The document summarizes several bone proteins found in the extracellular matrix. It discusses the roles of osteocalcin, osteopontin, osteonectin, BMP, and other proteins in bone formation, mineralization, and regulation of cellular activity. Osteocalcin is involved in mineralization and glucose metabolism. Osteopontin anchors osteoclasts to bone and regulates inflammation. Osteonectin binds tightly to hydroxyapatite and regulates mineralization. BMPs induce bone and cartilage formation through signaling pathways.

2. BONE PROTEINS IN
EXTRACELLULAR
MATRIX
Dr . Suman Mukherjee
MDS 1ST YR

3. Content
• Introduction
• Osteocalcin
• Osteopontin
• Osteonectin
• BMP
• BSP
• Thrombospondin
• DPD
• Matrix Gla protein
• CAP
• Amelogenin and Enamelin
• Bone resportion and formation
factors
• Bone markers
• Conclusion
• bibliography

4. Introduction
• Proteins are important conditioning factor in maintaining health of
periodontium.
• Other than collagen and proteoglycan several other protein component are
present in connective tissue.
• Most of the non collagenous proteins are produced by connective tissue cells.
• Proteins play important role in regulating cellular activity and tissue function.

5. Osteocalcin
• Osteocalcin, also referred to as bone γ-
carboxyglutamic acid (Gla) protein or BGP,
is a 46– 50 amino acid, secreted protein that
is produced primarily by osteoblasts.
• Smaller amounts are also be produced by
odontoblasts of the teeth and hypertrophic
chondrocytes.
• The protein was first isolated by Price et al
from bovine and human bone and shown to
represent the major fraction of Gla
containing protein in bone.

6. • A second Gla-protein, isolated later by the same group, was termed
matrix Gla protein or MGP. Together, these two proteins belong to a
distinct subgroup of the larger vitamin K dependent protein family, the
constituents of which are primarily involved in coagulation.
• The human osteocalcin gene, BGLAP, is located on chromosome 1 at
1q25-q31.

7. • Osteocalcin has routinely been used as a serum marker of
osteoblastic bone formation and believed to act in the bone matrix
to regulate mineralization, but new genetic and pharmacologic
evidence now points to a hormonal role for the protein.

8. Role of osteocalcin in mineralization
• Mature osteocalcin is secreted into the bone micro-environment and
then undergoes a conformational change that aligns its calcium-binding
Gla residues with the calcium ions in hydroxyapatite.
• This property was initially proposed as a mechanism that enables
osteocalcin to initiate the formation of hydroxyapatite crystals.
• However, subsequent work was more compatible with the notion that
osteocalcin functions as an inhibitor of bone mineralization.

9. • In support of this idea, osteocalcin inhibits the precipitation of calcium
salts from saturated solutions, and chronic treatment of rodents with
warfarin, an inhibitor of vitamin K-dependent γ-carboxylation, results
in over-mineralization and the premature closure of the growth plate.

10. • The precise role of osteocalcin within the bone matrix remains unclear,
and osteocalcin’s inhibitory effect on bone mineralization is likely to
be considerably lower than that of MGP since the removal of this gene
results in the calcification of the aorta and the progressive
mineralization of the growth plate.
• Other studies have led to the hypothesis that osteocalcin exerts a
mechanical function within the bone matrix.

11. • As a result of its ability to tightly bind hydroxyapatite and form a
complex with collagen through the matrix protein osteopontin ,
osteocalcin was proposed as means to bridge the matrix and mineral
fractions of bone tissue.
• Such an arrangement is compatible with the formation of dilatational
bands that are seen when bone fractures. In this situation, osteocalcin
and osteopontin might serve to prevent crack growth by stretching and
dissipating energy.

12. • As osteocalcin is one of the predominant non collagen proteins in bone
(2% of total bone protein), it has been suggested that osteocalcin is
involved in the mineralization process, is an effector molecule for
calcitriol (1,25- [OH],D,) and influences the movement and
proliferation of osteoclasts.
• Recent work from several groups have now clearly demonstrated a role
for osteocalcin in the regulation of glucose metabolism
• The discovery of osteocalcin’s role in regulating glucose metabolism
was independently established by Fulzele et al in the course of studies
designed to examine insulin actions in osteoblasts.

