Establishment and assessment of a new human embryonic stem cell-based biomarker assay for developmental toxicity screening.

A metabolic biomarker-based in vitro assay utilizing human embryonic stem (hES) cells was developed to identify the concentration of test compounds that perturbs cellular metabolism in a manner indicative of teratogenicity. This assay is designed to aid the early discovery-phase detection of potential human developmental toxicants. In this study, metabolomic data from hES cell culture media were used to assess potential biomarkers for development of a rapid in vitro teratogenicity assay. hES cells were treated with pharmaceuticals of known human teratogenicity at a concentration equivalent to their published human peak therapeutic plasma concentration. Two metabolite biomarkers (ornithine and cystine) were identified as indicators of developmental toxicity. A targeted exposure-based biomarker assay using these metabolites, along with a cytotoxicity endpoint, was then developed using a 9-point dose-response curve. The predictivity of the new assay was evaluated using a separate set of test compounds. To illustrate how the assay could be applied to compounds of unknown potential for developmental toxicity, an additional 10 compounds were evaluated that do not have data on human exposure during pregnancy, but have shown positive results in animal developmental toxicity studies. The new assay identified the potential developmental toxicants in the test set with 77% accuracy (57% sensitivity, 100% specificity). The assay had a high concordance (≥75%) with existing in vivo models, demonstrating that the new assay can predict the developmental toxicity potential of new compounds as part of discovery phase testing and provide a signal as to the likely outcome of required in vivo tests.

Histological and molecular structure characterization of annular collagen after intradiskal electrothermal annuloplasty.

The mechanism of pain relief of intradiskal electrothermal annuloplasty (IDET) in the treatment of lumbar diskogenic pain is uncertain. Theories include sealing of annular fissures via collagen denaturation and contraction. Prior studies offer conflicting qualitative data on the ability of IDET to denature collagen. The objective of the present study is to evaluate IDET treatment effect on annular collagen using quantitative data supplied by Fourier-transform infrared imaging spectroscopy. The posterior annulus of disks (n = 3) from an intact human cadaveric spine at room temperature were treated with two different radiothermal catheters using standard intradiskal electrothermal annuloplasty (IDET) heating protocols. Disks were dissected free with catheters in place and fixed in formalin. Channels created by the catheters were marked and catheters were removed. Tissue samples of treated areas adjacent to the channels and internal control areas from the same disk were stained for light microscopy and placed on barium sulfate windows for Fourier transform infrared imaging spectroscopy (FT-IRIS) analysis. Treated areas showed evidence of disruption in the fibrillar organization of annular collagen by light microscopy compared to intact stroma from control areas. Quantitative FT-IRIS analysis compared ratios of wavenumber regions known to be sensitive to collagen denaturation. Mean values for the ratios amide II/1,338 cm(-1) (137.21 +/- 25.84 treated, 76.94 +/- 16.77 control) and 1,640/1,660 cm(-1) (0.98 +/- 0.03 treated, 0.89 +/- 0.03 control) were significantly different between treated and control samples (p < 0.001), indicating a breakdown in collagen integrity. Separate analysis by catheter type suggests that catheter design may impact treatment effect.

Comparison of mineral quality and quantity in iliac crest biopsies from high- and low-turnover osteoporosis: an FT-IR microspectroscopic investigation.

Fourier-transform infrared microspectroscopy (FTIRM) allows analysis of mineral content, mineral crystal maturity and mineral composition at approximately 10-micron spatial resolution. Previous FTIRM analyses comparing 4-micron thick sections from non-decalcified iliac crest biopsies from women with post-menopausal osteoporosis, as contrasted with iliac crest tissue from individuals without evidence of metabolic bone disease, demonstrated significant differences in average mineral content (decreased in osteoporosis) and mineral crystal size/perfection (increased in osteoporosis). More importantly, these parameters, which vary throughout the tissue in relation to the tissue age in healthy bone, showed no such variation in bone biopsies from patients with osteoporosis. The present study compares the spatial and temporal variation in mineral quantity and properties in trabecular bone in high- and low-turnover osteoporosis. Specifically, six biopsies from women (n=5) and one man with high-turnover osteoporosis (age range 39-77) and four women and two men with low turnover osteoporosis (age range 37-63) were compared to ten "normal" biopsies from three men and seven woman (age range: 27-69). "High turnover" was defined as the presence of increased resorptive surface, higher than normal numbers of osteoclasts and greater than or equal to normal osteoblastic activity. "Low turnover" was defined as lower than normal resorptive surface, decreased osteoclast number and less than normal osteoblastic activity. Comparing variations in FTIR-derived values for each of the parameters measured at the surfaces of the trabecular bone to the maximum value observed in multiple trabeculae from each person, the high-turnover samples showed little change in the mineral: matrix ratio, carbonate: amide I ratio, crystallinity and acid phosphate content. The low-turnover samples also showed little change in these parameters, but in contrast to the high-turnover samples, the low-turnover samples showed a slight increase in these parameters, indicative of retarded, but existent resorption and formation. These data indicate that FTIR microspectroscopy can provide quantitative information on mineral changes in osteoporosis that are consistent with proposed mechanisms of bone loss.