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Transient expression of myofibroblast-like cells in rat rib fracture callus.

ABSTRACT: BACKGROUND AND PURPOSE: We have previously shown that early fracture callus of rat rib has viscoelastic and contractile properties resembling those of smooth muscle. The cells responsible for this contractility have been hypothesized to be myofibroblast-like in nature. In soft-tissue healing, force generated by contraction of myofibroblasts promotes healing. Accordingly, we tried to identify myofibroblast-like cells in early fibrous callus. ANIMALS AND METHODS: Calluses from rat rib fractures were removed 7, 14, and 21 days after fracture and unfractured ribs acted as controls. All tissues were analyzed using qPCR and immunohistochemistry. We analyzed expression of smooth muscle- and myofibroblast-associated genes and proteins including alpha smooth muscle actin (?SMA), non-muscle myosin, fibronectin extra domain A variant (EDA-fibronectin), OB-cadherin, connexin-43, basic calponin (h1CaP), and h-caldesmon. RESULTS: In calluses at 7 days post-fracture, there were statistically significant increases in expression of ?SMA mRNA (2.5 fold), h1CaP mRNA (2.1 fold), EDA-fibronectin mRNA (14 fold), and connexin-43 mRNA (1.8 fold) compared to unfractured ribs, and by 21 days post-fracture mRNA expression in calluses had decreased to levels approaching those in unfractured rib. Immunohistochemistry of 7 day fibrous callus localized calponin, EDA-fibronectin and co-immunolabeling of OB-cadherin and ?SMA (thus confirming a myofibroblastic phenotype) within various cell populations. INTERPRETATION: This study provides further evidence that early rat rib callus is not only smooth muscle-like in nature but also contains a notable population of cells that have a distinct myofibroblastic phenotype. The presence of these cells indicates that in vivo contraction of early callus is a mechanism that may occur in fractures so as to facilitate healing, as it does in soft tissue wound repair.

SUBMITTER: McDonald SJ

PROVIDER: S-EPMC3278664 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Transient expression of myofibroblast-like cells in rat rib fracture callus.

McDonald Stuart J SJ Dooley Philip C PC McDonald Aaron C AC Schuijers Johannes A JA Ward Alex R AR Grills Brian L BL

Acta orthopaedica 20120117 1

<h4>Background and purpose</h4>We have previously shown that early fracture callus of rat rib has viscoelastic and contractile properties resembling those of smooth muscle. The cells responsible for this contractility have been hypothesized to be myofibroblast-like in nature. In soft-tissue healing, force generated by contraction of myofibroblasts promotes healing. Accordingly, we tried to identify myofibroblast-like cells in early fibrous callus.<h4>Animals and methods</h4>Calluses from rat rib ...[more]

PMID: 22248170

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Project description:Rib fracture detection is time-consuming and demanding work for radiologists. This study aimed to introduce a novel rib fracture detection system based on deep learning which can help radiologists to diagnose rib fractures in chest computer tomography (CT) images conveniently and accurately. A total of 1707 patients were included in this study from a single center. We developed a novel rib fracture detection system on chest CT using a three-step algorithm. According to the examination time, 1507, 100 and 100 patients were allocated to the training set, the validation set and the testing set, respectively. Free Response ROC analysis was performed to evaluate the sensitivity and false positivity of the deep learning algorithm. Precision, recall, F1-score, negative predictive value (NPV) and detection and diagnosis were selected as evaluation metrics to compare the diagnostic efficiency of this system with radiologists. The radiologist-only study was used as a benchmark and the radiologist-model collaboration study was evaluated to assess the model's clinical applicability. A total of 50,170,399 blocks (fracture blocks, 91,574; normal blocks, 50,078,825) were labelled for training. The F1-score of the Rib Fracture Detection System was 0.890 and the precision, recall and NPV values were 0.869, 0.913 and 0.969, respectively. By interacting with this detection system, the F1-score of the junior and the experienced radiologists had improved from 0.796 to 0.925 and 0.889 to 0.970, respectively; the recall scores had increased from 0.693 to 0.920 and 0.853 to 0.972, respectively. On average, the diagnosis time of radiologist assisted with this detection system was reduced by 65.3 s. The constructed Rib Fracture Detection System has a comparable performance with the experienced radiologist and is readily available to automatically detect rib fracture in the clinical setting with high efficacy, which could reduce diagnosis time and radiologists' workload in the clinical practice.

