by Professor K.G. Neoh
Bacteria readily adhere on all types of surfaces and form biofilms. The
biofilm protects the colonizing microorganisms and thus, bacteria in a biofilm
can be several orders of magnitude more resistant to antibacterial agents than
their planktonic counterparts. Biofilms once formed are very difficult to
eradicate, and the implications of this resistance and persistence are clearly
manifested in biomaterials-associated infection.
A biofilm is an aggregate of microorganisms in which
cells are stuck to each other and/or to a surface. These adherent cells are
frequently embedded within a self-produced matrix of extracellular polymeric
substance (EPS). Biofilm EPS is a polymeric jumble of extracellular DNA,
proteins, and polysaccharides.
It has been estimated that medical device-associated infections are
responsible for ~50% of nosocomial infections1.
Antibiotic therapy for established implant infections tends to be prolonged and
yet may not be effective. Very often, it becomes necessary to remove and/or
revise the implant, at considerable expense and trauma to the patient.
The increasing use of antibiotics to combat infections is recognized as the
main cause for the emergence of antimicrobial resistance which has become a
major public-health problem worldwide2. For example,
methicillin-resistant Staphylococcus aureus (S. aureus) which was
suspected to cause sporadic infections in the early 1960s, has now reached
endemicity in many hospitals, with ~ 60% of hospital-acquired S. aureus
isolates in the USA, being resistant to methicillin3.
In view of the close association of biofilms with infections and the
difficulty in eradicating biofilms once they are established, a preventive
approach against biofilm formation is clearly a preferred strategy compared to
the administration of antimicrobial agents after the biofilm has been formed.
This approach forms a major rationale behind Professor Neoh Research Group's strategies for modifying
surfaces to resist bacteria adhesion and formation of biofilm, and/or kill the
bacteria during their initial attachment to the surface. Furthermore, for
orthopedic implants, strategies which can endow the surface with antibacterial
properties concomitantly with promotion of osseointegration would be highly
Much of our
work on biomaterial surface modification focuses on titanium and titanium
alloys due to their extensive use as implant materials in orthopaedic and dental
applications. One of the simplest ways to functionalize these surfaces is via
layer-by-layer (LbL) technique4. This technique is
based on the attractive electrostatic force between a charged surface and an
oppositely charged polyelectrolyte and the subsequent build up of oppositely
charged polyelectrolytes into a multilayer, typically with a film thickness
which ranges from tens to hundreds of nanometers.
It is a versatile and efficient, yet facile, technique, and a wide range of
materials including natural polymers, peptide and nanoparticles can be
incorporated into the layered films. We have constructed polyelectrolyte multilayers (PEMs) of
hyaluronic acid (HA) and chitosan (CS) on titanium (Figure 1) to inhibit
adhesion and growth of Escherichia coli (E. coli) and S.
Polyelectrolyte multilayers on titanium comprising hyaluronic acid and chitosan
with surface conjugated RGD.
Crosslinking between the HA and CS chains was introduced to impart greater
stability. The multilayers achieve high antibacterial efficacy through a
combination of the action of HA against bacterial adhesion and the bactericidal
properties of CS. Cell-adhesive arginine-glycine-aspartic acid (RGD) peptide can
then be conjugated on the surfaces of these PEMs, which results in significant
increase in proliferation and alkaline phosphatase activity of osteoblasts
cultured on these surfaces (by 100-200% over that of pristine titanium
substrates). Since no bacteria which bind directly to an RGD domain have been
identified6, the high antibacterial efficacy of the
multilayer was retained with about 80% reduction in the number of adherent
bacterial cells relative to that on pristine titanium.
Another method to achieve a selective biointeractive surface on titanium
which simultaneously enhances bone cell function while decreasing bacterial
adhesion involves the grafting of an intermediate antibacterial polymer layer
followed by the conjugation of a growth factor. An example of this concept is
illustrated in Figure 2.
