Josh B. Parker

Systemic Correction of Storage Disease in MPS I NOD/SCID Mice Using the Sleeping Beauty Transposon System

The Sleeping Beauty (SB) transposon system is a nonviral vector that directs transgene integration into vertebrate genomes. We hydrodynamically delivered SB transposon plasmids encoding human alpha-L-iduronidase (hIDUA) at two DNA doses, with and without an SB transposase gene, to NOD.129(B6)-Prkdc(scid) IDUA(tm1Clk)/J mice. In transposon-treated, nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with mucopolysaccharidosis type I (MPS I), plasma IDUA persisted for 18 weeks at levels up to several hundred-fold wild-type (WT) activity, depending on DNA dose and gender. IDUA activity was present in all examined somatic organs, as well as in the brain, and correlated with both glycosaminoglycan (GAG) reduction in these organs and level of expression in the liver, the target of transposon delivery. IDUA activity was higher in the treated males than in females. In females, omission of transposase source resulted in significantly lower IDUA levels and incomplete GAG reduction in some organs, confirming the positive effect of transposition on long-term IDUA expression and correction of the disease. The SB transposon system proved efficacious in correcting several clinical manifestations of MPS I in mice, including thickening of the zygomatic arch, hepatomegaly, and accumulation of foamy macrophages in bone marrow and synovium, implying potential effectiveness of this approach in treatment of human MPS I.

A Comparison Between a Retrograde Interference Screw, Suture Button, and Combined Fixation on the Tibial Side in an All-Inside Anterior Cruciate Ligament Reconstruction A Biomechanical Study in a Porcine Model

Background Effective soft tissue graft fixation to the tibial tunnel in all-inside anterior cruciate ligament reconstructions has been reported to be a problem and may lead to retrograde pullout at ultimate load testing. Hypothesis A combined retrograde bioabsorbable screw and cortical-cancellous suture button suspension apparatus would gain stiffness from the button and strength from the screw, thus providing for a larger pullout ultimate load, yield load, and stiffness when compared with either fixation alone in an all-inside anterior cruciate ligament reconstruction. Study Design Controlled laboratory study. Methods Eighteen porcine tibias (average bone mineral density of 1.46, measured by dual-energy x-ray absorptiometry scan) and 18 bovine extensor tendon allografts were divided into 3 groups retrograde bioabsorbable screw fixation, cortical-cancellous suture button suspension apparatus fixation, and combined fixation in the tibia, with 6 specimens per group. They were biomechanically tested with cyclic (500 cycles, 50-250 N, 1 Hz) and load-to-failure (20 mm/min) parameters. Results During cyclic testing, the retrograde screw—only group had a larger cyclic displacement (2.98 ± 2.28 mm) than the suture button with retrograde screw combination group (1.40 ± 0.34 mm). The combination fixation group also produced a higher cyclic stiffness (161.93 ± 61.81 N/mm) than the retrograde screw—only group (91.59 ± 43.26 N/mm). In load-to-failure testing, the retrograde screw with suture button combination group withstood significantly higher initial failure forces (873.87 ± 148.74 N) than the retrograde screw—only (558.44 ± 126.33 N) and suture button—only (121.76 ± 40.57 N) groups. Additionally, ultimate loads were also significantly higher for the combination group (1027 ± 157.11 N) than either the retrograde screw group (679.00 ± 109.44 N) or the suture button group (161.00 ± 29.27 N). The retrograde screw with suture button combination group showed significantly higher pullout stiffness (152.50 ± 46.37 N/mm) than either the retrograde screw—only group (78.31 ± 12.85 N/mm) or the suture button—only group (25.79 ± 9.30 N/mm). Conclusion Soft tissue grafts fixed with a combination of a retrograde screw and a suture button were able to withstand higher initial failure and ultimate failure loads and were also stiffer than grafts fixed with either a retrograde screw or a suture button alone. Clinical Relevance These findings may prove useful in providing additional stability when using an all-inside technique in a difficult case, or in a patient with poor bone stock, and may also be useful as an alternative to more commonly used tibial tunnel soft tissue fixation techniques.

