Epiphyseal Plate Chondrocytes Research Articles

Alcohol intake during pregnancy reduces offspring bone epiphyseal growth plate chondrocyte proliferation through transforming growth factor β-1 inhibition in the Sprague Dawley rat humerus.

Intrauterine alcohol exposure delays bone maturation and intensifies osteoporosis and fracture risk. As most studies emphasize the neurological aspects of intrauterine alcohol exposure, there is a lack of research on the implications pertaining to osseous tissue. Previous studies investigated these effects in fetuses, with limited studies on postnatal life. Postnatal studies are crucial since peak bone growth occurs during adolescence. This study aimed at assessing the effects of prenatal alcohol exposure on the humerus proximal and distal growth plate chondrocytes in 3-week-old rats. Sprague Dawley rats (n=9) were assigned to either the ethanol group (n=3), saline (n=3), and untreated (n=3) group and time-mated. Once pregnant, as confirmed by the presence of a copulation plug, the former 2 groups were treated with 0.015 ml/g of 25.2% ethanol and 0.9% saline. The untreated group received no treatment. The left humeri belonging to 6 pups per group were used. Serial sections were cut with a microtome at 5 µm thickness. These sections were stained with haematoxylin and eosin for assessment of normal morphology or immunolabeled with anti-Ki-67 and transforming growth factor β-1 (TGFβ-1) antibody. Prenatal alcohol exposure adversely effected the growth plate sizes and the number of cells in the proliferative zone. Fewer TGFβ-1 immunopositive and proliferative chondrocytes were found using the anti-Ki-67 antibody. This may explain the growth retardation in offspring exposed to gestational alcohol, showing that gestational alcohol exposure inhibits cell proliferation, aiding the diminished stature.

Lineage-specific differences and regulatory networks governing human chondrocyte development.

To address large gaps in our understanding of the molecular regulation of articular and growth plate cartilage development in humans, we used our directed differentiation approach to generate these distinct cartilage tissues from human embryonic stem cells. The resulting transcriptomic profiles of hESC-derived articular and growth plate chondrocytes were similar to fetal epiphyseal and growth plate chondrocytes, with respect to genes both known and previously unknown to cartilage biology. With the goal to characterize the regulatory landscapes accompanying these respective transcriptomes, we mapped chromatin accessibility in hESC-derived chondrocyte lineages, and mouse embryonic chondrocytes, using ATAC-sequencing. Integration of the expression dataset with the differentially accessible genomic regions revealed lineage-specific gene regulatory networks. We validated functional interactions of two transcription factors (TFs) (RUNX2 in growth plate chondrocytes and RELA in articular chondrocytes) with their predicted genomic targets. The maps we provide thus represent a framework for probing regulatory interactions governing chondrocyte differentiation. This work constitutes a substantial step towards comprehensive and comparative molecular characterizations of distinct chondrogenic lineages and sheds new light on human cartilage development and biology.

Leptin accelerates BMSC transformation into vertebral epiphyseal plate chondrocytes by activating SENP1-mediated deSUMOylation of SIRT3.

Bone marrow mesenchymal stem cells (BMSCs) are capable of multidirectional differentiation, and engrafted BMSCs can be used to replace damaged chondrocytes for treatment of intervertebral disc disease. However, chondroblast differentiation of implanted BMSCs is inhibited by the anoxic environment of the articular cavity. Here, we found that leptin enhanced the transformation of BMSCs into chondrocytes under hypoxic conditions. BMSCs isolated from mice were cultured in medium supplemented with leptin under hypoxia. The expression of MFN1/2 and OPA1 were increased only in BMSCs cultured in an anoxic environment. In addition, in hypoxic environments cell energy metabolism relies on glycolysis regulated by leptin, rather than by mitochondrial oxidation. The expression of the de-SUMOylation protease SENP1 was elevated, leading to SIRT3-mediated activation of PGC-1α; these processes were regulated by CREB phosphorylation, and promoted mitochondrial fusion and cell differentiation. The chondrogenic activity of BMSCs isolated from SIRT3-knockout mice was lower than that of BMSCs isolated from wildtype mice. Implantation of SIRT3-knockout murine-derived BMSCs did not significantly improve the articular cartilage layer of the disc. In conclusion, the hypoxic microenvironment promoted BMSC differentiation into chondrocytes, whereas osteoblast differentiation was inhibited. SENP1 activated SIRT3 through the deSUMOylation of mitochondria and eliminated the antagonistic effect of SIRT3 acetylation on phosphorylation. When phosphorylation activity of CREB was increased, phosphorylated CREB is then transferred to the nucleus, affecting PGC-1α. This promotes mitochondrial fusion and differentiation of BMSCs. Leptin not only maintains chondrogenic differentiation homeostasis of BMSCs, but also provides energy for differentiation of BMSCs under hypoxic conditions through glycolysis.

