Spheroid Culture System, a Promising Method for Chondrogenic Differentiation of Dental Mesenchymal Stem Cells.

The objective of the present work was to develop a three-dimensional culture model to evaluate, in a short period of time, cartilage tissue engineering protocols. The spheroids were compared with the gold standard pellet culture. The dental mesenchymal stem cell lines were from pulp and periodontal ligament. The evaluation used RT-qPCR and Alcian Blue staining of the cartilage matrix. This study showed that the spheroid model allowed for obtaining greater fluctuations of the chondrogenesis markers than for the pellet one. The two cell lines, although originating from the same organ, led to different biological responses. Finally, biological changes were detectable for short periods of time. In summary, this work demonstrated that the spheroid model is a valuable tool for studying chondrogenesis and the mechanisms of osteoarthritis, and evaluating cartilage tissue engineering protocols.

Influence of Periodontal Ligament Stem Cell-Derived Conditioned Medium on Osteoblasts.

Mesenchymal stem cells (MSC) are involved in the regeneration of various missing or compromised periodontal tissues, including bone. MSC-derived conditioned medium (CM) has recently been explored as a favorable surrogate for stem cell therapy, as it is capable of producing comparable therapeutic effects. This study aimed to evaluate the influence of periodontal ligament stem cells (PDLSC)-CM on osteoblasts (OB) and its potential as a therapeutic tool for periodontal regeneration. Human PDLSC were isolated and characterized, and CM from these cells was collected. The presence of exosomes in the culture supernatant was observed by immunofluorescence and by transmission electron microscopy. CM was added to a cultured osteoblastic cell line (Saos-2 cells) and viability (MTT assay) and gene expression analysis (real-time PCR) were examined. A cell line derived from the periodontal ligament and showing all the characteristics of MSC was successfully isolated and characterized. The addition of PDLSC-CM to Saos-2 cells led to an enhancement of their proliferation and an increased expression of some osteoblastic differentiation markers, but this differentiation was not complete. Saos-2 cells were involved in the initial inflammation process by releasing IL-6 and activating COX2. The effects of PDLSC-CM on Saos-2 appear to arise from a cumulative effect of different effective components rather than a few factors present at high levels.

Osteoarthritis of the Temporomandibular Joint: A Narrative Overview.

Background and Objectives: This study reviewed the literature to summarize the current and recent knowledge of temporomandibular joint osteoarthritis (TMJOA). Methods: Through a literature review, this work summarizes many concepts related to TMJOA. Results: Although many signaling pathways have been investigated, the etiopathogenesis of TMJOA remains unclear. Some clinical signs are suggestive of TMJOA; however, diagnosis is mainly based on radiological findings. Treatment options include noninvasive, minimally invasive, and surgical techniques. Several study models have been used in TMJOA studies because there is no gold standard model. Conclusion: More research is needed to develop curative treatments for TMJOA, which could be tested with reliable in vitro models, and to explore tissue engineering to regenerate damaged temporomandibular joints.

Mesenchymal stem cell-derived small extracellular vesicles as cell-free therapy: Perspectives in periodontal regeneration.

Mesenchymal stem cells (MSC) are involved in the regeneration of the different missing or compromised periodontal tissues. MSC-derived small extracellular vesicles (sEV) have recently been explored as a favorable substitution for stem cell therapy, as they are capable of producing therapeutic effects comparable to those of their parent cells, with advantages over cell therapy. The aim of this review is to evaluate the use of mesenchymal stem cells (MSC)-derived sEV as cell-free therapy in periodontal regeneration. A review of the scientific literature on sEV and their use in periodontal regeneration was performed. The main characteristics of sEV are described, and their mechanisms of action and potential biological effects in periodontal regeneration are studied. A summary of existing preclinical studies conducted in animals is performed. The results indicate that sEV derived from MSC are emerging as a promising new therapeutic tool in the field of periodontal regeneration and may become an ideal therapeutic option. In this review, we have summarized recent advances in this regard in order to better understand this newly emerging treatment. First results in vivo are promising and show a favorable potential for cell-free therapy in periodontal regeneration.

