DISSERTATIONS | TONG XIAOYU | CORTICAL BONE HISTOMORPHOMETRY | No 418
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ISBN 978-952-61-2494-0 ISSN 1798-5706
Dissertations in Health Sciences
THE UNIVERSITY OF EASTERN FINLAND
TONG XIAOYU
CORTICAL BONE HISTOMORPHOMETRY
Microarchitectural Heterogeneity across Anatomical Sites in Healthy Males
TONG XIAOYU
Histomorphometric examination of undecalcified bone specimens is a standard
tool to study metabolic bone diseases.
With regard to bone structural quality, histological analysis has focused mostly on the cancellous bone in female subjects
with postmenopausal osteoporosis. The understanding of cortical properties across
adulthood is limited. This study aimed to characterize the cortical microarchitecture
at different skeletal sites of healthy male subjects using quantitative cortical bone
histomorphometry.
Cortical Bone Histomorphometry - Microarchitectural Heterogeneity across
Anatomical Sites in Healthy Males
AUTHOR: TONG XIAOYU
Cortical Bone Histomorphometry - Microarchitectural Heterogeneity across
Anatomical Sites in Healthy Males
To be presented by permission of the Faculty of Health Sciences, University of Eastern Finland for public examination in the Auditorium MS302, Medistudia building, University of Eastern Finland,
Kuopio, on Friday, May 12th 2017, at 1 pm
Publications of the University of Eastern Finland Dissertations in Health Sciences
Number 418
Kuopio Musculoskeletal Research Unit, Institute of Clinical Medicine, School of Medicine, Faculty of Health Sciences, University of Eastern Finland
Kuopio 2017
Grano Oy Kuopio, 2017
Series Editors:
Professor Tomi Laitinen, M.D., Ph.D.
Institute of Clinical Medicine, Clinical Physiology and Nuclear Medicine Faculty of Health Sciences
Professor Hannele Turunen, Ph.D.
Department of Nursing Science Faculty of Health Sciences
Professor Kai Kaarniranta, M.D., Ph.D.
Institute of Clinical Medicine, Ophthalmology Faculty of Health Sciences
Associate Professor (Tenure Track) Tarja Malm, Ph.D.
A.I. Virtanen Institute for Molecular Sciences Faculty of Health Sciences
Lecturer Veli-Pekka Ranta, Ph.D. (pharmacy) School of Pharmacy
Faculty of Health Sciences
Distributor:
University of Eastern Finland Kuopio Campus Library
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ISBN (print): 978-952-61-2494-0 ISBN (pdf): 978-952-61-2495-7
ISSN (print): 1798-5706 ISSN (pdf): 1798-5714
ISSN-L: 1798-5706
Author’s address: Kuopio Musculoskeletal Research Unit (KMRU),
Institute of Clinical Medicine, Faculty of Health Sciences, University of Eastern Finland
KUOPIO FINLAND
Supervisors: Professor Heikki Kröger, M.D., Ph.D.
Department of Orthopaedics, Traumatology, and Hand Surgery
Kuopio University Hospital and Kuopio Musculoskeletal Research Unit, Clinical Research Center, School of Medicine, Faculty of Health Sciences, University of Eastern Finland
KUOPIO FINLAND
Professor Jukka Jurvelin, Ph.D.
Dean, Faculty of Science and Forestry
Department of Applied Physics, Faculty of Science and Forestry University of Eastern Finland
KUOPIO FINLAND
Associate Professor Hanna Isaksson, Ph.D.
Department of Biomedical Engineering, Department of Orthopaedics,
Lund University LUND
SWEDEN
Reviewers: Adjunct professor Riku Kiviranta, Ph.D.
Medical Biochemistry and Genetics, Institute of Biomedicine University of Turku
TURKU FINLAND
Professor Juha Tuukkanen, Ph.D.
MRC Oulu and Department of Anatomy and Cell Biology University of Oulu
OULU FINLAND
Opponent: Professor Ken Poole, Ph.D.
Department of Medicine
University of Cambridge and Addenbrooke's Hospital CAMBRIDGE
UNITED KINGDOM
Tong, Xiaoyu
Cortical Bone Histomorphometry - Microarchitectural Heterogeneity across Anatomical Sites in Healthy Males
University of Eastern Finland, Faculty of Health Sciences
Publications of the University of Eastern Finland. Dissertations in Health Sciences Number 418. 2017. 158 p.
ISBN (print): 978-952-61-2494-0 ISBN (pdf): 978-952-61-2495-7 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706
ABSTRACT:
Histological examination of undecalcified bone biopsy specimens is a valuable and well- established tool to study metabolic bone diseases. The investigations of bone structural characteristics have been mostly limited to cancellous bone in female subjects with postmenopausal osteoporosis. This study aimed to characterize the cortical bone microarchitecture at anatomical sites characteristically suffering fragility fractures (the femoral neck and the subtrochanteric femoral shaft) and non-fracture site (the iliac crest) in healthy males.
Undecalcified histological sections of the femoral neck, subtrochanteric femoral shaft and iliac crest obtained from male cadavers (n=20, aged 18–82 years,) were stained using modified Masson-Goldner stain. The complete samples were scanned, using a light microscope, and the histological boundaries were defined using a novel criteria developed in this study. This method made it possible to determine different histological zones, i.e., absolute cortical bone area, endocortical bone area, and cancellous bone area.
The reproducibility for identification of the criteria was studied. Cortical bone histomorphometry was performed with low (x50) and high magnification (x100) microscopy to examine the morphometric characteristics of cortical bone in selected skeletal sites. The variations of histomorphometric parameters occurring with age and in different anatomical regions were primarily investigated. The osteocytic parameters at the femoral neck and shaft were analyzed using phase contrast microscopy and epifluorescence. Moreover, the microarchitectural characteristics of the cortical bone and the osteocytic distribution were compared between these skeletal sites. Also, the comparison was made with the cortical properties measured by scanning acoustic microscopy.
Moderate to high reproducibility was revealed for low magnification parameters (e.g.
cortical bone area). The coefficient of variation (CV %) ranged from 0.02 to 5.61 in the intra-observer study and from 0.09 to 16.41 in the inter-observer study. Identification of the three histological areas revealed the best intra- and inter-observer reproducibility for the absolute cortex area. At the femoral neck, the cortical porosity was lower in the infero-anterior area than in the antero-superior area, supero-anterior area and supero- posterior area. The osteocyte lacunar number per cortical bone area was higher in the infero-anterior and antero-inferior areas, as compared to antero-superior, superio- posterior and postero-superior areas. At the femoral shaft, osteonal area per cortical bone area was lower and cortical porosities were higher in the posterior quadrant than in the other quadrants. At the iliac crest, the external cortex was thicker than the internal
cortex and many osteon structural parameters (e.g. mean osteonal perimeter, max osteonal diameter) were higher in the external cortex. When comparing different anatomical sites, the mean cortical width and osteon number per cortical bone area were highest in the femoral shaft, and the maximal osteonal diameter and mean wall width were highest in the external iliac crest. Many osteon structural parameters were lower in the femoral neck. The osteocyte lacunar number per cortical bone area at the femoral neck were higher than at the subtrochanteric femoral shaft.
This study presents novel criteria for the definition of histological boundaries in human cortical bone. Thereby, the cortical bone was more precisely separated from the trabecularized structures. At the femoral neck, subtrochanteric femoral shaft and iliac crest, it was demonstrated that the variations of cortical microarchitecture associated with age and were different in investigated anatomical region. There was variation in distribution of osteocytic parameters at the proximal femur and along with age. These findings extend the information, over that available in present reference data, on cortical microarchitecture in adult male skeleton. The present results may improve understanding of the underlying morphometric changes that might predispose to fragility fractures.
