Femke Mathot

Chapter 4 72 When considering whether differentiated or undifferentiated cells should be used upon translation into future human clinical studies, the extra costs and preparation time required for the differentiation of MSCs should be taken into account. Clinically, differentiation would lead to an extended period of 3 weeks between injury and repair. This must also be balanced with the hypothetical malignant potential of differentiated MSCs compared to undifferentiated MSCs. 72, 73 Limitations A limitation of the current in vitro setting is the lack of an effective tissue repair environment which is present in an in vivo setting. 70 However, an in vitro setting lends itself perfectly to evaluate the mechanisms in a thorough manner while limiting the number of animals used. Future in vivo animal studies are needed to corroborate these in vitro findings. Rat tissues were exclusively used in order to easily correlate the results to functional outcomes of nerve allografts seeded with undifferentiated and differentiated MSCs in an in vivo rat-model, which is the logical next step prior to the translation to human tissue. The current rat-model enables us to explore the underlying mechanism of action of differentiated versus undifferentiated MSCs in peripheral nerve repair, before these studies are translated to a more clinically applicable model. We did not examine the effect of changed gene expression profiles on secreted growth factors. Although ELISA analysis could have contributed to our findings, it is a costly technique and attempts to correlate mRNA expression to protein secretion have had variable success. 74 The gene expression levels of many different genes that are relevant for nerve regeneration have been examined and compared. The disadvantage of testing many genes at many different time points is that a statistical type I error is more likely to occur. Corrections were used to adjust for multiple comparisons, limiting the power of our findings. This numerical consideration emphasizes the significance of the findings that were statistically significant. We found several significant differences within and between groups over time. As expected, extracellular matrix genes like COL1A1 and COL3A1 were highly expressed, while the expression of most other genes was a hundredfold less. However, due to the different cellular function of the genes we examined, these differences do not define the relevance of the findings. All genes have their basic level of expression that was (significantly) influenced by the ECM of the decellularized nerve allograft. To make a valid expression comparison between differentiated versus undifferentiated cells, we deliberately displayed expression levels only as a ratio of our housekeeping gene (GAPDH), instead of calculating a ratio of their baseline gene expression (cells only). These limitations notwithstanding, gene expression profiles of cell types before and after interacting with the ECM of decellularized nerve allografts have not been described and compared to this extent before. The evaluation to three weeks after seeding enabled us to describe a potential expression cascade of both cell-types when used in combination with