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