Glucocorticoid effects on chondrogenesis, differentiation and apoptosis in the murine ATDC5 chondrocyte cell line.

Glucocorticoids (GC) are used extensively in children and may cause growth retardation, which is in part due to the direct effects of GC on the growth plate. We characterised the ATDC5 chondrocyte cell line, which mimics the in vivo process of longitudinal bone growth, to examine the effects of dexamethasone (Dex) and prednisolone (Pred) during two key time points in the chondrocyte life cycle - chondrogenesis and terminal differentiation. Additionally, we studied the potential for recovery following Dex exposure. During chondrogenesis, Dex and Pred exposure at 10(-8) M, 10(-7) M and 10(-6) M resulted in a significant mean reduction in cell number (28% vs 20%), cell proliferation (27% vs 24%) and proteoglycan synthesis (47% vs 43%) and increased alkaline phosphatase (ALP) activity (106% vs 62%), whereas the incidence of apoptosis was unaltered. Minimal effects were noted during terminal differentiation with both GC although all concentrations of Dex lowered apoptotic cell number. To assess catch-up growth the cells were incubated for a total of 14 days which included 1, 3, 7, 10 or 14 days exposure to 10(-6) M Dex, prior to the recovery period. Recovery of proteoglycan synthesis was irreversibly impaired following just one day exposure to Dex. Although cell number showed a similar pattern, significant impairment was only achieved following 14 days exposure. Irreversible changes in ALP activity were only noticed following 10 days exposure to Dex. In conclusion, GC have maximal effects during chondrogenesis; Dex is more potent than Pred and cells exposed to Dex recover but this may be restricted due to differential effects of GC on specific chondrocyte phenotypes.

Regulation of chondrocyte terminal differentiation in the postembryonic growth plate: the role of the PTHrP-Indian hedgehog axis.

Chondrocyte differentiation during embryonic bone growth is controlled by interactions between PTHrP and Indian hedgehog. We have now determined that the major components of this signaling pathway are present in the postembryonic growth plate. PTHrP was immunolocalized throughout the growth plate, and semiquantitative RT-PCR analysis of maturationally distinct chondrocyte fractions indicated that PTHrP, Indian hedgehog, and the PTH/PTHrP receptor were expressed at similar levels throughout the growth plate. However, patched, the hedgehog receptor, was more highly expressed in proliferating chondrocytes. Although all fractionated cells responded to PTHrP in culture by increasing thymidine incorporation and cAMP production and decreasing alkaline phosphatase activity, the magnitude of response was greatest in the proliferative chondrocytes. Bone morphogenetic proteins are considered likely intermediates in PTHrP signaling. Expression of bone morphogenetic protein-2 and 4--7 was detected within the growth plate, and PTHrP inhibited the expression of bone morphogenetic protein-4 and 6. Although organ culture studies indicated a possible paracrine role for epiphyseal chondrocyte-derived PTHrP in regulating growth plate chondrocyte differentiation, the presence within the postembryonic growth plate of functional components of the PTHrP-Indian hedgehog pathway suggests that local mechanisms intrinsic to the growth plate exist to control the rate of endochondral ossification.

Parathyroid hormone-related peptide expression in tibial dyschondroplasia.

Parathyroid hormone-related peptide (PTHrP) has a key role in the growth of long bones, as it is a negative regulator of growth plate chondrocyte terminal differentiation. We have examined the distribution and gene expression levels of PTHrP in the growth plates of broiler chickens with tibial dyschondroplasia (TD) in order to determine whether increased expression of PTHrP is responsible for the delayed chondrocyte differentiation that is characteristic of this skeletal disorder. PTHrP protein distribution and gene expression levels were assessed by immunocytochemistry and reverse transcriptase-polymerase chain reaction, respectively. In growth plates of normal birds, PTHrP was found to be distributed throughout all maturational zones of the growth plate. In cartilage proximal to the TD lesion, PTHrP immunostaining and the level of PTHrP gene expression were similar to that observed in normal birds. In contrast, many chondrocytes within the centre of the TD lesion stained poorly for PTHrP and this was reflected in the lower levels of PTHrP mRNA detected in lesion cells. These results suggest that alterations in PTHrP distribution and gene expression are not primarily responsible for the delayed chondrocyte differentiation and hypertrophy noted in dyschondroplasia, but are a result of secondary changes due to the pathology of the condition.

