Biomedical subjects

Rachael V Sugars

Publications and source records attributed to Rachael V Sugars.

7 recordsLinked to original sources

PubMed2007

Bone matrix formation in osteogenic cultures derived from human embryonic stem cells in vitro.

Bone matrix production and mineralization involves sophisticated mechanisms, including the initial formation of an organic extracellular matrix into which inorganic hydroxyapatite crystals are later deposited. Human embryonic stem (hES) cells offer a potential to study early developmental processes and provide an unlimited source of cells. In this study, four different hES cell lines were used, and two different approaches to differentiate hES cells into the osteogenic lineage were taken. Undifferentiated cells were cultured either in suspension, facilitating the formation of embryoid bodies (EBs), or in monolayer, and both methods were in the presence of osteogenic supplements. Novel to our osteogenic differentiation study was the use of commercially available human foreskin fibroblasts to support the undifferentiated growth of the hES cell colonies, and their propagation in serum replacement-containing medium. Characterization of the osteogenic phenotype revealed that all hES cell lines differentiated toward the mesenchymal lineage, because T-Brachyury, Flt-1, and bone morphogenetic protein-4 could be detected. Main osteoblastic marker genes Runx2, osterix, bone sialoprotein, and osteocalcin were up-regulated. Alizarin Red S staining demonstrated the formation of bone-like nodules, and bone sialoprotein and osteocalcin were localized to these foci by immunohistochemistry. Cells differentiated in monolayer conditions exhibited greater osteogenic potential compared to those from EB-derived cells. We conclude that in vitro hES cells can produce a mineralized matrix possessing all the major bone markers, the differentiation of pluripotent hES cells to an osteogenic lineage does not require initiation via EB formation, and that lineage potential is not dependent on the mode of differentiation induction but on a cell line itself.

Bone Matrix↗

PubMed2006

Transcriptome analysis of fetal metatarsal long bones by microarray, as a model for endochondral bone formation.

Endochondral bone formation is orchestrated by mesenchymal cell condensation to form cartilage anlagen, which act as a template for bone formation and eventual mineralization. The current study performed gene expression analysis to examine pre- and post-mineralization stages (E15 and E19) of endochondral bone formation, using fetal metatarsal long bones as a model. An extensive number of genes were differentially expressed, with 543 transcripts found to have at least 2-fold up-regulation and 742 with a greater than 2-fold down-regulation. A bioinformatics approach was adopted based on gene ontology groups, and this identified genes associated with the regulation of signaling and skeletal development, cartilage replacement by bone, and matrix degradation and turnover. Transcripts linked to skeletal patterning, including Hoxd genes 10-12, Gli2 and Noggin were considerably down-regulated at E19. Whereas genes associated with bone matrix formation and turnover, ACP5, MMP-13, bone sialoprotein, osteopontin, dentin matrix protein-1 and MMP-9 all were distinctly up-regulated at this later time point. This approach to studying the formation of the primary ossification center provides a unique picture of the developmental dynamics involved in the molecular and biochemical processes during this intricately regulated process.

Animals↗

PubMed2006

Substitution of bovine dentine sialoprotein with chondroitin sulfate glycosaminoglycan chains.

Dentine sialoprotein (DSP) represents 5-8% of all non-collagenous proteins present in the tooth, but, together with dentine phosphoprotein, has been shown to be vital for correct tooth formation. Recently, the existence of a highly glycosylated form of porcine DSP has been reported and it was shown to possess glycosaminoglycan (GAG) chains. The current investigation confirms that this is also the case for bovine DSP and has further characterized these carbohydrates. Dentine sialoprotein was purified from bovine dentine extracts by anion exchange chromatography and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and mass spectroscopy. An increase in molecular mass was observed, from 120 kDa to greater than 250 kDa, with a corresponding rise in anionic strength. Cellulose acetate electrophoresis and western blotting indicated the presence of chondroitin sulfate GAG chains within these dentine fractions. Further examination using sequential digestion with chondroitinase AC and N-glycosidase cleaved the samples first to 95 kDa and then to 80 kDa, respectively, confirming a high level of glycosylation. These results support the classification of bovine DSP as a proteoglycan, and that the carbohydrate substitutions may contribute to the functional properties of DSP.

Amino Acid Sequence↗

PubMed2006

Adsorption and interactions of dentine phosphoprotein with hydroxyapatite and collagen.

Dentine phosphoprotein (DPP) has been proposed to both promote and inhibit mineral deposition during dentinogenesis. The present study aimed to investigate the molecular interactions of DPP and dephosphorylated DPP (DPP-p) with hydroxyapatite (HAP). Bovine DPP was purified and dephosphorylated by alkaline phosphatase to obtain DPP-p. DPP and DPP-p adsorption to HAP was determined along with their ability, when free in solution or bound to collagen, to influence HAP-induced crystal growth. Absorption isotherms suggested that lower DPP concentrations (1.5-6.25 microg ml(-1)) demonstrated a reduced affinity for HAP compared with higher protein concentrations (12.5-50.0 microg ml(-1)). Dephosphorylated DPP had a much reduced affinity for HAP compared with DPP. Dentine phosphoprotein inhibited seeded HAP crystal growth, in a dose-dependent manner, whilst removal of the phosphate groups reduced this inhibition. When bound to collagen fibrils, DPP significantly promoted the rate of HAP crystal growth over 0-8 min. Conversely, DPP-p and collagen significantly decreased the rate of crystal growth over 0-18 min. These results indicate a major role for the phosphate groups present on DPP in HAP crystal growth. In addition, concentration-dependent conformational changes to DPP, and the interaction with other matrix components, such as collagen, are important in predicting its dual role in the mineralization of dentine.

