gms | German Medical Science

Introduction: Reconstruction of peripheral nerve lesions remains a major challenge in plastic surgery. Clinical strategies to treat extensive peripheral nerve injuries are currently limited to autologous nerve transplantations. The aim of this tissue engineering approach is the development of a bioartificial nerve graft on basis of a specifically designed cylindrical collagen scaffold providing biological, chemical and structural cues for regenerating axons. We present modifications of a specially designed collagen matrix with the attempt to achieve the standard of autologous nerve grafts.

Material and methods: In-vitro: Microstructured collagen scaffolds with longitudinally orientated pore channels (see Figure 1 [Fig. 1]) and an optimized cell-matrix interaction for glial cells served as guidance channels for sprouting axons. The collagen scaffolds were seeded with rat Schwann cells (SC’s) and biocompatibility was demonstrated by viability stainings (FDA & PI), XTT-proliferation assays, immunocytochemistry (S100, p75, GFAP, Vimentin) and scanning electron microscopy (SEM), morphometry analyses, as well as by in-vitro regeneration experiments with dorsal root ganglia (DRG’s).In-vivo: SC seeded and cell-free cylindrical collagen scaffolds were implanted into artificially created 2 cm defects of the rat sciatic nerve with a regeneration period of 6 and 12 weeks. Regeneration was evaluated by histological (semithin sections, toluidine blue staining and transmission electron microscopy [TEM]), morphometrical (number of fibers, G-ratio) and functional analyses (Static Sciatic Index [SSI]; nerve stimulation tests; muscle weight; motor endplate stainings). In addition, in-vitro and ex-vivo monitoring of SCs before and after implantation was performed using green fluorescent protein (GFP) and confocal laser scanning microscopy (cLSM).

Results: In-vitro, the novel cylindrical collagen scaffolds were sufficiently stable over the entire observation period and SC’s displayed their typical morphology also deep within the scaffolds and long term viability in vitro could be demonstrated. SEM demonstrated typical SC morphology with round-oval cell bodies and bipolar cell processes with an orientation along longitudinal channels within the matrix. In-vitro regeneration experiments with DRG’s showed orientated axons within longitudinal channels originating from sensible DRG neurons accompanied by SC’s. In-vivo, after 6 and 12 weeks, all nerve guides were well integrated into the host tissue. In the presence of a transparent smooth sheath and in the absence of scarring or adhesions. GFP-labeled SC’s could be detected up to 12 weeks after implantation. Intraoperative stimulation of the re-exposed and regenerated sciatic nerve were positive and resulted in toe spreading. Histological analyses presented myelinated and fascicular orientated axons without neuroma formations reaching the distal part of the nerve graft already after 6 weeks. TEM showed that these fascicles were surrounded by a perineurial sheath made by basal lamina forming perineurial cells. The diameter of such a perineurium corresponded the diameter of the original pore channels.

Conclusion: The developed cylindrical collagen scaffolds resulted in an advanced interaction between SC’s and the nerve guide in-vitro. Both in-vitro and in-vivo regeneration experiments revealed appropriate regeneration with axonal sprouting reaching the distal end of the guidance channels in an orientated manner.