METHODS: Primary cultures of young, pre-senescent, and senescent fibroblast cells were incubated with γ-tocotrienol for 24 h. The expression levels of ELN, COL1A1, MMP1, CCND1, RB1, and IL6 genes were determined using the quantitative real-time polymerase chain reaction. Cell cycle profiles were determined using a FACSCalibur Flow Cytometer.
RESULTS: The cell cycle was arrested in the G(0)/G(1) phase, and the percentage of cells in S phase decreased with senescence. CCND1, RB1, MMP1, and IL6 were upregulated in senescent fibroblasts. A similar upregulation was not observed in young cells. Incubation with γ-tocotrienol decreased CCND1 and RB1 expression in senescent fibroblasts, decreased cell populations in the G(0)/G(1) phase and increased cell populations in the G(2)/M phase. γ-Tocotrienol treatment also upregulated ELN and COL1A1 and downregulated MMP1 and IL6 expression in young and senescent fibroblasts.
CONCLUSION: γ-Tocotrienol prevented cellular aging in human diploid fibroblasts, which was indicated by the modulation of the cell cycle profile and senescence-associated gene expression.
METHODS: 15wt% of zirconia (ZrO2) as well as 30, 35, and 40wt% of beta-tricalcium phosphate (β-TCP) were compounded with PA 12, followed by the fabrication of filament feedstocks using a single screw extruder. The fabricated filament feedstocks were used to print the impact specimens. The melt flow rate, tensile properties of fabricated filament feedstocks, and 3D printed impact properties of the specimens were assessed using melt flow indexer, universal testing machine, and Izod pendulum tester, respectively. The microstructure of selected filament feedstocks and broken impact specimens were analysed using a field emission scanning electron microscope and universal testing machine. Human periodontal ligament fibroblast cells (HPdLF) were used to evaluate the cytotoxicity of the materials by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid) (MTT) assay.
RESULTS: Hybrid ceramics filled PA 12 indicated sufficient flowability for FDM 3D printing. The tensile strength of hybrid ceramics filled PA 12 filament feedstocks slightly reduced as compared to unfilled PA 12. However, the tensile modulus and impact strength of hybrid ceramics filled PA 12 increased by 8%-31% and 98%-181%, respectively. A significant increase was also detected in the cell viability of the developed composites at concentrations of 12.5, 25, 50 and 100mg/ml.
SIGNIFICANCE: The newly developed hybrid ceramics filled PA 12 filament feedstock with improved properties is suitable for an FDM-based 3D printer, which enables the creation of patient-specific craniofacial implant at a lower cost to serve low-income patients.
Methods: The genes were transferred into chondrocytes at passage-1 (P1) via lipofection. The post-transfected chondrocytes (SOX9-, TERT- and SOX9/TERT) were analysed at P1, P2 and P3. The non-transfected group was used as control. The 3D culture was established using the chondrocytes seeded in a disc-shaped PLGA/fibrin and PLGA scaffolds. The resulting 3D "cells-scaffolds" constructs were analysed at week-1, -2 and -3. The histoarchitecture was evaluated using haematoxylin and eosin, alcian blue and safranin o stains. The quantitative sulphated glycosaminoglycan (sGAG) content was measured using biochemical assay. The cartilage-specific markers expression were analysed via real-time polymerase chain reaction.
Results: All monolayer cultured chondrocytes showed flattened, fibroblast-like appearance throughout passages. Proteoglycan and sGAG were not detected at the pericellular matrix region of the chondrocytes. The sGAG content assay indicated the matrix production depletion in the culture. The cartilage-specific markers, COL2A1 and ACAN, were downregulated. However, the dedifferentiation marker, COL1A1 was upregulated. In 3D "cells-scaffolds" constructs, regardless of transfection groups, chondrocytes seeded in PLGA/fibrin showed a more uniform distribution and produced denser matrix than the PLGA group especially at week-3. Both sGAG and proteoglycan were clearly visualised in the constructs, supported by the increment of sGAG content, quantitatively. Both COL2A1 and ACAN were upregulated in SOX9/TERT-PLGA and SOX9/TERT-PLGA/fibrin respectively. While, COL1A1 was downregulated in SOX9/TERT-PLGA.
Conclusion: These findings indicated that the SOX9/TERT-transfected chondrocytes incorporation into 3D scaffolds facilitates the cartilage regeneration which is viable structurally and functionally.