X; RA2 =.579
.06-
'0
.055-
.05-
.045 -
0 *0 0
.04lJ0
.035 oS
03 -
. . .S .
.
.
. i .
.025
.025 .03 .035 .04
Ustrain L3
Y =.018 + .496 * .258
.
.045
.
.
! .
.05
.
.055
Figure 9: Correlations between L3 and L4 in biomechanical properties
Table
Uload
Udisp
4:
Correlat
Energy
Stiffness
Ustrain
'' ' '
Regression Equation
y = 17.29+0.872x
y = 1.392+0.859x
y = 817.7+0.597x
y = 0.081+0.502x
R z
L4 v. L3
0.762
0.723
0.579
0.256
0.258
Droperties
P-value
0.0010
0.0018
0.0106
0.1354
0.1338

3.4.3 Correlations Between Bone Morphology and Biomechanical Properties
Regression Analysis revealed significant correlations (Table 4, Fig. 12) between bone architecture and biomechanical properties, with minimum cross-sectional area
[minA] of the vertebral body correlating the best. Statistically significant correlations were found between Uload and total bone volume [BV], energy and BV, Uload and minA, stiffness and minA, and energy and minA. There were no statistically significant correlations between Udisp and BV, Ustrain and BV, and stiffness and BV. Similarly, there were no significant correlations between Udisp and minA and Ustrain and minA. In addition, energy was the only biomechanical property that when plotted with bone volume fraction [BV/TV] yielded statistically significant results.
Table 5: Correlations between bone morphology and biomechanical properties
Uload v. BV
Stiffness v. BV
Energy v. BV
Udisp v. BV
Ustrain v. BV
Uload v. minA
Stiffness v. minA
Energy v. minA
Udisp v. minA
Ustrain v. minA
Uload v. BV/TV
Stiffness V BV/TV
Energy v. BV/TV
Udisp v. BV/TV
Ustrain v. BV/TV
Regression Equation y = 33.119+8.768x
y = -32.41+ 100.215x
y = -3.063+ 1.343x
y = 0.1980-0.00 1x y = 0.044-3.224E-4x
y = -27.537+85.188x
y = -1020.7+1103.4x
y = 0.8010+7.264x
y = 0.2690-0.04 1x y = 0.060-0.0090x
y = 90.934+243.428x
y = 1445+142.13x
y = -3.661+67.85x
y = 0.119+0.186x
y = 0.027+0.041 x
0.393
0.219
0.652
0.014
0.015
0.790
0.564
0.406
0.243
0.248
0.151
0.002
0.830
0.119
0.120
P-value
0.0502
0.1728
0.0047
0.7427
0.7385
0.0006
0.0123
0.0475
0.1480
0.1431
0.2666
0.9676
0.0002
0.3295
0.3268
A :-x
:4C
*1
B .•
;z
0~
"Thx
13C
BV minA
Figure 10: Relationship between bone morphology and biomechanical properties
(A) Uload v. BV (B) Uload v. minA