Research progress on the mechanism of interleukin-1β on epiphyseal plate chondrocytes

Epiphyseal plate injury, a common problem in pediatric orthopedics, may result in poor bone repair or growth defects. Epiphyseal plate, also known as growth plate is a layer of hyaline cartilage tissue between the epiphysis and metaphyseal and has the ability to grow longitudinally. Under normal physiological conditions, the epiphyseal plate has a certain axial resistance to stress, but it is fragile in growth phase and can be damaged by excessive stress, leading to detachment or avulsion of the epiphysis, resulting in life-long devastating consequences for patients. There is an obvious inflammatory response in the phase of growth plate injury, the limited physiological inflammatory response locally favors tissue repair and the organism, but uncontrolled chronic inflammation always leads to tissue destruction and disease progression. Interleukin-1β (IL-1β), as representative inflammatory factors, not only affect the inflammatory phase response to bone and soft tissue injury, but have a potentially important role in the later repair phase, though the exact mechanism is not fully understood. At present, epiphyseal plate injuries are mainly treated by corrective and reconstructive surgery, which is highly invasive with limited effectiveness, thus new therapeutic approaches are urgently needed, so a deeper understanding and exploration of the pathological mechanisms of epiphyseal plate injuries at the cellular molecular level is an entry point. In this review, we fully introduced the key role of IL-1 in the progression of epiphyseal plate injury and repair, deeply explored the mechanism of IL-1 on the molecular transcript level and endocrine metabolism of chondrocytes from multiple aspects, and summarized other possible mechanisms to provide theoretical basis for the clinical treatment and in-depth study of epiphyseal plate injury in children.

Leptin differentially regulate cell apoptosis and cycle by histone acetylation in tibial and vertebral epiphyseal plates.

Leptin showed different apoptosis regulation effects on the chondrocytes from tibial and vertebral epiphyseal plates. However, the mechanism is still unclear. In this study, we tested the protein profile of tibial and vertebral epiphyseal plate chondrocytes with and without leptin stimulation by mass spectrometry and found that the histone acetylation level of tibial chondrocytes was decreased after leptin treatment, while increased in vertebral epiphyseal plates. COIP assay showed that leptin promoted H3, H4 histone acetylation by recruiting CREB binding protein (CBP)/P300 to activate histone acetyl transferases (HATs) activity in vertebral disc chondrocytes. But in tibial plate cartilage cells, leptin did not recruit CBP and p300, thus differently affect the apoptosis of epiphyseal plate chondrocytes. Through explored the mechanism of histone acetylation modulated by leptin, and its effect on cartilage cell apoptosis and cell cycle regulation, This provides a novel target therapy possibility therapeutic approach to for the related disease.

Leptin differentially regulates endochondral ossification in tibial and vertebral epiphyseal plates.

