(393av) Effects of PLGA Nano Patterns of Various Dimensions On Osteoblast and Osteosarcoma Cell Responses

Effects of PLGA Nano Patterns of Various
Dimensions on Osteoblast and Osteosarcoma Cell Responses

Yongchen Wang¹, Lijuan Zhang¹,
Linlin Sun², and Thomas J. Webster²

¹Department
of Chemistry, Brown University, Rhode Island 02912, USA

²School
of Engineering, Brown University, Rhode Island 02912, USA

Abstract:
The interactions between substrates and cells, and especially the use of
nanotopographies on the substrates to manipulate cell functions have been
widely studied. Among these studies, research on the effects of
nanotopographies on cancer cell behaviors can help develop new strategies for
cancer therapy. In this work, nano patterns were fabricated on a biocompatible
and biodegradable co-polymer, poly(lactic-co-glycolic acid) (PLGA), with a
template method ^{1, 2}. The sizes of nano patterns included 27nm,
190nm, 300nm, 400nm, and 520nm, and a flat surface feature was also made as a
control. The surface features, root-mean-square (RMS) roughness and wettability
of PLGA nano patterns were characterized. Both human healthy osteoblasts (OB)
and human cancerous osteosarcoma cells (OS) were cultured on these PLGA surface
features and the effects of PLGA nano patterns on their cell adhesion was
evaluated by 4 hour cell adhesion assays. In the cell adhesion assays, 27nm
PLGA nano patterns enhanced both osteoblast and osteosarcoma cell adhesion the
most compared with other surface features and there were relatively strong
correlations between either osteoblast density or osteosarcoma cell density and
the RMS roughness of PLGA nano patterns. More interestingly, 27nm PLGA nano
patterns exhibited a significant increase in osteoblast density compared with
osteosarcoma cell density cultured on the same surface features and therefore
could be further developed for a novel bone cancer therapy instead of the
traditional cancer therapy methods.

Experiments: The nano patterns were initially created by the self-assembly of
polystyrene (PS) beads of 27nm, 190nm, 300nm, 400nm, and 520nm. These patterns
were first transferred from PS beads to polydimethylsiloxane (PDMS) molds,
which polymerized in close contact with the PS beads, and then were transferred
from PDMS molds to PLGA films, which formed in close contact with the PDMS
molds after solvent volatilization. After synthesis, the surface features of
PGLA nano patterns were studied by AFM. RMS roughness values were calculated
from flattened z-sensor scan AFM images and the wettability were characterized
with a contact angle goniometer to measure the water contact angles of the PLGA
nano patterns.

Human healthy osteoblasts (CRL-11372,
ATCC) and human cancerous osteosarcoma cells (CRL-1427, ATCC)
were cultured in their respective complete culture medium under a standard
environment. The cell adhesion of both osteoblasts and osteosarcoma cells were
evaluated by culturing these two types of cells on the PLGA nano patterns for 4
hours and investigating the cell density by means of MTT assays. Cell numbers
were counted with a hemocytometer. All cell studies were accomplished in
triplicate and the data were analyzed with a one-tailed student t-test.

Results and Discussion: From height scan AFM images (Scanning size: 5µm°Á5µm), nano patterns of 27nm, 190nm, 300nm, 400nm and 520nm and a flat
PLGA surface were fabricated (Fig. 1). The RMS roughness and wettability of
different PLGA surface features were characterized (Table. 1). 27nm PLGA nano
patterns exhibited the largest RMS roughness and its surface was the most
hydrophobic. Flat PLGA surfaces showed the smallest RMS roughness and its surface
was the most hydrophilic.

Figure.
1. AFM images of PLGA films with (A) flat surfaces; (B) 27nm; (C) 190nm; (D)
300nm; (E) 400nm; and (F) 520nm nano patterns.

Table. 1. RMS roughness and water contact
angles of the flat PLGA film and PLGA films with 27nm, 190nm, 300nm, 400nm, and
520nm nano patterns.

For osteoblasts alone or osteosarcoma cells
alone, the 27nm nano patterns increased cell adhesion the most (Fig. 2). For
osteoblasts and osteosarcoma cells cultured on the same surface feature, only 27nm
nano patterns showed a significant osteoblast cell density increase compared
with osteosarcoma cell density. From the correlation plots between cell density
of osteoblasts or osteosarcoma cells, and the physical properties including RMS
roughness and wettability (Fig. 3; Table. 2), there were relatively strong
correlations between either osteoblast density or osteosarcoma cell density and
the RMS roughness of PLGA nano patterns, and relatively weak correlations
between either osteoblast density or osteosarcoma cell density and the
wettability of PLGA nano patterns. These results could be explained in respect
to several aspects. First, larger RMS roughness means larger surface area and
more sites for the adsorption of proteins regulating cell adhesion. Second, the
RMS roughness of 27nm PLGA nano patterns were the closest to the roughness of
the real bone matrix ³. Third, 27nm is the closest to the dimensions
of the extracellular matrix proteins participating in cell adhesion ^{4, 5}.
However, currently, the mechanism of the effects of PLGA nano patterns on the
cellular responses of osteoblasts and osteosarcoma cells is not known. Further
study is needed to fully understand this.

Figure. 2. 4 hr cell adhesion results of OB
and OS (N=3; seeding density: 3500 cells/cm²). The cell densities
are average values +/- S.E.M. For OB alone, *, ** and *** represents p<0.05
compared with the flat, 27nm and 300nm surface features, respectively; for OS
alone, ^, ^^, ^^^ and ^^^^ represents p<0.05 compared with the flat, 27nm,
190nm and 300nm surface features, respectively; and + represents p<0.05 for
OS cell density compared with OB density on the same surface features.

Figure. 3. Correlation plots between
osteoblast density/RMS values obtained from AFM, osteoblast density/ contact
angle, osteosarcoma cell density/RMS values obtained from AFM and osteoblast
cell density/contact angle.

Table. 2. R² values and R values
of the correlation plots between osteoblast density/RMS values obtained from
AFM, osteoblast density/contact angle, osteosarcoma cell density/RMS values
obtained from AFM, and osteosarcoma cell density/contact angle.

Conclusions: PLGA nano patterns of various dimensions could be fabricated with a
template method. For cell studies, 27nm PLGA nano patterns not only increased
the cell density most for osteoblasts alone or osteosarcoma cells alone, but it
also significantly increased osteoblast density compared with osteosarcoma cell
density cultured on the same surface features. It was also found that there were
relatively strong correlations between either osteoblast or osteosarcoma cell
densities, and RMS roughness of PLGA nano patterns. Thus, 27nm PLGA nano
patterns should be further studied as a potentially novel bone cancer therapy
with few adverse effects.

Acknowledgements: We would like to thank Hermman Foundation for their financial
support, and Vera Fonseca and Professor Eric Darling for their technical
support with AFM use.

References:

[1] Zhang L. et al. Inter J Nanomed. 5, 269,
2010.

[2] Zhang L. et al. J Biomed Mater Res A. 100A, 94, 2012.

[3] Liu, H. et al. J Biomed Mater Res A. 78A, 798, 2006.

[4] Elias, K. L. et al. Biomaterials. 23,
3279, 2002.

[5] Koteliansky, V. E. et al. Eur J
Biochem. 119, 619, 1981.