In orthopedic surgery, bone grafts are used for reconstructing bone defects caused by implant-associated complications, trauma, and tumors.1,2 Although autografts can be used, donor site morbidity can be avoided with the use of allografts.3–5 Bone grafts can be used as large structural bone grafts from postmortem donors or as bone chips from morselized femoral heads donated by living patients undergoing total hip arthroplasty. These bone chips are used to fill defects that require biomechanical stability, which can be achieved by compressing the chips into the defect site.2 Fresh frozen bone chips are preferred because they contain the original osteoconductive and osteoinductive proteins. However, fresh frozen bone chips can add the risk of local contamination.6,7 Surgery with bone allografts is complex and time consuming; therefore, it is prone to a higher infection rate (2.0%–2.5%).8–10 Additionally, the grafting impaction creates an avascular area where local circulation is disrupted. In the case of site infection, systemically administered antibiotics cannot reach the infected bone graft.11 In addition, because bone grafts act as a foreign material, biofilms can be formed on the surface, increasing antibiotic resistance.12,13Staphylococcus epidermidis and Staphylococcus aureus are the organisms that most commonly colonize foreign body surfaces.14
Antibiotics delivered from an implanted biomaterial potentially can be used to prevent infections, treating the surgical site without local or systemic toxicity. Certainly, it would be an advantage if these materials had osteoconductive and osteoinductive capacity, supporting bone healing without further surgery.15 Morselized bone allografts can be used as carriers if the material is impregnated with antibiotic solutions16,17 or mixed with antibiotic powders.18,19
Gentamicin sulfate is the most commonly used antibiotic for local application in orthopedic surgery, for example, mixed with bone cements.20,21 HERAFILL powder (Heraeus Medical GmbH, Wehrheim, Germany) is used in the composition of bioabsorbable beads and is composed of calcium sulfate, calcium carbonate, and hydrogenated triglyceride tripalmitate as a bonding additive. It contains 4 mg gentamicin sulfate, corresponding to 2.5 mg gentamicin base.
The goal of the current study was to evaluate HERAFILL powder as a bone void-filling material as well as an antibiotic carrier once mixed with allograft bone chips. The efficacy of the bone chips mixed with HERAFILL was measured by drug release tests and bacterial susceptibility with Bacillus subtilis, S epidermidis, and S aureus.
Materials and Methods
Preparation of Bone Chips
Bone tissue was obtained from the bone bank of the Innsbruck Medical University, Austria. Femur heads were removed during femoral head osteotomy from patients who had undergone hip replacement surgery. Throughout the procedure, the bone was rinsed and cooled with sterile 0.9% saline to prevent damage by heating. Cortical and cartilage tissues were removed from the femoral heads with a bone saw. Bone chips were prepared from the spongious tissue using a bone mill (Noviomagus Bone Mill, Spierings Medische Techniek BV, Nijmegen, The Netherlands). Bone chips were mixed to achieve homogeneous bone quality. All patients gave their written consent to allow use of the removed tissue for research purposes.
HERAFILL powder, composed of calcium sulfate, calcium carbonate, and hydrogenated triglyceride tripalmitate as a bonding additive containing 4 mg gentamicin sulfate, corresponding to 2.5 mg gentamicin base, was donated by Heraeus Medical GmbH (Wehrheim, Germany).
Mixing Bone Chips With HERAFILL
The following range was set for mixing: 1 g HERAFILL powder (1% gentamicin base; 1 mg gentamicin base) for each 1 g of bone chips (bone chips HERAFILL; w/w = 1:1). The bone chips were defrosted and added to tubes. HERAFILL powder was added to the bone chips. The bone chips with powder were mixed with a spatula and then vortexed for 1 minute (Figure 1).
Morselized bone chips mixed with HERAFILL powder (Heraeus Medical GmbH, Wehrheim, Germany).
Gentamicin Base Release
The release of gentamicin base from the bone chips mixed with HERAFILL was performed with phosphate-buffered saline, pH 7.4 (Sigma-Aldrich, Schnelldorf, Germany). For that purpose, 3 mL of phosphate-buffered saline was added to each tube containing bone chips mixed with HERAFILL. The tubes were vortexed for 1 minute and placed on a rocking table (Rocky Biometra, Goettingen, Germany; 20 cycles/min) at 37°C to simulate the static environment in which bone grafts are used.22–25 After 0, 1, 4, and 12 hours and 1, 2, 3, 4, 5, 6, and 7 days, the elution medium was completely removed and fresh phosphate-buffered saline was added. The elution was vortexed and stored at −20°C until the microbiologic tests were performed.
