Dataset Information

A preliminary, observational study using whole-blood RNA sequencing reveals differential expression of inflammatory and bone markers post-implantation of percutaneous osseointegrated prostheses

ABSTRACT: While the benefits of direct skeletal attachment of artificial limbs are well recognized, device failure due to infection and insufficient osseointegration remain obstacles to obtaining consistent, successful outcomes. Currently, the potential for device failure is assessed by clinical, radiographic evidence of bone atrophy, the presence of radiolucent lines at the bone-implant interface, and subjective pain and function scores. Our hypothesis is that measurable biological indices might add an objective means to assess trends toward bone and stomal healing. This longitudinal cohort study was undertaken to identify potential serological biomarkers suggestive of bone remodeling and the presence of stomal tissue inflammation. Ten unilateral transfemoral amputee Veterans, who were implanted with a percutaneous osseointegration (OI) system, were recruited to participate in this IRB-approved study. Venous blood samples were obtained from before the Stage 1 Surgery up to 1 year following the Stage 2 Surgery. Whole-blood RNA was extracted, sequenced, mapped, and analyzed. Of the significant differentially expressed (DEGs) genes (p<0.05) identified, four genes (IL12B, IL33, COL2A1, and SOST) were validated using qPCR. Enrichment analysis was performed to identify significant (p<0.01) Gene Ontology (GO) terms. Most DEGs were only detected at PoS1 immediately after the first surgery. Four significant genes identified, IL12B and IL33, related to inflammation, and COL2A1 and SOST associated with bone remodeling were identified with greater than 20 log fold-change. Whole-blood RNA-seq data from 10 patients who previously underwent percutaneous osseointegrated lower limb implantation revealed four genes of interest that are known to be involved in inflammation or bone remodeling. If verified in future studies, these genes may serve as markers for predicting optimal bone remodeling and stomal tissue healing following OI implantation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE194108 | GEO | 2022/04/04

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Osteogenesis Imperfecta (OI) is a genetic, rare disease characterized by bone fragility, with a wide range in the severity of clinical manifestations. The majority of the cases are due to mutations in COL1A1 or COL1A2 genes which code for the type I collagen molecule. Mesenchymal stem cells (MSCs), as the progenitors of the osteoblasts, the main type I collagen secreting cell type in the bone, have been proposed and tested as an innovative therapy for OI with promising but transient outcomes. In this study, we performed a phase I clinical trial based on reiterative infusions of histocompatible MSCs in two pediatric patients affected by severe and moderate OI. The aim of this study was to assess the safety and effectiveness of this cell therapy in non-immunosuppressed OI patients. The host response to MSCs was also evaluated by collecting the sera from OI patients before, along and after the cell therapy. The results of this clinical trial are twofold: the sequential administration of MSCs is safe and improve the bone parameters and quality of life of OI patients. Moreover, MSCs therapy elicits a pro-osteogenic paracrine response in patients, especially noticeable in that affected by severe OI. These results indicate the feasibility and potential of reiterative MSCs infusion for OI and highlight the paracrine response shown by OI patients as a consequence of MSCs treatment. Study design and patients: The Mesenchymal Stem Cell Therapy for the Treatment of Osteogenesis Imperfecta (TERCELOI) study (www.clinical trials.gov: # NCT02172885, EudraCT number: 2012-002553-38) is an independent multi-center Phase I clinical trial to evaluate the feasibility, safety and potential efficacy of infused sibling HLA-matched MSCs in non-immunosuppressed children with OI. PBMCs isolation and serum collection: Venous blood samples were collected before the cell therapy (basal serum), along the cell therapy (1 week, 1 month and 4 months after each MSCs infusion) and during the follow-up period (1 and 2 years after the fifth and last MSCs infusion). MLR assay: PBMCs isolated from patients were resuspended in PBS+FBS (5%) and stained with carboxyfluoroscein succinate-ester (CFSE) (Molecular Probes, USA). Bone mineral density: Bone mineral density (BMD) at the lumbar spine (LS) from L1 to L4, was measured by dual energy x-ray absorptiometry (DXA) on a whole-body scanner within a pediatric platform (Hologic QDR densitometer). Raw measurements were converted to Z-scores for analysis using reference standards for age and pubertal status. OI-MSCs isolation and characterization and culture: OI-MSCs were derived from discarded and donated bone fragments of OI pediatric patients undergoing corrective surgery. All patients suffered Type III-IV OI, with mutations in either COL1A1 or COL1A2 genes. The donation was approved by the Basque Clinical Research Ethics Committee. Briefly, bone chips were mechanically flushed with PBS and then cut into small pieces to extrude cells without the use of enzymes. The cut bone pieces were let undisturbed during 14 days until OI-MSCs migrated to cell culture plate. OI-MSCs were assessed for the expression of CD105, CD90 and CD73 and the absence of CD14, CD34, CD45, CD19 and HLA-DR. MSCs were also tested for their potential to differentiate to osteoblasts and adipocytes using specific cell culture media. OI-MSCs were cultured under standard growth medium (DMEM low glucose with glutamax (Gibco), penicillin/streptomycin (Gibco) and fetal bovine serum at 10% (Sigma-Aldrich, USA). Osteogenesis induction medium (OIM) was composed of standard medium plus ascorbic acid 0.2 mM, beta-glycerophosphate 10 mM, and dexamethasone 10 nM (all from Sigma-Aldrich). When specified, FBS was replaced by P01 and P02 serum samples at 2.5%. RNAseq and Q-PCR validation: OI-MSCs from 10 OI pediatric patients were seeded in 96-well plates (1 000 cells/well) and the following day cultured in the presence of OIM. Next, total RNA was isolated with the AllPrep kit (QIAGEN, USA). cDNA library (TrueSeq stranded Total cDNA library, Illumina, USA) and sequencing at HiSeq 4000 (PE100nt, 50 million reads/sample).

