In vitro effects of equine autologous conditioned serum and autologous equine serum on inflamed chondrocyte pellets
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ABSTRACT:
PROVIDER: PRJNA638808 | ENA |
REPOSITORIES: ENA
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Project description:The aim of the study was to investigate the effects of autologous equine serum (AES) incubated for 24 h and autologous conditioned serum (ACS) on inflamed equine chondrocyte pellets in vitro.
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Project description:Monolayer cultures of primary chondrocytes undergo morphological changes that have been broadly characterized as de-differentiation. Transcriptional profiling was used to evaluate changes in gene expression during this process. Articular cartilage was harvested from a single Standardbred horse; the chondrocytes were isolated and then maintained in monolayer culture for up to 14 days. Chondrocytes were collected on Day 1 and Day 14 of culture and snap frozen in liquid nitrogen. Total RNA was isolated, subjected to one round of linear RNA amplification, and then applied to an equine-specific cDNA microarray composed of 9322 elements. Three biological replicates were analyzed at each time point using a dye-swap experimental design. One hundred six transcripts were present at levels greater than or equal to five-fold higher in Day 1 chondrocytes relative to the cells collected at Day 14. Conversely, 68 transcripts were present at levels greater than or equal to five-fold higher in Day 14 chondrocytes. Northern blot hybridization validated the microarray data by confirming the down-regulation of steady state mRNA levels for type II procollagen and aggrecan core protein and the up-regulation of type I procollagen. Gene-by-gene and cluster analyses will be used to compare the process of “de-differentiation” in primary culture to the developmental stages of normal chondrocyte differentiation and to the fibrocartilage repair tissue that forms in osteoarthritic lesions. Keywords: articular cartilage, de-differentiation, horse, cDNA microarray This is a direct comparison between one and fourteen day cultured chondrocyte transcriptomes. Three sets of technical replicates were compared. For each comparison and its dye-swap, one Day 1 technical replicate was randomly compared to one Day 14 technical replicate.
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Project description:The pathology of failure in currently used biological treatments (cell therapies and microfracture) for cartilage repair or early osteoarthritis (OA) is poorly understood. We aimed to identify a reliable panel of biological predictors, present in synovial fluid, which can be used in the preoperative setting to optimise patient selection and therapy efficacy. The-long term aim is to reduce the number of patients who progress to end-stage OA and require knee replacement by making earlier, less invasive treatments more effective. In this first stage, we have taken synovial fluid aspirates from patients undergoing autologous chondrocyte implantation (ACI) at our centre in Oswestry, UK. Synovial fluids (SFs) were obtained from 14 ACI responders (mean Lysholm improvement of 33 (17-54)) and 13 non-responders (mean Lysholm decrease of 14 (4-46)) at the two stages of surgery (cartilage harvest and chondrocyte implantation). Dynamic range compression of synovial fluids coupled with label-free quantification mass spectrometry (MS), was used in an unbiased approach to identify predictive markers of ACI success or failure and to investigate the biological pathways involved in the clinical response to ACI.
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Project description:Monolayer cultures of primary chondrocytes undergo morphological changes that have been broadly characterized as de-differentiation. Transcriptional profiling was used to evaluate changes in gene expression during this process. Articular cartilage was harvested from a single Standardbred horse; the chondrocytes were isolated and then maintained in monolayer culture for up to 14 days. Chondrocytes were collected on Day 1 and Day 14 of culture and snap frozen in liquid nitrogen. Total RNA was isolated, subjected to one round of linear RNA amplification, and then applied to an equine-specific cDNA microarray composed of 9322 elements. Three biological replicates were analyzed at each time point using a dye-swap experimental design. One hundred six transcripts were present at levels greater than or equal to five-fold higher in Day 1 chondrocytes relative to the cells collected at Day 14. Conversely, 68 transcripts were present at levels greater than or equal to five-fold higher in Day 14 chondrocytes. Northern blot hybridization validated the microarray data by confirming the down-regulation of steady state mRNA levels for type II procollagen and aggrecan core protein and the up-regulation of type I procollagen. Gene-by-gene and cluster analyses will be used to compare the process of “de-differentiation” in primary culture to the developmental stages of normal chondrocyte differentiation and to the fibrocartilage repair tissue that forms in osteoarthritic lesions. Keywords: articular cartilage, de-differentiation, horse, cDNA microarray
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