histo 232 to 243
Bone Formation
 Endochondral ossification – cartilage is precursor of bone
o Route of formation of bones of extremities and parts of axial skeleton that bear weight (vertebrae)
 Intramembranous ossification – bone formed without cartilage first (membranes ossify)
o Route of development of flat bones of skull and face, mandible, and clavicle
 Because of remodeling that occurs later, initial bone tissue laid down by endochondral formation or by
intramembranous formation soon replaced
 Replacement bone established on preexisting bone by appositional growth and is identical despite formation
 Continued growth of long bones involves histogenesis of both endochondral and intramembranous bone
o Intramembranous bone occurs through activity of periosteal (membrane) tissue
Osteoporosis
 Most commonly occurring bone disease characterized by progressive loss of normal bone density accompanied
by deterioration of microarchitecture
 Caused by imbalance between osteoclast-mediated bone resorption and osteoblast-mediated bone deposition
 In healthy individuals osteoclast activity primarily regulated by PTH and to lesser degree by IL-1 and TNF
o Differentiation of osteoclast precursors is under influence of M-CSF and IL-6
o Estrogens (especially estradiol) inhibit formation of M-CSF and IL-6, limiting activity of osteoclasts
o In postmenopausal women, secretion of these cytokines increased, resulting in enhanced activity of
osteoclasts leading to intensified bone resorption
 3 general types of osteoporosis
o Type I primary osteoporosis – occurs in postmenopausal women; long-term effect usually more severe
than osteoporosis that develops in later years of life
o Type II primary osteoporosis – occurs in elderly individuals in 60s or 70s and is leading cause of serious
morbidity and functional loss in this age group
o Secondary osteoporosis – develops as result of drug therapy (i.e., corticosteroids) or disease processes
that may affect bone remodeling, including malnutrition, prolonged immobilization, weightlessness (i.e.,
with space travel), and metabolic bone diseases (i.e., hyperparathyroidism, metastatic cancers)
 Osteoporotic bone has normal histologic structure, but there is less tissue mass; results in weakened bones
 Individuals suffering from fractures at greater risk for death, not directly from fractures but from complications
of hospitalization because of immobilization and increased risk of pneumonia, pulmonary thrombosis, and
embolism
 Hormone replacement therapy reduces risk of fractures but causes greater risk of adverse cardiovascular
diseases and breast cancer
 Selective estrogen receptor modulators (SERMs), such as raloxifene, bind to estrogen receptors and act as
estrogen agonist in bone; in other tissues, it blocks estrogen receptor (acting as antagonist)
 Bisphosphonates like alendronate or risedronate inhibit osteoclastic activity by inducing apoptosis of osteoclasts
 Hormonal therapy includes use of human PTH (i.e., teriparatide) which promotes bone formation by increasing
osteoblastic activity and improving thickness of trabecular bone
 Neutralizing monoclonal antibodies against RANKL molecules (denosumab) reduce number of differentiating
osteoclasts by inhibiting their activation and survival, thus preventing bone resorption
Nutritional Factors in Bone Formation
 Calcium deficiency during growth causes rickets; bone matrix doesn’t calcify normally
o May be caused by insufficient amounts of calcium or vitamin D
o X-ray of child presenting with rickets presents bowed lower limbs and deformed chest and skull (often
having square appearance)
o If not treated, skeletal deformities and short stature may be permanent
 Osteomalacia – nutritional or vitamin deficiency in adults
 Vitamin D also needed for normal calcification as well as absorption of calcium in intestines
 Vitamin A deficiency suppresses endochondral growth of bone
 Vitamin A excess leads to fragility and subsequent fractures of long bones
 Vitamin C essential for synthesis of collagen; deficiency leads to scurvy; matrix produced in scurvy not calcifiable
Intramembranous Ossification
 In 8th week of gestation, mesenchymal cells migrate and aggregate in specific areas where bone destined to
form; condensation of cells initiates intramembranous ossification
o Mesenchymal cells differentiate into osteoprogenitor cells expressing Cbfa1 transcription factor;
