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Concept: Bone healing

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OBJECTIVES: To report on a series of dogs and cats with long bone fractures that occurred as a direct consequence of linear external skeletal fixation (ESF) application. METHODS: Retrospective study. Data from the medical records and radiographs of canine (n = 4) and feline (n = 7) cases were collected from three referral and three first opinion practices in the UK (1999 to 2011). RESULTS: Long bone fractures occurred following the application of linear ESF either while the ESF was in situ or after removal. All fractures occurred through either a pin tract or an empty drill hole. Pins associated with ESF-related fracture tended to be in the higher end of the recommended size range. The majority of cases had additional complicating factors such as multiple injuries, revision surgery, poor owner compliance with postoperative exercise restriction and the presence of empty drill holes. CLINICAL SIGNIFICANCE: In cases with features that could complicate outcome, careful attention should be paid to recommendations for ESF application. Leaving empty drill holes is suboptimal. The retrospective nature of the study, low numbers of, and diversity amongst, cases should be taken into consideration when interpreting the results from this study.

Concepts: Osteoporosis, Bone, Bone fracture, Fracture, Orthopedic surgery, Bone fractures, Bone healing, Fibrocartilage callus

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We evaluated both the outcome of using a locking plate as a definitive external fixator for treating open tibial fractures and, using finite element analysis, the biomechanical performance of external and internal metaphyseal locked plates in treating proximal tibial fractures. Eight open tibial patients were treated using a metaphyseal locked plate as a low-profile definitive external fixator. Then, finite element models of internal (IPF) as well as two different external plate fixations (EPFs) for proximal tibial fractures were reconstructed. The offset distances from the bone surface to the EPFs were 6cm and 10cm. Both axial stiffness and angular stiffness were calculated to evaluate the biomechanical performance of these three models. The mean follow-up period was 31 months (range, 18-43 months). All the fractures united and the mean bone healing time was 37.5 weeks (range, 20-52 weeks). All patients had excellent or good functional results and were walking freely at the final follow-up. The finite element finding revealed that axial stiffness and angular stiffness decreased as the offset distance from the bone surface increased. Compared to the IPF models, in the two EPF models, axial stiffness decreased by 84-94%, whereas the angular stiffness decreased by 12-21%. The locking plate used as a definitive external fixator provided a high rate of union. While the locking plate is not totally rigid, it is clinically stable and may be advisable for stiffness reduction of plating constructs, thus promoting fracture healing by callus formation. Our patients experienced a comfortable clinical course, excellent knee and ankle joint motion, satisfactory functional results and an acceptable complication rate.

Concepts: Bone, Bone fracture, Fracture, Finite element method, Finite element method in structural mechanics, Bone healing, Periosteum, Direct stiffness method

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BACKGROUND: The bone nonunion is an important complication of bone fracture repair. The existing models developed on small animal species prevent using osteosynthesis materials designed to be implanted in human bones. The goal of this study was to develop a nonunion process in a noncritical segmental tibial defect in sheep, a species analogous in size to humans. MATERIALS AND METHODS: The animals were divided into two groups of four animals each. In Group 1 (experimental), the defect was created by surgically stripping the periosteum from the edges of a distal tibial osteotomy, keeping the edges 5 mm apart, and placing an incomplete O-shaped silicone ring in the gap. Group 2 (control) was intervened with a simple fracture at the distal end of the tibia. In both groups an interlocking nail was used as a fixation system. Over 8 wk after surgery, radiographs and histologic and histomorphometric analyses were performed. RESULTS: The control group showed a typical bone repair process. In contrast, the experimental group showed a fracture line with rounded edges and a scarce callus formation. The bone callus showed reduced amount of bone formation and large content of fibrous tissue (P=0.001). CONCLUSIONS: These results indicate that our model developed an atrophic nonunion in sheep, a species having multiple similarities to humans, such as weight, size, bone structure, and bone remodeling process.

