Estudio doctrinal sobre la mediación familiar

Elucidating biomineralisation mechanisms directly from biological samples can be impractical, and the complex environments in which calcification takes place can make it difficult to understand the effects of variables to nucleation and growth.

In vitro and in silico experiments are essential to studying biomineralisation. The literature is vast and cannot be summarised easily; nonetheless a number of examples will be provided here.

Idealised solution experiments are perhaps the most commonly employed model to study biominerlisation. Bolze and co-workers showed that the initial solid produced in pure solutions is ACC which is found in a colloidal form [Bolzeet al., 2002]. Particles up to around 270 nm (in terms of colloidal diameter) were found with low polydispersity and a mass density lower than that of crystalline polymorphs. Later studies showed that ACC particles were stable up to around a size of 100 nm in diameter [Nudelmanet al., 2010].

Wanget al. used turbidity measurements to study the precipitation of cal- cium carbonate, which confirmed the limiting size to ACC stability of around 100 nm [Wanget al., 2012]. They showed that further growth above this size took place via aggregation of ACC, but that crystallisation to calcite in aggregates occurred. Va- terite was also found, and this transformed in time to calcite [Meldrum and Colfen, 2008]. The transformation presumably takes place via Ostwald’s step rule, where the emerging phase is the one nearest in stability to the original; a number of phases (with increasing stability) may be visited in a multi-step, energetically downhill path to the thermodynamically favoured one. The mechanism of transformation between states is not defined by Ostwald’s rule, and it is not true that this multi-step pathway to the most stable phase occurs for all materials [Threlfall, 2003]. Later experiments [Wang et al., 2013; Aziz et al., 2011] showed that there is a certain degree of ki- netic control to calcium carbonate precipitation, with changes in stirring rate, pH and temperature affecting particle size and the product of crystallisation. Under- standing the co-action between kinetic and thermodynamic control could be key to exploiting biomineralisation processes.

Nanoparticle computer simulations have provided a much needed under- standing of solid phases at length and time scales difficult to probe with experi- mental techniques. Molecular dynamics (MD) simulations by Cooke and Elliot have shown that calcite crystals containing less than forty formula units of calcium car-

bonate amorphise in solution, indicated by a change in pair distribution functions and carbonate orientation [Cooke and Elliott, 2007]. Calcite crystalline order was found to persist in solution for clusters containing more than forty formula units [Cooke and Elliott, 2007]. Tribelloet al. measured no energy barrier to the growth of ACC from free energy calculations of ion addition to nanoparticles, highlight- ing that calcification by the initial deposition of an amorphous phase is faster than classical crystallisation [Tribelloet al., 2009]. Quigley et al. have shown using free energy calculations that calcite particles are thermodynamically stable at sizes of 2 nm, with the amorphous phase only lower in energy due to confinement effects [Quigleyet al., 2011]. Unfortunately, the force fields employed in these simulations do not reproduce the solvation enthalpies of calcium carbonate, and therefore cannot predict the stabilities of phases in solution accurately [Raiteri et al., 2010]. Later work by Raiteri and Gale showed that hydrated ACC particles up to around 5 nm in diameter are more stable than crystalline phases [Raiteri and Gale, 2010]. These studies seem to contradict the experimental findings. However, it is plausible that metastable ACC up to_∼100 nm persists in solution before crystallisation.

Additive experiments allow scientists to understand the influence of certain functional groups on the mechanism of biomineralisation. Xiao and Yang studied the effect of hydroxyl, carboxyl and amine–rich organic molecules on the polymorph control of calcium carbonate when formed from constituent ions in solution [Xiao and Yang, 2011]. Amorphous particle intermediates (_∼200 nm in diameter) all contained up to around 13 wt% water and organic molecules. While carboxyl groups showed a preference for calcite formation, hydroxyl and amine–rich molecules led to the formation of aragonite. The polymorph control was observed during the formation of ACC, and the inclusion of organics did not affect crystallisation during the ACC_−→crystalline transition [Xiao and Yang, 2011].

Some organic molecules have been used to stabilise ACC in solution. Exam- ples include polydopamine [Wang and Xu, 2013], (poly)aspartate and DNA [Gower, 2008], and these additives generally work by kinetic control; coating ACC particles in organics can limit the extent to which they can grow, and so inhibit crystallisation [Nudelmanet al., 2010]. It is true that organic additives can influence many stages in biomineralisation, and Gebauer and co-workers have suggested a classification scheme according to how additives control crystal formation [Gebaueret al., 2009]. For example, polyaspartate was shown to bind to calcium, inhibit nucleation and to influence the local structure of the bound phase, making it a type I, III and V additive [Verchet al., 2011].

in the presence of organic monolayers [Harding et al., 2014]. This system mimics the deposition of solid phases onto an insoluble organic matrix, which takes place in living organisms. Experiments have shown [Han and Aizenberg, 2003, 2008] that self assembled monolayer (SAM) chain length can control the crystallisation front, and head groups can stabilise particular polymorphs. Amorphous calcium carbonate has been found to be initially deposited onto acidic monolayers, with subsequent crystal orientation being directed by the organic template [Fricke and Volkmer, 2007]. Indeed, the geometry of the the SAM head groups can be highly selective to the formation of particular crystallographic planes [Aizenberg et al., 1999b]. Simulations have shown that monolayer surface charge density is crucial to crystallisation; the lattice structure which closely matches SAM surface charge density is likely to be low in energy, and crystal propagation tends to follow this orientation [Freeman et al., 2008]. Aizenberg and colleagues showed [Aizenberg

et al., 1999a] that calcite growth can be controlled on patterned SAMs, with crystal nucleation rates affected by the polarity of monolayer head groups.