Project description:BACKGROUND:The pathophysiology of osteonecrosis of the femoral head (ONFH) is poorly understood, and the diagnosis is idiopathic in as many as 40% of patients. Genetic and epigenetic etiologies have been postulated, yet no single nucleotide polymorphisms (SNPs) with intuitive biologic implications have been elucidated. QUESTIONS/PURPOSES:(1) Do individuals with ONFH share common biologically relevant genetic variants associated with disease development? (2) What is the mechanism by which these SNPs may impact the expression or function of the affected gene or protein? METHODS:This retrospective genome-wide association study (GWAS) evaluated participants from the Mayo Clinic Biobank and Mayo Clinic Genome Consortium between August 2009 and March 2017. We included every patient with atraumatic ONFH in each of these respective registries and every control patient in a previous GWAS with an acceptable platform to perform statistical imputation. The study was performed in two phases, with an initial discovery cohort and a subsequent validation cohort. The initial discovery cohort consisted of 102 patients with ONFH and 4125 controls. A logistic regression analysis was used to evaluate associations between SNPs and the risk of ONFH, adjusted for age and sex. Seven SNPs were identified in a gene of biological interest, peroxisome proliferator-activated receptor gamma (PPARG), which were then evaluated in a subsequent validation cohort of 38 patients with ONFH and 464 controls. Age, sex, race, and previous steroid exposure were similar between patients with ONFH and controls in both the discovery and validation cohorts. Separate from the two-phase genetic investigation, we performed targeted pharmacosurveillance to evaluate the risk association between the use of antidiabetic thiazolidinediones, a class of PPARG agonists, and development of ONFH by referencing 9,638,296 patient records for individuals treated at Mayo Clinic. RESULTS:A combined analysis of the discovery and validation cohorts revealed that seven SNPs were tightly clustered adjacent to the 3' end of PPARG, suggesting an association with the risk of ONFH (p = 1.58 x 10-5.50 x10). PPARG gene-level significance was achieved (p = 3.33 x 10) when all seven SNPs were considered. SNP rs980990 had the strongest association with the risk of ONFH (odds ratio [OR], 1.95; 95% CI, 1.46-2.59; p = 5.50 x 10).The seven identified SNPs were mapped to a region near the PPARG gene and fell in a highly conserved region consisting of several critical transcription factor binding sites. Nucleotide polymorphisms at these sites may compromise three-dimensional chromatin organization and alter PPARG 3' end interactions with its 5' promoter and transcription start site. Pharmacosurveillance identified that patients who were exposed to thiazolidinediones had an increased relative risk of developing ONFH of 5.6 (95% CI, 4.5-7.1). CONCLUSIONS:We found that disruption of PPARG regulatory domains is linked to an increased risk of ONFH. Mechanistically, aberrant regulation of PPARG compromises musculoskeletal differentiation because this master regulator creates a proadipogenic and antiosteogenic state. Furthermore, PPARG alters steroid metabolism and vasculogenesis, processes that are inextricably linked with ONFH. Pharmacologically, predisposition to ONFH was further exposed with thiazolidinedione use, which upregulates the expression of PPARG and is known to alter bone metabolism. Collectively, these findings provide a foundation to perform confirmatory studies of our proposed mechanism in preclinical models to develop screening diagnostics and potential therapies in patients with limited options. LEVEL OF EVIDENCE:Level III, prognostic study.

Project description:Adhesion formation after flexor tendon repair remains a clinical problem. Early postoperative motion after tendon repair has been demonstrated to reduce adhesion formation while increasing tendon strength. It is hypothesized that during mobilization, tendon cells experience mechanical shear forces that alter their biology in a fashion that reduces scar formation but also activates key genes involved in tendon healing. To test this hypothesis, primary intrinsic tenocyte cultures were established from flexor tendons of 20 Sprague-Dawley rats and sheared at 50 rpm (0.41 Pa) using a cone viscometer for 6 and 12 hours. Total RNA was harvested and compared with time-matched unsheared controls using cDNA microarrays and Northern blot analysis. Microarray analysis demonstrated that mechanical shear stress induced an overall "antifibrotic" expression pattern with decreased transcription of collagen type I and collagen type III. Shear stress down-regulated profibrotic molecules in the platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor signaling pathways. In addition, shear stress induced an overall decrease in transforming growth factor (TGF)-beta signaling pathway molecules with down-regulation of TGF-beta2, TGF-beta3, TGF-RI, and TGF-RII expression. Moreover, sheared tendon cells increased expression of matrix metalloproteinases and decreased expression of tissue inhibitors of metalloproteinase, an expression pattern consistent with an antifibrotic increase in extracellular matrix degradation. However, up-regulation of genes implicated in tendon healing, specifically, vascular endothelial growth factor-A and several bone morphogenetic proteins. Interestingly, the known mechanoresponsive gene, TGF-beta1, also implicated in tendon healing, was differentially up-regulated by shear stress. Northern blot validation of our results for TGF-beta1, TGF-beta2, TGF-beta3, and collagen type I demonstrated direct correlation with microarray data. Groups of assays that are related as part of a time series. Keywords: time_series_design

