Cellular and Molecular Alterations Underlying Abnormal Bone Growth in X-Linked Hypophosphatemia.

X-linked hypophosphatemia (XLH), the most common form of hereditary hypophosphatemic rickets, is caused by inactivating mutations of the phosphate-regulating endopeptidase gene (PHEX). XLH is mainly characterized by short stature, bone deformities and rickets, while in hypophosphatemia, normal or low vitamin D levels and low renal phosphate reabsorption are the principal biochemical aspects. The cause of growth impairment in patients with XLH is not completely understood yet, thus making the study of the growth plate (GP) alterations necessary. New treatment strategies targeting FGF23 have shown promising results in normalizing the growth velocity and improving the skeletal effects of XLH patients. However, further studies are necessary to evaluate how this treatment affects the GP as well as its long-term effects and the impact on adult height.

The Formation of the Epiphyseal Bone Plate Occurs via Combined Endochondral and Intramembranous-Like Ossification.

The formation of the epiphyseal bone plate, the flat bony structure that provides strength and firmness to the growth plate cartilage, was studied in the present study by using light, confocal, and scanning electron microscopy. Results obtained evidenced that this bone tissue is generated by the replacement of the lower portion of the epiphyseal cartilage. However, this process differs considerably from the usual bone tissue formation through endochondral ossification. Osteoblasts deposit bone matrix on remnants of mineralized cartilage matrix that serve as a scaffold, but also on non-mineralized cartilage surfaces and as well as within the perivascular space. These processes occur simultaneously at sites located close to each other, so that, a core of the sheet of bone is established very quickly. Subsequently, thickening and reshaping occurs by appositional growth to generate a dense parallel-fibered bone structurally intermediate between woven and lamellar bone. All these processes occur in close relationship with a cartilage but most of the bone tissue is generated in a manner that may be considered as intramembranous-like. Overall, the findings here reported provide for the first time an accurate description of the tissues and events involved in the formation of the epiphyseal bone plate and gives insight into the complex cellular events underlying bone formation at different sites on the skeleton.

MAPK inhibition and growth hormone: a promising therapy in XLH.

X-linked hypophosphatemia (XLH) leads to growth retardation and bone deformities, which are not fully avoided by conventional treatment with phosphate and vitamin D analogs. Pediatric patients have been treated with growth hormone (GH), and recent findings suggest that blocking fibroblast growth factor 23 actions may be the most effective therapy, but its effects on growth are not known. We here report the effect of MAPK inhibition alone or associated with GH on growth and growth plate and bone structure of young Hyp (the XLH animal model) mice. Untreated Hyp mice were severely growth retarded and had marked alterations in both growth plate structure and dynamics as well as defective bone mineralization. GH accelerated growth and improved mineralization and the cortical bone, but it failed in normalizing growth plate and trabecular bone structures. MAPK inhibition improved growth and rickets and, notably, almost normalized the growth plate organization. The administration of a MAPK pathway inhibitor plus GH was the most beneficial treatment because of the positive synergistic effect on growth plate and bone structures. Thus, the growth-promoting effect of GH is likely linked to increased risk of bone deformities, whereas the association of GH and MAPK inhibition emerges as a promising new therapy for children with XLH.-Fuente, R., Gil-Peña, H., Claramunt-Taberner, D., Hernández-Frías, O., Fernández-Iglesias, Á., Alonso-Durán, L., Rodríguez-Rubio, E., Hermida-Prado, F., Anes-González, G., Rubio-Aliaga, I., Wagner, C., Santos, F. MAPK inhibition and growth hormone: a promising therapy in XLH.

Growth plate alterations in chronic kidney disease.

Growth retardation is a major feature of chronic kidney disease (CKD) of onset in infants or children and is associated with increased morbidity and mortality. Several factors have been shown to play a causal role in the growth impairment of CKD. All these factors interfere with growth by disturbing the normal physiology of the growth plate of long bones. To facilitate the understanding of the pathogenesis of growth impairment in CKD, this review discusses cellular and molecular alterations of the growth plate during uremia, including structural and dynamic changes of chondrocytes, alterations in their process of maturation and hypertrophy, and disturbances in the growth hormone signaling pathway.

