Background: Bacteria form biofilms on the surface of orthopaedic devices, causing persistent infections. Monitoring biofilm formation on bone grafts and bone substitutes is challenging due to heterogeneous surface characteristics. We analyzed various bone grafts and bone substitutes regarding their propensity for in-vitro biofilm formation caused by S. aureus and S. epidermidis.

Methods: Beta-tricalciumphosphate (β-TCP, Chro-nOsTM), processed human spongiosa (TutoplastTM) and PMMA (EndobonTM) were investigated. PE was added as a growth control. As test strains S. aureus (ATCC 29213) and S. epidermidis RP62A (ATCC 35984) were used. Test materials were incubated with defined bacterial solution (105 colony-forming units (cfu)/ml) at 37°C for 24 h without shaking. After 24 h, the test materials were removed and washed 3 times in PBS, followed by a standardised sonication protocol (Trampuz et al. 2007, NEJM). The resulting sonication fluid was plated in aliquots of 100μl onto aerobe blood agar with 5% sheep blood and incubated at 37°C with 5% CO2 for 24 h. Bacterial counts were enumerated and expressed as cfu/ml. Sonicated samples were transferred to a microcalorimeter (TA Instrument) and heat flow at 37°C was continuously monitored over a 24h period with a precision of 0.0001°C and a sensitiviy of 200μW. All experiments were performed in triplicates to calculate the mean ± standard deviation. ANOVA analysis was used for statistical calculations.

Results: For S. aureus bacterial counts (log10 cfu/ sample) were significantly higher (p< 0.001) for the porous (β-TCP 7.67 ± 0.17, Tutoplast 7.65 ± 0.15) than for the solid samples (PMMA 6.12 ± 0.18, PE 5.17 ± 0.22). Bacterial density (log10 cfu/surface) was 10^1–10^2 times higher for the S. epidermidis than for the S. aureus. In calorimetry the shape of the heat flow curves was characteristic for the individual strain and was not influenced by the test materials. The time to detection (TTD) was shortest for β-TCP for both strains and TTD was always shorter for S. aureus than S. epidermidis with corresponding material. Cfu/sample calculated from the calorimetric data was concordant with the standard culturing method.

Conclusion: Our results demonstrate biofilm formation with both strains on all tested materials. The calorimetry in all cases was able to detect quantitatively the amount of biofilm. Further studies are needed to see whether calorimetry is a suitable tool also to monitor approaches to prevent and treat infections associated with bone grafts and bone substitutes.

Purpose: A preliminary biomechanical test conducted on cadaver specimens validated a new technique for vertebral bone harvesting for anterior intervertebral grafting of the lumbar spine. A cylinder of autologous bone harvested from a neighboring vertebra was used for the intervertebralimplant. The harvesting site was filled with a bone substitute. The biomechanical tests confirmed good restoration of the vertebral body structure. An in vivo study was conducted in the baboon. A block of tricalcium-phosphate (beta-TCP) impregnated with transforming growth factor beta3 (TGF-beta3) was used to fill the bone gap. The purpose of the present study was: 1) to assess the efficacy of this in vivo technique on a primate model, 2) to validate the surgical technique.

Material and methods: The retroperitoneal approach was used to operate nine baboons. Eighteen bone cylinders were harvested. The harvesting hole was left empty or filled with a 15 mm diameter beta-TCP cylinder, or with a beta-TCP cylinder impregnated with TGR-beta3. Control scans were obtained at three and six months postoperatively. The baboons were sacrificed at 6 months and the vertebral bodies were removed for histology study.

Results: There was no evidence of fracture or loss of vertebral body height. The harvesting holes left empty did not fill, while osteointegration and substantial resorption of the bone substitute was observed in the two other groups. In the group with beta-TCP impregnated with TGF-beta3 the resorption of the cylinder was more complete and signs of prevertebral neoformation of subperiosteal bone, not observed in the beta-TCP group, was observed. There was no neoformation of bone in the spinal canal or in the foramens. The scans showed progressive resorption starting three months postoperatively in the beta-TCP group impregnated with TGF-beta3.

Conclusion: The beta-TCP block used is a very good bone substitute for the primate spine. TGF-beta3 accelerates bone resorption and induces neoformation of subperiosteal bone. The new surgical technique for local harvesting of vertebral bone was validated.