Isolation and Culture of Primary Osteocytes from the Long Bones of Skeletally Mature and Aged Mice

Stern, Amber Rath; Stern, Matthew M.; Dyke, Mark Van; Jähn, Katharina; Prideaux, Matthew; Bonewald, Lynda F.

doi:10.2144/0000113876

Cited by 182 publications

(107 citation statements)

References 46 publications

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“…Recent data from the Bonewald laboratory has demonstrated that seven to nine sequential digests are needed to release true osteocytes from mouse bones [27]. This is consistent with our data in Fig.…”

Section: Resultssupporting

confidence: 92%

Isolation and characterization of human osteoblasts from needle biopsies without in vitro culture

et al. 2013

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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.

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Section: Resultssupporting

confidence: 92%

Isolation and characterization of human osteoblasts from needle biopsies without in vitro culture

et al. 2013

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“…Primary bone cells were isolated as previously described with some modifications (Stern et al, 2012). Briefly, long bones were dissected from 5- or 22-month-old C57BL/6 male mice for cell death assay and from 3-month-old DMP1-GFP female mice for live imaging.…”

Section: Methodsmentioning

confidence: 99%

β-aminoisobutyric Acid, l-BAIBA, Is a Muscle-Derived Osteocyte Survival Factor

et al. 2018

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SUMMARY Exercise has beneficial effects on metabolism and on tissues. The exercise-induced muscle factor β-aminoisobutyric acid (BAIBA) plays a critical role in the browning of white fat and in insulin resistance. Here we show another function for BAIBA, that of a bone-protective factor that prevents osteocyte cell death induced by reactive oxygen species (ROS). L-BAIBA was as or more protective than estrogen or N-acetyl cysteine, signaling through the Mas-Related G Protein-Coupled Receptor Type D (MRGPRD) to prevent the breakdown of mitochondria due to ROS. BAIBA supplied in drinking water prevented bone loss and loss of muscle function in the murine hindlimb unloading model, a model of osteocyte apoptosis. The protective effect of BAIBA was lost with age, not due to loss of the muscle capacity to produce BAIBA but likely to reduced Mrgprd expression with aging. This has implications for understanding the attenuated effect of exercise on bone with aging.

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“…Although recent studies have described methods for extraction of primary osteocytes from mouse long bones,45 the yields are generally low and would not have been sufficient for our experimental purposes. Therefore, we have used the MLO‐Y4 cell line which serve as a good osteocyte model as they respond to mechanical stimulation by releasing prostaglandin‐E 2 ,46 ATP,47 and nitric oxide,48 integral to osteocytes’ orchestration of adaptive bone remodeling.…”

Section: Discussionmentioning

confidence: 99%

Osteocyte physiology and response to fluid shear stress are impaired following exposure to cobalt and chromium: Implications for bone health following joint replacement

et al. 2016

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The effects of metal ion exposure on osteocytes, the most abundant cell type in bone and responsible for coordinating bone remodeling, remain unclear. However, several studies have previously shown that exposure to cobalt (Co2+) and chromium (Cr3+), at concentrations equivalent to those found clinically, affect osteoblast and osteoclast survival and function. In this study, we tested the hypothesis that metal ions would similarly impair the normal physiology of osteocytes. The survival, dendritic morphology, and response to fluid shear stress of the mature osteocyte‐like cell‐line MLO‐Y4 following exposure to clinically relevant concentrations and combinations of Co and Cr ions were measured in 2D‐culture. Exposure of MLO‐Y4 cells to metal ions reduced cell number, increased dendrites per cell and increased dendrite length. We found that combinations of metal ions had a greater effect than the individual ions alone, and that Co2+ had a predominate effect on changes to cell numbers and dendrites. Combined metal ion exposure blunted the responses of the MLO‐Y4 cells to fluid shear stress, including reducing the intracellular calcium responses and modulation of genes for the osteocyte markers Cx43 and Gp38, and the signaling molecules RANKL and Dkk‐1. Finally, we demonstrated that in the late osteoblasts/early osteocytes cell line MLO‐A5 that Co2+ exposure had no effect on mineralization, but Cr3+ treatment inhibited mineralization in a dose‐dependent manner, without affecting cell viability. Taken together, these data indicate that metal exposure can directly affect osteocyte physiology, with potential implications for bone health including osseointegration of cementless components, and periprosthetic bone remodeling. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:1716–1723, 2017.

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Isolation and Culture of Primary Osteocytes from the Long Bones of Skeletally Mature and Aged Mice

Cited by 182 publications

References 46 publications

Isolation and characterization of human osteoblasts from needle biopsies without in vitro culture

Isolation and characterization of human osteoblasts from needle biopsies without in vitro culture

β-aminoisobutyric Acid, l-BAIBA, Is a Muscle-Derived Osteocyte Survival Factor

Osteocyte physiology and response to fluid shear stress are impaired following exposure to cobalt and chromium: Implications for bone health following joint replacement

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