pediatric bone biology & diseases (2/e): part 2

C H A P T E R<br /> <br /> 16<br /> <br /> Pediatric Bone Histomorphometry<br /> Frank Rauch<br /> Shriners Hospital for Children, Montreal, Quebec, Canada<br /> <br /> INTRODUCTION<br /> Bone biopsies can be useful for establishing a diagnosis in an individual patient with a bone disorder.<br /> They can also be used for investigating disease characteristics or treatment effects. Biopsy samples can be<br /> assessed qualitatively or quantitatively. The quantitative analysis of bone specimens is called bone<br /> histomorphometry.<br /> Bone histomorphometry is a key tool for studying<br /> bone tissue. Both the activity of bone metabolism and<br /> the amount and distribution of bone tissue can be<br /> analyzed with unsurpassed resolution. When tetracycline labeling is performed prior to biopsy, bone histomorphometry offers the unique possibility to study<br /> bone cell function in vivo. Importantly for pediatric<br /> use, the growth process does not directly interfere with<br /> the measurements.<br /> Bone histomorphometry is also an excellent educational tool. The insight derived from studying bone<br /> tissue can be used better to understand results of indirect methods, such as bone densitometry or biochemical<br /> markers of bone metabolism. Knowledge of bone tissue<br /> is crucial to put the disparate findings of molecular and<br /> cellular studies into perspective.<br /> Despite these advantages, bone histomorphometry is<br /> underused in pediatrics. This may be partly due to the<br /> fact that histomorphometry requires an invasive procedure to obtain a bone sample, is labor intensive, and<br /> needs special equipment and expertise. Other reasons<br /> may include overestimation of the utility of non-invasive bone diagnostics and lack of information about<br /> what bone histomorphometry does.<br /> Bone tissue is very hard and for that reason is more<br /> difficult to process than soft tissues. In routine<br /> pathology, bone tissue is therefore usually decalcified<br /> and thus converted into a soft tissue. However, this<br /> leads to the loss of important information about bone<br /> mineralization and bone cell activity. To assess metabolic<br /> <br /> Pediatric Bone, Second Edition DOI: 10.1016/B978-0-12-382040-2.10016-4<br /> <br /> bone disorders, it is therefore generally more informative to analyse samples undecalcified.<br /> This chapter summarizes the methodology of bone<br /> histomorphometry and highlights the tissue-level characteristics of normal and abnormal bone development.<br /> The aim is to open this field to the non-specialized<br /> reader with an interest in pediatric bone disorders.<br /> More detailed accounts of methodology can be found<br /> elsewhere [1,2].<br /> <br /> BASIC CONCEPTS<br /> Between birth and adulthood, bones undergo considerable increases in size. The most frequently assessed<br /> aspect of this process is longitudinal bone growth, as<br /> reflected by body height. The increase in bone length is<br /> mostly due a mechanism called endochondral ossification [3]. The primary effector cells of this process are<br /> growth plate chondrocytes. These cells continuously<br /> divide and synthesize a cartilaginous matrix which, in<br /> a stepwise process, is subsequently converted into<br /> osseous tissue. Longitudinal bone growth and endochondral ossification are a traditional focus of interest in pediatric research. However, histomorphometry does not<br /> usually deal with endochondral ossification and therefore this process is not described in more detail here.<br /> Bone histomorphometry mostly provides information on two other aspects of bone development, bone<br /> remodeling and bone modeling. These tissue-based<br /> mechanisms of bone development and maintenance<br /> have received far less attention in pediatrics than longitudinal bone growth. Bone created by endochondral<br /> ossification is continuously renewed by a process named<br /> remodeling [4]. Remodeling consists of successive cycles<br /> of bone resorption and formation on the same bone<br /> surface. The basic features of this process are identical<br /> for trabecular and cortical bone [4]. A group of osteoclasts removes a small quantity (“packet”) of bone tissue<br /> <br /> 383<br /> <br /> Copyright Ó 2012 Elsevier Inc. All rights reserved.<br /> <br /> 384<br /> <br /> 16. PEDIATRIC BONE HISTOMORPHOMETRY<br /> <br /> which, after a reversal phase, is replaced by a team of<br /> osteoblasts. The entire group of cells involved in this<br /> process is named remodeling unit or basic multicellular<br /> unit. The fact that osteoblast activity is linked to<br /> previous osteoclast action has been named “coupling”<br /> [4]. The difference in the amounts of bone which are<br /> removed and added in one remodeling cycle is called<br /> “remodeling balance”. The remodeling balance is typically close to zero so that there is no or little net effect<br /> on the amount of bone. However, the remodeling<br /> process renews the bone tissue and thereby prevents<br /> tissue damage from accumulating [5].