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Two positional characteristics of the ciliary axoneme--its location on the plasma membrane as it emerges from the cell, and its orientation in three-dimensional (3D) space--are known to be critical for optimal function of actively motile cilia (including nodal cilia), as well as for modified cilia associated with special senses. However, these positional characteristics have not been analyzed to any significant extent for primary cilia. This review briefly summarizes the history of knowledge of these two positional characteristics across a wide spectrum of cilia, emphasizing their importance for proper function. Then the review focuses what is known about these same positional characteristics for primary cilia in all major tissue types where they have been reported. The review emphasizes major areas that would be productive for future research for understanding how positioning and 3D orientation of primary cilia may be related to their hypothesized signaling roles within different cellular populations., (Copyright © 2011 Wiley Periodicals, Inc.)

Primary cilia have functions as sensory organelles integral to signal transduction and establishment of cell polarity. In articular cartilage the primary cilium has been hypothesized to function as an antenna to sense the biomechanical environment, regulate the secretion of extracellular matrix components, and maintain cellular positional information, leading to high tissue anisotropy. We used analysis of electron microscopy serial sections to demonstrate positional attributes of the primary cilium of adult equine articular chondrocytes in situ. Data for ~500 axonemes, comparing superficial to radiate chondrocytes from both load-bearing and non-load-bearing regions, were graphed using spherical co-ordinates θ, φ. The data demonstrate the axoneme has a definable orientation in 3D space differing in superficial and radiate zone chondrocytes, cells that differ by 90° in the orientation of their major axes to the articular surface. Axonemal orientation is more definable in load-bearing than in non-load-bearing areas. The position of emergence of the axoneme from the cell also is variable. In load-bearing regions of the superficial zone, extension of the axoneme is from the cellular side facing the subchondral bone. In radiate zone cells, axonemes extend from either face of the chondrocyte, that is, both toward the articular surface or toward the subchondral bone. In non-load-bearing regions this consistency is lost. These observations relate to current hypotheses concerning establishment of tissue anisotropy in articular cartilage during development, involving both migration of cells from the joint periphery and a restricted zone of division within the tissue resulting in the columnar arrangement of radiate zone cells., (Copyright © 2011 Wiley-Liss, Inc.)

Background: The fluoroquinolones are a relatively new class of antimicrobials with an appealing spectrum of activity. Their use in pediatric medicine is limited because of the concern over possible growth inhibition, as published reports have documented articular cartilage damage in animal models after their administration. These data, extrapolated to include the epiphyseal cartilage, suggest that these agents may reduce growth rates, but limited human data are at the least equivocal, if not strictly contradictory to such claims. Specific investigations into the effects of fluoroquinolones on epiphyseal plate cartilage and growth velocity have not been performed., Methods: Gatifloxacin and ciprofloxacin were used as representative agents of the fluoroquinolone class. Each drug was administered to experimental lambs over a 14-day interval at a dose designed to reflect those used in pediatric medicine. Recumbent versus standing intervals were used to monitor for arthropathy. Upon completion of fluoroquinolone administration, lambs underwent double fluorochrome labeling for determination of growth velocity. Gross and microscopic analysis of articular cartilage was performed to assess for pathologic changes. Age- and sex-matched lambs served as controls., Results: Neither gatifloxacin nor ciprofloxacin negatively affected growth velocity of the proximal tibial growth plate as measured by double fluorochrome labeling. In addition, no difference between experimental and control lambs in regard to recumbent versus standing intervals was noted. Examination of the articular cartilage failed to suggest chondrotoxicity., Conclusion: Fluoroquinolone antimicrobials do not affect growth velocity in the ovine model when administered along a dosing regimen that closely models that seen in pediatric medicine., Clinical Relevance: Fluoroquinolones may be acceptable for use in the pediatric population, as concerns over chondrotoxicity and growth inhibition may not be valid. These data suggest that expanded studies in lambs and other species, including humans, with differences in dosing and duration are justified to ultimately demonstrate clinical safety.

