Scanning electron microscopy of (A) control and (B) modified allograft surfaces following a 6-hour incubation with S aureus.
Courtesy of Constantinos Ketonis, MD, PhD


Published 9/1/2016
Peter Pollack

Stopping Biofilms Before They Form

Covalent attachment process may reduce risk of infection for tumor patients
Covalent attachment of antibiotics to allograft surfaces could inhibit bacterial colonization and help tumor patients resist infection for a far longer period than most current technologies, according to information from a study conducted by Constantinos Ketonis, MD, PhD, and his colleagues. Dr. Ketonis presented findings from his ongoing work in a scientific paper at the 2016 AAOS Annual Meeting.

"This is a continuation of a technology we've been working on for the last 8 to 10 years," he said. "We achieve covalent attachment of bioactive molecules onto various substrates, with the goal of preventing biofilm formation. We conducted our initial study with titanium alloy, but we have also looked at cobalt-chrome, stainless steel, and most recently, biologic substrates such as bone allograft. In the paper I presented to the Academy, we attached doxycycline to bone allografts."

High risk of infection
"The risk of infection is very high for patients with orthopaedic tumors who undergo massive bone resections and allograft implantations," said Dr. Ketonis. "Some studies suggest that 30 percent to 40 percent of such patients might become infected. They are usually immunocompromised and on chemotherapeutics; then they go on to radiation therapy. The soft-tissue bed is usually poor, and once it becomes infected, results can be catastrophic. Doxycycline is specifically appropriate for these patients, as a large percentage of them end up having polymicrobial infections, both gram-positive and gram-negative, and doxycycline is active against both."

Dr. Ketonis explained that bone allografts act as foreign materials, at least in the initial period following implantation. They lack a blood supply, have poor immune surveillance, and can serve as a highly porous haven for bacteria attachment and growth. Once the bacteria attach, they secrete biofilm, which helps the infection evade the immune system.

"Systemic antibiotics cannot penetrate the biofilm matrix," he said. "In this situation, there are few options other than to resect everything, which can be pretty catastrophic. In some cases, this can lead to resection of the allograft or amputation of the limb, and possibly even death."

To attach the antibiotic to the allograft, the researchers perform a light demineralization to expose the collagen on the surface of the allograft. Dr. Ketonis explained that the bone's high natural amine content serves as anchor points to attach linkers, which in turn support the structure to which the doxycycline is attached.

"An analogy is that this produces a condition as if the surface of the bone is covered by spears," said Dr. Ketonis. "Instead of waiting for the bacteria to attach and secrete biofilm, which forces us to address a much more difficult issue, we prevent the attachment of bacteria in the first place. If the bacteria cannot attach, they can't secrete biofilm."

95 percent reduction
To assess the efficacy of the technique, the research team conducted an in vitro study in which they coupled morcellized cancellous or cortical human bone allograft with two Fmoc-AEEA linkers and doxycycline. They used scanning electron microscopy (SEM) to assess bone topography, and immunofluorescence to confirm doxycycline coverage and uniformity. Excess, nonattached antibiotic was allowed to elute from the bone surface for at least 2 weeks before further assessment. They tested the covalent surface using Staphylococcus aureus (S aureus) and Escherichia coli, followed by either direct immunofluorescence and SEM visualization, or sonication and plating for bacterial counts.

"To test our surfaces, we challenge native, unmodified bone with bacteria and compare them with our antibiotic-modified surfaces," said Dr. Ketonis. "In our studies, if we inoculate with levels of bacteria that are typical of clinical inoculates, we don't find any bacterial attachment to our modified surfaces. So we use inoculates that are much higher than what we would expect to see clinically, just so we can get a meaningful count. Even with those much higher concentrations, we show reproducibly 95 percent or greater reduction in bacterial attachment on our surfaces compared to naïve allograft surfaces."

Dr. Ketonis noted that he is sometimes asked about the difference between the covalent bonding technique and elution technology. He explained that the primary difference is that the covalent bond does not allow the bioactive molecules to release from the surface of the bone.

"This is not an elution technology," he said. "It is a permanent, covalent attachment or tethering of the antibiotic to the bone. One problem of elution antibiotics is the creation of concentration gradients and the development of resistance. As the material elutes over time, the bacteria is exposed to sub-inhibitory concentrations of antibiotic. Some studies have suggested that may increase the risk of resistant bacterial strains. In addition, the elution is finite and the kinetics can be unpredictable, leading to high concentrations in the local environment and potential systemic interactions with other drugs, kidney compromise, and other issues.

"If you think of it mathematically, the covalent attachment produces an antibiotic concentration of zero just off the surface of the allograft, and an effectively infinite concentration on the surface. The surface remains protected, and free bacteria are taken care of by the immune system of the host."

Dr. Ketonis and his team's research is ongoing. Earlier versions of the technology using various bioactive compounds with both metal and allograft have been tested in rats and sheep; a rat study is underway to assess the effects of doxycycline and allograft in a living model.

Dr. Ketonis's coauthors are Isabelle Mortalena, DDS; John A. Abraham, MD; Javad Parvizi, MD, FRCS; Christopher S. Adams, PhD; and Noreen J. Hickok, PhD.

Peter Pollack is the electronic content specialist for AAOS Now. He can be reached at

Bottom Line

  • Covalent attachment of antibiotics to allograft surface may prevent the formation of biofilm.
  • At concentrations greater than would be seen clinically, the technique is associated with a 95 percent or greater reduction in bacterial attachment compared to naïve allograft surfaces.
  • Compared to elution technology, covalent attachment offers a permanent, highly localized effect.
  • Research is ongoing, with further testing in animal models planned.