Sex-specific Stone-forming Phenotype in Mice During Hypercalciuria/Urine Alkalinization.

Sex differences in kidney stone formation are well known. Females generally have slightly acidic blood and higher urine pH when compared with males, which makes them more vulnerable to calcium stone formation, yet the mechanism is still unclear. We aimed to examine the role of sex in stone formation during hypercalciuria and urine alkalinization through acetazolamide and calcium gluconate supplementation, respectively, for 4 weeks in wild-type (WT) and moderately hypercalciuric [TRPC3 knockout [KO](-/-)] male and female mice. Our goal was to develop calcium phosphate (CaP) and CaP+ calcium oxalate mixed stones in our animal model to understand the underlying sex-based mechanism of calcium nephrolithiasis. Our results from the analyses of mice urine, serum, and kidney tissues show that female mice (WT and KO) produce more urinary CaP crystals, higher [Ca2+], and pH in urine compared to their male counterparts. We identified a sex-based relationship of stone-forming phenotypes (types of stones) in our mice model following urine alkalization/calcium supplementation, and our findings suggest that female mice are more susceptible to CaP stones under those conditions. Calcification and fibrotic and inflammatory markers were elevated in treated female mice compared with their male counterparts, and more so in TRPC3 KO mice compared with their WT counterparts. Together these findings contribute to a mechanistic understanding of sex-influenced CaP and mixed stone formation that can be used as a basis for determining the factors in sex-related clinical studies.

Recombinant Human Keratinocyte Growth Factor Ameliorates Cancer Treatment-Induced Oral Mucositis on a Chip.

Oral mucositis (OM) is a severe complication of cancer therapies caused by off-target cytotoxicity. Palifermin, which is recombinant human keratinocyte growth factor (KGF), is currently the only mitigating treatment available to a subset of OM patients. This study used a previously established model of oral mucositis on a chip (OM-OC) comprised of a confluent human gingival keratinocytes (GIE) layer attached to a basement membrane-lined subepithelial layer consisting of human gingival fibroblasts (HGF) and human dermal microvascular endothelial cells (HMEC) on a stable collagen I gel. Cisplatin, radiation, and combined treatments are followed by a recovery period in the OM-OC to determine possible cellular and molecular mechanisms of OM under effects of KGF. Cancer treatments affected the keratinocyte layer, causing death and epithelial barrier loss. Both keratinocytes and subepithelial cells died rapidly, as evidenced by propidium iodide staining. In response to radiation exposure, cell death occurred in the apical epithelial layer, predominantly, within 24h. Cisplatin exposure predominantly promoted death of basal epithelial cells within 32-36h. Presence of KGF in OM-OC protected tissues from damage caused by cancer treatments in a dose-dependent manner, being more effective at 10 ng/mL. As verified by F-actin staining and the Alamar Blue assay, KGF contributed to epithelial survival and induced proliferation of GIE and HGF as well as HMEC within 120h. When the expression of eighty inflammatory cytokines is evaluated at OM induction (Day 12) and resolution (Day 18) stages in OM-OC, some cytokines are identified as potential novel therapeutic targets. In comparison with chemoradiation exposure, KGF treatment showed a trend to decrease IL-8 and TNF-a expression at Day 12 and 18, and TGF-ß1 at Day 18 in OM-OC. Taken together, these findings support the utility of OM-OC as a platform to model epithelial damage and evaluate molecular mechanisms following OM treatment.

Three-dimensional refractive index estimation based on deep-inverse non-interferometric optical diffraction tomography (ODT-Deep).

Optical diffraction tomography (ODT) solves an inverse scattering problem to obtain label-free, 3D refractive index (RI) estimation of biological specimens. This work demonstrates 3D RI retrieval methods suitable for partially-coherent ODT systems supported by intensity-only measurements consisting of axial and angular illumination scanning. This framework allows for access to 3D quantitative RI contrast using a simplified non-interferometric technique. We consider a traditional iterative tomographic solver based on a multiple in-plane representation of the optical scattering process and gradient descent optimization adapted for focus-scanning systems, as well as an approach that relies solely on 3D convolutional neural networks (CNNs) to invert the scattering process. The approaches are validated using simulations of the 3D scattering potential for weak phase 3D biological samples.

Stabilization of uric acid mixed crystals by melamine.