13. Other osteocalcin effects
• It builds strong bones
• Adjusts insulin and glucose levels
• Stimulate testosterone production
• May improve muscle strength
• May improve brain function

14. Low Osteocalcin
• Associated with insulin resistance and diabetes
• Low levels may indicate heart disease risk
• Atheroscelorosis
• Metabolic syndrome
• Obesity
• NAFLD (fatty liver)

15. High Osteocalcin
• May indicate osteoporosis
• Associated with diabetes in pregnancy
• Associated with increased risk of breast cancer.
• May indicate anemia

16. Osteopontin
• Osteopontin (OPN), a glycoprotein was first identified in 1986 in
osteoblasts. Osteopontin is a multifunctional protein, highly expressed
in bone.
• The prefix of word ‘osteo’ indicates that the protein is expressed in
bone, the suffix ‘pontin’ is derived from ‘pons’ the Latin word for
bridge that signifies osteopontin’s role as a linking protein.

17. • Osteopontin is also known as bone sialoprotein I (BSPI), early T-
lymphocyte activation (ETA-I), Urinary stone protein, Nephropontin
and Uropontin secreted phosphoprotein (SPP 1) and Rickettisia
resistance (Ric) K BPP (bone phosphoprotein) is a human gene
product.
• Osteopontin is composed of ~ 300 amino acids residues and has ~ 30
carbohydrate residues attached including ten sialic acid residues .The
protein is rich in acidic residues 30 – 36 % are either aspartic or
glutamic acid residues.

18. Chemical
Structure
• OPN is single chain
polypeptide composed of
about 300 amino acids. It
is expressed as a 33-kDa
nascent protein.
• OPN can go through post
translational
modifications.

19. • Specific splice variants of OPN are Osteopontin-a, Osteopontin -b and
Osteopontin-c
• OPN was originally isolated from bone and was later independently
identified as secreted phosphoprotein I (SppI) and early T-lymphocyte
activation 1 (Eta-1) (Senger et al. 1989; Patarca et al. 1989).

20. • OPN is a member of the SIBLING (Small Integrin-Binding Ligand, N-
linked Glycoprotein) family of proteins, which map to human
chromosome 4 (Fisher et al. 2001).
• OPN is synthesized as an approximately 32 kDa protein, but due to
extensive posttranslational modifications its apparent molecular mass
ranges from 45 to 75 kDa (Kazanecki et al. 2007).

21. • OPN possesses a negative charge due to a preponderance of acidic
amino acids and serine phosphorylation. OPN also contains calcium
binding sites and two putative heparin binding domains (Kon et al.
2008).
• OPN can interact directly with extracellular matrix proteins including
fibronectin (Mukherjee et al. 1995) and collagen type I (Chen et al.
1992; Martin et al. 2004)

22. Intracellular OPN
• An intracellular form of OPN (iOPN) has been reported to be
expressed in dendritic cells and macrophages (Shinohara et al. 2006,
2008a, b; Zohar et al. 2000).
• Studies by Shinohara et al. suggest that the intracellular form of OPN
is generated due to translation initiation downstream of the usual start
site in bone marrow-derived DCs (dendritic cells) and transfected 293T
cells.

23. Functions of
Osteopontin
• The putative functions of
Osteopontin are bone
mineralization, regulation
of immune cell function,
inhibition of calcification,
control of tumour cell
phenotype and cell
activation

24. Osteopontin – Role in
Biomineralisation
• Osteopontin has been implicated as an important factor in bone
remodeling.
• It plays a role in anchoring Osteoclasts to the mineral matrix of
bones .
• The inorganic part of bone is the mineral hydroxyapatite,
Ca(PO4)(OH)2. Loss of this mineral may lead to osteoporosis as
the bone is depleted for calcium if this is not supplied in the diet.

25. • OPN is expressed by both Osteoclasts and osteoblasts which are the
cells responsible for bone remodeling. During normal bone
mineralization, osteoclast derived OPN inhibits the formation of
hydroxyapatite.
• OPN expression is found in regions of dystrophic calcification

26. Osteopontin and Inflammation
• OPN appears to regulate innate immune cells and adaptive immune
cells at multiple levels.
• OPN functions as a Th 1(T – helper 1) cytokine, promotes cell-
mediated immune responses and plays a role in chronic inflammatory
diseases.
• Plasma OPN levels have been found associated with various
inflammatory diseases.

27. • OPN appears to regulate macrophage infiltration during the
inflammatory response.
• OPN is a multifunctional cytokine and adhesion protein that contains
an integrin-binding RGD sequences that interact with CD44v 6/7.
• OPN is chemotactic for various cell types notable monocytes/
macrophages, which are attracted to sites of infection and
inflammation
• OPN activates intracellular signaling pathways and regulates gene
expression as a consequence of its interactions with its various
receptors

28. Osteopontin and Cancer
• Osteopontin expression has been shown to enhance the invasive
potential of cancer cells and plays an important role in cancer
progression
• OPN was shown to be associated with recurrence of prostate cancer
within 72 months and
• OPN was suggested to be a clinically useful marker for predicting
biochemical recurrence as OPN is secreted by tumour cells.

