Biomineralization with engineered cellular systems
Hydroxyapatite (HAP) is the final product of bone biomineralization process and HAP formation is controlled by proteins, enzymes and small molecules secreted to extracellular matrix (ECM). Among these molecules, alkaline phosphatase (ALP) leads formation of HAP crystals and noncollagenous proteins control crystal nucleation and growth, and inhibit crystal formation. Osteocalcin (OCN) and osteopontin (OPN), are the most abundant noncollagenous proteins in ECM, which controls mineralization events. In this study, effect of OCN and OPN on HAP crystal formation was studied in order to achieve controlled crystal growth. In vitro biomineralization assays were conducted to understand the effect of OCN and OPN on the crystal structure of as formed minerals. While OCN decreases crystal growth rate and inhibit mineralization, which leads to more uniform crystal formation, OPN provides faster mineral formation with reduced Ca/P ratio. Moreover, a mammalian engineered cell line was constructed to achieve expression of bone extracellular matrix (ECM) proteins. For this purpose, genetic cassettes were produced to express OCN and OPN proteins, which are the most common non-collagen proteins that control bone mineral formation. By this way, production of bone type minerals with controlled size, shape and Ca/P ratio can be possible. Our system provides a truly biomimetic approach to HAP formation compared to chemical synthesis methods in literature. We believe our current findings will lead to innovative approaches for bone biomineralization in regenerative medicine and bone tissue engineering.