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DOE OSTI2021

In Vivo Entombment of Bacteria and Fungi during Calcium Oxalate, Brushite, and Struvite Urolithiasis

Human kidney stones form via repeated events of mineral precipitation, partial dissolution, and reprecipitation, which are directly analogous to similar processes in other natural and manmade environments, where resident microbiomes strongly influence biomineralization. High-resolution microscopy and high-fidelity metagenomic (microscopy-to-omics) analyses, applicable to all forms of biomineralization, have been applied to assemble definitive evidence of in vivo microbiome entombment during urolithiasis. Stone fragments were collected from a randomly chosen cohort of 20 patients using standard percutaneous nephrolithotomy (PCNL). Fourier transform infrared (FTIR) spectroscopy indicated that 18 of these patients were calcium oxalate (CaOx) stone formers, whereas one patient formed each formed brushite and struvite stones. This apportionment is consistent with global stone mineralogy distributions. Stone fragments from seven of these 20 patients (five CaOx, one brushite, and one struvite) were thin sectioned and analyzed using brightfield (BF), polarization (POL), confocal, super-resolution autofluorescence (SRAF), and Raman techniques. DNA from remaining fragments, grouped according to each of the 20 patients, were analyzed with amplicon sequencing of 16S rRNA gene sequences (V1–V3, V3–V5) and internal transcribed spacer (ITS1, ITS2) regions. Bulk-entombed DNA was sequenced from stone fragments in 11 of the 18 patients who formed CaOx stones, and the patients who formed brushite and struvite stones. These analyses confirmed the presence of an entombed low-diversity community of bacteria and fungi, including Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Aspergillus niger. Bacterial cells approximately 1 μm in diameter were also optically observed to be entombed and well preserved in amorphous hydroxyapatite spherules and fans of needle-like crystals of brushite and struvite. In conclusion, these results indicate a microbiome is entombed during in vivo CaOx stone formation. Similar processes are implied for brushite and struvite stones. This evidence lays the groundwork for future in vitro and in vivo experimentation to determine how the microbiome may actively and/or passively influence kidney stone biomineralization.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Understanding the setting and hardening process of wollastonite-based brushite cement. Part 2: Influence of the boron and aluminum concentrations in the mixing solution

The mixing solution of wollastonite-based brushite cement is a phosphoric acid solution containing metallic cations and borax. This work complements a previous study devoted to the influence of the H{sub 3}PO{sub 4} concentration, Ca/P and liquid-to-solid (l/s) ratios on the setting and hardening process of the binder by providing new insight into the role of aluminum and boron. Boron retards the setting and decreases the heat released during the process. It also contributes to reduce the macroporosity of the hardened material but yields to poor compressive strength. With aluminum in the mixing solution, the mechanical properties are greatly improved thanks to the precipitation of an amorphous aluminophosphate which increases the density of the cement matrix. But aluminum alone leads to fast setting. A joint addition of boron and aluminum to the mixing solution makes it possible to get a material with optimized properties both in fresh and hardened states.

36 MATERIALS SCIENCE↗

DOE OSTI2021

Impacts of Initial Ca/P on Amorphous Calcium Phosphate

Amorphous calcium phosphate (ACP) is a metastable phase in the crystallization pathway of calcium phosphates. Understanding its chemical and structural properties could provide critical insights into biomineralization mechanisms. Of particular interest is the impact of the initial Ca/P on the structure of ACP. Further, the influence of Ca/P on the size, precipitate chemistry, and transformation of ACP is crucial to understanding the metastability of amorphous phosphates. In situ pair distribution function (PDF) analysis results 5 min after mixing show that the Ca-P bonding geometry varies from predominantly monodentate, to mixed monodentate and bidentate, to predominantly bidentate as Ca/P increases from 0.2 to 5.0. This relationship is consistent across a pH range of 6-11. Samples with only monodentate Ca-P geometries transform directly to hydroxylapatite, while samples some or all bidentate geometries form brushite. Regardless of the initial ratio, the Ca/P of precipitates is close to 1.0. In situ small-angle X-ray scattering shows particle size increases with increasing Ca/P. This is the first evidence of structural variations in ACP and is directly linked to system chemistry. Finally, synthesis of ACP with monodentate Ca-P geometries is a promising method to control the crystallization pathway to form hydroxylapatite at circumneutral pH without stabilizing ions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2021

A high-efficiency self-healing cementitious material based on supramolecular hydrogels impregnated with phosphate and ammonium

High-efficiency self-healing cementitious materials using hydroxyapatites as healing product were described. Supramolecular hydrogels impregnated with phosphate and ammonium ions were designed. Morphology of the supramolecular hydrogel and its capability to release phosphate in solution, the structure of healing products and the self-healing efficiency were characterized with a range of analytical techniques. Cracks in the cementitious materials with width up to 1 mm are closed and completely healed. The healing product in the cracks without ammonium ions was brushite with a sponge-like morphology. Released ammonium ions reacted with calcium hydroxide in the cement matrix, increasing the concentration of soluble calcium ions up to 300 times that in reference pore solution. Ammonium ions facilitated the formation and aggregation of healing products which were converted into hydroxyapatites, improving the maximum healable crack width.

36 MATERIALS SCIENCE↗