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...
TRANSCRIPT
A PrimerA 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.
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.
Bone Histology and Histopathology for Clinicians
Bone Histology and Histopathology for Clinicians
Presented ByPresented By
Mayo ClinicDivisions of Endocrinology and Orthopedic Research
Mayo ClinicDivisions of Endocrinology and Orthopedic Research
The American College of EndocrinologyThe American College of Endocrinology
andand
Supported by an Educational Grant from:Supported by an Educational Grant from:
The American College of Endocrinology
The American College of Endocrinology
The authors acknowledge the many valuable contributions of others
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
• 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 McCarthy
• Dr. B.L. Riggs
• Dr. Jean Sibonga
• Peter Steiner,Illustration & Design
• Dr. Peter Tebben
• Dr. Robert Tiegs
• Dr. Russell Turner
• 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
Bone Histology and Histopathology for Clinicians©©
©2007 Mayo Foundation for Medical Education and Research©2007 Mayo Foundation for Medical Education and Research and licensed to and licensed to The American College of Endocrinology.The American College of Endocrinology.
This presentation provides basic This presentation provides basic instruction in bone histology, and in the instruction in bone histology, and in the histopathology of metabolic bone histopathology of metabolic bone diseases and related disorders. It was diseases and related disorders. It was prepared primarily for endocrine fellows, prepared primarily for endocrine fellows, endocrinologists, osteologists, and other endocrinologists, osteologists, and other physicians and scientists interested in physicians and scientists interested in metabolic and related bone diseases.metabolic and related bone diseases.
This presentation provides basic This presentation provides basic instruction in bone histology, and in the instruction in bone histology, and in the histopathology of metabolic bone histopathology of metabolic bone diseases and related disorders. It was diseases and related disorders. It was prepared primarily for endocrine fellows, prepared primarily for endocrine fellows, endocrinologists, osteologists, and other endocrinologists, osteologists, and other physicians and scientists interested in physicians and scientists interested in metabolic and related bone diseases.metabolic and related bone diseases.
• Normal Bone Microanatomy and Normal Bone Microanatomy and Histology Histology
• Bone Cells and Bone Bone Cells and Bone RemodelingRemodeling
• Basic Bone Histomorphometry Basic Bone Histomorphometry
• Normal Bone Microanatomy and Normal Bone Microanatomy and Histology Histology
• Bone Cells and Bone Bone Cells and Bone RemodelingRemodeling
• Basic Bone Histomorphometry Basic Bone Histomorphometry
Normal Bone Microanatomy
Corticalbone
Corticalbone
Cancellousbone
Cancellousbone
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 bonecortical bone which encloses an irregular medullary space containing cancellous bonecancellous bone, bone that is composed of branching networks of interconnecting bony trabecular elements.
From Gray’s Anatomy
Normal Bone Microanatomy
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.
Corticalbone
Corticalbone
Cancellousbone
Cancellousbone
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 differsTrabecular orientation differs
Variable corticalthickness
Variable corticalthickness
Anatomic Features of a Normal Transiliac Bone Biopsy
7.5mm
CortexCortex
TrabeculaeTrabeculae
Hematopoieticand fatty marrowHematopoietic
and fatty marrow
Normal Bone MicroanatomyDifferential Tissue Stains
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.
Unmineralized boneUnmineralized bone
Mineralized boneMineralized bone
Normal Bone Microanatomy and HistologyCortical Bone
Cortical boneCortical bone forms a relatively thick and dense outer wall and makes up about 80% of total skeletal mass.
Normal Bone Microanatomy and HistologyPeriosteum
The outer cortical surface is enveloped in the periosteumperiosteum, a connective tissue covering that contains cells that maintain, change, and repair the external cortical surface.
The periosteumperiosteum also contains blood vessels, sensory nerves, and dense fibrous tissue that is contiguous with the connective tissue elements of tendons, ligaments, and joint capsules
Normal Bone Microanatomy and HistologyPeriosteum
CortexCortex
PeriosteumPeriosteum
Normal Bone Microanatomy and HistologyBone 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 HistologyCancellous 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 cellsLining cells
EndosteumEndosteum
Normal Bone Microanatomy and Histology Cortical BSUs
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)(Cortex above (left) under incandescent and (right) polarized light)
Cortical (Haversian) BSUViewed in cross section under Polarized light
Trabecular BSUsTrabecular 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 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 amountof bone removed is precisely replaced and there is no net change in bone mass. Only bone architecture is changed.
