In-Vitro

Introduction: The inherent low oxygen tension in normal cartilage has implications on inflammatory conditions associated with osteoarthritis (OA). Biomechanical signals will additionally contribute to changes in tissue remodelling and influence the inflammatory response. In this study, we investigated the combined effects of oxygen tension and fibronectin fragment (FN-f) on the inflammatory response of chondrocytes subjected to biomechanical signals. Methods: Chondrocytes were cultured under free-swelling conditions at 1%, 5% and 21% oxygen tension or subjected to dynamic compression in an ex vivo 3D/bioreactor model with 29 kDa FN-f, interleukin-1beta (IL-1 beta) and/or the nitric oxide synthase (NOS) inhibitor for 6 and 48 hours. Markers for catabolic activity (NO, PGE(2)), tissue remodelling (GAG, MMPs) and cytokines (IL-1 beta, IL-6 and TNF alpha) were quantified by biochemical assay. Aggrecan, collagen type II, iNOS and COX-2 gene expression were examined by real-time quantitative PCR. Two-way ANOVA and a post hoc Bonferroni-corrected t-test were used to analyse data. Results: Both FN-fs and IL-1 beta increased NO, PGE(2) and MMP production (all P < 0.001). FN-f was more active than IL-1 beta with greater levels of NO observed at 5% than 1% or 21% oxygen tension (P < 0.001). Whilst FN-f reduced GAG synthesis at all oxygen tension, the effect of IL-1 beta was significant at 1% oxygen tension. In unstrained constructs, treatment with FN-f or IL-1 beta increased iNOS and COX-2 expression and reduced aggrecan and collagen type II (all P < 0.001). In unstrained constructs, FN-f was more effective than IL-1 beta at 5% oxygen tension and increased production of NO, PGE(2), MMP, IL-1 beta, IL-6 and TNF alpha. At 5% and 21% oxygen tension, co-stimulation with compression and the NOS inhibitor abolished fragment or cytokine-induced catabolic activities and restored anabolic response. Conclusions: The present findings revealed that FN-fs are more potent than IL-1 beta in exerting catabolic effects dependent on oxygen tension via iNOS and COX-2 upregulation. Stimulation with biomechanical signals abolished catabolic activities in an oxygen-independent manner and NOS inhibitors supported loading-induced recovery resulting in reparative activities. Future investigations will utilize the ex vivo model as a tool to identify key targets and therapeutics for OA treatments.

http://arthritis-research.com/content/15/5/R163 
http://www.ncbi.nlm.nih.gov/pubmed/24286132

Introduction: Lately, several new stem cell sources and their effective isolation have been reported that claim to have potential for therapeutic applications. However, it is not yet clear which type of stem cell sources are most potent and best for targeted therapy. Lack of understanding of nature of these cells and their lineagespecific propensity might hinder their full potential. Therefore, understanding the gene expression profile that indicates their lineage-specific proclivity is fundamental to the development of successful cell-based therapies. Methods: We compared proliferation rate, gene expression profile, and lineage-specific propensity of stem cells derived from human deciduous (SCD) and permanent teeth (DPSCs) over 5 passages. Results: The proliferation rate of SCD was higher (cell number, 25 × 106 cells/mL; percent colony-forming units [CFUs], 151.67 ± 10.5; percent cells in S/G2 phase, 12.4 ± 1.48) than that of DPSCs (cell number, 21 × 106 cells/mL; percent CFUs, 133 ± 17.62; percent cells in S/G2 phase, 10.4 ± 1.18). It was observed that fold expression of several pluripotent markers such as OCT4, SOX2, NANOG, and REX1 were higher (&gt;2) in SCD as compared with DPSCs. However, DPSCs showed higher expression of neuroectodermal markers PAX6, GBX2, and nestin (fold expression &gt;100). Similarly, higher neurosphere formation and neuronal marker expression (NF, GFAP) were found in the differentiated DPSCs into neuron-like cells as compared with SCD. Conclusions: This study thus demonstrates that both SCD and DPSCs exhibit specific gene expression profile, with clear-cut inclination of DPSCs toward neuronal lineage.

The post-natal dental pulp tissue contains a population of multipotent mesenchymal progenitor cells known as dental pulp stromal/stem cells (DPSCs), with high proliferative potential for self-renewal. In this investigation, we explored the potential of DPSCs to differentiate into pancreatic cell lineage resembling islet-like cell aggregates (ICAs). We isolated, propagated, and characterized DPSCs and demonstrated that these could be differentiated into adipogenic, chondrogenic, and osteogenic lineage upon exposure to an appropriate cocktail of differentiating agents. Using a three-step protocol reported previously by our group, we succeeded in obtaining ICAs from DPSCs. The identity of ICAs was confirmed as islets by dithiozone-positive staining, as well as by expression of C-peptide, Pdx-1, Pax4, Pax6, Ngn3, and Isl-1. There were several-fold up-regulations of these transcription factors proportional to days of differentiation as compared with undifferentiated DPSCs. Day 10 ICAs released insulin and C-peptide in a glucose-dependent manner, exhibiting in vitro functionality. Our results demonstrated for the first time that DPSCs could be differentiated into pancreatic cell lineage and offer an unconventional and non-controversial source of human tissue that could be used for autologous stem cell therapy in diabetes.

Introduction: Lately, several new stem cell sources and their effective isolation have been reported that claim to have potential for therapeutic applications. However, it is not yet clear which type of stem cell sources are most potent and best for targeted therapy. Lack of understanding of nature of these cells and their lineage-specific propensity might hinder their full potential. Therefore, understanding the gene expression profile that indicates their lineage-specific proclivity is fundamental to the development of successful cell-based therapies. Methods: We compared proliferation rate, gene expression profile, and lineage-specific propensity of stem cells derived from human deciduous (SCD) and permanent teeth (DPSCs) over 5 passages. Results: The proliferation rate of SCD was higher (cell number, 25 × 106 cells/mL; percent colony-forming units [CFUs], 151.67 ± 10.5; percent cells in S/G2 phase, 12.4 ± 1.48) than that of DPSCs (cell number, 21 × 106 cells/mL; percent CFUs, 133 ± 17.62; percent cells in S/G2 phase, 10.4 ± 1.18). It was observed that fold expression of several pluripotent markers such as OCT4, SOX2, NANOG, and REX1 were higher (>2) in SCD as compared with DPSCs. However, DPSCs showed higher expression of neuroectodermal markers PAX6, GBX2, and nestin (fold expression >100). Similarly, higher neurosphere formation and neuronal marker expression (NF, GFAP) were found in the differentiated DPSCs into neuron-like cells as compared with SCD. Conclusions: This study thus demonstrates that both SCD and DPSCs exhibit specific gene expression profile, with clear-cut inclination of DPSCs toward neuronal lineage.