High dietary fat and the development of osteoarthritis in a rabbit model.

OBJECTIVE: Osteoarthritis (OA) is associated with obesity, although this relationship remains unclear. Proposed etiologies of OA in obesity include mechanical loading of malaligned joints and possible toxicity of dietary fat. The hypothesis tested in the present study was that increased dietary fat worsens OA in both malaligned and normal joints, detected by biochemical and histological cartilage markers. METHOD: 83 New Zealand white rabbits were divided among two conditions related to OA: bowing of the knee and a 14%kcal vs 47.8%kcal fat diet. Rabbit weights and knee angles were compared throughout the experiment. At 28 and 38 weeks, intra-articular forces were measured, animals sacrificed, and knee cartilage examined for histological changes, glycosaminoglycan content, 35S uptake, and aggrecanase-1 expression. RESULTS: There were no differences in animal weights or intra-articular forces between the two diets. Despite increased fat content in their diet, animals on the 47.8%kcal fat diet did not gain excess weight. Representative histology showed atypical shearing of articular cartilage among animals on the high fat diet. Animals on the 47.8%kcal fat diet had suppression of protein synthesis compared to the 14%kcal fat diet: lower glycosaminoglycan content and aggrecanase-1 expression in all knee compartments at both times, and lower 35S uptake at 38 weeks. CONCLUSION: These results suggest dietary fat, independent of animal weight, results in altered chondrocyte function. Increased dietary fat was associated with changes in rabbit cartilage in vivo and appears to be a risk factor for the development of OA.

Collagenase and collagenase inhibitors in osteoarthritic and normal cartilage.

In advanced osteoarthritis, all of the cartilaginous components are lost from the joint surface. Although mechanisms exist for proteoglycan degradation, there is not known to be any system for removal of the collagen. This study suggests that the loss of the collagen components may be a function of articular cartilage collagenase. The enzyme in normal human cartilage is bound to an inhibitor and appears to be present in very small amounts. Attempts to demonstrate collagenase activity in ground human articular cartilage or in its lysosomal fraction were unsuccessful. 7-Day cartilage tissue cultures also failed to demonstrate the presence of the enzyme; but the same culture fluid, incubated with trypsin, showed significant degradation of collagen, suggesting that trypsin destroyed the inhibitor. 7-Day culture fluids were then chromatographed on a heparin-charged Sepharose 4B affinity column that had been activated with cyanogen bromide. This removed the inhibitor, and the chromatographed fluid from osteoarthritic cartilage released 42% of the incorporated counts of the collagen substrate, whereas normal cartilage released 10.1% and a trypsin control, 6.4%. Electrophoresis of the degradation products of the enzyme-collagen complex incubated at 37 degrees C revealed breakdown was complete to small dialyzable fragments, while at 25 degrees C larger fragments were split off.

Human osteoblasts in culture metabolize both 1 alpha, 25-dihydroxyvitamin D3 and its precursor 25-hydroxyvitamin D3 into their respective lactones.

