Bone Histology and Histopathology for Clinicians A Primer

Bone Histology and Histopathology for Clinicians A Primer Stephen F. Hodgson M. D. , M. A. C. E, F. A. C. P. Bart L. Clarke M. D. , F. A. C. E. , F. A. C. P. Robert Wermers M. D. , F. A. C. E. Theresa Hefferan, Ph. D. Michael Yaszemski. M. D. , Ph. D.

Presented By Mayo Clinic Divisions of Endocrinology and Orthopedic Research and The American College of Endocrinology

Supported by an Educational Grant from: The American College of Endocrinology

The authors acknowledge the many valuable contributions of others • • • Julie Burgess Glenda Evans Dr. Lorraine Fitzpatrick Dr. Hunter Heath Dr. Dan Hurley Donna Jewison Dr. Ann Kearns Dr. Kurt Kennel Dr. Sundeep Khosla • • • Dr. Rajiv Kumar Dr. James Mc. Carthy Dr. B. L. Riggs Dr. Jean Sibonga Peter Steiner, Illustration & Design • Dr. Peter Tebben • Dr. Robert Tiegs • Dr. Russell Turner

Bone Histology and Histopathology for Clinicians© This presentation provides basic instruction in bone histology, and in the histopathology of metabolic bone diseases and related disorders. It was prepared primarily for endocrine fellows, endocrinologists, osteologists, and other physicians and scientists interested in metabolic and related bone diseases. © 2007 Mayo Foundation for Medical Education and Research and licensed to The American College of Endocrinology.

Part I • Transiliac Bone Biopsy • Normal Bone Microanatomy and Histology • Bone Cells and Bone Remodeling • Basic Bone Histomorphometry

Transiliac Bone Biopsy Technique Play Skip

Normal Bone Microanatomy Cortical bone Cancellous bone From Gray’s Anatomy All bones of the human skeleton, though widely variable in function and shape, share a common anatomic organization. Grossly, they are composed of dense outer cortical bone which encloses an irregular medullary space containing cancellous bone, bone that is composed of branching networks of interconnecting bony trabecular elements.

Normal Bone Microanatomy Cortical bone Cancellous bone Undecalcified transiliac bone biopsies (right) are considered to be representative of all skeletal bone and are suitable for examining, measuring, and analyzing the microscopic features of cortical and cancellous bone. Also, with the appropriate use of absorbable fluorochrome agents, the dynamic changes that occur in bone can be assessed.

Normal Bone Microanatomy Variable cortical thickness Trabecular orientation differs Note that cortical thickness varies within individual bones. Also, note that trabeculae in the vertebral body are oriented vertically along lines of mechanical stress, whereas in the ilium they appear to be randomly oriented and are therefore said to be isotropic.

Anatomic Features of a Normal Transiliac Bone Biopsy Cortex Hematopoietic and fatty marrow 7. 5 mm Trabeculae

Normal Bone Microanatomy Differential Tissue Stains Mineralized bone Unmineralized bone A number of differential stains can be used to examine undecalcified tissue. Toluidine Blue stain (left) and Goldner Trichrome stain (right) will be used throughout this presentation, except as otherwise indicated. Each stain has characteristics that favor, or disfavor, its use. Either may be used for histomorphometric analysis.

Normal Bone Microanatomy and Histology Cortical Bone Cortical bone forms a relatively thick and dense outer wall and makes up about 80% of total skeletal mass.

Normal Bone Microanatomy and Histology Periosteum The outer cortical surface is enveloped in the periosteum, periosteum a connective tissue covering that contains cells that maintain, change, and repair the external cortical surface.

Normal Bone Microanatomy and Histology Periosteum The periosteum also contains blood vessels, sensory nerves, and dense fibrous tissue that is contiguous with the connective tissue elements of tendons, ligaments, and joint capsules Cortex Periosteum

Normal Bone Microanatomy and Histology Bone Structural Units (BSU) Both cortical and trabecular bones are composed of an assembly of individual bone structural units (BSU), also called osteons, each of which represents the structural end result of a focus of bone renewal (remodeling). Architecturally, cortical and trabecular BSUs are distinct. In cortical bone (left), BSUs may appear in cross section as concentric rings (lamellae), forming cylindrical - shaped structures. In cancellous bone (right), the lamellae are flat and appear stacked in saucer shaped depressions.

