Changes in chondrocyte gene expression following in vitro impaction of porcine articular cartilage in an impact injury model.

Our objective was to monitor chondrocyte gene expression at 0, 3, 7, and 14 days following in vitro impaction to the articular surface of porcine patellae. Patellar facets were either axially impacted with a cylindrical impactor (25 mm/s loading rate) to a load level of 2,000 N or not impacted to serve as controls. After being placed in organ culture for 0, 3, 7, or 14 days, total RNA was isolated from full thickness cartilage slices and gene expression measured for 17 genes by quantitative real-time RT-PCR. Targeted genes included those encoding proteins involved with biological stress, inflammation, or anabolism and catabolism of cartilage extracellular matrix. Some gene expression changes were detected on the day of impaction, but most significant changes occurred at 14 days in culture. At 14 days in culture, 10 of the 17 genes were differentially expressed with col1a1 most significantly up-regulated in the impacted samples, suggesting impacted chondrocytes may have reverted to a fibroblast-like phenotype.

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

BACKGROUND: Expression 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. RESULTS: Ten 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. CONCLUSIONS: BestKeeper, 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.

Median sternotomy closure in dogs: a mechanical comparison of technique stability.

OBJECTIVE: To evaluate the mechanical properties of canine median sternotomy closure using double-loop cerclage compared with other commonly used cerclage sternotomy closure patterns. STUDY DESIGN: Experimental study. SAMPLE POPULATION: Forty canine cadaveric sternal segments. METHODS: Median sternotomy-wire constructs were loaded to failure in a materials testing machine. Each construct was radiographed before and after testing. Specific wire configurations tested were: 2 single twist cerclage centered on the sternebrae, single twist figure of 8 pattern centered on the sternal synchondrosis, double twist figure of 8 pattern centered on the sternal synchondrosis, 2 double-loop cerclage centered on the sternebrae, and 1 double-loop cerclage centered on the sternal synchondrosis. Wire configurations were compared by craniocaudal translation of sternabrae on pre-test radiographs, and displacement during loading, and mode of failure, based on analysis of test video, and post-test radiographs. RESULTS: Double-loop cerclage did not provide superior mechanical stabilization of median sternotomy. Both figure of 8 patterns had the least displacement at higher loads. Two single twists that were centered on sternebrae were least able to prevent displacement at higher loads, caused the most craniocaudal translation before testing, and fractured more often at loads < or = 200 N compared with other configurations. CONCLUSION: Sternal wiring techniques stabilizing sternal synchondroses had the least displacement. Overall, figure of 8 cerclage appeared most suitable because they minimized displacement at high loads and facilitated accurate reduction during tensioning. CLINICAL RELEVANCE: In closing canine sternotomy, figure of 8 cerclage may be most suitable. Double-loop cerclage centered on the sternal synchondrosis should be avoided because of wire failure at high loads.

Biomechanical evaluation of suture pullout from canine arytenoid cartilages: effects of hole diameter, suture configuration, suture size, and distraction rate.

OBJECTIVE: To evaluate the mechanical properties of canine arytenoid cartilage-suture constructs. STUDY DESIGN: Experimental study. SAMPLE POPULATION: Eighty canine cadaveric larynges. METHODS: Arytenoid cartilage-suture constructs were loaded to failure on a materials testing machine. The effect of hole size, suture configuration, suture size, and rate of distraction on load at failure, displacement at failure, energy to failure, and construct stiffness were evaluated. Polypropylene sutures were used exclusively. Specific variables evaluated were: (1) hole size-SH needle, 22, 20, and 18 ga hypodermic needles; (2) suture configurations-single dorsal and ventral articular sutures, double sutures, horizontal mattress, locking loop, and single non-articular sutures; (3) suture size-1, 0, 2-0, and 3-0; and (4) distraction rate-0.83 and 36.66 mm/s. RESULTS: Hole size had no effect on any biomechanical variable. Double suture and horizontal mattress configurations had the highest median load and energy at failure. Single dorsal suture configurations that did not include the arcuate crest had the lowest median load at failure. Larger suture sizes tended to result in stiffer constructs. Cartilage-suture constructs behaved in a viscoelastic manner where load at failure, energy at failure, and stiffness increased when distraction rate was increased, whereas displacement at failure did not. Most constructs failed by suture pullout regardless of distraction rate, although 50% of horizontal mattress configurations failed by avulsion of the muscular process. CONCLUSION: Suture and hole sizes appear to have few effects on the biomechanical performance of arytenoid-suture constructs. Double-suture and horizontal mattress suture patterns had the best overall mechanical properties for arytenoid lateralization. Single-suture techniques, which do not incorporate the arcuate crest, were biomechanically inferior. CLINICAL RELEVANCE: Cumbersome large-diameter sutures offer no advantage over smaller sutures when performing arytenoid lateralization. The cross-sectional geometry of the muscular process should be taken into account when placing sutures in the arytenoid cartilages. Single-suture techniques that do not incorporate the arcuate crest should be avoided.

