A Micro-Comb Test System for In Situ Investigation of Infiltration and Crystallization Processes
The investigation of mineralization and demineralization processes is important for the understanding of many phenomena in daily life. Many crystalline materials are exposed to decay processes, resulting in lesions, cracks, and cavities. Historical artifacts, for example, often composed of calcium carbonate (CaCO 3 ), are damaged by exposure to acid rain or temperature cycles. Another example for lesions in a crystalline material is dental caries, which lead to the loss of dental hard tissue,
... ntal hard tissue, mainly composed of hydroxyapatite (HAp). The filling of such cavities and lesions, to avoid further mineral loss and enable or support the remineralization, is a major effort in both areas. Nevertheless, the investigation of the filling process of these materials into the cavities is difficult due to the non-transparency and crystallinity of the concerned materials. In order to address this problem, we present a transparent, inexpensive, and reusable test system for the investigation of infiltration and crystallization processes in situ, being able to deliver datasets that could potentially be used for quantitative evaluation of the infiltration process. This was achieved using a UV-lithography-based micro-comb test system (MCTS), combined with self-assembled monolayers (SAMs) to mimic the surface tension/wettability of different materials, like marble, sandstone, or human enamel. Moreover, the potential of this test system is illustrated by infiltration of a CaCO 3 crystallization solution and a hydroxyapatite precursor (HApP) into the MCTS. Minerals 2017, 7, 187 2 of 11 Sandstones, like Pietra Serena (PS), are composed of a mixture of oxides and carbonates and are characterized by a relatively high porosity [20, 21] . In addition, they are relatively soft and easy to handle, which is among the reasons why this material was often used in architecture. In comparison, marbles are materials containing varying degrees of carbonates, depending on the quarrying area, especially of calcium  . Carrara marble (CM) represents a class of rock with a carbonate content of over 80% and a generally lower porosity  . Due to the exquisite white color, CM was often used for statues and architecture, as well  . Over the centuries of their existence, historical monuments and artworks are exposed to many decay processes  . The porosity of the stones enables the infiltration of water, which can result in mechanical stress and, finally, in damage of the structural integrity of the stones due to thermal cycles and crystallization processes inside the pores. This also shows how important a better understanding of crystallization in confined environments is  . Additionally, chemical reactions-e.g., caused by acid rain-can lead to cracks and cavities, for example, due to the dissolution of CaCO 3 . To preserve historical artifacts by preventing further damage and restoring the integrity, several restoration formulations have been developed    . Similar problems related to acid-induced decay processes and subsequent mineral loss are a major challenge in dentistry-called dental caries. It is the most common chronic disease around the world affecting more than 95% of concerned adults [27, 28] . Caries result from bacteria, which form a biofilm on the teeth. These bacteria metabolize carbohydrates and produce acids as by-products  . The acidic metabolites demineralize the dental hard tissues-enamel and dentin-mainly composed of hydroxyapatite (HAp), resulting in the formation of lesions in the range of nanometers to hundreds of microns    . Due to the enormous number of diseased individuals, several different treatments for refilling the cavities have been established. The developed filling materials range from cements [33-35] over (nano)ceramics [36,37] to peptide-amorphous calcium phosphate composites [38, 39] . Nevertheless, the mode of action of many fillings is not completely understood. The investigation of the infiltration behavior of new filling materials in dental lesions, as well as in pores and cracks in historical artifacts, is a great challenge. Furthermore, the investigation of the underlying mechanism of the interactions with the substrates (stones and teeth) is crucial for the development and validation of the filling methods and materials. In both cases, the production of suitable tests systems (TSs) is complex and time consuming. The stone samples need to be aged artificially to create controlled and reproducible cavities and damage. This is usually achieved by multiple cycles of thermal shock or hot-cold cycling, purposeful infiltration of soluble salts, or treatment with acids    . For the dental TSs, the usage of artificial tooth lesions, made of bovine or human teeth, is the state of the art [39, 43, 44] . Therefore, the teeth need to be embedded in a polymer matrix, polished afterwards and etched with acids [39, 44, 45] . Both tooth and stone TSs, need to be replaced after each experiment, resulting in a high consumption of the TSs. Apart from the sophisticated preparation of the TSs, it is difficult to obtain an insight into the lesions due to the crystallinity of the TSs and non-transparency to visible light. The investigation of infiltration depth and crystallization processes of fillers with light microscopy or scattering techniques is hindered. In this paper, we present the fabrication of a UV-lithography-based, transparent, inexpensive, and reusable micro-comb test system (MCTS) for the investigation of infiltration and crystallization processes in situ by using common methods, like optical light microscopy (LM). The lesions of the MCTSs were varied from 10 µm to 100 µm in width. In addition, the surface tension/wetting properties of PS and CM were imitated, as well as human enamel, by using a mixture of different thiols in self-assembled monolayers (SAMs). Furthermore, we investigated the infiltration and crystallization of CaCO 3 and HAp in the MCTSs using an infiltration setup. Minerals 2017, 7, 187 3 of 11 Materials and Methods Materials Poly(acrylic acid) sodium salt (PAA, M w = 8000 g/mol, 45 wt % in water, M w = 15,000 g/mol, 35 wt % in water), calcium chloride dihydrate (CaCl 2 ·2H 2 O, ≥99%), 2-Amino-2-(hydroxylmethyl)-1,3propanediol (Tris, ≥99,9%), 2-Propanol (iPrOH, p.a.), and 11-mercapto-1-undecanol (MUO, 97%) were purchased from Sigma Aldrich (Taufkirchen, Germany). Disodium hydrogen phosphate (Na 2 HPO 4 , ≥98%) and ethanol (EtOH, p.a.) were purchased from Carl Roth (Karlsruhe, Germany). Potassium chloride (KCl, 99.5%), sodium hydrogen carbonate (NaHCO 3 , p.a.), and sodium hydroxide solution (NaOH, 0.1 N) were obtained from Merck Chemicals (Darmstadt, Germany). 1-Dodecanethiol (DDT, ≥98%) and poly(acrylic acid) (PAA, M w = 2000 g/mol, 63 wt % in water) were purchased from Acros Organics (Geel, Belgium). The photoresist SU-8 3050 and photo developer mr-Dev 600 were purchased from Micro Resist Technology (Berlin, Germany). All chemicals were used without further purification. All experiments were carried out using double-deionized water (18.2 MΩ) using a Milli-Q Direct 8 machine from Merck Millipore (Darmstadt, Germany).