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.
Bone Remodeling Bone Remodeling Sequence of Bone Cell ActivitySequence 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
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 RemodelingActivation
Lining cells produce collagenase, which exposes the mineralized bone surface for bone resorption
Lining cells produce collagenase, which exposes the mineralized bone surface for bone resorption
Bone RemodelingResorption
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).
(Mineralized bone)
(Marrow)
OsteoclastOsteoclast Sealedmicro-environment
Sealedmicro-environment Ruffled membrane
The basal surface of the osteoclast is rich in HCl and cathepsin transfer organelles and is called the ruffled membrane.
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
Resorption (Howship’s) lacunaeResorption (Howship’s) lacunae
Bone RemodelingResorption
Osteoclasts
Morphology
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).
OsteoclastsOsteoclasts Acid Phos (+)osteoclasts Acid Phos (+)osteoclasts
Osteoclasts
Variations
OsteoclastOsteoclastss
PrevalencePrevalence
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 Biochemical Effects of Bone RemodelingRemodeling
Markers of Bone ResorptionMarkers 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 Bone RemodelingRemodeling
ReversalReversal
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.
PreosteoblastsPreosteoblasts
ReversalReversalPre-Osteoblast Pre-Osteoblast
MaturationMaturation
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.
PreosteoblastsPreosteoblasts OsteoblastsOsteoblasts
Bone Bone RemodelingRemodeling
Bone FormationBone FormationUnmineralized osteoidUnmineralized osteoidUnmineralized osteoidUnmineralized 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 FormationBone FormationOsteoblasts Osteoblasts –– Effects of Cell Effects of Cell
AgeAge
…but flatten as they complete bone formation to eventually become lining cells
…but flatten as they complete bone formation to eventually become lining cells
Young osteoblasts appear cuboidal and robust…Young osteoblasts appear cuboidal and robust…
Type I CollagenType I Collagen
Type I collagen is a triple helical structure Type I collagen is a triple helical structure composed of two composed of two αα1 chains and one 1 chains and one αα2 chain. 2 chain. Collagen Collagen αα chains are characterized by Gly-X-Y chains are characterized by Gly-X-Y repeating triplets where X and Y are usually repeating triplets where X and Y are usually proline and hydroxyproline, respectively.proline and hydroxyproline, respectively.
Type I collagen is synthesized in a procollagen Type I collagen is synthesized in a procollagen form which undergoes post-translational form which undergoes post-translational hydroxylation and glycosylation of selective hydroxylation and glycosylation of selective residues. It further undergoes removal of terminal residues. It further undergoes removal of terminal sequences before being secreted in its mature sequences before being secreted in its mature form from the basilar surface of osteoblasts into form from the basilar surface of osteoblasts into the underlying extra cellular space.the underlying extra cellular space.
Type I collagen is a triple helical structure Type I collagen is a triple helical structure composed of two composed of two αα1 chains and one 1 chains and one αα2 chain. 2 chain. Collagen Collagen αα chains are characterized by Gly-X-Y chains are characterized by Gly-X-Y repeating triplets where X and Y are usually repeating triplets where X and Y are usually proline and hydroxyproline, respectively.proline and hydroxyproline, respectively.
Type I collagen is synthesized in a procollagen Type I collagen is synthesized in a procollagen form which undergoes post-translational form which undergoes post-translational hydroxylation and glycosylation of selective hydroxylation and glycosylation of selective residues. It further undergoes removal of terminal residues. It further undergoes removal of terminal sequences before being secreted in its mature sequences before being secreted in its mature form from the basilar surface of osteoblasts into form from the basilar surface of osteoblasts into the underlying extra cellular space.the underlying extra cellular space.
