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
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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.
Divisions of Endocrinology and Orthopedic Research
The American College of Endocrinology
College of Endocrinology
Dr. Lorraine Fitzpatrick
Dr. Hunter Heath
Dr. Dan Hurley
Dr. Ann Kearns
Dr. Kurt Kennel
Dr. Sundeep Khosla
Dr. Rajiv Kumar
Dr. James McCarthy
Dr. B.L. Riggs
Dr. Jean Sibonga
Peter Steiner,Illustration & Design
Dr. Peter Tebben
Dr. Robert Tiegs
Dr. Russell Turner
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.
Bone Cells and Bone Remodeling
Basic Bone Histomorphometry
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.
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.
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.Normal Bone Microanatomy
Trabecular orientation differs
Hematopoieticand fatty marrow
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.
Cortical bone forms a relatively thick and dense outer wall and makes up about 80% of total skeletal mass.
The outer cortical surface is enveloped in the periosteum, a connective tissue covering that contains cells that maintain, change, and repair the external cortical surface.
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
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.
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.
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.
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)
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.
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 amountof bone removed is precisely replaced and there is no net change in bone mass. Only bone architecture is changed.
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
Lining cells produce collagenase, which exposes the mineralized bone surface for bone resorption
Cells derived from circulating mononuclear phagocyte precursors are recruited to become bone resorbing pre-osteoclasts, which cannot be visually identified by standard microscopy. Pre-osteoclasts mature to become osteoclasts and attach to the exposed mineralized bone surface, to form an isolated and sealed micro-environment that is rich in both HCl and lysozomal enzymes (cathepsin).
The basal surface of the osteoclast is rich in HCl and cathepsin transfer organelles and is called the ruffled membrane.
Resorption (Howship’s) lacunae 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
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 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).
Acid Phos (+)osteoclasts
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.
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.
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.
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.
Osteoblasts secrete type I collagen, called osteoid, from their basal surfaces onto the previously resorbed surface. Osteoid forms the organic matrix of bone.
…but flatten as they complete bone formation to eventually become lining cells
Young osteoblasts appear
cuboidal and robust…
Type I collagen is a triple helical structure composed of two α1 chains and one α2 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.
Some osteoblasts become
entrapped in osteoid to
As bone mineralizes, osteocytes tend to become pyknotic but retain metabolic responsiveness to PTH and other stimuli
Osteocytes retain communication
with the surface and with other cells
through a system of microtubules
Osteoblasts secrete collagen matrix directly on the resorption lacunar surface. The resulting scalloped interface between old bone and new matrix is called the Reversal, or Cement Line
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.
Under conditions of rapid turnover, e.g., normal growth, fracture healing, or under some pathologic conditions as illustrated, osteoid is deposited in disorganized fashionand is called woven bone in contrast to lamellar bone.
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.
The reversal line defines the limit of bone erosion and the original site of bone formation.
The persistence of a serrated interface indicates that mineral deposition has not begun at this location.
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.
As early mineralization proceeds, the serrated 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 reversal line becomes obscured and the mineralization front becomes a smooth linear interface. When a flurorochrome labeling agent, such as 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 OsteoidThe Mineralization Front
New mineralized bone
Old Mineralized bone
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.
Single label 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
Osteocytic lacunaeand canaliculi
Mineralization of OsteoidFluorescent Labeling With Tetracycline:Fluorescence Microscopy
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.
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 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 33-year-old Woman
Fluorescent bands deep within mineralized trabeculae indicate previous incidental tetracycline exposure.
Between the BMU and bone marrow is a structure called the bone remodeling compartment (BRC).
Lining cells 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
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 RemodelingThe Bone Remodeling Compartment (BRC)
BRC 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
Cancellous Bone RemodelingThe Bone Remodeling Compartment (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.
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.
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 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 Sequence
to ultimately form inactive lining cells.
Cancellous Bone Remodeling 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 Remodeling Space
Increases in remodeling space (turnover) are associated with an increasing tendency for fracturing. It’s size is a limiting factor for increasing bone mass with drugs that reduce turnover (eg. Bisphosphonates)
The remodeling space (RS)refers to that volume of bone that has undergone resorption or will undergo formation and mineralization, and which therefore does not contribute to mineralized bone mass. The RS is directly related to bone turnover, and represents the skeleton’s potential for increasing bone volume, mass, and strength.
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 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.
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.
Fluorochrome labeling with tetracyclene (right) documents the circumferential closure of a cortical BMU (osteon).
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.
A complete cortical remodeling cycle requires from six to nine months. Circumferential closure at any point requires about three months.
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.
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.
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.
Trabecular bone volume, (Tb.V) is the relative volume of total cancellous bone measured (TV), expressed as %, that is occupied by trabeculae.
Tb.V is about 20% in women and 22% in men.
Tb.Vis related to cancellous bone mass. It declines with age and with bone loss
Tb.V is also commonly referred to as
Bone Volume / Total Volume, or
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)
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
Tb.Th = 1/Tb.S
Mean trabecular thickness, (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.
Trabecular number (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
In the ileum, average combined cortical width (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.
The average distance between visible labels, divided by the labeling interval, is the mineral apposition rate (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
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 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 Mineralizing Surface (MS)
MS is used in the calculations for bone formation rates, (BFR), activation frequency (Ac.F), and mineralization lag time (MLT).
Osteoid thickness (O.Th) is the mean thickness, in µm, of osteoid seams on cancellous surfaces.
Bone Histomorphometry 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 Osteoid Thickness (O.Th)
O.Th is normally <12.5 µm. Increased O.Th suggests abnormal mineralization (osteomalacia).
The time interval between osteoid secretion and its subsequent mineralization, in days, is known as the mineralization lag time (MLT).
MLT is a measure of mineralization competence and is normally less than 22 days in women and 27 days in men.
MLT is calculated as:
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.
Wall thickness is the average thickness of trabecular BSU.
(W.Th) is used to assess the overall balance between resorptionand formation.
Average thickness of BSU
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)
BFR = MAR(MS/BS)
Bone formation rates are expressed as: 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
Bone HistomorphometryBone Formation Rates
BFR/BV in (mm³/mm³/yr)
BFR/BS in (mm³/mm²/yr)
Alternatively, BFR/BS can be derived as:
BFR/BS = Ac.f x W.Th
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 (sL.S) 2.3% 2.4%
Double labeled surface (dL.S) 6.2% 3.0%
Parameter Female mean Male mean
Wall Thickness (W.Th) 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/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 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
End part I