Milca I. Aponte-Rom

1
Proliferation and Mineralization of Human
Osteosarcoma Cells on Different Polyarylates and
Polycarbonate Substrates
Milca I. Aponte-Román1, Dr. Robert Dubin2, Dr.
Joachim Kohn2, Dr. Adrian Mann1, Dr. David
Denhardt3, Departments of 1Materials Science
Engineering, and 3Cell Biology Neuroscience,
2Center for Biomaterials. Rutgers University,
The Sate University of New Jersey
Introduction
Results
The goal of this research was to characterize the
response of human osteosarcoma cells to polymer
substrate variation in vitro and to establish
correlation between cellular response and polymer
chemistry/structure. In this particularly
project mineralization by human osteosarcoma cell
line, Saos-2, when grown on various polymers was
examined. The biodegradable and biocompatible
polymers examined are members of the
"polyarylate" and polycarbonates combinatorial
library developed in the Dr. J. Kohns research
group at Rutgers University. Alizarin Red-S
assay was used to compare the mineralization on
each of the substrates.
Alizarin, is the main colorant found in the
madder plant Rubia tinctorum

1. Polyarylates

• Poly(DTE suberate)
• Poly(DTB suberate)
• Poly(DTH suberate)
• Poly(DTO suberate)
• Poly(DTD suberate)
• Poly(DTE succinate)
• Poly(DTH succinate)
• Poly(DTD succinate)
• Poly(DTE dodecandioate)
• Poly(DTH dodecandioate)
• Poly(DTD dodecandioate)
• Poly(DTH adipate)
• Poly(DTH sebacate)
• PLGA poly(DL-lactate co-glycolate)
• PLA poly(DL-lactate)
• TCP tissue culture plastic

Plate with polycarbonate-coated coverslips after
stained with alizarin red-S.
Substrates
Future Work
Polyarylate- and polycarbonate-coated glass
coverslips were prepared in Kohns lab.
Chemico-physical properties affecting polymer
flexibility, protein adhesion and degradation
were altered by the inclusion of DT,
poly(ethylene glycol) (PEG), and Iodine (I2) into
the backbone of the poly(DTE carbonate), and by
alterations in the pendent chain and diacid
componet of the polyarylate family of polymers.
This work was done in the Cell Biology
Neuroscience Department as a part of my
Integrative Graduate Education and Research
Traineeship (IGERT). The main idea was to rotate
through the laboratory of my interdisciplinary
secondary advisor, Dr. Denhardt, and learn about
cell culture. For now on I will be working
using a Near Field Scanning Optical Microscope
(NSOM) to nanopattern surfaces coated with Self
Assembled Monolayers (SAMs). The idea is to
functionalize the SAM with bioactive chemicals by
using the laser from the NSOM to locally initiate
a chemical reaction.
B. Polycarbonates

• Poly(DTE Carbonate)
• Poly(DTE-co-4 PEG lK Carbonate)
• Poly(DTE-co-8 PEG lK Carbonate)
• Poly(DTE-co-10 DT carbonate)
• Poly(DTE-co-10 DT-co-4 PEG lK Carbonate)
• Poly(DTE-co-10 DT-co-8 PEG lK Carbonate)
• Poly(I2 DTE Carbonate)
• Poly(I2 DTE-co-4 PEG lK Carbonate)
• Poly(I2 DTE-co-8 PEG lK Carbonate)
• Poly(I2 DTE-co-10 I2DT lK Carbonate)
• Poly(I2 DTE-co-10 I2DT-co-4 PEG lK Carbonate)
• Poly(I2 DTE-co-10 I2DT-c0-8 PEG lK Carbonate)
• 17. PLGA-resomer 506 (Boehringer Ingelheim)
• PLLA-resomer L 206 (B. I Chemicals)
• TCP tissue culture plastic

Cell Culture
Saos-2 cells were initially cultured on top of
polyarylate- and polycarbonate-coated coverslips
in HAMs F-12 medium supplemented with 10 fetal
bulvine serum, 10mM Hepes pH 7.5, penicillin
and streptomycin, and glutamine. At day 7,
10mM ?-glycerolphosphate, 10nM dexamethasone
(DEX), and 50?g/mL ascorbic acid were added to
induce mineralization, and the addition repeated
at every medium change until the end of
experiment. Cultures were grown at 37oC in a
humidified 5 CO2 atmosphere.
Discussion and Preliminary Conclusions

• All Saos-2 cultures on polyarylate-coated
  coverslips formed calcium phospahate mineral with
  minimal disparity between each other.
• All Saos-2 cultures on polycarbonate-coated
  coveslips formed calcium phosphate mineral.
  However, low light absorbance was obtained in
  polycarbonate substrates with 4 and 8 PEG (code
  2 and 3 from polycarbonate series). PEG in 8
  resulted in poor cell attachment and little
  spreading.
• In contrast, no significant variations were
  obtained on the polycarbonate cultures where PEG
  is also present in 4 and 8 (code 10, 11, 14,
  and 15 from polycarbonate series). When Iodine
  (I2) is incorporated it seems to suppress the
  effect of PEG. It is possible that the Iodine
  may work to suppress the PEG by steric hindrance
  rather than making the polymer more hydrophobic.
• These experiments need to be repeated before firm
  conclusions can be drawn.

Alizarin Red-S Assay
Acknowledgments
Alizarin red is a dye, which binds selectively to
calcium. Every week the medium of one plate was
removed and the culture wells were briefly washed
with PBS and stained for 10 min with 1 alizarin
red-S pH 4.14. Cultures were then rinsed with
water and PBS, to remove the stain not associated
with calcium mineral deposit. A de-staining
procedure was followed using 10 cetylpyridinium
chloride in 10nm sodium phospahete pH 7.0 for 15
min in order to measure the optical absorbance.
Absorbance measurements were done with a
microplate reader with 570nm.

• The author would like to express her gratitude
  to,
• Dr. Denhardt from the Department of Cell Biology
  Neuroscience, for letting me work in his lab
  and teaching me about cell culture.
• Dr.Robert Dubin, for providing us with the
  culture plates coated with polycarbonate and
  polyarylates, and valuable discussion.
• Christian Kazanecki, Melissa Weidner and Jennifer
  Luo for their help in cell culture and for
  sharing their lab with me.
• IGERT for supporting this work.