Melamine stabilizes heterogeneous nucleation of calcium crystals by increasing the retention time and decreasing the rate of dissolution. Stabilization of such mixed crystals limit the efficacy of non-invasive treatment options for kidney stones. Crystalline forms of uric acid (UA) are also involved in urolithiasis or UA kidney stones; however, its interactions with contaminating melamine and the resulting effects on the retention of kidney stones remain unknown. Since melamine augments calcium crystal formation, it provides an avenue for us to understand the stability of UA-calcium phosphate (CaP) crystals. We show here that melamine facilitates UA+CaP crystal formation, resulting in greater aggregates. Moreover, melamine induced mixed crystal retention through a time-dependent manner in presence and/or absence of hydroxycitrate (crystal inhibitor), indicating its abridged effectiveness as conventional remedy. CaP was also shown to modify optical properties of UA+CaP mixed crystals. Differential staining of individual crystals revealed enhanced co-aggregation of UA and CaP. The dissolution rate of UA in presence of melamine was faster than its heterogeneous crystallization form with CaP, although the size was comparatively much smaller, suggesting disparity in regulation between UA and CaP crystallization. While melamine stabilized UA, CaP and mixed crystals in relatively physiological conditions (artificial urine), the retentions of those crystals were further augmented by melamine, even in presence of hydroxycitrate, thus reducing treatment efficacy.

Digital Holographic Microscopy to Assess Cell Behavior.

Digital holographic microscopy is an imaging technique particularly well suited to the study of living cells in culture, as no labeling is required and computed phase maps produce high contrast, quantitative pixel information. A full experiment involves instrument calibration, cell culture quality checks, selection and setup of imaging chambers, a sampling plan, image acquisition, phase and amplitude map reconstruction, and parameter map post-processing to extract information about cell morphology and/or motility. Each step is described below, focusing on results from imaging four human cell lines. Several post-processing approaches are detailed, with an aim of tracking individual cells and dynamics of cell populations.

Programmable Physical Properties of Freestanding Chitosan Membranes Electrofabricated in Microfluidics.

Microfluidic-integrated freestanding membranes with suitable biocompatibility and tunable physicochemical properties are in high demand for a wide range of life science and biological studies. However, there is a lack of facile and rapid methods to integrate such versatile membranes into microfluidics. A recently invented interfacial electrofabrication of chitosan membranes offers an in-situ membrane integration strategy that is flexible, controllable, simple, and biologically friendly. In this follow-up study, we explored the ability to program the physical properties of these chitosan membranes by varying the electrofabrication conditions (e.g., applied voltage and pH of alginate). We found a strong association between membrane growth rate, properties, and fabrication parameters: high electrical stimuli and pH of alginate resulted in high optical retardance and low permeability, and vice versa. This suggests that the molecular alignment and density of electrofabricated chitosan membranes could be actively tailored according to application needs. Lastly, we demonstrated that this interfacial electrofabrication could easily be expanded to produce chitosan membrane arrays with higher uniformity than the previously well-established flow assembly method. This study demonstrates the tunability of the electrofabricated membranes' properties and functionality, thus expanding the utility of such membranes for broader applications in the future.

Oral mucositis on a chip: modeling induction by chemo- and radiation treatments and recovery.

Oral mucositis (OM) is a debilitating complication affecting roughly 70% of head and neck cancer patients receiving chemotherapy and/or radiation treatment. No broadly effective preventative treatment for OM exists. Therefore, anin vitromodel of cancer treatment-induced OM would aid studies into possible origins of the pathology and future drug targets to ameliorate it. In this study, we present a microfluidic oral mucosa triculture tissue construct consisting of a keratinocyte layer attached to a subepithelial fibroblast and endothelial cell-embedded collagen gel. To address the typically low stability of mucosal constructs in microfluidics, ruthenium-catalyzed photocrosslinking was implemented to strengthen the collagen gel and prevent the invasion of keratinocytes, thus maintaining tissue construct geometry and oral mucosa barrier function for over 18 d of culture. Next, the OM chip was exposed to cisplatin (day 10) and damaging radiation (day 11, ± cisplatin at day 10), mimicking damage from cancer therapy. Damage to and then recovery of the tissue layers and function were observed over days 11-18. Therefore, several important features of OM induction and resolution were modeled in microfluidic culture. The OM model on a chip allows for more sophisticated studies into mechanisms of OM and potential treatments.

Dual-modality digital holographic and polarization microscope to quantify phase and birefringence signals in biospecimens with a complex microstructure.

