Osteoblastic Cells From Rat Long Bone Ii: Adhesion to Substrata and Integrin Expression in Primary and Propagated Cultures

Abstract

Propagation in vitro of rat tibial osteoblasts (ROB) is accompanied by increased expression of the early osteogenic marker alkaline phosphatase (AP) and maturation of the osteogenic phenotype. In order to establish the pattern of the integrin expressed in ROB during progression to the mature osteoblastic phenotype, we have used biosynthetic, immunoblotting and immunohistochemical assays. We immunoprecipitated from osteoblasts, expanded for 1.5- and 7.5-doubling, alpha 5 beta 1, alpha v beta 3, alpha 3 beta 1, alpha 6 beta 1 and alpha 1 beta 1 integrin heterodimers; furthermore beta 5, alpha 2 and alpha 4 chains were detected by immunoblots and indirect immunofluorescence. alpha v, alpha 1, alpha 6 subunits in most cells, and beta 3 and beta 1 subunits in a minority, were found to be associated with adhesion plaques in osteoblasts of 1.5-, 4.5- and 7.5-doubling grown in the presence of FCS, while all other subunits stained diffusely all the cells. Adhesion to fibronectin (FN), laminin (LN), collagen type I (COL I) and III(COL III) by ROB at different doubling (1.5-11) was dependent on substratum concentration, and after 2.5 h at 55 nM 60% of the cells adhered to all substrata. Arg-Gly-Asp-Ser (RGDS) containing peptides inhibited adhesion of cells differentially, according to substratum; no dependence on extent of progation in vitro was observed. In conclusion, ROB cultured in vitro for 1.5- to 11-doubling had an unchanged pattern of expression of integrin subunits, heterodimer association and cellular distribution. Adhesion specificity and affinity were also unchanged. These results suggest that the phenotypic maturation, detected as an increase in AP expression, is not accompanied by major changes in the potential for cell-matrix interactions, and does not correspond to changes in the type of integrin subunits expressed by osteoblasts.

Detachment strength of human osteoblasts cultured on hydroxyapatite with various surface roughness. Contribution of integrin subunits

“…Osteoblasts generally express integrin subunits a 1 , a 2 , a 3 , a 4 , a 5 , a 6 , a V , b 1 , b 3 , and b 5 on various substrates [24]. For attachment to fibronectin and collagen type I, osteoblasts express integrin subunit b 1 .…”

Extracellular matrix‐mimetic adhesive biomaterials for bone repair

“…Alpha subunit expression data have been more inconsistent, with different combinations of α1, α2, α3, α4, α5, and αv subunits having been detected by immunohistochemistry in human and rat bone 30–34. The expression of the previously mentioned alpha subunits has also variously been determined by flow cytometry, immunoprecipitation, immunocytochemistry, and Northern blot analysis on primary bone cultures 29–31, 35–39. Although reports of alpha subunit and integrin heterodimer expression in osteoblasts have sometimes been contradictory, many studies have identified the α1β1, α2β1, α3β1, α5β1, αvβ3 integrins, and their subunits in osteoblasts and bone cultures 29, 33, 37, 38.…”

The Expression of Metalloproteinase-2, −9, and −14 and of Tissue Inhibitors-1 and −2 Is Developmentally Modulated During Osteogenesis In Vitro, the Mature Osteoblastic Phenotype Expressing Metalloproteinase-14

“…Changes ensuing from ECM proteolysis elicit cellular responses through integrin and nonintegrin receptors (56,61,70–72) and, in turn, integrin occupancy can affect differentiation and MMP expression (59,62,63) . During osteogenesis, the modulation of synthesis and deposition of the ECM and of the associated endogenous growth factors and cytokines change the osteoblast microenvironment.…”