Microtubules are potential regulators of growth-plate chondrocyte differentiation and hypertrophy.

Terminal differentiation of growth-plate chondrocytes is accompanied by the acquisition of a spherical morphology and a large increase in cell volume. These changes are likely to be associated with rearrangement of the cytoskeleton, but little information on this aspect of chondrocyte hypertrophy is available. We report a role for microtubules in the control of chondrocyte maturation and hypertrophy. Chick growth-plate chondrocytes were fractionated into five maturationally distinct populations by Percoll density gradient centrifugation, and agarose gel differential display analysis was performed. We identified a 1200 bp cDNA fragment derived from a transcript that was most highly expressed in the hypertrophic chondrocytes. After cloning and sequencing, FASTA and BLAST analysis revealed 100% identity to chick beta7-tubulin. Differential expression was confirmed in a reverse transcription-polymerase chain reaction (RT-PCR) assay using specific primers for a 343 bp fragment from the 3' untranslated region of beta7-tubulin. Beta7-tubulin was upregulated three-fold in fully hypertrophic chondrocytes compared with the other four fractions, which all had similar levels of expression. Immunocytochemical localization of beta-tubulin in chick growth-plate sections demonstrated little staining in the chondrocytes of the proliferating zone, but intense cytoplasmic staining was present in the large hypertrophic chondrocytes. In cell culture studies, the addition of colchicine (10(-6) mol/L) resulted in a higher rate of [3H]-thymidine uptake (36.0%; p < 0.001), but lower amounts of alkaline phosphatase activity (69.1%; p < 0.001), collagen (49.1%; p < 0.01), and glycosaminoglycan (43.3%; p < 0.01) accumulation within the cell-matrix layer. Further evidence for the involvement of microtubules in chondrocyte differentiation and hypertrophy was obtained by morphological assessment of colchicine-treated growth-plate explant cultures. A partial failure of chondrocyte hypertrophy was observed, although collagen type X immunoreactivity was noted within the interstitial matrix. Further studies are required to identify the exact role of microtubules in chondrocyte hypertrophy, but the results presented here suggest that upregulation of beta-tubulin may be required for increased microtubule synthesis during changes in cell size during the hypertrophic process. In addition, as cell-matrix interactions are required for chondrocyte maturation, microtubules may promote the differentiated phenotype as a result of their role in Golgi-mediated secretion of matrix proteins.

A novel integral membrane protein is differentially expressed in the chick growth plate and maps to chromosome 1.

The growth plate is a specialised region of cartilage located at the growing ends of long bones in higher vertebrates. It is responsible for longitudinal bone growth and is under the control of many local and systemic factors. The growth plate consists of an orderly arrangement of small proliferative and larger mature hypertrophic chondrocytes. This paper describes the isolation by differential display of a 988-bp cDNA fragment derived from a transcript that is more highly expressed in proliferating rather than hypertrophic chondrocytes of the chick growth plate. Using 3' RACE, a further 939 bp of cDNA sequence was obtained. The 1.9 kb sequence contains a 924-bp open reading frame encoding an unknown 308 amino acid protein. This protein has a putative transmembrane domain near its N-terminus and three dileucine motifs at its carboxy tail. This gene was expressed in all other tissues examined. A polymorphism was identified by SSCP analysis and the gene was mapped to the centromeric region of the short arm of chicken chromosome 1, close to the locus for autosomal dwarfism.

