Phosphorescent nanoparticles for quantitative measurements of oxygen profiles in vitro and in vivo.

We present the development and characterization of nanoparticles loaded with a custom phosphor; we exploit these nanoparticles to perform quantitative measurements of the concentration of oxygen within three-dimensional (3-D) tissue cultures in vitro and blood vessels in vivo. We synthesized a customized ruthenium (Ru)-phosphor and incorporated it into polymeric nanoparticles via self-assembly. We demonstrate that the encapsulated phosphor is non-toxic with and without illumination. We evaluated two distinct modes of employing the phosphorescent nanoparticles for the measurement of concentrations of oxygen: 1) in vitro, in a 3-D microfluidic tumor model via ratiometric measurements of intensity with an oxygen-insensitive fluorophore as a reference, and 2) in vivo, in mouse vasculature using measurements of phosphorescence lifetime. With both methods, we demonstrated micrometer-scale resolution and absolute calibration to the dissolved oxygen concentration. Based on the ease and customizability of the synthesis of the nanoparticles and the flexibility of their application, these oxygen-sensing polymeric nanoparticles will find a natural home in a range of biological applications, benefiting studies of physiological as well as pathological processes in which oxygen availability and concentration play a critical role.

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.

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.

Exercise mitigates the stunting effect of cold temperature on limb elongation in mice by increasing solute delivery to the growth plate.

Ambient temperature and physical activity modulate bone elongation in mammals, but mechanisms underlying this plasticity are a century-old enigma. Longitudinal bone growth occurs in cartilaginous plates, which receive nutritional support via delivery of solutes from the vasculature. We tested the hypothesis that chronic exercise and warm temperature promote bone lengthening by increasing solute delivery to the growth plate, measured in real time using in vivo multiphoton microscopy. We housed 68 weanling female mice at cold (16°C) or warm (25°C) temperatures and allowed some groups voluntary access to a running wheel. We show that exercise mitigates the stunting effect of cold temperature on limb elongation after 11 days of wheel running. All runners had significantly lengthened limbs, regardless of temperature, while nonrunning mice had shorter limbs that correlated with housing temperature. Tail length was impacted only by temperature, indicating that the exercise effect was localized to limb bones and was not a systemic endocrine reaction. In vivo multiphoton imaging of fluoresceinated tracers revealed enhanced solute delivery to tibial growth plates in wheel-running mice, measured under anesthesia at rest. There was a minimal effect of rearing temperature on solute delivery when measured at an intermediate room temperature (20°C), suggesting that a lasting increase in solute delivery is an important factor in exercise-mediated limb lengthening but may not play a role in temperature-mediated limb lengthening. These results are relevant to the study of skeletal evolution in mammals from varying environments and have the potential to fundamentally advance our understanding of bone elongation processes.

Microarray cluster analysis of irradiated growth plate zones following laser microdissection.

PURPOSE: Genes and pathways involved in early growth plate chondrocyte recovery after fractionated irradiation were sought as potential targets for selective radiorecovery modulation. MATERIALS AND METHODS: Three groups of six 5-week male Sprague-Dawley rats underwent fractionated irradiation to the right tibiae over 5 days, totaling 17.5 Gy, and then were killed at 7, 11, and 16 days after the first radiotherapy fraction. The growth plates were collected from the proximal tibiae bilaterally and subsequently underwent laser microdissection to separate reserve, perichondral, proliferative, and hypertrophic zones. Differential gene expression was analyzed between irradiated right and nonirradiated left tibia using RAE230 2.0 GeneChip microarray, compared between zones and time points and subjected to functional pathway cluster analysis with real-time polymerase chain reaction to confirm selected results. RESULTS: Each zone had a number of pathways showing enrichment after the pattern of hypothesized importance to growth plate recovery, yet few met the strictest criteria. The proliferative and hypertrophic zones showed both the greatest number of genes with a 10-fold right/left change at 7 days after initiation of irradiation and enrichment of the most functional pathways involved in bone, cartilage, matrix, or skeletal development. Six genes confirmed by real-time polymerase chain reaction to have early upregulation included insulin-like growth factor 2, procollagen type I alpha 2, matrix metallopeptidase 9, parathyroid hormone receptor 1, fibromodulin, and aggrecan 1. CONCLUSIONS: Nine overlapping pathways in the proliferative and hypertrophic zones (skeletal development, ossification, bone remodeling, cartilage development, extracellular matrix structural constituent, proteinaceous extracellular matrix, collagen, extracellular matrix, and extracellular matrix part) may play key roles in early growth plate radiorecovery.