National Library of Medicine Classification: WE 250, WE 850
Medical Subject Headings: Bone and Bones/anatomy and histology; Bone Diseases, Metabolic; Cancellous Bone; Femoral Fractures; Femur; Haversian System; Ilium; Male; Osteocytes; Osteoporosis; Porosity
Tong, Xiaoyu
Kortikaalisen luun histomorfometria - Mikroarkkitehtuuriset eroavaisuudet miesten luustossa Itä-Suomen yliopisto, terveystieteiden tiedekunta
Publications of the University of Eastern Finland. Dissertations in Health Sciences Numero 418. 2017. 158 s.
ISBN (print): 978-952-61-2494-0 ISBN (pdf): 978-952-61-2495-7 ISSN (print): 1798-5706 ISSN (pdf): 1798-5714 ISSN-L: 1798-5706
TIIVISTELMÄ:
Dekalsifioimattomien luunäytteiden histomorfometrinen tutkimus on vakiintunut menetelmä metabolisten luusairauksien tutkimisessa. Luun rakenneominaisuuksien tutkimus on rajoittunut lähinnä hohkaluuhun ja postmenopausaalisen osteoporoosiin naisilla. Tämän väitöskirjatutkimuksen tarkoituksena oli karakterisoida kortikaalisen luun mikrorakennetta luissa, joissa esiintyy tyypillisiä osteoporoottisia murtumia (reisiluun kaula ja subtrokanteerinen reisiluun varsi) sekä ei-murtumapaikoissa (suoliluun harju) luustoltaan terveillä miehillä.
Dekalsifioimattomat, koko luun poikkipinta-alan kattavat histologiset leikkeet miespuolisten kadaverien (n=20, ikä 18-82 vuotta) reisiluun kaulasta, subtrokanteerisesta femurin varresta ja suoliluun harjusta värjättiin modifoidulla Masson-Goldnerin värjäyksellä. Näytteet tutkittiin valomikroskopialla, skannattiin ja kortikaalisen luun histologinen rajaaminen suoritettiin uudella, tässä tutkimuksessa kehitetyllä menetelmällä.
Uusi menetelmä ja kriteeristö mahdollistaa kuoriluun histologisten luuvyöhykkeiden tarkan määrittämisen: absoluuttinen korteksi, endokortikaalinen luu ja hohkaluu. Uuden menetelmän toistettavuus määritettiin. Kortikaalisen luun histomorfometria suoritettiin mikroskooppisilla suurennoksilla x50 ja x100 valituilta luualueilta. Ensisijaisesti tutkittiin histomorfometristen luuparametrien vaihtelua iän ja anatomisten mittauspaikkojen suhteen. Osteosyyttiparametrit reisiluun kaulassa ja varressa analysoitiin faasikontrasti- ja epifluoresenssimikroskopialla. Lisäksi kortikaalisen luun mikrorakennetta ja osteosyyttien jakaantumaa verrattiin eri mittauspaikoissa.
Histologisia tuloksia verrattiin myös akustisella ultraäänellä saatuihin mittauksiin
Pienemmällä (x50) suurennoksella tehtyjen mittausten (esim. kortikaaliseen luun pinta-ala) toistettavuus todettiin kohtalaiseksi tai hyväksi. Mittaajan sisäinen toistettavuusvirhe (CV%) vaihteli 0.02 -5.61% ja mittaajien välinen virhe vastaavasti 0.09 – 16.41%. Parhain toistettavuus todettiin absoluuttisen korteksin määrittämisessä.
Reisiluun kaulassa kortikaalinen porositeetti oli vähäisempää infero-anteriorisella alueella, verrattuna antero-superioriseen, supero-anterioriseen ja supero-posterioriseen alueeseen. Osteosyyttilakunoiden määrä oli korkeampi infero-anteriorisesti ja antero- inferiorisesti, verrattuna antero-superioriseen, supero-posterioriseen ja postero- superioriseen alueeseen.
Reisiluun varren posteriorisessa neljänneksessä osteonaalinen alue oli pienempi ja luun porositeetti suurempi kuin muissa osissa.
Suoliluun harjussa ulompi korteksi oli paksumpi kuin sisempi korteksi. Useat osteonaaliset parametrit olivat suurempia ulommassa korteksissa.
Eri mittauspaikkoja vertailtaessa, keskimääräinen korteksin paksuun ja osteonien määrä oli korkein reisiluun varressa. Maksimaalinen osteonin läpimitta ja keskimääräinen osteonin seinämän leveys olivat suurimmat suoliluuharjun ulommalla korteksilla. Useat osteonin rakennetta kuvaavat parametrit olivat pienempiä reisiluun kaulassa. Osteosyyttilakunoiden määrä oli suurempi reisiluun kaulassa verrattuna reisiluun varteen.
Tässä tutkimuksessa esitetään uudet kriteerit kortikaalisen luun histologisten rajojen määrittämiselle. Tämä mahdollistaa kortikaalisen luun tarkemman erottelun hohkaluusta. Kortikaalisen luun mikrorakenteessa oli merkittävää vaihtelua sekä iän että anatomisen paikan mukaan (reisiluun kaula, reisiluun subtrokanteerinen reisiluun varsi ja suoliluun harju). Osteosyyttiparametreissa todettiin merkittävää vaihtelua reisiluun yläosassa sekä myös iän mukaan.
Tutkimuslöydökset lisäävät tietämystämme kortikaalisen luun mikrorakenteesta miehellä ja ne voivat myös parantaa käsitystämme luun histomorfometrisistä muutoksista, jotka altistavat osteoporoottisille murtumille.
Yleinen Suomalainen asiasanasto: aineenvaihdunta; luu; luunmurtumat; miehet; osteoporoosi; reisiluu
Acknowledgements
This study was carried out in Kuopio Musculoskeletal Research Unit (KMRU), University of Eastern Finland, Kuopio; Department of Applied Physics, University of Eastern Finland, Kuopio; and at the Department of Orthopaedics, Traumatology, and Hand Surgery, Kuopio University Hospital, Kuopio, during the years 2012 to 2017.
I would like to acknowledge my deepest gratitude for Professor Heikki Kröger, for your excellent guidance, caring, patience, kindness, and providing me with a warm atmosphere for doing research. The joy and enthusiasm you have were contagious and motivational for me, especially during tough times both in my study and my life. The perfect example you have provided as a successful doctor and scientist had impressed me greatly. How lucky I am to have you as my colleague, my friend and my Finnish father.
For the outstanding working efficiency and scientific passion, I would like to thank my supervisor Professor Hanna Isaksson. You let me experience the scientific research and related practical issues beyond the textbooks. Your contributions of time, inspiring ideas, and scientific writing skills have been beyond price. Further, I would like to thank my supervisor, Professor Jukka Jurvelin. You have taught me, both consciously and unconsciously, how rigorous research is done, making my PhD experience productive and stimulating.
The members of the Biophysics of Bone and Cartilage (BBC) have contributed immensely to my study. The group has been a source of friendships as well as good advice and collaboration. I am especially grateful for the collaboration with Markus Malo. You have been guiding my research, helping me to develop my background in biophysics, and bringing creative thoughts into our cross-disciplinary collaboration. I would also like to thank Roope Lasanen and Sami Väänänen for being always willing to help and give their best suggestions.
Many thanks for my reviewers, Professor Riku Kiviranta and Professor Juha Tuukkanen, for your professional comments and your kind help in improving this thesis.
I also would like to thank Ewen MacDonald for careful, efficient English revision of my thesis.
I gratefully acknowledge our excellent laboratory assistants. Warm thanks to Ritva Sormunen, who has always been wholeheartedly supportive and been ready to try new ways to improve sample quality. I appreciate all the invaluable collaboration with Sib- Labs and the time spent there. Thank you Virpi, Jari, Mikko for your sunny smiles and good conversations. Special thanks go to Arto Koistinen, who is always so helpful and provids me with his proficiency in operating new techniques and equipment.
