Osteopontin eluting microspheres decrease calcification in tissue engineered heart valves

Abstract

Introduction: Calcific degradation is a leading cause of heart valve failure for all bioprosthetic heart valve replacements, including tissue engineered heart valves. This study was performed to develop a drug delivery system that would provide local controlled release of osteopontin, the body’s principal regulator of ectopic calcification, in an attempt to decrease pathological calcification of tissue engineered heart valves. Methods: Osteopontin[OPN] was encapsulated in polylactide co-glycolide[PLGA] microspheres using spontaneous emulsification. Microsphere production was confirmed using SEM and a release profile was generated by fluorescense. Ovine heart valves were decellularized using Triton X and SDS. Microsphere attachment to the decellularized heart valve was assessed using flourimetry. Three decellularized heart valves[control, blank microspheres and OPN microspheres] were implanted subcutaneously into each of seven BL6 mice, the standard first line model for ectopic calcification. Results: Osteopontin microspheres[1-30um] were successfully fabricated (Fig 1). A release profile demonstrated typical release profile with an initial burst effect followed by steady state release. Microspheres coated with avidin exhibited increased binding to decellularized tissue using fluorimetry. In vitro release of a fluorescent agent from the microspheres attached to decellularized vascular tissue exhibited a consistent fluorescence of 3800 FIU from day 2 to 14. Identically treated avidin bound microspheres fluoresced at 14500. Avidin bound microspheres attached to biotinylated tissue fluoresced at 16500. The amount of ectopic calcification[mcg/mg] of subcutaneously implanted decellularized heart valve tissue after four weeks was determined using atomic absorption spectroscopy and standardized for specimen weight (see table). Decellularized tissue only (D), decellularized tissue with blank microspheres (B), and decellularized tissue with osteopontin microspheres (O). Conclusions: A microsphere drug delivery system which elutes osteopontin was created. The binding of microspheres to decellularized vascular matrix can be enhanced by coating the surfaces with avidin and biotin. Preliminary in vivo data suggests that calcification of matrices is significantly reduced when osteopontin microspheres are attached to the scaffolds in the subcutaneous implantation model. Decellularized tissue only (D), decellularized tissue with blank microspheres (B), and decellularized tissue with osteopontin microspheres (O).