Bacillus subtilis Assay for Estimation of Antibiotic Release Concentrations
Gentamicin base concentration in the elutions was determined using a conventional microbiologic agar diffusion B subtilis assay (test agar, pH 8.0).23,26 A 6-mm-diameter metal punch was used to make a hole at the center of each B subtilis agar plate into which 100 µL of each elution or 100 µL of 10-fold dilutions (10.000-0.01 mg/L) of gentamicin sulfate (for a standard curve) was added. The plates containing the samples were incubated at 37°C for 24 hours. After incubation, the diameter of the zone of inhibition formation (in centimeters) was measured for each plate with a ruler. The diameter was confirmed with a second measurement. The standard curve was obtained by logarithmic regression and used to predict the concentration of gentamicin base in each elution. This assay was carried out in triplicate.
Suspensions of both S aureus (ATCC 29913) and S epidermidis (ATCC 12228) at 2×105 (0.5 McFarland) were prepared and 10 µL was inoculated over Müller-Hinton agar plates. A 6-mm-diameter metal punch was used to make a hole at the center of each plate where 100 µL of each antibiotic elution sample was added. The plates were incubated at 37°C for 24 hours. After 24 hours, the zones of inhibition formation were measured with a ruler. These tests were performed in triplicate.
The average amount of gentamicin base released from bone chips mixed with HERAFILL at 0 to 12 hours was 99.66 mg/mL. On day 7, the gentamicin base released was 0.42 mg/mL (Figure 2).
Gentamicin base released from bone chips mixed with HERAFILL powder (Heraeus Medical GmbH, Wehrheim, Germany) from 0 hours to 7 days.
The elution released from bone chips mixed with HERAFILL promoted a zone of inhibition formation on S epidermidis and S aureus plates. However, S epidermidis is more susceptible than S aureus to gentamicin base delivered from bone chips mixed with HERAFILL (Figure 3).
Zone of inhibition formation on Staphylococcus epidermidis and Staphylococcus aureus plates by elution released from bone chips mixed with HERAFILL powder (Heraeus Medical GmbH, Wehrheim, Germany).
In this study, the authors evaluated the original powder used to produce the bone void-filling material HERAFILL beads as an antibiotic carrier once mixed with fresh bone grafts. The efficacy of the loaded bone chips was measured by drug release tests and bacterial susceptibility with B subtilis, S epidermidis, and S aureus. HERAFILL powder is composed of calcium sulfate, calcium carbonate, and tripalmitate as a bonding additive. The resulting amount of gentamicin is an adequate agent to prevent infection and reinfection.27
Loading bone chips with HERAFILL powder in this study was performed by mixing the bone chips with powder. The mixing was easily done manually. Some authors first dilute an antibiotic powder in a saline solution and then soak the bone grafts in this solution, keeping it for weeks or months before use.16,17 The authors believe that this is an efficient method for coating bone chips because the tissue would act as a sponge in absorbing the solution. Therefore, this approach could also be an alternative for long-term storage of the grafts with antibiotic solutions. However, according to Sorger et al,28 preservation of the grafts for up to 100 hours in an antibiotic solution might affect the mechanical stability of the bone. Based on the findings of Witsø et al17 and Parrish,29 mechanical testing of osteochondral and structural allografts impregnated with antibiotics in solution is needed before this option is used clinically.
Antibiotic-loaded cancellous bone is an alternative or supplement to nonbiologic material such as cement or metal in mega implants. For example, antibiotic-loaded bone chips can be used in revisions of aseptic and septic loosened hip and knee prostheses. In a clinical study of 2-stage revision arthroplasties, the reinfection rate was lower with the use of bone allografts impregnated with antibiotics than with the use of grafts without antibiotics.19 Antibiotic-containing allografts can also be used for nonhealed fractures and infected pseudarthroses.17 The frequency of antibiotic resistance after the use of antibiotic-loaded or impregnated bone allografts remains to be determined.
The amount of gentamicin base released from bone chips mixed with HERAFILL in this study reached 0.42 mg/mL on day 7. Although this concentration of gentamicin is low, based on the literature, this amount would be enough to reach the minimal inhibitory concentration required to kill S aureus and S epidermidis planktonic cells.30,31 In this way, for 7 days after implantation, the surgical site would be protected against bacterial infection.
This study confirmed the ability of bone grafts to act as antibiotic carriers once mixed with antibiotic-containing HERAFILL powder. Bone chips mixed with HERAFILL showed efficacy against S aureus and S epidermidis. Further studies with HERAFILL as bone void-filling material are needed.
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