Biocompatibility of chitosan-amorphous calcium phosphate composite membrane for bone regeneration

Objectives: Chitosan (CS) with calcium phosphate based materials has been used as tissue engineering scaffold due to its superior anti-bacterial activity, biocompatibility and osteoconductivity. Compare to hydroxyapatite or tricalcium phosphate, amorphous calcium phosphate (ACP) presented better cell adhesion and cell proliferation in vitro and the ability to enhance bone regeneration in vivo. In this study, to take the advantages of CS and ACP, we developed a novel composite membrane of CS-ACP for using in bone regeneration and evaluated the cellular response to the material.
Methods: Chitosan membrane was synthesized with 2% chitosan powder in acetic acid solution and then reacted with 1N NaOH to produce asymmetric porous structure. To produce the CS-ACP membrane, the chitosan membrane was first placed into 0.06M Na₂HPO₄ solution, and then into 0.1 M Ca(NO₃)₂•4H₂O solution. The characteristics of CS-ACP membrane were investigated via SEM, XRD and TEM. Furthermore, U2OS cell adhesion, proliferation and cytotoxicity to CS-ACP membrane were evaluated using SEM observation, WST-1 and LDH assays.
Results: After 5 minutes of reaction, SEM demonstrated the formation of spheres in the chitosan membrane, which were confirmed to be ACP by XRD and TEM. After 10 days of reaction, the spheres transferred into needle-like particles, which presented the characteristics of hydroxyapatite (HAP) via SEM, XRD and TEM evaluation. Compare to CS membrane, CS-ACP (reaction for 1 day) membrane presented better cell adhesion. WST-1 assay showed there was no significant difference in cell proliferation behavior among CS-ACP, CS and control groups. LDH assay demonstrated more celll death was found in CS-ACP and CS groups than control group, but no significant differences among the experimental groups and control group.
Conclusions: By using soaking method, we successfully developed CS-ACP membrane for bone regeneration application. Furthermore, the CS-ACP membrane is biocompatible based on the results of SEM observation, WST-1 and LDH test.