Collagen synthesis in normal and osteoarthritic human cartilage.

Collagen metabolism in osteoarthritic human articular cartilage was compared to that in normal cartilage and was also correlated with the degree of severity of the osteoarthritic lesion as determined by a histological-histochemical grading system. No correlation was apparent between the concentrations of DNA, hydroxyproline, and hydroxylysine and the degree of severity of the osteoarthritic lesion (except in far-advanced lesions). Similarly, there was no correlation in levels of these components in tissues from the normal vs. osteoarthritic group. The similarity of the values of the ratio hydroxylysine/hydroxyproline in osteoarthritic tissue compared with normal, and the lack of variation in these with increasing severity of the disease process argues against the possibility that osteoarthritis is associated with a major shift in the synthesis of type II collagen to type I. [3H]Proline incorporation into osteoarthritic cartilage was increased fourfold as compared to normal cartilage and varied with advancing histological-histochemical grade. Measurement of the specific activity of insolubilized hydroxyproline-containing material of the cartilage matrix, as an index of the turnover of collagen, showed a sixfold increase in osteoarthritic cartilage which also varied with grade. These data suggest that collagen synthesis in these tissues is substantially greater than in nonosteoarthritic tissues and varies directly with the severity of the disease process up to a point and then varies inversely as the lesion becomes more severe.

The glycosaminoglycans of normal and arthritic cartilage.

The cartilages from the hip joints of 13 normal and 15 osteoarthritic humans were analyzed for glycosaminoglycan content and distribution. The GAGs were separated by elution with CPC on a short cellulose column by the technique of Svejcar and Robertson after digestion of the tissue with pronase and papain. The eluates were identified by a variety of methods including determination of molar ratios, N-acetyl-hexosamine determinations after hyaluronidase treatment and thin-layer chromatography of unhydrolyzed and hydrolyzed GAGs. From the data obtained, it was demonstrated that cartilage from arthritic patients showed a significant increase in the concentration of chondroitin 4-sulfate and a significant decrease in keratan sulfate, with only slight changes in the total amount of GAG present. Calculations of the molar ratios showed variation in the sulfation with chondroitin 4-sulfate appearing in the "supersulfated" state in the arthritic cartilage. The data lead to speculation regarding the process of osteoarthritis, and it is concluded that the changes seen are more likely to represent an altered pattern of synthesis rather than selective degradation. Since the changes suggest a younger cartilage, a theory is advanced that the chondrocyte responds to the chronic stress of osteoarthritis by modulation to a chondroblastic phase.

Fluorescent antibody localization of the vitamin D-dependent calcium-binding protein in the oviduct of the laying hen.

Fluorescein isothiocyanate-conjugated sheep antirabbit gamma-globulin was used in the indirect fluorescent antibody technique to localize the vitamin D-dependent calcium-binding protein (CaBP) in the shell gland of the laying hen. Intense specific fluorescence, indicative of the presence of CaBP, was found only in the tubular gland cells and not in the surface epithelium. CaBP was primarily associated with the luminal surface and other cell boundaries, and in lesser concentrations within the cells except for occasional intracellular regions of high concentration. The magnum, isthmus and vagina were essentially negative. Since the concentrations of intestinal CaBP is highly and directly correlated with the efficiency of calcium absorption, the present results suggest that the tubular gland cells might have a primary role in the transport of calcium associated with egg shell formation.

Growth hormone-releasing hormone depletion in the female rat: similarities to aging.

The age-related decline in growth hormone (GH) secretion has been largely attributed to age-related degeneration of hypothalamic growth hormone-releasing hormone (GHRH)-producing neurons. GH decline has recently been linked to age-related bone changes in humans. Bone loss and decreased bone strength are common in aging rats and humans, but density of remaining mineral tissue is known to be increased. The effect of induced hypothalamic GHRH deficiency on bone was assessed, and similarities between bone changes encountered and those taking place in aging were identified. Female rats received monosodium glutamate (MSG) following birth, and they were euthanized at 19 weeks of age. Femurs from MSG-treated rats had greater mineral density (p < .05), greater mineral/matrix ratio (p < .01), lower mineral apposition rate (p < .005), and lower bone formation rate (p < .05). These results suggest that hypothalamic GHRH decline plays a substantial role in the development of bone pathology similar to that observed in aging individuals.

The association of lipid abnormalities with tissue pathology in human osteoarthritic articular cartilage.

