Histology Ch 8 224-243
Cells of Bone Tissue
5 cell types associated with bone tissue (osteoclast NOT from same lineage as others):
1. Osteoprogenitor Cells – derived from mesenchymal stem cells and functions in osteogenesis.
Responsive to stimuli that transorm them into bone-forming cells: osteoblasts, fibroblasts,
adipocytes, chondrocytes, and muscle cells
-Key factor in differentiation is core binding factor alpha-1 (CBFA1), which prompts phenotype
of OSTEOBLAST
-osteoprogenitor cell is a resting cell that can differentiate into an osteoblast and secrete matrix and are
found on external AND internal surface of bones, and comprise of periosteal cells that form innermost
layer of periosteum and endosteal cells that line the marrow cavities, the Haversian canals, and
Volkmann’s canals
2. Osteoblasts – differentiated bone-forming cell that secretes matrix, secreting both type I collagen
and bone matrix proteins (BMPs) which constitute initial unmineralized bone (osteoid).
-BMPs produced are Ca-binding proteins such as osteocalcin and osteonectin, multiadhesive
glycoproteins such as sialoproteins I, II, osteopontin, thrombospondin, and various
proteoglycans and aggregates, and alkaline phosphatase (clinical marker of osteoblast activity)
-osteoblast responsible for calcification of bone matrix through secretion of membrane-limited matrix
vesicles rich in ALP and actively secreted when cell produces bone matrix
-osteoblast processes communicate with other osteoblasts and osteocytes with gap junctions
3. Osteocytes – osteocyte is the mature bone cell enclosed by bone matrix that it previously secreted
as an osteoblast (called an osteocyte when osteoblast is completely surrounded)
-osteocytes are responsible for maintaining bone matrix, and participate in mechanotransduction,
where osteocyte responds to mechanical forces applied to bone, such as weightlessness or increased
mechanical loading; cell can synthesize new matrix or degrade matrix to help maintain calcium
homeostasis
-death of osteocytes through trauma, senescence, or aptoptosis results in bone resorption by
osteoclasts followed by repair by osteoblasts
-osteocytes occupies a lacuna (space) that conforms to shape of cell, and send cytoplasmic processes
through canaliculi in matrix to contact neighboring osteocytes, osteoblasts, and pericytes
-Quiescent osteocytes – little rER and diminished Golgi, more calcified matrix
-Formative osteocytes – evidence of matrix deposition and characteristic of osteoblasts
-Resorptive osteocytes – evidence of osteoclast, well developed golgi
-matrix metalloproteinases (MMPs) degrade matrix: called osteocytic osteolysis
4. Bone Lining Cells – derived from osteoblasts and cover bone that is not remodeling
-Called PERISOTEAL cells on bone surfaces and ENDOSTEAL on the inside
-gap junctions present where processes contact one another
-thought to maintain and nutritionally supply osteocytes embedded in bone matrix and regulate Ca
