Ch. 2 - Bone cells

Mature osteoblast fate after matrix production and mineralization

- 60-80% undergo apoptosis - 0-15% become flat bone-lining cells (could become blasts again, involved in Ca-exchange, may play role in regulatory bone response to environ changes, thought to form bone canopy [overarching bone remodeling surface] - 5-20% become osteocytes by terminal differentiation

Osteocyte: interesting facts

- 90% of all bone cells - former osteoblasts (transition takes 3-5 days) - reside in lacunae - connected to other cells via canaliculi (stellate/dendrite-like extensions projected into the canaliculi)

OPG

- A decoy receptor for RANKL - Ability for differentiation of osteoclasts is based on ratio of RANKL/OPG bc OPG and RANK are competing for RANKL - If more OPG, more RANKL/OPG interactions and less osteoclast differentiation activity - OPG - Soluble, not attached to pre-osteoclasts; out-competes RANK when present

Sealing zone

- A ring sealing clast to bone surface (helps attachment before resorption) - Podosomes (clast extensions) made of F actin (binding protein eventually forming ring to seal off resorption area (on edges of clast) - Integrin receptors on bone help seal - podosome have integrin receptors - osteopontin has RGF components on ECM (these are interns that bind to integrin receptor

Osteoclasts are polarized cells

- Apical membrane = in contact with bone (resorptive side of bone) / tentacle-like morphology called ruffled border - gives greater surface area (more resorp room) / sealing zones (SZ) make two prominent attachment points - Basolateral surface = non-resorptive surface; not ruffled

Runx2 (Cbfa1)

- Core-binding factor alpha-1 - Important transcrip factor - Experiments with Runx2 null mice (mice had non-calcified skeleton, suppressed bone formation) - Functions of the transcript factor - factor has role to get precursor to pre-osteoblast, and to get it to osteoblast / promotes transcrip. of collagen type 1, bone sialoprotein, osteocalcin, osteorectin

Osteoblast derivation

- Derived in MSCs - Reside in bone marrow and periosteum - Pluripotent cells (potential to become one of many cell types depending on signal) - Need specific signaling (SMAD, beta-catenin) to become osteoprogenitor cells

Canaliculi

- Fluid-filled (periosteocyte fluid) - Chemical signals can pass through this fluid

Bone cell lineages

- Hematopoietic stem cells (clasts) - Mesenchymal stem cells (MSCs) (blasts, cytes) - embryonic CT

Osteoclast morphology

- Large cells (bigger than blasts) - Multinucleate (fusion of pre-osteoclasts = nuclei retained) - Organelles (next slide) - Polarized cells (distinct surfaces) (next slide)

Mature osteoblast role in matrix mineralization

- Lay down protein framework where mineral is deposited on - Also initiates mineralization: alkaline phosphatase (ALP) -- enzyme critical in this role/hallmark of bone formation/can be IDed histologically/molecularly/cellularly

Osteocyte function

- Maintain bone mass and matrix - Mechanosensory cell - Secrete sclerostin (regulate blast and last activity)

Osteoclast: background

- Monocyte/macrophage family - In bone marrow - Pre-osteoclasts form in marrow - Travel to sites where resorption is needed - Factors triggering proliferation: macrophage stimulating facotr (C-fms receptor > M-CSF binds to this) - Interleukin-3

Osteoclast: first differentiation step in marrow

- Monocytes --> preosteoclasts - Then travel from marrow to tissue site where it can differentiate into clast - Differentiation regulated by two main factors: 1) M-CSE (macrophage colony stimulator) 2) RANKL (receptor activator of the nuclear factor kappa B ligand) - These secreted by stromal osteoblastic cells + osteocytes in order for clasts to mature

Osteoprogenitor cells to pre-osteoblasts

- Need Runx2 to become pre-osteoblast (elongated, spindly appearance / uninucleate)

Mature osteoblasts form functional synctytium (on bone surface)

