Micro-Computed Tomography Derived Anisotropy Detects Tumor Provoked Deviations in Bone in an Orthotopic Osteosarcoma Murine Model

<div><p>Radiographic imaging plays a crucial role in the diagnosis of osteosarcoma. Currently, computed-tomography (CT) is used to measure tumor-induced osteolysis as a marker for tumor growth by monitoring the bone fractional volume. As most tumors primarily induce osteolysis, lower bone fractional volume has been found to correlate with tumor aggressiveness. However, osteosarcoma is an exception as it induces osteolysis and produces mineralized osteoid simultaneously. Given that competent bone is highly anisotropic (systematic variance in its architectural order renders its physical properties dependent on direction of load) and that tumor induced osteolysis and osteogenesis are structurally disorganized relative to competent bone, we hypothesized that μCT-derived measures of anisotropy could be used to qualitatively and quantitatively detect osteosarcoma provoked deviations in bone, both osteolysis and osteogenesis, <i>in vivo</i>. We tested this hypothesis in a murine model of osteosarcoma cells orthotopically injected into the tibia. We demonstrate that, in addition to bone fractional volume, μCT-derived measure of anisotropy is a complete and accurate method to monitor osteosarcoma-induced osteolysis. Additionally, we found that unlike bone fractional volume, anisotropy could also detect tumor-induced osteogenesis. These findings suggest that monitoring tumor-induced changes in the structural property isotropy of the invaded bone may represent a novel means of diagnosing primary and metastatic bone tumors.</p></div

μCT analysis of extra-osseous extension of primary osteosarcoma.

<p>(A) Single-cell suspensions (1×10⁵) of murine osteosarcoma (K7M3) cells were injected into the tibia of mice (n = 30). Weekly radiographic evaluations were performed to monitor tumor growth. Representative X ray of tibias 3 and 5 weeks after tumor cell inoculation are shown. Scale bar indicates 1 mm. (B) Tumor growth was quantified by measuring the area define by soft tissue silhouettes in serial radiographs and recorded as the percent increase compared to the contralateral non-injected control tibia. *, P<0.01 compared to 1week post tumor injection group. (C) Representative 3D μCT images of osteosarcoma-injected tibias 3 or 5 weeks after injection with corresponding areas of measurement (metaphysis and diaphysis) are demonstrated. Scale bar indicates 1.0 mm. (D) Anisotropic values determined at the tibial metaphysis 3 (n = 5), 4 (n = 10) or 5 weeks (n = 15) after tumor injection (left side y-axis). Bone fraction volume of the tumor-laden tibial metaphysis determined by μCT normalized to the contralateral tumor free tibial metaphysis (right side y-axis indicates % relative decrease in bone fraction volume from 100%). (n = 5 at 3W; n = 12 at 4W; n = 14 at 5W) *, P<0.01 compared to no tumor injection tibias. (E) Anisotropic values determined at the tibial diaphysis 3 (n = 5), 4 (n = 10) or 5 weeks (n = 15) after tumor injection. *, P<0.01 compared to no tumor injection tibias.</p

Bone microstructural properties of no or tumor-injected tibias were analaysied by uCT.

<p>Mean±standard deviation (number of samples).</p><p>Time = weeks post tumor injection; BV/TV = bone volume fraction; Conn dens. = connectivity density.</p

Defining areas for quantitative analysis of primary tumor growth by μCT.

<p>Areas where primary tumor growth parameters were measured (tibial metaphysis and diaphysis) are demonstrated in a 3D reconstructed μCT image (A). (Detailed in <i>Methods</i>.) (B) Representative 2D, axial images of the metaphysis and diaphysis demonstrate delineation of cortical bone between the endosteum (dotted yellow line) and periosteum (solid yellow line). The intramedullary or trabecular compartment is that surrounded by the endosteum. Areas of osteolysis (*) and periosteal osteoblastic reaction (arrow) are seen in both metaphyseal and diaphyseal regions of this osteosarcoma-injected tibia. Scale bar indicates 1.0 mm. (C) Relative tibia metaphyseal bone fractional volume (BV/TV) and (D) anisotropic measurements from tibial metaphyses and (E) diaphyses of control and osteosarcoma-injected mice demonstrate a significant degree of osteolysis (decreased BV/TV) of trabecular bone and decreased bone organization (manifested by lower anisotropic values) in osteosarcoma-injected tibias. *, P<0.01 compared to no tumor injection tibias.</p

Imaging of primary osteosarcoma.

<p>Representative plain radiographs, histopathologic images, and 3D reconstructions from micro-computed tomography (μCT) of contralateral tumor free tibias. (A) and tibias injected with osteosarcoma cells (B). In tibias injected with osteosarcoma cells, trabecular bone in the medullary cavity is completely replaced by tumor, resulting in the osteolysis seen on radiographs (denoted by *). As tumor cells permeate and destroy cortical bone as they extend into the extra-osseous soft tissues, a vigorous osteoblastic periosteal reaction occurs, which corresponds to reactive woven bone deposition on the periosteal surface (arrows). Scale bar indicates 1.0 mm.</p

Degree of anisotropy measured by μCT as a means to quantify intra-osseous osteosarcoma.

<p>Representative examples of micro-computed tomography (μCT) (A) and histopathology (B) of control-injected and osteosarcoma-injected tibias is demonstrated. μCT analysis demonstrates marked osteolytic destruction of trabecular and cortical bone (indicated by *) and an associated periosteal reaction (yellow arrow) in the osteosarcoma-injected tibia. Martius scarlet blue-stained (MSB) sections (B) disclose the histologic correlates of the radiologic findings in osteosarcoma-injected tibias. Tumor cells replace the medullary cavity and permeate through native cortical bone (black arrow) resulting in pronounced osteolysis, as well as inciting a vigorous periosteal osteoblastic reaction (yellow arrow). The periosteal reaction results in a markedly disorganized pattern of bone deposition compared to normal bone. Anisotropy, a measure of bone organization demonstrated schematically in (C) might represent a novel parameter by which to quantify the local growth of primary osteosarcoma. Scale bar indicates 1.0 mm.</p

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

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

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

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