1 alpha, 25-Dihydroxyvitamin D3 [1 alpha, 25-(OH)2D3], the hormonal form of vitamin D3, is further metabolized in the kidney and intestine through the carbon 24 (C-24) oxidation pathway initiated by C-24 hydroxylation, and the carbon 23 (C-23) oxidation pathway initiated by C-23 hydroxylation. The C-24 oxidation pathway leading to the formation of calcitroic acid has been previously reported to be present in bone cells, but the C-23 oxidation pathway leading to the formation of 1 alpha, 25-(OH)2D3-26,23-lactone has not been described in bone cells, even though 1 alpha, 25-(OH)2D3-26,23-lactone is noted to have a significant effect on bone formation. Therefore, in the present study, we investigated the production of 1 alpha, 25-(OH)2D3-26,23-lactone in normal human osteoblasts, and our studies revealed that human osteoblasts possess the activity of both 24- and 23-hydroxylases constitutively. Thus, 1 alpha, 24(R),25-(OH)3D3, 1 alpha, 25-(OH)2-24-oxo-D3, 1 alpha, 23(S), 25-(OH)3-24-oxo-D3, 1 alpha, 23-(OH)2-24,25,26,27-tetranor D3, and calcitroic acid formed through the C-24 oxidation pathway and 1 alpha, 23(S),25-(OH)3D3 and 1 alpha, 25-(OH)2D3-26,23-lactone formed through the C-23 oxidation pathway were detected under basal conditions. Also, the synthesis of these metabolites was increased significantly when the cells were treated with 1 alpha, 25-(OH)2D3 (50 nM) for 24 h before incubation with the tracer. As 25-hydroxyvitamin D3 (25OHD3) follows similar side-chain modifications as 1 alpha, 25-(OH)2D3, the metabolism of 25OHD3 in normal human osteoblasts was studied under basal conditions. We found that 25OHD3 was also metabolized through both C-24 and C-23 oxidation pathways, resulting in significant synthesis of 24(R),25-(OH)2D3 along with 25OH-24-oxo-D3, 23(S),25-(OH)2-24-oxo-D3, 23(S),25-(OH)2D3, and 25OHD3-26,23-lactone. Under the same experimental conditions, we looked for 1 alpha, 25-(OH)2D3 synthesis, as earlier studies have shown production of 1 alpha, 25-(OH)2D3 in human bone cells. During a time-course study ranging from 1-24 h, we found that by 2 h, the 24(R), 25-(OH)2D3 concentration rose and accumulated considerably during the following 24 h, but 1 alpha, 25-(OH)2D3 did not accumulate at any time. However, other 1-hydroxylated metabolites, 1 alpha, 23(S),25-(OH)3D3, 1 alpha, 23(S),25-(OH)3-24-oxo-D3, as well as 1 alpha, 25-(OH)2D3-26,23-lactone were detected.(ABSTRACT TRUNCATED AT 400 WORDS)

Early diagnosis of disc-space infection using Gallium-67.

A 4-year-old boy had had progressive central lumbar pain and hamstring spasm. He had a normal lumbar-spine x-ray except for minimal L-5, S1 spondylolysis, but gave an abnormal gallium-67 scan in the region of the low lumbar spine. Eight weeks following intensive antibiotic therapy, confirmation of the diagnosis of disc-space infection was established by roentgenographic studies that demonstrated narrowing of the L 4-5 intervertebral disc space. A technetium-99m diphosphonate bone scan, performed concurrently with the gallium-67 study, was normal.

Low-dose transaxial tomography. An alternative to computed tomography for the evaluation of anteversion of the femur during childhood.

Low-dose transaxial tomography is a technique that can produce cross-sectional images of the hips and femurs in children to permit calculation of the angle of femoral anteversion. Transaxial tomography was compared with computed tomography in terms of measured radiation dose and image quality. Transaxial tomography was found to require at least 90% less radiation dose, and the images were judged to be acceptable for the determination of anteversion.

Effects of a delayed steroid injection on ligament healing using a rabbit medial collateral ligament model.

Corticosteroids are known to inhibit collagen synthesis in vitro as well as having a deleterious effect on ligament healing when applied immediately following injury. An acute injection of betamethasone into a transected rabbit medial collateral ligament significantly impaired the biomechanical and histological properties compared to non-injected transected ligaments. Differences in mechanical, histological and biochemical properties were observed up to 3 months following injury and an acute steroid injection. The present study explored the effects of a corticosteroid (betamethasone) injection 7 days following the initial injury. Biomechanical and histomorphometric analyses were carried determine if the previously observed deleterious effects of a corticosteroid injection immediately following injury can be linked to an interference in the inflammatory phase of healing due to the presence of the corticosteroid.

RGD-coated titanium implants stimulate increased bone formation in vivo.

