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Biomedical subjects

S B Doty

Publications and source records attributed to S B Doty.

At least 19 recordsLinked to original sources
PubMed1992

FT-IR microscopic mappings of early mineralization in chick limb bud mesenchymal cell cultures.

Chick limb bud mesenchymal cells differentiate into chondrocytes and form a cartilaginous matrix in culture. In this study, the mineral formed in different areas within cultures supplemented with 4 mM inorganic phosphate, or 2.5, 5.0, and 10 mM beta-glycerophosphate (beta GP), was characterized by Fourier-transform infrared (FT-IR) microscopy. The relative mineral-to-matrix ratios, and distribution of crystal sizes at specific locations throughout the matrix were measured from day 14 to day 30. The only mineral phase detected was a poorly crystalline apatite. Cultures receiving 4 mM inorganic phosphate had smaller crystals which were less randomly distributed around the cartilage nodules than those in the beta GP-treated cultures. beta GP-induced mineral consisted of larger, more perfect apatite crystals. In cultures receiving 5 or 10 mM beta GP, the relative mineral-to-matrix ratios (calculated from the integrated intensities of the phosphate and amide I bands, respectively) were higher than in the cultures with 4 mM inorganic phosphate or in the in vivo calcified chick cartilage.

Animals↗
PubMed1992

Studies of matrix vesicle-induced mineralization in a gelatin gel.

Matrix vesicles isolated from fourth-passage cultures of chondrocytes were tested for their ability to induce hydroxyapatite formation in a gelatin gel in order to gain insight into the function of matrix vesicles in in situ mineralization. These matrix vesicles did not appear to be hydroxyapatite nucleators per se since the extent of mineral accumulation in the gel diffusion system was not altered by the presence of matrix vesicles alone, and in the vesicle containing gels, mineral crystals were formed whether associated with vesicles or not. In gels with these matrix vesicles and beta-glycerophosphate, despite the presence of alkaline phosphatase activity, there was no increase in mineral deposition. This suggested that in the gel system these culture-derived vesicles did not increase local phosphate concentrations. However, when known inhibitors of mineral crystal formation and growth (proteoglycan aggregates [4 mg/ml], or ATP [1 mM], or both proteoglycan and ATP) were included in the gel, more mineral was deposited in gels with the vesicles than in comparable gels without vesicles, indicating that enzymes within these vesicles were functioning to remove the inhibition. These data support the suggestion that one function of the extracellular matrix vesicles is to transport enzymes for matrix modification.

Adenosine Triphosphate↗
PubMed1992

Studies of mineralization in tissue culture: optimal conditions for cartilage calcification.

The optimal conditions for obtaining a calcified cartilage matrix approximating that which exists in situ were established in a differentiating chick limb bud mesenchymal cell culture system. Using cells from stage 21-24 embryos in a micro-mass culture, at an optimal density of 0.5 million cells/20 microliters spot, the deposition of small crystals of hydroxyapatite on a collagenous matrix and matrix vesicles was detected by day 21 using X-ray diffraction, FT-IR microscopy, and electron microscopy. Optimal media, containing 1.1 mM Ca, 4 mM P, 25 micrograms/ml vitamin C, 0.3 mg/ml glutamine, no Hepes buffer, and 10% fetal bovine serum, produced matrix resembling the calcifying cartilage matrix of fetal chick long bones. Interestingly, higher concentrations of fetal bovine serum had an inhibitory effect on calcification. The cartilage phenotype was confirmed based on the cellular expression of cartilage collagen and proteoglycan mRNAs, the presence of type II and type X collagen, and cartilage type proteoglycan at the light microscopic level, and the presence of chondrocytes and matrix vesicles at the EM level. The system is proposed as a model for evaluating the events in cell mediated cartilage calcification.

Aggrecans↗
PubMed1992

Morphological studies of bone and tendon.