essential for osteoblast differentiation and expression of genes necessary for both intramembranous
and endochondral ossification
o Newly organized tissue at bone site becomes more vascularized, and aggregated mesenchymal cells
become larger and rounded
o Cytoplasm of osteoprogenitor cells changes from eosinophilic to basophilic, and clear Golgi area forms
 At this stage, it’s an osteoblast, which then secretes collagens (mainly type I), bone
sialoproteins, osteocalcin, and other components of bone matrix (osteoid)
 Osteoblasts in bone matrix become increasingly separate from one another as matrix produced, but remain
attached by thin cytoplasmic processes
 Because of abundant collagen content, bone matrix appears denser than surrounding mesenchyme, in which
intercellular spaces reveal only delicate CT fibers
 With time, matrix becomes calcified, and interconnecting cytoplasmic processes of bone-forming cells
(osteocytes) contained within canaliculi
 More surrounding mesenchymal cells in membrane proliferate, giving rise to population of osteoprogenitor cells
o Some osteoprogenitor cells come into apposition with initially formed spicules, become osteoblasts, and
add more matrix – appositional growth
o Spicules enlarge and become joined in trabecular network with general shape of developing bone
 Through continued mitotic activity, osteoprogenitor cells maintain numbers and provide constant source of
osteoblasts for growth of bone spicules
o New osteoblasts lay down bone matrix in successive layers, giving rise to woven bone
o Immature bone characterized internally by interconnecting spaces occupied by CT and blood vessels
Endochondral Ossification
 Begins with proliferation and aggregation of mesenchymal cells at site of future bone
 Under influence of FGFs and BMPs, mesenchymal cells initially express type II collagen and differentiate into
chondroblasts that produce cartilage matrix
 Initially, hyaline cartilage model with general shape of bone formed
o Once established, cartilage model grows by interstitial and appositional growth; increase in length of
cartilage attributed to interstitial growth and increase in width is result of addition of cartilage matrix
produced by new chondrocytes that differentiate from chondrogenic layer of perichondrium
surrounding cartilage mass
 Perichondrial cells in midregion of cartilage model no longer give rise to chondrocytes and produce osteoblasts
o CT surrounding this portion of cartilage no longer functionally perichondrium but periosteum
o Because cells in periosteum differentiating into osteoblasts, osteogenic layer found in periosteum
 Layer of bone forms around cartilage model; classified either as periosteal bone because of its location or
intramembranous bone because of its method of development
 In long bones, distinctive cuff of periosteal bone (bony collar) established around cartilage model in diaphyseal
portion of developing bone
 With establishment of periosteal bony collar, chondrocytes in midregion of cartilage model become
hypertrophic
o As chondrocytes enlarge, their surrounding cartilage matrix resorbed, forming thin irregular cartilage
plates between hypertrophic cells
o Hypertrophic cells begin to synthesize alkaline phosphatase; concomitantly, surrounding cartilage matrix
undergoes calcification (not mineralization of bone)
 Calcified cartilage matrix inhibits diffusion of nutrients, causing death of chondrocytes in cartilage model; with
death of chondrocytes, much of matrix breaks down, and neighboring lacunae become confluent, producing
increasingly large cavity
o With death of chondrocytes, much of matrix breaks down, and neighboring lacunae become confluent,
producing increasingly large cavity
o One or several blood vessels grow through thin diaphyseal bony collar to vascularize cavity
 Mesenchymal stem cells residing in developing periosteum migrate along penetrating blood vessels and
differentiate into osteoprogenitor cells in bone marrow cavity
 Hemopoietic stem cells (HSCs) gain access to cavity via new vasculature, leaving circulating to give rise to
marrow including all blood cell lineages
 As calcified cartilage breaks down and is partially removed, some remains as irregular spicules; when
osteoprogenitor cells come in apposition to remaining calcified cartilage spicules, they become osteoblasts and
begin to lay down bone matrix (osteoid) on spicule framework
