BONE LOSS AND ITS MEDIATORS

1. MECHANISM AND MEDIATORS
OF BONE DESTRUCTION
SHILPI AGARWAL JR–III
Department of Periodontology
King Geroge’s Medical University
Lucknow

2. CONTENTS
PART I
• Bone destruction in
periodontal disease
• Radius of action of
microbial toxins
• Rate of bone resorption
• Period of destruction
• Bone remodeling cycle
• Periodontal bone disease
- Bacteria derived factors
- Host derived factors
PART II
• Factors Determining Bone
Morphology in Periodontal
Disease
• Bone Destruction Patterns
in Periodontal Disease

3. INTRODUCTION
• The height and density of the alveolar bone are normally
maintained by an equilibrium between bone formation and
bone resorption
• The level of bone is the consequence of past pathologic
experiences, whereas changes in the soft tissue of the
pocket wall reflect the present inflammatory condition.

4. BONE DESTRUCTION IN PERIODONTAL
DISEASE IS CAUSED BY
EXTENSION OF GINGIVAL
INFLAMMATION
TRAUMA FROM OCCLUSION
SYSTEMIC DISORDERS

5. EXTENSION OF GINGIVAL INFLAMMATION
Periodontitis is always preceded by gingivitis, but not all
gingivitis progresses to periodontitis.
The inflammatory invasion of the bone surface and the initial
bone loss that follows mark the transition from gingivitis to
periodontitis.
In advanced stages of disease, the number of motile organisms
and spirochetes increases, whereas the number of coccoid rods
and straight rods decreases.

6. • Chronic gingivitis into a progressive periodontitis by placing
a silk ligature into the sulcus and tying it around the neck of
the tooth.
• This induced ulceration of the sulcular epithelium, a shift in
the inflammatory infiltrate from predominantly plasma cells
PMNs, and osteoclastic resorption of the alveolar crest.
Heijl L, Rifkin BR, Zander HA: Conversion of chronic gingivitis to
periodontitis in squirrel monkeys. J Periodontol 47:710, 1976.

7. HISTOPATHOLOGY
• Gingival inflammation extends along the collagen fiber
bundles and follows the course of the blood vessels through
the loosely arranged tissues around them into the alveolar
bone
• Interproximally, inflammation spreads to loose connective
tissue around the blood vessels, through the fibers, and then
into the bone through vessel channels that perforate the
crest of the interdental septum at the center of the crest

9. PATHWAYS OF INFLAMMATION
INTERPROXIMAL EXTENSION
1- GINGIVA TO BONE
2-BONE TO PDL
3-GINGIVA TO PDL
FACIAL AND LINGUAL EXTENSION
1-GINGIVA ALONG OUTER PERIOSTEUM
2-PERIOSTEUM INTO BONE
3-GINGIVA TO PDL
A B

10. RECREATION OF TRANSCEPTAL FIBRES
• Inflammation
destroys the gingival
and transseptal fibers
• There is a continuous
tendency to recreate
transseptal fibers
across the crest of the
interdental septum

11. SOME IMPORTANT points
• Enlargement of the marrow spaces
• Fatty bone marrow – Fibrous bone marrow
• Bone destruction in periodontal disease is not a process of
bone necrosis. It involves the activity of living cells along
viable bone.
• The amount of inflammatory infiltrate correlates with the
degree of bone loss but not with the number of osteoclasts.

12. RADIUS OF ACTION OF MICROBIAL
TOXINS
There is a range of effectiveness of about 1.5 to 2.5 mm within
which bacterial plaque can induce bone resorption
Waerhaug J., 1979
• Beyond 2.5 mm, there is no effect;
• Interproximal angular defects can appear only in spaces that
are wider than 2.5 mm, because narrower spaces would be
destroyed entirely

13. RATE OF BONE RESORPTION
Loe and colleagues
• 0.2 mm / year- facial surface
• 0.3 mm / year- proximal surface
• % OF
SUBJECTS
• LOSS OF
ATTACHMENT/
year
RAPID
PROGRESSION
• 8%
• 0.1 to 1mm
MODERATE
PROGRESSION
• 81%
• 0.05 to 0.5
MINIMAL
PROGRESSION
• 11%
• 0.05 to 0.09
Loss of attachment can be equated with loss of bone, although attachment
loss precedes bone loss by about 6 to 8 months

