Margaret A. McNulty

Disease progression and phasic changes in gene expression in a mouse model of osteoarthritis.

Osteoarthritis (OA) is the most common form of arthritis and has multiple risk factors including joint injury. The purpose of this study was to characterize the histologic development of OA in a mouse model where OA is induced by destabilization of the medial meniscus (DMM model) and to identify genes regulated during different stages of the disease, using RNA isolated from the joint “organ” and analyzed using microarrays. Histologic changes seen in OA, including articular cartilage lesions and osteophytes, were present in the medial tibial plateaus of the DMM knees beginning at the earliest (2 week) time point and became progressively more severe by 16 weeks. 427 probe sets (371 genes) from the microarrays passed consistency and significance filters. There was an initial up-regulation at 2 and 4 weeks of genes involved in morphogenesis, differentiation, and development, including growth factor and matrix genes, as well as transcription factors including Atf2, Creb3l1, and Erg. Most genes were off or down-regulated at 8 weeks with the most highly down-regulated genes involved in cell division and the cytoskeleton. Gene expression increased at 16 weeks, in particular extracellular matrix genes including Prelp, Col3a1 and fibromodulin. Immunostaining revealed the presence of these three proteins in cartilage and soft tissues including ligaments as well as in the fibrocartilage covering osteophytes. The results support a phasic development of OA with early matrix remodeling and transcriptional activity followed by a more quiescent period that is not maintained. This implies that the response to an OA intervention will depend on the timing of the intervention. The quiescent period at 8 weeks may be due to the maturation of the osteophytes which are thought to temporarily stabilize the joint.

Histopathology of naturally occurring and surgically induced osteoarthritis in mice.

OBJECTIVE The morphology of lesions in mouse models of osteoarthritis (OA) has not been comprehensively characterized, in part because current histological assessments of OA focus primarily on articular cartilage (AC). In the present study, sections of murine stifle joints with naturally occurring (aged animals) and surgically induced (destabilized medial meniscus, DMM) OA were examined using a newly developed histological grading scheme that includes quantitative measurements and semiquantitative grades to evaluate multiple joint tissues. DESIGN The data collected was analyzed using Principal Components Analysis (PCA); factor scores for each joint were generated. Individual parameters and factor scores were compared between surgical groups and among age groups. For comparison, the original Mankin Histological-Histochemical Grading System (HHGS) also was applied. RESULTS Overall, lesions were most severe in the medial tibial plateaus. Significant changes in AC and neighboring bone were identified in surgically induced models and in naturally occurring disease. Mean factor scores provided a comprehensive evaluation of joint changes. An important new finding was that chondrocyte cell death within the AC was a commonly identified lesion and its extent significantly increased with age. While the Mankin HHGS detected significant overall differences in OA severity between surgical groups, it was not sensitive in detecting age-related differences, nor did it provide information regarding changes in individual tissues. CONCLUSION These results demonstrate the utility of this newly developed murine OA grading scheme in identifying lesions in AC and in other joint tissues. Surgically induced changes were similar to those occurring naturally with aging.

Sclerostin Antibody Increases Bone Volume and Enhances Implant Fixation in a Rat Model

BACKGROUND Previous studies have demonstrated that sclerostin blockade is anabolic for bone. This study examined whether systemic administration of sclerostin antibody would increase implant fixation and peri-implant bone volume in a rat model. METHODS Titanium cylinders were placed in the femoral medullary canal of ninety male Sprague-Dawley rats. One-half of the rats (n=45) received murine sclerostin antibody (Scl-Ab, 25 mg/kg, twice weekly) and the other one-half (n=45) received saline solution. Equal numbers of rats from both groups were sacrificed at two, four, or eight weeks after the implant surgery and the femora were examined by microcomputed tomography, mechanical pull-out testing, and histology. RESULTS Fixation strength in the two groups was similar at two weeks but was 1.9-fold greater at four weeks (p=0.024) and 2.2-fold greater at eight weeks (p<0.001) in the rats treated with sclerostin antibody. At two weeks, antibody treatment led to increased cortical area, with later increases in cortical thickness and total cross-sectional area. Significant differences in peri-implant trabecular bone were not evident until eight weeks but included increased bone volume per total volume, bone structure that was more plate-like, and increased trabecular thickness and number. Changes in bone architecture in the intact contralateral femur tended to precede the peri-implant changes. The peri-implant bone properties accounted for 61% of the variance in implant fixation strength, 32% of the variance in stiffness, and 63% of the variance in energy to failure. The implant fixation strength at four weeks was approximately equivalent to the strength in the control group at eight weeks. CONCLUSIONS Sclerostin antibody treatment accelerated and enhanced mechanical fixation of medullary implants in a rat model by increasing both cortical and trabecular bone volume.