 First site where bone begins to form in diaphysis of long bone is primary ossification center
 Combination of bone (initially only thin layer) and underlying calcified cartilage is mixed spicule
 Calcified cartilage is more basophilic, whereas bone is distinctly eosinophilic
o With Mallory stain, bone stains deep blue, and calcified cartilage stains light blue
o Calcified cartilage no longer contains cells, whereas newly produced bone may reveal osteocytes in bone
matrix
 Spicules persist for short time before calcified cartilage component removed; remaining bone component of
spicule may continue to grow by appositional growth, thus becoming larger and stronger, or may undergo
resorption as new spicules formed
Growth of Endochondral Bone
 Endochondral bone growth begins in second trimester of fetal life and continues into early adulthood
 Growth in length of long bones depends on presence of epiphyseal cartilage
o During endochondral bone formation, avascular cartilage gradually replaced by vascularized bone tissue;
initiated by VEGF and accompanied by expression of genes responsible for production of type X collagen
and MMPs (enzymes responsible for degradation of cartilage matrix)
 Zones in epiphyseal cartilage (from most distal to diaphyseal center of ossification towards center) are
o Zone of reserve cartilage – exhibits no cellular proliferation or active matrix production
o Zone of proliferation – cartilage cells undergo division and organize into distinct columns; cells larger
than those in reserve zone and actively produce collagen (mainly types II and XI) and other cartilage
matrix proteins
o Zone of hypertrophy – contains greatly enlarged cartilage cells; cytoplasm accumulates glycogen
 Chondrocytes continue to secrete type I collagen while increasing secretion of type X collagen
 Hypertrophic chondrocytes also secrete VEGF, which initiates vascular invasion
 Cartilage matrix compressed to form linear bands between columns of hypertrophied cells
o Zone of calcified cartilage – hypertrophied cells begin to degenerate and cartilage matrix becomes
calcified, serving as initial scaffold for deposition of new bone
 Chondrocytes positioned in more proximal part of this zone undergo apoptosis
o Zone of resorption – zone nearest diaphysis; calcified cartilage in direct contact with CT of marrow cavity
 Small blood vessels and accompanying CT invade region previously occupied by dying
chondrocytes and form series of spearheads, leaving calcified cartilage as longitudinal spicules
 Invading blood vessels are source of osteoprogenitor cells, which differentiate into boneproducing cells
 As bone laid down on calcified spicules, cartilage resorbed, ultimately leaving primary spongy bone that
undergoes reorganization through osteoclastic activity and addition of new bone tissue, accommodating
continued growth and physical stresses placed on bone
 Shortly after birth, secondary ossification center develops in proximal epiphysis; cartilage cells undergo
hypertrophy and degenerate; calcification of matrix occurs, and blood vessels and osteogenic cells from
perichondrium invade region, creating new marrow cavity
 Later, another epiphyseal ossification center forms in distal end of bone (also secondary ossification center)
 With development of secondary ossification centers, only cartilage that remains from original model is articular
cartilage at ends of bone and transverse disc of cartilage (epiphyseal growth plate) that separates epiphyseal
and diaphyseal cavities
 Cartilage of epiphyseal growth plate responsible for maintaining growth process; for bone to retain proper
proportions and shape, both external and internal remodeling must occur as bone grows in length
o Proliferative zone of epiphyseal plate gives rise to cartilage on which bone later laid down
o Thickness of epiphyseal plate remains relatively constant during growth
o Amount of new cartilage produced (zone of proliferation) equals amount resorbed (zone of resorption)
o Resorbed cartilage replaced by spongy bone
o Actual lengthening of bone occurs when new cartilage matrix produced at epiphyseal plate
o Production of new cartilage matrix pushes epiphysis away from diaphysis, elongating bone
 Bone increases in width or diameter when appositional growth of new bone occurs between cortical lamellae
and periosteum; marrow cavity enlarges by resorption of bone on endosteal surface of cortex of bone