Project description:Inhibitors of Wnt signaling have been previously shown to be involved in prostate cancer (PC) metastasis; however the role of Sclerostin (Sost) has not yet been explored. Here we show that elevated Wnt signaling derived from Sost deficient osteoblasts (OBSOSTKO) promotes PC invasion while rhSOST has an inhibitory effect. In contrast, rhDKK1 promotes PC elongation and filopodia formation, morphological changes characteristic of an invasive phenotype. Furthermore, rhDKK1 was found to activate canonical Wnt signaling in PC3 cells as quantified by TOPFLASH reporter and b-catenin activity, suggesting that SOST and DKK1 have opposing roles on Wnt signaling in this context. Gene expression analysis of PC3 cells co-cultured with OBs exhibiting varying amounts of Wnt signaling identified CRIM1 as one of the transcripts up-regulated under highly invasive conditions. Following further analysis we found that CRIM1 increases PC3 invasion, complexes with b-catenin, and promotes cell-adhesion, suggesting that elevated Wnt signaling secreted from the bone may promote PC tropism by promoting CRIM1 expression and facilitating cancer cell invasion and adhesion to bone. We concluded that SOST and DKK1 have opposing effects on PC3 cell invasion and that bone-derived Wnt signaling positively contributes to the invasive phenotypes of metastatic cancer cells by activating CRIM1 expression and facilitating PC-OB physical interaction. As such, we investigated the effects of high concentrations of SOST in vivo. We found that PC3-cells overexpressing SOST injected via tail vein did not readily metastasize in NSG xenografts, suggesting that targeting the molecular bone environment may influence bone metastatic outcome in clinical settings. PC3 cells were cultured for 48 hours in specified conditions prior to RNA extraction and hybridization on Affymetrix Human Genome U133 Plus 2.0 Array. All experiments were performed in at least duplicates. Total RNA was collected from PC3 cells cultured in normal growth media, cocultured with WT mouse osteoblasts supplemented with recombinant human DKK1, cells cocultured with WT mouse osteoblasts supplemented with recombinant human SOST, or cocultured with SostKO mouse osteoblasts.

Dataset Information

A preliminary, observational study using whole-blood RNA sequencing reveals differential expression of inflammatory and bone markers post-implantation of percutaneous osseointegrated prostheses

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets