TECHNOLOGICAL INSTITUTE OF THE PHILIPPINES MANILA

363 P. CASAL ST., QUIAPO, MANILA

COLLEGE OF ENGINEERING AND ARCHITECTURE

CHEMICAL ENGINEERING DEPARTMENT

JOURNAL CRITIQUE IN MOLECULAR BIOLOGY

Development of Osteogenic Cell sheets for Bone Tissue Engineering Applications

SUBMITTED BY:
NARANJO, ZENY N.

SUBMITTED TO:
MR. DENNIS MACAPAGAL

SUMMARY:
Many Tissue engineering techniques have proven their ability when it comes to repairing or
regenerating organs. But in the case of bone tissue, the considered gold-standard technique
has not been able to address certain issues that can contribute to the effectiveness of that
technique. In bone tissues, it is very hard to mimic its structure because of its complex
properties. In this paper a new method has been proposed to address bone related issues.
Using cell sheet engineering technique combined with temperature responsive cell recovery
method the potential of CS engineering might open a new way to address bone related
issues.
CONTENT:
The field of Tissue Engineering has been known for years as one of the greatest pioneers
when it comes to Regenerative Medicine. From repairing damaged tissues or organs up to
replacing it, Tissue Engineering has proved its ability. Certain applications such as allografts
and bone grafting were common in the field of bone tissue engineering, because of the
number of bone related-illnesses are growing. But in using these strategies some issues
were encountered making it inefficient. In the article entitled Development of Osteogenic
Cell sheets for Bone Tissue Engineering Applications, a new strategy has been created to
overcome issues regarding bone tissue engineering.
Current strategies in bone tissue engineering proved to be ineffective in many cases
because, the desired responses or results were not achieved. Factors such as
immunogenicity of the material, scarce availability of tissues, morbidity issues, and lack of
integration in the host tissue makes this strategies performance inefficient. The authors of
the article proposed a new strategy where a cell sheet is created. The difference between
the other cell sheet techniques is that in this paper, thermoresponsive dishes were used in
recovering culture cells to be used. Other authors attempted to produce cell sheets but due
to the complexity of the structure of the bone, the results were not as good as the technique
used by the author. In this paper, it has been discussed that using the thermoresponsive
dishes will help improve the potential of osteogenic cell sheets in bone formation.
For the creation of osteogenic cell sheet, rat bone marrow stromal cells were obtained. The
bone marrow obtained is added in Histopaque 1083 and centrifuged to recover viable
mononuclear cells for the procedure. The cell fraction in the solution is obtained after
centrifugation and washed with PBS to remove the remaining Histopaque. The cells are then
cultured for 24 hours in a polystyrene dishes supplying with growth mediums such as fetal
bovine serum and basal medium. The solution is added with an antibacterial medium to

prevent contamination and exposed to a CO 2 humidified atmosphere to reduce media

evaporation and stabilize the pH. After that span of time, the adherent cells were removed
using trypsin-EDTA and seeded in the thermoresponsive dishes prepared. For three weeks
the cells were supplied with osteogenic medium to ensure that the cells are getting the right
amount of nutrients they needed.
The

thermoresponsive

dishes

were

prepared

by

spreading

solution

of

N-

isopropylacrylamide monomer and 2-propanol in a culture dish. To polymerize the solution in

the culture dish surface electron beam was used. After that, further refining were done such
as removal of ungrafted monomer using cold distilled water and dried with nitrogen gas.
Then it was sterilized with ethylene oxide gas before it was used.
After three weeks, cells were recovered from the dishes using polyvinylidene difluoride
membrane to immobilize the desired proteins for detection and implantation purposes.
Using 6-week old male nude mice, the cell sheets were.transplanted on its dorsal skin.
Control mice were also prepared by implanting silicone only for comparison purposes. The
animals were constantly supplied with food and water and after a week, 3 weeks, and 6
weeks of transplantation the animals were euthanized with CO 2 and the implants were
recovered for histological characterization.
For histological characterization, the recovered cell sheets and implanted samples were
embedded in paraffin without demineralization. To confirm the presence of minerals in the
samples staining was performed. Using alizarin red as primary staining agent followed by
hematoxylin. The slides should show pink or purple color when viewed in a microscope.
While for the detection of osteocalcin immunostaining is done using incubator with antiosteocalcin antibody. Using this antibody will determine if calcium minerals have formed in
the samples because this is where the calcium binds. Enzymes such as biotinylated
secondary antibody will be added and after that the sections were stained to analyze using
microscope.
Further analyses were done to determine if the osteogenic cells have fully developed. The
3D structure of the mineralized tissue was examined using non destructive techniques, X-ray
and microcomputed tomography. Also Calcium quantification was done using ocresolphthalein complexon method.

After the procedure and several test conducted the results have been evaluated. Calcium
deposits were positive in the cell sheet samples because of the intensity of the staining.
Osteocalcin the bone forming matrix has also tested positive in immunohistochemistry. While
the X-ray and Tomography analysis confirmed positive in the formation of tissue after
transplantation. And lastly the presence of mineralized new tissue was also positive using
the immunochemistry result of the osteocalcin formed.
As for the overall conclusion it has been noted that the desired bone characteristics were
obtained using this technique. This has become a new interesting field for bone tissue
engineering to be studied.
CRITIQUE:
In every experimental procedure or technique presence of flaws is inevitable. For this paper,
there are some few factors that have been observed that might affect the experimental
result. First is the usage of electron beam. Even though it is used at a low energy level or
energy dosage, the researcher should be mindful that at higher level it can either make the
exposed material breakdown. It can also lead to creation of free radicals that might combine
with the culture cells. Another is the usage of ethylene oxide gas. This gaseous sterilization
is first and foremost carcinogenic and mutagenic. It attacks the cellular proteins and nucleic
acids that lead to its mutation or necrosis. Finally, beside from being protested by animal
advocates due to the method of collection, the Fetal bovine serum has also proven some
disadvantages that can affect the procedure. The serum is a major source of viral
contaminants, meaning viruses, prions, etc. that can alter or affect the experiment. Second it
affects the genotypic and phenotypic cell stability. And lastly it can suppress cell spreading,
attachment, and differentiation, which are the key factors in tissue engineering. Some
alternatives were proposed instead of using the serum.