Osteoblast-Mesenchymal Stem Cell Coculture Drives <i>In Vitro</i> Osteogenesis in 3D Bioprinted Periosteum

The periosteum serves as a local source of osteoprogenitor cells and vasculature, therefore influencing the key processes of osteogenesis and neovascularization during bone healing. However, it is often not considered in traditional bone tissue engineering strategies. The periosteum consists of two stratified cell layers, including an inner cambium layer, which serves as a local source of osteoblasts (OBs) and osteoprogenitor cells, and an outer fibrous layer, which hosts vasculature, collagen fibers, and support cells. While several studies have investigated different methodologies to produce tissue-engineered periosteum (TEP) substitutes, few have evaluated the roles of specific cell types within the inner cambium layer and their patterning in 3D environments on underlying bone tissue development. Therefore, we sought to investigate whether mesenchymal stem cells (MSCs) alone, OBs alone, or a 1:1 mixture of the two would result in increased osteogenic differentiation of bone layer MSCs in a 3D bioprinted periosteum-bone coculture model <i>in vitro</i>. We first evaluated these effects in a 2D transwell model, demonstrating that OB-containing cultures, either alone or in a mixed population with MSCs, upregulated alkaline phosphatase activity and runt-related transcription factor 2 (<i>RUNX2</i>) expression. In the 3D bioprinted model, the mixed population showed higher levels of <i>RUNX2</i> expression and calcium deposition, indicating increased osteogenic differentiation within the bone layer. Results obtained from this study provide evidence that a mixed population of MSCs and OBs within the inner cambium layer of TEP can increase bone regeneration.