Engineering a Zone of Calcification that is Localized in Space and Mechanically Stiff

Introduction: Fabrication of hybrid skeletal tissue constructs often involves creation of a mineralized component, mimicking bone and the interface between soft tissue and bone. One of the key structural components of the interface to bone, and bone itself, is hydroxyapatite. Hydroxyapatite can be formed rapidly in vitro by controlled reaction of calcium (Ca) and phosphate (PO4) with the use of a double diffusion system. The mechanism limiting hydroxyapatite formation in this system is unclear, but may be the chemical reaction or the diffusive transport of reactants. Normally, the calcified interface functions as a mechanical transition with stiffness, associated with mineral content, intermediate between that of soft tissue and bone. We hypothesized that diffusion of calcium and phosphate into agarose, as a hydrogel tissue analog, enables the formation of a precipitated mineral in a diffusion-limited process and enhances hydrogel stiffness. The objectives of this study were to determine (1) the extent of calcification and the associated stiffening of the hydrogel, and (2) whether calcification was governed by a diffusion-limited or reaction-limited process.