Temperature alters solute transport in growth plate cartilage measured by in vivo multiphoton microscopy.

Solute delivery to avascular cartilaginous plates is critical to bone elongation, and impaired transport of nutrients and growth factors in cartilage matrix could underlie many skeletal abnormalities. Advances in imaging technology have revolutionized our ability to visualize growth plates in vivo, but quantitative methods are still needed. We developed analytical standards for measuring solute delivery, defined by amount and rate of intravenous tracer entry, in murine growth plates using multiphoton microscopy. We employed an acute temperature model because of its well-established impact on bone circulation and tested the hypothesis that solute delivery changes positively with limb temperature when body core and respiration are held constant (36 degrees C, 120 breaths/min). Tibial growth plates were surgically exposed in anesthetized 5-wk-old mice, and their hindlimbs were immersed in warm (36 degrees C) or cool (23 degrees C) saline (n = 6/group). After 30 min of thermal equilibration, we administered an intracardiac injection of fluorescein (50 microl, 0.5%) and captured sequentially timed growth plate images spanning 10 min at standardized depth. Absolute growth plate fluorescence was normalized to vascular concentrations for interanimal comparisons. As predicted, more fluorescein infiltrated growth plates at 36 degrees C, with standardized values nearly double those at 23 degrees C. Changing initial limb temperature did not alter baseline values, suggesting a sustained response period. These data validate the sensitivity of our system and have relevance to strategies for enhancing localized delivery of therapeutic agents to growth plates of children. Applications of this technique include assessment of solute transport in models of growth plate dysfunction, particularly chondrodysplasias with matrix irregularities.

Analyzing primary cilia by multiphoton microscopy.

In this chapter, a technique is outlined for the use of immunohistochemistry (IHC) followed by multiphoton microscopy (MPM) for the analysis of incidence, length, and 3D orientation of the axoneme of the primary cilium. Although the application presented specifically emphasizes localizations in tenocytes and chondrocytes, the technique is applicable to cells in a wide range of connective tissues. The primary advantages of utilizing MPM as opposed to TEM for these kinds of ciliary analyses are the rapidity of the technique for preparation of the samples and the ability to collect data from multiple cells simultaneously. Using MPM, the axoneme, basal body, and associated centriole can be visualized by specific IHC with localizing antibodies. However, the resolution achieved through TEM analyses allows the complex morphology of the primary cilium to be visualized, and this remains the primary advantage of TEM versus MPM. SHG, which occurs only with MPM, allows visualization of collagen fibrils and is particularly advantageous for localizing primary cilia associated with cells in connective tissues. This, and the deep penetration with less photobleaching, are the primary advantages of MPM compared to confocal microscopy. As with any microscopical technique, the protocol needs to be optimized for any given tissue. In particular, additional antigen retrieval techniques to enhance the unmasking of specific epitopes for antibody binding may be required for adaptation of this approach to other dense connective tissues with complex spatial organizations such as intervertebral disc or meniscus.

Histomorphometric evidence of growth plate recovery potential after fractionated radiotherapy: an in vivo model.

This study evaluated the hypothesis that early growth plate radiorecovery is evident by growth rate, histomorphometric and immunohistochemical end points after exposure to clinically relevant fractionated radiation in vivo. Twenty-four weanling 5-week-old male Sprague-Dawley rats were randomized into eight groups. In each animal, the right distal femur and proximal tibia were exposed to five daily fractions of 3.5 Gy (17.5 Gy) with the left leg serving as a control. Rats were killed humanely at 7, 8, 9, 10, 11, 14, 15 and 16 days after the first day of radiation exposure. Quantitative end points calculated included individual zonal and overall growth plate heights, area matrix fraction, OTC-labeled growth rate, chondrocyte clone volume and numeric density, and BrdU immunohistochemical labeling for proliferative index. Transient postirradiation reductions occurred early and improved during observation for growth rate, proliferative indices, transitional/hypertrophic zone matrix area fraction, proliferative height, and clonal volume. Reserve and hypertrophic zone height remained increased during the period of observation. The current model, using a more clinically relevant fractionation scheme than used previously, shows early evidence of growth plate recovery and provides a model that can be used to correlate temporal changes in RNA and protein expression during the early period of growth plate recovery.

