Date of Award
Doctor of Philosophy (PhD)
All living organisms utilize phosphorus (P) as an essential component of their cell membranes, DNA and RNA, and adenosine triphosphate. Bones, in addition to bearing loads, play an important role in balancing P levels in our bodies. In bones, a network of collagen templates and calcium phosphate (CaP) nanocrystals builds hierarchical levels, from nano- to macroscale. Within this architecture, the thermodynamic properties of CaP minerals are influential. Despite the importance of nucleation, growth, and crystallization in collagen structures for tissue development, little kinetic study of these processes has been conducted due to the limited in situ techniques for monitoring these nanoscale reactions in organic templates. In this dissertation, utilizing synchrotron-based in situ measurement techniques, kinetic aspects of collagen mineralization (i.e., nucleation, growth, and crystallization of CaP) were evaluated at multiple-length scales. Based on the kinetic observations, I sought a better understanding of the physicochemical properties of bones, and how they provide thermodynamic advantages for CaP mineralization.
First, I evaluated the nucleation kinetics and pathways for CaP minerals in collagen and their phase transformation from amorphous species to crystalline plates, using in situ small-angle X-ray scattering (SAXS). Collagen mineralization without an extrafibrillar nucleation inhibitor, polyaspartic acid (pAsp), led initially to spherical aggregates of CaP in the entire extrafibrillar space (extrafibrillar mineralization, EM). With time, the spherical aggregates transformed into plates at the outermost surface of the collagen matrices, preventing intrafibrillar mineralization (IM). However, mineralization with pAsp led directly to the formation of intrafibrillar CaP plates with a spatial distribution gradient through the depth of the matrix. We also experimentally obtained nucleation energy barriers to IM and EM by combining in situ SAXS observations and the modified classical nucleation theory. The addition of pAsp increases interfacial energies between nuclei and mineralization fluids. In contrast, the confined gap spaces inside collagen fibrils lower the energy barrier by reducing the reactive surface area of nuclei, decreasing the surface energy penalty for IM. The confined gap geometry, therefore, guides the 2D morphology and structure of bioapatite and changes the nucleation pathway by reducing the total energy barrier.
Next, we examined the influence of nanoscale nucleation on microscale CaP distribution in highly organized collagen matrices extracted from chicken tibia bones, and consequently on the matrices’ mechanical properties. The matrices were mechanically loaded during mineralization to understand the physicochemical responses of collagen, which are different from biological responses. We found that cyclic strain increased acellular nucleation rates by 33% compared to nucleation under static strain. The enhanced transport of mineralization fluids under cyclic strain delivers more nucleation precursors into the matrices, increasing the elastic modulus, strength, and resilience. To easily mimic the microenvironment generated by cyclic strain for engineering applications, we also applied pulsed electric stimulations. Low amplitude pulsed stimulation through the cathode enhanced the transport of ionic body fluid components, leading to successful mineralization of the inner surface of tube-like collagen scaffolds through a microscale channel.
Finally, we expanded our knowledge obtained from the biomineralization studies to an environmental remediation strategy. We proposed a sustainable P management strategy for nutrient-rich aqueous environments, inspired by bones’ ability to balance P in the human body. By seeding reactive CaP nuclei with an engineered crystalline structure in calcium alginate beads, we were able to control the energy barriers to CaP nucleation and manage an environmental aqueous P concentration as a function of pH. The CaP seeded beads effectively immobilized aqueous P in their macroscale organic/mineral networks, which have the potential to be reused as a slow-release fertilizer.
Our findings illuminate the importance of nanoscale nucleation reactions, macroscale mineral distributions, and macroscale mechanical properties. The findings also provide new insights into designing biomaterials with hierarchical structures. By improving the engineered control of properties of highly organized collagen matrices, and using cyclic strain condition and pulsed electrical stimulations, we can improve bone-fracture healing. Furthermore, designing CaP–organic composites provides promising solutions for environmental remediation. We expect that the bioinspired principles can also be applied to other fields of engineering requiring knowledge of chemistry at organic–mineral interfaces.
John Fortner, Daniel Giammar, Jill D. Pasteris, Stavros Thomopoulos,