Elsevier

Acta Biomaterialia

Volume 120, 15 January 2021, Pages 213-223
Acta Biomaterialia

Involvement of prenucleation clusters in calcium phosphate mineralization of collagen

https://doi.org/10.1016/j.actbio.2020.07.038Get rights and content

Abstract

Involvement of thermodynamically-stable prenucleation clusters (PNCs) in the biomineralization of collagen has been speculated since their existence was reported in mineralization systems. It has been hypothesized that intrafibrillar mineralization proceeds via nucleation of inhibitor-stabilized intermediates produced by liquid-liquid separation (aka. polymer-induced liquid precursors; PILPs). Here, the contribution of PNCs and PILPs to calcium phosphate intrafibrillar mineralization of collagen was examined in a model with a semipermeable membrane that excludes nucleation inhibitor-stabilized PILPs from reaching the collagen fibrils, using cryogenic electron microscopy of reconstituted fibrils and conventional transmission electron microscopy of collagen sponges. Molecular dynamics simulation with the Interface force field (IFF) was used to confirm the existence of PILPs with amorphous calcium phosphate and elucidate details of the dynamics. Furthermore, intrafibrillar mineralization of single collagen fibrils was experimentally observed with unstabilized PNCs when anionic/cationic polyelectrolytes were used to establish Donnan equilibrium across the semipermeable membrane. Molecular dynamics simulation verified PNC formation within the collagen intrafibrillar gap zones at the atomic scale and explained the role of external PILPs. The PILPs decrease the interfibrillar water content and increase the interfibrillar ionic concentration. Nevertheless, intrafibrillar mineralization of collagen sponges with PNCs alone was inefficacious, being constrained by competition from extrafibrillar mineral precipitation.

Statement of Significance

Compared with conventional PILP-based intrafibrillar mineralization, mineralization of collagen fibrils using unstabilized PNCs is constrained by competition from extrafibrillar mineral deposition. The narrow window of opportunity for PNCs to produce intrafibrillar mineralization provides a plausible explanation for the feasibility of nucleation inhibitor-free intrafibrillar apatite assembly during reconstitution of type I collagen.

Introduction

Different in vitro models have been developed for rationalizing the mechanisms of intrafibrillar mineralization of calcium phosphate (CaP) in collagen [1], [2], [3], [4], [5], [6], based on differential interpretations of the classical and nonclassical nucleation theories [7], [8], [9]. According to the classical nucleation theory, crystallization is predicated upon the stochastic association of ion clusters which are unstable with respect to dissolution. These precursors grow spontaneously by ion attachment and unit cell replication into crystals with definitive lattice structures upon overcoming the interfacial free energy barrier between the supersaturated solution and the aggregate [10]. However, phenomena such as involvement of disordered CaP phases in biogenic mineralization, presence of stable (metastable) prenucleation clusters (PNCs) in inorganic mineral salt solutions, and ordered aggregation of mesocrystals in crystal growth are not readily accountable by the classical nucleation theory [[11], [12]–13].

Two-step and multi-step nucleation models based on the non-classical crystallization pathway have been introduced to account for these phenomena [14,15]. According to these models, minerals do not crystallize directly into the most thermodynamically-stable phase. Instead, intermediate phases are formed in ionic species-crowded solutions, generating PNCs and subsequently, increasingly-densified hydrated intermediates, for participation in the crystallization chain of events [16]. This chain of events has also been identified in crystallization of CaP. Using cryogenic-electron microscopy (cryo-EM), Dey et al. [17] reported that surface-induced apatite precipitation from simulated body fluid was initiated by speciation beyond ion pairs, analogous to the “Posner's clusters” of highly-hydrated calcium phosphate in the classical crystallization pathway; the latter densified into metastable amorphous nanodroplets via liquid-liquid demixing and subsequently nucleate and grow into crystalline phases. Other forms of CaP prenucleation species have been reported in the literature [[18], [19], [20], [21]–22]. These PNCs may represent different stages of speciation development during densification into hydrated amorphous calcium phosphate (ACP). The philosophical discovery of PNC and its role in biomineralization appear to have bridged the gap between classical and non-classical nucleation theories [9,23].

