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Thesis Format



Doctor of Philosophy




Hutter, Jeffrey L.


Calcium oxalate crystals are found in kidney stones as either calcium oxalate monohydrate (COM) or calcium oxalate dihydrate (COD). COM crystals are the most abundant form as they are thermodynamically more stable than COD crystals under physiological conditions. Certain aspartic acid-rich molecules such as osteopontin (OPN) are known to affect stone formation by inhibiting COM and COD growth. We have studied COM {010} and COD {100} faces in the presence of OPN, poly-aspartic acid (poly-ASP) and synthetic peptides derived from OPN to investigate the inhibitor mechanism.

We observed that poly-ASP preferentially inhibits one particular direction of {010} faces on COM crystals, creating “finger-like” features, while growth continues in other directions. We find that these features appear at a threshold concentration of poly-ASP that varies with the length of the polymer. Attempts to model the threshold concentration suggest that this is due to a competition between adsorption of polymer to already-pinned steps and removal of poly-ASP by desorption and possibly coverage by advancing steps between strongly pinned steps.

Growth of {100} faces of COD crystals in the presence of synthetic peptides derived from OPN with different iso-electric points displayed varying levels of inhibition. The two principal directions of the elongated elliptical hillocks on {100} faces were inhibited to different degrees by the peptides. This preferential inhibition is most likely the result of different Ca2+ ion densities and structures of the two different step directions.

The effects of imaging on the growth itself were studied for the case of COM growth in the presence of OPN. In the presence of OPN molecules, a continuously-scanned area showed faster growth relative to a larger area scanned less frequently. This enhanced vertical growth in the continuously-scanned area was found to vary with the OPN concentration and calcium oxalate supersaturation. We modeled this effect using a modified version of the Cabrera-Vermilyea model (1958), in which steps are pinned by adsorbed impurities, resulting in a decreased propagation speed due to the resulting curvature. In our case, we assume adsorption directly to steps, rather than to terraces as in the Cabrera-Vermilyea model.

Summary for Lay Audience

Crystallization is an important field of study because many materials, both in nature and industry, are crystalline. In some cases, crystallization is undesired. One such example is the formation of kidney stones, which are comprised mainly of calcium oxalate. About 10% of the human population suffers from kidney stones at some point in their lives. We are using the atomic force microscope (AFM) to study the mechanism by which molecules found in urine prevent calcium oxalate crystallization.

Calcium oxalate crystals are found in kidney stones in two forms: calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD). Proteins and large molecules found in urine are known to intervene in the process of crystal formation. We have used poly-aspartic acid – an amino acid – and osteopontin (OPN) – a large protein found in urine – to affect the growth of COM and COD crystals synthesized in the laboratory. Using the AFM, we were able to observe crystal growth in the presence of these inhibitors and develop a model to explain the inhibition mechanism. Having a better understanding of COM and COD crystallization in the presence of proteins will lead to improved therapeutic measures to control kidney stones.

The AFM is a member of the scanning probe microscope family, which allows real-time investigation of surface properties at a wide range of length scales. During imaging, a sharp tip scans across the sample and records the topography by measuring forces between the tip and sample surface. We studied the effects of the interaction between the AFM tip and sample surface on crystallization. We find that in the absence of inhibitors, the interaction is negligible, and growth is unaltered. In the presence of OPN, we observed enhanced growth due to scanning, suggesting that the imaging process removes inhibitors from the surface. We propose a model for this effect, which has important implications for the interpretation of AFM observations of crystal growth and inhibition.

0.3mM_0.06pASP50.mov (16372 kB)
Supplementary movie Chapter 3

HighResolution_polyASP50.mov (7651 kB)
Supplementary movie Chapter 6