I greatly appreciate the support received through the collaborative work from our research group at Kuopio Musculoskeletal Research Unit. Thank you Inari, who kindly opened the door of bone histomorphometry for me. Without your meticulous, patient guidance and constant feedback this PhD would not have been achievable. Thank you Toni for providing me an excellent example as an outstanding researcher. My thanks also go to Henna, Seija, Pirkko and Marianna for your selfless help and the encouragement to keep me going on.
My deep appreciation also belongs to Joose Raivo, Seija Heikeinen, and Arja Afflekt for for their friendship and the warmth they extended to me and for always making me feel so relax.
I would also like to say a heartfelt thank you to my Mum Zhaoying, Dad Jianjun for always believing in me and encouraging me to follow my dreams. And my parents in law for helping in whatever way they could during this challenging period.
And finally to wife, Li Kang, who has been by my side throughout this period, living every single minute of it, and without whom, I would not have had the courage to embark on this journey in the first place. And to darling Aoxiang for being such a good little baby that past two months, and making it possible for me to complete what I started.
I would like to acknowledge financial support from Kuopio University Hospital, the CSC, the Finnish Cultural Foundation, the Sigrid Juselius Foundation, and the strategic funding of the University of Eastern Finland.
Kuopio May 2017
Tong Xiaoyu
List of the original publications
This dissertation is based on the following original publications:
I Tong XY, Malo M, Burton IS, Isaksson H, Jurvelin JS, and Kröger H.
Development of new criteria for cortical bone histomorphometry in femoral neck: intra- and inter-observer reproducibility. J Bone Miner Metab 33(1):109-18, 2015.
II Tong XY, Burton IS, Isaksson H, Jurvelin JS, and Kröger H. Cortical Bone Histomorphometry in Male Femoral Neck: The Investigation of Age- Association and Regional Differences. Calcif Tissue Int 96(4):295-306, 2015.
III Tong XY, Malo M, Burton IS, Isaksson H, Jurvelin JS, and Kröger H.
Histomorphometric and osteocytic characteristics of cortical bone in male subtrochanteric femoral shaft. J Anat. 2016. Submitted.
IV Tong XY, Burton IS, Isaksson H, Jurvelin JS, and Kröger H. Iliac crest histomorphometry and skeletal heterogeneity in men. Bone Rep 6:9-16, 2017.
The publications were adapted with the permission of the copyright owners.
Contents
1 INTRODUCTION 1
2 REVIEW OF LITERATURE 3
2.1 BONE BIOLOGY... ...3
2.1.1 Bone structure...3
2.1.2 Bone cells... ...4
2.1.3 Bone matrix... ...7
2.1.4 Bone mineralization...7
2.1.5 Bone formation, modelling and remodelling...8
2.1.6 Bone mass development with aging and between the sexes...9
2.2 BONE QUALITY... ...9
2.2.1 Assessment of bone quality...9
2.2.2 Bone histomorphometry...10
2.2.3 Scanning acoustic microscopy...14
2.3 OSTEOPOROSIS...14
2.3.1 Pathogenesis... ...14
2.3.2 Diagnosis...15
2.3.3 Treatment... ...15
3 AIMS OF THE STUDY 17
4 MATERIALS AND METHODS 19
4.1 SUBJECTS (STUDIES I-IV)...19
4.2 BONE BIOPSY AND HISTOLOGY... ...20
4.3 CRITERIA FOR DEFINITION OF HISTOLOGICAL BOUNDARIES...21
4.4 CORTICAL BONE HISTOMORPHOMETRY (STUDIES I-IV)...22
4.5 REPRODUCIBILITY ANALYSIS (STUDY I)...26
4.6 OSTEOCYTE ANALYSIS (STUDY III)...26
4.7 COMPARISON OF CORTICAL CHARACTERISTICS...27
4.8 STATISTICAL ANALYSIS (STUDIES I-IV)...27
5 RESULTS 29
5.1 REPRODUCIBILITY OF BOUNDARY DEFINITION (STUDY I)...29
5.1.1 Intra-observer reproducibility...29
5.1.2 Inter-observer reproducibility...29
5.2 AGE-ASSOCIATED CHANGES (STUDIES II-IV)...30
5.2.1 Femoral neck (study II)...30
5.2.2 Subtrochanteric femoral shaft (study III)...31
5.2.3 Iliac crest (study IV)...31
5.3 ANATOMICAL REGION-ASSOCIATED VARIATIONS (STUDIES II-IV)..31
5.3.1 Femoral neck (study II)...31
5.3.2 Subtrochanteric femoral shaft (study III)...32
5.3.3 Iliac crest (study IV)...33
5.4 OSTEOCYTE ANALYSIS...34
5.4.1 Age-associated variation (study III) ...34
5.4.2 Anatomical region-associated variation...34
5.4.3 Correlation with the cortical characteristics (study III)...34
5.5 CORTICAL CHARACTERISTICS IN COMPARISON...35
5.5.1 Differences in the cortical microarchitecture (study IV)...35
5.5.2. Differences in the osteocytic properties...36
5.5.3. Comparison between using histomorphometry and scanning acoustic microscopy...36
6 DISCUSSION 41
6.1 STRUCTURE BASED DEFINITION OF HISTOLOGICAL BOUNDARIES..41
6.2 AGE ASSOCIATED VARIATIONS………...42
6.2.1. Femoral neck...42
6.2.2 Iliac crest...42
6.3 ANATOMICAL REGION-ASSOCIATED VARIATIONS...43
6.3.1 Femoral neck...43
6.3.2 Subtrochanteric femoral shaft...43
6.3.3 Iliac crest...43
6.4 OSTEOCYTE ANALYSIS...44
6.4.1 Age associated variation...44
6.4.2 Anatomical region-associated variation...44
6.5 CORTICAL CHARACTERISTICS IN COMPARISON...45
6.5.1 Differences in the cortical microarchitecture...45
6.5.2 Differences in the osteocytic properties...45
6.5.3. Comparison between using histomorphometry and scanning acoustic microscopy...45
7 CONCLUSIONS 47
8 REFERENCES 49
ORIGINAL PUBLICATIONS I-IV
Abbreviations
1,25(OH)2D 1,25-dihydroxyvitamin D Ac.F Activation frequency AFF(s) Atypial femoral fracture(s) ANT Anterior
ANT. Ct.Wi Anterior cortical width ANOVA One-way analysis of
variance
ASBMR American Society for Bone and Mineral Research BFR/BS Bone formation rate BMD Bone mineral density BMI Bone mass index BMPs Bone morphogenetic
proteins
BMU Bone multicellular unit BP(s) Bisphosphonate(s) BS/BV Bone surface per bone
volume
BSU Bone structural unit BV Bone volume
BV/TV Bone volume fraction Ct.Ar Cortical bone area Ct.Ar/T.Ar Cortical bone area per
tissue area
Cn.Ar Cancellous bone area Cn.Ar/T.Ar Cancellous bone area
per tissue area Ct.Po Cortical porosity
CV Coefficient of variation DEXA Dual-energy X-ray
absorptiometry ERT Estrogen replacement
therapy
Ec.Ar Endocortical area Ec.Ar/T.Ar Endocortical area per
tissue area
Ec.Pm Endocortical perimeter E.Pm/Ec.Pm Erosion perimeter per
endocortical perimeter ES/BS Eroded surface GM-CSF Granulocyte-
macrophage colony stimulating factor H.Ar/Ct.Ar Haversian canal area
per cortical bone area H.Pm Haversian canal
perimeter
HR-CT High-resolution micro- computed tomography HR-MRI High-resolution
magnetic resonance imaging
IGFs Insulin-like growth factors
LAT Lateral
LAT. Ct.Wi Lateral cortical width
IL-1 Interleukin-1 IL-6 Interleukin-6 IL-11 Interleukin-11 INF Inferior
INF. Ct.Wi Inferior cortical width MAR Mineral apposition rate Max.On.Dm Max osteonal diameter M-CSF Macrophage colony
stimulating factor Md.V/TV Mineralized bone
volume
Mean Ct.Wi Mean cortical width MED Medial
MED. Ct.Wi Medial cortical width Min.On.Dm Min osteonal diameter MMA Methylmetacrylate MS/BS Mineralising surface per
bone surface
MS/OS Mineralising surface per osteoid surface
Mlt Mineralization lag time MS/OS Percentage of osteoid