29. OPN –Role in Vascular diseases
• Osteopontin is found to be expressed in smooth muscle cells (SMC)in the
atherosclerotic lesion in angiogenic endothelial cells and macrophages.
• OPN is also re-expressed in SMC cells associated with human restenotic
lesions.
• OPN modulates proliferation, migration and accumulation of SMC and
endothelial cells involved in repair ,remodeling processes of the vasculature.
• Findings suggest that OPN may be an important regulator of arterial mineral
deposition under conditions of injury and disease

30. Osteonectin
• Osteonectin was originally isolated by Termine et al. (1981)
• One of the intriguing features of osteonectin is its ability to bind tightly
to hydroxyapatite. This was first demonstrated by Termine et. al.
(1981).
• Osteonectin (ONN) is a protein that is concerned with regulating the
adhesion of osteoblasts and platelets to their extracellular matrix, as
well as early stromal mineralization.

31. • Osteonectin is an acidic, cysteine-rich
glycoprotein comprised of a single
polypeptide chain that can be broken into 4
domains:
• 1) an acidic region at the amino terminus,
• 2) a cysteine- rich area,
• 3) a hydrophilic region and
• 4) an E-F hand structure at the carboxy
terminus

32. • Osteonectin, also referred to as secreted protein acidic and rich in cysteine, or
SPARC, is a phosphoprotein that is the most abundant non collagenous
polypeptide expressed in bone.
• The mature protein binds selectively to hydroxyapatite, collagen fibrils, and
vitronectin at distinct sites and may allow proper organization of the bone
matrix through contacts with the cellular surface.
• Osteonectin also inhibits cellular proliferation through arrest of cells in the G1
phase of the cell cycle. It may regulate the activity of platelet-derived growth
factor, vascular endothelial growth factor, and FGF-2.

33. • Osteonectin, initially isolated from demineralized bone matrix, was named
according to its ability to bind to calcium, hydroxyapatite, and collagen, and
to nucleate hydroxyapatite deposition.
• The protein has also been called secreted protein, acidic and rich in cysteine
(SPARC), basement membrane -40, or “culture shock” protein .
• Osteonectin is an antiadhesive protein because it inhibits cell spreading,
induces rounding of cells, and disassembles focal adhesions . Other activities
of osteonectin include calcium-dependent binding to collagens and
thrombospondin, binding to platelet-derived growth factor-AB and -BB, and
regulation of cell proliferation and MMP expression.

34. • The gene SPARC or ON, which contains 10 exons, is located on
chromosome 5q31.3-q32.
• The gene is expressed at high levels in tissues undergoing
morphogenesis, remodeling, and wound repair. It is also made by cells
of osteoblastic lineage and the hypertrophic chondrocytes of the growth
plate.
• The SPARC gene is also expressed in several postnatal non-skeletal
tissues, including salivary and renal tubular epithelium

35. THE FUNCTION OF OSTEONECTIN
• Two lines of investigation shed light on the possible function of
osteonectin.
• The first line is supported by the work of Termine et al. (1981a,b) and
the work from (Romberg et al., 1985,1986) demonstrating the ability of
osteonectin to bind calcium, hydroxyapatite and collagen.
• Termine et al. (198la) were the first to suggest the notion that
osteonectin might serve as a nucleation site for the deposition of mineral
on collagen in lamellar bone formation.

36. • They have suggested a model in which osteonectin binds to collagen
fibrils at, presumably, specific sites, followed by mineral binding,
precipitation of mineral to form hydroxyapatite, and crystal
enlargement.
• The data obtained to date are consistent with this model, but further
information is required before it can be accepted as fact.

37. • The second line of evidence is composed of data from Stenner et al.
(1986) concerning osteonectin in platelets, Sage et al. (1984, 1986)
concering the 43K protein in cultured fibroblasts, endothelial cells and
other cell types, and the data reported by Mason et al. (1986a,b)
concerning SPARC.
• The secretion of osteonectin by platelets during activation, along with
the other properties of osteonectin have led us to suggest that
osteonectin may play a role in the anchoring of cells to sub-cellular
substrates (Stenner et al., 1986).

38. • Mason et al. (1986a,b) have also suggested that SPARC may play a role in the
establishment or modification of extracellular matrices, possibly during
embryonal development as well as during wound repair, a notion that fits in
well with one of the functions of platelets.
• However, while osteonectin is clearly a component of the extracellular bone
matrix, both SPARC (Mason et al., 1986) and the 43K protein (Sage et al.,
1986) do not appear to be incorporated into the matrix in cultured cells.
• Whether this is related to differences between the proteins, the tissues of origin,
or differences between cell culture vs in situ analysis, remains to be
demonstrated.