Bone FormationBone FormationOsteoblasts Become Osteoblasts Become
OsteocytesOsteocytes
Some osteoblasts become entrapped in osteoid to become osteocytes
Some osteoblasts become entrapped in osteoid to become osteocytes
OsteocytesOsteocytes
As bone mineralizes, osteocytesosteocytes tend to become pyknotic but retain metabolic responsiveness to PTH and other stimuli
As bone mineralizes, osteocytesosteocytes tend to become pyknotic but retain metabolic responsiveness to PTH and other stimuli
Bone FormationBone FormationOsteocytic Osteocytic CanaliculiCanaliculi
Osteocytes retain communicationwith the surface and with other cellsthrough a system of microtubules called canaliculi
Osteocytes retain communicationwith the surface and with other cellsthrough a system of microtubules called canaliculi
Bone Bone FormationFormation
Reversal LineReversal Line
Osteoblasts secrete collagen matrix directly on the resorption lacunar surface. The resulting scalloped interface between old bone and new matrix is called the ReversalReversal, or Cement Line Cement Line
Osteoblasts secrete collagen matrix directly on the resorption lacunar surface. The resulting scalloped interface between old bone and new matrix is called the ReversalReversal, or Cement Line Cement Line
Bone Bone FormationFormation
Lamellar BoneLamellar BoneUnder 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 Bone FormationFormation
Woven BoneWoven Bone
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.
Lamellar boneLamellar bone
Woven boneWoven bone
Biochemical Effects of Bone Biochemical Effects of Bone RemodelingRemodeling
Markers of Bone FormationMarkers 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 Bone FormationFormation
Reversal LineReversal Line
The reversal line defines the limit of bone erosion and the original site of bone formation.
The reversal line defines the limit of bone erosion and the original site of bone formation.
Bone Bone FormationFormation
Reversal LineReversal Line
The persistence of a serrated interface indicates that mineral deposition has not begun at this location.
The persistence of a serrated interface indicates that mineral deposition has not begun at this location.
Mineralization of Mineralization of OsteoidOsteoid
The Mineralization FrontThe 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.
As early mineralization proceeds, the serrated reversal linereversal line becomes obscured and the mineralization frontmineralization front becomes a smooth linear interface. When a flurorochrome labeling agent, such as tetracyclinetetracycline, 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 OsteoidMineralization of OsteoidThe Mineralization FrontThe Mineralization Front
Reversal lineReversal line
Mineralization frontMineralization front
Mineralization of OsteoidMineralization of OsteoidFluorescent Labeling With Fluorescent Labeling With
Tetracycline:Tetracycline:Fluorescence MicroscopyFluorescence Microscopy
Old Mineralized boneOld Mineralized bone
MarrowMarrowNew mineralized boneNew 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.
Label #1Label #1
#2#2
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.
Mineralization of OsteoidMineralization of OsteoidFluorescent Labeling With Tetracycline:Fluorescent Labeling With Tetracycline:
Fluorescence MicroscopyFluorescence Microscopy
Single labelSingle label
Osteocytic lacunaeand canaliculiOsteocytic lacunaeand canaliculi
Normal Iliac Bone Biopsy From aNormal Iliac Bone Biopsy From a33-year-old Woman33-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.
Fluorescent bands deep within mineralized trabeculae indicate previous incidental tetracycline exposure.
Normal Iliac Bone Biopsy From aNormal Iliac Bone Biopsy From a33-year-old Woman33-year-old Woman
Cancellous Bone RemodelingCancellous Bone RemodelingThe Bone Remodeling Compartment The Bone Remodeling Compartment
(BRC)(BRC)
Between the BMU and bone marrow is a structure called the bone bone remodeling compartment (BRC).
BRCBRC
Cancellous Bone RemodelingCancellous Bone RemodelingThe Bone Remodeling Compartment (BRC)The Bone Remodeling Compartment (BRC)
The BRC is lined by sinusoidal vascular structuressinusoidal vascular structures whose marrow interface is made up of lining cellslining cells that form a canopy over the remodeling site.
The BRC is lined by sinusoidal vascular structuressinusoidal vascular structures whose marrow interface is made up of lining cellslining cells that form a canopy over the remodeling site.
Lining cellsLining cells
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 RemodelingCancellous Bone RemodelingThe Bone Remodeling Compartment (BRC)The Bone Remodeling Compartment (BRC)
BRCBRC
Cancellous Bone RemodelingCancellous 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 Cancellous Bone RemodelingRemodelingSequenceSequence
Lead by osteoclastic resorption, the BMU moves across the surface of cancellous bone. Resorption is succeeded by formation, which eventually becomes new mineralized bone.
