Femke Mathot

General introduction 19 1 PREVIOUS RESEARCH Optimization of processed nerve allografts In studies performed prior to this thesis, the focus first was on optimization of nerve allograft processing/decellularization techniques. Histological analysis of Avance® nerve grafts revealed remaining cellular debris in the grafts, potentially leading to immunologic responses or blockage of ingrowing axons. In an effort to improve these aspects, elastase was added to the decellularization protocol. Elastase demonstrated to significantly reduce the amount of cellular debris and immunogenicity of nerve allografts. Subsequently, cold storage (4 ° C) of decellularized nerve allografts resulted in a less damaged ultrastructure of the grafts compared to frozen storage (-80 ° C) techniques. 64 Elastase-processed, cold stored allografts demonstrated improved functional outcomes in a rodent model compared to frozen stored allografts that were decellularized without elastase. In a bigger nerve defect model (rabbit) however, autografts still outperformed these optimized decellularized nerve allografts. 65 Mesenchymal stem cells To be able to test if MSCs can supplement decellularized allografts and conduits, a delivery technique needed to be defined that would not be traumatic to either the MSCs or the nerve substitute, in contradiction to previously described injection techniques. 66 Even distribution of MSCs and an efficient method were additional requirements. Dynamic seeding, by which MSCs are rotated in the presence of a decellularized nerve allograft, led to a nontraumatic, homogenously distributed attachment of MSCs to the surface of nerve allografts. 67 The process of decellularizing allografts and seeding them with MSCs in a rat model is illustrated in figure 3 . The interaction between MSCs and the extracellular matrix of the nerve allografts led to enhanced expressions of multiple trophic genes in vitro, which was partly confirmed by quantification of the produced trophic factors. 68 Taking this seeding strategy to an in vivo rodent model, it was shown that the seeded MSCs survived up to 29 days and that the MSCs did not migrate to surrounding tissues. 69 It also led to enhanced expressions of angiogenic factors. 70 It would therefore be interesting to not only study the long term functional outcomes after nerve repair with MSC-seeded allografts, but also to analyze if the MSCs improve vascularization of the nerve allografts. Figure 3. Nerve harvest, decellularization and seeding techniques. (Used with permission of Mayo Foundation for Medical Education and Research, all rights reserved.)