(625g) Eggshell Particle-Reinforced Hydrogels for Bone Tissue Engineering

More than two million bone graft procedures are performed each year globally to treat patient defects, including but not limited to aging, degeneration, cancer, accidents, diseases, and war injuries. Bone repair is crucial to restore patient functionality after the injury. Protein-based hydrogel scaffolds are widely used for bone tissue engineering applications due to their tunable physical, chemical, and biological properties. We hypothesized that incorporation of eggshell microparticles (ESP) into protein-based hydrogels can possibly enhance osteogenic differentiation and bone regeneration due to the superior biophysical properties of calcium carbonate over the other calcium-based minerals. Although they are readily available and cost-effective, the valuable potential of eggshells in bioengineering applications remains greatly understudied.

In this work, we developed ESP-reinforced gelatin-based hydrogels to obtain mechanically stable and biologically active three-dimensional (3D) scaffolds that can differentiate pre-mature cells into osteoblasts. This work is the first demonstration that eggshell particles can be used in a composite 3D construct for bone tissue engineering. We investigated the physical properties including swelling ratio, degradation, and mechanical properties of the composite hydrogels as well as their porosity. Pre-osteoblasts were encapsulated within the ESP-reinforced hydrogels to study their differentiation and evaluate mineral deposition by the cells. The ESP-reinforced gels were then subcutaneously implanted in a rat model to determine their biocompatibility and degradation.

The ESP-reinforced composite hydrogels have shown outstanding tunability in physical and biological properties holding substantial promise for engineering mineralized tissues (e.g. bone, cartilage, tooth, tendon). These 3D scaffolds enabled differentiation of pre-osteoblasts without the use of specialized osteogenic growth medium. The ESP-reinforced gels exhibited significant enhancement in mechanical properties and mineralization by pre-osteoblasts. Findings suggest that our novel composite hydrogel shows superior mechanical properties and indicates a favorable in vivoresponse.

In summary, ESP-reinforced hydrogels were successfully fabricated and evaluated for their suitability for osteogenic cell cultures in vitroand biocompatibility in vivo. The in vivosubcutaneous implantation experiments exhibited biodegradation potential of the ESP-reinforced hydrogels in 14 days as well as indicating no significant inflammatory response. The implants were easily accepted by the host, allowed for cellular migration and infiltration in 3D, and highly vascularized. Because ESP is an inexpensive and readily accessible material, we anticipate that it will now be incorporated into 3D scaffolds in a range of new biomedical applications.