Validation of a Radiography-Based Quantification Designed to Longitudinally Monitor Soft Tissue Calcification in Skeletal Muscle

<div><p>Introduction</p><p>Soft tissue calcification, including both dystrophic calcification and heterotopic ossification, may occur following injury. These lesions have variable fates as they are either resorbed or persist. Persistent soft tissue calcification may result in chronic inflammation and/or loss of function of that soft tissue. The molecular mechanisms that result in the development and maturation of calcifications are uncertain. As a result, directed therapies that prevent or resorb soft tissue calcifications remain largely unsuccessful. Animal models of post-traumatic soft tissue calcification that allow for cost-effective, serial analysis of an individual animal over time are necessary to derive and test novel therapies. We have determined that a cardiotoxin-induced injury of the muscles in the posterior compartment of the lower extremity represents a useful model in which soft tissue calcification develops remote from adjacent bones, thereby allowing for serial analysis by plain radiography. The purpose of the study was to design and validate a method for quantifying soft tissue calcifications in mice longitudinally using plain radiographic techniques and an ordinal scoring system.</p><p>Methods</p><p>Muscle injury was induced by injecting cardiotoxin into the posterior compartment of the lower extremity in mice susceptible to developing soft tissue calcification. Seven days following injury, radiographs were obtained under anesthesia. Multiple researchers applied methods designed to standardize post-image processing of digital radiographs (N = 4) and quantify soft tissue calcification (N = 6) in these images using an ordinal scoring system. Inter- and intra-observer agreement for both post-image processing and the scoring system used was assessed using weighted kappa statistics. Soft tissue calcification quantifications by the ordinal scale were compared to mineral volume measurements (threshold 450.7mgHA/cm³) determined by μCT. Finally, sample-size calculations necessary to discriminate between a 25%, 50%, 75%, and 100% difference in STiCSS score 7 days following burn/CTX induced muscle injury were determined.</p><p>Results</p><p>Precision analysis demonstrated substantial to good agreement for both post-image processing (κ = 0.73 to 0.90) and scoring (κ = 0.88 to 0.93), with low inter- and intra-observer variability. Additionally, there was a strong correlation in quantification of soft tissue calcification between the ordinal system and by mineral volume quantification by μCT (Spearman r = 0.83 to 0.89). The ordinal scoring system reliably quantified soft tissue calcification in a burn/CTX-induced soft tissue calcification model compared to non-injured controls (Mann-Whitney rank test: <i>P</i> = 0.0002, ***). Sample size calculations revealed that 6 mice per group would be required to detect a 50% difference in STiCSS score with a power of 0.8. Finally, the STiCSS was demonstrated to reliably quantify soft tissue calcification [dystrophic calcification and heterotopic ossification] by radiographic analysis, independent of the histopathological state of the mineralization.</p><p>Conclusions</p><p>Radiographic analysis can discriminate muscle injury-induced soft tissue calcification from adjacent bone and follow its clinical course over time without requiring the sacrifice of the animal. While the STiCSS cannot identify the specific type of soft tissue calcification present, it is still a useful and valid method by which to quantify the degree of soft tissue calcification. This methodology allows for longitudinal measurements of soft tissue calcification in a single animal, which is relatively less expensive, less time-consuming, and exposes the animal to less radiation than <i>in vivo</i> μCT. Therefore, this high-throughput, longitudinal analytic method for quantifying soft tissue calcification is a viable alternative for the study of soft tissue calcification.</p></div

Spearman Correlation of the STiCSS and μCT measurements.

<p>Spearman Correlation of the STiCSS and μCT measurements.</p

Soft Tissue Calcification Scoring System (STiCSS).

<p>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.</p

Flow Diagram of Post-Image Processing.

<p>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.</p

Cost, Time, and Radiation Exposure Analysis.

<p>Cost, Time, and Radiation Exposure Analysis.</p

Comparison of STiCSS Score and Mineral Volume Determined by <i>ex vivo</i> μCT.

<p>STiCSS scores correlated with mineral volume measurement determined by <i>ex vivo</i> 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 <i>ex vivo</i> μCT from the same samples.</p

Intra-observer analysis of Post-Image Processing.

<p>Intra-observer analysis of Post-Image Processing.</p

Radiographic Analysis and Quantification of Soft Tissue Calcification Following Burn/CTX Injury.

<p>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).</p

Dystrophic Calcification and Heterotopic Ossification Are Histologically Distinct Yet Radiographically Equivalent.

<p>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).</p

Unexpected timely fracture union in matrix metalloproteinase 9 deficient mice

<div><p>Immediately following a fracture, a fibrin laden hematoma is formed to prevent bleeding and infection. Subsequently, the organized removal of fibrin, via the protease plasmin, is essential to permit fracture repair through angiogenesis and ossification. Yet, when plasmin activity is lost, the depletion of fibrin alone is insufficient to fully restore fracture repair, suggesting the existence of additional plasmin targets important for fracture repair. Previously, activated matrix metalloproteinase 9 (MMP-9) was demonstrated to function in fracture repair by promoting angiogenesis. Given that MMP-9 is a defined plasmin target, it was hypothesized that pro-MMP-9, following plasmin activation, promotes fracture repair. This hypothesis was tested in a fixed murine femur fracture model with serial assessment of fracture healing. Contrary to previous findings, a complete loss of MMP-9 failed to affect fracture healing and union through 28 days post injury. Therefore, these results demonstrated that MMP-9 is dispensable for timely fracture union and cartilage transition to bone in fixed femur fractures. Pro-MMP-9 is therefore not a significant target of plasmin in fracture repair and future studies assessing additional plasmin targets associated with angiogenesis are warranted.</p></div