Vinoth Kumar S/O Jayaseelan
Email Address
denvkj@nus.edu.sg
Organizational Units
DENTISTRY
dept
DUKE-NUS MEDICAL SCHOOL
faculty
DENTISTRY
faculty
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Publication The effect of cyclic mechanical strain on the expression of adhesion-related genes by periodontal ligament cells in two-dimensional culture(2012) Saminathan, A.; Vinoth, K.J.; Wescott, D.C.; Pinkerton, M.N.; Milne, T.J.; Cao, T.; Meikle, M.C.; DENTISTRYBackground and Objective: Cell adhesion plays important roles in maintaining the structural integrity of connective tissues and sensing changes in the biomechanical environment of cells. The objective of the present investigation was to extend our understanding of the effect of cyclic mechanical strain on the expression of adhesion-related genes by human periodontal ligament cells. Material and Methods: Cultured periodontal ligament cells were subjected to a cyclic in-plane tensile deformation of 12% for 5s (0.2Hz) every 90s for 6-24h in a Flexercell FX-4000 Strain Unit. The following parameters were measured: (i)cell viability by the MTT assay; (ii)caspase-3 and -7 activity; and (iii)the expression of 84 genes encoding adhesion-related molecules using real-time RT-PCR microarrays. Results: Mechanical stress reduced the metabolic activity of deformed cells at 6h, and caspase-3 and -7 activity at 6 and 12h. Seventy-three genes were detected at critical threshold values <35. Fifteen showed a significant change in relative expression: five cell adhesion molecules (ICAM1, ITGA3, ITGA6, ITGA8 and NCAM1), three collagen α-chains (COL6A1, COL8A1 and COL11A1), four MMPs (ADAMTS1, MMP8, MMP11 and MMP15), plus CTGF, SPP1 and VTN. Four genes were upregulated (ADAMTS1, CTGF, ICAM1 and SPP1) and 11 downregulated, with the range extending from a 1.76-fold induction of SPP1 at 12h to a 2.49-fold downregulation of COL11A1 at 24h. Conclusion: The study has identified several mechanoresponsive adhesion-related genes, and shown that onset of mechanical stress was followed by a transient reduction in overall cellular activity, including the expression of two apoptosis 'executioner' caspases. © 2011 John Wiley & Sons A/S.Publication Appliance-induced osteopenia of dentoalveolar bone in the rat: Effect of reduced bone strains on serum bone markers and the multifunctional hormone leptin(2013-12) Vinoth, J.K.; Patel, K.J.; Lih, W.-S.; Seow, Y.-S.; Cao, T.; Meikle, M.C.; DENTISTRYTo understand, in greater detail, the molecular mechanisms regulating the complex relationship between mechanical strain and alveolar bone metabolism during orthodontic treatment, passive cross-arch palatal springs were bonded to the maxillary molars of 6-wk-old rats, which were killed after 4 and 8 d. Outcome measures included serum assays for markers of bone formation and resorption and for the multifunctional hormone leptin, and histomorphometry of the inter-radicular bone. The concentration of the bone-formation marker alkaline phosphatase (ALP) was significantly reduced at both time points in the appliance group, accompanied by a 50% reduction in inter-radicular bone volume; however, osteocalcin (bone Gla protein) levels remained unaffected. Bone collagen deoxypyridinoline (DPD) crosslinks increased 2.3-fold at 4 d only, indicating a transient increase in bone resorption; in contrast, the level of the osteoclast-specific marker, tartrate-resistant acid phosphatase 5b (TRACP 5b), was unchanged. Leptin levels closely paralleled ALP reductions at both time points, suggesting an important role in the mechanostat negative-feedback loop required to normalize bone mass. These data suggest that an orthodontic appliance, in addition to remodeling the periodontal ligament (PDL)-bone interface, may exert unexpected side-effects on the tooth-supporting alveolar bone, and highlights the importance of recognizing that bone strains can have negative, as well as positive, effects on bone mass. © 2013 Eur J Oral Sci.Publication Low temperature tolerance of human embryonic stem cells(2006) Boon, C.H.; Vinoth, K.J.; Liu, H.; Hande, M.P.; Cao, T.; DEAN'S OFFICE (DENTISTRY)This study investigated the effects of exposing human embryonic stem cells (hESC) to 4°C and 25°C for extended durations of 24h and 48h respectively. Cell survivability after low temperature exposure was assessed through the MTT assay. The results showed that hESC survivability