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Establishment of an artificial urine model in vitro and rat or pig model in vivo to evaluate urinary crystal adherence – Scientific Reports

Ureteral stents, which are used to prevent urinary tract obstruction in urological patients, are prone to obstruction due to urinary crystal adhesion1,2. This study describes an artificial urine model and an animal model to evaluate the effectiveness of new ureteral stents designed to prevent stone adhesion to reduce stent-related adverse events such as pain, infection, bloody urine, and irritative bladder symptoms21,22. In the development of novel biomaterials for stents, rapid and reproducible in vitro testing procedures are desired to facilitate the evaluation of potential materials. In vitro models have been used primarily to assess stent encrustation, durability, and bacterial resistance. While these models provide valuable insight into the underlying mechanisms of biofilm formation and incrustation, they are far from reflecting the true state of affairs within the urinary tract. Many authors argue that static models using artificial urine do not mimic complex human urinary tract models23. However, Dynamic models such as the Modified Robbins Device (MRD) and the Center for Disease Control (CDC) biofilm reactor are essential for prototype testing, but not for screening testing. In addition, contamination is a problem for dynamic models. In addition, the cost of experiments is high24,25. We suggest that our experimental models are a static model that is quick and easy to use as a screening test to evaluate new stent materials.

Crystal adhesion to ureteral stents is related to pH, solubility and supersaturation. Crystal formation depends on solubility and supersaturation, and the relationship between supersaturation and crystal nucleation and growth is defined by the equation proposed by Nyvlt26. Increasing supersaturation increases crystal nucleation and growth. Artificial urine with a pH of 6.00 at the start of the experiment is weakly acidic, within the reference range of human urine pH, and contains ammonium chloride and urea in its composition26.

Ammonium chloride contains ammonia. These slightly precipitated ammonium ions increase the pH of the solution, causing calcium oxalate and calcium phosphate to become supersaturated and crystals to precipitate on the surface of the ureteral stent27,28. The solubility of calcium oxalate is 6.7 mg/ L. The solubility of calcium phosphate is 3.1 mg/ L29. The three types of artificial urine contain calcium chloride, sodium oxalate and sodium dihydrogen phosphate above the solubility level. However, comparing crystal adhesion in artificial urine, model 1 has higher concentrations of calcium chloride compared to artificial urine models 2 and 3. Also, since artificial urine model 3 is based on the average urine composition of a healthy person, it has a lower Ca concentration than model 1 and 210,11,12. Pak et al. found that crystal growth of brushite increased with an increase in Ca concentration10, and indeed artificial urine model 1 had the most crystals attached to the ureteral stent during the 4-week period.

Evaluating the need for urea in the artificial urine model 1, the pH of artificial urine containing urea increased due to hydrolysis of urea contained in the artificial urine to produce carbon dioxide and ammonia. Also, Urea is isomeric to ammonium cyanate, producing a small amount of cyanide and ammonium ions in the artificial urine30. This causes a rapid increase in the pH of the artificial urine. Crystals grow from crystal nuclei on the surface of the stent, The urea-containing artificial urine had a strong pH increase, whereas the urea-free artificial urine, which had a slower pH increase, produced crystal nuclei on the surface of the ureteral stent as a starting point for crystal growth. Calcium oxalate crystals and calcium phosphate crystals account for the majority of renal stones31,32. We suggest that a gradual increase in the pH of the solution due to slightly precipitated ammonium ions leads to supersaturation of calcium oxalate crystals and calcium phosphate crystals and the precipitation of crystals on the surface of the ureteral stent26. However, If the pH and alkalinity of artificial urine containing urea increases too rapidly, the precipitation of magnesium ammonium magnesium phosphate crystals increases, conditions that are not suitable for calcium crystals precipitation33. Therefore, we suggest that conditions with a gradual increase in pH without urea are more suitable for calcium crystals formation than conditions with a rapid increase in pH, such as those containing urea.

When the starting pH of artificial urine was raised to pH 6.30, crystal formation seemed to be rapid and crystallization seemed to occur. When the pH was raised, the precipitation of calcium crystals accelerated, and the number of crystals adhering to the ureteral stent also increased. During the 5 week immersion, Ca on the ureteral stent immersed in artificial urine with a starting pH of pH 6.30 was also higher than in artificial urine with a starting pH of pH6.00, pH6.10 and pH6.20, indicating that crystal adherence to ureteral stents correlates with Ca.