Longitudinal bone growth is governed by a complex network of endocrine signals including leptin. In mouse, leptin deficiency leads to distinct phenotypes in bones of the limb and spine, suggesting the appendicular and axial skeletons are subject to differential regulation by leptin. We established primary cultures for the chondrocytes from tibial and vertebral epiphyseal plates. Cellular proliferation and apoptosis were analyzed for the chondrocytes that had been treated with various concentrations of leptin. Crucial factors for chondrocyte proliferation and differentiation, such as BMP7 and Wnt3, were measured in the cells treated with leptin alone or in combination with pharmacological inhibitors of STAT and ERK signaling pathways. Primary culture of tibial epiphyseal plate chondrocytes has greater proliferating capability compared with that of vertebral epiphyseal plate chondrocytes. Leptin could promote the proliferation of tibial epiphyseal plate chondrocytes, while its effect on vertebral epiphyseal plate chondrocytes was inhibitory. Consistently, apoptosis is inhibited in tibial but promoted in vertebral epiphyseal plate chondrocytes by leptin. Importantly, leptin differentially modulates chondrogenic signaling pathways in tibial and vertebral epiphyseal chondrocytes through STAT and ERK pathways. Leptin differentially regulates chondrogenic proliferation and differentiation in appendicular and axial regions of the skeletons. The signaling pathways in these two regions are also distinct and subject to differential regulation by leptin through the STAT pathway in tibial epiphyseal plate chondrocytes but through the ERK pathway in vertebral epiphyseal plate chondrocytes. Therefore, the regulation of leptin is multi-faceted in the distinct anatomical regions of the skeleton. Knowledge gained from this system will provide insights into the pathophysiological causes for the diseases related to bone development and metabolism.

Histology of Epiphyseal Plate of Adolescent Rat Stimulated by Laserpuncture

Epiphyseal plate was used for determining longitudinal bone growth. Laserpuncture was believed to stimulate height growth. We used 40 male Wistar rats which aged three weeks old and weighed more than 40 g as subjects. They randomly divided into group A or B and each group evenly divided into four subgroups which were a negative control and others applied with laser on GV20, ST 36 or combination of GV 20+ST 36 respectively. These acupoints were then stimulated using the laser. After treatment, mice were sacrificed, then tibias were taken for histology preparation processes. By light microscope, epiphyseal plate (EP) height (µm) and chondrocytes hypertrophy (CH) height were measured at six equidistant points, and the values were averaged to obtain a final result for each section. Collected data were analyzed using ANOVA test, and the significant value was set up p< 0.05. The mean of EC and CH were lower than control, but mean of ratio EP/CH were higher than control. However, ANOVA showed that there did not differ significantly (p=0.36).

The effect of mechanical stretch stress on the differentiation and apoptosis of human growth plate chondrocytes.

The study is aimed to investigate the effect of stretch stress with different intensities on the differentiation and apoptosis of human plate chondrocytes. In the present study, the human epiphyseal plate chondrocytes were isolated and cultured in vitro. Toluidine blue staining and type II collagen immunohistochemical staining were used to identify the chondrocytes. Mechanical stretch stresses with different intensities were applied to intervene cells at 0-, 2000-, and 4000-μ strain for 6h via a four-point bending system. The expression levels of COL2, COL10, Bax, Bcl-2, and PTHrp were detected by quantitative RT-PCR. Under the intervention of 2000-μ strain, the expression levels of COL2, COL10, and PTHrp increased significantly compared with the control group (P<0.05), and the expression level of PCNA was also increased, but the difference was not statistically significant (P>0.05). Under 4000-μ strain, however, the expression levels of PCNA, COL2, and PTHrp decreased significantly compared with the control group (P<0.05), and the expression level of COL10 decreased slightly (P>0.05). The ratio of Bcl-2/Bax gradually increased with the increase of stimulus intensity; both of the differences were detected to be statistically significant (P<0.05). In conclusion, the apoptosis of growth plate chondrocytes is regulated by mechanical stretch stress. Appropriate stretch stress can effectively promote the cells' proliferation and differentiation, while excessive stretch stress inhibits the cells' proliferation and differentiation, even promotes their apoptosis. PTHrp may play an important role in this process.

Assessment of Epiphyseal Plate Allograft Viability and Function After Ex Vivo Storage in University of Wisconsin Solution

