Download Bone Matrix

Bone
• Gross histology
• Bone matrix and bone cells (osteoblast, osteocyte and osteoclast)
• Regulation of osteoblast/osteocyte/osteoclast formation
• Endochondral and intramembranous bone formation
• Bone remodeling
Development and Maintenance of the Skeleton
Development Maintenance
Age
0-20
20-50
50+
BR  BF
Modeling
BR  BF
Remodeling
BF > BR
Uncoupled
BF = BR
Coupled
BF < BR
Uncoupled
Sequence BF  BR
Activity
Osteoporosis
Marks S.C. and Hermey, D.C. The Structure and Development of Bone; Principles of Bone Biology,
Blezikian, Raisz and Rodan, eds, 1996.
Chemical Composition of Bone
Body of Mandible
Flat (skull and pelvis) and long (femur, tibia) bones
Two types of bone macroscopically:
1. Compact bone: dense outer sheet covering flat bones and shaft of long bones;
mature bone
2. Trabecular/cancellous/spongy bone: present within the central marrow cavity
comprised of network of delicate bars and sheet of trabecular bone which branch
and intersect to form a sponge-like network
Periosteum: layer of dense connective
tissue surrounding bone. Has 2 layers:
Outer fibrous layer and inner layer next
to bone containing bone cells their
precursors and blood vessels
Endosteum: layer if thin connective tissue
lining the inner surface of bone facing
bone marrow
Microscopic appearance (both compact and trabecular)
Arranged as lamellae (microscopic layer)
3 distinct lamellae: circumferential concentric and interstitial
concentric
Concentric lamellae forms the basic metabolic unit of bone the osteon
(haversian system)
Bone Matrix
Bone Matrix is physiologically mineralized and is constantly
regenerated throughout life as a consequence of bone turnover
Bone consists various bone matrix proteins that play an integral
part in bone function
The predominant and basic building block of bone is type I
collagen
However, trace amounts of type III and V collagens are
also present
Association of type I collagen to mineralization
Type I Collagen
Most abundant protein in bone matrix (~ 85 - 90%
of organic matrix)
Provides elasticity, flexibility and serves as a scaffold
determining shape
Binds and orients other proteins that nucleate hydroxyapatite
deposition
Collagen undergo several post translational modifications
(hydroxylation of certain lysyl and hydroxylysyl residues), which
are measured in urine as a parameter for bone resorption
The animal model with defective type I collagen production has a
condition similar to osteogenesis imperfecta, where the bones are
mechanically weak and mineral crystals small
Type I collagen
19 types of collagen known so far in humans. Types 1, 2, 3 and 4 are
most common
Predominantly synthesized by fibroblasts
Rod-like protein. Type I collagen has a length of ~ 300 nm, 1.5 nm in
diameter and consists of a right handed triple helix of subunits
composed of two 1(1) chain and one 2(1) chain
There are 3 amino acids per turn and every 3rd amino acid is a G
Synthesized as type 1 procollagen that contain an additional 150 amino
acids at the N-terminal and 250 at the c-terminal
Normal
OI
The Origin and Location of Bone Cells
Marks S.C. and Hermey, D.C. The Structure and Development of Bone; Principles of Bone Biology,
Blezikian, Raisz and Rodan, eds, 1996.
Osteoprogenitor cells: Contribute to maintaining the
osteoblast population and bone mass. Located in the
periosteum and endosteum and differentiate into osteoblasts
Osteoblasts: synthesize the bone matrix on the bone forming
surfaces
Osteocytes: organized throughout the mineralized bone
matrix that support bone architecture
Osteoclasts: large multinucleated cells derived from fusion
of monocytes/macrophages and resorbs bone.
Osteoblasts are located on the periosteal and endosteal bone
surfaces and are responsible for bone formation through secretion
of the organic components of bone matrix
Functions of Osteoblasts
1. Main Function of osteoblast: Secretion of a complex
mixture of bone matrix proteins (osteoid)
Secretion of osteoid is unidirectional towards the bone surface
Osteoblasts are normally separated form the mineralized bone
matrix by a thin layer of unmineralized matrix (osteoid seam)
2. Regulate differentiation and activity of osteoclasts
2. Indirectly maintain calcium homeostasis
3. Indirectly responsible for mineralization of osteoid
Osteoblast Differentiation
Origin of Osteoblasts
Limited
self renewal
Unlimited
self renewal
osteoblasts
Runx2
Osx
PPAR2
Multipotential
Daughter
Tri-Bipotential
Cell
Progenitor
Cell
Stem Cell
Decreasing proliferation
PPAR2
adipocytes
chondroblasts
myoblasts
fibroblasts
Increasing differentiation
Adapted from Primer on the metabolic bone diseases and disorders of mineral metabolism.
