Biomedical Engineering Reference
In-Depth Information
conditions that favored cartilage tissue formation by influencing cell attachment, spreading
and the amount and composition of cartilaginous tissue that forms (Spiteri, Pilliar, and
Kandel 2006; Bhardwaj et al. 2001; Ciolfi et al. 2003). Not only porosity, but also surface
geometry and topography have been found to have positive effects on the behaviour of
chondrocytes (Bhardwaj et al. 2001).
Nanoscale topographic effects have been illustrated in nanostructured poly-lactic-co-glycolic
acid (PLGA)/nanophase Titania (TiO
2
) composites, which have elicited an enhanced
chondrocyte response compared to surfaces with a conventional or micrometer topography
(Savaiano and Webster 2004). We have recently reported on our hypothesis that the
nanotopographical cues, from porous nanotubular structured substrates made of TiO
2
,
already being an osseointegrating biomaterial (Bjursten 2009; Oh et al. 2006), may also be a
candidate for providing an alternative way to positively influence cartilage formation and
the cellular behaviour of cartilage chondrocytes.
4.1 Up-regulated chondrocyte synthesis of extracellular matrix components
The dimensions of the nanotubes were varied in order to determine if the size of the
nanotube diameters would play a role in the chondrocyte behaviour.
For this comparative
chondrocyte cell culture study, a commercially pure Ti surface, without surface modification
was used as a control, as it commonly used as implant material.
It is well known that chondrocytes, the primary cells of cartilage, are extremely active cells.
They produce a large amount of extracellular matrix (ECM) that is critical for the mechanical
properties and joint lubrication characteristics of cartilage. In the SEM micrographs in
Figure 4, the nanotubes substrates appear that they are inducing a positive response from
the chondrocytes because the cells begin synthesizing abundant ECM deposition and fibril
organization. In the SEM observations of chondrocytes a striking difference in the
production of ECM fibrils between the flat Ti without a nanostructure vs. TiO
2
nanotube
surfaces is revealed. Fibrils are abundant and extending from all areas of the chondrocyte
cell creating a dense network of ECM on the nanotube substrates.The flat Ti most likely
lacks surface structuring cues for signaling ECM fibril production and organization. One
possibility is that ECM protein formation into dense fibrils on the surface may be “nano-
inspired” to form on the nanotube structure because of the precise dimensions or fine scale
cues of the top surface (tip of the vertical wall) of TiO
2
nanotubes having a physically
confined geometry which could aid in fibril formation. It was demonstrated previously that
the nanotubes produced bio-active nanostructured formations of sodium titanate nanofibers
directly on the top of TiO
2
nanotube walls when the nanotubes were exposed to NaOH
solution (Oh S 2005). ECM proteins once secreted, in this study, may also self-assemble
according to the top-wall surface geometric nanocues.
In the lower panel of Figure 4, immunofluorescent images for collagen Type II (red color)
are illustrated for flat Ti vs. TiO
2
nanotubes. Both surfaces stained positive for collagen type
II, but there was large networks of connected bundles expressed across the surface of the
nanostructure.
When a morphological analysis was conducted, it was determined that nanotubes induce a
more spherical chondrocyte cell shape (data not shown). Fibroblastic shaped cells were
observed on the flat controls. The percentage of round shaped, spherical cells was
significantly lower for chondrocytes on the polystyrene, Ti, and the smallest diameter
(30nm) nanotube substrates compared to the larger diameter 50, 70, and 100nm TiO
2
