Bone substitute made from a Brazilian oyster shell functions as a fast stimulator for bone-forming cells in an animal model

Abstract

Despite their demonstrated biocompatibility and osteogenic properties, oyster shells have been reported as a potential alternative to other commonly used materials for bone substitution. This study evaluated whether an experimental bone substitute (EBS) made from a typical oyster shell of Northeastern Brazil (Crassostrea rhizophora) has effects on bone development using an animal model. Oysters were collected from a biologically assisted vivarium, and their inner layer was used for preparing an EBS. Chemical and surface characterization of EBS was performed using Individually Coupled Plasma Optical Emission Spectrometry (ICP-OES) and Scanning Electron Microscope (SEM), respectively. Seventy-two rats were randomly assigned to groups according to the treatment of bone defects created in the submandibular area: Negative Control (-C), Positive Control (+C; Bio-Oss®) and EBS. Euthanasia occurred at 7, 21, 42 and 56 days postoperatively. The bone pieces were stained with hematoxylin and eosin (H&E). The formation of bone tissue was evaluated histologically and histomorphometrically. Data were analyzed through the Kruskal-Wallis test and ANOVA considering a significant level of 5%. The main element found in EBS was calcium (71.68%), and it presented heterogeneity in the particle size and a porosity aspect at SEM analysis. Histological results revealed the absence of inflammatory cells in all groups, being that EBS presented the most accelerated process of bone formation with a statistically significant difference between this group and the +C and -C groups in the 21-day time-point (p < 0.05). After 21 days, the bone formation process was similar between all groups (p > 0.05), showing an immature lamellar bone pattern after 56 days of experimentation (p > 0.05). Within the limitations of this study, it was possible to conclude that EBS presented good biocompatibility and promoted fast stimulation for bone-forming cells in an animal model.

Fig 1

Surface characterization using SEM analysis of EBS at X2.500 (A) and X5.000 (C); and Bio-Oss® at X2.500 (B) and X5.000 (D).

Fig 2. Histological findings of bone defect after 21 days.

A and B represent the -C group with the presence of osteocytes (black arrow), osteoblasts (white arrow) and neoformed bone trabeculation (NB). C and D represent the +C group showing mesenchymal tissue (MT) and discret neoformed bone trabeculation (NB). E and F represent the EBS group where the predominance of MT and multifocal points of NB occupy the bone defect, presence of osteocytes and osteoblasts. B indicates Bio-Oss®; EBS indicates experimental bone substitute; * indicates the border of the bone defect. Tissues were stained with H&E and examined at 5x and 20x magnification (200 μm and 50 μm, repectively).

Fig 3. Bone formation score median between groups at different periods.

Different symbols (*,**) indicate statistically significant differences between groups at the same period.