BONE

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BONE

• ITS ALIVE!
• Organic Framework
• Osteoblasts, osteocytes, osteoclasts
• Collagen
• Other organic molecules
• Inorganic Salts
• calcium and phosphorus keep bone rigid
• Bone stores minerals and ions for body functions

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Bone begins as mesenchymal cells

• 4 bone cell types
• Progenitor cells
• Osteoblast
• Osteocyte
• Osteoclast

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MBOC
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Periosteum

• A thin connective tissue layer surrounding bone
• Contains the cells that are the source of bone
• Osteoprogenitor cells
• Must be preserved during surgery or bone will not
  re-grow

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Osteoprogenitor or Osteogenic cells

• Appearance
• pale staining,
• small, spindle shaped
• Location
• present on all non-resorbing surfaces
• periosteum

• Function
• give rise to osteoblasts in vascularized regions
• chondroblasts in avascular regions

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Osteoblasts

• Appearance
• large, nondividing cells,
• Active nucleus
• Lots of rER and golgi,
• cytoplasmic processes
• Form a monolayer covering all sites of active
  bone formation

• Function
• Highly polarized cells that synthesize and
  secrete organic constituents of bone matrix
  (osteoid)
• aid in calcification

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osteoblasts
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Two forms of bone

• Compact or Lamellar Bone
• Solid bone, no spaces
• except for those accommodating cells, processes
  and blood vessels
• Cortex of long bones

• Spongy, cancellous or trabecular bone
• Usually interior of bone
• Spaces exist between trabeculae (bridges) of bone
• Marrow found within the spaces
• Spine, ribs, jaw, wrist

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Bone requires extensive vascularization
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Compact bone
Cancellous bone
Not at same magnification
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Compact bone morphologydont memorize this

• Lacuna
• osteocyte home
• Haversian canal
• Central canal for blood vessels, etc
• Canaliculi
• Osteocyte processes
• Lamellae
• Concentric circles representing appositional bone
  deposition

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Cancellous bone
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Bone Ossification

• Involves both production of organic bone matrix
  and calcification
• Details of both processes to follow in week 17
• Two types of ossification
• Intramembranous
• Our cells are performing this type
• Endochondral
• Requires a cartilage model

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Intramembranous ossification
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Our Experiment

• Inducing bone marrow mesenchymal stem (D1 ORL
  UVA) cells to differentiate into bone
• Ascorbic acid-2-phosphate
• B Glycerol phosphate
• In 4 weeks will look for various phenotypes
• Production of alkaline phosphatase
• Sequestering/production of calcium
• In short, has bone been made?!

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D1 ORL UVA cells

• Cloned from a multipotent mouse bone marrow
  stromal precursor
• Can differentiate into osteocytes, chondrocytes,
  adipocytes in presence of appropriate stimulus
• Are homing cells
• When injected systemically, will home to the bone
  marrow, unless specifically placed in another
  location
• Can use various tracers, including GFP, to see
  this happen
• Can be used to treat osteoporosis, improve bone
  implant adherence, augment bone growth or repair.

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Differentiation and Expansion Media

• Expansion media
• DMEM, sodium bicarbonate, antibiotics and FBS
• To keep a stock plate going
• Differentiation media
• Use this on 6 well plates
• As above with
• ascorbic acid-2 phosphate
• A stable form of vitamin C
• Induces production of collagen
• B glycerol phosphate
• Induces production of alkaline phosphate

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General plan of experiment
3/24-25, plate 1 row of wells Change media on
3/27-28
3 week differentiation
3/30-4/1, plate next row of wells Change media on
4/3-4
2 week differentiation
4/7-8, plate next two rows of wells Change media
on 4/10-11 Top row has differentiation media,
next has expansion media
1 week differentiation
undifferentiated
You will also be keeping a stock plate each time
in expansion media
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Working with D1 ORL UVA cells

• Keep your media straight
• Stock plate gets expansion media,
• Wells get differentiation media
• Trypsinizing is slightly different
• Rinse 2x with PBS
• Add 1/2ml of TRED and DONT ASPIRATE
• Not necessary to place in incubator, but go
  ahead and watch under microscope
• To prevent clumping, dont slap plate
• Stop action of TRED by adding at least 3x amount
  of expansion media

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Working, continued

• Plating the well plates requires centrifugation
  to place cells in proper media
• Resuspend cells in expansion media and count!
• Plate proper amount on stock plate
• Place remaining cells into 15ml tube and
  centrifuge
• Resuspend to have 1 x 104 cells/ml
• Plate two mls/well
• Best to keep cells at 70 confluency or less on
  stock plate
• High confluency may induce changes

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Splitting your stock plate on the return day

• Instead of counting, split your plate
• Look at your plate before trypsinizing,
• if it is very confluent, do a 16 split,
• if it is of low confluency, do a 13 split
• Splitting is a quick way to renew your plate once
  you a comfortable working with your cells.
• Splitting is different from diluting
• It is MUCH easier, but dont get them confused!

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Simply, 15

• The volume (mls) you resuspend in is the second
  number
• The volume you take out and put in new plate is
  the first number
• If you resuspend in 5mls of media
• and you add 1ml of this suspension to your new
  plate,
• that is a 15 split
• you took 1/5th of the re-suspension
• Make up the difference in the new plate with the
  appropriate amount of media