Lian JB, Stein GS and Aubin JE., Bone formation: Maturation and functional activities of
osteoblast lineage cells, ASBMR, 2003
Runx2 is a bone-related transcription factor
that is essential for osteoblast differentiation
and bone formation
Wildtype
Otto et al., 1997
Runx2-/-
Osteoprogenitor Cells: Mesenchymal cells located on periosteal surfaces
and bone marrow stromal cells are a good source of osteoblasts which act
in concert with osteoclasts to model bone during growth and maintain
bone architecture during adulthood
Commitment of mesenchymal stem cells (MSCs) to tissue-specific types
is mediated by trancriptional regulators that serve as “master switches”
BMP2/4/7 induce transcription factors that mediate commitment of
early progenitors (stem cells) toward a osteoblast phenotype
In Vitro Development of Osteoblast Phenotype
Pre-osteoblast
Proliferation
> Osteoblast > Pre-osteocyte > Osteocyte
Matrix Maturation
Mineralization
Apoptosis
Histone
Collagen
TGF 
Msx-2
cFos/cJun
Id, Twist
Growth
Collagen
Alk Phos
Fra-2/JunD
Cbfa1
Dlx-5
MGP
Osteopontin
Bone SialoProtein(BSP)
Osteocalcin
Collagenase
Bax
Differentiation
Proliferation >
+
ECM
< Mineralization
The osteoblast developmental sequence: Lessons from cell culture studies
3 stages of bone cell differentiation are recognized according to the proteins and
mRNA expressed at different stages:
•
Proliferation and extracellular matrix (ECM) biosynthesis
•
ECM development, maturation and organization
•
ECM mineralization
Osteoblast Differentiation
1.
Proliferation and ECM biosynthesis: Increased mitotic activity with expression of
cell cycle and cell growth regulated genes that support proliferation
During this proliferation period, and fundamental to development of bone phenotype,
several genes associated with formation of the ECM (type I collagen, fibronectin,
and transforming growth factor- (TGF- ) are actively expressed and then gradually
down-regulated with collagen mRNA being maintained at low basal level during
subsequent stages of osteoblast differentiation
2.
Immediately after down-regulation of proliferation, proteins associated with the bone
cell phenotype are detected
Alkaline phosphatase levels increase 10-fold in the immediate post-proliferative phase
3.
With the onset of mineralization, other bone-related genes are induced: bone
sialoprotein (BSP), osteopontin and osteocalcin are increased which parallels
mineral deposition
Osteocytes: Terminally differentiated osteoblasts
Osteocytes: terminally differentiated osteoblasts that support bone structure and
metabolic function
Osteocytes develop by forming numerous cytoplasmic connections with adjacent
cells to ensure their viablity as the mineralizing osteoid renders the ECM impermeable
Osteocytes are present in lacunae and has numerous cellular extensions of filapodial
processes
Osteocytes express OCN, galectin-3 and CD44 (a cell adhesion receptor for hyaluronate
and several bone matrix proteins that are involved in cellular extensions)
Osteocytes are continuous with the lining osteoblasts and is necessary for transmitting
mechanosensory signals such as transducing stress signals (streching, bending) to
biological activity
Osteoclast
Multinucleated cell the originates from hematopoietic stem cells
Characterized by possessing tartrate-resistant acid phosphatase (TRAP) within its
cytoplasmic vesicles and vacuoles
Found against bone surface in hollowed depressions called Howship’s lacunae
RANK: receptor-activated nuclear factor κB and RANKL: RANK ligand
Ruffled border: Adjacent to resorbing
bone surface the osteoclast is closely
apposed to bone and has deep folds
called ruffled border.
Adjacent cytoplasm does not have any
organelles and is enriched in actin.
This zone is called the clear or sealing
Zone. This enables the osteoclast to
attach to mineralized surface and
creates an acidic microenvironment
which demineralizes bone and exposes
the organic matrix.
The matrix is degraded by enzyme acid
phosphatase and cathepsin B
Bone Formation
Endochondral bone formation
Intramembranous Bone Formation
Bone Remodeling
Five Phases of Remodeling
1. Activation of Osteoclasts
2. Resorption of Bone
3. Reversal Phase
4. Formation of Bone
Activation of Osteoblasts
Mineralization
5. Resting
Mineralization
Physiologic bone mineralization in mammals refers to the ordered
deposition of apatite on a type I collagen matrix
The mineral is an analog of the geologic material, hydroxyapatite
[Ca10(PO4)6(OH)2] - provides mechanical rigidity and load
bearing
strength to the bone composite
Bone mineral also contains numerous impurities (carbonate,
magnesium, acid phosphate) and vacancies (missing OH-) and is
usually referred to as a poor, crystalline, carbonate-substituted
apatite. These small imperfect crystals are more soluble that
geologic apatite, enabling bone to act as a reservoir for calcium,
phosphate, and magnesium ions
Steps in Mineralization:
1. Initiation
2. Propagation
3. ECM mineralization
Bone Mineral is deposited at discrete sites in collagenous matrix
As bone matures, the mineral crystals become larger and more perfect
(containing fewer impurities)
The increase in crystal dimension is due both to the actual addition of
ions to the crystals (crystal growth) and to aggregation of the crystals
Initiation
Not dependent on
alkaline phosphatase
Propagation
ECM mineralization
Dependent on alkaline phosphatase