Nerve growth factor (NGF) is the protein responsible for the development and maintenance of sensory skin innervation. Given the role of appropriate innervation in skin healing, NGF has been indicated as a possible prohealing treatment in pathological conditions characterized by nerve ending loss, such as chronic ulcers in diabetes, however its use as a therapeutic agent is limited by its hyperalgesic effect. We tested the effect of topical application of the non-algogenic NGF derivative hNGFP61S/R100E in two models of skin ulcer induced in dbdb diabetic mice, investigating healing time, skin histology, reinnervation and angiogenesis using morphological and molecular approaches. We showed that the topical administration of CHF6467, a recombinant human NGF in which an amino acid substitution (R100E) abolished the hyperalgesic effect usually associated with NGF, accelerates skin repair in experimental wounds (full-excision and pressure ulcer) induced in diabetic mice (dbdb). CHF6467-induced acceleration of wound healing was accompanied by increased reepithelization, re-innervation and re-vascularization, as assessed by histology, immunohistochemistry and image analysis. Bioinformatic analysis of differentially expressed genes and signaling pathways in the wound tissues showed that Akt-mTOR was the most regulated pathway. In spite of the transdermal absorption leading to measurable, dose-dependent increases in CHF6467 plasma levels, no systemic thermal or local mechanical hyperalgesia was observed in treated mice. When tested in vitro in human cell lines, CHF6467 stimulated keratinocyte and fibroblast proliferation and tube formation by endothelial cells. Collectively, these results support a possible use of CHF6467 as a pro-healing agent in skin lesions in diabetes. SIGNIFICANCE STATEMENT Topical application of CHF6467 accelerates re-innervation, neoangiogenesis and wound healing in diabetic mice in both full-thickness skin excision and pressure ulcer models through the Akt/mTOR pathway, and does not induce hyperalgesia.

The complexity of the central nervous system (CNS) requires researchers to consider all the variables linked to the interaction between the different cell inhabitants. On this basis, any in vitro study of the physiological and pathological processes regarding the CNS should consider the balance between the standardization of the assay and the complexity of the cellular system which mimics the in vivo microenvironment. One of the main structural and functional components of the CNS is the oligodendrocyte precursor cell (OPC), responsible for developmental myelination and myelin turnover and repair during adulthood following differentiation into mature oligodendrocytes. In the present brief research report, we describe a 3D culture tool (VITVO) based on an inert and biocompatible synthetic polymer material scaffold, functionalized with laminin coating, and tested as a new culture microenvironment for neural stem/precursor cell (NSPC) differentiation compared to standard 2D cultures. NSPCs spontaneously differentiate in the three neural lineages (neurons, astrocytes and OPCs), identified by specific markers, along the fibers in the 3D structure. Analysis of the mRNA levels for lineage differentiation markers reveals a higher expression compared to those seeded on a 2D surface, suggesting an acceleration of the differentiation process. We then focused on the oligodendroglial lineage, showing that in VITVO, mature oligodendrocytes exhibit a myelinating morphology, proven by 3D image elaboration, linked to a higher expression of mature oligodendrocyte markers. This preliminary study on an innovative 3D culture system is the first robust step in producing new microenvironment-based strategies to investigate in vitro OPC and oligodendrocyte biology.

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