PURPOSE: The purpose of this in vitro study was to analyze the toxicity of acrylate-based restorative composite resins filled with hydroxyapatite and a silica/hydroxyapatite combination.
MATERIAL AND METHODS: Five different restorative materials based on bisphenol A-glycidyl methacrylate (bis-GMA) and tri-ethylene glycol dimethacrylate (TEGDMA) were developed: unfilled (H0), hydroxyapatite-filled (H30, H50), and silica/hydroxyapatite-filled (SH30, SH50) composite resins. These were tested for in vitro cytotoxicity by using human bone marrow mesenchymal stromal cells. Surface morphology, elemental composition, and functional groups were determined by scanning electron microscopy (SEM), energy-dispersive x-ray spectroscopy (EDX), and Fourier-transformed infrared spectroscopy (FTIR). The spectra normalization, baseline corrections, and peak integration were carried out by OPUS v4.0 software.
RESULTS: Both in vitro cytotoxicity results and SEM analysis indicated that the composite resins developed were nontoxic and supported cell adherence. Elemental analysis with EDX revealed the presence of carbon, oxygen, calcium, silicon, and gold, while the presence of methacrylate, hydroxyl, and methylene functional groups was confirmed through FTIR analysis.
CONCLUSIONS: The characterization and compatibility studies showed that these hydroxyapatite-filled and silica/hydroxyapatite-filled bis-GMA/TEGDMA-based restorative composite resins are nontoxic to human bone marrow mesenchymal stromal cells and show a favorable biologic response, making them potential biomaterials.
MATERIALS AND METHODS: Diabetic ADSCs were treated with DFO and compared to normal and non-treated diabetic ADSCs for expression of HIF-1α, VEGF, FGF-2 and SDF-1, at mRNA and protein levels, using qRT-PCR, western blotting and ELISA assay. Activity of matrix metalloproteinases -2 and -9 were measured using a gelatin zymography assay. Angiogenic potential of conditioned media derived from normal, DFO-treated and non-treated diabetic ADSCs were determined by in vitro (in HUVECs) and in vivo experiments including scratch assay, three-dimensional tube formation testing and surgical wound healing models.
RESULTS: DFO remarkably enhanced expression of noted genes by mRNA and protein levels and restored activity of matrix metalloproteinases -2 and -9. Compromised angiogenic potential of conditioned medium derived from diabetic ADSCs was restored by DFO both in vitro and in vivo experiments.
CONCLUSION: DFO preconditioning restored neovascularization potential of ADSCs derived from diabetic rats by affecting the HIF-1α pathway.
Methods: hTenowere isolated from human hamstring tendon. Presence of insulin receptor beta (INSR-β) on normal tendon tissues and the hTeno monolayer culture were analyzed by immunofluorescence staining. The presence of Glucose Transporter Type 1 (GLUT1) and Glucose Transporter Type 4 (GLUT4) on the hTeno monolayer culture were also analyzed by immunofluorescence staining. Primary hTeno were treated with 0.008, 0.08, 0.8 and 8.0 µM of TNF-α, with and without insulin supplement. Outcome measures include 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) assay to determine the glucose uptake activity; colourimetric total collagen assay to quantify the total collagen expression levels; COL-I ELISA assay to measure the COL-I expression levels and real-time qPCR to analyze the mRNA gene expressions levels of Scleraxis (SCX), Mohawk (MKX), type I collagen (COL1A1), type III collagen (COL3A1), matrix metalloproteinases (MMP)-9 and MMP-13 in hTeno when treated with TNF-α. Apoptosis assay for hTeno induced with TNF-α was conducted using Annexin-V FITC flow cytometry analysis.
Results: Immunofluorescence imaging showed the presence of INSR-β on the hTeno in the human Achilles tendon tissues and in the hTeno in monolayer culture. GLUT1 and GLUT4 were both positively expressed in the hTeno. TNF-α significantly reduced the insulin-mediated 2-NBDG uptake in all the tested concentrations, especially at 0.008 µM. Total collagen expression levels and COL-I expression levels in hTeno were also significantly reduced in hTeno treated with 0.008 µM of TNF-α. The SCX, MKX and COL1A1 mRNA expression levels were significantly downregulated in all TNF-α treated hTeno, whereas the COL3A1, MMP-9 and MMP-13 were significantly upregulated in the TNF-α treated cells. TNF-α progressively increased the apoptotic cells at 48 and 72 h.
Conclusion: At 0.008 µM of TNF-α, an IR condition was induced in hTeno, supported with the significant reduction in glucose uptake, as well as significantly reduced total collagen, specifically COL-I expression levels, downregulation of candidate tenogenic markers genes (SCX and MKX), and upregulation of ECM catabolic genes (MMP-9 and MMP-13). Development of novel IR model in hTeno provides an insight on how tendon homeostasis could be affected and can be used as a tool for further discovering the effects on downstream molecular pathways, as the implication for diabetic tendinopathy.
METHODS: iPC clones were generated from two colorectal cancer (CRC) cell lines by retroviral transduction of the Yamanaka factors. The iPC clones obtained were characterized by morphology, expression of pluripotency markers and the ability to undergo in vitro tri-lineage differentiation. Genome-wide miRNA profiles of the iPC cells were obtained by microarray analysis and bioinformatics interrogation. Gene expression was done by real-time RT-PCR and immuno-staining; MET/EMT protein levels were determined by western blot analysis.
RESULTS: The CRC-iPC cells showed embryonic stem cell-like features and tri-lineage differentiation abilities. The spontaneously-differentiated post-iPC cells obtained were highly similar to the parental CRC cells. However, down-regulated pluripotency gene expression and failure to form teratoma indicated that the CRC-iPC cells had only attained partial pluripotency. The CRC-iPC cells shared similarities in the genome-wide miRNA expression profiles of both cancer and pluripotent embryonic stem cells. One hundred and two differentially-expressed miRNAs were identified in the CRC-iPC cells, which were predicted by bioinformatics analysis be closely involved in regulating cellular pluripotency and the expression of the MET/EMT genes, possibly via the phosphatidylinositol-3 kinases-protein kinase B (PI3K-Akt) and transforming growth factor beta (TGF-β) signaling pathways. Irregular and inconsistent expression patterns of the EMT vimentin and Snai1 and MET E-cadherin and occludin proteins were observed in the four CRC-iPC clones analyzed, which suggested an epithelial/mesenchymal hybrid phenotype in the partially reprogrammed CRC cells. MET/EMT gene expression was also generally reversed on re-differentiation, also suggesting epigenetic regulation.
CONCLUSIONS: Our data support the elite model for cancer cell-reprogramming in which only a selected subset of cancer may be fully reprogrammed; partial cancer cell reprogramming may also elicit an epithelial-mesenchymal mixed phenotype, and highlight opportunities and challenges in cancer cell-reprogramming.