Parathyroid hormone effect on facilitating stress fracture repair
Author Bakr, Mahmoud
Published 2019-11
Thesis Type Thesis (PhD Doctorate)
School School of Medical Science
DOI https://doi.org/10.25904/1912/3371
Copyright Statement The author owns the copyright in this thesis, unless stated otherwise.
Downloaded from http://hdl.handle.net/10072/389571
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PTH effect on facilitating stress fracture repair.
PhD Candidate:
Mahmoud Bakr. BDSc, MDSc (Oral Biology), ADC, Grad Cert High Ed, FHEA
Student number: s2808448
School of Medical Science
Griffith University
July 2019
Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy
Supervisors
Principal: Prof. Mark Forwood
Principal: Prof. Nigel Morrison
Associate: Prof. Ward Massey
Associate: A/Prof. Helen Massa
Contents
Acknowledgements ……………………………………………………………………………….....viii
List of figures ………………………………………………………………………………………...... x
List of tables ………………………………………………………………………………………....xvii
List of abbreviations …………………………………………………………………………...... xx
Abstract……………………………………………………………………..……………………....xxiii
Publications………………………………………………………………………………………...xxvii
1. Introduction:
1.1. Overview.…..……….…………………………………………………………………………....2
1.2. Aim of the study..….. …..……..………………………………………………………………....7
1.3. Hypotheses…………….…………………………………………………………………………7
1.4. Significance and Innovation…….…………………………..…………………………………...8
2. Review of literature:
2.1. Bone composition and microstructure: …………………………………………………….…..10
2.1.1. Types of bone…..…………………………………………………………………………10
2.1.2. Bone microstructure………………………………………………………………………11
2.1.3. Modeling and remodeling………………………………………………………………...12
2.1.4. Differences between human and rat bones……………………………………………….13
2.1.5. Functions and activities of bone………………………………………………………….15
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page ii
2.2. Bone cells……………………………………………………………………………………….16
2.2.1. Osteoblasts………………………………………………………………………………..16
2.2.2. Bone lining cells………………………………………………………………………….17
2.2.3. Osteocytes………………………………………………………………………………...18
2.2.4. Osteoclasts………………………………………………………………………………..19
2.2.5. Osteal tissue macrophages (OsteoMacs)…………………………………………………21
2.3. Factors affecting healing and bone remodeling…………………………………………………22
2.3.1. Mechanical loading and mechanostrat…………………………………………………….22
2.3.2. Remodeling cycle and activation………………………………………………………….23
2.4. Stress Fractures: ………………………………………………………………………………..28
2.4.1. Classification of bone stress injuries………………...…...………………………………28
2.4.2. Pathophysiology and epidemiology of stress fractures……...………………………...…30
2.4.3. Biological and molecular concepts of SFx healing………………...…………………….33
2.4.4. Management and treatment concepts of SFx……………………………………………..35
2.5. Parathyroid hormone (PTH):………………………………………………………………...…41
2.5.1. Background on PTH……………………………………………………………………....41
2.5.2. PTH as an anabolic agent………………………………………………………………....43
2.5.3. Mechanism of action of PTH…………………………………………………………..…48
2.6. Bisphosponates ……………………………………………………………………………..….54
2.6.1. Background on bisphosphonates……………………...………………………………….54
2.6.2. Bisphosphonates as antiresorptive agents………………………………………………..56
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page iii
2.7. Potential uses of PTH and bisphosphonates in SFx healing……………………………………60
2.7.1. PTH and SFx healing……………………………………………………………………..60
2.7.2. Combined ALN PTH tretament and SFx healing………………………………………...61
3. Methodology:
3.1. Treatments....……………………………………………...……………………………………64
3.2. Experimental design.……………………………………………...……………………………66
3.3. Histomorphometry..…………………………………………..……………………………...…68
3.4. Statistical analyses.………………………………………….……………………………….....74
4. Single PTH experiment:
4.1. Introduction……………………………………………………………………………………..77
4.2. Experimental design……………………………………………………………………………78
4.3. Results…..……………………..………………………………..………………………………79
4.3.1. Single PTH injection……………………………………………………………………...79
4.3.2. Woven bone parameters………………………………………………….………………79
4.3.3. Osteoclast parameters……………………………………………….……………………80
4.3.4. Healing parameters………………………………………………..……………………...82
4.3.5. Erosion (unhealed) parameters…...………………………………...……………………85
4.3.6. Porosity (BMU) parameters………...………………………………………………..…..86
4.4. Discussion (Single PTH experiment)…………………………………………….....………….91
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page iv
5. Daily PTH experiment:
5.1. Introduction……………………………………………………………………………………100
5.2. Experimental design…………………………………………………………………………..101
5.3. Results: ………………………..………………………………………….…………………..102
5.3.1. Daily PTH injections……………………………………………………………………102
5.3.2. Woven bone parameters………………………………………………………………...104
5.3.3. Osteoclast parameters…………………………………………………………….……..104
5.3.4. Healing parameters……………………………………………………………..……….105
5.3.5. Erosion (unhealed) parameters……………………………………………………...…..107
5.3.6. Porosity (BMU) parameters………………………………… …………………..……...107
5.3.7. Comparison between single and daily PTH injections………………………………….112
5.4. Discussion (Daily PTH experiment)…………………………………………..………………112
6. Combined ALN-PTH experiment:
6.1. Introduction……………………………………………………………………………………122
6.2. Experimental design…………………………………………………………………………..123
6.3. Results: ………………………..………………………………………………………….…..124
6.3.1. Combined ALN PTH treatment…………………………………………………………124
6.3.2. Woven bone parameters………………………………………………………………...125
6.3.3. Osteoclast parameters……………………………………………………………...……126
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page v
6.3.4. Healing parameters……………………………………………………………………...130
6.3.5. Erosion (unhealed) parameters………………………………………………………….132
6.3.6. Porosity (BMU) parameters………………………… …………………………..……...132
6.3.7. Comparison between daily PTH and combined ALN-PTH treatment..…..…………….136
6.4. Discussion (Combined ALN-PTH experiment)………………………………………………136
7. Geneal discussion and conclusions:
7.1. General discussion ……………………………………………………………………...….....152
7.2. Conclusions……………………………………………………………………………………155
7.3. Recommendations for future research…………………………………………………………156
8. References:.…………………………………………………………………………………..…...158
Appendix 1 – Summary of non-significant findings……………………………………………….233
Appendix 2 – Single PTH injection publication……………………………………………………238
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Acknowledgements
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page vii
I would like to thank all my supervisors for their continuous guidance and support during my PhD journey. I would like to also to thank the technical staff in the
School of Medical Science – Griffith University, especially Ms. Wendy Kellty and Mr. Bradley Paterson for their efforts in facilitating all animal experiments and laboratory work in this project.
The School of Dentistry and Oral Health – Griffith University and Head of School
– Professor Robert Love were very helpful in allowing a balanced academic workload while completing my PhD.
I am very thankful for Professor Mark Forwood who provided a great leadership throughout all stages of the project from start to completion.
Finally, it would not have been possible to complete this work without the support and understanding of my family who accepted my long working hours in the last five years.
Mahmoud Bakr
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List of figures
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page ix
Fig 1: A schematic summary of the pathways undertaken by PTH in order to produce its effect, adapted from Swarthout et al., 2002………………………………………………………….50
Fig 2: Showing PTHR1 intracellular signalling. After ligand coupling PTHR1 activates four different intracellular signalling cascades: a) GαS-adenylyl cyclise-cAMP-protein kinase A (PKA), b) Gαq-phospholipase C (PLC) β- inositol triphosphate-cytoplasmic Ca2+- protein kinase C18, c) Gα12/13-phospholipase D- transforming protein RhoA and d) β-arrestin-extracellular signal-regulated kinase
1/2(ERK1/2), adapted from Yavropoulou et al., 2017………………………………………51
Figure 3: Technique for SFx axial compression loading. Fig 3A: Forwood Laboratory, School of Medical Science, Griffith University, Australia. Fig 3B: courtesy of Orthopaedic Research laboratories, School of Medicine, Indiana University, USA. ……………………..………….67
Figure 4: (a): Photomicrograph of a transverse section of a rat’s ulna showing the standardized position chosen for histomorphometric analysis in this study along the SFx. To avoid any bias the slides selected for analysis were chosen at a position where the SFx (Green) was midway between the outer cortical margins (Red) and the inner medullary cavity (Yellow) – (Toluidine
Blue 2X). (b): A schematic diagram on a TRAP stained slide showing the boundaries of the
Basic Multicellular Unit (BMU) (Yellow) with osteoclast count (Green) and osteoclast perimeter (Black) – (TRAP 10X)……………………………………………………………..69
Figure 5: Showing histomorphometric analysis using Osteomeasure™. (a): Toludine Blue section showing a transverse section in the rat ulna that was selected for histomorphometric
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page x analysis of SFx. (b); TRAP stained section showing the histomorphometric analysis of the osteoclast number and perimeter.
Red Cortical area (Ct.Ar, mm2) Green Number of osteoclasts (N.Oc)
Yellow Woven bone area (Wo.B.Ar, mm2) Yellow Oc surface perimeter (OC.Pm, µm)
Light blue Woven bone Width (Wo.B.Wi, mm)
Dark red Length of stress fracture (SFx.Le, µm)
…………………………………………………………………..…………………………….72
Figure 6: Showing histomorphometric analysis of a Basic Multicellular Unit (BMU) using
Osteomeasure™ software.
Blue Porosity area (SFx.Po.Ar, µm2)
Light Blue Erosion area (SFx.E.Ar, µm2)
Yellow Length of remodeling unit along fracture line (BMU.Le, µm)
Purple Area of healed new bone (SFx.He.Ar, µm2)
………………………………………………………………………………………………...73
Figure 7: (a & b): Showing the significantly high woven bone apposition rates (red arrows point to the amount of woven bone in each section) during early remodeling phases (1 week post SFx loading) (a & b) when compared to late remodeling (6 weeks post SFx loading) (c & d)
(Toluidine Blue 2 X). Scale bar = 100µm…….……………….……………………………....80
Figure 8: (a): Showing the limited availability of osteoclasts in VEH group after 1 week (b):
Showing the significant increase in osteoclast in PTH group after 2 weeks when compared to the VEH group shown in (a) (TRAP 10X), (c & d): Showing the significant decrease in
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page xi osteoclasts with the progression in healing after 6 weeks in both VEH and PTH groups (TRAP
10X). Scale bar = 10µm.………………………………………………………………….….81
Figure 9: Histomorphometric variables of SFx healing (±SD). PTH positively influenced bone histomorphmetric indices along all stages of the bone remodeling cycle following the loading of stress fracture (SFx). The time points between 2 and 6 weeks also influenced all variables significantly. There was a significant interaction between time and the type of treatment (VEH or PTH) that affected osteoclasts number and perimeter after 2 weeks. *** = P≤0.01
(differences between PTH and VEH). ++ = P≤0.05, +++ = P≤0.01 (differences compared to the previous time point)……………………………………………………………..………...…..82
Figure 10: Progression of remodeling. (a & b): Shows absence of porosity (BMU) at the SFx entry point one week post loading in both VEH and PTH groups; (c & d): Development of porosity (BMU) area at the entry point of the SFx two weeks post loading in both VEH and
PTH groups; (e & f): Progression in healing and the gradual filling of the porosity (BMU) areas with new bone six weeks post loading in both VEH and PTH groups, with new bone formation also demonstrated along the course of the SFx (black arrows) in PTH group; (g & h): Shows the significant reduction in porosity (BMU) area 10 weeks post SFx loading in the PTH group
(h) when compared to VEH group (g) due to complete healing by direct remodeling starting at the entry of the SFx (Toluidine Blue 10X). Scale bar = 10µm. (i): A complete section showing the area of interest manginified in panels (a-h) including the entry point of SFx and BMU (black circle) (Toluidine Blue 2X). Scale bar = 100µm………………………………………………84
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Figure 11: A scatterplot with a line of bit fit showing the significant negative correlation between erosion perimeter and healing perimeter 6 weeks post SFx induction…………..……86
Figure 12: Histomorphometric variables of SFx healing (±SD). PTH increased the healing area/ mm2 of the cortical bone area 6 weeks after SFx. PTH also decreased the porosity (BMU) parameters 10 weeks after SFx due to progression in healing. Most of the derived and normalized variables were significantly influenced by the main effect of time between the 2 week and 6 week time points. However, there was no significant interaction between time and the type of treatment (VEH or PTH). +++ = P≤0.01 (differences compared to previous time point)………………………………………………………………………………………….87
Figure 13: Daily PTH injections for 14 days resulted in a significantly higher BV/TV within the distal femur than the daily VEH injection group after 2 weeks ***P.≤0.01 (Mean
±SD)………………………………………………………………………………………....103
Figure 14: Histomorphometric variables of SFx healing (±SD). There was no significant interaction between time and the type of treatment (Daily PTH or VEH). (a, b, c): Daily PTH injections for 14 days only improved woven bone apposition rate. Woven bone parameters were significantly less after 6 weeks when compared to the second week. (d, e, f): Osteoclast parameters were significantly less after 6 weeks when compared to the second week. (g, h, i):
Healing parameters improved significantly after 6 weeks when compared to the second week.
** = P≤0.05 (differences between PTH and VEH). +++ = P≤0.01 (differences compared to the previous time point)………………………………………………...………………………..106
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Figure 15: Histomorphometric variables of SFx healing (±SD). All of the standard, derived and normalized variables were significantly influenced by the main effect of time between the 2 week and 6 week time points. However, there was no significant interaction between time and the type of treatment (Daily PTH or VEH). +++ = P≤0.01 (differences compared to the previous time point)…………………………………………………………………………………...108
Figure 16: Histomorphometric variables of SFx woven bone callus, osteoclast and healing parameters (±SD). Combined ALN-PTH treatment was superior to ALN and VEH treatments in terms of woven bone sculpture and modeling throughout the healing process of SFx.
Cessation of ALN results in a significantly less woven bone width indicating a more rapid progression in healing. Cessation of ALN after 2 weeks resulted in a significantly greater osteoclast number. ALN and combined ALN-PTH treatment modes (cessation and continuation) resulted in less healing area and perimeter after 6 weeks when compared to VEH.
Most variables were significantly influenced by the main effect of time between the 2 week and
6 week time points. Furthermore, there was a significant interaction between the main effects of time and the treatment type on healing perimeter. ** = P≤0.05, *** = P≤0.01 (differences between treatment types). +++ = P≤0.01 (differences compared to the previous time point)………………………………………………………………………………………...128
Figure 17: (a): Showing normal osteoclastic activity in VEH groups, (c): suppressed osteoclastic activity in ALN groups, (e): higher osteoclastic activity in daily PTH group, and (i): higher osteoclastic activity in the ALN-PTH2 group during the resorptive phase of the remodeling cycle after 2 weeks. (g):
Continuation of ALN treatment in ALN-PTH1 resulted in suppression of osteoclastic activity along the
SFx despite the recruitment and availability of osteoclasts around the woven bone callus. (b, d, f, h &
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page xiv
J): After 6 weeks, osteoclastic activity was significantly less in all groups apart from some osteoclastic trials indicating past resorptive activity. Daily PTH group showed evidence of osteoclastic activity only around but not along the SFx due to progression in healing. (TRAP 10X). Scale bar = 10µm………129
Figure 18: (a) Showing the initiation of remodeling in VEH groups. (e) Daily PTH treatment accelerated remodeling at 2 weeks through the development of larger areas of porosity (BMUs) in comparison to all other groups. (g & i) The combined ALN-PTH treatments triggered the development of multiple porosity areas (BMUs) that were significantly larger when ALN treatment was ceased. (c): ALN treatment resulted in the development of smaller BMUs (d): After 6 weeks, healing was completely suppressed in ALN groups but continued normally in VEH (b) and Daily PTH groups (f). (h & j): In the combined ALN-PTH treatment, areas of erosion persisted after 6 weeks along the SFx despite the progression of healing in other areas, especially when ALN treatment was continued. Overall the percentage healing was high in ALN-PTH2 due to larger porosity areas developed under the effect of
ALN and larger healing areas after cessation of ALN. (Toluidine Blue 10X). Scale bar = 10µm……..131
Figure 19: Histomorphometric variables of SFx healing (±SD). Continuation of ALN in the combined ALN-PTH1 treatment mode resulted in a significantly less percentage fracture length occupied by erosion after two weeks when compared to VEH and cessation of ALN in the combined ALN-PTH2 treatment mode. Healing, porosity variables were significantly greater, while erosion variables were significantly less as a result of the main effect of time between the
2 week and 6 week time points. *** = P≤0.01 (differences between treatment types). +++ =
P≤0.01 (differences compared to the previous time point)………………………………….133
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List of tables
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page xvi
Table 1. Experimental design for single PTH, daily PTH and combined ALN-PTH experiments.
Number of rats per group (n)= 15 per group ……………………………………..……………65
Table 2: Experimental design of the single PTH experiment. Number of rats per group (n)= 15
………………………………………………………………………………………………...78
Table 3: A summary of the significant findings related to the effect of time, treatment as well as the interaction between treatment and time on different variables using a linear model, 2- way ANOVA statistical analysis……..……………………………………………………….88
Table 4: Experimental design of the daily PTH experiment. Number of rats per group (n)= 15.
……………………………………………………………………………………………….102
Table 5: A summary of the significant findings from the daily VEH and PTH groups related to the effect of time, treatment as well as the interaction between treatment and time on different variables using a linear model in a 2-way ANOVA statistical analysis………………………109
Table 6: Experimental design of the combined ALN PTH experiment. Number of rats per group
(n)= 15……………………………………………………………………………………….124
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Table 7: A summary of the significant findings from the ALN, ALN-PTH and VEH3 groups related to the effect of time, treatment as well as the interaction between treatment and time on different variables using a linear model in a 2-way ANOVA statistical analysis…………….134
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List of abbreviations
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page xix
AF Atrial Fibrillation
AFF Atypical femoral fracture
ALN Alendronate
BMC Bone mineral content
BMD Bone mineral density
BMP Bone morphogenic protein
BSI Bone stress injury
BMD Bone mineral density
BMU Basic multicellular unit
BP Bisphosphonate
BV Mineralized bone volume
CaSR Calcium Sensing Receptor
ESMO European Society of Medical Oncology
GPCR G-protein coupled receptor
IGF-1 Insulin-like growth factor – 1
ILs Interleukins
IL-6 Interleukin-6 i-PTH Intermittent parathyroid hormone
LIF Leukemia Inhibitory Factor
LIPUS Low intensity pulse ultrasound
LVDT Linear Variable Digital Transformer
MAR Mineral apposition rate
M-CSF Macrophage colony-stimulating factor
MCP-1 Monocyte chemotactic protein – 1
MRI Magnetic resonance imaging
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page xx
MSC Mesenchymal stem cell
MTX Methotrexate
NF Nanofibrous
NOF National osteoporosis foundation
NSAIDs Non-steroidal anti-inflammatory drugs
Ob Osteoblasts
Oc Osteoclasts
ONJ Osteoradionecrosis of jaw
OPN Osteopontin
PKA Protein kinase - A
PKC Protein kinase – C
PTH Parathyroid hormone
PTHdP PTH-derived peptide
PTHR1 Parathyroid hormone receptor – 1
PTHrP Parathyroid hormone related protein
RANK-L Receptor activator of nuclear factor kappa-B ligand
RCT Randomized control trial
SFx Stress fracture
TGF-β Transforming growth factor – β
TMJ Temporomandibular joint
TV Total bone volume
VDR Vitamin D receptor
VEGF Vascular endothelial growth factor
VEH Vehicle
ZA Zoledronic acid
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page xxi
Abstract
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page xxii
Stress fractures (SFx) result from repetitive cyclical loading of bone. They are frequent athletic injuries and underlie atypical femoral fractures following long-term bisphosphonate (BP) therapy. Parathyroid hormone (PTH) and BPs, including Alendronate (ALN), have opposing effects on bone dynamics. ALN is an antiresorptive agent that decreases bone turnover, and with prolonged doses of BP the healing of SFxs is delayed. The extent to which intermittent
PTH (iPTH) remains effective in the treatment of SFx in the presence of an ongoing BP treatment has not been tested. Starting 24 hours after SFx induction, the effect of a single PTH injection, and daily iPTH injections for 14 days, on the healing of SFx in the rat ulna was investigated. SFx was induced in 330 female Wistar rats (300 15 g) during a single loading session. In the single PTH injection experiment, rats were divided into two groups (n=60). A single PTH (8 μg/100g) or vehicle (VEH) saline injection was administered 24 hours after loading. Ulnae were examined 1, 2, 6 or 10 weeks following SFx.
In the daily iPTH experiment, rats were divided into two equal groups (n=30) and received either daily iPTH (8 μg/100g/day) for 14 days or equivalent daily VEH injections. In the combined ALN-PTH therapy experiment, rats were divided into five equal groups (n=30), received either, ALN (1 ug/kg/day) for the whole duration of the experiment (ALN1) or only until SFx initiation (ALN2); combined ALN-PTH with continuation (ALN-PTH1) or cessation
(ALN-PTH2) of ALN after SFx induction or an equivalent vehicle (VEH) as a control. Ulnae were examined 2 or 6 weeks following SFx. Two Toluidine Blue and TRAP-stained sections of the SFx were examined for histomorphometric analysis using Osteomeasure™ software.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page xxiii
In the single PTH experiment, a significant interaction was observed between the effects of time and type of treatment on osteoclast number (N.Oc) and perimeter (Oc.Pm). This resulted in osteoclast number and perimeter being significantly greater two weeks following PTH treatment (P≤0.001). Most histomorphometric variables were significantly influenced by the main effect of time between the second and sixth weeks. After 6 weeks, bone formation was the main activity in BMUs. A significant negative correlation (r= -0.602) was observed between erosion and healing perimeters in PTH group after 6 weeks (P= 0.038).
In the daily iPTH treatment, the results show that daily iPTH injections for 14 days increased
SFx remodeling by improving the woven bone architecture. This was evident in the form of a a significantly greater woven bone apposition rate (P = 0.049), woven bone area (P = 0.041) when compared to the daily VEH injection group after 2 weeks of SFx initiation. Compared to the results obtained from the single PTH experiment, daily iPTH injections for 14 days were not superior to a single PTH injection. Despite increasing the recruitment and availability of osteoclasts, daily iPTH injections are not responsible for the transportation of osteoclasts from the BMU to the narrow SFx line. This was demonstrated by the presence of osteoclastic activity within the surrounding woven bone callus that did not translate into a significant effect within the BMU or the SFx line.
In the combined ALN-PTH protocol, there was a significant interaction between the effects of time and treatment type on the woven bone width and woven bone apposition rate. Combined
ALN PTH treatment improved woven bone architecture. However, woven bone variables remained unaffected by the cessation or continuation of ALN. Cessation of ALN after SFx
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page xxiv induction effectively increased osteoclast number in ALN-PTH2 (ALN cessation) group when compared to ALN PTH1 (ALN continuation) group (P= 0.006), and VEH (P= 0.024) after two weeks. Furthermore, there was a significant interaction between the effects of time and treatment type on the number of osteoclasts per unit BMU are and length. The number of osteoclasts per unit BMU area and length was significantly greater in ALN cessation groups.
The combined ALN-PTH treatment was not superior to daily iPTH treatment
It was concluded that a single PTH injection increased osteoclastogenesis by the second week of the remodeling cycle in a SFx in vivo. Intermittent daily PTH injections (iPTH) for 14 days, starting 24 hours after the initiation of SFx, had the same effect on indices of SFx healing as a single PTH injection. The increased number of osteoclasts available around the SFx area was not beneficial. This was related to the limited area available for these cells to be transported from the BMU into the SFx line to produce a more significant effect. It was also concluded that intermittent short duration iPTH treatment effectively increased remodeling of SFx even after
BP treatment, provided that BP was ceased at the time of SFx. iPTH injections for 14 days were not sufficient to counteract the effect of a concurrent BP treatment. The results obtained from the current study could potentially help develop shorther iPTH treatment protocols for clinical management of SFxs. It also helps guide clinical decision making to cease BP treatment in cases of SFx. Furthermore, future research using the same treatment protocols from the current study augmented by Vit D, calicium supplements and/or mechanical loading could achieve better treatment outcomes.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page xxv
Publications
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page xxvi
The findings from the single PTH injection (Chapter 4) have been published in the Journal of
Orthopaedic Research (Appendix).
Bakr MM, Kelly WL, Brunt AR, Paterson BC, Massa HM, Morrison NA, Forwood MR. A
Single injection of PTH improves osteoclastic parameters of remodeling at a stress fracture site in rats. J Orthop Res. 37: 1172-82, 2019
The work from the daily iPTH and combined ALN-PTH experiments (Chapters 5 and 6) is ready for submision.
Bakr MM, Kelly WL, Brunt AR, Paterson BC, Massa HM, Morrison NA, Forwood MR.
Intermittent parathyroid hormone (PTH) accelerates stress fracture remodeling more effectively following cessation of bisphosphonate treatment.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page xxvii
Chapter 1
Introduction 1. Introduction
1.1. Overview:
Bone remodeling is necessary to maintain the integrity of cortical and cancellous bone throughout life. It turns over aged bone tissue, removes bone microdamage, reorganizes fracture callus and repairs stress fractures (SFx) (Frost, 1963; Hattner et al, 1965; Kidd et al., 2010). But dysregulated bone remodeling impairs bone quality and is central to many diseases of bone, such as osteoporosis and Paget’s disease (Burr and Martin, 1989). Repetitive loads can cause microstructural damage and remodeling activity in a positive feedback cycle that precipitates
SFx (Burr et al., 1997). Following the activation event, resorption is the first phase of remodeling and therefore contributes to repair of microdamage; but can also lead to SFx progression due to the initial transient porosity (Burr et al., 1997; Kidd et al., 2010).
SFx is a serious consequence of intensive training, accounting for 10% of cases in a typical sports medicine practice, with prevalence in athletes and recruits reported to range from 0.2% to as high as 49% (Kiuru et al., 2005). In runners, prevalence ranges from 10% to 31% with the tibia being the most affected bone in both sexes (Spitz and Newberg, 2002). During basic military training, the prevalence can be as high as 30% (Milgrom et al, 1985). Changes to training and management for Israeli recruits brought the SFx incidence down from as high as
40%, in some cohorts to about 10% in 2015 (Milgrom and Finestone, 2017)
Atypical diaphyseal fractures of the femur, in patients on long-term bisphosphonates (BPs), are also classified as SFxs (Odvina et al., 2005; Lenart et al., 2009; Neviaser et al., 2008). Atypical
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 2 femoral fractures (AFF), verified by x-ray, occur in 2 out of 100,000 patients per year for 2 years of alendronate (ALN) use, but increase to 78 out of 100,000 patients per year for 8 years of use, supporting an association with duration of ALN treatment (Andrews, 2011).
Nonetheless, these rates remain low relative to the rate for all hip fractures of 103/10,000 person per year (Shane et al., 2010).
In Australia, (Girgis and Seibel, 2011) reported that the prevalence of AFF in one region of
New South Wales (NSW, Australia) increased from 0.2/10,000 patient-years in the general population to 10/10,000 patient-years. in patients receiving BPs. These fractures contribute to the economic burden for health care costs, but significantly to morbidity and mortality for individual patients. In patients diagnosed with AFF, the mortality is up to 25% after 2 years; and of those patients who survive, 50% are not back to pre-injury function after 2 years (Girgis and Seibel, 2011). Atypical femoral fractures have attracted international attention, including a task force of the American Society of Bone and Mineral Research (Shane et al., 2010, Shane et al.,2014); and. in several parts of the world, regulatory action has enforced a special warning about these fractures in the Summary of Product leaflet of ALN, the most commonly used BP
(Compston 2010).
Intermittent PTH (iPTH) has an anabolic effect in bone (Sibai et al., 2011). Different molecular mechanisms of iPTH action have been suggested; either by increasing the number of osteoprogenitor cells (Aubin et al., 2002, Balani et al., 2017), inhibiting precursor cells of osteoblasts from undergoing apoptosis (Manogalas, 2000, Balani et al., 2017), and enhancing proliferation of preosteoblasts and transforming bone lining cells into osteoblasts (Dobnig and
Turner, 1995; Jilka et al., 2009, Kim et al., 2012). Other documented mechanisms by which
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 3 iPTH enhances healing include the increase in synthesis of bone matrix proteins; as well as an increase in osteoclastogenesis during the remodeling phase of repair (Nakajima et al., 2002). In addition, iPTH enhanced the early stages of endochondral bone repair by increasing chondrocyte recruitment and differentiation. Furthermore, the anabolic effect of iPTH is controlled by an increase in canonical Wnt signaling (Kakar et al., 2007)
Continuous (eg infusion) or intermittent presentation of PTH creates a significant difference in its action, often referred to as catabolic or anabolic (Hruska et al 1991). For example, continuous administration of PTH leads to an increase in osteoclast number and activity, resulting in bone loss (Mundy and Roodman, 1987), whereas intermittent administration leads to an increase in trabecular bone formation (Dempster et al., 1993; Finklestein et al., 1996).
This is most likely a result of the combination of enhanced osteoblast function (Hock, 1999) and inhibition of osteoblastic apoptosis (Jilka et al., 1999). iPTH treatment also enhances fracture healing (Campbell et al., 2015). Daily systemic treatment with iPTH (1-34) at a dose of 5-30µg/kg per day increases callus formation, and doses ranging between 10-200 µg/kg per day increase callus stiffness and strength (Alkhiary et al., 2004; Andreassen et al., 1999; Holzer et al., 1999; Komatsubara, 2005; Nakajima et al., 2002). In humans, preliminary data suggest that iPTH (1-34) at the usual dose for osteoporosis treatment (20µg/kg) accelerates fracture union (Aspenberg et al., 2010). To conclude, PTH can be considered as a double edge sword for regulation of bone metabolism and turnover with its anabolic and catabolic effects, and its complex signaling mechanisms (Qin et al., 2004).
The main therapeutic limitation of iPTH is that its use is restricted to two years due to an indication of increased risk of prostate cancer (Schneider et al., 2005) and osteosarcoma, observed in rats during preclinical testing (Vahle et al., 2002; Tashijan and Chabner, 2002; Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 4
Tashjian et al., 2006). The question is whether iPTH treatment remains effective if used concurrently with BP treatment. Sloan et al., 2010 studied the effect of iPTH and ALN treatment and found that iPTH may accelerate intracortical bone remodeling induced by microdamage and ALN may delay intracortical bone remodeling during SFx repair in rats. The study did not investigate mechanisms of the iPTH effect in SFx healing, whether SFx repair was complete following treatment, nor whether BP treatment blunted the iPTH anabolic effect for SFx healing. It is known, for example, that the BPs may blunt subsequent iPTH therapy
(Gasser et al., 2000, Gasser et al., 2005), but it is not a universal observation (Boonen et al
2008).
The use of iPTH has been investigated in a number of conditions including osteoporosis in men
(Kurland et al., 2000; Orwoll et al., 2004), fractures and bone mineral density in postmenopausal women with osteoporosis (Neer et al., 2011), cortical bone architecture in postmenopausal women with osteoporosis (Ascenzi et al., 2012), and hyperparathyroidism
(Rubin et al., 2011). BPs, including ALN, have a long track record in clinical trials for osteoporosis treatment (Cranney et al., 2002a; Cranney et al., 2002b) and fracture prevention
(Black et al., 2006). Furthermore, combined iPTH and ALN treatment has been tested in clinical trials investigating glucocorticoid induced osteoporosis (Saag et al., 2007; Saag et al., 2009) and bone mineral density (BMD) in postmenopausal osteoporosis (Black et al., 2003).
In this study, there is an opportunity to test alternative treatments to accelerate SFx healing, while osteoporosis treatment continues. This could prevent unusual SFxs, in patients who still need bone-sparing effectiveness of osteoporosis treatment. It is imperative, therefore, that effects of BP on iPTH therapy for SFx are determined. This project has the opportunity to
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 5 significantly advance knowledge of iPTH mechanisms in repair of SFx, and to determine its efficacy in the presence of a BP.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 6
1.2. Aim of the study:
The overall aim of the study was to determine the efficacy of iPTH treatment to accelerate indices of SFx healing in the rat ulna in the presence or absence of ALN.
1.3. Hypotheses:
Hypothesis 1 (Chapter 4)
1. A single treatment with PTH, 24 hours following SFx creation, will accelerate
histomorphometric indices of SFx healing.
Hypotheses 2 and 3 (Chapter 5)
2. A daily treatment with iPTH for 14 days, starting 24 hours following SFx, will
accelerate histomorphometric indices of SFx healing.
3. A daily treatment with iPTH for 14 days, starting 24 hours following SFx is more
superior than a single PTH injection.
Hypotheses 4 and 5 (Chapter 6)
4. Combined ALN-PTH treatment is more superior than daily iPTH in accelerating
histomorphometric indices of SFx healing.
5. Treatment with iPTH, will accelerate histomorphometric indices of SFx healing, even
in the presence of concurrent ALN treatment.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 7
1.4. Significance and innovation:
Effectively treating SFx requires knowledge of their etiology. While successive groups sought to demonstrate in vivo repair of microdamage (Burr et al., 1985; Mori and Burr, 1993); and its progression to SFx (Li et al 1985), the ulnar loading model in rats became an experimental standard to examine transitions from microdamage to SFx and subsequent repair (Bentolila et al. 1998; Hsieh et al 2002; Kidd et al 2010). Forwood’s group tracked directed remodeling of an ulnar SFx over a 10-week period. They identified stabilization of the of the periosteal exit point by woven bone, excavation of the full SFx line by resorption; and, completion of remodeling via new bone formation along the crack line (Kidd et al., 2010). This work also established extensive data characterizing gene expression over the duration of remodeling from initiation to conclusion. Subsequently, Kidd et al (2011) observed that healing of SFx was retarded by BP treatment via its action on the resorption and formation phases of remodeling.