Compromised epiphyseal plate function can result in limb deformities. Microvascular transplantation of an epiphyseal plate allograft is a potentially effective approach to reestablish longitudinal limb growth. For this procedure to become clinically useful, the technique for temporary ex vivo storage of allografts must be reliable. The goal of this study was to determine a time frame for which proximal tibial epiphyseal plate allografts could be stored in University of Wisconsin Preservation Solution (UWPS) and remain functional in vivo after microvascular transplantation. Proximal tibial epiphyseal plate allografts from skeletally immature female New Zealand White rabbits (10 to 12 wk of age) were used. Allografts (isolated on the popliteal arteriovenous pedicle) were stored ex vivo in cold UWPS for periods of up to 21 days. Chondrocyte viability, phenotype, and extracellular matrix composition of growth plate cartilage was assessed. Microvascular transplantations of nonstored or prestored (3 d) allografts were performed and analysis of bromodeoxyuridine and calcein incorporation was done to determine chondrocyte proliferation and new bone growth, respectively. In vitro analysis showed that, compared with control tissue, epiphyseal plate chondrocyte viability (P>0.05), organization, and collagen extracellular matrix was preserved up to 4 days in cold UWPS. Microvascular transplantation of nonstored epiphyseal plate allografts was successful. Despite care being taken to ensure vascular patency during the microvascular procedure, transplantation of prestored allografts failed due to absent flow in the larger vessels and in the allograft based upon the visualization of organized thrombus within the vascular pedicle, and absent flow within the composite graft itself. However, growth plate viability and function was detected in a peripheral region of a single allograft where partial blood flow had been maintained during the transplantation period. Ex vivo storage in cold UWPS for 3 days maintains growth plate chondrocyte viability and function in vivo. However, future studies must be directed toward investigating the direct effect of ex vivo storage on the integrity and function of the vascular pedicles.

The effect of Kashin‐Beck disease‐affected feed and T‐2 toxin on the bone development of Wistar rats

To investigate the effect of Kashin-Beck disease (KBD)-affected feed and T-2 toxin on the bone development of Wistar rats. Seventy-eight ablactation Wistal rats (50% male and 50% female) weighing approximately 65 g were obtained from Sichuan Medical Center (Chengdu, China), and were randomly assigned to three groups (Groups A, B and C). Group A had 24 rats and were fed with commercial rat feed (control); Group B had 30 rats and were fed with commercial rat feed and T2 toxin by intragastric administration; and Group C had 24 rats and were fed with the KBD-affected feed. The histological sections were stained with hematoxylin and eosin (H&E) and Masson dye. Weight gain was fastest Group A rats and Group C rats had the lowest weight gain (P < 0.05). There were no epiphyseal plate chondrocyte necroses in the control group at the first, second, and fourth weeks. In the T-2 toxin group, two rats had chondrocyte-focus necroses at the labrocyte cell zone at the second week. At the fourth week, six rats had chondrocyte-focus or lamellar necroses at the labrocyte cell zone. Three rats had focus necrosis at the proliferation cell zone, and there were three rats with penetration necrosis. In the KBD-affected group, one rat had chondrocyte-focus necrosis at the labrocyte cell zone at the second week and seven rats had chondrocyte-focus necrosis at the labrocyte cell zone at the fourth week. And at the same time, two rats had focus necrosis at the proliferation cell zone, three rats had lamellar necrosis at the labrocyte cell zone, four had focus necrosis at the labrocyte cell zone, and two rats had penetration necrosis. The epiphyseal plate Masson dye of the control group showed deep blue collogen coloration and in the KBD-affected group and T-2 toxin group, collogen showed a pale blue color, the drum dyeing was uneven, and the collogen was showed an absence of color in the region of the necrosis. With KBD-affected feed or T-2 toxin intervention, rats had focus necrosis and lamellar necrosis at the epiphyseal plate. KBD-affected feed rats had less weight gain than T-2 toxin intervention rats, which means there were other etiological factors in KBD-affected feed.

Lead and growth status of schoolchildren living in the copper basin of south-western Poland: Differential effects on bone growth

