Calcium Phosphate Crystals Research Articles

Investigation of Calcium and Magnesium Phosphate Crystals in Stones Treated with Diammonium Hydrogen Phosphate Conservation Product: Potential of Micro-Raman Spectroscopy

This study is aimed at investigating crystals (calcium and magnesium phosphates) formed due the interaction of an inorganic conservation treatment (diammonium hydrogen phosphate—DAP) with carbonatic (calcitic and dolomitic) stones through micro-Raman Spectroscopy. The addressed questions concern (i) the identification of magnesium and calcium phosphate minerals crystallized within dolomitic stone samples with a different degree of conservation state and treated with two different DAP solution molarities and (ii) the distinction of complex calcium phosphate mixtures (hydroxyapatite—HAP and octa calcium phosphate—OCP) crystallized within a calcarenite stone treated with DAP. A statistically relevant number of Raman spectra have been acquired in sample cross sections and curve fitting analysis has been performed for the in-depth interpretation of data. The outcomes indicate that Raman Spectroscopy is an effective alternative method for the identification of poorly crystalline calcium phosphates (not easily detectable with X-ray diffraction), even when scarcely present in mixture with magnesium phosphates. Evidence of the Raman analytical capability and high potential to distinguish HAP and OCP in calcitic stones are also presented and discussed.

Calcification alters the viscoelastic properties of tendon fascicle bundles depending on matrix content.

Tendon fascicle bundles are often used as biological grafts and thus must meet certain quality requirements, such as excluding calcification, which alters the biomechanical properties of soft tissues. In this work, we investigate the influence of early-stage calcification on the mechanical and structural properties of tendon fascicle bundles with varying matrix content. The calcification process was modeled using sample incubation in concentrated simulated body fluid. Mechanical and structural properties were investigated using uniaxial tests with relaxation periods, dynamic mechanical analysis, as well as magnetic resonance imaging and atomic force microscopy. Mechanical tests showed that the initial phase of calcification causes an increase in the elasticity, storage, and loss modulus, as well as a drop in the normalized value of hysteresis. Further calcification of the samples results in decreased modulus of elasticity and a slight increase in the normalized value of hysteresis. Analysis via MRI and scanning electron microscopy showed that incubation alters fibrillar relationships within the tendon structure and the flow of body fluids. In the initial stage of calcification, calcium phosphate crystals are barely visible; however, extending the incubation time for the next 14 days results in the appearance of calcium phosphate crystals within the tendon structure and leads to damage in its structure. Our results show that the calcification process modifies the collagen-matrix relationships and leads to a change in their mechanical properties. These findings will help to understand the pathogenesis of clinical conditions caused by calcification process, leading to the development of effective treatments for these conditions. STATEMENT OF SIGNIFICANCE: This study investigates how calcium mineral deposition in tendons affects their mechanical response and which processes are responsible for this phenomenon. By analyzing the elastic and viscoelastic properties of animal fascicle bundles affected by calcification induced via incubation in concentrated simulated body fluid, the study sheds light on the relationship between structural and biochemical changes in tendons and their altered mechanical response. This understanding is crucial for optimizing tendinopathy treatment and preventing tendon injury. The findings provide insights into the calcification pathway and its resulting changes in the biomechanical behaviors of affected tendons, which have been previously unclear.

Nanocrystal-induced chronic tubular-nephropathy in tropical countries: diagnosis, mitigation, and eradication