mineralizing N.On/Ct.Ar Osteon number per
cortical bone area N.Ot/Ct.Ar Osteocyte number per
cortical bone area N.Ot.Lc/Ct.Ar Osteocyte lacunar
number per cortical bone area
N.Po/Ct.Ar Pore number per
cortical bone area Ob.S/BS Osteoblast surface Oc.S/BS Osteoclast surface OPG Osteoprotegerin O.Pm/Ec.Pm Osteoid perimeter per
endocortical perimeter OS/BS Osteoid surface
On.Ar/Ct.Ar Osteonal area per cortical bone area On.Pm Osteonal perimeter O.Th Osteoid thickness OV/BV Osteoid volume PDGFs Platelet derived
growth factors Ps.Pm Periosteal perimeter PGE Prostaglandin E2 PMMA Polymethylmetacrylate POST Posterior
POST. Ct.Wi Posterior cortical width PTH Parathyroid hormone RANK Receptor activator of
nuclear factor-kB (NFkB) RANKL Receptor activator of
nuclear factor-kB ligand (NFkB ligand)
rh-PTH (1-34) recombinant 1-34 fragment of human PTH
rh-PTH (1-84) recombinant human intact PTH ROI Region of interest
SAM Scanning acoustic microscopy SD Standard deviation SERM(s) Selective estrogen-
receptor modulator(s) SR Strontium ranelate SUP Superior
SUP. Ct.Wi Superior cortical width T.Ar Tissue area
Tb.N Trabecular number Tb.Pf Trabecular pattern
factor
Tb.Sp Trabecular separation Tb.Th Trabecular thickness TGFβ Transforming growth
factor-beta
TRAP Tartrate-resistant acid phosphatase Tr.Z Transitional zone TV Tissue volume W.Th Wall thickness W.Wi Wall width
1 Introduction
Osteoporosis is a highly prevalent musculoskeletal disease characterized by micro- architectural deterioration of bone tissue and decreased bone mineral density (BMD), consequently resulting in increased bone fragility and susceptibility to fracture (NIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis, and Therapy 2000, Makridis et al. 2015). Osteoporotic fractures cause significant morbidity and mortality in the elderly, and increase the burden on health-care resources (Boonen, Singer 2008).
Impaired development of peak bone mass in early life and/or accumulated bone loss during aging can lead to osteoporosis (Hernandez, Beaupre & Carter 2003). The skeletal integrity is maintained by constant remodelling in adulthood (Clarke 2008). During the course of ageing, bone remodelling becomes imbalanced (Weinreb et al. 1989). The bone formation is affected by the reduction in the osteoblast differentiation, activity and life span (Jilka et al. 1998a, Jilka et al. 1998b), whereas the bone resorption is increased due to the rise of osteoclast activity (Teitelbaum 2000). This leads to a net decrease in bone mass (Jilka et al.
1996). Thus, a thorough understanding of the mechanism of bone cellular activity is important prior to treatment of osteoporosis. Moreover, changes in bone microarchitecture occurring during aging can lead to reduced bone strength that can only partly be accounted for by BMD (as measured using dual-energy X-ray absorptiometry) (Stone et al. 2003, Mayhew et al. 2005). An effective approach to evaluate bone microarchitectural characteristics may improve the ability to predict fracture risk.
Histological examination of undecalcified transiliac bone specimens has become a valuable and standard clinical and research tool to study the etiology, pathogenesis, and treatment of metabolic bone diseases (Recker et al. 2011). Typically, it has been used to estimate bone turnover rate at different bone surfaces, e.g., endocortical and cancellous surfaces (Power et al. 2003, Rauch, Travers & Glorieux 2007). In different studies, however, the definition of these surfaces varies and there are no unanimously accepted criteria to identify the boundaries between specific histological areas (Dempster et al. 2013). On the other hand, histological analysis of the structural characteristics attributable to age-related bone loss in healthy subjects has focused almost exclusively on the cancellous bone (Tsangari, Findlay & Fazzalari 2007). The understanding of properties of cortical bone across adulthood at the sites of fragility fractures are limited. Moreover, although the differences between the external and internal iliac crest cortices were interpreted as the effects of growth in children (Schnitzler, Mesquita & Pettifor 2009), it is still unknown if this asymmetry still exists after adulthood. Except for the cortical width (Castillo, Ubelaker &
Djorojevic 2012), structural characteristics of the cortical bone have rarely been evaluated and compared between different skeletal sites. There are limited data available on the structural heterogeneity of cortical bone. Further, fragility fractures, which increase with advancing age (Currey, Brear & Zioupos 1996), are more common in women with postmenopausal osteoporosis (Riggs, Khosla & Melton 2002, Seeman 2003). Thus, older women have been the subjects of most bone histomorphometric studies (Ma et al. 2014, Zebaze et al. 2010). No systematic investigation of the histomorphometric properties of the cortical bone has been conducted in males.
As the most common osseous cell in the adult skeleton (Bonewald 2007), osteocytes are mainly thought to be responsible for translating mechanical signals into biochemical signals, and therefore orchestrating the bone remodelling process (van Oers et al. 2008a, Tan et al.
2007). The variation in number and viability of the osteocyte may also be linked to bone brittleness and fragility (O'Brien et al. 2004, Noble 2005). So far, the association between osteocyte distribution and cortical bone microarchitecture remains unknown.
This study was aimed to close the above mentioned gaps by characterizing the quantitative properties of the cortical bone in healthy male. Changes in cortical microarchitecture were investigated by using bone histomorphometry combined with studies using scanning acoustic microscopy at specific skeletal sites. The osteocyte distribution at the sites in relation to fragility fracture was also determined.
2 Review of Literature
2.1. BONE BIOLOGY
2.1.1 Bone structure
At the gross level, bone can be broadly categorized into five categories: long bones (e.g., femur and tibia), short bones (e.g., carpal bones of the hand), flat bones (e.g., skull and scapula), irregular shaped bones (e.g., vertebra), and sesamoid bones (i.e. bones embedded in tendons). They are organized in two main structural types: cortical bone and cancellous bone (Seeman, Delmas 2006). By weight, cortical bone forms approximately 80 percent of the adult skeleton, and it provides high resistance to bending and torsion (Adler 2000). On the other hand, due to its higher porosity and therefore higher surface area, cancellous bone has a higher rate of metabolic activity and responds more rapidly to mechanical stimuli than cortical bone (Adler 2000, Buckwalter et al. 1996a). Both cortical and cancellous bone determine bone strength (Zebaze et al. 2014) and they are therefore critical in determining hip fracture resistance (Zimmermann et al. 2011, Carballido et al. 2013).
