The Validity of CMFDA and Ethd-1 in the Determination of Osteocyte Viability in the Diaphysis of Long Bones in a Sheep Model

Abstract

The determination of osteocyte viability is essential in bone research and clinical practice; however its determination at the time of sampling, without disruption of the bone matrix is difficult. The current clinical histological diagnosis of osteonecrosis requires the identification of empty osteocyte lacunae, which may take up to 4 weeks to completely develop. The aim of this study was to assess the efficacy of 5-chloromethylfluorescein diacetate (CMFDA) and ethidium homodimer-1 (EthD-1) to stain osteocyte viability at the time of sampling without disrupting the bony architecture in a sheep model. The methods involved harvesting adult sheep femoral and tibial mid-diaphyseal bone and staining 1 cm thick samples with different concentrations of CMFDA and EthD-1 for varying durations at different temperatures. Osteocyte viability and the effect on background staining were assessed. The results show that CMFDA and EthD-1 can successfully stain for osteocyte viability in situ at the time of sampling without destruction of the bone matrix. Optimal staining is achieved with a concentration of 2.5 μl CMFDA (25 μmol) and 5 μl EthD-1 (10 μmol) to 1ml DMEM (Dulbecco's modified Eagle's medium) for a duration of 4 hours at 4oC. These intermediate stain concentrations, low temperatures and short incubation periods limit background staining. We conclude that in contrast to analysing for empty osteocyte lacunae, CMFDA and EthD-1 can stain osteocytes for their viability from the time of injury and thus may aid in an earlier and more precise diagnosis of the percentage of osteocyte necrosis.