In tropical countries, a mysterious tubulo-interstitial chronic renal disease (CKD), unrelated to diabetes, hypertension, and immunological causes, manifested four decades ago. Approximately 25,000 primarily middle-aged male farmers succumb annually to this crystal-tubular nephropathy (CTN). Without any known causative factors, it was identified as CKD of unknown aetiology (CKDu). Because multiple factors contribute to causing it later, was changed to CKD of multi-factorial (CKDmfo). Despite no evidence, it was hypothesised to cause by agrochemicals or heavy metals in food or drinking contaminated water. However, current data suggest that the CKD-CTN is due to natural geogenic water contamination. Consumption of concentrated stagnant groundwater from deep-dug wells and tube wells containing hard water and fluoride, overdecades is necessary for its clinical manifestations. In all affected countries have prolonged annual dry seasons that led to the evopo-concentration of ions and minerals in groundwater, making hard water even more unpalatable, thus, peasants consume lesser amounts of water. They develop chronic dehydration from daily exposure to hot climatic conditions aggravated by regular alcohol intake. These conditions provide a highly conducive environment—a perfect storm for calcium phosphate (CaPO4) crystal formation in renal tissues. Our recent histological and preliminary electron microscopic data reveal deposition of CaPO4 crystals and nano-tubes in kidneys. While CaPO4 nano-minerals are unstable, the presence of fluoride ions stabilises and allows their growth. This new concept paves the path for highly cost-effective, straightforward local solutions to protect farm workers and eliminate the disease, without embarking on expensive medications, interventions, or building hospitals. Chronic dehydration-associated CKD–CTN is preventable by increased consumption of potable water. Increasing clean water consumption reduces CKD–CTN incidence, and associated morbidities and premature deaths. However, the damage becomes irreversible when the disease advances beyond CKD stage IIIB. The incidence of this deadly renal failure can be prevented by its education, lifestyle changes, and increased water consumption, not by treating the renal disease or expanding dialysis centres/hospitals, or transplantation services. Eradication of CKD-CTN cost significantly less than the current approach of treating affected persons and unnecessarily expanding health infrastructure. Since the manifestation of CKD-CTN is due to consuming naturally contaminated drinking water (with calcium containing hard water and fluoride), it is not difficult to remove these to prevent CKD-CTN: thus, international assistance is unwarranted for its eradication. The straightforward approaches described here will prevent CKD–CTN and save thousands of lives in affected farming communities.

Macrophage death in atherosclerosis: potential role in calcification.

Cell death is an important aspect of atherosclerotic plaque development. Insufficient efferocytosis of death cells by phagocytic macrophages leads to the buildup of a necrotic core that impacts stability of the plaque. Furthermore, in the presence of calcium and phosphate, apoptotic bodies resulting from death cells can act as nucleation sites for the formation of calcium phosphate crystals, mostly in the form of hydroxyapatite, which leads to calcification of the atherosclerotic plaque, further impacting plaque stability. Excessive uptake of cholesterol-loaded oxidized LDL particles by macrophages present in atherosclerotic plaques leads to foam cell formation, which not only reduces their efferocytosis capacity, but also can induce apoptosis in these cells. The resulting apoptotic bodies can contribute to calcification of the atherosclerotic plaque. Moreover, other forms of macrophage cell death, such as pyroptosis, necroptosis, parthanatos, and ferroptosis can also contribute by similar mechanisms to plaque calcification. This review focuses on macrophage death in atherosclerosis, and its potential role in calcification. Reducing macrophage cell death and/or increasing their efferocytosis capacity could be a novel therapeutic strategy to reduce the formation of a necrotic core and calcification and thereby improving atherosclerotic plaque stability.

Opacification of Hydrophilic Acrylic Intraocular Lenses: Overview of Laboratory Methods for Histological Analysis and Replication of IOL Calcification.

Opacification of intraocular lenses (IOLs) due to material changes is a serious complication that can compromise the good visual outcomes of uncomplicated cataract surgery. In hydrophobic acrylic IOLs, opacification can result from glistening formation, while in hydrophilic acrylic IOLs, there is a risk of calcification due to the formation of calcium phosphates within the polymer. Over time, various methods have been developed to investigate calcification in hydrophilic acrylic IOLs. The aim of this article is to provide an overview of standard histological staining and models used to simulate IOL calcification. Histological staining can be used to detect calcification and assess the extent of crystal formation. The development of in vivo and in vitro replication models has helped to identify the underlying pathomechanisms of calcification. In vivo models are suitable for assessing the biocompatibility of IOL materials. Bioreactors as an in vitro model can be used to investigate the kinetics of crystal formation within the polymer. The replication of IOL calcification under standardized conditions using electrophoresis allows for the comparison of different lens materials with respect to the risk of calcification. The combination of different analytical and replication methods can be used in the future to further investigate the pathomechanisms of calcium phosphate crystal formation and the influence of risk factors. This may help to prevent calcification of hydrophilic acrylic IOLs and associated explantation and complications.

