Cai Jiye, Chen Yao, Li Renqiang#, Xia Ke
(1 Department of Chemistry, Jinan University, Guangzhou 510632, China; #Department of Biotechnology, Jinan University, Guangzhou 510632, China)
Supported in part by Grants-in Aid for Scientific Research from Guangdong Province, China (990483), the Ministry of Education, China (00124), and the National Natural Science Foundation of China (60278014 and 30230350).
Abstract Crystallization of calcium oxalate with the self-assembly of organic matrix chondroitin sulfate film and aggregation of these crystals on the film were studied using atomic force microscope. Results showed that the formation and growth of calcium oxalate crystal were induced or controlled by chondroitin sulfate molecule, and further these crystals aggregated to form Liesegang loop on the film, which were due to the interaction between calcium oxalate and chondroitin sulfate, especially to the reaction of calcium ion with those functional groups. The formation of Liesegang loop of calcium oxalate was related to the concentrations of calcium oxalate and chondroitin sulfate. These results would be in favor of the understanding to the mechanism of mineral crystal growth controlled by organic matrix and to the formation process of urinary calculus in human body due to that the calcium oxalate is the main component of urinary calculus and the chondroitin sulfate is an important component in human urine.
Keywords Calcium oxalate, chondroitin sulfate, atomic force microscope, crystallization, Liesegang loop.
Studies on the formation and growth of mineral constituent crystal in mimic organic matrix system help us to understand the mechanism of calculus in human body. Some studies on this field, especially on the formation of mineral crystal induced by LB film, and on the Liesegang loop formed by these mineral crystal have been performed [1-3]. However, those organic substances existed in human body and their self-assembly process may be related to the crystallization and aggregation of mineral crystal. Therefore, it is helpful to use those organic substances existed in human body as the matrix to probe the formation and growth of mineral crystal.
Urinary calculus is a normal disease happened in human. There are mineral and organic constituents in the calculus, of which calcium oxalate is the main mineral component. It is still a puzzle about the mechanism to form the urinary calculus, so that in this study, the crystallization of calcium oxalate accompanying the self-assembly of chondroitin sulfate molecules and its aggregation induced by chondroitin sulfate film are imaged with atomic force microscope (AFM) due to chondroitin sulfate is an important organic component in human urine, which is not yet presented in the previous literatures. This study is evidently in favor of our probing to the formation mechanism of urinary calculus in human body.
2.1 CaC2O4 crystallization controlled by chondroitin sulfate
Figure 1 showed the AFM images of crystallization process and aggregation of calcium oxalate accompanying the self-assembly of chondroitin sulfate film on mica. Calcium oxalate small balls scattered at first (Fig. 1 A) and gradually aggregated to form the crystal (Fig. 1 B and C), which was evidently induced or controlled by chondroitin sulfate due to that these crystals showed regular and some regular hexagon crystals were formed (Fig. 1 D, E and F) but at the control without chondroitin sulfate, the growth of calcium oxalate crystal or the crystal shape lacked the regularity (Fig. 2). These indicated that chondroitin sulfate had a great effect on the formation of calcium oxalate crystal.
Fig. 1 AFM images of crystallization and aggregation of calcium oxalate accompanying the self-assembly of chondroitin sulfate film on mica. Scanning scopes: A. 5mm; B. 5mm; C. 15mm; D. 25mm; E. 15mm; F. 25mm.
Fig. 2 AFM images of crystallization and aggregation of calcium oxalate in purified water on mica. Scanning scopes: A. 5mm; B. 1.7mm; C. 7mm.2.2 Aggregation of CaC2O4 crystals on chondroitin sulfate film
After two days, crystals produced by the supersaturated solution of calcium oxalate on chondroitin sulfate film were imaged with AFM and shown in the Figure 3. The crystals aggregated to form many loops that relatively dispersed even on the film. These loops were almost in the same shape, composed of hexagon, quadrilateral or irregular crystals, but their diameters might display difference. The action of these calcium oxalate crystals was evidently a Liesegang phenomenon , it indicated also that the aggregation of calcium oxalate crystals was induced or controlled by chondroitin sulfate.Fig. 3 AFM images of aggregation of calcium oxalate crystals on chondroitin sulfate film.
Scanning scopes: A. 16mm; B. 10mm; C. 14mm.
The three-dimensional image to the loop and its height curve map were shown in Figure 4. It showed that the crystals size and their arrangement in the loop were relatively regular, which explained also the affecting role of chondroitin sulfate to the crystallization and aggregation of calcium oxalate.
Fig. 4 Three-dimensional AFM image of Fig. 3 B and its height curve map.
After further scanning in small scope at the center of the loops (Fig. 5), some calcium oxalate small balls were found to be embedded in the meshes formed by chondroitin sulfate film itself and not to participate the loop construction.
