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Cellular expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures.
J Cell Physiol 1994; 158(3):555-72JC

Abstract

Rat bone marrow stromal cells comprise a heterogeneous mixture of cell lineages including osteoblastic cells. When grown in the presence of ascorbic acid, beta-glycerophosphate and 10(-8) M dexamethasone, osteoprogenitor cells within the population divide and differentiate to form bone nodules (Maniatopoulos et al., 1988, Cell Tissue Res., 254:317-330; Aubin et al., 1990, J. Bone Miner. Res., 5:S81) providing a useful model to investigate temporal and spatial changes in expression of osteoblastic markers. Immunocytochemistry was combined with Northern blotting, enzymatic assay, and radioimmunoassay to analyze the expression of bone-related proteins during the growth and differentiation sequence. By mRNA levels, protein production and/or enzymatic activity, expression of osteocalcin, bone sialoprotein, and alkaline phosphatase increased concomitantly with the development of bone nodules, while osteopontin mRNA levels decreased and those of SPARC/osteonectin did not change significantly. In older cultures with mineralizing nodules, mRNA levels for alkaline phosphatase and bone sialoprotein, but not osteocalcin, declined. Immunolabeling revealed that cells in early cultures stained poorly for SPARC/osteonectin and strongly for thrombospondin. Later, SPARC/osteonectin staining increased in most cells, while thrombospondin staining could be seen in both matrix and in cells, but with marked intercellular variability in intensity. At all time points studied, osteoblasts within bone nodules stained homogeneously for thrombospondin and alkaline phosphatase, and with marked heterogeneity of intensity amongst cells for SPARC/osteonectin and osteocalcin. Labelling with RCC455.4, a monoclonal antibody raised against rat calvaria cells which intensely labels osteoblasts and osteocytes (Turksen et al., 1992, J. Histochem. Cytochem., 40:1339-1352), co-localized with osteocalcin. Alkaline phosphatase activity and the amount of osteocalcin determined by both radioimmunoassay and immunolabelling decreased in very late cultures, a time corresponding to appearance of fully mineralized nodules. These studies indicate that the bone marrow stromal cell system is a useful model to study the temporal and spatial expression of bone-related proteins during osteogenesis and formation, mineralization, and maturation of bone nodules. Further, immunolabelling at the individual cell and single bone nodule level allowed discrimination of marked variability of expression of osteoblast markers during the differentiation sequence.

Authors+Show Affiliations

Medical Research Council Group in Periodontal Physiology, University of Toronto, Ontario, Canada.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

8126078

Citation

Malaval, L, et al. "Cellular Expression of Bone-related Proteins During in Vitro Osteogenesis in Rat Bone Marrow Stromal Cell Cultures." Journal of Cellular Physiology, vol. 158, no. 3, 1994, pp. 555-72.
Malaval L, Modrowski D, Gupta AK, et al. Cellular expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures. J Cell Physiol. 1994;158(3):555-72.
Malaval, L., Modrowski, D., Gupta, A. K., & Aubin, J. E. (1994). Cellular expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures. Journal of Cellular Physiology, 158(3), pp. 555-72.
Malaval L, et al. Cellular Expression of Bone-related Proteins During in Vitro Osteogenesis in Rat Bone Marrow Stromal Cell Cultures. J Cell Physiol. 1994;158(3):555-72. PubMed PMID: 8126078.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Cellular expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures. AU - Malaval,L, AU - Modrowski,D, AU - Gupta,A K, AU - Aubin,J E, PY - 1994/3/1/pubmed PY - 1994/3/1/medline PY - 1994/3/1/entrez SP - 555 EP - 72 JF - Journal of cellular physiology JO - J. Cell. Physiol. VL - 158 IS - 3 N2 - Rat bone marrow stromal cells comprise a heterogeneous mixture of cell lineages including osteoblastic cells. When grown in the presence of ascorbic acid, beta-glycerophosphate and 10(-8) M dexamethasone, osteoprogenitor cells within the population divide and differentiate to form bone nodules (Maniatopoulos et al., 1988, Cell Tissue Res., 254:317-330; Aubin et al., 1990, J. Bone Miner. Res., 5:S81) providing a useful model to investigate temporal and spatial changes in expression of osteoblastic markers. Immunocytochemistry was combined with Northern blotting, enzymatic assay, and radioimmunoassay to analyze the expression of bone-related proteins during the growth and differentiation sequence. By mRNA levels, protein production and/or enzymatic activity, expression of osteocalcin, bone sialoprotein, and alkaline phosphatase increased concomitantly with the development of bone nodules, while osteopontin mRNA levels decreased and those of SPARC/osteonectin did not change significantly. In older cultures with mineralizing nodules, mRNA levels for alkaline phosphatase and bone sialoprotein, but not osteocalcin, declined. Immunolabeling revealed that cells in early cultures stained poorly for SPARC/osteonectin and strongly for thrombospondin. Later, SPARC/osteonectin staining increased in most cells, while thrombospondin staining could be seen in both matrix and in cells, but with marked intercellular variability in intensity. At all time points studied, osteoblasts within bone nodules stained homogeneously for thrombospondin and alkaline phosphatase, and with marked heterogeneity of intensity amongst cells for SPARC/osteonectin and osteocalcin. Labelling with RCC455.4, a monoclonal antibody raised against rat calvaria cells which intensely labels osteoblasts and osteocytes (Turksen et al., 1992, J. Histochem. Cytochem., 40:1339-1352), co-localized with osteocalcin. Alkaline phosphatase activity and the amount of osteocalcin determined by both radioimmunoassay and immunolabelling decreased in very late cultures, a time corresponding to appearance of fully mineralized nodules. These studies indicate that the bone marrow stromal cell system is a useful model to study the temporal and spatial expression of bone-related proteins during osteogenesis and formation, mineralization, and maturation of bone nodules. Further, immunolabelling at the individual cell and single bone nodule level allowed discrimination of marked variability of expression of osteoblast markers during the differentiation sequence. SN - 0021-9541 UR - https://www.unboundmedicine.com/medline/citation/8126078/Cellular_expression_of_bone_related_proteins_during_in_vitro_osteogenesis_in_rat_bone_marrow_stromal_cell_cultures_ L2 - https://doi.org/10.1002/jcp.1041580322 DB - PRIME DP - Unbound Medicine ER -