These data suggest a mechanism whereby inhibition of remodeling by BPs, retards SFx healing, contributing to the etiology of AFF. Optimizing the rat model of SFx has provided an innovative approach to examine focal remodeling with a known time course and precise anatomical location. This offers a ground-breaking opportunity to test potential therapeutic targets for treating SFx, including AFF; and to advance understanding of the mechanisms whereby iPTH might accelerate that healing.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 8
Chapter 2
Review of Literature
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 9
2. Review of literature
2.1. Bone Composition and Microstructure
2.1.1. Types of bone
Bone is a polyphase material composed of fibrous organic matrix permeated by large stores of mineral (inorganic) salts. It forms about 14 percent of the adult body weight (Crowther and
Mourad, 2002). Water accounts for about 20 percent of the wet weight of mature cortical bone, bone salts about 45 percent and organic substances account for the remaining 35 percent (Carter and Spengler, 1978). The organic component is primarily collagen (about 90-95 percent), glycosaminoglycans (one percent) and other non-collagenous proteins (five percent) (Frankel and Nordin, 2001). Periosteum covers the external surface of bone and consists of two layers: an outer fibrous layer and an inner more cellular and vascular layer. The inner osteogenic layer or cambium layer can form new bone while the outer layer forms part of the insertions of tendons, ligaments and muscles (Buckwalter and Cooper, 1987).
Bone is classified into either cortical or cancellous bone. Compact bone comprises 85% of the skeleton, is strong, solid, and very organized. Its basic structural unit is the Haversian system
(osteon), a cylindrical entity that runs along the long axis of the bone. It is comprised of concentric rings of matrix called lamellae, which contain small holes called lacunae with an osteocyte in each. The center of the Haversian system is an opening called the Haversian canal, which contains blood vessels and nerves. Haversian canals and lacunae are connected to each other by canaliculi that are oriented parallel to the horizontal axis of the bone. Cancellous
(spongy) bone makes up 15% of the skeleton, and most bones contain at least some of each
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 10 type. The vertebrae are predominately cancellous bones, and flat bones such as the skull and ilium also contain a greater percentage of this spongy bone. It lacks the Haversian system, and the lamellae are arranged instead as trabecular plates or rods that branch and connect with each other to form a meshwork. The pattern of meshwork is not random but is determined by the stress to which the bone is subjected (Crowther and Mourad, 2002).
Cortical and cancellous bone are essentially one material, the porosity of which varies over a wide range (Carter and Hayes, 1977). This parameter has an important influence on the mechanical properties of bone tissue. The porosity of cortical bone varies from five to 30 percent whilst that of cancellous bone ranges from 30 to 90 percent (Carter and Spengler, 1978;
Frankel and Nordin, 2001).
2.1.2. Bone microstructure
Developmentally, bone has been classified as either primary or secondary. Primary bone is formed by endochondral ossification or subperiosteal deposition (intramembranous ossification) and is the first bone formed in a region (Carter and Spengler, 1978). During growth, and subsequent remodeling, bone is continually resorbed in the presence of osteoclasts and deposited by osteoblasts (Frost, 1963). Bone formed by this process is termed secondary bone. The types of primary bone found in large mammals are circumferential lamellar bone, woven fibred bone and primary Haversian bone (primary osteons) (Currey, 2002). Woven fibred bone usually contains large irregularly shaped vascular spaces, with osteoblasts lining the surrounding surface. These osteoblasts deposit successive layers of new bone in a lamellar arrangement which progressively reduces the diameter of the vascular space. This process
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 11 forms a system of anastamosing tunnels termed primary osteons or primary Haversian systems
(Carter and Spengler, 1978; Currey, 2002).
As maturation continues, a small singular vascular canal surrounded by successive layers of circumferential lamellae is formed. In many small animals, such as the laboratory rat, the predominant vascular system is the non-Haversian canal (Enlow, 1962; Singh and Gunberg,
1970). Histologically, these bear superficial resemblance to primary osteons. They are easily distinguished, however, by the lack of cement lines or other sharply defined borders; and by the absence of circumferential lamellae (Singh and Gunberg, 1970). In humans, both Haversian and lamellar bone are interspersed throughout the diaphysis. But in the rat, Haversian bone is almost entirely absent, present only near the major nutrient vessels (Singh and Gunberg, 1970).
2.1.3. Modeling and remodeling
In the process of tunnelling and refilling described above, groups of cells function as organised units called "basic multicellular units" (BMU) by Frost in 1963. This name implies that cells in adult bone do not function independently as they might in other tissues, but form part of an organisation of cells that control and mediate bone homeostasis. BMU's, therefore are the functional units of bone in which osteoblasts and osteoclasts act in coordination, the process of which is termed "coupling" (Frost, 1963). Because BMU's act as a cellular unit, they resorb and form bone in "quantized" packets (Parfitt, 1979). The cycle of remodeling by which BMU's create quantized packets of bone follows well-defined phases which always begin with activation, followed in sequence by resorption and formation, and is sometimes called the A-
R-F sequence of remodeling (Frost, 1963).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 12
In the literature there has been a tendency to describe any process that involves resorption or formation of bone resulting from functional adaptation as "remodeling". According to Martin and Burr (1989), remodeling in the strictest sense refers to the replacement of bone via the A-
R-F sequence, and should be distinguished from "growth", as the enlargement of skeletal tissue without regard to changes in shape. Modeling on the other hand, involves the addition of bone to surfaces of lamellar bone without prior resorption (A-F), or the resorption of bone from lamellar surfaces without subsequent formation (A-R) (Martin and Burr, 1989). That is, only resorption or formation occurs at a given surface. Modeling is responsible therefore, for the alteration of bone shape during growth and for the movement of bone surfaces through space, for example during mechanical adaptations, termed cortical drift (Frost, 1987a).
2.1.4. Differences between human and rat bones
Animal fracture models especially in rats are very important to investigate the outcomes of fracture healing. They constitute about 38% of all animals used in fracture experiments
(O’Loughlin et al., 2008). However, results of biomechanical testing often show great variability and could skew the statistical significance of results. Therefore, efforts have been made to improve the validity of rat fracture models and standardizing biomechanical testing procedures in small animal studies by increasing the efficacy of mechanical testing and modifying the experimental setup (Leppanen et al., 2008; Reifenrath et al., 2014; Prodinger et al., 2018a).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 13
The mechanical properties of different bones in mice were tested and the radius was preferred for mechanical testing because of its high aspect ratio, minimal measurement error, and low variability (Schriefer et al., 2005). In another study, rat femora showed the most accurate and consistent radiological and biomechanical results (Prodinger et al., 2018b). In some experiments, an intraindividual variation between left and right bones in the same animal was observed and could be explained by the development of paw preferences in rats resulting in one favoured side (preferentially used for example as leading leg in climbing) and causing biomechanically relevant differences (Cunha et al., 2017; Prodinger et al., 2018a). The principle of handeness in animals might have been overlooked in the past and needs to be investigated in the future for standardization across different experiments in order to compare results from multiple studies and reach a consensus.
The rat skeleton shares many similarities with the human skeleton. For example, the temporomandibular joint (TMJ) shows that in general, the articular structure of rats was similar to that in humans. However, the rat TMJ had no articular eminence (Porto et al., 2010). The cellular phenotype of osteoblasts and osteoclasts is similar in rats and humans. Therefore, the effect of medication on bone and fracture healing in rats allows deductions about the human situation, designating these models ideal in exploring unknown or adverse effects on bone.
However, the absence of the Haversian system and the presence of permanently open growth plates in rodents raise concerns about some possible discrepancies and prevents direct translation of the results to human (Peric et al., 2015). In conclusion, selecting the proper animal fracture model (including the bone site) as well as the appropriate mechanical testing methods that includes loading the bones in an anatomically correct configuration and at the largest
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 14 possible aspect ratio (length/diameter), seems to be the gold standard to reduce the sample size, minimize errors and eliminate possible variation in results (Vashisthth, 2008).
2.1.5. Functions and activities of bone
In addition to providing strength, support for the body, and serving as the attachment for muscles to allow movement to occur, bone is extremely active on a metabolic level and is important in the regulation of several endocrine and mineral metabolic functions (Quarles,
2008). It is the major source of calcium; it buffers against acidosis and can adsorb toxins and heavy metals. Calcium, phosphate, and magnesium are minerals vital to the function of all cells
(Sarko, 2005).
Bone has the unique ability to heal without the formation of a fibrous scar. Bone healing and repair is a complex process that involves the regulation of certain growth factors and cytokines.
This correlates with the activation, proliferation and differentiation of certain bone cells
(Kasama et al., 2007; Stegen et al., 2015). Bone microstructure is an important regulator of bone function. For example, mesenchymal stem cell (MSCs) migration and osteoblast alignment can reconstruct bone microstructure, which guide an appropriate bone formation during bone remodeling and regeneration (Ozasa et al., 2018). Bone structure, activities and functions are dependent on complex interactions between cells, matrix, cell-derived factors, and systemic factors (Boskey and Posner, 1984).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 15
2.2. Bone cells
2.2.1. Osteoblasts
Osteoblasts are the bone matrix forming cells that could differentiate from a variety of progenitor populations such as bone marrow, neural crest, and periosteal cells. Despite their short life span (approximately two weeks) they play a vital role in bone formation. Osteoblasts have all the criteria of protein forming cells including abundant rough endoplasmic reticulum, mitochondria, golgi apparatus and secretory vesicles in order to form type-I collagen within the bone matrix (Kassem et al., 1992). Eventually osteoblasts become bone-lining or osteocytes that are responsible for the maintenance of the already formed bone matrix (Jilka et al., 1998;
Capulli et al., 2014; Bradley et al., 2019). At the end of their cycle, osteoblasts could also undergo the process of programmed cell apoptosis (Jilka et al., 1998). Certain agents reduce or inhibit osteoblast and osteocyte apoptosis. These include estrogen, selective estrogen receptor modulators, BPs, calcitonin, CD40 Ligand, Calbindin‐D28k, Monocyte Chemotactic
Proteins (MCP-1), MCP-3, and mechanical loading in the form of fluid flow shear stress
(Bonewald, 2007).
To maintain normal bone homeostasis, bone formation is linked to bone resorption. Failure of coupling between the two processes can lead to bone disorders and changes in bone mass (Sims and Martin, 2014). Therefore, osteoblasts interact with different cell types including osteoclasts through direct cell contact, cytokines and signals transmitted through the bone matrix (Tamma and Zallone, 2012; Chen et al., 2018). The exact mechanism of communication and interaction between osteoblasts and osteoclasts remains incompletely understood (Phan et al., 2004;
Mundy and Elefteriou, 2006). The correlation between osteoblasts and osteoclasts is
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 16 bidirectional. It was suggested earlier that osteoblasts can affect osteoclast formation (Rodan and Martin, 1981). Osteoblasts regulate differentiation of osteoclasts through expression of
Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL/TNFSF11) and monocyte colony stimulating factor (M‐CSF 1) (Takahashi et al., 1988). Osteoblasts also produce osteoprotegerin (OPG/TNFRSF11B) that blocks this interaction. Osteoclasts, in turn, produce several cytokines including WNTs, BMP6, and TGFβ to recruit osteoblast progenitors to the bone surface (Pederson et al., 2008). Later, studies demonsatrated that osteoclasts can also regulate osteoblast activity (Matsuoka et al., 2014; Wang et al., 2015).
2.2.2. Bone lining cells
Bone lining cells are dormant flattened osteoblasts that cover the inactive or non-remodeling surfaces (Miller et al., 1989; Matic et al., 2016). They appear thin with a flat nuclear profile; their cytoplasm displays few cytoplasmic organelles such as profiles of rough endoplasmic reticulum (Miller et al., 1989). Some bone lining cells exhibit cellular processes extending and gap junctions that separate adjacent bone lining cells and could extend between these cells and osteocytes (Miller et al., 1989; Aarden et al., 1994).
Bone lining cells could be reactivated under certain conditions and regain their secretory functions depending on the overall physiological status of bone and functional demands
(Donahue et al., 1995). Bone lining cells functions are not completely understood, they could prevent the direct interaction between osteoclasts and bone matrix to prevent bone resorption, they also participate in osteoclast differentiation, producing osteoprotegerin (OPG) and
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 17
RANKL (Mosley, 2000; Andersen et al., 2009). Furthermore, bone lining cells could be differentiated into active osteoblasts if treated with iPTH (Kim et al., 2012).
2.2.3. Osteocytes
Osteocytes make up over 90–95% of all bone cells in an adult human skeleton compared with
4–6% osteoblasts and around 1–2% osteoclasts (Franz-Odendaal et al., 2006). Osteocytes are derived from MSCs lineage through osteoblast differentiation. In this process, four recognizable stages have been proposed: osteoid-osteocyte, preosteocyte, young osteocyte, and mature osteocyte (Franz-Odendaal et al., 2006). They reside in lacunae and form an interconnected network through dendritic processes in canaliculi (Baud, 1968). Through this network, steocytes are believed to act as sensors, mediators of mechanical loading and respond by sending signals that control remodeling (Bonewald, 2011; Bonewald, 2019).
The mechanosensitive nature of osteocytes explains their role in remodeling in response to fatigue damage, they also allow transport of cell signaling molecules, nutrients and eliminate waste products (Klein-Nulend et al., 2003). Wnt/β‐catenin signaling pathway is responsible for osteocytes’ response to mechanical loading (Duan and Bonewald, 2016). Osteocytes communicate together and to the outer vascular and bone marrow spaces. They regulate phosphate and have been described as an endocrine gland through their network functions
(Dallas et al., 2013). Integrins are the most common adhesion molecules responsible for mediating the interaction between bone cells and bone matrix (Zimmerman et al., 2000).
Integrins are highly crucial for generating and amplifying the signals resulting from bone tissue deformation transduced by the mechanosensitive osteocyets (Wang et al., 2007).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 18
Osteocytes undergo apoptosis which can act as a chemotactic signal for osteoclast recruitment
(Noble et al., 1997; Verborgt et al., 2000; Kousteni et al., 2002; Vanderschueren et al., 2004;
Cardoso et al., 2009). Furthermore, osteoclasts are capable of engulfing apoptotic osteocytes
(Taniwaki and Katchburian, 1998; Boabaid et al., 2001; Cerri et al., 2003). In fact, osteocytes are known to act as orchestrators of the bone remodeling process, producing factors that influence osteoblast and osteoclast activities (Rochefort et al., 2010). Impaired osteocytic function results in bone diseases such as osteoporosis and glucocorticoid induced bone fragility in adults, as well as nonbone diseases such as renal problems, skeletal and cardiac muscle impairment (Bonewald, 2017).
2.2.4. Osteoclasts
Osteoclasts differentiate from the monocyte/macrophage lineage cell of hematopoietic origin.
Osteoclasts are multinucleated and can be generated in vitro from mononuclear monocyte/macrophage (Takahashi et al., 1994; Quinn et al., 1994; Udagawa, 2002; Boyle et al., 2003) Two cytokines are essential for osteoclastogenesis, the first is the receptor activator of nuclear factor‐κB ligand (RANKL) and the second being M‐CSF, also known as colony stimulating factor-1 (CSF‐1) (Suda et al., 1999; Theill et al., 2002; Udagawa, 2002; Ross et al.,
2005). Besides the well-established interaction between osteoclasts and osteoblasts, other cells types are also important for osteoclast biology and homeostasis. This cell to cell interaction occurs within the bone marrow and provides an interface for the emerging disci-pline of osteoimmunology (Takayanagi, 2007; Lorenzo et al., 2008; Takayanagi, 2009; Takayanagi et al., 2012).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 19
The mechanism of bone resorption by osteoclasts is unique as it requires physical intimacy between the resorptive cells and the bone surfaces. This creates an isolated mircoenviroment that is highly acidic through the creation of an electrogenic proton pump (H+-ATPase) and a
Cl– channel to a pH of ~4.5 (Boyle et al., 2003). This results in mobilization of the inorganic hydroxyapatite crystals and leads to the exposure of the organic matrix that is degraded later by enzymes such as collagenase and cathepsin K (Baron, 1989; Teitelbaum et al., 1995;
Teitelbaum, 2000; Teitelbaum, 2007; Vaananen et al., 2000).
During this process osteoclasts undergo a process of polarization this is facilitated by the actin cytoskeleton in which an F-actin ring consisting of a dense continuous zone of highly dynamic podosome is formed, followed by an area of membrane that develops into the ruffled border is isolated (Lakkakorpi et al., 1991; Saltel et al., 2004; Chabadel et al., 2007; Luxenburg et al.,
2007; Arana-Chavez, Bradaschia-Correa, 2009; Khan et al., 2013). The ruffled border is highly important to maintain an acidic zone to dissolve the hydroxyappetite crystals (Kornak et al.,
2001; Graves et al., 2008; Crockett et al., 2011). Within the ruffled border, protons and enzymes, such as tartrate-resistant acid phosphatase (TRAP), cathepsin K, and matrix metalloproteinase-9 (MMP-9) are secreted into a bay-like depression called Howship lacunae resulting in matrix degradation (Yamaza et al., 1998; Mulari et al., 2003; Ljusberg wt al., 2005;
Graces et al., 2008; de Souza Faloni et al., 2012). The resulting waste products are endocytosed across the ruffled border and transported to the plasma membrane with the help of the mechanical action by the ruffled border (Everts et al., 2002; Arana-Chavez and Bradaschia-
Correa, 2009).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 20
2.2.5. Osteal tissue macrophages (OsteoMacs)
Macrophages have a close association with bone (Hume et al., 1984) and osteoclasts
(Teitelbaum, 2007) as they share a common growth factor (CSF-1) that is specific to both cells
(Teitelbaum, 2007). They have been isolated from fracture repair sites and are thought to play a role in fracture healing (Andrew et al., 1994). Furthermore, they are known to be contributing to the pathogenesis of pathological conditions associated with bone such as osteoporosis (Cenci et al., 2000) and rheumatoid arthritis (Kaneko et al., 2001). A unique type of macrophages that is resident within osteal tissue was identified and named OsteoMacs (Chang et al., 2008).
OsteoMacs are present together with bone lining cells within both endosteum and periosteum.
They regulate osteoblast function and important role in bone dynamics (Chang et al., 2008).
With the concept of osteoimmunology rapidly emerging and demonstrating the links/interactions between bone and immune cells (Arron and Choi, 2000), it is highly important to recognize the role of OsteoMacs in bone homeostasis and ther interaction with other bone cells. Osteomacs are important for osteoblast mineralization in response to elevated extracellular calcium (Dvorak et al., 2004; Chang et al., 2008); and are present in bone modeling sites (Chang et al., 2008). They could be responsible for stimulating the coupling process that occur between osteoblasts and osteoclast in the bone remodeling process (Martin and Sims,
2005; Chang et al., 2008). Given their proximity to bone surfaces and the well-known ability of macrophages to detect and engulf dying cells (Henson and Hume, 2006), OsteoMacs are in a very good position to sense and respond to bone damage and apoptotic cell death that is essential for bone remodeling and osteoclast recruitment (Jilka et al., 2007). Finally, OsteoMacs
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 21 could have a wide range of clinical applications in bone regeneration (Batoon et al., 2017) and bone biomaterials (Miron and Bosshardt, 2016).
2.3. Factors affecting healing and bone remodeling
2.3.1. Mechanical loading and the mechanostrat
Bone undergoes constant remodeling to meet requirements of mineral homeostasis, and to renew ageing and damaged bone matrix (Jaworski, 1984; Mori and Burr, 1993). It helps achieve different functional goals including balance of minerals in the body, adaptation to mechanical loading to reduce the risk of fractures and allow for repair of mechanical damage that was caused by repetitive or cyclic loading (Burr, 2002). In order to achieve the above-mentioned functional goals, two different types of bone remodeling have been investigated, targeted (site- dependent) and non-targeted (Stochastic) remodeling (Martin and Burr, 1989; Parfitt, 1996;
Parfitt et al., 1996; Burr, 2002).
Remodeling involves bone resorption of existing bone by osteoclasts, followed by subsequent formation of new bone by osteoblasts (Scully and Besterman 1982; Lanyon et al., 1984;
Albright and Skinner, 1987; Albright, 1987; Reid et al., 1991; Morris et al., 1997). Regular activity helps maintain bone strength and quality through the activities of bone modeling and remodeling. Frost's mechanostat theory (Frost, 1987a) later referred to as the Utah Paradigm
(Frost, 2001) is unique because it distinguishes between modeling and remodeling processes, thresholds for activating lamellar or woven bone formation and can be applied to the etiology of osteopenia and osteoporosis. In the Utah Paradigm, bone adapts by different biological
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 22 processes within four mechanical usage windows, with thresholds defined by minimum effective strains (MES) at which adaptation occurs (Frost 1983).
Conversely, modeling can add cortical and trabecular bone, loading in the overload zone and remains off within, or below, the physiological loading zone. It also predicts addition of woven bone to surfaces in response to extreme loading in the pathological loading zone. Moreover, the threshold loads for activation, "set points", may be influenced by humoral or local factors (such as hormones, steroids or drugs) or metabolic bone diseases such that the adaptation effected by them mimics the response expected for the perceived change in mechanical loads (Frost 1987,
Burr and Martin, 1989).
2.3.2. Remodeling cycle and activation
As described above, these bone metabolic activities (modeling and remodeling) depend on the coordinated activity of osteoclasts, osteoblasts and osteal macrophages. Following an activation event, remodeling proceeds by initiation of resorption, a reversal phase characterized by presence of pre-osteoblasts, osteoblastic formation and then termination of the remodeling cycle. Activation events can be linked to disruption of the bone matrix, or to endocrine control of mineral metabolism. In 1960, Frost first described microcracks in bone in vivo (Frost, 1960).
This was followed by a long period of scepticism in the field until an association between microdamage and resorption spaces in vivo emerged in the 1980’s (Burr et al., 1985).
Subsequently, experimental evidence demonstrated that remodeling “targeted” microdamage caused by fatigue loading of bone (Mori and Burr 1993; Bentolila et al., 1998; Kidd et al 2010;
Hsieh and Silva, 2002; Silva et al., 2011). As Frost predicted, osteocyte apoptosis, direct
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 23 osteocyte damage or interruption of canalicular connections between cells by microcracks were reported stimuli for local remodeling responses to fatigue microdamage (Colopy et al, 2004;
Noble et al, 2003; Verborgt et al, 2000).
Furthermore, local remodeling sites are more frequently found in association with sites of microdamage. This shows a direct correlation between microdamage and activation of repair in the sense that remodeling events are targeted towards areas of microdamage (Burr et al., 1985;
Burr and Martin, 1993; Mori and Burr, 1993). In a rat model, it has been shown that intracortical remodeling could take place in response to fatigue loading in rats which do not usually exhibit intracortical remodeling in normal circumstances (Bentolila et al., 1998). The amount of targeted or damage-initiated remodeling is about 30% of the total bone remodeling (Mashiba et al., 2001b). It was hypothesized that BPs could minimise stochastic (non-targeted) remodeling through different mechanisms including the inhibition of new remodeling units, or termination of osteoclastic resorption (Parfitt et al., 1996). On the contrary another study suggested that BPs could not preferentially suppress one type of remodeling (stochastic) without affecting the other type (targeted) (Mashiba et al., 2000).
In addition to microdamage repair, remodeling fulfils calciotropic functions in response to endocrine regulation and release of factors in the bone microenvironment (Raggatt and
Partridge, 2010). These factors include, insulin growth factor-I (IGFI), parathyroid hormone
(PTH), tumour necrosis factor- ∝ (TNF- ∝) and interleukin-6 (IL-6) (Suda et al., 1999;
Takayanagi, 2006; Rucci, 2008). Regardless of the activation event, these different inputs provide signals to osteoblastic cells to increased surface expression of RANKL. The pre-
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 24 osteoclasts also present within the bone microenvironment, express RANK, which is a member of the Tumour necrosis factor (TNF) receptor family and more pertinently is the receptor for
RANKL (Yasuda et al., 1998). The interaction of RANK and RANKL, activates the transcription factor NF-κβ, which is an osteoclast transcription factor that drives the expression of genes that stimulate osteoclast formation and differentiation (Yasuda et al., 1998). More specifically, these genes activate mononuclear monocyte-macrophage osteoclast precursors, which invade the endosteum containing the lining cells on the bone surface and fuse multiple mononuclear cells to form multinucleated preosteoclasts (Kim et al, 2005).
As explained above, the remodeling process involves extensive cross talk between different types of bone cells and regulatory proteins through complicated interactive mechanisms (Kular et al., 2012). In the resorption phase, the now differentiated osteoclasts attach to the surface of the bone and dissolve the bone through a two-fold process. First, the inorganic bone constituent is corroded via acidification, and then the organic component is degraded by enzymatic lysosomes, such as cathepsin K (Teitelbaum, 2007; Rucci, 2008). Then, to prevent rapid excessive bone resorption, the osteoclasts undergo cell death, known as apoptosis (Rucci,
2008). This phase is synonymous with the catabolic pathway. The reverse phase is when bone resorption is transformed to bone formation. This involves cells that have similar properties to macrophages that remove the debris that remains after the acidification of inorganic bone and the enzymatic degradation of organic bone (Rucci, 2008). Directly following the reverse phase is the fourth and final formation phase. This entails osteoblast activity wherein new bone matrix is synthesized and matrix mineralization proceeds (Clarke, 2008). When bone formation is finalized, majority of the osteoblasts experience apoptosis and the remaining osteoblasts revert into bone lining cells or osteocytes (Clarke, 2008).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 25
As noted above, a link between microdamage and activated remodeling dates back to 1960
(Frost, 1960). It was hypothesized that damage related to fatigue stimulates bone remodeling in the form of a negative feedback loop (Frost, 1960; Frost, 1966; Enlow, 1977; Martin and Burr,
1982). When challenged by fatigue or repetitive cyclic loading, the number of resorption spaces increase in cortical bone (Burr et al., 1985; Mori and Burr 1993; Kidd et al 2010; Hsieh and
Silva, 2002; Silva et al., 2011). This may accelerate the production of a SFx as it decreases the modulus of elasticity (Scully and Besterman, 1982; Burr et al., 1997). If fatigue damage continues, this will stimulate woven bone formation on the external surface. This is an important mechanism to prevent the formation and/or progression of SFx, as it stabilizes the bone that is weakened by the internal remodeling (Johnson et al., 1963; Uhthoff et al., 1985).
In addition to the above, it was proposed that osteoporotic fracture may be a consequence of a positive feedback between damage accumulation and the increased remodeling space associated with repair (Burr et al., 1997).
A mathematical model for repair of fatigue damage and SFx was used to answer to the questions above as well as the answers to the questions of: (a) How does the half-life of fatigue damage
(the time required for half of a bolus of damage to be removed) compare with the duration of the remodeling cycle (b) Whether the porosity is associated with the remodeling response contributing to SFx, even as damage is repaired (Martin, 1995). The model adapted to increased loading by increasing remodeling to repair the additional damage and by adding new bone periosteally to reduce strain. However, if too much loading was encountered, the porosity associated with increased remodeling caused the system to become unstable; i.e., damage, porosity, and strain increased at a very high rate and without limit. It is proposed that this
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 26 phenomenon is the equivalent of a SFx and that its biological and mechanical elements are significant in the etiology of SFxs.
Activation of osteoclasts is necessary to initiate remodeling (Cochran et al., 1968). Once osteoclasts are activated, they start forming cutting cones via the secretion of proteolytic enzymes to create tunnels. Each osteoclastic cone resorbs about 3 times its volume and form tunnels that are 3-10mm deep (Engh et al., 1970; Enlow, 1977). New Haversian canals are formed and then filled with newly formed mineralized matrix formed by osteoblasts as well as immature lamellar bone (Parfitt, 1984; Albright and Skinner, 1987). This process of osteoblastic support of the new Haversian canals starts 10-14 days after the onset of the remodeling (Li et al, 1985). The conversion of the newly formed lamellar bone into mature matrix lags a week behind and may take up to 6 months or more (Engh et al., 1970; Enlow, 1977; Albright and
Skinner, 1987) during which secondary mineralization proceeds slowly (Bala et al., 2010). The early phases of resorption and formation result in a temporarily weakened bone during the third week after onset of remodeling (Scully and Besterman, 1982; Li et a.l, 1985). SFxs are more likely to develop during this period, especially if stresses continue to be applied (Matheson et al., 1987; Greaney et al., 1983).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 27
2.4. Stress fractures:
2.4.1. Classification of bone stress injuries:
Stress fractures are a part of the bone stress injury pathology continuum. The continuum includes (in order) accelerated remodeling, stress reactions, SFxs and complete bone fracture.
Classifying the degree of stress injuries in bone could be a challenging process (Fredericson et al., 1995). It was revealed that about 26 different classification systems for SFx exist (Miller et al., 2011). Based on radionuclide imaging, a scheme categorizing bone stress as a continuum starting with normal remodeling, followed by fatigue and bone exhaustion and, finally, cortical failure and fracture (Roub et al., 1979). Another classification defined the boundary between different types of stress injuries and characterized a stress reaction as a stress injury that only exhibits various degrees of remodeling, while a SFx was defined as a stress injury that could result in ultimate failure of the associated bone (Jones et al., 1989). In fact, most SFx cases could be classified as stress reactions. Therefore, there was a need for developing other classification systems that take into consideration clinical, radiographic findings as well as management plans.
A four stage scintigraphic grading system was developed according to lesion dimension, bone extension, and tracer accumulation. The grading system starts from Small, ill-defined lesion with mildly increased activity in the cortical region (grade I), progressing to a lesion that is larger than grade I, well-defined, elongated lesion with moderately increased activity in the cortical region (grade II). Grade I and II represent mild lesions that could progress to more severe lesions. Grade III lesions are wide fusiform lesion with highly increased activity in the cortico-medullary region, grade IV lesions are wide extensive lesion with intensely increased activity in the transcortico-medullary region (Zwas et al., 1987). Furthermore, MRI was used
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 28 to examine the system mentioned above and allowed more separation of different grades of stress injuries, which is very helpful in clinical management (Arendt and Griffiths, 1997). More recently, MRI grading system of stress injuries together with clinical risk factors were used as predictors to determine the time required for athletes to return to sport (Nattiv et al., 2015).
Once a stress injury has progressed to the stage of being a SFx, it could be further classified using different grading systems. Firstly, SFx were classified into high risk and low risk SFx which mainly relies on location only (Boden and Osbahr, 2000; Boden et al., 2001). More recently, the Kaeding–Miller classification system for SFx was introduced and has the advantages of being simple and easy to use. It also registers key clinical features while being widely applicable and reproducible (Kaeding and Miller, 2013). This classification system uses three variables, fracture grade, fracture location and the diagnostic image modality used. Grade
I of this classification system shows evidence of SFx with no evidence of a fracture line or pain.
Grade II is the same as Grade I with evidence of symptoms and pain. Grade III is a non- displaced fracture line. Grade IV is a displacd fracture line (<2mm). Grade V describes non- union SFx (Kaeding and Miller, 2013). As a gold standard classification for SFx has not been developed yet, the Kaeding-Miller classification system is clinically relevant, and allows a better description and communication for clinical and research purposes.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 29
2.4.2. Pathophysiology and epidemiology of stress fractures
Stress fractures (SFx) account for about 1%-7% of all athletic injuries (Boden et al., 2000;
Gerhmann et al., 2006). They account for 10% of cases in a typical sports medicine practice, with prevalence rates ranging from as low as 0.2% to as high as 49% (Kiuru et al., 2005). The main difference between SFx and acute fractures is related to the nature of loading. An acute fracture typically occurs due to a single maximal loading, while SFxs occur due to repetitive submaximal loading (Boden et al., 2000). The diagnosis of a SFx starts with a symptomatic patient who has a stress reaction or grade II or III bone stress injury that is proven by a bone scan or magnetic resonance imaging (MRI) without a true fracture line (Zwas et al., 1987;
Fredericson et al., 1995; Kaeding and Miller, 2013). If the repetitive loading continues, the bone reaction can change into grade IV bone stress injury or a true SFx (Fredericson et al., 1995;
Boden et al, 2001). This sequence of events explains the higher incidence of SFx amongst runners and military recruits (Bennell et al., 1996; Boden et al., 2000; Boden et al., 2001;
Brunker et al., 1996; Cline et al., 1998 and Gerhmann et al., 2006).
In the literature, several terms have been used to describe the symptoms and radiographic finding associated with bone remodeling and tibial SFxs. Examples include "Shin Splint"
(Slocum, 1967; Detmer, 1986), "medial tibial syndrome" (Clement, 1975; Mubarak et al., 1982;
Michael and Holder, 1985) and "medial tibial stress syndrome" (Puranen, 1974). This demonstrates that bone's reaction to stress can include a wide variety of physical findings and radiographic presentations (Fredericson et al., 1995, Anderson et al., 1997; Zwas et al., 1987).
