Effect of systemic calcium deficiency on the expression of transforming growth factor‐β in chick embryonic calvaria

Abstract

The developmental process of intramembranous ossification involves bone formation directly from mesenchymal differentiation without a cartilage intermediate. We have previously observed that systemic calcium deficiency in the developing chick embryo, produced by long-term shell-less culture, results in the appearance of chondrocyte-like cells in the calvarium, a parietal bone which normally develops via intramembranous ossification. This investigation aims to analyze the mechanism underlying this calcium deficiency-related, aberrant appearance of cartilage phenotype in the chick embryonic calvarium. In view of the reported involvement of transforming growth factor p (TGF-P) in osteogenesis and chondrogenesis, we have examined and compared here the expression of TGF-p in the chick embryonic calvaria of normal (in ovo development, NL), shell-less (SL), and calcium-supplemented SL (SL + Ca) embryos. TGF-f3 expression was analyzed at the mRNA level by blot and in situ cDNA hybridization, and at the protein level by immunohistochemistry and immunoblotting. The results presented here indicate that: 1) TGF-p is expressed in the chick embryonic calvarium by both periosteal cells and osteocytes, as revealed by in situ hybridization and immunohistochemistry; 2) TGF-P expression is significantly increased in SL calvarium compared to NL calvarium, at both protein and mRNA levels; 3) the number of TGF-f3 expressing cells increases in the SL calvarium, particularly along the central, subcambial core region of the bone; and 4) exogenous calcium repletion to the SL embryo affects the expression of TGF-P such that the pattern approaches that in the NL embryo. Taken together, these results indicate that altered TGF-f3 expression accompanies the aberrant appearance of cartilage phenotype caused by systemic calcium deficiency. We postulate that normal cellular differentiation along the osteogenic pathway during embryonic intramembranous ossification is crucially dependent on regulated TGF-0 expression. o 1992 Wiley-Liss, Inc.

Biocompatibility and bone mineralization potential of 45S5 Bioglass®-derived glass–ceramic scaffolds in chick embryos

“…Previous studies by several authors [22][23][24], as well as a pilot study from our laboratory, have reported that Ca repletion to the shell-less embryos in the form of two to three 5-6 cm 2 eggshell pieces or a daily addition of CaCO 3 suspension (10% w/v in water), applied as several spots, onto the CAM resulted in improved overall growth and increased calcification of the skeletal components, especially the endochondral long bones (femur and tibia). In the chick embryo, the Ca required for skeletal mineralization is mobilized from the CaCO 3 , in the form of calcite, stored in the eggshell by the CAM [22][23][24].…”

“…In the chick embryo, the Ca required for skeletal mineralization is mobilized from the CaCO 3 , in the form of calcite, stored in the eggshell by the CAM [22][23][24]. In our experimental model, the scaffold was implanted on the CAM at 10 days of embryonic development.…”

“…Compared with normal controls, calcium-deficient chick embryos exhibit a chondrogenic phenotype that is restored to an osteogenic phenotype upon calcium (Ca) repletion [22][23][24]. The shell-less chick embryo thus represents a unique experimental system for studying the relationship between Ca-containing biomaterials and skeletogenesis.…”

“…in ovo or ex ovo (shell-less), and both methods are in regular use. Chick embryos grown in shell-less culture are calcium-deficient by 9 days of incubation [22][23][24].…”

Pattern of expression of transforming growth factor-β4 mRNA and protein in the developing chicken embryo

“…In situ hybridization was performed essentially as described previously (McDonald and Tuan, 1988;Sato and Tuan, 1992). Sections were deparaffinized with xylene followed by Americlear (Polysciences, Warrington, PA) for 20 min each, hydrated through a graded series of ethanol (loo%, 95%, 70%, and 50%) for 1 min each, equilibrated with 2 x SSC for 1 min, and demineralized in 0.25 M HC1 for 30 min.…”