Development of a 3D human osteoblast cell culture model for studying mechanobiology in orthodontics.

OBJECTIVES: Mechanobiology phenomena constitute a major element of the cellular and tissue response during orthodontic treatment and the implantation of a biomaterial. Better understanding these phenomena will improve the effectiveness of our treatments. The objective of this work is to validate a model of three-dimensional (3D) culture of osteoblasts to study mechanobiology. MATERIALS AND METHODS: The hFOB 1.19 cell line was cultured either traditionally on a flat surface or in aggregates called spheroids. They were embedded in 0.8% low-melting agarose type VII and placed in a polyethylene terephthalate transwell insert. Compressive forces of 1 and 4 g/cm2 were applied with an adjustable weight. Proliferation was evaluated by measuring diameters, monitoring glucose levels, and conducting Hoechst/propidium iodide staining. Enzyme-linked immunosorbent assays focusing on the pro-inflammatory mediators interleukin (IL)-6 and IL-8 and bone remodelling factor osteoprotegerin were performed to evaluate soluble factor synthesis. quantitative reverse transcription-polymerase chain reaction was performed to evaluate bone marker transcription. RESULTS: The 3D model shows good cell viability and permits IL dosing. Additionally, three gene expression profiles are analysable. LIMITATIONS: The model allows analysis of conventional markers; larger exploration is needed for better understanding osteoblast mechanobiology. However, it only allows an analysis over 3 days. CONCLUSION: The results obtained by applying constant compressive forces to 3D osteoblastic cultures validate this model system for exploring biomolecule release and analysing gene transcription. In particular, it highlights a disturbance in the expression of markers of osteogenesis.

Enhancement of the biocompatibility by surface nitriding of a low-modulus titanium alloy for dental implant applications.

To enhance their longevity, dental implants must be highly biocompatible and must have a low elastic modulus close to that of the bone. They must also possess a high superficial hardness and a high corrosion resistance. For these reasons, a recently developed low-modulus Ti-27Nb alloy with nontoxic elements was treated by gas nitriding at high temperature in this study. A very thin nitrided layer of 0.5 µm in thickness followed by an enriched nitrogen zone was observed. Consequently, a very high hardness evaluated at about 1800 HV was obtained in surface, which represents an increase of 4-5 times the hardness of the non-nitrided alloy. This superficial hardness was experimentally observed to decrease up to 800 nm in depth from the surface to the core. The low modulus of Ti-27Nb (evaluated at 55 GPa, which is twice lower than the commercially pure titanium) was not affected by the surface nitriding treatment. A better corrosion resistance was observed and a significant decrease in ion release rates for the nitrided alloy (ion release of 1.41 ng/cm2 compared to the 163.58 ng/cm2 obtained for the commercially pure titanium at pH = 7.48 in artificial Carter-Brugirard saliva). The cytocompatibility was not compromised and the cell viability performed on human osteoblasts, fibroblastic cells, and epithelial cells was enhanced on the nitrided surface in comparison with the non-nitrided surface. These combined properties make the nitrided Ti-27Nb alloy a good candidate for dental implant applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1483-1490, 2019.

[Bone mechanobiology, an emerging field: a review]. / La mécanobiologie osseuse, un domaine émergeant : revue de littérature.

INTRODUCTION: Mechanobiology, at the interface between biology and biophysics, studies the impact of mechanical forces on tissues, cells and biomolecules. The application of orthodontic forces, followed by induced tooth displacement, is a striking example of its clinical application. OBJECTIVE: The purpose of this article was to compile a review of the literature on the subject of mechanobiology; from its detection at bone level to the presentation of stimulated intracellular pathways. MATERIALS AND METHODS: The literature search was conducted on the Pubmed database in April 2018, with associations of the terms "mechanobiology", "orthodontics", "cell culture", "physiopathology". RESULTS: Three major areas of research were selected: highlighting of the phenomenon and its application in the field of bone biology; the cellular effectors of mechanobiology and its clinical applications. The use of mechanobiology in dentofacial orthopedics opens up a new field of reflection for clinicians regarding future advances in orthodontics.

