Introduction
The combined incubation of a composite scaffold with bone marrow stromal cells in a perfusion bioreactor could make up a novel hybrid graft material with optimal properties for early fixation of implant to bone. The aim of this study was to create a bioreactor activated graft (BAG) material, which could induce early implant fixation similar to that of allograft. Two porous scaffold materials incubated with cells in a perfusion bioreactor were tested in this study.

Methods and Materials
Two groups of 8 skeletally mature female sheep were anaesthetized before aspiration of bone marrow from the iliac crest. For both groups, mononuclear cells were isolated, and injected into a perfusion bioreactor (Millenium Biologix AG, Switzerland). Scaffold granules (Ø~900-1500 µm, ~88% porosity) in group 1, consisted of hydroxyapatite (HA, 70%) with β-tricalcium-phosphate (β-TCP, 30%) (Danish Technological Institute, Denmark). The granules were coated with poly-lactic acid (PLA) 12%, in order to increase the mechanical strength of the material (Phusis, France). Scaffold granules (Ø~900-1400 µm, 80% porosity) in group 2 consisted of pure HA/β-TCP (FinCeramica, Italy). For both groups, cells were incubated in the bioreactor for 2 weeks. Fresh culture medium supplemented with dexamethasone and ascorbic-acid was added every third or fourth day.
Porous titanium alloy implants with diameter=length=10mm (Biomet®, USA) were inserted bilaterally in each of the distal femurs of the sheep; thus 4 implants in each sheep. The concentric gap (2 mm) surrounding the implant was filled with 1) BAG (autogenous), 2) granules, 3) granules+bone marrow aspirate (BMA, autologous) or 4) allograft. The sheep were euthanized after 6 weeks. Distal femurs were removed and implant-bone samples were divided in two parts. The superficial part was used for mechanical testing and micro-CT scanning, and the profound part for histomorphometry.
Push-out tests were performed on an 858 Bionix MTS hydraulic materials testing machine (MTS Systems Corporation, USA). Shear mechanical properties between implant and newly generated bone were calculated to assess implant fixation.
Results were assessed by One-way ANOVA. P-values less than 0.05 were considered significant.

Results
One sheep in group 1 had to be euthanized after 4 weeks (excluded). One implant in each group was loosened and could not undergo push-out test (excluded). Group 1: No significant differences regarding failure energy (kJ/m2, p=0.44) or ultimate shear strength (MPa, p=0.17) could be seen. Shear stiffness (MPa) was significantly higher for the allograft group (p=0.04). Group 2: No significant differences regarding failure energy (p=0.11) or shear stiffness (p=0.52) could be seen. Ultimate shear strength was significantly higher for allograft (p=0.04).
Results from µ-CT scanning and histomorphometry are pending.

Discussion and Conclusion
The present study shows a possible effect of bioreactor activated bone substitute on early implant fixation. We are currently working on bone microarchitecture surrounding implant and histomorphometry. These results will aid in determining if BAG could make up a promising alternative for allograft as bone graft material.