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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.
Fracture nonunion is a major complication of bone fracture regeneration and repair. The molecular mechanisms that result in fracture nonunion appearance are not fully determined. We hypothesized that fracture nonunion results from the failure of hypoxia and hematoma, the primary signals in response to bone injury, to trigger Bmp2 expression by mesenchymal progenitor cells (MSCs). Using a model of nonstabilized fracture healing in transgenic 5'Bmp2BAC mice we determined that Bmp2 expression appears in close association with hypoxic tissue and hematoma during the early phases of fracture healing. In addition, BMP2 expression is induced when human periosteum explants are exposed to hypoxia ex vivo. Transient interference of hypoxia signaling in vivo with PX-12, a thioredoxin inhibitor, results in reduced Bmp2 expression, impaired fracture callus formation and atrophic-like nonunion by a HIF-1α independent mechanism. In isolated human periosteum-derived MSCs, BMP2 expression could be induced with the addition of platelets concentrate lysate but not with hypoxia treatment, confirming HIF-1α-independent BMP2 expression. Interestingly, in isolated human periosteum-derived mesenchymal progenitor cells, inhibition of BMP2 expression by PX-12 is accomplished only under hypoxic conditions seemingly through dis-regulation of reactive oxygen species (ROS) levels. In conclusion, we provide evidence of a molecular mechanism of hypoxia-dependent BMP2 expression in MSCs where interference with ROS homeostasis specifies fracture nonunion-like appearance in vivo through inhibition of Bmp2 expression. Stem Cells 2016;34:2342-2353.
© 2016 AlphaMed Press.
Thrombin (coagulation factor IIa) is a serine protease encoded by the F2 gene. Pro-thrombin (coagulation factor II) is cut to generate thrombin in the coagulation cascade that results in a reduction of blood loss. Procoagulant states that lead to activation of thrombin are common in bone fracture sites. However, its physiological roles and relationship with osteoblasts in bone fractures are largely unknown. We herein report various effects of thrombin on mouse osteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed proteinase-activated receptor 1 (PAR1), also known as the coagulation factor II receptor. They also produced monocyte chemoattractant protein (MCP-1), tissue factor (TF), MCSF and IL-6 upon thrombin stimulation through the PI3K-Akt and MEK-Erk1/2 pathways. Furthermore, MCP-1 obtained from thrombin-stimulated MC3T3-E1 cells induced migration by macrophage RAW264 cells. All these effects of thrombin on MC3T3-E1 cells were abolished by the selective non-peptide thrombin receptor inhibitor SCH79797. We also found that thrombin, PAR-1, MCP-1, TF as well as phosphorylated AKT and p42/44 were significantly expressed at the fracture site of mouse femoral bone. Collectively, thrombin/PAR-1 interaction regulated MCP-1, TF, MCSF and IL-6 production by MC3T3-E1 cells. Furthermore, MCP-1 induced RAW264 cell migration. Thrombin may thus be a novel cytokine that regulates several aspects of osteoblast function and fracture healing.
Copyright © 2015 Elsevier Inc. All rights reserved.
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.
Previous studies demonstrate associations of low 25-hydroxyvitamin D (25(OH)D) concentrations with low bone mineral density (BMD) and fractures, motivating widespread use of vitamin D supplements for bone health. However, previous studies have been limited to predominantly White populations despite differences in the distribution and metabolism of 25(OH)D by race/ethnicity. We determined associations of serum 25(OH)D, 24,25-dihydroxyvitamin D (24,25(OH2)D3), and parathyroid hormone (PTH) with BMD among 1773 adult participants in the Multi-Ethnic Study of Atherosclerosis (MESA) in a staggered cross-sectional study design. Vitamin D metabolites were measured using liquid chromatography-mass spectroscopy and PTH using a 2-site immunoassay from serum collected in 2000-2002. Volumetric trabecular lumbar BMD was measured from computed tomography scans performed in 2002-2005 expressed as g/cm(3). We used linear regression and graphical methods to compare associations of vitamin D metabolite and PTH concentrations with BMD as the outcomes measure among White (n=714), Black (n=353), Chinese (n=249), and Hispanic (n=457) participants. Serum 25(OH)D and 24,25(OH2)D3 concentrations were highest among Whites and lowest among Blacks. BMD was greatest among Black participants. Higher serum 25(OH)D was only associated with higher BMD among Whites and Chinese participants (P-for-interaction=0.054). Comparing the lowest category of 25(OH)D (<20 ng/ml) to the highest (≥30 ng/ml), the adjusted mean difference in BMD was -8.1g/cm3 (95% CI -14.8, -1.4) for Whites; -10.2g/cm3 (-20.4, 0.0) for Chinese vs. 8.8 g/cm3 (-2.8, 20.5) for Black and -1.1g/cm3 (-8.3, 6.2) for Hispanic. Similar results were observed for serum 24,25(OH2)D3. Serum PTH was not associated with BMD. In a multi-ethnic population, associations of 25(OH)D with BMD were strongest among White and Chinese participants and null among Black and Hispanic participants. Further studies are needed to determine optimal biomarkers for bone health for multiple ethnic groups.
