Cartilage Bone Formation Research Articles

Effects of Astragalus Extract Mixture HT042 on Circulating IGF-1 Level and Growth Hormone Axis in Rats.

Astragalus extract mixture HT042 is a standardized functional food granted by the Korean FDA for promoting “Children’s Height Growth”. In this study, we determined whether HT042 affects circulatory Insulin-like growth factor-1 (IGF-1) after administration and investigated whether Growth hormone (GH), Growth hormone-releasing hormone receptor (GHRH-R), and Growth hormone secretagogue receptor (GHS-R) mRNAs are expressed in the pituitary, and whether Growth hormone-releasing hormone (GHRH) and Somatostatin (SST) are expressed in the hypothalamus. We also evaluated the growth effect of HT042 on endochondral bone formation. Male Sprague-Dawley rats in the control and HT042 groups were orally administered a single dose of the control and HT042, respectively, and those in the recombinant human GH (rhGH) group were subcutaneously injected with rhGH. Tetracycline was injected intraperitoneally 72 h prior to sacrifice to decide endochondral bone formation. To determine the endocrine or paracrine/autocrine mechanism, we evaluated the expression of local BMP-2 and IGF-1, an immunohistochemical study after HT042 administration. It was confirmed that the growth-promoting effect of HT042 can be contributed to the increase in serum IGF-1, which can be stimulated by GH secretion. Administration of HT042 modulated the activity of GHRH-R and GHR-S in the pituitary gland and promoted GH secretion, thereby changing longitudinal growth through GH/IGF-1 mediation. Results for GHRH and SST expression demonstrated that the hypothalamus can be influenced and mediated by HT042 through a complex neuroendocrine regulatory system. In addition, it was confirmed by oral administration for 10 days that HT042 increased bone formation in cartilage, which is important for height growth. The effect of HT042 could be owing to upregulation of local Bone morphogenetic protein-2 (BMP-2) and IGF-1 expression in the growth plate, which could be regarded as a GH-dependent autocrine/paracrine pathway, as well as circulatory IGF-1.

The splicing factor SRSF1 modulates pattern formation by inhibiting transcription of tissue specific genes during embryogenesis

Alternative splicing is a major mechanism regulating pattern of gene expression through the production of multiple mRNAs from a single gene transcript. Any misregulation can cause various human diseases and also have severe effects on embryogenesis. SRSF1 is one of the critical factors regulating alternative splicing at many stages of vertebrate development and any disturbance in SRSF1 leads to serious consequences. In current study, we investigated the effects of loss of the SRSF1 gene using antisense morpholino oligonucleotides (MO) in Xenopus embryogenesis. It is evident from the results of RT-PCR and whole-mount in situ hybridization that SRSF1 is a maternal gene having strong expression in head, eyes and central nervous system. Moreover, SRSF1 morphants exhibited malformed phenotypes, including miscoiled guts, heart and cartilage formation, edema in the head and heart, and small eyes. Especially, in SRSF1 morphants, bone cartilage formation was reduced in the brain and Nkx-2.5 expression was dramatically reduced in the heart of SRSF1 morphants. In addition, a dramatic reduction in functional chordin RNA in SRSF1 morphants was observed suggesting that chordin is one of the targets of SRSF1. Thus, we concluded that SRSF1 is an essential factor for pattern formation including heart, cartilage and germ layers through the regulation of specific genes.

Vascular calcification: mutual interaction between bone and blood vessel

Vascular calcification often associates with bone-cartilage formation. Artery sclerotic lesions accompany the expression of bone matrix proteins such as osteopontin, osteocalcin and matrix Gla protein and transcription factors including Runx2, osterix and Sox9. These lesions also express BMP, osteoprotegerin (OPG) and RANKL, which are important factor regulating bone formation and resorption. MGP-deficient mice exhibited extensive artery calcification as well as OPG-deficient mice. Thus, bone metabolism-related factors actively participate in vascular calcification, which had been interpreted as a passive calcification due to dystrophic calcification.

