RESUMO
During the differentiation cascade of growth plate chondrocytes, cells undergo as much as a 10-15-fold increase in volume. This volume increase, which occurs to different extents in growth plates growing at different rates, has been demonstrated to be the single most significant variable in understanding the quantitative aspects of the cellular kinetics of long bone growth. Our hypothesis is that this volume increase, which occurs through cell swelling by water imbibition, requires intracellular accumulation of osmolytes through activation or upregulation of membrane transport mechanisms. Significant intracellular accumulation of inorganic osmolytes, such as Na+, K+, and Cl-, is potentially disruptive to normal cellular metabolism, whereas intracellular accumulation of organic osmolytes is considered to be more compatible with metabolic function. Thus, we concentrated on determining the contributions of organic osmolytes--betaine, amino acids, inositol, and sorbitol--to volume increase. Pooled cryostat sections of young bovine growth plates were extracted followed by automated analysis for their content of amino acids. Analysis for betaine and the sugar alcohols was done by extraction and derivatization, followed by high-performance liquid chromatography (HPLC). Parallel stereological analyses correlated osmolyte changes to stages of chondrocytic differentiation, specifically comparing intracellular concentration and amount in proliferative vs. hypertrophic chondrocytes. Calculations demonstrated that, maximally, these organic osmolytes, in total, account for 6%-7% of the intracellular osmolytes required to sustain the volume increase, and that the most significant contribution is from betaine. This suggests that intracellular accumulation of organic osmolytes is not a primary strategy used by growth plate chondrocytes during volume increase of their terminal differentiation. The data also suggest that there is a differential regulation of transporters of these osmolytes such that intracellular concentrations are constantly modified as cells proceed through the differentiation cascade.
Assuntos
Condrócitos/citologia , Lâmina de Crescimento/citologia , Osteogênese/fisiologia , Equilíbrio Hidroeletrolítico/fisiologia , Aminoácidos/análise , Animais , Betaína/análise , Bovinos , Diferenciação Celular/fisiologia , Tamanho Celular/fisiologia , Condrócitos/química , Hipertrofia , Inositol/análise , Costelas/citologia , Costelas/crescimento & desenvolvimento , Sorbitol/análiseRESUMO
The role of bone morphogenetic protein-5 (BMP-5) in regulating chondrocytic activity during endochondral ossification was examined in the mouse proximal tibial growth plate. Short ear mice homozygous for the SEA/Gn point mutation in the coding region for BMP-5 (King, J. A. et al. Dev Biol 166:112122; 1994) and heterozygous long ear littermates were examined at 5 and 9 weeks of age (n = 9/group, four groups). Animals were injected with oxytetracycline to estimate the rate of growth and with bromodeoxyuridine to identify proliferative chondrocytes. Age-related changes in chondrocytic stereological and kinetic parameters were compared by image analysis of 1-microm-thick growth plate sections. The number of proliferative chondrocytes did not vary with age in either genotype, but proliferative phase duration increased significantly (approximately 67%) with age in the long ear mice, whereas no change was detected in the short ear mice. The number of hypertrophic chondrocytes increased significantly (approximately 27%) in the short ears, whereas this number decreased significantly (approximately 40%) in the long ears. There was a small, but significant, increase in hypertrophic phase duration (approximately 45%) in short ear mice, but no change was detected in the long ears. These results indicate that BMP-5 deficiency prevents age-related decelerations in chondrocytic proliferation and initiation of hypertrophic differentiation, suggesting a role of BMP-5 in inhibiting these processes.