movement into and out of bone
5. Osteoclasts – responsible for bone resorption, resting on bone where resorption is taking place.
Shallow resorption bay (howship’s lacuna) can be observed in bone directly under osteoclast
-tartrate-resistant acid phosphatase (TRAP) clinical marker for osteoclast activity
-osteoclasts are derived from fusion of mononuclear hematopeoietic progenitor cells under influence of
granulocyte/macrophage progenitor cells (GMP, CFU-GM)
-stromal cells in bone marrow secrete monocyte colony stimulating factor (M-CSF), TNF, and several
interleukins
-cells committed to becoming osteoclasts express c-fos and NF-kB and RANK, which interacts with
RANKL produced on stromal cell surface and is important in osteoclast differentiation
-in inflammation, T cells can produce RANKL to stimulate osteoclast bone resorption
-pathway can be blocked by osteoprotegerin (OPG) which serves as decoy for RANKL
-Newly formed osteoclasts undergo activation process to become bone-resorbing cells
-Osteoclasts exhibit 3 special regions when resorbing bone:
1. Ruffled Border – part of cell in contact with bone contains numerous deep membrane
infoldings forming microvillous structures that increase surface area for exocytosis of hydrolytic
enzymes and proteins as well as endocytosis of degradation products
2. Clear Zone – ring-like perimeter of cytoplasm adjacent to ruffled border that demarcates
bone area being resorbed, where resorption and degradation occurs and contains actin
filaments and vinculin/talin
-plasma membrane has ECM adhesion molecules – integrins
3. Basolateral region – function in exocytosis of digested material, TRAP found here
-osteoclasts resorb bone tissue by releasing protons and lysosomal hydrolases into constricted
microenvironment of extracellular space
-enzymes are cathepsin K, MMPs degrade collagen and other proteins
-before digestion can occur, bone matrix must be decalcified through acidification, so osteoclasts
contain carbonic anhydrase II, produces H2CO3 which dissociates to H+ and HCO3, and then ATPdependent proton pumps push H+ out of ruffled border to make pH 4 or 5
-chloride channels and proton pumps facilitate electroneutrality of ruffled border, and excess HCO3 is
removed via a Cl-HCO3 protein exchanger at basolateral membrane
-acidic environment initiates degradation of mineral component of bone to Ca, PO4, H2O
-phagocytic function of osteoclasts is regulated by factors such as parathyroid hormone (PTH) which
promotes bone resorption and calcitonin secreted by thyroid gland reduces osteoclast activity
-molecules that play a role in osteoclast activity are cathepsin K, carbonic anhydrase II, and TCIRG1
(proton pump)
-deficiency of these causes osteopetrosis – increased bone density and defective osteoclast
function
Clinical Correlation – Osteoporosis – means porous bone, most common bone disease characterized by
progressive loss of bone density with architecture deterioration
-caused by imbalance of osteoclast resorption and osteoblast deposition, resulting in decreased bone
mass, enhanced bone fragility, and increased risk of fracture
-osteoclasts regulated by PTH, IL-1 and TNF, M-CSF and IL-6. Female estrogens inhibit these cytokines to
limit osteoclast activity, so postmenopausal women are more at risk
-Three types of osteoporosis:
1. Type I Primary Osteoporosis – post-menopausal women, more severe than type II
2. Type II primary Osteoporosis – occurs in elderly in 70’s or 80’s
3. Secondary Osteoporosis – develops as a result of drug therapy or disease processes such as
malnutrition, prolonged immobilization, weightlessness
-osteoporotic bone has normal histology with less tissue mass; femoral head and neck fractures are
common, as well as compressed vertebral fractures
-treatment is with diet, vitamin D and calcium supplementation/exercise. In post-menopausal women,
hormone-replacement therapy is effective with estrogen and progesterone
-Selective estrogen receptor modulators are also treatment options (mimic estrogen)
-bisphosphonates inhibit osteoclastic activity by inducing apoptosis
-recombinant PTH is a therapy, and newer therapies target RANK, RANKL and OPG molecules such as
neutralizing monoclonal antibodies against RANKL
Bone Formation – development of bone is classified as endochondral or intramembranous
-called endochondral if cartilage serves as precursor to bone, or intramembranous if without
-bones of extremities and vertebrae develop from endochondral ossification, and flat bones of skull,
face, mandible, and clavicle develop by intramembranous ossification
Intramembranous Ossification – bone is formed by differentiation of mesenchymal cells into
osteoblasts. Mesenchymal cells differentiate into osteoprogenitor cells expressing CBFA1 to
differentiate into osteoblasts. Area becomes vascularized and mesenchymal cells become more
