Effects of High-Temperature-Pressure Polymerized Resin-Infiltrated Ceramic Networks on Oral Stem Cells

International audienceObjectivesThe development of CAD—CAM techniques called for new materials suited to this technique and offering a safe and sustainable clinical implementation. The infiltration of resin in a ceramic network under high pressure and high temperature defines a new class of hybrid materials, namely polymer infiltrated ceramics network (PICN), for this purpose which requires to be evaluated biologically. We used oral stem cells (gingival and pulpal) as an in vitro experimental model.MethodsFour biomaterials were grinded, immersed in a culture medium and deposed on stem cells from dental pulp (DPSC) and gingiva (GSC): Enamic (VITA®), Experimental Hybrid Material (EHM), EHM with initiator (EHMi) and polymerized Z100™ composite material (3M®). After 7 days of incubation; viability, apoptosis, proliferation, cytoskeleton, inflammatory response and morphology were evaluated in vitro.ResultsProliferation was insignificantly delayed by all the tested materials. Significant cytotoxicity was observed in presence of resin based composites (MTT assay), however no detectable apoptosis and some dead cells were detected like in PICN materials. Cell morphology, major cytoskeleton and extracellular matrix components were not altered. An intimate contact appeared between the materials and cells.Clinical SignificanceThe three new tested biomaterials did not exhibit adverse effects on oral stem cells in our experimental conditions and may be an interesting alternative to ceramics or composite based CAD—CAM blocks

Validation of Housekeeping Genes to Study Human Gingival Stem Cells and Their In Vitro Osteogenic Differentiation Using Real-Time RT-qPCR

International audienceGingival stem cells (GSCs) are recently isolated multipotent cells. Their osteogenic capacity has been validated in vitro and may be transferred to human cell therapy for maxillary large bone defects, as they share a neural crest cell origin with jaw bone cells. RT-qPCR is a widely used technique to study gene expression and may help us to follow osteoblast differentiation of GSCs. For accurate results, the choice of reliable housekeeping genes (HKGs) is crucial. The aim of this study was to select the most reliable HKGs for GSCs study and their osteogenic differentiation (dGSCs). The analysis was performed with ten selected HKGs using four algorithms: ΔCt comparative method, GeNorm, BestKeeper, and NormFinder. This study demonstrated that three HKGs, SDHA, ACTB, and B2M, were the most stable to study GSC, whereas TBP, SDHA, and ALAS1 were the most reliable to study dGSCs. The comparison to stem cells of mesenchymal origin (ASCs) showed that SDHA/HPRT1 were the most appropriate for ASCs study. The choice of suitable HKGs for GSCs is important as it gave access to an accurate analysis of osteogenic differentiation. It will allow further study of this interesting stem cells source for future human therapy

In vitro effects of two silicate-based materials, Biodentine and BioRoot RCS, on dental pulp stem cells in models of reactionary and reparative dentinogenesis.

Calcium silicate-based cements are biomaterials with calcium oxide and carbonate filler additives. Their properties are close to those of dentin, making them useful in restorative dentistry and endodontics. The aim of this study was to evaluate the in vitro biological effects of two such calcium silicate cements, Biodentine (BD) and Bioroot (BR), on dental stem cells in both direct and indirect contact models. The two models used aimed to mimic reparative dentin formation (direct contact) and reactionary dentin formation (indirect contact). An original aspect of this study is the use of an interposed thin agarose gel layer to assess the effects of diffusible components from the materials.The two biomaterials were compared and did not modify dental pulp stem cell (DPSC) proliferation. BD and BR showed no significant cytotoxicity, although some cell death occurred in direct contact. No apoptosis or inflammation induction was detected. A striking increase of mineralization induction was observed in the presence of BD and BR, and this effect was greater in direct contact. Surprisingly, biomineralization occurred even in the absence of mineralization medium. This differentiation was accompanied by expression of odontoblast-associated genes. Exposure by indirect contact did not stimulate the induction to such a level.These two biomaterials both seem to be bioactive and biocompatible, preserving DPSC proliferation, migration and adhesion. The observed strong mineralization induction through direct contact highlights the potential of these biomaterials for clinical application in dentin-pulp complex regeneration

Cytoskeleton and major extracellular matrix proteins were analyzed by immunofluorescence.

<p>Actin was observed in DPSC (A), DPSC with BD (B) and in DPSC with BR (C); vimentin and tubulin (D, E and F); and type 1 collagen and fibronectin (G, H and I). The observed decrease in type 1 collagen, when DPSC were in contact with biomaterials, was confirmed using RT-qPCR (5J), whereas protein secretion was not modified by the experimental conditions (Fig 5K). Scale Bar: 100μm.</p

Media used in the study.

<p>Media used in the study.</p

Primer sequences used for the study.

<p>Primer sequences used for the study.</p

Biomaterials and stem cells characteristics.

<p>SEM microphotographs of the powdered materials. Enamic (Fig 1A), EHM (Fig 1B), EHMi (Fig 1C) and Z100 (Fig 1D). FACS analysis of classical mesenchymal stem cells markers (CD29, 90, 105, 146 and STRO1), of hematopoietic cells (CD45) and of endothalium (CD31) for GSC (Fig 1E) and DPSC (Fig 1F). CFU-F assays of the GSC (Fig 1G). Differentiation staining of the GSC after 14 days: Oil Red O staining (Fig 1H) and Alizarin Red S staining (Fig 1I).Scale bar 100μm.</p

Composition and properties of the biomaterials.

<p>Composition and properties of the biomaterials.</p

Proliferation and cytoxicity analysis.

<p>Calcein AM viability assays for GSC cultured with the tested materials (Fig 2A-E). Cells microscopic morphology in the same conditions (Fig 2F-J). Scale bar 100μm. Figure 2K: Cellular proliferation analysis. Ki67 indexes graph for the 2 first days of culture in direct contact condition; MTT assays in direct contact or indirect contact for 21 days,. MTT cytotoxicity assays in direct and indirect contact conditions (Fig 2L).</p