Innovative Three-Dimensional Microscopic Analysis of Uremic Growth Plate Discloses Alterations in the Process of Chondrocyte Hypertrophy: Effects of Growth Hormone Treatment.

Chronic kidney disease (CKD) alters the morphology and function of the growth plate (GP) of long bones by disturbing chondrocyte maturation. GP chondrocytes were analyzed in growth-retarded young rats with CKD induced by adenine intake (AD), control rats fed ad libitum (C) or pair-fed with the AD group (PF), and CKD rats treated with growth hormone (ADGH). In order to study the alterations in the process of GP maturation, we applied a procedure recently described by our group to obtain high-quality three-dimensional images of whole chondrocytes that can be used to analyze quantitative parameters like cytoplasm density, cell volume, and shape. The final chondrocyte volume was found to be decreased in AD rats, but GH treatment was able to normalize it. The pattern of variation in the cell cytoplasm density suggests that uremia could be causing a delay to the beginning of the chondrocyte hypertrophy process. Growth hormone treatment appears to be able to compensate for this disturbance by triggering an early chondrocyte enlargement that may be mediated by Nkcc1 action, an important membrane cotransporter in the GP chondrocyte enlargement.

A simple method based on confocal microscopy and thick sections recognizes seven subphases in growth plate chondrocytes.

This manuscript reports a novel procedure to imaging growth plate chondrocytes by using confocal microscopy. The method is based on fixed undecalcified bone samples, in-block staining with eosin, epoxy resin embedding and grinding to obtain thick sections. It is simple, inexpensive and provides three-dimensional images of entire chondrocytes inside their native lacunae. Quantitative analysis of volume, shape and cytoplasm density of chondrocytes at different strata of the growth plate allowed to objectively grade chondrocytes of the growth plate in seven different clusters. These seven categories of chondrocytes were subsequently evaluated by immunohistochemistry of some well-defined molecular landmarks of chondrocyte differentiation. Furthermore, immunohistochemical analysis of proteins responsible for ionic changes and water transport allowing chondrocyte swelling during hypertrophy was also performed. Results obtained indicate that four subphases can be defined in the pre-hypertrophic zone and three subphases in the hypertrophic zone, a fact that raises that chondrocytes of the growth plate are less homogeneous than usually considered when different zones are defined according to subjective cell morphological criteria. Results in the present study provide a technological innovation and gives new insights into the complexity of the process of chondrocyte differentiation in the growth plate.

Marked alterations in the structure, dynamics and maturation of growth plate likely explain growth retardation and bone deformities of young Hyp mice.

Mechanisms underlying growth impairment and bone deformities in X-linked hypophosphatemia are not fully understood. We here describe marked alterations in the structure, dynamics and maturation of growth plate in growth-retarded young Hyp mice, in comparison with wild type mice. Hyp mice exhibited reduced proliferation and apoptosis rates of chondrocytes as well as severe disturbance in the process of chondrocyte hypertrophy disclosed by abnormal expression of proteins likely involved in cell enlargement, irregular chondro-osseous junction and disordered bone trabecular pattern and vascular invasion in the primary spongiosa. (Hyp mice had elevated circulating FGF23 levels and over activation of ERK in the growth plate.) All these findings provide a basis to explain growth impairment and metaphyseal deformities in XLH. Hyp mice were compared with wild type mice serum parameters, nutritional status and growth impairment by evaluation of growth cartilage and bone structures. Hyp mice presented hyphosphatemia with high FGF23 levels. Weight gain and longitudinal growth resulted reduced in them with numerous skeletal abnormalities at cortical bone. It was also observed aberrant trabecular organization at primary spongiosa and atypical growth plate organization with abnormal proliferation and hypertrophy of chondrocytes and diminished apoptosis and vascular invasion processes. The present results show for the first time the abnormalities present in the growth plate of young Hyp mice and suggest that both cartilage and bone alterations may be involved in the growth impairment and the long bone deformities of XLH.