Optical phase and birefringence signals occur in cells and thin, semi-transparent biomaterials. A dual-modality quantitative phase and polarization microscope was designed to study the interaction of cells with extracellular matrix networks and to relate optical pathlength and birefringence signals within structurally anisotropic biomaterial constructs. The design was based on an existing, custom-built digital holographic microscope, to which was added a polarization microscope utilizing liquid crystal variable retarders. Phase and birefringence channels were calibrated, and data was acquired sequentially from cell-seeded collagen hydrogels and electrofabricated chitosan membranes. Computed phase height and retardance from standard targets were accurate within 99.7% and 99.8%, respectively. Phase height and retardance channel background standard deviations were 35 nm and 0.6 nm, respectively. Human fibroblasts, visible in the phase channel, aligned with collagen network microstructure, with retardance and azimuth visible in the polarization channel. Electrofabricated chitosan membranes formed in 40 µm tall microfluidic channels possessed optical retardance ranging from 7 to 11 nm, and phase height from 37 to 39 µm. These results demonstrate co-registered dual-channel acquisition of phase and birefringence parameter maps from microstructurally-complex biospecimens using a novel imaging system combining digital holographic microscopy with voltage-controlled polarization microscopy.

Computational multi-wavelength phase synthesis using convolutional neural networks [Invited].

Multi-wavelength digital holographic microscopy (MWDHM) provides indirect measurements of the refractive index for non-dispersive samples. Successive-shot MWDHM is not appropriate for dynamic samples and single-shot MWDHM significantly increases the complexity of the optical setup due to the need for multiple lasers or a wavelength tunable source. Here we consider deep learning convolutional neural networks for computational phase synthesis to obtain high-speed simultaneous phase estimates on different wavelengths and thus single-shot estimates of the integral refractive index without increased experimental complexity. This novel, to the best of our knowledge, computational concept is validated using cell phantoms consisting of internal refractive index variations representing cytoplasm and membrane-bound organelles, respectively, and a simulation of a realistic holographic recording process. Specifically, in this work we employed data-driven computational techniques to perform accurate dual-wavelength hologram synthesis (hologram-to-hologram prediction), dual-wavelength phase synthesis (unwrapped phase-to-phase prediction), direct phase-to-index prediction using a single wavelength, hologram-to-phase prediction, and 2D phase unwrapping with sharp discontinuities (wrapped-to-unwrapped phase prediction).

Mueller matrix polarimetry and polar decomposition of articular cartilage imaged in reflectance.

Articular cartilage birefringence relates to zonal architecture primarily of type II collagen, which has been assessed extensively in transmission, through thin tissue sections, to evaluate cartilage repair and degeneration. Mueller matrix imaging of articular cartilage in reflection is of potential utility for non-destructive imaging in clinical and research applications. Therefore, such an imaging system was constructed to measure laser reflectance signals, calibrated, and tested with optical standards. Polar decomposition was chosen as a method to extract fundamental optical parameters from the experimental Mueller matrices, with performance confirmed by simulations. Adult bovine articular cartilage from the patellofemoral groove was found to have ∼0.93 radians retardance, low diattenuation of ∼0.2, and moderately high depolarization of 0.66. Simulations showed that variation in depolarization drives inaccuracy of depolarization and retardance maps derived by polar decomposition. These results create a basis for further investigation of the clinical utility of polarized signals from knee tissue and suggest potential approaches for improving the accuracy of polar decomposition maps.

Keratin 19 maintains E-cadherin localization at the cell surface and stabilizes cell-cell adhesion of MCF7 cells.

A cytoskeletal protein keratin 19 (K19) is highly expressed in breast cancer but its effects on breast cancer cell mechanics are unclear. In MCF7 cells where K19 expression is ablated,we found that K19 is required to maintain rounded epithelial-like shape and tight cell-cell adhesion. A loss of K19 also lowered cell surface E-cadherin levels. Inhibiting internalization restored cell-cell adhesion of KRT19  knockout cells, suggesting that E-cadherin internalization contributed to defective adhesion. Ultimately, while K19 inhibited cell migration and invasion, it was required for cells to form colonies in suspension. Our results suggest that K19 stabilizes E-cadherin complexes at the cell membrane to maintain cell-cell adhesion which inhibits cell invasiveness but provides growth and survival advantages for circulating tumor cells.

An Oral-mucosa-on-a-chip sensitively evaluates cell responses to dental monomers.

