## 4 Discussion
Moreover, though the overall number of colonies in the PN + HA group was not higher than in the PN alone group, the colony patter was distinctively different. Most colonies in the PN alone group were diffused, i.e., were formed by sparse cells, scattered quite far apart. On the contrary, PN + HA appeared to induce the formation of a higher number of dense or mixed colonies that control or PN alone, confirming that PN + HA, while possessing a pro-growth effect on cells, is also endowed with the capability to affect cell arrangement and alignment (Figure 2F,G). A further confirmation was obtained by Sirius red staining [24], which visualized collagen deposits (Figure 3A-C) and revealed that PN and PN + HA were able to progressively increase collagen deposition. PN + HA furthermore facilitated the formation of more numerous collagen-positive nodules with high cells density and multilayer cell stacking, which becomes visible at fluorescence microscopy (Figure 3F). It appears that, as expected, these high-density nodules were also centers of higher matrix formation. Though this may be in part due to the mere presence of more cells, we collected evidence that points at an increased production of collagen (Figure 3G,H). Even when adjusting by cell number, we observed an increased in collagen 1a1 expression by cells stimulated with PN and PN + HA, which in this latter case was also significantly higher than the control group, where no change in expression was measured. Moreover, if the expression of Collagen 3a1 did not appear to change in the control group or event tended to decrease in the PN group, there was a significant increased, adjusted by cell number, after stimulation with PN + HA (Figure 3H). This finding is of particular interest because Collagen 3a1 is an early product of wound healing and is known to play a role in orchestrating matrix assembly during regeneration [25]. It has actually been shown that an increased Collagen 1a1/3a1 ratio is conductive to healing impairment and keloid formation [26]. A sustained Collagen 3a1 production is therefore possibly predictive of reduced risk of abnormal wound healing. Furthermore, despite the presence of HA in the formulation, Alcian Blue staining did not show any suppression of proteoglycan production, as similar levels can be detected in control, PN and PN + HA groups (Figure 4). It cannot be excluded that some of the visible staining was actually obtained from exogenous HA from the hydrogel, but the net effect appears to be the presence of comparable levels of proteoglycans in the matrix. The effects of PN + HA biopolymer on wound healing can be gauged from the scratch assay (Figure 5), which is a well-established, simple test to assess the capability of an in vitro model to bridge an artificially induced gap in the cell layer [27]. An optimal wound closure requires a combination of a coordinated array of signals and cell activities, including cell growth and migration. Cells at the borders may well duplicate but will also need to migrate into the defect to adequately and quickly fill it. In an in vivo setting, a wound will cause the release of several mediated from the injured area, including molecules from damaged and ruptured cells [28]. Such compounds include ATPs and DNA from dying cells and these have been shown to act as important cues and stimulate wound repair. PN may be in part replicating this signaling, while also providing building blocks for duplicating cells, through a well-characterized salvage pathway. This alone appeared to improve scratch healing to a certain, albeit limited, extent, but the combination of PN and HA turned out to be critical to ensure a faster gap closure already after 48 h (Figure 2). This suggests that cell migration may also be facilitated by PN + HA, possibly by the presence of HA, itself a normal component of granulation tissue in wounds, which provides a provisional matrix over the gap that fibroblast can more easily cross.