The study analyzed the relationship between blood lead (BPb) levels and growth status of upper and lower body segments among schoolchildren living in the copper basin of south-western Poland. Schoolchildren 7-15 years of age (463 males, 436 females) living in the vicinity of copper smelters and refineries were tested for blood lead. Weight, height, symphyseal height (leg, lower segment length), and arm length were measured. Trunk (upper segment including head and neck) length was estimated as height minus symphyseal height. The BMI was calculated using weight/height2 (kg m-2). Mean blood lead in the total sample was 7.7 +/- 3.5 micro g dL-1 (2.0-33.9 micro g dL-1). Blood lead level was significantly and linearly related to reduced weight, height, trunk, leg, and arm lengths. The estimated reduction in height was 5 mm per 1 micro g dL-1 increase in blood lead (5.0 cm per 10 micro g dL-1). The reduction occurred in leg length (males, 64%; females 72%) compared to trunk length (males, 36%; females, 28%). Estimated reductions in arm length were 2.0 cm (males) and 2.5 cm (females) per 10 micro g dL-1 increase in blood lead. Hence, limb (leg and arm) lengths were markedly reduced. Linear skeletal growth was reduced with increased blood lead, even at levels below that considered for action by US CDC standards. The reduction in height occurred primarily in leg length. Arm length was also reduced, suggesting limb lengths in general were affected by blood lead. Results are consistent with lead-associated interruption of (1) major epiphyseal growth plate chondrocyte hyperplasia, hypertrophy, and interrupted matrix calcification as a primary cause of stunted long bone growth, and reduced stature; and (2) attenuated osteoblast activity (proliferation and migration) as a secondary cause of stunted growth in the study population.

Chondrocyte-Specific Knockout of the G Protein Gsα Leads to Epiphyseal and Growth Plate Abnormalities and Ectopic Chondrocyte Formation

G(s)alpha is a ubiquitously expressed G protein alpha-subunit that couples receptors to adenylyl cyclase. Mice with chondrocyte-specific ablation of the G(s)alpha gene had severe epiphyseal and growth plate abnormalities and ectopic cartilage formation within the metaphyseal region of the tibia. These results show that G(s)alpha negatively regulates chondrocyte differentiation and is the critical signaling mediator of the PTH/PTH-rP receptor in growth plate chondrocytes. G(s)alpha is a ubiquitously expressed G protein alpha-subunit that mediates signaling through G protein-coupled receptors to activate the cAMP/protein kinase A signaling pathway. Although studies suggest an important role for G(s)alpha in regulating growth plate development, direct in vivo results examining this role are lacking. The G(s)alpha gene was ablated in murine cartilage by mating mice with loxP sites surrounding the G(s)alpha promoter and first exon with collagen 2a1 promoter-Cre recombinase transgenic mice. Skeletal tissues were studied by gross and microscopic pathology, and gene expression was determined by in situ hybridization. Mice with complete chondrocyte-specific G(s)alpha deficiency (homozygotes) died within minutes after birth and had severe epiphyseal and growth plate defects with shortening of the proliferative zone and accelerated hypertrophic differentiation of growth plate chondrocytes, a phenotype similar to that of PTH/PTH-related peptide (PTHrP) receptor knockout mice. Indian hedgehog and PTH/PTHrP receptor expression in prehypertrophic chondrocytes was unaffected in mutant mice. PTHrP expression in periarticular cartilage was increased in the mutant mice, probably because of the closer proximity of Ihh-secreting chondrocytes to the periarticular zone. In addition, these mice developed ectopic cartilage at the anterior side of the metaphyseal region in the tibia. Mice with partial G(s)alpha deficiency (heterozygotes) exhibited no phenotype. These results show that G(s)alpha negatively regulates chondrocyte differentiation and is the critical signaling mediator of the PTH/PTHrP receptor in epiphyseal and growth plate chondrocytes.

Familial chondrodysplasia in Holstein calves.

After the release of a report from France on the occurrence of malformed calves genetically related to a Holstein sire, a study was performed to characterize the defect. Danish breeders were encouraged to submit defective progeny of the sire for laboratory examination. Four cases were submitted, whereas a fifth case was only reported. Lesions in affected calves were analogous, with disproportionate growth retardation characterized by fascial dysplasia and shortening of the vertebral column and the abaxial skeleton. Endochondral osteogenesis was disturbed with disorganization of epiphyseal plate chondrocytes, a lesion consistent with generalized chondrodysplasia. Based on morphology, the defect was grouped as a "Dexter bulldog type". A genetic etiology was suspected as cases occurred in a familial pattern. Genealogical evaluation revealed several common ancestors belonging to widely used breeding lines of US Holstein, but because of the extensive use of these sires, their presence in the pedigrees of affected calves might be accidental. Further studies are needed to determine the mode of inheritance.