Copyright © 2015 Elsevier Inc. All rights reserved.
The ubiquitin/proteasome system plays an important role in regulating the activity of osteoblast precursor cells. Proteasome inhibitors (PSIs) have been shown to stimulate the differentiation of osteoblast precursor cells and to promote bone formation. This raises the possibility that PSIs might be useful for enhancing fracture healing. In this study, we examined the effect of the local administration of PSI on fracture repair in rats. The effects of treatment on the healing of a fractured femur were assessed based on radiographs, micro-computed tomography (μCT) analysis, biomechanical testing, and histological analysis. PSI enhanced osteogenic differentiation in bone marrow- and periosteum-derived mesenchymal progenitor cells in vitro. Moreover, the local administration of PSI in vivo promoted fracture healing in rats, as demonstrated by an increased fracture callus volume in radiographs at 2 weeks post-fracture, and improved radiographic scores. By week 4, PSI treatment had enhanced biomechanical strength and mineral density in the callus as assessed using bending tests, and μCT, respectively. Histological sections demonstrated that PSI treatment accelerated endochondral ossification during the early stages of fracture repair. Although further investigations are necessary to assess its clinical use, the local administration of PSIs might be a novel, and effective therapeutic approach for fracture repair.
© 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
Bone fractures remain a serious health burden and prevention and enhanced healing of fractures have been obtained by augmenting either BMP or Wnt signaling. However, whether BMP and Wnt signaling are both required or are self-sufficient for anabolic and fracture healing activities has never been fully elucidated. Mice haploinsufficient for Dkk1 (Dkk1(+/-)) exhibit a high bone mass phenotype due to an up-regulation of canonical Wnt signaling while mice lacking Bmp2 expression in the limbs (Bmp2(c/c);Prx1::cre) succumb to spontaneous fracture and are unable to initiate fracture healing; combined, these mice offer an opportunity to examine the requirement for activated BMP signaling on the anabolic and fracture healing activity of Wnts. When Dkk1(+/-) mice were crossed with Bmp2(c/c);Prx1::cre mice, the offspring bearing both genetic alterations were unable to increase bone mass and heal fractures, indicating that increased canonical Wnt signaling is unable to exploit its activity in absence of Bmp2. Thus, our data suggest that BMP signaling is required for Wnt-mediated anabolic activity and that therapies aimed at preventing fractures and fostering fracture repair may need to target both pathways for maximal efficacy.
Copyright © 2015 Elsevier Inc. All rights reserved.
Underlying vascular disease is an important pathophysiologic factor shared among many co-morbid conditions associated with poor fracture healing, such as diabetes, obesity, and age. Determining the temporal and spatial patterns of revascularization following a fracture is essential for devising therapeutic strategies to augment this critical reparative process. Seminal studies conducted in the last century have investigated the pattern of vascularity in bone following a fracture. The consensus model culminating from these classical studies depicts a combination of angiogenesis emanating from both the intact intramedullary and periosteal vasculature. Subsequent to the plethora of experimental fracture angiography in the early to mid-20th century there has been a paucity of reports describing the pattern of revascularization of a healing fracture. Consequently the classical model of revascularization of a displaced fracture has remained largely unchanged. Here, we have overcome the limitations of animal fracture models performed in the above described classical studies by combining novel techniques of bone angiography and a reproducible murine femur fracture model to demonstrate for the first time the complete temporal and spatial pattern of revascularization in a displaced/stabilized fracture. These studies were designed specifically to i) validate the classical model of fracture revascularization of a displaced/stabilized fracture, ii) assess the association between intramedullary and periosteal angiogenesis and iii) elucidate the expression of VEGF/VEGF-R in relation to the classical model. From the studies, in conjunction with classic studies of angiogenesis during fracture repair, we propose a novel model (see abstract graphic) that defines the process of bone revascularization subsequent to injury to guide future approaches to enhance fracture healing. This new model validates and advances the classical model by providing evidence that during the process of revascularization of a displaced fracture 1) periosteal angiogenesis occurs in direct communication with the remaining intact intramedullary vasculature as a result of a vascular shunt and 2) vascular union occurs through an intricate interplay between intramembranous and endochondral VEGF/VEGF-R mediated angiogenesis.
