Femke Mathot

Chapter 10 172 ANGIOGENESIS & GENE EXPRESSION Our research was aimed to correlate the level of angiogenesis to the expression of angiogenic genes and to describe if the functional outcomes trace back to the demonstrated expression levels of the evaluated trophic genes. To answer these questions the correlations between each of the studied subjects (angiogenesis and gene expression, angiogenesis and functional outcomes, functional outcomes and gene expression) will be discussed sequentially. Angiogenesis is crucial for the supply of nutrients and trophic factors, but also influences the alignment of bands of Büngner. 30, 31 VEGF-a has been extensively studied and demonstrated to be an indispensable factor in the angiogenesis process and therefore also for nerve regeneration. 31, 44-46 When studying the VEGF-a gene expression of differentiated and undifferentiated MSCs in chapter 4 , it occurred that the VEGF-a expression of both cell-types was significantly enhanced after interacting with the extracellular matrix of the decellularized nerve graft. For undifferentiated MSCs, this was previously demonstrated in vitro and in vivo while using the same seeding strategy. 47, 48 The VEGF-a expression of differentiated MSCs was significantly higher than of undifferentiated MSCs directly after seeding (p=0.003), lower at the longer term time points and approached the expression of undifferentiated MSCs again at 21 days after seeding. Enhanced expression of VEGF-a directly after differentiating MSCs was demonstrated before 18 , but longitudinal VEGF-a expression after seeding has not been compared to this extent. Translating these findings to a long-term in vivo model, it was hypothesized that undifferentiated MSCs would enhance vascularization to an equal or even augmented level compared to differentiated MSCs. Although there were no significant differences concerning the level of revascularization between undifferentiated and differentiated MSCs in chapter 6 , only differentiated MSCs significantly improved vascularization of decellularized allografts. This partly contradicts the expectations based on the demonstrated VEGF-a expressions described in chapter 4 . In an attempt to explain the subjective discrepancy between the in vitro and in vivo findings one could argue that the time frame of both studies is completely different. The in vitro data suggested an increasing VEGF-a expression trend in differentiated MSCs from 14 days onwards, while the expression in undifferentiated MSCs slowly decreased from 7 days onwards. The hypothesis that VEGF-a expression of differentiated MSCs will overtake the expression of undifferentiated MSCs on the long term is supported by previous research reporting significantly higher VEGF-a expression in differentiated MSCs after 12 weeks in vivo, which matches our in vivo angiogenesis findings. 49 The long term follow up in vivo might as well have minimized the between group differences due to the superlative nerve regeneration capacities of rats 50, 51 which could be based on superposed revascularization capacities. 52 Another hypothetical explanation is the absence of environmental signals in the in vitro setting, what might have caused the differentiated MSCs to lose their differentiational state, leading to different gene expression patterns. The differentiational state of MSCs is better preserved in vivo by signals from the surrounding regenerating nerve. 53 Therefore, in vivo outcomes are believed to represent the effects of