39. BMP
• BMPs were discovered by Urist in 1960s, to date, about 20 BMP family
members have been identified and characterized.
• BPMs exert diverse biological processes ranging from early embryonic tissue
patterning to postnatal tissue homeostasis, which mainly through triggering
canonical signaling pathway where intracellular Smads play central roles in
delivering the extracellular signals to the nucleus.
• However, it is not very clear about the cellular and molecular mechanisms of
BMPs.

40. Structure
• All BMPs are secreted as precursor protein with a hydrophobic stretch
of about 50–100 amino acids. Every BMP contains seven cysteins, in
which six of these cysteins build a cystin knot and the seventh is used
for dimerization with a second monomer.

41. Function
• BMPs have been implicated in a variety of functions. They not only
induce the formation of both cartilage and bone, but also play a role in
a number of non-osteogenic developmental processes.
BMPs function in skeleton development
• As their name indicates, BMP molecules are capable of inducing
ectopic cartilage and bone formation, a process that mimics embryonic
endochondral bone formation

42. • Extensive studies demonstrate that BMPs are important factors regulating
chondrogenesis and skeletogenesis during normal embryonic
development.
• The BMPs with greatest osteogenic capacity are BMP-2, -4, -5, -6, -7,
and -9.

43. BMP GENE
LOCUS
FUNCTION RECEPTORS
AND
INHIBITORS
BMP-1 8P21 It is a metalloprotease that acts on procollagen I, II, and III. It is
involved in cartilage development
Type I receptors
BMP-2 20P12 Acts as a disulfide-linked homodimer and induces bone and
cartilage formation. It is a candidate as a retinoid mediator. Plays a
key role in osteoblast differentiation
ACVRIA,
ACVRIB,
ACVRIC,
ACVRLI-BMPR
(BMPRIA–
BMPRIB)
BMP-3 14P22 Induces bone formation Type II receptors

44. BMP GENE FUNCTION RECEPTORS
AND
INHIBITORS
BMP-4 14q22-
q23
Regulates the formation of teeth, limbs and bone from mesoderm. It
also plays a role in fracture repair
ACVR2A,
ACVR2B,
AMHR2,
BMPR2
BMP-5 6p12.1 Performs functions in cartilage development Type III
receptors
BMP-6 6p12.1 Plays a key role in joint integrity in adults Betaglycan
BMP-7 20q13 Plays a key role in osteoblast differentiation. It also induces the
production of SMADI. Also key in renal development and repair.

45. BMP GENE FUNCTION RECEPTORS
AND
INHIBITORS
BMP-8 1p35-p32 Involved in bone and cartilage development
BMP-9 Promoting chondrogenic differentiation of human multipotential
mesenchymal cells
Ligand inhibitors
BMP-10 2q14 Play a role in the trabeculation of the embryonic heart Cerberus,
Chordin, DAN,
Decorin,
Follistatin,
Gremlin, Lefty,
LTBPI
BMP-15 Xp11.2 May play a role in oocyte and follicular development Noggin, THBSI

46. • Bmp-2 is expressed in areas surrounding the initial cartilage
condensations, periosteal and osteogenic zones, while Bmp-4 is expressed
in perichondrium.
• Bmp-6 is expressed in hypertrophic chondrocytes. High levels of Bmp-7
mRNA have been observed in the perichondrium, but its expression is
absent in the zones of joint formation.
• BMP-2 and BMP-7 have significant importance in bone development and
the development of a wide array of tissues outside of bone.

47. • Bmp-6 mutant mice have delayed sternum ossification.
• BMP-3 represents an exception. Consistent with its opposite roles to
BMP-4 during mesoderm patterning, BMP-3 inhibits BMP-2-induced
osteogenic differentiation.
• Bmp-3 knockout mice exhibit increased bone density, which indicate
that BMP-3 antagonizes BMP signaling in vivo

48. Other function
• Accumulated evidence indicates that BMPs play an important role in
regulation of stem cell properties; however, their functions are different
in the different stem cell compartments
• BMPs may have pleiotropic functions including mutagenesis or
oncogenesis, thus, extensive studies have been reported in the field of
musculoskeletal oncology, especially osteosaroma

49. • During embryonic development, BMPs mediate programmed cell
death, or apoptosis, the process that removes unnecessary tissues,
thereby ensuring proper morphogenesis. BMP-4 was first demonstrated
to induce apoptosis in rhombomeric neural crest cells.
Receptors and molecular signaling
• Members of the BMP family bind to two distinct type II and type I
serine/threonine kinase receptors, both of which are required for signal
transduction

50. Potential medical application
• BMPs are multifunctional cytokines which are members of the transforming
growth factor-beta superfamily.
• They are the only signaling molecules which can singly induce de novo bone
formation at orthotopic and heterotopic sites and their osteoinductive potency
makes them clinically valuable as alternatives to bone graft.
• Several means of delivering BMPs to patients are undergoing evaluation
including systemic administration, gene transfer and local matrix delivery
vehicle implantation.

