Growth Plate Development Research Articles

Expression and release of insulin-like growth factor binding proteins in isolated epiphyseal growth plate chondrocytes from the ovine fetus.

Insulin-like growth factor-II (IGF-II) is an autocrine modulator of epiphyseal chondrogenesis in the fetus. The cellular availability of IGFs are influenced by the IGF-binding proteins (IGFBPs). In this study, we investigated the control of expression and release of IGFBPs from isolated epiphyseal growth plate chondrocytes from the ovine fetus by hormones and growth factors implicated in the chondrogenic process. Chondrocytes were isolated from the proliferative zone of the fetal ovine proximal tibial growth plate and maintained in monolayer culture at early passage number. Culture media conditioned by chondrocytes under basal conditions released IGFBPs of 24, 34, and 29 kDa, and a less abundant species of 39-43 kDa that were identified immunologically as IGFBP-4, IGFBP-2, IGFBP-5, and IGFBP-3, respectively. Messenger RNAs encoding each species were identified by Northern blot analysis within chondrocytes, as was mRNA encoding IGFBP-6. Exposure to IGF-I or IGF-II (13 or 26 nM) caused an increase in expression and release of IGFBP-3. The release of IGFBP-2 and IGFBP-5 were also potentiated without changes to steady state mRNA, and for IGFBP-5 this was due in part to a release from the cell membrane in the presence of IGF-II. Insulin (16.7 or 167 nM) selectively increased mRNA and the release of IGFBP-3, while cortisol (1 or 5 microM) inhibited both mRNA and release of IGFBP-2 and IGFBP-5. Transforming growth factor-beta1 (TGF-beta1) (0.1 or 0.2 nM) increased the expression and release of IGFBP-3, and caused an increase in mRNAs encoding IGFBP-2 and IGFBP-5. Neither growth hormone (GH), fibroblast growth factor-2, nor thyroxine (T(4)) had any effect on IGFBP expression or release. The results suggest that IGFBP expression and release within the developing growth plate can be modulated by IGF-II and other trophic factors, thus controlling IGF availability and action.

Ror2, encoding a receptor-like tyrosine kinase, is required for cartilage and growth plate development.

Receptor tyrosine kinases often have critical roles in particular cell lineages by initiating signalling cascades in those lineages. Examples include the neural-specific TRK receptors, the VEGF and angiopoietin endothelial-specific receptors, and the muscle-specific MUSK receptor. Many lineage-restricted receptor tyrosine kinases were initially identified as 'orphans' homologous to known receptors, and only subsequently used to identify their unknown growth factors. Some receptor-tyrosine-kinase-like orphans still lack identified ligands as well as biological roles. Here we characterize one such orphan, encoded by Ror2 (ref. 12). We report that disruption of mouse Ror2 leads to profound skeletal abnormalities, with essentially all endochondrally derived bones foreshortened or misshapen, albeit to differing degrees. Further, we find that Ror2 is selectively expressed in the chondrocytes of all developing cartilage anlagen, where it essential during initial growth and patterning, as well as subsequently in the proliferating chondrocytes of mature growth plates, where it is required for normal expansion. Thus, Ror2 encodes a receptor-like tyrosine kinase that is selectively expressed in, and particularly important for, the chondrocyte lineage.

RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism.

We have generated RANK (receptor activator of NF-kappaB) nullizygous mice to determine the molecular genetic interactions between osteoprotegerin, osteoprotegerin ligand, and RANK during bone resorption and remodeling processes. RANK(-/-) mice lack osteoclasts and have a profound defect in bone resorption and remodeling and in the development of the cartilaginous growth plates of endochondral bone. The osteopetrosis observed in these mice can be reversed by transplantation of bone marrow from rag1(-/-) (recombinase activating gene 1) mice, indicating that RANK(-/-) mice have an intrinsic defect in osteoclast function. Calciotropic hormones and proresorptive cytokines that are known to induce bone resorption in mice and human were administered to RANK(-/-) mice without inducing hypercalcemia, although tumor necrosis factor alpha treatment leads to the rare appearance of osteoclast-like cells near the site of injection. Osteoclastogenesis can be initiated in RANK(-/-) mice by transfer of the RANK cDNA back into hematopoietic precursors, suggesting a means to critically evaluate RANK structural features required for bone resorption. Together these data indicate that RANK is the intrinsic cell surface determinant that mediates osteoprotegerin ligand effects on bone resorption and remodeling as well as the physiological and pathological effects of calciotropic hormones and proresorptive cytokines.