Background: Disruption of the periosteum, whether traumatic or elective, has long been known to accelerate growth in the developing skeleton. However, the extent, timing, and mechanism of the resultant increase in growth velocity (if any) remain undefined. The primary research questions were: Does periosteal resection result in a change (increase) in growth velocity of a long bone at the growth plate? When does the effect start after the resection and for how long? Finally, which of several cellular mechanisms is most likely responsible for the change in growth velocity?, Methods: Five lambs underwent proximal tibial growth plate periosteal resection with subsequent measurement of growth velocity by implantable microtransducers or fluorochrome labeling. This former technique provided real-time growth velocity data with a resolution of about 10 microm (width of a proliferative zone chondrocyte). These measurements were accurate at up to 4 weeks postoperative, as verified by fluorochrome labeling, and radiographic measurement. Two lambs were continued on the study for an additional 3 weeks. Histomorphometric and stereological assessments of chondrocytic kinetic parameters were performed on control and experimental tibiae after euthanasia., Results: Periosteal resection increased growth velocity in every lamb, at every time point, and in a consistent and sustained manner. Histomorphometric correlation to this phenomenon indicated that the cellular basis of this acceleration was most likely the result of hypertrophic chondrocyte axial elongation rather than changes in chondrocyte proliferation, magnitude of hypertrophic chondrocytic swelling, or increased matrix production., Conclusions: Periosteal resection creates immediate and sustained acceleration of growth resulting from axial elongation of the hypertrophic chondrocyte. Although the increase in growth velocity was consistent, the absolute magnitude of the acceleration suggests that periosteal resection be considered as an adjunct to other primary procedures. Periosteal resection may serve as a useful clinical adjunct to provide a modest growth stimulus in cases of hemihypertrophy or angular limb deformity or to counteract the growth inhibition seen when performing distraction osteogenesis.

Differential growth is the phenomenon whereby growth plates in the same individual at the same time all have uniquely different axial growth velocities. Differential growth is clearly present in the adolescent skeleton. In this study we ask two questions. When and by what pattern does the phenomenon of differential growth begin? Second, to what extent are the development of differential growth velocities correlated with changes in hypertrophic chondrocyte volume and/or with changes in chondrocytic production/turnover? Four growth plates (proximal and distal radial; proximal and distal tibial) were studied at 24 different time points in Long-Evans rats between the 17th gestational day (when differential growth does not exist) and postnatal day 27 (when differential growth is well established). Growth velocities were measured using fluorochrome labeling. Using stereological methodology, multiple chondrocytic kinetic parameters were measured for all growth plates. Elongation of the proximal radial growth plate decreases relative to elongation in the other three growth plates in the late fetal phase. Differential growth is fully expressed at postnatal day 13 when the other three growth plates start to decrease daily elongation at different rates. Differential growth is primarily associated with differences in hypertrophic cell volume manifested when growth deceleration occurs. This study also illustrates that differential growth is superimposed on systemic regulators that affect all growth plates simultaneously. The most dramatic illustration of this is the sharp decline in growth velocity in all four growth plates that occurs perinatally., ((c) 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Purpose: Our current understanding of the rate and pattern of physeal closure is based on roentgenographic, magnetic resonance imaging, and qualitative histological studies. The purpose of this report is to provide a detailed histomorphometric/stereological analysis of a distal tibial human growth plate in the process of physiological epiphysiodesis., Methods: A human distal tibial growth plate was sampled in three regions (anterior, central, and posterior), with each region further separated medially, in the middle, and laterally. The regions were assessed for the location and extent of bony bar formation as well as for physeal height. Companion sections from optimally fixed tissue in the distal 100 microm of the hypertrophic zone were analyzed for hypertrophic chondrocytic volumes., Results: Physis closure started in the middle of the central region of the growth plate, with 46% of the volume in this area occupied by trans-physeal bridging bone. The growth plate was also narrowed with the lowest physeal heights evident in the middle of the central and anterior regions of the physis. Disruption of the regular columns of the physis was evident with the cells arranged in clusters with intervening areas of acellularity. The average hypertrophic cell volume was 5,900 microm(3) and did not significantly differ between different areas of the physis., Conclusions: This is the first characterization of closure in a human distal tibial growth plate via optimum fixation and stereological techniques. The studied physis was during the earliest phases of closure and provides stereological support that the distal tibial physis closes in a central to medial direction.

