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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
www.indiandentalacademy.com
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
www.indiandentalacademy.com
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)
www.indiandentalacademy.com
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
www.indiandentalacademy.com
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.
www.indiandentalacademy.com
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
www.indiandentalacademy.com
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 :
www.indiandentalacademy.com
Osteoclast (OCL)
www.indiandentalacademy.com
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.
Identification Features :
www.indiandentalacademy.com
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.
Identification Features :
www.indiandentalacademy.com
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.
Identification Features :
www.indiandentalacademy.com
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.
Identification Features :
www.indiandentalacademy.com
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
Identification Features :
www.indiandentalacademy.com
₪ 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.
www.indiandentalacademy.com
Osteoclast (OCL)
₪This clear zone or sealing zone
₪Attaches the cells to the mineralized surface
₪Isolate the microenvironment between them and
bone surface.
Identification Features :
www.indiandentalacademy.com
Osteoclast (OCL)
₪Lamina Limitans (electron dense interfacial matrix
layer) often observed between the sealing zone and
calcified tissue surface.
Identification Features :
₪ Immunocytochemical preparation of
osteopontin showing black dots of
osteopontin on the bone surface at sealing
zone.
www.indiandentalacademy.com
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.
Identification Features :
www.indiandentalacademy.com
Osteoclast (OCL)
₪Other ultrastructural characteristics of osteoclasts
include
₪Large numbers of lysosomes,
₪Numerous pleomorphic mitochondria,
₪Extensive golgi complexes located around the
nucleus
Identification Features :
www.indiandentalacademy.com
₪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.
Identification Features :
www.indiandentalacademy.com
Osteoclast (OCL)
₪ Cytochemically they are identified by possessing
TRAP (tartrate resistantant acid phosphatase) with
in the cytoplasmic vesicles and vacuoles.
Identification Features :
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.
www.indiandentalacademy.com
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:
www.indiandentalacademy.com
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:
www.indiandentalacademy.com
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:
www.indiandentalacademy.com
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:
www.indiandentalacademy.com
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:
www.indiandentalacademy.com
Osteoclast (OCL)
₪ Stromal cells and osteoblast modulate osteoclast
differentiation via
₪Various molecules
₪Direct Cell to cell interaction
origin:
www.indiandentalacademy.com
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:
www.indiandentalacademy.com
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:
www.indiandentalacademy.com
Osteoclast (OCL)
origin:
www.indiandentalacademy.com
Osteoclast (OCL)
origin:
www.indiandentalacademy.com
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.
www.indiandentalacademy.com
Osteoclast (OCL)
Differentiation markers
₪ 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)
Differentiation markers
₪ 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.
www.indiandentalacademy.com
Osteoclast (OCL)
Differentiation markers
₪ 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.
www.indiandentalacademy.com
Osteoclast (OCL)
Differentiation markers
₪ 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.)
www.indiandentalacademy.com
Osteoclast (OCL)
Differentiation markers
₪ 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),
www.indiandentalacademy.com
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
www.indiandentalacademy.com
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
www.indiandentalacademy.com
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.
www.indiandentalacademy.com
Osteoclast (OCL)
Mechanism of action of bone resorption :
₪ 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.
www.indiandentalacademy.com
Osteoclast (OCL)
Mechanism of action of bone resorption :
₪ 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)
Mechanism of action of bone resorption :
₪ Role of mineral
₪ Osteoclast activation requires contact with bone
mineral.
₪ Osteoclasts can resorb calcified cartilage, enamel,
dentine or cementum in addition to mineralized bone
www.indiandentalacademy.com
Osteoclast (OCL)
Mechanism of action of bone resorption :
₪ Role of mineral
₪ Uncalcified cartilage is not resorbed by osteoclasts
₪The osteoid or related unmineralized collagenous
matrix could prevent effective osteoclastic activation.
www.indiandentalacademy.com
Osteoclast (OCL)
Mechanism of action of bone resorption :
₪ 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.
www.indiandentalacademy.com
Osteoclast (OCL)
Mechanism of action of bone resorption :
₪ 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.
www.indiandentalacademy.com
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.
Mechanism of action of bone resorption :
www.indiandentalacademy.com
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),
Mechanism of action of bone resorption :
www.indiandentalacademy.com
Osteoclast (OCL)
₪ Osteoclast adhesion to bone
₪ 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).
Mechanism of action of bone resorption :
www.indiandentalacademy.com
Osteoclast (OCL)
₪ Osteoclast adhesion to bone
₪ OFA expression on bone cells is restricted to
osteoclasts and preosteoclasts.
Mechanism of action of bone resorption :
www.indiandentalacademy.com
Osteoclast (OCL)
₪ Osteoclast adhesion to bone
₪ 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
Mechanism of action of bone resorption :
www.indiandentalacademy.com
Osteoclast (OCL)
₪ Osteoclast adhesion to bone
₪ 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.
