Nicholas A. Mignemi

Micro-computed tomography assessment of the progression of fracture healing in mice

The mouse fracture model is ideal for research into the pathways of healing because of the availability of genetic and transgenic mice and the ability to create cell-specific genetic mutations. While biomechanical tests and histology are available to assess callus integrity and tissue differentiation, respectively, micro-computed tomography (μCT) analysis has increasingly been utilized in fracture studies because it is non-destructive and provides descriptions of the structural and compositional properties of the callus. However, the dynamic changes of μCT properties that occur during healing are not well defined. Thus, the purpose of this study was to determine which μCT properties change with the progression of fracture repair and converge to values similar to unfractured bone in the mouse femur fracture model. A unilateral femur fracture was performed in C57BL/6 mice and intramedullary fixation performed. Fractured and un-fractured contralateral specimens were harvested from groups of mice between 2 and 12 weeks post-fracture. Parameters describing callus based on μCT were obtained, including polar moment of inertia (J), bending moment of inertia (I), total volume (TV), tissue mineral density (TMD), total bone volume fraction (BV/TV), and volumetric bone mineral density (vBMD). For comparison, plain radiographs were used to measure the callus diameter (D) and area (A); and biomechanical properties were evaluated using either three-point bending or torsion. The μCT parameters J, I, TV, and TMD converged toward their respective values of the un-fractured femurs over time, although significant differences existed between the two sides at every time point evaluated (p<0.05). Radiograph measurement D changed with repair progression in similar manner to TV. In contrast, BV/TV and BMD increased and decreased over time with statistical differences between callus and un-fractured bone occurring sporadically. Similarly, none of the biomechanical properties were found to distinguish consistently between the fractured and un-fractured femur. Micro-CT parameters assessing callus structure and size (J, I, and TV) were more sensitive to changes in callus over time post-fracture than those assessing callus substance (TMD, BV/TV, and BMD). Sample size estimates based on these results indicate that utilization of μCT requires fewer animals than biomechanics and thus is more practical for evaluating the healing femur in the mouse fracture model.

Fibrinolysis is essential for fracture repair and prevention of heterotopic ossification.

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.

Signal transduction pathway analysis in desmoid-type fibromatosis: transforming growth factor-β, COX2 and sex steroid receptors.

Despite reports of sex steroid receptor and COX2 expression in desmoid‐type fibromatosis, responses to single agent therapy with anti‐estrogens and non‐steroidal anti‐inflammatory drugs are unpredictable. Perhaps combination pharmacotherapy might be more effective in desmoid tumors that co‐express these targets. Clearly, further understanding of the signaling pathways deregulated in desmoid tumors is essential for the development of targeted molecular therapy. Transforming growth factor–β (TGFβ) and bone morphogenetic proteins (BMP) are important regulators of fibroblast proliferation and matrix deposition, but little is known about the TGFβ superfamily in fibromatosis. A tissue microarray representing 27 desmoid tumors was constructed; 14 samples of healing scar and six samples of normal fibrous tissue were included for comparison. Expression of selected receptors and activated downstream transcription factors of TGFβ family signaling pathways, β–catenin, sex steroid hormone receptors and COX2 were assessed using immunohistochemistry; patterns of co–expression were explored via correlational statistical analyses. In addition to β–catenin, immunoreactivity for phosphorylated SMAD2/3 (indicative of active TGFβ signaling) and COX2 was significantly increased in desmoid tumors compared with healing scar and quiescent fibrous tissue. Low levels of phosphorylated SMAD1/5/8 were detected in only a minority of cases. Transforming growth factor–β receptor type 1 and androgen receptor were expressed in both desmoid tumors and scar, but not in fibrous tissue. Estrogen receptor–β was present in all cases studied. Transforming growth factor–β signaling appears to be activated in desmoid‐type fibromatosis and phosphorylated SMAD2/3 and COX2 immunoreactivity might be of diagnostic utility in these tumors. Given the frequency of androgen receptor, estrogen receptor–β and COX2 co‐expression in desmoid tumors, further assessment of the efficacy of combination pharmacotherapy using hormonal agonists/antagonists together with COX2 inhibitors should be considered.

