ISOLATION AND CULTURE OF HUMAN OSTEOBLASTS Peter Ma Tayyub Ali Muhammad Fahad Khan Kacper Rusiak Jaleed Ahmed
ISOLATION AND CULTURE OF HUMAN
OSTEOBLASTS
Peter Ma
Tayyub Ali
Muhammad Fahad Khan
Kacper Rusiak
Jaleed Ahmed
YOUTUBE VIDEO
http://www.youtube.com/watch?v=78RBpWSOl08#t=58
0.58s - 1.56s
IMPORTANCE OF OSTEOBLASTS
Osteoblasts are mononuclear cells that are vital to
human bone health
They assist the body in the areas of bone function,
repair and growth
Bone is a dynamic tissue that continually undergoes a
process called bone remodeling consists of the tissue
being broken down and then built back up
OSTEOBLASTS FUNCTION
Osteoblasts: (Greek: osteo “bone” and blast “germ”)
Osteoblasts main function is to synthesize and mineralize bone
allowing it to support the human body
Osteoblast cells have primarily been used in investigating the
skeletal systems:
the effects of inflammatory mediators, regulation of bone metabolism, and
the study of chemicals associated with physical trauma
APPLICATIONS
Osteoblasts have also been used in conjunction with carbon
nanotubes
Carbon nanotubes possess extraordinary properties in increasing
the strength of hydroxylapatite (HA)
HA is used in medical appliances placed within a patient’s body
to replace amputated bones or as a coating on prosthetic limbs
APPLICATIONS
Recent research has found how HA reinforced with carbon nanotubes would behave within the body through the use of osteoblasts cells
Figure 1: Flexible HA approximately having mineral
to organic matrix ratio of human bone Source:http://en.wikipedia.org/wiki/Hydroxylapatite
DISTRIBUTORS
There are two major distributors for osteoblasts cells;
Cell Applications Inc and PromoCell
The average price for one ampoule of more than 500,000
cells can go for approximately 700-900$
BONE CELLS
There are four different types of bone cells that continuously maintain bones.
These are: osteoclasts, osteoblasts, osteocytes, and lining cells.
Figure 2: Bone cells Source:Eijken,M. (2007). Human Osteoblast Differentiation and Bone Formation: Growth Factors,
Hormones, and Regulatory Networks
MESENCHYMAL STEM CELLS
Human osteoblast cells are a highly specialized type of
cell derived from mesenchymal stem cells (MSCs)
MSCs cells are multipotent cells that can differentiate
into a variety of cell types such as fibroblasts,
myoblasts
Mainly found in the bone marrow
MSCS CELLS POTENCY
Figure 3: MSCs cell differentiation Source: http://www.discoverymedicine.com
HOW OSTEOBLASTS FORM
There needs to be a certain signal for MSCs cells to
generate osteoblasts. The two important transcription
factors that play an important role in MSCs cells being
converted to osteoblasts are Runx and Osterix
Through tests done on mice, it was discovered if they had
a gene deficiency of Runx2 and Osx then they had defective
bone formation and lack of mature osteoblasts.
Once the MSCs cells are committed to become osteoblasts,
they are called osteoprogenitor cells
Skeletal Precursor
BMP & WNT Injection Osteoprogenitor
RUNX2
WNT Signalling
Mesenchymal Cells
Pre-ostioblast
RUNX2 & Osterix
Transcription Factors
Initiate OBG
Osteoblasts
DETECTION OF ALKALINE PHOSPHATE IN PROLIFERATING
OSTEOBLASTS
Prepare
Solutions &
Buffers
Fixation of
the Cells
Wash the
Cells Stain the
Cells Wash the
Cells
OSTEOBLAST MINERALIZATION
Seed
Osteoblast
Grow
Osteoblast
Differentiation
Culture of
Induced
Osteoblasts
Induce
Osteoblast
FERMENTATION OF OSTEOBLASTS
Osteogenic differentiation
-> Seed Mesenchymal stem cells
-> Grow the cells
-> Induce mesenchymal cells with differentiation medium
-> Incubate for 21 days
-> Change medium every 3 days to populate cell culture
OSTEOBLAST DETECTION
1. Prepare solution
2. Washing Cells
3. Fixation of Cells
4. Stain the cell
5. Wash and analyze
NON-FERMENTATION
-> Use osteoblasts to derive a new product
-> Mineralization of osteoblasts
-> Osteoblasts synthesize collagen, produce calcium and phosphate
-> hydroxyapatite mineral (Ca10[PO4]6[OH]2
-> Osteoblasts are used to create bone matrix
-> Mineral content in bone is hard to pinpoint
CANADIAN REGULATIONS
-Osteoblasts research done mostly on rat and dog osteoblast cells.
-Potential tumorigenic hazards associated with heavy metal
internalization in humans
-Testing of the bioengineered bones and cartilages as well as
osteoblasts injections still not allowed in humans.
-The use of human osteoblasts descended from mesenchymal stem
cells is much less controversial than the use of embryonic stem
cells
-The immunology issues (Physical activity, Age, Disease
environment)
The End