Osteoclast/ dental courses

INDIAN DENTAL ACADEMY
Leader in continuing Dental
Education
www.indiandentalacademy.com

References
₪Tencate,
s - Oral Histology
₪Development structure and Function
₪Orbans Oral Histology – 13th edition
₪Osteoclast Differentiation
₪ G. David Roodman
₪ Department of Medicine, Division of Hematology, University of Texas
Health Science Center and the Audie L. Murphy Memorial Veterans
Hospital, Research Service, San Antonio, Texas

References
₪Identification and characterization of osteoclast-like
cells and their progenitors in cultures of feline marrow
mononuclear cells
₪ KENNETH. IBBOTSON, G. DAVID ROODMAN, LINDA M . McMANUS, and
GREGORY R. MUNDY
₪ The Medical Service of the Audie L. Murphy Memorial Veterans
Administration Hospital and Departments of Medicine and Pathology of the
University of Texas Health Science Center, San Antonio, Texas 78284

References
₪OSTEOCLAST FUNCTION AND ITS CONTROL
₪ MONE ZAIDI, MICHAEL PAZIANAS, VIJAI S. SHANKAR,BRIDGET E. BAX,
CHRISTOPHER M. R. BAX, PETER J. R. BEVIS, CLIFFORD STEVENS*,
CHRISTOPHER L.-H. HUANGt, DAVID R. BLAKE*, BALJIT S. MOONGA
AND A. S. M. TOWHIDUL ALAM
₪ Department of Cellular and Molecular Sciences, Bone Research Unit, St
George's Hospital Medical School, London SW17 ORE,
₪ Bone and Joint Research Unit, the London Hospital Medical School, London
EC,
₪ Physiological Laboratory, University of Cambridge, Cambridge CB2 3EG
₪ (MANUSCRIPT RECEIVED 14 SEPTEMBER 1992, ACCEPTED 2 MARCH 1993)

Learning Objectives
• At the end of the seminar the learner should be able
to know about
₪ Osteoclast cells and its features
₪ Origin of Osteoclast cell
₪ Differentiating markers of osteoclast
₪ Mechanism of action of Osteoclast cell.www.indiandentalacademy.com

Osteoclast (OCL)
₪ Bone is a dynamic tissue that under goes
constant remodelling through balancing bone
formation and resorption.
₪Formation  Osteoblast cells
₪ Resorption  Osteoclast cells

Osteoclast (OCL)
₪ The osteoclast (OCL) is the primary bone
resorbing cell.
₪ The osteoclast were first recognised in 1873 by
Albert Kolliker as a multinuclear cell that digest
bone.
₪ It is a highly specialized multinucleated cell
whose primary function is to help in the control
of calcium homeostasis.

Osteoclast (OCL)
₪ Increase in the degree of multinucleation enhance
the capacity of OCL to resorb bone.
₪ More no. of nuclei  more bone resorption
₪ Less no. of nuclei  less bone resorption

Osteoclast (OCL)
₪ Compare to other bone cells and their respective
precursors, osteoclast are much larger cells.
₪ And thus because of their size they can be easily
identified under light microscope and often are seen
in clusters.
Identification Features :

Osteoclast (OCL)

Osteoclast (OCL)
₪ The osteoclast is an extremely large cell, ranging in
size from 50 -100 µm in diameter.
₪ It is multinucleated and may contain 2-100 nuclei per
cell, but usually contains between 10 and 20.

Osteoclast (OCL)
₪ Typically osteoclast are found against the surface of
bone, occupying the hollow out depression known as
HOWSHIP
,
s LACUNAE.
₪ These are the shallow trough which shows the
activity and mobility of osteoclast cell during active
resorption.

Osteoclast (OCL)
₪The osteoclast is located at
₪ Endosteal bone surfaces,
₪ Within the haversian system,
₪ And on periosteal surfaces beneath the
periosteum.
₪Osteoclasts are most abundant at sites of active
bone cell activity, such as metaphyses of growing
bone.

Osteoclast (OCL)
₪ In electron microscopy osteoclasts have several
unique ultrastructural features.
₪The characteristic ruffled border
₪Forms at the site where the osteoclast is attached
to bone matrix
₪Is a convolution of plasma membrane with many
long cytoplasmic processes.

