The next few weeks:

1
The next few weeks

• Th, April 17bone assays and migration
• Th, April 24come fill out evaluations for Joanne
  and lecture portion, exam review
• Bring your questions
• Mon, April 28 and Tues, April 29final exam, in
  class
• TA determined time feverish attempt to finish
  last lab report before 5pm

2
All of these phrases have appeared on
TA/instructor evaluations. Which two phrases have
NOT appeared on any of Joannes?

• You remind me of Audrey Hepburn.
• If I dont get an A, I will come to your house
  and poison your dog.
• Where do you get your clothes?
• Will you marry me?
• Obviously not me, but too funny not to include
• Strengths good chest and biceps.
• Weakness calves and hamstrings

• I wish all my professors were this enthusiastic.
• The color of the Powerpoints hurts my eyes .
• I dont complain about you to my friends like I
  do about my other TAs.
• Cute and funny
• Treats us like kindergarteners
• She is more like a junior college professor
  because she really cares that you learn the
  material
• Compliments in foreign languages

3
Histological staining of bone and osteoblast
lysate formation
4
Review of the cells and basic bone morphology

• Progenitor cells
• Can become fibroblasts of periosteum or
  chondroblasts or osteoblasts
• Continual production of new osteoblasts which
  replace old osteoblasts
• D1 ORL UVAs are these mesenchymal stem cells

• Osteoblasts 
• Lie along mineralized bone tissues irregular
  contours, shoulder to shoulder one cell deep.  
• Every day, add their own volume of new bone
  tissue to periosteal surfaces.  
• Every three days, new osteoblasts push older
  osteoblasts toward mineralized bone tissues and
  encase them.     

5
More bone cells

• Osteocytes
• Encased osteoblasts cannot add new bone tissue.  
  
• Can transfer nutrients from the periosteum to the
  new bone tissue
• When encased osteoblasts no longer function as
  bone tissue manufacturing osteoblasts, we call
  them osteocytes. 

• Osteoclasts
• Breakdown bone should it become too thick or if
  the body needs calcium

6
Osteoid

• Low proteoglycan content
• cant hold much water
• 10 of organic matrix
• Collagen types I and V
• 90 of organic matrix
• Osteonectin
• anchors collagen to bone mineral
• Osteocalcin
• binds calcium

Red-osteoid
EM of collagen
Blue arrows-osteoblasts Yellow arrows-osteoid
green/yellow- osteoid
7
Biologic Mineralization and Matrix Vesicles

• Occurs in extracellular matrices
• Inside and outside of collagen fibrils
• Biologic mineralization is a cell-regulated event
• Even though it is occurring extracellular
• Physiochemical factors are basic to the process
• Calcitonin, PTH, somatotropin

8

• Initiation of mineralization occurs only when
  CA2 and PO4- ions in the matrix exceed the
  normal threshold level

9
Several events are responsible for mineralization

• Binding of extracellular Ca2 by osteocalcin and
  other sialoproteins
• Creates a high local Ca2
• High Ca2 stimulates osteoblasts to secrete
  alkaline phosphatase which increases the local
  concentration of PO4- ions
• This stimulates further increases in the local
  Ca2

10
Mineralization events, contd

• Osteoblasts release small (50-200nm) matrix
  vesicles into the bony matrix by exocytosis
• Contain alkaline phosphatase and pyrophosphatase
  that cleave PO4- ions from other molecules of the
  matrix

11
Osteoblasts and alkaline phosphatase

• Osteoblasts give a strong cytochemical reaction
  for alkaline phosphatase
• AP is an ectoenzyme that hydrolyzes monophosphate
  esters at high pH.
• The enzyme disappears when cell ceases protein
  synthesis and is embedded in mineralized bone
  matrix to become an osteocyte

12
Mineralization of bone continued

• Eventually the local isoelectric point increases
  which results in the crystallization of CaPO4 in
  surrounding matrix vesicles
• CaPO4 crystals initiate matrix mineralization by
  formation and deposition of hydroxyapatite
  crystals Ca10(PO4)6(OH)2
• Along length of collagen fibers
• Crystals then surround osteoblasts
• Once initial crystals of hydroxyapatite have
  precipitated, they grow rapidly by accretion
  until they join neighboring crystals produced
  around other matrix vesicles
• Wave of mineralization sweeps through the osteoid

13
Bone Ossification

• Involves both production of organic bone matrix
  and calcification
• Two types of ossification
• Intramembranous
• Our cells are performing this type
• Endochondral
• Requires a cartilage model

14
Endochondral ossification-dont memorize
Marieb
15
Intramembranous ossification
16
(No Transcript)
17
Metabolic bone disorders-dont memorize
Normal Osteoporosis Osteomalacia
Green is mineralized bone, red that is not marrow
is unmineralized osteoid
18
Stem cell treatment leads to reduction of bone
formation in children

• A type of stem cell therapy called hematopoietic
  cell transplantation (HCT) often leads to a
  reduction in bone formation in children,
• HCT is used to treat cancers of the blood such as
  leukemia.
• Bone mineral density (BMD) is reduced after HCT
• The proportion of children with osteopenia -- a
  bone-thinning condition just short of
  osteoporosis -- increased from 18 percent at
  baseline to 33 percent at 1 year after HCT
• The level of osteocalcin, a blood marker of bone
  formation, may be predictive of recovery from the
  initial bone loss.
• Osteocalcin levels greater than 6.5 ng/L at 100
  days predicted recovery from the initial bone
  loss at 1 year.
• Bone resorption was not increased (work of
  osteoclasts), bone production was decreased
  (defective osteoblast functioning)
• SOURCE The Journal of Clinical Endocrinology and
  Metabolism, March 2006.

19
General plan of experiment
Von Kossa
lysates
3 week differentiation
2 week differentiation
1 week differentiation
undifferentiated
20
Creating Osteoblast Lysates

• Keep everything on ice or in freezer block!
• Start with undifferentiated and progress through
  1, 2, 3 week differentiated.
• Clean scraper in ethanol in between
• Clean sonicator tip in ethanol after use.
• Freeze these samples for next week.

21
Von Kossa Staining

• Fixing
• Neutral formalin buffer (NFB)
• Formaldehyde and salts
• Staining for ALP (red)
• Red Violet
• Naphthol, N,N Dimethylformamide, salts
• Staining for mineralized bone (brown/black)
• 2.5 Silver nitrate solution
• This is an environmental hazard, please dispose
  of pipets where indicated!!

22
Safety and considerations

• Add reagents to plate in fume hood
• Wear goggles and gloves
• Good idea to bring goggles since lab only has a
  few
• But you dont need lab coat
• Dispose of wastes properly
• Follow instructions and place wastes in labeled
  bottles