Robert W. Norrdin

Subchondral Bone Failure in an Equine Model of Overload Arthrosis

Gross examination of metacarpo-/metatarsophalangeal (fetlock) joints from racehorses revealed defects on the condylar surface that ranged from cartilage fibrillation and erosion to focal cartilage indentations and cavitation in subchondral bone characteristic of traumatic osteochondrosis. Because these lesions represented a spectrum of mechanically induced arthrosis in which microdamage is thought to play a role, a histologic study of sagittal sections was made to study the morphogenesis. Subchondral bone failure developed beneath a flattened section of the condyle where the margin of the sesamoid bone produces compression as well as shear on impact of the foot with the ground. Milder lesions had thickening of subchondral bone and underlying trabeculae. With advancing sclerosis an increased amount of osteocyte necrosis was present. Occasional vascular channels with plugs of matrix debris and cells were present just beneath the cartilage. There was increased prominence of subchondral vessels, and osteoclastic remodeling was seen in and around the sclerotic zone. Apparent fragmentation lines in the subchondral bone suggested increased matrix fragility. Irregular trabecular microfractures developed at a depth of a few millimeters. Increased vascularity with hemorrhage, fibrin, and fibroplasia could be seen in enlarged marrow spaces at this more advanced stage. The overlying articular cartilage was variably indented but remained largely viable with degeneration and erosion limited to the superficial layers. Focally, breaks in the calcified layer appeared to lead to collapse and cartilage infolding. In metacarpal condyles from experimental horses run on a treadmill, there were milder changes at the site. The subchondral bone was increased in volume and there was increased diffuse staining with basic fuchsin, but no increase in the number of microcracks was seen. The findings in the racehorses indicate that the equine fetlock condyle is a consistent site of overload arthrosis in which microfracture and failure in subchondral bone may occur. Controlled exercise in treadmill horses may provide a model in which to study the pathogenesis.

Early Increase in Osteoclast Number in Mice after Whole-Body Irradiation with 2 Gy X Rays

Abstract Willey, J. S., Lloyd, S. A. J., Robbins, M. E., Bourland, J. D., Smith-Sielicki, H., Bowman, L. C., Norrdin, R. W. and Bateman, T. A. Early Increase in Osteoclast Number in Mice after Whole-Body Irradiation with 2 Gy X Rays. Radiat. Res. 170, 388–392 (2008). Bone loss is a consequence of exposure to high-dose radiotherapy. While damage to bone vasculature and reduced proliferation of bone-forming osteoblasts has been implicated in this process, the effect of radiation on the number and activity of bone-resorbing osteoclasts has not been characterized. In this study, we exposed mice to a whole-body dose of 2 Gy of X rays to quantify the early effects of radiation on osteoclasts and bone structural properties. Female C57BL/6 mice (13 weeks old) were divided into two groups: irradiated and nonirradiated controls. Animals were killed humanely 3 days after radiation exposure. Analysis of serum chemistry revealed a 14% increase in the concentration of tartrate resistant acid phosphatase (TRAP)-5b, a marker of osteoclast activity, in irradiated mice (P < 0.05). Osteoclast number (+44%; P < 0.05) and osteoclast surface (+213%; P < 0.001) were elevated in TRAP-stained histological sections of tibial metaphyses. No significant change was observed in osteoblast surface or osteocalcin concentration or in trabecular microarchitecture (i.e. bone volume fraction) as measured through microcomputed tomography (P > 0.05). This study provides definitive, quantitative evidence of an early, radiation-induced increase in osteoclast activity and number. Osteoclastic bone resorption may represent a contributor to bone atrophy observed after therapeutic irradiation.

Systemic effects of prostaglandin E2 on vertebral trabecular remodeling in beagles used in a healing study

SummaryProstaglandin E2 (PGE2) at a dose of 10 mg/kg was administered orally to beagles used in a study of rib fracture and drill hole defect healing. Double fluochrome labels were given prior to surgical manipulation and before necropsy at 30 days. Bone remodeling was evaluated in trabecular bone of the fourth lumbar vertebra. There was a decrease in the number and extent of posttreatment labels (P<0.05) in the controls, with decreased mineral apposition rate (P<0.05) and decreased active bone formation rate (P<0.01). In dogs given PGE2 for 30 days following surgery, the extent of posttreatment labels (P<0.05) and bone formation rate (P<0.01) were increased. There was no difference found, however, in static morphometric parameters, including osteoid and osteoblast-covered surface, indicating that the stimulation of bone formation may have been transitory and matrix synthesis had declined. In another group of dogs given PGE2 for 5 days prior to surgical manipulation and between the first and second pretreatment labels, the extent of the double-labeled surface was increased (P<0.05) indicating an acute PG effect to sustain formation at remodeling sites. These studies show that PGE2 given orally has a systemic effect on bone remodeling in vertebral trabeculae that involves the stimulation of formation activity.