Identification and cloning of a novel phosphatase expressed at high levels in differentiating growth plate chondrocytes.

Growth plate chondrocytes progress through a proliferative phase before acquiring a terminally-differentiated phenotype. In this study we used Percoll density gradients to separate chick growth plate chondrocytes into populations of different maturational phenotype. By applying agarose gel differential display to these populations we cloned a cDNA encoding a novel 268 amino acid protein (3X11A). 3X11A contains two peptide motifs that are conserved in a recently identified superfamily of phosphotransferases. It is likely that 3X11A is a phosphatase, but its substrate specificity remains uncertain. 3X11A expression is upregulated 5-fold during chondrocyte terminal differentiation and its expression is approximately 100-fold higher in hypertrophic chondrocytes than in non-chondrogenic tissues. This suggests that 3X11A participates in a biochemical pathway that is particularly active in differentiating chondrocytes.

Ascorbic acid-induced chondrocyte terminal differentiation: the role of the extracellular matrix and 1,25-dihydroxyvitamin D.

Chondrocyte terminal differentiation is associated with cellular hypertrophy increased activity of plasma membrane alkaline phosphatase and the synthesis of collagen type X. The hypertrophic phenotype of cultured chondrocytes can be stimulated by ascorbic acid but the underlying mechanisms for this phenotypic change are unclear. As ascorbic acid is central to many hydroxylation reactions, the possibility was examined that its pro-differentiating effects are mediated by its effects on collagen and vitamin D metabolite formation. In vitro studies indicated that ascorbic acid-induced chondrocyte alkaline phosphatase activity was inhibited by the addition of both collagen and proteoglycan synthesis inhibitors. The addition of arginine-glycine-aspartic acid (RGD)-containing peptides also resulted in lower alkaline phosphatase activity. Chicks supplemented with dietary ascorbic acid had higher concentrations of both collagen and proteoglycans within their growth plates but the chondrocyte maturation rate was unaltered. No evidence was obtained to suggest that ascorbic acid-induced collagen production was mediated by lipid peroxidation. In addition, supplementation with dietary ascorbic acid resulted in higher serum 1,25-dihydroxyvitamin D3 concentrations and increased chondrocyte vitamin D receptor number. Ascorbic acid-treated chondrocytes maintained in vitro also had increased vitamin D receptor numbers but chondrocyte receptor affinity for 1,25-dihydroxyvitamin D3 was unaltered. These results indicate that ascorbic acid promotes both chondrocyte matrix production and 1,25-dihydroxyvitamin D3 synthesis, accompanied by upregulation of the vitamin D receptor. Thus, ascorbic acid may be causing amplification of the vitamin D receptor-dependent genomic response to 1,25-dihydroxyvitamin D, resulting in promotion of terminal differentiation. Strong evidence is provided to support the hypothesis that ascorbic acid-induced chondrocyte terminal differentiation is mediated by interactions between integrins and RGD-containing cartilage matrix proteins.

Distribution and quantification of pyridinium cross-links of collagen within the different maturational zones of the chick growth plate.

In order to assess alterations in the collagen network during endochondral ossification the pyridinium cross-links of collagen were quantified in sequential transverse sections through the chick growth plate. This was accomplished using both morphological (alkaline phosphatase (ALP) histochemistry and collagen type X immunostaining) and analytical (HPLC) analyses. In articular cartilage, pyridinoline concentrations were maximal in the deep mature zones. In contrast, the proliferating chondrocyte zone of the growth plate had approximately a 10-fold greater pyridinoline cross-link concentration than the mature hypertrophic zone. Deoxypyridinoline was first found in the prehypertrophic zone of the growth plate cartilage that reacted positively for ALP activity but before collagen type X was detected. However, deoxypyridinoline concentrations were highest in the most differentiated regions of the growth plate where it was the principal pyridinium cross-link. In tibial dyschondroplasia, where chondrocyte differentiation is arrested in the prehypertrophic zone, higher concentrations of both cross-links were found with increasing distance down the lesion. We conclude that the decrease in pyridinoline cross-link concentration down the growth plate may be an essential adaptation (via increased collagenase activity and collagen turnover) of the matrix for vascular invasion and osteoclastic resorption to occur.