A comprehensive histological assessment of osteoarthritis lesions in mice

Objective: Accurate histological assessment of osteoarthritis (OA) is critical in studies evaluating the effects of interventions on disease severity. The purpose of the present study was to develop a histological grading scheme that comprehensively and quantitatively assesses changes in multiple tissues that are associated with OA of the stifle joint in mice. Design: Two representative midcoronal sections from 158 stifle joints, including naturally occurring and surgically induced OA, were stained with H&E and Safranin-O stains. All slides were evaluated to characterize the changes present. A grading scheme that includes both measurements and semiquantitative scores was developed, and principal components analysis (PCA) was applied to the resulting data from the medial tibial plateaus. A subset of 30 tibial plateaus representing a wide range of severity was then evaluated by 4 observers. Reliability of the results was evaluated using intraclass correlation coefficients (ICCs) and area under the receiver operating characteristic (ROC) curve. Results: Five factors were retained by PCA, accounting for 74% of the total variance. Interobserver and intraobserver reproducibilities for evaluations of articular cartilage and subchondral bone were acceptable. The articular cartilage integrity and chondrocyte viability factor scores were able to distinguish severe OA from normal, minimal, mild, and moderate disease. Conclusion: This newly developed grading scheme and resulting factors characterize a range of joint changes in mouse stifle joints that are associated with OA. Overall, the newly developed scheme is reliable and reproducible, characterizes changes in multiple tissues, and provides comprehensive information regarding a specific site in the stifle joint.

Sclerostin Antibody Treatment Improves Implant Fixation in a Model of Severe Osteoporosis

BACKGROUND The mechanical fixation of orthopaedic and dental implants is compromised by diminished bone volume, such as with osteoporosis. Systemic administration of sclerostin antibody (Scl-Ab) has been shown to enhance implant fixation in normal animals. In the present study, we tested whether Scl-Ab can improve implant fixation in established osteoporosis in a rat model. METHODS We used an ovariectomized (ovx) rat model, in which we found a 78% decrease in trabecular bone volume at the time of implant surgery; sham-ovx, age-matched rats were used as controls. After placement of a titanium implant in the medullary cavity of the distal aspect of the femur, the rats were maintained for four, eight, or twelve weeks and treated biweekly with Scl-Ab or with the delivery vehicle alone. Outcomes were measured with use of microcomputed tomography, mechanical testing, and static and dynamic histomorphometry. RESULTS Scl-Ab treatment doubled implant fixation strength in both the sham-ovx and ovx groups, although the enhancement was delayed in the ovx group. Scl-Ab treatment also enhanced bone-implant contact; increased peri-implant trabecular thickness and volume; and increased cortical thickness. These structural changes were associated with an approximately five to sevenfold increase in the bone-formation rate and a >50% depression in the eroded surface following Scl-Ab treatment. Trabecular bone thickness and bone-implant contact accounted for two-thirds of the variance in fixation strength. CONCLUSIONS In this model of severe osteoporosis, Scl-Ab treatment enhanced implant fixation by stimulating bone formation and suppressing bone resorption, leading to enhanced bone-implant contact and improved trabecular bone volume and architecture. CLINICAL RELEVANCE Systemic administration of anti-sclerostin antibodies might be a useful strategy in total joint replacement when bone mass is deficient.

A Comparative Analysis of 7.0-Tesla Magnetic Resonance Imaging and Histology Measurements of Knee Articular Cartilage in a Canine Posterolateral Knee Injury Model A Preliminary Analysis

Background There has recently been increased interest in the use of 7.0-T magnetic resonance imaging for evaluating articular cartilage degeneration and quantifying the progression of osteoarthritis. Purpose The purpose of this study was to evaluate articular cartilage cross-sectional area and maximum thickness in the medial compartment of intact and destabilized canine knees using 7.0-T magnetic resonance images and compare these results with those obtained from the corresponding histologic sections. Study Design Controlled laboratory study. Methods Five canines had a surgically created unilateral grade III posterolateral knee injury that was followed for 6 months before euthanasia. The opposite, noninjured knee was used as a control. At necropsy, 3-dimensional gradient echo images of the medial tibial plateau of both knees were obtained using a 7.0-T magnetic resonance imaging scanner. Articular cartilage area and maximum thickness in this site were digitally measured on the magnetic resonance images. The proximal tibias were processed for routine histologic analysis with hematoxylin and eosin staining. Articular cartilage area and maximum thickness were measured in histologic sections corresponding to the sites of the magnetic resonance slices. Results The magnetic resonance imaging results revealed an increase in articular cartilage area and maximum thickness in surgical knees compared with control knees in all specimens; these changes were significant for both parameters (P <.05 for area; P<.01 for thickness). The average increase in area was 14.8% and the average increase in maximum thickness was 15.1%. The histologic results revealed an average increase in area of 27.4% (P <.05) and an average increase in maximum thickness of 33.0% (P 5 .06). Correlation analysis between the magnetic resonance imaging and histology data revealed that the area values were significantly correlated (P <.01), but the values for thickness obtained from magnetic resonance imaging were not significantly different from the histology sections (P > 1). Conclusion These results demonstrate that 7.0-T magnetic resonance imaging provides an alternative method to histology to evaluate early osteoarthritic changes in articular cartilage in a canine model by detecting increases in articular cartilage area. Clinical Relevance The noninvasive nature of 7.0-T magnetic resonance imaging will allow for in vivo monitoring of osteoarthritis progression and intervention in animal models and humans for osteoarthritis.

