Search

(1) Background: Three-dimensional cultures are useful tools to evaluate regenerative approaches in vitro, as they may mimic the spatial arrangement of cells more closely to natural tissues than routine 2D culture methods. (2) Methods: We investigated the effects of a polynucleotide, hyaluronic acid (PN, HA) compound on 3D spheroid cultures of primary gingival fibroblasts, by measuring their morphology over time, cell viability with Calcein-AM, a fluorescent marker, and cell growth potential by re-plating spheroids in attachment-permissive regular culture plates under routine conditions and following them up for 15 days. (3) Results: PN + HA induced an increase in spheroid size and perimeter and a decrease in spheroid circularity, as cells tended to grow and form small peripheral stacks around the spheroid. Levels of cell viability were also higher in this group. After re-plating, only the spheroids previously stimulated with PN + HA dissolved completely during the second week of culture and colonized the plate, thus indicating the retention of a higher level of viability by the cells forming the whole spheroid with this stimulus. (4) Conclusions: Taken together, our data support the idea that the combination of PN and HA has synergic effects on primary fibroblasts and promotes their viability, the growth of 3D cellular constructs, and the retention of a remarkable proliferative potential over the course of the experimental period, making it a promising compound for further investigations.