after exposure to 25°C and 4°C for 24h was 77.3 ± 4.8 % and 64.4 ± 4.4 % respectively (significantly different, P < 0.05). The corresponding survival rates after 48h exposure to 25°C and 4°C was 71.0 ± 0.5 % and 69.0 ± 2.3 % respectively (not significantly different, P > 0.05). Spontaneous differentiation of hESC after low temperature exposure was assessed by morphological observations under bright-field and phase-contrast microscopy, and by immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. hESC colonies were assigned into 3 grades according to their degree of spontaneous differentiation: (1) Grade A which was completely or mostly undifferentiated, (2) Grade B which was partially differentiated, and (3) Grade C which was mostly differentiated. In all low temperature exposed groups, about 95% of colonies remain undifferentiated (Grade A), which was not significantly different (P > 0.05) from the unexposed control group maintained at 37°C. Additionally, normal karyotype was maintained in all low temperature-exposed groups, as assessed by fluorescence in situ hybridization (FISH) of metaphase spreads with telomere and centromere-specific PNA probes. Further analysis with m-FISH showed that chromosomal translocations were absent in all experimental groups. Hence, hESC possess relatively high-tolerance to extended durations of low temperature exposure, which could have useful implications for the salvage of hESC culture during infrequent occurrences of incubator break-down and power failure. ©2006 Ivyspring International Publisher. All rights reserved.Publication Comparison of the response of human embryonic stem cells and their differentiated progenies to oxidative stress(2009-08-01) George, S.; Heng, B.C.; Vinoth, K.J.; Kishen, A.; Cao, T.; ORAL AND MAXILLOFACIAL SURGERY; RESTORATIVE DENTISTRYObjective: To investigate effects of oxidative stress on human embryonic stem cells (hESC) and their spontaneously differentiated fibroblastic progenies (at passage 5). Background Data: In ischemic disease models, high levels of free radicals and reactive oxygen species are critical factors in decreasing survivability and engraftment of transplanted/transfused cells. Hence, it is imperative to characterize response of hESC and their differentiated progenies to oxidative stress. Methods: Oxidative stress was induced either by (i) varying durations (0 to 40 min) of photodynamic treatment (diode laser, 664 nm, 30 mW) in the presence of 10 μM methylene blue as a photosensitizer, or by (ii) exposure to varying concentrations of hydrogen peroxide (0 to 50 μM) for a fixed duration of 40 min. Additionally, the effects of heat shock and mild oxidative stress preconditioning on oxidative stress response was also investigated. Results: Consistently higher survivability (MTT assay) of hESC was observed compared to their differentiated fibroblastic progenies, upon exposure to equivalent levels of oxidative stress. Further experiments demonstrated that heat-shock pretreatment (42°C for 90 min) did not enhance the resistance of either hESC or their differentiated progenies to oxidative stress (photodynamic treatment), but in fact had a slightly detrimental effect on their survivability upon subsequent exposure to oxidative stress. Similarly, preconditioning of both undifferentiated hESC and their differentiated progenies with low levels of oxidative stress also did not enhance cellular survivability upon subsequent exposure to much higher levels of oxidative stress induced by photodynamic treatment. Conclusions: Undifferentiated hESC are intrinsically more resistant to oxidative stress compared to their spontaneously differentiated fibroblastic progenies. © 2009, Mary Ann Liebert, Inc.Publication Engineering the periodontal ligament in hyaluronan-gelatin-type i collagen constructs: Upregulation of apoptosis and alterations in gene expression by cyclic compressive strain(Mary Ann Liebert Inc., 2015) Saminathan, Aarthi; Sriram, G; Vinoth, Jayasaleen Kumar Umar; Cao, Tong; Meikle, Murray C; RESTORATIVE DENTISTRYPublication Human embryonic stem cells may display higher resistance to genotoxic stress as compared to primary explanted somatic cells(2008-06-01) Vinoth, K.J.; Heng, B.C.; Poonepalli, A.; Banerjee, B.; Balakrishnan, L.; Lu, K.; Hande, M.P.; Cao, T.; ORAL AND MAXILLOFACIAL SURGERY; PHYSIOLOGYThe use of human embryonic stem (hES) cells in genotoxicity