# Sustainable Eggshell-Based Amorphous Calcium Phosphate Scaffolds and Membrane Protein Hydrogel for Regeneration in Rabbit Femoral and Calvarial Defects

## TL;DR

This paper explores using eggshell waste to create sustainable bone scaffolds that, when combined with a special hydrogel, improve bone regeneration in rabbits.

## Contribution

The novel contribution is the sustainable upcycling of eggshell waste into amorphous calcium phosphate scaffolds paired with a bioactive hydrogel for enhanced bone regeneration.

## Key findings

- ACP scaffolds combined with HA/P2 and SEPs showed the highest bone volume and trabecular number.
- Histology confirmed more extensive mineralized collagenous deposition and better bone–material fusion with SEPs.
- Eggshell-derived ACP scaffolds with SEP-enriched hydrogel significantly enhanced early bone regeneration in critical-sized defects.

## Abstract

Critical-sized bone defects require grafts with ideal
spatial supporting.
We upcycled eggshell waste sustainably into porous amorphous calcium
phosphate (ACP) scaffolds and evaluated their early bone-regeneration
performance alone and in combination with flexible and bioactive adjuncts in vivo. ACP scaffolds with ∼60% porosity were fabricated
by pressure-densification, using ice microspheres as space holders.
Before implantation, ACP scaffolds were vacuum-infiltrated with a
hyaluronic acid (HA) hydrogel to minimize internal voids. Bilateral
critical-size femoral and calvarial defects were created in New Zealand
White rabbits and reconstructed with one of four treatments: (1) Sham,
(2) ACP scaffold (ACP), (3) ACP infused with HA hydrogel containing
a proline-rich peptide P2 (ACP + HA/P2), or (4) ACP infused with HA/P2
supplemented with soluble eggshell membrane proteins (prepared by
our lab, ACP + HA/P2 + SEPs). After 6 weeks, micro-CT and histology
were performed. All ACP-containing groups showed greater defect filling
and mineralization than the Sham group. Micro-CT analysis demonstrated
that ACP + HA/P2 + SEPs achieved the highest bone volume and trabecular
number and the lowest trabecular separation. Histology confirmed more
extensive mineralized collagenous deposition on ACP scaffolds and
a more integrated bone–material fusion zone in this group.
In femoral defects with graft displacement, ACP scaffolds induced
cortical–scaffold bone bridging within the marrow, most evident
with SEPs. Eggshell-derived ACP scaffolds, particularly when paired
with SEP-enriched HA hydrogel, enhance early regeneration of critical-sized
bone defects. Upcycling the inorganic and organic constituents of
eggshell waste yields a complementary, single-origin biomaterials
package for sustainable bone repair.