Osteoclast (OCL)
₪The plasma membrane is closely apposed to the bone
surface with an adjacent organelle free area, the clear
zone, which is rich in actin like filaments
₪ Actin
₪ Vinculin
₪ Talin

₪ A many of cell membrane infolding making
up the ruffled border
₪ Sealing zone of osteoclast showing striation
and granularity representing the concentration
of contractile protein in this region.

Osteoclast (OCL)
₪This clear zone or sealing zone
₪Attaches the cells to the mineralized surface
₪Isolate the microenvironment between them and
bone surface.

Osteoclast (OCL)
₪Lamina Limitans (electron dense interfacial matrix
layer) often observed between the sealing zone and
calcified tissue surface.
₪ Immunocytochemical preparation of
osteopontin showing black dots of
osteopontin on the bone surface at sealing
zone.

Osteoclast (OCL)
₪This layer consist of RGD(arg - gly- aspartic acid ) containing
molecules
₪Bone sialoprotein
₪Osteopontin
₪ That facilitate the osteoclast adhesion and formation
of sealing zone via αvβ3 mediated mechanism.

Osteoclast (OCL)
₪Other ultrastructural characteristics of osteoclasts
include
₪Large numbers of lysosomes,
₪Numerous pleomorphic mitochondria,
₪Extensive golgi complexes located around the
nucleus

₪Osteoclast possess numerous golgi complexes located near the nuclei and mitochondriawww.indiandentalacademy.com

Osteoclast (OCL)
₪ The cytoplasm is characterized by dense granules.
₪In addition have many prominent vesicles, especially
in the region of the ruffled border.
₪The multiple nuclei are centrally located, but have a
highly variable shape and contain one prominent
nucleolus per nucleus.

Osteoclast (OCL)
₪ Cytochemically they are identified by possessing
TRAP (tartrate resistantant acid phosphatase) with
in the cytoplasmic vesicles and vacuoles.
Photomicrograph showing the red staining associated with the presence of
tartrate-resistant acid phosphatase (TRAP) in the cytoplasm of a multinucleated,
culture-generated mouse osteoclast. Bar 5 -20 mm.

Osteoclast (OCL)
₪ A variety of studies have clearly indicated that the
osteoclast is hematopoietic in origin and not bone
derived.
₪ But still the origin of the osteoclast is a subject of
intense investigation and controversy.
origin:

Osteoclast (OCL)
₪ Walker et al. reported that transplantation of spleen
and marrow cells into osteotropic mice results in
removal of the excess bone present in these animals.
origin:

Osteoclast (OCL)
₪ Marks has shown that transplantation of spleen cells
into the osteopetrotic rat can cure the disease.
₪ In contrast to untreated rats whose osteoclasts lack
ruffled borders, infusion of mononuclear spleen cells
resulted in the formation of osteoclasts with ruffled
borders.
origin:

Osteoclast (OCL)
₪ All these studies are conclude that the osteoclast
precursor is a mononuclear cell that is hematopoietic
in origin.
₪ The osteoclast forms by fusion of mononuclear
precursors rather than endomitosis.
origin:

Osteoclast (OCL)
₪ Multinucleated osteoclast arise from the hematopoietic
precursors of the monocyte /macrophage lineage
₪ Stromal cells in the marrow cavity and osteoblast
modulate the differentiation of osteoclast.
origin:

Osteoclast (OCL)
₪ Stromal cells and osteoblast modulate osteoclast
differentiation via
₪Various molecules
₪Direct Cell to cell interaction
origin:

Osteoclast (OCL)
₪ Stromal cells and oasteoblast expresses
RANKL(receptor-activated nuclear factorκB) over their plasma
membrane.
₪ RANKL binds to the RANK presented on the
progenitors of osteoclast.
origin:

Osteoclast (OCL)
₪ This attachment cause induction of signalling cascade
leading to induction of
₪Osteoclast differentiation
₪Fusion of osteoclast precursors cell
₪Leading to the increased survival of activity of
osteoclast
origin:

Osteoclast (OCL)
origin:

Osteoclast (OCL)
Differentiation markers
₪ Histochemical studies have shown that osteoclasts
contain a Tartrate-resistant acid phosphatase (TRAP)
activity that is found in
₪Lysosomes,
₪Golgi complexes,
₪Extracellular channels of the ruffled border,
₪ And the space between cells and bones.

