R McCulloch

Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage

BackgroundExpression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. The purpose of this study was to identify the most stable housekeeping genes in porcine articular cartilage subjected to a mechanical injury from a panel of 10 candidate genes.ResultsTen candidate housekeeping genes were evaluated in three different treatment groups of mechanically impacted porcine articular cartilage. The genes evaluated were: beta actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hydroxymethylbilane synthase, hypoxanthine phosphoribosyl transferase, peptidylprolyl isomerase A (cyclophilin A), ribosomal protein L4, succinate dehydrogenase flavoprotein subunit A, TATA box binding protein, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein—zeta polypeptide. The stability of the genes was measured using geNorm, BestKeeper, and NormFinder software. The four most stable genes measured via geNorm were (most to least stable) succinate dehydrogenase flavoprotein, subunit A, peptidylprolyl isomerase A, glyceraldehyde-3-phosphate dehydrogenase, beta actin; the four most stable genes measured via BestKeeper were glyceraldehyde-3-phosphate dehydrogenase, peptidylprolyl isomerase A, beta actin, succinate dehydrogenase flavoprotein, subunit A; and the four most stable genes measured via NormFinder were peptidylprolyl isomerase A, succinate dehydrogenase flavoprotein, subunit A, glyceraldehyde-3-phosphate dehydrogenase, beta actin.ConclusionsBestKeeper, geNorm, and NormFinder all generated similar results for the most stable genes in porcine articular cartilage. The use of these appropriate reference genes will facilitate accurate gene expression studies of porcine articular cartilage and suggest appropriate housekeeping genes for articular cartilage studies in other species.

Resistance to Subsidence of an Uncemented Femoral Stem After Cerclage Wiring of a Fissure

OBJECTIVE To compare: (1) the force required to initiate subsidence, and (2) the relative subsidence, of femoral stems implanted into intact femora, and then into the same femora in which an induced fissure had been stabilized by cerclage. STUDY DESIGN In vitro, mechanical study. SAMPLE POPULATION Femora (n=9) from 9 dogs. METHODS Femora were prepared for implantation of an uncemented stem. Stems were implanted with continuous and impact loading. After axial loading until a fissure occurred, the stems were extracted, and the fissure stabilized with double-loop cerclage. Stems were reimplanted, and reloaded to failure. RESULTS Mean±SD load to initiate subsidence in intact femora was 1706±584 N compared with 2379±657 N for cerclaged bones (P=.002). Mean relative subsidence of intact femora was 3.99±2.09 mm compared with 1.79±2.99 mm for cerclaged bones (P=.091). CONCLUSIONS The load to initiate subsidence is increased in femora that have fissured, then have been stabilized with double-loop cerclage, when compared with intact femora. The relative subsidence is not different between intact and stabilized specimens.

Progression of Gene Expression Changes following a Mechanical Injury to Articular Cartilage as a Model of Early Stage Osteoarthritis

An impact injury model of early stage osteoarthritis (OA) progression was developed using a mechanical insult to an articular cartilage surface to evaluate differential gene expression changes over time and treatment. Porcine patellae with intact cartilage surfaces were randomized to one of three treatments: nonimpacted control, axial impaction (2000 N), or a shear impaction (500 N axial, with tangential displacement to induce shear forces). After impact, the patellae were returned to culture for 0, 3, 7, or 14 days. At the appropriate time point, RNA was extracted from full-thickness cartilage slices at the impact site. Quantitative real-time PCR was used to evaluate differential gene expression for 18 OA related genes from four categories: cartilage matrix, degradative enzymes and inhibitors, inflammatory response and signaling, and cell apoptosis. The shear impacted specimens were compared to the axial impacted specimens and showed that shear specimens more highly expressed type I collagen (Col1a1) at the early time points. In addition, there was generally elevated expression of degradative enzymes, inflammatory response genes, and apoptosis markers at the early time points. These changes suggest that the more physiologically relevant shear loading may initially be more damaging to the cartilage and induces more repair efforts after loading.