14. PERIOD OF DESTRUCTION
• Episodic, intermittent manner, with periods of inactivity or
quiescence that alternate with destructive periods
T Lymphocytic
lesion
B Lymphocytic
(Plasma cell)
ACTIVE LESION
• Gram Negative
• Motile
• Loose
INACTIVE LESION
• Gram Positive
• Non- Motile
• Dense

15. THE BONE REMODELING CYCLE
OSTEOBLAST
• Periodontal pathogens,
specifically Aggregatibacter
actinomycetemcomitans -
stimulate apoptosis of
osteoblastic cells (Gadhavi
A.etal 2000).
• Osteoblast subsequently
becomes an Osteocyte
entrapped in the bone matrix,
dies via apoptosis, or becomes
a lining cell

16. OSTEOCYTE
• Most abundant bone cells
• Communicate with each
other via dendritic
process encapsulated in
canaliculi
• Play role in calcium
homeostasis ( mobilize
calcium from bone matrix
and transport them via
cell processes to
osteoblasts)

17. OSTEOCLASTS
• Osteoclasts are cells derived
from the hematopoietic
lineage
• Express osteoclastic enzyme,
tartrate-resistant acid
phosphatase (TRAP).
• 3 or more nuclei per cell.
• Site of resorption via blood
supply.
• Lifespan of an osteoclast - 2
weeks.
( Manolagas SC.2000).

18. OSTEOCLASTS continued
Other characteristic morphologic features
1. Ruffled membrane
2. Clear zone
3. The proton pump that regulates hydrogen ion concentration

20. BONE RESORPTION PROCESS
• Attachment of the osteoclast to the
bone matrix (Gay CV, Weber
JA.2000).
• This matrix attachment is mediated
by integrins
• Formation of the sealing zone that
enables the osteoclast to isolate a
microenvironment beneath it to
facilitate resorption.

21. Cell membrane of ruffled border contain transmembrane proton [H+]
pump complexes responsible for generating acidified enviroment
beneath the ruffled border

23. PERIODONTAL BONE DISEASE
Hallmarks of periodontal disease (PD)
 INFLAMMATION
 BONE LOSS

24. • The initial response to bacterial infection is a local
inflammatory reaction that activates the innate immune
system.
• Amplification of this initial localized response results in the
release of cytokines and other mediators and propagation of
inflammation through the gingival tissues. (Graves DT. and
Cochran D. 2003)

25. • The failure to encapsulate this ‘‘inflammatory front 'within
gingival tissue results in expansion of the response adjacent
to alveolar bone.
• The inflammatory process then drives the destruction of
connective tissue and alveolar bone that is the cardinal sign
of PD and mark the transient from gingivitis to periodontitis.

26. ROLE OF THE ‘‘INFLAMMATORY FRONT’’ IN
PERIODONTAL BONE RESORPTION
Inflammatory reaction → bone loss
depends on two critical factors.
(Graves DT. and Cochran D. 2003)
1. Concentration of inflammatory mediators to activate
pathways leading to bone resorption.
2. Penetration into gingival tissue to reach within a critical
distance to alveolar bone.

27. The substances that can induce inflammatory responses and bone
resorption in Periodontal disease come from 2 sources:
• Bacterial derived Factors
(Microbial Virulence Factor)
• Host derieved Factors.

28. BACTERIA DERIVED FACTORS
LIPOPOLYSACCHARIDE
• Large molecules composed of a lipid component (lipid A) and
a polysaccharide component.
• Found in the outer membrane of gram-negative bacteria, act
as endotoxins (LPS is frequently referred to as endotoxin).
• Elicit strong immune responses in animals.
• Immune system in animals recognise LPS via Toll like
receptors (TLRs)

29. CD14/TLR-4/MD-2/
complex + LPS
CD14 and
MD-2 LPS
TLR-4
Inflammatory mediators (most notably
cytokines) and the differentiation of immune
cells (e.g., dendritic cells)
Lipopolysaccharide.
•Increased vasodilation and vascular permeability,
•Recruitment of inflammatory cells by chemotaxis,
•Release of proinflammatory mediators by the leukocytes

30. LIPOTEICHOIC ACID
• Gram-positive cell walls, lipoteichoic acid (LTA), also
stimulates immune responses, although less potently than
LPS.
• LTA signals through TLR-2.
• Porphyromonas gingivalis has an atypical form of LPS and is
recognized by both TLR-2 and TLR-4 Dixon DR. et al., (2005).

