Dynamic hydrostatic pressure promotes differentiation of human dental pulp stem cells

Abstract

The masticatory apparatus absorbs high occlusal forces, but uncontrolled parafunctional or orthodontic forces damage periodontal ligament (PDL), cause pulpal calcification, pulp necrosis and tooth loss. Morphology and functional differentiation of connective tissue cells can be controlled by mechanical stimuli but effects of uncontrolled forces on intra-pulpal homeostasis and ability of dental pulp stem cells (DPSCs) to withstand direct external forces are unclear. Using dynamic hydrostatic pressure (HSP), we tested the hypothesis that direct HSP disrupts DPSC survival and odontogenic differentiation. DPSCs from four teenage patients were subjected to HSP followed by assessment of cell adhesion, survival and recovery capacity based on odontogenic differentiation, mineralization and responsiveness to bone morphogenetic protein-2 (BMP-2). HSP down-regulated DPSC adhesion and survival but promoted differentiation by increasing mineralization, in vivo hard tissue regeneration and BMP-2 responsiveness despite reduced cell numbers. HSP-treated DPSCs displayed enhanced odontogenic differentiation, an indication of favorable recovery from HSP-induced cellular stress.

Figure 1

Recovery capacity of HSP-treated DPSCs. HSP-treated DPSCs acquired round morphology, detached from glass discs and displayed fewer cells per unit area in a magnitude-dependent pattern as shown in representative images 2 hours after HSP application (A-D). There were fewer surviving HSP-treated DPSCs (n= 4 patients) within the first 12 to 24 hours after HSP treatment (E). HSP-treated cells continued to proliferate until confluence similar to control untreated cells (F and G, solid arrows) but HSP-treated cells partially lost adherence to the culture dish at the glass-plastic interface resulting in fewer surviving cells transitioning from the glass coverslip to tissue culture plastic (G, clear arrow). Real time PCR further showed a decrease in mRNA of one or both adhesion markers, ICAM-1 and VCAM-1, in HSP-treated DPSCs from 3 of 4 patients (H). [A to G are representative patterns displayed by all patients; mRNA levels were normalized to TATA-binding protein; differences in cell survival and mRNA levels between HSP-treated and control cells were not statistically significant].

Figure 2

HSP modulates DPSC in vitro differentiation and BMP-2 responsiveness. Representative Alizarin Red staining showed calcium accumulation occurred earlier at 2 weeks in HSP-treated DPSCs (A and B, arrows) while control DPSCs were yet to display similar calcium accumulation (C). Western blotting with anti-human DMP-1 or DSPP demonstrated individual variability in the expression of odontogenic markers in both control and HSP-treated DPSCs from four patients (D). But densitometric analysis of immunoreactive bands normalized to α-tubulin did not show any appreciable to differences between control and 2.5 MPa HSP (E). Real time PCR analysis showed that pre-treatment of DPSCs with HSP significantly enhanced DSPP levels in response to bone morphogenetic protein-2 (BMP-2) stimulation (mean of n = 4 patients) while DMP-1 was unchanged (data not provided). Enhanced BMP-2 response was attenuated to baseline level by noggin, a BMP antagonist (G). [*= statistically significant at p = 0.009].

Figure 3

HSP modulates in vivo differentiation and BMP-2 response of DPSCs. HSP-treated DPSCs retained ability to regenerate hard tissues within a bed of hydroxyapatite/tricalcium phosphate (HA) six weeks after transplantation. Relative to control DPSC (A), transplanted HSP-treated DPSCs regenerated appreciable hard tissue (arrows) and displayed highly cellular (B), partially cellular (C) and acellular (D) histological features surrounded by host fibrous tissue (FT).