Under normal circumstances, bone can withstand stresses applied either through muscle pull or the shock of a weight bearing extremity against the ground or an object. Mechanical stress is
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 30 defined as the force per unit area of the load-bearing bone (Scully and Besterman 1982; Brukner et al., 1999). Low levels of these forces can cause deformation known as strain (Albright and
Skinner, 1987). There are several factors that affect the stress-strain bone response including: direction of the load, geometric outline of bone, surrounding muscular contraction, microarchitecture and bone density (Brukner et al., 1999). In most daily activities when the forces are removed, bone elasticity is sufficient to return bone to its original shape without damage, this is called the elastic range (Daffner and Pavlov 1992; Zachazewski et al., 1996). In cases where forces exceed the elastic range and falls within the plastic range (Scully and
Besterman 1982; Zelko and De Plama, 1986), a lower load may cause permanent damage and greater deformation (Chamay and Schantz, 1972; Skinner and Cook, 1982). Therefore, in cases of excessive muscle pull in non-weight bearing bones, many will occur in the regions of highest stress in the bone (Wolf et al., 2017), but a stress reaction may develop in the bone-tendon junction (Brukner et al., 1996; Lord et al., 1996). On the other hand, weakness or fatigue of shock-absorbing muscles allows more forces to be transferred to bone and make it more susceptible to stress SFxs due to an increase in strain rate, magnitude and location on bone
(Matheson et al., 1987, Yoshikawa et al 1994; Fyhrie et al., 1998; Milgrom et al., 2007).
Several intrinsic and extrinsic factors have been associated with SFx when determining its etiology (Fredericson et al., 2005; Khan et al., 1994 and Lee et al., 2011). Intrinsic factors include anatomical and biological that is variable between patients such as poor vascular supply, poor bone density, postural abnormalities and abnormal hormonal levels. Conversely, extrinsic factors include nature of activity, techniques or new training programs, issues with equipment or footwear, nature of training surfaces as well as nutrition (Maitra 1997;
Korpelainen, 2001 and Gerhmann et al., 2006). In addition to the above, muscle fatigue has also been included as a possible factor for the development of SFx in several studies. This is Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 31 assumed to be due to the biomechanical changes that occur after muscle fatigue that leads to reduced energy absorption by the muscles and changes in loading patterns (Stanitski et al.,
1978; Weist et al., 2004; Yoshikawa et al., 1994). Finally, females have higher incidence of
SFx (Nattiv et al., 2000; Shaffer et al., 2006; Nieves et al., 2010). The reasons behind that include, a wider pelvis, a more common genu valgum and 25% less muscle mass in females, which results in a compensatory foot pronation and an increased angle created at the insertion of the quadriceps muscle due to the greater genu valgus known as the Q-angle, (Barrow et al.,
1988; Maitra et al., 1997). Also, women with a history of a second/third metatarsal SFx demonstrated differences in loading in the middle forefoot (Queen et al., 2009). This could be a possible risk factor for future SFx injuries. The female athlete triad of disordered eating, amenorrhea and osteopenia (Yeager et al., 1993) is also an important factor in the etiology of
SFx in females, this concept was more recently expanded to cover the relative energy deficiency that may underlie this syndrome (Mountjoy et al, 2014).
In young athletic women, susceptibility to fracture is increased as a result of disordered eating, amenorrhea and low bone mass, termed the “female triad” (Yeager et al., 1993). In a recent study, the risk factors associated with a bone stress injury (BSI), including stress reactions and
SFxs were identified in female adolescents and young adults participating in competitive or recreational exercise activities. It was reported that the risk of BSIs increased from approximately 15% to 20% for significant single risk factors to 30% to 50% for combined risk factors associated with the female athlete triad. These data support the notion that the cumulative risk for BSIs increases as the number of Triad-related risk factors accumulates
(Barrack et al., 2014). The incidence of SFx amongst different races is also variable. SFx is more common in white military recruits than African - American and Hispanics (Brudvig et al.,
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 32
1983; Gardner et al., 1988). This could be explained by the difference in the overall bone density in whites when compared to the other groups (Cohn et al., 1977).
2.4.3. Biological and molecular concepts of SFx healing
Stress fractures (SFx) are classified into high risk SFx that have a low potential for spontaneous healing and need operative treatment, and low risk SFx that usually heal with non-operative management. High risk SFx include, medial malleolus, talus, navicular, second and third metatarsal base, fifth metatarsal, femoral neck, anterior cortex of the tibia and sesamoid SFx
(Boden and Osbahr, 2000). Low risk SFx include calcaneus, cuneiforms, cuboid, general metatarsal, posteromedial tibia, fibula, pelvis, femoral shaft and lateral malleolus (Boden et al.,
2001; Mayer et al, 2014). Another classification for SFx includes two types: fatigue and insufficiency fractures. Fatigue fractures are caused by abnormal stresses to normal healthy elastic bone. On the contrary, insufficiency fractures result from normal stresses applied on bone that is mineral deficient or abnormally inelastic, examples include osteomalacia, osteoporosis and rheumatoid arthritis (Daffner and Pavlov, 1992; Umans and Pavlov, 1994;
Speed, 1998).
Healing of SFx is a multistage process that involves complex but well-organized steps following injury. The biological processes driving these stages are regulated by cell signaling molecules that include resorption and formation gene expression and expression related to angiogenesis, including: (1) RANKL, OPG, TNFα, Interlukins (IL) such as IL-1α, IL-6, and
IL-11 (regulation of bone resorption); (2) BMP-2, Runx-2 (transcription factor), insulin growth factor-1 (IGF-1), and sclerostin gene (SOST; bone formation); (3) VEGF (angiogenesis); (4)
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 33 stromal cell-derived factor-1 [SDF-1] (chemotaxis); and (5) cyclooxygenase [COX-1 and
COX-2] and osteopontin [OPN] (multiple potential functions in bone biology) (Wohl et al.,
2009; Kidd et al., 2010). The expression of these biological mediators is also found at sites that are distant from the injury and the RANKL/OPG ratio is initially reduced then increases very early in SFx corresponding with the bone remodeling than happens earlier than fracture callus
(Fazzalari, 2011).
Bone fatigue repair is a process similar to an abbreviated form of intramembranous fracture repair. This is characterized by rapid formation of periosteal woven bone due to increases in the expression of genes associated with angiogenesis, cell proliferation and osteoblastogenesis.
In addition, the response from the local vasculature precedes the osteogenic response to fatigue loading (Wohl et al., 2009). Furthermore, it was confirmed that woven bone formation induces over-expression of genes responsible for cell proliferation, angiogenesis and osteogenesis when compared to lamellar bone formation; and that these molecular changes precede woven bone formation (Mckenzie and Silva, 2011). Once woven bone formation stabilizes the SFx region, remodeling begins to repair the SFx line (Kidd et al., 2010)
One of the most important molecular regulators is monocyte chemotactic protein-1 (MCP-1 or
CCL2) which is a member of the CC chemokine superfamily that plays a critical role in the recruitment and activation of leukocytes during acute inflammation (Tangirala et al., 1997;
Kidd et al., 2010). MCP-1 is also found at the site of tooth eruption, a feature consistent with a role in osteoclastic biological activity (Wise et al., 2002). Furthermore, MCP-1 is induced by
RANKL and promotes osteoclast fusion into multinuclear cells (Kim et al., 2005). MCP-1 overexpression together with other members of CC chemokines subfamily is evident in bone
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 34 pathological conditions such as multiple myeloma (Choi et al., 2000), rheumatoid arthritis and osteoarthritis (Lisignoli et al., 1999; Lisignoli et al., 2002). It should be noted that increased osteoclastic activity and osteolysis is a common feature in all these bone conditions. They also share the same signaling pathway with PTH treatment and SFx.
Following treatment of rats with iPTH, MCP-1 was the most induced gene on array analysis, being induced by an order of magnitude greater than the next three candidate genes identified
(Li et al., 2007b, Li et al., 2007c). This relationship between iPTH treatment and MCP-1 suggests that iPTH may accelerate SFx healing via upregulation of MCP-1. It was reported that the chemokine MCP-1 is specifically regulated during the early phase of remodeling following
SFx initiation (Wu et al., 2013). MCP-1 mRNA and protein were localized in osteoblasts adjacent to the SFx. MCP-1 is secreted by osteoblasts and its receptor CCR2 can be found in osteoclasts (Kim et al., 2006). Therefore, it is a strong candidate as an important signaling molecule attracting osteoclasts and its precursors to the site of bone remodeling, especially around SFx when tested in rat ulnae (Wu et al., 2013). To confirm that, a dominant negative inhibitor of MCP-1 called (7ND) was used in cell cultures and showed that inhibition of MCP-
1 resulted in blockage of osteoclast differentiation (Morrison et al., 2014). Despite the extensive research and understanding of biological processes and molecular signaling, the causes of non-union and variability of fracture healing is not understood.
2.4.4. Management and treatment concepts of SFx
A cyclic management protocol was developed based on the understanding of bone remodeling and preventative strategies (Romani et al., 2002). The first priority in management is a period of an "active rest", where athletes are allowed to train in a pain free manner and prevent muscle
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 35 atrophy. Goals during this period were abbreviated in the R.E.S.T acronym, R= removal of the abnormal stress, E= exercise to maintain cardiovascular activity and prevent atrophy, S= Safe and pain free return to the previous activity, T= Time for bone maturity to catch up with increased remodeling (Zelko and De Palma, 1986). Management of SFx consists of three phases. Phase I allows time for healing of damaged blood vessels, revascularization of ischaemic tissues, maturation of periosteum and osteocytes (Clement et al., 1981). Phase II includes general conditioning and strengthening of the injured area. Finally, phase III will allow for gradual remodeling and return to normal activity (Romani et al., 2002). This protocol is different from all other 2 phase protocols that suggest complete removal of stress and gradual loading and increase in function (Andrish et al., 1974; Clement, 1975; Sallis and Jones, 1991;
Daffner and Pavlov, 1992; Umans and Pavlov, 1994; Reeder et al., 1996; Brukner et al., 1999).
It should be noted that in the 3-phase protocol, the periods of increased stress are alternated with periods of rest to allow bone maturation during remodeling because bone is weak at this stage (Romani et al., 2002).
In cases where SFx are low-risk, a modified 2-phase protocol is suggested. Phase 1 includes pain control through local physiotherapy, nonsteroidal anti-inflammatory medication, ice massage, and physical therapy modalities. Weight bearing is allowed for normal activities within the tolerance of pain. The offending activity, such as running, is discontinued and immobilization might be needed in some cases. Phase 2, graduated return to sport, begins when the athlete has been pain-free for one week (Warden et al., 2014) or up to 10 to 14 days
(Fredericson et al., 2006). Other modifications of programs to manage SFx healing have been proposed but are basically variations on these approaches (Ivkovic et al., 2006). Alternatively,
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 36 for some high-risk fractures, surgical fixation may be required due to the increased risk of delayed healing and nonunion (Aweid et al., 2013).
The success of management protocols demands compliance from the patient (Satterfield et al.,
1990). It should be noted that SFx management and rehabilitation requires a multidisciplinary approach consisting of the patient, coach, physician, athletic trainer, and a sport psychologist.
Early diagnosis is a crucial factor as continuing the vigorous training or activity may lead to adverse outcomes such as a displaced fracture (Geslien et al., 1976; Zelko and De Plama, 1986).
Differential diagnosis of SFx include Shin splints (Nielsen et al., 1991; Anderson et al., 1996), osteomyelitis (Sweet and Allman, 1971), compartment syndrome (Slocum, 1967) and tumor
(Sweet and Allman, 1971; Nielsen et al., 1991; Martin et al., 1993; Anderson and Greenspan,
1996). Finally, some preventative regimens are also necessary. For example, using a cyclic training group (Scully and Besterman, 1982), as well as limiting sports activities to one playing surface and one pair of shoes (Zelko and De Plama, 1986; Torg et al., 1987).
Therapeutic (non-operative) methods for management of SFx include BPs, bone stimulators, oral contraceptives, calcitonin, orthotics, calcium and vitamin D. (Mayer et al., 2014). It was hypothesized that BPs have the potential to decrease the progression of SFxs by suppressing bone remodeling through reduced osteoclastic function (Milgrom et al., 2004; Barrett et al.,
2007; Finestone et al., 2008). However, a prospective randomized trial of 324 military recruits showed no difference in the incidence of SFxs of the lower extremities between prophylactic risedronate and placebo (Milgrom et al., 2004). In the ulna loading model, the resorption phase of healing a SFx was significantly reduced, leading to reduced bone formation and adaptation to cyclic fatigue loading 4-6 weeks after SFx, and delayed healing due to inhibition of remodeling (Barrett et al., 2007; Sloan et al., 2010; Kidd et al., 2011). The 25-year experience
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 37 of the Israeli Army on prevention of SFxs showed sleep variables and training modifications, but not BP treatment, decreased the incidence of SFxs (Finestone et al., 2008; Milgrom and
Finestone 2017).
Low-intensity pulsed ultrasound (LIPUS) and nonsteroidal anti-inflammatory drugs (NSAIDs) were trialed to investigate their effect on SFx healing. Neither LIPUS nor NSAID influenced bone resorption, but each had significant and opposite effects on intracortical bone formation rate (Li et al., 2007a). These effects indicate that LIPUS may be used to facilitate SFx repair whereas NSAID may delay tissue level repair of SFxs. When used in combination, the beneficial LIPUS effect was not impaired by the detrimental NSAID effect (Li et al., 2007a).
With regards to bone stimulators, two different types are available, electromagnetic stimulators and ultrasound stimulators. Limited numbers of controlled studies are available to evaluate the efficacy of the different bone stimulators on the healing of SFx. One study found no significant difference in healing of SFx between electromagnetic simulators and placebo (Beck et al.,
2008). In a case series of athletes, LIPUS was effective in pain relief and early return to vigorous activity, without bracing, for the patients with posterior-medial SFxs (Brand et al., 1999).
Furthermore, LIPUS helped an elite athelete with a mid-tibial SFx to get back to light training after 3 weeks and full training activities in 4.5 weeks (Jensen, 1998). LIPUS showed some promising results in complicated healing disorders as in delayed healing or nonunions, especially in non-smokers (Mayr et al., 2000). In a military study, there was no significant difference in time to healing using low-intensity pulsed ultrasound (Rue et al., 2004). Another study confirmed the findings from Rue et al., 2004 and showed that LIPUS was not an effective treatment for the healing of lower limb bone stress injuries. It should be noted that the duration
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 38 of the latter study only 4 weeks in a small, mostly female population (Gan et al., 2014). With regards to the rat ulnar SFx model, low-intensity pulsed ultrasound alone produced better results than ultrasound and NSAIDs combined as well as controls (Li et al., 2007a). As noted above, despite showing some success, the results from different studies are not consistent and show different variabilities in cohorts and methods.
Results obtained from studies using hormone replacement therapy with oral contraceptives
(OCP) in prevention of SFxs are controversial. A randomized study of 150 young female runners with low-dose OCP showed SFx incidence was lower than controls in the OCP group, but was not significant (Cobb et al, 2007). A military study showed that OCP did not have a preventative effect on the incidence of SFx (Van Hal et al., 1982). Therefore, other factors such nutrition, calcium intake, energy status and body mass index can be used to predict improvement in bone mineral density and normal bone turnover (Cumming et al., 1996;
Fredericson et al., 2005; Cobb et al., 2007).
Calcium and vitamin D improve BMD in the elderly, postmenopausal women as well women undergoing breast cancer therapy, but it is not definitively proven to prevent SFx (Myburgh et al., 1990; Cline et al., 1998; Bennell et al., 2000; Ruffing et al., 2006; Lappe et al., 2008; Cobb et al., 2010; Nieves et al., 2010). A lower level of serum 25(OH)D concentration was possibly considered as a predisposing element for bone SFxs in young Finnish men (Ruohola et al.,
2006). In track and field athletes and military recruits, no significant difference was found with increased calcium and vitamin D intake and incidence of all types of SFx (Bennell et al., 2000).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 39
One of the largest studies on the topic showed that in female military recruits, 2000 mg of calcium and 800 IU of vitamin D daily had a 20% lower incidence of SFxs during basic training than those taking a placebo (Lappe et al., 2008). The recommended daily dose of calcium depends on age, while vitamin D intake is more controversial (American Academy of
Orthopaedic Surgeons, 2000; Greer et al., 2006; National Institutes of Health Office of Dietary
Supplements, 2009). In some studies, daily supplementation of 500 to 800 mg of calcium and
400 to 800 IU vitamin D improves BMD and decreases fracture risk in general significantly
(not specifically SFx) (Grados et al., 2003; Bischoff-Ferrari et al., 2005).
Other non-operative treatments have little data as regards to healing of SFx. For example, calcitonin inhibits osteoclasts during the remodeling process of healing of SFx, it improves
BMD and biomechanical properties to a limited extent. However, its significance in preventing or healing SFx is not known (Hullinger et al., 1944; Jiang et al., 2005; Li et al., 2005; Manabe et al., 2009). Furthermore, data regarding the use of non-steroidal anti- inflammatory drugs
(NSAIDs) in treatment of SFx is limited. It was suggested by Wheeler and Batt (2005) that to minimize risk, the use of NSAIDs should be avoided in the treatment of SFxs wherever possible until results of further research are available. This could be explained by NSAIDS either reversibly or irreversibly block the cyclooxygenase pathway, thereby inhibiting prostaglandin synthesis. From the available evidence it is still unclear whether this inhibition significantly impairs healing of SFxs in humans (Wheeler and Batt, 2005).
Certainly, data demonstrating a delay in healing of SFx in the rat ulna following ibuprofen treatment (Li et al., 2007a; Kidd et al.,2013) and healing for nonunions (Mayr et al., 2000) supports this cautious approach, but not all NSAIDs will have this effect (Kidd et al., 2013).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 40
The treatment of SFxs continues to advance with new emerging technologies. A recent review related to SFxs described new treatment modes including application of magnetic fields, hyperbaric oxygen therapy, growth factors and bone morphogenetic proteins, besides the classic therapies such as BPs, low intensity pulsed ultrasound and PTH (Astur et al., 2016).
2.5. Parathyroid hormone (PTH)
2.5.1. Background on PTH
PTH is a peptide that regulates calcium homeostasis in the body by its catabolic action on the skeleton (Hock et al., 2002). Paradoxically, when administered intermittently, it has an anabolic action on bone and has been used for treatment of osteoporosis (Rosen, 2004; Hodsman et al.,
2005). Furthermore, the use of iPTH to treat fractures (Andreassen et al., 1999; Holzer et al.,
1999; Andreassen et al., 2001; Jahng et al., 2002), localized osseous defects and accelerate SFx healing has been investigated (Sloan et al., 2010; Gomberg et al., 2011; Raghavan and
Chrisofides, 2012). In addition, other studies have investigated the role of iPTH in osseointegration of implants (Skripitz and Aspenberg, 2001; Reynolds et al., 2011). iPTH is most commonly delivered by injection, but local delivery methods are emerging and could expand the horizons of using iPTH in tissue engineering of bone (Wei et al., 2004). Examples include matrix-bound engineered active fragment of human parathyroid hormone PTH(1-34)
(Arrighi et al., 2009), an infusion pump for treatment of hyperparathyroidism (Cusano et al.,
2012), local pulsatile PTH delivery with a cell-free biomimetic nanofibrous (NF) scaffold for local bone regeneration (Dang et al., 2017a), a biodegradable device to strengthen bone (Dang et al., 2017b) or delivery of PTH-derived peptide (PTHdP) using apatite crystals (Yang et al.,
2018). Furthermore, exploration of the different mechanisms that PTH can affect the mesenchymal stem cells could have clinical application in bone regenerative medicine (Casado-
Diaz et al., 2019). Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 41
PTH was FDA-approved in the United States in 2002. Two different approaches are available, parathyroid hormone (1-34) (PTH 1-34), or teriparatide and full-length recombinant human parathyroid hormone (PTH 1-84) or abaloparatide, which is available for treatment of postmenopausal osteoporosis in many European countries (Migloire et al., 2012) and fracture healing (Peichl et al., 2011). PTH 1-84 has a slightly longer half-life than PTH 1-34 which is a disadvantage for treating osteoporosis and may account for the increase in hypercalcemia but may be advantageous in treating hypoparathyroidism (Cusano et al., 2013). Parathyroid hormone related protein (PTHrP) is widely expressed in normal tissues throughout development. PTH and PTHrP have significant homology in the N-terminal region, allowing them to bind to the same PTH - 1 receptor, though each protein favors a different conformational state, resulting in somewhat different downstream effects on calcium metabolism (Wysolmerski, 2012b).
The principal physiological action during continuous PTH exposure causes bone loss, or what is known as a catabolic effect. Intermittent PTH has been described as a double-edged sword for bone metabolism (Qin et al., 2004). Firstly, the hypothesis that iPTH exerts its anabolic actions via an increase in osteoblast differentiation was also supported by microarray data from osteoblastic cell lines (Qin et al., 2003). From numerous studies, it can be concluded that the mode of administration and associated signaling pathways are responsible for the paradoxical actions of the hormone (Qin et al., 2004).
2.5.2. PTH as an anabolic agent
A number of studies have confirmed the anabolic effect of iPTH and its correlation with the timing and duration of treatment (Dempster et al., 1993; Schiller et al., 1999; Neer et al., 2001;
Andreassen et al., 2004). Intermittent PTH results in increased bone formation, cortical bone Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 42 volume and width. It also improves bone architecture and mechanical properties in rodents, rabbits, primates and humans (Hirano et al., 2000; Burr et al., 2001; Dempster et al., 2001;
Mashiba et al., 2001a,b; Seeman et al., 2001; Zhou et al., 2003; Sato et al., 2004). Finally, iPTH promoted bone healing in ovariectomized and healthy rats, when it is applied during early stage of healing without having any adverse systemic effect (Komrakova et al., 2010).
A single PTH injection improved the bone mineral density (BMD) after 3 weeks in the tibial metaphysis of rats (Nishida et al., 1994). Intermittent PTH was proven to be effective in treatment of osteoporosis in post-menopausal women. It decreased the risk of vertebral and non-vertebral fractures, increased vertebral, femoral, and total-body bone mineral density (Neer et al., 2001), and also improved cortical bone microstructure (Ascenzi et al., 2012). In addition to the above, iPTH has been also investigated in the treatment of osteoporosis in men (Kurland et al., 2000; Orwoll et al., 2004), and hyperparathyroidism (Rubin et al., 2011). Similarly, iPTH enhanced mechanical properties after distraction osteogenesis in rats; an intervention used in both leg lengthening and bone transplantation with external fixtures in treatment of fractures and non-unions (Seebach et al., 2004). Intermittent PTH treatment at 30 µg/kg before and after osteotomy in rats accelerated the healing process as evidenced by earlier replacement of woven bone to lamellar bone, increased new cortical shell formation, and increased the ultimate load up to 12 weeks after osteotomy in rats (Komatsubara et al., 2005).
Because the effect of iPTH appears strongest in repairing cancellous, loaded bone with hematopoietic marrow, one could expect iPTH to have even stronger effect in orthopedic surgery than in the treatment of osteoporosis. Intermittent PTH can increase bone formation once it has been initiated, or in a repair process (Skripitz et al., 2000). Thus, iPTH cannot be
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 43 expected to be useful in non-unions, for example, although it might speed up fracture repair. It was suggested that iPTH could have an effect in the early postoperative period after osteosynthesis or joint replacement by stimulating new bone formation, thereby decreasing the risk of late loosening (Skripitz and Aspenberg, 2004). For example, in a published case report, surgical fixation, autografting augmented with BMP-7 was not successful until PTH was used
(Paridis and Karachalios, 2011).
In addition, several studies have shown that combining iPTH and mechanical loading had a synergistic effect that resulted in an increased osteoblastic activity and stimulation of bone formation indices on all bone surfaces. It also depressed bone resorption on endocortical surfaces leading to a higher periosteal bone formation rate, which created a larger cross- sectional area, more cortical bone area, and a thicker cortex (Carvalho et al., 1994; Tang et al.,
1997; Chow et al., 1998; Ma et al., 1999; Li et al., 2003; Hagino et al., 2001; Bakker et al.,
2003; Kim et al., 2003). This is in agreemrent with the theory that nonmechanical agents like hormone could change the strain thresholds (setpoints) for modeling or remodeling activity, resulting in changes within the skeleton’s response to mechanical loading to remove or add bone (Frost, 1987b; Frost, 1992).
While these studies suggested a synergistic effect of loading and PTH, Roberts et al, (2009) mapped the site-specific distribution of new bone apposition due to the combined treatments.
They noted specific regions of mechanically induced bone formation which were enhanced when hPTH-(1-34) was administered. This led to a conclusion that the effect of PTH was additive, rather than synergistic. This additive effect of PTH could be due to its activation of the PTHR1, mimicking local actions of PTHrP at this receptor (Stewart, 1996). However, there is also good evidence that its action influences intracellular calcium by activating L-type
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 44 calcium channels (Li et al., 2003). This finding of an additive PTH effect is of clinical relevance when weight bearing is used to enhance lower extremity fracture healing.
Furthermore, when comparing the effect of LIPUS and iPTH on fracture healing, iPTH increased fracture site mechanical strength and stiffness. This is due to iPTH affecting the callus bone mineral content (BMC) but not the total callus volume (TV). This may suggest that iPTH
(in contrast to LIPUS) could be beneficial in the healing of acute bone fractures (Warden et al.,
2009).
In a recent literature review and meta-analysis comparing the effectiveness of iPTH
Teriparatide, BPs, Raloxifene, Denosumab, and calcium and vitamin D, that included 116 studies and a total of 139,647 patients, iPTH Teriparatide had the highest reduction in vertebral fracture incidence, hip fracture, and non-vertebral fracture, and had the highest probability of being ranked most effective in all three categories (49%, 42%, and 79%, respectively) when compared to the other treatments (Murad et al., 2012). Another report that included randomized controlled trials (RCTs) concluded that Teriparatide is associated with a reduced risk of both vertebral and non-vertebral fractures in postmenopausal osteoporotic women (Crandall et al.,
2012). This conclusion was statistically justified by a previous clinical trial (Neer et al., 2001).
The clinical uses of iPTH in treatment of osteoporosis in men is tested in smaller scope, and might not have the statistical power to show a significant reduction in fracture incidence when compared to studies conducted on osteoporotic women, therefore markers such as BMD could be used instead for monitoring (Capriani et al., 2012). The first randomized trial examining the efficacy of Teriparatide (PTH 1-34) in men was conducted in 2000 (Kurland et al., 2000).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 45
The Teriparatide group showed BMD increases of 13.5% at the lumbar spine and 2.9% at the femoral neck after 18 months. A subsequent, larger but shorter trial involving 437 men with osteoporosis showed a 5.9% increase in lumbar spine BMD and 1.5% increase at the femoral neck BMD (Orwoll et al., 2003).
Furthermore, iPTH has proven to be effective in the treatment of glucocorticoid induced osteoporosis for both men and women (Saag et al., 2007; Saag et al., 2009). In addition to the above clinical applications, promising results have been obtained after using full length native
PTH (1-84) as well as Teriparatide (PTH 1-34) in treatment of hypoparathyroidism (Rubin et al., 2010; Sikjaer et al., 2011; Bilezikian et al., 2012a; Bilezikian 2012b; Rubin et al., 2012).
Serum and urinary calcium levels were improved and maintained during iPTH treatment
(Cusano et al., 2011). Recently an infusion pump was trialled successfully in the treatment of hypoparathyroidism using Teriparatide (Cusano et al., 2012). In relation to the present study, iPTH accelerates fracture healing through stimulating hard callus formation and improving the strength of the fracture site (Ellegaard et al., 2010; Aspenberg et al., 2010; Aspenberg and
Johansson, 2010; Peichl et al., 2011).
In dentistry, iPTH is being considered for treatment of mandibular fractures. For example, PTH
(1-34) might enhance the healing of mandibular fractures in the early phase (7-day period).
However, long-term administration (21-day period) showed no statistically significant differences between the control and experimental group, as assessed by radiographic densitometry (Rowsban et al., 2010).
The combined effect of iPTH and BMP-7 in metaphyseal bone healing was investigated and it resulted in increased callus total volume (TV), mineralized volume (BV), average cross-
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 46 sectional area, bone mineral content (BMC), increased callus total volume (TV), and mineralized volume (BV). This was explained by the difference between BMP-7 and iPTH in biological activities. BMP-7 appeared to be strictly anabolic, those of iPTH appeared to work in the context of coupled remodeling. It was proposed that the combined treatment enhanced healing via two mechanisms. First, BMP-7 promoted greater local stem cell recruitment and differentiation of this larger pool of osteoprogenitor cells. Second, the actions of iPTH were anabolic and catabolic, promoting osteoblast differentiation as well as remodeling of the newly formed bone tissues.
The combination of both agents led to greater bone volume as well as better microstructural organization and integration of this bone with the surrounding tissues (Morgan et al., 2008). It is worth mentioning that a limitation of the above-mentioned study was that it used only one time point (4 weeks after the defect) where healing is expected to be well advanced. Further studies are required to investigate all stages of healing separately to identify the stage/s where the combined treatment has the greatest effect. The combined effect of PTH and mechanical loading showed a synergistic effect on tibial cortical bone and load-induced bone adaptation
(Ma et al., 1999; Hagino et al., 2001; Li et al., 2003). Furthermore, a more recent study showed that iPTH treatment and mechanical loading have positive additive effects on periosteal bone formation (Meakin et al., 2017).
2.5.3. Mechanism of action of PTH
The mechanism of the anabolic effect of iPTH is not yet fully understood. The large increase in mineral apposition rate (Parfitt, 1987) is probably due mainly to an increase in osteoblast number (Li et al., 1999, Watson et al., 1999). Some of those new osteoblasts originated from the resting bone lining cells (Dobnig and Turner, 1995). In young rats, iPTH also increases the Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 47 differentiation of osteoprogenitor cells into osteoblasts. In 2-month-old rats, iPTH stimulates the proliferation and differentiation of osteoprogenitor cells in the bone marrow (Nishida et al.,
1994, Onyia et al., 1995, Balani et al., 2017). iPTH binds to receptors on osteoblasts and osteocytes and leads to increased expression of the early genes c-fos, c-myc, c-jun, and IL-6.
These early genes are known to be involved in cell proliferation (Onyia et al., 1995, Liang et al., 1999). Intermittent PTH not only initiates modeling but also postpones osteoblast apoptosis
(Jilka et al., 1999). Daily injections of iPTH attenuate osteoblast apoptosis in mice, thereby increasing osteoblast number, bone formation rate and bone mass; but they do not affect osteoclast number (Bellido et al., 2003). Moreover, inhibition of apoptosis occurs at the early stages of osteoblast differentiation, while induction of apoptosis in more mature osteoblasts, represents a mechanism for clearing old osteoblasts to renew supply of osteoblasts in the BMU
(Chen et al., 2002). Furthermore, both intermittent and continuous PTH treatment promotes the development of new blood vessels, increased vasculature on the adjacent bone surfaces via a
VEGF-dependent mechanism, by localizing capillaries near sites of new bone formation, and contributes to new bone formation as well as increased bone strength (Langer et al., 2009; Jilka et al., 2010; Towler, 2011).
The mitogenic iPTH effect may be mediated by regulators of bone remodeling such as
Transforming Growth Factor β (TGFβ), which is a potent mitogen for bone cells, regulated in part through PTH (Centrella et al., 1988). PTH also interacts with insulin-like growth factor I
(IGF-I) (Dempster et al., 1993). PTH enhances the IGF-I synthesis and the secretion of IGF-1 binding proteins in osteoblast-like cells, and IGF-I antibody decreases PTH-induced collagen synthesis (Canalis et al., 1989, McCarthy et al., 1989). On the other hand, it might be possible that a continuous elevation of PTH could exert anabolic effects on skeletal tissue if its catabolic component could be minimized via estrogen repletion (Shen et al., 2000). Two studies with Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 48
IGF-1-null mice indicated that IGF-I mediates the anabolic effects of PTH (Miyakoshi et al.,
2001; Bikle et al., 2002).
Binding of PTH to its membrane receptor PTH1R, a G-protein-coupled receptor (GPCR), activates two well-defined signal transduction pathways in osteoblasts. One is the protein kinase
A (PKA), the other one is the protein kinase C (PKC) (Karaplis and Goltzman, 2000). The in vivo daily injection of different PTH peptides indicates that only those peptides that activate the PKA pathway exhibit anabolic effects on bone, and peptides that activate the PKC pathway do not (Rixon et al., 1994; Armamento-Villareal et al., 1997). Activation of the PKA pathway is mediated by the Gas of the receptor, this cascade stimulates cAMP production, and subsequently PKA. Activation of the PKC pathway is a Gaq mediated response. PKC is activated through phospholipase C dependent and independent pathways, which leads to activation of osteoblastic bone formation (Jilka, 2007). Through these pathways PTH can mediate both bone formation and bone resorption (Silva and Bilezikian, 2015). The PKA and PKC pathways are summarized in Figure 1 and Figure 2 (Swarthout et al., 2002; Yavropoulou et al., 2017).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 49
Figure 1: A schematic summary of the pathways undertaken by PTH in order to produce its effect, adapted from Swarthout et al., 2002.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 50
Figure 2: Showing PTHR1 intracellular signalling. After ligand coupling PTHR1 activates four different intracellular signalling cascades: a) GαS-adenylyl cyclise-cAMP-protein kinase A (PKA), b) Gαq-phospholipase C (PLC) β- inositol triphosphate-cytoplasmic Ca2+- protein kinase C18, c) Gα12/13-phospholipase D- transforming protein RhoA and d) β-arrestin-extracellular signal-regulated kinase
1/2(ERK1/2), adapted from Yavropoulou et al., 2017.
PTH regulates Cbfa1, collagenase-3 (MMP-13) and osteocalcin. Cbfa1 (RUNX-2) plays a central role in osteoblast differentiation. Its synthesis correlates with osteogenesis during development. Targeted inactivation of Cbfa1 in mice generated animals that have no osteoblasts
(Komori et al., 1997). PTH strongly stimulates the activity of Cbfa1 at both the transcriptional and posttranslational level (phosphorylation) (Selvamurugan et al., 2000; Krishnan et al., 2003).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 51
The genes encoding collagenase-3, a matrix metalloproteinase, and osteocalcin, a bone-specific matrix protein, are specifically expressed by differentiating and mineralizing osteoblasts. PTH strongly increases their mRNA abundance (Partidge et al., 1987; Noda et al., 1988). Besides all of the above, iPTH regulates the expression of many genes associated with osteoblast differentiation, which suggests that iPTH is responsible for this process.
