Cornelia E. Farnum

Determination of proliferative characteristics of growth plate chondrocytes by labeling with bromodeoxyuridine.

SummaryPostnatal 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.

Enlargement of growth plate chondrocytes modulated by sustained mechanical loading

Background: Mechanical compression and distraction forces are known to modulate growth in vertebral growth plates, and they have been implicated in the progression of scoliosis. This study was performed to test the hypothesis that growth differences produced by sustained compression or distraction loading of vertebrae are associated with alterations in the amount of increase in the height of growth plate chondrocytes in the growth direction. Methods: Compression or distraction force of nominally 60% of body weight was maintained for four weeks on a caudad vertebra of growing rats by an external apparatus attached, by means of transcutaneous pins, to the two vertebrae cephalad and caudad to it. Growth of the loaded and control vertebrae was measured radiographically. After four weeks, the animals were killed and histological sections of the loaded and control vertebrae were prepared to measure the height of the hypertrophic zone (average separation between zonal boundaries), the mean height of hypertrophic chondrocytes, and the amount of increase in cell height in the growth direction. Results: Over the four weeks of the experiment, the growth rates of the compressed and distracted vertebrae averaged 52% and 113% of the control rates, respectively. The reduction in the growth rate of the compressed vertebrae was significant (p = 0.002). In the compressed vertebrae, the height of the hypertrophic zone, the mean chondrocyte height, and the amount of increase in cell height averaged 87%, 85%, and 78% of the control values, respectively, and all were significantly less than the corresponding control values. In the distracted vertebrae, these measurements did not differ significantly from the control values. The height of the hypertrophic zone and the mean chondrocyte height correlated with the growth rate (r 2 = 0.29 [p = 0.03] and r 2 = 0.23 [p = 0.06], respectively), when each variable was expressed as a proportion of the control value. The percentage changes in the measurements of the chondrocytic dimensions relative to the control values were smaller than the percentage changes in the growth rates, a finding that suggested that the rate of chondrocytic proliferation was also modulated by the mechanical loading. Conclusions: Mechanical loading of tail vertebrae in rats modulated their growth rate, which correlated with changes in the height of hypertrophic chondrocytes. The effects of compression were greater than those of distraction. Clinical Relevance: Information about the growth rate and chondrocytic response to mechanical loads in rat vertebrae undergoing mechanically modulated growth will be helpful in determining how human vertebral growth might respond to altered loading states during progression or treatment of scoliosis and other growth-related angular skeletal deformities.

Quantitative Three-Dimensional Analysis of Chondrocytic Kinetic Responses to Short-Term Stapling of the Rat Proximal Tibial 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.

Volume Increase in Growth Plate Chondrocytes During Hypertrophy: The Contribution of Organic Osmolytes

During the differentiation cascade of growth plate chondrocytes, cells undergo as much as a 10-15-fold increase in volume. This volume increase, which occurs to different extents in growth plates growing at different rates, has been demonstrated to be the single most significant variable in understanding the quantitative aspects of the cellular kinetics of long bone growth. Our hypothesis is that this volume increase, which occurs through cell swelling by water imbibition, requires intracellular accumulation of osmolytes through activation or upregulation of membrane transport mechanisms. Significant intracellular accumulation of inorganic osmolytes, such as Na+, K+, and Cl-, is potentially disruptive to normal cellular metabolism, whereas intracellular accumulation of organic osmolytes is considered to be more compatible with metabolic function. Thus, we concentrated on determining the contributions of organic osmolytes--betaine, amino acids, inositol, and sorbitol--to volume increase. Pooled cryostat sections of young bovine growth plates were extracted followed by automated analysis for their content of amino acids. Analysis for betaine and the sugar alcohols was done by extraction and derivatization, followed by high-performance liquid chromatography (HPLC). Parallel stereological analyses correlated osmolyte changes to stages of chondrocytic differentiation, specifically comparing intracellular concentration and amount in proliferative vs. hypertrophic chondrocytes. Calculations demonstrated that, maximally, these organic osmolytes, in total, account for 6%-7% of the intracellular osmolytes required to sustain the volume increase, and that the most significant contribution is from betaine. This suggests that intracellular accumulation of organic osmolytes is not a primary strategy used by growth plate chondrocytes during volume increase of their terminal differentiation. The data also suggest that there is a differential regulation of transporters of these osmolytes such that intracellular concentrations are constantly modified as cells proceed through the differentiation cascade.

Pericellular matrix of growth plate chondrocytes: a study using postfixation with osmium-ferrocyanide.

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.

Primary Cilia Are Highly Oriented with Respect to Collagen Direction and Long Axis of Extensor Tendon

Skeletal tissues adapt to their mechanical environments by modulating gene expression, cell metabolism, and extracellular matrix (ECM) architecture; however, the mechanosensory mechanisms for these processes are incompletely understood. Primary cilia have emerged as critical components of the cellular mechanosensory apparatus and have been hypothesized to participate in establishment of cellular and ECM orientation, but their function in skeletal tissues is just beginning to be examined. Here we focused on tendon, a tissue with an oriented matrix that is ideal for analysis of spatial relationships between primary cilia and the ECM. The objective of this study was to characterize the incidence and orientation of tenocyte primary cilia in their native ECM. Primary cilia, nuclei, and collagen were analyzed three‐dimensionally in immunofluorescently labeled rat extensor tendon using multiphoton microscopy and semiautomated morphometry. Primary cilia were observed in 64% of tenocytes. The cilia were highly oriented with respect to the ECM: cilia were aligned parallel to the collagen fibers and the long axis of the tendon. This study represents the first quantification of the in situ incidence and orientation of primary cilia in tendon.

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.

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 ∼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.

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.

Expression of p21CIP1/WAF1 in chondrocytes.

Abstract. During endochondral ossification, proliferative activity of chondrocytes is arrested and the cells undergo terminal hypertrophic differentiation. We examined the expression of the cyclin-dependent kinase inhibitor, p21CIP1/WAF1 in permanent cartilage (xyphoid and articular cartilage) and in cartilage undergoing endochondral ossification (growth plate, epiphyseal ossification centers, and costochondral junctions) to determine if p21 is up-regulated in chondrocytes during hypertrophic differentiation. Northern blot analyses demonstrated expression of p21 in chondrocytes undergoing endochondral ossification and from sites of permanent cartilage. Quantitative analyses of Northern data showed an association between expression of the hypertrophic-specific marker, collagen type X, and the level of 21 expression. In situ hybridization of rodent femoropatellar joints and costochondral junctions localized p21 mRNA to chondrocytes within both the proliferative and hypertrophic zones of the growth plates, in chondrocytes involved in formation of the epiphyseal ossification centers, and in articular chondrocytes. Immunohistochemical analyses of p21 expression in the same tissues demonstrated comparatively higher levels of p21 protein in postmitotic chondrocytes. These data suggest that p21 is active in cell cycle regulation in chondrocytes, and that increased p21 expression is associated with hypertrophic differentiation.