Abstract
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.
Highlights
Bony defects are a common problem in musculoskeletal surgery e.g. after tumor resection, infection, revision arthroplasty or trauma
Based on a combination of electro spraying of cells with electro spinning of nanofiber scaffold a process which was described for cardiovascular tissue engineering[48], osteosarcoma cell line[49], or MSC50 a spinning apparatus was constructed allowing for direct incorporation of cells via electro spraying into the electrospinning process
In case of the poly-(l-lactic acid) (PLLA) Col I Blend, this is mainly limited by the pore size of nanofiber scaffold[43,44]
Summary
Bony defects are a common problem in musculoskeletal surgery e.g. after tumor resection, infection, revision arthroplasty or trauma. Studies have shown that the physiological process of fracture healing is disturbed in 5–30% which results in delayed or incomplete recovery, depending on the treatment[1] To treat such defects, adequate bone replacement is needed. Multiple artificial scaffolds for bone replacement on the basis of ceramics, polymer foams, membranes, gels or composite materials are currently under investigation[7,8,9,10,11,12,13,14,15,16] In this context nanofiber-based scaffolds are of interest due to their ability to mimic the extracellular matrix to some extent[17,18,19,20,21,22,23]. The fiber diameter varies as a function of concentration, voltage, electrode spacing and flow rate between 100 and 730 nm[41]
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