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Summary: We isolate and characterize osteoblasts from humans without in vitro culture. These techniques should be broadly applicable to studying the pathogenesis of osteoporosis and other bone disorders. Introduction: There is currently no data regarding the expression of specific genes or pathways in human osteoblasts that have not been subjected to extensive in vitro culture. Thus, we developed methods to rapidly isolate progressively enriched osteoblast populations from humans and characterized these cells. Methods: Needle bone biopsies of the posterior iliac crest were subjected to sequential collagenase digests. The cells from the second digest were stained with an alkaline phosphatase (AP) antibody, and the AP+ cells were isolated using magnetic cell sorting. Results: Relative to AP− cells, the AP+ cells contained virtually all of the mineralizing cells and were enriched for key osteoblast marker genes. The AP+ cells were further purified by depletion of cells expressing CD45, CD34, or CD31 (AP+/CD45/34/31− cells), which represented a highly enriched human osteoblast population devoid of hematopoietic/endothelial cells. These cells expressed osteoblast marker genes but very low to undetectable levels of SOST. We next used high-throughput RNA sequencing to compare the transcriptome of the AP+/CD45/34/31− cells to human fibroblasts and identified genes and pathways expressed only in human osteoblasts in vivo, but not in fibroblasts, including 448 genes unique to human osteoblasts. Conclusions: We provide a detailed characterization of highly enriched human osteoblast populations without in vitro culture. These techniques should be broadly applicable to studying the pathogenesis of osteoporosis and other bone disorders. [ABSTRACT FROM AUTHOR]

Summary: Serum sclerostin levels are associated with cortical porosity, suggesting that changes in sclerostin production during growth may play a role in defining cortical structure. Introduction: Sclerostin, produced by osteocytes, is a potent inhibitor of Wnt signaling and bone formation. While sclerostin levels increase with age in adults and are higher in men compared to women, there is currently no information on changes in circulating sclerostin levels during growth in humans. Methods: We measured serum sclerostin levels in 6- to 21-year-old girls ( n = 62) and boys ( n = 56) and related these to trabecular and cortical bone microarchitectural parameters using high-resolution peripheral quantitative computed tomography and to markers of bone turnover. Results: Serum sclerostin levels were higher in boys as compared to girls and declined in both sexes following the onset of puberty. There was no consistent relationship between sclerostin levels and trabecular bone parameters in either sex. However, serum sclerostin levels were inversely associated with cortical volumetric bone mineral density and cortical thickness in girls and positively associated with the cortical porosity index in both girls and boys. Bone turnover markers were positively correlated with serum sclerostin levels in both sexes. Conclusion: The gender difference in serum sclerostin levels appears to be established during puberty, and sclerostin levels tend to decline in late puberty in both girls and boys. Serum sclerostin levels are associated with cortical porosity, suggesting that changes in sclerostin production during growth may play a role in defining cortical structure. [ABSTRACT FROM AUTHOR]