Crystalline Architecture of PCNL-Derived Fragments. The high- and super-resolution optical, laser, x-ray microscopy, and Raman spectroscopy conducted in the present study indicate that the original PCNL-derived fragments have a crystalline architecture that is consistent with those previously observed in CaOx kidney stones38–42. Following the approaches presented in these previous studies, the term “crystalline architecture” is herein used to refer to stone structure (crystal size, shape, intergrown morphologies), stratigraphy (crystal and organic matter layering), diagenetic phase transitions (post-depositional dissolution and recrystallization), and paragenesis (historical sequence of formational events). Therefore, the approaches and terminology of GeoBioMed38–42 have been directly adopted and applied in the present study. RL microscopy and x-ray micro-computed tomography (micro-CT) indicate that the original PCNL-derived fragments are irregular 4-12mm-diameter crystalline aggregates that exhibit SPL probe impressions and breakage patterns (Figs. 2A, B, SFigs. 1, 2). Each PCNL-derived and SPL-derived fragment in the present study is primarily a high-density calcium oxalate monohydrate (COM; Whewellite; CaC2O4 • H2O) cortex (COMC) composed of high-frequency alternations of organic-matter-rich nanolayers (peptides, proteins, and other cellular molecules) and COM mineral-rich nanolayers (Figs. 2B-F)47–50. All six of the PCNL-derived fragments were impacted to varying degrees by ultrasonic wave energy and intermittent shockwaves from use of an SPL probe. This includes breakage surfaces and notch formation on the exterior of some PCNL-derived fragments (Fig. 2A, B; SFigs. 1, 2), as well as rare occurrences of fine fracturing within COMC that are exclusively observed adjacent to the SPL notches (Fig. 3A).
Individual crystal faces (sector zones) within COMC are common and form as the result of disequilibrium precipitation, during which ions and organic matter are differentially incorporated on age-equivalent crystal growth faces (Figs. 3B, C, H)38–42, 51. Free-floating COM crystals (COMFF) are entombed, either individually or in clusters, on growing COMC concentric surfaces that seed the growth of radiating crystal bundles (COMB) that either truncate or redirect the growth of sector zones (Fig. 3C, F). Additionally, COMC commonly exhibits repeated in vivo events of crystal fracturing (cracking) and faulting (displacement across the fractures) to form laths with discontinuous layering (Fig. 3D, E). COMC also exhibits Ångstrom-scale dissolution and recrystallization (mimetic replacement, COMM) (Fig. 3E, G). The outermost margins of the PCNL-derived fragments are composed of calcium oxalate dihydrate (COD; Weddellite; CaC2O4 • 2H2O) crystals that were encrusted by COMC, experienced COD dissolution (CODD), and were partially filled with replacement COM (COMR) (Fig. 3H).
Fracture Geometries of SWL-Derived Particles. The crystalline architecture of the SWL-derived particles, as revealed by CAF and SRAF microscopy, provides a high-fidelity crystalline framework within which to characterize SWL-induced fracture geometries (Figs. 5, 6). Importantly, previous studies using scanning electron microscopy (SEM) on SWL-derived particles provided valuable information on external and internal CaOx crystal growth and fracturing morphologies47. However, the SEM tool is inherently limited compared to the detailed information on crystalline architecture that is provided by the CAF and SRAF microscopy38–42. SWL-induced fractures were observed in the present study to propagate in a variety of perpendicular, oblique, and concentric-parallel trajectories relative to the nanolayered crystalline architecture of the original PCNL-derived fragments (Figs. 5, 6; Supplementary Figs. 6–10). These observations have been integrated to establish a systematic nomenclature of fracture morphologies for SWL-derived particles that include: (1) rectangular particles with fracture margins formed perpendicular to the COMC concentric nanolayer stratigraphy (Figs. 5A), parallel to radiating sector zones (Figs. 6B, D), and cross-cutting COM mimetic replacement (COMM) crystals (Figs. 6G)47,52−54; (2) pointed particles that form from fractures perpendicular, oblique, and parallel to the COMC concentric nanolayer stratigraphy (Figs. 5B-H), which merge at angles of 60-120o to form arrowhead-like tips (Fig. 5B-H, 6A, D) that are similar in appearance to Hertzian-cone conchoidal fractures caused by surface radial tensile stress in silicates and metals54–56; (3) concentrically spalled particles created by inter- and/or intracrystalline fracturing along concentric organic matter-rich COMC nanolayers via cohesive-zone brittle microfracture spallation (Fig. 5F, G)35,52,53; and (4) irregular surface particles created by coalescing microfractures that cause irreversible fatigue damage and little plastic deformation during the application of cyclic tensile stress52–54, 57,58 at the margins of original COM crystal bundles that initially grow from COM free floating (COMFF) crystals after landing on growing COMC surfaces (Fig. 6B-H). These CAF- and SRAF-defined fracture categories enhance and improve upon observations completed in earlier studies exclusively using SEM47.
