Abstract
Novel biomaterials are being developed and studied, intended to be applied as bone graft substitute materials. Typically, these materials are being tested in in vitro setups, where among others their cytotoxicity and alkaline phosphatase activity (as a marker for osteoblastic differentiation) are being evaluated. However, it has been reported that in vitro tests correlate poorly with in vivo results and therefore many promising biomaterials may not reach the clinic as a bone graft substitute product. One of the reasons for the poor correlation, may be the minimal complexity of the in vitro tests, as compared to the in vivo environment. Ex vivo models, mimicking the natural tissue environment whilst maintaining control of culture parameters, may be a promising alternative to assess biomaterials for bone formation. Assess the possibility of an ex vivo culture platform to test biomaterials on their potential to stimulate new bone formation. Osteochondral plugs (cylinders n=10, Ø 10 mm, height 15 mm) were drilled from fresh porcine knees, from the slaughterhouse. A bone defect (Ø 6 mm) was created and which was filled with a biomaterial graft (S53P4 bioactive glass (n=3); collagen sponges loaded with BMP-2 (n=3, as positive control)) or kept empty (n=4). The explants were cultured in custom-made two-chamber bioreactors for six weeks (LifeTec Group BV). Cartilage and bone were physically separated, similar to the in vivo situation, by a sealing ring. The two tissues were cultured in separate compartments, allowing for specific culture medium for each tissue. Medium was changed every 2–3 days and weekly micro computed tomography (µCT) images were obtained to longitudinally monitor the formation of new bone. An MTT assay was performed on half of the samples after six weeks of culture. The other samples were fixed for histology, to determine which cells were present after six weeks. The MTT metabolic assay showed that a number of cells in the bone were viable after six weeks. The further away from the border, the fewer living cells were observed. The cells in the cartilage also survived. No significant bone formation was observed with µCT in either of groups, even though abundant bone formation was expected in the BMP-2 group. Explanations of the negative results of the positive group might be that too few viable cells remain after six weeks, or that the cells that are still present are not able to form bone. No significant bone formation was observed in the bone defects in osteochondral explants that were cultured with, or without, biomaterials for six weeks. However, the platform showed that it is capable to successfully culture osteochondral explants for six weeks.
Histology needs to be performed to evaluate which cells were present at the end of the culture and this will be compared to the cells present directly after drilling the explants.