Normal Bone Microanatomy and Histology Cancellous Bone Cancellous bone accounts for the remaining 20% of skeletal mass and consists of interconnecting trabecular plates that share the medullary space with hematopoietic and fatty marrow.

Normal Bone Microanatomy and Histology Endosteum The inactive or resting trabecular surface is covered by a thin endosteum which, like the contiguous cortical endosteum, has widely spaced flat lining cells that are believed to have osteogenic potential, and form a barrier between marrow and bone. Lining cells Endosteum

Normal Bone Microanatomy and Histology Cortical BSUs are laminated bony cylinders (seen in cross section above) that have central (Haversian) canals enclosing vascular structures, nerves, and a thin membranous lining (cortical endosteum) containing flat, inactive appearing lining cells. Cortical BSUs arise from Haversian and other communicating channels called Volkman’s canals. They are about 0. 4 mm in width and are several mm in length. They are oriented in a branching pattern and lie perpendicular to the long access of bone. (Cortex above (left) under incandescent and (right) polarized light)

Cortical (Haversian) BSU Viewed in cross section under Polarized light

Trabecular BSUs are laminated saucer-shaped structures that, though appearing somewhat variable in two-dimensional view, contain a relatively uniform volume of bone, each BSU representing a “quantum” of bone.

Bone Cells and Bone Remodeling All normal adult human bone undergoes renewal and repair through a process called bone remodeling. Teams of bone resorbing and bone forming cells form basic multicellular units (BMU) that function at discrete sites throughout the skeleton in a highly coordinated sequence of cellular activity. At any given remodeling site, bone resorption always precedes bone formation, resulting in the removal and subsequent replacement of a quantum of bone at each site. Under normal steady state conditions, the amount of bone removed is precisely replaced and there is no net change in bone mass. Only bone architecture is changed.

Bone Remodeling Sequence of Bone Cell Activity The sequential events of the bone remodeling cycle are driven by an evolution of cellular events that occurs over a time period of three to six months: • Activation – a quiescent bone surface becomes populated with cells that have been recruited from osteoclast precursors and are destined to become bone resorbing osteoclasts • Resorption – osteoclasts mature and remove a finite quantum of mineralized bone • Reversal – osteoclast activity and numbers decline and are replaced by pre-osteoblasts (bone forming cell precursors) • Formation – preosteoblasts become mature osteoblasts and secrete bone matrix, which subsequently undergoes mineralization

Bone Remodeling Activation Lining cells produce collagenase, which exposes the mineralized bone surface for bone resorption

Bone Remodeling Resorption (Mineralized bone) Osteoclast Sealed micro-environment Ruffled membrane (Marrow) Cells derivedsurface from circulating mononuclear phagocyte are recruited The basal of the osteoclast is rich in HCl precursors and cathepsin transferto become boneand resorbing pre-osteoclasts, which cannot be visually identified by organelles is called the ruffled membrane. standard microscopy. Pre-osteoclasts mature to become osteoclasts and attach to the exposed mineralized bone surface, to form an isolated and sealed microenvironment that is rich in both HCl and lysozomal enzymes (cathepsin).

Bone Remodeling Resorption (Howship’s) lacunae Mature osteoclasts move over the surface, removing mineral and organic components of mature bone simultaneously, leaving serrated footprints, or Howship’s lacunae, on the surface

Osteoclasts Morphology Osteoclasts Acid Phos (+)osteoclasts Osteoclasts have variable morphology. Though often appearing as large multinuclear cells, they may be small, appear mononuclear, and, except for their characteristic location within resorption lacunae, can be difficult to distinguish from fibroblasts, osteoblasts, and other cells. Positive identification may be made using acid phosphatase stains (right).

Osteoclasts Variations

Osteoclasts Prevalence In normal bone (left), osteoclasts are encountered infrequently, only about three being identified per 100 mm of trabecular surface, and therefore may be absent from an entire section. Under some pathologic conditions (e. g. , above right) the number, size, and activity of osteoclasts may increase.

