Coupled OPG-Fc on Decellularized Aortic Valves by EDC/NHS Attenuates Rat MSCs Calcification In Vitro

AbstractValve calcification commonly damages natural human heart valves and tissue-engineered heart valves (TEHVs), and no ideal intervention is available in clinical practice. It is increasingly considered that osteoprotegerin (OPG) inhibits vascular calcification. Herein we aimed to explore whether free OPG-Fc fusion protein or coupled OPG-Fc on decellularized aortic valves attenuates calcification. Calcification of rat bone marrow–derived mesenchymal stromal cells (MSCs) was induced by osteogenic differentiation media, and the effects of free OPG-Fc or OPG-Fc coupled on the decellularized porcine aortic heart valve leaflet scaffolds by coupling agents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) on calcification were observed. Mineralization of the extracellular matrix, alkaline phosphatase (ALP) activity, and expression of osteoblastic markers were assessed to determine the calcification kinetics. Our results indicated that the matrix calcium content and the ALP activity, as well as the mRNA expression levels of a bone morphogenetic protein-2 (BMP-2), osteopontin (OPN), and osteocalcin (OC), of the MSCs seeded on plates with free OPG-Fc or on the OPG-Fc–coupled scaffolds decreased compared with their control MSCs without coupled OPG-Fc. The results suggest that both free and immobilized OPG-Fc on the decellularized aortic valve scaffolds by EDC/NHS can attenuate the calcification of MSCs induced by osteogenic differentiation media, implying that OPG-Fc might be a new treatment or prevention strategy for the calcification of natural human heart valves and TEHVs in the future.