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 -tricalcium-phosphate (−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/-TCP (Fin Ceramica, 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. 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

The scarcity of mesenchymal stem cells (MSCs) in iliac crest bone marrow aspirate (ICBMA), and the expense and time in culturing cells, has led to the search for alternative harvest sites. The reamer-irrigation-aspirator (RIA) provides continuous irrigation and suction during reaming of long bones. The aspirated contents pass via a filter, trapping bony fragments, before moving into a ‘waste’ bag from which MSCs have been previously isolated. We examined the liquid and solid phases, performed a novel digestion of the solid phase, and made a comparative assessment in terms of number, phenotype and differentiation capacity with matched ICBMA.

The solid fraction from the filtrate was digested for 60 minutes at 37°C with collagenase. Enumeration was performed via the colony-forming unit fibroblast (CFU-F) assay. Passage (P2) cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages, and their phenotypes assessed using flow cytometry (CD33, CD34, CD45, CD73, CD90, and CD105).

MSCs from the RIA phases were able to differentiate at least as well as those from ICBMA, and all fractions had phenotypes consistent with other established sources. The median number of colonies for the three groups was: ICBMA = 8.5 (2 to 86), RIA-liquid = 19.5 (4 to 90), RIA-solid = 109 (67 to 200) per 200 μl. The mean total yield of cells for the three groups was: ICBMA = 920 (0 to 4275), RIA-liquid = 114 983 (16 500 to 477 750), RIA-solid = 12 785 (7210 to 28 475).

The RIA filtrate contains large numbers of MSCs that could potentially be extracted without enzymatic digestion and used for bone repair without prior cell expansion.