Biochemical Effects of Bone Remodeling Markers of Bone Resorption Osteoclastic resorption of mineralized bone releases minerals in support of mineral homeostasis, and products of collagenous protein degradation, including the inter- and intramolecular collagen cross links, into the circulation. The relative concentrations of cross links in blood or urine reflect the degree of bone resorbing activity and are considered to be “markers” of bone resorption.

Bone Remodeling Reversal Preosteoblasts As the resorptive phase wanes and is replaced by the reversal phase, resorption lacunae become populated by mononuclear pre-osteoblasts (cells that may be derived from recruited monocytes and circulating bone-forming cell precursors). Preosteoblasts are destined to become bone-forming osteoblasts. Osteoclasts ultimately undergo cell death, or apoptosis.

Reversal Pre-Osteoblast Maturation Preosteoblasts Osteoblasts Preosteoblasts (left) can be visually identified by their proximity to the resorption surface, clear cytoplasm, single nuclei, and (+)stain for alkaline phosphatase. They mature into osteoblasts (right), which appear as mononuclear cells with prominent nucleoli and deeply stained cytoplasm. Osteoblasts form a cellular monolayer on the resorption surface previously abandoned by osteoclasts.

Bone Remodeling Bone Formation Unmineralized osteoid Osteoblasts secrete type I collagen, called osteoid, from their basal surfaces onto the previously resorbed surface. Osteoid forms the organic matrix of bone.

Bone Formation Osteoblasts – Effects of Cell Age …but flatten as they complete bone formation to eventually become lining cells Young osteoblasts appear cuboidal and robust…

Type I Collagen Type I collagen is a triple helical structure composed of two α 1 chains and one α 2 chain. Collagen α chains are characterized by Gly-X-Y repeating triplets where X and Y are usually proline and hydroxyproline, respectively. Type I collagen is synthesized in a procollagen form which undergoes post-translational hydroxylation and glycosylation of selective residues. It further undergoes removal of terminal sequences before being secreted in its mature form from the basilar surface of osteoblasts into the underlying extra cellular space.

Bone Formation Osteoblasts Become Osteocytes Some osteoblasts become entrapped in osteoid to become osteocytes

Osteocytes As bone mineralizes, osteocytes tend to become pyknotic but retain metabolic responsiveness to PTH and other stimuli

Bone Formation Osteocytic Canaliculi Osteocytes retain communication with the surface and with other cells through a system of microtubules called canaliculi

Bone Formation Reversal Line Osteoblasts secrete collagen matrix directly on the resorption lacunar surface. The resulting scalloped interface between old bone and new matrix is called the Reversal, Reversal or Cement Line

Bone Formation Lamellar Bone Under normal conditions, collagen molecules establish covalent C-to-N cross links that result in both end-to-end and side-to-side alignment, forming mats of aligned and interconnected collagen molecules. Collagen mats periodically alternate their spatial orientation 90°, resulting in the layered or lamellar configuration seen in normal BSUs.

Bone Formation Woven Bone Under conditions of rapid turnover, e. g. , normal growth, fracture healing, or under some pathologic conditions as illustrated, osteoid is deposited in disorganized fashion and is called woven bone in contrast to lamellar bone. Woven bone Lamellar bone

Biochemical Effects of Bone Remodeling Markers of Bone Formation Osteoblasts secrete collagenous and noncollagenous proteins into circulation, including the C and N-terminal fragments of procollagen, alkaline phosphatase, and osteocalcin. Concentrations of these products in serum and urine serve as “markers” of bone formation and turnover.

Bone Formation Reversal Line The reversal line defines the limit of bone erosion and the original site of bone formation.

Bone Formation Reversal Line The persistence of a serrated interface indicates that mineral deposition has not begun at this location.

Mineralization of Osteoid The Mineralization Front Ten to fifteen days following secretion, osteoid undergoes maturational changes that prepare it for the initial deposition of calcium phosphate crystals. This occurs along an interface between mineralized and unmineralized bone, called the mineralization front.