<br /> Bone growth in width occurs through a different<br /> mechanism, called modeling [4]. Bone modeling<br /> involves the same set of effector cells as bone remodeling, osteoclasts and osteoblasts. However, while in<br /> remodeling both cells types are sequentially active on<br /> the same bone surface, osteoclasts and osteoblasts act<br /> on different surfaces during modeling. During bone<br /> growth in width, osteoblasts are typically located on<br /> the outer (periosteal) surface of a bone cortex, where<br /> they deposit bone matrix and later mineralize it.<br /> Thereby, the outer circumference of a long bone or<br /> a vertebral body is increased. At the same time, osteoclasts located on the inner (endocortical) surface of the<br /> cortex resorb bone, thus increasing the size of the<br /> marrow cavity. Since osteoblasts are active without<br /> interruption in bone modeling, much more rapid<br /> increases in the amount of bone tissue can occur than<br /> in bone remodeling. Osteoclasts usually remove less<br /> bone tissue than is deposited by osteoblasts during<br /> modeling [4]. Therefore, modeling leads to a net increase<br /> in the amount of bone tissue. For example, the difference<br /> between osteoblastic matrix deposition and osteoclastic<br /> bone resorption leads to cortical thickening. Modeling<br /> and remodeling are not just abstract concepts of bone<br /> metabolism, but are reflected in the histoanatomy of<br /> cortical bone.<br /> <br /> that the transiliac sample must be obtained under standardized conditions and with appropriate tools. It is<br /> essential that the sample is not fractured or crushed<br /> and contains two cortices separated by a trabecular<br /> compartment. These requirements are often quite difficult to meet in small or very osteopenic children.<br /> Bone specimens for histomorphometric evaluation<br /> are horizontal, full-thickness (transfixing) biopsies of<br /> the ilium from a site 2 cm posterior from the anterior<br /> superior iliac spine (Fig. 16.1). This bone is easily accessible, does not require extensive surgery, and is associated with few postoperative complications. Also, this is<br /> the only site for which pediatric histomorphometric<br /> reference data have been published [6]. It is important<br /> to note that horizontal transiliac samples are required<br /> for histomorphometric evaluation. Vertical samples<br /> (from the iliac crest downwards) cannot be used<br /> because of the presence of the growth plate. The transiliac sample must be obtained at a site well below the<br /> iliac crest growth plate. Specimens containing growth<br /> cartilage do not allow for a reliable quantitative analysis because turnover is very high and cortical thickness is very low in the bone adjacent to the growth<br /> plate.<br /> The usual bone biopsy instrument is the Bordier<br /> trephine (Fig. 16.2). The inner diameter of the trochar<br /> should be at least 5 mm. We are using 5 mm needles in<br /> children below 12 years of age and 6 mm needles in children 12 years or older, unless their height is below the<br /> third percentile.<br /> In children and adolescents, the biopsy procedure is<br /> usually performed under general anesthesia. This procedure does not have side effects other than transient local<br /> discomfort [7]. Patients are allowed to get out of bed<br /> after 3 hours and can usually be discharged on the<br /> same day. The operator’s experience is an important<br /> factor in obtaining an adequate sample and in keeping<br /> intervention-related morbidity to a minimum.<br /> <br /> METHODOLOGY<br /> Bone Biopsy<br /> Clinical Procedure<br /> Bone histomorphometry was first developed to study<br /> rib bone samples. This was soon abandoned because the<br /> ilium proved to be a much more convenient site for<br /> obtaining bone samples. In principle, histomorphometric analysis can be performed in any bone. In clinical<br /> pediatrics, however, the utility of samples from sites<br /> other than the ilium is limited because reference data<br /> are only available for the ilium.<br /> Quantitative bone histomorphometry requires an<br /> intact biopsy specimen of good quality. This implies<br /> <br /> FIGURE 16.1<br /> procedure.<br /> <br /> PEDIATRIC BONE<br /> <br /> Anatomic location for the transiliac bone biopsy<br /> <br /> 385<br /> <br /> METHODOLOGY<br /> <br /> FIGURE 16.2<br /> <br /> View of a 5-mm trephine for transiliac bone biopsy.<br /> <br /> Full histomorphometric analysis requires prior in<br /> vivo bone labeling. Dynamic parameters of bone cell<br /> function can only be measured when the patient has<br /> received two courses of tetracycline label prior to biopsy.<br /> Tetracycline compounds form calcium chelate<br /> complexes that bind to bone surfaces. These complexes<br /> are buried within the bone at sites of active bone formation, whereas they redissociate from the other bone<br /> surfaces once serum tetracycline levels decrease. The<br /> tetracycline trapped at formation sites can then be visualized under fluorescent light.