Inhibition of angiogenesis and suppression of colorectal cancer metastatic to the liver using the Sleeping Beauty Transposon System

BackgroundMetastatic colon cancer is one of the leading causes of cancer-related death worldwide, with disease progression and metastatic spread being closely associated with angiogenesis. We investigated whether an antiangiogenic gene transfer approach using the Sleeping Beauty (SB) transposon system could be used to inhibit growth of colorectal tumors metastatic to the liver.ResultsLiver CT26 tumor-bearing mice were hydrodynamically injected with different doses of a plasmid containing a transposon encoding an angiostatin-endostatin fusion gene (Statin AE) along with varying amounts of SB transposase-encoding plasmid. Animals that were injected with a low dose (10 μg) of Statin AE transposon plasmid showed a significant decrease in tumor formation only when co-injected with SB transposase-encoding plasmid, while for animals injected with a higher dose (25 μg) of Statin AE transposon, co-injection of SB transposase-encoding plasmid did not significantly affect tumor load. For animals injected with 10 μg Statin AE transposon plasmid, the number of tumor nodules was inversely proportional to the amount of co-injected SB plasmid. Suppression of metastases was further evident in histological analyses, in which untreated animals showed higher levels of tumor cell proliferation and tumor vascularization than animals treated with low dose transposon plasmid.ConclusionThese results demonstrate that hepatic colorectal metastases can be reduced using antiangiogenic transposons, and provide evidence for the importance of the transposition process in mediating suppression of these tumors.

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.

The 1:1 Versus the 2:2 Tunnel-Drilling Technique Optimization of Fixation Strength and Stiffness in an All-Inside Double-Bundle Anterior Cruciate Ligament Reconstruction—a Biomechanical Study

Background Double-bundle anterior cruciate ligament (ACL) reconstructions involve drilling 2 tibial tunnels separated by a narrow 2-mm bone bridge. The sequence of reaming and drilling the tibial tunnels for double-bundle ACL reconstructions has not been defined. Hypothesis Fixing a graft in the posterolateral ACL tibial tunnel before reaming the anteromedial tibial tunnel will reduce the number of complications, as compared with drilling both the anteromedial and posterolateral tunnels before graft fixation, when performing double-bundle ACL reconstructions. Study Design Controlled laboratory study. Methods Twelve porcine tibias were divided into 2 groups of 6 specimens. Fresh bovine extensor tendons grafts were fixed in 7-mm tunnels reamed using an inside-out method. Grafts were fixed in a retrograde fashion with 7-mm bioabsorbable retrograde screws. The tibias in group 1 were reconstructed by reaming and reconstructing the posterolateral tunnel before reaming and securing the graft for the anteromedial tunnel (ie, 1:1 method), whereas those in the second group were reconstructed by reaming both tunnels before graft fixation in either (ie, the 2:2 method). The specimens were biomechanically tested with cyclic and load-to-failure parameters. Results Cyclic testing revealed no significant difference between the 2 methods in displacement or stiffness. In load-to-failure testing, the 1:1 group withstood significantly higher initial failure loads and ultimate loads. Pullout displacement was significantly higher for the 1:1 group. Whereas no tibias in the 1:1 group sustained fractures, 4 from the 2:2 group demonstrated a bone bridge fracture. Conclusion Soft tissue ACL grafts fixed in the tibia with the 1:1 method withstood significantly higher initial and ultimate failure loads and were stiffer than the grafts fixed with the 2:2 method. Tibias fixed with the 1:1 method were also less susceptible to bone bridge fracture. Clinical Relevance The potential for a lower complication rate and greater pullout strength seen with the 1:1 method may prove useful to surgeons performing anatomic double-bundle ACL reconstructions, in addition to other procedures involving reconstructing 2 closely positioned tunnels, including anatomic posterolateral corner and medial collateral reconstructions.

