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Biomedical subjects

N Shepard

Publications and source records attributed to N Shepard.

At least 37 records · Page 2Linked to original sources
PubMed1981

Acridine orange stabilization of glycosaminoglycans in beginning endochondral ossification. A comparative light and electron microscopic study.

This study indicated that acridine orange, when combined with the initial fixative stabilized soluble matrix glycosaminoglycan in situ in areas where considerable glycosaminoglycan extraction is known to occur. Acridine orange was able to diffuse through bone into areas of undecalcified mineralizing cartilage and to bind with the glycosaminoglycans in these areas equally well as in growth plate cartilage matrix. Matrix staining was visible by light microscopy without further staining and was seen to vary territorially in intensity; although cellular definition was poor. This deficiency was overcome by the additional application of p phenylenediamine which stained the cells intensely. At the ultrastructure level, glycosaminoglycan was present as electron dense structures in the cartilage matrix. Preliminary X-ray microanalysis studies confirmed that the acridine orange stained structures contain sulphur; this finding extends the use of acridine orange further to quantitative analysis of glycosaminoglycan.

Acridine Orange↗
PubMed1981

Induction of bone resorption by parathyroid hormone in congenital malignant osteopetrosis.

A male patient, afflicted with malignant congenital osteopetrosis, was studied over a 5 year period. Hypocalcemia (less than 8 mg/dl) with lack of an appropriate increase in serum immunoreactive parathyroid hormone (iPTH) prevailed at all times. Under a calcium restricted diet, a 6-hour infusion of parathyroid extract normalized serum calcium, and increased the urinary hydroxyproline excretion suggesting that bone resorption had been induced. A second attempt to induce resorption was made by infusing a synthetic amino terminal fragment of bovine PTH over a period of 3 weeks at the dose of 1.5 units/kg/hr. This infusion evoked an increase in serum calcium (8.1 to 10.5 mg/dl), urinary calcium (0.03 to 0.65 mg/g creatinine) and urinary hydroxyproline (160 to 372 mg/g creatinine); and urinary hydroxyproline (160 to 372 mg/g creatinine); increases which were reversed by calcitonin administration. Iliac crest bone biopsies were obtained before and on the last day of the 3-week infusion. Quantitative comparison of the two specimens showed that, during PTH infusion, there was a 23% decrease in bone volume due to the increase in marrow space, a 93% increase in the number of osteoclasts and 136% increment in the osteoclastic resorption surface. Electron microscopic examination of the osteoclasts in the first tissue sample showed no evidence of ruffled borders, while in the second biopsy, numerous cytoplasmic processes indicative of resorptive activity were visible at the matrix-cell interface. It is proposed that, in our patient, the osteopetrotic phenotype is the consequence of an abnormality in the interaction between PTH and osteoclasts that may be related to the synthesis of a physiologically "defective" PTH.

25-Hydroxyvitamin D 2↗
PubMed1981

Ruthenium red- p-phenylenediamine staining of monolayers to facilitate handling and selection of specific cells for transmission electron microscopy.

The handling of monolayers for transmission electron microscopy has presented many problems, the main one being difficulty in visualizing the monolayers after polymerization of their plastic embedment following conventional glutaraldehyde-osmium fixation. The application of ruthenium red-p-phenylenediamine during processing intensely darkened cells which could be examined and photographed either in 95% ethanol or following Spurr monolayers when re-embedding, and permitted precise localization of monolayers within flat embedding molds when trimming and thin sectioning for transmission electron microscopy. Increased color density is the combined result of more complete retention of soluble elements during initial fixation by ruthenium red and the formation of a colored reaction product between the bound ruthenium red and osmium which is further intensified by p-phenylenediamine.

Cells, Cultured↗
PubMed1980

Healing of articular cartilage in intra-articular fractures in rabbits.

Intra-articular fractures were created in the distal end of the femur in adult rabbits. Fractures were then reduced in three different fashions: incomplete reduction, adequate reduction without compression of the fregments, and ideal reduction, which consisted of reduction with strong compression of the fracture fragments. Animals were killed at intervals ranging from seven weeks to one year, and the cartilage fractures were studied by light, transmission, and scanning electron microscopy, using proteoglycan stains. Cartilage fractures that were inadequately reduced or were adequately reduced without compression healed by fibrocartilage only. Fractures that were reduced with compression across the fragments healed with a tissue which, by light and electron microscopy, appeared to be hyaline cartilage, which showed the collagen and proteoglycan orientation and relationships of normal hyaline cartilage. The healing of these defects appears to be the result of a proliferation of cells emanating from the region of the tidemark, and perhaps of migration of chondrocytes from the surface into the lesion. We postulate that compression of the cartilage surfaces either creates a physical environment that allows certain chondrocytes to heal the defect with hyaline cartilage, or, by coapting the surfaces, prevents ingrowth of granulation tissue from the subchondral bone that might interfere with repair by hyaline cartilage. This study suggests a hitherto unappreciated capacity for hyaline cartilage to repair structural defects.