The expression of transforming growth factor-beta by cultured chick growth plate chondrocytes: differential regulation by 1,25-dihydroxyvitamin D3.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) and transforming growth factor-beta (TGF-beta) are both important regulators of chondrocyte growth and differentiation. We report here that 1,25(OH)2D3 differentially regulates the expression of the genes for TGF-beta 1 to -beta 3 and the secretion of the corresponding proteins in cultured chick chondrocytes. Confluent growth plate chondrocytes were serum-deprived and cultured in varying concentrations of 1,25(OH)2D3. Cells were assayed for TGF-beta mRNA and conditioned medium was assayed for TGF-beta activity and isoform composition. Active TGF-beta was only detected in 10(-8) M 1,25(OH)2D3-treated cultures (8.37 ng active TGF-beta/mg protein). There was a significant decrease in total (latent-active) TGF-beta activity in conditioned medium of 10(-12) M (23.4%; P < 0.05) and 10(-10) M (20.7%; P < 0.05) 1,25(OH)2D3-treated cultures but 10(-8) M 1,25(OH)2D3 significantly increased (30.9%; P < 0.01) TGF-beta activity. The amounts of TGF-beta 1, -beta 2 and -beta 3 isoforms produced were similar in control, 10(-10) or 10(-12) M 1,25(OH)2D3-treated cultures but the conditioned medium of 10(-8) M 1,25(OH)2D3-treated cultures contained significantly higher amounts of all three isoforms. Quantification of TGF-beta mRNA demonstrated differential control of TGF-beta gene expression with TGF-beta 1 and -beta 3 mRNA levels reduced by all concentrations of 1,25(OH)2D3 examined (10(-8), 10(-10) and 10(-12) M) whilst TGF-beta 2 mRNA concentrations were elevated. Our results indicated that 1,25(OH)2D3 regulates chick growth plate chondrocyte TGF-beta secretion and mRNA expression in a concentration-dependent and isoform-specific manner. This interaction may be important in the regulation of chondrocyte metabolism and endochondral bone growth.

Regulators of chondrocyte differentiation in tibial dyschondroplasia: an in vivo and in vitro study.

Tibial dyschondroplasia (TD) is a disorder of endochondral bone growth and results in the retention of a mass of unmineralized, avascular cartilage extending into the metaphysis. We have studied various parameters of chondrocyte differentiation, both in isolated chick chondrocytes and growth plate sections, in an attempt to determine whether the inhibition in chondrocyte differentiation seen in TD is a consequence of an inherent incapability of chondrocytes to differentiate terminally and mineralize. Results from in vitro experiments indicated that both normal and lesion chondrocytes synthesized a matrix that stained with antibodies to types II and X collagen and displayed foci of mineralization. Alkaline phosphatase activity in lesion chondrocytes was significantly increased in comparison to that in normal hypertrophic chondrocytes. In addition, normal and lesion chondrocytes in culture synthesized transforming growth factor-beta and 24,25(OH)2D3 but not 1,25(OH)2D3. There was no significant difference in the production rate of these growth regulators between normal and lesion chondrocytes. In contrast, in growth plate sections, alkaline phosphatase activity was markedly reduced in the lesion chondrocytes and sites of mineralization were not evident. Type II collagen was located throughout the growth plate and lesion, but type X collagen was not present within the lesion except at sites of vascularization. These results indicate that, in culture, lesion chondrocytes have the ability to differentiate terminally and mineralize, and suggest that the primary abnormality in TD is related to a developmental fault which is only operative in vivo. This may include a defect in cartilage vascularization and/or impairment of chondrocyte differentiation by mechanisms that have not yet been elucidated but may involve the abnormal production of regulatory factors.

