Improvement in the osteoblastic cellular response to a commercial collagen membrane and demineralized freeze-dried bone by an amino acid derivative: An in vitro study

Purpose: The objectives of this in vitro study were (1) to determine whether a commercially available collagen membrane (CM) or human demineralized freeze-dried bone (DFDB) particles adversely affected viability or function in cultured osteoblasts through oxidative stress, and, if so, (2) to determine whether N-acetyl cysteine (NAC) successfully prevented loss of viability and dysfunction in osteoblasts. Materials and methods: Rat calvaria-derived osteoblasts were seeded onto polystyrene and commercially available CM (Cytoplast^®) or DFDB (DynaGraft^™) with or without pretreatment with NAC solution. The osteoblastic response was evaluated using a flow cytometric cell viability assay, measurement of attached viable cell number, quantification of reactive oxygen species (ROS) and alkaline phosphatase (ALP) staining. Results: The percentage of viable cells on CM was <50% at 24 h after seeding. However, this increased to 70% by pretreatment with NAC. The numbers of attached osteoblasts on DFDB remained at 60% the level of that on polystyrene at 24 h after seeding, but increased to up to 90% the level of that on polystyrene with NAC pretreatment. Although collagen materials increased intracellular ROS generation 1.5-5 times that with polystyrene, this was significantly reduced by NAC pretreatment. The percentage of the ALP-positive area was consistently 7% or less on CM and DFDB at days 7 and 14, which was restored by NAC pretreatment up to 60% or more. Conclusions: Commercially available CM and DFDB impaired osteoblastic viability and function and markedly increased intracellular ROS, indicating an oxidative stress-mediated negative impact on osteoblasts. Pretreatment with NAC substantially alleviated these cytotoxic effects.