Assuntos
Desenvolvimento Ósseo/fisiologia , Proteínas Morfogenéticas Ósseas/deficiência , Proteínas Morfogenéticas Ósseas/fisiologia , Lâmina de Crescimento/fisiologia , Tíbia/fisiologia , Animais , Proteína Morfogenética Óssea 5 , Proteínas Morfogenéticas Ósseas/genética , Bromodesoxiuridina/metabolismo , Contagem de Células , Diferenciação Celular , Divisão Celular/fisiologia , Condrócitos/fisiologia , Modelos Animais de Doenças , Feminino , Lâmina de Crescimento/citologia , Hipertrofia/patologia , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos Knockout , Oxitetraciclina , Tíbia/citologiaRESUMO
The pericellular matrix surrounding chondrocytes from all zones of epiphyseal growth plate cartilage, as well as from articular, tracheal, and auricular cartilage, was examined using a number of variations of osmium ferrocyanide postfixation of aldehyde-fixed tissues. Comparisons were made with other fixative techniques, including ruthenium red, safranin O, and lanthanum nitrate, all of which have previously been reported to stabilize a variety of lacunar matrix components. An electron-dense material was preserved uniquely by osmium-ferrocyanide in the lacunar matrix of mid and late zone hypertrophying chondrocytes and was absent from all other zones of the growth plate as well as from the other types of cartilage examined. Because of its highly restricted distribution, this electron-dense material is hypothesized to represent a pericellular matrix component involved with either matrix calcification or metaphyseal capillary penetration. Several hypotheses are presented as to its specific composition.
Assuntos
Cartilagem/citologia , Ferrocianetos , Fixadores , Osmio , Animais , Cartilagem/ultraestrutura , Microscopia Eletrônica , Suínos , Porco MiniaturaRESUMO
A postembedment method for the localization of lectin-binding glycoconjugates was developed using Epon-embedded growth plate cartilage from Yucatan miniature swine. By testing a variety of etching, blocking, and incubation procedures, a standard protocol was developed for 1 micron thick sections that allowed visualization of both intracellular and extracellular glycoconjugates with affinity for wheat germ agglutinin and concanavalin A. Both fluorescent and peroxidase techniques were used, and comparisons were made between direct methods and indirect methods using the biotin-avidin bridging system. Differential extracellular lectin binding allowed visualization of interterritorial , territorial, and pericellular matrices. Double labeling experiments showed the precision with which intracellular binding could be localized to specific cytoplasmic compartments, with resolution of binding to the Golgi apparatus, endoplasmic reticulum, and nuclear membrane at the light microscopic level. This method allows the localization of both intracellular and extracellular lectin-binding glycoconjugates using fixation and embedment procedures that are compatible with simultaneous ultrastructural analysis. As such it should have applicability both to the morphological analysis of growth plate organization during normal endochondral ossification, as well as to the diagnostic pathology of matrix abnormalities in disease states of growing cartilage.
Assuntos
Metabolismo dos Carboidratos , Lâmina de Crescimento/metabolismo , Técnicas Histológicas , Lectinas , Porco Miniatura/metabolismo , Animais , Concanavalina A/metabolismo , Resinas Epóxi , Matriz Extracelular/metabolismo , Lâmina de Crescimento/ultraestrutura , Histocitoquímica , Microscopia Eletrônica , Microscopia de Fluorescência , Suínos , Aglutininas do Germe de TrigoRESUMO
The distribution of intracellular and extracellular lectin-binding glycoconjugates of the reserve cell zone of growth plate cartilage was studied in the distal radial growth plate of 4-week-old Yucatan swine using a postembedment method on Epon-embedded sections. Direct comparisons were made to articular, tracheal, and auricular cartilages not involved in endochondral ossification. All patterns of lectin binding that in the growth plate were restricted to the reserve cell zone were also patterns characteristic of tracheal, articular, and auricular cartilages. These included: (a) pericellular binding with peanut agglutinin (PNA) without prior digestion with neuraminidase; (b) pericellular binding with wheat germ agglutinin (WGA) at 24 h; (c) intracellular cytoplasmic binding to concanavalin A (CON-A), Lens culinaris agglutinin (LCA), and Lotus tetragonobolus agglutinin (LTA) after periodic acid oxidation; and (d) a lack of pericellular binding with CON-A and ricin agglutinin 1 (RCA-1) after periodic acid oxidation. We conclude that reserve zone chondrocytes lack specific phenotypic markers as defined by lectin-binding affinity that are found in the cellular zones of the growth plate that undergo calcification and vascularization. The reserve zone has identical lectin-binding affinities to the three structural cartilages used as controls. One interpretation of these results is that the reserve zone may not be involved directly in endochondral ossification, but may have a structural function in growth plate cartilage.