Articular cartilage is one of very few body tissues uniquely characterized as having substantial stores of lipid deposits. Lipid droplets are naturally accumulated by chondrocytes and individual fatty acids have been shown to have protective as well as deleterious effects on cartilage degradation in animal models of degenerative joint disease. As a means to better assess the role of lipids in human joint pathology, a comparative analysis of fatty acids was undertaken in small segments of osteoarthritic articular cartilage. The data were assessed in terms of chondrocyte synthetic activity and histological determination of disease severity. The distribution profile of individual fatty acids in normal and osteoarthritic specimens remained constant, with palmitic, oleic, and linoleic acids representing 85% of the total fatty acids. In contrast, levels of total fatty acids were markedly increased in association with increasing degree of lesion severity. Compared with tissue from normal-aged joints, grade 0 to 1 mild lesions had elevated levels of total fatty acids, essential fatty acids, and chondrocyte synthetic activity of 80%, 312%, and 393%, respectively. More severe tissue involvement (grade 6 to 9), was associated with even greater increases of 440%, 1,100%, and 1,150%, respectively. No change was noted in cholesterol content in any tissue. The accumulation of arachidonic acid was greater than the proportional increase in total fatty acid content and was primarily distributed into the neutral lipid fraction, where it constituted almost 62% of the fatty acid level in tissues of moderate lesion severity. There was an association of lipid accumulation in general and arachidonic acid in particular with histological severity.(ABSTRACT TRUNCATED AT 250 WORDS)

Colony-forming efficiency response of bone marrow stromal cells to acute blood loss.

Colony-forming efficiency (CFE) was used to monitor the proliferative response of alkaline phosphatase-positive rabbit bone marrow stromal cells to acute blood loss. The CFE of animals subjected to a 1% blood loss was 0.97 compared with 0.06 (p less than 0.01) in nonbled animals. Sera obtained from animals 10 days after an initial blood loss stimulated the CFE of marrow cultures from nonbled donors to the same degree as osteogenin. Erythropoietin and control sera (from nonbled animals) had no effect. Hence, acute blood loss and sera from bled animals stimulate proliferation of alkaline phosphatase-positive marrow stromal cell colonies. The agent(s) responsible is unknown but it is present in serum in response to blood loss. Confirmation of a specific effect on osteoprogenitor cells may warrant the designation "osteopoietin."

Pulsing direct current-induced repair of articular cartilage in rabbit osteochondral defects.

Osteochondral defects in the distal femoral condyles of rabbits exposed to a pulsing direct current exhibits an enhanced quality of repair. The signal, with a peak value of 2 microA repeating at 100 Hz, imposed an electric field in the tissue of 20-60 mV/cm2. Maximum efficacy was seen with a shorter period of exposure (40 vs. 160 h) initiated 48 h after surgery for 4 h/day. Repair tissue originated primarily from metaplasia of subchondral elements although hyperplasia of pre-existing chondrocytes at the margins of the defect could be detected. Defects in treated joints contained Safranin O staining material that was histologically similar to a disorganized hyaline cartilage. Central areas of the defects in control animals contained Safranin O-negative material that generally extruded over the surface as a pannus. The edges of nontreated defects also had characteristics of cartilaginous healing, stressing the importance of using serial sectioning techniques in this model of cartilage repair.

Stereological study of the developing distal femoral growth plate.

The distal femoral growth plate has a uniquely convoluted structure comprised of four mammillate processes. Factors contributing to the development of these processes and overall plate geometry were explored using three-dimensional image analysis of the canine distal femoral epiphysis. The growth plate at birth remains relatively flat until ossification of the epiphysis begins at 1 week of age. Epiphyseal ossification proceeds eccentrically, projecting in the medial-lateral and anterior-posterior directions. Growth plate activity indexed by [3H]thymidine labeling and plate thickness revealed regional differences in cell proliferation. This was measured as a decreased labeling index and thinning of the growth plate in areas capped by the ossifying epiphysis. The eccentric ossification pattern and associated variations in growth plate activity result in definition of an "intraphyseal" groove and medial-lateral oriented sulcus. The groove and sulcus bisect the plate into four quadrants, giving rise to a convoluted structure composed of four areas of plate elevations termed mammillary processes (MP). By 5 weeks, the pattern of ossification results in greater development of the MP in the anterior-medial quadrant and in decreasing order, in the posterior-medial, anterior, and posterior-lateral quadrants. By 10 weeks, a uniform rate of cell proliferation was observed coincident with completion of ossification of the epiphysis. The data suggest that localized variations in growth plate proliferation are associated with ossification of the epiphysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of aging on tensile mechanical properties of the rabbit distal femoral growth plate.

