Effects of Allogeneic Bone Substitute Configurations on Cell Adhesion Process In Vitro

To explore the potential effect of three allogenic bone substitute configurations on the viability, adhesion, and spreading of osteoblasts in vitro.

Freeze-dried cortical bone were ground and fractions were divided into three groups with different sizes and shapes, defined as bone fiber (0.1 mm × 0.1 mm × 3 mm), bone powder (0.45-0.9 mm), and bone granule group (3-6 mm). MC3T3-E1 cells were divided and co-cultured within groups to induce cell adhesion. The configuration of allogenic bone was captured by scanning electron microscopy and confocal laser scanning microscopy, and substrate roughness values were quantified. Cell adhesion rate was assessed using the hemocyte counting method, cell viability was determined by CCK-8 assay and live/dead staining, and cell morphology was visualized by Phalloidin and DAPI, and the mRNA expression of adhesion-related gene (vinculin) of different substitutes were determined with quantitative real-time polymerase chain reaction.

The roughness values of bone fiber, bone powder, and bone granule group were 1.878 μm (1.578-2.415 μm), 5.066 μm (3.891-6.162 μm), and 0.860 μm (0.801-1.452 μm), respectively (bone powder group compared with bone granule group, H = 18.015, P < 0.001). Similar OD values of all groups in CCK-8 assay indicated good biocompatibility of these substitutes (bone fiber, 0.201 ± 0.004; bone powder, 0.206 ± 0.008; bone granule group, 0.197 ± 0.006; and the control group, 0.202 ± 0.016, F = 0.7152, P > 0.05). In addition, representative cell adhesion rates at 24 h showed significantly lower cell adhesion rate in bone fiber group (20.3 ± 1.6%) compared to bone powder (29.3 ± 4.4%) and bone granule group (27.3 ± 3.2%) (F = 10.51，P = 0.009 and P = 0.034, respectively), but there was no significant difference between the latter two groups (P > 0.05). Interestingly, the expression of vinculin mRNA steadily decreased in a time-dependent manner. The vinculin expression reached its peak at 6 h in each group, and the vinculin levels in bone fiber, bone powder, and bone granule group were 2.119 ± 0.052, 3.842 ± 0.108, and 3.585 ± 0.068 times higher than those in the control group, respectively (F = 733.643, all P < 0.001). Meanwhile, there was a significant difference in the expression of target gene between bone powder and bone granule group (P = 0.006).

All allogenic bone substitutes presented an excellent cell viability. Moreover, bone powder and bone granule group were more likely to promote cell adhesion and spreading compared to bone fiber group.