Date of Graduation

8-2014

Document Type

Thesis

Degree Name

Master of Science in Biomedical Engineering (MSBME)

Degree Level

Graduate

Department

Biomedical Engineering

Advisor/Mentor

Wolchok, Jeffrey C.

Committee Member

McIntosh, Matthias C.

Second Committee Member

Zaharoff, David A.

Keywords

Biochemistry; Enzyme Inhibitor; Metalloproteinase

Abstract

Up to date, a plethora of protein based materials are used as implants to stimulate tissue regeneration or fillers to alleviate tissue or organ impairment. This includes glottal insufficiency, urinary bladder incontinence and especially in cosmetic industrial to improve facial contour. Once in vivo, protein-based materials are decomposed by cell secreted matrix metalloproteinases (MMP) and lose their volume within months. By introducing MMP inhibitor (MMPI), the extent of material degradation over time may be reduced. In this dissertation, the development of cell-based assay capable of identifying MMPI candidates for protein-based implant lifetime prolongation is described. To visualize the degradation, DQ-gelatin, (heavily fluorescence labeled gelatin that emits fluorescence signal proportional to its degradation) was used to represent the implant material. This gelatin was co-cultured with NIH-3T3 in a 96-well plate supplemented with growth media under standard tissue culture condition (5% CO2, 95% humidity at 37°C). Number of seeded cells, DQ-gelatin concentration and experiment run time were varied to optimize signal-to-noise ratio whilst taking into account more than 80% of seeded cells must remain viable. With optimized parameters, 0.8 million cells cultured on cell adhesion support scaffold in presence of 50 µg/mL DQ-gelatin for 5 days, the efficacy of BB-94 and TIMP-1 as synthetic and natural MMPI candidate were investigated. Both BB-94 and TIMP-1 were tested at different concentrations according to their IC50 and the approximated amount of MMPs in tissue fluid, 20-1000 nM and 0.1-2 µg/mL respectively, to determine their most efficient dosage. BB-94 and TIMP-1 demonstrated maximum potential at 72.59±4.75 % and 60.00±27.41 % at the concentration of 1000 nM and 2 µg/mL respectively. Statistical Analysis could not detect the significant difference from varying MMP inhibitor concentration, therefore, their concluded most efficient dosage in our experiment is the lowest concentration used for testing. Because our assay generated reliable statistically distinct signals and are capable of detecting quantitative inhibitory efficacy of MMP inhibitors, we believe our novel cell-based assay is a feasible method for MMP inhibitor screening that could better represent the complex degradation process of protein-based implants in biological systems than the current conventional enzyme-based methods.

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