The development of technologies to promote vascularization of engineered tissue would drive major developments in tissue engineering and regenerative medicine. Recently, we succeeded in fabricating three-dimensional (3D) cell constructs composed of mesenchymal stem cells (MSCs). However, the majority of cells within the constructs underwent necrosis due to a lack of nutrients and oxygen. We hypothesized that incorporation of vascular endothelial cells would improve the cell survival rate and aid in the fabrication of biomimetic bone tissues in vitro. The purpose of this study was to assess the impact of endothelial cells combined with the MSC constructs (MSC/HUVEC constructs) during short- and long-term culture. When human umbilical vein endothelial cells (HUVECs) were incorporated into the cell constructs, cell viability and growth factor production were increased after 7 days. Furthermore, HUVECs were observed to proliferate and self-organize into reticulate porous structures by interacting with the MSCs. After long-term culture, MSC/HUVEC constructs formed abundant mineralized matrices compared with those composed of MSCs alone. Transmission electron microscopy and qualitative analysis revealed that the mineralized matrices comprised porous cancellous bone-like tissues. These results demonstrate that highly biomimetic bone tissue can be fabricated in vitro by 3D MSC constructs incorporated with HUVECs.
Freeze‐dry processing improves the operability and stability of cell‐based biomaterials and facilitates sterilization for clinical application. However, there is no established freeze‐drying protocol for engineered tissues. Recently, we reported that biomimetic bone tissues can be fabricated using scaffold‐free three‐dimensional (3D) cell constructs with potential applications as bone graft materials. The purpose of this study was to assess the influence of freeze drying on the morphology and components of 3D cell constructs. Cell constructs freeze dried in phosphate buffered saline (PBS) maintained organic and inorganic components; whereas sodium citrate buffer (SCB)‐treated constructs had significantly lower amounts of calcium and bone‐related proteins. Alkaline phosphatase (ALP) activity in cell constructs was maintained by freeze drying in 10% sucrose‐containing PBS, whereas cell constructs treated with PBS without sucrose or with sucrose‐containing SCB showed significant reductions of ALP activity. In this study, we found that sucrose‐containing phosphate buffer was suitable for freeze drying to maintain minerals and protein functions within 3D cell constructs, whereas citrate buffer was inappropriate. The insights gained by this study may facilitate the development of novel cell‐based biomaterials fabricated by tissue engineering approaches and bone graft biomaterials.
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