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Forkhead box O1 (FOXO1) is upregulated during bone formation and in response to stimulation by bone morphogenetic proteins. Studies presented here examined the functional role of FOXO1 in a well defined culture system in which pre-osteoblastic cells undergo terminal differentiation in vitro. Mineralizing cultures of MC3T3-E1 cells were examined with or without FOXO1 knockdown by RNAi. Normal cells show the upregulation of FOXO1 and RUNX2 DNA binding activity, alkaline phosphatase activity, and mRNA levels of FOXO1, RUNX2, type 1 collagen, osteocalcin and MMP13 during formation of mineralizing nodules. In FOXO1 depleted cells each of these measurements was significantly reduced compared to values in control cells transfected with scrambled siRNA (P

To gain insight into the effect of diabetes on fracture healing, experiments were carried out focusing on chondrocyte apoptosis during the transition from cartilage to bone. Type 1 diabetes was induced in mice by multiple low-dose streptozotocin injections, and simple transverse fractures of the tibia or femur was carried out. Large-scale transcriptional profiling and gene set enrichment analysis were performed to examine apoptotic pathways on total RNA isolated from fracture calluses on days 12, 16, and 22, a period of endochondral bone formation when cartilage is resorbed and chondrocyte numbers decrease. Tumor necrosis factor α (TNF-α) protein levels were assessed by ELISA and caspase-3 by bioactivity assay. The role of TNF was examined by treating mice with the TNF-specific inhibitor pegsunercept. In vitro studies investigated the proapoptotic transcription factor FOXO1 in regulating TNF-induced apoptosis of chondrogenic ATDC5 and C3H10T1/2 cells as representative of differentiated chondrocytes, which are important during endochondral ossification. mRNA profiling revealed an upregulation of gene sets related to apoptosis in the diabetic group on day 16 when cartilage resorption is active but not day 12 or day 22. This coincided with elevated TNF-α protein levels, chondrocyte apoptosis, enhanced caspase-3 activity, and increased FOXO1 nuclear translocation (p < .05). Inhibition of TNF significantly reduced these parameters in the diabetic mice but not in normoglycemic control mice (p < .05). Silencing FOXO1 using siRNA in vitro significantly reduced TNF-induced apoptosis and caspase activity in differentiated chondrocytes. The mRNA levels of the proapoptotic genes caspase-3, caspase-8, caspase-9, and TRAIL were significantly reduced with silencing of FOXO1 in chondrocytic cells. Inhibiting caspase-8 and caspase-9 significantly reduced TNF-induced apoptosis in chondrogenic cells. These results suggest that diabetes causes an upregulation of proapoptotic genes during the transition from cartilage to bone in fracture healing. Diabetes increased chondrocyte apoptosis through a mechanism that involved enhanced production of TNF-α, which stimulates chondrocyte apoptosis and upregulates mRNA levels of apoptotic genes through FOXO1 activation. © 2010 American Society for Bone and Mineral Research.

BMD does not entirely explain an individual's risk of fracture. The purpose of this study was to assess whether specific differences in spatially resolved bone composition also contribute to fracture risk. These differences were assessed using Fourier transform infrared spectroscopic imaging (FTIRI) and analyzed through multiple logistic regression. Models were constructed to determine whether FTIRI measured parameters describing mineral content, mineral crystal size and perfection, and collagen maturity were associated with fracture. Cortical and cancellous bone were independently evaluated in iliac crest biopsies from 54 women (32 with fractures, 22 without) who had significantly different spine but not hip BMDs and ranged in age from 30 to 83 yr. The parameters that were significantly associated with fracture in the model were cortical and cancellous collagen maturity (increased with increased fracture risk), cortical mineral/matrix ratio (higher with increased fracture risk), and cancellous crystallinity (increased with increased fracture risk). As expected, because of its correlation with cortical but not cancellous bone density, hip BMD was significantly associated with fracture risk in the cortical but not the cancellous model. This research suggests that additional parameters associated with fracture risk should be targeted for therapies for osteoporosis.

Osteomalacia is a pathological bone condition in which there is deficient primary mineralization of the matrix, leading to an accumulation of osteoid tissue and reduced bone mechanical strength. The hypothesis that there are no qualitative or quantitative differences in osteomalacic bone mineral or matrix compared to disease-free bones was tested by examining unstained sections of polymethyl methacrylate (PMMA) embedded iliac crest biopsies using Fourier transform infrared imaging (FTIRI) at approximately 6-microm spatial resolution. Controls were seven female subjects, aged 36-57, without apparent bone disease. The experimental group consisted of 11 patients aged 22-72, diagnosed with osteomalacia. The spectroscopic parameters analyzed in each data set were previously established as sensitive to bone quality: phosphate/amide I band area ratio (mineral content), 1660/1690 cm(-1) peak ratio (collagen cross-links), and the 1030/1020 cm(-1) peak ratio (mineral crystallinity). The correspondence between spectroscopic mineral content (phosphate/amide I ratio) and ash weight was validated for apatite crystals of different composition and crystallite size. The FTIRI results from the biopsies expressed as color-coded images and pixel population means were compared with the nonparametric Mann-Whitney U test. There were no significant differences in the cortical parameters. Significant difference was found in the mineral content of the trabecular regions with a lower mean value in osteomalacia (P = 0.01) than in controls. Mineral crystallinity tended to be decreased in the trabecular bone (P = 0.09). This study supports the hypothesis that, in osteomalacia, the quality of the organic matrix and of mineral in the center of bone does not change, while less-than-optimal mineralization occurs at the bone surface. This study provides the first spectroscopic evaluation of whole bone mineral and matrix properties in osteomalacia, demonstrating that there are few differences in collagen cross-links between biopsies from patients with osteomalacia and from individuals without histological evidence of bone disease.