alcian-blue and Hypertrophy

In order to elucidate the role of β-catenin in hypertrophic cartilage zone of the growth plate, we deleted the β-catenin gene ctnnb1specifically from hypertrophic chondrocytes by mating ctnnb1(fl/fl) mice with BAC-Col10a1-Cre-deleter mice. Surprisingly, this resulted in a significant reduction of subchondral trabecular bone formation in BACCol10Cre; ctnnb1(Δ/Δ) (referred to as Cat-ko) mice, although Cre expression was restricted to hypertrophic chondrocytes. The size of the Col10a1 positive hypertrophic zone was normal, but qRT-PCR revealed reduced expression of Mmp13, and Vegfa in Cat-ko hypertrophic chondrocytes, indicating impaired terminal differentiation. Immunohistological and in situ hybridization analysis revealed the substantial deficiency of collagen I positive mature osteoblasts, but equal levels of osterix-positive cells in the subchondral bone marrow space of Cat-ko mice, indicating that the supply of osteoblast precursor cells was not reduced. The fact that in Cat-ko mice subchondral trabeculae were lacking including their calcified cartilage core indicated a strongly enhanced osteoclast activity. In fact, TRAP staining as well as in situ hybridization analysis of Mmp9 expression revealed denser occupation of the cartilage erosion zone with enlarged osteoclasts as compared to the control growth plate, suggesting increased RANKL or reduced osteoprotegerin (Opg) activity in this zone. This notion was confirmed by qRT-PCR analysis of mRNA extracted from cultured hypertrophic chondrocytes or from whole epiphyses, showing increased Rankl mRNA levels in Cat-ko as compared to control chondrocytes, whereas changes in OPG levels were not significant. These results indicate that β-catenin levels in hypertrophic chondrocytes play a key role in regulating osteoclast activity and trabecular bone formation at the cartilage-bone interface by controlling RANKL expression in hypertrophic chondrocytes.

Topics: Alcian Blue; Animals; beta Catenin; Cartilage; Cell Differentiation; Cell Line; Cell Separation; Chondrocytes; Collagen Type I; Fluorescent Antibody Technique; Gene Deletion; Growth Plate; Hypertrophy; Immunohistochemistry; In Situ Hybridization; Mice; Mice, Knockout; Osteoclasts; Osteogenesis; Platelet Endothelial Cell Adhesion Molecule-1; RNA, Messenger

Common in vitro protocols for chondrogenesis of mesenchymal stem cells (MSCs) induce an inadequate, hypertrophic differentiation cascade reminiscent of endochondral bone formation. We aimed to modify chondrogenic protocols in order to identify potent inducers, promotors, and inhibitors to achieve better chondrogenesis. Nine factors suspected to stimulate or inhibit chondrogenesis were used for chondrogenic in vitro induction of MSC. Differentiation was assessed by immunohistochemistry, alcian-blue staining, qRT-PCR, and quantification of alkaline phosphatase (ALP) activity. Pre-differentiated pellets were transplanted subcutaneously into SCID mice to investigate stable cartilage formation. Transforming growth factor (TGF)-beta was always required for chondrogenic differentiation and deposition of a collagen-type-II-positive extracellular matrix, while bone morphogenetic protein (BMP)-2, -4, -6, -7, aFGF, and IGF-I (10 ng/ml) were alone not sufficiently inductive. Each of these factors allowed differentiation in combination with TGF-beta, however, without preventing collagen type X expression. bFGF or parathyroid hormone-like peptide (PTHrP) inhibited the TGF-beta-responsive COL2A1 and COL10A1 expression and ALP induction when added from day 0 or 21. In line with a reversible ALP inhibition, in vivo calcification of pellets was not prevented. Late up-regulation of PTH1R mRNA suggests that early PTHrP effects may be mediated by a receptor-independent pathway. While TGF-beta was a full inducer, bFGF and PTHrP were potent inhibitors for early and late chondrogenesis, seemed to induce a shift from matrix anabolism to catabolism, but did not selectively suppress COL10A1 expression. Within a developmental window of collagen type II(+)/collagen type X(-) cells, bFGF and PTHrP may allow inhibition of further differentiation toward hypertrophy to obtain stable chondrocytes for transplantation purposes.

Topics: Alcian Blue; Alkaline Phosphatase; Animals; Biomarkers; Bone Morphogenetic Proteins; Cell Differentiation; Cell Shape; Cells, Cultured; Chondrocytes; Chondrogenesis; Collagen Type II; Collagen Type X; Coloring Agents; Fibroblast Growth Factor 1; Humans; Hypertrophy; Immunohistochemistry; Insulin-Like Growth Factor I; Intercellular Signaling Peptides and Proteins; Matrix Metalloproteinase 13; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, SCID; Parathyroid Hormone-Related Protein; Peptide Fragments; Phenotype; Receptor, Parathyroid Hormone, Type 1; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Staining and Labeling; Time Factors; Transforming Growth Factor beta

Determination of hyperplastic and hypertrophic changes of mucus-secreting cells in animal airways has been performed in the past by using histologic, immunologic, and/or molecular biologic approaches. Histologic techniques are tedious and time-consuming. The other approaches require specific antibodies and cDNA probes that have proved difficult to develop. Described here is a method for the rapid estimation of hyperplastic and hypertrophic changes of secretory epithelial cells in rat airways. The assay specifically measures acidic and neutral mucoproteins in a linear fashion from 0.5 microgram to at least 10 micrograms. Male Sprague-Dawley rats were exposed to metabisulfite mist (10% wt/vol) for 5 days/wk for 3 wk. The lungs were removed and homogenized in a phosphate-buffered solution containing reducing agents and protease inhibitors. The particulate matter was removed by centrifugation, and the soluble extract was applied to a column packed with Sepharose CL-6B. The material eluting in the void volume was applied to a PVDF membrane and stained for either acidic or neutral mucosubstances using Alcian blue or periodic acid-Schiff (PAS) staining, and the absorbance was read using a 96-well plate reader. Lungs from sodium metabisulfite-exposed animals showed a 7-fold and 3.5-fold increase in PAS-positive and Alcian blue-positive material, respectively. The increase in both PAS and Alcian blue staining was hyaluronidase and chondroitinase insensitive. The observed changes are consistent with morphometric measurements of mucus-containing cells in histologic sections of the tissues. This assay may be useful in determining which neurohumoral mediators might be involved in mucus cell hypertrophy and hyperplasia in animal models of chronic obstructive pulmonary disease.

Topics: Administration, Inhalation; Alcian Blue; Animals; Chromatography, Agarose; Epithelium; Histocytochemistry; Hyperplasia; Hypertrophy; Lung; Male; Mucins; Periodic Acid-Schiff Reaction; Proteoglycans; Rats; Rats, Sprague-Dawley; Staining and Labeling; Sulfites; Trachea