Absorption↗

PubMed2004

Modulation of collagen fibrillogenesis by dentinal proteoglycans.

Studies have identified different pools of proteoglycan (PG) species present within the unmineralized matrix of the predentine, the transitional phase at the predentine-dentine interface and the mineralized dentine. These PGs alter with respect to the chemical nature of their glycosaminoglycan (GAG) chains and as a result of extracellular processing of the macromolecule in the matrix. The present study has examined the influence of the PGs isolated from these phases and the influence of the attached GAG chains, upon their ability to control collagen fibrillogenesis. PGs isolated from the three phases were characterized and determined to contain a mixture of decorin and biglycan. Results have indicated that predentine PGs, which are substituted with a higher proportion of dermatan sulfate, significantly delayed fibril formation while ultimately promoting the formation of thicker fibrils. Removal of the GAG chains further delayed fibrillogenesis, leading to the formation of thinner fibrils, compared with the collagen-only control. PGs isolated from predentine-dentine, which contained a higher proportion of chondroitin sulfate, also significantly delayed fibrillogenesis, resulting in thicker collagen fibrils. GAG chains attached to the predentine-dentine interface PGs played a role in the timing of fibrillogenesis with fibril formation initiated at the same time as the collagen control, but yielding thicker fibrils. Dentine PGs significantly inhibited fibrillogenesis and fibril thickness over concentrations of 50-25 microg/mL protein. In conclusion, the PGs isolated from the distinct phases have indicated differing roles in the orchestrated organization of the extracellular matrix during dentinogenesis, with roles for both the core protein and attached GAG chains indicated.

Animals↗

PubMed2004

Nucleobindin is produced by bone cells and secreted into the osteoid, with a potential role as a modulator of matrix maturation.

Nucleobindin (Nuc), also known as CALNUC, is a Ca(2+)-binding protein, located in the nucleus, the Golgi apparatus and the endoplasmic reticulum (ER). The presence of a signal sequence in Nuc suggests secretion from the cell and it has been found in bone extracellular matrix. Within the present study, molecular biological and morphological methods were combined to evaluate the synthesis and distribution of Nuc in and around cells of rat metaphyseal and calvarial bone. Northern blot analysis and in situ hybridization of bone tissues confirmed that the protein was a product of bone cells. By electron microscopy, immunolabeling for Nuc was seen in osteoid of newly formed bone, on all surfaces facing the various bone cells and also in compact bone. Intracellularly, the gold particles were found in the rough ER of osteoblasts, which suggested synthesis of the protein by these cells. Compared to bone sialoprotein and osteopontin, Nuc demonstrated different localization pattern in bone trabeculae, with the majority of labeling restricted to nonmineralized osteoid. Moreover, the role of Nuc during the mineralization process was investigated in rat calvaria-derived primary osteoblasts grown under osteogenic conditions. Semiquantitative RT-PCR and Northern blot analysis showed Nuc expression to be low during cell proliferation, upregulated during differentiation and matrix maturation, but subsequently downregulated during mineralization. In summary, our data show that Nuc was synthesized by osteoblasts and osteocytes, and secreted into the osteoid, suggesting a role as a modulator of matrix maturation in the mineralization process in bone.

Animals↗

PubMed2002

The interaction of recombinant decorin with alpha2HS-glycoprotein-implications for structural and functional investigations.

Isolated protein preparations of the small leucine-rich proteoglycans (SLRPs) associated with mineralized tissues have provided important information in understanding their structural and functional interactions within extracellular matrices and their potential roles in mineralization. Two important SLRPs, decorin and biglycan, copurify following extraction and purification from mineralized tissues using standard procedures, and to overcome this problem decorin was synthesized within a mammalian expression system to obtain pure preparations. The expressed protein was purified from the culture medium using anion-exchange chromatography, and characterization confirmed the presence of a decorin-rich fraction. However, N-terminal sequencing revealed the additional presence of alpha2HS-glycoprotein (alpha2HSG), representing approximately 35% of the total purified fraction. The decorin-rich fraction was subjected to selected further purification techniques to separate decorin from alpha2HSG. Application of the sample at a low concentration (1 mg/ml) to a second anion-exchange procedure and elution over an expanded sodium chloride gradient resulted in a high degree of purity (98%), with a single protein isolate demonstrable by SDS-PAGE. Electroelution achieved partial purification ( approximately 89%), but immunoprecipitation with antibodies against the glycosaminoglycan chain and the polyhistidine tag failed to separate the two proteins. This study suggests there is a strong interaction between recombinantly produced decorin and alpha2 HSG and highlights the importance of the purification technique to the application of recombinantly produced proteins or those that have been extracted from mineralized tissues for use in structural and functional interactions.

Acetates↗