Evaluation of the osteoinductive potential of biomimetic scaffolds using precursor cells

Bone tissue engineering represents a promising approach for the treatment of critical-sized bone defects through the combination of biomaterials and cells. In this context, three-dimensional (3D) biomimetic scaffolds can provide a suitable environment to support cell proliferation and osteogenic differentiation. The aim of this thesis was to investigate the osteogenic response of MC3T3-E1 pre-osteoblastic cells cultured on a Mg-doped biphasic calcium phosphate (BCP) scaffold with a 60/40 HA/β-TCP composition, developed and produced by GreenBone Ortho®. The experimental study followed a stepwise approach, initially using two-dimensional (2D) culture to establish and optimize the experimental procedures before their application to the more complex 3D scaffold model. Osteogenic differentiation was investigated at both the transcriptional and functional levels through the analysis of osteogenic gene expression by quantitative PCR and the analysis of alkaline phosphatase (ALP) activity. In 2D culture, osteogenic stimulation induced a coordinated modulation of osteogenic gene expression and an increase in ALP activity, supporting the progression of osteogenic differentiation. The analysis was subsequently extended to the 3D scaffold model, where osteogenic stimulation resulted in a similar overall osteogenic response, characterized by the modulation of osteogenic markers at the transcriptional level. The functional assessment of ALP activity in the 3D scaffolds required optimization of the protein extraction procedure. The combination of freeze–thaw cycles and repeated sonication improved cell lysis and intracellular protein recovery, enabling reliable quantification of ALP activity in the 3D constructs. Overall, these findings demonstrate that the investigated Mg-doped BCP scaffold supports osteogenic differentiation of MC3T3-E1 cells in a 3D culture model and that osteogenic stimulation enhances the cellular response at both the transcriptional and functional levels.