Knowledge of human gingival cell responses to dental monomers is critical for the development of new dental materials. Testing standards have been developed to provide guidelines to evaluate biological functionality of dental materials and devices. However, one shortcoming of the traditional testing platforms is that they do not recapitulate the multi-layered configuration of gingiva, and thus cannot evaluate the layer-specific cellular responses. An oral mucosa-chip with two cell layers was previously developed as an alternative platform to assess the oral mucosa responses to dental biomaterials. The mucosa-chip consists of an apical keratinocyte layer attached to a fibroblast-embedded collagen hydrogel through interconnecting pores in a three-microchannel network. Here, cell responses in the mucosa-chip were evaluated against 2-hydroxyethyl methacrylate (HEMA), a common monomer used in restorative and aesthetic dentistry. The response of mucosal cell viability was evaluated by exposing the chip to HEMA of concentrations ranging from 1.56 to 25 mM and compared to cells in conventional well-plate monoculture. The co-cultured cells were then stained and imaged with epifluorescence and confocal microscopy to determine the layer-specific responses to the treatment. Mucosa-chips were demonstrated to be more sensitive to assess HEMA-altered cell viability than well-plate cultures, especially at lower doses (1.56 and 6.25 mM). The findings suggest that the mucosa-chip is a promising alternative to traditional platforms or assays to test a variety of biomaterials by offering a multi-layered tissue geometry, accessible layer-specific information, and higher sensitivity in detecting cellular responses.

Tuning the porosity of biofabricated chitosan membranes in microfluidics with co-assembled nanoparticles as templates.

Biopolymer membranes assembled in microfluidic devices offer many biological process- and analysis-related applications. One of the key characteristics of bio-fabricated membranes is their porosity, which regulates the transport of molecules, ions, or particles and contributes to their semi-permeability and selectivity. This study aims to tune the porosity of biofabricated chitosan membranes (CM) using incorporated nanoparticles as templates. CM with polystyrene nanoparticles (CM-np) were assembled by flow in microchannel networks. The membranes with incorporated nanoparticles were crosslinked with glutaraldehyde, and then the nanoparticles were dissolved with dimethyl sulfoxide. The in situ synthesized porous CM (pCM) were characterized with scanning electron microscopy and polarized light microscopy. Permeability tests confirmed the increased pore sizes of the pCM and enhanced permeability to macromolecules. Sharper static gradients in three-channel microfluidic devices were demonstrated with the pCM as compared to those with the original CM. The capability to customize the porosity of flow-assembled, freestanding and robust biopolymer membranes inside a microfluidic network is attractive and broadens the applications of these membranes in biomolecular and cellular studies.

Tracking Single Cells Motility on Different Substrates.

Motility is a key property of a cell, required for several physiological processes, including embryonic development, axon guidance, tissue regeneration, gastrulation, immune response, and cancer metastasis. Therefore, the ability to examine cell motility, especially at a single cell level, is important for understanding various biological processes. Several different assays are currently available to examine cell motility. However, studying cell motility at a single cell level can be costly and/or challenging. Here, we describe a method of tracking random cell motility on different substrates such as glass, tissue-culture polystyrene, and type I collagen hydrogels, which can be modified to generate different collagen network microstructures. In this study we tracked MDA-MB-231 breast cancer cells using The CytoSMARTTM System (Lonza Group, Basel, Switzerland) for live cell imaging and assessed the average cell migration speed using ImageJ and wrMTrck plugin. Our cost-effective and easy-to-use method allows studying cell motility at a single cell level on different substrates with varying degrees of stiffness and varied compositions. This procedure can be successfully performed in a highly accessible manner with a simple setup.

Pulsed focused ultrasound lowers interstitial fluid pressure and increases nanoparticle delivery and penetration in head and neck squamous cell carcinoma xenograft tumors.