The Soviet biosatellite COSMOS 2044 carried adult rats on a spaceflight that lasted 13.8 days and was intended to repeat animal studies carried out on COSMOS 1887. Skeletal tissue and tendon from animals flown on COSMOS 2044 were studied by light and electron microscopy, histochemistry, and morphometric techniques. Studies were confined to the bone cells and vasculature from the weight-bearing tibias. Results indicated that vascular changes at the periosteal and subperiosteal region of the tibia were not apparent by light microscopy or histochemistry. However, electron microscopy indicated that vascular inclusions were present in bone samples from the flight animals. A unique combination of microscopy and histochemical techniques indicated that the endosteal osteoblasts from this same mid-diaphyseal region demonstrated a slight (but not statistically significant) reduction in bone cell activity. Electron-microscopic studies of the tendons from metatarsal bones showed a collagen fibril disorganization as a result of spaceflight. Thus changes described for COSMOS 1887 were present in COSMOS 2044, but the changes ascribed to spaceflight were not as evident.

Adaptation, Physiological↗
PubMed1992

Size of metallic and polyethylene debris particles in failed cemented total hip replacements.

Reports of differing failure rates of total hip prostheses made of various metals prompted us to measure the size of metallic and polyethylene particulate debris around failed cemented arthroplasties. We used an isolation method, in which metallic debris was extracted from the tissues, and a non-isolation method of routine preparation for light and electron microscopy. Specimens were taken from 30 cases in which the femoral component was of titanium alloy (10), cobalt-chrome alloy (10), or stainless steel (10). The mean size of metallic particles with the isolation method was 0.8 to 1.0 microns by 1.5 to 1.8 microns. The non-isolation method gave a significantly smaller mean size of 0.3 to 0.4 microns by 0.6 to 0.7 microns. For each technique the particle sizes of the three metals were similar. The mean size of polyethylene particles was 2 to 4 microns by 8 to 13 microns. They were larger in tissue retrieved from failed titanium-alloy implants than from cobalt-chrome and stainless-steel implants. Our results suggest that factors other than the size of the metal particles, such as the constituents of the alloy, and the amount and speed of generation of debris, may be more important in the failure of hip replacements.

Chromium Alloys↗
PubMed1991

Bone particles from gallium-treated rats are resistant to resorption in vivo.

Gallium nitrate is a clinically effective agent for the treatment of cancer related hypercalcemia. The mechanism of action of this agent was investigated following development of a quantitative in vivo bone resorption assay modified from the method of Glowacki. In a preliminary study, the time course of resorption of 50 mg subcutaneous implants of bone powder in growing rats was followed by chemical analysis of mineral (ash and Ca) contents, enzymatic and histochemical assay of tartrate resistant acid phosphatase (TRAP) activity, and image analysis of changes in particle size using von Kossa stained sections. Day 21 was chosen as a single time point for the comparison of the extent of resorption of gallium-containing and control bone particles. Resorption of bone particles containing 0.39 micrograms Ga/mg bone was significantly inhibited relative to control particles. Mineral content (6.7 vs. 3.6 mg), Ca content (1.72 vs. 1.37 mg), and the percentage of the field covered by bone particles (12 vs. 9%) were greater in the animals which received gallium-containing bone particles. Similarly, the number of osteoclast-like cells and the TRAP activity in the gallium-containing bone particle implants at 21 days were increased relative to controls. These data indicate that gallium incorporation into bone matrix confers resistance to resorption.

Acid Phosphatase↗
PubMed1991

Requirement of vitamin C for cartilage calcification in a differentiating chick limb-bud mesenchymal cell culture.

Mesenchymal cells isolated from stage 21-24 chick limb-buds plated in a micro-mass culture differentiate to form chondrocytes and synthesize a calcifiable matrix. In the presence of inorganic phosphate (4 mM), hydroxyapatite mineral deposits around cartilage nodules. Ascorbic acid is, in general, an essential co-factor for extracellular matrix synthesis in culture, since it is required for collagen synthesis. In this study we demonstrate that in the absence of ascorbic acid supplementation in the mesenchymal cell cultures, mineral deposition (indicated by X-ray diffraction, measurement of Ca:hydroxyproline ratio, and 45Ca uptake) does not occur. Concentrations of 10-50 micrograms/ml ascorbate were compared to find the "optimal" concentration for cell mediated mineralization; 25 micrograms/ml was selected as optimal based on matrix appearance at the EM level and the rate of 45Ca uptake. High concentrations of ascorbic acid (greater than 75 micrograms/ml), while increasing the amount of hydroxyproline in the matrix synthesized, caused some cell death and hence less cell-mediated mineralization. This study demonstrates both the need for viable cells and a proper matrix for in vitro cell-mediated mineralization, and shows that varying the concentration of L-ascorbate (vitamin C) in the medium can have a marked effect on mineralization in vitro.