Osteoclast (OCL)
₪ Tartrate-resistant acid phosphatase appears to be a
marker for osteoclasts in bone sections.
₪ Other acid hydrolases are present in osteoclasts,
including
₪Leucine aminopeptidase,
₪β-d-galactosidase,
₪β-d-glucosidase.
All are non specific for the osteoclast.www.indiandentalacademy.com

Osteoclast (OCL)
₪ Calcitonin receptors have been suggested as a good
maker for mammalian osteoclast differentiation.
₪ This differentiate osteoclast cells from other cells of
the mononuclear phagocyte series.

Osteoclast (OCL)
₪ Gay and co-workers have examined carbonic-
anhydrase activity in chicken osteoclasts by electron
microscopy.
₪ By using histochemical technique they found positive
stains for carbonic-anhydrase in
₪Cytosol,
₪Golgi apparatus and other vesicles,
₪Plasma membrane and its Ruffled border,
₪Bone surface beneath the osteoclast.

Osteoclast (OCL)
₪ Monoclonal antibody techniques have examined the
surface phenotype of osteoclasts and identify unique
differentiation markers for osteoclasts.
₪121F
₪ 23c6 and 13c2 (directed against the vitronectin receptor
present on osteoclasts.)

Osteoclast (OCL)
₪ Other markers for osteoclasts are
₪Matrix Metalloproteinase (MMP)-9,
₪Cathepsin K,
₪Carbonic Anhydrase II (CAII),
₪a3 Subunit Of The Vacuolar [H+]-ATPase (proton
pump),
₪Chloride Channel 7 (ClC-7),

Osteoclast (OCL)
Factors affecting differentiation of OCL
₪ Factors affecting the differentiation of osteoclasts are
₪ 1,25-Dihydroxyvitamin D3
₪Parathyroid Hormone and Parathyroid Hormone-
Related Peptide
₪GM-CSF
₪CSF-1
₪Interleukin-3, -1, -6.
₪Transforming Growth Factor-α and -β

Osteoclast (OCL)
Factors affecting differentiation of OCL
₪ Factors affecting the differentiation of osteoclasts are
₪Tumor Necrosis Factor-α and -β
₪Gamma Interferon
₪Calcitonin
₪Osteoinductive Factor
₪1,25D3 Lactone
₪Prostaglandins

Osteoclast (OCL)
Mechanism of action of bone resorption :
₪ An osteoclast resorbs bone through the formation of a
sealing zone of close adhesion between itself and the
bone.
₪The adjacent plasma membrane is thrown into complex
folds, the 'ruffled border', which consists of finger-like
membrane projections.

Osteoclast (OCL)
₪ The 'ruffled border' and 'clear zone' constitute a
system for bone resorption unique to the osteoclast.
₪ The expression of these structures generally depends
upon the level of cellular activity.

Osteoclast (OCL)
₪ Thus, regulators of osteoclast activity rapidly increase
or decrease the size of the 'ruffled border' and 'clear
zone'.
₪Parathyroid hormone,
₪ 1,25-dihydroxy vitamin D3
₪ Prostaglandin E2
₪ Calcitonin www.indiandentalacademy.com

Osteoclast (OCL)
₪ Role of mineral
₪ Osteoclast activation requires contact with bone
mineral.
₪ Osteoclasts can resorb calcified cartilage, enamel,
dentine or cementum in addition to mineralized bone

Osteoclast (OCL)
₪ Role of mineral
₪ Uncalcified cartilage is not resorbed by osteoclasts
₪The osteoid or related unmineralized collagenous
matrix could prevent effective osteoclastic activation.

Osteoclast (OCL)
₪ Role of mineral
₪ Osteoclasts, normally resting in the periosteal and
endosteal mesenchyme,
₪ For its activation it must cross a cellular (osteoblast)
barrier and possibly a second osteoid barrier to gain
contact with mineral.

Osteoclast (OCL)
₪ Role of osteoblast
₪ The osteoblast is the primary target cell in hormonal
activation of the osteoclast as it possess specific
surface receptors for
₪parathyroid hormone
₪parathyroid hormone-related protein (PTHrP),
₪1,25-dihydroxyvitamin D3.