PTH indirectly regulates and increase osteoclastic activity (Teitelbaum, 2000). This was confirmed by observations of monocyte chemotactic protein -1 (MCP-1, or CCL2) which is induced by RANKL and promotes osteoclast fusion into multinuclear cells (Kim et al., 2005).
Furthermore MCP-1 is secreted by osteoblasts and its receptor CCR2 can be found in osteoclasts (Kim et al., 2006). MCP-1 is also important as a signaling molecule attracting osteoclasts and its precursors when needed for repair (Wu et al., 2013). This was confirmed recently through using a dominant negative inhibitor of MCP-1 called (7ND), which resulted in blockade of human osteoclasts differentiation (Morrison et al., 2014).
In addition, it was demonstrated that Insulin-like Growth Factor – 1 (IGF-1) is thought to be a mediator of the anabolic effects of iPTH on bone and has opposite effects on formation of trabecular and cortical bone in adult female rats (Tobias et al., 1992; Miyakoshi et al., 2001;
Bikle et al., 2002). Finally, it was revealed that iPTH causes mRNA’s rapid encoding of two particular cytokines, interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) (Horowitz, 1996;
Pollock et al., 2009; Cho et al., 2013). There has been longstanding preconceived notions regarding these cytokines is that they are factors that resorb bone, however a number of studies exposed that they can be linked to increased bone formation and the anabolic effect of iPTH
(Franchimont et al., 2005; Cho et al., 2013; Poulton et al., 2013).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 52
More recently, the role of parathyroid hormone related protein (PTHrP) and parathyroid hormone receptor-1 (PTHR1) has attracted a lot of interest in literature. Both PTH and PTHrP act equally on PTHR1 to induce bone formation and activate signaling through cyclic AMP
(cAMP) (Kemp et al., 1987). Osteocytes express PTHrP (Kartsogiannis et al., 1997), and osteocyte-derived PTHrP is highly important in cortical bone remodeling (Wang et al., 2013), bone formation, bone matrix strength but not osteoclastogenesis (Ansari et al., 2018).
Furthermore, osteocyte-derived PTHrP acts in paracrine/autocrine manner with both a PTHR1 receptor mediated action, which is similar to therapeutic PTH, and a PTHR1 endogenous receptor independent action on osteocyte network (Ansari et al., 2018). Both osteocytes PTHrP actions (receptor dependent/receptor independent) could be contrasting each other, which adds more to the complexity of PTH mechanism of action (Massfelder et al., 1997; Hochane et al.,
2013). A simplified mathematical two state receptor kinetics model for PTHR1 was designed to distinguish between the active and inactive receptors, as well as receptor-ligand complexes that might help develop a better understanding of this complex process (Potter et al., 2005). A recent review has highlighted the effects of PTHR1 on anabolic actions in the skeleton (Osagle-
Clouard et al., 2017).
To conclude, in the osteoblast, PTH initiates a complicated signaling network with two outputs: bone formation and bone resorption. How the input PTH signal is applied (pulsatile or continuous), but apparently not the amount of PTH, determines the ratio of these two outputs and subsequently determines the net result (bone gain or bone loss, anabolic or catabolic effects). In summary, activation of osteoblasts by PTH results in expression of genes important for the degradation of the extracellular matrix (collagenase-3), production of growth factors
(IGF-I), and stimulation and recruitment of osteoclasts (RANKL and IL-6) (Swarthout et al.,
2002).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 53
We believe that finding a simple but relevant model and study of these models should shed light on the mechanism of the two paradoxical functions of PTH. Besides testing the efficacy of different delivery methods, it could eventually lead to the discovery of newer generation drugs for osteoporosis besides the intranasal (Agu et al., 2004; Williams et al., 2018) and transdermal drug coated microneedle patch system methods of administration (Daddona et al., 2011). This could avoid the side effect of inducing hypercalcemia. Such drugs might also be used to promote fracture healing (Qin et al., 2004; Yukata et al., 2018). An alternative approach to intermittent injection of PTH is the use of an orally active compound that antagonizes the calcium-sensing receptor to stimulate transient increases in circulating PTH levels (Nemeth et al., 2001). To conclude, there is not a single pathway for iPTH action, instead PTH acts via multiple mechanisms that lead to a “crosstalk” between different bone cells and regulated by certain “coupling factors” that are regulated by other hormones (Osagle-Clouard et al., 2017).
The debate continues about the mechanism of PTH and its role in bone metabolism despite the plethora of research available.
2.6. Bisphosphonates (BPs):
2.6.1. Background on bisphosphonates
Bisphosphonates (BPs) are stable analogues of pyrophosphatase. They are deposited on bone surfaces within minutes or hours of uptake (Russell, 2006; Drake et al., 2008). The mode of action on the osteoclast is radically different between non-nitrogen containing BPs (first generation), where apoptosis is induces through forming a toxic analog of adenosine triphosphate, while nitrogen-containing BPs (2nd and 3rd generation) targets the enzyme farnesyl diphosphate synthase needed for post-translational modification of small GTP-binding proteins required for osteoclastic function (Russell et al., 2008; Reyes et al., 2016). Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 54
All BPs have an affinity for bone tissue, and more specifically to osteoclasts, because during bone resorption the acidic PH of the resorption lacuna of the osteoclasts causes an intracellular uptake of BPs, leading to the internalization of substantial amounts of these drugs (Martin and
Grill, 2000; Coxon et al., 2008). Independently of the pathway used to inhibit bone resorption, either inducing a cytotoxic effect in osteoclasts or inhibiting the production of farnesyl diphosphate, all of them lead to the apoptosis of osteoclasts (Reyes et al., 2016).
BPs are incorporated into the bone matrix and have a biological half-life of more than 10 years for both nitrogen and non-nitrogen containing BPs (Schneider, 2009). There have been concerns about the long-term use of BPs because of their potential for over-suppression of the normal process of bone turn over (Hirano et al., 2000; Mashiba et al., 2001a,b; Biovon et al.,
2002; Akkus et al., 2003; Ciarelli et al., 2003; O'Brien et al., 2005). This could lead to the accumulation of microdamage within the bone and eventually result in the development of SFxs
(eg AFFs) as result of a long-term inhibition of osteoclastic functions (Mashiba et al., 2000;
Allen and Burr, 2005). Other risk factors for development of SFx identified in literature included glucocorticoids and protein pump inhibitors (Giusti et al., 2010). In 2008, the National
Osteoporosis Foundation (NOF) in its clinical update reported " Results suggest that for most women, taking a 5-year “drug holiday” after being on ALN (5-1Ü mg/day) for 5 years does not increase fracture risk and might be advantageous.” (Watts et al., 2008). For women at high risk for vertebral fractures, continuing ALN for a total of 10 years is a reasonable clinical option.
The increase in bone mass affected by treatment reduces the risk of fracture. Yet, one year of
ALN treatment reduced the toughness of trabecular bone (its ability to resist microdamage formation) in beagle dogs at doses used for treatment in humans (Mashiba et al., 2001a;
Mashiba et al., 2001b; O'Neal et al., 2010). The overall effect of ALN treatment is a reduction in the prevalence of fractures.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 55
2.6.2. Bisphosphonates as antiresorptive agents
BPs are highly effective in the treatment of osteoporosis. Numerous large clinical trials have demonstrated their efficacy in reducing bone turnover, increasing bone mineral density and reducing vertebral and non-vertebral fracture risk in patients with osteoporosis (Cranney et al.
2002a, 2002b; Maricic, 2010). A recent study estimated that between 2001 and 2008, 144,670 low-energy fractures were prevented amongst women who took BPs in the United States (Siris et al. 2011). Another benefit for BPs in addition to treatment of osteoporosis and fracture prevention is relief of bone pain in Paget’s disease (Siris, 1994; Siris et al., 1996; Siris et al.,
1998; Reid et al., 2005), bone cancer (Multiple Myeloma) and bone metastasis (Berensen et al.,
1996; Berensen et al., 2001; Rosen et al., 2001). BPs could be used together with radiotherapy to decrease bone pain in cancer patients. As a result, the ESMO (European Society of Medical
Oncology) stated recently in their recommendations that zoledronic acids should be used in patients with metastatic breast or prostate cancer, and in selected individuals with lung, renal and other solid tumors with metastasis (Coleman et al., 2014).
BPs are also generally well tolerated and considered to have an excellent safety profile even at higher doses (Dunstan et al. 2007). One of the complications of prolonged use of BPs is femoral insufficiency fracture or atypical femoral fractures (AFF) (Girgis et al., 2010; Giusti et al.,
2010; Watts and Diab, 2010; Girgis and Seibel, 2011a,b,c; Blum et al., 2016; Koh et al., 2017;
Suh et al., 2019), a type of SFx. These fractures rarely heal spontaneously and most likely require surgery for displacement or persistent pain (Ha et al., 2010). In addition to the above, long term extra-skeletal side effects of BPs have been documented in literature including gastrointestinal (GI) effects, cardiovascular complications and some types of cancer (Reyes et al., 2016). Oral BPs have been associated with a number of GI adverse effects including ulcers, erosions, esophagitis, bleeding, pills fragments (Kim et al., 2014). A number have reported a Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 56 possible association between BPs and atrial fibrillation (AF). However, these associations were not confirmed after further analyses (Heckbert et al., 2008; Abrahamsen et al., 2009; Pazianas et al., 2010; Eriksen et al., 2014). A number of case studies have been published linking the possible risks of esophageal cancer with oral BPs (De Groen et al., 1996; Green et al., 2010;
Lin et al., 2013). However, meta-analyses of the above-mentioned observational studies could not confirm the association between oral BPs and esophageal cancer (Oh et al., 2012; Ghirardi et al., 2014).
In dentistry, the local delivery of BPs can improve implant fixation and avoid the osteonecrosis of the jaw (ONJ), another rare complication caused by the systemic use of BPs (Abtahi et al.,
2013). Surface coating of dental implants with BPs also improved stability and fixation in humans (Abtahi et al., 2010; Abtahi et al., 2012a). When BPs are released from the implant surface, the balance in bone turnover is shifted in the favor of bone formation through reduction of osteoclastic activity. The end result is a gain in the overall bone density, which can lead to an improved early fixation, reduction of failure rates and time needed between insertion of the dental implant and its loading (Wermelin et al., 2008). In addition to the above, treatment of periodontitis with a high concentration of BPs showed regeneration of part of the lost bone without developing ONJ in any of the cases (Pradeep et al., 2012; Sharma and Pradeep,
2012a,b).
There is evidence that systemic BPs (especially intravenous) used in high doses may lead to
ONJ under certain conditions. In particular, patients treated for oral cancer, or who had surgical procedures exposing bone to the oral environment, were the strongest associating risk factors
(Marx et al., 2005; Mavrokokki et al., 2007; Barasch et al., 2011; Fellows et al., 2011).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 57
Osteocyte death is not necessary for ONJ (Abtahi 2012b), the possible mechanism for ONJ is the impaired bone resorption, and possible changes to vascularity, caused by BPs that prevents the removal or separation between bacterially contaminated bone and adjacent healthy bone; leading to delayed healing (Abtahi et al., 2013).
For patients diagnosed with ONJ, the treatment is usually palliative focusing on elimination of pain and preventing progression of exposed bone. Aggressive debridement or surgical procedures are not indicated (Marx et al., 2005). The use of an intermittent low dose PTH improved bone regeneration and healing, however, the effect of PTH was delayed between 3-6 months after discontinuation of the BPs (Harper and Fung, 2007). Such delays in the response to PTH have been observed by others when it follows from BP treatment (Ma et al., 2003;
Lecart et al., 2004; Lau and Adachi, 2009).
Combination therapy of PTH plus BPs (ALN) impairs the anabolic function of PTH and its ability to enhance bone mineral density (BMD) (Black et al., 2003; Finklestein et al., 2003).
For example, PTH treatment for 2 months after an anti-resportive agent increased bone formation and mineralization rate, but less than that achieved with PTH treatment alone (Ma et al., 2003). The question is to what extent PTH can remain effective in the presence of an antiresorptive treatment.
The Forwood laboratory showed that a high dose of BPs (Risedronate) impaired healing of a large SFx line by reducing the volume of bone resorbed and replaced during remodeling (Kidd et al., 2011). However, formation of periosteal callus was not adversely affected. This woven
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 58 bone reaction can return the bone to its original strength after 2 weeks of SFx induction (Silva and Tohey, 2007; Uthgenannt et al., 2007). But there is little analysis available for the combined effects of a BP with PTH. In the ulna SFx model, PTH increased bone mineral content (BMC) significantly by 7% at 4 weeks and bone mineral density BMD and BMC significantly by 10% and 7% at 8 weeks compared to controls, whereas ALN did not change BMD or BMC (Sloan et al., 2010). PTH significantly stimulated bone formation by 114% at 2 weeks, increased intracortical resorption area by 23% at 4 weeks, and enhanced the ultimate force of the affected ulnae by 15% at 8 weeks (Sloan et al., 2010). Similar to Kidd et al (2011), ALN significantly suppressed bone formation rate by 44% compared to the control at 4 weeks (Sloan et al., 2010).
These data suggest that PTH could accelerate SFx remodeling, but the combined effects of PTH with BPs remain unknown.
To conclude, many questions remain unanswered when it comes to combination or cyclic therapy (Rosen, 2004). For example, could short-term PTH exposure be followed by long-term
BP treatment? More questions need to be answered with regards to the PTH signaling cascade that will influence future decisions and will direct future research to enhance SFx healing. The answers for these questions will most probably be specific to each repair scenario.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 59
2.7. Potential uses of PTH and bisphosphonates in SFx healing
2.7.1. PTH and SFx healing
Endogenous PTH leads to an increase in bone resorption via stimulation of osteoclasts. On the contrast, teriparatide could reach maximum serum concentrations in 30 minutes and return to negligible serum levels in a few hours. This allows stimulation of new bone formation by favoring stimulation of osteoblasts over osteoclasts (Deal and Gideon, 2003). The reports about the positive effects of teriparatide used rats with tibial fractures (Andreassen et al., 1999;
Andreassen et al., 2001). This was followed by studies on cynomolgus monkeys as their bone remodeling systems are very similar to humans (Manabe et al., 2006). Furthermore, the first human randomized double-blind study examined postmenopausal women with fractures of the distal radius and showed shortened time of fracture healing using teriparatide (Aspenberg et al.,
2010).
After the successful use of PTH in fracture healing, investigations were made regards the use of PTH in accelerating the healing of SFx. It was evident that PTH improves SFx healing in rat ulna (Sloan et al., 2010). Studies show the successful use of teriparatide in acclearating the healing of metatarsal (Raghavan and Christofides, 2012), femoral neck (Malhorta et al., 2013), lower extremity (Almirol et al., 2016) and bilateral sacral (Baillieul et al., 2017) SFxs.
Furthermore, teriparatide was used successfully to augment the healing of a delayed union SFx in a gymnast (Gende et al., 2019). However, it should be noted that in most of the above studies and case reports, PTH treatment was supplemented with vitamin D and calcium citrate which raises the question about the presence of a synergistic effect between PTH and other supplements.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 60
2.7.2. Combined ALN PTH treatment and SFx healing
The combined anti-resorptive and PTH treatments has been extensively investigated in literature for treatment of postmenopausal women with osteoporosis (Black et al., 2003;
Finkelstein et al., 2003; Cosman et al., 2005; Tsai et al., 2013; Cosman et al., 2017). In addition to treatment of postmenopausal osteoporosis, combination antiresorptive-anabolic (i-PTH) therapy has been tested to accelerate healing of closed fracture repair, reduce fracture risk, improve trabecular microarchitechture, enhance bone healing around implants and bone transplantation (Hashimoto et al., 2007; Arrington et al., 2008; Aspenberg et al., 2010; Li et al.,
2012; Ettinger et al., 2013; Casanova et al., 2016). In a murine closed fracture model, combination treatment of Zolendronate (ZA) and PTH increased callus strength (Casanova et al., 2016). Furthermore, mean lacuna volume was higher in the combination treatment when compared to VEH groups but was found to be the highest in PTH monotherapy group
(Casanova et al., 2016). This was attributed to osteocytic osteolysis, a phenomenon that is caused by PTH and was observed in a number of previous studies (Tazawa et al., 2004; Teti et al., 2009; Tommasini et al., 2012; Wysolmerski et al., 2012a).
It is evident that BP treatment delays the healing of SFx (Sloan et al., 2010; Kidd et al., 2011).
Prolonged BP treatment leads to accumulation of microdamage and development of SFxs that could progress to AFF (Mashiba et al., 2001b; Watts and Diab 2010; Gedmintas et al., 2013;
Adler, 2018; Black et al., 2019). Therefore, it is recommended that BPs are discontinued in cases of SFx or AFF (Larsen and Schmal, 2018) or replaced with Denosumab (Tsai et al.,
2017a; Tsai et al., 2017b). There are a lot of similarities between SFx and AFF in terms of etiology and possible management protocols (Schilcher et al, 2014).
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Teriparatide treatment has been used with great success in clinical AFF cases (Schilcher et al,
2014; Watts et al., 2017). However, questions were raised on whether the observed positive effect on healing was due to cessation of the antiresoptive medication, starting teriparatide treatment or a combination of both factors together (Watts et al., 2017). Teriparatide was used in conjunction with surgical intervention for treatment of AFF (Tsuchie et al., 2015; Miura et al., 2019). Furthermore, a recent case report showed healing of bilateral calcaneus due to long- term methotrexate (MTX) treatment in a patient suffering from systemic lupus erythematosus with a history of an unsuccessful prolonged BP treatment. A combined bone-specific denosumab-teriparatide treatment was used after discontinuation of BP and MTX (Rolvien et al., 2019). The current study is the first study to investigate the effect of iPTH treatment on the healing of SFx in the presence of a concurrent BP treatment.
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Chapter 3
Methodology
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3. Methodology:
The overarching aims of the study were to investigate if PTH treatment accelerated healing of
SFx, and if it remained effective in the presence BP treatment. The hypothesis that short-term
PTH treatment was sufficient to activate remodeling was tested through treatment of rats with
PTH as a single dose, and intermittently for 14 days. These durations are based on the protocol from the Partridge laboratory and elicit the minimum and maximum induction levels of MCP-
1 (Li et al., 2007c), respectively. For BP treatment, ALN was chosen because it is the compound with the most long-term clinical data and is associated with atypical femoral SFx. Rats were pre-treated for 14 days with ALN. At the time of SFx loading, ALN therapy was continued, concurrent with PTH; or stopped at the time of PTH treatment. Timing of outcome measures was based on published data from the Forwood laboratory for measurement of resorption (7 and 14 days) and formation at (6 and 10 weeks) in this rat ulnar loading model (Kidd et al.,
2010).
The Griffith University Animal Ethics Committee approved the experimental protocols
(MSC/02/13/AEC).
3.1. Treatments:
Human PTH-(1-34) peptide (Sigma-Aldrich) is dissolved in 0.9% saline with 1% rat heat- inactivated serum in a final volume of 200 μl and injected s.c. daily 8 μg/100g/day for 1 or 14 days. The duration of 14 days elicits the maximum induction levels of MCP-1 (Li et al., 2007c).
ALN in saline carrier was injected once daily 1.0 g/kg/d s.c. The dose is adjusted for the Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 64 metabolic rate of the rat and equivalent to the human clinical dose (Iwata et al., 2006). The initial study was conducted using a single PTH dose that was injected 24 hours after loading of
SFx and an equivalent single VEH injection. Ulnae were collected at 1, 2, 6 and 10 weeks. The results obtained from the initial study showed no differences or significant findings at the one- week and 10-week timelines. Therefore, a decision was made to conclude the remaining experiments (daily PTH and combined ALN PTH treatments) with only the two-week and six- week timelines. This is in agreement with the reduction principle of the ethical use of animals in research and testing (The three Rs principles) (Russell and Burch, 1959).
Table 1. Experimental design for single PTH, daily PTH, and combined ALN-PTH
experiments. Number of rats per group (n)= 15 per group.
Group 7day 14day 6wks 10wks
PTH1 15 15 15 15
VEH1 15 15 15 15
PTH2 N/A 15 15 N/A
VEH2 N/A 15 15 N/A
ALN1 N/A 15 15 N/A
ALN2 N/A 15 15 N/A
ALN-PTH1 N/A 15 15 N/A
ALN-PTH2 N/A 15 15 N/A
VEH3 N/A 15 15 N/A
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Treatment schedules:
PTH1=Single dose PTH 24h after SFx.
VEH1= control for PTH1.
PTH2= daily PTH injection post-SFx, up to 14 days.
VEH2= control for PTH2.
ALN1= daily ALN up to 6 weeks.
ALN2= Cessation of ALN at time of SFx, no treatment up to 6 weeks.
ALN-PTH1= daily ALN + 14 days of PTH treatment.
ALN-PTH2= Cessation of ALN at time of SFx, + 14 days of PTH treatment.
VEH3= control for ALN-PTH groups.
3.2. Experimental Design:
Female Wistar rats 12 weeks of age, weight (300 ± 15g) had an ulnar SFx induced. Rats were anesthetized with halothane and oxygen for loading. SFx was achieved in a single loading session of the forearm in axial compression at 18-20 N (Fig 3) using a Haversine wave at 2 Hz until an increase in displacement of 10% was reached, on average after ~8000 cycles (range =
4000-20,000 cycles), or about 60 minutes. This produced a remarkably standardized SFx.
Accounting for variability, anesthesia and recovery, 4-5 rats were loaded/day. Loading was performed in a loading device using a linear variable digital transformer (LVDT) to monitor displacement in the limb. Because of the natural ulnar curvature, axial compression is converted
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 66 into bending forces with the lateral cortex in tension and the medial in compression (Mosley et al, 1997). Loading involved cyclic compressive at 18-20 N load using a Haversine wave form.
A single injection of opioid analgesia (Buprenorphine 0.05 mg/kg s.c) was used following the loading sessions. Rats were housed in pairs and allowed unrestricted cage activity. Rats were euthanized at 1, 2, 6 or 10 weeks post-SFx for the single injection experiment, and 2 or 6 weeks for the daily PTH and ALN PTH experiments (Table 1).
Screw adjustment
LVDT Upper Loader
Lower Loader
Figure 3: Technique for SFx axial compression loading. Fig 3A: Forwood Laboratory, School of Medical Science, Griffith University, Australia. Fig 3B: courtesy of Orthopaedic Research laboratories, School of Medicine, Indiana University, USA.
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3.3. Histomorphometry:
Rats were anesthetized using sodium pentobarbital (Nembutal®, 50 mg.kg-1) and perfused intracardially with phosphate buffered saline until the tissue blanched, followed by 4% paraformaldehyde. Ulnae of all rats and femura (only for the daily PTH injection and daily
VEH injection groups) were dissected to remove soft tissues (leaving deep muscles intact), post-fixed in paraformaldehyde for 4 h at 4° C, decalcified in EDTA for 4 weeks, paraffin embedded and transverse sections, 5 m, cut in the region of the SFx. Approximately 300 serial sections were cut in the transverse plane using a microtome, sufficient to locate the region of the SFx. Adjacent serial sections were stained with toluidine blue and for tartrate resistant acid phosphatase (TRAP). As previously described (Kidd et al., 2011), two toluidine blue-stained sections from each bone were measured at a standard level along the SFx that is located midway between central medullary cavity and medial surface of the cortical margins (Fig 4). This procedure avoids bias in selecting the region of interest due to standardization of the position of SFx selected for examination and analysis.
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Figure 4: (a): Photomicrograph of a transverse section of a rat’s ulna showing the standardized position chosen for histomorphometric analysis in this study along the SFx. To avoid any bias the slides selected for analysis were chosen at a position where the SFx (Green) was midway between the outer cortical margins (Red) and the inner medullary cavity (Yellow) – (Toluidine
Blue 2X). (b): A schematic diagram on a TRAP stained slide showing the boundaries of the
Basic Multicellular Unit (BMU) (Yellow) with osteoclast count (Green) and osteoclast perimeter (Black) – (TRAP 10X).
Osteomeasure™ software was used for histomorphometric measurements. The area of a BMU was defined as the total area that had been resorbed. Within this BMU, the area filled with new bone formation was defined as healed area. A distinct cement line around a previously resorbed area of bone defined the healed area. The area that had been resorbed, but not yet filled by new bone formation, was defined as porosity. The total osteoclast number was counted along the
SFx. Neither the osteoclasts present within the entire cortical bone area, nor in the newly formed
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 69 woven bone callus were included in the osteoclast count.
Morphometric measures included standard variables (Figs 4, 5 and 6):
1- Cortical bone area (Ct.Ar, mm2).
2- Cortical bone perimeter (Ct.Pm, mm).
3- Woven bone area (Wo.B.Ar, mm2).
4- Woven bone perimeter (Wo.B.Pm, mm).
5- Woven bone Width (Wo.B.Wi, mm).
6- Length of stress fracture (SFx.Le, µm)
7- Length of remodeling unit along stress fracture line (BMU.Le, µm).
8- Porosity (BMU) area (SFx.Po.Ar, µm2).
9- Porosity (BMU) area perimeter (SFx.Po.Pm, µm).
9- Erosion (unhealed) area (SFx.E.Ar, µm2).
10- Erosion (unhealed) perimeter (SFx.E.Pm, µm).
11- Healing area (SFx.He.Ar, µm2).
12- Healing area perimeter (SFx.He.Pm, µm).
13- Number of Osteoclasts (N.Oc).
14- Osteoclasts surface perimeter (Oc.Pm, µm).
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From the standard variables measured above, derived variables were obtained:
1- Number of osteoclasts per µm2 of Basic Multicellular Unit (BMU) area (N.Oc/µm).
2- Number of osteoclasts per µm of Basic Multicellular Unit (BMU) length (N.Oc/µm).
3- Healing percentage = (SFx.He.Ar,µm2/SFx.Po.Ar x 100, µm2).
4- Woven bone apposition rate per day = Wo.B width X Wo.B perimeter/Number of days
In addition, the following derived variables were calculated to correct for variations in the total length of the fracture line.
1- Percentage fracture length occupied by bone formation (SFx.He.Pm, µm/SFx.Le, µm %)
2- Healing area per mm2 of the cortical bone area (SFx.He.Ar, µm2/ Ct.Ar, mm2)
3- Porosity (BMU) area per mm2 of the cortical bone area (SFx.Po.Ar, µm2 /Ct.Ar, mm2)
4- Percentage fracture length occupied by erosion (SFx.E.Pm, µm /SFx.Le, µm %)
5- Erosion area per mm2 of the cortical bone area (SFx.E.Ar, µm2/ Ct.Ar, mm2)
To confirm the efficacy of the daily iPTH treatment, the systemic effect of daily iPTH on the trabecular bone of the distal femur within the same SFx animal model, the percentage of mineralized bone volume within the total tissue volume (BV/TV %) was measured.
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(a) (b)
Figure 5: Histomorphometric analysis using Osteomeasure™. (a): Toludine Blue section showing a transverse section in the rat ulna that was selected for histomorphometric analysis of
SFx. (b); TRAP stained section showing the histomorphometric analysis of the osteoclast number and perimeter.
Red Cortical area (Ct.Ar, mm2) Green Number of osteoclasts (N.Oc)
Yellow Woven bone area (Wo.B.Ar, mm2) Yellow Oc surface perimeter (OC.Pm, µm)
Light blue Woven bone Width (Wo.B.Wi, mm)
Dark red Length of stress fracture (SFx.Le, µm)
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Figure 6: Showing histomorphometric analysis of a Basic Multicellular Unit (BMU) using
Osteomeasure™ software.
Blue Porosity area (SFx.Po.Ar, µm2)
Light Blue Erosion area (SFx.E.Ar, µm2)
Yellow Length of remodeling unit along fracture line (BMU.Le, µm)
Purple Area of healed new bone (SFx.He.Ar, µm2)
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3.4. Statistical Analyses:
Sample sizes were informed by the study for Risedronate treatment of SFx in rats (Kidd et al.,
2010). This work demonstrated that 15 rats/group is required to achieve statistical power of
80% for P < 0.05 to detect differences among groups for morphometric variables. Power analysis showed that 15 rats per group provides 80% power of detecting an effect size of PTH treatment of 0.6 Z-scores at an alpha level of 0.05. Similarly, adequate power is present to detect the effect of time (80% power to detect an effect of 0.72 Z-scores). For the possible interaction of time and PTH treatment, power is 80% to detect an interaction that has an effect size close to one Z score. Furthermore, for statistically non-significant findings, Cohen’s d was calculated to provide an objective assessment of the effect size (Cohen, 1988; Cohen, 1992; Sawilowsky,
2009).
Data was analyzed using a 2-way ANOVA using time and treatment group as the independent variables in experiment 1 (Single PTH Injection) and experiment 2 (Daily PTH Injections). In the presence of a significant statistical interaction, the main effects in the original 2-way
ANOVA were ignored, post-hoc pairwise comparisons were performed between individual groups and differences determined using Fishers LSD. In the presence of a non-significant statistical interaction, the ANOVA main effect for each independent variable (time or treatment group) was reported independently. In experiment 3 (Combined ALN PTH Treatment) a 2-way
ANOVA with time (2 weeks vs, 6 weeks) and specific treatment group (ALN1 vs ALN2 vs
ALN-PTH1 vs. ALN-PTH2 vs VEH) as the independent variables. Pearson correlation coefficient was used to determine any significant correlations between variables. Significance was accepted at P≤0.05.
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When comparisons were made between different treatment modes (Single PTH vs. Daily PTH) or (Daily PTH vs ALN PTH1 vs ALN PTH2), one-way ANOVA test was used with each treatment group as an independent variable. The Bonferroni correction method was used to adjust the level of significance for the family wise error rate. An adjusted alpha level of .0125 per test (.05/4) was used for multiple comaprisons in experiment 2 (Single PTH vs. Daily PTH).
Significance was accepted at P≤0.0125 in multiple comparisons for experiment 2. An adjusted alpha level of .008 per test (.05/6) was used for multiple comaprisons in experiment 3 (Daily
PTH vs ALN PTH1 vs ALN PTH2). Significance was accepted at P≤0.008 in multiple comparisons for experiment 3.
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Chapter 4
Single PTH Experiment
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4.1. Introduction:
Parathyroid hormone (PTH) could have an anabolic or catabolic effect on bone depending on the mode of administration (continuous vs. intermittent) (Qin et al., 2014). PTH has shown promising results in treatment of fracture healing (Ellegaard et al., 2010; Aspnberg et al., 2010;
Peichl et al., 2011; Campbell et al., 2015; Yukata et al., 2018), prevention of fractures as well as reduction of fracture risk in patients with osteoporosis (Neer et al., 2001; Capriani et al.,
2012; Crandall et al., 2012). Despite the positive therapeutic effects, most of the PTH treatment protocols are prolonged, can place a financial burden on patients and governments due to the high cost of PTH, they also raise concerns regards some reported side effects, observed in animals (Vahle et al., 2002; Jolette et al., 2006; Tashjian and Goltzman, 2008; Watanabe et al.,
2012). Therefore, the current experiment is highly important because it is the first to explore the effect of a single PTH injection on the healing of SFx. The ideal period to induce the maximum MCP-1 levels has been reported to be 14 days (Li et al., 2007c). If a single PTH injection is a sufficient to elict similar MCP-1 levels and shift the quantum (amount or net result) of remodeling towards bone formation, this would open the doors to a new paradigm shift in the research and treatment of SFxs with short duration iPTH or even single PTH injections, as well as the mechanism of anabolic PTH action which is not yet fully understood.
We hypothesized that a single treatment with PTH, 24 hours following SFx creation, would accelerate histomorphometric indices of SFx healing. If successful, this innovative approach of using a single PTH injection to accelerate the healing of SFxs could of high clinical relevance.
The findings from the current experiment could also be extended and further tested to be applied for the AFF model that shares a lot of properties with the current SFx animal model.
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4.2. Experimental design:
Table 2: Experimental design of the single PTH experiment. Number of rats per group (n)= 15.
1 week 2 weeks 6 weeks 10 weeks
Single PTH 15 15 15 15
Single VEH 15 15 15 15
This experiment included 120 female wistar rats. The experiment was conducted as outlined in chapter 3. Rats were treated with either a single PTH injection 24 hours after SFx loading (8
μg/100g) or an equivalent VEH injection. The data collected from this experiment was analyzed using a 2-way ANOVA with time and treatment group as the independent variables. In the presence of a significant statistical interaction, the main effects in the original 2-way ANOVA were ignored, post-hoc pairwise comparisons were performed between individual groups and differences determined using Fishers LSD. In the presence of a non-significant statistical interaction, the ANOVA main effect for each independent variable (time or treatment group) was reported independtely. Significance was accepted at P≤0.05.
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4.3. Results:
4.3.1. Single PTH injection:
There were no significant differences among groups in terms of cortical bone parameters, length of SFx, nor length of BMU along the SFx line and number of osteoclasts per µm of Basic
Multicellular Unit (BMU) length (Appendix 1a). Furthermore, there were no signicant changes to report one-week post-SFx induction as the initial phase of bone remodeling and healing has not started yet. Furthermore, the results obtained from the 10 weeks post-SFx induction group showed no significant changes when compared to the 6 weeks post-SFx group. Furthermore, results obtained form the 10 weeks post SFx induction group was almost identical to the VEH group at the same timeline, with the exception of porosity parameters.
