# Comparative evaluation of the effectiveness of a novel composite bone substitute synthesized from eggshell-derived hydroxyapatite and fish collagen in bone regeneration of critical-sized calvarial defects in Wistar rats

## TL;DR

A new bone substitute made from eggshells and fish collagen was tested in rats and showed good bone regeneration, though not as effective as a commercial product.

## Contribution

A novel composite bone graft combining eggshell-derived hydroxyapatite and fish collagen was developed and evaluated for bone regeneration.

## Key findings

- The composite graft showed significantly better bone regeneration than empty controls and eggshell-derived hydroxyapatite alone.
- Radiodensity was higher in composite graft-treated sites compared to controls and similar to a commercial graft (Bio-Oss).
- The composite graft demonstrated promising but not superior performance compared to a standard commercial bone graft material.

## Abstract

Large bone deformities pose significant challenges in regenerative periodontal surgeries. A healthy underlying bone and the osteoinductive effect of the surrounding environment are the primary factors that influence bone regeneration. Collagen and hydroxyapatite are universally used bone substitutes owing to their excellent biocompatibility and biodegradability. Innovations in the osteoinductive properties of hydroxyapatite and its use in different modified forms, combining it with other materials, may result in enhanced biomechanical properties. This research utilized domestic chicken eggshells to synthesize hydroxyapatite and fish scales to extract collagen. The novel composite graft was prepared by integrating both materials. The beneficial effects of the composite graft on the healing and regeneration of bone were assessed in the calvarial bone of Wistar rats both radiographically and histomorphometrically.

Hydroxyapatite was synthesized from domestic chicken eggshells using the chemical precipitation method. Collagen was extracted from the scales of Rohu fish. The novel composite material was prepared by direct mixing and lyophilization, with further mixing with glycerol to attain an appropriate consistency. After preliminary characterization, bone regeneration was assessed in surgically created critical bone defects in rats. Radiographic bone fill was assessed using cone beam computed tomography, which was followed by histomorphometry. The bone regenerative capacities of the two novel materials were compared with empty control sites and a commercially available standard bone graft (Bio-Oss).

The novel materials displayed significantly superior regenerative capacity both radiographically and histomorphometrically when compared to empty controls, but the regenerative ability was lower when compared to the standard bone graft material (Bio-Oss) over 90 days. A trend of increased new bone formation and significantly higher radiodensity was observed in the sites treated with the composite graft compared to the empty control (p < 0.0001) and eggshell-derived hydroxyapatite groups (p < 0.01). Moreover, Bio-Oss exhibited a marginal improvement in radiodensity compared to the composite graft (p < 0.05).

The composite graft demonstrated improved bone regeneration and radiodensity in the surgically created critical-sized defects. Hence, it can be considered a promising material for the regeneration of periodontal and peri-implant defects. Further laboratory and clinical studies may be required to confirm its osteoinductive properties.