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Inorganic Salts
calcium and phosphorus keep bone rigid Bone stores minerals and ions for body functions
MBOC
Periosteum
A thin connective tissue layer surrounding bone Contains the cells that are the source of bone
Osteoprogenitor cells
Function
give rise to osteoblasts in vascularized regions chondroblasts in avascular regions
Location
present on all nonresorbing surfaces periosteum
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
osteoblasts
Usually interior of bone Spaces exist between trabeculae (bridges) of bone Marrow found within the spaces Spine, ribs, jaw, wrist
Compact bone
Cancellous bone
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
Endochondral
Requires a cartilage model
Intramembranous ossification
Our Experiment
Inducing bone marrow mesenchymal stem (D1 ORL UVA) cells to differentiate into bone
Ascorbic acid-2-phosphate B Glycerol phosphate
Can be used to treat osteoporosis, improve bone implant adherence, augment bone growth or repair.
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
4/7-8, plate next two rows of wells 1 week differentiation Change media on 4/1011 Top row has undifferentiated differentiation media, next has expansion media will also be keeping a stock plate each time in expansion media You
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
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!
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