4.3.2. Woven bone parameters:
There was no significant interaction between the main effects of time and the type of treatment (VEH or PTH) on the woven bone parameters (Table 3). Woven bone area was greater in PTH group when compared to VEH group (P= 0.035). Woven bone apposition rate was significantly less after 2 weeks (P<0.001) when compared to the first week, it was also significantly less after 6 weeks (P<0.001) when compared to the second week (Fig 7 and 9a).
Woven bone width was significantly less after 6 weeks (P<0.001) when compared to the second week (Fig 9b). Woven bone perimeter was greater over time (P= 0.003). It was significantly greater after 2 weeks (P= 0.016) when compared to the first week (Fig 9c).
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Figure 7: (a & b): Showing the significantly high woven bone apposition rates (red arrows point to the amount of woven bone in each section) during early remodeling phases (1 week post SFx loading) (a & b) when compared to late remodeling (6 weeks post SFx loading) (c & d)
(Toluidine Blue 2 X). Scale bar = 100µm.
4.3.3. Osteoclast parameters:
There was a significant interaction between the main effects of time and the type of treatment
(VEH or PTH) on the number of osteoclasts (F= 2.751; P= 0.047) and osteoclast perimeter (F=
5.27; P= 0.02) (Table 3).The total osteoclast number and perimeter along the SFx were significantly greater (P<0.001) in PTH group at 2 weeks, compared to VEH (Figs 8d, 8e, 8f,
8g, 9d and 9e). The number of osteoclasts per µm2 basic multicellular unit (BMU) area was significantly less after 6 weeks (P<0.001) when compared to the second week (Fig 9f).
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Figure 8: (a): Showing the limited availability of osteoclasts in VEH group after 1 week (b): Showing the significant increase in osteoclast in PTH group after 2 weeks when compared to the VEH group shown in (a) (TRAP 10X), (c & d): Showing the significant decrease in osteoclasts with the progression in healing after 6 weeks in both VEH and PTH groups (TRAP 10X). Scale bar = 10µm.
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Figure 9: Histomorphometric variables of SFx healing (±SD). PTH positively influenced bone histomorphmetric indices along all stages of the bone remodeling cycle following the loading of stress fracture (SFx). The time points between 2 and 6 weeks also influenced all variables significantly. There was a significant interaction between time and the type of treatment
(VEH or PTH) that affected osteoclasts number and perimeter after 2 weeks. *** = P≤0.01
(differences between PTH and VEH). ++ = P≤0.05, +++ = P≤0.01 (differences compared to the previous time point).
4.3.4. Healing parameters:
There was no significant interaction between the main effects of time and the type of treatment
(VEH or PTH) on the healing parameters (Table 3).
Compared to VEH, healing area was 17.4% higher (P= 0.815) and healing perimeter was 27% higher (P= 0.464) in PTH groups, after 6 weeks. These differences represent moderate effect sizes using Cohen’s d (d= 0.395 and d=0.443, respectively) (Figs 10e, 10f, 10g and 10h). There Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 82 was no significant effect of PTH on percentage healing (P= 0.839 – Cohen’s d= 0.440) after 10 weeks when compared to VEH groups (Figs 10g, 10h and 10i). Furthermore, there were no significant differences between VEH and PTH groups when the percentage fracture length occupied by bone formation (SFx.He.Pm, µm/SFx.Le, µm %) was calculated to correct for variations in the total length of the SFx line. With regards to the main effect of time on healing parameters, it was found that healing area, healing perimeter and percentage healing increased significantly after 6 weeks when compared to the second week in both VEH and PTH groups
(P<0.001; P<0.001 and P= 0.05 respectively) (Figs 9g, 9h, 9i). The normalized healing area
(relative to SFx length) (SFx.He.Pm, µm/SFx.Le, µm %) was not statistically significant after
2 weeks when compared to the first week (P= 0.096), but increased significantly after 6 weeks
(P<0.001) when compared to the second week (Fig 12b). Finally, the healing area per mm2 of the cortical bone area (SFx.He.Ar, µm2/ Ct.Ar, mm2) increased significantly after 6 weeks when compared to the second week (P<0.001) (Fig 12a).
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Figure 10: Progression of remodeling. (a & b): Shows absence of porosity (BMU) at the SFx entry point one week post loading in both VEH and PTH groups; (c & d): Development of porosity (BMU) area at the entry point of the SFx two weeks post loading in both VEH and
PTH groups; (e & f): Progression in healing and the gradual filling of the porosity (BMU) areas with new bone six weeks post loading in both VEH and PTH groups, with new bone formation also demonstrated along the course of the SFx (black arrows) in PTH group; (g & h): Shows the significant reduction in porosity (BMU) area 10 weeks post SFx loading in the PTH group
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 84
(h) when compared to VEH group (g) due to complete healing by direct remodeling starting at the entry of the SFx (Toluidine Blue 10X). Scale bar = 10µm. (i): A complete section showing the area of interest manginified in panels (a-h) including the entry point of SFx and BMU (black circle) (Toluidine Blue 2X). Scale bar = 100µm.
4.3.5. Erosion (unhealed) parameters:
There was no significant interaction between the main effects of time and the type of treatment
(VEH or PTH) on the erosion parameters (Table 3).
A significant negative correlation (r= -0.602) was observed between erosion and healing perimeters in PTH group after 6 weeks (P= 0.038) (Fig 11) (Fig 12e). There were no significant differences between VEH and PTH groups in erosion area) (Fig 12d). This was also the case when the percentage fracture length occupied by erosion (SFx.E.Pm, µm /SFx.Le, µm %) and the erosion area per µm of the SFx length (SFx.E.Ar, µm2/ SFx.Le, µm) were normalized for
SFx length or cortical bone area (SFx.E.Ar, µm2/ Ct.Ar, mm2) (Figs 12c, 12f and Table 3).
With regards to the main effect of time on erosion (unhealed) parameters, there was a significant increase in erosion (unhealed) area and perimeter after 2 weeks (P= 0.008; P<0.001 respectively) when compared to the first week, this was followed by a significant decrease in erosion area and perimeter after 6 weeks (P<0.001) (Figs 12d and 12e). This was also consistent with corrected variables including the erosion (unhealed) area per mm2 of the cortical bone area and the percentage fracture length occupied by erosion, where a significant increase was observed after 2 weeks (P= 0.005; P<0.001 respectively) when compared to the first week in both VEH and PTH groups. This was followed by a significant decrease in both the erosion
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(unhealed) area per mm2 of the cortical bone area and the percentage fracture length occupied by erosion after 6 weeks (P<0.001) (Fig 12c, 12f).
Figure 11: A scatterplot with a line of best fit showing the significant negative correlation between erosion perimeter and healing perimeter 6 weeks post SFx induction.
4.3.6 Porosity (BMU) parameters:
There was no significant interaction between the main effects of time and the type of treatment
(VEH or PTH) on the erosion parameters (Table 3).
There were no significant differences in porosity area and porosity perimeters in PTH groups when compared to VEH groups (Fig. 10f). It should be noted that there was a 43% decrease
(P= 0.703–Cohen’s d= 0.597) in porosity area in PTH group after 10 weeks when compared to
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VEH group (Figs 10g, 10h, 12g and 12h). With regards to the main effect of time on porosity parameters, we found that porosity (BMU) area, perimeter and porosity (BMU) area per mm2 of the cortical bone area (SFx.Po.Ar, µm2 /Ct.Ar, mm2) increased significantly after 2 weeks
(P= 0.01; P= 0.0002 and P= 0.01 respectively) when compared to the first week (Figs 12g, 12h,
12i and Table 3).
Figure 12: Histomorphometric variables of SFx healing (±SD). PTH increased the healing area/ mm2 of the cortical bone area 6 weeks after SFx. PTH also decreased the porosity (BMU) parameters 10 weeks after SFx due to progression in healing. Most of the derived and normalized variables were significantly influenced by the main effect of time between the 2 week and 6 week time points. However, there was no significant interaction between time and the type of treatment (VEH or PTH). +++ = P≤0.01 (differences compared to previous time point).
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Table 3: A summary of the significant findings related to the effect of time, treatment as well
as the interaction between treatment and time on different variables using a linear model, 2-
way ANOVA statistical analysis.
Variable Effect F Significance
Treatment 4.577 .035
Woven Bone Area (Wo.B.Ar, mm2) Time .870 .459
Interaction (Treatment * Time) .977 .407
Treatment 2.652 .106
Woven Bone Width (Wo.B.Wi, mm) Time 13.41 <0.001
Interaction (Treatment * Time) .893 .448
Treatment 1.695 .196 Woven Bone Perimeter (Wo.B.Pm, Time 4.901 .003 mm) Interaction (Treatment * Time) .690 .560
Treatment .146 .703 Porosity (BMU) Area (SFx.Po.Ar, Time 10.73 <0.001 µm2) Interaction (Treatment * Time) 2.249 .087
Treatment .014 .906 Porosity (BMU) Area Perimeter Time 14.45 <0.001 (SFx.Po.Pm, µm) Interaction (Treatment * Time) 2.396 .073
Treatment .055 .815
Healing Area (SFx.He.Ar, µm2) Time 35.12 <0.001
Interaction (Treatment * Time) 1.125 .343
Healing Area Perimeter Treatment .540 .464
(SFx.He.Pm, µm) Time 36.83 <0.001
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 88
Interaction (Treatment * Time) 1.155 .331
Treatment .577 .449 Erosion (unhealed) Area Time 36.79 <0.001 (SFx.E.Ar, µm2) Interaction (Treatment * Time) .139 .936
Treatment .084 .772 Erosion (unhealed) Perimeter Time 33.15 <0.001 (SFx.E.Pm, µm) Interaction (Treatment * Time) .028 .994
Treatment 4.643 .034
Number of osteoclasts (N.Oc) Time 28.16 <0.001
Interaction (Treatment * Time) 2.751 .047
Treatment 6.923 .010 Osteoclasts surface perimeter Time 19.23 <0.001 (Oc.Pm, µm) Interaction (Treatment * Time) 5.276 .002
Treatment .041 .839
Healing percentage (He%) Time 4.165 .020
Interaction (Treatment * Time) 1.195 .310
Treatment 1.301 .257 Woven bone apposition rate per day Time 43.92 <0.001 (mm2/day) Interaction (Treatment * Time) .96 .412
Number of osteoclasts per µm2 of Treatment 3.660 .060
Basic Multicellular Unit (BMU) area Time 6.750 .002
(N.Oc/µm2) Interaction (Treatment * Time) .954 .391
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 89
Healing area per mm2 of the cortical Treatment .193 .661
bone area (SFx.He.Ar, µm2/ Ct.Ar, Time 34.617 <0.001
mm2) Interaction (Treatment * Time) 1.468 .228
Porosity (BMU) area per mm2 of the Treatment .008 .931
cortical bone area Time 10.19 <0.001
(SFx.Po.Ar, µm2 /Ct.Ar, mm2) Interaction (Treatment * Time) 2.203 .092
Treatment .612 .436 Erosion (unhealed) area per mm2 of Time 39.86 <0.001 the cortical bone area Interaction (Treatment * Time) .289 .833 (SFx.E.Ar, µm2/ Ct.Ar, mm2)
Percentage fracture length occupied Treatment .049 .826
by bone formation Time 56.36 <0.001 Interaction (Treatment * Time) .701 .554 (SFx.He.Pm, µm/SFx.Le, µm %)
Percentage fracture length occupied Treatment .090 .764 by erosion (SFx.E.Pm, µm /SFx.Le, Time 38.68 <0.001
µm %) Interaction (Treatment * Time) .038 .990
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 90
4.4. Discussion – Single PTH experiment:
We tested the hypothesis that short-term treatment is sufficient to increase and accelerate remodeling and accelerate healing of SFx. Following PTH treatment, osteoclast parameters were greater than controls in the resorption phase of bone remodeling that occupies the first two weeks after SFx initiation. Although both groups showed greater bone formation at six and ten weeks, the effect of PTH on osteoclastogenesis did not translate into significantly greater bone formation at these time-points. For example, there was no significant resolution of porosity (BMU filling) in the formation phase at six to ten weeks after SFx initiation. Timing of outcome measures was based on our published data for measurement of resorption (7 and 14 days) and formation at (6 and 10 weeks) in the same rat model (Kidd et al., 2010). The increase in healing parameters in the PTH group, though not significant, suggests that further investigation is merited, either in PTH dose or time points, to determine if there is a connection between the PTH mediated increase in OC number in the BMU and ultimate healing.
The results can be explained by understanding the mechanism of healing of SFxs, which occurs through direct remodeling and starts from the periosteal exit point of the SFx and progresses along the SFx line (Kidd et al., 2010). Basic Multicellular Units (BMUs) are formed in the first two weeks and osteoclasts play a significant role at this initial stage of healing (Kidd et al.,
2010). This is different from the repair process and healing of a complete fracture, which starts with an external as well as an internal callus, followed by endochondral ossification of the external callus. Only after this initial phase of stabilization, bone remodeling starts to replace the lamellar bone (Li et al., 1999). The rat model used in this study is a standardized and reliable model that has been used in previous studies to produce a consistent SFx (Kidd et al., 2010;
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 91
Kidd et al., 2011; Kidd et al., 2013) and facilitates the study of SFx remodeling. The time points chosen for histomorphometric analysis of SFx remodeling were carefully chosen based on published studies that showed that 2 weeks is the most representative time point for the early
(resorptive) phase of the remodeling cycle, while 6 weeks is the most representative time point for the late (formative) phase of the remodeling cycle. Current PTH treatment protocols are
18-24 months (Canalis et al., 2007) which equals to 4 weeks in a rat’s life span (Sengupta,
2013). The innovation in the present study was to reduce the treatment window of PTH to the shortest possible period through a single injection in comparison to 14 daily injections which is sufficient to induce a stable (plateau) serum PTH levels (Li et al., 2007b; Li et al., 2007c).
The observed increase in healing parameters correlated significantly with the reduction in erosion 6 weeks post-SFx. This correlation demonstrates a positive effect of a single PTH injection on the formation phase of SFx remodeling, independent of time in the linear model.
The main effect of time showed statistically significant improvement in healing parameters from 2 to 10 weeks. Although the loading protocol reliably produces a SFx line, there is variability in the number of cycles required to achieve the 10 percent displacement criterion.
This produces variability in the nature of the SFx and its healing that is reflected in the variability of histomorphometric data. Increasing the sample size could improve statistical power, however, the number of animals used in this experiment was based on the 3R principles of animal testing determined from a calculation of statistical power of 80% for p≤0.05 to detect differences among groups for morphometric variables from previous studies (Kidd et al., 2010).
A limitation of the current study is the modest change that was observed in healing and porosity variables. While we detected significant increases in osteoclastogenesis, the single dose of PTH may have been insufficient to augment the subsequent formation phase above that observed in Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 92 the VEH group. The power/sample size calculation for the project was performed a priori and understandably did not take into account possible variations in data distribution. Although not statiscally significant porosity parameters were less after 10 weeks. This is mechanically important during healing given the exponential relationship between bone strength and porosity
(Wachter et al., 2002).
At the time of SFx loading, rats used in the present study were 17 weeks of age. Previous work by Kidd et al., 2010 was used to characterize the current rat model. iPTH treatment increases bone formation at all ages, but the underlying cellular and molecular mechanisms facilitating the anabolic response to the hormone could differ by age (Friedl et al., 2007). Furthermore, the chosen age of 17 weeks also ensures that the rats are out of the adolescent growth spurt, which is about 40 days of age (Yingling et al., 2007). A potential limitation of the study was the fact that rats undergo continuous longitudinal skeletal growth and have other differences to the human skeleton. Furthermore, the age-dependent differences of the iPTH effect are mainly related to bone density and quality, which were not investigated in the present study.
Interestingly, PTH failed to increase union rates, callus size and strength in open fractures when compared to closed fracture (Tagil et al., 2010). This was suggested to be due to the lack of sufficient number of cells for recruitment in the area of open fractures, as well as the inability of PTH to recruit progenitor cells from potential surrounding tissues. Other agents with nonspecific anabolic effect such as BMP were suggested (Tagil et al., 2010). Nonetheless,
Kumabe et al. recently showed that triweekly administration of PTH (1–34) increased union rate and accelerated bone healing in a rat refractory fracture model, suggesting that systemic
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 93 administration of PTH (1–34) could become a novel and useful therapy for delayed union or non-union (Kumbae et al., 2017). This confirms the role of certain bone modulators such as monocyte chemotactic protein-1 (MCP-1) in recruitment of osteoclast precursors for bone remodeling (Li et al., 2007c). It also sheds more light on the suggested mechanism of action of
PTH.
In the present study, the significant increase in the number of osteoclasts along the SFx after 2 weeks may be explained by PTH induction of monocyte chemotactic protein-1 (MCP-1), which is responsible for differentiation and recruitment of osteoclast precursors in early remodeling phases (Kim et al., 2005, 2006; Wu et al., 2013; Morrison et al., 2014). Although we did not measure MCP-1 in this study, previous work demonstrated that MCP-1 is specifically regulated during SFx repair, and we hypothesize that iPTH may amplify that response (Wu et al., 2013).
Furthermore, the significant increase in the number of osteoclasts along the SFx after two weeks is supported by recent studies showing an increase in osteoclasts and osteoblasts after 4 weeks of daily PTH treatment (Vrahnas et al., 2016), and an increased influx of osteoclasts after iPTH treatment in mice (Meakin at al., 2017).
The mechanism of the anabolic action of PTH is supported by other factors including calcium availability and the calcium sensing receptor (CaSR) (Al-Dujaili et al., 2016). Osteoclasts are responsible for increasing the availability of free Ca++ ions during bone resorption (Silver et al., 1988). Free Ca++ concentration up to 40mM result in osteoblast migration, proliferation, survival, differentiation and mineralization which facilitate healing and new bone formation
(Dvorak and Riccardi., 2004; Marie, 2010; Riccardi et al., 2013). Also the downregulation of
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 94
Sclerostin has been linked to have a contributing effect to the anabolic PTH effect, through reduction of Sost expression which negatively regulated Wnt signaling (O’Brien et al., 2008;
Kramer et al., 2010).
There were no significant effects shown by a single PTH injection after one week. This is supported by previous research that demonstrated the initial stimulation of bone modeling is evident through an increase in bone formation markers such as aminoterminal propeptide of type I procollagen (P1NP) and osteocalcin (Bilezikian, 2008). This is followed by an increase in bone resorption markers such as serum cross-linked C-telopeptide (CTX) and urinary cross- linked N-telopeptide of type I collagen (NTX). This sequence gives rise to an anabolic window during which the osteoanabolic actions of PTH are optimized (Bilezikian, 2008). In the present study, the anabolic window was between 2-6 weeks after the initial PTH treatment. After 10 weeks the effect of a single PTH injection was similar to a VEH injection. This coincides with another study where a cyclic PTH regimen was utilized for 7 weeks and at the end of the experiment, the cyclic PTH regimen did not further effects beyond those achieved at 2 weeks
(Iida-Klein et al., 2006).
Over a 4-week period, Sloan et al showed that daily intermittent PTH (iPTH) treatment enhanced intracortical bone remodeling in the rat ulna (Sloan et al., 2010). This was demonstrated by increased resorption area and mineral apposition rate from 2 weeks after SFx
(Sloan et al., 2010). The accelerated repair shown by Sloan is consistent with the data from the current study, but their iPTH treatment regime was clearly more prolonged. In non-SFx models, therapeutic treatment with iPTH improves bone formation and mineral apposition rate after one
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 95 week of starting the PTH treatment; as well as increasing eroded bone surface and osteoclast number (Nishida et al., 1994). Similarly, iPTH enhanced mechanical properties after distraction osteogenesis in rats; an intervention used in both leg lengthening and bone transportation with external fixtures in treatment of fractures and non-unions (Seebach et al., 2004). Intermittent
PTH treatment at 30 µg/kg before and after osteotomy in rats accelerated the healing process as evidenced by earlier replacement of woven bone to lamellar bone, increased new cortical shell formation, and increased the ultimate load up to 12 weeks after osteotomy in rats
(Komatsubara et al., 2005).
The above observations from daily repeated iPTH are consistent with those from the single dose protocol, but clearly of greater magnitude. There were increases in healing area and perimeter after six weeks, as well as an increase in healing percentage after 10 weeks following a single
PTH injection. Though not statistically significant, we hypothesize that the increase in healing parameters could explain the decrease in the overall porosity after six weeks and demonstrate the shift in the remodeling cycle towards bone formation after PTH administration, which is in agreement with previous studies (Nishida et al., 1994; Bellido et al., 2003; Seebach et al., 2004;
Komatsubara et al., 2005; Sloan et al., 2010).
The recruitment and availability of osteoclasts is not only important for mediation of the early
(resorptive) phase of the bone remodeling cycle, it also has a significant effect on the PTH mediated anabolic effect observed during the late (formative) phase of the bone remodeling cycle (Vrahnas et al., 2016). This has been reported through osteoclastic expression of Bone
Morphogenic Proteins (BMPs) which also regulate the formation, activity and functions of osteoclasts (Kanatani et al., 1995; Onishi et al., 1998; Kaneko et al., 2000; Itoh et al., 2001;
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 96
Giannoudis et al., 2007). For example, BMP2 improves the activation of murine osteoclast precursors (Jensen et al., 2010); and BMP2 and BMP4 signaling facilitates the anabolic effect of PTH (Khan et al., 2016). In addition to the above, BMP2 and BMP7 are effective clinically in treatment of non-union fractures in long bone (Derner and Anderson, 2005; Conway et al.,
2014).
Therefore, in the present study, the significant increase in the number of osteoclasts in PTH treated groups may have been accompanied by an increase in BMPs expression, which in turn promoted the anabolic activity of PTH and healing, especially when PTH was unopposed by a concurrent ALN treatment. This would need experimental verification. This hypothesis is supported by the impairment of osteoblasts and osteoclasts function that was shown in cases of
BMP2/BMP4 deletion in KO mice (Khan et al., 2016), which would result in a reduction in the anabolic effect of PTH. In addition to the above, it has been proven through measuring bone mineral deposition and periosteal surface fluorescent labelling that iPTH could possibly have a simultaneous effect on both bone resorption and bone formation (Meakin et al., 2017).
The consensus in the available literature is that PTH augments BMP2 expression via CREB transactivation in osteoblasts, and that the anabolic effect of PTH is mediated by its ability to enhance BMP2 and BMP4 in osteoblasts and its precursors (Zhang et al., 2011; Kamiya et al.,
2012; Khan et al., 2016). Therefore, it seems reasonable in a clinical situation to recommend assessment of BMP2 and BMP4 as a predictor and a clinical indicator of treatment success in
SFx patients before prescribing or trialing PTH treatment. This is consistent with a study that
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 97 investigated osteoporotic treatment failures in patients with low bone mass and used
BMP2/BMP4 status as a clinical predictor of treatment success rate (Elraiyah et al., 2016).
PTH is under investigation for potential uses in tissue engineering and implant integration.
Innovations continue with development of a novel tissue engineering model using ectopic ossicles to investigate the anabolic effect of PTH, showing that cell implantation strategies can be responsive to PTH treatment (Pettway et al., 2005). This opens the way for many future clinical applications. The results obtained from the current work showed that a single PTH injection, 24 hours post SFx, augmented the remodeling response to a SFx in vivo, as evidenced in the osteoclastic parameters. This could be of potential benefits in minimizing the rates of failure of osseointegration in the fields of implantology and tissue engineering. The ease of administration of a single PTH injection also allows for multiple clinical applications in the future, where short-term intervention with PTH might be preferable and more economical.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 98
Chapter 5
Daily PTH Experiment
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 99
5.1. Introduction:
The previous chapter showed that a single PTH injection is capable of activating remodeling at a SFx site and shifting the quantum of remodeling towards bone formation. The most significant outcome from the single PTH injection experiment was the increase of osteoclastic parameters after 2 weeks from SFx induction (Bakr et al., 2019). This has been shown in literature with longer treatment periods and the mechanism of action was through the stimulation of the vitamin D receptor (VDR) and retinoid X receptors in osteoblasts by 1,25(OH)2D3 which increases M-CSF and RANKL production and indirectly stimulates osteoclastic bone resorption
(Crockett et al., 2011; Isthiaq et al., 2015).
Given the promising results from the single PTH injection experiment, the next step was to investigate the effect of daily iPTH for 14 days starting 24 hours after SFx induction on the histomorphometric indices of SFx healing. The duration of daily iPTH injections was based on previous protocols to elict the maximum MCP-1 and serum PTH levels (Li et al., 2007c). The hypothesis was that a daily treatment with iPTH for 14 days, starting 24 hours following SFx, will accelerate histomorphometric indices of SFx healing. It would be expected that iPTH daily injections for 14 days will have a similar or greater effect on SFx healing than a single PTH injection. In the literature, multiple studies investigated the efficacy of iPTH are based on prolonged treatment protocols for osteoporosis (Jiang et al., 2003; Ascenzi et al., 2012).
Furtehrmore, some studies investigated the effect of iPTH on the healing of SFxs in the rat ulna model (Kidd et al., 2010; Sloan et al., 2010) as well as healing of AFFs (Schilcher et al., 2014).
It was suggested that a minimum treatment period of 4 weeks is required to initiate a more accelerated pattern of bone resorption (Altman-Singles et al., 2017). Therefore, the experiment
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 100 described in this chapter fills gaps in the literature related to the efficacy of very short duration of iPTH to activate remodeling.
5.2. Experimental design:
This experiment included 60 female wistar rats. The experiment was conducted as outlined in chapter 3. Rats were treated with either daily PTH injections that started 24 hours after SFx loading (8 μg/100g/day) for 14 days or equivalent daily VEH injections. The data collected from this experiment was analyzed using a 2-way ANOVA with time and treatment group as the independent variables. In the presence of a significant statistical interaction, the main effects in the original 2-way ANOVA were ignored, post-hoc pairwise comparisons were performed between individual groups and differences determined using Fishers LSD. In the presence of a non-significant statistical interaction, the ANOVA main effect for each independent variable (time or treatment group) was reported independently. Significance was accepted at P≤0.05.
When comparisons were made between different treatment modes (Single PTH vs. Daily PTH), one-way ANOVA test was used with each treatment group as an independent variable. The
Bonferroni correction method was used to adjust the level of significance for the family wise error rate. An adjusted alpha level of .0125 per test (.05/4) was used for multiple comparisons.
Significance was accepted at P≤0.0125.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 101
Table 4: Experimental design of the daily PTH experiment. Number of rats per group (n)= 15.
2 weeks 6 weeks
Daily PTH 15 15
Daily VEH 15 15
5.3. Results
5.3.1. Daily PTH injections:
To confirm the efficacy of the daily iPTH treatment, we measured the systemic effect of daily iPTH on the BV/TV of trabecular bone of the distal femoral metaphysis within the same SFx animal model. The results showed that daily PTH injection group had a significantly higher
BV/TV within the distal femur than the daily VEH injection group after 2 weeks (P= 0.005).
After 6 weeks there were no significant differences in BV/TV within the distal femur between daily PTH and daily VEH groups (P= 0.265). (Table 5) (Fig 13).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 102
Figure 13: Daily PTH injections for 14 days resulted in a significantly higher BV/TV within the distal femur than the daily VEH injection group after 2 weeks ***P.≤0.01 (Mean ±SD).
There were no significant differences between daily PTH and daily VEH groups in terms of cortical bone area (CT.B.Ar, mm2) and cortical perimeter (Ct.Pm, mm), woven bone perimeter
(Wo.B.Pm, mm), and the length of SFx (SFx.Le, µm) within the rat ulna. (Appendix 1b)
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5.3.2. Woven bone parameters:
There was no significant interaction between the main effects of time and the type of treatment
(VEH or PTH) on the woven bone parameters (Table 5).
Woven bone apposition rate (Fig 14a) and woven bone area (Fig 14b) were significantly greater in daily PTH injection group when compared to the daily VEH injection group (P=
0.049 and P= 0.041 respectively). There were no statistically significant differences between daily PTH and daily VEH groups with regards to woven bone width (P= 0.173) (Fig 14c).
Woven bone parameters were significantly less after 6 weeks when compared to the second week in terms of woven bone area (P= 0.02), woven bone width (P<0.001) and woven bone apposition rate (P<0.001) (Figs 14a, 14b & 14c).
5.3.3. Osteoclast parameters:
There was no significant interaction between the main effects of time and the type of treatment
(VEH or PTH) on the osteoclast parameters (Table 5).
There were no significant differences between the daily PTH and the daily VEH injections groups in terms of osteoclast number or perimeter (P>0.2) (Figs 14d & 14e). With regards to the main effect of time, the number of osteoclasts and osteoclast perimeter were significantly less (P= 0.015 and P= 0.021 respectively) after 6 weeks when compared to 2 weeks (Figs 14d
& 14e). The number of osteoclasts per µm2 basic multicellular unit (BMU) area and the number of osteoclasts per µm basic multicellular unit (BMU) length were significantly less after 6 weeks when compared to the second week (P= 0.001 and P= 0.007 respectively) (Fig 14f).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 104
5.3.4. Healing parameters:
There was no significant interaction between the main effects of time and the type of treatment
(VEH or PTH) on the healing parameters (Table 5).
There were no significant differences in healing parameters between daily VEH and daily PTH injections groups (P>0.5). With regards to the main effect of time, healing area (P<0.001) (Fig
14g), healing perimeter (P<0.001) (Fig 14h), percentage healing (P<0.001) (Fig 14i), healing area per mm2 of the cortical bone area (P<0.001) (Fig 15a) and percentage fracture length occupied by bone formation (P<0.001) (Fig 15b) were significantly greater after 6 weeks when compared to the second week.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 105
Figure 14: Histomorphometric variables of SFx healing (±SD). There was no significant interaction between time and the type of treatment (Daily PTH or VEH). (a, b, c): Daily PTH injections for 14 days only improved woven bone apposition rate. Woven bone parameters were significantly less after 6 weeks when compared to the second week. (d, e, f): Osteoclast parameters were significantly less after 6 weeks when compared to the second week. (g, h, i):
Healing parameters improved significantly after 6 weeks when compared to the second week.
** = P≤0.05 (differences between PTH and VEH). +++ = P≤0.01 (differences compared to the previous time point).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 106
5.3.5. Erosion (Unhealed) parameters:
There was no significant interaction between the main effects of time and the type of treatment (VEH or PTH) on the erosion parameters (Table 5). There were no significant differences in erosion parameters between daily VEH and daily PTH injections groups. With regards to the main effect of time, the percentage fracture length occupied by erosion (P=
0.001) (Fig 15c), erosion area (P<0.001) (Fig 15d), erosion perimeter (P= 0.003) (Fig 15e), and the erosion area per mm2 of the cortical bone area (P<0.001) (Fig 15f) were significantly less after 6 weeks when compared to the second week.
5.3.6. Porosity (BMU) parameters:
There was no significant interaction between the main effects of time and the type of treatment
(VEH or PTH) on the porosity parameters (Table 5).
As a result of retention of osteoclasts for a longer period of time, porosity (BMU) area (P=
0.018) (Fig 15g), porosity (BMU) perimeter (P= 0.023) (Fig 15h) and porosity (BMU) area per mm2 of the cortical bone area (P= 0.010) (Fig 15i) were significantly higher after 6 weeks when compared to the second week.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 107
Figure 15: Histomorphometric variables of SFx healing (±SD). All of the standard, derived and normalized variables were significantly influenced by the main effect of time between the 2 week and 6 week time points. However, there was no significant interaction between time and the type of treatment (Daily PTH or VEH). +++ = P≤0.01 (differences compared to the previous time point).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 108
Table 5: A summary of the significant findings from the daily VEH and PTH groups related to
the effect of time, treatment as well as the interaction between treatment and time on different
variables using a linear model in a 2-way ANOVA statistical analysis.
