An increasing number of biomaterials are in development, seeking to mimic the natural cascade of events during fracture repair. However, these biomaterials need to be rigorously tested prior to clinical application. In vitro testing lacks the physiological environment, while in vivo studies do not always predict the patient response. Here we hypothesize that the chorioallantoic membrane (CAM) assay can be used to culture human living bone and we aim to examine the potential of this system to test biomaterials. Specifically, we examined the addition of a novel clay hydrogel, Laponite®, as a vehicle for growth factors to enhance bone formation. Empty-cored bone cylinders were extracted from freshly isolated human femoral heads and perfused with Laponite®, Laponite®-VEGF, Laponite®-BMP2 or Blank before culture over 7 days in vitro or on Green fluorescent protein-labelled chicks (GFP-CAM). Micro computed tomography (μCT) was conducted on the cylinders to quantify the change of bone volume, followed by histological examination. Histological analysis demonstrated the invasion of chick vasculature into the human tissue evidenced by the presence of avian capillaries in the bone marrow space. Immunohistochemical detection of GFP, collagen type II and Sox9 showed the presence of newly deposited collagenous matrix and cell condensations, which co-localized with avian cells (GFP+) on the CAM-implanted cylinders. μCT indicated a significant increase in bone volume compared to in vitro and control groups (P<0.001). Critically, addition of Laponite®-BMP2 resulted in the highest increase in bone volume (7.84%±5.2 S.D.), followed by Laponite®-VEGF (7.84%±5.4 S.D.), compared to Laponite® (3.99%±5.0 S.D.). These studies demonstrate the potential of the CAM to integrate human bone tissue and provide a surrogate blood supply to aid regeneration. This avian-human system in combination with the histological and μCT analysis provides a simple alternative preclinical model for the screening of novel biomaterials in bone tissue engineering.
14 May 2016 - 17 May 2016