#4000 ROLE OF ENDOTHELIAL CELLS IN VASCULAR CALCIFICATION

Abstract Background and Aims Vascular calcification (VC) represents a common pathological feature of cardiovascular disease and VC caused by hyperphosphatemia is one of the hallmarks of chronic kidney disease (CKD). Therefore, elucidating the pathogenesis of VC may have significant clinical benefits for CKD patients. Vascular smooth muscle cells challenged with high-phosphate (Pi) actively participate to VC by trans-differentiating into simil-osteoblastic cells that acquire the capability to deposit hydroxyapatite crystals in the extracellular matrix of tunica media in arteries. The role of endothelial cells (ECs) in high-Pi calcification has been poorly investigated until now. Nonetheless, following particular stimuli ECs can lose their endothelial characteristics and acquire several different phenotypes transdifferentiating with a process known as endothelial-to-mesenchymal transition (EndMT). Given the central role of VC for CKD patients wellbeing we decided to investigate whether ECs may have a role in VC process. Method We set up an in vitro calcification model with human aortic ECs (HAECs) challenged with 2, 2.5, 3, 3.5 mM high-Pi for 7 days. Calcium deposition, cell viability and cell trans-differentiation were evaluated. Results In our model we observed deposition of calcium phosphate crystals induced by high-Pi that resulted significant from 3 up to 3.5 mM Pi (Ctr 0.43 ± 0.01; 3 mM Pi 6.72 ± 1.15; 3.5 mM Pi 17.31 ± 1.88; micromol Ca++/mg protein; P < .01; Fig 1A-B). We found that calcification is mediated by Pi-influx, as demonstrated by the inhibition of calcium deposition by Phosphonoformic acid (PFA), an inhibitor of Na/Pi cotransporter Pit-1 (3.5 mM Pi 15.52 ± 1.97; 1 mM PFA 0.6 ± 0.05; micromol Ca++/mg protein; P < .0001; Fig 1A, C). In addition, in our model high-Pi did not show any significant toxic effect at every concentration tested compared to control cells. In Pi-treated ECs we observed a clear change in cell-morphology from cobblestone to spindle shaped, a recognized sign of EndMT. Moreover, we observed a change in extracellular matrix composition more similar to osteoblastic matrix. In facts, ECs treated with Pi were Alcian Blue positive, a staining that binds glycosaminoglycans contained in bone matrix, with blue granules of calcium-phosphate in the cytoplasm of treated cells. Starting from day 5 up to day 7 the transition was confirmed by an up-regulation of SNAIL and N-cadherin mRNA expressions, one of the master genes and one the markers of EndMT process, respectively. A modification of endothelial phenotype was also supported by a progressive decrease in protein expression of Von Willebrand factor and VE-Cadherin observed at day 9 of calcification (−53.7% and −32.6% respectively). Moreover, from day 5 till day 7 we found some osteoblastic differentiation signs such as a progressive increase of RUNX2 and BMP2 mRNA expression and an increase of protective MGP mRNA levels. Conclusion Our data demonstrate that HAECs are able to calcify. High-Pi induces trans-differentiation in simil-osteoblastic cells and a progressive loss of endothelial characteristics through the EndMT process. More studies are needed to better elucidate the role for ECs in CKD VC process.

Microstructural Properties and in vitro Dissolution of Microporous Bioactive 13-93B3 Glass Powders Synthesized via Solution Combustion Synthesis

This study investigates the bioactivity and microstructural properties of borate-based bioactive 13-93B3 glass produced by the solution combustion synthesis (SCS) method using urea and sucrose fuels at different reaction temperatures. The results show these glasses exhibit unique properties compared to conventionally-synthesized borate-based bioactive glasses. The glass particles produced have porous surfaces, with particle sizes below 100 nm. BET measurements reveal that the mesoporous structure of these glasses is characterized by high-specific surface areas, promoting a high ion release and hydroxyapatite layer formation in simulated body fluid (SBF). The crystallization of the amorphous calcium phosphate was investigated by 1H, 31P MAS, and 2D 31P{1H} heteronuclear correlation (HETCOR) spectroscopy, showing that it progresses linearly up to three days. The produced glasses were tested for their effects on the viability of 3T3 cells and were found to have no toxic effects. Therefore, the produced glasses are promising candidates for tissue engineering applications.