31. BACTERIAL ENZYMES AND NOXIOUS PRODUCTS
Noxious Products
• Ammonia (NH3)
• Hydrogen sulfide (H2S) → direct tissue damage
• Short-chain carboxylic acids
( butyric acid and propionic acid)
• Butyric acid induces apoptosis in T-cells, B-cells, fibroblasts, and
gingival epithelial cells.
• The short-chain fatty acids may aid P. gingivalis infection through
tissue destruction and may also create a nutrient supply for the
organism by increasing bleeding into the periodontal pocket

32. BACTERIAL ENZYMES
Proteases
1. Break down structural proteins such as collagen, elastin, and
fibronectin.
2. Provide peptides for bacterial nutrition.
• P. gingivalis produces two classes of cysteine proteases-
gingipains
1.Lysine specific gingipain Kgp
2.Arginine specific gingipains RgpA and RgpB
• gingipains modulate the immune system and disrupt
immune-inflammatory responses, leading to increased
tissue breakdown.

33. P. gingivalis and Aggregatibacter actinomycetemcomitans invade
the gingival tissues,including the connective tissues.
Fusobacterium nucleatum can invade oral epithelial cells, and
bacteria that routinely invade host cells may facilitate the entry
of noninvasive bacteria by coaggregating with them.
MICROBIAL INVASION.

34. MICROBIAL INVASION

35. FIMBRIAE
Bacterial fimbriae are important for modifying and stimulating
immune responses in the periodontium.
• P. gingivalis fimbriae stimulate immune responses, such as IL-6
secretion.
• Major fimbrial structural component of P. gingivalis, FimA -
stimulate nuclear factor (NF)- κβ and IL-8 in a gingival epithelial
cell line via TLR-2.
• Monocytes are also stimulated by P. gingivalis FimA, secreting
IL-6, IL-8, and TNF-α.

36. BACTERIAL DEOXYRIBONUCLEIC ACID AND
EXTRACELLULAR DEOXYRIBONUCLEIC ACID
DNA isolated from P. gingivalis, A. actinomycetemcomitans, and
Peptostreptococcus micros stimulates macrophages and gingival
fibroblasts to produce TNF-α and IL-6 in a dose-dependent
manner.
•Bacterial DNA stimulates immune cells via TLR-9, which
recognizes hypomethylated CpG regions of the DNA.
•Immune stimulation by bacterial DNA from subgingival species
could contribute to periodontal pathogenesis.

37. Host Derived Factors
MEDIATORS OF BONE RESORPTION
• STIMULATORS OF BONE RESORPTION
• INHIBITORS OF BONE RESORPTION

38. MEDIATORS OF BONE RESORPTION
STIMULATORS
• Interleukin-1 (IL-1)
• Interleukin-6 (IL-6)
• Tumor necrosis factor (TNF)
• Matrix metalloproteinases
• Prostaglandin E2 (PGE2)
• Parathyroid hormone
• Vitamin D3
• Receptor activator of NFκB
(RANK)
• RANK ligand (RANKL)
INHIBITORS
• Interferon gamma (IFN-γ)
• Interleukin-10
• Transforming growth factor
β
• Osteoprotegerin (OPG)
• Estrogens
• Androgen
• Calcitonin (CT)
• Bisphosphonates

39. INTERLEUKIN-1
• IL- 1 family of cytokines comprises atleast of 11 members
including IL-1α, IL-1β, IL-1Ra, IL-18, IL-1F5, IL-1F6, IL-1F7,
IL-1F8, IL-1F9, IL-1F10, IL-33
• Secreted by a variety of cells including macrophages, B
cells, neutrophils, fibroblasts, and epithelial cells.