Assuntos
Glicoconjugados/metabolismo , Lâmina de Crescimento/citologia , Lectinas/metabolismo , Animais , Animais Selvagens , Orelha , Glicoconjugados/análise , Lâmina de Crescimento/metabolismo , Histocitoquímica , Osteogênese , Especificidade da Espécie , Suínos , TraqueiaRESUMO
In the distal hypertrophic cell zone of growth plate cartilage, the penetration of metaphyseal vascular endothelial cells is into the noncalcified territorial and pericellular matrices. Cellular mechanisms that promote metaphyseal vascularization are understood poorly, partly because no study has addressed the question of the time sequence of cellular interactions at the chondro-osseous junction. The purpose of the present study is to make predictions about the relative and the real time duration of cellular events during vascular invasion, including an analysis of the time sequence of death of the terminal hypertrophic chondrocyte. The data from serial section analysis at the light microscopical level of tetracycline-labeled growth plates indicate that death of the terminal hypertrophic chondrocyte occurs in discrete morphological stages characterized by rapid cellular condensation followed, within minutes, by endothelial cell penetration into the vacated lacuna. Cellular condensation lasts approximately 45 min or 18% of the time a cell spends as a terminal chondrocyte. The data also demonstrate that chondrocytic death occurs prior to invasion by vascular endothelial cells and that the chondrocytic lacuna remains empty for as long as 15 min before an endothelial cell or blood vascular cell fills the space.
Assuntos
Endotélio Vascular/citologia , Lâmina de Crescimento/irrigação sanguínea , Animais , Sobrevivência Celular , Lâmina de Crescimento/citologia , SuínosRESUMO
The functional unit within the growth plate consists of a column of chondrocytes that passes through a sequence of phases including proliferation, hypertrophy, and death. It is important to our understanding of the biology of the growth plate to determine if distal hypertrophic cells are viable, highly differentiated cells with the potential of actively controlling terminal events of endochondral ossification prior to their death at the chondro-osseous junction. This study for the first time reports on the visualization of living hypertrophic chondrocytes in situ, including the terminal hypertrophic chondrocyte. Chondrocytes in growth plate explants are visualized using rectified differential interference contrast microscopy. We record and measure, using time-lapse cinematography, the rate of movement of subcellular organelles at the limit of resolution of this light microscopy system. Control experiments to assess viability of hypertrophic chondrocytes include coincubating organ cultures with the intravital dye fluorescein diacetate to assess the integrity of the plasma membrane and cytoplasmic esterases. In this system, all hypertrophic chondrocytes, including the very terminal chondrocyte, exist as rounded, fully hydrated cells. By the criteria of intravital dye staining and organelle movement, distal hypertrophic chondrocytes are identical to chondrocytes in the proliferative and early hypertrophic cell zones.
Assuntos
Cartilagem/patologia , Lâmina de Crescimento/patologia , Animais , Cartilagem/ultraestrutura , Movimento Celular , Sobrevivência Celular , Lâmina de Crescimento/ultraestrutura , Hipertrofia , Microscopia de Interferência , Filmes Cinematográficos , Organelas/fisiologia , Ratos , Ratos Endogâmicos , SuínosRESUMO
In this study, we tested the hypothesis that hypertrophic cell volume varies directly with the rate of longitudinal bone growth. The volume of hypertrophic chondrocytes (using stereological techniques) and longitudinal bone growth per 24 h (using oxytetracycline labeling techniques) were measured in the proximal and distal radial growth plates and the proximal and distal tibial growth plates of 21- and 35-day-old hooded rats and 21- and 35-day-old Yucatan pigs. We demonstrated a high coefficient of correlation (rats 0.98, pigs 0.83) between the final volume of hypertrophic chondrocytes and the rate of longitudinal bone growth over a wide range of growth rates and volumes of hypertrophic chondrocytes. In addition, we demonstrated a positive linear relationship between the rate of longitudinal bone growth and the final volume of hypertrophic chondrocytes. The slope of the regression line was different for rats than for pigs. The relationship was independent of the location of the growth plate in the animal and the age of the animal. The data suggest that mechanisms regulating volume changes in hypertrophic chondrocytes may exist and that chondrocytic volume increase is a major determinant of the rate of longitudinal bone growth. However, the relative contribution of cellular hypertrophy to longitudinal bone growth may be different in rats than in pigs.
