Effect of calcification on agarose gel stiffness and integration strength with Bone

The zone of calcified cartilage (ZCC) is critical for the normal attachment of articular cartilage to bone as well as to the biomimetic bioengineering of osteochondral tissue constructs. However, relatively few osteochondral tissue engineering approaches have created a tissue resembling and functioning like the ZCC. The implementation of a double diffusion system, wherein calcium (${Ca}^{2+}$) and phosphate (${{PO}_{4}}^{3-}$) ions are diffused toward each other, provides a method to induce local mineralization within a hydrogel. The objectives of the present study were to (1) estimate the diffusivity of ${Ca}^{2+}$ and ${{PO}_{4}}^{3-}$ within a 2% agarose gel, (2) characterize morphologically, chemically, and biomechanically the mineral structure formed with agarose using the double diffusion system, and (3) determine the feasibility of using the double diffusion system to create a mineral structure at the site of agarose attachment to subchondral bone (ScB), trabecular bone (TB), or porous titanium. The diffusion of Ca2+ and PO43- created calcified agarose consistent with the formation of hydroxyapatite (HA). From concentration profiles, the diffusion coefficients for ${Ca}^{2+}$ and ${{PO}_{4}}^{3-}$ in a 2% agarose gel were estimated to be ${6.4x10}^{-6}$ and ${1.3x10}^{-6}$ cm2/s, respectively. Using the double diffusion system, mineralization was visualized grossly as a broad precipitation band and by micro-CT scan as a toroidal structure The indentation stiffness of the gel was increased (+50%) to a peak coincident with the location of the peak precipitation band and chemical content of ${Ca}^{2+}$ and ${{PO}_{4}}^{3-}$. The integration strength between agarose and ScB (0.27 ± 0.02 N) was less than that between agarose and TB (0.73 ± 0.05 N). Application of the double diffusion did induced calcification locally at the targeted site of a porous titanium disc; however, it did not at either a ScB or TB target. These results may be applied to enhance formation of a biomimetic interface between hydrogel and a target porous rigid structure