Bacterial diskospondylitis associated with posterior paresis/paralysis in North American farmed mink (Mustela vison)

Posterior paresis/paralysis in farmed mink is responsible for significant morbidity and mortality, with individual farms reporting the loss of as many as 700 animals each year. Although this disease has been recognized by North American mink farmers for approximately 40 years, there are few published reports focusing on this entity. The objective of this study was to investigate the etiology and pathogenesis of the disease. Complete necropsy examinations were done on 40 clinically affected mink, ranging from 7 to 10 weeks of age, and on three normal animals in the same age range from two mink farms. Thirty-two of the 40 clinically affected animals had an isolated vertebral lesion characterized by bone lysis and proliferation that usually was centered on an intervertebral disk space in the midthoracic area. An inflammatory reaction, composed primarily of neutrophils, was present within the vertebral sections in 25 of the 40 affected animals (62.5%), and the presence of gram-positive cocci was confirmed in 8 of 10 animals (80%) in which bacterial organisms were observed histologically. Bacterial cultures from 15 affected animals yielded Streptococcus sp. from the intervertebral disk space in 13 of 15 (86.7%) animals and from heart blood in 6 of 8 (75%). A farm visit revealed no history or evidence of traumatic wounds as a source of infection in these animals, and the diet appeared to be adequate for skeletal development. We conclude that posterior paresis/paralysis in farmed mink is associated with bacterial diskospondylitis, likely occurring secondary to bacteremia/septicemia.

Tissue mineral concentrations and osteochondrosis lesions in prolific sows across parities 0 through 7.

Mortality in swine herds is often associated with lameness, and trace minerals are implicated in maintaining integrity of skeletal tissues. The objectives of this study were to determine if prolific sows displayed evidence of trace mineral depletion with age and to determine the prevalence of osteochondrosis (OC) lesions. Reduced mineral concentrations with age would support recommendations for an increase in the amount of dietary minerals. Tissue samples were collected from 66 sows selected to represent a cross-sectional profile of a prolific herd fed diets with inorganic sources of trace minerals fortified at concentrations typically found in commercial diets. Females ranged from nulliparous (parity 0) to parity 7 with a lifetime average of 12.9 ± 0.5 pigs born alive per litter. Minerals were assessed in humerus, scapula, ovary, liver, and muscle (psoas major) tissues. Percent bone ash increased (P < 0.05) with parity from 64 to 66% but differed among bone sections. The Ca (39.0%) and P (18.9%) concentrations in bone ash were essentially constant in all sections and parities. Bone Cu, Fe, Mn, and Zn concentrations varied among sections, but differences due to parity (P < 0.05) were only detected in Fe. Bone Fe decreased from approximately 49 μg/g ash in parity 0 and 1 sows to approximately 29 μg/g ash in parity 7, likely reflecting loss of hemopoietic tissue with age. No evidence was detected in liver for depletion of trace minerals across parity; however, liver Cu and Zn concentrations tended to increase with age. Liver Mn concentrations varied with parity, but no consistent trend with parity was evident. Ovary Cu and Mn concentrations varied dramatically as a function of the reproductive status, but no evidence was detected for depletion with parity. Articular surfaces of the distal scapula and proximal and distal humerus were evaluated grossly for prevalence of OC; bones were then sectioned to evaluate lesions in subchondral bone and physis. Incidence of OC lesions on the articular-epiphyseal cartilage complex varied among bone sites, but differences across parities were not detected. In a subset of sows with subchondral bone lesions, the lesions appeared severe enough to contribute to clinical lameness, particularly in the distal humerus site. However, none of the sows exhibited lameness at slaughter. As no reductions in mineral concentrations with age were detected, recommendations to increase dietary mineral supplementation with age were not supported.

