Spatiotemporal and Microscopic Analyses of Asymmetric Liesegang Bands: Diffusion-Limited Crystallization of Calcium Phosphate in a Hydrogel

Abstract

In the more than 100 years since the Liesegang phenomenon was discovered, intensive studies have been conducted to understand and control the characteristics of the periodic precipitation patterns in which the outer electrolyte diffuses into a hydrogel containing the inner electrolyte. Between fields of physics and chemistry, the periodicity of the precipitate has been investigated restrictively by spatial analyses and numerical simulations at macroscopic scales and it has been considered as a result of simple precipitation. In this work, calcium ion diffusion into gelatin hydrogels containing phosphate ions, a biomimetic system for bone formation, resulted in typical Liesegang patterns at macroscopic scales, but the asymmetric growth of the crystal was found in every single band at microscopic scales, which has not been observed or overlooked in the previous reports. The pattern consists of three characteristic bands: a continuous band, a split-fin band, and an intact-fin band. While the continuous band has a uniform crystal density, the split-fin and intact-fin bands have asymmetric crystal densities along the single band. We investigate the formation process of individual bands as well as the whole pattern by combining microscopic and spatiotemporal analyses based on the nucleation theory. Formation processes of asymmetric bands are explained by the unique stability and the diffusive property of amorphous precursors depending on the rate of calcium ion delivery. This is the first study to focus on the inhomogeneity of a single band in Liesegang patterns and the time-dependent mechanism of its growth.

Bioinspired Control of Calcium Phosphate Liesegang Patterns Using Anionic Polyelectrolytes

“…The opacity of each band was measured by the gray value obtained from the 8-bit digital images. In a control experiment, the typical pattern of Liesegang bands was observed as mentioned in our previous report . A continuous band was formed at the near reservoir, and periodic patterns consisted of asymmetric bands (Figure a).…”

“…The photograph was converted to an 8-bit image, and the lightness of each pixel was measured with the gray level using Image J. The spatiotemporal development of a single band was obtained from the setup for a cylindrical hydrogel, which was used in our previous work . Three-dimensional (3D) curves were recorded using a camcorder (HDR-CX360, Sony, Japan) and processed with the line profile function of MATLAB (R2017a, The Mathworks, Inc., USA).…”

“…In most previous studies, the Liesegang phenomenon has been numerically interpreted based on a simple precipitation mechanism. However, in the view of crystallization, the phenomenon could be interpreted as more complicated processes including nucleation, growth, and phase transformation . Furthermore, there is an increasing number of discoveries, suggesting that the phenomenon occurs through the attachment of amorphous intermediate particles larger than atoms/ions/molecules. − In a recent crystallization theory, it has been emphasized to investigate characteristic properties of amorphous intermediates, such as their stability, diffusivity, or reactivity. − In this context, the recent viewpoint of crystallization can give insight into the control and interpretation of the Liesegang phenomenon.…”

“…Recently, we found the inhomogeneity in single bands of CaP Liesegang patterns formed under slow and controlled diffusion in a gelatin medium. It was demonstrated that the overall structure of each band was dependent on the diffusive properties of ACP precursors . Although anionic polymers have been used in biomimetic approaches to generate complicated microstructures of biogenic materials, it has not been used to control the Liesegang phenomenon.…”

Diffusion-Controlled Crystallization of Calcium Phosphate in a Hydrogel toward a Homogeneous Octacalcium Phosphate/Agarose Composite

“…Considering a technical difficulty in measuring concentrations of ions and intermediates in a hydrogel, it is more feasible to calculate them based on simulation, as we reported in recent work. 48 Although it still remains a challenging issue, the quantitative analysis of crystallization in a hydrogel will be useful for fundamentally understanding the slow diffusion systems as well as precisely synthesizing organic−inorganic composites. Furthermore, this synthetic strategy using a regulated Ca 2+ flux can be an alternative method to produce homogeneous OCP/hydrogel composites, which produce integrated bonelike composites for biomedical applications.…”