Correlations between mechanical stress history and tissue differentiation in initial fracture healing

Acknowledgements
Paracrine signaling by bone-marrow-derived mesenchymal stem cells (MSCs) plays a major role in tissue repair. Although the production of regulatory cytokines by MSC transplantation is a critical modulator of tissue regeneration, we focused on exosomes, which are extracellular vesicles that contain proteins and nucleic acids, as a novel additional modulator of cell-to-cell communication and tissue regeneration. To address this, we used radiologic imaging, histological examination, and immunohistochemical analysis to evaluate the role of exosomes isolated from MSC-conditioned medium (CM) in the healing process in a femur fracture model of CD9−/− mice, a strain that is known to produce reduced levels of exosomes. We found that the bone union rate in CD9−/− mice was significantly lower than wild-type mice because of the retardation of callus formation. The retardation of fracture healing in CD9−/− mice was rescued by the injection of exosomes, but this was not the case after the injection of exosomes-free conditioned medium (CM-Exo). The levels of the bone repair-related cytokines, monocyte chemotactic protein-1 (MCP-1), MCP-3, and stromal cell-derived factor-1 in exosomes were low compared with levels in CM and CM-Exo, suggesting that bone repair may be in part mediated by other exosome components, such as microRNAs. These results suggest that exosomes in CM facilitate the acceleration of fracture healing, and we conclude that exosomes are a novel factor of MSC paracrine signaling with an important role in the tissue repair process.


This work focuses on exosomes, which are extracellular vesicles, as a novel additional modulator of cell-to-cell communication. This study evaluated the role of exosomes isolated from mesenchymal stem cell (MSC)-conditioned medium (MSC-CM) in the fracture-healing process of CD9−/− mice, a strain that is known to produce reduced levels of exosomes. Retardation of fracture healing in CD9−/− mice was rescued by the injection of MSC exosomes, but this was not the case after the injection of exosome-free CM. This study finds that MSC exosomes are a novel factor of MSC paracrine signaling, with an important role in the tissue repair process.

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Mesenchymal Stem Cell-Derived Exosomes Promote Fracture Healing in a Mouse Model

An experimental model of a contaminated open fracture has been developed. This model has been used to test the hypothesis that stable fixation of a contaminated open fracture will reduce its susceptibility to infection. The tibiae of male New Zealand white rabbits were fractured and then fixed with either a dynamic compression plate (stable group) or a loose-fitting intramedullary rod (unstable group). The fracture site was then inoculated with a standard inoculum of Staphylococcus aureus. There were 20 rabbits in the stable group and osteomyelitis developed in seven (35 per cent); in the unstable group 15 (71 per cent) out of 21 animals developed osteomyelitis. This difference in infection rates was statistically significant (P < 0.02). This experimental study supports the concept of stabilization of open fractures in man.

The prevention of infection in open fractures: an experimental study of the effect of fracture stability.

Angiogenesis is essential for the increased delivery of oxygen and nutrients required for the reparative processes of bone healing. Vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, has been implicated in this process. We have previously shown that hypoxia specifically and potently regulates the expression of VEGF by osteoblasts. However, the molecular mechanisms governing this interaction remain unknown. In this study, we hypothesized that the hypoxic regulation of VEGF expression by osteoblasts occurs via an oxygen-sensing mechanism similar to the regulation of the erythropoietin gene (EPO). To test this hypothesis, we examined the kinetics of oxygen concentration on osteoblast VEGF expression. In addition, we analyzed the effects of nickel and cobalt on the expression of VEGF in osteoblastic cells because these metallic ions mimic hypoxia by binding to the heme portion of oxygen-sensing molecules. Our results indicated that hypoxia potently stimulates VEGF mRNA expression. In addition, we found that nickel and cobalt both stimulate VEGF gene expression in a similar time- and dose-dependent manner, suggesting the presence of a hemelike oxygen-sensing mechanism similar to that of the EPO gene. Moreover, actinomycin D, cycloheximide, dexamethasone, and mRNA stabilization studies collectively established that this regulation is predominantly transcriptional, does not require de novo protein synthesis, and is not likely mediated by the transcriptional activator AP-1. These studies demonstrate that hypoxia, nickel, and cobalt regulate VEGF expression in osteoblasts via a similar mechanism, implicating the involvement of a heme-containing oxygen-sensing molecule. This may represent an important mechanism of VEGF regulation leading to increased angiogenesis in the hypoxic microenvironment of healing bone.

VEGF expression in an osteoblast-like cell line is regulated by a hypoxia response mechanism.

(1969). Reaming of the Medullary Cavity and its Effect on Diaphyseal Bone: A Fluorochromic, Microangiographic and Histologic Study on the Rabbit Tibia and Dog Femur. Acta Orthopaedica Scandinavica: Vol. 40, No. sup128, pp. 1-165.

Reaming of the medullary cavity and its effect on diaphyseal bone. A fluorochromic, microangiographic and histologic study on the rabbit tibia and dog femur.

The blood supply of healing undisplaced closed fractures of the radius and tibia in adult mongrel dogs was studied by means of microangiograms and histological preparations. The microangiograms were prepared from decalcified longitudinal slices across the fracture site, one millimeter in thickness. The corresponding histological preparations were made from the same slices of tissue. The vascular pattern at the site of fracture, at intervals from one day to eight weeks after fracture, was compared with the normal vascular pattern. Immediately after fracture there was a marked opening up of the existing arterial tree. With the advancement of healing, both medullary and periosteal circulations increased greatly by the development of new blood vessels; but the medullary arterial system, when intact, played the major role in the supply of blood to the uniting callus and in the revascularization of the necrotic cortex at the fracture site. The ascendency of the medullary blood supply increased as healing progressed. In the dog the normal configuration of the blood supply of the compacta is similar to that in man; the inner two-thirds of the cortex is supplied by branches of the nutrient artery. Our experimental findings in the dog support clinical experience. The periosteal circulation of long bones can take over when the medullary circulation has been interrupted by displacement of the fracture fragments or by surgery—as will be described in subsequent reports of experiments in progress. However, the medullary arterial supply, when available, dominates the vascular picture in rapid fracture healing.

Microangiography in Bone Healing

The blood supply of healing undisplaced closed fractures of the radius and tibia in adult mongrel dogs was studied by means of microangiograms and histological preparations The microangiograms were prepared from decalcified longitudinal slices across the fracture site, one millimeter in thickness

John C. Beer

Papers

Microangiography in Bone Healing

Microangiography in bone healing. II. Displaced closed fractures.