Most bone defects heal successfully, however, there is an increasing number of cases where bone self-healing is insufficient. Thus, there is a high need for scaffolds able to replace the clinical gold standard treatment autologous bone grafting, which entails donor site morbidity and lacks control over spatial architecture to match defect sites. Biofabrication offers great potential to produce constructs that provide control over shape, architecture and composition. Therefore, this study aims to develop a 3D-printable composite biomaterial-ink to fabricate patient-specific bone graft substitutes for bone regeneration. Based on the heterogenous nature of bone, the biomaterial ink combines inorganic osteoinductive calcium phosphate particles (CaP) with tyramine modified hyaluronic acid-Collagen type I (THA-Col) organic matrix for the delivery of chemically modified RNAs (cmRNAs) inducing nerve, vessel, and bone formation.
Biomaterial-ink formulations consisting of 17.5 mg/mL THA, 2.5 mg/mL Col with 1 U/mL horseradish peroxidase (HRP), and 0.02% w/v Eosin Y, were combined with a range of 0-30% w/v CaP of size 45-63 or 45-106 µm. 0.17 mM H2O2 was added for enzymatic pre-crosslinking, to create a viscoelastic gel with shear thinning properties. After extrusion of desired structure, further gelation was triggered by light crosslinking for 30 minutes (505 nm). 1% v/v Nanocapsules, as vectors for the cmRNA, were mixed into the pre-polymer solution, gelation and distribution of Nanocapsules within the ink was then analyzed. Composites were further characterized for printability, cohesion, swelling, degradability, and compression modulus. Printability of formulations was evaluated by printing a continuous strut, line spacing, lattice, and overhanging strut on a pillar structure. Further, biomaterial composites were assessed in vitro using a metabolic activity assay after 1, 3 and 7 days using human mesenchymal stem cells (hMSCs).
All formulations were viscoelastic and extrudable, with the formation of a continuous strut, good shape retention and without waviness. The addition of cmRNA vectors resulted in homogeneous dispersion within the matrix and did not influence the gelation mechanisms. All printed formulations retained their original weight and macroscopic shape when lyophilized and rehydrated. Additionally, formulations of THA-Col showed higher metabolic activity compared to THA alone. The range of identified formulations is being assessed for in vitro osteogenesis of hMSCs (viability, mineralization, alkaline phosphatase (ALP) production, gene expression, and protein production).
Here, a 3D-printable composite THA-Col/CaP biomaterial-ink was developed that is suitable for the combination with cmRNAs/vectors and holds significant potential as bone graft substitute for bone regeneration."