Blount disease is a deformity of the knee as a result of abnormal mechanical forces known to influence the growth of the physis. Despite existing studies on mechanical forces on chondrocyte cultures or limited growth plate specimens, very little information characterizes the whole growth plate to asymmetrical loading. In this study, we evaluate the response of 5 ovine proximal tibial growth plates to asymmetrical mechanical loading. Fresh proximal tibia specimens were mounted, and compressive forces were applied via a servohydraulic test frame (MTS Systems Corporation, Minneapolis, Minn) machine at standardized locations while transducers recorded the displacement at different locations. With this method, we demonstrate that loading (cyclical or static) on 1 edge of the tibial surface results in compression through the physis under the site of pressure. In addition, we record statistically significant tensile displacement opposite the compressed side (P < 0.001); this effect diminished as loading cell moved central on the tibial surface. We further show that growth plate topography influences the amount of tension and compression observed. From this study, we conclude that asymmetrical loading (such as that observed in Blount disease) may lead to compression (which retards growth) but also develops tension on the convex side (which may be a mechanism to increase deformity via Depelch phenomenon). The relationship of physeal architecture (more undulations-less physeal strain) may explain why greater deformity is observed on the tibial side of the knee in adolescent Blount disease than on the femoral side.

The rate and patterns of longitudinal bone growth are affected by many different local and systemic factors; however, uncompromised growth is usually considered to be smoothly continuous, with predictable accelerations and decelerations over periods of months to years. The authors used implanted microtransducers to document bone growth in immature lambs. Bone length measurements were sampled every 167 seconds for 21 to 25 days. The authors show that at least 90% of bone elongation occurs during recumbency and almost no growth occurs during standing or locomotion. The authors hypothesize that growth may also occur in children during rest or sleep, thus supporting the concept of nocturnal growth and perhaps a relationship to growing pains.

Background: Impairment of growth in children with chronic renal failure may be due, in part to the insensitivity to the actions of growth hormone by insulin-like growth factor-I (IGF-I) because of accumulations of IGF binding proteins. There are a few studies describing the changes that occur in the growth plate in renal failure. None of these studies has simultaneously compared the modifications in the expression of selected markers of endochondral bone formation in renal failure with mild or advanced secondary hyperparathyroidism., Methods: Forty-six rats that underwent 5/6 nephrectomy (Nx) were fed either standard rodent diet (Nx-control) or high phosphorus diet to induce advanced secondary hyperparathyroidism (Nx-phosphorus) for 4 weeks. Sections of the tibia were obtained for growth plate histomorphometry, immunohistochemistry studies, and in situ hybridization experiments for selected markers of endochondral bone formation., Results: Weight gain, gain in length, and tibial length were less in Nx animals. Serum parathyroid hormone (PTH) and phosphorus levels were higher and serum calcium levels were lower in the Nx-phosphorus group. The width of the growth plate was much shorter in the Nx-phosphorus group due to a decrease in both proliferative and hypertrophic zones. IGF-I protein and IGF binding protein-3 staining were diminished in both Nx groups without changes in the IGF-I receptor expression; the decline in IGF-I protein expression was much lower in the Nx-phosphorus group. PTH/PTH receptor protein (PTHrP) receptor mRNA transcripts decline and tartrate-resistant acid phosphastase (TRAP) staining increased only in the Nx-phosphorus group., Conclusion: The growth impairment in renal failure may be worsened by the severity of secondary hyperparathyroidism.