Assuntos
Desenvolvimento Ósseo/fisiologia , Cartilagem/patologia , Lâmina de Crescimento/crescimento & desenvolvimento , Animais , Feminino , Lâmina de Crescimento/citologia , Hipertrofia , Análise dos Mínimos Quadrados , Masculino , Oxitetraciclina , Rádio (Anatomia) , Ratos , Análise de Regressão , Suínos , TíbiaRESUMO
Differential elongation of growth plates is the process by which growth-plate chondrocytes translate the same sequence of gene regulation into the appropriate timing pattern for a given rate of elongation. While some of the parameters associated with differential growth are known, the purpose of this study was to test the hypothesis that eight independent variables are involved. We tested this hypothesis by considering four different growth plates in 28-day-old Long-Evans rats. Temporal parameters were provided by means of oxytetracycline and bromodeoxyuridine labeling techniques. Stereological parameters were measured with standard techniques. For all four growth plates, the calculated number of new chondrocytes produced per day approximated the number of chondrocytes lost per day at the chondro-osseous junction. This suggests that the proposed equations and associated variables represent a comprehensive set of variables defining differential growth. In absolute numbers, the proximal tibial growth plate produced about four times as many chondrocytes per day as the proximal radial growth plate (16,400 compared with 3,700). In the proximal tibia, 9% of growth is contributed by cellular division; 32%, by matrix synthesis throughout the growth plate; and 59%, by chondrocytic enlargement during hypertrophy. In the more slowly elongating growth plates, the relative contribution to elongation from cellular enlargement decreases from 59 to 44%, with a relative increase in contribution from matrix synthesis ranging from 32% in the proximal tibia 49% in the proximal radius. This study suggests that differential growth is best depicted as a complex interplay among cellular division, matrix synthesis, and cellular enlargement during hypertrophy. Differential growth is best explained by considering a set of eight independent variables, seven of which vary from growth plate to growth plate. Thus, this study confirms the importance of cellular hypertrophy during elongation and adds to our understanding of the importance of locally mediated regulatory systems controlling growth-plate activity.
Assuntos
Lâmina de Crescimento/citologia , Lâmina de Crescimento/fisiologia , Animais , Matriz Óssea/metabolismo , Ciclo Celular , Lâmina de Crescimento/metabolismo , Masculino , Rádio (Anatomia) , Ratos , Ratos Endogâmicos , Tíbia , Fatores de TempoRESUMO
Regulation of postnatal growth of long bones occurs in multiple levels of chondrocytic activity, including stem cell proliferation, proliferative zone cycling, and regulation of changes in chondrocytic shape during hypertrophy. The differentiation sequence of chondrocytes is the same in all growth plates, but rates of elongation at a single point in time and over a period of time differ widely among individual growth plates, which suggests that the rates of sequential gene activation and suppression in this phenotypic pattern can vary. The purpose of this study was to investigate, directly and in vivo, parameters of the cell cycle of proliferative chondrocytes in growth plates growing at widely different rates at a single point in time in order to analyze the relationship between cell cycle time, including the duration of each phase of the cell cycle (G1, S, G2, and M), and the rate of growth. The experimental design used repeated pulse labeling with bromodeoxyuridine and was analyzed using a regression model of time of pulse label with increasing labeling index. Total cell cycle time was calculated as the inverse of the slope of the relationship of the labeling index and the time between labels. The y intercept was the calculated labeling index at time zero. Multiple comparison contrasts were used to test for individual differences among four growth plates with growth rates ranging from approximately 50 to 400 microns per 24 hours from 28-day-old rats. The estimate of total cell cycle time for the proximal tibial growth plate was 30.9 hours. Cell cycle times for the other three growth plates were 34.0, 48.7, and 76.3 hours for the distal radius, distal tibia and proximal radius, respectively. Although the times for the proximal tibia and distal radius did not differ significantly, all other times were significantly different (p < 0.05). Almost all differences in total cell cycle time were attributable to significant differences in the length of the G1 phase. The S phase was estimated at 3.4-6.1 hours; the G2 phase, at 3.0 hours; and the M phase, at 0.5-0.6 hours. The current study suggests that regulation through cell cycle parameters, specifically in the G1 phase, may be involved in overall regulation of differential postnatal long bone growth. It has previously been established that increase and shape change of cellular volume during hypertrophy may be regulated at the level of individual growth plates and that both are significant in understanding differential growth of long bone at this level. By demonstrating that chondrocytes in the proliferating zone have different cell cycle times that are regulated primarily through differences in the duration of G1, this study suggests that, in addition to systemic controls of chondrocyte proliferation, local controls may modulate rates of proliferation of individual growth plates and thus may be another locally mediated regulator of differential growth.