Numerous studies have demonstrated that peptide modified surfaces influence short- and long-term cell responses such as attachment, shape and function in vitro. These responses are mediated via cell receptors known as integrins which bind specifically to short peptide sequences from larger proteins. Integrins transduce information to the nucleus through several cytoplasmic signalling pathways. Little is known, however, about the ability of peptide-coated surfaces to influence cell responses in vivo. The present study was designed to evaluate the quality and quantity of the new bone formed in response to titanium rods surface-coated with the peptide sequence Arg-Gly-Asp-Cys (RGDC) using gold-thiol chemistry and implanted in rat femurs. Histomorphometric analysis of cross-sections perpendicular to the implant long axis showed a significantly thicker shell of new bone formed around RGD-modified versus plain implants at 2 weeks (26.2 +/- 1.9 vs. 20.5 +/- 2.9 microm; P < 0.01). A significant increase in bone thickness for RGD implants was also observed at 4 weeks while bone surrounding controls did not change significantly in thickness (32.7 +/- 4.6 vs. 22.6 +/- 4.0 microm; P < 0.02). Mechanical pull-out testing conducted at 4 weeks revealed the average interfacial shear strength of peptide modified rods was 38% greater than control rods although this difference was not statistically significant. These pilot data suggest that an RGDC peptide coating may enhance titanium rod osseointegration in the rat femur. Long-term studies and evaluation of other peptides in larger animal models are warranted.

Degradative enzyme systems in osteoarthritic cartilage.

In osteoarthritis, despite increased matrix synthesis, there is a reduction of both major matrix components, proteoglycan and collagen. This study suggests that this is the result of enhanced degradative activity intrinsic to the cartilage. Because osteoarthritis is a focal disease, histologic controls were used to measure the severity in different areas of the cartilage and in different specimens. Neutral proteoglycanase and collagenase were both described in human cartilage, and their levels matched the severity of the disease as did acid phosphatase, a marker of lysosomal enzymes. Articular cartilage collagenase has an inhibitor in the cartilage and has negligible activity in normal cartilage. This was found not to be a lysosomal enzyme. A model of osteoarthritis was studied and found to have the same biochemical pattern as human disease. Using this method, inhibitors of degradative enzymes were used as a treatment. The chelator of metallic cations EDTA was found to have a significant effect on reduction of degradative enzyme activity and altered the arthritic process.

Drug-induced inhibition of proteoglycanase activity in the Hulth-Telhag model.

Evidence has accumulated that there are increased degradative enzymes in osteoarthritis responsible for joint destruction. These enzymes are metal-dependent, and inhibited by EDTA. EDTA was administered intra-articularly to rabbits in an experimental model of osteoarthritis. There was a 25% reduction in neutral proteoglycanase activity and 75% of the animals had a reduction in the severity of the arthritis as measured on a histologic-histochemical grading system. It is suggested that future chemotherapeutic studies on arthritis might focus on enzyme inhibition.

Treatment of proteoglycan aggregates with physeal enzymes reduces their ability to inhibit hydroxyapatite proliferation in a gelatin gel.

In vitro, cartilage proteoglycans (PGs) are effective inhibitors of hydroxyapatite formation and growth. Their inhibitory ability decreases with decreasing PG size and charge density. It has been suggested that the enzyme-mediated alteration in the size and conformation of PGs in the growth plate may similarly facilitate the calcification process. In this study, a gelatin gel system was used to monitor hydroxyapatite formation and growth in the presence of proteoglycan aggregates, before and after enzyme treatment. To reproduce the physeal degradation cascade, an enzyme preparation was used that contained all of the growth plate enzymes. At a concentration of 500 micrograms/ml, the untreated proteoglycan aggregates reduced the amount of mineral formed by 30%. When the aggregates were treated with the heat-inactivated enzyme, the same extent of inhibition was found. In contrast, treating the aggregates with the crude growth plate enzyme preparation removed all the inhibitory ability, such that 500 micrograms/ml of proteoglycan preparation yielded 10% more mineral than the controls. Treatment of the aggregates with chondroitinase ABC and trypsin, similarly removed all the inhibitory ability. These data, suggest that enzymatic degradation of proteoglycans may contribute to the regulation of growth plate calcification.

Partial purification and characterization of a proteoglycan-degrading neutral protease from bovine epiphyseal cartilage.

Degradation of the proteoglycan matrix is considered an essential step in the process of calcification in the growth plate. This laboratory has just described the presence of a protease in human growth plate cartilage that degrades proteoglycan at neutral pH. We report here the isolation, partial purification, and characterization of these proteoglycan-degrading neutral proteases of bovine epiphyseal cartilage. It appears that there is more than a single enzyme active at neutral pH. These enzymes are of low molecular weight (below 30,000), poorly charged, and inhibited by metal chelating agents. Activity is best restored in the presence of zinc. This represents the first characterization of this important enzyme group.