Bone elongation in the postnatal animal is a result of cellular activity during endochondral ossification. Growth plate chondrocytes undergo a differentiation cascade involving stem cell clonal expansion and cellular enlargement during hypertrophy. Nutritional status has a significant effect on rates of bone growth, and a period of accelerated growth will occur if nutritional stunting of growth in early childhood can be corrected. This study focuses on changes in rates of increase in bone length in a model of catch-up growth in 4-wk-old male rats. Animals fasted for 3 d reached a weight approximately 60% of the control littermates. By 28 d postfasting, fasted animals had regained weight to 95% of control levels. A 3-d fast caused an immediate and profound decrease in rate of growth in the proximal tibial growth plate to only 30% of that of control animals, while stopping growth in the distal tibial growth plate. During the rapid initial rate acceleration of bone elongation, growth rate in both growth plates reached that of the control littermates by 7 d postfasting. The proximal tibial growth plate then maintained rates that were 10-15% higher than control over the rest of the experimental period. By 10 d postfasting, the previously fasted animals were on the same weight/rate trajectory as the control littermates. Changes in elongation rates were reflected by dramatic changes in growth plate morphology in all cellular zones. This is the first study to directly correlate weight recovery during catch-up with growth rate responses at the level of the growth plate.

Although it has been demonstrated clinically that controlled compression across a growth plate will slow the rate of endochondral ossification and thus can be used to correct angular limb deformities, the cellular-based mechanism by which altered growth is achieved is poorly understood. This study used short-term uniaxial stapling of the rat proximal tibial growth plate as an experimental system to study chondrocytic responses in the growth plate that account quantitatively for the decreased rate of growth. Growth plates were labeled with oxytetracycline to measure bone growth, and with bromodeoxyuridine to analyze proliferative cell kinetics. Multiple indicators of chondrocytic activity, measured by stereological parameters, were analyzed using growth rate as the primary dependent variable. The unique feature of this analysis was the creation of three-dimensional reconstructions that allowed analysis of data in all directions with distance from the staple. A significant observation was that for the entire operated limb after both 3 and 6 days, all chondrocytic kinetic parameters were affected, indicating that proliferative and hypertrophic responses both act to decrease growth rate in response to stapling. This contradicted our hypothesis that proliferative and hypertrophic responses could occur independently, and that small changes in rate would be attributed primarily to the former and large changes to the latter. The data from this study also demonstrate that volume regulation during hypertrophy can be affected by a primarily mechanical perturbation. Because changes in hypertrophic cell number and volume throughout the growth plate that occur by day 3 remain similar at day 6, the initial modulation of chondrocytic volume and shape may represent the limit of the response while maintaining a growth plate capable of continued growth.

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.

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.

Background: It has been suggested that within the growth plate, the final volume and shape of hypertrophic chondrocytes are important variables in determining the rate of longitudinal bone growth. To better understand the organization and regulation of chondrocytic hypertrophy as related to longitudinal bone growth, the beginning and end, and the location and magnitude of chondrocytic volume and shape changes during the hypertrophic process were defined in the proximal tibial growth plate of 35-day-old rats., Methods: In this study we used two different approaches, a stereological analysis of chondrocytes in unbiasedly defined, narrow growth plate strata, and a serial section reconstruction and measurement of individual cells. In both experiments chondrocytes were preserved using optimal chemical fixation. Proliferating chondrocytes were identified using bromodeoxyuridine labelling, and the rate of longitudinal bone growth was determined using oxytetracycline labelling., Results: In both studies, immediately following cell division in the proliferative zone, chondrocytic volume gradually increased toward the mid-point of the growth plate. During this phase of about 30 hours, approximately 20% of the final cell volume was obtained. During the following 20 hours the remaining 80% was acquired. The estimated rate of cell volume increased changed from approximately 50 microns 3/hr during the first 30 hours to about 800 microns 3/hr during the last 20 hours. The increase in cell volume resulted in an increase in both the vertical and the horizontal chondrocytic diameters. Cell parameters did not change during the final five hours of the maturation process., Conclusions: In this study we demonstrated that chondrocytic enlargement starts immediately following cell division in the proliferative zone, and that chondrocytic enlargement consists of two morphologically distinguishable phases. The transition point between the first and the second phase of chondrocytic enlargement corresponded with the junction between the proliferative zone and the maturation zone.