Enlargement of growth plate chondrocytes modulated by sustained mechanical loading.

BACKGROUND: Mechanical compression and distraction forces are known to modulate growth in vertebral growth plates, and they have been implicated in the progression of scoliosis. This study was performed to test the hypothesis that growth differences produced by sustained compression or distraction loading of vertebrae are associated with alterations in the amount of increase in the height of growth plate chondrocytes in the growth direction. METHODS: Compression or distraction force of nominally 60% of body weight was maintained for four weeks on a caudad vertebra of growing rats by an external apparatus attached, by means of transcutaneous pins, to the two vertebrae cephalad and caudad to it. Growth of the loaded and control vertebrae was measured radiographically. After four weeks, the animals were killed and histological sections of the loaded and control vertebrae were prepared to measure the height of the hypertrophic zone (average separation between zonal boundaries), the mean height of hypertrophic chondrocytes, and the amount of increase in cell height in the growth direction. RESULTS: Over the four weeks of the experiment, the growth rates of the compressed and distracted vertebrae averaged 52% and 113% of the control rates, respectively. The reduction in the growth rate of the compressed vertebrae was significant (p = 0.002). In the compressed vertebrae, the height of the hypertrophic zone, the mean chondrocyte height, and the amount of increase in cell height averaged 87%, 85%, and 78% of the control values, respectively, and all were significantly less than the corresponding control values. In the distracted vertebrae, these measurements did not differ significantly from the control values. The height of the hypertrophic zone and the mean chondrocyte height correlated with the growth rate (r (2) = 0.29 [p = 0.03] and r (2) = 0.23 [p = 0.06], respectively), when each variable was expressed as a proportion of the control value. The percentage changes in the measurements of the chondrocytic dimensions relative to the control values were smaller than the percentage changes in the growth rates, a finding that suggested that the rate of chondrocytic proliferation was also modulated by the mechanical loading. CONCLUSIONS: Mechanical loading of tail vertebrae in rats modulated their growth rate, which correlated with changes in the height of hypertrophic chondrocytes. The effects of compression were greater than those of distraction. CLINICAL RELEVANCE: Information about the growth rate and chondrocytic response to mechanical loads in rat vertebrae undergoing mechanically modulated growth will be helpful in determining how human vertebral growth might respond to altered loading states during progression or treatment of scoliosis and other growth-related angular skeletal deformities.

Growth plate chondrocyte enlargement modulated by mechanical loading.

Mechanical compression and distraction forces are known to modulate growth in vertebral growth plates, and are implicated in the progression of scoliosis. The amount of cellular enlargement is thought to be a key variable correlating with differing growth at different anatomical sites. This work tested the hypothesis that growth differences produced by mechanical loading are associated with alterations in the amount of cell height increase in the growth direction. Compression force (3 animals) or distraction force (4 animals) of 60% bodyweight was maintained on a caudal vertebra of growing rats with an external apparatus attached to vertebrae adjacent to the experimental level via transfixing pins. Growth of loaded and control vertebrae was measured from weekly radiographs. After four weeks, animals were euthanized and the loaded vertebrae and adjacent control vertebrae were excised for histological sections from which the following were measured: height of the hypertrophic zone (the average separation between boundaries of the zone); Mean Hypertrophic chondrocytic height; slope of the linear regression of cell height vs. position in the zone, (the amount of cell enlargement). Distracted and compressed vertebra growth averaged 110% and 53% of control levels. The percentage changes in the measures of chondrocyte dimensions, relative to control values, were smaller than the percentage changes in growth velocity. Also, the effects were of smaller magnitude for distracted vertebrae than for compressed vertebrae. Growth plate zonal height, slope and mean cell height all correlated significantly (p<0.05) with growth rate (expressed as a percentage of control) (Pearson r = 0.69, 0.37 and 0.41 respectively. Thus mechanical loading of tail vertebrae modulated their growth rate, that in turn correlated with changes in the amount of hypertrophic chondrocyte height increase. The effects for compression were greater than for distraction.