Surface/Nano Engineering of Materials for Combating Biomaterials-Centered Infections and Enhancing Implant Integration

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.

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 promising.

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. aureus⁵.

Figure 1. 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 BMP-2

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 BMP-2.

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.

References

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 - 587, 2005
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, 2008.
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, 4135-4148, 2004
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, 1603-1611, 2009
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.