Pathology of the Vertebral Column of Horses with Cervical Static Stenosis

Specimens of ligamentum flavum, joint capsule, and dorsal lamina were collected at surgery or necropsy from 25 horses with cervical static stenosis. All horses had myelographic evidence of dorsal compression of the spinal cord caused by soft tissue and/or bone in the caudal cervical area, primarily at C6-7. Most horses also had radiographic evidence of degenerative joint disease of articular facets. Histologically 19 horses had osteosclerosis and cartilage retention in the dorsal lamina, and 24 horses had increased fibrocartilage at the ligamentum flavum attachment to dorsal lamina. The ligamentum flavum and joint capsule had fibrovascular tissue in 20 horses. Fibrocartilaginous tissue, old hemorrhage, and fat necrosis were not unusual. One horse each had a synovial cyst, eosinophilic granulomas in the joint capsule, and osteochondrosis of articular facets. These findings indicate that abnormal biomechanical forces or instability of articulations result in stretching and tearing of the ligamentum flavum and joint capsule with subsequent fibrovascular and fibrocartilaginous proliferation, osteosclerosis of the dorsal lamina, and osteophyte formation on the articular facets.

Calcified cartilage morphometry and its relation to subchondral bone remodeling in equine arthrosis

The calcified layer of articular cartilage is known to be affected by age and mechanical factors that may play a role in the development of arthrosis. Because these factors are also related to subchondral remodeling and sclerosis, a morphometric study was carried out in fluorochrome-labeled animals to determine whether the level of subchondral remodeling affected the thickness of the calcified cartilage layer and its irregularity and vascularity at the interface with subchondral bone. These parameters were also studied at a site of increased mechanical stress. The area and thickness of the calcified cartilage layer was determined in basic fuchsin-stained ground sections (120 microm). The irregularity of the chondro-osseous interface was expressed as the ratio of its length to that of the relatively straight tidemark (Int/Tid) and the number of abutting vessels with and without fluochrome labels were counted (N.Ves/Tid,%L.Ves/Tid). These were compared with single-labeled surface (sLS/BS, %) in subchondral bone, which was used as an index of remodeling. In a group of 12 horses, in which one carpus had an osteochondral fragment surgically created 10 weeks earlier, there was activation of subchondral remodeling in the third carpal bone opposite the fragment. An increase in %L.Ves/Tid (p < 0.01) at the interface was correlated with the increase in %sLS/BS in subchondral bone (r=0.431, p=0.035). The number of abutting vessels and the interface irregularity were not significantly changed on the fragmented side. In the metacarpal condyles from the fetlock joints of the same horses there were no differences associated with the surgically created fragment in the carpus and no correlation of %L.Ves/Tid with subchondral %sLS/BS. At a site where mechanical overload and traumatic osteochondrosis is known to occur on the palmar surface, the calcified cartilage was thinner, and the interface irregularity tended to be greater. These findings indicate that activated subchondral remodeling extends to involve the calcified layer, but the thickness and irregularity of the calcified cartilage are not consistently related to current subchondral remodeling. At sites of mechanical overload the calcified cartilage was thinner and the interface tended to be more irregular, suggesting previous increased remodeling.

Bone changes in mucopolysaccharidosis VI in cats and the effects of bone marrow transplantation: Mechanical testing of long bones

Mucopolysaccharidosis VI (MPS VI) is a genetic lysosomal storage disease in which a defect in aryl sulfatase B leads to accumulation of the glycosaminoglycan dermatan sulfate and abnormalities in the development of cartilage and bone. A feline model of this disease was used to evaluate the efficacy of bone marrow transplant (BMT) therapy. Long bones from MPS VI cats (N = 6) and MPS VI + BMT cats (N = 7) were compared with control cats (N = 11) and control + BMT cats (N = 5) in mechanical tests. Dissected femurs and tibias were subjected to three-point bending and a subgroup of tibias were tested with the mechanical response tissue analyzer (MRTA) in which vibration is used to measure tissue impedance. Cats with MPS VI had markedly decreased stiffness and strength in both bone (p < 0.01). There was no significant difference in the MPS VI + BMT group. In the tibias, there was also decreased stiffness and strength in the control + BMT group as compared to controls (p < 0.05). However, when cross-sectional area was used to normalize for bone size there was good correlation with strength in both femurs (r = 0.907, p < 0.01) and tibias (r = 0.915, p < 0.1), and there were no significant differences between groups in the modulus of elasticity. In the tibias, in which stiffness was measured by MRTA, there was significant correlation with three-point bending stiffness. These results indicate that, in cats with MPS VI, the decreases in stiffness and strength of long bones can be largely accounted for by the decrease in bone size (osteopenia) that is present.