Expression and activity of the CDK inhibitor p57Kip2 in chondrocytes undergoing hypertrophic differentiation.

Growth plates of p57-null mice exhibit several abnormalities, including loss of collagen type X (CollX) expression. The phenotypic consequences of p57 expression were assessed in an in vitro model of hypertrophic differentiation. Adenoviral p57 expression was not sufficient for CollX expression but did augment induction of CollX by BMP-2. During hypertrophic differentiation, chondrocytes pass from an actively proliferative state to a postmitotic, hypertrophic phenotype. The induction of growth arrest is a central feature of this phenotypic transition. Mice lacking the cyclin dependent-kinase inhibitor p57Kip2 exhibit several developmental abnormalities including chondrodysplasia. Although growth plate chondrocytes in p57-null mice undergo growth arrest, they do not express collagen type X, a specific marker of the hypertrophic phenotype. This study was carried out to investigate the link between p57 expression and the induction of collagen type X in chondrocytes and to determine whether p57 overexpression is sufficient for the induction of hypertrophic differentiation. Neonatal rat epiphyseal or growth plate chondrocytes were maintained in an aggregate culture model, in defined, serum-free medium. Protein and mRNA levels were monitored by Western and Northern blot analyses, respectively. Proliferative activity was assessed by fluorescent measurement of total DNA and by 3H-thymidine incorporation rates. An adenoviral vector was used to assess the phenotypic consequences of p57 expression. During in vitro hypertrophic differentiation, levels of p57 mRNA and protein were constant despite changes in chondrocyte proliferative activity and the induction of hypertrophic-specific genes in response to bone morphogenetic protein (BMP)-2. Adenoviral p57 overexpression induced growth arrest in prehypertrophic epiphyseal chondrocytes in a dose-dependent manner but was not sufficient for the induction of collagen type X, either alone or when coexpressed with the related CDKI p21Cip1. Similar results were obtained with more mature tibial growth plate chondrocytes. p57 overexpression did augment collagen type X induction by BMP-2. These data indicate that p57-mediated growth arrest is not sufficient for expression of the hypertrophic phenotype, but rather it occurs in parallel with other aspects of the differentiation pathway. Our findings also suggest a contributing role for p57 in the regulation of collagen type X expression in differentiating chondrocytes.

Characterization of a biomaterial with cartilage-like properties expressing type X collagen generated in vitro using neonatal porcine articular and growth plate chondrocytes

Objective The availability of cartilage with or without the potential to ossify and suitable for surgical restoration and resurfacing of joints is an important clinical problem in arthritis-related pathology, trauma and reconstructive surgery. Here, we designed experiments to generate a biomaterial with cartilage-like properties by culturing neonatal porcine articular and growth plate chondrocytes on a hydrogel substrate and to examine the biochemical and histological characteristics of the resulting tissue.Design Neonatal porcine epiphyseal and growth plate chondrocytes were cultured on poly(2-hydroxyethyl methacrylate) (polyHEMA)-coated dishes to prevent their adherence to plastic. We previously described that this procedure allows the maintenance of the chondrocyte-specific phenotype for ≥8 months. Chondrocytes were isolated by successive enzymatic digestions and cultured at high density (>2.0×107cells/ml) in DMEM with 10% FBS, 50μg/ml ascorbic acid, glutamine, vitamins, and antibiotics for up to 10 weeks on 60mm plastic culture dishes coated with polyHEMA. The tissues produced during culture were studied histologically and biochemically and were examined for cellular proliferation employing3H-thymidine incorporation and for their collagen production employing biosynthetic labeling with14C-proline and Western blot with specific antibodies. The expression of relevant collagen genes was examined employing RT-PCR.Results Within 24h of culture, isolated chondrocytes organized into well-formed clusters and in 2 weeks formed structures with gross appearance and consistence similar to those of natural cartilage. The wet weight of the tissue formed in vitro increased six-fold during the 10-week period of study. Cell proliferation measured by the incorporation of3H-thymidine increased during the first 3 weeks and reached a plateau in subsequent weeks. Histological examination showed that the cultures contained rounded chondrocytes embedded in an abundant cartilaginous extracellular matrix. The cartilage formed contained large amounts of collagen and sulfated proteoglycans as examined by staining with Masson's Trichrome and Alcian blue, respectively. Deposition of calcium in the deeper layers of the tissue was demonstrated with the von Kossa stain. Western analyses with specific antibodies showed that type II collagen was present from the first week and progressively increased in the cultures, whereas type X collagen was first detected at 4 weeks and increased with length of culture. When chondrocytes isolated from the growth plate were included, small amounts of type I collagen were detected in the medium of cultured biomaterial as expected. Type III collagen was not detected by Western blot over the 10-week period. High levels of type II and type X collagen gene expression were demonstrated by RT-PCR.ConclusionThese studies demonstrate the production in vitro of cartilage-like tissue with similar morphological, histochemical and biochemical characteristics to those of natural growth plate cartilage. The cartilage generated in vitro has the potential to be used in reconstructive surgery and in joint resurfacing and restoration of skeletal defects.