The temporal and spatial development of vascularity in a healing displaced fracture

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.

2010 Young Investigator Award winner: Therapeutic aprotinin stimulates osteoblast proliferation but inhibits differentiation and bone matrix mineralization.

Study Design. Analysis of the effect of antifibrinolytics on in vitro bone formation. Objective. As the direct effect of antifibrinolytics on bone formation is unknown, we examined whether antifibrinolytics routinely used in spine surgery, namely, aprotinin and aminocaproic acid, affect osteoblast function in vitro. Summary of Background Data. Antifibrinolytics are used in spine surgery to prevent intraoperative blood loss and decrease the need for transfusion. They are either delivered systemically or included as a component of most tissue sealants. Although the role of the fibrinolytic system in wound healing is well established, reports of indirect effects on normal bone biology are emerging. This suggests that the pharmacological targeting of this system may also influence skeletal mass and integrity. Methods. Osteoblast progenitor cells were cultured with therapeutic doses of aprotinin and aminocaproic acid. The effect of the antifibrinolytics on osteoblast development was determined by measuring cellular viability and proliferation, quantification of matrix mineralization, and genetic analysis of osteoblast differentiation markers. Protease inhibition profiles of the antifibrinolytics were determined by amidolytic chromogenic assays. Results. Therapeutic concentrations of aprotinin dose-dependently inhibited plasmins proteolytic activity, stimulated osteoblast proliferation, and inhibited osteoblast differentiation and matrix mineralization. Aprotinin inhibition of osteoblast differentiation and matrix mineralization could be recovered by removing aprotinin from culture or stimulating cells with bone morphogenetic protein-2 or plasmin. Conversely, aminocaproic acid inhibited plasmins proteolytic activity significantly less than aprotinin and had no effect on osteoblast proliferation, differentiation, or matrix mineralization in its therapeutic range. Conclusion. These findings demonstrate that the antifibrinolytics have drastically different effects on osteoblasts due in part to different efficacies of protease inhibition. Further, this work suggests that the fibrinolytic proteases and their inhibitors have great potential to regulate bone by affecting the processes that control osteoblast growth and differentiation.

Plasmin Prevents Dystrophic Calcification after Muscle Injury.

Extensive or persistent calcium phosphate deposition within soft tissues after severe traumatic injury or major orthopedic surgery can result in pain and loss of joint function. The pathophysiology of soft tissue calcification, including dystrophic calcification and heterotopic ossification (HO), is poorly understood; consequently, current treatments are suboptimal. Here, we show that plasmin protease activity prevents dystrophic calcification within injured skeletal muscle independent of its canonical fibrinolytic function. After muscle injury, dystrophic calcifications either can be resorbed during the process of tissue healing, persist, or become organized into mature bone (HO). Without sufficient plasmin activity, dystrophic calcifications persist after muscle injury and are sufficient to induce HO. Downregulating the primary inhibitor of plasmin (α2‐antiplasmin) or treating with pyrophosphate analogues prevents dystrophic calcification and subsequent HO in vivo. Because plasmin also supports bone homeostasis and fracture repair, increasing plasmin activity represents the first pharmacologic strategy to prevent soft tissue calcification without adversely affecting systemic bone physiology or concurrent muscle and bone regeneration.

Validation of a Radiography-Based Quantification Designed to Longitudinally Monitor Soft Tissue Calcification in Skeletal Muscle.

Introduction 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. Methods 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. Results 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: P = 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. Conclusions 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 in vivo μCT. Therefore, this high-throughput, longitudinal analytic method for quantifying soft tissue calcification is a viable alternative for the study of soft tissue calcification.

