Calcium oxalate (CaOx) crystallization under laminar flow conditions, relevant for kidney stone formation, was studied in a microfluidic device simulating the geometry of kidney collecting ducts. In a typical microfluidic experiment, two reactive solutions with designated concentrations of calcium (Ca) and oxalate (Ox) ions were brought into contact in a microfluidic channel to create a laminar co-current flow of the two streams. As the streams flow co-currently in the channel, diffusion takes place between the two streams across the channel width, resulting in reactive crystallization leading to CaOx nucleation and growth of CaOx crystals along the mixing front. We studied the growth of these crystals in artificial urine as a function of the fluid flow rate in the channel, the molar ratio of Ca : Ox in the medium and the presence of an organic protein, osteopontin (OPN), known to inhibit the growth of CaOx crystals. Three different flow velocities at a fixed molar ratio of Ca : Ox = 7.5 and four molar ratios of Ca : Ox at a fixed mean flow velocity of 0.035 m s−1 were tested. Lastly, three additive OPN concentrations were evaluated: 2.4 × 10−8 mol m−3, 6 × 10−8 mol m−3 and 8.4 × 10−8 mol m−3. The mean flow velocity did not alter the crystal growth of CaOx in the studied range, whereas altering the molar ratio of Ca : Ox had a high impact on the growth rate. In addition, the type of pseudopolymorph which nucleated appears to depend strongly on the molar ratio. At a low Ca : Ox ratio, both calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) nucleated simultaneously and the growth of the two pseudopolymorphic forms of CaOx crystals was observed. The lowest applied OPN concentration decreased the growth rate of COD, while higher concentrations of OPN slowed down the nucleation kinetics to a point that it completely inhibited the formation of any CaOx crystal in artificial urine within the investigated timeframe. COD was seen under all the conditions investigated, whilst COM was seen in experiments for Ca : Ox molar ratio values between 5 and 6. Our results were rationalized using finite element simulations supported by solution chemistry modelling.@en

Kidney stone formation is a global health problem with increasing prevalence. Stone formation is a physiochemical process involving crystallization of inorganic salts in the presence of biological constituents in the urinary system. To inhibit kidney stone formation, a better understanding of the underlying physicochemical mechanism of stone formation in the kidney is required. In this thesis, the solubility, nucleation and growth of calcium oxalate (CaOx), the most common inorganic constituent of kidney stones, were studied under different conditions such as ion concentration, pH value, and also the role of inhibitors in water or artificial urine was investigated. The first step towards this work was obtaining the solubility curve of calcium oxalate monohydrate (COM) in the solvent, such as ultrapure water and different buffers, to elucidate the physicochemical conditions which can cause the kidney stone formation (Chapter 2). Beside the solubility study, advanced technology to observe crystal formation in small scale and a very short time was needed. The volume, structure and flow properties inside the kidney inspired us to use microfluidic technology with comparable volume and flow rate. The developed microfluidic devices that mimic pathways in the human kidney were used to study the nucleation and growth of calcium oxalate crystals. The developed devices rendered an alternate perspective to the study of kidney stone formation and showed that microfluidics can provide precise, simple and fast detection of stone formation under various experimental conditions. Initially, the designed microfluidic device allowed us to build a testing platform for the study of nucleation kinetics of CaOx inside isolated environments provided by droplets. Preliminary experiments were performed by dissolving calcium chloride and sodium oxalate in ultrapure water. The aqueous solution, containing the ions, forms the droplet phase and oil were used as the continuous phase. Altering the pH values, as well as increasing the concentration of additives such as magnesium and osteopontin (OPN), were shown to slow down the nucleation kinetics, or even inhibit nucleation (Chapter 3). @en