Project description:Adhesion formation after flexor tendon repair remains a clinical problem. Early postoperative motion after tendon repair has been demonstrated to reduce adhesion formation while increasing tendon strength. It is hypothesized that during mobilization, tendon cells experience mechanical shear forces that alter their biology in a fashion that reduces scar formation but also activates key genes involved in tendon healing. To test this hypothesis, primary intrinsic tenocyte cultures were established from flexor tendons of 20 Sprague-Dawley rats and sheared at 50 rpm (0.41 Pa) using a cone viscometer for 6 and 12 hours. Total RNA was harvested and compared with time-matched unsheared controls using cDNA microarrays and Northern blot analysis. Microarray analysis demonstrated that mechanical shear stress induced an overall "antifibrotic" expression pattern with decreased transcription of collagen type I and collagen type III. Shear stress down-regulated profibrotic molecules in the platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor signaling pathways. In addition, shear stress induced an overall decrease in transforming growth factor (TGF)-beta signaling pathway molecules with down-regulation of TGF-beta2, TGF-beta3, TGF-RI, and TGF-RII expression. Moreover, sheared tendon cells increased expression of matrix metalloproteinases and decreased expression of tissue inhibitors of metalloproteinase, an expression pattern consistent with an antifibrotic increase in extracellular matrix degradation. However, up-regulation of genes implicated in tendon healing, specifically, vascular endothelial growth factor-A and several bone morphogenetic proteins. Interestingly, the known mechanoresponsive gene, TGF-beta1, also implicated in tendon healing, was differentially up-regulated by shear stress. Northern blot validation of our results for TGF-beta1, TGF-beta2, TGF-beta3, and collagen type I demonstrated direct correlation with microarray data. Groups of assays that are related as part of a time series. Computed

Project description:Adhesion formation after flexor tendon repair remains a clinical problem. Early postoperative motion after tendon repair has been demonstrated to reduce adhesion formation while increasing tendon strength. It is hypothesized that during mobilization, tendon cells experience mechanical shear forces that alter their biology in a fashion that reduces scar formation but also activates key genes involved in tendon healing. To test this hypothesis, primary intrinsic tenocyte cultures were established from flexor tendons of 20 Sprague-Dawley rats and sheared at 50 rpm (0.41 Pa) using a cone viscometer for 6 and 12 hours. Total RNA was harvested and compared with time-matched unsheared controls using cDNA microarrays and Northern blot analysis. Microarray analysis demonstrated that mechanical shear stress induced an overall "antifibrotic" expression pattern with decreased transcription of collagen type I and collagen type III. Shear stress down-regulated profibrotic molecules in the platelet-derived growth factor, insulin-like growth factor, and fibroblast growth factor signaling pathways. In addition, shear stress induced an overall decrease in transforming growth factor (TGF)-beta signaling pathway molecules with down-regulation of TGF-beta2, TGF-beta3, TGF-RI, and TGF-RII expression. Moreover, sheared tendon cells increased expression of matrix metalloproteinases and decreased expression of tissue inhibitors of metalloproteinase, an expression pattern consistent with an antifibrotic increase in extracellular matrix degradation. However, up-regulation of genes implicated in tendon healing, specifically, vascular endothelial growth factor-A and several bone morphogenetic proteins. Interestingly, the known mechanoresponsive gene, TGF-beta1, also implicated in tendon healing, was differentially up-regulated by shear stress. Northern blot validation of our results for TGF-beta1, TGF-beta2, TGF-beta3, and collagen type I demonstrated direct correlation with microarray data.

Dataset Information

CORR® ORS Richard A. Brand Award for Outstanding Orthopaedic Research: Engineering flexor tendon repair with lubricant, cells, and cytokines in a canine model.

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CORR® ORS Richard A. Brand Award for Outstanding Orthopaedic Research: Engineering flexor tendon repair with lubricant, cells, and cytokines in a canine model.

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