Novel Lesions of Bones and Joints Associated with Chikungunya Virus Infection in Two Mouse Models of Disease: New Insights into Disease Pathogenesis

Chikungunya virus is an arbovirus spread predominantly by Aedes aegypti and Ae. albopictus mosquitoes, and causes debilitating arthralgia and arthritis. While these are common manifestations during acute infection and it has been suggested they can recur in patients chronically, gaps in knowledge regarding the pathogenesis still exist. Two established mouse models were utilized (adult IRF 3/7 -/- -/- and wild-type C57BL/6J mice) to evaluate disease manifestations in bones and joints at various timepoints. Novel lesions in C57BL/6J mice consisted of periostitis (91%) and foci of cartilage of necrosis (50% of mice at 21 DPI). Additionally, at 21 DPI, 50% and 75% of mice exhibited periosteal bone proliferation affecting the metatarsal bones, apparent via histology and μCT, respectively. μCT analysis did not reveal any alterations in trabecular bone volume measurements in C57BL/6J mice. Novel lesions demonstrated in IRF 3/7 -/- -/- mice at 5 DPI included focal regions of cartilage necrosis (20%), periosteal necrosis (66%), and multifocal ischemic bone marrow necrosis (100%). Contralateral feet in 100% of mice of both strains had similar, though milder lesions. Additionally, comparison of control IRF 3/7 -/- -/- and wild-type C57BL/6J mice demonstrated differences in cortical bone. These experiments demonstrate novel manifestations of disease similar to those occurring in humans, adding insight into disease pathogenesis, and representing new potential targets for therapeutic interventions. Additionally, results demonstrate the utility of μCT in studies of bone and joint pathology and illustrate differences in bone dynamics between mouse strains.

Implant placement increases bone remodeling transiently in a rat model.

To examine bone remodeling following implant placement, 88 female Sprague–Dawley rats underwent either sham ovariectomy (sham‐ovx) or ovariectomy (ovx) at 4.5 months. At 11 months, 17 baseline control animals were euthanized, while 71 rats received bilateral intramedullary femoral implants. Implanted rats were randomized to 4‐, 8‐, or 12‐week follow‐up times. Microcomputed tomography was used to assess cortical area and trabecular architecture in all rats. Dynamic and static histomorphometry were performed in a subset to examine the trabecular and endocortical bone in the distal femoral metaphysis adjacent to the implant and the periosteal surface at the midshaft superior to the implant (n = 59). Implantation did not affect bone volume in either sham‐ovx or ovx rats compared to baseline controls. Implant placement significantly increased mineralizing surface, mineral apposition rate, and bone formation rate in both sham‐ovx and ovx rats at the trabecular and endocortical surfaces at four and sometimes 8 weeks, with a return to baseline values by 12 weeks. At the periosteal surface, implant placement increased bone formation at 4 weeks with a return to baseline levels by 8 weeks. Thus, implant placement increases bone remodeling transiently without affecting bone volume in sham‐ovx and ovx rats.

An anatomy precourse enhances student learning in veterinary anatomy

Veterinary anatomy is often a source of trepidation for many students. Currently professional veterinary programs, similar to medical curricula, within the United States have no admission requirements for anatomy as a prerequisite course. The purpose of the current study was to evaluate the impact of a week‐long precourse in veterinary anatomy on both objective student performance and subjective student perceptions of the precourse educational methods. Incoming first year veterinary students in the Louisiana State University School of Veterinary Medicine professional curriculum were asked to participate in a free precourse before the start of the semester, covering the musculoskeletal structures of the canine thoracic limb. Students learned the material either via dissection only, instructor‐led demonstrations only, or a combination of both techniques. Outcome measures included student performance on examinations throughout the first anatomy course of the professional curriculum as compared with those who did not participate in the precourse. This study found that those who participated in the precourse did significantly better on examinations within the professional anatomy course compared with those who did not participate. Notably, this significant improvement was also identified on the examination where both groups were exposed to the material for the first time together, indicating that exposure to a small portion of veterinary anatomy can impact learning of anatomical structures beyond the immediate scope of the material previously learned. Subjective data evaluation indicated that the precourse was well received and students preferred guided learning via demonstrations in addition to dissection as opposed to either method alone. Anat Sci Educ 9: 344–356.

Reduced Osteoarthritis Severity in Aged Mice With Deletion of Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that is elevated in the serum and synovial fluid of patients with osteoarthritis (OA). This study was undertaken to investigate the potential role of MIF in OA in human joint tissues and in vivo in mice with age‐related and surgically induced OA.