Alkaline Phosphatase↗
PubMed1990

Cosmos 1887: morphology, histochemistry, and vasculature of the growing rat tibia.

Light microscopy, electron microscopy, and enzyme histochemistry were used to study the effects of spaceflight on metaphyseal and cortical bone of the rat tibia. Cortical cross-sectional area and perimeter were not altered by a 12.5-day spaceflight in 3-month-old male rats. The endosteal osteoblast population and the vasculature near the periosteal surface in flight rats compared with ground controls showed more pronounced changes in cortical bone than in metaphyseal bone. The osteoblasts demonstrated greater numbers of transitional Golgi vesicles, possibly caused by a decreased cellular metabolic energy source, but no difference in the large Golgi saccules or the cell membrane-associated alkaline phosphatase activity. The periosteal vasculature in the diaphysis of flight rats often showed lipid accumulations within the lumen of the vessels, occasional degeneration of the vascular wall, and degeneration of osteocytes adjacent to vessels containing intraluminal deposits. These changes were not found in the metaphyseal region of flight animals. The focal vascular changes may be due to ischemia of bone or a developing fragility of the vessel walls as a result of spaceflight.

Alkaline Phosphatase↗
PubMed1987

Histomorphometric analysis of rat skeleton following spaceflight.

Male Sprague-Dawley rats were placed in orbit for 7 days aboard the space shuttle. Bone histomorphometry was performed in the long bones and lumbar vertebrae of flight rats and compared with data derived from ground-based control rats. Trabecular bone mass was not altered during the 1st wk of weightlessness. Strong trends were observed in flight rats for decreased periosteal bone formation in the tibial diaphysis, reduced osteoblast size in the proximal tibia, and decreased osteoblast surface and number in the lumbar vertebra. For the most part, histological indexes of bone resorption were normal in flight rats. The results indicate that 7 days of weightlessness are not of sufficient duration to induce histologically detectable loss of trabecular bone in rats. However, cortical and trabecular bone formation appear to be diminished during the 1st wk of spaceflight.

Animals↗
PubMed1985

Activation of osteoclasts and the repopulation of bone surfaces following hibernation in the bat, Myotis lucifugus.

In previous studies of the adult bat, Myotis lucifugus, bone loss that occurred during the hibernating period was attributed to osteocytic osteolysis, rather than osteoclastic activity. We have used histochemistry and light and electron microscopy to determine the functional state of the skeleton during the hibernating period. We find that during hibernation the marrow cavity of the long bones is filled with lipid deposits interspersed with vascular sinusoids containing mononuclear cells and red blood cells. The lipid deposits are found within fat cells and at extracellular sites. Hematopoietic cells are absent, osteoclasts are absent, and the bone surfaces are covered with quiescent bone-lining cells. Osteocytes retain their structural integrity and maintain canalicular systems with the bone lining cells and with other osteocytes. During the arousal period, osteoclasts reappear on the bone surface, followed in time by increased numbers of osteoblast-like cells. Perivascular cells undergo structural hypertrophy. Many mononuclear cells are now found in extravascular sites. The lipid content in the marrow is gradually reduced and replaced by hematopoietic cells. Each of these events occurs in a repeatable sequence that is related to the state of hibernation of Myotis lucifugus.

Animals↗
PubMed1985

Prosthetic synovitis.

