Computational protocol: Bone-Induced Chondroinduction in Sheep Jamshidi Biopsy Defects with and without Treatment by Subchondral Chitosan-Blood Implant

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Protocol publication

[…] Intact knee joints were scanned at a resolution of 45 µm (GE Locus eXplore, Milwaukee, WI) to observe global bone characteristics, and the condyles removed with a band saw, fixed in 4% paraformaldehyde/100 mM cacodylate pH 7.4, and rescanned at a resolution of 27 µm for detailed analysis of each hole. Scans were calibrated against water, air, and a hydroxyapatite-containing phantom (SB3, 1.82 g/cm3, Gammex, Middleton, WI) and reconstructed using GE Medical Systems eXplore Reconstruction Utility. A standardized morphometric analysis of hole depth, and cross-sectional area (at 6 distinct positions in each hole) was undertaken in GE Medical Systems eXplore MicroView version 2.2 using data sets repositioned in a coronal view for each hole. Hole diameter (D) was calculated based on the formula:D=2×(cross−sectionalarea)/3.14.A novel radial analysis method of bone repair in subchondral bone defects was developed. To generate the quantitative measures, the central axis of each Jamshidi hole was established in the trabecular bone region below the subchondral bone plate and 1 to 2 mm above the bottom of the hole. Linear measures were then performed within each image slice radially in 4 directions and extending 1.5 mm from the central axis to include the hole, the original sidewall of the hole, and several millimeters beyond the initial sidewall of the hole. Approximately 140 slices (at 27-µm increments) through a 3-mm deep region were analyzed in each hole from the center across 4 planes. ImageJ (version 1.42) was then used to create text files that contained the X, Y, and Z coordinates of each voxel in the µCT images along with their corresponding gray scale intensity value. A virtual ashing procedure from GE (MicroView) was used to turn the intensities to mineral content. Software was developed in Python (version 3.1.1) and Cran-R (www.r-project.org) to determine radial gradients in bone mineral density and bone volume fraction from the central axis of the hole to beyond the repair tissue-bone interface using the text files created in ImageJ. The bone threshold was calculated using Ng’s method. The average bone mineral density (g/cm3) and bone volume fraction (%) radiating from the center of the hole were obtained for individual Jamshidi holes for 1-day repair (N = 12 treated and control holes), and holes treated with implant versus controls for 3-week repair (N = 12) and 3-month repair (N = 12). [...] Analysis of variance was used to test for significant differences in histomorphometric analyses between sample groups (N = 4 for each chitosan treatment and contralateral control) and Student’s equal variance 2-tailed t tests with the Bonferroni–Holm correction were computed for parameters that showed significance with analysis of variance. The general linear model (Statistica, V6.2, StatSoft, Tulsa, OK) was used to test the effect of implant treatment on Vv for different cell types at 3 weeks postoperative (N = 12 treated vs. control holes), as well as the effect of implant treatment on bone volume fraction at 3 months postoperative with average bone volume fraction values at 3 systematic locations from the central axis as categorical predictors (0.5 mm, 0.75 mm and 1.0 mm, N = 12 treated vs. N = 12 control defects). Linear regression analysis analyzed percentage Safranin O–stained subchondral repair cartilage of all defects at 3 months versus average hole cross-sectional area (N = 24), hole depth at surgery, or hole depth at 3 months; P < 0.05 was considered significant. […]

Pipeline specifications

Software tools ImageJ, Statistica
Applications Miscellaneous, X-ray microtomography, Microscopic phenotype analysis
Organisms Ovis aries
Diseases Hematologic Diseases