PTHrP Expression in Chondrocytes, Regulation by TGF-β, and Interactions between Epiphyseal and Growth Plate Chondrocytes

Although PTHrP has been identified as a key regulator of chondrocyte differentiation in the growth plate, the factors directly regulating PTHrP expression have not been identified. Furthermore, while cells from the epiphysis are considered the physiologic source of PTHrP, the relative expression of PTHrP in epiphyseal and growth plate chondrocytes has not been defined. PTHrP expression was examined in chondrocytes isolated from 3- to 5-week-old chick long bones. The expression of PTHrP mRNA was 10-fold higher in epiphyseal chondrocytes compared to cells from the growth plate. Growth plate chondrocytes were isolated into populations with distinct maturational characteristics by countercurrent centrifugal elutriation and analyzed for PTHrP expression. The expression was highest in the least mature cells and progressively declined with the onset of maturation. The regulation of PTHrP expression was further examined in epiphyseal chondrocytes. Both TGF-β1 and cis-retinoic acid stimulation markedly increased PTHrP mRNA levels, while BMP-2 and PTHrP stimulation decreased the expression of this transcript. The effects of TGF-β1 (8.9-fold stimulation) and TGF-β3 (9.2-fold) were slightly greater than the effects of TGF-β2 (4.9-fold). The effect of TGF-β was dose-dependent and increases could be detected after 68 h of treatment. To analyze the paracrine effect of epiphyseal and growth plate chondrocytes on each other, these cells were placed in coculture and the mRNA from each of the populations was harvested separately after 24 h. Following coculture the PTHrP mRNA levels increased in the epiphyseal cells while the expression of type X collagen and Indian hedgehog transcripts decreased in growth plate chondrocytes. The results demonstrate potentially important paracrine interactions between these cell populations, possibly mediated by TGF-β and PTHrP.

Hypertrophy of growth plate chondrocytes in vivo is accompanied by modulations in the activity state and surface area of their cytoplasmic organelles.

The rate of longitudinal bone growth is regulated primarily by modulations in the activity of epiphyseal plate hypertrophic chondrocytes, these being manifested as changes in cell and matrix volume. It was the purpose of this study to ascertain whether the cytoplasmic organelles representing the cellular production apparatus, i.e. rough endoplasmic reticulum, Golgi apparatus and mitochondria, contribute to these changes by modulating their rate of activity or by increasing/decreasing the surface area and/or volume of their membranes. Using rats at different stages of growth, the surface areas and volumes of the three organellar systems were quantified in epiphyseal plate chondrocytes at the onset and termination of hypertrophy by ultrastructural stereology. Matrix synthesis during the same span was assessed by monitoring the production of its principal components, namely, fibrillar collagen (ultrastructural morphometry) and glycosaminoglycans (quantitative (35)S-autoradiography). Each organelle adapts to increases (21- to 35-day-old rats) and decreases (35- to 80-day-old rats) in growth rate by its own individual combination of the two alternative mechanisms, but modulations in the level of activity predominate over alterations in the surface area or volume of their membranes. These findings point to the danger of relying solely on data gleaned from a quantitative ultrastructural analysis of organellar parameters and emphasise the necessity of conducting functional assays in parallel, as performed here.