Overexpression of copper/zinc superoxide dismutase in the median preoptic nucleus improves cardiac function after myocardial infarction in the rat

Previous reports indicate that overexpression of copper/zinc superoxide dismutase (CuZnSOD), an intracellular superoxide (O2•−) scavenging enzyme, in the brain subfornical organ improves cardiac function in a mouse model of heart failure (HF). A downstream hypothalamic site, the MnPO, may act as a relay centre for O2•− to serve as a mediator in the pathophysiology of HF. To test the hypothesis that elevated O2•− in the MnPO contributes to the pathophysiology of HF and decreased cardiac function, we injected adenovirus encoding CuZnSOD (AdCuZnSOD, n=7) or control empty adenovirus vector (AdEmpty, n=7) into the MnPO of normal rats. Subsequently, rats were subjected to coronary artery ligation to create a myocardial infarct (MI) of the left ventricle. Cardiac function was monitored via echocardiography. Upon completion, rat brains were examined for CuZnSOD expression in MnPO via immunofluorescence and histopathological analyses of cardiac infarct size were conducted. Baseline (EF) ejection fractions (%) of AdCuZnSOD and AdEmpty rats were 73 ± 1 and 71 ± 1, respectively. Two weeks after MI, EF was significantly decreased in both groups of rats (AdCuZnSOD: 51 ± 3, AdEmpty: 46 ± 1). In contrast, by 4 weeks post MI, EF had improved to 64 ± 2 in AdCuZnSOD rats, yet was only 52 ± 1 in AdEmpty rats, and this was accompanied by lower plasma noradrenaline levels in AdCuZnSOD rats (0.49 ± 0.19 ng/mL) compared to AdEmpty rats (1.20 ± 0.32 ng/mL). In conclusion, despite decreases in EF early after MI, overexpression of CuZnSOD in the MnPO was related to an improvement in left ventricular function and concomitant decreased plasma noradrenaline levels 4 weeks post MI.

Bone Disease in the Common Marmoset Radiographic and Histological Findings

The common marmoset (Callithrix jacchus) is a New World primate that is used in biomedical research due to its small size and relative ease of handling compared with larger primates. Although bone disease in common marmosets is well recognized, there are very few detailed descriptions in the literature that cover the range of lesions seen in these animals. For all animals used to model human disease, it is important to be aware of background lesions that may affect the interpretation of study findings. This retrospective study details bone diseases encountered in marmoset breeding colonies at 2 different institutions. Affected marmosets at Johns Hopkins University had lesions compatible with diagnoses of rickets, fibrous osteodystrophy and osteopenia. Affected marmosets at the Wisconsin National Primate Research Center exhibited severe lesions of osteoclastic bone resorption and remodeling that had an unusual distribution and were not easily categorized into a known disease entity. The purpose of this report is to document these naturally occurring skeletal lesions of common marmosets and suggest an approach to evaluating skeletal disease in prospective studies of these animals that will allow the most accurate diagnoses.

Effect of intra-articular pressurization associated with arthroscopy on the immature physis: investigation in an animal model.

Background Despite the increased use of arthroscopy in pediatric orthopaedics, there is a paucity of data regarding the potential long-term effects of this procedure on the immature physis. The purpose of this study was to test the hypothesis that elevated intra-articular pressures used during arthroscopic surgery do not result in growth disturbances or morphologic alterations in the epiphyseal plate. Methods Twenty-seven 6-week-old skeletally immature New Zealand white rabbits were divided into experimental (n=21) and control groups (n=6). In the experimental group, a hydraulic pump was used to pressurize 1 randomly assigned knee joint per rabbit to intra-articular pressures of 120 mm Hg for 2 hours. In the control group, rabbits received a sham intervention. All rabbits were killed at 6 months of age (skeletal maturity), and their tissues were evaluated grossly, radiographically, and histologically. Data collection included gross measurements (femur and tibia lengths, evaluation of varus/valgus angulation, and knee joint range of motion) and histologic analyses to determine whether morphologic changes were present in the articular cartilage or physis. Confidence intervals were used to test for statistical equivalence. Results The pressurized and control groups had statistically equivalent gross measurements. No significant articular cartilage or physeal lesions were identified in histologic sections or radiographic studies. Conclusion This study provided no evidence that arthroscopic pressurization of the knee joint to 120 mm Hg for 2 hours significantly affected physeal growth in a skeletally immature rabbit model. Clinical Relevance This study provides the first direct evidence that arthroscopic pressurization of immature joints has no clinically significant adverse long-term effects. Therefore, novel uses of arthroscopy in pediatric patients should be explored without undue concern with regard to premature physeal closure.