Embedding Collagen in Multilayers for Enzyme-Assisted Mineralization: A Promising Way to Direct Crystallization in Confinement

Abstract

The biogenic calcium phosphate (CaP) crystallization is a process that offers elegant materials design strategies to achieve bioactive and biomechanical challenges. Indeed, many biomimetic approaches have been developed for this process in order to produce mineralized structures with controlled crystallinity and shape. Herein, we propose an advanced biomimetic approach for the design of ordered hybrid mineralized nano-objects with highly anisotropic features. For this purpose, we explore the combination of three key concepts in biomineralization that provide a unique environment to control CaP nucleation and growth: (i) self-assembly and self-organization of biomacromolecules, (ii) enzymatic heterogeneous catalysis, and (iii) mineralization in confinement. We use track-etched templates that display a high density of aligned monodisperse pores so that each nanopore may serve as a miniaturized mineralization bioreactor. We enhance the control of the crystallization in these systems by coassembling type I collagen and enzymes within the nanopores, which allows us to tune the main characteristics of the mineralized nano-objects. Indeed, the synergy between the gradual release of one of the mineral ion precursors by the enzyme and the role of the collagen in the regulation of the mineralization allowed to control their morphology, chemical composition, crystal phase, and mechanical stability. Moreover, we provide clear insight into the prominent role of collagen in the mineralization process in confinement. In the absence of collagen, the fraction of crystalline nano-objects increases to the detriment of amorphous ones when increasing the degree of confinement. By contrast, the presence of collagen-based multilayers disturbs the influence of confinement on the mineralization: platelet-like crystalline hydroxyapatite form, independently of the degree of confinement. This suggests that the incorporation of collagen is an efficient way to supplement the lack of confinement while reinforcing mechanical stability to the highly anisotropic materials. From a bioengineering perspective, this biomineralization-inspired approach opens up new horizons for the design of anisotropic mineralized nano-objects that are highly sought after to develop biomaterials or tend to replicate the complex structure of native mineralized extracellular matrices.

Enzymatic Approach in Calcium Phosphate Biomineralization: A Contribution to Reconcile the Physicochemical with the Physiological View

“…Moreover, by combining real-time monitoring and ex situ characterization techniques, we observed that the growth mechanism of the mineral was significantly impacted by the physicochemical conditions of the medium (pH and temperature). In another study, we embedded type I collagen within a multilayered film containing ALP in a way that mineralization was initiated in the vicinity of self-assembled collagen fibrils [ 134 ]. This led to an extensive formation of larger mineral particles, compared to the film without collagen, with higher surface coverage (see Figure 6 A,B insets).…”

“…This discrepancy suggests the existence of a different pathway when the mineralization is initiated by enzymes embedded in polyelectrolyte multilayers. Interestingly, the addition of type I collagen to the studied system induces noticeable effects [ 134 ]. For this purpose, collagen was embedded with ALP within the same multilayered film inside the nanopores.…”

“…( E , H ) Embedding type I collagen within the enzyme-based multilayers in 500 nm pore size leads to the formation of HAP crystals stacked onto each other and interconnected with collagen fibrillary nanostructures. Adapted from [ 134 ].…”

Self‐Activating Phosphorus‐Rich Substrate Import Enzyme Catalytic Domains for Bone Regeneration

“…[ 15 ] Further, the mineralization process is also guided by the interaction of enzymes and macromolecules, which control the morphology of mineral crystals and achieve orderly mineralization in various 3D networks. [ 15a,16 ] These bio‐macromolecular matrices are usually collagen and collagen‐mimicking polymers such as polysaccharides, alginate polyacrylamide, and oligo[poly(ethylene glycol) fumarate] hydrogel. [ 16–17 ] Due to the efficient catalytic activity of the enzymes, more uniform and orderly crystal growth has resulted in mineralizing fluid (MF), wherein CaGP is the main component.…”

“…[ 15a,16 ] These bio‐macromolecular matrices are usually collagen and collagen‐mimicking polymers such as polysaccharides, alginate polyacrylamide, and oligo[poly(ethylene glycol) fumarate] hydrogel. [ 16–17 ] Due to the efficient catalytic activity of the enzymes, more uniform and orderly crystal growth has resulted in mineralizing fluid (MF), wherein CaGP is the main component. [ 16a ]…”