Mineralization of Osteoid The Mineralization Front Mineralization front Reversal line As early mineralization proceeds, the serrated reversal line becomes obscured and the mineralization front becomes a smooth linear interface. When a flurorochrome labeling agent, such as tetracycline, tetracycline is present, it becomes incorporated into the mineralization front, leaving a clear linear record of the precise site where mineralization was occurring during tetracycline exposure.

Mineralization of Osteoid Fluorescent Labeling With Tetracycline: Fluorescence Microscopy Label #1 #2 Old Mineralized bone New mineralized bone Marrow Tetracycline is usually administered on two occasions separated by an interval of several days. The presence of well-resolved double labels indicates that normal bone mineralization was actively occurring over the labeling interval.

Mineralization of Osteoid Fluorescent Labeling With Tetracycline: Fluorescence Microscopy Single label Osteocytic lacunae and canaliculi The presence of a single label indicates that mineralization was occurring during only one labeling period. Note that osteocytic lacunae and canaliculi are visible under fluorescence.

Normal Iliac Bone Biopsy From a 33 -year-old Woman Recent double tetracycline labeling has resulted in multiple double- and single-fluorescent labels on the surfaces of trabeculae, marking the location of active bone mineralization.

Normal Iliac Bone Biopsy From a 33 -year-old Woman Fluorescent bands deep within mineralized trabeculae indicate previous incidental tetracycline exposure.

Cancellous Bone Remodeling The Bone Remodeling Compartment (BRC) BRC Between the BMU and bone marrow is a structure called the bone remodeling compartment (BRC).

Cancellous Bone Remodeling The Bone Remodeling Compartment (BRC) Lining cells The BRC is lined by sinusoidal vascular structures whose marrow interface is made up of lining cells that form a canopy over the remodeling site.

Cancellous Bone Remodeling The Bone Remodeling Compartment (BRC) BRC The BRC is thought to be a component of the BMU providing a local environment for regional cell signaling and the coordination of the coupling of formation to resorption.

Cancellous Bone Remodeling Though the remodeling cycle begins with osteoclastic resorption ands ends with osteoblastic formation and mineralization, osteoclasts and osteoblasts are otherwise simultaneously present in different regions of the same BMU during most of the active remodeling cycle.

Cancellous Bone Remodeling Sequence Early osteoblastic formation New mineralized bone Mineralization front Reversal line Osteoclastic resorption Lead by osteoclastic resorption, the BMU moves across the surface of cancellous bone. Resorption is succeeded by formation, which eventually becomes new mineralized bone.

Cancellous Bone Remodeling Sequence Osteoblasts flatten to ultimately form inactive lining cells.

Cancellous Bone Remodeling Space Increases The remodeling in remodeling space (RS)refers space (turnover) to that volume are associated of bone with that has an increasing undergone tendency resorptionfor orfracturing. will undergo It’sformation size is a limiting and mineralization, factor for and increasing which therefore bone does massnot with contribute drugs that to reduce mineralized turnover bone (eg. mass. The RS is Bisphonates) directly related to bone turnover, and represents the skeleton’s potential for increasing bone volume, mass, and strength.

Cancellous and Cortical Bone Remodeling Cancellous BMU Cortical BMU Cancellous bone remodeling (left) occurs over a trabecular surface, whereas cortical remodeling (right) occurs within a cylinder. Bone cell function and the sequence of cell activities are otherwise similar. Cancellous bone remodeling units occur in greater numbers, causing the cancellous bone turnover rate to be about tenfold that of cortical bone.

Cortical Bone Remodeling Osteoclasts Cutting cone Cortical BRUs originate from Haversian or Volkman’s canals, where osteoclasts excavate a resorption cavity called a cutting cone, which extends in a linear path through the cortex, forming a resorption tunnel.

Cortical Bone Remodeling Osteoblasts forming ostoid Closing cone Reversal zone Behind the advancing cutting cone is an irregular area somewhat devoid of active cells, the reversal zone, followed by an elongated tapering tunnel lined by osteoid and osteoblasts, which circumferentially refill the resorption tunnel, the closing cone.