- Networks working in unison of cells - Composed of: 1) adherens junctions - link blasts together; link microfilaments to microfilaments from other cells through plaques and transmem glycoproteins (cadherens) 2) tight junctions - mechanically held together; no material can squeeze between cells 3) gap junctions - connected electrically to cytes by blasts; composed of connexons (main connexon contains connexon 43) / connexons linking blast blast and blastcyte

Osteoclast organelles

- Numerous mitochondria to generate ATP used in resorption activites - Numerous vacuoles and vesicles in order to store digestive enzymes and chemicals

Bone cell functions

- Osteoclast resorb (destroy) bone - Osteoblast forms bone (form cells all around them > lacunae > into an osteocyte) - Osteocyte maintains bone; originally osteoblasts (they differentiated)

Sclerostin

- Osteocytes regulate bone formation through sclerostin - Increased bone formation associated with decreased sclerostin - inhibitor of bone formation

Mature osteoblast morphology

- Plump, cuboidal - Uninucleate (from one MSC) - Typical organelles (all ones necessary for protein synth) - Polarity: matrix-facing side (synthesis side), marrow-facing side (signal in marrow talks to blast with receptors on this side (most signal receps here) - Bone resorption and creation happen on bone surface

Mature osteoblast function in ECM protein production

- Produce collagen type 1, osteonectin, osteopontin, osteocalcin

RANK

- Receptor activator of nuclear factor kappa B - The receptor - Located on surface of pre-osteoclasts - When RANKL present, binds to RANK and causes pre-osteoclasts to differentiate into mature osteoclasts

Lacunocanalicular system

- Sets up good ability to communicate with other cytes and blasts - Extensions radiate through lacunae into canaliculi to communicate - Important role in maintaining bone mass bc environmental changes can be sensed by the periosteocyte fluid (also ion + hormone conc.) - Cytes could be mechanosensor (sense mechanical loading) bc sensing fluid flow changes in periosteocyte fluid (ex. change in freq. of mech. vibrations on bone - change fluid flow rate - fluid across surface creates shear force - detected by cytes which can respond

Osteocyte morphology

- Stellate or dendritic - Smaller than blasts (no need for big volume of organelles involved in protein synthesis but still have some nucleus, mito) - Buffer zone (space that isn't mineralized) in lacunae - lacunocanalicular system

Osterix (Osx)

- Works downstream of Cbfa1 - Critical for differentiation - Osx-null mice lacked bone formation

PEX

- early and late cytes - phosphate metabolsim

FGF23

- early and mature cytes - phosphate metabolism

DMP-1

- early and mature cytes - phosphate metabolism and mineralization

Plastin/fimbrin

- expressed in all cytes - dendrite branching

Podoplanin

- expressed in early, embedding sites - cyte dendrite formation

MMP14

- matrix degradation - canaliculi formation

Gene contribution to cytes forming the extensions

- ~50 extensions formed - CD44 - gene coding for plastin and fimbrin - Protein podoplanin

Osteoclast functioning in resporption

1. Osteoclast bound 2. In ruffled border region, class first demineralizes bone, degrades protein matrix 3. Using V-ATPase pumps and CIC-7 exposes bone to HCl 4. V-ATPase uses energy to pump H+ onto bone, creating a gradient for Cl- 5. CIC-7 pumps Cl- onto bone (H+ and Cl- make HCl which eats mineral) 6. Once demineralized, vacuoles and vesicles secrete hydrolyses and proteinases (enzymes) that degrade collagen and NCPs 7. Primary enzyme is cathepsin K, also MMPs

RANKL

Also secreted by osteoblast cells + osteocytes

Osteopontin RGF components

Arginine, glycine, aspartic acid

Cathepsin K

Collagenolytic enzyme (breaks down collagen)

MMPs

Matrix metalloproteinases; degrade protein matrix of bone

Bone cell types

Osteoclast, osteoblast, osteocyte

Factors triggering the differentiation of osteoclast precursors into osteoclasts

RANK, RANKL, Osteoprotegerin (OPG)

Mature osteoblast role in regulating osteoclast differentiation

RANK/RANKL/OPG