Concepts: Osteoporosis, Bone, Bone fracture, Skeletal system, Science, Orthopedic surgery, Bone healing, Fibrocartilage callus

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During the healing process after bone fracture, soft callus forms adjacent to the fracture site, is replaced by hard callus, and is finally remodeled to the original bone configuration. Although the cambium layer of the periosteum is reported to play an essential role in callus formation, we still lack direct in vivo evidence of this. To investigate the cell lineage of the soft callus, we analyzed the process of fracture healing in Prx1-Cre;ROSA26 reporter (R26R), Col1a1(3.6 kb)-Cre;R26R, Col1a1(2.3 kb)-Cre;R26R, Sox9-CreERT2;R26R, and Sox9-LacZ mice with X-gal staining. In the Prx1-Cre;R26R, in which the cells of the periosteum stained for X-gal before fracture, all cells in the soft callus were X-gal positive, whereas in the Col1a1(3.6 kb)-Cre;R26R mice, the cells in the periosteum before fracture stained for X-gal and the soft callus was partly composed of X-gal-positive cells. In contrast, in the Col1a1(2.3 kb)-Cre;R26R mice, in which the mature osteoblasts in the cambium layer of the periosteum were marked before fracture, no cells in the soft callus at the fracture site were X-gal positive. These results suggest that most of the cells in the soft callus are derived from the mesenchymal progenitors in the periosteum, and not from mature osteoblastic cells. Interestingly, in the Sox9-LacZ mice, Sox9-expressing X-gal-positive cells emerged in the periosteum adjacent to the fracture site 3 days after fracture. We demonstrated this by injecting tamoxifen into the Sox9-CreERT2;R26R mice for 3 days after fracture, so that these Sox9-expressing periosteal cells gave rise to cells in the soft and hard calli. Our findings show that the periosteal cells in which Sox9 expression is induced just after fracture are the major source of the chondrocytes and osteoblasts in the fracture callus.

Concepts: Osteoporosis, Bone, Bone fracture, Healing, Osteoblast, Bone healing, Periosteum, Fibrocartilage callus

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It is proposed that the external asymmetric formation of callus tissues that forms naturally about an oblique bone fracture can be predicted computationally. We present an analysis of callus formation for two cases of bone fracture healing: idealised and subject-specific oblique bone fractures. Plane strain finite element (FE) models of the oblique fractures were generated to calculate the compressive strain field experienced by the immature callus tissues due to interfragmentary motion. The external formations of the calluses were phenomenologically simulated using an optimisation style algorithm that iteratively removes tissue that experiences low strains from a large domain. The resultant simulated spatial formation of the healing tissues for the two bone fracture cases showed that the calluses tended to form at an angle equivalent to the angle of the oblique fracture line. The computational results qualitatively correlated with the callus formations found in vivo. Consequently, the proposed methods show potential as a means of predicting callus formation in pre-clinical testing.

Concepts: Osteoporosis, Bone fracture, Orthopedic surgery, Bone fractures, Bone healing, Osteology, Fibrocartilage callus, Catagmatic

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Loss of Sostdc1, a growth factor paralogous to Sost, causes the formation of ectopic incisors, fused molars, abnormal hair follicles, and resistance to kidney disease. Sostdc1 is expressed in the periosteum, a source of osteoblasts, fibroblasts and mesenchymal progenitor cells, which are critically important for fracture repair. Here, we investigated the role of Sostdc1 in bone metabolism and fracture repair. Mice lacking Sostdc1 (Sostdc1(-/-)) had a low bone mass phenotype associated with loss of trabecular bone in both lumbar vertebrae and in the appendicular skeleton. In contrast, Sostdc1(-/-) cortical bone measurements revealed larger bones with higher BMD, suggesting that Sostdc1 exerts differential effects on cortical and trabecular bone. Mid-diaphyseal femoral fractures induced in Sostdc1(-/-) mice showed that the periosteal population normally positive for Sostdc1 rapidly expands during periosteal thickening and these cells migrate into the fracture callus at 3 days post fracture. Quantitative analysis of mesenchymal stem cell (MSC) and osteoblast populations determined that MSCs express Sostdc1, and that Sostdc1(-/-) 5 day calluses harbor >2-fold more MSCs than fractured wildtype controls. Histologically a fraction of Sostdc1-positive cells also expressed nestin and α-smooth muscle actin, suggesting that Sostdc1 marks a population of osteochondral progenitor cells that actively participate in callus formation and bone repair. Elevated numbers of MSCs in D5 calluses resulted in a larger, more vascularized cartilage callus at day 7, and a more rapid turnover of cartilage with significantly more remodeled bone and a thicker cortical shell at 21 days post fracture. These data support accelerated or enhanced bone formation/remodeling of the callus in Sostdc1(-/-) mice, suggesting that Sostdc1 may promote and maintain mesenchymal stem cell quiescence in the periosteum.

Concepts: Bone, Fracture, Stem cell, Mesenchymal stem cell, Bone marrow, Stem cells, Skeletal system, Bone healing

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Failure of bone fracture healing occurs in 5% to 10% of all patients. Nonunion risk is associated with the severity of injury and with the surgical treatment technique, yet progression to nonunion is not fully explained by these risk factors.