Assuntos
Cartilagem/citologia , Ciclo Celular/fisiologia , Lâmina de Crescimento/citologia , Animais , Divisão Celular/fisiologia , Masculino , Ratos , Ratos Endogâmicos , Análise de Regressão , Tíbia/ultraestruturaRESUMO
PURPOSE: To determine if pentoxifylline, interleukin 1alpha, selenium and misoprostol can minimize damage to physeal longitudinal growth during single radiation dose exposure in an animal model. MATERIALS AND METHODS: Eighty-seven weanling Sprague-Dawley rats were randomized into 15 drug/dose groups. All groups received a single 17.5-Gy gamma-irradiation exposure to the right knee, the left limb serving as an internal control. Pentoxifylline was injected 30 min before exposure, sodium selenite and interleukin 1alpha 24 h before exposure and misoprostol 2 h before exposure. Positive controls received 17.5 Gy. At 6 weeks, animals were sacrificed, the hind limb lengths were measured and detailed histomorphometric analysis was performed. RESULTS: Statistically significant reductions (p < or = 0.03) in mean limb length discrepancy compared with irradiation alone were seen following administration of pentoxifylline (50 mg kg(-1)), interleukin 1alpha (15 mcg kg(-1)), selenium (5 mg kg(-1)) and misoprostol (20 mg kg(-1)). Histomorphometric endpoints and growth rate remained altered at 6 weeks despite treatment, but length discrepancy reduction was highly correlated with the appearance of regenerative clones. CONCLUSIONS: Each drug reduced the amount of anticipated growth arrest in the animal model and some compared favourably in magnitude with that previously demonstrated for the established radioprotectant drug amifostine. Restoration of growth appears related to appearance of regenerative clones.
Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Desigualdade de Membros Inferiores/prevenção & controle , Protetores contra Radiação/farmacologia , Animais , Desenvolvimento Ósseo/efeitos da radiação , Regeneração Óssea/efeitos dos fármacos , Regeneração Óssea/efeitos da radiação , Interleucina-1/farmacologia , Ossos da Perna/efeitos dos fármacos , Ossos da Perna/efeitos da radiação , Desigualdade de Membros Inferiores/etiologia , Masculino , Misoprostol/farmacologia , Modelos Animais , Pentoxifilina/farmacologia , Lesões Experimentais por Radiação/fisiopatologia , Ratos , Ratos Sprague-Dawley , Selênio/farmacologiaRESUMO
Regulation of growth of long bones occurs in cartilage growth plates, where proliferation of chondrocytes, matrix synthesis, and an increase in vertical height in the direction of growth all contribute to the final length of a bone. In this study, we tested the hypothesis that an increase in chondrocytic vertical height is a major variable that accounts for the decreased rate of growth of long bones in Scottish deerhound dogs that had pseudoachondroplasia. The diagnosis of pseudoachondroplasia is based, primarily, on the demonstration of alternating electron-dense and electron-lucent lamellae with a periodicity of 100 to 150 nanometers in dilated rough endoplasmic reticulum. These ultrastructural changes are similar to those seen in humans who have pseudoachondroplasia. In Scottish deerhounds that have the disease, growth of bone is approximately 65 per cent of that in normal animals. There were striking differences in the diameters of proliferating and hypertrophic chondrocytes in pseudoachondroplastic animals compared with normal animals. Specifically, the horizontal diameter of proliferating chondrocytes was 22.7 micrometers in normal animals and 11.3 micrometers in pseudoachondroplastic animals. The vertical diameter of proliferating chondrocytes was 4.8 and 7.6 micrometers in normal and pseudoachondroplastic animals. In the distal 100 micrometers of the hypertrophic zone, the mean horizontal diameter of hypertrophic chondrocytes was 29.6 and 19.1 micrometers and the mean vertical diameter was 22.8 and 18.6 micrometers in normal and pseudoachondroplastic animals. All these differences were statistically significant. The changes in vertical height resulted in a significant difference in the incremental difference in vertical height between chondrocytes from the proliferative and hypertrophic zones in normal animals (18.0 micrometers per chondrocyte) and pseudoachondroplastic animals (11.0 micrometers per chondrocyte). Each chondrocyte in the abnormal plates achieved only 61 per cent of the incremental difference of chondrocytes in normal plates. The mean cellular volume of chondrocytes in the hypertrophic zone was 13,050 cubic micrometers in the normal animals and 10,740 cubic micrometers in the pseudoachondroplastic animals. This difference was not statistically significant. These results are discussed in relation to current theories of the role of the shape and change in volume of chondrocytes in the regulation of longitudinal growth of bone.