Histologically, bone consists of two types: woven (primary) and lamellar (secondary) bone. The latter is present in both cortical and cancellous bone (Buckwalter et al. 1996a, Buckwalter 1994). Woven bone is characterized by disorganized pattern of collagen fibrils and high irregular osteocytes volume, which makes it potentially weak, compared to lamellar bone. It rarely persists in healthy adult skeleton, instead it is but typically resorbed and replaced by lamellar bone (Torzilli et al. 1981). In contrast, lamellar bone is formed in a more homogeneous pattern which includes relatively uniform-sized collagen fibrils and osteocytes. Mechanically, it is stronger than woven bone (Currey 1984). With tightly packed collagen fibrils in sheets, lamellar bone can form osteons, circumferential, interstitial lamellae in cortical bone and trabeculae in cancellous bone (Buckwalter 1994).
Osteon works as the bone structural unit (BSU) in cortical bone (Safadi et al. 2009). Its center is occupied by a 40-50 μm Haversian canal containing blood vessels, nerves, and connective tissue (Jepsen 2009). The central Haversian canal is surrounded by concentric layers of 20 to 30 osseous lamellae making the osteon wall approximately 70 to 100 μm thick. Osteons are densely packed together and separated only by interstitial lamellae, which are the remains of incompletely resorbed osteons (Marotti 1996) (Fig.1). Moreover, the longitudinal orientation of osteons enables the cortical bone to be more resistant to fracture when loaded in parallel than perpendicular to its long axis. The boundaries between osteons are named cement lines, which act as sheaths to prevent microcracks from extending transversely bone rapidly (Yeni et al. 1997, Nalla et al. 2004).
Figure 1. Schematic diagram shows osteons (Haversian system) in the cortical bone. The top view is obtained under a polarization microscopy indicating the concentric lamellae and osteocytes. Masson Goldner trichrome stain, magnification. 400x.
Osteons are formed by concentric lamellae surrounding central Haversian canals. They are linked transversally by Volkmann's canals creating an intracortical network. This network is also connected with the periosteum and bone marrow. The periosteum covers the external surfaces of bone and contributes significantly to the bone blood supply (Clarke 2008). Its cells (i.e. periosteal cells) are able to reconstruct bone in response to local or systemic stimuli (Buckwalter et al. 1995). Although the periosteum decreases both in thickness and osteogenic capacity with ageing, periosteal cells keep forming new bone throughout life, leading to increased diameter of diaphysis in long bones along ageing (Buckwalter et al. 1995, Orwoll 2003).
2.1.2 Bone cells
Bone cells comprise approximately 10 percent of the bone volume and function diversely in bone formation, resorption, repair, and mineral homeostasis (Clarke 2008, Buckwalter et al.
1995). They arise from two origins: mesenchymal stem cells and hematopoietic stem cells (Buckwalter et al. 1996a). The former in the bone marrow includes both osteogenic and adipogenic potential (Bennett et al. 1991), differentiating into the osteoblast. The latter gives rise to circulating or marrow monocytes in addition to osteoclasts (Hayase, Muguruma &
Lee 1997).
Osteoblasts
Osteoblasts are histologically characterized as plump cuboidal cells that are found as tightly organized on bone surfaces at the sites of active bone formation (Malluche, Faugere 1986) (Fig.2). They are the primary cells responsible for the osteogenesis and the mineralization, and their activities are marked by alkaline phosphatase release (Clarke 2008). Moreover, osteoblasts may respond to the autocrine and paracrine stimuli to mediate changes in bone size and shape, which could be modulated by a piezo-electric effect of the hydroxyapatite crystals (Safadi et al. 2009).
In addition to the production of type I collagen and proteoglycans, osteoblasts can produce a variety of non-collagenous proteins, including osteocalcin, osteopontin, bone sialoprotein, and osteonectin (Silvent et al. 2013). Besides, osteoblasts also secrete many cytokines and colony stimulating factors (CSF), such as interleukin-6 (IL-6), interleukin-11 (IL-11), granulocyte-macrophage colony stimulating factor (GM-CSF), and macrophage
colony stimulating factor (M-CSF) (Shiina-Ishimi et al. 1986, Felix, Fleisch & Elford 1989).
Further, osteoblasts are able to secrete numerous growth factors as well. They include transforming growth factor-beta (TGFβ), bone morphogenetic proteins (BMPs), platelet derived growth factors (PDGFs), and insulin-like growth factors (IGFs) (Khan, Bostrom &
Lane 2000). Mature osteoblasts possess receptors for PTH and 1,25(OH)2D, two hormones that play important roles in regulating bone metabolism and mineral homeostasis (Ohmori et al. 2000, Takeda et al. 1999). Thus, osteoblasts play a critical role in the constitution of the organic component of bone matrix, the regulation of bone remodelling, and the myelopoiesis (Buckwalter et al. 1996a, Liedert et al. 2005).
Figure 2. Histological image of osteoblasts. Osteoblasts (red arrows) are identified as plump cells that lie in line on top of the unmineralized bone, osteoid. Masson Goldner trichrome stain, magnification. 400x.
Osteocytes
Osteocytes are the most frequent cell type in bone tissue and represent approximately 90–
95% of all osseous cells in the adult skeleton (Buckwalter et al. 1995, Lanyon 1993). They are former osteoblasts that have become buried in the bone matrix, and they also express genes of the mesenchymal (Bonewald 2011). In the bone structure, osteocytes become individually enveloped in the extracellular matrix within spaces known as lacunae (Noble 2008) (Fig.3).
An interconnected system is established by osteocyte and their lacunae, enabling an extensive molecular exchange through its large surface area. It also provides a potential mechanism that contributes substantially to bone mineral homeostasis (Aarden, Burger &
Nijweide 1994, Teti, Zallone 2009). Functionally, osteocytes are mainly thought to be capable of translating mechanical cues into biochemical signals (van Oers et al. 2008a, Tan et al. 2007), and its density is suggested to reflect the activity of the bone remodelling process (Qiu et al. 2003, Hernandez, Majeska & Schaffler 2004).
Figure 3. Microscopic field with two separate image types captured: phase contrast (A) and fluorescent (B) images, respectively. Osteocyte lacunae (red arrows) and osteocytes (yellow arrows) are demonstrated. Masson Goldner trichrome stain, magnification. 200x.
Osteoclasts
Osteoclasts are the primary bone resorbing cells and are found at sites where resorption takes place, or within “resorbed” pits or cavities known as the Howship’s lacunae (Buckwalter et al. 1996a, Teitelbaum 2011) (Fig.4). They are present in groups in cancellous bone on the surface and in cortical bone digging Haversian canals (Raisz, Rodan 1998).
Osteoclasts can also tunnel through the cortical bone by creating channels (Buckwalter et al.
1996a). In vivo, the growth and differentiation factors of osteoclasts include for instance PTH, interleukin (IL-1), and PGE2, which stimulate the bone resorption and promote the osteoclast formation (Boyle, Simonet & Lacey 2003).
Receptor for activation of nuclear factor kappa B (NF-κB) ligand (RANKL), expressed by T cells, marrow stromal cells, and osteoblast lineage cells (Lacey et al. 1998, Burgess et al.
1999), stimulates the M-CSF-expanded precursors to commit to the osteoclast phenotype.
RANKL activity can be inhibited by the presence of decoy receptor osteoprotegerin (OPG), which is expressed by osteoblasts (Kostenuik, Shalhoub 2001). Thus, OPG gives osteoblasts a critical role in controlling the relationship between bone formation and bone resorption (Simonet et al. 1997). The RANKL/OPG ratio is a key regulator of the bone resorption process and is indicative of osteoclastogenic activity in various bone remodeling diseases (Matsuo, Irie 2008).
Osteoclasts are activated when they come in contact with the prepared mineralized bone surface (Blair, Athanasou 2004). Their ruffled border in the resorption lacuna secretes protons and proteases, which solubilize and digest the mineral matrix (Raisz, Rodan 1998).
Many bone diseases, including osteoporosis, are characterized by distinct bone loss.