As shown in the supplementary material, SEM–EDS analysis (C, O, Ca, P) suggested that calcium phosphate crystals were deposited. However, when combined with the fact that oxalic acid was found at 8.67 µg/cm, we consider that calcium oxalate as well as calcium phosphate was deposited simultaneously34.

In the rat experimental model, since the crystals found in urine on day 35 and on day 42 had the octahedral structure of calcium oxalate crystals35. This was because ethylene glycol was metabolized into hyperoxaluria, resulting in the formation of calcium oxalate crystals in the urine. However, ethylene glycol and some of its metabolites are nephrotoxic, so it was necessary to monitor body weight during the experiment14,19. Jarald et al. used 0.75% ethylene glycol and induced kidney stones in 28 days in their study, but this study used a lower concentration of 0.4% ethylene glycol and was able to induce kidney stones in 35 days14. Renal stones were induced even at low concentrations of ethylene glycol because the additional administration of calcium chloride promoted hypercalciuria and the deposition of calcium oxalate crystals36.

The pigs did not increase exponentially with respect to their weight, but we suggest this was due to the fact that they were fed a high fiber diet that did not affect the study design. Soria F et al. suggest that ideally, interventions should be carried out on about 30 kg models, as the dimensions of their urinary tract at that weight are comparable to a human adult15,16.

The increase in urinary crystals in the pig fed 5% hydroxyproline was due to hyperoxaluria, as hydroxyproline, a precursor of oxalic acid, was metabolized to oxalic acid, and Ca in the urine promoted calcium oxalate crystals17,18,19,20. The increase in urinary crystals in the pig fed 1% ethylene glycol and vitamin D was also due to hyperoxaluria, as ethylene glycol was metabolized to oxalic acid, and the additional administration of vitamin D promoted calcium oxalate crystals by increasing the rate of Ca absorption in the body. The fact that the maximum urinary Ca value was higher in the pig with 1% ethylene glycol and vitamin D also indicates that vitamin D promoted Ca absorption in the body14,28. The lack of correlation between urinary crystals and urinary Ca concentrations is due to the fact that when crystals were counted under a microscope, they were counted regardless of size, and because of weak magnification, magnesium ammonium phosphate crystals and other urinary crystals were counted.

The fact that Ca deposition was greater on the renal side than on the ureteral or bladder side is reasonable because most clinically important stones grow in the renal papillae34,37,38.

A model of crystal deposition in urine could be established for both hydroxyproline and ethylene glycol + vitamin D. Ethylene glycol + vitamin D may be more likely to increase urinary Ca concentrations and precipitate calcium oxalate crystals. However, ethylene glycol and some of its metabolites are nephrotoxic and body weight should probably be monitored during the experiment.

Artificial urine of several compositions is sensitive to pH, temperature and environment. Also, the crystals observed under the microscope were crystals adhering to the outside of the ureteral stent, and not all adhering crystals could be observed, as crystals normally adhere to the inside of the ureteral stent. However, the results correlated with the quantitative results for Ca on the ureteral stent in vitro, which is a useful comparison. The in vivo experiment was also valuable because it allowed the potabilization of a model in which crystals in the urine adhered to the stent surface in a short period of time, although the n was small. In addition, we have not tested the urine culture, and had no control group. In the future, the number of animal models will be increased and urine samples will be analyzed in more detail (in vivo pH, urinary leukocyte count, urinary bacterial count, presence of infection, etc.) with the check of urine culture and set of control group.

In conclusion, in vitro crystal adhesion to the surface of ureteral stents can be expected when the ureteral stent is immersed for a period of 5 weeks at an initial pH of 6.30 using the urea-free artificial urine model 1. In vivo, in the rat experimental model crystals were found in the urine after 35 days when 0.4% ethylene glycol and 1% ammonium chloride was administered. In the pig experimental model, urinary crystals increased when either 5% hydroxyproline or 1% ethylene glycol + vitamin D was administered, but urinary Ca was more likely to increase in the model with 1% ethylene glycol + vitamin D. This in vitro and in vivo experimental model can be used to evaluate the effectiveness of newly developed ureteral stents for preventing crystal adhesion, as part of the development of improved ureteral stents.