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.

Forelimb versus hindlimb skeletal development in the big brown bat, Eptesicus fuscus: functional divergence is reflected in chondrocytic performance in Autopodial growth plates.

The morphology of the chiropteran forelimb demonstrates musculoskeletal specializations for powered flight essentially unique among mammals, including extreme elongation of the distal skeletal elements. Recent studies have focused primarily on the relative timing and levels of gene expression during early stages of endochondral ossification in the chiropteran embryo for clues to the molecular basis of the evolutionary origins of flight in these species. The goal of the current study was to examine how elongation of skeletal elements of the forelimb autopod is achieved through a combination of cellular proliferation, cellular enlargement and matrix synthesis during a period of rapid postnatal growth in Eptesicus fuscus. Quantitative analyses were done of multiple performance parameters of growth plate chondrocytes during all phases of the differentiation cascade. Fourteen autopodial growth plates from the forelimb and hindlimb of one individual, as well as the proximal tibial growth plate, were collected and analyzed. Significant differences were seen in all performance parameters examined. Particularly striking were the differences between growth plates of the manus and pes in the size of the pool of chondrocytes in all cellular zones and rates of turnover of terminal cells. The magnitude of hypertrophy of chondrocytes in growth plates of the manus in E. fuscus far exceeded what has been reported previously in any species, even in rapidly elongating rodent long bones. Volume changes approaching x70 and height changes of 50-60 mum/cell (paralleling the direction of growth) occurred after proliferation in the most rapidly growing growth plates. The data demonstrate that final differences in lengths of homologous skeletal elements in the autopod of the forelimb and hindlimb of this species result not just from an initiating factor early in development, but from continued quantitative differences in chondrocytic performance during postnatal bone elongation as measured by multiple kinetic-based parameters.

Postnatal bone elongation of the manus versus pes: analysis of the chondrocytic differentiation cascade in Mus musculus and Eptesicus fuscus.

Bones elongate postnatally by endochondral ossification as cells of the cartilaginous growth plate undergo a differentiation cascade of proliferation, cellular hypertrophy and matrix synthesis. Interspecific comparisons of homologous bones elongating at different rates has been a useful approach for studying the dynamics of this process. The purpose of this study was to measure quantitative stereological parameters of growth plates of the third digit of the manus and pes of the laboratory mouse, and make comparisons to chondrocytic performance parameters in the homologous bones of the big brown bat, Eptesicus fuscus, where extremely rapid postnatal elongation of bones of the manus is associated with skeletal modifications for powered flight. Measurements were made across all zones of forelimb and hindlimb autopod growth plates by dividing each growth plate into strata of equal height (from thirteen 200-mum-high strata in the metacarpus to five 40-mum-high strata in phalangeal bones of the pes). Results indicate that all chondrocytic performance parameters known to quantitatively contribute to the elongation potential of a growth plate change together. A significant finding was that in growth plates of the chiropteran manus, final hypertrophic cell size and shape were achieved early in the zone of hypertrophy, indicating that interstitial expansion of the growth plate resulting from the incremental chondrocytic height increase in the direction of elongation was completed soon after the transition from the cessation of proliferation to the initiation of hypertrophy. This is unlike what has been reported in most mammalian growth plates previously analyzed, but is the situation in the proximal tibial growth plate of rapidly growing frogs and precocial birds. This suggests that a similar adaptation for stabilization of a rapidly elongating bone has evolved independently in three widely separated groups that have in common rapid growth in limbs to be used for early active, powered locomotion.

Connective tissue growth factor and insulin-like growth factor 2 show upregulation in early growth plate radiorecovery response following irradiation.