Influence of quantity of litter on nest box selection and nesting behaviour of domestic hens.

1. Twenty two hens (Gallus gallus domesticus) were given a free choice, for egg laying, between 4 nest boxes containing different quantities of wood shavings (litter): 3180 cm3 (large), 1060 cm3 (medium), 350 cm3 (small) and none (zero). 2. There was considerable individual variation in the quantity of litter selected for egg laying, but 17 hens laid 50% or more of their eggs in a single litter quantity. Hens selected the large quantity in preference to the small and zero. More searching, nest selection and nesting behaviours tended to be directed to the boxes with the largest quantities and also to the quantities that the hens finally selected for egg laying. 3. In a second experiment, the hens from experiment 1 which showed a preference for litter, and a further 22 naïve hens were given a choice of three nest boxes containing no litter and one containing one of 6 quantities. Over 30 trials there were 5 replicates using each of the three quantities used in experiment 1 plus 115 cm3, 40 cm3 and 10 cm3. 4. There was a wide range in the proportion of trials in which hens selected litter; for experienced hens the range was 0.43 to 0.97, but only two naïve hens were within this range, all others being between 0 and 0.43. 5. The pattern of choosing litter quantities was comparable but at different levels of preference in experienced and naïve hens and a contour map was derived enabling predictions to be made of the probability of a particular quantity being chosen. 6. The variability of individual responses suggest that no single type of nest box system can cater for the requirements of all hens.

Influence of motivational state on choice of food or a dustbathing/foraging substrate by domestic hens.

Domestic hens were trained to run a Y-maze and make an association between differently coloured doorways and access to food pellets or sand. The hens were tested for their choice of doorway when the goals were not visible from the choice point and when they were food or sand deprived. Hens made the choice appropriate to their deprivation state (correct choice) significantly more often for food than sand and were faster at choosing and entering the goal box when food deprived. In a follow up experiment, the goals were visible from the choice point. Again the hens chose correctly significantly more often when food than sand deprived and made the choice and entered the goal box faster when food deprived. Thus, failure to choose sand in the first experiment was not due to an inability to learn the association, but appears to result from a strong motivation to feed in the Y-maze, even when not food deprived, and a weak motivation to dustbathe or forage, even when sand deprived.

Haematological changes associated with food-related oral lesions in brown leghorn hens.

After 19 weeks on a mash diet nine of the 10 birds exhibited from two to 19 oral lesions. The majority of these lesions involved the total erosion of the buccal epidermis giving a total area of exposed dermis of 0.6 to 94.5 mm(2). This was accompanied by a significant reduction in the numbers of heterophils, monocytes and eosinophils. Birds with lesions also had significantly reduced concentrations of haemoglobin and may have been suffering from a mild form of microcytic normochromic anaemia without the complication of an extensive bone marrow involvement. These findings have implications for poultry welfare.

Behavioural consequences of partial beak amputation (beak trimming) in poultry.

1. The effects of beak trimming on 16-week-old Brown Leghorn hens, housed individually in battery cages, was assessed by comparing their behaviour after trimming with their behaviour before trimming and with the behaviour of a sham-operated control group. 2. In the short-term, times spent feeding, drinking and preening decreased. 3. In the long-term, times spent preening and pecking at the cage decreased and times spent standing inactive increased, with no signs of returning to pretreatment values after 5 weeks. 4. During the first three weeks, times spent feeding and drinking decreased and during the first two weeks, times spent sitting dozing increased, but after 5 weeks these had returned to near pre-treatment values. 5. It is argued that pain is the most probable cause of these behavioural changes. 6. The decrease in welfare to the individual bird caused by this pain will conflict with any increase in welfare to the flock brought about by beak trimming; this should be considered before any decision to beak trim is taken.