Bone sialoprotein keratan sulfate proteoglycan (BSP-KSPG) and FGF-23 are important physiological components of medullary bone.

Medullary bone is a specialized bone found in the marrow cavity of laying birds. It provides a significant contribution to the calcium supply for egg shell formation. Medullary bone is distinguished from cortical bone by the presence of large amounts of a keratan sulfate proteoglycan (KSPG). The aims of the present experiment are to confirm the identity of the core protein of KSPG, identify a marker of medullary bone metabolism, and determine whether changes in keratan sulfate (KS) concentration in blood are associated with the egg-laying cycle. Using two different isolation techniques- one specific for bone and another for blood- we have identified bone sialoprotein (BSP) to be the core protein of this KSPG. We also determined that the amount of keratan sulfate (KS) in laying hen blood fluctuates in synchrony with the egg-laying cycle, and thus can serve as a specific marker for medullary bone metabolism. During the course of this investigation, we also found FGF-23 (phosphatonin) to be expressed in medullary bone, in synchrony with the egg-laying cycle. Western blotting was used to demonstrate the presence of this peptide in both laying hen blood and medullary bone extracts. The importance of FGF-23 (phosphatonin) and parathyroid hormone in normalizing the dramatic changes in plasma calcium and phosphorus during the 24h egg-laying cycle is discussed.

Lack of a relationship between plasma fibroblast growth factor-23 and phosphate utilization in young chicks.

Fibroblast growth factor 23 (FGF-23) is proposed to be the hormone that controls phosphate (P) homeostasis in chickens. This study was initiated to investigate the effect of feeding young chicks diets that were either adequate (0.45%) or marginal (0.25%) in available P content on plasma FGF-23 levels. The dietary level of available P significantly (P ≤ 0.05) affected bone mineralization and bone length, but was without effect (P > 0.05) on growth rate and circulating FGF-23 concentrations. Substantial individual variation in bone mineralization and plasma FGF-23 levels was observed, and the correlation between these two variables was non-significant (P > 0.05). This suggested that there was no alteration in FGF-23 activity in response to suboptimal dietary P intake. The relationship of these observations to studies on the immunosuppression of FGF-23 activity is subsequentlydiscussed.

Effects of strontium on bone strength, density, volume, and microarchitecture in laying hens.

UNLABELLED: Strontium has been reported to have beneficial effects on bone. Treatment of laying hens, which are susceptible to osteoporosis and bone fracture, with strontium increased DXA measurements of BMD and BMC and microCT measurements of bone volume and microarchitecture and improved the mechanical performance of whole bone, but had no effect on the estimated material properties of the bone tissue. INTRODUCTION: Strontium (Sr) has been reported to dissociate bone remodeling and have positive influences on bone formation. We supplemented the diet of laying hens, which are susceptible to osteoporosis and bone fracture, with Sr to study the capacity of the element to improve bone mechanical integrity and resistance to fracture. MATERIALS AND METHODS: Increasing dosages of Sr (0, 3000, 4500, and 6000 ppm) were fed to 196 13-week-old pullets for 11 months. BMD and BMC, as measured by conventional and DXA methods, microarchitectural parameters derived from microCT, and structural and material properties as determined by three-point bending test, were studied. Calcium (Ca), phosphorus (P), and Sr levels in plasma and bone, as well as egg output, shell quality, and composition, were assessed. RESULTS: Sr concentrations in plasma and bone increased in a dose-dependent manner without affecting Ca and P. Treatment with Sr increased BMD and BMC as measured by DXA, increased cortical and medullary bone volume, trabecular thickness, number, and surface, and improved whole bone ultimate load, but had no effect on the estimated material properties of diaphyseal bone. Sr also increased the ash content of eggshells and did not affect egg output and shell quality. CONCLUSIONS: Sr supplementation induced large positive effects on bone density, volume, and microarchitecture as measured by radiographic methods. Sr treatment also improved the structural strength of diaphyseal bone but had no effect on the estimated material properties of the bone tissue.

