Development of hydrogel-based composite scaffolds containing eggshell particles for bone regeneration applications

This study describes the development and characterization of novel composite scaffolds, made of an alginate-chitosan hydrogel matrix containing eggshell (ES) particles, for bone tissue engineering applications. Scaffolds with ES particles, either untreated or treated with phosphoric acid to create a nanotextured particle surface, were compared to scaffolds without particles. Results indicate that the nanotexturing process exposed occluded ES proteins orthologous to those in human bone extracellular matrix. Scaffolds with ES or nanotextured ES (NTES) particles had a higher porosity (81 ± 4% and 89 ± 5%, respectively) than scaffolds without particles (59 ± 5%) (p = .002 and p < .001, respectively). Scaffolds with NTES particles had a larger median pore size (113 μm [interquartile range [IQ]: 88-140 μm]) than scaffolds with ES particles (94 μm [IQ: 75-112 μm]) and scaffolds without particles (99 μm [IQ: 74-135 μm]) (p < .001 and p = .011, respectively). The compressive modulus of the scaffolds with ES or NTES particles remained low (3.69 ± 0.70 and 3.14 ± 0.62 kPa, respectively), but these scaffolds were more resistant to deformation following maximum compression than those without particles. Finally, scaffolds with ES or NTES particles allowed better retention of human mesenchymal stem cells during seeding (53 ± 12% and 57 ± 8%, respectively, vs. 17 ± 5% for scaffolds without particles; p < .001 in both cases), as well as higher cell viability up to 21 days of culture (67 ± 17% and 61 ± 11%, respectively, vs. 15 ± 7% for scaffolds without particles; p < .001 in both cases). In addition, alkaline phosphatase (ALP) activity increased up to 558 ± 164% on day 21 in the scaffolds with ES particles, and up to 567 ± 217% on day 14 in the scaffolds with NTES particles (p = .006 and p = .002, respectively, relative to day 0). Overall, this study shows that the physicochemical properties of the alginate-chitosan hydrogel scaffolds with ES or NTES particles are similar to those of cancellous bone. In addition, scaffolds with particles supported early osteogenic differentiation and therefore represent a promising new bone substitute, especially for non-load bearing applications.