Variable Effect F Significance Treatment 4.42 .041 Woven Bone Area (Wo.B.Ar, mm2) Time 5.73 .020 Interaction (Treatment * Time) .715 .402
Treatment 1.915 .173
Woven Bone Width (Wo.B.Wi, mm) Time 37.520 <0.001
Interaction (Treatment * Time) .260 .612 Treatment .080 .778 Porosity (BMU) Area (SFx.Po.Ar, µm2) Time 12.540 .001 Interaction (Treatment * Time) .023 .879 Treatment .270 .606 Porosity (BMU) Area Perimeter Time 16.447 <0.001 (SFx.Po.Pm, µm) Interaction (Treatment * Time) .623 .434 Treatment .012 .914
Healing Area (SFx.He.Ar, µm2) Time 55.332 <0.001
Interaction (Treatment * Time) .002 .961 Treatment .121 .729 Healing Area Perimeter Time 32.562 <0.001 (SFx.He.Pm, µm) Interaction (Treatment * Time) .974 .329 Treatment .260 .613 Erosion (unhealed) Area Time 35.037 <0.001 (SFx.E.Ar, µm2) Interaction (Treatment * Time) .068 .796 Treatment .318 .576 Erosion (unhealed) Perimeter Time 15.063 <0.001 (SFx.E.Pm, µm) Interaction (Treatment * Time) .212 .674
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 109
Treatment .888 .350
Number of osteoclasts (N.Oc) Time 17.846 <0.001
Interaction (Treatment * Time) .546 .463 Treatment 1.197 .279 Osteoclasts surface perimeter Time 16.523 <0.001 (Oc.Pm, µm) Interaction (Treatment * Time) .570 .454 Treatment .022 .882
Healing percentage (He%) Time 11.034 <0.001
Interaction (Treatment * Time) .439 .511 Treatment 3.925 .049 Woven bone apposition rate per day Time 266.685 <0.001 (mm2/day) Interaction (Treatment * Time) 1.35 .251 Treatment 1.327 .257 Number of osteoclasts per µm2 of Basic Multicellular Unit (BMU) area Time 14.467 .001 (N.Oc/µm2) Interaction (Treatment * Time) .477 .494 Treatment 1.089 .302 Number of osteoclasts per µm of Basic Multicellular Unit (BMU) length Time 7.898 0.007 (N.Oc/µm) Interaction (Treatment * Time) 1.027 .316 Treatment .012 .912 Healing area per mm2 of the cortical bone area (SFx.He.Ar, µm2/ Ct.Ar, Time 34.032 <0.001 mm2) Interaction (Treatment * Time) .001 .975
Porosity (BMU) area per mm2 of the Treatment .105 .747 cortical bone area Time 15.710 <0.001 2 2 (SFx.Po.Ar, µm /Ct.Ar, mm ) Interaction (Treatment * Time) .065 .801 Treatment .287 .595 Time 35.657 <0.001 Erosion (unhealed) area per mm2 of the Interaction (Treatment * Time) .056 cortical bone area .814 (SFx.E.Ar, µm2/ Ct.Ar, mm2)
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 110
Treatment .080 .779 Percentage fracture length occupied by Time 33.717 <0.001 bone formation Interaction (Treatment * Time) 1.088 (SFx.He.Pm, µm/SFx.Le, µm %) .302
Treatment .577 .451 Percentage fracture length occupied by Time 16.657 <0.001 erosion (SFx.E.Pm, µm /SFx.Le, µm %) Interaction (Treatment * Time) .412 .524
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 111
5.3.7. Comparison between single and daily PTH injections:
A comparison between the single and daily modes of PTH administration was made to highlight if there were any significant differences. One-way ANOVA test was used in this comparison with each treatment group as an independent variable. Post hoc test results were used to compare only between the single injection mode vs. daily injections at the 2-week timepoint and the 6-week timepoint. Comparisons were not made between different treatment modes at different timelines nor between groups from the same treatment group at different timelines as it has been already discussed in this chapter.
There were no significant differences between single PTH and daily PTH injections at the adjusted level of accepted significance P≤0.0125.
5.4. Discussion – Daily PTH experiment:
Though observed in a different model, dose-dependent increases in bone formation in a fracture callus with increasing PTH doses were associated with a decrease in osteoclast numbers, and diminished bone remodeling (Milstery et al., 2017). It was demonstrated that higher doses of
PTH had no additional benefits on the mechanical stiffness of the callus. Therefore, it was concluded that lower doses of PTH and administration only during the early phases of callus formation might be advantageous to stimulate fracture healing (Milstrey et al., 2017). All of the above highlights the importance of the modeling effect that PTH has during the early stages of treatment. Research involving animal experiments showed that modeling accounted for only
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 112
20% of bone formation, while human studies demonstrated between 5% - 30% of new bone formation taking place at modeling surfaces (Ma et al., 2006; Rhee at al., 2013). SFxs heal by direct remodeling and the increased woven bone response that was induced by iPTH might activate periosteal osteoblasts but not in the SFx line and was not sufficient to allow for a significant increase in healing parameters in the current experiment after 6 weeks.
This could be explained by the hypothesis that the activity of osteoclasts does not contribute to anabolism of PTH yet they produce factors that are essential in the regulation of osteoblast activity, as well as maintaining the normal coupled response of resorption and formation in bone remodeling (Karsdal et al., 2007). For example, it was demonstrated that a combination of cathepsin K inhibitor with iPTH therapy increased bone formation, as it maintains osteoclast numbers while also reducing their activity, confirming that osteoblast activity alone is not sufficient enough to induce the anabolic effects of PTH in bone, but rather it must be in parallel with factors produced by osteoclasts (Yamane et al., 2009). Similarly, a study that investigated in-vivo PTH treatment increased in vitro osteoclastogenesis and resorption without altering the number of osteoclast precursors, through increasing the receptors’ activation and responsiveness of cells to RANKL and M-CSF, as well as producing osteoclasts that are more active in bone resorption, and capable of fusion and formation of larger osteoclasts in-vitro
(Jacome-Galazra et al., 2011). It is well known that osteoclasts and their precursors lack sensitive high affinity receptors (Fuller et al., 1998; Teittelbaum, 2000; Zaidi, 2007; Lupp et al., 2010). Furthermore, an intermittent mode of PTH administration lead to a transient elevation in OPG and decline in RANKL mRNA that only lasted for 3 hours after injection, before returning to normal levels (Ma et al., 2001). Therefore, due to the transient nature of
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 113
PTH on osteoclast precursors at the SFx site, the effect of a single PTH may have been as potent as daily injections for 14 days on osteoclastic parameters.
Finally, there is a delay in the rise of bone resorption markers which precede bone formation markers, thus providing an “anabolic window” that maximizes the short-term efficacy of PTH
(Rubin and Bilezikian, 2003). During the anabolic window, the net effect is always a positive balance between formation and resorption within the bone remodeling unit (BMU). Intermittent dosing has an effect on both remodeling- and modeling-based bone formation at cortical and cancellous surfaces (Amugongo et al., 2014; Grosso et al., 2015). Therefore, it was hypothesized that a PTH treatment period longer than 4 weeks might be required to activate a more accelerated pattern of bone resorption (Altman-Singles et al., 2017), allowing for larger
Basic Multicellular Units (BMUs) and wider SFx healing surfaces that allows for a better distribution of a larger number of osteoclasts that are already activated and recruited to the area as shown in the present study.
In the current experiment, the systemic effect of daily PTH injections was confirmed through investigating its effect on trabecular bone at a site that is distant from the SFx area (distal femur). The systemic effect of daily PTH injections was evident after 2 weeks through a significantly higher BV/TV in the distal femur. However, after 6 weeks, daily PTH injections for 14 days did not elicit the same systemic effect, consistent with activated modeling
(formation). This is consistent with the results obtained from the current experiment within the local bone areas around the SFx. It is also in alignment with a recent study that showed a higher bone volume with higher daily PTH doses as a result of a higher trabecular number in the
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 114 healing callus and a denser trabecular bone network, indicating increased bone formation
(Milstrey et al., 2017). This opens the possibilities for investigating the effects of weekly rather than daily PTH injections, which has been investigated previously but only in the treatment of
AFF (Tsuchie et al., 2018). It also highlights the different effects on bone surfaces, including the woven bone callus, and the remodeling required to heal the SFx line.
It is well known that iPTH increases osteoclastic parameters and bone resorption (Crockett et al., 2011; Ishtiaq et al., 2015). The initial study conducted with the single PTH injection confirmed the findings from the previous studies. In the current study, the increased number of osteoclasts demonstrated at the SFx site with iPTH injections for two weeks was similar, but not superior, to the effect shown with a single PTH injection. iPTH contributes to the recruitment and differentiation of osteoclasts at the SFX site. In addition, there was increased osteoclastic activity in the woven bone callus surrounding the SFx region. It could be argued, that at the SFx site, there is limited tissue space for osteoclasts to enter the crack line from the periosteal BMU, to initiate resorption of the SFx line. That is, regardless of increased osteoclastogenesis, stimulated by daily iPTH at the periosteal exit point of the crack, there is a limited resorption surface available for their osteoclast activity along the SFx crack. For this reason, a significant difference in SFx healing over the six weeks may not be manifested.
The effect of daily PTH injection for 14 days was significant in improving of woven bone area and apposition rate when compared to VEH. In a previous study PTH treatment resulted in an increase in trabecular thickness, microstructure and density, together with a decrease in trabecular separation (Bradbeer et al., 1992). In another study PTH improved not only woven
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 115 bone structure in the form higher density, volume and the tendency to form more plate-like structure, but also cortical bone structure, thickness as well as BMD (Jiang et al., 2003). A more recent study confirmed the same findings and showed an improved cortical bone microstructure, due to improvement of collagen fibers orientation and distribution, which increases the chances of Haversian system formation from woven bone that acts as a starting point for stabilization of SFx as well as prevention of the risk of future fractures in patients suffering from osteoporosis (Ascenzi et al., 2012).
Other factors including Ca++ concentration play an important role in bone remodeling through acting as a coupling mediator between osteoblasts and osteoclasts (Al-Dujaili et al., 2016). It has been demonstrated that CaSR have a larger effect on cortical bone than woven bone (Xue et al., 2012). Furthermore, healing was accelerated in acute as well as SFx and AFF with daily
PTH injections supplemented with vitamin / calcium citrate supplement (Gomberg et al., 2011;
Raghavan and Chrisofides, 2012; Blood et al., 2015; Koh et al., 2017). This suggests a role for
CaSR in the anabolic effects of PTH and is in agreement with the results from the current study where daily PTH injections improved woven bone apposition rate when compared to VEH.
Finally, PTH increases remodeling intensity, the net deposition of bone increases within each of the greater numbers of remodeling units as PTH promotes the differentiation, work and lifespan of osteoblasts in existing and newly created bone remodeling units (Ma et al., 2006), as well as increasing bone formation on quiescent bone surfaces (Linsday et al., 2006). The downstream of PTH effect on remodeling removes old fully calcified bone matrix and replaces it with new, less mineralized bone matrix and improves the overall fracture healing (Jobke et
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 116 al., 2011; Chiang et al., 2013). It should be noted that in all of the above studies, patients received a prolonged PTH treatment.
Despite the apparent positive effects of PTH treatment on bone remodeling, there has been some concerns regarding the quality of the new bone that is formed in response to PTH administration (Ejersted et al., 1994; Kneissel et al., 2001; Misof et al., 2003; Paschalis et al.,
2003; Fuchs et al., 2008). However, high resolution synchrotron-based Fourier Transform
Infrared Microspectroscopy (sFTIRM) and calcein labelling demonstrate that the new bone deposited during PTH treatment shows normal collagen maturation, crosslinking, fiber longitudinal stretch and mineral deposition, which remains unaltered with PTH treatment
(Vrahnas et al., 2016). Furthermore, intermittent PTH treatment together with loading had positive additive effects on cortical periosteal bone formation, which was time and age dependent (Sugiyama et al., 2008; Meakin et al., 2017).
The consensus amongst all studies in the literature is that PTH is a well stablished anabolic agent that has been used safely for more than a decade for both trabecular and cortical bone
(Capriani et al., 2012). It has been shown that trabecular thickness, microstructure and density increases with a decrease in trabecular separation (Bradbeer et al., 1992). Furthermore, the net effect of cortical bone thickness and structure is improved as shown in a number of studies
(Dempster et al., 2001; Hyldstrup et al., 2002; Uusi-Rasi et al., 2002; Jiang et al., 2003).
Although applied to a SFx repair, the results from the current study are consistent with the literature and demonstrate the validity of PTH as an anabolic agent even in smaller doses than the currently recommended clinical doses.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 117
Despite having a very similar effect to a single PTH injection, the significance of the present study of daily iPTH for 14 days is its ability to improve the outcomes of SFx healing with short duration treatment. This is highly important due to the higher cost of the current long treatment protocols as well as the concerns related to development of osteosarcoma in rats (Vahle et al.,
2002; Jolette et al., 2006; Tashjian and Goltzman, 2008; Watanabe et al., 2012) and to lesser extent in humans (Tashjian and Chabner, 2002; Harper et al., 2007; Mirabello et al., 2009;
Lubitz and Prasad, 2009; Subbiah et al., 2010). The difference between osteosarcoma incidence rates in rats and humans and humans could be explained simply through the higher doses used in rat models and the longer durations of exposure which could be virtually the rodent’s lifetime
(Capriani et al., 2012). Other explanations besides dosage and exposure times could be that rats as a species have continuous skeletal growth and modeling throughout its lifetime. The ever- growing skeleton in rats is considered as a developing organ that contains immature and potentially tumorgenic cells that could respond to PTH in an unexpected and uncontrolled manner resulting in osteosarcoma (Capriani et al., 2012).
Finally, local delivery systems for PTH targeting specific fractured or areas that are at risk could be a potential solution to avoid the systemic side effects of PTH (Kothari et al., 2011; Wang et al., 2018). Furthermore, PTH has been recommended as a solution for bone regeneration in large bony defects and fracture non-unions. This could be achieved through using a scaffold biomaterial to allow local application and delivery of PTH (Wojda and Donahue, 2018). The numerous and potential applications of PTH in bone regeneration and tissue engineering gives the potential for numerous research opportunities to fill the gap between local and systemic administration of PTH in a variety of unresolved clinical situations. Examples include high risk
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 118
SFxs that are managed operatively, including the use of additive compounds such as bone grafts, BMPs and possibly PTH.
Furthermore, the results from the current study showed that a single PTH injection had a positive effect on osteoclastic parameters indicative of remodeling activation (Bakr et al.,
2019). In this chapter we showed that daily PTH injections for 14 days were not superior to a single PTH injection, except in improving woven bone apposition rate. Other studies have recommended the use of iPTH once to three times per week as opposed to daily (Tsunori, 2015;
Kumbae et al., 2017; Ota et al., 2019). However, the ideal frequency of Teriparatide administration remains unknown because no clinical trials have examined PTH therapy in this context. Finally, the question remains open to discussion about the window where iPTH is ideal for usage and whether it is effective throughout all phases of the remodeling cycle or should be limited to some phases that are more responsive than others?
In conclusion, PTH can only increase the recruitment of and availability of bone cells, which was evident in woven bone area. However, PTH is not capable or responsible for the transportation of these cells from the larger woven bone area into the narrow SFx. This means that a limited number of cells can access the SFx line, particularly in the non-Haversian rat bone. In the increased osteoclastogenic environment, the nature of the SFx line limits the increase in remodeling at this site. Furthermore, daily PTH injections could have resulted in an early activation of remodeling which includes both a marked increase in the number of BMUs
(increased activation frequency) and a net excess of osteoclast resorbing activity resulting in a negative bone balance within each BMU. This is similar to the mechanism of bone density loss
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 119 in osteoporosis (Khosla et al., 2008) and could explain that the net result of healing in daily
PTH injections, was similar to a single PTH injection due to the increased amount of bone loss earlier in the remodeling cycle that needs to be compensated for later in the repair phase of the remodeling cycle. The current study was only limited to introducing PTH in the first two weeks in the remodeling cycle and looking at the early immediate effect of the treatment. Further treatment with PTH in the advanced healing stages (between 2 weeks and 6 weeks post SFx), could result in an additional boost in the treatment outcomes.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 120
Chapter 6
Combined ALN-PTH Experiment
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 121
6.1. Introduction:
The rat model used in the current study is successful and reliable for investigating stress SFx in vivo (Kidd et al., 2010; Kidd et al., 2011; Kidd et al., 2013; Bakr et al., 2019). Since many patients suffering from osteoporosis receive long-term BP treatment, they may be at higher risk for development of SFx, as well as more serious complications including AFF (Watts and Diab
2010; Blum et al., 2016; Kim et al., 2016a; Kim et al., 2016b; Koh et al., 2017; Adler, 2018;
Lim et al., 2018). Therefore, the aim of the current experiment was to investigate the efficacy of iPTH treatment to accelerate indices of SFx healing in the rat ulna in the presence or absence of ALN. We hypothesize that treatment with iPTH, will accelerate histomorphometric indices of SFx healing, even in the presence of concurrent BP treatment. This would be important to heal emerging SFx and prevent them from being displaced or propagate to AFF. There is plethora of studies that investigate the effect of combined therapy on osteoporosis, which is not the case for SFx and potential AFF (Miller and McCarthy, 2015). Therefore, the experiment discussed in this chapter investigates healing of early SFx using a combined treatment of ALN-
PTH and the effect of continuation or cessation of the ALN treatment.
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6.2. Experimental design:
For ease of reference, group abbreviations in this section refer to the following trreatments:
ALN1= daily ALN up to 6 weeks; ALN2= Cessation of ALN at time of SFx, no treatment up to
6 weeks; ALN-PTH1= daily ALN + 14 days of iPTH treatment; ALN-PTH2= Cessation of ALN at time of SFx, + 14 days of iPTH treatment; VEH= control for ALN-PTH groups.
This experiment included 150 female wistar rats. The experiment was conducted as outlined in chapter 3. Rats were pre-treated with daily ALN injections for 14 days. After SFx loading ALN was either ceased in half of the animals or continued in the other half. In the combined ALN-
PTH groups, daily PTH injections (8 μg/100g/day) for 14 days were administered starting 24 hours after SFx loading. VEH3 groups received equivalent VEH injections to the maximum number of injections that animals in the ALN-PTH1 group (ALN continuation) receive. The data collected from this experiment was analyzed using a 2-way ANOVA with time and treatment group as the independent variables. In the presence of a significant statistical interaction, the main effects in the original 2-way ANOVA were ignored, post-hoc pairwise comparisons were performed between individual groups and differences determined using
Fishers LSD. In the presence of a non-significant statistical interaction, the ANOVA main effect for each independent variable (time or treatment group) was reported independtely.
Significance was accepted at P≤0.05.
When comparisons were made between different treatment modes (Daily PTH vs. ALN PTH1 vs. ALN PTH2) one-way ANOVA test was used with each treatment group as an independent variable. The Bonferroni correction method was used to adjust the level of significance for the
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 123 family wise error rate. An adjusted alpha level of .008 per test (.05/6) was used for multiple comparisons. Significance was accepted at P≤0.008.
Table 6: Experimental design of the combined ALN PTH experiment. Number of rats per
group (n)= 15.
2 weeks 6 weeks
ALN1 15 15
ALN2 15 15
ALN-PTH1 15 15
ALN-PTH2 15 15
VEH3 15 15
6.3. Results:
6.3.1. Combined ALN PTH treatment:
There were no significant differences among groups in terms of cortical bone area (CT.B.Ar) and cortical perimeter (Ct.Pm), woven bone area (Wo.B.Ar, mm2), and the length of stress fracture (SFx.Le, µm) (Appendix 1c).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 124
6.3.2. Woven bone parameters:
There were no significant differences in woven bone area between different treatment groups
(Fig 16a). There was a significant interaction between the main effects of time and the type of treatment (ALN1 vs. ALN2 vs. ALN PTH1 vs. ALN PTH2 vs VEH) on woven bone width (F=
3.169; P= 0.016). Continuation or cessation of ALN had no effect on woven bone width 2 weeks post SFx induction. Woven bone width was significantly higher in the combined ALN
PTH1 treatment when compared to ALN1 (P= 0.006) after 2 weeks. It was also significantly higher in the combined ALN PTH2 treatment when compared to ALN2 (P<0.001) at the same timeframe. After 6 weeks, Woven bone width was significantly greater in continuous ALN-
PTH1 treatment when compared to ALN cessation in ALN-PTH2 treatment (P= 0.05) and
VEH group (P=0.016) (Fig 16b).
There was no significant interaction between the effects of time and treatment type on the woven bone perimeter (Table 7). With regards to the main effect of time on woven bone perimeter, it was significantly higher at 6 weeks post SFx inducation when compared to 2 weeks
(P= 0.008) (Fig 16c).
There was a significant interaction between the main effects of time and the type of treatment
(ALN1 vs. ALN2 vs. ALN PTH1 vs. ALN PTH2 vs VEH) on woven bone apposition rate (F=
4.127; P= 0.003). Woven bone apposition rate was significantly greater in combined treatment groups (ALN PTH1 and ALN PTH2) when compared to ALN treatment groups (ALN1 and
ALN2) after 2 weeks of SFx induction (P= 0.008 and P<0.001 respectively). Furthermore, woven bone apposition rate was significantly higher in ALN PTH1 and ALN PTH2 when
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 125 compared to VEH after 2 weeks of SFx induction (P= 0.002 and P<0.001 respectively). Finally, woven bone apposition rate decreased significantly in all groups after 6 weeks when compared to the second week (P<0.001) (Fig 16d).
6.3.3. Osteoclast parameters:
There was no significant interaction between the effects of time and treatment type on the the number of osteoclasts or osteoclast perimter (Table 7).
The number of osteoclasts was significantly higher in ALN PTH2 group when compared to
ALNPTH1 (P= 0.006), and VEH group (P= 0.024) (Fig 16e) (Fig 17). The number of osteoclasts and osteoclast perimeter were significantly less after 6 weeks when compared to 2 weeks post
SFx induction (P<0.001) (Figs 16e, 16f) (Fig 17).
There was a significant interaction between the main effects of time and the type of treatment
(ALN1 vs. ALN2 vs. ALN PTH1 vs. ALN PTH2 vs VEH) on the number of osteoclasts per unit porosity (BMU) area (F= 4.032; P= 0.005). The number of osteoclasts per unit porosity (BMU) area was significantly greater after two weeks following cessation of ALN (ALN2) when compared to continuous ALN1 treatment (P<0.001). Furthermore, the number of osteoclasts per unit porosity (BMU) area was significantly greater after two weeks of ALN cessation in ALN-
PTH2 group when compared to when ALN treatment was continued in ALN-PTH1 group (P=
0.03) (Fig 16g) (Fig 17). With regards to the single main effect of time, the number of osteoclasts per µm2 of BMU area was significantly less after 6 weeks when compared to the
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 126 second week in ALN cessation groups (ALN2 and ALN PTH2) when compared to ALN continuation groups (ALN1 and ALN PTH1) (P<0.001) (Fig 16g) (Fig 17).
There was a significant interaction between the main effects of time and the type of treatment
(ALN1 vs. ALN2 vs. ALN PTH1 vs. ALN PTH2 vs VEH) on the number of osteoclasts per unit
BMU length (F= 2.816; P= 0.028). The number of osteoclasts per unit BMU length was significantly greater two weeks after ceasing ALN (ALN-PTH2 group) when compared to ALN continuation in ALN-PTH1 group (P<0.001) (Fig 16g) (Fig 17). Furthermore, the number of osteoclasts per µm of BMU length was significantly less after 6 weeks post SFx induction when compared to the second week in ALN1 group (P= 0.005), ALN2 group (P= 0.001), ALN PTH2 group (P<0.001).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 127
Figure 16: Histomorphometric variables of SFx woven bone callus, osteoclast and healing parameters (±SD). Combined ALN-PTH treatment was superior to ALN and VEH treatments in terms of woven bone sculpture and modeling throughout the healing process of SFx.
Cessation of ALN results in a significantly less woven bone width indicating a more rapid progression in healing. Cessation of ALN after 2 weeks resulted in a significantly greater osteoclast number. ALN and combined ALN-PTH treatment modes (cessation and continuation) resulted in less healing area and perimeter after 6 weeks when compared to VEH.
Most variables were significantly influenced by the main effect of time between the 2 week and
6 week time points. Furthermore, there was a significant interaction between the main effects of time and the treatment type on healing perimeter. ** = P≤0.05, *** = P≤0.01 (differences between treatment types). +++ = P≤0.01 (differences compared to the previous time point).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 128
Figure 17: (a): Showing normal osteoclastic activity in VEH groups, (c): suppressed osteoclastic activity in ALN groups, (e): higher osteoclastic activity in daily PTH group, and (i): higher osteoclastic activity in the ALN-PTH2 group during the resorptive phase of the remodeling cycle after 2 weeks. (g):
Continuation of ALN treatment in ALN-PTH1 resulted in suppression of osteoclastic activity along the
SFx despite the recruitment and availability of osteoclasts around the woven bone callus. (b, d, f, h &
J): After 6 weeks, osteoclastic activity was significantly less in all groups apart from some osteoclastic trials indicating past resorptive activity. Daily PTH group showed evidence of osteoclastic activity only around but not along the SFx due to progression in healing. (TRAP 10X). Scale bar = 10µm
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 129
6.3.4. Healing parameters:
There was no significant interaction between the effects of time and treatment type on any of the healing parameters (Table 7). With regards to the main effect of time, all healing parameters were significantly greater 6 weeks post SFx induction when compared to the second week
(P<0.001) (Fig 18). This includes healing area (Fig 16h), healing perimeter (Fig 16i), healing percentage (Fig 19a), healing area per mm2 of the cortical bone area (Fig 19b), and the percentage fracture length occupied by bone formation (Fig 19c).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 130
Figure 18: (a) Showing the initiation of remodeling in VEH groups. (e) Daily PTH treatment accelerated remodeling at 2 weeks through the development of larger areas of porosity (BMUs) in comparison to all other groups. (g & i) The combined ALN-PTH treatments triggered the development of multiple porosity areas (BMUs) that were significantly larger when ALN treatment was ceased. (c): ALN treatment resulted in the development of smaller BMUs (d): After 6 weeks, healing was completely suppressed in ALN groups but continued normally in VEH (b) and Daily PTH groups (f). (h & j): In the combined ALN-PTH treatment, areas of erosion persisted after 6 weeks along the SFx despite the progression of healing in other areas, especially when ALN treatment was continued. Overall the percentage healing was high in ALN-PTH2 due to larger porosity areas developed under the effect of
ALN and larger healing areas after cessation of ALN. (Toluidine Blue 10X). Scale bar = 10µm.
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6.3.5. Erosion (unhealed) parameters:
There was no significant interaction between the effects of time and treatment type on any of the erosion parameters (Table 7).
With regards to the main effect of time, all erosion parameters were significantly less after 6 weeks of SFx induction when compared to the second week (P<0.001) (Figs 19d, 19e, 19f,
19g). The percentage fracture length occupied by erosion was significantly less in the ALN
PTH1 when ALN was continued in comparison to ALN PTH2 when ALN was ceased (P= 0.006) and VEH (P= 0.01) (Fig 19f).
6.3.6. Porosity (BMU) parameters:
There was no significant interaction between the effects of time and treatment type on any of the porosity parameters (Table 7).
There were no significant differences in porosity parameters between different treatment groups. With regards to the main effect of time, porosity area, perimeter and the porosity
(BMU) area per mm2 of the cortical bone area increased significantly after 6 weeks in all treatment groups when compared to the second week (P<0.001) (Figs 19h & 19i).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 132
Figure 19: Histomorphometric variables of SFx healing (±SD). Continuation of ALN in the combined ALN-PTH1 treatment mode resulted in a significantly less percentage fracture length occupied by erosion after two weeks when compared to VEH and cessation of ALN in the combined ALN-PTH2 treatment mode. Healing, porosity variables were significantly greater, while erosion variables were significantly less as a result of the main effect of time between the
2 week and 6 week time points. *** = P≤0.01 (differences between treatment types). +++ =
P≤0.01 (differences compared to the previous time point).
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Table 7: A summary of the significant findings from the ALN, ALN-PTH and VEH3 groups
related to the effect of time, treatment as well as the interaction between treatment and time on
different variables using a linear model in a 2-way ANOVA statistical analysis.
Variable Effect F Significance Treatment 6.403 <0.001 Woven Bone Width (Wo.B.Wi, mm) Time 49.783 <0.001 Interaction (Treatment * Time) 3.169 .016 Treatment 2.141 .079 Woven bone perimeter (Wo.B.Pm, mm) Time 7.279 .008 Interaction (Treatment * Time) 1.004 .408 Treatment .330 .858 Porosity (BMU) Area (SFx.Po.Ar, µm2) Time 31.318 <0.001 Interaction (Treatment * Time) .524 .718 Treatment .556 .695 Porosity (BMU) Area Perimeter Time 28.886 <0.001 (SFx.Po.Pm, µm) Interaction (Treatment * Time) 1.010 .405 Treatment .279 .891 Healing Area (SFx.He.Ar, µm2) Time 149.784 <0.001 Interaction (Treatment * Time) 1.277 .282 Treatment .418 .795 Healing Area Perimeter Time 164.804 <0.001 (SFx.He.Pm, µm) Interaction (Treatment * Time) 1.662 .163 Treatment .536 .710 Erosion (unhealed) Area Time 126.722 <0.001 (SFx.E.Ar, µm2) Interaction (Treatment * Time) 1.841 .125 Treatment 2.237 .069 Time 54.302 <0.001 Erosion (unhealed) Perimeter Interaction (Treatment * Time) 1.450 .221 (SFx.E.Pm, µm)
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 134
Treatment 2.431 .05 Number of osteoclasts (N.Oc) Time 41.695 <0.001 Interaction (Treatment * Time) 1.847 .124 Treatment 2.089 .086 Osteoclasts surface perimeter Time 42.468 ,0.001 (Oc.Pm, µm) Interaction (Treatment * Time) 1.730 .147 Treatment .238 .916 Healing percentage (He%) Time 46.449 <0.001 Interaction (Treatment * Time) .838 .505 Treatment 8.724 <0.001 Woven bone apposition rate per day Time 425.900 <0.001 (mm2/day) Interaction (Treatment * Time) 4.217 .003 Treatment 6.095 <0.001 Number of osteoclasts per µm2 of Basic Multicellular Unit (BMU) area Time 46.342 <0.001 (N.Oc/µm2) Interaction (Treatment * Time) 4.032 .005 Treatment 3.559 .009 Number of osteoclasts per µm of Basic Multicellular Unit (BMU) length Time 38.909 <0.001 (N.Oc/µm) Interaction (Treatment * Time) 2.816 .028 Treatment .289 .885 Healing area per mm2 of the cortical bone area (SFx.He.Ar, µm2/ Ct.Ar, Time 146.062 <0.001 mm2) Interaction (Treatment * Time) 1.300 .274
Porosity (BMU) area per mm2 of the Treatment .284 .888 cortical bone area Time 153.287 <0.001 2 2 (SFx.Po.Ar, µm /Ct.Ar, mm ) Interaction (Treatment * Time) .721 .579 Treatment .503 .733 Erosion (unhealed) area per mm2 of the Time 130.923 <0.001 cortical bone area Interaction (Treatment * Time) 1.946 .107 (SFx.E.Ar, µm2/ Ct.Ar, mm2)
Treatment .284 .888 Percentage fracture length occupied by
bone formation Time 153.287 <0.001 (SFx.He.Pm, µm/SFx.Le, µm %) Interaction (Treatment * Time) .721 .579
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 135
Treatment 2.576 .041 Percentage fracture length occupied by Time 45.254 <0.001 erosion (SFx.E.Pm, µm /SFx.Le, µm %) Interaction (Treatment * Time) 1.898 .115
6.3.7. Comparison between daily PTH and combined ALN-PTH
treatments:
A comparison between the daily PTH and combined ALN PTH treatment modes was made to
highlight if there were any significant differences. One-way ANOVA test was used in this
comparison with each treatment group as an independent variable. Post hoc test results were
used to compare only between the daily PTH mode vs. ALN PTH1 (continued ALN) vs. ALN
PTH2 (ceased ALN) at the 2-week timepoint and the 6-week timepoint. Comparisons were not
made neither between different treatment modes at different timelines nor between groups from
the same treatment group at different timelines as it has been already discussed in this chapter.
There were no significant differences between daily PTH and combined ALN PTH treatment
at the adjusted level of accepted significance P≤0.008.
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6.4. Discussion –
Combined ALN-PTH experiment:
This is the first investigation of the combined effect of intermittent PTH treatment with a concurrent or ceased ALN treatment on the overall remodeling of SFx. In this experiment, the histomorphometric variables for remodeling of the SFx were improved when ALN treatment ceased at the time of introducing PTH therapy. There have been inconsistent and controversial results in literature with regards to the combined anti-resorptive and PTH therapies in treatment of postmenopausal women with osteoporosis (Black et al., 2003; Finkelstein et al., 2003;
Cosman et al., 2005; Cosman et al., 2009; Finkelstein et al., 2010; Cosman et al., 2011; Cosman et al., 2013; Muschitz et al., 2013; Schafer et al., 2013; Tsai et al., 2013, Cosman et al., 2017).
For example, greater bone turnover was achieved in women with osteoporosis receiving anti- resorptive therapy (ALN) for 18 months after cessation of that therapy and switching to PTH for an additional period of 18 months (Cosman et al., 2009). These data supported the observations that BP treatment blunted the anabolic effect of PTH. In this Chapter, the cessation of ALN at the time of SFx initiation lead to a greater percentage healing at six weeks, consistent with those clinical observations. It has been suggested that the additive effect of the combined
ALN-PTH treatment is due to increased bone formation with a combined osteoclast inhibitory action, implying that the anabolic effect of PTH might be independent of resorption (Altman et al., 2014).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 137
In a recent study, PTH treatment increased BP turnover in fracture repair in rats (Murphy et al.,
2015). This leads to a lesser amount of BP being accumulated in bone, remobilization of BP and a decrease in BP load in fracture, leading to an overall improvement in healing (Murphy et al., 2015). A more recent review concluded that the optimum sequential combination of treatment is yet to be developed (Anastasilakis et al., 2018). However, a combination of
Teriparatide with Denosumab or zoledronic acid for postmenoapuasal women with osteoporosis was recommended. This is mainly due to the synergistic effect observed, especially with Denosumab, where a transitory increase of serum PTH and a decrease in intracortical porosity was observed (Seeman et al., 2011).
Perhaps greater healing of the SFx could be obtained in the current study if Denosumab was used instead of ALN. This is because the combination of Teriparatide and Denosumab permits continued Teriparatide-induced modeling-based bone formation to that in PTH monotherapy
(Ma et al., 2006; Tsai et al., 2013). It has been recommended that the class of antiresorptive medication should be reassessed for patients who are on BPs and develop, or are at risk of developing, SFx or AFF (Collinge and Favela, 2016). Further investigations are needed before translating these findings into clincal application and recommending that patients on ALN who suffer from a SFx should be temporarily switched to PTH monotherapy or combined denosumab-PTH therapy.