51. • The latter methodology is in advanced stages of development for application
in humans in the treatment of selected spinal fusions, fracture repairs,
craniomaxillofacial surgery and periodontal injury and disease
• The BMPs are also widely distributed in non-skeletal tissues such as nerve,
gastrointestinal tract, kidney, heart and lungs and they have a central role in
vertebrate and non-vertebrate organogenesis.
• Initial studies indicate that the BMPs have neuro, cardio and reno-protective
actions and it is likely that therapeutic indications for their use will extend
well beyond skeletal disease and injury.

52. • Several groups have completed phase III clinical trials and are awaiting FDA
approval for nonunion fracture healing and alveolar ridge augmentation. BMPs
have been approved by the FDA for use in nonunion fractures and spinal
fusions. Approval is currently pending for alveolar ridge augmentation.
• Over the next few years, we will see how widely used BMPs become in the
clinic.
• Recent comparisons indicate that use of BMP is at least as effective as
traditional treatments using allografts (bone from cadavers), and may have
fewer long-term side-effects. Cost analyses suggest that use of BMPs is less
costly than use of allografts.

53. BONE INDUCTION PRINCIPLE (BIP):
• The product of the interaction of the mesenchymal cells with the
surface of the implanted matrix is referred to as the bone induction
principle (BIP).
• Since the BIP occupies the space between the implanted matrix and the
extracellular substances secreted by the mesenchymal cells, it has a
three-dimensional structure.
• The BIP fills the pores of a millipore membrane, 125 μm, with a pore
size of 0.45μm.

54. BONE SIALOPROTEIN
• BSP, which is essentially unique to mineralized tissues, is expressed at
high levels coincident with denovo formation of bone (I Chen et al., 1991a,
1992) and cementum (MacNeil et al., 1995, 1996).
• Consequently, considerable interest has been generated in BSP as a
potential nucleator of hydroxyapatite and as a specific marker of osteoblast
and cementoblast differentiation.
• However, the cell attachment and cell signaling properties of BSP indicate
multiple roles for this protein in bone remodeling and also in several
pathologies.

55. • Bone sialoprotein constitutes from 8 to 12% of
the total non-collagenous proteins in bone and
cementum (Fisher et al., 1983, 1987, 1990),
with substantially lower amounts (- 1%) in
dentin (Fujisawa et al., 1993).
• It was first isolated from bovine cortical bone
by Herring and Kent (reviewed in Herring,
1972) as a 23-kDa glycoprotein with high sialic
acid content.
• Although it was the first bone sialoprotein to be
extensively studied, complete characterization
was compromised by difficulties in isolating the
protein in an intact form.

56. • Therefore, it was not until the development of dissociative extraction
procedures (Termine et al., 1981) that a more comprehensive
characterization was achieved.
• Newly deposited BSP was purified from 1-2 mm of the outer section of
fetal calf subperiosteal bone-a site of rapid bone growth (Fisher et al.,
1983).
• However, subsequent studies indicated that the preparation was likely
contaminated with a second sialoprotein, osteopontin (Franzen and
Heinegard, 1985)

59. Functions of BSP
• Studies on the temporo-spatial expression and tissue localization in
newly forming bone in vivo and in vitro, together with its structural
properties, indicate that BSP might initiate hydroxyapatite formation
during de novo bone formation.
• Consistent with a role in hydroxyapatite nucleation, BSP is able to
nucleate hydroxyapatite formation in a steady-state agarose gel system
under conditions of sub-threshold calcium phosphate supersaturation
(Hunter and Goldberg, 1993).

60. • That BSP is able to nucleate hydroxyapatite formation is also supported by the
association of BSP expression and pathological mineralization and BSP
expression have been reported in ankylosing periodontal ligament (Lekic et al.,
1997) and in ligaments of patients with pseudoxanthoma elasticum (Contri et al.,
1996).
• Notably, high expression of BSP is found at sites of rapid bone formation, as
occurs in de novo bone formation and fracture repair, where woven bone is
formed .
• The BSP localizes in association with the extrafibrillar mineral, which is
relatively abundant in woven bone (Bianco et al, 1993; Chen et al., 1 994) and
cementum (McKee et al, 1996a; Bosshardt et al., 1998).