Excessive osteoclastic bone resorption, due to either increase in number or activity, has been suggested to be the main cause. As a result, the osteoclast is the major target of therapeutic approaches to bone diseases (Parfitt 1984, Woo et al. 2006).
A
B
A
Figure 4. Histological image of osteoclasts. Osteoclasts are identified as multinucleated, irregular shaped, giant cells. Masson Goldner trichrome stain, magnification. 400x.
Lining cells
Bone lining cells are flattened, squamous cells found lying directly against the bone matrix in areas where there is no active bone formation. These cells can be regarded as quiescent osteoblasts (Hadjidakis, Androulakis 2006). They are also similar to the osteoprogenitor cells because they can be reactivated to become functional osteoblasts under conditions that warrant active bone formation, such as during bone remodelling and fracture repair (Safadi et al. 2009). Moreover, lining cells may have a role in attracting osteoclasts to specific sites and in stimulating the bone resorption (Chambers, Fuller 1985, Hauge et al. 2001).
Bone marrow cells
Besides bone cells, other cells are present in the bone microenvironment. It has been suggested that bone marrow cells play a role in the local regulation of bone remodelling and bone turnover (Carter 1990). Cells from the monocyte/macrophage and lymphoid lineages produce various substances such as cytokines and growth factors that directly or indirectly act on bone cell recruitment and activity.
2.1.3 Bone matrix
Bone matrix consists of four major components: mineral matrix (60-70%), organic matrix (20-30%), lipids, and water (10-15%) (Buck, Dumanian 2012). The mineral matrix is an inorganic matrix, which is predominantly in the form of hydroxyapatite and provides the majority of the bone’s stiffness. Imbalances in mineral matrix formation may lead to rickets, osteomalacia, and osteopetrosis (Glimcher 1998).
Organic matrix consists mainly of type I collagen, but also of small amounts of proteoglycans, glycoproteins, and many growth factors (e.g. osteonectin and IL-1). Organic matrix is generated by osteoblastic secretion and provides bone’s resistance to mainly tensile forces (Buckwalter et al. 1996a).
2.1.4 Bone mineralization
The process of bone mineralization is well regulated and takes place throughout life (Buckwalter et al. 1996a). The crystals of calcium phosphate are transformed from soluble calcium and phosphate, being laid down in precise amounts within the fibrous matrix (Parfitt 1998). A higher degree of mineralization increases the bone stiffness while a lower degree of mineralization increases the possibility for higher deformation when subjected to excessive force (Torzilli et al. 1981).
In patients with osteomalacia, an impairment of bone mineralization exists and the osteoid fails to mineralize (Parfitt 1998). The characteristic histomorphometric findings of this disease are an accumulation of osteoid reflected by increased osteoid thickness, surface, and volume. The high amount of unmineralized matrix leads to pathological and fragile fractures (Parfitt et al. 1985, Siris et al. 1987).
2.1.5 Bone formation, modelling, and remodelling
In the fetus, bone forms in one of two ways, i.e., endochondral ossification or intramembranous ossification. Importantly, the two types of embryonic bone formation mirror the two types of bone healing that occur in adults (Epker & Frost 1965).
Endochondral ossification refers to the predominant process involved in long bone formation and in longitudinal growth of long bones at the physeal plate (Erlebacher et al.
1995). It is initiated by the proliferation of chondrocytes into a cartilage framework with four distinct zones (resting zone, proliferative zone, hypertrophic zone, and calcification zone) (Ortega, Behonick & Werb 2004). The calcification zone, also named as the zone of dead chondrocytes, creates a barren matrix which promotes capillary ingrowth and migration of osteoprogenitor cells. The latter then differentiate into osteoblasts that produce bone (Shapiro 2008).
Bone is continuously turned over by modelling and remodelling (Buck, Dumanian 2012, Buckwalter et al. 1996b). Bone modelling is responsible for changes in bone shape and mass during growth (Hunziker 1994). Both the intramembranous and endochondral ossification contribute to the changes in size and shape of the craniofacial skeleton (Stool SE, Vig KWL, Peetrone JFA, Hymer B 2003). Bone remodelling, on the other hand, is mainly ongoing process during adulthood, and during the process, old bone tissue is replaced by new bone tissue (Hadjidakis, Androulakis 2006). Both modelling and remodelling allow adaptation and maintenance of the mechanical integrity of the skeleton (Buckwalter et al. 1996b).
Bone remodelling occurs on the surface of bone as well as within bone, via an important coordination of osteoclastic and osteoblastic function into a defined remodelling unit (Clarke 2008, Hadjidakis, Androulakis 2006). The basic multicellular unit (BMU) acts as the instrument of bone remodelling. It is a unique temporary anatomical structure, including osteoclasts, osteoblasts, associated blood vessels, nerves, and connective tissue (Parfitt 1994).
The number of active BMUs and the relative amounts of bone resorption and formation within individual BMUs determine the rate of bone turnover (Dempster DW 2006, Eriksen 1986). In particular, bone remodelling creates cortical bone that consists almost entirely of the osteons and the remnants of osteons which have been partially resorbed and replaced by cutting cones (Buckwalter et al. 1996b). Specifically, after osteoclasts have completed their resorptive activity, they move away from the site of the resorption. Soon afterwards, active osteoblasts cover the resorbed surface with osteoid seams which are then mineralized to complete the remodelling process (Hadjidakis, Androulakis 2006, Buckwalter et al. 1996b). The end result of each remodelling cycle at the cortex is the production of a new osteon (Clarke 2008). On the other hand, remodelling of trabecular, endosteal, and periosteal surfaces of bone resembles cortical bone remodelling, with the exception that the osteoclasts lie on the surface of the bone and excavate the Howship’s lacuna (Hadjidakis, Androulakis 2006, Brown et al. 1990). Moreover, remodelling at the endosteal and periosteal surfaces would result in alterations in the thickness and width of tubular bones (Safadi et al. 2009). In addition, the end result of remodelling at the cancellous bone is the formation of a hemiosteon (a trabecular packet) (Parfitt 1998).
The remodelling rate can be increased, for example, by thyroxin, PTH, 1,25(OH)2D. The remodelling rate decreases under the effect of estrogen, calcitonin, and glucocorticosteroids.
The mechanical conditions also modulate the remodelling rate (Christiansen, Hassager &
Riis 1998).
2.1.6 Bone mass development with aging and between the sexes
Bone mass changes dramatically with age (Matkovic 1991). It increases rapidly during adolescence and reaches the maximum level (i.e. peak bone mass) around 20-25 years of age.
Then, the skeletal mass will start to decline and may decrease to approximately half of the peak bone mass by the eighth or ninth decade of life (Matkovic 1992). However, bone size and shape stay more or less the same although the mass is declining (Christiansen, Hassager & Riis 1998). The pattern of age-related bone loss differs in cortical and cancellous bone. Cortical bone usually becomes more porous with advancing age and the cortices of long bones become thinner because the rate of endosteal resorption exceeds the rate of periosteal formation (Keshawarz, Recker 1984). In cancellous bone, characteristically, its trabeculae commonly decrease in number rather than in thickness, leading to a remarkable decline in cancellous bone density (Buckwalter et al. 1996b). The age-related loss of cortical and cancellous bone substantially increase skeletal fragility (Raisz, Seeman 2001).
The bone loss in men seems to follow a linear function and is relatively low, probably of the order of 3-5 percent per decade, which explains the rather low incidence of osteoporotic fractures in men (Christiansen, Hassager & Riis 1998). In women, bone loss before menopause is small and probably similar to that in men (Riggs et al. 1986). Around menopause, however, bone loss starts to accelerate, averaging 2% per year. This bone loss continues for many years after menopause and is still present in elderly women (Ensrud et al. 1995). It has been suggested that during a normal life span, women can lose nearly half of the maximum cancellous bone mass and approximately one-third of the maximum cortical bone mass whereas men lose approximately 30% less bone mass compared to women (Richelson et al. 1984). This explains why osteoporosis and its related fractures are much more common in women than in men.