Fig. 5 AFM images of calcium oxalate crystal embedded in the meshes of chondroitin sulfate film. Scanning scopes: A. 2.5mm; B. 1.5mm; C. 1.2mm.
From this study, the crystallization of calcium oxalate and its aggregation are evidently related to the self-assembly of chondroitin sulfate molecules or its film. Although this study cannot explain how the chondroitin sulfate molecules induce the crystallization of calcium oxalate and its aggregation, or the chemical reaction between them, based on the property of chondroitin sulfate molecule that is an acid mucopolysaccharide with electronegative due to the existence of a lot of anion groups, such as hydroxy, carboxy in the molecule, it may adsorb calcium cation at the specific place  and favor the formation of the crystal core . In addition, the interaction between calcium cation and oxygen or nitrogen atom in chondroitin sulfate molecule can fix calcium oxalate at the specific point on the film. These form the foundation to induce or control the formation and growth of calcium oxalate crystal. These controlled growth include the formation in size, shape and structure of the crystal. The comparison in the growth of the crystal between the sample (Fig. 1, calcium oxalate add chondroitin sulfate) and the control (Fig. 2, calcium oxalate add water) supports this explanation.
As the formation of mature crystal with certain size, it begins moving on the film driven by the diffusion force. During the moving, the crystal is also controlled or affected by the film owing to the interaction between crystal and chondroitin sulfate molecule. It is the chemical interaction and diffusion force, we consider, that drive the crystals to contact and connect each other, and aggregate regularly to form Liesegang loop. However, from our studies, the formation of Liesegang loop is related to the concentrations of calcium oxalate and chondroitin sulfate. Suitable concentration of chondroitin sulfate and supersaturated calcium oxalate are two key conditions to form Liesegang loop. When the concentration of chondroitin sulfate is just fit for forming reticulation (1.0mg/mL), it favors the formation of Liesegang loop of calcium oxalate. But if the concentration of calcium ion is not enough, Liesegang loop cannot form even if the concentration of chondroitin sulfate is suitable. The concentration relationship between calcium oxalate and chondroitin sulfate for Liesegang loop formation explain the complex process for the aggregation of mineral crystal induced or controlled by organic matrix, or the relatively strict condition to form urinary calculus in life body. These may mean that not only need enough mineral constituent but also the organic matrix has to be suitable for the formation and growth of calculus in life body.
On the other hand, those too small balls of calcium oxalate cannot move to aggregate due to they have been embedded in the meshes of the film as shown in Figure 5. These mean the existence of interaction between crystals and chondroitin sulfate molecules, and also that the crystal size is too small, the diffusion force of them is too weak to move on the film. This is to say that it needs enough concentration for mineral constituent for their aggregation in organic matrix system.
This study has presented a formation process of calcium oxalate mineral crystal and its characteristics with the existence of chondroitin sulfate organic matrix that is an important component of human urine, and also has given out the concentration relationship between calcium oxalate and chondroitin sulfate for the formation of Liesegang loop. These may help us to understand the formation process of urinary calculus in human body. Evidently, more detailed studies are needed due to the complex process for the formation and growth of mineral crystal induced or controlled by organic matrix although our study has started the step on this direction.
Chondroitin sulfate can induce or control the formation and growth of calcium oxalate crystal. These crystals may aggregate to form Liesegang loop on chondroitin sulfate film, but the formation of Liesegang loop needs suitable concentration of chondroitin sulfate and supersaturated calcium oxalate. The affecting role to calcium oxalate crystal by chondroitin sulfate is ascribed to their interaction produced mainly by calcium ion with those functional groups in chondroitin sulfate molecule.
Chondroitin sulfate was purchased from Sigma, and 0.25g of the compound was weighed and dissolved in 250 ml purified water. Calcium oxalate (in AR) of 0.6mol was made. A sample of 5mL of chondroitin sulfate and calcium oxalate, respectively, were put at the same time on the surface of fresh mica treated well by using NiCl2, dried naturally at room temperature, then imaged with atomic force microscope (AFM) once every four hours, which was for the probing to the growth of calcium oxalate crystal accompanying the self-assembly of chondroitin sulfate. In the control, chondroitin sulfate was replaced with purified water.
For the investigation of the action of these crystals on the film, due to that the crystals on the film in the treatment above was too dilute, so that the 5mL of supersaturated solution of calcium oxalate was put on the chondroitin sulfate film, imaged with AFM after two days.
AFM (Autoprobe CP Research, THERMO MICROSCOPE, USA) was performed with a APSC-0100 scanner, Si3N4 probe needle, microarm length 180mm, force constant 3.2 N/m, imaged with tapping mode. All images were treated with the Flatten Auto to remove the low-frequency noise in the scanning direction.