Physeal reconstruction using tissue donated from early postnatal limbs in a murine model.

Physeal reconstruction was performed in a murine model by transplanting corresponding postnatal tissue from 4-day-old C57B mice to resection defects. The site of the reconstruction, the murine distal femoral epiphysis, is completely cartilaginous and avascular at this stage of development. The tissue transplanted into the defect was demonstrated to have high kinetic activity by its incorporation of tritiated thymidine. The physeal reconstruction as performed restored only 25% of normal growth. While transplanting cell populations is feasible, the method will require a great deal of work before clinical application.

Electron microscopic analysis of articular cartilage proteoglycan degradation by growth plate enzymes.

To assess the effect of intracellular growth plate chondrocyte enzymes on proteoglycan structure, we examined enzyme-treated articular cartilage proteoglycans and untreated articular cartilage proteoglycans with the electron microscopic monolayer technique. The untreated proteoglycan monomers ranged in length from less than 20 nm to more than 700 nm, with a mean length of 224.5 +/- 101.6 nm in one experiment and 224.6 +/- 95.7 nm in a second experiment. Incubation with growth plate enzymes reduced proteoglycan monomers to fragments with lengths that varied from less than 5 nm to 143 nm, increased the variability in monomer length, and destroyed proteoglycan aggregates. The enzyme treated monomers had an average length of 29.5 +/- 17.9 nm in one experiment and 35.2 +/- 17.0 nm in a second experiment. The smallest common fragments were 15 nm long and would be expected to contain about 15 glycosaminoglycan chains. This experiment demonstrates that enzymes extracted from growth plate chondrocytes can degrade the chondroitin sulfate-rich region of proteoglycan monomer core proteins, produce a range of monomer fragment sizes with less than 2% of the fragments shorter than 5 nm or longer than 100 nm, increase the variability in monomer length, and degrade proteoglycan aggregates.

Comparative study of neutral proteoglycanase activity by growth plate zone.

The degradation of proteoglycans has been considered an essential step in the process of endochondral ossification. Neutral proteases, described in the growth plate, have been implicated in this process. If these neutral proteases are important in the endochondral process, their level of activity should be highest at the point where calcification is occurring. This study measures neutral protease activity with a quantitative viscometric assay throughout all zones of the bovine growth plate. The results demonstrate that the enzyme is present throughout all zones of the growth plate. However, enzyme levels in the lower hypertrophic and calcifying zones are significantly higher than anywhere else. The enzymes came from the cartilage cells and not from the invading vasculature. This implied proof of the role of neutral proteases in the endochondral process implies that by inhibiting this class of enzymes, one should be able to block endochondral ossification.

In vitro and in vivo effects of metal chelators on cartilage metabolism.

The effect of metallic chelating agents (EDTA, EGTA) on cartilage metabolism was studied both in vitro, on calf cartilage, and in vivo, in rabbits. The question asked was whether it was possible to affect neutral protease activity, and not also inhibit beneficial synthetic systems. In vitro, EDTA suppressed anabolic processes, while EGTA had no effect. However, EDTA in vivo did not suppress glycosaminoglycan or RNA production, but paradoxically stimulated them. At the same time, EDTA inhibited neutral protease activity in normal animals.

Weight-bearing alters the expression of collagen types I and II, BMP 2/4 and osteocalcin in the early stages of distraction osteogenesis.

This study was performed to investigate the effect of loading on the biology of newly forming bone during limb lengthening. Unilateral 2.0 mm femoral lengthenings were performed in 20 male Sprague Dawley rats. Half (n = 10) of the animals were allowed to bear weight freely, while the other half were prevented from weight-bearing via an ipsilateral through-knee amputation. The animals in each group were sacrificed after one (n = 5) or four (n = 5) days of consolidation (post-operative days seven and 10, respectively). In situ hybridization for osteocalcin and collagen I, and antibody staining for collagen II and BMP 2/4 were used to evaluate the molecular influence of loading. There was more new bone in the distraction gap of the weight-bearing animals than there was in the non-weight-bearing animals. BMP 2/4 expression, and the messages for collagen I and osteocalcin, were more abundant in tissue from the weight-bearing animals; collagen II was higher in the non-weight-bearing animals. This suggests that early regenerate tissue is capable of responding to loading, and that weight-bearing appears to stimulate intramembranous ossification. These findings support the concept of early weight-bearing after limb lengthening.

