In search of new strategies to prevent, cure and alleviate human kidney stone pathogenesis, recent cross-disciplinary studies have shown that calcium stones form as a result of repeating natural events of in vivo crystallization, dissolution, recrystallization, fracturing, faulting and microbiome interactions.^1–4 Importantly, these dynamic processes are governed by the same mechanisms of biological influence on mineral growth (biomineralization) and repeated post-formational physical, chemical and biological alteration (diagenetic phase transitions) that have taken place in natural Earth environments for billions of years (universal biomineralization).¹ Several studies over the past 80 years have observed kidney stone dissolution and recrystallization using brightfield and polarization microscopy (petrography), x-ray diffractometry, x-radiography and scanning electron microscopy.^5–10 Jensen showed that “calcium oxalate in urinary calculi is primarily deposited as dihydrate and is subsequently transformed to monohydrate by partial loss of water.”⁵ Subsequent studies referred to this process as crystal conversion, recrystallization, transition, modification or pseudomorphism. Much later, in vitro stone exposure to solvents resulted in “postulation of a dehydration process of primary Weddellite (calcium oxalate dihydrate, COD) crystals via dissolution and recrystallization to Whewellite (calcium oxalate monohydrate, COM).”⁹ However, during this time period the advanced multimodal fluorescence microscopy techniques available today were either yet to be invented or simply not envisioned to be applicable to kidney stone research. In addition, these earlier studies lacked the essential unifying concept that sequential diagenetic phase transitions are directly linked to thermodynamic potential of the physical, chemical and biological human host-urine-mineral-microbiome system as a whole.¹ In any case, it is clear that these previous studies were not employed to recognize, evaluate, quantify or effectively utilize 1) the 3-dimensional extent and distribution of in vivo dissolution and recrystallization throughout the entire volume of a given stone; 2) the possibility that multiple repeated events of partial dissolution and recrystallization have occurred throughout a stone’s formational history; and 3) the inherent potential that naturally occurring in vivo dissolution and recrystallization could be strategically exploited to guide the testing and development of new clinical stone therapies.

The full extent of naturally repeating dissolution and recrystallization during natural in vivo stone formation was not discovered and tracked until recently. This was only made possible when basic conceptual and technological approaches from geology, biology and medicine (GeoBioMed²) were merged with quantum advances in superresolution autofluorescence microscopy (SRAF; 140 nm-resolution) and x-ray microcomputed tomography (microCT, ∼3 μm-resolution).¹ Especially important in this regard is the SRAF excitation of organic matter (eg human and microbiome biomolecules, cell debris and extracellular polymeric substances) entombed within stone crystals, which records alternating sequences of organic matter-rich and mineral-rich nanolayers (figs. 1–3). This crystalline stratigraphy, seen in all mineralogical forms of calcium stones (fig. 4), creates a spatial and temporal history of calculus growth called a paragenetic sequence.¹ This contextual and historical framework has enabled identification and chronological reconstruction of repeated events of crystallization, dissolution, recrystallization, fracturing, faulting and microbiome entombment during stone formation.^1–4 This work has also documented that stone recrystallization, in its own right, is a complex process. One mode of recrystallization occurs as nm, μm, mm and at times cm-scale bulk fabric destructive dissolution to form irregular voids and crystal molds that are later infilled with mineral precipitation. A second mode involves Ångstrom-scale events of recrystallization that preserve, within the replacement crystals, the original intricate nm-scale growth fabrics of the crystals being replaced (mimetic replacement).

Figure 1. Structure and composition of human kidney stone fragment composed of hydroxyapatite (HAP), COD, and COM. Reflected light (A) and 3-dimensional volume rendered x-ray microCT image (B) of stone fragment in A; scale bars for A and B are 1 mm. Virtual microCT cross-section (C) taken along white line in B; scale bar is 600 mm. Circular polarization (360°) image (D). Paired SRAF (140 nm-resolution) image (E). Line of section from which petrographic thin section was prepared is shown in B. Scale bars for D and E are 500 mm. Original COD crystals were dissolved, leaving crystal molds (red SRAF epoxy) that are partially filled with fractured and faulted COM crystals. These are encrusted by nano-layered COM that in turn is truncated and encrusted by another nano-layered COM cortex. Modified and used by permission.¹

Figure 2. Naturally repeated events of in vivo dissolution, recrystallization, fracturing and faulting within human kidney stone fragment shown in fig. 1. Areas from which each SRAF (140 nm-resolution) image is enlarged are shown in fig. 1, E. Epoxy within pore space exhibits red autofluorescence. A through C, fractured and faulted COM (dark blue to green and brown SRAF) that grew in COD dissolved crystal molds (red SRAF) and were later encrusted with nano-layered COM (green, brown, blue and yellow SRAF). Scale bar for A is 100 mm; B and C is 10 mm. D, enlargement of area shown in fig. 1, E, which has been rotated 90° clockwise. Scale bar for D is 100 mm. Entire paragenetic sequence is exhibited, with crystal molds of original COD crystals partially filled with fractured and faulted COM and overlaid by nano-layered COM, which in turn was truncated and encrusted by another nano-layered COM cortex. Modified and used by permission.¹