Cortical Bone Remodeling Formation, Longitudinal, and Cross-Sectional Views #2 #1 Fluorochrome labeling with tetracyclene (right) documents the circumferential closure of a cortical BMU (osteon).

Cortical Bone Remodeling Completed Haversian canal Forming Haversian canal Bone formation eventually terminates, leaving a central Haversian canal which contains blood vessels, lymphatics, and connective tissue, elements that are contiguous with those of the periosteum, endosteum, and bone marrow.

Haversian Remodeling of Cortical Bone* Bone formation (osteoblasts) Resorption (osteoclast) Haversian canal Cutting cone Remodeled cortex

Cortical Bone Remodeling A complete cortical remodeling cycle requires from six to nine months. Circumferential closure at any point requires about three months.

Basic Bone Histomorphometry Stereologic Basis The measurement and analysis of bone structure and bone remodeling is called bone histomorphometry. It is usually performed on cancellous bone from transiliac biopsies. The isotropic (randomly oriented) nature of trabeculae in iliac bone is assumed, and allows two-dimensional measurements (area) to be converted to, and expressed as, three-dimensional (volume) measurements. This is a fundamental stereologic principle used in histomorphometry. Isotropy also implies that structures are viewed and measured at some random degree of obliquity. Therefore, a correction factor for obliquity (4/π) is used in all thickness measurements. * *For detailed discussion, see Recker, RR. Bone Histomorphometry: Techniques and Interpretation, CRC Press, 1983.

Basic Bone Histomorphometry Using computer graphics, multiple fields of known medullary area/volume are analyzed. Bone tissue volume (TV) is the sum of field volumes All trabeculae within each field are graphically outlined and trabecular bone volume (Tb. V), and total trabecular bone surface (Tb. S) are determined.

Basic Bone Histomorphometry Using computer graphics, multiple fields of known medullary area/volume are analyzed. Bone tissue volume (TV) is the sum of field volumes All trabeculae within each field are graphically outlined and trabecular bone volume (Tb. v), and total trabecular bone surface (Tb. S) are determined.

Bone Histomorphometry Trabecular Bone Volume (Tb. V) Trabecular bone volume, (Tb. V) Tb. V is the relative volume of total cancellous bone measured (TV), TV expressed as %, that is occupied by trabeculae. Tb. V is about 20% in women and 22% in men. Tb. Vis Tb. V related to cancellous bone mass. It declines with age and with bone loss

Bone Histomorphometry Trabecular Bone Volume (Tb. V) Note Tb. V is also commonly referred to as Bone Volume / Total Volume, or BV/TV

Bone Histomorphometry Surface Classification Total bone (trabecular) surfaces (Tb. S) are measured, subclassified by type, and each type of surface expressed as % of Tb. S, or as % of a specific surface type: Resorbing surface (RS) Osteoid surface (OS) Osteoblast surface(Ob. s) (as % osteoid surface)

Bone Histomorphometry Trabecular Separation (Tb. Sp) Trabecular Separation Tb. Sp is the mean distance in mm between trabeculae (measured by integrated computer graphics) Tb. Sp is a measure of trabecular connectivity Tb. Sp increases with trabecular bone loss

Bone Histomorphometry Trabecular Thickness (Tb. Th) Tb. Th = 1/Tb. S Mean trabecular thickness, (Tb. Th) Tb. Th is a measure of trabecular structure and is calculated as the reciprocal of Tb. S Tb. Th is reduced by aging and osteoporosis.

Bone Histomorphometry Trabecular Number (Tb. N) Trabecular number (Tb. N). Tb. N The number of trabeculae present per lineal mm Tb. N is calculated as Trabecular bone volume/Trabecular Thickness Tb. N is a measure of trabecular connectivity Tb. N decreases with bone loss

Bone Histomotphometry Cortical Thickness (Ct. Th) In the ileum, average combined cortical width (Ct. Th) Ct. Th in women and men is about 820 µm and 915 µm, respectively. Ct. Th correlates with dual photon absorptiometric (DPX) measurements of bone density.