screening can potentially overcome the deficiencies associated with using immortalized cell lines, primary explanted somatic cells, and live animal models. Hence this study sought to compare the responses of hES cells and primary explanted somatic cells (IMR-90 cells, human fetal lung fibroblasts) to genotoxic stress, to evaluate whether hES cells can accurately reflect the normal physiology of human somatic cells. The effects of mitomycin C (MMC) on the chromosomal stability of hESC and IMR-90 was assayed and compared by fluorescence in situ hybridization (FISH) with telomere-specific peptide nucleic acid and multicolor (m) FISH techniques. The results showed that, the percentage of aberrant cells increased from 6% in the untreated control to 57.5% at the higher dose of 0.06 μg/ml MMC (9.6-fold increase) group in the case of IMR-90 cells, whereas hES cells displayed a corresponding increase from 6% to 28% (4.6-fold increase). Telomere FISH ascertained that the main types of damage induced by MMC are chromosomal breaks and the loss of telomeric signals. No fusions were observed in all samples analyzed. This was further confirmed by mFISH, which showed that fusions and translocations were not the type of aberration induced by MMC, with no such aberrations being observed in all samples analyzed. Hence, hES cells of the H1 line are apparently more resistant to MMC-induced DNA damage, as compared to the IMR-90 cells. These results highlight possible intrinsic differences in response to damaging agents between hES cells and normal somatic cells. © Mary Ann Liebert, Inc. 2008.Publication Engineering three-dimensional constructs of the periodontal ligament in hyaluronan-gelatin hydrogel films and a mechanically active environment(2013-12) Saminathan, A.; Vinoth, K.J.; Low, H.H.; Cao, T.; Meikle, M.C.; DENTISTRYBackground and Objective: Periodontal ligament (PDL) cells in stationary two-dimensional culture systems are in a double default state. Our aim therefore was to engineer and characterize three-dimensional constructs, by seeding PDL cells into hyaluronan-gelatin hydrogel films (80-100 μm) in a format capable of being mechanically deformed. Material and Methods: Human PDL constructs were cultured with and without connective tissue growth factor (CTGF) and fibroblast growth factor (FGF)-2 in (i) stationary cultures, and (ii) mechanically active cultures subjected to cyclic strains of 12% at 0.2 Hz each min, 6 h/d, in a Flexercell FX-4000 Strain Unit. The following parameters were measured: cell number and viability by laser scanning confocal microscopy; cell proliferation with the MTS assay; the expression of a panel of 18 genes using real-time RT-PCR; matrix metalloproteinases (MMPs) 1-3, TIMP-1, CTGF and FGF-2 protein levels in supernatants from mechanically activated cultures with Enzyme-linked immunosorbent assays. Constructs from stationary cultures were also examined by scanning electron microscopy and immunostained for actin and vinculin. Results: Although initially randomly distributed, the cells became organized into a bilayer by day 7; apoptotic cells remained constant at approximately 5% of the total. CTGF/FGF-2 stimulated cell proliferation in stationary cultures, but relative quantity values suggested modest effects on gene expression. Two transcription factors (RUNX2 and PPARG), two collagens (COL1A1, COL3A1), four MMPs (MMP-1-3, TIMP-1), TGFB1, RANKL, OPG and P4HB were detected by gel electrophoresis and Ct values < 35. In mechanically active cultures, with the exception of P4HB, TGFB1 and RANKL, each was upregulated at some point in the time scale, as was the synthesis of MMPs and TIMP-1. SOX9, MYOD, SP7, BMP2, BGLAP or COL2A1 were not detected in either stationary or mechanically active cultures. Conclusion: Three-dimensional tissue constructs provide additional complexity to monolayer culture systems, and suggest some of the assumptions regarding cell growth, differentiation and matrix turnover based on two-dimensional cultures may not apply to cells in three-dimensional matrices. Primarily developed as a transitional in vitro model for studying cell-cell and cell-matrix interactions in tooth support, the system is also suitable for investigating the pathogenesis of periodontal diseases, and importantly from the clinical point of view, in a mechanically active environment. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.