The increasing prevalence of urolithiasis in industrialized societies triggered considerable interest in how various species found in urine regulate the nucleation and growth of common kidney stone constituents such as calcium oxalate (CaOx). Yet, the role macromolecules play in kidney stone formation is often overlooked due to their low concentration in urine. In this study, we investigate the nucleation kinetics of CaOx in artificial urine with droplet-based microfluidic induction time measurements at varying concentrations of oxalate and hyaluronic acid (HA), a polysaccharide commonly found in urine. The formation of two pseudo-polymorphic forms of calcium oxalate crystals, calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD), are carefully monitored using polarized light microscopy in induction time experiments. COM and COD nucleated concomitantly in artificial urine yet with distinct kinetics. Our results indicate that higher oxalate concentrations favor the formation of COD, the metastable form, over COM, the most stable form. Moreover, COD is also the fastest nucleating form in droplets under studied conditions. Furthermore, increasing the concentration of HA at fixed calcium and oxalate concentrations favored the nucleation of COM. We observed that in droplets where COM nucleated first, COD was not formed within the experimental time scale. However, in the droplets where COD appeared first, COM crystals were also observed later. We hope our findings shed light on the role macromolecules such as HA plays in dictating the pseudo-polymorphic form of CaOx and guide next generation treatments.@en

Detected kidney stone cases are increasing globally, yet knowledge on the conditions for stone formation is lacking. Experimental approaches mimicking the micro-environmental conditions present in vivo can help sci-entists untangle intertwined physiochemical and biological phenomena leading to kidney stone formation. As crystal nucleation often initiates at liquid-solid interfaces, the interface morphology plays a significant role in the rate of nucleation. Within the nephron, the functional unit of the kidney, four segments can be distinguished that contain different surface morphologies. Particularly, the cells lining these segments contain protrusions in the shape of nanopillars that vary in length, diameter and spacing. Exploiting the opportunities provided by organ- on-a-chip technology, we designed and manufactured a proof-of-principle microfluidic device proposed to in-crease our understanding of the relation between kidney surface morphology and kidney stone crystallization. We used two-photon polymerization to fabricate biocompatible surfaces that mimic the nephron morphologies with materials properties similar to those of biological structures. The fabricated cilia were incorporated in the microfluidic device, which was designed to observe in vitro crystallization of calcium oxalate under flow@en

A droplet-based microfluidic platform is presented to study the nucleation kinetics of calcium oxalate monohydrate (COM), the most common constituent of kidney stones, while carefully monitoring the pseudo-polymorphic transitions. The precipitation kinetics of COM is studied as a function of supersaturation and pH as well as in the presence of inhibitors of stone formation, magnesium ions (Mg2+), and osteopontin (OPN). We rationalize the trends observed in the measured nucleation rates leveraging a solution chemistry model validated using isothermal solubility measurements. In equimolar calcium and oxalate ion concentrations with different buffer solutions, dramatically slower kinetics is observed at pH 6.0 compared to pHs 3.6 and 8.6. The addition of both Mg2+ and OPN to the solution slows down kinetics appreciably. Interestingly, complete nucleation inhibition is observed at significantly lower OPN, namely, 3.2 × 10-8 M, than Mg2+ concentrations, 0.875 × 10-4 M. The observed inhibition effect of OPN emphasizes the often-overlooked role of macromolecules on COM nucleation due to their low concentration presence in urine. Moreover, analysis of growth rates calculated from observed lag times suggests that inhibition in the presence of Mg2+ cannot be explained solely on altered supersaturation. The presented study highlights the potential of microfluidics in overcoming a major challenge in nephrolithiasis research, the overwhelming physiochemical complexity of urine.

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Selective crystallization of polymorphs is highly sought after in industrial practice. Yet, state-of-the-art techniques either use laboriously engineered solid surfaces or strenuously prepared heteronucleants. We propose an approach where surfactants in solution self-assemble effortlessly into mesoscopic structures dictating the polymorphic outcome of the target solute. Sodium dodecyl sulfate (SDS) surfactant is used as a tailored additive to crystallize different polymorphic forms of a model active pharmaceutical ingredient, d-mannitol. Different mesoscopic phases of SDS template particular polymorphs: packed monolayers, micelles, and crystals favored the β, α, and δ forms of d-mannitol, respectively. A synergistic effect of topological templating and molecular interactions is proposed as the rationale behind the observed selective crystallization of polymorphs. This crystal engineering technique suggests that surfactant self-assemblies can be used as tailored templates for polymorphic control.@en