A temporary decrease in bone mineral density (BMD) was reported in a number of studies that investigated the use of combination anabolic/antiresorptive treatment protocols (Whitmarsh et al., 2015; Eriksen and Brown, 2016). This is a likely consequence of the transient cortical
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 138 porosity produced by PTH, and the shift to a reduction in the mean degree of mineralization of the bone matrix (Roschger et al., 2014). Nonetheless, the increased bone mass produced by iPTH at the periosteal surface ensures a maintenance of bone strength (Burr et al., 2001). It should be noted that the greater proportion of new bone formation is modeling-based rather than remodeling with combination ALN-PTH treatment mode. There is no direct and reliable data that favors one mechanism of bone formation over the other in SFx that heals by direct remodeling.
In cases of combined treatment, decreasing the frequency of anti-resorptive treatment results in an increased BMD (Cosman et al., 2011). It is possible that pretreatment with anabolic agents like PTH, followed by an antiresorptive agent, then continuing the combination therapy could improve treatment outcomes to increase bone mass (Cosman et al., 2017). However, while this could be applied to chronic conditions such as osteoporosis, it is not relevant to SFx, where the timing of injury cannot be predicted and remodeling is required to heal the SFx. In fact, porosity created by a “pre-”iPTH treatment could exacerbate SFx progression (Burr et al., 1997)
Following initiation of SFx, the results from the current study indicate that upon cessation of
ALN treatment, daily PTH injections for 2 weeks contributed to remodeling activation by increasing the recruitment and availability of osteoclasts needed for the initial (resorptive) phase of the bone remodeling cycle. Besides the significant increases in osteoclast number and perimeter, the number of osteoclasts per unit porosity (BMU) area was significantly greater after two weeks of cessation of ALN in ALN-PTH2 group (P= 0.03), compared to continuation of ALN (ALN-PTH1). This could be explained by the fact that BPs have the affinity to bone
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 139 tissues. The acidic PH environment created in Howship’s lacunae around the osteoclasts facilitates the uptake of BPs, which in addition to the inherent affinity of BPs to minerals eventually leads to apoptosis of osteoclasts (Russell et al., 2008). We observed that intermittent
PTH treatment was more effective in SFx repair when ALN treatment ceased.
This is consistent with studies that investigated the early healing of atypical femoral fractures
(AFF) that share similar properties to the SFx model in the current study (AFFs are discussed in more detail below). For example, in clinical studies healing of non-displaced AFF improved when BPs were ceased (Schilcher et al., 2011; Watts et al., 2017). There is debate as to whether the improved healing of AFF was due to the teriparatide treatment, the discontinuation of BPs or both factors combined (Watts et al., 2017). Furthermore, a reduction in the incidence of AFF by 70% per year was reported, when BPs were ceased. This was attributed to the osteoclastic bone resorption and repair of micro-cracks that follows the discontinuation of BP treatment
(Schilcher et al., 2015).
In a recent case report, discontinuation of BPs and treatment with Teriparatide resulted with a near complete radiographically verified healing of AFF, but unfortunately 12 months after the cessation of BPs, bilateral recurrence of AFF occurred, despite sequential use of Teriparatide and Denosumab (Ramchand et al., 2016). This illustrates the difficulty of managing cases of
SFx in association with antiresorptive therapy. That is, cessation of antiresorptive agents is favored for healing of SFx but compromises the structural integrity of bone in osteoporotic patients; whereas, continuation of antiresportive treatment may delay healing and lead to atypical fractures. In the absence of randomized controlled trials on this question, a task-force
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 140 of the American Society for Bone and Mineral Research (ASBMR) recommend discontinuation of BPs, adequate calcium and vitamin D, and consideration of Teriparatide for those who appear not to heal on conservative therapy (Shane et al., 2014).
Though observed in rats, the fact that a short period (2-6 weeks) of ALN discontinuation allowed the anabolic effect of PTH to be expressed is highly important. BPs are known to have a long residence time in bone, and to also be recycled, and return to other resorptive surfaces even after the treatment has ceased (Russell et al., 2008; Diab et al., 2013). Limited data is available from BP discontinuation clinical trials but are suggestive of a residual and continuous biological effect of BPs, especially ALN and zoledronic acid for a lengthy time period after discontinuation (Black et al., 2006; Black et al., 2012; Boonen et al., 2012). It was hypothesized that BP treatment causes tissue brittleness that initiates cracks, increases homogeneity of osteonal and interstitial structures, impairs targeted repair by BMUs and allows easier accumulation of microdamage at areas of maximum forces and mechanical loading (Donnelly et al., 2012; Ettinger et al., 2013; Demirtas and Ural, 2018). In the present study, cessation of
ALN for a period of 2-6 weeks allowed a significant increase in the overall healing percentage.
In this rat model, this is likely related to increased remodeling activation and the coupled bone formation that follows, rather than the matrix changes observed in osteonal bone.
The current clinical treatment protocols for osteoporosis are based on long-term drug administration with chosen periods of cessation called “drug holidays”. The evidence for the value of the drug holiday periods remain weak and a careful evaluation for the risks vs. benefits of cessation of BPs should be investigated thoroughly before making a decision (Ye et al.,
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 141
2010). In principle, this permits bone turnover to increase and allows normal skeletal repair mechanisms. The optimal length of a ‘drug holiday’ has not been established but existing data suggest 3-5 years with ALN, 3-6 years with zoledronate and 1-2 year with risedronate. A decision to recommence therapy should then probably be based on regular reassessment of bone mineral density and fracture risk (Anagnostis and Stevenson, 2015; Anagnostis et al., 2017).
New alternative drugs with a shorter duration of action like Denosumab (Reyes et al., 2016) or
Romosozumab (EVENITY™) that was developed as a humanized monoclonal antibody against
Sclerostin (Markham, 2019) could provide a more reversible option as an anti-resorptive agent and could potentially be beneficial for patients with SFxs to prevent the risk of fractures, or at least allow for a quicker healing in the case of development of an atypical femoral fracture
(AFF) due to earlier activation of remodeling.
The results obtained in the present study from daily PTH groups and ALN-PTH groups showed that daily PTH treatment alone or in combination with a prior ALN shifted the bone remodeling dynamics within the area of a SFx towards healing, and reduced resorption significantly. These results are consistent with a recent study that investigated the effect of i-PTH after a prolonged
ALN treatment on new bone formation and bone tissue heterogeneity (Altman-Singles et al.,
2017). Not only that the balance in bone remodeling was shifted in favor of formation, but also mechanical properties were improved and the heterogeneity of the newly formed bone was improved after switching to PTH following a prolonged ALN treatment (Altman-Singles et al.,
2017).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 142
It should be noted that there are some differences in methodology between the above-mentioned investigation and the present study. For example, their rats were ovariectomized and pretreated with ALN twice a week for 12 weeks compared to 14-days daily ALN pretreatment in the current study. In addition, ALN was followed by PTH treatment twice a week for 4 weeks compared to daily PTH injections for 14 days in the current study. From both studies, it can be recommended that PTH could be used as a possible treatment option for patients with prior BP treatment in cases of osteoporosis and/or SFx. Previous reports in the dental field showed accelerated bone regeneration and absence of ulceration in BP related osteonecrosis of the jaw
(ONJ) after 6-10 months of anabolic therapy with PTH (Ma et al., 2003; Lecart et al., 2004;
Marx et al., 2005; Harper and Fung, 2007; Lau and Adachi, 2009). In the present study, cessation of ALN and anabolic therapy with PTH for a shorter duration (6 weeks) has proven to shift remodeling positively towards healing.
The higher values for woven bone parameters observed in this chapter is consistent with other studies (Altman et al., 2014; Altman et al., 2017), and dispels the questions concerning efficacy of PTH in formation of new bone around sites that have been previously suppressed by BP treatment (Black et al., 2003; Finkelstein et al., 2003; Finkelstein et al., 2010). For example,
PTH therapy can enable new bone formation and replace the old bone matrix on a previously resorption-suppressed site caused by BP treatment (Kim et al., 2012). Furthermore, PTH when compared to other anabolic agents like strontium ranelate, has a long-term stimulating effect on bone formation and resorption markers in postmenopausal women with osteoporosis, previously treated with BPs (de Sousa et al., 2010).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 143
A combination of ALN + PTH treatment resulted in an increased woven bone width, perimeter and apposition rate regardless of whether ALN was continued or ceased after induction of SFx and PTH treatment. That is, ALN treatment had little inhibitory effect on woven bone formation, as previously observed (Kidd et al., 2011). PTH is well known for its dual effect on osteoblasts and osteoclasts. iPTH stimulates osteoblast proliferation and longevity (Jilka,
2007), it also indirectly affects osteoblastic maturation and functions through cells of the osteoblastic lineage (Teitelbaum, 2000). As a result of the above-mentioned consequences to
PTH treatment, there is an increase in RANK-L and monocyte colony stimulating factor (M-
CSF) which stimulate osteoclastic activity and proliferation (Teitelbaum, 2000). This includes activity during woven bone formation. The increase in woven bone callus is important for maintaining the strength and mechanical properties of the ulna, despite the intracortical resorption that follows microdamage and SFx induction (Sloan et al., 2010).
As the duration of antiresorptive treatments increased to 10 years or more, a debate started concerning the risks versus benefits of antiresorptive treatment, and the consequences of
“frozen bone” (Adler, 2018). One of the side-effects that emerged was a SFx in the proximal femoral diaphysis, which progressed to an atypical femoral fracture (AFF) (Watts and Diab
2010; Gedmintas et al., 2013; Saita et al., 2015; Blum et al., 2016; Kim et al., 2016a; Kim et al., 2016b; Alwahhabi and Alsuwaine, 2017; Koh et al., 2017; Adler, 2018; Lim et al., 2018;
Black et al., 2019). Recently, a case of fracture in the proximal ulna was also reported after prolonged BP treatment (Oh et al., 2018).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 144
It is now well accepted that the pathogenesis of AFF is its initiation as a SFx, due to accumulation of un-repaired microdamage following suppression of bone turnover by BPs
(Shane et al., 2014; Iwata et al., 2019). It is also acknowledged that initiation of the microdamage is facilitated by bone hypermineralization and accumulation of advanced glycation end-products that reduce the toughness of the bone matrix (Vashishth et al., 2001;
Seeman and Delmas, 2006; Seeman, 2009; Lloyd et al, 2017; Larsen and Schmal, 2018). This hypothesis is supported by the location of the AFF in areas of stress (lateral femur), and the fact that such fractures develop over a long period of time and are preceded by episodes of prodromal pain (Shane et al., 2014). These AFF-related SFx share remarkably similar features to the SFx produced in the rat ulna (Schilcher et al, 2014). Due to the similarities between SFx and AFF, we hypothesized that anabolic PTH treatment would accelerate SFx in the presence of BP treatment. That hypothesis is rejected by the data presented in this Chapter.
For every 1000 women at risk for fracture, 100 fractures would be prevented with BP treatment for three years. Even with a relative risk of AFF as high as 12 (compared to patients not on
BPs), only 1.25 AFF would be experienced in 1000 women treated for 3 years (Black and
Rosen, 2016). In the first instance this appears to be a low risk. However, osteoporosis management by BPs usually extends longer than three years, and there relatively few long-term clinical trials of greater than 10 years. This resulted in the recommendation that postmenopausal women treated with intravenous (IV) BPs for more than three years, or those treated with oral
BPs for more than five years should be assessed for continued high fracture risk. If they show a good response to BPs and are no longer at a high risk for fractures, they should be considered for a drug holiday and reassessed in another 2-3 years (Adler et al., 2016). Another
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 145 recommendation was to space the initial course of intravenous BPs over 5 years (Grey et al.,
2012).
The above-mentioned risks of AFF resulted in great controversy regarding the long-term use of
BPs for prevention of osteoporotic fractures in postmenopausal women. One view recommends that the current use of BPs as the first line of treatment should be continued (Borrelli et al.,
2014); the second view recommends discontinuation of BPs once AFF has been diagnosed
(Larsen and Schmal, 2018); and a third point of view encourages the rise of a new paradigm shift in the treatment of osteoporosis. Examples include a combination of Teriparatide and
Denosumab, starting treatment with Teriparatide followed by Denosumab (the DATA
SWITCH study) (Tsai et al., 2017a, b). Furthermore, Teriparatide resulted in fewer fractures in glucocorticoid induced osteoporosis when compared to ALN treatment (Saag et al., 2009).
Therefore, it could be possible to lower AFF risk by the proposed 7-year plan that includes pretreatment with Teriparatide followed by 5 years of BPs treatment and another sequential anabolic therapy after a drug holiday (Adler, 2018).
In addition to the above, Teriparatide was used in conjunction with surgical intervention for treatment of AFF (Tsuchie et al., 2015; Miura et al., 2019). A recent meta-analysis that included
251 patients with osteoporotic fractures suggested an improved fracture healing time and superior functional outcomes. This was especially shown in lower limbs due to the difference in biomechanics and weight-bearing that could supplement healing (Lou et al., 2016). Another meta-analysis of 380 fracture patients failed to show significant improvements in typical fracture healing in the absence of osteoporosis (Shi et al., 2016). More specific systematic
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 146 reviews related to AFF revealed positive results with Teriparatide, but the quality of evidence was not very high due to absence of randomized controlled trials (RCTs) (Im and Lee, 2015;
Black et al., 2019). Despite the above-mentioned success in treatment of AFF with Teriparatide, there has been some cases when a new contralateral lateral or bilateral AFF has developed despite the use of sequential Teriparatide and antiresorptive agents, such as strontium ranelate or denosumab (Ramchand et al., 2016; Nguyen et al., 2017). The significance of immediate treatment of AFF with Teriparatide has been highlighted in literature (Greenspan et al., 2018), which is consistent with the timing of PTH treatment chosen for the current study (24 hours after SFx initialtion). The above-mentioned results highlight the importance of the current findings related to SFx healing and notes the variability of treatment outcomes which could be related to the different animal model or unique clinical scenario of each individual case, as well as the different treatment protocols adopted in each study.
PTH has shown promising results in the treatment of BP related AFFs (Carvalho et al., 2011;
Huang et al., 2012; Saleh et al., 2012; Chiang et al., 2013; Lampropoulou-Adamidou et al.,
2013; Miller and McCarthy, 2015; Miyakoshi et al., 2015; Mastaglia et al., 2016; Yeh et al.,
2017; Watts et al., 2017; Tsuchie et al., 2018). In one of the case reports, despite successful healing of AFF using teriparatide, the authors questioned that it had a major role in the healing process. It was hypothesized that surgical fixation, vitamin D therapy, calcium and ALN discontinuation could have played a secondary role in the healing process (Gomberg et al.,
2011; Huang et al., 2012; Kim et al., 2016b; Larsen and Schmal, 2018). This aligns well with the results from the current study where more superior outcomes where observed when ALN was ceased. It also opens the horizon for further experiments using vitamin D and/or calcium supplements combined with PTH in healing SFXs.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 147
One recent study investigated the PTH mechanism of action on AFF healing and reported an improvement in stem cells functionality (proliferative and differentitive capacities), that augments the inherent anabolic effect of PTH on osteoblast precursors (Casado-Diaz et al.,
2019). It should be noted that in majority of the previous studies, PTH showed better results when BPs were prescribed at lower doses or stopped/excluded from the treatment regimen. This is consistent with the results obtained in the current study and highlights the similarities between the SFx animal model used in the present study and AFF model. It also opens the way for future clinical applications of the results obtained from the current in the field of AFF.
Due to the opposing effects of BPs and PTH on remodeling dynamics, a sequential therapy protocol could produce a greater synergistic clinical outcome than a monotherapy treatment protocol (Amugongo et al., 2014; Cosman et al., 2017). This concept was first proposed by
Frost (1979) in which he hypothesized that greater increases in bone mass could occur if coherent cells (osteoblasts and osteoclasts) in remodeling were manipulated in a sequence of activation, depression (of resorption, allowing formation to proceed), then a treatment free period allowing remodeling to progress. This was termed ADFR for activate-depress-free- repeat (Frost, 1979), with the repeat being used to achieve desired bone mass outcomes. This concept has been tested over the last two decades (Hodsman and Steer, 1993; Erben, 1996;
Dempster et al., 2001; Kobayashi et al., 2003; Linsday et al., 2006; Jee et al., 2007; Cusano and
Bilezikian, 2011). However, in a SFx model this might not be applicable as the timing of a SFx cannot be predicted. Furthermore, a further inhibition of the remodeling with another antiresorptive treatment might improve the mechanical properties of the newly formed bone.
Sequential treatment would also delay the overall healing and replacement of woven bone with cortical bone.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 148
It is proposed that antiresorptive therapy (ALN) act as a preservative agent for the newly formed lamellar bone during the post PTH treatment period, as they are retained in mineralized bone tissue with a multi-year half-life (Amugongo et al., 2014). This means that release of retained
BP principally from trabecular bone tissue by osteoclastic resorption results in a gradual (Black et al., 2006), rather than an abrupt (Bone et al., 2011) loss of anti-resorptive efficacy following treatment discontinuation. In the present study, cessation of ALN treatment for as little as 14 days allowed for greater anabolic action by PTH. This could be explained by the fact that SFx repair in this model heals by direct remodeling even without ALN or PTH treatment within 10 weeks, while other studies investigated osteoporosis treatment in postmenopausal women or ovariectomized rats (Cosman et al., 2005; Cosman et al., 2009; Cosman et al., 2011; Cosman et al., 2013; Amugongo et al., 2014). Cessation of ALN treatment and the gradual release of residual ALN from mineralized bone tissue rather than the active intake, allowed for the anabolic PTH action to predominate.
With regards to cortical bone parameters, the present study showed that daily PTH injections for 14 days increased significantly the cortical bone area when compared to ALN-PTH and
VEH groups. This is in agreement with a number of studies that showed that daily PTH treatment for 4 weeks increased cortical dimensions (Vrahnas et al., 2016), improved cortical bone structure (Jiang et al., 2003), increased bone formation within the femoral neck with predominantly cortical bone (Cosman et al., 2016) and increased cortical thickness after adding
PTH to a concurrent ALN treatment (Whitmarsh et al., 2015). All of the above findings support the evidence that resorption induced by PTH receptor signaling in osteocytes (Rhee et al., 2013) and PTHrP in the periosteum (Wang et al., 2013) are critical for full anabolism in cortical bone remodeling.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 149
To conclude, the results from the current study showed that daily PTH injections for 14 days increased SFx remodeling through improving the woven bone apposition rate, improving osteoclast and basic multicellular unit (BMU or porosity) parameters. Cessation of ALN after
SFx induction in the combined ALN PTH treatment was effective in increasing osteoclast parameters. Woven bone parameters remained unaffected by the cessation or continuation of
ALN. Furthermore, the results from the current study show that intermittent short duration PTH injections are effective in improving remodeling of SFx even after BP treatment.
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Chapter 7
General Discussion
Conclusions
Recommendations
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7.1 General discussion:
Stress fractures are a result of cylic repetitive loading that lead to accumulation of microdamage within bone matrix (Sallis and Jones, 1991; Speed, 1998; Shaffer et al., 2006). In loaded rat ulnae, increases in gene and protein expression of target molecules can be unequivocally related to the remodelling event initiated by SFx. The question is whether bone remodelling could be exploited to accelerate SFx repair. For example, there are few published data to demonstrate, in vivo, the signals involved in attraction of preosteoclastic cells to a site of remodelling. The specific upregulation of monocyte chemoattractant protein-1 (MCP-1) at a SFx site could be one such potential target (Wu et al, 2013). The current study opens the horizons for further exploration of the molecular biology of PTH action and the related chemokines. Up to date there are limited studies investigating the short term use of iPTH on SFx healing in vivo. The current study is the first to investigate the effect of a single PTH injection on SFx healing. It is also the first study to investigate the effect of iPTH on SFx healing in the presence of a concurrent BP treatment, or its cessation. After characterizing the animal SFx model, the results from the current study are promising with regards to short term iPTH and will help direct future research.
It is well established that SFx heal by direct remodelling (Kidd et al., 2010), and that BP treatment retards healing because of remodelling depression (Kidd et al., 2011). These data suggest a similar mechanism for the aetiology of AFFs. Consistent with the recommendations of the ASBMR task-force (Shane et al., 2010; Shane et al., 2014), we have optimized a rat model of SFx that provides an innovative approach to examine focal remodeling with a known time course and precise anatomical location. Specific activation of remodeling at the SFx could
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 152 be one approach for patients identified with femoral SFx. Parathyroid hormone (PTH) has anabolic actions, involving remodeling activation, which could potentiate SFx healing.
Evidence from studies of the rat ulna (Sloan et al 2010), and case studies in humans (Gomberg et al 2011) support the positive effect of PTH on SFx healing. Sloan et al did not investigate whether SFx repair was complete following treatment, nor whether BP treatment blunted the
PTH anabolic effect for SFx healing. The current study filled the gaps in literature and answered the question whether the anabolic effect of PTH remains effective if BP treatment continues.
The current results support the hypothesis that a single PTH injection is sufficient to augment remodeling indices of a SFx in vivo. The quantum of remodeling was evident in measurable variables of BMU dynamics in the resorption phase of SFx healing at 2 weeks post-loading, especially with the interaction between time and treatment type affecting the osteoclasts number and perimeter. During the formation phase of the remodeling cycle, 6 weeks post SFx loading, the main effect of time was very powerful over most histomorphometric variables in favor of healing. The effect of a single PTH injection reached a plateau level after 10 weeks post SFx loading, with the exception of reducing the porosity (BMU) parameters due to progression in healing. Although trends towards improved healing of the SFx were not significant, the current study opens the way for future research investigating the use of shorter PTH treatment regimens where activated remodeling would be favorable to healing of skeletal injury.
Furthermore, the current results show that daily PTH injections for 14 days, starting 24 hours after SFx induction were sufficient to improve woven bone architecture. In the combined ALN
PTH therapy, there was a significant interaction between the main effects of time and the type
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 153 of treatment (ALN1 vs. ALN2 vs. ALN PTH1 vs. ALN PTH2 vs VEH) on woven bone width, woven bone apposition rate and the number of osteoclasts per unit porosity (BMU) area. Woven bone width and apposition rate were significantly higher in the combined ALN PTH treatment groups when compared to ALN only. This was evident regardless of the continuation or cessation of ALN. After 6 weeks, woven bone width was significantly less in combined ALN
PTH2 treatment where ALN was ceased when compared to continuation of ALN in the combined ALN PTH1 treatment. Furthermore, ceasing ALN after SFx induction increased ostecoclastic activity after two weeks. Finally, the combined ALN-PTH treatment was not superior to daily PTH injections per se. Since SFxs heal by direct remodeling, early activation of remodeling by PTH, combined with cessation of ALN, allows for better clinical outcomes through overcoming the remodeling suppression initially induced by BPs. This is supported by a number of studies in the literature related to AFF healing in osteoporosis patients after a long- term BP treatment (Gomberg et al., 2011; Compston 2010; Compston, 2012).
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 154
7.2 Conclusions:
From the findings of the present study, the following was concluded:
1- A single PTH injection 24 hours after SFx induction increases osteoclastic parameters
in the SFx rat model. This supports the first hypothesis of the current study.
2- Daily iPTH injections for 14 days starting 24 hours after SFx induction is equally
effective with regards to osteoclastic and healing parameters when compared to a single
PTH injection in the SFx rat model. This supports the second hypothesis of the current
study.
3- Daily iPTH injections for 14 days starting 24 hours after SFx induction is not superior
to a single PTH injection in acceleration of hitomorphometric indices of SFx healing. It
only results in an improvement in woven bone area and woven bone apposition rate
when compared to a single PTH injection in the SFx rat model. This partially rejects the
third hypothesis of the current study.
4- Combined ALN-PTH is not superior to daily iPTH injections in the SFx rat model. This
rejects the fourth hypothesis of the current study.
5- Cessation of ALN after SFx induction in the combined ALN-PTH treatment results in
better treatment outcomes when compared to continuation of ALN after SFx induction
in the rat model. This rejects the fifth hypothesis of the current study.
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 155
7.3 Recommendations for future research:
1- Investigate the possibility of incrementally increasing the number of iPTH doses starting 24 hours after induction of SFx in order to determine an effective number of iPTH doses that could possibly improve the positive outcomes with regards to the SFx healing parameters after 6 weeks.
2- Investigate the effect of administration of a single weekly dose of iPTH injections for 10 weeks starting 24 hours after induction of SFx, which could prevent the effect of PTH treatment from reaching a plateau level after 10 weeks, due to the prolonged frequency of injections when compared to daily injections. This could potentially improve the treatment outcomes when compared to daily iPTH injections for 14 days.
3- Investigate the possibility of combining the daily PTH injections for 14 days, 24 hours after induction of SFx with vitamin / calcium supplements in order to obtain a more superior effect on SFx histomorphometric remodeling indices when compared to the single dose of PTH.
4- Investigate the use of additional sequential ALN treatment to allow for secondary mineralization to occur after healing and prevent the recurrence of SFx.
5- Investigate the effect of combining intermittent PTH treatment (Single or Daily injections) and/or combined ALN-PTH therapy with other treatment modalities such as ultrasound or mechanical loading.
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Chapter 8 References
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Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 232
Appendix 1
Summary of non-significant findings
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 233
Appendix 1a: Showing a summary of non-significant findings from the single PTH experiment.
Variable Effect F Significance Treatment .058 .811 Cortical Bone Area (CT.B.Ar, mm2) Time 3.883 .052 Interaction (Treatment * Time) .037 .847
Treatment .281 .597 Cortical perimeter (Ct.Pm, mm) Time 3.808 .057 Interaction (Treatment * Time) .002 .962 Treatment 3.436 .067 Length of Stress Fracture (SFx.Le, µm) Time .019 .819 Interaction (Treatment * Time) 1.538 .218 Treatment 3.476 .065 Length of remodeling unit along stress Time 2.196 .141 fracture line (BMU.Le, µm) Interaction (Treatment * Time) .421 .518 Treatment 3.546 .062 Number of osteoclasts per µm of Basic Multicellular Unit (BMU) length Time 2.028 .157 (N.Oc/µm) Interaction (Treatment * Time) .900 .345
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 234
Appendix 1b: Showing a summary of non-significant findings from the daily PTH experiment.
Variable Effect F Significance Treatment .739 .394 Cortical Bone Area (CT.B.Ar, mm2) Time 4.293 .054 Interaction (Treatment * Time) .150 .700
Treatment .792 .378 Cortical perimeter (Ct.Pm, mm) Time 5.601 .052 Interaction (Treatment * Time) .200 .657 Treatment .008 .930 Woven Bone Perimeter (Wo.B.Pm, mm) Time 2.536 .118 Interaction (Treatment * Time) .114 .737 Treatment .279 .600 Length of Stress Fracture (SFx.Le, µm) Time 1.789 .187 Interaction (Treatment * Time) .339 .563 Treatment 1.089 .302 Number of osteoclasts per µm of Basic Multicellular Unit (BMU) length Time 7.898 .07 (N.Oc/µm) Interaction (Treatment * Time) 1.027 .316
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 235
Appendix 1c: Showing a summary of non-significant findings from the combined ALN PTH experiment.
Variable Effect F Significance Treatment 1.800 .133 Cortical Bone Area (CT.B.Ar, mm2) Time 2.615 .108 Interaction (Treatment * Time) .968 .427
Treatment 1.738 .146 Cortical perimeter (Ct.Pm, mm) Time 1.785 .184 Interaction (Treatment * Time) .989 .416 Treatment 1.302 .273 Woven Bone Area (Wo.B.Ar, mm2) Time .094 .759 Interaction (Treatment * Time) 1.454 .220 Treatment .454 .769 Length of Stress Fracture (SFx.Le, µm) Time .205 .651 Interaction (Treatment * Time) .692 .598
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 236
Appendix 2
Single PTH injection publication
Effect of PTH on facilitating stress fracture healing. PhD Thesis - Mahmoud Bakr Page 237
Single Injection of PTH Improves Osteoclastic Parameters of Remodeling at a Stress Fracture Site in Rats
Mahmoud M. Bakr ,1,2 Wendy L. Kelly,1 Athena R. Brunt,1 Bradley C. Paterson,1 Helen M. Massa,1 Nigel A. Morrison,1 Mark R. Forwood1 1School of Medical Sciences and Menzies Health Institute Queensland, Griffith University, Queensland 4222, Australia, 2School of Dentistry and Oral Health, Griffith University, Queensland 4222, Australia
Received 19 January 2018; accepted 17 February 2019 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.24262
ABSTRACT: Stress fractures (SFx) result from repetitive cyclical loading of bone. They are frequent athletic injuries and underlie atypical femoral fractures following long-term bisphosphonate (BP) therapy. We investigated the effect of a single PTH injection on the healing of SFx in the rat ulna. SFx was induced in 120 female Wistar rats (300 15 g) during a single loading session. A single PTH (8 mg.100g 1) or vehicle (VEH) saline injection was administered 24 h after loading. Rats were divided into four groups (n ¼ 15) and ulnae were examined 1, 2, 6, or 10 weeks following SFx. Two Toluidine Blue and TRAP-stained sections of the SFx were examined for histomorphometric analysis using OsteomeasureTM software. An increase in osteoclast number (N.Oc) and perimeter (Oc.Pm) was observed two weeks following PTH treatment (p < 0.01). At 6 weeks, bone formation was the main activity in BMUs. At 10 weeks, the proportion of healing along the SFx line remained 50% greater in PTH groups (p ¼ 0.839), leading to a 43% reduction in the porosity area of BMU (p ¼ 0.703). The main effect of time was a significant variable along the entire SFx remodeling cycle, with significant interactions between time and treatment type affecting (N.Oc) (p ¼ 0.047) and (Oc.Pm) (p ¼ 0.002). We conclude that a single PTH injection increases osteoclastogenesis by the second week of the remodeling cycle in a SFx in vivo. ß 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res
Keywords: stress fracture; parathyroid hormone; bone remodeling; healing; ulna
Bone remodeling is necessary to maintain the integrity effect of PTH is regulated by an increase in canonical of cortical and cancellous bone throughout life. It turns Wnt signalling.9 over aged bone tissue, removes bone micro-damage, re- Intermittent PTH treatment also enhances fracture organizes fracture callus and repairs stress fractures.1 healing. Daily systemic treatment with PTH (1–34) at Stress fracture (SFx) is a serious consequence of a dose of 5–30 mg/kg per day increases callus forma- intensive training, accounting for 10% of cases in a tion, and doses ranging between 10 and 200 mg/kg per typical sports medicine practice, with prevalence in day increase callus stiffness and strength.8,10–13 In athletes and recruits reported to range from 0.2% to as humans, preliminary data suggest that PTH (1–34) at high as 49%.2 Stress fractures also occur in clinical the usual dose for osteoporosis treatment (20 mg/kg) settings where the remodeling response is insufficient accelerates fracture union.14 To conclude, PTH is a to prevent progression of fatigue microdamage to a double-edge sword for regulation of bone metabolism stress fracture. For example, this is observed in and turnover with its anabolic and catabolic effects, atypical femoral fractures associated with long-term and its complex signaling mechanisms.15,16 antiresorptive therapy.3 We previously demonstrated that SFx heal by direct Parathyroid hormone (PTH) has an anabolic effect remodeling.1 Periosteal woven bone stabilizes the in bone.4 Different molecular mechanisms of PTH region, resorption excavates the SFx line, and new action have been suggested; either by increasing the bone formation completes the repair. We have also number of osteoprogenitor cells,5 inhibiting precursor optimized a rat model of SFx that provides an innova- cells of osteoblasts from undergoing apoptosis,6 and tive approach to examine focal remodeling with a enhancing proliferation of preosteoblasts and trans- known time course and precise anatomical location. forming bone lining cells into osteoblasts.7 Other We have extensive data characterizing histology and documented mechanisms by which PTH enhances gene expression over the duration of remodeling from healing include the increase in synthesis of bone initiation to conclusion.1 matrix proteins; as well as an increase in osteoclasto- Parathyroid hormone (PTH) increases bone remod- genesis during the remodeling phase of repair.8 In eling with a net-positive effect on bone mass; with addition, PTH enhances the early stages of endochon- osteoblast number and activity increased, primarily at dral bone repair by increasing chondrocyte recruit- remodeling sites. With some evidence that PTH accel- ment and differentiation.9 Furthermore, the anabolic erates fracture healing,17 these observations suggest that PTH could also accelerate SFx healing in cases of non-healing or suppressed remodeling. Proof of princi- Conflict of interest: None. ple for accelerating SFx healing with PTH exists for Grant sponsor: National Health and Medical Research Council; the rat ulna.18 However, this was achieved using daily Grant number: Forwood APP1049190. PTH injections for the entire eight-week duration of Correspondence to: Mahmoud M. Bakr (T: +61756780752; F:+61756780789 the experiment. We hypothesize that a single PTH E-mail: m.bakr@griffith.edu.au) injection administered 24 h post SFx loading could be # 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. sufficient to increase remodeling at the SFx site during
JOURNAL OF ORTHOPAEDIC RESEARCH1 MONTH 2019 1 2 BAKR ET AL. the resorptive and formative phases of the remodeling strated that 15 rats/group is required to achieve statistical cycle. The aim would be to activate the normal healing power of 80% for p < 0.05 to detect differences among groups response with activated remodeling, supported by a for morphometric variables. Because the SFx is produced sound woven bone callus. Once initiated, prolonged unilaterally, treatment differences were tested among PTH treatment would not be necessary, since BMU progres- and Vehicle groups, and not using contralateral controls. sion should continue to termination. We hypothesized PTH group received a single subcutaneous (S.C) injection of human PTH-(1–34) peptide (Sigma–Aldrich) dissolved in that a single treatment with PTH could accelerate SFx 0.9% saline with 1% rat heat-inactivated serum in a final by initiating a quantum of remodeling greater than volume of 200 ml with a dose of (8 mg/100g body weight) that initiated by the SFx itself. This would be achieved twenty four hours after loading of SFx. VEH group received by increasing the availability of osteoclasts and reduc- an equivalent dose of saline. Rats were euthanized at 1, 2, 6, tion of porosity. The aim of this study was to deter- and 10 weeks post-SFx. mine the efficacy of a single PTH injection in accelerating SFx healing in the rat ulna. Tissue Processing Rats were anesthetized and perfusion-fixed in 4% parafor- MATERIALS AND METHODS maldehyde. Ulnae were dissected, post-fixed for 4 h at 4˚C, One hundred and twenty female Wistar rats (300 15 g, decalcified in 14% aqueous EDTA for 4 weeks, paraffin Animal Resource Centre, Perth, WA) had an ulnar stress embedded and transverse sections, 5 mm thickness, cut in the fracture induced. Rats were anesthetized with halothane and region of the SFx. Approximately 300 serial sections were oxygen for loading. As previously described,1 SFx was cut, to locate the region of the SFx. Adjacent serial sections achieved in a single loading session of the forearm in axial were stained with toluidine blue and for tartrate resistant compression, which produces a remarkably standardized acid phosphatase (TRAP).1 Two toluidine blue-stained sec- SFx. Loading was performed in a loading device using an tions from each bone were measured at a standard point LVDT to monitor displacement in the limb. Because of the along the SFx (middle point between outer cortical margin natural ulnar curvature, axial compression is converted into and central medullary cavity) as shown in Figure 1. TRAP bending forces with the lateral cortex in tension and the staining was performed on 2 sections for counting of osteo- medial in compression. Loading involved cyclic compressive clasts. The staining solution (pH 5.0) consisted of 0.1 M Tris– loading at 18–20 N load and 2 Hz cycle frequency. This HCl, 17.5 mg/ml naphthol AS-BI phosphoric acid (Sigma), produces strains in the ulnar in the range of 4000–4500 128 mg/ml hexasotized pararosaniline (Sigma) reacted with micro-strain.1 Loading was stopped at a predetermined point 128 mg/ml of sodium nitrite and 1 mg/ml of sodium tartrate. when increased displacement reaches 10%. This level of The sections were incubated with the stain in a 37˚C oven for stiffness loss consistently produces stress fractures after 60 min and were counterstained with Mayer’s hematoxylin 8000 cycles, on average, or about 60 min (range ¼ 4000– and mounted in Cytoseal permanent mounting media. 20,000). Opioid analgesia (single injection, buprenorphine 0.05 mg.kg 1 s.c.) was used following loading sessions. Rats Histomorphometry were housed in pairs and allowed unrestricted cage activity OsteomeasureTM software was used for histomorphometric in between loading sessions. The Griffith University Animal measurements. As previously described,1 histomorphometry Ethics Committee approved the experimental protocols was performed at a standard level along the SFx where the (MSC/02/13/AEC). fracture line was half way between the medial cortical Rats were divided equally into parathyroid (PTH) and margin and the medullary cavity of the bone in transverse vehicle (VEH) treated groups (n ¼ 15 each). Sample sizes section (Fig. 1). This eliminates bias in the selection of the were informed by a previous study that investigated Risedr- region of interest (ROI). The area of a BMU was defined as onate treatment using the same rat model.19 It was demon- the total area that had been resorbed. Within this BMU, the
Figure 1. To eliminate bias in selecting the ROI, the standard position of SFx for measurement was located midway between the outer cortical margin and the central medullary cavity (small arrows). SFx ¼ stress fracture.