Concepts: Osteoporosis, Bone, Bone fracture, Collagen, Orthopedic surgery, Distraction osteogenesis, Bone healing, Osteology

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The heparin-binding growth and differentiation factor midkine (Mdk) is proposed to negatively regulate osteoblast activity and bone formation in the adult skeleton. As Mdk-deficient mice were protected from ovariectomy (OVX)-induced bone loss, this factor may also play a role in the pathogenesis of postmenopausal osteoporosis. We have previously demonstrated that Mdk negatively influences bone regeneration during fracture healing. Here, we investigated whether the inhibition of Mdk using an Mdk-antibody (Mdk-Ab) improves compromised bone healing in osteoporotic OVX-mice. Using a standardized femur osteotomy model, we demonstrated that Mdk serum levels were significantly enhanced after fracture in both non-OVX and OVX-mice, however, the increase was considerably greater in osteoporotic mice. Systemic treatment with the Mdk-Ab significantly improved bone healing in osteoporotic mice by increasing bone formation in the fracture callus. On the molecular level, we demonstrated that the OVX-induced reduction of the osteoanabolic beta-catenin signaling in the bony callus was abolished by Mdk-Ab treatment. Furthermore, the injection of the Mdk-Ab increased trabecular bone mass in the skeleton of the osteoporotic mice. These results implicate that antagonizing Mdk may be useful for the therapy of osteoporosis and osteoporotic fracture-healing complications.

Concepts: Osteoporosis, Hormone replacement therapy, Bone, Bone fracture, Hip fracture, Skeletal system, Menopause, Bone healing

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Fractures heal predominantly through the process of endochondral ossification. The classic model of endochondral ossification holds that chondrocytes mature to hypertrophy, undergo apoptosis and new bone forms by invading osteoprogenitors. However, recent data demonstrate that chondrocytes transdifferentiate to osteoblasts in the growth plate and during regeneration, yet the mechanism(s) regulating this process remain unknown. Here, we show a spatially-dependent phenotypic overlap between hypertrophic chondrocytes and osteoblasts at the chondro-osseous border in the fracture callus, in a region we define as the transition zone (TZ). Hypertrophic chondrocytes in the TZ activate expression of the pluripotency factors [Sox2, Oct4 (Pou5f1), Nanog], and conditional knock-out of Sox2 during fracture healing results in reduction of the fracture callus and a delay in conversion of cartilage to bone. The signal(s) triggering expression of the pluripotency genes are unknown, but we demonstrate that endothelial cell conditioned medium upregulates these genes in ex vivo fracture cultures, supporting histological evidence that transdifferentiation occurs adjacent to the vasculature. Elucidating the cellular and molecular mechanisms underlying fracture repair is important for understanding why some fractures fail to heal and for developing novel therapeutic interventions.

Concepts: Bone, Cartilage, Tissues, Endochondral ossification, Intramembranous ossification, Osteoblast, Chondrocyte, Bone healing

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Bone formation during fracture repair inevitably initiates within or around extravascular deposits of a fibrin-rich matrix. In addition to a central role in hemostasis, fibrin is thought to enhance bone repair by supporting inflammatory and mesenchymal progenitor egress into the zone of injury. However, given that a failure of efficient fibrin clearance can impede normal wound repair, the precise contribution of fibrin to bone fracture repair, whether supportive or detrimental, is unknown. Here, we employed mice with genetically and pharmacologically imposed deficits in the fibrin precursor fibrinogen and fibrin-degrading plasminogen to explore the hypothesis that fibrin is vital to the initiation of fracture repair, but impaired fibrin clearance results in derangements in bone fracture repair. In contrast to our hypothesis, fibrin was entirely dispensable for long-bone fracture repair, as healing fractures in fibrinogen-deficient mice were indistinguishable from those in control animals. However, failure to clear fibrin from the fracture site in plasminogen-deficient mice severely impaired fracture vascularization, precluded bone union, and resulted in robust heterotopic ossification. Pharmacological fibrinogen depletion in plasminogen-deficient animals restored a normal pattern of fracture repair and substantially limited heterotopic ossification. Fibrin is therefore not essential for fracture repair, but inefficient fibrinolysis decreases endochondral angiogenesis and ossification, thereby inhibiting fracture repair.

Concepts: Bone fracture, Coagulation, Fibrin, Platelet, Fibrinolysis, Initiation, Intramembranous ossification, Bone healing