Injectable photocrosslinkable hyaluronic acid hydrogels incorporated with platelet lysate enhance the dentinogenic differentiation of human dental pulp stem cells

摘要

Introduction: The restoration of the dentine-pulp complex (comprising mineralized and soft tissues), resulting from pulpitis, deep caries, trauma, thermic stimuli and iatrogenic procedures is challenging and has a huge impact in the healthcare systems. Pulp capping has been the most widely used therapy to repair pulp damage. However the current materials employed, namely calcium hydroxide, promote cellular necrosis due to their alkaline pH. Moreover, the reparative dentin formed often present defects result on procedure failure. In the present study was proposed an alternative photopolimerizable hydrogel system based on hyaluronic acid (HA) and platelet lysate (PL). This versatile system could be injected into any defect shape, closely interacting with the defect surfaces, and crosslinked in situ for the further controlled release of the platelet-origin cytokines, with mitogenic and morphogenic properties, in a controlled manner. Materials and Methods: HA hydrogels incorporating PL were prepared after photocrosslinking of methacrylated HA (me-HA) solution, previously dissolved in PL. Hydrogels produced with me-HA dissolved in PBS were used as control. The crosslinking reaction was triggered by excitation of the photoinitiator Irgacure 2959 by UV light (Omnicure series 2000 EXFO S2000-XLA), producing stable hydrogels. HDPSCs were isolated by enzymatic digestion from the dental pulp tissue extracted from permanent teeth. The expression of mesenchymal stem cell-associated markers CD 44, CD 90 e CD 105 was assessed by flow cytometry in hDPSCs cultures at passages 3 to 6. The effect of the hydrogel incorporated with PL on the hDPCs was analyzed. Cells were seeded at a cell density of 5×104/sample. The metabolism, quantified by alamar blue assay, and cell proliferation, quantified as a function of dsDNA, of cells cultured 7,14 and 21 days in in osteogenic/odontogenic medium were analyzed. The hDPCs differentiation was followed overtime in terms of ALP activity and calcium deposition. Also the expression of the osteogenic/odontogenic genes ALP, RUNX2 and COLIA1 was quantified by rt-PCR analysis. Results and Discussion: The metabolism as well as population of hDPCs was increased over time in the study group. The adhesion sites provided by the fibrinogen present in PL, as well as the mitogenic properties of PL cytokines, might explain the observed results. The expression of the osteogenesis transcription factor RUNX2, the extracellular matrix protein COLIA1 and ALP genes was enhanced in the cells seeded onto the hydrogels incorporated with PL. The ALP activity in cellular lysates, increased after 14 days in culture, corroborated the ALP gene expression results. Moreover, calcium deposition was observed after 21 days, in the study group. Conclusions: Data demonstrates that the hyaluronic acid hydrogels incorporating platelet lysate increase the metabolism and stimulate the dentin matrix deposition by hDPSCs, providing clear evidence of the potential of the newly developed system in the regeneration of damaged pulp/dentin tissue.