| Dystrophic calcification has been the long-standing major cause of bioprosthetic heart valve failure, and has been well studied in terms of the underlying causative mechanisms. Such understanding has yielded several anti-calcification strategies involving biomaterial modification at the preparation stage: chemical alteration, extraction of calcifiable components, or material modification with small-molecule anti-calcific agents. However, newer therapeutic opportunities are offered by the growing illustration of the pathology as a dynamic, actively regulated process involving several gene products, such as osteopontin (OPN), matrix-gla protein (MGP) and glycosaminoglycans (GAGs). Osteopontin, a multi-functional matricellular glycosylated phosphoprotein has emerged as a prime candidate for the role of an in vivo inhibitor of ectopic calcification with two putative mechanisms: crystal poisoning and mineral-dissolution. The full therapeutic realization of its potential necessitates a better understanding of the mechanisms of anti-calcification by osteopontin, as well as appropriate in vivo models in which to evaluate its efficacy, potency and molecular mechanisms.; In this work, we pursued the development and characterization of a reliable in vivo model with the OPN-null mouse to simulate the calcification of bioprosthetic valve material, namely glutaraldehyde-fixed bovine pericardium (GFBP) tissue. Subsequently, we used the calcification model to evaluate hypotheses based on the anti-calcific potential of osteopontin. Several modes of administering exogenous OPN to the implant site in OPN-null mice were explored, including soluble injected OPN, OPN covalently immobilized on the biomaterial, and OPN adsorbed onto the biomaterial. An investigation of the structure-function aspects of the anti-calcific ability of OPN was also pursued in the in vivo model. The OPN-null mouse was also used as an in vivo test-bed to evaluate the anti-calcific potential of other biomolecules, namely hyaluronic acid (HA) and natural reducing agents, such as glutathione.; Direct rescue of the calcification phenotype in the OPN-null mice was achieved by administration of exogenous OPN, providing strong evidence of OPN's ability to mitigate ectopic calcification. Significant reduction in calcification was observed on administering OPN in soluble injected form and also when immobilized (adsorbed) onto the biomaterial. Mechanistic insights were also gained, since maximal anti-calcific effect was offered by OPN only when the protein had adequate phosphorylation as well as a functional RGD domain—suggesting synergy between these two structural elements and also a “threshold effect” for the degree of phosphorylation. In addition, the OPN-null in vivo calcification model was employed to gain evidence for the anti-calcific potential of covalently-immobilized hyaluronic-acid (HA) and the natural reducing agent glutathione. |