Figure 3. Structure and composition of a human kidney stone fragment composed of amorphous calcium phosphate (ACP), HAP and COD. A, SRAF (140 nm-resolution) image of concentrically layered ACP-HAP stone fragment. Scale bar for A is 750 mm. B and C, initial stages of formation of planar concentrically layered COD crystals are diagenetic phase transitions from amorphous light yellow to white SRAF ACP material to HAP spherules that coalesce to form euhedral COD crystals with well-developed sector zones. Scale bar for B is 50 mm; C is 20 mm. Images are displayed with best-fit intensity profiles and/or after gamma correction of 0.45. Modified and used by permission.¹

Figure 4. X-ray microCT (∼3–4 mm-resolution) images of human kidney stones illustrating extensive repeated in vivo events of dissolution and recrystallization. A and B, stone composed of original apatite and COD, which are partially dissolved and replaced by COM. C and D, stone composed of original COD, which is partially dissolved and replaced by COM. E and F, stone composed of predominantly original COD, which is partially dissolved and replaced by COM. G and H, stone composed of original calcium carbonate (CaCO³) and COD and partially replaced by COM. Virtual cross-sections shown in B, D, F and H were taken from corresponding line of section of whole stone fragment rendered in 3 dimensions shown in A, C, E and G. Jet black inside each image is background and dissolution/recrystallization pore space left with crystal molds after dissolution, while individual mineral phases are represented by variable shades of gray indicative of differences in density. Scale bars for A, B, C, D, E, G and H are 1 mm. Scale bar for F is 400 mm.

GeoBioMed has yielded unexpected new insights and hypotheses into the process of urolithiasis as recorded by the mineralogy, crystalline structure, stratigraphy and diagenetic alteration of calcium stones and their fragments.^1–4 This has led to development of a new classification scheme within the context of a stone’s paragenetic sequence. Importantly, quantification of SRAF and microCT images reveals that on average more than 50% of the total volume of calcium stones have undergone repeated naturally occurring in vivo dissolution and recrystallization (figs. 1–4).¹ It is especially striking to contrast the large scale bulk stone impact of natural dissolution and recrystallization (figs. 1–4) with the minute nm to μm-scale extent of dissolution observed on single COD crystal faces that have been induced with supplements such as citrate and hydroxycitrate.¹¹ GeoBioMed has also identified that tens to hundreds nm-diameter calcium phosphate and hydroxyapatite spherules coalesce and undergo diagenetic phase transitions to form concentrically well-formed planar (euhedral) and sector zoned COD crystals under disequilibrium conditions (fig. 3).¹ Furthermore, measurements of nanolayered COD and COM intracrystalline stratigraphy indicate that stone growth rates are lower than those observed in other natural and engineered environments of biomineralization. These analyses have also shown that layering frequencies in kidney stones are orders of magnitude higher than in these other environments. Therefore, far from being inert crystalline aggregates, calcium stones are dynamic bioreactors that experience a variety of growth rates and layering frequencies throughout their formational history, which will provide clues in future studies about the processes that form them.¹

GeoBioMed has identified multiple basic processes and mechanisms regarding stone growth that can now be used to direct and inform the next generations of stone diagnostics and treatments. In specific, GeoBioMed findings lay the groundwork for in vitro and in vivo experimentation to identify previously unexplored targets for clinical urolithiasis therapies, as well as to study other forms of biomineralization within the human body.¹ Future testing will include in vitro experiments of stone growth within microfluidic testbeds (GeoBioCell).¹ This will permit nm, μm, mm and cm-scale manipulation of gradients and fluctuations in urine solution chemistry, pH, flow hydraulics and human and microbiome biomolecules, as well as manipulation of microbiome phylogenetic, morphological and metabolic diversity. Some examples of the immense range of future experimentation targets include disruption of diagenetic phase transitions, dissolving stone fragments in vivo, establishment of rapid clinical stone diagnostics, reduction of recurrence rate from stone fragments of variable sizes, testing of biomolecular inhibitors and promoters, harnessing the potential for strategic nano-scale water storage and release, and even diagnosis and control of stone growth in astronauts during space travel. A documentary highlighting the development and application of GeoBioMed approaches to urolithiasis is presented as a Mayo Clinic Heritage Film entitled “A World in a Grain of Sand: New Developments in Kidney Stones” (https://history.mayoclinic.org/books-films/heritage-films.php).