4.1 Effect of ZOL Treatment Timing on LA
4.1.1 Effect of OVX on Vertebral Trabecular Bone
Few studies have investigated the effects of bisphosphonate treatment using ZOL in rats with established bone loss and compared the recovering therapy with preventative treatment. This study, therefore, determined the effect of OVX and early and late ZOL treatment on the microstructure of trabecular bone in the L4 and CD6 in OVX rats. OVX tended to induce changes in bone morphology in L4, although differences between OVX and the remaining groups were not always significant. OVX caused a decreased BV/TV,
ConnD and TbNr and an increased TbSp and SMI. SMI ranges from 0 to 1; an increasing value indicates a change from plate-like to more rod-like bone which agrees with the
(5-7 literature on the effects of OVX on rat vertebrae.
'
24)
4.1.2 Effect of ZOL on Vertebral Trabecular Bone
The inhibiting effects of bisphosphonates preventing further thinning of trabeculae after the onset of osteoporosis, coupled with ongoing bone formation, are thought to increase or at minimum maintain bone microstructure in humans and animals, which agrees with results presented above.(5-7,15,26) Indeed, early ZOL administration immediately following OVX prevented major changes in structural parameters, and thus inhibited the development of osteoporosis with significantly higher BV/TV and lower
TbSp than in the OVX group. Late ZOL therapy at 8 weeks post OVX allowed for osteoporosis development and showed trends for recovering effect on bone mass and structure; no significant differences between the OVX and OVX+lateZOL groups were seen except in BV/TV and SMI. While there were no significant differences in TbNr,
ZOL tended to inhibit the loss of trabeculae resulting from OVX, with early treatment being more effective. In addition, there were no significant differences between the
OVX+earlyZOL and OVX+lateZOL groups, but trends showed a slightly more favorable bone microstructure for early treatment over late ZOL therapy.
4.1.3 Effect of ZOL on Vertebral Cortical Bone
ZOL treatment had no effect on cortical thickness in the L4. Such data, however, cannot be verified with current literature, for not only are the effects of OVX on cortical bone controversial, but also unknown is the effect of ZOL treatment. Differences in opinion may arise from variations in age at OVX and the rat strains used.
(23) It is known that endosteal bone resorption increases with aging and estrogen-deficiency. To partially compensate for the loss of endocortical bone mass, periosteal bone formation may occur in young rats undergoing OVX.
1 pronounced when rats undergo OVX at later ages (although this may be straindependent).
While periosteal bone formation results in bones of increased radial dimensions, bone loss coupled with formation does not necessarily result in a change in overall cortical thickness or loss of strength.
4.2 Comparison of the Effect of OVX and ZOL in CD6 and L4
The effect of OVX and ZOL on vertebral bone microstructure indeed differed between loaded [L4] and unloaded bones [CD6]. A two-factor ANOVA revealed an interaction between vertebral site and treatment group; significant differences were found for BV/TV and TbSp in the group*site analysis. For BV/TV, differences between treatment groups were significant independent of site. For both BV/TV and TbSp, differences between sites were significant independent of treatment group. These findings
Michal Ruchelsman
Page: 15 out, oft 2'ý Jý1.111',Ir 2_0)07

suggest that mechanical loading plays a role in a vertebra's response to OVX and ZOL therapy. Such deductions are supported by current literature. Mechanical loading and estrogen stimulate bone remodeling and regulate bone homeostasis in the rat-withbackpack-model.(
2
0) Estrogen depletion as in an OVX rat causes bone loss to result in a loaded vertebrae. Similarly, when a normally loaded vertebra is not subjected to loading, bone loss also results. However, the load in the backpack experienced by the rat was high enough to elicit an osteogenic response sufficient to compensate for the ovariectomyinduced bone loss.(20) It is therefore hypothesized that a less loaded bone such as CD6 will show little response to OVX. Since ZOL inhibits bone resorption, little will change in CD6 as a result of ZOL treatment.
4.3 Secondary Study
In this study, changes merely in trabecular bone microstructure were determined.
The primary objectives are furthered by Julienne Brouwers by conducting compression tests on L3 and fatigue loading on L4 to determine the extent that biomechanical properties change as a result of OVX and ZOL treatment timing. Compression testing was initially performed on L3 and L4 of ten secondary rats to help determine fatigue loading parameters and to assist with the design of future studies. Other motivation includes determining whether the results of one study on L3 can be of use in a study on
L4.
Findings indicate that the bone microarchitecture and biomechanical properties in L4 can in fact be predicted based on those for L3 as evidenced by strong, significant correlations (p<0.05). L4 had lower values for ultimate load, stiffness, and energy to failure than L3. However, L4 showed a more favorable bone microstructure, with higher values for bone volume fraction, structure model index and minimum area than L3. There were also good, significant correlations between bone morphology (specifically, bone volume and minimum area) and compressive biomechanical properties, suggesting that to a certain extent, bone morphology is a good indication of bone strength. Such results are seemingly puzzling; given the strong relationship between bone architecture and biomechanical properties, should L4 show a more favorable bone microstructure than L3,
L4 is expected to also have higher values for ultimate load, strength, energy. However, simple regression analysis showed poor, insignificant correlations between BV/TV and
Uload and other associated biomechanical properties. Only energy v. BV/TV yielded a strong and significant correlation. Data thus indicates that total bone mass, including contribution by both cortical and trabecular bone and not bone volume fraction alone, is a reasonable predictor of bone strength. Surely, the importance of cortical bone when a vertebra is loaded is also suggested.
4.4 Limitations of the study
4.4.1 Primary Study
Due to relatively high standard deviations, small sample sizes and differences at baseline, trends were seen between the treatment groups but few significant differences were reached. High standard deviations may be attributed to the use of retired breeder rats, which vary in response to OVX. Eight animals had to be removed from the study due to an unsuccessful response to ovariectomy, decreasing the number of animals used in data analysis. The average bone volume fraction at the start of the study was higher in groups with lower animal numbers [OVX and OVX+lateZOL], (' ) further decreasing the ability to observe significant differences between groups.
MIichal Ruchelsmnan
JaIRM.1v 1-0077

