The distribution of fibronectin in rat tooth and periodontal tissues: an immunofluorescence study using a monoclonal antibody

The distribution of fibronectin (FN) in longitudinal, buccolingual sections of decalcified adult rat periodontium and teeth was studied by indirect immunofluorescence using a monoclonal antibody. FN was present in virtually all regions of the periodontium, including the gingiva, periodontal ligament, many blood vessel walls, alveolar bone, incisor and molar predentine and dentine, and molar acellular and cellular cementum. The cementum of the incisor, ameloblasts, stratum intermedium and stellate reticulum, and the connective tissue of the pulp and the surface of ondontoblasts facing the pulp in the incisor and molar were not labeled for FN. FN distribution was not always uniform either within a given connective tissue or between different connective tissues of the same organ.     

Stanniocalcin 1 (STC1) protein and mRNA are developmentally regulated during embryonic mouse osteogenesis: the potential of stc1 as an autocrine/paracrine factor for osteoblast development and bone formation.

STC1, a mammalian homologue of stanniocalcin (STC) which plays a major role in calcium/phosphate homeostasis in fish, has been recently isolated. We have characterized the spatiotemporal distribution of STC1 mRNA and protein during mouse embryonic development generally and osteogenesis specifically. Northern blotting analysis of whole embryos showed that STC1 mRNA is highly and differentially expressed during embryogenesis. By in situ hybridization, STC1 mRNA was detected early in mesenchymal condensations and was then found to be highly expressed in perichondrial cells, periosteal cells, and then osteoblasts during endochondral bone formation. In bones forming by intramembranous ossification, STC1 mRNA was not detected until osteogenic cells appeared. The cellular distribution of STC1 protein closely corresponded to that of its mRNA, but the protein was also detected in hypertrophic chondrocytes. In the MC3T3-E1 osteogenic cell model, STC1 protein and mRNA were detectable throughout proliferation and differentiation stages but levels were relatively higher late during nodule formation/mineralization phases. For comparison, STC1 mRNA was also found in epithelial cells of both embryonic and adult intestine that had not previously been described among tissues responsive to calcium/phosphate transport. These results suggest that STC1 is expressed in a time- and cell-specific manner and may play an autocrine/paracrine role during osteoblast development and bone formation.     

Synthesis of collagen, collagenase and collagenase inhibitors by cloned human gingival fibroblasts and the effect of concanavalin A

Individual clones of human gingival fibroblasts that differ in morphology and growth characteristics have been found to synthesize collagen (types I, III and V), collagenase and collagenase inhibitors, and to be capable of degrading native collagen mats. Although collagenase activity was normally low, synthesis of the enzyme could be stimulated ten-fold by Concanavalin A. These results demonstrate that individual fibroblasts have the ability to both synthesize and degrade collagen.     

Phenotypic differences in subclones and long-term cultures of clonally derived rat bone cell lines

Previous studies with clonally derived populations of cells have shown that cells released from embryonic rat calvaria by enzymatic digestion are heterogeneous with respect to their hormone responsiveness, morphology, and production of matrix components [Aubin JE et al; J. Cell Biol 92:452, 1982]. Several of these clonal populations have been used to study the effects of long-term culture and inter- and intraclonal cell heterogeneity. During continuous subculture, marked changes in collagen synthesis were observed in two clonal populations. Both of these clones were originally responsive to parathyroid hormone (PTH) and synthesized primarily type I collagen with small amounts of type III and V collagens, although one clone (RCJ 3.2) had a fibroblastic morphology whereas the second clone (RCB 2.2) displayed a more polygonal shape. Following routine subculture over 3 yr, clone RCB 2.2 was found to synthesize exclusively alpha 1(I)-trimer and not other interstitial collagens. When the same cells were maintained at confluence for 1-2 wk, however, they also synthesized type III collagen. Whereas RCJ 3.2 did not show such dramatic changes in collagen synthesis after long-term subculture, two subclones derived from RCJ 3.2 were found to synthesize almost exclusively either type III collagen (RCJ 3.2.4.1) or type V collagen (RCJ 3.2.4.4). Immunocytochemical staining indicated that both subpopulations also produced type IV collagen, laminin, and basement membrane proteoglycan, proteins that are typically synthesized by epithelial cells. The differences in collagen expression by the various clonal cell populations were accompanied by qualitative and quantitative differences in other secreted proteins and differences in cell morphology. The results demonstrate both the inter- and intraclonal heterogeneity of connective tissue cells and their diverse potentiality with respect to extracellular matrix synthesis.     