Accurate Calcium Oxalate Monohydrate (COM) solubility measurements are essential for elucidating the physiochemical mechanism behind the formation of kidney stones, nephrolithiasis. Yet the reported solubility values of COM in ultrapure water, arguably the simplest solvent relevant for nephrolithiasis, vary significantly depending on implemented method. To address this variation, we present an experimental study of the solubility of COM validated by a model based on the Debye–Hückel theory describing the solution chemistry and the complex formation. We also carefully monitor potential pseudopolymorphic/hydrate transitions during the solubility measurements with in-situ and ex-situ methods. Our results indicate that the solubility of COM in ultrapure water is a weak function of temperature. However, the measured solubility varies significantly in buffer solutions across physiologically relevant pH values at body temperature. The proposed model explains observed trends as a combined effect of ionic strength, protonation reactions, and soluble complex formation. Moreover, it predicts solubility of COM in buffer solutions remarkably well using our measurements in ultrapure water as input, demonstrating the consistency of presented approach. The presented study parleying experiments and modelling provides a solid stepping stone to extend the physiochemical understanding of nephrolithiasis to more realistic solutions laden with biological complexity.@en

Hypothesis: Our ability to dictate the colloid geometry is intimately related to self-assembly. The synthesis of anisotropic colloidal particles is currently dominated by wet chemistry and lithographic techniques. The wet chemical synthesis offers limited particle geometries at bulk quantities. Lithographic techniques, on the other hand, provide precise control over the particle shape, although at lower yields. In this respect, two-photon polymerization (2PP)¹ has attracted growing attention due to its ability to automatically fabricate complex micro/nano structures with high resolution. Experiments: We manufacture precisely designed colloids with sizes ranging from 1 µm to 10 µm with 2PP and optimize the process parameters for each dimension. Moreover, we study the shape dependent Brownian motion of these particles with video microscopy and estimate their diffusion coefficients. Findings: We observe that increasing the geometrical anisotropy leads to a pronounced deviation from the analytically predicted diffusion coefficient for disks with a given aspect ratio. The deviation is attributed to stronger hydrodynamic coupling with increasing anisotropy. We demonstrate, for the first time, 2PP manufacturing of colloids with tailored geometry. This study opens synthesis of colloidal building blocks to a broader audience with limited access to cleanrooms or wet-chemistry know-how.

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We detail the analysis of centrifugal homogenization process by a hydrodynamic model and the model-guided design of a low-cost centrifugal homogenizer. During operation, centrifugal force pushes a multiphase solution to be homogenized through a thin nozzle, consequently homogenizing its contents. We demonstrate and assess the homogenization of coarse emulsions into relatively monodisperse emulsions, as well as the application of centrifugal homogenization in the mechanical lysis of mpkCCD mouse kidney cells. To gain insight into the homogenization mechanism, we investigate the dependence of emulsion droplet size on geometrical parameters, centrifugal acceleration, and dispersed phase viscosity. Our experimental results are in qualitative agreement with models predicting the droplet size. Furthermore, they indicate that high shear rates kept constant throughout operation produce more monodisperse droplets. We show this ideal homogenization condition can be realized through hydrodynamic model-guided design minimizing transient effects inherent to centrifugal homogenization. Moreover, we achieved power densities comparable to commercial homogenizers by model guided optimization of homogenizer design and experimental conditions. Centrifugal homogenization using the proposed homogenizer design thus offers a low-cost alternative to existing technologies as it is constructed from off-the-shelf parts (Falcon tubes, syringe, needles) and used with a centrifuge, readily available in standard laboratory environment.

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This in vitro study aimed to evaluate the bleaching efficacy and color stability of nonthermal atmospheric pressure plasma (NAPP)-activated 40% hydrogen peroxide (HP) and deionized water (DW). Methods: Two hundred-forty bovine enamel−dentin samples were stained with a coffee solution before bleaching. Samples were randomly divided into 12 (n = 20): (1) HP for 5 minute (HP-5), (2) HP-10, (3) HP-20, (4) Plasma-HP-5, (5) P-HP-10, (6) P-HP-5/HP-15, (7) P-HP-10/HP-10, (8) P-DW-5, (9) P-DW-10, (10) DW-5, (11) DW-10, and (12) DW-20. After bleaching, the samples were re-stained for 48 h using red wine. The color differences measured using a dental spectrophotometer after bleaching and after re-staining were reported as ΔE1 and ΔE2, respectively. The data were analyzed using Kruskal−Wallis and Mann−Whitney U tests (p< 0.05).@en

This in vitro study aimed to evaluate the effect of nonthermal atmospheric pressure plasma (NAPP)-activated hydrogen peroxide (HP) and deionized water (DW) on enamel microhardness and enamel surface morphology.
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