Biomedical subjects

Mikael Wendel

Publications and source records attributed to Mikael Wendel.

10 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

Differential regulation of osteoadherin (OSAD) by TGF-beta1 and BMP-2.

Osteoadherin (OSAD) is a member of the small leucine rich-repeat proteoglycan (SLRP) family. SLRPs are normally found in extracellular matrices, but OSAD is the only member restricted to mineralized tissues. We investigated the promoter region of OSAD by in silico analysis and found that the proximal promoter region contains sites for Smad-3, Smad-4, and AP-1. All are effectors of TGF-beta family signalling. We tested sensitivity of the promoter to the two TGF-beta family members TGF-beta1 and BMP-2. We found TGF-beta1 to down regulate OSAD, while BMP-2 up regulates OSAD. As a consequence of how OSAD is regulated by TGF-beta1 and BMP-2 and its temporal expression pattern in osteoblasts and bone development, we can conclude OSAD as an early marker for terminally differentiated matrix producing osteoblasts.

Animals↗

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↗

PubMed2005

Heat-induced retrieval of immunogold labeling for nucleobindin and osteoadherin from Lowicryl sections of bone.

The main purpose of this study was to examine whether antigens can be retrieved by heating Lowicryl sections of paraformaldehyde-fixed (PFF) tissues. Thus the intensity of the immunogold signal for two bone proteins (Nucleobindin (Nuc) and osteoadherin (OSAD)) was compared in retrieved and non-retrieved sections of PFF rat bone. As an additional experiment, the effect of antigen retrieval (for Nuc) in sections of tissue primary stabilized by high pressure freezing with subsequent freeze substitution (HPF-FS) was studied. Finally, the tissue distribution patterns of Nuc labeling were compared in non-retrieved HPF-FS sections to that of retrieved and non-retrieved PFF sections. Antigen retrieval in Lowicryl sections of PFF tissues showed significantly enhanced labeling intensity for both proteins in all compartments where they are known to occur. Retrieved PFF Lowicryl sections showed only minor ultrastructural differences compared to non-retrieved ones. Retrieval of HPF-FS sections exhibited no enhancement of labeling but rather a slight reduction, which was significant in the cytoplasm and in cartilage. Furthermore, striking ultrastructural differences were observed in retrieved HPF-FS sections compared to non-retrieved ones with loss of coherence and structure in sections subjected to heating. Comparison of the distribution patterns of Nuc in the sections of PFF and HPF-FS tissues showed discrepancy in most compartments. Antigen retrieval by heating Lowicryl sections of PFF tissues significantly enhances immunogold labeling in all cell compartments where the bone proteins are known to occur. However, the procedure may distort the tissue distribution pattern of bone proteins.

Acrylic Resins↗

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↗

PubMed2004

Organized development from human embryonic stem cells after injection into immunodeficient mice.

Information concerning the development and differentiation of human embryonic stem (hES) cells in vivo is limited. The present study has focused on the in vivo outcome and differentiation of the hESC line HS181, after injection into SCID/beige mice. hES cell-derived teratomas were explored using histological evaluation and by the identification of markers for differentiated cells and tissues. The analyses identified predominant differentiation along a neuronal lineage, the formation of bone/cartilage and epithelia. Fluorescent in situ hybridization (FISH) analysis with a human-specific probe showed the teratomas to be mainly of human origin, with the most organized areas being exclusively human. Importantly, the study revealed interactions between mouse and human tissues, most notably in the formation of vessels. Both mouse and human cells contributed to specific microstructures in which mouse cells could be observed to take on the appropriate histiotypic appearance. Hence, HS181 cells were able to develop into defined mature tissues, supporting the relevant use of this hES cells model for studies of early human development, given the use of appropriate controls for host contribution. Although extensive mitotic activity implicated progenitor cell activity, no detectable multipotent or malignant areas were observed during the observation period. Persisting undifferentiated hESC were not detected.

Animals↗

PubMed2003

Calreticulin--an endoplasmic reticulum protein with calcium-binding activity is also found in the extracellular matrix.

