Dataset Information

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

ABSTRACT: 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.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.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.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.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.

SUBMITTER: Fujita K

PROVIDER: S-EPMC4216562 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Fujita K K Roforth M M MM Atkinson E J EJ Peterson J M JM Drake M T MT McCready L K LK Farr J N JN Monroe D G DG Khosla S S

Osteoporosis international : a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA 20131011 3

<h4>Summary</h4>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.<h4>Introduction</h4>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 an ...[more]

PMID: 24114401

Similar Datasets

Project description:ObjectivesTotal joint arthroplasty is one of the most frequent and effective surgeries today. However, despite improved surgical techniques, a significant number of implant-associated infections still occur. Suitable in vitro models are needed to test potential approaches to prevent infection. In the present study, we aimed to establish an in vitro co-culture setup of human primary osteoblasts and S. epidermidis to model the onset of implant-associated infections, and to analyze antimicrobial implant surfaces and coatings.Materials and methodsFor initial surface adhesion, human primary osteoblasts (hOB) were grown for 24 hours on test sample discs made of polystyrene, titanium alloy Ti6Al4V, bone cement PALACOS R®, and PALACOS R® loaded with antibiotics. Co-cultures were performed as a single-species infection on the osteoblasts with S. epidermidis (multiplicity of infection of 0.04), and were incubated for 2 and 7 days under aerobic conditions. Planktonic S. epidermidis was quantified by centrifugation and determination of colony-forming units (CFU). The quantification of biofilm-bound S. epidermidis on the test samples was performed by sonication and CFU counting. Quantification of adherent and vital primary osteoblasts on the test samples was performed by trypan-blue staining and counting. Scanning electron microscopy was used for evaluation of topography and composition of the species on the sample surfaces.ResultsAfter 2 days, we observed approximately 104 CFU/ml biofilm-bound S. epidermidis (103 CFU/ml initial population) on the antibiotics-loaded bone cement samples in the presence of hOB, while no bacteria were detected without hOB. No biofilm-bound bacteria were detectable after 7 days in either case. Similar levels of planktonic bacteria were observed on day 2 with and without hOB. After 7 days, about 105 CFU/ml planktonic bacteria were present, but only in the absence of hOB. Further, no bacteria were observed within the biofilm, while the number of hOB was decreased to 10% of its initial value compared to 150% in the mono-culture of hOB.ConclusionWe developed a co-culture setup that serves as a more comprehensive in vitro model for the onset of implant-associated infections and provides a test method for antimicrobial implant materials and coatings. We demonstrate that observations can be made that are unavailable from mono-culture experiments.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets