Project description:Understanding the effects of biomechanical loading on human growth plate cartilage, which are unknown so far, could uncover diagnostic and therapeutic avenues. To address this, we used rare human growth plate biopsies obtained from children undergoing epiphysiodesis. These biopsies were exposed to a precisely controlled mechanical loading application using a microloading device. Subsequently, the biopsies were cultured for 24 hours post-mechanical loading, followed by RNA-sequencing analyses to decipher the molecular responses.
Project description:The aim of the current study was to identify molecular markers for articular cartilage that can be used for the quality control of tissue engineered cartilage. Therefore a genom-wide expression analysis was performed using RNA isolated from articular and growth plate cartilage, both extracted from the knee joints of minipigs. Keywords: Native material or primary cells isolated from articular cartilage and growth plate cartilage
Project description:The aim of the current study was to identify molecular markers for articular cartilage that can be used for the quality control of tissue engineered cartilage. Therefore a genom-wide expression analysis was performed using RNA isolated from articular and growth plate cartilage, both extracted from the knee joints of minipigs. Keywords: Native material or primary cells isolated from articular cartilage and growth plate cartilage Articular and growth plate cartilage were taken for RNA extraction and hybridization on Affymetrix microarrays. Furthermore chondrocytes from each type of cartilage were isolated and cell culture was started and terminated at day 10 or day 20. Total RNA from cultivated cells was extracted, and hybridization on Affymetrix microarrays was performed.
Project description:Cartilage originates from mesenchymal cell condensations that differentiate into chondrocytes of transient growth plate cartilage or permanent cartilage of the articular joint surface and trachea. MicroRNAs fine-tune the activation of entire signaling networks and thereby modulate complex cellular responses, but so far only limited data are available on miRNAs that regulate cartilage development. Here we characterize an miRNA which promotes the biosynthesis of a key component in the RAF/MEK/ERK pathway in cartilage. Specifically, by transcriptome profiling we identified miR-322 to be upregulated during chondrocyte differentiation. Among the various miR-322 target genes in the RAF/MEK/ERK pathway, only Mek1 was identified as a regulated target in chondrocytes. Surprisingly, an increased concentration of miR-322 stabilizes Mek1-mRNA to arise protein levels and dampen ERK1/2 phosphorylation, while cartilage-specific inactivation in mice linked the loss of miR-322 to decreased MEK1 levels and increased RAF/MEK/ERK pathway activation. Such mice died perinatally due to tracheal growth restriction and respiratory failure. Hence, a single miRNA can stimulate the production of an inhibitory component of a central signaling pathway to impair cartilage development.
Project description:Articular and growth plate cartilage have comparable structures consisting of three distinct layers of chondrocytes, suggesting similar differentiation programs and therefore similar gene expression profiles. To address this hypothesis and to explore transcriptional changes that occur during the onset of articular and growth plate cartilage divergence, we used microdissection of 10-day-old rat proximal tibial epiphyses, microarray analysis, and bioinformatics to compare gene expression profiles in individual layers of articular and growth plate cartilage. We found that many genes that were spatially upregulated in intermediate/deep zone of articular cartilage were also spatially upregulated in resting zone of growth plate cartilage (overlap greater than expected by chance, P < 0.001). Interestingly, superficial zone of articular cartilage showed an expression profile with similarities to both proliferative and hypertrophic zones of growth plate cartilage (P < 0.001 each). Additionally, significant numbers of known proliferative zone markers (3 out of 6) and hypertrophic zone markers (27 out of 126) were spatially upregulated in superficial zone compared to intermediate/deep zone (more than expected by chance, P < 0.001 each). In conclusion, we provide evidence that intermediate/deep zone of articular cartilage has a gene expression profile more similar to resting zone of growth plate cartilage, whereas superficial zone has a gene expression profile more similar to proliferative and hypertrophic zones.
Project description:Ollier disease is a rare, non-hereditary disorder which is characterized by the presence of multiple enchondromas (EC), benign cartilaginous neoplasms arising within the medulla of the bone, with an asymmetric distribution. The risk of malignant transformation towards central chondrosarcoma (CS) is increased up to 35%. The etiology of Ollier disease is unknown. We therefore undertook genome-wide expression profiling using Illumina Beadarray v3.0 for 7 enchondromas of 6 patients and compared them with controls in order to find differentially expressed genes in these benign tumors. The goal of this study was to find differentially expressed genes between enchondromas, a benign cartilage forming tumor occuring in the metaphyses of bone, and controls. We have used samples of normal growth plate and articular cartilage as the controls. Seven enchondromas were compared to two growth plate and four cartilage samples.