4.4.2 Secondary Study
The use of two different rat strains and differences in treatments may have lead to variations in findings for correlations between L3 and L4 in terms of bone morphology and biomechanical properties. However, since comparisons were made between the two vertebrae within one rat, treatment received and conditions exposed to are likely to have played a minor role. When sawing the vertebral body, variations in location were also likely to have occurred, possibly incorporating parts of the growth plate. Further, the extent to which pedicles were clipped was not always uniform among the rats. pCT images revealed that when differences existed, they were not always observable by eye.
Leaving too much remaining pedicles result in the ability to withstand higher loads, while clipping too far can result in compromising the cortical bone which greatly contributes to structure and strength of the vertebral body. In addition, cutting plano-parallel ends for the vertebral body is essential for accurate results. Lastly, the precision of the machinery and variations in mechanical behavior of the vertebrae as indicated by differences in shape of the generated force-displacement curves present further limitations.
Michal Ruchtel smian Page 17 out of
'
222 Janiuary 2007

This study investigated the effect of early and late treatment of zoledronate treatment in both loaded [L4] and unloaded [CD6] vertebrae on bone microstructure using [tCT in adult, estrogen-deficient rats. OVX resulted in a loss of trabecular bone mass and compromised microstructure. In the L4 vertebrae, a single injection of ZOL at
OVX inhibited these changes. Although differences in bone volume fraction and associated bone microstructure between early and late ZOL treatment were not statistically significant by end-of-study measurements, early ZOL showed slightly favorable trends. But while results from L4 showed the favorable use of early ZOL therapy, no significant differences were observed between treatment groups in the CD6;
CD6 showed no response to OVX and ZOL, suggesting that the response to OVX and bisphosphonate therapy may be mediated in part by mechanical loading. Results from the secondary study yielded strong correlations in the properties between L3 and L4.
Correlations between bone microarchitecture and biomechanical properties also showed that total bone volume and minimum area are reasonable predictors of biomechanical properties. Overall, the findings demonstrate that ZOL is not only effective at reversing and inhibiting ovariectomy-induced defects in bone architecture, but also reveal the importance of the time-point of treatment.
All research was conducted at Orthopedic Biomechanics Lab, Dept. of Orthopedic
Surgery, BIDMC and Harvard Medical School. A Special thanks to Mary Bouxsein, Ph.D
and Julienne Brouwers and the rest of the Lab for all of their assistance and guidance throughout. Also, thank you to Professor Kimberly Hamad-Schifferli for serving as my faculty advisor in the Department of Mechanical Engineering, MIT.

