Whole genome expression analysis reveals differential effects of collagen scaffold on peripheral nerve regeneration

The peripheral nervous system (PNS) could regenerate after an injury, by modulating molecular patterns that partially resemble the ones of the developmental stage. Nonetheless, even regenerated peripheral axons are never fully myelinated and never show complete functional recovery. Thus, several bioengineering approaches have been proposed for the PNS repair, trying to recreate the best molecular environment to enhance PNS regeneration. Herein, we tested in vivo the biological impact of a micro- patterned collagen scaffold (MPCS) over 10-mm critical size defects in the adult rat sciatic nerve. We have investigated the mechanisms regulating the effects of the micro-patterned collagen scaffold on the regenerating sciatic nerve by microarray-based whole genome profiling. Peripheral nerves from healthy rats and rats undergoing sciatic nerve crush injury only were used as controls. 3 time points were analyzed (8, 25 and 40 days after implant/crush). A total of 22523 probes were examined. We found that MPCS up-regulated a total of 132 RNA species at 25 days, as compared to crush nerve, wherein quite a few genes were involved in neurogenesis, organ morphogenesis and/or axiogenesis. Moreover, the co-expression network built from the 48 RNA species upregulated exclusively by MPCS had 41 nodes and 80 interactions. Five genes involved in neurogenesis (Ndn, Dbi, Cspg4, Rxrg, Bmp7) and two genes involved in organ morphogenesis (Rdh10, Bhlhb5) were found to be part of the co- expression network. Interestingly, we found that MPCS induced the expression of molecules involved in Wnt pathway and RXR signalling, known players in the development of neuronal circuits and nervous system remyelination. Analysis of transcription factor binding sites showed high enrichment of binding sites for transcription factors MEF2 and GATA, both involved in developmental processes. By gene expression analysis, we provide molecular evidence that the optimal micro-patterning of the proposed collagen scaffold plays a key role in turning the inner micro-environment hospitable for development. MPCS could coordinate recapitulation of the major pathways regulating nerve development and morphogenesis, leading to a morphologically normal nerve.