JOURNAL OF ORTHOPAEDIC RESEARCH1 MONTH 2019 SINGLE PTH INJECTION AUGMENTS REMODELING 3 area filled with new bone formation was defined as healed line and number of osteoclasts per mm of Basic area. A distinct cement line around a previously resorbed Multicellular Unit (BMU) length (Appendix table). area of bone defined the healed area. The area that had been resorbed, but not yet filled by new bone formation, was Woven Bone Parameters defined as porosity. Morphometric measures included stan- Woven bone area has increased in PTH group after dard, derived and correction variables (Fig. 2a–c and Table 1). 6 weeks when compared to VEH group at the same Statistical Analysis time line (p ¼ 0.037). Woven bone apposition rate Sample sizes were informed by our previous study for 1 decreased significantly with time after 2 weeks (p ¼ 1.9 Risedronate treatment of rats. This demonstrated that 15 5 rats/group was required to achieve statistical power of 80% 10 ) when compared to the first week, it also p ¼ 5 for p < 0.05 to detect differences among groups for morpho- decreased further after 6 weeks ( 2.5 10 ) when metric variables. In the current study, power analysis compared to the second week in both VEH and PTH showed that 15 rats per group provides 80% power of groups (Fig. 4a). Woven bone thickness decreased detecting an effect size of PTH treatment of 0.6 Z-scores at significantly with time after 6 weeks (p ¼ 2.5 10 4) an alpha level of 0.05. Similarly, adequate power was present when compared to the second week in both VEH and to detect the effect of time (80% power to detect an effect of PTH groups (Fig. 4b). Woven bone perimeter increased 0.72 Z-scores). For the possible interaction of time and PTH significantly with after 2 weeks (p ¼ 0.016) when treatment, power is 80% to detect an interaction that has an compared to the first week in both VEH and PTH effect size close to one Z score. Furthermore, for statistically groups (Fig. 4c). There was no significant interaction non-significant findings, Cohen’s d was calculated to provide 20–22 between the main effects of time and the type of an objective assessment of the effect size. All data was normally distributed and analyzed using a 2- treatment (VEH or PTH) on the woven bone parame- way ANOVA and differences determined using Fishers LSD. ters (Table 2). Pearson’s correlation coefficient was used to determine any significant correlations between variables. Significance was Osteoclast Parameters accepted at p < 0.05. The total osteoclast number was counted along the SFx. Neither the osteoclasts present within the entire RESULTS cortical bone area, nor in the newly formed woven There were no significant differences among groups in bone callus were included in the osteoclast count. terms of cortical bone parameters, length of stress There was a significant increase in total osteoclast fracture, nor length of BMU along the stress fracture number and perimeter along the SFx (p ¼ 7 10 4 and
Figure 2. (a): Histomorphometric analysis using OsteomeasureTM. Red Cortical area (Ct.Ar, mm2), Yellow Woven bone area (Wo.B. Ar, mm2), Light blue Woven bone Width (Wo.B.Wi, mm), Dark red Length of stress fracture (SFx.Le, mm) (Toluidine Blue 2X), (b): Showing histomorphometric analysis using OsteomeasureTM. Green Number of osteoclasts (N.Oc), Yellow Osteoclast surface perimeter (OC.Pm, mm) (TRAP 10X), (c): Showing histomorphometric analysis of a Basic Multicellular Unit (BMU) using OsteomeasureTM software. Blue Porosity area (SFx.Po.Ar, mm2), Light Blue Erosion area (SFx.E.Ar, mm2), Yellow Length of remodeling unit along fracture line (BMU.Le, mm), Purple Healing area (SFx.He.Ar, mm2), (e): Showing the significant increase in osteoclast in PTH group after 2 weeks when compared to the VEH group shown in (d) (TRAP 10X), (f and g): Showing the significant decrease in osteoclasts with the progression in healing after 6 weeks in both VEH and PTH groups (TRAP 10X). Scale bar ¼ 10 mm.
JOURNAL OF ORTHOPAEDIC RESEARCH1 MONTH 2019 4 BAKR ET AL.
Table 1. A Summary of all Histomorphometric Variables Measured Using OsteomeasureTM Software
Variable Type Description Abbreviation Standard Cortical bone area (Ct.Ar, mm2) Cortical bone perimeter (Ct.Pm, mm) Woven bone area (Wo.B.Ar, mm2) Woven bone perimeter (Wo.B.Pm, mm) Woven bone Width (Wo.B.Wi, mm) Length of stress fracture (SFx.Le, mm) Length of remodeling unit along stress fracture line (BMU.Le, mm) Porosity (BMU) area (SFx.Po.Ar, mm2) Porosity (BMU) area perimeter (SFx.Po.Pm, mm) Healing area (SFx.He.Ar, mm2) Healing area perimeter (SFx.He.Pm, mm) Erosion (unhealed) area (SFx.E.Ar, mm2) Erosion (unhealed) perimeter (SFx.E.Pm, mm) Number of osteoclasts (N.Oc) Osteoclasts surface perimeter (Oc.Pm, mm) Derived Number of osteoclasts per mm2 of Basic Multicellular Unit (BMU) area (N.Oc/mm2) Number of osteoclasts per mm of Basic Multicellular Unit (BMU) length (N.Oc/mm). Healing percentage ¼ Healing area/Porosity area 100 (He%) Woven bone apposition rate per day ¼ Woven Bone Width Woven Bone (Wo.B.App) perimeter/Number of days Correction Percentage fracture length occupied by bone formation (SFx.He.Pm, mm/SFx. Le, mm%) Healing area per mm2 of the cortical bone area (SFx.He.Ar, mm2/Ct.Ar, mm2) Porosity (BMU) area per mm2 of the cortical bone area (SFx.Po.Ar, mm2/Ct.Ar, mm2) Percentage fracture length occupied by erosion (SFx.E.Pm, mm/SFx.Le, mm%) Erosion (unhealed) area per mm2 of the cortical bone area (SFx.E.Ar, mm2/Ct.Ar, mm2) p ¼ 1 10 5 respectively) in PTH group at 2 weeks, after 6 weeks. Though non-significant, the these differ- compared to VEH (Fig. 2d–g and 4d and e). Examining ences represent moderate effet sizes using Cohen’s d the effect of time on the number of osteoclasts and (d ¼ 0.395 and d ¼ 0.443, respectively) (Fig. 3e, 3f and osteoclast perimeter, there was a significant increase 4g and 4h). A 53.4% increase was observed in percent- after 2 weeks (p ¼ 1.5 10 13 and p ¼ 2.94 10 10, age healing (p ¼ 0.839–Cohen’s d ¼ 0.440) after respectively) in both VEH and PTH groups when 10 weeks when compared to VEH groups (Fig. 3g, 3h compared to the first week (Fig. 4d and e). Further- and 4i). Furthermore, there were no significant differ- more, there was a significant decrease in the number ences between VEH and PTH groups when the of osteoclasts and osteoclast perimeter after 6 weeks percentage fracture length occupied by bone formation (p ¼ 1.35 10 9 and p ¼ 3 10 6, respectively) when (SFx.He.Pm, mm/SFx.Le, mm %) was calculated to compared to 2 weeks in both VEH and PTH groups correct for variations in the total length of the SFx (Fig. 4d and e). There was also a significant interaction line. With regards to the main effect of time on healing between the main effects of time and the type of parameters, healing area, healing perimeter and per- treatment (VEH or PTH) on the number of osteoclasts centage healing increased significantly after 6 weeks (F ¼ 2.751; p ¼ 0.047) and osteoclast perimeter when compared to the second week in both VEH and (F ¼ 5.27; p ¼ 0.02) (Table 2). The number of osteo- PTH groups (p ¼ 1.28 10 8; 1.53 10 9 and p ¼ 0.05, clasts per mm2 basic multicellular unit (BMU) area respectively) (Fig. 4g–i). The normalized healing area decreased significantly after 6 weeks (p ¼ 5.8 10 4) (relative to SFx length) (SFx.He.Pm, mm/SFx.Le, mm when compared to the second week in both VEH and %) increased without being statistically significant PTH groups (Fig. 4f). after 2 weeks when compared to the first week (p ¼ 0.096), but increased significantly after 6 weeks Healing Parameters (p ¼ 1.01 10 11) when compared to the second week Compared to VEH, increases of 17.4% and 27% were in both VEH and PTH groups (Fig. 5b). Finally, the observed in healing area (p ¼ 0.815) and healing healing area per mm2 of the cortical bone area (SFx. perimeter (p ¼ 0.464) of PTH groups, respectively, He.Ar, mm2/Ct.Ar, mm2) increased significantly after
JOURNAL OF ORTHOPAEDIC RESEARCH1 MONTH 2019 SINGLE PTH INJECTION AUGMENTS REMODELING 5
Table 2. A Summary of the Significant Findings Related to the Effect of Time, Treatment as Well as the Interaction Between Treatment and Time on Different Variables Using a Linear Model, 2-Way ANOVA.
Variable Effect F Significance
Woven Bone Area (Wo.B.Ar, mm2) Treatment 4.577 .035 Time .870 .459 Interaction (Treatment Time) .977 .407 Woven Bone Width (Wo.B.Wi, mm) Treatment 2.652 .106 Time 13.41 1.9 10 7 Interaction (Treatment Time) .893 .448 Woven Bone Perimeter (Wo.B.Pm, mm) Treatment 1.695 .196 Time 4.901 .003 Interaction (Treatment Time) .690 .560 Porosity (BMU) Area (SFx.Po.Ar, mm2) Treatment .146 .703 Time 10.73 3 10 6 Interaction (Treatment Time) 2.249 .087 Porosity (BMU) Area Perimeter (SFx.Po.Pm, mm) Treatment .014 .906 Time 14.45 6.45 10 8 Interaction (Treatment Time) 2.396 .073 Healing Area (SFx.He.Ar, mm2) Treatment .055 .815 Time 35.12 1.13 10 15 Interaction (Treatment Time) 1.125 .343 Healing Area Perimeter (SFx.He.Pm, mm) Treatment .540 .464 Time 36.83 3.27 10 16 Interaction (Treatment Time) 1.155 .331 Erosion (unhealed) Area (SFx.E.Ar, mm2) Treatment .577 .449 Time 36.79 3.35 10 16 Interaction (Treatment Time) .139 .936 Erosion (unhealed) Perimeter (SFx.E.Pm, mm) Treatment .084 .772 Time 33.15 4.84 10 15 Interaction (Treatment Time) .028 .994 Number of osteoclasts (N.Oc) Treatment 4.643 .034 Time 28.16 2.39 10 13 Interaction (Treatment Time) 2.751 .047 Osteoclasts surface perimeter (Oc.Pm, mm) Treatment 6.923 .010 Time 19.23 5.84 10 10 Interaction (Treatment Time) 5.276 .002 Healing percentage (He%) Treatment .041 .839 Time 4.165 .020 Interaction (Treatment Time) 1.195 .310 Woven bone apposition rate per day (mm2/day) Treatment 1.301 .257 Time 43.92 2.63 10 18 Interaction (Treatment Time) .96 .412 Number of osteoclasts per mm2 of Basic Multicellular Treatment 3.660 .060 Unit (BMU) area (N.Oc/mm2) Time 6.750 .002 Interaction (Treatment Time) .954 .391 Healing area per mm2 of the cortical bone area (SFx.He. Treatment .193 .661 Ar, mm2/ Ct.Ar, mm2) Time 34.617 1.63 10 15 Interaction (Treatment Time) 1.468 .228 Porosity (BMU) area per mm2 of the cortical bone area Treatment .008 .931 (SFx.Po.Ar, mm2 /Ct.Ar, mm2) Time 10.19 6 10 6 Interaction (Treatment Time) 2.203 .092 Erosion (unhealed) area per mm2 of the cortical bone area Treatment .612 .436 (SFx.E.Ar, mm2/Ct.Ar, mm2) Time 39.86 3.93 10 17 Interaction (Treatment Time) .289 .833 Percentage fracture length occupied by bone formation Treatment .049 .826 (SFx.He.Pm, mm/SFx.Le, mm%) Time 56.36 1.44 10-21 Interaction (Treatment Time) .701 .554 Percentage fracture length occupied by erosion (SFx.E. Treatment .090 .764 Pm, mm /SFx.Le, mm%) Time 38.68 8.85 10 17 Interaction (Treatment Time) .038 .990
JOURNAL OF ORTHOPAEDIC RESEARCH1 MONTH 2019 6 BAKR ET AL.
Figure 3. Progression of remodeling. (3a and b): Shows absence of porosity (BMU) at the SFx entry point one week post loading in both VEH and PTH groups; (3c and d): Development of porosity (BMU) area at the entry point of the SFx two weeks post loading in both VEH and PTH groups; (3e and f): Progression in healing and the gradual filling of the porosity (BMU) areas with new bone 6 weeks post loading in both VEH and PTH groups, with new bone formation also demonstrated along the course of the SFx (black arrows) in PTH group; (3g and h): Shows the significant reduction in porosity (BMU) area 10 weeks post SFx loading in the PTH group (h) when compared to VEH group (g) due to complete healing by direct remodeling starting at the entry of the SFx (Toluidine Blue 10X). Scale bar ¼ 10 mm.
Figure 4. Histomorphometric variables of SFx healing ( SEM). PTH positively influenced bone histomorphmetric indices along all stages of the bone remodeling cycle following the loading of stress fracture (SFx). The time points between 2 and 6 weeks also influenced all variables significantly in both VEH and PTH groups. There was a significant interaction between time and the type of treatment (VEH or PTH) that affected osteoclasts number and perimeter after 2 weeks. p 0.05, p 0.01 (differences between PTH and VEH). þþp 0.05, þþþp 0.01 (differences compared to the previous time point).
JOURNAL OF ORTHOPAEDIC RESEARCH1 MONTH 2019 SINGLE PTH INJECTION AUGMENTS REMODELING 7
Figure 5. Histomorphometric variables of SFx healing ( SEM). PTH increased the healing area/mm2 of the cortical bone area 6 weeks after SFx. PTH alsodecreased the porosity (BMU) parameters 10 weeks after SFx due to progression in healing. Most of the derived and normalized variables were significantly influenced by the main effect of time between the 2 week and 6 week time points. However, there was no significant interaction between time and the type of treatment (VEH or PTH) with the exception of osteoclasts number and perimeter after 2 weeks. p 0.05, p 0.01 (differences between PTH and VEH). þþp 0.05, þþþp 0.01 (differences compared to previous time point).
6 weeks when compared to the second week (p ¼ 1.27 and the percentage fracture length occupied by ero- 10 8) in both VEH and PTH groups (Fig. 5a). There sion, where a significant increase was observed after was no significant interaction between the main effects 2 weeks (p ¼ 0.005; p ¼ 1.27 10 15, respectively) when of time and the type of treatment (VEH or PTH) on compared to the first week in both VEH and PTH the healing parameters (Table 2). groups. This was followed by a significant decrease in both the erosion (unhealed) area per mm2 of the Erosion (Unhealed) Parameters cortical bone area and the percentage fracture length Erosion perimeter decreased by 47.7% (p ¼ 0.772– occupied by erosion after 6 weeks (p ¼ 4.2 10-14; Cohen’s d ¼ 0.340) in the PTH group after 6 weeks 1.82 10 13, respectively) (Fig. 5c and f). when compared to VEH, but was not significant. A significant negative correlation (r ¼ 0.602) was ob- Porosity (BMU) Parameters served between erosion and healing perimeters in There were no significant differences in porosity area PTH group after 6 weeks (p ¼ 0.038) (Fig. 5e). There and porosity perimeters in PTH groups when com- were no significant differences between VEH and PTH pared to VEH groups (Fig. 3f). It should be noted that groups in erosion area (Fig. 5d). This was also the case there was a 43% decrease (p ¼ 0.703–Cohen’s when the percentage fracture length occupied by d ¼ 0.597) in porosity area in PTH group after 10 weeks erosion (SFx.E.Pm, mm /SFx.Le, mm %) and the when compared to VEH group (Fig. 3g, 3h and 5g and erosion area per mm of the SFx length (SFx.E.Ar, mm2/ 5h). With regards to the main effect of time on porosity SFx.Le, mm) were normalized for SFx length or parameters, porosity (BMU) area, perimeter and po- cortical bone area (SFx.E.Ar, mm2/Ct.Ar, mm2) rosity (BMU) area per mm2 of the cortical bone area (Fig. 5c and f and Table 2). With regards to the main (SFx.Po.Ar, mm2/Ct.Ar, mm2) increased significantly effect of time on erosion (unhealed) parameters, there after 2 weeks (p ¼ 0.01; p ¼ 0.0002 and p ¼ 0.01, respec- was a significant increase in erosion (unhealed) area tively) when compared to the first week in both VEH and perimeter after 2 weeks (p ¼ 0.008; 4.29 10 18, and PTH groups (5g, 5h, 5i and Table 2). respectively) when compared to the first week, this was followed by a significant decrease in erosion area DISCUSSION and perimeter after 6 weeks (p ¼ 2.2 10 13; We tested the hypothesis that short-term treatment is 5.8 10 12 respectively) (Fig. 5d and e). This was also sufficient to increase and accelerate remodeling, and consistent with corrected variables including the ero- accelerate healing of SFx. There were significant sion (unhealed) area per mm2 of the cortical bone area increases in osteoclast parameters in the resorption
JOURNAL OF ORTHOPAEDIC RESEARCH1 MONTH 2019 8 BAKR ET AL. phase of bone remodeling that occupies the first two Although not significant, a decrease of 43% in porosity weeks after SFx initiation. Although both groups (10 weeks) would be mechanically important during showed increasing bone formation at six and ten healing given the exponential relationship between weeks, the effect of PTH on osteoclastogenesis did not bone strength and porosity.23 For the non-significant translate into significantly greater bone formation at changes Cohen’s d values were between 0.39 and 0.59, these time-points. For example, there was no signifi- demonstrating moderate clinically relevant effect sizes cant resolution of porosity (BMU filling) in the forma- according to Cohen20,21 and Sawilowky.22 tion phase at six to ten weeks after SFx initiation. The mechanism of the anabolic effect of PTH is not Timing of outcome measures was based on our pub- yet fully understood. The large increase in mineral lished data for measurement of resorption (7 and 14 apposition rate24 is probably due mainly to an increase days) and formation at (6 and 10 weeks) in the same in osteoblast number.25,26 It is likely that some of rat model.1 The increase in healing parameters in the those new osteoblasts originated from periosteal pre- PTH group, though not significant, suggests that osteoblasts.7 At the periosteum, PTH exerts pro-differ- further investigation is merited, either in PTH dose or entiating and pro-survival effects on post-mitotic pre- time points, to determine if there is a connection osteoblasts.27 Targeting the latter cells is an effective between the PTH mediated increase in OC number in mechanism for increasing osteoblast number in perios- the BMU and ultimate healing. teal bone where the production of osteoblasts from The observed increase in healing parameters corre- replicating progenitors is slow, and the rate of apopto- lated significantly with the reduction in erosion sis is less than that of cancellous bone.27 PTH binds to 6 weeks post-SFx. This correlation demonstrates a receptors on osteoblasts and osteocytes and leads to positive effect of a single PTH injection on the forma- increased expression of the early genes c-fos, c-myc, c- tion phase of SFx remodeling, independent of time in jun, and IL-6. These early genes are involved in cell the linear model. The main effect of time showed proliferation.28–30 Intermittent PTH not only initiates statistically significant improvement in healing pa- remodeling, but also postpones osteoblast apoptosis.31 rameters from 2 to 10 weeks. In addition to the above, Daily injections of PTH attenuate osteoblast apoptosis the nature of the rat model used could lead to a degree in mice, thereby increasing osteoblast number, bone of variability in the data collection despite the strict formation rate and bone mass; but they do not affect measures used for standardization of the SFx loading osteoclast number.32 procedures and the point of SFx located and used for Over a 4-week period, Sloan et al. showed that daily histomorphometric analysis. Increasing the sample intermittent PTH (iPTH) treatment enhanced intra- size could have led to a more significant and powerful cortical bone remodeling in the rat ulna.18 This was statistical scenario, however, the number of animals demonstrated by increased resorption area and min- used in this experiment was in accordance with the 3R eral apposition rate from 2 weeks after SFx.18 The principles of animal testing and has been used success- accelerated repair shown by Sloan is consistent with fully to achieve statistical power of 80% for p < 0.05 to our data, but their iPTH treatment regime was clearly detect differences among groups for morphometric more prolonged. In non-SFx models, therapeutic treat- variables in previous studies.1 In the current study, ment with iPTH improves bone formation and mineral the sample size was determined based on the power apposition rate after one week of starting the PTH analysis which showed that 15 rats per group provided treatment; as well as increasing eroded bone surface 80% power of detecting an effect size of PTH treatment and osteoclast number.33 Similarly, iPTH enhanced of 0.6 Z-scores at an alpha level of 0.05. Similarly, mechanical properties after distraction osteogenesis in adequate power was present to detect the effect of rats; an intervention used in both leg lengthening and time (80% power to detect an effect of 0.72 Z-scores). bone transportation with external fixtures in treat- For the possible interaction of time and PTH treat- ment of fractures and non-unions.34 Intermittent PTH ment, power is 80% to detect an interaction that has treatment at 30 mg/kg before and after osteotomy in an effect size close to one Z score. When interactions rats accelerated the healing process as evidenced by between time and treatment were not present, main earlier replacement of woven bone to lamellar bone, effects were presented. Therefore, 15 rats per group increased new cortical shell formation, and increased were sufficient to detect differences between variables the ultimate load up to 12 weeks after osteotomy in among groups in the current study. rats.13 A limitation of the current study is the modest The above observations from daily repeated iPTH change that was observed in healing and porosity are consistent with those from our single dose protocol, variables. While we detected significant increases in but clearly of greater magnitude. There were increases osteoclastogenesis, the single dose of PTH may have in healing area and perimeter after six weeks, as well been insufficient to augment the subsequent formation as an increase in healing percentage after 10 weeks phase above that observed in the VEH group. The following a single PTH injection. Though not statisti- power/sample size calculation for the project was cally significant, we hypothesize that the increase in performed a priori and understandably did not take in healing parameters explains the decrease in the to account possible variations in data distribution. overall porosity after six weeks and demonstrate the
JOURNAL OF ORTHOPAEDIC RESEARCH1 MONTH 2019 SINGLE PTH INJECTION AUGMENTS REMODELING 9 shift in the remodeling cycle towards bone formation future, where short-term intervention with PTH after PTH administration, which is in agreement with might be preferable and more economical. previous studies.13,18,32–34 Interestingly, PTH failed to increase union rates, CONCLUSION callus size and strength in open fractures when Our results support the hypothesis that a single PTH compared to closed fracture.35 This was suggested to injection is sufficient to augment remodeling of a SFx be due to the lack of sufficient number of cells for in vivo. The quantum of remodeling was evident in recruitment in the area of open fractures, as well as measurable variables of BMU dynamics in the resorp- the inability of PTH to recruit progenitor cells from tion phase of SFx healing at 2 weeks post-loading, potential surrounding tissues. Other agents with non- especially with the interaction between time and specific anabolic effect such as BMP were suggested.35 treatment type affecting the osteoclasts number and Nonetheless, Kumabe et al. recently showed that tri- perimeter. During the formation phase of the remodel- weekly administration of PTH (1–34) increased union ing cycle, 6 weeks post SFx loading, the main effect of rate and accelerated bone healing in a rat refractory time was very powerful over most histomorphometric fracture model, suggesting that systemic administra- variables in favor of healing. The effect of a single tion of PTH (1–34) could become a novel and useful PTH injection reached a plateau level after 10 weeks therapy for delayed union or non-union.36 This con- post SFx loading, with the exception of reducing the firms the role of certain bone modulators such as porosity (BMU) parameters due to progression in monocyte chemotactic protein-1 (MCP-1) in recruit- healing. The increase in healing parameters in the ment of osteoclast precursors for bone remodeling.37 It PTH group, though not significant, suggests that also sheds more light on the suggested mechanism of further investigation is merited, either in PTH dose or action of PTH. In the present study, the significant time points, to determine if there is a connection increase in the number of osteoclasts along the SFx between the PTH mediated increase in OC number in after 2 weeks may be explained by PTH induction of the BMU and ultimate healing. This opens the way for monocyte chemotactic protein-1 (MCP-1), which is future research investigating the use of shorter PTH responsible for differentiation and recruitment of treatment regimens where activated remodeling would osteoclast precursors in early remodeling phases.38–41 be favourable to healing of skeletal injury. Although we did not measure MCP-1 in this study, our previous work demonstrated that MCP-1 is specifically AUTHORS’ CONTRIBUTIONS regulated during SFx repair, and we hypothesize that Study design: MB, WK, NM and MF. Study conduct: MB, iPTH may amplify that response.40 WK, AB, BP, and MF. Data collection: MB, WK and AB. The rats used in the present study were 17 weeks of Data analysis: MB, HM, NM and MF. Data interpretation: age at the time of SFx loading. This was based on MB, HM, NM and MF. Drafting manuscript: All authors. previous work by Kidd et al.1 that characterized the Revising manuscript: All authors. Approval of the final version of the manuscript: All authors. Responsibility of the current rat model. iPTH treatment increases bone integrity of data analysis: MB, WK, NM and MF. All authors formation at all ages, but the underlying cellular and have read and approved the final submitted manuscript. molecular mechanisms facilitating the anabolic re- 42 sponse to the hormone could differ by age. The age- ACKNOWLEDGMENTS dependent differences of the iPTH effect are mainly This project was reviewed and granted ethical approval by related to bone density and quality, which were not Griffith University Animal Ethics Committee (GU Ref No: MSC/ investigated in the present study. Furthermore, the 02/13/AEC). We thank Ms. Gemma Diessel for technical support chosen age of 17 weeks also ensures that the rats are in all stages of the project. We also thank Prof. Ward Massey for out of the adolescent growth spurt. his feedback and input in the initial stages of the project. This PTH is under investigation for potential uses in project was funded by National Health and Medical Research tissue engineering and implant integration. 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APPENDIX
VEH 1 PTH 1 VEH 2 PTH 2 VEH 6 PTH 6 VEH 10 PTH 10 Variable/Group wk wk wks wks wks wks wks wks
Woven Bone Area (Wo.B.Ar, mm2) Mean 0.776 1.15 0.739 0.796 0.611 1.12 0.709 0.757 SEM 0.09 0.3 0.087 0.052 0.028 0.274 0.087 0.122 Woven Bone Width (Wo.B.Wi, mm) Mean 280.1 329.6 327.3 320.4 206.5 263.1 193.1 200.3 SEM 17.7 32.7 25 27.34 16.6 36.6 9.3 7.7 Woven Bone Perimeter (Wo.B.Pm, mm) Mean 8.42 9.19 10.06 10.1 9.87 11.08 10.5 10.41 SEM 0.66 0.63 0.54 0.27 0.46 0.52 0.39 0.47 Porosity (BMU) Area (SFx.Po.Ar, mm2) Mean 0 0 0.019 0.018 0.017 0.029 0.039 0.021 SEM 0 0 0.008 0.004 0.003 0.009 0.008 0.006 Porosity (BMU) Area Perimeter (SFx.Po.Pm, mm) Mean 0 0 0.723 0.836 0.825 1.32 1.39 0.841 SEM 0 0 0.234 0.155 0.13 0.366 0.273 0.215 Healing Area (SFx.He.Ar, mm2) Mean 0 0 0.0043 0.0042 0.047 0.064 0.069 0.057 SEM 0 0 0.0026 0.0013 0.0093 0.0147 0.0118 0.0093 Healing Area Perimeter (SFx.He.Pm, mm) Mean 0 0 0.409 0.627 2.71 3.71 3.16 2.75 SEM 0 0 0.181 0.166 0.484 0.759 0.501 0.412 Erosion (unhealed) Area (SFx.E.Ar, mm2) Mean 0 0 0.015 0.014 0.013 0.011 0 0 SEM 0 0 0.006 0.003 0.005 0.006 0 0 Erosion (unhealed) Perimeter (SFx.E.Pm, mm) Mean 0 0 0.525 0.501 0.052 0.027 0 0 SEM 0 0 0.164 0.091 0.02 0.019 0 0 Number of osteoclasts (N.Oc) Mean 0 0 13.62 25.00 3.28 4.16 3.82 5.1 SEM 0 0 3.79 2.86 0.818 1.09 0.929 1.22 Osteoclasts surface perimeter (Oc.Pm, mm) Mean 0 0 0.293 0.803 0.139 0.157 0.147 0.172 SEM 0 0 0.184 0.091 0.037 0.049 0.036 0.045 Healing percentage (He%) Mean 0 0 22.6 23.3 276.4 220.6 176.9 271.4 SEM 0 0 0.055 0.046 2.57 0.702 0.362 1.97 Woven bone apposition rate per day (mm2/day) Mean 355.71 462.35 239.43 232.87 49.06 72.73 29.46 29.98 SEM 43.03 84.39 23.62 21.92 5.32 13.56 2.22 1.95 Number of osteoclasts per mm2 of Basic Multicellular Unit (BMU) area (N.Oc/mm2) Mean 0 0 949.74 1773.01 273.51 278.35 145.33 766.88 SEM 0 0 319.47 568.77 110.08 86.47 30.71 373.29 Healing area per mm2 of the cortical bone area (SFx.He.Ar, mm2/ Ct.Ar, mm2) Mean 0 0 0.0028 0.0032 0.0315 0.0461 0.0467 0.0384 SEM 0 0 0.0057 0.0038 0.021 0.0386 0.0288 0.0249 Porosity (BMU) area per mm2 of the cortical bone area (SFx. Po.Ar, mm2 /Ct.Ar, mm2) Mean 0 0 0.0126 0.0136 0.0123 0.0219 0.0264 0.0148 SEM 0 0 0.0054 0.0033 0.0025 0.0075 0.0058 0.004 Erosion (unhealed) area per mm2 of the cortical bone area (SFx.E.Ar, mm2/ Ct.Ar, mm2) Mean 0 0 0.098 0.0104 0.0049 0.0045 0 0 SEM 0 0 0.003 0.006 0.006 0.006 0 0 Percentage fracture length occupied by erosion (SFx.E.Pm, mm /SFx.Le, mm%) Mean 0 0 32.08 31.16 3.18 1.18 0 0 SEM 0 0 9.4 5.1 1.08 0.84 0 0
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