Aberrations of cell cycle and cell death in normal development of the chick embryo growth plate

The epiphyses of femurs from 7.5–15 day chicken embryos were studied by electron microscopy. Several forms of aberrant cell cycles were present: (1) in the perichondrium, polyploid metaphases, segmentating large (giant) cells, and mitotic catastrophe (midway between mitosis and apoptosis) were observed; (2) in the resting zone, premature chromosome condensation was found; (3) in the proliferative zone, approximately 5% of divisions were aberrant, representing most often mitosis restitution from metaphase and more seldom from the anaphase; (4) in all layers, ‘dark chondrocytes’ representing a premortal form of hypersecretory cells undergoing often a-mitotic nuclear segmentation were present. Many of the aberrations of cell cycle were combined with cell death. These deviations omitting or adapting the cell cycle check-points represent evidently the normal epigenetic mechanisms of development and repair. At the same time, by origin and appearances they seem very close to the loss of the growth control displayed by malignant tumours. This connection is briefly analysed in view of some current concepts of carcinogenesis.

Expression of bone-specific genes by hypertrophic chondrocytes: implication of the complex functions of the hypertrophic chondrocyte during endochondral bone development.

Endochondral bone formation is one of the most extensively examined developmental sequences within vertebrates. This process involves the coordinated temporal/spatial differentiation of three separate tissues (cartilage, bone, and the vasculature) into a variety of complex structures. The differentiation of chondrocytes during this process is characterized by a progressive morphological change associated with the eventual hypertrophy of these cells. These cellular morphological changes are coordinated with proliferation, a columnar orientation of the cells, and the expression of unique phenotypic properties including type X collagen, high levels of bone, liver, and kidney alkaline phosphatase, and mineralization of the cartilage matrix. Several studies indicate that hypertrophic chondrocytes also express osteocalcin, osteopontin, and bone sialoprotein, three proteins which until very recently were widely believed to be restricted in their expression to osteoblasts. Recent studies suggest that the hypertrophic chondrocytes are regulated by the calcitropic hormones, morphogenic steroids, and local tissue factors. These considerations are based on the regulation by 1,25 (OH)2D3 and retinoids of the cartilage specific genes as well as osteopontin and osteocalcin expression in hypertrophic chondrocytes. They are also based on the effects on growth plate development caused by 1) transgenic ablation of autocrine/paracrine regulators such as PTHrP and of the transcriptional regulator c-fos and 2) naturally occurring genetic mutations of the FGF receptor. These studies further suggest that specific transcriptional factors mediate exogenous regulatory signals in a coordinated manner with the development of bone. While it has been widely demonstrated that the majority of hypertrophic chondrocytes undergo apoptosis during terminal stages of the developmental sequence, their response to specific exogenous regulatory signals and their expression of bone-specific proteins give rise to questions about whether all growth chondrocytes have the same developmental fates and have identical functions. Furthermore, specific questions arise as to whether there are similar mechanisms of regulation for commonly expressed genes found in both cartilage and bone or whether these genes have unique regulatory mechanisms in these different tissues. These recent findings suggest that hypertrophic chondrocytes are functionally coupled during endochondral bone formation to the recruitment of osteoblasts, vascular cells, and osteoclasts.

Effect of Lead on Bone and Cartilage in Sexually Mature Rats: A Morphometric and Histomorphometry Study

The effect of exposure to lead on the longitudinal development of the femur and of its cartilage growth plate was studied in rats. A group of forty-five 50-day-old female Wistar rats was divided into a control group of 20 rats and an experimental group of 25 rats fed a diet supplemented with 17 mg of lead acetate per kilogram of feed for 50 days. On Day 50 all rats were killed and their right femurs were dissected. The femurs were cleaned of soft tissue and femoral lengths were measured with a Vernier caliper and thickness of growth cartilage (GPC-Th μm) by histomorphometry. Final body weights were significantly (P< 0.05) lower in the control group than in the rats given the lead-supplemented diet. Femur length did not differ between groups. Histomorphometry of the femur showed that the thickness of growth cartilage was higher (P< 0.05) in the control group. These findings suggested lead-induced inhibition of growth plate development. The growth plate may be one of the key target tissues accounting for the adverse effects of chronic lead exposure on skeletal development.