SWL-Derived Particle Size Distributions. During the course of the 72-hour H2O porosity saturation of the original PCNL-derived fragments59, the weight of both 106F2 and 106F4 increased approximately 10% (Fig. 7A). The larger PCNL-derived fragment 106F2 was exposed to six sequential ex vivo 100-shock SWL treatments with a consistent Level 3 intensity at a rate of 90 shocks per minute (Fig. 7). The smaller PCNL-derived fragment 106F4 was subjected to only two 100-shock treatments at the same shockwave intensity and rate (Fig. 7). The SWL-derived particles produced by each 100-shock treatment were sieved with the 2mm-mesh net in the calibration container of the lithotripter, weighed, and imaged (SFig. 3; STab. 1). The weight percent of SWL-derived particles captured in the 2mm-mesh net was observed to decrease from approximately 50–85% after the first 100-shock treatment and eventually reach a 0% decrease after progressive treatments. This is consistent with previous observations of weight loss with increasing shock wave treatments48. The SWL-derived particle fractions small enough to pass through the 2mm-mesh net were trapped on a 0.47µm filter, imaged, quantified, and classified according to the Wentworth grain size scale (Figs. 7B, C)46,60,61. These analyses indicate that the SWL-derived particles from both 106F2 and 106F4 that passed through the 2mm-mesh net are in size classes that range from very fine silt (4.998µm) to very fine pebbles (2.926mm) (Figs. 7B, C; STab. 2). The SWL-derived particles from both 106F2 and 106F4 exhibit right-skewed (positive) normal size frequency distributions (after Garcia, 2008) with size class modes of fine sand (Figs. 7B, C)62. While small clay-sized (< 4µm-diameter) SWL-derived particles were not detected in the present study despite the use of 0.47µm filters (Fig. 7), it is possible that some unknown amount of this fine grain size class may have been lost while decanting between sequential stages of SWL experimentation.
Implications
Experimental results in the present study indicate that each SWL treatment of CaOx PCNL-derived fragments produced 5µm- to 2mm-diameter SWL-derived particles that range on the Wentworth grain size scale from very fine silts through sands and very fine pebbles (mode = 125–250µm-diameter fine sands; Fig. 7). The majority of these SWL-derived particles are significantly below the 3-4mm-diameter detection limit of clinical non-contrast computer tomography scans20,63,64. As discussed here, results from the present study, as well as inference made from previous studies, indicate that these small SWL-derived particles are likely to increase the chance for post-SWL treatment stone recurrence65–68. Therefore, the common clinical practice of using negative computed tomography screens to identify and declare patients as “kidney stone free” with “clinically insignificant residual fragments”17,18,24,29,67,69−74, should be fundamentally reevaluated.
In natural environmental waters, the primary factors controlling the rate and extent of mineral precipitation and dissolution reactions include mineralogical stability, fluid saturation state, and the amount of surface area available per unit mass of mineral grains43–45, 75. Furthermore, fine crystalline structure (crystalline architecture) and crystal aggregate grain size play influential roles in both the primary (original) crystallization and secondary physical, chemical, and biological alteration (diagenesis)42,76,77 of the kidney stone deposits. The increase in total surface area per gram, as both grain size and volume decrease, can be approximated as:
(1) \(\text{A} = \frac{\lambda }{\rho *V}=\left(\frac{\beta }{\rho }\right){r}^{-1}\) (Walter and Morse, 1984)45 where:
A = specific surface area (SSAp) of each particle per unit mass (g)
\(\lambda\) = particle surface area (also known as Ap) per unit mass (g), which is approximated as a sphere using a spherical radius (r) derived from grain size: Ap= \(4\pi {r}^{2}\)
V = volume of each particle (also known as Vp) as a function of equivalent spherical radius (r): Vp=\(\frac{4}{3}\pi {r}^{3}\)
\(\rho\) = bulk density of material (COM)78 = 2.12 g/cm3
\(\beta\) = shape factor for sphere or cube, which is 3 based on surface to volume ratios and assumes geometrically equidimensional particles
These estimates permit the size frequency distributions of the SWL-derived particles, as measured in the present study (Fig. 7), to be used to quantitatively estimate the impact of effective reactive surface area on post-SWL recurrence (Fig. 8). This simple approximation dramatically illustrates that a decrease in SWL-derived particle size is accompanied by an exponential increase in the total surface area available for ensuing sequential events of crystal growth and/or dissolution (diagenetic phase transitions)42,76,77 (Fig. 8). Previous experimental studies of fine-grained marine carbonate skeletal components (coral, echinoid, and algal skeletons), suggest that textural microstructure (referred to as crystalline architecture in the present study38,42), surface roughness, and grain size combine to influence total reactive surface area45,79. However, the dense relatively non-porous interior crystalline architecture of the CaOx kidney stone grains observed in the present study (Figs. 2–6) suggest that the total exterior surface area is a reasonable first-order approximation of reactive surface area in both PCNL-derived fragments and SWL-derived particles (Fig. 8).
