Conference on bone

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Conference on bone

• VIBS 443 and VIBS 602

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FUNCTIONS OF CARTILAGE

• EVOLUTIONARY - EMRYONIC MODEL FOR BONES

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CELLS OF CT

• FIBROBLASTS
• MESENCHYMAL
• CELLS and RBC
• ADIPOSE CELLS
• MACROPHAGE
• PLASMA CELLS
• MAST CELLS and WBC
• CHONDROBLASTS
• CHONDROCYTES
• OSTEOBLASTS
• OSTEOCYTES
• OSTEOCLASTS

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HYALINE CARTILAGE

• EPIPHYSEAL PLATE, RESPONSIBLE FOR LONGITUDINAL
  GROWTH OF LONG BONES

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ENDOCHONDRAL (SPONGY/ CANCELLOUS) BONE FORMATION

• CARTILAGE MODEL
• CENTERS OF OSSIFICATION
• PRIMARY CENTER OF OSSIFICATION
• DIAPHYSIS
• SECOND CENTER OF OSSIFICATION
• CENTER OF EACH EPIPHYSIS

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MITOSIS

• DEFINED - CELL DIVISION IN WHICH DUPLICATED
  CHROMOSOMES DIVIDE INTO DAUGHTER CELLS THAT HAVE
  IDENTICAL CHROMATIN CONTENT

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Developing ovarian follicle has many cells in
mitosis
mitosis
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Radioactive precursors is the source of
radioactivity in final cellular
product------------------------------------------

• Product Precursor

Protein (general) amino acids
DNA thymidine
RNA uridine
Collagen (sp. protein) proline and lysine (sp.
a.a.)
Cartilage matrix 35 SO4
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Tritiated Thymidine labeling of nuclei
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DO THE PULSE CHASE EXPERIMENT
LOOK FOR THE TEMPORAL APPEARANCE OF SILVER
GRAINS IN THE PHOTOGRAPHIC EMULSION OVER
ORGANELLES OF INTEREST
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EVIDENCE FOR PROTEIN PATHWAY

• TEMPORAL APPEARANCE OF RADIOACTIVE PROTEINS IN
  DIFFERENT ORGANELLES
• (produced from radioactive precursors
  e.g., labeled AA)
• Detected by
• AUTORADIOGRAPHY -
• VISUAL
• CELL FRACTIONATION - BIOCHEMICAL

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EVIDENCE FOR PROTEIN PATHWAY

• AUTORADIOGRAPHY
• PROCEDURE TO LOCALIZE A PRODUCT (e.g., PROTEIN)
  WITHIN A CELL OR GEL THAT IS SELF-RADIOACTIVE DUE
  TO THE CELLS INCORPORATION OF RADIOACTIVE
  PRECURSORS (e.g., RADIOACTIVE AMINO ACIDS) INTO
  THAT PRODUCT THAT IS VISUALIZED IN A
  PHOTOGRAPHIC EMULSION.

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GENERAL ORGANIZATION OF CARTILAGE

• CHONDROCYTES / CHONDROBLASTS

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239
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ANGIOGENESIS
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ANGIOGENESIS growth of blood vessels
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• MESENCHYMAL
• CELLS
• Endothelial cells
• Smooth muscle cells
• Fibroblasts

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COMPACT BONE

• REMODELING OSTEOCLASTS DIGEST BONE,
  OSTEOBLASTS FROM ENDOSTEUM REPLACES IT

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CELLS OF BONE

OSTEOBLAST OSTEOCYTE
OSTEOCLAST
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BONE REPAIR
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Expected events with bone formation

• Clot
• Migration if mesenchymal cells
• Proliferation
• Differentiation
• Calcification
• Entry of blood vessels,
• osteoclasts, and osteoblasts
• Colonization of bone marrow

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Cellular/tissue processes involved

• Chemotaxis
• Mitosis
• Cell differentiation
• Cell death
• Angiogenesis

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Considerations

• How to set up an experiment to test the temporal
  changes in transplant growth and development?
• How to set up an assay for each event in bone
  formation to evaluate the temporal changes
  expected in transplants placed in the back skin
  of rats?
• What can be concluded about the cellular and
  tissue processes involved in the general
  transplant or transplants receiving specific
  extracted components?

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CONFERENCE ON BONE FORMATION

• Because your brother was born with a severe
  facial disfigurement, you decided to become a
  plastic surgeon. You dreamed of developing a
  magic powder that you could mix and form into a
  mode, that when implanted would stimulate the
  formation of bone of exactly the right shape and
  structure that you needed for your reconstructive
  surgery. Being more than a dreamer, you decided
  to study bone formation and figure out how to
  transform your dream into reality.
• Just for kicks, you demineralized some rat bone
  fragments (treat with 0.5M HCl, then extract with
  distilled water, ethanol ether) to kill all the
  endogenous cells (such that new bone growth would
  result from stimulation of host cells and not
  proliferation of cells from the implant itself)
  and extract most of the calcium and lipids, and
  then implanted these fragments under the skin of
  another rat. Subcutaneous implantation of
  demineralized bone matrix in rats was found to
  induce factors in the host resulting in the
  biosynthesis and differentiation of cartilage,
  bone, and hematopoietic marrow at the site of the
  implant.
• 1. Design an experiment to test the sequence of
  events and the time course required for new bone
  formation.
• A. Describe the expected morphology of each stage
  of new bone formation occurring at the site of
  the implant.

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• B. If you injected radioactive thymidine (cell
  division) or sulfate (metabolic differentiation)
  at the site of the implant of developing bone and
  then made sections of the implanted material
  followed by autoradiography, which cells and/or
  structures of the developing bone would have
  incorporate either label?
• 2. In order to try to isolate and characterize
  some of these factors, you extract the
  demineralized bone with high salt and SDS,
  conditions which should extract many proteins.
  You then implant the bone residue to see if it
  still promotes bone formation. What stages of
  bone formation might be blocked, and how could
  you test where it is blocked? If you hypothesize
  that your extraction procedure has removed a
  chemotactic factor necessary for recruitment of
  mesenchymal cells toward the site of the implant,
  what tests could you perform to prove your
  hypothesis?
• 3. Your brother's bone disease is postulated to
  have been due to an overproduction of
  osteoclasts. Using bone fragments and marrow
  obtained by a biopsy, what morphological
  characteristics would you expect his adult bone
  to have? What results might occur if you
  implanted demineralized bone fragments from the
  biopsy into rats? How could you explain your
  result if the bone implant from the biopsy
  material resulted in normal bone formation? What
  tests might you perform if you suspect that the
  matrix of your brother's bone is the problem?

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HISTOGENESIS OF BONE

• ENDOCHONDRAL OSSIFICATION
• DEPOSITION OF BONE MATRIX ON A PREEXISTING
  CARTILAGE MATRIX
• CHARACTERISTIC OF LONG BONE FORMATION

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ENDOCHONDRAL BONE GROWTH

• IN WIDTH - EXTENSION OF COMPACT BONE BY
  APPOSITIONAL GROWTH
• IN LENGTH - EPIPHYSEAL PLATE
• CARTILAGE GROWTH
• CALCIFICATION OF CARTILAGE
• SPONGIOSA
• PRIMARY SPONGIOSA
• CARTILAGE CORE
• SECONDARY SPONGIOSA - OSTEOID CORE