| Tailoring tissue engineering strategies to match patient and repair site specific bone regeneration needs, offers to improve clinical outcomes. The silk-scaffold based in-vitro bone regeneration system differentiating human mesenchymal stem cells (hMSCs) to osteoblast-like cells [1], was used as a tool to elucidate the impact of microenvironment and scaffold features on osteogenesis and metabolism. The osteogenic outcomes and metabolic parameters were assessed at varying conditions of amino acid (lysine and proline) concentration, oxygen level and scaffold degradability. Based on these results and known pathways, a metabolic model was derived to explain the increased rate of bone regeneration in the presence of 5x lysine and proline supplements as well as low (5%) oxygen levels. The variations in osteogenic rate were directly related to silk scaffold degradability, thus reiterating the importance of matrix features in guiding cell function and differentiation. Further studies reported the first in vitro coculture of human-specific osteoblast-like cells and osteoclast-like cells differentiated on 3D silk scaffolds to resemble the holistic bone remodeling process. This study confirmed differentiation of human osteoclast-like cells from THP-1 human monocytic cell line in the presence of PMA and RANKL, in 2D tissue culture flasks as well as 3D silk scaffolds. This system provided a more realistic estimate of bone regeneration and was used as a drug screening platform using commercially available bisphosphonates. Additionally, these osteogenic cells demonstrated active ECM remodeling by degrading silk fibroin films. Gene expression of cell-specific MMPs and integrins was evaluated. When compared to the other cell types, osteoclast-like cells resulted in the largest degradation pits and expressed significantly higher levels of MMP1 and MMP2, thereby implying these proteases in osteoclastic resorption of silk films. |