Thyroid hormone inhibits growth and stimulates terminal differentiation of epiphyseal growth plate chondrocytes.

As a continuation of our studies on mineralization in epiphyseal growth plate (GP) chondrocyte cultures, the effects of tri-iodothyronine (T3) in both beta-glycerophosphate-containing, serum-free (HL-1) and beta-glycerophosphate-free, serum-containing medium (DATP5) were studied. The GP cells responded to T3 in a serum-, stage-, and dosage-dependent manner. Added at graded levels (0.1-10.0 nM) to preconfluent cultures (from day 7) in both HL-1 and DATP5, T3 caused progressive decreases in protein, collagen, and DNA synthesis but increased mineral deposition. In postconfluent cultures, these effects of T3 were generally muted. In preconfluent cultures, proteoglycan (PG) levels were not significantly affected in DATP5, although in HL-1 they were decreased by approximately 50%. In postconfluent cultures, T3 increased PG levels in DATP5 but had no effect in HL-1. In HL-1, alkaline phosphatase (ALP) activity was progressively increased by 200-500% in both pre- and postconfluent cultures. In DATP5 in preconfluent cultures, T3 initially stimulated but later suppressed ALP; in postconfluent cultures, T3 also transiently increased ALP but did not suppress activity upon longer exposure. The inhibitory effects of T3 on protein, PG, and DNA levels of GP chondrocytes suggest that in vivo its effects on bone growth must occur primarily after cellular proliferation. Apparently by binding to the 50 kDa thyroxine-binding globulin, which cannot penetrate the PG barrier, accessibility of T3 to GP chondrocytes is limited until the time of vascular penetration when its stimulatory effects on ALP and mineral deposition become critical for continued bone development.

Retinoid-induced epiphyseal plate closure in guinea pigs.

Vitamin A and its derivatives (retinoids) have been known to cause premature epiphyseal closure in humans as an unwanted side effect of chronic treatment. The purpose of the present study was to determine if guinea pigs could serve as an animal model of retinoid-induced epiphyseal plate closure, and to utilize this model to study the mechanism. Weanling male Hartley guinea pigs were treated ip via osmotic pump for up to 14 days with vehicle or 0.50 to 5.5 mg/kg/day of the retinoic acid receptor (RAR)-selective agonist AGN 190121. Histopathological examination of the proximal tibia of AGN 190121-treated guinea pigs revealed a dose-dependent disruption of the epiphyseal plate. The natural retinoids all-trans-retinoic acid and 13-cis-retinoic acid also induced epiphyseal plate closure in guinea pigs when administered by ip injection for 10 days. Prominent histological features of retinoid-induced epiphyseal closure included the loss of basophilic staining in the extracellular matrix of epiphyseal plate chondrocytes and the invasion of the epiphyseal plate by osteoclasts. To determine if the epiphyseal closure detected histologically was reversible, guinea pigs were treated for 6 days with the RAR-selective agonist (E)-4[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)propen- 1-y1]benzoic acid (TTNPB) or vehicle, and groups of guinea pigs were euthanized on Day 7 or 57. TTNPB but not vehicle treatment caused histological evidence of epiphyseal closure at both time points, and significant bone elongation between Day 7 and Day 57 was detected only in vehicle-treated animals. Epiphyseal closure and other toxic effects of TTNPB were blocked by cotreatment of guinea pigs with a fivefold molar excess of AGN 193109, an RAR antagonist. Taken together, these data demonstrate the utility of the guinea pig as an animal model of retinoid-induced epiphyseal closure and suggest that RAR activation is necessary and sufficient for this activity.

Mechanism of longitudinal bone growth and its regulation by growth plate chondrocytes.

Growth plate chondrocytes play a pivotal role in promoting longitudinal bone growth. The current review represents a brief survey of the phenomena involved in this process at the cellular level; it delineates the contributions made by various activities during the course of the chondrocyte life cycle, notably proliferation and hypertrophy, and illustrates how the relative contributions may be modulated according to the particular needs of an organism at critical phases of growth. The cellular mechanisms by which a few well characterized growth-promoting substances exert their influences are discussed in the light of recent findings pertaining to epiphyseal plate chondrocytes in vivo.