INTRODUCTION: The growth plate response following radiotherapy is poorly understood. In particular, little is known about the changes in growth plate growth factors and cytokines following irradiation. The hypothesis was that a limited number of growth factors and cytokines play a role in growth plate proliferative and hypertrophic chondrocyte radio-recovery. METHODS: The right limbs of 6 rats were irradiated (17.5 Gy), leaving the left limbs as controls. Limbs were harvested 1 (n = 3) and 2 (n = 3) weeks later. Microarrays were constructed from chondrocytes obtained by laser microdissection from the proliferative zone (PZ) and the hypertrophic zone (HZ) of normal and irradiated tibia growth plates. Real-time PCR was used to confirm the expression of parathyroid hormone receptor 1 (Pthr1), connective tissue growth factor (CTGF), insulin-like growth factor I receptor (IGF1R), insulin-like growth factor II (IGF2), interleukin 17beta (IL17b) and chemokine ligand 12 (CXCL12). RESULTS AND CONCLUSIONS: IGF2 is upregulated in the PZ and CTGF is upregulated in both the PZ and HZ 1 week after irradiation, prior to the histomorphometric appearance of growth plate recovery in this immature animal radiation model, supporting their role in stimulating early return of the growth plate. By 2 weeks after irradiation, a number of growth factors and cytokines, including CTGF and Pthr1 in both zones, CXCL12 and its receptor in the PZ, and IL17b and bone morphogenetic protein 2 in the HZ, show upregulation, suggesting a possible later role in radiorecovery. The effects of irradiation on Pthr1, CTGF, IGF2 and CXCL12 in PZ and Pthr1, CTGF, IL17b and IGF1R in the HZ determined by microarray and real-time RT-PCR was highly correlated (r = 0.797, p < 0.05 in the PZ and r = 0.875, p < 0.01 in the HZ, respectively).

Growth plate zonal microarray analysis shows upregulation of extracellular matrix genes and downregulation of metalloproteinases and cathepsins following irradiation.

Although the growth plate matrix area fraction increases after irradiation, extracellular matrix (ECM) gene expression in this context has not been studied. The hypothesis was that normally expressed ECM genes would be upregulated after irradiation. The right limbs of six Sprague-Dawley 5-week-old rats were irradiated with the left limbs as controls. Half of the animals were harvested after 1 week and half after 2. Microarray was conducted from normal and irradiated tibial growth plate proliferative zone (PZ) and hypertrophic zone (HZ) chondrocytes separated by laser microdissection at each time point. In situ hybridization (ISH) and real-time polymerase chain reaction (PCR) were used to confirm expression of selected genes. At 1 and 2 weeks after irradiation, both normally expressed ECM genes and others not highly expressed in the normal growth plate showed upregulation. Metalloproteinases and cathepsins were downregulated. PZ gene expression after irradiation exhibited features of the normal HZ, suggesting premature terminal differentiation. ECM genes not highly expressed in the normal growth plate included several members of the small leucine-rich proteins and the ezrin-radixin-moesin family. The effects of irradiation on cathepsin K (Ctsk), integrin binding sialoprotein (Ibsp), and procollagen II alpha 1 (Col2a1), as determined by ISH and real-time PCR, were highly correlated with the microarray results. Accumulation of matrix following radiation injury to the growth plate correlated well with changes in gene expression. Upregulation of genes not normally highly expressed in the noninjured growth plate suggests their importance in the injury and repair response.

Solute transport in growth plate cartilage: in vitro and in vivo.

Bone elongation originates from cartilaginous discs (growth plates) at both ends of a growing bone. Here chondrocytes proliferate and subsequently enlarge (hypertrophy), laying down a matrix that serves as the scaffolding for subsequent bone matrix deposition. Because cartilage is generally avascular, all nutrients, oxygen, signaling molecules, and waste must be transported relatively long distances through the tissue for it to survive and function. Here we examine the transport properties of growth plate cartilage. Ex vivo, fluorescence photobleaching recovery methods are used in tissue explants. In vivo, multiphoton microscopy is used to image through an intact perichondrium and into the cartilage of anesthetized mice. Systemically introduced fluorescent tracers are monitored directly as they move from the vasculature into the cartilage. We demonstrate the existence of a relatively permissive region at the midplane of the growth plate, where chondrocytes transition from late proliferative to early hypertrophic stages and where paracrine communication is known to occur between chondrocytes and cells in the surrounding perichondrium. Transport in the living mouse is also significantly affected by fluid flow from the two chondro-osseus junctions, presumably resulting from a pressure difference between the bone vasculature and the cartilage.