Urolithiasis pada Kucing Snow di K and P Clinic Surabaya

Urolithiasis is a disorder in the form of crystals or stones in the urinary tract such as the urinary bladder that occurs in both male and female animals. The condition of the formation of calculi due to supersaturation in urine consisting of one or several types of minerals. A case of urolithiasis in a Persian cat named Snow, female, 40 months old, 3.76 Kg, solid white hair color, came to the K and P Clinic and a physical examination was performed by palpation of the bladder which was filled with urine but not distended. Complete blood count (CBC) examination using blood samples showed leukocytosis, lymphocytosis, monocytosis, and thrombocytosis. The results of blood chemistry tests using blood serum samples showed hypoglycemic conditions and an increase in blood urea nitrogen (BUN) values. Calcium phosphate crystals are found on microscopic examination of the urine. Urinalysis test showed abnormal values ​​for ascorbic acid, microalbumin, protein, calcium, leukocytes, creatinine, blood, and protein to creatinine ratio. The formation of calculi looks like a hyperechoic mass and the presence of an acoustic shadow can be seen on ultrasonography (USG) examination of the urinary bladder. The handler given is cystotomy surgery.
 Keywords: Urolithiasis, calcium phosphate, ultrasound.

Hydrophobic surface properties of hydrophilic acrylic lenses do not protect against calcification

Opacification through calcification of hydrophilic acrylic intraocular lenses is aserious complication of cataract surgery, which usually results in explantation of the lens. In the process of calcification, the intraocular lens material plays acrucial role: calcification only occurs in hydrophilic acrylic lenses. Hydrophobic acrylic lenses show no crystal formation within the polymer. Hydrophilic acrylic lenses from some manufacturers have hydrophobic surface properties. The question arises as to what influence these surface properties have on the risk of calcification. The present study investigated whether the hydrophobic surface properties of hydrophilic acrylic lenses can prevent calcification. Using an electrophoretic in vitro model of calcification, two hydrophilic lenses with hydrophobic surface properties were compared to two hydrophilic lenses and ahydrophobic negative control to determine the risk of calcification. The lenses were then analyzed by optical microscopy, Alizarin Red and Von Kossa staining, scanning electron microscopy (SEM) and energy dispersive X‑ray spectroscopy (EDX). All four hydrophilic lens models showed calcification within the polymer. No difference was found between the hydrophilic lenses and the hydrophilic lenses with hydrophobic surface properties in terms of crystal formation. The hydrophobic negative control showed no calcification. The investigation conducted in this study under standardized conditions could show that hydrophobic surface properties of hydrophilic acrylic lenses do not protect against calcium phosphate crystal formation within the polymer. There also is arisk of calcification in these lens models.

Enhydra fluctuans Lour. aqueous extract inhibited the growth of calcium phosphate crystals: An in vitro study

Enhydra fluctuans Lour. is consumed by various tribes in their ethnomedicinal practices for the treatment of kidney stones and urinary problems. However, no scientific studies were conducted to evaluate its effect on crystal growth. Hence, the present study proposed to investigate the effect of aqueous extract of whole plant of standardized E. fluctuans (AEEF) on the growth of calcium phosphate (brushite) crystals. Attempts were also made to evaluate the effect of in vitro free radical scavenging and antimicrobial activities of AEEF. In vitro studies indicated that AEEF (50, 100, and 200 µg/mL) exhibited an inhibitory role on brushite crystal growth. The average length of the deposited brushite crystals was decreased by citric acid (1molL−1) and AEEF (200, 100, and 50 µg/mL) till day 8. Results showed that AEEF (200 and 100 µg/mL) and citric acid significantly (P<0.001) decreased the crystal bed as compared to the control on day 8 of the study. AEEF showed an antimicrobial effect against Staphylococcus aureus and exhibited antioxidant activity. In conclusion, the aqueous extract of the whole plant of Enhydra fluctuans was found effective in the inhibition of brushite crystals. Further, in vivo studies along with molecular studies and bioactivity-guided fractionation are required to strengthen its antilithiatic effect along with identifying the bioactive compounds and the mechanism of action involved therein.

Ultrasound Guided Arthroscopic Removal of Calcific Tendonitis: A Minimum of 2-Year Followup.