Expression of bone-specific genes by hypertrophic chondrocytes: Implications of the complex functions of the hypertrophic chondrocyte during endochondral bone development

Endochondral bone formation is one of the most extensively examined developmental sequences within vertebrates. This process involves the coordinated temporal/spatial differentiation of three separate tissues (cartilage, bone, and the vasculature) into a variety of complex structures. The differentiation of chondrocytes during this process is characterized by a progressive morphological change associated with the eventual hypertrophy of these cells. These cellular morphological changes are coordinated with proliferation, a columnar orientation of the cells, and the expression of unique phenotypic properties including type X collagen, high levels of bone, liver, and kidney alkaline phosphatase, and mineralization of the cartilage matrix. Several studies indicate that hypertrophic chondrocytes also express osteocalcin, osteopontin, and bone sialoprotein, three proteins which until very recently were widely believed to be restricted in their expression to osteoblasts. Recent studies suggest that the hypertrophic chondrocytes are regulated by the calcitropic hormones, morphogenic steroids, and local tissue factors. These considerations are based on the regulation by 1,25 (OH)2D3 and retinoids of the cartilage specific genes as well as osteopontin and osteocalcin expression in hypertrophic chondrocytes. They are also based on the effects on growth plate development caused by 1) transgenic ablation of autocrine/paracrine regulators such as PTHrP and of the transcriptional regulator c-fos and 2) naturally occurring genetic mutations of the FGF receptor. These studies further suggest that specific transcriptional factors mediate exogenous regulatory signals in a coordinated manner with the development of bone. While it has been widely demonstrated that the majority of hypertrophic chondrocytes undergo apoptosis during terminal stages of the developmental sequence, their response to specific exogenous regulatory signals and their expression of bone-specific proteins give rise to questions about whether all growth chondrocytes have the same developmental fates and have identical functions. Furthermore, specific questions arise as to whether there are similar mechanisms of regulation for commonly expressed genes found in both cartilage and bone or whether these genes have unique regulatory mechanisms in these different tissues. These recent findings suggest that hypertrophic chondrocytes are functionally coupled during endochondral bone formation to the recruitment of osteoblasts, vascular cells, and osteoclasts.

Receptors for parathyroid hormone and parathyroid hormone-related peptide: from molecular cloning to definition of diseases.

The parathyroid hormone/parathyroid hormone-related peptide receptor belongs to a distinct family of G protein-coupled receptors, the members of which usually signal through at least two second messenger systems, adenylate cyclase and phospholipase C. The parathyroid hormone/ parathyroid hormone-related peptide receptor is most abundantly expressed in bone, kidney and growth-plate chondrocytes, and, at lower levels, in a variety of fetal and adult tissues. To search for human diseases that are caused by parathyroid hormone/parathyroid hormone-related peptide receptor defects, genomic DNA of patients with pseudohypoparathyroidism type Ib and of patients with Jansen's metaphyseal chondrodysplasia was screened for mutations in all coding exons of the receptor gene. Inactivating parathyroid hormone/parathyroid hormone-related peptide receptor mutations were excluded in patients with pseudohypoparathyroidism type Ib. However, a receptor mutation that causes agonist-independent, constitutive cAMP accumulation was identified in a patient with Jansen's metaphyseal chondrodysplasia, a rare form of short-limbed dwarfism associated with hypercalcemia despite normal or low concentrations of parathyroid hormone and parathyroid hormone-related peptide. These findings allow the conclusion to be drawn that parathyroid hormone/parathyroid hormone-related peptide receptors mediate the endocrine actions of parathyroid hormone, which are required for the control of calcium homeostasis and the autocrine-paracrine actions of parathyroid hormone-related peptide, which are required for normal growth-plate development.

Ｃｏｎｇｅｎｉｔａｌ Ｃｏｎｔｒａｃｔｕｒａｌ Ａｒａｃｈｎｏｄａｃｔｙｌｙと下垂体機能低下症による両側大腿骨頭すべり症を合併した１例

A 30-year-old male was seen at our institution with right hip pain. He had noticeable arachnodactyly, multiple contractures, dolichostenomelia, and abnormality of the external ears. He had not developed secondary sexual characteristics. Internal rotation of the hip was limited and Drehman's sign was positive bilaterally. Roentgenograms showed incomplete development of the epiphyseal growth plate and posterior slipping of both upper femoral epiphyses. Clinical and laboratory findings revealed congenital contractural arachonodactyly associated with slipped capital femoral epiphyses due to primary hypopituitarism. We began hormonal therapy and performed Southwick's osteotomy through the lesser trochanter to the patient's right hip and in situ fixation to the left hip.

