Three-Dimensional Printed Scaffolds with Multipotent Mesenchymal Stromal Cells for Rabbit Mandibular Reconstruction and Engineering.

Multipotent mesenchymal stromal cells (MSC) derived from both the bone marrow and adipose tissue possess the ability to differentiate into multiple cell lineages, regulate the immune function by secreting numerous bioactive paracrine factors, and hold great potential in cell therapy and tissue engineering. When combined with three-dimensional (3D) scaffolds, MSC can be used for bone defect reconstruction and engineering. This protocol describes the isolation of bone marrow mesenchymal stromal cells (BMMSC) and adipose-tissue derived stem cells (ADSC) from rabbits for subsequent seeding on tissue-engineered 3D-printed scaffolds and transplantation into a rabbit-model with the goal of repairing large osseous mandibular defects (one quarter of the lower jaw is removed surgically). Steps to demonstrate the three cell differentiation lineage potentials of BMMSC and ADSC into osteocytes, adipocytes, and chondrocytes are described. A modified cell seeding method using syringes on scaffold is detailed. Creating a large mandibular bone defect, the rapid prototyping method to print a customized 3D-scaffold, the scaffold implantation procedure in rabbits, and microcomputed tomography (micro-CT) analysis are also described.