61. • The presence of BSP in cementum and reversal lines (Chen et al.,
1994; McKee et al., 1996b) indicates a potential role in mediating cell
attachment to mineralized surfaces.
• In this regard, BSP and OPN have similar properties, although they
differ in temporal expression during bone formation (I Chen et at.,
1992)

62. Thrombospondin
• Thrombospondin-1 is the first and most studied naturally occurring
protein inhibitor of angiogenesis.
• TSP1 was the first naturally occurring protein inhibitor of angiogenesis
to be identified; its anti-angiogenic effects have since been established
in a multitude of experimental models and linked to specific epitopes
in the multi-domain, multi-functional TSP1 molecule.

63. • TSP1 is the first identified, and therefore best studied thrombospondin
family representative, its structure is thus considered as prototype for the
other family members.
• In the thrombospondin family, another member, TSP2 has a similar domain
structure and, non-surprisingly, its functions significantly overlap with those
of TSP1. Specifically, both TSP1 and TSP2 potently inhibit angiogenesis

64. • Thrombospondin exists in a number of tissues and is synthesized by
several cell types.
• Thrombospondin has a trimeric or pentameric structure. It
demonstrates its functions at the cell surface and in the extracellular
matrix to encourage cell attachment, spreading, and migration.
• Thrombospondin also is a key for the proper organization of collagen
fibrils in the skin and cartilage.

65. Periostin
• Periostin is a lately identified protein which is evidenced to be present in periodontal
ligament.
• It has been named so as it was found initially in periosteum.
• The biochemical structure of perostin is a disulfied linked protein that favors osteoblast
attachment and spreading. It is critical for mediating epithelial-mesenchymal complex
that is necessary for formation of periodontium.
• Periostin is assumed to play a role in response of periodontal ligament to mechanical
stress. It is believed to be focused more in areas of pressure than in areas of tension.
• Hence periostin, therefore thought to be important for maintenance of periodontal
ligament

66. Deoxypyridinoline (DPD)
• DPD is formed by reaction of side-chains of two hydroxylysine molecules and
one lysine molecule, and pyridinoline (PYD) is formed by reaction of side-
chains of three hydroxylysine molecules (both compounds have inborn
immunogenetics and fluorescence).
• DPD is found mostly in bones, not so much in dentine, while PYD is located in
bone collagen fibrils and cartilage and to a lesser extent in other tissues (tendons,
ligaments, blood vessel walls).

67. • Since the bones have the most intensive remodeling, they are
considered as the most important source. Deoxypyridinoline is
considered a specific resorption marker because it is formed during
collagen maturation. (not during biosynthesis and therefore it appears
only as a resorption product of the mature matrix).

68. Matrix gla-protein
• Matrix Gla-protein (MGP) is a small secretory protein that can undergo
two types of posttranslational modification: γ-glutamate carboxylation and
serine phosphorylation.
• The protein was first described in 1983 by Price et al. who purified it from
the bovine bone matrix.
• Among the proteins involved in vascular calcium metabolism, the vitamin
K-dependent matrix Gla-protein (MGP) plays a dominant role.

69. • Although on a molecular level its mechanism of action is not completely
understood, it is generally accepted that MGP is a potent inhibitor of arterial
calcification.
• Its pivotal importance for vascular health is demonstrated by the fact that
there seems to be no effective alternative mechanism for calcification
inhibition in the vasculature.
• An optimal vitamin K intake is therefore important to maintain the risk and
rate of calcification as low as possible.With the aid of conformation-specific

70. • antibodies MGP species in both tissue and the circulation have been detected
in the healthy population, and significant differences were found in patients
with cardiovascular disease (CVD).
• Using ELISA-based assays, uncarboxylated MGP (ucMGP) was
demonstrated to be a promising biomarker for cardiovascular calcification
detection.
• These assays may have potential value for identifying patients as well as
apparently healthy subjects at high risk for CVD and/or cardiovascular
calcification and for monitoring the treatment of CVD and vascular
calcification.

71. Cementum attachment protein (CAP)
• Is consistently found in acellular cementum.
• It is biochemically thought to be involved in communication pathways
that are thought to play a role in the development of cementoblasts
from their precursor cells.
• Apart from this CAP is also thought to provide attachment to the
periodontal ligament fibers.

72. Ameloblastin
• Ameloblastin (AMBN) and enamelin (ENAM) are two of the 23 secretory
calcium-binding phosphoprotein (SCPP) genes on human chromosome 4q.
• They are members of the proline and glutamine rich group.
• Both ameloblastin and enamelin function specifically during tooth formation,
and probably both are only critical for dental enamel formation.
• Both genes have been shown to degenerate in mammals that have lost the
ability to make dental enamel during evolution.