Epiphyseal replacement using developing tissue donors in a murine model: a combined histologic and radiographic study.

Epiphyseal transplantation has long been a goal of orthopaedic surgeons. While microvascular surgery has raised hopes that this goal could be achieved, factors other than blood supply also appear capable of affecting the function of the epiphysis. Basic research into the biology of the epiphysis appears to be required. This would be facilitated with a model of epiphyseal transplantation using a small mammal. The purpose of this experiment was to develop such a model in the mouse. Developing CD1 mouse or Lewis rat limb tissue was used to replace knee tissue that had been resected from CD1 postnatal mouse hosts. Donor tissue ranged from 14-day embryonic mouse to 9-day postnatal mouse or 18- and 19-day fetal rat, which has a gestation similar to the mouse. The murine tissue is known to be avascular prior to the sixth postnatal day. The limbs were analyzed radiographically and histologically. The results show that epiphyseal replacement could be studied using developing tissue donors in a murine model. The results suggest that donor tissue prior to vascularization and tissue combinations with the least developmental time mismatch (the least heterochronicity) produced relatively the best, although still abnormal epiphyses.

Kinetic and biochemical heterogeneity in vertebrate chondroepiphyseal regions during development.

The purpose of this study was to see if kinetic and biochemical heterogeneity could be documented in vertebrate chondroepiphyseal regions as they develop from mesenchymal condensations to cartilage. The kinetics of developing proximal and distal femoral chondroepiphyseal regions were studied from early limb bud stage to newborn animals in chicks, mice, and rabbits with thymidine autoradiography. Proteoglycan synthesis in the proximal femoral chondroepiphyseal region of the rabbit was studied with radioactive sulfate incorporation at 28 days of gestation and at 1 and 4 days after birth. The results indicated that these kinetic and biochemical characteristics of the developing chondroepiphyseal regions became heterogeneous very early in development. This early programming of populations of cells for division and for different biochemical functions existed during the fetal period when heterogeneity has been described histologically but has not been well documented.

Instability of the distal tibiofibular syndesmosis after bimalleolar and trimalleolar ankle fractures.

We studied the late results after bimalleolar and trimalleolar ankle fractures in thirty-four patients after an average follow-up of four years. Twenty-one patients had had open reduction and internal fixation of the medial malleolus only and thirteen, internal fixation of both the medial malleolus and the lateral malleolus. Twenty-four lesions were supination-external rotation fractures; six, pronation-external rotation; and four, supination-adduction fractures. All initial and post-reduction roentgenograms were evaluated, and the patients were re-evaluated two to seven years after fracture. Re-evaluation included physical examination as well as standardized and stress roentgenograms of both ankles. Criteria were developed for measuring the width of the syndesmosis and assessing the late roentgenographic, subjective, and objective results, as well as any late instability of the syndesmosis and osteoarthritis. Significant correlations were found between: (1) the adequacy of the reduction of the syndesmosis and late arthritis, (2) the adequacy of the initial reduction of the syndesmosis and the late stability of the syndesmosis, (3) the late stability of the syndesmosis and the final outcome, and (4) the adequacy of the reduction of the lateral malleolus and that of the syndesmosis. Based on the findings in this small series and on the evidence published in the literature, we concluded that adequate reduction of the syndesmosis is necessary to achieve a stable ankle following supination-external rotation and pronation-external rotation fractures of the ankle, and that the reduction of the syndesmosis will be unsatisfactory if the lateral malleolus is not well reduced.

Lateral hamstring transfer and gait improvement in the cerebral palsy patient.

A retrospective analysis of twenty-three spastic patients who underwent forty-three transfers of the semitendinosus muscle to the lateral intramuscular septum and of the semimembranosus muscle to the biceps is presented. Decreased knee-flexion deformity as well as improved walking function were achieved in 91 per cent. An unsatisfactory result was associated with complications of the procedure. Only one knee of the forty-three that were operated on showed late genu recurvatum. This procedure appears to be both effective and relatively free of late comlications.