Axonemal positioning and orientation in three-dimensional space for primary cilia: what is known, what is assumed, and what needs clarification.

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.

The effect of periosteal resection on tibial growth velocity measured by microtransducer technology in lambs.

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.

The effect of fluoroquinolone antibiotics on growing cartilage in the lamb model.

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.

Orientation of primary cilia of articular chondrocytes in three-dimensional space.

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.

Histomorphometric analysis of an adolescent distal tibial physis prior to growth plate closure.

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.

Age and pattern of the onset of differential growth among growth plates in rats.

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.

Mechanical behavior of the lamb growth plate in response to asymmetrical loading: a model for Blount disease.

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.

Bone elongation in rats with renal failure and mild or advanced secondary hyperparathyroidism.

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.

Effect of short-term fasting on bone elongation rates: an analysis of catch-up growth in young male rats.

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.

Growing pains: are they due to increased growth during recumbency as documented in a lamb model?

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.