Project description:Fracture healing requires controlled expression of thousands of genes. Only a small fraction of these genes have been isolated and fewer yet have been shown to play a direct role in fracture healing. The purpose of this study was threefold: (1) to develop a reproducible open femur model of fracture healing that produces consistent fracture calluses for subsequent RNA extraction, (2) to use this model to determine temporal expression patterns of known and unknown genes using DNA microarray expression profiling, and (3) to identify and validate novel gene expression in fracture healing. In the initial arm of the study, a total of 56 wild-type C57BL/6 mice were used. An open, stabilized diaphyseal femur fracture was created. Animals were killed at 1, 5, 7, 10, 14, 21, and 35 days after surgery and the femurs were harvested for analysis. At each time point, fractures were radiographed and sectioned for histologic analyses. Tissue from fracture callus at all stages following fracture yielded reproducibly large amounts of mRNA. Expression profiling revealed that genes cluster by function in a manner similar to the histologic stages of fracture healing. Based on the expression profiling of fracture tissue, temporal expression patterns of several genes known to be involved in fracture healing were verified. Novel expression of multiple genes in fracture callous tissue was also revealed including leptin and leptin receptor. In order to test whether leptin signaling is required for fracture repair, mice deficient in leptin or its receptor were fractured using the same model. Fracture calluses of mice deficient in both leptin or leptin receptor are larger than wild-type mice fractures, likely due to a delay in mineralization, revealing a previously unrecognized role of leptin signaling in fracture healing. This novel model of murine fracture repair is useful in examining both global changes in gene expression as well as individual signaling pathways, which can be used to identify specific molecular mechanisms of fracture healing.

Project description:PurposeGubernaculum-cremaster complex development is hormonally regulated and abnormal in a cryptorchid rat model. Using cell tracking techniques and imaging we studied myogenic phenotypes and fates in the fetal rat gubernaculum-cremaster complex.Materials and methodsEmbryonic day 17 gubernaculum-cremaster complexes were labeled with CellTracker™ or the DNA synthesis marker EdU (5-ethynyl-2'-deoxyuridine), or immobilized in Matrigel® and grown in culture. Embryonic day 17 to 21 gubernaculum-cremaster complex sections and cells were imaged using wide field and deconvolution immunofluorescence microscopy, and muscle and/or myofibroblast specific antibodies. Deconvolved image stacks were used to create a 3-dimensional model of embryonic day 21 gubernaculum-cremaster complex muscle.ResultsPAX7 (paired box 7) positive and myogenin positive muscle precursors were visible in a desmin-rich myogenic zone between muscle layers that elongated and became thicker during development. Gubernaculum-cremaster complex inner mesenchymal cells expressed desmin and αSMA (α smooth muscle actin) at lower levels than in the myogenic zone. After pulse labeling with CellTracker or EdU mesenchymal cells became incorporated into differentiated muscle. Conversely, mesenchymal cells migrated beyond Matrigel immobilized gubernaculum-cremaster complexes, expressed PAX7 and fused to form striated myotubes. Mesenchymal gubernaculum-cremaster complex cell lines proliferated more than 40 passages and showed contractile behavior but did not form striated muscle. Our 3-dimensional gubernaculum-cremaster complex model had 2 orthogonal ventral layers and an arcing inner layer of muscle.ConclusionsOur data suggest that mesenchymal cells in the peripheral myogenic zone of the fetal gubernaculum-cremaster complex contribute to formation of a distinctively patterned cremaster muscle. Nonmyogenic, desmin and αSMA positive gubernaculum-cremaster complex mesenchymal cells proliferate and have a myofibroblast-like phenotype in culture. Intrinsic mechanical properties of these divergent cell types may facilitate perinatal inversion of the gubernaculum-cremaster complex.

Dataset Information

Transient expression of myofibroblast-like cells in rat rib fracture callus.

Publications

Transient expression of myofibroblast-like cells in rat rib fracture callus.

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