## 1 Introduction  
Tissue regeneration is the ambitious goal of a rich research field that aims at recreating lost tissues using a wide array of approaches. Tissue regeneration and engineering have therefore prompted the generation of an abundant literature that covers many, if not all, of their possible clinical applications, including the regeneration of periodontal tissues, i.e., the tissues that support teeth, including bone, periodontal ligament and gingiva [1]. Periodontal surgery has greatly benefited from the introduction of regenerative treatments, which help regain the important support tissues lost in common pathologic processes, such as periodontitis. Although some regenerative techniques are already available in periodontitis and routinely used, limitations in their predictability are still present and fuel novel lines of investigation on better scaffold materials and improved biological cues [2]. One such possible bioactive compound is polynucleotides, a DNA derivative that has shown promising results in numerous in vitro cell models, e.g., gingival fibroblasts [3] and in clinical trials [4; 5; 6; 7]. An extensive number of studies have been published providing a solid rationale for polynucleotides as \"bioexctivating primers\" by promoting the physiological restoration of tissues [8]. Polynucleotides have been shown to act on cells via different mechanisms including the supply of nucleotides to support DNA duplication, and thus cell viability, cell growth, wound healing, and synthesis of matrix components [8]. The complexity of tissue regeneration, however, far exceeds the simple need to promote cell survival and growth. The notion that tissues are elements formed by the ordered arrangement of cells and matrix into complex 3D constructs is central to these efforts [9]. This implies that cells should also be provided with specific signals that may act as instructions to control the geometric assembly of cells, such as matrix components. Consistent with this idea, a compound of polynucleotides and hyaluronic acid, a common molecule in extracellular matrices, has been proposed to facilitate overall tissue regeneration [10; 11; 12; 13] as well as to specifically improve periodontal wound healing [14]. The rationale of this compound is therefore to provide a stimulus to support viability while mimicking the extracellular matrix and thus providing provisional support during tissue formation. Such an endeavor, however, requires appropriate tools for testing. It has long been acknowledged that 2D cultures, in spite of their simplicity, do not represent a reliable model of tissue physiology and are not an ideal environment to investigate tissue engineering [15]. Cells in tissues do not grow, in the majority of cases, as two-dimensional sheets, but develop in 3D matrices, where the interactions between cells, the diffusion of soluble mediators, and the access to nutrients radically differ from the conditions present in a standard culture plate [16]. This also entails that cell reactions to stimuli in a tissue differ from standard 2D cultures, hence the need to set up in vitro models that are closer to in vivo conditions [17]. Among the plethora of 3D culture protocols that are screened and optimized in the literature, spheroids play an important role, as they are a potent culture model that yet proves quite simple to be managed and can be easily standardized [18]. We set off to use the spheroid model to investigate the regenerative potential of a polynucleotide-hyaluronic acid compound on gingival fibroblasts, because, unlike other scaffold-based models (e.g., Matrigel [19]), spheroids do not require a scaffold and are therefore suitable to investigate different stimuli that already include a scaffold molecule-hyaluronic acid, in this case.