OsteoclasticresorptionOsteoclasticresorption
Reversal lineReversal line
Early osteoblasticformationEarly osteoblasticformation
Mineralization frontMineralization front
Newmineralizedbone
Newmineralizedbone
Osteoblasts flattento ultimately form inactive lining cells.
Osteoblasts flattento ultimately form inactive lining cells.
Cancellous Bone RemodelingCancellous Bone RemodelingSequenceSequence
Cancellous Bone RemodelingCancellous Bone RemodelingRemodeling SpaceRemodeling Space
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.
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)
Cancellous and Cortical Bone Remodeling Cancellous and Cortical Bone Remodeling
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.
Cancellous BMUCancellous BMU Cortical BMUCortical BMU
Cortical Bone Remodeling Cortical Bone Remodeling
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.
Cutting coneCutting cone
OsteoclastsOsteoclasts
Cortical Bone Remodeling Cortical Bone Remodeling
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.
Reversal zoneReversal zoneClosing coneClosing cone
Osteoblasts forming ostoidOsteoblasts forming ostoid
Cortical Bone RemodelingCortical Bone RemodelingFormation, Longitudinal, and Cross-Sectional ViewsFormation, Longitudinal, and Cross-Sectional Views
Fluorochrome labeling with tetracyclene (right) documents the circumferential closure of a cortical BMU (osteon).
#2#2
#1#1
Cortical Bone RemodelingCortical Bone Remodeling
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.
Completed Haversian canalCompleted Haversian canal
Forming Haversian canalForming Haversian canal
Haversian Remodeling of Cortical Bone*Haversian Remodeling of Cortical Bone*
Cutting coneCutting cone
Resorption(osteoclast)Resorption(osteoclast)
Haversian canalHaversian canal
Bone formation(osteoblasts)Bone formation(osteoblasts)
Remodeled cortexRemodeled cortex
Cortical Bone RemodelingCortical Bone Remodeling
A complete cortical remodeling cycle requires from six to nine months. Circumferential closure at any point requires about three months.
Basic Bone Basic Bone HistomorphometryHistomorphometryStereologic BasisStereologic 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.*
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.
*For detailed discussion, see Recker, RR. Bone Histomorphometry:Techniques and Interpretation, CRC Press, 1983.
Basic Bone Basic Bone HistomorphometryHistomorphometry
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 Basic Bone HistomorphometryHistomorphometry
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 Bone Histomorphometry Trabecular Bone Volume Trabecular Bone Volume
(Tb.V)(Tb.V)
Trabecular bone volume, (Tb.VTb.V) is the relative volume of total cancellous bone measured (TVTV), expressed as %, that is occupied by trabeculae.
Tb.VTb.V is about 20% in women and 22% in men.
Tb.VTb.Vis related to cancellous bone mass. It declines with age and with bone loss
Bone Histomorphometry Bone Histomorphometry Trabecular Bone Volume Trabecular Bone Volume
(Tb.V)(Tb.V)
Note
Tb.V is also commonly referred to as
Bone Volume / Total Volume, or
BV/TV BV/TV
Bone Bone HistomorphometryHistomorphometry
Surface ClassificationSurface 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 HistomorphometryBone HistomorphometryTrabecular Separation Trabecular Separation
(Tb.Sp)(Tb.Sp)
Trabecular Separation Tb.SpTb.Sp is the mean distance in mm between trabeculae (measured by integrated computer graphics)
Tb.SpTb.Sp is a measure of trabecular connectivity
Tb.SpTb.Sp increases with trabecular bone loss
Bone Histomorphometry Bone Histomorphometry Trabecular Thickness Trabecular Thickness
(Tb.Th)(Tb.Th)
Mean trabecular thickness, (Tb.ThTb.Th) is a measure of trabecular structure and is calculated as the reciprocal of Tb.S
Tb.Th Tb.Th is reduced by aging and osteoporosis.