 As bones elongate, remodeling required
 When individual achieves maximal growth, proliferation of new cartilage within epiphyseal plate terminates
o When proliferation of new cartilage ceases, cartilage that has already been produced in epiphyseal plate
continues to undergo changes that lead to deposition of new bone until there is no cartilage left
o At this point, epiphyseal and diaphyseal marrow cavities become confluent (epiphyseal closure)
o Only remaining cartilage found on articular surfaces of bone
o Vestigial evidence of site of epiphyseal plate reflected by epiphyseal line consisting of bone tissue
Development of Osteonal (Haversian) System
 Osteons typically develop in preexisting compact bone
 Compact bone may be formed from fetal spongy bone by continued deposition of bone on spongy bone
spicules; may be deposited directly as adult compact bone (e.g., circumferential lamellae of adult bone); or may
be older compact bone consisting of osteons and interstitial lamellae
 Internal remodeling – process in which new osteons formed
 During development of new osteons, osteoclasts bore tunnel (resorption cavity) through compact bone
o Resorption cavity has dimensions of new osteon
o When osteoclasts have produced appropriately sized cylindrical tunnel by resorption of compact bone,
blood vessels and their surrounding CT occupy the tunnel
o As tunnel occupied, new bone deposition on its wall begins almost immediately
o Osteoclast resorption and osteoblast synthesis constitute bone-remodeling unit
o Bone-remodeling unit consists of advancing cutting cone (resorption canal) and closing cone
 Tip of cutting cone consists of advancing osteoclasts closely followed by advancing capillary loop
and pericytes; also contains numerous dividing cells that give rise to osteoblasts, additional
pericytes, and endothelial cells
 Cutting cone constitutes small fraction of length of bone-remodeling unit, thus is much less
frequently seen than closing cone
o Osteoclast’s canal establishes diameter of future osteonal (Haversian) system
o After diameter of future Haversian system established, osteoblasts begin to fill canal by depositing
organic matrix of bone (osteoid) on its walls in successive lamellae
o With time, bone matrix in each lamellae becomes mineralized; as successive lamellae of bone deposited
from periphery inward, canal ultimately attains relatively narrow diameter of adult osteonal canal
 Compact adult bone contains Haversian systems of varying age and size
o Younger Haversian systems less completely mineralized than older systems; undergo secondary
mineralization that continues to a point even after osteon has been fully formed
 In adult, deposition balances resorption; in aged, resorption often exceeds deposition, leading to osteoporosis
Biologic Mineralization and Matrix Vesicles
 Mineralization occurs in ECM of bone, cartilage, and dentin, cementum, and enamel of teeth
o Matrices of structures except enamel contain collagen fibrils and ground substance; mineralization is
initiated in same time within collagen fibrils and ground substance surrounding them
o In enamel, mineralization and fact that physicochemical factors are basic to process, biologic
mineralization is cell-regulated event
 Mineralization involves secretion of matrix vesicles into bony matrix
o Places where mineralization initiated, local concentration of Ca2+ and PO4 ions in matrix must exceed
normal threshold level
 Events responsible for mineralization
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Binding of extracellular Ca2+ by osteocalcin and other sialproteins creates high local concentration of
Ca2+
High Ca2+ concentration stimulates osteoblasts to secrete alkaline phosphatase (ALP), which increases
local concentration of PO4 ions; high PO4 concentration stimulates further increases in Ca2+
concentration where mineralization will be initiated
At stage of high extracellular Ca2+ and PO4, osteoblasts release small matrix vesicles into bony matrix by
exocytosis; matrix vesicles contain ALP and pyrophosphatase that cleave PO4 ions from other molecules
of matrix
Matrix vesicles that accumulate Ca2+ and cleave PO4 ions cause local isoelectric point to increase, which
results in crystallization of CaPO4 in surrounding matrix vesicles
CaPO4 crystals initiate matrix mineralization by formation and deposition of hydroxyapatite crystals in
matrix surrounding osteoblasts