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dc.contributor.advisorLove, Robert
dc.contributor.advisorDias, George
dc.contributor.authorSajeev, Jeeson
dc.date.available2014-11-09T20:36:31Z
dc.date.copyright2014
dc.identifier.citationSajeev, J. (2014). Influence of keratin preparations on cementoblast OCCM-30 and fibroblast L929 cells (Thesis, Doctor of Clinical Dentistry). University of Otago. Retrieved from http://hdl.handle.net/10523/5124en
dc.identifier.urihttp://hdl.handle.net/10523/5124
dc.description.abstractAim: Keratin preparations have been shown to have bioactive effects on various cells including osteoblasts and neurons and offer the potential as a biomaterial in tissue regeneration. The aim of this study was to assess the in vitro effect of keratin preparations on the viability and proliferation of cementoblast OCCM-30 and fibroblast L929 cells and on the mineralisation capability of the OCCM-30 cells. Method: Cementoblast OCCM-30 cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% Fetal bovine serum (FBS) and 1% antibiotic-antimycotic. The fibroblast L929 cells were grown in minimum essential media (MEM) with 10% FBS and 1% antibiotic-antimycotic. The cells of L929 and OCCM-30 were assessed for viability in growth media supplemented with 10 mg/ml, 1 mg/ml and 0.1 mg/ml of keratin with a LIVE/DEAD assay™ Kit using confocal laser scanning microscope. Cell proliferation was tested with similar keratin concentrations using an alamarBlue® proliferation assay at 0, 24, 48 and 72 h. The proliferation assay showed that 10 mg/ml was toxic for the cementoblast cell, hence the concentration was ruled out for further testing by mineralisation assays. Alkaline phosphatase assay (SensoLyte® pNPP alkaline phosphatase assay kit) was performed for the OCCM-30 cells with keratin concentrations of 1 mg/ml, 0.1 mg/ml and the control (nil keratin) at 3, 6, and 10 d. Alizarin red assay was performed at 14 d for 1 mg/ml, 0.1 mg/ml and the control group (nil keratin). Results: The viability assay showed concentrations at 1 mg/ml or greater of keratin proved toxic to the fibroblast L929 cells. However, OCCM-30 cells showed similar viability to the control group with 1 mg/ml of keratin solution. Keratin concentration >1 mg/ml showed a toxic effect on OCCM-30 cells. The proliferation assay for L929 cells showed 10 mg/ml and 1 mg/ml were toxic compared to 0.1 mg/ml and 0.01 mg/ml keratin groups (P < 0.0001) in that they were unable to proliferate in the presence of 10 mg/ml and 1 mg/ml keratin. Cell proliferation of the control group was significantly higher than 10 mg/ml (P < 0.0001) and 1 mg/ml (P < 0.0001) while cell proliferation in the presence of 0.1 mg/ml and 0.01 mg/ml keratin was not significantly different (P > 0.05) to the control group. The proliferation assay for cementoblast OCCM-30 cells showed keratin concentration at 10 mg/ml prevented its proliferation and was significantly lower than all other keratin groups tested (P < 0.0001). Keratin concentrations at 1 mg/ml and lower did not hinder the proliferation of the cementoblast OCCM-30 cells with similar proliferation as the control (P > 0.05). The alkaline phosphatase (ALP) activity of cementoblast OCCM-30 cells exposed to keratin 1 mg/ml showed no signs of ALP activity at any of the time points tested. There was significantly higher OCCM-30 cell ALP activity with the control and 0.1 mg/ml groups compared with the 1 mg/ml group at 6 d and 12 d (P < 0.0001). The alizarin red assay was consistent with the ALP activity of the OCCM-30 cells with significantly lower levels of calcium staining observed between 1 mg/ml of keratin concentration and each of the remaining groups at day 14 (P < 0.0001), but there were no significant differences between 0.1 mg/ml and the control group (P > 0.05). Conclusion: Keratin preparations were shown to allow normal cell proliferation of fibroblast L929 and cementoblast OCCM-30 cells and normal mineralisation of the cementoblasts. The results show promise that keratin could be developed as an endodontic regeneration scaffold material.
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectOCCM-30
dc.subjectL929
dc.subjectkeratin
dc.titleInfluence of keratin preparations on cementoblast OCCM-30 and fibroblast L929 cells
dc.typeThesis
dc.date.updated2014-11-08T04:11:25Z
dc.language.rfc3066en
thesis.degree.disciplineDepartment of Oral Rehabilitation
thesis.degree.nameDoctor of Clinical Dentistry
thesis.degree.grantorUniversity of Otago
thesis.degree.levelDoctoral
otago.interloanyes
otago.openaccessAbstract Only
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