osteoblastic and secreting type I collagen, bone sialoproteins, osteocalcin, and other BMPs
-newly formed bone appears in histologic sections as small, irregularly shaped spicules and trabeculae
-in appositional growth – spicules enlarge and join in trabecular network with general shape of
developing bone, and osteoprogenitor cells provide source of osteoblasts for growth of bone spicules
Endochondral Ossification – begins with proliferation and aggregation of mesenchymal cells at site of
future bone, and under influence of FGFs and BMPs, they express type II collagen and differentiate into
chondroblasts to produce cartilage matrix
-initially a hyaline cartilage model with general shape of bone is formed, and once established, it grows
by interstitial and apposition growth
-increase in length of cartilage model is due to interstitial growth, and width is a result of
addition of cartilage matrix produced by new chondrocytes from layer of perichondrium
-first sign of ossification is appearance of a cuff of bone around cartilage model; this is when osteoblasts
are first produced, and CT around cartilage is no longer a perichondrium, it is now called a periOSTEUM,
and an osteogenic layer can now be identified
-in long bones, a bony collar is established around cartilage model in diaphysis
-with establishment of periosteal bony collar, chondrocytes in midregion of cartilage model become
hypertrophic, enlarging and resorbing surrounding cartilage, begin to synthesize alkaline phosphatase
and surrounding matrix undergoes calcification
-Calcified cartilage matrix inhibits diffusion of nutrients, causing death of chondrocytes in cartilage
model
-Mesenchymal stem cells migrate into cavity along the growing blood vessels and differentiate into
osteoprogenitor cells; hematopoietic stem cells gain access to new vasculature and enter bone marrow
to produce blood cell lineages
-first site where bone begins to grow in diaphysis of long bone is called primary ossification center, and
combination of bone and underlying calcified cartilage is called mixed spicule
Growth of Endochondral Bone – begins in 2nd trimester of fetal life and continues to adulthood (12th
week of gestation)
-growth in length of long bones depends on presence of epiphyseal cartilage
-during endochondral formation of bone, avascular cartilage is gradually replaced by vascularized bone
tissue through vascular endothelial growth factor (VEGF) and expression of genes for type X collagen
and MMPs (degrade cartilage)
-the Zones of epiphyseal cartilage beginning with zones most distal to diaphyseal center of ossification
and proceeding toward center are:
1. Zone of Reserve Cartilage – exhibits no cell proliferation or matrix production
2. Zone of Proliferation – adjacent to reserve cartilage, cartilage undergoes division and
organization into columns, produce type II and XI collagen
3. Zone of Hypertrophy – enlarged cartilage cells that are metabolically active and continue
secreting collagen type I and increasing type X collagen, also secrete VEGF
4. Zone of Calcified Cartilage – hypertrophied cells begin to degenerate and cartilage matrix
becomes calcified , which serves as scaffold for new bone deposition
5. Zone of Resorption – zone nearest the diaphysis, here calcified cartilage is in direct contact
with connective tissue of marrow; small blood vessels and CT invade region previously occupied
by dying chondrocytes
-Bone deposition occurs on cartilage spicules in the same manner as described for formation of initial
ossification center
-as bone is laid down on calcified spicules, cartilage is resorbed leaving primary spongy bone which
undergoes reorganization through osteoclastic activity and addition of new bone tissue
-a secondary ossification center develops at proximal epiphysis after birth; cartilage cells undergo
hypertrophy and degenerate; calcification of the matrix occurs, and blood vessels and osteogenic cells
from perichondrium invade region to create marrow cavity
-only the cartilage that remains from original model is articular cartilage at the ends of bone and
a transverse disc of cartilage known as epiphyseal growth plate separates the epiphyseal and
diaphyseal cavities
-cartilage of the epiphyseal growth plate is responsible for maintaining growth processes
-proliferative zone of epiphyseal growth plate gives rise to cartilage on which bone is later laid down
-thickness of epiphyseal plate remains constant during growth
-amount of new cartilage produce (zone of proliferation) equals amount resorbed (zone of resorption)
-resorbed cartilage is replaced by spongy bone
-lengthening of bone occurs when new cartilage matrix is produced at epiphyseal plate, which pushes
epiphysis away from diaphysis to elongate the bone
-bone increases in width or diameter when appositional growth of new bone occurs between cortical