Tb.ThTb.Th = 1/Tb.STb.ThTb.Th = 1/Tb.S
Bone Histomorphometry Bone Histomorphometry Trabecular Number Trabecular Number
(Tb.N)(Tb.N)
Trabecular number (Tb.NTb.N). The number of trabeculae present per lineal mm
Tb.NTb.N is calculated as Trabecular bone volume/Trabecular Thickness
Tb.NTb.N is a measure of trabecular connectivity
Tb.NTb.N decreases with bone loss
Bone HistomotphometryBone Histomotphometry Cortical Thickness Cortical Thickness
(Ct.Th)(Ct.Th)In the ileum, average combined cortical width (Ct.ThCt.Th) in women and men is about 820 µm and 915 µm, respectively. Ct.ThCt.Th correlates with dual photon absorptiometric (DPX) measurements of bone density.
Bone Histomorphometry Bone Histomorphometry
Mineral Apposition RateMineral Apposition Rate(MAR)(MAR)
The average distance between visible labels, divided by the labeling interval, is the mineral apposition rate (MARMAR) in µm/day, the avarage rate at which new bone mineral is being added on any actively forming surface. MARMAR 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
MARMAR =Interlabel distance
Label interval Label interval
MARMAR =Interlabel distance
Label interval Label interval
Bone HistomorphometryBone Histomorphometry Mineralizing Surfaces Mineralizing Surfaces
(MS)(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
MS is used in the calculations for bone formation rates, (BFRBFR), activation frequency (Ac.FAc.F), and mineralization lag time (MLTMLT).
Bone HistomorphometryBone HistomorphometryMineralizing Surface (MS)Mineralizing Surface (MS)
Bone HistomorphometryBone HistomorphometryOsteoid Thickness Osteoid Thickness
(O.Th)(O.Th)
Osteoid thickness (O.ThO.Th) is the mean thickness, in µm, of osteoid seams on cancellous surfaces.
O.Th is normally <12.5 µm. Increased O.Th suggests abnormal mineralization (osteomalacia).
Bone HistomorphometryBone HistomorphometryOsteoid Thickness (O.Th)Osteoid Thickness (O.Th)
Bone Histomorphometry Bone Histomorphometry Mineralization Lag Time (MLT)Mineralization Lag Time (MLT)
The time interval between osteoid secretion and its subsequent mineralization, in days, is known as the mineralization lag time (MLTMLT).
MineralizationMineralization
SecretionSecretion
Bone Histomorphometry Bone Histomorphometry
Mineralization Lag TimeMineralization Lag Time
MLTMLT is a measure of mineralization competence and is normally less than 22 days in women and 27 days in men.
Bone Histomorphometry Bone Histomorphometry Mineralization Lag Time Mineralization Lag Time
(MLT)(MLT)
MLTMLT is calculated as:
MLTMLT is calculated as:
O.Th
MAR
MS
Osx
Bone Histomorphometry Bone Histomorphometry Activation: Activation Frequency Activation: Activation Frequency
(Ac.f)(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.fAc.f). Ac.fAc.f is a measure of bone turnover and is expressed in years.
Bone Histomorphometry Bone Histomorphometry
Wall Thickness (W.Th)Wall Thickness (W.Th)
Wall thickness is the average thickness of trabecular BSU.
(W.ThW.Th) is used to assess the overall balance between resorptionand formation.