40. INTERLEUKIN-1
IL-1α and IL-1 β are equally potent in stimulating bone
resorption .
The effects of IL-1 probably occur by two mechanisms.
1. Stimulation of the production and release of PGE2, there
by stimulating bone resorption.
2. Direct action of IL-1 on osteoclasts independent of PGE2
receptor.

41. INTERLEUKIN-1β
• IL-1β plays a key role in inflammation and immunity.
• Activate innate immune response.
• Induces the synthesis and secretion of other mediators that
contribute to inflammatory changes e.g. synthesis of PGE2,
platelet activating factor (PAF) and nitrous oxide (NO).
• IL-1β increases the expression of ICAM-1 on endothelial cells
and stimulates secretion of chemokines CXCL-8, thereby
facilitating the infiltration of neutrophils into affected tissues.

42. INTERLEUKIN-1β continued..
IL-1β role in adaptive immunity:
• Regulates the development of antigen presenting cells, such
as dendritic cells.
• Stimulates IL-6 secretion by macrophages (which in turn
activates B cells).
• Enhances antigen-mediated stimulation of T-cells.
Ben- Sasson Sz., 2009

43. INTERLEUKIN-1α
• IL-1α - an intracellular protein.
• It act as a signalling cytokine
• Biologically active IL-1α is expressed and mediates
inflammation only when released from necrotic cells, acting as
an “alarmin” to signal the immune system during cell and
tissue damage.

44. INTERLEUKIN-18
• IL-18 interacts with IL-1β and shares many of the
proinflammatory effects of IL-1β.
• Produced by stimulated monocytes and macrophages.
• IL-18 results in proinflammatory responses, including
activation of neutrophils.

45. INTERLEUKIN-18 continued..
Interacts IL-12 and IL-15
Induce IFN-γ
Inducing Th1 cells
Activate cell mediated
Immunity.
Absence of IL-12
Induces IL-4 and Th2
Regulates Humoral
(Antibody-mediated)
Immunity
INTERLEUKIN-18

46. INTERLEUKIN-6
Interleukin-6 and Related Cytokines
• IL-6, IL-11, leukemia-inhibitory factor (LIF), and oncostatin M
• Share common signalling pathways via signal transducers
glycoprotein gp 130.
Heinrich PC 2003
• IL-6 secretion is stimulated by cytokines such as IL-1β and TNF-α
• Produced by a range of immune cells, including T-cells, B cells,
macrophages and dendritic cells, as well as resident cells such as
keratinocytes, endothelial cells, and fibroblasts.

47. INTERLEUKIN-6 continued..
• IL-6 is also secreted by osteoblasts and stimulates bone
resorption and development of osteoclasts Ishimi Y and
Kurihara N (1990)
• IL-6 stimulates T-cell differentiation and function,
• Regulation of the balance of T-cell subsets, particularly the
activation of Th17 cells (a subset of T cells that produce IL-
17) and the balance with regulatory T cells (Treg cells)
Bettelli E., 2006

48. TNF-α
• TNF-α is a key inflammatory mediator in periodontal
disease, and it shares many of the cellular actions of IL-1β
• TNF-α stimulates the development of osteoclasts and limits
tissue repair by induction of apoptosis in fibroblasts.
• TNF-α and IL-1β act directly on cells of the osteoclast
lineage to stimulate bone resorption (Takahashi
N.etal.,2002)

49. PROSTAGLANDINS
• Prostaglandin E2 (PGE2)
vasodilatation and induces
cytokine production by a
variety of cell types.
• PGE2 is produced by various types of
cells and most significantly in the
periodontium by macrophages
and fibroblasts.
• Has a major role in
contributing to the tissue
damage that characterizes
periodontitis.
IL-1β
TNF-α
LPS
COX-2
increased
production
of PGE2 in
inflamed
tissues.
osteoclastic bone
resorption
Induction of MMPs