ENDOTHELIAL DYSFUNCTION AND DEFECTIVE SMOOTH MUSCLE CONTRACTILITY IN ANEURYSMAL FIBULIN-4 DEFICIENT MICE: 8A.05

Objective: Using Fibulin-4 knockdown mice (Fibulin-4R/R), we previously showed that the dosage of Fibulin-4 can determine the severity of aneurysm formation. Strikingly, even a modest reduction in expression of Fibulin-4 in the heterozygous Fibulin-4+/R mice occasionally resulted in small aneurysm formation. Aortic pulse pressure was 2 to 3-fold higher in Fibulin-4R/R mice, resulting from increased aortic stiffness. Here, we analyzed the altered biological pathways in aneurismal disease at the molecular, cellular and functional level. Methods: Aorta transcriptome changes of Fibulin-4+/R and Fibulin-4R/R aortas were performed using ANOVA and Ingenuity Pathway Analysis. Histology was applied to verify cellular abnormalities. Functional analysis occurred by measuring isometric forces of thoracic aortas in vitro and aortic pressures with a pressure transducer catheter in vivo. Results: Ingenuity Pathway Analysis identified three major dysregulated pathways, including TGFβ signaling, immune response and specifically Ca2+ signaling genes involved in the maintenance of contractile function. Histological analysis and á-smooth muscle actin immunostaining showed loss of smooth muscle cells (SMCs), which coincided with cartilage bone formation, already in Fibulin-4+/R animals. Severe loss of SMCs and increase of extracellular matrix depositions resulted in overall thickening of the aortic wall in Fibulin-4R/R mice. In addition, Fibulin-4R/R mice showed increased endothelial damage, endothelial cellular proliferation, as evidenced by BrdU labeling and thrombus formation, indicative for endothelial dysfunction. In vitro, maximum contractility was determined for phenylephrine and compared to KCl (100 mM). KCl responses showed a reduced contractile capacity of ascending aortas in Fibulin-4R/R mice correlating with a reduction of SMCs in this area. In descending aortas, phenylephrine-induced contractility decreased from 54 ± 7% in wildtypes to 13 ± 2% in Fibulin-4R/R mice. Endothelium-dependent vasorelaxation by acetylcholine, following preconstruction with U46619, was strongly reduced in Fibulin-4R/R mice. Finally, with reducing expression of fibulin-4, diastolic blood pressure decreased from 63 ± 3 in Fibulin-4+/+ to 60 ± 5 in Fibulin-4+/R and 41 ± 7 in Fibulin-4R/R mice. Conclusion: Our results uncover a role for Fibulin-4 in the maintenance of SMC contractile function and endothelial responsiveness.

Bone formation in cartilage produced by transplanted epiphyseal chondrocytes.

Chondrocytes were isolated from rat epiphyseal cartilage, cultured in vitro, and exposed to exogenous tracers which accumulated in their lysosomes. The cells were then injected into the posterior tibial muscle of animals from the same outbred strain, where they reconstructed calcifying hyaline cartilage. The mineralization of the tissue was followed by ingrowth of blood capillaries from the host bed. Macrophage-like cells surrounding the vessels phagocytized degenerated chondrocytes and unmineralized matrix, whereas multinucleated chondroclasts removed some of the mineralized cartilage matrix. Mesenchyme-like cells accompanying the invading vessels attached to the remaining septa of calcified cartilage matrix and developed into osteoblasts depositing bone matrix on the surface of these septa. The apparent lack of inherent tracer labeling of the lysosomes in the different bone cells indicate that they were derived from the host. No signs of transformation of chondrocytes into bone cells were observed. When isolated rat epiphyseal chondrocytes were injected into the wall of the hamster cheek pouch, calcifying cartilage was reconstructed without signs of subsequent ossification. Transplantation of cartilage reconstructed in the hamster into the dorsal muscles of rats was, however, followed by formation of bone by a sequence analogous to that described above. Such an osteogenetic response was also obtained when the cartilage had been devitalized before transplantation. These experiments show that calcified cartilage, developing in or grafted into an intramuscular site, is able to induce and serve as a substrate for endochondral bone formation, similar to that occurring during normal development. They further indicate that bone induction by calcified cartilage does not require the presence of living chondrocytes.

The Distribution of Alkaline Phosphatase in the Skull of the Developing Trout

1. The histological and cytological distribution of alkaline phosphatase in developing trout has been studied with special reference to membrane and cartilage bone formation in the skull. 2. Nuclear phosphatase is widely distributed in the youngest stages examined, but decreases as differentiation proceeds. 3. Extracellular phosphatase is always associated with ossification or fibre formation. 4. No deposition of calcium salts in the absence of phosphatase was observed. 5. Alkaline phosphatase is probably connected with histogenesis in general apart from its special function in calcification.