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Acondroplasia/patologia , Desenvolvimento Ósseo , Acondroplasia/fisiopatologia , Animais , Cartilagem Articular/ultraestrutura , Cães , Feminino , Lâmina de Crescimento/ultraestrutura , Masculino , Microscopia Eletrônica , Microscopia de PolarizaçãoRESUMO
Electron microscopy was used to diagnose primary ciliary dyskinesia in a litter of English pointer dogs and in a golden retriever dog. A technique of membrane solubilization, fixation, and negative staining with glutaraldehyde tannic acid identified abnormally constructed central and B microtubules in respiratory cilia from dogs with primary ciliary dyskinesia. Shortened outer dynein arms commonly associated with primary ciliary dyskinesia actually represents the absence of a specific subset of the three most peripheral components of the whole outer dynein arm structure.
Assuntos
Transtornos da Motilidade Ciliar/veterinária , Doenças do Cão/patologia , Animais , Cílios/patologia , Transtornos da Motilidade Ciliar/patologia , Cães , Feminino , Masculino , Microscopia Eletrônica , Traqueia/patologiaRESUMO
A contributing factor to the lack of understanding the cause of osteochondritic syndromes has been incomplete knowledge of the morphology of lesions in subclinical stages of the disease. In osteochondritic growth plate cartilage from growing swine, the morphology of the pericellular matrix surrounding hypertrophic zone chondrocytes is abnormal and is characteristic of a matrix in which the ordered interactions of matrix macromolecules with each other and with the plasma membrane have been altered. In the present study, ultrastructural histochemical techniques were used to analyze the nature of macromolecular interactions in the pericellular matrix in normal growth plate cartilage, and selective enzyme digestions of normal growth plate cartilage were used to simulate the morphology found in osteochondritic lesions. Results showed that a pericellular macromolecular material which was both ferrocyanide positive and trypsin sensitive was essential for stabilizing the cell membrane/pericellular interface in normal growth plates. The highly variable morphology of this same material in osteochondritic lesions was simulated by hyaluronidase digestion. Since similar pericellular matrix abnormalities have not been described in other diseases of growth plate cartilage, they may represent a matrix abnormality unique to the vascularization failure of osteochondritic syndromes. Our ability to simulate the ultrastructural morphology of subclinical osteochondritic lesions enhances the potential for understanding the macromolecular changes found in the pericellular matrix of osteochondritic cartilage. Based on these results, a new hypothesis is presented for the early sequence of events in the pathogenesis of osteochondrosis.
Assuntos
Lâmina de Crescimento/ultraestrutura , Osteocondrite/veterinária , Doenças dos Suínos/patologia , Suínos/anatomia & histologia , Animais , Histocitoquímica , Microscopia Eletrônica , Osteocondrite/patologiaRESUMO
Chondrocytes from the distal ulnar physis of 1- and 5-month-old boars were studied by transmission electron microscopy. Among reserve, proliferating, and early hypertrophic chondrocytes, 2 distinct cell types (dark cells and light cells) were recognized, and in the distal proliferative and early hypertrophic regions, dark cells seemed to be involved in matrix vesicle production. Late hypertrophic light cells seemed to follow 2 lines of maturation. One pathway resulted in cells with a stellate shape, typical of hypertrophic cells in several species other than swine. A 2nd pathway resulted in large oval cells with water-clear cytoplasm. These cells appeared to become encased in a lamina of fine fibrils interspersed with irregularly shaped electron-dense material. The functional significance of these 2 pathways remains unclear.