Liquid-liquid separation of densified, hydrated PNCs by spinodal decomposition represents an alternative route of mineralization [[24], [25]–26]. Such a process is notably identified in calcium carbonate mineralization systems. Phase separation occurs spontaneously via diffusion, in the absence of a thermodynamic barrier, when the concentration of the increasingly densified PNCs crosses the unstable spinodal within the miscibility gap of an isobaric phase diagram (temperature vs composition) of a binary solvent-solute system [27]. In the absence of a stabilizing agent, this alternative mode of liquid densification readily coalesces into a hydrated liquid mineral intermediate that eventually solidifies upon the loss of hydration water. Introduction of a polyelectrolyte additive does not alter the liquid-liquid binodal limit of the solvent-solute system, but probably inhibits the sequestration of hydration water or stabilizes the nanodroplets colloidally against aggregation or dehydration [28]. Liquid-liquid phase separation intermediates that are stabilized by matrix proteins or their biomimetic nucleation-inhibiting analogs [29] have been coined polymer-induced liquid precursors (PILPs) [30]. The existence of PILPs has been challenged in a recent cryo-EM study [31], however, cryo-TEM is arguably a snapshot procedure that does not account for the dynamic nature of these intermediates. Unmistakably, PILP is revolutionary as a crystallization concept for calcium carbonate, CaP-based mineralization systems and other non-calcium based systems [32,33]. For CaP-based mineralization systems, polyelectrolyte stabilization of amorphous CaP mineralization precursors is important for intrafibrillar mineralization of collagen because the process does not occur in the absence of polymeric process-directing agents except under extreme hyperosmolarity [5,8].

Because PNCs are the smallest aggregates identified from CaP mineralization systems and are formed in solutions irrespective of the presence of nucleation inhibitors [3,17], one may envisage them, or their increasingly-densified hydrated intermediates, as the primary precursor species in the intrafibrillar mineralization process. This is logical from a bottom-up nano-engineering perspective because PNCs (~0.87 nm in diameter) [17] are smaller than the spacings between triple helical strands (~1.5 nm) within a collagen microfibril [34]. Individual entities can enter those spaces directly without relying on the larger shape-adaptable, polyelectrolyte-stabilized PILP particulates [35].

Despite the perceivably well-defined role of PNC as solute precursors in crystallization [36], the relative contribution of PNC in intrafibrillar mineralization of collagen fibrils by CaP systems is unknown. A major hurdle that undermines such investigations is the lack of an appropriate experimental model that distinguishes the thermodynamically-stable PNCs from nucleation inhibitor-stabilized PILPs produced via spinodal decomposition. This hurdle was resolved experimentally in the present study by using an in vitro model of a semipermeable membrane to establish Donnan equilibrium, and to restrict the passage of cationic or anionic polyelectrolyte nucleation inhibitors through the semipermeable membrane to produce stabilized PILPs during collagen biomineralization. The experimental results were validated using molecular dynamics simulation (MDS), which allow critical insights into PNC speciation and growth [28,[37], [38], [39]–40]. Specifically, we utilized the Interface Force Field (IFF) along with CHARMM36 that contains calcium phosphate models with quantitative validation of aqueous interfacial properties across a range of pH values [[41], [42]–43]. The hypothesis tested was that collagen intrafibrillar mineralization may be achieved using hydrated PNCs within normal physiological range, under the condition that inhibitors establish electrical neutrality and osmotic equilibrium across a semipermeable membrane.

Section snippets

Materials

Poly(allylamine) hydrochloride (PAH, Mw 17.5 kDa), calcium chloride dihydrate (CaCl2•2H2O), sodium phosphate dibasic (Na2HPO4) and fluorescamine were purchased from MilliporeSigma (St. Louis, MO, USA). Poly-l-aspartic acid sodium salt (PAsp, Mw 27 kDa) was purchased from Alamanda Polymers (Huntsville, Alabama, USA). All chemicals were used as received. Dialysis tubing kit with molecular weight cut-off (MWCO) of 500 Da was purchased from Spectrum Laboratories, Inc. (Rancho Dominguez, CA, USA).

Results and discussion

The rationale of the present in vitro model was to use a semipermeable membrane to establish Donnan equilibrium, and to restrict the passage of cationic or anionic polyelectrolyte nucleation inhibitors through the membrane to produce stabilized PILPs during collagen biomineralization. To fulfill this experimental setup, GPC was used to select the membrane MWCO. Based on the GPC results (SI-2), dialysis tubing with 500 Da MWCO was used for subsequent experiments. Calcium ions (40.08 Da) and

Conclusion

The mechanism of intrafibrillar mineralization of collagen with PNCs alone is unraveled by cryo-EM and molecular dynamics simulation using a model system with a semipermeable membrane that helped establish Donnan equilibrium. The detailed dynamics of PNCs of calcium phosphates and atomic-level insights into the structure of PILPs are revealed by simulations in all-atom resolution in agreement with the experimental data. Compared with conventional PILP-based intrafibrillar mineralization,

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The present work was supported by grants 81722015, 81870805, 81970959 and 81720108011 from National Nature Science Foundation of China and grant 2020TD-033 from the Shaanxi Key Scientific and Technological Innovation Team. We acknowledge support from the National Science Foundation (DMREF 1623947 and CBET 1530790, OAC 1931587, CMMI 1940335). This work also utilized the Summit supercomputer, a joint effort of the University of Colorado Boulder and Colorado State University, which is supported by

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