## 4 Discussion  
Regeneration is a potent capability of living organisms to recreate parts of tissue that have been lost and thus restore their own integrity [20]. However, several circumstances, such as defect extension or the presence of disease, may impair this process and call for adjunctive treatments [21]. Under such conditions, tools may be needed to improve cell growth and healing potential. Biomaterials, one of the mainstays of most regenerative strategies, have proved to be able to provide excellent support to healing tissues, although they often lack the capability to impart specific cues to obtained desired responses from  
Figure 4: **(A–C)** Microphotographs of gingival fibroblasts cultured with vehicle **(A)**, PN **(B)** or PN + HA **(C)** and stained with Alcian Blue. Magnification 10\(\times\), Bar = 100 \(\upmu\)m.  
Figure 5: **(A–C)** Microphotographs of gingival fibroblasts, after scratch, cultured with vehicle **(A)**, PN **(B)** or PN + HA **(C)** and stained with Giemsa. Magnification 4\(\times\) (Bar = 100 \(\upmu\)m). **(D)** Graph showing the amount of healing area (scratch area covered by cells) at time = and after 48 or 96 h. *** \(p<\) 0.001.  
cells [22]. Recently, biomimetic approaches have become object of in-depth investigation, as they rely on principles of activity that are physiologically found in organisms [5]. The present study reports on the responses of primary gingival fibroblasts to a hyaluronic acid biopolymer enriched with polynucleotides (PN + HA). PN is a compound containing a mixture of DNA fragments that has accrued a conspicuous body of evidence regarding its capability to enhance cell vitality in several in vitro and in vivo studies and can count on a long clinical experience of use as healing support therapy, especially for wounds or ulcers [14; 15]. Our data confirmed that PN are effective in promoting cell growth in gingival fibroblasts (Figure 1), with consistent results across several assays. Addition of HA appeared to exert a marginal effect on growth, which was observed at direct count and, partially, at luminescence, although MTT noticeably failed to pick this up (Figure 1). Most of the available literature reports that 100 \(\upmu\)g/mL is the optimal dose of polynucleotides for in vitro and in vivo applications and our data confirm that gingival fibroblasts too achieve their peak response at that concentration. Microscopic observations confirmed the impression of a higher cellular density in the presence of PN + HA hydrogel than in the control group or with PN alone, consistently with direct count data. Microphotographs may also offer a clue as to why metabolic viability assays like MTT failed to observe a further increase in cell number in the PN + HA group. MTT is a very common and well established cellular assay that is based on the conversion of MTT reagent to a blue compound formazan, which can then be quantified, and thus relies on the cellular metabolic activity [23]. Microphotographs show that cells in the PN + HA group tended to form nodules and to stack up to form multilayers. Although our cell model was a commercially available primary cell model that tended to grow in monolayers, it can be hypothesized that the presence of HA may have facilitated the formation of nodules, thanks to a more abundant biopolymer matrix, and thus of cell-rich areas that did not provide a linear MTT response. Indeed, our clonogenic assay showed that PN and PN + HA significantly improved colony number as compared to vehicle, which can be easily explained by their effects on cell growth (Figure 2), and which possibly became even more visible in a challenging situation for cells in CFU-assay. In a clonogenic assays, cells are seeded at very low density [19], where paracrine stimulation from neighboring cells is very poor and PN may have thus provided just enough stimulation to allow the formation of 3-4 times the number of colonies as in the control group.