A large-deformation, finite-element study of chondrodiatasis in the canine distal femoral epiphyseal plate

A large-deformation, finite-element analysis was conducted to model stress fields around the developing growth plate as a first approach to comprehend the clinical application of traction for limb lengthening procedures. The model chosen was a cross section through the distal femoral growth plate of a 14-week-old dog. The chosen section passed through two of the conformational bends (mammillary processes) formed by the natural convolutions of the physis. Three different loading conditions were applied to the distal femoral epiphyseal model. The model also examined the effects of different values of Young's modulus of the growth-plate cartilage. The cortical bone in all cases, experienced the highest stresses. In the growth plate, the highest principal stresses occurred where the physis intersects cortical bone. There were localized stresses that were higher than those that caused fracture in a rabbit model [Guse et al., J. Orthop. Res. 7, 667–673 (1989)]. Results also indicated the following: a small tilt of 0.1 in loading application increases the maximum principal stresses and the von Mises stresses in certain regions of the growth plate by about 8%; the shearing streses in the growth plate are sensitive to Young's modulus of the growth plate; and traction pins that do not grip the cancellous bone in the epiphysis will increase the regions of high principal stress in the growth plate.

Spinal deformities in deferoxamine-treated homozygous beta-thalassemia major patients.

A new constellation of spinal changes are observed in homozygous beta-thalassemia major (HBT) patients receiving deferoxamine (DF), an iron-chelating drug used in combination with transfusion therapy in certain anemic syndromes. In a retrospective study of 22 HBT patients who were receiving DF therapy, morphological deformities (decreased spinal height, increased thoracic kyphosis, vertebral flattening and elongation anteriorly, and disk calcification) were found in 16 of 22 patients. These changes are believed to be caused by interference with spinal growth-plate development. Investigation of DF-dose correlation supports the conclusion that the spinal changes were DF-induced. Spinal changes observed in DF-treated patients differ both morphologically and pathogenetically from earlier reports of vertebral deformities occurring as a sequel to compensatory marrow hyperplasia in poorly transfused patients.

Immunohistochemical characterisation of the costo-chondral junction in sudden infant death syndrome (SIDS)

A number of studies have concluded that infants who die of SIDS have evidence of significant growth retardation. Although there appears to be considerable overlap with control infants, reduced weight gain, poor neurological development and specific abnormalities of the bone growth plate have all been reported. This raises the possibility that SIDS infants may have been physiologically compromised for some time prior to death. Alteration of growth plate morphology may, however, be influenced by primary abnormalities of proteoglycan composition, given that growth plate organisation depends on the intimate interaction of chondrocytes with extracellular matrix. The following study was undertaken to characterise the distribution of major growth plate proteoglycans in a group of SIDS infants compared to control infants. Sections of rib and adjacent costal cartilage were taken at autopsy from 9 SIDS infants and 8 age- matched controls (4 asphyxial deaths, 2 traumatic deaths, 1 poisoning and 1 drowning). Following decalcification and routine processing, immunohistochemical staining was performed for chondroitin (ch)-4-SO 4 , ch-6-SO 4 , keratan SO 4 and over- sulphated ch-SO 4 with antibodies 2-B-6, 3-B-3, 5-D-4 and 7-D-4 using standard methods. The tested proteoglycans showed either a uniform or zonal distribution in the growth plate in SIDS infants, with an identical pattern of distribution in controls. This preliminary finding suggests that the abnormal growth plate morphology reported in a percentage of infants dying of SIDS may well be a secondary phenomenon , not related to primary skeletal dysfunction. In summary, the distribution of proteoglycans within the extracellular matrix of the costochondral junction in SIDS infants in this study appeared normal.