The term "prosthetic synovitis" is applied to reactive changes resulting from a synovial-like membrane formed between a failed prosthesis (noninfected) and the bone interface. This report is the result of light-microscopic and clinical examination of more than 100 specimens obtained at surgery of failed previous hip replacements. The morphology and cell distribution of those tissues removed at surgery in 51 noninfected cemented total hip operations allowed a quantitative estimate of surface cell population by a "touch imprint" technique; qualitative and quantitative estimate (scale, 1 to 4+) of cell population and foreign body materials by light microscopy; and electron microscopy and biochemical analysis of selected samples. Histologic examination included the following cell population, in decreasing order of frequency: acidophilic histiocytes (95%); giant cells (80%); fibronoid material (80%); lymphocyte and plasma cells (26%); and neutrophils (8%). Microscopic examination showed that the largest particles of acrylic cement and shards of high-density polyethylene appeared to be walled off by connective tissue capsules. The majority of smaller particles were incorporated into the histiocyte/macrophage or giant cell population. Histochemistry indicated that these particles elicited "foci" of cellular activity within the synovial-like membrane. This increased activity included the appearance of increased endogenous peroxidase activity in those macrophages within the "foci"; increased betagalactosidae activity among these histiocytes; and a localization of acid phosphates activity within giant cells along the borders of inclusions within the cell cytoplasm. We conclude that wear products resulting from total hip arthroplasty, including the bone cement, can induce increased lysosomal and proteolytic activity within the histiocyte and giant cell populations. It may be important to emphasize that there were "reactive foci" within the membrane and that the entire membrane, even though infiltrated with macrophages, did not respond uniformly to the presence of prosthetic debris. We advance a theory that the first step toward a distractive phenomenon at the interface is micromotion between the cement and bone. Micromotion may be caused by removal of subchondral plate during total hip replacement, leading to fatigue and loss of trabeculae and resultant increase motion, thus bone loss. Bone loss may be the direct result of mechanical injury, increased osteoclasis, or direct lysis of bone by various enzymes released by the interface membrane.

Acetabulum↗
PubMed1984

Tubular microfibrils in the glomeruli of membranous nephropathy.

The presence of tubular microfibrils in the mesangium and capillary walls of the renal glomeruli is a rare occurrence that has been reported by others in different glomerular disease processes. The two cases presented in this report demonstrated such microfibrils in membranous glomerulonephropathy, an association previously described in only one other case. These microfibrils differed in size and structural characteristics from collagen fibers, amyloid fibrils, and cryoglobulin microtubules.

Adult↗
PubMed1981

Morphological evidence of gap junctions between bone cells.

Cell membrane specializations occur at contact sites between adjacent osteoblasts and osteoblasts and osteocytes. These junctions have been described by other investigators as being important in preventing the extracellular movement of material around bone cells. Previously we described how certain small proteins circumvented the osteoblast population and rapidly penetrated the canalicular-osteocyte system. In the present study we used lanthanum colloid as an extracellular marker, the lanthanum readily penetrated the bone cell junctions and the extracellular space of bone. Morphologically, these junctions were not "tight" or "occluding" structures, but resembled "gap" junctions. These gap junctions contained elements which formed intercellular bridges between adjacent cells but also maintained a 2 nm space between cells that contained extracellular fluid. These gap junctions may have an important function in the control or coordination of bone cell activity throughout a given volume of bone.

Aging↗
PubMed1980

Problems inherent in obtaining the alkaline phosphatase reaction.

Problems encountered in the histochemical localization of alkaline phosphatase activity are discussed and solutions presented. The purpose is to achieve a reaction that can be studied by light and electron microscopy and to distinguish alkaline glycerophosphatase from inorganic pyrophosphatase activity. Details are presented concerning fixatives, fixation times, incubation media, enzyme inhibitors, activators, and associated techniques that can be used to obtain optimal histochemical results.

Alkaline Phosphatase↗
PubMed1979

Raman microprobe studies of two mineralizing tissues: enamel of the rat incisor and the embryonic chick tibia.

The laser-Raman microprobe developed at the National Bureau of Standards has been applied to the study of the mineralization process in rat incisor enamel and embryonic chick tibia. Cryostat sections were prepared from fresh frozen tissues and allowed to air dry. In these mineralizing tissues two forms of phosphorus compounds have been observed: (1) an inorganic phase identified as apatitic phosphate and (2) an organic phosphate. The distribution of these components from the mineralizing front to regions of higher mineralization has been determined with a spatial resolution of approximately 15 micrometer. The studies suggest the existence of a carbonate, with a Raman band corresponding to that of the mineral huntite, Mg3Ca(CO3)4, and found in regions of low phosphate mineral content.

Amelogenesis↗
PubMed1977

Inorganic trimetaphosphatase as a histochemical marker for lysosomes in light and electron microscopy.

A new cytochemical method is presented for the light and electron microscopic localization of lysosomes in mineralized and soft tissues. Inorganic trimetaphosphate is used as substrate in a lead chelate incubation medium at pH 3.9. Lysosomes in several tissues are strongly reactive, and reaction product is frequently present in Golgi saccules and GERL. The reaction can be differentiated from acid glycerophosphatase activity, is relatively insensitive to fixation and demineralization procedures, and the reaction is often complete after short incubation times.

Animals↗