Characterization of the non-collagenous proteins in avian cortical and medullary bone.

Northern blotting, RT-PCR, and Western blotting techniques were used to characterize the matrix constituents of avian cortical and medullary bone. Extracts of bone tissue were found to contain multiple isoforms of bone sialoprotein (BSP), osteopontin (OPN), osteonectin (ON), osteocalcin (OC), and dentin matrix protein-1 (DMP-1). Only single transcripts were observed with Northern blotting; therefore it was concluded that the isoforms were due to differences in post-translational modifications. Since medullary bone is rich in keratan sulfate (KS), RT-PCR was used to investigate the expression of known keratan sulfate-containing proteoglycans (KSPGs). Although this tissue was found to express lumican and osteoglycin/mimecan, there was little evidence to suggest that these proteoglycans were a major source of the keratan sulfate glycosaminoglycans. Treatment of medullary bone extracts with keratanase resulted in the appearance of a BSP immunoactive band of approximately 59 kDa. However, it was not possible to isolate and identify the intact keratan sulfate proteoglycan.

Effect of fibroblast growth factors 1, 2, 4, 5, 6, 7, 8, 9, and 10 on avian chondrocyte proliferation.

It has been demonstrated that fibroblast growth factor receptors are key regulators of endochondral bone growth. However, it has not been determined what fibroblast growth factor ligand(s) (FGFs) are important in this process. This study sought to determine whether FGFs 1, 2, 4, 5, 6, 7, 8, 9, and 10 were capable of stimulating avian chondrocyte proliferation in vitro. We have found that FGFs 2, 4, and 9 strongly stimulate avian chondrocyte proliferation while FGFs 6 and 8 stimulate proliferation to a lesser extent. RT-PCR indicates that FGF-2 and FGF-4 are expressed in the postnatal avian epiphyseal growth plate (EGP) while FGF-8 and FGF-9 are not. Thus, FGF-2 and FGF-4 stimulate chondrocyte proliferation and are both present in the EGP. This suggests that FGF-2 and FGF-4 may be important ligands, in vivo, for the regulation of endochondral bone growth. These observations coupled with our observation that multiple avian FGF receptors (Cek1, Cek2, Cek3, and FREK) are expressed in proliferative chondrocytes highlights the complexity of FGF signaling pathways in postnatal endochondral bone growth.

Short-term zinc deficiency inhibits chondrocyte proliferation and induces cell apoptosis in the epiphyseal growth plate of young chickens.

The purpose of this study was to investigate the effect of zinc deficiency on chondrocyte proliferation, differentiation and apoptosis in the epiphyseal growth plate of juvenile chickens. Newly hatched broiler chickens were fed either a low zinc (10 mg/kg diet) or a zinc-adequate (68 mg/kg diet) soy protein-based purified diet. Cell proliferation in the growth plate was evaluated with bromodeoxyuridine (BrdU) labeling. Apoptosis was assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. Chondrocyte differentiation was evaluated with immunostaining of osteonectin as a marker of maturation. As early as d 3 of feeding, zinc deficiency significantly inhibited chondrocyte proliferation, promoted cell differentiation and induced cell apoptosis in the growth plate. These effects were manifested primarily in areas remote from the blood supply. Immunostaining for local growth factors such as insulin-like growth factor-1 (IGF-1), parathyroid hormone-related protein (PTHrP) and fibroblast growth factor-2 (FGF-2) did not reveal any differences between growth plates of zinc-deficient and zinc-adequate chickens after 3 d of feeding. By d 7, severe growth plate lesions characterized by reduced cellularity and abnormally shaped cells were formed in areas remote from blood vessels. Immunoreactive IGF-1, PTHrP and FGF-2 were all greatly reduced in the lesion. However, the growth rate and food intake of zinc-deficient chickens were not different from those of the controls during the 7-d experiment. Therefore, a direct effect of zinc deficiency on proliferation, differentiation, and apoptosis of growth plate chondrocytes was indicated.