Figure 2. Titanium
surface grafted with carboxymethyl chitosan with conjugated
The titanium surface is first functionalized with dopamine7 which serves as the anchor for the grafting of a
carboxymethyl chitosan (CMCS) layer. This is then followed by the conjugation of
bone morphogenetic protein-2 (BMP-2) to the CMCS-grafted surface8. Bacteria adhere readily to the pristine titanium surface
as can be seen from the viable bacterial cells stained green in Figure 3a. The
CMCS layer provides antibacterial properties, and the number of viable cells on
the CMCS-functionalized titanium surface (with and without conjugated BMP-2) was
significantly less than that on the pristine Ti (Figure 3b).
Figure 3. Fluorescence
microscopy images of (a) pristine Ti, and (b) Ti functionalized with CMCS and
conjugated BMP-2, under green filter after immersion in a PBS suspension of
S. aureus (106 cells/ml) for 6 h.
While the CMCS has no significant effect on osteoblasts cultured on the
modified substrates, the conjugated BMP-2 retained its effectiveness in
promoting the osteogenic functions of these cells as indicated by increased cell
attachment (Figure 4), alkaline phosphatase activity and calcium mineralization.
An advantage of such functionalized surfaces for in vivo applications is that
the BMP-2 remained immobilized on the substrate surface where it is needed and
is not released. This would minimize the risk of undesirable effects arising
from the growth factor at locations beyond the implant site in the body.
Figure 4. Confocal
laser scanning microscopy images of osteoblasts cultured for 24 h on surfaces of
(a) pristine Ti, and (b) Ti functionalized with CMCS and conjugated
Our group is currently applying the same concept to address
one of the key challenges in bone healing and regeneration which is to ensure
adequate blood supply to meet the metabolic demands of recovery. Vascular
endothelial growth factor (VEGF) immobilized on an intermediate polymer layer
can promote the survival and proliferation of endothelial cells and also induce
the differentiation of human mesenchymal stem cells into endothelial
cells9. The effects from the co-immobilization of
VEGF and BMP-2 are currently being investigated. Thus, application of these
surface functionalization strategies to implants can potentially be very useful
for accelerating vasculature formation and new bone tissue formation.
1. R.O. Darouiche, "Antimicrobial coating of devices for
prevention of infection: Principles and protection", International Journal of
Artificial Organs 30, 820-827, 2007.
2. H .Goosens, M. Ferech,
R. Vander Stichele, M. Elseviers, "Outpatient antibiotic use in Europe and
association with resistance: a cross-national database study", Lancet 365, 579 -
3. J. Chastre, "Evolving problems with resistant
pathogens", Clinical Microbiology and Infection 14 (Suppl. 3), 3-14, 2008.
4. G. Decher, "Fuzzy nanoassemblies: Toward layered polymeric
multicomposites", Science 277, 1232, 1997.
5. P. H. Chua, K. G.
Neoh, Z.L. Shi, E. T. Kang, "Structural stability and bioapplicability
assessment of hyaluronic acid-chitosan polyelectrolyte multilayers on titanium
substrates", Journal of Biomedical Materials Research A 87, 1061-1074,
6. L. G. Harris, S. Tosatti, M. Wieland, M. Textor, R. G.
Richards, "Staphylococcus aureus adhesion to titanium oxide surfaces coated with
non-functionalized and peptide-functionalized
poly(L-lysine)-grafted-poly(ethylene glycol) copolymers", Biomaterials 25,
7. X. Fan, L. Lin, J. L. Dalsin, P. B.
Messersmith, "Biomimetic anchor for surface-initiated polymerization from metal
substrates", Journal of American Chemical Society 127, 15843-15847, 2005.
8. Z.L. Shi, K. G. Neoh, E. T. Kang, C. K. Poh, W. Wang, "Surface
functionalization of titanium with carboxymethyl chitosan and immobilized bone
morphogenetic protein-2 for enhanced osseointegration", Biomacromolecules 10,
9. C. K Poh, Z.L. Shi, T. Y. Lim, K. G. Neoh,
W. Wang, "The effect of VEGF functionalization of titanium on endothelial cells
in vitro", Biomaterials 31, 1578-1585, 2010.
Copyright AZoNano.com, Professor K.G. Neoh (National University