50. MATRIX METALLOPROTEINASES
• MMPs are secreted in inactive form and are activated by the
proteolytic cleavage of a portion of the latent enzyme. This is
achieved by proteases, such as cathepsin G, produced by
neutrophils.
• Inhibitors of MMPs in tissues include
tissue inhibitors of metalloproteinases(TIMPs)
the most important in periodontal disease is TIMP-1.
• Key inhibitors of MMPs found in the serum include
1. glycoprotein α1-antitrypsin
2. α2 macroglobulin

51. MATRIX METALLOPROTEINASES
• Predominant MMPs in periodontitis are MMP-8 and MMP-9
secreted by neutrophils → degrade type I collagen ,most
abundant in periodontal ligament.
• MMPs - 9 and 14 are critical for osteoclast access to
resorption sites.
• MMPs -13 present in resorption lacunae, functions to
remove collagen remnants leftover by osteoclasts.
• MMPs-2,9,13 and 14 are important in osteoblastic bone
formation.
• MMPs-14 contributes to normal bone homeostasis.

53. MATRIX METALLOPROTEINASES
• MMPs are also inhibited by the tetracycline class of
antibiotics.
• Systemic adjunctive drug treatment of doxycycline for
periodontitis exploits the anti-MMP properties of this
molecule.
• This has led to the development of sub-antimicrobial
formulation of doxycycline to inhibit collagenase activity.

54. PARATHYROID HORMONE (PTH)
• PTH acts on both bone-resorbing cells and bone-forming
cells. The net effect of the hormone depends on whether it
is administered continuously or intermittently.
• When administered continuously, it increases osteoclastic
bone resorption and suppresses bone formation.
• When administered in low doses intermittently, its major
effect is to stimulate bone formation, a response that has
been called the anabolic effect of PTH Neer RM et al.,(2001).

55. 1, 25-DIHYDROXYCHOLECALCIFEROL
(VITAMIN D 3)
• The active metabolites of vitamin D3 have complex effects
on calcium homeostasis and bone regulation.
• 1,25(OH)2 D3 stimulates osteoclastic bone resorption in vitro
and in vivo.
• It increases both osteoclast number and activity, with an
increase in ruffled border size and clear-zone volume

56. RANK
• Receptor activator of
nuclear factor-kappa
(RANK) also known as
TRANCE Receptor
• It is a member of the tumor
necrosis factor receptor
(TNFR) molecular sub
family.
• RANK - osteoclasts
precursors and mature
osteoclasts.
• RANK is the receptor for
RANK- Ligand (RANK-L).

57. RANKL
• Receptor activator of nuclear
factor-kappa B ligand (RANKL)
(also known as osteoprotegerin-
ligand,OPG-L,)
• It is a cell surface protein present
on bone marrow stromal cells,
osteoblasts, and fibroblasts.
• RANKL interacts with its receptor,
RANK, an interaction essential for
osteoclast differentiation,
activation and bone resorption.

58. RANKL
• RANKL is expressed by a number
of other cell types, including
fibroblasts and T and B
lymphocytes.
• RANKL is expressed at low levels in
fibroblasts; its expression is
induced in response to toxin from
Aggregatibacter
actinomycetemcomitan (Lerner
UH.2006).

59. OPG
• Osteoprotegerin (OPG) is a
soluble decoy receptor for RANKL.
• OPG binds to RANKL and inhibits
the differentiation of osteoclast
by blocking the binding of RANK
Ligand to RANK. Boyle WJ.et
al.,2003.
• Expressed by bone marrow
stromal cells, osteoblasts, and
periodontal ligament fibroblasts.

60. Induction of osteoclastogenesis

61. Reduced osteoclastogenesis

62. RANK/RANK-L/OSTEOPROTEGERIN SYSTEM
• Osteoprotegerin is a natural inhibitor of osteoclast
differentiation and activation.
• Any interference with this system can shift the balance
towards increased bone formation or resorption.
• Pro-inflammatory cytokines- IL-1 and TNF-α ,regulate the
expression of RANKL and OPG.
• IL-1β

63. RANKL
RANK
Osteoclast
differentiation
Binding of RANKL to RANK results in osteoclast differentiation and
activation and thus bone resorption.
Bone
resorption

