Chitlac-coated Thermosets Enhance Osteogenesis and Angiogenesis in a Co-culture of Dental Pulp Stem Cells and Endothelial Cells

Dental pulp stem cells (DPSCs) represent a population of stem cells which could be useful in oral and maxillofacial reconstruction. They are part of the periendothelial niche, where their crosstalk with endothelial cells is crucial in the cellular response to biomaterials used for dental restorations. DPSCs and the endothelial cell line EA.hy926 were co-cultured in the presence of Chitlac-coated thermosets in culture conditions inducing, in turn, osteogenic or angiogenic differentiation. Cell proliferation was evaluated by 3\u2013[4,5\u2013dimethyl\u2013thiazol\u20132\u2013yl\u2013]\u20132,5\u2013diphenyl tetrazolium bromide (MTT) assay. DPSC differentiation was assessed by measuring Alkaline Phosphtase (ALP) activity and Alizarin Red S staining, while the formation of new vessels was monitored by optical microscopy. The IL-6 and PGE2 production was evaluated as well. When cultured together, the proliferation is increased, as is the DPSC osteogenic differentiation and EA.hy926 vessel formation. The presence of thermosets appears either not to disturb the system balance or even to improve the osteogenic and angiogenic differentiation. Chitlac-coated thermosets confirm their biocompatibility in the present co-culture model, being capable of improving the differentiation of both cell types. Furthermore, the assessed co-culture appears to be a useful tool to investigate cell response toward newly synthesized or commercially available biomaterials, as well as to evaluate their engraftment potential in restorative dentistry

Nitric Oxide-mediated cytotoxic effect induced by zoledronic acid treatment on Human Gingival Fibroblasts

Zoledronic acid (ZA) belongs to bisphosphonates (BPs), drugs administered to treat resorptive bone diseases. Although ZA is largely used in the clinical practice, significant adverse effects of ZA, such as osteonecrosis of the jaw (ONJ), were recorded. The aim of this work was to evaluate the role of Nitric Oxide (NO) in the in vitro response of Human Gingival Fibroblasts (HGFs) to 1, 5, 10 and 100μM ZA. HGFs morphology was evaluated through phase contrast microscopy and live/ dead staining; MTT and ELISA assays were applied to measure cell viability, Collagen Type I and IL6 secretion. ROS production and mitochondrial membrane potential were evaluated by flow cytometry; NO production and NOS activity by spectrophotometric analysis; eNOS and nNOS expression by fluorescence microscopy. Viable fibroblasts are evidenced in control sample while floating dead cells and cells close to detachment phase in ZA treated sample along with decreased level of Collagen Type I. Control sample shows higher number of viable cells respect to ZA treated one and ROS production increases when ZA is added. Released NO in ZA treated sample appears higher and NO overproduction is related to increased nNOS activity. IL 6 secretion level is higher in ZA treated sample than in control one. Our results suggest ROS involvement in NO overproduction, due to nNOS recruitment, both at low and high doses. In turn, NO release seems to be able to trigger the inflammatory response only when high doses are administered

Redox control of IL-6-mediated dental pulp stem-cell differentiation on alginate/hydroxyapatite biocomposites for bone ingrowth

Composites and porous scaffolds produced with biodegradable natural polymers are very promising constructs which show high biocompatibility and suitable mechanical properties, with the possibility to be functionalized with growth factors involved in bone formation. For this purpose, alginate/hydroxyapatite (Alg/HAp) composite scaffolds using a novel production design were successfully developed and tested for their biocompatibility and osteoconductive properties in vitro. Redox homeostasis is crucial for dental pulp stem cell (DPSC) differentiation and mineralized matrix deposition, and interleukin-6 (IL-6) was found to be involved not only in immunomodulation but also in cell proliferation and differentiation. In the present study, we evaluated molecular pathways underlying the intracellular balance between redox homeostasis and extracellular matrix mineralization of DPSCs in the presence of composite scaffolds made of alginate and nano-hydroxyapatite (Alg/HAp). Prostaglandin-2 (PGE2) and IL-6 secretion was monitored by ELISA assays, and protein expression levels were quantified by Western blotting. This work aims to demonstrate a relationship between DPSC capacity to secrete a mineralized matrix in the presence of Alg/HAp scaffolds and their immunomodulatory properties. The variation of the molecular axis Nrf2 (nuclear factor erythroid 2-related factor 2)/PGE2/IL-6 suggests a tight intracellular balance between oxidative stress responses and DPSC differentiation in the presence of Alg/HAp scaffolds