Cocktail-party effect in king penguin colonies

The king penguin, Aptenodytes patagonicus, breeds without a nest in colonies of several thousands of birds. To be fed, the chick must recognize the parents in a particularly noisy environment using only vocal cues. The call an adult makes when seeking the chick is emitted at a high amplitude level. Nevertheless, it is transmitted in a colonial context involving the noise generated by the colony and the screening effect of the bodies, both factors reducing the signal-to-noise ratio. In addition, the adult call is masked by a background noise with similar amplitude and spectral and temporal characteristics, enhancing the difficulty for the chick in finding its parents. We calculate that the maximum distance from the caller at which its signal can be differentiated from the background noise (signal-to-noise ratio equal to 1) should not exceed 8 to 9 m in a feeding area. But our tests show that, in fact, chicks can discriminate between the parental call and calls from other adults at a greater distance, even when call intensity is well below that of the noise of simultaneous calls produced by other adults. This capacity to perceive and extract the call of the parent from the ambient noise and particularly from the calls of other adults, termed the ''cocktail-party effect'' in speech intelligibility tests, enhances the chick''s ability to find its parents.     

Isolation of monoclonal antibodies recognizing rat bone-associated molecules in vitro and in vivo.

Knowledge of the number and kinds of differentiation steps that characterize cells of the osteoblast lineage is inadequate. To further analyze osteoblast differentiation, we generated a series of monoclonal antibodies (MAb) to osteogenic cells. Spleen cells from mice immunized with whole-cell populations enriched for expression of osteoblast-associated properties or bone formation in vitro were fused with the SP2/0 myeloma cell line. Supernatants from growing hybridomas were screened by indirect immunofluorescence on frozen sections of a portion of 21-day fetal rat heads that included the calvaria bone, periosteum, muscle, fibrous connective tissue, and skin. Six MAb were selected with bone-associated staining and limited ability to label other tissues. Either cell surface or cytoplasmic molecules were recognized by five of the MAb; one recognized a molecule detectable both in the cytoplasm, on the cell surface, and in the extracellular matrix. Of the antibodies selected, one identified both preosteoblasts and osteoblasts and has been found to be against alkaline phosphatase. The others recognized the mature osteoblasts, osteocytes, and chondrocytic cells. The pattern and distribution of the labeling in vivo extended to primary cells and cell lines in vivo. These results support earlier observations on molecules differentially expressed by cells at different stages of the osteoblast lineage and extend the available cell surface and cytoplasmic epitopes identifiable as marker molecules.     

Positive and negative immunoselection for enrichment of two classes of osteoprogenitor cells.

The number of identifiable stages and expression of differentiation markers in cells of the osteoblast lineage are not well understood. In the present study, a mAb, designated rat bone marrow (RBM) 211.13, was prepared that stained selectively the osteogenic and preosteoblastic cells along the surfaces of bone in calvariae, femurs, and metatarsals. The staining was cell surface associated and coincided with that for alkaline phosphatase (APase) detected histochemically. Only cells positive for APase activity by biochemical assay and not those without APase activity (e.g., fetal rat skin) stained with RBM 211.13. By immunoblotting, RBM 211.13 recognized a band coinciding with APase activity on nonreducing/nondenaturing gels, and RBM 211.13 precipitated a protein which on reduced gels migrated with an apparent molecular mass of approximately 80 kD. RBM 211.13 labeling was abolished by phosphatidylinosital-specific phospholipase C, known to release APase from the cell surface. All of these data support the concept that RBM 211.13 recognizes the bone isoenzyme of APase. RBM 211.13 was used to sort by flow cytometry the APase-positive and APase-negative cells from mixed fetal rat calvaria (RC) cell populations. The osteoprogenitors we identified earlier that form bone nodules in vitro (Bellows, C. G., J. E. Aubin, J. N. M. Heersche, and M. E. Antosz. 1986. Calcif. Tissue Int. 36:143-154; Bellows, C. J., J. N. M. Heersche, and J. E. Aubin. 1990. Dev. Biol. 140:132-138) were found within the APase-positive pool. By immunopanning, RC cells were separated into APase-enriched (APase-positive, adherent) and APase-depleted (APase-negative, nonadherent) populations. The APase-positive fraction was enriched two-to-threefold for bone-forming osteoprogenitors compared to unfractionated cells, while the APase-negative population formed very few nodules under the same conditions. Both populations responded to the glucocorticoid dexamethasone (DEX) with an increase in bone nodule formation. However, the fold stimulation in bone formation in the APase-negative population was approximately 30-fold, while the fold stimulation in the APase-positive population was only approximately 5-fold. These data suggest that APase expression can be used for immunoselection to fractionate osteoblastic populations into an APase-positive population and a population initially APase-negative, that virtually all osteoprogenitors forming bone in vitro in the absence of added glucocorticoids reside in the APase-positive pool, and that the only osteoprogenitors present in the APase-negative pool are those requiring DEX to differentiate.