Ectopic bone formation analyses.

<p>A: Bone formation capability of muscle-derived cells. Representative histological sections of a scaffold loaded with BMSCs or MuSCs cultured with both dexamethasone and BMP-2. Scale bar: 1 mm (left panels), 200 μm (middle panels) and 50 μm (right panels). Black arrows indicate new bone formation in the scaffold. Black arrow heads indicate osteocytes and green arrow heads indicate bone lining cells. B: Newly formed bone, T: β-TCP, P: Porous area. B: Recruitment of cells residing in muscle tissue to participate in BMP-2-induced ectopic bone formation. Cells labeled prior to local BMP-2 administration were detected in the newly formed woven bone area. Representative histological sections were stained with H&E and evaluated for i-QD fluorescence. The black arrows in the H&E image show the locations of fluorescently labeled cells indicated with white arrows in the fluorescent image. Scale bar: 200 μm. B: Newly formed bone, T: β-TCP, P: Porous area. C: Augmentation of ectopic bone formation by dexamethasone. C-1, 2: Representative histological sections of an excised scaffold that had been loaded with BMP-2 alone and a scaffold that had been loaded with dexamethasone and BMP-2. The sections were stained with H&E and immunostained for osteocalcin. Black arrows indicate new bone formation in the scaffold. Red arrows indicate osteocalcin positive staining area. Scale bar: 1 mm (top panels of C-1), 200 μm (bottom panels of C-1) and 50 μm (C-2). B: Newly formed bone, T: β-TCP, P: Porous area. C-3: Quantification of bone formation at 3 weeks after transplantation. The Y axis indicates the bone formation ratio calculated as total bone area/total scaffold area. Each bar represents the mean with the standard deviation (SD). *denotes <i>P</i> < 0.05.</p

Dexamethasone pretreatment and osteogenic induction with combined dexamethasone and BMP-2 treatment enhance the osteogenic differentiation of BMSCs and MuSCs.

<p>A: Schematic representation of the cell culture protocol. Gross images of ALP staining (ALP) and Von Kossa staining (VK) of BMSCs (B) and MuSCs (D). Quantitative analysis of the mRNA expression of ALP and osteocalcin in BMSCs (C) and MuSCs (E). The fold change in gene expression was normalized to that of BM-Dex-AG or Mu-Dex-AG. Bars show the mean and SEM. Statistical significance was confirmed between the BM and BM-Dex groups (ALP: p = 0.015, OCN: p = 0.023) and between the Mu and Mu-DEX groups (ALP: p = 0.019, OCN: p = 0.015). Effects of combinations of differentiation reagents were significant for ALP in BM-Dex (p = 0.032) and Mu-DEX (p = 0.019) and for OCN in Mu-DEX (p = 0.024).</p

RT-PCR primers used in this study.

<p>RT-PCR primers used in this study.</p

Colony formation assay of BMSCs and MuSCs.

<p>A: Schematic representation of the colony formation unit assay protocol. Cells seeded at P2 were allowed to form single-cell-derived colonies with or without 10^-7 M dexamethasone for 7 days before osteogenic induction. ND-ND indicates normal growth medium at P0, P1, and P2. ND-D indicates normal growth medium at P0 and P1 and dexamethasone-containing medium at P2. D-ND indicates dexamethasone-containing medium at P0 and P1 and normal growth medium at P2. D-D indicates dexamethasone-containing medium at P0, P1, and P2. Gross images of BMSCs (B) and MuSCs (D) in each dish stained by ALP and crystal violet. Quantification of the total colony number and fraction of ALP-positive colonies (%) among total colonies in BMSCs (C) and MuSCs (E). *** denotes P < 0.001 as determined by Student’s t-test.</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

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

Western blot analyses of the SMAD1/5/8 and phosphorylation of SMAD 1/5.

<p>Western blot analyses of P-SMAD 1/5, SMAD1/5/8 and α-tubulin expression in BMSCs (A) and MuSCs (B) under four different osteogenic induction conditions with or without dexamethasone.</p

Dexamethasone affects cell proliferation during osteogenic differentiation.

<p>The absorbance at 585 nm was measured for dye extracted from the wells, and ratios relative to the standard are presented in the graphs. A: BM, B: BM-Dex, C: Mu, and D: Mu-Dex</p

Cost, Time, and Radiation Exposure Analysis.

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