Mechanism of action of bone resorption :
www.indiandentalacademy.com
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.
Mechanism of action of bone resorption :
www.indiandentalacademy.com
Osteoclast (OCL)
₪ Mineral dissolution
₪ Acridine Orange fluorescence was increased by
₪Parathyroid hormone
₪Prostaglandin E2,
₪ And decreased by calcitonin
Mechanism of action of bone resorption :
www.indiandentalacademy.com
Osteoclast (OCL)
₪ Mineral dissolution
₪ 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.
Mechanism of action of bone resorption :
www.indiandentalacademy.com
Osteoclast (OCL)
₪ Mineral dissolution
₪ 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).
Mechanism of action of bone resorption :
www.indiandentalacademy.com
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.
Mechanism of action of bone resorption :
www.indiandentalacademy.com
Osteoclast (OCL)
₪ Role of Carbonic anhydrases
₪ 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.
Mechanism of action of bone resorption :
www.indiandentalacademy.com
Osteoclast (OCL)
₪ Role of Carbonic anhydrases
₪ Meanwhile, basolateral exchange of HCO3-ions for Cl-
by anion exchanger provides Cl- ions required for the
intense acidification occurring in the resorption lacuna.
Mechanism of action of bone resorption :
www.indiandentalacademy.com
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'.
Mechanism of action of bone resorption :
www.indiandentalacademy.com
Osteoclast (OCL)
₪ Matrix degradation
₪ 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.
Mechanism of action of bone resorption :
www.indiandentalacademy.com
Osteoclast (OCL)
₪ Matrix degradation
₪ The three types of collagenolytic cysteine-proteinases
found in bone
₪cathepsins L,
₪cathepsins B,
₪cathepsins N.
Mechanism of action of bone resorption :
www.indiandentalacademy.com
Osteoclast (OCL)
₪ Matrix degradation
₪ 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)
Mechanism of action of bone resorption :
www.indiandentalacademy.com
Osteoclast (OCL)
www.indiandentalacademy.com
www.indiandentalacademy.com
www.indiandentalacademy.com
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Osteoclast/ dental courses

  • 1. INDIAN DENTAL ACADEMY Leader in continuing Dental Education www.indiandentalacademy.com
  • 2. 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 www.indiandentalacademy.com
  • 3. 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 www.indiandentalacademy.com
  • 4. 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) www.indiandentalacademy.com
  • 5. 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
  • 6. Osteoclast (OCL) ₪ Bone is a dynamic tissue that under goes constant remodelling through balancing bone formation and resorption. ₪Formation  Osteoblast cells ₪ Resorption  Osteoclast cells www.indiandentalacademy.com
  • 7. 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. www.indiandentalacademy.com
  • 8. 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 www.indiandentalacademy.com
  • 9. 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 : www.indiandentalacademy.com
  • 11. 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. Identification Features : www.indiandentalacademy.com
  • 12. 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. Identification Features : www.indiandentalacademy.com
  • 13. 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. Identification Features : www.indiandentalacademy.com
  • 14. 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. Identification Features : www.indiandentalacademy.com
  • 15. 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 Identification Features : www.indiandentalacademy.com
  • 16. ₪ 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. www.indiandentalacademy.com
  • 17. Osteoclast (OCL) ₪This clear zone or sealing zone ₪Attaches the cells to the mineralized surface ₪Isolate the microenvironment between them and bone surface. Identification Features : www.indiandentalacademy.com
  • 18. Osteoclast (OCL) ₪Lamina Limitans (electron dense interfacial matrix layer) often observed between the sealing zone and calcified tissue surface. Identification Features : ₪ Immunocytochemical preparation of osteopontin showing black dots of osteopontin on the bone surface at sealing zone. www.indiandentalacademy.com
  • 19. 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. Identification Features : www.indiandentalacademy.com
  • 20. Osteoclast (OCL) ₪Other ultrastructural characteristics of osteoclasts include ₪Large numbers of lysosomes, ₪Numerous pleomorphic mitochondria, ₪Extensive golgi complexes located around the nucleus Identification Features : www.indiandentalacademy.com
  • 21. ₪Osteoclast possess numerous golgi complexes located near the nuclei and mitochondriawww.indiandentalacademy.com
  • 22. 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. Identification Features : www.indiandentalacademy.com
  • 23. Osteoclast (OCL) ₪ Cytochemically they are identified by possessing TRAP (tartrate resistantant acid phosphatase) with in the cytoplasmic vesicles and vacuoles. Identification Features : 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. www.indiandentalacademy.com
  • 24. 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: www.indiandentalacademy.com
  • 25. 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: www.indiandentalacademy.com
  • 26. 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: www.indiandentalacademy.com
  • 27. 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: www.indiandentalacademy.com