Alterations in the growth plate associated with growth modulation by sustained compression or distraction.

Sustained mechanical load is known to modulate endochondral growth in the immature skeleton, but it is not known what causes this mechanical sensitivity. This study aimed to quantify alterations in parameters of growth plate performance associated with mechanically altered growth rate. Vertebral and proximal tibial growth plates of immature rats and cattle, and rabbit (proximal tibia only) were subjected to different magnitudes of sustained loading, which altered growth rates by up to 53%. The numbers of proliferative chondrocytes, their rate of proliferation, and the amount of chondrocytic enlargement occurring in the hypertrophic zone were quantified. It was found that reduced growth rate with compression and increased growth rate with distraction were associated with corresponding changes in the number of proliferative chondrocytes per unit width of growth plate, and in the final (maximum) chondrocytic height in the hypertrophic zone (overall correlation coefficients 0.38 and 0.56 respectively). According to multiple linear regression coefficients for these two variables (0.72 and 1.39 respectively), chondrocytic enlargement made a greater contribution to altered growth rates.

Restoration of growth plate function following radiotherapy is driven by increased proliferative and synthetic activity of expansions of chondrocytic clones.

Radiation therapy encompassing an active epiphysis can negatively impact the potential for bone growth by disrupting cell-cycle progression and accelerating apoptosis and terminal differentiation in physeal chondrocytes. Despite functional derangement following radiation exposure, the irradiated growth plate retains a capacity for regeneration and recovery of growth. The purpose of this study was to characterize the initial sequence of events leading to functional growth recovery in irradiated weanling rat growth plates. We hypothesized that growth in an irradiated epiphysis would be partially restored due to the expansion of chondrocytic clones. Stereological histomorphometry was used to compare chondrocytic cell and matrix turnover between the first and second week following irradiation, and to determine the relative contribution of each of the cellular and extracellular matrix (ECM) compartments to growth. We found that restoration of growth in the irradiated limb was strongly associated with the proliferative activity and production of ECM by these chondrocytic clones, as they expand in average volume, but not in numerical density. We conclude that chondrocytes forming expansive clones and exhibiting increased mitotic and matrix synthesis activity initiate the early restoration of function in the irradiated growth plate, and would be a logical target for strategies to restore full growth potential.

Combination radioprotectors maintain proliferation better than single agents by decreasing early parathyroid hormone-related protein changes after growth plate irradiation.

Our hypothesis was that combinations of radioprotectors would be more effective than individual agents in minimizing the effects of radiation on the growth plate after single-fraction hind-limb irradiation of Sprague-Dawley rats. At 2 days postirradiation, the decrease in parathyroid hormone-related protein and parathyroid hormone receptor 1 expression in the irradiated growth plate transitional and hypertrophic zones was reversed in both of the combination groups but persisted in the groups treated with the individual drugs. By 2 weeks, positive findings unique to the combination-treatment animals included greater mean proliferation in the irradiated growth plate than on the contralateral side, smaller limb length discrepancies, reversal of the increased overall matrix area fraction, and reversal of the usual deficiency in Indian hedgehog staining in the irradiated hypertrophic zone. While all treatments had a positive effect in reversing the decrease in B-cell leukemia 2 protein and coincident increase in Bax previously observed 2 weeks postirradiation, the two combination groups had a more robust effect. Combinations of radioprotectors may achieve their beneficial additive effects in the growth plate by decreasing the usual early drop in parathyroid hormone-related protein and parathyroid hormone receptor 1 after irradiation, resulting in a cascade of parathyroid hormone-related protein-mediated events.

In vivo delivery of fluoresceinated dextrans to the murine growth plate: imaging of three vascular routes by multiphoton microscopy.

