A Preliminary Study Of The Ability Of Human Dental Pulp Fibroblasts To Trigger Nemosis

Dental pulp inflammation has long been perceived as a negative factor leading topulp disruption. Previous studies have suggested that the inflammatory reaction might be aprerequisite for the burst of progenitors implicated in pulp repair. To investigate themigration of human dental pulp stem cells (hDPSCs) in response to human dental pulpfibroblasts (HDPFs) nemosis, an in vitro model of nemosis-induced inflammation inthree-dimensional culture was used in this study.1. Human dental pulp fibroblasts (HDPF) in three-dimensionalcultures exhibit spheroid formation.The formation of fibroblast clusters in nemosis was initiated by seeding10,000fibroblasts in one well of a U-bottomed,96-well plate coated with agarose or in ahanging-drop culture. An in vitro model of nemosis-induced inflammation in athree-dimensional culture was used in this study. We observed HDPF spheroid formation and discovered that cell-cell adhesion between HDPFs led to necrosis. Cell death detectionand cell counting kit-8assays showed reduced numbers of live cell and increased levels ofcell membrane leakage in HDPF spheroids.Using scanning electron microscopy and transmission electron microscopy, a surfacemorphology analysis and an ultrastructural examination of HDPFs were performed.Monolayer HDPFs exhibited a normal structure; the cells were intact, with abundantorganelles and an integral nucleus. However, as culturing time progressed, the HDPFspheroids exhibited mitochondrial swelling, expansion of the endoplasmic reticulum,chromatin margination, chromatin dispersal with irregular clumping, and, eventually,karyopyknosis. Ultrastructural examination of the HDPF spheroids indicated that theyexhibited the characteristics of necrosis, implying that cell-cell adhesion of human dentalpulp fibroblasts triggers necrosis.2. Human dental pulp fibroblasts in three-dimensional culturesexpress proinflammatory factors and chemokines.One hallmark of nemosis is the massive induction of COX-2and IL-8. We thereforemeasured COX-2protein expression in HDPF spheroids using Western blot analysis andIL-8release in HDPF spheroids using an ELISA. Because COX-2induces the release ofPGE2, which plays an important role in pulp inflammation, we measured the extent ofPGE2release using an ELISA. Western blot analysis showed that the COX-2proteinexpression in HDPF spheroids increased during the course of the incubation period. TheELISA results showed that the amount of PGE2and IL-8secreted by HDPF spheroidsgradually increased with increasing culture time. However, in the HDPF monolayer group,PGE2was released at basal levels throughout the incubation time and showed nosignificant increase at any time point. There were only low detectable amounts of IL-8released from the monolayer HDPFs. Of note, no external stimuli were present in thethree-dimensional cultures. The secretion of proinflammatory cytokines and chemokineswas triggered only through direct cell-to-cell adhesion rather than by external stimuli,implying that the release of proinflammatory cytokines and chemokines was programmed. 3. The effect of human dental pulp fibroblasts in three-dimensionalcultures on human dental stem cell (hDPSC) migration.We have demonstrated that nemotic fibroblasts might be a source of chemokines andcytokines and thereby might induce stem cell migration; however, there is currently noevidence to support this hypothesis. Putative nemosis-targeted cell types include cancer,epithelial, endothelial and immune cells. The paracrine interplay between fibroblasts innemosis and stem cells has not yet been reported. The development of dentin-pulpengineering strategies requires an investigation of stem cell potential and the interactionsbetween stem cells and non-stem “supportive” cells.We utilized an in vitro model of nemosis-induced inflammation in three-dimensionalculture to study the migration of hDPSCs in response to HDPF nemosis. A transwellmigration assay showed that the migration counts of hDPSCs cultured in conditionedmedium obtained from the monolayer were lower than those cultured in conditionedmedium obtained from the spheroids. Thus, conditioned medium obtained from HDPFspheroids could promote more hDPSC migration than that obtained from normaltwo-dimensional adherent HDPFs. This model might provide a potential research tool forstudying interactions between fibroblasts and stem cells, and studies concerningnemosis-targeted stem cells might help treat pulp inflammation.