  • 28. 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: www.indiandentalacademy.com
  • 29. Osteoclast (OCL) ₪ Stromal cells and osteoblast modulate osteoclast differentiation via ₪Various molecules ₪Direct Cell to cell interaction origin: www.indiandentalacademy.com
  • 30. 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: www.indiandentalacademy.com
  • 31. 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: www.indiandentalacademy.com
  • 34. 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. www.indiandentalacademy.com
  • 35. Osteoclast (OCL) Differentiation markers ₪ 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
  • 36. Osteoclast (OCL) Differentiation markers ₪ 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. www.indiandentalacademy.com
  • 37. Osteoclast (OCL) Differentiation markers ₪ 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. www.indiandentalacademy.com
  • 38. Osteoclast (OCL) Differentiation markers ₪ 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.) www.indiandentalacademy.com
  • 39. Osteoclast (OCL) Differentiation markers ₪ 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), www.indiandentalacademy.com
  • 40. 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 www.indiandentalacademy.com
  • 41. 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 www.indiandentalacademy.com
  • 42. 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. www.indiandentalacademy.com
  • 43. Osteoclast (OCL) Mechanism of action of bone resorption : ₪ 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. www.indiandentalacademy.com
  • 44. Osteoclast (OCL) Mechanism of action of bone resorption : ₪ 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
  • 45. Osteoclast (OCL) Mechanism of action of bone resorption : ₪ Role of mineral ₪ Osteoclast activation requires contact with bone mineral. ₪ Osteoclasts can resorb calcified cartilage, enamel, dentine or cementum in addition to mineralized bone www.indiandentalacademy.com
  • 46. Osteoclast (OCL) Mechanism of action of bone resorption : ₪ Role of mineral ₪ Uncalcified cartilage is not resorbed by osteoclasts ₪The osteoid or related unmineralized collagenous matrix could prevent effective osteoclastic activation. www.indiandentalacademy.com
  • 47. Osteoclast (OCL) Mechanism of action of bone resorption : ₪ 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. www.indiandentalacademy.com
  • 48. Osteoclast (OCL) Mechanism of action of bone resorption : ₪ 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. www.indiandentalacademy.com
  • 49. 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. Mechanism of action of bone resorption : www.indiandentalacademy.com
  • 50. 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), Mechanism of action of bone resorption : www.indiandentalacademy.com
  • 51. Osteoclast (OCL) ₪ Osteoclast adhesion to bone ₪ 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). Mechanism of action of bone resorption : www.indiandentalacademy.com
  • 52. Osteoclast (OCL) ₪ Osteoclast adhesion to bone ₪ OFA expression on bone cells is restricted to osteoclasts and preosteoclasts. Mechanism of action of bone resorption : www.indiandentalacademy.com
  • 53. Osteoclast (OCL) ₪ Osteoclast adhesion to bone ₪ 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 Mechanism of action of bone resorption : www.indiandentalacademy.com
  • 54. Osteoclast (OCL) ₪ Osteoclast adhesion to bone ₪ 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. Mechanism of action of bone resorption : www.indiandentalacademy.com
  • 55. 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. Mechanism of action of bone resorption : www.indiandentalacademy.com
  • 56. Osteoclast (OCL) ₪ Mineral dissolution ₪ Acridine Orange fluorescence was increased by ₪Parathyroid hormone ₪Prostaglandin E2, ₪ And decreased by calcitonin Mechanism of action of bone resorption : www.indiandentalacademy.com
  • 57. Osteoclast (OCL) ₪ Mineral dissolution ₪ 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. Mechanism of action of bone resorption : www.indiandentalacademy.com
  • 58. Osteoclast (OCL) ₪ Mineral dissolution ₪ 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). Mechanism of action of bone resorption : www.indiandentalacademy.com
  • 59. 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. Mechanism of action of bone resorption : www.indiandentalacademy.com
  • 60. Osteoclast (OCL) ₪ Role of Carbonic anhydrases ₪ 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. Mechanism of action of bone resorption : www.indiandentalacademy.com
  • 61. Osteoclast (OCL) ₪ Role of Carbonic anhydrases ₪ Meanwhile, basolateral exchange of HCO3-ions for Cl- by anion exchanger provides Cl- ions required for the intense acidification occurring in the resorption lacuna. Mechanism of action of bone resorption : www.indiandentalacademy.com
  • 62. 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'. Mechanism of action of bone resorption : www.indiandentalacademy.com
  • 63. Osteoclast (OCL) ₪ Matrix degradation ₪ 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. Mechanism of action of bone resorption : www.indiandentalacademy.com
  • 64. Osteoclast (OCL) ₪ Matrix degradation ₪ The three types of collagenolytic cysteine-proteinases found in bone ₪cathepsins L, ₪cathepsins B, ₪cathepsins N. Mechanism of action of bone resorption : www.indiandentalacademy.com
  • 65. Osteoclast (OCL) ₪ Matrix degradation ₪ 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) Mechanism of action of bone resorption : www.indiandentalacademy.com