Crystallization of calcium phosphate oriented by self-assembling diblock copolymers, in solution and at the air-water interface

Living organisms produce crystalline structures called biogenic crystals or biominerals, of various shapes and properties. Among them, mechanical properties, such as resistance to stress or elasticity, are often far from those of the corresponding species typically crystallized in the laboratory. For example, the fabrication of bones and teeth (calcium phosphate), shells (calcium carbonate), as well as the dissolution of unwanted materials such as kidney stones (calcium oxalate), essentially involves proteins that induce or inhibit nucleation, or favor the growth of crystals following a particular orientation. Compounds affecting crystal growth are gathered under the term “crystallization additives”.
Calcium phosphate (CaP) is the main component of mammal bone. The demand for long-lasting, high-quality bone implants makes it interesting to quantitatively understand CaP nucleation, growth and degradation at various interfaces. A tremendous amount of work has already been dedicated to the controlled fabrication of CaP from aqueous and organic solution. Commercially available bone cements are typically viscous mixtures of CaP and water enabling a rapid solidification upon injection into a fracture. It appears that composition control is critical, since subtle differences may profoundly affect the cement behavior in vivo.
We therefore chose to focus on this particular material and test its interactions with synthetic polymers, presenting different template structures and chemical groups to the CaP, thus enabling us to study in a systematic way (pH, concentration, maturation time), interactions pathways that were different in nature or strength, as well as occurring at different steps during the course of the crystallization (nucleation, growth, maturation).
The synthetic polymers were chosen among the diblock copolymers family, because this allowed an interesting tandem where one block is responsible for interacting with the crystals while the other block determines the secondary structure of the polymer phase, its aggregation and possibly the shape of the resulting mineral at the micrometer scale.
This work presents three studies of CaP crystallization control by polymeric additives, under controlled conditions, in aqueous solution or at the air-water interface. In the perspective of medical applications, all chosen blocks are to some extent biocompatible, except the hydrophobic block holding the third polymer at the air-water interface. Three polymers were chosen in order to reflect the diversity of the interaction pathways:
1) poly(ethylene oxide)-block-poly(2-methyl-2-oxazoline), a neutral dihydrophilic block copolymer, in solution. We present the first characterization of well-defined self-assembly in presence of a mere difference in hydrophilicity as aggregation driving force instead of hydrophobic interactions. As far as the mineralization of CaP is concerned, no significant influence was detected and this work may serve as a control study for the polybasic copolymer that has poly(ethylene oxide)-block-poly(2-methyl-2-oxazoline) as synthetic precursor, poly(ethylene oxide)-block-poly(ethylene imine). The self-assembly study grew well beyond the PhD, implying a wider range of techniques, and is now soon to be published.
2) polyethylene oxide-block-polyvalerolactone, a neutral amphiphilic block copolymer, in solution. Here, the PEO block is combined to a biodegradable, rather crystalline, hydrophobic block, at different degrees of polymerization. Despite having no particular affinity to CaP, PVL and its copolymers with PEO have found some interest as nanocontainers for drug delivery. However, again no significant influence was detected. This study suffered from the polymer high crystallinity. In later studies this problem was addressed by preferring the polycaprolatone variant.
3) poly(n-butylacrylate)–block–poly(acrylic acid), a charged amphiphilic block copolymer, at the air-water interface. Previously studied in solution, the polymer offers several tuning possibilities and the acrylic acid function is known to interact strongly with CaP. This approach was to our knowledge the first study on CaP mineralization of polymeric Langmuir films. We found that various conditions may be easily simulated in terms of charge surface density, supersaturation or pH, and result in various outcomes ranging from quick nucleation and growth without hierarchy to slow formation of nearly crystalline hexagonal array of uniform particles with identical particle sizes even at very long range (over 30 µm).
The outcome of this study is interesting because it demonstrates that even a rather flexible matrix like our polymer film at the air-water interface leads to uniform particles. Moreover, the film also acts as a tool for the 2D arrangement of the resulting particles in a near-crystalline order. The implication for biomineralization is that even rather flexible scaffolds swollen with water are able to regulate mineralization on the atomic (crystal phase), the nanoscopic (particle size and shape) and the sub-micron to micron scale (2D arrangement) of the precipitate. As a result, the current work could serve as a model for biological mineralization, which is more closely related to nature than films made from e.g. detergents or other low molecular mass compounds.