Bone Histomorphometry Mineral Apposition Rate (MAR) MAR = Interlabel distance Label interval The average distance between visible labels, divided by the labeling interval, is the mineral apposition rate (MAR) MAR in µm/day, the avarage rate at which new bone mineral is being added on any actively forming surface. MAR is the basic measurement and calculation on which all dynamic estimates of bone formation are based. It is usually expressed as the adjusted appositional rate ((Aj. Ar) or MAR (MS/BS ) – see next slide

Bone Histomorphometry Mineralizing Surfaces (MS) Total mineralizing surfaces (MS) include all double and ½ of single-labeled surfaces. MS is expressed relative to total bone surface or, MS = total labeled surface / BS

Bone Histomorphometry Mineralizing Surface (MS) MS is used in the calculations for bone formation rates, (BFR), BFR activation frequency (Ac. F), Ac. F and mineralization lag time (MLT). MLT

Bone Histomorphometry Osteoid Thickness (O. Th) Osteoid thickness (O. Th) O. Th is the mean thickness, in µm, of osteoid seams on cancellous surfaces.

Bone Histomorphometry Osteoid Thickness (O. Th) O. Th is normally <12. 5 µm. Increased O. Th suggests abnormal mineralization (osteomalacia).

Bone Histomorphometry Mineralization Lag Time (MLT) Secretion Mineralization The time interval between osteoid secretion and its subsequent mineralization, in days, is known as the mineralization lag time (MLT). MLT

Bone Histomorphometry Mineralization Lag Time MLT is a measure of mineralization competence and is normally less than 22 days in women and 27 days in men.

Bone Histomorphometry Mineralization Lag Time (MLT) MLT is calculated as: O. Th MAR x MS Os

Bone Histomorphometry Activation: Activation Frequency (Ac. f) The average time that it takes for a new remodeling cycle to begin on any point on a cancellous surface is called the activation frequency (Ac. f). Ac. f is a measure of bone turnover and is expressed in years.

Bone Histomorphometry Wall Thickness (W. Th) W. Th= Average thickness of BSU Wall thickness is the average thickness of trabecular BSU. (W. Th) W. Th is used to assess the overall balance between resorption and formation.

Bone Histomorphometry Bone Formation Rates Bone formation rates (BFR/BV and BFR/BS) are the calculated rates at which cancellous bone surface and bone volume are being replaced annually. They are derived from estimates of: Mineral Appositional Rate (MAR), (interlabel distance (4/π) (labeling interval) in µm/Day x 365. Relative Mineralizing Surface (MS), Bone Surfaces (BS) or Bone Volume (BV) or BFR = MAR(MS/BS) BFR= MAR(MS/BV)

Bone Histomorphometry Bone Formation Rates Bone formation rates are expressed as: BFR/BV in (mm³/yr) BFR/BS in (mm³/mm²/yr) Alternatively, BFR/BS can be derived as: BFR/BS = Ac. f x W. Th

Bone Histomorphometry Normal Mean Values Parameter Female mean Male mean Cortical thickness (Ct. Th) 823 µm 915 µm Cancellous bone volume (BV/TV) 21. 8% 19. 7% Osteoid thickness (O. Th) 12. 3 µm 11. 1 µm Osteiod surface (OS) 8. 4% 6. 5% Osteoblast/osteoid interface (Ob. s/OS) 22. 1% 14. 4% Osteoclasts/trabecular surface(N. Oc/BS) 3. 0/100 mm 3. 5/100 mm Eroded surface (ES) 2. 3% 1. 5% Single labled surface (s. L. S) 2. 3% 2. 4% Double labeled surface (d. L. S) 6. 2% 3. 0%

Bone Histomorphometry Normal Mean Values Parameter Female mean Wall Thickness (W. Th) Male mean 49. 8 µm Mineral Apposition Rate (MAR) 0. 88 µm/d 0. 89 µm/d Bone formation Rate Surface (BFR/BS) (mm³/mm²/yr) 0. 019 0. 009 Volume (BFR/BV) (mm³/yr) 0. 250 0. 131 Mineralization Lag Time (M. Lt) 21. 1 d 27. 6 d Activation Frequency (Ac. f) 0. 42 y

Bone Histology and Histopathology for Clinicians End part I