The endosteal surface of the bone pieces from the cortical stub squares was sanded until flat and VAN linked to the primary amines displayed on the ECM proteins of bone allograft (Fig. 5A-B). Covalent tethering of VAN to allograft did not alter the surface topography as seen by SEM (Fig. 5C-D). The microarchitecture of modified and control bone appeared similar by containing a dense collagen fiber meshwork, osteocytic lacunae with the Haversian and Volkmann's canals being preserved.
Human fetal osteoblast-like cell cultured on VAN-allograft (48 hrs) retained normal morphology and distribution. Cell shape and distribution were very similar between the control and VAN-modified allograft with cells appearing to align along the underlying collagenous matrix (Fig. 8). Cell density appeared similar on both the control and VAN-bone, suggesting the VAN-bone is sufficiently like the parent bone substrate to support bone ingrowth.
Allograft bone is used frequently to replenish bone loss that may be encountered during revision joint arthroplasty . With the rise in the number of complex revision arthroplasty cases and the increase of aging population, the demand for bone grafts will continue. Worldwide, bone grafts are used in approximately 2.2 million orthopaedic procedures annually, generating a $2.5 billion per year industry . Unfortunately, the prevalence of allograft-related infection rates remains relatively high with a reported incidence of 4% to 12% compared to periprosthetic joint infection rates of 1% to 3% [11, 26, 41]. This study was designed to investigate the phenomenon of allograft infection by conducting a series of experiments. We determined if (1) increasing initial S. aureus inoculation of bone allograft resulted in a proportionate increase in colonization; (2) addition of antibiotics to solution could decrease allograft colonization; (3) covalent tethering of antibiotic to allograft altered its ability to prevent bacterial colonization; and (4) covalent modification altered the topography of the allograft or its biological properties in allowing osteoblast-like cell adhesion.
The most important limitation of this study is that it is an in vitro attempt to describe an in vivo phenomenon. As a result, it necessarily deals with the time frames and ideal environment associated with bacterial colonization under laboratory conditions. Second, much of the data that we present depends on visual assessment of bacterial colonization on surfaces. With the special properties of adherent/biofilm bacteria, detection of these bacteria using the stains and SEM, as we have described, are methods that circumvent the need for biofilm dissolution and bacterial detachment required for accurate counts and are thus, of necessity, observational. However, these methods can only sample limited areas on the surface and hence cannot offer the assurance that there are not pockets of allograft that appear different. Unfortunately, currently available more quantitative methods, ie, resuspension of adherent bacteria and subsequent plating are plagued by problems such as poor recovery of bacteria . Third, we assessed colonization by one organism and one antibiotic construct. In a situation where infection is establishing, it may not be rare to find multiple microorganisms. We limited the experimental design to the most common infecting organism, namely Staphylococcus aureus, which we believe represents a “model organism” for allograft associated infections. However, we acknowledge other organisms may behave differently. Fourth, the concentrations of vancomycin used for addition to solution was based on our experience with these experiments over the past few years and appears to be ˜10X the MIC for the planktonic S. aureus. It was our aim to stay within clinically useful levels while avoiding concentrations that could become toxic to mammalian cells.
We found small increases in initial bacterial inoculate resulted in a disproportionately large increase in bacterial colonization of allograft surfaces. The bacteria readily attached to, and colonized, cortical allografts in vitro. From our subsequent electromicrograph analysis, it appears that bacterial colonization was potentiated by the porous nature of bone that provides bacteria with topographically protected niches where they can attach, proliferate, and form a mature biofilm. Successful bacterial colonization can be achieved with very small inoculate sizes when foreign bodies are present . For example, the initial inoculate of bacteria required to infect an incision site decreases by 4 logs when sutures are in place . In our experiments, colonization was achieved on bone with the lowest inoculate tested (100 cfu) and the attached bacteria harvested dramatically increased for every subsequent 10-fold increase in dose. However, a much higher inoculum of 104 cfu was chosen to challenge the modified allograft in order to capture the clinical scenario when larger number of bacteria may be present.
Systemic or local antibiotics alone are unlikely to eradicate an established infection around a prosthesis or an allograft . We attempted to recapitulate this situation through introduction of antibiotics after establishment of bacterial colonization on the allograft. As observed clinically, these bathing antibiotics, while clearing the nonadherent bacteria, were unable to affect the adherent bacteria, much of which remained encased in a biofilm slime. Importantly, bacteria were able to attach to bone surfaces even when inoculated in an antibiotic-rich medium. Electron microscopy demonstrated that S. aureus attached and initiated biofilm formation on cortical allografts within 6 hours. By 12 and 24 hours, the surface was covered with the thick biofilm glycocalyx, entrenching and protecting the underlying bacteria from environmental challenges including solution antibiotics [21, 33, 35]. We suggest in vivo colonization of allografts during establishment of infection can occur preferentially in protected niches, although the timeframe of these events is likely to be longer than what we describe here. To prevent establishment of infection during the immediate postsurgical period, prophylactic antibiotic regimens have been adopted that involve the administration of antibiotics intravenously for 2 to 14 days and orally for up to 16 weeks [15, 24, 29]. Similarly, to prevent initial infection, physicians have empirically impregnated bone allografts with antibiotics before implantation, achieving high local concentrations of antibiotic during the initial elution . Our in vitro data support these processes, and even though they are important means to prevent establishment of infection, they are imperfect. Unfortunately, as with supplementation of PMMA cement with antibiotics, elution kinetics from allograft are largely variable, in which high local antibiotic concentrations can have deleterious effects on host cells thereby retarding graft incorporation. Additionally, once antibiotic is depleted, the graft is again susceptible to bacterial colonization. Therefore, although use of allograft has yielded excellent results, its use in cases of infection has been controversial as a result of the possible increased risk of reinfection [8, 9, 18].
In an attempt to address these problems, we turned to an antibiotic-modified cortical allograft as an approach to providing an antibacterial surface that would effectively resist infection. Despite subjecting the modified allograft to bacterial inoculate three to four orders of magnitude higher than what one expects to encounter in a clinical scenario [4, 17], the allograft exhibited excellent efficacy in preventing colonization by S. aureus. The process of modification did not appear to affect the microarchitecture of bone or its biological activity, at least based on limited cellular adhesion studies that were performed here. The surface demineralization that we used is not uncommon in allograft preparations and reportedly enhances bone incorporation and ingrowth [27, 28]. The solvents used during the chemical modification also appeared to access the niches and allow coupling of allograft throughout.
The observation that osteoblast-like cells were able to adhere to the surface of the modified allograft as readily as the control allograft is encouraging. Previous experiments in our laboratory and those of others have shown antibiotics in higher concentrations can have deleterious effects on mammalian cells [2, 40]. Therefore, toxicity studies must be performed with the introduction of any novel bioactive construct or drug delivery system [10, 36, 38]. When the cells on our antibiotic-laden construct were visualized, careful observations failed to identify any differences among their density, morphology, distribution, and cell-cell associations on either control or VAN-modified allograft. Although a detailed assessment of biocompatibility, including measurements of osteoblastic maturation, is required to fully characterize the effect of the VAN-surface on osteoblastic function, these initial findings are suggestive that the VAN-bone should be biocompatible. Furthermore, because the antibiotic is permanently bonded to the surface of the allograft, no systemic toxicity is expected in vivo.
We thank the Musculoskeletal Transplant Foundation for providing samples for this work as well as for their generous support of these studies.
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