Background: We have developed a novel technique for managing rotator cuff calcific tendonitis, involving arthroscopic debridement of calcific tendonitis with localization assistance from a breast biopsy needle under ultrasound guidance. While we have demonstrated encouraging results at six-month follow-up, the medium-term outcomes and the long-term outcomes of this technique at 2 years or beyond are unknown. The aim of this paper was to determine if this technique was successful in resolving symptoms after two years and beyond. Study Design: Retrospective Cohort Study. Methods: Patients who underwent arthroscopic debridement of calcific tendonitis with localization assistance from a breast biopsy needle under ultrasound guidance by a senior surgeon were evaluated using patient-rated pain scores and functional status with the use of the Likert scales and via examiner-rated shoulder range-of-motion and strength at the pre-operative visit, at 1, 6, 12, and 24 weeks post-operatively, and long-term at a mean of 249 weeks after surgery. Results: At a mean follow-up period of 4.8 years (range, 2-10 years), 31 patients (33 shoulders) experienced significant improvement in the severity of pain at rest, with overhead activities, and during sleep compared to their pre-operative presentation (p < 0.001). The patient experienced less frequent pain during activities and sleep, and a decreased frequency of extreme pain (p < 0.001). Passive range of abduction (p = 0.003), forward flexion (p < 0.001), and supraspinatus strength (p = 0.018) improved compared to the presurgical presentation. Out of 27 patients, 24 patients (89%) had complete resolution of calcific tendonitis, and 26 patients (96%) had an intact rotator cuff. Conclusion: Arthroscopic debridement of calcific tendonitis with localization assistance from a breast biopsy needle under ultrasound guidance was very effective. Patients had significant pain relief, improved range of motion, and a reduction in stiffness at a mean post-operative period of 4.8 years. Patients had a significant reduction in residual calcification, and rotator cuff integrity was largely preserved by long-term follow-up. What is known about this subject: Calcific tendonitis of the rotator cuff is one of the most painful and debilitating disorders of the shoulder. This condition is characterized by the deposition of calcium-phosphate crystals within the rotator cuff tendons. Arthroscopic debridement and excision of rotator cuff calcifications have proven to be efficacious treatments with regards to clinical and functional outcomes in the short and medium term. Identifying the calcific lesion intra-operatively, however, can prove to be challenging. Furthermore, inadequate excision of the calcific deposit has been shown to have poorer clinical outcomes. We designed a technique that utilizes the assistance of ultrasound to guide a localization-biopsy wire to the calcific lesion. This technique aids in precisely identifying the location of the lesion intra-operatively to optimize accuracy in removing the maximum amount of calcific deposit possible. A short-term follow-up study by us has demonstrated successful outcomes with regards to the return of function and relief of pain. However, there have been no studies evaluating the effectiveness of this particular technique beyond six months. What this study adds to current knowledge: At a mean of 4.8 years, arthroscopic debridement of calcific tendonitis, using our technique, was successful in relieving the severity and frequency of pain with overhead activities, pain at rest, and pain during sleep, as well as improving range of motion.

Unravelling the Basic Calcium Phosphate crystal-dependent chondrocyte protein secretome; a role for TGF-β signaling

Basic Calcium Phosphate (BCP) crystals play an active role in the progression of osteoarthritis (OA). However, the cellular consequences remain largely unknown. Therefore, we characterized for the first time the changes in the protein secretome of human OA articular chondrocytes as a result of BCP stimulation using two unbiased proteomic analysis methods. Isolated human OA articular chondrocytes were stimulated with BCP crystals and examined by Quantitative Reverse Transcription PCR (RT-qPCR) and enzyme-linked immune sorbent assay (ELISA) after twenty-four and forty-eight hours. Forty-eight hours conditioned media were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) and an antibody array. The activity of BCP dependent Transforming Growth Factor Beta (TGF-β) signaling was analyzed by RT-qPCR and luciferase reporter assays. The molecular consequences regarding BCP-dependent TGF-β signaling on BCP-dependent Interleukin 6 (IL-6) were investigated using specific pathway inhibitors. Synthesized BCP crystals induced IL-6 expression and secretion upon stimulation of human articular chondrocytes. Concomitant induction of catabolic gene expression was observed. Analysis of conditioned media revealed a complex and diverse response with a large number of proteins involved in TGF-β signaling, both in activation of latent TGF-β and TGF-β superfamily members, which were increased compared to non-stimulated OA chondrocytes. Activity of this BCP driven TGF-β signaling was confirmed by increased activity of expression of TGF-β target genes and luciferase reporters. Inhibition of BCP driven TGF-β signaling resulted in decreased IL-6 expression and secretion with a moderate effect on catabolic gene expression. BCP crystal stimulation resulted in a complex and diverse chondrocyte protein secretome response. An important role for BCP-dependent TGF-β signaling was identified in development of a pro-inflammatory environment.