Evaluation of Functional SiO2 Nanoparticles Toxicity by a 3D Culture Model.

CONTEXT: as a kind of non-metal oxide SiO2 NPs have been extensively used in biomedicine, pharmaceuticals and other industrial manufacturing fields, such as DNA delivery, cancer therapy… Our group had developed a method based on microemulsion process to prepare SiO2 NPs incorporating photonic or magnetic nanocrystals and luminescent nanosized inorganic metal atom clusters. However, the toxicity of nanoparticles is known to be closely related to their physico-chemical characteristics and chemical composition. OBJECT: it is therefore of interest to investigate the toxicity of these novel SiO2 NPs to the cells that may come in contact. MATERIALS AND METHODS: the potential toxic effect of the functional @SiO2 NPs containing Mo6 clusters with or without gold nanoparticles was investigated, at concentrations 1 µg/mL, 10 µg/mL and 100 µg/mL each, on three different cell lines. Cell viability was measured by the MTT test in monolayer's culture whereas the cytotoxicity in spheroid model was examined by the APH assay. In a second time, oxidative-stress-induced cytotoxicity was investigated through glutathione levels dosages. RESULTS: the results indicated that both A549 and L929 cell lines did not exhibit susceptibility to functional @SiO2 NPs-induced oxidative stress unlike KB cells. DISCUSSION: SiO2 NPs containing CMB may become toxic to cultured cells but only at a very high dosage level. Therefore, this toxicity depends on cell lines and more, on the model of cell cultures. The selection of appropriate cell line remains a critical component in nanotoxicology. CONCLUSION: these results are relevant to future applications of SiO2 gold-cluster NPs in controlled release applications.

Spheroid model study comparing the biocompatibility of Biodentine and MTA.

The primary objective of this study was to assess the biological effects of a new dentine substitute based on Ca3SiO5 (Biodentine™) for use in pulp-capping treatment, on pseudo-odontoblastic (MDPC-23) and pulp (Od-21) cells. The secondary objective was to evaluate the effects of Biodentine and mineral trioxide aggregate (MTA) on gene expression in cultured spheroids. We used the acid phosphatase assay to compare the biocompatibility of Biodentine and MTA. Cell differentiation was investigated by RT-qPCR. We investigated the expression of genes involved in odontogenic differentiation (Runx2), matrix secretion (Col1a1, Spp1) and mineralisation (Alp). ANOVA and PLSD tests were used for data analysis. MDPC-23 cells cultured in the presence of MTA had higher levels of viability than those cultured in the presence of Biodentine and control cells on day 7 (P = 0.0065 and P = 0.0126, respectively). For Od-21 cells, proliferation rates on day 7 were significantly lower in the presence of Biodentine or MTA than for control (P < 0.0001). Col1a1 expression levels were slightly lower in cells cultured in the presence of MTA than in those cultured in the presence of Biodentine and in control cells. Biodentine and MTA may modify the proliferation of pulp cell lines. Their effects may fluctuate over time, depending on the cell line considered. The observed similarity between Biodentine and MTA validates the indication for direct pulp-capping claimed by the manufacturers.

In vitro effects of Choukroun's platelet-rich fibrin conditioned medium on 3 different cell lines implicated in dental implantology.

OBJECTIVE: The aim of this work was to determine the relevance of Choukroun's platelet-rich fibrin (PRF) in dental implantology by determining the in vitro effects of soluble factors released by PRF clot. We used 3 different cell lines implicated in dental implantology: osteoblast, keratinocyte, and fibroblast. METHODS: Cellular viability, cell proliferation, and gene expression were analyzed using PRF conditioned medium. Three different cells lines were used: SaOS2 (osteoblast), MRC5 (fibroblast), and KB (epithelial cell). RESULTS: The sulforhodamine B assay showed a significant increase in cell number for the undiluted and 1:3 diluted conditioned medium after 24 and 48 hours. There was no effect for the 1:9 dilution. Cell cycle analysis by flow cytometry confirmed the viability test results. After 48 hours, PRF conditioned medium induced gene expression in osteoblasts. Expression of osteopontin and osteocalcin, late osteogenic markers, was observed using reverse transcriptase-polymerase chain reaction (RT-PCR). CONCLUSIONS: This study establishes a model to evaluate, in vitro, the effects of soluble growth factors released by PRF clot. Our work confirmed PRF is useful in stimulating tissue healing and bone regeneration. This work should recommend Choukroun's PRF in numerous implantology clinical applications.