Human growth plate development in the fetal and neonatal period

The development of the normal human upper tibial growth plate was studied at autopsy in 46 stillborns and 79 newborns of 20-41 weeks gestational age. During this time period, the histology of this plate evolves from a highly cellular structure with relatively poor columnar organization and matrix development to the well known structure seen later in postnatal life. The thickness of the growth plate, assessed in the area surrounding the longitudinal tibial axis, decreases continuously from 1.15 mm on the 20th week to 0.6 mm on the 38th week. This decrease results from losses of both matrix and cellular components, mostly of the latter. However, the relative fraction of area occupied by the matrix significantly increased (12%) and matrix area per cell increased 1.5 times over the last half of gestation, indicating a maturation process of the plate towards a more matrix-oriented structure with age. In this maturation process the number of cells per unit area does not change and the average size of the cells appears to decrease. Plate thickness does not decrease further in the final 3 weeks of pregnancy and increases in early neonatal life; this has no apparent influence on the tibial growth rate. In the period under study the relative anatomical participation of the upper tibial growth plate decreases from approximately 4% of the radiographic length of the tibia on the 20th week to less than 1% at term. Present data will provide fetal and neonatal growth plate standards needed to obtain a better understanding of this structure during both normal and abnormal conditions.

Analysis of growth plate abnormalities following intraosseous infusion through the proximal tibial epiphysis in pigs.

Intraosseous infusion has become an increasingly popular technique for vascular access in critically ill or injured children. Continuing acceptance of this procedure by physicians and possibly by prehospital personnel may lead to inadvertent placement through the immature growth plate. In our study, we intentionally penetrated the epiphyseal plate with the intraosseous needle and infused fluids in order to observe what complications, if any, might arise from this procedure. Twenty pigs 3 to 4 weeks old had a bone marrow aspiration needle introduced into the medullary cavity through the tibial epiphysis under fluoroscopic visualization. Sodium bicarbonate at 2 mEq/kg (n = 10) or 0.9 normal saline at 2 mL/kg (n = 10) was infused through the intraosseous needle. Radiographs of the involved growth plates were taken at two months and six months after infusion. No growth disturbances or growth plate abnormalities were detected clinically or radiographically through the rapid growth phase of the porcine tibia. Therefore, we believe that intraosseous infusion is a safe method of alternative vascular access associated with no significant growth defects despite injury to the developing growth plate from placement and infusion.

Tibial epiphyseal development: a cross-sectional histologic and histomorphometric study in the New Zealand white rabbit.

Sequential histomorphometric studies on the developing rabbit tibia from birth to skeletal maturity demonstrate that growth plate height lessens as longitudinal growth diminishes. Differing rates of development proximally and distally are documented. Distally, growth plate height, width, and area and total epiphyseal area peak or reach near maximum values by 3 weeks, whereas proximally, they do so by 8 weeks (except for height, which also peaks at 3 weeks). The distal growth plate is being obliterated by 16 weeks, at which time the proximal growth plate remains well structured and open. The distal tibia and fibula develop as one tissue mass. The articular cartilage and epiphyseal cartilage are continuous from birth, whereas a single ossification center and a single growth plate are present by 8 weeks. The data point to the presence of intrinsic growth plate, as well as systemic, control mechanisms affecting skeletal growth. Knowledge of temporal and quantitative features of epiphyseal and growth plate development will greatly aid in the elucidation of the underlying controls.

Blood vessels in the developing growth plate of the equine distal radius and metacarpus

Microangiography, histology and corrosion casts were used to study vessels in the distal radial and metacarpal growth plates of 45 foals. Transphyseal vessels run from epiphysis to metaphysis, and their morphology suggesls that they may temporarily be involved in nourishment of lhe metaphyseal side of the growth plate.

Observations of growth plate development in achondroplastic (cn/cn) mice.

The autosomal recessive gene for achondroplasia (cn) in the homozygous condition inhibits the growth of the cartilaginous skeleton in mice. Changes in the width of the proximal growth plate of the tibia and the caudal vertebrae and in the number of proliferative and hypertrophic cells were recorded in normal and achondroplastic (cn/cn) mice during the first 30 neonatal days. In normal mice as in achondroplastics, the width of the cartilaginous growth plate decreased progressively with increasing age. The proliferative zone of cn/cn mice was similar in structure to that of normal mice and showed no significant difference. Hypertrophied cartilage was less wide and there were fewer hypertrophic cells at the epiphyseal plates than found in normal mice. The data of the present study show that retarded longitudinal bone growth in cn/cn mice is due to reduced hypertrophy of the chondrocytes.

Morphological studies of the canine intervertebral disc. The assignment of the beagle to the achondroplastic classification

The morphological development of the epiphyseal growth plate and intervertebral discs of newborn and nine-month-old beagles, greyhounds and dachshunds have been studied. On the basis of the cellular and matrix development of these canine tissues together with the known characteristics associated with achondroplasia it is concluded that the beagle should be assigned to the chondrodystroid breed classification.