Nanocarriers offer a promising approach to significantly improve therapeutic delivery to solid tumors as well as limit the side effects associated with anti-cancer agents. However, their relatively large size can negatively affect their ability to efficiently penetrate into more interior tumor regions, ultimately reducing therapeutic efficacy. Poor penetration of large agents such as nanocarriers is attributed to factors in the tumor microenvironment such as elevated interstitial fluid pressure (IFP) and fibrillar collagen in the extracellular matrix. Our previous studies reported that pretreatment of solid tumor xenografts with nondestructive pulsed focused ultrasound (pFUS) can improve the delivery and subsequent therapy of a variety of therapeutic formulations in different tumor models, where the results were associated with expanded extracellular spaces (ECS), an increase in hydraulic conductivity, and decrease in tissue stiffness. Here, we demonstrate the inverse relationship between IFP and the penetration of systemically administered nanoparticle (NP) probes, where IFP increased from the tumor periphery to their center. Furthermore, we show that pretreatment with pFUS can safely reduce IFP and improve NP delivery; especially into the center of the tumors. These results coincide with effects generated in the fibrillar collagen network microstructure in the ECS as determined by quantitative polarized light microscopy. Whole tumor and histomorphometric analysis, however, did not show significant differences in collagen area fraction or collagen feature solidity, as well as tumor cross-sectional area and aspect ratio, as a result of the treatments. We present a biophysical model connecting the experimental results, where pFUS-mediated cytoarchitectural changes are associated with improved redistribution of the interstitial fluid and lower IFP. The resulting improvement in NP delivery supports our previous therapeutic studies and may have implications for clinical applications to improve therapeutic outcomes in cancer therapy.

Morphology, Motility, and Cytoskeletal Architecture of Breast Cancer Cells Depend on Keratin 19 and Substrate.

Cancer cells gain motility through events that accompany modulation of cell shape and include altered expression of keratins. However, the role of keratins in change of cancer cell architecture is not well understood. Therefore, we ablated the expression of keratin 19 (K19) in breast cancer cells of the MDA-MB-231 cell line and found that cells lacking K19 become more elongated in culture, with morphological reversion toward the parental phenotype upon transduction of KRT19. Also, the number of actin stress fibers and focal adhesions were significantly reduced in KRT19 knockout (KO) cells. The altered morphology of KRT19 KO cells was then characterized quantitatively using digital holographic microscopy (DHM), which not only confirmed the phenotypic change of KRT19 KO cells but also identified that the K19-dependent morphological change is dependent on the substrate type. A new quantitative method of single cell analysis from DHM, via average phase difference maps, facilitated evaluation of K19-substrate interactive effects on cell morphology. When plated on collagen substrate, KRT19 KO cells were less elongated and resembled parental cells. Assessing single cell motility further showed that while KRT19 KO cells moved faster than parental cells on a rigid surface, this increase in motility became abrogated when cells were plated on collagen. Overall, our study suggests that K19 inhibits cell motility by regulating cell shape in a substrate-dependent manner. Thus, this study provides a potential basis for the altered expression of keratins associated with change in cell shape and motility of cancer cells. © 2020 International Society for Advancement of Cytometry.

Modulating the properties of flow-assembled chitosan membranes in microfluidics with glutaraldehyde crosslinking.

Flow-assembled chitosan membranes are robust and semipermeable hydrogel structures formed in microfluidic devices that have been used for important applications such as gradient generation and studying cell-cell signaling. One challenge, however, remains unresolved. When a polydimethylsiloxane (PDMS) microchannel with a flow-assembled, deprotonated chitosan membrane (DCM) is treated with anti-adhesion agents such as Pluronic F-127 to prevent biomolecular and cellular adsorption on PDMS, the interaction between DCM and PDMS is compromised and the DCM easily delaminates. To address this challenge, DCMs in microfluidics are crosslinked with glutaraldehyde to modulate their properties, and the altered properties of the glutaraldehyde treated chitosan membrane (GTCM) are investigated. First, the GTCM's acidic resistance was confirmed, its mechanical robustness against hydrostatic pressure was significantly improved, and it remained intact on PDMS after Pluronic treatment. Second, crystallization in DCM and GTCM was investigated with quantitative polarized light microscopy (qPLM), which revealed that GTCM's optical retardance and anisotropy were lower, implying less molecular alignment than in DCM. Finally, membrane permeability was tested with FITC-labeled dextran transport experiments, which showed that the transport across GTCM was slightly higher than that across DCM. Overall, glutaraldehyde-crosslinked chitosan membrane has better acidic resistance, higher strength under Pluronic treatment, and less molecular microalignment, while its semi-permeability is retained. This study demonstrates how glutaraldehyde crosslinking can be used to modify and improve biopolymer membrane properties for broader applications, such as in an acidic environment or when Pluronic passivation is needed.

Microstructural densification and alignment by aspiration-ejection influence cancer cell interactions with three-dimensional collagen networks.