73. • Ameloblasts undergo pathological changes, such as mineralization of
the cell layer, disorganization, and apoptosis.

74. Amelogenin
• Amelogenin protein is the major component of the continuously secreted
enamel extracellular matrix that controls the mineralization of enamel
crystals.
• amelogenin is essential for the sustained growth in the length of the enamel
crystallites and it to play a critical role in helping to form the complex
prismatic architecture of normal enamel

75. Enamelin
• Enamelin, the largest known enamel protein, is a minor component of the
matrix (1 to 5%) and is absolutely essential for formation of normal enamel
tissue.
• Porcine enamelin is secreted as a 186-kDa (1104 aa) glycoprotein.
• This acidic glycoprotein, like amelogenin, is processed immediately following
secretion producing intermediate products (155 kDa, 145 kDa, 89 kDa) that
are not stable and found only near the enamel surface.

76. • One stable proteolytic intermediate fragment that accumulates to about 1% is
the 32kDa enamelin, which has a strong affinity to adsorb onto the enamel
crystals and is highly conserved across species.
• Mutations in ENAM gene results in defective enamel, specifically hypoplastic
form of autosomal dominant amelogenesis imperfecta (AI), and a diverse
group of genetically altered conditions.
• While the presence of enamelin was shown to be critical for the mineralization
of normal enamel, details on the molecular mechanisms of its functions in
controlling enamel crystal formation are still lacking.

77. Determinants of skeletal homeostasis and bone
mass. Schematic representation of the servo
system that maintains bone mass at steady-state
levels. Physiological (blue) and pharmacological
(orange) stimulators and inhibitors of bone
formation and resorption are listed. The relative
impact, where known, is represented by the
thickness of the arrows.

78. Bone Markers
• Parallel with better understanding of biochemical processes in bone and isolation
and characterization of cellular components of skeletal matrix, the number of
new potential biochemical markers of bone formation and resorption is
increasing. Generally, markers are classified into the following groups
• a. Enzyme activity markers of bone formation (connected with osteoblast
activity) and of bone resorption (connected with osteoclast activity);
• b. Bone matrix proteins and resorption products of organic skeletal matrix,
which are released into circulation during bone formation and resorption;
• c. Inorganic skeletal matrix markers (calcium, phosphorus which, above all,
reflect calcium-phosphorus homeostasis).

79. • Biological factors like tissue specificity, effect of change in liver or kidney
function on marker clearance, biological rhythm of the marker due to
standardization of physiological sampling time, immobilization, etc.
• Pre-analytical factors sample storage procedures, i. e. time and temperature,
sample freezing and thawing, anticoagulant effect, etc.
• Analytical specificity and accuracy microheterogeneity of markers as, e. g. ,
degree of ALP glycosylation, possibility of marker resorption into several different
fragments as in case of osteocalcin, bias in methods due to non-harmonized
calibrations, specificity of antibodies and, enzyme activity inhibitors.
• Diagnostic validity the question of differences between markers considering their
diagnostic sensitivity and specificity

80. BONE
FORMATION
MARKERS

81. BONE
RESORPTIO
N MARKER

84. Conclusion
• The two sialoprotein, osteopontin and BSP are important for initiation of
mineralization, where BSP probably acts as crystal nucleator (sodek et.al. 1992)
• Excessive mineralization is prevented by osteocalcin and osteonectin, whose
ability to slow down crystal growth may regulate the growth and size of HA
crystal.
• Osteopontin, BSP and Osteocalcin seem to have dual roles, they are important
for both mineralization and resorption, whereas (so far) the only function of
osteonectin in bone seems to be as a negative regulator of crystal growth.

85. Bibliography
• URIST, M.R.; SILVERMAN, B.F.; BURING, K.; DUBUC, F.L.; and ROSENBERG, J.M.:
The Bone Induction Principle, Clin Orthop 53: 243-283, 1967.
• YEOMANS, J.D., and URIST, M.R.: Bone Induction by Decalcified Dentin Implanted into
Oral Osseous and Muscle Tissues, Arch Oral Biol 12:999-1008, 1967
• Addadi L, Weiner S (1985). Interactions between acidic proteins and crystals:
stereochemical requirements in biomineralization. Proc Natl Acad Sci USA 82:4110-4114.
Aplin HM, Hirst KL, Crosby AH, Dixon MI (1995).
• Mapping of the human dentin matrix acidic phosphoprotein gene (DMPI) to the
dentinogenesis imperfecta type II critical region at chromosome 4q21. Genomics
30:347349.