Regression of a Primary Pseudo-Tumoral Calcinosis Progressing During 16 Years under Combined Treatment with Acetazolamide and Aluminum Hydroxide: A Case Report

Primary pseudo-tumoral calcinosis is a rare genetic disease due to a disorder of phosphorus metabolism characterized by the formation and deposition of calcium phosphate crystals in the peri-articular regions. The treatment is based on the reduction of hyperphosphatemia by hygienic and nutritional measures, hypophosphatemic drugs and surgery. We report the case of a 19-year-old patient followed since the age of 3 years for a primary pseudo-tumoral calcinosis that was resistant to surgery and that had well progressed with aluminum hydroxide and acetazolamide. Treatment combining aluminum hydroxide and acetazolamide with hygienic and nutritional measures seems to be an interesting therapeutic option for this pathology.

MP05-16 THE DEVELOPMENT OF A THREE-DIMENSIONAL KIDNEY SPHEROID MODEL TO STUDY CALCIUM-BASED CRYSTAL FORMATION

You have accessJournal of UrologyCME1 Apr 2023MP05-16 THE DEVELOPMENT OF A THREE-DIMENSIONAL KIDNEY SPHEROID MODEL TO STUDY CALCIUM-BASED CRYSTAL FORMATION John Chmiel, Wongsakorn Kiattiburut, Aaron Kwong, Priyanka Prabhu, Hassan Razvi, Leonard Luyt, Jeremy Burton, and Jennifer Bjazevic John ChmielJohn Chmiel More articles by this author , Wongsakorn KiattiburutWongsakorn Kiattiburut More articles by this author , Aaron KwongAaron Kwong More articles by this author , Priyanka PrabhuPriyanka Prabhu More articles by this author , Hassan RazviHassan Razvi More articles by this author , Leonard LuytLeonard Luyt More articles by this author , Jeremy BurtonJeremy Burton More articles by this author , and Jennifer BjazevicJennifer Bjazevic More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003216.16AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: An estimated 10% of North Americans suffer from nephrolithiasis. With rising incidence rates, there is a strong need for models to study the pathophysiology of stone disease. In vitro cell culture models are essential for mechanistic and high-throughput analysis of stone formation and drug treatment; however, there are no suitable models that adequately represent the pathophysiology of stone disease. Advancements in cell culture have given rise to three-dimensional cysts, which are hollow spheroid structures that have apical to basal polarization. These culture systems have similar physiochemical environments to in vivo models and can be cultured for the extended times required for stone formation. Here, we developed a novel 3D kidney cyst model that can generate calcium-based kidney stones. METHODS: Madin-Darby canine kidney (MDCK) cells were cultured in suspension in growth factor-reduced Matrigel to generate hollow spheroid cysts. Crystal formation was induced by culturing cysts in a custom crystal-forming media for two weeks. Immunofluorescent staining was used for characterizing and visualizing the three-dimensional structure. Alendronate-fluorescein, a fluorescently labelled bisphosphonate that specifically binds to calcium-containing crystals, was used along with plain polarized light to visualize the crystal structures. RESULTS: Stains for cellular components and structures demonstrated that the luminal spheroids were successfully formed under the culture conditions. The spheroids also have basal-to-apical polarization suggesting that crystals will form on the inside lumen of the hollow cyst. Birefringent structures resembling calcium oxalate dihydrate and calcium phosphate crystals were observed embedded along the apical face of the spheroid and protruding into the lumen. Alendronate-fluorescein staining confirmed the presence of calcium-containing crystals in the lumen of the cyst. CONCLUSIONS: We have demonstrated that hollow MDCK spheroids cultured with crystal-forming media can generate calcium-containing crystals in the spheroid lumen. This model is the first in vitro tissue culture-based model to reliably develop calcium-based crystals and can be used to investigate their pathogenesis or to perform high-throughput drug screening. Furthermore, it paves the way for personalized medicine by building the foundation for a cell system where renal tissue can be harvested and cultured to identify individual intrinsic risk factors that might contribute to stone development. Source of Funding: Canadian Institutes of Health Research (CIHR) CGS-D, The Northeastern Section of the American Urological Association (NSAUA), Lawson Health Research © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e49 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information John Chmiel More articles by this author Wongsakorn Kiattiburut More articles by this author Aaron Kwong More articles by this author Priyanka Prabhu More articles by this author Hassan Razvi More articles by this author Leonard Luyt More articles by this author Jeremy Burton More articles by this author Jennifer Bjazevic More articles by this author Expand All Advertisement PDF downloadLoading ...

