Table 1.
Cost, Time, and Radiation Exposure Analysis.
Fig 1.
Flow Diagram of Post-Image Processing.
Prior to quantification, all digital radiographs underwent post-image processing to ensure appropriate resolution and contrast settings to allow for comparisons between images. The flow diagram demonstrates the stepwise procedure for processing images with ImageJ.
Fig 2.
Soft Tissue Calcification Scoring System (STiCSS).
STiCSS is an ordinal scale [0–4] developed for quantifying the varying degrees of soft tissue calcification from radiographic images of the lower extremity. Representative images of each STiCSS score are provided along with the operational definition designated to each score. Yellow dotted lines outline the area of interest for soft tissue calcification (the posterior compartment of the lower extremity), while the listed percentages correlate to the extent of soft tissue calcification within each sample image.
Table 2.
Intra-observer analysis of Post-Image Processing.
Table 3.
Intra-observer Error on STiCSS.
Table 4.
Inter-observer Analysis of the STiCSS.
Fig 3.
Comparison of STiCSS Score and Mineral Volume Determined by ex vivo μCT.
STiCSS scores correlated with mineral volume measurement determined by ex vivo μCT at a threshold of 450.7mgHA/cm3. Correlation was examined using scores from 5 independent observers (A-E) who individually scored 28 images using the STiCSS scale. Scores were then individually plotted against mineral volume measurements obtained by ex vivo μCT from the same samples.
Table 5.
Spearman Correlation of the STiCSS and μCT measurements.
Fig 4.
Radiographic Analysis and Quantification of Soft Tissue Calcification Following Burn/CTX Injury.
A) Radiographic images of C57BL6 mice that either received a CTX injury alone (N = 8 mice, 16 samples) or a burn injury with a CTX injection (N = 10 mice, 20 samples). B) Graphical representation of radiographic images quantified using the STiCSS. Data represents both the left and right leg of each individual animal. Median and interquartile ranges are shown. Mann-Whitney rank test (p<0.0001, ****) demonstrated significant differences between control (CTX injury alone) and the burn injury group (CTX/Burn Injury).
Table 6.
Sample Size Calculator.
Fig 5.
Dystrophic Calcification and Heterotopic Ossification Are Histologically Distinct Yet Radiographically Equivalent.
Radiographic and histological images of mice that received a CTX muscle injury and developed dystrophic calcification or heterotopic ossification within the posterior compartment of the lower extremity. Radiographically, dystrophic calcification and heterotopic ossification, two states of soft tissue calcification, are indistinguishable. Nevertheless, the STiCSS can be applied to both processes even though the precise state of mineralization cannot be determined. Histologically, dystrophic calcification and heterotopic ossification can be easily discriminated by their distinct histological characteristics apparent by H&E and Von Kossa (stain for mineralized tissue) staining. Dystrophic calcification is defined histologically by the presence of amorphous, unorganized, calcium phosphate crystals interspersed with necrotic debris at the site of tissue injury (yellow arrow heads). Heterotopic ossification is characterized by mineralized mature bone (red asterisk), which may be associated with a central medullary cavity with intratrabecular hematopoiesis (green arrow heads).