86. • Duncan H, Frost H M, Villaneuva A R, Sigler J W. The osteoporosis of rheumatoid arthritis.
Arthritis Rheum 1965; 8: 943-54.
• Kennedy A C, Lindsay R. Bone involvement in rheumatoid arthritis. Clin Rheum Dis 1977;
3: 403-20.
• Satyanarayana.U, Chakrapani.U, Proteins and amino acids , Text book of biochemistry
,Books and allied Ltd;3:43.
• Hoffman R. Bone formation and resorption around developing teeth transplanted into the
femur. Am J Anat 1966;118(1):91–102.
• Schroeder HE. Handbook of microscopic anatomy. Vol. 5. The periodontium. Berlin:
Springer-Verlag, 1986: 12– 323.

87. Thank You

88. PREVIOUS SEMINAR
QUESTIONS

89. difference between Bacterial and
human collagenase
• Bacterial collagenase has a wide range of specificity i.e. it can degrade
the stable native helix collagen structure under denaturing condition
under specific Ph and temperature.
• True bacterial collagenases are consensually described as enzymes that
cleave helical regions of fibrillar collagen molecules under
physiological conditions (Harrington, 1996).
• The destruction of collagen fibers by collagenases is one of the unique
characteristics of periodontal disease (Choi et al., 2012).
Bacterial collagenases - A review. Duarte AS, Correia A, Esteves AC Crit Rev Microbiol. 2016;42(1):106-26. doi:
10.3109/1040841X.2014.904270. Epub 2014 Apr 22.

90. MMP-8 extensively used ?
• it is the active form of MMP-8 that initiates collagen destruction then the
ability to measure this activity in GCF should improve our understanding of
the role of MMP-8 in health and disease and in due course facilitate
biomarker development for periodontal tissue destruction.
• MMP-8 has an important role in the initial stages of collagen
degradation. It has also been shown to be highly discriminatory for the
site-specific diagnosis of periodontitis and the longitudinal monitoring
of periodontitis
Matrix metalloproteinase-8 activity in gingival crevicular fluid: development of a novel assay.
McCrudden, M. T. C., Irwin, C. R., El Karim, I., Linden, G. J., & Lundy, F. T. (2016). Matrix metalloproteinase-8
activity in gingival crevicular fluid: development of a novel assay. Journal of Periodontal Research.
DOI: 10.1111/jre.12423

91. • In periodontitis, neutrophils attracted to sites harbouring bacterial biofilms are a
major source of MMPs, particularly MMP-8 (released from specific/secondary
granules) and MMP-9 (from secretory/tertiary granules).
• MMP-8 released from neutrophils has been characterised to unwind the gross
triple helix conformation of collagen and thus initiate collagen destruction,
whereas MMP9 can only degrade collagen which has already been denatured by
the collagenases
• The MMPs have emerged as a family of proteolytic enzymes worthy of
investigation as both diagnostic aids and as therapeutic targets in periodontitis

92. Chemokines vs Cytokines
• Cytokines and chemokines are two immune-modulating agents, which are involved in
the mediating and modulating of the responses of the immune system.
• Several types of cytokine superfamilies are identified: chemokines, ILs, INFs, CSFs,
TNFs and TGFs. They only differ in the function they perform in the body.
• Chemokines produce a concentration gradient, guiding other leukocytes to the site of
infection. The main difference between cytokines and chemokines is that cytokines are
small protein substances which are secreted by cells in the body, affecting other
cells whereas chemokines are one of the superfamilies of cytokines, containing
chemotactic activity.

93. Cytokines Chemokines
Immune modulating agents made up from
proteins .
Superfamily of cytokines which mediates
chemotaxis.
Involved in both cellular and antibody
mediated immunity in the body.
Involved in directing the cells in the immune
system to the site of infection.
Chemokines, Ils, IFs, CSFs, TNFs and TGFs
are the types of cytokines in the body.
CC chemokines, CXC chemokines, C
chemokines and CX3C chemokines are the
structural types of chemokines .
Cytokines generally act autocrine and
paracrine mechanisms.
Chemokines act on the receptor associated G
protein.
Cytokines induce the formation of other
cytokines creating ( "amplification cascade").
chemokin act through receptors related to G -
protein

95. Use of Hyaluronic Acid
• Treat aging skin
• Osteoarthiritis – decrease joint stiffness and pain
• Cataract – injecting into eye during cataract surgery
• To treat Mouth sores
• Dry eyes – Hyalistil
• Eye trauma – treat detach retina and other eye disorders
• Healing skin wounds and burns

96. SERUM
INHIBITO
RS OF
MMPS

97. Collagenase Mmps
• MMP 1, 8, 13 and 18

98. BONE PROTEINS IN
EXTRACELLULAR
MATRIX
Dr . Suman Mukherjee
MDS 1ST YR