<br /> Figure 16.3 shows the labeling schedule. The tetracycline compound used is tetracycline-HCl (brand names<br /> differ from country to country, e.g. TetracyclineÒ;<br /> SumycinÒ, AchromycinÒ, and several other brand<br /> names) at a dose of 20 mg/kg body weight per day,<br /> <br /> FIGURE 16.3 Schedule for prebiopsy tetracycline labeling.<br /> <br /> PEDIATRIC BONE<br /> <br /> 386<br /> <br /> 16. PEDIATRIC BONE HISTOMORPHOMETRY<br /> <br /> with a maximum dosage of 1500 mg per day. The daily<br /> amount is given orally in two doses. The drug is given<br /> for two days in both label courses. The two courses are<br /> separated by an interlabel time of 10 days. Bone biopsy<br /> is performed 4e6 days after the last administration of<br /> tetracycline.<br /> Although children and adolescents generally tolerate<br /> tetracycline double labeling well, some side effects<br /> might be observed, such as allergic reactions, vomiting,<br /> and photosensitivity. Administering the drug after<br /> meals can diminish gastrointestinal side effects. It is<br /> important that these meals do not include milk or other<br /> dairy products because tetracycline complexes with<br /> calcium contained in the food and is not absorbed<br /> adequately. Sun exposure must be avoided while taking<br /> tetracyclines. Tetracycline use is generally not recommended for children younger than 9 years of age<br /> because discoloration of teeth may occur. However, the<br /> previously mentioned schedule appears to be safe in<br /> this respect. At the Montreal Shriners Hospital, it has<br /> been used for more than 300 biopsies in children<br /> younger than 9 years of age and tooth discoloration<br /> has never been observed.<br /> Sample Processing<br /> The biopsy sample should be placed into a fixative as<br /> soon as possible after the procedure. The fixation<br /> process aims at the preservation of bone tissue constituents by inactivating lysosomal enzymes. The choice of<br /> fixative and temperature at which the sample should<br /> be kept depends on the planned staining techniques.<br /> For routine histomorphometry, 70% ethanol or 10% buffered formalin at room temperature can be used. The<br /> duration of fixation should be at least 48 hours but<br /> should not exceed 10 days because the tetracycline<br /> labels are washed out when fixation is too long.<br /> Following fixation, the specimen is dehydrated in absolute ethanol and embedded in methylmethacrylate.<br /> Cutting mineralized bone requires a special microtome. For each specimen, two to five series of undecalcified, 6e10-mm-thick consecutive sections should be cut<br /> at least 150 mm apart. The sections are then deplastified<br /> to allow for optimal staining. The most widely used<br /> staining methods for histomorphometric analysis are<br /> toluidine blue and Masson Goldner trichrome. The<br /> sections that will be used for fluorescence microscopy<br /> are mounted unstained. An appropriately large sample<br /> area must be available to obtain representative<br /> measures. Therefore, at least two sections of a biopsy<br /> should be available for each type of analysis in order<br /> to obtain a measurable tissue area of 40e50 mm2.<br /> Measurement Procedure<br /> The actual histomorphometric analysis requires<br /> a high-quality microscope that is suitable for fluorescence<br /> <br /> microscopy. In the early years of the technique, histomorphometric measurements were performed by manual or<br /> point-counting techniques. These methods involved the<br /> use of a grid placed in the microscope eyepiece. This<br /> has been replaced by computerized systems that allow<br /> for automation of the analysis process. Whatever method<br /> is used, histomorphometric analysis is time consuming<br /> because even the most advanced systems rely on the<br /> operator’s judgment to identify correctly the individual<br /> histoanatomical components.<br /> <br /> HISTOMORPHOMETRIC MEASURES<br /> Definitions<br /> Histomorphometric measures follow a standardized<br /> and well-defined terminology that was introduced in<br /> 1987 [8]. Articles published before that time are often<br /> quite difficult to read because many authors used<br /> private nomenclature. An introductory overview of the<br /> most important histomorphometric terms is given<br /> here. More detailed information can be found in the<br /> 1987 terminology report [8].<br /> In histomorphometry, bone is defined as bone matrix,<br /> whether mineralized or not. Unmineralized bone is<br /> called osteoid, whereas mineralized bone does not<br /> have a special designation. Bone and the associated<br /> soft tissue, such as bone marrow, are referred to as<br /> tissue. Osteoblasts are cells on bone surfaces that are<br /> producing and secreting bone matrix currently. Flat cells<br /> of the osteoblast lineage that cover quiescent nonperiosteal bone surfaces are referred to as lining cells.<br /> The term osteoclast is restricted to multinucleated cells<br /> that are currently in contact with a bone surface and<br /> are actively resorbing bone.