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 degrees 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 degrees 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 x 10(4) cells/ml and placed in cold storage for 1-14 d. After cold storage the cultures were then incubated at 37 degrees C for 1-20 d. A set of replicate plates was also challenged with 10 ng/ml endotoxin upon incubation at 37 degrees 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.