In growing mammals there is a positive linear relationship between the mean hypertrophic chondrocyte volume and the rate of bone elongation. This suggests that the control of chondrocytic volume in the growth plate, is a major determinant in controlling bone elongation in mammals. In the present study the existence of such a relationship was tested for in birds. A scheme of fluorochrome labelling was devised to enable direct measurement of bone elongation per unit time. Four weight-bearing growth plates from two-week-old mallard ducklings and the corresponding four growth plates from two-week-old leghorn chicks were studied. Growth plate cartilage was fixed in the presence of ruthenium hexamine trichloride and embedded in Epon araldite. Estimates of mean cell volume, v(chondr), and mean cubic intercept (l3) were calculated by applying the stereological relationship: v(chondr) = (pi/3) x (l3). Regression analysis revealed a positive linear relationship between the two parameters, rate of bone elongation and mean hypertrophic cell volume in both species (squared correlation statistics: 65 per cent for mallards, 54 per cent for leghorns). There was a wide range in rates of bone elongation among growth plates studied (318 to 1418 microns 24 h-1 for mallards, 77 to 445 microns 24 h-1 for leghorns) and compared to mammals (such as rabbits, rats, swine and dogs), a small range in mean cell volume (2709 to 4786 micron3 for mallards, 3663 to 5719 micron3 for leghorns).(ABSTRACT TRUNCATED AT 250 WORDS)

Histological and ultrastructural evaluation of the ends of long bones of juvenile dinosaurs from the Upper Cretaceous Two Medicine Formation of Montana revealed the preservation of growth plates. Growth plates are discs of cartilage present near the ends of growing long bones that generate bone elongation. Comparison of the fossils with modern taxa demonstrated homology of the growth plate in birds and dinosaurs. The presence of an avian-type growth plate in dinosaurs adds a shared derived anatomical character corroborating inclusion of birds within the Dinosauria. Additionally, possession of a growth plate, which in birds is capable of producing rapid determinate long bone growth, implies that an avian developmental pattern may have been present in these dinosaurs.

Postnatal bone growth occurs by the process of endochondral ossification in cartilaginous growth plates at the ends of long bones. The rate and extent of long bone growth is determined by a combination of chondrocytic proliferation, matrix production, and increase in chondrocytic height in the direction of growth during cellular enlargement. In this study, single pulse and/or repeated pulse labeling with the thymidine analog bromodeoxyuridine (BrdU) was used to study the role of cellular proliferation in controlling long bone growth. Variables studied included progression of the label over time following a pulse, and patterns and progression of the label over time following repeated pulse labeling for 24 and 48 hours. Examination was made of the proliferative characteristics of chondrocytes, the spatial pattern of cellular proliferation, and cell cycle kinetics. With respect to the spatial pattern of proliferative chondrocytes, results suggest that chondrocytes within a column are more synchronized with each other than are chondrocytes in different columns. This is consistent with the concept that each column represents a clonal expansion of a stem cell, which may proceed independently from adjacent columns. Despite this apparent heterogeneity, all chondrocytes in the proliferative zone complete at least one cell cycle in 24-28 hours. This estimate of the cell cycle time is significantly shorter than previous estimates of cell cycle times in similar growth plates. Our results also suggest that chondrocytes entering the cell cycle in the proximal part of the growth plate spend an average of 4 days in the proliferative cell zone, representing approximately four cellular divisions. After leaving the cell cycle, an additional 48 hours is required for the label to reach the terminal chondrocyte, which represents the time required to complete hypertrophy. These data are important when considering hypotheses concerning both the role of controls on proliferation in the determination of overall rate of long bone growth, as well as the interplay between proliferation and hypertrophy in regulating the overall amount of growth achieved by a given growth plate.

