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

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

Vascular quantification of healing fractures of WT and MMP-9 deficient mice.

<p>Quantitative assessment of microfil based angiography surrounding the healing fracture. No statistical differences in vascularity were identified between wild type and MMP-9 deficient mice at any time point for either vascular volume (mm³) or vessel thickness (mm). 7d- WT: N = 4, MMP-9: N = 3; 10d- WT: N = 4, MMP-9: N = 3; 14d- WT: N = 5, MMP-9: N = 6; 21d- WT: N = 4, MMP-9: N = 6; 28d- WT: N = 7, MMP-9: N = 6. Individual time points were assessed by non-parametric t-tests. Alpha = 0.05.</p

MMP-9 is not required for endochondral fracture healing.

<p>A) Both MMP-9 deficient and WT littermates possessed abundant soft cartilage at 7 and 10 dpf. At 21 dpf, hard tissue callus dominated in MMP-9 deficient and WT littermates. B) Quantification of the total callus volume and % soft tissue fracture callus demonstrated that there was no significant difference between MMP-9 deficient mice and WT littermates at any time assessed. While the % soft tissue callus area appears to trend higher in MMP-9 deficient mice at 14 DPI as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198088#pone.0198088.ref005" target="_blank">5</a>], there is marked variability between MMP-9 deficient mice.</p

Revascularization of the fracture is unaffected by a loss of MMP-9.

<p>Visual representation of microfil based angiography overlaid with radiographic images. No differences in vascularity were observed between wild type and MMP-9 deficient mice at any time point.</p