Fracture Healing in Protease-Activated Receptor-2 Deficient Mice

Protease‐activated receptor‐2 (PAR‐2) provides an important link between extracellular proteases and the cellular initiation of inflammatory responses. The effect of PAR‐2 on fracture healing is unknown. This study investigates the in vivo effect of PAR‐2 deletion on fracture healing by assessing differences between wild‐type (PAR‐2+/+) and knock‐out (PAR‐2−/−) mice. Unilateral mid‐shaft femur fractures were created in 34 PAR‐2+/+ and 28 PAR‐2−/− mice after intramedullary fixation. Histologic assessments were made at 1, 2, and 4 weeks post‐fracture (wpf), and radiographic (plain radiographs, micro‐computed tomography (µCT)) and biomechanical (torsion testing) assessments were made at 7 and 10 wpf. Both the fractured and un‐fractured contralateral femur specimens were evaluated. Polar moment of inertia (pMOI), tissue mineral density (TMD), bone volume fraction (BV/TV) were determined from µCT images, and callus diameter was determined from plain radiographs. Statistically significant differences in callus morphology as assessed by µCT were found between PAR‐2−/− and PAR‐2+/+ mice at both 7 and 10 wpf. However, no significant histologic, plain radiographic, or biomechanical differences were found between the genotypes. The loss of PAR‐2 was found to alter callus morphology as assessed by µCT but was not found to otherwise effect fracture healing in young mice.

Abstract P20: Severe Injury Leads to Plasmin Consumption Below a Critical Threshold Required to Heal Soft Tissue Injury

PURPOSE: Pannexin 3 (Panx3) is a gap junction protein. We have previously shown that Panx3 plays multiple channel functions: 1) as a hemichannel to regulate intracellular ATP/cAMP levels between cells and the extracellular space, 2) as an ER calcium channel to regulate calcium flux within the cell, and 3) a gap junction to exchange ions and small molecules between cells. However, the role of Panx3 in skin tissue regeneration, proliferation, and/or differentiation is unclear. Here, we demonstrate that Panx3 plays a role in the skin wound healing process by controlling the inflammatory response, epidermal-mesenchymal transition (EMT), keratinocyte proliferation, and collagen deposition.

The hyperglycemic byproduct methylglyoxal impairs anticoagulant activity through covalent adduction of antithrombin III.

INTRODUCTION The blood coagulation system is a tightly regulated balance of procoagulant and anticoagulant factors, disruption of which can cause clinical complications. Diabetics are known to have a hypercoagulable phenotype, along with increased circulating levels of methylglyoxal (MGO) and decreased activity of the anticoagulant plasma protein antithrombin III (ATIII). MGO has been shown to inhibit ATIII activity in vitro, however the mechanism of inhibition is incompletely understood. As such, we designed this study to investigate the kinetics and mechanism of MGO-mediated ATIII inhibition. METHODS MGO-mediated ATIII inhibition was confirmed using inverse experiments detecting activity of the ATIII targets thrombin and factor Xa. Fluorogenic assays were performed in both PBS and plasma after incubation of ATIII with MGO, at molar ratios comparable to those observed in the plasma of diabetic patients. LC-coupled tandem mass spectrometry was utilized to investigate the exact mechanism of MGO-mediated ATIII inhibition. RESULTS AND CONCLUSIONS MGO concentration-dependently attenuated inhibition of thrombin and factor Xa by ATIII in PBS-based assays, both in the presence and absence of heparin. In addition, MGO concentration-dependently inhibited ATIII activity in a plasma-based system, to the level of plasma completely deficient in ATIII, again both in the presence and absence of heparin. Results from LC-MS/MS experiments revealed that MGO covalently adducts the active site Arg 393 of ATIII through two distinct glyoxalation mechanisms. We posit that active site adduction is the mechanism of MGO-mediated inhibition of ATIII, and thus contributes to the underlying pathophysiology of the diabetic hypercoagulable state and complications thereof.