2.2 BONE QUALITY
Bone quality refers to all properties that combined determine how well the skeleton can resist fracture, such as bone architecture, geometry, composition, accumulation of microdamages, and the rate of bone turnover (Seeman, Delmas 2006, Licata 2009). All these properties are highly inter-linked, and it is therefore difficult to separately determine their effect on bone quality. Still, development of approaches to evaluate bone quality aims to assess the effects of ageing and metabolic bone diseases on the structure, composition, and mechanical properties of the skeleton (Rowe, Shapiro 1998, Hernandez, Keaveny 2006).
2.2.1 Assessment of bone quality
Methods for bone quality assessment play an increasingly important role in understanding metabolic bone diseases, e.g. osteoporosis (Garnero, Delmas 2004, Johnell et al. 2002). In the past decades, considerable efforts have been expended in the development of methods for assessing bone quality in order to provide early detection and possible monitoring of this disease. These methods have increased our understanding of how bone quality contributes to bone strength in metabolic bone diseases.
Bone turnover
Bone turnover is a major determinant of bone quality and is assessed in clinical practice by measurement of biochemical markers of resorption and formation (Garnero et al. 1996, Gerdhem et al. 2004). Serum based bone markers cannot discriminate between turnover changes of a specific skeletal category (i.e. cancellous bone vs. cortical bone), but reflect the overall changes of bone resorption and formation. These markers are of unequal specificity and sensitivity, and their circulation can be influenced by factors other than bone turnover, such as their metabolic clearance (Christiansen, Hassager & Riis 1998). As a result, each marker requires careful selection subject to specific clinical situations. Bone turnover can
also be assessed by a histomorphometric method, which is done by the determination of the activation frequency (Ac.f [N/year]). It indicates the probability for a new remodelling cycle to be initiated at any point of the trabecular surface (Monier-Faugere, Chris Langub &
Malluche 1998).
There are considerable intra-individual variations in bone turnover throughout the skeleton, i.e. bone turnover in one skeletal site may not reflect turnover at other sites (Monier-Faugere, Chris Langub & Malluche 1998, Eventov et al. 1991). Thus, it is not surprising that there may be differences between the biochemical markers and the histomorphometry in assessment of bone turnover. On the other hand, inter-structural variations in bone turnover exist as well. It has been suggested that the cancellous bone makes up to 80% of the turnover, while the cortex makes up to only 20% of the turnover (Christiansen, Hassager & Riis 1998).
In normal young adults, coupling between bone resorption and formation results in metabolic balance. However, uncoupling between formation and resorption will result either in negative or positive bone balance at the remodelling site (Recker 1992). If bone turnover is high, the minute changes observed in the bone remodelling sites are amplified (Monier-Faugere, Chris Langub & Malluche 1998). For example in the case of negative bone balance, bone loss is greater in individuals with high bone turnover.
Assessment of bone microarchitecture
Cortical and cancellous bone architectures both make an important contribution to bone strength (Koch 1917, Skedros, Baucom 2007). Changes of cortical width and porosity with age may increase the skeletal fragility and the risk of fractures (Schaffler, Choi & Milgrom 1995, McCalden et al. 1993). In cancellous bone, the size and shape of trabeculae, and their connectivity and orientation may contribute to stress transfer and skeletal stiffness (van der Linden et al. 2001, Aaron et al. 2000).
Bone architectural features can be assessed with three- and two- dimensional approaches (Compston 2006). The former includes, e.g. high-resolution micro-computed tomography (HR-CT) and high-resolution magnetic resonance imaging (HR-MRI) while the latter refers to histological sections of bone biopsy analyses.
Assessment of microdamage
Microdamage in bone consists of microcracks and microfractures. However, the relationship between them is unknown (Burr, Radin 2003, Taylor, Lee 2003). At the microscopic level, microcracks represent the physical separation of bone matrix which grow longitudinally along the lamellar interfaces of the bone (Boyce et al. 1998). Their density increases with aging (Frank et al. 2002) and their variation manifests either impact loading or longer-term fatigue-related processes (Dai et al. 2004). Since microcracks frequently arise from osteocyte lacunae under fatigue loading (Reilly 2000), both osteocyte density and microcrack length have been considered to be a potential determinant of bone quality (Ma et al. 2008). During aging microcracks accumulate and they are commonly observed in the cancellous bone of healthy individuals (Cheng et al. 1997). Compared with vertebrae, healing of microfractures is less common at sites typical to hip fracture, such as the femoral neck (Vernon-Roberts, Pirie 1973). Therefore, microfractures may increase the bone brittleness and fragility fracture risk (Burr, Radin 2003, Qiu et al. 2005).
Assessment of microdamage is predominantly carried out by applying histological techniques (Burr, Hooser 1995, Huja et al. 1999), e.g. en bloc staining and fluorescence microscopy.
2.2.2 Bone histomorphometry
Histological examination of undecalcified transilial bone biopsy specimens provides quantitative information on bone remodelling and microarchitecture. It is considered to be an ideal tool for assessing the quality of bone, an increasingly important issue (Recker 1994).
The aim of bone histomorphometry is to assign numerical values to the various structural elements constituting bone in order to quantitatively evaluate potential differences between groups of patients, healthy individuals, or changes occurring after certain treatments (Parfitt 1983, Dempster, Shane, 2001). In the microscope, the bone section appears as a two- dimensional image consisting of profiles of bone tissue, bone cells, and bone marrow (Compston 2005). The primary measurements include parameters related to length, width, and area. Based on the stereological principles on the relationships between the two- dimensional and the three-dimensional parameters, the length, width, and area are transformed to surface, thickness, and volume, respectively (Dempster et al. 2013).
Bone biopsy
Bone biopsy is the basis for the histomorphometric analysis. At the present time, bone biopsy represents the most informative diagnostic tool and a significant research method in metabolic bone diseases (Weinsten 2008). Bone biopsies are used to measure bone quality, in particular the degree of mineralization and microarchitectural derangements, to assess bone turnover and bone balance, and to analyze the effects of treatment on bone structure and bone turnover (Kulak, Dempster 2010).
In order to perform a reliable analysis bone samples with adequate size should include both cortical and cancellous bone (Monier-Faugere, Chris Langub & Malluche 1998).
Traditionally, the iliac crest has been selected as the biopsy specimen since this site is easily accessible and is associated with fewer postoperative complications (Podenphant et al.
1986). The biopsy is performed as an outpatient minor surgery. It is safe and generally well tolerated (Wand et al. 1992). Bone biopsy can be obtained in a vertical or horizontal direction. The former approach allows assessment of the subcortical cancellous bone and the deep cancellous bone without size restrictions. The latter provides information on the external and internal cortices, although the sample size is limited by the thickness of the iliac bone (Monier-Faugere, Chris Langub & Malluche 1998).
Fracture related skeletal sites (e.g. the proximal femur) have also been investigated. The bone distribution in the proximal femur acts as a significant determinant of fracture risk (Recker 1994). For example, it has been suggested that, in cases the femoral neck fracture, the predominant feature is a localized thinning of the cortical bone and increased cortical porosity (Johannesdottir et al. 2013). Thus, the biopsy of related sites may aid to better understand the mechanism of hip fractures.
