The effects of cold storage and endotoxin challenge on osteoblast viability and interleukin-6 production

Autogenous hip marrow is an excellent source of pluripotential cells for regenerative procedures. However, before this treatment modality can be employed a method to attenuate osteoclast activity must be developed. The shock of cold storage (4°C) is thought to abate osteoclast activity through the downregulation of osteolytic cytokines produced by osteoblasts. The objective of this study was to evaluate the effects of cold storage (4°C) and endotoxin challenge on bone cell culture viability and interleukin-6 (IL-6) production. These cells (osteoblasts) were primarily harvested from murine calvaria utilizing sequential digestions, separated by density gradient and combined. Twelve-well cell culture plates were inoculated with 2×10⁴ cells/ml and placed in cold storage for 1-14 d. After cold storage the cultures were then incubated at 37°C for 1-20 d. A set of replicate plates was also challenged with 10 ng/ml endotoxin upon incubation at 37°C for 4 consecutive days. Cells were evaluated daily for alkaline phosphatase activity. Cell culture supernatants were also collected daily and batch assayed for IL-6 production. Cell cultures did not survive more than 48 h of cold storage. There was a decrease in IL-6 secretion in all refrigerated cultures and a significant decrease in those cells refrigerated for 48 h versus control cultures (p<0.05). Replicate cultures treated with endotoxin secreted significantly increased amounts of IL-6 in both the control cultures and the cultures exposed to 24 h of cold storage versus non-endotoxin-treated control cultures (p<0.05). These observations suggest that after 48 h of cold storage autogenous marrow may be safe to use because of the dramatic decrease in IL-6 production by osteoblasts.