<br /> A transiliac biopsy specimen consists of two cortices<br /> separated by a cancellous compartment (Fig. 16.4). The<br /> terms cancellous and trabecular are usually used interchangeably. The outer delimitation of the cortex is called<br /> the periosteal surface, and the inner border is the endocortical surface (Fig. 16.4). Osteonal and Volkmann<br /> canals are lined with intracortical surfaces. The bone<br /> surfaces in the cancellous compartment are referred to<br /> as cancellous (or trabecular) surfaces. Intracortical,<br /> endocortical, and trabecular surfaces are in continuity<br /> and together form the endosteal surface or envelop. In<br /> the literature, there is confusion regarding the latter<br /> term because many authors use the term endosteal<br /> surface when in fact referring to the endocortical<br /> surface.<br /> Histomorphometric measurements are performed in<br /> two-dimensional sections. This may cause conceptual<br /> problems because bone is a three-dimensional organ.<br /> What is perceived and measured as an area in the<br /> <br /> PEDIATRIC BONE<br /> <br /> 387<br /> <br /> HISTOMORPHOMETRIC MEASURES<br /> <br /> FIGURE 16.4<br /> <br /> Schematic representation of<br /> a bone biopsy section. The different types of<br /> bone surfaces are indicated.<br /> <br /> histologic section in fact reflects a volume. In order to<br /> highlight the three-dimensional nature of bone, threedimensional terminology is favored when reporting histomorphometric results. For example, the percentage of<br /> unmineralized bone is measured in the two-dimensional<br /> bone slice as osteoid area relative to total bone area.<br /> However, the result of this ratio is reported as osteoid<br /> volume per bone volume. This is done simply by<br /> convention, and it should not be mistaken as an actual<br /> three-dimensional measurement.<br /> <br /> Histomorphometric Parameters<br /> Terminology for most histomorphometric parameters<br /> follows a standardized scheme: source e measurement/<br /> referent. Source refers to the type of bone that is<br /> measured (e.g. cancellous or cortical). Since analyses<br /> are often limited to cancellous bone, the source prefix<br /> is usually omitted, as long as there is no possibility of<br /> confusion. Measurement is the type of parameter that<br /> is determined. Histomorphometric data are usually not<br /> given as absolute values but are related to each other.<br /> This is what is meant by “referent”. Most parameters<br /> are related to a surface area or a volume.<br /> Histomorphometric parameters can be classified<br /> into four categories (Table 16.1): structural parameters,<br /> static bone formation parameters, dynamic formation<br /> parameters, and static bone resorption parameters.<br /> Dynamic parameters can only be determined when<br /> tetracycline labeling is performed prior to obtaining<br /> the biopsy. There are no dynamic parameters of bone<br /> resorption, which is one of the main shortcomings of<br /> histomorphometry.<br /> Structural Parameters<br /> The overall size of an intact biopsy specimen is<br /> expressed as core width (C.Wi) (Fig. 16.5), which is the<br /> <br /> mean distance between the two periosteal surfaces of<br /> the sample. C.Wi thus reflects the thickness of the ilium,<br /> but also depends on the angle between biopsy needle<br /> and ileal surface. Ideally, the needle should be perpendicular to the ilium, but this is not always easy to<br /> achieve.<br /> Cortical width (Ct.Wi) is determined as the mean<br /> distance between the periosteal and endocortical<br /> surfaces of each cortex. Usually, results from both<br /> cortices are combined. Determination of Ct.Wi is not as<br /> straightforward as the widespread use of this parameter<br /> in radiological techniques might suggest. Indeed, there<br /> is often a smooth transition from cortical to cancellous<br /> bone, and two observers may disagree where the border<br /> between the two compartments should be drawn.<br /> Bone volume per tissue volume (BV/TV) of trabecular bone is the combined volume of mineralized and<br /> unmineralized bone matrix relative to the total volume<br /> of the trabecular compartment (see Fig. 16.5). BV/TV<br /> can also be measured in cortical bone, but many authors<br /> prefer to express cortical results as cortical porosity<br /> (Ct.Po), which is simply the complement of BV/TV in<br /> cortical bone (Ct.Po ¼ 100%  BV/TV). The two<br /> surface-to-volume ratios (BS/BV and BS/TV) are important for establishing the link between bone surfacebased cellular activity and the effect on the amount of<br /> bone. For example, the differences in turnover of cortical<br /> and cancellous bone mostly reflect differences in the<br /> bone surface-to-volume ratio, whereas the surfacerelated activity of bone cells is quite similar [9].<br /> In trabecular bone, BV/TV can be schematically<br /> divided into two separate components: mean trabecular<br /> thickness (Tb.Th) and trabecular number (Tb.N). Tb.N is<br /> equivalent to the number of trabeculae that a line<br /> through the cancellous compartment would contact<br /> per millimeter length. The mean distance between two<br /> trabeculae, trabecular spacing (Tb.Sp) or trabecular<br /> <br /> PEDIATRIC BONE<br /> <br />