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

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. Bony defects are a common problem in musculoskeletal surgery e.g. after tumor resection, infection, revision arthroplasty or trauma. In general, 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. As this affects approximately 500,000 surgeries in Europe and approximately 2.2 million surgeries per year worldwide, bone is the second most transplanted tissue 2,3. Although replacement with autologous bone is the "golden standard", this procedure is limited by availability of transplant material and ster...

Advancements and Future Perspectives in Cell Electrospinning and Bio‐Electrospraying

“…These findings indicate the potential future application of cell‐seeded nanofiber scaffolds for major bone deformities. [ 10 ]…”

“…MSCs [4] PLGA electrospinning 15 kV No Osteoblast-like MG63 cells [5] Lecithin and PEO, and alginate 0.16 kV mm −1 No C2C12 myoblast [6] Alginate and PEO 10.5 kV Yes, topological cue C2C12s [8] Fibrinogen, and 0.2% PEO, electrospinning 4.5 kV Yes, myogenic medium HUVECs [9] Alginate and PEO, electrospraying 0.075 kV mm −1 No Stem cells isolated from rat femora [10] PLLA and PLLA-collagen type-I nanofiber scaffolds (PLLA Col I Blend), electrospinning 25 kV yes, PLLA MC3T3 cells [11] PVA, electrospinning 8 kV No…”

Nanofiber Carriers of Therapeutic Load: Current Trends

“…To overcome these shortcomings, cell-loaded scaffolds were produced by direct incorporation of cells during NF fabrication. The combination of co-axial cell electrospraying during NF multi-jet electrospinning was optimized, with cell survival and the number of trapped cells strongly dependent on the collecting electrode type, spraying rate, spinning distance and number of spraying devices [ 170 ]. Although possible biological incompatibility between polymer composition (collagen) and used cell line (MSC) or organic solvent residues could be responsible for lower cell density and metabolic activity, combined electrospray/spinning process did not influence MSC differentiation into osteoblasts.…”

Scaffold-free cell-based tissue engineering therapies: advances, shortfalls and forecast

“…Traditional scaffold-based tissue engineering strategies employ a cytocompatible, biodegradable and mechanically stable natural or synthetic in origin polymeric scaffold with a fully interconnected porous network for efficient transport and exchange of oxygen, nutrients and metabolites 13 , 14 . Although very many scaffold conformations (e.g., hydrogels 15 , 16 , sponges 17 , 18 , fibres 19 , 20 , films 21 , 22 ) have been developed, and have demonstrated safety and efficacy in preclinical setting and phase I clinical trials as cell delivery vehicles, only a handful of them constitute a Food and Drug Administration (FDA)/European Medicines Agency (EMA) approved device (Table 1 ). This limited technology transfer from laboratory benchtop to clinical applicability has been attributed to component (e.g., limited understanding of the mechanism of action of the various device components; device components do not comply with regulatory frameworks; toxicity issues) and process (e.g., too complex to allow for large-scale efficient and reproducible manufacturing; too long to be profitable) limitations.…”