Zinc in microscopic calcifications isolated from thyroid fine needle aspiration may serve as a biomarker of thyroid nodule malignancy: A promising proof-of-concept.

Thyroid nodules (TNs) are common neck ultrasonography (US) findings, yet only 5-10% of these nodules harbor thyroid cancer (TC). When US characteristics are consistent with an intermediate or high suspicion for TN malignancy, fine needle aspiration for cytology (FNAC) is indicated. The main limitation of FNAC is that cytological results can be indeterminate in up to 30% of cases, necessitating reevaluation through repeated FNAC, expensive molecular testing, or diagnostic thyroid lobe resection. As such, there is a need for further refinement of current diagnostic algorithms for TNs without subjecting patients to additional invasive procedures. As calcifications detected during thyroid US are considered a high-risk feature for malignancy, we used the material remaining following routine thyroid FNAC to isolate microscopic calcifications (MCs). We then characterized the elemental composition, morphology, and crystal phases of these MCs, ultimately revealing differences between the MCs from benign and malignant TNs. Specifically, thyroid MCs were identified as calcium phosphate crystals containing varying levels of magnesium, sodium, iron, and zinc. MCs obtained from malignant TNs, mainly papillary thyroid carcinoma, were composed of sub-micrometer spherical particles, whereas MCs from benign TNs consisted of faceted particles. While samples from most patients with a final diagnosis of malignant TNs (50% of them with indeterminate cytology) harbored zinc-containing MCs, zinc was largely absent in MCs from benign TNs (23% with indeterminate or non-diagnostic cytology). Together, these data suggest that the presence of zinc in MCs isolated from samples collected during routine FNAC may potentially offer value as a biomarker of TN malignancy. STATEMENT OF SIGNIFICANCE: As up to 40% of patients assessed for thyroid malignancy do not receive a definite diagnosis following thyroid nodule (TN) fine needle aspiration (FNA), there is a pressing need to improve the accuracy of current diagnostic algorithms. Chemical analyses of microscopic calcifications (MCs) may serve as a diagnostic target. We developed a straightforward protocol to chemically characterize MCs from excess material collected from TNs during routine FNA and found that these MCs differed between benign and malignant TNs. Specifically, zinc in TN-derived MCs may indicate a higher nodule malignancy risk, thus increasing the diagnostic accuracy of the FNA procedure, reducing the need for recurrent biopsies and diagnostic surgical procedures, and decreasing the costs, uncertainty, and stress faced by affected patients.

Bioinformatics analysis reveals the potential role of matrix metalloproteinases in immunity and urolithiasis.

The pathogenesis of urolithiasis remains unclear, making the development of medications for treatment and prevention stagnant. Randall's plaques (RPs) begin as interstitial calcium phosphate crystal deposits, grow outward and breach the renal papillary surface, acting as attachment for CaOx stones. Since matrix metalloproteinases (MMPs) can degrade all components of extracellular matrix (ECM), they might participate in the breach of RPs. Besides, MMPs can modulate the immune response and inflammation, which were confirmed to be involved in urolithiasis. We aimed to investigate the role of MMPs in the development of RPs and stone formation. The public dataset GSE73680 was mined to identify differentially expressed MMPs (DEMMPs) between normal tissues and RPs. WGCNA and three machine learning algorithms were performed to screen the hub DEMMPs. In vitro experiments were conducted for validation. Afterwards, RPs samples were classified into clusters based on the hub DEMMPs expression. Differentially expressed genes (DEGs) between clusters were identified and functional enrichment analysis and GSEA were applied to explore the biological role of DEGs. Moreover, the immune infiltration levels between clusters were evaluated by CIBERSORT and ssGSEA. Five DEMMPs, including MMP1, MMP3, MMP9, MMP10, and MMP12, were identified between normal tissues and RPs, and all of them were elevated in RPs. Based on WGCNA and three machine learning algorithms, all of five DEMMPs were regarded as hub DEMMPs. In vitro validation found the expression of hub DEMMPs also increased in renal tubular epithelial cells under lithogenic environment. RPs samples were divided into two clusters and cluster A exhibited higher expression of hub DEMMPs compared to cluster B. Functional enrichment analysis and GSEA found DEGs were enriched in immune-related functions and pathways. Moreover, increased infiltration of M1 macrophages and enhanced levels of inflammation were observed in cluster A by immune infiltration analysis. We assumed that MMPs might participate in RPs and stone formation through ECM degradation and macrophages-mediated immune response and inflammation. Our findings offer a novel perspective on the role of MMPs in immunity and urolithiasis for the first time, and provide potential biomarkers to develop targets for treatment and prevention.

