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Invertebrate organisms that use calcium carbonate extensively in the formation of their hard tissues have the ability to deposit biominerals with control over crystal size, shape, orientation, phase, texture, and location. It has been proposed by our group that charged polyelectrolytes, like acidic proteins, may be employed by organisms to direct crystal growth through an intermediate liquid phase in a process called the polymer induced liquid precursor (PILP) process. Recently, it has been proposed that calcium carbonate crystallization, even in the absence of any additives, follows a non classical, multi step crystallization process by first associating into a liquid precursor phase before transition into solid amorphous calcium carbonate (ACC) and eventually crystalline calcium carbonates. In order to determine if the PILP process involves the promotion, or stabilization, of a naturally occurring liquid precursor to ACC, we have analyzed the formation of saturated and supersaturated calcium carbonate bicarbonate solutions using Ca 2+ ion selective electrodes, pH electrodes, isothermal titration calorimetry, nanoparticle tracking analysis, 13 C T 2 relaxation measurements, and 13 C PFG STE diffusion NMR measurements. These studies provide evidence that, in the absences of additives, and at near neutral pH (emulating the conditions of biomineralization and biomimetic model systems), a condensed phase of liquid like droplets of calcium carbonate forms at a critical concentration, where it is stabilized intrinsically by bicarbonate ions. In experiments with polymer additive, the data suggests that the polymer is kinetically stabilizing this liquid condensed phase in a distinct and pronounced fashion during the so called PILP process. Verification of this precursor phase and the stabilization that polymer additives provide during the PILP process sheds new light on the mechanism through which biological organisms can exercise such control over deposited CaCO 3 biominerals, and on the potential means to generate in vitro mineral products with features that resemble biominerals seen in nature.

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We have put forth the hypothesis that collagen is mineralized during bone formation by means of a polymer-induced liquid-precursor (PILP) process, in which a liquid-phase mineral precursor could be drawn into the gaps and grooves of the collagen fibrils by capillary action, and upon solidification, leave the collagenous matrix embedded with nanoscopic crystallites of hydroxyapatite. This hypothesis is based upon our observations of capillarity seen for liquid-phase mineral precursors generated with calcium carbonate. Here, we demonstrate proof-of-concept of this mechanism by mineralizing Cellagen sponges (type I reconstituted bovine collagen) in the presence of a liquid-precursor phase to calcium carbonate. Scanning electron microscopy (SEM) was used to examine the mineralized collagen, which in combination with selective etching studies, revealed the extent to which the mineral phase infiltrated the collagenous matrix. A roughly periodic array of disk-like crystals was found to be embedded within the collagen fibers, demonstrating that the mineral phase spans across the diameter of the fibers. Some of the morphological features of the mineralized fibers in our in vitro model system are similar to those seen in natural bone (albeit of a different mineral phase), lending support to our hypothesis that these non-equilibrium morphologies might be generated by a PILP process. SEM provides a different perspective on the morphology of bone, and has been useful here for examining the extent of mineralization in composite structures generated via the PILP process. However, further investigation is needed to examine the nanostructural arrangement of the crystallites embedded within the collagenous matrix.

It was hypothesized that applying the polymer-induced liquid-precursor (PILP) system to artificial lesions would result in time-dependent functional remineralization of carious dentin lesions that restores the mechanical properties of demineralized dentin matrix. 140 µm deep artificial caries lesions were remineralized via the PILP process for 7–28 days at 37°C to determine temporal remineralization characteristics. Poly-L-aspartic acid (27 KDa) was used as the polymeric process-directing agent and was added to the remineralization solution at a calcium-to-phosphate ratio of 2.14 (mol/mol). Nanomechanical properties of hydrated artificial lesions had a low reduced elastic modulus (ER = 0.2 GPa) region extending about 70 μm into the lesion, with a sloped region to about 140 μm where values reached normal dentin (18–20 GPa). After 7 days specimens recovered mechanical properties in the sloped region by 51% compared to the artificial lesion. Between 7–14 days, recovery of the outer portion of the lesion continued to a level of about 10 GPa with 74% improvement. 28 days of PILP mineralization resulted in 91% improvement of ER compared to the artificial lesion. These differences were statistically significant as determined from change-point diagrams. Mineral profiles determined by micro x-ray computed tomography were shallower than those determined by nanoindentation, and showed similar changes over time, but full mineral recovery occurred after 14 days in both the outer and sloped portions of the lesion. Scanning electron microscopy and energy dispersive x-ray analysis showed similar morphologies that were distinct from normal dentin with a clear line of demarcation between the outer and sloped portions of the lesion. Transmission electron microscopy and selected area electron diffraction showed that the starting lesions contained some residual mineral in the outer portions, which exhibited poor crystallinity. During remineralization, intrafibrillar mineral increased and crystallinity improved with intrafibrillar mineral exhibiting the orientation found in normal dentin or bone.