Effect of hydroxyapatite and beta-tricalcium phosphate nanoparticles on promonocytic U937 cells.

OBJECTIVES: Nanoparticles from implanted materials are reported as the main cause of implant failure. Monocytes are among the first cells to colonize the inflammatory site. We evaluated the biological effects of bone substitutes presented to U937 cells in vitro as micron- or nanometer-sized particles. METHODS: The HA (550 nm) and beta-TCP (550 nm) nanoparticles were incubated with U937 cells. Cell cycle modification, specific antigens expression, and the extent of cell death were determined. RESULTS: Firstly, by using the sulforhodamine B (SRB) test and the annexin V-FITC analysis by flow cytometry, our results provide evidence of the absence of cytotoxicity, and show that nanoparticles do not induce more apoptosis than microparticles in U937 cells. Secondly, although morphologic evidence of stimulation of U937-cells was found by confocal microscopy, neither bone substitute altered the distribution of the cells into different phases of the cell cycle (Kit Cycle Test Plus DNA). These results suggest that nanoparticles do not cause promonocyte maturation in macrophages. Thirdly, the flow cytometry results showed no differences in the expression of the adherence and activation markers. SIGNIFICANCE: The results suggest that nanoparticles do not promote the differentiation of promonocytic U937 cells into macrophages and do not induce an enhanced inflammatory response.

Development of a three-dimensional model for rapid evaluation of bone substitutes in vitro: effect of the 45S5 bioglass.

The evaluation of innovative bone substitutes requires the development of an optimal model close to physiological conditions. An interesting alternative is the use of an immortalized cell line to construct multicellular spheroids, that is, three-dimensional (3D) cultures. In this study, a modified hanging drops method has resulted in the generation of spheroids with a well-established human fetal osteoblasts line (hFOB 1.19), and tests have been focused on the effect of 45S5 bioglass ionic dissolution products in comparison with two-dimensional (2D) cultures. Depending on cell culture type, quantitative analysis (cell proliferation, viability/cytotoxicity, and cellular cycle) and qualitative analysis (electron microscopy and genes expression) showed a differential effect. Cell proliferation was enhanced in 2D-conditioned cultures in accordance with literature data, but decreased in 3D cultures submitted to the same conditions, without change of gene expression patterns. The decrease of cell proliferation, observed in conditioned spheroids, appears to be in agreement with clinical observations showing the insufficiency of commercially available bioglasses for bone repairing within nonbearing sites, such as periodontal defects or small bone filling, in general. Therefore, we suggest that this model could be adapted to the screening of innovative bioactive materials by laboratory techniques already available and extended monitoring of their bioactivity.

Gingival organotypic culture and langerhans cells: a tool for immunotoxicologic experiments.

Langerhans cells (LCs) are dendritic cells localized in epidermis and mucosal tissues, where they are responsible for triggering the immune response. To study LCs in the oral epithelium, organotypic cultures were prepared using gingival explants. Immunochemical techniques using anti-CD1a, anti-HLA-DR, and anti-Langerin antibodies were used to detect and quantify LCs at various times. Observations were made by light and confocal microscopy. Quantification studies showed that there is a statistically significant drop in LC numbers in the epithelial tissue after 96 h of incubation. Gingival organotypic cultures thus are a good model for studying the migration of LCs and their involvement in contact hypersensitivity and periodontal diseases. The model offers potential utility as a tool for the study of periodontal tissue in the presence of different stimuli and for conducting immunotoxicologic experiments.