Ocular-chondrodysplasia in Labrador Retriever dogs is characterized by short-limbed dwarfism and ocular abnormalities. The purposes of the present study were to develop morphological criteria to define the matrix and/or chondrocytic abnormalities associated with this chondrodysplasia, and to test the hypothesis that ineffective matrix-directed cellular swelling was associated with the decreased longitudinal bone growth in these animals. The proximal and distal radial growth plates were collected from four affected animals of the same litter. Stereological techniques were used to analyze both cellular shapes and cellular volume changes in the hypertrophic zone. The pathological changes seen in these growth plates varied between animals and included disorganization of cellular columns with abnormal extent of calcification. Chondrocytes of all zones contained two types of abnormal cellular inclusions classified as light and dark, based on the intensity of eosinophilic staining. Both types of inclusions contained material that resembled the surrounding extracellular matrix, varying only in the apparent hydration of the contents. It could be demonstrated that light inclusions were located in the peripheral cytoplasm and connected to the extracellular matrix through narrow channels. By contrast, dark inclusions were membrane bound and perinuclear. Chondrocytes with multiple, large inclusions appeared to be undergoing degenerative changes. Although the final volume achieved by hypertrophic chondrocytes was consistent with that of normal growth plates, there was a high level of variability of chondrocytic shape and evidence of premature cellular condensation in the maturation zone. The severity of the dwarfism correlated both with the extent of chondrocytic changes and the severity of the ocular lesions.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

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.

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.

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.

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.

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.

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.

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.

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.

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.

Conjunctival goblet cells (GCs) were quantitated to establish baseline values for density and distribution of these cells in healthy canine eyes. From each of 18 sites, tissue was collected, sectioned at 2 micron, and stained with periodic acid Schiff stain. Within each sampling site, 500 epithelial cells (GCs, squamous, polygonal, and basal epithelial cells) were counted and the ratio of GCs to total epithelial cells was computed as an index of goblet cell density or goblet cell index (GCI). A heterogenous distribution of canine conjunctival goblets cells was demonstrated. Lower nasal fornix (LNf) and adjacent sites, lower middle fornix (LMf) and lower nasal tarsal (LNt), had the highest mean densities of goblet cells. In contrast, GCs were essentially absent from the upper and lower bulbar areas. Remaining sites had intermediate GCIs. Sex differences in GCIs were noted for LNf and LNt sites. Mean tear film breakup times (BUTs) were determined, and, for normal beagle dogs, were 19.38 (+/- 4.80 secs) OS and 19.96 (+/- 5.01 secs) OD. The similarities between canine and human conjunctival goblet cell distributions support the use of the dog for studying the conjunctival mucous system.

The blood vasculature of the bovine jejunum and proximal part of the ileum was studied in 20 mature dairy cows at slaughter. The cranial mesenteric artery and vein supplied the jejunum and ileum, and their major branches were present in all specimens and supplied similar regions of the intestinal tract. Proximal branches of the cranial mesenteric artery were pancreatic arteries, caudal pancreaticoduodenal artery, middle colic artery, and ileocolic artery. A large collateral branch arose from the proximal segment of the cranial mesenteric artery, anastomosing with the continuation of the cranial mesenteric artery distally along the jejunum. Jejunal arteries arose from the continuation of the cranial mesenteric artery, forming a series of anastomosing arches. Straight vessels arising from these arches did not branch or anastomose before entering the serosal layer of the intestine. The proximal part of the ileum was supplied by branches from the continuation of the cranial mesenteric artery; these branches anastomosed with the mesenteric ileal (ilei mesenterialis) artery, a branch of the ileocolic artery. The venous supply paralleled the arterial supply in all specimens.

Using a procedure of membrane solubilization followed by tannic acid fixation-staining, we found abnormally constructed central and B microtubules in cilia from dogs with primary ciliary dyskinesia. We also found that a ciliary abnormality commonly associated with primary ciliary dyskinesia, "shortened outer dynein arms," actually represents the absence of a specific set of the 3 peripheral subunits that make up the outer dynein arms. We believe that this is the first time that these have been reported. Because the proximal subunits of the outer dynein arm are present and attached to the A microtubule, we argue that in these patients there is no structural abnormality of the A microtubule or the A-tubule attachment sites for outer and inner arms and radial spokes. Also, because a majority of cilia in these patients were motile, we believe that the data from this study support the concept that the outer dynein arms are not required for some motion but probably required for normal motility; however, the central microtubules, central microtubular complex, and probably the B tubules are essential for motility.

