Cellular expression of bone‐related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures

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.

Bone induction by BMP‐2 transduced stem cells derived from human fat

“…OC is another late bone marker only secreted by osteoblasts [20] and also signals terminal osteoblast differentiation [7]. OC was not found in any cultures at four and seven days.…”

Sustained In Vitro Expansion of Bone Progenitors Is Cell Density Dependent

“…Identifying a relationship between population growth, nodule formation, and expression of surface or other osteoblast markers is the object of much study [15,16,28,[49][50][51][52]. To this end, separation of the input RC population into four purified fractions (ALP showed that nodules are able to form within each fraction [53].…”

The effects of patient age on human osteoblasts' response to Ti–6Al–4V implants in vitro

“…CA) and the area of nodules was measured using Sigmascan software (Jandel Corporation, San Rafael, CA). The number of nodules was counted in all groups with four patients in each [30][31][32][33][34][35][36][37][38] group and two separate experiments (with different subjects) were performed.…”

“…After cells reach confluence, nodules of multilayered osteoblasts form in scattered foci. The nodules stain for alkaline phosphatase [4,42], and are the regions where bone extracellular matrix proteins are deposited [36] and mineralization occurs. Previous studies have shown that the mineralized nodules resemble woven bone that is produced in vivo [4,8,13,20,42].…”