Journal
EUROPEAN CELLS & MATERIALS
Volume 12, Issue -, Pages 16-25Publisher
SWISS SOCIETY FOR BIOMATERIALS, AO RESEARCH INSTITUTE
DOI: 10.22203/eCM.v012a02
Keywords
bone; three dimensional; viability; methods
Ask authors/readers for more resources
Biocompatibility studies are carried out either in two dimensional monolayer culture or in animal studies. Bone organ cultures are therefore required in order to reduce the number of animal studies performed, while at the same time ensuring a more natural environment than that provided by monolayer culture of isolated cells. Due to the three dimensional nature of bone explants, assays that determine the distribution of viable cells are required, however dense mineralised bone is not easily penetrated by soluble factors. We sought to compare a number of non-radioactive viability methods in order to assess their suitability for use with cancellous bone. Fluorescent live/dead staining, MTT activity and lactate dehydrogenase detection were all investigated on either whole bone explants (9.5 mm in diameter, 5 mm high) or on sections of explants. All these assays are routinely used in 2 dimensional cell culture systems, yet each required modifications to be suitable for use with cancellous bone. Factors such as penetration of reagent, incubation time, assay temperature and ease of determining viable cells were all compared. It was demonstrated that penetration of the reagents into whole cores was a major problem which easily led to artefacts that could be overcome by preparing 250 mu m unfixed sections. Fluorescent live/dead staining had extra complications caused by the autofluorescence of the bone generating a high signal to noise ratio, making assessment of osteocyte viability impossible. MTT staining was difficult to interpret due to the punctate nature of the stain. We found that lactate dehydrogenase staining of 250 mu m thick unfixed sections led to excellent viability determination of osteocytes within the mineralised matrix. It also maintained marrow structure. Decreasing the viscosity of the LDH assay solution used in published methods led to a greatly improved penetration into the calcified matrix. Quantification of thick sections is aided by using the autofluorescence of the bone to highlight the darkly stained osteocytes against the fluorescing bone.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available