Our study revealed that three methods could successfully prepare monosodium urate (MSU) crystals, but ease of preparation varies, the degree of inflammation caused by them is different. The prepared MSU crystals were basically consistent with clinical MSU crystals or commercial MSU crystals in terms of shape and degree of inflammation, except at individual time points. The degree of inflammation induced by MSU crystals prepared by neutralization and alkalization methods is greater than by the acid titration method, but the quantity of MSU crystals obtained by the alkaline method was higher and less time-consuming. Overall, MSU crystals prepared by the alkaline method should be recommended to study the molecular mechanism of gout in animal and cellular models.

Monosodium urate (MSU) monohydrate (NaC5H3N4O3.H2O), in which a urate molecule binds to sodium and a water molecule, is one of the most common forms of crystalline urate, the main sediment in gouty arthritis [18]. MSU crystals viewed by microscope were needle-shaped, with a triclinic structure containing three unequal axes, none of which are perpendicular to the others [19]. The general physicochemical process of MSU crystallization formation is similar to that of other crystal formation processes, and the formation conditions depend on the urate concentration and other related factors, such as the solubility limit, approximately 6.8 mg/dL (405 µmol/L) under normal physiological conditions [20, 21]. Beyond this concentration, supersaturation causes the formation of MSU crystals to begin, with further changes in urate solubility or other excitation events and further expansion of crystallization occurring, depending on local conditions. Multiple factors have been found to influence the formation of MSU crystals, including temperature, sodium and cation concentrations, pH, mechanical stress, cartilage composition, uric acid-binding antibodies, and cartilage and synovial fluid components [21,22,23]. When the conditions affecting MSU solubility change (e.g., increased concentration and/or decreased temperature), MSU molecules accumulate in solution [24]. Given the importance of MSU crystals in studying the pathogenesis of gout diseases, the preparation method of urate crystals is very important.

The preparation of MSU crystals is often the first step in studying gout-related mechanisms [25]. In most experiments, a small number of MSU crystals used natural tophi extracted from the human body, and most of them were artificially prepared and synthesized. It is necessary to explore an efficient and facile method to prepare MSU crystals. However, there are few previous studies on the elaboration of the characteristics of different MSU crystal preparation methods and comparing their advantages and disadvantages. Our study reported three main urate preparation methods: neutralization, alkaline, and acid methods [13, 17, 26,27,28]. This study intends to compare these three different preparation methods, from their degree of simplicity, the uniformity of uric acid crystal length, crystal acquisition rate, and the degree of the induced inflammatory response, to obtain simple, efficient MSU crystals with better inflammatory effect.

We compare the lengths and quantities of MSU crystals prepared from the three MSU crystal preparation methods. The mouse foot pad and air pouch model were used to compare the differences in the inflammatory response induced by crystals from the three different MSU preparation methods in mice. The results showed that Urate crystals were prepared using the same quality of uric acid, with the highest quality obtained by alkaline titration, followed by the acid method, and the lowest by neutralization. The lengths of MSU crystals prepared by the three preparation methods were classified according to < 50 μm, 50 ~ 100 μm, 100 ~ 150 μm, 150 ~ 200 μm and > 200 μm, and the proportion of MSU crystals in each length interval in the three preparation methods was counted. The uniformity of urate crystals prepared by acid titration is relatively poor, and the crystals with large lengths account for more, the proportion of the crystals larger than 200 μm and shorter than < 50 μm was significantly higher than the remaining two groups. The uniformity of MSU crystals prepared by the alkali titration and neutralization methods was better. And the average length of MSU crystals obtained by acid titration is the longest, and no significant difference was observed in the shape of crystals obtained with the other two methods. In an in vivo study, compared with injecting larger MSU crystals into the knee, injecting smaller MSU crystals into the knee of dogs resulted in a stronger inflammatory response, higher phagocytosis rate, and increased leukocyte infiltration [29]. Ashika Chhana also found that human cartilage homogenate will increase the formation of MSU crystals, promote the formation of smaller crystals, and enhance the inflammatory response [30]. These studies suggest that altering the size of MSU crystals may affect the inflammatory response elicited [21, 30]. In this study, the window of inflammation triggered by acid titration preparation is shorter compared to other forms of crystal preparation. Certain previous studies have shown that the window of inflammation induced by MSU crystals is not exactly the same. TNF-α level in cell culture supernatants reached peak at 15 or 18 h, and MSU crystal-induced IL-1β secretion levels peaked at 6 or 8 or 48 h [16, 31,32,33] .

There are some limitations to this study. Firstly, the current study weakly investigated the inflammation triggered by MSU crystals, leaving out the inflammasome pathway, the key intracellular signaling activated by these crystals, and later time points of investigation. Secondly, the detection indicators are not comprehensive enough.

In conclusion, by comparing three urate crystal synthesis methods, the alkali titration method has high repeatability, good uniformity, simple production procedures, few steps, is less time-consuming, and the induced inflammation model is stable. The window of inflammation triggered by acid titration preparation is shorter compared to other forms of crystal preparation. But it needs to show the differences among the time points in the groups. It has an important significance for the in vivo studies, especially those testing anti-inflammatory drugs. It will help us to better study the mechanism of gout and formulate treatment strategies in the future. However, the differences in the gouty inflammation induced by MSU prepared by different methods remain poorly studied. Exploring a simple, efficient, time-saving, and highly inflammatory MSU crystal is significant for animal and cell experiments. At present, we only discuss the above three MSU crystal preparation methods, and we may find better MSU crystal preparation methods in the future.