Eggshells derived Nano-hydroxyapatite doped with Si and Zn as novel bone regenerative material in critical-sized bone defects – in vitro and in vivo study

Abstract Hydroxyapatite Nano-particles are the most abundant inorganic compound found in bone tissue. Nano-hydroxyapatite (N-HA) was synthesized by different methods to be used as bone substitution material due to its favorable biocompatibility and biological activity. To mimic the natural structure of bone inorganic phase, N-HA has been doped with several ions to enhance its degradation and cell interaction. In this work, using the wet chemical precipitation method, N-HA was synthesized from eggshells as a bio-waste source and doped with silicon (Si+ 4) and zinc (Zn+ 2). Spherical Nano-sized particles of hydroxyapatite (HA) were obtained with a high surface area and an interconnected porous structure. The average particle size was 239 nm, and the average porosity of 20%. The substitution of Si+ 4 and Zn+ 2 into N-HA crystal lattice affected physical features by decreasing the particle size and crystallinity. The crystal size of N-HA decreased from 61 nm to 46 nm after the dual doping. Doping ions also improved the biological behavior of N-HA by increasing the cell viability of N-HA in vitro. Synthesis of N-HA from eggshells seemed to affect the phase purity of the material, a phase of whitlockite (WH) appeared in N-HA powder even after was sintered at a high temperature of 1200○c. The in vivo results demonstrated the ability of Si –Zn doped HA to well integrate into bone tissue and induce bone regeneration in critical-sized bone defects. Histological analysis showed that the mean value of newly-formed bone area (total bone area/total area) measured via histomorphometric reached 69%±0.015% after 12 weeks in grafted defects. Furthermore, significant differences in bone formation were found between grafted and control samples. We present our study to be a starting point for more investigation into the ability to synthesize a biomimicry bone substitution martial that meets the renewable requirements in the bone regeneration field.