## 4 Discussion  
It has been previously shown that the combination of PN and HA exhibits emerging properties that clinically support tissue healing and protection [14, 22, 23, 24, 25]. These have been investigated using in vitro models that have shown that a combination of PN and HA promotes cell viability and matrix synthesis in human gingival fibroblasts in regular 2D cultures [26]. However, we concede that standard cultures can only offer very partial insights into the biological properties of such compounds [27] and we were thus prompted to pursue a more thorough investigation of these molecules, resorting to spheroids, a more complex and more challenging in vitro condition. To obtain spheroids, we cultured fibroblasts on special round-bottomed multiwell-plates, whose surfaces had been specifically treated to inhibit cell attachment. Cells, thus unable to adhere to the substrate, attached only to one another and formed spheroids very quickly. Our data suggest that in the absence of stimuli only the cells that were originally seeded formed spheroids, and this culture condition did not promote cell growth, as the size of the spheroid remained unchanged over the course of the experiment (Figure 1). The size of control spheroids tended to slightly decrease over time, and this may be easily interpreted as a consequence of cell death. This hypothesis is supported also by the presence of abundant cell debris around control spheroids. Taken together, it can be therefore assumed that gingival fibroblasts seeded into the plates in the absence of external stimuli promptly formed spheroids but were unable to further grow. Although most cells apparently survived, a sizable fraction of the initially plated cells died and detached from spheroids. The spheroid condition is possibly quite challenging for primary fibroblasts, as these did not appear to proliferate even in the presence of PN or HA alone, from what can be inferred from spheroid size measurements. In contrast, the addition of a combination of PN and HA supported an increase in spheroid size, and thus, possibly, an increase in  
Figure 5: Microphotographs of spheroids stimulated with (**A**) vehicle, (**B**) Polynucleotides, (**C**) Polynucleotides and Hyaluronic acid, (**D**) Hyaluronic acid, after re-plating into regular culture plates and cultured for 15 days. Spheroids that had been previously cultured in PN + HA dissolved completely after replating and their cells colonized the new plate. Spheroids previously cultured under different conditions were mostly retained, although some of their cells did spread out and grew on the new plates. Magnification 4\(\times\). Bar = 50 \(\upmu\)m.  
cell number, although alternative explanations cannot be ruled out. It is possible that the increase in spheroid size is caused by the deposition of abundant extracellular matrix, which would be consistent with our previous data [3; 26], or a combination of both phenomena. Our present data on spheroid circularity, however, suggest that gingival fibroblasts may have grown within PN + HA-treated spheroids, or they have migrated and rearranged. The decrease in spheroid circularity index in PN + HA samples from day 5 on (Figure 2B) corresponds to the observed apparition of little cell stacks at the periphery of the spheroids (Figure S1), which indicate that the cells in the outer layers of the cluster did have the tendency to grow or move out of the spheroid. It has been shown that spheroids are characterized by strong inequalities in the distribution of their nutrients and oxygen conditions, and, as a consequence, of cell activity, with peripheral cells more viable than those in the center, as confirmed by our confocal data [21]. This may have been brought about by a possible scaffolding activity of hyaluronic acid [28], which may have provided cells with a provisional matrix on which they could reposition themselves, in combination with the action of PN, which is known to promote cell viability in several cell models [11].