In Vitro Mineralization Models: Examining The Formation Of Calcium Phosphate In A Hydrogel Based Double Diffusion System.
In biomineralization, both crystal nucleation and growth are under tight regulation. The components required for biomineralization are: the controlled delivery of reagents required for crystal growth to the site of mineralization, a nucleating substrate, a growth medium (often a hydrogel-like matrix), and growth-modifying elements (often acidic biomacromolecules). Using synthetic analogs of components found in biology, in vitro models can be created to study various aspects of biomineralization. Depending on what one wants to model, the type of in vitro system can vary greatly: from solution growth of hydroxyapatite using a constant composition set-up, to in vitro mineralization using cells, to synthetic growth in hydrogels. Traditionally, crystal growth in hydrogels is a technique used by crystallographers to grow large crystals. For those seeking to model biomineralization, hydrogels are also excellent models of the extracellular matrix (ECM) microenvironment. In this thesis, I use an optimized hydrogel-based double diffusion system (DDS) (Chapt. 1) to explore interesting questions in biomineralization such as: What are the effects of various types of hydrogels (in which ions have different diffusivities) on both the crystal morphology and degree of mineralization in an environment where the rate of diffusion is controlled by changing the experimental setup (Chap. 2)? What i is the role of substrates in mineralization within an ECM-like matrix, and how can such a substrate be fabricated and introduced into a hydrogel-based DDS (Chap. 3)? What are the effects regulating gradients of inhibitors, using enzymes strategically placed in a DDS, on mineral formation within an ECM-like environment (Chap. 4)? By evaluating the DDS in the context of classical diffusion theory, an optimized system was designed and tested (Chap. 1). Using an innovative layered hydrogel design, differences in ion diffusivities within different hydrogels were eliminated. These experiments showed that both collagen and gelatin gels produce similar crystal morphology, while both agarose and collagen have increased mineral content over that of gelatin (Chap. 2). Placing porous silicon substrates into the DDS, revealed that cooperative behavior of proteins and substrates has an effect on both the morphology and the quantity of mineral in the hydrogel (Chap. 3). Finally, by modulating a gradient of mineral inhibitors, mineral gradients within the hydrogel are formed. The resulting "sharpness" in the mineral/hydrogel interface is proportional to the steepness of the inhibitor gradient (Chap. 4). Taken together these results provide insight into the formation of calcium phosphate in biological systems. ii
Hydrogel; Hydroxyapatite; Biomineralization
Estroff, Lara A.
Ober, Christopher Kemper; Boskey, Adele
Materials Science and Engineering
Ph. D., Materials Science and Engineering
Doctor of Philosophy
dissertation or thesis