Previous studies have reported that calreticulin (CRT), a calcium-binding and chaperoning protein, is expressed only in the endoplasmatic reticulum, nucleus and at the cell surface. In this study we clearly show that odontoblasts and predentin matrix contain CRT. To our knowledge, this is the first time CRT has been described in the extracellular matrix. The expression of CRT was studied by immunohistochemistry, ultrastructural immunocytochemistry and in situ hybridization in developing rat teeth. CRT was detected as a 59-kDa protein in rat pulp cell culture medium and dentin extracellular matrix extract by Western blotting. The presence of the protein was shown in rat odontoblasts and predentin with immunohistochemistry. At the ultrastructural level, the labeling was distributed in the rat odontoblasts, ameloblasts and predentin. Northern blotting showed the presence of CRT mRNA in rat molars, which was confirmed by in situ hybridization in odontoblasts and ameloblasts. We now present the first convincing evidence that CRT is found in extracellular matrix where it may play an important role in mineralization.

Ameloblasts↗

PubMed2003

Differential expression of osteopontin and bone sialoprotein in bone metastasis of breast and prostate carcinoma.

Breast and prostate cancer often metastasise to the skeleton. Interestingly, the histopathological characteristics of the bone lesions that arise from these two cancer types differ. Breast tumours give rise to metastases in the skeleton with a mixed lytic/sclerotic pattern, whereas a predominantly sclerotic pattern is seen in metastases from prostate tumours. Osteopontin (OPN) and bone sialoprotein (BSP) are bone matrix proteins that have been implicated in the selective affinity of cancer cells for bone. In the present study, 21 patient cases with skeletal metastasis and their respective primary tumours (12 with breast cancer, 9 with prostate cancer) were investigated by immunohistochemistry in order to assess the level of OPN and BSP. Moderate to strong OPN expression was found in 42% of all breast tumours and in 56% of all prostate tumours. Significantly more breast cancer bone metastases exhibited high OPN expression, 83%, as compared with prostate tumour bone metastases, 11% (P = 0.0019). In contrast, moderate to strong BSP expression was found in 33% of breast tumours and in 89% of prostate tumours. In the bone lesions, only 33% of breast tumour metastases showed moderate/strong BSP expression compared to 100% of prostate tumour metastases (P = 0.0046). This divergent pattern of OPN/BSP expression could be an important determinant for the different characteristics of these two types of bone metastasis, i.e., lytic vs. sclerotic, consistent with the proposed role of OPN in differentiation and activation of osteoclasts and of BSP as a stimulator of bone mineralisation.

Adult↗

PubMed2003

Identification, distribution and expression of osteoadherin during tooth formation.

Osteoadherin (OSAD) is a keratan sulfate-containing proteoglycan, belonging to the small leucine-rich proteoglycan (SLRP) family. In bone OSAD has been localized in primary spongiosa within the bovine fetal rib growth plate. Moreover, in situ hybridization has shown expression of OSAD in osteoblasts close to the cartilage and bone border in the growth plate of rat femur. mRNA expression has also detected OSAD in mature osteoblasts on the surface of bone trabeculae. We have identified OSAD in extracts of bovine dentin, and the identity was verified by N-terminal sequencing. Western blot analysis detected two bands in bovine bone and dentin at 85 kDa and 60 kDa. Northern blot analysis of RNA samples from 5-d-old-rat tooth and femur showed a 1.9-kb transcript for OSAD in both tissues. OSAD was located to the mineralized dentin matrix, cementum and surrounding alveolar bone by immunohistochemistry, and in situ hybridization showed OSAD to be highly expressed during early crown formation in the entire odontoblast cell layer, in the area of Hertwig's epithelial root sheath, in the cells of the newly formed mantle dentin, and in the odontoblasts at the fissures. Ultrastructural studies indicated that OSAD might be associated with collagen fibrils. Thus, OSAD may play an important role during tooth development and biomineralization of dentin.

Alveolar Process↗