Bioactive coatings on 3D printed scaffolds for bone regeneration: Use of Laponite™ to deliver BMP-2 for bone tissue engineering – progression throughin vitro, chorioallantoic membrane assay and murine subcutaneous model validation

Fracture non-union occurs as a consequence of various factors, leading to the development of potentially substantial bone defects. Biomaterial-based approaches for bone regeneration aim to explore alternative strategies to repair non-healing fractures and critical-sized bone defects. Thus, rigorous assessment of the ability to translate biomaterials towards clinical use is vital. Growth factors induce an effect on cells to change their phenotype, behaviour and initiate signalling pathways, leading to an effect on matrix deposition and tissue formation. Bone morphogenetic protein-2 (BMP-2) is a potent osteogenic growth factor, with a rapid clearance time in vivo necessitating clinical use of high doses, with potential deleterious side-effects. This work explored the potential for Laponite™ nanoclay coating of poly(caprolactone) trimethacrylate (PCL-TMA900) scaffolds to bind BMP-2 for enhanced osteoinduction. In vitro experiments confirmed the cytocompatibility of the PCL-TMA900 scaffolds and effective osteogenic differentiation of C2C12 myoblast cells in response to the Laponite/BMP-2 coating. The chorioallantoic membrane (CAM) assay verified PCL-TMA900 scaffold material biocompatibility and ability to support angiogenesis. A murine subcutaneous implantation model assessed heterotopic bone formation in response to the Laponite/BMP-2 coating, when used immediately post-coating and after 24 hours of room temperature storage, to evaluate a delayed use manner. The Laponite/BMP-2 coated PCL-TMA900 scaffolds implanted showed consistent, significant bone formation over the study period compared to the uncoated PCL-TMA 900 scaffold and BMP-2 only coated control scaffolds in vivo , indicating the ability of Laponite to bind the BMP-2 to the PCL-TMA900 scaffold. Bone formed peripherally around the Laponite/BMP-2 coated scaffold, with no aberrant bone formation observed. The Laponite/BMP-2 coating was found to retain its bioactivity after storage for 24 hours prior to use in vivo , however this was not to the same volume or reliability of bone formation as when used immediately post-coating. To take these studies forward, the Laponite/BMP-2 coating warrants examination in a critical-sized bone defect model to assess efficacy in an osseous site.