Interactive effects of LPS and dentine matrix proteins on human dental pulp stem cells.

AIM: To investigate the combinatorial effects of lipopolysaccharide (LPS) and extracted dentine matrix proteins (eDMP) on regenerative and inflammatory responses in human dental pulp stem cells (DPSCs). METHODOLOGY: Culture media were supplemented with several concentrations of LPS, eDMP and combinations of both. Cell viability was assessed over 1 week by MTT assay; cell survival was evaluated after 24 h and 7 days by flow cytometry. The expression of mineralization-associated marker genes was determined by real-time quantitative polymerase chain reaction (RT-qPCR). To analyse the inflammatory response, secretion of interleukin 6 (IL-6) was quantified in the initial and the late phase of cell culture by enzyme-linked immunosorbent assay (ELISA). Data were treated nonparametrically and Mann-Whitney U-tests were performed to compare all experimental groups (α = 0.05). RESULTS: Whereas LPS had no impact on viability, eDMP led to a concentration-dependent decrease, which was significant after 7 days (P ≤ 0.024). A moderate decline of cell survival induced by LPS was detected after 48 h (P ≤ 0.026), whereas eDMP was able to reverse this effect. eDMP alone caused increased expression of tested marker genes, LPS had no regulatory effect. Combined eDMP and LPS induced an upregulation of collagen type I and osteocalcin, whereas expression levels of dentine matrix acidic phosphoprotein and dentine sialophosphoprotein were similar to the control. IL-6-secretion was increased by LPS over time. eDMP markedly elevated initial production of IL-6 (P ≤ 0.002), but suppressed LPS-induced cytokine production in the later phase. CONCLUSIONS: Lipopolysaccharide did not affect cell viability but interfered with odontoblast-like cell differentiation of DPSCs. Proteins from the dentine matrix may have a protective effect, attenuate the detrimental impact of LPS and thus play an important role during pulp repair.

Dentine matrix proteins: isolation and effects on human pulp cells.

AIM: To establish a simplified and efficient protocol for the isolation and concentration of matrix proteins from human dentine, and to assess the effects of extracted dentine matrix proteins (eDMP) on the behaviour of human pulp cells. METHODOLOGY: Matrix proteins were isolated from human dentine, purified, concentrated and characterized with protein and enzyme-linked immunosorbent assays (ELISA). Culture media were supplemented with eDMP in different concentrations, referred to as eDMP 1-10 000, to assess viability and proliferation of human pulp cells by DNA and MTT assays; apoptotic events were quantified by flow cytometry. Chemotactic effects of eDMP were assessed in a modified Boyden chamber assay. Expression levels of odontoblastic marker genes in pulp cells cultured with eDMPs were determined by real-time quantitative PCR, and the ability to induce mineralization was demonstrated by alizarin red staining. Nonparametric statistical analysis was performed to pairwise compare different groups at all time-points (Mann-Whitney U-test, α = 0.05). RESULTS: High concentrations of eDMP exhibited significant antiproliferative effects (P ≤ 0.023) after 5 (eDMP 1000) and 7 days (eDMP 500) without affecting cell viability. Apoptosis was barely influenced (P ≥ 0.089). eDMP exerted a concentration-dependent chemotactic stimulus on dental pulp cells with statistical significance already at low dosage (P = 0.006 at eDMP 10). Changes in gene expression indicated a differentiation into odontoblast-like cells, which was corroborated by findings of mineral nodule formation. CONCLUSIONS: A novel, effective and time-saving protocol for isolation and concentration of dentine matrix proteins is presented. As eDMP stimulates chemotaxis, differentiation and mineralization without affecting viability, endogenous dentine matrix proteins might be valuable for approaches to regenerate or engineer dental pulp.

Three-dimensional culture of dental pulp stem cells in direct contact to tricalcium silicate cements.

OBJECTIVES: Calcium silicate cements are biocompatible dental materials applicable in contact with vital tissue. The novel tricalcium silicate cement Biodentine™ offers properties superior to commonly used mineral trioxide aggregate (MTA). Objective of this study was to evaluate its cytocompatibility and ability to induce differentiation and mineralization in three-dimensional cultures of dental pulp stem cells after direct contact with the material. MATERIALS AND METHODS: Test materials included a new tricalcium silicate (Biodentine™, Septodont, Saint-Maur-des-Fossés, France), MTA (ProRoot® MTA, DENSPLY Tulsa Dental Specialities, Johnson City, TN, USA), glass ionomer (Ketac™ Molar Aplicap™, 3M ESPE, Seefeld, Germany), human dentin disks and polystyrene. Magnetic activated cell sorting for to the surface antigen STRO-1 was performed to gain a fraction enriched with mesenchymal stem cells. Samples were allowed to set and dental pulp stem cells in collagen carriers were placed on top. Scanning electron microscopy of tricalcium silicate cement surfaces with and without cells was conducted. Cell viability was measured for 14 days by MTT assay. Alkaline phosphatase activity was evaluated (days 3, 7, and 14) and expression of mineralization-associated genes (COL1A1, ALP, DSPP, and RUNX2) was quantified by real-time quantitative PCR. Nonparametric statistical analysis for cell viability and alkaline phosphatase data was performed to compare different materials as well as time points (Mann-Whitney U test, α = 0.05). RESULTS: Cell viability was highest on tricalcium silicate cement, followed by MTA. Viability on glass ionomer cement and dentin disks was significantly lower. Alkaline phosphatase activity was lower in cells on new tricalcium silicate cement compared to MTA, whereas expression patterns of marker genes were alike. CONCLUSIONS: Increased cell viability and similar levels of mineralization-associated gene expression in three-dimensional cell cultures on the novel tricalcium silicate cement and mineral trioxide aggregate indicate that the material is cytocompatible and bioactive. CLINICAL RELEVANCE: The tested new tricalcium silicate cement confirms its suitability as an alternative to MTA in vital pulp therapy.