Tensile tests to failure were performed on the distal femoral growth plate of rabbits. The measured tensile forces, together with animal weight and dimensional measurements of the femur, were used to develop a noninvasive method of predicting growth failure loads and to examine the effects of age on the tensile strength of the growth plate. Growth plate failure load correlated with rabbit weight, (r = 0.93), femoral length, (r = 0.91), and contoured growth plate surface area, (r = 0.74). Regression analysis for these relationships allowed for failure load prediction if animal weight, femoral length, or growth plate surface area was known. Nominal stress (failure load/surface area) was calculated and correlated with animal weight (age) for each distal femoral growth plate utilizing two surface area estimating techniques: growth plate surface contour and growth plate cross-sectional enlargement. A strong association was found with either technique. The growth and maturation of growth plate cartilage is of major importance in pediatric orthopaedics. This study demonstrate that the age-dependent increases in tensile mechanical properties of the growth plate do not result merely from growth plate enlrgement or conformational changes. The changing tensile mechanical properties of the growth plate may explain why transphyseal distraction used clinically has produced varied and somewhat unpredictable results. Our study suggests that dose-response investigations are needed to define safe and optimal transphyseal distraction tensions before further clinical application is pursued.

Biochemical and metabolic abnormalities in articular cartilage from osteoarthritic human hips. III. Distribution and metabolism of amino sugar-containing macromolecules.

Since 1960, numerous studies have supported the thesis that the synthetic activity of articular chondrocytes is increased in osteoarthritis, but several recent reports have challenged this concept. To clarify this problem fully and also to define further the products of this increased synthesis, three experiments were performed in which the distribution and rates of synthesis of amino sugar-containing macromolecules in normal and osteoarthritic cartilage from the human femoral head were assessed by biochemical analysis and studies of the incorporation of 3H-glucosamine and 35SO4. The biochemical data obtained clearly demonstrated the previously noted significant decrease in hexosamine content in osteoarthritic tissue. This decrease was principally due to a diminution in glucosamine concentration and correlated inversely with the severity of the disease process (as measured by a previously described histological-histochemical grading system). Metabolic studies showed a marked increment in the rates of incorporation of 3H-glucosamine into both the glucosamine and the galactosamine fractions of the cartilage. The increased synthesis correlated directly in a non-linear fashion with the severity of the disease. The ratio of the rate of incorporation of 3H-glucosamine into the glucosamine fraction to the rate of its incorporation into the galactosamine fraction was the same in normal and osteoarthritic samples, suggesting that the decline in glucosamine concentration was not related to a qualitative alteration of synthetic activity.

Development of an osteogenic bone-marrow preparation.

The osteogenic effect of bone marrow was tested in rabbits, using chambers that had been implanted in the peritoneal cavity (ectopic site) and in a delayed-union model (orthotopic site). Osteogenesis was accelerated in both sites after concentration of marrow elements by centrifugation, but not after unit gravity sedimentation. Chambers that were implanted with marrow that had been processed by simple and isopyknic centrifugation demonstrated a more pronounced increase in deposition of calcium compared with whole-marrow implants of equal volume (101 compared with 193 per cent). Orthotopic grafting of a rabbit delayed-union model with whole marrow and marrow that had been processed with simple centrifugation significantly increased osteogenesis, as measured biomechanically and biochemically. Significantly improved healing was evident radiographically at five weeks after grafting with bone marrow that had been concentrated by simple centrifugation.

Characterization of chondrocytes from bovine articular cartilage: I. Metabolic and morphological experimental studies.

Articular chondrocytes were enzymatically isolated from mature bovine cartilage and separated by density zone-velocity sedimentation in a gradient. For cells from different levels of the gradient, size and staining characteristics were determined and rates of incorporation of tritiated cytidine and radiosulphate were measured as indicators of RNA and sulphated glycosaminoglycan synthesis, respectively. The data clearly show that the chondrocyte population is composed of cells that vary continuously in size and metabolic activity from one limit to another. The largest cells also demonstrated the greatest RNA production while the smallest cells had the least. There was, however, no such differentiation of sulphated proteoglycan production.