These evaluations permit the effect of SWL-derived particle size and associated surface area to be evaluated in the context of surface- and transport-control chemical reactivity regimes43,44. As a result, SWL-derived particles that are below the size limit of detection by clinical non-contrast computed tomography scanners (Fig. 8) could still have an extremely high potential to serve as a nidus for crystal growth and stone recurrence80. In addition, these fine SWL-derived particles (Figs. 7, 8) would have a high affinity for adherence to extracellular mucus linings on renal tissues such as glycosaminoglycan hyaluronans81. This would serve to reduce the effectiveness of post-SWL irrigation18, as well decrease spontaneous passage under variable infundibulopelvic angles16. This also further supports the suggestion by Brain et al. (2021) that smaller SWL-derived particle size fractions should not be discounted when evaluating a patient’s risk for stone recurrence24. Therefore, as noted earlier, terms such as “clinically insignificant fragments” should be avoided moving forward.
The results of the present study have important implications for all applications of shock wave lithotripsy, which has been in clinical use since the 1980s and has spurred extensive controlled experimentation to better understand mechanisms of fragmentation, improve efficiency of the treatment, and reduce post-SWL stone recurrence82. The majority of this work has been completed on artificial composites (begostones or phantom stones) suspended in water61,83−90. Furthermore, other studies have shown that COM is more resistant to SWL-generated fragmentation than is COD48. However, this previous work used “physical properties” and did not consider the influence of original CaOx kidney stone crystalline architecture, size frequency distributions (as opposed to acoustic comminution that is based on grain averages), fracture geometries, and the influence of reactive surface area on recurrence.
Regarding fracture patterns, the original CAF- and SRAF-defined crystalline architecture of each of the PCNL-derived fragments that were subjected to ex vivo SWL treatment in the present study, creates a geometric framework within which SWL-induced fracture propagation patterns can be identified. These crystalline fabrics range from large hundreds of mm-diameter single crystals to tens of nm-diameter nanocrystals that form concentric nanolayers91. Further, these SWL-derived particles exhibit rectangular, pointed, concentrically spalled, and irregular particle geometries. Similar to arrow heads produced from chert (flint)92, these irregular fragments have razor sharp edges that can pierce, land on to, cut into, and damage sensitive tissues throughout the kidney, bladder, and urethra, which could further contribute to ensuing stone recurrence28,42.
In conclusion, the influence of SWL-derived particle size frequency distributions, fracture patterns, and reactive surface area now opens the way for many new directions of experimental research into the development of therapies meant to reduce post-SWL kidney stone recurrence. For example, factors that influence diagenetic phase transitions and reactive surface area for each Wentworth size class of SWL-derived particles can now be tested using controlled experimentation of urine-stone-microbe-renal tissue interactions within microfluidic testbeds such as the GeoBioCell38–42, 77. Of specific utility is the small size of SWL-derived particles, which fit easily into GeoBioCell microfluidic flow chambers and channels, and therefore permit real-time quantitative tracking of diagenetic phase transitions controlled by a wide variety of interacting physical, chemical, and biological processes41,91,93. Potential examples include39,42: (1) real-time systematic analysis of the effect of microbiome- and host human-derived protein catalysis by promotional and inhibitory macromolecules (i.e., anionic proteins and glycosaminoglycans) on crystal aggregation and cell attachment10,11,31,42,49,94; (2) controls on crystal growth morphology, mineralogy, chemistry, aggregation, layering, dissolution, and recrystallization; and (3) the influence of organic acids such as citric, oxalic and formic acids excreted by specific bacteria and Ffungi entombed in CaOx kidney stones on stone dissolution. Furthermore, new GeoBioCell microfluidic testbeds themselves could be designed and constructed with 3D chamber printing, silica etching, and renal cell 3D printing of microfluidic flow channels in multiple configurations to simulate the actual renal hydrology, anatomy, and physiology41,42. In addition, effects of specific plant extracts and anti-oxidants such as hydroxy citrate can now be systematically tested with respect to post-SWL diagenetic phase transitions, cellular control, urine supersaturation, and flow rate, and their effect on CaOx kidney stone recurrence80,95−99.