Sample preparation
After the specimen is obtained, the biopsy is fixed in the ethanol for 24-48 hours depending on the sample size. The fixation aims to inhibit postmortem changes without removing the mineral from bone (Recker 1994). Then the sample is dehydrated thoroughly with absolute ethanol, since all plastic monomers commonly used in embedding are not miscible with water. Further, the sample is embedded in methylmethacrylate (MMA), the final hardness of which approximates to the hardness of bone (Trueba et al. 2003). These embedding mediums are nonflammable, suitable in hardness for cutting, and also available for dissolution after cutting. After embedding, a microtome is used to cut the sample and various cutting fluids are used to keep the block moist (Monier-Faugere, Chris Langub &
Malluche 1998). Then, the sections are typically further stained. The most frequently used stains include the modified Masson-Goldner trichrome stain, the solochrome cyanine stain, and von Kossa's stain (Goldner 1938). Different stains are chosen for different purposes in discriminating the calcified bone from uncalcified matrix (Rentsch et al. 2014). Unstained sections are used for polarization and fluorescence microscopy.
Quantitative histological analysis
Currently, the procedures are carried out by semiautomatic measurements and computerized-assisted histomorphometric analysis. Completely automatic measurements
are limited to discrimination of cellular details, detection of woven versus lamellar bone, and recognition of erosion surfaces (Monier-Faugere, Chris Langub & Malluche 1998). Thus, the fully automatic assessment of bone structure might not be possible until video cameras, staining techniques, and computerized image-analysis capabilities are innovatively improved.
In cancellous bone analysis, the determination of bone architecture should be done at low magnification (e.g. x20) in order to include the greatest possible number of trabeculae, whereas specific details, e.g. bone cell count, should be performed using high magnification (e.g. x200). In each desired field, the operator characterizes the bone structure with different types of measurements such as bone volume, osteoid surface, erosion surface, osteoblast interface, osteoclast interface, cell counts and tetracycline labeling (Table 1). Various measurements are represented by different colors to allow verification. Related parameters are automatically computed by the software that implement principles of stereology (Dempster et al. 2013).
Cortical bone histomorphometry is traditionally less used than the histomorphometric analysis of cancellous bone, especially in sites other than iliac crest (Tsangari, Findlay &
Fazzalari 2007, Vedi, Kaptoge & Compston 2011). However, the recent interest in the mechanical properties (i.e. strength, elasticity, geometry) of cortical bone, e.g. when assessing fracture risk, has stimulated evaluation of morphometric changes in cortical bone both before and after a fragility fracture (Koivumaki et al. 2012, Rho et al. 2002). In practice, the cortical component should be primarily separated from the endocortical and cancellous bone to enable measurements of the macro-structural parameters (e.g. area, width, and perimeter) under low magnification (e.g. x50). Then the cortical bone is evaluated under the bright light and polarization microscopy using higher magnification (e.g. x100) in order to characterize the microarchitecture including osteons, microcracks, and cortical pores.
Table 1. Frequently used cancellous bone histomorphometry parameters and definition.
Cancellous Parameters Definition
Structural Parameters (Units)
Bone volume fraction BV/TV (%) Percent of marrow space occupied by mineralized and unmineralized bone Trabecular thickness Tb.Th (μm) Average thickness of trabeculae Trabecular number Tb.N (N/mm) Number of trabecular silhouettes Trabecular separation Tb.Sp (μm) Average distance between trabeculae Static Formation Parameters (Units)
Osteoid volume fraction OV/BV (%) Percent of bone volume consisting of unmineralized bone, i.e., osteoid
Osteoid surface OS/BS (%) Percent of bone surface covered by osteoid Osteoid thickness O.Th (μm) Average thickness of osteoid seams
Osteoblast surface Ob.S/BS (%) Percent of bone surface ocupied by osteoblasts Wall thickness W.Th (μm) Average thickness of bone tissue that has been
deposited at a remodelling site Static Erosion Parameters (Units)
Eroded surface ES/BS (%) Percent of bone surface presenting resoption surface Osteoclast surface Oc.S/BS (%) Percent of bone surface ocupied by osteoclasts Dynamic Formation Parameters (Units)
Mineralizing surface MS/BS (%) Percent of bone surface presenting mineralizing activity Mineralizing surface MS/OS (%) Percent of osteoid surface mineralizing
Mineral apposition rate MAR (μm/d) Distance between the two tetracycline courses divided by the length of the labeling interval
Mineralization lag time Mlt (d) Time interval between deposition and mineralization of bone matrix
Bone formation rate BFR/BS (μm3/
μm2/year) Amount of bone formed per year on a given bone surface Activation frequency Ac.F (N/year) Frequency of appearance of new remodelling units at one
location per year
2.2.3 Scanning acoustic microscopy
Quantitative scanning acoustic microscopy (SAM) images the acoustic impedance of inhomogeneous materials with a spatial resolution comparable to that of other micro imaging modalities. The liquid couplant (e.g. water) transmits the acoustic waves from the ultrasound transducer to the specimen (Hube et al. 2006). Both fresh and fixed specimens can be studied by SAM. Besides superficial properties, SAM is able to measure the internal and subsurface structures including optically opaque materials (Bumrerraj, Katz 2001).
Very commonly, the amplitude of the reflected ultrasound pulse is measured with SAM to quantitate the information of material density and elastic properties in the probing direction (Lakshmanan, Bodi & Raum 2007). The tissue elastic coefficient can be estimated from the acoustic impedance measurements (Preininger et al. 2011). Because the age- dependent variation in tissue mechanical properties (e.g. elasticity) and microarchitecture (e.g. cortical porosity) directly influences the tissue acoustic properties (Hofmann et al. 2006, Rohrbach et al. 2012), SAM has been used for assessing the spatial variation in elastic properties and bone structure over the complete cross-sectional samples (Katz, Meunier 1993).
2.3 OSTEOPOROSIS
Osteoporosis is a systemic skeletal disorder characterized by low bone mineral density (BMD) and the microarchitectural deterioration of bone tissue. Consequently, ostoporosis results in fractures occurring in the absence of trauma or in response to only low force trauma (force equal to or less than a fall from a standing height) (NIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis, and Therapy 2000). Fragility fractures, in particular hip fractures, are a significant cause of morbidity and mortality in the elderly. They also produce a growing economic burden on health-service resources all over the world (Raisz, Seeman 2001, Cooper, Melton 1992). In most cases fragility fractures are preceded by a long and silent period during which the bones become progressively more fragile (Greenspan et al. 2012).
2.3.1 Pathogenesis
The precise pathogenesis of the accelerated bone loss is unknown (Ito et al. 2011). It has been suggested that bone loss is related to the cessation of gonadal steroid production.
Whenever there is a decline in gonadal steroids (estrogen in women and androgen in men) as a result of a disease or therapy, there is a transient, approximately 5-7 years long period of increased bone remodelling and accelerated bone loss. However, it is unlikely that estrogen deficiency has direct effects on the skeleton. There is compelling evidence that estrogen modulates the local production of cytokines, IL-1, and IL-6, which increase bone resorption (Roodman 1993, Horowitz 1993, Abrahamsen et al. 2000). It is not yet known whether these may act directly on the skeleton or mature bone cells.
Although estrogen status and heredity are major determinants of premenopausal bone loss, very little is known about the cellular mechanisms underlying age-related bone loss (Khosla 2013). In women, bone loss may begin before there is any decline in ovarian estrogen production, while in men it may also precede the age-related decline in androgen production (Kleerekoper, Avioli 1998). Since the process of age-related bone loss is slow while the physiological changes are subtle, it is difficult to formulate and substantiate any single hypothesis to account for the age-related bone loss. As suggested by different studies, the age-related decline in renal production of calcitriol, elevation in PTH, negative skeletal balance, and possibly other factors may together lead to osteoporosis (Khosla 2013). A relatively tenable hypothesis is that late consequences of estrogen deficiency rather than age-related processes, are the principal causes of the secondary hyperparathyroidism and increased bone resorption in postmenopausal women (McKane et al. 1997, Lindsay et al.