The biology of human chondrosarcoma. I. Description of the cases, grading, and biochemical analyses.

Tissues from sixty-nine cartilage tumors in sixty-six individuals were obtained at the time of operation and each lesion was graded as benign (seventeen) or as a low-grade (thirty-three) or high-grade (nineteen) chondrosarcoma according to histological and roentgenographic criteria. The material obtained was analyzed by biochemical techniques for its content of water, ash, DNA, total protein (composed of collagen and so-called excess protein), and carbohydrate. In addition, proteoglycan subunit was isolated and the chain lengths of chrondroitin sulphate and keratan sulphate were determined. Analysis of the data showed that for the most part the tumors differed only quantitatively from articular cartilage controls, with the principal variations noted in water, ash, protein, and collagen content. Sugar concentrations were highly variable, but analysis of the proteoglycan subunit showed a distribution of glycosaminoglycans characteristic of immature articular cartilage. Marked shortening of the keratan sulphate chains was noted without significant alteration in the chain lengths of chondroitin sulphate, The wide variations in pattern suggest that the cartilage tumors are not biochemically homogeneous and therefore probably do not represent a single group of genetic errors.

A large-deformation, finite-element study of chondrodiatasis in the canine distal femoral epiphyseal plate.

A large-deformation, finite-element analysis was conducted to model stress fields around the developing growth plate as a first approach to comprehend the clinical application of traction for limb lengthening procedures. The model chosen was a cross section through the distal femoral growth plate of a 14-week-old dog. The chosen section passed through two of the conformational bends (mammillary processes) formed by the natural convolutions of the physis. Three different loading conditions were applied to the distal femoral epiphyseal model. The model also examined the effects of different values of Young's modulus of the growth-plate cartilage. The cortical bone in all cases, experienced the highest stresses. In the growth plate, the highest principal stresses occurred where the physis intersects cortical bone. There were localized stresses that were higher than those that caused fracture in a rabbit model [Guse et al., J. Orthop. Res. 7, 667-673 (1989)]. Results also indicated the following: a small tilt of 0.1 degree in loading application increases the maximum principal stresses and the von Mises stresses in certain regions of the growth plate by about 8%; the shearing stresses in the growth plate are sensitive to Young's modulus of the growth plate; and traction pins that do not grip the cancellous bone in the epiphysis will increase the regions of high principal stress in the growth plate.

Mineral and matrix contributions to rigidity in fracture healing.

The purpose of this study was to investigate the relationships among selected properties of fracture callus: bending rigidity, tissue density, mineral density, matrix density and mineral-to-matrix ratio. The experimental model was an osteotomized canine radius in which the development of the fracture callus was modified by electrical stimulation with various levels of direct current. This resulted in a range of values for the selected properties of the callus, determined post mortem at 7 weeks after osteotomy. We found that the rigidity (R) of the bone-callus combination obeyed relationships of the form R = axb, where x is the tissue density, mineral density, matrix density or the mineral-to-matrix ratio of the repair tissue. These are analogous to power-law relationships found in studies of compact and cancellous bone. The results suggest that fracture callus at 7 weeks after osteotomy in canine radius behaves more like immature compact bone than cancellous bone in its mechanical and physicochemical properties. The present study demonstrates the feasibility of developing non-invasive in vivo densitometric methods to monitor fracture healing, since models may be developed that can predict mechanical properties from densitometric data. Further studies are needed to develop a refined model based on experimental data on the mechanical and physicochemical properties and microstructure of fracture callus at different stages of healing.

Electrophysiology of direct current stimulation of fracture healing in canine radius.

Electrophysiological mechanisms involved in the electrical stimulation of fracture healing remain largely unknown. The purpose of the present study was to establish relationships between osteogenetic response and intraosseous measures of electrical dose in experimental fractures (osteotomies) of canine radii stimulated by direct currents. The response was determined postmortem at seven weeks after osteotomy by measuring the bending rigidity and four physicochemical properties: tissue density, mineral density, matrix density, and mineral-to-matrix ratio. The currents measured in bone ranged from 0.1 to 17 microA. Three regions of enhanced osteogenetic response were observed at approximately 1, 7, and 13 microA, separated by regions of unstimulated response. Evidence presented in this paper suggests that enhanced response resulted mainly from electrical modulation of early events in the fracture repair sequence.