Effect of nozzle diameter and cross-linking on the micro-structure, compressive and biodegradation properties of 3D printed gelatin/collagen/hydroxyapatite hydrogel

Since the existing polymeric hydrogel inks lack printability, shape fidelity and the desired mechanical properties for bone tissue regeneration, a hydrogel comprised of gelatin (G), collagen (C), and hydroxyapatite (H) nanoparticles is utilized for extrusion-based 3D printing. The rheological characterization of the composite GCH inks was performed to evaluate their printability, while the cuboid column model scaffolds were printed at a printing speed of 8 mm/s by using different needle inner diameters (500 μm/21G, 250 μm/25G, and 200 μm/27G), followed by carbodiimide induced crosslinking for 12 or 24 h. The samples were tested for their micro-structure, swelling, compression and stiffness performance, before and after incubation in an HBSS solution for up to 14 days. The wall diameter of a 3D printed scaffold decreases and pore size increases primarily with the decreasing inner diameter of the nozzles, and secondarily by increasing the crosslinking time. This was supported with their swelling capacity and the creation of new CaP crystals on the scaffold walls' top surfaces by the time of incubation, necessary for cells’ adhesion, proliferation and growth.The compressive modulus and stiffness of the scaffolds increases proportionally with the increase of their wall diameter and the time of crosslinking, and is inversely proportional to their pores size. The scaffold with the smaller pores provides superior modulus and stiffness, even after 14 days of incubation in the physiological solution (i.e. from ∼0.94 to ∼0.71 MPa and from 17 to 20 kPa to 5–9 kPa, respectively). This is comparative with the reported values for gelatin-based composites, and in the range for hard tissue regeneration at non-load bearing sites, as well as cartilage applications.

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 limits 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.

Competition and Neutralization: Thermally Induced Crystallization and Phase Evolution of Amorphous Calcium Phosphate with Cosubstitution of Larger and Smaller Divalent Cations.

The presented study evaluated the effect of cosubstitution of larger and smaller divalent cations on the thermally induced crystallization of amorphous calcium phosphate (ACP). The predesigned combinations of larger (Sr2+ and Ba2+) and smaller (Mg2+, Cu2+, and Co2+) divalent cations were carried out and their effects on the thermodynamic equilibrium between α/β-tricalcium phosphate (TCP) were outlined. The coexistence of larger and smaller divalent cations shielded the formation of α-TCP and shifted the thermodynamic equilibrium toward the β-TCP, which implied that the smaller cations dominated the crystalline phase. However, the retarded crystallization induced by the larger cations still remained and allowed ACP to maintain its amorphous nature partly or completely until a higher temperature.

A Proteomic Screen to Unravel the Molecular Pathways Associated with Warfarin-Induced or TNAP-Inhibited Arterial Calcification in Rats

Arterial media calcification refers to the pathological deposition of calcium phosphate crystals in the arterial wall. This pathology is a common and life-threatening complication in chronic kidney disease, diabetes and osteoporosis patients. Recently, we reported that the use of a TNAP inhibitor, SBI-425, attenuated arterial media calcification in a warfarin rat model. Employing a high-dimensionality unbiased proteomic approach, we also investigated the molecular signaling events associated with blocking arterial calcification through SBI-425 dosing. The remedial actions of SBI-425 were strongly associated with (i) a significant downregulation of inflammatory (acute phase response signaling) and steroid/glucose nuclear receptor signaling (LXR/RXR signaling) pathways and (ii) an upregulation of mitochondrial metabolic pathways (TCA cycle II and Fatty Acid β-oxidation I). Interestingly, we previously demonstrated that uremic toxin-induced arterial calcification contributes to the activation of the acute phase response signaling pathway. Therefore, both studies suggest a strong link between acute phase response signaling and arterial calcification across different conditions. The identification of therapeutic targets in these molecular signaling pathways may pave the way to novel therapies against the development of arterial media calcification.