Lopes Anabela, Lopes-Lima Manuel, Ferreira Jorge, Araújo Sandra, Hinzmann Mariana, Oliveira José, Rocha António, Domingues Bernardo, Bobos Iulius, Machado Jorge
ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Laboratório de Fisiologia Aplicada, Rua de Jorge Viterbo Ferreira No. 228, 4050-313, Porto, Portugal.
J Membr Biol. 2014 Jun;247(6):501-14. doi: 10.1007/s00232-014-9660-1. Epub 2014 Apr 8.
The present work proposes to analyse the results obtained under in vitro conditions where cellulose artificial membranes were incubated with biological fluids from the freshwater bivalve Anodonta cygnea. The membranes were mounted between two half 'Ussing chambers' with different composition solutions in order to simulate epithelial surfaces separating organic fluid compartments. The membrane surfaces were submitted to two synthetic calcium and phosphate solutions on opposite sides, at pH 6.0, 7.0 or 9.0 during a period of 6 hours. Additional assays were accomplished mixing these solutions with haemolymph or extrapallial fluid from A. cygnea, only on the calcium side. A selective ion movement, mainly dependent on the membrane pore size and/or cationic affinity, occurred with higher permeability for calcium ions to the opposite phosphate chamber supported by calcium diffusion forces across the cellulose membrane. In general, this promoted a more intense mineral precipitation on the phosphate membrane surface. A strong deposition of calcium phosphate mineral was observed at pH 9.0 as a primary layer with a homogeneous microstructure, being totally absent at pH 6.0. The membrane showed an additional crystal phase at pH 7.0 exhibiting a very particular hexagonal or cuttlebone shape, mainly on the phosphate surface. When organic fluids of A. cygnea were included, these crystal forms presented a high tendency to aggregate under rosaceous shapes, also predominantly in the phosphate side. The cellulose membrane was permeable to small organic molecules that diffused from the calcium towards the phosphate side. In the calcium side, very few similar crystals were observed. The presence of organic matrix from A. cygnea fluids induced a preliminary apatite-brushite crystal polymorphism. So, the present results suggest that cellulose membranes can be used as surrogates of biological epithelia with preferential ionic diffusion from the calcium to the phosphate side where the main mineral precipitation events occurred. Additionally, the organic fluids from freshwater bivalves should be also thoroughly researched in the applied biomedical field, as mineral nucleators and crystal modulators on biosynthetic systems.
本研究旨在分析在体外条件下获得的结果，即将纤维素人工膜与淡水双壳贝类天鹅绒蚬的生物体液一起孵育。将膜安装在两个装有不同成分溶液的半“尤斯室”之间，以模拟分隔有机液区室的上皮表面。在6小时内，将膜表面分别置于pH值为6.0、7.0或9.0的两种合成钙和磷酸盐溶液的相对两侧。仅在钙侧，将这些溶液与天鹅绒蚬的血淋巴或外套腔液混合进行了额外的试验。发生了选择性离子移动，主要取决于膜孔径和/或阳离子亲和力，钙离子通过纤维素膜的钙扩散力对相对的磷酸盐腔室具有更高的渗透性。一般来说，这促进了磷酸盐膜表面更强烈的矿物沉淀。在pH值为9.0时观察到磷酸钙矿物的强烈沉积，形成具有均匀微观结构的初级层，而在pH值为6.0时则完全没有。在pH值为7.0时，膜在磷酸盐表面呈现出额外的晶相，呈现出非常特殊的六边形或乌贼骨形状。当加入天鹅绒蚬的有机体液时，这些晶体形式呈现出以玫瑰状聚集的高度倾向，也主要在磷酸盐侧。纤维素膜对从小分子有机物从钙侧向磷酸盐侧扩散是可渗透的。在钙侧，观察到的类似晶体很少。天鹅绒蚬体液中有机基质的存在诱导了磷灰石-透钙磷石晶体多晶型的初步形成。因此，目前的结果表明，纤维素膜可以用作生物上皮的替代物，钙离子优先向发生主要矿物沉淀事件的磷酸盐侧进行离子扩散。此外，淡水双壳贝类的有机体液作为生物合成系统中的矿物成核剂和晶体调节剂，在应用生物医学领域也应进行深入研究。