Assuntos
Cartilagem/ultraestrutura , Epífises/ultraestrutura , Suínos/anatomia & histologia , Animais , Cartilagem/crescimento & desenvolvimento , Epífises/crescimento & desenvolvimento , Hipertrofia , Masculino , Microscopia Eletrônica , Suínos/crescimento & desenvolvimento , Ulna/crescimento & desenvolvimento , Ulna/ultraestruturaRESUMO
In this study, lectin-binding techniques are applied to growth-plate cartilage to analyze the intracellular localization of lectin-binding glycoconjugates of chondrocytes in situ. The binding of ten fluorescein-conjugated lectins is analyzed on 1-micron-thick Epon-embedded, nondecalcified sections of growth plates from Yucatan swine. Comparisons are made to intracellular binding in chondrocytes of tracheal, articular, and auricular cartilage. Ear epithelium, tracheal epithelium, and kidney are used as positive control tissues for the specificity of lectin binding. Only the mannose-binding lectins had affinity for the RER and nuclear envelope. Eight lectins reacted within the Golgi complex with characteristic patterns which ranged from localized fine linear strands to extensive vesicular accumulations. When cartilage slabs were exposed before embedment to the ionophore monensin to disrupt intracellular transport through the Golgi, it was possible to define differential subcompartments of the Golgi complex, based upon sites of sugar addition. Also, it was possible to characterize the cytoplasmic deposits of reserve-zone chondrocytes which were positive with concanavalin A as glycogen, based upon their sensitivity to amylase. This method allows resolution at the light-microscopic level of lectin-binding glycoconjugates with localization to specific organelles. Patterns of intracellular binding were consistent with biochemical data relating to the subcellular localization of processing steps of complex carbohydrates prior to secretion.
Assuntos
Cartilagem/citologia , Epífises/citologia , Fluoresceínas/metabolismo , Lectinas/metabolismo , Animais , Cartilagem/metabolismo , Concanavalina A/metabolismo , Retículo Endoplasmático/metabolismo , Fluoresceína , Glicogênio/metabolismo , Complexo de Golgi , Manose/metabolismo , Microscopia Eletrônica , Monensin/farmacologia , Suínos , Aglutininas do Germe de TrigoRESUMO
Recent biochemical and morphologic evidence supports the concept that hypertrophic chondrocytes of growth plate cartilage are fully viable cells that play a major functional role in controlling endochondral ossification. However, events associated with chondrocyte death remain unknown. In this study we assess the viability of terminal hypertrophic chondrocytes in situ in an organ culture system viewed simultaneously with rectified Nomarski interference contrast optics and with vital staining under fluorescence optics. Second, we use two methods of optimal chemical fixation at the ultrastructural level to define morphologically distinct stages of the terminal hypertrophic chondrocyte, which we interpret as the stages preceding chondrocyte death. An analysis of serial sections at the light microscope level showed that terminal chondrocytes were found, with different probabilities, in three morphologically distinguishable stages. Seventy-five percent of all profiles were fully hydrated cells with an intact plasma membrane making direct contact with the pericellular matrix, a morphology identical to that of living terminal chondrocytes viewed in Nomarski optics. Approximately 1% of terminal chondrocytes, while still in a fully hydrated state, consistently made a direct asymmetrical contact of the plasma membrane with the last transverse septum. In 24% of the profiles, terminal chondrocytes were found as condensed cells that retained their attachment to the last transverse septum. The stages were not characteristic of chondrocytes positioned more proximally in the growth plate. We hypothesize that a condensed morphology eventually characterizes each hypertrophic chondrocyte, and we relate these observations to current hypotheses concerning the mechanism of death of hypertrophic chondrocytes.
