Dentinogenesis imperfecta type II (DGI-II) lacks intrafibrillar mineral with severe compromise of dentin mechanical properties. A Dspp knockout (Dspp–/–) mouse, with a phenotype similar to that of human DGI-II, was used to determine if poly-L-aspartic acid [poly(ASP)] in the “polymer-induced liquid-precursor” (PILP) system can restore its mechanical properties. Dentin from six-week old Dspp–/– and wild-type mice was treated with CaP solution containing poly(ASP) for up to 14 days. Elastic modulus and hardness before and after treatment were correlated with mineralization from Micro x-ray computed tomography (Micro-XCT). Transmission electron microscopy (TEM)/Selected area electron diffraction (SAED) were used to compare matrix mineralization and crystallography. Mechanical properties of the Dspp–/– dentin were significantly less than wild-type dentin and recovered significantly (P < 0.05) after PILP-treatment, reaching values comparable to wild-type dentin. Micro-XCT showed mineral recovery similar to wild-type dentin after PILP-treatment. TEM/SAED showed repair of patchy mineralization and complete mineralization of defective dentin. This approach may lead to new strategies for hard tissue repair.

Acid-etched dentin samples with a zone of demineralized dentin were remineralized via the polymer-induced liquid-precursor (PILP) process. Poly-L-aspartic acid was used as the polymeric process-directing agent. Samples were incubated in the mineralization solution for 1- 4 weeks. Dentin samples remineralized by the PILP process presented a surface morphology very similar to the intact mineralized dentin’s architecture, in contrast to samples mineralized via the conventional nucleation and growth method (without polymer additive), which led to a superficial crust of randomly organized mineral crystals. Energy dispersive x-ray spectroscopy analysis of the PILP-mineralized samples showed the presence of calcium and phosphate ions at high levels. Since no hydroxyapatite (HA) clusters were observed on the surface of the PILP-mineralized samples, we could conclude the signal was produced from the mineral embedded within the dentin matrix. TEM and diffraction analyses suggest that both intrafibrillar and interfibrillar remineralization occurred in the demineralized dentin matrix.

A Polymer-Induced Liquid-Precursor (PILP) process for mineralization of calcium carbonate has been studied in-situ by laser light scattering. Static and dynamic light scattering data were obtained from CaCl2 solutions containing poly(aspartic acid). Under these conditions calcium carbonate mineralizes through a liquid droplet precursor phase when the solution is exposed to the decomposition products of ammonium carbonate. Our measurements probe the integrated scatterer mass and the apparent hydrodynamic radius Rh,app of the droplets as they nucleate and coalesce. The data reveal three stages in the formation of the PILP phase: an early stage of droplet growth to Rh,app ≈ 250 nm; a mid-time stage of fluctuations and polydispersity in particle size; and a final growth period where Rh,app increases from 350 nm to the micron scale. Aggregation of precursor droplets, rather than atom-by-atom growth, is the dominant mechanism of mineral formation under these conditions. With respect to biomineralization, this first observation of 100-nm-scale droplets is significant, implying a possibility to mineralize from the liquid phase within the nanoscale compartments in which many biominerals form.

Our biomimetic approach for fabricating organic-inorganic composites with structures similar to biominerals is based on a novel mineralization process, called the Polymer-Induced-Liquid-Precursor (PILP) process. This process enables the deposition of non-equilibrium mineral morphologies of calcite under low-temperature and aqueous-based conditions [1], including patterned thin films of calcite. We have recently found that when a surplus of acidic polymer is added, the patterned mineral films act as a secondary template for directing new crystal outgrowths, which form into complex morphologies of calcite with time, such as fibrous mats and “horsetails”. Two interdependent factors, the polymer and Ca-ion concentration, which change the local solution environment over time, appear to modulate the creation of these different structures. Such observations may provide clues for unraveling the long-standing mystery of how biological systems fabricate their sophisticated and complex morphologies.

Intrafibrillar mineralization of type-I collagen with hydroxyapatite (HA) is the basis of the complex biological composite known as bone, which from a material science perspective is a fascinating example of an interpenetrating bioceramic composite. Using a polymer-induced liquid-precursor (PILP) process, collagen substrates were highly infiltrated with a liquid-phase mineral precursor to calcium carbonate (CaCO3). At sections of partially mineralized collagen, banded mineral patterns were observed perpendicular to the collagen fibrils, while other fibrils were completely mineralized. An acid etch, used to preferentially remove superficial mineral, further revealed such banded patterns in fully mineralized samples. Removal of the collagen matrix with a dilute hypochlorite solution showed an interpenetrating mineral phase, with mineral disks that spanned the diameter of the pre-existing collagen fibrils, supporting our hypothesis that intrafibrillar mineralization can be achieved via capillary action applied to a liquid-phase mineral precursor.

Our biomimetic approach for patterned crystallization is based on the combination of the Micro-Contact Printing technique and a novel mineralization process, called the Polymer-Induced-Liquid-Precursor (PILP) process, which enables the deposition of mineral films under low-temperature and aqueous-based conditions. We demonstrate that a liquid-phase mineral precursor is deposited onto specific areas templated with self-assembled monolayers of alkanethiolate on gold, and then the patterned calcitic films grow under constrained conditions via transformation of the PILP phase, leading to control over the location and morphology of calcitic films.