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Bony defects are a common problem in musculoskeletal surgery. Replacement with autologous bone grafts is limited by availability of transplant material. Sterilized cancellous bone, while being osteoconductive, has limited osteoinductivity. Nanofiber scaffolds are currently used for several purposes due to their capability of imitating the extracellular matrix. Furthermore, they allow modification to provide functional properties. Previously we showed that electrospun nanofiber scaffolds can be used for bone tissue regeneration. While aiming to use the osteoinductive capacities of collagen type-I nanofibers we saw reduced scaffold pore sizes that limited cellular migration and thus colonization of the scaffolds. Aim of the present study was the incorporation of mesenchymal stem cells into the electrospinning process of a nanofiber scaffold to produce cell-seeded nanofiber scaffolds for bone replacement. After construction of a suitable spinning apparatus for simultaneous electrospinning and spraying with independently controllable spinning and spraying devices and extensive optimization of the spinning process,  in vitro  and  in vivo  evaluation of the resulting scaffolds was conducted. Stem cells isolated from rat femora were incorporated into PLLA (poly-l-lactide acid) and PLLA-collagen type-I nanofiber scaffolds (PLLA Col I Blend) via simultaneous electrospinning and –spraying. Metabolic activity, proliferation and osteoblastic differentiation were assessed  in vitro . For  in vivo  evaluation scaffolds were implanted into critical size defects of the rat scull. After 4 weeks, animals were sacrificed and bone healing was analyzed using CT-scans, histological, immunhistochemical and fluorescence evaluation. Successful integration of mesenchymal stem cells into the scaffolds was achieved by iteration of spinning and spraying conditions regarding polymer solvent, spinning distance, the use of a liquid counter-electrode, electrode voltage and spinning duration.  In vivo  formation of bone tissue was achieved. Using a PLLA scaffold, comparable results for the cell-free and cell-seeded scaffolds were found, while the cell-seeded PLLA-collagen scaffolds showed significantly better bone formation when compared to the cell-free PLLA-collagen scaffolds. These results provide support for the future use of cell-seeded nanofiber scaffolds for large bony defects.

Direct incorporation of mesenchymal stem cells into a Nanofiber scaffold – in vitro and in vivo analysis