A stereological expression relating the mean number of organelle profiles per cell profile, Np, to the mean number of organelles per cell, Nc, is first derived and then tested for cilia in tangenital articular chondrocytes where Nc is known to be 1.0. Nc derived stereologically for this organelle-cell model is 1.19. This close approximation of the estimate to a known value provides empirical evidence of the usefulness of the derived stereological expression.

The incidence of abnormal tracheal cilia from each of seven normal healthy dogs was estimated using samples prepared for TEM and SEM from the dorsal, lateral, and ventral aspects of the 3rd, 12th, and 24th tracheal rings. Some samples were demembranated with Triton X-100 and stained with tannic acid to visualize microtubular protofilaments. From each region of each ring of each dog on which TEM was done, 500 transversely sectioned cilia were observed, 27,000 cilia overall. Abnormal numbers of central microtubules and abnormal numbers of peripheral microtubules occurred in all samples in about 2% of all cilia. Compound cilia were consistently observed in samples from the 3rd ring and were not observed in any samples from the 24th ring. Therefore, before abnormal ciliary ultrastructure may be associated with disease, the rate of occurrence must exceed that in appropriate control individuals. Additionally, compound cilia, which have previously been associated with a variety of diseases, obviously occur in normal individuals; however, their observation may be a function of biopsy site selection.

The incidence and structure of equine and murine chondrocytic cilia were studied using serial sections and transmission electron microscopy. Overall, 96% of all equine chondrocytes and 100% of all murine chondrocytes had one cilium. The structure of these cilia included rootlets, basal feet, alar sheets, and an axoneme of nine peripheral doublets which progressively bent and terminated as they coursed towards the tip of the ciliary shaft. Together with the previous studies on neonatal and adult canine chondrocytic cilia, we conclude that the structure and incidence of chondrocytic cilia does not vary among species, regions within a joint, cell types, or age groups.

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.

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.

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.

An 11-year-old Dalmatian was examined and treated for bilateral nasal discharge and cough of 6 months' duration. Response to medical treatment and surgical intervention was unsatisfactory. Histologic examination of lung tissue revealed chronic severe catarrhal bronchitis and bronchiolitis with bronchiectasis. Histologic findings and barium sulfate bronchography indicated abnormal mucociliary clearance in the respiratory tract. Electron microscopy revealed abnormalities or deletions of outer and/or inner dynein arms in 26% of the ciliary profiles from the affected dog. Similar abnormalities were not found in 500 ciliary profiles from age- and gender-matched control dogs.

In growing swine, ossification failure due to osteochondrosis has an incidence of nearly 100% in the distal ulna of animals at six months of age, yet the etiology of the disease is understood poorly. In this study, the ultrastructure of the chondrocyte and its pericellular matrix is analyzed in normal growth plates and in growth plates with lesions characteristic of osteochondrosis using aldehyde primary fixatives and osmium-ferrocyanide as the secondary fixative. Chondrocytes in lesion areas fail to undergo normal hypertrophic cell maturation, and they have an accumulation of rough endoplasmic reticulum, lipid droplets and mitochondria. These morphological changes are interpreted to be both variable and nonspecific for osteochondrosis. Within the pericellular matrix of chondrocytes from lesion areas, the most striking abnormality is the presence of a highly condensed matrix with an accumulation of large, irregularly shaped deposits of electron dense material. These morphological alterations are characteristic of a matrix which either is not secreted normally, or in which the highly ordered interactions of diverse macromolecules has been lost. These pericellular matrix changes have not been described in other diseases of growing cartilage. They may be significant in the failure of metaphyseal vascular penetration of the pericellular matrix which is characteristic of osteochondrosis.

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.

Compared with neutrophils from healthy dogs, neutrophils from 2 dogs with primary ciliary dyskinesia had increased distance of random migration, but fewer of the neutrophils migrated. The affected dogs had an increase in the numbers of Staphylococcus aureus phagocytized. Lymphocyte blastogenesis in the affected dogs in response to standard mitogens was considered to be normal.