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Objectives: To implement a dentin slice model of mesenchymal stem cells derived from dental tissues in a fibrin-agarose construct for dental pulp regeneration. Material and Methods: MSCs derived from different oral cavity tissues were combined with a fibrin-agarose construct at standard culture conditions. Cell viability and proliferation tests were assayed using a fluorescent cell dye Calcein/Am and WST-1 kit. The proliferation assay was evaluated at 24, 48, 72, and 96 hours. Also, we assessed the dental pulp stem cells (DPSCs) cell morphology inside the construct with histological stains such as Hematoxylin and Eosin, Masson's trichrome, and Periodic acidSchiff. In addition, we elaborated a tooth dentin slice model using a culture of DPSC in the fibrinagarose constructs co-adhered to dentin walls. Results: The fibrin-agarose construct was a biocompatible material for MSCs derived from dental tissues. It provided good conditions for MSCs' viability and proliferation. DPSCs proliferated better than the other MSCs, but the data did not show significant differences. The morphology of DPSCs inside the construct was like free cells. The dentin slice model was suitable for DPSCs in the fibrin-agarose construct. Conclusion: Our findings support the dentin slice model for future biological use of fibrin-agarose matrix in combination with DPSCs and their potential use in dental regeneration. The multipotency, high proliferation rates, and easy obtaining of the DPSCs make them an attractive source of MSCs for tissue regeneration.
Objetivos: Implementar un modelo de dentina con células madre mesenquimales derivadas de tejidos dentales en una constructo de fibrina-agarosa para la regeneración de la pulpa dental. Material y Métodos: Las MSC derivadas de diferentes tejidos de la cavidad oral se combinaron con una construcción de fibrina-agarosa en condiciones de cultivo estándar. Las pruebas de viabilidad y proliferación celular se ensayaron utilizando un kit de colorante celular fluorescente Calcein/Am y WST-1. El ensayo de proliferación se evaluó a las 24, 48, 72 y 96 horas. Además, evaluamos la morfología celular de las células madre de la pulpa dental (DPSC) dentro de la construcción con tinciones histológicas como hematoxilina y eosina, tricrómico de Masson y ácido peryódico de Schiff. Además, elaboramos un modelo de rebanadas de dentina dental utilizando un cultivo de DPSC en las construcciones de fibrina-agarosa coadheridas a las paredes de la dentina. Resultados: La construcción de fibrina-agarosa fue un material biocompatible para las MSC derivadas de tejidos dentales. Proporcionó buenas condiciones para la viabilidad y proliferación de las MSC. Las DPSC proliferaron mejor que las otras MSC, pero los datos no mostraron diferencias significativas. La morfología de las DPSC dentro de la construcción era como la de las células libres. El modelo de corte de dentina fue adecuado para DPSC en la construcción de fibrina-agarosa.Conclusión: Nuestros hallazgos respaldan el modelo de corte de dentina para el futuro uso biológico de la matriz de fibrina-agarosa en combinación con DPSC y su uso potencial en la regeneración dental. El multipotencial, las altas tasas de proliferación y la fácil obtención de las DPSC las convierten en una fuente atractiva de MSC para la regeneración de tejidos.
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Humanos , Sefarosa/química , Células Madre/química , Materiales BiocompatiblesRESUMEN
The clinical benefit of therapies using Mesenchymal Stem Cells (MSCs) is attributable to their pleiotropic effect over cells and tissues, mainly through their secretome. This paracrine effect is mediated by secreted growth factors and extracellular vesicles (EV) including small EV (sEV). sEV are extra-cellular, membrane encompassed vesicles of 40 to 200 nm diameter that can trigger and signal many cellular responses depending on their cargo protein and nucleic acid repertoire. sEV are purified from cell culture conditioned media using several kits and protocols available that can be tedious and time-consuming, involving sequences of ultracentrifugations and density gradient separations, making their production a major challenge under Good Manufacturing Practices (GMP) conditions. We have developed a method to efficiently enrich cell culture media with high concentrations of sEV by encapsulating cells in semipermeable cellulose beads that allows selectively the release of small particles while offering a 3D culture condition. This method is based on the pore size of the capsules, allowing the release of particles of ≤ 200 nm including sEV. As a proof-of-principle, MSCs were encapsulated and their sEV release rate (sEV-Cap) was monitored throughout the culture and compared to sEV isolated from 2D seeded cells (sEV-2D) by repetitive ultracentrifugation cycles or a commercial kit. The isolated sEV expressed CD63, CD9, and CD81 as confirmed by flow cytometry analysis. Under transmission electron microscopy (TEM), they displayed the similar rounded morphology as sEV-2D. Their corresponding diameter size was validated by nanoparticle tracking analysis (NTA). Interestingly, sEV-Cap retained the expected biological activities of MSCs, including a pro-angiogenic effect over endothelial cells, neuritic outgrowth stimulation in hippocampal neurons and immunosuppression of T cells in vitro. Here, we successfully present a novel, cost, and time-saving method to generate sEV from encapsulated MSCs. Future applications include using encapsulated cells as a retrievable delivery device that can interact with the host niche by releasing active agents in vivo, including sEV, growth factors, hormones, and small molecules, while avoiding cell clearance, and the negative side-effect of releasing undesired components including apoptotic bodies. Finally, particles produced following the encapsulation protocol display beneficial features for their use as drug-loaded delivery vehicles.
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AIM: To evaluate the impact of two non-surgical periodontal treatment modalities on metabolic and periodontal clinical parameters in subjects with type 2 diabetes mellitus (T2DM) and poor glycaemic control and chronic periodontitis. MATERIAL AND METHODS: A randomized controlled clinical trial was conducted. Ninety-three T2DM subjects with glycosylated haemoglobin (HbA1c) > 7% were randomly assigned to one of two groups receiving scaling with root planing in multiple sessions quadrant-by-quadrant (Q by Q) or within 24 hr (one stage). Periodontal parameters, HbA1c, glycaemia blood levels (FPG) and C-reactive protein (CRP) values were assessed at baseline and at 3 and 6 months post-therapy. RESULTS: At 6 months, HbA1c had decreased by 0.48% in the Q by Q group and by 0.18% in the one-stage group (p = 0.455). After therapy, subjects with an initial HbA1c < 9% showed an increase of 0.31% (p = 0.145), compared with a decrease of 0.88% (p = 0.006) in those with an initial HbA1c ≥ 9%. Periodontal parameters improved significantly (p < 0.0001) post-therapy, with similar results for both treatment modalities. CONCLUSION: Periodontal therapy had the greatest impact on HbA1c reduction on patients with an HbA1c > 9% regardless of treatment modality. Both modalities resulted in significant improvements in periodontal parameters.
