The objective of the proposed bioengineering research partnership (BRP), located at the University of Florida, is to examine two key issues relevant to urolithiasis; 1) the effects of acidic biopolymers and lipid membranes on nucleation, growth and aggregation of calcium oxalate (CaOx) crystals in an artificial urinary environment; and 2) the injurious effects of a liquid-phase mineral precursor on tubular epithelial cells grown in culture. With regard to 1), many investigators have examined the promotory and inhibitory effects of acidic glycoproteins on crystal growth and aggregation. Our work differs in that a primary focus will be to investigate the relevance of a recently discovered polymer-induced liquid-precursor (PILP) process to pathological biomineralization. The PILP process generates non- equilibrium crystal morphologies which exhibit features similar to crystals found in kidney stones, such as for example, stratified spherulites. Mineral films and coatings are also deposited by the process, and repetitive depositions might lead to concentrically laminated structures, such as those commonly observed in composite stones. In addition, the interfacial aspects of this liquid-liquid phase separation process lead to a pronounced aggregation tendency of crystals. Lastly, we hypothesize that the presence of this cementatious mineral precursor in the urinary tract could influence the attachment and retention of crystals to renal epithelial cells; or the highly ionic precursor phase could cause cell injury or death, leading to the release of modulatory factors or membrane fragments, which could promote heterogeneous nucleation and/or aggregation of crystals. The proposed work consists of ten Specific Aims which fall under four topical areas: crystal-macromolecule, crystal- crystal, crystal-lipid, and crystal-cell interactions. The bioengineering techniques to be used include measurement of interparticle forces by Atomic Force Microscopy, measurement of long-range interactions between submicron CaOx particles and mimetic lipid membranes with an optical trap force transducer, and nucleation of crystals and PILP phase on mimetic lipid membranes using Langmuir monolayers. This 5-year project will enable us to assess the relevance of the PILP process to pathological calcification, as well as to perform a comparative analysis with the more traditional concepts pertaining to the role of lipids and acidic biopolymers in stone formation, and will contribute to the development of bioengineering techniques that are new to the field of stone research. The long-range clinical goal of this BRP is to provide a more effective means of diagnosis, treatment, and long-term prevention of renal calculi.
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