Renal Failure in Perinatally Irradiated Beagles

Of 99 beagles receiving 270-435R60 Co gamma radiation (bilateral, whole-body; 10 min exposure time) at 55 days in utero or 2 days of age and allowed to survive beyond 70 days, 15 have subsequently ...

Bone Mineral Density in Feline Mucopolysaccharidosis VI Measured Using Dual-Energy X-Ray Absorptiometry

Dual-energy X-ray absorptiometry (DXA) was used to determine the in vivo bone mineral density (BMD) and bone mineral content (BMC) of lumbar vertebrae in six cats affected with the inherited lysosomal storage disease mucopolysaccharidosis VI (MPS VI). DXA was also performed on MPS cats that had a bone marrow transplant (BMT) and total body irradiation (TBI) (MPS+BMT;n=7), normal cats that had a bone marrow transplant, and TBI (control+BMT; n=8) and normal cats (control; n=14). Following euthanasia, one of the lumbar vertebrae that had been scanned (L5) was harvested and bone volume (BV/TV%) was determined by histomorphometry. The in vivo BMD and BMD measurements were compared with the BV/TV%. There was a greater BMD and BMC in the MPS+BMT cats compared with the MPS cats but the difference was not statistically significant. However, there was a greater BV/TV% in the MPS+BMT cats compared with the MPS cats and the difference was significant (P=0.0152). Correlation between the noninvasive in vivo DXA measurements of BMD and BMC and the BV/TV% was significant (r2=0.767, P<0.0001; r2=0.504, P<0.0001). Noninvasive in vivo DXA was a rapid and precise method for measuring the lumbar BMD and BMC in cats and it correlated well with histomorphometric determination of bone mass. Further, the response of inherited storage diseases such as MPS VI to therapy, such as BMT, could be monitored in a longitudinal fashion using DXA.

Hepatic failure and hemochromatosis of Salers and Salers-cross cattle.

Hemochromatosis is rare in domestic mammals. Five clinical cases and one preclinical case of hemochromatosis were diagnosed in Salers and Salers-cross cattle. Clinical disease developed between 9 and 22 months of age. Animals were healthy until weaning but then lost weight, developed rough hair coats, and lost incisor teeth. In two animals, hemochromatosis was identified by liver biopsy, biochemical evidence of hepatic injury, and/or elevated transferrin saturation values. At necropsy, carcasses were thin, with firm dark brown livers and lymph nodes, soft bones, and brown-colored small bowel. The principal histologic changes were hepatocellular siderosis and periportal, bridging, and perivenular fibrosis. Siderocalcinosis involved collagen, elastin, reticulin, and basement membrane components in liver, lymph nodes, spleen, duodenum, and kidney. Hepatic iron concentrations in clinically affected cattle were 1,500–10,500 μg/g wet weight (reference range for cattle = <300 μg/g). Ultrastructurally, the heaviest intrahepatic deposition was in hepatocytes, which contained large intracytoplasmic siderosomes. Iron deposition in bone was associated with osteopenia. Genetic analysis indicated a common ancestral bull in the pedigrees of five of six affected cattle; no pedigree was available for the remaining animal. Four dams of five affected animals were phenotypically normal and had histologically normal livers. Test mating of four cows to the ancestral bull resulted in a female calf that developed clinicopathologic and histologic evidence of preclinical hemochromatosis by 40 days of age. It was not possible to establish the pattern of inheritance because of the small number of pedigrees from affected cattle.

Correlation of quantitative computed tomographic subchondral bone density and ash density in horses.

The purpose of this study was to compare subchondral bone density obtained using quantitative computed tomography with ash density values from intact equine joints, and to determine if there are measurable anatomic variations in mean subchondral bone density. Five adult equine metacarpophalangeal joints were scanned with computed tomography (CT), disarticulated, and four 1-cm(3) regions of interest (ROI) cut from the distal third metacarpal bone. Bone cubes were ashed, and percent mineralization and ash density were recorded. Three-dimensional models were created of the distal third metacarpal bone from CT images. Four ROIs were measured on the distal aspect of the third metacarpal bone at axial and abaxial sites of the medial and lateral condyles for correlation with ash samples. Overall correlations of mean quantitative CT (QCT) density with ash density (r=0.82) and percent mineralization (r=0.93) were strong. There were significant differences between abaxial and axial ROIs for mean QCT density, percent bone mineralization and ash density (p<0.05). QCT appears to be a good measure of bone density in equine subchondral bone. Additionally, differences existed between axial and abaxial subchondral bone density in the equine distal third metacarpal bone.