Osteoclast (OCL)
₪ It is proven that
₪cell contact
₪soluble products released by the osteoblast
is required for osteoblast-induced osteoclastic
stimulation to take place.
₪ The nature of this soluble factor is still unclear 0.5-1
kDa to over 25 kDa.

Osteoclast (OCL)
₪ Osteoclast adhesion to bone
₪ In order to resorb bone, the osteoclast must
effectively adhere to the bone surface.
₪ It contain characterized adhesive receptor, osteoclast
functional antigen (OFA),

Osteoclast (OCL)
₪ OFA comprises of
₪140 kDa δ-chain (immunologically cross-reacts with the
vitronectin δ-chain receptor)
₪85 kDa β-chain related to the platelet gp III-a
molecule (β3, integrin).

Osteoclast (OCL)
₪ OFA expression on bone cells is restricted to
osteoclasts and preosteoclasts.

Osteoclast (OCL)
₪ Integrins, such as the vitronectin receptor, are
transmembrane proteins that act as linkers between
₪Extracellular ligands and cytoskeletal components,
such as vinculin, talin, δ-actinin and F-actin

Osteoclast (OCL)
₪ The ligands in bone containing the RGD (arginine-
glycine-aspartic acid) tripeptide recognition sequence
that could potentially bind to OFA.
₪ These include
₪Osteopontin,
₪Thrombospondin
₪Bone sialoprotein.

Osteoclast (OCL)
₪ Mineral dissolution
₪ Osteoclasts secrete acid ,lactate and citrate and thus
maintain a low pH (values of 4.5 ) in the resorptive
hemivacuole.
₪ Active H+ion secretion from the osteoclast was
demonstrated by using Acridine Orange as an indicator
of extracellular acidification.

Osteoclast (OCL)
₪ Acridine Orange fluorescence was increased by
₪Parathyroid hormone
₪Prostaglandin E2,
₪ And decreased by calcitonin

Osteoclast (OCL)
₪ It has been reported that the apical membrane of the
'ruffled border' contain proton pumps that may be
involved in the acidification of the extracellular
resorptive hemivacuole.

Osteoclast (OCL)
₪ Two types of proton pumps have been implicated in
the acidification process.
₪ V-type or Electrogenic vacuolar type proton pump
(H+-ATPase){predominantly involved}
₪P-type plasma membrane pump
(H+- K+-ATPase).

Osteoclast (OCL)
₪ Role of Carbonic anhydrases
₪ The proton pump in the osteoclast is supplied by
protons generated by the action of carbonic anhydrase
isoenzyme II
₪ Immunocytochemical techniques have localized the
enzyme to the 'ruffled border' of active osteoclasts.

Osteoclast (OCL)
₪ It catalyses the conversion of CO2 (generated from
cell respiration) to HCO3
- and H+.
CO2  HCO3
- + H+
₪ Shows that bone resorption is critically dependent
upon CO2 concentration.

Osteoclast (OCL)
₪ Meanwhile, basolateral exchange of HCO3-ions for Cl-
by anion exchanger provides Cl- ions required for the
intense acidification occurring in the resorption lacuna.

Osteoclast (OCL)
₪ Matrix degradation
₪ Osteoclastic lysosomal enzymes are destined for
secretion and transported in a specific direction via
primary lysosomes and are released into the resorptive
hemivacuole at the 'ruffled border'.

Osteoclast (OCL)
₪ Lysosomal hydrolases, exhibiting optimal activity at an
acid pH, resorb the organic matrix of bone.
₪ Collagen can be digested by a number of lysosomal
cysteine-proteinases in the acid environment.

Osteoclast (OCL)
₪ The three types of collagenolytic cysteine-proteinases
found in bone
₪cathepsins L,
₪cathepsins B,
₪cathepsins N.

Osteoclast (OCL)
₪ Increased levels of acid phosphatase activity in plasma
occur during physiological and pathological states of
increased bone remodelling.
₪ This isoenzyme is not inhibited by dextro-rotatory
tartaric acid, (a competitive inhibitor of prostatic and lysosomal acid
phosphatases)

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