Salivary histatins promote cell spreading and metabolic activity

Large volume bone defects can result from various diseases, causes delayed repair of healing, a rapid and stable attachment of osteogenic cells to the surface of the implant material is a first highly important step. Histatin-1 is currently recognized as a highly versatile peptide, capable of facilitating cell migration and adhesion in multiple cell types. This thesis mainly describes our studies of the activity of Histatin-1 and its variants on osteogenic cells. This would help to draw a conclusion whether we can apply the peptide histatins to the repair of tissue defects related to osteogenic cells. For this reason, we used simple and stable in vitro models to determine the effect of histatins. This thesis mainly describes our studies of the activity of Hst1 and its variants on osteogenic cells. Osteogenic cells were seeded on bio-inert glass slides and titanium surfaces with or without the presence of histatin-1 in dose-dependent or time-course assays. One scrambled, six truncated histatin-1 variants and D-histatin-1 were also evaluated. Cell spreading was analyzed using a well-established point-counting method. Confocal laser scanning microscopy (CLSM) revealed that fluorescent labeled Hst1 (F-Hst1) and D-histatin-1 (F-DHst1) were taken up into the intracellular space of cells. In chapter 2 we report the stimulatory effect of histatin-1 on surface adherence and spreading of osteogenic cells in vitro, as well as the signaling pathways involved. It was found that histatin-1 significantly promoted the spreading of osteogenic cells on both bio-inert substrates and titanium and sandblasted, large grit, acid-etched implant (SLA) surfaces, while truncated histatin-1 and scrambled histatin-1, did not. Furthermore, it was found that both ERK1/2 and p-38 inhibitors abolished these cell-promoting activities, suggesting that both ERK1/2 and p38 signaling pathways were involved. Fluorogenic (F)- histatin-1 was taken up by the cells and localized within the vicinity of the nuclei. In chapter 3 we reported the effect of co-administration of all-trans retinoic acid (ATRA), an active metabolite of vitamin-A, and histatin-1 on the spreading and osteogenic activities of osteogenic cells on bio-inert glass surfaces. Short-term co-administration of ATRA and histatin-1 cells resulted in significantly higher surface spreading of osteogenic cells compared to either ATRA or Hst1 alone. Retinoic acid receptors α, β antagonists, such as ER-50891 and LE-135, both nullified these effects of ATRA. In chapter 4, the uptake dynamics and identification of subcellular targets of histatin-1, histatin-2 and histatin-5 in epithelial cells (HO1N1, a human buccal epithelial carcinoma cell line) were investigated. Flow cytometry with fluorescence-labeled peptides revealed that cellular uptake of F- histatin-1 was substantially enhanced compared to those of F- histatin-2 and F- histatin-5. Confocal laser scanning microscopy (CLSM) revealed that both F- histatin-1 and F- histatin-1 associated with mitochondria and the endoplasmatic reticulum, whereas F- histatin-1 targeted exclusively to the mitochondria. No association with lysosomes or the Golgi apparatus was observed with any of the three fluorogenic peptides. In chapter 5, it was found that D- histatin-1 promoted the spreading of osteogenic cells on both bio-inert substrates and titanium SLA surfaces in a significantly higher efficacy than Hst1. No intracellular accumulation of F-D- histatin-1 was detected, while histatin-1 distributed in the vicinity of nuclei after administration to the cells.