Assuntos
Lâmina de Crescimento/citologia , Animais , Membrana Celular/ultraestrutura , Sobrevivência Celular , Fixadores , Microscopia Eletrônica , Microscopia de Fluorescência , Técnicas de Cultura de Órgãos , SuínosRESUMO
In the distal hypertrophic zone of growth-plate cartilage, the pericellular matrix surrounding individual chondrocytes and the territorial matrix uniting chondrocytes into columnar groups are invaded by metaphyseal endothelial cells prior to osteogenesis. In the present study, lectin-binding glycoconjugates were analyzed in these two matrix compartments of growth-plate cartilage from Yucatan swine. Nine lectin-fluorescein conjugates were tested by a postembedment method on 1-micron-thick, nondecalcified, Epon-embedded sections. Chondrocytes in all cellular zones were surrounded by a pericellular matrix which showed positive binding for peanut agglutinin (PNA), ricin agglutinin (RCA-I), and soybean agglutinin (SBA). Binding by these lectins was sensitive to digestion with hyaluronidase, chondroitinase, and trypsin. Pericellular glyconconjugtes that bind RCA-I and concanvalin A (CONA) after periodic acid oxidation, and which were sensitive to trypsin but not to chondroitinase or hyaluronidase, were present in the hypertrophic cell zone. Within the territorial matrix, binding of lectins specific for galactose, N-acetylgalactosamine, and fucose showed gradients of intensity which became maximal at the last transverse septum. Lectin-binding histochemistry more precisely differentiated the microheterogeneity of glycoconjugate distribution within these two matrix compartments than has been possible with other histochemical techniques. Lectin-binding affinity is a potentially useful technique by which to isolate cartilage matrix macromolecules unique to specific cellular zones of the growth plate.
Assuntos
Lâmina de Crescimento/metabolismo , Lectinas/metabolismo , Animais , Metabolismo dos Carboidratos , Membrana Celular/metabolismo , Condroitinases e Condroitina Liases , Concanavalina A/metabolismo , Matriz Extracelular/metabolismo , Lâmina de Crescimento/anatomia & histologia , Histocitoquímica , Hialuronoglucosaminidase , Ricina/metabolismo , Suínos , TripsinaRESUMO
Chondrocytes of the cartilaginous growth plate are found in a spatial gradient of cellular differentiation beginning with cellular proliferation and ending with cellular hypertrophy. Although it is recognized that both proliferation and hypertrophy contribute significantly to overall bone growth, mechanisms acting on the chondrocyte to control the timing, the rate, and the extent of hypertrophy are poorly understood. Similarly, mechanisms acting on the terminal chondrocyte to cause its death at the chondro-osseous junction have not been investigated. In this study we examine the condensation of terminal hypertrophic chondrocytes in proximal and distal radial growth plates of Yucatan swine at 4 weeks of age. The animals were raised in a controlled environment where activity and feeding patterns were synchronized to a given time in the light/dark cycle. We analyzed cellular condensation both as a function of circadian rhythms in a 24-hr time period, and as a function of overall rate of growth. The data suggest that the magnitude of circadian influences on long bone growth is significantly damped at the level of the hypertrophic chondrocyte compared to that seen by previous investigators studying circadian influences on chondrocytic proliferation. Secondly, the condensation of hypertrophic chondrocytes at the chondro-osseous junction varies inversely with rate of growth in length of the bone. At any time period, a higher percentage of terminal chondrocytes in the condensed form was found in the slower-growing of the two growth plates. We relate these findings to current hypotheses concerning controls of chondrocytic hypertrophy and possible controls over the timing of hypertrophic cell death.
Assuntos
Lâmina de Crescimento/citologia , Suínos/crescimento & desenvolvimento , Animais , Desenvolvimento Ósseo/fisiologia , Contagem de Células , Divisão Celular , Ritmo Circadiano/fisiologia , Feminino , Lâmina de Crescimento/patologia , Lâmina de Crescimento/fisiologia , Hipertrofia , Masculino , GravidezRESUMO
The arrangement of C-tubule protofilaments was studied in basal bodies of respiratory epithelial cilia using tannic acid staining techniques. At the proximal end of the basal body, the C tubule consisted of 10 protofilaments arranged analogously to the 10 protofilaments of the B tubule. In the region of the basal foot, 5 of the C protofilaments sequentially terminated. First, the C10 and C9 filaments terminated. Then the C1 and C8 filaments terminated, and finally the C7 filament terminated leaving a curved sheet of 5 filaments C2-C6 coursing through the distal third of the basal body. This study validates previous suggestions that the C tubule is incomplete distally, and this study clearly demonstrates the extent and consistency of this incompleteness. This morphological study also raises again the question of the function of the C tubules of basal bodies in light of both the incomplete nature of the C tubule and the apparent minor role of the C tubule in providing attachment sites for the basal foot and the alar sheets.