W.Th=Average thickness of BSUW.Th=Average thickness of BSU
Bone Bone HistomorphometryHistomorphometry
Bone Formation RatesBone Formation RatesBone 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 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 formation rates are expressed as:Bone formation rates are expressed as:
BFR/BV in (mm³/mm³/yr)
BFR/BS in (mm³/mm²/yr)
BFR/BV in (mm³/mm³/yr)
BFR/BS in (mm³/mm²/yr)
Alternatively, BFR/BS can be derived as:Alternatively, BFR/BS can be derived as:
BFR/BS = Ac.f x W.ThBFR/BS = Ac.f x W.Th
Bone HistomorphometryBone HistomorphometryBone Formation RatesBone Formation Rates
Bone HistomorphometryBone HistomorphometryNormal Mean ValuesNormal Mean Values
ParameterParameter Female meanFemale mean Male meanMale mean
Cortical thickness (Ct.Th)Cortical thickness (Ct.Th) 823 823 µmµm 915 µm915 µm
Cancellous bone volume (BV/TV)Cancellous bone volume (BV/TV) 21.8%21.8% 19.7%19.7%
Osteoid thickness (O.Th)Osteoid thickness (O.Th) 12.3 µm12.3 µm 11.1 µm11.1 µm
Osteiod surface (OS)Osteiod surface (OS) 8.4%8.4% 6.5%6.5%
Osteoblast/osteoidOsteoblast/osteoid interface (Ob.s/OS) interface (Ob.s/OS) 22.1%22.1% 14.4%14.4%
Osteoclasts/trabecularOsteoclasts/trabecular surface(N.Oc/BS) surface(N.Oc/BS) 3.0/100 mm3.0/100 mm 3.5/100 mm3.5/100 mm
Eroded surface (ES)Eroded surface (ES) 2.3%2.3% 1.5%1.5%
Single labled surface (sL.S)Single labled surface (sL.S) 2.3%2.3% 2.4%2.4%
Double labeled surface (dL.S)Double labeled surface (dL.S) 6.2%6.2% 3.0%3.0%
ParameterParameter Female meanFemale mean Male meanMale mean
Cortical thickness (Ct.Th)Cortical thickness (Ct.Th) 823 823 µmµm 915 µm915 µm
Cancellous bone volume (BV/TV)Cancellous bone volume (BV/TV) 21.8%21.8% 19.7%19.7%
Osteoid thickness (O.Th)Osteoid thickness (O.Th) 12.3 µm12.3 µm 11.1 µm11.1 µm
Osteiod surface (OS)Osteiod surface (OS) 8.4%8.4% 6.5%6.5%
Osteoblast/osteoidOsteoblast/osteoid interface (Ob.s/OS) interface (Ob.s/OS) 22.1%22.1% 14.4%14.4%
Osteoclasts/trabecularOsteoclasts/trabecular surface(N.Oc/BS) surface(N.Oc/BS) 3.0/100 mm3.0/100 mm 3.5/100 mm3.5/100 mm
Eroded surface (ES)Eroded surface (ES) 2.3%2.3% 1.5%1.5%
Single labled surface (sL.S)Single labled surface (sL.S) 2.3%2.3% 2.4%2.4%
Double labeled surface (dL.S)Double labeled surface (dL.S) 6.2%6.2% 3.0%3.0%
Bone HistomorphometryBone HistomorphometryNormal Mean ValuesNormal Mean Values
ParameterParameter Female meanFemale mean Male meanMale mean
Wall Thickness Wall Thickness (W.Th)(W.Th) 49.8 49.8 µmµm
Mineral Apposition Rate Mineral Apposition Rate (MAR)(MAR) 0.88 0.88 µm/dµm/d 0.89 µm/d0.89 µm/d
Bone formation RateBone formation RateSurface (BFR/BS) Surface (BFR/BS) (mm³/mm²/yr)(mm³/mm²/yr) 0.0190.019 0.0090.009
Volume (BFR/BV) Volume (BFR/BV) (mm³/mm³/yr)(mm³/mm³/yr) 0.2500.250 0.1310.131
Mineralization Lag Time Mineralization Lag Time (M.Lt)(M.Lt) 21.1 d21.1 d 27.6 d27.6 d
Activation Frequency (Ac.f) Activation Frequency (Ac.f) 0.42 y0.42 y
ParameterParameter Female meanFemale mean Male meanMale mean
Wall Thickness Wall Thickness (W.Th)(W.Th) 49.8 49.8 µmµm
Mineral Apposition Rate Mineral Apposition Rate (MAR)(MAR) 0.88 0.88 µm/dµm/d 0.89 µm/d0.89 µm/d
Bone formation RateBone formation RateSurface (BFR/BS) Surface (BFR/BS) (mm³/mm²/yr)(mm³/mm²/yr) 0.0190.019 0.0090.009
Volume (BFR/BV) Volume (BFR/BV) (mm³/mm³/yr)(mm³/mm³/yr) 0.2500.250 0.1310.131
Mineralization Lag Time Mineralization Lag Time (M.Lt)(M.Lt) 21.1 d21.1 d 27.6 d27.6 d
Activation Frequency (Ac.f) Activation Frequency (Ac.f) 0.42 y0.42 y
Bone Histology and Histopathology for Clinicians
Bone Histology and Histopathology for Clinicians
End part I