Animals↗
PubMed1978

Changes in proteoglycan and collagen in cartilage in rheumatoid arthritis.

Articular cartilage from thirteen patients with rheumatoid arthritis was studied by both light and electron microscopy using recently developed staining techniques for proteoglycan. Normally proteoglycan is concentrated about active chondrocytes, but in the cartilage from these patients it was found to be diminished about most of the chondrocytes while it was increased around a few. Fragmentation of collagen and phagocytosis of the fragments by chondrocytes were also noted. This dissolution of collagen was associated with loss of proteoglycan in the matrix. These findings are consistent with the variable nature of the rheumatoid destructive process and appear to be morphological reflections of biochemical changes which formerly were thought to occur in this disease only at the cartilage-pannus junction.

Arthritis, Rheumatoid↗
PubMed1977

The localization of articular cartilage proteoglycan by electron microscopy.

Rabbit articular cartilage was fixed with glutaraldehyde containing Ruthenium Red or Safranin O proteoglycan localization is easily obtained. Ruthenium Red stained proteoglycan is easily visualized by electron microscopy. Previous digestion with papain prevented staining by either technique confirming that the material was indeed proteoglycan. Using these methods of proteoglycan identification the sites of attachment to collagen are shown as well as the deposition of proteoglycan about cells of differing vitality and the relationship of proteoglycan to cell membranes.

Animals↗
PubMed1977

The use of ruthenium and p-phenylenediamine to stain cartilage simultaneously for light and electron microscopy.

p-Phenylenediamine was incorporated during the dehydration of cartilage blocks fixed with ruthenium red, glutaraldehyde and osmium. The addition of p-phenylenediamine permitted the visualization by light microscopy of cellular detail not obtained with ruthenium red alone. Ruthenium red is a widely used method for retaining and localizing proteoglycan for electron microscopy but its inability to produce sufficient density within the cells for light microscopy prevented simultaneous visualization by these two modes. The addition of p-phenylenediamine staining overcame this shorcoming without interfering with the ultrastructural detail and both stains can also be used before the making of radioautographs.

Animals↗
PubMed1976

Simultaneous localization of proteoglycan by light and electron microscopy using toluidine blue O. A study of epiphyseal cartilage.

The simultaneous localization of proteoglycan by light and electron microscopy was demonstrated by fixing epiphyseal cartilage in a glutaraldehyde toluidine blue O solution. Sections cut for light microscopy viewing and those cut for electron microscopy required no further staining, although, in the latter case, staining with uranyl acetate and lead improved the overall contrast. By this technique, electron-dense structures were seen concentrated about the cells which were actively synthesizing matrix, and these structures appeared to bind collagen fibrils. Similar structures were not seen in conventionally fixed tissue. They could also not be identified when the specimens were previously incubated with the proteoglycan-digesting enzyme, papain, prior to toluidine blue O fixation. The toluidine blue O fixation method, unlike conventional fixation and staining, retained proteoglycan in the pericellular areas of actively synthesizing cells and made it visible by light and electron microscopy. It appears that proteoglycans is both precipitated and stained by the presence of toluidine blue O during fixation.

Animals↗
PubMed1976

Metabolic recovery of articular cartilage after intra-articular injections of glucocorticoid.

The recovery of adult rabbit articular cartilage after nine weekly intra-articular injections of hydrocortisone acetate was investigated using biochemical, autoradiographic, and tracer methods. After the cessation of steroid injections, the rates of synthesis of proteoglycans and collagen increased by up to 900 per cent. The increase occurred because of accelerated metabolism of existing cells and an increase (caused by mitosis) in the number of functioning chondrocytes. The proteoglycan concentration only returned to normal values after six months.

Animals↗
PubMed1976

The resurfacing of adult rabbit articular cartilage by multiple perforations through the subchondral bone.

The repair of articular cartilage removed from the femora of adult rabbits at the knee was studied. When multiple perforations were made through the subchondral bone a cartilaginous material, staining heavily with safranin O, first filled the holes. This material resembled hyaline cartilage, and by twelve months there was complete resurfacing of the joint, although the material lost its hyaline appearance after eight months and at one year it resembled dense collagenous tissue.

Animals↗
PubMed1975

Alterations of rabbit articular cartilage by intra-articular injections of glucocorticoids.

The destruction of rabbit articular cartilage after intra-articular injections of hydrocortisone acetate was investigated using histological, biochemical, and tracer methods. Fissures and cysts increased in number as increasing amounts of hydrocortisone were given. A linear decrease of hexosamine to less than 50 per cent after twelve injections was accompanied by insigificant changes in deoxyribonucleic acid and hydroxyproline content. The synthesis of proteoglycans and proteins was reduced to one-third, while the production of collagen dropped to less than one-fifth. The changes in thymidine incorporation were not significant. Based on these data, a model indicating the sequence of events which leads to joint destruction after intra-articular injections of glucocorticoid is proposed.

Animals↗