Copyright © 2014 Elsevier Inc. All rights reserved.
BACKGROUND - Debate exists over the safety of rigid intramedullary nailing of femoral shaft fractures in skeletally immature patients. The goal of this study was to describe functional outcomes and complication rates of rigid intramedullary nailing in pediatric patients.
METHODS - A retrospective review was performed of femoral shaft fractures in skeletally immature patients treated with trochanteric rigid intramedullary nailing from 1987 to 2009. Radiographs made at initial injury, immediately postoperatively, and at the latest follow-up were reviewed. Patients were administered the Nonarthritic Hip Score and a survey.
RESULTS - The study population of 241 patients with 246 fractures was primarily male (75%) with a mean age of 12.9 years (range, eight to seventeen years). The majority of fractures were closed (92%) and associated injuries were common (45%). The mean operative time was 119 minutes, and the mean estimated blood loss was 202 mL. The mean clinical follow-up time was 16.2 months (range, three to seventy-nine months), and there were ninety-three patients with a minimum two-year clinical and radiographic follow-up. An increase of articulotrochanteric distance of >5 mm was noted in 15.1% (fourteen of ninety-three patients) at a minimum two-year follow-up; however, clinically relevant growth disturbance was only observed in two patients (2.2%) with the development of asymptomatic coxa valga. There was no femoral head osteonecrosis. Among the 246 fractures, twenty-four complications (9.8%) occurred. At the time of the latest follow-up, 1.7% (four of 241 patients) reported pain. The average Nonarthritic Hip Score was 92.4 points (range, 51 to 100 points), and 100% of patients reported satisfaction with their treatment.
CONCLUSIONS - Rigid intramedullary nailing is an effective technique for treatment of femoral shaft fractures in pediatric patients with an acceptable rate of complications.
LEVEL OF EVIDENCE - Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.
Copyright © 2014 by The Journal of Bone and Joint Surgery, Incorporated.
BACKGROUND - Flexible intramedullary nailing (IMN) has become a popular technique for the management of unstable or open forearm fractures. Recent publications have suggested an increased incidence of delayed union and poor outcomes in older children and adolescents. The objective of this study was to review forearm fractures treated with IMN, comparing the rate of complications and outcomes between the 2 age groups. Our hypothesis was that IMN is an effective technique with a similar rate of complications in both age groups.
METHODS - An Institutional Review Board-approved retrospective review was conducted of pediatric forearm fractures treated from 1998 to 2008 at a single institution. Over the study time period, 4161 pediatric forearm fractures were managed nonoperatively (92%) and 353 were treated operatively with plate, cross-pin, or intramedullary fixation (8%). Patients with inadequate follow-up, cross-pin, or plate fixation were excluded. Medical records were reviewed for indications and complications. Complications were graded with a modification of the Clavien-Dindo classification. Outcomes were judged by a new grading system.
RESULTS - A total of 205 forearm fractures treated with IMN in 203 patients were identified. The mean age was 9.7 years (range, 1.7 to 16.2 y) and mean follow-up was 42 weeks. Operative indications were failure of closed treatment in 165 (80%) and open fracture in 40 (20%). Mean time from injury to IMN was 5.9 days (range, 0 to 25 d). Single bone IMN was performed in 40 of 185 both bone fractures (26%); there were 20 single-bone forearm fractures treated with IMN. Open reduction was required in 61/165 (37%) of closed fractures. Asymptomatic delayed union (grade 1 complication) was observed in 9 fractures (4%). More severe complications were noted in 17% (grade 2 to 4 complications). Postoperative compartment syndrome occurred in 3 isolated forearm fractures with a significant younger mean age (6.0 vs. 10 y, P=0.031). Overall, complications were significantly more frequent in children older than 10 years of age (25/101) as compared with younger children (13/104, P=0.031). In particular, delayed union was more common in children over the age of 10 years (9/101 vs. 1/104, OR=9.99, P=0.009). Outcomes were good or excellent in 91% of fractures. There was no statistical association of patient age with a fair or poor outcome.
CONCLUSIONS - IMN is an effective technique for pediatric forearm fractures with good to excellent outcomes in 91%. Complications are not infrequent with this technique, with complications of grade 2 to 4 severity in 17%. There was a 2-fold increase in the rate of complications in children over the age of 10 years. Compartment syndrome was more common in younger children. Patients and families should be counseled about the risks preoperatively.