Macromolecular Crowding in the Development of Bone Tissue-Engineered Substitutes

Introduction: Bone injuries and pathophysiologies dramatically rise among the young and physically active and aging and physically inactive populations. Tissue engineered bone substitutes are considered a potential alternative to traditional bone grafts due to their multifunctional capacity and unlimited supply. Unfortunately, the development of bone tissue engineered medicines requires prolonged in vitro cultures, which are associated with cellular phenotype losses and very high manufacturing costs. Although macromolecular crowding has been shown to enhance and accelerate extracellular matrix deposition in a diverse range of cell populations, its potential in bone engineering has yet to be assessed. Herein, we ventured to assess the potential of macromolecular crowding in the development of scaffold-free and scaffold-based bone tissue engineered medicines. Materials & Methods: Randomly orientated poly-e-caprolactone (PCL) and poly-e-caprolactone-calcium phosphate (PCL-CaP) scaffolds were developed, and their structural and physicochemical properties were assessed using scanning electron microscopy, x-ray diffraction, differential scanning calorimetry, tensile testing and compression testing. Scaffold-free and scaffold-based bone tissue engineered medicines were developed using primary human osteoblasts and carrageenan as macromolecular crowding agent. After 3, 7 and 14 days in culture, basic cell function (via morphology, viability, metabolic activity and proliferation), protein deposition (via electrophoresis, immunofluorescence and histology) and mineralisation (via alkaline phosphatase activity, calcium deposition and gene expression) were assessed. Results: Structural analysis revealed that electrospun scaffolds with uniform morphology were fabricated. X-ray diffraction confirmed the successful incorporation of the CaP into the electrospun scaffold, which also resulted in enhanced mechanical properties. No significant differences in cell morphology, viability, metabolic activity and proliferation were found between the groups. Protein deposition analysis made apparent that macromolecular crowding significantly increased protein deposition at all time points. Macromolecular crowding also improved mineralisation in all groups. Conclusions: This work advocates the use of macromolecular crowding in the development of bone tissue engineered medicines. Acknowledgments: This worked has emanated from research supported by Research Ireland (grant number 19/FFP/6982). This work has also received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant number 866126).