Project description:Affymetrix Chicken Gene 1.0 ST Array (ChiGene-1_1-st) profiles were generated from growth plate cartilage derived from the distal femur and proximal tibia of embryonic chickens at three developmental stages (day 12, 13 and 15) corresponding to stages prior to and immediately after the acqusition of mechano-responsive cartilage growth in the femur but not tibia (which does not respond to mechanical stimuli until later stages). Additionally, array profiles were generated from growth plate cartilage from the distal femur and proximal tibia of E15 chicks which were pharmacologically immobilized by decamethonium bromide treatment, which reduces longitudinal growth of the femur only at this stage.
Project description:Short-read NGS technology (SOLIDTM, Life Technologies) was used to establish a comprehensive repertoire of miRNA expressed in either equine cartilage or subchondral bone. Undamaged cartilage and subchondral bone samples from 10-month Anglo-Arabian foals affected by osteochondrosis (OC) were analyzed and compared with samples from healthy foals. Samples were also subjected or not to an experimental mechanical loading to evaluate the role of miRNAs in the regulation of mechano-transduction pathways. Epiphyseal cartilage and subchondral bone miRNome were defined, including about 300 new miRNAs. Differentially expressed miRNAs were identified between bone and cartilage from healthy and OC foals, as well as after the experimental mechanical loading, suggesting that miRNAs play a role in equine OC physiopathology and in the cellular response to biomechanical stress in cartilage and bone.
Project description:Articular and growth plate cartilage have comparable structures consisting of three distinct layers of chondrocytes, suggesting similar differentiation programs and therefore similar gene expression profiles. To address this hypothesis and to explore transcriptional changes that occur during the onset of articular and growth plate cartilage divergence, we used microdissection of 10-day-old rat proximal tibial epiphyses, microarray analysis, and bioinformatics to compare gene expression profiles in individual layers of articular and growth plate cartilage. We found that many genes that were spatially upregulated in intermediate/deep zone of articular cartilage were also spatially upregulated in resting zone of growth plate cartilage (overlap greater than expected by chance, P < 0.001). Interestingly, superficial zone of articular cartilage showed an expression profile with similarities to both proliferative and hypertrophic zones of growth plate cartilage (P < 0.001 each). Additionally, significant numbers of known proliferative zone markers (3 out of 6) and hypertrophic zone markers (27 out of 126) were spatially upregulated in superficial zone compared to intermediate/deep zone (more than expected by chance, P < 0.001 each). In conclusion, we provide evidence that intermediate/deep zone of articular cartilage has a gene expression profile more similar to resting zone of growth plate cartilage, whereas superficial zone has a gene expression profile more similar to proliferative and hypertrophic zones. 10-day-old rat proximal tibial epiphyses were manually microdissected into articular cartilage superficial (SZ) and intermediate/deep (IDZ) zones and growth plate cartilage resting zone (RZ) for total RNA extraction and hybridization on Affymetrix microarrays. We used 10-day-old animals because, at this age, the secondary ossification center has recently begun to form and divides the epiphysis into articular cartilage distally and growth plate cartilage more centrally. The 4 SZ samples were taken from animals 5-8, respectively, whereas the 4 IDZ and 4 RZ samples were each taken from animals 1-2, 3-4, 5-6, and 7-8, respectively.
Project description:Kinase activity of cGMP-dependant, type II, protein kinase (PRKG2) is required for the proliferative to hypertrophic growth transition of growth plate chondrocytes during endochondral ossification. Loss of PRKG2 function in rodent and bovine models results in dwarfism. We compared growth plate cartilage gene expression profiles of PRKG2R678X/R678X, dwarf and PRKG2R678X/+, unaffected Angus cattle using microarray technology to discover pathways regulated by PRKG2. Calves used in this analysis were produced by the mating between a dwarf carrier sire and two dams- a mother (carrier) and daughter (dwarf) pair. Each dam produced three calves by embryo transfer, which were born in two calving groups. Growth plate cartilage was harvested from the tibia of each animal at approximately 215 days of age. Total RNA was extracted by a modified Trizol protocol. The bovine cDNA microarray (GEO Accession: GPL8813) was used for this analysis. Samples were labeled with Cy3 or Cy5 and hybridized by dye swapping the dwarf (4 dwarf animals- 2 per dam/calving group) and unaffected individuals (2 unaffected animals- 1 per dam/ calving group) such that each dwarf was compared to the unaffected full-sib produced in the same calving group. Each cDNA array was analyzed at three different PMT levels (PMT70,80 and 90), resulting in 12 total image files for statistical analysis.