Extracellular matrix microstructure and mechanics are crucial to breast cancer progression and invasion into surrounding tissues. The peritumor collagen network is often dense and aligned, features which in vitro models lack. Aspiration of collagen hydrogels led to densification and alignment of microstructure surrounding embedded cancer cells. Two metastasis-derived breast cancer cell lines, MDA-MB-231 and MCF-7, were cultured in initially 4 mg/ml collagen gels for 3 days after aspiration, as well as in unaspirated control hydrogels. Videomicroscopy during aspiration, and at 0, 1, and 3 days after aspiration, epifluorescence microscopy of phalloidin-stained F-actin cytoskeleton, histological sections, and soluble metabolic byproducts from constructs were collected to characterize effects on the embedded cell morphology, the collagen network microstructure, and proliferation. Breast cancer cells remained viable after aspiration-ejection, proliferating slightly less than in unaspirated gels. Furthermore, MDA-MB-231 cells appear to partially relax the collagen network and lose alignment 3 days after aspiration. Aspiration-ejection generated aligned, compact collagen network microstructure with immediate cell co-orientation and higher cell number density apparently through purely physical means, though cell-collagen contact guidance and network remodeling influence cell organization and collagen network microstructure during subsequent culture. This study establishes a platform to determine the effects of collagen density and alignment on cancer cell behavior, with translational potential for anticancer drug screening in a biomimetic three-dimensional matrix microenvironment, or implantation in preclinical models.

Microfluidic fabrication of stable collagen microgels with aligned microstructure using flow-driven co-deposition and ionic gelation.

The controlled biofabrication of stable, aligned collagen hydrogels within microfluidic devices is critically important to the design of more physiologically accurate, longer-cultured on-chip models of tissue and organs. To address this goal, collagen-alginate microgels were formed in a microfluidic channel by calcium crosslinking of a flowing collagen-alginate solution through a cross-channel chitosan membrane spanning a pore allowing ion diffusion but not convection. The gels formed within seconds as isolated islands in a single channel, and their growth was self-limiting. Total gel thickness was controlled by altering the concentration of calcium and collagen-alginate flow rate to reach an equilibrium of calcium diffusion and solution convection at the gel boundary, for a desired thickness of 30-200 µm. Additionally, less calcium and higher flow produced greater compression of the gel, with regions farther from the pore compressing more. An aligned, stable collagen network was demonstrated by collagen birefringence, circumferential texture orientation, and little change in gel dimensions with de-chelation of calcium from alginate by prolonged flow of EDTA in the channel. Resultant gels were most stable and only slightly asymmetric when formed from solutions containing 8 mg ml-1 collagen. Diffusion of 4 kDa and 70 kDa fluorescently-labeled dextran indicated size-dependent diffusion across the gel, and accessibility of the construct to appropriately-sized bioactive molecules. This work demonstrates the physicochemical parameter control of collagen gel formation in microfluidic devices, with utility toward on-chip models of dense extracellular matrix invasion, cancer growth and drug delivery to cells within dense extracellular matrix bodies.

Matting Calcium Crystals by Melamine Improves Stabilization and Prevents Dissolution.

Melamine induces calcium phosphate (CaP) and calcium oxalate (CaOx) crystal formation; however, the physicochemical mechanism is not clear. Recently, we found that melamine has a discriminatory effect on CaP, CaOx, and CaP + CaOx (Mixed) crystal dissolution. Thus, to delineate the mechanism, we examined crystal interactions through birefringence analysis and found that CaP becomes increasingly birefringent when bound to melamine, while the birefringence of CaOx decreases when it forms CaOx-melamine cocrystals. We also confirmed the feasibility of such melamine-CaP/CaOx co-crystallization at the nanomicromolar range. Interestingly, ammeline, which is a similar triazine, did not accelerate CaP/CaOx/Mixed crystal formation and growth, indicating the specificity of crystal interaction by melamine. Furthermore, melamine stabilizes the CaP/CaOx/Mixed crystals when exposed to a crystal inhibitor (etidronic acid) or dissolution agents (citrate analogues), while it induces crystal growth by increasing crystal retention, suggesting melamine's interference with conventional dissolution remedies. Morphological and elemental analysis of melamine-CaP/CaOx/Mixed co-crystals using scanning electron microscopy further revealed that melamine harbors such crystals by creating a nucleation site. Finally, we confirmed the physiological relevance of melamine exposure using artificial urine to show the induction, stabilization, and retention of mixed crystals in the presence of crystal-inhibitor/dissolution agent and thus established potential causes of recurrence of kidney stones.