Conference on bone
VIBS 443 and VIBS 602
FUNCTIONS
OF CARTILAGE
EVOLUTIONARY EMRYONIC MODEL
FOR BONES
CELLS OF CT
FIBROBLASTS
MESENCHYMAL
CELLS and RBC
ADIPOSE CELLS
MACROPHAGE
PLASMA CELLS
MAST CELLS and WBC
CHONDROBLASTS
CHONDROCYTES
OSTEOBLASTS
OSTEOCYTES
OSTEOCLASTS
HYALINE
CARTILAGE
EPIPHYSEAL PLATE,
RESPONSIBLE FOR
LONGITUDINAL GROWTH
OF LONG BONES
ENDOCHONDRAL (SPONGY/
CANCELLOUS) BONE FORMATION
CARTILAGE MODEL
CENTERS OF OSSIFICATION
PRIMARY CENTER OF
OSSIFICATION
• DIAPHYSIS
SECOND CENTER OF
OSSIFICATION
• CENTER OF EACH EPIPHYSIS
MITOSIS
DEFINED - CELL DIVISION IN WHICH DUPLICATED
CHROMOSOMES DIVIDE INTO DAUGHTER CELLS
THAT HAVE IDENTICAL CHROMATIN CONTENT
Developing ovarian
follicle
has many cells
in mitosis
mitosis
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
Tritiated
Thymidine
labeling of nuclei
DO THE :
PULSE – CHASE
EXPERIMENT
LOOK FOR THE :
TEMPORAL
APPEARANCE
OF SILVER
GRAINS IN THE
PHOTOGRAPHIC
EMULSION OVER
ORGANELLES OF
INTEREST
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
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 CELL’S
INCORPORATION OF
RADIOACTIVE PRECURSORS
(e.g., RADIOACTIVE AMINO
ACIDS) INTO THAT PRODUCT
THAT IS VISUALIZED IN A
PHOTOGRAPHIC EMULSION.
GENERAL ORGANIZATION OF
CARTILAGE
CHONDROCYTES /
CHONDROBLASTS
239
ANGIOGENESIS
ANGIOGENESIS –
growth of blood
vessels
MESENCHYMAL
CELLS
Endothelial cells
Smooth muscle cells
Fibroblasts
COMPACT BONE
REMODELING – OSTEOCLASTS DIGEST BONE,
OSTEOBLASTS FROM ENDOSTEUM REPLACES IT
CELLS OF
BONE
OSTEOBLAST
OSTEOCYTE
OSTEOCLAST
BONE REPAIR
Expected events with
bone formation
•
•
•
•
•
•
Clot
Migration if mesenchymal cells
Proliferation
Differentiation
Calcification
Entry of blood vessels,
osteoclasts, and osteoblasts
• Colonization of bone marrow
Cellular/tissue processes involved
•
•
•
•
•
Chemotaxis
Mitosis
Cell differentiation
Cell death
Angiogenesis
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?
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.
•
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?
HISTOGENESIS OF BONE
ENDOCHONDRAL
OSSIFICATION
DEPOSITION OF BONE
MATRIX ON A
PREEXISTING
CARTILAGE MATRIX
CHARACTERISTIC OF
LONG BONE
FORMATION
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