We have covalently immobilized chitosan onto a titanium (Ti) surface to manage implant-related infection and poor osseointegration, two of the major complications of orthopedic implants. The Ti surface was first treated with sulfuric acid (SA) and then covalently grafted with chitosan. Surface roughness, contact angle and surface zeta potential of the samples were markedly increased by the sulfuric acid treatment and the subsequent chitosan immobilization. The chitosan-immobilized Ti (SA-CS-Ti) showed two novel antimicrobial roles: it a) prevented the invasion and internalization of bacteria into the osteoblast-like cells, and b) significantly increased the susceptibility of adherent bacteria to antibiotics. In addition, the sulfuric acid-treated Ti (SA-Ti) and SA-CS-Ti led to significantly increased (P<0.05) osteoblast-like cell attachment, enhanced cell proliferation, and better osteogenic differentiation and mineralization of osteoblast-like cells.

The ability of human neutrophils to clear newly attached Staphylococcus aureus bacteria from a serum-coated glass surface was examined in vitro using time-lapse confocal scanning laser microscopy. Quantitative image analysis was used to measure the temporal change in bacterial biomass, neutrophil motility, and fraction of the surface area policed by neutrophils. In control experiments in which the surface was inoculated with bacteria but no neutrophils were added, prolific bacterial growth was observed. Neutrophils were able to control bacterial growth but only consistently when the neutrophil/bacterium number ratio exceeded approximately 1. When preattached bacteria were given a head start and allowed to grow for 3 h prior to neutrophil addition, neutrophils were unable to maintain control of the nascent biofilm. In these head-start experiments, aggregates of bacterial biofilm with areas of 50 μm2 or larger formed, and the growth of such aggregates continued even when multiple neutrophils attacked a cluster. These results suggest a model for the initiation of a biofilm infection in which a delay in neutrophil recruitment to an abiotic surface allows surface-attached bacteria time to grow and form aggregates that become protected from neutrophil clearance. Results from a computational model of the neutrophil-biofilm surface contest supported this conceptual model and highlighted the stochastic nature of the interaction. Additionally, we observed that both neutrophil motility and clearance of bacteria were impaired when oxygen tension was reduced to 0% or 2% O2.

In order to evaluate the anti-infective efficacy of the titanium implant materials, two co-culture systems, a low-bacteria/osteoblast (L-B) and a high-bacteria/osteoblast system (H-B), were established. Untreated (UN-Ti), sulfuric acid-treated (SA-Ti), and chitosan immobilized titanium (SA-CS-Ti) materials were developed and evaluated. Bacteria and osteoblast behaviors, including initial attachment (evaluated at 30 minutes), adhesion (evaluated at 4 hours), and osteoblast spreading on each material surface were evaluated using quantification assays, scanning electron microscopy (SEM), and confocal microscopy. Quantification analysis at 30 minutes showed significantly higher number of osteoblast present on SA-CS-Ti in both L-B (10,083±2,626) and H-B (23,592±2,233) than those on the UN-Ti (p<0.05). SEM observation and confocal microscopy results showed more surface area was occupied by adhered osteoblasts on SA-CS-Ti than UN-Ti and SA-Ti in both co-culture systems at 30 minutes. At all time points, SA-CS-Ti had the lowest level of bacterial adhesion compared to UN-Ti and SA-Ti in both co-culture systems. A significantly (p<0.05) lower number of bacteria were recovered from SA-CS-Ti (2,233±681) in the H-B system compared to UN-Ti (5,367±1,662) and SA-Ti (4,533±680) at 4 hours. Quantitative and qualitative co-culture results show the great potential of chitosan immobilization onto implant materials to prevent implant-associated infections.

Biomaterial-related infections (BRIs) have become a major challenge in the field of orthopedic implants. In this study, we delved into the problem of BRI and attempted to reduce the possibility of BRI incidence via surface modification of titanium (Ti) with chitosan (SA-CS-Ti). To comprehensively evaluate the anti-infection potential of SA-CS-Ti, we first constructed a postoperative infection (POI) model with varying concentrations of bacteria (10 CFU/sample and 10 CFU/sample) and a constant number of SaOS-2 cells (10 /sample). Then, we biologically characterized the interactions between the SaOS-2 cells, bacteria, and different Ti implants using the POI model. The results from the osteoblastic cell and bacterial attachment tests demonstrated that the SA-CS-Ti surfaces exhibit superior osteogenic behavior relative to other Ti surfaces studied while showing significant anti-infective activities in the POI model with a low infection ratio (bacteria: cell ratio of 0.001:1) 30 min after infection. Additionally, the SA-CS-Ti surfaces showed significantly reduced (p < 0.05) bacteria proliferation compared to the control Ti surfaces (UN-Ti), demonstrating their antifouling property. The significantly increased (p < 0.05) sensitivity of Staphylococcus. aureus adhered to the SA-CS-Ti surfaces against cefazolin (1 mg/L treatment) and gentamicin (10 mg/L and 100 mg/L treatment) in the coculture system augmented potential of SA-CS-Ti to be used as orthopedic implants. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 586-594, 2016.

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