lab 9 bone_experiment

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					                    BONE
• IT’S 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
Bone begins as mesenchymal cells



                    • 4 bone cell types
                       –   Progenitor cells
                       –   Osteoblast
                       –   Osteocyte
                       –   Osteoclast
MBOC
                    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
 Osteoprogenitor or Osteogenic cells
• Appearance
                          • Function
  – pale staining,
                            – give rise to
  – small, spindle
                              osteoblasts in
    shaped
                              vascularized
• Location                    regions
  – present on all non-     – chondroblasts in
    resorbing surfaces        avascular regions
  – periosteum
                   Osteoblasts
• Appearance                • Function
  – large, nondividing        – Highly polarized
    cells,                      cells that synthesize
  – Active nucleus              and secrete organic
  – Lots of rER and             constituents of bone
    golgi,                      matrix (osteoid)
  – cytoplasmic processes     – aid in calcification
  – Form a monolayer
    covering all sites of
    active bone formation
osteoblasts
              Two “forms” of bone

• Compact or Lamellar • Spongy, cancellous
  Bone                  or trabecular bone
  – Solid bone, no spaces     – Usually interior of
     • except for those         bone
       accommodating cells,   – Spaces exist between
       processes and blood
       vessels
                                trabeculae (bridges) of
                                bone
  – Cortex of long bones
                              – Marrow found within
                                the spaces
                              – Spine, ribs, jaw, wrist
Bone requires extensive vascularization
Compact bone                   Cancellous bone




               Not at same magnification
Compact bone morphology
     don’t memorize this
                 • Lacuna
                    – osteocyte home
                 • Haversian canal
                    – Central canal for blood
                      vessels, etc
                 • Canaliculi
                    – Osteocyte processes
                 • Lamellae
                    – Concentric circles
                      representing
                      appositional bone
                      deposition
Cancellous bone




         trabeculae
marrow
              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
Intramembranous ossification
          Our Experminent
• 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?!
              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.
  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
            General plan of experiment
3/27-28, plate 1 row of
wells
Change media on 3/30-
31                                          3 week differentiation
4/3-4, plate next row of
wells
Change media on 4/6-7                       2 week differentiation

4/10-11, plate next two
rows of wells
Change media on 4/13-                         1 week differentiation
14
Top row has                                    undifferentiated
differentiation media,
next has expansion
media will also be keeping a stock plate each time in expansion media
  You
     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 DON’T ASPIRATE
   – Not necessary to place in incubator, but go ahead and watch
     under microscope
   – To prevent clumping, don’t slap plate
   – Stop action of TRED by adding at least 3x amount of
     expansion media
               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
        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 1:6 split,
      • if it is of low confluency, do a 1:3 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 don’t get them confused!
                    Simply, 1:5
• 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 1:5 split
   –   you took 1/5th of the re-suspension
• Make up the difference in the new plate with the
  appropriate amount of media

				
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posted:8/12/2012
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