Alleviation of commercial collagen sponge-and membrane-induced apoptosis and dysfunction in cultured osteoblasts by an amino acid derivative

Purpose: The objectives of this in vitro study were to determine whether the commercial collagen material used in bone augmentation procedures induces oxidative stress-mediated adverse effects on the viability and function of osteoblasts and to determine whether N-acetyl cysteine (NAC), an antioxidant amino acid derivative, can alleviate these effects. Materials and Methods: Commercial collagen sponge (Collaplug) and membrane (BioGide) were treated with NAC. Rat calvaria-derived osteoblasts were directly seeded on these materials with or without NAC pretreatment. Cytotoxic evaluation was performed by flowcytometric cell viability assay, confocal laser microscopic analysis of attached cell morphology and reactive oxygen species (ROS) localization, and alkaline phosphatase staining. Results: Cell viability was less than 40% on both collagen sponge and membrane 24 hours after seeding and increased to 50% with NAC pretreatment. Cell death was characterized by apoptosis. Colonization of attached cells was sparse on the untreated sponge and membrane on day 1, and the cells were round, small, and filled with intense and closely packed intracellular ROS. In contrast, NAC-pretreated material had dense cell colonies consisting of well-spread osteoblasts and fully developing cytoskeleton and cellular processes with little ROS generation. On day 7 of culture, NAC-pretreated collagen sponge and membrane yielded an expanded alkaline phosphatase-positive area occupying 60% and 80% of the surface area, respectively, whereas the untreated collagen materials had limited alkaline phosphatase activity (7% or less). Conclusions: Commercial collagen sponge and membrane induced considerable cell death, impaired initial function, and generated extraordinary intracellular ROS in attached osteoblasts, whereas NAC pretreatment substantially ameliorated these effects. The potential benefits of NAC’s detoxifying capacity on bone regeneration using collagen matrix materials in an animal model should be confirmed with further study.