64. RANKL
OPG
Inhibits
differentiation
of osteoclasts
OPG has the opposite effect, inhibits differentiation of osteoclasts
Inhibit
bone
resorption

65. RANKL
OPG
BALANCE BETWEEN OPG
AND RANKL ACTIVITY CAN
THEREFORE DRIVE BONE
RESORPTION OR BONE
FORMATION

66. • RANKL/OPG ratio of 3.33 : 1.89 severe
chronic localized periodontitis
• RANKL/OPG ratio 1.8 : 4.0 in healthy
gingiva
(Crotti T.etal.,2003)

67. INHIBITORS OF BONE RESORPTION
• INTERFERON γ
Interferon γ is a cytokine produced by activated T lymphocytes that
inhibits bone resorption Gowen M et al 1986
 It has an effect on bone resorption that is opposite to that of IL-1
and TNF-α.
 Gamma interferon is more effective in inhibiting IL-1 or TNF-α
than systemic hormones like PTH or 1,25-(OH)2 D3.

68. INHIBITORS OF BONE RESORPTION
INTERLEUKIN 1 RECEPTOR ANTAGONIST(IL-1Ra)
• IL-1Ra has structural homology to IL-1β, and binds to the IL-1
receptor (IL-1R1).
• No signal transduction, therefore IL-1Ra antagonizes the
action of IL-1β.
• IL-1Ra is important in regulating inflammatory responses -
anti-inflammatory cytokine.
• IL-1Ra levels GCF and tissues of patients with
periodontal disease -role in immunoregulation in periodontitis
Roberts FA.et al., (1997).

69. INHIBITORS OF BONE RESORPTION
INTERLEUKIN 10
• Possess immunosuppresive properties.
• Produced by Treg cells, monocytes and B cells.
• Suppresses cytokine secretion from Th1 cells, monocytes
and macrophages.

70. TRANSFORMING GROWTH FACTOR -β
• It is a growth factor that functions as a cytokine and has
immunoregulatory roles such as
1. Regulation of T-cell subsets and the action of Treg cells
2. It plays a role in repair and regeneration.
• TGF- β levels are higher in the the GCF and periodontal
tissues of patient with periodontitis and gingivitis.

71. CALCITONIN
• Calcitonin is a potent
inhibitor of bone resorption
but, in pharmacologic use,
has limited application since
patients become refractory
to its inhibitory action,
phenomenon referred to as
Escape.
Altkorn D.etal 2001
INHIBITORS OF BONE RESORPTION

72. INHIBITORS OF BONE RESORPTION
• ESTROGEN
Inhibits bone resorption as evidenced
by the increase in osteoporosis that
ensues with estrogen deficiency after
menopause.
Mechanisms of action :
promote the programmed cell death of
osteoclasts and hence reduce their
period of activity Rodan GA etal.,(2000).

73. INHIBITORS OF BONE RESORPTION
BISPHOSPHONATES
• Bisphosphonates bind to the bone surface and act directly
on osteoclasts to inhibit their resorptive activity and to
promote their apoptosis.
• Bisphosphonates also affect protein production in
osteoblasts (Stronski SA.etal.,1988).

74. BISPHOSPHONATES
• Alendronate reduced the risk of progressive alveolar bone loss.
• A protective effect of alendronate has seen in periodontal
patients with type 2 diabetes (Rocha M et al., 2001).

75. Bone Destruction Caused by Trauma
from Occlusion
• TFO can produce bone destruction in the absence or
presence of inflammation
• In the absence of inflammation-- increased compression and
tension of the PDL and increased osteoclasis of alveolar
bone
• These changes are reversible in that they can be repaired if
the offending forces are removed.
• Persistent TFO results in funnel-shaped widening of the
crestal portion of the PDL with resorption of the adjacent
bone

76. CONCLUSION
• All of these scenarios would benefit from a better
understanding of prognostic features of our patients’
susceptibility to bone loss.
• Improved understanding of prognosis will likely arise
through better molecular identification of disease
susceptibility genes.
• As these 2 goals converge, we will be in a much better
position to effectively intervene in the osseous destruction
associated with periodontal diseases.

77. Thank you