lamellae and periosteum
-at maximal growth, proliferation of new cartilage in epiphyseal growth plate terminates and epiphyseal
and diaphyseal marrow becomes confluent, called epiphyseal closure. Vestigial evidence of epiphyseal
plate is called epiphyseal line
Clinical Correlation – Nutritional Factors in Bone Formation – Ca deficiency causes rickets, where bone
matrix does not calcify normally and may be caused by insufficient dietary calcium or vitamin D needed
for Ca absorption: xray shows bowed lower lumbs, deformed chest and skull
-in adults ,the same nutritional or vitamin deficiency leads to osteomalacia
-vitamins A and C also affect bone: vitamin A suppresses endochondral growth of bone and excess leads
to fragility and fractures. Vitamin C is essential for collagen synthesis and deficiency leads to scurvy;
post-menopausal women = osteoporosis
Development of Osteonal (Haversian System) – osteons develop in preexisting compact bone
-compact bone can take several forms: formed from fetal spongy bone by continued bone deposition; it
may be deposited directly as adult compact bone; or it may be older compact bone consisting of osteons
and interstitial lamellae
-new osteons forming is called internal remodeling
-during development of new osteons, osteoclasts bore a tunnel, a resorption cavity through compact
bone
-this tunnel has dimensions of new osteon
-once osteoclasts produced a tunnel, blood vessels and CT come into the tunnel and new bone
deposition on its wall begins immediately
-osteoclast resorption and osteoblast synthesis constitute a bone-remodeling unit consisting of cuttong
zone (resorption canal) and a closing zone
-tip of cutting zone consists of advancing osteoclasts closely followed by an advancing capillary loop and
pericytes, as well as osteoblasts
-osteoclasts drill a hole 200um in diameter to establish future Haversian system
-once diameter is established of haversian system, osteoblasts fill canal by depositing organic bone
matrix (osteoid) on its walls in successive lamellae
-compact adult bone contains Haversian systems of varying age and size
-in adult, deposition balances absorption; in aged, resorption often wins out to develop osteoporosis
Biologic Mineralization and Matrix Vesicles – biologic mineralization is cell- regulated extacellular event
and occurs in the ECM of bone depositing collagen and ground substance
-mineralization involves secretion of matrix vesicles into bony matrix
-places where mineralization of bone is initiated, Ca, PO4 ions must exceed normal threshold
-binding of extracellular Ca by osteocalcin and other sialoproteins creates high Ca concentration
-high Ca stimulates osteoblast to secrete alkaline phosphatase to increase PO4 ions which further
increases Ca stimulation
-osteoblasts release small matrix vesicles into bony matrix by exocytosis containing ALP and
pyrophosphatase to cleave PO4 from other molecules of matrix
-mastrix vesicles that accumulate Ca and cleave PO4 cause crystallization of CaPO4 in vesicles
-CaPO4 crystals initiate matrix mineralization by formation of hydroxyapatite crystals in matrix around
osteoblasts
Physiologic Aspects of Bone – bone serves as reservoir for body calcium which is essential for health
and life; regulation of calcium regulated by PTH (raise blood levels) and calcitonin (lower blood levels)
-PTH stimulates osteocytes and osteoclasts to resorb bone, allowing Ca release into blood, constituting
osteocytic osteolysis
-PTH also reduced excretion of Ca by kidney and stimulates Ca absorption by small intestine
-PTH stimulates kidney to excrete PO4 produced by bone resorption
-Calcitonin inhibits bone resorption to inhibit effects of PTH on osteoclasts
-Bone can repair itself after injury – initial response to a fracture is neutrophil recruitment followed by
macrophages to clean up the mess, then fibroblasts and capillaries proliferate and grow into injury; new
loose CT (granulation tissue) is formed and becomes denser, and cartilage forms in parts of it; The
dense CT and cartilage grow to produce a soft callus to help stabilize and bind together the fractured
bone
-callus forms and osteoprogenitor cells divide into osteoblasts to deposit new bone on outer surface of
bone at some distance from fracture, and continues to grow into fracture until it reaches the callus.
-osteogenic buds from new bone invade callus and begin depositing new bone inside, becoming a bony
callus, which calcifies and is replaced by bone similar to endochondral ossification
-takes 6-12 weeks in healthy people
-Other hormones involved are Growth Hormone, which stimulates growth of epiphyseal cartilage and
bone by directly acting on osteoprogenitor cells
-chondrocytes in epiphyseal growth plates are regulated by insulinlike growth factor I (IGF-I) produced in
liver in response to GH