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1. Brouwers 2006 World Conference of Biomechanics, Germany
2. Brouwers, JEM, Ruchelsman M, v. Rietbergen B, Bouxsein M 2007 Orthopedic
Research Society Conference, San Diego
3. Ederveen AGH, Spanjers CPM, Quaijtaal JHM, Kloosterboer HJ 2001 Effect of 16 months of treatment with Tibolone on bone mass, turnover, and biomechanical quality in mature ovariectomized rats. J Bone Miner Res 16:1674-81
4. Frost HM, Jee WSS 1992 On the rat model of human osteopenias and osteoporosis.
Bone Miner 18:227-36
5. Glatt M, Pataki A, Evans GP, Hornby SB, Green JR 2004 Loss of vertebral bone and mechanical strength in estrogen-deficient rats is prevented by long-term administration of zoledronic acid. Osteoporosis Int. 15(9):707-15
6. Glatt M 2001 The bisphosphonate zoledronate prevents vertebral bone loss in mature, estrogen-deficient rats as assessed by micro-computed tomography. Eur Cell Mater 1:
18-26
7. Hornby SB, Evans GP, Hornby SL, Pataki A, Glatt M, Green JR 2003 Long-term zoledronic acid treatment increases bone structure and mechanical strength of long bones of ovariectomized adult rats. Calcif Tissue Int. 72(4):519
8. Jiang Y, Zhao J, Genant HK, Dequeker J, Geusens P 1997 Long-term changes in bone mineral and biochemical properties of vertebrae and femur in aging, dietary calcium restricted, and/or estrogen-deprived/-replaced rats. J Bone Miner Res 12:
820-831
9. Kalu D 1991 The ovariectomized rat model of postmenopausal bone loss. J Bone
Miner Res 15: 175-92
10. Ke HZ, Jee WS, Zeng QQ, Li M, Lin BY, 1993 Prostaglandin E2 increased rat cortical bone mass when administered immediately following ovarietomy. Bone
Miner 21(3): 189-201
11. Muller R, Hannan M, Smith SY, Bauss F 2004 Intermittent Ibandronate preserves bone quality and bone strength in the lumbar spine after 16 months of treatment in the ovariectomized cynomolgus monkey. J
12. Little DG, McDonald M, Sharpe IT, Peat R, Williams P, McEvoy T 2005 Zoledronic acid improves femoral head sphericity in a rat model of perthes disease. J Orthop Res
23(4): 862-8
13. Pan B, To LB, Farrugia AN, Findlay DM, Green J, Evdokiou A, Lynch
K, Atkins GJ, Zannettino AC 2004 The nitrogen-containing bisphosphonate, zoledronic acid, increases mineralization of human bone-derived cells in vitro. Bone
34(1): 112-23
14. Peng Z, Tuukkanen J, Zhang H, Vaananen HK 1999 Alteration in the mechanical competence and structural properties in the femoral neck and vertebrae of ovariectomized rats. J
15. Recker R, Masarachia P, Santora A, Howard T, Chavassieux P, Arlot M, Rodan G,
Wehren L, Kimmel D 2005 Trabecular Bone microacrchitecture after aledronate treatment of osteoporotic women, Curr Med Res Opin 21(2): 185-94
16. Reid IR,
Devogelaer JP, Kaufman JM, Jaeger P, Body JJ, Brandi ML, Broell J, Di Micco R,
Genazzani AR, Felsenberg D, Happ J, Hooper MJ, Ittner J, Leb G, Mallmin H,
Murray T, Ortolani Rubinacci A, Saaf M, Samsioe G, Verbruggen L, Meunier PJ
2002 Intravenous zoledronic acid in postmenopausal women with low bone mineral density. Engl J Med 346(9): 653-61
T; 1 T) :• : !t•L :•i•t • ' I

17. Rhee Y, Won YY, Baek MH, Kim SK 2004 Maintenance of increased bone mass after human parathyroid hormone with sequential zoledronate treatment in ovariectomized rats J Bone Miner Res. 19: 931-7
18. Spector M, Fall 2006 MIT course HST.522 Lecture Slides
19. Theriault R 2003 Zoledronic Acid (Zometa) use in bone disease. Expert Rev
Anticancer Ther 3(2): 157-66
20. Tromp AM, Bravenboer N, Tanck E, Oostlander A, Holzmann PJ, Kostense PJ, Roos
JC, Burger Eh, Huiskes R, Lips P 2006 Additional Weight Bearing during Exercise and Estrogen in the Rat: The Effect on Bone Mass, Turnover, and Structure. Calcif
Tissue Int. 79(6): 404-15
21. Turner CH, Roeder RK, Wieczorek A, Foroud T, Liu G, Peacock M 2001 Variability in skeletal mass, structure, and biomechanical properties among inbred strains of rats.
J Bone Miner Res 16: 1532-9
22. Turner RT, Vandersteenhoven JJ, Bell NH 1987 The effects of ovariectomy and 17 beta-estradiol on cortical bone histomorphometry in growing rats. J Bone Miner Res
2(2): 115-22
23. Weber K, Kaschig C, Reinhold G 2004 1-alpha-Hydroxyvitamin D
2 and 1-alphahydroxyvitamin D
3 have anabolic effects on cortical bone, but induce intracortical remodeling at toxic doses in ovariectomized rats. Bone 35(3): 704-710
24. Wronski TJ, Dann LM, Scott KS, Cintron M 1989 Long-term Effect of ovariectomy and aging on the rat skeleton. Calcif Tissue Int 45: 360-366
25. Yamazaki I, Yamaguchi H 1989 Characteristics of an ovariectomized osteopenic rat model. J Bone Miner Res 4:13-22
26. http://www.nof.org/osteoporosis/diseasefacts.htm (2006 22 September).
27. http://cacons.org/education/edu06.htm (2006 19 September).
28. http://recdpoll.pharmacy.ualberta.ca/drugbank/index.html (2006 October 18)
29. http://wvvw.sandiego-spine.com (2007 January 2)
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