Bone elongation by endochondral ossification occurs through the differentiation cascade of chondrocytes of cartilaginous growth plates. Molecules from the systemic vasculature reach the growth plate from three different directions: epiphyseal, metaphyseal, and a ring vessel and plexus associated with the perichondrium. This study is an analysis of the real-time dynamics of entrance of fluoresceinated tracers of different molecular weights into the growth plate from the systemic vasculature and tests the hypothesis that molecular weight is a key variable in the determination of both the directionality and the extent of tracer movement into the growth plate. Multiphoton microscopy was used for direct in vivo imaging of the murine proximal tibial growth plate in anesthetized 4- to 5-week-old transgenic mice with green fluorescent protein linked to the collagen II promoter. Mice were given an intracardiac injection of either fluorescein (332.3 Da) or fluoresceinated dextrans of 3, 10, 40, 70 kDa, singly or sequentially. For each tracer, directionality and rate of arrival, together with extent of movement within the growth plate, were imaged in real time. For small molecules (up to 10 kDa), vascular access from all three directions was observed and entrance was equally permissive from the metaphyseal and the epiphyseal sides. Within our detection limit (a few percent of vascular concentration), 40 kDa and larger dextrans did not enter. These results have implications both for understanding systemic and paracrine regulation of growth plate chondrocytic differentiation, as well as variables associated with effective drug delivery to growth plate chondrocytes.

Radioprotectant combinations spare radiation-induced damage to the physis more than fractionation alone.

PURPOSE: The aim of this study was to determine if fractionation and individual or combinations of radioprotectants could minimize damage to physeal longitudinal growth in an animal model to any greater extent than fractionation alone. MATERIALS AND METHODS: Sixty-three weanling male Sprague-Dawley rats were randomized into seven equal groups. Five groups received a total 25 Gy radiation exposure in three equal fractions to the right knee with the left as non-irradiated control. For each group, pentoxifylline, misoprostol, and amifostine were given individually and amifostine was also given in combination with each of the other drugs prior to the radiation fractions. One group each received 25 Gy in one or three fractions without radioprotection. At six weeks, limb lengths and histomorphometry were assessed. RESULTS: The single fraction of 25 Gy caused a mean tibial length discrepancy of 24.4%. Fractionation decreased this to 18.8% (p < 0.001). Beyond fractionation alone, the mean femoral length discrepancies were significantly decreased by each of the added individual and combination radioprotectant drugs (p < 0.0004). The smallest absolute femoral length discrepancy (11%) was achieved with fractionation and the combination of amifostine and misoprostol. CONCLUSIONS: Radioprotectants may be beneficial in growth plate radioprotection, alone or in combination.

Modulation of vertebral and tibial growth by compression loading: diurnal versus full-time loading.

PURPOSE: This study was designed to determine whether the amount of endochondral growth response to mechanical compression and the underlying growth mechanism differed with night-time or day-time loading, relative to full-time loading. METHODS: Mechanical compression (nominally 0.1 MPa stress) was applied across tibial and tail vertebral growth plates of growing Sprague-Dawley rats. Four groups of animals (five per group) were used: 24/24 h (full-time loading); 12/24 h (day-loading); 12/24 h (night-loading); and 0/24 h (sham instrumented). Contralateral tibiae and adjacent vertebrae served as within-animal controls. The animals were euthanized after eight days. Growth plates were processed for quantitative histology to measure 24-h growth, total and BrdU-positive proliferative zone chondrocyte counts, and hypertrophic chondrocytic enlargement in the growth direction. RESULTS: Growth as a percentage of within-animal control averaged 82% (full-time); 93% (day-loading); 90% (night-loading); 100% (sham) for vertebrae. For proximal tibiae it averaged 70% (full-time); 84% (day-loading); 86% (night-loading); 89% (sham). Reduced amount of hypertrophic chondrocytic enlargement explained about half of this effect in full-time loaded growth plates, but was not significantly altered in half-time loaded growth plates. The remaining variation in growth was apparently explained by reduced total numbers of proliferative zone chondrocytes. These findings indicate that sustained compression loading suppressed growth more than intermittent loading at both anatomical locations.