Morpho - functional evaluation and potential application of biomaterials in regenerative therapies: human periodontal ligament fibroblasts in vitro model

Regenerative procedures in oral and maxillofacial surgeries are still a challenge for researchers and clinicians. Understanding the biological and morphological reaction of human cells towards regenerative biomaterials is essential to choose the most performing biomaterials for specific clinical situations. This research project aims to investigate the biological and morphological reaction of hPLFs toward different classes of biomaterials: respectively, APCs, dentinal derivates, and DBBM. The first study provided a biological and morphological analysis of APCs obtained with different protocols, in culture with hPLFs in standard conditions (37° C - 5 % CO2). The study design included the evaluation of L-PRF, CGF, and APG in contact with the hPLF cell line after 24 h, 72 h and 7 days of in vitro culture. Subsequently, hPLFs were cultured in standard conditions in contact with different dentinal derivates (TT, DDP, SG, and BIOS) and the evaluations were performed after 24 h, 72 h, and 7 days of in vitro culture. Finally, this research project also provides a biological and morphological evaluation of hPLFs cultures in contact with different DBBM particles, treated with a low-temperature protocol, by performing the bio-morphological evaluations after 24 h, 72 h, and 7 days of in vitro culture. The presented three studies shared a similar experimental design: in fact, the biological reaction of hPLFs towards these different classes of biomaterials was evaluated by XTT assays to assess cell proliferation and viability. Furthermore, the morphological reaction was evaluated by LM, SEM, and CLSM examinations. As regards APCs, the XTT assay showed an interesting response in the growth curve towards CGF. This biological data was confirmed by LM and SEM evidence, with the best results for the CGF and L-PRF. Moreover, dentinal derivates (SG, DDP, and TT) induced in hPLFs a significant growth, as suggested by XTT assays. Morphological evidence confirmed this biological data: densely packed cells, characterized by a modified shape, cytoplasmatic extensions (DDP and TT) and the thickening of the cellular membrane (SG and BIOS) were respectively detected by LM and SEM. Furthermore, CLSM detected a progressive increase and a dynamic expression of proliferative and cytoskeletal markers (vinculin, actin, and integrin), suggesting a cytoskeletal reaction to the tested biomaterials. Regarding DBBM, the XTT assays showed a proliferation growth curve in different DBBM particles treated with a low-temperature protocol, with a statistical significance between the different experimental materials and the negative control. Morphological observations highlighted significant morphological changes in hPLFs, characterized by a polygonal shape (LM), surface reactions with the thickening of the membrane (SEM), and expression of actin (CLSM). In conclusion, a significant onset of the growth characterized the hPLFs exposed to the selected classes of biomaterials, making them promising options for periodontal regeneration interventions.